Evaluation of probiotic characteristics of newly isolated Lactobacillus spp.: Immune modulation and longevity

International Journal of Food Microbiology 148 (2011) 80–86

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Evaluation of probiotic characteristics of newly isolated Lactobacillus spp.: Immune modulation and longevity Jin Lee c, Hyun Sun Yun c, Kyu Won Cho a, Sejong Oh d, Sae Hun Kim c, Taehoon Chun a, Bongjoon Kim b,⁎, Kwang Youn Whang a,⁎⁎ a

Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 136-701, Republic of Korea CJ Foods R&D Center, CJ CheilJedang Corporation, Seoul, 152-051, Republic of Korea Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul, 136-701, Republic of Korea d Division of Animal Science, Chonnam National University, Gwangju, 500-757, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 9 August 2010 Received in revised form 11 April 2011 Accepted 6 May 2011 Available online 13 May 2011 Keywords: Lactobacilli Probiotic characteristics Immune modulation C. elegans

a b s t r a c t In the current study, the probiotic potential of approximately 350 strains of lactic acid bacteria isolated from Korean infant feces and Kimchi was investigated. Common probiotic properties of the bacterial strains, such as acid tolerance, bile tolerance and adhesion to human intestinal epithelial cells (HT-29 cells), were examined. Some strains were found to have immune modulatory and antimicrobial properties. Antagonistic activity against a panel of pathogenic bacteria was found to be strain dependent. To evaluate the immune modulatory activity of the strains, lymphocyte interferon (IFN)-γ secretion was determined in conjunction with cell proliferation. Some strains of Lactobacillus gasseri, L. fermentum and L. plantarum exhibited increased IFN-γ levels and lymphocyte proliferation. To evaluate the effects of these immune modulating lactobacilli on host life span, Caenorhabditis elegans was used as an in vivo model. Nematodes that were supplied heat-killed lactobacilli as a food source exhibited obvious differences in life span compared with those fed Escherichia coli OP50. The mean life span (determined as mean percent survival) of worms fed L. plantarum CJLP133 and L. fermentum LA12 was 13.89% and 13.69% greater, respectively, than that of control nematodes after 21 days (P = 0.036 and 0.043, respectively). In addition, some of safety profiles, including hemolytic type, gelatin hydration and degradation of urea, were found to be positive. These newly identified lactobacilli hold promise for use as probiotic agents, feed additives and/or in food applications. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Probiotic lactobacilli have been known for centuries to promote human health and prevent human disease. Lactobacillus is a wellcharacterized genus in the lactic acid bacteria (LAB) group, which is composed of 100 recognized species (Claesson et al., 2008). A large number of studies have demonstrated the probiotic potential of various lactobacilli (Guarner and Schaafsma, 1998; Ouwehand et al., 2002). Even though there has been a long history of safe consumption of probiotic lactobacilli in traditional food, several criteria should be examined before they are used as probiotic agents or in industrial-

Abbreviations: Con A, concanavalin A; LPS, lipopolysaccharide; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PHA, phytohemaglutinin. ⁎ Correspondence to: B. Kim, CJ Foods R&D Center, CJ CheilJedang Corporation, 636, Guro-dong, Guro-gu, Seoul 151-051, Republic of Korea. Tel.: + 82 2 2629 5263; fax: + 82 2 2629 5368. ⁎⁎ Correspondence to: K.Y. Whang, Division of Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea. Tel.: + 82 2 3290 3056; fax: + 82 2 3290 3499. E-mail addresses: [email protected], [email protected] (B. Kim), [email protected] (K.Y. Whang). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.05.003

grade food products. Probiotic lactobacilli should be resistant to gastric acid and bile acid, and also colonize the intestinal and/or genital mucosa to reside in the gastrointestinal tract (Jacobsen et al., 1999; Johansson et al., 1993). There were a lot more criteria above and beyond. Lactobacilli have been shown to inhibit pathogenic bacteria such as Escherichia coli, Listeria monocytogens, Salmonella spp., and others (Axelsson et al., 1989; Jacobsen et al., 1999). Other clinically proven health effects have been reported for lactobacilli, such as cholesterol reduction, diarrhea prevention, enhancement of lactose intolerance symptoms, anticancer effects and immunomodulatory effects, all of which are considered functional aspects of probiotic criteria (Andersson et al., 2001; McFarland, 2000). Recently, immune modulation by probiotics was recognized as an important aspect of host gut health (Pagnini et al., 2010). It has been suggested that the health benefits of some probiotics used in functional foods and pharmaceutical preparations are due to the capacity of these microorganisms to stimulate the host immune system (Perdigón et al., 2002). Defense responses to pathogenic bacteria and microbial antigens have been shown to promote gut maturation and integrity. The ability of non-pathogenic lactobacilli to inhibit various pathogenic bacteria has also been shown in several

