KR100196680B1 - A lactobacillus pentosus lsdm which is seperated 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, Lactococcus cremoris and Lactococcus lactis have been mainly used as lactic acid bacteria used to make cottage cheese, and other gas producing strains (Emmons & Beckett, J. Dairy Sci). , 67, 2192, 1984), Lactococcus themophilus 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, The Korean Journal of Food Science and Technology, 26 (3), 199, 1994), by subsidiary materials (Lee et al., Journal of the Korean Society for Industrial Microbiology, 20 (1), 102,1994), have been predominantly changed over time. 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), Isolation of Acid- and Bile-resistant Lactic Acid Bacteria (So, Korea University) Although there have been studies on doctoral dissertations, 48, 1994), 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 lactic acid bacteria in dairy products, but Jqgdish isolated from plant material silage. It has been suggested that lactic acid bacteria can be used in dairy products (Jagdish, Liverstock-adviser, 10 (6), 33, 1986).

In addition, the conventionally discovered Lactobacillus pentosus (Lactobacillus pentosus) is a gram-positive, rod-like, conditional hetero lactic acid fermentation bacteria, has the effect of inhibiting the antibacterial action and food poisoning bacteria, but is used in fermented sausage and dry sausage, etc. Due to its poor ability, mixed bacteria are mainly used rather than single strains, and so far, no cases have been used as inoculation 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 the lactic acid bacteria inoculated into reduced skim milk.

2 is a graph showing the protein degradation 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 of unflavored cottage cheese made with Lactobacillus pentosus LSDM.

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

FIG. 6 is a chromatogram showing fragrance components in unadded cream cheeses prepared in 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 method of the examples described later.

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, 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, 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, the antibiotic resistance test was used to observe the viability to measure the minimum inhibitory concentration (MIC) value, and the resistance to penicillin-G in the β-lactam series is much more resistant than the mixed strain, while the mixed strain for the novo bicosocin 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. Bile resistance was measured by testing the growth of lactic acid bacteria in MRS medium. As a result, resistance to bile 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

[Separation of Microorganisms]

After separating Kimchi juice from Kimchi sample, dilute total microorganisms to 10 ⁶ -10 ⁷ cfu / ml level with dilution peptone, and inoculate it with MN medium containing NaN ₃ (Table 1) by 0.1ml planar 35 Incubated for 48 hours at ℃. Then, each colony was purely separated in an electric MRS medium, and the colony that turned yellow was tentatively selected as lactic acid bacteria. The selected strains were plated with platinum three times in the previous MRS medium, and the strain was purely isolated by aerobic culture, inoculated in 10% reduced skim milk (Difco Laboratories, USA), and confirmed the coagulation at 35 ° C.

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)) and the results are summarized in Table 2 below. . These strains are gram positive and bacillus, do not grow well with or without oxygen, and do not produce ammonia from gas and arginine from glucose, resulting in Streptobacterium in the genus Lactobacillus. It was confirmed to belong. On the other hand, 49 kinds of sugar fermentation tests were conducted using the API 50CHL Kit (API bioMerieux, France) to identify the species. As a result, acid was generated from 24 species such as lactose, and the result was entered into the ATB identification system. It was confirmed that Bacillus pentosus (Lactobacillus pentosus). During the passage of skim milk coagulation strains isolated from kimchi, natural mutant strains with a rapid skim milk coagulation rate were named Lactobacillus pentosus LSDM, and on September 10, 1996, to the Korean Bacillus Association (KFCC). Deposited under accession number KFCC-10913.

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

Example 3

Microbial Growth Test

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 into a 1 ml pipette with 150 ml of 10% reduced skim milk, incubated for 24 hours at 30 ° C for 3 hours, and each sample was diluted in 0.1% peptone solution. agar) poured into a flat plate and hardened, and then cultured at 30 ° C. for 48 hours to count. In addition, the pH change at the time of inoculating 5% of lactic acid bacteria into 10% reduced skim milk was measured, and the result is 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 the inoculation bacterium by cutting the curd to reach pH 4.8 in the cheese cheese manufacturing process.

Example 4

[Proteolysis of Microorganisms]

Proteolytic 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 showed 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 double dilution method and the minimum inhibitory concentration (MIC) value was measured, and the results are shown in Table 3. .

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

As can be seen in Table 3, L. pentosus LSDM is a mixed strain having a minimum inhibitory concentration of 20 units / ml, 0.5 units / ml of L. lactis, 1.0 units / ml of the β-lactam family for penicillin-G. While much more resistant, Novobiocin was 0.15 μg / ml, showing very low resistance compared to 6.0 μg / ml L. lactis 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.

*: Standard color is specified in steps from 0 to 5, where 0 is negative and 5 is maximum intensity. 1 is 5 nanomole (nanomole), 2 is 10 nanomole, 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 the methods of Gilliland and Walker. , 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 tolerance 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), the results are shown in Table 5.

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 at pH 1 and all died after 1 hour.

Example 9

[Antibacterial 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.

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

* Measured after 6 hours incubation at 37 ℃

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

* Measured after 6 hours incubation at 37 ℃

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

* Measured after 6 hours incubation at 37 ℃

As can be seen in Tables 6-8, L. pentosus LSDM was 42,59% against Escherichia coli and 50.59% Staphylococcus oreus (Salphyellaocphi) against Salmonella typhi. 54.84% inhibition against aureus). For Escherichia coli, L. pentosus LSDM and Staphylococcus Oreus and Salmonera typhi showed higher inhibitory effects.

Example 10

[Analysis of fragrance components]

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, and the results are shown in Figures 4, 5, 6 degrees. As can be seen in Figures 4, 5, and 6, the fragrance components of the cheese using L. pentosus LSDM were all 18 separations, of which the similarity was more than 90% 3-hydroxy-2-butanone, Acetic acid and phenolic 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 prior art lactic acid and flavor generation ability, and is conjugated to produce Korean cottage cheese.

Claims (1)

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

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