KR102605089B1 - Microbial cryoprotectant agent containing Helianthus tuberosus extract and improving method of storage stability - Google Patents
Microbial cryoprotectant agent containing Helianthus tuberosus extract and improving method of storage stability Download PDFInfo
- Publication number
- KR102605089B1 KR102605089B1 KR1020230013649A KR20230013649A KR102605089B1 KR 102605089 B1 KR102605089 B1 KR 102605089B1 KR 1020230013649 A KR1020230013649 A KR 1020230013649A KR 20230013649 A KR20230013649 A KR 20230013649A KR 102605089 B1 KR102605089 B1 KR 102605089B1
- Authority
- KR
- South Korea
- Prior art keywords
- genus
- lactic acid
- acid bacteria
- potato extract
- pork potato
- Prior art date
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000003860 storage Methods 0.000 title claims abstract description 15
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- 240000008892 Helianthus tuberosus Species 0.000 title claims description 6
- 235000003230 Helianthus tuberosus Nutrition 0.000 title claims description 6
- 239000002577 cryoprotective agent Substances 0.000 title description 8
- 239000003795 chemical substances by application Substances 0.000 title 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 116
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- 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
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/10—Products from fruits or vegetables; Preparation or treatment thereof of tuberous or like starch containing root crops
- A23L19/12—Products from fruits or vegetables; Preparation or treatment thereof of tuberous or like starch containing root crops of potatoes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
-
- 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
- A23V2250/00—Food ingredients
- A23V2250/50—Polysaccharides, gums
- A23V2250/502—Gums
- A23V2250/5062—Inulin
-
- 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
- A23V2300/00—Processes
- A23V2300/14—Extraction
-
- 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
- A23V2300/00—Processes
- A23V2300/48—Ultrasonic treatment
-
- 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
Abstract
본 발명은 돼지감자 추출물을 포함하는 미생물 동결건조 보호용 조성물 및 이를 이용한 미생물 저장 안정성 개선 방법에 관한 것으로, 본 발명의 조성물 및 이를 이용한 방법은 식품용 유산균을 안전하게 장기보관할 수 있고, 신규 동결보호 소재로서 미생물의 생존율을 증가 시키는데 우수한 효과가 있다.The present invention relates to a freeze-dried protective composition for microorganisms containing pork potato extract and a method for improving microbial storage stability using the same. The composition of the present invention and the method using the same can safely store food-grade lactic acid bacteria for a long time and serve as a new freeze-protection material. It has an excellent effect in increasing the survival rate of microorganisms.
Description
본 발명은 돼지감자 추출물을 포함하는 미생물 동결 보호용 조성물 및 이를 이용한 미생물 저장 안정성 개선 방법에 관한 것이다.The present invention relates to a composition for microbial freeze protection containing a pork potato extract and a method for improving microbial storage stability using the same.
유산균은 당류를 에너지원으로 사용하여 유산을 생성하는 세균으로 사람이나 포유동물의 소화관, 구강 및 질 등에서 발견되며, 각종 발효식품에도 널리 분포되어 있다. 유산균은 인류가 가장 오랫동안 광범위하게 이용하고 있는 미생물 중 하나로서, 사람이나 동물의 장에 해로운 물질을 생성하지 않으며 장내에서 부패를 방지하는 순기능을 가진 미생물이다. 특히, 감염에 대한 저항성, 면역 증진 작용 등의 유익한 작용을 하는 것으로 알려져 있어서, 유산균을 포함하는 프로바이오틱스 (Probiotics)의 개발이 활발히 진행중이다.Lactic acid bacteria are bacteria that produce lactic acid using sugars as an energy source. They are found in the digestive tract, oral cavity, and vagina of humans and mammals, and are widely distributed in various fermented foods. Lactic acid bacteria are one of the microorganisms that have been widely used by mankind for the longest time. They do not produce substances harmful to the intestines of humans or animals and have the positive function of preventing decay in the intestines. In particular, the development of probiotics containing lactic acid bacteria is actively in progress because it is known to have beneficial effects such as resistance to infection and immunity enhancement.
이러한 유산균의 지속적인 안정성 유지를 위한 장기 보존 방법으로는 현재 분무건조와 동결건조 방법을 사용하고 있으며, 동결건조는 유산균 균체를 냉동 건조시킴으로서 저장하는 방법으로 대부분의 유산균을 효과적으로 장기 보존할 수 있는 방법이다. 다만, 동결건조 방법은 오염방지, 저장, 간편성의 장점을 가지고 있으나, 동결건조 과정에서 물리적이고, 생화학적인 스트레스로 세포의 활성과 생존율에 많은 영향을 미치게 되므로 유산균의 생존율을 최대한 높일 수 있는 방법이 요구되고 있다.Spray drying and freeze-drying are currently used as long-term preservation methods to maintain the continued stability of lactic acid bacteria. Freeze-drying is a method of storing lactic acid bacteria by freeze-drying them and is a method that can effectively preserve most lactic acid bacteria for a long period of time. . However, the freeze-drying method has the advantages of preventing contamination, storage, and simplicity, but the physical and biochemical stress during the freeze-drying process greatly affects the activity and survival rate of cells, so there is no method that can maximize the survival rate of lactic acid bacteria. It is being demanded.
