Probiotic characteristics of lactic acid bacteria isolated from ... - iubmb

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Probiotic characteristics of lactic acid bacteria isolated from kimchi J.-H. Chang1,2, Y.Y. Shim3, S.-K. Cha2 and K.M. Chee1 1 School of Life Sciences and Biotechnology, Korea University, Seoul, South Korea 2 Traditional Foods Research Center, Korea Food Research Institute, Kyunggi-do, South Korea 3 Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, SK, Canada

Keywords cytotoxicity, ELISA, kimchi fermentation, lactic acid bacteria, Lactobacillus acidophilus KFRI342, probiotics. Correspondence Youn Young Shim, Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place Saskatoon, SK S7N 0X2, Canada. E-mail: [email protected] Kew M. Chee, School of Life Sciences and Technology, Korea University, Anam-dong, 21 Sungbuk-gu, Seoul 136-701, South Korea. E-mail: [email protected]

2009 ⁄ 0936: received 28 May 2009, revised 18 November 2009 and accepted 30 November 2009 doi:10.1111/j.1365-2672.2009.04648.x

Abstract Aims: The present work was aimed at identifying strains of lactic acid bacteria (LAB) from kimchi, with properties suitable for use as starter cultures in yogurt fermentation. Methods and Results: A total of 2344 LAB strains were obtained from two different sources, one group consisted of commercial LAB strains from kimchi, and the second group consisted of those strains isolated from various types of kimchi. The LAB strains from both groups were screened for resistance to biological barriers (acid and bile salts), and the four most promising strains were selected. Further analysis revealed that KFRI342 of the four selected strains displayed the greatest ability to reduce the growth of the cancer cells, SNU-C4. The in vivo efficacy of strains in quinone reductase induction assay was evaluated, and the extent of DNA strand breakage in individual cells was investigated using the comet assay. Strain KFRI342 was identified as Lactobacillus acidophilus by 16S rRNA sequence analysis, showed protection against tumour initiation and imparted immunostimulation as well as protection against DNA damage. Conclusions: Strain KFRI342, which showed probiotic characteristics reducing cancer cell growth, could be a suitable starter culture for yogurt fermentation because of its strong acid production and high acid tolerance. Significance and Impact of the Study: This is the first report to describe a bacterium, isolated from kimchi, Lact. acidophilus KFRI342 which has the probiotic characteristics and the acid tolerance needed for its use as a starter culture in yogurt fermentation.

Introduction The lactic acid bacteria (LAB) a component of several fermented foods including dairy products have long been consumed by people. LAB are the focus of intensive international research for their essential role in most fermented foods. Their ability to produce various antimicrobial compounds, (Temmerman et al. 2002), antitumoural activity (Hilde et al. 2003), inhibition of pathogenic species (Salminen et al. 1998), protection against colon cancer (Al-Rwaily et al. 2005), stimulation of the immune system (Isolauri et al. 2001), stabilization of gut 220

microflora (Gibson et al. 1997) and alleviation of lactose intolerance (de Vrese et al. 2001) is of high value. At present, exports of kimchi have been rising, and it has been developed as a global cultural product by its registration with CODEX in 2001 (Kim et al. 2006). Further, one of the most important factors in human longevity is the control of tumours, and a variety of LAB originating from yogurt has been reported to possess antitumour effects as well as antimutagenic activities (Hirayama and Rafter 2000). In Korea, LAB are utilized as starter micro-organisms in Korean-fermented vegetable kimchi. Korean kimchi is

