Campus Monterrey
School of Engineering and Sciences
High-Protein Diets Effect on Metabolic Profiles, Gut Microbiota and Inflammation Markers in a Murine Model.
A thesis presented by
Laura Bárcena Lozano
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 3rd, 2020
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This thesis project is dedicated to my sister and my parents.
Muchas gracias por haberme dado la oportunidad de poder llegar hasta donde estoy hoy, por poder haber estudiado y disfrutado de los momentos gracias a vuestro
esfuerzo y a vuestros ánimos que me han impulsado a seguir adelante.
Gracias por enseñarme los valores de viajar, compartir, respetar, investigar y aprender a valerme por mí misma.
I also dedicate this work to my friends. You have been side by side to me in the laughter but also in the complicated moments of this year. This journey has been more
beautiful and fun having you near me.
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I mainly thank the University of Oviedo and Tecnológico de Monterrey the chance to be able to attend my studies in the MBI program.
I thank CONACYT for the economic support given that has given me the opportunity of stay in México.
I thank the CICUAL Committee for allowing the experimental procedure at the Vivarium of Tecnológico the Monterrey.
I thank Nutriomics and Emerging Tecnologies group at Tecnológico de Monterrey for their acceptance and for the allowance to work with them.
At the same time, I thank the workers of the Vivarium for their help every day of the experimental procedure, but specially to the MVZ Mirna Castañeda and Paulo Martínez, who without their guidance, the project would not have been possible.
I thank the CIDPRO department for the allowance to work with them and for the brought help.
I thank IBQ Fátima Alvarado Monroy, for the assistance with the immunological analyses.
I thank the students and workers at the Nutriomics group of Dra. Yolanda Arlette Santacruz López; I wouldn’t have been able to complete this work without you. I give special thanks to my laboratory and thesis partner Mayra Sánchez, that has been working next to me in every step of the project, through every obstacle and winning. Also, to Felipe López, for the optimum support in every step of the project.
Last but not least, I thank Dra. Yolanda Arlette Santacruz López for the guidance and help through the master’s thesis, and for making it possible to perform this research.
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and Inflammation Markers in a Murine Model by
Laura Bárcena Lozano
ABSTRACT
Dietary food is a key factor that limits the composition of microbial communities in the gut. Extreme diets cause a gut microbiota dysbiosis, modifying immunological markers and being able to produce inflammation in diverse organs. Specialized diets for losing weight and gaining muscle mass, and a raising economical support to obtain meat products in the market have doubled the recommended protein consumption amongst the population. The aim of this work was to elucidate the effects over metabolic profiles, gut microbial communities and inflammation markers that a high-protein diet, vegetal- and animal- based, can cause in a murine model. 27 male mice of 17 weeks of life (Mus musculus C57BL/6) divided in 3 groups: 1) vegetal, 2) animal and 3) standard were fed ad libitum with a high-protein diet (25- 30 %) for 7.5 weeks, following the directions of CICUAL and the Vivarium of Tecnológico de Monterrey. Mice were weighted every week. After the experimental phase, epididymal fat was measured in every group. Also, cecum samples were analysed by qPCR to evaluate the changes in gut microbiota (total bacteria, Bifidobacterium, Lactobacillus, Enterobacteria). Blood samples were collected to obtain serum, and the inflammation markers TNF-α, IL-6 and IL-10 and were analysed by Milliplex® MAP technology and the CRP by ELISA. The statistical tool Minitab® was used to process the results through ANOVA and transforming the data when necessary. Vegetal-based protein diet individuals had more epididymal fat than the rest of the groups. Moreover, they showed a higher IL-10 production as well as the CG. Nevertheless, microbial communities were compromised in the animal-based protein diet, showing signs of dysbiosis, although not presenting excessive production of inflammatory cytokines. Therefore, metabolism of a protein excess with similar amino acid profile may have negative consequences in amino acid utilization and formation of by-products, gut microbiota profiles and inflammation in gut depending on the protein source.
Keywords: Amino acids, Gut microbiota, High-protein diet, inflammation.
VI SCFA Short Chain Fatty Acids
qPCR Quantitative Polymerase Chain Reaction ELISA Enzyme-Linked Immunosorbent Assay
CICUAL Comité Interno para el Cuidado y Uso de los Animales de Laboratorio
CIDPRO Centro de Investigación y Desarrollo de Proteínas CRP C- Reactive Protein
MCP-1 Monocyte Chemoattractant Protein- 1 IL-6 Interleukin- 6
IL-10 Interleukin- 10
TNF-α Tumoural Necrosis Factor - α WHO World Health Organization FEN Fundacón Española de Nutrición FAO Food and Agriculture Organization
FIRA Fideicomisos Instituidos en Relación con la Agricultura SAGARPA Secretaría de Agricultura y Desarrollo Rural
TLR Toll-like Receptor NLR Nod-like Receptor
MALT Mucosa-Associated Lymphoid Tissue GALT Gut-Associated Lymphoid Tissue ILF Isolated Lymphoid Follicles
MAMPs Microbe-Associated Molecular Patterns IL-1 Interleukin- 1
CG Control Group
VII VPD Vegetal Protein Diet DC Dendritic Cells NK Natural-Killer cells
BCAAs Branched-Chain Amino Acids PBS Phosphate Buffered Saline
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Figure 1. Weight increase of the three diet groups 28
Figure 2. Microorganism communities in cecum 35
Figure 3. Inflammation markers in serum samples 37
Figure 4. SCFAs measured in colon 39
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Table 1. Overall effects on gut microbiota of high-protein diets 11
Table 2. Diets formulation 21
Table 3. Amino acid composition of protein extracts 22
Table 4. Physicochemical properties of the pellets 23
Table 5. PCR characteristics of bacterial groups 26
Table 6. Metabolic profiles 30
Table 7. Total amino acidic content in feed per Kg of diet 32
Table 8. Microorganism quantity in cecum 35
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Abstract V
Abbreviations and Acronyms VI
List of Figures VIII
List of Tables IX
Chapter I. Introduction 1
Chapter II. Literature Review
2. 1. Diets: Characteristics and Requirements in Humans 3
2. 2. Gut Microbiota 6
2. 3. Immune System and High Protein Diets 11
2. 4. Short Chain Fatty Acids 15
Chapter III. Hypotheses and Objectives
3. 1. Hypotheses 17
3. 2. General Objective 17
3. 3. Specific Objectives 17
Chapter IV. Materials and Methods
4. 1. Animals and Stay Conditions 18
4. 2. Experimental Design 18
4. 3. Protein Extraction 19
4. 4. Diet Preparation 20
4. 5. Quality Control 23
4. 6. Animal Sample Extraction 24
4. 7. Quantitative Polymerase Chain Reaction 25
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4. 9. Gas Chromatography 27
4. 10. Statistical Analyses 27
Chapter V. Results and Discussion
5. 1. Metabolic Profiles 28
5. 2. Microbial Communities in the Gut 34
5. 3. Inflammation Markers 36
5. 4. SCFAs 38
Chapter VI. Conclusions 41
Chapter VII. Future Perspectives 42
Chapter VIII. References A
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I. INTRODUCTION
Nowadays, there is a continuous rise in protein consume all over Western countries (World Resources Institute, 2016), mostly induced by the economical availability of foods rich in protein such as meat and legumes (Salter, 2018; Charania &
Li, 2019) and the latest trends focused on weight loss (Astrup et al., 2015) and muscle mass gain (Morton et al., 2018). The focus over the consume of high protein diets due to supplementation with mixtures of hydrolysates powders or by augmenting the percentage of protein containing nutrients (Pasiakos et al., 2014; O’Bryan et al., 2020), has taken center stage by many researchers.
