Isolation, Identification, and Evaluation of New Lactic Acid Bacteria

1919 Journal of Food Protection, Vol. 79, No. 11, 2016, Pages 1919–1928 doi:10.4315/0362-028X.JFP-16-096 Published 2016 by the International Association for Food Protection This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Isolation, Identification, and Evaluation of New Lactic Acid Bacteria Strains with Both Cellular Antioxidant and Bile Salt Hydrolase Activities In Vitro SHUANG XU,1 TAIGANG LIU,2* CHIRAZ AKOREDE IBINKE RADJI,1 JING YANG,1 1Key

AND

LANMING CHEN1*

Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), China Ministry of Agriculture, College of Food Science and Technology, and 2College of Information Technology, Shanghai Ocean University, 999 Hu Cheng Huan Road, Shanghai 201306, People’s Republic of China MS 16-096: Received 3 March 2016/Accepted 9 July 2016

ABSTRACT In this study, we analyzed Chinese traditional fermented food to isolate and identify new lactic acid bacteria (LAB) strains with novel functional properties and to evaluate their cellular antioxidant and bile salt hydrolase (BSH) activities in vitro. A sequential screening strategy was developed to efficiently isolate and obtain 261 LAB strains tolerant of bile salt, acid, and H2O2 from nine Chinese traditional fermented foods. Among these strains, 70 were identified as having 2,2-diphenyl-1-picrylhydrazyl radical scavenging and/or BSH activity. These strains belonged to eight species: Enterococcus faecium (33% of the strains), Lactobacillus plantarum (26%), Leuconostoc mesenteroides (14%), Pediococcus pentosaceus (6%), Enterococcus durans (9%), Lactobacillus brevis (9%), Pediococcus ethanolidurans (3%), and Lactobacillus casei (1%). The pulsed-field gel electrophoresis genome fingerprinting profiles of these strains revealed 38 distinct pulsotypes, indicating a high level of genomic diversity among the tested strains. Twenty strains were further evaluated for hydroxyl radical scavenging activity, reducing power, and ferrous ion chelating activity exerted by both viable intact cells and/or intracellular cell-free extracts. Some strains, such as L. plantarum D28 and E. faecium B28, had high levels of both cellular antioxidant and BSH activities in vitro. These strains are promising probiotic components for health-promoting functional foods. Key words: Bile salt hydrolase activity; Cellular antioxidant activity; Chinese traditional fermented foods; Lactic acid bacteria; Probiotics; Pulsed-field gel electrophoresis genotyping

New strains of lactic acid bacteria (LAB) with novel functional properties are of interest to both academic institutions and the food industry. LAB are generally recognized as safe food-grade microorganisms. Numerous studies have revealed their beneficial effects on human health, such as maintaining balance in the gastrointestinal microbial community, acting against pathogenic microorganisms, and enhancing innate and adaptive immune responses (5, 29, 37). Oxidative stress may be involved in the pathogenesis of various human diseases, such as cancer, diabetes, cardiovascular disease, Alzheimer’s disease, rheumatoid arthritis, and cataracts (30), resulted from an imbalance between production and elimination of reactive oxygen species and free radicals (14). Superoxide anions, hydroxyl radicals (HRs), and transitional metal ions (iron and copper) are known as free radicals, which are very unstable and react rapidly with cellular molecules leading to cell or tissue damage (1). Although enzymatic and nonenzymatic antioxidative defense systems have been * Authors for correspondence. Tel: (86) 21-61900623; Fax: (86) 2161900623; E-mail: [email protected] (T.L.). Tel: (86) 2161900504; Fax: (86) 21-61900365; E-mail: [email protected] (L.C.).

found in living organisms (29), LAB are an emerging source of food-grade cellular antioxidants to combat oxidative stress (38). A high level of serum total cholesterol is considered a risk factor for cardiovascular disease, a leading cause of death in many countries (22). Thus, reducing serum cholesterol concentrations by improving the diet should decrease the incidence and mortality of ischemic heart disease and atherosclerosis (13). In some in vivo clinical trials, consumption of probiotic LAB reduced systemic cholesterol and blood lipid concentrations (4). For example, Lactobacillus reuteri can have a hypocholesterolemic effect both therapeutically and preventively (36). Although the functional mechanisms remain largely unknown, research has indicated that the ability of probiotic lactobacilli to reduce serum cholesterol concentrations in vivo is correlated with bile salt hydrolase (BSH) activity (3, 41). An increasing amount of evidence supports the clinical application of probiotics in the prevention and/or treatment of human diseases (9). Some strains of the LAB Lactobacillus, Bifidobacterium, Enterococcus, and Oenococcus possess antioxidative activity (34, 39), whereas others have BSH activity (9, 10, 21). However, to our knowledge, none

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valuable probiotic candidates (2). China has many kinds of fermented foods that have been made by traditional manufacturing procedures for hundreds or even thousands of years, and these foods contain a robust microflora and very rich LAB diversity (5). In our recent research, bacteria in some popular Chinese traditional fermented food were identified using denatured gradient gel electrophoresis fingerprint profiles and with Illumina Miseq III sequencing analysis of the 16S rRNA gene. The resulting data revealed abundant LAB in the fermented food samples (C.A.I.R., personal communication). In the present study, for the first time we aimed to develop a sequential screening strategy (Fig. 1) to efficiently isolate LAB strains tolerant to bile salts, acid, and H2O2 from Chinese traditional fermented foods. We hoped to also obtain new LAB strains with both high BSH activity and cellular antioxidant activity by further evaluation of their HR scavenging (HRS) activity, reducing power (RP), and ferrous ion chelating (FIC) activity exerted by viable intact cells (ICs) and/or intracellular cell-free extracts (CFEs). These LAB strains with novel functional properties could be used in health-promoting new functional foods.

