Cellular fatty acid composition and exopolysaccharide contribute to bile tolerance in Lactobacillus brevis strains isolated from fermented Japanese pickles.

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Cellular fatty acid composition and exopolysaccharide contribute to bile tolerance in Lactobacillus brevis strains isolated from fermented Japanese pickles Shigenori Suzuki, Hiromi Kimoto-Nira, Hiroyuki Suganuma, Chise Suzuki, Tadao Saito, and Nobuhiro Yajima

Abstract: Bile tolerance is a fundamental ability of probiotic bacteria. We examined this property in 56 Lactobacillus brevis strains isolated from Japanese pickles and also evaluated cellular fatty acid composition and cell-bound exopolysaccharide (EPS-b) production. The bile tolerance of these strains was significantly lower in modified de Man – Rogosa – Sharpe (MRS) medium (without Tween 80 or sodium acetate) than in standard MRS medium. Aggregating strains showed significantly higher bile tolerance than nonaggregating strains in MRS medium, but there was no significant difference in the modified MRS media. The relative octadecenoic acid (C18:1) content of the 3 most tolerant aggregating and nonaggregating strains was significantly higher when bile was added to MRS. In MRS without Tween 80, the relative C18:1 content was only marginally affected by addition of bile. In MRS without sodium acetate, only the 3 most tolerant nonaggregating strains increased their relative C18:1 content in the presence of bile. Meanwhile, culture in MRS without sodium acetate reduced EPS-b production in aggregating strains. In conclusion, both EPS-b and cellular fatty acid composition play important roles in bile tolerance of pickle-derived L. brevis. Key words: Lactobacillus brevis, bile tolerance, fatty acid composition, exopolysaccharide. Résumé : La tolérance a` la bile est un attribut fondamental des bactéries probiotiques. Nous avons examiné cet attribut chez 56 souches de Lactobacillus brevis isolées de cornichons japonais, et avons évalué leur composition cellulaire en acides gras et leur production d’exopolysaccharides associés a` la cellule (EPS-b). La tolérance a` la bile chez ces souches était significativement inférieure dans un milieu de Man – Rogosa – Sharpe (MRS) modifié (sans Tween 80 ni acétate de sodium) par rapport a` un milieu MRS normal. Les souches agrégables étaient significativement plus tolérantes a` la bile que les souches non agrégables dans le milieu MRS, mais aucune différence n’a été relevée dans le milieu MRS modifié. Le contenu relatif en acide octadécènoïque (C18:1) des trois souches agrégables et non agrégables les plus tolérantes était significativement plus élevé lorsque l’on ajoutait de la bile au MRS. Dans du MRS sans Tween 80, le contenu relatif C18:1 n’a été que marginalement affecté par l’ajout de bile. Dans du MRS sans acétate de sodium, seules les trois souches non agrégables les plus tolérantes ont augmenté leur contenu relatif C18:1 en présence de bile. Du même coup, la culture en MRS sans acétate de sodium a fait baisser la production d’EPS-b chez les souches agrégables. En conclusion, les EPS-b et la composition en acides gras jouent un rôle important dans la tolérance a` la bile chez les L. brevis issus de cornichons. [Traduit par la Rédaction] Mots-clés : Lactobacillus brevis, tolérance a` la bile, composition en acides gras, exopolysaccharides.

Introduction Lactobacillus brevis is a hetero-fermentative lactic acid bacterium (LAB) that is widely isolated from dairy products, pickles, silage, feces, and intestinal tracts of humans and animals (Hammes and Hertel 2009). Several studies (Rönkäa et al. 2002; Meira et al. 2012) have proposed some L. brevis strains as candidate probiotics. Lactobacillus brevis KB290, which was isolated from Japanese pickles, has probiotic functions such as intestinal regulation (Nobuta et al. 2009) and immunomodulation (Kishi et al. 1996; Fukui et al. 2013; Waki et al. 2014), and is used as an intervention in irritable bowel syndrome (Murakami et al. 2012). Fuller (1989) defined probiotics as a live microbial feed supplement that benefits the host animal by improving its intestinal microbial balance. For probiotic use, LAB must tolerate digestive

juice and bile (Joint FAO/WHO Expert Consultation 2001). Bile can kill microorganisms by acting as a biological detergent and dissolving membrane fatty acids (Begley et al. 2005). Therefore, bile tolerance is an essential property for probiotics (Dunne et al. 2001). Many studies on probiotics have revealed that cell properties, such as cellular fatty acid composition (Murga et al. 1999; Kimoto et al. 2002) and exopolysaccharides (EPS) (Lebeer et al. 2007), play important roles in bile tolerance. Alteration of cellular fatty acid composition by bile treatment can affect lipid fluidity (Begley et al. 2005). Cell-bound EPS (EPS-b) also contributes to the bile tolerance of L. brevis KB290 (Suzuki et al. 2013), and Fukao et al. (2013) reported a relationship between cellular aggregation and EPS-b in this strain. However, the bile tolerance mechanisms of L. brevis species relating to cell structure still require further clarification.

