KR102517668B1 - Composition cryopreservation of lactic acid bacteria comprising enzyme treated aloe vera and a method for cryopreservation of lactic acid bacteria using the same - Google Patents
Composition cryopreservation of lactic acid bacteria comprising enzyme treated aloe vera and a method for cryopreservation of lactic acid bacteria using the same Download PDFInfo
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 166
- 241000894006 Bacteria Species 0.000 title claims abstract description 87
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Classifications
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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
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3571—Microorganisms; Enzymes
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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
- 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
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
-
- 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
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/40—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by drying or kilning; Subsequent reconstitution
- A23L3/44—Freeze-drying
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- A23Y2220/00—
Abstract
본 발명은 효소처리 알로에 베라를 포함하는 유산균 동결건조 보호용 조성물, 이를 포함하는 유산균 동결건조 보호제 및 이를 이용한 동결건조 보호 방법에 관한 것으로, 본 발명에 따르면 본 발명에 따른 효소처리 알로에 베라는 유산균을 동결건조 시 동결로 인한 화학적 및 물리적 스트레스로부터 보호하고 동결된 유산균의 활성을 보존할 수 있으므로, 동결건조 보호제로서 유용하게 이용될 수 있다.The present invention relates to a composition for protecting lactic acid bacteria by freeze-drying containing enzyme-treated aloe vera, a freeze-drying protective agent for lactic acid bacteria containing the same, and a freeze-drying protection method using the same. Since it can protect from chemical and physical stress caused by freezing during drying and preserve the activity of frozen lactic acid bacteria, it can be usefully used as a lyophilization protecting agent.
Description
본 발명은 유산균 동결건조 보호용 조성물에 관한 것으로, 더욱 구체적으로는 효소처리 알로에 베라를 포함하는 유산균 동결건조 보호용 조성물에 관한 것이다.The present invention relates to a composition for protecting lactic acid bacteria by freeze-drying, and more particularly, to a composition for protecting lactic acid bacteria by freeze-drying containing enzyme-treated aloe vera.
유산균은 발효 결과물로서 주로 유산을 생성하는 세균으로서, WHO에서는 '충분한 양을 섭취했을 때 건강에 좋은 효과를 주는 살아있는 균'으로 정의하고 있다. 현재까지 알려진 유산균의 효과 및 기능으로는 장내균총의 안정화, 유해세균의 정착억제에 따른 부패산물 생성감소 및 질병 예방, 면역 활성화 작용, 항암작용, 콜레스테롤 저하, 유당 불내증의 경감, 변비억제 등이 있다. 이처럼 유산균의 다양한 효과 및 기능으로 인하여 유산균을 이용한 다양한 제품들이 연구개발 및 출시되고 있다.Lactobacillus is a bacterium that mainly produces lactic acid as a result of fermentation, and is defined by the WHO as 'a live bacterium that exerts a beneficial effect on health when ingested in sufficient amounts'. The effects and functions of lactic acid bacteria known so far include stabilization of the intestinal flora, reduction of decay product production and disease prevention due to inhibition of settlement of harmful bacteria, immune activation, anticancer activity, cholesterol lowering, lactose intolerance relief, and constipation inhibition. . As such, due to the various effects and functions of lactic acid bacteria, various products using lactic acid bacteria are being researched, developed, and released.
제품에 이용되는 유산균은 일반적으로 생균제 및 종균제로서 활용하고 있는데, 유산균을 생균제 및 종균제로 활용하기 위해서는 생산된 유산균체 분말의 활성과 생존을 오랫동안 유지하는 것이 필수적으로 보증되어야 한다. 동결건조 방법은 이를 달성하기 위한 방법 중 하나로서 일반적으로 사용되고 있는 방법이다. 동결건조 방법은 유산균 균체를 냉동 건조시킴으로서 저장하는 방법으로 유산균의 활성을 오랜 기간 동안 유지할 수 있을 뿐만 아니라 취급, 저장, 상품화 및 실제 적용성에서 편리함이 있다. 다만, 동결건조 방법은 동결건조 과정에서 물리적 및 생화학적인 스트레스로 유산균의 활성 및 생존율에 영향을 미치게 되므로, 유산균의 활성을 보존하고 생존율을 향상시키기 위한 방법이 요구되고 있다. Lactic acid bacteria used in products are generally used as probiotics and inoculum. In order to use lactic acid bacteria as probiotics and inoculum, it is essential to maintain the activity and survival of the produced lactic acid bacteria powder for a long time. The lyophilization method is a method generally used as one of the methods for achieving this. The freeze-drying method is a method of storing lactic acid bacteria by freeze-drying them, and not only can maintain the activity of lactic acid bacteria for a long period of time, but also has convenience in handling, storage, commercialization, and practical applicability. However, since the freeze-drying method affects the activity and survival rate of lactic acid bacteria due to physical and biochemical stress during the freeze-drying process, a method for preserving the activity of lactic acid bacteria and improving the survival rate is required.