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in vitro methods (Delgado et al., 2007; Jacobsen et al., 1999; Perdigón et al., 2002). Caenorhabditis elegans is a small soil nematode that feeds on bacteria. It is used extensively as an experimental model system for studying bacterial infection and/or longevity extension (Ikeda et al., 2007; Kurz and Tan, 2004). Ikeda et al. (2007) recently reported that LAB may contribute to host defenses and prolong the life span of C. elegans. In the current study, the probiotic activity of newly isolated lactobacilli from Korean infant feces and the traditional Korean fermented vegetable Kimchi was characterized, including acid tolerance, bile tolerance and adhesion to human intestinal epithelial cells. Some of the tested strains exhibited immune modulatory properties and antimicrobial activity, and were able to extend survival in a host organism. The results of this combined analysis demonstrated that L. gasseri, L. fermentum and L. plantarum, which are predominant in Asian populations, have potential health promoting effects. These newly identified probiotic lactobacilli hold promise for use as probiotics in functional food applications and/or as feed additives. 2. Materials and methods 2.1. Isolation and preliminary identification LAB stains were isolated from Korean infant feces and Kimchi, a traditional Korean fermented vegetable. For enrichment of lactobacilli, all samples were cultured anaerobically (Macs Mics Jar Gassing System, Don Whitley Scientific Ltd., UK) on modified MRS agar (pH 5.0) at 37 °C for 48 hours (h). Single colonies were picked from the plates and culture purified on MRS agar. The isolates were gram-stained and characterized by carbohydrate utilization pattern using an API 50 CH system (BioMeriux, France). For long-term storage, stock cultures were maintained at −80 °C in MRS broth containing 15% glycerol. All strains were subcultured three times prior to experimental analysis. 2.2. Species level identification In total, 209 isolates were identified by 16S rDNA sequence analysis. Chromosomal DNA from each strain was extracted and the 16S rRNA gene was amplified using universal primers. The PCR primer sequences were as follows: forward primer, 5′-AGAGTTTGATCCTGGCTCAG-3′; reverse primer, 5′-GGTTACCTTTGTTACGACTT-3′ (Bioneer, Korea). The thermal cycling parameters were denaturation at 94 °C for 5 minutes (min), followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 50–55 °C for 1 min, polymerization at 72 °C for 40 s and a final polymerization step at 72 °C for 5 min. Amplified products were purified for sequencing using a Gel Extraction Kit (Intron, Korea). The sequences of the final products were analyzed by an ABI 377 automated DNA sequencer (Perkin Elmer, USA). Sequence homologies were examined by comparing the obtained sequences with those in the DNA Databases (http://www.ncbi.nlm.nih.gov/BLAST). 2.3. pH and bile tolerance Tolerance to low pH and bile content was assessed as described by Jacobsen et al. (1999), with minor modifications. The ability of the strains to grow at low pH was evaluated in acidified MRS broth (final pH 2.5) containing 1000 unit/ml of pepsin (Sigma, USA). The tolerance of the strains to bile (Oxgall) was determined in MRS broth containing 0.3% oxgall (Sigma, USA). Ten milliliters of each type of modified MRS was inoculated with a bacterial suspension to a final cell concentration of approximately 1.0 × 10 7 CFU/ml. pH tolerance was evaluated by measuring survival after 3 h of incubation at 37 °C. Bile tolerance was evaluated by measuring survival after 24 h of incubation at 37 °C. In these experiments, 100 μl of was plated in duplicate onto MRS agar.