Received 18 January 2014. Accepted 28 January 2014. S. Suzuki, H. Suganuma, and N. Yajima. Research and Development Division, Kagome Co., Ltd., 17 Nishitomiyama, Nasushiobara, Tochigi 329-2762, Japan. H. Kimoto-Nira and C. Suzuki. Functional Biomolecules Research Group, NARO Institute of Livestock and Grassland Science, Tsukuba, Ikenodai 2, Ibaraki 305-0901, 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: 183–191 (2014) dx.doi.org/10.1139/cjm-2014-0043

Published at www.nrcresearchpress.com/cjm on 12 February 2014.

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Table 1. Lactobacillus brevis strains examined in this study. Strain

Origin

JCM1059T KB290 KB392 KB605 KB606 KB643 KB667 KB876 KB885 KB905 KB910 KB911 KB1029 KB1030 KB1040 KB1041 KB1053 KB1055 KB1056 KB1071 KB1072 KB1076 KB1095 KB1096 KB1104 KB1131 KB1140 KB1141 KB1142 KB1143 KB1145 KB1149 KB1186 KB1218 KB1257 KB1258 KB1260 KB1265 KB1272 KB1277 KB1282 KB1388 KB1389 KB1407 KB1409 KB1415 KB1416 KB1418 KB1420 KB1427 KB1440 KB1448 KB1452 KB1567 KB1584 KB1663

Human feces Suguki (turnip pickles) Spontaneous mutant isolated from KB290 Shiba-zuke (eggplant pickles) Shiba-zuke Shiba-zuke Shiba-zuke Takana-zuke (cruciferous pickles) Takana-zuke Takana-zuke Takana-zuke Takana-zuke Akakabu-zuke (turnip pickles) Akakabu-zuke Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Suguki Asotakana-zuke (cruciferous pickles) Pesora-zuke (eggplant pickles) Pesora-zuke Pesora-zuke Pesora-zuke Pesora-zuke Pesora-zuke Pesora-zuke Pesora-zuke Shakushina-zuke (cruciferous pickles) Shakushina-zuke Kuki-zuke (aroid pickles) Kuki-zuke Kuki-zuke Kuki-zuke Kuki-zuke Kuki-zuke Kuki-zuke Kuki-zuke Kuki-zuke Kuki-zuke Tsudakabu-zuke (turnip pickles) Tsudakabu-zuke Hinona-zuke (cruciferous pickles)

Tween 80 (polyoxyethylene sorbitan monooleate: CAS Registry No. 9005-65-6) consists of oleic acid (octadecenoic acid; C18:1), which provides cellular fatty acids (Partanen et al. 2001) and promotes growth by improving membrane permeability (Taoka et al. 2011). Corcoran et al. (2007) reported that Tween 80 in de Man – Rogosa – Sharpe (MRS) medium enhanced the C18:1 content and bile tolerance of Lactobacillus rhamnosus GG. It has been reported that the growth environment of lactobacilli affects their fatty acid composition, which in turn appears to affect their bile tolerance (Kimoto-Nira et al. 2012). A previous

study reported briefly that there are positive correlations among the amounts of EPS-b, the growth indices in artificial digestive juices, and the cellular aggregation in L. brevis isolated from Japanese pickles (Suzuki et al. 2014). In this study, features of cell structure, such as cellular fatty acid composition and EPS-b production, were investigated to elucidate the mechanisms of bile tolerance in L. brevis isolated from various Japanese pickles.

