Note Isolation and Identification of Lactic Acid Bacteria from Xiaoshan

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Isolation and Identification of Lactic Acid Bacteria from Xiaoshan Pickle Radish, a Traditional Fermented Vegetable

1,2* 1,2 Yan Chen and Tiejin Ying

1Department of Food Science and Nutrition, College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, PR China 2Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, PR China

Received September 24, 2015 ; Accepted September 6, 2016

The lactic acid bacterial flora during processing of Xiaoshan pickle radish were investigated. The samples were pickled with the product from three different markets by spontaneous fermentation. The average pH value varied from 6.8 ± 0.1 to 4.6 ± 0.2. There was no significant difference between the number of bacteria and pH value in samples from different product sites. A total of 387 gram-positive and catalase-negative isolates were obtained. All isolates were identified as Lactobacillus sakei and Leuconostoc lactis by physiological tests and 16S rRNA gene sequencing. Leuc. lactis was the dominated species in the initial stages of fermentation, but in late stages L. sakei had a remarkly increasing and the percentage were 0.0%, 16.7%, 50.0%, 81.8%, 80.0%, 83.3% and 100.0% respectively from stage “A”(before washing with clean water) , ‘‘B’’ (after washing with clean water), ‘‘C’’ (before first curing), ‘‘D’’(after first curing), ‘‘E’’ (before second curing), ‘‘F’’ (after second curing) to “G” (product ready-to-eat).

Keywords: identification, lactic acid bacteria, Xiaoshan pickle radish,fermentation

Introduction present in the raw materials. The quality is unstable as the process Xiaoshan pickle radish (Raphanus sativus L.) or “Xiaoshan depends on season, climate, raw material properties, manual luobogan” is one of the traditional fermented vegetables and a operation and so on. The current fermentation is still a traditional famous local specialty of Zhejiang province in China (Fig.1). The household technology without addition of any commercial starter product has been widely consumed with its special flavor. Xiaoshan cultures. As a result, it is difficult to ensure an adequate level of pickle radish is made from radish cultivar of “Yidaozhong”. After hygiene and quality uniformity. It is thus important to screen the washing with clean water, the roots are cut into uniform strips, dominate microorganism of Xiaoshan pickle radish with beneficial each with peel. Then they are wilted with sunlight for 3 to 5 days. function and desirable properties to be used as starter cultures, Salt particle is added to about 3% by quality and well kneaded, which will improve the quality and safety of the final product and then the mixture is pressed tightly layer by layer into vat. It is left standardizes the production process. for frist stage curing at room temperature for 3 days and then sun Lactic acid bacteria (LAB) play an important role in vegetable dried for 2 to 3 days.To form the final product, the second stage fermentation, which cause rapid acidification of the raw material. curing is carried out with addition of 1.5% salt. The product will be They contribute to the taste, flavor and texture of fermented ready-to-eat after 7 days fermentation and 2 to 3 days drying. products and inhibit food spoilage bacteria and pathogenic bacteria The traditional products of Xiaoshan pickle radish was based by producing organic acids, mainly lactic acid, bacteriocins, on spontaneous fermentation initiated by the natural microflora ethanol, aroma compounds, exopolysaccharides, several enzymes

*To whom correspondence should be addressed. E-mail: [email protected] 130 Y. Chen & T. Ying

‘‘E’’ (product before second curing); stage ‘‘F’’ (product after second curing); stage ‘‘G’’ (product ready-to-eat). Enumeration and isolation of lactic acid bacteria 10 g sample was transferred to 90 mL 0.85% sterile physiological saline and _ _ serial dilutions (10 1 _ 10 9) of each sample were prepared. LAB were detected and isolated on MRS agar (Hangzhou microbial

