Influence of Isolation Temperature on Isolating Diverse Lactic Acid Bacteria from Kimchi and Cultural Characteristics of Psychrotrophs

Hye In Ko; Chang Hee Jeong; Se-Jin Park; So-Rim Kim; Jong-Bang Eun; Tae-Woon Kim

doi:10.4014/jmb.2303.03047

Articles Service

Metrics

CrossRef [3]
View[667]
Download[271]

Research article

Food Microbiology and Biotechnology (FMB) | Fermentation and Food Technology

Influence of Isolation Temperature on Isolating Diverse Lactic Acid Bacteria from Kimchi and Cultural Characteristics of Psychrotrophs

Hye In Ko^1,2†, Chang Hee Jeong^1,3†, Se-Jin Park², So-Rim Kim^1,2, Jong-Bang Eun², and Tae-Woon Kim^1*

¹Technology Innovation Research Division, World Institute of Kimchi, Gwangju 61755, Republic of Korea
²Department of Integrative Food, Bioscience and Biotechnology, Chonnam National University, Gwangju 61186, Republic of Korea
³Honam National Institute of Biological Resources, Mokpo 587262, Republic of Korea

Correspondence to:

Tae-Woon Kim, korkimchiman@wikim.re.kr

^†These authors contributed equally to this work.

Received: March 30, 2023; Revised: April 28, 2023; Accepted: May 2, 2023

J. Microbiol. Biotechnol. 2023; 33(8): 1066-1075

Published August 28, 2023 https://doi.org/10.4014/jmb.2303.03047

Abstract

Kimchi is a traditional Korean fermented vegetable that is stored and fermented at low temperatures. However, kimchi lactic acid bacteria (LAB) are typically isolated under mesophilic conditions, which may be inappropriate for isolating the diverse LAB. Therefore, this study investigated the suitable conditions for isolating various LAB from kimchi. Here, LAB were isolated from four kimchi samples using MRS, PES, and LBS media and varying isolation temperatures (30, 20, 10, and 5°C). Then, MRS was selected as the suitable medium for LAB isolation. A comparison of culture–dependent and culture–independent approaches indicated that 5°C was not a suitable isolation temperature. Thus, the number and diversity of LAB were determined at 30, 20, and 10°C using 12 additional kimchi samples to elucidate the effect of isolation temperature. With the exception of two samples, most samples did not substantially differ in LAB number. However, Leuconostoc gelidum, Leuconostoc gasicomitatum, Leuconostoc inhae, Dellaglioa algida, Companilactobacillus kimchiensis, Leuconostoc miyukkimchii, Leuconostoc holzapfelii, and Leuconostoc carnosum were isolated only at 10 and 20°C. The growth curves of these isolates, except Leu. holzapfelii and Leu. carnosum, showed poor growth at 30°C. This confirmed their psychrotrophic characteristics. In Weissella koreensis, which was isolated at all isolation temperatures, there was a difference in the fatty acid composition of membranes between strains that could grow well at 30°C and those that could not. These findings can contribute to the isolation of more diverse psychrotrophic strains that were not well isolated under mesophilic temperatures.

Keywords

Kimchi, lactic acid bacteria, psychrotrophs, isolation temperature

Graphical Abstract

Introduction

Kimchi is a traditional Korean food made by fermenting various vegetables. It is produced via fermentation of salted vegetables with various spices and ingredients, including garlic, red pepper powder, ginger, green onion, and salt-fermented seafood. Although there are various microorganisms derived from several raw materials, lactic acid bacteria (LAB) dominate kimchi during the fermentation process [1]. The most common LAB genera in kimchi are Leuconostoc, Lactobacillus, Weissella, Lactococcus, and Pediococcus [1, 2]. In general, the dominant species are Leuconostoc mesenteroides, Leu. citreum, Leu. gasicomitatum, Leu. carnosum, Leu. gelidum, Lactilactobacillus sakei, W. koreensis, and Weissella cibaria [1, 3]. The microbial community in kimchi varies depending on the fermentation stage. Various microbial communities exist in the early and middle stages of fermentation, with Leu. mesenteroides as the predominant species [2]. Dominant species at the late stage of fermentation include Lactiplantibacillus plantarum, L. sakei, and W. koreensis [1]. The microbial community affects the sensory properties of kimchi, which are attributable to the different metabolisms and metabolites of each strain [4]. These microbial community changes occur as the pH decreases because of organic acid produced by LAB during kimchi fermentation [5].

Temperature is also one of the critical factors affecting the microbial community in kimchi, owing to the differences in the optimal cultural temperature among bacterial strains [6‒9]. Hence, the fermentation temperature of kimchi influences its LAB flora, leading to a difference in the kimchi sensory characteristics [1, 10].

Kimchi is generally prepared at home during early winter, kept at room temperature for 1‒2 days, and stored at approximately 0°C to inhibit excessive fermentation. Alternatively, it is fermented at 4°C until consumption [11, 12]. Furthermore, commercially available kimchi is generally stored below 10°C for distribution [13]. When it is shipped to the consumer, the refrigerated truck temperature is maintained between 0‒5°C [13]. Then, consumers store and ferment the commercial kimchi at approximately 0°C before consumption [13]. These kimchi fermentation conditions serve as the bacteria cultural environment, making them an important part of the strain isolation process.

In a previous study, a LAB strain was isolated from yogurt at a medium temperature (37°C), which is the appropriate cultural temperature for these strains (32‒45°C) [14, 15]. Furthermore, thermophilic bacteria were cultured at a high temperature (50°C) [17]. In addition, a high salinity medium is used to isolate halophilic microbial species from high salt environments [16]. This is a fundamental prerequisite for isolating strains. Desirable conditions are required to successfully isolate microbes from kimchi, with consideration for its fermentation temperature.

Extensive isolation and identification of more LAB strains from kimchi could considerably contribute to our understanding of the role of each species in the kimchi fermentation process. Although low temperatures are suitable environments for some species of Leuconostoc belonging to psychrotrophic LAB [1, 3, 18], the current methods for isolating LAB from kimchi usually apply mesophilic isolation conditions (30°C) [19, 20]. Hence, it is necessary to investigate the suitable conditions to isolate more diverse LAB from kimchi.

