IJFS#1317_bozza Ital. J. Food Sci., vol. 31, 2019 - 233 PAPER INFLUENCE OF EXOPOLYSACCHARIDE ON THE GROWTH OF LACTIC ACID BACTERIA H. TSUDA*1,2, S. OKUDA2, T. HARAGUCHI2 and K. KODAMA2 1Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki city, Aomori 036-8561, Japan 2Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562, Nanatsuka, Shobara City, Hiroshima 727-0023, Japan *Corresponding author: Tel.: +89172362111; Fax: +81172393750 E-mail address: tsudah@hirosaki-u.ac.jp ABSTRACT The highest production of exopolysaccharides (EPSs) by Lactobacillus buchneri GM3701 and Lb. plantarum RB-3 was 216 and 79.0 mg/L, when incubated in 10% glucose media at 25°C for 6 d and 5% glucose media at 25°C for 4 d, respectively. The EPSs consisted of mainly glucose. Bacterial growth in the media supplemented with the EPSs was investigated using various bacteria, including Lactobacillus, Staphylococcus and Escherichia strains. The EPS enhanced the growth of Lb. farciminis HM2001. This result suggests that the growth of some lactic acid bacteria can be enhanced by the supplementation with an EPS produced by Lactobacillus strains. Keywords: exopolysaccharide, growth enhancement, Lactobacillus, yeast extract Ital. J. Food Sci., vol. 31, 2019 - 234 1. INTRODUCTION Exopolysaccharides (EPSs) are long-chain carbohydrate polymers that are released by a wide range of microorganisms, including fungi and bacteria (DONOT et al., 2012). EPSs are present outside of the cell wall, and they exhibit great diversity, not only in their sugar composition but also in their linkage, branching, and substitution (CHAPOT-CHARTIER and KULAKAUSKAS, 2014). EPSs can be bound or unbound to the cell wall, and cell- bound EPSs are distinguished into capsular polysaccharides (CPS) (CAGGIANIELLO et al., 2016). The physiological role of bacterial EPSs is not yet completely understood. EPSs may be associated with cell protection against unfavourable environmental conditions, like desiccation, the presence of oxygen or toxic compounds, low temperatures, high osmotic pressures, and bacteriophage attack, and they may contribute to the uptake of metal ions, biofilm formation, and cell adhesion mechanisms (CAGGIANIELLO et al., 2016; CERNING, 1990; SANCHEZ et al., 2006). On the other hand, LIU et al. (2017) reported that EPSs produced by Lactobacillus plantarum inhibited the biofilm formation of Pseudomonas, Escherichia, Salmonella, and Staphylococcus. Generally, it is thought to be very unlikely that bacteria can use EPSs as an energy source; however, there are some studies that have reported that EPSs are degraded by lactic acid bacteria (LAB). PHAM et al. (2000) reported that EPSs produced by Lactobacillus rhamnosus were degraded by the enzymes of this strain and that some reducing sugars were liberated. Additionally, some Bifidobacterium strains can breakdown plant cell wall polysaccharides (VAN DEN BROEK et al., 2008). Furthermore, the growth of LAB and Bifidobacterium strains were enhanced by supplementing the cultures with the EPS produced by lactobacilli (HONGPATTARAKERE et al., 2012; KORAKLI et al., 2002; RUIJSSENAARS et al., 2000; TSUDA and MIYAMOTO, 2010). It is unclear if this enhancement was because of the utilization of monosaccharides degraded from the EPSs. To begin with, there are only a few reports about the influence of EPSs on the growth of LAB, and more studies are necessary to better understand the influence of EPSs on the growth of LAB. We should point out that crudely purified EPSs may contain mannan from the yeast extract in media, and the mannan may be used by some bacteria. In this study, EPSs, produced by a Lactobacillus strain, were investigated for their yields and monosaccharide components. Furthermore, the influence of EPSs on growth was evaluated using Lactobacillus, Streptococcus, Staphylococcus, Aerococcus, and Escherichia strains. 