Instituto Tecnológico y de Estudios Superiores de Monterrey

Instituto Tecnológico y de Estudios Superiores de Monterrey

Campus Monterrey

School of Engineering and Sciences

DEVELOPMENT OF A MILK CHOCOLATE ADDED WITH FISH OIL AND PROBIOTICS: PHYSICOCHEMICAL CHARACTERIZATION,

SENSORY ACCEPTABILITY AND ITS EFFECT ON COGNITIVE SKILLS OF RATS

A thesis presented by

Paulinna Faccinetto Beltrán

Submitted to the

School of Engineering and Sciences

in partial fulfillment of the requirements for the degree of

Master of Science In Biotechnology

Monterrey Nuevo León, December 4^th, 2020.

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Instituto Tecnológico y de Estudios Superiores de Monterrey

School of Engineering and Sciences

The committee members, hereby, certify that have read the thesis presented by Paulinna Faccinetto Beltrán and that it is fully adequate in scope and quality as a partial requirement for the degree of Master of Science in Biotechnology,

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Dr. Daniel Alberto Jacobo Velázquez Tecnológico de Monterrey School of Engineering and Sciences

Principal Advisor

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Dra. Arlette Santacruz López Tecnológico de Monterrey

School of Engineering and Sciences Co-advisor

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Dra. Carmen Hernández Brenes Tecnológico de Monterrey

School of Engineering and Sciences Committee Member

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Dr. Rubén Morales Menéndez Dean of Graduate Studies School of Engineering and Sciences Monterrey Nuevo León, December 4^th, 2020.

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Dra. Esther Pérez Carrillo Tecnológico de Monterrey

School of Engineering and Sciences Committee Member

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Dr. Jacinto Bañuelos Pineda Universidad de Guadalajara

University Center for Biological and Agricultural Sciences

Committee Member

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Declaration of Authorship

Paulinna Faccinetto Beltrán declare that this thesis titled, “Development of a milk chocolate added with fish oil and probiotics: physicochemical characterization, sensory acceptability and its effect on cognitive skills of rats” and the work presented in it are my own. I confirm that:

• This work was done wholly or mainly while in candidature for a Master of Science degree at this University.

• Where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated.

• Where I have consulted the published work of others, this is always clearly attributed.

• Where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work.

• I have acknowledged all main sources of help.

• Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself.

___________________________

Paulinna Faccinetto Beltrán Monterrey Nuevo León, December 4^th, 2020

@2020 by Paulinna Faccinetto Beltrán All rights reserved

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DEDICATION

I dedicate this thesis to my parents, Alejandro Faccinetto and Rosa Amelia Beltrán because they have always been my example to follow. Without your support, trust, guidance, wisdom, and love, I would not have gotten this far. Thanks to you, I have become the person and professional that I am today. This life won't be enough to give you everything back. I also dedicate it to my brothers Giancarlo and Luis Jorge for always being by my side.

Likewise, I dedicate it to Ramona, who accompanied me in the worst moments, someone who gave me everything without asking for anything in return. Finally, I dedicate this thesis to God for being a very significant source of love and impulse in my life.

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ACKNOWLEDGEMENTS

First, I would like to thank my advisor, Dr. Daniel Jacobo, for his guidance, patience, and unconditional support throughout the process. Likewise, I want to thank you for your positive attitude, pushing me to give my best effort, and always giving me the best example.

Thank you for that and much more.

Likewise, I thank my co-advisor, Dr. Arlette Santacruz, for all her support and teaching throughout the master's degree. Thank you very much for helping me with probiotics microencapsulation, microbiota analysis, and samples of the rat analysis tissues.

I also thank Felipe López for always being on the lookout in the laboratory, for your help, the laughs, and the qPCR analysis of the rat microbiota.

To the committee member Dr. Esther Pérez, I thank you immensely for being so present during the rheology analysis of the formulated chocolates. Likewise, thank you very much for your immense support and for all the teaching you gave me in the food area.

I would also like to thank another member of my committee, Dr. Carmen Hernández, for her support in the area of fatty acid analysis. Likewise, I thank Martin for supporting me with analyzing and quantifying fatty acids in the chocolate samples.

To Dr. Jacinto Bañuelos for allowing me to use the facilities during behavior and memory analysis of rats. Also, I thank Octavio Aguirre since, without his support and guidance, the behavioral analysis would not have been carried out. I thank Laura for supporting me with the rats' maintenance and for teaching me about their handling. Thank you very much for all the teaching to the veterinary department of the University of Guadalajara.

To Ing. Norma Orozco for her help and teaching during formulating and making the chocolates. Likewise, thanks to the staff within the Mexican Institute of Confectionery in San Luis Potosí.

I thank the research groups of the Tecnológico de Monterrey, the Bioprocess and Synthetic Biology group and the Nutriomics group, and the VitaFort research foundation.

Thanks to CONACyt for scholarship # 923811 and Tecnológico de Monterrey for the academic scholarship.

A deep thank you to my parents for all the support throughout this long journey and process. I thank you for always trusting me, pushing me to progress, for your love, your

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immense wisdom, and your example. Also, I thank my aunt Alicia and the Estrada Silva family for their support, love, and company during my stay in Guadalajara.

To my fellow masters Esteban Guardiola and Fernanda Palafox, for always being through best and worse. For supporting me in difficult times and for making my study days more joyful and enjoyable. Above all, I thank Esteban for teaching me so many things to encourage me and his remarkable friendship. Also, I thank Yessica Pantoja for her support in the bioprocess lab.

Daniela, Alejandro, Amaly, and Doña Eva, to my roommates from Monterrey, for all the adventures and joys. Likewise, for teaching me a lot in a short time and for becoming a family so quickly. Above all, I thank my roommate, partner, and friend Rebeca Gómez not only for being with me during the master's degree process but for teaching me so many things about probiotics and food. I also thank her immensely for teaching me to be a better person, for her positivism, for pushing me always to improve, and for letting me be part of his life.

Finally, a big thank you to my best friends Jocelyn Robles, Carolina Carrasco, Gerardo Inzunza, Marianne Ibarra, Alejandra Rosas, and Melissa Egurrola for always looking out for me, for their words of encouragement, and their support. Thanks for everything.

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DEVELOPMENT OF A MILK CHOCOLATE ADDED WITH FISH OIL AND PROBIOTICS: PHYSICOCHEMICAL CHARACTERIZATION,

SENSORY ACCEPTABILITY AND ITS EFFECT ON COGNITIVE SKILLS OF RATS

BY

PAULINNA FACCINETTO BELTRÁN

ABSTRACT

Nowadays, new trends have been developed where society seeks the consumption of healthier foods in order to improve their health and consequently life expectancy. These new eating habits can help prevent neurodegenerative diseases such as Alzheimer's and Parkinson's. Industries seek consumer satisfaction through the development of new- generation, wide-range products, which are called functional foods. These foods have a very high potential to provide both support and prevention for these conditions. The health of future generations is one of the factors that must be taken into account today, because childhood is a very important stage for their cognitive development. To satisfy this type of market, the development of functional foods added with bioactive components that help to improve memory and that in turn have a pleasant flavor have been increasing. An example may be the use of milk chocolate as a vehicle for compounds that have been found to improve cognitive decline; highlighting omega-3 (ω3) and probiotics.