High protein diets have been demonstrated to favor weight loss, and enhance muscle mass formation, although it seems to depend on the origin and quantity of the protein (Phillips, 2016). Nevertheless, the effects caused in overall health depending on the origin of the protein and the quantity administered seem to be at least contradictory.
On the one hand, animal proteins are generally related to protein synthesis in muscle an improved metabolic status (Gilbert et al., 2011), although they may cause systemic inflammation (Mei et al., 2011). On the other hand, vegetal proteins have been seen to induce a healthier microbiota profile and better immune function in the digestive tract (Singh et al., 2017). Again, there have been found differences even inside the animal-protein group and vegetal protein group depending on the exact origin and amino acid composition of the proteins (Bech-Nielsen et al., 2012).
The origin and amino acid composition of the proteins would influence the digestibility, absorption and the utilization of the amino acids in the host, and therefore influence fat metabolism, muscle metabolism (Arentson-Lantz et al., 2019), microbiota profiles by the availability of nutrients and changes in the lumen environment (Bibbò et al., 2016), and hence the immunomodulatory properties of gut microbiota and their byproducts in the mucosal and epithelial immune system (Kolodziejczyk et al., 2019).
In México, black beans and read meat are foods with a high protein content (16- 25 %) (Raya-Pérez et al., 2014; Ahmad et al., 2018) that are highly consumed (Grajales- García et al., 2012). Black beans have been related to beneficial effects (Mudryj et al., 2014), consume de red meat on the contrary, has been related to cancer and instability in gut (Zhao et al., 2017). Although, there are discrepancies on the effects that only their protein extracts may cause (Gilbert et al., 2011).
In this project, a high protein diet (25 - 30 %) derived from protein extracts from black beans and corn and read meat with a similar amino acid composition have been
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administered to young adult mice (Mus musculus C57BL/6) for 7.5 weeks. Metabolic profiles, gut microbiota composition, inflammatory markers and short chain fatty acids were measured. Both of the groups showed differences between them and compared to the control group (20 % protein from various origins). Metabolic profiles get improved with animal protein and show lipid accumulation in the group with plant protein.
Nevertheless, signs of dysbiosis appear with the animal protein, although the vegetal protein group have lower quantities of probiotic bacteria. Immune system was more activated in the control and vegetal group, rather than the animal protein group. Short chain fatty acid accumulation in colon was higher in the control group, due to a higher carbohydrate rate in the diet, and lower in the plant protein group.
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II. LITERATURE REVIEW
2. 1. Diets: characteristics and requirements in humans.
Reaching a consensus over the finest and healthier diet has always been the major of the challenges and goal to reach to many nutritionists over decades. Macro and micronutrient intake and their effects over human health are constantly being reevaluated as new benefits get elucidated in every research made (Solon-Biet et al., 2014).
Generally, the recommended macronutrient percentage to be taken in the diet is focus on the quantity of fat, protein and carbohydrates, including fiber, that should be daily consumed. These can be summarized in no more than 30 % of fat, between 55 and 60 % of carbohydrates including from 21 to 38 g of fiber per day and a 10 to 15 % of protein in an adult’s diet with normal physical activity as stated by the Spanish Nutrition Foundation (FEN) the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO). It is necessary to adapt it to the individual’s requirements as it is crucial for the health maintenance (Burd et al., 2019). Nevertheless, cultural and economic variables have a direct impact on achieving the consume of the rates and the quality and origin of the previously mentioned macro and micronutrients (Dernini & Berri, 2015). One of the major changes in the consume rates is related to the protein intake. The European Food Safety Authority (2019) recommends consumption from 0.66 g to 0.83 g of a high-quality protein/ kg of the person’s weight per day.
Therefore, an appropriate quantity of protein would be of 44 g per day for women and 56 g per day for men. However, the quantity of protein taken per person per day in these Western countries is above 87 g with no distinction between men and women (World Resources Institute, 2016). The protein amount that should be taken per person per day is being doubled, risking the probabilities of inducing a pathophysiological condition (Carbone & Pasiakos, 2019).
2. 1. 1. Protein rich foods increased availability and consumption.
Meat based products and those rich in protein like legumes have been consumed worldwide over the years with an increasing tendency (FAO, 2013). This trend could be explained inherently due to the culture in a certain region, the development of intensive practices in farming and technological innovations and its economical ease for the reachability of a wider number of regions (Salter, 2018; Charania & Li, 2019). Moreover, in the named Western societies there is an existent tendency between young adults of significantly augmenting the protein intake on a daily basis in order to losing weight
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(Astrup et al., 2015; Eisenstein et al., 2002) or gaining muscular mass (Samal & Samal, 2017; Morton et al., 2018). They manage to reach that purpose by increasing the percentage of protein-rich foods in the diet or by taking supplements (Pasiakos et al., 2014; O’Bryan et al., 2020).
Meat consume in Mexico is a clear example of the above-mentioned tendency in the augment of consumption protein-rich foods as there has been a rise in a 52 % from 1993 to 2013 (FAO, 2020a). This derives in the rise of supply of protein, that has increased to almost 100 g per person per day as the last estimated (FAO, 2020a). From that estimation, animal protein constitutes the 44 % and more than 50 % is to plant protein (FAO, 2020b). Between plant protein sources, black beans (Phaseolus vulgaris L.) constitute a crucial source, and they are highly produced and consumed in México, being in the third place of the world’s consumers in 2017, as gathered by FIRA (2019a).
Black bean per capita consume in México is of 10.4 kg per year (FIRA, 2019a) and the total amount is actually of more than 1,100 million tones, as estimated by SAGARPA (2017a). Therefore, beans represent a major nutrient consumed in this country. Also, cereals like corn are very important in Mexicans’ diet and economy, as they are the seventh producers in the world and the fifth consumers (FIRA, 2019b), having a per capita intake of 196 kg per year (SAGARPA, 2017b).
In reference to the consume of animal protein, México is the seventh country in the world’s ranking of bovine meat consumption (by the year 2018). Mexicans consumed an annual mean of approximately 9 kg of bovine meat in 2018 (FIRA, 2019c), making it an essential product in the population’s diet. From 16 % to 25 % of the composition of the black bean in grams is protein (Peña-Betancourt & Conde-Martínez, 2012; Raya- Pérez et al., 2014), and 21 % in beef cuts (Ahmad et al., 2018). It can be concluded that these food types correspond an important source of dietary nutrients in the Mexican diet (Grajales-García et al., 2012).
2. 1. 2. Dietary protein metabolism.