Materials and methods Bacterial strains and growth conditions The bacterial strains used in this study were L. brevis JCM1059T (isolated from human feces), L. brevis KB290, L. brevis KB392 (a spontaneous mutant of KB290, nonaggregating strain, Fukao et al. 2013), and 53 strains of L. brevis isolated from Japanese pickles (Table 1). All strains were cultured at 30 °C in MRS broth (Oxoid, Hampshire, UK) and stored at –80 °C in MRS broth containing 15% (v/v) glycerol (Wako, Tokyo, Japan). Prior to use, cultures were thawed at 30 °C, inoculated at 1% (v/v) into fresh MRS, and cultured at 30 °C for 24 h. Assessment of aggregation Aggregation ability was assayed as previously reported (Suzuki et al. 2014). Briefly, the aggregability of the tested strain was judged when the cells became agglomerated following vigorous mixing for 10 s in medium using an agglomerator mixer (Se-08: TAITEC, Saitama, Japan). After stirring, the mixture culture was poured onto a plate and observed on a color illuminator (Fujifilm, Tokyo, Japan). Estimation of bile tolerance Bile tolerance of the strains was measured according to a previously described method, with minor modifications (Kimoto et al. 2002). Briefly, 10 g of Bacto Oxgall (Becton, Dickinson and Company, Annapolis, Maryland, USA) was dissolved in 100 mL of distilled water and autoclaved at 121 °C for 15 min. Sterile distilled water or the Oxgall solution (150 ␮L) was added to 5 mL of MRS broth, prepared according to de Man et al. (1960), or modified MRS broth (mMRS-A, without Tween 80; mMRS-B, without sodium acetate) to a final concentration of 0.3% (v/v) (Kimoto et al. 2002). Peptone, Lab-Lemco powder, and yeast extract were purchased from Oxoid. Glucose, Tween 80, sodium acetate trihydrate, triammonium citrate, magnesium sulfate heptahydrate, and manganese sulfate tetrahydrate were purchased from Wako. Dipotassium hydrogen phosphate was purchased from Kanto Chemical (Tokyo, Japan). Each broth was inoculated with 50 ␮L of the fresh culture and then incubated at 30 °C. After 24 h, the optical density (OD) of the cultures was measured using a spectrophotometer (U-2910, Hitachi, Tokyo, Japan; wavelength 620 nm) against an uninoculated broth control. Bile tolerance was calculated as follows: bile tolerance (%) = (OD of culture with bile / OD of culture without bile) × 100. Cellular fatty acid composition Because lipids are found predominantly in the membranes of Gram-positive bacteria (Kimoto-Nira et al. 2009), for the purposes of this study, the cell membrane fatty acid composition was considered to be approximately equal to the total cellular fatty acid composition, and total cellular lipids were extracted. Briefly, 50 mL of L. brevis cultured in MRS, mMRS-A, or mMRS-B was centrifuged at 8000g for 20 min at 5 °C, and the resulting cell pellets were washed 3 times with distilled water. The fatty acids were extracted from the cells and then methyl-esterified according to the method of Smittle et al. (1974). The methyl esters were separated on a gas chromatograph (GC-17A and GCMS-QP5050, both Shimadzu, Kyoto, Japan) according to the method of Dionisi et al. (1999). Fatty acid methyl esters were tentatively identified by comparing their retention times and molecular masses with those of Bacterial Acid Methyl Ester Mix (Sigma-Aldrich, St. Louis, Missouri, USA). Published by NRC Research Press

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Table 2. Bile tolerance of tested Lactobacillus brevis strains that showed most, least or lower tolerance in MRS, mMRS-A, and mMRS-B media. MRS

Aggregating Most tolerant


Least tolerant

Nonaggregating Most tolerant

Least or lower tolerant

mMRS-A

mMRS-B

Strain

Bile tolerance (%)

Strain

Bile tolerance (%)

Strain

Bile tolerance (%)

KB290 KB1389 KB1584 Mean

70.8 66.9 70.4 69.4


58.6 55.4 54.7 56.2


35.5 35.5 35.2 35.4


61.5 62.5 57.7 60.6


49.5 51.9 48.5 50.0


33.4 29.3 33.4 32.0


70.9 72.7 74.3 72.6


59.3 57.1 53.5 56.6

JCM1059T KB1277 KB1427 Mean

56.1 59.8 57.6 57.8


31.2 38.7 34.3 34.7


29.7 29.5 31.7 30.3

KB605* KB1029* KB1076* Mean

30.7 29.9 28.5 29.7

Note: Table 2 is a summary of Supplementary Table S11, showing the three strains with the highest and lowest bile tolerance in the 3 types of MRS media following bile treatment. MRS, de Man – Rogosa – Sharpe medium; mMRS-A, MRS without Tween 80; mMRS-B, MRS without sodium acetate. *Strains with lower bile tolerance.

Fig. 1. Mean bile tolerance of tested Lactobacillus brevis strains in standard and modified MRS (de Man – Rogosa – Sharpe) medium. Open bars, MRS; shaded bars, mMRS-A (MRS medium without Tween 80); solid bars, mMRS-B (MRS medium without sodium acetate). Aggregating (9 strains) and nonaggregating (47 strains) L. brevis strains were tested. Results are expressed as means, and error bars represent standard deviations. Bars with different letters are significantly different (Tukey, P < 0.05).

EPS extraction EPS-b from aggregating strains KB290, KB1389, and KB1584 was extracted according to a previously described method (Suzuki et al. 2013). Briefly, each culture was serially diluted (10-fold) with sterile saline containing 0.1% (m/v) agar, then dilutions were poured onto MRS agar plates and incubated at 30 °C for 48 h. Resulting bacterial colonies were counted. A 100 mL aliquot of each culture was centrifuged at 8000g for 10 min at 5 °C, and the

1

pellet was washed twice with sterile saline. The cell pellet was resuspended in 5 mL of 50 mmol/L EDTA (Dojindo, Kumamoto, Japan), and the suspension was stirred gently for 4 h at 5 °C. The mixture was then centrifuged as described above. Two volumes of chilled ethanol (99.5%, Wako) were added to the supernatant, and the mixture was incubated at 5 °C overnight. The mixture was centrifuged (16 000g, 20 min, 5 °C), the pellet was resuspended in 10 mL of distilled water, and the suspension was 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. The dialysate was lyophilized and the total amount of EPS was estimated according to the phenol – sulfuric acid method (Dubois et al. 1959). Results are expressed as glucose equivalents per 109 colony-forming unit counts. Statistical analysis Statistical analysis was performed using SPSS (IBM, version 15.0J for Windows, SPSS, Inc., Chicago, Illinois, USA), and statistical significance was defined as P < 0.05. Bacterial growth in various media with and without bile was determined as the mean of 2 independent experiments. The results of fatty acid composition assessments are presented as means of 2 independent experiments. EPS-b quantities are presented as means ± standard deviation (n = 3). Growth, bile tolerance, and EPS-b production in the various media were analyzed using Tukey’s test, and differences in cellular fatty acid compositions between media with and without bile were tested using a Student’s t test.