The first phase of this research consisted on developing a a milk chocolate added with fish oil and probiotics. The physicochemical characterization [rheology, texture, water activity (aw), instrumental color] and sensory acceptability test of the formulations obtained were carried out. Two different concentrations of FO were added to chocolate, which provided 76.0±5.2 mg and 195.8 ± 6.5 mg of ω3 polyunsaturated fatty acids (PUFAs) per serving size (12 g). Likewise, chocolates were added with probiotics (L. plantarum 299v and L. rhamnosus GG) associated with improvement of cognitive function. Chocolates added with both concentrations of fish oil showed an adequate probiotic bacteria counts (>1x10⁶ CFU per serving size). Likewise, rheology, texture, aw and instrumental color showed that the treatment that contained a lower concentration of ω3 PUFAs added with probiotics presented a similar behavior as compared with the control. Likewise, this treatment showed

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adequate overall sensory acceptability (> 7, meaning that it was moderately liked by consumers). Chocolate with the highest concentration of FO showed a low overall acceptability (< 6, around 5.5, meaning that the chocolate neither like or dislike). Therefore, the chocolate formulation containing probiotics and the lower concentration of FO (with 76.0±5.2 of ω3 PUFAs per serving size) was selected to evaluate its effect on the development of cognitive skills in rats.

In the second phase of this thesis, the effect of chocolate consumption, added with FO and probiotics, on the development of cognitive skills in rats and on the microbial content (Lactobacillus, Bifidobacterium, Enterobacteriaceae and total bacteria) in the cecum was evaluated. Cognitive skills in rats was determined by the memory and behavior tests using the Barnes open maze test of the individuals in the short (day 1 to 4) and long term (at day 7). Results showed that the great majority of latency times and errors of the Chocolate group improved with respect to the control. Likewise, the combination of chocolate together with fish oil and probiotics showed a positive effect on the memory of rats compared with the effect of the bioactive components added alone. Blood sugar levels, epididymal adipose tissue weight, and brain weight were not affected by the treatments. On the other hand, the consumption of probiotics alone or in combination with chocolate decreased the bacterial count of Enterobacteria, whereas Lactobacillus and Bifidobacteria counts were not affected.

Results shown in this thesis showed that chocolate could be used as an adequate vehicle of omega 3 PUFA and probiotics. Furthermore, this chocolate excerted positive effect on the cognitive skills of rats, representing a potential functional food to be introduced in the nutraceutical market. Further studies could be directed performing clinical trials to confirm the positive effect of the formulations on the cognitive development in children.

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DESARROLLO DE UN CHOCOLATE CON LECHE ADICIONADO CON ACEITE DE PESCADO Y PROBIÓTICOS:

CARACTERÍZACIÓN FISICOQUÍMICA, PROPIEDADES, ACEPTABILIDAD SENSORIAL Y SU EFECTO EN LAS

HABILIDADES COGNITIVAS DE RATAS

POR

PAULINNA FACCINETTO BELTRÁN

RESUMEN

Hoy en día, se han desarrollado nuevas tendencias donde la sociedad busca el consumo de comidas más saludables con el fin de mejorar su salud y por consecuencia la esperanza de vida. Estos nuevos hábitos de alimentación pueden ayudar a prevenir enfermedades neuro-degenerativas como lo son el Alzheimer y Parkinson. Las industrias buscan la satisfacción de los consumidores por medio del desarrollo de productos de nueva generación, de amplia gama,a los cuales les llaman alimentos funcionales. Estos alimentos tienen un potencial muy alto para brindar tanto apoyo como prevención a dichas afecciones.

La salud de las generaciones futuras es uno de los factores que hay que tener en cuenta hoy en día, debido a que la niñez e una etapa muy importante para su desarrollo cognitivo.

Para satisfacer este tipo de mercado se han buscado el desarrollo de alimentos funcionales que tengan componentes bioactivos que ayuden a mejorar la memoria y que a su vez tengan una sabor agradable. Un ejemplo puede ser el uso del chocolate con leche como vehículo de compuestos que se han encontrado que mejoran el declive cognitivo;

resaltando al omega-3 (ω3) y a los probióticos.

La primera fase de esta investigación consisitió en el desarrollo de un chocolate de leche adicionado con aceite de pescado y probióticos. Se llevó a cabo la caracterización fisicoquímica [reología, textura, actividad de agua (aw), color instrumental] y el análisis sensorial de las diferentes formulaciones. Dos diferentes concentraciones de FO se añadieron al chocolate, proporcionando 76.0±5.2 mg y 195.8 ± 6.5 mg de ácidos grasos poliinsaturados ω3 ( PUFAs) por porción. Igualmente, se añadieron cepas probióticas (L.

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plantarum 299v y L. rhamnosus GG) a los chocolates, previamente asociados con la mejora del desarrollo cognitivo. Los chocolates adicionados con ambas concentraciones de aceite de pescado mostraron un recuento de bacterias probióticas adecuado (>1x10⁶ CFU por porción). Así mismo, la reología, textura, aw y el color instrumental mostraron que el tratamiento con menor concentración de ω3 PUFAs añadido con probióticos presentó un comportamiento similar al control. También este tratamiento mostró una aceptabilidad sensorial global adecuada (>7, significando que es preferido moderadamente por los consumidores). El chocolate con una concentración mayor de FO mostró un aceptabilidad global baja (<6, alrededor de 5.5., significando un gusto neutro por el chocolate). Por lo tanto, la formulación de chocolate con probióticos y una concentración menor de FO (76.0±5.2 de ω3 PUFAs por porción) fue selecionado para evaluar su efecto en el desarrollo de las funciones cognitivas en ratas.

En la segunda fase de esta tesis, el efecto del consumo del chocolate añadido con FO y probióticos fue evaluado en el desarrollo cognitivo en ratas y en el contenido microbiano (Lactobacillus, Bifidobacterium, Enterobacteriaceae y bacterias totales) obtenido a partir del cecum. Las habilidades cognitivas en las ratas fueron determinadas por una prueba de memoria y comportamiento utiliazando la prueba del laberinto abierto de Barnes de los individuos en un corto (día 1 a 4) y largo plazo (día 7). Los resultados mostraron que la gran mayoría de los tiempos de latencia y errores cometidos del grupo Chocolate mejoraron con respecto al control. Así mismo, la combinación del chocolate en conjunto de aceite de pescado y probióticos mostró un efecto positivo en la memoria de las ratas comparado con el efecto de los compuestos bioactivos evaluados por separado. El nivel de azúcar, el peso del tejido adiposo epididimal y el peso del cerebro no fueron afectados por los tratamientos. Por otro lado, el consumo de probióticos por si solo o en combinación con el chocolate disminuyó el conteo de Enterobacteria, mientras que los conteos de Lactobacillus y Bifidobacteria no fueron afectados.

Los resultados de esta tesis mostraron que el chocolate puede ser utilizado como un vehículo adecuado de omega 3 PUFA y probióticos. Además, este chocolate ejerció un efecto positivo en las habilidades cognitivas de ratas, representando un alimento funcional con potencial para ser introducido en el mercado de los nutracéuticos. Estudios adicionales pueden ser dirigidos en realizar pruebas clínicas para confirmar el efecto positivo de las formulaciones en el desarrollo cognitivo en niños.