Dietary protein is essential for daily energy intake, maintenance of cells and muscles and creation of macrostructures in the body (Dangin et al., 2002). It is the most satiating macronutrient (Mackie & Macierzanka, 2010), and its absorption depends on the mixture of nutrients it has been taken with, the health-status of the individual and the quantity and source of protein (Danging et al., 2002). In the stomach, they get hydrolyzed by pepsin and when reached the small intestine, other enzymes, bile acids and phospholipids, digest, denaturize and change the properties of the dietary proteins (Mackie & Macierzanka, 2010). The small intestine is where polypeptides are mostly
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processed (90 - 95 %), and turn to free amino acids, dipeptides and tripeptides.
Remaining non-processed or non-absorbed ones end in the large intestine. It is in the enterocytes where peptides transform into single amino acids and get used in the own cells or enter the circulatory system (Bhutia & Ganapathy, 2018; Have et al., 2007). In its assimilation in the body, the amino acid composition takes part and a minimum protein intake to stimulate its synthesis in the body (>1.5 g/ (kg diet x day)) (Dangin et al., 2002).
Gut barrier cells metabolize up to 30 – 50 % of the absorbed proteins, for the protein turnover, and the remaining 50 % is sent to the portal-vein system. The intestinal mucosa is a tissue where the highest rates of protein synthesis take part (Have et al., 2007).
The mixture of macronutrients in the meal and the composition of amino acids of the proteins produce a significant variability in the percentages of protein, peptide and amino acid metabolism, as well as the effects they entail (Have et al., 2007).
A high-quality protein, defined as the one that contains the richest amino acid composition, contributes better on the absorption and availability of amino acids in serum (Have et al., 2007). Generally, animal-origin proteins would contribute better to these tasks rather than vegetal-origin proteins due to the amino acid composition (Lonnie et al., 2018). Thermogenesis, fat reduction and muscle gain, are also dependent of the amino acidic composition of the proteins taken in the diet, and the quantity, quality and speed processing of the protein (Westerterp-Plantenga et al., 2006). Proteins change the speed of gastric emptying, that determine its availability in the intestine. Slow-speed proteins like casein have a slow and more continuous release into the gut and favor the anabolism of muscles. On the contrary, high-speed proteins have a more rapid release of amino acids into the portal system and cells, favoring the gluconeogenesis and oxidation processes (Have et al., 2007). The process of digestion of proteins also depends on the protein source and structure; casein precipitates in the stomach, where it gets hydrolyzed, while other type of proteins as soy protein, is soluble and is a high- speed protein that rapidly reaches the intestine, where it gets digested (Jahan-Mihan et al., 2011). It also needs to be taken into account that is difficult to rate and quantify the absorption of dietary proteins and their comparison, as the gut receives amino acids from two sources: the diet in the gut lumen and the synthesized and carried by the systemic circulation (Have et al., 2007). In the distal colon is where the higher fermentation, or putrefaction of single amino acids and peptides happen. Microorganisms take part in this crucial process, were the help digesting these molecules and also produce important compounds for the individual’s absorption (Riaz-Rajoka et al., 2017).
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2. 1. 3. Secondary effects on overall health of specialized diets.
It is well known the overall effects that some extreme diets may cause over metabolic profiles, gut microbiota, and their relationship with some diseases (Kolodziejczyk et al., 2019). These could be summarized in rich in fiber, rich in fat and rich in protein content. An important quantity of researchers show that a high fiber diet could be extremely beneficial for the health, as it reduces pro-inflammatory proteins expression (Morowitz et al., 2017), increases the number of beneficial bacteria (So et al., 2018), improves glucose tolerance (Boulangé et al., 2016) and supports hosts’ immunity against harmful gut pathogens (Sivaprakasam et al., 2016). Otherwise, a high-fat diet has been related to harmful effects for the host’s health such as weight gain, adipose liver (Heijden et al., 2015), permeabilization of the intestinal barrier (Sonnenburg &
Bäckhed, 2016) and a decrease of bacterial diversity (Boulangé et al., 2016). General effects of these two types of diet have been able to be elucidated and specified.
On the contrary, there is a lack of studies that could grant transparency over the concrete effects that a diet rich in protein origin may cause (Azadbakht et al., 2013;
Cuenca-Sánchez et al., 2015). In fact, most of the studies reduce the calorie intake made by proteins (Bisanz et al., 2019), plus depending on the protein origin, their overall effects seem to be distinct from one another (Kostovcikova et al., 2019). A study performed by Cui & Kim (2018) stated that a diet rich in animal protein increased inflammation in the gut and induced colitis, while a rich in plant protein showed no progress on inflammation.
Also, there is prove that a high protein diet from beef origin in obese mice, increase systemic inflammation by measuring key inflammatory markers as TNF-α, IL1β, IL-6 (Ijaz et al., 2018). Plus, a diet with exceeded protein quantity is proved to induce lower life expectancy (Solon-Biet et al., 2014). On the other hand, a study performed with mung bean isolate reduced inflammatory processes in mice liver, while high concentration of casein or soy protein wouldn’t (Watanabe et al., 2017). Moreover, some other toxic and harmful by-products of protein digestion get released to the colon lumen as phenols and indoles, ammonia, amines and thiols and hydrogen sulfide compounds, that function as carcinogens, mutagenic, and as cellular toxins, respectively (Macfarlane & Macfarlane, 2012; Yao et al., 2015).
2. 2. Gut microbiota.
Human beings live in a continuous symbiosis with microorganisms in different parts of their bodies (Karkman et al., 2017), and most of their population are found in the digestive tract (Rajilić-Stojanović & de Vos, 2014). The abundance and diversity of the
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microbial populations in the same individual are not stable during our life (Costello et al., 2009). They evolve and vary with human’s age, physical activity, health status, diet and an infinite number of ambient variables (Rinninella et al., 2019a).
Gut microorganism communities are formed by more than 35000 bacterial and fungi species, and each region of the gut is inhabited by different genera in response to a diversity of existent microenvironments (Jandhyala et al., 2015). Predominant microorganism phyla alongside the intestine of healthy individuals are Firmicutes and Bacteroidetes, representing the 90 % of gut microbiota (Rinninella et al., 2019a) and followed by Actinobacteria and Verrucomicrobia (Carding et al., 2015). In the small intestine genera as Clostridium, Streptococcus, Lactobacillus, Proteobacteria and Enterococcus can be found. Following the anatomic structure, the cecum region in the large intestine is more diverse than the previous regions (Hayashi et al., 2005). Cecum is mainly populated by Roseburia, Ruminococcus, Fecalibacterium, Butyvibrio and Fusobacteria (Jandhyala et al., 2015), although some authors have found high amounts of Lactobacillus and E. coli (Marteau et al., 2001). Finally, in the ascending, transverse and descending colon regions there can be found Clostridium, Prevotella, Streptococcus, Enterobacteria, Enterococcus, Lactobacillus, Ruminococus and Eubacterium amongst others (Jandhyala et al., 2015). Generalizing into the most represented phyla, Bacteroidetes is higher in the first regions of the intestine, but diminish in the large intestine, where Firmicutes outstands (Wang et al., 2005). Studies performed by Swidsinski et al. (2005) in mice show that there are also differences in the organization of the microbial communities in the gut lumen, as some are more attached to the mucosa and others stay outer to the luminal area. They found that Lactobacillus, Enterococcus, Clostridia and Proteobacteria were more attached to the mucosa, while others as Bifidobacterium, Streptococcus, Prevotella, and the general phyla Bacteroides, Firmicutes and Actinobacteria were only found in the feces part inside the gut. Plus, in mice with inflammation in colon, Bacteroides, Clostridium coccoides and Eubacterium rectale were found invading the tissue.
