Inhibitory effect of lactic acid bacteria isolated from kimchi against murine norovirus

Food Control 109 (2020) 106881

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Inhibitory effect of lactic acid bacteria isolated from kimchi against murine norovirus

T

Dong Joo Seoa, Day Jungb, Soontag Jungb, Daseul Yeob, Changsun Choib,∗ a b

Department of Food and Nutrition, Gwangju University, Gwangju, 61743, Republic of Korea Department of Food and Nutrition, Chung-Ang University, Anseong, 17546, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Norovirus Fermented food Kimchi Lactic acid bacteria Food safety

Human norovirus causes gastroenteritis through a fecal-oral route. However, there are no effective therapeutic agents owing to the lack of culture systems. Alternatively, antiviral food extracts and beneficial microorganisms could be developed as antiviral agents. Here, we investigated the inhibitory effects of 142 lactic acid bacteria (LAB) or LAB-free filtrate from kimchi products against murine norovirus-1 (MNV-1) in RAW264.7 cells. LAB strains were identified through 16S rRNA gene sequencing. Cells were pre-treated with LAB or LAB-free filtrate, and co-treated with LAB and MNV-1 at 5 °C for 21 days. Among the 56 coccus-shaped LAB, pre-treatment of Pediococcus pentosaceus (CAU170229-2 and CAU170230-3) and Weissella cibaria (CAU170231-1 and CAU170231-3) significantly reduced MNV-1 by 1.93 ± 0.21–3.49 ± 0.43 to log10 PFU/mL. Among the 86 rodshaped LAB, pre-treatment of Lactobacillus sakei (CAU170208-2 and CAU170210-4) and Lactobacillus curvatus (CAU170210-2 and CAU170235-3) significantly decreased MNV-1 by 1.42 ± 0.26–1.70 ± 0.63 log10 PFU/mL. LAB-free filtrates decreased MNV-1 by 0.26 ± 0.07–0.50 ± 0.18 to log10 PFU/mL, and MNV-1 co-treated with LAB slightly reduced MNV-1 by 0.56 ± 0.07–0.60 ± 0.09 to log10 PFU/mL. Thus, W. cibaria, P. pentosaceus, L. curvatus, and L. sakei from kimchi are promising anti-noroviral candidates.

1. Introduction Human norovirus (HuNoV), belonging to the family Caliciviridae, is a non-enveloped positive-sense single-stranded RNA virus. HuNoV is a prevalent enteric pathogen causing viral gastroenteritis through the fecal-oral route worldwide. HuNoV is highly resistant to environmental fluctuations such as temperature, acid, and surface properties (Buckley et al., 2018; Cannon et al., 2006). HuNoV culture systems have been developed using intestinal or immune cell lines, and infection animal models have been used to investigate the biochemical properties and viability of HuNoV (Takanashi et al., 2014). Although the human intestine enteroid could successfully be used for the cultivation of HuNoV, this system is limited as the viral replication depends on specific viral genotypes and titers (Ettayebi et al., 2016). This lack of an effective culture system for HuNoV has greatly hindered the ability to gain viral control on foods, environments, and facilities.

Several recent studies have demonstrated the antiviral activity of food extracts and compounds against enteric viruses. Ginseng and grape seed extract inhibited hepatitis A virus (HAV) and feline calicivirus (FCV) (Lee et al., 2013; Su & D'Souza, 2011). Phytochemicals such as flavonoids and ginsenosides reduced murine norovirus (MNV), FCV, and HAV (Lee et al., 2013; Seo et al., 2016). In addition, lactic acid bacteria (LAB) isolated from fermented foods and their bacteriocins have been evaluated for their potential to inhibit RNA and DNA viruses (Maragkoudakis, Chingwaru, Gradisnik, Tsakalidou, & Cencic, 2010; Todorov et al., 2010). Their antiviral mechanism of action has been suggested to involve immune enhancement and cell protective effects. As HuNoV is an enteric food-borne virus causing gastroenteritis in the intestinal tract, an anti-noroviral effect by interaction with enteric bacteria such as LAB may be possible. LAB are non-sporing, non-respiring, catalase-negative, and acidtolerant gram-positive bacteria that are well known as beneficial enteric