유산균의 높은 생존율과 저장 안정성을 위한 동결 보호제로는 디메틸설폭사이드, 글리세롤, 혈청, 혈청 알부민 등의 화합물이 사용되지만 식품용에는 적합한 소재가 아니다. 또한, 식품용 동결 보호제 소재로 폴리사카라이드, 탈지유, 탈지 대두분, 설탕, 포도당 등이 사용되지만 생존율에서 큰 효용을 보이지 못하고 있는 실정이다.Compounds such as dimethyl sulfoxide, glycerol, serum, and serum albumin are used as cryoprotectants for the high survival rate and storage stability of lactic acid bacteria, but they are not suitable for food use. In addition, polysaccharides, skim milk, skim soybean flour, sugar, and glucose are used as food cryoprotectant materials, but they do not show much effectiveness in terms of survival rate.
따라서, 유산균을 장기보관하기 위해 동결건조 과정에서 발생할 수 있는 손상으로부터 유산균을 보호하면서 인체에 무해한 새로운 동결 보호제 개발이 필요한 실정이다.Therefore, in order to store lactic acid bacteria for a long time, there is a need to develop a new cryoprotectant that is harmless to the human body while protecting the lactic acid bacteria from damage that may occur during the freeze-drying process.
본 발명자들은 유산균을 장기 보관하면서 인체에 무해한 동결 보호용 조성물을 개발하고자 예의 연구 노력하였다. 그 결과, 돼지감자 추출물이 유산균을 동결 건조시 유산균의 생존율을 높이는데 우수한 효과가 있음을 확인하였다.The present inventors made extensive research efforts to develop a freeze protection composition that is harmless to the human body while storing lactic acid bacteria for a long period of time. As a result, it was confirmed that the pork potato extract had an excellent effect in increasing the survival rate of lactic acid bacteria when freeze-dried.
이에, 본 발명의 목적은 돼지감자 (Helianthus tuberosus) 추출물을 포함하는 미생물 동결 보호용 조성물을 제공하기 위한 것이다.Accordingly, the purpose of the present invention is to provide a composition for microbial freeze protection containing a pork potato ( Helianthus tuberosus ) extract.
본 발명의 다른 목적은 돼지감자 추출물과 미생물을 혼합하여 동결하는 단계를 포함하는 미생물 저장 안정성 개선 방법을 제공하기 위한 것이다.Another object of the present invention is to provide a method for improving microbial storage stability, comprising mixing pork potato extract and microorganisms and freezing them.
본 발명의 또 다른 목적은 돼지감자 추출물과 미생물을 혼합하여 동결하는 단계를 포함하는 미생물 동결 보호용 조성물 제조 방법을 제공하기 위한 것이다.Another object of the present invention is to provide a method for producing a composition for freezing protection of microorganisms, including the step of mixing pork potato extract and microorganisms and freezing them.
이에, 본 발명자들은 인체에 무해하면서 미생물의 동결 보호효과가 뛰어난 동결 보호용 조성물을 찾고자 노력한 결과, 돼지감자 추출물을 포함하는 미생물 동결 보호용 조성물을 제조하여 본 발명을 완성하게 되었다.Accordingly, the present inventors attempted to find a composition for cryoprotection that is harmless to the human body and has excellent cryoprotection effect against microorganisms, and as a result, they completed the present invention by preparing a composition for cryoprotection of microorganisms containing pork potato extract.
이하, 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는 돼지감자 (Helianthus tuberosus) 추출물을 포함하는 미생물 동결 보호용 조성물이다.One aspect of the present invention is a composition for microbial freeze protection containing an extract of pork potato ( Helianthus tuberosus ).
본 명세서 상 "돼지감자"는 국화과에 속하는 여러해살이 풀로써, 돼지감자 (Jerusalem artichoke, wild sunflower)라고 부르며, 북아메리카 원산이다. 학명은 Helianthus tuberosus로 기재한다. 돼지감자는 이눌린을 포함하고 있으며, 돼지감자의 이눌린 성분은 천연 인슐린의 효능이 있어, 혈당을 낮추며, 콜레스테롤을 개선해 주며, 수용성 식이섬유로 프리바이오틱스 효과 및 배변 활동에 도움이 된다. 돼지감자는 탄수화물과 단백질, 비타민 C, 칼륨, 철, 무기질 등이 풍부하며, 면역력을 높여주고, 피로 해소에 좋으며, 배변 활동을 도와 장 건강에 좋다. 열량이 낮고 독소를 배출한다.In this specification, "pork potato" is a perennial herb belonging to the Asteraceae family, called pork potato (Jerusalem artichoke, wild sunflower), and is native to North America. The scientific name is Helianthus tuberosus . Pork potatoes contain inulin, and the inulin component of pork potatoes has the effect of natural insulin, lowers blood sugar, improves cholesterol, and is a soluble dietary fiber that has a prebiotic effect and helps bowel movements. Pork potatoes are rich in carbohydrates, protein, vitamin C, potassium, iron, and minerals. They increase immunity, are good for relieving fatigue, and help bowel movements, which are good for intestinal health. It is low in calories and excretes toxins.