MATERIALS AND METHODS

FIGURE 1. Flow chart of the sequential screening strategy developed in this study.

of the LAB strains have both of these two very important functional properties. Once ingested, all probiotics must cope with the extremely low pH (from 1 to 3 before to .6.0 after food consumption) environment in the human stomach and bile in the gastrointestinal tract before reaching the sections of the tract where they can have various beneficial effects (5, 7, 35). Therefore, acid resistance and bile tolerance are considered basic criteria for screening potential probiotic strains (25, 28, 44). Although hydrogen peroxide (H2O2) is a relatively weak oxidant, it is highly diffusive, has a long lifetime (16), and contributes to oxidative damage either directly or as a precursor of HR (23). H2O2 (1 mmol/liter) has been used to evaluate the resistance of Lactobacillus plantarum KCTC 3099 from kimchi to oxidative stress (23). Traditional fermented foods, particularly those made in nonindustrialized countries, are a great potential source of

Samples. Nine popular Chinese traditional foods derived from dairy, vegetables, and soybeans were collected from local manufacturers in various regions of China from July to September 2014 (Table 1). Milk cake is also called rubing in China, and kimchi was made from Chinese cabbage for individuals of Korean ancestry living in Jilin Province. Direct plating to screen for bile salt–tolerant LAB strains. LAB strains were screened for bile salt tolerance using the direct plate assay described by Chou and Weimer (7) with some modifications. Each sample (20 g) was homogenized in four volumes of phosphate-buffered saline (PBS, pH 7.4) in a lab blender (BagMixer, Interscience, Paris, France). Microbial cells in supernatant were appropriately diluted and spread on de Man Rogosa Sharpe (MRS; Beijing Land Bridge Technology, Beijing, China) agar, pH 6.8, containing 0.03 g/liter bromocresol green (Sangon Biological Engineering Technology and Services, Shanghai, China) and 0.15% oxgall bile salt (Beijing Land Bridge Technology). All plates were incubated under anaerobic conditions (Mitsubishi AnaeroPak System, Pack-Anaero, Mitsubishi Gas Chemicals, Tokyo, Japan) at 378C for 48 h. Colonies with visible color change (from green to yellow) and surrounded by a clear zone were randomly selected for evaluation by Gram staining and catalase reaction tests according to the methods described by Chen

TABLE 1. Samples collected in the People’s Republic of China Sample

Source

Milk tofu Milk cake (rubing) Dongbei kimchi Kimchi Pickled turnip brine Sichuan kimchi Guizhou kimchi Pickled taro lotus stem Stinky soybean

ESEN Inner Mongolia Special Flavour Handmade Food Shop, Chifeng, Inner Mongolia Dali Yang Shi Local Product Taobao Shop, Dali, Yunan Province Countryside Agricultural Product Healthy Shopping, Tieli, Heilongjiang Province Yang Bian Han Bai Shop, Yanji, Jilin Province Long Nan Shu Wei Shan Zhen Trade Co., Mianyang, Sichuang Province Sichuan Pao Cai (Taobao), Chengdu, Sichuang Province Ju Xiang Chilli and Tea Wholesale Shop, Guiyang, Guizhou Province Gua Gua Farm, Enshi, Hebei Province Ning Meng Special Product Jiu Wei Farmer Shop, Huaian, Jiangshu Province


