KR20030097235A - Medium for lactic acid producing bacteria not affecting taste, color and flavour of product - Google Patents

Abstract

PURPOSE: A medium for activating functional lactic acid bacteria which do not affect characteristic taste, color and flavor of a product is provided, thereby rapidly activating lactic acid bacteria and producing high concentration of bacteriocin. CONSTITUTION: A medium for activating functional lactic acid bacteria which do not affect characteristic taste, color and flavor of a product comprises a minimal medium and at least one compound selected from MnSO4, glucose, alanine, glycine and asparagine, wherein the minimal medium comprises K2HPO4, KH2PO4, MgSO4 7H2O, C6H5Na3O7, (NH4)2SO4 and distilled water; and the medium comprises (a) 0.5 to 5 wt.% of K2HPO4, 0.1 to 5 wt.% of KH2PO4, 0.1 to 5 wt.% of MgSO4 7H2O, 0.01 to 5 wt.% of C6H5Na3O7, 0.01 to 1 wt.% of (NH4)2SO4 and 79 to 99.28 wt.% of distilled water; and (b) at least one compound is selected from the group consisting of 0.01 to 1 wt.% of MnSO4, 0.005 to 1 wt.% of glucose, 0.005 to 1 wt.% of alanine, 0.005 to 1 wt.% of glycine and 0.005 to 1 wt.% of asparagine.

Description

Functional lactobacillus activation medium without affecting the inherent taste, color and aroma {MEDIUM FOR LACTIC ACID PRODUCING BACTERIA NOT AFFECTING TASTE, COLOR AND FLAVOUR OF PRODUCT}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a functional lactic acid bacteria activation medium that does not affect the inherent taste, color and aroma of the product, and more particularly, to an activation medium capable of activating the lactic acid bacteria in a short time to produce a high concentration of bacteriocin.

[Private Technology]

Bacteriocin is a natural nontoxic preservative produced by microorganisms and is an antibacterial substance produced by many gram-positive bacteria and gram-negative bacteria. Bacteriocin is composed of proteins or carbohydrates, and when ingested in the human body, it is decomposed by proteolytic enzymes of the digestive organs and is a natural preservative that is nontoxic and non-residual to the human body.

Bacteriocin, which is currently of most interest in the industry, is mainly produced by lactic acid bacteria, and is produced in five major genera ( Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Camobacterium ) that form a group of lactic acid bacteria.

Bacteriocin improves shelf life of fermented milk, fermented alcoholic beverages, canned products, refrigerated, frozen products, red pepper paste, miso, tofu, lactic acid bacteria fermentation products, and prevents rancidity and deterioration of traditional foods (kimchi, medicinal herbs, Takju, etc.). Bacteriocin also maintains activity at high temperatures, is stable at a wide range of pH, and is non-toxic, colorless, and odorless. Therefore, such bacteriocin is highly regarded as a natural preservative that can replace a chemical synthetic preservative, and is actually used as a means to secure food safety with minimal heat treatment and low temperature distribution.

Bacteriocin production mainly consists of lactic acid bacteria, MRS (bacto proteose peptone No.3 10 g, bacto beef extract 10 g, bacto yeast extract 5 g, bacto dextrose 20 g, polysorbate 80 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium sulfate General lactic acid bacteria medium such as 0.1 g, manganese sulfate 0.05 g, dipotassium phosphate 2 g / L) and cultured in a medium consisting of Whey and the addition of a beef extract, winsim bacteria from the culture It consists of a method of separation by separation and filtration. However, in the case of general lactic acid bacteria medium, not only is it difficult to produce a high concentration of bacteriocin in a short time, and when the lactic acid bacteria culture medium is dried, it is difficult to apply to various foods due to poor color, taste and aroma due to media components such as yeast extract and meat extract.

In order to solve the problems of the prior art, an object of the present invention is to provide a medium capable of producing a high concentration of bacteriocin in a short time.

It is another object of the present invention to provide a medium capable of activating lactic acid bacteria in a short time.

In addition, an object of the present invention is to provide a culture medium containing a minimum component capable of activating the lactic acid bacteria in a short time.

In another aspect, the present invention is to provide a lactic acid bacteria culture medium that does not change the color, taste and aroma of the food when the lactic acid bacteria culture medium is added to the food dried.

