Biotechnological Approaches to Sustainable Development Goals 3031333691, 9783031333699

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Patrick Omoregie Isibor Paul Akinduti Solomon U. Oranusi Jacob O. Popoola   Editors

Biotechnological Approaches to Sustainable Development Goals

Biotechnological Approaches to Sustainable Development Goals

Patrick Omoregie Isibor  •  Paul Akinduti Solomon U. Oranusi  •  Jacob O. Popoola Editors

Biotechnological Approaches to Sustainable Development Goals

Editors Patrick Omoregie Isibor Department of Biological Sciences Covenant University Ota, Ogun State, Nigeria

Paul Akinduti Department of Biological Sciences Covenant University Ota, Ogun State, Nigeria

Solomon U. Oranusi Department of Biological Sciences Covenant University Ota, Ogun State, Nigeria

Jacob O. Popoola Department of Biological Sciences Bowen University Iwo, Osun State, Nigeria

ISBN 978-3-031-33369-9    ISBN 978-3-031-33370-5 (eBook) https://doi.org/10.1007/978-3-031-33370-5 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023, Corrected Publication 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

The International Biotechnology Conference, Exhibition, and Workshop (IBCEW) is a biennial conference sponsored by the Department of Biological Sciences at Covenant University in Ota, Ogun State, Nigeria. The theme of the 2022 conference was “Biotechnological Tools to Achieving Sustainable Development Goals.” IBCEW 2022 was an extension of a hands-on training workshop successfully held in 2021 in collaboration with Inqaba Biotech Limited, Nigeria. This book is a compilation of the IBCEW 2022 proceedings, which emphasize Covenant University’s sustainable economy and society goals. The title of the book Biotechnological Approaches to Sustainable Development Goals reflects the conference’s theme, aimed at documenting modern biotechnological techniques in a pedagogically compelling fashion. The book comprises five sections emanating from the conference’s sub-themes: Part I - Food and Sustainable Agriculture Part II - Climate Change and the Environment Part III - Health Pandemic and Biotechnology Part IV - Biotechnology for Sustainable Economy Part V - Industrial Biotechnology and SDGs All chapters were peer-reviewed in a double-blind editorial system. Ota, Ogun State, Nigeria   Iwo, Osun State, Nigeria

Patrick Omoregie Isibor Paul Akinduti Solomon U. Oranusi Jacob O. Popoola

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Contents

Part I Food and Sustainable Agriculture Mycotoxin Occurrence and Risk Assessment in Infants and Young Children (IYC) Formulated Foods in Southwest Nigeria���������    3 Comfort Adebukola Adelodun, Solomon U. Oranusi, Dango Zilpah George, Paul Akinduti, and Yemisi Dorcas Obafemi In Vitro Antifungal Activity of Bauhinia monandra (Kurz) Leaf Extracts Against Fungal Pathogens Isolated from Spoilt Musa paradisiaca L. ��������������������������������������������������������������������   17 Margaret Ikhiwili Oniha, Michelle Kaosisochukwu Aniebonam, Eze Frank Ahuekwe, Stephen Oluwanifise Oyejide, and Olugbenga Samson Taiwo Effect of Aspergillus fumigatus MT899185 Phytase Addition on the Nutritional and Phytate Content of Formulated Cowpea-Based Poultry Feed������������������������������������������������������������������������������������������������������   27 Adeola Elizabeth Onibokun, Angela Obiageli Eni, and Solomon U. Oranusi Histology, Condition Factor, and Bioaccumulation Analysis of Clarias gariepinus (Burchell, 1822) Exposed to MC-LR��������������������������   37 Patrick Omoregie Isibor, Onwaeze Oritseweyinmi Ogochukwu, David Osagie Agbontaen, Paul Akinduti, Adagunodo Theophilus Aanuoluwa, Obafemi Yemisi, Dedeke Gabriel Akinwumi, and Akinsanya Bamidele Morphological Trait Variation and Correlation Analysis in Landraces of Southern Nigerian Fluted Pumpkin (Telfairia occidentalis Hook. F.) ����   53 Oluwadurotimi S. Aworunse, Jacob O. Popoola, Lawrence S. Fayeun, Eze Frank Ahuekwe, and Olawole O. Obembe

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Contents

Part II Climate Change and the Environment Comparative Analysis of the Trophic Status, Length-Weight Relationship, Gastro-somatic Index and Bioaccumulation of Trace Metals in Wild and Captive Clarias gariepinus ��������������������������������������������   85 Patrick Omoregie Isibor, Onwaeze Oritseweyinmi Ogochukwu, Iloh Ekene Valerie, Taiwo Olugbenga Samson, Paul Akinduti, and Obafemi Yemisi Occurrence and Characteristics of Microplastics in the Surface Water and Sediment of Lagos Lagoon, Nigeria��������������������������������������������  103 Fadekemi O. Akinhanmi, Opeyemi I. Ayanda, and Gabriel A. Dedeke  ssessment of the Effectiveness of Chlorination for Drinking Water A Treatment����������������������������������������������������������������������������������������������������������  119 Olubukola Oziegbe, Olusola Ojo-Omoniyi, Eze Frank Ahuekwe, Obinna C. Nwinyi, Paul Eyinnaya Atulegwu, and Etitua Julius Oziegbe Part III Health Pandemic and Biotechnology Lack of Association of CYP2C9:c.430C>T and SCN1A:c.3184A>G Polymorphisms with Epilepsy Risk or Drug Resistance in Childhood Epilepsy Syndromes in Lagos State, Nigeria��������������������������  131 Ibitayo Abigail Ademuwagun, Solomon Oladapo Rotimi, and Ezekiel Adebiyi Computational Approaches Toward Prevention and Surveillance of Lassa Fever in Developing Countries��������������������������������������������������������  145 Gift Nzubechi Elughi, Margaret Ikhiwili Oniha, Bowofoluwa Sharon Abimbola, Kesioluwa Eunice Orukotan, Eze Frank Ahuekwe, and Paul Akinduti Antibacterial Efficacy of Thymus vulgaris Essential Oil Against Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Urinary Tract Infections ����������������������������������������������������������������������������  159 S. O. Egwuatu, T. M. Obuotor, O. S. Taiwo, W. E. Ike, A. E. Ojo, Patrick Omoregie Isibor, O. F. Adeniji, F. M. Oyeyipo, O. A. Awotoye, and Paul Akinduti Antibacterial Efficacy of Syzygium aromaticum Essential Oil Against Extended Spectrum Beta-Lactamase-Producing Escherichia coli in Urinary Tract Infections ����������������������������������������������������������������������������  173 S. O. Egwuatu, O. S. Taiwo, T. M. Obuotor, M. I. Oniha, O. Oziegbe, S. O. Adebajo, W. E. Ike, F. M. Oyeyipo, A. O. Kuye, and Paul Akinduti  rowth and Haemato-Biochemical Responses of All-Male Tilapia, G Oreochromis niloticus, to Diets Containing Fermented Cassava Leaf Meal����������������������������������������������������������������������������������������������������������  187 Oluwagbenga O. Olude and Paul Akinduti

Contents

ix

Implication of Age-Demography of Mycobacterium tuberculosis Infection Among HIV-Seropositive and HIV-Seronegative Individuals ����  205 A. D. Akinyosoye, M. I. Oniha, T. J. Oduselu, J. A. Akinbo, and Paul Akinduti Comparative Phytochemical Contents and Antioxidant Activities of Tapinanthus cordifolius and Irvingia wombolu Leaf Extracts������������������  215 Chike-Ekwughe Amarachi, Aliyu Najeeb Olamilekan, Aliyu Adamu, Adebayo Abiodun Humphery, and Ogunlana Olubanke Olujoke The Central Metabolism Model of Anopheles gambiae: A Tool for Understanding Malaria Vector Biology ��������������������������������������  229 Eunice O. Adedeji, Olubanke O. Ogunlana, Segun Fatumo, Olufemi T. Aromolaran, Thomas Beder, Rainer Koenig, and Ezekiel Adebiyi Part IV Biotechnology for Sustainable Economy Plant Microbiome Engineering: Principles, Methods, and Current Trends ����������������������������������������������������������������������������������������  251 Kesioluwa Eunice Orukotan, Gift Nzubechi Elughi, Bowofoluwa Sharon Abimbola, Abimbola David Akinyosoye, Eze Frank Ahuekwe, and Olubukola Oziegbe  ater Purification Potentials of Crustacean Chitosan��������������������������������  269 W Patrick Omoregie Isibor, Paul Akinduti, Oniha Margaret Ikhiwili, Adagunodo Theophilus Aanuoluwa, and Obafemi Yemisi Dorcas Part V Industrial Biotechnology and SDGS Omics and Mutagenesis: Molecular Optimization Strategies for Strain Improvement in Biosurfactant Production����������������������������������  291 Bowofoluwa Sharon Abimbola, Eze Frank Ahuekwe, Kesioluwa Eunice Orukotan, Abimbola David Akinyosoye, Fadekemi Akinhanmi, and Obinna C. Nwinyi Comparison of Two Extraction Methods to Obtain Quality Genomic DNA from Eggplants (Solanum sp.)����������������������������������������������  305 Ajiboye I. Babafemi, Olatunde Temitope, Jacob O. Popoola, and Omonhinmin A. Conrad Correction to: Morphological Trait Variation and Correlation Analysis in Landraces of Southern Nigerian Fluted Pumpkin (Telfairia occidentalis Hook. F.)����������������������������������������������������������������������  C1 Oluwadurotimi S. Aworunse, Jacob O. Popoola, Lawrence S. Fayeun, Eze Frank Ahuekwe, and Olawole O. Obembe Index������������������������������������������������������������������������������������������������������������������  317

About the Editors

Patrick  Omoregie  Isibor  is currently a Lecturer in the Department of Biological Sciences at Covenant University. He received his Ph.D. and M.Sc. in Environmental Quality Management from the University of Benin and a B.Sc. in Zoology from Ambrose Alli University. Dr. Isibor’s research interests include ecotoxicology, hydrobiology, bioaccumulation, biosequestration, biodiversity conservation, and aquatic ecology. He is a member of the Association for  Environmental Impact Assessment of Nigeria (AEIAN), the International Association of Risk and Compliance Professionals (IARCP), and the African Society for Toxicological Sciences (ASTS). He is an Editor for the African Journal of Health, Safety, and Environment and a reviewer for several reputable international journals. Paul Akinduti  is a Lecturer and researcher with the Microbiology Unit of the Department of Biological Sciences at Covenant University. He received his Ph.D. in Medical Microbiology and Bacteriology from Olabisi Onabanjo University. He is a Fellow of the Medical Laboratory Science Council of Nigeria and a recipient of the World Academy of Sciences and Bill and Melinda Gates Foundation Travel Awards. Dr. Akinduti’s areas of research interest include antimicrobial agents, antimicrobial resistance, molecular biology, bacterial pathogenesis, antimicrobial susceptibility testing, bacterial antibiotic resistance, bacteriology, antibacterial activity, bacterial drug resistance, xi

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About the Editors

diagnostic microbiology, molecular bacteriology, molecular microbiology, foodborne diseases, molecular typing, microbial genetics, bacterial toxins, bacterial conjugation, and pathogenesis. Solomon U. Oranusi  is a Professor of Microbiology at Covenant University, where he has also served as Sub-Dean of the School of Postgraduate Studies. Prof. Solomon holds a B.Sc. degree in Microbiology from the University of Nigeria Nsukka (UNN) and an M.Sc. and Ph.D. in Microbiology from Ahmadu Bello University (ABU) Zaria. He is a Fellow of the Institute of Medical Laboratory Science Council of Nigeria and a Public Analyst with MIPAN. His research interests include biogas and digestate bio-fertilizer production from food and agricultural wastes (waste-to-wealth), food safety, HACCP, and public and environmental health. Professor Oranusi is a member of several academic and professional organizations, including the Institute of Public Analysts of Nigeria (IPAN), the Association of Industrial Microbiologists of Nigeria (AIMN), the Nigerian Institute of Food Science and Technology (NIFST), the Institute of Medical Laboratory Sciences of Nigeria (IMLSN), and the American Society for Microbiology (ASM). He has published more than 130 peer-reviewed scientific articles and is a Reviewer for several international journals. He has served as an examiner for postgraduate theses and dissertations and as an assessor for the promotion of applicants to the professorial cadre of academic institutions. Jacob O. Popoola  is an Associate Professor of Pure and Applied Biology at Bowen University. He was previously a researcher and Senior Lecturer in the Department of Biological Sciences at Covenant University, where he conducted research in applied ethnobotany, plant genetics, and molecular biology. Dr. Popoola received his Ph.D. in Biology (Plant Genetics and Biotechnology) from Covenant University. His current projects include sequencing diversity of Moringa oleifera towards genetic improvement, biosystematic studies of miscellaneous neglected underutilized legumes as future food security, and characterization of root and tubers for utilization.

List of Figures

Fig. 1 Fig. 2 Fig. 3

Frequency of occurrence of fungal isolates from Musa paradisiaca���������������������������������������������������������������������   22 Mean zones of inhibition for antifungal activity of aqueous leaf extract of B. monandra against fungal isolates��������������������������   23 Mean zones of inhibition for antifungal activity of ethanol leaf extract of Bauhinia monandra against fungal isolates���������������   24

Fig. 1

Phytate contents of formulated cowpea-based feed and control group (Feed 1). Key: Feed 1 = commercial poultry feed, feed 2 = cowpea-based feed formulated with addition of Aspergillus fumigatus phytase MT899185, feed 3 = cowpea-based feed formulated with addition of commercial phytase, feed 4 = cowpea-based feed without the addition of phytase�����������   31

Fig. 1

H & E-stained microscopic view of dissected liver tissue of fish. (a) shows the photograph of normal catfish liver, while (b) showed a slight debris (black arrow), and (c) exhibited necrosis (yellow arrow), and discoloration (black arrow), while (d) exhibited necrosis (black arrow), severe discoloration (yellow arrow) and vacuolization (blue arrow). Scale bar = 25 μm��������������������������������������������������������   42 H & E-stained microscopic view of dissected gill tissue of fish shows retained gill architectural morphology in Eleyele River (a). Fishes from Pond A (b) showed no observable alterations. Fish samples from Pond B (c) showed necrosis (black arrow), while those from Iju River (d) showed desquamation (black arrow) and hyperplasia and epithelial lifting at the secondary lamellae (red arrow). Scale bar = 25 μm�����������������������������������������������������������������������������   46 H & E-stained microscopic view of the skeletal muscle tissue of fish showed no observable change in the skeletal muscle tissue of fish from Eleyele River (a) and Pond A (b). Fish samples harvested from Pond B (c) showed slight

Fig. 2

Fig. 3

xiii

xiv

List of Figures

observable lesions (white arrow) amidst the muscle fibres, while those harvested from Iju River (d) showed swelling of skeletal muscle fibres (white arrow), degenerated cell (black arrow) and oedema (yellow arrow). Scale bar = 25 μm���������   47 Fig. 1 Fig. 2 Fig. 3

Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7

Fig. 1 Fig. 2

A two-dimensional scatter plot of PC1 and PC2 based on quantitative trait vector loadings of the Telfairia occidentalis landraces�������������������������������������������������������������������������������������������   65 Dendrogram resulting from cluster analysis of the 32 Telfairia occidentalis landraces based on Bray-Curtis similarity coefficient�������������������������������������������������������������������������   66 Qualitative traits’ percentage distribution in the 32 Telfairia occidentalis landraces. FLC = flower colour, LAPS = leaf apex shape, VPG = vine pigmentation, SDC = seed colour and PPC = pulp colour���������������������������������������   71 Trophic status of samples of C. gariepinus in the pond��������������������   90 Trophic status of C. gariepinus specimens in Eleyele River������������   91 Trophic status of Clarias gariepinus samples in Ado-Odo River�����   91 Gastro-somatic index of Clarias gariepinus samples from sample sites���������������������������������������������������������������������������������������������������   93 Length-weight relationship of catfish in pond����������������������������������   93 Length-weight relationship of catfish from Eleyele River����������������   94 Length-weight relationship of Clarias gariepinus samples in Ado-Odo River������������������������������������������������������������������������������   94 Concentration of trace metals across sample sites����������������������������   95 Plastic wastes in the Lagos Lagoon��������������������������������������������������  105 Location of the study area and sampling sites in the Lagos Lagoon������������������������������������������������������������������������������������  106 Comparison of physicochemical properties of the Lagos Lagoon across the sampling stations��������������������������������������  109 Microplastic abundance in water (per L) and sediment (per kg) at the four sampling stations�����������������������������������������������  110 Morphotypes of microplastics in surface water and sediment samples����������������������������������������������������������������������������������������������������  110 Microscopic images of microplastics: (a) blue fiber, (b) blue fragment, (c) film, (d) foam, (e) blue fiber, (f) cellophane������������������������������������������������������������������������������������  111 ATR-FTIR spectra (blue, measured spectrum; red, reference spectrum from the OpenSpecy spectral database) of analyzed microplastics: (a) polychloroprene, (b) polyethylene, (c) polyvinylidene chloride-acrylonitrile������������������������������������������  112 A flowchart for the development of vaccine pipeline using reverse vaccinology�����������������������������������������������������������������  150 Structure of random forest����������������������������������������������������������������  151

List of Figures

Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 1 Fig. 2 Fig. 3 Fig. 1

Fig. 2

Fig. 1 Fig. 2 Fig. 3 Fig. 1

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Screening for ESBL production using double-disk synergy test������  165 Antimicrobial testing of Thymus vulgaris essential oil ESBL-producing E. coli isolates�������������������������������������������������������  166 Minimum inhibitory concentrations (MIC). Key: MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration�����������������������������������������������������������������  166 Minimum bactericidal concentration (MBC) exhibited by Thymus vulgaris essential oil against the ESBL-producing Escherichia coli���������������������������������������������������������������������������������  167 The time rate of kill of the essential oil of Thymus vulgaris on ESBL-producing Escherichia coli. MIC 1, minimum inhibition concentration 1; MIC 2, minimum inhibition concentration������������  168 Gas chromatography-mass spectroscopy (GC-MS) analysis showing peaks of components of Thymus vulgaris essential oil (TVEO)���������������������������������������������������������������������������������������������  168 Molecular docking of ESBL-producing Escherichia coli synthase with the components of Thymus vulgaris oil������������������������������������  169 The time kill rates of the essential oil of Syzygium aromaticum on ESBL-producing Escherichia coli. MIC 1 minimum inhibition concentration 1, MIC 2 minimum inhibition concentration�������������  181 Gas chromatography-mass spectroscopy (GC-MS) analysis showing peaks of components of Syzygium aromaticum essential oil����������������������������������������������������������������������������������������  182 Molecular docking of ESBL-producing Escherichia coli with the components of Syzygium aromaticum oil���������������������������  182 Serum (a) total protein (b) globulin (c) albumin of all-male tilapia, Oreochromis niloticus-­fed diets containing graded levels of fermented cassava leaf meal. Data are expressed as mean ± SEa,b. Means with different superscript vary significantly (p T and SCN1A (c.3184A>G) genotypes and alleles in drug-responsive and drug-resistant patients����������������������������������������������������������������  137 Distribution of E. coli among patients attending secondary and tertiary health facilities���������������������������������������������������������������  164 Antibiotic susceptibility profile in ESBL-producing E. coli�������������  164 Distribution of beta-lactamase and ESBL-producing E. coli among male and female���������������������������������������������������������  165 Components of Thymus vulgaris essential oil����������������������������������  167 Distribution of E. coli among patients attending Idi-aba and State Hospital Ijaiye�������������������������������������������������������������������  177 Antibiotic susceptibility profile��������������������������������������������������������  178 Distribution of beta-lactamase and ESBL-producing E. coli among male and female��������������������������������������������������������������������  178 Screening for ESBL production using double disc synergy test�������  179 Antimicrobial testing of Syzygium aromaticum essential oil on ESBL-producing E. coli isolates��������������������������������������������������  180 Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) exhibited by Syzygium aromaticum essential oil against the ESBL-producing Escherichia coli����������������������������������������������������  181 Components of Syzygium aromaticum oil����������������������������������������  183 Composition (g/kg) of experimental diets����������������������������������������  190 Amino acid composition (g/kg ingredient) of fermented cassava leaf meal (CLM) and fermented cassava leaf meal (FCLM)�����������������������������������������������������������������������������  193 Growth performance and nutrient utilization of all-male Oreochromis niloticus fingerlings fed the experimental diets�����������  194

List of Tables

Table 4 Table 5

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Haemato-biochemical parameters of all-male Oreochromis niloticus fed the experimental diets�����������������������������  197 Carcass composition of all-male tilapia, Oreochromis niloticus fingerlings fed with fermented cassava leaf meal�����������������������������  197

Table 1

Preliminary phytochemical screening of I. wombolu and T. cordifolius extracts�����������������������������������������������������������������  220

Table 1

Network statistics of curated An. gambiae central metabolism model�����������������������������������������������������������������������������  237 Metabolic genes in An. gambiae central metabolism model predicted as essential genes by choke point analysis�������������  241 Predicted essential metabolic genes observed to be upregulated in midgut of P. berghei-­infected An. gambiae��������������  243

Table 2 Table 3 Table 1

Characteristics of tyrosine and serine family integrases�������������������  257

Table 1

Classification of biosurfactants, producing microbial genera, and examples�������������������������������������������������������������������������������������  293 Strengths and deficiencies of omics-based techniques���������������������  299

Table 2 Table 1 Table 2

Accessions used to study comparative DNA extraction in eggplants (Solanum species)���������������������������������������������������������  307 Summary of results from DNA extraction methods�������������������������  308

Part I

Food and Sustainable Agriculture

Mycotoxin Occurrence and Risk Assessment in Infants and Young Children (IYC) Formulated Foods in Southwest Nigeria Comfort Adebukola Adelodun, Solomon U. Oranusi, Dango Zilpah George, Paul Akinduti, and Yemisi Dorcas Obafemi

1 Introduction Nutrition in children is essential for the first 2 years to promote optimal growth, health, and development, as hypothesized by the concept of “Developmental Origins of Health and Disease” (DOHaD) (Adeyeye, 2016). However, depending on the age of the children, meals throughout this time might be diverse and complex. Exclusively consumed breast milk, baby formula, breast milk combined with complimentary meals, and exclusively consumed complementary foods are among the diet categories. Examples of complementary foods include cereal-based, oilseed-­ based, and fruit-based products. Unfortunately, these foods may harbor many fungi metabolites known as mycotoxins (Chilaka & Mally, 2020). Mycotoxins are toxic secondary metabolites in food produced by some fungal species (Piacentini et al., 2019). Aflatoxins (Afs), zearalenone (ZEN), ochratoxins (OTAs), fumonisins (FBs), citrinin (CIT), trichothecenes (TCs), and Alternaria toxins are among the 300 to 400 mycotoxins discovered to date (JECFA, 2008). Species of Aspergillus, Alternaria, Fusarium, Penicillium, and Claviceps are responsible for producing these mycotoxins (Mandy & Nyirenda, 2018). Mycotoxins can contaminate food products during any crop life and process cycle. However, different fungi and forms of toxin synthesis predominate at certain phases. Fusarium, Alternaria, and Claviceps species thrive during the pre-harvest stages of crops such as wheat, corn, and barley on-field and produce mycotoxins. In contrast, Aspergillus and Penicillium species thrive during storage (Ezekiel et al., 2020b). Consumption of mycotoxin-contaminated foods by infants and young children (IYC) remains a severe problem, particularly in economically growing countries in Sub-Saharan Africa (SSA), such as Nigeria, Burkina Faso, and Tanzania (Chukwudi C. A. Adelodun · S. U. Oranusi (*) · D. Z. George · P. Akinduti · Y. D. Obafemi Department of Biological Sciences, Covenant University, Ota, Ogun State, Nigeria e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_1

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et al., 2021). Mycotoxins in these foods are inevitable worldwide because they are not easily eliminated during food processing due to their heat stability and ability to withstand physical and chemical treatments (Alshannaq & Yu, 2017). However, the concentration level of these mycotoxins in food consumed by IYC is a significant cause of concern because mycotoxins have been linked with developmental problems and health risks such as neurodevelopmental disorders in children. This review examines mycotoxin occurrence in foods consumed by IYC and the associated risk following consuming contaminated foods.

2 The Implication of Mycotoxins in IYC Food Formulations As indicated above, mycotoxin occurrence in food is unavoidable worldwide; however, mycotoxin levels in foods for the IYC population are relevant to public health, considering their sensitivity and vulnerability (Zhang et  al., 2020). Furthermore, the IYC developing immune system and organs may predispose them to toxicity. Mycotoxins have been linked to developmental problems in children, such as neurodevelopmental disorders. Mycotoxin exposure is associated with various symptoms, including vomiting, dizziness, nausea, and long-term degenerative diseases, due to its nephrotoxic, neurotoxic, genotoxic, estrogenic, hepatotoxic, teratogenic, immunotoxic, and carcinogenic properties (Piacentini et al., 2019; Javed et al., 2021). As a result, some countries have established stricter regulatory standards or guidelines than those for the general public to prevent IYC exposure to major mycotoxins, as shown in Table 1.

3 Mycotoxin Occurrence in Cereal-Based Products Cereals are one of the most common sources of mycotoxin contamination (Hernández et al., 2021). However, the contamination of these products by mycotoxins is of significant relevance for IYC due to their vulnerability to toxicity induced by mycotoxins. First foods fed to children at the weaning stage (6–23 months) in Nigeria and some Sub-Saharan countries consist of “transitional” foods, which are essentially porridges mainly consisting of cereals, thereby increasing the risk of diverse mycotoxin exposure among these children (Mahato et  al., 2019), (Ezekiel et al., 2020a). In a study by Ojuri et  al. (2018), 137 industrially processed and household-­ formulated complementary food samples fed to Nigerian IYC were evaluated. The authors reported 19 different mycotoxins in the household-formulated complementary food (Ogi) with 55.2% aflatoxin occurrence rate. The same study reported 28 (Arce-López et al., 2020) mycotoxins in 84 industrially processed nut- and cereal-­ based complementary food samples. Some of the mycotoxins recorded include Afs, FBs, DON, ochratoxin A (OTA), NIV, MON, BEA, CTN, and ZEN. In cereal-based infant products in Burkina Faso, aflatoxins, ochratoxins, and fumonisins were

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Table 1  Global regulatory standards for mycotoxins for infants’ and young children’s foods

Countries European Union

Food products Cereal-based and infant foods

Infants’ formula

Brazil

China

Apple-based and general baby foods Cereal-based and infant foods

Maize-based and infant foods Infant formula Cereal-based and infant foods

United States

Apple juice Apple-based products Cereal-based and infant foods

Indonesia

Baby foods

Mycotoxins Aflatoxin Fumonisins Ochratoxin Deoxynivalenol T-2/HT-2 Zearalenone Tenuazonic acid AFM1

Limits (μg/kg) 0.1 200 0.5 200 15 20 – 0.025

PAT

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Aflatoxin Fumonisins Ochratoxin Deoxynivalenol T-2/HT-2 Zearalenone Tenuazonic acid Fumonisins (FB1 + FB2) Total aflatoxin Aflatoxin Fumonisins Ochratoxin Deoxynivalenol T-2/HT-2 Zearalenone Tenuazonic acid PAT PAT Aflatoxin Fumonisins Ochratoxin Deoxynivalenol T-2/HT-2 Zearalenone Tenuazonic acid Aflatoxin Fumonisins Ochratoxin Deoxynivalenol T-2/HT-2 Zearalenone Tenuazonic acid

1 – 2 200 – 20 – 200 1 0.5 200 5 – – 60 – 0.05 0.025 2 – – 1000 15 – – – – 0.5 200 – – –

References Piacentini et al. (2019)

Piacentini et al. (2019) Piacentini et al. (2019) Piacentini et al. (2019)

Javed et al. (2021) Javed et al. (2021) Piacentini et al. (2019)

Javed et al. (2021) Javed et al. (2021) Javed et al. (2021)

Ezekiel et al. (2020a, b)

(continued)

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Table 1 (continued)

Countries Food products South Korea Cereal-based baby foods

Nigeria

Infant foods

Mycotoxins Aflatoxin Fumonisins Ochratoxin Deoxynivalenol T-2/HT-2 Zearalenone Tenuazonic acid Aflatoxin B1

Limits (μg/kg) References 0.1 Javed et al. (2021) – 0.5 0.2 – 20 NA 0 Ojuri et al. (2018)

NA no available data on mycotoxin regulation for foods consumed by IYC in Sub-Saharan Africa

reported at a 73.4% occurrence rate (Ware et al., 2017). These two researchers also reported a higher level of AFB1 and other aflatoxins exceeding the EU maximum tolerance limits for mycotoxins in cereal products (Ojuri et al., 2018). The production of these mycotoxins in cereal and cereal products is highly dependent on environmental factors such as temperature and moisture content during harvesting and storage (Milani, 2013). For example, aflatoxin producers are favored by warm conditions; therefore, drought can cause the cracking of groundnut pods, leading to the growth of A. flavus and A. parasiticus, resulting in significant aflatoxin accumulation. Ochratoxins grow in conditions of relatively high moisture content; therefore, storage of contaminated cereal crop under high humid conditions favors the production of ochratoxins (Milani, 2013). Also, different physical, chemical, and biological processing methods could contribute to distribution of mycotoxins in cereal-based products (Sarmast et al., 2021). Fermentation, cooking, roasting, baking, and milling are the most common processing methods used in the food industries in the production of cereal-based products. These methods may lead to the redistribution, reduction, or increase in the mycotoxin content of these products (Sarmast et al., 2021). However, it has been reported that some of these processing technologies, such as baking, have received more attention due the decontamination or reduction in mycotoxin level in cereals such as wheat, corn, and rice (Sarmast et al., 2021).