In this study, LAB species that are isolated from kimchi based on the isolation temperature and culture medium were identified. In addition, the culture characteristics of strains that were isolated at low temperatures and exhibited poor growth under mesophilic conditions were confirmed.

Materials and Methods

Preparation and Physicochemical Properties of Kimchi Samples

Kimchi samples were obtained from homemade kimchi (HM) and seven local processing plants in Korea. Here, the name of the company where the kimchi was purchased is used as the sample name (Kimchi-town (KT), Han-sang-gung (HSG), Tae-seo (TS), Tae-baek (TB), Ye-so-dam (YSD), I-nam-jang (INJ), Ggot-soon-yi (GSY)). KT was fermented at 4°C for 29 days and sampling was done on Days 1 (KT 1), 15 (KT 2), and 30 (KT 3) to confirm suitable isolation conditions. Next, kimchi samples were fermented at 10°C (10HSG, 10TS, 10TB, 10YSD, 10INJ, and 10GSY) and 4°C (4HSG, 4TS, 4TB, 4YSD, 4INJ, and 4GSY) for 1 and 2 weeks, respectively. Each sample was subsequently ground and filtered through sterile gauze to obtain kimchi soup. The pH was measured using a pH meter (STARA1117, Thermo Fisher Scientific Inc., USA). The titratable acidity was calculated in terms of lactic acid using 0.1 N NaOH until the sample pH reached 8.3. Finally, soluble solid contents and salinity of the kimchi samples were measured using a refractometer (PAL-1, Atago Co., Japan) and a salt meter (PAL-ES2, Atago Co.).

Isolation and Identification of LAB from Kimchi

To investigate the appropriate isolation condition range of LAB from kimchi, the kimchi soup of HM, KT 1, KT 2, and KT 3 were serially diluted and spread on De Man Rogosa Sharp (MRS), phenylethyl alcohol sucrose (PES, peptone 5.0 g/l; yeast extract 0.5 g/l; sucrose 20.0 g/l; ammonium sulfate 2.0 g/l; magnesium sulfate haptahydrate 0.5 g/l; potassium dihydrogen phosphate 1.0 g/l; agar 15.0 g/l), and Lactobacillus selection (LBS, pancreatic digest of casein 10.0 g/l; yeast extract 5.0 g/l; dextrose 20.0 g/l; monopotassium phosphate 6.0 g/l; ammonium citrate 2.0 g/l; sodium acetate hydrate 25.0 g/l; magnesium sulfate 0.575 g/l; manganese sulfate 0.12 g/l; ferrous sulfate 0.034 g/l; Tween 80 1.0 g/l; agar 15.0 g/l) agar. The media were then incubated at 30, 20, 10, and 5°C for 24‒48, 48‒72, 144‒168, and 336–360 h, respectively. In addition, samples were spread on plate count agar (PCA, tryptone 5.0 g/l; yeast extract 2.5 g/l; dextrose 1.0 g/l; agar 15.0 g/l) and incubated at 35°C for 24–48 h. To confirm suitable isolation temperatures for isolating diverse LAB, 10INJ, 10GSY, 10YSD, 10HSG, 10TB, 10TS, 4INJ, 4GSY, 4YSD, 4HSG, 4TB, and 4TS were also serially diluted, spread on MRS, and incubated at 30, 20, and 10°C for 24‒48, 48‒72, and 144‒168 h, respectively. Subsequently, microbial counts were recorded (log CFU/ml). Next, approximately 30 colonies were chosen at random and subcultured in MRS agar. The isolates were identified by amplifying the 16S rRNA gene of bacteria using universal primers 27F (5¢-AGA GTT TGA TCM TGG CTC AG-3¢) and 1492R (5¢-TAC GGY TAC CTT GTT ACG ACT T-3¢).

Next generation Sequencing (NGS)

DNA was extracted according to the manufacturer’s instructions using a DNeasyPowerSoil Kit (Qiagen, Germany). The extracted DNA was quantified using a Quant-IT PicoGreen assay kit (Invitrogen, USA). The sequencing libraries were prepared according to the Illumina 16S metagenomic sequencing library protocols to amplify the V3 and V4 regions. The first PCR product was purified using AMPure beads (Agencourt Bioscience, USA). Following purification, 2 μl of the first PCR product was amplified for final library construction containing the index using the NexteraXT indexed primer (Illumina, USA). The PCR product was purified using AMPure beads. Then, the final purified product was quantified using qPCR according to the qPCR quantification protocol guide (KAPA library quantification kits for Illumina sequencing platforms) and qualified using the TapeStation D1000 ScreenTape system (Agilent Technologies, Germany). Finally, the paired-end (2 × 300 bp) sequencing was performed through Macrogen systems (Korea) using the MiSeq platform (Illumina).

Growth Curve of Kimchi LAB Isolated at Low Temperatures

To investigate the growth curve of LAB isolated at low isolation temperatures, including Leu. gelidum (from 4HSG, KCKM P0052), Leu. gasicomitatum (from 4TS, KCKM P0057), Leu. inhae (from 10HSG, KCKM P0061), D. algida (from 10YSD, KCKM P0083), C. kimchiensis (from 10YSD, KCKM P0044), and W. koreensis (from 4HSG, KCKM P0054), bacterial strains were incubated in MRS broth at 20°C for 24 h. Next, they were inoculated in MRS broth and incubated at 10, 15, 20, and 25°C. Each strain was incubated at 10 and 15°C for 96 h, or at 20 and 25°C for 72 h. The cultures were sampled every 24 h, serially diluted, spread on MRS agar, and incubated at 20°C for 48–72 h to count colony forming units. Finally, the corresponding growth curve was prepared.

Bacterial Membrane Fatty Acid Composition

To determine the difference in thermal adaptation between W. koreensis that did not grow well at 30°C (KCKM P0035, KCKM P0054) and that which grew well at the same temperature (KCKM 0130), strains were cultured at different temperatures to analyze their membrane fatty acid composition. W. koreensis KCKM P0035 and W. koreensis KCKM P0054 were cultured at 10°C for 144‒168 h, 20°C for 48‒72 h, and 28°C for 24‒48 h. Similarly, W. koreensis KCKM 0130 was cultured at 20°C for 48‒72 h, 28°C for 24‒48 h, and 33°C for 24‒48 h. Next, cells were harvested using a 4 mm loop and stored at -80°C before analysis. These cells were analyzed according to the Sherlock Microbial Identification System (MIS) using the Agilent 6890N(G1530N) gas chromatograph and Sherlock version 6.1.