Abbreviations: CFU, colony-forming unit; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; FCV, feline calicivirus; HAV, hepatitis A virus; HuNoV, human norovirus; IFN, interferon; IL, interleukin; LAB, lactic acid bacteria; MNV-1, murine norovirus-1; MRS, de Man, Rogosa and Sharpe; NO, nitric oxide; PBS, phosphate-buffered saline; PFU, plaque-forming units; RV, rotavirus; TGEV, transmissible gastroenteritis virus; TNF, tumor necrosis factor; VSV, vesicular stomatitis virus ∗ Corresponding author. Department of Food and Nutrition, School of Food Science and Technology, Chung-Ang University, Anseong, 17546, Republic of Korea. Tel: +82-31-670-4589, Fax: +82-31-676-8741. E-mail address: [email protected] (C. Choi). https://doi.org/10.1016/j.foodcont.2019.106881 Received 1 May 2019; Received in revised form 1 September 2019; Accepted 5 September 2019 Available online 12 September 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.

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2.4. 16S rRNA analysis

bacteria (Salminen & Von Wright, 2004). LAB mainly produce lactic acid as the end point of carbohydrate metabolism. The major LAB genera include Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weissella. LAB are essential for the production of fermented foods such as soy products (natto, doenjang, and kochujang), dairy products (yogurt, cheese, and butter), fish products (joetgal), vegetable products (kimchi, pickle, and sauerkraut), and meat products (sausage and salami) (Ray & Joshi, 2014). Kimchi is one of the most traditional and representative Korean fermented vegetable foods. Various types of LAB are produced during kimchi fermentation and ripening, such as those of the genera Weissella, Lactobacillus, and Leuconostoc (Park, Jeong, Lee, & Daily, 2014). LAB strains isolated from kimchi and their metabolites were previously identified to have antioxidant, antitumor, antiobesity, antimutagenic, and antimicrobial effects (Park et al., 2014). However, the viral inhibitory effects of kimchi-derived LAB have not been investigated to date. Accordingly, the aim of the present study was to isolate LAB from kimchi products and investigate their inhibitory effects against MNV-1 through the pre-treatment of LAB or LAB-free filtrate on murine macrophages (RAW264.7 cells) and the co-treatment of LAB and MNV-1.

For identification of the LAB isolated from kimchi, the genomic DNA was extracted from LAB growing on MRS agar (Kang et al., 2018). The DNA was amplified targeting the 16S rRNA gene region with the primer set 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) using BioFACT™ 2X Taq PCR Master Mix 1 (ST301-50h). After gel electrophoresis and purification (HiGene™ Gel & PCR Purification System, GP104-100) of the polymerase chain reaction template, the 16S rRNA gene was amplified with the sequencing primers 518F (5′-CCA GCA GCC GCG GTA ATA C-3′) and 805R (5′-GAC TAC CAG GGT ATC TAA TC-3′) using BigDye™ Terminator v3.1 Cycle Sequencing Kit. The sequences of 16S rRNA genes were analyzed using a DNA sequencer (ABI 3730XL DNA Analyser) and DNA STAR Lasergene software. 2.5. Preparation of LAB and the LAB-free filtrate LAB and LAB-free filtrates were prepared according to methods described in previous studies (Aboubakr, El-Banna, Youssef, AlSohaimy, & Goyal, 2014; Maragkoudakis et al., 2010). In brief, the LAB were propagated in MRS broth for 24–48 h at 37 °C. Two milliliters of LAB was centrifuged at 2,000 g for 15 min. The LAB pellets were washed twice with phosphate buffered saline (PBS) and resuspended in serum-free and phenol red-free DMEM (GIBCO, A14430-01). The total LAB in 1 mL of the culture solution were counted by plating 10-fold serially diluted LAB on MRS agar. The optical density of the viable LAB in 1 mL was measured at 620 nm, and the number of LAB was determined by reference to a standard curve. The final concentration of LAB was prepared at 9 log10 colony forming units (CFU)/mL. According to previous studies, we used 7 log10 CFU/mL as the final concentration (Bae et al., 2018; Lee et al., 2012). To prepare the LAB-free filtrate, 2 mL of LAB (9 log10 CFU/mL) was centrifuged at 2,000 g for 15 min. The supernatant was collected in a 15 mL conical tube and neutralized to pH 7 with 1 M NaOH. The LAB-free filtrate was then prepared by filtering through a 0.2 μm pore-size filter.