On the other hand, not only the quantity of the microorganisms is crucial but also the diversity, as it has been proven that in inflammatory diseases there is found lower gut microbial richness (Valdés et al., 2018). As an example, a high amount of Bacteroides, Prevotella and Ruminococcus, as well as Lactobacillus, Bifidobacterium or Akkermansia contribute on the maintenance and improvement of gut homeostasis (Jandhyala et al., 2015; Hills et al., 2019).
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2. 2. 1. Importance of microorganisms in our body.
Gut microbiota is involved in the regulation of the gut-related health system, through the maintenance of a balance and homeostasis in the gut lumen (Kho & Lal, 2018). Recently, it has been proven that not only is related to the gut health, but also to the general nervous, immune and endocrine systems (Westfall et al., 2017). The interaction between the systems is known between the scientific community as the Gut- Brain-Microbiota axis (Zhuang et al., 2018), therefore, changes in one of the axes can affect the others, having an impact in the individual’s health (Jiang et al., 2017).
Generally, they remain in a thin equilibrium as long as there is stability in the axis and in the environmental factors, beneficially contributing to the individual’s health (Theilmann et al., 2017). Each type of bacteria produces different molecules that can be beneficial or harmful to the organism, and therefore, a different composition or a change in the gut microbiota pattern translates in a different ratio of the metabolites they excrete, that in the end result in different effects on the organism (Walker & Lawley, 2013). There is also no need to say that diversity is important, as it is related with metabolic health and the previous mentioned metabolite ratio (Sonnenburg & Bäckhed, 2016).
Numerous examples have been described of a beneficial relationship in digestion. Probiotics produce bacteriocins that maintain pathogenic or commensal bacteria in low proportion (Anjum et al., 2014) plus benefit from specific adhesion mechanisms to allow it (Monteagudo-Mera et al., 2019). SCFAs produced in gut by probiotic bacteria contribute on the maintenance of colonocytes and the mucus layer (Martin et al., 2018). Moreover, they produce vitamins and contribute in the metabolism of bile salts (Sharma et al., 2020). Specific secreted metabolite examples are those as the following: Bifidobacterium excretes complex vitamin B and lactic acid, that maintain the immune system active. Lactobacillus and Bacteroidetes in general produce the SCFAs in higher proportion. As mentioned before, they have antioxidant activity in diverse organs throughout the body, maintain the mucosa and are energy sources for colonocytes. Streptococcus, also with a high presence in colon, produce folic acid with anticancer activity (Das et al., 2020).
On the other hand, it has been reported that when there is a dysbiosis in the composition of microbial communities, intestinal barrier disrupts, individual’s metabolic profiles worsen and low-grade inflammation appears in the gut and other organs (Martin et al., 2018). Disruptions or harmful effects in the gut activate a response from the host’s immune system, involving pro-inflammatory cytokines produced by macrophages that reach the damaged site and induce a low-grade inflammation and permeability in blood
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vessels to try to repair the injured region (Levy et al., 2017). Although, the chronic exposure to the effects involving inflammatory and immune system processes can lead to chronic inflammatory diseases (Carding et al., 2015) such as, Irritable Bowel Syndrome and Inflammatory Bowel Disease (Kostic et al., 2014), metabolic syndrome (Carding et al., 2015) and cancer (De Almeida et al., 2019). Diverse authors define and recognize a dysbiosis when there is a loss of diversity, loss of probiotics and commensal bacteria and an unexpected and quick rise in the quantity of pathogenic bacteria. Some also state specific bacterial groups as definers of the dysbiosis such as Fusobacteria and Enterobacteria (Brüssow, 2019). Studies made with patients undergoing gastrointestinal disorders showed a bigger decompensation in the microorganism concentration ratio than the healthy individuals (Saffouri et al., 2019).
2. 2. 2. Intestinal microbiota and nutrition.
Due to the different microenvironments and microorganisms housed in the intestine, the fermentation, absorption and transformation of nutrients take place in defined parts. The majority of nutrient transport to the portal system occurs in the small intestine (Kiela & Grishan, 2017), although is in the large intestine where the most important fermentation of fibre and putrefaction of proteins occur due to the presence of certain microorganisms (Rowland et al., 2018), and therefore the transport of the by- products. Fermentation of dietary fibre as polysaccharides and oligosaccharides (Hugenholtz et al., 2013) slightly starts in the ileum, moving forward to the large intestine where the microorganism fully digest it (Williams et al., 2017). The cecum region is the site of a major fermentation of fibre as its removal shows a significant decrease in the detection of dietary fibre by products plus a lower bacterial diversity in the continuing colon regions (Brown et al., 2018). In studies in mice altering the diet for four weeks, microbiota patterns in lower ileum were altered severely augmenting the Lactobacillus quantity over a prebiotic component in the feed mixture (Kawakami, 2020).
Putrefaction of peptides occurs mostly in the transverse and distal colon, and some of the microorganisms involved in the process are species from Bacteroides, Eubacterium, Propionibacterium, Clostridium (Rowland et al., 2018), Streptococcus and Lactobacillus. When metabolized by anaerobic bacteria they produce terminal H- compounds, SCFAs or gases (Davila et al., 2013).
In conclusion, depending on the food intake of percentages of macronutrients, microorganism rates specialised in digesting certain types of molecules will get modified (Tan et al., 2014).
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2. 2. 3. Effects of excessive dietary protein over gut microbiota.
An excessive intake of dietary protein above the recommended quantity per day produces in some cases detrimental changes over gut microorganism communities. A summary of the effects over gut microbiota can be seen in Table 1. In rats fed with a high-protein diet from animal origin for 6 weeks, it was found that probiotic microorganisms were decreased, and genera from the Enterobacteria family was increased (Mu et al., 2017; Mu et al., 2016). Excessive dietary protein also has different results depending on the site of the large bowel. The diet rich in protein also have different effects over the cecum communities and the changes in the rest of the colon, as there were found no changes in diversity in cecum, but they did in colon (Liu et al., 2014). It has also been proven that depending on the percentage administered with the same protein origin, the effects in the same microorganism quantities change (McAllan et al., 2014). With the intake of a 20 % of whey extract Bifidobacterium increased more than in a 20% of only casein, but in a 40% of whey extract its population got reduced lower than with the 20 % of casein. The effects caused by different origins with a normal dose of protein (20 %) was stated by Zhu et al. (2015). They administered 5 types of protein extracts, ended grouping them in 3 clusters due to the common effects on gut microbiota though. They were red meat (beef and pork), white meat (chicken and fish) and others (casein and soy). The Bacteroidetes: Firmicutes ratio was altered having more Firmicutes percentage the ones with white meat food and the soy ones had more Bacteroidetes. There also were concrete differences in bacterial genera, proving that specific bacterial patterns were modified as well.
