KR19980066785A - Lactobacillus pentosus LSDM Isolated from Kimchi - Google Patents

Abstract

The present invention relates to the production of inoculation bacteria for cottage cheese. More specifically, the present invention relates to a Lactobacillus pentosus LSDM, a novel lactic acid bacterium capable of producing lactic acid and odors to be suitable for the manufacture of cottage cheese. Lactic acid bacteria of the present invention is superior to the lactic acid fermentation bacteria found in the conventional lactic acid and flavor generation ability, and is suitable for producing Korean-style cottage cheese of a new taste and texture.

Description

Lactobacillus pentosus LSDM Isolated from Kimchi

The present invention relates to a novel lactic acid bacteria isolated from kimchi. More specifically, the present invention relates to Lactobacillus pentosus LSDM with lactic acid and odor producing ability to be suitable for the manufacture of cottage cheese.

In general, lactobacillus used to prepare the cottage cheese, Lactococcus cremoris and Lactococcus lactis has been mainly used, and other gas producing strains (Emmons Beckett, J. Dairy Sci. , 67, 2129, 1984), Lactococcus thermophilus and Lactobacillus bulgaricus (Panday et al., Indian J. Dairy Sci., 42 (3), 567, 1989) have been reported.

However, studies on lactic acid bacteria of kimchi by fermentation temperature during Kimchi fermentation (Lee, et al., Korean Journal of Industrial Microbiology, 20 (1), 102, 1992; Park et al., Institute of Basic Science, Inha University, 11, 16, 1990; Min and Kwon, Journal of the Korean Society of Food Science and Technology, 16 (4), 443, 1984), Chinese cabbage varieties (Lee, 26 (3), 239, 1994), by salinity (High, Korean Journal of Food Science and Technology, 26 (3), 199, 1994) By subsidiary material (Lee et al., Korea Journal of Industrial Microbiology, 20 (1), 102, 1994).

In addition, the addition of inoculation bacteria for the homogenization of kimchi (Choi et al., Korean Journal of Industrial Microbiology, 17, 403, 1989; Lee and Kim, Korean Journal of Nutrition and Nutrition, 17 (4), 342, 1988), Isolation of bacteriocin-producing lactic acid bacteria (Pak et al., Korean Journal of Agrochemicals, 26 (1), 35, 1983; Journal of Korean Society of Food Science and Technology Symposium, 43, 1994) Doctoral dissertations (48, 1994) have been studied, but there are no cases of lactic acid bacteria isolated from kimchi and used in dairy products.

Overseas Changes of Lactic Acid Bacteria during Fermentation of Sauerkraut (Stamer et al, J. Milk Food Technol., 34, 521, 1971) and Isolation of Lactic Acid Bacteria from Botanical Gardens such as Vegetables, Grains, Roots, and Legumes (Nout Rambouts, J. Applied Bacteriology Symposium Supplement, 73, 1368, 1992), but there have been no cases of using these lactic acid bacteria in dairy products, except that Jagdish has been isolated from silage, a plant material. It has been suggested for use in this dairy product (Jagdish, Liverstock-adviser, 10 (6), 33, 1986).

In addition, conventionally found Lactobacillus pentosus (Lactobacillus pentosus) is a gram-positive, rod-like, conditional hemolytic fermentation lactic acid bacteria, has the effect of inhibiting the antibacterial action and food poisoning bacteria, but is used in fermented sausages and dry sausage, etc. Due to the poor production ability, mixed bacteria are mainly used rather than single strains, and there have been no cases of inoculating bacteria of fermented milk products and cottage cheese.

Thus, the present inventors found that the new lactic acid bacteria isolated from kimchi are excellent in lactic acid and fragrance generating ability and suitable for the preparation of Korean-style cottage cheese, thus completing the present invention.

After all, the main object of the present invention is to provide a novel Lactobacillus pentosus LSDM isolated from kimchi.

1 is a graph showing the pH change when lactic acid bacteria are inoculated into reduced skim milk.

2 is a graph showing the protein degradation ability between lactic acid bacteria in reduced skim milk.

3 is a graph showing the growth of lactic acid bacteria in MRS medium containing bile and L-cysteine.