et al. (6). Only gram-positive, catalase-negative LAB strains were selected as candidates for further analysis. Screening for acid-tolerant LAB strains. LAB strains were screened for acid tolerance according to the method described by Chou and Weimer (7) with slight modifications. Strains were individually inoculated into 96-well bacterial culture plates containing either MRS broth acidified with hydrochloric acid to pH 3.5 or nonacidified MRS broth at pH 6.8. After being incubated anaerobically at 378C for 48 h, the strain cultures with little or no reduction in optical density in MRS broth at pH 3.5 compared with corresponding cultures in MRS broth at pH 6.8 were selected for further analysis. Cell density was determined at 600 nm using a multimode microplate reader (Synergy, BioTek Instruments, Winooski, VT). Screening for H2O2-resistant LAB strains. LAB strains were screened for H2O2 tolerance according to the method described by Lee et al. (23) with slight modifications. Strains were individually inoculated into MRS broth (pH 3.5) containing 1 mmol/liter H2O2 and incubated anaerobically at 378C for 48 h. The cultures with the most growth were chosen for further analysis. Preparation of viable ICs and intracellular CFEs of LAB strains. ICs and CFEs of LAB strains were prepared according to the method described by Lin and Yen (27). Strains were individually incubated anaerobically at 378C for about 18 h, and 1 ml of each cell culture (109 CFU/ml) was harvested by centrifugation at 6,000 3 g for 10 min. Cell pellets were quickly washed twice with PBS (pH 7.4) and resuspended in deionized water. To obtain CFEs, cell pellets were followed by ultrasonic disruption using the Bioruptor UCD 200 and Bioruptor water cooler (Diagenode, Denville, NJ) at 48C. Cell debris was removed by centrifugation at 8,000 3 g for 10 min at 48C, and the resulting supernatant was stored at 208C. BSH activity assays. LAB strains with BSH activity were screened using the direct plate assay as described by Dashkevicz and Feighner (8). The MRS agar plates were supplemented with 0.5% (wt/vol) taurocholic acid (TCA) and 0.37 g/liter CaCl2. BSH activity was indicated by precipitation of TCA into the agar medium below and around colonies. The BSH activity of LAB strains was quantified using the ninhydrin assay described by Dong et al. (9) with slight modifications. Appropriately diluted sample (10 ll) was added to 180 ll of reaction buffer (0.1 mol/liter sodium phosphate, pH 6.0), 10 ll of TCA (10 mmol/liter final concentration), and 10 ll of liquid paraffin. The reaction was carried out at 378C for 30 min, and 200 ll of 15% (wt/vol) trichloroacetic acid was immediately added. The sample was then centrifuged to remove the precipitate. For the second reaction, 100 ll of the supernatant was thoroughly mixed with 1.9 ml of ninhydrin reagent (0.5 ml of 1% [wt/vol] ninhydrin in 0.5 mol/liter sodium citrate buffer [SCB, pH 5.5], 1.2 ml of glycerol, and 0.2 ml of SCB) and boiled for 14 min. After cooling the tubes for 3 min in tap water, the absorbance at 570 nm was measured, and a standard curve was prepared with glycine. To reduce oxidation of the enzyme, 10 mmol/liter dithiothreitol was routinely added to CFEs. One unit of BSH activity was defined as the amount of enzyme that liberated 1 lmol/liter amino acid from the TCA substrate in 1 min (expressed as units per milligram of protein). Protein concentrations were measured using the Bradford protein assay kit (Sangon Biological).

NEW BIOACTIVE LACTIC ACID BACTERIA

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Antioxidative activity assays: DPPH radical scavenging. The ability of strains to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals (DPPH-RS) was measured according to the method of Lin and Chang (26) with slight modifications. A sample of 800 ll of IC or CFE was mixed with 1 ml of freshly prepared DPPH solution (0.2 mmol/liter in ethanol; Sigma-Aldrich, St. Louis, MO) and allowed to react for 30 min in the dark at room temperature. The control contained deionized water instead of the sample solution. The scavenged DPPH radical was then monitored by measuring the decrease in absorbance (A) at 517 nm. The DPPH-RS activity was defined as follows: DPPH-RS (%) ¼ [(1 Asample)/Acontrol] 3 100%. Antioxidative activity assays: HRS. The ability of strains to scavenge HR was determined according to the methods described by Gutteridge (12) and Jeong et al. (17). The reaction mixture contained 100 ll of FeSO4 (5 mmol/liter), 500 ll of deionized water, 100 ll of sodium salicylate (5 mmol/liter in ethanol), and 200 ll of IC or CFE. The reaction was started by adding 100 ll of H2O2 (3 mmol/liter) and was incubated at 378C for 15 min. The absorbance of the generated hydroxylated salicylate complex was measured at 510 nm, and HRS activity was calculated using the following formula: HRS (%) ¼ [(Asample Acontrol)/(Ablank Acontrol)] 3 100%, where Acontrol is the absorbance of the control solution containing sodium salicylate, FeSO4, and H2O2 and Ablank is the absorbance of the blank solution containing sodium salicylate and FeSO4. Antioxidative activity assays: RP and FIC. The RP and FIC activity assays were performed according to the methods described by Lin and Yen (27) and Lee et al. (24), respectively. 16S rRNA gene sequence analysis. LAB strains identified by routine biochemical test kits (Hangzhou Tianhe Microorganism Reagent Co., Hangzhou, China) were further subjected to 16S rRNA gene sequence analysis. Genomic DNA of the LAB strains was individually prepared using the Biospin bacteria genomic DNA extraction kit (BSC12S1, Bioer Technology, Hangzhou, China) according to the manufacturer’s instructions. PCRs for amplification of bacterial 16S rRNA genes were performed as described previously (18). DNA fragments from the amplicons were sequenced by Sangon Biological, and sequences were analyzed as described previously (43). PFGE analysis. Pulsed-field gel electrophoresis (PFGE) was performed according to the methods described by He et al. (15) and Xu et al. (40). Genomic DNA of the LAB strains was individually isolated using the CHEF bacterial DNA plug kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. Each agarose plug was prepared by mixing an equal volume of the cell culture and the melted 2% CleanCut agarose provided with the kit. The prepared plug was then placed in 250 ll of lysozyme buffer with 10 ll of lysozyme stock provided with the kit and incubated at 378C overnight with gentle shaking. Genomic DNA was digested using the restriction endonucleases AscI or SmaI (New England BioLabs, Ipswich, MA) according to the manufacturer’s instructions. The resulting DNA fragments were resolved using the CHEF Mapper system (Bio-Rad) with 0.53 Tris-borate-EDTA (Sangon Biological). Electrophoresis was performed at 6 V/cm at 148C with a field angle of 1208 using 1% SeaKem Gold agarose gel (Lonza, Basel, Switzerland). AscIdigested and SmaI-digested genomic DNA fragments were resolved by applying single run steps of 17.5 h with a pulse ramping from 2.98 to 12.91 s and 17.5 h with a pulse ramping from