ª 2010 The Authors Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 220–230

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the product of LAB fermentation of more than 100 types of vegetables, and various species of LAB are involved in kimchi fermentation, including those in the Leuconostoc and Lactobacillus genera (Mheen and Kwon 1984). A typical adult Korean consumes an average of 50–200 g of kimchi per day, and its market in Korea exceeded 130 million USD in 2002 (Kim and Chun 2005). There are many types of kimchi, and there has been increasing consumption of factory-made products with a 15–20% yearly rise in sales. More than 200 kinds of kimchi are made at home using traditional methods. Kimchi fermentation is initiated by various endogenous micro-organisms in the raw materials. During this process, the number of LAB increases with the onset of acid production and then remains the dominant micro-organism in properly fermented kimchi (107–109 CFU g)1) (Lee et al. 1992). The LAB in kimchi are either psychrophilic or psychrotrophic, facultative anaerobic organisms that are highly resistant to salts. In a study by Mheen and Kwon (1984), it was discovered that the best flavour of kimchi resulted when the pH was 4Æ2, and the lactic acid on the third day after fermentation at 20C reached 0Æ6% (v ⁄ v) (Mheen and Kwon 1984). Probiotic cultures applied in yoghurt need to be particularly resistant to acidic conditions, because the pH of this type of products is at least 4Æ5 and often much lower (Vinderola et al. 2000). Freshly isolated LAB of human origin are often sensitive to acidic conditions (Park et al. 2002). In contrast, LAB that are currently used in fermented milk products are more resistant to acidic conditions and, therefore, may be more suitable if they also display desirable effects on the host (Cha et al. 2008a). This suggests a potential for Lactobacilli isolated from kimchi to be used for yogurt production. The requirements include resistance to gastric acid and physiological concentrations of bile in yogurt fermentation as probiotic (Gilliland et al. 1984). Therefore, research aimed at identifying strains of LAB from kimchi with properties suitable for use as starter cultures in yogurt fermentation is necessary. In addition to being acceptable for yogurt fermentation, these bacteria may have antitumoural properties. There are studies to suggest that LAB play an important role and function in the host’s immunoprotective system by increasing specific and nonspecific mechanisms to have an antitumour effect (Kato et al. 1983; Schiffrin et al. 1995). Many of the antitumour activities attributed to LAB cultures have been suggested to involve an enhanced function of the immune response. More work needs to be carried out to identify the specific strains and strain characteristics responsible for antitumour effects and the mechanisms by which these effects are mediated. Therefore, in this study, we attempted to identify strains of LAB from kimchi with properties suitable for

Anticancer activity of Lactobacillus strains isolated from kimchi

use as an antitumoural starter cultures in yogurt fermentation. The first group of strains, group A, consisted of 135 commercial strains of LAB, isolated from kimchi. Group B consisted of 2209 strains of LAB, isolated from various types of kimchi. The screening criteria to preselect these strains involved assessing the capacity of the strains to undergo milk fermentation and to assess their resistance to biological barriers (acid and bile salts). We describe the evaluation of the cytotoxicity assay of these strains using CHO-K1 and SNU-C4 cell lines. In addition, the in vivo efficacy of strains in quinone reductase (QR) induction assay was evaluated, and the extent of DNA strand breakage in individual cells was investigated using the comet assay. Materials and methods Materials The chemicals and cell culture materials were obtained from the following sources; pentoxifylline, low melting point agarose, Roswell Park Memorial Institute (RPMI) 1640 medium, minimum essential medium (MEM) Eagle’s, foetal bovine serum (FBS), ethidium bromide (EtBr), ethylenediaminetetraacetic acid (EDTA), disodium salt, Tris–buffer, sodium bicarbonate, trypsin, phosphatebuffered saline with Tween 20 [PBST; 0Æ01 mol phosphate buffer with 138 mmol NaCl, 2Æ7 mmol KCl, 0Æ05% (w ⁄ v) Tween 20, pH 7Æ4], phosphate–citrate buffer tablets (50 mmol phosphate–citrate buffer, pH 5Æ0, one tablet per 100 ml)1), bile salts and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenylte-trazolium bromide (MTT) were purchased from Sigma (St Louis, MO, USA). The 96-Well microtitre plates were obtained from Nunc Co. (Roskilde, Denmark). Cell lines CHO-K1 (hamster ovary), SNU-C4 (human colon cancer) and Hepa-1c1c7 (Hepa) cell lines were obtained from Korean Cell Line Bank (Seoul, South Korea). The colon adenocarcinoma cell line HT-29 and the mouse macrophage cell line RAW 264.7 were obtained from American Type Culture Collection (Rockville, MD, USA). Strains For this study, a total of 2344 LAB strains were investigated; 135 commercial LAB strains were isolated from kimchi and were called as Lactobacillus acidophilus (n = 14), Lactobacillus brevis (n = 7), Lactobacillus casei ssp. casei (n = 26), Lactobacillus plantarum (n = 20), Lactobacillus curvatus (n = 3), Lactobacillus delbrueckii