유산균 동결건조 시 유산균을 보호하여 세포 손상을 막기 위하여 동결건조 보호제를 일반적으로 사용하고 있으며, 동결건조 보호제는 미생물의 종류 및 보호제 성분에 따라 서로 다른 영향을 미칠 수 있기 때문에 미생물의 종류 및 그 특성에 맞는 적절한 동결건조 보호제의 사용이 필요하며, 이에 따라 다양한 동결건조 보호제의 개발이 이루어지고 있다. 구체적으로, 한국등록특허 제1665888호는 유산균 동결건조 보호제로서 찹쌀풀을 이용하고 있고, 한국등록특허 제1816802호는 미생물 동결건조 보호제로서 글루코오스 및 갈락토오스를 포함하는 세포외 다당을 이용하고 있으며, 한국등록특허 제1866197호는 동결건조 보호제로서 호박분말을 이용하고 있다. 그러나 종래에 효소처리 알로에 베라를 동결건조 보호제로서 이용하는 구성에 대해서는 알려진 바 없다.Freeze-drying protective agents are generally used to prevent cell damage by protecting lactic acid bacteria during freeze-drying of lactic acid bacteria. Since freeze-drying protective agents can have different effects depending on the type of microorganism and the components of the protective agent, it depends on the type of microorganism and its characteristics. It is necessary to use an appropriate lyophilization protection agent, and accordingly, various lyophilization protection agents are being developed. Specifically, Korean Patent No. 1665888 uses glutinous rice paste as a freeze-drying protecting agent for lactic acid bacteria, and Korean Patent No. 1816802 uses extracellular polysaccharides including glucose and galactose as a freeze-drying protecting agent for microorganisms. Patent No. 1866197 uses pumpkin powder as a freeze-drying protection agent. However, conventionally, there is no known configuration using enzyme-treated aloe vera as a lyophilization protection agent.
한편, 알로에 베라는 백합과(科)의 알로에속(屬)에 속하는 다년생 식물로서 고대 이집트 왕조 시대부터 질병을 막기 위해 사용되었다고 알려져 있다. 알로에 베라는 면역력 증강, 위, 장 및 피부의 건강 및 배변활동에 효과적이기 때문에 화장품 원료, 건강기능식품, 식품 등 다양한 용도로 활용되고 있다. 최근에는 알로에 베라가 균주를 캡슐화하여 동결건조 할 경우, 균주의 생존율이 증가한다는 연구가 진행된 바 있으나, 알로에 베라 자체를 동결 건조 보호제로 이용하는 것은 실질적으로 유산균을 보호하는 효과가 미미하기 때문에 알로에 베라를 동결건조 보호제로 이용하기 위한 방법의 모색이 필요하다.Meanwhile, Aloe Vera is a perennial plant belonging to the Aloe genus of the Liliaceae family and is known to have been used to prevent diseases since the ancient Egyptian dynasty. Since aloe vera is effective for immunity enhancement, stomach, intestine and skin health and bowel movement, it is used for various purposes such as cosmetics raw materials, health functional foods, and food. Recently, a study has been conducted that the survival rate of the strain increases when aloe vera is encapsulated and freeze-dried. It is necessary to find a method for using it as a freeze-drying protective agent.
이에, 본 발명자들은 상기 종래기술들의 문제점들을 극복하기 위하여 예의 연구노력한 결과, 효소처리 알로에 베라의 경우, 유산균 동결건조 과정에서 유발되는 세포 손상으로부터 유산균을 보호할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors have made intensive research efforts to overcome the problems of the prior art, and as a result, in the case of enzyme-treated aloe vera, it was confirmed that lactic acid bacteria can be protected from cell damage caused by the lactic acid bacteria freeze-drying process, and the present invention was completed. I did.
따라서, 본 발명의 주된 목적은 유산균 동결건조 과정에서 유발되는 세포 손상으로부터 유산균을 보호할 수 있는 효소처리 알로에 베라를 포함하는 유산균 동결건조 보호용 조성물을 제공하는 데 있다.Accordingly, a main object of the present invention is to provide a composition for protecting lactic acid bacteria from freeze-drying, including enzyme-treated aloe vera, capable of protecting lactic acid bacteria from cell damage caused by freeze-drying of lactic acid bacteria.
본 발명의 다른 목적은 상기 유산균 동결건조 보호용 조성물을 포함하는 유산균 동결건조 보호제를 제공하는 데 있다.Another object of the present invention is to provide a lactic acid bacteria freeze-drying protective agent comprising the composition for protecting the lactic acid bacteria freeze-dried.
