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ARTICLE Growth and bile tolerance of Lactobacillus brevis strains isolated from Japanese pickles in artificial digestive juices and contribution of cell-bound exopolysaccharide to cell aggregation Shigenori Suzuki, Hiroyuki Honda, Hiroyuki Suganuma, Tadao Saito, and Nobuhiro Yajima
Abstract: Cell-bound exopolysaccharide (EPS) of the aggregable strain Lactobacillus brevis KB290 isolated from traditional Japanese pickles has been reported to protect against the effects of bile. However, there are no reports of bile tolerance mechanisms for other L. brevis strains that have aggregability. To elucidate the mechanism of bile tolerance of L. brevis KB290, we found 8 aggregable L. brevis strains out of 121 L. brevis strains isolated from traditional Japanese fermented pickles. We estimated their growth in artificial digestive juice and the amount of cell-bound EPS. We found 3 types of aggregation for these strains: filiform (5 mm). There was no significant difference in growth between nonaggregable and aggregable strains in the artificial digestive juice. The large floc strains selected from the aggregation strains showed significantly higher growth in the artificial digestive juice than nonaggregable strains. In medium and large floc strains, cell-bound EPS, mainly consisting of glucose, N-acetylglucosamine, and N-acetylmannosamine, were observed. The amount of EPS and each strain’s growth index showed a positive correlation. We conclude that aggregable L. brevis strains were also protected by cell-bound EPS. Key words: Lactobacillus brevis, digestive juice tolerance, cell aggregation, cell-bound exopolysaccharides, monosaccharides. Résumé : On a rapporté que les exopolysaccharides (EPS) associés a` la cellule de la souche agrégable Lactobacillus brevis KB290, isolée de cornichons japonais traditionnels, peuvent protéger contre les effets de la bile. Or, il n’y a aucun rapport portant sur les mécanismes de tolérance a` la bile chez d’autres souches de L. brevis ayant la faculté de s’agréger. Afin d’élucider le mécanisme de tolérance a` la bile chez L. brevis KB290, nous avons repéré 8 souches de L. brevis agrégables sur 121 souches de L. brevis isolées de cornichons japonais traditionnels fermentés. Nous avons estimé leur croissance dans un suc digestif artificiel et mesuré la quantité d’EPS associés a` la cellule. Nous avons constaté 3 types d’agrégation chez ces souches : filiforme (< 1 mm), floculation moyenne (1–5 mm) ou floculation large (> 5 mm). Il n’y avait pas de différence significative entre les souches agrégables et non agrégables au chapitre de la croissance dans le suc digestif artificiel. Les souches agrégables a` floculation large ont poussé plus rapidement que les souches non agrégables dans le suc digestif artificiel. Chez les souches a` floculation moyenne et large, on a constaté que les EPS associés a` la cellule étaient principalement constitués de glucose, N-acétylglucosamine et N-acétylmannosamine. On a établi une corrélation positive entre la quantité d’EPS chez les souches et leur indice de croissance. Nous tirons la conclusion que les souches agrégables de L. brevis sont également protégées par des EPS associés a` la cellule. Mots-clés : Lactobacillus brevis, tolérance au suc digestif, agrégation cellulaire, exopolysaccharides associés a` la cellule, monosaccharides.
Introduction Lactic acid bacteria are found in many environments, including the gastrointestinal tract of animals, dairy and meat products, pickles, soil, and plants. “Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” are called probiotics (Joint FAO/WHO Working Group 2002). Various capabilities are required to confer the probiotic benefits to their host, such as tolerance to low pH, digestive enzymes, and bile salts, to survive passage through the gastrointestinal tract and grow in the gut (Joint FAO/WHO Expert Consultation 2001). Bile, which contains bile salt, cholesterol, phospholipids, and the pigment biliverdin, is synthesized in the liver and secreted into the duodenum (Hofmann 1999). The main function of bile in
the digestive tract is to act as a biological detergent, which emulsifies and solubilizes fats and lipids for absorption (Begley et al. 2005). This function affects not only fats and lipids in foods but also the cell membrane lipid of the intestinal microbiota. Bile dissolves membrane lipids of microorganisms and causes dissociation of integral membrane proteins (Heuman et al. 1996). This solubilization results in leakage of the microorganisms’ contents and death (Begley et al. 2005). This mechanism contributes to a physicochemical defense of the host. In other words, bile tolerance is an essential property of probiotics. Many studies of probiotics have revealed that cellular fatty acid composition (Murga et al. 1999; Kimoto et al. 2002), exopolysaccharide (EPS; Lebeer et al. 2007; Suzuki et al. 2013), and an efflux pump (Gueimonde et al. 2009) are important parts of bile tolerance mechanisms.
Received 28 October 2013. Revision received 14 January 2014. Accepted 14 January 2014. S. Suzuki, H. Honda, H. Suganuma, and N. Yajima. Research and Development Division, Kagome Co., Ltd., 17 Nishitomiyama, Nasushiobara Tochigi, 329-2762, Japan. T. Saito. Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba, Sendai 981-8555, Japan. Corresponding author: Shigenori Suzuki (e-mail: [email protected]). Can. J. Microbiol. 60: 139–145 (2014) dx.doi.org/10.1139/cjm-2013-0774
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Table 1. Pickle information and Lactobacillus brevis strain numbers used in this study.