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ssp. delbrueckii (n = 6), Lactobacillus fermentum (n = 4), Leuconostoc lactis ssp. lactis (n = 25), Lactococcus lactis ssp. lactis biovar diacetylactis (Lact. diacetylactis, n = 2), Weissella paramesenteroides (n = 2), Leuconostoc mesenteroides ssp. mesenteroides (n = 18) and Lactobacillus sakei (n = 8). The other 2209 LAB isolates were obtained from 140 kinds of kimchi. All cultures were maintained as frozen stocks at )70C. Prior to each of the experiments, bacteria were propagated twice in de Man–Rogosa–Sharpe (MRS) broth (Difco Lab., Detroit, MI, USA) at 37C for 24 h. All strains of LAB were kindly provided by Korea Food Research Institute (KFRI, Kyunggi-do, South Korea). Lactobacillus acidophilus KFRI34 was deposited with a patent in Korean Collection for Type Culture (Daejeon, South Korea) on June 2005 (Cha et al. 2008a).

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and deoxynucleoside triphosphate (dNTP; Takara, Shiga, Japan) sequenced using an ABI 3730 XL DNA analyzer (Applied Biosystems, Foster City, CA, USA) in combination with a BigDye terminators ver. 3.1 cycle sequencing kit (Applied Biosystems). PCR products were sequenced with forward and reverse primers, 518F (5¢-CCA GCA GCC GCG GTA ATA CG) and 800R (5¢-TAC CAG GGT ATC TAA TCC). PCR was performed with one cycle of denaturation for 3 min at 94C, followed by 35 cycles of denaturation for 45 s at 94C each, annealing for 1 min at 55C and extension for 1 min at 72C. The obtained DNA sequence was deposited in the GenBank database under accession number AY773947. The nucleotide sequence for the 16S rRNA was compared with the registered sequences in the GenBank databases using Blast (http://www.ncbi.nlm.nih.gov/BLAST/).

Isolate identification To identify the isolated LAB, a API 50 CHL carbohydrate test kit (BioMe´rieux Co., Marcy I’Etoile, France) and 16S rRNA sequence analysis were used. For API 50 CHL kit, LAB maintained on modified MRS media were dissolved in the 5 ml solvent, adjusted for their number with McFarland standard no. 4 (Densima; BioMe´rieux) and smeared on API 50 CHL media. Subsequently, the media were filled with mineral oil, cultured at 37C for 48 h and confirmed by API Lab plus software (BioMe´rieux). DNA extraction was conducted as modified by Keegan et al. (2003). Maintenance medium was removed from the wells by aspiration and discarded, and the infected monolayers were washed three times with 500 ll of sterile PBS. Cell monolayers were harvested in 200 ll of 10 mmol Tris, 1 mmol EDTA buffer (pH 8Æ0). Resuspended cells were transferred to microcentrifuge tubes and collected by centrifugation for 10 min at 8000 g. The supernatant was removed by aspiration, and the pellet was rinsed in a 200 ll aliquot of GeneAmp polymerase chain reaction (PCR) buffer II (Applied Biosystems, Melbourne, Vic., Australia). Samples were pelleted for 10 min at 10 000 g, and the supernatant was discarded. The pellet was resuspended in 50 ll of InstaGene matrix (Bio-Rad, Regents Park, NSW, Australia), vortexed briefly and heated for 10 min at 56C followed by 100C for 20 min. The sample was centrifuged at 17 000 g to pellet the InstaGene matrix, and a 10 ll aliquot of the supernatant was used in the PCR assay. A fragment of the 16S rRNA gene was amplified according to the method described by Piutti et al. (2003) using primers 27F (5¢-AGA GTT TGA TCM TGG CTC AG) and 1492R (5¢-TAC GGH TAC CTT GTT ACG ACT T). PCR products of the expected size (c. 1400 bp) were purified with the Montage PCR96 Cleanup kit (Millipore, Billerica, MA, USA) to remove unincorporated PCR excess primers 222

