CN1317385C - 干酪乳杆菌Bd-II菌株及其在降低血脂方面的应用 - Google Patents
干酪乳杆菌Bd-II菌株及其在降低血脂方面的应用 Download PDFInfo
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- CN1317385C CN1317385C CNB031289959A CN03128995A CN1317385C CN 1317385 C CN1317385 C CN 1317385C CN B031289959 A CNB031289959 A CN B031289959A CN 03128995 A CN03128995 A CN 03128995A CN 1317385 C CN1317385 C CN 1317385C
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/245—Lactobacillus casei
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S435/822—Microorganisms using bacteria or actinomycetales
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- Y10S435/856—Lactobacillus casei
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- Health & Medical Sciences (AREA)
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
本发明公开了一种干酪乳杆菌(Lactobacillus casei)Bd-II CGMCCNO.0849,及其在降低血脂方面的应用。
Description
技术领域
本发明涉及一种乳杆菌属的新菌株,以及该菌株在降低血脂方面的应用。
背景技术
人和动物的肠道,传统发酵食品等栖居着大量的有益菌群,在营养物质的消化与吸收、机体的生长与发育、免疫与拮抗等方面均发挥着重要的生理作用。近年来,微生态学家发现益生菌群中的乳杆菌、双歧杆菌以及肠球菌等与胆固醇的代谢有直接的关系。国外大量的体内和体外研究都发现和证实了某些益生乳酸菌具有降低人体或动物的血清胆固醇含量的功效。而我国传统食品中有着丰富的尚未被西方科学家发现的此类菌种资源,有待进一步开发利用。
发明内容
本发明人针对上述课题,研究了以我国传统食品泡菜和腌制苜蓿中的微生物群为目标菌群,应用高胆固醇培养基,从中筛选具有降低胆固醇功效的益生乳酸菌,并进一步研究其在调节血脂方面的应用。
因而本发明的目的是提供一种干酪乳杆菌Bd-II;
本发明的另一目的是公开上述干酪乳杆菌Bd-II在降低血脂方面的应用。
本发明的干酪乳杆菌(Lactobacillus casei)Bd-II制备过程如下:将泡菜汁或苜蓿汁作为样品冷藏,测定其pH值,并接种于MRS平板(Merck公司购得)且进行总菌落计数;然后采取上述样品,根据上述测定结果用pH 6.8的PBS缓冲溶液浸泡混匀并梯度稀释;
吸取合适的稀释度(每毫升含有100个左右细菌)100微升涂布于LBS平板,进行培养;
挑取典型菌落转接到MRS平板进一步纯化;
挑取理想单菌落转接于MRS液体进行增菌,获得乳酸菌菌株并冷藏保存;
将上述新鲜的乳酸菌菌株以体外模型初步筛选具有降胆固醇功能的益生菌(USMAN and A.HOSONO.Bile tolerance,taurocholate deconjugation,andbinding of cholesterol by lactobacillus gasseri strains.J.Dairy Sci.199982:243-248.),进行菌种鉴定,其中可获得干酪乳杆菌Bd-II。
该干酪乳杆菌Bd-II的微生物学特性示于表1。
表1干酪乳杆菌Bd-II的微生物学特性