3.1 Fungi Associated with Mycotoxin Production Fungi known for the production of mycotoxins are generally referred to as toxigenic fungi of which Aspergillus, Fusarium, and Penicillium are the three main genera. Other genera include Alternaria and Claviceps (Greeff-Laubscher et al., 2020). Aflatoxins are chemical derivatives of difuranocoumarin produced by a polyketide pathway by Aspergillus species: Aspergillus flavus, Aspergillus parasiticus, and the rare Aspergillus nomius (Luttfullah & Hussain, 2011). AFs are one of the most potent toxic substances that are found in a wide range of crops especially

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grains and nuts which are commonly used for the preparation of children’s food. The six major aflatoxins occur naturally and are significant contaminants of a wide variety of foods and feeds among the identified 20 are AFB1, AFB2, AFG1, AFG2, AFMI, and AFM2 (De Ruyck et al., 2015). Fumonisins were discovered in 1988 following an outbreak of equine leukoencephalomalacia in South Africa in 1970 (Marasas, 2001). Fumonisins are mycotoxins produced by Fusarium, Fusarium verticillioides, and Fusarium proliferatum mostly in corn and corn products. Deoxynivalenol (DON, vomitoxin) is a subtype mycotoxin belonging to the Type B non-macrocyclic trichothecenes commonly found in wheat, barley, and corn that have been infected by the mold Fusarium graminearum and Fusarium culmorum (Schothorst & van Egmond, 2004). They are mostly found in Fusarium head blight (FHB) disease in cereals including oats, barley, wheat rye, and maize and less frequently in rice, sorghum, and triticale (Pascari et al., 2019). T-2 toxin is representative of a large group of trichothecenes. It belongs to the Type A chemical class of non-macrocyclic trichothecenes. The principal fungus responsible for the production of T-2 toxin is F. sporotrichioides (Council for Agricultural Science, 2003). Ochratoxins were the first group of mycotoxins to be found after the discovery of the AFs. OTA is a secondary fungal metabolite of two major genera Aspergillus and Penicillium with the primary producing species of Aspergillus section Circumdati, Aspergillus niger, Penicillium verrucosum, Penicillium thymicola, and Penicillium nordicum (Marin et al., 2013). Ochratoxin (OT) can be grouped into three different subtypes, namely, A, B, and C (Alhamoud et al., 2019). Zearalenone is a subtype mycotoxin produced by Fusarium species: Fusarium graminearum (Gibberella zeae) and Fusarium culmorum. Zearalenone has estrogenic and anabolic activity, and its main mechanism is by mimicking the impacts of the female estrogen hormone, disrupting conception, ovulation, and fetal development at levels above 1 mg/ kg (Calori-Domingues et al., 2016). ZEN is found in corn, wheat, barley, oats, sorghum, and sesame. ZEA mycotoxin subtype is classified as Group 3 carcinogens (IARC, 1993).

3.2 Comparative Mycotoxin Level in Breast Milk and Infant Formula Breast milk has been reported to contain mycotoxins in various investigations, particularly in the European Union (Bogalho et al., 2018). In the EU, the mycotoxins OTA and AFM1 are the most extensively explored in breast milk, but OTAs appear to be the main mycotoxins detected in human breast milk in the region (Hernández et al., 2021). While AFB1 and AFM1 dominated breast milk samples from the SSA, most of which exceeded EU regulatory restrictions on processing infant/children’s foods (Alegbe et al., 2017). In studies by Alegbe et al. (2017) and Adejumo et al. (2013), the incidence of AFM1 in Nigerian breast milk samples was 82%. AFM1 has also been reported in breast milk samples in Tanzania, Cameroon, Kenya, and Sudan, among others in Sub-Saharan Africa (Chilaka & Mally, 2020). Another

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study from Nigeria observed low-level occurrence of AFM1 in breast milk samples using thin-layer chromatography (TLC) (Atanda et al., 2007). Meanwhile, the sensitivity of the analytical approach may have impacted the results reported by these authors. In another study in Nigeria on breast milk samples, eight (Zhang et  al., 2020) mycotoxins, AFM1, OTA, OTB, BEA, enniatins (ENN B), sterigmatocystin (STG), alternariol methyl ether (AME), and dihydrocitrinone (DCIT), were discovered (Ezekiel et al., 2020a, b). Infant formula is a supplement to the IYC diet and meets their daily nutritional needs. It can also expose this age group to mycotoxin contamination, as mycotoxins have been found in infants’ formula in many regions. The prevalence of OTAs and AFB1 was assessed among 14 common infant formula branded Italian products (Meucci et al., 2010). While 133 (72%) samples were positive for OTA mycotoxin, only two (1%) were positive for AFM1. According to Ojuri et al. (2018), mycotoxins were found multiple times in 17 baby formula samples regularly fed to IYC in Nigeria, as reported by Chilaka and Mally (2020). The most common toxin is ZEN, found in 23.5% of animals (range: 0.4–5.4 g/kg), followed by BEA (17.5%, range: 0.1–13.4 g/kg). Trichothecenes (DON, T-2 toxins (T-2), moniliformin (MON), and nivalenol) were found in 11.8% of the samples, while AFB1 and AFB2 were observed in one sample at 4.2 g/kg and 0.5 g/kg, respectively (Chilaka & Mally, 2020).

3.3 Mycotoxin Contamination in Fruit-Based and Oilseed Products Vegetables, fruits, pulses, and oilseeds have been used to produce food for IYC (Westland & Crawley, 2018). Their usage has been supported, particularly in developing regions like Sub-Saharan Africa, to fight the incidence of malnutrition-­ associated health issues. However, due to the high chemical stability of mycotoxins, they can contaminate fruits and vegetables and then be transferred to processed products (Sengling Cebin Coppa et al., 2019). While other parts of the world have data on contamination of IYC foods in fruits and vegetables, Sub-Saharan Africa lacks information, which indicates that the region needs more research to determine the level of mycotoxin contamination in these products. Fusarium mycotoxin in processed soybean flour, a Nigerian weaning food, was discovered in a study by Chilaka (2017). The frequency of ZEN (81%) was found in the analytical samples, as well as FBs (28%) and HT-2 toxin (25%), and 15-acetyldeoxynivalenol ­(15-ADON) (31%). In another study by Ojuri et al. (2018), mycotoxins, including AF (80%), MON (60%), BEA (80%), and 3-nitropropionic acid (20%), were observed in five Nigerian peanut butter samples in concentrations of 5.4  g/kg, 6.5–13.6 g/kg, 0.6–4.1 g/kg, 2.2–3.5 g/kg, respectively.

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3.4 Risk Assessment of Mycotoxin-Contaminated Foods Consumed by IYC The threat posed by mycotoxins to IYC depends on the degree of harm caused by mycotoxins and the degree to which they are exposed to these toxins. It is crucial to understand exposure patterns in areas where mycotoxin contamination is of public health concern (Tshalibe, 2019). To assess risks, mycotoxin exposure in the general public (or a subset of the public) is compared to health-based recommendations such as tolerable daily intake (TDI). The greatest dose that has no impact in the most sensitive species tested is used to calculate a TDI, multiplied by a safety or uncertainty factor (Wokorach et al., 2021). These investigations are usually carried out in countries with a significant risk of exposure to a specific mycotoxin. However, reports on contamination of supplemental foods for IYC from Nigeria are scarce. In addition, using the numerical point estimation approach, no effort has been made to describe public health risks associated with chronic mycotoxin dietary exposure through complementary foods in IYC (Ojuri et al., 2018). In a study by Ojuri et al. (2018), a structured questionnaire was used to collect data on daily complementary food consumption. LC-MS/MS was used to determine mycotoxin occurrence in the foods consumed by this population. Several mycotoxins were found to have high exposure estimations significantly higher than the toxicology reference point, suggesting that Nigerian infants and young children may be exposed to significant doses of hazardous secondary metabolites and mycotoxins such as AFs, FB, OTA, and CIT, regularly posing a severe health risk to this population. Mycotoxin exposure assessment can be carried out using two methods, dietary exposure assessment and mycotoxin exposure assessment using biomarkers (Gratz et al., 2019). In recent years, biomarker analysis has been used to quantify mycotoxin exposure and determine exposure based on mycotoxin levels in food (Arce-López et al., 2020). Unlike the occurrence and dietary intake methods that often lead to underexposure or overestimation, biomarker analysis covers the ingestion of mycotoxins from all dietary sources and routes of exposure. In exposure assessment studies, aflatoxin albumin and aflatoxin DNA adducts have been used (Degen, 2011). However, recently available biomarkers that can be used as an individual measure for aflatoxin exposure (e.g., aflatoxin/albumin adducts in blood serum) are limited in terms of individual assessment of aflatoxin exposure because they only show AF exposure during the marker’s lifetime (about 22  days for serum) (Henry et  al., 2002). Urinary biomarkers for AFB1 include AFM1, AFP1, and AFQ1 while non-­ metabolized ZEN + α-zearalenol (α-ZEL) + β-zearalenol (β-ZEL) 1 is the biomarker for ZEN, non-metabolized FB1 for FB1, and DON + de-epoxy-deoxynivalenol-1 (DOM-1)  +  15-acetyl-DON (15-Ac-DON) for DON (Franco et  al., 2019). These biomarkers are usually deconjugated in urine samples by digestion with β-glucuronidase or sulfatase to increase concentration and detection (Franco et al., 2019). Despite the lack of data on mycotoxin biomarker analyses in Sub-Saharan Africa, a few studies have reported high levels of mycotoxin exposure in IYC (Mollay et al., 2020).

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4 Public Health Risk Associated with Mycotoxin Contaminated IYC Foods Scientists have explored the possibility of a link between the development of IYC and exposure to mycotoxin, particularly to fumonisins and aflatoxins. The relationship between child developmental impairment and AF dietary exposure in 242 children was examined in Kenya (Gong et al., 2016). The authors reported an association between the children exposed to aflatoxin and the prevalence of wasting in the population. A similar study reported a higher level of AF-lysine in children with acute malnutrition and impaired growth than in healthy children in Nigeria (McMillan et al., 2018). Although the specific mechanism by which mycotoxins affect infant growth is uncertain, they have an impact. A study (Smith et al., 2015) proposed two ways in which mycotoxin can cause stunting and induction of environmental enteric dysfunction, disrupting the insulin-like growth factor 1 (IGF1) axis. This IGF supports most growth hormone’s growth-promoting activities, which is crucial in a child’s development (Hellström et al., 2016). In a study of 199 Kenyan schoolchildren, AF-albumin level was inversely related to IGF-binding protein-3 (IGFBP3) and IGF1 (Chilaka & Mally, 2020). The immunosuppressive effects of Afs render babies more vulnerable to sickness, nutrition absorption, and appetite loss (Alassane-Kpembi et  al., 2017). Aflatoxins can also cause intestinal ischemia (damage) by blocking protein synthesis, resulting in stunted growth in children (Chilaka & Mally, 2020). This process is also thought to be used by other mycotoxins, such as DON, to restrict growth. On the other hand, FB may inhibit IYC growth by inhibiting ceramide synthase, a crucial enzyme that produces sphingolipids, causing sphingolipid metabolism to be disrupted (Chilaka & Mally, 2020). Sphingomyelin is a prevalent sphingolipid in mammalian cell membranes. It is essential in forming lateral structures for Toll-like receptors, insulin receptors, and class A and B scavenger receptors. Sphingomyelin is also involved in cell signaling, and FBs were reported to affect intestinal barrier function via altering the sphingoid base-1 phosphate signaling pathway (Álvarez et al., 2020). Autistic spectrum disorder (ASD) is a lifelong neurodevelopmental syndrome characterized by social deficits, problems with interactions with others, and unusually constrained and repetitive behaviors (Salim et al., 2020). According to scientific data, multiple interacting genetic variables appear to have a role in the etiology of ASD (Morris et  al., 2018). Epidemiologists have looked into the possible links between neurodevelopmental anomalies and mycotoxin exposure in IYC.  The effects of mycotoxin exposure on the development of ASD symptoms in a group of 110 children in an Italian hospital, including 52 autistic children and 58 healthy children (31 children were siblings, while 27 were unrelated children), was studied. The researchers discovered a significant link between ochratoxin levels in autistic children’s serum (p = 0.0002) and urine (p = 0.0017) compared to unrelated healthy children.

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Although the dietary system and toxin exposure levels are comparable, the researchers noted that the average number of mycotoxins discovered in children with ASD was lower than in their siblings, which they attributed to possible changes in the toxin’s biotransformation and metabolism (Salim et al., 2020). Furthermore, a significant correlation was discovered with OTAs in the urine when comparing patients with ASD to healthy children, unrelated individuals, and siblings (p = 0.0081). Still, there was no significant difference between the children with ASD and their siblings. Based on extensive scientific evidence from epidemiological studies, aflatoxins (AFG1, AFG2, AFB1, and AFB2) have been classified as Group 1 human carcinogens. Group 2B includes AFM1, FB1, FB2, and OTA, which is also categorized as a possible carcinogen in humans, although human data is still inconclusive (Yip et al., 2017). AFB1 is the most reported potent Group 1 human carcinogen causing hepatocellular carcinoma (HCC). AFB1 induces HCC-causing mutation in the P53 gene, which plays a vital role in protecting humans against cancer. The mutation of this P53 gene by AFB1 allows the cell to increase out of control, leading to cancer (Marin-Kuan et al., 2008). Aflatoxins cause breast cancer which produces tumors in the breast. Fumonisins also have a potential carcinogenic effect on humans. Consuming cereals contaminated by fumonisins has been associated with esophageal cancer in humans. Fumonisins inhibit N-acetyltransferase, a key enzyme in the metabolism of sphingolipids, causing the decrease of complex sphingolipids and a rapid increase in sphinganine. Increased accumulation of sphinganine nephrotic and hepatic toxicity due to free sphingoid-based results in apoptosis and abnormal cell proliferation (Kew, 2015; Khanna & Verma, 2018). Furthermore, ochratoxin has been reported to have carcinogenic effects on humans. According to Tesfamariam et al. (2020), OTA carcinogenicity is caused by interconnected epigenetic processes, including activation of specific cell signaling pathways, oxidative stress, and protein synthesis inhibition. Although childhood HCC is rare, childhood HCC instances have been recorded in SSA (Cameron & Warwick, 1977; Kew et al., 1982), with the majority of cases being attributed to hepatitis B virus (HBV) exposure. Despite the lack of evidence linking infant AF exposure to HCC risk in Sub-Saharan Africa, early prenatal exposure to carcinogens, as revealed by the detection of AFM1 in breast milk, transplacental transfer of AFs, and the frequency of these mycotoxins in food for IYC, is a cause for concern (Chilaka & Mally, 2020).

5 Conclusion The occurrence of mycotoxins in foods for IYC, irrespective of food category, is highlighted in the few studies in Sub-Saharan, Africa. However, there is a limitation in epidemiological studies on the occurrence, exposure, and risk assessment of mycotoxin contamination in foods for IYC in Sub-Saharan, Africa, particularly in Nigeria. As a result, more epidemiological studies are needed to highlight other

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potential health risks associated with mycotoxin exposure in Sub-Saharan Africa and gain better knowledge of the association between mycotoxin exposure and health issues that have previously been reported. While further study is necessary, it is critical to emphasize the need to raise public knowledge about mycotoxin exposure, incidence, and potential health repercussions and use collaborative efforts to develop policies and programs to fight the threat posed by mycotoxin contamination.

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In Vitro Antifungal Activity of Bauhinia monandra (Kurz) Leaf Extracts Against Fungal Pathogens Isolated from Spoilt Musa paradisiaca L. Margaret Ikhiwili Oniha, Michelle Kaosisochukwu Aniebonam, Eze Frank Ahuekwe, Stephen Oluwanifise Oyejide, and Olugbenga Samson Taiwo

1 Introduction Foods are organic compounds sourced from plants and animals ingested to provide moisture, protein, fat, carbohydrate, minerals and other organic compounds. Plantains are important food crops that serve as staple foods for human consumption as well as a source of income generation for the economically growing countries. Musa paradisiaca, the cooking banana and sometimes referred to as plantain, is a member of the Musaceae family with origin from Southeast Asia (Nwaiwu et al., 2012). It is also cultivated across tropic and subtropical climates of the globe (Vu et al., 2018). It is the fourth most significant food crop in the world with Cameroon as the world leading producer that exports approximately 4.31 million tons yearly (FAO, 2019). Musa paradisiaca contains diverse important nutritional and economic values. Compared to other fruits and vegetables, plantains have greater total dietary fibre content, particularly in hemicelluloses (Imam & Aktar, 2011). According to Sojinu et al. (2021), plantains, both ripe and unripe, are beneficial in the treatment of some ailments and diseases (such as goitre) due to their high concentration of essential nutrients, antioxidants and other biologically active components. Economically, different parts are utilized as food, fodder and compost (Agama-Acevado et al., 2016), biorefinery (Martinez-Ruano et al., 2018), food packaging and production of acids.

M. I. Oniha (*) · M. K. Aniebonam · E. F. Ahuekwe · O. S. Taiwo Department of Microbiology, Covenant University, Ota, Ogun State, Nigeria e-mail: [email protected] S. O. Oyejide Department of Cell Biology and Genetics, University of Lagos, Akoka, Lagos State, Nigeria © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_2

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Plantains are susceptible to pests and diseases which are major constraints in production. Different microbial strains are found to infest and deteriorate foods including plantain due to the ability to invade and infect plants pre and postharvest. Fungi are the most crucial and common pathogens responsible for crop diseases. These fungi infect a wide range of fruits and vegetables during storage and transportation (Abdullah et al., 2016). Fungal invasion in plantain can be due to mycotoxins or the extracellular enzymes that aid in spoilage. Studies have shown that in fruits and herbs, an extensive range of different fungal species are responsible for characteristic problems that include features, nutritional merit, organoleptic traits and deficient shelf life with some cases of allergic or toxic disorders among consumers caused indirectly by fungi from the production of allergens or mycotoxins. Some phytopathogenic fungi associated with the deterioration of plantain include Fusarium sp., Aspergillus flavus, Aspergillus niger, Colletotrichum sp. and Penicillium sp. (Umeh et al., 2017; Ejimofor et al., 2022; Hassen et al., 2022). Synthetic fungicides have become ingrained throughout agriculture. Over 110 novel fungicides have been produced since the discovery of the first synthetic fungicide, phenylmercury acetate, in 1913, thus reducing loss in production yield. In spite of their benefits, the extensive use of fungicides has posed some risks to the environment and human health because of their toxicity and long-lasting impacts. It has also amounted to the development of resistant strains (Ons et al., 2020). Plants are the richest, most cost-effective and guaranteed alternative source of antimicrobials and contain different phytochemicals. These ensure plants subsist as aetiologies of pharmaceutics used in traditional medicine and medicinal preparations (Ncube et al., 2018). Extensive studies on many medicinal plants are in view with a focus on discovering more potent and less toxic compounds from them. Bauhinia monandra is a tropical plant greatly employed in traditional medicine particularly in the management of diabetes. Additionally, the seed extracts have been shown to have hypoglycaemic, antioxidant, antimicrobial, anti-inflammatory, anti-nociceptive and antimicrobial properties (Aderogba et  al., 2006; Ajiboye et al., 2015; Solomon et al., 2016). With antimalarial, antiviral, antibacterial, antifungal, antidiarrheal and antispasmodic properties that have been studied, several Bauhinia species are also used as traditional medicines around the world (Onyije et al., 2012). The leaves of B. purpurea Linn, B. forficata Link and B. monandra Kurz are extensively used to cure diabetes in Brazil. Despite many reports about the pharmacological properties of its different species, only few reports have been published regarding its antimicrobial activity. There is a paucity of information in the research about the antifungal activity of B. monandra. Hence, this study was conducted to evaluate the in vitro antifungal activity of B. monandra leaf extracts against some phytopathogenic fungi.

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2 Materials and Methods 2.1 Fungal Isolates and Inoculum Quantification A total of 24 healthy and randomly spoilt plantain fingerlings that were obtained from Agbara, Iju and Ota markets (being major collection and retailing points) in Nigeria were labelled accordingly. These samples were immediately conveyed in Ziploc bags to the microbiology laboratory for analysis. The plantain fingerlings were subjected to a 2-minute surface sterilization in 1% hypochlorite and three thorough rinses in sterile distilled water. The samples were then aseptically placed on sterile filter papers to drain excess moisture. A sterile scalpel was used to cut out four pieces from the margin of the lesion, and the mycoflora from the plantain samples were isolated using PDA supplemented with 1 mL of 10% chloramphenicol after incubation at 25 °C for 5–7 days (Jha, 1995). The fungal isolates were phenotypically characterized by microscopy based on the observations for hyphal type, septation of hyphae (wall type of hyphae), spore colour, shape and arrangement and rhizoids or foot cells as described in Barnett and Hunter’s atlas of mycology and compendium of fungus (1987).

2.2 Plant Collection and Identification Leaves of Bauhinia monandra were harvested for use from the Covenant University Ota, Ogun State, Nigeria, between February and April 2022 in a sterile polythene bag and transported to the biology laboratory for identification by a botanist in the Biological Sciences Department. Authentication of the already identified plant species (voucher specimen number: Bm/Bio/H822) was conducted at the Herbarium Section of the Forestry Research Institute of Nigeria (FRIN), Ibadan, Nigeria, and issued a forestry herbarium identification number of B. monandra-FHI No: 112777.

2.3 Preparation of Aqueous and Ethanolic Extracts The mature disease-free leaves were rinsed to eliminate dust and other foreign particles and air-dried under a shade at ambient temperature for 3 weeks (Vinoth et al., 2011). Dried leaves were ground into powder using a sterile blender and preserved in airtight bottles at room temperature (25–30 °C) until use. The powdered leaves were extracted in aqueous and ethanol solvents by soaking 200 g of the leaves in 1000 mL of each of the aqueous and ethanol solutions to macerate for 2–3 days while stirring frequently to ensure that all soluble materials were dissolved. Following maceration, the combinations were filtered using Whatman’s No. 1 filter paper and muslin cloth. A rotary evaporator was used to concentrate the filtrates that

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were produced, and they were then stored at 4 °C for later use. After the solvents were recovered under pressure, slurried extracts were obtained (Oniha et al., 2021). The crude extracts obtained were then kept at 4 °C for subsequent use. The concentrated extracts were later dissolved in appropriate volumes of dimethyl sulfoxide (DMSO) to make varying crude concentrations (1000 mg/mL, 500 mg/mL, 250 mg/ mL, 125 mg/mL and 62.5 mg/mL where 1000 mg/mL was used for the antifungal assay) for antifungal assessment. All the stock solutions were stored in sterile capped bottles, labelled accordingly and stored at 4 °C for analysis. Each antifungal test was carried out in three replicates against each fungal isolate.

2.4 Phytochemical Screening of Plant Extracts Qualitative chemical tests were conducted to check for the presence of anthraquinones, tannins, saponins, steroids, cardiac glycosides, flavonoids, terpenoids and alkaloids according to the method of Sofowora (1993).

2.5 In Vitro Antifungal Activity of Crude Extracts of Bauhinia monandra A loopful of the fresh subcultures of each of the fungal isolates was picked from the Petri plate using a sterile inoculating loop and transferred into sterile McCartney bottles containing 1  mL of distilled water to match 0.5 McFarland standards. Antifungal activity of the crude extracts was conducted using the agar-well diffusion method as illustrated by Cheesbrough (2006) after the potato dextrose agar medium (Oxoid) was prepared according to the manufacturer’s instructions. After this, the standard dose was prepared by dissolving 1000 mg of crude extract in 1 mL of DMSO (1:1) for both the aqueous and ethanolic extracts (stock concentration for the antifungal test). Ketoconazole (100 mg/mL), the antifungal agent, was used as the positive control, while DMSO served as the negative control. The standardized test organisms were used to seed the surfaces of the agar plates. Using cork borers, wells were made in the agar and 0.2 mL of each concentration of the plant extract was introduced. The plates were incubated after being allowed for an hour to allow the extract to fully diffuse into the agar pores. Over the course of 3–5 days of incubation at room temperature, the plates were examined for zones of inhibition, which were quantified and reported accordingly. The negative and positive control plates contained no extracts but ketoconazole (positive) and DMSO (negative), respectively. All the sensitivity tests were carried out in duplicates and the mean zones of inhibition were recorded appropriately. MFC was defined as the lowest extract concentration that showed no visible growth after incubation time, and MIC was the lowest concentration showing minimal growth. The diameters measured from the zones of inhibition of the in vitro antifungal activity were analysed as the average of two replicates.

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2.6 Data Analysis Significant differences between and within the averages of treatments and controls were analysed using ANOVA at p  ≤  0.05 and post hoc tests. Statistical analyses were computed using the SPSS version 20 software package.

3 Results and Discussion Spoilage of foods results from diverse microbiological, physical or chemical activities, and these mechanisms are not always mutually exclusive since rotting caused by one process might lead to another (Sahu & Bala, 2017). Diverse microorganisms can be found to infest and contaminate perishable foods including a plantain (Sahu & Bala, 2017). There is a high possibility that these microorganisms came in contact with plantain during cultivation, harvesting, storage and transportation to the market, thereby infecting the plant at both pre- and post-harvest stages. These microorganisms are associated with soil, air and contaminated environments. The contamination of plantain by fungi generally affects its economic value. Consequently, these contaminated plantains can affect the health of the general consumers. Additionally, diverse phytopathogenic fungi that infect plantains and oranges vary depending on the species and environmental factors. From this study, the results of the isolation and characterization of mycofloral pathogens based on their cultural and microscopic features from the contaminated plantain samples are shown in Table  1 and include Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Geotrichum sp., Rhizopus sp. and Alternaria sp. The results of this study corroborate the findings of Abdullah et al. (2016), Ejimofor et al. (2022) and Sani Table 1  Preliminary identification of fungal pathogens Code P1, 2 and 3

Cultural features Black cottony growth with yellow colour on the reverse of the plate

P1, 2 and 3

Lemon green powdery growth with yellow colour on the reverse of the plate Brown powdery growth with yellow colour on the reverse of the plate Grey mycelial strands with brown tips

P1, 2 and 3 P1 and 2

Microscopic features Radiate conidial heads, septate hyphal strands with black spores suspended in a conidial sac attached to conidiophores with the spores arranged in chains Hyaline septate hyphae with bluish-green spores suspended in a conidial sac

Brown spores contained in a conidial sac arranged from inside out in chains and suspended in a hyaline conidiophore Thick-walled non-septate hyphal strands bearing brown oval-shaped spores contained in a sporangium

Key: P1 represents Agbara market, (P2) Iju market, and (P3) Oja-Ota market

Preliminary identification Aspergillus niger

Aspergillus flavus

Aspergillus fumigatus Rhizopus

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Frequency of occurrence of isolates from samples in %

M. I. Oniha et al. 30 25 20 15 10 5 0

Aspergillus Aspergillus Aspergillus Alternaria Rhizopus sp Geotrichum niger fumigatus flavus

Mucor

Fungi isolated from samples

Fig. 1  Frequency of occurrence of fungal isolates from Musa paradisiaca Table 2  Phytochemical screening of B. monandra leaf extracts Tests Carbohydrates Tanins Saponins Glycosides Alkaloids Phenols Terpenoids Cardiac glycosides Flavonoids Coumarins Steroids Anthocyanins Acids

Aqueous + + + − − − − + + − − − −

Ethanol − − − − − − + + − + + − −

Key: (+), present and (−), absent

and Kasim (2019) that published similar strains of phytopathogenic fungi in their studies. The frequency of fungi isolated from the plantain samples is presented in Fig. 1. Results of qualitative phytochemical screening showed the presence of carbohydrates, cardiac glycosides, phenols, tannins, saponins, anthocyanin, betacyanin, coumarins and terpenoids in the leaf extracts. Carbohydrates, tannins, saponins, flavonoids, cardiac glycosides and flavonoids were present in the aqueous extract only, while terpenoids, cardiac glycosides, coumarins and steroids were present in the ethanolic extract only (Table 2). Alkaloids, phenol, anthocyanin and acid were

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Mean zones of inhibition (mm)

35 30 25 20

Aspergillus niger

15

Aspergillus fumigatus Aspergillus flavus

10

Rhizopus Geotrichum

5 0

Concentration of extracts Fig. 2  Mean zones of inhibition for antifungal activity of aqueous leaf extract of B. monandra against fungal isolates

absent in both extracts. Our findings in this study support the reports of Ajiboye et al. (2015) that revealed the presence of flavonoids, tannins, steroids, terpenoids, saponins, cardiac glycosides and phenols in the leaf extract. Gizaw et  al. (2022) stated that phytochemicals are responsible for the antifungal activity exhibited by plant extracts. The results of the antifungal assessment of the aqueous and ethanolic extracts at different concentrations are presented in Figs. 2 and 3. Our findings revealed that both extracts had varying inhibitory activities against the fungal isolates at different concentrations. Secondary metabolites from medicinal plants have antimicrobial properties in which their active ingredients negatively affect the development and metabolism of microorganisms. Their screening can offer a substitute for creating chemical fungicides that are reasonably safe and economical (Nxumalo et al., 2021). The observed antifungal activity of the leaf extracts was dependent on both the concentration and polarity of the extraction solvent used, thus reducing concentrations with a corresponding reduction in the zones of inhibition. However, there is a paucity of information on the antifungal activity of B. monandra against some fungal species isolated in this study compared to other species of the genus which include B. variegate and B. racemosa with similarities in morphology and phytochemicals.

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Mean zones of inhibition

30 25 20

Aspergillus niger Aspergillus fumigatus

15

Aspergillus flavus 10

Geotrichum Rhizopus

5 0

Concentration of extracts

Fig. 3  Mean zones of inhibition for antifungal activity of ethanol leaf extract of Bauhinia monandra against fungal isolates

4 Conclusion The findings of this study emphasized the need for preventing both pre- and post-­ harvest fungal diseases, with a focus on the high rate of spoiling in plantain production. Varying degrees of antifungal activity were observed in both extract types of B. monandra tested against the fungal pathogens depending on the extract type and concentrations. Thus, extracts of medicinal plants possess antifungal potentials that will aid in the prevention of fungal infections in plantains, thereby greatly minimizing spoilage and fungal infection. Further studies need to be conducted on extracts of B. monandra to fully uncover its antimicrobial properties as well as focus on the development of novel antifungal plant extracts as pre- and post-harvest antifungal agents.

References Abdullah, Q., Mahmoud, A., & Al-Harethi, A. (2016). Isolation and identification of fungal post-­ harvest rot of some fruits in Yemen. PSM Microbiology, 1(1), 36–44. Adeniji, T. A., Tenkouano, A., Ezurike, J. N., Ariyo, C. O., & Vroh-Bi, I. (2010). Value-adding post-harvest processing of cooking bananas (Musa spp. AAB and ABB genome groups). African Journal of Biotechnology, 9(54), 9135–9141.

