Lactococcus lactis subsp. lactis has been isolated from the intestines of marine fish and is a candidate probiotic for aquaculture. In order to use the bacterium as a probiotic, properties such as bile tolerance need to be assessed. Here, we compared bile tolerance in L. lactis strains derived from different sources. Three L. lactis subsp. lactis strains from marine fish (MFL), freshwater fish (FFL) and cheese starter (CSL) were used along with an L. lactis subsp. cremoris strain from cheese starter (CSC). The four strains were grown under various culture conditions: MRS broth containing bile salts/acids; MRS agar containing Oxgall; and PBS containing fish bile. Survival/growth of the strains in the presence of sodium cholate and sodium deoxycholate varied in the order MFL, CSL > CSC > FFL; in the presence of sodium taurocholate, the order was MFL > CSL > CSC > FFL. In liquid media containing various concentrations of Oxgall, survival of the strains was observed in the order MFL > CSL > FFL and CSC. The survival of MFL was not affected by bile collected from the goldfish (Carassius auratus subsp. auratus) or the pufferfish (Takifugu niphobles) although the other strains showed significant inhibition of growth.
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Bile tolerance, Lactic acid bacteria, Lactococcus lactis, Probiotics.
Beneficial microbes for humans and for various livestock species have been investigated for their abilities to modulate the composition of the intestinal microbiota for better growth, digestion, immunity and disease resistance in the host . These microorganisms are defined as ï¿½probioticsï¿½ . The use of probiotics in aquaculture offers the possibility of controlling intestinal microflora to eliminate opportunistic pathogen(s) that may cause serious disease; aquatic animals are inevitably in constant and intimate contact with their surrounding environment and are sensitive to changes in its composition . The manipulation of the intestinal microflora through dietary supplementation with probiotics may provide a therapeutic modality to ameliorate any adverse effects of the antibiotics and drugs used in aquaculture [4, 5].
Microorganisms belonging to several groups, including Saccharomyces, Clostridium, Bacillus, Enterococcus, Lactobacillus, Shewanella, Leuconostoc, Lactococcus, Carnobacterium, and Aeromonas, have been used as probiotics in fish culture . Various criteria, such as colonization of the intestinal tract of the host and safety have been suggested for selecting probiotic microorganisms with health effects that are beneficial to the host [7, 8]. In order to survive in and colonize the intestinal tract of the host, candidate probiotic microorganisms need to show high tolerance to gastric acid and bile [9, 10]. Bile secreted in the intestinal tract can reduce bacterial survival by destroying the cell membranes of the microorganisms. The principal components of these membranes are lipids and fatty acids and any modification to these may affect not only cell permeability and viability, but also interactions between the bacterial membrane and the environment [11, 12]. Investigation of tolerance to bile acids in terrestrial animals has shown that survival in the presence of bile acids such as chenodeoxycholic, ursodeoxycholic, cholic, deoxycholic or lithocholic acid varies among microbial species and is also affected by the nutrition of the host . To date, however, the tolerance of microbial species to fish bile has not been evaluated; moreover, the physiological concentrations of the components of fish bile have yet to be determined .
Recently, we isolated Lactococcus lactis subsp. lactis strains from the intestinal tracts of the Grass Puffer (Takifugu niphobles), a marine fish species  and the Amur catfish (Silurus asotus), a freshwater species . These strains showed unique phenotypic traits, for example, halotolerance, compared to those from traditional sources such as cheese starter, suggesting that these fish strains show adaptation to the environmental conditions of the host [14, 15]. Therefore, we predicted that L. lactis subsp. lactis strains derived from different sources would show variation in their tolerance to bile acids and fish bile. In this study, we compared the bile tolerance of the L. lactis strains derived from marine fish, freshwater fish and cheese starter culture.
Materials and methods
Bacterial strains and culture conditions
Three L. lactis subsp. lactis strains were used here; these were obtained from laboratory collections originally derived from the grass puffer T. niphobles (MFL strain ), the Amur catfish S. asotus Linnaeus (FFL strain ) and the cheese starter culture O-114 (CSL strain ). The L. lactis subsp. cremoris strain derived from cheese starter culture O-114 (CSC strain ) was also included in the study. All strains were cultured at 25ï¿½C in MRS broth (BD, NJ, USA) or on MRS-agar plates under aerobic conditions. MRS broth and agar plates were prepared with 50% artificial seawater instead of distilled water.
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Bile acid/salt tolerance
The four L. lactis strains were pre-cultured at 25ï¿½C for 24 hours in MRS broth. Bacterial cultures (approximately 109 CFU) of each strain were then inoculated into MRS broth containing various concentrations of sodium cholate, sodium taurocholate or sodium deoxycholate (Sigma-Aldrich, MO, USA) as described by Yokota et al. . Cell growth in each broth was measured using optical density at 660 nm (OD660) after a 24 h incubation period.
The four L. lactis strains were used to prepare suspensions of 108ï¿½109 CFU ml-1 in MRS broth and 100 ï¿½l aliquots of suspension were immediately used to inoculate MRS-agar plates containing various concentrations (0ï¿½10%) of Oxgall (BD, NJ, USA) as described by Klaenhammer and Kleeman . After incubation at 25ï¿½C for 3 days, bacterial numbers were determined by counts of viable cells on the MRS-agar plates.
Fish bile sensitivity
Bacterial suspensions of approximately 109 CFU ml-1 were prepared in PBS using each of the four L. lactis strains. Bile was collected from the grass puffer (body weight 91.3ï¿½1.52 g) and goldfish (Carassius auratus subsp. auratus; body weight 8.9ï¿½0.34 g) by puncturing the gallbladder. The collected samples of bile were stored at -20ï¿½C until use. A 90 ï¿½l aliquot of bacterial suspension was inoculated into sterile PBS (pH 7.6) or into sterile PBS (pH 7.6) containing 10% (v/v) fish bile, and incubated for 1.5 h at 37ï¿½C as described by Nikoskelainen et al. . After incubation, samples were serially diluted in sterile PBS, inoculated onto MRS-agar plates, and bacterial numbers were determined by viable counts on the plates.