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Pickle (Japanese name)
Prefecture in Japan
Main materialsa
No. of L. brevis strains
Fermented leaf and stem vegetable Takana-zuke Fukuoka Nozawana-zuke Nagano Asotakana-zuke Kumamoto Shakushina-zuke Saitama Hinona-zuke Shiga
Cruciferous vegetable (Brassica juncea var. integrifolia) and salt (4%–7%) Cruciferous vegetable (Brassica rapa var. hakabura) salt (3%–5%) Cruciferous vegetable (Brassica juncea var. integrifolia) and salt (1%–10%) Green pak choi (Brassica chinensis var. rosularis), salt (3%–5%), and soy sauce Cruciferous vegetables (Brassica rapa var. akana), salt (3%–6%), and rice-bran
19 4 2 2 1
Fermented turnip Suguki Tsudakabu-zuke Akakabu-zuke
Turnip (Brassica rapa var. neosuguki) and salt (3%–5%) Turnip (Brassica rapa var. glabra), salt (2%–5%), and rice-bran Turnip (Brassica rapa var. glabra), cucumber (Cucumis sativus), and salt (3%–5%)
38 5 4
Eggplant (Solanum melongena), Japanese basil (Perilla frutescens var. crispa), and salt (5%–8%) Eggplant, red pepper (Capsicum annuum), and salt (5%–10%)
15
Stem of aroid (Colocasia esculenta), Japanese basil, and salt (3%–5%)
16
Kyoto Shimane Gifu
Fermented eggplant Shiba-zuke Kyoto Pesora-zuke
Yamagata
Fermented stem vegetable Kuki-zuke Mie and Wakayama aInitial
15
salt concentration information was indicated by the pickle providers.
Lactobacillus brevis KB290 is a lactic acid bacterium isolated from traditional Japanese fermented pickles, Suguki, and is well known as a novel probiotic strain (Nobuta et al. 2009; Murakami et al. 2012). Our recent studies revealed that the bile tolerance mechanism of L. brevis KB290 is related to unique aggregability (Fukao et al. 2013) and cell-bound EPS (Suzuki et al. 2013). Because there are no reports on bile acid resistance resulting from aggregation of strains other than KB290, it is not clear whether resistance by EPS is ubiquitous or whether the mechanism is unique to KB290. Elucidation of this issue will provide more information on the bile tolerance mechanism of L. brevis KB290. This may have implications for the use of L. brevis strains isolated from fermented pickles as a probiotics. We investigated L. brevis strains isolated from various traditional Japanese fermented pickles, found the aggregable strains, and studied the contribution of cell-bound EPS to their tolerance of artificial digestive juice.
Estimation of the growth index in the artificial digestive juices The growth index of tester strains in the artificial digestive juice was measured as previously reported (Suzuki et al. 2013). Each tester strain culture (1 mL) was added to a tube filled with artificial gastric juice (9 mL) and kept at 37 °C for 3 h. The reaction mixture (3 mL) was dispensed into tubes filled with artificial intestinal juice (9 mL) and incubated at 37 °C for 7 h. For bacterial colony counts, each tester strain culture and digested sample was serially diluted (10-fold) with dilution solution (saline containing 0.1% (m/v) agar), poured into MRS agar (OXOID), and incubated at 30 °C for 48 h. We calculated the growth index as previously reported (Suzuki et al. 2013):
Materials and methods
Estimation of the survivability of strains in bile salt We estimated the survival of KB290, KB392, and ethylenediaminetetraacetic acid (EDTA)-treated strains in bile salts as previously reported (Suzuki et al. 2013). Briefly, we centrifuged the cultures (8000g, 10 min, 5 °C), washed the precipitates twice with sterile saline, and resuspended them well in sterile phosphatebuffered saline (PBS; Nissui, Tokyo, Japan) or EDTA (Dojindo, Kumamoto, Japan) solution. The mixture was gently stirred for 4 h at 5 °C. The mixture was centrifuged as before, suspended in 10 mL of PBS, and then centrifuged again. The precipitates were washed in PBS, suspended in filtered (pore size, 0.20 m) PBS with or without 0.2% (m/v) bile salts, and incubated for 3 h at 37 °C. The colonies were counted as aforementioned. Survivability was calculated as follows:
Bacterial strains and culture conditions A total of 121 L. brevis strains isolated from traditional Japanese fermented pickles were used from our stock culture collection (Table 1). All strains had been identified as L. brevis by 16S rRNA sequencing and carbohydrate fermentation. Lactobacillus brevis JCM1059T (isolated from human feces) and L. brevis KB290 (isolated from Suguki; deposited into JCM as JCM17312) were also used as negative and positive controls, respectively. We cultured all strains at 30 °C in de Man – Rogosa – Sharpe (MRS) medium (Oxoid, Hampshire, UK) and stored them at –80 °C in MRS medium containing 15% (v/v) glycerol (Wako, Tokyo, Japan) until use. We thawed the frozen MRS culture containing 15% (v/v) glycerol to 30 °C, inoculated the strains at 1% (v/v) in fresh MRS medium for subcultures, and then cultured them again in the same way for 24 h. Assessment of aggregability Aggregability was assayed as previously reported (Fukao et al. 2013) with minor modifications. Aggregability of the tester strain was judged when the cells became agglomerated after vigorous mixing for 10 s in MRS medium using an agglomerator mixer (Se-08: TAITEC, Saitama, Japan). After stirring, we poured the mixed culture onto a blank plate, and observed it on a color illuminator (Fujifilm, Tokyo, Japan). We used L. brevis JCM1059T and KB290 as references. We defined aggregation floc size as follows: filiform, 5 mm.
