Effect of Lactic Acid Fermentation on Antioxidant Properties
Food Sci. Biotechnol. 19(3): 641-646(2010)
DOI 10.1007/s10068-010-0090-2
Effect of Lactobacillus plantarum as a Starter on the Food Quality and Microbiota of Kimchi
Kieun Lee and Y eonhee Lee
Received: 12 October 2009 / Revised: 4 February 2010 / Accepted: 10 February 2010 / Published Online: 30 June 2010
? KoSFoST and Springer 2010
Abstract Lactobacillus plantarum has been reported to be responsible for acid increase at the later stage of kimchi fermentation and considered not inappropriate as a starter of kimchi. If functional L. plantarum strain can survive in large quantity in kimchi during fermentation, it may endow new functionality to kimchi. After 12 day fermentation at 4o C, L. plantarum, Leuconostoc mesenteroides, Weissella cibaria, Weissella confusa, and Lactobacillus sakei were the most prevalent ones in kimchi without a starter. In kimchi with a starter, L. plantarum was detected from the 1 day fermentation and throughout 25 day fermentation. There was no difference in pH between 2 kinds of kimchi. The acid content after the 21 day increased more in kimchi with starter when most kimchi is consumed before this time. Survival of a starter strain throughout the whole fermentation suggested the possible use of various functional lactic acid-producing bacterias (LABs) to kimchi endowing a new beneficial function to kimchi.
Keywords:lactic acid-producing bacteria, kimchi, Lactobacillus plantarum, fermentation, starter
Introduction
Kimchi is a name for various Korean traditional fermented vegetable mainly Chinese cabbages and raddish. After Chinese cabbages were soaked in salt water, they were mixed with red pepper, green onion, garlic, and fermented fish. During fermentation, various organic acids including lactic acid were produced and lowered the pH producing the good taste which is various on each region and season. Microbiota in kimchi depends on the ingredients (1), fermentation temperature (2), salt concentration (3), and the composition of microbiota affects the kinds of acid produced and the taste of kimchi. People have believed that a starter, except Leuconostoc which appeared at the first stage of fermentation of kimchi and the most important one for kimchi, could not survive during kimchi fermentation while decreases the taste and quality of kimchi. Leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus brevis, Pediocococcus cerevisiae, Lactobacillus sakei have been reported to involve in kimchi fermentation (4). At the first stage of fermentation, Leuconostoc spp. is the main bacteria, followed by Pediococcus spp.Then at the later stage of fermentation, the number of Lactobacillus spp. increased (5). Leuconostoc spp. is mainly used for a starter for kimchi while L. plantarum has been reported to be responsible for acid increase at the later stage of kimchi fermentation and considered not inappropriate as a starter of kimchi. However, functional L. plantarum strain can survive in large quantity in kimchi during fermentation without change the food quality of kimchi, it may endow new functionality to kimchi. To test the survival ability of L. plantarum and its effect on kimchi, succession of microbiota in kimchi with and without a starter-L. plantarum was analyzed with various methods. Kimchi microbiota has been mainly studied with isolation of bacteria on selective medium (6), mainly deMan-Rogosa Sharpe (MRS) agar (7), identification with Gram-staining, and catalase test and then API 50CH or CHL. Additional methods often used are microbial identification system (8,9), multiplex polymerase chain reaction (PCR) (10), and sequencing of 16S ribosomal DNA (11). Recently, various faster and easier molecular methods have been developed and used. These are single strand conformation polymorphism (SSCP) (12), temporal
Kieun Lee, Yeonhee Lee (
)
Culture Collection of Antimicrobial Resistant Microbes, Department of Biology, Seoul Women’s University, Seoul 139-774, Korea
Tel:+82-2-970-5664 ; Fax:+82-2-970-5829
E-mail:yhlee@swu.ac.kr
RESEARCH ARTICLE
642K. Lee and Y. Lee
temperature gradient gel electrophoresis (TTGE) (13), and denaturing gradient gel electrophoresis (DGGE) (14). Among these, DGGE can identify various fastidious or non-culturable microorganisms based on the principle that mobility in denaturant gradient is dependent on temperature. After it was first developed, DGGE has been successfully applied to analyze terrestrial sample (15), deep sea water (16), or hot spring (17). DGGE has been frequently used to analyze non-fastidious microorganisms in a short period time (18). And specific bacterial primers instead of universal primers have been successfully used to analyze a specific group of microorganisms. For example, group specific primers have been successfully used to analyze lactic acid-producing bacteria (LABs) in dairy products (14).
