Notes
Isolation and Identification of Lactic Acid Bacteria from Xiaoshan Pickle Radish, a Traditional Fermented Vegetable
2017 年 23 巻 1 号 p. 129-136
詳細
2017 年 23 巻 1 号 p. 129-136
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).
Xiaoshan pickle radish (Raphanus sativus L.) or “Xiaoshan luobogan” is one of the traditional fermented vegetables and a famous local specialty of Zhejiang province in China (Fig. 1). The product has been widely consumed with its special flavor. Xiaoshan pickle radish is made from radish cultivar of “Yidaozhong”. After washing with clean water, the roots are cut into uniform strips, each with peel. Then they are wilted with sunlight for 3 to 5 days. Salt particle is added to about 3% by quality and well kneaded, then the mixture is pressed tightly layer by layer into vat. It is left for frist stage curing at room temperature for 3 days and then sun dried for 2 to 3 days.To form the final product, the second stage curing is carried out with addition of 1.5% salt. The product will be ready-to-eat after 7 days fermentation and 2 to 3 days drying.
Picture of Xiaoshan pickle radish.
The traditional products of Xiaoshan pickle radish was based on spontaneous fermentation initiated by the natural microflora present in the raw materials. The quality is unstable as the process depends on season, climate, raw material properties, manual operation and so on. The current fermentation is still a traditional household technology without addition of any commercial starter cultures. As a result, it is difficult to ensure an adequate level of hygiene and quality uniformity. It is thus important to screen the dominate microorganism of Xiaoshan pickle radish with beneficial function and desirable properties to be used as starter cultures, which will improve the quality and safety of the final product and standardizes the production process.
Lactic acid bacteria (LAB) play an important role in vegetable fermentation, which cause rapid acidification of the raw material. They contribute to the taste, flavor and texture of fermented products and inhibit food spoilage bacteria and pathogenic bacteria by producing organic acids, mainly lactic acid, bacteriocins, ethanol, aroma compounds, exopolysaccharides, several enzymes and other microbial growth-inhibiting substances (Leroy et al., 2004; Chen et al., 2010). Various LAB have been identified from different Chinese traditional fermented foods (Liu et al., 2011). Pickles can be used as good screening sources for the isolation of valuable microorganisms (Hiraga et al., 2008). Chen et al. (2006) indicated that Pediococcus pentosaceus and Tetragenococcus halophilus are responsible for the fermentation of suan-tsai (fermented leaf mustard). Yu et al. (2012) analyzed 36 pickle samples from 6 different regions in Sichuan province and identified as Enterococcus thailandicus, Lactobacillus alimentarius, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus sakei, Lactobacillus spicheri, Leuconostoc lactis and Pediococcus ethanolidurans. However, very limited document concerning the LAB of Xiaoshan pickle radish have been reported until now. Zou et al. (2007) reported that L1 isolated from Xiaoshan pickle radish slices was identified as Lactobacillus sp. by phenotypic methods, but did not discriminate isolates at the species level.
The objective of this paper was to isolate and identify the predominant lactic acid bacteria present during Xiaoshan pickle radish fermentation. This information can be taken as the basis of the development of starter cultures and may pave the way for large-scale commercial production.
Sample collection Three batches of “Yidaozhong” radishes were purchased from Hangzhou Gudang agricultural products market (GD), Xiaoshan Desheng agricultural products market (DS) and Hangzhou agricultural and sideline products logistics center (AS). Spontaneous fermentation occurred at average temperature of 25°C by traditional technology (Fig. 2). The pH values of the pickle juice were determined using a calibrated portable pH-meter (pHS-25, shengci, Shanghai, China).
Flowchart depicting for fermentation of Xiaoshan pickle radish.
Samples were taken at the end of each stage of production: stage “A” (product before washing with clean water); stage “B” (product after washing with clean water); stage “C” (product before first curing); stage “D” (product after first curing); stage “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°C 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 isolated from plates. The selected colonies were purified by replating on MRS agar plates from a single colony at random for further identification. This procedure was repeated several times. Only gram-positive, catalase-negative strains were selected. Purified strains of LAB were preserved in MRS broth using 15% (v/v) glycerol at −20°C.
