KR20090036343A - Method for using high osmotic pressure tolerant strain as natural flavor - Google Patents
Method for using high osmotic pressure tolerant strain as natural flavor Download PDFInfo
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- KR20090036343A KR20090036343A KR1020070101469A KR20070101469A KR20090036343A KR 20090036343 A KR20090036343 A KR 20090036343A KR 1020070101469 A KR1020070101469 A KR 1020070101469A KR 20070101469 A KR20070101469 A KR 20070101469A KR 20090036343 A KR20090036343 A KR 20090036343A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/88—Taste or flavour enhancing agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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Abstract
Description
본 발명은 락토바실러스 속 유산균에 속하는 내염성 균주를 천연 식품 첨가제로 사용하는 식품의 풍미 향상 방법에 관한 것으로, 더욱 상세하게는 내염성이고 글루타미네즈 활성이 40 U/㎎ 이상인 락토바실러스 속 유산균을 식품 제조과정에 첨가하는 단계를 포함하는 식품의 풍미 향상 방법에 관한 것이다.The present invention relates to a method for improving the flavor of food using a salt tolerant strain belonging to the Lactobacillus genus Lactobacillus as a natural food additive. It relates to a method for improving the flavor of food comprising the step of adding to the process.
현재 각종 식품 첨가제로 화학조미료인 MSG(Monosodium glutamate; 모노소듐 글루타민산염)가 널리 사용되고 있으며, 그 소비량은 꾸준히 증가하고 있다. 글루타민은 버섯, 육류, 김, 토마토 등 자연 식품에 단백질의 일부분으로 존재하며, 글루타민을 자연식품에서 섭취했을 때에는 부작용이나 병적 증세가 나타나지 않지만, 화학적인 방법으로 합성한 MSG를 식품 첨가제로 사용하였을 때 경우에 따라 독성을 띄기도 한다(Tiziana P et al ., Food Chemistry 104(4):1712-1717, 2007). 그리고 화학조미료(MSG)를 과다섭취함으로써, MSG에 민감한 사람들에게서 '중국 음식점 증후군(Chinese Restaurant Syndrome)', '암과의 관련성'(고온에서는 발암물질로 변 할 수 있음), '천식과의 관련성' 등이 나타난다고 보고된 바 있다. 이 같은 현상은 서구화된 식생활로 화학조미료의 사용이 늘어나면서, 영양섭취에 중요한 천연식품을 덜 사용하게 됨에 따라 나타나게 되었다.Currently, MSG (monosodium glutamate), a chemical seasoning, is widely used as a food additive, and its consumption is steadily increasing. Glutamine exists as a part of protein in natural foods such as mushrooms, meat, seaweed, and tomatoes, and no side effects or pathological symptoms occur when glutamine is consumed in natural foods.However, when chemically synthesized MSG is used as a food additive Sometimes toxic (Tiziana P et. al . , Food Chemistry 104 (4): 1712-1717, 2007). And by overingesting MSG, Chinese Restaurant Syndrome, cancer's association with cancer (which can turn into carcinogens at high temperatures) and asthma in people sensitive to MSG Has been reported. This phenomenon has emerged due to the increasing use of chemical seasonings in westernized diets and less natural foods that are important for nutrition.
이에, 최근 식품 첨가제나 성분으로서 인체 내 건강에 이로움을 주는 천연 식품 첨가제에 대한 관심이 대두하고 있다. 식생활의 서구화에 따라 각종 성인병 발병의 주요 원인이 식생활에 있다는 것이 밝혀지면서 생명체(동물, 식물, 미생물)와 효소로부터 인체의 생리기능을 향상시키는 기능성 물질을 찾는 연구가 함께 이루어지고 있다. 그 중에서 음식의 맛을 증진시킬 수 있는 상업적으로 중요한 효소로 알려져 있는 글루타미네이즈(glutaminase) 효소를 대표적인 예로 들 수 있다.Therefore, recently, interest in natural food additives that benefit human health as a food additive or ingredient has emerged. With the westernization of diet, it is revealed that the main cause of various adult diseases is in the diet, and research is being conducted to find functional substances that improve the physiological functions of the human body from living organisms (animals, plants, microorganisms) and enzymes. Among them, glutaminase enzyme, which is known as a commercially important enzyme that can enhance the taste of food, is a representative example.
글루타미네이즈는 L-글루타민을 L-글루타민산염으로 탈아민화시키는 촉매역할을 하며, 식품에 첨가하였을 때 맛을 증진시키는 효과를 가지고(Hodson & Linden, Physiology & Behavior 89(5):711-717, 2006), 일반적으로 간장과 같은 발효식품에 맛을 제공하는 중요한 효소로 알려져 있다. 상기 글루타민산염의 소듐 염 성분은 음식에 첨가하였을 때 맛을 내는 역할을 하며, 효모나 곰팡이뿐만 아니라 박테리아를 포함한 미생물 어디에나 포함되어있어, 발효식품의 첨가제로 널리 쓰인다(Naohisa M et al., J. Bioscience & Bioengineering, 2005). 글루타민산염 생성에 가장 큰 역할을 수행하는 글루타미네이즈는 대체로 온도나 열에 대한 안정성을 보이며, 염에 대한 내성이 높아 식품가공과정에서의 환경 변화 조건에 견딜 수 있기 때문에(Lapufade P et al ., Appl Environ Microbiol 64:2485-2489, 1998) 식품첨가제로서 사용이 가능하다. 글루타민의 물질대사에는 많은 효소들이 관계하 는데 그 중에서 글루타민산염 생성에서 글루타미네이즈가 가장 큰 역할을 한다.Glutaminase acts as a catalyst for the deamination of L-glutamine into L-glutamate and has a taste enhancing effect when added to foods (Hodson & Linden,Physiology & Behavior 89 (5): 711-717, 2006), generally known as important enzymes that provide taste to fermented foods such as soy. The sodium salt component of glutamate serves to taste when added to food, and is contained not only in yeast or mold but also in microorganisms including bacteria, and is widely used as an additive in fermented foods (Naohisa Met al.,J. Bioscience & Bioengineering, 2005). Glutaminase, which plays the biggest role in the production of glutamate, is generally stable to temperature and heat, and has high salt resistance, which can withstand environmental changes in food processing (Lapufade Pet al .,Appl Environ Microbiol 64: 2485-2489, 1998) as food additives. Many enzymes are involved in the metabolism of glutamine, of which glutamines play the largest role in the production of glutamate.
