Production, properties, and industrial food application of lactic acid ...

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Abstract Exopolysaccharides (EPS)-producing lactic acid bacteria (LAB) are industrially important microorganisms in the development of functional food ...
Appl Microbiol Biotechnol DOI 10.1007/s00253-015-7172-2

MINI-REVIEW

Production, properties, and industrial food application of lactic acid bacteria-derived exopolysaccharides Emanuele Zannini 1 & Deborah M. Waters 1 & Aidan Coffey 2 & Elke K. Arendt 1

Received: 12 August 2015 / Revised: 8 November 2015 / Accepted: 11 November 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Exopolysaccharides (EPS)-producing lactic acid bacteria (LAB) are industrially important microorganisms in the development of functional food products and are used as starter cultures or coadjutants to develop fermented foods. There is large variability in EPS production by LAB in terms of chemical composition, quantity, molecular size, charge, presence of side chains, and rigidity of the molecules. The main body of the review will cover practical aspects concerning the structural diversity structure of EPS, and their concrete application in food industries is reported in details. To strengthen the food application and process feasibility of LAB EPS at industrial level, a future academic research should be combined with industrial input to understand the technical shortfalls that EPS can address. Keywords Lactic acid bacteria . Exopolysaccharides . Food fermentation . EPS industry applications

Introduction Lactic acid bacteria (LAB) have traditionally been associated with food and feed fermentations. Their uses around the world include improving the preservation, organoleptic characteristics, and nutritional values of a large variety of food and beverages products. LAB are generally considered beneficial * Elke K. Arendt [email protected] 1

School of Food and Nutritional Sciences, University College Cork, Western Road, Cork, Ireland

2

Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland

microorganisms with some strains known to have health-promoting (probiotic) attributes. However, other genera (Streptococcus, Lactococcus, Enterococcus and Carnobacterium) also contain species or strains that are recognised human and animal pathogens. A thorough understanding of the taxonomy, metabolism, and genetics of LAB is thus necessary to fully utilise the technological, nutritional, and health-promoting aspects of LAB while avoiding potential risks Von Wright and Axelsson (2012). Several LAB produce polysaccharides that occur as cell wall constituents (peptidoglycan) are released from the cell. The latter are either permanently attached to the surface of the microbial cell in form of capsules (capsular polysaccharide, CPS) or secreted into the environment as exopolysaccharides (EPS) (Chapot-Chartier et al. 2011). The first report of EPS formation by wine-spoiling LAB dates back to Pasteur (Pasteur 1861), as cited by Leathers (2002); Orla-Jensen (1943) described EPS formation from sucrose by Leuconostoc spp., mesophilic lactobacilli, and pediococci and indicated the role of EPS formation in the spoilage of apple cider and beer. Two phenotypes of EPS-producing strains exist. The Bropy^ phenotype forms a long filament when an inoculation loop is placed into the EPS-covered colony and then slowly withdrawn, while the Bmucoid^ phenotype strain appears as shiny and smooth colonies growing on suitable agar plates (Dierksen et al. 1997; Rühmann et al. 2015). According to Knoshaug et al. (Knoshaug et al. 2007), the interaction of the ropy polysaccharide with other cellular components, such as the intrinsic protein or capsular polysaccharides (CPS) that remain attached to the cell walls, is responsible for the change in viscosity during growth while also providing the desirable ropiness evident when the fluid is manipulated. The exact nature of this interaction remains to be determined. The roles of these EPS in their natural environment

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are complex and still unclear. EPS is likely to play a role in cellular recognition (De Vuyst and Degeest 1999; Looijesteijn et al. 2000, 2001), quorum-sensing control (He et al. 2015), in exchange genetic information, and in the protection of the microbial cell integrity in an ecosystem against hostile environments (desiccation, osmotic stress, pH) and antimicrobial factors (bacteriophages, phagocytosis, predation by protozoa, metal ions, nisin, lysozyme, cleaning agents, ethanol, and antibiotics) (Chapot-Chartier et al. 2011; Flemming and Wingender 2010; Ruas-Madiedo et al. 2002). EPS also have a key role in biofilm formations and surfaces adhesion enabling the colonisation of different environments (De Vuyst et al. 2001; Dertli et al. 2015; Flemming and Wingender 2010; Walter et al. 2008) (Table 1). Since most EPS-producing bacteria lack the genes involved in their own EPS degradation (Badel et al. 2011; Kim et al. 1998; Patel et al. 2010, 2012; Kim and Fogler 1999), it is questionable that EPS serve as a food reserve (Cerning 1990). Other species such as Streptococcus mutans and Streptococcus sobrinus can degrade the EPS dextran by producing dextranase, and S. mutans can also utilise oligosaccharides as a nutrient (Colby and Russell 1997). Additionally, EPS enzyme degradation was reported for Streptococcus thermophiles and Lactobacillus rhamnosus during prolonged fermentations (Degeest et al. 2002; Pham et al. 2000). Pham et al. (2000) found that Lactobacillus rhamnosus R showed a large spectrum of glycohydrolases (α-D-glucosidase, β-D-glucosidase, α-D-galactosidase, β-D-galactosidase, β-D-glucuronidase, and some traces of α-L-rhamnosidase). When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars lowering the viscosity up to 33 % within 27 h of incubation. Even though the EPS alone did not serve as a carbon source, Russo et al. (2012) found a positive effect on L. plantarum WCFS1 and L. acidophilus NCFM growth when the β-D-glucan isolated from P. parvulus 2.6. was added in a glucose-containing chemically defined medium. EPS can also act as substrate for other organisms in complex ecosystems. In this regard, Salazar et al. (2008) showed that EPS synthesised by intestinal bifidobacteria could act as fermentable substrates for microorganisms in the human gut environment, promoting shifts in short chain fatty acids (SCFA) profiles and changes in relationships among intestinal microbial populations. EPS-producing LAB are industrially important microorganisms in the development of functional food products and are used as starter cultures or coadjutants to develop fermented foods such as yoghurt, cheese, and cereal-based products (Badel et al. 2011; Jolly et al. 2002; Patel Ap 2013; Ruas-Madiedo et al. 2002; Tieking et al. 2003) because of their viscosity and mouthfeel enhancement properties. In contrast, alcoholic beverages such as beers, ciders, and wines are