Identification of lactic acid bacteria
Biochemical and physiological characteristics Further identification of gram-positive and catalase-negative isolates was performed by using the following physiological tests: growth at temperatures of 10, 15 and 45°C in MRS broth for 5 days, growth at pH value of 4.5 and 9.0 in MRS broth for 3 days, growth at NaCl concentrations of 2.0%, 4.0% and 6.5% (w/v) in MRS broth for 3 days (Kozaki et al., 1992). NH3 production from arginine was determined at 35°C for 24 h in MRS broth with 0.3% L-arginine (Harrigan et al., 1966). Gas production (CO2) from glucose was investigated by growing the bacteria in MRS broth that contained inverted Durham tubes (Tohno et al., 2013). The type of D and L isomers of lactic acid produced from glucose was assayed in modified MRS broth using a commercial kit (Hoffman La Roche Diagnostic, Mannheim, Germany) (Mathara et al., 2004). Sugar fermentation patterns were evaluated according to the methods described by Kozaki et al (1992). The results were checked according to the information supplied in Bergey's Manual of Systematic Bacteriology (1984).
16S rDNA sequencing and molecular identification Total genomic DNA was extracted from 5 mL samples of overnight cultures grown in MRS broth at 37°C by Genomic DNA Prep Kit (Tiangen, Beijing, China). PCR amplified using the universal primer pairs 27F:5′-AGAGTTTGATCCTGGCTCAG-3′ and 1541R:5′-AAGGAGGTGATCCAGCC-3′. All PCR reactions were carried out in 50 µL reaction volumes containing: 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 50 ng of bacterial DNA, 60 pM of each primer, 0.2 mM each dNTPs, and 2.5 U of Taq polymerase (Solarbio, Beijing, China). PCR amplifying procedure was as follows: 5 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 58°C, 2 min at 72°C and then 10 min at 72°C for final extension. The PCR products were subjected to gel electrophoresis in 1% agarose gel, followed by staining with ethidium bromide and visualization under UV light. Amplified 16S rRNA was isolated from the agarose gel using a Gel Extraction Kit (Tiangen, Beijing, China). The sequencing of purified products was performed by Shanghai Sangon Biosciences Corporation of China.
The nucleotide sequences of the 16S rRNA gene of all the isolates were analyzed and determined by the BLAST program on the NCBI website (i). The alignments were analyzed to construct a phylogenetic tree and to compare similarities among the sequences by the neighbor-joining method using MEGA software version 6.0.
Nucleotide sequence accession numbers
The represent 16S rDNA sequences were deposited in the GenBank at the NCBI with the accession ID KP901103 to KP901104.
Statistical analysis Each experiment was repeated three times. The numbers of colonies were converted to Log cfu/g. Analysis of variance (ANOVA) was performed using SAS software (version 9.1, SAS Institute, N.C., USA) by Duncan's multiple range test. A significant difference was defined as p < 0.05.
pH of samples and enumeration of acid-producing bacteria
Table 1 shows the counts of acid-producing bacteria and pH value in samples during seven different fermentation stage from three markets in Hangzhou. The result indicated that there had no distinct association between production sites with pH value and bacteria quantity. The pH value of three market showed the same change, with an average drop from 6.8 ± 0.1 to 4.6 ± 0.2. There were no significant differences between the pH value and acid-producing bacteria counts of the same fermentation stage in three market.
| Items | Market | Fermentation stage | ||||||
|---|---|---|---|---|---|---|---|---|
| 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.
The acid-producing bacteria counts were low in all the batches during stages “A” “B” and “C” for the fermentation of pickle radish had not started. In the next stage a rise in bacteria counts was observed after first curing. The viable counts varied from 2.47 ± 0.32 to 5.41 ± 0.28 Log (cfu/g), 2.55 ± 0.06 to 5.54 ± 0.06 Log (cfu/g) and 2.51 ± 0.08 to 5.59 ± 0.32 Log (cfu/g) respectively. In stages “E”, the bacteria counts were significantly increased. The acid-producing bacteria counts reached the peak after the second curing. In the later stage “G”, the population decreased to a equivalent level with stage “E”.
The average pH values in three market of samples at stage “C” were found to be higher than other samples. In the next stage “D”, the measured values significantly decreased (p < 0.05), with the same trend in stage “E”. Samples of stage “F” and “G” had a longest fermentation time and the lowest pH values.