2. Materials and methods 2.1. Cells and viruses RAW264.7 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Hyclone Laboratories, Logan, UT, USA). Strain MNV-1 was kindly provided by professor H. W. Virgin of the University of Washington and was used as a surrogate of HuNoV. Viruses were propagated in RAW264.7 cells for 24 h at 37 °C in a 5% CO2 incubator and prepared at a titer of 6 log10 plaque-forming units (PFU)/mL (Bae et al., 2018; Lee, Yoo, Ha, & Choi, 2012; Shearer, Hoover, & Kniel, 2014).

2.6. Antiviral assay 2.2. Kimchi products 2.6.1. Pre-treatment of RAW264.7 cells with LAB or LAB-free filtrate The effect of LAB pretreatment against MNV-1 was observed according to slight modifications of previously reported assays (Lee et al., 2011; Maragkoudakis et al., 2010). RAW 264.7 cells (0.5 × 105/well) in DMEM containing 10% FBS were overlaid in a 24-well plate overnight. The cells were inoculated with LAB (7 log10 CFU/mL) when reaching 85–90% confluence. After 90 min of treatment, the RAW264.7 cells were washed with PBS twice, and 200 μL of serum-free and phenol red-free DMEM was added to each well. MNV-1 was serially diluted 10-fold in PBS and added to each well. Each well was then overlaid with 1X DMEM and 1X agarose gel (Sigma, Milwaukee, WI, USA), 5% FBS, and 1% penicillin-streptomycin, and incubated for 48 h at 37 °C in a 5% CO2 incubator. Plaques were counted after staining with 1.5% crystal violet solution. Similarly, the LAB-free filtrate diluted with 1X DMEM (1:10 dilution) was added to the cells at 85–90% confluence. After 90 min, the RAW264.7 cells were washed with PBS twice, and 200 μL of serum-free, phenol red-free DMEM was added to each well. MNV-1 was serially diluted 10-fold in PBS and added to each well. The MNV-1 titer was determined according to the plaque assay as described above.

A total of 42 kimchi products were purchased from local and online markets from April 24, 2017 to November 14, 2017. These kimchi products could be classified into the following 20 types: four gat kimchi (Indian mustard leaves), four kkakdugi (diced radish), three yeolmu kimchi (young radish), three chonggak kimchi (ponytail radish), three baechu kimchi (Chinese cabbage), three baek kimchi (whitish baechu), three pa kimchi (green onion), three pogi kimchi (cabbage), two dongchimi (radish water), two geotjeori kimchi (salad-type), two mul kimchi (watery), two mat kimchi (cut cabbage), one buchu kimchi (leek), one kannip kimchi (perilla leaf), one oi-sobagi (stuffed cucumber), one mukeunji (long-term-aged), one nabak kimchi (slicedradish), one baek yeolmu mul kimchi (young radish watery), one ulgari-baechu kimchi (winter-grown cabbage), and one seokbakji (radish). All kimchi products were immediately used for LAB isolation and stored at 5 °C until further analysis. 2.3. LAB isolation Each of the 42 kimchi products was fermented for 4 weeks at 5 °C. LAB strains were isolated once a week. In brief, the kimchi juice was plated onto de Man, Rogosa and Sharpe (MRS) agar (Difco Laboratories, Detroit, MI, USA) and maintained for 24 h at 37 °C in a 5% CO2 incubator. Each colony that formed on the MRS agar plate was grown in MRS broth (Difco Laboratories, Detroit, MI, USA) and stored in a cryovial with a 50% glycerol stock solution at −70 °C until used in the antiviral assay.

