Trends in Sustainable Chocolate Production 3030901688, 9783030901684

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Charis M. Galanakis Ed.

Trends in Sustainable Chocolate Production

Trends in Sustainable Chocolate Production

Charis M. Galanakis Editor

Trends in Sustainable Chocolate Production

Editor Charis M. Galanakis Galanakis Laboratories Chania, Greece

ISBN 978-3-030-90168-4    ISBN 978-3-030-90169-1 (eBook) https://doi.org/10.1007/978-3-030-90169-1 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 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

Chocolate is an extremely popular delicacy that is continuously consumed worldwide by people of all ages due to its sensory characteristics. On the other hand, the awareness of modern consumers about the link between healthy eating and well-­ being is changing their consuming habits in foods that generate positive organoleptic feelings and are also supported by sustainable supply chains. Besides, sustainability is becoming an essential item for the food industry worldwide as resources become more restricted and demand grows. However, most books on the market cover chocolate manufacture, without considering sustainable practices of production, consumption, and market aspects. Food Waste Recovery Group (www.foodwasterecovery.group) has developed several activities, including consultation reports to different governmental, industrial, and research bodies, workshops, webinars, e-courses, publications, a new open-access journal (Discover Food, Springer Nature), and multiple books in the broad fields of food, nutrition, bioresources, and environment. Following these efforts, the current book fills the knowledge transfer gap between academia and industry by covering all the essential aspects of the chocolate industry (manufacture, functionality, sustainability of the supply chain, commercialization aspects, and market characteristics) in one reference. The ultimate goal is to support the scientific community, professionals, and enterprises that aspire to develop a sustainable chocolate sector. The book consists of ten chapters. Chapter 1 deals with the state-of-the-art harvesting and post-harvest handling of cacao and provides an update on chocolate manufacturing and the impacts of processing on chocolate’s sensory and functional quality. Chocolate production implies an extensive post-harvest process of the cacao beans, the seeds of the tree Theobroma cacao L. All the steps from bean harvesting into the chocolate bar’s development affect chocolate properties, providing to the final product unique sensory qualities that will attract consumers. Chapter 2 provides a comprehensive overview of the origins, processing, quality control, and flavor development of cocoa beans. Significant cocoa flavor markers are theobromine, caffeine, catechin, epicatechin, and proanthocyanidins. However, cocoa flavor notes are built upon intricate combinations of amino acids, alcohols, v

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phenols, volatile acids, esters, aldehydes, ketones, lactones, terpenoids, minerals, glycated and polymeric substances. The chocolate industry and cocoa products undergo intense competition and evolution, which demands new food products. In Chapter 3, the ways to improve the functional characteristics of the chocolate products are reviewed. Calorie reduction in chocolates using sugar and cocoa butter alternatives and its effect on chocolate quality is also denoted. In addition, the influence of processes applied for cocoa and chocolate productions on polyphenol composition and antioxidant activity is summarized. The growing demand for functional food from sustainable plant-based produce motivates food scientists to develop new types of chocolate products containing vegetal extract. The incorporation of vegetal extract in the chocolate formulation is intentionally aimed to improve the health-promoting properties of the chocolate, such as phenolic content and antioxidant activity. Herbs and spices are some other ingredients with potential use in the chocolate formula. To this line, Chapter 4 presents the various bioactive compounds in cocoa, the impact of chocolate processing on the bioactive compounds of cocoa, enhancing the bioactive components of chocolate by incorporating different vegetal extracts, and the consumer perception of chocolate and chocolate with vegetal extracts. Chapter 5 focuses on the phase transition of tempered and non-tempered dark chocolate processed with cocoa from different geographical origins, using differential scanning calorimetry, rheometry, and thermography. Cocoa butter plays a vital role in the appearance and texture of chocolate, and the polymorphic forms in the final product depend on the tempering process. The fatty acids profile does not present significant differences related to the geographical origin, with higher values for palmitic acid, stearic acid, and oleic acid. However, the phase transition is influenced by the tempering process. Since consumers have become aware of different cocoa genotypes and their origins, which caused an expanding market of premium chocolates with single-origin cocoa beans, cocoa beans may be subjected to adulteration due to the high demand for superior quality products. Therefore, for the accurate discrimination of the cocoa beans, three major analytical approaches can be implemented: (1) chemical approaches, (2) biomolecular approaches, and (3) isotopic approaches. In Chapter 6, these three approaches are reviewed together with the recent literature on traceability of cocoa origin. Nowadays, the increase in the global demand for chocolate production changes the production systems of this agri-food product. Although chocolate production is increasing daily, this volume production leads to a substantial environmental cost due to changes in production systems. Given the awareness of the irreparable ecological consequences, there is currently significant interest in producing and consuming sustainable food products such as chocolate. To better understand the environmental impacts of the life cycle of chocolate, Chapter 7 provides an overview of the environmental impacts of chocolate production throughout its life cycle. Also, it reviews and compares the environmental problems of chocolate production and presents a clear picture of future perspectives which should be considered in the production of chocolate.

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Chapter 8 examines the discourse of corporate sustainability efforts for cocoa sourcing, using Ghana as an example. Taking nothing away from the sincerity or insincerity of corporate sustainability efforts, the chapter underlines the disconnect of values, motivations, and benefits between corporate sustainability for now and local people’s sustainability in perpetuity that threatens the UN’s Sustainable Development Goals (SDGs) generally. Chapter 9 identifies the more widely used sustainability labels in chocolate and investigates the relation between chocolate labeling and purchase intention and perception. The results conclude that sustainability labeling information influences consumers’ purchase decisions and sensory scores. Hence, many consumers worldwide are willing to pay extra money for cocoa and chocolate manufactured following ethical principles and, thus, with sustainability labels on the packaging. Finally, Chapter 10 characterizes the chemical composition of the residual cocoa biomass and explores the existent valorization strategies. Based on the composition (polyphenols, organic acids, methylxanthines, etc.) of this by-product, valuation strategies applied to different fields, such as the food industry, human health, cosmetics, and bioremediation, are proposed for each one. These advances would help improve some socio-economical and environmental indicators and promote the sustainability of the world’s cocoa production chain. Conclusively, the current book is expected to assist food scientists and technologists, researchers and professionals working in the edge of the food and environmental field, and agriculturalists and food engineers, who seek to improve the efficiency of production systems. It also concerns specialists working in the chocolate industry, from farm to fork. It could also be purchased by University Libraries and Institutes all around the world to be used as a textbook and/or ancillary reading in under-graduates and post-graduate level multi-discipline courses dealing with sustainable food systems, agricultural and environmental science, and food processing. At this point, I would like to acknowledge and thank all authors for accepting my invitation. Their dedication to the project, timeline, and editorial guidelines are highly appreciated. I would also like to thank the acquisition editor Daniel Falatko, book manager Arjun Narayanan, and all the production team of Springer Nature for their help during this book’s preparation. Last but not least, I have a message for all the readers. This kind of book project is a collaborative effort containing hundreds of thousands of words, and it may contain errors. Constructive comments and even criticism are always welcome, so do not hesitate to get in touch with me to suggest any changes. Chania, Greece

Charis M. Galanakis

Contents

1 State-of-the-Art Chocolate Manufacture����������������������������������������������    1 Marcela Hernández-Ortega, Carla Patricia Plazola-Jacinto, and Lourdes Valadez-Carmona 2 The Taste Development of Cocoa Bean: Evidence from the Tropical Rain Forest to the Table ��������������������������   41 Miftakhur Rohmah, Kartika Sari, and Anton Rahmadi 3 Improving Functionality of Chocolate ��������������������������������������������������   75 Nevzat Konar, Ibrahim Palabiyik, Ömer Said Toker, Arifin Dwi Saputro, and Haniyeh Rasouli Pirouzian 4 Improving the Functionality of Chocolate by Incorporating Vegetal Extracts����������������������������������������������������������  113 Dimas Rahadian Aji Muhammad, Dwi Larasatie Nur Fibri, and Sangeeta Prakash 5 Impact of Geographical Origin on Chocolate Microstructure, Phase Transition, and Fat Bloom������������������������������������������������������������  153 João Dias, António Panda, Ana Partidário, Nuno Alvarenga, João Lita da Silva, Teresa Cordeiro, and Pedro Prazeres 6 Making Cocoa Origin Traceable������������������������������������������������������������  189 Senem Kamiloglu, Perihan Yolci-Omeroglu, and Omer Utku Copur 7 Environmental Impacts of Chocolate Production and Consumption����������������������������������������������������������������  229 Homa Hosseinzadeh-Bandbafha and Mohammadali Kiehbadroudinezhad 8 Chocolate Industry Sustainable Sourcing Practices ����������������������������  259 Julia Bello-Bravo, Anne Namatsi Lutomia, John W. Medendorp, and Barry Robert Pittendrigh

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9 Sustainability Labeling in the Perception of Sensory Quality and Consumer Purchase Intention of Cocoa and Chocolate ��������������  291 Marta Puchol-Miquel, José Manuel Barat, and Édgar Pérez-Esteve 10 Valuation Strategies for the Biomass Generated While Producing and Transforming Cocoa into Chocolate ����������������  325 Jesús Anthony Gutiérrez Chávez, José Manuel Barat Baviera, and Édgar Pérez-Esteve Index������������������������������������������������������������������������������������������������������������������  351

Chapter 1

State-of-the-Art Chocolate Manufacture Marcela Hernández-Ortega , Carla Patricia Plazola-Jacinto and Lourdes Valadez-Carmona

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Abstract  Chocolate production implies an extensive post-harvest process of the cacao beans, the seeds of the tree Theobroma cacao L. All the steps from bean harvesting into the chocolate bar’s obtention (cocoa origin, composition and manufacturing procedure) will affect chocolate properties, providing to the final product unique sensory qualities that will attract consumers. Cocoa products are worldwide consumed because they are recognized as a significant source of polyphenols, molecules with essential health benefits. The current consumers’ concern about their wellness leads them to change the purchasing behavior and looking for new beneficial health-related products. In this context, and in addition to its sensorial properties chocolate rich in cacao content is an exceptional carrier to deliver bioactive compounds such as flavonoids, tannins, peptides, fiber, and some probiotics among others; making the chocolate a good healthy product. For this reason, this chapter aims to provide an update on the harvesting, post-harvest handling of cacao, chocolate manufacturing, and how each process impacts the sensorial and functional quality of chocolate. Keywords  Cacao bean · Chocolate · Epicatechin · Methylxanthines · Mood M. Hernández-Ortega Departamento de Nutrición, Facultad de Ciencias de la Salud, Universidad Anáhuac México, Huixquilucan, Estado de México, Mexico e-mail: [email protected] C. P. Plazola-Jacinto Departamento de Ingeniería Bioquímica, Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Unidad Profesional Adolfo López Mateos, Ciudad de México, Mexico L. Valadez-Carmona (*) Departamento de Biotecnología y Bioquímica, Centro de Investigación en Ciencias Biológicas Aplicadas, Universidad Autónoma del Estado de México, Toluca, Estado de México, Mexico e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 C. M. Galanakis (ed.), Trends in Sustainable Chocolate Production, https://doi.org/10.1007/978-3-030-90169-1_1

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1.1  Introduction Cacao (Theobroma cacao L.) belongs to Sterculiaceae family and is also called “Food of God”. Cacao is widely distributed in tropical countries mainly in those of the equatorial region such as Cote d’Ivory (32.2%), Ghana (19.3%), Indonesia (16.4), Brazil (6.2), Cameroon (6.1), Nigeria (5.6%), and others with less production (Dinarti et al., 2015; Li et al., 2019). Cacao is economically significant because it is the raw material for chocolate, liquor, cocoa powder and cacao butter manufacture for confectionery, food and cosmetic industries (Dinarti et  al., 2015; Rojas et al., 2020). According to the World Cocoa Foundation ICCO, annual global cacao production was more than four million tons (Kongor et al., 2016; Valadez-Carmona et al., 2017) of which 90% are produced by five to six million farmers in developing countries. The cacao tree is a perennial and non-climacteric tree with five-year generation time, after this the tree has continuously production throughout the year. Cacao tree grows best in hot and moisture conditions; in drought conditions vegetative and reproductive functions are depressed (Kongor et  al., 2016). Cacao pods emerge from flowers as an extension; the ovules of 15 to 17 old week pods begin solidifying until a developed enlarge and deepen seeds/beans of violet color during the last eight weeks (Li et al., 2019). When the pods are mature the cacao fruit is constituted by the shell, mucilage, and beans (Fig. 1.1). The shell is made up of 3 well differentiated layers; (1) the exocarp which is spongy and soft; (2) the mesocarp, comprises of hardy semi-­ woody cells which vary according to the genotype; and (3) the endocarp which is smoothly and fleshy attached to the mucilage. The cacao fruit contain around 30 to 40 beans embedded in a mucilaginous pulp which are extracted from the pod. Farming practices and genus of cacao influence the beans’ quality parameters such as size, shape, appearance, and exterior color (Araujo et al., 2019; Armengot et al., 2020; Gutiérrez, 2017; Kongor et al., 2016). In fact, harvesting factors such as the genotype, geographic and edaphoclimatic conditions as well as the maturity stage influence on physical, nutritional, and phytochemical characteristics of cacao beans. While post-harvesting processing such as fermentation, drying and toasting influences flavor and aroma development.

Fig. 1.1  Structure of cacao fruit

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(Pérez et al., 2009). Besides cacao’s economic importance as a crop, chocolate and cacao derived products have health-related benefits confirmed in in vitro studies. The health benefits such as anti-carcinogenic, vasodilatory, antidiabetic, and antiatherogenic properties have been attributed to the bioactive compounds like polyphenols mainly to the catechin family, which exhibits antioxidant properties (Ioannone et al., 2015; Nguyễn et al., 2018; Rojas et al., 2020; Gutiérrez & Pérez, 2015; Nazaruddin et al., 2006). The differences between cacao varieties and origin of the cacao beans influence strongly the aroma, phenolic content, volatile compounds, mineral and biochemical composition, pH, total acidity, simple carbohydrates, lipids, proteins, alkaloids.

1.2  Cacao Cultivar Systems Cacao production is estimated over four million tones around the world (Araujo et al., 2019), and 90% is produced by smallholders (Dinarti et al., 2015). There are two principal cultivar systems that have been used to make cacao: (1) shaded, which is the traditional cacao cultivar system and produces high quality product, and (2) full-sun monocultures which improves the cacao production. Both cultivar systems have benefits to the farmers, thus, to select the plan that fits better to the farmer is necessary to consider all the variables involved in the production (Pérez-Neira et al., 2020; Somarriba & López Sampson, 2018; Useche & Blare, 2013). Agroforestry is defined as “the intentional integration of trees and shrubs into crop and animal farming systems to create environmental, economic, and social benefits” (Muschler, 2016). In this context cacao might be produced using different agroforestry practices. It differs in the crop husbandry cover intensity and allows combining forestry and agricultural production on the same area. These practices are classified as follows: (1) forest-like systems in which cacao is included in the natural forest ecosystem, (2) mix shade systems in which are included various shade trees that varies in size, (3) productive shade systems, (4) specialized shade systems, (5) open sun, and (6) no-shade systems; the two latter decrease the forest cover and intensified the agricultural land use leading to a deforestation (Somarriba & López Sampson, 2018). When diverse varieties of leguminous, fruit trees, palm, and timber trees are included in the agroforestry system they provide natural nitrogen fixing to nourish the soil (Armengot et al., 2020; Muschler, 2016; Useche & Blare, 2013; Wartenberg et al., 2020; Wickramasuriya & Dunwell, 2018). The agroforestry creates right microclimate conditions such as reduction of wind speed and light availability, increment of relative humidity and buffered temperatures of the cultivar area with environmental benefits such as the nutrient cycle, pest and disease regulation, and biodiversity conservation (Wartenberg et al., 2020). Besides, it also provides a social, cultural, and economic impact to the farmers and the surrounding’s rural livelihoods; all of these benefits derived from the architecture and spatial arrangement of cacao trees that mimics the native forest and limits the land to other agricultural uses (Clough et  al., 2009; Pérez-Neira et  al., 2020; Useche & Blare, 2013).

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Agroforestry systems can offer both (1) planned biodiversity, which is associated with the crops and may vary according to farmer management; and (2) the associated biodiversity in which is included flora, fauna, and microorganisms that may colonize the land from surroundings (Useche & Blare, 2013). It is estimated that shaded cacao cultivar represents 85% of the cultivars in Latin America. The agroforestry cacao system synergizes the associated biodiversity and the planned biodiversity creating a welcoming environment and additional species. However, this practice has diminished and currently it represents only 31% of the total global cultivated area (Somarriba & López Sampson, 2018; Somarriba et al., 2012). In contrast both the increasing demand of chocolate, and the decrease in the productivity of aging trees had incentive the exploitation of land converting most of the forest into cacao farmlands (Hebbar et al., 2020). This, transformed the traditional shaded cultivar to full-sun monocultures, which have higher yields and profits than shade cultivars in a short period (Pérez-Neira et al., 2020, Useche & Blare, 2013). The implementation of full-sun monoculture has some benefits to the farmers as high yield productivity in short term (kg and/or $ ha−1), due to the modernization of the production systems and the labor inputs. However, these benefits go along with some drawbacks as the increase and dependence on non-renewable energy consumption and the loss of the genetic variability due to the replacement of local varieties for genetically uniform types (Pérez-Neira et al., 2020, Useche & Blare, 2013). This loss in genetic variability increases the vulnerability of the crop to environmental changes, weeds, pest and pathogens development, exacerbating the use of herbicides and pesticides for their management and synthetic fertilizers for nutrient deficiency (Pérez-Neira et al., 2020; Useche & Blare, 2013). The consumers’ recent environmental degradation awareness has changed their purchasing behavior and demands organic, green and sustainable products with fair trades. This market tendency indirectly has renewed the interest of producers to reinvert their time in agroforestry practices such as periodical removal of diseased pods, drainage systems maintenance and pruning of cacao trees (Armengot et al., 2020). This tendency in the market has also indirectly renewed the producers’ interest to reinvert their time in agroforestry practices by higher cacao prices that compensate for the extra labor. Therefore, the development of eco-labels, specific certifications and denomination of origin with strict environmental production standards encourage farmers to access to an alternative, distinctively and certified market niches, improving their economy besides reforesting their agricultural areas (Armengot et  al., 2020; Somarriba & López Sampson, 2018; Useche & Blare, 2013). Contrary to the farmers’ beliefs, acceptable cultivar management practices either in agroforestry or monocultures impact on the cacao yield, reducing the number of the diseased pod when a periodical removal of the diseased pods are done. Armengot et al. (2020) observed that frosty diseased pods lower than 18% were cut in the sporulation phase. These practices may avoid deforestation of the land and may increase the farmers’ environmental non-market benefits. Although the economic benefits, yet the small farmers’ have to consider several variables to choose what type of agricultural systems adapted best to their necessities. Various authors proposed and analyzed different models (Hayes, 2008; LeClair,

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2008). Nonetheless, only consumer-worker utility models were taking into account and, the relationship producer-consumer-worker were not included in the development of a decision model. These models did not consider the effect of the price premium on the producers and did not consider the ecological impact. Therefore, Useche et al. developed a farm-household model in which they considered the market benefits of the production and the associated environmental non-market benefits to the production. Useche and Blare (2013), focused on the planned biodiversity effects on household behavior, emphasizing the non-market ecological economy’s valuing the biodiversity and environmental questions. Useche and Blare (2013) model approaches consider estimating both the crash crop and the production of planned biodiversity. This model was applied to Ecuadorian cacao production observing a compensatory effect in different product markets, labor market limitations, and differences in market and shadow wages derived from the environmental benefits associated with planned biodiversity management.