Growth index ⫽ (bacterial count after incubation with artificial intestinal juice × 4)/ (bacterial count of strain culture × 0.1)
Survivability ⫽ bacterial count after incubation with bile salts/bacterial count after incubation with PBS EPS extraction, purification, and analysis We extracted cell-bound EPS (EPS-b) from the bacterial surface as previously described (Suzuki et al. 2013). Briefly, we centrifuged 100 mL of the cultured strain (8000g, 10 min, 5 °C) and washed the precipitate twice with sterile saline. The precipitate was well suspended in 50 mL of 50 mmol/L EDTA and stirred gently for 4 h at 5 °C. After EDTA treatment, we added 2 volumes of chilled ethanol (Wako) to the supernatant, kept the mixture at 5 °C overnight, and centrifuged (16 000g, 30 min, 5 °C). The precipitate was Published by NRC Research Press
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Fig. 1. Observation of aggregation floc of Lactobacillus brevis sp. strains after mixing. (A) Filiform floc aggregation, (B) medium floc aggregation, and (C) large floc aggregation.
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Fig. 2. Growth indices of Lactobacillus brevis strains used in this study. (A) Nonaggregable strains (113 strains) and aggregable strains (8 strains); (B) nonaggregable strains (113 strains) and large floc (4 strains). Columns are expressed as means, and error bars represent standard deviations.
Fig. 3. Survivability of tester strains with (open bars) or without (closed bars) EDTA (ethylenediaminetetraacetic acid) treatment in bile salts. Columns are expressed as means, and error bars represent standard deviations (n = 3). *, indicates a significant difference between JCM1059T and tester strains (P < 0.05). †, indicates a significant difference between untreated and EDTA treated (P < 0.05).
suspended in 10 mL of distilled water and dialyzed against 3 L of distilled water using a 6–8 kDa dialysis membrane (Spectra/Por, VWR International, Radnor, Pennsylvania, USA) for 2 days with 3 water changes per day. We lyophilized the dialysate and analyzed the constituent sugars using a 4-aminobenzoic acid ethyl ester labeling kit (J-Oil Mills, Tokyo, Japan) (Yasuno et al. 1997) according to the manufacturer’s instructions. We estimated the total amount of EPS carbohydrate using a phenol – sulfuric acid method (Dubois et al. 1956). We expressed the results as glucose equivalents per 109 culture strain cells. Statistical analysis Statistical analysis was performed with SPSS (IBM, version 15.0J for windows, SPSS, Inc., Chicago, Illinois, USA), and statistical significance was accepted at the P < 0.05 level of probability. Estimation of the growth index in the artificial digestive juices was analyzed using Student’s t test, and correlation between EPS amount and growth index was tested using Spearman’s rank correlation.
Results Aggregability of L. brevis Lactobacillus brevis JCM1059T showed no aggregability, and KB290 showed large floc aggregation (Fig. 1). Aggregability was shown by L. brevis KB1131, KB1140, and KB1145 from Suguki (fermented turnip), and also by strains obtained from other kinds of Japanese pickle: KB1282 from Pesora-zuke (fermented eggplant), KB1388 and KB1389 from Shakushina-zuke, and KB1567 and KB1584 from Tsudakabu-zuke (both fermented leaf and stem vegetable; Figs. 1A–1C). Among these strains, KB1131 and KB1140 formed filiform aggregation (Fig. 1A), KB1145 and KB1567 formed medium floc aggregation (Fig. 1B), and KB1282, KB1388, KB1389, and KB1584 formed large floc aggregation (Fig. 1C). Growth index of nonaggregable and aggregable L. brevis in artificial digestive juice The growth index means of 113 nonaggregable strains and 8 aggregable strains (Figs. 1A–1C) were 2.22 ± 1.04 and 2.82 ± 0.83, Published by NRC Research Press
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Fig. 4. High-performance liquid chromatography (HPLC) chromatogram of hydrolyzed cell-bound exopolysaccharides extracted from aggregable strains. Monosaccharide standards: 1, galactose; 2, mannose; 3, glucose; 4, N-acetylmannosamine; 5, N-acetylglucosamine; 6, fucose; 7, N-acetylgalactosamine.
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Table 2. Found aggregable Lactobacillus brevis strains. Strain
Isolated from:
Aggregation form
Growth indexa
KB1140 KB1131 KB1145 KB1567 KB1282 KB1584 KB1388 KB1389 KB290 JCM1059T
Suguki Suguki Suguki Tsudakabu-zuke Pesora-zuke Tsudakabu-zuke Shakushina-zuke Shakushina-zuke Positive control Negative control
Filiform Filiform Medium floc Medium floc Large floc Large floc Large floc Large floc Large floc Nonaggregation
1.93 2.13 2.28 2.52 2.58 2.99 3.95 4.20 2.32 1.48
aValues
are the means of 2 independent experiments.