FIG. 4 is a chromatogram showing the fragrance components of unflavored cottage cheese made with Lactobacillus pentosus LSDM.

FIG. 5 is a chromatogram showing the fragrance components of unflavored cottage cheese made with Lactococcus lactis.

FIG. 6 is a chromatogram showing the fragrance components of the unadded cream cheese in the mixed culture.

The novel lactic acid bacteria of the present invention were isolated from kimchi, and examined the morphological, physiological and biochemical properties according to the invention of the examples described below.

The present inventors first separated kimchi juice from kimchi sample, diluted with peptone dilution, and cultured in MRS medium, and then, each colony was purely separated and selected as a lactic acid bacterium. The selected strains were plated in MRS medium, and then aerobicly cultured and purified. As a result of identifying purely isolated strains, the strains were Gram-positive, bacilli, well grown with or without oxygen, and negative for catalase and motility. In addition, it was grown at 15 °, but not at 45 ° C., and did not generate ammonia from glucose and gas from glucose, but belonged to Streptobacterium in the genus Lactobacillus. . On the other hand, as a result of conducting 49 kinds of sugar fermentation tests to confirm the species name, acid was generated from 24 species such as lactose, and the results were inputted into the ATB identification system and examined. As a result, the lactic acid bacteria of the present invention were lactobacillus pentosus ( Lactobacillus pentosus (Lactobacillus pentosus) was finally identified, and during the passage of the skim milk coagulation strains isolated from kimchi, natural mutant strains with a rapid rate of skim milk coagulation were named Lactobacillus pentosus LSDM.

As a result of testing the growth of lactic acid bacteria by measuring the number of viable cells and pH, the growth rate was almost the same as that of commercial strains. As a result of measuring the protein degradation of lactic acid bacteria, overall protein degradation was weaker than that of commercial strains, but after 18 hours The rapid increase of up to 24 hours showed a similar level of proteolytic activity when cultured up to 24 hours. In addition, by measuring the growth inhibitory concentration (MIC) value through the antibiotic resistance test, the resistance to penicillin-G in the β-lactam series is much more resistant than the mixed strain, while the mixed strain for the Novobiocin Very low resistance compared to Enzyme activity test results showed high enzyme activity in β-galactosidase, β-glucosidase, α-glucosidase and N-acetyl-β-glucosaminidase. As a result of measuring the growth of lactic acid bacteria in MRS medium, bile resistance was stronger than that of commercial strains, and pH resistance was stronger than that of commercial strains. From the above results, it was confirmed that Lactobacillus pentosus LSDM of the present invention is a lactic acid bacterium suitable for the manufacture of cottage cheese. The Lactobacillus pentosus LSDM of the present invention was deposited on September 10, 1996 by the Korean Society of Seedling Association (KFCC) under Accession No. KFCC-10913.

Hereinafter, in the present specification, the Lactobacillus pentosus LSDM is referred to as L. pentosus LSDM for convenience.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1 Isolation of Microorganisms

After separating kimchi juice from kimchi sample, dilute total microorganisms to the level of 10 ⁶ -10 ⁷ cfu / ml with dilution peptone, and inoculate 0.1 ml of MRS medium (Table 1) to which NaN ₃ was added by 35 Incubated for 48 hours at ℃. Then, each colony was purely separated in an electric MRS medium, and the colony that turned yellow was selected as a potential lactic acid bacterium. The selected strains were plated with platinum three times in the previous MRS medium, followed by aerobic incubation and pure separation. Purely isolated strains were inoculated in 10% reduced skim milk (Difco Laboratories, USA), and confirmed the coagulation at 35 ℃.