본 발명의 다른 목적은 효소처리된 알로에 베라를 이용한 유산균 동결건조 보호 방법을 제공하는 데 있다.Another object of the present invention is to provide a method for protecting lactic acid bacteria by freeze-drying using enzyme-treated aloe vera.
본 발명의 한 양태에 따르면, 본 발명은 효소처리 알로에 베라를 포함하는 유산균 동결건조 보호용 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a composition for protecting lactic acid bacteria freeze-dried containing enzyme-treated aloe vera.
본 발명의 유산균 동결건조 보호용 조성물에 있어서, 상기 효소는 알로에 베라의 다당류(polysaccharide)를 올리고당(oligosaccharide)로 생성하기 위한 효소일 수 있으며, 바람직하게는 베타-만난아제(β-mannanase)이다.In the composition for protecting lactic acid bacteria from freeze-drying of the present invention, the enzyme may be an enzyme for converting polysaccharides of Aloe vera into oligosaccharides, and is preferably beta-mannanase.
본 발명의 용어 ‘효소처리 알로에 베라’는 알로에 베라의 다당류가 효소처리에 의해 올리고당으로 변환된 것으로, 좀 더 상세하게는 알로에 베라의 주요 다당체 성분인 아세만난(Acemannan)의 β-1, 4 결합부위가 베타-만난아제에 의해 끊어지면서 올리고당 형태로 분해된 알로에 베라이다.The term 'enzyme-treated aloe vera' of the present invention refers to the conversion of polysaccharides of aloe vera into oligosaccharides by enzymatic treatment, and more specifically, the β-1, 4 linkage of acemannan, the main polysaccharide component of aloe vera. It is aloe vera that has been decomposed into an oligosaccharide form as the site is cut by beta-mannanase.
본 발명의 유산균 동결건조 보호용 조성물에 있어서, 상기 효소처리는 알로에 베라 중량(g) 당 10,000unit/g 내지 100,000unit/g, 바람직하게는 40,000unit/g 내지 60,000unit/g, 가장 바람직하게는 50,000unit/g 농도의 효소를 알로에 베라와 혼합하여 효소반응 시킬 수 있다. 상기 효소의 농도가 10,000unit/g 미만일 경우 충분한 효소처리가 이루어지기 어려우며, 100,000unit/g을 초과할 경우 효율성 및 비용적인 측면에서 바람직하지 못하다.In the lactic acid bacteria freeze-drying protective composition of the present invention, the enzyme treatment is 10,000 unit / g to 100,000 unit / g, preferably 40,000 unit / g to 60,000 unit / g, and most preferably 50,000 per weight (g) of aloe vera. Unit/g concentration of enzyme can be mixed with aloe vera for enzymatic reaction. If the concentration of the enzyme is less than 10,000 unit / g, it is difficult to perform sufficient enzyme treatment, and if it exceeds 100,000 unit / g, it is not preferable in terms of efficiency and cost.
본 발명의 유산균 동결건조 보호용 조성물에 있어서, 상기 효소반응 시간은 20분 내지 60분, 바람직하게는 20분 내지 50분, 가장 바람직하게는 30분 동안 시킬 수 있다. 상기 효소반응 시간이 20분 미만일 경우 효소반응이 효과적으로 일어나지 않아 동결건조 보호 효과가 다소 떨어지며, 60분 초과할 경우 효소반응이 과하게 일어나 동결건조 보호 효과가 전혀 나타나지 않는다.In the composition for protecting lactic acid bacteria by freeze-drying of the present invention, the enzyme reaction time may be 20 to 60 minutes, preferably 20 to 50 minutes, and most preferably 30 minutes. When the enzymatic reaction time is less than 20 minutes, the enzymatic reaction does not occur effectively, and the freeze-drying protection effect is somewhat reduced.
본 발명의 유산균 동결건조 보호용 조성물에 있어서, 상기 효소반응 온도는 20℃ 내지 80℃, 바람직하게는 30℃ 내지 70℃, 가장 바람직하게는 70℃에서 효소반응 시킬 수 있다. 상기 효소반응 온도가 20℃ 미만일 경우 효소반응이 효과적으로 일어나지 않아 동결건조 보호 효과가 다소 떨어지며, 80℃ 초과할 경우 효소의 변성으로 효소반응이 일어나지 않아 동결건조 보호 효과가 전혀 나타나지 않는다.In the composition for protecting lactic acid bacteria by freeze-drying of the present invention, the enzyme reaction temperature may be 20 ° C to 80 ° C, preferably 30 ° C to 70 ° C, most preferably 70 ° C. When the enzyme reaction temperature is less than 20 ° C., the enzyme reaction does not occur effectively, so the freeze-drying protection effect is somewhat reduced.