In this study, L. plantarum PL62 was added before fermentation and the succession of microbiota during kimchi fermentation was analyzed with isolation methods on media and DGGE to observe whether L. plantarum PL62 survive during kimchi fermentation. L. plantarum PL62 produces conjugated linoleic acids with anti-cancer and anti-obesity acitivities (19). If L. plantarum PL62 could survive, LABs with a new beneficial activity can be used as a starter for a neutraceutical kimchi.
Materials and Methods
Preparation of kimchi sample Chinese cabbages kimchi was bought from the market (poggikimchi, Chongga, Seoul, Korea) and L. plantarum PL62 (19) was added to kimchi to a final concentration of 108/g. During the whole fermentation period for 4 weeks, both kimchi with and without a starter were tightly sealed and kept at 4o C. After a portion (50g) of kimchi was taken at various times, kimchi was broken in a stomacher (Model 400 Stomacher; Seward, London, England) with 3 times of 10min run and 10min rest. The supernatant (20mL) was serially diluted in saline to assay the colony forming unit (CFU) and kept in 40% glycerol stock at ?70o C for the further study.
Assay of CFU and identification of LABs in kimchi Serially diluted kimchi supernatant were inoculated on MRS containing 0.002% bromophenol blue to differentiate each LAB (20). After 48hr incubation at 37o C, the number of colonies appeared on the media were counted and the morphology of each colony was observed under a microscope. Each colony was isolated and identified with 16S rRNA sequencing by Sanger’s dideoxynucleotide chain termination method using universal primers as described by others (21) in an ABI prism 310 Genetic Analyzer (PE Applied Biosystems, Foster City, GA, USA). Sequences were compared with the data in GenBank (http:/ /https://www.360docs.net/doc/7917793594.html,/).Amplified rRNA gene restriction analysis Amplified rRNA gene restriction analysis (ARDRA) was performed to differentiate Weissella confusa from Weissella cibaria which was used to be named as Weissella kimchii (22). PCR was performed with W0121(5'-CGTGGGAAACCT ACCTCTTA-3') and W0823(5'-CCCTCAAACA TCTAG CAC-3') as designed by Jang et al. (23) and the reaction was constituted with an initial denaturation at 94o C for 5min, 30 cycles of 30sec at 94o C, 30 sec at 61o C, and 1min at 72o C, and a final extension at 72o C for 7min. PCR products were digested with Mnl (MBI Fermentas; Hanover, MD, USA) and DNA fragments were separated on a 2% agarose gel.
Assay of pH and acid content pH of kimchi was measured with a pH meter (Suntex, Taipei, Taiwan). The amount of 0.1 N NaOH to make pH of kimchi to 8.4 was measured and then it was calculated into the amount of lactic acid.
PCR-DGGE An aliquot of kimchi sample (200μL) stored at ?70o C in 40% glycerol was centrifuged at 387×g in a microcentrifuge. The supernatant was transferred to a new tube and centrifuged at 22,250×g for 20min and washed 3 times with sterile water. The bacterial pellet was dispersed in 30μL sterile water and heated at 100o C for 10 min to prepare DNA template for PCR. PCR was performed with Lac1 (5'-AGCAGTAGGGAA TCTTCCA-3') and GCLac2 (5'-CGCCCGGGGCGCGCCCCGGGCG GCCCGGGGGCACCGGGGGA TTYCACCGCTACACA TG-3') as designed by Walter et al. (24) and the reaction was constituted with an initial denaturation at 94o C for 2 min, 40 cycles of 30 sec at 94o C, 1 min at 61o C, and 1 min at 68o C, and a final extension at 68o C for 10min. DGGE was performed using a 10% acrylamide in a Dcode system and run at 50V for 16 hr at 60o C following the guideline by the manufacturer. Denaturant concentration of a gel was formed from 30 to 60% of formamide and 7.0M urea. Markers for DGGE were prepared by PCR of each type strain-L. plantarum A TCC 14917, L. sakei A TCC 15521, W. confusa A TCC 10881, Weissella koreensis KCCM 41516, W. cibaria A TCC 3746, Leuc. mesenteroides A TCC 8293. After electrophoresis, bands were visualized with ethidium bromide.
Sequencing of DNA bands from DGGE Each band was cut from the gel, macerated with a razor, and put in sterile water. DNA was extracted from the supernatant with ethanol to remove urea and used for PCR with Lac1-GCLac2. After fragments with 400 bp were confirmed on a gel, each DNA fragment was extracted with a Qiagen PCR purification kit (Qiagen, Hilden, Germany) and used for the sequencing PCR with Sanger’s method (25). DNA
Kimchi Starter with a New Function643 was dissolved in template suppression reagent (TSR) and
analyzed in ABI prism 310 Genetic Analyzer (PE Applied
Biosystems). The resulting sequences were compared with
the data in GenBank (https://www.360docs.net/doc/7917793594.html,/).
