Note Isolation and Identification of Lactic Acid Bacteria from Xiaoshan
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_ Food Science and Technology Research, 23 (1), 129 136, 2017 Copyright © 2017, Japanese Society for Food Science and Technology doi: 10.3136/fstr.23.129 http://www.jsfst.or.jp Note 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.