Instituto Tecnológico y de Estudios Superiores de Monterrey Campus Monterrey School of Engineering and Sciences

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Instituto Tecnológico y de Estudios Superiores de Monterrey Campus Monterrey

School of Engineering and Sciences

Study of vegetal and animal high protein-based diets on a human gastrointestinal ex vivo model over gut microbiota composition (Probiotics and Enterobacteria) and metabolites

profile (Biogenic amines and Fatty acids).

A thesis presented by Karen Castaño Sánchez

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, June 11^th, 2020

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Instituto Tecnológico y de Estudios Superiores de Monterrey Campus Monterrey

School of Engineering and Sciences

The committee members, hereby, certify that have read the dissertation presented by Karen Castaño Sánchez and that it is fully adequate in scope and quality as a partial requirement for the degree of Master of Science in Biotechnology.

______________________________

Dra. Yolanda Arlette Santacruz López Tecnológico de Monterrey School of Engineering and Sciences

Principal Advisor

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Dr. Sergio Serna Saldívar Tecnológico de Monterrey School of Engineering and Sciences

Co-Advisor

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Dra. Cristina Chuck Hernández Tecnológico de Monterrey School of Engineering and Sciences

Committee Member

______________________________

Dr. Rubén Morales Menéndez Dean of graduate Studies School of Engineering and Sciences

Monterrey Nuevo León, June 11^th, 2020

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Declaration of Authorship

I, Karen Castaño Sánchez, declare that this dissertation titled, “Study of vegetal and animal high protein-based diets on an human gastrointestinal ex vivo model over gut microbiota composition (probiotics and enterobacteria) and metabolites profile (SCFA and biogenic amines)” and the work presented in it are my own. I confirm that:

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

• Where any part of this dissertation 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 dissertation is entirely my own work.

• I have acknowledged all main sources of help.

• Where the dissertation 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.

___________________________

Karen Castaño Sánchez Monterrey Nuevo León, June 11^th, 2020

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iii Dedication

To my parents Perla Sánchez and Saúl Castaño, my brothers Grecia, Isaac and to my best friend Mayra. Thanks for the support, patience and love.

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iv Acknowledgments

I would like to express my thanks to:

Dra. Arlette for her patience, advices, and for giving the opportunity to work with her and learn for my errors.

Dra. Cristina for its kindness, patience, knowledge and, always be ready to help in difficult times.

Dr. Serna for its trust, patience, time and economic support.

Dra. Beatriz Acosta, Felipe López, and Dr. Erick Heredia for its patience, knowledge and support.

Dra. Perla Ramos, Dra. Sara Garza, Abraham Díaz and Andrea Lara for its kindness and material support.

Dra. Marisela González Ávila , Dr. Ricardo García, and Dr. Sebastian Gradilla, for its trust, knowledge, kindness, and friendship during my research state in CIATEJ, Guadalajara.

Finally, I would like to thank CONACYT for Ciencia Básica project and scholarship (CVU 884718); To Tecnológico de Monterrey for the academic scholarship and to NutriOmics group for their economic support.

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v

“Study of vegetal and animal high protein-based diets on a human gastrointestinal ex vivo model over gut microbiota composition (Probiotics

and Enterobacteria) and metabolites profile (Biogenic amines and Fatty acids).”

by

Karen Castaño Sánchez

Abstract

For the last years, the trend of high protein diets (HPD) has been widely adopted by the general population as well as by recreational and professional athletes as a strategy to lose weight and gain muscle mass. Nevertheless, during this type of diet a relatively high amount of protein could reach the large intestine, increasing protein fermentation by gut microbiota, which in contrast to carbohydrate fermentation, potentially leads to dysbiosis because of the concomitant reduction of beneficial microbial metabolites (e.g. Short-chain fatty acids) and the increase in the production of harmful ones, such as biogenic amines. These compounds derived from amino acid decarboxylation, have mainly been investigated in fermented foods. However, recently its production by human isolated bacteria has been discovered. The objectives of this work were to study the effect of animal and vegetal high protein diets on probiotics (Bifidobacterium and Lactobacillus) and Enterobacteria concentrations as well as the evaluation of short and branched chain fatty acids, and biogenic amines (putrescine, tyramine, cadaverine, and histamine), on an ex vivo model of the human gastrointestinal tract. For this purpose, three human digestive simulators (ARIS, CIATEJ) were inoculated with human microbiota, and after that, fed with three diets for a period of 24 days. The diets consisted of two high-protein diets (50% protein, 30%, carbohydrates, and 20% fat), either with an animal or vegetal protein, and a control diet (19% protein, 56 % carbohydrates, and 25% fat). The vegetal protein isolate consisted of a mixture of black bean protein, obtained through alkaline extraction and acid precipitation procedure, and corn

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vi protein (zein). Whereas, the animal protein was obtained from freeze-dried and defatted (with hexane) beef. The amino acid profile of the dehydrated beef was used to formulate a vegetal protein with a similar amino acid profile. Samples from the digestors corresponding to the large intestine were collected every 72 h and stored at -20°C until gas chromatography analysis (short and branched chain fatty acids), HPLC analysis (biogenic amines), and Real-time qPCR (bacteria concentration).

The production, at the end of the fermentation (24 day), of short chain fatty acids (SCFA) and branched-chain fatty acids (BCFA) was higher in the section of the descending colon of the three diets evaluated. In this regard, acetic and butyric acid were the main SCFA produced in the animal protein-based diet. Whereas, butyric acid was the main SCFA in the vegetal protein-based diet. On the other hand, both diets led to similar concentrations of BCFA, caproic, and valeric acid from which the last one, was the acid mostly produced in both diets. Regarding biogenic amines ,both high protein diets lead to similar concentrations, and the main amines were putrescine and cadaverine. Finally, the probiotics concentrations ended (24 day) with similar concentrations in both colon segments of the three diets, and the same was observed with the concentration of Enterobacteria. In this respect, among the bacteria evaluated, the concentrations of Enterobacteria showed the highest decrease in comparison to the probiotics in both colon segments of the three diets.

Keywords: High protein diets, Animal protein-based diet, Vegetal protein-based diet, Lactobacillus, Bifidobacteria, Enterobacteria, Short chain fatty acids, Biogenic amines.

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vii

Abbreviations

AC Ascending colon

APD Animal protein-based diet

ARIS Automatic and Robotic Intestinal System BA Biogenic amines

BCFA Branched chain fatty acids CAD Cadaverine

DAO Diamino oxidase DC Descending colon

EFSA European Food Safety Authority GLP-1 Glucagon like peptide 1

HIS Histamine

HGM Human gut microbiota HPD High protein diets

IC⁵⁰ Half of the strongest cytotoxic effect LOAEL Lowest observed adverse effect MAO Monoamine oxidase

MCM Multistage continuous model NOAEL No observed adverse effect level PAS Polyamines

PUT Putrescine

PYY Peptide tyrosine tyrosine

RDA Recommended dietary allowance SCFA Short chain fatty acids

SPD Spermidine SPM Spermine TYR Tyramine

VPD Vegetal protein based diet

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viii

List of figures

Figure II-1.ARIS... 13 Figure IV-1. General process of Alkaline extraction- acid precipitation applied for black bean protein obtention. ... 29 Figure IV-2. Manual version of the ARIS model. ... 32

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ix

List of tables

Table II-1. Gut microbiota metabolites during human HPD. ... 9

Table IV-1. Diets provided to the ARIS. ... 28

Table IV-2. Reactor conditions of the ARIS. ... 33

Table IV-3. PCR mix used for each DNA sample ... 34

Table IV-4. Primers for the 16s rRNA gene of the bacteria evaluated. ... 35

Table IV-5. Elution gradient of HPLC analysis. ... 37

Table V-1. Balck bean protein purity and recovery obtained by Ultrafiltration. ... 39

