KR100630907B1 - Lactobacillus sporogenes cu 4109 - Google Patents
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- KR100630907B1 KR100630907B1 KR1020050073745A KR20050073745A KR100630907B1 KR 100630907 B1 KR100630907 B1 KR 100630907B1 KR 1020050073745 A KR1020050073745 A KR 1020050073745A KR 20050073745 A KR20050073745 A KR 20050073745A KR 100630907 B1 KR100630907 B1 KR 100630907B1
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- glutathione
- concentration
- whey protein
- cell
- lactobacillus sporogenes
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Abstract
Description
도 1은 본 발명에 의한 락토바실러스 스포러제네스 CU 4109의 균체내 글루타티온 농도의 측정 결과를 나타낸 그래프.1 is a graph showing the results of measuring the concentration of glutathione in cells of Lactobacillus sporogenes CU 4109 according to the present invention.
도 2는 본 발명에 의한 락토바실러스 스포러제네스 CU 4109의 ICR 마우스에서 간세포내 글루타티온 농도의 측정 결과를 나타낸 그래프.Figure 2 is a graph showing the measurement results of hepatocyte glutathione concentrations in ICR mice of Lactobacillus sporogenes CU 4109 according to the present invention.
도 3은 전립선세포(PC-3M-MM2)세포배양시 첨가된 카세인(casein) 및 유청단백질(WPI) 가수분해물(500ug/ml)의 글루타티온(GSH) 농도의 상승효과 측정 결과를 나타낸 그래프.Figure 3 is a graph showing the results of measuring the synergistic effect of glutathione (GSH) concentration of casein and whey protein (WPI) hydrolyzate (500ug / ml) added during prostate cell (PC-3M-MM2) cell culture.
도 4는 산화제 처리에 의한 세포사멸효과 및 유청단백질에 의한 생존율 상승효과의 측정 결과를 나타낸 그래프.Figure 4 is a graph showing the measurement results of the cell death effect by oxidant treatment and the survival rate synergistic effect by whey protein.
도 5는 유청단백질 처리에 의한 산화제에 의한 DNA 손상 발생 정도가 낮아 세포 사멸율이 현저하게 저하되는 결과를 나타낸 그래프.5 is a graph showing a result that the rate of cell death is significantly lowered due to the low degree of DNA damage caused by the oxidizing agent by whey protein treatment.
도 6은 In vivo (mouse) 실험에서 유청단백질의 세포내 glutathione sulphydryl(GSH) 농도 상승효과 측정 결과를 나타낸 그래프.Figure 6 is a graph showing the results of measuring the synergistic effect of intracellular glutathione sulphydryl (GSH) concentration of whey protein in the experiment in vivo (mouse).
도 7은 Plaque forming cell 계수법에 의한 유청단백질에 의한 IgM 농도 증가에 따른 면역증강 효과를 나타낸 그래프.Figure 7 is a graph showing the immune enhancing effect of the increase in IgM concentration by whey protein by Plaque forming cell counting method.
도 8은 비타민 C 첨가에 의한 유청단백질의 세포내 글루타티온 상승효과 측정 결과를 나타낸 그래프.8 is a graph showing the results of measuring intracellular glutathione synergy of whey protein by vitamin C addition.
도 9는 유청단백질과 Will 동결건조 분말 및 L. casei 세포동결건조분말의 TBA 방법에 의한 리놀레산(linoleic acid)산화 억제 활성을 비교한 결과를 나타낸 그래프.Figure 9 shows whey protein and Will lyophilized powder and L. casei A graph showing the results of linoleic acid oxidation inhibitory activity by the TBA method of the freeze-dried powder.
본 발명은 락토바실러스(Lactobacillus)속의 새로운 유산균인 락토바실러스 스포러제네스 CU 4109에 관한 것으로서, 보다 상세하게는 항산화 활성이 우수한 것을 특징으로 하는 포자형성능력이 있는 유산균인 락토바실러스 스포러제네스 CU 4109(기탁기관: 농업생명공학연구원, 기탁번호: KACC 91129, 기탁일: 2004. 07. 22.)에 관한 것이다. The present invention relates to a lactobacillus sporogenes CU 4109, a new lactic acid bacterium of the genus Lactobacillus, more specifically, a lactobacillus sporogenes CU 4109 (Lactobacillus sporogenes CU 4109), which is characterized by excellent antioxidant activity. Depositary Organization: Institute of Agricultural Biotechnology, Deposit Number: KACC 91129, Deposited Date: July 22, 2004).
지구상 대부분의 생명체는 공기 중의 산소를 호흡하여 산화시켜 얻어지는 에너지를 이용하여 생명을 유지하는데, 이런 산소가 필요한 대사과정에서 산소의 과잉섭취, 스트레스나 다른 원인에 의해서 산소가 비정상적으로 증가하여 불가피하게 세포를 파괴시키는 독성물질들이 부산물로 만들어지는데 이것을 활성산소라고 한다. 활성산소는 생체 조직을 공격하여 세포를 산화, 손상시키는 주범이며 유해산소 라고도 한다. 한편 병원체나 이물질을 제거하기 위한 생체방어 과정에서도 O2, H2O2와 같은 활성산소가 대량 발생하며 이들의 강한 살균 작용을 통해서 병원체로부터 인체를 보호하는 작용을 하기도 한다. Most of life on earth uses the energy obtained by breathing and oxidizing oxygen in the air to maintain life. The oxygen is abnormally increased due to the excessive intake of oxygen, stress or other causes during the metabolic process that requires oxygen. Toxic substances that destroy it are made into byproducts, which are called free radicals. Free radicals attack biological tissues, oxidizing and damaging cells, and also called harmful oxygen. On the other hand, a large amount of active oxygen such as O 2 , H 2 O 2 is also generated in the biological defense process to remove pathogens and foreign substances, and also act to protect the human body from pathogens through their strong sterilization action.
그러나 환경오염과 화학물질, 자외선, 혈액순환장애, 스트레스 등으로 과잉 생산된 활성산소는 인체의 정상적인 DNA와 세포, 조직을 공격하여 세포나 세포소기관에 손상을 초래하기도 하며 조직내의 단백질과 핵산에 산화반응에 의한 변화를 야기시켜 단백질의 기능 저하를 초래하고 단백질의 가수분해 반응을 과도하게 상승시킴으로 질병발생과 노화 등 유해한 작용을 하게 된다. 또한 핵산에도 손상을 주는데 핵산 염기의 변형, 핵산 염기의 유리, 결합의 절단, 당의 산화 분해 등을 초래하여 돌연변이나 암의 원인이 되기도 한다. 현재 우리가 앓고 있는 질환 중 약90%정도가 활성산소와 관련이 있다고 하며, 그 질환에는 주로 암, 동맥경화, 당뇨병, 뇌졸중, 심근경색, 간염, 신장염, 교원병, 아토피성 피부염, 파키슨병 외에 자외선과 방사선에 의한 질병 등이 있다. However, free radicals produced due to environmental pollution, chemicals, ultraviolet rays, blood circulation disorders, stress, etc. attack the normal DNA, cells and tissues of the human body, causing damage to cells and organelles, and oxidizing proteins and nucleic acids in tissues. It causes a change by the reaction, leading to a decrease in the function of the protein and excessively increasing the hydrolysis reaction of the protein has a harmful action such as disease occurrence and aging. It also damages nucleic acids, causing modification of nucleic acid bases, release of nucleic acid bases, cleavage of bonds, and oxidative degradation of sugars, which can cause mutations and cancer. About 90% of the diseases we are currently suffering from are related to free radicals, which include cancer, arteriosclerosis, diabetes, stroke, myocardial infarction, hepatitis, nephritis, collagen disease, atopic dermatitis, Parkinson's disease, and ultraviolet rays. And diseases caused by radiation.
