Effect of dairy matrices on the survival of Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei during in vitro and in vivo digestion.

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Milk proteins as source of bioactive peptides

Despite there are numerous sources like soy, and meat, milk and dairy products are the best precursors of bioactive peptides (Korhonen 2009). The functionality of these proteins is evident when we think that milk is the main aliment in newborns that are provided by an immature digestive system, and who therefore depend completely of the proteins present in breast milk (immunoglobulins, lysozyme, lactoperoxidase, lactoferrin, etc.) and the immunocompetent cells (macrophages, lymphocytes, T and B cells, etc.) to fight potential infections. In addition with the growth factors, that have an important role on the development of the intestinal and immune system (Martínez Augustin & Martínez de Victoria, 2006).
It is important at this point to make a distinction between the bioactive proteins naturally present in milk (like immunoglobulins) and the bioactive peptides that are released from these native proteins after digestion. Bioactive peptides are the resulting product of a breakdown of proteins by enzymes, which exert a specific bio-function only after release from the original protein (Kitts & Weiler, 2003). Some of bioactive peptides derived from caseins reveal multifunctional properties; for instance, peptides from the sequence 60-70 of β-casein show immunostimulatory, opioid and inhibition of the angiotensin converting enzyme (ACE) activities at the same time, probably because these regions in the primary structure of caseins contain overlapping peptides sequences, which exert different biological effects. These regions have been considered as “strategic zones” which are partially protected from proteolytic breakdown because of its high hydrophobicity and the presence of proline residues (Korhonen & Pihlanto, 2003; Meisel, 2005).
In dairy products, the bioactive peptides can be released through the proteolytic action of the natural present enzymes in milk. However the most productive way to liberate these peptides is during food processing, via proteolysis by microbial enzymes from the lactic acid bacteria or secondary starters (Choi et al., 2012).

Bioactive peptides in cheeses

Cheese is a very complex food matrix containing numerous peptides released by a proteolysis especially during a ripening period. Proteolysis is a complex and important biochemical event that occurs during cheese manufacture, especially during ripening. Despite the type of cheese, the main objective of proteolysis is the degradation of complex proteins into peptides and amino acids.
In most ripened cheeses, proteolysis can be summarized as follows: Initial hydrolysis of caseins is catalyzed by residual coagulant, plasmin, cathepsin D (in some cases) and somatic cell proteinases releasing large (water insoluble) and intermediate-sized peptides (water soluble). Those peptides are subsequently hydrolyzed by the coagulant and enzymes from the starter and nonstarter flora of the cheese releasing small peptides and free amino acids (Fox & McSweeney, 1996; McSweeney & Sousa, 2000). In this regard, numerous studies in different cheeses have identified the biological activity of a wide range of peptides released during proteolysis, establishing that the type and quantity of these peptides is dependent mostly of the starter culture used and the ripening conditions employed (Choi et al., 2012; Gobbetti, Stepaniak, De Angelis, Corsetti, & Di Cagno, 2002; Gupta et al., 2009; Gupta et al., 2013).
Some of the bioactivities identified include the antibacterial activity in water extracts of Mozzarella, Gouda, Swiss and Cheddar cheeses (Pritchard et al., 2010; Theolier et al., 2014); cytomodulatory properties in buffala Mozzarella cheeses (De Simone et al., 2009) and lyophilized extracts from middle aged Gouda cheese (Meisel & Günther, 1998); antioxidant activity in water extracts of Cheddar cheeses (Pritchard et al., 2010) or anti-hypertensive activity in Cheddar cheeses added with probiotics (Ong et al., 2007) or in water extracts of Mexican fresh cheeses (Paul & Van Hekken, 2011; Torres-Llanez et al., 2011) mong others activities (Table 1.1).