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2.4. Adhesion assay The ability of the strains to adhere to human epithelial cells was investigated according to the method of Kim et al. (2009). Monolayers of HT-29 intestinal epithelial cells were prepared in RPMI1640 (Sigma, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, USA) in 24-well tissue culture plates (Sarstedt, Germany) at a concentration of 4 × 10 4 cells/well. The cells were incubated with approximately 1 × 10 7 CFU/ml of the strain to be tested. After 2 h of incubation at 37 °C, the monolayers were washed six times with PBS. Adherent bacteria were detached by repeatedly pipetting with chilled sterile water, diluted in quarter strength Ringer's solution and then enumerated by counting on MRS agar plates. The assay was repeated twice for every strain and counts were performed in duplicate. 2.5. Inhibition of intestinal pathogens To measure the inhibitory effects of the lactobacilli on intestinal pathogens, a spot-on-lawn method was used. Six representative intestinal pathogens were used for the analysis. E. coli O157: H7 ATCC 43894 and Salmonella typhimurium KCCM 11806 were purchased. The other pathogens were previously isolated in the Dairy Food Microbiology Laboratory at Korea University (Seoul, Korea; Table 2), and consisted of Enterococcus faecalis, Staphylococcus aureus, Yersinia enterocolitica and L. monocytogenes. Bacteria were grown in brain heart infusion (BHI) broth (Difco, USA) at 37 °C for 18 h. Then, 1 ml of an overnight indicator bacterial culture (ca. 1 × 10 9 CFU/ml) was mixed with 100 ml of BHI agar (1.2%) and poured into a culture plate. Test cultures of lactobacilli were spotted (40 μl) on the surface of the agar plate containing 0.2% glucose and then incubated for 12 h at 37 °C to enable the spots to develop. The inhibitory effect of non-cultured MRS was used as a negative control. After incubation, inhibition zones were determined. A clear zone (halo) of more than 2 mm around the spot was scored as positive. Each test was performed three times. 2.6. Lymphocyte proliferation assay The effects of the strains on immune cell proliferation were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) cell proliferation assay (Promega, USA). Lymphocytes were freshly isolated from spleen cells of B6 mice and then plated in a 96-well flat bottom tissue culture plate at a concentration of 1 × 10 6 cells/well in 200 μl of RPMI-1640 (supplemented with 10% FBS). After 24 h incubation, test bacterial cells were added to each well in serum-free media and the plates were incubated in a humidified 5% CO2 atmosphere at 37 °C. The test bacterial cells were prepared as follows. After culture in MRS broth at 37 °C for 18 h, the bacterial cell suspension was centrifuged at 8000 ×g for 10 min and washed twice with PBS. Then, each bacterial cell count was adjusted to 6 × 10 6 CFU/ml. After 72 h, the culture medium was removed from each well and 20 μl of MTT solution (2 mg/ml) was added. After 1 h, colored formazan crystals were dissolved in 100 μl of dissolving solution. The plates were scanned on a multi-well scanning spectrophotometer (ELISA reader) at 490 nm to determine the optical density (OD) of each well. Each test was performed in triplicate. As a positive control, lymphocytes were cultured in the presence of concanavalin A (Con A, 10 μg/ml), phytohemaglutinin (PHA, 10 μg/ml), or lipopolysaccharide (LPS, 10 μg/ml); cells cultured in the absence of bacteria or mitogen were used as negative controls. 2.7. Determination of interferon (IFN)-gamma production Live and heat-killed bacteria were used in these experiments. Live bacteria were prepared using the same method described

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earlier, but the volume of the bacterial suspension was adjusted to a density of 10 5 CFU/ml to 10 8 CFU/ml. Heat-killed cell were prepared as follows. After culture in MRS broth at 37 °C for 18 h, each grown bacterial cell were centrifuged at 8000 × g for 10 min and washed twice with PBS. The volume of the bacterial suspension was then adjusted to a density of 10 5 CFU/ml to 10 8 CFU/ml. Lastly, cells were heat-killed at 70 °C for 10 min and then stored in aliquots at − 20 °C. The level of IFN-γ in the culture supernatants was measured using the enzyme-linked immunosorbent assay (ELISA). Briefly, splenic lymphocytes from B6 mice were plated in a 96-well flat bottom tissue culture plate at a concentration of 1 × 10 6 cells/well in 200 μl of RPMI-1640 (supplemented with 10% FBS). The bacterial cells were added at various concentrations (10 5 to 10 8 CFU/well) and then the plates were incubated in a humidified 5% CO2 atmosphere at 37 °C. After 3 days, culture supernatants were collected and the amount of IFN-γ released was quantified by ELISA. In the ELISA, microtiter plates were coated with 5 μg/ml of purified antiIFN-γ antibody (BD Bioscience, no. 554409, USA) for 4 h at room temperature, and then the wells were washed three times with PBS. After blocking with 1% bovine serum albumin (BSA) in PBS, samples (50 μl of culture supernatant) were added to each well in triplicate. The plates were incubated for 2 h at room temperature, after which they were washed five times with PBS. Bound IFN-γ was detected by incubation with a biotinylated anti-IFN-γ antibody (BD Bioscience, no. 554410, USA) for 2 h. The wells were washed with PBS, and then freshly prepared streptavidin-conjugated horseradish peroxidase (Vector, no. SA-5004, USA) and substrate (3,3′,5,5′tetramethylbenzidine) (KPL, no. 50-76-03, USA) were added. The reactions were stopped by the addition of 100 μl of the stop solution (3 M HCl). The OD of each well was measured at 450 nm using an ELISA reader (Molecular Device, USA). For the positive control, lymphocytes were cultured with Con A (10 μg/ml), PHA (10 μg/ml) or LPS (10 μg/ml); cells cultured in the absence of bacteria or mitogen were used as negative controls.