1.3  Factors That Affect the Cacao Beans’ Quality Several physical and biochemical indicators are used to evaluate the quality of cacao beans: size, amount, color, acidity, amount and type of volatile compounds and polyphenols, among others (Kongor et al., 2016). These indicators are affected by environmental growth conditions (soil chemical composition, temperature, moisture, etc.), maturity grade, postharvest treatment, and chocolate manufacture processing.

1.3.1  Environmental Factors The type of cultural system influences the quality of cacao, it has been reported that soil and climatological conditions have positive or negative impacts on the cacao quality such as flavor, aroma, and biochemical composition of cacao beans, which determined the kind of market in which will be sold. The shade cacao cultivars in humid tropics contribute positively to carbon storage, nutrient cycle, and reforestation. Cacao trees grow in coarse particle soils rich in nutrients and a depth of 1.5 m, allowing them to develop a sound root system (Kongor et al., 2016). The soil structure is commonly as aggregates important on soil organic matter storage (SOM) which protects the organic matter compounds from a rapid degradation. The remaining plants, animals, and microorganisms are the principal organic matter found in soil which, is degraded gradually, releasing the nutrients to be uptake by cocoa trees (Wartenberg et  al., 2020). Besides the soil structure the balance of cationic and anionic compounds in the soil is vital to avoid nutritional problems that affect cacao quality. The cation exchange capacity (CEC)

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indicates the soil’s ability to absorb and release cations (e.g., Ca+, Mg2+, and K+). According to the International Cocoa Organization ICCO (2013), the optimal total nitrogen-total phosphorus ratio should be 1.5. It has been reported that shade cacao cultivars increase C concentrations by 6%, compared to open cultivars. Wartenberg et  al. (2020) observed that C was much higher under rambutan (3.6  g C kg−1 ± 1.1 g C kg−1) compared to other species (jackfruit, guava, mango and coconut). Similar effects were observed on N and P concentration, however the increase in C, N and P may vary depending on the type of shade tree species. In this context the soil’s water retention and drainage properties are also crucial for cacao tree growth due to it is susceptible to lack of water. Another factor to consider is the pH of the soil that influences the solubility of minerals and nutrients. A pH in a range of 5-0-7.5 is ideal for cacao tree growth (Dogbatse et al., 2020; ICCO, 2013). However, a too acid (pH  8) soil has to be avoided. A temperature rate of 31–35 °C is the optimal for photosynthesis in cocoa (Balasimha et al., 1991; Hebbar et al., 2020; Yapp, 1992); a rainfall rate between 1400- and 2000-mm year−1 is sufficient to maintain a profitable growth of cacao. Besides C, N and P soil’s concentration the carbon dioxide (CO2), rainfall, and temperature are crucial to seedling and productivity. Hebbar et al. (2020) evaluated under controlled conditions the interaction effect of the CO2, high temperatures and water deficit on growth, photosynthesis, and WUE observed that elevated CO2 concentration in the cultivars have a positive effect on the plant’s height; 550 and 700  ppm CO2 produce plants of 1.64  m and 1.71  m respectively. Similar effects were observed to photosynthesis which increased 10% at 550  ppm and 29% at 700 ppm CO2. In contrast a water deficit at 50% negatively affects the plant height, it decreased 6% and 19% at 550 and 700 ppm CO2 respectively. Besides, Dogbatse et al. (2020) studied the growth and nutrient uptake of different acidic soils in Ghana. All the grounds evaluated were sandy clay loam and were adequate to hold water, thus, maintaining the moisture needed for the plant. However, their pH was acid ranging from 4.21 to 5.66 attributable to low exchangeable cations mainly Ca, which may mitigate the toxicity cause by Al concentrations. The soils had high concentrations of both P and K, probably due to the soil’s pH (5.5) and to the high levels of clay in the soils respectively (Dogbatse et al., 2020). These findings highlight the importance of the soil characteristics such as structure and chemical composition for cacao cultivars that may enhance growth, yield, and flavor and aroma compounds development.

1.3.2  Genetic Factors The type of cultivar, climate, and the soil where cacao grows influence the cacao bean quality, genetic, and variety are also important factors to be considered (Gutiérrez, 2017). The cacao tree is a diploid specie (2n = 2x = 20), with genotypes range from 411  Mb to 494  Mb. Ten genetically differentiated populations of the cacao tree have been identified (Amelonado, Contamana, Criollo, Curaray, Guiana,

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Iquitos, Marañon, Nacional, Nanay and Purús) (Argout et al., 2017; Hämälä et al., 2020; Schwarzkopf et al., 2020; Wickramasuriya & Dunwell, 2018). All of them originating from diverse locations in Central and South America. Guiana shows lower genetic diversity among the others and presents high similarity to Marañon than either Nanay or Iquitos varieties. These ten populations are significantly different among them due to strong signatures of differentiation. Criollo and Amelonado varieties are characterized for being highly self-fertilized, contrary to Iquitos and National Ecuadorian cacao which have low self-fertilization frequency. (Schwarzkopf et  al., 2020). Although cacao is genetically diverse and more than 14,000 varieties are known; the main commercial species to manufacture chocolate are Forastero, Trinitario, and Criollo (Gutiérrez, 2017; Rojas et al., 2020; Aprotosoaie et al., 2016). The principal differences among the commercial varieties are the geographic origin, fruit morphology and flavor characteristics. Criollo (Theobroma cacao L. ssp. cacao Cuat) is cultivated since pre-Columbian times in Central America, mainly by the Mayans. Nowadays, Criollo trees are grown only in Central America, Venezuela (largest producer), Madagascar, Sri Lanka, and Samoa. Some physical characteristics of Criollo cacao are pod yellow or red when is ripe, large, rounded beans, and white-colored cotyledons. Generally, Criollo cacao is susceptible to both pest damage and climatic changes producing low yields. Criollo cacao is highly appreciated by national and international chocolatiers and the chocolate industry to manufacture fine chocolate due to its aroma and flavor characteristics. The latter may develop flavors like mild, nutty, earthy, flowery, or tea-like (Aprotosoaie et al., 2016; Gutiérrez, 2017; Gutiérrez & Pérez, 2015). Forastero (Theobroma cacao L. ssp. Shaerocarpum Cuat), is a variety cultivated in West Africa and South America. It is integrated by several subvarieties, being Amelonado the most known. The group is divided into bulk, necessary and ordinary sub-groups used to produce different cacao derived products. Bulk Forastero represents over 90% of the world production and has large genetic variability it is used for breeding over the world (Aprotosoaie et al., 2016). Trinitario is a hybrid resulted from Criollo and Forastero varieties. It is cultivated in South America, Central America and in the West Indies. Producers used Trinitario because it is less susceptible to diseases and has higher yields than the other varieties. Trinitario is characterized by a robust raw chocolate and some wine-like flavors (Aprotosoaie et al., 2016). In this context, Schwarzkopf et al., (2020) evaluated the recombination rates in distinct cacao populations. They observed an overlapping in the recombination hotspots location across the populations; moreover, their results supported the hypothesis of increased recombination rates in domesticated plants such as Criollo cacao (Schwarzkopf et al., 2020). Derived the increased demand for cacao and the sustainable production, governments have developed programs to improve cacao production through rehabilitation, intensification, and propagation by cacao clones. These programs are focused on the use of disease-resistant and high-yield clones. In this context, the chocolate industry and international organizations subsidized by the Indonesian government have implemented breeding programs to produce superior clones of cacao through

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embryogenesis. Significantly 53 farmer selections from Sulawesi were analyzed for genetic identity and parentage; finding that farmer selections are comprised of hybrids for three groups of cacao germplasm: Trinitario and two Upper Amazon Forastero groups (Dinarti et  al., 2015). In 2004 ICCO developed the program “Cocoa germplasm utilization and conservation: a global approach” in collaboration with different countries (Brazil, Cameroon, Cote D’Ivoire, Ecuador, Ghana, Malaysia, Nigeria, Papua New Guinea, Trinidad And Tobago, and Venezuela). The objectives were international hybrid selection, germplasm conservation, and enhancement particularly for pest and disease-resistant cacao varieties. The project results were the obtention of promising local clones used for breeding, and successful germplasm enhancement for Phytophthora pod rot resistance in Trinidad and Tobago.

1.3.3  Harvesting and Post Harvesting Processing 1.3.3.1  Maturity Processing is any operation that transforms agricultural products into a commercial item. During chocolate production, cacao goes through a multi-step process (Fig. 1.2) including harvesting, beans separation from the pods, fermentation, and sometimes roasting, contributing to the development of flavor and aroma of cacao beans. (Gutiérrez, 2017; Tee et al., 2019; Gutiérrez & Pérez, 2015). A cacao pod too ripe may increase the risk of rotting and seed germination, while a pod too unripe may negatively affect the fermentation process. Therefore, to achieve premium cacao flavor from the beans, the pods have to be harvested at the right time. The maturity stage of cacao is determined by its external pod coloration which, varies according to the genotypes. Thus, coloration is a decisive criterion to taking account by experienced farmers during harvesting to identify the mature cacao. Unripe pods are green, while full ripe pods present different tones. Fully ripe pods may be colored in yellow tones (citrine), orange (amber), red (ruby), violet (amethyst) while, some others might stay in green tones (Gutiérrez, 2017, Tee et  al., 2019, Gutiérrez & Pérez, 2015). In practice, the harvest of cacao is a non-standardized process done about 5  months after the emerging of the pod and coloration changes. However, if the harvested pods are yet in the ripe stage of maturity, it may conduct an over-fermentation; firstly, an anaerobic fermentation inside the pod and aerobic fermentation once the beans are extracted from the pod. This over-fermentation causes excessive proteolysis damaging the precursors of flavor. The quantification of the secondary metabolites responsible for pigmentation may be a rapid and non-destructive method helpful for cacao pods maturity determination; however, it may be laborious and costly to be done by farmers (Tee et al., 2019). Some researchers have sought techniques that allow establishing homogeneous and standardized ripeness indicators to harvest cacao. Rojas et  al. (2020)

1  State-of-the-Art Chocolate Manufacture Fig. 1.2 General chocolate making process

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evaluated physicochemical parameters in three clones cultivated in different Colombia zones to identify potential indicators of cacao maturity. The fruit shape, size, weight, color, moisture, pH, total soluble solids, and titratable acidity. Results point out that as the degree of ripeness progresses, groove depth and apex width are higher; the coloration intensity increased, and total soluble solids increased. While moisture and pH decrease. Each of the clones evaluated presented differences in the parameter through the ripeness process, meaning that ripeness indicators have to be implemented for each cacao variety. On the other hand, Tee et al. (2019) explored a multiparametric fluorescence sensor to estimate some of these metabolites, such as anthocyanin, flavonol, chlorophyll, and nitrogen, during 5 months across the pod development to determine the optimum harvest period. The non-destructive fluorescence method consisted of scanning the pods with a fluorimeter with six light-emitting diode sources in the UV-A (370  nm) with blue (470  nm), green (516  nm), and red (635  nm) spectral regions. The results showed that flavonols are accumulated in the pods as they developed, contrary to chlorophyll and nitrogen balance, which decrease as they reach maturity. They conclude that both pods and beans harvested at 4 months present quality as good as those harvested at 5 months (Tee et al., 2019). Despite the ripeness stage is critical to harvest cacao high quality, there is no a standardized method that enables farmers to know the exact time to harvest the fruit. Therefore, for developing a technique to identify the optimal maturity stage of cacao, more studies are needed. 1.3.3.2  Fermentation Process Raw beans are naturally astringent and bitter in an unpleasant way, the fermentation process lead to biochemical changes necessary for flavor and aroma precursors formation that contribute to the chocolate flavor profile (Santos et  al., 2020). Fermentation begins since the selected pods are manually open to remove cacao beans. This process has to be precise to avoid damaging the beans (Aprotosoaie et  al., 2016; Gutiérrez, 2017; Rivera-Fernández et  al., 2012; Nazaruddin et  al., 2006). During fermentation, the cocoa beans undergo physical changes such as the loss of mucilage and their soft and compact texture (Gutiérrez & Pérez, 2015). In practice, the fermentation is an artisanal and non-controlled process; beans are placed in wooden boxes, sacks, baskets, trays, heaps and plastic containers and subjected to different conditions, which may increase the non-volatile acidity, affecting the quality of the beans (Aprotosoaie et al., 2016; Rivera-Fernández et al., 2012). The length of fermentation may vary depending on the cacao variety, Criollo require 2 or 3 days to fully fermented, while to the Forastero it may take 5–8 days (Rivera-Fernández et al., 2012). The degree of fermentation is measured comparing the color of cut beans to the Munsell color chart. Variations on the fermentation conditions affect the pH, titratable acidity, temperature, and enzymatic activity, leading to biochemical changes in phenolics, alkaloids, and nitrogenous compounds

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influencing the flavor and aroma development (Rivera-Fernández et  al., 2012, Nazaruddin et al., 2006). The mucilaginous pulp (40% of the total fresh weight) that surrounds the cacao beans is the first one to be subjected to the biochemical changes during fermentation due to its richness in glucose, fructose, sucrose, salts, pectin, organic acids, and proteins, hydrolyzed by microorganisms during fermentation. The enzymatic activity varies among the cacao genotypes. Genotypes with high endoprotease and aminopeptidase activity produce better cacao flavor. The pH also influences the enzyme activity; some of the enzymes involved in fermentation are inactivated at a too acid pH, reducing the flavor precursors production. The associated microbial community of cacao beans fermentation is integrated by yeast, bacteria (lactic, acetic, and Bacillus species), and filamentous fungi. Moreover, the microbial activity determines some structural changes promoting the cell constituents (substrates and enzymes) movement (Aprotosoaie et  al., 2016; Santos et  al., 2020; Leal Jr et al., 2008). At the first stage of the fermentation process, pulp sugars are hydrolyzed and transformed into ethanol and lactate by yeast and lactic bacteria. However, it is relevant to consider the pulp fraction since an excess of it may decrease the oxygen diffusion, thus, extending the fermentation time, increasing the lactate and ethanol production, and the acidity. Constant removal of the beans during fermentation or partial removal of pulp may reduce this unfavorable effect. Afterward, the second phase of fermentation begins (48–96  h), the aeration increasing, yeast activity is inhibited, and lactic and acetic bacteria are established. All of the biochemical changes performed by microorganisms provoke the loss of membrane permeability, causing the cotyledons’ death (Aprotosoaie et al., 2016; Gutiérrez & Pérez, 2015; Leal Jr et al., 2008). At the final stage of fermentation, the substrates are entirely consumed thus, the production of acetic acid ceases increasing the pH up to 5, leading to protein total degradation by endogenous proteases (Aprotosoaie et al., 2016; Leal Jr et al., 2008). The cacao protein composition contains albumin (14–52%) and vicilin (7S)- class globulin (23–43%), which start their hydrolysis after 2-days of fermentation beginning. Albumin is partially hydrolyzed, close to 57% whereas, vicilin (7S)- class globulin is highly hydrolyzed ~90% by aspartic endoprotease and carboxypeptidase. Thus, hydrophobic amino acids (leucine, alanine, phenylalanine, and tyrosine) involved in aroma precursors formation are released (Hue et al., 2016). Regarding cacao bioactive compounds (Fig. 1.3) three groups are distinguished: (1) flavan-3-ols also known as catechins family (ca. 37%) comprised by (−)-epicatechin, (−)-catechin, (−)-epigallocatechin, (+)-gallocatechin, (+)-epicatechin and (+)-catechin; (2) proanthocyanidins (ca. 58%), comprised by condensed dimers, trimers or oligomers of flavan-3,4-diols being epicatechin the main extension, and (3) anthocyanins (ca.4%), formed by 3-β-D galactosidyl cyanidin and 3-α-L arabinosidyl cyaniding (Gültekin-Özgüven et al., 2016; Lemarcq et al., 2020; Nazaruddin et al., 2006; Rusconi & Conti, 2010; Schinella et al., 2010; Valadez-Carmona et al., 2017). During fermentation, catechins are oxidized by polyphenol oxidase, and their amount is reduced up to 10–20% higher values are considered a sign of non-­ adequate fermentation. The oxidation of catechins turned the violet cotyledons into

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Fig. 1.3  Principal bioactive compounds of cacao beans

the brown characteristic color of chocolate. Proanthocyanidins content decrease 3 to 5 times while anthocyanidins disappear. During the aerobic phase of fermentation, polyphenols are condensed into high molecular insoluble tannins, protein –polyphenol complexes are oxidized. Carbonyl-amino complexes are concentrated reducing the astringency, while alkaloids promote the bitterness development (Gültekin-­ Özgüven et  al., 2016; Nazaruddin et  al., 2006; Valadez-Carmona et  al., 2017; Plazola-Jacinto et al., 2019). The pH, temperature and oxygen diffusion contribute to microbial succession throughout the fermentation process. Acidy pH and richness of sugar content allow yeast growing during the first stage. Yeast is the responsible of ethanol production due to pectinolytic enzyme activity. An increase in temperature and aeration contributes to the yeast replacement by lactic acid bacteria (LAB). LAB oxidizes the ethanol to lactic acid, afterwards acetic acid bacteria (AAB) become the dominating microbiota in the fermenting mass and oxidizes the acid lactic to acetic acid. At the end of fermentation spore-forming bacteria growth along filamentous fungi appears on the bean’s surfaces. Some of the microorganisms identified from cacao fermentation are: Kluyveromyces marxianus, Saccharomyces cerevisiae var. chevalieri, Candida rugopelliculosa, and Kluyveromyces thermotolerans, Hanseniaspora guilliermondii, Pichia kudriavzevii, Lactobacillus plantarum, Lactobacillus fermentum, Acetobacter pasteurianus and Gluconobacter frateurii. The sequence of the microorganisms’ appearance is not well defined yet, it may act simultaneously during fermentation process and even being different depending of the region of origin (Gutiérrez, 2017). The general activities and composition of each group of microorganisms involved in cacao beans fermentation and responsible for aroma and flavor precursors production are: Yeast  The anaerobic stage of fermentation is conducted by yeast hydrolyzing sugars to produce alcohol and citric acid decomposition to increase pH from 3.5 to 4.2. Some of the yeast involved in cacao bean fermentation process are Saccharomyces cerevisiae, Hanseniaspora guilliermondii, H. uvarum, Pichia kluyveri, P. membra-

