KR20080078460A - Novel lactrobacillus buchneri and use thereof - Google Patents

Abstract

A novel lactic bacteria is provided to be used variously to prepare an antiflatulent agent, a probiotics, a food additive and a fermentation product and be also be used as a starter for fermenting kimchi. A Lactobacillus buchneri having high gamma-amino butyric acid(GABA) production capability is deposited as a deposition no. KCCM10835P. An antiflatulent composition, a probiotics composition, a feed composition, a food additive composition or a fermented product such as kimchi comprises Lactobacillus buchneri or a culture material thereof. To prepare fermented products, the Lactobacillus buchneri or the culture material thereof is used as a starter. A method for culturing the Lactobacillus buchneri comprises a step of culturing the Lactobacillus buchneri in a medium consisting of carbon source, nitrogen source, vitamin and mineral at a temperature of 10-30 deg.C for 24-60 hours.

Description

New Lactobacillus buchneri and use thereof

1 is a reverse phase HPLC graph showing a detection curve of gamma-aminobutyric acid (GABA) present in kimchi.

2 is a graph quantifying the GABA content of kimchi according to the aging period using reverse phase HPLC.

(A: Jongga House Kimchi, B: Ilmi Flavored Kimchi, C: Jongga House Pork Belly)

Figure 3 is a thin layer chromatography (TLC) results of qualitative analysis of the GABA production amount of Lactobacillus bootsneri according to the present invention.

(1: J-1, 2: E-2, 3: E-1, 4: O-03, 5: O-04, 6: H-W2, 7: P-2, 8: P-1, 9 : H-W1, 10: O-05)

Figure 4 is a graph comparing the GABA production capacity of Lactobacillus bootsneri according to the present invention and the GABA production capacity of known lactic acid bacteria.

(A: Lactobacillus casei, B: Lactobacillus bootsneri of the present invention, C: Lactobacillus brevis)

The present invention relates to a novel Lactobacillus bootsneri and its use, and more particularly, to Lactobacillus buchneri and gamma-aminobutyric acid (GABA) high performance.

In general, kimchi contains vitamin A, vitamin B, vitamin C and essential amino acids, and has a large amount of fiber, which is known to be effective in chronic diseases such as constipation and atherosclerosis, and also prevents cancer.

On the other hand, gamma-aminobutyric acid (GABA; gamma-aminobutyric acid) is an amino acid present in the brain of a mammal and is a neurotransmitter produced from glutamate by glutamate decarboxylase. In particular, it activates the movement of blood vessels to lower blood pressure (Kayahara H. and Sgiura D., Food & Development ., 36 (6): 4-6, 2001), and improves brain function to improve mental stability and learning ability. It is known to increase (Ishikawa K. and Saito S., Psychopharmacology , 56: 127-131, 1978; Akaga T., Food & development , 36 (6): 7-9, 2001). Currently, in addition to the study of GABA-containing tea, many studies have reported that lactic acid bacteria isolated from fermented foods such as rice and soybeans and kimchi produce high concentrations of GABA, and technology to increase GABA content in foods by applying the isolated lactic acid bacteria to actual foods. Many have also been reported (see Korean Patent Nos. 10-0478491, 10-2006-0006342 and 10-2004-0096975). Kimchi containing a large amount of GABA has been developed as a part of improving the taste and functionality of Kimchi (Korean Patent Application No. 2001-14638), but indirectly adding soy protein or soybeans Since it was produced, there was a problem that the taste of kimchi may be lost by soybean or soybean.

The present inventors found that the higher the GABA content as the kimchi is aged, isolating lactic acid bacteria having high GABA performance from kimchi aged more than 6 months, using this to produce kimchi to produce a functional kimchi with a high GABA content Was completed.

Accordingly, it is an object of the present invention to provide a lactic acid bacterium having high GABA performance and its use.

In order to achieve the above object, the present invention provides a Lactobacillus bootsneri or culture thereof having high GABA performance.

In order to achieve the other object of the present invention, the present invention provides a composition for formal, probiotic, feed and food additives containing the Lactobacillus bootsneri or its culture.

In addition, the present invention provides a fermentation product containing the Lactobacillus bootsneri or its culture in order to achieve another object of the present invention.

In addition, the present invention provides a method for producing a fermentation product using the Lactobacillus bootsneri or its culture as a starter.

In another aspect, the present invention provides a culture so that the Lactobacillus bootsneri can be used as a starter in kimchi.

Furthermore, the present invention provides a method for culturing the Lactobacillus bootsneri.

Hereinafter, the present invention will be described in detail.

Lactobacillus bootsneri of the present invention is characterized by the high performance of GABA as a novel lactic acid bacteria isolated from kimchi aged for 6 months or more.