2. MATERIAL AND METHODS 2.1. Bacterial strains In the present study, 22 bacterial strains, including Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Staphylococcus, Aerococcus, Paenibacillus, and Escherichia coli, were used (Table 1). The strains were isolated from Wagyu milk, Japanese pickles and fermented sushi at our laboratory unless otherwise stated (TSUDA et al., 2012; TSUDA, 2015). The lactic acid bacteria were incubated in TYG broth (10 g/L tryptone, 5.0 g/L yeast extract, 5.0 g/L glucose, 1.0 g/L Tween 80, and 0.1 g/L L-cysteine HCl monohydrate, pH 6.8 ± 0.2). The other strains were also incubated in TYG broth to compare the growth rate. All strains were stored in 10% reconstituted skim milk at -20°C. An inoculum of 1% was used for all tests. Ital. J. Food Sci., vol. 31, 2019 - 235 Table 1. Strains used in the present study. Species Strain No. Source Lactobacillus reuteri PUHM1004 Wagyu milk Lb. coryniformis SAB01 Japanese pickles Lb. sakei subsp. sakei SAB04 Japanese pickles Lb. delbruckii subsp. bulgaricus NBRC 13953 * Lb. alimentarius EM2001 Fermented sushi Lb. casei HM3701 Fermented sushi Lb. buchneri GM3701 Fermented sushi Lb. farciminis HM2001 Fermented sushi Lb. acidipiscis JAM3706 Fermented sushi Lb. plantarum JAB2001 Fermented sushi Lb. plantarum RB3 Japanese pickles Lb. plantarum PUHM1023 Wagyu milk Enterococcus faecalis PUHM1006 Wagyu milk Lactococcus lactis PUHM1014 Wagyu milk Streptococcus thermophilus NBRC 13957 * Str. salivarius AB3002 Fermented sushi Str. pluranimalium PUHM1022 Wagyu milk Aerococcus viridans PUHM5301 Wagyu milk Staphylococcus auricularis PUHM5201 Wagyu milk Sta. aurigulas PUHM5203 Wagyu milk Paenibacillus turicensis PUHM5101 Wagyu milk Escherichia coli NBRC 102203 * *: NBRC: NITE Biological Resource Center. 2.2. Effects of the incubation conditions on exopolysaccharide (EPS) production Lb. buchneri GM3701 and Lb. plantarum RB-3 were used as EPS-producing LAB. The EPS productivities were tested using the 22 strains in Table 1, and the cultures of the two strains showed ropiness. Therefore, strains GM3701 and RB-3 were selected as EPS producers. The ropiness was confirmed by inserting a sterile wire loop and pulling ropes from the media. A clear zone area using the Indian ink method was used as a simplified indicator of the EPS yield. Twenty microliter of LAB culture was put on a glass slide, and a few drops of Indian ink were added and mixed. A cover slip was placed over the mixture, and then, the prepared slide was observed microscopically with immersion oil. A clear zone area for each individual cell was obtained as follows: the cell area was subtracted from the clear zone area, and then, the obtained figures were divided by the cell numbers. This assay was performed with at least 10 clumps. The effect of the incubation temperature on EPS production was investigated at 25, 30, and 37°C. Glucose, fructose, sucrose, and lactose were supplemented in TY broth (10 g/L tryptone, 5.0 g/L yeast extract, 1.0 g/L Tween 80, and 0.1 g/L L-cysteine HCl monohydrate, pH 6.8 ± 0.2) as carbon sources at 25, 50, and 100 g/L. All assays were performed at least three times. Ital. J. Food Sci., vol. 31, 2019 - 236 2.3. Preparation of the EPSs A modified version of the method from LINDSAY et al. (2003) was used to prepare EPSs from bacterial culture. EPSs in the bacterial culture were precipitated with two volumes of cold ethanol, followed by stirring for 1 h at 4°C. The precipitated EPSs were collected by suction filtration, and the collected EPSs were dissolved in deionized water. The EPSs were again precipitated with two volumes of cold ethanol and subsequently lyophilized. Furthermore, EPSs from the TYG broth were prepared using the same method. The lyophilized EPSs were analysed for their carbohydrate and protein content. The total amount of carbohydrates in the lyophilized EPSs was determined with the phenol- sulphuric acid method using glucose as the standard (DUBOIS et al., 1956). The protein content was determined using the protein-dye binding method with bovine serum albumin as the standard (BRADFORD, 1976). All assays were performed at least three times. 2.4. Monosaccharide analysis of the EPSs The lyophilized EPSs were hydrolysed in 2 M trifluoroacetic acid (TFA) at 120°C for 2 h. After hydrolysis, the water and TFA were removed with a centrifugal concentrator (CC- 181, TOMY, Tokyo, Japan), and the dry sample was dissolved in water. The hydrolysed EPS solution was spotted onto silica gel thin layer chromatography plate (Merck, Tokyo, Japan), along with standard solutions for glucose, galactose, mannose, arabinose, xylose, and rhamnose. The plate was developed in 1-butanol:2-propanol:H2O (3:12:4), dried, sprayed with a phenol-sulfuric solution, and then heated at 110°C for 15 min to visualize brown spots (Adachi, 1965; Huebner et al., 2007). The monosaccharides of the EPSs were further analysed as follows. The sugar composition was determined by high pressure liquid chromatography with refractive-index detection (column: Sugar-D (Nacalai tesque, Kyoto, Japan); mobile phase: 85% acetonitrile; flow rate: 1.0 mL/min; temperature: 30°C). Glucose, mannose, N-acetylglucosamine, arabinose, xylose, and rhamnose were used as the standards. 