인간의 구강계, 소화계 및 비뇨 생식계의 정상균총으로 서식하는 락토바실러스 속 유산균(Lactobacillus spp.)은 젖산을 생성하여 pH를 산성화시켜 병원성을 무력화시키며, 락토바실러스 속 유산균에 의해 생성된 항생물질인 박테리오신(bacterocin)은 여러 가지 병원성 균의 생육을 억제한다(Goossens D et al ., Dig Dis Sci 37:44-50, 2005; Hiller SL et al ., Clin Infect Dis 16:S273-281, 1991; Karine V et al ., J Bacteriol 70:2057- 2064, 2002; Ouwehand AC et al ., Antonie van Leeuvenhoek 82:279-289, 2002). 이와 같은 특징을 갖는 락토바실러스 속 유산균은 인간과 동물의 식이요법 치료제와 정장제로 사용되는 프로바이오틱스(probiotics)로 많은 연구가 되어있다(Talarico TL et al ., Antimicrob Agents Chemother 32:1854-1858, 1988; Wolf BW et al., Food Chem Toxicol 36:1085-1094, 1998). Lactobacillus genus Lactobacillus inhabiting normal flora of human oral, digestive and urogenital systems spp.) produces lactic acid to acidify the pH to neutralize pathogenicity, and bacteriocin, an antibiotic produced by Lactobacillus lactic acid bacteria, inhibits the growth of various pathogenic bacteria (Goossens D et. al . , Dig Dis Sci 37: 44-50, 2005; Hiller sl et al . , Clin Infect Dis 16: S 273-281, 1991; Karine V et al . , J Bacteriol 70: 2057-2064, 2002; Ouwehand AC et al . , Antonie van Leeuvenhoek 82: 279-289, 2002). Lactic acid bacteria of the genus Lactobacillus having such characteristics have been studied as probiotics used as dietary treatments and formal medicines for humans and animals (Talarico TL et. al . , Antimicrob Agents Chemother 32: 1854-1858, 1988; Wolf BW et al. , Food Chem Toxicol 36: 1085-1094, 1998).
프로바이오틱스 균주의 대표적인 예로, 락토바실러스 플란타룸 ( Lactobacillus plantarum ), 락토바실러스 에시도필러스( Lactobacillus acidophilus ), 락토바실러스 헬베티쿠스 ( Lactobacillus helveticus ), 락토바실러스 불가리쿠 스( Lactobacillus bulgaricus ), 락토바실러스 람노수스 ( Lactobacillus rhamnosus ) 및 락토바실러스 델브루에키이( Lactobacillus delbrueckii ) 같은 몇몇 종은 염에 내성이 높고, 열에 대한 안정성도 높은 것으로 연구되었다(Erwin G et al., J Bacteriol 180:4718-4723, 1998; Jaya P et al ., Appl Envir Microbiol 69:917-925, 2003). 이에 락토바실러스 속 유산균은 동물의 사료, 거름, 우유 및 유제품뿐만 아니라 다양한 식품에 천연 첨가제로 사용되어 식품의 안정성, 점성 및 분산성 등에 중요한 효과를 제공하며, 동/식물성 식품의 섭취 시 저작에도 중요한 역할을 한다. 그 중에서 특히 락토바실러스 불가리쿠스를 이용한 유제품 산업이 많이 이용되고 있다.Representative examples of probiotic strains, Lactobacillus Planta Room ( Lactobacillus plantarum), Lactobacillus Ecija FIG filler's (Lactobacillus acidophilus ), Lactobacillus Helveticus (Lactobacillus helveticus ), Lactobacillus Bulgari ( Lactobacillus) bulgaricus ), Lactobacillus Ramno Seuss (Lactobacillus rhamnosus), and Lactobacillus del Brewer ekiyi (Lactobacillus Some species , such as delbrueckii ) , have been studied to be resistant to salts and to high thermal stability (Erwin G et al. , J Bacteriol 180: 4718-4723, 1998; Jaya P et. al . , Appl Envir Microbiol 69: 917-925, 2003). The lactobacillus genus Lactobacillus is used as a natural additive in a variety of foods, as well as animal feed, manure, milk and dairy products, providing important effects on food stability, viscosity and dispersibility, and important for chewing when ingesting animal / vegetable foods. Play a role. Among them, especially Lactobacillus The dairy industry using Bulgari is widely used.
이에 본 발명자들은 MSG를 대체할 미생물 유래의 글루타민산염을 생성하기 위해, 프로바이오틱스로 널리 알려져 이용되고 있는 락토바실러스 속 유산균으로부터 내염성이며 높은 글루타미네이즈 활성을 나타내는 균주들을 선별하고 상기 균주들의 최적활성 조건과 안정성을 측정함으로써 본 발명을 완성하였다.In order to produce glutamate derived from microorganisms to replace MSG, the present inventors screened strains showing saline and high glutamine activity from Lactobacillus sp. Lactobacillus genus, which is widely known and used as probiotics, The present invention was completed by measuring stability.
본 발명의 목적은 식품 내 글루타민산염을 생성하는 데 있어, 내염성이며 높은 글루타미네이즈 활성을 띄는 프로바이오틱스인 락토바실러즈 종을 천연 식품 첨가제로 이용하는 식품을 제조하는 방법을 제공하는 것이다.SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing a food product using lactobacillus species, which is a probiotic that is salt-tolerant and has high glutamine activity in producing glutamate in food, as a natural food additive.
상기 목적을 달성하기 위하여, 본 발명은 내염성이고 글루타미네즈 활성이 40 U/㎎ 이상인 락토바실러스 속 유산균을 식품 제조과정에 첨가하는 단계를 포함하는 식품의 풍미 향상 방법을 제공한다.In order to achieve the above object, the present invention provides a method for improving the flavor of food comprising the step of adding lactic acid bacteria of the genus Lactobacillus salt-resistant and glutamine activity more than 40 U / mg.
또한, 본 발명은 내염성이고 글루타미네즈 활성이 40 U/㎎ 이상의 락토바실러스 속 유산균인 락토바실러스 플란타룸 KLB13 균주(수탁번호: KCTC 11185BP)을 제공한다.The present invention is salt tolerant and glue Tommy Jimenez activity is more than 40 U / ㎎ Lactobacillus genus Lactobacillus is Lactobacillus Planta room KLB13 strain (Accession Number: KCTC 11185BP) is provided.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 내염성이고 글루타미네즈 활성이 40 U/㎎ 이상인 락토바실러스 속 유산균을 식품 제조과정에 첨가하는 단계를 포함하는 식품의 풍미 향상 방법을 제공한다.The present invention provides a method for improving the flavor of food comprising the step of adding lactobacillus sp. Lactobacillus which is salt tolerant and glutamine activity is 40 U / mg or more in the food manufacturing process.
상기 식품은 고염식품인 것으로 상기 고염식품에는 장류, 젖갈류 및 장아찌 류 등이 포함된다. 또한, 상기 장류는 간장, 고추장 및 된장 등이 있다.The food is a high salt food, and the high salt food includes jangjang, lactose and pickles. In addition, the soy sauce includes soy sauce, red pepper paste and miso.