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TABLE 2. BSH activity and DPPH-RS activity of 70 LAB strains DPPH-RS activity (%) Strain

Enterococcus faecium B18 E. faecium B19 E. faecium B20 E. faecium B21 E. faecalis B22 E. faecium B24 E. faecium B27 E. faecium B28 Lactobacillus plantarum B5 L. plantarum B8 L. plantarum B13 Leuconostoc mesenteroides B4 Pediococcus pentosaceus B26 E. faecium C10 E. durans C11 E. durans C12 E. faecium C13 E. durans C15 E. durans C17 E. faecium C20 E. faecium C3 E. faecium C4 E. faecium C5 E. faecium C7 E. durans C8 E. faecium C9 P. pentosaceus C1 Lactobacillus brevis D6 L. brevis D11 L. brevis D13 L. brevis D14 L. brevis D15 L. brevis D20 L. plantarum D10 L. plantarum D24 L. plantarum D25 L. plantarum D27 L. plantarum D28 L. plantarum D29 P. ethanolidurans D31 Lactobacillus casei K17 L. plantarum K10 L. mesenteroides K16 P. ethanolidurans K2 P. pentosaceus K18 E. faecium M16 P. pentosaceus M2 E. faecium P1 E. faecium P12 E. faecium P2 E. faecium P7 E. faecium P8 L. plantarum T10 L. plantarum T28 L. plantarum T29 L. plantarum T9 L. mesenteroides T18 L. mesenteroides T20 E. durans V18

IC

36.38 39.80 25.64 25.34 39.65 39.80 37.04 43.16 42.56 30.28 39.22 42.92 39.36 43.30 31.05 29.20 34.97 21.06 27.81 36.89 44.80 38.25 42.74 43.95 40.81 44.52 48.58 32.46 27.09 19.24 39.8 53.28 44.08 25.13 37.68 47.28 35.22 34.86 39.22 25.13 53.67 50.71 56.94 48.73 42.39 49.50 51.00 28.99 33.12 45.30 32.55 37.84 49.43 51.92 49.15 39.81 51.78 53.35 33.90

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

CFE

0.38 0.25 0.13 0.25 0.38 1.01 1.89 0.37 0.88 1.13 1.13 0.38 0.50 0.49 1.54 0.86 0.31 1.01 0.25 0.88 0.12 0.19 0.74 0.06 0.37 0.80 0.25 0.19 0.88 0.31 0.06 0.06 0.50 0.63 0.25 0.38 0.06 0.38 0.75 0.63 0.69 1.17 0.50 1.07 0.88 0.86 0.860 0.49 1.11 1.48 0.12 0.50 1.23 0.37 1.67 0.49 0.06 0.99 0.12

15.07 14.31 10.55 11.68 14.31 13.94 12.81 16.76 25.80 18.63 32.20 28.08 27.70 30.62 31.33 15.08 34.86 16.14 21.67 25.91 33.10 31.68 30.86 34.39 31.21 30.86 27.44 29.19 11.86 8.29 27.87 45.74 36.53 14.5 15.25 21.09 25.99 18.64 38.42 9.23 50.06 45.82 48.88 44.41 38.65 38.42 45.95 13.66 10.60 31.45 17.9 13.55 38.75 44.76 45.23 38.99 46.88 51.59 12.72

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

BSH activity (U/mg)

1.18 1.42 1.18 0.65 1.18 1.18 1.30 1.82 0.86 0.33 0.56 1.49 0.33 0.89 0.82 1.81 1.14 0.54 1.47 1.43 0.20 0.54 1.02 1.08 0.82 1.08 1.95 1.30 0.56 1.82 0.65 1.63 1.18 0.65 0.56 0.65 1.96 2.59 0.98 0.86 0.54 0.54 1.59 0.54 0.20 1.49 1.73 1.34 0.93 0.01 1.14 0.74 1.34 0.20 0.01 1.08 1.63 0.93 0.35