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Aderogba, M., Ogundaini, A., & Eloff, J. (2006). Isolation of two flavonoids from Bauhinia Monandra (KURZ) leaves and their antioxidative effects. African Journal of Traditional, Complementary and Alternative Medicines, 3, 59–65. Agama-Acevedo, E., Sañudo-Barajas, J. A., Vélez De La Rocha, R., González-Aguilar, G. A., & Bello-Perez, L. A. (2016). Potential of plantain peels flour (Musa paradisiaca L.) as a source of dietary fibre and antioxidant compound. CyTA-Journal of Food, 14(1), 117–123. Ajiboye, A. T., Musa, M. D., Otun, K. O., Jimoh, A. A., Bale, A. T., Lawal, S. O., & Arowona, M. T. (2015). The studies of antioxidant and antimicrobial potentials of the leaf extract of the Bauhinia monandra plant. Natural Product and Chemical Research, 3(4), 1000180. Barnett, H.  I., & Hunter, B.  B. (1987). Illustrated genera of imperfect fungi (3rd ed., p.  320). Macmillan Publishing Company. Cheesbrough, M. (2006). District laboratory practice in tropical countries. District laboratory practice in tropical countries (Vol. 2, pp. 47–54). Cambridge University Press. Ejimofor, F.  C., Johnson, O.  O., & Enoch, N.  N. (2022). Fungi associated with rot in plantain and its effects on nutritional value of the fruit. Journal of Global Ecology and Environment, 16(2), 37–42. FAO. (2019). Food and Agriculture Organization of the United Nations (pp. 1–4). Battling Black Sigatoka Disease in the Banana Industry. Gizaw, A., Marami, L. M., Teshome, I., Sarba, E. J., Admasu, P., Babele, D. A., Dilba, G. M., Bune, W. M., Bayu, M. D., Tadesse, M., & Abdisa, K. (2022). Phytochemical screening and in vitro antifungal activity of selected medicinal plants against Candida albicans and Aspergillus niger in West Shewa Zone, Ethiopia. Advances in Pharmacological and Pharmaceutical Sciences, 28(2022), 3299146. Hassen, N. I., Badaluddin, N. A., Mustapha, Z., & Zawawi, D. D. (2022). Identification and prevention of microbial contaminants in Musa paradisiaca tissue culture. Malaysian Applied Biology, 51(5), 129–143. Imam, M. Z., & Akter, S. (2011). Musa paradisiaca L. and Musa sapientum L. A phytochemical and pharmacological review. Journal of Applied Pharmaceutical Science, 1(5), 14–20. Jha, D. K. (1995). Laboratory manual on seed pathology (pp. 13–30). Vikas Publishing House Private Ltd. Martínez-Ruano, J. A., Caballero-Galván, A. S., Restrepo-Serna, D. L., & Cardona, C. A. (2018). Techno-economic and environmental assessment of biogas production from the banana peel (Musa paradisiaca) in a biorefinery concept. Environmental Science and Pollution Research, 25(36), 35971–35980. Ncube, N. S., Afolayan, A. J., & Okoh, A. I. (2018). Assessment techniques of antimicrobial properties of natural compounds of plant origin: Current methods and future trends. African Journal of Biotechnology, 7(12), 1797–1806. Nxumalo, K. A., Aremu, A. O., & Fawole, O. A. (2021). Potentials of medicinal plant extracts as an alternative to synthetic chemicals in postharvest protection and preservation of horticultural crops: A review. Sustainability, 13(11), 5897. Nwaiwu, I. U., Eze, C. C., Amaechi, E. C. C., & Osuagwu, C. O. (2012). Problems and prospects of large scale plantain banana (Musa spp) production in Abia State, Nigeria. International Journal of Basic and Applied Sciences, 1(4), 322–327. Oniha, M. I., Eni, A. O., Akinnola, O. O., Omonigbehin, E. A., Ahuekwe, E. F., & Olorunshola, J.  F. (2021). In vitro antifungal activity of extracts of Moringa oleifera on phytopathogenic fungi affecting Carica papaya. Open Access Macedonian Journal of Medical Sciences, 9, 1081–1085. Ons, L., Bylemans, D., Thevissen, K., & Cammue, B.  P. A. (2020). Combining biocontrol agents with chemical fungicides for integrated plant fungal disease control. Microorganisms, 8(12), 1930. Onyije, F. M., & Avwioro, O. G. (2012). Effect of ethanolic extract of Bauhinia monandra leaf on the liver of alloxan-induced diabetic rats. Journal of Physiology and Pharmacology, 2(1), 59–63.

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Sahu, M., & Bala, S. (2017). Food processing, food spoilage and their prevention: An overview. International Journal of Life-Sciences Scientific Research, 3, 10.21276/ijlssr.2017.3.1.1. Sani, M. A., & Kasim, M. (2019). Isolation and identification of fungi associated with postharvest deterioration of banana (Musa paradisiaca L.). Pharmacology Online, 2, 347–354. Sojinu, O.  S., Biliaminu, N.  T., Mosaku, A.  M., Makinde, K.  O., Adeniji, T.  H., & Adeboye, B. M. (2021). The implications of ripening agents on chemical compositions of plantain (Musa paradisiaca). Heliyon, 7(6), e07123. Solomon, S., Muruganathan, N., & Senthamilselvi, M.  M. (2016). Anti-oxidant and anti-­ inflammatory activity of Bauhinia tomentosa (flowers). Indo-American Journal of Pharmaceutical Research, 6, 4321–4326. Umeh, S. O., Okafor, U. C., & Okpalla, J. (2017). Effect of microorganisms and storage environments on ripening and spoilage of plantain (Musa paradisiaca) fruits sold in Eke Awka market. World Wide Journal of Multidisciplinary Research and Development, 3(10), 186–190. Vinoth, S., Kanna, R., P, Packiaraj, G., & Narayanasamy, J. (2011). Evaluation of phytochemical, antimicrobial and GC-MS analysis of extracts of Indigofera trita L.F. SPP. subulata (vahl ex poir). International Journal of Agricultural Research, 6(4), 358–367. https://doi.org/10.3923/ ijar.2011.358.367 Vu, H. T., Scarlett, C. J., & Vuong, Q. V. (2018). Phenolic compounds within banana peel and their potential use a review. Journal of Functional Foods, 40, 238–248.

Effect of Aspergillus fumigatus MT899185 Phytase Addition on the Nutritional and Phytate Content of Formulated Cowpea-Based Poultry Feed Adeola Elizabeth Onibokun, Angela Obiageli Eni, and Solomon U. Oranusi

1 Introduction Vigna unguiculata (L.) Walp (cowpea) is a crop plant which belongs to the family Fabaceae. It is a widely produced legume with more than 8.9 million tonnes produced worldwide in 2019 and Africa producing more than 8.6 million tonnes (FAOSTAT, 2021). Nigeria, the principal grower and consumer of cowpea, accounts for 42% and 40% of Africa’s and global cowpea production, respectively (FAOSTAT, 2021). Cowpea contains large amounts of protein, several vitamins and minerals. It also contains potassium with significant amounts of calcium, magnesium, phosphorous, vitamin A and C, thiamine, riboflavin, niacin, vitamin B6 and pantothenic acid (Alayande, 2012). However, its use is limited due to the presence of anti-nutrients such as phytate. Phytate (myo-inositol hexakisphosphate; IP6) is a major anti-­ nutrient found in plant-based animal feeds accounting for about two thirds of the total phosphorus in plant-based feed (Lee et al., 2020). Useful cations like calcium, iron, manganese, potassium, zinc and magnesium bind firmly to the negatively charged phosphate in phytic acid. This binding makes the cations insoluble which affects their bio-availability (Handa et al., 2020). Since phytate is the most popular storage form of phosphorus in plants, the phosphorus requirement of animals is provided for by supplementing with an inorganic phosphate (Lamid et al., 2018). Another alternative to this may be the inclusion of exogenous phytase to the animal feed (Dahiya & Singh, 2014). Microbial phytase is the most commonly used exogenous enzyme in the feed for monogastric animals (Dersjant-li et al., 2015). The A. E. Onibokun (*) · A. O. Eni Department of Biological Sciences, College of Science and Technology, Covenant University, Ota, Ogun State, Nigeria e-mail: [email protected] S. U. Oranusi Department of Biological Sciences, Covenant University, Ota, Ogun State, Nigeria © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_3

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presence of phytase in livestock feed improves the nutrients for livestock since phytate is regarded as an anti-nutrient (Lamid et  al., 2018). Poultry consumption is projected to increase to about 230% in 2030 compared to other livestock which is estimated to reach about 40–80% of what they were in 2010 (Gad et  al., 2020). Alluding to this report, poultry which accounted for about 37% of the global meat industry in 2017 is expected to produce about 331 million tonnes of meat by 2028 (Oladokun & Adewole, 2020). Feed accounts for the major cost in poultry production. The requirements for energy and protein make up about 95% of this cost, while supply of vitamins and other feed additive accounts for about 3–4% and 1–2%, respectively (Zentek & Goodarzi Boroojeni, 2020). Besides cereals (which is mainly corn), the protein source commonly used is soybean meal. Availability and supply of soybean meal is limited in many parts of the world. Also, soybean production comes with its attendant challenges, such as importation cost and genetic modification which have not been widely accepted. The abundance of cowpea in Nigeria and its high nutritional status make it a suitable alternative to soybean in animal feed production (Zentek & Goodarzi Boroojeni, 2020). This study was conducted to determine the effect of A. fumigatus MT899185 on the nutritional and phytate content of cowpea-based poultry feed.

2 Materials and Methods 2.1 Phytase Production A previously isolated phytase-producing Aspergillus fumigatus MT899185 with suitable pH and temperature stability was induced for quantitative phytase production by submerged fermentation (SmF). One millilitre spore suspension containing fungal spores was inoculated into 100 mL phytase screening broth (PSB) in 250 mL Erlenmeyer flasks and incubated in a 150 rpm shaking water bath at 30  °C for 5  days. Cell-free supernatant was obtained from the fermentation medium by filtration using a Whatman No. 4 filter paper and a separating funnel (Qasim et al., 2016).

2.2 Phytase Assay The fungal phytase activity was determined by incubating 1 mL of the culture filtrate (crude phytase) in 1 mL of 0.2 M sodium acetate buffer (pH 5.5) containing 0.5% sodium phytate as substrate in two separate test tubes marked as the experimental tubes. In another tube, marked as the control tube, 1  mL of the prepared substrate was added without the crude phytase. Both the experimental and control tubes were incubated at 37 °C for 30 min. The reaction in all the tubes was terminated by addition of 1 mL of trichloroacetic acid (15% [w/v]). Thereafter, 1 mL of crude phytase was added to the control tube only. After the addition of 2 mL of a

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colouring reagent (3.66  g of FeSO4·7H2O, 0.5  g of (NH4)6Mo7O24·4H2O and 1.6 mL of concentrated H2SO4 in 50 mL of distilled water), the sample was chilled on ice water followed by incubation for 10 min at 30 °C. The released phosphate was determined at 750 nm using a UV-Vis spectrophotometer (Lee et al., 2005). The protein quantification was carried out according to the method of Lowry, Rosebrough, Farr and Randal (1951).

2.3 Feed Formulation Corn and cowpea were ground to pass through 1 mm sieve mesh using a laboratory mill before formulation (Farhadi et al., 2019). All experimental feeds were formulated according to Cobb 500 breeder recommendations (Walters et al., 2019). In all cases, de-hulled soybean was replaced with cowpea and phytase supplementation was done as reported by Al-Harthi et al. (2020).

2.4 Determination of Proximate Composition of Formulated Feeds Proximate composition of feed samples was determined following the method of AOAC (1990) for moisture, crude fibre and protein, while ash and lipid contents were determined according to the method of James (1995) and Ilodibia et al. (2014), respectively. The carbohydrate content was determined by the difference between 100 and the sum of all other values (protein, fibre, ash, fats and moisture contents) as expressed below: CrudeCHOcontent  %   100  %Moisture  %Ash  %Fat  %Crude fiber  %Protein

Determination of Gross Energy Gross energy was calculated based on the following formula (Owolabi et al., 2012): Gross energy  KJ per 100 g dry matter    crude protein  16.7    crude lipid  37.7    crude carbohydrates 16.7  Determination of Dry Matter The dry matter of each feed sample was calculated by deducting the moisture content of each feed from 100. Mathematically, it is expressed as:

Dry matter  %   100   %moisture 



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2.5 Determination of Phytate Content Phytate content was determined according to the method of Fategbe et al. (2021) with some modifications. Each feed sample (1 g) was weighed into 250 mL beakers, to which 50 mL of concentrated hydrochloric acid (HCL) was added to soak the samples and allowed to stand for 3 h. After 3 h, each mixture was filtered through a double layer of Whatman No. 1 filter paper. The filtrate from each sample was dispensed into a clean 250 mL beaker and topped up with 53.3 mL of distilled water to 100  mL.  An aliquot of 5  mL of 0.3% ammonium thiocyanate solution was then added into each filtrate mixture as an indicator and titrated with 0.00495  g/mL iron(III) chloride solution. The end point was slightly brownish yellow which persisted for 5 min. The phytate content in each extract was expressed as a proportion of the sample in mg/g using the calculation below: Phytate  mg / g   Titre value  0.00495  1000





where titre value = value obtained from end point of titration experiment 0.00495 = concentration of iron(III) chloride solution in g/mL 1000 = conversion factor of concentration in g/mL to mg/mL

3 Results 3.1 Phytase Production and Assay The phytase produced by 1 mL spore suspension showed a total activity of 619 U/ mL (Table 1).

3.2 Proximate Composition of Feed Samples Feed 1 (control feed) had the highest moisture (13.95%) and carbohydrate (38.19%) contents, but had the least ash (11.50%), crude fibre (3.06%) and protein (29.36%) contents. Among the experimental feeds (feeds 2–4), feed 2, which was the feed formulated with the laboratory-produced phytase from Aspergillus fumigatus MT899185, had the highest crude fibre content (7.14%) and protein content

Table 1  Quantitative assessment of phytase produced by A. fumigatus MT899185 S/N 1

Sample code 2A

Sample location Ota

Number of spores/mL 2 × 107

Total activity (units/mL) 619

Total protein (mg/mL) 177

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Table 2  Proximate compositions of different feed treatments with varying phytase compositions Sample Feed 1 Feed 2 Feed 3 Feed 4

MC (%) 13.95 11.69 12.49 12.66

AC (%) 11.50 18.63 22.23 20.71

FC (%) 3.32 2.98 3.48 3.52

CFC (%) 3.06 7.14 5.77 6.01

PC (%) 29.36 39.50 34.47 35.29

CHO (%) 38.19 20.56 21.56 21.81

DM (%) 86.05 88.31 87.51 87.34

GE (kJ/100 g) 1253.3 1014.3 1066.9 1086.3

0 .8

0 .6

0 .4

0 .2

4 e e F

e e F

d

d

2 d F

e

e

d e e F

3

0 .0

1

P h y t a t e C o n c e n t r a t io n ( m g /g )

MC moisture content, AC ash content, FC fat content, CFC crude fibre content, PC protein content, CHO carbohydrate content, DM dry matter, GE gross energy

Fig. 1  Phytate contents of formulated cowpea-based feed and control group (Feed 1). Key: Feed 1 = commercial poultry feed, feed 2 = cowpea-based feed formulated with addition of Aspergillus fumigatus phytase MT899185, feed 3 = cowpea-based feed formulated with addition of commercial phytase, feed 4 = cowpea-based feed without the addition of phytase

(39.50%) but had the least moisture (11.69%), ash (18.63%), fat (2.98%) and carbohydrate (20.06%) contents (Table 2).

3.3 Phytate Composition of Feed Samples Feed 4 which was not supplemented with phytase had the highest phytate content (0.594 mg/g), while feed 2 which was supplemented with the phytase produced by Aspergillus fumigatus MT899185 had the lowest phytate content (0.198  mg/g). Feeds 1 and 3 had phytate contents of 0.346  mg/g and 0.396  mg/g, respectively (Fig. 1).

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3.4 Economics Local market prices were used to determine the cost per 25 kg bag of feed formulated as described in Table 1. A 25 kg bag of formulated feed amounted to a total value of three thousand one hundred and fifty-five naira (N3,155) compared to the cost of 25  kg bag of commercial which was four thousand five hundred naira (N4,500) as at the time of purchase for this study. Supplementation of feed with cowpea reduced feed cost per 25 kg bag by 29.9% which amounted to a reduction of one thousand three hundred forty-five naira (N1,345) per 25 kg bag.

4 Discussion In this study, the production of phytase by A. fumigatus MT899185 was observed after 5 days of incubation at 30 °C. This is similar to previously published reports on phytase production (Coban & Demirci, 2014; Qasim et al., 2016; Neira-Vielma et al., 2018). This may be due to the inducible nature of the enzyme; hence, a prolonged lag phase may be required by the fungi to produce the enzyme. The use of submerged fermentation (SmF) in this study is corroborated by the report of Jain, Sapna and Singh, (2016) where they highlighted the fact that submerged fermentation is associated with ease in the recovery of product (Sethi et al., 2016). Although all formulated feeds in this study met the minimum protein requirement for poultry which is 15% (Osman et al., 2019), the protein content for feed 2, which was formulated with the laboratory-produced phytase from Aspergillus fumigatus MT899185, was the highest (39.50%). This result is different from the one obtained from feed meal made from sunflower shell waste, where a protein content of 16.8% was reported (Osman et al., 2019). Also, Al-Arif et al. (2020) reported protein composition between 21.88% and 22.39% of feed derived from varying compositions of fermented rice bran This suggests that protein content of cowpea may be higher when compared with the protein source in feed 1, most likely soybean which is the globally utilized protein source in poultry nutrition (Jlali et  al., 2020; Zentek & Goodarzi Boroojeni, 2020; Wang et al., 2021). Protein is a vital nutrient component needed in animal nutrition for the growth and tissue regeneration, and it also serves as a very fundamental building block for the animal cells (Osman et al., 2019). The observed increase in carbohydrate content of the control feeds is suggestive of an increase in the amount of corn, but this did not however reflect in the analysed crude fibre content for feed 1 (3.06%) when compared with feeds 2, 3 and 4 with 7.14%, 5.77% and 6.01% crude fibre, respectively. Besides protein, carbohydrates are the most important requirement in poultry feed. This is because they serve as the main source of energy to the birds as other alternative sources of energy, e.g. fats and oils, are less digestible (Lamot et  al., 2019). In this study, the fat content of the feed groups 1–4 ranged between 2.98% and 3.52% (Table 2). This result was in tandem with the results for fat content of feeds reported by Brannan, Livingston and van Rensburg (2021) for starters (3.43%), growers (3.50%) and finishers (4.50%). The

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fat content in the feed formulated with laboratory phytase from A. fumigatus MT899185 was the lowest (2.98%) when compared to the other three feeds. Although fat has been known to serve as an alternative source of energy and to alleviate heat stress in poultry, excess fat could form complexes with available micronutrient, e.g. calcium, and may make the calcium unavailable and decrease the effective energy provision from the feed (Wang et al., 2021). Comparing the results obtained, feed 2 showed reduction in phytate content with a corresponding increase in protein content compared to feed 4 (untreated feed) which had higher phytate and lower protein. The results agree with the study of Qasim et al. (2016), where they demonstrated that a reduction in phytate led to a corresponding increase in protein content of various soy-based products. Esmaeilipour et al. (2013) noted earlier that pre-treatment of feed with microbial phytase may influence phytate hydrolysis even before feeding, thus resulting in the degradation of the phytate. Furthermore, phytates are known to complex with proteins (Balwani et al., 2017); therefore, reduction in the phytate content of the formulated feed may have resulted in the breakdown of pre-formed phytate-protein complexes, thus making more proteins available. Previous report also supports the enzymatic digestion of feed substrate (in this case phytate) prior to the ingestion of the feed by the animals (Dailin et al., 2018).

4.1 Conclusion The use of fungal phytase as an animal feed additive for phytate reduction is well established. Phytate reduction in animal feed has been associated with improved nutrient utilization in monogastric animals. The cowpea-based feed formulated with phytase from A. fumigatus MT899185 was able to reduce the phytate content of the locally formulated cowpea-based feed. Furthermore, all the cowpea-based feeds formulated during this study showed higher protein content than the commercial poultry feed. Finally, the cost of producing the formulated cowpea-based feed was lower than the commercial poultry feed. This study has therefore demonstrated the possibility of utilizing cowpea as a protein source in poultry feed.

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Lamid, M., Al-Arif, A., Asmarani, O., & Warsito, S.  H. (2018). Characterization of phytase enzymes as feed additive for poultry and feed. IOP Conference Series: Earth and Environmental Science, 2018, 012009. https://doi.org/10.1088/1755-­1315/137/1/012009 Lamot, D. M., Sapkota, D., Wijtten, P. J. A., Van Den Anker, I., et al. (2019). Diet density during the first week of life: Effects on growth performance, digestive organ weight, and nutrient digestion of broiler chickens. Poultry Science, 98, 789–795. https://doi.org/10.3382/ps/pey002 Lee, D.-H., Choi, S.-U., & Hwang, Y.-I. (2005). Culture conditions and characterizations of a new phytase-producing fungal isolate, Aspergillus sp. L117. Mycobiology, 33(4), 223–229. Lee, S. A., Lupatsch, I., Gomes, G. A., & Bedford, M. R. (2020). An advanced Escherichia coli phytase improves performance and retention of phosphorus and nitrogen in rainbow trout (Oncorhynchus mykiss) fed low phosphorus plant-based diets, at 11 °C and 15 °C. Aquaculture, 516, 734549. https://doi.org/10.1016/j.aquaculture.2019.734549 Lowry, O., Rosebrough, N., Farr, A., & Randal, R. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265. https://doi. org/10.1007/978-­94-­007-­0753-­5_100521 Neira-Vielma, A. A., Aguilar, C. N., Ilyina, A., Contreras-Esquivel, J. C., et al. (2018). Purification and biochemical characterization of an Aspergillus niger phytase produced by solid-state fermentation using triticale residues as substrate. Biotechnology Reports, 17, 49–54. https://doi. org/10.1016/j.btre.2017.12.004 Oladokun, S., & Adewole, D. I. (2020). In ovo delivery of bioactive substances: An alternative to the use of antibiotic growth promoters in poultry production—A review. Journal of Applied Poultry Research, 29(3), 744–763. https://doi.org/10.1016/j.japr.2020.06.002 Osman, N. S., Khamil, I. A. M., & Sapawe, N. (2019). Proximate analysis of animal feed pellet formulated from sunflower shell waste. Materials Today: Proceedings, 19, 1796–1802. https:// doi.org/10.1016/j.matpr.2019.11.218 Owolabi, A. O., Ndidi, U. S., James, B. D., & Amune, F. A. (2012). Proximate, antinutrient and mineral composition of five varieties (improved and local) of cowpea, Vigna unguiculata, commonly consumed in samaru community, Zaria-Nigeria. Advance Journal of Food Science and Technology, 4(2), 70–72. Qasim, S. S., Shakir, K. A., & Al-Shaibani, A. B. (2016). Purification of phytase produced from a local fungal isolate and its application in food systems. The Iraqi Journal of Agricultural Sciences, 47, 112–120. Sethi, B. K., Jana, A., Nanda, P. K., Dasmohapatra, P. K., et al. (2016). Production of α-amylase by Aspergillus terreus NCFT 4269.10 using pearl millet and its structural characterization. Frontiers in Plant Science | www.frontiersin.org. [Online], 1, 639. https://doi.org/10.3389/ fpls.2016.00639 Walters, H. G., Coelho, M., Coufal, C. D., & Lee, J. T. (2019). Effects of increasing phytase inclusion levels on broiler performance, nutrient digestibility, and bone mineralization in low-phosphorus diets. Journal of Applied Poultry Research 28(4), 1210–1225. https://doi.org/10.3382/ japr/pfz087 Wang, Y., Wang, W., Li, L., Gou, Z., et al. (2021). Effects and interaction of dietary calcium and nonphytate phosphorus for slow-growing yellow-feathered broilers between 56 and 84 d of age. Poultry Science, 100(5), 101024. https://doi.org/10.1016/j.psj.2021.101024 Zentek, J., & Boroojeni, G. F. (2020). (Bio)Technological processing of poultry and pig feed: Impact on the composition, digestibility, anti-nutritional factors and hygiene. Animal Feed Science and Technology 268, 114576. https://doi.org/10.1016/j.anifeedsci.2020.114576 .

Histology, Condition Factor, and Bioaccumulation Analysis of Clarias gariepinus (Burchell, 1822) Exposed to MC-LR Patrick Omoregie Isibor, Onwaeze Oritseweyinmi Ogochukwu, David Osagie Agbontaen, Paul Akinduti, Adagunodo Theophilus Aanuoluwa, Obafemi Yemisi, Dedeke Gabriel Akinwumi, and Akinsanya Bamidele

1 Introduction Many animals are kept in captivity in farms, zoological gardens, conservation centres, research laboratories and homes for aesthetic, commercial or subsistence purposes. Some reports claim that captive animals are often healthier, more viable and more fecund than their free-living conspecifics. But other reports have argued otherwise. Species susceptibility is a noteworthy factor in biodiversity conservation. Regardless of the environment, anthropogenic activities interfere with the interactions between the biotic and abiotic components (Zegura et al., 2011). Fishes are a vital component of the human diet; they are however highly susceptible to anthropogenic perturbations. Inorganic nutrients such as nitrogen and phosphorus are essential for the primary productivity of aquatic ecosystems (Isibor, 2017a). However, excess of these nutrients, occasioned by anthropogenic activities, mainly unregulated application of artificial agricultural fertilizers, has been broadly reported (Isibor & Imoobe, 2017; Isibor, 2017a). Such unregulated use of artificial

P. O. Isibor (*) · O. O. Ogochukwu · P. Akinduti · O. Yemisi Department of Biological Sciences, Covenant University, Ota, Ogun State, Nigeria D. O. Agbontaen Department of Public Health, University of South Wales, Cardiff, UK A. T. Aanuoluwa Department of Physics, Covenant University, Ota, Ogun State, Nigeria D. G. Akinwumi Department of Pure and Applied Zoology, Federal University of Agriculture, Abeokuta, Nigeria A. Bamidele Department of Zoology, University of Lagos, Lagos, Nigeria © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_4

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fertilizers often causes eutrophication of the receiving water bodies, ultimately resulting in fish kills and severe health implications. Tools such as comparative methods have been employed to investigate the fundamental biological causes of species specificity. Advancing the study would help to improve husbandry and enclosure design, as well as reveal relationships between susceptibilities to poor captive welfare and vulnerability to anthropogenic threats in the natural habitat (Zegura et al., 2011; Ralls & Ballou, 2013). Clarias gariepinus is a benthopelagic opportunistic teleost that is mostly demanded in aquaculture due to its high commercial value as a delicacy in the sub-­ Saharan region. In Nigeria, it has become a major source of income, making a significant impact on the diet of the populace as an affordable source of animal protein and the per capita income generated in the country. Whether in captivity or natural habitat, monitoring the nutrient level of the aquatic environment is essential for fish productivity. Studies have shown that eutrophication impacts the foraging skills, detection and avoidance of predators, the success of mating, fertility and fecundity of fish eggs (Zupa et al., 2017). Reproductive dysfunctions in males and females of several fish species have been linked to eutrophication (Corriero et al., 2007; Newcombe et al., 2012; Zupa et al., 2017). Atresia of vitellogenic oocytes and impeded oocyte maturation have been attributed to insufficient pituitary luteinizing hormone, which might occur in a perturbed aquatic environment (Rosenfeld et al., 2012). The most common dysfunctions in females include gonadal underdevelopment (Paerl & Otten, 2013); hampered/halted oocyte maturation upon completion of vitellogenesis (Corriero et al., 2007; Newcombe et al., 2012) or absence of spawning. Spermatozoan deformities, inexpressible low-count semen in males (Zupa et al., 2017) and increased apoptosis have been reported in captive-reared male fish (Zupa et al., 2017). These dysfunctions have been associated with altered or destroyed spawning environments, nutritional deficiencies and ultimately, exposure to cyanobacteria (Mylonas et al., 2010). Advanced knowledge of the impacts of eutrophication on catfish might help improve its domestication (Rodríguez-Barreto et al., 2014. This is a sustainable tool that has the potential to enhance aquaculture production and viability, thus contributing to food security as well as to the reduction in fishing pressure on the wild fish populations which are already perturbed by several other human activities, some of which cause deleterious incidences like eutrophication (STECF, 2014). Eutrophication of aquatic ecosystems supports cyanobacteria blooms particularly in warm conditions. Cyanobacteria (blue-green algae) contain cells which produce biotoxins known as microcystins. Reports have shown the high susceptibility of many organisms to microcystins (Jos et  al., 2005; Isibor, 2017a). Toxic Microcystis cells have been recovered in the intestine of many aquatic vertebrates. Some literature described cyanobacteria as an important component of tropical cichlid’s and cyprinid’s diet (Isibor, 2017b). Toxicological evidence of MC-LR has been provided in the haematological studies (Isibor, 2017a) and oxidative stress analysis (Jos et  al., 2005; Isibor, 2017b). Microcystins cause odour and limit dissolved oxygen and nutrients available to organisms in aquatic ecosystem systems (Balasubramanian & Pavagadhi, 2013).