Results and Discussion
CFU change during fermentation When CFUs were
assayed at various times by counting the number of
colonies appeared on MRS agar containing bromophenol
blue, CFU in kimchi without a starter increased from
4.0×106 CFU/mL after 1 day to 6.0×107 CFU/mL after 5
day fermentation. In the case of kimchi with a starter, CFU was 1.8×108 CFU/ mL at the 12 day fermentation. At 12-16 day fermentation, CFU of LAB in kimchi without a starter was little larger than CFU of LABs in kimchi with a starter. CFUs in kimchi with and without a starter become similar after 25 day fermentation (Fig. 1). Detection of various LABs at various times of fermentation Each colony selected on MRS agar containing bromophenol blue was identified by 16SrRNA sequencing (Table 1). After 5 day fermentation, kimchi without a starter produced dark blue colonies of W. confusa. As fermentation continues, dark blue, big, and flat colony of W. cibaria and dark blue, little, and convex colony of W. confusa appeared as shown in Table 2. After 12 day fermentation, kimchi without a starter produced colonies of L. plantarum. Kimchi with a starter continuously produced colonies with typical morphology (large, white, a blue center, convex) of a starter strain
-L. plantarum PL62 from 1 day fermentation until the end of fermentation. These colonies were identified as L. plantarum with 16S rRNA sequencing.Colonies with characteristics of L. plantarum appeared from the 1 day of fermentation and appeared continuously during fermentation only in kimchi with a starter but not from kimchi without a starter (Table 3).
After 12 day fermentation, kimchi with a starter produced various colonies including light blue, small, and flat colonies of Leuc. mesenteroides,white with blue center and convex colonies of L. plantarum, dark blue with dark blue center and flat colonies of
L. sakei appeared even after
30 day fermentation. In the case of W. cibaria and W.
confusa which produce similar looking colonies, they were
differentiated from each other with ARDRA (Fig. 2). In
this study, Weissella spp., which has been rarely reported in
Fig. 1. CFU of lactic acid-producing bacteria during kimchi
fermentation at 4o C. ●, Kimchi without a starter; ○, Kimchi
with a starter
Table 1. Colony morphology of each species on MRS containing bromophenol blue
Colony Morphology Identification Genbank No.% Identity
Dark blue, big, and flat W. cibaria AJ42203199
Light blue, small, and flat Leuc. mesenteroides AY67524999
Dark blue and little convex W. confusa AY34156998
White with blue center and convex L. plantarum AY735404100
Dark blue with darker center and flat L. sakei AY204898100
644
K. Lee and Y. Lee
kimchi, appeared at the first stage of fermentation and these were W. confusa/ W. cibaria (formerly called as W.kimchii ). These 2 species cannot be differentiated from each other with DGGE but only with ARDRA.pH change during fermentation Both pHs in kimchi with and without a starter were pH 6.0 at the 1 day fermentation and then pH became pH 4.15 (without a starter) and pH 4.12 (with a starter) which is close to pH 4.2 which gives the best taste at 12 day fermentation.After 25 day fermentation, pHs in both kimchi decreased to 4.0.
Change in acidity Acidity in kimchi without a starter increased to 0.5-0.6% (the best acidity for taste) after 21-28 day and after then acidity decreased. Acidity in kimchi with a starter increased to 1.0% after 24 day fermentation and then decreased. Acidity in kimchi with a starter is higher than the kimchi without a starter. This may be due to the L. plantarum which is known to produce a large amount of lactic acid at the later stage of kimchi fermentation (5).
PCR with group specific primers-DGGE of kimchi When LABs were analyzed with DGGE (Fig. 3), no bands appeared from kimchi without a starter until 4 day fermentation. A band for W. confusa/W. cibaria had appeared from 5 day fermentation and the size of this band
decreased as fermentation continued. From 8 day until 30day fermentation, a band for Leuc. mesenteroides appeared. In the case of L. sakei/W. koreensis and L.plantarum , they both appeared from 12 day until 30 day fermentation. In the case of kimchi with a starter, a band with the same size of L. plantarum A TCC 14917 appeared from the 1 day fermentation. The band size decreased as fermentation continued and it was identified as L.plantarum with sequencing. Other bands selected on DGGE gel were identified by 16S rRNA sequencing (Table 4). LABs detected in DGGE are shown in Table 5.
Table 2. Colonies appeared on MRS containing bromophenol blue
1 day
5 day
8 day
12 day
16 day
25 day
30 day
Kimchi
Kimchi+PL 62
Table 3. LABs in kimchi detected on MRS containing bromophenol blue
1 day
5 day
8 day 12 day 16 day 25 day 30 day Kimchi
L. plantarum L. plantarum L. plantarum
L. plantarum
L. sakei
L. sakei W. confusa
W. confusa W. confusa,W. cibaria
W. confusa,W. cibaria
W. confusa,W. cibaria
Leu. mesenteroides
Leu. mesenteroidesLeuconostoc spp.