본 발명의 일 실험예에 따르면, 일반적으로 사용되는 동결건조 보호제 또는 동결건조 보호제로서 효소처리되지 않은 알로에 베라를 이용하여 유산균을 동결건조 시키는 경우보다 동결건조 보호제로서 본원발명에 따른 효소처리된 알로에 베라를 이용하여 유산균을 동결건조 시키는 경우 유산균 생균수가 더 많은 것을 확인하였다(실험예 1 및 실험예 2 참조). 이러한 결과는 효소처리된 알로에 베라를 동결건조제로 이용할 경우, 유산균 동결건조 시 발생되는 데미지를 최소화함으로써 유산균을 보호할 수 있음을 보여준다.According to an experimental example of the present invention, the enzymatically treated aloe vera according to the present invention is used as a freeze-drying protective agent rather than freeze-drying lactic acid bacteria using non-enzyme-treated aloe vera as a generally used freeze-drying protective agent or freeze-drying protective agent. When lactic acid bacteria were freeze-dried using, it was confirmed that the number of viable lactic acid bacteria was higher (see Experimental Example 1 and Experimental Example 2). These results show that when enzyme-treated aloe vera is used as a lyophilization agent, lactic acid bacteria can be protected by minimizing damage caused during freeze-drying of lactic acid bacteria.
본 발명의 다른 한 양태에 따르면, 본 발명은 효소처리 알로에 베라를 포함하는 유산균 동결건조 보호용 조성물을 포함하는 유산균 동결건조 보호제를 제공한다.According to another aspect of the present invention, the present invention provides a lactic acid bacteria freeze-drying protecting agent comprising a composition for protecting against lactic acid bacteria freeze-drying containing enzyme-treated aloe vera.
본 발명의 용어 ‘동결건조 보호제’는 동결건조 시 유산균을 동결로 인한 화학적 및 물리적 스트레스로부터 보호하고 동결된 유산균의 활성을 보존하는 기능성 제품을 의미한다.The term 'freeze-drying protective agent' of the present invention refers to a functional product that protects lactic acid bacteria from chemical and physical stress caused by freezing during freeze-drying and preserves the activity of frozen lactic acid bacteria.
본 발명에 따른 동결건조 보호제는 알로에 베라를 특정 효소로 처리하여 제조된 효소처리 알로에 베라를 이용할 수 있으며, 상기 효소는 알로에 베라의 다당류(polysaccharide)를 올리고당(oligosaccharide)으로 생성하기 위한 효소, 특히 베타-만난아제(β-mannanase)가 바람직하다.The freeze-drying protection agent according to the present invention may use enzyme-treated aloe vera prepared by treating aloe vera with a specific enzyme, and the enzyme is an enzyme for generating polysaccharide of aloe vera into oligosaccharide, particularly beta -Mannanase (β-mannanase) is preferred.
본 발명에 따른 동결건조 보호제는 효소처리 알로에 베라 이외에도 종래에 동결보호제로서 알려진 어떠한 성분도 추가로 포함될 수 있으며, 예컨대 당류, 아미노산, 펩타이드, 젤라틴, 글리세롤, 당알콜, 유청, 알긴산, 아스코르빈산, 효모 추출물, 탈지유, 트레할로스 및 마늘 파쇄물로부터 선택되는 하나 이상의 성분을 더 포함할 수 있다.In addition to the enzyme-treated aloe vera, the freeze-drying protection agent according to the present invention may further contain any components conventionally known as cryoprotectants, such as sugars, amino acids, peptides, gelatin, glycerol, sugar alcohol, whey, alginic acid, ascorbic acid, and yeast extract. , skim milk, trehalose, and garlic shavings.
본 발명의 다른 한 양태에 따르면, 본 발명은 알로에 베라를 효소로 처리하여 효소처리된 알로에 베라를 수득하는 단계, 상기 수득한 효소처리된 알로에 베라를 유산균과 혼합하는 단계 및 상기 혼합물을 동결건조 하는 단계를 포함하는 유산균 동결건조 보호 방법을 제공한다.According to another aspect of the present invention, the present invention includes the steps of treating aloe vera with an enzyme to obtain the enzyme-treated aloe vera, mixing the obtained enzyme-treated aloe vera with lactic acid bacteria, and freeze-drying the mixture. It provides a lactic acid bacteria freeze-drying protection method comprising the step.