2.6.2. Co-treatment of LAB and MNV-1 To examine the co-treatment effect of LAB against MNV-1, the storage temperature was set to 5 °C because kimchi is commonly stored at 0–5 °C. MNV-1 was mixed with an equal volume of each LAB isolate. The final concentration of each LAB and MNV-1 was 7 log10 CFU/mL and 6 log10 PFU/mL, respectively. MNV-1 not treated with LAB was 2

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RAW264.7 cells significantly decreased MNV-1 by 1.11 ± 0.11 to 3.49 ± 0.43 log10 PFU/mL (P < 0.05; Table 2). In addition, there were significant differences in the inhibitory ability among the LAB isolates, even among the same species (P < 0.05). For example, pretreatment with W. cibaria CAU170231-1, CAU170231-3, and CAU170276-4 showed different degrees of inhibition, as did treatment with P. pentosaceus CAU170229-2, CAU170230-3, and CAU170208-4. P. parvulus CAU170209-1, W. paramesenteroides CAU170276-3, W. confusa CAU170278-3, and W. hellenica CAU170277-4 only slightly decreased MNV-1 compared to the other strains. Among the 86 Lactobacillus strains, the pre-treatment of L. curvatus (CAU170210-2, CAU170235-3, CAU170206-2, and CAU170207-1), L. sakei (CAU170208-2, CAU170210-4, CAU170206-1, and CAU1702124), L. plantarum (CAU170212-4), and L. coryniformis subsp. torquens (CAU170209-2) in RAW264.7 cells significantly decreased MNV-1 by 1.70 ± 0.63 to 1.06 ± 0.28 log10 PFU/mL. Compared with each strain, the pretreatment of W. cibaria CAU170231-1 reduced MNV-1 two times more than L. curvatus CAU170210-2 (3.49 ± 0.43 log10 versus 3.49 ± 0.43 log10 PFU/mL).

used as a positive control. MNV-1 and LAB titers were measured for 0, 1, 3, 5, 7, 14, and 21 days of co-culture. The MNV-1 and LAB mixture (100 μL) was centrifuged at 2,000 g for 15 min, and the MNV-1 titer was observed with a plaque assay as described above. The LAB titer was determined on MRS agar by plating the MNV-1 and LAB mixture (100 μL) serially diluted in 0.1% peptone water (Oxoid, Basingstoke, UK). 2.7. Statistical analysis Three replicates were used for plaque assay of each data. Each experiment was repeated three times independently. Data were analyzed using one-way analysis of variance and Duncan's multiple range test in Statistical Analysis System (SAS 9.1; Cary, NC, USA). Significant differences were indicated with P-values less than 0.05. 3. Results 3.1. LAB isolated from kimchi products A total of 481 LAB strains was isolated from the 42 kimchi products according to different fermentation stages for 4 weeks of culture. Among them, 19 species were identified by the 16S rRNA analysis, which belong to the genera Lactobacillus, Leuconostoc, Pediococcus, and Weissella (Table 1). Leu. mesenteroides and L. sakei were most prevalent species in kimchi products used in this study. As kimchi is fermented by heterofermentative LAB (Jung et al., 2011), it is known that Leuconostoc, Lactobacillus, and Weissella were the dominant LAB genera and Lactococcus and Pediococcus are minor LAB genera by metagenomics analysis of kimchi. Depending on the fermentation stage, various LAB species were identified like previous study. As strains repeatedly isolated from same kimchi product at different fermentation stage were excluded, 142 LAB isolates were selected for anti-noroviral assay: Lactobacillus (n = 86), Leuconostoc (n = 21), Pediococcus (n = 10), and Weissella (n = 25).

