KR101263567B1 - Preparation Method of Fermented Medicinal-Herb Composition And Fermented Medicinal-Herb Composition Using The Same - Google Patents
Preparation Method of Fermented Medicinal-Herb Composition And Fermented Medicinal-Herb Composition Using The Same Download PDFInfo
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- KR101263567B1 KR101263567B1 KR1020100120022A KR20100120022A KR101263567B1 KR 101263567 B1 KR101263567 B1 KR 101263567B1 KR 1020100120022 A KR1020100120022 A KR 1020100120022A KR 20100120022 A KR20100120022 A KR 20100120022A KR 101263567 B1 KR101263567 B1 KR 101263567B1
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- lactobacillus
- bacteria
- acid
- cfu
- herbal
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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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
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
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- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
본 발명은 한방발효조성물 제조방법 및 이에 따른 한방발효 조성물에 관한 것으로, 보다 구체적으로는 (S1) 제조한약인 방풍통성산을 준비하는 단계; (S2) 락토바실러스균을 준비하는 단계; 및 (S3) 준비된 한약에 락토바실러스균을 접종하고 30~45℃에서 발효하는 단계;를 포함하는 한방발효 조성물 제조방법에 대한 것이다. 본 발명에 따르면 한약, 특히 방풍통성산의 다양한 생리활성 기능은 가지고 있으면서 방풍통성산의 항산화 효과, 항균효과 및 알레르기 억제 효과 등을 더욱 증진시킨 한방발효 조성물을 제공할 수 있다.
이에 따라 상기한 본 발명에 따른 한방발효 조성물은 알레르기 개선, 피부노화 및 병원성 미생물에 의한 피부질환의 개선에 효과가 있을 것으로 기대되므로 화장료 조성물, 약학 조성물, 식품 조성물 등에 함유되어 유용하게 이용될 수 있다.The present invention relates to a method for producing an herbal fermentation composition and a herbal fermentation composition according to the present invention, and more specifically, (S1) preparing a wind-breaking acid which is a Chinese medicine; (S2) preparing the Lactobacillus bacteria; And (S3) inoculating Lactobacillus bacteria on the prepared Chinese medicine and fermenting at 30-45 ° C .; According to the present invention, it is possible to provide an herbal fermentation composition which further enhances the antioxidant, antimicrobial and allergic effects of the windproof tonic acid while having various bioactive functions.
Accordingly, the herbal fermentation composition according to the present invention is expected to be effective in improving allergies, skin aging and skin diseases caused by pathogenic microorganisms, and thus may be usefully contained in cosmetic compositions, pharmaceutical compositions, and food compositions. .
Description
본 발명은 식품, 화장품, 제약 산업 분야에서 이용될 수 있는 한방발효 조성물의 제조방법 및 이에 따른 한방발효 조성물에 관한 것이다.
The present invention relates to a method for producing an herbal fermentation composition that can be used in the food, cosmetics, and pharmaceutical industries, and thus to a herbal fermentation composition.
최근 고령화 사회의 도래로 웰빙 트랜드가 사회 전반에 뿌리내리고 있으며, 이는 식품, 화장품, 주거 환경 등의 모든 분야에서 천연 식물성 소재 및 전통적인 민간 요법으로 전래되는 한약에 대한 관심을 증폭시켰다.
With the advent of an aging society in recent years, well-being trends are taking root throughout society, which has amplified the interest in herbal medicines that are introduced as natural vegetable materials and traditional folk remedies in all fields such as food, cosmetics and residential environment.
상대적으로 천연 식물소재의 제품화가 용이한 화장품 산업 분야에서 오래전부터 이를 원료로 사용한 시도들이 있어 왔으며, 기능식품 및 예방의학, 치료의학 분야에서도 동양의 식물성 약재(Herbal medicine)가 미국 및 유럽에서까지 허가 및 제품화되기 시작하면서, 천연 식물소재 및 한약의 인체 유용성에 대한 관심이 증가하였다.
In the cosmetics industry where natural plant materials are relatively easy to commercialize, there have been attempts to use them as raw materials for a long time, and oriental herbal medicines are approved in the US and Europe for functional foods, preventive medicine and therapeutic medicine. And as it begins to be commercialized, there is an increasing interest in the human usefulness of natural plant materials and herbal medicines.
천연 식물소재 또는 한약재는 다양한 생리활성 물질과 항산화 물질을 함유하고 있어 항노화, 항암, 항염, 면역기능 개선 등 다양한 효과를 보이지만, 추출된 이후에는 불안정하고 때론 자극적이어서, 인체에 독성을 띄는 경우도 종종 있다. 따라서, 최근에는 천연 추출물을 안정화시키거나, 독성을 줄이거나, 안정한 유도체로 전환시켜 효과를 높이려는 시도가 많이 진행되고 있으며, 그 일환으로 미생물이나 효소를 이용한 생물학적 전환(Biological transformation) 방법이 개발되고 있으며, 대표적인 방법으로 발효를 예로 들 수 있다.
Natural plant material or herbal medicine contains various biologically active substances and antioxidants, so it shows various effects such as anti-aging, anti-cancer, anti-inflammatory, and immune function improvement, but it is unstable and sometimes irritating after extraction, so it is toxic to human body. Often. Therefore, in recent years, many attempts have been made to stabilize the natural extracts, reduce their toxicity, or convert them into stable derivatives to increase their effectiveness. As a part, biological transformation methods using microorganisms or enzymes have been developed. As a representative method, fermentation may be mentioned.
발효는 미생물이 가지고 있는 효소를 이용하여 유기물을 분해시켜서 인간 생활에 유용하게 사용되는 물질을 제조하는 과정으로서, 인체에 유용한 물질을 만들어내는 대표적인 발효 미생물로는 유산균, 효모 등이 있고 다양한 식품들이 발효 과정에 의해서 생산된다. 또한, 발효는 유용 물질을 만들어 내는 것 이외에도 발효에 관여하는 유산균이 장 내에서 면역 및 독소제거의 역할을 담당하기도 한다. 더 나아가, 장내 미생물에 의한 발효를 통해 섭취된 성분은 분해되어 인체 세포에 흡수가 용이한 저분자 물질로 전환되거나, 불안정하거나 불활성인 형태에서 활성 형태로 전환되어 흡수되기도 하며, 이러한 결과는 장내 미생물에 의한 생물전환의 중요성을 단적으로 보여주는 것이라 볼 수 있다.
Fermentation is a process of producing substances that are useful for human life by decomposing organic substances using enzymes of microorganisms. Representative fermentation microorganisms that produce substances useful for human body include lactic acid bacteria and yeast, and various foods are fermented. Produced by the process. In addition to fermentation, in addition to producing useful substances, lactic acid bacteria involved in fermentation may play a role in immunity and detoxification in the intestine. Furthermore, the components ingested through fermentation by the intestinal microorganisms are decomposed and converted into low-molecular substances that are easily absorbed by human cells, or converted into unstable or inactive forms in the active form, which is absorbed by the intestinal microorganisms. This shows the importance of biotransformation.
따라서, 유산균 및 효모 등의 미생물 발효에 의해서 천연 식물소재 및 한약의 효능을 극대화시키거나, 안정화 문제를 해결하기 위한 시도는 다양한 식품, 화장품, 약품 등의 개발에 있어서 중요한 의미를 갖는다 하겠다.
Therefore, attempts to maximize the efficacy of natural plant material and herbal medicine or to solve the stabilization problem by microbial fermentation such as lactic acid bacteria and yeast have important meanings in the development of various foods, cosmetics, medicines and the like.
방풍통성산(防風通聖散)은 중국의 “의명대론” 및 한국의 “동의보감” 등에 기록되어 있는 처방으로서, 활석, 감초, 석고, 황금, 길경, 방풍, 당귀, 청궁, 적작약, 대황, 마황, 박하, 연교, 망초, 형개, 백출, 치자로 구성되어 있다. 상기 방풍통성산은 자가중독물이 쌓여있는 것을 피부, 비뇨기 및 소화기 등을 통하여 배설하고 해독하는데 유용하며, 비만성졸중 체질에 사용되는 경우가 많고, 체내의 식독, 수독, 풍독, 열독 등에도 처방된다. 이외에도 비만증, 습관성 변비, 고혈압, 만성신장염, 단독, 치질, 축농증, 당뇨병 등에도 응용될 수 있다. 또한, 중풍, 동맥경화, 염증성 피부질환에 사용되고 있으며, 지질저하 효과, 진통, 소염, 항균효과, 알레르기 억제 및 면역 효과, 간기능 증강효과 등 다양한 질환들에서 응용될 수 있다.
Windbreak Tongseongsan is a prescription recorded in China's theory of medicine, and Korea's "Consensus Bogam". Talc, licorice, gypsum, gold, Gilgyeong, windproof, Dongguk, Cheongung, Red Peony, rhubarb, ephedra, It is composed of peppermint, duct, forget-me-not, mold opening, extraction and gardenia. The wind-breaking acid is useful for excretion and detoxification of accumulated self-toxic substances through skin, urinary and digestive organs, and is often used for constitution of obesity, and is prescribed for food poisoning, poisoning, poisoning, heat poisoning and the like in the body. In addition, it can be applied to obesity, habitual constipation, hypertension, chronic nephritis, alone, hemorrhoids, sinusitis, diabetes and the like. In addition, it is used in stroke, arteriosclerosis, and inflammatory skin diseases, and may be applied to various diseases such as lipid lowering effect, analgesic, anti-inflammatory, antibacterial effect, allergy suppression and immune effect, and liver function enhancing effect.