본 명세서 상 "동결보호"는 유산균의 활성을 그대로 보존하기 위해 동결 건조하여 보관할 때 유산균 조직을 결빙으로부터 보호하는 것을 의미한다. 본 출원에서 "동결건조 (Freeze drying)"는 용기의 온도를 급격하게 낮추어 건조시키고자 하는 재료를 얼린 다음, 용기 내부의 압력을 진공으로 만들어 재료에 포함된 고체화된 용매를 바로 수증기로 승화시켜 건조하는 방법이다. 동결 건조는 열에 민감한 물질의 손상을 최소화하고, 효과적으로 장기 보존할 수 있는 방법으로서 오염 방지, 저장, 수송, 경제성 측면에서 유용하다. 하지만, 유산균의 동결 건조 과정에서 유산균의 활성과 생존율이 급격하게 감소하게 되고, 동결시 얼음 입자가 만들어지면서 유산균 세포의 막 구조를 손상시키는 문제점이 있다. 이를 개선하기 위해 유산균이 손상되거나 사멸하지 않고 재수화 시 그 기능을 회복할 수 있도록 동결 건조시 함께 첨가하는 물질을 "동결 보호제"라고 지칭하며, 본 발명에서는 "동결 보호용 조성물"이라고 지칭하였다.As used herein, “freeze protection” means protecting lactic acid bacteria tissue from freezing when freeze-dried and stored in order to preserve the activity of lactic acid bacteria. In this application, "Freeze drying" refers to freezing the material to be dried by rapidly lowering the temperature of the container, then creating a vacuum inside the container to immediately sublimate the solidified solvent contained in the material into water vapor and drying it. This is how to do it. Freeze drying is a method that minimizes damage to heat-sensitive materials and allows effective long-term preservation, and is useful in terms of pollution prevention, storage, transportation, and economic efficiency. However, during the freeze-drying process of lactic acid bacteria, the activity and survival rate of lactic acid bacteria decrease rapidly, and ice particles are created during freezing, which damages the membrane structure of lactic acid bacteria cells. To improve this, the substance added during freeze-drying so that the lactic acid bacteria are not damaged or killed and can recover their function upon rehydration is referred to as a “freeze protection agent,” and in the present invention, it is referred to as a “freeze protection composition.”
본 발명에 있어서 동결 보호용 조성물은 액상태로 제제화될 수 있다. 식품 제조를 위해 액상의 유산균을 배양하여 사용하기 보다는 동결된 형태 또는 동결건조된 분말의 형태로 제조하여 보급하는 것이 식품의 품질 향상 및 품질의 안정성 향상을 위해 유리하다.In the present invention, the composition for cryoprotection may be formulated in liquid form. Rather than cultivating and using liquid lactic acid bacteria for food production, it is advantageous to manufacture and distribute them in frozen or freeze-dried powder form to improve food quality and quality stability.
본 발명에 있어서 돼지감자 추출물은 물, 탄소수 1 내지 4의 저급 알코올 또는 이들의 혼합 용매에 의해 추출된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pork potato extract may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, but is not limited thereto.
본 발명에 있어서 탄소수 1 내지 4의 저급 알코올은 에탄올 (Ethanol) 및 이소프로판올 (Isopropanol)로 이루어진 군에서 선택된 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the lower alcohol having 1 to 4 carbon atoms may be one or more selected from the group consisting of ethanol and isopropanol, but is not limited thereto.
본 발명에 있어서 미생물은 유산균인 것일 수 있다.In the present invention, the microorganism may be a lactic acid bacterium.
본 발명에 있어서 유산균은 류코노스톡 (Leuconostoc)속, 락토바실러스 (Lactobacillus)속, 락토코커스 (Lactococcus)속, 비피도박테리움 (Bifidobacterium)속, 클로스트리듐 (Clostridium)속, 코리네박테리움 (Corynebacterium)속, 엔테로코커스 (Enterococcus)속, 스트렙토코커스 (Streptococcus)속, 카르노박테리움 (Carnobacterium)속, 아토포비움 (Atopobium)속, 테트라제노코쿠스 (Tetragenococcus)속, 와이셀라 (Weissella)속, 오에노코커스 (Oenococcus)속 및 바고코커스 (Vagococcus)속으로 이루어진 군에서 선택되는 하나 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the lactic acid bacteria include Leuconostoc genus, Lactobacillus genus, Lactococcus genus, Bifidobacterium genus, Clostridium genus, Corynebacterium ( Corynebacterium genus , Enterococcus genus, Streptococcus genus, Carnobacterium genus, Atopobium genus, Tetragenococcus genus, Weissella genus, It may be one or more selected from the group consisting of the genus Oenococcus and the genus Vagococcus , but is not limited thereto.
본 발명에 있어서 유산균은 류코노스톡 메센테로이데스 (Leuconostoc mesenteroides)인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the lactic acid bacteria may be Leuconostoc mesenteroides , but are not limited thereto.
본 발명에 있어서 돼지감자 추출물은 이눌린 (Inulin)을 유효성분으로 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pork potato extract may contain inulin as an active ingredient, but is not limited thereto.
본 발명에 있어서 이눌린은 총 돼지감자 추출물 함량 대비 5 내지 50 중량%, 5 내지 45 중량%, 5 내지 40 중량%, 5 내지 35 중량%, 5 내지 30 중량%, 5 내지 25 중량%, 10 내지 50 중량%, 10 내지 45 중량%, 10 내지 30 중량%, 10 내지 25 중량%, 예를 들어, 10 중량% 포함된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, inulin is 5 to 50% by weight, 5 to 45% by weight, 5 to 40% by weight, 5 to 35% by weight, 5 to 30% by weight, 5 to 25% by weight, and 10 to 10% by weight, based on the total pork potato extract content. It may contain 50% by weight, 10 to 45% by weight, 10 to 30% by weight, 10 to 25% by weight, for example, 10% by weight, but is not limited thereto.