Cheese as a vehicle for viable microorganisms

The total microbial population in a cheese ecosystem is in average 108 –109 viable cells/g of cheese, and that the annual consumption is 24 kg per French, it is traduced in an annual consumption of 2.4 x1012 and 2.4 x 1013 viable cells through the consumption of cheeses (Adouard et al., 2015). This raises the interests of cheese as a dairy matrix that can act as a vehicle for viable cells to enter in the digestive tract.
In this field, there are many questions that actually remain about the fate of eaten microorganisms in the gastro-intestinal (GI) tract. When a food microorganism is capable to reach the epithelial surfaces in the gastrointestinal tract, it automatically interacts with the intestinal microbiota. From this interaction results the beneficial effects exert by probiotics. However this interaction might not be exclusive for recognized probiotic microorganisms, cheese microorganisms could also exert some kind of effects on the intestinal microbiota as will be discussed in the next paragraph.

Potential effect of cheese microorganisms on human health

Interaction between cheese and intestinal microbiota could result in beneficial effects for the host. Lay et al., (2004) tested the effect of the Camembert microbiota on rats, finding an important decrease of azoreductase activity and an increase in mucolytic activity. Ibrahim et al., (2010) tested the immunomodulatory effect of commercial probiotic cheese in elderly patients and, further the effect of the probiotics on the host, they observed an increase in phagocytosis activity related to the consumption of control Gouda cheese. They attributed this immune response to the starter strains of the control cheese. Additionally, authors suggested that immune response of the host could be enhance according to the matrix used, founding higher response when cheese was used as probiotic carrier than previous studies from other authors using skim milk. Adouard et al., (2015), tested the immunomodulatory effect of different cheese-ripening microorganisms. Authors found that some of those dairy microorganisms exert a significant production of interleukin 10 (IL-10) that resulted even higher than the one observed for a well-known probiotic strain identified as B. longum Bb536.
Thus, from this point of view and without attempting to give the name or characteristics of probiotic to these microorganisms, we can notice the potential of cheese microbiota on health; and the importance of cheese as a dairy matrix carrier of live microorganism.
However and, as we previously established, in order to exert any kind of biological activity, cheese microbiota (and bioactive peptides) need to pass through stomach and tolerate its high acidity, before going through an intestinal stress mainly related to the effects of bile salts (Sumeri et al., 2012). It therefore appears essential to study their subsequent survival to the stress induced by the digestion.


Arginine Deiminase (ADI) pathway

This system has 4 components, ADI (EC, catabolic ornithine transcarbamoylase (cOTC, EC, carbamate kinase (CK, EC and a membrane transport protein. It converts arginine to L-citrulline and ammonia. Citrulline is further degraded forming ATP and L-ornithine. The released ammonia gives protection to LAB against acid damage by increasing the pH in the cytoplasm (Hutkins & Nannen, 1993). Additionally ADI pathway produces extra ATP that enhances the expulsion of protons by H+-ATPase complex. Lactobacillus sanfranciscensis utilize this ADI pathway during sourdough fermentation resulting in in an increase in ornithine production (De Angelis & Gobbetti, 2004).

Metabolism of urea

Helicobacter pylori, a well-known acid resistant bacteria, capable to live in the human stomach and cause gastric ulcers, has the ability to use urease to produce ammonia and bicarbonate to maintain a pH of around 6 inside its periplasmic space, even when the external conditions are around pH 2 (Adouard et al., 2015; Sachs, Weeks, Wen, Marcus, & Scott, 2005). This enzyme has been found in S. termophilus and the absence of it has been associated with the lack of ability of some strains to survive the in vitro digestive stress (Uriot et al., 2016).
Those microorganisms that are capable to survive the acid conditions in stomach can reach the duodenum, where they need to face a new stress caused mostly by the bile acids.

Bacteria stress in the intestine

Bile acids are synthesized in the liver from cholesterol and secreted from the gall bladder into the duodenum in the conjugated form. Later in the colon these acids suffer modifications such as deconjugation, dehydroxylation, dehydrogenation and deglucuronidation, through microbial metabolism. Both conjugated and deconjugated bile acids exhibit antibacterial activity, but the deconjugated forms are more lethal (Dunne et al., 2001).
Gram-positive bacteria are more sensitive to these salts than gram-negative. However, bile tolerance is also strain specific (Begley et al., 2005; Li, 2012). In gram-negative bacteria, the outer membrane constitutes an excellent hydrophobic barrier against bile. Still bile salts can go through the membrane and disturb the membrane characteristics such as charge, hydrophobicity and lipid fluidity because of their detergent properties (Begley et al., 2005). Bile salts can generate oxygen free radicals, alter RNA secondary structure, induce DNA damage and activate DNA repair related enzymes.(Li, 2012).
However, some bacteria are highly resistant to the stress caused by the bile salts/acid in the intestine. Some of the mechanisms observed by these bacteria are related to the expulsion of the bile by pumps, enzyme actions to metabolize the bile, production of specific proteins related to the membrane synthesis, etc.

