KR20170087397A - Strain isolated from infant feces and method to produce fermented milk having antioxidative activity using the same - Google Patents
Strain isolated from infant feces and method to produce fermented milk having antioxidative activity using the same Download PDFInfo
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- KR20170087397A KR20170087397A KR1020160144394A KR20160144394A KR20170087397A KR 20170087397 A KR20170087397 A KR 20170087397A KR 1020160144394 A KR1020160144394 A KR 1020160144394A KR 20160144394 A KR20160144394 A KR 20160144394A KR 20170087397 A KR20170087397 A KR 20170087397A
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Abstract
본 발명은 유아 분변에서 분리한 균주 및 이를 이용한 항산화능을 갖는 발효유의 제조방법에 관한 것이다. 본 발명의 균주를 프로바이오틱스로 사용하는 경우, 발효액에서 높은 항산화 효과가 우수하여, 이를 발효유 등을 포함하는 건강식품의 제조에 유용하게 사용할 수 있다. The present invention relates to a strain isolated from infant feces and a method for producing fermented milk having antioxidant ability. When the strain of the present invention is used as a probiotic, the antioxidant effect of the fermentation broth is excellent, and it can be usefully used for the production of health foods containing fermented milk and the like.
Description
본 발명은 유아 분변에서 분리한 균주 및 이를 이용한 항산화능을 갖는 발효유의 제조방법에 관한 것이다.The present invention relates to a strain isolated from infant feces and a method for producing fermented milk having antioxidant ability.
생균제(Probiotics)는 숙주의 장내 미생물 균형을 유지시킴으로써 유익한 작용을 하는 미생물을 총괄하며, 인체 의약품인 정장제나 유산균 제제, 사료첨가제 및 건강기능식품 등으로 사용되고 있다. 일반적으로 생균제로 사용되는 미생물들은 안전하며, 섭취시 생체 내 저해환경 즉 위장의 위산, 담낭의 담즙 및 소장에서 분비되는 각종 소화효소에 저항력을 갖고, 대장과 직장에 도달해 증식하고 정착하는 능력을 갖는다[FAO/WHO, 2002; Homma 1998].또한, 생균제는 장내 균총의 정상화, 유해 세균의 정착을 억제함에 따른 부패 산물의 생성 감소, 질병예방, 항암작용, 면역의 활성화, 혈중 콜레스테롤 저하 및 항산화 효과 등 균체 성분 자체로 체내에서 유용한 기능을 전제로 한다.Probiotics are used to maintain beneficial microorganisms in the intestinal microflora, and are used as formulases, lactic acid bacteria, feed additives, and health functional foods. In general, the microorganisms used as probiotics are safe, and they are resistant to the in vivo inhibitory environment such as gastric acid in the stomach, gallbladder bile and small intestine in ingestion, and have ability to proliferate and settle by reaching the colon and rectum (FAO / WHO, 2002; In addition, the probiotics are effective in normalizing the intestinal microflora, reducing the production of decay products due to inhibition of harmful bacteria, preventing disease, anticancer activity, activation of immunity, lowering of cholesterol and antioxidant effect of blood, Useful functions are premised.
프리바이오틱스(Prebiotics)는 장내에 서식하고 있는 미생물들의 성장과 활력에 선택적으로 작용하여 숙주에게 유익한 영향을 주는 비소화성 식품 성분들을 총칭하는 물질로서, 생균제에 기질로 작용하여 궁극적으로는 숙주의 건강을 향상시키는 기능을 한다. 최근까지 셀룰로오스, 헤미셀루로오스, 리그닌 및 펙틴 등의 식이섬유와 비소화성 탄수화물인 올리고당등이 있으며, 이들은 특정균주에 대하여 선택적인 성장을 촉진시키며, 장관 내 미생물 균총의 성장과 증식에 관여한다. 프리바이오틱스는 협의의 의미로는, 주로 비소화성 당류들이 이에 해당한다. 그러나 올리고당(oligosaccharide)은 식중독병원균을 포함하여 비 프로바이오틱스 미생물에도 영향을 줄 수 있기 때문에 최근에는 프리바이오틱스의 개념이 좀 더 광범위한 의미로 발전되고 있는 실정이다. 기본적으로 프리바이오틱스는 프로바이오틱스의 장내 증식을 촉진하는 것으로 알려져 있으며, 최근에는 식품 내 신바이오틱스 제제를 첨가한 후 쥐에서 뚜렷한 장내 균총의 개선 효과가 있음을 확인한 연구결과들이 보고되었다. 또한, IBD와 같은 염증성 장 질환의 경우, 특정 프리바이오틱스를 대사할 수 있는 프로바이오틱스를 처리한 결과 염증성 사이토카인의 감소 효과가 발견되어, 신바이오틱스에 대한 활발한 연구가 진행되고 있는 상황이며, 다양한 질병에 있어서 신바이오틱스의 접근이 이루어지고 있는 실정이다. 하지만, 아직까지 대부분의 신바이오틱스 연구는 비소화성의 올리고당을 활용한 것이 대부분이며, 비탄수화물 계열의 프리바이오틱스와 프로바이오틱스의 시너지 효과를 평가한 연구는 전무한 실정이다. Prebiotics is a generic term for non-digestible food ingredients that act selectively on the growth and vitality of microorganisms in the intestines and which has a beneficial effect on the host. It acts as a substrate for the probiotic agent and ultimately, . Until recently, dietary fibers such as cellulose, hemicellulose, lignin and pectin, and oligosaccharides such as non-degradable carbohydrates, etc., have been shown to promote selective growth of certain strains and participate in the growth and proliferation of intestinal microflora. Prebiotics, in the sense of the agreement, are mainly non-reducing sugars. However, since oligosaccharides can affect nonbiobiotic microorganisms including foodborne pathogens, the concept of prebiotics has recently been developed in a broader sense. Prebiotics is known to promote intestinal proliferation of probiotics. In recent years, research results have been reported confirming that intestinal microflora are improved in rats after addition of new biotics in food. In the case of inflammatory bowel disease such as IBD, treatment of probiotics capable of metabolizing certain prebiotics has been found to reduce inflammatory cytokines, and active research on synbiotics has been under way. The approach of synbiotics in diseases is being done. However, most of the synbiotics studies still use non-digestible oligosaccharides, and no studies have evaluated the synergistic effects of non-carbohydrate-based prebiotics and probiotics.
이에 본 발명자는 유아 분변에서 균주를 분리하고, 상기 분리된 균주 및 프리바이오틱스 소재인 식물추출물를 이용하여 제조된 발효액에서 항산화 효과 및 면역증진 효과가 우수함을 확인하여, 상기 균주와 프리바이오틱스 소재간의 시너지 효과를 확인함으로써, 본 발명을 완성하게 되었다.Thus, the present inventors have isolated the strain from infant feces and confirmed that the fermentation broth prepared by using the isolated strain and the plant extract as a prebiotic material is excellent in antioxidative and immuno-enhancing effects, By confirming the synergy effect, the present invention has been completed.
본 발명의 목적은 유아 분변으로부터 분리된 균주를 제공함에 있다.It is an object of the present invention to provide a strain isolated from infant feces.
본 발명의 다른 목적은 상기 균주의 배양산물을 제공함에 있다.Another object of the present invention is to provide a culture product of the strain.
본 발명의 다른 목적은 상기 균주 또는 이의 배양산물을 유효성분으로 포함하는 생균제 조성물을 제공함에 있다.Another object of the present invention is to provide a biocidal composition comprising the strain or a culture product thereof as an active ingredient.
본 발명의 또다른 목적은 상기 균주 또는 이의 배양산물을 유효성분으로 포함하는 장기능 개선용 식품조성물을 제공함에 있다.It is still another object of the present invention to provide a food composition for improving intestinal function comprising the strain or a culture product thereof as an active ingredient.
본 발명의 또다른 목적은 상기 균주 또는 이의 배양산물을 이용한 항산화용 발효유의 제조방법을 제공함에 있다.Another object of the present invention is to provide a method for producing fermented milk for antioxidation using the strain or a culture product thereof.
따라서, 본 발명은 유아 분변으로부터 분리된 락토바실러스 가세리 SRK545(Lactobacillus gasseri SRK545) 균주(수탁번호 KCCM11767P), 락토바실러스 가세리 SRK559(Lactobacillus gasseri SRK559) 균주(수탁번호 KCCM11768P) 또는 락토바실러스 가세리 SRK575(Lactobacillus gasseri SRK575) 균주(수탁번호 KCCM11769P)를 제공한다.Therefore, the present invention relates to a method for producing Lactobacillus gasseri SRK545 strain (Accession No. KCCM11767P), Lactobacillus gasseri SRK559 strain (Accession No. KCCM11768P) or Lactobacillus gasseri SRK575 Lactobacillus gasseri SRK575) (Accession No. KCCM11769P).
또한, 본 발명은 상기 균주, 상기 균주의 배양물, 상기 배양물의 농축물, 상기 배양물의 건조물, 상기 균주의 발효물 및 이들의 조합으로 이루어진 군에서 선택된 어느 하나를 유효성분으로 포함하는 생균제 조성물을 제공한다.The present invention also provides a method for producing a prodrug, comprising the step of culturing the above-mentioned strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, a fermented product of the strain, to provide.
