KR101575119B1 - An improving agent for preservation of Lactobacillus sp lactic acid bacterium comprising L. plantarum BBG L30 and preparation method of the same - Google Patents
An improving agent for preservation of Lactobacillus sp lactic acid bacterium comprising L. plantarum BBG L30 and preparation method of the same Download PDFInfo
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Abstract
Description
본 발명은 병원성 미생물의 항균활성을 가진 신규한 Lactobacillus plantarum BBG L30을 포함하는 Lactobacillus 속 유산균의 보존성을 증진시킨 보존제 및 그 제조방법에 관한 것이다.
The present invention relates to a preservative enhancing the preservability of Lactobacillus sp. Lactic acid bacteria comprising a novel Lactobacillus plantarum BBG L30 having antimicrobial activity against pathogenic microorganisms, and a method for preparing the same.
유산균(lactic acid bacterium)은 사람이나 동물의 장(腸)과 발효식품 등에서도 쉽게 발견되는 미생물로서 다양한 효능을 가지고 있는 안전한 미생물로(Orrhge, K. et al., 2000, Bifidobacteria and lactobacilli in human health, Drugs Exptl. Clin. Res., 26, 95-111). 장내 균총의 안정화, 유해세균의 정착 억제에 따른 부패산물 생성 감소 및 질병 예방, 면역 활성화 작용, 항암작용, 콜레스테롤 저하 등 숙주동물에 많은 도움을 준다. 유산균이 여러 부패성 미생물 및 병원성 미생물에 대하여 생육억제 작용을 갖는 것은 몇 가지 대사적인 특성 때문인데 젖산균은 대사산물로서 항균활성 인자인 organic acid, hydrogen peroxide, reuterin, diacetyl, acetaldehyde, bacteriocin 등을 생산하기 때문이다(Fuller, K., 1989, Probiotics in man and animals, J. Appl. Bacteriol., 66, 365-378).Lactic acid bacterium is a safe microorganism that can be easily found in the intestines and fermented foods of humans and animals. It is a safe microorganism with various efficacies (Orrhge, K. et al., 2000, Bifidobacteria and lactobacilli in human health , Drugs Expt. Clin. Res., 26, 95-111). It is very helpful for host animal such as stabilization of intestinal microflora, reduction of decay product formation due to inhibition of harmful bacteria, prevention of diseases, immune activation, anticancer effect and cholesterol lowering. Lactic acid bacteria have several metabolic characteristics that inhibit the growth of various pathogenic microorganisms and pathogenic microorganisms because lactic acid bacteria produce organic acid, hydrogen peroxide, reuterin, diacetyl, acetaldehyde, bacteriocin, etc. as metabolic products (Fuller, K., 1989, Probiotics in man and animals, J. Appl. Bacteriol., 66, 365-378).
특히 전통발효 식품에 주로 존재하는 Lactobacillus plantarum 유산균은 발효 중 젖산을 생성하여 유해한 미생물의 성장을 저해하고 식품의 풍미를 증진시키며 다양한 생리활성물질을 생산하는 것으로 알려져 있다(Ammor, M. S. and B. Mayo, Selection criteria for lactic acid bacteria to be used as functional starter cultures in dry sausage production: an update. Meat Sci., 138-1462, 2007). 또한, Lactobacillus plantarum은 식품의약품안전청에서 건강기능성 프로바이오틱스로 분류되어 인체 건강에 유익한 유산균으로 분류되며 Lactobacillus plantarum을 고지혈 마우스에 급여한 결과, 분변에서 유산균수가 증가하였고 혈청 내 콜레스테롤 수치를 감소시켰으며(Nguyen, T. D. et al., Characteristics of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects. Int. J. Food Microbiol, 113, 358-361, 2007) Lactobacillus plantarum AF1이 생성하는 항균 물질에 의한 항미생물 활성 범위는 항진균 활성 외에도 식중독균주를 포함한 그람 양성 및 음성 세균들에 강한 저해활성이 나타났다(Bae, M. S. and S. C. Lee, Preparation and characteristics of Kimchi with added styela clava. Korean J. Food Cookery, Sci., 24, 573-579, 2008).In particular, Lactobacillus plantarum lactic acid bacteria, which are mainly present in traditional fermented foods, are known to produce lactic acid during fermentation, thereby inhibiting the growth of harmful microorganisms, enhancing flavor of foods, and producing various physiologically active substances (Ammor, MS and B. Mayo, Selection criteria for lactic acid bacteria to be used as starter cultures in dry sausage production: an update. Meat Sci., 138-1462, 2007). Lactobacillus plantarum was classified as a healthy functional probiotics by the Korea Food & Drug Administration (KFDA) and classified as a beneficial lactic acid bacteria. Lactobacillus plantarum was fed to hyperlipidemic mice, resulting in an increase in the number of lactic acid bacteria in feces and a decrease in serum cholesterol levels (Nguyen, Antimicrobial activity of Lactobacillus plantarum AF1 produced by antimicrobials ( Lactobacillus plantarum AF1, TD, et al., Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects) In addition to antifungal activity, Gram-positive and negative bacteria, including food poisoning bacteria, showed strong inhibitory activity (Bae, MS and SC Lee, Preparation and characteristics of kimchi with added styela clava. -579, 2008).