The data on viable cell numbers was subjected to a two-way ANOVA in order to compare the effects of the L. lactis strain and bile type; the analysis was followed by a Tukey-Kramer post-hoc test, using StatView ver. 5.0 (SAS Institute Inc., NC, USA).
Tolerance of L. lactis strains to various bile acids/salts
To compare the tolerances of the four L. lactis strains to specific bile acids/salts, the bacteria were cultured in various concentrations of sodium cholate, sodium deoxycholate and sodium taurocholate. Sodium deoxycholate was found to have an approximately 1000-fold greater inhibitory effect on growth than sodium taurocholate and approximately 100-fold greater effect than sodium cholate (Figure 1). Growth curves for the four L. lactis strains varied in the cultures containing sodium cholate or sodium deoxycholate in the order MFL ï¿½ CSL > CSC > FFL, whereas in the presence of sodium taurocholate, the order was MFL > CSL > CSC > FFL.
Oxgall tolerance of the four L. lactis strains
To investigate variation in bile tolerance among the four strains, cultures were initiated in liquid media containing a range of Oxgall concentrations. Survival in the media varied among the strains: the MFL strain could survive in media containing up to 9% Oxgall and the CSL strain up to 3%; however, the FFL and CSC strains failed to form colonies if the medium contained more than 0.6% Oxgall (Figure 2).
Effects of fish bile on the growth of the four L. lactis strains
To evaluate whether any of the four L. lactis strains are suitable for use in aquaculture, we investigated their respective tolerances to bile using liquid media containing 10% bile from either the grass puffer or goldfish. The relative numbers of viable cells was not significantly altered in the presence of either grass puffer or goldfish bile in cultures of the MFL strain compared to the control (Figure 3). By contrast, the other strains showed significant reductions in the relative viable counts to 17ï¿½47% in the presence of grass puffer bile and to 4ï¿½17% in the goldfish bile (P < 0.05) compared to the control.
Microorganisms that are used as probiotics provide numerous beneficial effects by modulating various biological systems in the host . Lactococci have generally been shown to be safe for humans as is the case for lactobacilli , and they also do not appear to be pathogenic to aquatic organisms with the exception of L. garvieae infection in aquaculture [21-23]. L. lactis has been reported to be among the predominant lactic acid bacteria in freshwater fish used for aquaculture . Recently, bacteria that were classified in the genus Lactococcus were observed at a high frequency in the intestinal tract of fish  and, subsequently, the L. lactis subsp. lactis strains were isolated . There are no other reports on the isolation of L. lactis from marine environments, although it is expected to colonize the intestinal tract of marine fish.
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The different characteristics of L. lactis strains from the various sources indicate that each strain has adapted to its particular environment, suggesting that it will be necessary to carry out a careful selection of the strain depending on the purpose of its use as a probiotic in aquaculture. The freshwater fish-derived strain (FFL) has several unique characteristics such as low salt tolerance and intolerance of culture at 40ï¿½C under aerobic conditions, while the marine fish-derived strain (MFL) has higher halotolerance than other strains reported to date . In addition, there is uncertainty over the conditions required for the occurrence of the L. lactis subsp. lactis strain derived from marine fish: no colonies of this marine fish-derived strain formed when intestinal contents were directly inoculated onto agar plates [14, 16]. Successful colony formation in this strain requires a pre-culture in MRS broth for several days . This alteration in culture condition requirements suggests that this strain may have entered a VBNC or related state [14, 16].
In this study, comparison of tolerance of L. lactis subsp. lactis strains to several bile acid/salt components demonstrated that all strains had higher tolerance of sodium cholate and sodium taurocholate than of sodium deoxycholate, although the MFL and CSL strains showed highest tolerance to all tested bile acids/salts. A similar pattern was observed in tolerance to Oxgall. Kimoto-Nira et al.  demonstrated that the bile resistance of lactococci was altered by cellular fatty acid composition, such as the amount of hexadecanoic acid or octadecenoic acid (positive), and of hexadecanoic acid or C-19 cyclopropane (negative). Thus, it is possible that a similar difference in fatty acid composition might be present among the L. lactis strains used here. Determination of the relative fatty acid composition of the cellular membranes in these strains will be performed in a future study.
We also identified differences in the effects of bile from two different fish species on the growth of the L. lactis strains; although MFL did not appear to be affected by bile from either fish species, the other strains showed a significant decrease in viable cell counts. The rate of inhibition in these three strains was higher for goldfish bile than grass puffer bile. It has been reported that the bile of Cyprinidae contains cytotoxic cyprinol and cyprinol sulfate as the principal bile acids [27, 28]. Our results suggest that the marine fish-derived strain had a higher tolerance of these toxic substances, allowing the strain to survive in a wider range of fish intestinal tract environments.
In conclusion, L. lactis strains obtained from different sources showed differences in their respective bile tolerances, with that of the MFL strain derived from marine fish being higher than those of the other strains. Tolerance of Oxgall was also greatest in the MFL strain. The strains also showed differences in their sensitivities to fish bile: the survival of MFL was unaffected by grass puffer bile, whereas the other strains showed significant growth inhibition. Our results suggest that the MFL strain could of value as a probiotic in aquaculture.
This study was supported, in part, by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS) (S.I.), a Grant-in-Aid for Scientific Research (C) from JSPS (H.S.), and Research Grants for 2011 from the Nihon University College of Bioresource Sciences (S.I.).