Homo EPS produced by LAB
Lactic acid bacteria (LAB) are used in many fermented foods, particularly fermented dairy products such as cheese, buttermilk, and fermented milks. LAB produce lactic acid, carbon dioxide, and diacetyl/acetoin that contribute to the flavor, texture, and shelf life of fermented foods. Some LAB produce exopolysaccharide (EPS), and generally, EPS play a major role as natural texturizer in the industrial production of yoghurt, cheese, and milk-based desserts. Recently, EPS produced by LAB have received increasing attention, mainly because of their health benefits. In particular, immune stimulation, antimutagenicity, and the antitumor activity of fermented dairy products prepared with EPS-producing LAB or EPS themselves have been investigated [1-4].
EPS are polysaccharides secreted from the cell, or produced on the outer cell by extracellular enzymes. EPS from LAB are divided into two classes, homo- and hetero-EPS. Homo-EPS are composed of one type of monosaccharide, whereas hetero-EPS consist of regular repeating units of 3-8 different carbohydrate moieties synthesized from intracellular sugar nucleotide precursors . The biosynthesis of homo-EPS and hetero-EPS are different. Homo-EPS are made from sucrose using glucansucrase or levansucrase [6-7], and the synthesis of hetero-EPS involves four major steps, sugar transportation, sugar nucleotide synthesis, repeating unit synthesis, and polymerization of the repeating units . The major physiological function of EPS is believed to be biological defenses against various stresses such as phage attack, toxic metal ions, and desiccation , and it is very unlikely that bacteria use EPS as an energy source. However, some potentially probiotic LAB strains have been reported to degrade EPS produced by the other LAB strains [10-11].
The term "probiotic" was first proposed by Fuller , and its definition was further refined to "Live microorganisms which when consumed in adequate amounts as part of food confer a health benefit on the host" . Probiotic LAB thus represent a class of live food ingredients that exert a beneficial effect on the health of the host. Beneficial microorganisms in the intestine are enhanced by “prebiotics,” which are defined as "nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and activity of one or a limited number of bacterial species already resident in the colon, and thus improving host health" .
Most of the current prebiotics are low molecular weight except for inulin. As long carbohydrate chains are metabolized more slowly than the short ones, and polysaccharides thus exert prebiotic effects in more distal colonic regions compared to oligosaccharides, which are more rapidly digested in the proximal colon . Therefore, EPS produced by LAB can be used as prebiotics. This chapter reviews the physicochemical properties, genetics, and bioactivities of the EPS produced by LAB.
2. Chemical composition of EPS
Some LAB can produce EPS that are either secreted to the environment or attached to the cell surface forming capsules. EPS are classified into two groups: homo-EPS, consisting of a single type of monosaccharide (α-D-glucans, β-D-glucans, fructans, and others represented by polygalactan) and hetero-EPS, composed of different types of monosaccharides, mainly D-glucose, D-galactose, L-rhamnose, and their derivatives .
The differences arise between the homopolysaccharides mainly because of the features of their primary structure such as the pattern of main chain bonds, molecular weight, and branch structure. Two important groups of homo-EPS are produced by LAB; (i) α-glucans, mainly composed of α-1,6- and α-1,3-linked glucose residues, namely dextrans, produced by
The formation of dextran from sucrose has been recorded for
Mutan is the glucan synthesized by various serotypes of
|Homo-EPS||Main linkage(branching linkage)||Organism|
Alternan has alternate α-1,6 and α-1,3 linkages, and this structure is thought to be responsible for its distinctive physical properties including high solubility and low viscosity. These characteristics provide this glucan with a potential commercial application as a low viscosity texturizer in foods.
Levan is an EPS produced from sucrose. It is fructan composed of β-2,6-linked fructose molecules with some β-2,1-linked branches. Incidentally, inulin is a fructan composed of β-2,1-linked fructose molecules with some β-2,6-linked branches.
The chemical composition of hetero-EPS shows wide variablity. Hetero-EPS are polymerized repeating units mainly composed of D-glucose, D-galactose, and L-rhamnose. The composition of the monosaccharide subunits and the structure of the repeating units are considered not to be species-specific, except in case of
The quantities of hetero-EPS produced by LAB vary greatly. EPS production is 50-350 mg/l for
Fermentation conditions using undefined media have been improved to maximize yields. However, a chemically defined medium containing a carbohydrate source, mineral salts, amino acids, vitamins, and nucleic acid bases is more suitable for investigating the influence of different nutrients on LAB growth and EPS biosynthesis. The total yield of EPS produced by LAB depends on the composition of the medium (carbon and nitrogen sources) and the growth conditions, i.e., temperature, pH, and incubation time.
