KR101540843B1 - manufacturing method of complex kimchi lactic - Google Patents

Abstract

The present invention relates to a method for producing a compounded Kimchi lactic acid bacteria and a method for producing the same, which are prepared by mixing two or more strains of lactic acid bacteria isolated from kimchi and maximizing the function of lactic acid bacteria proliferation and lactic acid production, to provide.

Description

Technical Field [0001] The present invention relates to a method for manufacturing a complex kimchi lactic acid bacterium, which comprises culturing separately a Kimchi-cultivated Weissella koreensis strain 521 and a lactobacillus saccharum 171 strain,

The present invention relates to a method for producing a fermented soybean oil, which comprises mixing a strain extracted from Kimchi and maximizing the function of lactic acid bacteria proliferation and lactic acid production, The present invention relates to a method for producing a complexed Kimchi lactic acid bacterium.

Lactic acid bacteria refers to bacteria that produce lactic acid through carbohydrate fermentation. These lactic acid bacteria are widely used in the production of fermented foods such as yoghurt, cheese, sour crout (German kimchi) and sausage. Live lactic acid bacteria (live lactic acid bacteria) are classified as one of probiotics which is very good for health by promoting long-term health. Generally, probiotics lower serum cholesterol levels, improve gastrointestinal function, strengthen immunity, inhibit colorectal cancer.

In recent years, studies on food have been shifting from studies of basic functions (energy sources, constituents) to research on various phenomena related to human health, There is an explosive interest in foods that inhibit non-communicable chronic diseases. As a result, a new concept of functional food is attracting attention. Functional foods include probiotics, and probiotics are thought to have a significant effect on digestive microbiota and physical health. The probiotic market is still growing. Traditionally, probiotics have been used in yogurt or fermented dairy products. However, nowadays, as more consumers are looking for nonproduct probiotic products, probiotics are being used as additives in drinks, tablets, capsules, or lyophilized form.

Fruits and vegetables, including minerals, vitamins, dietary fiber, and antioxidants, do not contain dairy allegen (allergen). Plant-originated lactic acid bacteria (POLAB) are known as plant-originated lactic acid bacteria, and vegetable lactic acid bacteria have been extracted from uncooked plant fermented foods and from oriental rather than western countries. Kimchi, one of the Korean traditional foods, is attracting attention as a functional food. Kimchi is a mixture of salt and various vegetables and fermented for a reasonable period at ambient temperature. Due to the nature of the lactic acid-fermented vegetable product that should be ingested, kimchi is now a source of good lactic acid bacteria. The lactic acid bacteria found in Kimchi are psychrotrophic facultative anaerobic organism (salt - resistant anaerobic organism). Probiotics used in yogurt production are particularly required to be resistant to acidic conditions. This is because the pH in the yogurt is usually 4.5 or less. Most of the lactic acid bacteria isolated from the first person were very sensitive to acid conditions. However, Kimchi lactic acid bacteria can survive under extreme conditions where nutrients are deficient and antimicrobial substances are present around them. In addition, vegetable lactic acid bacteria including Kimchi have excellent ability to utilize the nutrients given around and can produce various bioactive substances. Therefore, lactic acid bacteria isolated from Kimchi can be used for functional foods. Foods, including probiotics, inhibit constipation caused by irregular eating habits, stress and excessive dieting, and are available for product development such as antioxidant effects, blood pressure maintenance, constipation, intestinal microflora, and anti-obesity.

Mixed cultures of lactic acid bacteria have been recognized as effective for certain fermentations. Currently, mixed cultures of lactic acid bacteria are used in the production of dairy products such as cheese and fermented milk. In this connection, the symbiotic relationship of various bacteria has already been proved.

However, in the past, a number of strains were mixed to produce a functional food as in the case of Kimchi Lactobacillus Patent Registration No. 10-1014872 (2011.02.15), and as in the case of Registration No. 10-0707102 (2006.09.05.), However, only the functional use of lactic acid bacteria such as Kimchi for inhibiting the proliferation of microorganisms has been made, and a patent for the mixed culture of lactic acid bacteria has not been presented yet.

In order to solve the above-mentioned problems, the method for producing a complex Kimchi lactic acid bacterium obtained by cultivating Weissella corynsis strain 521 isolated from kimchi of the present invention and Lactobacillus saccharum 171 strain comprises two or more And the strain is mixed and cultured to improve the growth ability and lactic acid production ability.

The strains isolated from the kimchi of the present invention are Weissella koreensis 521 and Lactobacillus sakei 171, and the ratio of mixed culture was Weissella koreensis 521: Lactobacillus casei 1 : 0.3 volume ratio.

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The present invention is a useful invention that can be used for various products by maximizing the functions of lactic acid bacteria proliferation and lactic acid production by culturing the strains extracted from Kimchi.

FIG. 1 is a graph showing the mean value and standard deviation of changes in cell growth, glucose consumption, and lactic acid production during the cultivation of lactic acid bacteria of Kimchi.
FIG. 2 is a graph showing the mean value and standard deviation of changes in cell growth, glucose consumption, lactic acid production, and total protein consumption by kimchi lactic acid bacteria alone and mixed culture.