또한, 본 발명은 상기 균주, 상기 균주의 배양물, 상기 배양물의 농축물, 상기 배양물의 건조물, 상기 균주의 발효물 및 이들의 조합으로 이루어진 군에서 선택된 어느 하나를 유효성분으로 포함하는 장기능 개선용 식품 조성물을 제공한다.The present invention also relates to a method for improving the intestinal function comprising an effective ingredient selected from the group consisting of the strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, the fermented product of the strain, ≪ / RTI >
또한, 본 발명은 상기 균주를 이용한 항산화용 발효유를 제공한다.The present invention also provides an antioxidant fermented milk using the strain.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 균주는 유아의 분변에서 분리되었으며, 분리된 균주의 분자생물학적 특성을 16S rDNA 유전자 염기서열로 분석하여 상기 분리된 균주가 락토바실러스 가세리 (Lactobacillus gasseri) 와 높은 상동성을 보이는 신규한 균주로 동정하였다. 상기 균주들을 락토바실러스 가세리 SRK545(Lactobacillus gasseri SRK545) 균주, 락토바실러스 가세리 SRK559(Lactobacillus gasseri SRK559) 및 락토바실러스 가세리 SRK575(Lactobacillus gasseri SRK575)로 명명하였고, 한국미생물보존센터(국외)에 2015년 9월 11일자로 기탁하여 각각 수탁번호 KCCM11767P, 수탁번호 KCCM11768P 및 수탁번호 KCCM11769P을 부여받았다.The strain of the present invention was isolated from the feces of an infant and the molecular biological characteristics of the isolated strain were analyzed by 16S rDNA gene sequence to find that the isolated strain exhibited high homology with Lactobacillus gasseri Respectively. Was named as the above strain Lactobacillus joined Lee SRK545 (Lactobacillus gasseri SRK545) strains, Lactobacillus joined Lee SRK559 (Lactobacillus gasseri SRK559) and Lactobacillus joined Lee SRK575 (Lactobacillus gasseri SRK575), 2015 years in the Korea Culture Center of Microorganisms (Overseas) September 11, respectively, and were granted accession number KCCM11767P, accession number KCCM11768P, and accession number KCCM11769P, respectively.
본 발명의 균주를 프로바이오틱스로 사용하는 경우, 발효액에서 높은 항산화효과가 존재하여, 이를 발효유 등을 포함하는 장기능 개선용 건강식품의 제조에 유용하게 사용할 수 있다.When the strain of the present invention is used as a probiotic, there is a high antioxidative effect in a fermentation broth, which can be usefully used for the production of a health food for improving bowel function including fermented milk and the like.
본 발명의 다른 양태에 따르면, 본 발명은 상기 균주의 배양물, 상기 균주의 발효물, 상기 배양물의 농축물, 배양물의 건조물 및 이들의 조합으로 이루어진 군에서 선택된 어느 하나인 배양산물을 제공한다. 상기 배양산물은 균주를 제거한 것 일수 있고, 또는 배양물을 원심 분리하여 수득한 상층액일 수 있다. 또한, 상기 배양산물은 배양물의 농축물 및 상기 배양물의 건조물을 모두 포함할 수 있다. 또한, 본원발명의 발효물은 상기 균주를 이용하여 발효를 시킨것으로, 액체 및 고체를 포함하며, 발효액을 포함하는 넓은 개념으로 사용하였다.According to another aspect of the present invention, there is provided a cultured product selected from the group consisting of a culture of the strain, a fermentation product of the strain, a concentrate of the culture, a dried product of the culture, and a combination thereof. The cultured product may be obtained by removing the strain, or may be a supernatant obtained by centrifuging the culture. In addition, the cultured product may include both a concentrated product of the cultured product and a dried product of the cultured product. The fermentation product of the present invention is fermented using the strain, and includes liquid and solid, and is used in a broad concept including a fermentation broth.
본 발명의 다른 양태에 따르면, 본 발명의 균주, 상기 균주의 배양물, 상기 균주의 발효물, 상기 배양물의 농축물, 상기 배양물의 건조물 및 이들의 조합으로 이루어진 군에서 선택된 어느 하나를 유효성분으로 포함하는 생균제 조성물을 제공한다.According to another aspect of the present invention, there is provided a method for producing a microorganism, which comprises culturing a strain of the present invention, a culture of the strain, a fermentation product of the strain, a concentrate of the culture, a dried product of the culture, Lt; / RTI >
본 명세서에서 사용되는 용어 "생균제" 또는 "프로바이오틱스(probiotics)"는 인간을 포함한 동물의 위장관 내에서 유해 미생물에 대한 저항성 증진을 통한 숙주의 장내 미생물 환경을 개선하여 숙주의 건강에 유익한 영향을 주는 살아있는 미생물을 의미한다. 프로바이오틱스는 단일 또는 복합 균주 형태로 사람이나 동물에 건조된 세포 형태나 발효 산물 형태로 급여될 수 있다. 본 발명의 균주가 생균제로 사용하는 경우, 프리바이오틱스 소재와 상승작용을 일으킬 수 있고, 상기 프리바이틱스 소재는 제한되지는 않지만, 식물 추출물이 바람직하다. 더욱 바람직하게는 꾸지뽕잎 추출물 또는 뽕잎 추출물이다. As used herein, the term " probiotic "or" probiotics "refers to a living organism that improves intestinal microbial environment by enhancing resistance to harmful microorganisms in the gastrointestinal tract of an animal, including humans, Means microorganisms. Probiotics may be fed in human or animal-derived cell or fermented product form in the form of single or complex strains. When the strain of the present invention is used as a probiotic agent, a synergistic effect with the prebiotic material can be caused. The prebiotic material is not limited, but a plant extract is preferable. More preferably, it is a leaf extract or mulberry leaf extract.
본 발명의 추출물은 발효에 적합하도록 가공하여 사용할 수있는데, 일 예로 꾸지뽕잎 또는 뽕잎추출물을 사용할 수 있다. 본 발명의 추출물은 꾸지뽕잎 또는 뽕잎에 추출용매를 처리하여 수득하는 경우에는, 다양한 추출용매가 이용될 수 있다. 바람직하게는, 극성 용매 또는 비극성 용매를 이용할 수 있다. 극성 용매로서 적합한 것은, (i) 물, (ii) 알코올(바람직하게는, 메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-프로판올, 노말-부탄올, 1-펜탄올, 2-부톡시에탄올 또는 에틸렌글리콜), (iii) 아세트산, (iv) DMFO(dimethyl-formamide) 및 (v) DMSO(dimethyl sulfoxide)를 포함한다. 비극성 용매로서 적합한 것은, 아세톤, 아세토나이트릴, 에틸 아세테이트, 메틸 아세테이트, 플루오로알칸, 펜탄, 헥산, 2,2,4-트리메틸펜탄, 데칸, 사이클로헥산, 사이클로펜탄, 디이소부틸렌, 1-펜텐, 1-클로로부탄, 1-클로로펜탄, o-자일렌, 디이소프로필 에테르, 2-클로로프로판, 톨루엔, 1-클로로프로판, 클로로벤젠, 벤젠, 디에틸 에테르, 디에틸 설파이드, 클로로포름, 디클로로메탄, 1,2-디클로로에탄, 어닐린, 디에틸아민, 에테르, 사염화탄소 및 THF를 포함한다. 보다 바람직하게는, 본 발명에서 이용되는 추출용매는 (a) 물, (b) 탄소수 1-4의 무수 또는 함수 저급 알코올(메탄올, 에탄올, 프로판올, 부탄올 등), (c) 상기 저급 알코올과 물과의 혼합용매, (d) 아세톤, (e) 에틸 아세테이트, (f) 클로로포름, (g) 부틸아세테이트, (h) 1,3-부틸렌글리콜, (i) 헥산 및 (j) 디에틸에테르를 포함한다. 가장 바람직하게는, 본 발명의 추출물은 물추출물이다. 본 명세서에서 사용되는 용어 '추출물'은 상술한 바와 같이 당업계에서 조추출물(crude extract)로 통용되는 의미를 갖지만, 광의적으로는 추출물을 추가적으로 분획(fractionation)한 분획물도 포함한다. 즉, 본 발명의 추출물은 상술한 추출용매를 이용하여 얻은 것뿐만 아니라, 여기에 정제과정을 추가적으로 적용하여 얻은 것도 포함한다. 예컨대, 상기 추출물을 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 통과시켜 얻은 분획, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획도 본 발명의 추출물에 포함되는 것이다. 본 발명에서 이용되는 추출물은 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로 제조될 수 있다The extract of the present invention can be used by processing to be suitable for fermentation. For example, the extract of Cucumis japonica or Mulberry leaf can be used. When the extract of the present invention is obtained by treating the leaves or mulberry leaves with an extraction solvent, various extraction solvents may be used. Preferably, a polar solvent or a non-polar solvent can be used. Suitable polar solvents are (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, Or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). Suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- But are not limited to, pentane, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2- chloropropane, toluene, 1- chloropropane, chlorobenzene, benzene, diethyl ether, diethylsulfide, Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF. More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.) (E) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether. . Most preferably, the extract of the present invention is a water extract. As used herein, the term " extract " means that it is used in the art as a crude extract as described above, but broadly includes fractions obtained by further fractionating the extract. That is, the extract of the present invention includes not only those obtained by using the above-mentioned extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained by the purification method is also included in the extract of the present invention. The extract used in the present invention can be produced in powder form by an additional process such as vacuum distillation and lyophilization or spray drying
상기 용어 "프리바이오틱스"는 장내에 서식하고 있는 미생물들의 성장에 선택적으로 작용하여 숙주에게 유익한 영향을 주는 비소화성 식품 성분들을 총칭하는 물질을 의미한다.The term "prebiotics" refers to a substance collectively referred to as non-degradable food ingredients that selectively affect the growth of microorganisms in the intestines and have a beneficial effect on the host.