이러한 조직 내부에서 일어날 수 있는 산화반응을 방지하거나 그 속도를 현저하게 느리게 해주어 우리 몸이 노화되고 손상되는 것을 막아주는 물질을 항산화제(antioxidants)라고 한다. 항산화제는 인체 내에 자연적으로 존재하는 것과 외부에서 투여해 주는 것으로 나눌 수 있다. 인체 내에 자연적으로 존재하는 항산화제로는 카탈라제(catalase), 슈퍼옥사이드 디스뮤타제(Superoxide Dismutase, SOD), 글루타티온 페록시다제(glutathione peroxidase) 등의 효소와 요산, 빌리루빈 등이 있으며 외부에서 투여해 주는 것으로 비타민 E, C, 베타카로틴이 있으며 미네랄 중에는 셀레니움이 대표적이다. 그 밖에도 멜라토닌 같은 호르몬, 녹차에 많이 들어 있는 플라보노이드 성분과 적포도주에 들어 있는 폴리페놀, 벌집을 구성하는 성분의 하나인 프로폴리스 등이 대표적인 항산화제들이다. Antioxidants are substances that prevent or slow down the oxidation reactions that can occur inside these tissues, preventing the body from aging and damaging. Antioxidants can be divided into those which exist naturally in the human body and those administered externally. Antioxidants that exist naturally in the human body include enzymes such as catalase, superoxide dismutase (SOD) and glutathione peroxidase, and uric acid and bilirubin. There are vitamins E, C, and beta-carotene, among which minerals are selenium. Other antioxidants include hormones such as melatonin, flavonoids found in green tea, polyphenols found in red wine, and propolis, one of the components of honeycomb.
유산균은 동물의 장내에 분포하여 유해 미생물 생장의 억제, 이상 발효의 치료, 혈중 콜레스테롤 저하, 면역 기능의 증진 등의 효과를 나타내며, 이러한 유산균 즉, 락토바실러스 애시도필러스(Lactobacillus acidophilus), 락토바실러스 카세이(Lactobacillus casei), 비피도박테리움(Bifidobsvyrtium)속 같은 균주를 활용한 발효 유제품 및 정장제가 상용화되어 널리 음용 또는 식용되고 있다. 최근에는 유산균의 면역 활성 작용, 항암 작용에 관한 연구들이 보고되고 있는데 이와 관련하여 유산균에 의한 활성산소의 항산화작용이 기대되고 있다. Lactobacillus is distributed in the intestines of animals and shows the effects of inhibiting the growth of harmful microorganisms, treatment of abnormal fermentation, lowering blood cholesterol, enhancing immune function, such as Lactobacillus acidophilus , Lactobacillus Fermented dairy products and formal preparations utilizing strains such as Lactobacillus casei and Bifidobsvyrtium genus have been commercialized and widely consumed or edible. Recently, studies on the immunological activity and anticancer activity of lactic acid bacteria have been reported. In this regard, antioxidative activity of active oxygen by lactic acid bacteria is expected.
그러나 실제로 항산화 활성을 가진 유산균이 발견되어 식품 등에 응용된 예가 거의 없으므로 본 발명의 목적은 TBA 이용한 유산균의 지방산 산화 억제 활성 측정, 체내 글루타티온 농도 측정, 간내 글루타티온 농도 측정을 통해 체내에서 질병발생과 노화 등 유해한 작용을 나타내는 생체내 활성산소의 발생을 방지하기 위해 산화반응을 방지하거나 그 속도를 현저하게 느리게 해주는 탁월한 항산화 활성을 나타내는 유산균주 및 이를 유효성분으로 함유하는 항산화 식품을 제공하는 것이다. However, since lactic acid bacteria have actually been found to have antioxidant activity, and there are few examples applied to foods, an object of the present invention is to measure fatty acid oxidation inhibitory activity of lactic acid bacteria using TBA, measurement of glutathione concentration in the body, concentration of glutathione in the liver, measurement of disease occurrence and aging in the body, and the like. In order to prevent the generation of free radicals in vivo exhibiting harmful action, to provide a lactic acid strain and an antioxidant food containing an excellent antioxidant activity that prevents the oxidation reaction or significantly slows down the rate.
이에, 상기와 같은 제반 문제점들을 해소하기 위하여 안출된 본 발명은 항산화 활성이 우수한 락토바실러스 스포러제네스 CU 4109 (Lactobacillus sporogenes CU 4109)를 제공함으로써 지방산 리놀레산을 이용한 산화 과정 중에서 트리바르비투르산(tribarbituric acid, TBA)을 이용한 산화 억제 활성이 높을 뿐 아니라 선발된 균주 중에서 균체내 함유 글루타티온 농도 및 실험동물 ICR 마우스를 이용한 급여실험에서 간 글루타티온 함유율을 현저하게 상승시킴으로써 항산화 활성이 매우 우수한 새로운 유산균 및 이를 유효성분으로 함유하는 항산화 식품을 제공하는데 그 목적이 있다. Accordingly, the present invention devised to solve the above problems is to provide Lactobacillus sporogenes CU 4109 ( Lactobacillus sporogenes CU 4109) excellent antioxidant activity in the oxidation process using fatty acid linoleic acid (tribarbituric acid) , Lactic acid bacteria and its active ingredient, which have high antioxidant activity by increasing the antioxidant activity using TBA) and significantly increasing the concentration of glutathione in the cells among the selected strains and the liver glutathione content in feeding experiments using experimental animal ICR mice. Its purpose is to provide antioxidant foods containing.
상기와 같은 목적 달성을 위한 본 발명은 락토바실러스 스포러제네스 CU 4109(기탁기관: 농업생명공학연구원, 기탁번호: KACC 91129, 기탁일: 2004. 07. 22.)를 제공하는데 그 특징이 있다. The present invention for achieving the above object is characterized by providing Lactobacillus sporagenes CU 4109 (depositment institution: Institute of Agricultural Biotechnology, Accession No .: KACC 91129, date of deposit: 2004. 07. 22.).
바람직하게는 상기 락토바실러스 스포러제네스 CU 4109는 항산화 활성이 우수한 것을 특징으로 한다. Preferably, the Lactobacillus sporogenes CU 4109 is characterized by excellent antioxidant activity.
또한 본 발명의 또다른 목적은 상기 락토바실러스 스포러제네스 CU 4109를 유효성분으로 함유하는 것을 특징으로 하는 항산화 식품을 제공하는데 있다. In addition, another object of the present invention to provide an antioxidant food, characterized in that containing the Lactobacillus sporogenes CU 4109 as an active ingredient.
이하, 본 발명의 구성을 좀 더 상세히 설명하면 다음과 같다. Hereinafter, the configuration of the present invention in more detail as follows.
본 발명에 따른 신균주 락토바실러스 스포러제네스 CU 4109의 분리 및 동정과 정은 다음과 같다. The isolation and identification process of the new strain Lactobacillus sporogenes CU 4109 according to the present invention is as follows.
신선한 원유를 엠알에스(MRS)액체 배지에서 37℃에서 24시간 배양한 후 0.02% 아지드 나트륨(sodium azide)이 함유된 엠알에스(MRS)플레이트(plate)에 도말한 후 37℃에서 48시간동안 배양하여 형성된 균락 중에서 선발된 균락을 엠알에스(MRS)액체 배지에 다시 배양하여 이 균주에 대한 바실러스속의 동정에 가장 많이 이용되고 있는 API kit를 이용하여 특성 실험 및 동정을 수행하였다.Fresh crude oil was incubated for 24 hours at 37 ° C. in MRS liquid medium and plated on a MRS plate containing 0.02% sodium azide for 48 hours at 37 ° C. The selected fungi from the fungi formed by culturing were re-incubated in MRS (MRS) liquid medium to perform characterization experiments and identification using the API kit which is most commonly used for identification of Bacillus genus for this strain.
본 발명에 따른 유산균의 특성은 다음과 같다.The characteristics of the lactic acid bacteria according to the present invention are as follows.
1) 균의 형태학적 특성1) Morphological characteristics of bacteria
① 운동성: 있음 ① Mobility: Yes
② 포자형성능: 있음 ② spore forming ability: Yes
2) 호모락틱(Homolactic) 발효 : +2) Homolactic fermentation: +
3) 카탈라제(Catalase) : -3) Catalase:-
4) 시토크롬(cytochromes) : -4) Cytochromes:-
5) 질산염 환원능력 : -5) Nitrate Reduction Capability:-
6) 인돌(Indole) 생성 : -6) Indole generation:-
7) 테이코산(Teichoic acid) : +7) Teichoic acid: +
8) 메나퀴논(Menaquinons) : +8) Menaquinons: +
이와 같은 균의 형태학적, 생리적 및 생장 특성에 근거하여 본 발명의 균주는 락토바실러스 스포러제네스로 동정하였고, 본 발명자들은 이를 락토바실러스 스포 러제네스 CU 4109로 명명하였다. 본 발명자는 선별된 균주인 바실러스속 균주인 락토바실러스 스포러제네스 CU 4109를 부다페스트 조약하의 국제기탁기관인 농업생명공학연구원에 2004년 7월 22일 기탁하였다(기탁번호: KACC 91129).Based on the morphological, physiological and growth characteristics of such bacteria, the strain of the present invention was identified as Lactobacillus sporogenes, and we named it Lactobacillus sporogenes CU 4109. The present inventors deposited the selected strain Bacillus strain Lactobacillus sporogenes CU 4109 on July 22, 2004 to the Institute of Agricultural Biotechnology, an international depository institution under the Budapest Treaty (Accession Number: KACC 91129).
