RESEARCH ARTICLE International Microbiology 20(2):75-84 (2017) doi:10.2436/20.1501.01.287. ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org Characterization of high exopolysaccharide- producing Lactobacillus strains isolated from mustard pickles for potential probiotic applications Jing-Yao Huang,1 Cheng-Yen Kao,2 We-Sin Liu,1 Tony J. Fang1,3* 1Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan, 2Department of Biotechnology and Laboratory Science in Medicine, School of Biomedical Science and Engineering, National Yang Ming University, Taipei, Taiwan, 3Food Industry Research and Development Institute, Hsinchu, Taiwan Received 27 March 2017 · Accepted 8 May 2017 Summary. The aim of this study was to characterize high exopolysaccharide (EPS)-producing lactic acid bacteria (LAB) isolated from mustard pickles in Taiwan for potential probiotic applications. Among 39 collected LAB strains, four most productive EPS-producing strains were selected for further analysis. Comparative analyses of 16S rDNA genes rpoA and pheS sequences demonstrated that these strains were members of Lactobacillus plantarum-group (LPG). NCD 2, NLD 4, SLC 13, and NLD 16 showed survival rates of 95.83% ± 0.49%, 95.07% ± 0.64%, 105.84% ± 0.82%, and 99.65% ± 0.31% under simulated gastrointestinal conditions, respectively. No cytotoxic effects on macrophage RAW 264.7 cells were observed when they were treated with a low dose (1 μg/ml) of stimulants extracted from the tested LAB strains. The production of nitric oxide in RAW 264.7 cells incubated with various LAB stimulants showed a dose-dependent increase. Among the four strains, SLC 13 showed higher inhibitory activity on growth of Enterococcus faecalis (BCRC 12302) and Yersinia enterocolitica (BCRC 10807). NLD 4 showed strong inhibitory activity against Escherichia coli O157:H7 (ATCC 43894) as compared with the other three strains. In summary, our results suggest that Lactobacillus pentosus SLC 13 may be a good candidate for probiotic appli- cations and for development of antibacterial compounds. [Int Microbiol 20(2):75-84 (2017)] Keywords: Lactobacillus spp. · exopolysaccharide · probiotics Introduction (LAB) group) and Bifidobacteriumare the most common pro- biotics used for human nutrition. LAB generally have many useful properties, including tolerance to gastric acid and bile The market of dietary supplements that promote health has salts, tolerance to antimicrobial agents, improvement of im- increased since 1990s. Among them, probiotics are defined as mune responses, gastrointestinal adsorption, and stability du- live microorganisms that could confer a health benefit on the ring processing [30]. Moreover, fermentation by the LAB host when adequate amounts are administered [18]. Currently, group is a natural bioprocessing technology for production of the genera Lactobacillus (a member of the lactic acid bacteria foods such as milk, vegetables, yogurt, cheese, meat, and co- coa beans. LAB-mediated fermentation thus improves nutri- *Corresponding author: Tony J. Fang tion and preserves qualities of food and beverage products for E-mail: [email protected] long periods [7]. 76 INT. MICROBIOL. Vol. 20, 2017 HUANG ET AL. The beneficial effects of LAB and its derivative exopo- 6000 ×g for 15 min after incubation for 48 h, and the resulting supernatant lysaccharide (EPS), include the prevention and treatment of was collected carefully. An aliquot of the supernatant (1 ml) was mixed with ° diarrheal disease, prevention of infections, antitumor activity, 4 ml of 95% ethanol, and then incubated at 4 C for 24 h. The precipitated EPS was centrifuged at 9000 ×g for 15 min, and the supernatant was dis- immunomodulation, prevention and treatment of allergies, carded. The precipitate of pure EPS was dried in the oven at 60 °C for 24 h. and alleviation of lactose intolerance [10,22,29,30,31,37]. In The production of EPS was analyzed by the phenol-sulfuric method using addition, the EPS produced by LAB is an important source of glucose as a reference standard [35]. Among collected isolates, EPS yields natural alternatives to various chemical compounds and plays were ranging from 0 to 0.43 ± 0.04 g/l. Four high EPS-producing LAB strains a critical role in the production of fermented dairy products in were further identified by means of the API 50 CH (BioMérieux, Marcy Asia, Eastern Europe, and Northern Europe. However, the l’Etoile, France) strips and gene sequences (16S rDNA, rpoA, and pheS genes) to the species level [4]. characteristics and production of EPS by LAB show a great variety, which depends on the type of LAB strains, culture DNA techniques. Mini Qiagen columns and a QiaAmp DNA extraction conditions, and composition of the medium [14]. kit (Qiagen, Valencia, CA) were used for chromosomal DNA extraction. PCR Mustard pickles, a vegetable processing product that is was carried out according to