performed using Minitab release 14 (Minitab Inc., USA). Student's t-test was performed to determine statistical differences among groups. Table 1 pH and bile tolerance and adhesive properties of 59 lactobacilli. Lactobacillus strainsa

Growth/survivalb

Adhesionc

pH 2.5

Oxgall 0.3%

−/+ −/+ −/+

+/+ +/+ −/+

6.40 ± 0.02 5.28 ± 0.00 6.94 ± 0.04

Isolates from Korean infant feces L. gasseri CJLF1 −/+ L. gasseri CJLF6 −/+ L. gasseri CJLF20 −/+ L. gasseri CJLF21 −/+ L. gasseri CJLF22 −/+ L. gasseri CJLF23 −/+ L. gasseri CJLF34 −/+ L. gasseri CJLF35 −/+ L. gasseri CJLF36 −/+ L. gasseri CJLF37 −/+ L. gasseri CJLF48 −/+ L. gasseri CJMF3 −/+ L. gasseri CJMF15 −/+ L. gasseri CJMF18 −/+ L. gasseri CJMF19 −/+ L. gasseri CJMF29 −/+ L. gasseri CJMF31 −/+ L. gasseri CJMF36 −/+ L. gasseri CJMF41 −/+ L. fermentum LA12 −/+ L. fermentum CJMA3 −/+ L. fermentum CJMA7 −/+ L. fermentum CJMA9 −/+ L. fermentum CJMA10 −/+ L. fermentum CJMA11 −/+ L. plantarum CJMA1 −/+ L. plantarum CJMA2 −/+

+/+ −/+ −/+ −/+ −/+ +/+ +/+ +/+ −/+ −/+ −/+ +/+ −/+ −/+ −/+ ND/ND ND/ND −/+ ND/ND −/+ −/+ +/+ −/+ +/+ +/+ −/+ −/+

6.70 ± 0.04 6.49 ± 0.12 6.29 ± 0.05 6.57 ± 0.08 6.09 ± 0.09 6.68 ± 0.10 6.50 ± 0.01 6.65 ± 0.03 5.72 ± 0.22 6.00 ± 0.17 6.01 ± 0.16 6.85 ± 0.03 5.66 ± 0.40 6.18 ± 0.06 6.02 ± 0.04 5.95 ± 0.19 6.14 ± 0.16 5.80 ± 0.68 6.57 ± 0.13 5.82 ± 0.20 6.56 ± 0.24 4.44 ± 0.09 4.65 ± 0.01 4.51 ± 0.04 4.36 ± 0.16 7.10 ± 0.22 6.66 ± 0.31

Isolates from Korean Kimchi L. brevis CJD1 L. brevis CJD2 L. brevis CJD44 L. brevis CJD45 L. brevis CJD48 L. brevis CJD49 L. brevis CJD54 L. brevis CJD55 L. brevis CJD56 L. brevis CJNR27 L. brevis CJNR28 L. plantarum CJLP56 L. plantarum CJLP133 L. plantarum CJLP243 L. plantarum CJNR02 L. plantarum CJNR04 L. plantarum CJNR16 L. plantarum CJNR26 L. plantarum CJNR37 L. plantarum CJNR54 L. plantarum BJ53 L. plantarum BJ54 L. plantarum BJ57 L. plantarum BJ58 L. plantarum BJ135 L. plantarum BJ203 L. plantarum BJ217 L. plantarum BJ224 L. plantarum BJ242

+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ −/+ −/+ −/+ +/+ −/+ +/+ +/+ +/+ −/− −/+ +/+ −/+ −/+ −/− −/+ +/+ −/+ −/+ −/+