본 발명에 따른 균주, 이의 배양물, 이의 발효물, 상기 배양물의 농축물, 상기 배양물의 건조물 및 이들의 조합으로 이루어진 군에서 선택된 어느 하나인 배양산물은 항산화능과 면역증진이 우수하며, 안전하며 독성 없는 프로바이오틱스로서 유용하게 이용될 수 있다. 따라서, 본 발명의 본 발명에 따른 균주, 이의 배양물, 이의 발효물, 상기 배양물의 농축물, 상기 배양물의 건조물 및 이들의 조합으로 이루어진 군에서 선택된 어느 하나인 배양산물은 생균제, 특히 인체 의약품인 정장제나 유산균 제제, 사료첨가제 및 건강식품에 유용하게 사용될 수 있다. The culture product according to the present invention, which is selected from the group consisting of a strain, a culture thereof, a fermentation product thereof, a concentrate of the culture, a dried product of the culture, and a combination thereof, is excellent in antioxidant ability and immunity, It can be usefully used as a non-toxic probiotic. Accordingly, the culture product according to the present invention, which is any one selected from the group consisting of a strain, a culture thereof, a fermentation product thereof, a concentrate of the culture, a dried product of the culture, and a combination thereof is used as a probiotic agent, And can be usefully used in formulations, lactic acid bacteria, feed additives, and health foods.
본 발명의 다른 양태에 따르면, 본 발명은 본 발명의 균주, 상기 균주의 배양물, 상기 균주의 발효물, 상기 배양물의 농축물, 상기 배양물의 건조물 및 이들의 조합으로 이루어진 군에서 선택된 어느 하나를 유효성분으로 포함하는 장 기능 개선용 식품 조성물은 식품, 음료 또는 발효유이다.According to another aspect of the present invention, there is provided a method for producing a microorganism, which comprises culturing a strain of the present invention, a culture of the strain, a fermentation product of the strain, a concentrate of the culture, a dried product of the culture, The food composition for improving intestinal function contained as an active ingredient is food, beverage or fermented milk.
또한, 상술한 성분 이외에도 공지의 첨가제로서 미각을 돋구기 위하여 매실, 레몬향, 파인애플향 또는 허브향과 같은 천연향료나 천연과즙, 클로르필린, 플라보노이드 등의 천연색소 및 감미 성분인 과당, 벌꿀, 당알코올, 설탕 등과 구연산, 구연산나트륨 같은 산미제를 혼합하여 사용할 수 있다. 또한, 본 발명의 균주 또는 이의 배양산물을 포함하는 식품으로 차, 젤리, 즙, 엑기스, 음료 등의 다양한 형태로 가공될 수 있으며, 본 발명의 식품은 인체에 부작용이 없으면서 복용이 용이하고 장기간 보관이 가능하다. 본 발명의 균주 또는 이의 배양산물을 식품 첨가물로 사용할 경우, 상기 각각의 균주 또는 이의 배양산물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 본 발명의 균주 또는 이의 배양산물을 식품 전체 100 중량부에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없으므로 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다. 상기 식품의 종류에는 특별한 제한은 없다. 본 발명의 균주 또는 이의 배양산물을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함하는 낙농제품, 각종 수프, 음료수, 차, 드링크제, 알코올음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다. 본 발명의 건강 음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토오스, 슈크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 균주 또는 이의 배양산물 100 ㎖당 일반적으로 0.01 내지 0.04 g, 바람직하게는 약 0.02 내지 0.03 g 이다. 본 발명의 상기 균주 또는 이의 배양산물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 프로바이오틱스는 천연 과일 주스, 과일 주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하지 않지만 본 발명의 생균제는 100 중량부 당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above-mentioned ingredients, known additives include natural flavors such as plum, lemon flavor, pineapple flavor or herb flavor, natural flavors such as natural fruit juice, chlorpillin and flavonoid, sweeteners such as fructose, honey, sugar alcohol , Sugar or the like and an acid agent such as citric acid or sodium citrate may be mixed and used. The food of the present invention can be processed into various forms such as tea, jelly, juice, extract, beverage and the like. The food of the present invention is easy to take while having no adverse effect on the human body, This is possible. When the strain or the culture product of the present invention is used as a food additive, each of the strains or the culture product thereof may be directly added, used together with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the strain or the culture product of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of the whole food. However, in the case of long-term consumption intended for health and hygiene purposes or health control purposes, the amount may be less than the above range, and there is no problem in terms of safety. Therefore, the active ingredient may be used in an amount of more than the above range. There is no particular limitation on the kind of the food. Examples of the food to which the strain or the culture product of the present invention can be added include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, , Beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense. The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. It is generally 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the strain or the culture product thereof. The strain or culture product of the present invention may be used as a nutrient, a vitamin, an electrolyte, a flavoring agent, a coloring agent, a pectic acid and its salt, an alginic acid and its salt, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, , Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the probiotics of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the proportion of such additives is not critical, the probiotics of the present invention are generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.
본 발명은 상기 균주 또는 이의 배양산물을 포함하는 발효유를 제공할 수 있다. 상기 균주 또는 이의 배양산물은 발효유 100 중량부 당 0,01 내지 50 중량부인 것이 바람직하나 이에 한정되는 것은 아니며 당해업계의 당업자가 제조시 중량부를 조절할 수 있다. The present invention can provide a fermented milk comprising the strain or a culture product thereof. The strain or the culture product thereof is preferably 0.01 to 50 parts by weight per 100 parts by weight of the fermented milk, but is not limited thereto, and the weight of the strain or the culture product thereof can be controlled by those skilled in the art.
또한, 본 발명은 상기 균주 또는 이의 배양산물을 이용한 발효유 제조방법을 제공한다. 이하, 발효유 제조방법을 상세히 설명한다.The present invention also provides a method for producing fermented milk using the strain or a culture product thereof. Hereinafter, a method for producing fermented milk will be described in detail.
제 1)단계는 프리바이오틱스 소재를 우유(milk)에 첨가하는 단계로, 프리바이오틱스 소재로 사용한 것은 제한되지 않으나, 바람직하게는 식물 추출물, 더욱 바람직하게는 꾸지뽕잎 또는 뽕잎 추출물임이 바람직하고, 상기 추출물은 동결건조된 것임이 바람직하다. 또한, 우유에 첨가시 상기 추출물의 농도는 0.1 내지 10 중량% 바람직하게는 0.1 내지 1 중량%일 수 있다. 또한, 1)단계 이후, 균질화 단계를 거칠 수 있다.The first step is a step of adding the prebiotic material to the milk. It is preferable that the prebiotic material is used as a prebiotic material, but it is preferably a plant extract, more preferably a leaf or mulberry leaf extract, The extract is preferably lyophilized. In addition, when added to milk, the concentration of the extract may be 0.1 to 10% by weight, preferably 0.1 to 1% by weight. Also, after step 1), a homogenization step can be carried out.
제 2)단계는 혼합물을 저온살균하는 단계로, 저온살균의 바람직한 온도는 70℃ 내지 90℃이고, 시간은 10분 내지 20분이다. The second step is a step of sterilizing the mixture, wherein the preferred temperature for the pasteurization is 70 ° C to 90 ° C and the time is 10 minutes to 20 minutes.
제 3)단계는 상기 락토바실러스 가세리 락토바실러스 가세리 SRK545(Lactobacillus gasseri SRK545) 균주(수탁번호 KCCM11767P), 락토바실러스 가세리 SRK559(Lactobacillus gasseri SRK559) 균주(수탁번호 KCCM11768P) 및 락토바실러스 가세리 SRK575(Lactobacillus gasseri SRK575) 균주(수탁번호 KCCM11769P)로 이루어진 군중 어느 하나 이상의 균주 또는 이의 배양산물을 접종하여 발효시키는 단계로, 상기 균주는 혼합물 총 질량의 1 내지 10 중량% 더욱 바람직하게는 2 내지 5 중량%일 수 있다. 이 후, 상기 혼합물은 30℃ 내지 50℃에서, 24 내지 100시간 발효할 수 있다.The third step is a step of isolating the Lactobacillus gasseri SRK545 strain (Accession No. KCCM11767P), the Lactobacillus gasseri SRK559 strain (Accession No. KCCM11768P) and the Lactobacillus gasseri SRK575 Lactobacillus gasseri SRK575 strain (Accession No. KCCM11769P), or a culture product thereof, and the strain is fermented by inoculation with 1 to 10% by weight, more preferably 2 to 5% by weight, Lt; / RTI > Thereafter, the mixture can be fermented at 30 ° C to 50 ° C for 24 to 100 hours.
본 발명의 균주를 프로바이오틱스로 사용하는 경우, 발효액에서 높은 항산화 효과가 우수하여, 이를 발효유 등을 포함하는 건강식품의 제조에 유용하게 사용할 수 있다. When the strain of the present invention is used as a probiotic, the antioxidant effect of the fermentation broth is excellent, and it can be usefully used for the production of health foods containing fermented milk and the like.