Statistical Analysis

All physicochemical properties and bacterial counts of kimchi samples were analyzed in triplicates. In addition, SPSS v.27 (SPSS Inc, USA) was used to perform one-way analysis of variance and Duncan’s multiple range tests. Results are expressed as the mean ± standard deviation; p < 0.05 was considered statistically significant.

Results

Fermentation Conditions and Physicochemical Properties of Kimchi Samples

The fermentation conditions and physicochemical properties of kimchi samples are shown in Table 1. HM, KT 1 (initial fermentation phase), KT 2 (middle fermentation phase), and KT 3 (final fermentation phase) kimchi were used for LAB isolation. Moreover, 10INJ, 10TB, 10GSY, 10YSD, 10TS, 10HSG, 4INJ, 4TB, 4GSY, 4YSD, 4TS, and 4HSG were used to investigate appropriate temperature for the isolation of various LAB. The fermentation period and temperature range of all samples were 1‒250 days at 0‒10°C, respectively. The range of pH, titratable acidity, salinity, and soluble solid contents in kimchi samples were 4.01‒5.78, 0.42‒1.22%, 1.49‒2.40%, and 8.10‒10.20 °Brix, respectively.

Table 1 . The fermentation conditions and physicochemical properties of kimchi samples.

Sample	Fermentation period (day)	Fermentation temperature (°C)	pH	Titratable acidity (%)	Salinity (%)	Soluble solid contents (°Brix)
HM	250	0 ± 2	4.85 ± 0.02	0.72 ± 0.04	1.89 ± 0.01	10.17 ± 0.15
KT 1	1	4 ± 1	5.78 ± 0.06	0.42 ± 0.00	1.49 ± 0.14	10.20 ± 0.61
KT 2	15	4 ± 1	4.32 ± 0.04	1.06 ± 0.04	1.70 ± 0.05	10.00 ± 0.30
KT 3	29	4 ± 1	4.07 ± 0.10	1.22 ± 0.03	1.60 ± 0.09	8.43 ± 0.12
10HSG	7	10 ± 1	4.40 ± 0.01	0.92 ± 0.05	1.87 ± 0.08	8.20 ± 0.36
10TS	7	10 ± 1	4.13 ± 0.03	0.90 ± 0.04	1.62 ± 0.09	9.30 ± 0.30
10TB	7	10 ± 1	4.16 ± 0.02	0.91 ± 0.04	2.15 ± 0.09	8.17 ± 0.31
10YSD	7	10 ± 1	4.01 ± 0.01	0.93 ± 0.01	2.40 ± 0.05	8.43 ± 0.32
10INJ	7	10 ± 1	4.14 ± 0.04	0.91 ± 0.04	1.77 ± 0.02	9.34 ± 0.62
10GSY	7	10 ± 1	4.25 ± 0.01	0.96 ± 0.02	1.65 ± 0.01	8.17 ± 0.47
4HSG	14	4 ± 1	4.40 ± 0.02	0.88 ± 0.07	1.61 ± 0.06	8.37 ± 0.40
4TS	14	4 ± 1	4.22 ± 0.02	0.88 ± 0.03	1.54 ± 0.07	9.73 ± 0.49
4TB	14	4 ± 1	4.31 ± 0.01	0.77 ± 0.03	1.74 ± 0.07	7.53 ± 0.25
4YSD	14	4 ± 1	4.13 ± 0.03	0.78 ± 0.04	2.06 ± 0.02	8.10 ± 0.46
4INJ	14	4 ± 1	4.27 ± 0.03	0.83 ± 0.02	1.64 ± 0.03	9.37 ± 0.31
4GSY	14	4 ± 1	4.48 ± 0.02	0.93 ± 0.06	1.63 ± 0.06	9.87 ± 0.46

Generally, pH in the initial fermentation phase was >5. However, it decreased to ≤4 after the late phase [11]. Further, the titratable acidity of the initial fermentation phase was below 0.4% and increased to >1% at the final phase. The changes in these physicochemical properties are indicative of the variety in fermentation conditions of the kimchi samples.

Viable Cell Counts on Different Media and Isolation Temperatures

To investigate suitable culture media and temperatures, the LAB from HM, KT 1, KT 2, and KT 3 kimchi samples was evaluated under three types of LAB selective media and four isolation temperature conditions (Table 2). The temperature used to isolate LAB from kimchi was defined as the isolation temperature to avoid confusion with the culture or incubation temperature of the isolated strains. The LAB counts of HM, KT 1, KT 2, and KT 3 ranged from 7.44‒8.08 log CFU/ml, 6.17‒7.06 log CFU/ml, 8.52‒8.89 log CFU/ml, to 8.06‒8.58 log CFU/ml, respectively. The LAB counts in HM and KT 1 showed a slight difference depending on the isolation temperature. Furthermore, the highest LAB count was observed in MRS agar. Many components of culture medium can affect the growth of each strain, and MRS, LBS, and PES media have different compositions. These media contain compounds that promote LAB growth, such as carbohydrates, nitrogen sources, and Mg salt. MRS and LBS use dextrose as a carbon source. In contrast, PES is a selective medium for Leuconostoc sp. and uses sucrose because these species release dextransucrase in high-sucrose media. This enzyme subsequently converts sucrose into dextran [21]. The kinds of carbohydrates and nitrogen sources that promote growth in each strain differ. Thus, the growth of strains found in the kimchi samples could be promoted in a medium with a favored nutrient source [22]. These results demonstrate that MRS is the most suitable isolation medium.

Table 2 . Bacterial count of four kimchi samples using different culture media.