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LIST OF FIGURES

3.1. Shear stress (A) and apparent viscosity (B) of chocolate samples added with fish oil

and probiotics with temperature 35ºC. . . .29

3.2. Changes in Storage modulus G’ (A) and Loss modulus G’’(B) in the frequency sweep test of chocolate samples (Temperature 35ºC). . . 30

3.3. Effect of heating and cooling on the rheological behavior of chocolate samples.. . . 32

4.1. Representation of the Barnes Maze test (BM) test labyrinth and protocol. . . 45

4.2. Total latency of rats using the Barnes Maze test. . . . . . .. . . 48

4.3. Latency until the first exploration of rats using the Barnes Maze test.. . . 50

4.4. Latency in the escape zone of rats using the Barnes Maze test. . . 51

4.5. The latency of the first exploration in the escape hole (Primary latency) of rats using the Barnes Maze test. . . .. . . . . . 53

4.6. The total number of errors using the Barnes Maze test (considering error when the rat introduced its nose into a different hole than the escape box). . . 54

4.7. The number of errors in the escape zone using the Barnes Maze test. . . .. . . . 56

4.8. Total distance traveled (cm) using the Barnes Maze test. . . .. . . . . . 57

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LIST OF TABLES

2.1. Chocolate formulations used as delivery system of nutraceuticals. . . .6 2.2. Effect of Probiotics in neurodegenerative diseases. . . .. . . 15

3.1. Fatty acid profile content (mg fatty acid per 100 g sample FW^-1) of fish oil source and milk chocolate added with probiotics and fish oil samples. . . .. . . 27

3.2. Instrumental color CIE LAB values and water activity (aw) of milk cholate added with probiotics and fish oil. . . 33

3.3. Texture parameters values of milk chocolate added with probiotics and fish oil. . . .36

3.4. Sensory acceptability values of chocolate samples added with fish oil and probiotics. . . . . . 37

4.1. Treatments composition of rat diets.. . . 42

4.2. Effect of treatments in glucose concentration, encephalon, and epididymal weight after Barnes Maze test. . . . . 58

4.3. RT-PCR microbial quantification after Barnes Maze test . . . .. . . .. . . 59

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CONTENTS

ABSTRACT vi

LIST OF FIGURES xi

LIST OF TABLES xii

CHAPTER 1. GENERAL INTRODUCTION . . . 1

1.1 Introduction. . . . . . . .1

1.2 Hypothesis. . . .2

1.3 General Objectives. . . 2

1.4 Specific objectives. . . . . . 3

1.5 Thesis structures. . . 3

CHAPTER 2. LITERATURE REVIEW. . . .4

2.1. Chocolate as a delivery system for nutraceuticals. . . .4

2.1.1. Chocolate and the effect of flavonoid content. . . 4

2.1.2. Chocolate as a delivery system of nutraceuticals . . . .. . . 5

2.1.3. Quality parameters of chocolate. . . 7

2.1.4. Future trends on the use of chocolate as delivery system of nutraceuticals for bioactive compounds delivery in chocolate. . . .. . 8

2.2. Nutraceuticals and cognition. . . 8

2.2.1. Importance of cognition and its relation to diet. . . .8

2.2.2. Animal model of cognitive function. . . .10

2.2.3. Role of omega-3 fatty acids on cognitive development. .. . . . ..12

2.2.4. Role of probiotics in the gut-brain axis. . . . . . .13

2.2.4.1. Methods to stabilize probiotics. . . .16

CHAPTER 3. PHYSICOCHEMICAL PROPERTIES AND SENSORY ACCEPTABILITY OF A NEXT GENERATION FUNCTIONAL CHOCOLATE ADDED WITH OMEGA-3 AND PROBIOTICS. . . 17

3.1. Synopsis. . . .17

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3.2. Introduction. . . . . . 17

3.3. Materials and methods. . . .19

3.3.1. Chemicals and active ingredients. . . .. . .19

3.3.2. Probiotic microencapsulation and viability determination. . . . .20

3.3.3. Milk chocolate production and determination of probiotics viability. . . .. . .20

3.3.4. Fatty acid profile. . . .21

3.3.5. Rheological analysis. . . .. . . .21

3.3.6. Texture analysis, surface color, and water activity determinations. . . .. . . .22

3.3.7. Sensory acceptability test. . . .22

3.3.8. Statistical analysis. . . .23

3.4. Results and discussion. . . .23

3.4.1. Viability of probiotics during microencapsulation and chocolate making process . . . . . . .23

3.4.2. Fatty acids profile. . . . . . .24

3.4.3. Rheological analysis. . . .. . . 26

3.4.4. Surface color, water activity and texture analysis. . . . . . .32

3.4.5. Sensory acceptability test of chocolates added with fish oil and probiotics. . . .36

3.5. Conclusions. . . .38

CHAPTER 4. EFFECT OF MILK CHOCOLATE ADDED WITH FISH OIL AND PROBIOTICS ON SHORT AND LONG TERM MEMORY PERFORMANCE OF RATS. . . .39

4.1. Synopsis . . . .39

4.2. Introduction. . . .40

4.3. Materials and methods. . . .. . . .41

4.3.1. Chemicals and active ingredients. . . .. . . .41

4.3.2. Probiotic microencapsulation and viability determination. . . . .41

4.3.3. Rat individuals and diets for cognitive tests. . . . .. . . 42

4.3.4. Chocolate elaboration and determination of probiotics viability. . . . . .. . . .42

4.3.5. Experimental model and rat manipulation. . . .43

4.3.6. Barnes Maze and behavioral test. . . .43

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4.3.7. Blood sugar levels, epididymal adipose tissue weight, and brain

weight determinations . . . .46

4.3.8. Microbial quantification by RT-PCR. . . .46

4.3.9. Statistical analysis. . . . . . .47

4.4. Results . . . .. . . .. . . 47

4.4.1. Barnes Maze (BM) and behavioral test analysis . . . .47

4.4.1.1. Total latency test. . . .47

4.4.1.2. Latency time until first exploration . . . 50

4.4.1.3. Latency in the escape zone . . . .52

4.4.1.4. Latency of the first exploration in the escape hole (primary latency) . . . 54

4.4.1.5. Total number of errors . . . 56

4.4.1.6. Total number of errors in the escape zone . . . 58

4.4.1.7. Total distance . . . 60

4.4.2. Blood sugar levels, epididymal adipose tissue weight, and brain weight analysis . . . .62

4.4.3. Microbial quantification by RT-PCR. . . . . . . . 63

4.5. Discussion . . . .63

4.5.1. Barnes Maze and behavioral test analysis . . . .63

4.5.2. Blood sugar levels, epididymal adipose tissue weight, and brain weight analysis. . . .68

4.5.3. Microbial quantification by RT-PCR . . . . . . .. . . . .69

4.6. Conclusions. . . .. . . 69

CHAPTER 5. GENERAL CONCLUSIONS AND RECOMMENDATIONS. . . .71

REFERENCES. . . . . . .73

VITA. . . . . . .117

CHAPTER 1

GENERAL INTRODUCTION

1.1. Introduction

Today, society seeks new eating habits and lifestyles to prevent various diseases such as diabetes, cardiovascular disease, obesity, and neurodegenerative diseases. For this reason, the food industry has strived to develop next-generation functional foods that can meet these demands. Next-generation functional foods added with bioactive ingredients have a high potential because they can reduce chronic degenerative diseases while providing proper nutrition (Santana-Gálvez, Cisneros-Zevallos & Jacobo-Velázquez, 2019;

Jacobo-Velázquez, Santana-Gálvez, & Cisneros-Zevallos, 2020). Likewise, many researchers focus on identifying functional food ingredients as product fortifiers to provide health benefits beyond the nutritional value of a product. Some of the bioactive ingredients used in the most significant proportion are fibers, vitamins, plant extracts, carotenoids, probiotics, and omega-3 (ω3) polyunsaturated fatty acids (PUFAs) (Fernandes, Coelho &

Salas-Mellado, 2019).