도 1은 균주를 이용하여 제조된 발효액의 가수분해 정도를 나타낸 도이다.
도 2는 본 발명의 균주와 프리바이오틱스 소재의 첨가 및 종류에 따른 발효액의 항산화 활성 효과를 ABTS를 이용하여 확인한 도이다.
도 3은 본 발명의 균주와 프리바이오틱스 소재의 첨가 및 종류에 따른 발효액의 항산화 활성 효과를 DPPH를 이용하여 확인한 도이다.
도 4는 본 발명의 균주와 프리바이오틱스 소재의 첨가 및 종류에 따른 발효액의 항산화 활성 효과를 FRAP를 이용하여 확인한 도이다.
도 5는 본 발명의 균주와 프리바이오틱스 소재의 첨가 및 종류에 따른 발효액의 항산화 활성 효과를 폴리페놀 함량 결과를 이용하여 확인한 도이다.
도 6은 본 발명의 균주와 프리바이오틱스 소재의 첨가 및 종류에 따른 발효액의 항산화 활성 효과를 플라보노이드 함량결과를 이용하여 확인한 도이다.
도 7은 본 발명의 선별된 균주인 545, 559 및 575 균주를 농도별로 희석한 경우, 프리바이오틱스 첨가 발효액의 농도별 항산화 활성 효능을 ABTS를 이용하여 확인한도이다.
도 8은 본 발명의 선별된 균주인 545, 559 및 575 균주를 농도별로 희석한 경우, 프리바이오틱스 첨가 발효액의 농도별 항산화 활성 효능을 DDPH를 이용하여 확인한도이다.
도 9는 본 발명의 선별된 균주인 545, 559 및 575 균주를 농도별로 희석한 경우, 프리바이오틱스 첨가 발효액의 농도별 항산화 활성 효능을 FRAP를 이용하여 확인한도이다.
도 10은 본 발명의 선별된 균주인 545, 559 및 575 균주를 농도별로 희석한 경우, 프리바이오틱스 첨가 발효액의 농도별 항산화 활성 효능을 폴리페놀 함량을 통하여 확인한도이다.
도 11은 본 발명의 선별된 균주인 545, 559 및 575 균주를 농도별로 희석한 경우, 프리바이오틱스 첨가 발효액의 농도별 항산화 활성 효능을 플라보노이드 함량을 통하여 확인한도이다.
도 12는 MRM method를 이용한 표준용액의 TIC 결과를 나타낸 도이다.
도 13은 MRM method를 이용한 꾸지뽕잎 첨가 발효액의 TIC 결과를 나타낸 도이다.
도 14는 MRM method를 이용한 뽕잎 첨가 발효액의 TIC 결과를 나타낸 도이다.
도 15는 발명의 선별된 균주인 545, 559 및 575 균주를 이용하여 제조된, 꾸지뽕잎을 첨가한 발효액의 Phenolic acid의 함량 변화를 나타낸 도이다.
도 16은 본 발명의 선별된 균주인 545, 559 및 575 균주를 이용하여 제조된, 뽕잎을 첨가한 발효액의 Phenolic acid의 함량 변화를 나타낸도이다.
도 17은 본 발명의 선별된 균주인 545, 559 및 575 균주를 이용하여 제조된, 꾸지뽕잎을 첨가한 발효액의 Rutin hydrate와 polyphenol 배당체의 함량 변화를 나타낸 도이다.
도 18은 본 발명의 선별된 균주인 545, 559 및 575 균주를 이용하여 제조된, 뽕잎을 첨가한 발효액의 Rutin hydrate와 polyphenol 배당체의 함량 변화를 나타낸 도이다.
도 19는 본원 발명의 균주를 이용하여 발효유를 제조하는 과정을 나타낸 도이다.FIG. 1 is a graph showing the degree of hydrolysis of a fermentation broth prepared using a strain.
FIG. 2 is a graph showing the antioxidative activity effect of the fermentation broth according to the addition and kinds of the strains and prebiotics of the present invention using ABTS.
FIG. 3 is a graph showing the antioxidative activity effect of the fermentation broth according to the addition and kind of the strains and prebiotics of the present invention using DPPH.
FIG. 4 is a graph showing the antioxidative activity effect of the fermentation broth according to the addition and kind of the strain and prebiotic material of the present invention using FRAP.
FIG. 5 is a graph showing the antioxidative activity effect of the fermentation broth according to the addition and type of the strain and prebiotic material of the present invention, using polyphenol content results.
FIG. 6 is a graph showing the antioxidative activity effect of the fermentation broth according to the addition and type of the strains and prebiotics of the present invention, using the results of the flavonoid contents.
FIG. 7 shows the antioxidant activities of prebiotic-supplemented fermentation broth obtained by diluting 545, 559 and 575 strains selected according to the present invention with ABTS.
FIG. 8 is a graph showing the antioxidant activity of each of 545, 559 and 575 strains selected by the present invention by DDPH according to the concentrations of prebiotics-added fermentation broth.
FIG. 9 is a graph showing the antioxidative activity of antibiotic activity of prebiotic-supplemented fermentation broth obtained by diluting 545, 559, and 575 strains selected by the present invention with FRAP.
FIG. 10 is a graph showing the antioxidative activity of the 545, 559 and 575 strains according to the present invention, when the strains were diluted by concentration, according to the concentration of the fermentation broth containing the prebiotics.
FIG. 11 is a graph showing the antioxidative activity of prebiotics-added fermentation broth when the 545, 559 and 575 strains selected according to the present invention were diluted by concentration, through the content of flavonoids.
12 shows a TIC result of the standard solution using the MRM method.
13 is a diagram showing the TIC results of the fermentation broth of cucumber leaf using the MRM method.
14 is a diagram showing the TIC result of the fermentation broth with mulberry leaf using the MRM method.
FIG. 15 is a graph showing changes in content of phenolic acid in fermentation broth containing cucumber leaf, prepared using 545, 559 and 575 strains selected from the strains of the present invention.
16 is a graph showing changes in content of phenolic acid in a fermentation broth supplemented with mulberry leaves prepared using
17 is a graph showing changes in the content of Rutin hydrate and polyphenol glycoside in the fermented broth added with cucumber leaf, prepared using
FIG. 18 is a graph showing changes in the contents of Rutin hydrate and polyphenol glycoside in fermentation broth containing mulberry leaves prepared using 545, 559 and 575 strains selected from the strains of the present invention.
19 is a view illustrating a process for producing fermented milk using the strain of the present invention.
[실시예 1]프로바이오틱스로 이용가능한 균주의 선별, 동정 및 상기 균주를 이용하여 제조된 발효액의 특성 평가 [Example 1] Selection and identification of strains usable as probiotics and characterization of fermentation broths prepared using the strains
1. 균주를 이용한 발효액 제조 1. Preparation of fermentation broth using strain
프리바이오틱스 신규소재인 꾸지뽕잎 또는 뽕잎을 동결건조한 후, 60℃로 가온한 우유에 농도가 0.2%가 되도록 첨가하고, 교반기로 1,000 rpm에서 약 15분간 균질화 하였다. 이 후, 95℃ 항온수조에서 약 15분간 살균하였고, 상온에서 식힌 후(cooling), 상기 균주 19종을 농도 3%가 되도록 접종하고, 41℃ 배양기에서 48시간 배양하였다. 상기 과정을 거쳐 제조된 발효액을 균주의 선별에 이용하였다.The new material of prebiotics, cucumber leaf or mulberry leaf, was lyophilized, added to milk at a temperature of 60 ° C to a concentration of 0.2%, and homogenized at 1,000 rpm for about 15 minutes with a stirrer. Thereafter, the cells were sterilized in a constant temperature water bath at 95 ° C for about 15 minutes, cooled at room temperature, inoculated with 19 strains at a concentration of 3%, and cultured in a 41 ° C incubator for 48 hours. The fermentation broth prepared through the above procedure was used for the selection of strains.
2. 발효액의 pH특성 평가 2. Evaluation of pH of fermentation broth
미생물학적 발효특성을 평가하기 위하여, 상기 실시예 1.1에서 제조한 발효액을 이용하여 pH를 측정하였다. 보다 구체적으로, 1.1의 발효액의 발효 전, 후의 pH를 Thermo 사의 ORION STAR A211 pH meter를 이용하여 측정하여, 이의 결과를 표 1에 나타내었다.In order to evaluate the microbiological fermentation characteristics, the pH was measured using the fermentation liquid prepared in Example 1.1 above. More specifically, the pH of the fermentation broth of 1.1 before and after fermentation was measured using a Thermo ORION STAR A211 pH meter, and the results are shown in Table 1.
상기 표 1에 나타난 바와 같이, 프리바이오틱 소재를 첨가하지 않은 대조군과 프리바이오틱스를 첨가한 그룹들 간의 발효 시간에 따른 pH가 큰 차이를 보였다. 하지만, 프리바이오틱스 소재 간 차이는 거의 없음을 확인하였다. As shown in Table 1, there was a large difference in pH depending on the fermentation time between the control group to which the prebiotic material was not added and the group to which the prebiotics were added. However, it was confirmed that there is almost no difference between the prebiotics materials.