본 발명에서는 한약, 특히 방풍통성산의 다양한 생리활성 기능은 가지고 있으면서 방풍통성산의 항산화 효과, 항균효과 및 알레르기 억제 효과 등을 더욱 증진시킬 수 있는 방법을 연구하고 이를 완성하였다.In the present invention, while studying a variety of physiological activity of the herbal medicine, especially windproof tonic acid has been studied and completed a method that can further enhance the antioxidant, antibacterial and allergic inhibitory effect of windproof tonic acid.
본 발명은 한약, 특히 방풍통성산의 다양한 생리활성 기능은 가지고 있으면서 방풍통성산의 항산화 효과, 항균효과 및 알레르기 억제 효과 등을 더욱 증진시킬 수 있는 한방발효 조성물 제조방법을 제공하고자 한다.
The present invention is to provide a herbal fermentation composition manufacturing method that can further enhance the antioxidant, antimicrobial and allergic effect of the windproof tonic acid while having a variety of physiological activity of the herbal medicine, especially windproof tongseong.
이에 본 발명은 바람직한 제1구현예로서 (S1) 제조한약인 방풍통성산을 준비하는 단계; (S2) 락토바실러스균을 준비하는 단계; 및 (S3) 준비된 방풍통성산에 락토바실러스균을 접종하고 30~45℃에서 발효하는 단계;를 포함하는 한방발효조성물 제조방법을 제공한다.
Therefore, the present invention provides a windproof tongsan acid prepared as a preferred first embodiment (S1); (S2) preparing the Lactobacillus bacteria; And (S3) inoculating Lactobacillus bacteria in the prepared wind-proof tonic acid and fermentation at 30 ~ 45 ℃; provides a method for producing a fermentation composition comprising a.
상기 구현예에 의한 한방발효조성물 제조방법에서, (S1) 단계에서의 방풍통성산은 1~5℃, 2,000~5,000×g으로 원심분리하여 침전물을 제거한 것일 수 있다.
In the method of producing a herbal fermented composition according to the above embodiment, the wind-breaking acid in step (S1) may be one to 5 ℃, 2,000 ~ 5,000 × g by centrifugation to remove the precipitate.
상기 구현예에 의한 한방발효조성물 제조방법에서, (S1) 단계에서의 방풍통성산은 pH를 4~7로 조정하여 준비되는 것일 수 있다.
In the method of producing a herbal fermented composition according to the embodiment, the wind-breaking acid in step (S1) may be prepared by adjusting the pH to 4-7.
상기 구현예에 의한 한방발효조성물 제조방법에서, (S1) 단계에서의 방풍통성산에 1~5%(w/v)의 설탕을 첨가한 것인 한방발효조성물 제조방법을 제공한다.
In the method of manufacturing herbal fermentation composition according to the above embodiment, it provides a method of producing an herbal fermentation composition in which 1-5% (w / v) of sugar is added to the wind-breaking acid in step (S1).
상기 구현예에 의한 한방발효조성물 제조방법에서, (S2) 단계에서의 락토바실러스균은 락토바실러스 브레비스 ( Lactobacillus brevis ), 락토바실러스 카제이 ( Lactobacillus casei ), 락토바실러스 델브루에키 서브스피시즈 불가리쿠스 ( Lactobacillus delbrueckii subsp . bultaricus ), 락토바실러스 퍼멘텀 ( Lactobacillus fermentum ), 락토바실러스 헬베티쿠스 ( Lactobacillus helveticus ), 락토바실러스 플란타럼 ( Lactobacillus plantarum )으로 이루어진 군에서 선택된 2종 이상인 한방발효조성물 제조방법을 제공한다.
In Oriental fermented composition production method according to this embodiment, the Lactobacillus bacteria in (S2) step is Lactobacillus brevis (Lactobacillus brevis), Lactobacillus casei (Lactobacillus casei), Lactobacillus del Brewer station sub-species Bulgaria kusu (Lactobacillus delbrueckii subsp . bultaricus), Lactobacillus momentum spread (Lactobacillus fermentum), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus Planta column (Lactobacillus plantarum) to provides a process for producing fermented herbal composition how two or more member selected from the group consisting of.
상기 구현예에 의한 한방발효조성물 제조방법에서, (S2) 단계에서의 락토바실러스균은 균수가 1×106~1×108CFU/㎖으로 조절된 것일 수 있다.
In the herbal fermentation composition production method according to the embodiment, the Lactobacillus bacteria in step (S2) may be the number of bacteria adjusted to 1 × 10 6 ~ 1 × 10 8 CFU / ㎖.
상기 구현예에 의한 한방발효조성물 제조방법에서, (S2) 단계에서의 락토바실러스균은 0.8×106~5×106CFU/㎖의 락토바실러스 브레비스 ( Lactobacillus brevis ) 및 락토바실러스 카제이 ( Lactobacillus casei ), 0.8×107~5×107CFU/㎖의 락토바실러스 델브루에키 서브스피시즈 불가리쿠스 ( Lactobacillus delbrueckii subsp . bultaricus ) 및 락토바실러스 플란타럼 ( Lactobacillus plantarum ), 0.8×108~5×108CFU/㎖의 락토바실러스 퍼멘텀 ( Lactobacillus fermentum ) 및 락토바실러스 헬베티쿠스 ( Lactobacillus helveticus )를 포함하는 것일 수 있다.
In Oriental fermented composition production method according to this embodiment, the Lactobacillus bacteria in (S2) phase is 0.8 × 10 6 ~ 5 × 10 6 CFU / ㎖ Lactobacillus brevis (Lactobacillus brevis) of and Lactobacillus casei (Lactobacillus casei ), 0.8 × 10 7 ~ 5 × 10 7 CFU / ㎖ of Lactobacillus del brewer sub-species Echinacea Bulgaria kusu (Lactobacillus delbrueckii subsp. bultaricus) and Lactobacillus Planta column (Lactobacillus plantarum), 0.8 × 10 8 ~ 5 × 10 8 CFU / ㎖ Lactobacillus momentum spread (Lactobacillus of It may be one containing fermentum), and Lactobacillus helveticus (Lactobacillus helveticus).
상기 구현예에 의한 한방발효조성물 제조방법에서, (S2) 단계에서의 락토바실러스균은 0.8×106~5×106CFU/㎖의 락토바실러스 브레비스 ( Lactobacillus brevis ) 및 락토바실러스 카제이 ( Lactobacillus casei ), 0.8×107~5×107CFU/㎖의 락토바실러스 헬베티쿠스 ( Lactobacillus helveticus ) 및 락토바실러스 플란타럼 ( Lactobacillus plantarum ), 0.8×108~5×108CFU/㎖의 락토바실러스 퍼멘텀 ( Lactobacillus fermentum ) 및 락토바실러스 델브루에키 서브스피시즈 불가리쿠스 ( Lactobacillus delbrueckii subsp . bultaricus )를 포함하는 것일 수 있다.
In Oriental fermented composition production method according to this embodiment, the Lactobacillus bacteria in (S2) phase is 0.8 × 10 6 ~ 5 × 10 6 CFU / ㎖ Lactobacillus brevis (Lactobacillus brevis) of and Lactobacillus casei (Lactobacillus casei ), 0.8 × 10 7 ~ 5 × 10 7 CFU / ㎖ of Lactobacillus helveticus (Lactobacillus helveticus) and Lactobacillus Planta column (Lactobacillus plantarum), 0.8 × 10 8 ~ 5 × 10 8 CFU / ㎖ of Lactobacillus buffer momentum (Lactobacillus fermentum) and Lactobacillus del brewer sub-species Echinacea Bulgaria kusu (Lactobacillus delbrueckii subsp . bultaricus ).
상기 구현예에 의한 한방발효조성물 제조방법에서, (S3) 단계에서는 락토바실러스 브레비스 ( Lactobacillus brevis ), 락토바실러스 델브루에키 서브스피시즈 불가리쿠스 ( Lactobacillus delbrueckii subsp . bultaricus ) 및 락토바실러스 헬베티쿠스 ( Lactobacillus helveticus )를 접종한 후 4~5일째에 락토바실러스 퍼멘텀 ( Lactobacillus fermentum ) 및 락토바실러스 플란타럼 ( Lactobacillus plantarum )을 접종하는 것인 한방발효조성물 제조방법을 제공한다.
Herbal composition in the fermentation production process according to this embodiment, (S3) step, Lactobacillus brevis (Lactobacillus brevis), species Lactobacillus del brewer sub-station Bulgaria kusu (Lactobacillus delbrueckii subsp . bultaricus), and Lactobacillus helveticus (Lactobacillus helveticus) the momentum (Lactobacillus inoculated after Lactobacillus bacteria spread in 4-5 days fermentum ) and Lactobacillus Provides that the herbal composition of the fermentation method of inoculating Planta Rum (Lactobacillus plantarum).
상기 구현예에 의한 한방발효조성물 제조방법에서, 각각의 락토바실러스균은 균수를 1×108CFU/㎖로 조절한 후 접종되는 것일 수 있다.
In the method of manufacturing herbal fermentation composition according to the embodiment, each of the Lactobacillus bacteria may be inoculated after adjusting the number of bacteria to 1 × 10 8 CFU / ㎖.
상기 구현예에 의한 한방발효조성물 제조방법에서, (S3) 단계에서는 락토바실러스균을 방풍통성산에 대하여 0.1~1%(v/v)의 양으로 접종되는 것일 수 있다.
In the method of producing a herbal fermented composition according to the embodiment, in the step (S3) may be inoculated with Lactobacillus bacteria in an amount of 0.1 ~ 1% (v / v) to the windproof acid.
본 발명은 바람직한 제2구현예로서 상기의 제조방법에 의하여 제조되어, 항균 및 항알레르기 활성을 나타내는 한방발효조성물을 제공한다.