Fig. 5. Correlation between exopolysaccharide (EPS) amounts and growth indices of aggregable Lactobacillus brevis strains (r = 0.78, Spearman, P < 0.05).
respectively (P = 0.086, Fig. 2A), although there was a statistically significant difference between means of these nonaggregable strains and 4 large floc aggregation strains (Fig. 1C) (P < 0.05, Fig. 2B, Table 2). Bile tolerance All large floc aggregation strains showed significantly higher survivability than L. brevis JCM1059T in bile salts, but EDTA treatment reduced the bile tolerance of all strains except for JCM1059T (Fig. 3). EPS analysis Monosaccharide constituents of EPS-b extracted from the aggregable L. brevis strains were analyzed. The chromatograms of hydrolyzed EPS-b showed 2 major peaks for L. brevis KB290 corresponding to glucose and N-acetylglucosamine (Fig. 4). The chromatograms showed 1 major peak for L. brevis KB1131 and KB1140, corresponding to glucose; however, other aggregable strains showed 2 or more major peaks corresponding to glucose, N-acetylglucosamine, and N-acetylmannosamine (Fig. 4). No EPS-b was extracted from JCM1059T (data not shown). Correlation between EPS-b amount and growth index The crude EPS-b amounts and growth indices of 9 aggregable strains (2 filiform strains, 2 medium floc aggregation strains,
4 large floc aggregation strains, and L. brevis KB290) in MRS showed a significant positive correlation (Fig. 5, r = 0.78, P < 0.05).
Discussion Lactobacillus brevis strains are often isolated from pickles, dairy products, and animal feces (Hammes and Hertel 2009). However, there is no information about the aggregability by vigorous mixing of L. brevis. In this study, we successfully found 8 aggregable strains from 121 L. brevis strains isolated from traditional Japanese fermented pickles. The aggregable strains were isolated not only from Suguki, which was reported as the isolation source of L. brevis KB290 (Kishi et al. 1996), but also from other pickles, such as fermented eggplant, fermented leaf and stem vegetable, and fermented turnip, which were from different production areas. These results suggested that aggregable L. brevis strains exist universally in other fermented pickles. In this study, we found 3 types of aggregation forms: large floc type similar to L. brevis KB290, medium floc, and filiform type. So we also considered that the 3 types of aggregation might bring about the growth differences in the artificial digestive juice as well as EPS production and EPS constitution of these strains. EPS enveloping the cell has been reported as protective toward bile for Bifidobacterium animalis (Ruas-madiedo et al. 2009), intestinal lactobacilli (Stack et al. 2010), and L. rhamnosus (Koskenniemi Published by NRC Research Press
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et al. 2011). We previously reported that plasmid pKB290-1 was involved in cell aggregation, growth in artificial digestive juice, and EPS-b production by KB290 (Fukao et al. 2013; Suzuki et al. 2013). Removal of EPS-b by EDTA or homogenization made KB290 sensitive to bile salt and artificial digestive juice (Suzuki et al. 2013). The mean growth index for large floc aggregation strains was significantly higher than for nonaggregation strains. In addition, EDTA treatment made large floc aggregation strains sensitive to bile salt, and the growth index of aggregation strains appeared to be dependent on floc size. Thus, we considered that EPS-b contributed to bile tolerance in aggregable L. brevis strains. Furthermore, constituents and amount of EPS might differ with aggregation type. In some bacteria it has been reported that cell-surface EPS plays a role in cell aggregation (Bahat-Samet et al. 2004). We already reported that EPS-b of L. brevis KB290 contained glucose and N-acetylglucosamine (approximately 1:1) (Suzuki et al. 2013) and that pKB290-1 was involved in the aggregation of KB290 (Fukao et al. 2013). In this study, EPS-b of filiform aggregation strains showed lower N-acetylglucosamine and N-acetylmannosamine ratios (less than 20% glucose ratio) than other aggregation strains. In contrast, medium and large floc aggregation strains showed higher N-acetylglucosamine and N-acetylmannosamine ratios (more than 100% glucose ratio). These results suggest that aggregation type might be controlled by the presence of acetylated monosaccharide. Koskenniemi et al. (2011) suggested that bile stress induced EPS production and resulted in a thicker EPS layer in Lactobacillus rhamnosus GG. Transmission electron microscopy observations revealed that EPS-b enveloped KB290 and that EDTA and homogenization removed the EPS-b from the cell (Suzuki et al. 2013). We considered that the amount of EPS-b might affect growth in the artificial digestive juice. Furthermore, the amount of EPS-b might be regulated by the amounts of N-acetylglucosamine and N-acetylmannosamine. Future studies on the EPS could provide more insight into the aggregation mechanisms of the L. brevis strains. We conclude that aggregable L. brevis strains from traditional Japanese fermented pickles were protected from bile by cellbound EPS and that the aggregation form might be regulated by constituent monosaccharides. For further clarification of bile tolerance mechanisms of L. brevis strains, another well-known bile tolerance mechanism such as cell membrane fatty acid composition (Begley et al. 2005) might be considered.
Acknowledgements We are grateful to Kunihiko Satoh (Kagome Co., Ltd.) for his valuable support. We thank Takuro Inoue and Erika Sasaki (Kagome Co., Ltd.) for their technical assistance.