spoiled by EPS-producing LAB. In wine, this spoilage can occur either during vilification or after bottling and leads to an alteration known as Bropiness^ or Boilness^, characterised by a viscous, thick texture, and oily feel, which although not appreciably altering the taste, renders the products unpleasant to the palate causing considerable economic loss (Gindreau et al. 2001). More recently, EPS were used as depollution agents, and there was a growing interest in their biological functions like antitumour, antioxidant, or prebiotic activities (Liu et al. 2010). Microbial polysaccharides with nutraceutical potential and bioactive properties have been also investigated in detail during the last few decades. EPS from LAB exhibited various biological activities such as probiotic functionality Patel et al. (2012), antitumour (Kitazawa et al. 1991), a cholesterol-lowering (Kitazawa et al. 1991), and immunomodulatory (Hosono et al. 1997; Surayot et al. 2014) effects. Regarding the health-promoting benefits of LAB and bifidobacteria EPS, two reviews on the immunomodulatory activity and prebiotic effects were recently published by Ryan et al. (2015) and Salazar et al. (2015). Chemical composition of LAB EPS According to the chemical composition and biosynthesis mechanisms, LAB EPS are classified into two distinct groups: homopolysaccharides (HoPS) and heteropolysaccharides (HePS). Homopolysaccharides HoPS are mainly synthetised extracellularly from an existing sucrose molecule, which act as donor of the corresponding monosaccharide by action of extracellular enzyme belonging to the glycosyl hydrolase (GH) family using sucrose as the glycosyl (fructose or glucose) donor (Leemhuis et al. 2013; Van Hijum et al. 2004, 2006); α-glucans and β-fructans are formed by glucansucrases (GS; GH family 70) and fructansucrases (FS; GH family 68), respectively (Cantarel et al. 2009). Glucansucrases are able to use the energy of the osidic bond (also called glycosidic linkages) of sucrose to catalyse the transfer of a corresponding glycosyl moiety (Fig. 1). Depending on the linkage type and the position of the carbon involved in the bond, HoPS can be classified as α-D glucans (dextran, mutan, reuteran, and alternan) and β-D glucans, whereas those containing fructose are fructans (levan and inulin-types) Ruas-Madiedo and de los Reyes-Gavilan (2005). Glucans and fructans are found most frequently among the homopolysaccharides, and they are both applied as ingredient in the food industry (Anwar et al. 2010; Buchholz and Seibel 2008). Moreover, a fourth group of HoPS, polygalactans, composed of a pentameric repeating unit of galactose has also been described (Gruter et al. 1992) (Table 2).

Appl Microbiol Biotechnol Table 1 Some of the roles ascribed to exopolysaccharides in biofilms

Process

Functional roles of exopolysaccharides to biofilms

Adhesion

EPS facilitate the initial steps in the colonisation of surfaces (abiotic and biotic) and long-term attachment of biofilms EPS enable intra-cell bridging through temporary immobilisation of the bacterial population and the subsequent development of high cell densities and cell–cell recognition Hydrophilic EPS have a high water retention capacity maintaining a hydrated microenvironment and enabling survival of the microbes in desiccated environments Neutral and charged EPS form a hydrated polymer network mediating the mechanical stability of biofilms (often in conjunction with multivalent cations), determining biofilm architecture, and allowing cell–cell communication EPS may serve as a source of carbon, nitrogen, and phosphorus containing compounds for utilisation by the biofilm ecosystem

Bacterial cell aggregation

Water retention

Cohesion of biofilms

Nutrient source Protective barrier

EPS confer resistance to non-specific and specific host defences during infection, as well as giving tolerance to various antimicrobial agents, protecting cyanobacterial nitrogenase from the harmful effects of oxygen, and also against some phagocytic protozoa

Organic compound and inorganic ion sorption

Charged and hydrophobic EPS mediate accumulation of nutrients from the environment, and sorption of xenobiotics and recalcitrant materials. They promote polysaccharide gel formation resulting in ion exchange, mineral formation, and the accumulation of toxic metal ions (thus collectively contributing to environmental detoxification)

Binding of enzymes

Non-glycolytic extracellular enzyme interaction with EPS leads to retention, stabilisation, and accumulation of enzymes preventing loss of valuable biocatalysts Lipopolysaccharides (isoprenoid glycosyl carrier lipids), which form lipoglycoconjugates, mediate the releases cellular material as a result of metabolic turnover

Export of cell components

Sink for excess energy

EPS stores excess carbon in conditions of unbalanced carbon to nitrogen ratios

From Nwodo et al. (2012)