본 발명의 유산균은 배양물 또는 동결건조된 분말 형태로 제공될 수 있다. 이와 같이 본 발명의 유산균 자체 또는 본 발명의 균주가 포함된 배양물은 항산화 효과가 기대되며 또한 항산화 식품, 항산화 제제로 사용될 수 있는 것이다. Lactic acid bacteria of the present invention may be provided in the form of a culture or lyophilized powder. As such, the lactic acid bacteria of the present invention or the culture containing the strain of the present invention are expected to have an antioxidant effect and can also be used as antioxidant foods and antioxidant agents.
항산화 활성을 나타내는 성분 중 세포내에서 중요한 기능을 나타내는 분자로 글루타티온 설프하이드릴(glutathione sulphydryl)의 중요성은 널리 인정되고 있으며 글루타티온 설프하이드릴(glutathione sulphydryl)을 형성하는 전구물질 중에서 중요한 것은 아미노산인 시스테인(cysteine)이 있다. 항 함유 아미노산인 시스테인(cysteine)이 풍부하게 함유되어 이루어진 우유단백질 중에는 유청단백질이 있으며 유청단백질을 순수하게 분말화 한 물질로서 유청단백질 파우더(whey protein isolate)가 있다.Among the components that exhibit antioxidant activity, the importance of glutathione sulphydryl as a molecule showing important functions in cells is widely recognized, and among the precursors forming glutathione sulphydryl, the important amino acid is cysteine ( cysteine). Whey protein is a protein that contains abundant anti-amino acid cysteine, and whey protein isolate is a pure powder of whey protein.
이하, 실시예 및 비교예를 통하여 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples.
<실시예 1><Example 1>
TBA 이용한 유산균의 지방산 산화 억제 활성 측정 시험Determination of Fatty Acid Oxidation Inhibitory Activity of Lactobacillus Using TBA
1) 균체 추출액(Intracellular cell-free extract)의 제조1) Preparation of Intracellular Cell-free Extract
MRS 액상배지에 본 발명의 락토바실러스 스포러제네스 CU 4109 균주를 접종하여 배양 후, 10,000rpm에서 10분간 원심 분리하여 균체 펠렛를 얻었다. 상기 균 체 펠렛을 생리식염수로 2회 세척하고, 0℃에서 20분간 초음파장치(Ultra sonicator)로 분쇄하였다. 분쇄된 균체를 10,000rpm에서 10분간 원심분리 후, 균체 추출액(Intracelluar cell-free extraction, IE)을 얻었다. After inoculating MRS liquid medium with Lactobacillus sporogenes CU 4109 strain of the present invention, the cells were centrifuged at 10,000 rpm for 10 minutes to obtain cell pellets. The cell pellet was washed twice with physiological saline, and ground at 0 ° C. for 20 minutes by an ultra sonicator. The pulverized cells were centrifuged at 10,000 rpm for 10 minutes, and cell extracts (Intracelluar cell-free extraction, IE) were obtained.
2) 리놀레산의 과산화 측정(TBA method)2) Measurement of peroxidation of linoleic acid (TBA method)
lml의 리놀레산(linoleic acid), 0.2ml의 트윈 20(tween 20), 19.7ml의 탈이온수(deionized water)를 혼합하여 20ml의 리놀레산 현탁액을 제조하였다. 리놀레산 현탁액 lml에 0.2ml의 아스코르베이트(ascorbate, 0.01%), 0.2ml의 황산 제1철(FeSO4, 0.01%), 0.5ml의 인산완충액(phosphate buffer solution, 0.02M, pH7.4) 및 상기에서 제조한 균체 추출액(IE) 0.4ml를 혼합하여 섞은 후 37℃에서 12시간 반응시켰다. 상기 반응액 2ml에 0.2ml의 트리클로로아세트산(TCA, 4%), 0.2ml의 부틸레이티드 하이드록시 톨루엔(BHT, 0.4%), 2ml의 트리바르비투르산(TBA, 0.8%)을 첨가한 후 100℃에서 30분간 정치하고, 식힌 후에 2ml의 클로로포름(chloroform)을 첨가하여 추출한 후 532nm에서 광학밀도(OD)값을 측정하였다. 측정한 광학밀도(OD)를 이용하여 본 발명에 의한 유산균의 지방산 산화 억제율(%)을 측정하였으며, 그 식은 다음과 같다. 20 ml of linoleic acid suspension was prepared by mixing 1 ml of linoleic acid, 0.2 ml of
지방산 산화 억제율(inhibition rate, %) = 1-(유산균처리구의 OD/ 대조군의 OD)×100%Fatty Acid Inhibition Rate (%) = 1- (OD of Lactic Acid Bacteria / OD of Control) × 100%
대조군 : 유산균을 첨가하지 않은 구 Control: sphere without added lactic acid bacteria
유산균처리구 : 유산균을 첨가한 구Lactic Acid Bacteria Treatment Zone
비교예Comparative example 1내지1 to 2(TBA 이용한 유산균의 산화 억제 활성 측정시험) 2 (oxidation inhibitory activity measurement test of lactic acid bacteria using TBA)
상기 실시예 1과 동일한 방법 및 조건으로 MRS 액상배지에서 배양한 락토바실러스 스포러제네스 CU 4222 및 락토바실러스 스포러제네스 CU 4223에 의한 지방산 산화 억제 활성을 측정하여 각각 비교예 1 및 2로 하여 실시예 1의 지방산 산화 억제 활성과 비교하였다. 그 결과 실시예1의 락토바실러스 스포러제네스 CU 4109 균체 추출액의 지방산 산화 억제 활성율(%)은 30.5%의 높은 억제활성을 보였으나 락토바실러스 스포러제네스 CU 4222(비교예1)은 8%, 락토바실러스 스포러제네스 CU 4223(비교예2)은 9.2%로 현저히 낮은 활성을 나타내었다.Fatty acid oxidation inhibitory activity by Lactobacillus sporogenes CU 4222 and Lactobacillus sporogenes CU 4223 cultured in MRS liquid medium by the same methods and conditions as in Example 1 were measured and compared to Comparative Examples 1 and 2, respectively. It was compared with fatty acid oxidation inhibitory activity of 1. As a result, the fatty acid oxidation inhibitory activity (%) of Lactobacillus sporogenes CU 4109 cell extract of Example 1 showed a high inhibitory activity of 30.5%, but Lactobacillus sporogenes CU 4222 (Comparative Example 1) was 8%, Lactobacillus sporogenes CU 4223 (Comparative Example 2) showed a significantly lower activity of 9.2%.
<실시예 2><Example 2>
균체내Intracellular 글루타티온 농도 측정 시험 Glutathione Concentration Test
1) 균체 추출액(Intracellular cell-free extract)의 제조1) Preparation of Intracellular Cell-free Extract
MRS 액상배지에 본 발명의 락토바실러스 스포러제네스 CU 4109 균주를 37℃에서 16시간 배양 후, 10,000rpm에서 10분간 원심 분리하여 균체 펠렛를 얻었다. 상기 균체 펠렛을 생리식염수로 2회 세척 후, 습윤된 균체 펠렛 10mg을 0℃에서 20분간 초음파장치(Ultra sonicator)로 분쇄하였다. 분쇄된 균체를 10,000rpm에서 10분간 원심분리 후 균체 추출액(Intracelluar cell-free extraction, IE)을 얻었다. Lactobacillus sporogenes CU 4109 strain of the present invention in an MRS liquid medium was incubated for 16 hours at 37 ℃, centrifuged for 10 minutes at 10,000rpm to obtain a cell pellet. After washing the cell pellets twice with physiological saline, 10 mg of the wet cell pellets was pulverized with an ultra sonicator at 0 ° C. for 20 minutes. The pulverized cells were centrifuged at 10,000 rpm for 10 minutes to obtain cell extracts (Intracelluar cell-free extraction, IE).
