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Aji-narezushi is a traditional lactic acid-fermented fish. In this study, we screened for lactose-utilizing, acidophilic, bile-resistant and cholesterol-lowering lactic acid bacteria (LAB) from aji-narezushi for use as starter strains for fermented foods, as well as for use as probiotics. Of the 301 LAB isolates, 277 fermented lactose, and among these, 171 grew in de Man, Rogosa and Sharpe broth adjusted to pH 3·5. Thirty-four of the isolates were grown in a broth containing 3% (w/v) bile. All of the isolates were lactobacilli. Seven isolates that demonstrated cholesterol-lowering activity in ethanolic solution were selected. All of the isolates were identified as Lactobacillus plantarum. Lactobacillus plantarum AN6 showed the highest cholesterol-lowering activity. AN6 was more resistant to acid, salt and bile than the type strain NBRC15891T. One-half of the cholesterol-lowering effect remained after boiling AN6 for 10 min. The Fourier transform infrared (FT-IR) analysis indicated that the content of cell wall polysaccharides in AN6 is higher than ones in the type strain. These results indicate that Lact. plantarum AN6 can be used as a profitable starter organism and probiotic.
Significance and Impact of the Study
Lactobacillus plantarum AN6 was isolated from aji-narezushi. Cholesterol-lowering activity of AN6 was higher than ones of the type strain. Cell surface of AN6 was rough. FT-IR analysis indicated that the content of cell wall polysaccharides of AN6 is higher than ones in the type strain. These results indicate that AN6 can be used as a new profitable starter and probiotic.
Hypercholesterolaemia is a major human health problem, playing a role in cardiovascular disease (Aso et al. 2005) as well as various other diseases such as diabetes, osteoporosis, Alzheimer's disease, immune response disorders and cancers (Martens et al. 2008; Chen et al. 2011; Pelton et al. 2012). Therefore, many reports have dealt with the subject of cholesterol, particularly with respect to low-density lipoprotein (LDL) cholesterol, and the cholesterol-lowering effect of various chemicals and food materials (Nijjar et al. 2010). Among food materials, the cholesterol-lowering effect of dietary fibres, including soluble and indigestible polysaccharides such as pectin from fruits and alginate from brown algae, is well known (Kuda et al. 1997; Theuwissen and Mensink 2008).
Certain lactic acid bacteria (LAB) and Bifidobacterium are regarded as probiotics, having health-promoting activity associated with metabolism and immunity (Rijkers et al. 2010). The cholesterol-lowering effect of some probiotics has been reported. For example, Lactobacillus fermentum, Lact. delbrueckii ssp. bulgaricus, Lact. acidophilus, Lact. casei and Bifidobacterium animalis have shown cholesterol-lowering activity in vitro (Liong and Shah 2005; Alhaj et al. 2010; Tok and Aslim 2010). Furthermore, the serum cholesterol-lowering effect of LABs in animals and/or humans was also reported (Sridevi et al. 2009; Lee et al. 2010). On the other hand, Yoon et al. (2011) reported that Lact. rhamnosus and Lact. plantarum promote cholesterol excretion in human enterocyte-like Caco-2 cells.
In modern Japanese cuisine, sushi is made from vinegar-flavoured rice combined with seafood. It is believed that contemporary sushi originated from a dish consisting of salted and long-fermented fish called narezushi. The earliest recorded reference to sushi in Japan appeared in the Yoro-Ritsuryo, a code of governing rules issued in AD 718, and apparently refers to narezushi (Kuda et al. 2009). As noted in previous studies, narezushi products have characteristics typical of foods prepared using lactic acid fermentation as they contain lactic acid (>2% (w/w)), have a low pH (<4·5) and have a moderately high salinity (3–7%). We previously isolated a lactic acid strain, Leuconostoc mesenteroides 1RM3, from a narezushi product, having inhibitory effects against Listeria infection and antioxidant properties, with tolerance to bile, acid and salt (Kanno et al. 2012; Nakamura et al. 2012).
In this study, to identify probiotic LAB exhibiting cholesterol-lowering effects for use as probiotics and/or starter strains, we screened for acid- and bile-resistant LAB from narezushi made with horse mackerel. Then, the cholesterol- and bile acid-lowering activities of the selected LAB were determined in vitro. Furthermore, the acid resistance and cholesterol-lowering effects of the selected strains were compared with those of the type strain.
Results and discussion
Isolation, screening and identification
A total of 301 isolates were derived from four aji-narezushi samples with the MRS and GAM agar. Among the isolates, 277 were able to ferment lactose; 171 of these isolates were able to grow in MRS broth adjusted to pH 3·5. Finally, 34 grew well in MRS broth containing 0·3% (w/v) bile. In the presence of 0·05% (w/v) cholesterol solution, these LAB decreased about 11 to 61% of the cholesterol. Seven isolates exhibiting high cholesterol-lowering activity were used for the next experiment.