상기 락토바실러스 속 유산균은 건강한 한국 여성의 질에서부터 분리된 것으로 내염성이며 상기 종의 글루타미네이즈는 높은 활성을 나타낸다. 상기 락토바실러스 속 유산균은 1.3M 이상으로 NaCl이 포함된 배지에서 성장가능한 내염성 균주이며, 40 U/mg 이상의 높은 글루타미네이즈 활성을 갖는다. 상기 1.3M 이상의 내염성 균주에는 KLB13, KLB14, KLB19, KLB20, KLB21, KLB30, KLB83, KLB95 및 KLB96의 균주들이 포함된다(표 1 참조). 또한 40 U/mg 이상의 높은 글루타미네이즈 활성을 갖는 균주에는 KLB1, KLB3, KLB6, KLB13, KLB21, KLB33, KLB39, KLB70 및 KLB96의 균주들이 포함된다(표 2 참조). 아울러, 1.3M 이상의 내염성 균주이며 40 U/mg 이상의 높은 글루타미네이즈 활성을 갖는 균주에는 KLB13, KLB21 및 KLB96의 균주들이 포함된다(도 3 참조).The lactobacillus genus Lactobacillus is isolated from the vagina of healthy Korean women and is salt tolerant, and glutamine of the species exhibits high activity. The Lactobacillus genus Lactobacillus is a salt tolerant strain that can be grown in a medium containing NaCl at 1.3M or more, and has a high glutamine activity of 40 U / mg or more. The salt resistant strains of 1.3M or more include strains of KLB13, KLB14, KLB19, KLB20, KLB21, KLB30, KLB83, KLB95, and KLB96 (see Table 1). In addition, strains having high glutamine activity of 40 U / mg or more include strains of KLB1, KLB3, KLB6, KLB13, KLB21, KLB33, KLB39, KLB70 and KLB96 (see Table 2). In addition, strains of more than 1.3M salt-resistant strain and high glutamine activity of 40 U / mg or more include the strains of KLB13, KLB21 and KLB96 (see Figure 3).
KLB13 및 KLB96의 균주들은 락토바실러스 플란타룸(Lactobacillus plantarum)으로 동정 되었고, KLB21은 락토바실러스 크리스파투스(Lactobacillus crispatus)으로 동정 되었다(도 4 참조). 락토바실러스 플란타룸은 그 유래가 식물로부터 분리되었고, 김치와 같은 한국 발효 음식에 널리 사용되며 각종 식품 첨가제로 널리 인증받고 있어, 상기 락토바실러스 플란타룸 KLB13(이하, KLB13; 수탁번호: KCTC 11185BP) 및 락토바실러스 플란타룸 KLB96(이하, KLB96)의 균주들은 적량, 적정한 방법으로 식품에 첨가하여 사용할 수 있음은 당연할 것이다.Strains of KLB13 and KLB96 have been identified as Lactobacillus Planta Room (Lactobacillus plantarum), KLB21 was identified as Lactobacillus Creative Spa tooth (Lactobacillus crispatus) (see Fig. 4). Lactobacillus Plantarum is isolated from plants, widely used in Korean fermented foods such as kimchi and widely certified as a variety of food additives, the Lactobacillus Planta Room KLB13 (hereinafter KLB13; Accession No .: KCTC 11185BP) and Lactobacillus Flags that lanthanide room KLB96 (hereinafter, KLB96) of the strains can be used in addition to the food in a suitable amount, an appropriate method is to be granted.
상기 KLB13 및 KLB96의 균주의 글루타미네이즈의 최적 NaCl 농도는 5% NaCl이다(도 5 참조). 또한, 상기 KLB13 균주의 글루타미네이즈 활성의 최적 온도는 50℃이고, KLB96 균주의 글루타미네이즈 활성의 최적 온도는 40 내지 50℃이다(도 6 참조). KLB13 균주는 60℃에서도 70% 이상의 글루타미네이즈 활성 안정성을 나타내고, KLB96 균주는 50℃까지 70% 이상의 글루타미네이즈 활성 안정성을 나타낸다(도 7 참조). 아울러, KLB13 및 KLB96 균주의 글루타미네이즈의 최적 pH는 pH 7이다(도 8 참조). KLB13 및 KLB96 균주 모두 pH 5 내지 9의 넓은 범위에서 높은 안정성을 나타낸다(도 9 참조).The optimal NaCl concentration of glutaminases of the strains of KLB13 and KLB96 is 5% NaCl (see FIG. 5). In addition, the optimum temperature of glutamine activity of the KLB13 strain is 50 ℃, the optimum temperature of glutamine activity of the KLB96 strain is 40 to 50 ℃ (see Figure 6). The KLB13 strain exhibited at least 70% glutamine activity stability even at 60 ° C., and the KLB96 strain exhibited at least 70% glutamine activity stability up to 50 ° C. (see FIG. 7). In addition, the optimal pH of glutamine of KLB13 and KLB96 strains is
본 발명의 KLB13 및 KLB96 균주 즉, Lactobacillus plantarum의 글루타미네이즈의 특성을 다른 미생물 유래(Lactobacillus rhamnosus , Aspergillus oryzae , Micrococcus luteus)의 글루타미네이즈의 특성과 비교하였다. 간장 발효시 염 농도인 3M NaCl에서는 KLB13 및 KLB96 균주를 포함한 Lactobacillus spp.의 글루타미네이즈는 다른 미생물 유래 글루타미네이즈 보다 높은 활성을 유지하였다. 최적 반응온도에 대해서는 Aspergillus oryzae(30℃)를 제외한 균주의 글루타미네이즈가 40 내지 50℃에서 최적 활성을 나타냈다. 또한, Lactobacillus spp. 유래 글루타미네이즈의 최적 pH는 pH 7.0인데 반해 나머지 미생물들의 글루타미네이즈는 염기성 pH에서 최고 활성을 보였다. 상기 KLB13 및 KLB96 균주는 타 균주에 비해 좀 더 넓은 범위의 pH에서 안정성을 나타내었다(표 3 참조).KLB13 and KLB96 strains of the present invention, namely Lactobacillus Plantarum Glutaminase Properties from Other Microorganisms ( Lactobacillus rhamnosus , Aspergillus oryzae , Micrococcus luteus ) were compared with those of glutamine. Lactobacillus containing KLB13 and KLB96 strains at 3M NaCl, the salt concentration during soybean fermentation The glutamine of spp. maintained higher activity than that of other microorganisms. Aspergillus for optimum reaction temperature Glutaminase of strains excluding oryzae (30 ° C.) showed optimal activity at 40-50 ° C. Also, Lactobacillus spp. The optimal pH of the derived glutamine was pH 7.0, whereas the glutamine of the remaining microorganisms showed the highest activity at basic pH. The KLB13 and KLB96 strains showed stability at a wider range of pH than other strains (see Table 3).