HoPS have a main backbone structure with variable degrees of branching and linkage sites, which differ among bacterial strains (De Vuyst and Degeest 1999; Monsan et al. 2001). HoPS have high-molecular weights in the order of 105–106 Da (Ruas-Madiedo et al. 2002a). Concerning the carbon involved in the linkage, glucan are subdivided into dextran α-1→6 (α-1→3), mutan α-1→3 (α-1→6), reuteran α-1→4, and alternan α-1→6/α-1→3) while fructans are subdivided into levan ß-2→6 (ß-2→1) and inulin-like ß-2→ Glucansucrase

Glucan+Fructose

Sucrose Fructansucrase

Glucansucrase

Fructan+Glucose

Glucooligosaccharide+Fructose

Sucrose + Acceptor Fructansucrase

Fructooligosaccharide+ Glucose

Fig. 1 Biosynthetic pathways leading to EPS synthesis in LAB

1 (ß-2→6) (Table 2). The bound in parenthesis represents the branching linkage. Dextran The term dextran refers to a large group of α-glucans in which the main backbone chain consists of α-1→6 glycosidic linkages. Dextran may also be branched through various secondary linkages such as α-1→2, α-1→3, and α-1→4 (Monsan et al. 2001). Pasteur was the first who described the microbial origin of the gelification process observed in cane sugar syrup (Pasteur 1861). This class of α-glucans was later assigned the name dextran due to it positive rotatory power (Monsan et al. 2001). In 1943, the corresponding enzyme was subsequently named dextransucrase (Hehre and Sugg 1942; Hestrin et al. 1943). The synthesis of dextran from sucrose was recorded for Leuconostoc mesenteroides subsp. mesenteroides. Nevertheless, the ability to synthesise dextran can be lost when serial refreshments are performed in media with increasing amount of salt.

Appl Microbiol Biotechnol Table 2

Types of homopolysaccharides produced by lactic acid bacteria and their predominant linkages

HoPS

α-Glucans Dextran

Main linkagea (branching linkage)

Species

α-1→6 (α1→ 3)

(Galle et al. 2010; Korakli and Vogel 2006; Kralj Lactobacillus reuteri, Lactobacillus casei, Lactobacillus sakei, et al. 2004; Ruas-Madiedo et al. 2002; Tieking Lactobacillus fermentum, Lactobacillus parabuchneri, et al. 2005) Lactobacillus curvatus, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc mesenteroides subsp. dextrtanicum, Streptococcus mutans, Streptococcus downei, Streptococcus sobrinus Streptococcus salivarius, Streptococcus gordonii Weisella cibaria Lactobacillus reuteri, Streptococcus mutans Streptococcus downei, (Giffard et al. 1991; Kralj et al. 2004; RuasStreptococcus sobrinus Madiedo et al. 2002)

References

Mutan

α-1→3 (α1→ 6)

Alternan

α-1→3/α1→6 Leuconostoc mesenteroides

(Giffard et al. 1991; Korakli and Vogel 2006; Kralj et al. 2004; Ruas-Madiedo et al. 2002

Reuteran

α-1 4 (α1 6)

(Kralj et al. 2004)

Others

α-1 3/α-1→6 Leuconostoc citreum NRRL B-1355 (α-1→2/α-1 3)

(Bounaix et al. 2010; Smith et al. 1998)

ß-1→3

Pediococcus damnosus, Pediococcus parvulus, Lactobacillus diolivorans G77, Lactobacillus sp.

(Duenas-Chasco et al. 1998; Duenas-Chasco et al. 1997; Werning et al. 2006)

Levan

ß-2→6 (ß-2→ 1)

Lactobacillus reuteri, Lactobacillus sanfranciscensis, Leuconostoc (Giffard et al. 1993; Korakli et al. 2000; Korakli mesenteroides, Streptococcus sobrinus, Streptococcus salivarius and Vogel 2006; Ruas-Madiedo et al. 2002

Inulinlike

ß-2→1 (ß-2→ 6)

Lactobacillus reuteri, Leuconostoc citreum, Streptocossus mutans

(Olivares-Illana et al. 2003; Rosell and Birkhed 1974; van Hijum et al. 2001; van Hijum et al. 2004)

Lactococcus lactis subsp. cremoris H414

(Gruter et al. 1992)

Lactobacillus reuteri

ß-Glucan ß-glucan Fructans

Polygalactan α-Gal/ß-Gal a

At least 50 % of the respective linkage

Kim et al. (2003) investigated the size distribution and the de gree o f branc hing o f dextra ns syn thesised by L. mesenteroides B-512FMCM dextransucrase, using different processing parameters (concentration of sucrose, 0.1– 4.0 M, pH values of 4.5–6.0, and different temperatures of 4–45 °C). They found that with increasing concentrations of sucrose, from 0.1 to 4.0 M, Leuconostoc mesenteroides B-512FMCM dextransucrase gave a decreasing amount of high-molecular weight dextran (>106 Da) with a concomitant increase in low-molecular weight dextran. The degree of branching increased from 5 % for 0.1 M sucrose to 16.6 % for 4.0 M sucrose while the temperature had very little effect on the size of the dextran, which was >106 Da, but it had a significant effect on the degree of branching, which was 4.8 % at 4 °C and increased to 14.7 % at 45 °C. Both the molecular weight and the degree of branching were not significantly affected by different pH values between 4.5 and 6.0. The degree and nature of the branch points are depending on the producing microorganism (Jeanes et al. 1954) and/or enzymes intervention. For example, biosynthesis of glucans by Lactobacillus sp. leads to the linear dextran whereas

