Apart from their basic metabolic activities, bacteria are also capable of synthesizing many biopolymers which differ in structure and chemical properties and thus in their functions in the cells. Considering their localization, biopolymers may be divided into intracellular (a small group with limited applications) and extracellular (a large group with wide applicability) ones. One of the classes of extracellular biopolymers includes exopolysaccharides (EPS). The EPS may be secreted outside a bacterial cell or may be produced as a capsule bound with external cellular membranes [66]. The EPS serve various functions in bacterial cells, like: protecting them against adverse effects of the environment (e.g., high or low temperature, high or low pH, and toxic metal ions) and against biological factors (e.g., phage attack), or helping them to colonize the environment (they are constituents of biofilms). It is presumed that EPS do not serve as a source of energy to bacterial cells, though some probiotic strains of lactic acid bacteria (LAB) were shown to be capable of EPS degradation [75]. Some LAB strains used in the dairy industry to produce fermented milks, e.g., yoghurt, kefir, sour milk and other fermented milk drinks, are able to synthesize EPS (the so-called EPS(+) strains). The application of EPS(+) strains may have highly positive effects on the rheological properties and quality of the manufactured fermented products [7, 12]. The EPS produced by lactic acid bacteria during formation of the casein curd of milk are capable of water retention and thereby inhibit syneresis in fermented drinks. In addition, by reacting with proteins, they may contribute to the reinforcement of the casein network, which improves the rheological properties, quality of the final product, and cheese yield. The use of adjunct EPS(+) starter cultures improves the smoothness, viscosity and stability of a yoghurt gel and of other fermented milks [4, 22, 32]. The character of changes induced in fermented products by the presence of EPS is determined by the chemical composition and structure of these compounds, including e.g., their molecular weight, type of bonds and the presence of side chains. The rheological properties of EPS-containing food products are also affected by the time of their most active production by LAB during food manufacture [21, 22, 67, 75]. The most active LAB strains were shown to produce EPS at even 3 g/l [58]. The EPS display also some health-promoting properties. An increase in the viscosity of EPS-containing products is believed to extend the time of their gastrointestinal passage, which may be beneficial for temporary gut colonization by LAB [15]. In addition, many studies have shown the immunomodulatory, hypocholesterolemic, anti-carcinogenic, and anti-ulcerous activities of EPS [24, 26, 42, 43, 50, 56, 59].
The EPS are high-molecular, long-chain linear biopolymers with side chains, which are constituted by carbohydrate units linked with α- and β-glycosidic bonds. They may be secreted by a cell to the extracellular space and remain bound with its surface thus forming a capsule (CPS – capsular exopolysaccharides). EPS may also be released to the external environment in the form of slime exopolysaccharides. Other types of polysaccharides include, e.g., cell wall polysaccharides (CWPS) linked with ionic or covalent bonds with the peptidoglycan layer on the cell’s surface [88]. Taking into account the EPS structure, they may be divided into homopolysaccharides (HoPS) and heteropolysaccharides (HePS). Molecules of HoPS consist of successively repeated monosaccharides of one type (e.g., D-glucose or D-fructose), and include two major groups: glucans (dextran, mutan, alternan, reuteran, curdlan) and fructans (levan, inulin-type fructans) [63, 67, 88]. In turn, HePS are built of sub-units containing 3 to 8 monosaccharides: D-glucose, D-galactose, L-fructose, L-rhamnose or, alternatively, acids: D-glucuronic, L-glucuronic and D-mannuronic. The HePS may also contain amino sugars, like, e.g., N-acetyl-D-glucosamine or N-acetyl-D-galactosamine [7, 67]. Molecular weights of HePS range from 104 to 6×106 Da [7].