Table V-2. Black bean protein purity and recovery obtained by Diafiltration. ... 39

Table V-3. Aminoacid profile of the bean protein, zein, and beef concentrates. 42 Table V-4. Aminoacid profile of the protein concentrates used for the diets tested in the ex vivo gastrointestinal model.. ... 43

Table V-5. Characterization of the protein sources (dry basis). ... 45

Table V-6. Bacteria concentrations (Log) of Lactobacillus, Bifidobacteria, Enterobacteria and Total bacteria after 24 days of fermentation in the ascending colon. 47 Table V-7. Bacteria concentrations (Log) of Lactobacillus, Bifidobacteria, Enterobacteria and Total bacteria after 24 days of fermentation in the descending colon. 48 Table V-8. SCFA and BCFA concentrations (mM) in the ascending colon at the end of fermentation (24 day). ... 50

Table V-9. SCFA and BCFA concentrations (mM) in the descending colon at the end of fermentation (24 day). ... 51

Table V-10 Biogenic amines concentrations (ppm) in the descending colon at the last day of fermentation (24 day). ... 54

Table VIII-1.pH values of the three diets evaluated in the ARIS. ... 81

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x

I. Introduction

Since the early 90's, high protein diets (protein intake above the recommended values or > 35% of dietary energy) have been widely used by the general population as a strategy to lose weight. In this regard, high protein diets (HPD) have been shown to improve the lipid profile of overweight people. Also, professional and nonprofessional bodybuilders have adopted HPD with the objective of increase fat oxidation as well as improve gains and preserve lean muscle mass. HPD are considered not a risk for individuals with normal renal function. However, in healthy individuals changes in urine biochemistry have been observed, which in the long term can end up in the development of kidney stones and bone losses. In this regard, the gradual introduction of protein during this regime, reduction of red meat intake, and supplementation of alkaline salts ( e.g. calcium, potassium, and magnesium) are recommended to decrease the incidence of such effects. Nevertheless, studies on the new organ, which consist of the trillion bacteria that reside in the large intestine of every human being, have shown that HPD decreases the abundance of probiotic bacteria as well as the production of short-chain fatty acids, which are considered one of the most beneficial metabolites derived from gut bacteria. Moreover, protein fermentation can give rise to a wide variety of toxic compounds such as sulfides, ammonia, phenols, and amines. In this respect, biogenic amines are potentially toxic compounds produced by decarboxylation of amino acids. These compounds are mainly known for their content in contaminated and intentionally fermented foods (e.g. cheese and wine). Nevertheless, recent research found that isolated strains from human feces can produce biogenic amines. In this regard, human gut microbiota is a community of bacteria; thus, the study of metabolites also must be evaluated in this way and considering human physiological conditions For ethical reasons and difficulty of accessing the human large intestine, metabolites released by gut microbiota have relied on the study of urine and feces. However, these samples also contain human metabolites. Besides, the kinetics of

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2 production cannot be evaluated as most of the microbial metabolites are rapidly absorbed by colonic tissue, yielding these samples as a proximal approach to the microbiota activity. In response to these limitations, ex vivo systems that simulate the human gastrointestinal tract, including the large intestine, are now available.

These systems allow sampling at any time; thus, giving the opportunity to the evaluate the kinetics of metabolite production by gut microbiota in a noninvasive manner, and in an environment very similar to the human large intestine.

The general objective of this work was the evaluation of the effect on microbiota composition ,and also the production kinetics of fatty acids and biogenic amines during high protein diets using the ex vivo ARIS model.

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II. Literature review

2.1 Dietary protein

Proteins are essential macronutrients for mammals whose main function is to provide amino acids. These molecules are conformed by one or more polypeptides, the last ones consisting on chains of amino acids formed by the linkage between the α-carboxyl group of one amino acid and the α-amino group of another amino acid (Hou et al., 2017). After protein breakdown during the digestive process, the released amino acids participate in important physiological roles such as nutrients transport (Gotto, 1990; Kono & Arai, 2015), synthesis of body proteins, nutrients oxidation, immune function, reproduction, and lactation (Wu, 2009, 2016). Amino acids can be obtained through the ingestion of dietary proteins from animal sources (e.g. eggs, milk, meat, fish and poultry), and plant sources (e.g. fruits, legumes, and grains) (Hoffman & Falvo, 2004). Moreover, nowadays there are marketed protein supplements based on animal sources (e.g. beef, whey, and casein) (Valenzuela et al., 2019), as well as from plants sources (e.g. rice, lupin, hemp, and soy)(Gorissen et al., 2018).

According to World Health Organization, and the Food and Agriculture Organization of the United Nations (FAO), the Recommended Dietary Allowance (RDA) of high-quality protein is 0.83 g • Kg⁻¹ • day⁻¹ for adults of both sexes and all ages (Marckmann et al., 2015). In this respect, plant and animal proteins contribute with ~ 65% and 35% of global protein intake, respectively (Wu, 2016). However, the opposite occurs in developed countries such as Europe and the USA, where dietary protein mainly comes from animal sources (55-71%), and particularly from red meat ,which contributes between ~16-35% of the animal protein intake. Regarding plant protein, this one mainly come from cereals which contribute with 40-70% of this protein intake (De Gavelle et al., 2017). The quality of protein is generally determined by the ability of a protein to meet the demand for amino acids while considering its digestibility and utilization by the body (Banaszek et al., 2019). In this respect, in

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4 contrast to animal protein, most of plant proteins are deficient in essential amino acids (methionine and lysine) (Burd et al., 2019).Nevertheless, mixtures of plant proteins can be done in order to complement the aminoacid profile of ingested plant protein (Young & Pellett, 1994). Similarly, despite plant proteins contains antinutritional factors that impair protein digestibility (e.g. trypsin inhibitors, tannins, and phytates) (Lynch et al., 2018), treatments such as cooking, germination and fermentation treatments can lead to a reduction of this ones (Ibrahim et al., 2002).Dietary proteins are not only a source of amino acids, as these ones are associated with other nutrients. In this regard, vegetal proteins are accompanied by a variety of dietary fibers, vitamins (B1, B2, B5, B6, and B9), minerals (Ca, Mg, K, Fe ,Zn, P), carotenoids, and flavonoids (Margier et al., 2018). Also, animal proteins contains heme iron, zinc, selenium, vitamin D, and vitamin B12 (Salter, 2018). In addition, in contrast to vegetal proteins, animal proteins, specially red meat, are accompanied by saturated fats, sodium, heme, and N-nitroso compounds which increase the risk of cardiovascular disease, obesity (Alisson-Silva et al., 2016), as well as colorectal cancer(Ijssennagger et al., 2015).

High protein diets

Over the past years, HPD have become a method used by the general population not only to fulfill protein nutritional requirements but to trigger weight reduction. In this respect, HPD have been shown to decrease serum triglycerides, low density lipoprotein, as well as increase HDL cholesterol concentrations in overweight individuals (Mateo-Gallego et al., 2017; Pasiakos et al., 2015). HPD are defined either when protein intake is above > 1.2 g • Kg⁻¹ • day⁻¹ (Ko et al., 2017), or when its intake represents more than 35% of total energy (Antonio et al., 2014). This dietary trend started in the late 90’s, with the publication of Atkins' Diet Revolution in 1972, then 20 years later the book was republished as Dr Atkins' New Diet Revolution, which became the bestselling diet book in history by selling 12 million copies (Lenzer, 2003). To date, the mostly known HPD include the Atkins, South beach, Stillman and the Zone, in which the ingestion of protein is 2.3, 2.6 , 4.3 ,and 2.3 g • Kg⁻¹ • day⁻¹, respectively (Pesta & Samuel, 2014). These diets aim to

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5 consume high amounts of protein and low amount of carbohydrates in order to lose weight. The basis behind HPD relies in that diet-induced thermogenesis increases more after protein intake as this one requires 20-30% of its available energy to be expended on its metabolism (i.e. gluconeogenesis, urea synthesis), whereas carbohydrates and fat only requires 5-10% (Leidy et al., 2015; Schutz, 2011).