한편, 유산균 Lactobacillus plantarum을 산업적으로 대량 생산하기 위해서는 저렴하면서 위생적이고 보존성이 증진된 배지가 절대적으로 필요하다. 하지만, 유산균 배양에는 고가의 MRS broth가 주로 사용되고 있으며 Lactobacillus plantarum 유산균을 효율적으로 배양할 수 있는 맞춤형 배지에 대한 기술적 진보는 매우 미흡한 실정이다.On the other hand, in order to industrially mass produce the lactic acid bacteria Lactobacillus plantarum , it is absolutely necessary to provide a medium which is inexpensive, hygienic and improved in preservability. However, expensive MRS broth is mainly used for culturing lactic acid bacteria, and technological advances for a customized culture medium capable of efficiently culturing Lactobacillus plantarum lactic acid bacteria are insufficient.
Lactobacillus plantarum의 관한 선행기술로서 배지조성물이 대한민국 등록특허 제10-0818360호에 개시된 바 있고, 식물성 배지에서 Lactobacillus plantarum의 배양을 위한 배지 최적화가 정은지 외 7명의 연구논문(Korean Society for Biotechnology and Bioengineering Journal 27, 347-351, 2012)에 개시된 바 있다. Lactobacillus plantarum BBG L30 균주와 관련된 본 발명자들의 선출원 대한민국 등록특허 제10-1431250호에는 균주의 분리방법이 개시되어 있다. 그러나 상기문헌 어디에도 본 발명자들이 분리한 신규 Lactobacillus plantarum BBG L30 균주를 포함하는 Lactobacillus 속 유산균의 보존성을 증진시킨 배지조성물 및 그 제조방법에 대하여는 개시된 바 없다.
As a prior art related to Lactobacillus plantarum , a medium composition has been disclosed in Korean Patent No. 10-0818360, and the medium optimization for the cultivation of Lactobacillus plantarum in a vegetable medium is described in Jung Eunji et al., 7 papers (Korean Society for Biotechnology and Bioengineering Journal 27 , ≪ / RTI > 347-351, 2012). Korean Patent No. 10-1431250 discloses a method for isolating a strain, which is related to Lactobacillus plantarum BBG L30. However, in the above-mentioned literatures, the Lactobacillus genus containing the novel Lactobacillus plantarum BBG L30 strain isolated by the present inventors A culture medium in which the preservability of a lactic acid bacterium is enhanced and a preparation method thereof have not been disclosed.
따라서 본 발명의 목적은 병원성 미생물의 항균활성을 가지는 신규한 락토바실러스 플란테리움 BBG L30 균주를 선별 분리하고 그 보전성을 증진시킨 신규한 배지 조성물을 제공하는데 있다.Accordingly, an object of the present invention is to provide a novel culture composition in which a novel Lactobacillus plantarum BBG L30 strain having antimicrobial activity of a pathogenic microorganism is selectively isolated and its integrity is improved.
본 발명의 다른 목적은 상기 락토바실러스 플란테리움 BBG L30 균주를 포함하는 Lactobacillus 속 유산균의 보존성을 증진시킨 보존제 조성물을 제공하는데 있다.
Another object of the present invention is to provide a preservative composition for enhancing the shelf life of Lactobacillus sp. Lactic acid bacteria comprising the Lactobacillus plantarum BBG L30 strain.
본 발명의 상기 목적은 병원성 미생물에 대하여 항균활성을 가지는 유용한 균주를 선별하는 단계와; 상기 선별된 균주의 형태적, 물리적 특성을 확인하는 단계와; 상기 선별된 균주를 동정하는 단계와; 상기 선별된 균주 배양액에 보존제를 첨가하여 균주의 보존성을 확인하는 단계와; 상기 선별된 균주 배양액에 보존제 첨가농도, 저장온도, 저장시간에 따른 균수 변화를 측정하는 단계를 통하여 달성하였다.
The above object of the present invention can be achieved by a method for screening a pathogenic microorganism, which comprises selecting a useful strain having an antimicrobial activity against a pathogenic microorganism; Confirming the morphological and physical characteristics of the selected strain; Identifying the selected strain; Adding a preservative to the selected culture medium to confirm the preservation of the culture; The concentration of the preservative, the storage temperature, and the storage time of the selected strains were measured.