본 발명에 있어서 유산균 동결 보호용 조성물은 "돼지감자 추출물"을 제외하고도, 종래에 동결보호제로 알려진 성분을 추가로 포함할 수 있다. 즉, 본 출원에서 유산균 동결 보호용 조성물에 추가로 포함되는 "동결 보호제"는 "돼지감자 추출물"을 제외하고 해당 기술분야에서 통상적으로 사용되는 동결 보호 효능을 가지는 물질을 의미한다. 상기 동결 보호제는 시판되는 것을 구입하여 이용할 수 있다. 상기 동결 보호제는 트레할로스, 탈지유, 당류, 아미노산, 펩티드, 젤라틴, 글리세롤, 당알콜, 유청, 알긴산, 아스코르빈산, 효모 추출물 등을 사용할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the composition for lactic acid bacteria cryoprotection may further include ingredients conventionally known as cryoprotectants, excluding "pork potato extract". That is, in this application, the “cryoprotectant” additionally included in the lactic acid bacteria cryoprotection composition refers to a substance having a cryoprotective effect commonly used in the relevant technical field, excluding “pork potato extract”. The cryoprotectant can be purchased and used commercially. The cryoprotectant may include, but is not limited to, trehalose, skim milk, sugars, amino acids, peptides, gelatin, glycerol, sugar alcohol, whey, alginic acid, ascorbic acid, yeast extract, etc.
본 발명의 다른 일 양태는 돼지감자 추출물과 미생물을 혼합하여 동결하는 단계를 포함하는 미생물 저장 안정성 개선 방법이다.Another aspect of the present invention is a method of improving microbial storage stability comprising mixing pork potato extract and microorganisms and freezing them.
본 발명의 또 다른 일 양태는 돼지감자 추출물과 미생물을 혼합하여 동결하는 단계를 포함하는 미생물 동결 보호용 조성물 제조 방법이다.Another aspect of the present invention is a method for producing a composition for freezing protection of microorganisms, comprising the step of mixing pork potato extract and microorganisms and freezing them.
본 발명에 있어서 돼지감자 추출물은 물, 탄소수 1 내지 4의 저급 알코올 또는 이들의 혼합 용매에 의해 추출된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pork potato extract may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, but is not limited thereto.
본 발명에 있어서 탄소수 1 내지 4의 저급 알코올은 에탄올 (Ethanol) 및 이소프로판올 (Isopropanol)로 이루어진 군에서 선택된 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the lower alcohol having 1 to 4 carbon atoms may be one or more selected from the group consisting of ethanol and isopropanol, but is not limited thereto.
본 발명에 있어서 미생물은 유산균인 것일 수 있다.In the present invention, the microorganism may be a lactic acid bacterium.
본 발명에 있어서 유산균은 류코노스톡 (Leuconostoc)속, 락토바실러스 (Lactobacillus)속, 락토코커스 (Lactococcus)속, 비피도박테리움 (Bifidobacterium)속, 클로스트리듐 (Clostridium)속, 코리네박테리움 (Corynebacterium)속, 엔테로코커스 (Enterococcus)속, 스트렙토코커스 (Streptococcus)속, 카르노박테리움 (Carnobacterium)속, 아토포비움 (Atopobium)속, 테트라제노코쿠스 (Tetragenococcus)속, 와이셀라 (Weissella)속, 오에노코커스 (Oenococcus)속 및 바고코커스 (Vagococcus)속으로 이루어진 군에서 선택되는 하나 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the lactic acid bacteria include Leuconostoc genus, Lactobacillus genus, Lactococcus genus, Bifidobacterium genus, Clostridium genus, Corynebacterium ( Corynebacterium genus , Enterococcus genus, Streptococcus genus, Carnobacterium genus, Atopobium genus, Tetragenococcus genus, Weissella genus, It may be one or more selected from the group consisting of the genus Oenococcus and the genus Vagococcus , but is not limited thereto.
본 발명에 있어서 유산균은 류코노스톡 메센테로이데스 (Leuconostoc mesenteroides)인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the lactic acid bacteria may be Leuconostoc mesenteroides , but are not limited thereto.
본 발명에 있어서 돼지감자 추출물은 이눌린 (Inulin)을 유효성분으로 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pork potato extract may contain inulin as an active ingredient, but is not limited thereto.
본 발명에 있어서 이눌린은 총 돼지감자 추출물 함량 대비 5 내지 50 중량%, 5 내지 45 중량%, 5 내지 40 중량%, 5 내지 35 중량%, 5 내지 30 중량%, 5 내지 25 중량%, 10 내지 50 중량%, 10 내지 45 중량%, 10 내지 30 중량%, 10 내지 25 중량%, 예를 들어, 10 중량% 포함된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, inulin is 5 to 50% by weight, 5 to 45% by weight, 5 to 40% by weight, 5 to 35% by weight, 5 to 30% by weight, 5 to 25% by weight, and 10 to 10% by weight, based on the total pork potato extract content. It may contain 50% by weight, 10 to 45% by weight, 10 to 30% by weight, 10 to 25% by weight, for example, 10% by weight, but is not limited thereto.