A relationship between balanced diets and brain development have been established because essential compounds found in food are critical to growth. Childhood is a necessary stage for cognitive development because it can predict future characteristics such as academic achievement, success, and how they fit into society (MAL-ED Network Investigators, 2018). The incorporation of bioactive ingredients like ω3 PUFAs in children diet is essential because they play a crucial role in cognitive development (Cerdó, Ruíz, Suárez & Campoy, 2017). ω3 PUFAs such as docosahexaenoic acid (C22:6, DHA) and eicosapentaenoic acid (C20:5, EPA) are fundamental for early-stage development and growth. These fatty acids' deficiencies affect brain performance, inducing behavior, and cognitive impairments (Spencer, Korosi, Layé, Shukitt-Hale & Barrientos, 2017). Likewise, it has been shown that there is a relationship between the cognition of the brain with peripheral functions through the interaction of the central nervous system in the brain and the enteric nervous system in the intestine (Cerdó, Ruíz, Suárez & Campoy, 2017).

Therefore, targeting gut microbiota is essential for the development of strategies for health care. In this context, the use of probiotics (Prob) may be potential in the treatment of neurological problems (Cerdó, Ruíz, Suárez & Campoy, 2017). For this reason, the addition

2 of probiotic strains such as L. plantarum 299v and L. rhamnosus GG has been found to improve cognitive functioning in patients with major depression and with children with communication disorders (Rudzki et al., 2019; Rianda, Agustina, Setiawan & Manikam, 2019). These neurodegenerative disorders are typically evaluated in animal models were short and long memory are assessed by spatial memory. One method that can provide essential information about spatial learning and memory impairments in rodents is the Barnes maze test (Luna et al., 2018; Gawel et al., 2019).

The development of new products containing bioactive ingredients can be challenging because various parameters need to be considered, such as the vehicle, the compound's safety, product stability, and possible interactions between the ingredients (Fernandes, Coelho & Salas-Mellado, 2019). The acceptability of these foods depends mainly on the vehicle used to carry the main compound (Cencic & Chingwaru; 2010). For this reason, milk chocolate is a feasible option to administrate bioactive compounds because it has been confirmed to be a suitable vehicle for carrying both omega-3 fatty acids and probiotics (Annunziata, Vecchio & Kraus, 2016; Konar et al., 2018; Foong, Lee, Ramli, Tan,

& Ayob, 2013).

Many studies have revealed that one of the critical factors during the acquisition of functional foods is taste (Annunziata, Vecchio & Kraus., 2016). Therefore, the quality of chocolate is an essential consideration to fulfill the consumer's expectations. Thus sensory characteristics can be changed by controlling processing variables such as viscosity, texture, and moisture (Gonçalves & Lannes, 2010).

1.2. Hypothesis

The present thesis hypothesis was that the addition of fish oil and probioticis on a milk chocolate formaltion will result in a production with physicochemical properties and sensory acceptability similar to the control and will improve rats' cognitive skills.

1.3. General Objective

To development a milk chocolate bar incorporated with L. plantarum 299v, L.

rhamnosus GG, and fish oil (ω3 fatty acids) and evaluate its physiochemical properties, sensory acceptability and its effect on rat’s cognitive skills.

3 1.4. Specific objectives

To evalate the hypothesis presented herein, five specific objectives were established:

1) To develop a milk-chocolate bar incorporated with microencapsulated probiotics and omega-3.

2) To characterize the fatty acids profile of milk chocolate bar.

3) To characterize physicochemical properties of the milk-chocolate bar.

4) To determine the sensory acceptability of milk-chocolate by hedonic scale tests.

5) To determine the effect of the milk chocolate incorporated with probiotics and omega-3 in memory and cognitive impairment using the Barnes Maze memory test with a rat animal model.

1.5. Thesis structure

This thesis is divided into four different chapters. Chapter 1 present a general introduction to the thesis. Chapter 2 shows a literature review with the primary information about cognitive development and its importance in children. Also, it describes the importance of functional foods and the use of components such as probiotics and omega-3 for cognitive development. Chapter 3 describes the elaboration process of a next-generation functional chocolate. Also, the microencapsulation technique of probiotics and analysis of the fatty acids profile is shown. Likewise, the physicochemical and sensory analysis of the new functional chocolate bar is evaluated. Chapter 4 presents the effect of fish oil, probiotics, and milk chocolate vehicle on the microbiota, adipose tissue, cognitive and brain development in male rats. Finally, Chapter 5 summarizes a series of general conclusions and recommendations for future studies.

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CHAPTER 2

LITERATURE REVIEW Nutraceuticals and cognition

2.1. Chocolate as a delivery system of nutraceuticals 2.1.1. Chocolate and the effect of flavonoid content

Chocolate is generally known as a confectionery product, but humans have consumed it for many years because of its health-promoting properties. Indigenous people in Mesoamerica initially consumed chocolate for medical uses as a remedy or a vehicle to deliver medicines. Kuna Indians in Panama consume daily 850.5 g of cocoa base beverage, which provided approximately 1,880 mg of procyanidins. This tribe presents lower hypertension prevalence and lower rates of diabetes, cancer, and strokes. Therefore, it has been hypothesized that the high consumption of this beverage is, in part, responsible for the lower prevalence of these diseases among the islanders (Katz, Doughty & Ali, 2011). Almost 150 uses have been documented for medicinal treatment in Europe, including the improvement of digestion, stimulation of the nervous system, anti-depressant, and reinforcing mental performance (Katz, Doughty & Ali, 2011; Jalil & Ismail, 2008).

These health benefits are mainly attributed to the antioxidant properties of polyphenols found in cocoa. The cocoa bean is one of the primary sources of dietary phenolics with a 12-18% total dry weight (Meng, Jalil & Ismail, 2009). The main compounds that provide most of the antioxidant activities in cocoa products are the flavanols procyanidins such as catechin and epicatechin (Katz, Doughty & Ali, 2011; Meng, Jalil &

Ismail, 2009). These compounds have the potential to prevent chronic denerative diseases such as cardiovascular diseases. Also, according with a meta-analysis cocoa-rich food intake reduces blood pressure (Meng, Jalil & Ismail, 2009). Cocoa and chocolate benefits that contribute to the cardiovascular system, contributing to normal blood flow, have been linked to cognitive performance. Animal studies have shown that flavonoids interact directly with molecular targets in the brain generating an antioxidative effect. This effect suggests neuroprotection by improving brain tissue and certain regions in charge of memory, learning, and cognition (Sokolov, Pavlova, Klosterhalfen & Enck, 2013).