Besides, HPD cause an increase in postprandial fullness by increasing both, the concentration in plasma levels of the appetite regulation hormones, peptide YY (PYY) and glucagon like peptide 1 (GLP-1) (Ghazzawi & Mustafa, 2019).

HPD are also widely used by recreational and professional bodybuilders for gain lean muscle mass. In this respect, protein ingestion can be up to 2.5 g • Kg⁻¹ • day⁻¹ in the offseason, whereas in the competition phase, the protein intake varies between 2.4 - 4.3 g • Kg⁻¹ • day⁻¹ (Ribeiro et al, 2019). Nevertheless there are cases where bodybuilders have extreme protein intakes (20-50 g of protein • Kg⁻¹ • day⁻¹) (Ramadan et al., 2015). Likewise, recreational gym users ingest protein supplements to enhance performance and strength maintenance (Tsitsimpikou et al., 2011). Nevertheless, these individuals can ingest more protein than the one they actually need (Thomas et al., 2019). In this regard, excess protein supplementation in most countries is feasible as this one is unregulated and unsupervised by public health authorities, additionally in the general population, recommendations about protein supplementation is mainly provided by non-health professionals (Kamper &

Strandgaard, 2017).

Safety of high protein diets

Protein byproducts such as urea, ammonia, and sulfate, can efficiently be processed by the liver and kidney for its subsequent removal from the body (Wu, 2016).

However, during HPD these metabolites can exceed the metabolic capacity oh these organs (Ko et al., 2017). For example, most popular HPD are low in carbohydrates;

therefore, more amino acids must be converted to glucose to provide energy, and as a consequence there is a higher production of such byproducts Additionally,

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6 individuals with low history of protein intake have a reduced expression of hepatic urea-cycle enzymes for ammonia detoxification; thus, the sudden start of HPD can lead to higher ammonia concentrations (Wu, 2016). In this regard, higher concentration of this compound in the blood is considered as a causative agent of hepatic encephalopathy, and also has been correlated with other neural diseases such as Alzheimer’s disease, amyotrophic lateral sclerosis, and Huntington’s disease (Griffin & Bradshaw, 2019). Additionally, although urea is considered a nontoxic metabolite (Wu, 2016), a recent study showed that excess urea caused DNA damage and increased the risk of bladder tumorigenesis in rats fed with a HPD (40% protein) (Liu et al., 2016).

Despite the above mentioned risks, HPD have been mainly known for its potential repercussion in renal biochemistry, and bone losses (Tipton, 2011). The mechanism is explained by the sulfuric acid generated from the oxidation of sulfur- based amino acids (i.e. cysteine and methionine), which increases the acidity of urine, and as a consequence, kidneys remove calcium from bones to neutralize the protein-induced acidosis (Mardon et al., 2008). A study done on this subject by Reddy et al. (2002), found an increased acidosis and calcium concentration in urine, as well as a decreased serum osteocalcin concentration in 10 healthy subjects that followed an Atkins type diet for 6 weeks. Moreover, the authors pointed out the risk of kidney stones formation in the long term if such diet continued, as the urine pH of these individuals decreased from 6 to 5.5, which is close to the value of 5.35, where uric acid precipitates to form uric acid stones. Moreover, this value was also close where calcium oxaloacetate stones can precipitate (pH 4) (Carvalho, 2018). In this respect, the effect of protein on urinary biochemistry depend not only the amount of protein, but also on the intrinsic and additional dietary elements. For example, in comparison to vegetal protein, animal protein has a higher content of purines ( uric acid precursors) (Choi et al., 2005), as well as sulfur amino acids (Ko et al., 2017).

Besides, it contains a higher amount of branched chain amino acids, which can increase the rate of kidney glomerular filtration (Kontessis et al., 1995), a mechanism that aims to eliminate excess nitrogenous by-products by increasing the blood flow in to the kidneys (Ramadan et al., 2015).

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7 On the other hand, a diet rich in fruits and vegetables has been suggested to counter arrest protein induced acidosis, as these sources have a high content of organic alkalizing salts such as potassium, magnesium oxalate, and citrate (Aparicio et al., 2013). In this respect, studies have shown that supplementation or adequate intake of calcium or potassium during HPD can lead to normal urine pH values as well as reduced calcium excretion in rats (Mardon et al., 2008), healthy individuals (Bowen, et al., 2004; Dawson-Hughes et al., 2004; Feskanich et al., 1996), and athletes (Kim et al., 2011). Additionally in athletes, it has been hypothesized that the higher demand of calcium by skeletal muscle during training, allows higher calcium retention (Poortmans & Dellalieux, 2000).

Nowadays, HPD are considered safe for people with normal renal function (Wu, 2016), but detrimental in patients with hepatic dysfunction or those susceptible to renal diseases such as patients with chronic kidney disease, diabetes, hypertension, and cardiovascular disease (Cuenca-Sánchez et al., 2015). In this respect, increased glomerular filtration has been shown to be detrimental for patients with Type 1, and Type 2 diabetes where it increases the risk of diabetic nephropathy and albuminuria (Bjornstad et al., 2015; Moriya et al., 2012). Moreover, in these individuals insulin resistance already increases urinary calcium excretion (Finkielstein & Goldfarb, 2006). On the other hand, in adults with normal renal function, increased glomerular filtration is reported to occur when protein intake is >

2 g • Kg⁻¹ • day⁻¹(Wu, 2016), and it has been suggested as a normal adaptation that do not necessary means a decline in renal function (Cuenca-Sánchez et al., 2015; Martin et al., 2005).

2.2 Protein metabolites derived from gut microbiota

Although most dietary protein digestion is equally efficient (~90%), between 12-18 g of proteins and peptides are transferred from the small intestine to its metabolization in the large intestine (Dallas et al., 2017). Various factors influence this protein flux such as HPD (Diether & Willing, 2019), saturation of the digestive capacity (Geypens et al., 1997), co-ingestion of substances that delay gastric emptying (Burd et

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8 al.,2019), renal failure and antiacids (Dallas et al., 2017), cooking temperatures and also the type of protein (animal or vegetal) (Poelaert et al.,2017). Once in the large intestine, peptides can undergo fermentation or decomposition in a process known as putrefaction, which in contrast to fermentation, it can be harmful to the host (Kaur et al., 2017). Indeed, the descending colon, the main site where putrefaction occurs, has the highest incidence of ulcerative colitis, inflammatory bowel disease, and colorectal cancer(Tuncil et al., 2017).

The main gut bacteria that can undergo putrefaction are Clostridium perfringens, Desulfovibrio, Peptostreptococcus, Acidaminococcus, Veillonella, Pro pionibacterium, Bacillus, Bacteroides , and Staphylococcus (Dallas et al., 2017).