The present invention provides a herbal fermentation composition prepared by the above production method as a second preferred embodiment, exhibiting antibacterial and antiallergic activity.
상기 구현예에 의한 한방발효조성물은 슈도모나스 애루지노사 ( Pseudomonas aeruginosa ), 스타필로코쿠스 아우레우스 속. 아우레우스 ( Staphylococcus aureus subsp . aureus ) 및 프로피오니박테리움 아크네스 ( Propionibacterium acnes )에 대하여 항균활성을 나타내는 것일 수 있다.
Herbal composition according to the embodiment, the fermentation Pseudomonas her labor Rouge (Pseudomonas aeruginosa), Staphylococcus brother in-house records. Aureus ( Staphylococcus aureus subsp . aureus ) And Propionic sludge may be an indication of an antimicrobial activity against tumefaciens arc Ness (Propionibacterium acnes).
본 발명은 바람직한 제3구현예로서 상기 한방발효 조성물을 함유하는 식품 조성물을 제공한다.
The present invention provides a food composition containing the herbal fermentation composition as a third preferred embodiment.
본 발명은 바람직한 제4구현예로서 상기 한방발효 조성물을 함유하는 약학 조성물을 제공한다.
The present invention provides a pharmaceutical composition containing the herbal fermentation composition as a fourth preferred embodiment.
본 발명은 바람직한 제5구현예로서 상기 한방발효 조성물을 함유하는 화장료 조성물을 제공한다.
The present invention provides a cosmetic composition containing the herbal fermentation composition as a fifth preferred embodiment.
본 발명에 따르면 한약, 특히 방풍통성산의 다양한 생리활성 기능은 가지고 있으면서 방풍통성산의 항산화 효과, 항균효과 및 알레르기 억제 효과 등을 더욱 증진시킨 한방발효 조성물을 제공할 수 있다. 상기한 본 발명에 따른 한방발효 조성물은 알레르기 개선, 피부노화 및 병원성 미생물에 의한 피부질환의 개선에 효과가 있을 것으로 기대되므로 화장료 조성물, 약학 조성물, 식품 조성물 등에 함유되어 유용하게 이용될 수 있다.
According to the present invention, it is possible to provide an herbal fermentation composition which further enhances the antioxidant, antimicrobial and allergic effects of the windproof tonic acid while having various bioactive functions. The herbal fermentation composition according to the present invention is expected to be effective in improving allergies, skin aging and skin diseases caused by pathogenic microorganisms, and thus may be usefully contained in cosmetic compositions, pharmaceutical compositions, food compositions, and the like.
도 1은 본 발명의 바람직한 일 실시예에 따라 5일간 배양된 한방발효 조성물(락토바실러스균)을 BPB가 첨가된 lactobacilli MRS 배지에 분주 후 도말하고 3일간 배양한 모습을 나타내는 사진이다.
도 2는 본 발명의 실시예 1과 실시예 2의 락토바실러스균의 성장곡선을 나타내는 그래프이고, 도 3은 본 발명의 실시예 3의 락토바실러스균의 성장곡선을 나타내는 그래프이다.
도 4는 본 발명의 실시예 3에 따른 한방발효 조성물의 배양기간에 따른 pH 변화를 나타내는 그래프이고, 도 5는 본 발명의 실시예 1 및 실시예 2에 따른 한방발효 조성물의 배양기간에 따른 pH 변화를 나타내는 그래프이다.
도 6은 본 발명의 바람직한 실시예에 따른 각 시료의 배양기간에 따른 DPPH 소거능을 나타내는 그래프이고, 도 7은 양성 대조구인 BHA의 농도에 따른 DPPH 소거능을 나타내는 그래프이다.
도 8은 본 발명의 바람직한 실시예에 따른 각 시료의 배양기간에 따른 SOD 유사 활성을 나타내는 그래프이다.
도 9는 본 발명의 바람직한 실시예에 따른 각 시료의 여드름균을 유발시키는 프로피오니박테리움 아크네스 ( Propionibacterium acnes )에 대한 항균활성을 나타내는 사진이다.
도 10은 RBL-2H3 세포주를 대상으로 한 한약(방풍통성산)의 농도에 따른 세포 독성을 나타내는 그래프이고, 도 11은 RBL-2H3 세포주를 대상으로 하여 음성 대조구, 방풍통성산 및 실시예 3의 한방발효 조성물의 처리 시간에 따른 세포 독성을 나타내는 그래프이다.
도 12는 RBL-2H3 세포주를 대상으로 하여 본 발명에 따른 실시예 3의 한방발효 조성물의 β-헥소사미니다아제(β-hexosaminidase)의 방출 억제 효과를 나타내는 그래프이다.
도 13은 RBL-2H3 세포주를 대상으로 하여 본 발명에 따른 실시예 3의 한방발효 조성물의 pH에 대한 β-헥소사미니다아제(β-hexosaminidase)의 방출 억제 효과를 나타내는 그래프이다.1 is a photograph showing the culture of herbal fermentation composition (Lactobacillus bacteria) cultured for 5 days in accordance with a preferred embodiment of the present invention after dispensing in lactobacilli MRS medium to which BPB is added and cultured for 3 days.
Figure 2 is a graph showing the growth curve of Lactobacillus bacteria of Example 1 and Example 2 of the present invention, Figure 3 is a graph showing the growth curve of Lactobacillus bacteria of Example 3 of the present invention.
Figure 4 is a graph showing the pH change according to the culture period of the herbal fermentation composition according to Example 3 of the present invention, Figure 5 is a pH according to the culture period of the herbal fermentation composition according to Examples 1 and 2 of the present invention Graph showing change.
6 is a graph showing DPPH scavenging ability according to the culture period of each sample according to a preferred embodiment of the present invention, Figure 7 is a graph showing DPPH scavenging ability according to the concentration of BHA, a positive control.
8 is a graph showing the SOD-like activity according to the culture period of each sample according to a preferred embodiment of the present invention.
Figure 9 is a photograph showing the antibacterial activity on Propionibacterium sludge tumefaciens arc Ness (Propionibacterium acnes) that cause acne bacteria of the samples according to an embodiment of the present invention.
10 is a graph showing the cytotoxicity according to the concentration of the Chinese medicine (windproof tonic acid) for the RBL-2H3 cell line, Figure 11 is a negative control, windproof tonic acid and herbal fermentation of Example 3 for the RBL-2H3 cell line It is a graph showing the cytotoxicity over the treatment time of the composition.
12 is a graph showing the inhibitory effect of β-hexosaminidase release of the herbal fermentation composition of Example 3 according to the present invention on RBL-2H3 cell line.
13 is a graph showing the inhibitory effect of β-hexosaminidase on the pH of the herbal fermentation composition of Example 3 according to the present invention for RBL-2H3 cell line.
이하, 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 한방발효 조성물 제조방법 및 이에 따른 한방발효 조성물에 관한 것으로, 한약인 방풍통성산에 락토바실러스균을 접종하고 발효시켜 방풍통성산의 다양한 생리활성 기능은 가지고 있으면서 방풍통성산의 항산화 효과, 항균효과 및 알레르기 억제 효과 등을 더욱 증진시킨 한방발효 조성물을 제공한다.
The present invention relates to a method for producing an herbal fermentation composition and a herbal fermentation composition according to the present invention, inoculated and fermented with lactobacillus bacteria in windproof tonic acid, which is a Chinese medicine, while having various physiological activity functions of windproof tonic acid, and antibacterial effect and antibacterial effect. It provides an herbal fermentation composition further enhanced allergic inhibitory effect.
이를 위하여 본 발명은 (S1)제조한약인 방풍통성산을 준비하는 단계; (S2) 락토바실러스균을 준비하는 단계; 및 (S3) 준비된 방풍통성산에 락토바실러스균을 접종하고 30~45℃에서 발효하는 단계;를 포함하는 한방발효조성물 제조방법을 제공한다.
To this end, the present invention comprises the steps of preparing a wind-breaking acid (S1) a pharmaceutical preparation; (S2) preparing the Lactobacillus bacteria; And (S3) inoculating Lactobacillus bacteria in the prepared wind-proof tonic acid and fermentation at 30 ~ 45 ℃; provides a method for producing a fermentation composition comprising a.
상기 (S1) 단계에서의 한약인 방풍통성산(활석, 감초, 석고, 황금, 길경, 방풍, 천궁, 당귀, 적작약, 대황, 마황, 박하, 연교, 망초, 형개, 백출 및 치자)은 방풍통성산의 효능에 크게 벗어나지 않는 범위에서 몇 가지 약재를 가감시켜 제조할 수도 있다. 구체적인 예로는 활석 6.37g, 감초 4.5g, 석고, 황금 및 길경 각각 2.62g, 방풍, 천궁, 당귀, 적작약, 대황, 마황, 박하, 연교 및 망초 각각 1.68g, 형개, 백출 및 치자 각각 1.31g 및 생강 5g으로 이루어진 한약재로 제조한 것이 바람직하다. Windproof Tongseong acid (Talc, licorice, gypsum, golden, Gilgyeong, windproof, Cheongung, Donkey, Red Peony, rhubarb, ephedra, peppermint, yeonkyo, forget-me-not, hyunghwa, Baekchul and Gardenia) in the step (S1) of the windproof Tongsan It can also be prepared by adding or subtracting some herbs within a range that does not deviate significantly from the efficacy. Specific examples include talc 6.37g, licorice 4.5g, gypsum, gold and Gilyeong 2.62g, windproof, cheongung, donkey, red peony, rhubarb, ephedra, peppermint, pontoon and forget-me-not 1.68g respectively, hyunghyeol, baekrye and gardenia respectively. It is preferable to prepare a herbal medicine consisting of 5g ginger.