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ARTICLE Growth and bile tolerance of Lactobacillus brevis strains isolated from Japanese pickles in artificial digestive juices and contribution of cell-bound exopolysaccharide to cell aggregation Shigenori Suzuki, Hiroyuki Honda, Hiroyuki Suganuma, Tadao Saito, and Nobuhiro Yajima
Abstract: Cell-bound exopolysaccharide (EPS) of the aggregable strain Lactobacillus brevis KB290 isolated from traditional Japanese pickles has been reported to protect against the effects of bile. However, there are no reports of bile tolerance mechanisms for other L. brevis strains that have aggregability. To elucidate the mechanism of bile tolerance of L. brevis KB290, we found 8 aggregable L. brevis strains out of 121 L. brevis strains isolated from traditional Japanese fermented pickles. We estimated their growth in artificial digestive juice and the amount of cell-bound EPS. We found 3 types of aggregation for these strains: filiform (5 mm). There was no significant difference in growth between nonaggregable and aggregable strains in the artificial digestive juice. The large floc strains selected from the aggregation strains showed significantly higher growth in the artificial digestive juice than nonaggregable strains. In medium and large floc strains, cell-bound EPS, mainly consisting of glucose, N-acetylglucosamine, and N-acetylmannosamine, were observed. The amount of EPS and each strain’s growth index showed a positive correlation. We conclude that aggregable L. brevis strains were also protected by cell-bound EPS. Key words: Lactobacillus brevis, digestive juice tolerance, cell aggregation, cell-bound exopolysaccharides, monosaccharides. Résumé : On a rapporté que les exopolysaccharides (EPS) associés a` la cellule de la souche agrégable Lactobacillus brevis KB290, isolée de cornichons japonais traditionnels, peuvent protéger contre les effets de la bile. Or, il n’y a aucun rapport portant sur les mécanismes de tolérance a` la bile chez d’autres souches de L. brevis ayant la faculté de s’agréger. Afin d’élucider le mécanisme de tolérance a` la bile chez L. brevis KB290, nous avons repéré 8 souches de L. brevis agrégables sur 121 souches de L. brevis isolées de cornichons japonais traditionnels fermentés. Nous avons estimé leur croissance dans un suc digestif artificiel et mesuré la quantité d’EPS associés a` la cellule. Nous avons constaté 3 types d’agrégation chez ces souches : filiforme (< 1 mm), floculation moyenne (1–5 mm) ou floculation large (> 5 mm). Il n’y avait pas de différence significative entre les souches agrégables et non agrégables au chapitre de la croissance dans le suc digestif artificiel. Les souches agrégables a` floculation large ont poussé plus rapidement que les souches non agrégables dans le suc digestif artificiel. Chez les souches a` floculation moyenne et large, on a constaté que les EPS associés a` la cellule étaient principalement constitués de glucose, N-acétylglucosamine et N-acétylmannosamine. On a établi une corrélation positive entre la quantité d’EPS chez les souches et leur indice de croissance. Nous tirons la conclusion que les souches agrégables de L. brevis sont également protégées par des EPS associés a` la cellule. Mots-clés : Lactobacillus brevis, tolérance au suc digestif, agrégation cellulaire, exopolysaccharides associés a` la cellule, monosaccharides.
Introduction Lactic acid bacteria are found in many environments, including the gastrointestinal tract of animals, dairy and meat products, pickles, soil, and plants. “Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” are called probiotics (Joint FAO/WHO Working Group 2002). Various capabilities are required to confer the probiotic benefits to their host, such as tolerance to low pH, digestive enzymes, and bile salts, to survive passage through the gastrointestinal tract and grow in the gut (Joint FAO/WHO Expert Consultation 2001). Bile, which contains bile salt, cholesterol, phospholipids, and the pigment biliverdin, is synthesized in the liver and secreted into the duodenum (Hofmann 1999). The main function of bile in
the digestive tract is to act as a biological detergent, which emulsifies and solubilizes fats and lipids for absorption (Begley et al. 2005). This function affects not only fats and lipids in foods but also the cell membrane lipid of the intestinal microbiota. Bile dissolves membrane lipids of microorganisms and causes dissociation of integral membrane proteins (Heuman et al. 1996). This solubilization results in leakage of the microorganisms’ contents and death (Begley et al. 2005). This mechanism contributes to a physicochemical defense of the host. In other words, bile tolerance is an essential property of probiotics. Many studies of probiotics have revealed that cellular fatty acid composition (Murga et al. 1999; Kimoto et al. 2002), exopolysaccharide (EPS; Lebeer et al. 2007; Suzuki et al. 2013), and an efflux pump (Gueimonde et al. 2009) are important parts of bile tolerance mechanisms.
Received 28 October 2013. Revision received 14 January 2014. Accepted 14 January 2014. S. Suzuki, H. Honda, H. Suganuma, and N. Yajima. Research and Development Division, Kagome Co., Ltd., 17 Nishitomiyama, Nasushiobara Tochigi, 329-2762, Japan. T. Saito. Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba, Sendai 981-8555, Japan. Corresponding author: Shigenori Suzuki (e-mail: [email protected]). Can. J. Microbiol. 60: 139–145 (2014) dx.doi.org/10.1139/cjm-2013-0774
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Table 1. Pickle information and Lactobacillus brevis strain numbers used in this study.