Glucans – being representatives of HoPS – are divided into α-D-glucans and β-D-glucans [63]. The production of α-D-glucans (e.g., dextran, mutan, alternan, reuteran) is assisted by dextransucrase which is an extracellular enzyme synthesized by, among others, bacteria of the
One of the most extensively described HePS is kefiran, built of mannose, glucose and galactose in approx. ratio of 1:5:7 [87]. Ability to produce kefiran was reported for:
The capability to synthesize EPS by LAB varies and depends on many factors, and is species- and strain-specific. EPS production by lactic bacilli (
EPS production is determined by the growth stage of bacteria, composition of culture medium (type of carbon and nitrogen sources, and presence of other nutrients), temperature, and pH, and/or by the presence of adjuvant microflora [2, 3, 62, 72, 82, 86, 92, 93] (Tab. I). The concentration of produced EPS is largely affected by conditions of growth of bacteria which synthesize them, whereas the monosaccharide composition of most of the EPS does not depend on the available source of carbon. Interesting – especially from the perspective of practical application – seems to be the fact that the same LAB strain may produce different EPS under various growth conditions [39].
Factors affecting EPS synthesis by lactic acid bacteria
Factors affecting EPS synthesis by LAB | References |
---|---|
Species/strain | [41, 58, 60, 79, 81, 83, 89] |
Growth stage of bacteria | [12, 13, 93] |
Temperature | [12, 13, 49, 62, 72, 82, 92, 93] |
pH of medium | [12, 39, 82, 93] |
Time of incubation | [12–14, 53, 82, 93] |
Culture medium composition (e.g., source of nitrogen and carbon) | [69, 72, 82, 83, 86, 92, 93] |
Presence of adjuvant microflora | [2, 3] |
Ample studies have addressed the effect of culture medium composition on the concentration of EPS produced by LAB [69, 72, 82, 86, 92]. The
Another factor influencing EPS synthesis by LAB is temperature. Many studies have shown the highest production of EPS at the so-called sub-optimal temperature, i.e. at few °C lower than the optimal temperature for growth of a given LAB species, by both mesophilic and thermophilic species [62, 72]. The overproduction of EPS at the sub-optimal temperature is a likely response of a bacterial cell to the physiological stress induced by the decreased temperature, especially in species or strains defective in proteolytic activity (e.g.,
Another factor influencing LAB ability to produce EPS is the time of incubation. Zhang
The optimal pH value for EPS production varies between species and between LAB strains, however usually reaches around 6 [12]. The intensive growth and maximum capability to produce EPS by
One of the factors which influence EPS synthesis by some LAB strains is the simultaneous presence of other LAB in the culture medium. This issue is of high importance as mixed cultures constituted by several strains of the same LAB species or by different species are usually used in the industrial practice. Mechanisms of their interactions may be based on cooperation (e.g.,
The capability of LAB to produce EPS in milk during fermentation is an especially important trait for the dairy industry as these compounds increase the apparent viscosity and improve the texture and mouthfeel of the dairy products as well as inhibit syneresis even at their low concentrations (from 0.1 to 0.4 g/l) [17]. The presence of EPS synthesized by LAB strains has a significant effect on changes in various properties of dairy products, including: yoghurt, kefir and many other fermented milk drinks, sour cream and cheeses [15, 28, 32, 46].
The consumption of milk desserts, yoghurts and snacks is observed to successively increase in the United States and also in EU Member States. Products of this type contain additives which affect their rheological properties; but, on the other hand, they have to meet consumers demands for natural and healthy foods [54]. In Great Britain, the addition of stabilizers is regulated by law, e.g., the addition of starch and other stabilizers to yoghurts should not exceed 1% and 0.5%, respectively. It seems that in this respect the EPS(+) strains of LAB may arise interest of the dairy industry. The use of EPS(+) starters strongly inscribes itself into the “clean label” trend which is rather a permanent and irreversible trend that needs to be taken into account by food producers [57].