L. reuteri 180 synthetised a branched EPS (Uzochukwu et al. 2001; Van Leeuwen et al. 2008, 2009). Additionally, the degree of branching is depending on enzyme conformation. On such regard, Torino et al. (2015) reported that a mutation of the gene encoding for the glucansucrase increased the α-(1,6) and decreased α-(1,4) linkages. A single enzyme can also catalyse the synthesis of several types of linkages, thus allowing, on its own, the formation of a branched polymer Neely and Nott (1962). On the contrary, certain bacterial strains have showed to produce dextrans of various structures, and this was attributed to the excretion of different dextransucrases by the microorganism (Côté and Robyt 1982a, b; Figures and Edwards 1981; Zahnley and Smith 1995). Native dextrans, partially degraded dextrans, and their derivatives have immense commercial value in pharmaceutical (lubricants and carriers), medical (antithrombotic, anticoagulant, and osmotic agents, and iron dextran for the treatment of anaemic deficiencies), and in food and chemical industries (adjuvant, emulsifier, carrier and stabiliser, and preservative enzyme cryoprotectant) (Aebischer et al. 2001;

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Bhavani and Nisha 2010; Goulas et al. 2004; Novak 1957; King and Speert 2002; Pucci and Kunka 1990; William and Joseph 1959). As a food ingredient, dextran was initially studied in the 1950s primarily as a thickener. Dextran has been listed as generally regarded as safe (GRAS) by the Food and Drug Administration (FDA) for use in animal feeds and medicines (21CFR582.1275) and indirectly in human foods as food packaging materials (21CFR186.1275) (FDA 2013). In 2001, the European Commission authorised the commercialisation of a Leuconostoc mesenteroides dextran preparation as a novel food ingredient in bakery applications up to level of 5 % to improve the softness, crumb texture, and loaf volume. According to the official decision statement, it is considered to have nutritional properties similar to starch (Byrne 2001). Alternan Alternan is a branched glucan with a unique backbone structure of alternating α-1→6 and α-1→3-D-glycosidic linkages (Côté 2002; Leathers et al. 2010). These types of linkages are thought to be responsible for its peculiar physical characteristic, that differ from commercial dextran, including high solubility, low viscosity, and a remarkable resistance to microbial and enzymatic hydrolysis should be added here (Leathers et al. 2003; Côté et al. 1997; Vandamme et al. 2002a; Côté 2009). Leuconostoc mesenteroides NRRL B-1355 was first reported to be an alternan-producing strain Côté and Robyt (1982a, b). Other two strains of Leuconostoc mesenteroides NRRL B-1501 and NRRL B-1498 are also known to produce alternan via alternansucrase; the enzyme that converts sucrose to alternan and fructose (Côté and Robyt 1982a, b; Jeanes et al. 1954). The gene that encodes alternansucrase also has been cloned and sequenced (Argüello-Morales et al. 2000). While naturally occurring strains of Leuconostoc mesenteroides that produce alternan also produce dextran as a troublesome contaminant, genetically improved strains for production of alternan with little or no dextran development have been isolated (Kim and Robyt 1994; Leathers et al. 1997, Leathers et al. 1995; Smith et al. 1994, 1998). Reuteran Reuteran is the name given to a specific α-glucan produced by the species Lactobacillus reuteri and is generally associated with fermented milk products. Reuteran is a glucan containing (α1→4) and (α1→6) glycosidic bonds Meng et al. (2015) with no repeating units present (van Leeuwen et al. 2008). Two strains of Lactobacillus reuteri LB121 and ATCC 55730 were identified as reuteran producers, and the corresponding enzyme reuteransucrase involved in the synthesis of this α-glucan has been characterised (Kralj et al. 2002). Reuteran, like other novel glucans, may play a role in the

thickening of fermented dairy foods. Additionally, due to its water solubility, reuteran may found an interesting application in bakery sector also (Arendt et al. 2007). Mutan Mutan is a glucan synthetised through mutansucrase by various serotypes of Streptococcus mutans. Additionally, different strains belonging to the species Leuconostoc and Lactobacillus are able to produce mutan (Côté and Skory 2012; Côté et al. 2013; Waldherr et al. 2010). Mutans contain mainly α–1→3 glycosidic bond (Leemhuis et al. 2013) that is responsible for its insolubility in water. Mutan polysaccharides are involved in the adhesion of oral flora microorganisms on the tooth surface to form a dental plaque (Hamada and Slade 1980; Pleszczyńska et al. 2015). ß-Glucan (1,3)-β-D-glucans from several bacteria and fungi constitute a group of naturally occurring polysaccharides with a main chain of (1,3)-linked β-glucopyranosyl units. It can be linear or branched with either (1,6)- or (1,2)-linked side chains of varying length and distribution (Torino et al. 2015). LAB sysnthesis of β- D -glucans occurs intracellularly by membrane-associated glycosyltransferase (Torino et al. 2015). Although its mechanism of action is not yet fully understood, it does not need sucrose as substrate (Werning et al. 2006). Lactobacillus spp. G-77 secretes two exopolysaccharides when grown on a glucose media. One of them was found to be a (1–3)-β-D-glucan, identical to that described for the EPS from Pediococcus damnosus 2.6 (Duenas-Chasco et al. 1998). The second HoPS was a dextran-type polysaccharides with α–1,6 glycosyl linkages and α–1→2 branching of a single D-glucose unit. When produced by beverage spoilage organisms, these ß-glucans cause an undesirable thickening which has been reported during cider (Duenas-Chasco et al. 1997) and wine (Gindreau et al. 2001) production leading to Boiliness^ or Bropiness^. Conversely, (1,3)-β-D-glucans are considered as biological response modifiers, and numerous publications describe their biological activities and therapeutic uses (Sletmoen and Stokke 2008). Their biological effects are influenced by their degree of branching, chain length, and tertiary structure (Zhang et al. 2005). Fructans Microbial fructans are high molecular mass polymers, which consist of chains of fructosyl units connected through β-(2→ 1) or β-(2→6) linkages (Ernst et al. 1998) and depending on the linkage type of the main chain, they are called inulin and