본 발명은 병원성 미생물의 항균활성을 가진 신규한 Lactobacillus plantarum BBG L30 균주를 제공하는 효과가 있을 뿐 아니라 Lactobacillus 속 유산균의 보존성을 증진시킨 보존제 조성물을 제공하는 뛰어난 효과가 있다.
The present invention, as well as the effect to provide a novel Lactobacillus plantarum strain L30 BBG with the antimicrobial activity of pathogenic microorganisms in Lactobacillus There is an excellent effect of providing a preservative composition having improved preservability of lactic acid bacteria.
도 1은 본 발명 Lactobacillus 속 신규한 균주 BBG L30의 유전자 염기서열 분석결과를 바탕으로 L30 균주가 락토바실러스 플란테리움 균주임을 나타내는 계통모식도이다.
도 2는 본 발명 Lactobacillus plantarum BBG L30의 단일균주 배양액에 sodium malate 첨가량과 저장기간에 따른 유산균주의 변화를 나타낸 그래프이다.BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a systematic diagram showing that the L30 strain is a Lactobacillus plantarum strain based on the result of a gene sequencing analysis of a novel strain BBG L30 of the genus Lactobacillus of the present invention. FIG.
Fig. 2 is a photograph of the Lactobacillus plantarum BBG L30 of the present invention This is a graph showing the change of lactic acid bacteria depending on the amount of sodium malate added and the storage period in a single strain culture solution.
이하, 본 발명을 실시예와 도면에 의거 더욱 상세하게 설명한다. 그러나 하기 실시예는 본 발명을 예시하기 위한 것에 불과하며 본 발명의 권리범위를 한정하는 것으로 의도되지는 않는다.
Hereinafter, the present invention will be described in more detail with reference to embodiments and drawings. However, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
<실험예 1> 새로운 균주의 분리<Experimental Example 1> Separation of a new strain
(1) 균주 분리 및 배양(1) Isolation and Culture of Strain
본 발명자들이 보유한 균주와 새로운 균주 분리를 위해 산토양, 하우스토양, 돼지분변 등에서 각각 시료를 채취한 후 시료 1 g에 멸균된 생리식염수(0.85% NaCl) 9 mL에 현탁 시킨후 MRS agar 배지에 103 ~ 106 희석한 희석액 0.1 mL를 첨가하여 유리봉으로 도말하여 2일 배양한 후 항균활성 균주를 순수 분리하였다.
In order to isolate a new strain from the strains possessed by the present inventors, samples were collected from mountain soils, house soil, and pig feces, and suspended in 9 mL of sterilized physiological saline (0.85% NaCl) in 1 g of the sample. After adding 0.1 mL of diluted diluted solution ( 3 ~ 10 6), it was plated with a glass rod and incubated for 2 days. Then, the antimicrobial active strain was purified.
(2) 항균활성 검정(2) Antimicrobial activity test
병원성 미생물 Salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium은 LB(Luria Bertani media broth) agar 배지에 35℃에서 계대배양하여 사용하였다. 상기 분리한 균주의 병원성 미생물의 항균활성을 통한 균주 선발을 하였다. 상기 계대배양한 병원성 미생물 Salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium을 LB 배지에 전 배양하여, LB agar에 현탁액(1.0×106 cfu/ml) 10 μL를 퍼지지 않게 주입하고 도말한 후 직경 5 mm의 Cokr Borer를 이용하여 구멍을 내었다. 상기 구멍에 MRS 배지에서 1일 배양한 배양액을 시린지 필터(0.2 μL)로 여과하여 각각 200 μL를 주입하고 35℃에서 20시간 배양후에 clear zone의 지름을 측정하였다.The pathogenic microorganisms Salmonella gallinarum , Salmonella pullorum , and Salmonella typhimurium were used in subculture on LB (Luria Bertani media broth) agar medium at 35 ℃. The strains were selected through the antimicrobial activity of the pathogenic microorganisms of the isolated strains. The pathogenic microorganisms Salmonella gallinarum , Salmonella pullorum , and Salmonella typhimurium were pre-cultured in LB medium, and 10 μL of suspension (1.0 × 10 6 cfu / ml) was injected into LB agar without spreading. Borer was used to drill holes. The culture was cultured in MRS medium for 1 day. The sperm was filtered with a syringe filter (0.2 μL), and 200 μL of each was injected. The diameter of the clear zone was measured after culturing at 35 ° C. for 20 hours.
실험결과, [표 1]에 나타낸 바와 같이 Salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium의 3종에 대해 모두 항균활성이 나타나는 것은 BBG L1, BBG L30, BBG L39 균주로 나타났으며 이 중에서 BBG L30 균주를 분리하였다.