또한, 균주 번호 508, 524, 545, 559, 575 및 589의 균주를 선별하여, 이 후 실험에 활용하였다.In addition, strains of
[표 1][Table 1]
3. 발효에 따른 가수분해 정도 확인3. Determination of degree of hydrolysis by fermentation
균주를 이용하여 제조된 발효액의 가수분해 정도를 도 1에 나타내었다.The degree of hydrolysis of the fermentation broth prepared using the strain is shown in Fig.
도 1에 나타난 바와 같이, 유아분변 유래 균주를 이용하여 48시간 동안 발효한 경우, 식물 추출물 유래 프리바이오틱스 첨가 시 가수분해 정도가 증가함을 확인하였다. 균주별로 가수분해 정도의 차이는 크게 나타나지 않았으나, 꾸지뽕잎 추출물을 첨가하였을 때 가수분해 정도가 크게 증가함을 확인하였다.As shown in FIG. 1, when fermentation was conducted for 48 hours using a strain derived from infant feces, it was confirmed that the degree of hydrolysis was increased upon addition of prebiotics derived from plant extracts. The degree of hydrolysis was not significantly different for each strain but the addition of cucurbit leaf extract significantly increased the degree of hydrolysis.
4. 균주의 분자생물학적 동정4. Molecular biology of the strain
16s DNA 서열분석을 통하여, 실시예 1.2에서 선별한 균주를 동정하였다. 보다 구체적으로, 상기 6종의 균주를 MRS broth에서 배양 후, 채취하여 DNA를 분리하였다. 이 후, PCR을 이용하여 DNA stock을 제조한 후, PCR 분석(Analyzed PCR), PCR 산물의 추출(purify PCR) 및 전기영동단계를 거쳐 동정하였다. 이의 결과를 표 2에 나타내었다.16s DNA sequence analysis, the strains selected in Example 1.2 were identified. More specifically, the six strains were cultured in MRS broth and then harvested to isolate DNA. Then, DNA stocks were prepared using PCR, and then analyzed by PCR, analyzed by purify PCR, and electrophoresis. The results are shown in Table 2.
[표 2][Table 2]
표 2에 나타난 바와 같이, 유아 분변에서 분리된 균주는 모두 락토바실러스 가세리(Lactobacillus gasseri)로 분류되었고, 이 중 545, 559, 575 균주 각각을 락토바실러스 가세리(Lactobacillus gasseri) SRK545, 락토바실러스 가세리 SRK 559, 락토바실러스 가세리 SRK575로 명명하여, 한국미생물보존센터에 2015년 9월 11일자로 기탁하여, 수탁번호 KCCM11767P, KCCM11768P, KCCM11769P를 부여받았다.As shown in Table 2, all strains isolated from infant feces were classified as Lactobacillus gasseri, and 545, 559 and 575 strains were classified as Lactobacillus gasseri SRK545,
5.발효액 내 펩타이드 프로파일링 5. Peptide profiling in fermentation broth
MALDI-TOF/MS를 이용하여, 식물추출물 유래 프리바이오틱스 및 유아분변 유래 프로바이오틱스를 이용하여 제조된 발효액 내에 생성되는 펩타이드의 분석하였다. 이의 결과를 표 3에 나타내었다.MALDI-TOF / MS was used to analyze the peptides produced in the fermentation broth prepared using plant extract-derived prebiotics and infant feces-derived probiotics. The results are shown in Table 3.
표 3에 나타난 바와 같이, 총 18개의 펩타이드를 확인하였다. 균주별로 생성된 펩타이드는 큰 차이가 없었으며, 주로 β-casein에서 유래된 펩타이드가 많이 확인되었음을 확인하였다. 발효로 인해 생성된 펩타이드가 항산화 활성 증진에 영향에 줄 수 있을 것이라 판단된다.As shown in Table 3, a total of 18 peptides were identified. There was no significant difference in the peptides produced by each strain, and it was confirmed that many peptides derived mainly from β-casein were found. Peptides produced by fermentation may be expected to influence the antioxidant activity.
[표 3][Table 3]
7. 선별된 균주를 이용하여 제조된 발효액의 첨가 프리바이오틱스 소재별 균수 측정 7. The addition of prebiotics material by measuring the number of bacteria of the selected strains produced by using the fermentation broth
선별 균주와 프리바이오틱스 소재를 첨가하여 제조된 발효유의 균수 측정 결과를 하기 표 4에 나타내었다. 꾸지뽕잎과 뽕잎 모두 575 > 559 > 545 순으로 균 수가 증가하였다. 하지만 프리바이오틱스 소재를 첨가하지 않고 우유와 균주만을 발효한 경우, 발효 후 균 수가 0.2 내지 0.4 log / CFU mL 정도 감소하는 경향을 보였으나 발효 전, 후 차이가 크게 나타나지 않음을 확인하였다.Table 4 shows the results of the measurement of the number of bacteria in the fermented milk prepared by adding the strain and the prebiotics. The number of bacilli increased in the order of 575> 559> 545. However, in the case of fermentation of only milk and strain without adding the prebiotic material, the number of bacteria after fermentation tended to decrease by 0.2 to 0.4 log / CFU mL, but it was confirmed that there was not much difference between before and after fermentation.
[표 4][Table 4]
[실시예 2]본 발명의 균주를 이용하여 제조된 발효액의 항산화 활성 확인[Example 2] Confirmation of antioxidant activity of a fermentation broth prepared using the strain of the present invention
1.ABTS assay 1.ABTS assay
먼저, 에탄올에 7 mM의 ABTS (2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt)를 녹인 용액 A 와 증류수에 2.45 mM 포타슘 퍼설페이트(potassium persulfate)를 녹인 용액 B를 잘 혼합한 후, 상온에서 12 내지 16 시간 정치하여 활성산소(radical)를 생성시키고, 이를 743 nm에서 흡광도가 1.4가 되도록 증류수로 희석하여 ABTS reagent를 제조하였다. 이 후, 샘플 20 μL(발효액)를 180 μL ABTS reagent와 반응시킨 후, 6분간 정치하였고, 734 nm에서 흡광도를 측정하였다. First, a solution A in which 7 mM ABTS (2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt) was dissolved in ethanol and a solution B in which 2.45 mM potassium persulfate was dissolved in distilled water After mixing well, the mixture was allowed to stand at room temperature for 12 to 16 hours to produce active radicals. The ABTS reagent was prepared by diluting it with distilled water to an absorbance of 1.4 at 743 nm. Subsequently, 20 μL of the sample (fermentation broth) was reacted with 180 μL ABTS reagent, allowed to stand for 6 minutes, and absorbance was measured at 734 nm.
2.DPPH assay2. DPPH assay
0.3 mM DPPH (2,2 Diphenyl 1 picrylhydrazyl)를 메탄올에 가시광선을 제거한 상태에서 오랜시간 용해시킨 후, 517 nm의 흡광도에서 0.9 내지 1.0의 흡광도를 갖도록 증류수를 이용하여 희석하여 DPPH reagent를 제조하였다. 여기에 샘플 용액 50 μL 와 DPPH reagent 150 μL를 혼합한 후, 암조건에서, 37℃, 30 분간 반응 시킨 후, 517 nm에서 흡광도를 측정하였다.The DPPH reagent was prepared by dissolving 0.3 mM DPPH (2,2
3.FRAP assay3.FRAP assay
FRAP(Ferric Reducing Ability of Plasma) reagent를 하기와 같은 방법으로 제조하였다. 먼저, 0.31 g의 아세트 나트륨 삼수화물(sodium acetate trihydrate)에 1.6 mL의 빙초산(glacial acetic acid)을 첨가한 후, 증류수를 첨가하고 100 mL로 부피를 조절하여 용액 A를 제조하였다. 또한, 10 mM의 TPTZ(2,4,6-tripyridyl- s-triazine)를 10 mL 40 mM의 HCl과 혼합하여 용액 B를 제조하였다. 또한, FeCl36H2O (Iron(III) Chloride hexahydrate) 20 mM을 10 mL 증류수에 녹인 용액 C를 제조하였다. 이 후, 용액 A, B, C를 혼합하여 FRAP reagent를 제조하였다. 샘플 6 μL와 FRAP reagent 180 μL를 혼합한 뒤, 37℃에서 30분간 반응을 진행한 후, 593 nm에서 흡광도를 측정하였다. 대조구로 샘플과 동량의 증류수를 FRAP reagent와 반응시켜 blank값으로 삼았다. FeSO4 7H2O (iron(II)sulfate heptahydrate)를 0 내지 1.0 mM의 농도로 조정하여 이를 593 nm 파장대에서 흡광도를 측정하였고, 해당 흡광도를 이용하여 직선의 검량곡선을 작성하고, 샘플의 항산화력을 FeSO47H2O의 농도에 대비하여 나타내었다.Ferric Reducing Ability of Plasma (FRAP) reagent was prepared as follows. First, 1.6 mL of glacial acetic acid was added to 0.31 g of sodium acetate trihydrate, and then distilled water was added thereto, and the volume was adjusted to 100 mL to prepare Solution A. In addition, Solution B was prepared by mixing 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) with 10 mL of 40 mM HCl. In addition, a solution C in which 20 mM of FeCl 36H 2 O (Iron (III) Chloride hexahydrate) was dissolved in 10 mL of distilled water was prepared. After that, FRAP reagent was prepared by mixing solutions A, B, and C. After mixing 6 μL of the sample and 180 μL of the FRAP reagent, the reaction was carried out at 37 ° C. for 30 minutes and the absorbance was measured at 593 nm. As a control, samples and the same amount of distilled water were reacted with FRAP reagent to make a blank value. FeSO 4 7H 2 O (iron (II) sulfate heptahydrate) was adjusted to a concentration of 0-1.0 mM, and the absorbance was measured at a wavelength of 593 nm. A calibration curves were prepared using the absorbance and the antioxidant activity of FeSO 4 7H are shown against the concentration of O 2.