TABLE 1. OVERALL EFFECTS ON GUT MICROBIOTA OF HIGH-PROTEIN DIETS Study
Model Protein Origin of protein
Time of diet intake
Gut microbiota
changes References Wistar adult
rats
45 % Casein 6 weeks ↓ Roseburia
↓ Prevotella
↑ E. coli
↓ Akkermansia
↓ Bifidobacterium
Mu et al. 2017
Wistar adult rats
45 % Casein 6 weeks ↓ Actinobacteria
↑ E. coli
↑ Shigella
↑ Enterococcus
↑ Streptococcus
↓ Bifidobacterium
Mu et al. 2016
Wistar adult rats
53 % Whole milk
proteins
15 days ↓ Clostridium
Lui et al. 2014
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Sprague- Dawley
20 % * Beef and
pork
90 days ↑ Proteobacteria
↓ Roseburia
↓ Prevotella
Zhu et al. 2015 Chicken and
fish
↑ Lactobacillus
↑ Firmicutes
↓ Bacteroidetes
↑ Actinobacteria
Soy ↑ Bacteroidetes
BALB/c mice 51 % Soy 35 days ↑ Bacteroidetes
Kostovcikova et al. 2019
50 % Wheat
gluten
↑ Bifidobacterium
↑ Lactobacillus
↑ Staphylococcus
↑ E. coli
C57BL/6J 40 % Whey
Protein
21 weeks ↑ Lactobacillus
↓ Bifidobacterium
↓ Clostridium
McAllan et al.
2014
C57BL/6J 258 g/kg diet
Casein 12 weeks ↑ Lactobacillus
↑ Actinobacteria
Ijaz et al. 2018 251 g/kg
diet
Beef ↓ Akkermansia
↑ Clostridium 252 g/kg
diet
Soy ↑ Proteobacteria
Human adults (obese)
139 g
(duplicated)
Chicken and fish
4 weeks ↑ Lactobacillus
↑ Firmicutes
↓ Bacteroidetes
↑ Actinobacteria
Russell et al.
2011
Percentages of protein quantity in the diet are specified, compared with the protein sources and the extent of the specialized food intake. Except from humans the rest of them were ad libitum.
There are common patterns as the rise of E. coli, although differences in the tendency of probiotics. *Non-high protein.
2. 3. Immune system and high protein diets.
2. 3. 1. Inflammation response: overall process and markers.
Diet, gut microbiota and immune system are highly related (Lobionda et al., 2019). It has been studied that a variety of molecules produced in the digestive tract are able to increase or diminish inflammatory processes by inducing or inhibiting the production of pro- and anti- inflammatory markers (Sonnenburg & Bäckhed, 2016).
The immune system is formed by a complex communicating network of molecules and reactions from the innate and adaptive response (Branzk & Diefenbach, 2018), including those related to the inflammation process (Turner et al., 2014).
Inflammation occurs in response to a tissue damage, presence of toxic compounds or
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infection in the body, allowing a rapid neutralization and back to the homeostasis of the injured tissue (Nathan, 2002), due to the production of immunoglobins and cytokines (Noakes & Michaelis, 2013). Generally, an inflammation results beneficial, as it allows the exacerbation of the harmful effects of having an infection or a injure, although a long exposure can lead to worsening the situation and contribute in the appearance of inflammation-related chronic diseases (Nathan, 2002; Germolec et al., 2018). Reached a threshold by the molecules involved in the first steps of inflammation, its resolution starts by the action of anti-inflammatory mediators (Barton, 2008).
The inflammatory response begins with the recognition of the pathogens by Toll- like receptors (TLRs) and Nod-like receptors (NLRs) (Motta et al., 2015; Noakes &
Michaelis, 2013) and the triggering of the pro-inflammatory signal cascade (Patel &
Chatterjee, 2017; Dowling & Dellacasagrande, 2016; Barton, 2008). This ligand-receptor union activates several proteins in macrophages and dendritic cells (DCs) that induce the expression of genes related to pro-inflammatory cytokines (Chen et al., 2018; Vidya et al., 2017) such as Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) (Tanaka et al., 2018; Turner et al., 2014). Once the inflammatory setting is displayed, cytokines, chemokines as MCP-1 (Bianconi et al., 2018), and other proinflammatory mediators like C-reactive protein are activated (Schrödl et al., 2016). It is then, when morphological and permeability changes in blood vessels are produced, allowing the migration of cells to the infected site (Luster et al., 2015), producing the inflammatory status. They are all guided by thresholds and gradients (Bachmann et al., 2006) and interact between one another; IL-6 and TNF-α provoke the production of MCP-1 (Rose-John, 2012) and CRP (Giudice & Gangestad, 2018). CRP itself, can also enhance the production of MCP-1 (Sproston & Ashworth, 2018). Plus, MCP-1 activates T-cells, that induce the pro- inflammatory cytokine production (Bianconi et al., 2018). Prolonged high levels of this molecules produce a chronic inflammatory status in various organs like liver or gut and therefore injuries in the tissues (Gabay, 2006; Sugimoto et al., 2016; Chen et al., 2018;
Germolec et al., 2018).
Ending the response, anti-inflammatory molecules regulate the “back-to- homeostasis” state, implying lipid-derived mediators, several immune cells and cytokines (Fullerton & Gilroy, 2016). Reached a certain threshold of pro-inflammatory mediators and time, the production and expression of anti-inflammatory molecules increases, and the final stage of the immunological response starts (Headland & Norling, 2015). They are able to block internal and external proinflammatory molecules and enhancers (Serhan, 2007), end with the recruitment of cells and eliminate neutrophils (Ortega- Gómez et al., 2013; Sugimoto et al., 2016). As said, IL-10 is an anti-inflammatory
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cytokine expressed by DCs, macrophages, granulocytes, NK cells and B- and T-cells (Rutz & Ouyang, 2016). It acts inhibiting the activity of pro-inflammatory mature T-cells and limiting the expression of pro-inflammatory molecules like IL-6 and TNF-α (Zhang &
An, 2007) by DCs and macrophages. Its expression is induced by the recognition of MAMPs by TLRs in macrophages, and levels of certain pro-inflammatory cytokines and transcription factors that induce its expression in the previous mentioned cells (Rutz &
Ouyang, 2016). The reduction of IL-10 production is also regulated by other molecules and the levels of IL-10 itself, as it has a negative feedback loop (Saraiva & O’Garra, 2010). Nevertheless, there have been reported clinical cases where IL-10 has a dual effect, meaning that it can stimulate the production of other pro-inflammatory cytokines on T, CD4 and CD8 cells (Lauw et al., 2000; Mülh et al., 2013).
2. 3. 2. Immune system in the digestive tract.
