P1: SFK/UKS BLBS102-c35 P2: SFK BLBS102-Simpson March 21, 2012 14:9 Trim: 276mm X 219mm Printer Name: Yet to Come 35 Biochemistry and Probiotics fats made this ingredients as a very good source of prebiotic components for human body as well as yogurt and probiotics bacteria (Boye et al 2010) Enriching milk with prebiotic supplements (Capela et al 2006) enhances the stability of probiotics in yogurt, but very few studies have been conducted with soy or pulses Addition of soy protein isolates in yogurt does not improve stability during storage (Pham and Shah 2009) but some pulses (Zare et al 2011) Food fermentations with probiotics have mainly been conducted on dairy and soy substrates Pulses contain many of the carbohydrates of soy but there are also various other oligosaccharides (Table 35.3) Therefore, from a carbohydrate perspective, growth of probiotics in pulses should require similar enzymatic profiles to those required in soy; however, a wider range of cultures can theoretically develop in some pulses due to the presence of verbascose or other oligosaccharides Cereal-based products also offer many possibilities for the development of probiotic-based (Farnworth 2004) Cereals mainly have starch Assimilation of starch typically requires amylases and maltases Vegetables are also frequently used as substrates for lactic acid fermentations and are thus potential matrices for probiotic cultures (Farnworth 2004) Sucrose is often found in these matrices, but a wide variety of substrates are encountered In celery, for example, mannitol is the main carbon-based substrate but original polysaccharides are also discovered (Thimm et al 2002) It would take too long to examine each plant-based matrix for the required enzymes Following are the points that must be emphasized: Carbohydrate substrates vary as a function of the food matrix (Table 35.3) and, thus enzymatic requirements vary accordingly There is variability in the ability of probiotic bacteria to use the carbohydrates (Tables 35.1 and 35.2); a strain selection process is required BIOCHEMISTRY AND STABILITY DURING STORAGE IN FOODS Protection Against Oxygen Probiotic bacteria are sensitive to many processes (heating, freezing, aeration) associated with food production or storage conditions (Champagne et al 2005) But they are also sensitive to some conditions that occur during storage The two most important are the presence of oxygen and high acidity of the food In both cases, some enzymatic systems may enhance the survival of the cultures to these stressful conditions Since the intestines offer an anaerobic environment, many strains are not adapted to growth in aerobic conditions Technological adaptations can be made: microencapsulation, addition of antioxidants, packaging conditions (Talwalkar and Kailasapathy 2004, Jimenez et al 2008) But some strains possess enzymatic systems that enhance their stability in foods containing oxygen The major problem associated with the presence of oxygen is that hydrogen peroxide is produced in various metabolic 679 pathways The two major enzyme systems that eliminate this toxic by-product of oxygen are NADH oxidase and NADH peroxidase (Shimamura et al 1992, Talwalkar and Kailasapathy 2003) These systems are inducible Thus, when cultures are gradually exposed to peroxide, an increased synthesis of these enzymes occurs NADH oxidase and NADH peroxidase function optimally at pH 5.0 (Talwalkar and Kailasapathy 2003), which suggests that the protection to oxygen would be higher in acid foods (yogurt, fruit juices) than in neutral foods (unfermented milk, vegetables) Not surprisingly, it was shown that when a probiotic culture was added at the beginning of a yogurt fermentation process, it was more stable during storage than when it was added directly during the finished product (Hull et al 1984) One hypothesis was that the cultures were gradually exposed to H2 O2 produced by L delbrueckii ssp bulgaricus during fermentation and that enzymes were synthesized that were subsequently useful during storage Indeed, when catalase was added in the products having direct inoculation of the probiotic culture in the finished product, an increased stability during storage was noted, while this was not the case in those where the probiotic culture was added earlier in the manufacture (Hull et al 1984) It can also be argued that the gradual exposure to acid during fermentation would also be involved in the beneficial effect of inoculation at the beginning of the fermentation Thus, enzymatic adaptation in acid environments needs to be addressed Protection Against Acid Most LAB and probiotic cultures have optimum growth rates between pH 5.5 and 6.6 Consequently, when they are exposed for short or long periods to pH levels below 5.5 (cheese, yogurt, fruit juices, stomach) acid diffuses into the cells and reduces the efficiency of the enzymatic processes Death may ultimately result Indeed, numerous studies report high viability loses of probiotics exposed to the acid of the stomach (Mainville et al 2005) as well as during storage in acid foods (Champagne et al 2005) In the latter situation, there is often a correlation between the poststorage pH in yogurts and the survival of probiotic bacteria (Kailasapathy et al 2008) It is well known that LAB try to maintain an intracellular pH (pHi ) constant in these acid environments, but it is not always possible As a result, pHi may drop and generate a variety of responses (O’Sullivan and Condon 1997) In many cases, an acid tolerance response (ATR) occurs that significantly increases the ability of cells to survive a short exposure (a few hours) to high acid environments In addition, cross-protection occurs and ATR helps protect against short-term stresses caused by heating, H2 O2 , salt, and ethanol (O’Sullivan and Condon 1997) The effect of the matrix medium on pHi varies between strains and if affected by incubation time (Nannen and Hutkins 1991) Therefore, ATR is not generalized amongst the LAB Unfortunately, most ATR studies were tested on short exposures to acidity, such as passage through the GI tract, but little is known on the enzymatic factors that will improve stability to weeks of exposure to acid There seems to be a statistically significant correlation between stability during storage in a fruit drink and the ability of the strains to grow at pH 4.2, but that the relationship was not P1: SFK/UKS BLBS102-c35 P2: SFK BLBS102-Simpson March 21, 2012 14:9 Trim: 276mm X 219mm 680 Printer Name: Yet to Come Part 6: Health/Functional Foods strong (R2 = 0.49) (Champagne and Gardner 2008) More data are needed for the enzymatic activities associated with resistance to long-term exposure to acid foods BIOCHEMISTRY AND HEALTH FUNCTIONALITY Survival to Gastric Acidity Many bacteria not survive passage to the conditions of the GI tract and, as mentioned previously, one of the most important criteria for functionality of probiotic bacteria is the survival to acid conditions of the stomach (Prasad et al 1998, Mainville et al 2005) Proton-translocating ATPases are the enzymes that are principally involved in maintaining a high pHi , as they contribute to excrete protons outside the cell (De Angelis and Gobbetti 2004) There are other enzymatic systems as well The arginine deiminase pathway, which involves three enzymes, contributes to maintaining pHi by producing ammonia as well as ATP for the ATPases (De Angelis and Gobbetti 2004) It must be kept in mind that there must be a source of ATP for the protontranslocating enzymes to function Therefore, acid resistance of probiotic cells in the stomach can also be linked to the presence of a fermentable sugar in the medium (Corcoran et al 2005) This shows that numerous enzymatic systems interact to prevent a big drop in pHi , when the bacteria produce lactic acid during their fermentation or when the cells are exposed to an exocellular environment having a low pH As mentioned previously, cells can be adapted to enhance their resistance to acid This ATR has been shown to be effective on subsequent short-term acid stresses such as those that occur when cells are exposed to gastric solutions As a result, enzymatic adaptations (H+ -ATPases or others) improve the survival of probiotics to passage in the GI tract (Matto et al 2006) In summary, it can be assumed that survival of probiotics to passage in the stomach requires at least two sets of enzymes: (1) those that can assimilate sugar or amino acid substrates and generate ATP and (2) those that excrete protons outside the cell to help maintain an acceptable pHi Lactose Maldigestion Lactase insufficiency indicates that the concentration of the βgal in the cells of the small intestine mucosa is very low As a result, hypolactasia causes insufficient digestion of lactose in the GI tract This phenomenon is alternatively called lactose malabsorption, lactose maldigestion, or lactose intolerance by various authors In addition to the intrinsic intestinal lactase activity and its determinants, other parameters that affect lactose digestion include ethnic origin and age Lactase activity is high at birth, decreases in childhood and adolescence, and remains low in adulthood The symptoms of lactose intolerance are increased breath hydrogen, flatulence, abdominal pain, and diarrhoea An official recognition of the benefit of yogurt in lactose digestion in the GI tract has been granted (EFSA 2010) and evidence points toward a reduction of symptoms of intolerance in lactose maldigesters The synthesis of β-gal by the yogurt starter