Sample	Temperature (°C)	Count (log CFU/ml)
Sample	Temperature (°C)	PCA	MRS	PES	LBS
HM	30	7.42 ± 0.05	7.60 ± 0.03^a	7.44 ± 0.06^a	7.51 ± 0.05^a
	20		7.91 ± 0.03^b	7.93 ± 0.04^b	7.49 ± 0.06^a
	10		8.04 ± 0.09^c	7.87 ± 0.03^b	7.64 ± 0.03^b
	5		8.08 ± 0.10^c	7.93 ± 0.06^b	7.54 ± 0.03^a
KT1	30	7.06 ± 0.09	7.02 ± 0.15^b	6.99 ± 0.08^b	6.40 ± 0.05^b
	20		7.06 ± 0.12^b	7.03 ± 0.09^b	6.43 ± 0.12^b
	10		7.06 ± 0.03^b	6.96 ± 0.10^b	6.22 ± 0.09^a
	5		6.19 ± 0.16^a	6.24 ± 0.15^a	6.17 ± 0.10^a
KT2	30	8.78 ± 0.06	8.85 ± 0.05^a	8.82 ± 0.08^a	8.64 ± 0.08^b
	20		8.87 ± 0.02^a	8.75 ± 0.02^a	8.52 ± 0.04^a
	10		8.87 ± 0.01^a	8.82 ± 0.09^a	8.59 ± 0.02^ab
	5		8.89 ± 0.01^a	8.88 ± 0.05^a	8.53 ± 0.04^a
KT3	30	8.22 ± 0.14	8.56 ± 0.05^a	8.44 ± 0.06^a	8.29 ± 0.11^b
	20		8.52 ± 0.09^a	8.43 ± 0.13^a	8.51 ± 0.04^c
	10		8.58 ± 0.05^a	8.52 ± 0.05^a	8.20 ± 0.15^ab
	5		8.48 ± 0.02^a	8.43 ± 0.07^a	8.06 ± 0.06^a

Viable Cell Counts at Different Isolation Temperatures

To investigate the suitable isolation temperature for various kimchi LAB, the number of colonies was counted at different isolation temperatures of 12 kimchi samples on MRS agar (Table 3). In only 4INJ and 4HSG, the number of colonies cultured at 20°C and 10°C was substantially higher than that at 30°C. As there was no significant difference between 10 of the 12 kimchi samples, analysis of the diversity of microorganisms isolated according to the isolation temperature was needed to determine the appropriate isolation temperature.

Table 3 . Lactic acid bacteria count of 12 kimchi samples at different isolation temperatures.

Sample	Temperature (°C)
Sample	30	20	10
10INJ	8.86 ± 0.22^a	9.03 ± 0.03^a	8.96 ± 0.11^a
10TB	8.95 ± 0.04^a	8.91 ± 0.08^a	8.95 ± 0.02^a
10GSY	9.03 ± 0.09^a	9.14 ± 0.06^a	9.12 ± 0.07^a
10YSD	8.68 ± 0.03^a	8.78 ± 0.07^a	8.79 ± 0.09^a
10TS	9.23 ± 0.07^a	9.25 ± 0.03^a	9.19 ± 0.05^a
10HSG	8.68 ± 0.17^a	8.61 ± 0.30^a	8.92 ± 0.11^a
4INJ	8.75 ± 0.05^a	8.87 ± 0.01^b	8.86 ± 0.05^b
4TB	8.35 ± 0.31^a	8.50 ± 0.15^a	8.56 ± 0.10^a
4GSY	8.91 ± 0.03^a	8.99 ± 0.02^a	8.93 ± 0.16^a
4YSD	8.22 ± 0.16^a	8.62 ± 0.47^a	8.39 ± 0.02^a
4TS	8.90 ± 0.08^a	9.08 ± 0.07^a	9.03 ± 0.15^a
4HSG	8.19 ± 0.02^a	8.58 ± 0.08^b	9.42 ± 0.13^c

Comparative Culture-Dependent and Culture-Independent Analyses

To determine the ideal isolation temperature for the LAB flora in kimchi, the culture-dependent and culture-independent results were compared. First, culture-independent analysis was performed. The results from kimchi samples are shown at genera and species levels in Fig. 1. As shown in Fig. 1A, Latilactobacillus sp. was the predominant genus in nine kimchi samples (HM, 10TS, 10YSD, 10INJ, 10GSY, 4TS, 4YSD, 4INJ, 4GSY) and Weissella sp. was the predominant genus in six samples (KT 2, KT 3, 10HSG, 10TB, 4HSG, 4TB). In KT 1, Lactococcus sp. was the predominant genus at the initial fermentation phase. However, other genera were also of similar relative abundance. Moreover, Leuconostoc sp. and Trichocoleus sp. showed the highest abundance in most of the samples. The species with the highest mean abundances were W. koreensis (30.52%), L. sakei (26.98%), T. caatingensis (12.62%), L. graminis (10.49%), Leu. gelidum (4.11%), Leu. mesenteroides (2.93%), Lac. cremoris (1.29%), and D. algida (1.18%) (Fig. 1B). During the culture-dependent analysis, 1,464 different LAB strains were isolated from kimchi samples. Information on strains isolated from kimchi at different temperatures is given in Tables S1–S16. Comparison of the diversity of 16 kimchi samples at different isolation temperatures was conducted using culture-dependent and culture-independent methods. To visualize the difference in the microbial community structure for some kimchi samples, relative abundance of species was represented in a heat-map style (Fig. 2). L. graminis was of high relative abundance in some kimchi samples (10YSD, 10INJ, 10GSY, 4YSD, and 4INJ) when analyzed through the culture-independent method, whereas it was not isolated by culture-dependent methods. This may be due to inappropriate culture conditions for this strain. The following LAB strains were not detected through the culture-independent method: Lac. lactis, W. cibaria, and Leu. miyukkimchii isolated using the culture-dependent method in KT 1; Leu. Holzapfelii; 10HSG, 10TS, and 10GSY, L. curvatus; 10HSG, 10TS, 10TB, 10YSD, 10INJ, 10GSY, 4YSD, 4INJ, and 4GSY Leu. pseudomesenteroides; Leu. Inhae; Leu. falkenbergense; Companilactobacillus heilongjiangensis; Enterococcus casseliflavus; Leu. Lactis; and Leu. carnosum isolated in KT 1; KT 2; 10HSG, 10TB, 10YSD, 10GSY, 4TS, and 4GSY; 10HSG, 4HSG, 4YSD, and 4GSY; 4HSG, 4TB, and 4GSY; 10HSG; 4HSG; 4TB; and 4GSY.