또한, 본 발명은 내염성이고 글루타미네즈 활성이 40 U/㎎ 이상의 락토바실러스 속 유산균인 락토바실러스 플란타룸 KLB13 균주(수탁번호: KCTC 11185BP)을 제공한다.The present invention is salt tolerant and glue Tommy Jimenez activity is more than 40 U / ㎎ Lactobacillus genus Lactobacillus is Lactobacillus Planta room KLB13 strain (Accession Number: KCTC 11185BP) is provided.
상기 KLB13 균주(수탁번호: KCTC 11185BP)의 글루타미네이즈의 최적 NaCl 농도는 5% NaCl이고(도 5 참조), 글루타미네이즈 활성의 최적 온도는 50℃이다(도 6 참조). 또한 KLB13 균주는 60℃에서도 70% 이상의 글루타미네이즈 활성 안정성을 나타내고(도 7 참조), 글루타미네이즈의 최적 pH는 pH 7이다(도 8 참조). 아울러, KLB13 균주는 pH 5 내지 9의 넓은 범위에서 높은 안정성을 나타낸다(도 9 참조).The optimal NaCl concentration of glutamine of the KLB13 strain (Accession No .: KCTC 11185BP) is 5% NaCl (see FIG. 5), and the optimum temperature of glutamine activity is 50 ° C. (see FIG. 6). In addition, the KLB13 strain exhibited at least 70% glutamine activity stability even at 60 ° C (see FIG. 7), and the optimal pH of glutamine was pH 7 (see FIG. 8). In addition, the KLB13 strain shows high stability over a wide range of pH 5-9 (see Figure 9).
본 발명의 내염성 락토바실러스 속 유산균을 천연 식품 첨가제로 사용하여 글루타민으로부터 글루타민산염을 생산하는 방법은 기존의 인공 식품 첨가제에 의한 부작용이나 병적 증세를 회피할 수 있다.The method of producing glutamate from glutamine using the salt-resistant Lactobacillus sp. Lactic acid bacteria of the present invention as a natural food additive can avoid side effects and pathological symptoms caused by existing artificial food additives.
이하, 본 발명을 실시예 및 비교예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by Examples and Comparative Examples.
단, 하기 실시예 및 비교예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 비교예에 한정되는 것은 아니다.However, the following Examples and Comparative Examples are merely illustrative of the present invention, the contents of the present invention is not limited to the following Examples and Comparative Examples.
<< 실시예Example 1> 내염성 균주 선별 1> Flameproof Strain Screening
<1-1> 균주 및 배양 조건<1-1> Strains and Culture Conditions
본 연구에 사용한 균주는 한국여성의 질로부터 분리한 락토바실러스 속 유산균을 대상으로 실험을 수행하였다.The strains used in this study were tested on Lactobacillus spp. Isolated from the vagina of Korean women.
질 도말로부터 유산균을 분리하기 위하여 유산균 선택배지인 로고사(Rogosa) 아가 플레이트에 스트리킹(streaking) 한 후에 이 플레이트를 37℃, 24~48시간 동안 촛불명(candle jar) 또는 혐기성 배양실(anaerobic chamber)에서 즉시 배양하였다. 단일 콜로니를 얻기 위하여 여러 번 로고사 아가 플레이트에 스트리킹하여 단일 콜로니를 얻었다. 각 시료들에 코드 번호(KLB)를 임의로 지어준 후 그람-염색, 카탈라아제 검사(catalse test) 및 현미경 관찰을 통하여 유산균임을 증명하였다.After streaking on the Lactobacillus selection medium, the Rogosa agar plate, to separate the lactic acid bacteria from the vaginal smear, the plates are placed in a candle jar or anaerobic chamber at 37 ° C for 24 to 48 hours. Incubated immediately at. Several single streaks were obtained on Logo agar plates to obtain a single colony. Each sample was randomly assigned a code number (KLB), and then proved to be a lactic acid bacterium by Gram-staining, catalase test, and microscopic observation.
상기 방법으로 분리된 유산균을 MRS 배지(1 ℓ 기준으로 글루코오스 20 g, 펩톤 No.3 10 g, 비프 추출액 10 g, 효모 추출액 5 g, 소듐 아세테이트 5 g, 암모늄 시트레이트 2 g, K2HPO4 2 g, 트윈 80 1 g, FeSO4·7H2O 35 mg, MgSO4 575 mg, MnSO4·7H2O 120 mg)를 사용하여 37℃에서 12시간 동안 혐기적 조건으로 정치배양 하였다.The lactic acid bacteria isolated by the above method was added to MRS medium (20 g of glucose, 10 g of peptone No. 3, 10 g of beef extract, 10 g of yeast extract, 5 g of sodium acetate, 2 g of ammonium citrate, K 2 HPO 4). 2 g,
<1-2> 염도 스트레스 <1-2> salinity stress MICMIC 실험 Experiment
실시예 1-1의 방법으로 배양된 락토바실러스 속 유산균을 대상으로 높은 내염성의 균주를 선별하고자 하였다.A high flame resistant strain was screened against Lactobacillus spp. Cultured by the method of Example 1-1.
페트리 디쉬를 기울인 상태에서 액체상태의 3M NaCl이 포함된 1.5% 아가 MRS 배지를 부어서 굳힌 후, 페트리 디쉬를 수평이 되게 한 후, 2%의 락토바실러스 속 유산균이 접종된 0.7% 아가 MRS 배지를 그 위에 부어 굳힌다. 37℃ 인큐베이터에서 24 시간 배양한 후, 전체 페트리 디쉬의 길이(A)와 미생물이 자린 길이(B)를 측정하여(도 2) 수학식 1에 의해 각 균주의 내염성을 계산하였다.Pour the 1.5% agar MRS medium containing 3M NaCl in liquid state while tilting the Petri dish, harden it, level the Petri dish, and then inject 0.7% agar MRS medium inoculated with 2% Lactobacillus spp. Pour over and harden. After incubation for 24 hours in a 37 ℃ incubator, the length (A) of the total petri dishes and the length of the microorganisms (B) was measured (Fig. 2) to calculate the flame resistance of each strain by the equation (1).
그 결과, 0.5M에서부터 1.5M에까지 내염성이 다양하게 나타났음을 확인하였고, 계산 값이 1.3M 이상 되는 내염성이 높은 KLB13, KLB14, KLB19, KLB20, KLB21, KLB30, KLB83, KLB95 및 KLB96의 9 개 균주를 선정하였다(표 1).As a result, it was confirmed that various salt resistance was shown from 0.5M to 1.5M, and nine strains of highly saltproof KLB13, KLB14, KLB19, KLB20, KLB21, KLB30, KLB83, KLB95, and KLB96 were calculated to have a calculated value of 1.3M or more. Were selected (Table 1).