Figure 1a is a photograph showing the degree of growth inhibition of Helicobacter filament by the lactic acid bacteria culture filtrate obtained by culturing in the activation medium of the present invention, Figure 1b is the growth of Helicobacter filament is not inhibited by the medium not inoculated with lactic acid bacteria Control picture,

2 is a graph showing the growth inhibitory ability against Helicobacter bacteria when the lactic acid bacteria culture filtrate was diluted,

Figure 3 is a photograph showing the degree of growth inhibition of Helicobacter Philo when the lactic acid bacteria culture filtrate is diluted to 1% with distilled water and then subjected to a well test,

4 is a photograph comparing the color difference between the culture filtrate prepared after culturing the lactic acid bacteria in MRS (A) and the culture filtrate (B) prepared after culturing the lactic acid bacteria in the activation medium.

In order to achieve the above object, the present invention provides a lactic acid bacteria activation medium comprising a minimum medium and at least one compound selected from the group consisting of MnSO ₄ , glucose, alanine, glycine and asparagine.

In another aspect, the present invention (a) at least one compound selected from the group consisting of MnSO ₄ , glucose, alanine, glycine and asparagine to prepare an activation medium and (b) inoculating lactic acid bacteria in the medium and It provides a bacteriocin production method comprising the step of culturing to prepare a culture.

In another aspect, the present invention provides a bacteriocin prepared by the above method for producing bacteriocin.

In another aspect, the present invention (a) at least one compound selected from the group consisting of MnSO ₄ , glucose, alanine, glycine and asparagine to prepare an activation medium and (b) inoculating lactic acid bacteria in the medium and It provides a lactic acid bacteria active solution prepared by the method comprising the step of culturing to prepare a culture.

In another aspect, the present invention (a) mixing the at least one compound selected from the group consisting of MnSO ₄ , glucose, alanine, glycine and asparagine in a minimal medium to prepare an activation medium, (b) inoculating the medium to lactic acid bacteria It provides a lactic acid bacteria active solution prepared by the method comprising the step of culturing to prepare a culture and (c) obtaining a culture filtrate from the culture.

Hereinafter, the present invention will be described in detail.

The present inventors produced lactic acid bacteria activating medium capable of propagating lactic acid bacteria in a short time and producing a high concentration of bacteriocin.

The lactic acid bacteria activation medium of the present invention is characterized in that it contains at least one compound selected from the group consisting of MnSO ₄ , glucose, alanine, glycine and asparagine in a minimal medium.

The minimal medium was _{K 2 HPO 4, KH 2 PO} 4, MgSO 4 .7H 2 0, C 6 H 5 Na 3 O 7, (NH 4) 2 SO ₄ and distilled as comprising a, preferably from 0.5 to 5 % by weight of K ₂ HPO _4, 0.1 to 5% by weight of KH ₂ PO _4, 0.1 to 5% by weight of MgSO ₄ .7H ₂ 0, 0.01 to 5% by weight of _{C 6 H 5 Na 3 O 7} , 0.01 to 1 Wt% (NH ₄ ) ₂ SO ₄ and 79 to 99.28 wt% distilled water.

The MnSO ₄ contained in the lactic acid bacteria activation medium of the present invention is preferably 0.01 to 1% by weight, and preferably 0.005 to 1% by weight of glucose, alanine or glycine, respectively.

The most preferred active lactic acid bacteria culture medium _{K 2 HPO 4, KH 2 PO} 4, MgSO 4 .7H 2 0, C 6 H 5 Na 3 O 7, (NH 4) 2 SO 4, MnSO 4, distilled water, glucose, alanine, glycine And asparagine.

By inoculating the lactic acid bacteria into the lactic acid bacteria activating medium of the present invention, the lactic acid bacteria active liquid can be prepared. Lactobacillus active liquid of the present invention means both lactic acid bacteria culture solution or lactic acid bacteria culture filtrate.

The lactic acid bacteria are preferably all kinds of lactic acid bacteria producing conventional bacteriocin, and may be inoculated with powdered lactic acid bacteria, lyophilized lactic acid bacteria, cultured lactic acid bacteria or lactic acid bacteria culture solution. Preferably, 1 × 10 ⁸ to 5.0 × 10 ⁸ cfu / ml of lactic acid bacteria are added to the liquid medium, and then incubated at 30 to 40 ° C. for 5-10 hours.

The lactic acid bacteria culture filtrate of the present invention can be obtained by removing the lactic acid bacteria from the lactic acid bacteria culture medium, in which the usual practice is centrifugation or filtration. However, the method for obtaining lactic acid bacteria filtrate is not limited thereto.

The lactic acid bacterium active liquid of the present invention contains a high concentration of bacteriocin and can be used for food additives, preservatives or food poisoning.