Fig. 1. The relative abundances of microbes, as determined through metagenomic analysis. A: genus level; B: species level (representing >1%)

Fig. 2. A heat map of the microbial community in culture-dependent and culture-independent analyses of four kimchi samples. The color intensity in each panel shows the percentage in the sample, according to the color key (right). The sample names are represented as isolation temperatures, and the result of culture-independent analysis is represented as the sample microbiome.

Isolated strains and relative abundance were different depending on the isolation temperature. More strains from nine of the 16 kimchi samples were isolated at <30°C. Moreover, five samples showed minimal difference in the number of isolated strains at varying isolation temperatures. Contrastingly, more strains were isolated at 30°C in the remaining two samples. Leu. gelidum, Leu. gasicomitatum, Leu. inhae, Dellaglioa algida, Companilactobacillus kimchiensis, Leu. miyukkimchii, Leu. holzapfelii and Leu. carnosum were not isolated from all samples at 30°C (Fig. 3). Although W. koreensis generally grows well under mesophilic conditions [23], some strains did not grow well at 30°C.

Fig. 3. A Venn diagram of lactic acid bacteria isolated from 16 kimchi samples using a culture-dependent method based on isolation temperature.

Cultural Characteristics of Psychrotrophic Strains

The culture characteristics of psychrotrophic kimchi LAB strains that were isolated at low temperatures and did not grow well at 30°C were investigated. Leu. gelidum (KCKM P0052), Leu. gasicomitatum (KCKM P0057), Leu. Inhae (KCKM P0061), D. algida (KCKM P0083), C. kimchiensis (KCKM P0044), and W. koreensis (KCKM P0054) were cultured at 10, 15, 20, and 25°C (Fig. 4). The growth curves of these isolates showed good growth at all the aforementioned temperatures, confirming their psychrotrophic characteristics.

Fig. 4. The growth curve of six selected strains at different culture temperatures (10, 15, 20, and 25°C).

Membrane Fatty Acid Composition of W. koreensis

Among the strains isolated at 10, 20, and 30°C, W. koreensis was divided into strains that grew well at 30°C (KCKM 0130) and those that did not grow (KCKM P0035) or slightly grew (KCKM P0054). Thus, to determine the difference in cultural characteristics between psychrotrophic and mesophilic W. koreensis, we compared changes in membrane fatty acid composition according to culture temperature of isolates with low isolation temperature (KCKM P0035 and KCKM P0054) and mesophilic strains (KCKM 0130) (Table 4). There was no specific trend in the membrane fatty acid composition of W. koreensis KCKM P0035 as the culture temperature increased. Contrastingly, the membrane fatty acid composition of W. koreensis KCKM 0130 changed with increasing culture temperature. Similarly, the C16:0, C16:1 ω7c/C16:1 ω6c ratio increased as the culture temperature increased. However, C14:0, C18:1 ω9c, and C19:0 decreased with increasing culture temperature. Moreover, as the culture temperature increased, the saturated fatty acid ratio increased, whereas the unsaturated fatty acid ratio decreased. W. koreensis KCKM P0054 showed a similar trend to W. koreensis KCKM 0130. However, the ratio of unsaturated fatty acids was almost unchanged as the temperature increased.

Table 4 . Comparison of relative membrane fatty acid composition between mesophilic and psychrotrophic Weissella koreensis at different culture temperatures.

	KCKM P0035			KCKM P0054			KCKM 0130
Fatty acid (%)	10°C	20°C	28°C	10°C	20°C	28°C	20°C	28°C	33°C
Saturated
C_12:0	1.06±0.06	0.69±0.06	1.20±0.11	2.00±0.03	2.05±0.27	1.49±0.43	1.97±0.32	2.56±0.39	1.92±0.39
C_14:0	1.18±0.02	1.21±0.08	1.22±0.02	1.75±0.02	1.33±0.05	1.32±0.02	1.45±0.08	1.45±0.14	1.21±0.16
C_16:0	10.68±0.11	11.29±0.05	10.51±0.40	9.63±0.11	10.54±0.12	13.22±0.12	10.80±0.22	10.81±0.41	16.53±0.82
C_17:0		0.19±0.03
C_18:0	1.40±0.05	1.18±0.02	1.49±0.09	1.54±0.02	1.61±0.06	1.06±0.09	1.44±0.04	1.59±0.07	0.96±0.24
Unsaturated
C_18:1 ω9c	80.40±0.25	80.10±0.54	80.36±0.28	78.90±0.54	78.85±1.07	78.70±0.85	78.99±0.69	76.92±1.21	75.19±1.74
C_18:3 ω6c							0.39±0.04	0.46±0.06	0.38±0.11
Branched-chain fatty acid
C_19:0 iso	2.78±0.05	2.48±0.28	2.74±0.29	3.10±0.09	2.95±0.27	1.72±0.05	3.44±0.20	3.20±0.15	1.63±0.58
Hydroxy fatty acids
C_17:0 2OH	2.07±0.09	1.75±0.03	1.96±0.16	2.64±0.07	2.00±0.16	1.29±0.13	1.12±0.19	1.32±0.11	1.05±0.41
C_16:1 2OH								0.63±1.10
Summed feature
C_16:1 ω7c/C_16:1 ω6c	0.42±0.16	1.10±0.88	0.77±0.13	0.65±0.17	0.99±0.34	1.20±0.56	0.40±0.70	1.08±0.96	1.88±0.42
Total saturated fatty acid	14.32±0.11	14.56±0.18	14.42±0.22	14.93±0.08	15.53±0.48	17.08±0.41	15.66±0.60	16.41±0.95	20.62±1.06
^aTotal unsaturated fatty acid	80.82±0.09	81.21±0.37	80.87±0.19	79.34±0.15	79.51±0.61	79.91±0.31	79.78±0.61	78.46±1.02	76.69±0.86