the manufacturer’s instruction using Taq poly- fermented by LAB and yeast, is an important dietary dish in merase (Promega, Madison, WI, USA). Taiwan. Traditional processing of mustard pickles is as fo- DNA sequencing and phylogenetic analysis. The sequence of llows: whole mustard vegetables (Brassica juncea) are pac- 16S rDNA gene, the housekeeping genes rpoA (the gene encoding the DNA- ked in layers with addition of solar salt to each vegetable la- dependent RNA polymerase alpha-subunit), and pheS (the gene encoding the yer. Followed by placement of a large rock on top of the con- phenylalanyl-tRNA synthase alpha-subunit) were used for phylogenetic anal- tainer, the mustard inside the container is slowly pressed and ysis and species identification. Primers and conditions for PCR amplification fermented. In this study, we aimed to isolate and characterize of the 16S rDNA, rpoA, and pheS genes were described previously [4]. To LAB strains with potential probiotic applications from mus- draw a phylogenetic tree of the 16S rDNA, rpoA, and pheS genes, sequences were first aligned using the CLC Workbench (CLC sequence viewer 7.0, tard pickles. To our knowledge, this is the first report of cha- CLC Bio/Qiagen, Aarhus, Denmark), and the aligned file was then subjected racterization of EPS-overproducing LAB strains from mus- to the neighbour-joining method to draw a phylogenetic tree with bootstrap tard pickles in Taiwan. analysis of 1000 replicates. Random amplified polymorphic DNA (RAPD)–PCR amplifi- Materials and methods cation. Three different primers (primers B, 5′-AACGCGCAAC-3′; primer E, 5′-GGCGTCGGTT-3′; and primer F, 5′-GGCCACGGAA-3′) for RAPD– PCR analysis used in this study were described previously [5]. In brief, the Sampling and isolation of lactic acid bacteria. Fifteen mustard PCR mixtures were made in a volume of 50 μl containing 100 ng of DNA, pickle samples for LAB isolation were collected in traditional markets (Beipu 10 pmol of primer, 0.15 mM each deoxynucleoside triphosphate, reaction Township, Hsinchu County, 4 samples and Meinong District, Kaohsiung buffer with MgCl , and 1 U of Taq DNA polymerase. The cycling program City, 3 samples) and a mustard pickle production center (Dapi Township, 2 consisted of 1 cycle of 94 °C for 5 min; 8 cycles of 94 °C for 30 s, 36 °C for Yunlin County, 8 samples) in Taiwan. The samples were transported to the 1 min, and 72 °C for 90 s; 35 cycles of 94 °C for 20 s, 36 °C for 30 s, and laboratory at room temperature for isolating LAB in batches within 24 h of 72 °C for 90 s; and a final cycle of 72 °C for 3 min. The PCR products were acquisition. electrophoretically separated in 1% agarose gels. The RAPD profiles were MRS (de Man, Rogosa, and Sharpe) agar plates were used for LAB iso- used to discriminate between the different isolates. lation. Each mustard pickle sample was crushed and mixed with phosphate- buffered saline (PBS) buffer. A serial dilution of the suspension was spread Tolerance of simulated gastrointestinal conditions. To eval- onto the surface of MRS agar plates. Samples were then incubated under an- uate the survival of LAB strains in an environment that mimics in vivo human aerobic conditions at 37 °C for 48 h. Six colonies were randomly selected upper-gastrointestinal tract conditions, an in vitro methodology was used. For from each MRS agar plates (total 90 colonies) and initially tested for acid acid tolerance analysis, 0.1 ml of a LAB culture medium from an overnight production, catalase activity, bacterial motility, Gram staining, and cell mor- culture was added to 9.9 ml of PBS (pH = 3). The mixture was incubated at phology. Among 90 colonies, a total of 39 LAB strains were isolated from 37 °C with agitation (80 rpm) for 3 h. One milliliter of the culture medium mustard pickles. The selected strains were stored at –80 °C in MRS broth was mixed with 9 ml of PBS, followed by serial dilution and spreading on containing 16% glycerol until testing. MRS agar plates. The number of surviving bacteria was measured by the plate counts after 48-h incubation under anaerobic conditions. The survival Exopolysaccharide production analysis. The EPS production of rate of LAB strains under acidic conditions was calculated according to the isolated LAB strains was evaluated according to a previous study with a following equation: modification [17]. In brief, the liquid fermented by LAB was centrifuged at Survival rate (%) = [A1(Log CFU/ml)/A0(Log CFU/ml)] × 100, where A1 EPS-PRODUCING LAB FOR PROBIOTIC APPLICATION INT. MICROBIOL. Vol. 20, 2017 77 is the viable count of LAB after 3 h at pH 3.0, and A0 is the viable count of Cell viability testing and nitric oxide (NO) production. For LAB at 0 h. the treatment of RAW 264.7 cells, LAB stimulants were prepared from an For analysis of bile salt tolerance, after acid treatment for 3 h, the surviv- overnight LAB culture.