The lactic acid bacteria active solution of the present invention may be prepared by a method comprising the steps of (a) preparing a lactic acid bacteria activation medium, (b) inoculating and culturing the lactic acid bacteria in the medium, or (a) lactic acid bacteria It may be prepared by a method comprising the steps of preparing an activation medium, (b) inoculating and incubating the lactic acid bacteria in the medium, and (c) obtaining a culture filtrate from the culture.

The present invention can be used for the production of a culture filtrate containing live or dead bacteria of lactic acid bacteria at the same time, food additives, preservatives, food poisoning bacteria prevention of high adhesion inhibitory and bactericidal.

In another aspect, the present invention is to prepare a culture medium by incorporating at least one compound selected from the group consisting of MnSO ₄ , glucose, alanine, glycine and asparagine in a minimal medium, inoculated with lactic acid bacteria in the medium to prepare a culture. It provides a method for producing bacteriocin, and the bacteriocin prepared therefrom.

The bacteriocin production method of the present invention may further include obtaining a culture filtrate from the culture or separating the bacteriocin from the culture or the culture filtrate. The bacteriocin separation method can be carried out by conventional methods. For example, bacteriocin may be separated by ammonium sulphate precipitation, molecular sieve chromatography (molecular sieve column chromatography).

The lactic acid bacteria activating medium of the present invention not only enables the production of high concentrations of bacteriocin by activating the lactic acid bacteria in a short time, but also does not harm the intrinsic taste, color and aroma of the food when the lactic acid bacteria culture medium or the filtrate is used as a food additive.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example: Activation Medium

(1) Composition of Activation Medium

_{K 2 HPO 4 7g / l,} KH 2 PO 4 2g / l, MgSO 4 .7H 2 0 1g / l, sodium sites rate 0.5g / l and _{(NH 4) 2 SO 4 0.1} g / l, to 100 mL with water To the minimum medium, 0.2g / l MnSO ₄ , 1% glucose, 100 ug / ml alanine, 100 ug / ml glycine or 100 ug / ml asparagine was added to prepare the activation medium of Table 1 below. The activation medium was titrated to pH 7.0 with HCl, and used by autoclaving.

Example Activation medium composition Example 1 Minimum medium + glucose + alanine + glycine + asparagine Example 2 Minimum medium + MnSO ₄ + glucose + alanine + glycine + asparagine Example 3 Minimum medium + MnSO ₄ + glucose Example 4 Minimum medium + MnSO ₄ + Alanine Example 5 Minimum medium + MnSO ₄ + glycine Example 6 Minimum medium + MnSO ₄ + Asparagine Example 7 Minimum medium + MnSO ₄ + glucose + alanine Example 8 Minimum medium + MnSO ₄ + glucose + glycine Example 9 Minimum medium + MnSO ₄ + glucose + asparagine Example 10 Minimum medium + MnSO ₄ + glucose + alanine + glycine Example 11 Minimum medium + MnSO ₄ + glucose + alanine + asparagine Example 12 Minimum medium + MnSO ₄ + glucose + glycine + asparagine Example 13 Minimum medium + MnSO ₄ + Alanine + Glycine Example 14 Minimum medium + MnSO ₄ + Alanine + Asparagine Example 15 Minimum medium + MnSO ₄ + glycine + asparagine Example 16 Minimum medium + MnSO ₄ + Alanine + Glycine + Asparagine Example 17 Minimum Medium + MnSO ₄ Comparative example Minimum badge Control MH Badge

MH medium used as a control is Muller Hinton; beef extract 3.0 g, acid hydrolysate of casein 17.5 g, starch 1.5 g.

Experimental Example 1: Effect Verification of Activation Medium I

Lactobacillus Confluence PL 9001 (KCCM-10245), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus Confluence PL 9004 (KCCM-10248), Lactobacillus Fermentum PL 9005 (KCCM-10250), Lactobacillus Permanent PL 9006 (KCCM-10251), Lactobacillus plantarum 9010 and Lactobacillus plantarum 9011 were used in the experiment. Each lactic acid bacteria was MRS medium (Difco, bacto proteose peptone No.3 10 g, bacto beef extract 10 g, bacto yeast extract 5 g, bacto dextrose 20 g, polysorbate 80 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium 0.1 g of sulfate, 0.05 g of manganesesulfate, and 2 g / L of dipotassium phosphate were incubated overnight at 37 ° C. Lactic acid bacteria or lyophilized Lactobacillus 5.0 X 10 ⁸ cfu / ml was added to the activation medium and 5-10 at 30 ° C. Time activated.

Lactobacillus culture filtrate and the same amount of MH medium was mixed and inoculated therein E. coli O157, a representative food poisoning bacteria. After 6 hours of incubation the growth of E. coli O157 was measured by OD.