The EPS(+) LAB strains are applied as adjunct starters in the manufacture of fermented products or are incorporated into mixed starters (Tab. II). The effect of EPS on the rheological properties of fermented milk is more tangible and yields better outcomes when EPS are synthesized
The use of EPS(+) lactic bacteria in the production of various fermented milk products
LAB species | Products |
---|---|
| buttermilk, kefir, Nordic ropy milks |
| buttermilk, kefir, dahi, Nordic ropy milks, reduced-fat Cheddar cheeses |
| buttermilk, kefir, dahi, Nordic ropy milks |
| yoghurt, dahi, Nordic ropy milks, fresh cheeses, Mozzarella cheese, Feta cheese |
| kefir, sour cream |
| kefir, sour cream, Nordic ropy milks |
| kefir, sour cream, Nordic ropy milks |
| fermented milks, yoghurt |
| fermented milks |
| yoghurt, Bulgarian buttermilk, Nordic ropy milks |
| kefir, kumys, Nordic ropy milks |
| acidophilus milk, kefir |
| fermented milks |
| probiotic yoghurt, fermented milks |
| probiotic yoghurt |
| probiotic yoghurt |
| probiotic yoghurt |
| kefir, acid-rennet cheeses |
| kefir |
| kefir |
| kefir |
| kefir |
| kefir |
Viscosity of milk gels formed during fermentation by the EPS(+) LAB strains depends not only on the quantity of EPS products but also, to a significant extent, on their primary structure (stiffness of the EPS backbone), molecular weight, molecule rotation and charge [21, 22, 74, 75], character of bonds inside the molecule, and potential presence of side chains [21, 22, 46, 47]. At the same molecular weight, EPS molecules with a linear structure occupy a larger volume in solution compared to the branched EPS, owing to which they have a greater impact on increasing the viscosity of a solution. Also the stiffness of the EPS backbone contributes to the increase in the viscosity of the EPS-containing solutions by preventing potential deformations of the molecules. In milk gels, the presence of EPS with high molecular weight, stiff and only slightly branched has a positive impact on their viscosity and stability, and on their reduced syneresis [21]. The high degree of branching and the flexibility of the backbone leads to the “compactness” of the EPS, which results in a decreased viscosity of the solution. The microstructure of a milk gel formed with LAB strains producing EPS of this type is similar to the microstructure of a gel made with EPS(–) cultures [22].
Another factor affecting the rheological properties of milk gels produced with ESP(+) LAB strains is the charge of the EPS molecule. The use of strains synthesizing anionic EPS in starter cultures enabled achieving milk gels with higher values of the elastic modulus (
The EPS may influence the formation of a casein gel structure by acting as a “bond”, and their effect on the protein matrix and structure depends on their concentration, interactions with proteins and characteristics of a molecule [22, 70]. They may positively co-act with milk proteins, thus increasing values of the viscoelastic moduli and firmness of milk gels [70]. This may be caused by electrostatic interactions between casein and EPS, which besides protein-protein interactions may additionally reinforce the structure of a casein gel [6, 21, 23, 70]. Interactions between milk proteins and EPS in a complicated system, like, e.g., fermented milk, are poorly recognized. Research addressing this problem need to take into account the differences in the mechanisms of formation of these compounds during fermentation compared to the process aided with stabilizers (modified starches and pectins) added to milk prior to its souring. Some studies related to the protein-EPS interactions were conducted in model systems, wherein purified EPS preparations were added to milk before fermentation [23]. However, milks produced in this way had different, less beneficial rheological properties than the fermented milks in which EPS were synthesized by LAB
A largely significant aspect from the practical perspective is the capability of milk gels to “recover” after stirring and pumping during production of e.g., stirred yoghurts. Studies addressing the effect of stirring milk gels produced using EPS(+) strains and for comparison using EPS(–) strains demonstrated that although the EPS-containing gels were indeed more compact, a greater decrease was noted in the value of their elastic modulus (
Yoghurt is a type of fermented milk manufactured using starter cultures:
The final consistency of the natural yoghurt is a result of the effects of the milk protein complex, lactic acid, and potentially EPS produced by yoghurt cultures. Desired rheological properties of yoghurt include: hardness, firmness, cohesiveness, smoothness, viscosity, and stability, which when taken all together signify lack of susceptibility to syneresis. The set yoghurt with a high level of syneresis is usually perceived by consumers as having a defect, although this is a natural phenomenon in this product [4, 34]. In the industrial practice, syneresis is reduced through increasing contents of dry matter components in processing milk to 14% (w/w) with dry dairy ingredients (skim milk powder, whey protein isolate, whey protein concentrate, sodium- or calcium caseinates) or by using stabilizers [80]. Unfortunately, the use of these additives always increases production costs of yoghurts, and the addition of stabilizers (gelatin, modified starches, gums) may negatively affect yogurt perception by potential consumers. Some countries have imposed bans or reductions in stabilizers use in yoghurt production. Yoghurts manufactured with strains capable of EPS production are less susceptible to syneresis, have higher viscosity and water holding capacity as well as smoother and creamy texture and decreased granularity. It may be concluded that the use of EPS(+) LAB strains in the production of yoghurts offers the possibility of limiting or eliminating the necessity of applying texture-forming additives [4, 27, 32–34, 70].
The microstructure of yoghurt is built of a casein matrix with incorporated fat globules. Spaces in the gel are filled with serum and LAB cells. The cultures applied in yoghurt production synthesize both capsular EPS and EPS secreted in the form of slime outside the cell. The capsular EPS are in direct contact with cells which use them for incorporation into the protein matrix. Different EPS(+) strains have various effects on the rheological properties of yoghurts. The stirred yoghurts produced using starter cultures synthesizing slime EPS were shown to be more viscous than these produced using cultures synthesizing capsular EPS or using EPS(–) starter cultures [4]. The yoghurt cultures may be divided into three groups: cultures incapable of EPS production, cultures producing capsular EPS, and cultures capable of producing both capsular and slime EPS [16, 34]. The cultures synthesizing slime EPS are generally believed to positively affect yoghurt consistency; however, the overproduction of these EPS leads to the manufacture of products with undesired mucosity, clearly perceptible in the mouth [19, 80]. The capsular EPS are usually not produced in excessive amounts as the size of the capsule is limited by the size of the bacterial cell. Bacterial capsules loosen gel microstructure in yoghurt, thus making its consistency smoother. Starter cultures synthesizing capsular EPS and lacking slime formation, produce yoghurts which are more viscous, more stable and less susceptible to syneresis compared to yoghurts produced by cultures incapable of synthesizing capsular EPS. In addition, the capsules retard diffusion of lactic acid from the cells, thus lead with time to the arrest of acid production by the cells. This may prevent the over-souring of yoghurt [31, 34]. A comparison of the microstructure of milk fermented using EPS(–) cultures (
A study aimed at elucidating the role of EPS in modeling the structure of yoghurt was conducted with LAB strains capable and incapable of EPS production. Viscosity was always higher in yoghurts manufactured with EPS(+) strains. Importantly, the EPS do not impart their own taste nor aroma to the product, but only improve its texture. The use of LAB cultures producing slime EPS was shown to enable reduction of soy protein isolate or concentrate added during the production of stirred yoghurt [4]. Partial or complete substitution of EPS(–)
A weak correlation was demonstrated between yoghurt texture and EPS concentration [71]. The major factors which affect the texture of EPS-containing yoghurts are interactions of these compounds with casein which vary depending on the EPS structure and product acidity (pH) [7]. In turn, Ruas-Madiedo
To determine the effect of EPS addition on the microstructure and rheological properties of yoghurts, purified EPS were added to milk intended for yoghurt production (in concentrations of 0.01–0.03%. Viscosity, water holding capacity, hardness, and microstructure of yoghurts were strongly dependent on EPS concentration. The best water holding capacity (i.e. the least syneresis) was found in the yoghurt produced with 0.01% EPS. In turn, the 0.03% addition of EPS caused greater syneresis – likewise in EPS-free yoghurt. Also in terms of rheological properties, the best turned out to be the yoghurt with 0.01% addition of EPS [91]. In turn, yoghurt produced with yoghurt cultures and the EPS(+)