6 0.002 6 0.004 6 0.015 6 0.018 6 0.005 6 0.002 6 0.003 6 0.002 NDa ND ND ND ND 0.050 6 0.005 ND 0.080 6 0.003 ND 0.014 6 0.001 0.009 6 0.001 0.007 6 0.001 0.031 6 0.002 0.007 6 0.001 0.040 6 0.003 0.003 6 0.001 0.017 6 0.004 0.007 6 0.001 ND 0.017 6 0.003 0.039 6 0.005 0.007 6 0.002 0.007 6 0.001 ND 0.008 6 0.001 0.066 6 0.004 0.076 6 0.003 0.073 6 0.005 ND 0.114 6 0.009 0.070 6 0.011 0.042 6 0.004 ND ND ND 0.002 6 0.001 ND 0.174 6 0.005 ND 0.014 6 0.001 0.007 6 0.002 0.004 6 0.001 0.011 6 0.001 0.009 6 0.001 ND ND ND ND ND ND 0.031 6 0.005 0.078 0.105 0.494 0.415 0.087 0.090 0.090 0.035

Origin

Stinky soybean Stinky soybean Stinky soybean Stinky soybean Stinky soybean Stinky soybean Stinky soybean Stinky soybean Stinky soybean Stinky soybean Stinky soybean Stinky soybean Stinky soybean Rubing Rubing Rubing Rubing Rubing Rubing Rubing Rubing Rubing Rubing Rubing Rubing Rubing Rubing Dongbei kimchi Dongbei kimchi Dongbei kimchi Dongbei kimchi Dongbei kimchi Dongbei kimchi Dongbei kimchi Dongbei kimchi Dongbei kimchi Dongbei kimchi Dongbei kimchi Dongbei kimchi Dongbei kimchi Kimchi Kimchi Kimchi Kimchi Kimchi Pickled turnip brine Pickled turnip brine Guizhou kimchi Guizhou kimchi Guizhou kimchi Guizhou kimchi Guizhou kimchi Milk tofu Milk tofu Milk tofu Milk tofu Milk tofu Milk tofu Sichuan kimchi


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TABLE 2. Continued DPPH-RS activity (%) Strain

IC

6 6 6 6 6 6 6 6

CFE

6 6 6 6 6 6 6 6

BSH activity (U/mg)

0.011 6 0.001 ND ND ND 0.015 6 0.002 ND 0.006 6 0.002 ND

E. faecium V24 L. mesenteroides V3 L. mesenteroides V4 L. mesenteroides V9 L. mesenteroides V12 L. mesenteroides V13 L. mesenteroides V21 L. plantarum Y12

17.50 33.54 36.75 35.75 40.16 36.57 44.52 43.43

L. plantarum Y14

41.67 6 0.93

33.57 6 0.35

ND

L. plantarum Y19

41.76 6 0.31

34.63 6 0.61

ND

L. plantarum Y26

48.73 6 0.13

38.75 6 0.82

ND

a

0.82 0.01 0.74 0.43 0.63 0.86 0.43 0.63

16.96 27.33 28.40 21.33 32.16 27.09 23.67 33.33

1.22 0.41 1.14 1.14 0.35 0.89 1.87 0.82

Origin

Sichuan kimchi Sichuan kimchi Sichuan kimchi Sichuan kimchi Sichuan kimchi Sichuan kimchi Sichuan kimchi Pickled taro lotus stem Pickled taro lotus stem Pickled taro lotus stem Pickled taro lotus stem

ND, not detected.

0.75 to 12.5 s, respectively. A DNA molecular marker (2.03 to 194.0 kb; New England BioLabs) was used as a reference. The PFGE bands were visualized, recorded, and analyzed as described previously (15). Statistical analysis. Data analysis was carried out using SPSS software (version 19.0, IBM, Armonk, NY). A one-way analysis of variance was used to determine the significance of differences between means at P 0.05. Critical difference values were used to perform multiple comparisons between means. All experiments were conducted in triplicate, and data presented are means 6 standard deviations of triplicate results.

RESULTS Isolation and sequential screening for LAB strains with tolerance to bile salt, acid, and H2O2. LAB strains tolerant of bile salt were isolated from the nine sample foods using the selective MRS agar plates. About 96 colonies derived from each sample were randomly picked from the MRS agar plates (pH 6.8) containing the oxgall bile salt (0.15%) and were identified as LAB based on the results of biochemical tests. These LAB strains were then individually inoculated into 96-well plates containing the acidified MRS broth (pH 3.5) and cultured anaerobically at 378C for 48 h. Cultures of 572 strains had little or no reduction in their cell density compared with corresponding cultures grown in the nonacidified MRS broth (pH 6.8). These 572 LAB strains that were tolerant of both bile salt and acid were then exposed to 1 mmol/liter H2O2. Of the 261 strains also resistant to the H2O2, the majority (67%) originated from fermented vegetable samples followed by 22% from fermented dairy samples and 11% from fermented soybean samples. These 261 LAB strains showing tolerance to all three stressors were chosen for further analysis. Screening for LAB strains with BSH and DPPH-RS activity. The 261 multitolerant LAB strains were qualitatively evaluated for BSH activity by the direct plate assay.