상기 방풍통성산은 1~5℃에서 2000~5000×g으로 원심분리하여 침전물을 제거한 후 이용하는 것이 바람직하며, pH를 4~7로 조정하는 것이 바람직하다.The wind-breaking acid is preferably used after centrifugation at 2000 ° C. to 5000 × g at 1 ° C. to remove the precipitate, and the pH is adjusted to 4-7.
또한, 상기 방풍통성산에는 미생물의 생육을 증대시키기 위하여 설탕(특히 brown sugar)을 방풍통성산에 대하여 1~5%(w/v)의 양으로 첨가하는 것이 바람직하다.
In addition, it is preferable to add sugar (especially brown sugar) to the windproof acid in an amount of 1 to 5% (w / v) with respect to the windproof acid.
그리고, (S2) 단계에서는 상기 방풍통성산을 발효시키기 위한 락토바실러스균을 준비한다. 상기 락토바실러스균은 락토바실러스 브레비스 ( Lactobacillus brevis ), 락토바실러스 카제이 ( Lactobacillus casei ), 락토바실러스 델브루에키 서브스피시즈 불가리쿠스 ( Lactobacillus delbrueckii subsp. bultaricus ), 락토바실러스 퍼멘텀 ( Lactobacillus fermentum ), 락토바실러스 헬베티쿠스 ( Lactobacillus helveticus ), 락토바실러스 플란타럼 ( Lactobacillus plantarum )으로 이루어진 군에서 선택된 2종 이상을 포함하는 것이 바람직하다. 또한, 락토바실러스균은 균수를 1×106~1×108CFU/㎖으로 조절하여 접종하는 것이 균의 생장에 바람직하다.
And, in step (S2) to prepare the Lactobacillus bacteria for fermenting the wind-proof acid. The Lactobacillus bacteria are Lactobacillus brevis (Lactobacillus brevis), Lactobacillus casei (Lactobacillus casei), Lactobacillus del Brewer station sub-species Bulgaria kusu (Lactobacillus delbrueckii subsp. Bultaricus), Lactobacillus buffer momentum (Lactobacillus fermentum), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus To include a combination of two or more selected from the group consisting of Planta column (Lactobacillus plantarum) is preferred. In addition, Lactobacillus bacteria are preferably inoculated by controlling the number of bacteria at 1 × 10 6 to 1 × 10 8 CFU / ml.
본 발명에서는 락토바실러스균을 복합배양하는 것이 바람직하다. 본 발명에서의 복합배양은 균수를 달리한 여러 종의 락토바실러스균을 접종하고 발효시키는 경우와 생육조건에 따라 여러 종의 락토바실러스균을 시기를 달리하여 접종하고 발효시키는 경우 등을 의미한다. In the present invention, it is preferable to complex culture the Lactobacillus bacteria. The complex culture in the present invention refers to the case of inoculating and fermenting several species of Lactobacillus bacteria with different numbers of bacteria and the case of inoculating and fermenting several kinds of Lactobacillus bacteria at different times according to growth conditions.
본 발명에서의 복합배양을 위한 락토바실러스균의 바람직한 구체적 예로는 0.8×106~5×106CFU/㎖의 락토바실러스 브레비스 ( Lactobacillus brevis ) 및 락토바실러스 카제이 ( Lactobacillus casei ), 0.8×107~5×107CFU/㎖의 락토바실러스 델브루에키 서브스피시즈 불가리쿠스 ( Lactobacillus delbrueckii subsp . bultaricus ) 및 락토바실러스 플란타럼 ( Lactobacillus plantarum ), 0.8×108~5×108CFU/㎖의 락토바실러스 퍼멘텀 ( Lactobacillus fermentum ) 및 락토바실러스 헬베티쿠스 ( Lactobacillus helveticus )를 포함하는 것이다. Preferred specific examples of the Lactobacillus bacteria for complex culture in the present invention is 0.8 × 10 6 ~ 5 × 10 6 CFU / ㎖ Lactobacillus brevis (Lactobacillus brevis) and Lactobacillus casei (Lactobacillus casei) of, 0.8 × 10 7 ~ 5 × 10 7 CFU / ㎖ of Lactobacillus del brewer sub-species Echinacea Bulgaria kusu (Lactobacillus delbrueckii subsp . bultaricus ) and Lactobacillus Planta column (Lactobacillus plantarum), 0.8 × 10 8 ~ 5 × 10 8 CFU / ㎖ Lactobacillus momentum spread (Lactobacillus of intended to include fermentum), and Lactobacillus helveticus (Lactobacillus helveticus).
또는 0.8×106~5×106CFU/㎖의 락토바실러스 브레비스 ( Lactobacillus brevis ) 및 락토바실러스 카제이 ( Lactobacillus casei ), 0.8×107~5×107CFU/㎖의 락토바실러스 헬베티쿠스 ( Lactobacillus helveticus ) 및 락토바실러스 플란타럼 ( Lactobacillus plantarum ), 0.8×108~5×108CFU/㎖의 락토바실러스 퍼멘텀 ( Lactobacillus fermentum ) 및 락토바실러스 델브루에키 서브스피시즈 불가리쿠스 ( Lactobacillus delbrueckii subsp . bultaricus )를 포함할 수도 있다.
Or 0.8 × 10 6 ~ 5 × 10 6 CFU / ㎖ of Lactobacillus brevis (Lactobacillus brevis) and Lactobacillus casei (Lactobacillus casei), 0.8 × 10 7 ~ 5 × 10 7 CFU / ㎖ of Lactobacillus helveticus ( Lactobacillus helveticus ) and Lactobacillus Planta column (Lactobacillus plantarum), 0.8 × 10 8 ~ 5 × 10 8 CFU / ㎖ Lactobacillus momentum spread (Lactobacillus of fermentum) and Lactobacillus del brewer sub-species Echinacea Bulgaria kusu (Lactobacillus delbrueckii subsp. may include bultaricus).
그리고, (S3) 단계에서는 준비된 방풍통성산에 상기 준비된 락토바실러스균을 접종하고 30~45℃에서 발효시킨다. 이때, 락토바실러스균은 방풍통성산에 대하여 0.1~1%(v/v)의 양으로 접종하는 것이 바람직하다.And, in step (S3) inoculated with the prepared Lactobacillus bacteria in the prepared wind-breaking acid and fermented at 30 ~ 45 ℃. At this time, the Lactobacillus bacteria is preferably inoculated in an amount of 0.1 ~ 1% (v / v) with respect to windproof acid.
본 발명의 복합배양을 위한 바람직한 또 다른 구체적 예로는, 각각 균수를 1×108CFU/㎖로 조절한 락토바실러스 브레비스 ( Lactobacillus brevis ), 락토바실러스 델브루에키 서브스피시즈 불가리쿠 스 ( Lactobacillus delbrueckii subsp . bultaricus ) 및 락토바실러스 헬베티쿠스 ( Lactobacillus helveticus )를 방풍통성산에 접종한 후, 상기 접종일로부터 4~5일째에 각각 균수를 1×108CFU/㎖로 조절한 락토바실러스 퍼멘텀 ( Lactobacillus fermentum ) 및 락토바실러스 플란타럼 ( Lactobacillus plantarum )을 각각 0.1~0.3%(v/v)씩 접종하여 발효시키는 방법이 있다.
Another preferred specific examples for the composite culture of the present invention, each of the number of
상기와 같이 제조된 본 발명의 한방발효 조성물은 발효하기 전의 한약(방풍통성산)보다 항산화 활성이 더욱 우수하고, 바실루스 서브틸리스 ( Bacillus subtilis ) PCI 219, 슈도모나스 애루지노사 ( Pseudomonas aeruginosa ) KCTC 2004, 스타필로코쿠스 아우레우스 속. 아우레우스 ( Staphylococcus aureus subsp . aureus ) KCTC 1916 등에 항균활성을 보였으며, 특히 여드름을 유발시키는 프로피오니박테리움 아크네스 ( Propionibacterium acnes ) KCTC 3314에 항균활성을 나타내었다. 프로피오니박테리움 아크네스 ( Propionibacterium acnes ) KCTC 3314에 대한 항균활성은 발효하기 전의 방풍통성산에는 없던 것이다. 또한, 60%에 가까운 알레르기 억제 효과를 나타내었다. 따라서, 본 발명에 따른 한방발효 조성물은 알레르기 개선, 피부노화 및 병원성 미생물에 의한 피부질환의 개선에 효과가 있을 것으로 기대된다.
The herbal fermentation composition of the present invention prepared as described above is more excellent in antioxidant activity than Chinese herbal medicine (windproof through acid) before fermentation, Bacillus subtilis ( Bacillus subtilis ) PCI 219, Pseudomonas her labor Rouge (Pseudomonas aeruginosa ) KCTC 2004, Staphylococcus brother in-house records. Aureus ( Staphylococcus aureus subsp . aureus), KCTC 1916, etc. propynyl sludge tumefaciens arc Ness (Propionibacterium acnes which showed antimicrobial activity, in particular causing acne)
이하, 본 발명의 구성을 실시예를 통하여 보다 상세히 설명하나, 본 발명의 범위가 하기 실시예로 한정되는 것은 아니다.
Hereinafter, the configuration of the present invention in more detail through examples, but the scope of the present invention is not limited to the following examples.
실시예Example
한약 선정 및 제조Chinese medicine selection and manufacturing
하기 표 1에서와 같이, 활석 6.37g, 감초 4.5g, 석고, 황금 및 길경 각각 2.62g, 방풍, 천궁, 당귀, 적작약, 대황, 마황, 박하, 연교 및 망초 각각 1.68g, 형개, 백출 및 치자 각각 1.31g, 생강 5g을 배합한 한약인 방풍통성산은 대구 약령시 내의 한의원에서 제조 의뢰하였다.