These bacteria can use amino acids to produce short-chain fatty acids, branched- chain fatty acids (i.e. isobutyric and isovaleric acid), ammonia, phenols, and sulfides, p-cresol, and indoles (Shen et al., 2010). Some of these metabolites have proven in vitro its toxicity. For example, ρ-cresol can reduce colonocyte proliferation and cell respiration (Andriamihaja et al., 2015), as well as lead to DNA damage (Al Hinai et al., 2019). Also, hydrogen sulfide can reduce butyrate oxidation in colonocytes (De Preter et al., 2012). Moreover, hydrogen sulfide has been associated with the observed destabilization of the intestinal mucus layer in inflammatory bowel patients (Ijssennagger et al., 2016).

Putrefaction can be attenuated by the availability of carbohydrates, as the last ones are the preferred energy source by most of the human gut bacteria. Besides, carbohydrate fermentation leads to a decreased luminal pH, which can inhibit the growth of putrefactive bacteria (Kårlund et al., 2019). Nevertheless, when carbohydrates are low and protein is available in higher concentrations, the rate of putrefaction increases (Dallas et al., 2017). Indeed SCFA, the main product of carbohydrate fermentation, have been found to be decreased during human high protein diets. Moreover, this type of diet has been shown to lead to the production of putrefactive toxic metabolites (Table II-1).

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9 Table II-1. Gut microbiota metabolites during human HPD.

Population Diet

P:C:F Period Metabolites Bacteria Reference

5 healthy

individuals Control (15:47:38) (24:45:31) HPD

2 weeks:

1 week Control 1 week HPD

Urinary phenol and p-cresol, and fecal

ammonia HPD>Control

NA (Geypens et

al., 1997)

19 Healthy

obese men Control

(13:52:35) HPLC (30:4:66)

HPMC (30:35:35)

8 weeks+3 days:

3 days Control 4 weeks HPMC 4 weeks HPLC

Butyrate, Acetate and propionate Control>HPMC>HPLC

Roseburia, Eubacterium

rectale Bifidobacterium,

Lactobacillus Control>HPMC>HP

LC

(Duncan et al., 2007)

17 Obese men

men Control

(15:65:20) HPMC ( 35:45:20)

HPLC (46:7:47)

9 weeks 1week control 4 weeks HPMC

4 weeks HPLC

Acetate and butyrate Control=HPMC>HPLC

Propionate Control>HPMC=HPLC

BCFA

HPMC =HPLC>Control

Roseburia, Eubacterium

rectale Control=HPMC>

HPLC

(Russell et al., 2011)

P: Protein (%); C: Carbohydrate (%); F: Fat (%).

HPLC: High protein low carbohydrate; HPMC: High protein medium carbohydrate.

NA: Not analized.

“=” Not statistically different; “>” statistically higher.

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10 2.3 Human gastrointestinal microbiota.

Human beings cohabitate in a symbiotic relationship with bacteria, fungi, viruses, archaea, and protozoans (Barko et al., 2018). The microorganisms that reside in the human gastrointestinal tract are collectively known as the human gut microbiota (HGM), in which the predominant microorganisms are bacteria. The number of bacteria inhabiting the gastrointestinal tract has been estimated to be more than 10¹⁴ colony-forming unit per gram of content (CFU/g), representing ∼10 more times the number of human cells and over 100 times the amount of genomic content (Thursby

& Juge, 2017).

The gastrointestinal tract is the body site with the highest amount of bacteria, as the total bacteria that habitat the skin, oral, nasal cavity, and vagina count for no more than 10¹² CFU/g (Yu, 2018). The density of bacteria in the gastrointestinal tract is not homogenous as these bacteria start increasing from the stomach to the large intestine, where due to the larger transit time, anaerobic environment, less secretion of antimicrobials as well as bile acids, is the site which harbors the largest amount of bacteria (Donaldson et al., 2016). Some of the main factors that shape HGM include the mode of delivery (cesarean and vaginal), diet during infancy (breast milk or formula), diet , prebiotics and probiotics consumption, use of antibiotics, and age (Jandhyala et al., 2015).From these factors, dietary changes appear to explain 57%

of the total structural variation in gut microbiota (Del Chierico et al., 2014).

The bacteria of gastrointestinal tract mainly belong to the phyla, Firmicutes, Bacteroides, Actinobacteria, Proteobacteria, Fusobacteria, Verrucomibria, Tenericutes, and Lentisphaerae (Rinninella et al., 2019). To date, over 1057 bacteria species have been identified (Lagier et al., 2018), each human being harboring ~160 distinct species (Yu, 2018). During the early stages of development, the HGM is dominated by two main phyla, Actinobacteria and Proteobacteria and by around 2.5 years of age, the composition, diversity and functional capabilities of the infant microbiota resemble those of adult microbiota (Cussotto et al., 2019) , where 90%

the gut bacteria of the gut now mainly belong to the phyla Firmicutes (49–76 %) and Bacteroidetes (16-23%) (Matsuoka & Kanai, 2015). Among these phyla the most

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11 abundant of bacteria include, Bacteroides, Clostridium, Eubacterium, Veillonella, Ruminococcus,Bifidobacterium, Fusobacterium, Lactobacillus, Peptostreptococcus, and Peptococccus (Rajilić-Stojanović & de Vos, 2014). Besides, the intestinal gut also harbors conditional pathogens (e.g. Enterococcus and Enterobacteria) (Shen et al., 2018), and pathogens from the phyla Proteobacteria (e.g., Campylobacter jejuni, Salmonella enterica, Vibrio cholera, and Escherichia.coli) and Bacteroidetes (e.g. Bacteroides fragilis), which in healthy conditions represents only ~ 0.1% of the HGM (Jandhyala et al., 2015). Similarly, conditional pathogens are found in low concentrations but these ones only become harmful under certain conditions (e.g.

dysbiosis) (Shen et al., 2018).

During a healthy state, the large intestine mainly maintains a mutualistic relationship with the Firmicutes: Clostridium coccoides and Clostridium leptum group (10-40%), as well as with Bacteroides (Lopetuso et al., 2013). The Clostridium coccoides includes species belonging the genera Butyrivibrio, Clostridium, Coprococcus, Dorea, Eubacterium, Lachnospira, Roseburia, and Ruminococcus, whereas the Clostridium leptum, includes Faecalibacterium prausnitzii and certain species of Eubacterium and Rumicococcus (Nagao-Kitamoto & Kamada, 2017).

Some of these genera are butyric acid-producing bacteria. Moreover, Faecalibacterium prausnitzii and Roseburia are usually considered as probiotic microorganisms (Shen et al., 2018). In this regard, other important probiotic members found in healthy individuals include Lactobacillus and Bifidobacterium, which provide specialized functions essential for to the maintenance of health including: 1. Reduction of levels of circulating Lipopolysaccharides (Rodes et al., 2013). 2. Downregulation of pro inflammatory molecules (e.g., TNF-α, IL-6,IL-8). 3.

Competition with putrefactive and pathogenic bacteria. 4. Increase the activity of antioxidant enzymes (e.g. superoxide dismutase, and catalase) (Molska & Reguła, 2019). 5. Production of vitamins (vitamin K and most of the water‐soluble B vitamins) (Gu & Li, 2016). 6. Upregulation of expression of tight junctions. 7. Reduction of symptoms of lactose intolerance. 8. Production of antibacterial molecules (e.g.

bacteriocins, hydrogen peroxide, and lactic acid). 9. Stimulation of mucin production by goblet cells. 10. Increased secretion of IgA (do Carmo et al., 2018).