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Pickle (Japanese name)
Prefecture in Japan
Main materialsa
No. of L. brevis strains
Fermented leaf and stem vegetable Takana-zuke Fukuoka Nozawana-zuke Nagano Asotakana-zuke Kumamoto Shakushina-zuke Saitama Hinona-zuke Shiga
Cruciferous vegetable (Brassica juncea var. integrifolia) and salt (4%–7%) Cruciferous vegetable (Brassica rapa var. hakabura) salt (3%–5%) Cruciferous vegetable (Brassica juncea var. integrifolia) and salt (1%–10%) Green pak choi (Brassica chinensis var. rosularis), salt (3%–5%), and soy sauce Cruciferous vegetables (Brassica rapa var. akana), salt (3%–6%), and rice-bran
19 4 2 2 1
Fermented turnip Suguki Tsudakabu-zuke Akakabu-zuke
Turnip (Brassica rapa var. neosuguki) and salt (3%–5%) Turnip (Brassica rapa var. glabra), salt (2%–5%), and rice-bran Turnip (Brassica rapa var. glabra), cucumber (Cucumis sativus), and salt (3%–5%)
38 5 4
Eggplant (Solanum melongena), Japanese basil (Perilla frutescens var. crispa), and salt (5%–8%) Eggplant, red pepper (Capsicum annuum), and salt (5%–10%)
15
Stem of aroid (Colocasia esculenta), Japanese basil, and salt (3%–5%)
16
Kyoto Shimane Gifu
Fermented eggplant Shiba-zuke Kyoto Pesora-zuke
Yamagata
Fermented stem vegetable Kuki-zuke Mie and Wakayama aInitial
15
salt concentration information was indicated by the pickle providers.
Lactobacillus brevis KB290 is a lactic acid bacterium isolated from traditional Japanese fermented pickles, Suguki, and is well known as a novel probiotic strain (Nobuta et al. 2009; Murakami et al. 2012). Our recent studies revealed that the bile tolerance mechanism of L. brevis KB290 is related to unique aggregability (Fukao et al. 2013) and cell-bound EPS (Suzuki et al. 2013). Because there are no reports on bile acid resistance resulting from aggregation of strains other than KB290, it is not clear whether resistance by EPS is ubiquitous or whether the mechanism is unique to KB290. Elucidation of this issue will provide more information on the bile tolerance mechanism of L. brevis KB290. This may have implications for the use of L. brevis strains isolated from fermented pickles as a probiotics. We investigated L. brevis strains isolated from various traditional Japanese fermented pickles, found the aggregable strains, and studied the contribution of cell-bound EPS to their tolerance of artificial digestive juice.
Estimation of the growth index in the artificial digestive juices The growth index of tester strains in the artificial digestive juice was measured as previously reported (Suzuki et al. 2013). Each tester strain culture (1 mL) was added to a tube filled with artificial gastric juice (9 mL) and kept at 37 °C for 3 h. The reaction mixture (3 mL) was dispensed into tubes filled with artificial intestinal juice (9 mL) and incubated at 37 °C for 7 h. For bacterial colony counts, each tester strain culture and digested sample was serially diluted (10-fold) with dilution solution (saline containing 0.1% (m/v) agar), poured into MRS agar (OXOID), and incubated at 30 °C for 48 h. We calculated the growth index as previously reported (Suzuki et al. 2013):
Materials and methods
Estimation of the survivability of strains in bile salt We estimated the survival of KB290, KB392, and ethylenediaminetetraacetic acid (EDTA)-treated strains in bile salts as previously reported (Suzuki et al. 2013). Briefly, we centrifuged the cultures (8000g, 10 min, 5 °C), washed the precipitates twice with sterile saline, and resuspended them well in sterile phosphatebuffered saline (PBS; Nissui, Tokyo, Japan) or EDTA (Dojindo, Kumamoto, Japan) solution. The mixture was gently stirred for 4 h at 5 °C. The mixture was centrifuged as before, suspended in 10 mL of PBS, and then centrifuged again. The precipitates were washed in PBS, suspended in filtered (pore size, 0.20 m) PBS with or without 0.2% (m/v) bile salts, and incubated for 3 h at 37 °C. The colonies were counted as aforementioned. Survivability was calculated as follows:
Bacterial strains and culture conditions A total of 121 L. brevis strains isolated from traditional Japanese fermented pickles were used from our stock culture collection (Table 1). All strains had been identified as L. brevis by 16S rRNA sequencing and carbohydrate fermentation. Lactobacillus brevis JCM1059T (isolated from human feces) and L. brevis KB290 (isolated from Suguki; deposited into JCM as JCM17312) were also used as negative and positive controls, respectively. We cultured all strains at 30 °C in de Man – Rogosa – Sharpe (MRS) medium (Oxoid, Hampshire, UK) and stored them at –80 °C in MRS medium containing 15% (v/v) glycerol (Wako, Tokyo, Japan) until use. We thawed the frozen MRS culture containing 15% (v/v) glycerol to 30 °C, inoculated the strains at 1% (v/v) in fresh MRS medium for subcultures, and then cultured them again in the same way for 24 h. Assessment of aggregability Aggregability was assayed as previously reported (Fukao et al. 2013) with minor modifications. Aggregability of the tester strain was judged when the cells became agglomerated after vigorous mixing for 10 s in MRS medium using an agglomerator mixer (Se-08: TAITEC, Saitama, Japan). After stirring, we poured the mixed culture onto a blank plate, and observed it on a color illuminator (Fujifilm, Tokyo, Japan). We used L. brevis JCM1059T and KB290 as references. We defined aggregation floc size as follows: filiform, 5 mm.