본 발명의 유산균 동결건조 보호 방법에 있어서, 상기 알로에 베라를 효소로 처리하는 단계에서의 효소는 알로에 베라의 다당류(polysaccharide)를 올리고당(oligosaccharide)으로 생성하기 위한 효소일 수 있으며, 바람직하게는 베타-만난아제(β-mannanase)이다.In the freeze-drying protection method for lactic acid bacteria of the present invention, the enzyme in the step of treating the aloe vera with an enzyme may be an enzyme for generating polysaccharides of aloe vera into oligosaccharides, and preferably beta- It is a β-mannanase.
본 발명의 유산균 동결건조 보호 방법에 있어서, 상기 효소처리는 알로에 베라 중량(g) 당 10,000unit/g 내지 100,000unit/g, 바람직하게는 40,000unit/g 내지 60,000unit/g, 가장 바람직하게는 50,000unit/g 농도의 효소를 알로에 베라에 혼합하여 20℃ 내지 80℃, 바람직하게는 30℃ 내지 70℃, 가장 바람직하게는 70℃에서 20분 내지 60분, 바람직하게는 20분 내지 50분, 가장 바람직하게는 30분 동안 효소반응 시킬 수 있다. 상기 효소의 농도가 10,000unit/g 미만일 경우 충분한 효소처리가 이루어지기 어려우며, 100,000unit/g을 초과할 경우 효율성 및 비용적인 측면에서 바람직하지 못하다. 또한, 상기 효소반응 온도가 20℃ 미만이거나 효소반응 시간이 20분 미만일 경우 효소반응이 효과적으로 일어나지 않아 동결건조 보호 효과가 다소 떨어지며, 효소반응 온도가 80℃ 초과하거나 효소반응 시간이 60분 초과할 경우 효소반응이 과하게 일어나 동결건조 보호 효과가 전혀 나타나지 않으므로 바람직하지 못하다.In the lactic acid bacteria freeze-drying protection method of the present invention, the enzyme treatment is performed in an amount of 10,000 unit/g to 100,000 unit/g, preferably 40,000 unit/g to 60,000 unit/g, and most preferably 50,000 units/g per weight (g) of aloe vera. A unit/g concentration of enzyme is mixed with aloe vera at 20 ° C to 80 ° C, preferably 30 ° C to 70 ° C, most preferably 70 ° C for 20 minutes to 60 minutes, preferably 20 minutes to 50 minutes, most Preferably, the enzymatic reaction may be performed for 30 minutes. If the concentration of the enzyme is less than 10,000 unit / g, it is difficult to perform sufficient enzyme treatment, and if it exceeds 100,000 unit / g, it is not preferable in terms of efficiency and cost. In addition, when the enzyme reaction temperature is less than 20 ℃ or the enzyme reaction time is less than 20 minutes, the enzyme reaction does not occur effectively, the freeze-drying protection effect is somewhat reduced, and when the enzyme reaction temperature exceeds 80 ℃ or the enzyme reaction time exceeds 60 minutes It is undesirable because the enzyme reaction occurs excessively and the freeze-drying protective effect is not shown at all.
본 발명의 유산균 동결건조 보호 방법에 있어서, 효소처리된 알로에 베라를 유산균과 혼합하는 단계에서 유산균 배양액 : 효소처리 알로에 베라의 혼합비율은 1:0.5~1%(v/v), 바람직하게는 1: 0.6~0.8%(v/v)이다. 효소처리 알로에 베라가 0.5% 미만일 경우 동결건조 중 발생 되는 데미지에 의한 보호가 제대로 이루어지지 않아 균주의 활성 및 감소가 발생하여 장기보존 안정성이 낮으며, 효소처리 알로에 베라가 1% 초과할 경우 균주의 장기보관 안정성은 높으나 장기보존 시 당 성분의 증가로 단단하게 굳어지는 제형상의 문제가 발생할 수 있으므로, 적절하지 못하다.In the freeze-drying protection method for lactic acid bacteria of the present invention, in the step of mixing enzyme-treated aloe vera with lactic acid bacteria, the mixing ratio of lactic acid bacteria culture medium: enzyme-treated aloe vera is 1:0.5 to 1% (v/v), preferably 1 : 0.6~0.8% (v/v). If the amount of enzyme-treated aloe vera is less than 0.5%, the protection from damage during freeze-drying is not properly performed, resulting in the activity and reduction of the strain, resulting in low long-term storage stability. Long-term storage stability is high, but it is not appropriate because it may cause a problem with the formulation that hardens due to the increase in sugar content during long-term storage.
본 발명의 유산균 동결건조 보호 방법에 있어서, 상기 동결건조 온도는 -50℃ 내지 -20℃에서 이루어질 수 있으며, 동결 건조는 당업계에서 통상적으로 사용하는 방법을 통해 이루어질 수 있다.In the lactic acid bacteria freeze-drying protection method of the present invention, the freeze-drying temperature may be made at -50 ° C to -20 ° C, and freeze-drying may be performed through a method commonly used in the art.