본 발명에 있어서 돼지감자 추출물은 60 내지 120 ℃, 60 내지 110 ℃, 60 내지 100 ℃, 60 내지 90 ℃, 70 내지 120 ℃, 70 내지 110 ℃, 70 내지 100 ℃, 70 내지 90 ℃, 80 내지 120 ℃, 80 내지 110 ℃, 80 내지 100 ℃, 예를 들어, 90 ℃의 온도로 추출하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pork potato extract is 60 to 120 ℃, 60 to 110 ℃, 60 to 100 ℃, 60 to 90 ℃, 70 to 120 ℃, 70 to 110 ℃, 70 to 100 ℃, 70 to 90 ℃, 80 to 80 ℃. Extraction may be performed at a temperature of 120°C, 80 to 110°C, 80 to 100°C, for example, 90°C, but is not limited thereto.
본 발명에 있어서 돼지감자 추출물은 30 내지 90분, 30 내지 80분, 30 내지 70분, 30 내지 60분, 40 내지 90분, 40 내지 80분, 40 내지 70분, 40 내지 60분, 50 내지 90분, 50 내지 80분, 50 내지 70분, 50 내지 60분, 예를 들어, 60분 동안 추출하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pork potato extract is 30 to 90 minutes, 30 to 80 minutes, 30 to 70 minutes, 30 to 60 minutes, 40 to 90 minutes, 40 to 80 minutes, 40 to 70 minutes, 40 to 60 minutes, 50 to 50 minutes. Extraction may be performed for 90 minutes, 50 to 80 minutes, 50 to 70 minutes, or 50 to 60 minutes, for example, 60 minutes, but is not limited thereto.
본 발명에 있어서 돼지감자 추출물은 5초 내지 200초, 5 내지 190초, 5 내지 180초, 10초 내지 200초, 10초 내지 190초, 10초 내지 180초, 예를 들어, 180초 동안 초음파 처리가 된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pork potato extract is ultrasonicated for 5 seconds to 200 seconds, 5 to 190 seconds, 5 to 180 seconds, 10 seconds to 200 seconds, 10 seconds to 190 seconds, 10 seconds to 180 seconds, for example, 180 seconds. It may have been processed, but is not limited to this.
본 발명에 있어서 초음파 처리단계는 주파수가 20 내지 50 kHz, 20 내지 45 kHz, 20 내지 40 kHz, 25 내지 50 kHz, 25 내지 45 kHz, 25 내지 40 kHz, 30 내지 50 kHz, 30 내지 45 kHz, 30 내지 40 kHz, 35 내지 50 kHz, 35 내지 45 kHz, 35 내지 40 kHz, 예를 들어, 40 kHz인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the ultrasonic treatment step has a frequency of 20 to 50 kHz, 20 to 45 kHz, 20 to 40 kHz, 25 to 50 kHz, 25 to 45 kHz, 25 to 40 kHz, 30 to 50 kHz, 30 to 45 kHz, It may be 30 to 40 kHz, 35 to 50 kHz, 35 to 45 kHz, 35 to 40 kHz, for example, 40 kHz, but is not limited thereto.
본 발명의 또 다른 일 양태는 동결 형태의 유산균 제제 또는 동결건조 유산균 분말 제제의 제조에 있어서, 멸균수 내에 현탁된 유산균에 대해 동결 보호용 조성물로 돼지감자 추출물을 첨가하여 동결 또는 동결건조하는 것을 포함하는 유산균의 동결 보호 방법, 즉, 동결건조 유산균 분말의 제조에 있어서 유산균의 동결보호를 위한 돼지감자 추출물의 용도를 제공한다. 상기 용도를 구현하기 위한 구성은 앞서 설명한 바와 같다.Another aspect of the present invention includes adding pork potato extract as a freeze-protection composition to lactic acid bacteria suspended in sterilized water and freezing or freeze-drying them in the production of a frozen lactic acid bacteria preparation or a freeze-dried lactic acid bacteria powder preparation. A method for freeze-protecting lactic acid bacteria, that is, the use of pork potato extract for freeze-protection of lactic acid bacteria in the production of freeze-dried lactic acid bacteria powder, is provided. The configuration for implementing the above purpose is the same as described above.
본 발명은 돼지감자 추출물을 포함하는 미생물 동결 보호용 조성물 및 이를 이용한 미생물 저장 안정성 개선 방법에 관한 것으로, 본 발명의 조성물 및 이를 이용한 방법은 식품용 유산균을 안전하게 장기보관할 수 있고, 신규 동결보호 소재로서 미생물의 생존율을 증가 시키는데 우수한 효과가 있다.The present invention relates to a composition for microbial freeze protection containing pork potato extract and a method for improving microbial storage stability using the same. The composition of the present invention and the method using the same can safely store food-grade lactic acid bacteria for a long period of time and can serve as a new freeze protection material for microorganisms. It has an excellent effect in increasing the survival rate.
도 1은 본 발명의 일 실시예에 있어서 돼지감자 추출물의 추출 온도 및 추출 시간에 따른 이눌린 (Inulin) 함량을 비교하여 나타낸 그래프이다.
도 2는 본 발명의 일 실시예에 있어서 돼지감자 추출물의 당 분석 결과를 나타낸 그래프이다.
도 3은 본 발명의 일 실시예에 있어서 동결 보호소재군의 종류에 따른 유산균의 생존율을 비교하여 나타낸 그래프이다.
도 4는 본 발명의 일 실시예에 있어서 돼지감자 추출물에 초음파 처리 시간에 따른 유산균의 생존율을 비교하여 나타낸 그래프이다.