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12 Dysbiosis is a harmful condition in which there is no longer a mutualistic relationship between the host and its microbiota. Indeed, this state has been associated with the set, maintenance and severity of several diseases such as obesity (Yu, 2018), colorectal cancer (Rinninella et al., 2019), type 2 diabetes (Allin et al., 2015), inflammatory bowel disease (Matsuoka & Kanai, 2015), and celiac disease (Serena et al., 2019). This state manifests with the reduction of symbiotic mutualistic bacteria as well as from beneficial bacterial species such as Lactobacillus and/or Bifidobacteria. As a consequence, there is an increase of pathogenic and putrefactive bacteria (e.g., Bacteroides) (Gagliardi et al., 2018).

2.4 Ex vivo digestion

Metagenomic studies have expanded the knowledge about the genomic content, the diversity of microbial communities, as well as its taxonomic abundance (Shakya,et al., 2019). Nevertheless, genomics approaches cannot show the kinetics of metabolites production during the fermentation process (Niccolai et al., 2019). For example, genes for the biosynthesis of amino acids have been identified in the gut bacteria Lactococcus lactis and Staphylococcus aureus. Nevertheless, it still requires the addition of the amino acids for their growth (Portune et al., 2016). In this regard, because of the difficulties to gain access to the human large intestine, most of the information about gut microbiota metabolites produced, have been obtained using biological end products (e.g. feces, urine, and plasma), however these samples also contains metabolites derived from human cells (Chen et al., 2019).

Moreover the extraction procedure used can fail in the capture of some metabolites (Yang et al., 2019).

Nowadays, there are available Multistage Continuous Models (MCM) where gut microbiota metabolites can be studied excluding the activity of human enzymes.

MCM consist of bioreactors that can simulate the chemical conditions of the main 5 sections of the human gastrointestinal tract (i.e. stomach, small intestine, ascendant colon, transverse colon, and descendant colon). These systems allow essential

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13 features of the gastrointestinal tract can be controlled including anaerobiosis, the luminal pH, temperature, transition time, mixing (peristalsis or stirring), digestive enzymes, bile salts and microbiota from a population of interest. One example of a MCM is the ARIS (Automatic and Robotic Intestinal System) (García-Gamboa et al., 2020 ).

Figure II-1. ARIS. From left to right. Stomach, small intestine, ascending colon, transverse colon and descending colon.

2.5 Short chain fatty acids

Short-chain fatty acids (SCFA) are aliphatic saturated monocarboxylate fatty acids with total carbon atom numbers going from 1 to 6 (Schönfeld & Wojtczak, 2016). To date, SCFAs are considered the major microbial products derived from the anaerobic metabolism of dietary fiber (Amylase-resistant starch and non-starch polysaccharides), and to a lesser extent, from dietary protein (Liu et al., 2018). The main produced SCFA include acetate, propionate, and butyrate representing 90–

95% of the SCFA present in the large intestine. The other 5% corresponds to valerate, caproate, and formate (Feng et al., 2018). SCFA are produced in an estimated millimolar ratio of 3:1:1 (Acetate: Propionate: Butyrate), and it has been estimated that the total concentration of SCFAs decreases from 70 to 140 mM in the

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14 proximal colon to 20 to 70 mM in the distal colon (den Besten et al., 2013b). An important physiological consequence is that the colonic pH is lower in the ascending colon (pH 5.5–6.5) where fermentation is highest compared to the pH in the distal colon (pH 6.5–7.0) (Andoh, 2016).

Besides the type of substrate, SCFA production depends on transit time, the site of fermentation and the different bacterial species present in the gut microbiota (Silva et al., 2020) from which, by following different pathways, acetate and propionate are produced by the phylum Bacteroidetes whereas Firmicutes are the primary contributors of butyrate (Chakraborti, 2015). Soon after dietary fiber has been metabolized to monosaccharides, these ones are converted into pyruvate via the Embden-Meyerhof-Parnas pathway, Entner-Doudoroff pathway, or Pentose phosphate pathway (Wolfe, 2015). Thereafter, the pyruvate can be used to produce SCFA (Koh et al., 2016).

Physiological role of SCFA

SCFA can be oxidized and contribute to 5–10% of human daily energy requirements.

Besides, SCFA can act as precursors for lipids and carbohydrates synthesis (Sukkar et al.,2019). One of the human cells that mainly rely on SCFA are epithelial cells, where butyrate can contribute to almost 70% of its energy requirements(Gonçalves

& Martel, 2013). Indeed, more than 85% of the butyrate that is generated after fermentation in the gut is immediately metabolized by epithelial cells (Duncan et al., 2007). Interestingly, in ruminants butyrate supply almost all its energy requirements (70%) (Miguel et al., 2019).

Regarding SCFA in the rest of the body, propionate can enter the tricarboxylic cycle as succinyl-CoA to be converted to oxaloacetate, the precursor for hepatic gluconeogenesis (Rahim et al., 2019). Whereas, acetate and butyrate can enter the tricarboxylic cycle as acetyl CoA and be oxidized for energy production (Sukkar et al., 2019). Moreover, acetate and butyrate can act a substrate for lipogenesis (e.g

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15 palmitate), or cholesterol synthesis (den Besten et al., 2013a). In this regard, a higher concentration of propionate in comparison to acetate, can inhibit the conversion of acetate to cholesterol and fat (Oliphant & Allen-Vercoe, 2019).

Beyond its energy contribution, there is now overwhelming evidence of the beneficial regulatory effect of SCFA including, but not limited to: 1. Pathogen exclusion, mainly Gram-negative Enterobacteriaceae, including the pathogens Salmonella spp. and Escherichia coli (Andoh, 2016). 2 . Contribution to mineral absorption by increasing its solubility in the lumen (Ca and Mg ) (Alexander et al., 2019). 3 . Enhancement of the release of the antimicrobial RegIIIγ by gut epithelial cells (butyrate) (Zhao et al., 2018). 4 . Increased secretion of IgA by plasma cells (acetate) (Wu et al., 2017). 5 . Enhancement of macrophages antimicrobial capacity against pathogens (Salmonella, Escherichia Coli, and Staphylococcus aureus) by triggering the release of calprotectin (butyrate) (Schulthess et al., 2019). 6 .Decrease plasma serum cholesterol by increasing fecal excretion of non-conjugated bile acids, and its uptake by the liver (Zhao et al., 2017). 7 . Proliferation of regulatory FOXP3 T cells as well as increase the production of anti-the inflammatory cytokine IL-10 (Zhu et al., 2019). 8. Increase leptin secretion and reduce adipose size in adipose tissue (McNabney & Henagan, 2017). 9. Increase mitochondrial biogenesis in skeletal muscle (González Hernández et al., 2019).

2.6 Biogenic amines

Biogenic amines (BA) are biologically nitrogenous organic compounds found in foods, mammals and plants, that can be produced from decarboxylation of its respective amino acids. The structure of BA can be aromatic and heterocyclic amines (e.g. histamine, tyramine, and serotonin); aliphatic (e.g. putrescine, cadaverine, spermine, spermidine); or aliphatic volatile amines (e.g., ethylamine) (Erdag et al., 2018). BA are mostly known for its content in fermented foods such as cheese, meat, fermented vegetables and wine. In this regard, its presence is associated with poor hygienic practices during the manufacturing process. Important factors for BA production in food include low oxygen (Dapkevicius et al., 2000), pH,

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16 temperature, proteolysis and the presence of microorganisms with decarboxylase activity including bacteria from the genus Lactobacillus, Pseudomonas, Enterobacteria and Enterococcusus (Doeun et al., 2017). Bacteria decarboxylases are induced by the presence of the amino acid precursor under acidic environments.

During the decarboxylation reaction there is a consumption of a proton; thus, this reaction alkalinizes the cytoplasm relative to external medium (Marcobal et al., 2012). In the absence of this system the accumulation of protons can lead to aggregation of intracellular proteins (Moreau, 2007).