As shown in Table 1 below, Talc 6.37g, Licorice 4.5g, Gypsum, Golden and Gilt 2.62g respectively, Windproof, Cheongung, Angelica, Red Peony, Rhubarb, Ephedra, Peppermint, Yeongyo and Forget-me-not 1.68g, Hungae, Baekchul and Gardenia, respectively. Bangpung Tongseong-san, a blended Chinese medicine, was manufactured and ordered by a Korean medical clinic in Yangnyeong-si, Daegu.
그리고, 제조된 방풍통성산은 4℃, 4,000×g에서 10분간 원심분리하여 침전물을 제거한 후 pH를 6.3으로 조정하고 121℃에서 15분간 멸균하였다.
The prepared wind-breaking acid was centrifuged at 4 ° C. and 4,000 × g for 10 minutes to remove precipitates, and then the pH was adjusted to 6.3 and sterilized at 121 ° C. for 15 minutes.
락토바실러스균의Lactobacillus 준비 Ready
상기 준비된 방풍통성산을 발효시키기 위한 유용미생물로서 락토바실러스균을 선정하였으며, 하기 표 2에서와 같이 한국생명공학연구원 유전자원센터 유전자은행(KCTC)에서 분양받아 락토바실리 MRS 배지(proteose peptone No.3 10g, beef extract 10g, yeast extract 5g, dextrose 20g, Polyoxyethylene sorbitan monooleate 1g, ammonium citrate 2g, maganessium sulfate 0.1g, manganesse selfate 0.05g, dipotassium phosphate 2g, sodium acetate 5g/L, pH 6.5, Difco Co.)에 계대배양한 후 균체를 20% 글리세롤이 포함된 저장액에 넣어 -70℃에 보관하였으며, 실험에 사용하기 전 계대배양을 실시하였다.
Lactobacillus bacteria were selected as a useful microorganism for fermenting the prepared wind-permeable acid, and were distributed in the Korea Institute of Biotechnology Genetic Resource Center Gene Bank (KCTC), as shown in Table 2, and lactobacillus MRS medium (proteose peptone No. 3 10g). , beef extract 10g, yeast extract 5g, dextrose 20g, Polyoxyethylene sorbitan monooleate 1g, ammonium citrate 2g, maganessium sulfate 0.1g, manganesse selfate 0.05g, dipotassium phosphate 2g, sodium acetate 5g / L, pH 6.5, Difco Co. After incubation, the cells were placed in a stock solution containing 20% glycerol and stored at -70 ° C. Subcultures were performed before use in experiments.
KCTC 3635 Lactobacillus delbrueckii subsp. bulgaricus
KCTC 3635
락토바실러스균의Lactobacillus 접종 및 발효 Inoculation and Fermentation
준비된 방풍통성산에 하기 표 3에 나타낸 바와 같이 균수를 달리하여 락토바실러스균 stock 용액을 제조한 후 각각 0.1%(v/v)씩 접종하였다.
As shown in Table 3 below, the prepared windproof acid was prepared by injecting lactobacillus bacteria stock solution by varying the number of bacteria, and inoculated by 0.1% (v / v).
KCTC 3635 Lb. delbrueckii subsp. bulgaricus
KCTC 3635
상기에서, 실시예 3은 접종시기를 달리한 것으로 stock 용액을 1×108 CFU/㎖로 제조한 후 Lb . brevis KCTC 3498, Lb . delbrueckii subsp . bulgaricus KCTC 3635 및 Lb . helveticus KCTC 3545를 접종한 후 5일째에 Lb . fermentum KCTC 3112 및 Lb . plantarum KCTC 3108을 접종한 것이다.
In the embodiment, for example, 3 Lb after preparing a stock solution that, unlike the time of inoculation to 1 × 10 8 CFU / ㎖. brevis KCTC 3498, Lb. delbrueckii subsp . bulgaricus KCTC 3635 and Lb. helveticus Lb. 5 days after inoculation with KCTC 3545 . fermentum KCTC 3112 and Lb. plantarum was inoculated with KCTC 3108.
상기와 같이, 락토바실러스균을 접종한 실시예 1, 실시예 2 및 실시예 3의 한약들을 7일 동안 발효시켜 본 발명의 한방발효 조성물을 제조하였다. 도 1은 5일간 배양된 실시예 3의 한방발효 조성물(락토바실러스균)을 BPB가 첨가된 락토바실리 MRS 배지에 100㎕ 분주 후 도말한 결과를 나타내는 사진이다. 여기에서, A는 방풍통성산만으로 배양한 것이고, B는 방풍통성산에 설탕(brown sugar)을 1%(w/v) 첨가하여 배양한 것이며, C는 방풍통성산에 설탕(brown sugar)을 5%(w/v) 첨가하여 배양한 것이다.
As described above, the herbal medicines of Examples 1, 2 and 3 inoculated with Lactobacillus bacteria were fermented for 7 days to prepare herbal fermentation compositions of the present invention. 1 is a photograph showing the results obtained after dispensing the herbal fermentation composition (Lactobacillus bacteria) of Example 3 incubated for 5 days in 100 μl of lactobacillus MRS medium to which BPB was added. Here, A is cultured with only windproof acid, B is cultured by adding 1% (w / v) of brown sugar to windproof acid, and C is 5% (brown sugar) w / v) was added and cultured.
또한, 도 2는 실시예 1과 실시예 2의 락토바실러스균의 성장곡선을 비교하여 나타내는 그래프이고, 도 3은 실시예 3의 락토바실러스균의 성장곡선을 나타내는 그래프이다. 도 3에서 보는 바와 같이 실시예 3의 락토바실러스균은 발효 후기에도 발효 초기와 유사하게 생육하는 것을 알 수 있다.
In addition, Figure 2 is a graph showing the growth curve of the Lactobacillus bacteria of Example 1 and Example 2, Figure 3 is a graph showing the growth curve of the Lactobacillus bacteria of Example 3. As shown in Figure 3 it can be seen that the Lactobacillus bacteria of Example 3 grow similarly to the initial fermentation even in the late fermentation.
실험예Experimental Example
pHpH 측정 Measure
한방발효 조성물(발효한약)에서 공시균주의 생육정도는 방풍통성산에 균을 접종하고 배양일수별로 100 ㎕를 취해 락토바실리 MRS한천배지에 도말하여 생균수를 측정한 후 확인하였다. 공시균주의 산 생성여부는 방풍통성산에 공시균주를 접종하고 배양일수별로 배양액을 취하여 10분간 1,000 ×g에서 원심분리 후 상등액의 pH를 측정하여 확인하였다.
The growth rate of the test strains in the herbal fermentation composition (fermented Chinese herbal medicine) was inoculated with the bacterium in the wind-breakable acid, 100 μl of each culture day and plated on lactobacillus MRS agar medium to determine the number of viable cells. Acid production of test strains was confirmed by inoculating the test strains into the wind-breakable acid and taking the culture solution for each culture day and centrifuging at 1,000 × g for 10 minutes to determine the pH of the supernatant.
실시예 1, 실시예 2 및 실시예 3에서 배양 1일째 균주의 산 생성은 방풍통성산에 5% 설탕을 첨가한 배지에서 배양한 경우에 더욱 우수하였다. 실시예 1에서 균주의 산 생성은 1% 설탕을 첨가하여 7일간 배양하였을 때 pH 3.49로 가장 높았으며, 실시예 2에서 균주의 산 생성은 5% 설탕을 첨가하여 7일간 배양하였을 때 pH 3.52로 가장 높았다. 실시예 1과 실시예 2에서 한약만으로 배양시 산 생성은 각각 pH 4.84와 pH 4.78로 유사하였으며(도 5), 실시예 3에서 한약만으로 배양시에는 pH 3.87로 산 생성이 우수하였다(도 4).
In Example 1, Example 2 and Example 3, the acid production of the strain on the first day of culture was more excellent when cultured in a medium in which 5% sugar was added to windproof acid. In Example 1, the acid production of the strain was the highest at pH 3.49 when cultured for 7 days with 1% sugar addition, and the acid production of the strain in Example 2 was pH 3.52 when cultured for 7 days with 5% sugar addition. Highest. In Example 1 and Example 2, the acid production was similar to pH 4.84 and pH 4.78, respectively, in culture with only Chinese medicine (FIG. 5). In Example 3, the acid production was excellent at pH 3.87 when only in Chinese medicine (FIG. 4). .
항산화 활성Antioxidant activity
전자공여능Electron donating ability
전자공여능은 Blois의 방법(Blois 1985)을 변형하여 실시하였다. DPPH는 실험 시작 전 메탄올에 녹인 후 0.3 mM이 되도록 하여 사용하였다. DPPH 용액과 멸균수로 희석된 시료(한약 또는 발효한약)를 1:1로 혼합하여 30분간 실온에서 반응시킨 후 517 ㎚에서 흡광도를 측정하였다. 음성 대조구는 시료대신 메탄올을 첨가하여 측정하였으며, 양성 대조구는 기존의 항산화제로 알려진 BHA로 측정하였다. 전자공여능은 시료첨가구와 음성대조구의 흡광도를 구하여 다음과 같이 백분율로 나타내었다.
Electron donating ability was performed by modifying the method of Blois (Blois 1985). DPPH was used after dissolving in methanol before the start of the experiment to 0.3 mM. Samples diluted with DPPH solution and sterile water (medicinal herbs or fermented herbs) were mixed 1: 1 and reacted at room temperature for 30 minutes, and then absorbance was measured at 517 nm. The negative control was measured by adding methanol instead of the sample, and the positive control was measured by BHA known as an antioxidant. The electron donating ability of the sample adder and the negative control was obtained as a percentage as follows.