도 5는 본 발명의 일 실시예에 있어서 돼지감자 추출물, 초음파 처리를 한 돼지감자 추출물 및 동결 보호소재군의 종류에 따른 유산균의 생존율을 비교하여 나타낸 그래프이다.Figure 1 is a graph showing comparison of the inulin content according to the extraction temperature and extraction time of the pork potato extract in one embodiment of the present invention.
Figure 2 is a graph showing the sugar analysis results of the pork potato extract in one embodiment of the present invention.
Figure 3 is a graph comparing the survival rate of lactic acid bacteria according to the type of freeze protection material group in one embodiment of the present invention.
Figure 4 is a graph showing the survival rate of lactic acid bacteria according to the sonication time in the pork potato extract in one embodiment of the present invention.
Figure 5 is a graph showing the survival rate of lactic acid bacteria according to the types of pork potato extract, ultrasonicated pork potato extract, and freeze-protection material group in one embodiment of the present invention.
이하, 본 발명을 실시예를 통해 상세히 설명한다. 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the present invention and the scope of the present invention is not limited to the following examples.
제조예 1. 동결 보호용 조성물 제조Preparation Example 1. Preparation of composition for freeze protection
1-1. 돼지감자 추출물의 제조1-1. Preparation of pork potato extract
증류수에 파쇄된 건조 돼지감자 (대한민국, 오허브사)를 10 %(w/v) 혼합한 후, 30, 50, 70 및 90 ℃의 항온수조에서 5, 30 및 60분 추출한 후, 실린지 필터로 여과하고 HPLC를 이용하여 구체적인 성분을 분석하였다. After mixing 10% (w/v) of crushed dried pork potatoes (Oherb, Korea) in distilled water, extraction was performed in a constant temperature water bath at 30, 50, 70, and 90°C for 5, 30, and 60 minutes, and then filtered using a syringe filter. It was filtered and specific components were analyzed using HPLC.
1-2. 돼지감자 추출물의 성분 분석1-2. Component analysis of pork potato extract
돼지감자 추출물의 성분 분석은 HPLC-RID를 활용하여 진행하였고 컬럼은 REZEX monosaccharide RPM, 용매는 증류수를 사용하였다. 상기한 온도 및 시간의 조건으로 각각 추출한 돼지감자 추출물의 이눌린 함량을 비교하여 도 1에 나타내었다.Component analysis of the pork potato extract was conducted using HPLC-RID, REZEX monosaccharide RPM column was used, and distilled water was used as the solvent. The inulin content of the pork potato extract extracted under the above-mentioned temperature and time conditions is compared and shown in Figure 1.
도 1에서 확인할 수 있듯이, 돼지감자 추출물에서 이눌린 함량이 가장 높은 것은 90 ℃ 및 60분 조건으로 추출하였을 때 였으며, 이때 37.3 mg/ml이 추출되었다.As can be seen in Figure 1, the highest inulin content in the pork potato extract was when extracted under conditions of 90°C and 60 minutes, and at this time, 37.3 mg/ml was extracted.
실시예 1. 돼지감자 추출물 당 성분 분석Example 1. Analysis of sugar components in pork potato extract
이눌린 함량이 가장 가장 높은 것으로 확인된 90 ℃ 및 60분 조건으로 추출한 돼지감자 추출물의 당 성분 분석을 진행하였다. 90 ℃로 예열된 열수에 파쇄된 돼지감자를 10 %(w/v)로 혼합한 다음 90 ℃의 항온수조에서 60분간 추출하였다. 추출물은 실린지 필터로 여과한 후 HPLC-RI Detector를 이용하여 분석하였다. 칼럼은 Shodex asahipak NH2P-50 4E, 용매는 아세토니트릴과 증류수를 7:3으로 혼합하여 사용하고, 그 결과를 도 2에 나타내었다.Sugar content analysis was conducted on the pork potato extract extracted at 90°C and 60 minutes, which was found to have the highest inulin content. Shredded pork potatoes were mixed at 10% (w/v) in hot water preheated to 90°C and then extracted in a constant temperature water bath at 90°C for 60 minutes. The extract was filtered through a syringe filter and then analyzed using an HPLC-RI Detector. The column used was Shodex asahipak NH2P-50 4E, and the solvent was a mixture of acetonitrile and distilled water in a ratio of 7:3, and the results are shown in Figure 2.
도 2에서 확인할 수 있듯이, 돼지감자 추출물의 당 분석 결과 주요 성분으로 설탕 (당 중합도 2, 27.1 mg/mL)과 올리고당 (당 중합도 3-10, 29.11 mg/mL) 그리고 고분자의 당 (당 중합도 11 이상, 8.3 ml/mL)로 구성되었다. 이 중 이눌린은 올리고당 및 고분자 당에 해당되며 추출물의 주요한 성분이기 때문에 동결보호 소재로의 개발 가능성이 높다고 판단하였다.As can be seen in Figure 2, as a result of sugar analysis of the pork potato extract, the main components are sugar (degree of sugar polymerization 2, 27.1 mg/mL), oligosaccharide (degree of sugar polymerization 3-10, 29.11 mg/mL), and polymer sugar (degree of sugar polymerization 11). above, 8.3 ml/mL). Among these, inulin corresponds to oligosaccharides and high molecular sugars and is a major component of the extract, so it was judged to have a high possibility of being developed as a cryoprotection material.