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Histopathology of fish liver and gills may provide a better understanding of the interactions between a stressor such as MC-LR and anatomical structures and functions (Hardy et al., 2015). Pathogens produce pathological changes in fish such as necrosis in the liver, tubular damage of the kidney and gill lamellar abnormalities (Abdel-Moneim et al., 2012). Therefore, histopathological studies are necessary for the description and evaluation of potential lesions in aquatic animals exposed to various infections and toxicants in aquaculture (Hardy et al., 2015). Histopathological investigations may provide a direct translation of toxicity of MC-LR through the determination of cellular changes that may occur in target organs such as the gills, muscles and liver (Khoshnood, 2015). A histological investigation may therefore prove to be a cost-effective tool to determine the health of organisms, hence reflecting the health of an entire aquatic ecosystem. In the environment, it has been observed that there are many quantitative relationships between the structure and biological activity of chemicals established in aquatic systems. It has been reported that cyanobacteria have the potential of disrupting the growth, development, histology, reproduction and general well-being of fish (Deng et al., 2010; Svircev et al., 2015). After ingestion or absorption, MCs are transported into the cells of humans, mice, rats and fish by organic anion-transporting polypeptides (Fischer et al., 2010; Steiner et al., 2014). MCs may be accumulated in various organs of fish organs, zooplankton, mussels and molluscs (Pham & Utsumi, 2018). These aquatic organisms may pose severe health risks to humans who consume them (Peng et al., 2010). To protect human health, it is imperative to control cyanotoxin concentrations in natural water bodies, fishponds and portable tanks (Hardy et al., 2015). Different exposure routes to MC-LR have elicited varied toxicities in fish tissues (Akindele, 1996; OEHHA, 2009). Fischer and Dietrich (2000) earlier observed sub-lethal effects such as dissociation and degeneration of hepatocytes in carps exposed to 130–300 μg/kg MC-LR. They observed loss of contact between hepatocytes, vacuolization and exfoliation of tubular epithelial cells in the kidney and dilation of Bowman’s capsule in Cyprinus carpio fed 400 μg/kg MC-LR through gavage. Histopathological evidence has also been recorded at 500  μg/kg in Oreochromis sp. injected intraperitoneally (Atencio et al., 2008). Eutrophication of aquatic ecosystems fosters cyanobacteria blooms particularly in warm conditions. In previous studies, microcystins have been detected in aquatic organisms such as mussels (Wilson et al., 2008), crustaceans and fish (Khan et al., 2011; Oyedeji, 2019). The detected concentrations mostly ranged between 0.01 and 100 μg/g tissue (Hardy et al., 2015). The liver is the first to experience the toxicity of MCs; however, sufficient MCs can pass via the liver to other organs such as muscles, kidneys and the brain (Fischer & Dietrich, 2000). Clarias gariepinus, being a priced exotic species in Nigeria, and the choice of most aquaculturists and fish consumers, qualifies as a candidate for this investigation. The variability of algal growth in the natural habitat and aquacultural systems necessitates a comparative evaluation of its bioaccumulation and toxicity between wild and captive Clarias gariepinus. Chia et al. (2009) detected MC-LR concentrations of 5.89, 4.80, 4.50, 4.48, 2.40 and 0.60 μg/L in selected Nigerian ponds. They reported the bioaccumulation of the toxicants by Clarias gariepinus and postulated

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health hazards in the consumers. Furthermore, organic ponds that utilize fertilizers for primary productivity may be more susceptible to elevated Microcystis than farms that depend on formulated feeds. The study is therefore aimed at using a multidimensional/integrated approach in the analysis of MC-LR between captive and wild C. gariepinus.

2 Materials and Methods The chosen study sites were Eleyele River, located at Northeast Ibadan, Oyo State (7°25′57″N, 3°51′54″E); Iju River, Southern Ado-Ota, Ogun State (6°34′59″N, 3°8′59″E); and two intensive fish ponds (labelled A and B) in Ado-Odo Local Government of Ogun State, Nigeria. The Eleyele River was characterized by minimal municipal wastes disposed around its catchment area, while the Iju River is located at the centre of a highly industrialized Ota town. The industries include recycling, manufacturing, food and pharmaceutical industries. Fish farm A was fed mainly on artificial feed, while fish farm B was an organic fish farm fed with mainly poultry droppings.

2.1 Collection and Analysis of Water and Fish Samples We collected 3 L of surface water samples from the four study sites at eight different spots (100 cm apart) in 250 mL dark sampling bottles (Isibor and Imoobe, 2017) and preserved them in a chest cooler with ice. Samples were transported immediately to the laboratory for further analysis. The average pH, temperature and DO at Eleyele River (6–8; 29 ± 2 °C; 7.5 mg/L), Iju River (6–7.5; 28 ± 3 °C; 4.5 mg/L), Pond A (6–9; 25 ± 2 °C; 5.2 mg/L) and B (6–7.2; 28 ± 0.4 °C; 5.8 mg/L) were read using a mercuryin-glass thermometer and Electric Probe Hydro-lab water quality meter (HANNA HI 9813 GRO), respectively. Twenty (20) juvenile Clarias gariepinus (16.82 ± 1.23 g; 10.11 ± 3.17 cm) samples were obtained from each of the four study sites, making a total of 80 fish. The lengths and weights of the fish were measured using a meter rule and Ohaus electronic weight balance (Model number ARC 120), respectively. The log-transformed length-weight relationships of the fish were determined by plotting a linear regression analysis and scatter diagrams of length and weight.

2.2 Collection, Sorting, Counting and Identification of Cyanobacteria Cells in Water Analysis of cyanobacteria samples was carried out using subsamples of 100  mL concentrates from collected water samples using the integrated hose pipe sampler. These subsamples were fixed with 0.1  mL of Lugol’s solution to precipitate and

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preserve algae (APHA, 1998). Laboratory analysis of cyanobacteria was done using the procedures of APHA (1998). Cyanobacteria biomass (cells/mL) was determined using the drop count technique as described by Chen et al. (2009).

2.3 Extraction of Microcystic Cells and Microcystin-LR In the laboratory, the culture of the algal samples was centrifuged at 3500 rpm for 2 h to compress algal cells into pellets. The cell pellet was then lyophilized for 48 h using a freeze-drying system following the guidelines of Lawrence and Menard (2001). The lyophilized cells were extracted three times using 10 mL 0.1 M of acetic acid and 20  mL of a mixture of methanol and chloroform (volume 1:1). The mixture was then sonicated in an ultrasound bath for 15 min, and then stirred for 30 min at room temperature, after which it was centrifuged again at 4500 rpm for 15 min. Microcystin congeners were identified in the cyanobacterial cell extracts using a Varian 9012 equipped with a Varian ProStar 330 Diode Array Detector. Identification was done based on the UV spectra of the congeners and by using commercial microcystin-LR, MC-RR and MC-YR standards provided by Merck Pharma, Nigeria, in Microcystin Plate Kit (Cat. No. 20-0087). The total microcystin constituent of the sample analysed yielded 94% MC-LR congener (6600  μg/kg dry wt.).

2.4 Extraction of MC-LR from Fish Tissues The livers and gills of the fish were eviscerated for histological examination following the procedures of Feist et al. (2015), as modified by Aaron et al. (2017). They were placed in a sample buffer (10 mL of buffer/g tissue) containing 10 mM Tris-­ HCl, 140 mM NaCl, 5 mM EDTA, Triton X-100 (1%), 1 mM PMSF and 1 mM DTT.  The tissues were minced and homogenized using a Dounce Homogenizer 3431-E20 (Thomas Technological Service, USA). Each homogenate was divided into aliquots, one of which served as a control. Homogenates were incubated with 1, 10 and 100 μg MC-LR/g tissue. MC-LR incubation took place in continuously rotating glass vials at 30 °C for 20 h to achieve a representative amount of covalently bound microcystin complexes (Akindele, 1996; Chen et al., 2009). Four or five different homogenates of pooled tissue samples were used for each MC-LR concentration. Microcystin congeners were identified in the cyanobacterial cell extracts using a Varian 9012 equipped with a Varian ProStar 330 Diode Array Detector.

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2.5 Histology of Fish Tissues Haematoxylin and eosin stains were used in the histopathological analysis of the liver, gill and muscle tissues excised from the fish samples. The microscopic view of the dissected liver, gill and muscle tissues of the fish captured from the rivers and fish farms was presented as evidence of MC-LR toxicity (Fig. 1).

2.6 Growth Exponent and Condition Factor The length-weight relationship of the fish was calculated using Le Cren’s cube law (Le-Cren, 1951).:

W = aLb

where W is the weight of fish (g), L is the observed total length (cm), ‘a’ is the regression intercept of the plotted graph and ‘b’ is the regression slope or growth exponent.

Fig. 1  H & E-stained microscopic view of dissected liver tissue of fish. (a) shows the photograph of normal catfish liver, while (b) showed a slight debris (black arrow), and (c) exhibited necrosis (yellow arrow), and discoloration (black arrow), while (d) exhibited necrosis (black arrow), severe discoloration (yellow arrow) and vacuolization (blue arrow). Scale bar = 25 μm

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Logarithmic transformation of Cube’s law: Log  W = Log  a + b Log L Fulton’s condition factor (K) was calculated thus as K   W  100   L3





where W = weight of fish (g) and L = length of fish (cm).

2.7 Statistical Analysis The descriptive statistics were presented as mean ± SD, which were subjected to ANOVA at a significance of p  Pond B> Pond A> Eleyele River releasing MC-LR of 2.04  ±  0.12, 1.24 ± 0.02, 0.26 ± 0.00 and 0.02 ± 0.00 μg/L, respectively. This attendant difference in Microcystis biomass between Iju River and other sites is attributable to the effluents from abattoirs around the catchment areas which are discharged into the river. The wastewater contains animal blood, droppings, a large volume of intestinal and stomach contents and other organic wastes which can stimulate the primary productivity of the aquatic environment, thereby resulting in an algal bloom. Similarly, the algal growth in Pond B can be linked to the poultry droppings used as

Table 1  Concentrations of MC-LR in water (μg/L) and tissues (μg/kg) of C. gariepinus Aquatic environment Eleyele River Iju River Pond A Pond B

Microcystis cells/ mL × 103 28 ± 2.62d 286 ± 8.12a 48 ± 1.82c 72 ± 6.92b

Water 0.02 ± 0.00 2.04 ± 0.12a 0.26 ± 0.00 1.24 ± 0.02a

Liver 0.01 ± 0.00 1.82 ± 0.02a 0.04 ± 0.00 0.08 ± 0.01

Gill 0.01 ± 0.00 1.19 ± 0.02a 0.24 ± 0.00 1.48 ± 0.01a

BFl/w 0.5 0.9 0.1 1.9a

BFg/w 0.5 0.6 0.9a 1.2a

Keys: Emboldened numbers are significant. Numbers with different superscripts are significantly different (0.05), while same superscripts are not. BFl/w bioaccumulation from water to the liver, BFg/w bioaccumulation from water to gills

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feed in the pond. This may also have stimulated algal growth in the pond, but to a lesser extent as Iju River received a larger volume and variety of organic wastes compared to the pond. Remains of artificial fish feed which have settled at the bottom of the tank in farm A may stimulate algal growth when they undergo disintegration and degradation. This extent may however be lower than that of animal droppings as seen in Iju River and Pond B.  Eleyele River is chiefly polluted by heavy metals, PCBs and aromatic hydrocarbons which may account for its relatively low algal content (Olayinka et  al., 2017; Adegbola et  al., 2021; Adewale et al., 2022). The MC-LR concentrations detected in the water (2.04  ±  0.12), fish liver (1.82 ± 0.02) and fish gill (1.19 ± 0.02) of Iju River were higher than the established limit of WHO (1 μg/L). High levels in the gill could be because it is the first organ to encounter biotoxins in polluted water, while the high concentration in the liver may be due to the detoxification role of the organ. Previous data have implicated Microcystis in the bioaccumulation of MC-LR in fish tissues (Fischer & Dietrich, 2000; Akan et al., 2012); hence, MC-LR accumulation in Iju River fish tissues is expected. From the high amount of microcystin cells in the Iju River, one might expect significant water-to-tissue bioaccumulation. However, this was not the case, and this is likely due to the larger and flowing nature of the natural habitat. The tolerable limit for MC-LR in fish tissue was set at 0.04 μg/kg bw/day (WHO, 1998). The liver and gills of catfish from Iju River and gills from Pond B exceeded this limit. In Pond B, significantly high concentrations were only detected in the water and fish gill (1.24 ± 0.02, 1.48 ± 0.01), indicating significant water-to-gill accumulation. The significant bioaccumulation of MC-LR in the liver and gills of fish from Pond B, higher than bioaccumulation in the Iju River, is evidence for the prognosis of greater bioaccumulation in controlled environments.

3.2 Impacts of MC-LR on Fish Condition Factor Results indicated a steady decrease in the slope (b) in proportion to increasing concentration of MC-LR, suggesting the toxicity of MC-LR. Although the regression coefficients of all fish groups were within expected range, the values however decreased with increased concentration of MC-LR (Table 2). Table 2  Condition factors and regression indices of Clarias gariepinus exposed to MC-LR Group Pond A Eleyele River Pond B Iju River

Regression intercept (a) −0.712 0.874

Regression slopes (b) 3.812a 2.918b

Regression coefficient (r) 0.955 0.923

Mean condition factors (K) 1.32 ± 0.034 1.02 ± 0.022

Qualitative analysis Good Fair

0.743 0.456

1.432c 1.225c

0.913 0.903

0.824 ± 0.062 0.78 ± 0.024*

Poor Poor

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Results showed that the fish from Pond A were considered good quality due to their robustness. This was characterized by the growth exponent (b) of the fish, which was positively allometric (Table 2). The ones harvested from Eleyele River exhibited slightly negative allometry, which suggests some slenderness. The fishes were thus considered fair in quality. Conversely, the fish individuals from Pond B and Iju River were considered poor qualities since they had low growth exponents which indicate much slenderness. Furthermore, the condition factors of fish individuals from Eleyele River and Pond A were significantly higher than those from Iju River and Pond B. The poor health conditions of fish in Iju River and Pond B are proportional to the amount of Microcystis cells. Robustness in fish from Pond A is attributable to the fair conditions in the water coupled with regular feeding of the fish, unlike in the natural habitat where the fish has to fend for itself, hence the slightly lower than growth exponent in Eleyele River fish than the benchmark of 3.

3.3 Histopathological Implications of MC-LR 3.3.1 Histopathology of the Liver Light microscopy showed that the liver tissues of all fish samples from each area elicited corresponding levels of structural alterations. A control sample showing healthy catfish liver was used for a comparative analysis. Fish from Eleyele River exhibited no observable tissue alterations (Fig. 1a), while fish from Pond A (Fig. 1b) showed a slight debris compared to control. Fish from Pond B (Fig.  1c) showed necrosis and discoloration, while fish from Iju River (Fig. 1d) showed even greater tissue degeneration (Fig. 1). Furthermore, the study of the tissue sections suggests that Microcystis cells released substantial MC-LR which was bioaccumulated in the fish tissues, thereby causing observable alterations. The bioaccumulation in the catfish corroborates the histopathological effects in the liver and gills (Rosenfeld et al., 2012). No observable alteration occurred in the liver section of fish in Pond A, while in Pond B, necrosis, characterized by relatively large black spots (Neumann et al., 2000), and discoloration, characterized by whitish growths (STECF, 2014; Rodríguez-Barreto et al., 2014; Shubin et al., 2016), were observed. The liver sections of fish from Iju river showed more severe tissue damages such as necrosis, severe discoloration and vacuolization, characterized by larger whitish space (Shubin et al., 2016), compared to that of Eleyele River which showed no significant alterations. 3.3.2 Histopathology of the Gills H & E-stained microscopic view of dissected gill tissues showed that fishes from Eleyele (Fig. 2a) and Pond A (Fig. 2b) exhibited no observable alterations and were not different in appearance to the samples observed in the control. Fishes in Pond B

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Fig. 2  H & E-stained microscopic view of dissected gill tissue of fish shows retained gill architectural morphology in Eleyele River (a). Fishes from Pond A (b) showed no observable alterations. Fish samples from Pond B (c) showed necrosis (black arrow), while those from Iju River (d) showed desquamation (black arrow) and hyperplasia and epithelial lifting at the secondary lamellae (red arrow). Scale bar = 25 μm

(Fig. 2c) showed necrosis, while fishes in Iju River (Fig. 2d) showed desquamation, hyperplasia and epithelial lifting at the secondary lamellae. 3.3.3 Histopathology of the Muscle The fish samples from Eleyele River and Pond A showed normal arrangements of muscle fibres and bundles (Fig. 3a and b, respectively). There were slight lesions observed in the muscle fibres of fish from Pond B (Fig. 3c). Fish captured from Iju River showed more severe damages such as deformities, degenerated muscle fibres and oedema, particularly within the perimysium. In a similar manner, the histopathology of fish gill in Pond A and Eleyele River showed no alterations, while Pond B showed necrosis, while gills from Iju River showed more severe alterations such as hyperplasia, characterized by growths around the gill lamella (Lack et al., 1990; Ralls & Ballou, 2013; Dey et al., 2015), and lamella epithelial lifting, characterized by distorted lamella (Mylonas et  al., 2010; Lowe et al., 2012; Dey et al., 2015; Khoshnood, 2015). MC-LR concentration-­ based histopathological alterations observed in this study are similar to the observations of Jos et  al. (2005) on tilapia. High concentration of MC-LR and high

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Fig. 3  H & E-stained microscopic view of the skeletal muscle tissue of fish showed no observable change in the skeletal muscle tissue of fish from Eleyele River (a) and Pond A (b). Fish samples harvested from Pond B (c) showed slight observable lesions (white arrow) amidst the muscle fibres, while those harvested from Iju River (d) showed swelling of skeletal muscle fibres (white arrow), degenerated cell (black arrow) and oedema (yellow arrow). Scale bar = 25 μm

bioaccumulation in fish from Iju River and Pond B, respectively, may result in extreme hyperplasia leading to complete lamellar fusions that may hamper transportation across the gill’s epithelium and compromise its efficiency. It was earlier reported that hypertrophy and hyperplasia of chloride cells may be directly linked to MC-LR action. Current observations credit the conclusion of Lujić et al. (2015) on gill alterations mediated by MCs. Vacuolations observed in the liver and hyperplasia observed in the gills, both of fish in Iju River are attributable to tendencies of tumour formation as a homeostatic remediation of MC-LR toxicity. The observation conforms to the findings on silver carp fed naturally with toxic MCs blooms (Fischer & Dietrich, 2000; Bouaicha et al., 2005; Paerl & Otten, 2013; Isibor, 2017a), toxicity reports on tilapia exposed to both MC-LR and MC-RR (Corriero et al., 2007; Atencio et al., 2008; Mylonas et al., 2010; Rathod et al., 2011; Newcombe et al., 2012; Svirčev et al., 2013; Drobac et  al., 2016), and impact of MCs on the kidney of omnivorous crucian carp (Carassius auratus) (Li et al., 2013). In vivo studies of the toxic effects of microcystins on the ultrastructures of hepatocytes in omnivorous common carps and carnivorous rainbow trouts also showed evidences of hyperplasia and vacuolations (Campos &

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Vasconcelos, 2010; Newcombe et  al., 2012; Paerl & Otten, 2013; Svirčev et  al., 2013; Parase & Mengunphan, 2015; Isibor, 2017b). From a molecular perspective, tumour production occurs through the inhibition of protein phosphatases 1 and 2A, which are basically the two key enzymes in cellular regulation. Precisely, the apoptotic mechanism by which microcystins destroy the liver is by inhibition of protein through removal of a phosphate from a protein molecule. This toxicity mechanism justifies classification of microcystins as possible human carcinogens (Grosse et al., 2006; Humpage & Burch, 2007; Parase & Mengunphan, 2015; Zupa et  al., 2017).  Microcystins are a group of toxic cyclic peptides produced by certain species of cyanobacteria, also known as blue-green algae. These toxins have been known to have significant impacts on various organisms, including catfish. In this discussion, we will explore the effects of microcystins on the histology (tissue structure), condition factor and DNA of catfish. This study has demonstrated the ability of microcystins to cause severe damage to the histology of catfish. When exposed to microcystins, catfish may experience changes in their organs, especially the liver. Histopathological studies showed that microcystin exposure can lead to liver and gill lesions, necrosis (cell death), fibrosis (excessive tissue repair) and haemorrhages. These histological changes indicate significant muscle damage, which is often the primary target organ of microcystin toxicity in fish. Furthermore, microcystins can also affect other organs such as the kidney, gills and intestine. Histological observations have revealed alterations in these tissues, including inflammation, degeneration and disruption of normal cellular architecture. These changes can impair the normal functioning of these organs, leading to various physiological disturbances in catfish. The condition factor is a parameter used to assess the overall health and well-being of fish. It is calculated by dividing the fish’s weight by its length. Changes in the condition factor indicate alterations in the fish’s physiology and health status. Exposure to microcystins has been found to affect the condition factor of catfish. This conforms to several other studies which have reported a decrease in the condition factor in catfish exposed to microcystins (Khan et al., 2011; Akan et al., 2012; Isibor and Imoobe, 2017; Oyedeji, 2019). This decline in condition factor is often associated with reduced food intake, impaired digestion and liver damage caused by microcystin toxicity. The decrease in the condition factor suggests that catfish exposed to microcystins may experience compromised health and reduced fitness. It is worth noting that the severity of the impact of microcystins on catfish depends on factors such as the concentration and duration of exposure, the specific cyanobacterial species producing the toxins and the individual susceptibility of the fish species. Microcystins can have profound effects on the histology, condition factor and DNA of catfish. These toxins can induce histological changes, particularly in the liver, as well as impact other organs. Additionally, exposure to microcystins can lead to a decrease in the condition factor, indicating compromised health. Understanding the impacts of microcystins on catfish is crucial for assessing the ecological and health risks associated with cyanobacterial blooms in aquatic environments.

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4 Conclusion The study has provided data for informed decisions for the management of the incidence of fish contamination from microcystins in aquaculture facilities and natural aquatic habitats. Data presented is useful for aquacultural management.

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Morphological Trait Variation and Correlation Analysis in Landraces of Southern Nigerian Fluted Pumpkin (Telfairia occidentalis Hook. F.) Oluwadurotimi S. Aworunse, Jacob O. Popoola, Lawrence S. Fayeun, Eze Frank Ahuekwe, and Olawole O. Obembe

1  Introduction Fluted pumpkin (Telfairia occidentalis Hook F.) is one of the nutritionally and economically relevant cucurbitaceous seed and leaf vegetables commonly cultivated in Southern Nigeria. The plant is indigenous to West Africa and some regions of Central Africa. It is a fast-growing creeping vine that branches luxuriantly, producing extensive foliage cover when pruned (Fayeun et al., 2016, 2018). Fluted pumpkin is a dioecious perennial crop that produces very large pepo fruits with numerous sizeable brown or black seeds (Nwangburuka et al., 2014; Umeoka & Ogbonnaya, 2016; Fayeun et  al., 2018). The perennial habit of the plant confers an all-year-­ round availability that contributes to small-holder farmer’s revenue annually (Fayeun et  al., 2012a, 2018). Fluted pumpkin is touted as one of the indigenous The original version of this chapter was revised. The correction to this chapter is available at https://doi.org/10.1007/978-3-031-33370-5_21 O. S. Aworunse · E. F. Ahuekwe Department of Biological Sciences, Covenant University, Ota, Nigeria J. O. Popoola Department of Biological Sciences, Bowen University, Iwo, Osun State, Nigeria L. S. Fayeun Department of Crop, Soil and Pest Management, The Federal University of Technology, Akure, Nigeria O. O. Obembe (*) Department of Biological Sciences, Covenant University, Ota, Nigeria UNESCO Chair on Plant Biotechnology, Plant Science Research Cluster, Department of Biological Sciences, Covenant University, Ota, Nigeria e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023, Corrected Publication 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_5

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vegetables that can help to combat poverty, generate foreign exchange and provide sustainable employment (Fayeun & Odiyi, 2015), thereby realising the goals 2 and 3 of United Nations sustainable development agenda. The leaves and seeds form an integral component of many indigenous cuisines (Omimakinde et  al., 2018). Consumers prefer fluted pumpkin over other vegetables because the green quality and good texture of the leaves can be maintained in soups for longer periods (Fayeun et al., 2012a). More so, the folkloric use of the leaf extract to remedy diabetes, high blood pressure, convulsion and anaemia is well recognised (Ehiagbonare, 2008; Fayeun et al., 2012a, b; Nwangburuka et al., 2014). Fluted pumpkin leaves are rich in protein, carbohydrates, vitamins, crude fibres, iron, calcium, potassium, copper, cobalt, phenols, flavonoids and terpenes (Omimakinde et al., 2018; Ochokwu et al., 2021). Oils derived from the seeds serve as feedstock for the manufacture of pomade and margarine (Nwangburuka et al., 2014). In addition to the unsaturated fatty acids which represent a significant percentage of the seed-derived oils, the seeds of fluted pumpkin are good sources of protein, fibre, calcium, iron, zinc and vitamins (China et al., 2021). Fluted pumpkin growers are faced with problems of low and unpredictable yield, poor quality of harvest and reduced pest and disease tolerance due to the unavailability of improved cultivars. Considering that the crop provides nutrition for over 100 million people, coupled with its industrial and economic potential, the need to develop improved cultivars becomes more relevant than ever (Fayeun & Odiyi, 2015). In order to efficiently select and breed for improved or new genotypes, breeders must leverage available variation in germplasm collections (Fayeun et al., 2018). While phenotypic and genotypic characterisations constitute the most commonly used approaches for accessing variations (Shitta et al., 2022), phenotypic assessment remains a fundamental step in the description, classification and evaluation of plant germplasms to enhance utility in breeding programmes (N’Da, 2016; Kumari et  al., 2017; Nelimor et  al., 2019). Variation in phenotypic characters has been extensively applied in the identification of unique traits, duplicates and selection of lines with desirable attributes (Shitta et  al., 2022). Furthermore, phenotypic traits offer the advantage of being easily distinguishable, thus representing an easily accessible level of diversity to breeders and farmers (N’Da, 2016). Landraces of fluted pumpkin constitute an exploitable genetic resource for the development of improved varieties. Fluted pumpkin germplasms have been largely characterised using vegetative, fruit and seed attributes (Fayeun & Odiyi, 2015; Nwangburuka et  al., 2014; Fayeun et  al., 2016). However, broadening the set of traits to include floral/reproductive characters in the phenotypic evaluation could guarantee maximal utilisation in modern breeding programmes. In this study, 32 landraces of fluted pumpkin were obtained from 12 states across Southern Nigeria, where the vegetable is well known and densely cultivated, and assessed for morphological variability in quantitative and qualitative traits, including floral/reproductive traits.

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2 Materials and Methods 2.1 Germplasm Collection Thirty-two fruit genetic resources of Telfairia occidentalis fruits were obtained from 12 states in Southern Nigeria (Southwest, South-south and Southeast). The states included Abia, Anambra, Imo, Enugu, Ondo, Ogun, Oyo, Lagos, Delta, Rivers, Edo and Cross Rivers. The areas of collection of the fruits were separated by a distance of at least 5 km apart (Wada et al., 2021). Only fruits without any form of damage were selected. The seeds were transported while still in the fruit in order to preserve them before planting. In transit, the fruits were maintained under aerated conditions. The fruits were assigned unique codes which highlighted the species name and areas of collection. Passport data of the landraces are presented in Table 1.

2.2 Experimental Site and Climatic Condition On-field study was conducted at the experimental farm of the Department of Biological Sciences, Covenant University, Ota, Ogun State, Nigeria. The farm is located within longitude N6°40′25.272″, latitude E3°9′22.80288″ and an altitude of 47 m above sea level. Geomorphology of the site is plain level, with well-drained soil. The climate is of humid type, with a bimodal rainfall pattern marked by an extended rainy season that begins in April, peaks in June through mid-July, troughs slightly in late July through early August in what is commonly known as ‘August break’ and then peaks again in September (Chineke et al., 2010). This is followed by a dry season that extends from October through March. The average total sunshine period is about 7 h/day which can be extended in September through April (Alake & Alake, 2016). The average annual rainfall fluctuates between 1000 and 2000 mm, with a mean ambient temperature of around 27 °C. The field soil represents a sandy loam texture. The experimental site was previously under paw-paw production.

2.3 Land Preparation, Experimental Design and Layout The experiment was established from August 2020 to May 2021. The field was cleared and then tilled with a disc-ploughed to a 0.5 m depth. Subsequently, harrowing was conducted to pulverise and smoothen out the soil surface. Stumps and debris were removed before making ridges (plots) for the study. The experiment was laid out in a randomised complete block design (RCBD) with three replications. The experimental area had a measurement of 32 m × 30 m (960 m2), and each replication measured 32 m × 10 m (320 m2). Replications incorporated 32 experimental plots (to accommodate the 32 landraces of T. occidentalis in a randomised manner), each comprising a single row ridge that measured 10 m × 1 m (10 m2) as

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Table 1  Passport data of the 32 Telfairia occidentalis landraces used for genetic diversity assessment S/N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Landrace number ToOg001 ToOg002 ToOg003 ToOg004 ToLg001 ToLg002 ToON001 ToOn002 ToOn003 ToOy001 ToDt001 ToDt002 ToDt003 ToEd001 ToEd002 ToEd003 ToEd004 ToEd005 ToRv001 ToRv002 ToRv003 ToCr001 ToCr002 ToIm001 ToIm002 ToIm003 ToEn001 ToEn002 ToEn003 ToAn001 ToAn002 ToAb001

Area of collection Abeokuta Ifor Ota Idiroko Imota, Ikorodu Badagry Ita-Olorun community Okitipupa Akure Ibadan Okpara Inland Ubiaruku Ugbogo Ehor Ugbowo New Benin Abudu Ikpoba-Okha Choba Ahoada Etche Ogoja Anang Ikeduru Owerri Ngor-Okpala Nsukka Ezeagu Awgu Afor Nnobi Igbo-Ukwu Ohafia

State Ogun Ogun Ogun Ogun Lagos Lagos Ondo Ondo Ondo Oyo Delta Delta Delta Edo Edo Edo Edo Edo Rivers Rivers Rivers Cross Rivers Cross Rivers Imo Imo Imo Enugu Enugu Enugu Anambra Anambra Abia

Origin Unknown Unknown Ota Cotonou Unknown Unknown Unknown Unknown Unknown Elegoro park Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Ahoada Unknown Calabar Calabar Unknown Owerri Unknown Nsukka Unknown Unknown Ogidi Unknown Unknown

recommended by Odiyi et al. (2014). The replications were separated by 1 m alleys to reduce inter-replication competition.