<< 실시예Example 2> 2> 글루타미네이즈Glutamine 활성 측정 Active measurement
실시예 1의 방법으로 선별된 내염성 균주 9 개 균주를 포함한 본 발명의 락토바실러스 속 유산균 107 개 균주 중 총 64 개 균주의 글루타미네이즈 활성을 측정하였다.Glutaminase activity of a total of 64 strains of 107 strains of Lactobacillus sp. Lactobacillus of the present invention including 9 strains of saline tolerant strains selected by the method of Example 1 was measured.
도 1에서 설명하였듯이 글루타민은 글루타미네이즈에 의해 글루타민산염으로 탈아민화되고, 글루타민산염은 다시 조효소 NAD+ 및 글루탐산탈수소효소(glutamate dehydrogenase; GLDH)에 의해 2-옥소글루타르산과 NADH로 변환된다. 상기 과정에서 NAD+에서 NADH로의 변화는 분광학적으로 결정할 수 있다. 즉, NADH는 340 nm인 파장의 빛에 흡수 극대값을 가진다. 글루탐산탈수소효소(glutamate dehydrogenase)는 글루타민산염이 산화되고 이것이 가수분해되어 2-옥소글루타르산과 암모니아를 생성하는 반응을 촉매 하는 탈수소효소로, 반응에 피리딘 뉴클레오티드를 필요로 하는 유일한 탈수소효소이다(Kleerebezem M et al ., Proc Natl Acad Sci USA 100:1990-1995, 2003; Makarova K et al ., Proc Natl Acad Sci USA 103:15611-15616, 2006). 이에 글루탐산탈수소효소의 활성을 측정함으로써 글루타미네이즈의 활성을 간접적으로 측정하였다.As illustrated in FIG. 1, glutamine is deamined to glutamate by glutamine, and glutamate is converted into 2-oxoglutaric acid and NADH by coenzyme NAD + and glutamate dehydrogenase (GLDH). The change from NAD + to NADH in the process can be determined spectroscopically. In other words, NADH has an absorption maximum in light having a wavelength of 340 nm. Glutamate dehydrogenase is a dehydrogenase that catalyzes the reaction of glutamate to be oxidized and hydrolyzed to produce 2-oxoglutaric acid and ammonia. It is the only dehydrogenase that requires pyridine nucleotides in the reaction (Kleerebezem M). et al . , Proc Natl Acad Sci USA 100: 1990-1995, 2003; Makarova K et al . , Proc Natl Acad Sci USA 103: 15611-15616, 2006). The activity of glutamine was indirectly measured by measuring the activity of glutamate dehydrogenase.
<2-1> 시료 준비<2-1> Sample Preparation
MRS 배지를 사용하여 37℃에서 12시간 동안 혐기적 조건으로 정치배양 한 세포 배양액 5 mL를 13,000 rpm에서 원심분리한 후 PBS 완충액으로 현탁하여 세척하였다. 다시 원심분리한 후 상등액을 제거하고 12 mL의 0.75M 생리식염수로 현탁한 후, 20%로 앰피실린을 첨가하고 10초 간격의 펄스를 주어 2분간 소니케이터로 세포를 파쇄하였다. 세포 파쇄 과정은 얼음 상에서 수행하였다.5 mL of cell cultures which were incubated in anaerobic conditions at 37 ° C. for 12 hours using MRS medium were centrifuged at 13,000 rpm, and then washed with PBS buffer. After centrifugation again, the supernatant was removed, suspended in 12 mL of 0.75M saline, and ampicillin was added at 20%, and the cells were disrupted with a sonicator for 2 minutes with a pulse of 10 seconds. Cell disruption process was performed on ice.
<2-2> <2-2> 글루타미네이즈Glutamine 활성 측정 Active measurement
37℃에서 5분 동안 L-글루타민 용액과 아세테이트 완충액을 평형시킨 후, 실시예 2-1의 방법으로 준비한 시료 0.2 mL을 첨가하고 다시 10분간 37℃에서 반응시켰다. 0.75N 과염소산(perchloric acid) 용액으로 반응을 중지시켰고, 1.5N 소듐 아세테이트로 중화시켰다. 8,000 rpm에서 5분 동안 원심분리 한 후, 상등액 50 ㎕를 취해서 0.25M 하이드록실아민(hydroxylamine) 완충액(pH 8.0, 20mM EDTA), 10mM NAD+ 용액, ddH2O 및 770 U/㎖ GLDH(Sigma - Aldrich, USA)혼합물에 첨가하였다. 37℃에서 30분간 반응시킨 후 340 nm 파장에서 흡광도를 측정하였다. 수학식 2에 의해 부피 활성(U/㎖)을 계산하였다. A 시료는 상기 방법에 의해 측정된 흡광도를 의미하고, A0는 상기 방법에서 시료의 투입과 과염소산의 투입 순서를 바꾸어서 측정한 대조군의 흡광도를 의미하였다.After equilibrating the L-glutamine solution and the acetate buffer for 5 minutes at 37 ° C, 0.2 mL of the sample prepared by the method of Example 2-1 was added and reacted again at 37 ° C for 10 minutes. The reaction was stopped with 0.75N perchloric acid solution and neutralized with 1.5N sodium acetate. After centrifugation at 8,000 rpm for 5 minutes, 50 μl of supernatant was taken and 0.25M hydroxyllamine buffer (pH 8.0, 20mM EDTA), 10mM NAD + solution, ddH 2 O and 770 U / ml GLDH (Sigma- Aldrich, USA) was added to the mixture. After reacting for 30 minutes at 37 ℃ absorbance was measured at a wavelength of 340 nm. Volume activity (U / ml) was calculated by
질량 활성(U/mg)을 계산하고자 단백질 양을 Bradford 방법에 의해 측정하였다. 수학식 3에 의해 질량 활성(U/mg)을 계산하였다. C 는 시료 내의 글루타미네이즈의 함량(㎎/㎖)을 의미하였다.Protein amount was determined by Bradford method to calculate mass activity (U / mg). Mass activity (U / mg) was calculated by
그 결과, 총 43 개의 균주가 글루타미네이즈 활성을 나타내었고, 그 중 활성이 높은 KLB1, KLB3, KLB6, KLB13, KLB21, KLB33, KLB39, KLB70 및 KLB96의 9 개 균주를 선정하였다(표 2).As a result, a total of 43 strains showed glutamine activity, and 9 strains of KLB1, KLB3, KLB6, KLB13, KLB21, KLB33, KLB39, KLB70, and KLB96 having high activity were selected (Table 2).
또한 상기 결과와 실시예 1의 결과를 비교함으로써 내염성이면서 글루타미네이즈 활성이 높은 KLB13, KLB21 및 KLB96의 3 개 균주를 선정하였다(도 3)In addition, three strains of KLB13, KLB21, and KLB96, which had high salt resistance and glutamine activity, were selected by comparing the results with those of Example 1 (FIG. 3).