Hereinafter, the configuration of the present invention will be described in more detail.

Example  1: Materials and Methods

1-1: Identification of Strain from Kimchi

First, 50 g of Chinese cabbage kimchi was put into a sterilie filter bag, and 1 ml of the liquid sample was taken, diluted by decidual dilution method, plated on a 100 ul agar plate, and cultured in a 37 ° C incubator for 48 hours.

Ten single colonies were randomly selected for each sample, and the isolates were inoculated on a BCP agar plate by a flat plate method, and cultured in a 37 ° C incubator for 24 hours.

After contact with the MRS broth and incubation at 37 ° C for 16 hours, 80% glycerol stock was prepared and stored in an ultra-low temperature freezer (-70 ° C).

To isolate the lactic acid bacteria of the isolate, it was commissioned to Macrogen Co., Ltd.

The isolates were identified using the 16S rRNA method and were 99% homologous with the standard strains. The strains isolated from the above results were named as follows.

Leuconostoc mesenteroides JEI

Lactobacillus sakei 171

Leuconostock kimchi 132

Weissella koreensis 521

1-2: Strain and Growth Conditions

The four lactic acid bacteria used in the present invention were Leuconostoc mesenteroides JE1, Leuconostoc kimchi 132, Lactobacillus sakei 171, Weissella koriensis 521 Weissella koreensis 521) (hereinafter, the names of the respective strains are referred to as JEI, 132, 171, and 521, respectively).

The four strains identified above were isolated from Korean kimchi and stored at -80 ° C in de Man-Rogosa-Sharpe (MRS) medium supplemented with 20% glycerol, respectively. The strains were cultured in MRS medium supplemented with glucose at 37 ℃ for 32 hours under anaerobic conditions. Pure or mixed culture experiments were performed in a 500 ml Erlenmeyer flask containing 100 ml of MRS medium. (150 rpm, pH 6.5, 30 [deg.] C). The final inoculation concentration of each strain was 1 ml.

Specifically, 1 ml of 521 strains and 0.1, 0.2, 0.3, 0.4, 0.5 or 1 ml of 171 strains were mixed in the mixed culture.

1-3: Bioreactor fermentation  ( Bioreactor  Fermentation)

Fermentation was carried out in a fermentor (Fermentec Inc. Co. Daejeon, Korea) in anaerobic conditions (31 working volume, agitation 150 rpm, temperature 37 ° C). The pH was always maintained at 5.5 by automatic addition of 5N NaOH solution and the fermentation time was approximately 31 hours. Unless otherwise stated, the fermentation medium contains seeds of 5% MRS liquid culture. Each experiment was repeated three times.

1-4: Analysis method

Free amino acids were quantified by taking the culture supernatant at designated time intervals. Cultured samples were filtered by membrane (0.45 μm, GD; Millipore, [Bedford, USA]) and hydrolyzed by 6 M HCl. (Reaction at 110 [deg.] C in vacuum for 24 hours). Then, amino acid quantification was performed using a Hiach model L8800A automated amino acid analyzer (Hitachi, Japan). Growth of bacteria was measured using a spectrophotometer (600 nm). Fermentation broth (fermentation broth) was collected by centrifugation (20,000 × g , 10 min), and the supernatant was collected and the concentration of lactic acid and glucose was analyzed by HPLC equipped with a refractive index detector. The column used for the analysis was Aminex HPX-87H (Bio-Rad Co., USA) and the chromatography was carried out at 40 ° C using 0.01 N NH ₂ SO ₄ as eluent. The flow rate was 0.6 ml / min.

Example  2: Flask fermentation  (Flask fermentation)

First, fermentation was carried out through pure culture of each strain. 171 strains showed the highest culture turbidity (OD ₆₀₀ ? 1.36) during the culture period. (Fig. 1A). Since amino acid and peptide production by protein hydrolysis is a very good nutrient source for bacterial growth, 171 strains tested showed the highest proteolytic activity. That is, it can be seen that the 171 strain has a much higher growth rate than the strains having different growth rates.

On the other hand, 132 strains showed the lowest growth rate. Figure 1B shows the glucose consumption curve according to growth. All of the strains tested had conspicuously consumed glucose until 8 hours, but still retained 2.9-5.63 (g / l) of glucose in the medium after 32 hours of culture. 132 strain, glucose consumption decreased rapidly after 8 hours, and JEI, 521 and 171 strains remained in the same amount of glucose at 32 hours. From the viewpoint of lactic acid production (Fig. 1C), the maximum abortion amount after culturing for 32 hours was 12.5 g / l in 171 strains. On the other hand, JEI, 521, and 132 strains were 9.73, 9.7, and 8.54 g / l, respectively.