3.3. Pre-treatment effect of LAB-free filtrates against MNV-1 in RAW 264.7 cells Pre-treatment of LAB-free filtrates in RAW264.7 cells also significantly reduced MNV-1, but the reduction of MNV-1 was much lower than that induced by the LAB. Ten filtrates of the 56 coccus-shaped LAB isolates significantly decreased MNV-1 (P < 0.05; Table 3), but there was no statistical significance among LAB isolates. W. paramesenteroides (CAU170280-4 and CAU170276-3) filtrates showed the greatest reduction of MNV-1 by 0.50 ± 0.18 and 0.48 ± 0.07 log10 PFU/mL, followed by P. pentosaceus (CAU170230-3 and CAU170234-1) filtrates. L. parvulus, Leu. mesenteroides, and W. cibaria also slightly reduced MNV-1. While P. pentosaceus CAU170230-3, P. parvulus CAU170209-3, and W. cibaria (CAU170231-1 and CAU170276-4) decreased MNV-1 by both LAB and LAB-free filtrate, the other isolates showed the antinoroviral effect by either LAB or LAB-free filtrates (Tables 2 and 3). Among the 86 rod-shaped LAB isolates, filtrates of L. paracasei, L. plantarum, L. sakei, L. pentosus, and L. curvatus showed slight reduction of MNV-1 by 0.37 ± 0.06 to 0.26 ± 0.07 (P < 0.05; Table 3), and there were no significant differences in the inhibitory effects among isolates. Like coccus-shaped LAB isolates, L. sakei (CAU170208-2, CAU170206-1, and CAU 170212-4), L. plantarum CAU 170280-2, and L. curvatus CAU170206-2 decreased MNV-1 by both LAB and LAB-free filtrate. While some filtrates of L. paracasei CAU170214-2, L. sakei (CAU170202-2, CAU 170210-1, and CAU170206-3), and L. pentosus CAU170230-2 slightly decreased MNV-1, the pretreatment of their bacterium did not show the anti-noroviral effect significantly (Tables 2 and 3).

3.2. Pre-treatment effect of LAB against MNV-1 in RAW 264.7 cells Among the 56 coccus-shaped LAB isolates identified, the pre-treatment of W. cibaria (CAU170231-1, CAU170231-3, and CAU170276-4), P. pentosaceus (CAU170229-2, CAU170230-3, and CAU170208-4), P. parvulus (CAU170209-1), W. paramesenteroides (CAU170276-3), W. confuse (CAU170278-3), and W. hellenica (CAU170277-4) isolates in Table 1 Lactic acid bacteria isolated from kimchi products. Genus

Type strain

Number of isolates

Lactobacillus

Lactobacillus brevis Lactobacillus coryniformis subsp. Torquens Lactobacillus curvatus Lactobacillus fermentum Lactobacillus heilongjiangensis Lactobacillus parabuchneri Lactobacillus paracasei Lactobacillus pentosus Lactobacillus plantarum Lactobacillus sakei Leuconostoc citreum Leuconostoc mesenteroides Pediococcus parvulus Pediococcus pentosaceus Weissella cibaria Weissella confusa Weissella hellenica Weissella paramesenteroides Weissella soli

7 1 8 1 1 1 4 5 3 55 1 20 3 7 14 3 1 6 1 142

Leuconostoc Pediococcus Weissella

Total

3.4. Co-treatment effect of LAB against MNV-1 The co-treatment of LAB isolates and MNV-1 at 5 °C reduced MNV-1 slightly during the 21 days period (P < 0.05; Fig. 1). Among the 56 coccus-shaped LAB, P. pentosaceus (CAU170230-3) and W. paramesenteroides (CAU170280-4) significantly reduced MNV-1 by 0.60 ± 0.09 and 0.58 ± 0.12 log10 PFU/mL in 21 days of co-culture, respectively. Among the 86 rod-shaped LAB, L. pentosus (CAU170234-4) and L. sakei (CAU170212-4) reduced MNV-1 by 0.57 ± 0.12 and 0.56 ± 0.07 log10 PFU/mL, respectively. In addition, MNV-1 not treated with LAB naturally reduced by 0.26 ± 0.07 log10 PFU/mL after 21 days (P < 0.05), but there was a significant difference between the LAB-treated MNV-1 group and non-treated MNV-1 group on day 21 (P < 0.05). Overall, there was an approximate 0.5 log10 CFU/mL reduction of LAB isolates after 21 days of co-culture with MNV-1. 3