Table 1 Composition of MRS medium used for microbial separation in the present invention

Example 2 Identification of Microorganisms

The taxonomy of microorganisms was performed according to the method of Hammes et al. (The Prokaryotes, 1563-1578, 2nd Edition, Springer-Verlag Co. (1992)). The results are summarized in Table 2 below. These strains were both gram positive and bacillus, well grown with or without oxygen, and negative for catalase and motility. In addition, while growing at 15 ℃, did not grow at 45 ℃, ammonia is not produced from gas and arginine from glucose (glucose), it was confirmed that belongs to Streptobacterium of the genus Lactobacillus (Lactobacillus) . On the other hand, 49 kinds of sugar fermentation tests were conducted using the API 50CHL Kit (API bioMerieux, France) to identify the species. Acids were generated from 24 species such as lactose, and the results were inputted into the ATB identification system. It was confirmed that Lactobacillus pentosus (Lactobacillus pentosus). During the passage of skimmed milk coagulation strains isolated from Kimchi, natural mutant strains with rapid clotting speed were named as Lactobacillus pentosus LSDM, and were assigned to KFCC on September 10, 1996. Deposited under accession number KFCC-10913.

Table 2 Morphological, Physiological and Biochemical Properties of Lactic Acid Bacteria (L. pentosus LSDM)

In order to confirm whether L. pentosus LSDM isolated by the present inventors is suitable as an inoculation bacterium for cottage cheese, Lactococcus lactis (Ractococcus lactis, Rhone-poulenc, France), Lactococcus, which is a commercial strain for cheese as a control. Mixed cultures of Lactococcus lactis and Lactococcus cremoris (Redone-poulenc, France) were tested in 10% reduced skim milk.

Example 3 Growth Test of Microorganisms

The growth of lactic acid bacteria was tested by measuring the number of live bacteria and pH. The number of viable cells was inoculated with 1 drop of lactic acid bacteria in a 150 ml of 10% reduced skim milk with a 1 ml pipette, incubated for 24 hours at 30 ° for 3 hours, each sample diluted in 0.1% peptone solution, and plate count agar (BCP plate count agar) poured on a plate and hardened, followed by 48 hours of incubation at 30 ° C. In addition, the pH change when the lactic acid bacteria were inoculated with 10% reduced skim milk 5% was measured, and the results are shown in FIG. As can be seen in Figure 1, the growth rate was almost the same as the commercial strain, and it was confirmed that it was suitable as inoculation bacteria because it took 6 hours to cut the curd to reach pH 4.8 during the cottage cheese manufacturing process.

Example 4 Protein Degradation Test of Microorganisms

Protein degradation of lactic acid bacteria was measured by lactic acid bacteria in 10% reduced skim milk according to Hull's method (Hull, J. Dairy Sci., 30, 881, 1947), and the results are shown in FIG. It was. As can be seen in FIG. 2, L. pentosus LSDM had a weaker overall protein degradability compared to commercial strains, but showed a sharp increase from 18 hours to 24 hours. Showed.

Example 5 Antibiotic Resistance Test

In the antibiotic resistance test, tryptic soy broth (Difco, USA) was used to observe the growth by a 2-fold dilution method, and the minimum inhibitory concentration (MIC) value was measured, and the results are shown in Table 3. .

Table 3 Comparison of antibiotic susceptibility of L. pentosus LSDM, L. lactis and mixed strains (*)

*: The lowest inhibitory concentration was expressed as (µg / ml) for # labeled antibiotics and (units / ml) for the remaining antibiotics.

As can be seen in Table 3, L. pentosus LSDM has a minimum inhibitory concentration of 20 units / ml for penicillin-G in the β-lactam family, a mixed strain having 0.5 units / ml of L. lactis, 1.0 units / ml. While much more resistant, novobiocin was 0.15 µg / ml, which was much lower than L. lactis, 6.0 µg / ml, and 12.0 µg / ml mixed strain.

Example 6 Enzyme Activity Test

Enzyme activity test was carried out by diluting the strain incubated for 18 hours at 30 ℃ in MRS liquid medium to prepare a sample of 10 ⁵ -10 ⁶ cfu / ml level using API ZYM Kit (API bioMerieux, France) After incubation at 30 ° C. for 5 hours, the enzyme was reacted. Enzyme activity was expressed by a value of 0 to 5, comparing the standard color table, the results are shown in Table 4.

Table 4 Comparison of enzymatic activities of L. pentosus LSDM, L. lactis and mixed strains (*)

*: Standard colors are specified in steps from 0 to 5, with 0 being negative and 5 representing the maximum intensity. 1 is 5 nanomole (nanomole), 2 is 10 nano water, 3 is 20 nanomole, 4 is 30 nanomole, 5 is 40 nanomole or more enzyme activity.