2) 글루타티온(GSH) 농도 측정2) Glutathione (GSH) concentration measurement
상기에서 제조한 균체 추출액(IE)의 글루타티온(GSH) 농도를 GSH assay kit (calbiochem, Cat. No. 354102. USA)을 이용하여 3회 반복 측정하였다. The glutathione (GSH) concentration of the cell extract (IE) prepared above was measured three times using a GSH assay kit (calbiochem, Cat. No. 354102. USA).
글루타티온(GSH) 농도 측정시 0.2N 염산에 12mM 농도로 발색제가 용해된 R1용액과 글루타티온(GSH)을 포함한 메르캅탄류(mercaptan) 간에 티오에테르(thioether)를 생성하는 1차반응이 일어나고, 글루타티온(GSH)이 존재하는 상태에서 pH13의 알칼리 조건을 조성하기 위하여 R2용액(30% NaOH)을 가하면 티오엔(Thioene)이 생성되는 2차 반응이 일어난다. 티오엔(Thioene)의 최고 흡광이 일어나는 400nm에서 흡광도를 측정하여 글루타티온(GSH) 농도를 측정한다.When measuring the concentration of glutathione (GSH), a primary reaction occurs in which thioether is produced between R1 solution in which a coloring agent is dissolved in 0.2 N hydrochloric acid at a concentration of 12 mM and mercaptans including glutathione (GSH), and glutathione (GSH). In the presence of GSH), when a R2 solution (30% NaOH) is added to form an alkaline condition of pH 13, a secondary reaction occurs in which thioene is produced. Glutathione (GSH) concentration is measured by measuring the absorbance at 400 nm where the highest absorption of thioene occurs.
200㎕의 균체 추출액(IE)을 취하고 버퍼용액을 가하여 총량이 900㎕가 되게 한 후 50㎕의 R1용액을 가한 후 철저히 혼합하고 다시 50㎕의 R2용액을 가한 후 철저히 혼합하였다. 이 용액을 어두운 조건에서 25℃에서 10분간 정치한 후 400nm에서 흡광도를 측정한 후 다음 공식에 의하여 글루타티온(GSH) 농도를 계산하였다.200 μl of cell extract (IE) was taken, and a buffer solution was added to make the total amount 900 μl. Then, 50 μl of R1 solution was added and thoroughly mixed. Then, 50 μl of R2 solution was added and mixed thoroughly. After the solution was allowed to stand at 25 ° C. for 10 minutes in dark conditions, the absorbance was measured at 400 nm, and glutathione (GSH) concentration was calculated by the following formula.
〔 GSH 〕= 
〔 GSH 〕; 글루타티온 농도(umol/g)[GSH]; Glutathione Concentration (umol / g)
A :시료가 포함되었을 때 측정된 흡광도A: Absorbance measured when sample included
Ao :시료가 포함되지 않았을 때 측정된 흡광도Ao: absorbance measured when no sample is included
E : 표준물질을 400nm에서 측정하였을 때 몰흡광계수(molar extiction coefficient)E: molar extiction coefficient when the reference material is measured at 400 nm
D :시료의 희석비율D: Dilution ratio of the sample
비교예Comparative example 3내지3 to 11( 11 ( 균체내Intracellular 글루타티온 농도 측정시험) Glutathione concentration test)
상기 실시예 2와 동일한 방법 및 조건으로 MRS 액상배지에서 배양한 락토바실러스 스포러제네스 CU 4105, CU 4104, CU 4102, CU 4101, CU 4106, CU 4107, CU 4110, CU 4108 및 CU 4103의 균체내 글루타티온 농도를 측정하여 각각 비교예 3 내지 11로 하여, 실시예 2의 글루타티온 농도와 비교하여 도 1에 나타내었다. 그 결과, 실시예1의 락토바실러스 스포러제네스 CU 4109 균체내 글루타티온 농도는 8.6 μ㏖/g로 높은 수치를 나타내었는데 반해, 락토바실러스 스포러제네스 CU 4105(비교예 3)는 6.4μ㏖/g, CU 4104(비교예 4)는 5.6μ㏖/g, CU 4102(비교예 5)는 5.3μ㏖/g, CU 4101(비교예 6)은 6.2μ㏖/g, CU 4106(비교예 7)은 6.5μ㏖/g, CU 4107(비교예 8)은 6.4μ㏖/g, CU 4110(비교예 9)은 7.8μ㏖/g, CU 4108(비교예 10)은 7.1μ㏖/g 및 CU 4103(비교예 11)은 5.2μ㏖/g로서 실시예1에 비해 낮은 균체내 글루타티온 농도를 나타내었다. 특히 CU 4102(비교예 5)와 CU 4103(비교예 11)의 균체내 글루타티온 농도는 각각 5.3μ㏖/g, 5.2μ㏖/g로서 실시예2의 락토바실러스 스포러제네스 CU 4109 균체내 글루타티온 농도인 8.6 μ㏖/g보다 통계적인 유의성이 확연히 인정되는 수준으로 차이를 보여 세포내 항산화 활성 인자의 농도가 높은 것으로 확인되었다.Lactobacillus sporogenes CU 4105, CU 4104, CU 4102, CU 4101, CU 4106, CU 4107, CU 4110, CU 4108 and CU 4103 cultured in MRS liquid medium in the same manner and conditions as in Example 2 The glutathione concentrations were measured, and Comparative Examples 3 to 11, respectively, were compared with the glutathione concentrations of Example 2 and shown in FIG. As a result, the concentration of glutathione in the Lactobacillus sporogenes CU 4109 cells of Example 1 was 8.6 μmol / g, whereas the Lactobacillus sporogenes CU 4105 (Comparative Example 3) was 6.4 μmol / g. , CU 4104 (Comparative Example 4) is 5.6 μmol / g, CU 4102 (Comparative Example 5) is 5.3 μmol / g, CU 4101 (Comparative Example 6) is 6.2 μmol / g, CU 4106 (Comparative Example 7) Silver 6.5 μmol / g, CU 4107 (Comparative Example 8) 6.4 μmol / g, CU 4110 (Comparative Example 9) 7.8 μmol / g, CU 4108 (Comparative Example 10) 7.1 μmol / g and CU 4103 (Comparative Example 11) exhibited a lower cell concentration of glutathione as 5.2 μmol / g compared to Example 1. In particular, the concentration of glutathione in the cells of CU 4102 (Comparative Example 5) and CU 4103 (Comparative Example 11) was 5.3 μmol / g and 5.2 μmol / g, respectively, and the concentration of glutathione in the Lactobacillus sporogenes CU 4109 cell of Example 2 was measured. The difference in the level of statistical significance was significantly higher than that of 8.6 μmol / g.
<실시예 3><Example 3>
간내Liver 글루타티온 농도 측정 시험 Glutathione Concentration Test
6주령의 ICR mouse male (28±7주령)를 12hr/day로 조명하고, 온도는 22~23℃, 습도는 40~50%의 사육조건에서 무단백질 마우스 분말사료와 함께 본 발명의 락토바실러스 스포러제네스 CU 4109 균주를 동시에 급여한 후 실험동물의 간장 세포내의 글루타티온 농도를 측정하였다. 이때 락토바실러스 스포러제네스 CU 4109 균주의 양을 20g, 100g, 180g, 200g으로 변화를 주어 투여하였으며 그 결과를 도 2에 나타내었다. The 6-week-old ICR mouse male (28 ± 7 weeks of age) was illuminated at 12hr / day, temperature was 22-23 ° C., and humidity was 40-50% with no protein mouse powder feed under the Lactobacillus spores of the present invention. Glutathione concentrations in the hepatic cells of the experimental animals were measured after simultaneous feeding of the Rujenes CU 4109 strain. At this time, the amount of Lactobacillus sporogenes CU 4109 strain was administered by changing to 20g, 100g, 180g, 200g and the results are shown in FIG.