^aTotal unsaturated fatty acid: unsaturated + C_16:1 ω7c/C_16:1 ω6c

Discussion

This study investigated the appropriate conditions for isolating diverse LAB species from kimchi. Further, the cultural characteristics of psychrotrophs that did not grow well at medium temperature (30°C) were investigated. The diversity of LAB differed based on the isolation temperature of kimchi samples. In particular, Leu. gelidum, Leu. gasicomitatum, Leu. inhae, D. algida, C. kimchiensis, Leu. miyukkimchii, Leu. holzapfelii, and Leu. carnosum could not be isolated at 30°C. These findings indicate that 30°C is an unsuitable temperature for C. kimchiensis, Leu. miyukkimchii, and Leu. carnosum growth, despite the known optimum cultural temperature being approximately 30°C [23-25]. In a previous study, the appropriate cultural temperature of Leu. cremoris was 24‒27°C [26]. Additionally, Leu. gelidum and D. algida are typical psychrotrophic LAB [27, 28]. Leu. inhae also could not grow at 30°C in previous studies [7, 29]. Leuconostoc spp. can survive under cold stress conditions by expressing heat stress proteins [30]. Weissella spp. can also grow at low temperatures, and W. serratia growth is inhibited at >15°C [31]. Further, Mäkelä et al. [32] attempted to isolate ropy slime-forming strains, namely Lactobacillus spp. and Leuconostoc spp., at 30°C, 20°C, and 15°C. Most ropy slime forming strains were detected at 20°C and 15°C. Thus, the isolation temperature at 30°C seemed unsuitable for isolation of these ropy colony strains. Therefore, it may be inappropriate to isolate phychrotrophic LAB from kimchi under only mesophilic conditions (30°C).

Some LAB strains, such as Lac. lactis and L. plantarum were not isolated at 5°C. Furthermore, the culture period (14 days) was delayed at 5°C compared to that of conditions maintained at other isolation temperatures. These results showed low efficiency of 5°C in strain incubation. The suitable growth range for Lac. lactis is 27‒33°C [33, 34]. Therefore, 5°C was not a suitable isolation temperature for the diverse LAB from kimchi. The results of culture-dependent analysis obtained at 10°C and 20°C were considered more similar to culture-independent assay results than those obtained at 5°C and 30°C. This study found that 10°C and 20°C are suitable temperatures for isolating various psychrotrophic LAB, owing to growth retardation of strains at 5°C and inhibition of psychrotrophic LAB growth at 30°C.

Some LAB, including W. confusa, were identified using the culture-independent method but could not be identified through culture-dependent analysis. This is because the culture-independent method can detect the nucleotide sequences of dead microbiota with intact cells or those not cultured in the medium [35]. In contrast, some strains, including W. cibaria and Lac. Lactis, were identified through culture-dependent methods rather than the culture-independent method. Similar to our results, previous studies reported that some populations were missed by culture-independent methods due to their low numbers, which might be increased by culture-dependent approaches [36-38]. In the culture-dependent results, some of the Leuconostoc spp. were isolated at 10°C and 20°C whereas L. plantarum, C. heilongjiangensis, and E. casseliflavus were isolated only at 30°C. L. plantarum is a mesophilic bacterium [39, 40]. Therefore, it may have been isolated only at 30°C. As these strains were isolated only once from all samples, more samples are needed to confirm the isolation temperature of these strains. In this study, some mesophilic strains, including W. koreensis, Leu. carnosum, C. kimchiensis, and Leu. miyukkimchii, did not grow well at 30°C. Therefore, the difference in thermal tolerance between these strains must be investigated. The membrane fatty acid composition and adaptation of LAB under stress conditions are closely related [41]. For example, increased thermal tolerance was seen with the decrease of unsaturated fatty acids or the increase of saturated fatty acids in bacterial cell membrane fatty acids [41, 42]. In this study, an increased culture temperature of W. koreensis resulted in a decrease in the unsaturated fatty acid ratio of the thermal resistant strain. However, there was no difference in the strain with low thermal resistance. Furthermore, the saturated fatty acid ratio was increased with culture temperature in thermal resistant strains. An increased saturated fatty acid ratio or a decreased unsaturated fatty acid ratio enhances the thermal resistance of bacteria [41, 43]. In our study, W. koreensis KCKM 0130, whose growth limit was >30°C, could have decreased the unsaturated fatty acid content while increasing the saturated fatty acid content to adapt to the high culture temperature. Moreover, low thermal adaptation of W. koreensis KCKM P0035 and W. koreensis KCKM P0054, whose growth limit was <30°C, was possibly due to the low metabolism related to the change in membrane fatty acid composition. A medium temperature (approximately 30°C) is typically applied to isolate LAB from kimchi. However, more diverse strains were isolated at the isolation temperatures of 10°C and 20°C than at 30°C from most kimchi samples in this study. This indicates that only 30°C is insufficient for the isolation of more diverse LAB from kimchi.

Unlike kimchi, some fermented vegetables are fermented at room temperature using fewer ingredients [44, 45]. Therefore, kimchi fermented at temperatures below 10°C using various ingredients is expected to contain diverse psychrotrophic lactic acid bacteria.

Additionally, most psychrotrophic LAB isolated in this study may be key players in kimchi fermentation, with Leu. gelidum as one of the dominant strains [1]. However, recent studies suggest that psychrotrophic LAB, particularly Leu. gelidum, are food spoilage organisms that reduce the quality of fermented foods [18, 27, 46]. Thus, it is necessary to further investigate the influence of psychrotrophic strains on kimchi fermentation.

Our results suggest that the culture media composition, incubation time, and temperature in the isolation procedures are important for isolating diverse LAB from kimchi. Hence, mesophilic conditions (approximately 30°C) and isolation temperatures of 10–20°C in MRS medium should be used in parallel to isolate diverse LAB, including psychrotrophs, from kimchi. These findings can contribute to the isolation of more diverse LAB strains from kimchi.

Supplemental Materials

jmb-33-8-1066-supple.pdf

Acknowledgements

This work was supported by the World Institute of Kimchi (grant number KE2303-1), funded by the Ministry of Science and ICT.