Results and discussion Aggregation of L. brevis in MRS medium Nine (KB290, KB1131, KB1140, KB1145, KB1282, KB1388, KB1389, KB1567, and KB1584) of the 56 L. brevis strains showed aggregation (Table S11). These results reproduced the aggregating features of L. brevis strains (Suzuki et al. 2014).

Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjm-2014-0043. Published by NRC Research Press

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Fig. 2. Mean fatty acid composition of (A) the most bile-tolerant aggregating strains, (B) the most tolerant nonaggregating strains, and (C) the least tolerant nonaggregating strains cultured in MRS (de Man – Rogosa – Sharpe medium). Open bars, without bile; hatched bars, with bile. Fatty acid methyl esters are designated by the number of carbon atoms to the left of the colon, and the number of double bounds to the right. NI, percentage represented by several unidentified peaks on chromatograms. Results are expressed as means, and error bars represent standard deviations. *, indicates a significant difference between the groups treated with and without bile (P < 0.05). †, C19:0 includes C19-cyc fatty acid.

Published by NRC Research Press

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Fig. 3. Mean fatty acid composition of (A) the most bile-tolerant aggregating strains, (B) the most bile-tolerant nonaggregating strains, and (C) the least bile-tolerant nonaggregating strains cultured in mMRS-A (de Man – Rogosa – Sharpe medium without Tween 80). Shaded bars, without bile; hatched bars, with bile. Fatty acid methyl esters are designated by the number of carbon atoms to the left of the colon, and the number of double bounds to the right. NI, percentage represented by several unidentified peaks on chromatograms. Results are expressed as means, and error bars represent standard deviations. *, indicates a significant difference between groups treated with bile and without bile (P < 0.05). †, C19:0 includes C19-cyc fatty acid.


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Fig. 4. Mean fatty acid composition of (A) the most bile-tolerant aggregating strains, (B) the most bile-tolerant nonaggregating strains, and (C) the lower bile-tolerant nonaggregating strains cultured in mMRS-B (de Man – Rogosa – Sharpe medium without sodium acetate). Solid bars, without bile; hatched bars, with bile. Fatty acid methyl esters are designated by the number of carbon atoms to the left of the colon, and the number of double bounds to the right. NI, percentage represented by several unidentified peaks on chromatograms. Results are expressed as means, and error bars represent standard deviations. *, indicates a significant difference between the groups treated with and without bile (P < 0.05). †, C19:0 includes C19-cyc fatty acid.


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Fig. 5. Quantification of cell-bound expolysaccharide of aggregating strains of Lactobacillus brevis KB290, KB1389, and KB1584 incubated in MRS (de Man – Rogosa – Sharpe medium) and modified MRS. Open bars, MRS; shaded bars, mMRS-A (MRS without Tween 80); solid bars, mMRS-B (MRS without sodium acetate). Results are expressed as means, and error bars represent standard deviations (n = 3). *, indicates a significant difference between media (P < 0.05).

Bile tolerance in various media The bile tolerance data for individual strains is shown in Table S11. For both aggregating and nonaggregating strains, Tween 80 and sodium acetate were not essential components for growth. The mean bile tolerance of aggregating strains cultured in MRS was significantly higher (P < 0.05) than that of aggregating strains cultured in mMRS-B and nonaggregating strains cultured in all media (Fig. 1). The highest mean bile tolerance of nonaggregating strains was observed when strains were cultured in MRS, followed by culture in mMRS-A, and mMRS-B. Aggregating strains cultured in mMRS-A tended to have lower bile tolerance than when cultured in MRS, but this was not statistically significant (P = 0.076). Our previous study (Suzuki et al. 2013) revealed that EPS-b envelops the cell surface of KB290 and protects it against bile treatment. Another study found that the 8 aggregating strains also had EPS-b, which mainly consists of glucose, N-acetylglucosamine, and N-acetylmannosamine (Suzuki et al. 2014). In this study, we hypothesized that the 8 aggregating strains might also be enveloped by EPS-b, which would explain why mean bile tolerance was higher than in nonaggregating strains when cultured in MRS. This observation may be affected by the potentially limiting conditions of using MRS medium in vitro; therefore, further investigation using digestive fluids in vivo is required. Koser (1968) reported that Tween 80 is not essential for L. brevis growth in basal medium. However, supplementation with Tween 80 or oleic acid enhanced bile tolerance of lactococci (Kimoto et al. 2002) and L. rhamnosus GG (Corcoran et al. 2007) by altering cellular fatty acid composition. Furthermore, Guirard et al. (1946) predicted that sodium acetate in the medium contributes to membrane fatty acid synthesis in lactobacilli. Thus, we propose that the reduction in bile tolerance of both aggregating and nonaggregating strains in both modified MRS media was a result of the limitation of these components, which suppressed alteration of the membrane fatty acid composition. Cellular fatty acid composition To evaluate the alteration of cellular fatty acid composition of L. brevis strains, the 3 aggregating and 3 nonaggregating strains that showed the highest bile tolerance in MRS, mMRS-A, or mMRS-B were selected, as were the 3 nonaggregating strains that showed