During the whole fermentation process, the increase of bacteria counts linked to the decrease in pH values. The decrease in pH could be due to the gradual increase in fermenting microorganism population and accumulation of the organic acid consequently. The results are in agreement with the conclusion of many studies (Nche et al., 1994; Muyanja et al., 2003; Owusu-Kwarteng et al., 2012).
Identification of lactic acid bacteria
Identification of LAB isolates by physiological characteristics
A total 387 of gram-positive, catalase-negative isolates were obtained from 21 batches of samples. They were divided into two groups based on several morphological and physiological characters (Table 2).
| Characteristics | Group | |
|---|---|---|
| I (252 isolates) | II (135 isolates) | |
| Cell shape | rod | coccus |
| Gas from glucose | - | + |
| Lactic acid isomera | DL | D |
| NH3 from arginine | - | - |
| Growth at temperature (°C) | ||
| 10 | 0b | 0 |
| 15 | 252 | 135 |
| 45 | 252 | 135 |
| Growth at pH value | ||
| 4.5 | 252 | 135 |
| 9.0 | 252 | 135 |
| Growth in NaCl (w/v) | ||
| 2.0% | 252 | 135 |
| 4.0% | 252 | 135 |
| 6.5% | 252 | 0 |
| Acid from | ||
| D-Arabinose | 252 | 0 |
| D-Cellobiose | 252 | 135 |
| Esculin | 252 | 135 |
| D-Galactose | 252 | 135 |
| Gluconate | 252 | 0 |
| D-Mannitol | 0 | 0 |
| D-Mannose | 252 | 135 |
| D-Melezitose | 0 | 0 |
| Salicin | 252 | 135 |
| D-Lactose | 252 | 135 |
| D-Raffinose | 0 | 135 |
| L-Rhamnose | 0 | 0 |
| Ribose | 252 | 135 |
| D-Sorbitol | 252 | 0 |
| L-Sorbose | 0 | 0 |
| D-Sucrose | 252 | 135 |
| D-Trehalose | 252 | 0 |
| D-Xylose | 0 | 135 |
Group I was composed of 252 isolates. These isolates exhibited growth at 15 and 45°C, but not 10°C. They produced DL-lactic acid but no NH3 from arginine and no gas from glucose. They could ferment acid from D-arabinose, D-cellobiose, esculin, D-galactose, D-mannose, gluconate, salicin, D-lactose, ribose, D-sorbitol, D-sucrose and D-trehalose. Those strains were identified as Lactobacillus sakei due to their characteristics.
Group II was composed of 135 coccus-shaped isolates, which produced D-lactic acid. These isolates exhibited growth at 15 and 45°C, but not growth at 10°C and in the presence of 6.5% NaCl. They could produce gas from glucose but no NH3 from arginine. They fermented D-cellobiose, esculin, D-galactose, D-mannose, salicin, D-lactose D-raffinose, ribose D-sucrose and D-xylose. These isolates were assigned to genus Leuconostoc lactis.
Lactobacillus sakei was isolated from all fermentation stage except stage “A”, while Leuc. lactis was the dominant LAB isolated during stage “A” and stage “B”. The isolates of L. sakei increased from stage “C”, be equal with Leuc. lactis. From stage “D”, L. sakei was more prevalent than Leuc. lactis. In stage “G”, Leuc. lactis was no longer detected. From stage “A” to “G”, the percentage of L. sakei were 0.0%, 16.7%, 50.0%, 81.8%, 80.0%, 83.3% and 100.0%.
Regional similarities and differences in diversity were observed in the current study. Owusu-Kwarteng et al. (2012) reported Lactobacillus fermentum and Weisella confusa were isolated in all production sites and almost at all fermentation stages during traditional fura (fermented millet) processing in Ghana, the other LAB bacteria species which comprised a minor proportion of the total LAB occurred occasionally and in an irregular pattern among the production sites. Tsuda et al. (2012) collected nine funazushi (fermented crucian carp and rice) samples from different sites of Japan and found the LAB microflora of five homemade funazushi showed a number of differences with each other. In this study, the LAB counts of the radishes collected from different market did not show significant difference and it seemed that the LAB belonged to the same species. This may be territorial restriction because the “Yidaozhong” radishes were representative local variety of Xiaoshan in Hangzhou, the chance of products obtaining from the same source was high.