Example No addition of lactic acid bacteria culture medium Addition of Lactic Acid Bacteria Culture Example 1 0.037 0.11 Example 2 0.081 0.043 Example 3 0.023 0.016 Example 4 0.029 0.012 Example 5 0.059 0.022 Example 6 0.059 0.036 Example 7 0.048 0.004 Example 8 -0.008 0.002 Example 9 0.088 0.048 Example 10 0.056 0.024 Example 11 0.059 0.032 Example 12 0.069 0.043 Example 13 0.047 0.022 Example 14 0.062 0.03 Example 15 0.071 0.035 Example 16 0.099 0.018 Example 17 0.061 0.019 Comparative example 0.015 0.041 Control 0.221 0.188

As shown in Table 2, the culture filtrate obtained by culturing in the activation medium of Examples 1 to 17 was confirmed to inhibit the growth of E. coli O157. In particular, it was confirmed that the activation medium of Example 2, Example 16 and Example 17 is very effective for the proliferation of lactic acid bacteria. However, in the case of the minimal medium, the addition of the lactic acid bacterium filtrate was found to promote the growth of E. coli O157 rather than the use of only the minimum medium, which is the control medium.

Experimental Example 2: Effect Verification of Activation Medium II

Lactobacillus was cultured in the activation medium in the same manner as in Experimental Example 1, the culture filtrate was obtained to check the Helicobacter Philophyll growth inhibitory ability.

PL9001 viable cell powder was inoculated into Example 2 and Comparative Medium at 10 ⁸ cfu / ml, and then incubated at 37 ° C. for 5 hours. Centrifugation (15000 rpm, 10 min) to obtain a supernatant, diluted 1/2 with sterile MQ and then measured the urease (urease) component by the indophenol method. Since the measured value is proportional to the urease activity derived from Helicobacter Philophyll, it is possible to know the amount of Helicobacter Philobacteria present on the sample. Through this principle, the effect of Helicobacter growth inhibition according to the treatment of PL strain culture filtrate on Helicobacter Philophyllium was measured.

badge OD ₆₃₀ Comparative example 3.25 Example 2 1.48

In Table 3 it can be seen that the culture filtrate obtained by culturing in the medium of Example 2 inhibits the growth of Helicobacter pylori. In other words, the activation medium of the present invention significantly increases the proliferation of lactic acid bacteria and the production of bacteriocin.

Experimental Example 3: Effect Verification of Activation Medium III

Lactobacillus Confluence PL 9001 (KCCM-10245), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus Confluence PL 9004 (KCCM-10248), Lactobacillus Fermentum PL 9005 (KCCM-10250), Lactobacillus Fermentum PL 9006 (KCCM-10251), Lactobacillus plantarum 9010 and Lactobacillus plantarum 9011 were respectively inoculated into the medium of Example 2, incubated at 37 ° C for 24 hours, and then centrifuged to separate the supernatant. The supernatant was prepared as a culture filtrate.

Helicobacter growth inhibition experiment by PL strain culture filtrate was performed. Wells were made using Pasteur pipettes sterilized in Brucella broth. Staining Helicobacter Philophyllum on a solid medium, adding a filtrate to the pores, incubating in a carbon dioxide incubator (5% to 10% CO ₂ ), and two days later. The diameter was measured.

1 is a photograph showing the degree of growth inhibition of Helicobacter filament by the lactic acid bacteria culture filtrate cultured in the activation medium of the present invention, Figure 1a is the culture filtrates cultured in the medium of Example 2 (Lactobacillus compass PL 9001 ( KCCM-10245), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus confluence PL 9004 (KCCM-10248), Lactobacillus latement PL 9005 (KCCM-10250), Lactobacillus latement PL 9006 (KCCM- 10251), Lactobacillus plantarum 9010, and Lactobacillus plantarum 9011) were found to inhibit the growth of Helicobacter pylori. On the other hand, helicobacter pylori growth was not inhibited by lactic acid bacteria-inoculated medium as shown in FIG.

Therefore, the activation medium of the present invention can be used not only as a medium for the production of bacteriocin of various kinds of lactic acid bacteria, but also can be used for the production of lactic acid bacteriocin-containing culture solution.

In addition, the lactic acid bacteria culture filtrate was diluted in the medium at 1%, 5% and 10%, and inoculated with Helicobacter bacteria. After overnight, O.D. of Helicobacter bacteria grown in the place where the activating solution was not added was measured, and the difference from the growth value (O.D.) when the activating solution was not added was converted into inhibitory power.