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nifaciens, P. fermentans, I. orientalis, Candida torulopsis, C. silvae, C. zemplinina, C. diversa, C. stellimalicola, and Schizosaccharomyces spp. Lactobacilli  This group of bacteria acts in the second stage of the fermentation when increase the aeration, the LAB activity is not limited to produce lactic acid by metabolizing glucose they also participate in the assimilation of citric acid to rise pH.  Little LAB has identified from cacao beans fermentation, some of them are Lactobacillus, Leuconostoc, Lactococcus, and Pediococcus genera Lactobacillus collinoides, L. mal’ı, L. hilgardii, L. fermentum, and L. plantarum species. Acetic Acid Bacteria  The AAB transforms the ethanol produced by yeast into acetic acid during aerobic stage, this group of microorganism acts after increase in temperature and aeration. Some species found during cacao fermentation are: Acetobacter pasteurianus, A. aceti, A. syzygii, A. tropicalis, A. malorum, Gluconobacter oxydans, and G. xylinus. However, these species have shown insufficient activity in natural conditions. Thus, an artificial inoculum of yeast with pectinolytic enzymes would be an option when cacao beans are rich in pulp to small or large-scale process (Leal Jr et al., 2008). Under laboratory scale S. cerevisiae var. chevalieri and Kluyveromyces fragilis have shown good pectinolytic activity improving fermentation and final product quality. Therefore, to improve the cacao beans fermentation, the use of specific strains has been evaluated. Kluyveromyces marxianus hybrid strain with high pectinolytic activity was inoculated at a rate equivalent to 2.6 × 106 cell kg−1 of cacao beans (Aprotosoaie et al., 2016; Camu et al., 2008; Hansen et al., 2000). The fermentation with K. marxianus showed better sensorial attributes, less acidity, more degradation of proteins and beans with appropriate brown color than naturally fermented beans. Sande Santos evaluated the effect of different strains on cacao Scavina fermentation, finding that Candida parapsilosis, Torulaspora delbrueckii, and Pichia kluyveri are the ones with relevant activity on flavor precursors production. However, along with the cacao processing in temperate and humid conditions, fungi may proliferate and produce several secondary metabolites such as mycotoxins that contaminate the cacao beans or their derived products (chocolates or powders) (Akinfala et al., 2020; Copetti et al., 2013). Ochratoxin A (OTA) is one of the mycotoxins detected in cacao derived products. OTA is produced by Aspergillus carbonarius and Aspergillus niger, and Penicillium fungi genera. OTA exhibits immunotoxicity, neurotoxicity, and teratogenic effects that affect kidney and liver health (Brera et al., 2011; Copetti et al., 2013; Mishra et al., 2015). In 2001, the joint FAO/WHO Expert Committee on Food Additives (JECFA 2001) established the tolerable maximum limit intake at 100  ng/kg body weight. In 2003 the Italian Ministry of Health claims a maximum limit for OTA in cacao powder and cacao derived products at 0.5 μg/kg and 2 μg/kg respectively, contrary to the European Union that declares cacao-derived products as non-significant contributors of OTA intake. Brera et  al. (2011), evaluated 300 samples of cacao and chocolate-based

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products for sale in the Italian market, finding that 179 were positive for OTA presence. Still, the content was below the Italian limits. Since the results suggested that OTA exposure through cacao-derived products consumption is not a significant health concern the Italian Superior Council of Health decided to adopt the European legislation. On the other hand, Akinfala et al. (2020) assessed the fungal profile and fungal metabolites patterns of Nigerian cacao beans. Citrinin mycotoxin was detected on fermented beans, and unexpectedly OTA was not detected. It is the first time that citrinin was detected in fermented beans, and it is a health concern due to as same as OTA is also a potent nephrotoxin. More than 60% of cacao-derived products present OTA at different levels, consequently these products may not be exported or inclusive may be rejected if exceeds the permissible limits established in legislations. Thus, it is necessary the analysis for OTA during all the processing stages of cacao beans and cacao derived products (Mishra et al., 2015). The current analytical techniques, used to detect OTA are high performance liquid chromatography (HPLC) adapted with fluorescence detectors, gas chromatography-mass spectrometry, enzyme linked immunosorbent assay (ELISA), and thin layer chromatography (TLC). However, OTA on-site detection is difficult to perform by farmers or small producers. In this context, a new technique for OTA detection was developed by Mishra. The use of an electrochemical impedimetric aptasensor allowed a rapid detection and quantification for OTA. The aptasensor showed 0.15 ng/mL as the limit of detection (LOD) besides good selectivity and reproducibility. Therefore, a suitable and sensitive analytical technique selection joint of good sampling may facilitate the OTA detection in all process stages of cacao. 1.3.3.3  Drying After fermentation, the cocoa beans must be dried to (1) reduce the moisture extending the shelf life, (2) avoid the spoiling, (3) complete the oxidation began in fermentation, and (4) facilitate their transport to industry. The oxidation of polyphenols is achieved during drying; polyphenols are oxidized to quinones which condensates with free amino acids and sulfhydryl groups developing brown polymers. After drying, the cacao beans are dried about 7 days to reduce moisture by 7–8%, and the water activity must be below 0.7 to avoid spoiling during storage. The drying process should be slow to reduce astringency, bitterness and, acidity; too fast drying generates beans highly acid (Gültekin-Özgüven et  al., 2016; Gutiérrez & Pérez, 2015). Sun-dried is the traditional method to dry cacao beans because it develops a more marked chocolate flavor. However, it sundried takes a long time, low production rate, and may cause environmental contamination. Therefore, to avoid contamination artificial drying with acceptable manufacturing practices must be implemented. Therefore, hot air drying is an optional method that avoid exposition of environmental conditions, whereas the drying temperature is controlled and standardized.

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Once the drying process ends cacao beans are cleaned, selected and kept in sacks around 60–65  kg in a ventilated warehouse, then transported to continue the processing.

1.4  Chocolate Manufacture and Factors Affecting Its Quality 1.4.1  Roasting Roasting process goals are (1) to reduce the moisture content (  0.05). However, with an S. cerevisiae inoculum, the lowest total theobromine was obtained in fermented cocoa. The low content of theobromine indicates that theobromine degradation occurs more frequently during the alcoholic fermentation stage (Sandhya et al., 2016). One possible explanation is the presence of fungi capable of degrading theobromine, such as those found in cocoa husk fermentation (Adamafio et al., 2012). The use of various inoculants during the cocoa fermentation process resulted in different FTIR profiles, particularly in the identification zone, which was observed between 1500 and d. 600  cm−1. Additionally, Rahmadi et  al. (2020) discuss the detection peaks observed in the FTIR identification zone, which are thought to originate from the bonds of compounds such as RCOO, -OH, OCOCH3, SO2, NO2, COO-, ester, and PO- bonds, as well as benzene, POOH, C-Cl (alkyl), and CH=CH. The difference in percent transmitance (%T) in the identification zone of the FTIR spectrum of small-scale fermented cocoa after normalization to the %T value of two FTIR spectra of commercial samples taken from PTPN XIII is shown in Fig.  2.2. The consistency of observations of cocoa fermentation results using S. cerevisiae and A. aceti inoculants was determined using these two spectrograms (Fig. 2.3). Cocoa contains not only theobromine but also significant amounts of caffeine (Batista et  al., 2016). Changes in fermented cocoa relative to caffeine standards must be monitored, particularly in the FTIR identification zone between 600 and 1500 cm−1 (Fig. 2.4). Caffeine identification peaks were discovered at the following wavenumbers: 1483, 1454, 1431, 1402, 1358, 1325, 1285, 1236, 1188, 1072, 1026, 972, 928, 862, 800, 743, 698, 642, and 611 cm−1. The identification peaks can be ascribed to vibrations of covalent bonds composed of = C=N- (conjugated, cyclic), =N-N=O, -CH3, =CH2, C-N=O, -OH, =C-O-C-, -CH=CH2, three neighboring aromatic C-H, and ethylene (Rahmadi et al., 2019).

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As with the theobromine functional groups, the absorbance value of the FTIR spectrum in the caffeine identification zone was compared to the absorbance of fermented and commercial Cocoa FTIR spectra. Compared to fermented cocoa used in this study, industrial cocoa had the most significant absorbance difference in caffeine value. A mixed culture of A. aceti and S. cerevisiae in the fermentation process results in cocoa’s difference in caffeine absorbance that is most similar to commercial cocoa. However, the quantification of caffeine levels in fermented cocoa using an induction inoculum requires confirmation (Fig. 2.4).

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2.5  Controls on the Taste and Quality of Cocoa Beans 2.5.1  Plant Cultivars More than 14,000 varieties of T. cacao have been identified with significant genetic diversity (McShea et  al., 2008). However, the main varieties of cocoa species

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Table 2.7  Summary of flavor compounds in cocoa bean products Compound class Alkaloid Polyphenols/ flavonoids Amino acids and peptides

Volatiles Minute compounds

Major constituents Methylxanthines, i.e., theobromine, caffeine Epicatechin and pro-anthocyanidins

Example of flavor/taste Bitterness (theobromine, caffeine), slight alkaline and soapy (caffeine) Bitterness, astringency

Alanine, valine, leucine, phenylalanine, tyrosine

Sweet (alanine), mixed sweet and bitter (valine, phenylalanine), bitter (tyrosine), extremely bitter and unpleasant odor (leucine) Pyrazines, esters, alcohol, phenols, Specific flavors (floral, fruity, herbal, acids nutty, bitter, sweet, acid, etc.) After taste and pleasantness, astringency Aldehydes, ketones, lactones, enhances the specific flavor (saltiness, terpenoids, mineral, glycated and polymeric substances, contaminants bitter, umami, etc.)

commonly grown commercially in large plantations and used as industrial raw materials are Forastero, Criollo, Trinitario, and Nacional (Giacometti et al., 2015). The Forastero group has a strong chocolate base flavor and is classified as bulk, base, or ordinary cocoa. Forastero cocoa, or bulk cocoa with over 90% of world cocoa production, is used for cocoa mass, cocoa powder, cocoa butter, and milk/ dark chocolate (Fowler, 2009). The second variety, Criollo (Theobroma cacao L. ssp. cacao Cuat.), is now rare due to poor tolerance to insects and pests. Criollo cocoa is highly aromatic and with distinctive mild, nutty, earthy, flowery, or tea-like flavors (Ziegleder, 1990). Venezuela is the largest producer of Criollo cocoa (Jahurul et al., 2013). Trinitario is a hybrid of the Criollo and Forastero varieties with the advantage of being more resistant to disease than other varieties (Jahurul et  al., 2013). This variety presents a strong chocolate base character and a distinctive wine-like taste (Giacometti et  al., 2015). The Nacional variety is grown only in Ecuador. The Nacional variety cocoa has the characteristic Arriba flavor with a floral, spicy, and green aroma (Afoakwa et al., 2008).

2.5.2  Initial Starter During the Fermentation Stage The final quality of the bean in traditional fermentation is determined by the initial microflora transferred from cacao pods, equipment, and fermentation boxes (Schwan, 1998). Thus, preventive measures to minimize natural spoilage can include cleaning workers’ hands, equipment, and the cocoa pod splitting environment and removing unwanted plant parts and soils from fermentation boxes (Minifie, 1980; Schwan, 1998). Insufficiently fermented cocoa beans are identified by their purple or slaty color, bitter and astringent flavor, and lack of chocolate flavor. Slaty and purple beans occur due to insufficient mixing and drying of the beans before the fermentation

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process is completed. Thus, it is critical to transfer cocoa beans from batch to batch repeatedly over a defined period (e.g., 24 h) to maintain optimal fermentation conditions (Ardhana & Fleet, 2003).

2.5.3  Drying Open-air (solar) drying, by far the most popular method for cocoa bean drying, has a prolonged moisture removal rate and is prone to dust and insect contamination. However, mechanical drying is not economically viable in small-scale plantations due to the high capital and operating costs (McDoom et al., 1999). The other drying system parameter is that the hot air temperature should not exceed 60 °C; otherwise, the bean will brown excessively and develop a caramel flavor (Lynch, 1992). A multistage solar drying system was introduced, which allows for a 40%–50% reduction in drying time without compromising cocoa quality (Amir et al., 1991). Another approach, which utilizes forced air circulation and a solar energy-based heater, can reduce moisture levels from 98.8 to 8% within 18 h (McDoom et al., 1999), effectively eliminating the possibility of microbial growth. 2.5.3.1  Drying Controls 1. Humidity level Because most cocoa bean producers are in tropical countries with high humidity, the drying process becomes critical for producing high-quality beans and preventing mold growth (Amir et al., 1991). However, the moisture content limit for cocoa beans is around 7%. (Deus et al., 2018). Therefore, excessive drying can result in brittle shells, whereas excessive moisture promotes fungal growth. 2. Activation of enzymes Peroxidase activity significantly increases during the drying stage. Excessive peroxidase production is induced during the bean’s biosynthesis by stress (Kristensen & Rasmussen, 1996). Regardless of their loss of germination capability, fermented beans retain the ability to produce the enzyme. Active peroxidase can alter the bean’s quality during storage, notably bitterness and astringency (Sakharov & Ardila, 1999). As a result, it was suggested that an additional threeday incubation at 30 °C would inactivate the enzyme.

2.5.4  Storage and Mixing The critical moisture content is 8%, while the optimal moisture content for fermented bean storage is between 6–6.5% (Minifie, 1980) Off-flavor development can occur during the shipment of cacao products from various climatic regions,

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depending on the packaging. While cocoa beans lose relative humidity during pallet packaging but do not develop noticeable off-flavors, they may develop noticeable off-flavors during bulk container transportation due to the increasing relative humidity (Sharp, 1979). Additionally, fermented cocoa is classified according to its flavor intensity, with the Java (Indonesia) cocoa bean classified as mild. The industry blends species and varieties according to established formulae to create high-quality chocolate (Minifie, 1999).

2.5.5  Roasting Pyrazines are formed during fermentation and give the cocoa bean its distinctive flavor (Castro-Alayo et al., 2019). The unique cocoa aroma (alcohols, carboxylic acids, aldehydes, ketones, esters, and pyrazines) results from complex biochemical and chemical reactions occurring during the post-harvest processing of raw beans, as well as from a variety of factors such as the cocoa genotype, chemical composition of raw seeds, environmental conditions, farming practices, processing, and manufacturing stages (Aprotosoaie et al., 2016). Heat affects the pyrazine content, primarily via the Maillard reaction’s mechanism (de Brito et al., 2001). As a result of roasting, the concentrations of sugars and amino acids are reduced (Mermet et al., 1992). The leading indicators of roasted cocoa beans are tetramethyl-pyrazine and 2,5-dimethyl-pyrazine (van der Wal et al., 1971). These distinctive compounds are produced during a 20–30 min heat treatment at 150 °C. Apart from improving the flavor, roasting is intended to alter the color of the bean. Maillard reactions, polyphenolic oxidation and polymerization, protein degradation, and dextrin formation from starch vary the pigments (Minifie, 1980; Haslam, 1982; Krysiak, 2006). The final shade of cocoa is primarily determined by the ratio of anthocyanins to hydro-cinnamic acid derivatives (Serra-Bonvehi & Coll, 1997; Krysiak, 2006). 2.5.5.1  Controls That Are Critical During Roasting Excessive roasting may result in burnt odors and flavor loss. Thus, the roasting process is highly dependent on the time and temperature combination. At 135–150 °C for 15–45 min, the optimal and desired color and flavor formation occurs (Krysiak, 2006). However, in secondary toxic metabolites produced by mycotoxigenic molds, this time and temperature combination is insufficient to render the toxins inactive (Tran-Dinh et al., 1999).

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2.5.6  Alkalization Before alkalization, in the winnowing stage, the roasted cocoa beans are then separated from their shells. Subsequently, cocoa nibs, cocoa mass, or cocoa liquor are mixed with food-grade alkaline, natural cocoa, or baking powder, where the polyphenols are polymerized to reduce astringency and darken the cocoa’s color, and reduce the bitter-sour taste. Another objective of cacao alkalization is to increase the solubility of cocoa mass or cocoa nibs in solution (Giacometti et  al., 2015). Historically, this mixing process has been called Dutching (Nair Prabhakaran, 2010). 2.5.6.1  Controls That Are Critical During Alkalization Heat treatment often involves the alkalization of cocoa powder, suggesting a degree of protein denaturation and irreversible starch modification leading to off-flavor. Also, alkalization may increase difficulty in producing cocoa liquor or finer material during the grinding stage. Therefore, finding the sweet spot between heat temperature and time during alkalization leads to a more advantageous characteristic of cocoa flavor and color (Dyer, 2003).

2.5.7  Conching Conching is a long-term heat treatment that aims to soften the taste and texture of chocolate (McShea et al., 2008). Overall, off-flavors from cocoa nibs, cocoa mass, and cocoa liquor were reduced after going through the conching stage (Afoakwa et al., 2008). The heating temperature for conching to occur begins at 40 °C (Torres-­ Moreno et al., 2012). However, conching is usually carried out at 70 °C or higher (Afoakwa et al., 2008). 2.5.7.1  Controls That Are Critical During Conching Successful conching depends on consistent temperature and stirring. Factors affecting the final chocolate quality in the conching stage are temperature, agitation, duration, and aeration. When mixed, the chocolate core material exerts shear thickening properties. Therefore three stages of conching may be required that are producing thin film made of cocoa fat, removal of moisture, and liquefaction and homogenization of chocolate paste (Gutierrez, 2017).

