Applications of Microbial Exopolysaccharides in the Food Industry

Exopolysaccharides (EPSs) are high molecular weight polysaccharides secreted by microorganisms in the surrounding environment. In addition to the favorable benefits of these compounds for microorganisms, including microbial cell protection, they are used in various food, pharmaceutical, and cosmetic industries. Investigating the functional and health-promoting characteristics of microbial EPS, identifying the isolation method of these valuable compounds, and their applications in the food industry are the objectives of this study. EPS are used in food industries as thickeners, gelling agents, viscosifiers, and film formers. The antioxidative, anticancer, prebiotic, and cholesterol-lowering effects of some of these compounds make it possible to use them in functional food production.

Microbial exopolysaccharides in the Food compound for animal feeds, medicines, and human foods by Food and Drug Administration (FDA) (37). The European Commission allows using Leuconostoc mesenteroides dextran in the bakery to improve the softness, crumb texture, and loaf volume (38). Oil recovery enhancement (39), biodegradable coatings or films (40), and biosensors for the analysis of different biointeractions (41) are some other uses of dextran. Dextran reveals high water solubility and produces low viscosity solutions, so it can be added to foods at high concentrations without excessive viscosity. Adding dextran can raise the glass transition temperature of ice cream mixes and stabilize the final product. It prevents sugar crystallization, increases moisture retention, retards oxidation, and maintains the flavor and appearance of various foodstuffs (42,43). It also has some medical benefits, such as blood coagulation, treatment of hypovolemia, and management of iron deficiency anemia (44).

b. Pullulan
Pullulan is a neutral, non-toxic, non-mutagenic, and noncarcinogenic water-soluble polysaccharide consisting of maltotriose repeating units (45). It is considered a GRAS powder, which can be used as a replacement for starch in pasta or baked products (46,47). Pullulan is a candidate for packaging film in the food industry due to its high solubility in cold and hot water, mechanical strength, and resistance to pH changes. Pullulan films are colorless, tasteless, biodegradable, oxygen impermeable, high adhesive (48,49), flexible (50), highly impermeable to oxygen and oil (51,52), and heatsealable (52). Its physical characteristics are dependent on the composition, for instance, adding xanthan and locust bean gums reduces the mechanical properties of the pullulan film (53). However, Gounga et al proposed a whey protein isolate pullulan as a coating to keep the fresh chestnut fruits from moisture loss and color changes (54). Pullulan-based edible films can also serve as a carrier for flavors and antimicrobial substances. The pullulan films incorporated with meadowsweet flower extract (52) and sweet basil extract (55) can retard the growth of Rhizopus arrhizus on the apples without changing the color during storage. The number of Staphylococcus aureus, Aspergillus niger, and Saccharomyces cerevisiae in baby carrot was reduced at least by 3 log CFU/g using pullulan films containing caraway essential oil (CEO). The slow release of included antimicrobial agents from the film matrix increases the bacterial lag phase, decreases microbial growth rate in food, and improves its quality (56). Incorporation of pullulan film with sakacin A, essential oils (oregano and rosemary), or nanoparticles (zinc oxide or silver) was useful against pathogenic microorganisms such as S. aureus, L. monocytogenes, E. coli O157: H7, and S. typhimurium and improved the safety of refrigerated, fresh, or processed meat and poultry products (57,58).
Pullulan is resistant to mammalian amylases and is considered as a dietary fiber in human nutrition (51). It can be applied as an additive in low-calorie foods. It is predominantly metabolized by bifidobacteria (Ryan, Fitzgerald, and van Sinderen, 2006), and as a prebiotic, it increases the number of bifidobacteria and lactobacilli in feces (51,59  the acidifying activity of these bacteria in the presence of pullulan which reduced the number of E. coli (60).