culture is considered as the probioactive component involved in the positive effects on intestinal functions and colonic microflora, and reduced sensitivity to symptoms Unbroken bacterial cell walls act as a mechanical protection of lactase during gastric transit and also affect the discharge of the enzyme into the small intestine; they consequently influence the efficiency of the system It must also be mentioned that reduced amounts of lactose are often found in cheeses due to lactose utilization by starter microorganisms (Kilara and Shahani 1975, De Vrese et al 2001) Lactose catabolism mainly occurs by β-gal derived from the yogurt starters in fermented milk processing The optimum activity of streptococcal β-gal is at a neutral pH and at 55◦ C in presence of buffer Activity of β-gal is stimulated in presence of Mg2+ and oxgall (0.15 mL/100mL), while ethylenediaminetetraacetic acid (EDTA) causes inhibition Thermal denaturation occurs at 60◦ C, although stability can be enhanced by the addition of bovine serum albumin (Chang and Mahoney 1994) Therefore, it can be expected that yogurt drinks heated at high temperatures to enable storage at room temperature would not contain active β-gal and therefore not present the enzymatic functionality In comparison with yogurt cultures, other probiotic bacteria usually promote lactose digestion in the small intestine less efficiently However, it is suggested that some probiotic bacteria may act by preventing symptoms of intolerance in the large intestine in addition to (or rather than) by improving lactose digestion in the small intestine (De Vrese et al 2001) It is noteworthy that yogurt cultures are generally not included in lists of probiotic bacteria (Stanton et al 2003, CFIA 2009), presumably because they generally highly survive the gastric transit nor grow in the intestines, but some authors consider them as candidates (Santosa et al 2008) Blood Cholesterol Level High cholesterol in serum is associated with the incidence of human cardiovascular diseases (Othman et al 2011) One of the health-promoting benefits of probiotics is their ability to reduce blood cholesterol, which was observed in humans (Xiao et al 2003) and animals (Du Toit et al 1998, Nguyen et al 2007) However, some studies have been negative (Simons et al 2006) This points to a specific biological activity that is variable between cultures Bile salt hydrolase (BSH) in probiotics renders them more tolerant to bile salts, and it is believed that this activity also helps to reduce the blood cholesterol level of the host (Taranto et al 1997) Indeed, oral administration of encapsulated BSH reduced serum cholesterol levels in mice by 58% (Sridevi et al 2009) It is thought that bile salt deconjugation affects its enterohepatic circulation (Kim and Lee 2008), but the BSH hypothesis has not yet been completely demonstrated or elucidated Three different types of BSH from various Bifidobacterium strains have been identified (Kim et al 2004, Kim and Lee 2008) Data show that higher BSH activity can be acquired (Noriega P1: SFK/UKS BLBS102-c35 P2: SFK BLBS102-Simpson March 21, 2012 14:9 Trim: 276mm X 219mm Printer Name: Yet to Come 35 Biochemistry and Probiotics et al 2006) It is unknown if cells that are suddenly exposed to bile salts upon entrance to the duodenum can increase their BSH level and increase their functionality Thus, BSH activity in the GI tract can potentially be enhanced by culture preparation methodologies prior to consumption It must be kept in mind, however, that the production of secondary deconjugated bile salts through BSH activity may have undesirable effects Contradictory data suggest that colon cancer could be enhanced (Patel et al 2010), while another study suggests that Lactobacillus reuteri could have protective properties through precipitation of the deconjugated bile salts and a physical binding of bile salts by the bacterium, thereby making the harmful bile salts less bioavailable (De Boever et al 2000) Therefore, more data are needed to better ascertain the desirability of BSH activity Gas Discomforts It is considered that oligosaccharides that are not broken down by the human GI enzymes may be responsible for gas production in the GI tract (Yamaguishi et al 2009) Since they are not assimilated in the small intestine, these oligosaccharides end up in the colon where they are fermented by the microbiota (LeBlanc et al 2008) Unfortunately, the consumption of beans has been associated with gas production in the GI tract (Machaiah et al 1999) if consumed in sufficient amount Fortunately, symptoms reduce on frequent and continuous consumption (O’Donnell and Fleming 1984), but means to reduce discomforts are nevertheless desirable Legumes can be good substrates for the growth of