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2.6  Cocoa Mass Flavor Cocoa mass typically comprises powder containing 15–35% of fat. The main flavor components of cocoa powder are esters, pyrazines, alcohols, carboxylic acids, ketones, and aldehydes  (Table  2.7). These compounds are mainly formed during roasting because of carbohydrate and protein degradations, glycations, and polymerizations. Tetra-methyl pyrazine, isobutyl benzoate, and linalool are major aromatic constituents in cocoa mass (Alasti et al., 2019). Regardless of lowering acidic and astringency, the intense Dutching process may increase bitterness in the final cocoa mass product. Therefore, each cocoa processing house has a specialty in the Dutching process to obtain the cocoa mass’s intended final taste and aroma (Table 2.8).

2.7  Cocoa Butter Flavor Cocoa butter’s flavor is determined by the protein and fat content of the cocoa powder. The cocoa bean’s quality, the region in which it is grown, the cultivar of the cocoa plant, and the post-harvest technology all affect the taste of cocoa butter. Due to the nature of the plants, which are more resistant to diseases and pests, criollo cultivars are generally more widely developed worldwide. Cocoa powder derived from the Criollo cultivar contains approximately 30–40% protein and 46–56% fat. Among the numerous physical and chemical properties, the refractive index, melting point, iodine number, saponification number, and composition of fatty acids are critical for the food, pharmaceutical, and cosmetic industries. Table 2.9 summarizes the significant characteristics of brown fat. Brown fat’s most distinctive rheological profile is its melting characteristics, where it tends to be solid (set), complex (hard), and brittle at temperatures less than 27 °C (brittle). On the other hand, Brown fat melts readily at temperatures between 33 and 37 °C (Lipp et al., 2001).

2.7.1  Composition of Fatty Acids Brown fat is primarily made up of the saturated fatty acids of palmitic (C16) and stearic (C18). When the two are combined, they can account for up to 90% of the total fatty acids in cocoa butter (CB). Additionally, double-chain fatty acids such as oleic (C18:1) and linoleic (C18:2) are present in significant amounts in cocoa butter, accounting for up to 39% of the total fatty acids. Chocolate is considered a healthy food due to its balanced composition, which is unique in the food processing industry. The following table summarizes the fatty acid composition of cocoa butter and cocoa butter equivalents (CBE).

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Table 2.8  Summary of flavor development in cocoa bean products Factor and process responsible for flavour development Plant cultivars/ Innate characteristics genotype and quality of seeds/ plant Farming Growth of the plant, soil nutrition and contaminant uptake, integrated pest management, fruit development, and maturity Post Pod storage, pod harvesting opening, pulp collection Fermentation Successive microbial growth and enzymatic pulp and bean degradation depends on initial or starter microbes Drying

Heating by sunlight/ mechanical dryers, initial fermentation termination, drainage of soluble components

Major compound formation and Mechanism of flavor development breakdown Gene up/downregulation of flavor Alkaloids, components polyphenols, amino acids Climate and soil condition affects Sugar, alkaloids, polyphenols, amino micronutrient uptakes and enhances gene up/downregulation acids, mineral, and contaminant of flavor components uptakes

Initial microbial contamination, leading to the slight difference in successive microbial fermentation Enzymatic processes and secondary metabolites give a characteristic to flavor development

Sugar, alkaloid, polyphenol, amino acids, microbial mass, secondary metabolites, alcohols, acidic volatiles Glycated amino Non-heat tolerance chemical acids, acidic and breakdown terminations, initial Maillard reaction (polymerization, alcoholic soluble compounds, glycation, sugar arrangement), peptides acidic and alcoholic soluble component drainage, further secondary metabolite formations, and heat-tolerance enzymatic processes (continued)

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Table 2.8 (continued) Factor and process responsible for flavour development Roasting Stirred heat treatment at high temperature and final fermentation termination Alkalization Originally recognized as Dutching, alkaline treatment of cocoa mass or liquor Blending Combining to produce consistent flavor Milling Separation of cocoa mass and liquor Conching Heat treatment combined with mixing, agitating, aerating of liquid chocolate Pressing Separation of cocoa butter and cocoa powder

Major compound formation and Mechanism of flavor development breakdown The enhanced degree of Maillard Alkaloids, reaction, destruction of nutrients, polyphenols, amino acids, polymeric evaporation, and conversion of substances, volatile acids volatiles, and Acidic and astringency flavor minute compounds reductions, alkaloid and polyphenolic component degradation, flavor dispersal The physical mechanism of flavor mixing The physical mechanism of flavor separation Stability improvement of characteristic flavor by heated treatment, reduction, and conversion of acids and volatile compounds The physical mechanism of flavor separation

Triacylglycerol (TAG) is mostly symmetrical in brown fat. This symmetry refers to the fact that the fatty acid groups on the first and third chains of glycerol are identical. The most prevalent symmetrical composition found in brown fat was palmitate-­ oleate-­ palmitate (POP), followed by stearate-oleate-stearate (SOS) and palmitate-linoleate-palmitate (PLP). The brown fat composition with the most asymmetrical composition was palmitate-oleate-stearate (POS), followed by other combinations of palmitate, oleic, linoleic, and stearate (Table 2.3). The composition of TAG has a significant effect on the crystallization pattern of brown fat, which can have a glossy appearance under certain conditions (Lipp et al., 2001).

2.7.2  Flavor Release and Crystallization of Chocolate Fat Chocolate-fat products are widely used in the confectionery industry, which manufactures various candies. Additionally, brown fat is used as a raw material for premium ice cream, imparts a glossy finish to bakery products, and is combined with other natural materials to create various dairy products. The crystallization characteristics of brown fat dictate the final product’s appearance, glossy, dense, or full. Brown fat exhibits three distinct crystallization characteristics: temper, feather, and individual. Generally, the appearance of the temper

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crystallization characteristic is preferred over the other two. Three crystal types form at the same temperature range, 26 °C, but have significantly different melting points. The tempered crystal melts at 33.4 °C and appears as tiny dots generally evenly distributed under a microscope with a magnification of 120 times. Individual crystals melt and exhibit the characteristics of dense crystals and less dense crystals. They appear as lines of varying sizes with a distribution that goes under the microscope. Individual crystals melt at a higher temperature than feather crystals (approximately 32.4–35.1  °C), with characteristic crystals that appear elongated, feather-like, and dense but can be seen in irregular grooves (Dimick & Manning, 1987). The fatty acid composition of TAG dictates the crystal pattern that forms, with symmetrical fatty acids (such as POP and SOS) forming tempered and feather crystal patterns. For this temper and feather crystal pattern formation, the ideal fatty acid composition is 14.5–15.2% POP, 45.5–49.5% POS, and 27.5–29.0% SOS (Dimick & Manning, 1987) (Table 2.10).

2.8  Conclusion The cocoa flavor depends on several aspects from farm to table. Compared to other fermented foods, cocoa fermentation is an ad hoc/spontaneous process that varies significantly across the globe. The process of producing high-quality cocoa beans begins at the very beginning with cocoa plantations. The quality assurance starts from cocoa pods grown from certified seeds in well-managed plantations. Additionally, the post-harvest processing of raw beans may affect the taste, as do the numerous influences of cocoa genotype, raw bean chemical composition, environmental conditions, agricultural practices, processing, and manufacturing stages. This is an essential factor to consider and implement properly to maintain the quality of cocoa beans. In post-harvest, three factors determine the prime quality of cocoa: healthy cocoa pods, successful fermentation, and rapid and precise drying. Fermentation, roasting, alkalization, and conching are critical steps in the flavor Table 2.9  Physical and chemical characteristics of brown fat on cultivar Criollo Composition Total fat content (g/100 g cocoa beans) Refractive index (ND40) Melting point (°C) Iodine number (g I2/100 g brown fat) Saponification number (mg KOH/g brown fat)

Score 46.08–56.42 1.455–1.457 34.5–36 32.5–34.73 193.02–195.89

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Table 2.10  Composition of fatty acids in cocoa butter (CB) and cocoa butter equivalent (CBE) Fatty acid composition C14:0 C16:0 C18:0 C18:1 (trans) C18:1 (cis) C18:2 C20:0

CB (%/100 g) 0–0.09 24.78–26.91 32.86–37.68 n/d 32.70–37.08 1.09–3.36 0.82–1.10

Commercial CBE (%/100 g) 0–0.79 18.31–58.79 5.45–44.31 0.00–2.41 31.49–35.60 0.71–3.77 0.36–1.64

Lipp et al. (2001). n/d = not detected

development of cocoa beans because the aroma of cocoa is determined by volatile compounds such as pyrazines and aldehydes formed during the roasting of beans from the aroma of precursors produced in the beans via safe enzymatic reactions and heat-related reactions. The lengthy chain of cacao products from farmers to processing companies also requires government attention and strengthened regulations to improve cocoa’s quality.

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temperature on volatile compounds in cocoa. Food Chemistry, 132(1), 277–288. https://doi. org/10.1016/j.foodchem.2011.10.078 Rodriguez-Campos, J., Escalona-Buendía, H. B., Orozco-Avila, I., Lugo-Cervantes, E., & Jaramillo-­ Flores, M. E. (2011). Dynamics of volatile and non-volatile compounds in cocoa (Theobroma cacao L.) during fermentation and drying processes using principal components analysis. Food Research International, 44(1), 250–258. https://doi.org/10.1016/j.foodres.2010.10.028 Roelofsen, P.  A. (1958). Fermentation, drying, and storage of cacao bean. Advances in Food Research, 8, 225–296. Rohan, T. A. (1963). Processing of raw cocoa for the market. Food and Agriculture Organization of the United Nation. Saheeda, M., Sukhab, D., & Aveena, R. (2017). Comparison of the drying behavior of fermented cocoa (Theobroma cacao L.) beans dried in a cocoa house, greenhouse and mechanical oven. In Proceedings of the international symposium on cocoa research (ISCR) (pp. 13–17). Sakharov, I. Y., & Ardila, G. B. (1999). Variations of peroxidase activity in cocoa (Theobroma cacao L.) bean during their ripening, fermentation and drying. Food Chemistry, 65, 51–54. Samah, O. A., Ibrahim, N., Alimon, H., & Abdul-Karim, M. I. (1993). Fermentation studies of stored cocoa bean. World Journal of Microbiology and Biotechnology, 9, 603–604. Sandhya, M.  V. S., Yallappa, B.  S., Varadaraj, M.  C., Puranaik, J., Rao, L.  J., Janardhan, P., & Murthy, P.  S. (2016). Inoculum of the starter consortia and interactive metabolic process in enhancing quality of cocoa bean (Theobroma cocoa) fermentation. LWT-Food Science and Technology, 65, 731–738. https://doi.org/10.1016/j.lwt.2015.09.002 Schwan, R. F. (1998). Cocoa fermentations conducted with a define coctail inoculum. Applied and Environmental Microbiology, 64(4), 1477–1483. Schwan, R. F., Vanetti, M. C. D., Silva, D. O., Lopez, A., & Moraes, C. A. (1986). Characterization and distribution of aerobic, spore-forming bacteria from cacao fermentation in Bahia. Journal of Food Science, 51, 1583–1584. Schwan, R. F., & Wheals, A. E. (2004). The microbiology of cocoa fermentation and its role in chocolate quality. Critical Review in Food Science and Nutrition, 44(4), 205–221. Serra-Bonvehi, J., & Coll, F. (1997). Evaluation of bitterness and astringency of polyphenolic compounds in cocoa powder. Food Chemistry, 60(3), 365–370. Sharp, A.  K. (1979). Prevention of condensation damage to cocoa bean shipped in containers. Journal of Stored Products Research, 15, 101–109. Tafuri, A., Ferracane, R., & Ritieni, A. (2004). Ochratoxin A in Italian marketed cocoa products. Food Chemistry, 88, 487–494. Thompson, S.  S., Miller, K.  B., & Lopez, A.  S. (2007). Cocoa and coffee. In M.  P. Doyle, L.  R. Beuchat, & T.  J. Montville (Eds.), Food microbiology: Fundamentals and frontiers. ASM Press. Torres-Moreno, M., Tarrega, A., Costell, E., & Blanch, C. (2012). Dark chocolate acceptability: Influence of cocoa origin and processing conditions. Journal of the Science of Food and Agriculture, 92, 404–411. Tran-Dinh, N., Pitt, J. I., & Carter, D. A. (1999). Molecular genotype analysis of natural toxigenic and non-toxigenic isolates of Aspergillus flavus and A.parasiticus. Mycological Research, 103(11), 1485–1490. Urbańska, B., & Kowalska, J. (2019). Comparison of the total polyphenol content and antioxidant activity of chocolate obtained from roasted and unroasted cocoa beans from different regions of the world. Antioxidants, 8(8). https://doi.org/10.3390/antiox8080283 van der Wal, B., Kettenes, D. K., Stoffelsma, J., Sipma, G., & Semper, A. T. J. (1971). New volatile components of roasted cocoa. Journal of Agriculture and Food Chemistry, 19, 276–280. Varga, J., Kevei, E., Rinyu, E., Teren, J., & Kozakiewicz, Z. (1996). Ochratoxin production by Aspergillus species. Applied and Environmental Microbiology, 62(12), 4461–4464. Voigt, J., Heinrichs, H., Voigt, G., & Biehl, B. (1994). Cocoa-specific aroma precursors are generated by proteolytic digestion of the vicilin-like globulin of cocoa seeds. Food Chemistry, 50(2), 177–184. https://doi.org/10.1016/0308-­8146(94)90117-­1

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Chapter 3

Improving Functionality of Chocolate Nevzat Konar, Ibrahim Palabiyik, Ömer Said Toker, Arifin Dwi Saputro, and Haniyeh Rasouli Pirouzian

Abstract  Top trends in the food industry are mainly related with improvement of functionality of the products. Food manufacturers and researchers mainly focus on this subject and it is aimed to produce the functional products with quality characteristics similar to conventional ones. In the present chapter, the ways to improve functional characteristics of the chocolate products were reviewed. Calorie reduction in chocolates with using sugar and cocoa butter alternatives and its effect on chocolate quality was mentioned. In addition, the influence of processes applied for cocoa and chocolate productions on polyphenol composition and antioxidant activity was also addressed. Studies related with dietary fiber usage as a prebiotics and their effects on chocolate quality and probiotic chocolates and bioaccessibility of probiotics were also summarized in the chapter. It can be highlighted that functional N. Konar (*) Department of Food Engineering, Faculty of Agriculture, Eskisehir Osmangazi Univiersity, Eskisehir, Turkey I. Palabiyik Department of Food Engineering, Faculty of Agriculture, Tekirdag Namik Kemal Univiersity, Tekirdag, Turkey e-mail: [email protected] Ö. S. Toker Department of Food Enginering, Faculty of Chemical and Metallurgical, Yildiz Technical University, Istanbul, Turkey A. D. Saputro Department of Agricultural and Biosystems Engineering, Faculty of Agricultural Technology, Universitas Gadjah Mada, Yogyakarta, Indonesia e-mail: [email protected] H. R. Pirouzian Department of Food Science and Technology, Faculty of Nutrition and Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 C. M. Galanakis (ed.), Trends in Sustainable Chocolate Production, https://doi.org/10.1007/978-3-030-90169-1_3

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characteristics of the chocolate or derived products can be improved by enrichment and adjusting production process parameters. Chocolate is a good functional compound carrier since it is lovely consumed by people of all age throughout the world. Keywords  Chocolate · Cocoa · Functional food · Sugar-free · Polyphenols · Probiotics

3.1  Introduction In recent years, foods with health benefits have been attracted to the attention of consumers due to awareness of them about the close relationship between diet and health. Mostly, in this pandemic period, due to COVID 19, functional foods and foods, providing beneficial effects on immune system are preferred by people of all ages throughout the world. Therefore, food manufacturers and researchers have focused on developing functional foods that have similar quality characteristics with conventional products. In this case, the consumption prevalence of the foods is essential for transporting functional compounds into the body. In this respect, chocolate is one of the most suitable food product considering its consumption rate among people of all ages and production process where severe heat-processed are not performed. Chocolate is a product loved by many people and composed of cocoa mass, sugar, milk-based components, and emulsifiers depending on the chocolate type. In the food market, four types of chocolate are available: dark, milk, white, and ruby. Due to unique flavor characteristics, chocolate is consumed in different forms, such as compound chocolate. Chocolate also has beneficial effects on health since it is rich in phenolic compounds. However, some consumers have drawbacks about the consumption of chocolate due to high-calorie value. Therefore, lowering the calorie of chocolate and chocolate-based products has been attracted attention. In this chapter, possible fat and sugar reduction ways and their substitutes were mentioned. Also, enrichment of the chocolate with probiotics, polyphenols, dietary fibers, and prebiotics was summarised. Regarding probiotics, probiotic strains used in chocolate, advantages of chocolate as a probiotic delivering agent, the viability of them in chocolate storage, bioaccessibility of them, and usage effects of them on the quality of the end product were addressed. Chocolate, except for white type, naturally includes phenolic compounds due to cocoa-rich in bioactive compounds. The importance of variety and origin of cocoa beans, post-harvest processes applied for cocoa production, and the conching process on the phenolic compound composition was also mentioned. In addition, usage of dietary fibers and prebiotics on the quality was also quoted in this chapter. It can be highlighted that chocolate and chocolate-based products are a promising tool for delivering bioactive compounds since people lovelily consume it. The concern about the high calorie value of the products can be eliminated by using fat

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and sugar substitutes. The chocolate matrix and production process are also suitable for bioactive compounds easily degraded due to external factors.

3.2  T  rends in the Manufacturing of Reduced Calorie Chocolate Chocolate is one of the most popular confectionary products globally, consumed by people of all ages in society. Even though chocolate provides a health benefit, the popularity of chocolate is mainly due to its unique texture, aroma, and taste during its consumption (Konar et al., 2016; Tao et al., 2016; Aidoo et al. 2014a, b). High consumer demand for chocolate and its derivative products is attributed to those characteristics. Chocolate is a dense suspension of solid particles containing sugar, cocoa solids, and milk powder (relying on its type) dispersed in cocoa butter or its substitutes. The proportion of solid particles and cocoa butter are approximately 70% and 30%, respectively (Kiumarsi et al., 2017; Sarfarazi & Mohebbi, 2020). Chocolate is not consumed as a staple food. However, it is consumed considerably (Do et al., 2007; Afoakwa, 2010). Therefore, its consumption results in a high-calorie intake since dark chocolate; for instance, conventionally contains approximately 40–50% of sugar and about 30–40% of fat. Recently, foods such as chocolates that contain low sugar, low fat, and fat replacer are becoming more popular in the society (Faridah & Aziah, 2012; Saputro et al., 2017a; Konar et al., 2018). Health-conscious people give a lot of attention to calorie reduction in their diet. This phenomenon encourages chocolate makers, food technologists, and researchers to create low or reduced calorie chocolate. Nevertheless, reducing the amount of sugar and fat or replacing them with their alternatives are very challenging. The biggest challenge of doing this is maintaining the quality characteristics of the chocolate (Aidoo et al., 2013; Saputro et al., 2017b).