도 2에 도시된 바와 같이 간내 글루타티온 농도가 8μ㏖/g인 대조군에 비해 본 발명의 락토바실러스 스포러제네스 CU 4109 급여구의 경우 20g에서 9.5μ㏖/g, 100g에서 9.36μ㏖/g, 180g에서 9.15μ㏖/g, 200g에서 9μ㏖/g의 간내 글루타티온 농도를 나타내어 통계적인 유의성이 인정되는 수준으로 그 농도가 높았다.As shown in FIG. 2, the lactobacillus sporogenes CU 4109 feed group of the present invention was 20 g at 9.5 μmol / g, 100 g at 9.36 μmol / g, and 180 g compared to the control group having an intrahepatic glutathione concentration of 8 μmol / g. Intrahepatic glutathione concentrations of 9.15 μmol / g and 200 g of 9 μmol / g were shown, indicating that statistical significance was high.
또한 유청단백질분말이 세포내에서 글루타티온(GSH) 농도를 상승시키는 효과를 나타내는지와 지방산의 산화반응에서 산화 억제 활성을 나타내는지를 확인하기 위하여 전립선 세포배양법에 의한 실험과 쥐를 이용한 생체 실험 및 리놀레산 산화과정에서 억제 활성측정법을 이용하였다.In addition, to determine whether whey protein powder has an effect of increasing the concentration of glutathione (GSH) in the cell and its antioxidant activity in the oxidation reaction of fatty acids, experiments by prostate cell culture, in vivo experiments in rats and linoleic acid oxidation Inhibition activity assay was used.
<실시예4>Example 4
전립선세포(PC-3M-MM2)세포배양시Prostate Cell (PC-3M-MM2) Cell Culture 첨가된 카세인(casein) 및 Added casein and 유청단백질Whey Protein (( WPIWPI ) 가수분해물(500ug/ml)의 글루타티온() Glutathione (500ug / ml) GSHGSH ) 상승효과 측정시험) Synergy test
전립선 세포를 상피세포용 성장인자와 소 갑상선 추출물이 첨가된 배지에서 37℃, 5% 탄산가스가 함유된 배양기에서 배양하였다. 상기 배양한 세포를 96-well plate의 한 well 당 106cells/ml의 농도로 투입한 후 24시간 배양하고, 가수분해 처리한 유청단백질 추출액(WPI) 500㎍/㎖ 과 카세인500㎍/㎖, 글루타티온 합성 억제제인 부티오닌 설폭시민(Buthioneine sulfoximine , BSO) 500μM을 배지로 대치한 후 24시간 더 배양하였다. 처리구의 글루타티온농도는 glutathione assay kit를 이용하여 측정하였다.Prostate cells were cultured in an incubator containing 37 ° C. and 5% carbon dioxide in a medium to which epidermal growth factor and bovine thyroid extract were added. The cultured cells were inoculated at a concentration of 10 6 cells / ml per well of a 96-well plate and incubated for 24 hours. 500 µg / ml hydrolyzed whey protein extract (WPI) and 500 µg / ml casein, Butionine sulfoximine (BSO), a glutathione synthesis inhibitor, was replaced with 500 μM in culture and further incubated for 24 hours. Glutathione concentration of the treatment was measured using glutathione assay kit.
그 결과를 도 3과 [표1]에 나타내었다.The results are shown in FIG. 3 and [Table 1].
[표1]과 도 3에서 확인할 수 있는 바와 같이 세포배양 체계에서 유청단백질의 첨가 효과를 대조군와 비교할 때 글루타티온 농도가 6.04μ㏖/g인 대조군에 비해 유청단백질 추출액(WPI)의 경우 8.73μ㏖/g의 간내 글루타티온 농도를 나타내어 통계적인 유의성이 매우 높은 수준으로 차이를 나타내는 것으로 나타났으며 글루타티온 농도가 6.71μ㏖/g인 카세인의 경우보다도 유의성이 높은 것으로 나타났다. As can be seen in Table 1 and FIG. 3, the effect of whey protein addition in the cell culture system was 8.73 μmol / w for the whey protein extract (WPI) compared to the control group having a glutathione concentration of 6.04 μmol / g compared to the control group. The intracellular glutathione concentration of g was shown to show a statistically significant difference, and the significance was higher than that of casein with a glutathione concentration of 6.71 μmol / g.
<실시예5>Example 5
산화제 처리에 의한 세포사멸효과 및 Apoptosis effect by oxidant treatment and 유청단백질에Whey protein 의한 생존율 상승효과측정 Survival rate increase effect
세포를 96-well plate의 한 well 당 106cells/㎖의 농도로 seeding 하여 24시간 배양하였다. 24시간 배양 후 100mM의 가수분해 처리한 유청단백질 추출액(WPI), 100mM의 카세인, 100mM의 t-부틸 하이드로페록사이드(t-butyl hydroperoxide, TBHP), 500μM의 가수분해 처리한 유청단백질 추출액(WPI)과 부티오닌 설폭시민(BSO)의 혼합물, 500μM의 부티오닌 설폭시민(BSO)을 각각 처리한 후 24시간 더 배양하였다. 상기와 같이 48시간 배양 후 MTT 색소를 첨가하여 4시간을 추가로 정치하였다. 그 후 배지를 제거하고 MTT 색소의 산화에 의하여 생성된 프로마잔(fromazan) 결정을 0.04M 염산을 함유한 아이소프로판올(isopropanol) 100㎕에 용해한 후 620nm에서 흡광도를 측정하여 생존세포수를 측정하고 생존율을 구하였다. 세포의 생존률은 MTT assay를 통해 확인하였다. 그 결과를 도 4와 [표2]에 나타내었다. Cells were seeded at a concentration of 10 6 cells / ml per well of a 96-well plate and incubated for 24 hours. After 24 hours of incubation, 100 mM hydrolyzed whey protein extract (WPI), 100 mM casein, 100 mM t-butyl hydroperoxide (TBHP) and 500 μM hydrolyzed whey protein extract (WPI) And a mixture of butionine sulfoximine (BSO) and 500 μM butionine sulfoximine (BSO), respectively, and further incubated for 24 hours. After 48 hours of incubation as described above, the MTT dye was added for 4 hours. Thereafter, the medium was removed, and dissolved from 100 mg of isopropanol containing 0.04 M hydrochloric acid was dissolved from 100 mg of isopropanol produced by oxidation of the MTT pigment, and the absorbance was measured at 620 nm to measure the viable cell number. Was obtained. Cell viability was confirmed by MTT assay. The results are shown in FIG. 4 and [Table 2].
도 4와 상기 [표2]에서 확인할 수 있는 바와 같이 전립선 세포배양체계에서 산화제 처리에 의하여 세포사멸이 생성하는지 여부를 측정하기 위하여 산화제로 t-부틸 하이드로페록사이드(t-butyl hydroperoxide, TBHP)을 사용하였고 그 농도는 100mM 농도로 처리하였을 때 38.1%가 사멸하여 그 생존율이 61.9%로 저하되었음을 알 수 있었다. 정상적인 세포 배양 배지에 항산화제 t-부틸 하이드로페록사이드(TBHP)가 첨가되었을 때 생존율이 61.9%로 저하되며 배지에 글루타티온 합성억제제인 부티오닌 설폭시민(BSO)이 처리되었을 때에는 그 생존율이 33.7%까지 저하되었으나 유청단백질 및 카세인 처리에 의하여 글루타티온 농도가 유지된 세포의 경우 생존율이 86.7%까지 높게 유지되는 유의성이 있는 차이를 보이고 있음을 알 수 있었다.As shown in FIG. 4 and Table 2, t-butyl hydroperoxide (TBHP) was used as an oxidant to determine whether apoptosis was generated by oxidant treatment in a prostate cell culture system. The concentration was 38.1% when treated at 100mM concentration and the survival rate was lowered to 61.9%. Survival was reduced to 61.9% when antioxidant t-butyl hydroperoxide (TBHP) was added to normal cell culture media and 33.7% survival when butionine sulfoximine (BSO), a glutathione inhibitor, was treated in the medium. However, it was found that there was a significant difference that the survival rate of the cells maintained at the glutathione concentration by the whey protein and casein treatment was maintained to 86.7%.