<2-3> 균주 동정<2-3> Strain Identification
실시예 2-2의 방법으로 선별된 KLB13, KLB21 및 KLB96 균주를 RFLP(restriction fragment length polymorphism)를 이용하여 동정하였다.KLB13, KLB21 and KLB96 strains selected by the method of Example 2-2 were identified using RFLP (restriction fragment length polymorphism).
각 균주들을 대상으로 정방향 프라이머(서열번호 1: CCCAAGCTTAGAGTTTGATCCTGGCTCAG) 및 역방향 프라이머(서열번호 2: ACGCGTCGACAAGGAGGTGATCCAGCC)를 이용하여 전변성: 94℃에서 55분, 35 회 반복(변성: 94℃에서 1분, 어닐링 56℃에서 30초, 신장: 72℃에서 2분) 최종신장: 72℃에서 5분의 조건으로 16S PCR을 수행한 후 상기 PCR 반응 산물을 여러 종류의 제한효소(AluI, HaeIII, HinfI, MspI, CfoI; Roche, 스위스)로 각각 반응시켰다. 전기영동으로 상기 제한효소들에 의해 잘려진 패턴을 확인하고, RFLP 분석용 프로그램[NTSYS-pc (numerical taxonomy system of multivariate statustical programs); Applied Biostatistics Inc, USA]을 이용하여 비슷하게 나온 패턴별로 그룹을 지정해주었다.For each strain, using a forward primer (SEQ ID NO: CCCAAGCTTAGAGTTTGATCCTGGCTCAG) and a reverse primer (SEQ ID NO: 2: ACGCGTCGACAAGGAGGTGATCCAGCC), total denaturation: 55 min at 94 ° C, 35 repetitions (denaturation: 1 min at 94 ° C, annealing 56) 30 sec at ℃, height: 72 ℃ in 2 min.) final height: after performing the 16S PCR at 72 ℃ under the conditions of 5 minutes enzyme the PCR reaction products several types of limitation (Alu I, Hae III, Hinf I, Msp I, Cfo I; Roche, Switzerland). Electrophoresis confirmed the pattern cut by the restriction enzymes, and used for RFLP analysis [NTSYS-pc (numerical taxonomy system of multivariate statustical programs); Applied Biostatistics Inc, USA] was used to group by similar pattern.
그 결과, KLB13과 KLB96 균주는 Lactobacillus plantarum으로 동정 되었고, KLB21은 Lactobacillus crispatus로 동정 되었다(도 4). 또한, 상기 KLB13 균주의 16s rDNA 염기서열(서열번호 3)을 분석한 결과 Lactobacillus plantarum으로 동정 되었다.As a result, the strains KLB13 and KLB96 were Lactobacillus. plantarum was identified as KLB21 Lactobacillus It was identified as crispatus (FIG. 4). In addition, Lactobacillus as a result of analyzing the 16s rDNA sequence (SEQ ID NO: 3) of the KLB13 strain It was identified as plantarum .
<< 실시예Example 3> KLB13 및 KLB96 균주의 3> KLB13 and KLB96 strains 글루타미네이즈Glutamine 최적 활성 조건 Optimal active conditions
<3-1> <3-1> NaClNaCl 농도에 따른 According to concentration 글루타미네이즈Glutamine 최적 활성 조건 Optimal active conditions
실시예 2-2의 방법으로 KLB13 및 KLB96 균주의 글루타미네이즈 활성을 측정함에 있어서, L-글루타민 용액과 아세테이트 완충액을 평형 시킨 후, 실시예 2-1의 방법으로 준비한 시료 0.2 ㎖을 첨가한 상태의 NaCl 농도가 각각 0, 5, 10, 15, 20, 25가 되도록 하였다. 그 후 37℃에서 10분간 반응한 후, NaCl 농도에 변화를 준 시료와 대조군인 글루타미네이즈에 대한 반응을 멈추게 하는 과염소산을 먼저 첨가한 시료를 흡광도 625 ㎚에서 각각 측정하였다. 수학식 2 및 수학식 3에 의해 부피 활성(U/㎖) 및 질량 활성(U/㎎)을 계산하였다.In measuring glutamine activity of KLB13 and KLB96 strains by the method of Example 2-2, after equilibrating the L-glutamine solution and the acetate buffer, 0.2 ml of the sample prepared by the method of Example 2-1 was added. NaCl concentrations were set to 0, 5, 10, 15, 20, and 25, respectively. After reacting at 37 ° C. for 10 minutes, a sample in which NaCl concentration was changed and a sample to which perchloric acid was first added to stop the reaction to glutamine were measured at absorbance 625 nm. Volumetric activity (U / ml) and mass activity (U / mg) were calculated by the equations (2) and (3).
그 결과, KLB13 및 KLB96 균주 모두 5% NaCl에서 글루타미네이즈 활성이 가장 높았고, 10 내지 25%까지 NaCl 농도가 증가함에 따라서 글루타미네이즈 활성 감소에 있어서 KLB96 균주가 KLB13 균주보다 그 감소폭이 더 컸다(도 5).As a result, both KLB13 and KLB96 strains had the highest glutamine activity at 5% NaCl, and KLB96 strain had a greater reduction than KLB13 strain in reducing glutamine activity as NaCl concentration increased from 10 to 25% ( 5).
<3-2> 반응 온도에 따른 <3-2> according to the reaction temperature 글루타미네이즈Glutamine 최적 활성 조건 Optimal active conditions
실시예 2-2의 방법으로 KLB13 및 KLB96 균주의 글루타미네이즈 활성을 측정함에 있어서, L-글루타민 용액과 아세테이트 완충액을 평형 시킨 후, 실시예 2-1의 방법으로 준비한 시료 0.2 ㎖을 첨가한 상태에서 20, 30, 40, 50, 60 및 70℃에서 10분간 반응시켰다. 각 온도별로 반응시킨 시료와 대조군인 글루타미네이즈에 대한 반응을 멈추게 하는 과염소산을 먼저 첨가한 시료를 흡광도 625 ㎚에서 각각 측정하였다. 수학식 2 및 수학식 3에 의해 부피 활성(U/㎖) 및 질량 활성(U/㎎)을 계산하였다.In measuring glutamine activity of KLB13 and KLB96 strains by the method of Example 2-2, after equilibrating the L-glutamine solution and the acetate buffer, 0.2 ml of the sample prepared by the method of Example 2-1 was added. 10 minutes at 20, 30, 40, 50, 60 and 70 ℃. The samples reacted at each temperature and the samples to which perchloric acid was first added to stop the reaction to glutamine were measured at absorbance 625 nm, respectively. Volumetric activity (U / ml) and mass activity (U / mg) were calculated by the equations (2) and (3).
그 결과, KLB13 균주는 50℃에서 글루타미네이즈 활성이 가장 높았고, KLB96 균주는 40℃에서 가장 높은 활성을 나타냈지만 50℃에서도 비슷한 수준의 높은 활성을 나타냈다(도 6).As a result, the KLB13 strain had the highest glutamine activity at 50 ° C., and the KLB96 strain had the highest activity at 40 ° C., but showed similar levels of high activity at 50 ° C. (FIG. 6).