the lowest bile tolerance in MRS or mMRS-A, and the 3 nonaggregating strains that showed lower bile tolerance in mMRS-B (Table 2). The least tolerant aggregating strains were not selected because the bile tolerance values of the aggregating strains were marginally different (Table 2). In MRS, bile treatment significantly (P < 0.05) affected the mean relative content of tetradecanoic acid (C14:0), hexadecanoic acid (C16:0, only for Fig. 2A), hexadecenoic acid (C16:1), octadecanoic acid (C18:0), C18:1, and nonadecanoic acid (C19:0) in the most tolerant aggregating and nonaggregating strains (Figs. 2A and 2B). In contrast, the fatty acid composition of the least tolerant strains was not affected by bile (Fig. 2C). When the most tolerant aggregating strains were cultured in mMRS-A, bile affected the proportion of C14:0 and C16:1 (Fig. 3A). In the most tolerant nonaggregating strains, the proportions of C14:0 and C19:0 were most affected by bile treatment (P < 0.05) (Fig. 3B). In the least tolerant nonaggregating strains, only the proportion of C16:1 was affected by bile treatment (Fig. 3C). When cultured in mMRS-B, bile treatment significantly affected the proportions of C14:0, C16:0, and C19:0 in the most tolerant aggregating strains (Fig. 4A) (P < 0.05), as well as the proportions of C18:1 and C19:0 in the most tolerant nonaggregating strains (Fig. 4B) (P < 0.05), and the proportions of C14:0, C16:0, C16:1, C18:0, and C19:0 in the lower tolerant nonaggregating strains (Fig. 4C). Cellular fatty acids are a major component of cell membrane phospholipids and contribute to membrane fluidity (Spector and Yorek 1985). In lactobacilli, dodecanoic acid (C12:0), C14:0, C16:0, C16:1, heptadecanoic acid (C17:0), C18:0, C18:1, octadecadienoic acid (C18:2), and C19:0 are well-known cellular fatty acids (Rizzo et al. 1987), and their ratios are affected by environmental stresses (Guerzoni et al. 2001). In particular, bile induces the alteration of unsaturated fatty acid components of LAB and bifidobacteria (Murga et al. 1999; Taranto et al. 2003; Ruiz et al. 2007; Kimoto-Nira et al. 2009). C18:1 is a major cell membrane component of lactobacilli; it improves cell membrane fluidity (Guerzoni et al. 2001), which makes the membrane easier to repair if it is damaged by the emulsification and solubilization actions of bile (Taranto et al. 2003). In this study, the relative content of C18:1 in the most tolerant aggregating (mean bile tolerance = 69.4%) and nonaggregating strains (mean bile tolerance = 72.6%) cultured in MRS was significantly Published by NRC Research Press