Author Contributions

Hye In Ko: Formal analysis, methodology, and writing - original draft preparation

Chang Hee Jeong: Formal analysis, methodology, and writing - original draft preparation

Se-Jin Park: Methodology

So-Rim Kim: Methodology

Jong-Bang Eun: Data curation

Tae-Woon Kim: Conceptualization, supervision, writing - review & editing, project administration, and funding acquisition

References

Jung JY, Lee SH, Jeon CO. 2014. Kimchi microflora: history, current status, and perspectives for industrial kimchi production. Appl. Microbiol. Biotechnol. 98: 2385-2393.
Kim JY, Park SE, Kim EJ, Seo SH, Whon TW, Cho KM, et al. 2022. Long-term population dynamics of viable microbes in a closed ecosystem of fermented vegetables. Food Res. Int. 154: 111044.
Lee ME, Jang JY, Lee JH, Park HW, Choi HJ, Kim TW. 2015. Starter cultures for kimchi fermentation. J. Microbiol. Biotechnol. 25: 559-568.
Lee SH, Whon TW, Roh SW, Jeon CO. 2020. Unraveling microbial fermentation features in kimchi: from classical to meta-omics approaches. Appl. Microbiol. Biotechnol. 104: 7731-7744.
Lee JJ, Choi YJ, Lee MJ, Park SJ, Oh SJ, Yun YR, et al. 2020. Effects of combining two lactic acid bacteria as a starter culture on model kimchi fermentation. Food Res. Int. 136: 109591.
Hong SP, Lee EJ, Kim YH, Ahn DU. 2016. Effect of fermentation temperature on the volatile composition of kimchi. J. Food Sci. 81: C2623-C2629.
Pothakos V, Snauwaert C, De Vos P, Huys G, Devlieghere F. 2014. Psychrotrophic members of Leuconostoc gasicomitatum, Leuconostoc gelidum and Lactococcus piscium dominate at the end of shelf-life in packaged and chilled-stored food products in Belgium. Food Microbiol. 39: 61-67.
Shimodate K, Honda H. 2022. Isolation and identification of psychrotrophic lactic acid bacteria in godo, the traditional fermented soy food in Japan. J. Gen. Appl. Microbiol. 68: 219-224.
Song HS, Lee SH, Ahn SW, Kim JY, Rhee JK, Roh SW. 2021. Effects of the main ingredients of the fermented food, kimchi, on bacterial composition and metabolite profile. Food Res. Int. 149: 110668.
Lee D, Kim S, Cho J, Kim J. 2008. Microbial population dynamics and temperature changes during fermentation of kimjang kimchi. J. Microbiol. 46: 590-593.
Kim EJ, Seo SH, Park SE, Lim YW, Roh SW, Son HS. 2020. Initial storage of kimchi at room temperature alters its microbial and metabolite profiles. LWT 134: 110160.
Moon SH, Kim EJ, Kim EJ, Chang HC. 2018. Development of fermentation· storage mode for kimchi refrigerator to maintain the best quality of kimchi during storage. Korean J. Food Sci. Technol. 50: 44-54.
Kim JY, Kim BS, Kim JH, Oh SI, Koo J. 2020. Development of dynamic model for real-time monitoring of ripening changes of kimchi during distribution. Foods 9: 1075.
Nami Y, Bakhshayesh RV, Manafi M, Hejazi MA. 2019. Hypocholesterolaemic activity of a novel autochthonous potential probiotic Lactobacillus plantarum YS5 isolated from yogurt. LWT 111: 876-882.
Yang S, Yan D, Zou Y, Mu D, Li X, Shi H, et al. 2021. Fermentation temperature affects yogurt quality: a metabolomics study. Food Biosci. 42: 101104.
Baati H, Amdouni R, Gharsallah N, Sghir A, Ammar E. 2010. Isolation and characterization of moderately halophilic bacteria from Tunisian solar saltern. Curr. Microbiol. 60: 157-161.
Chamkha M, Mnif S, Sayadi S. 2008. Isolation of a thermophilic and halophilic tyrosol-degrading Geobacillus from a Tunisian hightemperature oil field. FEMS Microbiol. Lett. 283: 23-29.
Pothakos V, Snauwaert C, De Vos P, Huys G, Devlieghere F. 2014. Monitoring psychrotrophic lactic acid bacteria contamination in a ready-to-eat vegetable salad production environment. Int. J. Food Microbiol. 185: 7-16.
Lee KW, Shim JM, Park SK, Heo HJ, Kim HJ, Ham KS, et al. 2016. Isolation of lactic acid bacteria with probiotic potentials from kimchi, traditional Korean fermented vegetable. LWT 71: 130-137.
Won SM, Chen S, Park KW, Yoon JH. 2020. Isolation of lactic acid bacteria from kimchi and screening of Lactobacillus sakei ADM14 with anti-adipogenic effect and potential probiotic properties. LWT 126: 109296.
Lule V, Singh R, Behare P, Tomar SK. 2015. Comparison of exopolysaccharide production by indigenous Leuconostoc mesenteroides strains in whey medium. Asian J. Dairy Food Res. 34: 8-12.
Petrut S, Rusu E, Tudorache IS, Pelinescu D, Sarbu I, Stoica I, et al. 2019. Influence of various carbon sources on growth and biomass accumulation of some lactic acid bacteria strains. Rev. Chim. 70: 2434-2438.
Kim J, Kim JY, Kim MS, Roh SW, Bae JW. 2013. Lactobacillus kimchiensis sp. nov., isolated from a fermented food. Int. J. Syst. Evol. Microbiol. 63: 1355-1359.
Lee SH, Park MS, Jung JY, Jeon CO. 2012. Leuconostoc miyukkimchii sp.nov., isolated from brown algae (Undaria pinnatifida) kimchi. Int. J. Syst. Evol. Microbial. 62: 1098-1103.
Raimondi S, Spampinato G, Candeliere F, Amaretti A, Brun P, Castagliuolo I, et al. 2021. Phenotypic traits and immunomodulatory properties of Leuconostoc carnosum isolated from meat products. Front. Microbiol. 12: 730827.
Cooper RK, Collins EB. 1978. Influences of temperature on growth of Leuconostoc cremoris. J. Dairy Sci. 61: 1085-1088.