Acid tolerance Resistance to acidic conditions was tested by growing LAB in MRS broth adjusted to pH 2Æ0 and 3Æ0 using 0Æ1 N HCl at 30C. Active cultures of LAB strains were inoculated (2Æ0%, v ⁄ v) with pH-adjusted MRS broth, incubated at 37C for 72 h and compared to McFarland standard no. 4 (Densima; BioMe´rieux). Growth was recorded as positive (+), and no growth was recorded as negative ()). Bile tolerance The ability of the strains to grow in bile salt was determined. Briefly, Lactobacilli MRS broth was prepared with and without 0Æ1, 0Æ3, 0Æ5, 0Æ8 and 1Æ0% (w ⁄ v) of bile salt, dispensed in 10-ml volumes and sterilized by autoclaving at 121C for 15 min. For each culture to be tested, one tube of each media was inoculated with 0Æ2 ml of a freshly prepared MRS broth culture. The inoculated media were incubated at 37C in a water bath and were measured after each 24 h when compared to a control culture (without bile salts). The optical density was measured at 600 nm using a UV absorbance (Varian 634, Palo Alto, CA, USA). The growth times required for turbidity to reach an optical density of 0Æ3 were determined. Milk fermentation capacity One hundred microlitre aliquots of skim milk (Difco Lab.) media (10%, w ⁄ v skim milk in water) was pasteurized at 85C for 15 min and cooled down to 40C. Individual probiotic bacteria were inoculated (2%, v ⁄ v) and incubated at 37C under anaerobic conditions. The cultured milks were incubated until the onset of coagulation (c. 38 h). Flasks containing probiotic-fermented milk were obtained and stored at 4C.


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MTT assay The MTT assay was carried out as described by Yu and Li (2006) with some modifications. The CHO-K1 and SNU-C4 cells (5 · 104 cells per ml) were cultured in RPMI 1640 medium supplemented with 10% (v ⁄ v) heatinactivated FBS. Twenty microlitres of each of the four LAB strains and the same volume of the sterile PBS buffer (pH 7Æ4) alone as the control were added to wells in 96-well microtitre plates containing 180 ll of single cell suspension of the CHO-K1 or SNU-C4. Cultures were maintained in a humidified incubator at 37C in an atmosphere of 5% CO2 and 95% air, and the medium was exchanged with fresh medium every 48 h. MTT was dissolved in the sterile PBS (pH 7Æ4) to obtain a concentration of 5 mg ml)1. Fifty microlitres of MTT solution was added to each well, and the plates were incubated for additional 4 h at 37C, followed by aspiration of the MTT solution in medium. To achieve solubilization of the formazan crystal formed in viable cells, 150 ll of DMSO was added to each well. Absorbance was measured with an automatic enzyme-linked immunosorbent assay (ELISA) reader (THERMOmax; Molecular Devices Co., Sunnyvale, CA, USA) at 570 nm with DMSO as blanks for cytotoxicity calculation. % Cytotoxicity = [(Control OD Control OD] 100