Kimchi +PL 62
L. plantarum L. plantarum L. plantarum
L. plantarum L. plantarum L. plantarum L. sakei L. sakei L. sakei L. sakei W. confusa
W. confusa
W. confusa,W. cibaria W. confusa,
W. cibaria
Leu. mesenteroidesLeu. mesenteroidesLeu. mesenteroidesLeu. mesenteroidesLeu. mesenteroides
Fig. 2. Differentiation of W. cibaria from W. confusa with amplified 16S ribosomal DNA restriction analysis. Lane 1, 1 kb marker; Lane 2, W. confusa A TCC 10881; Lane 3, W. cibaria KCCM 3746; Lane 4, Weissella from 16 day fermentation; Lane 5,Weissella from 12 day fermentation; Lane 6, 1 kb marker
Kimchi Starter with a New Function 645
The composition of microbiota affects the kinds of acid
produced and the taste of kimchi. Until today, Leuc.mesenteroides , L. plantarum , L. brevis , P . cerevisiae , and L.sakei have been reported to involve in kimchi fermentation.At the first stage, Leuconostoc spp. is the major bacteria,followed by Pediococcus spp. Then at the later stage of fermentation, Lactobacillus spp. increased (5). Major LABs in kimchi fermented at 10, 20, or 30o C were W.confusa , Leuc. citreum , L. sakei , and L. curvatus (26) while Leuc. mesenteroides, L. plantarum, W. confusa, W. kimchii,and L. sakei were major LABs in kimchi fermented at 4o C in this study. We presumed that the composition of microflora in kimchi show changes due to the fermentation temperature in addition to season and the major ingredients (1).
In general, L. plantarum which appeared at the later stage of kimchi fermentation and is considered to be responsible for increase in acidity. This is why L.plantarum is not a good candidate for a kimchi starter.People have believed that a starter cannot survive during the whole fermentation process. In contrast to this belief,this study showed that the starter added to kimchi survived during the whole kimchi fermentation.
In this study, L. plantarum, W. confusa , W. cibaria, L.sakei, and Leu. mesenteroides were major LABs in kimchi while L. brevis and P . cerevisiae were not detected either by the culture method or DGGE. Our results from DGGE
coincided with the results from previous reports that Leu.mesenteroides appeared at the initial stage and L.plantarum appeared at the later stage (5). The result, that Leu. mesenteroides was not detected with DGGE even though it was detected on MRS, must be due to the presence of the large amounts of L. plantarum at the start compared to that of Leu. mesenteroides . The differences in kinds of LABs in kimchi in our results and others’ might be due to the fermentation temperature.
The survival of L. plantarum PL62 during fermentation suggested that a functional probiotic can be added to various fermented foods making functional foods-such as kimchi, pickle, sausages, etc.
Acknowledgments This work was supported by a research grant from Seoul Women’s University (2009).
Fig. 3. DGGE profile of kimchi. a, L. plantarum A TCC 14917; b, L. sakei A TCC 15521; c, W. confusa A TCC 10881; d, W. koreensis KCCM 41516; e, W. cibaria A TCC 3746; f, Leuc. mesenteroides A TCC 8293; g, Unknown band
Table 4. Identification of bands obtained by DGGE analysis Band Closest relatives Accession No.% Sequence similarity
a'L. plantarum
EF439680.197b'Uncultured Weissella spp .AY421869.199f'Uncultured Leuconostoc spp .AY42192999c'Uncultured bacterium GQ468078.199g'Uncultured bacterium DQ818931.197h'
L. pentosus
EU483102.1
100
Table 5. LABs in kimchi detected with DGGE
1 day
5 day
8 day
12 day 16 day 25 day 30 day Kimchi
L. plantarum L. plantarum L. plantarum L. plantarum W. koreensis/L. sakei
W. koreensis/L. sakei
W. koreensis/L. sakei
W. koreensis/L. sakei
W. confusa/W. cibaria
W. confusa/W. cibaria W. confusa/W. cibaria
Leuconostoc spp.Leuconostoc spp.
Leuconostoc spp.Leuconostoc spp.Leuconostoc spp.Kimchi+PL 62
L. plantarum L. plantarum L. plantarum
L. plantarum L. plantarum L. plantarum W. koreensis/L. sakei
W. koreensis/L. sakei
W. koreensis/L. sakei
W. koreensis/L. sakei
W. confusa/W. cibaria
W. confusa/W. cibaria
646K. Lee and Y. Lee
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