It has been found that flavanols and their metabolites can cross the blood-brain barrier, generating beneficial brain function changes, through angio- and neuro-genesis improving brain function by stimulating blood circulation to the brain. Likewise, it is believed

5 that flavanols that penetrate the brain accumulate in memory and learning regions, generating a positive impact on neuroprotection and cognition (Nehlig, A., 2013; Sokolov, Pavlova, Klosterhalfen & Enck, 2013). Therefore, it is believed that this neurological effect occur in two different ways. In the first one, it is believed that flavonoids interact with various signaling pathways such as the mitogen-activated protein (MAPK), extracellular-signal- regulated (ERK), and phosphoinositide 3-kinase (PI3-kinase/Akt) signaling cascade (Sokolov, Pavlova, Klosterhalfen & Enck, 2013). These cascades generate gene expression to establish long-term memory. Likewise, flavonoids modulate transcription factors and promote the expression of brain-derived neurotrophic factor (BDNF), a critical molecule used in neurogenesis, synaptic growth, and neuronal survival (Sokolov, Pavlova, Klosterhalfen &

Enck, 2013). Substantial evidence showed that rats deprived of neural injections of BDNF declined cognitive performance compared to the control using a water memory test (Morris water maze). These findings illustrate the importance of the involvement of BDNF in cognition processes of memory and learning (Piepmeier & Etnier, 2015).

In the second pathway, flavonoids present in chocolate, inhibits atheromatous plaque adhesion molecules that cause inflammation by facilitating the production of nitric oxide (NO) signaling molecules. The decrease of inflammation helps to relax the tissue of blood vessels, improving vascular function. This vasodilation helps brain blood flow throughout the central and peripheral nervous system. Thus, glucose and oxygen supply to neurons and waste metabolites remotion in the brain are enhanced (Sokolov, Pavlova, Klosterhalfen & Enck, 2013). Because NO is implicated in memory modulation, neuronal nitric oxide synthase (nNOS) inhibition leads to cognitive impairment. Recent studies evaluated memory and cognitive functions using rats with nNOS knockout in the Morris maze water test. Results showed impaired spatial performance of rats that may be attributed or linked directly to nNOS/NO knockout (Kirchner et al., 2004).

2.1.2. Chocolate as a delivery system of nutraceuticals

Changes in the confectionery industry have led to new products that provided good taste and health benefits, known as functional foods. The combination of consumer demands and confectionary products can be satisfied by adding bioactive components such as omega-3 polyunsaturated fatty acids (PUFAs, i.e. EPA/DHA) to a chocolate matrix (Konar et al., 2018). Likewise, most active ingredients used in the food industry need to protect the food matrix from passing through gastrointestinal tracts. Beneficial microorganisms, known as probiotics, are used in the food industry, generally in dairy vehicles, to maintain their

6 viability. However, many people do not consume these products because of lactose intolerance, allergies, or new food trends. For this reason, the use of chocolate is a good alternative as a vehicle of bioactive ingredients (Silva et al., 2017). Some examples of functional foods developed with chocolate as a delivery system are shown in Table 2.1.

On the other hand, when adding bioactive compounds to food formulations, the flavor is essential. Chocolate is a well-suited vehicle for masking unpleasant flavors because of its organoleptic characteristics (Kumar & Sharma, 2012). These vehicles are suitable and palatable for infants and children because they prefer sweet-tasting foods (Kumar & Sharma, 2012; Vasani & Shah, 2016). Therefore, it is found that milk chocolate is one of the most preferred for children and to high number of adults (Konar et al., 2018).

Table 2.1. Chocolate formulations used as delivery system of nutraceuticals.

Chocolate type and

presentation Active ingredient Results References

Semisweet Solid bar

Probiotics Lactobacillus acidophilus LA3 and Bifidobacterium animalis subsp. lactis BL

C1.

Bacterial viability (up to 120 days); panelists

acceptance (n=100). Silva et al. (2017)

Milk

Solid bar Omega-3 fatty acids (EPA/DHA) in different presentations: powder, microencapsulated

powder, oil and triglyceride from

different origins (microalgae and

fish).

The use of milk-chocolate as a delivery agent of EPA/DHA is confirmed; the microencapsulated form had the highest

acceptance.

Konar et al. (2018)

White Solid bar

Different origins affected after taste, overall acceptance, and color saturation; sensory

evaluation within acceptable limits.

Toker et al. (2018a)

Dark Solid bar

Successful fortification with EPA/DHA without causing adverse effects on quality;

microencapsulated form does not strongly affect quality.

Toker et al. (2018b)

Dark Solid bar

Probiotic Lactobacillus plantarum isolated from fermented cocoa beans.

Dark chocolate suitable carrier for probiotics with survival rate; no changes in

physicochemical properties.

Foong et al. (2013)

Dark

Chocolate masses and solid bar

Probiotics Lactobacillus caseii and Lactobacillus paracasei (lyophilized).

No significant differences in sensory properties compared to Lactobacillus-free

batches.

Nebesny, Żyżelewicz, Motyl, &

Libudzisz, (2007)

7 2.1.3. Quality parameters of chocolate

Chocolate is one of the most consumed confectionary foods because of the taste, sensory pleasure, and positive effect on emotions. Because chocolate is a complex multiphase system, refining and conching are critical phases to determine the consistency and particle size of the product to reach a specific texture and good sensory qualities (El- kalyoubi, Khallaf, Abdelrashid, & Mostafa, 2011). The milk chocolate system comprises solid particles (cocoa, sugar, and milk powder) dispersed in the fat phase (cocoa butter). The composition of these ingredients affects the final product sensory experience and rheological behavior as a fluid mass. To obtain high-quality products, the determination of these properties in chocolate manufacture must be well-defined to obtain the right palatable products and fulfill consumers' preferences (Glicerina, Balestra, Dalla Rosa, & Romani, 2015).

The rheology or flow behavior analysis of chocolate is of major relevance for quality control. This can be explained by the fact that if chocolate viscosity is too low, the texture would not be optimal, and if it is too high, bubbles may appear in the molded tablet. Also, the physicochemical properties not only affect viscosity but also affect the flavor. In other words, because consumers can perceive flow properties, the taste depends on the order and rate of contact related to the viscosity and melt rate. Chocolate rheology is usually quantified by yield stress and apparent viscosity parameters. Yield stress gives you information about the transition behavior from elastic to viscous deformation. In other words, it analyzes the change of chocolate from a pseudo-solid to a pseudo-liquid state; this pseudo-liquid state is related to minimum shear stress values. Rheological properties affect the final texture of chocolates, which plays a crucial role in the confectionery industry's elaboration process (Gonçalves & Lannes, 2010). On the other hand, molten chocolate has moisture content mainly from cocoa solids and sugar particles, which may increase friction and apparent viscosity values. Consequently, it is crucial to remove or avoid as much free water as possible to maintain the chocolate's flow properties and avoid non-desirable microbial growth (Afoakwa, Patersona & Fowler, 2007; Rezaei & Vandergheynst, 2010).