Under conditions of higher temperatures and slower growth, the production of the polymer per cell in
The effects of alterations to the nitrogen and carbon sources used in EPS production have also been investigated. According to early reports, neither LAB growth nor EPS production was specifically linked to the presence of casein or whey proteins in the growth medium. Garcia et al.  reported that EPS production by
It has been shown that an optimal ratio between the carbon and nitrogen is absolutely necessary to achieve high EPS yields . The production of EPS by
3. EPS biosynthesis by LAB
3.1. Homo EPS biosynthesis
Homo EPS are synthesized outside the cell by specific glycosyltransferase (GTF) or fructosyltransferase (FTF) enzymes (commonly named glucansucrases or fructan-sucrases). Homo-EPS producing LAB also use extracellular GTF enzymes to synthesize high-molecular mass α-glucans from sucrose. This process uses sucrose as a specific substrate, and the energy required for the process comes from sucrose hydrolysis. There is no energy requirement for EPS-production other than for enzyme biosynthesis because EPS synthesis by GTF or FTF does not involve active transport processes or the use of activated carbohydrate precursors. Therefore, large amounts of sucrose can easily be converted to EPS.
Glucan synthesis reactions catalysed by GTF can be written as follows (Fig. 1):
sucrose + H2O → glucose + fructose
sucrose + acceptor carbohydrate → oligosaccharide + fructose
sucrose + glucan (n) → glucan (n+1) + fructose
Although GTF enzymes have a high degrees of similarity, lactobacilli produce a broad spectrum of glucans, including polymers with α-1,6 linkages (dextran), α-1,3 linkages (mutan), and both α-1,6 and α-1,4 linkages (alternan). The relative molecular weight of glucans from lactobacilli range from 1 × 106 Da to 5 × 107 Da . In addition, GTF enzymes are not saturated by their substrate, and transfer reactions exceed the sucrose hydrolysis under sucrose concentrations above 100 mM .
The GTF enzymes of streptococci are generally produced constitutively. In contrast, the GTF enzymes of
The fructan synthesis reaction catalyzed by FTF can be written as follows:
sucrose + H2O → fructose + glucose
sucrose + acceptor carbohydrate → oligosaccharide + glucose
sucrose + fructan (n) → fructan (n+1) + glucose
Fructans generally have a relative molecular weight exceeding 5 × 106 Da. Similar to GTFs, FTFs are not saturated by their substrate, namely, sucrose, and transfer reactions exceed the rate of sucrose hydrolysis for sucrose concentrations above 200 mM . FTFs such as Lev, Inu, and LevS from lactobacilli exhibit pH optima of between 5.0 and 5.5. The optimum temperature for enzymes from the thermophilic
3.2. Hetero EPS biosynthesis
Hetero EPS are not synthesized by extracellular enzymes, but are instead synthesized by a complex sequence of interactions involving intracellular enzymes. EPS are made by polymerization of repeating units, and these repeating units are built by a series of addition of sugar nucleotides at the cytoplasmic membrane. Sugars are the starting materials for the synthesize sequence. LAB strains can utilize various monosaccharides and disaccharides as energy sources, via some well-studied sugar uptake systems include primary transport systems, direct coupling of sugar translocation to ATP hydrolysis via a transport-specific ATPase; secondary sugar transport systems, coupling of sugar transport to the transport of ions or other solutes, both as symport and antiport transport systems; and group translocation systems, coupling of sugar transport to phosphorylation via the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS; Fig. 2) . Polysaccharides must be hydrolyzed before uptake. For example, starch is hydrolyzed by α-amylase, and the raction products are subsequently hydrolyzed by the enzymes described above.
After the addition of a hetero-EPS repeating unit, the unit is exported through the cell membrane and becomes polymerized into the final hetero-EPS. Hence, several enzymes and proteins are involved in the biosynthesis and secretion of heterotype EPS, and the enzymes and proteins involved in these processes may not be unique to hetero-EPS anabolism.