2.5. Influence of the EPSs on bacterial growth Growth in media with EPSs as the sole carbon source was tested with the 22 strains. Glucose medium was used as a control. The growth rate was determined using a modified version of the method from Tsuda and Miyamoto (2010). Briefly, the tested strains were inoculated into TY broth (10 g/L tryptone, 5.0 g/L yeast extract, 1.0 g/L Tween 80, and 0.1 g/L L-cysteine HCl monohydrate, pH 6.8 ± 0.2) containing 0.2% (w/v) glucose and the lyophilized EPSs, and the cultures were incubated for 24 h at 37°C. The optical density at 660 nm (OD660) of the culture was measured at 0 and 24 h. All assays were performed at least three times. The growth rate against glucose was determined using the following equation: Growth rate = (Log OD660 of TYE at 24 h – Log OD660 of TYE at 0 h) / (Log OD660 of TYG at 24 h – Log OD660 of TYG at 0 h) TYE: TY broth containing the EPSs produced by strains GM3701 or RB-3 TYG: TY broth containing glucose Ital. J. Food Sci., vol. 31, 2019 - 237 2.6. Statistical analysis To identify differences, a one-way analysis of variance (ANOVA) was applied to the means, and the Student-Newman-Keuls test (P<0.05) was applied using Statview 5.0 software (SAS Institute, Cary, NC, USA). 3. RESULTS AND CONCLUSIONS 3.1. Effects of the incubation conditions on EPS production Lb. buchneri GM3701 and Lb. plantarum RB-3 are EPS-producing LAB strains. Ropiness was confirmed with a loop, and the clear zone surrounding the cell was confirmed by the Indian ink method. The effects of the incubation temperature and carbon source, which included glucose, fructose, sucrose, and lactose (100 g/L), on EPS production were investigated with Lb. buchneri GM3701 and Lb. plantarum RB-3 (Fig. 1). Concerning strain GM3701, the EPS yield was higher at 25 and 30°C than at 37°C for all of the sugars (P<0.05). There were no differences among the four tested sugars at 25°C, and the EPS yield in the glucose media was higher at 30°C than in the sucrose or lactose media (P<0.05). Concerning strain RB-3, the EPS yield was higher at 25 and 30°C than at 37°C for all of the sugars (P<0.05). The EPS yield in the glucose media was higher than in the sucrose media, and there were no differences among the four tested sugars at 30°C (P<0.05). From these results, it was presumed that a suitable incubation temperature and sugar for EPS production by strains GM3701 and RB-3 were 25°C and glucose, respectively. Therefore, these incubation conditions were applied in the series of tests. Subsequently, the effect of the carbon concentration (25, 50, or 100 g/L) on EPS production was investigated with glucose at 25°C (Fig. 2). EPS production by GM3701 was higher at 100 g/L glucose after 5, 6, and 7 days than at 25 or 50 g/L (P<0.05), and the production by RB-3 was higher at 50 and 100 g/L glucose after 4 days than at 25 g/L (P<0.05). The EPS yields are likely to decrease after reaching a maximum, as many studies have reported, and this is caused by enzymes, such as glycohydrolase, that are produced by bacteria (Pham et al., 2000). However, it is unclear whether the degraded EPSs were used for growth in that paper. 3.2. Characteristics of the EPSs The EPS-producing strain was incubated at the above condition, and then, the EPSs were purified by ethanol precipitation and lyophilized. Similarly, the EPSs from the TYG broth were lyophilized. The polysaccharide (PS) yield from the TYG broth was 50.8 mg/L, and the EPS yields from strains GM3701 and RB-3 were 340 and 146 mg/L, respectively (Table 2). The carbohydrate and protein contents in these lyophilized EPSs are shown in Table 2. All of the EPSs contained more than 76% carbohydrates and less than 9.6% protein. These results confirmed that the lyophilized EPSs were not proteinaceous slime. The monosaccharide analysis of the EPSs was done using seven monosaccharides that are known as constituents of EPSs (glucose, galactose, mannose, N-acetylglucosamine, arabinose, xylose, and rhamnose) as standards. The PSs from the TYG broth consisted mainly of mannose. Ital. J. Food Sci., vol. 31, 2019 - 238 Figure 1. Effects of the incubation temperature (1: 25°C, 2: 