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Table 2 Log reduction of MNV-1 in RAW 264.7 cells pre-treated with kimchi lactic acid bacteria (LAB) isolates. Shape

LAB strain

Source

Isolate number

Reduction(log10 PFU/mL)

Coccus

Weissella cibaria Weissella cibaria Pediococcus pentosaceus Pediococcus pentosaceus Pediococcus parvulus Weissella paramesenteroides Pediococcus pentosaceus Weissella cibaria Weissella confusa Weissella hellenica

Pa kimchi Pa kimchi Geotjeori kimchi Chonggak kimchi Nabak kimchi Pa kimchi Baek kimchi Pa kimchi Gat kimchi Ulgari-baechu kimchi

CAU170231-1 CAU170231-3 CAU170229-2 CAU170230-3 CAU170209-1 CAU170276-3 CAU170208-4 CAU170276-4 CAU170278-3 CAU170277-4

3.49 2.12 2.02 1.93 1.47 1.42 1.35 1.28 1.14 1.11

± ± ± ± ± ± ± ± ± ±

0.43a 0.16b 0.30bc 0.21bcd 0.68cde 0.51cde 0.22de 0.10e 0.02e 0.11e

Rod

Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus

Baechu kimchi Baek kimchi Baechu kimchi Yeolmu kimchi Kkakdugi Yeolmu kimchi Yeolmu kimchi Baek kimchi Nabak kimchi Baek yeolmu mul kimchi

CAU170210-2 CAU170208-2 CAU170210-4 CAU170235-3 CAU170280-2 CAU170206-1 CAU170206-2 CAU170207-1 CAU170209-2 CAU170212-4

1.70 1.54 1.49 1.42 1.32 1.22 1.18 1.07 1.06 1.06

± ± ± ± ± ± ± ± ± ±

0.63a 0.58a 0.60a 0.26a 0.30a 0.49a 0.17a 0.34a 0.35a 0.28a

curvatus sakei sakei curvatus plantarum sakei curvatus curvatus coryniformis subsp. torquens sakei

Data are presented as mean ± SD of triplicate experiments. Different letters in the column indicate statistically significant differences of coccus or rod-shaped strains.

4. Discussion

kimchi products were studied in previous study (Jung et al., 2011). L. sakei, Leu. citreum, Leu. kimchii, Leu. lactis, Leu. mesenteroides, W. cibaria, and W. confuse were isolated during the early fermentation stages of kimchi. L. alimentarius, L. brevis, L. curvatus, L. pentosus, L. sakei, Leu. citreum, Leu. gelidum, Leu. lactis, W.cibaria, W. confuse, and W. koreensis were present in middle stages of kimchi fermentation. L. curvatus, L. plantarum, L. sakei, L. brevis, Leu. citreum, Leu. gelidum, Leu. mesenteroides, P. cerevisiae, W. cibaria, W. confuse, and W. koreensis were isolated in late stages of kimchi fermentation (Jung et al., 2014; Kim et al., 2016; Lee, 1991). L. sakei, Leu. citreum, Leu. mesenteroides, W. confuse, and W. cibaria are associated with all stages of kimchi ripening. Although Leu. kimchii, Leu. gelidum, L. Alimentarius, L. koreensis, and P. cerevisiae were not isolated in this study, L. sakei was the most predominant in various kimchi followed by Leu. mesenteroides, W. cibaria, L. curvatus, P. pentosaceus, and L. brevis (Jung et al., 2014; Lee, 1991). In previous studies, the antiviral effect of L. casei, L. rhamnosus, L. curvatus, L. gasseri, L. plantarum, L. paracasei, and Bifidobacterium longum