Nowadays, there is a concern about the presence of BA in food. For example, putrescine and cadaverine can react with nitrites to yield the carcinogenic compounds Nitrosopyrrolidine and N-nitrosopiperidine, respectively (del Rio et al., 2019). In addition, its elimination from food product is difficult because BA, and decarboxylating enzymes are heat stable (Gillman, 2016). Generally, the intake of dietary BA causes no adverse reactions because humans possess amine oxidases, such as Monoamine Oxidase (MAO) and Diamine oxidase (DAO) for BA degradation (Mah et al., 2019). DAO activity is elevated in the small intestine, kidney, placenta,stomach, duodenum and colon (Fernández-Reina et al., 2018; Mateescu et al., 2017). Whereas, MAO is mainly found in the liver, nervous system, liver, muscle, and intestinal mucosa (Marcobal et al., 2012). Nevertheless, the detoxification process can be impaired during pharmacological treatment with MAO inhibitors which are frequently used to relief pain, stress , depression (Erdag et al., 2018), and also to treat patients with Parkinson’s disease (Flanagan et al., 2013).

Additionally, substances such as acetaldehyde, can block the activity of DAO and MAO (Doeun et al., 2017). Similarly, DAO degradation capacity can be impaired by the deficiency of its cofactors vitamin B6, vitamin C, copper, and zinc (Kovacova- Hanuskova et al., 2015). Besides, during pathologies like Ulcerative Colitis and Chron’s disease, there is a decrease in DAO activity (Mateescu et al., 2017).

Additionally, cadaverine and putrescine can facilitate the transport of histamine through the intestinal wall , and also inhibit the activity of DAO and histamine N- methyltransferase; thus, increasing its rate of absorption into the blood stream (del Rio et al., 2019). Moreover these amines can also block the activity of MAO

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17 (Karovicova et al., 2006).

To date little is known concerning the potential of human gut microbiota to produce BA and the in vivo influence of microbial-derived BA. In this respect, a recent study done by Pugin et al. (2017) found that human isolated strains belonging to the genera Bifidobacterium, Clostridium, Enterococcus, Lactobacillus, Pediococcus, Streptococcus, Enterobacter, Escherichia, Klebsiella, Morganella and Proteus , were are able to produce cadaverine, putrescine, spermine, spermidine, histamine, and tyramine after the provision of their amino acids precursors.

Polyamines

Polyamines (PAS) are molecules ubiquitous in all mammals and plants essential for basal cell function (Handa et al., 2018). PAS consists on aliphatic amines that contain more than two amino groups. The major PAS include Spermidine (Aminopropyl tetramethylene diamine), Spermine (Diaminopropyl tetramethylene diamine), and in less amount its precursor Putrescine (Tetramethylenediamine) (Büyükuslu, 2015). PAS crystals were first reported in 1674 by Antonie van Leeuwenhoek in human semen samples, but it was until 1924 when Rosenheim elucidated its structure and also synthesized them (Bachrach, 2010). Intracellular concentration of PAS have been reported to be 0.3~0.5 mM for Putrescine (PUT) , 3.9~5.9 mM for Spermidine (SPD) and 1.2~1.9 mM for Spermine (SPM) (Park & Kim, 2012), whereas luminal concentrations of PUT and SPD in healthy humans are between 0.5 to 1 mM (Tofalo et al., 2019). In mammals, the PAS pool can be obtained from endogenous sources (de novo synthesis from amino acids), or exogenous sources (e.g. diet and gut microbiota). Other minor contributions come from pancreatic-biliary secretion and villous extrusion from sloughed epithelial cells (Timmons et al., 2012).

PAS from dietary origin are considered the primary source of luminal PAS (Büyükuslu, 2015). Indeed, human milk becomes the first supply of exogenous PAS.

It has been estimated that babies can consume up to 3357– 4700 nmol of PAS per day from the milk of healthy mothers (Ali et al, 2013). Thereafter in life, fermented

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18 foods and the Mediterranean diet with high fruit and vegetables, become the most significant source of luminal PAS (Soda, 2019). In this regard there is no regulation or stablished legal limits for PAS in foods (del Rio et al., 2019). Recently, Del río et al. (2019), reported that the concentration of PUT required to achieve half of the strongest cytotoxic effect (IC⁵⁰) in the human colon adenocarcinoma cell line HT29 after 24 h of exposure was 3505 mg/kg. Whereas, the NOAEL and LOAEL were 441 mg/kg and 882 mg/kg , respectively. According to the authors, the cytotoxic mode of action of PUT was by causing cell necrosis.

Previous research administrating radiolabeled PAS in rats have demonstrated that these molecules are absorbed by passive diffusion for epithelial cells and distributed into the systemic circulation to reach various tissues such as lungs, spleen, heart, brain, and liver. Additionally, these authors found that, in comparison to PUT, SPM and SPM reach portal circulation in higher concentrations as PUT is rapidly oxidized by DAO in the small intestine (Bardócz et al., 1993; Kobayashi et al., 2003). Five years later, it was discovered that PUT can be converted to succinate in order to serve as an energy source for epithelial cells(Bardocz et al, 1998) Physiological role of Polyamines

PAS are molecules that at physiological pH carry a positive charge on their primary and secondary amino groups. This cationic nature allows them to interact an regulate pleiotropic functions on negatively charged molecules including, DNA, RNA , proteins , phospholipids (Büyükuslu, 2015), and also with free radicals (Miller- Fleming et al., 2015). For example, but not limited to, PAS can regulate growth, proliferation and differentiation of cells (Ali et al., 2011) by driving DNA replication, transcription, and translation (Korovina et al., 2012). Indeed, the oncogene Myc , which is activated in nearly 70% of all human cancers, upregulate synthesis of PAS in order to increase the proliferation rate (Phanstiel IV, 2018). Moreover, cancerous cells enhance the uptake of extracellular PAS to sustain the transcription of proteins that degrade the tissue matrix; thus, accelerating the process of metastasis (Soda, 2011).

Most of intracellular PAS are bound to ribosomes (Miller-Fleming et al., 2015)

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19 .In fact, PAS participate on a process called Hypusination, which is the posttranslational modification of the eukaryotic translational initiation factor elF5A.

During this process the aminobutyl group of SPD is added to a lysine residue of elF5A to form a protein called hypusine (Nε-(4-amino-2-hydroxybutyl)-lysine) (Wolff

& Park, 2015). Hypusine, binds to ribosomes and is used to facilitate peptide bond formation on the consecutive proline residues of mRNAs that code for 158 genes that code for proteins essential for human physiology such as DNA transcription, actin cytoskeleton, RNA splicing, extracellular protein, and signaling receptors (Mandal et al., 2014). However, the number of these genes could be higher as recently it has been found that proteins involved in the tricarboxylic acid cycle and oxidative phosphorylation also need hypusine for its proper translation (Puleston et al., 2019). To date elF5A is considered the only protein to be hypusinated (Bekebrede et al., 2020).

In mammals, PAS are essential since the first moment of life. Children receiving milk with less SPM and SPD show a high prevalence for the development of allergies, which has been attributed to the role of PAS during intestinal maturation (Peulen et al., 1998). Studies in mice have found that depletion of PAS impairs embryonic development (Nishimura et al., 2002), and its supply is essential for early development of the immune system and intestinal nutrient absorption (Romo et al., 2017). Moreover, depletion of PAS impairs the expression of E-cadherin, an essential protein that prevents paracellular flux and maintains epithelial barrier function (Liu et al., 2009).