In relation to the immune system’s functions in the gut, the gut-associated lymphoid tissue (GALT) is, roughly said, the most important and bigger immune tissue in the body, as it is exposed to continuous threats present in the lumen (Childs et al., 2019). It is part of the general mucosa-associated lymphoid tissue (MALT) and it is mainly formed by Peyer’s patches and isolated lymphoid follicles (ILFs) (Brandtzaeg, 2013). It tightly works with the mucosa, the epithelial barrier and the cells conforming it, and the lamina propia against foreign bodies (De Santis et al., 2015). Epithelial barrier avoids permeability to bacteria and large molecules due to the tight junctions the colonocytes have between them, plus it secretes substances to maintain the mucus layer’s homeostasis (Peterson & Artis, 2014). In the lamina propia, ILFs and immune cells and macrophages get activated by the continuous presence of microbe-associated molecular patterns (MAMPs), proteins and toxins that reach the barrier and manage to get through it. Plus, it offers immune activation and tolerance to commensal bacteria (Rinninella et al., 2019b). The inflammation lays low as long as the foreign bodies don’t surpass the capacities of the host’s response systems. As explained before, deficiencies in one of the intermediates may cause severe processes of inflammation; the excessive amount of PAMPs or toxic compounds above the limits of regeneration by the immune system, lead to a continuous expression of pro-inflammatory mediators, that end with irremediable damages (Friedrich et al., 2019). Several studies reviewed by Saraiva and O’Garra (2008) indicate the importance of maintaining the levels of IL-10 in line in the gut, as it helps maintaining gut homeostasis and avoids inflammation in the colon region.
Studies gathered by Fukata & Arditi (2013) conclude that receptors constitute a crucial control point of inflammation in gut, as they contribute maintaining mucosal homeostasis.
They also state that the lack of receptors plus the absence of IL-10 derive in colitis. In
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other words, anti-inflammatory molecules in the gut are able to avoid inflammatory diseases, though excessive pro-inflammatory mediators, gut dysbiosis and failures in the balanced immune microenvironments lead to diseases in the digestive tract (Friedrich et al., 2019).
In relation to the diet, some macro- and micronutrients are essential to develop an appropriate immune response; certain compounds or by products (or the lack of them) may increase inflammation and injuries, and cause an impaired immune response (Chen et al., 2018; Childs et al., 2019). Fiber, fats (Chaterjee & Bagchi, 2017), peptides and biogenic amines (Moughan & Rutherfurd-Markwick, 2013), and specific micro- (Gombart et al., 2019) and macro-nutrients (Chakaroun et al., 2020) produce changes both directly and by their processing, that change the microenvironment of the gut lumen. Between the consequences, they alter the ratios of microbial communities or release toxic compounds that harm colonocytes and the mucosal barrier (Tremaroli & Bäckhed, 2012) as hydrogen-sulfide molecules (Jantchou et al., 2010).
Associated to the GALT, attach to the mucosa, commensal microbial communities take part in the processing of the above-mentioned molecules (MacDonald, 2005). They also protect the barrier limiting the number pathogenic organisms, producing anti-inflammatory and energetic molecules, and stimulating the immune system (Hansen
& Sams, 2018).
2. 3. 3. Effects of dietary protein over GALT.
Single amino acids modulate colonocyte metabolism and gut immunity (Ma & Ma, 2018). Therefore, an increment in dietary amino acid intake, both by supplements or augmenting the protein quantity in diet can directly affect colonic mucosal microenvironment (Kong et al., 2018). Deficiencies in dietary protein intake are related to malnutrition and immune system’s compromising (Ruth & Field, 2013). Besides, an excessive quantity of dietary protein translates in the presence of more byproducts of its processing such as ammonia, that targets mucosal integrity (Scott et al., 2013). Derived from that, there are changes in microbial communities and an intestinal barrier disfunction, being able to produce systemic inflammation (Cui & Kim, 2018; Christ et al., 2019). In addition, the reduction of the probiotic ratio in gut microorganism communities is associated with a reduction of the production of SCFAs and therefore a suppression of the inhibition of pro-inflammatory cytokines (De Rosa et al., 2015; Wang et al., 2019).
High protein diets of beef origin increased some pro-inflammatory cytokines and negatively affected systemic inflammation (Ijaz et al., 2018) while other pro-inflammatory cytokines were not affected. However, a plant based high protein diet reduced TNF-α
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levels (Kostovcikova et al., 2019). Llewellyn et al. (2018) found that with the increment of casein percentage in the diet, mice would have increased levels of TNF-α and IL-6 and worsen colitis severity in mice. Studies made with dietary protein hydrolysates and peptides have shown both beneficial and detrimental immunomodulatory effects in intestinal homeostasis, inflammation and weight management depending on the origin of the original protein (Kiewiet et al., 2018). Hence, it puts again into question the dual effects on health varying the origin of the protein and its digestibility (Gilbert et al., 2011).
2. 4. Short Chain Fatty Acids.
Fermentation of mainly soluble dietary fibers like galactooligosaccharides and fructooligosaccharides in colon by microorganisms produce diverse molecules as byproducts such as SCFAs (acetate, propionate and butyrate) (Tan et al., 2014;
Sivaprakasam et al., 2016; Fernando et al., 2017) and certain gases (CO2, H2 and CH4) (Scott et al., 2013). They can also be formed by fermentation of residual peptides in colon in lesser amounts (Macfarlane & Macfarlane, 2012). Butyrate is the one containing more activity, but the one that is produced in less amount (15 %), followed by propionate (25 %) and acetate (60 %) (Morrison & Preston, 2016; Sonnenburg & Bäckhed, 2016;
Rinninella et al., 2019b). They regulate systemic homeostasis and colonocyte metabolism (as fluid and electrolyte uptake), as they are an energy source for epithelial cells (Morrison & Preston, 2016; Fernando et al., 2017). The molecules are also able to stimulate colonic blood flow and mucosal proliferation in the lumen of the gut (Martin et al., 2018), and prevent from pathogenic species’ proliferation (Tan et al., 2014;
Jandhyala et al., 2015). Therefore, they are indispensable to maintain the well- functioning and balance on the gut environment.
Several microorganisms are responsible of the formation of these molecules in colon. Acetate and propionate have been studied to be mostly produced by the Bacteroidetes phylum, but butyrate is a byproduct of Firmicutes (Kelly et al., 2015). At a more specific level, acetate is highly produced by Enterobacteria, Bifidobacterium, Lactobacillus and Streptococcus. Besides, propionate is a byproduct of Propionibacteria and Prevotella between others, and butyrate by Roseburia, Faecalibacteria and Butyvibrio. Moreover, they are all produced by the genera Clostridium (Macfarlane &
Macfarlane, 2012).
They are mostly metabolized in the gut epithelium, but part of them get internalized into the blood system by diffusion and through several transporting systems (Morrison & Preston, 2016; Sivaprakasam et al., 2016) and get transported to different
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organs of the body (Westfall et al., 2017). Propionate and acetate are the ones that get transported to the blood system, although butyrate is retained in the colonocytes where it serves as energy source and contributes in the maintenance of anoxygenic levels in the mucus layer, enhancing barrier function and propitiating a well immune system function (Kelly et al., 2015). Outside the gut they are able to cause effects on pancreas, enteric neurons, adipocytes and innate immune cells in general due to the interaction with the receptors that host these type of cells (Sivaprakasam et al., 2016). Butyrate, for example is able to repress the production of proinflammatory molecules TNF-α, IL-1 and nitric oxide (Tan et al., 2014). Nevertheless, acetate and propionate are related to the glucose metabolism, as acetate is related to cholesterol and lipid formation, while propionate is involved in gluconeogenesis (Rinninella et al., 2019b). There are also reports that SCFAs can act over inflammatory processes in other organs such as the brain (Srivastav et al., 2019), the lungs or the liver (Sivaprakasam et al., 2016).