In the present invention, it was confirmed that the GABA content is higher in the old kimchi aged more than 6 months compared to the kimchi not aged (see FIG. 2), was isolated from the lactic acid bacteria having a high GABA high performance from kimchi 6 months or more. As a result of the identification of the isolated lactic acid bacteria, it was confirmed that the strain belonging to Lactobacillus bootsneri, the strain was deposited on January 10, 2007 to the Korea Culture Center of Microorganisms (Accession No. : KCCM10835P).

Lactobacillus casei strain, which is a common lactic acid bacterium, produces little GABA, and Lactobacillus brevis, which is known to have a conventional GABA producing ability, produces about 0.17 mg / ml. The strain produces 13.45 mg / ml of GABA has a GABA production capacity of about 10 times or more compared to conventional lactic acid bacteria (see Figure 4).

Therefore, the lactic acid bacteria of the present invention can be used as a lactic acid bacteria having high GABA performance, for the purpose of improving health of humans and animals, that is, for formal use, probiotics or feed compositions. The composition may include a crushed cell wall fraction, live bacteria, dead bacteria, dry bacteria or culture of Lactobacillus bootsneri of the present invention as an active ingredient, and may further include an excipient or a carrier. The culture includes a culture solution itself cultured in a liquid medium, a filtrate (centrifuged supernatant) from which the strain is removed by filtration or centrifugation of the culture solution. The content of Lactobacillus bootsneri in the composition may vary depending on the use and formulation of the composition. Formal or probiotic compositions according to the invention may be prepared and administered in a variety of formulations and methods. For example, Lactobacillus bootsneri or its culture may be mixed with carriers and flavorings commonly used in the pharmaceutical arts for tablets, troches, capsules, elixirs, syrups. It may be prepared and administered in the form of syrup, powder, suspension or granule. As the carrier, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent and the like can be used. The mode of administration may be oral, parenteral or application, but preferably oral administration. In addition, the dosage may be appropriately selected depending on the absorbency, inactivation rate and excretion rate of the active ingredient in the body, age, sex, condition, etc. of the recipient. In addition, the feed composition according to the invention can be prepared in the form of fermented feed, compound feed, pellet form and silage (silage) and the like. The fermented feed may be prepared by fermenting an organic material by adding the Lactobacillus sakei and various microorganisms or enzymes of the present invention, the blended feed by mixing the various types of general feed and Lactobacillus bootsnery of the present invention Can be prepared. The feed in the form of pellets may be prepared by formulating the fermented feed or blended feed into a pellet machine, and silage may be prepared by fermenting the green feed with Lactobacillus bootsnery according to the present invention.

Lactobacillus bootsneri or its culture according to the present invention can be used as a food additive for food such as kimchi, beverages, baby food. In addition, the Lactobacillus bootsnery of the present invention can be used as a starter (starter) for the production of fermentation products. The fermented product includes cheese, kimchi, fermented reproductive products and the like. Fermentation products using the Lactobacillus bootsneri of the present invention can be prepared according to conventional methods known in the art. For example, after treating the grain powders such as brown rice and yulmu and the Lactobacillus bootsneri according to the present invention or 2-3 kinds of mixed lactic acid bacteria including the same, fermented at an appropriate temperature, nutrition of various agricultural products such as white rice, glutinous rice, sorghum, etc. Fermented reproductive products can be prepared by appropriate mixing to provide excellent balance and palatability.

In particular, Lactobacillus bootsneri according to the present invention can be used to prepare kimchi. Preferably, salted cabbage is mixed with common kimchi seasonings such as red pepper powder, garlic, ginger, leek, radish, and sugar, and then kimchi may be prepared by adding a culture of Lactobacillus bootsnery of the present invention. The culture of the Lactobacillus bootsneri of the present invention is preferably added 0.5 to 5% by weight, and most preferably 1% by weight based on the total weight of kimchi.

Lactobacillus bootsnery of the present invention is excellent in GABA generating ability, kimchi prepared with lactic acid bacteria of the present invention can be produced high functional kimchi with a high GABA content to reduce blood pressure and relax the effect.

Lactobacillus butnery according to the present invention can be cultured in large quantities by a conventional method for culturing the genus Lactobacillus. As the culture medium, a medium consisting of a carbon source, a nitrogen source, vitamins, and minerals may be used. For example, MRS (Man-Rogosa-Sharp) medium or Chinese cabbage juice medium may be used. The cabbage juice medium can be used by sterile after crushing and juice the pickled cabbage medium. Cultivation of the microorganism is possible under the culture conditions of the common Lactobacillus microorganisms, for example, can be incubated for 24 to 60 hours at 10 ℃ to 30 ℃. More preferably, it is preferable to incubate at 30 ° C for about 48 hours. Centrifugation or filtration may be performed to remove the culture medium in the culture and recover only the concentrated cells, which steps may be performed by those skilled in the art as needed. The concentrated cells may be frozen or lyophilized according to a conventional method so as not to lose their activity.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Reference Example  1>

Kimchi and Pasta  Kimchi GABA  Content comparison

“Kongga House Kimchi” (pH 3.87) sold by Jongga House as a general Kimchi with less than 4 months of ripening, and “Ilmi Flavored Kimchi” (pH 3.48) sold by Ilmi Foods, Chongga Pork Belly Porridge ”(pH 3.48) was purchased and the respective GABA content was quantified using reverse phase high performance liquid chromatography (HPLC).