As shown in Table 1, all of the three strains of Salmonella gallinarum , Salmonella pullorum, and Salmonella typhimurium were found to be BBG L1, BBG L30, and BBG L39 strains. Among them, BBG L30 strains were isolated Respectively.
NoBBG
No
(mm) Salmonella gallinarum
(mm)
(mm) Salmonella pullorum
(mm)
(mm) Salmonella typhimurium
(mm)
[주] L : lactic acid bacteria[Note] L: lactic acid bacteria
<실험예 2> 본 발명 신규한 ≪ Experimental Example 2 > L. plantarum L. plantarum BBG L30 균주의 항균 스펙트럼 조사Antimicrobial spectrum of BBG L30 strain
(1) 병원성 미생물의 배양(1) Culture of pathogenic microorganisms
병원성 미생물 Bacillus cereus, Candida albicans, Clostridium perfrigens, Escherichia coli, Haemophillus parasuis, Haemophillus simmunus, Listeria monocytogens, Mammheimia haemolytica type A, Pseudomonas aeruginosa, Pasteurella multocida type A, Salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium Staphylococcus aereus를 LB agar에 35℃에서 계대배양하여 사용하였다.
The pathogenic microorganisms Bacillus cereus, Candida albicans, Clostridium perfringens, Escherichia coli, Haemophillus parasuis, Haemophillus simmunus, Listeria monocytogens, Mammheimia haemolytica type A, Pseudomonas aeruginosa, Pasteurella multocida type A, Salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium Staphylococcus aereus were cultured on LB agar Followed by subculture at 35 ° C.
(2) 본 발명 L. plantarum BBG L30 균주의 항균 스펙트럼 조사(2) Antimicrobial spectrum of L. plantarum BBG L30 strain of the present invention
상기 계대배양한 병원성 미생물 Bacillus cereus, Candida albicans, Clostridium perfrigens, Escherichia coli, Haemophillus parasuis, Haemophillus simmunus, Listeria monocytogens, Mammheimia haemolytica type A, Pseudomonas aeruginosa, Pasteurella multocida type A, Staphylococcus aereus을 LB 배지에 전 배양하여, LB agar에 현탁액(1.0×106 cfu/ml) 10 μL를 퍼지지 않게 주입하고 도말한 후 직경 5 mm의 Cokr Borer를 이용하여 구멍을 내었다. 상기 구멍에 MRS 배지에서 1일 배양한 락토바실러스 플란테리움 BBG L30 균주 배양액을 시린지 필터(0.2 μL)로 여과하여 각각 200 μL를 주입하고 35℃에서 20시간 배양후에 clear zone의 지름을 측정하였다.The pathogenic microorganisms Bacillus cereus, Candida albicans , Clostridium perfigens , Escherichia coli , Haemophillus parasuis , Haemophillus simmunus , Listeria monocytogenes, Mammheimia haemolytica type A, Pseudomonas aeruginosa , Pasteurella multocida type A and Staphylococcus aereus were pre- 10 μL of the suspension (1.0 × 10 6 cfu / ml) was injected into the LB agar without spreading, and after pouring, holes were made with a 5 mm diameter Cokr Borer. The culture broth of Lactobacillus plantarum BBG L30 cultured in MRS medium for one day was filtered with a syringe filter (0.2 μL), and 200 μL of each was injected into the well. After the incubation at 35 ° C. for 20 hours, the clear zone diameter was measured.
병원성 미생물인 Bacillus cereus, Candida albicans, Clostridium perfrigens, Escherichia coli, Haemophillus parasuis, Haemophillus simmunus, Listeria monocytogens, Mammheimia haemolytica type A, Pseudomonas aeruginosa, Pasteurella multocida type A에 대하여 본 발명 BBG L30 균주의 항균 스펙트럼을 조사하였다.Antimicrobial spectrum of the present invention BBG L30 strain was investigated against pathogenic microorganisms such as Bacillus cereus, Candida albicans, Clostridium perfringens, Escherichia coli, Haemophilus parasuis, Haemophillus simmunus, Listeria monocytogens, Mammheimia haemolytica type A , Pseudomonas aeruginosa and Pasteurella multocida type A.
실험결과 [표 2]에서 나타낸 바와 같이 본 발명 L. plantarum BBG L30 균주의 Bacillus cereus, Candida albicans를 제외한 9종류의 병원성 미생물에 대해 항균효과가 높게 나타남을 확인하였다.
As shown in Table 2 , it was confirmed that the antimicrobial effect of 9 kinds of pathogenic microorganisms except Lactobacillus plantarum BBG L30 of the present invention except Bacillus cereus and Candida albicans was high.
