JOURNAL OF MEDICINAL FOOD J Med Food 18 (10) 2015, 1–8 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2015.3509

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Effect of Nanometric Lactobacillus plantarum in Kimchi on Dextran Sulfate Sodium-Induced Colitis in Mice Hyun Ah Lee,1,2 Yeon-Ju Bong,3 Hyunung Kim,4 Ji-Kang Jeong,3 Hee-Young Kim,3 Kwang-Won Lee,2 and Kun-Young Park1,3 1

Kimchi Research Institute and 3Department of Food Science and Nutrition, Pusan National University, Busan, Korea. 2 Division of Food Bioscience and Technology, College of Life Science and Biotechnology, Korea University, Seoul, Korea. 4 Biogenics Korea Co., Ltd., Seoul, Korea. ABSTRACT Nanometric Lactobacillus plantarum (nLp) is a processed form of Lab. plantarum derived from kimchi and is 0.5–1.0 lm in size. This study was undertaken to determine the effect of nLp and kimchi plus nLp (K-nLp) on a dextran sulfate sodium (DSS)-induced mouse model of colitis. Animals fed nLp or K-nLp had longer colons, but lower colon weights per unit length than DSS controls. In addition, nLp- or K-nLp-fed animals showed lower levels of proinflammatory cytokines and inflammatory genes in serum and in colon tissues, lower populations of total bacteria, but higher populations of lactic acid bacteria in feces, and lower activities of fecal b-glucosidase and b-glucuronidase. Furthermore, these suppressive activities of nLp on colitis were equivalent to or higher than those of naive Lab. plantarum. Consequently, nLp was found to exhibit anticolitic effects, and the addition of nLp to kimchi was found to enhance the protective activity of kimchi against DSSinduced colitis. These results suggest that nLp might be an effective substitute for live probiotics and be useful as a functional ingredient with the anticolitic activity by the probiotic and food processing industries.

KEY WORDS:  anticolitis  Balb/c mouse  kimchi  nanometric Lactobacillus plantarum

and interferon (IFN)-a levels (indicators of Th1 induction) are significantly increased at a lactic acid bacteria (LAB) particle size of *1 lm. Furthermore, the ability of Lab. plantarum to induce IL-12 is improved by the nanometric process.6 IL-12 has been frequently reported to promote Th1 immunity and to induce IFN-c, leading to anti-inflammatory activity and tumor suppressive effects.7 Furthermore, it has been suggested that nLp modulates the host immune system more than intact LAB. However, few studies have examined the effects of nanometric LAB on colitis in vivo. Kimchi is a traditional Korean fermented food, which is produced by fermentation mainly using LAB, and has been reported to have many beneficial effects, such as, anticancer, antioxidant, and antiobesity effects.8 Kimchi LABs exhibit diverse health-promoting properties, mainly in the gastrointestinal tract, that is, they attenuate the activities of b-glucosidase and b-glucuronidase in the colon, decrease inflammatory responses, and protect against colitis and colorectal cancer development. Ulcerative colitis is an inflammatory bowel disease characterized by inflammatory ulcers in the colon mucous membrane, diarrhea, and enterohemorrhage9; patients with ulcerative colitis are at higher risk of colorectal cancer development.10 In the present study, we evaluated the antiinflammatory effects of nLp in a mouse model of dextran

INTRODUCTION

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robiotics are live bacteria that exert beneficial effects on health and consist mainly of strains of lactobacilli and bifidobacteria.1 In particular, Lactobacillus plantarum (Lab. plantarum) is a major strain used for dairy, meat, and vegetable fermentation,2 and its various beneficial characteristics on intestinal health have been reported, such as its abilities to alleviate diarrhea, constipation associated with irritable bowel syndrome, and to protect against intestinal infections.3 Despite the definition that probiotics are live microorganisms, several biological responses have been reported for heat-killed cells or fractionated cellular components, such as cytoplasmic extracts, exopolysaccharides, and lipoteichoic acid. These responses include immunomodulatory effects and the ameliorations of colitis and allergies.4 Nanometric Lab. plantarum (nLp) derived from kimchi is a shrunken, dead granular form of Lab. plantarum of size 0.5–1.0 lm and has been registered in the DNA Data Bank of Japan. According to a previous study,5 interleukin (IL)-12 Manuscript received 28 April 2015. Revision accepted 15 July 2015. Address correspondence to: Kun-Young Park, PhD, Kimchi Research Institute, Pusan National University, Busan 609-735, Republic of Korea, E-mail: [email protected]