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levan, respectively (Seibel and Buchholz 2010). They are synthesised from sucrose by the activity of fructosyltransferases (FTF). Fructan production has been reported for the genera Streptococcus, Leuconostoc, Lactobacillus, and Weissella (Monsan et al. 2001; Tieking et al. 2003; van Geel-Schutten et al. 1998). The amount of fructan produced, type of linkages, molecular mass, and degree of branching depend on the enzymes involved in the synthesis as well as on the sucrose concentration, the presence of acceptor molecules, and their concentrations (Ruas-Madiedo and de los Reyes-Gavilan 2009). The enzymes catalysing the synthesis of these two polymers are inulosucrase and levansucrase, respectively. Most of the FTF characterised to date produce fructan of levan-type EPS, and only a few were reported to produce inulin. Indeed, only individual strains of Lactobacillus and Leuconostoc and Streptococus were reported to produce inulin or to posses inulosucrase-encoding genes (Anwar et al. 2008; Olivares-Illana et al. 2003; Schwab et al. 2007; Van Hijum et al. 2001. Levan Levan is a non-toxic, biologically active, extracellular polysaccharide that can be produced by both plants and microorganisms. It is a sugar polymer composed of fructose with 2, 6-linkages (Melo et al. 2007). Among LAB, levan is produced by strains from the oral flora, such as Streptococcus salivarius, Streptococcus mutans (Giffard et al. 1993; Shiroza and Kuramitsu 1988), Leuconostoc mesenteroides NRRL B-512 F, Lactobacillus sanfranciscensis LTH 2590, and Lactobacillus reuteri LB 121 (Patel et al. 2012). To date, few studies on this relatively new class of fructan showed that oligosaccharides produced by in vitro partial acid hydrolysis of levan could be utilised by different pure cultures that include Bifidobacterium adolescentis, B. longum, B. breve, B. pseudocatenulatum, Lactobacillus plantarum, and Pediococcus pentosaceus (Kang et al. 2002; Marx et al. 2000). Korakli et al. (2003) studied the formation of levan and kestose during growth of L. sanfrancisciensis, a typical LAB dominating traditionally prepared wheat and rye sourdoughs Hammes et al. (1996). They observed that in static pH fermentation, the yield of levan increased with increasing sucrose concentrations, and appreciable levels of 1-kestose were observed only when the sucrose concentration exceeded 50 g L-1. When Lactobacillus sanfrancisciensis LTH 2590 was used to ferment a wheat dough containing 60 g Kg−1 of sucrose, the formation of more than 5 g Kg−1 of levan was observed (Tieking and Ganzle 2005). Levan is naturally present in various food products and thus, is regularly consumed in very small amounts by humans.

However, it has been relatively ignored as a functional food ingredient due to its limited resources and very low content, until recently. Levan from L. sanfranciscensis LTH 2590 exhibits prebiotic effects (Korakli et al. 2003. Levan has attracted attention for its antitumour properties (Yoo et al. 2004), cholesterol-lowering properties, and application as an eco-friendly adhesive (Patel et al. 2012). The behaviour of levan in solution has also been studied (Kasapis et al. 1994). Levan is valuable in terms of its water-holding capacity. At a concentration higher than 1 % (w/v), a levan solution will display film-forming characteristics on a smooth surface. Inulin-type Inulin-type EPS are fructans or fructooligosaccharides containing β-1→2 osidic bonds. Lactobacillus johnsonii NCC 533 produces high-molecular mass inulin from sucrose by using an inulosucrase enzyme (Anwar et al. 2008). Streptococcus mutans strain JC2, Leuconostoc citreum CW28, and Lactobacillus reuteri 121 are some other LAB which produce inulins. These are acting as prebiotics in humans and animals (Patel et al. 2012). Plant-based inulin and fructooligosaccharides (FOS) were originally suggested as prebiotics that selectively stimulate bifidobacteria, intestinal microorganisms considered to be beneficial and that are extensively used as probiotics (Masco et al. 2005). Experimental evidence confirmed the bifidogenic effect of inulin and FOS (Gibson et al. 1995; Kruse et al. 1999; Wang and Gibson 1993). Additionally, inulin-type fructooligosaccharides synthesise butyrate, which nourish the enterocytes, prevent pathogenic adherence, and decrease pH of lumen (Sartor 2004). Inulin-type fructans can be also employed as vehicles for targeted drug delivery in treating colon cancer (Pool-Zobel 2005). Heteropolysaccharides HePS are produced by a variety of mesophilic (Lactococcus lactis subsp. lactis, L. lactis subsp. cremoris, Lactobacillus casei, Lb. sake, and Lb. rhamnosus) and thermophilic (Lb. acidophilus, Lb. delbrueckii subsp. bulgaricus, Lb. helveticus, and Streptococcus thermophilus) LAB. LAB are composed of a backbone of repeated subunits that are branched (at positions C2, C3, C4, or C6) or unbranched, and consist of three to eight monosaccharides, (D-glucose, D-galactose, and L-rhamnose) derivatives of monosaccharides ( N -acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc) or glucuronic acid (GlcA)) or substituted monosaccharides (such as phosphate, acetyl, and glycerol) (De Vuyst and Degeest 1999; Ruas-Madiedo et al. 2002a) (Fig. 2). The thermophilic HePS-producing LAB have recently received renewed interest, since they play a key role in the rheology, texture and