[주] 0: no inhibition, +: very slight inhibition(1-5mm), ++: moderate inhibition(6-10), +++: heavy inhibition(11- 15mm)+++: heavy inhibition (11- 15mm), ++: moderate inhibition (-6-10), +++: heavy inhibition
<실험예 3> 본 발명 ≪ Experimental Example 3 > L. plantarum L. plantarum BBG L30 균주의 특성 및 동정Characterization and identification of BBG L30 strain
(1) 본 발명 균주의 특성(1) Characteristic of the strain of the present invention
본 발명 L. plantarum BBG L30 균주의 생태학적 특성은 Bergey's Mannual of Systematic Bacteriology를 참고하여 조사하였으며 생화학적 실험은 API50 CHL medium Kit system을 이용하여 조사하였고, 16S rDNA 유전자 염기서열을 분석하였다.The ecological characteristics of the L. plantarum BBG L30 strain of the present invention were examined with reference to Bergey's Mannual of Systematic Bacteriology. Biochemical experiments were carried out using an API50 CHL medium kit system, and 16S rDNA gene sequences were analyzed.
실험결과, [표 3]에 나타낸 바와 같이 BBG L30 균주는 gram(+) 균으로 판명되었으며, 분리된 유산균주는 citrate를 이용하였으며 glucose로부터 산을 분비하였다.
As shown in Table 3, the BBG L30 strain was identified as gram (+) bacterium. The isolated lactic acid bacteria were citrate and secreted acid from glucose.
(2) 본 발명 균주의 당 이용성 특성(2) The glycosylation property of the strain of the present invention
당이용성 실험은 API kit system을 이용하여 조사하였으며, 본 발명 L. plantarum BBG L30 균주는 glucose 및 galactose와 같은 다양한 유기물을 이용하는 것으로 나타났다(표 4).The glucose tolerance test was conducted using an API kit system, and the L. plantarum BBG L30 strain of the present invention was found to use various organic substances such as glucose and galactose (Table 4).
(3) 본 발명 균주의 동정(3) Identification of the strain of the present invention
본 발명 신규한 L. plantarum BBG L30 균주를 2일간 배양하여 Benzyl chloride법을 변형한 방법을 이용하여 DNA를 추출하였다. 16S rDNA의 PCR 증폭산물은 PCR Product Purification Kit(Qiagen)를 사용하여 정제하였으며, PCR 정제산물은 Genetic analyer 310A(Applied Biosystems)을 사용하여 염기서열을 분석하였다. 염기서열은 NCBI/Genebank와 Ribosomal Database Project II(RDP II)의 database에서 상동성 검색을 수행하고, CLUSTAL X 프로그램(Thompson et al., 1994) 및 PHYLIP 프로그램(Felsenstein, 1993)을 이용하여 계통학적 위치를 확인하였으며, 균주를 최종적으로 동정하였다.The DNA of the novel L. plantarum BBG L30 strain of the present invention was cultured for 2 days and then transformed with the benzyl chloride method. The 16S rDNA PCR products were purified using the PCR Product Purification Kit (Qiagen). The PCR products were analyzed using a genetic analyzer 310A (Applied Biosystems). The nucleotide sequence was used to perform homology searches in NCBI / Genebank and Ribosomal Database Project II (RDP II) databases and to identify phylogenetic sites using the CLUSTAL X program (Thompson et al., 1994) and the PHYLIP program (Felsenstein, 1993) And the strain was finally identified.
16s rDNA 균주 동정결과 도 1에서 보는 바와 같이 BBG L30 균주는 Lactobacillus plantarum으로 동정되었으며, 따라서 균주 이름을 L. plantarum BBG L30로 명명하였다. 상기 본 발명 균주는 농업생명공학연구원 유전자 은행에 2014년 05월 28일자 특허균주로 기탁하였다(KACC91952P).
As shown in Fig. 1, the BBG L30 strain was identified as Lactobacillus plantarum , and thus the name of the strain was named L. plantarum BBG L30. The strain of the present invention was deposited at the GenBank Institute for Agricultural Biotechnology as a patented strain on May 28, 2014 (KACC91952P).
<실험예 4> 본 발명 ≪ Experimental Example 4 > L. plantarumL. plantarum BBG L30 균주 배양액의 보존성 향상 실험 Conservation improvement experiment of BBG L30 strain culture
본 발명 항균활성이 강한 L. plantarum BBG L30 균주를 배양한 액에 citric acid, carboxymethyl cellulose, dextrin, fructose, galactose, glycerol, glucose, lactose, mannose, mineral oil, potassium hydroxide, potassium sodium tatrate, soluble starch, sodium acetate, sodium alginate, sodium hyroxide, sodium malate, sodium succinate, soluble starch, trehalose를 총 중량대비 각 0.5, 2.0중량부를 첨가하여 25℃에서 30일 보관후에 미생물 생균수 변화를 측정하여 이를 생존률(%)로 표시하였다.Lactic acid, mannose, mineral oil, potassium hydroxide, potassium sodium tatrate, soluble starch, lactic acid, dextrin, fructose, galactose, glycerol, glucose, lactose, and lactose in a culture of L. plantarum BBG L30, sodium alginate, sodium hy- droxide, sodium malate, sodium succinate, soluble starch and trehalose were added to each 0.5, 2.0 parts by weight of the total weight. After storage for 30 days at 25 ° C, the viable cell counts were measured, Respectively.