Kefir is a traditional, slightly sparkled fermented milk, popular in countries of Eastern Europe. It contains ca. 0.1–1.0% ethanol, depending on the fermentation activity of yeast. In the traditional method of kefir production, kefir grains containing homo- and heterofermentative LAB, yeast and acetic acid bacteria are added to milk. Cells of bacteria constituting kefir grains are built into the EPS matrix [16]. Today, however, kefir starters are used in kefir production instead of kefir grains which were filtered from the final product after completed fermentation. Although kefir starters contain yeast, they are incapable of fermenting lactose. For this reason, modern industrial kefirs are slightly saturated with CO2 and contain trace amounts of alcohol. A recent trend assumes simplification of production technologies of fermented milk drinks, e.g., kefir or buttermilk, mainly owing to the concerns for their stability and economy. These practices may, however, lead to the deterioration of flavor and biodiversity of these products. A few of EPS(+) LAB strains, e.g.,
The EPS(+) cultures are also applied for the production of dahi – a traditional yoghurt made based on buffalo, cow or goat milk, popular throughout South Asian countries, such as, Bangladesh, India, Nepal, Pakistan, Sri Lanka, etc. [66]. The fat content of dahi is usually between 3.5–8%, but dahi assortment includes also its low-fat milk versions. Unfortunately, like for yoghurt and other dairy products, the low fat content of dahi has a negative effect on its quality, including lack of flavor, weak body and unstable texture [80]. The use of EPS(+)
Selected strains of mesophilic LAB (
Results of studies conducted so far have demonstrated the use of EPS(+) LAB strains to be a good alternative for the production of low-fat cheeses [12, 82]. The high water holding capacity of EPS has a positive effect on increasing viscosity and improving the texture and consistency of such cheeses [16, 25]. In many countries, some varieties of ripening rennet cheeses are produced in the low-fat version to meet demands of consumers who prefer low-caloric foods. A frequent defect of these cheeses is their little intense taste and rubbery, dry and grainy texture. A challenge faced by producers is to maintain their mouthfeel and texture similar to these of the full-fat cheeses, by modifying the production technology. Such attempts have been described in literature and involved the use of EPS(+) LAB [16, 20].
Traditional fresh Egyptian cheese (Karish) manufactured by acid coagulation of skim milk often shows texture defects typical of low-fat cheeses. In the microscope image, Karish cheese produced with the addition of EPS(+) cultures of LAB had a strongly porous structure and EPS were visible as clusters of fibers inside large pores. In turn, cheeses made using mutants of the same strains but incapable of producing EPS had a compact structure with small pores. The curd formed with the use of EPS(+) cultures was less rigid and more susceptible to deformations, compared to the curd formed using EPS(–) cultures [1, 31–33]. Fresh cheeses made with the use of EPS(+) cultures had smoother consistency than those produced without the addition of these cultures. Such improvement of textural properties may increase consumer acceptance of low-fat products. In addition, the use of EPS(+) cultures for the manufacture of fresh cheeses may increase consumer acceptance of cheeses containing fruits or vegetables owing to their improved spreadability and smoothness [1]. Hahn
Mozzarella cheese is manufactured with the use of thermophilic starter cultures including
The application of LAB synthesizing EPS may be a potential means for increasing the water content and improving the textural properties of low-fat Cheddar type cheeses. Considering that LAB have the GRAS status (i.e. are Generally Recognized As Safe for health on the basis of use for a long time), the use of an EPS(+) strain for the production of fermented foods is more propitious than the use of polysaccharides synthesized by other bacteria (e.g., dextran, gellan, pullulan, xanthan, alginates) [11]. Costa
Feta cheese manufactured using the EPS(+)cultures has an open microstructure with large pores which are partly or completely filled with a crosslinked structure, e.g., water with suspended EPS. In turn, Feta cheese produced with EPS(–) cultures has a compact structure. Aggregates of casein micelles in the cheese made with EPS-producing culture appeared to be more fused than these in the cheese made with the EPS-non-producing cultures [31, 32]. In earlier studies, Hassan
Investigations on the effect of EPS on the quality of cheeses were mainly conducted with products having a reduced or low fat content, while little is known about EPS effect on the quality of full-fat cheeses. Most likely, this effect was not investigated as the full-fat cheeses are soft and smooth enough, and free of defects typical of the reduced-fat cheeses.
