Elsevier

Carbohydrate Polymers

Volume 170, 15 August 2017, Pages 52-59
Carbohydrate Polymers

Lactobacillus plantarum CIDCA 8327: An α-glucan producing-strain isolated from kefir grains

https://doi.org/10.1016/j.carbpol.2017.04.053Get rights and content

Highlights

  • L. plantarum 8327 produces different EPS when grown in milk or semidefined medium.

  • The EPS synthetized by L. plantarum 8327 grown in milk was characterized as an α-glucan.

  • The glucan has a main chain with α-(1  4) glycosidic linkages branched at position O-3.

  • This is the first report about glucan production in milk by Lactobacillus.

Abstract

Lactobacillus plantarum CIDCA 8327 is an exopolysaccharide (EPS)-producer strain isolated from kefir with promising properties for the development of functional foods. The aim of the present study was to characterize the structure of the EPS synthesized by this strain grown in skim milk or semidefined medium (SDM). Additionally, genes involved in EPS synthesis were detected by PCR. L. plantarum produces an EPS with a molecular weight of 104 Da in both media. When grown in SDM produce an heteropolysaccharide composed mainly of glucose, glucosamine and rhamnose meanwhile the EPS produced in milk was composed exclusively of glucose indicating the influence of the sugar source. FTIR spectra of this EPS showed signals attributable to an α-glucan. Both by 1H NMR and methylation analysis it was possible to determine that this polysaccharide is a branched α-(1  4)-d-glucan composed of 80% linear α-(1  4)-d-glucopyranosyl units and 19% (1  4)-d-glucopyranosyl units substituted at O-3 by single α-d-glucopyranosil residues.

Introduction

Among the “Food-Grade” biopolymers obtained from natural sources, exopolysaccharides (EPS) synthesized by lactic acid bacteria (LAB) have focused the attention of researchers and manufacturers since these EPS contribute to the rheology of the fermented product and –on account of EPS potential health promoting properties- may also contribute to the development of functional foods (Das, Baruah, & Goyal, 2014; Patten & Laws, 2015).

EPS produced by LAB present a wide range of compositions, structures, molecular masses and conformations depending on the strain. The EPS can stay attached to the cell surface (capsular) or can be released to the culture media (Patten & Laws, 2015). High molecular weight polysaccharides are widely used in the food industry as stabilizers, emulsifiers, and to improve texture and viscosity. The functionality of these polymers is originated from the structural differences in the sugar subunits, which is also the reason of the great diversity among bacterial EPS and novel EPS structures among LAB (Mozzi et al., 2006, Patten and Laws, 2015). Complex genetic mechanisms of EPS production, carbohydrate source, incubation temperature and time, or pH of the culture medium were reported to affect in situ EPS production levels as well as their conformational characteristics, sugar linkages, and molecular mass (Ibarburu et al., 2015).

Many EPS synthesized by LAB have demonstrated to elicit some biological effect (Patten & Laws, 2015). It has been reported that some EPS can have immunomodulatory (Hidalgo-Cantabrana et al., 2012; Medrano, Racedo, Rolny, Abraham, & Pérez, 2011; Notararigo et al., 2014) and antitumoral activity in vivo (Wang et al., 2014), as well as an antagonistic effect against some intestinal pathogens in vitro (Medrano, Hamet, Abraham, & Pérez, 2009; Živković et al., 2016), among other health benefits. Aditionally, the prebiotic effect of several EPS of LAB has been demonstrated in vitro (Korakli, Gänzle, & Vogel, 2002) and in vivo (Hamet, Medrano, Pérez, & Abraham, 2016).

Lactobacillus plantarum is a versatile microorganism that can be found in a wide range of habitats such as dairy, meat, and many plant fermentations, and it can reach high cell densities which are desirable for industrial applications. Different L. plantarum strains are able to produce heteropolysaccharides after grown in glucose or lactose (Dilna et al., 2015; Tallon, Bressollier, & Urdaci, 2003; Wang et al., 2010, Zhang et al., 2013; Zhang, Liu, Tao, & Wei, 2016) or homopolysaccharides: galactanes when lactose is the unique sugar source (Wang et al., 2014) or glucans when sucrose is the sugar source (Das & Goyal, 2013).