Since it was reported that the norovirus surrogates were reduced during the kimchi fermentation, there have been some efforts to find antiviral factors in kimchi (Lee et al., 2012; Park et al., 2015). But several norovirus outbreaks occurred by the consumption of the tainted kimchi served in school meals. Based on the epidemiology, the causative food was the fresh kimchi which has very short fermentation time. Most of commercial kimchi products has very short fermentation time to give fresh taste. However, traditional kimchi served in household is fermented for a long time at low temperature and have different flavor and taste rather than fresh kimchi (Park et al., 2015). This study isolated many different LAB from commercial kimchi products depending on the various stage of fermentation. Among them, 16S rRNA sequencing identified 19 species belong to the genera Leuconostoc, Lactobacillus, and Weissella, and Pediococcus. As heterofermentative LAB contribute kimchi fermentation, the microbiome of

Table 3 Log reduction of MNV-1 in RAW 264.7 cells pre-treated with kimchi lactic acid bacteria (LAB)-free filtrates. Shape

LAB strain

Source

Isolate number

Reduction (log10 PFU/mL)

Coccus

Weissella paramesenteroides Weissella paramesenteroides Pediococcus pentosaceus Pediococcus pentosaceus Pediococcus parvulus Leuconostoc mesenteroides Leuconostoc mesenteroides Weissella cibaria Weissella cibaria Leuconostoc mesenteroides

Kkakdugi Pa kimchi Chonggak kimchi Mul kimchi Nabak kimchi Baechu kimchi Baek kimchi Pa kimchi Pa kimchi Pogi kimchi

CAU170280-4 CAU170276-3 CAU170230-3 CAU170234-1 CAU170209-3 CAU170210-3 CAU170207-2 CAU170231-1 CAU170276-4 CAU170211-3

0.50 0.48 0.47 0.45 0.35 0.33 0.32 0.31 0.31 0.31

± ± ± ± ± ± ± ± ± ±

0.18a 0.07a 0.06a 0.13a 0.16a 0.25a 0.15a 0.07a 0.07a 0.07a

Rod

Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus

Mat kimchi Kkakdugi Kkakdugi Baek kimchi Baek yeolmu mul kimchi Yeolmu kimchi Chonggak kimchi Yeolmu kimchi Baechu kimchi Yeolmu kimchi

CAU170214-2 CAU170280-2 CAU170205-2 CAU170208-2 CAU170212-4 CAU170206-1 CAU170230-2 CAU170206-2 CAU170210-1 CAU170206-3

0.37 0.34 0.32 0.32 0.32 0.32 0.32 0.32 0.27 0.26

± ± ± ± ± ± ± ± ± ±

0.06a 0.12a 0.15a 0.15a 0.15a 0.15a 0.15a 0.15a 0.14a 0.07a

paracasei plantarum sakei sakei sakei sakei pentosus curvatus sakei sakei

Data are indicated as mean ± SD of triplicate experiments. Different letters in the column show statistically significant differences of coccus or rod-shaped strains. 4

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0.7

Fig. 1. Reduction of MNV-1 co-treated with LAB for 21 days at 5 °C. A) Pediococcus pentosaceus (CAU170230-3), B) Weissella paramesenteroides (CAU170280-4), C) Lactobacillus pentosus (CAU170234-4), D) Lactobacillus sakei (CAU170212-4). Circles and triangles indicate the non-treated MNV-1 group and LAB-treated MNV-1 group, respectively. Data are the mean ± SD. Asterisks show a significant difference between the non-treated MNV-1 group and LAB-treated MNV-1 group (p < 0.05). Different letters indicate statistically significant differences between each non-treated MNV-1 group and LAB-treated MNV-1 group (p < 0.05).