3.2.1  Sugar Sugar or table sugar is the most familiar word for sucrose. Sucrose is a disaccharide composed of chemically linked monosaccharides glucose and fructose extracted from sugar cane or sugar beet (Goldfein & Slavin, 2015). Sugar has a primary function as a sweetening agent. Besides this, sugar also contributes to the rheological properties, textural characteristics, color, taste, and aroma of foods and beverages. Sugar also acts as a bulking and preserving agent (Di Monaco et al., 2018; Aidoo et al., 2013; Afoakwa et al., 2007; Saputro et al., 2018; Saputro et al., 2017a). With these functions, sucrose plays an essential role in foods. Sugar-sweetened beverages, sweet yogurt, sweet bakery, ice cream, baked food, dessert, muffin, candy,

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jam, and chocolate are several high-calorie foods which are made with “help” from sugar (Grabitske & Slavin, 2008; Sethupathy et al., 2020; Struck et al., 2016; Di Monaco et al., 2018; Martínez-Cervera et al., 2011). The specific and unique characters of these foods are triggered and formed by the presence of sugar. However, health issues related to high sugar levels and calories force consumers to limit sugar consumption and find other alternatives. Diabetic and obesity are the primary concern (Furlán et al., 2017b; Sarfarazi & Mohebbi, 2020). Hence, due to these adverse effects, World Health Organization (WHO) directives state that sugar should not represent 10% of the daily caloric intake (Di Monaco et  al., 2018; Mooradian et al., 2017). The aforementioned level can be reduced to 5% (Mooradian et al., 2017). 3.2.1.1  Trends in Sucrose Replacement Nowadays, trends in the (partial or full) replacement of sucrose with low-calorie sweeteners such as bulk sweetener, high intensity (potency) sweetener, and low digestible carbohydrate, which have healthier effects, have been growing (Kiumarsi et al., 2017; Aidoo et al., 2013; de Melo et al., 2009). Several researchers and food technologist comprehensively reviewed the profile of sugar-free bulk sweetener, high-intensity sweetener, and low digestible carbohydrate (Aidoo et  al., 2013; Clemens et al., 2016; Grabitske & Slavin, 2008; Shoaib et al., 2016; Chattopadhyay et al., 2014; Martyn et al., 2018; Shankar et al., 2013; Grembecka, 2015; Mooradian et al., 2017). These sweeteners have health benefits because they provide a sweet taste with low/without calories or “glycemic effects” (Meyer-Gerspach et al., 2016). Besides, review the replacement of sucrose with Non-centrifugal Sugars (NCS), a technical name of the unrefined sugar cane juice (Jafffe, 2012), and palm sap sugar (Saputro et al., 2019b), which is claimed to have a health benefit have also been published. The presence of “impurities,” especially minerals and antioxidants, is responsible for this claim. Bulk sweeteners are commonly referred to as sugar replacers. These types of sweeteners can substitute, to a great extent, both sweetness and physical bulking properties of sucrose (Kroger et al., 2006). The most common bulk sweeteners used in chocolate are sugar alcohols (polyols), such as erythritol, isomalt, lactitol, maltitol, mannitol, sorbitol xylitol (Grembecka, 2015; Aidoo et al., 2013). The degree of sweetness of polyols varies from half to about as sweet as sucrose (Aidoo et al., 2013). High-intensity sweeteners comprise of substances with a very intense sweetness. This sweetener can be used in small amounts to replace the sweetness of a more significant part of sucrose with a degree of sweetness hundreds to thousands of times higher than sucrose (Kroger et al., 2006). The use of high-intensity sweetener needs to be combined with other bulk sweeteners and low-digestible due to the absence of bulking property (Cikrikci et al., 2016). Several high-intensity sweeteners commonly used in chocolate are stevia and thaumatin, Acesulfame K, aspartame, sucralose, saccharin (Aidoo et al. 2014a, b; Shah et al., 2010; Morais et al., 2014). Fiber or fiber-like ingredients is known as low-digestible carbohydrate

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(Aidoo et al., 2013). Low-digestible carbohydrates are incompletely absorbed in the small intestine but partly fermented by bacteria in the large intestine (Grabitske & Slavin, 2008, 2009). The most popular low-digestible carbohydrates used in chocolate manufacturing are inulin, polydextrose, and resistant maltodextrin (Kiumarsi et al., 2017; Rezende et al., 2015; Aidoo et al. 2014a, b; Belscak-Cvitanovıc et al., 2015; Sarfarazi & Mohebbi, 2020). Inulin and polydextrose also act as prebiotic substances that provide advantages in chocolate (Konar et al., 2016). Understanding the functionality of sugar in food is very important when reducing or removing sugar from the product (Goldfein & Slavin, 2015). Sucrose provides bulk properties and has functional attributes that are difficult to duplicate. Therefore, the use of alternative sweetener changes the quality attributes of chocolate to some extent, not only in the quality attributes of chocolate but also in the sensorial properties. 3.2.1.2  Q  uality Characteristics of Chocolate Sweetened with Alternative Sweetener Reformulation of sugar in chocolate is commonly done by partially or fully replacing sucrose with an alternative sweetener. Replacement of sucrose affects physical characteristics, requiring a reformulation to achieve the desired characteristics of chocolate. The first thing to do is deciding of which sweetener and what concentration of sweetener that results in the highest similarity to the products formulated with sucrose in terms of sweetness intensity and quality characteristics of the products (Morais et al., 2014). In addition, combining a high-intensity sweetener which has a very high degree of sweetness with low digestible carbohydrate which has a bulky character is a must to create chocolate with characteristics as close as possible to the one sweetened with sucrose (Sarfarazi & Mohebbi, 2020). With regard to the use of bulk sweetener, the incorporation of a single polyol to meet all requirements of chocolate is very difficult. Therefore, a combination of several polyols as a chocolate sweetener is the most logical option (Rad et al. 2019b). The acceptability of chocolate by the consumer is determined by several characteristics, including aroma, taste, fineness, melting profile, flow behavior/rheological properties, textural characteristics, and appearance (color and glossiness) (Hinneh et al., 2019; Saputro et al., 2018; Saputro et al., 2019a). Hence, the replacement of sucrose with alternative sweetener should consider these aspects as the main parameters. In general, a well-defined impact of the use of alternative sweeteners on chocolate properties cannot be entirely concluded since studies about sucrose replacement were performed in various formulations, ingredients, processing methods and chocolate types. Nevertheless, in general, studies reported that alternative sweeteners, to some extent, influence the rheological behavior of chocolate (Aidoo et al., 2017; Farzanmehr & Abbasi, 2009; Shah et  al., 2010; Nebesny & Żyżelewıcz, 2005; Sokmen & Gunes, 2006; Saputro et al., 2017c), textural properties (Aidoo, 2015; Belscak-Cvitanovıc et al., 2015; Farzanmehr & Abbasi, 2009; Saputro et al., 2017c),

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fineness of chocolate (Belscak-Cvitanovıc et al., 2015), melting properties (Aidoo et al., 2017; Shah et al., 2010), and color of chocolate (Aidoo, 2015; Konar, 2013; Saputro et al., 2017a). To the best of our knowledge, there is no study investigating the impact of alternative sweeteners on the aroma profile of chocolate, except studies conducted by Saputro et al. (2017b) and Saputro et al. (2018). Several results of the latest researches are resumed, reporting the impacts of alternative sweeteners on dark, milk, and white chocolate characteristics as well as its preference by the consumer. Kiumarsi et al. (2020) reported that dark chocolates made with higher ratios of modified inulin exhibited a higher plastic viscosity, flow behavior index, and elastic modulus. After three months of storage, the melting point and crystallinity of chocolate formulated with an intermediate proportion of modified inulin exhibited similar/constant values. Sensory analysis revealed that good texture and color (appearance) could be obtained by increasing inulin content. In Cikrikci et al. (2016) study, the partial replacement of sucrose with stevia in dark chocolates presented similar plastic viscosity and yield stress values with control chocolates. Hardness values also supported these consequences. With regard to the sensory analysis, it was reported that dark chocolate exhibited ideal acceptance by having similar statistical results to control. Rad et al. (2019a) reported that Casson viscosity and Casson yield stress were significantly affected by the type of bulk sweetener (isomalt, xylitol, and maltitol). In addition, chocolate sweetened with alternative sweetener exhibited lower Tonset and higher enthalpy than chocolate reference. The fineness of chocolate sweetened with alternative sweetener increased due to particle aggregation. Based on sensory analysis, xylitol remarkably improved the overall acceptability. Furlán et al. (2017a) investigated the influence of sucralose (Su) and stevia (St) on the physicochemical properties of sucrose-sugar white chocolate. The results showed that partial or full replacement of sucrose by stevia and sucralose changed the stability, texture, moisture content, and percentage bloom formation. Chocolate sweetened with 75%St + 25%Su exhibited minimum bloom formation and minor color changes during storage. Based on their work, Konar et al. (2018) reported that the incorporation of inulin DP with the presence of probiotic resulted in significant effects on quality characteristics of chocolate other than rheological properties, such as texture, water activity, thermal profile, and color. To achieve the desired chocolate characteristics, adjustment of processing variables, and alternative sweetener proportions are frequently applied to the low/ reduced-calorie chocolate manufacturing. With these methods, good standard sucrose-sweetened chocolate quality can be approached.to some extent.

3.2.2  Cocoa Butter Commonly, chocolate contains 30–40% of fat (Do et  al., 2007; Saputro et  al., 2019a), making chocolate a high-calorie food (Prosapio & Norton, 2019). Due to several health problems caused by high-fat consumption such as heart-related disease, overweight, and obesity, trends in reducing the amount of fat in chocolate is

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nowadays increasing (Rezende et  al., 2015; Tao et  al., 2016; Lee et  al., 2009). Likewise, trends in decreasing fat content also occur in other food products such as ganache (Kim et al., 2017), butter (Emadzadeh et al., 2012), milk drink (Mıttal & Bajwa, 2012), dairy dessert (Arcia et al., 2011) and cookie (Zoulias et al., 2000). The efforts to reduce fat in chocolate face technological challenges (Saputro et al., 2019a). This happens since reducing fat by reducing the amount and replacing fat with other materials (Grabitske & Slavin, 2008; Do et  al., 2007) changes the characteristics of chocolate. The main characteristics of fat reduction are rheological and textural (Beckett, 2001; Servais et al., 2002; Do et al., 2007). The success of making reduced-fat chocolate with desirable properties will be significant progress in the production of low/reduced-calorie chocolate. 3.2.2.1  Trends in Reduced Fat Manufacturing Cocoa butter is the main fat used in chocolate. It has a relatively simple triacylglycerol (TAG) composition with the main TAG being 1,3-­dipalmitoyl-­2-­oleoyl-­glycer ol (POP), rac-palmitoylstearoyl-2-oleoyl-glycerol (POS), and 1,3-stearoyl-2-oleoylglycerol (SOS) (De Clercq et al., 2016; Campos et al., 2009). The TAG composition creates chocolate with melting temperature around body temperature, in the range of 23–37 °C (Afoakwa et al., 2007). Cocoa butter mainly determines rheological and textural properties, glossiness, snap, and melting behavior of chocolate (De Clercq et al., 2016; Norton et al., 2009). Considering several characteristics influenced by cocoa butter, reducing the amount of cocoa butter has many aspects to be considered. Cocoa butter (fat) reduction/replacement leads to two primary concerns. The first concern is the difficulty in the processing, including the handling of molten chocolate during molding and tempering and its application in food products (enrobing, panning) (Saputro et al., 2017a). The second concern is the loss of eating quality due to different (in-mouth) melting properties, hard texture, and difficulty swallowing (Do et al., 2007; Beckett, 2001). To some extent, the amount of fat also influenced the color of chocolate since it defined how well cocoa butter covered the solid particles (Afoakwa et al., 2008) and determined the roughness of the chocolate surface (Briones et al., 2006). Various ingredients are been found to reduce the fat content of chocolate (Do et al., 2007, 2010), while several approaches are used to replace the cocoa butter such as, hydrocolloid, hydrogel, xanthan gum, guar gum, and inulin (Lee et  al., 2009; Francis & Chidambaram, 2019; Syafiq et al., 2014; Amir et al., 2013; Rezende et al., 2015). Another replacement technique is to replace the fat phase by a water-­ in-­oil emulsion (Prosapio & Norton, 2019; Norton & Fryer, 2012; Norton et  al., 2009). However, the aforementioned approaches change the chocolate characteristics. Therefore, some adjustments to maintain the acceptable chocolate characteristics need to be performed. Reducing the proportion of cocoa butter in chocolate should be simultaneously done by adjusting the particle size distribution (Mongia & Ziegler, 2000; Do et al.,

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2007) and adding surfactant (Do et al., 2010; Schantz & Rohm, 2005) to maintain the desired chocolate properties. Do et al. (2007) stated that suitable viscosity of reduced-fat chocolate could be obtain by applying the principles of particulate suspensions rheology. By creating bimodal particle size distribution (PSD), the viscosity of concentrated suspension can be decreased to some extent. Bimodal particle size distribution can be intentionally made by mixing small particles with the larger ones (Saputro et al., 2017c; Mongia & Ziegler, 2000). The small particles fill the voids/gaps between the large ones, reducing the amount of cocoa butter required to fill the voids. In this case, small particles act as a lubricant for the larger ones, thus decreasing the viscosity (Do et al., 2007; Servais et al., 2002). More “free” cocoa butter is then available for the flow, which further decreases the chocolate viscosity (Saputro et al., 2017c). The presence of large particles and the small ones in chocolate can be recognized from the PSD curve. Chocolate with a “uniform” particle size has a propensity to exhibit a unimodal PSD curve. On the other hand, chocolate, which contains large and small particle sizes, shows a multimodal PSD. In addition to the particle size distribution adjustment, the use of surfactant, e.g., lecithin, polyglycerol polyricinoleate (PGPR) reduces the viscosity of chocolate (Schantz & Rohm, 2005). This phenomenon occurs because the surfactant lowers the interfacial tension between sugar particles (hydrophilic ingredient) and the cocoa butter phase (lipophilic component). Substituting cocoa butter with other ingredients usually is not only chosen to create reduced-fat chocolate, but also to gain additional benefits. The addition of fiber, which acts as a bulking agent, is highly needed to create a skeleton for chocolate formulated with high-intensity sweetener (Rezende et al., 2015). Moreover, the addition of fiber also provides prebiotic substances that are good for our health. The incorporation of hydrogel into chocolate creates heat resistant chocolate (Francis & Chidambaram, 2019). However, since fat proportion is reduced, thus acceptable tuning formulation and processing methods should be done to deal with this condition. The use of water-in- cocoa butter emulsion seems to have a better potency to create reduced-fat chocolate without changing many chocolate characteristics (Norton et al., 2009; Norton & Fryer, 2012). However, making a stable water-in-cocoa butter emulsion with a considerable amount of water is also challenging (Norton et al., 2009). Furtherer attention should be considered during the manufacturing process of chocolate because emulsion destabilization may occur. The main drawback of this method is that chocolate made with this type of emulsion is prone to sugar bloom (Do et al., 2007). 3.2.2.2  Quality Characteristics of Reduced Cocoa Butter Chocolate Several results of researches on the cocoa butter-reduced chocolate are summarized, reporting the characteristics of chocolate produced. Do et  al. (2007) studied the features of the chocolate model produced by dispersing sugar in several different fat levels. The sugars used were mixtures of coarse and fine sugar. They reported that

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optimizing PSD at a critical amount of fat (22%) resulted in decreased viscosity of molten chocolate and hardness of crystallized chocolate. A drop in the viscosity was attributed to the reduction of inter-particle contact. A decrease in the hardness was associated with the particle skeleton of the chocolate, which was less interconnected. De Graef et  al. (2011) applied oscillatory rheology to measure the yield stress of chocolate with 36.7% of total fat. They reported that the addition of PGPR decreased the yield stress of chocolate. The yield stress continued to decline as the concentration of PGPR increased by up to 0.8%. A different trend was observed when lecithin was used in the use of lecithin. Initially, it was reported that yield stress decreased by up to 0.2% of lecithin addition. However, the yield stress increased, starting from 0.4% of lecithin addition. The latter trend occurred due to the formation of reverse micelles in cocoa butter and the formation of self-­association of lecithin, possibly as multilayers around sugar (Afoakwa et al., 2007). This phenomenon commonly occurs in high-fat content chocolate. Therefore, in the fat reduced chocolate, the use of surfactants is very useful in decreasing the viscosity. Several works studied the addition of other ingredients to replace cocoa butter and its effect on the chocolate quality. Lee et al. (2009) reported that the replacement of cocoa butter with β-glucan-rich hydrocolloid (C-trim30) dramatically increased the viscosity of molten chocolate, a softer texture of the chocolate was obtained with the addition of C-trim30 up to 10%. In their study, Francis and Chidambaram (2019) added a hybrid hydrogel into chocolate to create fat-reduced and heat resistant chocolate. They reported that glossy appearance, less surface roughness, highest melting resistance, and required beta V form polymorphism were achieved by adding hydrogel up to 50% (v/v). Amir et al. (2013) reported that due to the replacement of cocoa butter with xanthan/guar gum blend, the hardness of chocolate increased. The hardness of the chocolate increased as the proportion of the gum in the chocolate increased. However, the replacement of cocoa butter with gum did not significantly affect the melting point (p > 0.05). Based on their study, Rezende et al. (2015) found that an increase in inulin and β-glucan contents (from 0 to 10 g/100 g chocolate) created chocolate with a higher viscosity and more resistant to flow. The effects were more pronounced for b-glucan. The substitution of up to 50% of the formulation of cocoa butter resulted in products with good acceptance. There is minimal new research on the application of water-in-cocoa butter emulsion as a cocoa butter replacer. One of them is research carried out by Prosapio and Norton (2019). In their study, the chocolate was made by mixing the emulsion with cocoa powder, icing sugar, and milk powder. Afterward, the hardness, thermal behavior, and water activity of chocolate were analyzed. The optimum mixing speed and mixing temperature of the ingredients were 300 rpm and 25 °C, respectively. The hardness and thermal behavior seem in the range of fine chocolate, similar to the reference. However, to have a better understanding, other chocolate characteristics such us color, glossiness, fineness, flow behavior, and aroma still need to be analyzed.