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sulfate sodium (DSS)-induced acute colitis and compared these effects with those of pure Lab. plantarum (pLp). Furthermore, to examine the potential applications of nLp in LAB-containing foods as functional ingredients, we compared the effects of kimchi and of kimchi with added nLp on colon health in a mouse model of DSS-induced colitis. MATERIALS AND METHODS

Histopathological analysis Segments of colon (from the ileocecal junction to the anal verge) were fixed and embedded in paraffin using standard procedures, and the sections (5 lm) so obtained were stained with hematoxylin and eosin.13 Colon tissues were examined and imaged using a Zeiss Axioskop 2 plus microscope equipped with an AxioCam MRc5 CCD camera (Carl Zeiss MicroImaging, Thornwood, NY, USA).

Sample preparation nLp was obtained directly from the manufacturer (Biogenics Korea Co., Ltd., Seoul, Korea). It is manufactured by incubating Lab. plantarum for 20 h under pH control, followed by incubation at high temperature (40C), high salinity (1.0%, w/w), and low pH (pH 5.0) for 4 h. It is then sterilized (80C, 10 min) and nanodispersed by high-pressure homogenization. The morphologies of pLp and nLp were examined under a scanning electron microscope (S-2460N; Hitachi High-Tech. Corp., Tokyo, Japan). Kimchi was manufactured using a slight modification of a previously described recipe11 as naturally fermented kimchi and with added nLp at 2.0 · 1010 CFU/kg kimchi. Kimchi samples were fermented for 3 weeks at 4C until the optimal ripeness (pH 4.3), when pressed kimchi juice was collected and sampled. Animal study Male Balb/c mice (weight 18–20 g, 6-week-old) were purchased from Samtaco Bio Korea (Osan, Korea) and DSS from MP Biochemicals (Solon, OH, USA). After a 7-day adaptation period, mice were randomly allocated to seven groups (n = 10): treatment naive controls (the normal group), DSS controls (the control group), a low-dose pLp group (2.0 · 1010 CFU/kg body weight daily) (the pLp-L group), a low-dose nLp group (2.0 · 1010 CFU/kg body weight daily) (the nLp-L group), a high-dose nLp group (2.0 · 1011 CFU/ kg body weight daily) (the nLp-H group), a naturally fermented kimchi without nLp group (the K group), or nLp (2.0 · 1010 CFU/kg kimchi) added to the naturally fermented kimchi group (the K-nLp group). Animals in the nLp-L, pLp-L, and nLp-H groups were orally administered nLp or pLp suspended in 0.2 mL of phosphate-buffered saline (PBS) daily for 14 days, and kimchi juice (5 g/kg body weight daily) was orally administered for 14 days to animals in the K and K-nLp groups. The normal and control groups were orally administered equal volumes of PBS daily. On the experimental day 7, ulcerative colitis was induced by treating animals (except normals) with 2% (w/w) DSS in drinking water for 7 days.12 All mice were sacrificed 7 days after initiating the induction of colitis on day 14 of the study, when blood and colons were collected for analysis. Animals were maintained under controlled conditions throughout (22C – 2C, RH 55% – 5% under a 12-h light cycle). Feed (AIN-93G; DooYeol Biotech, Seoul, Korea) and water were supplied ad libitum. The study protocol was approved by the Institutional Animal Care and Use Committee (PNUIACUC; approval number PNU-2012-0184) of Pusan National University (Busan, Korea).