실험결과, [표 5]에 나타낸 바와 같이 본 발명 L. plantarum BBG L30 균주 배양액에 아무것도 첨가하지 않고 본 발명 첨가물 대신 같은 양의 물을 첨가한 대조군은 30일 후 생존률이 0.80%(control)로 매우 낮게 나타났다. 균주 보존 효과가 가장 뛰어난 것은 소디움 말레이트(sodium malate)로 6.60%의 생존률을 나타냈으며, 프럭토오스, 만노스, 소디움 아세테이트, 소디움 석시네이트 등이 무첨가군 보다 약 8배의 효과가 나타났고 이 중 가장 바람직한 유산균주 액상배양액 보존제로서 sodium malate 첨가 농도는 2중량부로 결정하였다.
As shown in Table 5, the control group to which the same amount of water was added instead of the additive of the present invention without adding anything to the L. plantarum BBG L30 culture medium of the present invention showed a survival rate of 0.80% (control) after 30 days Respectively. Sodium malate showed the highest survival rate of 6.60%, and fructose, mannose, sodium acetate and sodium succinate showed about 8 times more effective than non - additive group. The most preferred concentration of sodium malate as a preservative for the lactic acid bacterium liquid culture medium was determined to be 2 parts by weight.
<실험예 5> 본 발명 신규한 ≪ Experimental Example 5 > L. plantarumL. plantarum BBG L30 균주 배양액에 보존제 첨가후 온도 및 시간 차이에 의한 생균수 변화 Changes in the number of viable cells due to temperature and time difference after adding preservative to BBG L30 strain culture
본 발명에 따라 제품 생산 시 저장 온도와 저장기간에 따른 생균수 변화가 나타날 것으로 판단 되어 보존성이 가장 우수한 sodium malate 배양액 총 중량대비 2중량부를 첨가하여 온도변화(4, 10, 18, 25, 35℃)와 시간변화(7, 14, 21, 28일)에 따른 생균수를 체크를 하여 역시 생존률(%)을 측정하였다.According to the present invention, 2 parts by weight of the total weight of the sodium malate culture solution having the best preservability is added, and the temperature changes (4, 10, 18, 25, 35 ° C ) And the number of viable cells according to time changes (7, 14, 21, 28 days) were also checked to determine survival rate (%).
실험결과, [표 6]에서 나타낸 바와 같이 Lactobacillus plantarum BBG L30 균주를 18℃ 이하에서 보관한 시료는 25℃ 이상에서 보관한 시료보다 생존률이 높았으며 무처리구에 비하여 총 중량대비 sodium malate 2중량부 처리구가 생존률을 높았다.
As shown in Table 6, the survival rate of Lactobacillus plantarum BBG L30 strain stored at 18 ° C or lower was higher than that stored at 25 ° C or higher. In comparison with the untreated control, 2% The survival rate was high.
[주] [week]
ND: not detected, 10ND: not detected, 10
33
cfu/ml.cfu / ml.
<실험예 6> 본 발명 신규한 ≪ Experimental Example 6 > L. plantarumL. plantarum BBG L30 균주 배양액에 보존제 첨가 후 농도 및 시간 차이에 따른 생균수 변화 Changes in the number of viable cells by concentration and time difference after adding preservative to BBG L30 strain
본 발명 L. plantarum BBG L30 균주의 보존효과 확인 및 최적농도 설정을 위하여, 배양액 총 중량대비 sodium malate를 0, 1, 2, 3, 4, 5중량부를 첨가하여 0, 7, 14, 21, 28, 60, 90, 120, 240일 동안 확인하였다.1, 2, 3, 4 and 5 parts by weight of sodium malate were added to the total weight of the culture broth to determine the preservation effect and optimum concentration of L. plantarum BBG L30 strain of the present invention at 0, 7, 14, 21, 28 , 60, 90, 120, and 240 days.
실험결과, 도 2에 나타낸 바와 같이 유산균주의 균체농도(log CFU/ml)는 sodium malate 첨가량이 증가할수록 유산균주의 보존효과는 증가하는 것을 확인하였으나 배양액 총 중량대비 10중량부 이상 높아져도 유산균주에 보존효과가 증가하지 않는 것을 확인하였다.As shown in FIG. 2, the concentration of lactic acid bacteria (log CFU / ml) increased as the amount of sodium malate was increased, but the preservation effect of the lactic acid bacteria was increased. However, It was confirmed that the effect did not increase.