30°C, 3: 37°C) and carbon source on EPS production by Lb. buchneri GM3701 (A) and Lb. plantarum RB-3 (B). Bars represent the standard deviation from the mean (n=3). ♦: glucose, ■: fructose, ▲: sucrose, ×: lactose. Ital. J. Food Sci., vol. 31, 2019 - 239 Figure 2. Effect of the glucose concentration on EPS production by L. buchneri GM3701 (A) and L. plantarum RB-3 (B). Bars represent the standard deviation from the mean (n=3). ◊: 25 g/L, □: 50 g/L, ∆: 100 g/L. Table 2. EPS yields and the carbohydrate and protein concentrations in the lyophilized EPSs. Strain EPS yield Carbohydrate Protein Composition of the EPS (%) (mg/L) (%) (%) Glucose Galactose Mannose Rhamnose GM3701 340 76.0 9.6 73.2 - 23.7 trace* RB-3 146 83.2 3.5 27.2 - 58.6 14.1 TYG broth 50.8 83.0 2.8 12.0 - 82.8 - *: trace means less than 10%. The correct yields and monosaccharide components of the EPSs produced by the LAB strains were estimated by subtracting the PS values, while taking the carbohydrate concentration into consideration (Table 3). The calculated EPS yields for the strains GM3701 and RB-3 were 216 and 79.0 mg/L, respectively. Glucose was found to be a major component of the EPS produced by strain GM3701, and glucose, mannose, and rhamnose Ital. J. Food Sci., vol. 31, 2019 - 240 were found to be the predominant sugar residues in the EPS produced by the strain RB-3 (Table 3). Glucose and rhamnose are typical components of many EPSs produced by LAB. The quantities of the hetero-EPSs produced by LAB vary greatly. The production of EPS is 50-350 mg/L for Str. thermophilus, 80-600 mg/L for Lc. lactis subsp. cremoris, 60-150 mg/L for Lb. delbrueckii subsp. bulgaricus, 50-60 mg/L for Lb. casei (CERNING, 1995), and approximately 140 mg/L for Lb. plantarum (STAAF et al., 2000; TSUDA and MIYAMOTO, 2010). The highest recorded yields for hetero-EPSs are 2775 mg/L for Lb. rhamnosus RW- 9595M (MACEDO et al., 2002) and 2500 mg/L for Lb. kefiranofaciens WT-2B (MAEDA et al., 2004). The EPS yields from Lb. buchneri GM3701 and Lb. plantarum RB-3 were 216 and 79.0 mg/L, respectively, and these values were thought to be a normal value for Lactobacillus EPSs. Table 3. EPS yields and monosaccharide components of lyophilized EPSs, obtained by subtracting the values for the EPSs from the TYG broth. Strain EPS yield Composition of the EPS (%) (mg/L) Glucose Mannose Rhamnose GM3701 216 70.9 trace* trace RB-3 79.0 41.7 29.0 28.3 *: trace means less than 10%. The PS from the TYG broth was thought to be mannan. It is well known that purified EPSs are contaminated with the mannan from the yeast cells in yeast extract. Glucose and rhamnose are the usual components of many EPSs produced by LAB (CAGGIANIELLO et al., 2016; DONOT et al., 2012; SANCHEZ et al., 2006), and the mechanisms of glucose incorporation into the polysaccharide chain are well known (DE VUYST et al., 2001). There are some reports about EPSs composed of glucose and mannose that are produced by Lactobacillus (HASHIGUCHI et al., 2011; SANCHEZ et al., 2006). 3.3. Growth enhancement by the EPSs The growth rates of the EPSs against glucose for the 22 strains are shown in Fig. 3. The highest growth rate was observed with Lb. farciminis HM2001 (P<0.05). All of the 22 tested strains showed an OD660 of more than 0.3 when they were incubated in TYG broth for 24 h. The growth rates of the EPSs against glucose were calculated, and all of the tested strains showed a growth rate of less than 0.1, except for strain HM2001. The EPSs produced by strains GM3701 and RB-3 showed growth rates of 0.146 and 0.113 with strain HM2001, respectively. Although the monosaccharide composition of the EPSs was different between the two EPSs (Table 3), the growth of Lb. farciminis HM2001 was enhanced by supplementation with either of the EPS produced by the LAB. The growth enhancement of this strain did not occur following supplementation with the PS from the TYG broth (data not shown). This suggested that the EPSs produced by Lb. buchneri GM3701 and Lb. plantarum RB-3 enhanced the growth of strain HM2001. On the other hand, the EPSs produced by Lb. plantarum RB-3 did not enhance the growth of the three tested Lb. plantarum strains. Ital. J. Food Sci., vol. 31, 2019 - 241 Figure 3. Growth rates of the EPSs against glucose for the 22 strains. Therefore, no species specificity was shown for growth enhancement by EPSs in this study. 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