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body, and mouthfeel of fermented dairy drinks (Ryan et al. 2015; Fox et al. 2015; Ravyts et al. 2011). HePS synthesis differs from HoPS synthesis since the precursor repeating units are formed intracellularly and isoprenoid glycosyl carried lipids are involved in the process (Cerning 1990). The repeating units are translocated across the membrane and subsequently polymerised extracellularly. Additionally, the biosynthesis and secretion of the LAB HePS occur at different phases of growth, and the amount and type are regulated by fermentation conditions (De Vuyst and Degeest 1999). Structurally, HePS may be ropy or mucoid. Under optimal culture condition, a HePS yield of 0.15–0.6 gl−1 may occur (Cerning 1995). The molecular mass of these HePS polymers ranges between 1.0 × 104 and 6.0 × 106 Da (De Vuyst and Degeest 1999). The producer strain, growth conditions (pH, temperature, incubation time, oxygen tension, and turbidity), and medium composition (carbon, nitrogen sources, and other nutrients) can influence the polymer yield and the monosaccharide subunits composition (De Vuyst and Degeest 1999; Degeest et al. 2001; Grobben et al. 2000; Looijesteijn et al. 2000; Torino et al. 2000, 2015). HePS have a great variability in structures (Fig. 1), and contrary to HoPS, their concentrations in broth can easily reach >100 mgL−1 (Duboc and Mollet 2001). The composition of the monosaccharide subunits and the structure of the repeating units are not species-specific, except i n c a s e o f L a c t ob a c i l l u s k e f i r a n o f a c i e n s s u bs p . kefiranofaciens. This species, isolated from kefir grain, a fermented dairy food from the north Caucasus region, produces large amounts of water-soluble polysaccharides called kefiran. This consists of approximately equal proportions of glucose and galactose (Micheli et al. 1999) and is able to improve visco-elastic properties of acid milk gels. Additionally, kefiran is reported to have antimicrobial (Rodrigues et al. 2005) activity and the ability to significantly reduce blood pressure and the serum cholesterol levels (Maeda et al. 2004). Kefiran is reported to confer protective immunity, maintain intestinal homeostasis, enhance IgA (immunoglobuline A) level at both the small and large intestine level, and influence the systemic immunity through the release of cytokines into the blood (Lemieux et al. 2006; Mora-Gutierrez 2014; Park et al. 2004; Piermaria et al. 2009; Stanton et al. 2014). Application of LAB EPS in food industries To date, polysaccharides recovered from plant, algae, and animal sources (e.g., starch, galactomannans, pectin, carrageenan, and alginate) are still the major contributors to the overall hydrocolloid market with xanthan gum being the only significant bacterial EPS, which accounts for 6 % of the total market value (Imeson 2010). This is mainly due to the higher prices of

Fig. 2 Characteristics of heteropolysaccharides produced by Lactobacillus sp.

bacterial polysaccharides, which are a consequence of the high value of the carbon sources commonly used and of the associated downstream costs. However, the existing supplies for plant and seaweeds polysaccharides are either not sufficiently reliable, are of variable consistency, or the quantities available are not sufficient to match demand. On the contrary, polysaccharides of microbial origin can be prepared in reliable quantities using conventional biotechnological processes (Laws et al. 2001) . The regulatory status of safety associated with the majority of lactobacilli attracts a lot of industrial interest due to the simplified regulatory hurdles to application in food products. The genus contains GRAS (generally recognised as safe) or QPS (qualified presumption of safety) bacteria, giving them exemption from risk for the application in the human health market. In this context, there is no need to eliminate biomass before the consumption of polysaccharide and low yield explain why the polysaccharide is seldom used as a purified additive. Some bacterial EPS can directly replace polysaccharides extracted from plants (e.g., guar gum or pectin) or algae (e. g., carrageenan or alginate) in traditional applications (Freitas et al. 2011), because of their improved physical properties. Conversely, other bacterial EPS possess unique properties that can launch a range of new commercial opportunities (e.g., bacterial cellulose or levan) (Kumar et al. 2007; Ullrich 2009). Beside the technological properties, LAB EPS might also contribute to human health (Fig. 3) as prebiotics or due to a n t i t u m o u r, a n t i u l c e r , i m m u n o m o d u l a t i n g , o r cholesterol-lowering activities (De Vuyst and Degeest 1999). However, no ESFA (European Food Safety Authority) or FDA health claims on LAB EPS have been approved. In the yoghurt business, there is an increasing consumer interest for stirred yoghurt products with a smooth and creamy texture obtained by mild homogenization of the milk coagulum after