probiotics (Chopra and Prasad 1990), but strain selection is required (Farnworth et al 2007) Although some probiotic cultures only use the short-chain carbohydrates in legumes (Desai et al 2002), the prospect of introducing probiotics into a high-fiber bean product is a very promising one It is unknown at the present time, if the carbohydrates in beans can indeed have a positive impact on survival and growth in the GI tract In addition to the health benefits, probiotics may also have the potential of reducing problems associated with digestibility Various studies in animals (LeBlanc et al 2008) and in humans (Nobaek et al 2000, Di Stefano et al 2004) have shown the potential of probiotics to reduce the symptoms of gas discomforts There appears to be a link between the synthesis of α-gal, which hydrolyze the oligosaccharides in beans, and the bioactivity of the cultures (LeBlanc et al 2008) As a result, a product (Proviva Fruit Drink) has received an opinion by the Swedish Nutrition Foundation (SNF 2003) “that an effect to decrease flatulence is reasonably well documented.” This was apparently the first such specific health benefit recognition for probiotic bacteria The culture used was Lactobacillus plantarum 299v However, a similar petition with L rhamnosus GG was not granted (EFSA 2008) This benefit of probiotics on sugar metabolism in the GI tract through α-gal resembles that of β-gal for lactose maldigestion mentioned previously It is unknown to what extent live cells are required for the health benefit to occur 681 Other Benefits For probiotic bacteria, the main purpose of proteases is for nutrition By hydrolysing proteins in the medium, the cells gain access to peptides and amino acids for the synthesis of their own proteins However, there are instances where peptides show biological activities Milk caseins have particularly been assessed in this respect (Korhonen and Pihlanto 2006) Thus, milk proteins contain peptidic angiotensin I-converting enzyme (ACE) inhibitors, which can be released by proteolysis during milk fermentation by some strains of L helveticus (Leclerc et al 2002) Hydrolysis of soy proteins by probiotic lactobacilli has also revealed the presence of ACE inhibitors (Donkor et al 2005) Through the ACE inhibition, peptides exert antihypertensive activity It is important to stress that the functionality of probiotics is linked to the substrate on which growth has occurred That is why the concept of probioactive not only includes bioactive compounds synthesized by the probiotic cells as such, such as exopolysaccharides, but also includes probioactives that are specifically the result of transformation of the food substrate This link between probiotic functionality and the food matrix may partially explain why there are contradictory data on the benefits of probiotics in the literature An ACE inhibition activity recorded in fermented milk may unfortunately not extend to fermented fruits or vegetables low in proteins The production of peptides is not the only way lactic cultures can affect blood pressure Certain strains of Lactococcus lactis convert glutamic acid to gamma-amino butyric acid (GABA) thanks to glutamate decarboxylase (Inoue et al 2003, Minervini et al 2009) High levels of free glutamate are rarely found in foods, and proteolysis is generally required to release glutamate from proteins before GABA production can occur The activity of glutamate decarboxylase tends to be higher in acid environments (Komatsuzaki et al 2005) Therefore, fermented milk or soy beverages seem particularly well suited for GABA production Foods naturally contain antioxidants and bioactive compounds, but some are linked to sugars or proteins When combined with sugars or other food ingredients, some of these bioactive compounds have less biological activity Therefore, deconjugation of the bioactives and their release into the product are desirable Two examples serve to illustrate this particular concept: (1) isoflavones in soy and (2) quercetin in onions Antioxidants are bioactive compounds that are quite varied in chemical In foods, examples include carotenoids, terpenes, anthocyanins, isoflavones, and flavonoids like quercetin Quercetin is of interest because of its high antioxidant capacity, that is, 229% higher than that of ascorbic acid (Kim and Lee 2004) In onions, quercetin is mainly found in three forms: (1) quercetin diglucoside (Qdg), (2) quercetin monoglucoside (Qmg), and (3) free quercetin Fermentation of red onions by lactic cultures substantially increased the proportion of Qmg (Bisakowski et al 2007), that may have a positive effect as fractions containing higher ratios of Qmg:Qdg have been reported to have higher antioxidant activity (Makris and Rossiter 2001) The enzymes responsible for this