실시예 2. 유산균 동결건조 후 생존율 검정Example 2. Survival rate test after freeze-drying lactic acid bacteria
돼지감자 추출물의 유산균 동결보호능을 평가하기 위해 대조군으로 트레할로스 (Trehalose)와 탈지유 (Skim milk)를 사용하여 평가하였다. 실시예 1과 동일한 방법으로 추출한 돼지감자 추출물과 트레할로스, 탈지유를 각각 증류수에 10 %(w/v)로 혼합하고 121 ℃에서 15분간 멸균하여 준비하였다. 유산균으로는 류코노스톡 메센테로이데스 (Leuconostoc mesenteroides) WiKim33을 대상으로 동결보호능 평가를 진행하였다. 400 mL MRS broth에 유산균을 접종한 후, 30 ℃에서 24시간 배양하고, 원심 분리한 후, 20 mL의 멸균증류수로 농축하였다. 그 다음, 농축한 유산균 5 mL을 동량의 동결 보호소재군으로 준비한 돼지감자 추출물, 트레할로스 및 탈지유에 혼합한 후 - 80 ℃에서 4시간 동결 후 40시간 동결건조를 진행하였다. 동결 건조된 유산균은 10 mL의 멸균(증류)수로 수화 후 희석하여 MRS agar plate에 도말 및 30 ℃에서 24 내지 48시간 배양 후 생존율 검정을 분석하여 그 결과를 도 3에 나타내었다.To evaluate the lactic acid bacteria cryoprotective ability of the pork potato extract, trehalose and skim milk were used as controls. Pork potato extract, trehalose, and skim milk extracted in the same manner as in Example 1 were each mixed at 10% (w/v) in distilled water and sterilized at 121°C for 15 minutes. As a lactic acid bacterium, Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) WiKim33 was tested for cryoprotective function. After inoculating lactic acid bacteria into 400 mL of MRS broth, it was cultured at 30°C for 24 hours, centrifuged, and concentrated with 20 mL of sterile distilled water. Next, 5 mL of concentrated lactic acid bacteria were mixed with the same amount of pork potato extract, trehalose, and skim milk prepared from the freeze-protection material group, and then frozen at -80°C for 4 hours and then freeze-dried for 40 hours. The freeze-dried lactic acid bacteria were hydrated and diluted with 10 mL of sterilized (distilled) water, smeared on an MRS agar plate, cultured at 30°C for 24 to 48 hours, and then analyzed for survival rate. The results are shown in Figure 3.
도 3에서 확인할 수 있듯이, 동결 보호소재를 무처리한 경우 균주의 생존율은 29.6 %을 나타내었으며, 동결보호소재로 사용되는 트레할로스 및 탈지유를 처리한 후 균주의 생존율은 각각 73.0 % 및 58.7 %를 나타내었다. 반면, 돼지감자 추출물을 적용한 균주의 생존율은 82.8%로 처리하지 않았거나, 다른 동결보호소재로 처리하였을 때 보다 높은 생존률을 나타내었다.As can be seen in Figure 3, when the freeze protection material was not treated, the survival rate of the strain was 29.6%, and after treatment with trehalose and skim milk, which are used as freeze protection materials, the survival rates of the strain were 73.0% and 58.7%, respectively. It was. On the other hand, the survival rate of the strain to which the pork potato extract was applied was 82.8%, which was higher than that when not treated or treated with other cryoprotective materials.
실시예 3. 초음파 처리 시간에 따른 돼지감자 추출물 적용 유산균 생존율 평가Example 3. Evaluation of lactic acid bacteria survival rate applied with pork potato extract according to sonication time
돼지감자 추출물의 동결보호능을 증진시키기 위해 초음파 처리를 하였다. 돼지감자 추출물을 초음파 수조에 넣고 10초, 30초, 60초 및 180초 초음파 처리를 하여 동결건조 보호소재를 준비하였다. 류코노스톡 메센테로이데스 (Leuconostoc mesenteroides) WiKim33에 초음파 처리하지 않은 돼지감자 추출물, 각각 10초, 30초, 60초 및 180초로 초음파 처리를 한 돼지감자 추출물을 유산균에 처리한 후 생존율을 분석하였다. 실시예 2와 동일하게 대조군으로는 아무것도 처리하지 않은 무처리구를 비교하였다.To improve the cryoprotective ability of the pork potato extract, ultrasonic treatment was performed. The pork potato extract was placed in an ultrasonic bath and ultrasonicated for 10 seconds, 30 seconds, 60 seconds, and 180 seconds to prepare freeze-dried protective material. Leuconostoc mesenteroides The survival rate was analyzed after treating non-sonicated pork potato extract and sonicated pork potato extract for 10 seconds, 30 seconds, 60 seconds, and 180 seconds respectively in WiKim33 with lactic acid bacteria. As in Example 2, an untreated group that was not treated with anything was compared as a control group.