Furthermore, to back up the behavior of chocolate, human senses are used to evaluate the product's tasting notes. For this reason, sensory evaluation is also a key element to evaluate the elaboration process of chocolate to ensure high-quality products and to reach consumer's preferences (Muñoz & Gutiérrez, 2015).

8 In general, ingredient composition and processing techniques are the major factors affecting chocolate quality parameters, including physicochemical properties, rheological behavior, and sensory perception (El-kalyoubi, Khallaf, Abdelrashid & Mostafa, 2011).

2.1.4. Future trends on the use of chocolate as delivery system of nutraceuticals

Health and disease prevention through food is a growing demand for healthcare that has stimulated the development of smart foods. These foods are characterized by the addition of nutraceuticals in their formulations. However, one of the major challenges is that bioactive compound incorporation may have poor dispensability in food matrices, generate a rare taste, and physicochemical instability. For these reasons, it is essential to develop smart foods that have targeted and controlled releases of the compounds; hence, increasing their bioavailability in the gastrointestinal tract. An example of new technology is combining delivery systems with nanosized bioactive compounds' encapsulation (Donsì & Ferrari, 2020). These compounds provide health promoting-properties; some examples are b- carotene, lycopene, b-glucans, w-3 PUFAs, probiotics, isoflavones, among others.

Nanotechnology has shown a solution to improve the bioavailability and stability of these nutrients during processing. An example is w-3 PUFAs nanocapsules developed to mask odors and taste of fish oil. Also, these nanocapsules have shown excellent results in protecting live probiotics. Therefore, nanotechnology can aid in transforming chocolate into smart food by increasing nutrient content and ensuring the bioavailability of the bioactive compounds to the body that may be lost during shelf-life (Neethirajan & Jayas, 2011).

Recently, milk and dark chocolate bars were fortified using nanocapsules of carotenoids from Spirulina plantensis that generally taste bitter. However, by using nanotechnology no significant differences were found for aroma, taste, and texture from the fortified chocolates as compared with the control. This investigation showed that using nanocapsules of bioactive compounds may be suitable to combine the delivery of nutraceuticals with a chocolate vehicle (Ekantari et al., 2019).

2.2. Nutraceuticals and cognition

2.2.1. Importance of cognition and its relation to diet

Cognitive ability refers to an individual's ability to have memory, communicate, make decisions, plan, and pay attention. This process results from various brain communications

9 that allow information to be processed (Pitt & Leung, 2016). Cognitive functions decline over time, causing a shift in attention span and problem-solving. Some changes that have been found to be present and increase in degenerative diseases are alpha- and beta-amyloid plaques, tau pathology, vascular lesions, hippocampal shrinkage, among others (Howes, Perry, Vásquez-Londoño & Perry, 2020). Nowadays, there is a big concern to enhance cognitive development through childhood to decrease the risk of unwanted adult outcomes (Feinstein & Bynner, 2004). Childhood is a crucial stage for cognitive development because it has been shown to predict future characteristics of their life, such as academic achievement, success, and how they fit into society. Targeting early childhood stages can be a critical window to assess the factors that can affect brain function. There are approximately 200 million children worldwide who cannot achieve their fullest cognitive potential that can be associated with the economic growth in a country. In other words, it can implicate the deficit of the income in their future, affecting national development. For this reason, ensuring the optimal cognitive development of the children can help for social and economic growth. These can also benefit generational poverty by breaking the cycle (MAL- ED Network Investigators, 2018).

A poor diet and lack of physical activity can lead to current and future health problems in children. Early childhood is a crucial stage for cognitive development, being a time of vulnerable diet deficiencies for children (Tandon et al., 2016). This period is defined from birth to eight years old, where brain develops at its peak (UNESCO, 2019). A combination of serious factors such as marginal diets and malnutrition is thought to affect cognitive development (MAL-ED Network Investigators, 2018). The connection between nutrition and brain development falls into the main building blocks that the diet can provide. These blocks are critical to enhance enzyme function in the brain, the synthesis of DNA, the metabolism of hormones, neurotransmitters, cell proliferation, among others (Nyaradi, Li, Hickling, Foster, & Oddy, 2013).

Likewise, it has been found that a correct diet that contains specific food groups can reduce the incidence and prevalence of neurodegenerative disorders. These specific food groups are rich in micronutrients and are called nutraceuticals. These compounds can generate a synergistic effect between the food components and generate a neuroprotective effect (Mecocci, Tinarelli, Schulz & Polidori, 2014). One of the examples of nutraceuticals that influence brain hemodynamics is cocoa flavanols such as catechin and epicatechin. It has been found to aid blood flow, thereby improving the vision and performance of cognitive tasks. Likewise, other flavanols such as quercetin, myricetin, and kaempferol generate

10 positive cognitive development effects by helping neuronal repair. On the other hand, other nutraceutical compounds such as the carotenoid lycopene have been associated with improving healthy subjects' cognitive quality. A large part of these phytochemicals have an effect against cognitive impairment, but more research is needed to make comparative analyzes between them. Also, more research is needed to have information about the intervention, dosage, and metabolic bioavailability that these compounds may have. Further investigations should consider the use of combinations of two or more of these antioxidant compounds to evaluate their possible synergistic effects on cognitive development (Mecocci, Tinarelli, Schulz & Polidori,2014).

It has been shown that people who are supplemented with multi-nutrients have cognitive improvements due to the synergies between the compounds. Therefore, it has been found that those patients who have been supplemented with multi-nutrients that contained ω3 PUFAs as the main component that showed beneficial effects on cognition.

Therefore, it has been suggested that diets high in ω3 PUFAs, such as EPA and DHA) can reduce the risk of cognitive impairment or even Alzheimer's disease (Baleztena et al., 2018) On the other hand, a clear relationship between the gut microbiota and brain/

cognitive development has been reported, indicating a link between the gut-brain-axis where inflammation or the microbiota imbalance can generate a severe influence on neurodegenerative disorders of the central nervous system (Akbari et al., 2016). Recent studies have shown that probiotics have beneficial effects in anxiety models. In addition, probiotics positively affects behavioral functions in patients with multiple sclerosis and Alzheimer's disease. Therefore, it is of scientific relevance to study the role of probiotic bacteria in behavioral functions of the central nervous system (Hadizadeh, Hamidi & Salami, 2019).

2.2.2. Animal models of cognitive function

Specific animal models can help to assess behavior and cognition, specifically for neurodegenerative diseases. Tests such as the Morris water maze (MWM) and the Barnes maze (BM) test can be used. Depending on the type of animal models, different characteristics can be evaluated, such as recognition memory, episodic memory, emotional memory, spatial memory, among others (Wahl et al., 2017). Spatial memory is of high significance because it allows obtaining, retaining, and recovering knowledge about the environment. Likewise, spatial memory helps to map direct routes and remember goal locations that can help with adaptive problems. This type of memory participates in a series

11 of acquisitive and consolidative events where the hippocampus plays an essential role.

Spatial memory is considered as part of episodic memory and also part of long-term memory.

Long-term memory indicates the retention of information for at least 24 h, extending for weeks or months (Luna et al., 2018).