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increased by bile treatment (P < 0.05) (Figs. 2A and 2B). In contrast, the C18:1 content of the least tolerant nonaggregating strains (mean bile tolerance = 34.7%) was not affected when cultured in MRS with bile (Fig. 2C). On the other hand, bile did not alter cellular C18:1 content in mMRS-A (Fig. 3). Moreover, the bile tolerance of the most tolerant aggregating and nonaggregating strains was reduced in mMRS-A (Table 2; aggregating mean bile tolerance = 56.2%, nonaggregating mean bile tolerance = 56.6%). Thus, alteration of C18:1 content by Tween 80 might contribute to bile tolerance of L. brevis. However, the most tolerant aggregating strains did not show an increase in C18:1 when cultured in mMRS-B (Fig. 4A) and showed lower bile tolerance (Table 2; mean bile tolerance = 35.4%) than in MRS or mMRS-A. Pollack and Tourtellotte (1967) reported that sodium acetate contributed to synthesis of long-chain fatty acids in microorganisms. Therefore, sodium acetate might also directly contribute to alterations in C18:1 levels or possibly support the action of Tween 80 in this process. In addition, KB290, KB1389, and KB1584 lost their aggregability when incubated in mMRS-B (data not shown). A previous study (Suzuki et al. 2013) revealed that EPS-b of KB290 is important for growth in artificial digestive juice and for bile tolerance, as compared with KB392 (a nonaggregating strain). Thus, we hypothesize that another component related to sodium acetate, such as EPS-b synthesis, might contribute to bile tolerance. Quantification of crude EPS-b The amounts of crude EPS-b extracted from L. brevis KB290, KB1389, and KB1584 incubated in MRS or mMRS-A were marginally different (Fig. 5). The amount of crude EPS-b that was extracted from these strains was significantly lower when the strains were cultured in mMRS-B than in the other media (Fig. 5) (P < 0.05). Enhancement of EPS production by L. brevis strains as a result of sodium acetate supplementation has not been previously reported. Sodium acetate is essential for the growth of the EPSproducing strain Lactobacillus delbrueckii subsp. bulgaricus (Grobben et al. 1998). Iino et al. (2002) also reported that sodium acetate affected activity of enzymes in the glycolytic pathway of Lactobacillus sakei, which might contribute to EPS production. Thus, we concluded that sodium acetate might contribute to EPS synthesis in aggregating L. brevis strains. In conclusion, both EPS-b and cellular fatty acid composition play important roles in the bile tolerance of pickle-derived L. brevis strains. This is the first report of the bile tolerance mechanisms of pickle-derived L. brevis strains other than KB290. The elucidation of whether EPS synthesis depends on sodium acetate and the discovery of the cause of bile tolerance reduction in nonaggregating strains cultured in mMRS-B are required to clarify the bile tolerance mechanisms of L. brevis strains. Further studies of these bile tolerance mechanisms may help improve viability in the host gastrointestinal tract and provide more insight into the application of L. brevis strains, including KB290, for probiotic use.

Acknowledgements We are grateful to Kunihiko Sato (Kagome Co., Ltd.) for his valuable support. We thank Kentaro Yamamoto and Sachiyo Igari (Kagome Co., Ltd.) for their technical assistance.

References Begley, M., Gahan, C.G.M., and Colin, H. 2005. The interaction between bacteria and bile. FEMS Microbiol. Rev. 29(4): 625–651. doi:10.1016/j.femsre.2004.09. 003. PMID:16102595. Corcoran, B.M., Stanton, C., Fitzgerald, G.F., and Ross, R.P. 2007. Growth of probiotic lactobacilli in the presence of oleic acid enhances subsequent survival in gastric juice. Microbiology, 153: 291–299. doi:10.1099/mic.0.28966-0. PMID:17185558. de Man, J.C., Rogosa, M., and Sharpe, M.E. 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23(1): 130–135. doi:10.1111/j.1365-2672.1960. tb00188.x. PMID:22484087. Dionisi, F., Golay, P.A., Elli, M., and Fay, L.B. 1999. Stability of cyclopropane and