5.15 ± 0.08 5.66 ± 0.03 5.33 ± 0.08 5.44 ± 0.07 4.91 ± 0.03 4.96 ± 0.10 5.04 ± 0.01 5.34 ± 0.18 5.23 ± 0.07 5.42 ± 0.12 5.94 ± 0.12 6.45 ± 0.13 7.85 ± 0.02 6.32 ± 0.27 5.20 ± 0.04 5.78 ± 0.40 5.25 ± 0.16 6.42 ± 0.21 6.38 ± 0.50 7.27 ± 0.11 5.94 ± 0.18 6.13 ± 0.33 5.42 ± 0.14 4.72 ± 0.16 6.80 ± 0.18 4.97 ± 0.01 5.71 ± 0.48 5.48 ± 0.06 6.42 ± 0.20

Documented strains L. rhamnosus GG L. brevis KB290 L. plantarum L299v

2.8. C. elegans killing assay The influence of selected lactobacillus strains on longevity in C. elegans was determined using the killing assay described by Ikeda et al. (2007), with some modifications. Briefly, L4-stage adult worms were exposed to a solution of sodium hypochlorite-sodium hydroxide to isolate fresh eggs, as previously described (Mylonakis et al., 2002). The egg suspension was spread on Nematode Growth Medium (NGM) agar plates (30 mm diameter) and then the plates were incubated overnight at 25 °C to allow hatching. After hatching, the suspension of L1-stage worms was subjected to centrifugation at 600 rpm for 1 min. The supernatant was removed, and the remaining worms were transferred onto fresh NGM plates covered with E. coli OP50, an international food standard. The plates were incubated at 25 °C for 2 days to allow the worms to reach the L4-stage. The killing assays were initiated with the 3-day adult healthy nematodes. Worms (12 each) were transferred to one plate covered with lawns of each lactobacillus strain, and then the plates were incubated at 25 °C. The number of live worms per plate was determined every 24 h by light microscopy (Olympus CH30, Japan) for the duration of the experiment (21 days). A worm was considered dead when it failed to respond to a gentle touch with a worm picker. Each experimental group (test lactobacillus strains fed group) was tested in duplicate and the control group (OP 50 fed group) was tested in quadruplicate. In the killing assay, we used glp-4 mutant worms instead of wild-type Bristol N2 worms. Mutant glp-4 worms have a normal morphology and brood sizes at 15 °C, but do not make gonads and are unable to produce eggs at 25 °C (Mylonakis et al., 2002). Thus, it is possible to score the number of live and dead worms. Statistical analysis was

a

−/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+ −/+

16s rDNA sequenced having over 99% homology. +, growth of Nlog 0.5 and/or survival; −, no growth or survival compared with initial inoculums. ND, not determined. c Average log no. of adhering lactobacilli in HT-29 cell after 2 h incubation. Initial inoculums at approximately 1 × 107 CFU/ml (log 7.0). b

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2.9. Safety evaluation Hemolysis and colony morphology were recorded after growth for 48 h on 5% sheep blood agar plates (Gibco, USA). Urease activity was

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determined in urea agar base (Difco, USA) dissolved in medium (pH 6.9). Gelatin liquefaction was determined in chilled nutrient gelatin tubes at 25 °C (room temperature) for at least 72 h. All of the parameters of bacterial safety were evaluated as described previously (Isenberg, 1992).

Table 2 Inhibition of six intestinal pathogens by 59 lactobacilli. Inhibitiona En. faecalisb

E. coli

Staphy. aureus

Y. enterocolitica

S. typhimurium

L. monocytogenes

+ − +

+ − +

+ − +

++ − ++

++ − +

++ − −

Isolates from Korean infant feces L. gasseri CJLF1 L. gasseri CJLF6 L. gasseri CJLF20 L. gasseri CJLF21 L. gasseri CJLF22 L. gasseri CJLF23 L. gasseri CJLF34 L. gasseri CJLF35 L. gasseri CJLF36 L. gasseri CJLF37 L. gasseri CJLF48 L. gasseri CJMF3 L. gasseri CJMF15 L. gasseri CJMF18 L. gasseri CJMF19 L. gasseri CJMF29 L. gasseri CJMF31 L. gasseri CJMF36 L. gasseri CJMF41 L. fermentum LA12 L. fermentum CJMA3 L. fermentum CJMA7 L. fermentum CJMA9 L. fermentum CJMA10 L. fermentum CJMA11 L. plantarum CJMA1 L. plantarum CJMA2