Seven typical isolates were identified using the API 50 CHL system and analysis of 16S rDNA gene sequences using BLAST. All of the isolates were identified as Lact. plantarum. According to the saccharide fermentation pattern in the API test, these isolates were divided to three groups. Some reports dealing the cholesterol-lowering property of Lact. plantarum in vivo have already been published (Nguyen and Lee 2007); however, few have reported on this activity in vitro.
Bile acid- and cholesterol-lowering activities of the selected strains
Cells of the seven selected Lact. plantarum isolates and the type strain were incubated with 0·1% (w/v) glycocholic acid, taurocholic acid and cholesterol ethanolic solutions for 60 min. As shown in Fig. 1, the isolate AN6 showed the highest cholesterol- and glycocholic acid-lowering ability. On the other hand, the type strain showed the highest taurocholic acid-lowering ability, although this type strain could not decrease glycocholic acid. Based on this result, we selected isolate AN6 for further study.
There are some reports dealing with the bile salt hydrolase of Lact. plantarum. Cebeci and Gürakan (2003) reported that a number of Lact. plantarum strains can survive in MRS broth adjusted to pH 4·0 and containing taurocholic acid; in contrast, they cannot survive in broth containing glycocholic acid. It is thought that the sensitivity and specific hydrolase differ between the strains, possibly affecting their probiotic activities.
Comparison of Lactobacillus plantarum AN6 and NBRC15891T growth
As shown in Fig. 2a, although both Lact. plantarum strains could survive in MRS broth adjusted to pH 2·5, the survival rate of AN6 was higher than that of the type strain NBRC15891T. Figure 2b shows the growth of the strains in MRS broth containing 5% (w/v) bile; the growth rate of AN6 was higher than that of the type strain. Based on the visual observation, AN6 grew in MRS broth containing 10% (w/v) NaCl, while the type strain did not. These results indicate the utility of AN6 as a probiotic, as well as in salted and fermented foods such as cheese, butter and pickles.
Boiling for 10 min decreased the cholesterol-lowering activity by about 60% of the type strain (Fig. 2c). On the other hand, most of the activity of AN6 remained even after the boiling. Therefore, we think that the cholesterol-lowering activity of AN6 was correlated with membrane or outer membrane compounds (Capozzi et al. 2011), rather than the bile-related enzymes (Sridevi et al. 2009).
Auto-aggregation and representative ATR spectra
After 48 h of incubation, the sediment volume of AN6 cells was larger than that of the type strain (Fig. 3a). Turbidity of AN6 culture was lower than that of the type strain. AFM observation showed rough surface of AN6 cells (Fig. 3b).
Representative ATR spectra (4000–550 cm−1) of the AN6 and the type strain are shown in Fig. 3c. The absorbance of AN6, particularly in 1200–900 cm−1, was higher than ones of the type strain. Furthermore, the crude outer cell extract from AN6 showed large absorbance also in 1200–900 cm−1. According to the review by Alvarez-Ordóñez et al. (2011), the absorbance in 1200–900 cm−1 reveals the occurrence of carbohydrates and polysaccharides in the cell wall but also the influence of nucleic acid.
It is well known that alterations in cell characteristics occur during growth. There are reports that the resistance of stationary-phase Lact. plantarum cells to acid, heat and other stresses is associated with membrane function (Capozzi et al. 2011; Ricciardi et al. 2012). These membrane characteristics, particularly in the polysaccharides in the cell wall and/or exopolysaccharide, may affect the cholesterol-lowering property. Study of the extraction, purification and characterization of AN6 exopolysaccharide is in progress now.
In conclusion, we screened for lactose-utilizing, acidophilic, bile-resistant and cholesterol-lowering lactic acid bacteria (LAB) in aji-narezushi. Lactobacillus plantarum AN6 showed the highest cholesterol-lowering activity. Resistance to acid, salt and bile was higher in AN6 than in the type strain of Lact. plantarum. Most of the lowering effect remained after boiling AN6 for 10 min. The Fourier transform infrared (FT-IR) analysis indicated that the content of cell wall polysaccharides in AN6 is higher than ones in the type strain. These results suggest AN6 can be used as a new starter organism and probiotic.
Materials and methods
Isolation and screening
Four aji-narezushi products were purchased from a retail fish store in Anamizu, Ishikawa Prefecture, Japan, and used in the current study. These products were made in small fisheries and were fermented spontaneously, without the addition of microbial starter or fungal malt. Samples (5 g) were homogenized with 45 ml of phosphate-buffered saline (pH 7·2, PBS; Nissui, Tokyo, Japan) containing 0·1% (w/v) agar for 30 s. Decimal dilutions of the homogenate (0·05 ml) were spread on Gifu anaerobic medium (GAM) agar plates (Nissui) for total bacterial counts and de Man, Rogosa and Sharpe (MRS) agar plates (Oxoid, Basingstoke, UK) for LAB. These agar plates were incubated anaerobically at 30°C using the AnaeroPack system (Mitsubishi Gas Chemical, Tokyo, Japan) for 3 days.