试验项目 | 结果 | 试验项目 | 结果 |
革兰氏染色细胞形态形成芽孢接触酶氧化酶在空气中生长兼性厌氧生长O/F试验产生乳酸碳水化合物产气葡萄糖葡萄糖酸钠碳水化合物产酸 | 阳性杆状---++发酵+-+ | 碳水化合物产酸(续)海藻糖木糖果糖核糖棉子糖乳糖松三糖水杨素鼠李糖七叶灵甘露糖 | +-++++++-++ |
葡萄糖葡萄糖酸钠纤维二糖***糖蔗糖 | +++-+ | 甘露醇麦芽糖山梨醇蜜二糖半乳糖 | +++-+ |
上述操作均在无菌条件下进行。
本发明的干酪乳杆菌Bd-II除具有一般乳杆菌的有益作用外,将该菌株应用于调节血脂方面,如开发功能性的乳制品,作为人类膳食补充剂,还起到辅助降低人们血清胆固醇浓度的功效。
通过借助以下实施例将更详细说明本发明。以下实施例仅为说明性的,本发明并不受这些实施例限制。
本发明的干酪乳杆菌Bd-II已于2002年12月6日在地址为中国北京市海淀区中关村北一条13号中国科学院微生物研究所内的中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏,并收到保藏号CGMCCNO.0849。
具体实施方式
实施例1样品采集
样品为:
泡菜汁:卷心菜泡菜,菜叶和汁水一起均质得到的匀浆;
苜蓿汁:采集苏州天平山农家的腌制苜蓿,取5克,加入10毫升无菌水均质得到的匀浆;
实施例2集和乳酸菌筛选
将上述样品装入无菌罗口瓶,旋紧,4℃冷藏。24小时内测定泡菜汁和苜蓿汁的pH,并接种于MRS平板测定其总菌落计数,然后按以下步骤处理。
实施例3LBS平板分离
取泡菜汁和苜蓿汁,根据实施例2中所测定的结果,无菌条件下用PBS缓冲溶液(pH 6.8)梯度稀释,吸取合适的稀释度溶液(每毫升含有100个左右细菌)100微升涂布于LBS平板,平板倒置放入厌氧培养箱,37℃厌氧培养36-72小时,观察菌落生长情况,平板4℃保存。
实施例4菌株镜检
无菌操作,接种环挑取实施例3所得典型菌落培养物,涂于载玻片,制片并革兰氏染色,油镜观察菌体形态,挑取***作为目的菌株。
实施例5菌株平板纯化
无菌条件下,接种环挑取实施例4所初步确定的目的菌落,在MRS平板上划线分离,37℃倒置厌氧培养24-36小时,挑取单菌落进行镜检,直至确定所得的菌株已经纯化。
实施例6菌株的增菌过程
挑取实施例5所得的理想单菌落,转接到1毫升MRS液体,37℃静置培养24小时左右,再按1%(体积比)的接种量将培养液转接到新的MRS液体,37℃静置培养24小时,再在液体MRS中重复扩增2次,所得培养液4℃保存。
实施例7以体外模型初步筛选具有降胆固醇功能的益生菌
15毫升无菌具塞离心管加入10毫升以无菌鸡蛋黄液为胆固醇源的高胆固醇培养液MRSO-CHOL,按1%(体积比)的接种量接入实施例6获得的新鲜的菌株,37℃培养24小时。培养液12,000×g离心10分钟,按照邻苯二甲醛测定胆固醇的方法测定上清夜中胆固醇的含量,以未接种的MRSO-CHOL作为对照,计算出各个实验菌株对胆固醇的降低率(S.E.Glliland,C.R.Nelson,C.Maxwell.Applied and EnviromentalMicrobiology,1985,49:377~381.),从中选出具有较好的降低胆固醇功效的菌株,结果见表2。
胆固醇的降低率计算公式为:
其中A为各实验菌株发酵上清液OD550nm[波长550nm(纳米)处的吸收光度值];
C为空白对照的OD550nm;
D为胆固醇降低率。
表2降胆固醇益生乳酸菌初步筛选结果
胆固醇降低率(%) | 菌株数(株) | 比例(%) | 来源 | |
泡菜 | 腌制苜蓿 | |||
40以上30-4020-3020以下 | 691338 | 9.0913.6419.7057.28 | 45212 | 22816 |
其中,Bd-II具有较高的胆固醇降低率,达到45.17%,对应的胆固醇降低量为231.95微克/毫升。另外还有5株菌株的胆固醇降低率在40%以上。
实施例8Bd-II菌种鉴定
经中国科学院微生物研究所检测鉴定,该Bd-II的微生物学特性如上述表1示。
实施例9菌体悬浮液制备方法