Kimchi broth was filtered through a 0.45, 25 mm syringe filter and used as a sample. Kimchi samples are derivatized by AccQ-Tag method and analyzed under linear gradient conditions. As the HPLC column, Nova-PakTM C18, 4um column was used. GABA was detected at about 38 minutes, and AABA (Alpha-aminobutyric acid, alpha-aminobutyric acid) was used as an internal standard for accurate quantification (see FIG. 1).

As shown in Figure 2, as a result of quantitative analysis by HPLC was able to confirm the significantly higher GABA content in the old kimchi compared to the normal kimchi, when the high of the GABA content in the kimchi of B company of the low pH of the two kimchi of A company and B company As kimchi matures, the GABA content increases. Accordingly, further experiments for separating high GABA-producing bacteria were separated and identified from kimchi 6 months old or older.

< Example  1>

Lactobacillus Bootsnery  Isolation of Strains

<1-1> Isolation of Lactic Acid Bacteria from Kimchi

The broth of the kimchi manufactured in a conventional manufacturing method kimchi old kimchi was aged 7 months was filtered through a sterilized 15 minutes at 121 ℃ gauze and the filtrate in 121 ℃ with 0.9% sodium chloride solution was sterilized 15 minutes to about 10 ^-1 Dilute to a concentration of 10 ⁻⁸ . 55 g of MRS and 2% agar were dissolved in 1 L of distilled water, and then diluted with Kimchi broth in MRS medium to which 0.002% bromophenol blue was added and incubated at 30 ° C. for 48 hours.

Among the bacteria grown on MRS medium, Lactobacillus selective medium, circular, light blue or white bacteria are classified as Lactobacillus. Therefore, among the bacteria grown on the MRS medium, the circular, light blue or white bacteria were selected and inoculated separately in MRS broth dissolved in MRS 55g in 1 L of distilled water and incubated at 30 ° C. for 48 hours. 55 g of MRS and 2% agar were dissolved in 1 L of distilled water, sterilized and plated in a cultured MRS medium. After dissolving 30 g of TSB (Tryptic Soy Broth) in 1 L of distilled water, 1% MSG (monosodium glutamate) was added to TSB broth, and the above cultured strains were incubated at 30 ° C. for 48 hours to isolate 73 strains.

After the TLC plate was cut to a size suitable for the size of the development tube, one drop of the culture supernatant of each strain isolated above at 1 cm from the bottom was placed in the TLC development tube, and the entire solution was developed for about 3 hours. The composition of the developing solution was butanol: acetic acid: water = 1: 1: 1 (v / v). After the developed TLC plate was taken out and dried sufficiently, the ninhydrin solution was sprayed, and then dried at 105 ° C. for 10 minutes to confirm the reddish purple position of GABA.

As a result of the above-described thin layer chromatography (TLC), gamma-aminobutyric acid (GABA) -producing microorganisms were primarily selected. The amount of Gaba produced in 10 strains was quantified by the same method as high performance liquid chromatography (HPLC) described in Reference Example 1, and is shown in Table 1 below. Among the bacteria described in Table 1, H-W3 strain having the highest Gaba production capacity was finally selected.

H-W1 H-W2 H-W3 H-W4 H-W5 H-WB1 H-WB2 H-WB3 H-WB4 H-BW GABA content (mg / ml) 2.14 2.48 2.86 2.49 1.58 2.71 2.59 2.14 2.57 2.11

The H-W3 strain selected above was inoculated with 0.5 g of TSB broth and grown for 24 hours at 30 ° C. 1 ml of TSB broth containing the fully grown strain was placed in a sterile tube and centrifuged at 8000 rpm for 10 minutes to obtain cells. After mixing TSB broth with 15% glycerol well, 1ml was added to the cells, mixed well with a vortex mixer, and immediately frozen. Frozen strains were then used as starters for lactic acid bacteria inoculation.