16S rRNA gene sequence identification and phylogenetic analysis To confirm the species, the nucleotide sequences of the 16S rRNA gene of all the tested strains were analyzed and determined. The 16S rDNA sequences of L1 from group I and L2 from group II were submitted to NCBI. The sequences were then deposited to Genbank and assigned accession No KP901103 to KP901104.
Some type strains were chosen to infer a possible phylogenetic classification. Phylogenetic tree analysis in figure 3 and figure 4 was performed to show the relationship of 16S rRNA gene sequences between the isolates and related type strains by using MEGA software. Isolate L1 formed a distinct phyletic line that is associated with the L. sakei subsp. sakei in the neighbor-joining analysis and supported by a bootstrap value of 71%. Its closest neighbor was the type strain of Lactobacillus sakei subsp. sakei JCM1157T, with 100% homology in their 16S rRNA gene sequences. L2 was most closely related to Leuconostoc lactis KCTC3528T, supporting the 100% value from bootstrap analysis of the phylogenetic tree, and showing 99% similarity in their 16S rRNA gene sequences.
Phylogenetic tree of L1with the 16S rRNA gene by neighbor-joining (NJ) method. Bootstrap values based on 1000 replications are given at the nodes.
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.
Leuconostoc lactis, as heterofermentative LAB, produce lactic acid, ethanol, and carbon dioxide from glucose through the 6-phosphogluconate/phosphoketolase pathway under anaerobic conditions (Jung et al., 2014). Leuconostoc lactis had been found in fermented Chinese foods of stinky tofu (soybean curd), sour mifen (rice noodle), sour milk (Liu et al., 2011). Similar to our results, 1 strain isolated from traditional pickles in Sichuan were accurately identified as Leuc. lactis (Yu et al., 2012). It has been reported that Leuconostoc species with heterofermentative properties are predominant during the early kimchi fermentation period because they are less acid-tolerant (Kim et al., 2005; Jung et al., 2014). Cho et al. (2006) found that Leuconostoc species predominated during the first half of the fermentation stage in kimchi . After day 30, the numbers of Leuc. citreum and Leuc. lactis cells decreased rapidly. The similar observation was made by Lee et al. (2015).
Lactobacillus sakei is specifically adapted to the meat matrix (Chaillou et al., 2009), but in recent years this species commonly occurs in fermented vegetables. The strain C2 isolated from traditional Chinese fermented cabbage was identified as L. sakei (Gao et al., 2010). Wouters et al. (2013) reported that L. sakei was always present in higher percentages in the fermentation of green leek parts than white leek parts. Lactobacillus species having facultative or homolactic fermentative properties were predominant during late kimchi fermentation when the pH of the fermentation mixture was low (Jung et al., 2014).
There were many reports concerning the LAB in Chinese pickles, but little documented studies concerned the semi-dried fermented vegetables. The semi-dried fermentation vegetables were usually dehydrated with wind and then were pickled with the salt spreading on the surface instead of soaking in the brine. Xiaoshan pickle radish is a kind of semi-dried fermented vegetables. In our study two LAB species, Leuc. lactis and L. sakei, were found and characterized, which were similar to those isolated from other pickles. But the species of LAB seems fewer than other traditional pickles. For example, 970 bacterial isolates of 15 species were identified from kimchi (Cho et al., 2006). 119 representative strains belonging to 5 genera and 18 species were isolated from suan-tsai and fu-tsai (fermented leaf mustard) (Chao et al., 2009). That may be because semi-dried fermented vegetables were pickled with higher salt content. The fermentation of semi-dried vegetables was weak than other pickles (Chen, 2011). The contribution of these species to the fermentation of Xiaoshan pickle radish remains to be investigated.
This is the first report describing the distribution and varieties of LAB that exist in Xiaoshan pickle radish. The results of this study would offer useful information contributing to the development of starter cultures with predictable characteristics.
In this study, the LAB in spontaneously fermented Xiaoshan pickle radish was studied by conventional methods combined with molecular biological methods. A shift in the LAB community and pH value was observed over time. The LAB counts increased whereas the pH decreased. The LAB counts of the radishes collected from different market did not show significant difference and it seemed that the LAB belonged to the same species. The two kinds of microorganism were identified as Leuc. lactis and L. sakei. Leuc. lactis was the dominated species in the initial stages of fermentation, but eventually gave way to the dominance of L. sakei.