将本发明的干酪乳杆菌Bd-II(CGMCC No.0849)接入10%脱脂奶,37℃静置培养16小时,计数;根据计数结果用脱脂奶做适当的稀释、悬浮得到如表3中所需的菌液浓度。Bd-II死菌悬浮液制备方法:悬浮108cfu/ml(每毫升中所含菌体个数)Bd-II脱脂奶置于100℃水浴加热15分钟,冷却,冰箱保存待用。
应用实施例降血清胆固醇功能性实验(体内实验,动物实验)
动物模型在益生乳酸菌降低血清胆固醇功能性实验研究中得到越来越广泛的应用(郑建仙.功能性食品(第三卷).北京:中国轻工业出版社,1999.9.408-412.)。高脂高胆固醇饲料诱发动物脂代谢紊乱,血脂水平发生变化:血清胆固醇(TC)和甘油三酯(TG)水平以及低密度脂蛋白胆固醇(LDL-C)含量升高,高密度脂蛋白胆固醇(HDL-C)含量降低。降血清胆固醇功能性实验通常做法是,饲喂高脂高胆固醇饲料的同时,饲喂实验动物实验样品,通过测定动物血清脂类含量的变化来确定实验样品对动物血脂的影响效果。
本发明筛选得到的益生乳酸菌是干酪乳杆菌(Lactobacillus casei)Bd-II(CGMCC No.0849),该菌株在体外实验中具有较高的降低胆固醇浓度的能力。
本发明采用SD大鼠,在常规饲料中添加重量比为1%的胆固醇、5%的猪油和0.25%的猪胆盐来诱发SD大鼠高胆固醇血症。同时饲喂悬浮干酪乳杆菌的脱脂牛奶,最后检测SD大鼠血清胆固醇等血脂含量,来判断该菌株是否具有降低血清胆固醇的功效。
1、实验动物和动物房条件
雄性SD大鼠(8周龄),40个。常规饲料喂养3天,随机分成5组,每组8个,分笼喂养。
动物房条件:温度22±2℃,湿度56±5%,1周换2-3次锯末。控制12小时光照,12小时黑暗。
饲料:常规饲料由中科院上海实验动物中心提供;
高胆固醇饲料:常规饲料添加重量比为1.0%的胆固醇,5%的猪油以及0.25%猪胆酸盐,委托中科院上海实验动物中心加工;
所有的饲料进动物房前辐射灭菌。
2、益生乳酸菌Bd-II功能性动物实验方法
(1)实验动物分组和饲喂安排
40只SD大鼠随机分成5组,饲喂不同的饲料和菌体悬浮液,具体的分组如表3所示。
表3实验动物分组和饲喂计划
实验分组a | 日常饲喂b | 灌胃3毫升/个/天 |
1.空白对照2.高脂对照3.高脂SKMc对照4.Bd-II活菌5.Bd-II死菌 | 常规饲料+水高脂饲料+水高脂饲料+水高脂饲料+水高脂饲料+水 | 水水SKM悬浮2.5×108cfu/ml活菌Bd-II的SKM悬浮2.5×108cfu/ml死菌Bd-II的SKM |
注:a每组8个SD大鼠
b饲喂实验共持续21天
cSKM——灭菌10%脱脂奶
(2)实验动物的日常管理和饲喂
SD大鼠按表3随机分成1-5个组别,除空白对照组1饲喂常规饲料外,2-5组均饲喂高脂饲料。按照表3中方案,每日上午8点对SD大鼠灌胃3毫升/个/天。
(3)大鼠血清采集和血脂浓度的检测
21天饲喂实验结束后,禁食12小时,***深度麻醉,摘眼球取血,装入无菌离心管,离心(3000×g,10分钟)收集血清,送上海华山医院检验中心测定血清中总胆固醇(TC),甘油三酯(TG),高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)的含量。
(4)统计分析
同组数据单因素方差分析。
不同组之间t检验,p<0.05差异有统计学意义,p<0.01和p<0.001差异有高度的统计学意义。
3、结果分析
21天饲喂实验结束后,禁食12小时,收集大鼠血清,送上海华山医院进行血清脂类分析,测定TC、TG、HDL-C和LDL-C含量,计算AI(动脉粥样硬化因子)。具体结果见表4。
表4大鼠血脂的测定结果
组别 | TC(mmol/L) | TG(mmol/L) | HDL(mmol/L) | LDL(mmol/L) | AI(LDL/HDL) |
1 | 1.4±0.29*** | 0.68±0.20* | 1.02±0.21* | 0.38±0.16*** | 0.38±0.18*** |
2 | 2.56±0.23 | 0.97±0.19 | 0.79±0.04 | 1.77±0.20 | 2.23±0.19 |