Figure 2 is a graph showing the growth inhibitory ability against Helicobacter bacteria when the lactic acid bacteria culture filtrate is diluted. In Figure 3, the growth inhibition of Helicobacter bacteria was the strongest in the culture filtrate diluted 10%, but the inhibitory activity was also observed in 1% mixed solution. At this time, the medium without addition of the culture filtrate was O.D. = 6.0, and O.D. = 6.5 when water was added instead of the culture filtrate.

In addition, as a result of further confirming the Helicobacter bacteria growth inhibition ability of the culture filtrate diluted 1% by a well test, it was confirmed that the growth of Helicobacter Philosophy even when diluted 1%.

Experimental Example 4: Verification of taste, color and aroma of the culture filtrate prepared using the lactic acid bacteria activation medium

The lactic acid bacteria were removed from the culture solution obtained by culturing the lactic acid bacteria in the medium of Example 2, and the culture filtrate was lyophilized. Then, the taste, color and aroma of the lyophilized culture filtrate were verified. In addition, it was compared with the taste, color and aroma of the culture filtrate prepared by the same method in the medium of Comparative Example.

MRS Badge Activation badge flavor Poke insipidity color partridge Colorless incense Save water Odorless

In Table 4, the activation medium of the present invention exhibits tasteless, colorless and odorless so as not to impair the aesthetics of the food even when used as an additive of other food.

Figure 4 shows the culture filtrate prepared after culturing the lactic acid bacteria in MRS (A) and the culture filtrate prepared after culturing the lactic acid bacteria in the activation medium (B). Comparing the color, A showed yellowish brown color, but B showed colorlessness.

As mentioned above, the lactic acid bacteria activating medium of the present invention activates lactic acid bacteria in a short time to enable the production of high concentrations of bacteriocins, and does not harm the intrinsic taste, color and aroma of the food when the lactic acid bacteria culture filtrate is used as a food additive. Do not. Therefore, the lactic acid bacteria culture filtrate of the present invention contains a high concentration of bacteriocin, and can be used for food additives, preservatives or food poisoning prevention.

Claims (11)

A lactic acid bacteria activation medium comprising a minimal medium and at least one compound selected from the group consisting of MnSO ₄ , glucose, alanine, glycine and asparagine. The method of claim 1 wherein the minimal medium is _{K 2 HPO 4, KH 2 PO} 4, MgSO 4 .7H 2 0, C 6 H 5 Na 3 O 7, (NH 4) 2 SO ₄ and distilled water and comprises Lactic acid bacteria activation medium. The method of claim 1, wherein the lactic acid bacteria activation medium (a) 0.5 to 5% by weight of K ₂ HPO _4, 0.1% to 5% of MgSO of KH ₂ PO _4, 0.1 to 5% by weight of ₄ .7H ₂ 0, 0.01 to 5% by weight of C ₆ H ₅ Na ₃ O ₇ , 0.01-1 wt% (NH ₄ ) ₂ SO ₄ and 79-99.28 wt% distilled water and (b) one type from the group consisting of 0.01 to 1 wt% MnSO ₄ , 0.005 to 1 wt% glucose, 0.005 to 1 wt% alanine, 0.005 to 1 wt% glycine and 0.005 to 1 wt% asparagine Lactic acid bacteria activation medium comprising a compound selected above. (a) preparing an activation medium by mixing at least one compound selected from the group consisting of MnSO ₄ , glucose, alanine, glycine and asparagine in a minimal medium; And (b) inoculating and culturing the lactic acid bacteria in the medium to prepare a culture; Bacteriocin production method comprising a. The production method according to claim 4, wherein the lactic acid bacteria of step (b) is powdered lactic acid bacteria, lyophilized lactic acid bacteria or cultured lactic acid bacteria. The method of claim 4, further comprising separating bacteriocin from the culture after step (b). The method of claim 4, further comprising obtaining a culture filtrate from the culture after the step (b). The method of claim 7, further comprising the step of separating the bacteriocin from the culture filtrate. A bacteriocin prepared by the method for producing bacteriocin according to any one of claims 4 to 8. (a) preparing an activation medium by mixing at least one compound selected from the group consisting of MnSO ₄ , glucose, alanine, glycine and asparagine in a minimal medium; And (b) inoculating and culturing the lactic acid bacteria in the medium to prepare a culture Lactic acid bacteria active liquid prepared by a method comprising a. (a) preparing an activation medium by mixing at least one compound selected from the group consisting of MnSO ₄ , glucose, alanine, glycine and asparagine in a minimal medium; (b) inoculating and culturing the lactic acid bacteria in the medium to prepare a culture; And (c) obtaining a culture filtrate from the culture; Lactic acid bacteria active liquid prepared by a method comprising a.