따라서, 보존효과가 좋은 sodium malate를 유산균주 배양액 및 액상제품에 총 중량대비 1.0 ~ 5.0중량부 처리시 보존 효과를 극대화되었으며, 10℃이하의 저온 저장시 가장 바람직하였다.
Therefore, the preservation effect of sodium malate, which has good preservation effect, was maximized when 1.0 to 5.0 parts by weight of lactic acid bacteria culture liquid and liquid product were added to the total weight.
<비교예 1> ≪ Comparative Example 1 & Lactobacillus 속 Lactobacillus genus 유산균주 배양액에 본 발명 보존제 첨가 후 생균수 변화Changes in the number of viable cells after adding the preservative of the present invention to the lactic acid bacteria culture medium
본 발명과 동일하게 Lactobacillus brevis HLJ59 유산균주 배양액에 총 중량대비 5중량부의 sodium malate를 첨가한 후 10℃에서 0, 30, 60, 90, 120, 240일 동안 생균수 변화를 측정하였다.In the same manner as in the present invention, 5% by weight of sodium malate was added to the Lactobacillus brevis HLJ59 lactic acid bacteria culture medium, and then the change in viable cell counts was measured at 10 ° C for 0, 30, 60, 90, 120 and 240 days.
실험결과, [표 7]에 나타낸 바와 같이 본 발명 보존제는 Lactobacillus brevis HLJ59 유산균주에 대한 보존효과도 유의적으로 높게 나타나는 것을 확인하였다.
As a result, as shown in Table 7, it was confirmed that the preservative of the present invention had a significantly higher preservation effect on Lactobacillus brevis HLJ59 lactic acid bacteria.
<실시예 1> 본 발명 유산균주의 보존제 조성물 제조Example 1 Preparation of preservative composition for lactic acid bacteria of the present invention
Salmonella gallinarum 외 병원성 미생물에 대한 항균 효과가 높은 본 발명 Lactobacillus plantarum BBG L30 균주와 Lactobacillus brevis HLJ59 균주를 MRS 배지에서 각각 1.0×109cfu/ml로 배양하였다. 상기 배양액에 총 중량대비 sodium malate 1.0 ~ 5.0중량부를 첨가하여 본 발명 보존제가 첨가된 보존제 조성물을 제조하였다.
In the present invention, which has high antimicrobial activity against Salmonella gallinarum and pathogenic microorganisms, Lactobacillus plantarum BBG L30 strain and Lactobacillus brevis HLJ59 strain were cultured on MRS medium at 1.0 × 10 9 cfu / ml, respectively. 1.0 to 5.0 parts by weight of sodium malate was added to the above culture solution to prepare a preservative composition to which the preservative of the present invention was added.
본 발명은 병원성 미생물의 항균활성을 가진 신규한 락토바실러스 플란테리움 BBG L30 균주를 제공하는 효과가 있을 뿐 아니라, 상기 유산균주의 보존제 조성물을 제공하여 Lactobacillus plantarum BBG L30 균주를 포함하는 다양한 Lactobacillus 속 유산균주의 배양액 뿐만 아니라 액상제품의 저장기간을 증진시켜 기능적, 품질 및 측면에서 유산균주의 가치를 향상시킬 수 있는 뛰어난 효과가 있으므로 식품산업상 매우 유용한 발명인 것이다.
The present invention in a variety of Lactobacillus containing a novel Lactobacillus strain Lactobacillus plantarum L30 flange rim Solarium BBG BBG L30 strain, as well as the effect, to provide the lactic acid preservative composition which provides care with the antimicrobial activity of pathogenic microorganisms It is an extremely useful invention in the food industry because it has an excellent effect of improving the storage period of the liquid product as well as the culture medium of the lactic acid bacteria and improving the value of the lactic acid bacteria in terms of function, quality and aspect.