c. Curdlan
Curdlan is a neutral and an acidic linear glucan with a few intra-or inter-chain (1→6)-linkages (13). It is a colorless, odorless, tasteless, and indigestible (61) compound that is used in the medical (drug encapsulation, modulation of immune responses, etc) and food industries (62). Although it is insoluble in water, two types of gel can be produced after heating the aqueous suspension. Curdlan gel strength depends on the heating temperature, time of heat-treatment, and concentration of curdlan. Two types of gel including a low-set gel (thermo-reversible gel formed between 55-80ºC) and a high-set gel (thermo-irreversible gel formed above 80ºC) can be produced. The latter is much more stable during retorting, deep-frying, and cycles of freeze-thawing (13). It is approved as a stabilizer and texturizer in the food industry by the FDA (63). Wu et al suggested the use of thermoreversible curdlan gel as a gel binder and dietary fiber in fish meat gel-based products (64). It increases the chewiness, gumminess, adhesiveness, and viscosity of an emulsified meatball (65) and improves the quality of tofu, noodles, and surimi because of its exclusive resilience and strength through heating and after freezing-thawing. Dense cross-links between curdlan and the fish proteins during heating improve the textural and rheological properties of Alaska pollock surimi gel (66).
Curdlan can reduce fat absorption and moisture loss during deep-frying (67) because it forms a reversible thermal gel that can capture water and makes it a barrier against oil and moisture. There are no digestive enzymes for curdlan in the upper alimentary tract; it can be considered as a fat mimetic by itself or in combination with other hydrocolloids (68,69). Using curdlan in the non-fat sausage as a fat mimetic improves the texture and flavor of sausage, similar to the 20% fat sausage (69).
Curdlan has the potential to use as an edible and biodegradable film for food packaging. Konjac glucomannan/curdlan blend films (70) fish gelatine/ curdlan blend films (71), and curdlan/chitosan membranes (72) have been found to show excellent waterproofing properties. The latter case also shows an antimicrobial effect.

d. Alternan
Altrenan is a long-chain homopolysaccharide produced by the alternansucrase enzyme from sucrose (14). Due to its high solubility, low viscosity, and high resistance to enzymatic hydrolysis, it is used as a low viscosity bulking agent in foods. It can also serve as a prebiotic to form symbiotic food (44).

e. Reuteran
Reuteran is a water-soluble α-glucan produced by reuteransucrase. It can improve the quality of gluten-free sourdough and sorghum bread, characterized by a softer crumb, extended shelf life, and prebiotic activity (16,74).

f. Scleroglucan
Scleroglucan is a water-soluble neutral homopolymer, which dissolves in both cold and hot water. Salt concentrations and extreme pH conditions (2.5-12) have no impact on solution viscosity. Its solution is thermostable (stable for 20 hours at 120°C) and shows pseudoplastic behavior with a high yield value. It is a good emulsifier and stabilizer (dressings and ice creams) and can improve the quality of frozen or heat-treated foods. However, it is not approved by food safety legislation in Europe and the USA (75).

g. Cellulose
Cellulose is a GRAS homopolysaccharide produced by a broad range of bacterial species, including Komagataeibacter (former Gluconacetobacter), Agrobacterium, Rhizobium, Salmonella, and Sarcina. Komagataeibacter is the most active strain in cellulose production with high yield and purity (1,18). The chemical composition of bacterial cellulose is indistinguishable from the plant one; however, it is free of hemicellulose, lignin, and pectin, which simplifies its extraction. Bacterial cellulose shows a higher water holding capacity and longer drying time (75), both of which make it a good candidate for use in food systems (1,76,77).
Bacterial cellulose as a thickener and gelling agent has several applications in increasing water binding capacity of surimi (78), improving the gel strength of tofu (79), replacement of fat in meatballs (80), emulsion and foam stabilization of ice cream (81) and immobilization of probiotic bacteria (82). As a dietary fiber, it can help to reduce food calories and improve body health.