2.4 Cultivation and Management Seeds were extracted from the fruits and washed in water to remove attached fruit pulp. The seeds were then air-dried at room temperature for 24  h to drain-off water. For each landrace, three seeds were manually sown per hole at a depth of

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2 cm (Oluwole et al., 2020) and at a spacing of 1 m between rows and 1 m among plants within a row (Odiyi et al., 2014). Seedlings were thinned to one per hole at the first-­true-­leaf stage so that each experimental plot contained ten plant stands (Alake & Alake, 2016). Although the study was conducted under rain-fed conditions, the experimental plots were irrigated to field capacity when necessary, using sprinklers. The plots were kept weed-free by manual weeding every 4 weeks. Pest and disease control were carried out as necessary using the Zeforce pesticide according to the manufacturer’s instruction every 8  weeks. Fertiliser (NPK 15:15:15) was applied at 4 and 8 weeks post sowing using the localised placement method in the soil close to the base of plant at a rate of 30  g/stand (Ezenwata et al., 2019).

2.5 Data Collection Five vigorous plants per landrace were tagged, and used in measurements and observations of morphological traits, with the exclusion of border plants at the extremes of each plot. Pods were harvested at full maturity (192–273  days) and weighed and then seeds were extracted, cleaned and weighed. Data were obtained on 31 morphological characters comprising seed, fruit pod, floral/reproductive and vegetative traits. Of these traits, 26 were quantitative, while 5 were qualitative. Morphological characterisation was performed using descriptors adapted from Cucumis melo (IPGRI, 2003), Moringa oleifera (Popoola et  al., 2016) and those previously reported for T. occidentalis (Fayeun & Odiyi, 2015). Quantitative characters were based on measurements, while qualitative characters were based on visual observation. A comprehensive description of all morphological characters considered in the study is presented in Table 2.

2.6 Data Analysis 2.6.1 Quantitative Morphological Trait Variability Data was evaluated by performing descriptive and multivariate analyses. Quantitative morphological data was subjected to one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) software (IBM SPSS Statistics for Windows version 25, IBM Corp, Armonk, NY). Significant differences in the means of the quantitative characters across all the 32 landraces were tested at p  Cr > Cd. There was a close relationship between the contamination of the fish intestines and the ages of the fish samples in Eleyele River and the artificial feed pond. This trend was not observed in the Ado-Odo River.

4 Discussion According to Abdul et al. (2017) and Abiona et al. (2019), Abraham et al. (2018), the seasonal diversity of food items can impact fish-eating preferences, diet and feeding intensity. Low gastro-somatic index might be due to an inverse connection between feeding activity and nutrient storage, which is reliant on gonadal development. When an adequate amount of energy has been accumulated in gut tissues, the time corresponding to the initiation of gonadal growth, high food consumption is generally detected during the post-spawning period, when most of the energy accumulated has been used up for gonadal growth, and low feeding habit occurs when enough energy has been amassed in gut tissues, the time corresponding to the initiation of gonadal growth. That is, a fish’s physiological condition determines how much it eats. Fish may also change their gut shape in reaction to available food supplies, according to Prati et al. (1971) demonstrating a high degree of plasticity

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(Smith et al., 1999; Zaki et al., 2010; Tuset et al., 2006; Sevcikova et al., 2011; Wartenberg et al., 2011; Tijani et al., 2012). The gastro-somatic index explains the feeding intensity. Factors affecting the feed intensity include stress and pollutants, feed type and sizes, hormones, physiological status of fish, etc. (Eriegha & Ekokotu, 2017). Fish from Eleyele River had the highest feeding intensity because they fed moderately well in relation to their body weight. The fishes in artificial feed pond did not have a high feeding intensity probably because of farm management practices. The river in Ado-Odo is highly polluted and it contains a variety of suspended particles and trace metals. This is a factor contributing to the low feeding intensity in the Ado-Odo River. The feeding habit, geographical location, species richness or abundance, sex, immune status, age distribution and sizes are also essential factors to consider in evaluating the well-being of fish species. Assessing the length-weight relationship of fish species helps determine the exponential growth rate of the fish by investigating how weight changes as the fish get longer (Abdel-Warith et al. 2020; Ayanda et al., 2020). Length-weight correlation simply infers that as fish get longer, they get heavier faster. Length-weight is also anchored on conditional factors, including feeding habit, season, distribution, maturity of the gonad, sex and annual fluctuations in environmental conditions (Akinsanya & Kuton, 2016). Generally, an estimation of the length-weight value of a fish implies the well-being of that fish (Akinsanya & Kuton, 2016). This estimation shows that fishes with increased weight at a given length are in better condition than fishes with less weight (Akinsanya & Kuton, 2016). It is significantly influenced by both biotic and abiotic environmental factors and may be used to indicate the health of the aquatic ecosystem in which fish reside. The evaluation of the health profile of a fish species is an index of its access to food, growth and development and feeding habits (Akinsanya & Kuton, 2016). According to Le Cren, when b = 3, growth is isometric, when b   3, growth is positively allometric. From the results of this work, the b values obtained from the graphs indicate a negative allometric growth rate (retarded growth) among the fish species from the controlled (captive) and uncontrolled (wild) environments. This indicates that the fish do not develop symmetrically (Moiseenko, 2005; Mona et al., 2013; Mutlu & Kurnaz, 2017) or get thinner as they grow longer. The current findings conform to the works of (Nwali et al., 2015; Ogbuagu et al., 2015; Oguguah et al., 2017; Ojelabi et al., 2018). In Eleyele Lake, Ayoade et al. (2018) discovered negative allometric development trends for Sarotherodon melanothe. The regression sum of squares from all locations except Eleyele River indicates that the length has a poor influence on the weight of the fish, which is a poor relationship between both parameters. Eleyele River has a moderate value for its regression sum of squares (56.89%). The length has a moderate influence on the weight. The length-weight relationship followed the trend. In previous literature (Akinyemi et al., 2014), the river is used as a point of reference because of its integrity. There is a probability that something may be going wrong in the environment because of the anthropogenic activities occurring in that area. In the artificial pond, this may be because of poor farm management

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practices, e.g. unclean water, etc. The fish farmer is probably interested in overfeeding the fish for more profit, but the decomposition of artificial feed given to the fish may increase nitrogen concentration in the water, which impacts the health of the fish, making it unhealthy. Allochthonous factors such as eutrophication may also contribute to the unhealthiness of the fish (Olowu et al., 2010; Okocha & Adedeji, 2011; Olagbemide, 2017; Oyeleke et al., 2018).

5 Recommendations and Conclusion In the Ado-Odo River, bioaccumulation may be a result of the constant pollution from numerous manufacturing industries in the catchment area. Ibadan is developing rapidly and the rate of emigration to Ibadan is on the rise. The increasing anthropogenic activities might have contributed to the pollution of the Eleyele River. Captive fishes showed positive allometry although farm management practices may also pose a threat to the well-being of the fishes here. The fishes from the uncontrolled environments were not in good condition because these rivers are not regulated. In alignment with Sustainable Development Goal (SDG) 6 which is aimed at clean water and sanitation, and SDG 14 which focuses on life underneath the water, renewed commitments from the government and stakeholders are imperative for developing regulatory frameworks that guide the discharge of industrial effluents into water bodies. Constant monitoring and surveillance of trace metals are necessary in water bodies.

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Occurrence and Characteristics of Microplastics in the Surface Water and Sediment of Lagos Lagoon, Nigeria Fadekemi O. Akinhanmi, Opeyemi I. Ayanda, and Gabriel A. Dedeke

1 Introduction Plastic production and use has been steadily increasing in recent years due to the high demand for plastic products (Plastics Europe, 2020). In 2017, global plastic production nestled at 348 million metric tons with an estimated 9% annual increase (Verla et al., 2019a). Their unique features including lightweight, low cost, flexibility, and durability have contributed to this high worldwide demand. Consequently, improper disposal and their nonbiodegradable nature present plastics as a threat to global aquatic and terrestrial ecosystems. Plastic debris accumulates in landfills and the world’s oceans and builds up in the environment as litter (Wang et al., 2019; Barbosa et al., 2020). While plastic debris is in the environment, they undergo fragmentation and abrasion to form tiny particles known as microplastics (1–1000 μm), which are more reactive and potentially toxic to all life-forms (González-Pleiter et  al., 2019). Microplastics (MPs) may also originate from the direct release of small plastic particles such as microbeads used in industrial processes, clothing, and personal care products (Andrady, 2011). Other primary sources of MPs include abrasives used in offshore maintenance and sandblasting at shipyards, microplastic dust from industrial processes like polishing and molding plastic products, and dust from plastic degradation at home, and textile fibers from laundry machines (Fred-­ Ahmadu et  al., 2021). In addition, secondary sources of MPs could also include plastic debris from inadequate waste handling; macroplastic debris from loss of fishing trawls, nets, and ropes; and plastic waste directly thrown into the water bodies during recreation and boating. Furthermore, leachate from landfills and F. O. Akinhanmi (*) · O. I. Ayanda Department of Biological Sciences, Covenant University, Ota, Nigeria G. A. Dedeke Department of Pure and Applied Zoology, Federal University of Agriculture, Abeokuta, Nigeria © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_7

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wastewater treatment plants (WWTP) are primary repositories of MPs (Golwala et al., 2021). MPs gain entry into aquatic systems primarily via surface/litter runoffs, plastic production processes, WWTP effluent, single-use plastics, poor waste disposal, and poor waste management practices (Lebreton et  al., 2017; Chaukura et  al., 2021; Turan et al., 2021; Benson et al., 2022). Hence, much global plastic debris and MPs reach the ocean via streams, rivers, and other inland waters (Lebreton et al., 2017). These contaminants do not biodegrade readily, leading to persistent pollution (Agboola & Benson, 2021). The persistence of MPs in the environment could result in them acting as organic-vector contaminants, wreaking havoc on aquatic and human life. Furthermore, there have been reports that they consist of leachable inorganic additives that are usually not covalently bonded to the plastic particles with a likelihood of getting desorbed into the ambient aqueous environment (Benson & Fred-Ahmadu, 2020; Jeong & Choi, 2020; Tanaka et al., 2019). The concerns about microplastic pollution stem from their increasing ubiquity in global ecosystems, particle toxicity, and chemical toxicity to aquatic biota and humans, as revealed in many studies (Enyoh et al., 2019a; Jambeck et al., 2015; Verla et al., 2019b). Recently, there has been increasing awareness of the menace of microplastic pollution in the Nigerian scientific and social community (Egbuna et al., 2021). Part of the major contributing factors to MP pollution in Nigeria is the volume of estimated municipal waste being generated, which is about 35  million tons, with 10–15% plastic waste (Ecolex, 2020). In fact, the country currently ranks sixth among countries with mismanaged plastic waste (Plastic Atlas, 2020), as against previously ranked ninth in the world (Obiezu, 2019). This is an indication of the rise in plastic waste in Nigeria. Furthermore, poor waste handling practices and minimal recycling eventually result in plastic waste ending up in the environments, i.e., the water, sediments and fauna, and landfills (leaching into underground water), where they may take several years to break down, degrade, and decompose (Egbuna et  al., 2021). In addition to the current plastic waste load in the environment is the indiscriminate disposal of face masks and single-use personal protective equipment enforced in response to the COVID-19 pandemic. MPs have been reportedly found in the surface water, sediments, and aquatic organisms in African ecosystems, with few studies originating from Nigeria (Akindele et al., 2019; Oni et al., 2020). In 2019, Enyoh et al. published a maiden report on the abundance, distribution, and composition of macro debris and microplastics from five rivers in Southeastern Nigeria. The reported abundance ranged from 440 to 1556 particles/L, with the distribution of plastic types being PET (29%), PE (22%), PVC (16%), PP (14%) and others (6%). In addition to reports on microplastic occurrence in the aquatic environment, studies have shown the presence of microplastics in the stomach of commercial fish species (Adeogun et  al., 2020), and gastropods have been used as bioindicators of microplastic pollution (Akindele et al., 2019) in Southwest Nigeria. Lagos Lagoon is a coastal wetland bordering the urban agglomeration of the most populous and largest city in Southern Nigeria. The rapidly growing population, coupled with the tremendous economic growth in Lagos city, has fairly increased both consumption growth and an upsurge in municipal waste generation.

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Fig. 1  Plastic wastes in the Lagos Lagoon

This situation is worsened by poor and inadequate waste management practices, allowing plastic waste into the city’s water bodies (Fig. 1). Lagos Lagoon is also a socio-economically important water system where some inhabitants of the Lagos metropolis generate their livelihood while providing transport services, places of recreation and abode, dumpsite for industrial and residential discharges, and a natural cushion to balance forces within the ecosystem (Omoyajowo et al., 2021). The occurrence and types of microplastics in the water and sediment from the Lagos Lagoon was reported by Olarinmoye et al. (2020). MPs’ abundance ranged from 139 to 303 particles/L in water and 310–2319 particles/kg in sediment, predominantly fibers in all samples followed by fragments and films. In addition, the presence and distribution of MPs in sediments of five beaches in Lagos, Nigeria, in order of abundance was reportedly polyethylene, polystyrene and polypropylene (Fred-Ahmadu et al., 2021). In spite of these reports, there’s still a need to further establish new invasions of MPs in the surface water and sediment around major sources of aquatic organisms in the Lagos Lagoon. Here, this study aims at presenting data of the presence and characteristics of microplastics in four stations, namely, Epe (EP), Makoko (MK), SagboKoji (SK), and Badagry (BD).

2 Materials and Methods 2.1 Study Area The study area is the Lagos Lagoon (Fig. 1), a coastal wetland found within the Southwestern region of Nigeria, bordering the Atlantic Ocean (Nwankwoala, 2012). The Lagos Lagoon complex, the fourth biggest lagoonal system in the Gulf of Guinea, spreads from Cotonou in the Republic of Benin to the edges of the Niger

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Fig. 2  Location of the study area and sampling sites in the Lagos Lagoon. (Source: Google Earth Pro 2021)

Delta in Nigeria along its 257 km path spanning more than 50 km in length and 3–13  km in width (Badejo et  al., 2014). Between latitudes 3°24′01.49″E and 6°23′49.69″N is where the main portion of the lagoon complex is, serving as the study site (Fig. 2). It is a vast region of shallow water, with depths ranging from 0.3 to 3 m in most parts, rising to a maximum of 5 m in the major Lagoon and 25 m in some dredged areas of the Lagos harbor (Ajao, 1996). Four different stations were sampled along the Lagos Lagoon (SagboKoji and Makoko), while further to the west (Badagry) and the last station to the east (Epe) (Fig.  2). The sampling stations SagboKoji and Makoko are located inside the Lagoon. SagboKoji, an island inhabited by local fishermen and Makoko on the west, is a local community characterized by pile houses. Badagry lies on the north bank of Porto Novo Creek, close to Lagos city and Epe located on the northside of the Lekki Lagoon.

2.2 Sampling Surface water and sediment samples were retrieved between February and March 2022, using composite sampling procedures to establish sample representativeness. Five sites were randomly sampled in each sampling station and their coordinates were recorded with a GPS. Equal volumes (2 L) of surface water samples were collected with a metal bucket at a depth of 10 cm, stored in pre-cleaned glass jars, and preserved with ethanol (40%). Sediment samples were retrieved with a Van Veen grab at 2–5 cm depth, stored in pre-cleaned glass jars, and preserved with ethanol

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(40%). Environmental parameters that describe the lagoon water quality were measured and recorded in situ using a multiparameter water analysis meter (HORIBA U-52, Advanced Techno Co., USA).

2.3 Microplastic Extraction from Water and Sediment Water samples were digested for 12 h with hydrogen peroxide (30% H2O2) and filtered using a membrane ultrafiltration setup. The pore size of the membrane filter was a 0.45 μm (Millipore). The filter paper was immediately transferred to a glass petri dish and allowed to dry prior to further analysis. Microplastics in surface water samples were estimated as particles per liter. Sediment samples were oven-dried at 40 °C for 12 h, while the wet and dry weights of the sediment samples were noted. 200  g of dried sediment samples were measured, mixed homogeneously with 400 mL of distilled water, and left to settle for 24 h in 500 mL glass conical flasks. 100 ml of a saturated solution of zinc chloride (ZnCl2) (1.5 g/cm3) was added and mixed homogeneously for 15  min. This was left to stand overnight and filtered using a stack of sieves. All extracted microplastics were transferred to a petri dish and allowed to dry prior to further analysis. Microplastics in sediment samples were estimated as particles per kilogram dry sediment.

2.4 Identification of Microplastics in Water and Sediment Samples 2.4.1 Visual Identification Visual identification was carried out with optical microscopy (Olympus CH, XSZ-107BN, USA). Standard criteria for the identification of speculative microplastics were adopted after Norén (2007). Size, color (black, blue, red, green, transparent), and shape (fiber, fragment, film) were observed and recorded. 2.4.2 FTIR Analysis Agilent FTIR (Germany) was used to characterize numerous plastic particles that were typical of the diverse morphotypes found in water and sediment. To achieve clean spectra, the FTIR measurements were carried out in the wavenumber range of 4000–650  cm−1, with eight co-added scans and a spectral resolution of 4  cm−1. Using the web tool OpenSpecy, the generated spectra were evaluated, and the polymer type was determined by contrasting the spectra with those in the reference library.

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2.5 Quality Control External sources of microplastic contamination were avoided by limiting contact with plastic products and always covering samples with aluminum foil. Only glass containers with tight-fitting lids were used to collect and keep samples. Background contamination was reduced by utilizing only cotton aprons and nitrile gloves. Before usage, all of the glassware was carefully cleaned and wrapped in aluminum foil. Ten pieces of nylon plastic pulled from the silt were chopped, mixed with water samples, and put through the same extraction procedure to test the method’s recovery effectiveness. The recovery rates varied from 95% to 100%. No significant contamination was found because procedure blanks with solely saturated ZnCl2 solution were evaluated together with the samples.

2.6 Statistical Analysis Data generated will be presented as mean ± standard deviation (SD) and will be analyzed using SPSS IBM version 25.0 computer program (SPSS Inc. Chicago, Illinois, USA). All data will be preliminarily tested for normality (Kolmogorov-­ Smirnov and Shapiro-Wilk test) and homogeneity (Levene test) prior to analysis. Variations in the means will be subjected to one-way analysis of variance (ANOVA) and Duncan multiple range test (DMRT). Statistical significance will be established at p T and SCN1A:c.3184A>G Polymorphisms with Epilepsy Risk or Drug Resistance in Childhood Epilepsy Syndromes in Lagos State, Nigeria Ibitayo Abigail Ademuwagun, Solomon Oladapo Rotimi, and Ezekiel Adebiyi

1 Introduction One of the distinguishing features of childhood epilepsy syndromes is refractoriness to drugs. This may be due to the presence of single nucleotide polymorphisms which have the potential to impact the pharmacokinetics of anti-seizure medications (ASMs) or influence the binding of drugs to the ion channels or receptors which are the primary sites of action of many ASMs (Balestrini & Sisodiya, 2018). Like other xenobiotics, the biotransformation of ASMs such as phenobarbital, primidone, topiramate, valproic acid, phenytoin, felbamate and carbamazepine occurs in two major stages, including oxidative catalysis by cytochrome P450s and phase II conjugation enzymes (Perucca, 2006). Most ASMs are oxidised by at least three major CYP enzymes, namely, CYP2C9, CYP2C19 or CYP3A4 (López-García et al., 2017; Silvado et al., 2018). The gene, CYP2C9,

I. A. Ademuwagun (*) Covenant University Bioinformatics Research (CUBRe), Covenant University, Ota, Ogun State, Nigeria Biochemistry Department, Covenant University, Ota, Ogun State, Nigeria e-mail: [email protected] S. O. Rotimi Biochemistry Department, Covenant University, Ota, Ogun State, Nigeria E. Adebiyi Covenant University Bioinformatics Research (CUBRe), Covenant University, Ota, Ogun State, Nigeria Computer and Information Sciences Department, Covenant University, Ota, Ogun State, Nigeria Division of Applied Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_9

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which maps to the chromosomal region, 10q24.2, is a highly versatile gene in drug metabolism, oxidising at least 15% of all treatment drugs. It is highly polymorphic, having 60 or more different polymorphic alleles (Dai et  al., 2014). Amongst these genes, three alleles predominate, namely, CYP2C9*1 (also known as the wild-type allele), CYP2C9*2 and CYP2C9*3 which are mutant alleles which impose reduced therapeutic impacts of the gene (Sánchez-­Diz et al., 2009; Soga et  al., 2004). The CYP2C9*2 polymorphism, also denoted as CYP2C9 c.430c>T allele, encodes a mutant protein which bears cysteine at position 144 in place of a charged arginine residue, resulting from a single nucleotide base pair substitution of cytosine for thymine at position 430 of the CYP2C9 gene. This variation results into a less functional enzyme (Eltalal et  al., 2021; Pratt et  al., 2019). Genetic polymorphism that influences structure-function relationship of these genes may affect the therapeutic value derived from ASM metabolism (Wilkinson, 2005). Another category of a highly polymorphic gene known to be involved in pharmacoresistance is SCN1A, the gene encoding the alpha subunit of voltage-gated sodium channel type 1 (Nav1.1). The sodium ion channels are critical for the depolarisation phase of action potentials in neurons; hence, they are the primary targets of several ASMs acting as channel blockers such as phenytoin, lamotrigine, mexiletine, lidocaine and so on (Dokken & Fairley, 2021). Thousands of pathogenic variants and population-based polymorphisms have been found in SCN1A alone (Xie et  al., 2020). Amongst the known SCN1A polymorphisms that modulate the structure-­ function relationship of Nav1.1 is a missense variation that changes a hydroxy group containing threonine residue at position 1067 to a neutral alanine residue, due to a nucleotide substitution of adenine to guanine at position 3184. The polymorphism affects channel gating properties and reduces the effectiveness of sodium channel blockers (Wang et al., 2014). The alteration in response to ASM administration has been linked to pharmacogenetics which play a crucial role in altering pharmacodynamics and pharmacokinetics, hereby influencing ASM tolerance, potency and toxicity (Franco & Perucca, 2015; Silvado et  al., 2018). And due to differences in inter-individual response to drugs, there has been a rising need to identify and genotype key genetic biomarkers responsible for low therapeutic effects of ASMs. For example, known polymorphic forms of CYP2C9 occur to varying extents in Caucasians, black Africans, black Americans, white Americans, Asians and North American and Brazilian races (Dagenais et  al., 2017; Jose et  al., 2005; Soga et  al., 2004; Sullivan-Klose et  al., 1996). Since racial and ethnic differences arise due to genetic polymorphisms, pharmacogenetics must be studied in the context of individual populations. To the best of our knowledge, this is the first study to report CYP2C9 and SCN1A drug response-­associated polymorphisms in Nigerian children with epilepsy syndromes.

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2 Patient Recruitment and Methods This research was approved by the Health Research and Ethics Committee (HREC) at the Lagos State University Teaching Hospital (LASUTH), Ikeja,  Lagos State, Nigeria, with permit number LREC/06/1754 and the Covenant University Health Research and Ethics Committee (CHREC), Covenant University, Ota, Ogun State, Nigeria, with permit number CHREC/50/2020. Participants’ information form was given to each patient’s parent, following proper education on the study objectives and benefits. Informed consent was obtained using a written consent form, signed by patients’ parents or legal guardians, in the presence of a research investigator and a witness. The sampling phase of this research was done at the Lagos State University Teaching Hospital (LASUTH), Ikeja, Lagos State, Nigeria. The participants who attended this study were neonates, infants and children (within the ages of 0–10 years), having clinical diagnosis of infantile and childhood epilepsy syndromes according to the ILAE guidelines.

2.1 Molecular Investigations Using polymerase chain reaction (PCR), the CYP2C9 gene region hosting the c.430C>T polymorphism was amplified as described by Eltalal et al. (2021). Two sets of forward and reverse primers were used to genotype the variant. The primers used are as follows: Forward primer 1 Forward primer 2 Reverse primer 1 Reverse primer 2

5′-GGAGGATGGAAAACAGAGAC-3′ 5′-GATATGGCCACCCCTGA-3′ 5′-AGGAGCATTGAGGACC-3′ 5′-GCTTCCTCTTGAACAC-3′

Pre-heating of the lid was done at 105 °C, followed by initial denaturation at 94 °C for 5 min; primer annealing and extension was done for 35 cycles at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s; and the final chain extension was done at 72 °C for 7 min. The PCR amplicons were resolved on a 3% agarose gel electrophoresis and CYP2C9 c.430C>T homozygous and heterozygous alleles appeared as follows: (i) Homozygous wild-type allele (CC) was detected with the band sizes of 243 and 367 bp. (ii) Homozygous mutant allele was detected with the band sizes of 156 and 367 bp. (iii) Heterozygous allele was detected with the band sizes of 156, 243 and 367 bp.

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2.2 Genotyping of Sodium Channel Alpha 1 Subunit Isoform Variant SCN1A c.3184 A>G Using Restriction Fragment Length Polymorphism The SCN1A c.3184 A>G mutation was genotyped in cases and controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique as previously described by El Fotoh et al. (2016). In a PCR setup containing a total of 25 μl reaction mixture, the genetic region carrying the c.3184 A>G polymorphism was selectively amplified using the following forward and reverse primers: Forward primer 5′-TGCACAAAGGAGTAGCTTATG-3′ Reverse primer 5′-AGTCAAGATCTTTCCCAATTTCAG-3′

The PCR reaction mixture contained 12.5  μl OneTaq Quick-Load 2X Master Mix with Standard Buffer, 1 μl forward primer, 1 μl reverse primer 0.5 μl nuclease-­ free water and 10 μl template DNA. Pre-heating of the lid was done at 105 °C, followed by initial denaturation at 95 °C for 5 min; primer annealing and extension was done for 40 cycles at 94 °C for 30 s, 57 °C for 1 min and 72 °C for 60 s; and the final chain extension was done at 72 °C for 7 min. Using PvuII restriction enzyme, the PCR amplicons were digested for 18 h at 37 °C followed by enzyme inactivation for 15 min at 65 °C. The digested PCR amplicons were resolved on a 3% agarose gel electrophoresis and SCN1A homozygous and heterozygous alleles appeared as follows: “A” allele had 168 bp, “G” allele had 145 and 23 bp while “AG” allele had 168, 145 and 23 bp, respectively.

2.3 Statistical Analysis GraphPad Prism version 8.0.0 for Windows, (GraphPad Software, San Diego, California, USA) was used to analyse participants’ data. All descriptive variables were presented as mean ± standard deviation (SD), while all categorical data were presented as percentages. Two groups were compared using a t-test for parametric datasets. Bio-Rad Image Lab software for Windows (Version 6.1) was used to analyse gel pictures for polymorphism studies.

3 Results This study evaluated the genotypes and allele frequencies of CYP2C9*2 polymorphism and SCN1A A>G polymorphisms in a group of 22 children affected with epilepsy syndromes, and 23 age- and sex-matched controls. Amongst these were 13

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drug-resistant and 9 drug-responsive cases. Demographic and clinical data of the 22 cases sampled reveal a prevalence of male patients (63.6%) over female patients (36.4%) (Table  1). The mean age of the patients at clinical presentation was 26 months, while the median age was 15 months, with the youngest person being 1 month old and the oldest patient being 106 months old. Seizure semiology revealed that most of the patients experienced generalised seizures (86.4%) over focal seizures (13.6%). Clinical diagnosis of patients varied between Landau-Kleffner syndrome (LKS) (9.1%), Lennox-Gastaut syndrome (LGS) (13.6%), infantile spasms (IS) (72.7%) and unknown diagnosis (4.5%). Patients with developmental delay (63.6%) were also higher than those without developmental delay (27.3%). Table 1  Demographic and clinical data of cases and controls Characteristics Sex Male Female Age at clinical presentation (months) Mean ± SD Min Max Range Seizure type Generalised Focal Diagnosis LKS LGS IS Unknown Developmental milestone delay Present Absent NA Milestone regression Present Absent NA Response to ASMs Responsive Resistant/refractory Seizure relapse after initial response to drugs Yes No

n

%

14 8

63.60 36.40

26 ± 5.9 1 106 105 19 3

86.40 13.60

2 3 16 1

9.10 13.60 72.70 4.50

14 6 2

63.60 27.30 9.10

15 5 2

68.20 22.70 9.10

9 13

40.90 59.10

8 4

66.70 33.30

Abbreviations: ASM anti-seizure medications, IS Infantile Spasms,  LGS Lennox-Gastaut syndrome, LKS Landau-Kleffner syndrome

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Likewise, the presence of milestone regression after seizure onset was determined. There was a higher incidence of developmental regression (68.2%) in the studied group than those without regression (22.7%).

3.1 Association Studies of CYP2C9(c.430C>T) and SCN1A(c.3184A>G) Genetic Polymorphisms with Drug Response in Cases and Controls Two known drug response-associated SNPs, CYP2C9:c.430C>T and SCN1A:c.3184A>G, were evaluated in cases vs controls and drug-responsive vs drug-resistant patients, using tetra-primer PCR (for CYP2C9) and PCR-RFLP (for SCN1A). As shown in Table 2, for SCN1A:c.3184A>G, there was a prevalence of wild-type (AA) reference genotype in both cases (63.64%) and controls (73.91%), while homozygous mutant genotype (GG) was not observed in either cases or controls. The wild-type allele (A) predominated over the mutant allele (G) in the cases (81.82%) and controls (86.96%). Association studies show that there was no statistically significant difference (P > 0.05) between the wild-type (AA) genotype and heterozygous AG genotype (P  =  0.467; odds ratio  =  0.6176; confidence interval = 0.1903–2.166) amongst cases and controls. Our studies on wild-type A and mutant G allele reveal a lack of association between the allelic distributions of wild-­ type A and mutant-type G alleles in cases and controls (P  =  0.5014; odds ratio = 0.675; confidence interval = 0.2026–2.021) (Table 2).