Of these cultures, 41 strains (15.7%) produced a clear zone of precipitation around colonies on the MRS agar plates containing TCA. These strains were selected for the further quantitative analysis of BSH activity. BSH activity (0.002 to 0.494 U/mg) was observed among the 41 LAB strains (Table 2). The highest BSH activity (0.078 to 0.494 U/mg) was found in LAB strains derived from the stinky soybean sample. The DPPH-RS activity of the 261 LAB strains also was measured. The scavenging activity of the ICs differed greatly depending on the strain, ranging from 1.69 to 56.94%. About 3.1% of the tested strains had very high DPPH-RS activity (.50%), whereas 33.3% of the strains had low activity (,10%). The 29 strains with a DPPH-RS value of .30% were selected for further analysis (Table 2). The CFEs of the 70 LAB strains (41 with BSH activity and 29 with high DPPH-RS activity) also differed in their DPPH-RS, ranging from 8.29 to 51.59% (Table 2). Nevertheless, higher values were observed for the ICs than for their corresponding CFEs, indicating that the ICs of the LAB strains had a higher DPPH-RS activity than did the CFEs.

Identification of the 70 LAB strains by 16S rRNA gene sequence analysis. 16S rRNA genes were individually amplified from genomic DNA of the 70 LAB strains by PCR, and the DNA sequences of the amplicons were determined. BLAST analysis of the obtained sequences against public databases revealed high sequence identities (.97%) with eight known species of LAB (Table 2): Enterococcus faecium (33%, 23 strains), L. plantarum (26%, 18 strains), Leuconostoc mesenteroides (14%, 10 strains), Pediococcus pentosaceus (6%, 4 strains), Enterococcus durans (9%, 6 strains), Lactobacillus brevis (9%, 6 strains), Pediococcus ethanolidurans (3%, 2 strains), and Lactobacillus casei (1%, 1 strain). The obtained 16S rRNA gene

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FIGURE 2. SmaI PFGE genotyping of the 39 LAB strains in group I identified in this study.

sequences have been deposited in the GenBank database under accession numbers KU301237 through KU301306. The L. casei and P. pentosaceus strains had no BSH activity, whereas most of the E. faecium strains (95.7%, 22 of 23 strains) had BSH activity. In this study, strains with DPPH-RS values (both ICs and CFEs) of .30% were considered high activity strains, and 32 LAB strains met this criterion (Table 2). High DPPH-RS activity was most common in L. plantarum (61.1%, 11 of 18 strains), followed by L. mesenteroides (40.0%, 4 of 10 strains) and E. faecium (39.1%, 9 of 23 strains).

Phylogenetic relationships of the 70 LAB strains. To track the relatedness of the 70 identified LAB strains, we obtained genome fingerprinting profiles by PFGE analysis. Seven strains could not be evaluated under the PFGE conditions used in this study. All the tested strains fell into two groups, I and II, based on the SmaI and AscI PFGE profiles, respectively. Group I included 39 strains: 29 of Enterococcus and 10 of Leuconostoc. Based on a significant difference of one DNA band of 10 to 190 kb on the PFGE gels, cluster analysis of the SmaI PFGE profiles of group I revealed 30 pulsotypes (Fig. 2) that clustered at 87% similarity, a cutoff value suggested for identifying isolates

belonging to the same epidemic strain (33). Within group I, the majority of the pulsotypes (80%, 24 of 30) had a single genome fingerprint pattern (strain), and the others contained two or three strains. All strains shared 35 to 100% similarity and were assigned to four distinct clusters (Fig. 2; clusters A through D), indicating that the identified Enterococcus and Leuconostoc strains in group I differed considerably, with remarkable genetic diversity in the tested samples. Among the 39 identified LAB strains in group I, 29 (74.4%) had BSH activity and 15 (38.5%) had high (.30%) DPPH-RS activity (both ICs and CFEs). The majority of the BSH active strains (79.3%, 23 of 29 strains) were grouped in clusters C and D, which were derived from the rubing, stinky soybean, and Guizhou kimchi samples tested in this study. Of these strains, the highest BSH activity (0.494 U/ mg) was detected in strain E. faecium B20, which was recovered from the stinky soybean sample. Most of the strains with high DPPH-RS activity were grouped in cluster B, originating from the rubing sample (Fig. 2). However, L. mesenteroides K16 in cluster A also had high DPPH-RS activity in both CFE (48.88%) and IC (56.94%). Group II included 24 identified LAB strains of Lactobacillus. The cluster analysis of the AscI PFGE profiles derived from group II revealed 18 pulsotypes with


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FIGURE 3. AscI PFGE genotyping of the 24 LAB strains in group II identified in this study.

DNA fragments ranging from 10 to 190 kb (Fig. 3). Of these, 72.2% (13 of 18 strains) had unique genome fingerprints. All the strains in group II were grouped into four major clusters (Fig. 3, clusters A through D) at a 50 to 100% similarity level, indicating high genomic diversity among the group II strains. Within group II, strains with BSH activity and high DPPH-RS activity clustered separately; the former were grouped mostly in clusters B and C, and the latter were mostly in cluster A (Fig. 3). L. brevis D15 in cluster B that

originating from Dongbei kimchi had very high DPPH-RS activity (CFE: 45.74%; IC: 53.28%).