Pothakos V, Nyambi C, Zhang BY, Papastergiadis A, De Meulenaer B, Devlieghere F. 2014. Spoilage potential of psychrotrophic lactic acid bacteria (LAB) species: Leuconostoc gelidum subsp. gasicomitatum and Lactococcus piscium, on sweet bell pepper (SBP) simulation medium under different gas compositions. Int. J. Food Microbiol. 178: 120-129.
Veselá H, Dorotíková K, Dušková M, Furmančíková P, Šedo O, Kameník J. 2022. The pork meat or the environment of the production facility? The effect of individual technological steps on the bacterial contamination in cooked hams. Microorganisms 10: 1106.
Kim E, Cho EJ, Yang SM, Kim MJ, Kim HY. 2021. Novel approaches for the identification of microbial communities in kimchi: MALDI-TOF MS analysis and high-throughput sequencing. Food Microbiol. 94: 103641.
Salotra P, Singh DK, Seal KP, Krishna N, Jaffe H, Bhatnagar R. 1995. Expression of DnaK and GroEL homologs in Leuconostoc mesenteroides in response to heat shock, cold shock or chemical stress. FEMS Microbiol. Lett. 131: 57-62.
Wang D, Chen G, Tang Y, Li H, Shen W, Wang M, et al. 2020. Effects of temperature on paocai bacterial succession revealed by culture-dependent and culture-independent methods. Int. J. Food Microbiol. 317: 108463.
Mäkelä PM, Korkeala HJ. 1992. The ability of the ropy slime-producing lactic acid bacteria to form ropy colonies on different culture media and at different incubation temperatures and atmosphere. Int. J. Food Microbiol. 16: 161-166.
Lee DA, Collins EB. 1976. Influences of temperature on growth of Streptococcus cremoris and Streptococcus lactis. J. Dairy Sci. 59: 405-409.
Adamberg K, Kask S, Laht TM, Paalme T. 2003. The effect of temperature and pH on the growth of lactic acid bacteria: a pH-auxostat study. Int. J. Food Microbiol. 85: 171-183.
Lu Y, Tan X, Lv Y, Yang G, Chi Y, He Q. 2020. Physicochemical properties and microbial community dynamics during Chinese horse bean-chili-paste fermentation, revealed by culture-dependent and culture-independent approaches. Food Microbiol. 85: 103309.
Kesmen Z, Yetiman AE, Gulluce A, Kacmaz N, Sagdic O, Cetin B, et al. 2012. Combination of culture-dependent and cultureindependent molecular methods for the determination of lactic microbiota in sucuk. Int. J. Food Microbiol. 153: 428-435.
Nguyen DTL, Van Hoorde K, Cnockaert M, De Brandt E, De Bruyne K, Le BT, et al. 2013. A culture-dependent and-independent approach for the identification of lactic acid bacteria associated with the production of nem chua, a Vietnamese fermented meat product. Food Res. Int. 50: 232-240.
Zhadyra S, Han X, Anapiyayev BB, Tao F, Xu P. 2021. Bacterial diversity analysis in Kazakh fermented milks Shubat and Ayran by combining culture-dependent and culture-independent methods. LWT 141: 110877.
Nwachukwu U, George-Okafor U, Ozoani U, Ojiagu N. 2019. Assessment of probiotic potentials of Lactobacillus plantarum CS and Micrococcus luteus CS from fermented milled corn-soybean waste-meal. Sci. Afr. 6: e00183.
Kim J, Lee MH, Kim MS, Kim GH, Yoon SS. 2022. Probiotic properties and optimization of gamma-aminobutyric acid production by Lactiplantibacillus plantarum FBT215. J. Microbiol. Biotechnol. 32: 783-791.
Min B, Kim K, Li V, Cho S, Kim H. 2020. Changes in cell membrane fatty acid composition of Streptococcus thermophilus in response to gradually increasing heat temperature. J. Microbiol. Biotechnol. 30: 739-748.
Shin Y, Kang CH, Kim W, So JS. 2019. Heat adaptation improved cell viability of probiotic Enterococcus faecium HL7 upon various environmental stresses. Probiotics Antimicrob. Proteins 11: 618-626.
Álvarez-Ordóñez A, Fernández A, López M, Arenas R, Bernardo A. 2008. Modifications in membrane fatty acid composition of Salmonella typhimurium in response to growth conditions and their effect on heat resistance. Int. J. Food Microbiol. 123: 212-219.
Tlais AZA, Lemos Junior WJF, Filannino P, Campanaro S, Gobbetti M, Di Cagno R. 2022. How microbiome composition correlates with biochemical changes during sauerkraut fermentation: a focus on neglected bacterial players and functionalities. Microbiol. Spectr. 10: e0016822.
Moore JF, DuVivier R, Johanningsmeier SD. 2021. Formation of γ-aminobutyric acid (GABA) during the natural lactic acid fermentation of cucumber. J. Food Compost. Anal. 96: 103711.
Andreevskaya M, Jääskeläinen E, Johansson P, Ylinen A, Paulin L, Björkroth J, et al. 2018. Food spoilage-associated Leuconostoc, Lactococcus, and Lactobacillus species display different survival strategies in response to competition. Appl. Environ. Microbiol. 84: e00554-18.

Article Tools

Article

Research article

J. Microbiol. Biotechnol. 2023; 33(8): 1066-1075

Published online August 28, 2023 https://doi.org/10.4014/jmb.2303.03047

Influence of Isolation Temperature on Isolating Diverse Lactic Acid Bacteria from Kimchi and Cultural Characteristics of Psychrotrophs

Hye In Ko^1,2†, Chang Hee Jeong^1,3†, Se-Jin Park², So-Rim Kim^1,2, Jong-Bang Eun², and Tae-Woon Kim^1*

¹Technology Innovation Research Division, World Institute of Kimchi, Gwangju 61755, Republic of Korea
²Department of Integrative Food, Bioscience and Biotechnology, Chonnam National University, Gwangju 61186, Republic of Korea
³Honam National Institute of Biological Resources, Mokpo 587262, Republic of Korea

Correspondence to:Tae-Woon Kim, korkimchiman@wikim.re.kr

^†These authors contributed equally to this work.

Received: March 30, 2023; Revised: April 28, 2023; Accepted: May 2, 2023