broth, China) supplemented with 1% CaCO3. 0.1 mL aliquots of the dilutions at sampling points ‘‘A’’, ‘‘B’’, ‘‘C’’, ‘‘D’’, ‘‘E’’, ‘‘F’’and ‘‘G’’were spread onto the surface of MRS agar in triplicate and were incubated at anaerobic incubator (YQX-II, cimo, Shanghai, China) at 37℃ for 48 h. Plates with 30 _ 300 colonies were enumerated. Colonies of acid-producing bacteria, identified by a clear zone around each colony, were randomly Fig. 1. Picture of Xiaoshan pickle radish. isolated from plates. The selected colonies were purified by and other microbial growth-inhibiting substances (Leroy et al., replating on MRS agar plates from a single colony at random for 2004; Chen et al., 2010). Various LAB have been identified from further identification. This procedure was repeated several times. different Chinese traditional fermented foods (Liu et al., 2011). Only gram-positive, catalase-negative strains were selected. Pickles can be used as good screening sources for the isolation of Purified strains of LAB were preserved in MRS broth using 15% valuable microorganisms (Hiraga et al., 2008). Chen et al. (2006) (v/v) glycerol at _20℃. indicated that Pediococcus pentosaceus and Tetragenococcus Identification of lactic acid bacteria halophilus are responsible for the fermentation of suan-tsai Biochemical and physiological characteristics Further (fermented leaf mustard). Yu et al. (2012) analyzed 36 pickle identification of gram-positive and catalase-negative isolates was samples from 6 different regions in Sichuan province and identified performed by using the following physiological tests: growth at as Enterococcus thailandicus, Lactobacillus alimentarius, temperatures of 10, 15 and 45℃ in MRS broth for 5 days, growth Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus at pH value of 4.5 and 9.0 in MRS broth for 3 days, growth at NaCl plantarum, Lactobacillus pentosus, Lactobacillus sakei, concentrations of 2.0%, 4.0% and 6.5% (w/v) in MRS broth for 3

Lactobacillus spicheri, Leuconostoc lactis and Pediococcus days (Kozaki et al., 1992). NH3 production from arginine was ethanolidurans. However, very limited document concerning the determined at 35℃ for 24 h in MRS broth with 0.3% L-arginine

LAB of Xiaoshan pickle radish have been reported until now. Zou (Harrigan et al., 1966). Gas production (CO2) from glucose was et al. (2007) reported that L1 isolated from Xiaoshan pickle radish investigated by growing the bacteria in MRS broth that contained slices was identified as Lactobacillus sp. by phenotypic methods, inverted Durham tubes (Tohno et al., 2013). The type of D and L but did not discriminate isolates at the species level. isomers of lactic acid produced from glucose was assayed in The objective of this paper was to isolate and identify the modified MRS broth using a commercial kit (Hoffman La Roche predominant lactic acid bacteria present during Xiaoshan pickle Diagnostic, Mannheim, Germany) (Mathara et al., 2004). Sugar radish fermentation. This information can be taken as the basis of fermentation patterns were evaluated according to the methods the development of starter cultures and may pave the way for large- described by Kozaki et al (1992). The results were checked scale commercial production. according to the information supplied in Bergey’s Manual of Systematic Bacteriology (1984). Materials and Methods 16S rDNA sequencing and molecular identification Total Sample collection Three batches of “Yidaozhong” radishes genomic DNA was extracted from 5 mL samples of overnight were purchased from Hangzhou Gudang agricultural products cultures grown in MRS broth at 37℃ by Genomic DNA Prep Kit market (GD), Xiaoshan Desheng agricultural products market (DS) (Tiangen, Beijing, China). PCR amplified using the universal and Hangzhou agricultural and sideline products logistics center primer pairs 27F:5´-AGAGTTTGATCCTGGCTCAG-3´ and (AS). Spontaneous fermentation occurred at average temperature 1541R:5´-AAGGAGGTGATCCAGCC -3´. All PCR reactions of 25℃ by traditional technology (Fig.2). The pH values of the were carried out in 50 μL reaction volumes containing: 10 mM pickle juice were determined using a calibrated portable pH-meter Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 50 ng of bacterial (pHS-25, shengci, Shanghai, China). DNA, 60 pM of each primer, 0.2 mM each dNTPs, and 2.5 U of Samples were taken at the end of each stage of production: Taq polymerase (Solarbio, Beijing, China). PCR amplifying stage ‘‘A’’ (product before washing with clean water); stage ‘‘B’’ procedure was as follows: 5 min at 94℃, 35 cycles of 1 min at (product after washing with clean water); stage ‘‘C’’ (product 94℃, 1 min at 58℃, 2 min at 72℃ and then 10 min at 72℃ for before first curing); stage ‘‘D’’(product after first curing); stage final extension. The PCR products were subjected to gel Lactic Acid Bacteria from Xiaoshan Pickle Radish 131