One method that can provide you with critical information about spatial learning and memory impairments in rodents is the BM test. This test, compared to the MWM test, assumes learning in a positive environment. This means that the rodent has to solve the maze using positive factors such as food, water, and shelter. The BM test starts from the assumption that the animal that stands in the middle of a circular surface must learn and remember where the safe shelter is located, also called an escape box. The apparatus consists of a circular platform with 18-20 holes on the periphery. This can be white, gray, or black as long as it allows the animal to be seen by the video program used. Likewise, to avoid certain aromatic distractions, it must be made of an easy-to-clean material. Also, it has a black box escape under one of the holes because rats prefer dark environments for shelter. As extra visuospatial cues, different figures can be placed at visible heights so that the animals can have points of comparison and location (Gawel et al., 2019).

The BM test begins with a rodent habituation phase, where it is introduced to the environment and is shown where the escape box is. A series of consecutive tests are then performed to evaluate spatial learning and spatial memory (Gawel et al., 2019). Then, acquisition training consists of two trials a day (up to 10 days), three minutes per trial, and 45 minutes between both daily trails. Before starting each trial, animals must be placed in the platform's center and cover 15 s by a start chamber. At the end of every trial, the platform must be cleaned with 70% ethanol (Pitts, 2018). Variants of the protocol have been carried out, but in general, this test evaluates spatial memory (short and long term) and working memory (Gawel et al., 2019). One of this test's main characteristics is that it is less stressful for the animals and less physically demanding than the MWM test. On the other hand, some disadvantages are that the test can be influenced by anxiety, aromatic cues, low motivation to explore, or low exploratory activities (Gawel et al., 2019; Rosenfeld & Ferguson, 2014).

The information obtained to evaluate the performance of the animals during the BM test is extensive. For example, the primary latency (time) to locate the escape hole for the first time, the total latency to locate the escape hole, the latency of the first scan in any hole, and the latency in which the animal remains in the escape zone (90º from the escape hole).

Likewise, it is essential to evaluate the number of errors made before entering the escape hole, those that are committed in the escape zone, and the total distance traveled (Pitts,

12 2018). In this case, it is very important to include multiple parameters. As a non-punishment test, rats tend to explore the platform with great freedom; therefore, the trials can be prolonged. Analyzing several parameters provides better information about the individual's capacity for memory and learning (Rosenfeld & Ferguson, 2014).

2.2.3. Role of omega-3 fatty acids on cognitive development

Omega-3 fatty acids (ω3) are PUFAs of 18-22 carbons with a double bond at the n- 3 position. Mammalian cells cannot synthesize these compounds, so it is usually obtained through the diet. The essential bioactive ω3’s PUFAs are EPA (C20: 5n-3) and DHA (C22:

6) (Robinson et al., 2010). These fatty acids have been found to play a crucial role in maintaining cognitive function in individuals through their neuroprotective properties (Dyall, 2015; Robinson et al., 2010). Populations with a higher intake of DHA have been found to have a lower risk of developing cognitive decline (Orellana et al., 2018). Likewise, it has been found that ω3 PUFAs have a beneficial effect on neuronal function, oxidation, inflammation, and cell death. The ω3 PUFAs are the major constituents of neuronal membranes, having DHA in a higher proportion up to 60%. DHA is more rapidly incorporated into gray matter, where neuron cell bodies are located, so it is essential to develop useful brain function. Therefore, it is important the adequate intake of DHA during childhood to have normal brain function, neurotransmission, vision, and synapsis plasticity (Robinson et al., 2010).

Fatty acids, such as ω3, have been found to form an essential part of children and infants' development because these compounds participate in severe neural processes, such as regulating membrane fluidity and gene expression (Rangel-Huerta & Gil, 2018).

Specifically, the chain length and high unsaturation of DHA provide fluidity and flexibility to the neurons' membrane. This allows and facilitates the transduction of signals inside the cell (Orellana et al., 2018). Therefore, it has been found that the imbalance or deficiency of these fatty acids is associated with the limited or poor development of a child. This can be translated into deficiencies in language, communication, motor development, verbal fluency, and problem-solving. Previous investigations showed that the supplementation of ω3 PUFAs at the age of 4 and 8 years generated improved academic performance evaluation (Rangel-Huerta & Gil, 2018). Likewise, it has been found that the accumulation of DHA in the brain begins in the uterus during pregnancy. This is typically generated during the second stage when the growth of gray matter in the brain accelerates (Rangel-Huerta & Gil, 2018). It has also been found that there is a rapid accumulation of DHA in a child's brain in

13 the last trimester of pregnancy and the in first two years of life. Likewise, other researchers found that the brain's gray matter accumulates the highest amount of DHA up to five years of age. Therefore, ω3 fatty acid supplementation during the early stages of a child and during pregnancy may be crucial for their future cognitive development (Orellana et al., 2018).

Various investigations have associated ω3 PUFAs with glial cells (microglia), having an anti-inflammatory effect. Microglia are glial cells of myeloid origin whose role is to maintain homeostasis in the brain, a crucial state of organisms because it plays an essential role in brain functions such as cognition and mood (Layé et al., 2018, Li et al., 2008). These cells are crucial due to their various functions in regulating brain development and pathological conditions that occur through inflammatory and non-inflammatory pathways.

Brain homeostasis detailed mechanisms have not been found, but these fatty acids have been shown to play an essential role in neuroinflammation cascades that help regulate microglial signaling (Layé et al., 2018)

2.2.4. Role of probiotics in the gut-brain axis

The human intestine is the site with the highest number of cells in the human body.

It has been reported that the intestinal microbiota is involved in various physiological processes, including the activation of the central nervous system (CNS), and there is evidence of communication of the gut-microbiota-brain (GMB) axis (Cheng, Liu, Wu, Wang,

& Tsai, 2019). The microorganisms present in the intestine affect the immune system's growth and regulation; therefore, it can help in the communication of the immune system of the CNS. These could be attributed to the synthesis of neuroactive molecules and metabolites that can modulate the pathogenesis of various neurodegenerative diseases such as Parkinson's, Alzheimer's, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis.

Intestinal dysbiosis may be behind these diseases by increasing pro-inflammatory cytokines, facilitating the pathogenesis of these disorders. Various studies have revealed that probiotic intake can help intestinal integrity by improving the mentioned disorders (Roy Sarkar &

Banerjee, 2019).

Despite the great diversity of research that exists, there are still no effective therapies for neurodegenerative diseases that decrease the cognition and functional abilities, such as Alzheimer’s. In recent studies, neuroinflammation and oxidative stress have also been associated with an essential role in the pathogenesis of this Alzheimer. These characteristics are influenced by the intestinal microbiota and probiotics (Agahi et al., 2018).

Therefore, new therapeutic solutions such as the use of probiotics have emerged to regulate

14 the microbiota-gut-brain axis, reducing Alzheimer's symptoms such as inflammatory reactions, oxidative stress, Beta-amyloid plaque deposition, and cognitive functions. Recent reports have found that neuroinflammation caused by Alzheimer disease is accompanied by an increase in pro-inflammatory cytokines such as TNF-α interleukin 6 (IL-6), which has been found in serum and brain tissue of Alzheimer's patients (Wong, Kobayashi, & Xiao, 2018).

Even though the exact mechanism of how probiotics act in neurodegenerative diseases is unknown, a large number of studies have shown that probiotics such as Lactobacillus and Bifidobacterium help improve memory deficits and cognitive development.