항산화 활성 측정방법 중 DPPH 라디칼 소거능은 비교적 간단하여 대량으로 측정이 가능하며, 실제 항산화활성과 연관성이 높은 방법이다. DPPH 라디칼을 갖는 물질 중에서 비교적 안정한 화합물로 에탄올이나 메탄올에서 보라색으로 발색되지만 항산화 활성을 갖는 물질과 만나게 되면 항산화 물질이 DPPH의 라디칼을 소거시켜 탈색된다. 전자공여 작용은 활성 라디칼에 전자를 공여하여 지방질 산화를 억제시키는 척도로 사용되며, 인체 내에서 활성 라디칼에 의한 노화를 억제하는 작용의 척도로도 이용되고 있다. DPPH radical scavenging ability of the antioxidant activity measurement method is relatively simple and can be measured in large quantities, and is highly related to antioxidant activity. It is a relatively stable compound among DPPH radicals, which is colored purple in ethanol or methanol, but when it encounters a substance having antioxidant activity, the antioxidant discolors by eliminating radicals of DPPH. Electron donating action is used as a measure of inhibiting lipid oxidation by donating electrons to active radicals, and is also used as a measure of inhibiting aging by active radicals in the human body.
DPPH의 소거능으로 전자공여능을 검토한 결과 도 6에서 보는 바와 같이 100배 희석하였을 때 한약(발효하지 않은 방풍통성산)은 31.7%로 나타났으며, 본 발명의 한방발효 조성물은 모두 70% 이상으로 나타났다. 락토바실러스균의 접종시기를 다르게 하여 한약만으로 배양한 경우(실시예 3) 배양 7일째 DPPH 소거능이 94.43%로 가장 높았으며(도 6, 3A), 도 7에서 보는 바와 같이 양성 대조구인 BHA의 0.5mM에 해당하는 DPPH 소거능을 갖는 것으로 나타났다.
As a result of examining the electron donating ability by DPPH scavenging ability, as shown in FIG. 6, when diluted 100-fold, the herbal medicine (not fermented windproof acid) was found to be 31.7%, and the herbal fermentation compositions of the present invention were all 70% or more. . When the inoculation time of the Lactobacillus bacteria was inoculated differently (Example 3), the DPPH scavenging ability was the highest as 94.43% at 7 days of culture (Fig. 6, 3A), and as shown in Fig. It was shown to have DPPH scavenging ability corresponding to mM.
SODSOD 유사활성Similar activity
SOD 유사 활성은 Marklund의 방법에 따라 실시하였다(Marklund and Marklund 1974). 각 시료 용액 0.2 ㎖에 Tris-HCl의 완충용액(50 mM Tris, 10 mM EDTA, pH 8.5) 2.6 ㎖와 7.2 mM pyrogallol 0.2 ㎖ (Sigma Co.)를 가하여 25℃에서 10분간 반응시킨 후 1.0 N HCl 0.1 ㎖를 가하여 반응을 정지시키고 반응액 중 산화된 피로가롤(pyrogallol)의 양을 420 ㎚에서 측정하였다. SOD 유사활성은 시료용액의 실험구와 대조구의 흡광도 감소율로 나타내었다.
SOD-like activity was performed according to Marklund's method (Marklund and Marklund 1974). To 0.2 ml of each sample solution, 2.6 ml of Tris-HCl buffer (50 mM Tris, 10 mM EDTA, pH 8.5) and 0.2 ml of 7.2 mM pyrogallol (Sigma Co.) were added and reacted at 25 ° C. for 10 minutes, followed by 1.0 N HCl. 0.1 ml was added to stop the reaction, and the amount of oxidized pyrogallol in the reaction solution was measured at 420 nm. SOD-like activity was expressed by the decrease in absorbance of the experimental and control groups of the sample solution.
한약의 SOD 유사 활성은 도 8에서 보는 바와 같이 36.34%로 확인되었다. 복합배양된 한약(본 발명의 한방발효 조성물)의 SOD유사 활성은 한약보다 높았으며, 1%와 5%로 설탕을 첨가하였을 때 더 높았다. 락토바실러스균의 접종시기를 달리하여 배양된 한방발효 조성물(실시예 3)은 배양일수에 따라 SOD유사 활성이 높아졌다(도 8, 3C).
SOD-like activity of the Chinese medicine was found to be 36.34% as shown in FIG. The SOD-like activity of the co-cultured Chinese herbal medicine (the herbal fermentation composition of the present invention) was higher than that of Chinese medicine and was higher when sugar was added at 1% and 5%. Herbal fermentation compositions (Example 3) cultured by varying the inoculation time of Lactobacillus bacteria increased SOD-like activity according to the days of culture (Fig. 8, 3C).
항균활성 측정Antimicrobial Activity
한약 및 한방발효 조성물의 항균 활성은 agar diffusion법으로 측정하였다. 검정균주는 Gram 양성균인 바실루스 서브틸리스 ( Bacillus subtilis ) PCI 219, 스타필로코쿠스 아우레우스 속. 아우레우 스 ( Staphylococcus aureus subsp . aureus ) KCTC 1916과 프로피오니박테리움 아크네스 ( Propionibacterium acnes ) KCTC 3314, Gram 음성균인 대장균( Escherichia coli ) KCTC 1682, 슈도모나스 애루지노사 ( Pseudomonas aeruginosa ) KCTC 2004와 살모넬라 티피뮤리움 ( Salmonella typhimurium ) KCTC 2504를 사용하였다. 또한 효모는 칸디다 알비칸스 ( Candida albicans ), 크립토코쿠스 네 오포르만스 ( Cryptococcus neoformans )을 사용하였으며, 곰팡이는 아스퍼질러스 니가 ( Aspergillus niger ), 아스퍼질러스 오리재 ( Aspergillus oryzae )을 사용하였다.The antimicrobial activity of the herbal and herbal fermentation compositions was measured by the agar diffusion method. The test strain was Bacillus subtilis, a Gram-positive bacterium. subtilis ) PCI 219, Staphylococcus brother in-house records. Lei's brother (Staphylococcus aureus subsp . aureus ) KCTC 1916 and propionic sludge tumefaciens arc Ness (Propionibacterium acnes)
세균은 LB배지(peptone 10 g, yeast extract 5 g, sodium chloride 5 g/ℓ, pH 6.8), 영양 배지(beef extract 3 g, peptone 5 g/ℓ, pH 6.8, Difco Co.)와 Reinforced Clostridial(RC) 배지(tryptose 10 g, beef extract 10 g, yeast extract 3 g, dextrose 5 g, sodium chloride 5 g, soluble starch 1 g, cysteine hydrochloride 0.5 g, sodium acetate 3 g/ℓ, pH 7.6)에 계대배양하였으며, 효모는 YM배지(yeast extract 3 g, malt extract 3g, dextrose 10 g, peptone 5 g/ℓ, pH 7.6)에 계대배양 한 후 4℃에서 보관하여 사용하였다. 곰팡이는 PDA배지(potato starch 4 g, dextrose 20 g/ℓ, pH 5.6, Difco Co.)에 계대배양하여 포자현탁액을 제조한 후 -20℃에서 보관하여 사용하였다. Bacteria were LB medium (peptone 10 g, yeast extract 5 g, sodium chloride 5 g / l, pH 6.8), nutrient medium (beef extract 3 g, peptone 5 g / l, pH 6.8, Difco Co.) and Reinforced Clostridial ( RC) Subcultured in medium (tryptose 10 g, beef extract 10 g, yeast extract 3 g, dextrose 5 g, sodium chloride 5 g, soluble starch 1 g, cysteine hydrochloride 0.5 g, sodium acetate 3 g / L, pH 7.6) Yeast was passaged in YM medium (yeast extract 3 g, malt extract 3 g, dextrose 10 g, peptone 5 g / l, pH 7.6) and stored at 4 ° C. The fungus was subcultured on a PDA medium (potato starch 4 g, dextrose 20 g / l, pH 5.6, Difco Co.) to prepare a spore suspension, and stored at -20 ° C.
항균 활성은 배양액을 4℃, 1,000 ×g에서 10분간 원심분리 후 상등액 20 ㎕를 페이퍼 디스크(paper disc)( = 6 ㎜, Whatman Co.)에 첨가하여 건조시킨 후 검정균주가 든 평판배지 위에 얹고 세균은 37℃에서 1일간 배양하였으며, 효모와 곰팡이는 28℃에서 각각 2일, 4일간 배양하여 생성된 클리어 존(clear zone)의 유무 및 크기를 통해 판단하였다.
Antibacterial activity was centrifuged for 10 minutes at 4 ℃, 1,000 × g, 20 μl of the supernatant was added to a paper disc (= 6 mm, Whatman Co.) and dried and placed on a plate containing the black strain. Bacteria were incubated at 37 ° C. for 1 day, and yeast and fungi were determined based on the presence and size of clear zones generated by incubation at 28 ° C. for 2 days and 4 days, respectively.
항균활성을 검토한 결과, 한약(발효하지 않은 방풍통성산)은 모든 시험균주에 항균활성이 없었으며, 본 발명의 한방발효 조성물(복합배양된 방풍통성산)은 바실루스 서브틸리스 ( Bacillus subtilis ) PCI 219, 슈도 모나스 애루지노사 ( Pseudomonas aeruginosa ) KCTC 2004, 스타필로코쿠스 아우레우스 속. 아우레우스 ( Staphylococcus aureus subsp . aureus ) KCTC 1916에 항균활성을 보였으며, 여드름을 유발시키는 프로피오니박테리움 아크네스 ( Propionibacterium acnes ) KCTC 3314에 항균활성을 보였다(표 4, 도 9).