Fructans
The fructans are made from sucrose by fructosyltransferase enzyme and can be separated into two groups of levantype and inulin-type.
Levan is water and oil-soluble polymer and insoluble in almost all organic solvents (86). It has low intrinsic viscosity and does not dissolve or swell in water at room temperature. It is resistant to amylase and invertase (43,87). It has some beneficial applications in medicine such as a plasma volume expander (88), anti-obesity agent (89), antitumor agent (90), and hyperglycaemic inhibitor (91). Levan can be used as a thickener, emulsifier, stabilizer, film-forming agent, encapsulating agent, and carrier for flavor in the food industry (92).
A study on animals showed that the intake of levan can stimulate the growth of lactic acid bacteria and increases their number in the feces (83). Levan heptose can also cause an increase in the fecal counts of Bifidobacterium sp. (93).
Levan can be used for film packaging; however, pure levan films are too brittle for practical use due to the lack of long flexible moieties in levan, which can be solved by the addition of plasticizers (84). Using more than 10 wt% glycerol plasticizer can reduce the fragility of the films (94). Levan-based films are good oxygen barriers (84). Usually, biopolymer nanocomposites have greater properties than the corresponding pure biopolymers. Due to the high molecular weight, and the highly branched and dense globular structure of levan, significant intermolecular entanglement is not possible. At the same time, using exfoliated montmorillonite clay blended with levan facilitates the hydrogen bonding between levan (hydroxyl groups) and montmorillonite, which leads to the formation of transparent, elastic, and strong film (95).

b. Inulin
Inulin-type EPS are fructooligosaccharides which have many applications in the food industry. It can increase the viscosity of water, which is dependent on the molecular weight and temperature (10). It can be used as a fat replacer in sausages (96,97) and non-fat functional dairy foods (98) and also a sugar replacer in chocolate (99). Generally, inulin gels are based on the interactions occurring between dissolved inulin chains. High molecular weight inulins are better gel formers than their lower molecular weight counterparts (10).
Inulin is a soluble fiber fermented by intestinal bacteria, resulting in the generation of large amounts of shortchain fatty acids; therefore, it can be used as a prebiotic in human and animal foodstuffs (100). Besides, it is effective in reducing food calories and blood triglycerides, lowering the risk of irritable bowel diseases, and preventing colon cancer (101,102).

Heteropolysaccharides
Heteropolysaccharides consist of various types of monosaccharides. The most widely used varieties in the food industry are listed below.

Kefiran
Kefiran is a water-soluble branched glucogalactan which consists of about equal amounts of D-galactose and D-glucose residues (103). It is excreted from kefir grains and is a potential food-grade thickener in fermented dairy products. It improves the rheological properties and viscosity of acidified milk and yogurt, which can be intensified by heat treatment (104). The viscosity of kefiran is lower than some polysaccharides such as locust bean or guar gum (105) and higher than some dextrans (106).
At low concentrations (less than 1 g/L), it shows the Newtonian behavior, while at higher concentrations, the pseudoplastic or shear-thinning flow is seen. Kefiran can form a translucent gel during cryogenic treatment (freezing, frozen storage, and thawing) (107) and transparent edible films. The plasticizers such as glycerol and sorbitol at low concentrations are needed to decrease the stiffness of this polysaccharide-based film (103,108). Kefiran film has a good water vapor barrier property. An excessive amount of glycerol (25 g/100 g) reduces the water vapor permeability, improves flexibility, and decreases the glass transition temperature of films. Kefiran films are soluble in water, which correlates with water temperature and glycerol addition (103,108). Using γ radiation (up to 9 kGy) can improve surface hydrophobicity, water sensitivity, and water vapor permeability of kefiran film; however, it changes the color of films (109). Probiotic organisms (Lactobacillus plantarum CIDCA 8327 and Kluyveromyces marxianus CIDCA 8154) can be incorporated into edible kefiran films, which can increase the resistance of organisms to acid (110). These features of plasticized kefiran films improve their potential uses, especially in the food industry.
Surveys show the role of kefiran in controlling blood pressure, lowering serum cholesterol and sugar levels, increasing fecal wet weight in constipated rats (111), promoting antimicrobial activity, and improving wound healing properties (112).