Fig. 2. Flowchart depicting for fermentation of Xiaoshan pickle radish. electrophoresis in 1% agarose gel, followed by staining with during stages ‘‘A’’ ‘‘B’’and ‘‘C’’ for the fermentation of pickle ethidium bromide and visualization under UV light. Amplified 16S radish had not started. In the next stage a rise in bacteria counts rRNA was isolated from the agarose gel using a Gel Extraction Kit was observed after first curing. The viable counts varied from (Tiangen, Beijing, China). The sequencing of purified products was 2.47 ± 0.32 to 5.41 ± 0.28 Log (cfu/g), 2.55 ± 0.06 to 5.54 ± performed by Shanghai Sangon Biosciences Corporation of China. 0.06 Log (cfu/g) and 2.51 ± 0.08 to 5.59 ± 0.32 Log (cfu/g) The nucleotide sequences of the 16S rRNA gene of all the respectively. In stages ‘‘E”, the bacteria counts were significantly isolates were analyzed and determined by the BLAST program on increased. The acid-producing bacteria counts reached the peak the NCBI website (i). The alignments were analyzed to construct a after the second curing. In the later stage ‘‘G”, the population phylogenetic tree and to compare similarities among the sequences decreased to a equivalent level with stage ‘‘E”. by the neighbor-joining method using MEGA software version 6.0. The average pH values in three market of samples at stage ‘‘C’’ Nucleotide sequence accession numbers were found to be higher than other samples. In the next stage “D”, The represent 16S rDNA sequences were deposited in the the measured values significantly decreased (p < 0.05), with the GenBank at the NCBI with the accession ID KP901103 to same trend in stage “E”. Samples of stage “F” and “G” had a KP901104. longest fermentation time and the lowest pH values. Statistical analysis Each experiment was repeated three times. During the whole fermentation process, the increase of bacteria The numbers of colonies were converted to Log cfu/g. Analysis of counts linked to the decrease in pH values. The decrease in pH variance (ANOVA) was performed using SAS software (version could be due to the gradual increase in fermenting microorganism 9.1, SAS Institute, N.C., USA) by Duncan’s multiple range test. A population and accumulation of the organic acid consequently. The significant difference was defined as p < 0.05. results are in agreement with the conclusion of many studies (Nche et al., 1994; Muyanja et al., 2003; Owusu-Kwarteng et al., 2012). Results and Discussion Identification of lactic acid bacteria pH of samples and enumeration of acid-producing bacteria Identification of LAB isolates by physiological characteristics Table 1 shows the counts of acid-producing bacteria and pH A total 387 of gram-positive, catalase-negative isolates were value in samples during seven different fermentation stage from obtained from 21 batches of samples. They were divided into two three markets in Hangzhou. The result indicated that there had no groups based on several morphological and physiological distinct association between production sites with pH value and characters (Table 2). bacteria quantity. The pH value of three market showed the same Group I was composed of 252 isolates. These isolates exhibited change, with an average drop from 6.8 ± 0.1 to 4.6 ± 0.2. There growth at 15 and 45℃, but not 10℃. They produced DL-lactic acid were no significant differences between the pH value and acid- but no NH3 from arginine and no gas from glucose. They could producing bacteria counts of the same fermentation stage in three ferment acid from D-arabinose, D-cellobiose, esculin, D-galactose, market. D-mannose, gluconate, salicin, D-lactose, ribose, D-sorbitol, The acid-producing bacteria counts were low in all the batches D-sucrose and D-trehalose. Those strains were identified as 132 Y. Chen & T. Ying

Table 1. pH changes and acid-producing bacteria counts during Xiaoshan pickle radish fermentation in 3 sites of Hangzhou