should identified in further study. Also, the antiviral activity of them should be evaluated according to various concentrations and treatment times in cells. For the first time, this study reported that the pre-treatment of Weissella spp. and Pediococcus spp. isolated from kimchi showed 2-fold higher antiviral activity against MNV-1 than that of Lactobacillus spp. However, these strains only reduced MNV-1 less than 0.5 log10 CFU/mL when they were co-treated at 5 °C as the typical storage temperature of kimchi. When the co-treatment effect of LAB and MNV-1 was minimal, the direct inhibitory mechanism such as interference of virus infectivity or direct attachment onto LAB was not likely. Taken together, the antiviral mechanism of MNV-1 by the pre-treatment of Weissella spp. and Pediococcus spp. may seemed to indirect. In other study, W. cibaria JW15 isolated from kimchi demonstrated the strong immunomodulatory effects by stimulating NF-κB, IL-1β, TNF-α, and NO in macrophages (Lee et al., 2013). In addition, P. pentosaceus OZF isolated from human breast milk was evaluated as a promising probiotic strain to enhance the immune response (Osmanagaoglu, Kiran, Yagci, & Gursel, 2013). As the antiviral effect of Weissella spp. and Pediococcus spp. has not been well investigated up to date, the immunomodulation by Weissella spp. and Pediococcus spp. should be investigated as a potential anti-noroviral mechanism. As P. pentosaceus, L. curvatus, and L. sakei are all included on the Qualified Presumption of Safety list compiled by the European Food Safety Authority, they could be used as potential candidates for application in the fermented food industry (Garrote et al., 2010). Some strains of W. cibaria, P. pentosaceus, L. curvatus, and L. sakei are known to produce bacteriocins (Salminen & Von Wright, 2004). Similarly, the effects of LAB isolated from kimchi applied in other fermented foods

against transmissible gastroenteritis virus, and rotavirus, human immunodeficiency virus, herpes simplex virus, influenza virus, vesicular stomatitis virus were reported (Hoang et al., 2015; Ivec et al., 2007; Maragkoudakis et al., 2010; Martín, Maldonado, Fernández, Rodríguez, & Connor, 2010). The pre-treatment of cells with LAB filtrate/supernatant, live LAB, or killed LAB, co-treatment of LAB and virus, and virus absorption to LAB were used for the antiviral assay (Botić, Klingberg, Weingartl, & Cencič, 2007). Depending on the LAB strains, their antiviral mechanisms of LAB and probiotic bacteria were proposed as follows: (1) the degradation of viral proteins, (2) the cellular protective effect, (3) the direct attachment on cells, (4) the immunomodulation, and (5) the interaction with cell receptors. As LAB metabolites containing organic acid, hydrogen peroxide, carbon dioxide, amino acids, and bioactive peptides such as bacteriocin have been as antimicrobial agents, previous study reported that L. plantarum LBP-K10 filtrate from kimchi reduced influenza A H3N2, and its antiviral active component was identified as cyclic dipeptides (Kwak et al., 2013; Šušković et al., 2010). When the pre-treatment of B. longum, L. plantarum, and L. reuteri metabolites inhibited the virus infectivity more than 60%, the production of antiviral metabolites may be associated with specific LAB strain and the antiviral activity of them were observed only at a high concentrate (Botić et al., 2007). On the contrary, the pre-treatment of a probiotic mixture supernatant of L. acidophilus, L. rhamnosus, Bifidobacterium bifidum, L. salivarius, and S. thermophilus was not effective to reduce MNV-1 and Tulane virus (Shearer et al., 2014). Compared with the other studies, the lactobacillus isolates from kimchi or its LAB-free filtrate could be candidate strain to reduce norovirus surrogate. Considering that the LAB or LAB-free filtrate was not concentrated, the active metabolites from each LAB strain 5

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have been evaluated in previous studies. To date, kimchi-derived probiotic strains such as W. koreensis HO20, L. plantarum Lb41, L. plantarum DKL119, and L. brevis 340G have been used for the production of rice cake, cottage cheese, and dairy products, respectively (Cho, Hong, & Kim, 2013; Choi, Park, & Yoon, 2013; Jeon, Son, Jeewanthi, Lee, & Paik, 2016; Seo et al., 2013). Thus, these strains could be applied as starters or supplements in the food industry. Their biochemical characteristics, safety properties such as virulence factors and antibiotic resistance, and sensory qualities should be investigated in further study.

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