전술한 바와 같이, 본 발명에 따른 효소처리 알로에 베라는 유산균을 동결건조 시 동결로 인한 화학적 및 물리적 스트레스로부터 보호하고 동결된 유산균의 활성을 보존할 수 있으므로, 동결건조 보호제로서 유용하게 이용될 수 있다.As described above, the enzyme-treated aloe vera according to the present invention protects lactic acid bacteria from chemical and physical stress caused by freezing during freeze-drying and can preserve the activity of the frozen lactic acid bacteria, so it can be usefully used as a freeze-drying protective agent. .
도 1 및 도 2는 동결건조 보호제에 따른 생균수를 확인한 결과이다.
도 3은 동결건조 보호제에 따른 장기 보존성을 확인한 결과이다.1 and 2 are the results of confirming the number of viable cells according to the lyophilization protection agent.
Figure 3 is the result of confirming the long-term preservation according to the lyophilization protective agent.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.
실시예 1: 효소처리 알로에 베라를 이용한 동결건조 유산균의 제조Example 1: Preparation of freeze-dried lactic acid bacteria using enzyme-treated aloe vera
준비된 알로에 베라에 50,000unit/g 농도로 β-Mannanase를 혼합하여 pH 2~7, 30~80℃ 조건하에서 교반기로 교반하여 혼합한다. 효소와 함께 혼합된 알로에 베라를 pH 3으로 조정하고, 70℃에서 30분간 교반기로 혼합하면서 알로에 베라를 효소처리 한다. 효소처리 30분 후 약 20℃정도까지 냉각 과정을 거쳐서 효소 반응을 정지시켜서 효소처리된 알로에 베라를 준비한다.Mix β-Mannanase with the prepared aloe vera at a concentration of 50,000 units/g and mix by stirring with a stirrer under conditions of pH 2~7 and 30~80℃. The pH of the aloe vera mixed with the enzyme was adjusted to 3, and the aloe vera was enzymatically treated while mixing with a stirrer at 70° C. for 30 minutes. After 30 minutes of enzymatic treatment, the enzymatic reaction is stopped through a cooling process to about 20° C. to prepare enzymatically treated aloe vera.
유산균으로서 당사의 기탁 균주인 락토코쿠스 락티스(Lactococcus lactis) VI-01, 락토바실러스 파라카제이(Lactobacillus paracasei) VI-02, 락토바실러스 플랜타럼(Lactobacillus plantarum) VI-05, 락토바실러스 퍼멘텀(Lactobacillus fermentum) VI-06 및 락토바실러스 람노서스(Lactobacillus rhamnosus) VI-13 균주 각각을 MRS 배지를 이용하여 종배양을 실시한 뒤 종배양액을 약 1% 접종하여 배양한다. 배양은 37℃에서 20rpm의 교반속도 조건하에서 배양하여 유산균 배양액을 준비하였다. 상기 유산균 배양액에 미리 준비한 효소처리 알로에 베라를 1:0.7(유산균 배양액:효소처리 알로에 베라)(v/v) 비율로 혼합한다. 혼합물을 -35℃ 내지 -45℃에서, 바람직하게 -40℃에서 동결건조하였다.As a lactic acid bacterium, our deposited strain Lactococcus lactis lactis ) VI-01, Lactobacillus paracasei VI-02, Lactobacillus plantarum VI-05, Lactobacillus fermentum VI-06 and Lactobacillus rhamnosus ) After performing seed culture using MRS medium for each of the VI-13 strains, about 1% of the seed culture medium is inoculated and cultured. The culture was cultured at 37 ° C. under conditions of an agitation speed of 20 rpm to prepare a culture medium of lactic acid bacteria. The previously prepared enzyme-treated aloe vera is mixed with the lactic acid bacteria culture medium at a ratio of 1:0.7 (lactic acid bacteria culture medium:enzyme-treated aloe vera) (v/v). The mixture was lyophilized at -35°C to -45°C, preferably at -40°C.
비교예 1: 동결건조 유산균의 제조 (1)Comparative Example 1: Preparation of freeze-dried lactic acid bacteria (1)
상기 실시예 1과 동일한 조건에서 동결건조 유산균을 제조하되, 동결건조 보호제로서 효소처리 알로에 베라가 아닌 당업계에서 동결건조 보호제로 이용되는 트레할로오스를 사용하였다.Lyophilized lactic acid bacteria were prepared under the same conditions as in Example 1, but trehalose, which is used as a lyophilization protection agent in the art, was used as a lyophilization protection agent, not enzyme-treated aloe vera.