useful bioconversion in onions are glucosidases These enzymes contribute to enhancing the biological P1: SFK/UKS BLBS102-c35 P2: SFK BLBS102-Simpson March 21, 2012 14:9 Trim: 276mm X 219mm 682 Printer Name: Yet to Come Part 6: Health/Functional Foods Table 35.4 Some Enzymatic Activities Which May be Linked to the Functionality of Probiotic Bacteria Enzyme β-Galactosidase NADH oxidase and NADH peroxidase ATPases Arginine deiminase pathway Function Lactose hydrolysis in food matrix Lactose hydrolysis in the GI tract Eliminate H2 O2 Excretion of protons (H+ ) from cells to outer medium Production of NH3 and ATP Bile salt hydrolase Deconjugation of bile salts α-Galactosidase Hydrolysis of stachyose and raffinose in GI tract Hydrolysis of caseins, or soy proteins Proteases Glutamate decarboxylase Glucosidases Conversion of glutamate to GABA Deconjugates isoflavones or quercetin from glycosylated forms Functionality Species Improved growth in milk Wide-ranging Improved lactose hydrolysis in GI tract for population suffering of lactose maldigestion Improved stability in presence on oxygen Reduced intracellular drop in pH and increased survival to gastric transit Reduced intracellular drop in pH and increased survival to gastric transit Lowers blood cholesterol level Yogurt starters Streptococcus thermophilus L delbrueckii ssp bulgaricus Reduces gas discomforts linked to consumption of beans Produces ACE inhibitory peptides, which are antihypertensive Lowering of blood pressure Higher levels of the more bioactive forms of antioxidants (cancer, bone metabolism, etc.) Lactobacilli and bifidobacteria—strain variable Many LAB (Streptococcus, Lactococcus and Lactobacillus spp) Lactobacilli Bifidobacterium ssp often show high activity, but many Lactobacillus ssp cultures are also active L plantarum, B longum L helveticus Lactococcus lactis, L paracasei L helveticus, L acidophilus, B longum, L plantarum GI, gastro-intestinal; ACE, acetyl choline esterase; L., Lactobacillus; B., Bifidobacterium; LAB, lactic acid bacteria; GABA, gamma-amino butyric acid value of other foods as well For example, isoflavones are encountered in soy products The native forms of the isoflavone in soy are mainly daidzein, genistin, and glycitein that are the glycosylated forms These β-glucosides, are not readily bioavailable in humans as they are unable to be absorbed through the intestinal tract Hydrolysis of β-glucosides by bacterial βglucosidases, produces the active and more readily metabolized aglycone forms (Setchell et al 2002) Many lactobacilli and bifidobacteria possess glucosidases Thus, the data show that soy fermentation with selected strains contributes to increasing the levels of bioactive aglycons (Champagne et al 2010) In summary, many enzymatic activities of lactic cultures contribute to the synthesis, or simply to the release, of probioactives in foods (Table 35.4) CONCLUSION Many biochemical processes are critical in the functionality of probiotic bacteria From a technological perspective, it can be foreseen that cells will be adapted to enhance a specific en- zymatic activity that will promote their growth or their stability during storage Minor acid, osmotic, and thermal stresses have been proposed for this purpose (De Angelis and Gobbetti 2004) As knowledge of the enzymatic systems involved in health functionality of probiotics increases, the requirement for viable cells might gradually be reduced Thus, the critical element for the health effect may be limited to the enzymatic components, for example, the quantity of the probioactive The use of pure enzymes might eventually be considered in substitution to probiotics, but only if the enzymes are stable in the matrix and if they survive passage to the GI conditions Even if it is enzymelinked, functionality might still require that the enzymes be delivered through a cell, viable or not, in order to protect them against inactivation in the adverse conditions, they will be exposed to in the food or the GI tract Therefore, more research is needed not only on the biochemical processes involved in the functionality of probiotic bacteria, but also on factors that will enable the “delivery” of the biochemical activity at the proper site P1: SFK/UKS BLBS102-c35 P2: SFK BLBS102-Simpson March 21, 2012 14:9 Trim: 276mm X 219mm Printer Name: Yet to Come 35 Biochemistry and Probiotics REFERENCES Accolas JP et al 1971 Study of interactions between various mesophilic and thermophilic lactic bacteria, in connection with the manufacture of cheese Lait 51: 249 Araya M et al 2002 Guidelines for the evaluation of probiotics in food Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food, London (ON, Canada) April 30 and May Available at 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