Analysis of serum proinflammatory cytokine levels Proinflammatory cytokine levels (tumor necrosis factor [TNF]-a, IL-1b, IL-6, and IFN-c) in serum were determined using appropriate enzyme-linked immunosorbent assay kits (ELISA MAX Deluxe Sets; Biolegend, San Diego, CA, USA), and optical densities were measured at 450 nm. Reverse transcription polymerase chain reaction To confirm the expressions of TNF-a, IL-1b, IL-6, IFN-c, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) in colonic tissues, we performed reverse transcription polymerase chain reaction (RT-PCR) using a slight modification of a previously described method.14 Fecal microorganism counts and fecal enzyme activities Fresh fecal samples (1 g) were added to PBS (9 mL) and centrifuged at 13,000 g for 30 min. Microorganism counts and fecal enzyme activity levels were determined using supernatants. To determine total bacterial and LAB counts, fecal samples were homogenized and diluted with sterile saline solution, plated on a plate count agar (Difco, Detroit, MI, USA) or MRS agar (Difco, Detroit, MI, USA), respectively, and incubated at 37C.15 b-Glucosidase and b-glucuronidase activities were measured with released p-nitrophenol, as previously described with slight modification.16,17 Amounts of p-nitrophenol released were determined using a UV-2401PC spectrophotometer

FIG. 1. Scanning electron micrograph of nanometric Lactobacillus plantarum (nLp) (A) and intact Lactobacillus plantarum (B).

ANTICOLITIS EFFECTS OF NANOMETRIC LAB. PLANTARUM Table 1. Body Weight Changes of Balb/c Mice with Dextran Sulfate Sodium-Induced Colitis

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RESULTS AND DISCUSSION Body weights, colon lengths, and colon weights/unit length

Body weight (g)/day Group 1

Normal Control 3 pLp-L 4 nLp-L 5 nLp-H 6 K 7 K-nLp 2

Initial

Day 6

Day 12

Day 15

19.2 – 0.9NS 19.3 – 0.5 19.0 – 0.5 19.2 – 0.5 19.6 – 0.9 19.1 – 0.8 19.4 – 0.2

20.6 – 1.1NS 20.7 – 0.8 20.5 – 0.9 20.9 – 0.7 21.1 – 0.7 20.9 – 0.9 20.4 – 0.8

21.2 – 0.9NS 20.6 – 1.1 20.9 – 1.0 20.9 – 1.2 21.2 – 1.0 20.9 – 0.6 20.8 – 1.0

21.4 – 1.1a 18.7 – 1.6c 19.9 – 1.4bc 19.4 – 1.7bc 20.3 – 1.1ab 19.2 – 1.0bc 19.5 – 1.3bc

The values are presented as means – SDs (n = 10). a–c Means with different letters in the same column were significantly different (P < .05) by Duncan’s multiple range test. 1 Group received phosphate-buffered saline (PBS) without any treatment. 2 Group received PBS containing 2% dextran sulfate sodium (DSS). 3 Group received low dose (2 · 1010 CFU/kg) of pure L. plantarum (pLp) and 2% DSS. 4 Group received low dose (2 · 1010 CFU/kg) of nanometric L. plantarum (nLp) and 2% DSS. 5 Group received high dose (2 · 1011 CFU/kg) of nLp and 2% DSS. 6 Group received naturally fermented kimchi and 2% DSS. 7 Group received naturally fermented kimchi with added nLp and 2% DSS. NS, not significantly different.

(Shimadzu, Kyoto, Japan) at 405 nm. A p-nitrophenol production rate of 1 lmol/min was defined as 1 unit. Statistical analysis Results are expressed as means – standard deviations. One way analysis of variance (ANOVA) and Duncan’s multiple range test were used to determine the significances of intergroup differences. The analysis was conducted using PASW statistics version 18 (IBM Co., Armonk, NY, USA), and statistical significance was accepted for P values of

Effect of Nanometric Lactobacillus plantarum in Kimchi on Dextran Sulfate Sodium-Induced Colitis in Mice.

Nanometric Lactobacillus plantarum (nLp) is a processed form of Lab. plantarum derived from kimchi and is 0.5-1.0 μm in size. This study was undertake...
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