실험예 1: 생균수 확인 (1)Experimental Example 1: Confirmation of viable cell count (1)
상기 실시예 1 및 비교예 1에서의 제조된 동결건조 유산균의 생균수를 측정하였다. 간략하게는 동결건조가 완료된 원분말에서 10배씩 단계 희석하여 준비한 뒤 MRS agar plate에 1ml을 도말하여 37℃ 배양기에서 24시간 이상 배양한 뒤 콜로니가 확인되었을 때 콜로니를 직접계수하고 희석배수를 적용하여 생균수를 확인하였다. 생균수를 측정한 결과는 표 1에 나타내었으며, 실시예 1의 유산균 생균수를 100으로 기준으로 하여 비교예 1의 생균수를 비율로 나타낸 결과를 도 1에 나타내었다.The viable cell count of the freeze-dried lactic acid bacteria prepared in Example 1 and Comparative Example 1 was measured. Briefly, prepare by diluting step by step 10 times from the lyophilized original powder, spread 1ml on MRS agar plate, incubate for more than 24 hours in a 37℃ incubator, and when colonies are confirmed, directly count colonies and apply the dilution factor The viable cell count was confirmed. The results of measuring the number of viable cells are shown in Table 1, and the results of expressing the number of viable cells in Comparative Example 1 as a ratio based on the number of viable cells of Example 1 being 100 are shown in FIG.
그 결과, 실시예 1과 같이 효소처리한 알로에 베라를 동결보호제로서 사용하였을 때의 유산균 생균수가 알로에 베라를 동결보호제로서 사용하지 않은 경우보다 더 많은 것으로부터 효소처리된 알로에 베라가 동결건조 중 발생되는 데미지를 최소화할 수 있음을 확인하였다.As a result, as in Example 1, when the enzymatically treated aloe vera was used as a cryoprotectant, the number of lactic acid bacteria viable was higher than when the aloe vera was not used as a cryoprotectant. It was confirmed that damage can be minimized.
비교예 2: 동결건조 유산균의 제조 (2)Comparative Example 2: Preparation of freeze-dried lactic acid bacteria (2)
상기 실시예 1과 동일한 조건에서 동결건조 유산균을 제조하되, 동결건조 보호제로서 효소처리를 하지 않은 알로에 베라를 동결건조 보호제를 사용하였다.Lyophilized lactic acid bacteria were prepared under the same conditions as in Example 1, but Aloe vera, which was not enzymatically treated, was used as a lyophilization protection agent.
실험예 2: 생균수 확인 (2)Experimental Example 2: Confirmation of viable cell count (2)
상기 실시예 1 및 비교예 2에서의 제조된 동결건조 유산균의 생균수를 측정하였다. 실험방법은 실험예 1과 동일하게 진행하였다. 생균수를 측정한 결과는 표 2에 나타내었으며, 실시예 1의 유산균 생균수를 100으로 기준으로 하여 비교예 2의 생균수를 비율로 나타낸 결과를 도 2에 나타내었다.The viable cell count of the freeze-dried lactic acid bacteria prepared in Example 1 and Comparative Example 2 was measured. The experimental method was carried out in the same way as in Experimental Example 1. The results of measuring the number of viable cells are shown in Table 2, and the results showing the number of viable cells in Comparative Example 2 as a ratio based on the number of viable cells in Example 1 being 100 are shown in FIG.
그 결과, 실시예 1과 같이 효소처리한 알로에 베라를 동결보호제로서 사용하였을 때의 유산균 생균수가 효소처리하지 않은 알로에 베라를 동결보호제로서 사용한 경우보다 더 많은 것으로부터 효소처리된 알로에 베라가 효소처리되지 않은 알로에 베라보다 동결건조 중 발생되는 데미지를 더 최소화할 수 있음을 확인하였다.As a result, as in Example 1, when enzymatically treated aloe vera was used as a cryoprotectant, the number of lactic acid bacteria viable was greater than when non-enzymatically treated aloe vera was used as a cryoprotectant. It was confirmed that damage generated during freeze-drying could be more minimized than untreated aloe vera.
실험예 3: 장기 보존성 확인Experimental Example 3: Confirmation of long-term preservation
유산균 동결 보호제로서 사용한 효소처리 알로에 베라가 유산균 장기보존에 미치는 영향을 확인하기 위하여 동결 보호제로서 효소처리 알로에 베라를 이용하여 동결 건조된 유산균과 동결 보호제로서 트레할로오스를 이용하여 동결 건조된 유산균의 생존율을 확인하였으며, 그 결과를 도 3a 내지 도 3e에 나타내었다.Lactic acid bacteria freeze-dried using enzyme-treated aloe vera as a cryoprotectant and trehalose as a cryoprotectant were tested to determine the effect of enzyme-treated aloe vera on long-term preservation of lactic acid bacteria. The survival rate was confirmed, and the results are shown in FIGS. 3a to 3e.