3 | 2.44±0.36 | 0.90±0.10 | 0.92±0.10 | 1.52±0.41 | 1.70±0.58 |
4 | 1.94±0.40** | 0.91±0.32 | 0.83±0.17 | 1.11±0.34** | 1.38±0.52** |
5 | 2.03±0.48* | 0.82±0.27 | 0.84±0.09 | 1.19±0.45 | 1.42±0.50** |
其中,*表示与组2相比P<0.05,差异有统计学意义;**表示与组2相比P<0.01,差异有高度的统计学意义;***表示与组2相比P<0.001差异具有高度统计学意义。
从表4可以看出,空白对照组(组1)与高脂对照组(组2)的TC、LDL和AI差异具有高度的统计学意义,TG和HDL差异具有统计学意义,说明实验动物的高血脂模型建立成功。
从表4还可以看出,高脂脱脂奶对照组(组3)与高脂对照组(组2)相比差异没有统计学意义,说明单独的脱脂奶培养基不具有降低血脂的作用。
从表4还可以看出,Bd-II活菌组(组4)与高脂对照组(组2)相比,能非常显著地降低TC、LDL和AI,TG和HDL差异没有统计学意义,说明Bd-II具有显著的降血脂功能,并能降低心血管疾病的风险。Bd-II死菌组(组5)与高脂对照组(组2)相比,TC显著降低,AT非常显著降低,TG、HDL和LDL差异没有统计学意义,说明Bd-II死菌也有降血脂的功能,并也能降低心血管疾病的风险。
Claims (2)
1、一种干酪乳杆菌(Lactobacillus casei)Bd-II CGMCC NO.0849。
2、权利要求1所述的干酪乳杆菌在制备降低血脂产品中的应用。
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AU2003255088A AU2003255088A1 (en) | 2003-05-30 | 2003-08-04 | Lactobacillus casei bd-ii strain and used to reduce blood cholesterol |
PCT/CN2003/000623 WO2004106498A1 (fr) | 2003-05-30 | 2003-08-04 | Souche de lactobacillus casei bd-ii, et son utilisation pour la reduction du taux de cholesterol sanguin |
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CN1119914A (zh) * | 1994-05-13 | 1996-04-10 | 成都市酿造公司调味品研究所 | 一种利用纯种乳酸菌生产泡菜的工艺 |
WO2001089541A1 (fr) * | 2000-05-25 | 2001-11-29 | Compagnie Gervais Danone | Utilisation de lactobacillus casei dans des peptides immunostimulantes |
WO2002018542A1 (en) * | 2000-09-01 | 2002-03-07 | Probi Ab | New strains of lactobacillus paracasei |
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WO2004106498A1 (fr) | 2004-12-09 |
CN1552845A (zh) | 2004-12-08 |
US20060127380A1 (en) | 2006-06-15 |
AU2003255088A1 (en) | 2005-01-21 |
US7270994B2 (en) | 2007-09-18 |
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