Once the organism reaches adulthood, the endogenous synthesis of PAS gradually starts decreasing (Bupp et al., 2018). However, the biosynthesis of PAS is maintained at relatively high rates in rapidly proliferating tissues,and those receiving growth-promoting hormonal stimuli (Pegg, 2016). Among the tissues that require relatively high amount of PAS, is the gastrointestinal tract which renews every 2-5 days (Gao et al., 2013).

PAS are recently began to be considered as essential molecules similar to vitamins because of its important contribution to health; thus, exogenous supplementation with PAS has been suggested (Madeo et al., 2019). In this respect,

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20 one of the most relevant studies to date, is that oral administration of SPD, retarded senescence and increased the chronological lifespan of nematodes, flies, yeast as well as of human peripheral blood mononuclear cells, by the upregulation of autophagy genes (Eisenberg et al.,2009).

Biosynthesis and catabolism of Polyamines

De novo synthesis of polyamines starts in with the conversion or arginine to ornithine by the enzyme arginase (Lefèvre et al., 2011). The amount of intracellular PAS is tightly regulated; thus, when SPD and SPM levels are high, the enzyme ornithine decarboxylase antizyme enhances degradation of ornithine decarboxylase by the ubiquitination of this one to the 26S proteasome(Ignatenko et al., 2011). In addition, ornithine decarboxylase antizyme increases polyamine efflux and decreasing polyamine uptake (Linsalata et al.,2014). In parallel, when PAS concentration is high, the constitutively expressed zinc dependent enzyme N1- Spermidine/Spermine- Acetyltransferase (SSAT) transfers acetyl groups onto SPD and SPM in order to reduce its charge; Thus, these ones no longer can bind to acidic molecules and are available to be either exported out of the cell or oxidized in the peroxisome (Pegg, 2008). The system regulating the concentration of PAS is important as upregulation of PAS has been observed in patients with asthma (Ilmarinen et al., 2014); type 2 diabetes (Fernandez-Garcia et al., 2019); obesity (Codoñer-Franch et al., 2011);

vaginosis (Nelson et al., 2015); alzheimer (Inoue et al., 2013), and colon cancer (Wallace & Caslake, 2001).

Besides, excess catabolism of PAS can reduce metabolic functions by depleting intracellular acetyl-CoA levels (Bekebrede et al., 2020), it also increases oxidative stress through the production of hydrogen peroxide, reactive oxygen species, as well by driving up the regulation of genes involved in the oxidative stress response such as p53, A170 stress-induced protein and cytoskeletal keratin 18 (Cerrada-Gimenez et al., 2011). Other harmful metabolites produced after PAS catabolism, include acrolein and 3 amino propanal (Sindhu, 2016). Acrolein can deplete cellular glutathione levels by forming the acrolein adduct S-(2-aldehydo- ethyl) glutathione (Singh et al., 2010). Indeed acrolein has been found to be in higher

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21 concentrations in patients with chronic kidney disease (Sindhu, 2016). Whereas, 3- Aminopropanal can damage RNA, DNA, proteins and cell membranes (Casero Jr &

Pegg, 2009).

Cadaverine

Cadaverine (1,5-diaminopentane) is also a polyamine, which is synthesized from the amino acid lysine instead of ornithine. In contrast to PAS, cadaverine (CAD) has not been found under physiological conditions (Bekebrede et al., 2020); thus, exogenous sources such as from fermented foods and possibly the gut microbiota are the main contributors (Pugin et al., 2017). CAD is one of the most abundant biogenic amines in fermented foods, where the genus Enterobacteria is the major microbial group correlated its production (Sahin-Ercan et al., 2016). Recently, Del Río et al. (2019), reported that the concentration of CAD required to achieve the half of the strongest cytotoxic effect (IC⁵⁰) in the human colon adenocarcinoma cell line HT29 after 24 h of exposure was 4161 mg/kg. Whereas, the NOAEL and LOAEL were 255 mg/kg and 511 mg/kg respectively, according to the authors the cytotoxic mode of action of cadaverine was by causing cell necrosis.

As a BA, CAD is used as a bacterial mechanism against acidic conditions.

The biosynthesis of this amine has been more studied in E. Coli , in which the CAD operon includes, a lysine decarboxylase, a lysine/cadaverine antiporter and a regulatory protein .This operon is activated at acidic pH (pH< 6.8) in a lysine rich environment (>5 mmol/L) (Ma et al., 2017).

Tyramine

Tyramine (TYR) is a vasoactive monoamine found in animals, plant ,and bacteria (Andersen et al., 2019). Humans beings can obtain this BA from the endogenous decarboxylation of tyrosine by the aromatic amino acid decarboxylase (Anwar et al., 2012), or from exogenous sources such as diet and gut microbiota (Pugin et al., 2017). TYR name come from the Greek word tyros which means

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22 cheese, the first source in which TYR was detected (Sathyanarayana & Yeragani, 2009). Nowadays, TYR has been found in various types of foods, but mainly in fermented foods where bacteria from the genus Lactobacillus, Enterococcus, Leuconostoc, and Lactococcus have been associated with its production (Andersen et al., 2019). In this regard, the activity of bacteria tyramine decarboxylase is reported to be optimum at pH 5 and its activity increases with the abundance of TYR in the media (Marcobal et al., 2012).

In humans, endogenously produced TYR form part of the trace amines , which consist on β-phenylethylamine, tryptamine, octopamine and synephrine (Anwar et al., 2012). These molecules have a structure similar to the endogenous biogenic amines dopamine, norepinephrine, serotonin, and histamine. However, trace amines only represents < 1% of these amines (Zucchi et al., 2006) . Trace amines are stored in synaptic vesicles and after release, these ones have affinity towards trace amine- associated receptors. Recently, Raab et al. (2016) reported that the trace amino acid receptor 1, is highly expressed in human cells from the pancreas and small intestine (duodenum and jejunum). Also, after exposure of cells from these organs with an agonist of this receptor, there was an increase in insulin secretion as well as production of PYY and GLP-1. According to the authors, this could be novel mechanism to exert physiological effects by the trace amines found in food. In this respect, among trace amines, TYR is one of the most potent activator of the trace amino acid receptor 1 (Pei et al., 2016).

Nevertheless, TYR has been mostly known for its activity as a false neurotransmitter (Khan & Nawaz, 2016). In this regard, TYR can stimulate sympathetic nerve terminals by displacing norepinephrine from the synaptic vesicles, which then activates adrenoreceptors (α and β1) in the myocardium, and blood vessels leading to an increase in heart contractility (Molenaar et al.,2007), as well as a rise in blood pressure (Finberg & Gillman, 2011; Zucchi et al., 2006).

Indeed, foods with a high content of TYR have been associated with migraine, stroke, heart attack, and increased respiration. As these effects were first reported after cheese consumption, especially aged cheeses, the overall episode is called

“tyramine reaction “or “cheese reaction” (Andersen et al., 2019). The degree of these

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23 effects greatly depends on MAO activity. Two MAO isozymes exist, A and B. MAO- A deaminates serotonin in the nervous system and dietary monoamines in the gastrointestinal system. MAO-B is found predominantly in liver and muscle and deaminates dopamine (Marcobal et al., 2012). Tyramine is a substrate for either form of MAO, being MAO-A, the one with more affinity to tyramine. (Finberg & Gillman, 2011).