Fermentation stage Items Market A B C D E F G pH GD ND ND 6.8 ± 0.2Aa 6.0 ± 0.3Ab 5.8 ± 0.2Ac 4.6 ± 0.1Ad 4.7 ± 0.2Ad DS ND ND 6.7 ± 0.1Aa 5.8 ± 0.2Ab 5.7 ± 0.3Ac 4.5 ± 0.2Ad 4.5 ± 0.2Ad AS ND ND 6.8 ± 0.1Aa 5.9 ± 0.3Ab 5.7 ± 0.2Ac 4.7 ± 0.2Ad 4.6 ± 0.3Ad Log (cfu/g) GD 2.47 ± 0.32Ad 2.44 ± 0.17Ad 2.43 ± 0.83Ad 5.41 ± 0.28Ac 6.35 ± 0.17Ab 8.51 ± 0.21Aa 6.27 ± 0.33Ab DS 2.55 ± 0.06Ad 2.50 ± 0.05Ad 2.85 ± 0.22Ad 5.54 ± 0.06Ac 6.14 ± 0.42Ab 8.33 ± 0.44Aa 6.46 ± 0.71Ab AS 2.51 ± 0.08Ad 2.46 ± 0.23Ad 2.73 ± 0.57Ad 5.59 ± 0.32Ac 5.98 ± 0.63Ab 7.96 ± 0.12Aa 5.93 ± 0.63Ab

Each value represents mean ± S.D. (n = 3); values with the different lowercase letters in the same row mean significant difference at 0.05 level of each fermentation stage; values with the different capital letters in the same column mean significant difference at 0.05 level of each market; ND means not detect.

Table 2. Phenotypic characteristic of LAB isolated from Xiaoshan Lactobacillus sakei due to their characteristics. pickle raish Group II was composed of 135 coccus-shaped isolates, which Group produced D-lactic acid. These isolates exhibited growth at 15 and Characteristics I (252 isolates) II (135 isolates) 45℃, but not growth at 10℃ and in the presence of 6.5% NaCl. They could produce gas from glucose but no NH from arginine. Cell shape rod coccus 3 They fermented D-cellobiose, esculin, D-galactose, D-mannose, Gas from glucose - + Lactic acid isomera DL D salicin, D-lactose D-raffinose, ribose D-sucrose and D-xylose.

NH3 from arginine - - These isolates were assigned to genus Leuconostoc lactis . Growth at temperature (°C) Lactobacillus sakei was isolated from all fermentation stage 10 0b 0 except stage “A”, while Leuc. lactis was the dominant LAB 15 252 135 isolated during stage “A” and stage “B”. The isolates of L. sakei 45 252 135 increased from stage “C”, be equal with Leuc. lactis. From stage Growth at pH value “D”, L. sakei was more prevalent than Leuc. lactis. In stage “G”, 4.5 252 135 Leuc. lactis was no longer detected. From stage “A” to “G”, the 9.0 252 135 percentage of L. sakei were 0.0%, 16.7%, 50.0%, 81.8%, 80.0%, Growth in NaCl (w/v) 2.0% 252 135 83.3% and 100.0%. 4.0% 252 135 Regional similarities and differences in diversity were observed 6.5% 252 0 in the current study. Owusu-Kwarteng et al. (2012) reported Acid from Lactobacillus fermentum and Weisella confusa were isolated in all D-Arabinose 252 0 production sites and almost at all fermentation stages during D-Cellobiose 252 135 traditional fura (fermented millet) processing in Ghana, the other Esculin 252 135 LAB bacteria species which comprised a minor proportion of the D-Galactose 252 135 total LAB occurred occasionally and in an irregular pattern among Gluconate 252 0 the production sites. Tsuda et al. (2012) collected nine funazushi D-Mannitol 0 0 D-Mannose 252 135 (fermented crucian carp and rice) samples from different sites of D-Melezitose 0 0 Japan and found the LAB microflora of five homemade funazushi Salicin 252 135 showed a number of differences with each other. In this study, the D-Lactose 252 135 LAB counts of the radishes collected from different market did not D-Raffinose 0 135 show significant difference and it seemed that the LAB belonged to L-Rhamnose 0 0 the same species. This may be territorial restriction because the Ribose 252 135 “Yidaozhong” radishes were representative local variety of D-Sorbitol 252 0 Xiaoshan in Hangzhou, the chance of products obtaining from the L-Sorbose 0 0 same source was high. D-Sucrose 252 135 D-Trehalose 252 0 16S rRNA gene sequence identification and phylogenetic D-Xylose 0 135 analysis To confirm the species, the nucleotide sequences of the 16S rRNA gene of all the tested strains were analyzed and a L: L-lactic acid, DL: DL-lactic acid, D: D-lactic acid. b Number of positive strains. determined. The 16S rDNA sequences of L1 from group I and L2 from group II were submitted to NCBI. The sequences were then Lactic Acid Bacteria from Xiaoshan Pickle Radish 133