<실시예6>Example 6
Single cell electrophoresis(Single cell electrophoresis CommetCommet assay)방법에 의한 산화제에 의한 DNA 손상발생과 DNA damage caused by oxidant by the method 유청단백질Whey Protein 처리세포와의 비교 Comparison with treated cells
Comet assay는 comet assay kit(TRIVIGEN, U.S.A. Cat#4250-050-k)를 이용하여 수행하였다Comet assay was performed using the comet assay kit (TRIVIGEN, U.S.A. Cat # 4250-050-k)
전립선 세포(1×105cell/ml)에 10mM의 t-부틸 하이드로페록사이드(TBHP)와100mM의 t-부틸 하이드로페록사이드(TBHP)를 첨가하여 37℃에서 1시간 정치시킨 후, 상기 전립선 세포(1×105cell/ml)와 용해된 저융점 아가로스(agarose)를 42℃를 유지하면서 1:10의 비율로 혼합 후, 즉시 comet slide의 circle 위에 75㎕를 채우고 4℃의 어두운 곳에서 젤(Gel)을 형성시켰다. 상기 젤이 형성된 슬라이드(slide)를 세포막용해용액(Lysis solution)에 4℃에서 1시간 동안 담구어 세포막을 용해하였다. 상기 용해시킨 슬라이드들을 버퍼용액(300mM NaOH, 1mM EDTA, pH13)으로 슬라이드 표면 위 0.7cm까지 가득 채운 후 20분간 수평상태에서 전기영동을 실시하였다. 상기 전기영동 조건은 25V/300mA로 20분간 실시하였다. 전기영동 후 70% 에탄올에 5분간 담근 후 희석된 SYBR Green(1:10,000) 50㎕로 염색 한 후 공기 중에서 자연건조 하였다. 상기 염색 후 자연 건조된 슬라이드는 형광현미경(250배)으로 관찰하였다. 그 결과를 도 5에 나타내었다. 10 mM t-butyl hydroperoxide (TBHP) and 100 mM t-butyl hydroperoxide (TBHP) were added to prostate cells (1 × 10 5 cells / ml) and allowed to stand at 37 ° C. for 1 hour. (1 × 10 5 cell / ml) and dissolved low melting agarose (agarose) at a ratio of 1:10 while maintaining 42 ° C, immediately fill 75 μl on a circle of comet slide and place it in a dark place at 4 ° C. Gel was formed. The gel-formed slides were immersed in Lysis solution at 4 ° C. for 1 hour to dissolve the cell membranes. The dissolved slides were filled with a buffer solution (300 mM NaOH, 1 mM EDTA, pH 13) to 0.7 cm above the slide surface and subjected to electrophoresis for 20 minutes in a horizontal state. The electrophoresis conditions were carried out at 25V / 300mA for 20 minutes. After electrophoresis, it was soaked in 70% ethanol for 5 minutes, stained with 50 μl of diluted SYBR Green (1: 10,000), and air dried in air. After the dyeing, the naturally dried slides were observed with a fluorescence microscope (250 times). The results are shown in FIG.
도 5에 도시된 바와 같이 전립선세포(PC-3M-MM2)를 산화제 t-부틸 하이드로페록사이드(TBHP)를 1mM 농도로 처리한 결과 산화제가 DNA 분자에 손상을 나타내는 것을 확인할 수 있었으며 산화제가 처리되지 않은 대조군 및 유청단백질 추출액(WPI)을 처리하여 세포내 글루타니온 농도가 높아진 세포에서는 DNA손상의 정도가 낮아 세포사멸율이 현저하게 저하되는 결과를 확인할 수 있었다.As shown in FIG. 5, when the prostate cells (PC-3M-MM2) were treated with oxidant t-butyl hydroperoxide (TBHP) at a concentration of 1 mM, it was confirmed that the oxidant showed damage to DNA molecules and the oxidant was not treated. The control group and the whey protein extract (WPI) were treated with high concentrations of intracellular glutanion, resulting in a significant decrease in apoptosis due to low DNA damage.
<실시예7>Example 7
In In vivovivo (mouse) 실험에서 (mouse) in the experiment 유청단백질의Whey protein 세포내Intracellular glutathioneglutathione sulphydrylsulphydryl (( GSHGSH ) 농도 상승효과측정) Concentration synergy effect measurement
면양 적혈구(5×106)를 면역화한 이후 5일 경과 후 비장세포중의 글루타티온함유율(μ㏖/g/wet tissue)을 카세인과 분무건조된 유청단백질(SDWPI) 및 동결건조된 유청단백질 분말(FDWPI)을 각각 총 사료 100g에 대하여 20g의 비율로 3주간 급여한 이후 비장 세포내 글루타티온 함유율을 측정하였다. 그 결과를 도 6에 나타내었다.5 days after immunization of sheep red blood cells (5 × 10 6 ) Glutathione content (μmol / g / wet tissue) in splenocytes was treated with casein, spray-dried whey protein (SDWPI) and lyophilized whey protein powder (FDWPI) at a rate of 20 g for 100 g total feed, respectively, for 3 weeks. Afterwards, the glutathione content in the spleen cells was measured. The results are shown in FIG.
도 6에 도시된 바와 같이 ICR mouse에 카세인을 급여한 대조군과 유청단백질을 급여한 실험군에 대하여 비장에서 측정된 글루타티온 함유율을 측정한 결과 대조군 보다 실험군에서 글루타티온 함유율이 유의성이 인정되는 수준으로 높게 나타났다. 또한 유청단백질을 분말화 하는 방법에 의한 글루타티온 농도의 차이를 알아보기 위하여 분무건조된 유청단백질(SDWPI)과 동결건조된 유청단백질(FDWPI)을 ICR 쥐에 급여하여 비장 세포내 글루타티온 함유율을 비교한 결과 그 차이는 나타나지 않았다. As shown in FIG. 6, the glutathione content measured in the spleen of the control group fed the casein and the whey protein was fed to the ICR mouse, and the glutathione content was higher in the experimental group than the control group. In addition, we compared the content of glutathione content in spleen cells by spraying whey protein (SDWPI) and freeze-dried whey protein (FDWPI) in ICR rats. The difference did not appear.
<실시예 8><Example 8>
Plaque forming cell 계수법에 의한 Plaque forming cell counting method IgMIgM 농도 증가에 따른 면역증강 효과 Immunity Enhancing Effect with Increased Concentration
3주간 실험사료를 급여한 후 쥐에 PBS로 2회 세척하여 농축한 5×106 면양 적혈구세포를 정맥 주사한다. 면양혈액으로부터 적혈구만을 분리하여 사용하였다. After feeding the experimental feed for 3 weeks, the rats were washed twice with PBS and intravenously injected with 5 × 10 6 sheep red blood cells. Only red blood cells were isolated from sheep blood.
IgM plaque forming cell은 Cunningham(1968)의 방법에 준하여 실시하였다. 면역화 5일째에 쥐로부터 비장을 적출한 후 pastle로 50-mash stainless steel screen을 이용하여 세포를 염류 평형용액(BSS)에 채집을 한다. 10% 농도의 가열에 의하여 불활성화시킨 송아지 혈청을 함께 첨가하여 비장세포(spleen cell)를 세척한 후에 염류 평형용액과 함께 15㎖로 만든다. 면양 적혈구는 PBS로 2회 세척하여 20% 농도로 농축한다. 보체 성분으로서 염류 평형용액에 1/10로 희석한 Guinea pig serum을 사용한다. 사용할 때까지 모든 용액은 얼음물에 보관하여야 한다.IgM plaque forming cell was performed according to the method of Cunningham (1968). On
0.05㎖의 비장세포 용액과 0.15㎖의 면양 적혈구, 0.75㎖의 보체용액을 혼합한다. 상기 혼합액을 미리 준비된 slide chamber에 넣고 파라핀왁스(paraffin wax) 또는 바세린(vaselin)으로 밀봉한다. 37℃에서 45∼60분 동안 배양을 시킨 다음, 각각의 샘플에서 plaque가 형성이 되면 이 플레이크의 개수를 센다.0.05 ml of splenocyte solution, 0.15 ml of sheep red blood cells, and 0.75 ml of complement solution are mixed. The mixture is placed in a slide chamber prepared in advance and sealed with paraffin wax or vaselin. Incubate at 37 ° C for 45-60 minutes, then count the flakes once plaque is formed in each sample.
비장에 대한 Total plaque forming cell에 대한 측정은 각 sample에 형성된 plaque forming cell에 PFC를 측정하기 위해 만든 혼합액과 실제 chamber에 들어간 양만큼의 비율을 곱하고, 여기에 300을 곱하여 나타낸다(Cunningham 과 Szenberg, 1968). 분석을 통해 얻어진 결과의 통계처리는 SAS(Window V 8.07)를 이용하여 분석하였으며 처리 평균간의 유의성은 Duncan`s multiple range test에 의하여 검정하였다. 그 결과를 도 7에 나타내었다.The measurement of the total plaque forming cell for the spleen is expressed by multiplying the ratio of the mixed solution made to measure PFC with the amount actually entered into the chamber, and multiplying it by 300 (Cunningham and Szenberg, 1968). ). Statistical processing of the results obtained was analyzed using SAS (Window V 8.07) and the significance between treatment means was tested by Duncan's multiple range test. The results are shown in FIG.