Table 2  Association studies of CYP2C9:c.430C>T and SCN1A (c.3184A>G) genotypes and alleles in cases and controls Mutations Cases SCN1A (c.3184A>G) AA 14 (63.64%) AG 8 (36.36%) GG 0 (0%) CYP2C9 (c.430C>T) CC 5 (22.73%) CT 11 (50%) TT 6 (27.27%) SCN1A (c.3184A>G) A 36 (81.82%) G 8 (18.18%) CYP2C9 (c.430C>T) C 21 (47.73%) T

Controls

Test

P-values

17 (73.91%) Reference genotype 6 (26.09%) X2 = 0.5541 P = 0.467 0 (0%) – –

Odds ratio (CI)

0.6176 (0.1903–2.166) –

4 (17.39%) Reference genotype 17 (73.91%) X2 = 0.7346 P = 0.3914 1.932 (0.4075–7.298) 2 (8.7%) X2 = 0.7012 P = 0.4024 0.4167 (0.0626–3.350) 40 (86.96%) Reference allele 6 (13.04%) X2 = 0.452 P = 0.5014 OR = 0.675 (0.2026–2.021)

25 Reference allele (54.35%) 23 (52.27%) 21 (45.65%) X2 = 0.3945 P = 0.530

OR = 0.767 (0.3387–1.706)

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In the case of CYP2C9:c.430C>T (Table 2), there was a prevalence of heterozygous genotype (CT) in both cases (50%) and controls (73.91%). Allelic distribution studies show a prevalence of mutant allele (T) (52.27%) in cases, while the wild-­ type (C) allele predominated in controls (54.35%). Association studies show that there was no statistically significant difference (P > 0.05) between the carriers of wild-type (CC) reference genotype and the heterozygous (CT) genotype (P = 0.3914; odds ratio 1.932; confidence interval 0.4075–7.298) in cases and controls. Also, the distribution of CC and homozygous mutant TT genotypes in cases and controls was not statistically different (P  =  0.4024; odds ratio  =  0.4167; confidence interval = 0.0626–3.350). Our studies on wild-type C and T allele also revealed a lack of association between the allelic distributions of wild-type C and mutant-type T alleles in cases and controls (P  =  0.530; odds ratio  =  0.767; confidence interval = 0.3387–1.706). Table 3 presents the association studies of SCN1A (c.3184A>G) and CYP2C9 (c.430C>T) polymorphisms with drug response in drug-responsive and drug-­ resistant patients. There was a prevalence of wild-type (AA) reference genotype in both drug-responsive (66.7%) and drug-resistant (61.5%) patients over other genotypes, while homozygous mutant genotype (GG) was not observed in either group. Likewise, the wild-type allele (A) predominated over the mutant allele (G) in the drug-responsive group (83.3%) and drug-resistant group (80.77%). Association studies show that there was no statistically significant difference (P > 0.05) between the wild-type genotype and the heterozygous AG genotype (P  =  0.8058; odds Table 3  Association studies of CYP2C9:c.430C>T and SCN1A (c.3184A>G) genotypes and alleles in drug-responsive and drug-resistant patients Drug-­ Mutations responsive SCN1A (c.3184A>G) AA 6 (66.7%) AG 3 (33.3%) GG 0 (0%) CYP2C9 (c.430C>T) CC 1 (11.1%) CT 5 (55.6%) TT 3 (33.3%) SCN1A (c.3184A>G) A 15 (83.30%) G 3 (16.70%) CYP2C9 (c.430C>T) C 7 (38.9%) T

11 (61.1%)

Drug-­ resistant 8 (61.5%) 5 (38.5%) 0 (0%)

4 (30.8%) 6 (46.2%) 3 (23%)

Test

P-value

Odds ratio (C.I)

Reference genotype X2 = 0.0604 P = 0.8058 OR = 1.25 (0.2515–6.165) Reference genotype X2 = 0.9503 P = 0.3296 0.30 (0.02–3.8) X2 = 1.061 P = 0.3031 0.25 (0.01–2.803)

21 (80.77%) Reference allele 5 (19.33%) X2 = 0.047 P = 0.8284 OR = 1.190 (0.2411–5.024) 14 (53.85%) Reference allele 12 (46.15%) X2 = 0.9538

P = 0.3288 OR = 0.5455 (0.1543–1.738)

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ratio = 1.25; confidence interval = 0.2515–6.165). Our studies on wild-type A and G allele also reveal a lack of association between the allelic distributions of wild-­ type A and mutant-type G alleles in (P = 0.8284; odds ratio = 1.190; confidence interval = 0.2411–5.024) (Table 3). In the case of CYP2C9 (c.430C>T) (Table 3), there was a prevalence of heterozygous genotype (CT) in both drug-responsive (55.6%) and drug-resistant (46.2%) cases. Allelic distribution studies show a prevalence of mutant allele (T) (61.1%) in drug-responsive group, while the wild-type (C) allele predominated in drug-­resistant group (53.85%). Association studies show that there was no statistically significant difference (P > 0.05) between the carriers of wild-type (CC) reference genotype and the heterozygous (CT) genotype (P = 0.3296; odds ratio = 0.30; confidence interval = 0.02–3.8) in drug-responsive and drug-resistant groups. Also, the distribution of CC and TT genotypes in drug-responsive and drug-resistant cases was not statistically different (P = 0.3031; odds ratio = 0.25; confidence interval = 0.01–2.803). Our studies on wild-type C and T allele also reveal a lack of association between the allelic distributions of wild-type C and mutant-type T alleles in drug-responsive and drug-resistant patients (P  =  0.3288; odds ratio  =  0.5455; confidence interval = 0.1543–1.738).

4 Discussion Many populations of the world have continued to use genetic and biomarker investigations to detect epileptic patients with higher predisposition to develop chemoresistance due to genetic polymorphisms (Coe et  al., 2012). In a report by Grover et al. (2010), at least 20% of patients’ variability in therapeutic response was attributable to genetics. Our first results show the clinical profiling of patients recruited into this study. We observed a prevalence of generalised seizures over seizures of focal onset. Consistent with our finding was the report of 94 children, ages 3 months to 2 years having epileptic encephalopathies, where Kulsoom et al. (2018) reported the prevalence of generalised tonic-clonic seizures accompanied with myoclonic jerks. Generalised seizures are the most prevalent kinds of seizures observed in childhood epilepsy (Table 1). We also noticed a prevalence of patients with infantile spasms over other sub-phenotypes of epilepsy syndromes (Table  1). This finding was consistent with reports of Yuskaitis et al. (2018). Amongst the epileptic spasms occurring in infancy, infantile spasms account for an incidence rate of 2–4 out of 10,000 live births, making them one of the commonest encephalopathies in this age category (Yuskaitis et al., 2018). Children affected with IS often fail to attain the optimal developmental milestone at the appropriate time, and in many instances, the attained milestones may regress with poor management of seizures. In this study, regression of development was a statistically significant finding. This characteristic has been repeatedly reported in many children populations amongst patients with epilepsy (Gombolay, 2022; Walker, 2022). This is due to the fact that early-onset seizures may gradually distort neuronal morphogenesis, hereby hampering

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developmental activities related to speech, co-ordination or motor functions. We also observed a higher frequency of drug resistance than responsiveness amongst our study cohort (Table 1). This finding corresponds with literature, as poor response to drugs is a major feature in childhood epilepsy syndromes (Specchio et al., 2022). Genetic studies have continued to show the relationship between response to ASM and genetic polymorphisms in drug-metabolising genes and ion channel genes. One notable class of enzymes metabolising ASMs is cytochrome P450s (CYP450s). They metabolise different classes of ASMs ranging from phenytoin to carbamazepine, to levetiracetam, topiramate and valproic acid (Lopez-Garcia et al., 2014). Reduction in efficacy of the recommended doses of ASMs or worsened drug interactions has been observed due to polymorphisms in CYP450 genes (Thorn et  al., 2012). For instance, the negative impact of some classes of drugs such as antidepressants, antibiotics and anticancer drugs which are known to inhibit the action of CYP2C9 is aggravated in the presence of mutant forms of this gene, specifically CYP2C9*2 and *3 (Silvado et al., 2018; Thorn et al., 2012; Zhou et al., 2010). Due to the essential role of CYP2C9 in metabolism of ASMs, understanding of the impact of polymorphisms on this efficiency of this gene becomes a crucial task, as this may help in administering well-suited medications to patients. On a worldwide scale, CYP2C9c.430C>T polymorphism has had higher predominance in persons of European descent (Sánchez-Diz et al., 2009) and absent or extremely low in Asians and Africans (Hamdy et  al., 2002; Soga et  al., 2004). While the CYP2C9c.430C>T polymorphism has been shown to impede the metabolic capacity of the enzyme (Maqbool et al., 2022) and modulate its effective therapeutic dose, the SCN1A c.3184A>G polymorphism had been reported to reduce channel-gating properties of the Nav1.1 protein and consequently, the effectiveness of sodium channel blockers (Wang et al., 2014). Our careful selection of these two polymorphisms was due to their roles in epilepsy pharmacoresistance from literature. In this study, we investigated the genotypes of CYP2C9 c.430C>T alleles in drug-responsive vs drug-resistant patients, and in cases vs controls. Our study observed no association between epilepsy risk or drug resistance and the CYP2C9 c.430C>T genotypes or alleles in both cases and controls and in drug-resistant and drug-responsive groups, respectively (Tables 2 and 3). These findings are in keeping with the reports of Seven et  al. (2014) who genotyped 132 cases and 50 control groups in Turkey and realised no statistically significant difference between cases and controls. Maqbool et al. (2022) studied CYP2C9 genetic polymorphism associated with response to antiepileptic medications in Pakistani population but realised the absence of CYP2C9 (c.430C>T) variation. Using Sanger sequencing, the authors rather detected a novel, likely pathogenic mutation in CYP2C9 (c.374G>A) in the patients who showed refractoriness to drugs. Furthermore, our report is consistent with the findings of Lakhan et  al. (2011) who reported the genetic studies on CYP2C9 polymorphism in Indian epileptic population (a total of 402 patients) with drug-resistant epilepsy and found no significant association between CYP2C9*2 mutant genotypes with multiple drug-resistant epilepsy. However, our study was not in tandem with the reports of Eltalal et  al. (2021) who reported a prevalence of mutant genotypes in drug-resistant than in drug-responsive patients. Another study

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by Chaudhary et al. (2016) interestingly revealed an association of wild-type alleles (C) and genotype (CC) with epilepsy in an Indian population. Such unexpected findings in genotyping studies emphasise the presence of genotype variability in epilepsy pathogenesis in various populations. Our observation in this study may be due to the interplay of other classes of CYP450 genes, which are known to metabolise ASMs, amongst which are CYP2C19 and CYP3A4, or other CYP2C9 alleles. Generally, these various reports per population in epilepsy polymorphism studies are often due to the phenomenon of “inter-individual variability” in genomics studies. Regarding the studies of drug resistance in epilepsy, there is a gradual expansion of focus from the CYP450s to ion channels which serve as main target sites for many ASMs such as phenytoin and sodium valproate (Löscher et al., 2009). One of these is the SCN1A gene which encodes the Nav1.1 protein channel. This protein plays a crucial role in the depolarisation phase of action potentials. This necessitated our investigation of the SCN1A c.3184A>G polymorphism in the cases vs controls and drug-responsive vs drug-resistant cases. Our study did not find a significant difference in the genotypes and alleles of cases vs controls and drug-­ resistant vs drug-responsive patients (Tables 2 and 3). In a report by El Fotoh et al. (2016), patients who were heterozygous for the sodium channel alpha 1 gene (SCN1A c.3184A>G) polymorphism were less likely to respond to ASMs compared to those who carried the wild-type variant. These sodium ion channel genes are critical for eliciting action potential in neurons of the CNS (Ademuwagun et al., 2021). Thousands of pathogenic variants have been identified in epileptic patients having SCN1A polymorphisms, and many functional studies relate their molecular pathogenesis to dysregulation in action potential generation in both excitatory and inhibitory neurons (Makinson et al., 2016; Veeramah et al., 2012; Xie et al., 2020). It is well known that structure-modulating mutations may impact functions of sodium ion channels, preventing proper drug-receptor interactions which are required for sodium channel blockers to fully elicit their roles. Our reports are not in line with the findings of El Fotoh et al. (2016). El Fotoh et al. (2016) and Lakhan et  al. (2009) reported an association between heterozygous genotype of SCN1A c.3184A>G polymorphism and a higher epilepsy risk in an Indian population. Similar to our findings, Lakhan et al. (2009) observed no association between the G allele of the SCN1A c.3184A>G polymorphism and epilepsy risk or drug resistance. On the other hand, El Fotoh et al. (2016) discovered an association between the G allele and epilepsy risk and epilepsy pharmacoresistance. A contradictory finding was recorded in a group of epileptic children from Slovenia, where Bertok et al. (2017) reported that the SCN1A c.3184A>G polymorphism was rather associated with reduced epilepsy risk and faster seizure remission in affected infants. Comparing these varied reports with our findings exposes the phenomenon of inter-­ individual genetic variability, a critical feature in population genetics, which necessitates the practice of individualised genomic and personalised medicine (Sardo et al., 2022). In the genetic studies of ion channels and their roles in epilepsy, researchers are beginning to reveal the presence of disease modifier genes and variations in

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different populations which may cause some polymorphisms to be linked with pathogenesis in some persons, while the same mutations in other gene environments may not be associated with disorders (Ademuwagun et al., 2021). Also, the identification of the phenomenon of inter-population variation in risk alleles of drugmetabolising genes and ion channel genes is gradually expanding the focus epilepsy pharmacogenomics in different regions of the world. Aside from the inconsistencies in reports from literature regarding genetic polymorphisms of epilepsy risk and pharmacogenomics, one limitation with our study may have risen due to the relatively lower sample size than other reported studies. There is a possibility for larger studies with larger samples to detect a link between the investigated polymorphisms with epilepsy risk.

4.1 Conclusion In summary, our study did not identify a significant association between CYP2C9:c.430C>T or SCN1A:c.3184A>G polymorphisms and epilepsy risk or pharmacoresistance within the studied group. In population genetics, inter-­ population genetic variation accounts for the varied reports in single nucleotide polymorphism studies amongst various populations. Hence, an understanding of the important role of genetic polymorphisms in drug response and metabolism is crucial in making better treatment decisions and proper disease management.

4.2 Recommendations for Future Studies We recommend future investigations on genetic polymorphisms within the context of one specific drug administration amongst children with epilepsy syndromes in Nigeria so as to profoundly delineate how genetic variations may influence the metabolism and efficacy of the drug, rather than using cohorts administered multiple ASMs. Furthermore, it is imperative to investigate larger cohort sizes in Nigeria, so as to improve our understanding of the roles of many genetic polymorphisms that may affect patient response to ASMs and their risk to epilepsy.

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Computational Approaches Toward Prevention and Surveillance of Lassa Fever in Developing Countries Gift Nzubechi Elughi, Margaret Ikhiwili Oniha, Bowofoluwa Sharon Abimbola, Kesioluwa Eunice Orukotan, Eze Frank Ahuekwe, and Paul Akinduti

1 Introduction Among West African nations, Lassa fever is a notable ailment which has considerably played a significant part in the region’s death and morbidity rate, particularly in Nigeria (Azeez-Akande, 2016). The Lassa mammarenavirus, which causes Lassa fever, is transmitted by the multi-mammate rat, Mastomys natalensis, which is the primary rodent vector (Yaro et al., 2021). Two missionary nurses who died in Nigeria in 1969 were the first to identify the virus as Lassa fever (Uwishema et al., 2021; Greenky et al., 2018). Many infectious diseases have epidemiological effects, like Lassa fever (Asogun et al., 2019). Understanding the possible factors of the propagation of the virus can help reduce the disease burden. A well-managed Lassa fever epidemic can aid resources management that would otherwise be useful for economic advantage toward the control and prevention of the LF spread (Kofman et  al., 2019;  Chauhan et  al., 2020). Lassa fever is considered to be a zoonotic illness, which spreads through the consumption of food or the usage of household objects that have been contaminated with the feces or urine of infected rats (Murphy and Ly 2021). High-level transmission of Lassa fever is by either a primary or a secondary contact with infected patients or materials (Tambo et al., 2018). Primary transmission is evident when the virus is spread from the rodent G. N. Elughi · B. S. Abimbola · K. E. Orukotan Department of Biological Sciences, Covenant University, Ota, Nigeria M. I. Oniha · E. F. Ahuekwe Department of Biological Sciences, Covenant University, Ota, Nigeria Biotechnology Research Cluster, Covenant University, Ota, Nigeria P. Akinduti (*) Department of Biological Sciences, Covenant University, Ota, Ogun State, Nigeria e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_10

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carrier to a man via consumption of poorly cooked or prepared rodent meat and urine, blood, and other bodily fluid contact (Bonwitt et  al., 2016). Secondary transmission is inevitable in poor resource setting having direct contact of the infected individual with uninfected populace via blood, body fluid, or sexual contact (Tambo et al., 2018). Nosocomial transmission is commonly observed through the transfer from the healthcare personnel to patients. This is another secondary transmission route with high implication on public health (Saka et  al., 2017; Tambo et al., 2018). Due to poor infection control and preventive practices, transmission of LF has led to a considerable morbidity and death from healthcareassociated infections (Saka et  al., 2017). The mortality and morbidity rates of Lassa fever are higher when the disease is transmitted unchecked (Buba et  al., 2018; Onah et al., 2020). The ability to quickly diagnose Lassa fever cases has been hampered by nonspecific clinical presentation and insufficient data on the virus infection rates, propagation, and location, in the West African region, even though the region has witnessed periods of outbreaks and fluctuating transmission (Hallam et al., 2018). A comprehensive study between 1969 and 2017 reported 21 outbreaks of Lassa fever in Nigeria linked to an estimated 6000 infected patients, 800 confirmed cases, and more than 700 deaths (Okwor et al., 2018). About 80% of individuals infected with Lassa virus (LASV) are asymptomatic or only have a minor condition, whereas the other 20% have severe multisystem dysfunction. More than 15–20% of patients admitted to the hospital because of Lassa fever die, resulting in fear and catastrophic socioeconomic consequences. In addition, the death rate of pregnant mothers and their unborn children is rising (Goh and Singh 2022;  Olayiwola & Bakarey, 2017). Approximately one-third of survivors may develop moderate to severe loss of hearing that may be irreversible. Contrary to Ebola virus disease, there is currently no developed vaccine (Lukashevich et al., 2019). If an early diagnosis is established, rehydration can increase the survival of the patients (Duvignaud et al., 2020). Presently, no antiviral drug has been licensed by the FDA or the European Medicines Agency for the treatment of LF, but ribavirin has recorded successful clinical management and is now the common therapy for Lassa fever despite its side effects and absence of selectivity in clinical trials (Eberhardt et al., 2019).

2 Epidemiological Implication of Lassa Fever to Public Health Lassa fever infectious epidemiology may take into account demographic data such as age, religion, gender, ethnicity, and marital status. The spread of Lassa fever in most communities are driven by human-rodent contact or vice versa. A key factor in rodent infestation and spread of Lassa fever in developing countries is poor housing condition. Lassa fever has been documented to affect people of all ages, including children, adults, and the elderly (Ireye et  al., 2021). Other considerable

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socioeconomic variables involved in the transmission of Lassa fever in rural areas include education and income as well as access to healthcare. Viral isolation in cell culture is the conventional method of isolating Lassa virus but it is not practical in rural areas because of the requirement for bio-safety level 4 biocontainment (Happi et al., 2019). Therefore, this makes diagnosis of LASV difficult in developing countries. To date, rodent management and behavioral modifications in humans have been the means of controlling Lassa fever in highly endemic areas (Bonwitt et al., 2016). These are poor methods for the prevention of Lassa fever as they are not effective.

3 Conventional Approaches to Prevention of Lassa Fever Preventing transmission of the Lassa virus from Mastomys to human is possible by avoiding rodent interaction, especially in areas where outbreaks occur. (i)  Use of Traps and Poisons The use of rat poison and routine traps has been in existence for years as a method of eradicating and controlling rodent in several households. Though the method is cheap and easily available, it portrays a lot of danger to human health as the food can be contaminated via the poisoned rats which could find their way back to the food store. Also, if rodents are killed, some will still definitely find their way back to human dwelling to look for food due to the fact that rodents are familiar with human foods (Abdullahi et al., 2020). This makes this method not holistic for prevention of LF. (ii)  Continuous Health Education, Awareness, and Sensitization The awareness campaign and sensitization strategies are meant to educate the public on the dangers of LF, including its spread, symptoms, and treatment options. Awareness and education of people about the dangers of Lassa fever, through several media outlets that include radio, social media, and television, has been reported (Wogu, 2018). However, in some developing countries especially in remote areas, these media outlets are not available to everyone, making it not a suitable method for developing countries. (iii)  Regular Closure of Doors, Windows, and Breeding Sites There must be a high level of local commitment to successful rodent-control techniques in rural areas (Mari Saez et al., 2018). However, the ability of individuals to construct rodent-proof homes is one of the main obstacles to rodent-control strategies against LF in developing countries, making this method unsatisfactory. (iv)  Proper Hygiene Practices Dirty and untidy environments including open solid waste dump area, compounds that are bushy, and disorderly household objects are ideal for these rodents. They are drawn to garbage dumps as they are steady source of food, which aids in the growth and reproduction of their offspring (Aigbiremolen et al., 2017). It is impossible to

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completely eradicate this rodent reservoir because of the widespread distribution of Mastomys throughout Africa (Gobir et al., 2019). (v)  Improve Hospital Infection Control Healthcare practitioners have a higher chance of contracting Lassa fever than those in any other profession (Ilesanmi et  al., 2021). Exposure to cases that are either confirmed or suspected puts them at much higher risk of contracting LF (Ilori et al., 2019). Healthcare workers should have regular training on the use of personal protective equipment (PPE) and infection control practices (ICP) while treating patients to avoid further spread of Lassa fever (Jin et al., 2020).

4 Modern Approaches to Prevention and Surveillance of Lassa Fever Bioinformatics has proven to be very helpful in the rapid combat against emerging and re-emerging diseases (Maljkovic Berry et al., 2020), of which Lassa fever is not an exemption. Due to insufficient and inadequate prevention methods, modern in silico approaches help to detect, characterize, and identify rapidly the particular variant responsible for an outbreak. It also helps to evaluate the risk. The use of computer technology to collect, analyze, store, and disseminate biological data and information and its application is highly essential for the prevention and preparedness for the future outbreaks (López-López et al., 2020; Pereira et al., 2020). (i)  Nucleic Acid Detection Infectious disease detection and surveillance is becoming increasingly dependent on bioinformatics methodologies and tools (Maljkovic Berry et al., 2020). Many diseases are characterized, identified, and typed using bioinformatics techniques. LASV can be prevented when early detection and diagnosis is performed with the use of real-time PCR. This has the capacity to track the activity of the PCR while it takes place (i.e., in real time). As a result, information is gathered during PCR instead of after the completion of PCR. This will prevent further spread from infected subject to others.

4.1 In Silico Approaches for Prevention (i)  Variant Genome Sequencing Genomic sequencing has revolutionized viral mutational changes and characterization with understanding of the genetic code presented by virus variants. Comparative outcome of sequences from different variants aids in tracking the spread, mutation, and potential impact on public health (Grubaugh et al., 2019). As the virus replicates and spreads through a population, it evolves and changes. Whole genome sequencing of Lassa virus is necessary to get relevant data about the variants of interests and concern (Kafetzopoulou et al., 2019). Sequencing is usually done using next-generation sequencing (NGS) platforms that include Illumina and Oxford Nanopore. There are

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many advantages of using NGS, including its high throughput and scalability, as well as its quick turnaround times (Brinkmann et al., 2019). (ii)  In Silico Drug Detection and Development Many computational methods have drastically reduced the expense and time of developing new drugs because of the rapid growth of computer software, algorithms, and hardware. Molecular docking can be applied to predict different binding mechanisms of ligand in the space of target molecule which can be used to develop more efficient, precise, and effective drug candidates for Lassa fever. Using a large amount of LASV genomic data, bioinformatics can help to identify the most important genes, which can then be used to identify potential target proteins for drug screening and design (Lin et al., 2020). Prevention of Lassa fever is possible with the aid of computational drug development. In order to examine the interaction between two molecules, molecular docking is commonly employed in the drug screening and design fields. Molecular docking has been used to access the effectiveness of antimalarial, anticancer, and antidiabetic drugs. Ligand-protein complexes are predicted and ligand binding affinities are estimated by fitting ligands into the binding sites of potential LASV protein using computer simulations (Jakhar et al., 2020). An important part of the hypothesis of “inducing fit” is that ligands and receptors recognize each other based on spatial form matching and energy matching (Jamal et al., 2020). For Lassa fever drug design and understanding how it works, it is essential to determine the optimal binding conformation of small-molecule ligands and protein receptors in complex structures (Torres et al., 2019). (iii)  In Silico Approach to Vaccine Development Vaccinations are the most effective means of preventing diseases. Production of vaccines using genomic data with the help of a computer without the need to culture microorganisms is referred to as reverse vaccinology (Michalik et al., 2022). It was first used to develop a vaccine against Neisseria meningitidis (group B). Reverse vaccinology analyzes the complete pathogen protein library to choose the best candidate antigens for a vaccine. In this way, vaccines previously impossible or difficult to manufacture can be developed and new antigens can be discovered toward improving current vaccine options (Ong et  al., 2021). The entire genome of the Lassa virus is sequenced to detect precise antigens, then the genes are expressed to produce protein, and each identified potential protein is screened for immune response (Heinrich et al., 2020). Antigens that can elicit an immune reaction and also the specific epitopes that are recognized by the immune system can be predicted using these computational approaches (Fig. 1).

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Sequencing of virus genome

Screening for vaccine potency in animal models e.g Monkey, Chimpanzee (in-vivo) and ELISA (in-vitro)

Prediction of potential antigens

Vaccine protein synthesis

Recombinant protein expression

In-silico, in-vitro and in-vivo screening for immune response

Vaccine development, clinical trial and Deployment

Fig. 1  A flowchart for the development of vaccine pipeline using reverse vaccinology

4.2 In Silico Approaches for Surveillance 4.2.1 Statistical Modeling Recently, the majority of the approaches that are used to undertake an analysis of a disease risk, outbreaks, and predictions involved the use of data mining technology which is now integrated into surveillance and prediction of outbreak. Predictive modeling and simulations in epidemiology aid in understanding Lassa fever spread, as well as prevention or control (Akhmetzhanov et al., 2019). (i)  Random Forest Modeling Based on decision trees, a random forest is utilized for modeling behavior and prediction analysis (Cheng et al., 2019). There are numerous decision trees in the random forest, each one reflecting a different classification of data. The Lassa fever prediction that receives the most votes is used as the basis for the random forest method, which examines each instance separately (Zhao et al., 2018). The random forest method is able to manage massive quantities of data because of its capacity to cope with a huge number of variables (Fig. 2). (ii)  Bayesian Model Bayesian models are statistical models in which all uncertainty is represented by probabilities, both in terms of the output and the input of the model (parameters) (Helleckes et al., 2022).

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Fig. 2  Structure of random forest

Approximate Bayesian computation was used to estimate epidemiological and demographic trends of LASV and its natural habitat which is the rodent M. natalensis, using data over the period on capture of rodents. Key characteristics, including the density-dependent mortality, frequency of horizontal transmission, and the seasonal patterns of precipitation that affect birth rates, can be accurately predicted to track Lassa fever spread using simulated sets of data (Nuismer et al., 2020). 4.2.2 Use and Application of Geographic Information System (GIS) in LF Surveillance GIS is a branch of computer science that makes use of geographic data and tabular information to visualize, analyze, and evaluate real-world issues. Its spatial indices have helped to store data that can be used to investigate and track the spread of LF through mapping (Ali, 2020). GIS has the potential to be a powerful weapon in the fight against Lassa fever and emerging diseases as well as other global health issues. New tools for the management and prevention of a wide variety of diseases have been made available as a result of the advent and advancement of geographic information systems (Thomas, 2018). All of the places within a defined distance of a point including all the streets that connect through an area can be rapidly identified

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and mapped using GIS. It is an ideal platform for monitoring and surveillance of Lassa fever among the population, hospitals, and different settlements (Bhatt & Joshi, 2012) particularly remote communities. Several tools and systems have been developed to help visualize disease data over time and location (Saran et al., 2020). Geographic information system generates dynamic mapping that can be used to analyze Lassa fever disease rate, disease tracking, disease clusters, and disease connection with network analysis, environmental parameters, and more (Saran et al., 2020). It is possible to use a GIS to better understand how Lassa virus spread and how to reduce the danger of an epidemic. GIS is an excellent tool for epidemiological studies because of its ability to analyze, manipulate, integrate, and present enormous amounts of environmental data in a seamless manner (Dong, 2017). As a result of these advantages, GIS technology holds a significant impact in public health as a tool for surveillance of LF. It is possible that the dynamic transmission of Lassa fever could be better represented and analyzed using geospatial technology and services when it occurs. (i)  Hotspot and Coldspot Analysis Risk of Lassa fever can be calculated by converting the risk levels found by several geosurveillance systems or surveys. At assessing the risks, a condition is defined that indicates an irregular accumulation in geographic data, and a predicted value linked to that condition is provided. Hotspot refers to an area wherein elevated levels of disease prevalence and incidence are concentrated, while coldspot refers to an area where low levels are concentrated (Carter et al., 2019). Now that GIS technology has progressed, it is common to employ the hotspot detection approach in LASV surveillance (Pribadi et al., 2021). (ii) Visualization Visualization can be used to highlight how disease trends have changed through time. Using GIS-enabled graphics, it is possible to show how Lassa fever spreads and retreats through time and space. Through mapping and visualization, it is possible to investigate the spatial correlations and patterns of Lassa fever with a wide variety of parameters including the environment and census. The results of conventional statistical analysis could be explored in unique and interesting ways through the use of visualization. (iii)  Contact Tracing There is a pressing need for a digital platform for recording real-time accurate data about Lassa fever patients, the diseases, diagnosis, medical records, and treatment as well as people they come in contact with in order to discover clusters and trigger notifications. This data may be useful in identifying Lassa fever risk factors and for making predictions about the next group of people to be infected with LF (Ejembi et al., 2019). (iv) Clustering Clustering involves the analysis of spatial-temporal patterns of the transmission of infectious illnesses like Lassa fever and detection of other disease-related factors connected with diverse spread which can be beneficial in clarifying the diseases’

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transmission process (Saran et al., 2020). This analysis on space-time patterns is a sort of surveillance system for Lassa fever which comprises observation of localization, recognizing the outbreak clusters of active cases, isolation of the infectious agent, and relative hazard evaluation of affected sites at an early stage. Clustering aids in the prompt implementation of Lassa fever prevention and management strategies, as well as the efficient distribution of available resources (Saran et al., 2020; Mitjà et al., 2020). (v)  Infectious Disease Surveillance Interventions can be adopted early to avoid further spread of Lassa fever outbreak which is the aim of infectious disease surveillance. Several epidemiology and statistical methodologies with geographical elements are required for these types of monitoring activities when looking into Lassa fever, ideally from small geographic areas (Kirby et al., 2017). Due to the fact that epidemics like Lassa fever tend to begin in smaller regions and then spread to other parts of the country if it is not managed, there are other strategies that demand rigorous regulation and cannot be applied to small regions (WHO, 2018). (vi)  Google Earth Engine Google Earth Engine can be used by researchers to track surface changes, identify patterns, and measure inconsistencies of Lassa fever. It can also be used for analyzing, monitoring, and displaying alterations in the Earth’s surface. Google Earth Engine offers a time-lapse video tool (Piégay et al., 2020). Data from a range of aerial imaging platforms, satellite, environmental variables, climate and weather forecast topography, and socioeconomic information are all housed in the database engine. This resource is accessible to the public. Google Earth Engine can help in monitoring the spread of Lassa fever.