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Example  3: Improved lactic acid production by co-cultivation of symbiosis

Table 1 below shows the changes in amino acids in the medium depending on the incubation time of the four strains. 171 strains have been shown to inhibit the growth of unwanted amino acids [alanine, valine, leucine, methionine, threonine, isoleucine, lysine, , glutamic acid (glutamic acid), arginine, proline, phenylalanine, tyrosine, glycine, glutamine, tryptophan . On the other hand, serine (cystine), cystine (cystine), and asparagine (asparagine) all consumed during 24 hour culture. The amino acids consumed above are essential amino acids in the growth of 171 strains. 132 strains secreted alanine, valine, serine, threonine, arginine, phenylalanine, and tyrosine. Conversely, JEI and 521 strains decreased the amount of amino acids in the medium as a whole. In particular, lucine, proline (proline), phenylalanine, tyrosine, aspagine, glutamine, and tryptophan were found to be essential amino acids in these strains when compared with a reduced amount.

Here, the initial amount of amino acids in fresh MRS medium was set at 100%.

No singn: 90 - 100

-: 90 - 80%, -: 80 - 70%

---: 70 - 50%, ----: less than 50%

+: 110 - 130%, ++: 130 - 150%

+++: 150-200%, ++++: over 200%

Example  4: Improvement of lactic acid production by mixed culture of symbiosis

In this example, mixed cultures of 171 and 521 strains showing a great difference in growth, lactic acid and amino acid secretion were tried. Proteolytic 171 strain showed a high growth rate by 521 strains and showed that it could efficiently produce amino acids consumed by 521 strains. Therefore, the present inventors expected that the promotion of the protein hydrolysis system during the mixed culture would increase the secretion of essential amino acids necessary for the growth of strains 171 and 521, thereby increasing the growth of both strains. We investigated the lactic acid production ability and growth during fermentation by single and mixed cultivation. Due to the difference in the efficiency of protein hydrolysis and the possibility of dominance of one strain to another strain, the mixing ratio was controlled by the existing experimental results. 521 strains were inoculated with 0.1, 0.2, 0.3, 0.4, and 0.5 ml of 171 strains in a fixed amount of 1 ml inoculum. As shown in Table 2, when 1 ml of 521 strain and 0.3 ml of 171 strain were cultured, the highest growth rate and lactic acid production ability (32 hours incubation) were observed when compared with the single culture of 521 strains. Higher growth rates and protein hydrolysis and amino acid secretion observed in mixed cultures were associated with increased protein hydrolysis efficiency. 171 strains were proteolytic strains, and the resulting protein hydrolysates were used by two strains in the mixed culture. Thus, the present inventors concluded that 521, a non-proteolytic strain, promotes growth due to the presence of 171 strains.

Example  5: Comparison of Fermentation Efficiency in Single and Mixed Culture

Fermentation efficiencies of single and mixed cultures were investigated using a fermenter. All of the strains showed similar exponential growth during the first 10 hours, and thereafter, the cell concentration of 171 strains increased and that of 521 strains and 171 strains increased. This phenomenon was not observed in the independent cultures of 521 strains (see Fig. 2A). A low amount of glucose was consumed in the mixed culture (see FIG. 2B). The lactic acid production ability was compared in the culture of each strain (see FIG. 2C). The advantage of mixing cultures is clearly demonstrated in lactic acid production capacity. Higher amounts of lactic acid were produced in the mixed culture. The lactic acid production capacity for glucose was 521, 171 and 69.1%, 85.5% and 96.2%, respectively. Figure 2D shows the protein changes in the culture medium during the independent culture and the mixed culture of 521 and 171. The amount of protein increased during the initial 2 hour fermentation, but then decreased continuously until the fermentation time of 32 hours. Initial protein hydrolysis began to appear after 12 hours of culture (protein consumption was 2.68 mg / l in 521 strains, 1.3 mg / l in 171 strains, and 1.12 mg / l in mixed cultures). Thereafter, protein consumption did not change significantly from 12 hours to 32 hours. Mixed cultures showed lower protein consumption than single culture of each strain. The advantage of low nutrient consumption in mixed cultures was clearly demonstrated. Proteins were hydrolyzed by extracellular proteases produced by the proteolytic 171 strain accumulated in culture medium alone and in mixed culture. Thus, the growth of non-proteolytic 521 strains was promoted by the appearance of 171 strains. Simultaneous cultivation of both strains could increase mutual growth and lactic acid production. In conclusion, the present inventors have found that fermentation efficiency is higher in a mixed culture than in a single culture. The symbiotic relationship between these two strains overcomes the growth limit of one strain and also improves the ability of lactic acid production.

Claims (4)

delete delete 50 g of Chinese cabbage kimchi was pulverized, and only the liquid portion was separated and cultured in a 37 ° C. incubator for 48 hours. The strain Weissella koreensis 521 and Lactobacillus sakei 171 were added to glucose The cells were cultured in MRS medium at 37 ° C for 32 hours under anaerobic conditions. Weissella koreensis 521 and Lactobacillus sakei 171 were mixed at a volume ratio of 1: 0.3, MRS broth was mixed and cultured in a 500 ml Erlenmeyer flask at a stirring speed of 150 rpm at a pH of 6.5 and 30 ° C. The cultured Weissella corynis strain 521 and Lactobacillus casei 171 were cultured in a mixed culture A method for producing a complex kimchi lactic acid bacterium. delete

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