+ − + + + + + + − − − + − − − − − − − + − − − − − ++ −

+ − + + + + + + − − − + − − − − − − − + + − − − − ++ +

+ − − − − + + + − − − + − − − − − + + + + − − − − ++ −

+ − ++ ++ ++ + + ++ − − − + − − − − − + + ++ ++ + + + ++ ++ +

+ − ++ ++ ++ + + + − − − + − − − − − − − ++ + − + − + ++ +

+ − − − − − + − − − − − − − − − − − − + + ++ + + + ++ ++

Isolates from Korean Kimchi L. brevis D1 L. brevis D2 L. brevis D44 L. brevis CJD45 L. brevis CJD48 L. brevis CJD49 L. brevis CJD54 L. brevis CJD55 L. brevis CJD56 L. brevis CJNR27 L. brevis CJNR28 L. plantarum CJLP56 L. plantarum CJLP133 L. plantarum CJLP243 L. plantarum CJNR02 L. plantarum CJNR04 L. plantarum CJNR16 L. plantarum CJNR26 L. plantarum CJNR37 L. plantarum CJNR54 L. plantarum BJ53 L. plantarum BJ54 L. plantarum BJ57 L. plantarum BJ58 L. plantarum BJ135 L. plantarum BJ203 L. plantarum BJ217 L. plantarum BJ224 L. plantarum BJ242

− − − − − − − − − − − + + ++ − − − − − − + + − + + + + + +

− − − − − − − − − − − + + + + + + + + + − − − + + − + − +

− − − − − − − − − − − + + + − − − ++ ++ ++ + − + ++ − − + + ++

− − − − − − − − − − − ++ ++ + − − + + − + + + − + + + + − +

− − − − − − − − − + − + + + − − − + + ++ − − − + + + + ++ +

− − − − − − − − − − − + − − + + + ++ ++ + − − − − − − − + −

Lactobacillus strains Documented strains L. rhamnosus GG L. brevis KB290 L. plantarum L299v

a

c

+, halo of inhibition between 2 and 5 mm; ++, halo of 5 mm inhibition and above; −, no inhibition. Abbreviations: En. faecalis, Enterococcus faecalis; E. coli, Escherichia coli O157:H7 ATCC 43894; Staphy. aureus, Staphylococcus aureus; Y. enterocolitica, Yersinia enterocolitica; S. typhimurium, Salmonella typhimurium KCCM 11806; L. monocytogenes, Listeria monocytogenes. c L. rhamnosus GG and L. brevis KB290 were utilized as positive and negative controls. b

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3. Results 3.1. pH and bile tolerance Of the approximately 350 strains tested, 118 survived following 2 h of incubation at pH 2.5 with 1000 unit/ml pepsin, but none seemed to have replicated. Bile tolerance over 24 h was observed for several strains. Taken together, 59 lactobacillus strains (including three documented strains) demonstrated strong survival under acid and bile conditions (Table 1). 3.2. Adhesion assay The ability of the 59 lactobacillus strains with high pH and bile tolerance to adhere to HT-29 epithelial cells was examined (Table 1). Considerable variation among the strains was observed. Among the tested strains, 29 strongly adhered to the epithelial cells, with adhesive properties higher than or similar to the positive control, L. rhamnosus GG. The remaining 30 strains exhibited moderate to low adhesive properties. 3.3. Inhibition of intestinal pathogens Inhibition of six pathogenic bacterial strains by the lactobacilli was highly variable (Table 2). Many of the strains tested showed weak or no inhibition of the pathogenic bacteria, but some strains, including L. fermentum LA12 and CJMA3, and L. plantarum CJMA1, CJLP56, CJLP133, CJLP243, CJNR26, CJNR37, CJNR54, BJ58, BJ217, BJ224 and BJ242 broadly inhibited pathogenic bacteria. No inhibitory effects of MRS on the six pathogenic strains were observed. 3.4. Cell viability The MTT assay is a colorimetric assay that measures mitochondrial function, which serves as an index of live, metabolically active cells (Ivec et al., 2007). Freshly isolated splenic lymphocytes were treated with lactobacilli for 72 h, and cell viability was assessed using the MTT assay. The effects of the strains on lymphocyte viability varied; some lactobacilli had no effect, while others promoted lymphocyte proliferation (Fig. 1). L. fermentum LA12, and L. plantarum CJMA1, CJLP56, CJLP133, CJLP243, CJNR26 and BJ53 were more effective in

Fig. 2. IFN-γ production by splenocytes following the addition of live lactobacillus strains (a) and heat-killed lactobacillus strains (b). Bacteria were tested over a range of 105 to 108 CFU/ml. Con A (concanavalin A, 10 μg/ml), PHA (phytohemaglutinin, 10 μg/ml) and LPS (lipopolysaccharide, 10 μg/ml) were used as positive controls. Data represent the means± standard deviation of triplicate assays.