4. Total polyphenol content assay4. Total polyphenol content assay
총 폴리페놀 함량은 Folin-Denis법에 의해 비색 정량하였다. 샘플 25 μL, folin-ciocalteu's phenol reagent 62.5 μL을 20%의 탄산나트륨 용액(sodium carbonate solution) 312.5 μL에 차례로 가하여, 교반한 후, 40분 방치하였다. 반응액은 분광광도계(spectrophotometer; UV-1601, SHIMADZU, Japan)를 이용하여 765 nm에서 흡광도를 측정하였다. 표준 용액은 갈산(gallic acid) 용액(0 내지 100 mg)를 사용하였다.Total polyphenol content was determined by colorimetry by the Folin-Denis method. 25 μL of the sample and 62.5 μL of the folin-ciocalteu's phenol reagent were sequentially added to 312.5 μL of a 20% sodium carbonate solution, and the mixture was left to stand for 40 minutes. The absorbance of the reaction solution was measured at 765 nm using a spectrophotometer (UV-1601, SHIMADZU, Japan). The standard solution was a gallic acid solution (0-100 mg).
5. Total flavonoid assay5. Total flavonoid assay
플라보노이드 함량을 평가하기 위하여, 플라보노이드와 메탈 이온과 킬레이트(chelate)를 형성하면, 특유의 발색반응이 일어남을 이용하여. 시료 속 플라보노이드의 정성 및 정량을 분석하는 원리를 이용하였다. 먼저, 0.2g의 AlCl36H2O(Aluminum (Ⅲ) chloride hexahydrate)을 10ml의 에탄올에 녹여 2%의 AlCl36H2O 용액을 제조한 후, 샘플 100 μL와 상기 용액 100 μL를 10분간 반응시켜 430 nm에서 흡광도를 측정하였다. 표준물질은 퀘르세틴(Quercetin) 용액 (0 내지 100 mg)을 이용하였다.In order to evaluate the content of flavonoid, flavonoid, metal ion and chelate are formed, and specific color reaction occurs. The principle of analyzing the qualities and quantities of flavonoids in the sample was used. First, 0.2 g of AlCl 3 6H 2 O (Aluminum (III) chloride hexahydrate) was dissolved in 10 ml of ethanol to prepare a 2% AlCl 3 6H 2 O solution. Then, 100 μL of the sample and 100 μL of the solution were reacted for 10 minutes And the absorbance was measured at 430 nm. Quercetin solution (0-100 mg) was used as a standard substance.
6. LC-MS/MS를 이용한 꾸지뽕잎과 뽕잎 추출물 첨가 발효액의 폴리페놀 분석6. Polyphenol analysis of fermentation broth containing cucumber leaf and mulberry leaf extract by LC-MS / MS
꾸지뽕잎 또는 뽕잎 추출물을 첨가하는 경우, 상업균주를 이용하여 5시간 발효한 발효액의 폴리페놀 및 본 발명에서 선별한 균주(505, 508, 524, 545, 559, 575, 589)를 이용하여 48시간 발효한 발효액의 폴리페놀 함량을 분석하였다.In case of adding cucumber leaf or mulberry leaf extract, polyphenol of the fermentation broth fermented for 5 hours using a commercial strain and strains (505, 508, 524, 545, 559, 575, 589) The polyphenol content of fermented fermentation broth was analyzed.
각 균주를 이용하여 제조된 발효액을 동결건조시킨 샘플을 정확히 1g 취하여 7 mL, 50% ethanol 0.05M H3P04 가 포함된 에탄올 용액을 가하여 섞은 후, 섞어준 샘플을 20분간 상온에서 초음파처리를 통해 추출하였다. 이 후, 5000 rpm에서 30분간 원심분리하여 상등액을 취하였고, 상등액을 0.2 μm 필터를 이용하여 필터링한 후, LC-MS로 분석하였다. 표준 용액(Standard stock solution)을 네오-클로로겐산(Neo-chlorogenic acid), 클로로겐산(chlorogenic acid), 디3,4-하이드록시히드로신난산(3,4-Dihydroxyhydrocinnamic acid), 카페인산(Caffeic acid), 루틴 수화물(Rutin hydrate), 퀘르세틴-3-갈락토사이드(Quercetin-3-galactoside), 퀘르세틴-3-글루코사이드(Quercetin-3- glucoside), 캄페롤-3-갈락토사이드(Kaempferol-3-galactoside), 캄페롤-3-루티노사이드(Kaempferol-3-rutinoside) 또는 캄페롤-3-글루코사이드(Kaempferol-3-glucoside)를 섞어 1 mg/mL로 제조 후, 10%의 ACN(acetonitrile)을 이용하여 10, 50, 100 및 1000 μg/mL로 희석하여 사용하였다. LC-MS는 Waters사의 UPLC-Acquity Binary pump, ZEVO-TQ를 이용하여 실험하였고, 0.1%의 포름산 용액, 0.1%의 포름산이 포함된 ACN을 A 및 B용매로 각각 사용하였다. 칼럼(Column)은 Acquity UPLC BEH C18 1.7μm (2.1x100mm)를 사용하였다. Gradient 조건은 표 5과 같으며, LC-MS 세부조건은 표 6와 같다. 또한, 상기 10가지 표준 용액에 대한 Tune 결과, 하기 표 7과 같은 조건에서 가장 높은 감도를 얻을 수 있었다. 이를 통하여 전체적인 MRM 방법을 확립하였고, 정량 시 이 조건을 이용하였다. Take exactly 1 g of the lyophilized sample of the fermentation broth prepared with each strain, add 7 mL of 50% ethanol with 0.05 M H 3 PO 4 in ethanol, mix the sample, and ultrasonicate for 20 minutes at room temperature. And extracted. The supernatant was then centrifuged at 5000 rpm for 30 minutes, and the supernatant was filtered through a 0.2 μm filter and analyzed by LC-MS. Standard stock solution is a mixture of neo-chlorogenic acid, chlorogenic acid, 3,4-dihydroxyhydrocinnamic acid, caffeic acid, Rutin hydrate, Quercetin-3-galactoside, Quercetin-3-glucoside, Kaempferol-3-galactoside, , Kaempferol-3-rutinoside, or Kaempferol-3-glucoside were mixed to prepare 1 mg / mL of the solution. Then, 10% ACN (acetonitrile) 10, 50, 100, and 1000 μg / mL, respectively. The LC-MS was tested with UPLC-Acquity Binary pump, ZEVO-TQ from Waters, and ACN containing 0.1% formic acid solution and 0.1% formic acid was used as A and B solvents, respectively. Acquity UPLC BEH C18 1.7 mu m (2.1 x 100 mm) was used as a column. Gradient conditions are shown in Table 5, and LC-MS detailed conditions are shown in Table 6. As a result of the tune for the above 10 standard solutions, the highest sensitivity was obtained under the conditions shown in Table 7 below. Through this, the whole MRM method was established and this condition was used for quantification.
[표 5][Table 5]
[표 6][Table 6]
[표 7][Table 7]
7. 결과 종합7. Results synthesis
본원 발명의 균주와 프리바이오틱스 소재의 첨가 및 종류에 따른 발효액의 항산화 활성 평가를 위해, DPPH, FRAP, ABTS의 결과를 도 2 내지 도 4에 나타내었다. The results of DPPH, FRAP and ABTS are shown in Figs. 2 to 4 for the evaluation of the antioxidative activity of the fermentation broth according to the addition of the strains and the prebiotics of the present invention and the kinds thereof.
도 2 내지 도 4에 나타난 바와 같이, 접종 직후의 발효액보다 48시간 발효 후 발효액의 항산화 활성이 높음을 확인하였다. 특히, 프리바이오틱스 소재로써 첨가한 꾸지뽕잎 및 뽕잎의 발효액이 우유 발효액이 항산화 활성이 4 내지 7배 정도 높음을 확인하였다. 또한, 프리바이오틱스 소재 간 항산화 활성의 차이를 보면, ABTS, DPPH, FRAP 실험 결과 모두 꾸지뽕잎을 첨가한 발효액이 뽕잎을 첨가한 발효액보다 항산화 활성이 높음을 확인하였다. 또한, 총 7개의 균주 가운데, 545, 559, 575 균주가 다른 균주에 비하여 활성이 높게 나타났으며 이는 ABTS, DPPH, FRAP 모두 유사한 결과를 나타냄을 확인하였다.As shown in Figs. 2 to 4, it was confirmed that the antioxidant activity of the fermentation broth after 48 hours fermentation was higher than that of the fermentation broth immediately after inoculation. Especially, it was confirmed that the fermentation broth of cucumber leaf and mulberry leaf added as a prebiotic material had antioxidative activity of about 4 to 7 times higher than that of fermented milk. The results of ABTS, DPPH and FRAP showed that the fermentation broth containing cucumber leaves has higher antioxidant activity than the fermented broth containing mulberry leaves. Among the seven strains, 545, 559 and 575 strains showed higher activity than the other strains, indicating that ABTS, DPPH and FRAP showed similar results.