From the incubated MRS and GAM agar plates, a total of 301 isolates were inoculated into 3 ml of 1/2-GAM ‘Nissui’ (Kuda et al. 2005) with 1% (w/v) lactose (Wako Pure Chemical, Osaka, Japan). After incubation at 37°C for 3 days, the acid production was determined by the addition of BTB-MR reagent (Kanno et al. 2012). The lactose-utilizing strains were inoculated into MRS broth containing 0·3% (w/v) of bile (Oxgall; Sigma-Aldrich Co., St. Louis, MO, USA), and the turbidity was assessed by visual observation after incubation at 37°C for 3 days. Lactose-utilizing and bile-resistant isolates were inoculated into MRS broth adjusted to pH 3·5 and incubated at 37°C for 2 days, and growth was subsequently determined.
Thirty-four lactose-utilizing, bile-resistant and acidophilic isolates were inoculated into 20 ml of MRS broth and incubated at 37°C for 48 h. After centrifugation (2200 g for 10 min at 4°C), cell turbidity was adjusted to OD600 = 4·0 with 60% (v/v) ethanol. Ethanol (0·01 ml) containing 5% (w/v) cholesterol (Wako Pure Chemical) was added to the cell suspension (1 ml, n =3). After incubation at 37°C for 60 min, the remaining cholesterol was determined using a commercial kit (Cholesterol E-test Wako; Wako Pure Chemical).
Identification and comparison with type strain
Seven bile-resistant and acidophilic isolates exhibiting cholesterol-lowering potential were identified using biochemical methods (API 50 CHL system; bioMérieux, Marcy-l'Etoile, France) and 16S rRNA gene sequence analysis as previously reported (An et al. 2010; Kuda et al. 2010).
Cholesterol- and bile acid-lowering activity (glycocholic and taurocholic acids, Sigma-Aldrich) of the seven selected isolates and the type strain (Lactobacillus plantarum NBRC15891T), in the 0·1% (w/v) solutions, were determined as previously discussed with the commercial kits (Cholesterol E-test Wako, and Total Bile Acid Test Wako). A strain, which showed the most cholesterol- and glycocholic acid-lowering ability, was used in further experiments.
For the acid resistance test, the strains were inoculated (8·2 log CFU ml−1) into MRS broth adjusted to pH 3·0 and incubated at 37°C for 24 h. The survival number was determined with GAM agar plates. For the bile resistance test, 0·1 ml of the pre-incubated culture was inoculated into 10 ml of MRS broth containing 5% (w/v) Oxgall. During incubation at 37°C for 24 h, growth was determined at OD600. For the salt tolerance test, 0·1 ml of the pre-incubated culture was inoculated into 10 ml of MRS broth containing 10% (w/v) NaCl. After 24 h incubation at 37°C, turbidity was visually determined. To examine the effect of heating on the cholesterol-lowering activity, the cell suspension prepared with PBS (OD600 = 4·0) was heated in boiling water for 10 min. After the centrifugation, the cholesterol-lowering activity of the pellet was determined as above.
Microscopic observation and FT-IR spectroscopy
Auto-aggregation, a selected isolate and the type strain in MRS culture incubated at 37°C for 48 h, was observed by eyes. Then, the washed cells were semi-dried on a cover glass, and then the dried residues were observed using an atomic force microscope (AFM: SPM-1000; Shimadzu, Kyoto, Japan) with a contact mode probe (Kuda et al. 2012).
For the preliminary analysis of the bacterial cells, we used a Fourier transform infrared (FT-IR) spectroscopy method. After incubation at 37°C for 24 h with MRS broth, the cells were collected by centrifugation (2200 g for 10 min at 4°C). The cells were washed with the PBS and then distiled water. The washed cells were dried for two hours in a clean bench with ventilation. The dried cells were positioned in direct contact with an infrared attenuated total reflection (ATR) diamond attached on a Thermo Nicolet iS5 FT-IR spectrometer (Thermo Electron Corp., San Jose, CA, USA). ATR spectra were recorded from 40 000 to 550 cm−1 at a resolution of 2 cm−1. Each strain cell was positioned three times on the ATR and scanned at least four times for the each positioned cells. OMNIC software (Thermo Electron Corp) was used for analyses (Al-Qadiri et al. 2008). To consider the outer cell compounds, crude polymers of the AN6 cells were extracted in boiling water for 20 min and precipitated by the alcohol precipitation method (Kuda et al. 2002). Then the dried extract was analysed by the FT-IR.
This research was partially supported by the Ministry of Education, Science, Sports and Culture, Japan: Grant-in-Aid for Scientific Research (C), 25450300, 2013–2015.