Xanthan
Xanthan is a high molecular weight, water-soluble, neutral, and non-toxic gum. This GRAS (38) heteropolysaccharide consists of repeating pentasaccharide units of D-glucose, D-mannose, and D-glucuronyl acid residues (molar ratio of 2:2:1) and variable proportions of O-acetyl and pyruvyl residues which can form a highly viscous solution in cold or hot water at low concentrations. It is resistant to enzymatic degradation and pH and temperature changes (113).
There are different opinions regarding the antioxidant properties of xanthan. Gawlik-Dziki revealed the strong antioxidative effect of xanthan gum (114). However, Sun et al stated that adding xanthan to whey protein isolate (WPI) stabilized oil-in-water emulsions prevented the antioxidant activity of WPI due to its interaction with xanthan, followed by the acceleration of lipid oxidation (115).
Xanthan is primarily used in the food industry due to its viscosifying and stabilizing properties. Its solution shows a shear-reversible pseudoplastic behavior. The high molecular weight xanthan shows high Newtonian viscosity at lower shear rates due to the formation of complex superstructures through hydrogen bonding. By increasing the shear rate, the network separates, and individual macromolecules are aligned in the shear direction; therefore, the viscosity decreases (116). Synergistic interactions between xanthan and plant galactomannans (such as locust bean and guar gum) at room temperature result in enhanced viscosity (117). Low concentrations of xanthan (up to 3 g/L) do not affect the yogurt viscosity, while as the concentration increases, the viscosity increases (118). The viscosity of xanthan strongly depends on salt or sugar concentration in the solution (105).
Thickening and emulsion stabilizing effects of xanthan are due to the formation of a fragile gel-like structure in the continuous phase of the emulsion, which prevents the oil droplets from creaming. However, due to the weak gel structure, xanthan alone cannot stabilize the emulsion unless it is combined with the proteins. Exclusively, adding xanthan to oil/water emulsions stabilized with lupin and soy protein isolates enhances the emulsion stability, which is associated with an increase of protein at the interface, and builds a polysaccharide network in the water phase (119). It can also reduce the oil uptake in deep frying foods (120).

Gellan
Gellan gum is a high molecular weight anionic polysaccharide composed of a tetrasaccharide backbone consisting of 2 β-D-glucose, L-rhamnose, D-glucuronic acid, and acyl (glyceryl and acetyl) substituents (29). It is available in a substituted or unsubstituted form. The polymer is produced from two acyl substituents present in the 3-linked glucose; namely, L-glyceryl positioned at O(2) and acetyl at O(6) (121). It is resistant to heat and relatively to pH. As the gellan gum is relatively non-toxic, it is approved by the FDA for use in foods (122).
It acts as a stabilizer, binder, thickener, and perfect gelling agent in different types of foods (123). Gellan gum is insoluble in cold water but can disperse in milk and reconstituted milk. A gel is produced rapidly by heating and cooling gellan solutions in the presence of cations. The rheological characteristics of gel depend on the level of acyl substituent. The low acyl one requires acid (H + ) and ions such as calcium (Ca 2+ ), magnesium (Mg 2+ ), sodium (Na + ), and potassium (K + ) to produce the gel. Divalent cations are more efficient than monovalent ions (121). Gellan gum can be used as a gelling agent in desserts and jams to provide gelatin with mouth-feel characteristics and a more potent gel (at a lower concentration) compared to pectin.
Interaction between gellan (negative charge) and milk protein (positive charge) leads to protein precipitation. Therefore its use in the solutions/gels of milk proteins is not reasonable unless by neutralizing the negative charges (124). However, its interaction with casein and lactoglobulins increases the yield of cheese and reduces the loss of proteins in whey. Both types of gellan can be used in a stirred yogurt; however, using the low-acyl type gives a lumpy consistency to the yogurt, which must be thoroughly mixed to achieve a smooth texture. High-acyl gellan is the only form that can be used in set yogurts (121). Adding low-acyl gellan can increase the heat stability of fermented cream so that it keeps the structure after being added to hot foods (125). It can also be used as a bulking agent in the ice cream, texture, and flavor release in jellies and improve the efficiency of other hydrocolloids in confections (125). Combinations of low acyl gellan and carrageenan can be used to produce gelatin-free confectionery which is suitable for halal (121).
Gellan film has excellent oil barrier properties, and conversely, it is a poor moisture barrier, which can be improved by adding lipids (126). Coating foods with gellan can reduce fat absorption during deep-frying, resulting in a reduction of fat in the final product (120).
Konjac glucomannan-gellan gum blend films are suitable for the release of active agents such as nisin. They were found to have antimicrobial activity against Staphylococcus aureus, which can be enhanced by increasing the content of gellan gum (127). A composite film composed of the gellan and cassava starch shows relatively good mechanical and barrier properties (128). Gellan film can act as a carrier of vitamin C (129) and as a matrix for encapsulation of heat-sensitive and probiotic bacteria (130) and essential fatty acids in the food (131).