5 Conclusion Controlling rodents, maintaining good cleanliness in the home, and storing food properly are all very important but not holistic for the prevention and surveillance of Lassa fever. However, computational approach offers a substantial amount of potential for the prevention and surveillance of Lassa fever in developing countries, although its application is really low due to high cost of equipment and materials and lack of expertise. Also, early detection and diagnosis will aid in the prevention of Lassa fever spread but this is still difficult in most developing countries. Furthermore, it is possible to use a variety of geospatial approaches provided by GIS to examine data in order to discover geographical and temporal correlations between events and outbreaks as well as to identify populations at risk or to track the spread of Lassa fever. It is still a matter of concern for public health that there are no approved drugs or vaccines for Lassa fever, despite the fact that there has

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been an increase in the number of people who have tested positive for the disease and died from it. As a result, there is an urgent need to develop drugs that can prevent and treat LF using the in silico approaches.

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Antibacterial Efficacy of Thymus vulgaris Essential Oil Against Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Urinary Tract Infections S. O. Egwuatu, T. M. Obuotor, O. S. Taiwo, W. E. Ike, A. E. Ojo, Patrick Omoregie Isibor, O. F. Adeniji, F. M. Oyeyipo, O. A. Awotoye, and Paul Akinduti

1 Introduction Over the years, antibiotics have been administered toward the treatment of several infections caused by bacterial pathogens. But for many years now, there has been the misuse of these chemical agents which has resulted in resistance to many of them globally (Chiang, 2018; Saipriya et al., 2018). Beta-lactam drugs are the main drug of choice for the treatment of infections caused by Gram-negative bacteria, and close to 65% of the antibiotics have been used all over the world (Patel, 2018). Resistance can be conferred on b-lactams in several ways including generation of efflux pumps, changes in the outer membrane porins produced, and the production of B-lactamase enzymes that inactivates S. O. Egwuatu · T. M. Obuotor · W. E. Ike · A. E. Ojo Department of Microbiology, College of Bioscience Federal University of Agriculture, Abeokuta, Ogun State, Nigeria O. S. Taiwo Department of Microbiology, College of Bioscience Federal University of Agriculture, Abeokuta, Ogun State, Nigeria Department of Biological Sciences, Covenant University, Ota, Nigeria P. O. Isibor · P. Akinduti (*) Department of Biological Sciences, Covenant University, Ota, Ogun State, Nigeria e-mail: [email protected] O. A. Awotoye Department of Biological Sciences, Covenant University, Ota, Nigeria O. F. Adeniji Department of Biological Sciences, Bells University of Technology, Ota, Ogun State, Nigeria F. M. Oyeyipo Department of Microbiology, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_11

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antibiotics (Eiamphungporn et  al., 2018). Extended-spectrum-β-lactamases are enzymes that have the ability to inactivate B-lactam antibiotics such as penicillins and aztreonam, including first, second-, third-, and fourth-generation cephalosporins (Ye, 2018). Primarily, they are encoded by the B-lactamase gene families; the TEM, CTX-M, and SHV. Enterobacteriaceae expresses these genes, majorly the Escherichia coli, and they occupy the universe which differs predominantly from one region to another (Indica et al., 2021). Urinary tract infection (UTI) is an infection that affects the urinary tract. It can be cystitis (bladder infection in which the pathogens colonize the lower urinary tract) or pyelonephritis (kidney infection which develops in the upper urinary tract) (Anna et al., 2021); the second most common form of infection in the human body is that of the urinary tract aside the flu and common cold. It is mostly more in females than the male, about 10–20% of women have UTI at certain point in their life, and majority have the infection over and over again after treatment (Jaysee John et al., 2020). UTI can also be grouped based on the anatomical and functional status of urinary tract (Jaysee John et al., 2020). Recently, natural products and medicinal plants have gained a lot of attention because of their antimicrobial properties. Thymus vulgaris (thyme) is a flowering plant which belongs to the family Lamiaceae and grows to 15–30 cm (6–12 in) tall by 40 cm (16 in). They have been accessed for several biological and therapeutic abilities; some of the Health benefits of Thymus vulgaris (thyme) include the following: diuretic, anthelmintic, antibroncholytic, antitussive, anthelmintic, antispasmodic, carminative, and its expectorant (Luna-Guevara et al., 2021).

2 Materials and Methods Study Setting and Design  This is a study of patients who presented with urinary tract infection at State Hospital, Ijaiye, and Federal Medical Center Idi-aba both in Abeokuta, Ogun State. Abeokuta is the capital and largest city in Ogun State, which is located at 79.39N32054″E. The state is situated Southwest of Ibadan, Oyo State, and North of Lagos, located on an altitude of about 159 m above sea level. It has a hot humid weather with annual rainfall of 963.3. Isolates Collection  Urine isolates were collected from the medical microbiology department of the hospitals and clinics and were transported in nutrient agar medium. Identification of Escherichia coli  Following aseptic technique, isolates were subcultured on eosin methylene blue (EMB) medium (Merck, Germany). The plates were incubated at 37 °C aerobically for 48 h, after which they were checked for bacterial growth. All isolates were then identified by their colony morphology, staining characters, pigment production, motility, and standard biochemical procedures (IMiVC).

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Raw Materials and Chemicals  Dry thyme (Thymus vulgaris) was purchased from Lafenwa market in Abeokuta, Ogun State, Nigeria, and was identified in the Herbarium of the Department of Botany, Obafemi Awolowo University, Ile Ife, Nigeria. Chemicals  Fetal bovine serum (FBS), L-glutamine, Dulbecco’s modified Eagle’s (DMEM) medium, dimethyl sulfoxide (DMSO), doxorubicin, penicillin, streptomycin, and sulforhodamine-B stain (SRB), (3-(4,5-dimethylthiazol-2-yl)-2,5-­ diphenyltetrazolium bromide), and all other chemicals and reagents used in this study were of analytical grade and purchased from Chemistry Department of Federal University of Agriculture, Abeokuta (Ogun State, Nigeria). Isolation of Essential Oils (EOs)  The essential oil of Thymus vulgaris (thyme) was extracted following hydrodistillation method using Clevenger’s apparatus for 3 h (Lamaty et al., 2018). Identification of Essential Oils  Using gas chromatography (GC), analysis approximately 5 μl of each pure volatile oil was applied. A GC analysis was performed using Hewlett-Packard model 5890 with a flame ionization detector (FID) and a fused silica capillary column DB-5 (60 m × 0.32 mm. id). Briefly, the oven temperature was maintained initially at 50 °C for 5 min and then programmed from 50 to 250 °C at a rate of 4 °C·min−1. Helium was used as the carrier gas, at flow rate of 1.1 mL·min−1. The injector and detector temperatures were 220 and 250 °C, respectively. The retention indices (Kovats index) of the separated volatile components were calculated using hydrocarbons (C7–C21, Sigma-Aldrich Co.). Antimicrobial Susceptibility Test Antimicrobial susceptibility of the isolates was performed by the Kirby-Bauer disk diffusion method on the Mueller-Hinton agar media (Merck, Germany). Using commercially available antibiotic disks (Mast, UK), including ofloxacin, streptomycin, Septrin, chloramphenicol, ciprofloxacin, amoxicillin, ampicillin, pefloxacin, ceftazidime, and gentamicin. The diameter of inhibition zone was measured for each antibiotic disk, and the results were defined in accordance with the CLSI guidelines (CLSI, 2017).

2.1 Antibacterial Activity Assay of Thymus vulgaris Essential Oils The agar cup diffusion method of Akinpelu and Onakoya (2006) was employed. An overnight culture of the organisms was standardized to contain approximately 108 cfu/ml, and this was then inoculated into 20 ml of molten nutrient agar. The culture medium was allowed to set. Thereafter, a sterile cork borer (8.0  mm

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diameter) was used to punch wells in the seeded nutrient agar. The agar plugs were removed with a flamed and cooled wire loop. The essential oil (Thymus vulgaris) was poured into the wells in different plates. The plates were thereafter incubated at 37 °C for 24 h, and the zone of inhibition was measured. Also the typed cultures were tested with oil extract to determine susceptibility. The experiment was repeated in triplicates. 2.1.1 Phenotypic Identification of ESBL-Producing Strains Detection of ESBL-producing organisms was performed by double-disk synergy test (DDST) method following CLSI recommendations. In brief, a suspension of 0.5 McFarland turbidity standards was cultured on Mueller-Hinton agar. A pair of antibiotic disks containing ceftazidime (30  μg) with ceftazidime/clavulanic acid (30/10 μg) was placed on Mueller-Hinton agar medium center to center, at a distance of 20 mm apart from each other. The plates were incubated for 24 h at 37 °C, and the diameter of inhibition zone was measured according to CLSI guidelines. An increase of ≥5 mm in the zone diameter around the clavulanic acid combination disks versus the same disks alone confirmed the presence of ESBL. 2.1.2 Ethical Considerations All ethical aspects of this research have been completely observed by the authors. It was approved by Research and Ethics Committee of the State Hospital Ijaiye, Abeokuta, Ogun State. All experiments were performed in accordance hospital. Informed consent was obtained from all participants or their legal guardians before the study, and their information remained confidential. 2.1.3 Determination of Minimum Inhibitory Concentrations (MIC) of the Essential Oils on Bacterial Isolates The method used in this study to determine the inhibitory activity of Thymus vulgaris oil was similar to that described by Akinpelu and Onakoya (2006) with a few modifications. This assay was done to determine the lowest concentration of the oil that will inhibit microbial growth. Twofold dilution of the essential oil (Thymus vulgaris) was prepared based on the concentration that inhibited growth in the agar well susceptibility test. Ten tubes were obtained, and 2  ml of sterile Tween 20 (reconstituting solvent) was dispensed into each tube except the first tube which contained 4 ml of the oil. 2 ml of the oil was removed from the first tube containing 4 ml into the second tube containing 2 ml of Tween 20 and mixed thoroughly; thereafter 2 ml of the mixture was taken and poured into the third tube and mixed properly. 2 ml of that mixture was transferred serially up to the tenth tube, and 2 ml was removed and kept. These test tubes containing 2 ml of different concentrations (100,

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50, 25, 12.5, 6.25, 3.125, 1.5625, 0.7825, 0.390, 0.195  mg/ml v/v), respectively, were later poured into sterile molten nutrient agar and mixed properly. This agar-­ extract mixture was thereafter transferred into petri dishes and allowed to set. The plates were then seeded with the standardized test organisms and incubated at 37 °C for 72 h. Controls were set up as inoculated nutrient agar plates without the extract as negative control and inoculated nutrient agar plates with extract as positive control. 2.1.4 Determination of Minimum Bactericidal Concentrations (MBC) of the Essential Oils on Bacterial Isolates Based on the MIC results obtained, the concentrations of the essential oils that showed no growth were subcultured onto sterile nutrient agar plates and incubated for 48 h for bactericidal activity. The MBC was taken as the least concentration that did not show any growth on the incubated nutrient agar plates. 2.1.5 The Time-Kill Kinetics The killing rate of Thymus vulgaris oil on ESBL-producing Escherichia coli was carried out using the method as described by Odenholt et  al. (2001) with a few modifications. The standardized bacterial suspension (0.5 ml) was serially diluted in (10−1, 10−2, 10−3, 10−4, 10−5, 10−6, 10−7, 10−8, 10−9, and 10−10); these dilutions were then seeded into nutrient agar plates using the pour plate technique. Heterogeneous plate count was then carried out to determine the viable microbial population count in each dilution factor to serve as reference for the rate of kill. Following this, 2 ml of the standardized bacterial suspension was added to 18 ml of the essential oil at a concentration equal to MIC × 1 value and MIC × 2 values. The mixture was thoroughly shaken together, and exactly 0.5 ml of the mixture was transferred immediately into 4.5 ml of already prepared sterile 3% Tween 80 nutrient broth, and the suspension was thoroughly mixed. This served as the portion taken at 0 min as this was done at 15 min interval for 2 h. Exactly 0.5 ml was taken from each suspension and serially diluted up to 10−6 in 4.5 ml sterile normal saline. Then, 0.5 ml of the final dilution factor was transferred into labeled nutrients agar plates. This plate was incubated at 37 °C for 24 h. This time at which the least number of viable counts was obtained was recorded as the time it will take the essential oil to kill the organism. 2.1.6 Molecular Ducking Three-dimensional (3D) structures of the component of the essential oils of Thymus vulgaris was drawn using Chem3D Pro 12.0. The generated structures were saved as pdb format. Target proteins and enzymes from site of action on Escherichia coli

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was identified and obtained from the protein data bank, and “Edu Pymol” software was used to screen and prepare the proteins for docking. The binding sites of the proteins were also identified using this software. The radius of the binding site used for this study was set at 8.0 Armstrong. “Auto vina” version 4 was therefor used to characterize the binding site of the protein, position the ligand (oil) into the binding site (orienting), and evaluate the strength of interaction of the ligand-receptor complexes (“scoring”) using the binding energies obtained.

3 Results Infection demography: Out of the 229 urine isolates collected from the patients, 159 (69.4%) isolates with significant growth for UTI and 70 (30.6%) showed no growth. Among patients from secondary health facility, proportion of male and female patients with UTI had a higher rate of E. coli distribution of 39.0% and 90.0%, respectively, compared to male and female patients attending tertiary health facility as is shown in Table  1. Table  2 showed the result of the antibiotic susceptibility Table 1  Distribution of E. coli among patients attending secondary and tertiary health facilities

Urine sample Significant growth Nonsignificant growth Total

Urine culture 229 159 70 229

Idi-aba Male % 28 (12.6) 18 (11.3) 10 (13.8) 28

Female % 72 (31.4) 52 (33.9) 20 (27.7) 72

State hospital Male % Female % 39 (16.5) 90 (39.3) 19 (11.9) 68 (42.7) 20 (27.7) 22 (30.5) 39 90

Table 2  Antibiotic susceptibility profile in ESBL-producing E. coli ATB OFX S SXT CH CPX AMP A PEF CN CT

N 159 159 159 159 159 159 159 159 159 159

Resistance n1 (%) 78 (49.1) 68 (42.8) 98 (61.6) 26 (16.4) 32 (21.1) 122 (76.7) 135 (84.9) 76 (47.8) 118 (74.2) 147 (92.4)

Sensitivity n2 (%) 81 (50.9) 91 (57.2) 61 (38.4) 133 (83.6) 67 (61.5) 37 (23.3) 24 (15.1) 83 (52.2) 41 (25.8) 12 (7.5)

Key: N total number of isolates, ATB antibiotics, OFX ofloxacin, S streptomycin, SXT Septrin, CH chloramphenicol, CPX ciprofloxacin, AM amoxicillin, A ampicillin, PEF pefloxacin, CN gentamycin, CT ceftazidime, ATB antibiotics

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Table 3  Distribution of beta-lactamase and ESBL-producing E. coli among male and female Characteristic Gender Male Female Age group ≤11 12–21 22–31 32–41 42–51 52–61

Beta-lactamase N %

ESBL N

%

14 35

28.6 71.4

11 19

36.6 63.3

1 3 27 13 2 3

2.0 6.1 55.1 26.5 4.0 6.1

1 1 18 7 1 2

3.3 3.3 60.0 23.3 3.3 6.6

Proportion of isolates positive to ESBL

40 35 30 25 20 15 10 5 0

ESBL-positive

ESBL-Negative

Fig. 1  Screening for ESBL production using double-disk synergy test

profile of ESBL-producing E. coli. The ESBL-E. coli had highest resistance to ceftazidime (92.4%), ampicillin (84.9%), and amoxicillin (76.7%), while high sensitivity to chloramphenicol (83.6%) and ciprofloxacin (61.5%) was observed. However, in Table 3 distribution of beta-lactamase and ESBL-producing E. coli was found to be higher in female patients (71.4%) and 63.3% compared to the male patients 28.6% and 36.6%, respectively. Highest rate of ESBL-E. coli was recorded among the age group 22–31 years (60.0%) and 32–41 years (23.3%). Figure 1 shows the phenotypic confirmation of the ESBL producers with highest rate of 30/49 isolates positive to ESBL and 19/49 were negative. Proportion of E. coli isolates with degree of zone of inhibition to antimicrobial activity of Thymus vulgaris essential oil showed high inhibition zone by two

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Zones of Inhibition AST

25 20 15 10 5 0

a(n=3)

b(n=3)

c(n=2)

d(n=12)

e(n=1)

f(n=2)

g(n=6)

h(n=2)

Isolates ID Fig. 2  Antimicrobial testing of Thymus vulgaris essential oil ESBL-producing E. coli isolates

MIC Zones of Inhibition

0.25 0.2 0.15 0.1 0.05 0

a(n=1)

b(n=9)

c(n=4)

d(n=8)

e(n=3)

f(n=3)

g(n=2)

Isolates ID Fig. 3  Minimum inhibitory concentrations (MIC). Key: MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration

isolates followed by six isolates as shown in Fig. 2, and same level of inhibition at the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) is shown in Figs. 2 and 3. Figure 3 shows the time rate of kill of the essential oil of Thymus vulgaris on ESBL-producing Escherichia coli with observed decreasing rate of bacteria count with time, and Table 4 shows the components of Thymus vulgaris essential oil revealing the gas chromatography-mass spectrometry (GC-MS) analysis and molecular ducking of the ESBL-producing with the components of Thymus vulgaris oil (Figs. 4, 5, 6, and 7).

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Table 4  Components of Thymus vulgaris essential oil Compound Ƿ-Cymenene, terpinolene, African-1-en, Ɓ-bourbonene, Thymohydroquinone, terpinolene, α-humulene, Cis-Ɓ-guaiene, cuparene, eudesmol Ƴ-eudesmol, eudesm-3-en-7-ol Calamenene B, thymol acetate, α-copaene, limonene Ɓ-Piene α-terpineol Borneol acetate Ƴ-Muurolene α-Cadinene Camphor, caryophyllene Ƴ-Cadinene, α-thujene Oct-1-en-3-ol, carvacrol methyl ether, α-cadinene Myrcene Camphene. Terpinen-4-ol. Thymol methyl ether Borneol, α-pinene 1.8-Cineole Carvacrol Ɓ-Caryophyllene Linalool Ƴ-Terpene p-Cymene Thymol

Total oil (%) Retention indices 0.1 1075,1082,1365,1386,1509,1082, 1455,1488,1498,1618,1650,1659

0.2

1517,1329,1356,1025

0.3

978 11796 1270 1474,1520

0.5 0.6 1.0 1.1 1.2 1.3 1.9

1,123,1578 1,507,932 962,1226,1534 987 950 1,215,1,164 1,150,936

2.1 2.3 3.1 3.7 5.2 29.1 38.1

1024 1278 142 1,086 1,051 1,015 1267

Zones of Inhibition of MBC

2.5 2 1.5 1 0.5 0

a(n=1)

b(n=4)

c(n=6)

d(n=2)

e(n=6)

f(n=7)

g(n=1)

h(n=1)

Isolates ID Fig. 4  Minimum bactericidal concentration (MBC) exhibited by Thymus vulgaris essential oil against the ESBL-producing Escherichia coli

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Fig. 5  The time rate of kill of the essential oil of Thymus vulgaris on ESBL-producing Escherichia coli. MIC 1, minimum inhibition concentration 1; MIC 2, minimum inhibition concentration

Fig. 6  Gas chromatography-mass spectroscopy (GC-MS) analysis showing peaks of components of Thymus vulgaris essential oil (TVEO)

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Fig. 7  Molecular docking of ESBL-producing Escherichia coli synthase with the components of Thymus vulgaris oil

4 Discussion A wide range of microbial pathogens cause urinary tract infections which are secondary infection, and it is a global challenge (Behzadi et  al., 2019). Among the Enterobacteriaceae family, E. coli is an important causal agent of urinary tract infection which is facilitated through the use of adhesion, pilli, fimbriae, and P1 blood group genotype receptor, aiding its attachment to the of urothelium (Shrestha et al., 2016). The present study identified high rates of Escherichia coli (159/229), and this rate was similarly reported by Soo et  al. (2021) implicating Escherichia coli in 80–90% of UTI cases and cause of cystitis and pyelonephritis. The recorded antibiotic susceptibility pattern of E. coli revealed high rate of resistance to ceftazidime, Septrin, ampicillin, amoxicillin, and gentamicin, while the least resistance was recorded against chloramphenicol, which could be linked to the misuse of antibiotics. Several studies similarly reported the E. coli high resistance to ampicillin (88.3%), and low susceptibility to amikacin, imipenem, and meropenem (Mohammed et al., 2016; Ahmed et al., 2019; Mohammad et al., 2021). In this study, rates of identified ESBL-producing E. coli found in UTI was lower to other findings, and this could influence by differences in geographical area, time of sample collection, and the diagnostic technique used (Nanoty et al., 2018). The antibacterial activity of the Thymus vulgaris (thyme) essential oil had high inhibitory potency against all the isolates. Reported bioactive components in thyme essential oil have been proven effective in inhibiting and altering the integrity of their cell membranes (Jafri & Ahmad, 2021). Observed bactericidal of MBC/MIC ratio is 4, and bacteriostatic ˃4 further shows the antimicrobial effect of Thymus vulgaris essential oil (Keepers et  al., 2014). This is further confirmed by time-kill kinetic studies showing a continuous decrease in the cell population as the time of exposure to the oil increases at

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different concentrations of the oil used. This is in agreement with the report of Boakye et al. (2016) who reported a gradual decrease in cell population for the first 3 h of exposure of the oil. Nevertheless, the rate of cell population decreased faster in MIC × 2 than in MIC × 1, and this agrees with the report of Forogh (2021) indicating a higher concentration causing significant antibacterial effect. The bactericidal antimicrobial agents have the ability to kill or eliminate the cell at a shortest period of time, while bacteriostatic agents only inhibit the growth or multiplication of microbes giving the immune system of the host time to clear the microbes from the system (Luis et al., 2021). The identified compounds from gas chromatography-mass spectrometry (GC-­ MS) analysis showed high level thymol, p-cymene, and Y-terpene, which serve as the major components of the essential oil. On the contrary, Mancini et al. (2014) earlier reported carvacrol and caryophyllene oxide as major constituent of the essential oil. However, El Hattabi et al. (2016) reported that the major constituent of the essential oil as carvacrol, p-cymene, and E-caryophyllene. The difference in the chemical composition of the essential oil could be due to the geographical origin, genetic factors, plant material, and harvesting season (Dugo et al., 2014). The molecular docking showed the components of the essential oil as potential lead molecules in the inhibition of bacterial growth and thus justifies its use in traditional folklore medicine. Alpha terpinolene, gamma terpinolene, and T-cadinol had the highest negative binding energy revealing its high potential for the inhibition of the extended-spectrum beta-lactamase synthesis in Escherichia coli.

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Antibacterial Efficacy of Syzygium aromaticum Essential Oil Against Extended Spectrum Beta-Lactamase-Producing Escherichia coli in Urinary Tract Infections S. O. Egwuatu, O. S. Taiwo, T. M. Obuotor, M. I. Oniha, O. Oziegbe, S. O. Adebajo, W. E. Ike, F. M. Oyeyipo, A. O. Kuye, and Paul Akinduti

1 Introduction Syzygium aromaticum (clove) antimicrobial and antioxidant properties that has made it to stand out among other spices (Batiha et al., 2020, Shrivastav et al., 2019) that are used to resolve gastrointestinal disorders, inflammation, and toothache (El-Shouny et  al., 2020; Oluwasina et  al., 2019). The result of previous studies showed the antibacterial activity of Syzygium aromaticum essential oil on multidrug-­ resistant bacteria are commonly affirmed through phenotypic screening (Shrivastav et al., 2019; Faujdar et al., 2020). Syzygium aromaticum is one of the spices that have been applied globally for the sake of health as well as the preservation of foods, and this is majorly because of its antithrombotic, antioxidant, antimutagenic, anti-ulcerogenic, antimicrobial and anti-inflammatory properties (Okmen et al., 2018). Extended-spectrum Ɓ-lactamases (ESBLs) are a group of plasmid-mediated, diverse, complex, and evolving enzymes posing a major therapeutic challenge in the S. O. Egwuatu (*) · T. M. Obuotor · S. O. Adebajo · W. E. Ike Department of Microbiology, College of Bioscience Federal University of Agriculture, Abeokuta, Ogun State, Nigeria O. S. Taiwo Department of Microbiology, College of Bioscience Federal University of Agriculture, Abeokuta, Ogun State, Nigeria Department of Biological Science, Covenant University, Ota, Nigeria M. I. Oniha · O. Oziegbe · A. O. Kuye Department of Biological Science, Covenant University, Ota, Nigeria P. Akinduti Department of Biological Science, Covenant University, Ota, Ogun State, Nigeria F. M. Oyeyipo Department of Microbiology, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_12

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treatment of both community-based and hospitalized patients. They have the ability to hydrolyze third-generation cephalosporins and aztreonam but are inhibited by clavulanic acid (Rawat & Nair, 2010). The significance of ESBL production by Enterobacteriaceae family particularly, Escherichia coli and Klebsiella pneumoniae is great as this leads to high rate of antimicrobial resistance (Pana and Zaoutis 2018). Ambler classified B-lactamases based on amino acid homology into class A which includes the TEM-1, 2, SHV-1, ESBLs, and KPC; class B which includes MBLs, NDM, IMP, and VIM; class C which includes ampC and CMY; and class D which includes the OXA (Sawa et al., 2020). E. coli are common bacteria which normally exist innocuously in the gut (intestines). The ESBL-producing strains produce extended-spectrum beta-lactamases (ESBLs), and this makes them resistant to cephalosporin antibiotics, as well as a number of other classes of antibiotics thereby making these infection much challenging to treat (Akinduti et al., 2015). Among the highly rated causes of healthcare-related infections are urinary tract infections (UTIs) of a high economic cost and are associated with high morbidity rate (Smith et al., 2019). This study therefore was conducted to determine the antibiotic efficacy of Syzygium essential aromaticum essential oil against extended spectrum beta-lactamase producing Escherichia coli in urinary tract infections in Abeokuta, Ogun State.

2 Materials and Methods Study setting and design: This is a study of patients who presented with urinary tract infection at State Hospital, Ijaiye, and Federal Medical Center Idi-aba both in Abeokuta, Ogun State. Abeokuta is the capital and largest city in Ogun State, is located at 79.39N 32054″E. The state is situated Southwest of Ibadan, Oyo State, and North of Lagos, located on an altitude of about 159 m above sea level. It has a hot humid weather with annual rainfall of 963.3. Ethical considerations: All ethical aspects of this research have been completely observed by the authors. It was approved by research and ethics committee of the State Hospital Ijaiye, Abeokuta, Ogun State. All experiments were performed in accordance with relevant guidelines and regulations in the hospital. Informed consent was obtained from all participants or their legal guardians before participating in the study, and their information remained confidential. Isolates collection: Bacterial isolates obtained from urine were collected from the Medical Microbiology Department of the hospital and was transported in nutrient agar medium. Identification of Escherichia coli: Following aseptic techniques, isolates were subcultured on eosin methylene blue (EMB) medium (Merck, Germany). The plates were incubated at 37 °C aerobically for 48 hours, after which they were checked for bacterial growth. All isolates were then identified by their colony morphology,

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staining characteristics, pigment production, motility, and standard biochemical procedures (IMiVC). Raw materials and chemicals: Dry clove (Syzygium aromaticum) was purchased from Lafenwa market in Abeokuta, Ogun State, Nigeria, and was identified in the Herbarium of the Department of Botany, Obafemi Awolowo University, Ile Ife, Nigeria. Chemicals: Fetal bovine serum (FBS), L-glutamine, Dulbecco’s modified Eagle’s (DMEM) medium, dimethyl sulfoxide (DMSO), doxorubicin, penicillin, streptomycin, and sulforhodamine-B stain (SRB), (3-(4,5-dimethylthiazol-2-yl)-2,5-­ diphenyltetrazolium bromide, and all other chemicals and reagents used in this study were of analytical grade and purchased from Chemistry Department of the Federal University of Agriculture, Abeokuta (Ogun State, Nigeria). Extraction of essential oils: The essential oil of Syzygium aromaticum (clove) was extracted following hydrodistillation method using Clevenger’s apparatus for 3 hours (Lamaty et al., 1987). Identification of components of essential oils: Using gas chromatography (GC) analysis, approximately 5 μl of each pure volatile oil was applied. A GC analysis was performed using Hewlett Packard model 5890 with a flame ionization detector (FID) and a fused silica capillary column DB-5 (60 m × 0.32 mm. id). The oven temperature was maintained initially briefly at 50 °C for 5 minutes, and then programmed from 50 to 250 °C at a rate of 4 °C·min−1. Helium was used as the carrier gas, at flow rate of 1.1 mL·min−1. The injector and detector temperatures were 220 and 250 °C, respectively. The retention indices (Kovats index) of the separated volatile components were calculated using hydrocarbons (C7–C21, Sigma-Aldrich Co.) as references (Adams, 1995). Antimicrobial susceptibility test: Antimicrobial susceptibility of the isolates was performed using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar media (Merck, Germany). Commercially available antibiotic disks (Oxoid, UK) including ofloxacin, streptomycin, Septrin, chloramphenicol, ciprofloxacin, amoxicillin, ampicillin, pefloxacin, ceftazidime, and gentamicin were tested. The diameter of inhibition zones was measured for each antibiotic disk, and the results were defined in accordance with the CLSI guidelines (CLSI, 2017). Phenotypic identification of ESBL-producing strains. Detection of ESBL-­ producing organisms was performed by double disc synergy test (DDST) method following CLSI recommendations. A bacterial suspension of 0.5 McFarland turbidity standards was cultured on Mueller-Hinton agar. A pair of antibiotic disks containing ceftazidime (30 μg) with ceftazidime/clavulanic acid (30/10 μg) was placed on Mueller-Hinton agar medium center to center, at a distance of 20 mm apart from each other. The plates were incubated for 24 hours at 37 °C, and the diameter of inhibition zone was measured. According to CLSI guidelines, an increase of ≥5 mm in the zone diameter around the clavulanic acid combination disks versus the same disks alone confirmed the presence of ESBL.