Growth index ⫽ (bacterial count after incubation with artificial intestinal juice × 4)/ (bacterial count of strain culture × 0.1)
Survivability ⫽ bacterial count after incubation with bile salts/bacterial count after incubation with PBS EPS extraction, purification, and analysis We extracted cell-bound EPS (EPS-b) from the bacterial surface as previously described (Suzuki et al. 2013). Briefly, we centrifuged 100 mL of the cultured strain (8000g, 10 min, 5 °C) and washed the precipitate twice with sterile saline. The precipitate was well suspended in 50 mL of 50 mmol/L EDTA and stirred gently for 4 h at 5 °C. After EDTA treatment, we added 2 volumes of chilled ethanol (Wako) to the supernatant, kept the mixture at 5 °C overnight, and centrifuged (16 000g, 30 min, 5 °C). The precipitate was Published by NRC Research Press
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Fig. 1. Observation of aggregation floc of Lactobacillus brevis sp. strains after mixing. (A) Filiform floc aggregation, (B) medium floc aggregation, and (C) large floc aggregation.
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Fig. 2. Growth indices of Lactobacillus brevis strains used in this study. (A) Nonaggregable strains (113 strains) and aggregable strains (8 strains); (B) nonaggregable strains (113 strains) and large floc (4 strains). Columns are expressed as means, and error bars represent standard deviations.
Fig. 3. Survivability of tester strains with (open bars) or without (closed bars) EDTA (ethylenediaminetetraacetic acid) treatment in bile salts. Columns are expressed as means, and error bars represent standard deviations (n = 3). *, indicates a significant difference between JCM1059T and tester strains (P < 0.05). †, indicates a significant difference between untreated and EDTA treated (P < 0.05).
suspended in 10 mL of distilled water and dialyzed against 3 L of distilled water using a 6–8 kDa dialysis membrane (Spectra/Por, VWR International, Radnor, Pennsylvania, USA) for 2 days with 3 water changes per day. We lyophilized the dialysate and analyzed the constituent sugars using a 4-aminobenzoic acid ethyl ester labeling kit (J-Oil Mills, Tokyo, Japan) (Yasuno et al. 1997) according to the manufacturer’s instructions. We estimated the total amount of EPS carbohydrate using a phenol – sulfuric acid method (Dubois et al. 1956). We expressed the results as glucose equivalents per 109 culture strain cells. Statistical analysis Statistical analysis was performed with SPSS (IBM, version 15.0J for windows, SPSS, Inc., Chicago, Illinois, USA), and statistical significance was accepted at the P < 0.05 level of probability. Estimation of the growth index in the artificial digestive juices was analyzed using Student’s t test, and correlation between EPS amount and growth index was tested using Spearman’s rank correlation.
Results Aggregability of L. brevis Lactobacillus brevis JCM1059T showed no aggregability, and KB290 showed large floc aggregation (Fig. 1). Aggregability was shown by L. brevis KB1131, KB1140, and KB1145 from Suguki (fermented turnip), and also by strains obtained from other kinds of Japanese pickle: KB1282 from Pesora-zuke (fermented eggplant), KB1388 and KB1389 from Shakushina-zuke, and KB1567 and KB1584 from Tsudakabu-zuke (both fermented leaf and stem vegetable; Figs. 1A–1C). Among these strains, KB1131 and KB1140 formed filiform aggregation (Fig. 1A), KB1145 and KB1567 formed medium floc aggregation (Fig. 1B), and KB1282, KB1388, KB1389, and KB1584 formed large floc aggregation (Fig. 1C). Growth index of nonaggregable and aggregable L. brevis in artificial digestive juice The growth index means of 113 nonaggregable strains and 8 aggregable strains (Figs. 1A–1C) were 2.22 ± 1.04 and 2.82 ± 0.83, Published by NRC Research Press
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Fig. 4. High-performance liquid chromatography (HPLC) chromatogram of hydrolyzed cell-bound exopolysaccharides extracted from aggregable strains. Monosaccharide standards: 1, galactose; 2, mannose; 3, glucose; 4, N-acetylmannosamine; 5, N-acetylglucosamine; 6, fucose; 7, N-acetylgalactosamine.
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Table 2. Found aggregable Lactobacillus brevis strains. Strain
Isolated from:
Aggregation form
Growth indexa
KB1140 KB1131 KB1145 KB1567 KB1282 KB1584 KB1388 KB1389 KB290 JCM1059T
Suguki Suguki Suguki Tsudakabu-zuke Pesora-zuke Tsudakabu-zuke Shakushina-zuke Shakushina-zuke Positive control Negative control
Filiform Filiform Medium floc Medium floc Large floc Large floc Large floc Large floc Large floc Nonaggregation
1.93 2.13 2.28 2.52 2.58 2.99 3.95 4.20 2.32 1.48
aValues
are the means of 2 independent experiments.