<110> BIGBIOGEN CO., LTD <120> An improving agent for preservation of Lactobacillus sp lactic acid bacterium comprising L. plantarum BBG L30 and preparation method of the same <130> 01 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1461 <212> DNA <213> Lactobacillus plantarum BBG L30 <400> 1 ggacggcgtg ctataatgca gtcgaacgaa ctctggtatt gattggtgct tgcatcatga 60 tttacatttg agtgagtggc gaactggtga gtaacacgtg ggaaacctgc ccagaagcgg 120 gggataacac ctggaaacag atgctaatac cgcataacaa cttggaccgc atggtccgag 180 cttgaaagat ggcttcggct atcacttttg gatggtcccg cggcgtatta gctagatggt 240 ggggtaacgg ctcaccatgg caatgatacg tagccgacct gagagggtaa tcggccacat 300 tgggactgag acacggccca aactcctacg ggaggcagca gtagggaatc ttccacaatg 360 gacgaaagtc tgatggagca acgccgcgtg agtgaagaag ggtttcggct cgtaaaactc 420 tgttgttaaa gaagaacata tctgagagta actgttcagg tattgacggt atttaaccag 480 aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc 540 ggatttattg ggcgtaaagc gagcgcaggc ggttttttaa gtctgatgtg aaagccttcg 600 gctcaaccga agaagtgcat cggaaactgg gaaacttgag tgcagaagag gacagtggaa 660 ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct 720 gtctggtctg taactgacgc tgaggctcga aagtatgggt agcaaacagg attagatacc 780 ctggtagtcc ataccgtaaa cgatgaatgc taagtgttgg agggtttccg cccttcagtg 840 ctgcagctaa cgcattaagc attccgcctg gggagtacgg ccgcaaggct gaaactcaaa 900 ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa 960 gaaccttacc aggtcttgac atactatgca aatctaagag attagacgtt cccttcgggg 1020 acatggatac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080 cccgcaacga gcgcaaccct tattatcagt tgccagcatt aagttgggca ctctggtgag 1140 actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200 cctgggctac acacgtgcta caatggatgg tacaacgagt tgcgaactcg cgagagtaag 1260 ctaatctctt aaagccattc tcagttcgga ttgtaggctg caactcgcct acatgaagtc 1320 ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380 caccgcccgt cacaccatga gagtttgtaa cacccaaagt cggtggggta accttttagg 1440 aaccagccgc ctaagtgaca g 1461 <110> BIGBIOGEN CO., LTD <120> An improving agent for preservation of Lactobacillus sp lactic acid bacterium comprising L. plantarum BBG L30 and preparation method of the same <130> 01 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1461 <212> DNA <213> Lactobacillus plantarum BBG L30 <400> 1 ggacggcgtg ctataatgca gtcgaacgaa ctctggtatt gattggtgct tgcatcatga 60 tttacatttg agtgagtggc gaactggtga gtaacacgtg ggaaacctgc ccagaagcgg 120 gggataacac ctggaaacag atgctaatac cgcataacaa cttggaccgc atggtccgag 180 cttgaaagat ggcttcggct atcacttttg gatggtcccg cggcgtatta gctagatggt 240 ggggtaacgg ctcaccatgg caatgatacg tagccgacct gagagggtaa tcggccacat 300 tgggactgag acacggccca aactcctacg ggaggcagca gtagggaatc ttccacaatg 360 gggaaagtc tgatggagca acgccgcgtg agtgaagaag ggtttcggct cgtaaaactc 420 tgttgttaaa gaagaacata tctgagagta actgttcagg tattgacggt atttaaccag 480 aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc 540 ggatttattg ggcgtaaagc gagcgcaggc ggttttttaa gtctgatgtg aaagccttcg 600 gctcaaccga agaagtgcat cggaaactgg gaaacttgag tgcagaagag gacagtggaa 660 ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct 720 gtctggtctg taactgacgc tgaggctcga aagtatgggt agcaaacagg attagatacc 780 ctggtagtcc ataccgtaaa cgatgaatgc taagtgttgg agggtttccg cccttcagtg 840 ctgcagctaa cgcattaagc attccgcctg gggagtacgg ccgcaaggct gaaactcaaa 900 ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa 960 gaaccttacc aggtcttgac atactatgca aatctaagag attagacgtt cccttcgggg 1020 acatggatac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080 cccgcaacga gcgcaaccct tattatcagt tgccagcatt aagttgggca ctctggtgag 1140 actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200 cctgggctac acacgtgcta caatggatgg tacaacgagt tgcgaactcg cgagagtaag 1260 ctaatctctt aaagccattc tcagttcgga ttgtaggctg caactcgcct acatgaagtc 1320 ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380 caccgcccgt cacaccatga gagtttgtaa cacccaaagt cggtggggta accttttagg 1440 aaccagccgc ctaagtgaca g 1461
Claims (6)
Lactobacillus plantarum Lactobacillus main culture medium or total, based on the weight of sodium acetate in the diluted solution, sodium succinate, sodium hydroxide and the sodium maleate any one of 1.0 to the Lactobacillus characterized in that the 5.0 parts by weight was added to prepare selected from plantarum Lactobacillus primary preservative Composition.
5. The Lactobacillus plantarum Lactobacillus strain preservative composition according to claim 4, wherein the Lactobacillus plantarum lactic acid bacteria is L. plantarum BBG L30 (KACC 91952P).
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