As can be seen in Table 4, L. pentosus LSDM showed high enzyme activity in β-galactosidase, β-glucosidase, α-glucosidase and N-acetyl-β-glucosaminidase. .

Example 7 Bile Resistance Test

Bile tolerance was measured by testing the growth of lactic acid bacteria in MRS medium containing 0.3% bile and 0.05% L-cysteine (Gilliland Walker, J. Dairy Sci), according to Gilliland and Walker's method. , 73, 905, 1990), and the results are shown in FIG. As can be seen in FIG. 3, the time taken to increase the OD value by 0.3 was 2.08 hours for L. pentosus LSDM, 4.32 hours for L. lactis, and 5.68 hours for mixed strains. Was strong.

Example 8 pH Resistance Test

The pH resistance test was measured according to the method of Clark et al. (Clark et al., Culture Dairy Products J., 28 (4), 11, 1993), and the results are shown in Table 5.

[Table 5] Comparison of lactic acid bacteria resistance after 3 hours in hydrochloric acid solution (unit: CFU / ml (Log number))

¹⁾ Initial log number before pH treatment: 8.80

²⁾ Initial log number before pH treatment: 8.70

³⁾ Initial log number before pH treatment: 8.72

As can be seen in Table 5, all lactic acid bacteria survived up to 3 hours at pH 2 and pH 3, but the log decreased at 0 hours in pH 1 and all died after 1 hour.

Example 9 Antibacterial Activity Test

Antimicrobial activity was measured according to the method of Gilliland and Speck (Gilliland Speck, J. Food Prot., 40 (12), 820, 1977), and the results are shown in Table 6.

Table 6 Inhibition of Food Poisoning Bacteria of L. pentosus LSDM in MRS Medium (*)

* Initial number of L. pentosus LSDM: 3.6 × 10 ⁷ CFU / ml

^a measurement after 6 hours incubation at 37 ° C

Table 7 Inhibition of Food Poisoning Bacteria of L. lactis in MRS Medium (*)

Initial number of L. lactis: 1.1 × 10 ⁸ CFU / ml

^a measurement after 6 hours incubation at 37 ° C

Table 8 Inhibition of Food Poisoning Bacteria in Mixed Strains in MRS Medium (*)

* Initial number of mixed cultures: 1.4 × 10 ⁶ CFU / ml

^a measurement after 6 hours incubation at 37 ° C

As can be seen in Tables 6-8, L. pentosus LSDM was 42.59% for Escherichia coli, 50.59% for Salmonella typhi, Staphylococcus aureus ), 54.84% of inhibition. L. pentosus LSDM for Escherichia coli and mixed strain for Staphylococcus oreus and Salmonella typhi showed higher inhibitory effects, respectively.

Example 10 Analysis of aroma component

On the other hand, in order to compare the aroma components of the cottage cheese prepared using these three strains, the aroma components collected by the dynamic headspace method (gas chromatograph mesSpectrometer (MSD, Hewlett-Packard 5972 series, USA, the results of the analysis are shown in FIGS. 4, 5, and 6. As can be seen in Figures 4, 5, and 6, the fragrance components of the cheese using L. pentosus LSDM were all separated by 18 separation atmospheres, of which the similarity was 90% or higher than 3-hydroxy-2-butanone, Acetic acid and phenol were separated. 11 strains of cheese using commercial strains were isolated. Among them, L. lactis contained acetic acid, 2-ethyl-1-hexanol and phenol, and mixed strains of 3-hydroxy-2butanone and phenol.

As described in detail above, the present invention provides Lactobacillus pentosus LSDM, which is a lactic acid fermentation bacterium isolated from kimchi. Lactic acid bacteria of the present invention is superior to the lactic acid fermentation bacteria found in the conventional lactic acid and flavor generation ability, it is suitable for producing Korean cottage cheese.

Claims (1)

Lactobacillus pentosus LSDM (KFCC-10913) isolated from kimchi.