As a result of examining the antimicrobial activity, Chinese herbal medicine (not fermented windproof acid) did not have antimicrobial activity in all test strains, and the herbal fermentation composition (complex cultured windproof acid) of the present invention was Bacillus subtilis ( Bacillus). subtilis ) PCI 219, pseudo Monastir her labor Rouge (Pseudomonas aeruginosa ) KCTC 2004, Staphylococcus brother in Les funny. Aureus ( Staphylococcus aureus subsp . aureus ) Showed antimicrobial activity on KCTC 1916, propionyl sludge tumefaciens arc Ness (Propionibacterium acnes) that cause
Strains
Strains
(days) Incubation time
(days)
PCI 219 Bacillus subtilis
PCI 219
KCTC 1916 Staphylococcus aureus subsp. aureus
KCTC 1916
KCTC 3314 Propionibacterium acnes
A: 한약(방풍통성산), B: 한약 + 1% 설탕, C: 한약 + 5% 설탕
A: Chinese medicine (windproof acid), B: Chinese medicine + 1% sugar, C: Chinese medicine + 5% sugar
세포독성 확인Cytotoxicity check
세포 독성은 미토콘드리아 탈수소효소 활성(Mitochondrial dehydrogenase activity)을 나타내는 MTT colorimetric reduction assay를 수행하여 검정물질이 세포의 생존에 미치는 효과를 Mosmann이 보고한 방법에 따라 측정하였다(Mosmann 1983). 지시세포인 RBL-2H3 세포주는 96 웰 플레이트에 웰당 1×105 cells 밀도로 분주하여 24시간 배양을 한 후 배지를 제거하고 FBS가 첨가되지 않은 배지를 분주하여 24시간 배양을 하였다. 세포배양 후 0.5 ㎎/㎖의 MTT시약(Generay Biotech Co.)을 10 ㎕씩 well에 첨가하여 4시간 배양을 하였으며, 잔여 MTT시약과 배지를 제거하고 DMSO와 EtOH를 1:1로 혼합하여 100 ㎕씩 분주한 후 침전물을 충분히 용해시켰다. 세포 독성은 ELISA reader를 사용하여 540 ㎚의 측정 파장을 기준으로 하여 흡광도를 측정한 후 확인하였다.
Cytotoxicity was measured according to the method reported by Mosmann by the MTT colorimetric reduction assay showing mitochondrial dehydrogenase activity (Mosmann 1983). The RBL-2H3 cell line, which is an indicator cell, was cultured in a 96 well plate at a density of 1 × 10 5 cells per well, cultured for 24 hours, and then cultured for 24 hours by removing the medium and dispensing the medium without FBS. After incubation, 10 mg of MTT reagent (Generay Biotech Co.) of 0.5 mg / ml was added to the wells and incubated for 4 hours. The remaining MTT reagent and the medium were removed, and DMSO and EtOH were mixed 1: 1. After dispensing, the precipitate was sufficiently dissolved. Cytotoxicity was confirmed after measuring the absorbance on the basis of the measurement wavelength of 540 nm using an ELISA reader.
RBL-2H3 세포주를 대상으로 하여 한약(발효하지 않은 방풍통성산)의 세포 독성을 확인한 결과를 도 10에 나타내었다. 한약의 처리시간은 일정하게 하고, 한약의 첨가량만을 다르게 하여 세포 독성을 확인한 결과 높은 농도(300 ㎕/㎖)로 한약을 첨가하여도 세포 독성이 없음을 확인하였다. The results of confirming the cytotoxicity of the herbal medicine (non-fermented perforated acid) against the RBL-2H3 cell line are shown in FIG. 10. The treatment time of the Chinese medicine was constant, and the cytotoxicity was confirmed by changing only the amount of Chinese medicine added, and it was confirmed that there was no cytotoxicity even if the Chinese medicine was added at a high concentration (300 μl / ml).
한방발효 조성물의 세포 독성 확인 시 처리농도는 10 ㎕/㎖로 하였으며, 처리시간은 30분, 1시간, 3시간, 6시간과 12시간으로 하였다. 도 11에서 보는 바와 같이 발효한약을 세포주에 30분, 1시간과 3시간 처리하였을 때는 세포 독성이 없었으나, 6시간 이후부터 세포에 영향을 주었다.
When the cytotoxicity of the herbal fermentation composition was confirmed, the treatment concentration was 10 μl / ml, and the treatment time was 30 minutes, 1 hour, 3 hours, 6 hours, and 12 hours. As shown in FIG. 11, when the fermented medicinal product was treated for 30 minutes, 1 hour and 3 hours in the cell line, there was no cytotoxicity, but the cells were affected after 6 hours.
β-β- 헥소사미니다아제(β-hexosaminidase)의Of hexosaminidase (β-hexosaminidase) 방출 억제 효과 Emission suppression effect
β-헥소사미니다아제(β-hexosaminidase)의 측정(Yamada et al. 2007)을 위해 RBL-2H3 세포주는 10% FBS, 1% 항생제(antibiotics)를 포함하는 D-MEM배지와 37℃의 5% CO2를 포함한 포화습도 공기조건에서 배양하여 80～90% 컨플루언시(confluency)에 도달하면 세포수를 5×105 cells/㎖로 조절하여 24 well plate에 분주 후 IgE를 0.5 ㎍/㎖로 첨가하여 24시간 배양하였다. 배양 후 배지를 제거하고 500 ㎕ 시라가니언(Siraganian) 완충액(119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 25 mM PIPES, 40 mM NaOH, pH 7.2)으로 2회 세척한 다음 5.6 mM glucose, 1 mM CaCl2 및 0.1% BSA (Sigma Co.)가 포함된 시라가니언(Siraganian) 완충액을 160 ㎕ 첨가하여 10분간 배양하였다. IgE (Sigma Co.)로 감작된 세포에 농도별로 시료를 처리하여 일정시간 배양한 후 항원(DNP-KLH, Alpha diagnostic Intl Inc.)을 1 ㎍/㎖로 첨가하여 10분간 반응시켰다. 반응 종결을 위해 10분간 얼음에 꽂아둔 후 반응 혼합물을 500 ×g에서 10분간 원심분리 하여 상등액을 분리하였다. 96 웰 플레이트에 상등액과 기질(1 mM nitrophenyl-N- acetyl-β-D-glucosaminide, Sigma Co.)을 각각 25 ㎕씩 첨가하여 37℃에서 1시간 배양한 후 0.1 M Na2CO3/NaHCO3를 200 ㎕ 첨가하여 반응을 정지시켜 ELISA reader로 405 nm에서 측정하였다. RBL-2H3 세포주에서 β-헥소사미니다아제(β-hexosaminidase)의 방출 억제는 다음과 같이 계산하였다.
RBL-2H3 cell line for measurement of β-hexosaminidase (Yamada et al. 2007), D-MEM medium containing 10% FBS, 1% antibiotics and 5% at 37 ° C. CO2After cultivating in saturated humidity air condition including 80 ~ 90% confluency, the cell number is 5 × 105 After adjusting to cells / ml and dispensing into a 24 well plate IgE was added to 0.5 ㎍ / ㎖ and incubated for 24 hours. After incubation, the medium was removed and 500 μl Siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl).2, 25 mM PIPES, 40 mM NaOH, pH 7.2) twice, followed by 5.6 mM glucose, 1 mM CaCl2 And 160 μl of Siraganian buffer containing 0.1% BSA (Sigma Co.) was added and incubated for 10 minutes. After culturing for a predetermined time by treating the samples by concentration to the cells sensitized with IgE (Sigma Co.), the antigen (DNP-KLH, Alpha diagnostic Intl Inc.) was added to 1 ㎍ / ㎖ and reacted for 10 minutes. After the reaction was completed for 10 minutes on ice, the reaction mixture was centrifuged at 500 xg for 10 minutes to separate the supernatant. Supernatant and substrate (1 mM nitrophenyl-N-25 Ml of acetyl-β-D-glucosaminide, Sigma Co.) was incubated at 37 ° C for 1 hour, and then 0.1 M Na2CO3/ NaHCO3To 200 μl was added to stop the reaction, and measured at 405 nm by ELISA reader. Inhibition of release of β-hexosaminidase in the RBL-2H3 cell line was calculated as follows.
Control : 항체와 항원 첨가, 시료 미첨가; Control: Add antibody and antigen, no sample;
Treated : 항체, 항원 및 시료 첨가; Treated: addition of antibodies, antigens and samples;
Blank : 시료와 기질만 첨가; Blank: Add sample and substrate only;
Spontaneous : 항체, 항원 및 시료 미첨가
Spontaneous: Antibody, Antigen, and No Sample
시험물질의 알레르기 억제 효과를 측정하는 방법 중에서 IgE로부터 감작된 비만세포에서 유리된 히스타민의 양을 측정하는 방법은 비만세포에서 히스타민의 농도가 낮고 복잡한 여러 단계를 수행하여야 하기 때문에 큰 편차를 나타내기 쉽다. 최근에는 높은 농도로 세포 내에 존재하며, 탈 과립 시 화학적 매개체들과 함께 방출되는 β-헥소사미니다아제(β-hexosaminidase)를 측정하는 방법이 널리 이용되고 있다(Choi 2002). Among the methods for measuring the allergic inhibitory effect of a test substance, the method of measuring the amount of histamine released from mast cells sensitized by IgE is likely to show a large deviation due to the low concentration of histamine in the mast cells and the complicated steps. . Recently, a method of measuring β-hexosaminidase, which is present in cells at high concentration and released with chemical mediators during degranulation, has been widely used (Choi 2002).