또한, KLB13 균주는 60℃에서도 70% 이상의 글루타미네이즈 활성 안정성을 나타냈으나, KLB96 균주는 40℃까지는 KLB13 균주와 비슷한 활성 안정성을 보이다가 60℃에서는 50%까지 활성 안정성이 하락하였다(도 7).In addition, the KLB13 strain showed more than 70% glutamine activity stability even at 60 ℃, KLB96 strain showed similar activity stability to the KLB13 strain up to 40 ℃, the activity stability was reduced to 50% at 60 ℃ (Fig. 7 ).
<3-3> <3-3> pHpH 에 따른 In accordance 글루타미네이즈Glutamine 최적 활성 조건 Optimal active conditions
실시예 2-2의 방법으로 KLB13 및 KLB96 균주의 글루타미네이즈 활성을 측정함에 있어서, L-글루타민 용액과 아세테이트 완충액을 평형 시킨 후, 실시예 2-1의 방법으로 준비한 시료 0.2 ㎖을 첨가한 후, 각 시료의 pH가 3, 5, 7, 9 및 11이 되도록 보정하였다. 이때 사용한 완충용액은 pH 3과 pH 5로의 보정을 위해서 0.1M 시트리산(citric acid)을 사용하였고, pH 9 와 11로의 보정을 위서는 Tris-HCl을 사용하였다. pH 7로의 보정을 위해서 Tris-HCl 완충용액과 1N NaOH를 이용하여 보정하였다.In measuring the glutamine activity of the KLB13 and KLB96 strains by the method of Example 2-2, after equilibrating the L-glutamine solution and the acetate buffer, 0.2 ml of the sample prepared by the method of Example 2-1 was added thereto. , PH of each sample was corrected to 3, 5, 7, 9 and 11. The buffer solution used was 0.1M citric acid (ctric acid) to calibrate to
그 후 37℃에서 10분간 반응한 후, 각 pH별 시료와 대조군인 글루타미네이즈에 대한 반응을 멈추게 하는 과염소산을 먼저 첨가한 시료를 흡광도 625 ㎚에서 각각 측정하였다. 수학식 2 및 수학식 3에 의해 부피 활성(U/㎖) 및 질량 활성(U/㎎)을 계산하였다.Then, after reacting for 10 minutes at 37 ℃, each sample was added at the absorbance of 625 nm, the sample for each pH and the perchloric acid to first stop the reaction to glutamine. Volumetric activity (U / ml) and mass activity (U / mg) were calculated by the equations (2) and (3).
그 결과, KLB13 및 KLB96 균주 모두 pH 7에서 글루타미네이즈 활성이 가장 높았고, 낮은 pH 보다 높은 pH에서 좀 더 높은 활성을 보였다. 또한, KLB13 균주가 pH 조건 변화에 따라 나타낸 활성이 KLB96 균주보다 더 높았다(도 8). As a result, both KLB13 and KLB96 strains had the highest glutamine activity at
또한, KLB13 및 KLB96 균주 모두 pH 5 내지 9의 넓은 범위에서 높은 안정성을 나타냈고, 낮은 pH(pH 3)에서는 활성이 소실된 것에 반해 높은 pH(pH 11)에서는 50% 정도의 활성을 유지하는 것으로 나타났다(도 9).In addition, both KLB13 and KLB96 strains showed high stability in a wide range of
<< 비교예Comparative example > > 글루타미네이즈Glutamine 특성 비교 Property comparison
본 발명의 KLB13 및 KLB96 균주 즉, Lactobacillus plantarum의 글루타미네이즈의 특성을 다른 미생물 유래의 글루타미네이즈의 특성과 비교하였다.KLB13 and KLB96 strains of the present invention, namely Lactobacillus The characteristics of glutamine in plantarum were compared with those of other microorganisms.
Aspergillus oryzae(Naohisa MK et al ., J. Bioscience & Bioengineering, 100: 576-578, 2005)와 Micrococcus luteus(Naohisa MK et al ., J. Bioscience & Bioengineering, 100: 576-578, 2005) 및 Lactobacillus rhamnosus(Alexandra WZ et al ., J Mol Catal 32:862-867, 2002)의 세 균주를 선택하여 기질, 내염성, 최적 온도, 최적 pH 및 pH 안정성을 비교하였다. Aspergillus oryzae (Naohisa MK et al . , J. Bioscience & Bioengineering , 100: 576-578, 2005) and Micrococcus luteus (Naohisa MK et al . , J. Bioscience & Bioengineering , 100: 576-578, 2005) and Lactobacillus rhamnosus (Alexandra WZ et al . , J Mol Three strains of Catal 32: 862-867, 2002) were selected to compare substrate, flame resistance, optimal temperature, optimal pH and pH stability.
그 결과, 간장 발효시 염 농도인 3M NaCl에서는 Lactobacillus spp.의 글루타미네이즈가 다른 미생물 유래 글루타미네이즈 보다 높은 활성을 유지함을 알 수 있었다. 최적 반응온도에 대해서는 Aspergillus oryzae(30℃)를 제외한 균주의 글루타미네이즈가 40 내지 50℃에서 최적 활성을 나타냈다. 또한, Lactobacillu spp. 유래 글루타미네이즈의 최적 pH는 pH 7.0인데 반해 나머지 미생물들의 글루타미네이즈는 염기성 pH에서 최고 활성을 보였다. 본 발명에 의해 선별된 두 균주는 타 균주에 비해 좀 더 넓은 범위의 pH에서 안정성을 나타내었다(표 3).As a result, Lactobacillus at 3M NaCl, the salt concentration during fermentation Glutaminase of spp. was found to maintain higher activity than other microorganism-based glutamines. Aspergillus for optimum reaction temperature Glutaminase of strains excluding oryzae (30 ° C.) showed optimal activity at 40-50 ° C. In addition, Lactobacillu spp. The optimal pH of the derived glutamine was pH 7.0, whereas the glutamine of the remaining microorganisms showed the highest activity at basic pH. Two strains selected by the present invention showed stability at a wider range of pH than other strains (Table 3).
도 1은 글루타민 대사작용 과정과 주요 효소의 반응을 나타낸 도이다.1 is a diagram showing a glutamine metabolism process and a reaction of a major enzyme.
도 2는 사면 배지를 응용한 내염성 측정 방법을 나타낸 도이다.Figure 2 is a diagram showing a method for measuring flame resistance using a slope medium.
도 3은 본 발명의 내염성 균주들의 글루타미네이즈 활성을 비교한 도이다.Figure 3 is a diagram comparing the glutamine activity of the salt tolerant strains of the present invention.