도 7에 도시된 바와 같이 유청단백질 2종류(FDWPI, SDWPI)와 소디움케이신(sodium caseinate)을 대비하여 IgM plaque 형성세포수를 계수하여 그 통계적인 유의성을 표시한 결과, 동결건조된 유청단백질(FDWPI)이 분무 건조된 유청단백질(SDWPI)보다 통계적인 유의차가 인정되는 수준의 면역증강 반응을 보였으며 분무 건조된 유청단백질(SDWPI)은 소디움케이신(sodium caseinate)보다 통계적으로 유의성이 인정되는 수준의 면역증강 반응이 있음을 알 수 있었다.As shown in FIG. 7, IgM plaque-forming cell numbers were counted against two types of whey proteins (FDWPI, SDWPI) and sodium caseinate, and the statistical significance thereof was displayed. FDWPI) showed an immunopotentiation level of statistically significant difference than that of spray-dried whey protein (SDWPI), and spray-dried whey protein (SDWPI) was statistically significant than sodium caseinate. It can be seen that there is an immune boosting reaction of.
<실시예 8><Example 8>
비타민 C 첨가에 의한 By adding vitamin C 유청단백질의Whey protein 세포내Intracellular 글루타티온 상승효과 측정 Measuring glutathione synergy
면양 적혈구(5×106)를 면역화한 이후 5일경과 후 분무건조된 유청단백질 분말(SDWPI) 및 동결건조된 유청단백질 분말(FDWPI), L-ascorbic acid 50mg/d (A-1), L-ascorbic acid 100mg/d (A-2)를 총 사료 100g에 대하여 각각 20g의 비율로 쥐에 3주간 급여한 이후 비장세포 중의 글루타티온 함유율(μ㏖/g/wet tissue)을 측정 하여 그 결과를 도 8에 나타내었다.5 days after immunization of sheep red blood cells (5 × 10 6 ) Spray dried whey protein powder (SDWPI) and lyophilized whey protein powder (FDWPI), L-ascorbic acid 50 mg / d (A-1) and L-
도 8에 도시된 바와 같이 첨가물질 A-1, A-2에 의하여 나타나는 결과가 ICR mouse에서 급여되었을 때 나타나는 비장세포중의 글루타티온을 측정한 결과이며 A-2의 첨가에 의하여 대조군(SDWPI과 FDWPI)보다 유의성이 높은 수준의 글루타티온 상승효과를 나타낼 수 있는 것으로 제시되었다. As shown in FIG. 8, the results indicated by the additives A-1 and A-2 are the results of measuring glutathione in splenocytes when fed from ICR mice, and the control group (SDWPI and FDWPI) by addition of A-2. It was suggested that the glutathione synergy may be significantly higher than).
<실시예 9>Example 9
유청단백질의Whey protein 총 항산화 활성 Total antioxidant activity
유청단백질 분말을 일정한 농도로 용해한 후 리놀레산(linoleic acid) 산화 system에서 항산화 활성을 측정하기 위하여 다음 방법으로 측정하였다.Whey protein powders were dissolved at a constant concentration and then measured by the following method to determine the antioxidant activity in linoleic acid oxidation system.
1)시료조제1) Sample preparation
동결건조 후 분말화 되어있는 시료, 유산균주 동결건조분말(casei), 발효유 동결건조분말(Will), 유청단백질 분말(WPI)을 각각 2%, 4%, 6%, 8%, 10%로 희석한 것을 0.2㎛ 여과지로 여과한다. After lyophilization, the powdered sample, lactic acid strain lyophilized powder (casei), fermented milk lyophilized powder (Will) and whey protein powder (WPI) were diluted to 2%, 4%, 6%, 8% and 10%, respectively. One was filtered with a 0.2 μm filter paper.
2)실험방법2) Experimental method
① 리놀레산(linoleic acid) 0.1㎖를 96%농도 에탄올 9.9㎖에 첨가한다. ① Add 0.1 ml of linoleic acid to 9.9 ml of 96% concentration ethanol.
② 리놀레산(linoleic acid)이 첨가된 에탄올 용액 0.5㎖를 0.85%농도 염화나트륨 용액 62.5㎖에 첨가한다. ② Add 0.5 ml of ethanol solution with linoleic acid to 62.5 ml of 0.85% sodium chloride solution.
③에탄올 용액이 첨가된 염화나트륨 용액 0.4㎖에 1%농도 SDS(sodium dodecyl sulphate) 용액 4㎕와 sample 45㎕를 첨가하고, 0.01% 황산제1철(FeSO4)용액 70㎕를 첨가한다.③ 4 μl of 1% sodium dodecyl sulphate (SDS) solution and 45 μl of sample are added to 0.4 ml of sodium chloride solution to which ethanol is added, and 70 μl of 0.01% ferrous sulfate (FeSO 4 ) solution is added.
④ 37℃에 60분간 배양한다. ④ Incubate for 60 minutes at 37 ℃.
⑤ 배양 후 0.4% BHT(butylated hydroxytoluene) 용액 310㎕와 0.02M의 아세테이트 버퍼(acetate buffer, pH3.5) 0.5㎖를 첨가한 후 0.8% TBA(thiobarbituric acid)용액 13㎕를 첨가한다. ⑤ After incubation, 310 μl of 0.4% BHT (butylated hydroxytoluene) solution and 0.5 ml of 0.02M acetate buffer (pH 3.5) are added, and 13 μl of 0.8% TBA (thiobarbituric acid) solution is added.
⑥ 80℃의 water bath에 40분간 가온처리한다. ⑥ Warm up in water bath at 80 ℃ for 40 minutes.
⑦ 방냉 후 5M 염산을 0.5㎖ 첨가한다. ⑦ After cooling, add 0.5 ml of 5M hydrochloric acid.
⑧ 1-부탄올(1-butanol) 1.7㎖을 첨가한 후 3000×g에 10분간 원심분리하여 상층액을 추출한다. ⑧ Add 1.7 ml of 1-butanol and centrifuge at 3000 × g for 10 minutes to extract the supernatant.
⑨ 상층액 추출 후 OD534값을 측정한다.⑨ Measure the OD 534 value after extracting the supernatant.
*계산식= (1-sample OD534/control OD534)×100* Calculation Formula = (1-sample OD 534 / control OD 534 ) × 100
유청단백질과 Will 동결건조 분말 및 L. casei 세포동결건조분말을 각각 2-10% 수준으로 용해하여 용액상태에서 TBA 방법에 의한 리놀레산(linoleic acid)산화 억제 활성을 비교한 결과는 도 9에 나타내었다.Whey Protein and Will Lyophilized Powder and L. casei The results of comparing the linoleic acid oxidation inhibitory activity by the TBA method in the solution state by dissolving the lyophilized dry powder at 2-10% levels are shown in FIG. 9.
유청단백질의 경우는 10% 수준의 농도에서 가장 높은 리놀레산 산화억제활성을 보이는 반면 건조된 성분구성에 따라 그 억제 수준의 차이를 보이는 것으로 나타났다. Whey protein showed the highest linoleic acid oxidation inhibitory activity at the concentration of 10%, while the difference in inhibition level was different depending on the dried ingredients.