또한, 도 5에 발효액 내 페놀함량을 나타내었고, 도 6에 플라보노이드 함량을 나타내었다.5 shows the phenol contents in the fermentation broth, and FIG. 6 shows the flavonoid contents.
도 5에 나타난 바와 같이, 총 폴리페놀 함량 실험 결과에서 꾸지뽕잎을 첨가한 발효액과 뽕잎을 첨가한 발효액이 비슷한 경향으로 폴리페놀을 함유하고 있음을 확인하였다. 특히, 꾸지뽕잎을 첨가한 발효액이 더 높은 활성을 가지고 있는 것을 확인하였다.As shown in FIG. 5, the total polyphenol contents of the fermented broth containing mulberry leaf and mulberry leaf were similar to those of polyphenol. Especially, it was confirmed that the fermentation broth added with cilantro leaves has higher activity.
도 6에 나타난 바와 같이, 꾸지뽕잎을 첨가한 발효액이 뽕잎을 첨가한 발효액보다 모든 균주에서 3배 이상의 플라보노이드를 함유하고 있음을 확인하였다.As shown in FIG. 6, it was confirmed that the fermentation broth containing cucurbit leaf contains 3 times more flavonoid than all fermented broth containing mulberry leaf.
상기 결과를 토대로, 항산화 활성 효능이 높은 3개의 균주 (545, 559, 575)를 선별하여 이후 농도별 항산화 활성 측정 및 생리활성 효능 실험을 추가적으로 진행하였다.Based on the above results, three strains (545, 559, and 575) having high antioxidant activity were selected and then the antioxidant activity and the physiological activity test were further performed.
선별된 균주 545, 559, 575 균주를 농도별로 희석하여 제조된 발효액의 농도별 항산화 활성 효능을 도 7 내지 도 11에 나타내었다.7 to 11 show the antioxidative activity effects of the selected strains 545, 559 and 575 according to the concentrations of the fermentation broth prepared by diluting the strains.
도 7에 나타난 바와 같이, ABTS를 수행한 경우, 꾸지뽕잎 또는 뽕잎 첨가시, 프로바이오틱스 발효액 모두 농도 의존적으로 항산화 활성이 감소함을 확인하였다.우유의 경우, 발효 전과 후가 차이가 없었으나, 꾸지뽕잎 발효액의 경우, 575 균주의 항산화 활성 효과가 가장 컸으며, 뽕잎 발효액의 경우, 559 균주가 항산화 활성 효능이 큼을 확인하였다.As shown in Figure 7, in the case of ABTS, the antioxidative activity of the probiotic fermentation broth was decreased in a concentration-dependent manner when cucumber leaves or mulberry leaves were added. In the case of milk, there was no difference between before and after fermentation, In the case of fermented broth, 575 strains had the highest antioxidant activity and 559 strains of mulberry leaf fermented broth showed antioxidant activity.
도 8에 나타난 바와 같이, DPPH를 수행한 경우, 꾸지뽕잎 발효액의 경우, 100mg/mL 농도에서는 88% 이상, 50 mg/mL 농도에서 85% 이상의 항산화 활성을 효능을 보임을 확인하였고, 뽕잎 발효액의 경우, 100 mg/mL에서는 88%, 50mg/mL에서는 70% 이상의 항산화 활성 효과를 보임을 확인하였다. 농도가 낮아짐에 따라 꾸지뽕잎 발효액과 뽕잎 발효액의 활성 차이가 나타남을 확인하였고, 꾸지뽕잎 발효액에 더 활성이 높음을 확인하였다.As shown in FIG. 8, when DPPH was performed, it was confirmed that the cucumber leaf fermentation broth exhibited an antioxidative activity of 88% or more at a concentration of 100 mg / mL and 85% or more at a concentration of 50 mg / mL. , It was confirmed that the antioxidant activity was 88% at 100 mg / mL and 70% at 50 mg / mL. As the concentration decreased, the activity difference between the fermented broth and the mulberry leaf fermented was found to be higher than that of the fermented broth.
도 9에 나타난 바와 같이, FRAP를 수행한 경우, 꾸지뽕잎 발효액이 뽕잎 발효액보다 100mg/mL 농도에서 높은 활성을 보였으며, ABTS, DPPH와 달리 575번의 FRAP 활성이 높음을 확인하였다.As shown in FIG. 9, when FRAP was carried out, the fermented broccoli fermentation broth showed higher activity than the mulberry leaf fermentation broth at a concentration of 100 mg / mL, and the FRP activity of 575 was higher than that of ABTS and DPPH.
또한, 도 10에 나타난 바와 같이, 총 폴리페놀 함량에서 559 또는 575 균주를 이용한 꾸지뽕잎 발효액에서 발효 후 폴리페놀 함량이 발효 전 총 폴리페놀 함량의 6배 정도 높음을 확인하였고, 545 균주를 이용한 꾸지뽕잎 발효액에서 4배 이상의 총 폴리페놀 함량이 증가함을 확인하였다. 뽕잎 발효액의 경우, 발효 전보다 발효 후 총 폴리페놀 함량이 2배 이상 증가함을 확인하였고, 559 균주를 이용하여 제조된 발효액의 총 폴리페놀 함량이 다른 균주에 비하여 상대적으로 높음을 확인하였다.Also, as shown in FIG. 10, it was confirmed that the polyphenol content of the cucumber leaf fermentation broth using 559 or 575 strains in the total polyphenol content was 6 times higher than the total polyphenol content before fermentation after fermentation, and 545 strains The total polyphenol content of the leaf fermentation broth was increased by 4 times or more. In the case of mulberry leaf fermented liquid, the total polyphenol content increased more than two times after fermentation before fermentation, and the total polyphenol content of the fermented liquid prepared using 559 strain was higher than that of the other strains.
도 11에 나타난 바와 같이, 총 플라보노이드 함량의 경우, 꾸지뽕잎 발효액의 발효 후 함량이 14 g/ 100 g 이상으로 뽕잎 발효액의 폴리페놀 함량에 비하여 5배가량 높은 함량을 갖은 것을 확인하였따. 특히, 꾸지뽕잎 발효액의 경우, 545, 575 균주의 플라보노이드 함량이 높은 것을 확인하였고, 뽕잎 발효액의 경우, 545 균주의 총 플라보노이드 함량이 높은 것을 확인하였다.As shown in FIG. 11, it was confirmed that the total flavonoid content was higher than that of the mulberry leaf fermentation broth by about 5 times as much as that of the fermented broccoli leaf fermentation broth of 14 g / 100 g or more. Especially, it was confirmed that flavonoid contents of 545 and 575 strains were higher in the fermented broccoli leaf, and 545 strains were higher in mulberry leaf fermentation broth.
또한, 도 12 내지 18에 MRM method를 이용하여 발효액을 분석한 결과를 나타내었다(도 12는 표준용액에서의 크로마토그램을 나타내었고, Neo-chlorogenic acid, Chlorogenic acid, 3,4-Dihydroxyhydrocinnamic acid, Caffeic acid, Quercetin-3-galactoside, Quercetin-3-glucoside, Kaempferol-3-galactoside, Kaempferol-3-rutinoside, Kamepferol-3-glucoside 순으로 확인 피크가 확인됨; 도 13: 꾸지뽕잎을 첨가한 발효액의 TIC 결과; 도 14: 뽕잎을 첨가한 발효액의 TIC 결과; 도 15: 꾸지뽕잎을 첨가한 유아 분변 유래 균주를 이용하여 제조된 발효액의 Phenolic acid의 함량 변화를 나타냄 도 16: 뽕잎을 첨가한 유아 분변 유래 균주를 이용하여 제조된 발효액의 Phenolic acid의 함량 변화를 나타냄; 도 17: 꾸지뽕잎을 첨가한 유아 분변 유래 균주를 이용하여 제조된 발효액의 Rutin hydrate와 polyphenol 배당체의 함량 변화 나타를 나타냄; 도 18: 뽕잎을 첨가한 유아 분변 유래 균주를 이용하여 제조된 발효액의 Rutin hydrate와 polyphenol 배당체의 함량 변화를 나타냄)12 to 18 show the result of analysis of the fermentation broth using the MRM method. (FIG. 12 shows the chromatogram in the standard solution. Neo-chlorogenic acid, chlorogenic acid, 3,4-dihydroxyhydrocinnamic acid, Caffeic 3-glucoside, Kaempferol-3-glucoside, Kaempferol-3-galactoside, Kaempferol-3-rutinoside and Kamepferol-3-glucoside in the order of ascorbic acid, quercetin-3-galactoside, quercetin- Fig. 14: TIC results of fermentation broth with mulberry leaf; Fig. 15: Changes in content of phenolic acid in fermentation broth prepared from infant feces derived from cucumber leaves Fig. 16: Figure 17: Changes in the contents of Rutin hydrate and polyphenol glycosides in fermentation broths prepared from infant feces derived from cucumber leaves, showing the change in content of phenolic acid in the fermented broth 18: Changes in the contents of Rutin hydrate and polyphenol glycosides in fermentation broths prepared from infant feces derived from mulberry leaves.