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3.3  E  nhancing Cocoa Polyphenols Concentrations and Antioxidant Activity: Downstream Processes Many studies are demonstrating the health benefits of cocoa polyphenols for humans, and presently, the consumption of dark chocolate is suggested as a polyphenol-­rich product. Polyphenols, as one of the bioactive ingredients, are known for antioxidant properties. The high content of the antioxidant activity of cocoa makes it to be used as a healthy product because antioxidants could prevent cancer, heart diseases, and modulate the immune system. Thus health-promoting effects of cocoa could be summarized into three essential classes: protection of the cardiovascular system, anti-oxidative properties, and anti-carcinogenic properties (Patel & Watson, 2018; Seem et al., 2019). Cocoa is rich in polyphenols, basically catechins (flavan-3-ols) and proanthocyanidins. Phenolic substances contain 12–18% of the total weight of dried cocoa nibs (Lamuela Raventos et al., 2005). Phenolic compounds play a crucial role in contributing to the sensory attributes of cocoa and cocoa products. They are responsible for the bitter taste of beans and influence the digestibility and stability of raw cocoa beans. Polyphenols are divided into three groups: proanthocyanidins (58%), catechins (37%), and anthocyanins (4%). The predominate catechin is (−)–epicatechin representing 35% of total catechin, responsible for color changes and astringent after-taste of beans. Procyanidins of cocoa are identified by dimers, trimers, or flavan-3,4-diol oligomers. Other polyphenols include flavones and phenolic acids. The composition of polyphenols and organoleptic properties of different species of cocoa beans depends on genotype, origin, ripeness degree, and beans’ technological processing (Giacometti et  al., 2014). The distinguished polyphenol level also could be influenced by the type of extraction applied, the duration of the procedure, and the solvent used in the research (Payne et al., 2010).

3.3.1  T  he Impact of Variety and Origin of Cocoa Beans on Polyphenol Content The most common cocoa tree cultivars are Forastero (bulk grade), Criollo (fine grade), and Trinitario (fine grade) cultivars (Beckett, 2008). Forastero variety is strong and resistant to adverse environmental conditions, and the flavor and taste of these beans are much less aromatic than the fine varieties. The Criollo beans possess a unique flavor and aroma. Trinitario provides high-quality beans. The Forastero variety contains a higher concentration of polyphenol compared to the Criollo variety (Oracz et al., 2015). According to Oracz et  al. (2015), cocoa beans of the Forastero cultivar from Brazil owned the highest contents of flavonols, flavan-3-ols, and anthocyanins; however, samples of the Trinitario cultivar from Papua New Guinea possessed the lowest content of polyphenols. In the study of Bruna et al. (2009), the polyphenol

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content of cocoa beans husks was strongly dependent on geographical origin. The beans originated from Madagascar were identified by a higher content of polyphenolic compounds, and the cocoa beans with Ecuador origin were characterized by the lowest content of polyphenols. Carrillo et al. (2014) reported that the lower the altitude, the higher the contents of flavan-3-ols, epicatechin, and polyphenols in the cocoa beans. Some studies showed that some environmental conditions such as rainfall, sun exposure, and soil type influence the accumulation of polyphenols in plants. Humidity, temperature, nutrient availability, and the use of fertilizers also influence the accumulation of polyphenols in plants (Chang et al., 2009). Research by Elwers et al. (2009) stated that soil fertilization might lead to a considerable reduction in the content of total polyphenols, anthocyanins, and flavan-3-ols.

3.3.2  P  ost-Harvest Processes Affecting the Polyphenol Content of Cocoa and Chocolate The content and composition of phenolic compounds affect the sensory properties (color, taste, and aroma) of cocoa and cocoa products. Pre-treatment techniques applied for cocoa beans and technological processes result in considerable losses of these desired bioactive compounds. Oxidation and polymerization of phenolic compounds are the main reasons for the degradation of these compounds during the various post-harvest processing and storage (Nazaruddin et al., 2006). Manufacturers are searching for novel technologies and solutions that would reduce these losses. Therefore it seems necessary to analyze the changes in the content of polyphenols from both health and consumer acceptance. 3.3.2.1  The Fermentation Process Fermentation is the primary process that determines the qualitative properties of cocoa products. It initiates some desirable microbiological, biochemical, and physicochemical transformations in cocoa beans. During fermentation, enzymes degrade the proteins, phenols, and sugars, and this would form aroma components improving the taste and color of the cocoa beans. Yeast, acetic acid bacteria, and lactic acid bacteria exert protective effects on the contents of polyphenols; however, aerobic spores and molds demonstrate adverse effects (Giacometti et al., 2014). The concentration and relative proportions of the different polyphenols in beans are related to the degree of fermentation. Longer fermentation leads to a more significant decrease in the level of flavanols. During fermentation, 70%, 90%, and 93% decrease was observed in the polyphenols content (−) epicatechin and anthocyanins, respectively (Efraim et  al., 2011). The fermentation process ends in a pH decrease, which stimulates enzymatic activity such as polyphenolic oxidase. During

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this period, the precursors are degraded by the action of enzymes. Polyphenols are oxidized to create quinine and 2-quinone. Polyphenols undergo oxidation, polymerization, and also interact with proteins. Elwers et  al. (2009) reported a greater decrease in catechin levels during fermentation and drying of Criollo beans in comparison to other cultivars. This reduction was responsible for the mild taste of chocolate. Sabahannur et al. (2018) investigated phenol content and antioxidant activity of cocoa beans after fermentation and roasting. The highest phenol level was found in cocoa beans without fermentation, and there was a 98% phenol reduction after fermentation and roasting. 3.3.2.2  The Drying Process The primary purposes of drying are to reduce the water content and to decrease microbial growth in cocoa beans. During this stage, the polyphenol levels decrease considerably due to enzymatic browning processes. This stage has a crucial role in reducing beans, bitterness, and acidity and improving skin color and taste characteristics. The non-enzymatic and enzymatic processes are the main procedures for color changes in the cocoa beans. Among the non-enzymatic reactions are hydrolysis of anthocyanins into anthocyanidins and the later polymerization with simple catechins to create complex tannins. Maillard reaction, another non-enzymatic process, happens during drying and involves the reaction of reducing sugars and proteins to form brown polymeric compounds (Suazo et  al., 2014). Moreover, the enzymatic browning involves the formation of quinones from phenolic compounds and the later formation of brown to black polymers under the action of the polyphenol oxidase. The reduction of polyphenols is also associated with the migration of these compounds with the evaporated water. It seems that natural sun drying of beans ends in far better effects, and optimization of drying time could be carried out using models created by Garcia-Alamilla et  al. (2007) or Hii et  al. (2006). Also, manufactures may merge some cocoa batches to obtain a uniform mixture, and after mixing, standardization of process parameters can be performed to achieve the desirable properties of the mixture. Alean et al. (2016) studied the influence of the drying process on cocoa polyphenols. Results indicated that the least decomposition of polyphenols was recorded at a temperature of 40 °C, with a 45%; however, the higher pollution of polyphenols was found at a temperature of 60 °C. It was finalized that the drying temperature, time, moisture of grain, and polyphenols volatility were the main factors for cocoa phenols degradation. The mentioned factors influenced the irreversible oxidative processes of polyphenols. Moreover, high temperatures and long times of drying would affect phenols degradation due to cellular destruction. It can be concluded that the drying process should be performed at a controlled temperature to maintain the reactions that are responsible for the aroma and flavor of the finished product.

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3.3.2.3  The Alkalization Process Alkalization of cocoa beans is performed by sodium or potassium carbonates leading to better solubility of cocoa and establishing distinct color. Investigations have indicated that pH changes affected the polyphenols level in cocoa powder. Increasing pH leads to high losses of polyphenols content (epi-catechin, catechin, and quercetin) and alters their composition. Polyphenols oxidation and polymerization under alkaline conditions are the main reasons for their loss (Giacometti et  al., 2014). Some chocolate producers have eliminated the alkalization process to save the bioactive compounds. 3.3.2.4  The Roasting Process Roasting is one of the crucial stages influencing the sensory attributes (taste, color, and texture) of cocoa and cocoa products. High temperatures (110–160  °C) for 5–120 min are conducted during this process. High temperatures and also prolonged times lead to epimerization of (−)-epicatechin to (−)-catechin and of (+)-catechin to (+)-epicatechin. In the case of procyanidins, more complex processes occur. In the early times of the roasting stage, procyanidins with high molecular weight decreases and then increase due to the polymerization of low-molecular-weight compounds in the next step (Kothe et  al., 2013). It seems low temperatures and short times of roasting would better protect the level of polyphenols. Changes in the concentration and composition of phenolic compounds during roasting are a result of the high temperature and the presence of oxygen, which speed up oxidative processes (Bordiga et al., 2015). Żyżelewicz et al. (2016) studied the influence of roasting conditions (temperature, time, relative humidity, and flow rate of air) on the polyphenol content of cocoa beans and nibs with different particle sizes and chocolates. Results demonstrated that the degradation of phenols was lower when they were roasted in the air with increased relative humidity. The airflow rate of v = 0.5 m/s and RH = 0.3% ended in lower degradation of the polyphenol level in comparison to the flow rate of v = 1 m/s. The loss of flavanols in the process of chocolate preparation was not high. Maillard reactions may lead to the production of new antioxidants due to polymeric reactions. The oxidation and condensation of single polyphenols to complex tannins may contribute to the increase in the antioxidant activity because it has been stated that tannins have a higher scavenging power than simple phenolic compounds (Hagerman et al., 1998). Oracz and Nebesny (2016) reported that low-temperature with moist air could be applied to enhance the phenols levels of roasted cocoa beans. As a result, it can be concluded that standardizing different roasting parameters such as time, temperature, humidity, and flow rate of air would allow manufactures to produce chocolates with increased content of bioactive compounds such as polyphenols (Żyżelewicz et al., 2016).

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3.3.2.5  The Conching Process Conching is necessary to heat treatment (over 40 °C), contributing to the final texture and taste of chocolate. During the conching process, depending on the time/ temperature combination due to high temperatures, degradation of bioactive compounds such as phenolic compounds is expected (Gültekin-Ozgüven et al., 2016). As a result, it can be finalized that losses of polyphenols depend mostly on the technological processes situations. Di Mattia et al. (2014) studied the influence of long time conching (LTC) and short time conching (STC) on procyanidins level and antioxidant properties of dark chocolate. The results showed that the procyanidins concentration and pattern were considerably influenced by the various process conditions. At the end of the conching, the STC-chocolates indicated a higher content of monomers compared to the LTC-samples, which, in turn, resulted more polymerized. Both STC and LTC samples showed comparable phenolic content and antioxidant power; however, chocolates collected during LTC presented a significant improvement of the radical scavenging properties. It can be concluded that the conching process, exclusively the LTC process, showed an improvement of the antiradical and reducing properties of chocolate. Recently, new cocoa processing techniques have been developed to reduce the losses of polyphenols. The impact of various drying techniques (sun-dried, oven-­ dried, and freeze-dried) and process temperature on the polyphenol concentration in fermented cocoa beans were studied by Hii et al. (2009). The results indicated that the freeze-dried and oven-dried at air temperature 70 °C reduced the polyphenols’ loss in comparison to the sun drying method. It seemed that the freeze-dried method leads to a reduction of enzyme activity of polyphenol oxidize. Also, applying high temperature permits shortening of the drying time, which assists the protection of polyphenols. The positive influence of heat processing in the water at 95  °C in 5  min performed before the drying process maintained at 80 °C was reported by Cienfuegos-­ Jovellanos Fernandez et al. (2010). The heat treatment of fresh cocoa beans in water at a 95 °C for 5 min leads to polyphenol oxidizes inactivation. A significant impact of post-harvest processing confirmed the importance of the process parameters optimization for the limitation of phenolic compound oxidation. High temperature applied during cocoa beans processing has a destructive effect on phenol compounds. Therefore, manufacturers attempt to change the technological process and also the composition of raw materials, so they could preserve the valuable compounds that have a positive impact on human health. The suggestions include using high cocoa chocolate containing 70% to even 99% of cocoa ingredients and also using raw chocolate subjected to a low-temperature treatment, which has a protective effect on polyphenols. As a result, the impact of the process on the bioactive ingredients content and the properties preferred by consumers should be taken into account.

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3.3.3  The Type of Chocolate The chocolate formulation and level of non-fat cocoa solids are the main factors affecting the polyphenols content. Dark chocolate is an excellent source for flavonoids, but antioxidant capacity and flavonoid content are influenced during chocolate manufacturing (Gültekin-Ozgüven et  al., 2016). Moreover, cocoa beans varieties and their processing parameters are important for affecting the polyphenols content. For example, chocolates produced from Forastero cocoa beans are less bitter than chocolates produced with Trinitario or Criollo cocoa beans (del Brunetto et al., 2007). The technique of chocolate production is a key factor in determining the polyphenols level. New technology has been introduced for the production of raw chocolate. In this novel method, chocolate is subjected to low temperatures (below 45 °C), and the roasting process is eliminated for cocoa beans. This technique increases the valuable compounds (polyphenols) in chocolate (Żyżelewicz et al., 2018) that have a positive effect on human health. Raw chocolates are obtained from cocoa beans that are only fermented and dried (not roasted). In this type of chocolate, the alkalization process is omitted; therefore, the conching is frequently extended to up to four days, and this makes it feasible to obtain a finished product that has a structure similar to conventional chocolates (Albak & Tekin, 2014). Moreover, the reduction of polyphenols in chocolates is related to the amount of cocoa mass used in the product formulation. It seems that the dilution effect is responsible for these differences. Some ingredients, for example, lecithin and sucrose, lead to a dilution effect and also may negatively interact with phenolic compounds.

3.4  D  ietary Fibres and Prebiotics as Bulking Agents and Bioactive Compounds Dietary fibers are known as plant foods that our bodies can not digest or absorb. Some of them have a low to moderate effect on our gut microbiota. At the same time, prebiotics is selectively used by resident microbes in our body. Both dietary fibers and prebiotics are beneficial for health, and some accepted health benefits of prebiotics are improved mineral absorption, modulate immune system and satiety, promote metabolic health. The examples of fiber prebiotics are inulin, fructo-­ oligosaccharides (FOS), galacto-oligosaccharides (GOS), resistant starch, polydextrose, xylo-oligosaccharide (XOS), and isomalto-oligosaccharide (IMO). Some common examples of non-fiber prebiotics include lactulose, bioactive compounds such as, polyphenols and polyunsaturated fatty acid. There are some studies showing that cocoa itself has a prebiotic effect due to both cocoa fibers and its polyphenol content. The main monomers present in cocoa, (+)-catechin and (−)-epicatechin, were shown to inhibit the growth of Clostridium

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histolyticum and promote the growth of Eubacterium rectale–C.  Coccoides, Lactobacillus spp. and Bifidobacterium spp. (Tzounis et al., 2008). In animal models, cocoa polyphenols significantly reduced Bacteroides, Staphylococcus genus, and C. histolyticum subgroup after young rats took 10% cocoa for 6 weeks (Massot-­ Cladera et al., 2012). According to a human study investigating the effect of consuming high cocoa flavanol on microbiota composition in 22 healthy volunteers gut, 4  weeks daily intake of a drink with 494  mg flavanols increased the growth of Lactobacillus spp., and Bifidobacterium spp. compared to a 29 mg flavanol containing drink (Tzounis et al., 2011). In another study, cocoa fiber was fermented into short-chain fatty acids like butyrate and acetic acid by beneficial bacteria, which inhibited the growth of harmful microbes. Moreover, cocoa antioxidants were also fermented into smaller absorbable units; therefore their bioavailability was increased by beneficial bacteria (Moore et al., 2014). Polydextrose and inulin have been the most widely studied prebiotic sources in chocolate products. Application of these ingredients affected rheological properties, physical qualities, and microstructure of chocolates. When solely polydextrose was used as a bulking agent, large crystals were formed with less inter-particle spaces, which increased Casson yield stress. However, Casson plastic viscosity increased, and Casson yield stress decreased when inulin was used solely in the formulation as a sugar replacer. This was resulting from the fact that inulin addition caused large crystals with wide space between particles (Afoakwa, 2016). In order to reduce sugar, dextrin, inulin, and oligofructose can be incorporated into the chocolate formulation. In one patent (De Brouwer et al., 2015), a chocolate pproduct was developed by replacing 30% of the sucrose content with a combination of dietary fibres consisting of dextrin, inulin, and oligofructose. It was found that reduced sugar chocolate formulations generally suffer from some deficiencies, including high viscosity, difficult processability, and poor mouthfeel and taste profile. Therefore, soluble corn fiber (SCF) can be an appropriate alternative to eliminate some of these disadvantages. SCF can be obtained from corn starch by enzymatic hydrolysis; therefore, it can be described as gluco-­ oligosaccharides. Glucose sub-units linked by a-1,4, and a-1,6 glycosidic linkages are the main constituents of this fiber. In one patent (Price & Bingley, 2019), SCF can be used in the chocolate formulation up to 35% with a degree of polymerization less than 10. It was claimed that when SCF was used to replace sugar, the viscosity of chocolate decreased, which is generally not observed for known reduced sugar chocolate compositions. Therefore, there was no need for fat addition, as low viscosity did not cause processing problems.