Appl Microbiol Biotechnol

fermentation. However, the homogenization strongly affects the rheology of the coagulum and facilitates undesirable serum separation (syneresis) since the network formed by the gel is broken. Several solutions were proposed to improve the texture of fermented milk products and reduce syneresis (Rohm and Kovac 1994). In order to overcome this problem, a well-known technological solution is to improve the product quality by increasing milk solids such as fat, proteins (Rohm and Schmid 1993), or sugars (sucrose, fructose), or by adding stabilisers such as pectin, starch, alginate, gelatin, when permitted by national legislation. However, these approaches do not address an increasing consumer demand for products with low (or reduced) fat, low sugar, low cost, and with as few food additives as possible. An answer to this challenge is to incubate the starter cultures at sub-optimal growth temperatures, which encourage EPS production, and/or to take advantage of the EPS produced naturally by LAB used as starter culture in the fermentation. The success of EPS application in the food industry is generally dictated by its ability to bind water, interact with proteins, and to increase the viscosity of the milk serum phase. EPS may act as texturisers and stabilisers, and consequently, avoid the use of food additives (Duboc and Mollet 2001). Although the mechanism of the interactions between EPS and milk constituents in fermented products is poorly understood, the viscosity and the charge of the EPS determine largely the physical properties of the end product. EPS produced by LAB are taste-neutral; however, since a fermented milk product becomes more viscous, its residence time in the mouth and time of contact with the palate and taste receptors is increased. As a result, taste perception is increased through an improved volatilization of the intrinsic yoghurt flavours. The benefits of EPS are detectable at extremely low concentrations. The aim is to obtain an appealing visual appearance (gloss) of a product, to prevent syneresis, to have a creamy and firm texture, and to give a pleasant mouthfeel (Duboc and Mollet 2001). In part-skim Mozzarella cheese production, encapsulated EPS S. thermophilus was successfully used to enhance Mozzarella cheese moisture level, yield, and melt properties without deleteriously compromising the whey process recovery steps in terms of whey viscosity or ultra-filtration concentration time (Broadbent et al. 2001). The application of levan in beverages with particular emphasis on its solution properties (Kasapis et al. 1994) has demonstrated that the viscosity of a levan solution is stable during heating and in sodium chloride. In addition, the viscosity of levan is influenced by acidic conditions (pH 2), but stable in the range of pH 4–10 Kim et al. (1998). In the food industry, levan is used as a stabiliser, an emulsifier, a formulation aid, surface-finishing agent, an encapsulating agent, and a carrier of flavours and fragrances (Han 1990; Han and Clarke 1990). Levan is useful in terms of its water-holding capacity (Table 3). At greater than 1 % (w/v)

Fig. 3 Schematic representation of the possible health-promoting properties of LAB EPSs

concentration, a levan solution will show film-forming characteristics on an appropriate smooth surface. Levan solution (below 1 %) can also be used in coatings or as bio thickener (De Vuyst et al. 2001) The enzymatic or chemical hydrolytic products of levan may be used in the food industry as sweeteners or dietary fibre, for example β-(2,6)-linked fructofuranosyl oligosaccharides (Han 1990). Dextran is the collective term given to a group of bacterial polyglucan composed of chains of D-glucose units connected by α–(1→6) linkages. These polysaccharides are synthesised by a number of bacterial species. The synthesis occurs extracellularly and is catalysed by a species-specific enzyme, dextransucrase. In the food industry, dextran is currently used as thickener for jam and ice cream. It prevents crystallisation of sugar, improves moisture retention, and maintains flavour and appearance of various food items (Table 3). Dextran represent the primarily polysaccharide characterizing kefir grains that host the microbial consortium, a stable association of different LAB, acetic acid bacteria, and yeasts, responsible of the production of water kefir (Pidoux 1989; Pidoux et al. 1988); Waldherr et al. (2010) identified a strain of Lactobacillus hilgardii producing large amounts of the granule-forming dextran in water kefir. The origin of water kefir remains unclear. There are some descriptions of similar grains called Bginger beer plants^ (Ward 1892) or BCalifornia bees^, BAfrican bees^, BAle nuts^, BBalm of Gilead^, and BJapanese Beer Seeds^(Kebler 1921). Pidoux called them BSugary kefir grains^ in order to differentiate them from the grains used for fermenting milk (Pidoux 1989; Pidoux et al. 1990). The use of dextran in bread technology is not widely spread even if its impact on bread quality (volume and texture) was shown. A patented process has been developed to obtain a sourdough rich in dextran using L. mesenteroides spp. (deposit number LMGP-16878) strain able to produce a sufficient amount of high-molecular weight dextran (106 Da) assuring a significant impact on bread volume (Lacaze et al. 2007; Vandamme et al.