HRS activity, RP, and FIC activity of the LAB strains. Based on the results obtained with the other screening experiments, 20 LAB strains with high BSH and/ or DPPH-RS activity were chosen for further analysis of HRS activity, RP, and FIC activity (Table 3). In the tested strains, high HRS activity was exerted by both ICs (36.95 to 79.93%) and CFEs (64.48 to 85.45%). However, unlike the

TABLE 3. HRS activity, RP, and FIC activity of 20 LAB strains HRS activity (%) Strain

E. durans C8 E. durans C12 E. durans C15 E. durans C17 E. durans V18 E. faecium B18 E. faecium B19 E. faecium B20 E. faecium B21 E. faecium B22 E. faecium B24 E. faecium B27 E. faecium B28 E. faecium C3 E. faecium C5 E. faecium C10 E. faecium M16 E. faecium P1 E. faecium P8 L. plantarum D28

ICs

36.95 58.66 68.53 68.51 67.65 56.91 67.32 58.18 79.93 45.18 47.92 51.80 61.36 70.18 47.70 60.04 70.61 73.14 67.11 71.93

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

RP (lmol/liter cysteine) CFEs

1.87 0.95 0.19 0.50 1.00 2.05 1.48 1.66 2.00 1.06 1.33 1.33 0.68 1.00 1.51 1.66 1.06 1.98 0.87 1.25

76.24 72.48 85.45 84.97 81.94 68.97 80.85 73.58 83.88 74.30 64.48 64.73 80.24 77.21 83.27 79.52 66.06 73.70 85.21 82.30

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

ICs

0.21 0.21 0.36 0.21 0.76 0.76 1.11 1.17 0.84 1.47 0.92 0.96 1.11 1.64 0.63 1.28 0.84 0.42 0.56 0.21

61.93 42.28 37.45 36.54 69.59 17.80 45.11 18.91 17.50 23.64 28.38 32.31 121.59 107.68 19.11 15.78 20.22 12.66 27.67 224.51

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

FIC activity (mg/liter), CFEs

CFEs

1.84 2.92 3.02 0.61 2.01 2.58 1.06 0.87 1.81 2.06 1.32 1.98 2.15 5.86 1.75 1.49 3.20 1.21 1.92 7.60

18.70 17.74 18.22 17.74 29.22 12.00 16.74 8.66 7.22 5.79 7.70 10.09 34.40 25.87 10.09 14.40 10.57 10.57 11.05 52.65

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

1.66 2.99 2.49 1.66 2.99 0.83 1.43 0.83 2.19 1.66 0.83 2.19 0.83 0.83 1.66 0.83 0.83 0.83 2.49 5.17

39.13 13.57 37.29 18.97 43.21 2.11 2.45 10.38 9.88 18.45 16.21 6.32 13.48 30.70 36.50 19.50 3.75 8.96 15.94 9.62

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

5.97 1.27 0.60 1.19 1.50 1.55 1.18 0.76 1.72 0.91 1.81 0.68 0.79 1.27 1.39 0.60 1.57 2.78 0.23 0.60

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DPPH-RS activity, the CFEs had higher HRS activity than did their corresponding ICs. Two strains had the highest HRS activity with either CFEs or ICs: E. faecium B21 (IC: 79.93%, CFE: 83.88%) and E. durans C15 (CFE: 85.45%, IC: 68.53%). The tested LAB strains also had various degrees of RP (Table 3). Remarkably, their ICs showed higher RP (1.09- to 4.26-fold) than the corresponding CFEs. Extremely high RP was detected from the IC of L. plantarum D28 (224.51 lmol/liter cysteine) compared with other strains (12.66 to 121.59 lmol/liter cysteine). The CFE of L. plantarum D28 also had much higher RP (52.65 lmol/liter cysteine) than did the other strains (5.79 to 34.40 lmol/liter cysteine). The CFEs of the tested LAB strains were also investigated for FIC activity, which differed greatly depending on the strain, ranging from 2.11 to 43.21 mg/ liter (Table 3). Four strains had very high FIC activity compared with that of the other strains: E. durans V18 (43.21 mg/liter), E. durans C8 (39.13 mg/liter), E. durans C15 (37.29 mg/liter), and E. faecium C5 (36.50 mg/liter).