3.5  Using Chocolate as Probiotic Delivering Agent Today, there is a consensus on the importance of intestinal microflora on the health and well-being of humans and animals (Genevois et al., 2016). Some members of this microflora play a key role in obtaining energy from components that escape

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from the upper part of the digestive system, providing essential vitamins and promoting the intestinal immune system. Therefore, it can even be described as an additional organ of the body (Possemiers et al., 2010). The most important of these microorganisms are defined as probiotics. Probiotics are living microorganisms and provide health benefits to their hosts in their adequate intake (Roobab et al., 2020; Eor et al., 2019). They play a major role in satisfying the expectations of consumers emerged in the twenty-first century, as well as providing essential nutrients for foods and improving their health-­supporting properties (Lalicic-Petronijevic et al., 2017). Therefore, interest in probiotics and food, including probiotics, has increased significantly in recent years (Skekh et al., 2020). Preventing infected diarrhea, lowering blood cholesterol levels, controlling intestinal infections, reducing lactose intolerance symptoms, and antitumor/anticancer agent effects can be stated as health benefits of probiotics (Kemsaworsd et al., 2016). They also improve the immune system and provide additional protection against pathogens (Silva et al., 2016). However, the health benefits of probiotics can only be achieved by choosing the proper strain and carrier product (Coman et al., 2012). The main strains in probiotic formulations are especially Lactobacillus and Bifidobacteria (Erdem et al., 2014). Also, Streptococcus sp., Enterococcus spp. and Saccharomyces boulardii are other probiotic microorganisms (Roobab et al., 2020). Some Bacillus (B. subtilis, B. pumilus, B. coagulans, B. cereus, and B. indicus) species also stand out with probiotic properties. Those with spores of this type draw attention due to their resistance to gastrointestinal environment conditions (Erdem et al., 2014). Traditional carriers of probiotics are dairy products (Valencia et  al., 2016). However, there are some reservations about the use of dairy products. Probiotic bacteria are generally delivered to consumers through dairy products such as yogurt, ice cream, and cheese (Eor et al., 2019). However, a significant part of the world population has lactose intolerance and milk allergy problems (Silva et al., 2017). The incidence of lactose intolerance is high in many populations around the world (Coman et  al., 2012; Succi et  al., 2017). The disadvantage is the relatively short shelf life of these products and the necessity of cold chain in transport and storage (Lalicic-Petronijevic et al., 2015; Nambiar et al., 2018; Succi et al., 2017). In addition, some researchers have found that probiotics have relatively low viability in fermented milk products (Succi et al., 2017). Probiotic carrier foods other than dairy products are also important due to the widespread veganism. Among the probiotic foods that are alternative to dairy products, chocolate is the most remarkable, widely consumed product, and popular in all age groups (Nambiar et  al., 2018). Also, the fact that chocolate contains compounds with antioxidant activity can provide an advantage to create healthy combinations (Nambiar et al., 2018). Another important factor is that confectioneries with probiotic properties have high commercialization potential (Konar et al., 2018). The use of chocolate as a probiotic carrier as an alternative to dairy products can produce solutions to potential problems such as lactose intolerance, cholesterol content, presence of allergic milk proteins, cold preservation requirement, short shelf life (Lalicic-Petronijevic

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et al., 2017). In this section, the importance of chocolate as a sustainable probiotic food from technology, consumer characteristics, and product quality perspectives is discussed.

3.5.1  Probiotic Strains in Chocolate An important criterion for effectiveness, efficiency, and success in the development of probiotic food is strain selection (Valencia et al., 2016; Nambiar et al., 2018). For example, in the study of Nambiar et  al. (2018), the strain was selected based on higher survivability in low pH and high bile concentration, antibiotic susceptibility, antimicrobial activity. In addition, the benefits of probiotics have been extensively studied and supported by scientific and clinical findings and results, but these benefits vary depending on the species and strains (Lalicic-Petronijevic et al., 2015). This is also valid for chocolate. The factors affecting the survival of probiotics in the chocolate structure were determined to be the strain of probiotics and the type of chocolate and storage conditions (Kemsaworsd et al., 2016). This applies to both probiotic stability and storage stability during the process. For example, L. acidophilus suffered a lower loss of viability in the white chocolate process than L. paracasei (Konar et al., 2018). In addition, dose selections were made by taking into consideration the health declaration conditions, therapeutic effect, loss of viability in the process and storage, and losses in the gastrointestinal tract. Probiotic chocolate studies encounter varying levels of inoculation in a wide range (6.0–10.0 log CFU/g) (Table 3.1). However, in general, it can be stated that the product should have a total alive probiotic load at the level of 9.0 log CFU/serving size throughout its shelf life. The serving size value is accepted as 25  g for chocolate (Konar et al., 2016). Especially Lactobacillus and Bifidobacteria species are used in probiotic chocolate development studies. Among these, the most common is L. acidophilus, L. casei, L. Paracasei, and B. lactis. There are also studies in which Stretococcus thermophilus was used in chocolate formulation (Lalicic-Petronijevic et al., 2017; Eor et al., 2019). A probiotic strain used in limited numbers was Bacilllus indicus (Erdem et al., 2014). Strain selection is generally based on sustainable supply and use in probiotic dairy products. Another approach in probiotic chocolate development studies is the use of different species or strain mixtures. With this approach, diversity can be provided in the potential health effects of different strains, as well as differences in resistance between strains can be exploited for viability. Another approach often used in probiotic product development is synbiotics, where a prebiotic fiber is involved in the formulation. In synbiotic chocolate formulations, the type of probiotic, as well as the choice of prebiotic substance and its properties (e.g., DP) should be considered for the level of viability (Konar et al., 2018). The prebiotic substance commonly used in chocolate formulations is inulin. This oligosaccharide is specifically used for metabolic activity and development by selective fermentation in the colon by Bifidobacteria (Sarfarazi & Mohebbi, 2020).

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Table 3.1  Probiotic chocolate studies Probiotic L. bacillus plantarum HM47 L. helveticus CNCM I-1722 and B. longum CNCM I-3470 (9:1) L. acidophilus LA3 B. animalis subsp. lactis BLC1 L. rhamnosus GG L. paracasei F19 L. casei DG L. reuteri DSM17938 L. acidiophilus NCFM B. animalis subsp. lactis HN019 L. acidophilus La-14 L. paracasei Lpc-37 L. acidophilus NCFM B. lactis HN019 L. acidophilus NCFM B. lactis HN019

Physical Viability Inoculation Chocolate Form Count Conditions ~9 log Milk Spray >1.0 × 108 CFU/g 25 °C, CFU/g dried 180 days

Reference Nambiar et al. (2018)

9 log Milk CFU/13.5 g Dark

Freeze dried

5.7 × 108 CFU/g 7.3 × 108 CFU/g

Prosess stability

Possemiers et al. (2010)

8 log CFU/g

Freeze dried

7.3 log CFU/g

25 °C, 120 days

Silva et al. (2017)

18 °C, 90 days

Succi et al. (2017)

Semi sweet

7.7 log CFU/g

8-9 log CFU/g

Dark (%80a)

Freeze dried

~8.2 log CFU/g

~8.4 log CFU/g ~8.2 log CFU/g ~5.4 log CFU/g 8-10 log CFU/g

9 log CFU/25 g

~9 log CFU/g

Milk

Dark (57 and 72%a) White

Milk

Dark Milk

Dark

Freeze dried

>6.61 log CFU/g

15 °C–7 M, Klindt-­ Toldam et al. 20– 30 °C–4 M, (2016) 15 °C–3 M (Total 14 M)

Freeze dried

6.74 log CFU/25 g

15 °C, 90 days

Konar et al. (2018)

Freeze dried

7.74 log CFU/25 g 8.56–8.32 log CFU/g

4–20 °C, 180 days

Lalicic-­ Petronijevic et al. (2015)

6.92–6.86 log CFU/g 8.49–7.77 log/ CFU/g

4–20 °C, 120 days 4–20 °C, 180 days

6.84–6.52 log/ CFU/g

4–20 °C, 120 days (continued)

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Table 3.1 (continued) Probiotic L. acidophilus LH5 (LA) S. thermophilus ST 3 (ST) B. breve BR2 (BB) L. acidophilus LH5 (LA) S. thermophilus ST 3 (ST) B. breve BR2 (BB) L. acidophilus LH5 (LA) S. thermophilus ST 3 (ST) B. breve BR2 (BB) L. brevis FERM BP-4693 L. brevis NITE BP-134 L. casei 01 L. acidophilus LA5 L. casei 01 L. acidophilus LA5 L. casei 01 L. acidophilus LA5

Physical Inoculation Chocolate Form ~9 log Milk Freeze CFU/g dried

Viability Count LA = ST > BB LA = ST > BB

Conditions 4 °C, 360 days 20 °C, 360 days

Dark

LA > ST > BB LA > ST = BB

4 °C, 360 days 20 °C, 360 days

Semi sweet

LA > ST > BB LA > ST > BB

4 °C, 360 days 20 °C, 360 days

Reference Lalicic-­ Petronijevic et al. (2017)

6 log CFU/g

Milk

Freeze dried

nd

na

Yonejima et al. (2015)

6.6 × 109 CFU/g 8.1× 109 CFU/f

White

Spray dried

2.4 log CFU/gb

4 °C, 60 days 25 °C, 60 days

Kemsaworsd et al. (2016)

6.6× 109 CFU/g 8.1× 109 CFU/f

Milk

6.6× 109 CFU/g 8.1× 109 CFU/f

Dark

8.7 log CFU/gb

2.2 log CFU/gb 8.8 log CFU/gb

2.0 log CFU/gb 8.8 log CFU/gb

4 °C, 60 days 25 °C, 60 days 4 °C, 60 days 25 °C, 60 days (continued)

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3  Improving Functionality of Chocolate Table 3.1 (continued) Probiotic Ba. indicus HU36 S. thermophilus MG 510 L. plantarum LRCC 5193 L. paracasei IMC501 and L. rhamnosus IMC502 (1:1)

Physical Inoculation Chocolate Form 6.08 log Dark Freeze CFU/g dried 2.3– Milk Freeze 2.5 × 1010 dried CFU/mL

9 log Milk CFU/100 g

Freeze dried

Viability Count 5.54 log CFU/g nd

4.5 × 107 CFU/g 1.5× 107 CFU/g

Conditions Process stability nd

Reference Erdem et al. (2014) Eor et al. (2019)

Process Coman et al. stability (2012) Room temperature, 6 M

nd not determined, na not applicable, L Lactobacillus, B Bifidobacterium, Ba Bacillus, S Streptococcus, M months a Cocoa percentage b Viability decrease

However, the low viability of Bifidobacteria from other probiotic species is remarkable (Table 3.1). In addition, the level of inulin DP affects the quality properties and probiotic viability of chocolate dark (Konar et al., 2017), milk (Toker et al., 2017), and white (Konar et al., 2018) designated for chocolate. Various factors, such as pH, water activity, storage conditions, and process affect probiotic viability in foods. Research has focused on improving this stability and ensuring control release in the gut. Among these studies, encapsulation applications come to the fore (Silva et  al., 2016). One way probiotics can be protected from environmental conditions and regulate the passage and distribution of the gastrointestinal tract is microencapsulation (Lalicic-Petronijevic et  al., 2017). Microencapsulation potentially reduces cell damage and death as it protects bacteria from harsh conditions and mild thermal applications (Nambiar et  al., 2018). In terms of chocolate technology, developing new products with minimal changes in process conditions will create important advantages. In this case, encapsulation techniques should be used that can eliminate or minimize the factors that negatively affect the probiotic stability in chocolate composition and process. In addition, another factor is the end product quality properties. Also, functional foods have relatively higher costs than conventional ones is a disadvantage for their accessibility. Therefore, low-cost coating agents and methods should be identified that will be compatible with product composition and properties in encapsulation. Another point is that probiotics maintain their viability in the storage process until they are used in the chocolate formulation and have physical properties suitable for inoculation or dosing in existing production methods and systems. There are several methods, such as emulsification, spray drying, and spray cooling, and coacervation to obtain dried viable cells which can be used in food

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products. The most widely used method for probiotics is freeze-drying (Skekh et al., 2020). Spray drying among these methods provides significant cost advantages (Nambiar et al., 2018). However, lactic acid bacteria are exposed to stress conditions such as high air temperature and rapid dehydration in the spray drying process (Su et al., 2019). Lyophilization is an expensive process that requires long processing time. As another method, the fluidized bed drying method may provide an advantage in probiotic encapsulation due to mild temperature application and shorter process time. Today, commercial probiotic bacteria are usually supplied as lyophilized in the food industry (Genevois et al., 2016). Lyophilized bacteria are also widely used in probiotic chocolate development studies (Table 3.1). The number of studies using probiotics prepared with spray dryer is quite limited (Kemsaworsd et  al., 2016; Nambiar et al., 2018). Spray drying is a low-cost technology that can be used in probiotic production. However, this method may result in loss of viability due to the high temperature, shear effects, osmotic pressure, and dehydration of the cells. Vacuum drying can also be used in microorganism protection as a simple, useful, and low-cost method (Genevois et al., 2016). Another approach for the probiotic addition method is encapsulation technology. The physical properties of the microcapsules may cause significant changes in probiotic viability and chocolate quality. Microcapsule size and size-distribution homogeneity may affect the packaging of solid particles in the chocolate microstructure, causing fat bloom development. Structural changes that may cause migration of TAGs with a low melting temperature in the microstructure may result in fat bloom. Silva et al. (2016) used a fluidized bed and lyophilization technique in L. paracasei encapsulation. Microcapsules obtained with fluidized bed have a spherical shape and robust structure, while lyophilized microcapsules are porous and fragile. In addition, while the probiotic microcapsules obtained with the fluidized bed have a homogeneous size distribution, particles with a larger range have been obtained as a result of lyophilization (Silva et al., 2016). Kemsaworsd et al. (2016) determined that L. casei and L. acidophilus powders obtained by the spray dryer technique have significant differences in particle size distribution and morphology. While L. casei microcapsules have a smaller and smooth surface, those belonging to L. acidophilus have been found to exhibit spherical and rough surface properties. However, low porosity, large size microcapsules, and solid structure contribute to probiotic viability by preventing water absorption (Silva et al., 2016). The use of different encapsulating agents can lead to different properties in surface morphology, size, dissolution, and bulk density. Therefore, it is useful to determine the microencapsulation method based on the probiotic strain. In designing this process, it should be kept in mind that probiotic microcapsules will be added at the stage where the chocolate mass will be exposed to minimal mechanical effects. In other words, the microcapsules added will be included in the chocolate formulation without being subjected to any size regulation (e.g. refinining or conching processes).

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In particular, Bifidobacteria and Lactobacillus microencapsulation studies reveal that microcapsules can be designed to improve product quality. Besides the product quality, the protection of the viability of probiotics is of great importance. Therefore, attention should be paid to the selection of materials that can resist gastric fluid in probiotic microencapsulation. For example, water sublimation can cause porosity in microcapsules obtained by lyophilization. This causes a decrease in stability against environmental conditions (Silva et al., 2016). Su et al. (2019) found that components such as whey protein isolates and calcium-enriched skim milk provide extracellular protection to cells by acting as a physical shield in the microencapsulation of lactic acid bacteria with a spray dryer. Another approach is to carry out the immobilization of probiotics in fibers of different plant origin to improve their stability and viability in storage (Genevois et al., 2016). Therefore, probiotic survival can be maintained in stomach pH values with innovative microstructure designs (Roobab et al., 2020).

3.5.2  Advantages as Probiotic Delivering Agent For developing novel foods it is necessary to retain all the functional characteristics without any significant losses during storage. This also applies to probiotic foods. Also, dose and viability are important criteria for probiotic activity. The health benefits of probiotics strongly depend on these microorganisms (Skekh et al., 2020). Therefore, probiotics must remain alive in food processing and throughout the gastrointestinal tract, and the food industry is exploring alternative foods that can be carriers for these microorganisms (Silva et  al., 2017). The main factors affecting probiotic viability in confectionery can be stated as water activity, temperature, temperature fluctuations, UV toxicity, oxygen exposure, sugar concentration, osmotic pressure, acidity, pH, storage temperature, presence of other microorganisms, and mechanical shearing (Valencia et al., 2016; Konar et al., 2018; Nambiar et al., 2018; Skekh et al., 2020). There are three main advantages in the use of chocolate as a probiotic carrier; (1) the process does not cause extreme reductions in probiotic viability, (2) longer shelf life than conventional probiotic products, (3) more efficient protection and colonization of certain chocolate components (especially cocoa butter, cocoa origin polyphenols, proteins of milk origin and sugar) during the process and storage. Since chocolate production contains stages that can cause damage to probiotic cells, the most appropriate inoculation stage is post-tempering (Succi et  al., 2017). Thus, these microorganisms are prevented from being exposed to negative temperature effects (Silva et al., 2017). Conching from previous stages of chocolate production has long-term moderate to severe heat treatment and shear effect. Also, oxygen exposure at this stage negatively affects probiotic viability. The disadvantages of the addition of probiotics in the tempering phase are that the microcapsules cannot be effectively coated with the fat phase with the risk of homogeneous distribution of probiotics in the chocolate mass. Higher viability levels may be achieved by using

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alternative methods for probiotic inoculation or by applying alternative process methods such as seeding technique in pre-crystallization. Chocolates, mainly those with a higher concentration of cocoa solids, are known as a rich source of polyphenols, such as catechins, anthocyanins, prochianidines, and flavonol glycosides (Lalicic-Petronijevic et al., 2017). Polyphenols are one of the most important food ingredients with antioxidant activity. Oxidative stress is one of the main factors that cause the death of probiotics in foods (Silva et  al., 2017). Therefore, the antioxidant activity of the phenolic compounds found in cocoa solids can minimize cell death by reducing oxidative stress (Maukonen & Saarela, 2015). However, different results regarding the polyphenol—probiotic interaction have been reached in probiotic chocolate studies. Kemsaworsd et al. (2016) emphasized that dark and milk chocolate came out of white chocolate before carrying probiotics due to the advantage of cocoa solids for oxygen toxicity. Since white chocolate does not contain cocoa solids, it does not contain flavonoids such as catechin, epicatechin, procyanidins, and is disadvantageous in terms of antioxidant activity (Sarfarazi & Mohebbi, 2020). However, Lalicic-Petronijevic et al. (2015) found no significant difference between milk (27% cocoa) and dark (75% cocoa) chocolates. One of the prominent product features in today’s chocolate consumption is the consumption of chocolates with high cocoa content. These products have also been used as probiotics carriers. The sensory properties of dark chocolate with different cocoa contents did not change significantly and negatively after various probiotics (Succi et al., 2017). In addition to being the most effective probiotics in health protection, Bifidobacteria are more negatively affected by oxygen exposure due to their anaerobic nature. Therefore, they experience stability problems. Although there are polyphenol antioxidant compounds in chocolate composition, some studies have emphasized that these antioxidants do not have a strong enough effect on Bifidobacteria (Lalicic-Petronijevic et al., 2017; Crittenden, 2004; Tripathi & Giri, 2014; Gaudreau et al., 2013). Another feature of polyphenols to be considered is that these compounds also have antimicrobial activity. Possemiers et al. (2010) found that the levels of probiotic viability in milk chocolate were higher than dark chocolate for both L. helveticus and B. longum. This difference was associated with the presence of more polyphenols in dark chocolate and polyphenols showing antimicrobial activity. While the antimicrobial effect of polyphenols is in product composition (pre-­ digestion and storage), it is a negligible factor due to low water activity. However, depending on the change of environmental conditions in the digestive process, antimicrobial activity effects may be seen on the probiotics of polyphenols. Therefore, digestion simulations are important in product development studies. In addition, with the increase of polyphenol bioaccessibility during digestion, antimicrobial activity on probiotics may increase (Klindt-Toldam et al., 2016). However, Succi et al. (2017) stated that polyphenols did not have a negative effect on the viability of L. rhamnosus, L. paracasei, L. casei, and L. reuteri in dark chocolate samples containing 50-85% cocoa and found that dark chocolate provided good protection to probiotics in the simulated gastrointestinal passage. In evaluating these results, it