To sum up, the production of SCFAs is determined by the microbial communities’’
composition, that at the same time is influence by the intake of macronutrients in the diet.
Hence, inflammatory processes in gut are kept below harmful level limits and the probability of the appearance of chronic inflammation and tissue damage is diminished.
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III. HYPOTHESES AND OBJECTIVES
3. 1. Hypotheses.
A diet rich in protein from plant and animal origin induces significant changes in the microbial communities on the gut and modifies the production of SCFAs, reflected in disparities over metabolic profiles and inflammation markers.
3. 2. General objective.
To evaluate the effects over metabolic profiles, gut microbiota and immune system of an animal (beef meat) and plant (Phaseolus vulgaris L. and Zein) based high- protein diet on a murine model.
3. 3. Specific objectives.
3. 3. 1. To evaluate the changes in weight and fat increment and gut microbiota by qPCR that a diet rich in animal and vegetal protein causes.
3. 3. 2. To measure the levels of CRP, TNF-α and the cytokines IL-10 and IL-6 in both diets through and immunological analysis and analyse their increment.
3. 3. 3. To quantify the short chain fatty acids produced in the colon of the individuals derived from the ingested protein (acetate, propionate and butyrate).
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IV. MATERIALS AND METHODS
4. 1. Animals and stay conditions.
30 mice belonging to Mus musculus C57BL/6 were purchased from Círculo de ADN S.A., with 9 weeks of life in the Vivarium of Tecnológico de Monterrey, where the experimental procedure was performed. They were weighted (E-LQ Series, Torrey®, Monterrey, Nuevo León México) individually and divided by their weight on cages of 3 and 4 individuals to adjust them to a similar metabolic state, reduce the decompensation of weigh and avoid unjust fights between them. Thus, each group of diet had 10 mice per group. The diet groups were the following: high in vegetal protein (VPD), in animal protein (APD) and a control diet (CD). The weight average of each group was similar, to reduce the variation of the experiment between the three different types of diet.
The cages were microisolator cages type III. They were placed on racks with a sterilized air-flow system that connected with the cage, creating and isolated environment in each of the cages. The cages were cleaned and replaced each week by the Vivarium technicians. They were equipped with a water dispenser and enough food of each type of diet, so they fed ad libitum. Each cage had a translucent red polycarbonate house-like-structure cage for the comfort of the individuals and the allowance of visualization of the individuals. The light cycles at the Vivarium were from 8am to 8pm. The humidity was approximately of 40 - 55 % during the stay, and the temperature varied between 21 and 23 ºC, controlled and measured by the Vivarium staff.
Additionally, the enclosures were equipped with materials to provide the environmental enrichment needed to reduce stress and avoid fights, such as cardboard cylinders of the rolls of toilet paper and the other times cloth and paper pieces, that were tied to the roof of the cages to simulate lianas and swings they could play with. The materials were alternated and changed each week to keep the surprise effect and exploring component of the environmental enrichment. They were sterilized on an autoclave (121 ºC, 20 min, 15 psi, model SM510, Yamato Scientific America Inc., Santa Clara, California, USA) and dried in a stove (141A, Felisa®, Guadalajara, Jalisco, México) at 60 ºC.
4. 2. Experimental design.
An animal protocol was approved by CICUAL (Comité Interno para el Cuidado y Uso de los Animales de Laboratorio), with the number 2019-013.
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Three types of diets were given to the mice: a control diet (CD) granted by the Vivarium of Tecnológico de Monterrey, a high animal-origin protein diet (APD) and a high vegetal-origin protein diet (VPD) cooked in the Centre of Biotechnology and CIDPRO Department (Centro de Investigación y Desarrollo de Proteínas) at Tecnológico de Monterrey.
The experimental procedure was started when mice reached the adult age according to The Jackson Laboratory, with 17 weeks of life. Diet was given to the mice for a total number of 53 days to the VPD group and 55 days to the APD group. The discrepancy of the duration of the diet was due to the distribution of the euthanasia dates.
Mice were marked each three or four days, and their physical appearance was examined in search of wellbeing markers: absence of aggression marks and wounds, brightness and abundance of hair along the body, opened eyes, rigidity of the whiskers and active and explorative behaviour.
Mice were individually weighted (E-LQ Series, Torrey®, Monterrey, Nuevo León México) each weak at the same time-slot (Fridays 11:30 am). The measuring system of the bascule only showed the results in even numbers, having an error of ±1 in the results.
Food intake was measured by counting the number of pellets and grams consumed in each cage (E-LQ Series, Torrey®, Monterrey, Nuevo León México), to approximately control the grams consumed per type of diet per mouse (without taking into account the crumbs and feed dust fallen to the floor of the cage), to keep in control that they were feeding. The food was replaced each week to ensure the maintenance of the quality and chemical and physical properties of the components.
Samples were collected at the end of the experimental design. The euthanasia of the mice was performed in different days, to reduce psychological stress of the workers involved and ensure the wellbeing of the mice. It took three days in the course of two weeks; one day per diet group, starting with the vegetal origin and finishing with the control group, and it took 20 to 25 minutes per individual.
4. 3. Protein extraction.
4. 3. 1. Animal protein extraction.
“Pulpa negra” beef meat was purchased in a local market (H-E-B®). The meat was chopped up in pieces of less than a centimetre using a blender (model 450-10, Osterizer®). It was dried in a dehydrating oven for 16 hours at 50 ºC. The dried meat was introduced in plastic zipper bags (14.9 x 16.5 cm, Reynolds®) wrapped with aluminium foil to avoid the humidity and stored at -80 ºC until use. The dry meat was later prepared to be defatted with hexane. It was tempered in a water bath (LWB-122D, Digital water
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bath, LabTech®, Guadalajara, Jalisco, México) at a temperature of 25 ºC, still covered with the aluminium foil to keep it dry, for approximately an hour. After, 250 g of dry meat (Mettler Toledo S.A., Columbus, Ohio, USA) were introduced in 1 L glass bottles (VWRTM, Radnor, Pennsylvania, USA) along with 750 ml of hexane (1:3) and left in agitation at 130 rpm and 20 ºC, for 24 hours (Model 1217, VWRTM, Radnor, Pennsylvania, USA). Hexane was removed and the meat filtered and spread over metal trays covered in aluminium foil under an extraction hood and left for approximately 5 or 6 hours, until the protein extract was completely dry. The meat protein extract was stored in sterile bags (115 x 230 mm, Whirl-Pak®) -covered in aluminium foil to avoid the entering of humidity- at -80 ºC until use.
4. 3. 2. Plant protein extraction.