Among the health benefits of EPS produced by some strains of L. plantarum it can be mentioned antioxidant activity (Zhang et al., 2013), antagonic activity against Bacillus cereus enterotoxin (Zhang et al., 2016), and antitumoral activity (Wang et al., 2014).

Kefir is a traditional beverage obtained by fermentation of milk with kefir grains that contain a wide diversity of lactic and acetic acid bacteria and yeasts immersed in a matrix composed of protein and the polysaccharide kefiran (Garrote, Abraham, & De Antoni, 2001). Kefiran production was associated to Lactobacillus kefiranofaciens though another lactobacilli isolated from kefir were described to produce EPS after growth in milk (Hamet, Piermaria, & Abraham, 2015; Wang, Zhao, Tian, Yang, & Yang, 2015).

L. plantarum CIDCA 8327 is a facultative heterofermentative Lactobacillus isolated from kefir grains (Garrote et al., 2001). This strain presents a hydrophilic surface and a moderate adhesion to intestinal cells (Caco-2 cell line), while it had a strong inhibitory activity against Salmonella typhimurium, S. enterica, S. gallinarum, S. sonnei and Escherichia coli (Golowczyc et al., 2008). Besides, it is able to grow in the presence of bile salts and survives after one hour of exposure to pH 2.5 (Golowczyc et al., 2008). Moreover, some studies demonstrate that this strain is able to produce organic acids such as lactic and acetic acid, and substances of low molecular weight with antifungal properties after growth in whey media (Londero et al., 2011). In addition this strain is able to grow in milk and produces in situ an EPS of low molecular mass (Hamet et al., 2015).

The above mentioned characteristics, that turn L. plantarum CIDCA 8327 into a promising starter to be potentially included in functional foods, prompted us to study the production and chemical composition of the EPS synthesized in situ by this strain.

Section snippets

Strains and growth conditions

L. plantarum CIDCA 8327 isolated originally from kefir grains (Garrote et al., 2001) was stored at −80 °C in sterile skim milk and reactivated in MRS broth at 30 °C for 24 h (De Man, Rogosa, & Sharpe, 1960). After that, L. plantarum was grown in UHT skim milk (Composition g/L: Protein 32, Fat 15, lactose 47; ashes 6.). La Serenisima, General Rodriguez, Argentina or in a semidefined medium (SDM) (Marieta, Ibarburu, Dueñas, & Irastorza, 2009) for 21 h or 96 h, depending on the determination. The SDM

L. plantarum CIDCA 8327 contains polysaccharide polymerase genes associated with surface/exopolysaccharide production

L. plantarum strains that produce EPS contain several gene clusters involved in the synthesis of the biopolymer (Remus et al., 2012). To detect the presence of polysaccharide polymerase genes in the EPS-producing L. plantarum CIDCA 8327 strain, several cps primers were designed based on the L. plantarum WCFS1 complete genome sequence (GenBank accession number AL93526) (Table 1). In this strain 4 gene clusters (designated cps1, cps2, cps3 and cps4) independently contribute to the overall

Discussion

The functional aspects of fermented foods are mostly related to the concept of probiotic bacteria however the microbial production of functional molecules, such as bioactive EPS, is targeted (Leroy & De Vuyst, 2016). Kefir grains are an interesting source of EPS-producing bacteria (Hamet et al., 2015, Moura de Paiva et al., 2016). Herein we demonstrated that L. plantarum CIDCA 8327 isolated from kefir is able to produce EPS in milk or SDM, whose composition depends on the growth conditions.

Acknowledgments

MVG is a member of the Researcher Career of CICPBA. PP, MM and AGA are members of Scientific Career of CONICET. AIP and MTD are supported by Universidad del Pais Vasco. AP is researcher from CSIC.

This study was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), CONICET and UNLP, the Spanish Ministry of Science and Innovation (AGL2012-40084-C03), the Basque Government (no. IT866-13). M.G.Ll. acknowledges the ‘Gobierno Vasco, Dpto. Agricultura, Pesca y

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