inducing lymphocyte proliferation than the positive control (10 μl/ml LPS). 3.5. IFN-γ production induced by lactobacillus strains The production of IFN-γ by cultured splenocytes following the addition of live or heat-killed lactobacilli was examined. The levels of secreted IFN-γ in culture supernatants were strain-dependent, and

Fig. 1. The effects of selected lactobacillus strains on lymphocyte proliferation by using the MTT colorimetric assay. Lymphocyte proliferation was measured as optical density at 490 nm. The results are expressed relative to stimulation with 10 μl/ml LPS, which was set at 1.00. Con A (concanavalin A, 10 μg/ml), PHA (phytohemaglutinin, 10 μg/ml) and LPS (lipopolysaccharide, 10 μg/ml) were used as positive controls. Data represent the means ± standard deviation of triplicate assays.

Fig. 3. Effect of different lactobacillus strains on the life span of C. elegans. Worms were fed a diet of E. coli OP50 for 3 days after hatching and then transferred to a diet of the indicated lactobacillus strain. LR GG (L. rhamnosus GG), LP CJLP133 (L. plantarum CJLP133) and LF LA12 (L. fermentum LA12) were tested against E. coli OP50, which is the international food standard for C. elegans. (n) indicates the total number of tested worms. *Data were analyzed by Student's t-test after 21 days to determine statistical significance.

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were induced over bacterial cell concentrations ranging from 10 5 to 10 8 CFU/ml (Fig. 2a and b). Some strains of L. gasseri (CJLF1, CJLF23, CJLF34, CJLF35 and CJLF3) and L. plantarum (CJLP133, CJLP243, CJNR26 and BJ53) induced more IFN-γ cytokine release than the positive controls (L. rhamnosus GG and L. brevis KB290). 3.6. C. elegans killing assay The effect of heat-killed lactobacilli on nematode survival was examined to further assess the beneficial effects of the newly isolated strains. The life spans of nematodes fed certain strains of heat-killed lactobacilli were obviously different than those fed only E. coli OP50 (Fig. 3). Among the strains tested, nematodes that were supplied with L. plantarum CJLP133 and L. fermentum LA12 as a food source had a prolonged mean life span relative to nematodes fed with other strains, including L. rhamnosus GG. In these experiments, nematodes were given approximately 10 6 CFU of heat-killed lactobacilli. The mean life span (in terms of survival percentage) of worms fed L. plantarum CJLP133 and L. fermentum LA12 was 82.14% and 81.94%, respectively, whereas that of worms fed OP50 was 68.25% after 21 days. The mean life span of worms was 13.89 % and 13.69% greater, respectively, than that of the control nematodes (P = 0.036 and 0.043, respectively). 3.7. Safety evaluation All of the selected lactobacilli were shown to be safe based on the results of the three major standardized tests. The strains appeared to have α-type or γ-type hemolysis (data not shown), and positive results were consistently obtained for urease activity and gelatin liquefaction (data not shown). 4. Discussion A panel of in vitro experiments was conducted to evaluate the probiotic properties of newly isolated lactobacillus strains. Tolerance to low pH and bile content, which are reflective of the acidic environment of the stomach and upper part of the intestines, is fundamental to probiotic activity. Similarly, the ability to adhere to the epithelium is a highly important parameter for improved probiotic activity. This latter attribute is closely related to the stability and continuity of bacterial residency and function in the intestine (Dunne et al., 2001). In the current study, HT-29 cells were used as an in vitro model of epithelial cell adherence. Considerable variation in adherence to HT-29 cells among the strains was observed. As a positive control, the strains were compared to L. rhamnosus GG, which has been shown in several previous studies to bind to enterocytes (Andersson et al., 2001; Ossowski et al., 2010). We identified 29 isolates that adhered strongly to HT-29 epithelial cells. These strains exhibited higher or similar adherence when compared to the control strain, L. rhamnosus GG. The remaining strains exhibited moderate to low adherence. The antimicrobial activity of probiotics against pathogenic bacteria has been broadly studied (Axelsson et al., 1989; Jacobsen et al., 1999). Here, the newly isolated strains from Korean infant feces, L. gasseri and L. fermentum, exhibited high antimicrobial activity against the enteropathogenic bacteria Y. enteroclitica and S. typhimurium. In addition, several L. plantarum strains isolated from Korean Kimchi displayed strong antimicrobial effects against S. aureus. Other pathogenic bacteria such as E. faecalis, E. coli and L. monocytogenes were only slightly inhibited. The effects of lactobacillus strains on cell proliferation were examined using the MTT assay, which is a well recognized tool for evaluating cell viability. Treatment of cells with probiotic bacteria alone did not result in decreased cell viability, and some of the lactobacilli stimulated cell proliferation. To further evaluate the potential probiotic activity of selected lactobacilli, we examined their effect on