Appl Microbiol Biotechnol Table 3 EPS

Food industrial application of functional exopolysaccharides and oligosaccharides from LAB LAB-producing EPS

Uses

References

Dextran Leuconostoc mesenteroides In bakery products, dextran improves softness, NRRL B-512F crumb texture, loaf volume, and may compensate Leuconostoc mesenteroides the low protein content of wheat flour. NRRL B-640 In confectionary, dextran can be used as stabiliser Leuonostoc citreum NRRL B-742 for confectionery preventing crystallisation, improves Leuconostoc citreum NRRL moisture retention, increases viscosity, and maintains B-1355 flavour. Its use is also suggested in soft drinks, flavour Leuconostoc citreum NRRLB-1299 extract, milk beverages, and icing compositions. Streptococcus mutans 6715 In ice cream, dextran can be used as stabilisers (2–4 %) Weisella cibaria MG1 conferring beneficial properties on viscosity. In frozen and dried foods, dextran can be used for stabilising vacuum, air dried, and freeze-dried or frozen foods (fish products, meat, vegetables, and cheese). Dextran can be also use as coating agent to protect food from oxidation and other chemical changes and also help to preserve texture and flavour. In non-alcoholic wort-based beverages, dextran can provide desirable textural properties. Levan can be uses as a source of di-fructofuranoses, Levan Steptococcus salivarius fructose, and fructooligosaccharides, as a stabiliser, Steptococcus mutans an emulsifier, a formulation aid, surface-finishing Leuconostoc mesenteroides agent, an encapsulating agent, a carrier of flavours, NRRL, B-512 F colour and fragrances, and as fat substitute (functional Lactobacillus sanfranciscensis chewing gum composition) LTH 2590 In bakery products, levans can be used as bread Lactobacillus reuteri LB 121 improvers according to their ability to increase volume, retard staling and to improve texture and taste of breads Kefiran L. lactis subsp. lactis In fermented milk like Kefir (traditional self-carbonated L. lactis subsp cremoris slightly alcoholic fermented milk from Eastern L. mesenteroides subsp. Europe), kefiran is the “glue” of the grains and dextranicum confers a slimy texture to the product by increasing Steptococcus thermophilus the water binding reducing at the same time the water flow in the matrix space Inulins Lactobacillus johnsonii NCC 533 Inulin can be used as a prebiotic, as a sugar replacer (especially in combination with high intensity Streptococcus mutans JC2 sweeteners), as a fat replacer and texture modifier in Leuconostoc citreum CW28 low-fat dairy products improving the mouthfeel. Lactobacillus reuteri 121 Especially, long-chain inulin addition to low-fat yoghurt resulted in enhanced creaminess. This effect also occurs in low-fat cheese, in yoghurt ice cream, in chocolate mousse, and in custards

2002a, b. The sourdough obtained permits to improve freshness, crumb structure, mouthfeel, and softness of all kinds of baked good from wheat-rich dough products to rye sourdough breads. Besides its functionality as a prebiotic, inulin can also be used for its technological properties, and it often delivers dual benefit: an improved organoleptic quality and a better-balanced nutritional compositions (Franck 2002). In food production, inulin enhances the quality of the food products acting as texturiser, emulsion stabiliser, and partial fat replacer. Inulin has a neutral bland taste, without any off-flavour or persistence. It combines easily with other food

(William and Joseph 1959, McCurdy et al., 1994, Vandamme et al. 2002a, Naessens et al., 2005, Bounaix et al., 2010, Zannini et al., 2013)

(Han, 1990, Vijn & Smeekens, 1999, Song et al., 2000, Jang et al. 2001, Vincent et al. 2005, Van Geel-Schutten, 2006, Park, 2007)

(Duboc and Mollet, 2001)

(Koca and Metin, 2004, Guven et al., 2005, Hennelly et al., 2006, Kip et al., 2006, Tárrega & Costell, 2006, Cardarelli et al., 2008, Paseephol et al., 2008, Guggisberg et al., 2009)

ingredients without altering delicate flavours. It is discreetly soluble in water (maximum 10 % at room temperature) and brings a rather low viscosity (less than 2 mPa.s for a 5 % w/w solution in water). Additionally, because of its gelling characteristics, inulin has a remarkable capacity to replace fat, particularly in products such as processed cheese, dairy spread cheese, and butter-like products (Franck 2002). Future trends The molecular bases for structure–function relationships of EPS in food systems become clearer over the last decade. As

Appl Microbiol Biotechnol

this research continues, the food industry can expect to see more widespread application of EPS and EPS/LAB symbiotic cultures in ways that provide added value and innovation to food and beverage products for years to come. Additionally, the production of more than one EPS-type by single strains may have strong potential for development of future food applications as they potentially offer synergistic effects on texture (dextran, ropy CPS) and nutritional improvement (levan, inulin, OS). This multiple EPS-type-producing LAB can also address the need for using natural additives to comply with food legislation that imposes severe constraints on materials that can be used. The LAB EPS biotechnological application in food and beverages industries will be dependent on biotechnological developments from high yields producing strains obtaining with all desirable properties linked to their use in food area. On such regard, future work needs to (i) determine the mechanisms responsible for the control and regulation of EPS biosynthesis, both at the level of genes and of proteins, to (ii) reduce their industrial costs by apply metabolic engineering for obtaining high yields necessary to reach commercialization, and to (iii) define the functionality of individual strains and the polymers or oligosaccharides derived in order to enable their deliberate use in food and beverages applications. However, the design of tailor-made EPS with desired/specific functionalities in food and beverages remains challenging. On such regards, future academic research should be combined with industrial input to understand the technical shortfalls that EPS can address. However, this must be partnered with an understanding of the economic and regulatory restrictions imposed on the food and beverage industry by the EPS fermentation process and other competing technologies. Acknowledgements Funding for this research was provided under the Irish National Development Plan, through the Food Institutional Research Measure, administered by the Department of Agriculture, Fisheries and Food, Ireland. This publication reflects only author’s views, and the community is not liable for any use that may be made of the information contained in this publication. Compliance with ethical standards Ethical statement This article complies with ethical standards and does not contain any studies with human participants performed by any of the authors. Conflict of interest The authors declare that they have no competing interests.

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