이러한 방법을 이용하여 항산화 및 항균 활성이 우수한 실시예 3으로 β-헥소사미니다아제(β-hexosaminidase)의 방출 억제 효과를 측정한 결과 도 12에서 보는 바와 같이 배양일수에 따라 알레르기 억제 효과는 증가하지 않고 유사하였다. 한약(방풍통성산)만으로 배양한 것은 배양 5일째에 56.81%, 1% 갈색 설탕(brown sugar)이 첨가된 한약은 배양 3일째 53.93%, 1% 갈색 설탕(brown sugar)이 첨가된 한약은 배양 3일째 52.41%로 높았다. As a result of measuring the release inhibitory effect of β-hexosaminidase in Example 3 having excellent antioxidant and antimicrobial activity using this method, the allergic inhibitory effect did not increase with culture days as shown in FIG. 12. Similar without. Cultured only with Chinese herbs (wind-permeable acid), 56.81% on
β-헥소사미니다아제 방출에 있어 pH의 영향은 도 13에서 보는 바와 같이 크지 않았다. pH 5.4인 한약의 β-헥소사미니다아제의 방출 억제 효과는 68%로 나타났으며, 발효한약의 β-헥소사미니다아제의 방출 억제 효과는 57%로 나타났다.
The effect of pH on β-hexosaminidase release was not large as shown in FIG. 13. The inhibitory effect of β-hexosaminidase on herbal medicines of pH 5.4 was 68%, and the inhibitory effect of β-hexosaminidase on fermented herbs was 57%.
통계분석Statistical analysis
상기의 실험예에서, 통계분석은 SigmaStat를 이용하여 분산분석을 실시하였으며, 시험 구간의 평균간 유의성 검정은 SNK(Student-Newman-Keuls) 테스트에 따른 다중검정방법으로 5% 수준에서 실시하였다. In the above experimental example, the analysis of variance was performed using SigmaStat, and the significance test between the mean of the test sections was performed at the 5% level by the multi-test method according to the Student-Newman-Keuls (SNK) test.
이상 살펴본 바와 같이, 본 발명은 방풍통성산 및 락토바실러스균을 이용한 것으로 구체적으로는 균수를 달리하여 접종하거나 시기를 달리하여 한약(방풍통성산)에 접종한 후 발효시키는 복합배양 방법을 통해 여러 종의 락토바실러스균이 잘 생육할 수 있도록 하였다. 이로 인해 본 발명은 방풍통성산의 다양한 생리활성 기능은 가지고 있으면서 발효하기 전의 한약(방풍통성산)보다 항산화 활성이 더욱 우수하고, 바실루스 서브틸리스(Bacillus subtilis) PCI 219, 슈도모나스 애루지노사(Pseudomonas aeruginosa) KCTC 2004, 스타필로코쿠스 아우레우스 속. 아우레우스(Staphylococcus aureus subsp. aureus) KCTC 1916 등에 항균활성을 보였으며, 특히 여드름을 유발시키는 프로피오니박테리움 아크네스(Propionibacterium acnes) KCTC 3314에 항균활성을 나타내었다. 또한, 60%에 가까운 알레르기 억제 효과를 나타내었다. 따라서, 본 발명은 화장품 산업, 제약 산업, 식품 산업 등에 매우 유용한 발명이다.As described above, the present invention uses windproof tonic acid and lactobacillus bacteria. Specifically, various kinds of lactose are inoculated by different inoculations or different time periods by inoculation into Chinese medicine (windproof tonic acid) and then fermented through a complex culture method. Bacillus bacteria were allowed to grow well. For this reason, the present invention has a variety of physiologically active functions of the windproof acid, while having better antioxidant activity than the herbal medicine (windproof acid) before fermentation, Bacillus subtilis PCI 219, Pseudomonas aeruginosa (Pseudomonas aeruginosa) KCTC 2004, genus Staphylococcus aureus. Aureus (Staphylococcus aureus subsp. Aureus) showed antimicrobial activity on KCTC 1916 and the like, and particularly on the propionibacterium acnes
Claims (16)
(S2) 락토바실러스균을 준비하는 단계; 및
(S3) 준비된 방풍통성산에 락토바실러스균을 접종하고 30~45℃에서 발효하는 단계;를 포함하는 한방발효 조성물 제조방법으로서,
상기 (S2) 단계에서의 락토바실러스균은 균수가 1×106~1×108CFU/㎖이고,
상기 (S2) 단계에서의 락토바실러스균은 0.8×106~5×106CFU/㎖의 락토바실러스 브레비스(Lactobacillus brevis) 및 락토바실러스 카제이(Lactobacillus casei), 0.8×107~5×107CFU/㎖의 락토바실러스 델브루에키 서브스피시즈 불가리쿠스(Lactobacillus delbrueckii subsp. bultaricus) 및 락토바실러스 플란타럼(Lactobacillus plantarum), 0.8×108~5×108CFU/㎖의 락토바실러스 퍼멘텀(Lactobacillus fermentum) 및 락토바실러스 헬베티쿠스(Lactobacillus helveticus)를 포함하거나, 또는
상기 (S2) 단계에서의 락토바실러스균은 0.8×106~5×106CFU/㎖의 락토바실러스 브레비스(Lactobacillus brevis) 및 락토바실러스 카제이(Lactobacillus casei), 0.8×107~5×107CFU/㎖의 락토바실러스 헬베티쿠스(Lactobacillus helveticus) 및 락토바실러스 플란타럼(Lactobacillus plantarum), 0.8×108~5×108CFU/㎖의 락토바실러스 퍼멘텀(Lactobacillus fermentum) 및 락토바실러스 델브루에키 서브스피시즈 불가리쿠스(Lactobacillus delbrueckii subsp. bultaricus)를 포함하는 것인 한방발효 조성물 제조방법.
(S1) preparing a wind-breaking acid which is a Chinese medicine;
(S2) preparing the Lactobacillus bacteria; And
(S3) inoculate the Lactobacillus bacteria in the prepared windproof acid and fermentation at 30 ~ 45 ℃; as an herbal fermentation composition manufacturing method comprising,
Lactobacillus bacteria in the step (S2) is the number of bacteria 1 × 10 6 ~ 1 × 10 8 CFU / ㎖,
Lactobacillus bacteria in the step (S2) is Lactobacillus brevis ( Lactobacillus brevis ) and Lactobacillus casei ( Lactobacillus casei ) of 0.8 × 10 6 ~ 5 × 10 6 CFU / ㎖, 0.8 × 10 7 ~ 5 × 10 7 CFU / ㎖ of Lactobacillus del Brewer station sub-species Bulgaria kusu (Lactobacillus delbrueckii subsp. bultaricus) and Lactobacillus Planta column (Lactobacillus plantarum), 0.8 × 10 8 ~ 5 × 10 8 CFU / ㎖ Lactobacillus buffer momentum (Lactobacillus of fermentum ) and Lactobacillus helveticus , or
Lactobacillus bacteria in the step (S2) is Lactobacillus brevis ( Lactobacillus brevis ) and Lactobacillus casei ( Lactobacillus casei ) of 0.8 × 10 6 ~ 5 × 10 6 CFU / ㎖, 0.8 × 10 7 ~ 5 × 10 7 Lactobacillus helveticus and Lactobacillus plantarum at CFU / mL, Lactobacillus plantarum , Lactobacillus fermentum at 0.8 × 10 8 to 5 × 10 8 CFU / mL and Lactobacillus delbru Eki Subspices Bulgaricus ( Lactobacillus delbrueckii subsp.bultaricus ) comprising a method of producing a herbal fermentation composition.
1~5℃, 2,000~5,000×g으로 원심분리하여 침전물을 제거한 것인 한방발효 조성물 제조방법.
According to claim 1, Wind-breaking acid in the step (S1)
Herbal fermentation composition manufacturing method is to remove the precipitate by centrifugation at 1 ~ 5 ℃, 2,000 ~ 5,000 × g.
The method of claim 1, wherein the wind-breaking acid in step (S1) is prepared by adjusting the pH to 4-7.
The method of claim 1, wherein 1-5% (w / v) of sugar is added to the wind-breaking acid in step (S1).
락토바실러스 브레비스 ( Lactobacillus brevis ), 락토바실러스 델브루에키 서브스피시즈 불가리쿠스 ( Lactobacillus delbrueckii subsp . bultaricus ) 및 락토바실러스 헬베티쿠스 ( Lactobacillus helveticus )를 접종한 후, 상기 접종일로부터 4~5일째에 락토바실러스 퍼멘텀 ( Lactobacillus fermentum ) 및 락토바실러스 플란타럼 ( Lactobacillus plantarum )을 접종하는 것인 한방발효 조성물 제조방법.
The method of claim 1, wherein in step (S3)
Lactobacillus brevis ( Lactobacillus brevis ), Lactobacillus del brewec sub-spices Bulgaricus ( Lactobacillus) delbrueckii subsp . bultaricus) and of Lactobacillus helveticus (then inoculated with Lactobacillus helveticus), to inoculate the inoculation Lactobacillus buffer momentum (Lactobacillus fermentum) and Lactobacillus Planta column (Lactobacillus plantarum) in 4-5 days from the date of herbal Fermentation composition preparation method.
The method of claim 9, wherein each of the Lactobacillus bacteria inoculated after adjusting the number of bacteria to 1 × 10 8 CFU / ㎖.
The method of claim 1, wherein in step (S3) Lactobacillus bacteria are inoculated in an amount of 0.1 ~ 1% (v / v) to the windproof acid.
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