도 4에서 확인할 수 있듯이, 무처리구는 31.5%의 유산균의 생존율을 나타내었고, 돼지감자 추출물을 처리한 실험군은 81.4%을 나타내었다. 반면, 돼지감자 추출물에 10초 초음파 처리한 실험군은 83.5%, 30초 초음파 처리한 실험군은 88.4%, 60초 초음파 처리한 실험군은 92.4%, 180초 초음파 처리를 한 실험군은 91.8%의 생존율을 나타내었다. 이는 돼지감자 추출물을 초음파로 처리하는 시간이 경과함에 따라 동결보존하고자 하는 미생물의 생존율이 증가하는 경향을 보이며, 초음파 처리 시간을 60초로 한 실험군에서 가장 높은 생존율을 나타내었다.As can be seen in Figure 4, the untreated group showed a survival rate of lactic acid bacteria of 31.5%, and the experimental group treated with pork potato extract showed 81.4%. On the other hand, the experimental group that sonicated the pork potato extract for 10 seconds showed a survival rate of 83.5%, the experimental group that sonicated the extract for 30 seconds showed a survival rate of 88.4%, the experimental group that sonicated the pork potato extract for 60 seconds had a survival rate of 92.4%, and the experimental group that sonicated the pork potato extract for 180 seconds showed a survival rate of 91.8%. It was. This shows that the survival rate of microorganisms to be cryopreserved tends to increase as the time for treating the pork potato extract with ultrasonic waves elapses, and the highest survival rate was shown in the experimental group where the ultrasonic treatment time was 60 seconds.
초음파 처리 시간에 따라 돼지감자 추출물에 함유된 단당 (glucose, fructose)의 함량이 감소하는 것을 확인할 수 있다. 단당은 유산균에 직접 적용하여 동결건조를 했을 때 생존율에 부정적인 영향을 주되, 초음파를 처리하여 돼지감자 추출물 내의 단당 함량을 감소시키면 유산균이 동결 건조 후 생존율을 높일 수 있음을 확인할 수 있었다.It can be seen that the content of simple sugars (glucose, fructose) contained in the pork potato extract decreases depending on the sonication time. It was confirmed that simple sugars have a negative effect on the survival rate when applied directly to lactic acid bacteria and freeze-drying them, but when the simple sugar content in the pork potato extract is reduced by ultrasonic treatment, the survival rate of lactic acid bacteria can be increased after freeze-drying.
실시예 4. 유산균 저장 안정성 평가Example 4. Lactic acid bacteria storage stability evaluation
실시예 1 내지 3에서 평가한 트레할로스, 탈지유, 돼지감자 추출물 및 60초간 초음파 처리된 돼지감자 추출물의 유산균 저장 안정성을 평가하였다. 각각의 돼지감자 추출물, 초음파 처리된 돼지감자 추출물을 유산균에 처리한 후 동결건조 후에 - 20 ℃에 저장하여 24주간의 생존율을 평가하였다.The lactic acid bacteria storage stability of trehalose, skim milk, pork potato extract, and pork potato extract sonicated for 60 seconds evaluated in Examples 1 to 3 was evaluated. Each pork potato extract and sonicated pork potato extract were treated with lactic acid bacteria, freeze-dried, and stored at -20°C to evaluate the survival rate for 24 weeks.
도 5에서 확인할 수 있듯이, 무처리된 유산균은 동결건조 직후 29.6%의 생존율을 보였으며, 저장 24주 경과 이후 18.9 %의 생존율을 나타내었다. 그리고 돼지감자 추출물이 처리된 유산균은 동결건조 직후 82.9%, 24주 경과 80.2 %의 생존율을 나타내었다. 또한, 60초 초음파 처리를 한 돼지감자 추출물은 동결건조 직후 95.2 %, 저장 24주 경과 88.8%의 생존율을 나타내어 가장 높은 생존율을 나타내었다. 동일한 돼지감자 추출물이라도 초음파 처리 유무에 따라 유산균의 생존율이 동결건조 직후에는 12.3%, 저장 24주 경과 후에는 8.6% 증가된 것을 확인할 수 있었다.As can be seen in Figure 5, untreated lactic acid bacteria showed a survival rate of 29.6% immediately after freeze-drying, and a survival rate of 18.9% after 24 weeks of storage. And lactic acid bacteria treated with pork potato extract showed a survival rate of 82.9% immediately after freeze-drying and 80.2% after 24 weeks. In addition, the pork potato extract treated with ultrasonic waves for 60 seconds showed the highest survival rate, with a survival rate of 95.2% immediately after freeze-drying and 88.8% after 24 weeks of storage. Even for the same pork potato extract, it was confirmed that the survival rate of lactic acid bacteria increased by 12.3% immediately after freeze-drying and by 8.6% after 24 weeks of storage depending on whether or not ultrasonic treatment was used.
Claims (18)
상기 돼지감자 추출물을 60 내지 180초 동안 초음파 처리하는 단계;
상기 돼지감자 추출물은 추출물 총 함량 대비 5 내지 50 중량%의 이눌린(Inulin)을 유효성분으로 포함하는,
미생물 동결 보호용 조성물 제조 방법.Preparing a pork potato extract by extracting pork potatoes ( Helianthus tuberosus ) with hot water at a temperature of 60 to 120° C. for 30 to 90 minutes; and
Sonicating the pork potato extract for 60 to 180 seconds;
The pork potato extract contains 5 to 50% by weight of inulin as an active ingredient based on the total extract content,
Method for producing a composition for microbial cryoprotection.
을 혼합하여 동결하는 단계를 포함하는 미생물 저장 안정성 개선 방법.A composition for microbial freeze protection prepared by the method according to any one of claims 1, 3 to 5; and a microorganism according to any one of claims 3 to 5;
A method of improving microbial storage stability comprising mixing and freezing.
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