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immune function by measuring lymphocyte proliferation and IFN-γ production. As a result, we identified distinct and typical patterns of IFN-γ release. It is even observed when the lymphocytes were treated with heat-killed bacterial preparations, which contained no bacterial metabolites, and were strain-dependent. These results suggest that the surface properties of some of lactobacilli elicit an immune response, as reflected by the release of IFN-γ and lymphocyte proliferation. IFN-γ has multiple functions in the host. It is released mainly during cellular immune responses, and is involved in antitumor and anti-infection responses (Kato et al., 1999). IFN-γ, a Th1type cytokine, inhibits the production of Th2-type cytokines such as IL-4 and IL-5. Perdigón et al. (2002) examined the immune responses to various lactobacillus strains and found that different LABs stimulate different levels of various cytokines such as TNF-α, IFN-γ, or IL-12. Also, it is well established that the cell wall structure of non-pathogenic Gram-positive bacteria acts as an excellent inducer of immune responses, as do pathogenic Gram-negative bacteria (Heumann et al., 1994; Perdigón et al., 2002). Several previous studies have reported that live as well as heat-killed LAB has immunostimulatory properties (Fujiwara et al., 2004; Sashihara et al., 2006). The population of dead bacteria in the host gastrointestinal tract could in fact be larger than that of live cells (Bucio et al., 2005; Heumann et al., 1994). The relationship between exposure to bacteria and aging was recently investigated using C. elegans as a model system. Ikeda et al. (2007) reported that LAB could contribute to host defenses and prolong the life span of C. elegans. Over a century ago, Metchnikoff (1908) demonstrated that consumption of fermented milk was closely related to longevity in Bulgarians. He believed that beneficial bacteria could balance the intestinal environment, preventing colonization by pathogens and thereby improve host health and prolong life span. Here, we found that the mean survival rate of nematodes supplied with heat-killed L. plantarum CJLP133 and L. fermentum LA12 as a food source was 82.14% and 81.94%, respectively, while for worms fed E. coli OP50, it was 68.25% after 21 days. This was a statistically significant effect, with P values of less than 0.05. Further molecular studies are needed to elucidate the precise mechanism of the prolonged life span in C. elegans; however, our results suggest that these newly identified probiotic lactobacilli may have beneficial effects on the health of the host. They also provide additional support for the use of the nematode as an appropriate in vivo model for screening probiotics. In conclusion, several new, beneficial probiotic lactobacilli were characterized in terms of immune modulation and longevity. These lactobacillus strains hold promise for use as probiotic agents, feed additives and/or in food applications. Acknowledgments This work was supported by the CJ CheilJedang Corporation. References Andersson, H., Asp, N.G., Bruce, A., Roos, S., Wadström, T., Wold, A.E., 2001. Health effects of probiotics and prebiotics: a literature review on human studies. Scandinavian Journal of Nutrition 45, 58–75. Axelsson, L.T., Chung, T.C., Dobrogosz, W.G., Lindgren, S.E., 1989. Production of a broad spectrum antimicrobial substance by Lactobacillus reuteri. Microbial Ecology in Health and Disease 2, 131–136. Bucio, A., Hartemink, R., Schrama, J.W., Verreth, J., Rombouts, F.M., 2005. Survival of Lactobacillus plantarum 44a after spraying and drying in feed and during exposure to gastrointestinal tract fluids in vitro. Journal of General and Applied Microbiology 51, 221–227. Claesson, M.J., Van Sinderen, D., O'Toole, P.W., 2008. Lactobacillus phylogenomics — towards a reclassification of the genus. International Journal of Systematic and Evolutionary Microbiology 58, 2945–2954. Delgado, S., O'Sullivan, E., Fitzgerald, G., Mayo, B., 2007. Subtractive screening for probiotic properties of Lactobacillus species from the human gastrointestinal tract in the search for new probiotics. Journal of Food Science 72, M310–M315. Dunne, C., O'Mahony, L., Murphy, L., Thornton, G., Morrissey, D., O'Halloran, S., Feeney, M., Flynn, S., Fitzgerald, G., Daly, C., Kiely, B., O'Sullivan, G.C., Shanahan, F., Collins, J.K.,

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