Sugars taken into the cell are converted into sugar nucleotides. Iintracellular monosaccharides are converted to sugar nucleotide substrates for polymerization reactions, including UDP (uridine diphosphate), dNTP (thymidine diphosphate), and GDP (guanosine diphosphate). Such polymerization reactions are catalyzed by glycosyl pyrophosphorylases.
Glu-1P (Gal-1P) + UTP → UDP-Glu (UDP-Gal) + pyrophosphate
UDP-glucose is then converted to UDP-galactose by epimerases such as UDP-glucose-4-epimerase. This reaction is reversible.
UDP-glucose ↔ UDP-galactose
Glycosidic linkages are formed on membranes in the cytoplasm. A sugar moiety is transferred to C55-polyprenyl phosphate, a carrier lipid and component of the membrane, by priming glycosyl transferases. This transfer triggers the addition of a repeating unit to the hetero-EPS molecule. Disruption of the priming glycosyl transferase gene generates non-EPS-producing mutants . Thus, priming glycosyl transferases are thought to be crucial for EPS biosynthesis. The addition of the repeating unit is completed by the action of glycosyl transferase on the sugar residue attached to C55-polyprenyl phosphate. Therefore, the type and number of glycosyl transferases available determine the range of repeating units in hetero-EPS. C55-polyprenyl phosphate is also involved in bacterial cell wall biosynthesis, and therefore, cell wall biosynthesis and EPS synthesis compete for this substrate. The repeating unit is exported through the bacterial membrane, and is polymerized to become a hetero-EPS (Fig. 3).
3.3. Instability of EPS production
The instability of hetero-EPS production has been reviewed by de Vuyst et al. . Briefly, a loss in the ability to produce slime may be caused by repeated subculture of bacterial strains or incubation at high temperatures. The loss of plasmids from ropy mesophilic LAB strains is generally the reason for loss of slime production. On the other hand, thermophilic LAB, namely,
Priming glycosyl transferases are thought to be crucial for EPS biosynthesis and disruption of the priming glycosyl transferase gene generates non-EPS-producing mutants. Tsuda et al. generated the EPS-producing mutant strain 301102S from the non-EPS-producing
4. Polysaccharides and oligosaccharides for colon health
EPS produced by LAB have various functional roles in human or animal health including immunomodulatory properties, antiviral activity, antioxidant activity, and antihypertensive activity [1, 55, 71, 72], and have also been used as food additives for texture improvement. These properties have been extensively reviewed [8, 9, 56, 73, 74]. Besides these properties, prebiotics based on LAB and oligosaccharides have other health benefits. Prebiotics are usually non-digestible oligosaccharides that selectively stimulate the growth and activity of a limited number of bacterial species in the colon, such as bifidobacteria and lactobacilli, and therefore, improve host health. Detrimental bacteria may form substances such as ammonia, hydrogen sulfide, indles, and amines that are noxious to the host. However, beneficial bacteria such as bifidobacteria and lactobacilli inhibit the proliferation of detrimental bacteria, and their cell components stimulate the host immune system . Gastrointestinal microflora consist of approximately 1014 colony forming units (cfu)/g of various types of both detrimental and beneficial bacteria, and the numbers and composition vary greatly along the gastrointestinal tract. The balance of the gastrointestinal micro flora influences different aspects of host health such as bowel movement, tympanites flatulence, and the absorption of nutrients. Many factors may upset this balance, including stress, consumption of antibiotics, infection, food poisoning, and the natural ageing process. To redress this balance, the growth and activities of beneficial bacteria may be enhanced by specific ingredients in foods.
Various oligosaccharides have been identified as prebiotics, that can increase the number of
The food for specified health use (FOSHU) system was introduced in Japan in 1991. FOSHU refers to foods containing ingredients that provide health benefits and have officially approved physiological effects on the human body. FOSHU is intended to be consumed for the maintenance or promotion of health or for special health uses, for example, to control conditions such as blood pressure or blood cholesterol. To be defined as FOSHU, it is important to assess the safety of the food as well as the effectiveness of health promotion, and this assessment must be approved by the Ministry of Health, Labour and Welfare in Japan. At present (2012), 990 foods are recognized as FOSHU, and of these, 86 provide gastrointestinal health benefit. Foods for balancing gastrointestinal micro flora contain galactosylsucrose, soy oligosaccharides, lactulose, GOS, FOS, isomalto-oligosaccharides, raffinose, xylo-oligosaccharides, mannobiose, and brewer's yeast cell wall as functional ingredients.