DISCUSSION In the present study, a number of LAB strains were isolated from samples of nine Chinese traditional fermented foods. A sequential screening strategy (Fig. 1) was developed, and 261 LAB strains with tolerance to bile salt, acid, and H2O2 were obtained. To our knowledge, strains with these characteristics have not been described previously. The DPPH-RS assay is widely used to evaluate antioxidant activity because of its simplicity, rapidity, sensitivity, and reproducibility compared with other methods (42). In this study, this assay was used to determine the cellular antioxidant activity of the 261 multitolerant LAB strains. Of these 261 strains, 32 had high DPPH-RS activity exerted by both ICs and CFEs. Consistent with previous reports based on fewer LAB strains (26, 38), our data (Table 2) provide direct evidence of the higher scavenging activity (1.09- to 4.26-fold) exerted by ICs than by CFEs. The 261 LAB strains were also examined for their BSH activity, and 41 BSH-positive strains were identified by qualitative and quantitative assays. Of these, the highest percentage of BSH-positive strains were Enterococcus (93.1%, 27 of 29 strains), followed by Lactobacillus (40%, 10 of 25 strains) and Pediococcus and Leuconostoc (33.3 to 20%). High percentages of BSH-positive Enterococcus strains were also reported previously (10). In the present study, E. faecium B20 had the highest BSH activity (0.494 U/mg), which was also higher than that of many LAB strains reported previously. For example, Kumar et al. (21) reported that L. plantarum 91 had relatively high BSH activity (0.299 U/mg) against total cholesterol among 15 tested Lactobacillus strains (0.071 to 0.299 U/mg). In the present study, the phylogenetic relationships of the 70 LAB strains with BSH activity and high DPPH-RS activity were investigated by PFGE molecular typing analysis. The SmaI PFGE and AscI PFGE profiles revealed a total of 38 pulsotypes. The tested strains with BSH and antioxidant activities were distributed among the PFGE clusters of the SmaI and AscI profiles, suggesting the


absence of a relationship between the SmaI or AscI restriction sites in the bacterial genomes and the sites where the sequences responsible for the functional properties are located. No correlation was found between the LAB species and the particular functional properties of the tested strains. The high intraspecies heterogeneity of the LAB strains was also observed in previous studies (40). In this study, the high level of genetic diversity within the LAB species increases the potential of finding new strains with novel functional properties, because organoleptic characteristics are usually strain specific (31). Our data also indicated that the presence of LAB strains with different functional properties differed depending on the type of food sample, perhaps due to the selective pressure of the ecosystem where the fermented food was made (11). HR, which is the most reactive oxygen radical, can react with all biomacromolecules in living cells and can induce severe cell damage. Therefore, HRS plays a critical role in reducing cell oxidative damage (42). The HRS activity of LAB strains has been reported in previous studies (32, 42). Ren et al. (32) reported that the HRS activity exerted by the ICs of nine Lactobacillus strains isolated from fermented food and human intestine samples was 10.37 to 94.26% in vitro. In the present study, the 20 LAB strains with high BSH and/or DPPH-RS activity also had high HRS activity exerted by both CFEs (64.48 to 85.45%) and ICs (36.95 to 79.93%). The RP of compounds has been considered an important indicator for their potential antioxidative activity. Some enzymes (e.g., catalase, NADH oxidase, and NADH peroxidase) and nonenzymatic compounds (e.g., glutathione, ascorbate, and a-tocopherol) can minimize the generation of reactive oxygen species and control the transitional metal ions to prevent the oxidation reaction (42). Cysteine has been used as the standard for expression of the RP in a number of previous studies (27, 34, 39, 42). Consistent with previous research (34), in our study ICs of the tested LAB strains had higher RP (1.09- to 4.26-fold) than did their corresponding CFEs. A relatively high RP was detected from the ICs of L. plantarum D28 (224.51 lmol/ liter cysteine), which was also higher than that yielded by the wine LAB Oenococcus oeni strain SD-1f (182.50 lmol cysteine/liter) in a previous report (34). Transitional metal ions can catalyze the generation of reactive oxygen species, which in turn oxidize unsaturated lipids in cells (20). Among these metal ions, iron is the most abundant and highly reactive in formation of reactive oxygen species. Iron also can react with H2O2 and superoxide anion to produce HR by the Fenton reaction (19). Various levels of FIC activity in CFEs of LAB strains have been reported previously (24, 27, 34). In the present study, the FIC activity of CFEs of the tested LAB strains ranged from 2.11 to 43.21 mg/liter; E. durans V18 had the highest activity (43.21 mg/liter). Physiological chelators, such as EDTA, diethylenetriaminepentaacetic acid, penicillamine, and desferrioxamine, in the CFEs of LAB strains might contribute to the FIC activity by capturing metal ions and thus preventing oxidation damage. This study is the first investigation of a sequential screening strategy to efficiently isolate 261 LAB strains with


tolerance to bile salt, acid, and H2O2 from nine Chinese traditional fermented foods. Of these 261 strains, 70 also had DPPH-RS and/or BSH activity and were identified as belonging to eight LAB species: E. faecium, L. plantarum, L. mesenteroides, P. pentosaceus, E. durans, L. brevis, P. ethanolidurans, and L. casei. The SmaI and AscI PFGE genome fingerprinting profiles revealed 38 pulsotypes, indicating a high level of genomic diversity among the tested strains. Some strains, such as E. faecium B28 and L. plantarum D28, had high cellular antioxidant and BSH activity in vitro, and their functions in vivo will be investigated in our future research.

ACKNOWLEDGMENTS This study was supported by grants from the Shanghai Municipal Education Commission (B-9500-10-0004, 13YZ098, and ZZhy12028) and the National Nature Science Foundation of China (31271830).

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