Apart from the sensory benefits stemming from the EPS presence in dairy products, many EPS(+) LAB strains exhibit traits of probiotics [12, 16]. The probiotic activity of LAB strains is believed to be partly associated with the activity of biopolymers they produce [84]. The probiotic effect is then due not only to the activity of viable microorganisms, but also to the activity of their metabolites, including EPS (the so-called postbiotics). Beside the prebiotic effect of EPS [78], they are also claimed to have antibacterial activities [35, 41] and many health-promoting properties like: anti-carcinogenic, antioxidative, immunomodulatory, and reducing blood cholesterol [24, 26, 42, 43, 50, 51, 56, 59, 64, 65, 79, 84].
The mechanism of the anti-carcinogenic activity of EPS has not been fully elucidated, yet. According to one of the theories, EPS induce apoptosis of cancer cells by removing reactive oxygen species from their mitochondria [24, 51, 94]. The oxidative stress plays a key role in cancer pathogenesis. Levels of antioxidants and reactive oxygen species are correlated with the development and malignant transformation of cancer cells [37, 51]. Given that the EPS are capable of promoting antioxidative transformations and removing reactive oxygen species in cancer cells, they may as well inhibit their proliferation [51]. An EPS isolated from
The slime EPS produced by
Furthermore, EPS isolated from
Apart from the longstanding health benefits resulting from the use of EPS(+) strains in the manufacture of dairy products, the EPS may also beneficially affect consumer physiology. Presumably, increased viscosity of EPS-containing fermented milk may prolong the time of its retention in the gastrointestinal tract, which is beneficial for, e.g., temporary gut colonization by probiotic bacteria. Another example of the putative health benefits of some EPS is their degradation in the colon to short-chain fatty acids (SCFAs) by enteric microflora. The SCFAs, and butyric acid in particular, provide energy to intestinal epithelium cells, and some of them prevent colon cancer [95].
In the food industry, the role of LAB capable of producing EPS may increase considering their effects on the rheological and textural properties of fermented food products. The EPS synthesized by LAB differ in their chemical composition and structure. Their production is relatively low (up to 0.1%), however they improve the consistency, stability and the widely understood quality of the final product. LAB capable of EPS production may find application in the manufacture of fermented milk products in countries in which the use of stabilizers is either limited or banned by law. The application of adjunct EPS(+) cultures in the production of fermented milks allows reducing the addition of milk powder and other thickening agents and offers vast possibilities for diversifying production, inscribing into the “clean label” trend, and meeting consumer demands for health-promoting and/or dedicated foods. EPS(+) cultures could also be applied in cheesemaking, especially in the case of low-fat fresh cheeses. Such a solution would prevent whey syneresis, but simultaneously give the sensation of some “fatness”. Besides technological benefits, the use of EPS(+) LAB for the manufacture of fermented dairy products has a positive effect on human health, involving mainly elongation of fermented milk retention in the gastrointestinal tract and thereby promoting gut colonization by probiotic bacteria. The EPS are usually prebiotics, however they also exhibit anti-ulcer, immunomodulatory, anti-carcinogenic activities and reduce blood cholesterol.