GOS are well-known type of prebiotic oligosaccharides found in human milk. The concentration of oligosaccharides is 100 times higher in human breast milk than in bovine milk . Many studies have shown that breast-fed infants have intestinal microflora dominated by bifidobacteria. The reason for this phenomenon is thought to be that the oligosaccharides in breast milk, including GOS, can reach the upper gut without being digested where the bifidobacteria can utilize them. At present, GOS is produced by the enzymatic treatment of lactose by β-galactosidase. GOS produced in this manner usually have degrees of polymerization (DP) between 2 and 10. Furthermore, the type of glycosidic linkage is determined by the reaction conditions: final products usually possess β-1,2, β-1,3, or β-1,4 linkages. GOS is given a caloric value of 2 kcal/g in Japan and Europe for food-labelling purpose.
The effect of GOS on defecation has been studied in healthy volunteers. Defecation frequency was significantly increased, and faeces became significantly softer after the subjects drank a beverage containing 5.0 g of GOS, on a daily basis. Therefore, consumption of a beverage containing 5.0 g of GOS can improve defecation in individuals with a tendency for constipation . Ishikawa et al. reported that the number of faecal bifidobacteria increased significantly after subjects consumed 2.5 g of GOS/day for 3 weeks . GOS utilization by enterobacteria was further investigated in vitro. The trisaccharide forms of GOS were utilized by
The use of beneficial bacteria or their enzymes in the synthesis of prebiotics may be a good way to produce prebiotics with high specificity. Rabiu reported that five different GOS were produced using β-galactosidase extracted from five different
FOS is used as a generic term for all β-2,1 linear fructans with a variable DP. Inulin and oligofructose are common forms of FOS that are widely found in nature. Chicory inulin has a DP of 2-60, and the product of its partial enzymatic hydrolysis is oligofructose or FOS with a DP of 2-10.
The effect of FOS intake on intestinal microflora was studied in humans. The number of bifidobacteria in faeces was significantly increased during the FOS intake (1 g/d) period, and a significant increase in stool frequency and a softening effect on stool were observed . FOS increased the level of bifidobacteria in faeces, whereas that of bacteroides, clostridia, and fusobacteria decreased in subjects that were fed FOS (15 g/d) for 15 days . Another study measured the increase in number of
4.3. Selection of high-efficiency prebiotics
It is not clear which oligosaccharides are the most suitable substrates for the selective growth of specific beneficial species or strains. Several research group have suggested useful methods to investigate the potential prebiotic activity of oligosaccharides [88-92]. Potential prebiotic activities were determined on the basis of the changes in the growth of beneficial and undesirable bacteria, such as bifidobacteria, lactobacilli, clostridia, and bacteroides. Such methods can evaluate the ability of specific strains to utilize a particular prebiotic, and a comparison of the prebiotic activities of oligosaccharides by using these methods could help in the choice of prebiotics for improving the gastrointestinal microflora on an individual basis. However, it is important to understand that only a limited group of bacteria can be chosen from the gastrointestinal microflora by using these methods, and that polysaccharides and oligo-saccharides are fermented by numerous species in the gastrointestinal tract.
Oligosaccharides produced by beneficial bacteria or their enzymes may enhance the growth of beneficial bacteria. A novel GOS mixture produced using
The dietary fiber, arabinoxylan is the predominant hemicellulose from cereals and exhibits prebiotic activity . The addition of water-unextractable arabinoxylans increased the population of bifidobacteria and bacteroides in a medium inoculated with faecal slurry. Polysaccharides are not usually utilized by microorganisms. Remarkably, however,
Poly- and hetero-oligosaccharides produced by LAB may be potential prebiotics. Studies on the production of polysaccharides and oligosaccharides by enzymes in beneficial microorganisms may lead to the production of highly selective prebiotics, although in vitro evaluation may be difficult because of degradation and utilization of polysaccharides by various microorganisms in the gastrointestinal tract. Administration of synbiotic food containing a combination of a probiotic bacterial strain and the prebiotic sugar produced by that strain could be effective in improving human health.
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