도 4는 RFLP(Restriction fragment length polymorphism)를 이용한 KBL 13 균주 및 KLB96 균주의 동정결과를 나타낸 도이다.4 is a diagram showing the results of identification of
도 5는 NaCl 농도에 따른 글루타미네이즈 활성을 나타낸 도이다.5 is a diagram showing glutamine activity according to NaCl concentration.
도 6은 온도에 따른 글루타미네이즈 최적 활성을 나타낸 도이다.6 is a diagram showing the glutamine activity according to the temperature.
도 7은 온도에 따른 글루타미네이즈 안정성을 나타낸 도이다.7 is a diagram illustrating glutamine stability with temperature.
도 8은 pH에 따른 글루타미네이즈 최적 활성을 나타낸 도이다.8 is a diagram showing the optimum glutamine activity according to pH.
도 9는 pH에 따른 글루타미네이즈 안정성을 나타낸 도이다.9 is a diagram showing glutamine stability according to pH.
<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Method for using high osmotic pressure tolerant strain as natural flavor <130> 7P-08-41 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> 16s PCR Forward primer <400> 1 cccaagctta gagtttgatc ctggctcag 29 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 16s PCR Reverse primer <400> 2 acgcgtcgac aaggaggtga tccagcc 27 <210> 3 <211> 1250 <212> DNA <213> Artificial Sequence <220> <223> 16S rDNA of Lactobacillus sp. KLB 213 sequeces <400> 3 cgcgttggga gctctcccat atggtcgacc tgcaggcggc cgcgaattca ctagtgattc 60 ccaagcttag agtttgatcc tggctcagga cgaacgctgg cggcgtgcct aatacatgca 120 agtcgaacga actctggtat tgattggtgc ttgcatcatg atttacattt gagtgagtgg 180 cgaactggtg agtaacacgt gggaaacctg cccagaagcg ggggataaca cctggaaaca 240 gatgctaata ccgcataaca acttggaccg catggtccga gcttgaaaga tggcttcggc 300 tatcactttt ggatggtccc gcggcgtatt agctagatgg tggggtaatg gctcaccatg 360 gcaatgatac gtagccgacc tgagagggta atcggccaca ttgggactga gacacggccc 420 aaactcctac gggaggcagc agtagggaat cttccacagt ggacgaaagt ctgatggagc 480 aacgccgcgt gagtggagaa gggtttcggc tcgtaaaact ctgttgttaa agaagaacat 540 atctgagagt aactgttcag gtattgacgg tatttaacca gaaagccacg gctaactaca 600 agcattccgc ctggggagta cgcccgcaag gctgaaactc aaaggaattg acgggggccc 660 gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt 720 gacatactat gcaaatctaa gagattagac gttcccttcg gggacatgga tacaggtggt 780 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 840 ccttattatc agttgccagc attaagttgg gcactctggt gagactgccg gtgacaaacc 900 ggaggaaggt ggggatgacg tcaatcatca tgccccttat gacctgggct acacacgtgc 960 tacaatggat ggtacaacga gttgcgaact cgcgagagta agctaatctc ttaaagccat 1020 tctcagttcg gattgtaggc tgcaactccc tacatgaagt cggaatcgct agtaatcgcg 1080 gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg 1140 agagtttgta acacccaaag tcggtggggt aaccttttag gaaccagccg cctaaggtgg 1200 gacagatgat tagggtgaag tcgtaacaag gtagccgtag gagaacctgc 1250 <110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Method for using high osmotic pressure tolerant strain as natural flavor <130> 7P-08-41 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> 16s PCR Forward primer <400> 1 cccaagctta gagtttgatc ctggctcag 29 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 16s PCR Reverse primer <400> 2 acgcgtcgac aaggaggtga tccagcc 27 <210> 3 <211> 1250 <212> DNA <213> Artificial Sequence <220> <223> 16S rDNA of Lactobacillus sp. KLB 213 sequeces <400> 3 cgcgttggga gctctcccat atggtcgacc tgcaggcggc cgcgaattca ctagtgattc 60 ccaagcttag agtttgatcc tggctcagga cgaacgctgg cggcgtgcct aatacatgca 120 agtcgaacga actctggtat tgattggtgc ttgcatcatg atttacattt gagtgagtgg 180 cgaactggtg agtaacacgt gggaaacctg cccagaagcg ggggataaca cctggaaaca 240 gatgctaata ccgcataaca acttggaccg catggtccga gcttgaaaga tggcttcggc 300 tatcactttt ggatggtccc gcggcgtatt agctagatgg tggggtaatg gctcaccatg 360 gcaatgatac gtagccgacc tgagagggta atcggccaca ttgggactga gacacggccc 420 aaactcctac gggaggcagc agtagggaat cttccacagt ggacgaaagt ctgatggagc 480 aacgccgcgt gagtggagaa gggtttcggc tcgtaaaact ctgttgttaa agaagaacat 540 atctgagagt aactgttcag gtattgacgg tatttaacca gaaagccacg gctaactaca 600 agcattccgc ctggggagta cgcccgcaag gctgaaactc aaaggaattg acgggggccc 660 gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt 720 gacatactat gcaaatctaa gagattagac gttcccttcg gggacatgga tacaggtggt 780 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 840 ccttattatc agttgccagc attaagttgg gcactctggt gagactgccg gtgacaaacc 900 ggaggaaggt ggggatgacg tcaatcatca tgccccttat gacctgggct acacacgtgc 960 tacaatggat ggtacaacga gttgcgaact cgcgagagta agctaatctc ttaaagccat 1020 tctcagttcg gattgtaggc tgcaactccc tacatgaagt cggaatcgct agtaatcgcg 1080 gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg 1140 agagtttgta acacccaaag tcggtggggt aaccttttag gaaccagccg cctaaggtgg 1200 gacagatgat tagggtgaag tcgtaacaag gtagccgtag gagaacctgc 1250
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KR101293692B1 (en) * | 2011-09-19 | 2013-08-06 | 한경호 | Production Method of Clear Soy Source, The Clear Soy Source Produced by thereof Method, and cultivation method of lactobacillus |
KR101337512B1 (en) * | 2010-07-30 | 2013-12-06 | 주식회사 웰빙엘에스 | The fermented Myungran using vegetable origin lactic acid bacteria and salt |
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KR20160141581A (en) | 2015-06-01 | 2016-12-09 | 주식회사 네이처웍스 | Fermented additive for food, a korean sauce prepared by using the same and method for production of the korean sauce |
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JP4921721B2 (en) | 2005-03-14 | 2012-04-25 | ヤマモリ株式会社 | Production method of functional food |
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KR101337512B1 (en) * | 2010-07-30 | 2013-12-06 | 주식회사 웰빙엘에스 | The fermented Myungran using vegetable origin lactic acid bacteria and salt |
KR101293692B1 (en) * | 2011-09-19 | 2013-08-06 | 한경호 | Production Method of Clear Soy Source, The Clear Soy Source Produced by thereof Method, and cultivation method of lactobacillus |
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