conjugated linoleic acids during fatty acid quantification in lactic acid bacteria. Lipids, 34(10): 1107–1115. doi:10.1007/s11745-999-0462-8. PMID:10580338. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., and Smith, F. 1959. Colorimetric method for determination of sugars and related substance. Anal. Chem. 28(3): 350–356. doi:10.1021/ac60111a017. Dunne, C., O’Mahony, L., Murphy, L., Thornton, G., Morrissey, D., O’Halloran, S., et al. 2001. In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. Am. J. Clin. Nutr. 73(2 Suppl.): 386S–392S. PMID:11157346. Fukao, M., Oshima, K., Morita, H., Toh, H., Suda, W., Kim, S.W., et al. 2013. Genetic analysis by deep sequencing of the probiotic Lactobacillus brevis KB290 harboring nine plasmids reveals genomic stability. PLoS One, 8(3): e60521. doi:10.1371/journal.pone.0060521. PMID:23544154. Fukui, Y., Sasaki, E., Fuke, N., Nakai, Y., Ishijima, T., Abe, K., and Yajima, N. 2013. Effect of Lactobacillus brevis KB290 on the cell-mediated cytotoxic activity of mouse splenocytes: a DNA microarray analysis. Br. J. Nutr. 110(9): 1617–1629. doi:10.1017/S0007114513000767. PMID:23544404. Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bacteriol. 65(5): 365–378. PMID:2666378. Grobben, G.J., Chin-Joe, I., Kitzen, V.A., Boels, I.C., Boer, F., Sikkema, J., et al. 1998. Enhancement of exopolysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus NCFB2772 with a simplified defined medium. Appl. Environ. Microbiol. 64: 1333–1337. PMID:16349540. Guerzoni, M.E., Lanciotti, R., and Cocconcelli, P.S. 2001. Alteration in cellular fatty acid composition as a response to salt, oxidative and thermal stress in Lactobacillus helveticus. Microbiology, 147: 2255–2264. PMID:11496002. Guirard, B.M., Snell, E.E., and Williams, R.J. 1946. The nutritional role of acetate for lactic acid bacteria; the response to substances related to acetate. Arch. Biochem. 9: 361–379. PMID:21023612. Hammes, W.P., and Hertel, C. 2009. Family I. Lactobacillaceae. In Bergey’s manual of systematic bacteriology. 2nd ed. Vol. 3. The Firmicutes. Edited by P. De Vos, G.M. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.-H. Schleifer, and W.B. Whitman. Springer, New York, USA. pp. 465–511. Iino, T., Uchimura, T., and Komagata, K. 2002. The effect of sodium acetate on the growth yield, the production of L- and D-lactic acid, and the activity of some enzymes of the glycolytic pathway of Lactobacillus sakei NRIC 1071T and Lactobacillus plantarum NRIC 1067T. J. Gen. Appl. Microbiol. 48: 91–102. doi:10. 2323/jgam.48.91. PMID:12469305. Joint FAO/WHO Expert Consultation. 2001. Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria. Córdoba, Argentina. Kimoto, H., Ohmomo, S., and Okamoto, T. 2002. Enhancement of bile tolerance in lactococci by Tween 80. J. Appl. Microbiol. 92: 41–46. doi:10.1046/j.13652672.2002.01505.x. PMID:11849326. Kimoto-Nira, H., Kobayashi, M., Nomura, M., Sasaki, K., and Suzuki, C. 2009. Bile resistance in Lactococcus lactis strains varies with cellular fatty acid composition: analysis by using different growth media. Int. J. Food Microbiol. 131: 183–188. doi:10.1016/j.ijfoodmicro.2009.02.021. PMID:19339076. Kimoto-Nira, H., Suzuki, S., Yakabe, T., and Suzuki, C. 2012. Relationships between fatty acid composition and bile tolerance in lactobacillus isolates from plants and from non-plant materials. Can. J. Microbiol. 58(12): 1396–1404. doi:10.1139/cjm-2012-0442. PMID:23210997. Kishi, A., Uno, K., Matsubara, Y., Okuda, C., and Kishida, T. 1996. Effect of the oral administration of Lactobacillus brevis subsp. coagulans on interferon-␣ producing capacity in humans. J. Am. Coll. Nutr. 15(4): 408–412. doi:10.1080/07315724. 1996.10718617. PMID:8829098. Koser, S.A. 1968. Lactobacillus. Chap. 21. In Vitamin requirements of bacteria and yeasts. Charles C. Thomas, Springfield, Ill., USA. pp. 340–365. Lebeer, S., Verhoeven, T.L.A., Vélez, V.M.P., Vanderleyden, J., and De Keersmaecker, S.C.J. 2007. Impact of environmental and genetic factors on biofilm formation by the probiotic strain Lactobacillus rhamnosus GG. Appl. Environ. Microbiol. 73(21): 6768–6775. doi:10.1128/AEM.01393-07. PMID:17827316. Meira, S.M., Helfer, V.E., Velho, R.V., Lopes, F.C., and Brandelli, A. 2012. Probiotic potential of Lactobacillus spp. isolated from Brazilian regional ovine cheese. J. Dairy Res. 79(1): 119–127. doi:10.1017/S0022029911000884. PMID:23171587. Murakami, K., Habukawa, C., Nobuta, Y., Moriguchi, N., and Takemura, T. 2012. The effect of Lactobacillus brevis KB290 against irritable bowel syndrome: a placebo-controlled double-blind crossover trial. Biopsychosoc. Med. 6(1): 16. doi:10.1186/1751-0759-6-16. PMID:22863114. Murga, M.L.F., Bernik, D., de Valdez, G.F., and Disalvo, A.E. 1999. Permeability and stability properties of membranes formed by lipids extracted from Lactobacillus acidophilus grown at different temperatures. Arch. Biochem. Biophys. 364(1): 115–121. doi:10.1006/abbi.1998.1093. PMID:10087172. Nobuta, Y., Inoue, T., Suzuki, S., Arakawa, C., Yakabe, T., Ogawa, M., and Yajima, N. 2009. The efficacy and the safety of Lactobacillus brevis KB290 as a human probiotics. Int. J. Probiotics Prebiotics, 4(4): 263–270. Partanen, L., Marttinen, N., and Alatossava, T. 2001. Fats and fatty acids as growth factors for Lactobacillus delbrueckii. Syst. Appl. Microbiol. 24: 500–506. doi:10.1078/0723-2020-00078. PMID:11876356. Pollack, J.D., and Tourtellotte, M.E. 1967. Synthesis of saturated long chain fatty Published by NRC Research Press


Suzuki et al.