Black beans (Phaseolus vulgaris L.) donated by the Nutriomics group of the Centre of Biotechnology were left for 24 hours in water (1:4 w/v) at 4ºC to soften the hull and facilitate the removal by hand. The coatless beans were dried in an oven with convective air at 60ºC for 18 hours. When dried, they were mashed and grinded until obtaining a homogeneous and thin flour with a blender (model 450-10, Osterizer®). The protein extraction was performed through the alkaline extraction and isoelectric precipitation methods. Black bean flour was mixed with distilled water in a proportion 1:10 (black bean flour:water). Immediately, it was adjusted to pH 9 with 6 N NaOH (LAQUA ph1200, Horiba Scientific Ltd., Kyoto, Japan) and HCl 15 %, and heated to 52 ºC for 1 h under stirring. Later, it was centrifuged 20 minutes at 20 ºC at 3500 rpm (IEC CL40R, Thermo ScientificTM, Waltham, Massachusetts, USA). The supernatant was kept and adjusted to pH 4.5 with 15 % HCl and was again centrifuged for 35 minutes at 4 ºC at 3500 rpm. The precipitated protein was rescued from the bottles and stored in zip-lock bags at -80 ºC. With a minimum of 24 hours at -80 ºC, the protein extract was lyophilised (model 7755040, LabconcoTM, Kansas City, Missouri, USA) for 72 hours. Extract of corn protein (Zein) was directly purchased from Fisher ScientificTM.
4. 4. Diet preparation.
Diets were formulated following the steps of the report of the American Institute of Nutrition written by Reeves et al. (1993) with some variations as explained in Table 2.
Dry ingredients were put under UV light for 15 minutes for one side, and other 15 minutes on the other side. They were firstly mixed without water or oil, then the water was added and the liquid Vitamin B12 and it was thoroughly mixed again. Finally, the soy oil was added to the mixture, as it gives the mixture some hardness, and well
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kneaded. Small pieces of approximately (2.5 x 1.5 x 1 cm) were made and placed in the oven trail.
Pellets were cooked on a convective air oven (TS-12657NH, Jet Fryer®) for approximately an hour -depending on the quantity of pieces introduced in the oven-.
Feed was stored at 4 ºC in the Centre of Biotechnology and transported to the Vivarium when needed.
TABLE 2. DIETS FORMULATION
Ingredients APD (g/ kg of diet) VPD (g/ kg of diet)
Potato starch (J.R. Foods) 265.6 265.6
Animal protein extract 385 -
Vegetal protein extract (Black bean and corn, 9:1) - 385
Maltodextrin (J.R. Foods) 155 155
Sucrose 55 55
Soybean oil (Nutrioli) 40 40
Microcrystalline cellulose (Sigma-Aldrich®) 50 50
Mineral Mix AIN-76M (MP Biomedicals) 35 35
Vitamin Mix AIN-76M (MP Biomedicals) 10 10
L-cysteine (Sigma-Aldrich®) 1.8 1.8
Choline bitartrate (Sigma-Aldrich®) 2.5 2.5
Tert-butylhydroquinone (Sigma-Aldrich®) 8 mg 8 mg
B12 Vitamin (Grossman®) 10 mg 10 mg
Selenium (GNC) 100 μg 100 μg
The CD group diet is the PicoLab® Rodent Diet 20 5053. Vitamin B12 was added to equal the quantity of the renewed mixes AIN-93M. Selenium was added to complement the lack of it in the mineral mixes.
The quantity of protein in the diets and the source percentage in the VPD were adjusted following the results obtained of the protein extracts analysis about the presence of protein and the amino acid composition. Both diets were design to have the most similar amino acid composition, that can be seen in Table 3. The amino acidic composition of the protein extracts of the design diets were given by an external laboratory. The total protein quantity in each of the extracts varies, therefore the amino- acid quantity is comparable between the groups to the extracts in general and does not equally respond to the same protein.
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TABLE 3. AMINO ACID COMPOSITION OF PROTEIN EXTRACTS Essential amino
acids
Beef protein extract
Black bean protein extract
Corn protein extract (Zein)
Lysine 6.82 6.56 0.15
Leucine 6.34 7.94 16.58
Isoleucine 3.97 4.73 3.60
Cysteine 0.82 0.61 0.66
Threonine 3.38 3.52 2.30
Methionine 2.00 1.17 1.35
Tryptophan 1.09 1.15 0.22
Phenylalanine 3.24 5.61 5.90
Arginine 4.87 5.12 1.22
Histidine 3.01 2.72 1.25
Valine 4.05 5.38 3.34
Non-essential amino acids
Proline 2.91 3.54 8.62
Glutamic acid 11.47 13.02 20.60
Aspartic acid 7.04 10.27 4.40
Glycine 3.34 3.48 1.21
Tyrosine 3.44 3.51 4.33
Serine 2.49 4.58 4.05
Alanine 4.44 3.65 8.10
Non-proteinogenic amino acids
Ornithine 0.08 0.04 0.02
Taurine 0.07 0.03 0.01
Lanthionine 0.28 0.19 0.38
TOTAL PROTEIN
CONTENT Per 100 g 75.15 86.82 88.29
Some pellets of different lots were aleatory selected to perform various bromatological analyses described in Table 4.
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TABLE 4. PHYSICOCHEMICAL PROPERTIES OF THE PELLETS
Diet type Moisture Ash Protein
CD 10.0 6.1 20
APD 12.4 ± 0.2 3.9 ± 0.2 25.7 ± 5.7
VPD 9.3 ± 0.1 3.5 ± 0.1 30.5 ± 7.6
Moisture was calculated by a thermogravimetric analysis. Constant-weight metal dishes were weighted (ML54, Mettler Toledo S.A., Columbus, Ohio, USA) and 5 grams of grinded feed samples were added. The dishes with the samples, performed by triplicate, were introduced in a drying oven at 110ºC (VWRTM, Radnor, Pennsylvania, USA) for three and a half hours. They were placed in a desiccator to cool for five minutes and weighted. The process was repeated until the weight remained constant. The moisture content was calculated by the following equation:
!"#$%&'( *"+%(+% % = (/#$ℎ!+ $2345("! ) − (/#$ℎ!+ $2345(#!)
82345("! 9 100
Being:
xw = weight of the correspondent material.
sample0 = wet sample.
sample1 = dry sample.
4. 5. Quality control.
A microbiological analysis was made of every lot of pellets cooked, to ensure the absence of microorganisms on the mouse feed.
Before the packaging of the pellets, 10 g of pellets were aleatory selected, and mixed with 90 ml of PBS 1x (Phosphate Buffered Saline) (BioxonTM, Oaxaca, México) previously prepared and autoclaved as indicated by the manufacturer. 100 μl of the dilutions 10-1 and 10-2 were cultured in PDA (Potato Dextrose Agar) (DifcoTM) plates and nutrient agar plates by duplicate. Nutrient agar cultured plates were incubated (1535, Sheldon Manufacturing Inc., Cornelius, Oregon, USA) at 37 ºC for 24 and 48 hours, and PDA cultured plates were incubated at 25 ºC for 72 and 120 hours (1535, Sheldon Manufacturing Inc., Cornelius, Oregon, USA).
Pellets were given to the animals when no UFCs were found on the plates.