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should be taken into consideration that different probiotics have different sensitivity to the antimicrobial activity of polyphenols (Klindt-Toldam et al., 2016). With this sensitivity difference, the probiotic strain, which will be carried by chocolate, should be determined by considering the potential antioxidant and antimicrobial activity, process, and storage. The fat phase is of great importance to the characteristics of chocolate. This phase is formed by cocoa butter. It has been determined that different lipid matrices can protect probiotics from gastrointestinal fluids (Silva et al., 2016). The lipid fraction of cocoa butter has a protective effect on probiotics during storage (Klindt-­ Toldam et  al., 2016). The buffering effect of the fat phase and amount at its contribution to the protection of probiotics from external influences (Nambiar et al., 2018). For example, Valencia et  al. (2016) determined that cocoa butter protects B. longum against adverse environmental conditions. Since cocoa butter protects bacterial cells from water and H + ions, it contributes to probiotic viability (Silva et al., 2017). In addition, it was found that this fraction protects probiotics more than the environmental factors in the upper parts of the gastrointestinal system compared to the samples where the only microencapsulation is applied (Possemiers et  al., 2010). Probiotic cell walls have a hydrophobic profile. This can be associated with the release of microorganisms into the intestine during fat digestion. In addition, the fact that cocoa butter has a buffering effect on passage through the stomach affects the level of viability (Maillard & Landyut, 2008; Lalicic-Petronijevic et al., 2017). Therefore, cocoa butter—probiotic interaction may provide an advantage for viability (Nambiar et al., 2018). Another major chocolate ingredient is sugar. Although it is defined by some researchers as having an inert effect on quality parameters other than sugar sensory properties, we can state that it is the determining components inflow, texture, melting properties, and even affects the crystallization behavior of cocoa butter (Rasouli Pirouzian et al., 2020). This ingredient is important in the development of probiotic chocolate because sugar helps to buffer the probiotic through the gastrointestinal tract (Ronadheera et al., 2010). In addition, sugar (>40%) protects lactic acid bacteria in the passage of the gastrointestinal tract and during storage (Skekh et al., 2020; Silva et al., 2017). However, negative consumer attitude, which can be encountered due to the high energy content of chocolate and its products, increases the interest in low-calorie and sugar-free products (Kiumarsi et al., 2020). Therefore it seems necessary to study the effects of bulking agents and sugar alternatives on probiotic viability. The main ingredients of the chocolate mass are cocoa butter, cocoa solids, and sugar. However, in the production of milk and white chocolate, in addition, milk-­ based ingredients (milk powder, milk fat, demineralized whey powder, etc.) are used. Also, the most preferred type of chocolate in consumption is milk chocolate. For this reason, it is important to use chocolate containing ingredients of milk origin as a probiotic distribution and carrier. As a result of some previous studies, it has been determined that milk chocolate is more advantageous in preserving probiotic viability than dark chocolate. This situation is especially associated with a higher protein content of milk chocolate and

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buffering capacity of milk proteins (Statpathy et  al., 2020; Klindt-Toldam et  al., 2016). In addition, cocoa butter lipid fraction and milk proteins have a synergistic interaction on probiotic viability in the gastric system. This interaction may lead to higher viability of probiotics found in milk chocolate than dark chocolate (Klindt-­ Toldam et al., 2016). Milk powder and demineralized whey powder can be used interchangeably in a chocolate formulation. Synthesis of amino acids, purines, pyrimidines, and some carbohydrates, enzyme cofactors, is associated with nitrogen use of microorganisms. Whey powder may be a source of nitrogen as well as carbon to microorganisms. For example, whey can be used in fermentation applications for the production of L. casei (Genevois et al., 2016). In this case, the influence of whey on survival and metabolic activity of probiotic culture should be considered. Chocolate characteristics include low moisture content ( 0.01)

chocolates (Table 5.3) and allow to perceive the polymorphic status of chocolate (Afoakwa et al., 2009b). Correlating the melting temperature of chocolates with the melting temperature of the cocoa butter polymorphs reported in literature, it is possible to confirm that in tempered chocolates the endothermic peak corresponds mainly to crystalline form β(V) (melting temperature range from about 32 °C to 34 °C), and in untempered chocolates are present not only type β(V), but also type β(VI) crystals (melting temperature range from about 34 °C to 36 °C) (Keller et al., 1996; Lawler & Dimick,

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b)

a)

Brazil Cuba

Heat flow (mW)

S. Domingue

S. Thomé

Venezuela

exo

20

30

Temperature (oC)

40

20

30

Temperature (oC)

40

Fig. 5.6  Thermograms of (a) tempered chocolates and (b) untempered chocolates scanned at a heating rate of 5 °C.min−1. All plots were vertically displaced for a better visualization Table 5.3  Calorimetric parameters associated with the melting process of untempered and tempered chocolate of the tested samples Brazil

Tempered Untempered

Cuba

Tempered Untempered

Venezuela

Tempered Untempered

Saint Domingue

Tempered Untempered

São Thomé

Tempered Untempered

Tonset (°C) 30.7 (0.7) 30.1 (2.0) 30.0 (1.1) 30.7 (1.1) 32.2 (0.7) 29.4 (0.5) 29.8 (1.5) 30.2 (0.7) 30.8 (1.2) 30.8 (1.0)

Tpeak (°C) 32.8 (0.0) 33.7 (0.4) 33.2 (0.6) 34.0 (0.3) 33.7 (0.8) 33.9 (0.7) 32.9 (0.5) 33.8 (0.2) 33.6 (0.6) 33.4 (0.1)

Tend (°C) 34.6 (0.3) 35.5 (0.1) 35.1 (0.6) 36.0 (0.2) 35.7 (1.1) 36.4 (0.7) 35.7 (0.2) 36.0 (0.2) 35.5 (0.3) 36.2 (0.3)

Tindex (°C) 3.9 5.4 5.1 5.3 3.5 7.0 5.9 5.8 4.7 5.4

ΔHmelt (J/g) 40.97 (1.1) 45.32 (0.3) 47.06 (3.1) 51.03 (2.5) 52.13 (2.2) 54.05 (0.3) 48.46 (0.5) 50.65 (1.0) 39.92 (4.9) 49.93 (0.6)

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2008; Buscato et al., 2018; Pirouzian et al., 2020). However, if seen in more detail, in tempered chocolates it is also possible to observe a slight shoulder in the temperature range of crystalline form β(VI), although much less intense than the one observed in untempered chocolates (Fig. 5.6b). This observation is compatible with polymorphic transition from β(V) to β(VI) that can occur after long time storage (Afoakwa et al., 2008; Vilgis, 2015; da Silva et al., 2017) and/or by oscillatory storage temperatures, even in well-tempered chocolates (Afoakwa et al., 2008; Altimiras et al., 2007; Lonchampt & Hartel, 2006; Zhao et al., 2018). In fact, previous works have reported that polymorphic transformation from β(V) to β(VI) crystals is extremely slow at 16 °C, but much quicker at 27 °C (Zhao et al., 2018). The results of melting enthalpy of tempered chocolate presented lower values than untempered chocolate (Table 5.3), coherent with previous works (Afoakwa et al., 2009a). Untempered chocolates were only molten at 45  °C, without tempering. The absence of this pre-crystallization process induced an uncontrolled crystallization, with the emergence of seeds of unstable polymorphs, like β′(IV), that quickly convert to the more stable crystalline forms (β(V) and β(VI). This causes a fat bloom (Lonchampt & Hartel, 2006), with a strong negative impact on the mechanical properties (hardness and stickiness) (Afoakwa et al., 2009b) and visual appearance (dark brown spots on a light brown background) (Kinta & Hatta, 2005, 2012). Interestingly, although fat bloom is always associated to polymorphic transition from β(V) to β(VI) forms, not all well-tempered chocolates that undergo this polymorphic transition exhibit visual bloom (Lonchampt & Hartel, 2004; da Silva et al., 2017). The appearance of fat bloom on the chocolate surface can only be observed in case of a significant growth of the crystal size (Ziegler, 2009), characterized by a dull and whitish external appearance with needlelike crystals of fat deposited on the surface (Kinta & Hatta, 2012).

5.3.3  Rheological Tests The main importance of rheometry on phase transitions in foods is their sensitivity to indicate changes in modulus properties that occur in amorphous food materials above glass transition temperature (Mahato et al., 2019; Jothi et al., 2020; Roos, 1995), namely in mechanical properties (i.e., storage modulus, loss modulus) or loss tangent (tan δ), due to their ability to observe relaxation times of molecular rotations and energy loss, which are related to viscosity (Roos, 1995). Small amplitude oscillatory measurements during solidification were performed using a controlled shear-strain rheometer through a temperature sweep from 32 to 10 °C where different profiles were observed, affected by both tempering process and origin of chocolate (Figs. 5.7 and 5.8). The solidification of both tempered and untempered chocolate was not significantly affected by temperature from 32  °C to 21.9  °C, which can be considered as the “liquid-like” region where log G* is independent from temperature and no significant variations in the structure were observed. Even

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Fig. 5.7  Complex modulus of untempered chocolate, for different origins, as a function of temperature

Fig. 5.8  Complex modulus of tempered chocolate, for different origins, as a function of temperature

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though, tempered chocolate presented a slight increase in log G* (Pa) from 3.7 to 4.3 between these temperatures, excepting Saint Domingue with lower values, from 3.3 to 3.7 (Fig. 5.8). Approximately, from 11 °C (untempered chocolate) or 13 °C (tempered chocolate) to 21.9 °C temperature, the log G* values exhibit a decreasing behaviour which is consistent with the solidification temperature of form γ(I), the most unstable polymorphic form (Le Révérend, 2009). In both cases, final log G* values ranged between 7.6 and 7.9 revealed to have no influence from origin, but a smoother variation was observed in tempered chocolate (Figs. 5.7 and 5.8). The kinetic of log G* was similar to the evolution of the fraction of solid fat during solidification, observed in previous studies (Engmann & Mackley, 2006a; Vereecken et al., 2007; Tang & Marangoni, 2007), which may indicate a cause-effect relation. Thus, complex modulus kinetic may be related with the profile of free fatty acids of chocolate but especially with the arrangement of triglycerides as the solid fat matrix is formed (Engmann & Mackley, 2006a, b). In fact, results showed that hardening process occurs between temperatures HTi and HTf (Table 5.4), consequence of the fat crystallisation process and polymorphic forms (Afoakwa, 2010). The obtained values were not affected by origin nor tempering method. It is important, however, to deepen knowledge in this area for a better understanding of hardening process of chocolate and to determine the glass transition temperature. Hardness is effective when analysing the firmness of chocolate (Jin et al., 2019) and was expressed as the maximum force measured to fracture chocolate samples stored at 20  °C for 20  days. The results are presented in Table  5.5 and is quite noticeable the higher values on tempered chocolate ranging from 30.63 N (Brazil) to 63.97 N (Saint Domingue), while untempered chocolate ranged from 14.01 N (Brazil) to 20.90 N (Venezuela). According to Glicerina et al. (2013), higher hardness values support the presence of an extremely hard and consistent structure increasing the resistance to the compression, which may be due to a higher cocoa solids or solid fat content (Do et al., 2007). However, the large difference between the measured values for hardness between some origins, like Brazil and Saint Domingue, cannot simply be justified by the cocoa content in the manufacturer’s declaration (Table 5.1), thus other causes may influence hardness such as tempering process, particle size distribution (Afoakwa et al., 2007) or TAG profile (Beckett, Table 5.4  Initial (HTi) and final (HTf) hardening temperatures during solidification Origin Cuba São Thomé Venezuela Saint Domingue Brazil

Tempered HTi (°C) 21.9 21.9 21.9 21.9 21.9

HTf (°C) 13.1 13.1 13.2 13.2 13.2

Untempered HTi (°C) 21.9 21.9 20.9 21.9 20.9

HTf (°C) 10.7 11.0 10.7 10.9 11.0

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Table 5.5  Image and texture properties of chocolate after 20 days storage time Tempered Y Peak histogram 70.2b 69.3a (1.1) (1.5) São Thomé 70.6b 70.0 (2.0) (1.7) Venezuela 47.7a 47.3 (1.8) (5.8) Saint Domingue 44.8a 40.3 (1.0) (1.2) Brazil 44.5a 43.0b (0.2) (2.6)

Origin Cuba

Hardness (N) 32.24a (5.85) 40.88 (3.05) 46.98b (7.12) 63.97c (4.23) 30.63a (3.68)

Untempered Y Peak histogram 132.7c 100.3a (2.3) (53.0) 136.9c 79.0a (3.3) (5.3) 90.7a 57.0a (1.3) (3.6) 100.9b 59.3 (2.3) (0.6) 97.7ab 59.7a (0.9) (1.5)

Hardness (N) 15.73ab (2.11) 15.44ab (1.37) 20.90b (4.12) 18.36b (2.38) 14.01a (2.17)

In each column, means with at least one common letter are not significantly different (p > 0.01)

2008). In fact, previous studies have reported the heterogeneity of the microstructure of cocoa butter due to the pre-crystallisation process, where seeded chocolate with cocoa butter in the form β(VI) presented a denser structure than non-seeded chocolate (Svanberg et al., 2011). Additionally, other studies refer the lower SOS/ SOO ratio as the cause for lower hardness values (Beckett, 2008). The hardness values obtained in the present study for tempered chocolate are similar to the literature (Glicerina et  al., 2013; Afoakwa et  al., 2008), however other works present higher hardness values but samples are not comparable (Ostrowska-Ligęza et al., 2019).

5.3.4  Infrared Thermography The use of infrared thermography during the solidification allowed the evaluation of the temperature at the surface of tempered (Fig. 5.9) and untempered (Fig. 5.10) chocolates. It is important to note that the initial temperature of untempered chocolate was 40 °C, while in tempered chocolate was 32 °C. Generically, all runs using tempered chocolate presented the following stages (Fig. 5.9): (1) initial temperature of 32 °C; (2) temperature decrease until Tlow; (3) temperature increase until Thigh, and; (4) temperature decrease until equilibrium with environment. Such “kink” has been reported previously (Tewkesbury et  al., 2000) and is a result of latent heat release after crystallisation of TAG, causing a slight increase on surface temperature. In fact, no relation was observed between Tlow or Thigh and the free fatty acids profile (Table 5.2), nevertheless the effect of TAG profile must not be disregarded. According to previous authors, this kind of curve may be classified as “under-tempered” (Letourneau et  al., 2005; Afoakwa et al., 2009b), which is usually related with higher temperatures during tempering process and where a part of the stable crystals are re-melted prior to cooling,

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Fig. 5.9  Temperature profile of tempered chocolate, from different origins, during solidification

Fig. 5.10 Temperature profile of untempered chocolate, from different origins, during solidification

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resulting in the formation of unstable fat crystals (Afoakwa et al., 2009b). However, in the present study, no impact was observed on texture or appearance after 20 days storage time. Overall, Thigh presented similar results for almost all origins, around 21.8 °C, excepting Saint Domingue (22.8 °C). The obtained values are comparable with previous works on dark chocolate 52% (Rothkopf & Danzl, 2015), where a Thigh around 20.6 °C and a Tlow around 18.9 °C were observed. Additionally, a close relation was observed between Thigh and HTi (Table 5.4). As presented in Fig. 5.10, such profile was not observed in untempered chocolate. Although some models have been proposed to explain phase transition in tempered chocolate, the process in untempered chocolate is still unknown. Based on published data, it is expected that tempered chocolate will start faster the crystallisation process around polymorphic form β(V) and the following immobilization of double and triple chains of TAG. On the other hand, HTf of untempered chocolate (Table 5.4) presented lower values than tempered chocolate. It is then expected, a higher Brownian motion and thermophoresis effects in untempered chocolate during solidification and, as consequence, a higher convection coefficient in heat transfer (Ganji & Malvandi, 2016). Although experimental data is required, these mechanisms may partially explain the faster cooling rates observed in untempered chocolate. Previous studies revealed that untempered chocolate produced no stable fat crystals during solidification as a higher energy was released during crystallization, resulting in consistent cooling of the fat matrix, thus no inflexion point was observed (Afoakwa et al., 2008).

5.3.5  Digital Image Analysis It is widely known the impact of untempered chocolate on the visual and structural properties of chocolate (Afoakwa, 2010; Beckett, 2008), affecting quality during storage and usually known as “fat bloom”. Such phenomenon occurs when fat crystals at chocolate surface affect the reflection of incident light and can be observed as a whitish/greyish film covering the entire chocolate, thus making the appearance unacceptable for consumers (Afoakwa, 2010). However, the exact involved mechanisms in such process is not yet fully known (Quevedo et al., 2013). Colorimeter has been widely used to evaluate the visual aspects of chocolate (da Silva et al., 2017; Ashida et al., 2020), but the small opening area (normally 2–5 cm2) present obvious limitations when non-homogeneous colour patterns arise (Briones & Aguilera, 2005). Actually, image processing presents itself as a reliable tool for visual analysis (Zhao et al., 2018; Briones & Aguilera, 2005; Nopens et al., 2008), however a standardized lighting is crucial to minimize gloss or shades, enhancing repeatability and comparison between samples. The luminance (Y) is a calculated nondimensional parameter from RGB of digital images obtained from computer vision and used to measure the formation of white and greyish spots, typical of fat bloom. Table 5.5 shows the Y values of chocolate after 20 storage days at 20 °C for both tempered and untempered chocolate. The option for such storage time prior image analysis was due to the lag period

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observed and measured in previous studies using different techniques (Bricknell & Hartel, 1998; Miquel & Hall, 2002) and which is accepted as the time period for the onset of bloom. The Y results of tempered chocolate from Brazil, Venezuela and Saint Domingue presented results significantly different between 44.5 and 47.7 (p