Alginate
The alginates are linear anionic biocompatible polysaccharides produced from seaweed and bacteria (132). Intake of alginates as dietary fiber can decrease the intestinal absorption and destructive potential of gastrointestinal luminal contents, increase satiety, modulate the colonic microflora, and promote the colonic barrier function (133). It is used as a viscosity regulator, stabilizer, and packaging material in the food industry, and has applications in wound healing, drug delivery, and cell microencapsulation in medical sciences (32,(133)(134)(135)(136). It is well known that the M/G ratio, the degree of acetylation, and the molecular weight determine their rheological properties (137). As the gelling properties are linked to the G subunits interacting with divalent ions, such as calcium, increasing the G-blocks leads to the formation of stronger gels with higher viscosity in the presence of Ca 2+ (87).

Viilian
The viilian is the linear heteropolysaccharide isolated from a ropy fermented milk product "viili" and is composed of glucose, galactose, rhamnose, and phosphate with a molar ratio of 2:2:1:1, respectively (31). Viilian decreases the syneresis of fermented milk products. It can be used as a thickener in food systems and is also correlated to the lowering of serum cholesterol levels in rats (138).

Acetan
The acetan (or xylinan) is an anionic heteropolysaccharide produced by Acetobacter xylinum. It is a good viscosifier and gelling agent in sweet confectionery products (139).
The main applications of various EPSs in the food system are summarized in Table 2.

Isolation and Purification of EPS
Due to the favorable effects of EPS mentioned above, in recent years, interest in the isolation of these compounds and their use in different industries has increased. The isolation method should not affect the chemical and physical properties of the polysaccharides (180). Microbial    Harvesting the pellicles (centrifugation) Washing with water to remove the residual culture medium Lyse the microbial cells (alkali at 80°C) Filtration (remove the dissolved materials) Neutralization with 5% acetic acid and rinsing Washed with deionized water 184 EPS production occurs during the bacterial growth stages. The quality, molecular characteristics, and yield of EPS depend on the nutrient status and bacterial growth condition. Therefore, choosing the appropriate culture medium is the first step in isolating an adequate amount of high-quality EPS. An optimal balance between carbon (for energy production) and nitrogen (for cell synthesis) is needed to achieve high yields (181). Various media were used to culture EPS-producing LAB, most of which are skim milk and whey-based media (182). The concentration and type of simple sugars in the culture media affect the EPS yield (181). The simplest method of EPS isolation involves three stages of centrifugation (for cell removal), dialysis against water, and lyophilization. In some cases, ethanol precipitation may be used before dialysis to concentrate the EPS. As the culture media components become more complex, the extraction method becomes more sophisticated.

Microbial exopolysaccharides in the Food
For example, in high-protein environments, it may be necessary to reduce protein levels by trichloroacetic acid, proteases, or a combination of both. Other techniques such as membrane filtration (microfiltration, ultrafiltration, and diafiltration) may be used to purify the EPS (183). Table 3 presents the extraction process of some important microbial EPSs. The isolation method has an impact on the total amount of EPS obtained; therefore, different methods should be analyzed to determine the best method for isolation of EPS.

Conclusion
Nowadays, the ability of microorganisms to produce EPS has been the focus of attention. These natural compounds have different applications in various industries, including the food industry. The rapid growth of microorganisms, high productivity rate, and safety approval of EPS have enabled them to be used as inexpensive compounds to improve the texture, sensory, and nutritional attributes of foods and make functional food to treat some human diseases especially gastrointestinal disorders and metabolic syndromes.