<1-2> Identification of Selected Bacteria

16s rDNA sequence of the strain isolated in <1-1> was determined and compared with the disclosed strain. SEQ ID NO: 1 (AgAgTTTgATCMTGGCTCAg) was used as a forward primer for PCR, and SEQ ID NO: 2 (TACggYTACCTTgTTACgACTT) was used as a reverse primer. 16s rDNA sequence for identifying the strain was examined through the following process. MRS broth was used as a medium, and bacteria were incubated at 30 ° C. In the 16s rDNA set, the denaturation step was performed at 95 ° C for 5 minutes, the amplification step was 35 cycles (94 ° C 45 seconds, 55 ° C 60 seconds, 72 ° C 60 seconds), and the extension step was 75 ° C for 10 minutes. The amplified DNA was developed on a 1% (w / v) agarose gel and analyzed for 16s rDNA sequences. As a result, it was confirmed that the DNA was 99% identical to the 16S rDNA sequences of Lactobacillus bootsneri (results not shown). .

 Therefore, the present inventors deposited the Lactobacillus butneri isolated in the present invention to the Korean Culture Center of Microorganisms as of January 10, 2007 (Accession No .: KCCM10835P).

< Example  2>

Of lactic acid bacteria of the present invention GABA  Generation amount comparison

In order to confirm the degree of GABA production of Lactobacillus bootsneri of the present invention, Lactobacillus casei (KCCM 12452) and Lactobacillus brevis (Lactobacillus brevis, KCCM 11509) known to have a conventional GABA production capacity were used as a control. Each lactic acid bacterium was inoculated with 10 ml of MRS broth to which 50 mM glutamate was added. After incubating at 30 ° C. for 3 days, GABA was quantified by HPLC in the same manner as described in Reference Example 1, and the results are shown in FIG. 4.

As shown in Figure 4, the Lactobacillus bootsneri of the present invention was confirmed to have a GABA production capacity of about 10 times or more compared to the Lactobacillus brevis known to have a conventional Lactobacillus casei strain and GABA production capacity.

< Production Example >

Preparation of Kimchi Using Isolated Strains

GABA high-performance bacteria isolated in <Example 1> was added to the pickled cabbage with kimchi seasoning when preparing kimchi.

First, cut the Chinese cabbage in half, add 8% of natural salt, put it in a pickling container filled with 7% salt water, and soak it for 4-5 hours (picking cabbage), then wash the pickled cabbage under running water and put it on the dehydration table. Stacked and naturally dehydrated for 24 hours in a refrigerator at 4 ℃, remove the bottom of the dehydrated cabbage was carried out a pre-treatment process of the cabbage cut to a size of 3 ~ 5cm. Looking at the blending process of kimchi seasoning, with respect to 100 parts by weight of cabbage, 17-18 parts by weight of red pepper powder, 18-19 parts by weight of anchovy sake, 7-8 parts by weight of salted shrimp, 6-7 parts by weight of garlic, 0.5-1 A kimchi seasoning mixing process was performed by blending 5 parts by weight, 5 parts by weight of ionic starch syrup, 0.5 parts by weight of sugar, and 0.65-0.69 parts by weight of sodium L-glutamate (MSG).

Pickled Chinese cabbage, isolated in Example 1 Lactobacillus bootsneri was added in an amount of 10 ⁵ to 10 ⁷ cfu / ml, and kimchi seasoning was added to make the kimchi evenly, followed by fermentation at 10 to 30 ° C.

As described above, the novel Lactobacillus bootneri of the present invention is excellent in GABA generation ability. Therefore, the lactic acid bacteria of the present invention can be used in various forms for the preparation of formal preparations, probiotics, feed additives, food additives and fermented products. In particular, when the kimchi is prepared using the Lactobacillus bootsneri according to the present invention as a starter, it is possible to produce a functional kimchi having a large amount of GABA having a blood pressure lowering and neuroleptic effect.

<110> PARK, Hyun Jin <120> Novel Lactobacillus buchneri and use <130> NP07-0005 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 tacggytacc ttgttacgac tt 22  

Claims (10)

Lactobacillus buchneri (Accession No .: KCCM10835P) or a culture thereof having high gamma-aminobutyric acid (GABA) performance. Claim 1 lactobacillus bootsneri or a formal composition containing the culture thereof. A probiotic composition comprising the Lactobacillus butneri of claim 1 or a culture thereof. Feed composition containing the Lactobacillus bootsneri or a culture thereof of claim 1. Claim 1 Lactobacillus bootsnery or food additive composition containing the culture thereof. A fermentation product comprising the Lactobacillus bootsneri of claim 1 or a culture thereof. The fermented product according to claim 6, wherein the fermented product is kimchi. The method for producing a fermented product using the Lactobacillus bootsneri or its culture as a starter of claim 1. The method of claim 9, wherein the fermented product is kimchi. The method of culturing Lactobacillus bootsneri, characterized in that the culture for 10 to 30 ℃, 24 to 60 hours in a medium consisting of carbon source, nitrogen source, vitamins and minerals.

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