Fig. 3. Phylogenetic tree of L1with the 16S rRNA gene by neighbor-joining (NJ) method. Bootstrap values based on 1000 replications are given at the nodes. deposited to Genbank and assigned accession No KP901103 to associated with the L. sakei subsp. sakei in the neighbor-joining KP901104. analysis and supported by a bootstrap value of 71%. Its closest Some type strains were chosen to infer a possible phylogenetic neighbor was the type strain of Lactobacillus sakei subsp. sakei classification. Phylogenetic tree analysis in figure 3 and figure 4 JCM1157T, with 100% homology in their 16S rRNA gene was performed to show the relationship of 16S rRNA gene sequences. L2 was most closely related to Leuconostoc lactis sequences between the isolates and related type strains by using KCTC3528T, supporting the 100% value from bootstrap analysis of

MEGA software. Isolate L1 formed a distinct phyletic line that is the phylogenetic tree, and showing 99% similarity in their 16S 134 Y. Chen & T. Ying

Fig. 4. Phylogenetic tree of L2 with the 16S rRNA gene by neighbor-joining (NJ) method. Bootstrap values based on 1000 replications are given at the nodes. rRNA gene sequences. kimchi . After day 30, the numbers of Leuc. citreum and Leuc. Leuconostoc lactis, as heterofermentative LAB, produce lactic lactis cells decreased rapidly. The similar observation was made by acid, ethanol, and carbon dioxide from glucose through the Lee et al. (2015). 6-phosphogluconate/phosphoketolase pathway under anaerobic Lactobacillus sakei is specifically adapted to the meat matrix conditions (Jung et al., 2014). Leuconostoc lactis had been found (Chaillou et al., 2009), but in recent years this species commonly in fermented Chinese foods of stinky tofu (soybean curd), sour occurs in fermented vegetables. The strain C2 isolated from mifen (rice noodle), sour milk (Liu et al., 2011). Similar to our traditional Chinese fermented cabbage was identified as L. sakei results, 1 strain isolated from traditional pickles in Sichuan were (Gao et al., 2010). Wouters et al. (2013) reported that L. sakei was accurately identified as Leuc. lactis (Yu et al., 2012). It has been always present in higher percentages in the fermentation of green reported that Leuconostoc species with heterofermentative leek parts than white leek parts. Lactobacillus species having properties are predominant during the early kimchi fermentation facultative or homolactic fermentative properties were predominant period because they are less acid-tolerant (Kim et al., 2005; Jung et during late kimchi fermentation when the pH of the fermentation al., 2014). Cho et al. (2006) found that Leuconostoc species mixture was low (Jung et al., 2014). predominated during the first half of the fermentation stage in There were many reports concerning the LAB in Chinese Lactic Acid Bacteria from Xiaoshan Pickle Radish 135 pickles, but little documented studies concerned the semi-dried Chen, Y.S., Wu, H.C., Liu, C.H., Chen, H.C., and Yanagida, F. (2010). fermented vegetables. The semi-dried fermentation vegetables Isolation and characterization of lactic acid bacteria from jiang-sun were usually dehydrated with wind and then were pickled with the (fermented bamboo shoots), a traditional fermented food in Taiwan. J. salt spreading on the surface instead of soaking in the brine. Sci .Food Agric., 90, 1977-1982. Xiaoshan pickle radish is a kind of semi-dried fermented Chen ,Y.S., Yanagida, F., and Hsu, J.S. (2006). Isolation and vegetables. In our study two LAB species, Leuc. lactis and L. sakei, characterization of lactic acid bacteria from suan-tsai (fermented were found and characterized, which were similar to those isolated mustard), a traditional fermented food in Taiwan. J. Appl. Microbiol., from other pickles. But the species of LAB seems fewer than other 101, 125-130. traditional pickles. For example, 970 bacterial isolates of 15 species Cho, J., Lee, D., Yang, C., Jeon, J., Kim, J., and Han, H. (2006). Microbial were identified from kimchi (Cho et al., 2006). 119 representative population dynamics of kimchi, a fermented cabbage product. FEMS strains belonging to 5 genera and 18 species were isolated from Microbiol. Lett., 257, 262-267. suan-tsai and fu-tsai (fermented leaf mustard) (Chao et al., 2009). Gao, Y. R., Jia, S. R., Gao, Q., and Tan, Z. L. (2010). 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