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Assay of Antibacterial Activity of Syzygium aromaticum Essential Oils The agar well diffusion method of Akinpelu and Onakoya (2006) was employed. An overnight culture of the organisms was standardized to contain approximately 108 cfu/ml, and this was then inoculated into 20 ml of molten nutrient agar. The culture medium was allowed to set. Thereafter, a sterile cork borer (8.0 mm diameter) was used to punch wells in the seeded nutrient agar. The agar plugs were removed with a flamed and cooled wire loop. The essential oil (Syzygium aromaticum) was poured into the wells in different plates. The plates were thereafter incubated at 37 °C for 24 hours, and the zone of inhibition was measured. E. coli was tested with oil extract to determine susceptibility to serve as control. The experiment was repeated in triplicates. Determination of Minimum Inhibitory Concentrations (MIC) of the Essential Oils on Bacterial Isolates The method used in this study to determine the inhibitory activity of Syzygium aromaticum oil was similar to that described by Akinpelu and Onakoya (2006) with a few modifications. This assay was done to determine the lowest concentration of the oil that will inhibit microbial growth. Twofold dilution of the essential oil (Syzygium aromaticum) was prepared based on the concentration that inhibited growth in the agar well susceptibility test. Ten tubes were obtained, and 2 ml of sterile Tween 20 (reconstituting solvent) was dispensed into each tube except the first tube which contained 4 ml of the oil. 2 ml of the oil was removed from the first tube containing 4 ml into the second tube containing 2 ml of Tween 20 and mixed thoroughly; thereafter, 2 ml of the mixture was taken and poured into the third tube and mixed properly. 2 ml of that mixture was transferred serially up to the tenth tube, and 2 ml was removed and kept. These test tubes containing 2 ml of different concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.7825, 0.390, 0.195  mg/ml  v/v), respectively, were later poured into sterile molten nutrient agar and mixed properly. This agar-­ extract mixture was thereafter transferred into petri dishes and allowed to set. The plates were then seeded with the standardized test organisms and incubated at 37 °C for 72  hours. Controls were set up as inoculated nutrient agar plates without the extract as negative control. Determination of Minimum Bactericidal Concentrations (MBC) of the Essential Oils on Bacterial Isolates Based on the MIC results obtained, the MBC was taken as the least concentration that did not show any growth on the incubated nutrient agar plates. Determination of Rate of Kill The killing rate of Syzygium aromaticum oil on ESBL-producing Escherichia coli was carried out using the method described by Akinduti et al. (2021) with a few modifications. The standardized bacterial suspension (0.5 ml) was serially diluted in sterile normal saline to obtain ten dilutions; these dilutions were then seeded into nutrient agar plates using the pour plate technique. Heterogeneous plate count was then carried out to determine the viable microbial population count in each dilution factor to serve as reference for the rate of kill. Following this, 2  ml of the

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standardized bacterial suspension was added to 18 ml of the essential oil at a concentration equal to MIC ×1 value and MIC ×2 values. The mixture was thoroughly shaken together, and exactly 0.5 ml of the mixture was transferred immediately into 4.5 ml of already prepared sterile 3% Tween 20 nutrient broth, and the suspension was thoroughly mixed. This served as the portion taken at 0 minute as this was done at 15 minutes interval for 2 hours. Exactly 0.5 ml was taken from each suspension and serially diluted up to 10−6 in 4.5 ml sterile normal saline. Then, 0.5 ml of the final dilution factor was transferred into labeled sterilized pre-sterilized molten nutrients agar plates. This plate was incubated at 37 °C for 24 hours. The exact time at which the minimum viable count was obtained was recorded as the time it will take the antimicrobial agent to kill the organism.

3 Results A total of 229 urine isolates were collected during the study period from the State Hospital and Federal Medical Center Idi-aba both in Abeokuta Ogun State. Table 1 shows the result of the patients attending Idi-aba and State Hospital Ijaiye both in Abeokuta, Ogun State; 159 urine samples had significant growth while 70 samples were nonsignificant with 69.4% and 30.6%. Table 2 shows the result of the antibiotic susceptibility profiles of the isolates; five classes of antibiotics were used, and they are aminoglycoside, chloramphenicol, fluoroquinolones beta-lactams, and sulfonamides. Table 3 shows the distribution of beta-lactamase and ESBL-producing E. coli among male (14) patients and female (15) patients. Table 4 shows the phonotypic confirmation of the ESBL producers with double disc synergy test; 30 out of the 49 isolates that were screened were confirmed to be ESBL producers. Table 5 shows the result of the antimicrobial testing of Syzygium aromaticum essential oil on ESBL-producing E. coli with isolate numbers 2, 10, 15, 16, and 18 having the highest zone of inhibition of 14 mm and the lowest value which is 8 mm found in number 14. Table 5 shows the result of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).

Table 1  Distribution of E. coli among patients attending Idi-aba and State Hospital Ijaiye Urine culture Urine sample Significant growth Nonsignificant growth Total

229 159 70 229

In-patients Male 28 (12.6) 18 (11.3) 10 (13.8) 28

Female 72 (31.4) 52 (33.9) 20 (27.7) 72

Out-patients Male 39 (16.5) 19 (11.9) 20 (27.7) 39

Female 90 (39.3) 68 (42.7) 22 (30.5) 90

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Table 2  Antibiotic susceptibility profile ATB OFX S SXT CH CPX AMP A PEF CN CT

Resistance n1 78 (49.1) 68 (42.8) 98 (61.6) 26 (16.4) 32 (21.1) 122 (76.7) 135 (84.9) 76 (47.8) 118 (74.2) 147 (92.4)

N 159 159 159 159 159 159 159 159 159 159

Sensitivity n2 81 (50.9) 91 (57.2) 61 (38.4) 133 (83.6) 67 (61.5) 37 (23.3) 24 (15.1) 83 (52.2) 41 (25.8) 12 (7.5)

Table 3  Distribution of beta-lactamase and ESBL-producing E. coli among male and female Characteristic Gender Male Female Age group ≤11 12–21 22–31 32–41 42–51 52–61

Beta-lactamase N %

ESBL N

%

14 35

28.6 71.4

11 19

36.6 63.3

1 3 27 13 2 3

2.0 6.1 55.1 26.5 4.0 6.1

1 1 18 7 1 2

3.3 3.3 60 23.3 3.3 6.6

Two isolates (E19 and E30) showed the highest susceptibility to MIC 6.26 mg/ mL and lowest susceptibilty at MIC 0.0373 mg/ml, while minimum bactericidal concentration of 0.75 mg/ml to two isolates E15 and E30. Figure 1 shows the time rate of kill of the essential oil Syzygium aromaticum on ESBL-producing Escherichia coli. Table 6 shows the components of Syzygium aromaticum essential oil. Gas chromatography-­mass spectroscopy (GC-MS) analysis and molecular docking of the ESBL-producing with the components of Syzygium aromaticum essential oil (Figs. 2 and 3).

4 Discussion Among the most frequently isolated pathogens in clinical samples are the Enterobacteriaceae, and they are involved in causing infections with Escherichia coli being the most prevalent (Kalisvar et al., 2021). Antimicrobial agents such as the beta-lactams, aminoglycosides, and quinolones often used for treatment of

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Table 4  Screening for ESBL production using double disc synergy test

Isolates E1 E2 E3 E4 E5 E6 E7 E8 E24 E25 E31 E32 E33 E34 E35 E36 E37 E38 E39 E41 E42 E31 E32 E34 E35 E36 E37 E39 E41 E42 E52 E53 E56 E57 E58 E59 E60 E61 E62 E63

Ceftriaxone + clavulanic acid (Zone of inhibition in mm) 17 14 15 14 10 13 10 10 18 18 13 19 12 17 14 12 16 13 18 12 17 17 12 17 11 12 16 15 14 17 19 13 14 15 18 16 12 14 13 16

Ceftriaxone (Zone of inhibition in mm) 9 7 11 8 7 6 7 6 6 6 9 8 9 11 11 8 9 7 10 9 10 9 8 10 6 8 8 10 9 10 10 10 8 8 9 9 10 9 9 12

ESBL type (DDST zone of inhibition ˃5 mm) Positive Positive Negative Positive Negative Positive Negative Negative Positive Positive Negative Positive Negative Positive Negative Positive Positive Positive Positive Negative Positive Positive Negative Positive Positive Negative Positive Positive Positive Positive Positive Negative Positive Positive Positive Positive Negative Positive Negative Negative (continued)

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Table 4 (continued)

Isolates E64 E65 E66 E67 E68 E69 E70 E72 E73

Ceftriaxone + clavulanic acid (Zone of inhibition in mm) 16 13 18 15 13 11 12 18 14

Ceftriaxone (Zone of inhibition in mm) 5 10 14 11 4 10 10 11 11

ESBL type (DDST zone of inhibition ˃5 mm) Positive Negative Positive Negative Positive Negative Negative Positive Negative

Table 5  Antimicrobial testing of Syzygium aromaticum essential oil on ESBL-producing E. coli isolates Zones of inhibition (mm) 8 ± 0.0 9 ± 0.0 11 ± 0.0 11 ± 0.0 12 ± 0.0 13 ± 0.0 14 ± 0.0

Isolates codes E14 E19, E20, E23 E3, E6, E9, E11, E21, E25, E26, E27, E30 E1, E4, E8, E12, E17, E22 E29, E24, E7, E5, E13 E28 E2, E10, E15, E16, E18

infections caused by ESBL-producing E. coli which are obviously becoming less effective because of the resistance the ESBL confers on the organism (Karimov et al. 2021). Uncontrolled and excessive use of antibiotics particularly in areas with poor hygienic practices in developing countries is among the many factors that contribute to antibiotic resistance (Olumide et al., 2021). Therefore, proper knowledge of pathogen as well as the antibiotic susceptibility profile is important toward proper and accurate use of antibiotics. The isolation and characterization of ESBL-producing Escherichia coli in the State Hospital Ijaiye, and Federal Medical Center Idi-aba both in Abeokuta were carried out in order to highlight its importance and check the efficacy of the Syzygium aromaticum essential oil on them. Out of the 229 urine isolates, 159 (69%) were identified as E. coli with 109 resistant to 2 or 3 classes of antibiotics with high resistance to ceftazidime, Septrin, ampicillin, amoxicillin, and gentamycin and least resistance to chloramphenicol. This correlates with the report of Ahmed et al. (2019) whose study showed high resistance of E. coli to ampicillin. In another study by Mohammed et al. (2016), E. coli isolates were extremely resistant to ampicillin but highly sensitive to amikacin, imipenem, and meropenem. Thirty out of the total number of organisms that were multidrug-resistant were confirmed ESBL producers by double disk synergy test. ESBLs have been found in various members of the Enterobacteriaceae and other Gram-negative bacilli (Giwa et al., 2018).

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Fig. 1  The time kill rates of the essential oil of Syzygium aromaticum on ESBL-producing Escherichia coli. MIC 1 minimum inhibition concentration 1, MIC 2 minimum inhibition concentration Table 6  Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) exhibited by Syzygium aromaticum essential oil against the ESBL-producing Escherichia coli MIC zone of inhibition (mg/ml) 0.097 0.75

MBC zone of inhibition (mg/ml) 0.75 1.53

0.0373

Isolate ID E9 E1, E7, E8, E13, E18, E20, E21, E27 E16

0.195

E14, E17

3.25

0.375

E15, E23

6.25

1.56

E4, E5, E6, E12, E26, E28 E10, E22, E24 E2, E3, E11, E25, E29 E19, E30

12.5

1.95 3.25 6.25

1.56

25

Isolate ID E9 E7, E8 E10, E13, E18, E22, E27 E1, E4, E5, E12, E21, E24, E26, E28 E2, E3, E6, E11, E25, E29 E14, E15, E16, E17, E20, E23 E15, E30

Key: MIC minimum inhibitory concentration, MBC minimum bactericidal concentration

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Fig. 2  Gas chromatography-mass spectroscopy (GC-MS) analysis showing peaks of components of Syzygium aromaticum essential oil

Fig. 3  Molecular docking of ESBL-producing Escherichia coli with the components of Syzygium aromaticum oil

The antibacterial activity of the Syzygium aromaticum (clove) essential oil on E. coli which showed that Syzygium aromaticum essential oil had lower potency against some isolates was tested. Similar antibacterial pattern of Syzygium aromaticum activity against S. aureus was reported in a study done by Abdullah et  al. (2015). Thosar et al. (2013) also reported strong inhibitory effects of clove essential oil at low concentrations against all organisms tested as compared with other oils such as lavender and peppermint oil. Eugenol a major component of clove essential oil has been proven effective in combating several pathogens such as S. typhi, P. mirabilis (Muniz et al., 2021), E. coli, S. aureus, and Pseudomonas aeruginosa by altering the integrity of their cell membranes (Huma et al., 2021). The GC-MS analysis for Syzygium aromaticum essential oil identified eight different compounds with three as the major components of the essential oil (Table 7). It was observed that eugenol (80.98%) was present in the highest concentration followed by phenol, 2 methoxy 4-(2-propenyl)-acetate (11.52%) and caryophyllene (6.00%). This correlates with the report of Azir Uddin et al. (2017) who identified

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Table 7  Components of Syzygium aromaticum oil Retention Peak time 1 8.516

Area 332,046

2 3 4 5 6 7

10.380 10.926 12.038 12.655 12.964 13.345

545,178 711,895 267,599,342 19,831,590 2,669,130 38,054,291

8

14.065

716,568 100.00

Area % Height 0.10 184,687 0.16 0.22 80.98 6.00 0.81 11.52

464,325 519,309 48,501,469 15,801,026 2,254,411 22,317,370

0.22 619,633 100.00 100.00

Height % 0.20 0.51 0.57 53.50 17.43 2.49 24.62

0.68 100.00

AH Name 1.80 6-Methyl-2-heptanol, acetate 1.17 Methyl salicylate 1.37 Phenol,4-(2propenyl) 5.52 Eugenol 1.26 Caryophyllene 1.18 Humulene 1.71 Phenol, 2methoxy-4(2propenyl)-acetate 1.16 Caryophyllene

some of the compounds found in his study as 3-Allyl-6-methoxyphenol (69.44%), eugenol acetate (10.79%), 2-pentanone 4-hydroxy-4-methyl (7.78%), and caryophyllene (6.80%). However, there are some variations in the compounds from Azir Uddin et  al. (2017); these variations could be due to many factors such as geographical origin, genetic factors, plant material, and harvesting time (Dugo et al., 2014). Antimicrobials are considered as bactericidal if the MBC/MIC ratio is ≤4 and bacteriostatic if ˃4 (Keepers et al., 2014). The ratio obtained for the test organisms with Syzygium aromaticum essential oil were less than 4 for some organisms, while others were greater than 4. This shows that bactericidal and bacteriostatic action of the oil is organism specific; this was confirmed by time-kill kinetic studies. The rate of kill of the Syzygium aromaticum essential oil as depicted in this study indicates a continuous decrease in the cell population as time of exposure increases for both concentrations of the MIC used. This is in line with the reports of Boakye et  al. (2016) who reported a gradual decrease in cell population for the first 3 hours of exposure of the oil to the isolates. However, the rate of cell population decreased faster in MIC ×2 than in MIC ×1; this is in agreement with the report of Mohammadi (2021) which states that the higher the concentration, the higher the antibacterial effect of the agent against organisms. Bacteriostatic antibiotic agents only inhibit the growth or multiplication of microbes giving the immune system of the host time to clear the microbes from the system, while the ability of the essential oil to kill or eliminate microorganisms at the shortest period of time is taken as the bactericidal activity in antibiotics Luis et al. (2021). Molecular docking conducted in this study revealed that the components of the essential oil are potential lead molecules in the inhibition of bacterial growth and thus justifies its use in traditional folklore medicine. Caryophyllene oxide, caryophyllene, and humulene had the highest negative binding energy and was able to compete favorably for the binding site of the organism with cefotamine antibiotics which served as the standard thus the highest potential for the inhibition of the ESBLs from the Escherichia coli.

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Growth and Haemato-Biochemical Responses of All-Male Tilapia, Oreochromis niloticus, to Diets Containing Fermented Cassava Leaf Meal Oluwagbenga O. Olude and Paul Akinduti

1 Introduction Increasing human population projected at about 10 billion by 2050 (United Nations, 2022) and stagnated production from capture fisheries (FAO, 2020; Boyd et  al., 2022) have put more responsibility on aquaculture as a way of avoiding future animal protein intake crisis. Aquaculture is expected to almost double its current production figures by 2050 in order to meet fish demand and ensure protein security, especially among low-income countries with high poverty index (Boyd et  al., 2022). However, quality feed and feed ingredients, which are key factors that drive expansion of aquaculture, remain scarce and expensive, slowing down aquaculture production in many countries including Nigeria (Bene et  al., 2015; Udo & Umanah, 2017). Further, some conventional ingredients used in aquaculture production are not locally sourced; for which reason they are subject to the vagaries of foreign exchange and thus aggravate the existing trade imbalance in many developing countries like Nigeria. Although, appreciable progress have been made in downplaying the use of conventional feed ingredients, with some of the researched non-­conventional alternatives being used at commercial level (Zlaugotne et al., 2022); the dynamics of human nutrition, especially in view of escalating human population and technological advancement, has made it such that ingredients that were basically or largely previously relevant in animal nutrition are now being considered in human diets. The situation underscores the significance of further search for substitute feed resources, especially those ones that O. O. Olude (*) Department of Marine Sciences, Faculty of Science, University of Lagos, Akoka, Lagos, Nigeria e-mail: [email protected] P. Akinduti Department of Biological Sciences, Covenant University, Ota, Ogun State, Nigeria © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. O. Isibor et al. (eds.), Biotechnological Approaches to Sustainable Development Goals, https://doi.org/10.1007/978-3-031-33370-5_13

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are wastes or by-products. Unlike soybean, the alternative should be largely irrelevant in human diet, locally sourced, less prohibitive and with minimal carbon footprint (Zlaugotne et al., 2022). Magbanua and Ragaza (2022) recently detailed some of the plant-derived feed ingredients researched as replacement for the conventional fish meal in tilapia diet. However, not a few factors have been advanced for poor utilization of many of those alternatives, especially those that are plantderived. Incomparable crude protein content and other nutritional deficiencies relative to the conventional ones, nutritional stress factors in form of high crude fibre, indigestible carbohydrates such as non-starch polysaccharides and antinutrients such as phytates, tannins and enzyme inhibitors among many others are some of the factors constraining the utilization of alternatively-sourced ingredients in practical fish feed (Deng et al., 2015; Olude & Sahu, 2016; Magbanua & Ragaza, 2022). These challenges have necessitated the development of sustainable and innovative approaches in utilizing the available cheaper alternatives in feed formulation so as to encourage a more responsible and resilient aquafeed industry. Solid-state fermentation, a low-technology and low-­energy technique that grows micro-organism on substrate with little or no free flowing water, offers great solution in transforming waste into value-added products at cheap cost. It has been used severally to valorize unconventional ingredients through increased crude protein content, enhanced amino acid profile, increased nutrient availability and digestibility and reduced crude fibre and antinutrients (Lateef et  al., 2008; Olude et al., 2020; Okoth et al., 2022; Terefe et al., 2022). Cassava is a staple crop primarily cultivated for its root, and it is the seventh most important crop in the world (Hue et al., 2012). Its propensity to grow well in marginal soil and adverse weather conditions ensured its prominence and spread in many countries of the world with Nigeria, Democratic Republic of Congo, Thailand, Ghana, Indonesia and Brazil being the top producers (Ikuemonisan et al., 2020). Cassava production has grown consistently over decades at about 3% rate with production in year 2021 being in excess of 300 million tonnes with about 351 million tonnes projected for year 2027 (FAO, 2018; IMARC Group, 2022). Cassava leaf, a by-product of cassava cultivation, is reported to be similar in yield to that of the tuber and is mostly left to decay on farmlands after harvest of the tuber (Hue et al., 2012). Using Ghana as a case study, Poku et al. (2018) also alluded to the huge, renewable, yet grossly underutilized biomass from cassava value web whose development could trigger a competitive bio-economy. The leaf’s crude protein content is averagely 21% (Hue et al., 2012) which is incomparable to more than 40% usually found in soybean meal (Banaszkiewicz, 2011); for which reason it cannot effectively substitute soybean meal (SBM) in fish diet. Limited utilization of sundried cassava leaf meal associated with residual antinutrients was observed in rohu, Labeo rohita fingerlings, when it was incorporated as a substitute for a carbohydrate component (de-oiled rice bran, DORB; Olude et al., 2021). Marked reduction in crude fibre and antinutrients and an enhanced crude protein of sweet potato leaf meal was reported by Meshram et al. (2018) after fermenting with fungus, Chaetomium globosum. Their study also reported that the improved nutritional value of sweet potato

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leaf meal translated to total substitution of DORB in feed for L. rohita fingerlings. To our knowledge, there is no previous report on the utilization of fermented cassava leaf meal (FCLM) in fish diet. We envisaged that solid-state fermentation of cassava leaf meal using Chaetomium globosum will improve its nutritional composition and thus its feeding value to tilapia, Oreochromis niloticus fingerlings. Thus, the present study assessed the effects of substituting soybean meal with fermented cassava leaf meal on growth, feed efficiency and health of all-male tilapia, Oreochromis niloticus fingerlings.

2 Materials and Methods 2.1 Preparation of Diet Based on preliminary laboratory trial, to determine the parameters of fermentation with optimum crude protein yield, sundried cassava leaf meal was fermented with Chaetomium globosum (MTCC-4179) at concentration of 3 × 105 cell/g and temperature 28 °C for 144 h following the method described by Meshram et al. (2018). Prior to incubation with C. globosum, the leaf’s moisture content was adjusted to 50% with distilled water and autoclaved for 15 min at 121 °C and 15 psi to ensure a sterile medium. Thereafter, fermentation was stopped by drying the leaf in hot-air oven at 50 °C for 24 h. 0, 100, 200 and 300 g/kg of the FCLM were used to substitute soybean meal in four (FCLM0, FCLM100, FCLM200, FCLM300) isonitrogenous (35% crude protein) and iso-energetic (18.53 kJ/g) diets. The diets also contained fish meal, groundnut cake and maize meal among other micro-ingredients and feed additives (Table 1). The quantity of each ingredient required to formulate 1 kg of each diet was weighed using a top-loading balance (Camry, EK5350), thoroughly mixed with a little volume of warm water to form a dough, steam-cooked in an autoclave and pelleted through 2 mm die using improvised hand pelletizer. The pellets were air-dried for 2 days at room temperature and packed in properly labelled airtight cellophane bags before the start of the experiment.

2.2 Experimental Fish and Procedure All-male Oreochromis niloticus fingerlings were procured from a commercial fish farm (Choice Fisheries Consults Ltd., Lagos, Nigeria) and transported in oxygen bags to fish nutrition experimental unit of the Department of Marine Sciences situated in the Botanical garden of the University of Lagos, Nigeria. They were kept in a holding tank and fed with control diet for 28  days acclimatization period. Afterwards, the fish were weighed (average weight, 6.21 ± 0.05 g) into 12 rectangular plastic tanks (0.52 × 0.33 × 0.21) m3 at the rate of 6 fish per tank. The tanks were

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Table 1  Composition (g/kg) of experimental diets Ingredients FCLM0 FCLM100 Fish meal 100 100 Groundnut cake 175 220 Soybean meal 300 200 FCLM 0 100 Maize meal 314.8 269.8 Vegetable oil 60 60 a Vit/min. premix 20 20 CMC 10 10 Methionine 10 10 Lysine 10 10 BHT 0.2 0.2 Proximate composition of the diets (g/kg) Moisture 91.7 93.5 Crude protein 303.2 306.6 Ether extract 128.1 119.1 Total ash 87.3 85.2 Crude fibre 77.1 85.2 NFE 312.6 310.4 ¶ Gross energy (kJ/g) 18.94 18.77 Antinutrients in the diet (mg/kg) Tannins – 6.56 HCN – 38.32

FCLM200 100 264 100 200 225.8 60 20 10 10 10 0.2

FCLM300 100 306.8 0 300 183.0 60 20 10 10 10 0.2

95.1 302.1 110.3 101.1 97.2 294.2 18.24

98.5 303.0 101.4 91.7 97.7 307.7 18.15

CLM 81.5 242.1 66.3 103.0 185.3 321.8 17.06

FCLM 79.0 286.9 96.9 89.0 147.8 300.4 18.3

13.12 76.64

19.68 114.96

115.3 460.0

65.6 383.2

Radar vitamin/mineral premix supply/100 g diet, vitamin A palmitate, 1000 IU; cholecalciferol (D), 1000 IU; α-tocopherol acetate (E), 1.1 mg; menadione (K), 0.02 mg; thiamine B1, 0.63 mg; riboflavin (B2), 0.5  mg; pantothenic acid, 1.0  mg; pyridoxine (B6), 0.15  mg; cyanocobalamin (B12), 0.001 mg; nicotinic acid, 3.0 mg; folic acid, 0.1 mg; choline, 31.3 mg; ascorbic acid (C), 0.1 mg; ferrous sulphate, 0.05 mg; copper sulphate, 0.25 mg; manganese sulphate, 6.00 mg; cobalt chloride, 0.5 mg; zinc sulphate, 5.0 mg; sodium selenite, 0.02 mg. ¶ Calculated gross energy (kJ/g) = (Crude protein × 23.64) + (Ether extract × 39.8) + (Total carbohydrate × 17.15)/100 CMC carboxymethyl cellulose, BHT butylated hydroxytoluene, FM fish meal, SBM soybean meal, GNC groundnut cake, FCLM fermented cassava leaf meal a

previously disinfected with salt water, filled with borehole water to 2 3 of their volume and covered with perforated lids to prevent the fish from jumping out of the tank. The water in the tanks was aerated with air compressor (Hailea, ACO 300A) fitted with air hose and air stone. The four diets were randomly assigned and fed twice daily at 3% body weight offered in two equal installments at 09:00 and 17:00 h for 60 days to triplicate groups of tilapia fish in the tanks. The fish were fed every day except when they were batch-weighed (every 15th day) with a sensitive electronic balance (Camry model EK5350) and the quantity of feed adjusted to the new weight. Water in the rearing tanks was changed regularly, and faecal matter was siphoned out to sustain optimum water quality. The monitored pH (7.18–7.25),

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dissolved oxygen (7.8–9.0 mg/L) and temperature (28.1–29.0 °C) were observed to be within acceptable limits for tilapia (Boyd, 1998).

2.3 Chemical Analyses Sundried cassava leaf meal (CLM), FCLM and other major feed ingredients were analysed for proximate composition according to the standard method of AOAC (1990); nitrogen-free extract was calculated as the difference in the original sample weight and the sum of other proximate composition (moisture, crude protein, ether extract, crude fibre and ash) of the sample. The amino acid profiling of CLM and FCLM followed the chromatographic (GC2010 Plus Instrument, Shimadzu, Tokyo, Japan) procedure of Husek (1991). Tannin was estimated by the spectrophotometric method of Makkar et al. (2007), while the titrimetric method of AOAC (1995) was used in quantifying the cyanide content. Prior to the commencement of the feeding trial, four fish were sacrificed for the determination of initial proximate carcass composition (AOAC, 1990), whereas three fish per replicate were sacrificed at the completion of the feeding trial for the same purpose.

2.4 Performance Parameters and Survival The parameters of growth, nutrient utilization and survival were calculated using the following relationships:

Mean weight gain  MWG,g   Wf – Wi



Percentage weight gain  PWG,%    Wf  Wi  / Wi   100



Specific growth rate  SGR,% / day   100  ln Wf  ln Wi  /  t 



where Wf and Wi are final and initial live fish weights (g), respectively, while t is the rearing period

Feed conversion ratio  FCR   Total dry feed fed  g  / Total wet weight gain  g 



Protein efficiency ratio  PER   Wet weight gain  g  / Amount of protein fed  g  Protein productive value  PPV ,%    Final fish body protein  Initial fish body protein  / Crude protein fed   100



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2.5 Sample Collection, Haematological and Serum Biochemical Parameters At the end of the experiment, three fingerlings per replicate were sedated with clove oil and bled through the caudal vein using 1 mL disposable hypodermic needle as described in Gbore et  al. (2010). The haematological parameters of packed cell volume (PCV), haemoglobin, red blood cell (RBC) count, white blood cell (WBC) count as well as WBC differentials and blood indexes such as mean corpuscular haemoglobin concentration (MCHC), mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) of the experimental fish were determined at the Central Research Laboratory of Lagos University Teaching Hospital (LUTH) using automatic analyser (MINDRAY, BC 3200). Similarly, based on the principle of colorimetry, serum biochemical parameters of total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), glucose, triglycerides, cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were carried out with ERBA MANNHEIM (XL 200/640) automatic biochemical analyser.

2.6 Statistical Analysis of Experimental Data The data derived from the experiment were subjected to one way analysis of variance (ANOVA), and when the differences in means were significant (p