Tyramine is considered the most toxic BA. Nevertheless, no legal limits have been stablished for tyramine in foods products. In this regard, the European Food Safety Authority (EFSA) has stablished that an intake of 600 mg of tyramine per meal is not likely to cause adverse effects in healthy individuals not taking MAO inhibitors but those taking these ones , should not take more than 6 mg per meal (Andersen et al., 2019). In this respect, recently, Linares et al. (2016), reported that the concentration of tyramine required to achieve half of the strongest cytotoxic effect (IC⁵⁰) in HT29 cells was 439 mg/kg. Whereas, the NOAEL and LOAEL were 247 mg/kg and 302 mg/kg, respectively. According to the authors the cytotoxic mode of action of tyramine was by causing cell necrosis. In addition, another study found that TYR concentrations found in food (250-1000 mg/Kg) decreased the expression of DNA repair proteins (del Rio et al., 2018).

Histamine

Histamine (2-[4-imidazolyl]ethylamine) consists of a molecule that primarily acts as an immune modulator, and neurotransmitter in the human body. Due to its presence various tissues, its name come the Greek word histos which means tissue (De Benedetto et al., 2015). Histamine (HIS) can be endogenously synthesized by various types of cells (i.e. Macrophages, eosinophils, neutrophils, dendritic cells, platelets, neurons, gastric mucosal cells, mast cells, and basophils).The process of synthesis starts in the Golgi apparatus with the decarboxylation of the amino acid histidine by the enzyme histidine decarboxylase. In this regard, mast cells and basophiles are the only cells that can synthesize and store histamine in intracellular granules (Brzezińska-Błaszczyk, 2010).Indeed these cells are the major producers

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24 of HIS (Fernández-Reina et al., 2018).

The release of HIS from this granules is called degranulation, and is triggered by allergens, extreme temperatures, lipopolysaccharides (Mateescu et al., 2017), as well as citrus fruit, peanuts, chocolate, and spinach (Maintz & Novak, 2007).During degranulation, T and B cells recognize the stimuli and lead to the production of specific IgE antibodies which then bind to high affinity FcεRI receptors of histamine storage cells, mainly basophils and mast cells, triggering the release of histamine and other proinflammatory mediators that lead to an inflammatory allergic response, which may include bronchoconstriction, mucus hypersecretion, and vasodilation (Thangam et al., 2018).

Dietary intake and Histamine Legislation

Apart from endogenous production, HIS can be obtained from the diet, especially from fermented foods such as anchovy sauce, sand lance sauce, squid and clam paste (Xiang et al., 2019). In this respect, the EFSA and Food and Drug Administration (FDA) have legislated histamine concentration in foods. However, this legislation only applies for fish products, in which the EFSA established maximum histamine concentrations of 200 mg/kg and 400 mg/kg for scombroid-like fish and fish products, respectively. Whereas, the FDA has suggested that histamine concentrations in fish of over 500 mg/kg can represent a danger to health (Linares et al., 2016). In this respect the most known histamine intoxication is Scombroid fish which is caused by the ingestion of high amounts of HIS from fish of the Scomberesocidae family (e.g. mackerel, bonito, albacore, and skipjack), as well as other fishes (e.g. sardine, and bluefish) (Feng et al., 2016).

Physiological role of histamine

The systemic effect of HIS is influenced by 4 different histamine receptor expressed in a variety of cell types (brain, immune cells, airways , cardiac tissue,

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25 and smooth muscle) (Thangam et al., 2018), as well as on the ability of the host to metabolize and degrade excessive histamine by the activity of DAO and intracellular histamine-N-methyltransferase (Mateescu et al., 2017). A defect in the efficacy of these enzymes, can lead to Histamine intolerance, also known as food histaminosis or food histamine sensitivity, which in contrast to histamine intoxication, can be triggered by low concentrations of histamine (Sánchez-Pérez et al., 2018).

Alterations in HIS metabolism can lead to the development of anaphylaxis, peptic ulcer, atopic dermatitis, asthma, neurological disorders and rheumatoid arthritis (Pino-Ángeles et al., 2012). Similarly, histamine intolerance and histamine intoxication, can lead to a pseudo allergy, which is called in that way because it triggers symptoms similar to an allergic response, including abdominal pain, diarrhea, vomiting, urticaria, dermatitis, pruritus, rhinitis, nasal congestion, asthma, arrhythmias, and headaches (Sánchez-Pérez et al., 2018).

Recently, Linares et al. (2016), reported that the concentration of histamine required to achieve half of the strongest cytotoxic effect (IC⁵⁰) in HT29 cells after 24 of exposure was 2890 mg/kg. Whereas the NOAEL and LOAEL were 333 mg/kg and 445 mg/kg, respectively. According to the authors the cytotoxic mode of action of histamine was by causing cell apoptosis. Moreover, Del rio. (2018), found that histamine caused DNA fragmentation and downregulation of the gene coding for carnitine palmitoyl transferase 2.

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III. Hypothesis and objectives

3.1 Hypothesis

Diets high in animal or vegetable protein could generate dysbiosis in a healthy human gut microbiota in an ex vivo model, and also cause the production of biogenic amines

3.2. General Objectives

Study the population dynamics of the healthy human gut microbiota and establish the potential production of metabolites during a diet high in animal and plant protein using an ex vivo human intestinal digestion system.

3.3. Specific Objectives

a) Extraction and characterization of red meat and black bean protein for the design of high protein diets with a similar amino acid profile.

b) Ex vivo digestion of the animal and vegetal high protein diets on the manual version of the simulator of the human gastrointestinal tract ARIS.

c) Quantification, after 24 days of fermentation, of Lactobacillus, Bifidobacteria, and Enterobacteria (ascending and descending colon), by Real time PCR.

d) Quantification, after 24 days of fermentation, of short and branched chain fatty acids (ascending and descending colon), as well as biogenic amines (descending colon), by gas chromatography and liquid chromatography, respectively.

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IV. Methodology

The present work was divided into three stages. The first one consisted in the design oh high protein diets followed by the obtention of the protein fraction of black bean (Phaseolus vulgaris L) , and beef. The second one consisted in the evaluation of the diets in the ex vivo human gastrointestinal model ARIS. Finally, the third stage consisted in the quantification of bacterial groups by real time qPCR, as well as the determination of biogenic amines and fatty acids by high-performance liquid chromatography and gas chromatography, respectively.

4.1 Reagents

Sodium Hydroxide, Hydrochloric Acid and Hexane were purchased from DEQ (Development of Chemical Specialties, Mexico). Zein, Sodium Bicarbonate, Ethyl Ether, Acetone, Dansyl Chloride, Ammonium Hydroxide( ≥25% NH3 in H2O), Volatile Free Fatty Acid Mix,Tyramine (≥98.5%), Cadaverine (≥96.5%), Putrescine (≥98.5%), and Histamine (≥97.0%), were purchased from Sigma Aldrich (St. Louis, MO), HPLC grade Acetonitrile and HPLC Water were purchased from CTR Scientific (Monterrey, NL). Yeast supplement of Saccharomyces Cerevisiae was obtained from (Tecnica industrial Rosanco, (Orizaba, Veracruz).

4.2 Development of control and high protein diets

In this work, the ARIS standard diet (19% protein, 56 % carbohydrates, and 25% fat) from the research laboratory CIATEJ (Guadalajara, Jalisco) was used as a control diet, and modified in order to design a high protein diet (50% protein, 30%, carbohydrates, and 20% fat). The amount of each of the sources used to accomplish the macronutrients stablished for the diets, are shown in Table IV-1. The source of protein of the control diet was a commercial yeast supplement from Saccharomyces

Instituto Tecnológico y de Estudios Superiores de Monterrey Campus Monterrey School of Engineering and Sciences

Abstract

Abbreviations

List of figures

List of tables

Table of contents

I. Introduction

II. Literature review

III. Hypothesis and objectives

IV. Methodology