Individually or in combination, both probiotic species generate a positive change in cognitive function. On the other hand, it has been found that there is a multifactorial influence depending on the strains and the doses used (Den et al., 2020). In Table 2.2, studies on probiotic strains that have shown to exert a beneficial effect in models with cognitive problems are summarized.

15 Table 2.2. Effect of Probiotics in neurodegenerative diseases.

Microbial strains Results Study

Lactobacillus

plantarum PS128 (10^9 CFU/mL)

Ingestion alleviated motor deficits and neurotoxicity. Inhibited neurodegenerative process of PD model.

↑Neurotrophic factors, AOX levels

↓Glial hyperactivation, neuroinflammation, and oxidative stress

Liao et al.

(2020)

Lactobacillus helveticus R0052 and Bifidobacterium

longum R0175 (10^9 CFU/mL)

Alleviated apoptosis in the hippocampus induced by LPS exposed rats.

↓LPS-induced caspase-3 activation ↑Procaspase-3 expression

↓Cleaved caspase-3 in target tissues

Mohammadi et al. (2019)

Lactobacillus plantarum MTCC1325

(12x10^8 CFU/mL)

Delayed neurodegeneration in AD induced rat model.

↑Reversion of ATPase enzymes to normal levels (Helps to ATP brain homeostasis)

Mallikarjuna et al. (2016)

Lactobacillus paracasei KW3110

↓ Prevalence of IFN-γ -producing inflammatory CD4-positive T cells

↓ Serum levels of pro-inflammatory cytokines ↓Retinal inflammation by pro-inflammatory cytokine-producing

macrophage

Morita et al.

(2018)

Lactobacillus rhamnosus (JB-1)

↓ GABAAα2 mRNA expression in the prefrontal cortex

↓ Reduced stress-induced corticosterone

↓Anxiety/ depression behaviors

Bravo et al.

(2011)

Lactobacillus helveticus strain NS8

(10^9 CFU/mL)

↓ Neuroinflammation

↑Cognitive function

↓ Anxiety-like behavior

Luo et al.

(2014)

Improved chronic stress-like behavior and cognitive dysfunction.

↓ Plasma corticosterone

↑ IL-10

Liang et al.

(2015)

Lactobacillus Plantarum 299v (10x10^9 CFU)

Improved cognitive performance in MDD patients.

↓ Kynurenine (neurotoxic on CNS) concentration

Rudzki et al.

(2019)

Lactobacillus rhamnosus

GG (10^10 CFU) ↑Cognitive function ↑ Scores total cognition Sanborn et al. (2018)

Bifidobacterium breve A1 (2X10^10 CFU, 24 weeks)

↑Cognitive function in MCI model assessed in elderly participants

↑ MMSE and DSST test scores

(Kobayashi et al., 2019)

Bifidobacterium longum (NK46) (1X10^9

CFU/day)

Alleviated cognitive decline Suppressed:

NF-κB activation, TNF-α expression, amyloid-β, β/γ-secretases, caspase-3 expression, and amyloid-β accumulation in the

hippocampus

↓ LPS blood levels

Lee et al.

(2019)

Abbreviations: CFU; colony-forming units; AOX, Antioxidant; PD, Parkinson's disease; LPS, Lipopolysaccharide; AD, Alzheimer's disease; IFN-γ, interferon-gamma; MDD, Mayor Depression disease MCI, mild cognitive impairment; MMSE, Mini Mental State Examination test; DSST, Digit Symbol Substitution Test; NF-κB, Nuclear factor κB; TNF-α, Tumor necrosis factor α.

16 2.2.4.1. Methods to stabilize probiotics

Over time, the demand for food products containing probiotic strains has increased.

On the other hand, using these beneficial microorganisms is difficult from a technological perspective, because of their demanding nutrition and sensitivity to the environment they are subjected to during industrial processes. These changes can also generate unwanted attributes in the final product, reducing its shelf-life. Some parameters that must be considered to preserve the viability of probiotics are the pH, water activity temperature (aw), oxygen content, presence of other microorganisms, among others (Gueimonde & Sánchez, 2012).

Probiotics must be protected appropriately, not only to survive during industrial processes but also in the intestinal tract's acidic environment. Therefore, probiotic encapsulation techniques, such as microencapsulation, have been developed to maintain their stability. Microencapsulation is a process where probiotic cells are incorporated into an encapsulating matrix to protect them against damaging factors from the environment. This technology allows protection against low pH and bile salts from the gastrointestinal tract (Shori, 2017). The most used techniques for the microencapsulation of probiotics in large scales are freeze-drying and spray-drying (Iravani, Korbekandi & Mirmohammadi,2015).

The encapsulation of probiotics employing spray-drying has several advantages, such as maintaining optimal humidity in the powders. Likewise, in large scales production, this process is fast and low cost. Also, powders obtained from the process result in small particle size maintaining adequate cortex protection (Jantzen, Göpel, & Beermann, 2013).

Because hot air and rapid dehydration are used in this process, lactic acid bacteria need compounds that protect them, such as whey protein, prebiotics, granular starch, soluble fiber, and trehalose (Su et al., 2019; Liu et al., 2015 ).

17

CHAPTER 3

PHYSICOCHEMICAL PROPERTIES AND SENSORY ACCEPTABILITY OF A NEXT GENERATION FUNCTIONAL

CHOCOLATE ADDED WITH OMEGA-3 AND PROBIOTICS

3.1. Synopsis

Omega 3 (ω3) fatty acids and probiotics (L. plantarum 299v and L. rhamnosus GG) are nutraceuticals related to enhancing cognitive development in children. In this study, a functional milk chocolate formulation was developed to serve as a vehicle for both nutraceuticals. Probiotics (Prob) and fish oil (FO) were added during the tempering process and chocolate formations with either 76.0±5.2 mg (FO1) or 195.8±6.5 mg (FO2) ω3 fatty acids and >1x106 CFU of probiotics per chocolate portion (12 g) was obtained. The physicochemical properties (rheological analysis, texture, surface instrumental color, !_!, texture, and fatty acid profile), and sensory acceptability of chocolate formulations were determined. Prob and FO addition generated a decrease in L* and white index (WI) values.

Results showed a decrease in !_! for all treatments except for Prob+FO2. Hardness values decreased, considering all treatments except for Prob. Rheological parameters of FO1 and Prob+FO1 presented the most similar behavior as compared with the control. Sensory acceptability test indicated that when chocolate formulations were added with either Prob or FO1, the product's overall acceptability was not affected compared with the control; whereas when both nutraceuticals were added combined Prob+FO1 the product showed adequate overall acceptability (>7, like moderately). FO2 formulation with and without Prob was not considered adequate to maintain chocolate stability parameters and sensory acceptability.

Results indicated that milk chocolate is a suitable vehicle for delivering fish oil and probiotics, which are essential to enhance cognitive development in children.

3.2. Introduction.

Today, society seeks new eating habits and lifestyles to improve their life expectancy.

These new eating habits can help to prevent various diseases such as diabetes, cardiovascular disease, obesity, Alzheimer's, Parkinson's, among others. For this reason, the food industry has strived to develop next-generation functional foods that can meet these