Protein concentration was determined by staining with crystal violet in an identical set of test plates. Induction of QR activity was calculated from the ratio of specific enzyme activities of compound-treated cells in comparison with a solvent control. Immunostimulating activity assay The production of nitrogen oxide (NO) was determined by measuring the optical density at 550 nm. Sodium nitroprusside (10 mmol l)1) in PBS (pH 7Æ4) was mixed with different concentrations of extract (2–10 mg ml)1) dissolved in methanol and incubated at 25C for 30 min, followed by removal of 1Æ5 ml of the incubation solution and dilution with 1Æ5 ml of Griess reagent [1% sulfanilamide, 2% phosphoric acid, and 0Æ1% N-(1-naphthyl)ethylenediamine dihydrochloride]. The absorbance of the chromophore formed during diazotization of the nitrite with sulfanilamide and subsequent coupling with N(1-naphthyl)-ethylenediamine dihydrochloride was measured at 546 nm along with a control. The level of the cytokines, human interleukin (IL)-1a was measured by ELISA. Strain was co-cultured with a murine macrophage RAW 264.7 cell line at the concentration of 5 · 105 cells per ml in a 96-well flat-bottomed tissue culture plate.

Sample OD)/

Quinone reductase activity assay To determine the ability of the LAB to induce QR, cultured Hepa-1c1c7 mouse hepatoma cells were used, as described by Chang et al. (1997). Briefly, the cells were cultured in a MEM supplemented with 10% FBS (v ⁄ v), 100 U ml)1 penicillin and 100 lg ml)1 streptomycin at 37C for 24 h in a humidified 5% CO2 incubator. The cells were plated at densities of 1 · 106 ml)1 in 60-mm culture dishes. To the culture media, 100 lmol of LAB strains was added directly and incubated for 48 h. The control cells were treated only with the sterile PBS buffer (pH 7Æ4). Then 200 ll of mixed solution containing 25 ll 50 mmol menadione and 25 ml reaction mixture [25 mmol Tris ⁄ HCl (pH 7Æ4), 0Æ67 mg BSA per ml)1), 0Æ01% Tween 20, 5 lmol FAD, 30 lmol NADP, 1 mmol glucose 6-phosphate and 2 U glucose-6-phosphate dehydrogenase per ml and 0Æ3 mg MTT per ml] was added to each well. After 5 min incubation at 25C, 50 ll 0Æ3 mmol dicoumarol in 5 mmol potassium phosphate (pH 7Æ4) was added to stop further reaction and then UV absorbance at 610 nm was measured. QR activity was determined by measuring the NADPH-dependent menadiol-mediated reduction of MTT to a blue formazan.

Comet assay The alkaline comet assay was performed to determine the oxidative DNA damage according to the method of Singh et al. (1988) with a little modification. The number of cultured HT-29 (human-derived colon) cells were adjusted as 1 · 105 cells per ml (900 ll), and the cells were incubated with 40 and 400 lg ml)1 concentrations of KFRI342 (10 ll) for 15 min at 121C. After pre-incubation, the cells were centrifuged at 600 g for 5 min and washed using 10 ml PBS. The cell cultures were also treated for 5 min with 10 ll H2O2 (final concentration 200 lmol) as a positive control and with 10 ll PBS on the ice, followed by washing with PBS and centrifugation. Slides were imaged using image analysis (Kinetic Imaging; Komet 5.0, Wirral, UK) and fluorescence microscope (Leica DMLB, Wetzlar, Germany), which determined the percentage of fluorescence in the tail (tail intensity; 50 cells from each of two replicate slides). Statistical analysis Analysis of variance was used to determine and compare the probiotic characteristics of LAB. Least significant differences were calculated using the general linear model procedure in sas software (SAS Institute Inc., Cary, NC, USA). The MTT assay results were expressed as mean


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(%) ± SD. For comet assay, the mean values of the DNA damage (tail moment) from each treatment were compared using one-way analysis of variance (anova) with Duncan’s multiple range tests. Statistical analyses were performed with the statistical program the spss for windows (SPSS Inc., Chicago, IL, USA). P-value of