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

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.

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.

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).

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.
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 7.44-8.08log CFU/ml, 6.17-7.06log CFU/ml, 8.52-8.89log CFU/ml, to 8.06-8.58log 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.

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.

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 C 16:0 , C 16:1 ω7c/C 16:1 ω6c ratio increased as the culture temperature increased.However, C 14:0 , C 18:1 ω9c, and C 19:0 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.

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][24][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 culturedependent approaches [36][37][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.

Fig. 2 .
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.

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