acids from sodium acetate-1-C14 by Mycoplasma. J. Bacteriol. 93(2): 636–641. PMID:6020566. Rizzo, A.F., Korkeala, H., and Mononen, I. 1987. Gas chromatography analysis of cellular fatty acid and neutral monosaccharides in the identification of lactobacilli. Appl. Environ. Microbiol. 53(12): 2883–2888. PMID:3435147. Rönkäa, E., Malinena, E., Saarelab, M., Rinta-Koskic, M., Aarnikunnasa, J., and Palva, A. 2002. Probiotic and milk technological properties of Lactobacillus brevis. Int. J. Food Microbiol. 83(1): 63–74. doi:10.1016/S0168-1605(02)00315-X. PMID:12672593. Ruiz, L., Sánchez, B., Ruas-Madiedo, P., de los Reyes-Gávilan, C.G., and Margolles, A. 2007. Cell envelope changes in Bifidobacterium animalis ssp. lactis as a response to bile. FEMS Microbiol. Lett. 274: 316–322. doi:10.1111/j.15746968.2007.00854.x. PMID:17651391. Smittle, R.B., Gilliland, S.E., Speck, M.L., and Walter, W.M., Jr. 1974. Relationship of cellular fatty acid composition to survival of Lactobacillus bulgaricus in liquid nitrogen. Appl. Environ. Microbiol. 27(4): 738–743. PMID:4363555. Spector, A.A., and Yorek, M.A. 1985. Membrane lipid composition and cellular function. J. Lipid Res. 26: 1015–1035. PMID:3906008. Suzuki, S., Yakabe, T., Suganuma, H., Fukao, M., Saito, T., and Yajima, N. 2013.

191

Cell-bound exopolysaccharides of Lactobacillus brevis KB290: protective role and monosaccharide composition. Can. J. Microbiol. 59(8): 549–555. doi:10. 1139/cjm-2013-0115. PMID:23898998. Suzuki, S., Honda, H., Suganuma, H., Saito, T., and Yajima, N. 2014. 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. Can. J. Microbiol. 60(3): 139–145. doi:10.1139/cjm-20130774. Taoka, Y., Nagano, N., Okita, Y., Izumida, H., Sugimoto, S., and Hayashi, M. 2011. Effect of Tween 80 on the growth, lipid accumulation and fatty acid composition of Thraustochytrium aureum ATCC 34304. J. Biosci. Bioeng. 111: 420–424. doi:10.1016/j.jbiosc.2010.12.010. PMID:21216665. Taranto, M.P., Fernandez, M.M.L., Lorca, G., and de Valdez, G.F. 2003. Bile salts and cholesterol induce changes in the lipid cell membrane of Lactobacillus reuteri. J. Appl. Microbiol. 95(1): 86–91. doi:10.1046/j.1365-2672.2003.01962.x. PMID:12807457. Waki, N., Yajima, N., Suganuma, H., Buddle, B.M., Luo, D., Heiser, A., and Zheng, T. 2014. Oral administration of Lactobacillus brevis KB290 to mice alleviates clinical symptoms following influenza virus infection. Lett. Appl. Microbiol. 58(1): 87–93. doi:10.1111/lam.12160. PMID:24329975.


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.

Intraspecies cellular fatty acids heterogeneity of Lactobacillus plantarum strains isolated from fermented foods in Ukraine.

Lactobacillus brevis strains from fermented aloe vera survive gastroduodenal environment and suppress common food borne enteropathogens.

Lactobacillus furfuricola sp. nov., isolated from Nukadoko, rice bran paste for Japanese pickles.

Multiple Genome Sequences of Exopolysaccharide-Producing, Brewery-Associated Lactobacillus brevis Strains.

Technological properties and probiotic potential of Lactobacillus fermentum strains isolated from West African fermented millet dough.

Probiotic potential of lactobacillus strains isolated from sorghum-based traditional fermented food.

Complete Genome Sequence of Lactobacillus fermentum MTCC 25067 (Formerly TDS030603), a Viscous Exopolysaccharide-Producing Strain Isolated from Indian Fermented Milk.

Cellular fatty acid composition of isolates from Legionnaires disease.

Cellular fatty acid composition of Francisella tularensis.

Cellular fatty acid composition of Plesiomonas shigelloides.

In vitro evaluation of the probiotic and functional potential of Lactobacillus strains isolated from fermented food and human intestine.

Antibacterial activity of Lactobacillus sake isolated from dry fermented sausages.

In vitro screening of selected probiotic properties of Lactobacillus strains isolated from traditional fermented cabbage and cucumber.

Technological Potential of Lactobacillus Strains Isolated from Fermented Green Olives: In Vitro Studies with Emphasis on Oleuropein-Degrading Capability.

Draft Genome Sequence of Lactobacillus farciminis NBRC 111452, Isolated from Kôso, a Japanese Sugar-Vegetable Fermented Beverage.

Heterogeneity between and within Strains of Lactobacillus brevis Exposed to Beer Compounds.

Investigation of antibacterial, acid and bile tolerance properties of lactobacilli isolated from Koozeh cheese.

Fatty acid composition of breast milk from Nigerian and Japanese women.

Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese.

Draft Genome Sequence of Lactobacillus collinoides CUPV237, an Exopolysaccharide and Riboflavin Producer Isolated from Cider.

Physiological Characteristics and Production of Folic Acid of Lactobacillus plantarum JA71 Isolated from Jeotgal, a Traditional Korean Fermented Seafood.

Effect of the fatty acid composition of acclimated oenological Lactobacillus plantarum on the resistance to ethanol.

Characterization of an exopolysaccharide produced by Lactobacillus plantarum YW11 isolated from Tibet Kefir.