유청단백질 분말과 L. casei CU 4114를 탈지유 배지에 배양한 후 동결건조에 의하여 분말화 한 성분과 발효유 Will을 분말화 한 분말을 5%로부터 10% 까지 각 농도에서의 액체가 갖는 리놀레산 산화 체계에서 산화반응 억제 활성을 측정한 결과로서 5% 이하의 농도에서 나타내는 특징은 비교적 탈지유성분이 높은 비율로 함유된 L. casei 분말이 동등한 농도의 유청단백질이나 Will 완제품 분말보다 월등히 높은 항산화 활성을 나타내었다. 이는 균체성분에 의한 항산화 활성이 높은데 기인된 것으로 판단된다. 그러므로 배양 후 배지성분을 완전히 세척에 의하여 제거한 순수한 세포성분을 동결 건조한 후 단백질 성분과 비교 시험이 필요할 것으로 판단되며 또한 위의 실험결과 L. casei 의 세포내용물에는 특징적으로 높은 함유율의 글루타티온(GSH)이 내포되었다는 사실과 연관성이 있는 것으로도 그 원인을 추적할 수 있다. Whey protein powder and L. casei CU 4114 were incubated in skim milk medium and then lyophilized in powder and powdered powdered fermented milk Will from 5% to 10% in linoleic acid oxidation system. As a result of measuring the inhibitory activity of oxidation reaction, the characteristic at the concentration below 5% is L. casei containing a relatively high percentage of skim milk components. The powders showed significantly higher antioxidant activity than equivalent whey protein or Will finished powders. This is believed to be due to the high antioxidant activity by the cell components. Therefore, after culturing, pure cell components removed by complete washing of media components are required to be lyophilized and compared with protein components. In addition, the contents of L. casei cells were characterized by high content of glutathione (GSH). The cause can also be traced back to the fact that it is implicit.
유청단백질 분말은 그 농도가 증가함에 따라 점진적으로 항산화 활성도 강화되는 현상을 나타낸다. 10%수준의 용액상태에서는 세가지 종류의 분말에 대한 총 산화 억제 활성은 유사한 수준에 도달한다는 점은 특이한 의미를 가질 것으로 판단된다.Whey protein powders gradually increase their antioxidant activity as their concentration increases. In the 10% solution state, the total antioxidant activity of the three types of powder reaches a similar level is considered to have a unique meaning.
<실시예 10><Example 10>
락토바실러스Lactobacillus 스포러제네스Sporogenes CU 4109를 유효성분으로 함유하는 것을 특징으로 하는 발효유의 제조 Preparation of fermented milk characterized by containing CU 4109 as an active ingredient
제조공정은 다음과 같다.The manufacturing process is as follows.
(1)요구르트 믹스(mix) 제조(1) yogurt mix production
탈지분유, 탈지유, 포도당을 용해하여 총 고형분이 20% 수준이 되도록 조정한 후 다음 공정이 진행되도록 이송한다.The skim milk powder, skim milk, and glucose are dissolved to adjust the total solids to a level of 20% and then transferred to the next process.
(2)용해 및 가열처리(2) melting and heat treatment
60℃정도까지 가열하면서 용해상태로 전환 시킨 후 90℃에서 30분간 가열처리하여 기질 중에 존재하는 비포자 형성 미생물과 품질에 영향을 미칠 수 있는 효소를 불활성화시킨다. 냉각하여 37℃까지 온도를 조정한다.After conversion to the dissolved state while heating up to 60 ℃ about 30 minutes heat treatment at 90 ℃ to inactivate the spore-forming microorganisms present in the substrate and enzymes that can affect the quality. Cool to adjust the temperature to 37 ° C.
(3)선별된 특수균주 starter 준비 및 접종(3) Preparation and inoculation of selected special strain starter
항산화 활성이 특이한 것으로 확인된 바 있는 락토바실러스 스포러제네스 CU 4109 균주를 별도로 스타터(starter)균주로 준비한 후 기질의 3% 수준이 되는 양으로 확보한 후 이를 냉각된 상태의 요구르트 믹스의 담긴 발효탱크에 접종한다.Fermentation tank containing lactobacillus sporogenes CU 4109 strain, which has been found to have an unusual antioxidant activity, was prepared as a starter strain and secured in an amount of 3% of the substrate, and then cooled in a yogurt mix in a cooled state. Inoculate.
(4)발효(4) fermentation
37℃에서 48시간 배양하여 최종 TA가 0.6% 수준에 도달하도록 조정하고 과산도상태로 진행되지 않도록 발효과정 중에 TA를 확인하고 발효를 적정시기에 종결시킨다.Incubate at 37 ° C for 48 hours to adjust the final TA to reach 0.6% level. Check the TA during the fermentation process so that it does not progress to superacidity and terminate the fermentation at the appropriate time.
(5)냉각 및 균질(5) cooling and homogeneous
30℃ 정도로 냉각 후 이 온도에서 12시간 정도 유지하여 숙성시키고 150kg/cm2 조건에서 균질 한다.After cooling to 30 ° C, it is kept at this temperature for 12 hours to mature and homogenized at 150kg / cm 2 condition.
(6) 시럽혼합 및 과일 첨가 성분 첨가(6) Syrup Mixing and Fruit Additives
설탕 시럽을 제조한 후 가열 살균하고 1:1 중량비율로 혼합하고 과일 퓨레(puree)를 적절한 양 첨가하여 기호특성을 높인다.Sugar syrup is prepared, heat sterilized, mixed at a weight ratio of 1: 1, and fruit puree is added in an appropriate amount to enhance the taste characteristics.
(7)항산화 기질 성분 용해 열처리 및 첨가성분 혼합 후 섞어줌(7) Dissolve heat treatment of antioxidant substrate components and mix with additives
항산화 기질 성분으로 유청단백질 순수 분리물(WPI)을 용해하고 저온살균처리하여 병원성 세균을 파괴한 후 보조 항산화 물 아스코빈 산(L-ascorbic acid)을 첨가하고 혼합한다. 이를 상기 (6)의 혼합물에 첨가하여 연속 용해장치를 이용하여 철저히 섞어 완제품을 얻는다.WPI is dissolved as an antioxidant substrate component, and pasteurized to destroy pathogenic bacteria, and then supplemented and mixed with supplemental antioxidant ascorbic acid (L-ascorbic acid). This is added to the mixture of (6) and thoroughly mixed using a continuous dissolution apparatus to obtain a finished product.
(8)충전 및 포장 (8) filling and packing
특수 디자인된 용기에 충전 후 포장한다.Packed in a specially designed container.
(9)냉장 (9) refrigeration
5℃로 유지되는 냉장고에 제품을 보관하면서 소비자의 요구에 따라 배달한다.The product is stored in a refrigerator maintained at 5 ° C and delivered according to the needs of consumers.
이상에서 설명한 바와 같이 본 발명에 의한 락토바실러스 스포러제네스 CU 4109(Lactobacillus sporogenes CU 4109)는 질병발생과 노화 등 유해한 작용을 나타내는 생체내 활성산소의 발생을 방지하기 위해 산화반응을 방지하거나 그 속도를 현저하게 느리게 해주는 항산화 활성을 나타내는 효과가 탁월하며, 이는 지방산 리놀레산을 이용한 산화 과정 중에서 트리바르비투르산(tribarbituric acid, TBA)을 이용한 산화 억제 활성이 높을 뿐 아니라 선발된 균주 중에서 균체내 함유 글루타 티온 농도 및 실험동물 ICR 마우스를 이용한 급여실험에서 간 글루타티온 함유율을 현저하게 상승시킴으로써 확인할 수 있다. As described above, Lactobacillus sporogenes CU 4109 according to the present invention prevents the oxidation reaction or prevents the rate of oxidation in order to prevent the generation of free radicals in vivo which exhibits harmful effects such as disease occurrence and aging. The antioxidant activity of remarkably slowing down is excellent, which is not only high in antioxidant activity using tribarbituric acid (TBA) in the oxidation process using fatty acid linoleic acid, but also glutathione contained in bacteria among selected strains. It can be confirmed by significantly increasing the concentration of hepatic glutathione in the concentration test and the feeding experiment using the experimental animal ICR mouse.
그러므로 본 발명과 같이 항산화 활성을 나타내는 포자형성능력이 있는 유산균주 및 이를 유효성분으로 함유하는 항산화 식품을 식음용 하였을 때 항산화 활성을 기대할 수 있다.Therefore, when the lactic acid bacteria having sporulation ability exhibiting antioxidant activity and antioxidant foods containing the same as an active ingredient, such as the present invention can be expected to antioxidant activity.
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| KR100483961B1 (en) | 2001-11-02 | 2005-04-18 | 김영만 | Water soluble Tea Composition Comprising Lactobacilus sporogenes |
| KR100524766B1 (en) | 2003-12-16 | 2005-10-31 | 김대옥 | Cube sugar including lactic ferments and preparation method thereof |
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| KR100483961B1 (en) | 2001-11-02 | 2005-04-18 | 김영만 | Water soluble Tea Composition Comprising Lactobacilus sporogenes |
| KR100524766B1 (en) | 2003-12-16 | 2005-10-31 | 김대옥 | Cube sugar including lactic ferments and preparation method thereof |
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