그 결과, 트레할로오스를 동결보호제로 사용하였을 때는 시험샘플 보관 6개월까지 최초 균수 대비 약 20% 이상의 균수가 감소하는데 반해 효소처리한 알로에 베라를 동결보호제로서 사용하였을 때 장기보존에 따른 유산균의 활성을 계속 유지 시켜 주는 것으로 나타났다.As a result, when trehalose was used as a cryoprotectant, the number of bacteria decreased by about 20% or more compared to the initial number of bacteria for up to 6 months of storage of the test sample, whereas when enzymatically treated aloe vera was used as a cryoprotectant, It has been shown to remain active.
Claims (11)
상기 효소는 베타-만난아제(β-mannanase)인 것을 특징으로 하는 유산균 동결건조 보호용 조성물.
A composition for protecting lactic acid bacteria by freeze-drying containing enzyme-treated aloe vera,
The enzyme is beta-mannanase (β-mannanase) Lactic acid bacteria freeze-drying protection composition, characterized in that.
상기 효소처리는 알로에 베라 중량(g) 당 10,000unit/g 내지 100,000unit/g 농도의 효소를 알로에 베라와 혼합하여 효소반응 시키는 것을 특징으로 하는 유산균 동결건조 보호용 조성물.
According to claim 1,
The enzyme treatment is a composition for protecting lactic acid bacteria freeze-drying, characterized in that the enzymatic reaction by mixing the enzyme at a concentration of 10,000 unit / g to 100,000 unit / g per weight (g) of aloe vera with aloe vera.
상기 효소반응 시간은 20분 내지 60분인 것을 특징으로 하는 유산균 동결건조 보호용 조성물.
According to claim 3,
Lactic acid bacteria freeze-drying protection composition, characterized in that the enzyme reaction time is 20 minutes to 60 minutes.
상기 효소반응 온도는 20℃ 내지 80℃인 것을 특징으로 하는 유산균 동결건조 보호용 조성물.
According to claim 3,
The enzyme reaction temperature is 20 ℃ to 80 ℃ lactic acid bacteria freeze-drying protective composition, characterized in that.
상기 효소는 베타-만난아제(β-mannanase)인 것을 특징으로 하는 유산균 동결 건조보호제.
A lactic acid bacteria freeze-drying protection agent comprising the lactic acid bacteria freeze-drying protection composition according to claim 1,
The enzyme is beta-mannanase (β-mannanase), characterized in that the lactic acid bacteria freeze-drying protection agent.
2) 상기 수득한 효소처리된 알로에 베라를 유산균과 혼합하는 단계; 및
3) 상기 혼합물을 동결건조 하는 단계;를 포함하는 유산균 동결건조 보호 방법으로서,
상기 1) 단계에서의 효소는 베타-만난아제(β-mannanase)인 것을 특징으로 하는 유산균 동결건조 보호 방법.
1) treating aloe vera with an enzyme to obtain an enzymatically treated aloe vera;
2) mixing the obtained enzyme-treated aloe vera with lactic acid bacteria; and
3) freeze-drying the mixture; as a lactic acid bacteria freeze-drying protection method comprising,
Lactic acid bacteria freeze-drying protection method, characterized in that the enzyme in step 1) is beta-mannanase (β-mannanase).
상기 1) 단계에서의 효소처리는,
알로에 베라 중량(g) 당 10,000unit/g 내지 100,000unit/g 농도의 효소를 알로에 베라에 혼합하여 20℃ 내지 80℃에서 20분 내지 60분 동안 효소반응 시키는 것을 특징으로 하는 유산균 동결건조 보호 방법.
According to claim 7,
Enzyme treatment in step 1),
Lactic acid bacteria freeze-drying protection method characterized by mixing an enzyme at a concentration of 10,000 unit / g to 100,000 unit / g per aloe vera weight (g) with aloe vera and enzymatically reacting at 20 ° C to 80 ° C for 20 minutes to 60 minutes.
상기 2) 단계에서의 유산균 배양액 : 효소처리 알로에 베라의 혼합비율은 1:0.5~1%(v/v)인 것을 특징으로 하는 유산균 동결건조 보호 방법.
According to claim 7,
Lactic acid bacteria culture solution in step 2): Lactic acid bacteria freeze-drying protection method, characterized in that the mixing ratio of enzyme-treated aloe vera is 1: 0.5 to 1% (v / v).
상기 3) 단계에서의 동결건조 온도는 -50℃ 내지 -20℃인 것을 특징으로 하는 유산균 동결건조 보호 방법.According to claim 7,
Lactic acid bacteria freeze-drying protection method, characterized in that the freeze-drying temperature in step 3) is -50 ℃ to -20 ℃.
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