Efflux pumps

Outflow of bile salts from the bacterial cytoplasm is without doubts the best-characterized and probably the most important mechanism of bile salt resistance.
Efflux pumps are enzymes active transporters localized in the cytoplasmic membrane of cells. The best characterized one it’s identified as AcrB which gives the resistance to E. coli against solvents, detergents, antibiotics and bile salts (Gunn, 2000). AcrB is formed by: a transporter (efflux) protein (AcrB) an accessory protein (i.e. AcrA) and an outer-membrane (for example TolC), located in the outer membrane protein channel. AcrB captures its substrate either from within the phospholipid bilayer of the inner membrane, or from the cytoplasm, and then transport then to the extracellular medium through TolC, which forms a channel in the outer membrane. AcrA protein works as the intermediate between TolC and AcrB. This pump utilizes the energy of the proton motive force (PMF) (Piddock, 2006). In gram-positive bacteria there are other efflux pumps with only one component, instead of three which can realize the same function as AcrB., for instance MexAB-OprM in P. aeruginousa (Sun, Deng, & Yan, 2014).

Table of contents :

1. Literature review
1.1 Functional Foods
1.2 Bioactive peptides
1.3 Milk proteins as source of bioactive peptides
1.4 Bioactive peptides in cheeses
1.4.1 Antioxidant
1.4.2 Antihypertensive
1.5 The case of Mexican cheeses
1.5.1 Cotija cheese
1.5.2 Fresh goat cheese
1.6 Cheese microbiota
1.7 Cheese as a vehicle for viable microorganisms
1.8 Potential effect of cheese microorganisms on human health
1.9 Bacterial stress in the stomach
1.9.1 Protein expression
1.9.2 Transport protein activation
1.9.3 Metabolism modifications Arginine Deiminase (ADI) pathway Metabolism of urea
1.10 Bacteria stress in the intestine
1.10.1 Efflux pumps
1.10.2 Bile salt hydrolases
1.10.3 Extracellular polysaccharides
1.11 Effect of a food matrix in the microorganism survival
1.11.1 Dairy matrices
1.11.2 Dairy matrix effect on microorganism survival
1.12 Mechanisms for matrix protection effect
1.12.1 Preadaptation effect
1.12.2 Macrostructure effects
1.12.3 Microstructure effects
2. Results and Discussion
2.1: Angiotensin converting enzyme inhibitory and antioxidant peptides release during ripening of Mexican Cotija hard cheese.
2.1.1 Presentation of the first article published in the Journal of Food Research
2.1.2 Article: Angiotensin converting enzyme inhibitors and antioxidant peptides release during ripening of Mexican Cotija hard cheese
Material and Methods
Results and Discussion
2.1.3 Complementary results Evolution of pH Moisture evolution Protein evolution Evolution of nitrogenous fractions during ripening Nitrogenous fractions analysis by RP-HPLC Antioxidant activity ACE inhibitory activity
2.2: Antioxidant and angiotensin converting enzyme inhibitory activity in fresh goat cheese prepared without starter culture. A preliminary study.
2.2.1 Presentation of the second article published in the CyTA Journal of Food
2.2.2 Article: Antioxidant and angiotensin converting enzyme inhibitory activity in fresh goat cheese prepared without starter culture. A preliminary study
Materials and Methods
Results and Discussion
2.3 Final remarks of section 2.1 and 2.2
2.4 Effect of dairy matrices on the survival of Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei during in vitro and in vivo digestion.
2.4.1 Presentation of the third article in preparation
2.4.2 Article: Effect of dairy matrices on the survival of Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei during in vitro and in vivo digestion .
Material and Methods
Results and Discussion
3. General discussion
4. Conclusions and perspectives


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