Fig. 5. Correlation between exopolysaccharide (EPS) amounts and growth indices of aggregable Lactobacillus brevis strains (r = 0.78, Spearman, P < 0.05).
respectively (P = 0.086, Fig. 2A), although there was a statistically significant difference between means of these nonaggregable strains and 4 large floc aggregation strains (Fig. 1C) (P < 0.05, Fig. 2B, Table 2). Bile tolerance All large floc aggregation strains showed significantly higher survivability than L. brevis JCM1059T in bile salts, but EDTA treatment reduced the bile tolerance of all strains except for JCM1059T (Fig. 3). EPS analysis Monosaccharide constituents of EPS-b extracted from the aggregable L. brevis strains were analyzed. The chromatograms of hydrolyzed EPS-b showed 2 major peaks for L. brevis KB290 corresponding to glucose and N-acetylglucosamine (Fig. 4). The chromatograms showed 1 major peak for L. brevis KB1131 and KB1140, corresponding to glucose; however, other aggregable strains showed 2 or more major peaks corresponding to glucose, N-acetylglucosamine, and N-acetylmannosamine (Fig. 4). No EPS-b was extracted from JCM1059T (data not shown). Correlation between EPS-b amount and growth index The crude EPS-b amounts and growth indices of 9 aggregable strains (2 filiform strains, 2 medium floc aggregation strains,
4 large floc aggregation strains, and L. brevis KB290) in MRS showed a significant positive correlation (Fig. 5, r = 0.78, P < 0.05).
Discussion Lactobacillus brevis strains are often isolated from pickles, dairy products, and animal feces (Hammes and Hertel 2009). However, there is no information about the aggregability by vigorous mixing of L. brevis. In this study, we successfully found 8 aggregable strains from 121 L. brevis strains isolated from traditional Japanese fermented pickles. The aggregable strains were isolated not only from Suguki, which was reported as the isolation source of L. brevis KB290 (Kishi et al. 1996), but also from other pickles, such as fermented eggplant, fermented leaf and stem vegetable, and fermented turnip, which were from different production areas. These results suggested that aggregable L. brevis strains exist universally in other fermented pickles. In this study, we found 3 types of aggregation forms: large floc type similar to L. brevis KB290, medium floc, and filiform type. So we also considered that the 3 types of aggregation might bring about the growth differences in the artificial digestive juice as well as EPS production and EPS constitution of these strains. EPS enveloping the cell has been reported as protective toward bile for Bifidobacterium animalis (Ruas-madiedo et al. 2009), intestinal lactobacilli (Stack et al. 2010), and L. rhamnosus (Koskenniemi Published by NRC Research Press
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et al. 2011). We previously reported that plasmid pKB290-1 was involved in cell aggregation, growth in artificial digestive juice, and EPS-b production by KB290 (Fukao et al. 2013; Suzuki et al. 2013). Removal of EPS-b by EDTA or homogenization made KB290 sensitive to bile salt and artificial digestive juice (Suzuki et al. 2013). The mean growth index for large floc aggregation strains was significantly higher than for nonaggregation strains. In addition, EDTA treatment made large floc aggregation strains sensitive to bile salt, and the growth index of aggregation strains appeared to be dependent on floc size. Thus, we considered that EPS-b contributed to bile tolerance in aggregable L. brevis strains. Furthermore, constituents and amount of EPS might differ with aggregation type. In some bacteria it has been reported that cell-surface EPS plays a role in cell aggregation (Bahat-Samet et al. 2004). We already reported that EPS-b of L. brevis KB290 contained glucose and N-acetylglucosamine (approximately 1:1) (Suzuki et al. 2013) and that pKB290-1 was involved in the aggregation of KB290 (Fukao et al. 2013). In this study, EPS-b of filiform aggregation strains showed lower N-acetylglucosamine and N-acetylmannosamine ratios (less than 20% glucose ratio) than other aggregation strains. In contrast, medium and large floc aggregation strains showed higher N-acetylglucosamine and N-acetylmannosamine ratios (more than 100% glucose ratio). These results suggest that aggregation type might be controlled by the presence of acetylated monosaccharide. Koskenniemi et al. (2011) suggested that bile stress induced EPS production and resulted in a thicker EPS layer in Lactobacillus rhamnosus GG. Transmission electron microscopy observations revealed that EPS-b enveloped KB290 and that EDTA and homogenization removed the EPS-b from the cell (Suzuki et al. 2013). We considered that the amount of EPS-b might affect growth in the artificial digestive juice. Furthermore, the amount of EPS-b might be regulated by the amounts of N-acetylglucosamine and N-acetylmannosamine. Future studies on the EPS could provide more insight into the aggregation mechanisms of the L. brevis strains. We conclude that aggregable L. brevis strains from traditional Japanese fermented pickles were protected from bile by cellbound EPS and that the aggregation form might be regulated by constituent monosaccharides. For further clarification of bile tolerance mechanisms of L. brevis strains, another well-known bile tolerance mechanism such as cell membrane fatty acid composition (Begley et al. 2005) might be considered.
Acknowledgements We are grateful to Kunihiko Satoh (Kagome Co., Ltd.) for his valuable support. We thank Takuro Inoue and Erika Sasaki (Kagome Co., Ltd.) for their technical assistance.
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