도 13 내지 18에 나타난 바와 같이, 유아분변 유래 균주를 이용하여 발효한 결과, Phenolic acid 중 Neo-chlorogenic acid, Chlorogenic acid, Caffeic acid의 함량은 감소하였고, 3,4-Dihydroxyhydroinnamic acid의 함량은 대폭 증가함을 확인하였다. Chlorogenic acid는 발효로 인해 대사되는데 에스테르 결합의 손실로 인하여 caffeic acid가 생성이 되고, 또한, caffeic acid 내 이중결합의 감소로 인하여 3,4-Dihydroxyhydro cinnamic acid를 형성한다. 상기와 같이, 꾸지뽕잎 또는 뽕잎을 첨가하여 발효하는 경우, 꾸지뽕잎 및 뽕잎 내에 존재하는 phenolic acids가 발효특성 및 항산화 활성 증진에 영향을 준 것 판단된다.As shown in FIGS. 13 to 18, the content of neo-chlorogenic acid, chlorogenic acid, and caffeic acid in phenolic acid decreased while the content of 3,4-dihydroxyhydroinnamic acid was significantly increased Respectively. Chlorogenic acid is metabolized by fermentation. Caffeic acid is formed due to loss of ester bond, and 3,4-Dihydroxyhydro cinnamic acid is formed due to decrease of double bond in caffeic acid. As described above, when fermenting with the addition of cucumber leaves or mulberry leaves, the phenolic acids present in the leaves and the mulberry leaves were judged to have an influence on the fermentation characteristics and the antioxidant activity enhancement.
또한, 유아 분변 유래 균주를 이용하여 발효한 결과, Rutin hydrate와 배당체 (Quercetin-3-glucoside, Kaempferol-3-rutinoside, Kaempferol-3-glucoside)의 경우에는 발효 전후 큰 차이를 보이지 않음을 확인하였다. As a result of fermentation using infant fecal-derived strains, it was confirmed that there was no significant difference between fermentation and rutin hydrate in the case of quercetin-3-glucoside, kaempferol-3-rutinoside and kaempferol-3-glucoside.
[실시예 3]유아 분변 분리균주를 이용한 항산화 기능성을 갖는 발효유 제조[Example 3] Production of fermented milk having antioxidative function using infant feces isolates
1. 현탁액 제조1. Manufacture of suspension
본 발명의 균주를 이용하여 발효유를 제조하기 위하여, 유산균 현탁액 제조 MRS broth에 본 발명의 유아 분변 분리 유산균을 접종하여 18시간 배양하였다. 배양 후, 원심분리를 이용하여 상등액을 버리고 펠렛(pellet)만 취하여 2번 세척하고, 펠렛을 1/10로 농축하여 현탁액을 제조하였다. To produce fermented milk using the strain of the present invention, the lactic acid bacteria isolated from infant feces of the present invention was inoculated into MRS broth of lactic acid bacterium suspension preparation and cultured for 18 hours. After the incubation, the supernatant was discarded by centrifugation, and the pellet was washed twice by taking only the pellet, and the pellet was concentrated to 1/10 to prepare a suspension.
2. 발효유 제조2. Production of fermented milk
하기 표 8의 배합 비율에 따라 계량 및 교반(무가당은 정백당, 향 제외)하였다. 유가당의 경우, 향을 투입하여 자재가 섞이도록 교반하였다. 이 후, 95℃, 5분동안 살균하고, 41℃ 내지 43℃로 조정하여 냉각하고, 현택액을 배양 종균접종 후, 41℃로 배양 후, pH 4.4 내지 4.5에서 배양을 종료하였다.And were weighed and stirred according to the blending ratio shown in Table 8 below. In case of oil sugar, fragrance was added, and the mixture was stirred to mix the materials. Thereafter, the mixture was sterilized at 95 DEG C for 5 minutes, adjusted to 41 DEG C to 43 DEG C, cooled, and incubated at 41 DEG C after inoculation with the suspension, followed by termination of the culture at pH 4.4 to 4.5.
[표 8][Table 8]
3. 발효유의 유산균수 측정3. Measurement of the number of lactic acid bacteria in fermented milk
본 발명의 균주 및 꾸지뽕잎을 이용하여 제조한 발효유의 유산균수를 측정하였다. 이의 결과를 표 9에 나타내었다. The number of lactic acid bacteria in the fermented milk prepared using the strain of the present invention and cucumber leaves was measured. The results are shown in Table 9.
표 9에 나타난 바와 같이, 발효 후 유산균 수는 배양 전보다 2 내지 3 log/CFU 증가함을 확인하였다. 이는 천연 식용 소재 꾸지뽕 농축액이 프리바이오틱스로서 작용하여 유산균수를 증가시킨 것으로 판단된다. 또한, 상업 균주와 유아 분변 유래 선별 균주의 상승효과로 인하여 총 유산균수가 증가한 것으로 판단된다. As shown in Table 9, it was confirmed that the number of lactic acid bacteria after fermentation increased by 2 to 3 log / CFU before the culture. This suggests that the natural edible material Coconut concentrate increased the number of lactic acid bacteria by acting as prebiotics. In addition, the total number of lactic acid bacteria was increased due to the synergistic effect of commercial strains and selective strains derived from infant feces.
기탁기관명 : 한국미생물보존센터(국외)Name of depository: Korea Microorganism Conservation Center (overseas)
수탁번호 : KCCM11767PAccession number: KCCM11767P
수탁일자 : 2015911Checked on: 2015911
기탁기관명 : 한국미생물보존센터(국외)Name of depository: Korea Microorganism Conservation Center (overseas)
수탁번호 : KCCM11768PAccession number: KCCM11768P
수탁일자 : 2015911Checked on: 2015911
기탁기관명 : 한국미생물보존센터(국외)Name of depository: Korea Microorganism Conservation Center (overseas)
수탁번호 : KCCM11769PAccession number: KCCM11769P
수탁일자 : 2015911Checked on: 2015911
Claims (5)
1 ~ 10 중량%의 락토바실러스 가세리 SRK545(Lactobacillus gasseri SRK545) 균주(수탁번호 KCCM11767P), 락토바실러스 가세리 SRK559(Lactobacillus gasseri SRK559) 균주(수탁번호 KCCM11768P), 및 락토바실러스 가세리 SRK575(Lactobacillus gasseri SRK575) 균주(수탁번호 KCCM11769P)로 이루어진 군에서 선택된 어느 하나의 균주를 포함하는 항산화 및 면역강화용 발효유.
0.1 to 10% by weight of leaf extract or mulberry leaf extract; And
1 to 10% by weight of Lactobacillus gasseri SRK545 strain (Accession No. KCCM11767P), Lactobacillus gasseri SRK559 strain (Accession No. KCCM11768P), and Lactobacillus gasseri SRK575 ) Strain (Accession No. KCCM11769P). The antioxidant and immune enhancing fermented milk according to claim 1,
상기 균주는 상기 균주의 배양물, 상기 배양물의 농축물, 상기 균주의 발효물, 및 배양물의 건조물로 이루어진 군에서 선택된 어느 하나를 더 포함하는 것을 특징으로 하는 항산화 및 면역강화용 발효유.
The method according to claim 1,
Wherein the strain further comprises any one selected from the group consisting of a culture of the strain, a concentrate of the culture, a fermentation product of the strain, and a dried product of the culture.
2) 상기 혼합물을 70 ~ 90 ℃에서 10 ~ 20분 동안 저온살균하는 단계; 및
3) 상기 저온살균된 혼합물에 1 ~ 10 중량%의 락토바실러스 가세리 SRK545(Lactobacillus gasseri SRK545) 균주(수탁번호 KCCM11767P), 락토바실러스 가세리 SRK559(Lactobacillus gasseri SRK559) 균주(수탁번호 KCCM11768P), 및 락토바실러스 가세리 SRK575(Lactobacillus gasseri SRK575) 균주(수탁번호 KCCM11769P)로 이루어진 군에서 선택된 어느 하나의 균주를 접종하여 발효시키는 단계;를 포함하는 항산화 및 면역강화용 발효유의 제조방법.
1) adding 0.1 to 10% by weight of leaf extract or mulberry leaf extract to milk to obtain a mixture;
2) pasteurizing the mixture at 70-90 ° C for 10-20 minutes; And
3) To the above pasteurized mixture, 1 to 10% by weight of Lactobacillus gasseri SRK545 strain (Accession No. KCCM11767P), Lactobacillus gasseri SRK559 (Accession No. KCCM11768P) and Lactobacillus gasseri SRK559 And a step of inoculating and fermenting a strain selected from the group consisting of Lactobacillus gasseri SRK575 (Accession No. KCCM11769P) and fermenting the fermented milk.
상기 발효는 30 ~ 50 ℃에서 24 ~ 100 시간 동안 수행되는 것을 특징으로 하는 항산화 및 면역강화용 발효유의 제조방법.
The method of claim 3,
Wherein the fermentation is carried out at 30 to 50 DEG C for 24 to 100 hours.
상기 균주는 상기 균주의 배양물, 상기 배양물의 농축물, 상기 균주의 발효물, 및 배양물의 건조물로 이루어진 군에서 선택된 어느 하나를 더 포함하는 것을 특징으로 하는 항산화 및 면역강화용 발효유의 제조방법.The method of claim 3,
Wherein the strain further comprises any one selected from the group consisting of a culture of the strain, a concentrate of the culture, a fermentation product of the strain, and a dried product of the culture.
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