CN1244686C - 干酪乳杆菌lc2w菌株及其在制备抗高血压产品中的应用 - Google Patents
干酪乳杆菌lc2w菌株及其在制备抗高血压产品中的应用 Download PDFInfo
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- CN1244686C CN1244686C CNB031294502A CN03129450A CN1244686C CN 1244686 C CN1244686 C CN 1244686C CN B031294502 A CNB031294502 A CN B031294502A CN 03129450 A CN03129450 A CN 03129450A CN 1244686 C CN1244686 C CN 1244686C
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Abstract
本发明公开了一种干酪乳杆菌(Lactobacillus casei)LC2W CGMCCNO.0828,及其在抗高血压方面的应用。
Description
技术领域
本发明涉及一种新的乳杆菌菌株,以及该菌株在生产和制造具有抗高血压(和/或降血压)作用产品方面的应用。
背景技术
根据世界卫生组织(WHO)规定,对成人而言,如果静息血管收缩压(SBP)超过140mmHg即属于高血压,当静息SBP超过160mmHg后,即必须采取药物治疗措施,而静息SBP在120-140mmHg属于血压偏高的人群。据统计,在西方工业化国家,有15%以上的人群患有高血压。统计数据表明,中国的2002年的高血压患者的人数超过1.3亿,占总人口的10%。在高血压人群中,由于各种原因,仅有少数人采取了药物治疗措施。
高血压会引起患者心、脑、肾等器官损害,并与糖、脂质代谢紊乱等有密切关系,可明显降低患者生活质量。抗高血压药物的应用,应不仅仅局限于简单的降压,还必须考虑降低血压波动性(BPV),增强压力感受反射敏感性(BRS),改善心率变异性(HRV)及血压昼夜节律,从而防治高血压并发症,提高高血压患者的生活质量。
常用的降血压药物有利尿降压药物、β-受体阻滞剂、钙拮抗剂、影响血管紧张素II形成的药物如ACEI(血管紧张素转化酶抑制剂)和血管紧张素II受体拮抗剂等,这些药物有些具有使用范围局限性,如塞利洛尔是一种长效的β-受体阻滞剂,其减慢心率作用较轻,对老年人及心率偏慢的高血压患者较为合适;而有些抗高血压药物在降压的同时可能会给患者到来某些副作用,如国内目前临床上广泛使用的第一代钙拮抗剂,会加重传导性减弱和负性肌力作用,使高血压患者心脏病发作的危险增高,还可引起反射性交感神经兴奋,导致心肌耗氧增加和加重心律失常,不能有效降低发病和死亡率等。
ACEI与血管紧张素I转化酶结合,从而抑制血管紧张素II(Ang II)生成,导致缓激肽分解减慢,结果血管舒张,血压下降。过氧化脂质会损伤细胞膜导致细胞死亡,而卡托普利(一种属于ACEI、用于治疗高血压的临床药物)可显著降低过氧化脂质,有利于降低高血压并发症。对早期高血压患者进行ACEI为基础的抗高血压治疗能使心血管疾病的发病和死亡的危险性降低。新型的ACEI药物如苯那普利、培哚普利、赖诺普利等具有心肌修复作用,并防止修复性纤维化形成。此外,ACEI可扩张肾小球动脉,故能有效降低肾小球内毛细血管压,从而降低肾脏高灌注,减少白蛋白排泄。与其它几类抗高血压药物相比,只有ACEI能减少尿蛋白和改善肾功能。但有25%服用ACEI的患者会发生持续性干咳、有的甚至不能耐受,被迫停药。
部分特定的微生物如瑞士乳杆菌(Lactobacillus helveticus)和啤酒酵母(Saccharomyces cerevisiae)及瑞士乳杆菌(Lactobacillus helveticus)LBK-16在以乳为基料的基质中生长时,可通过所分泌的蛋白酶的水解作用,将乳蛋白中一些特定肽片段释放出来,这些片段具有抑制血管紧张素I转化酶(ACE)的作用或者阿片肽活性,从而在体内产生降压作用,如瑞士乳杆菌和啤酒酵母在发酵乳的过程中所产生的VPP(缬氨酰-脯氨酰-脯氨酸)和IPP(异亮氨酰-脯氨酰-脯氨酸)(两者对ACE抑制作用的IC50分别为9uM和5uM),含有这两种小肽的发酵乳在SHR(自发性高血压大鼠)和高血压患者体内具有显著的降压作用,给SHR按5ml/Kg体重单次口服该发酵乳后,SHR的SBP可降低25mmHg;高血压患者每天口服这种发酵乳95ml,连续8周后,血管收缩压(SBP)可降低14.1±3.1mmHg。在部分微生物生长的过程中,还能合成某些物质,这些物质在SHR体内也具有降血压作用,例如干酪乳杆菌YIT9018细胞自溶物中分离到一种分子量为180,000Dalton(道尔顿)的多糖-糖肽复合物,当按1mg/Kg体重的剂量给SHR和RHR(肾性高血压大鼠)单次口服该化合物6-12小时后,这两种高血压大鼠的SBP分别下降10-20mmHg。目前从实验性高血压动物模型以及少量的人体实验结果表明,采用某些特定的微生物对乳进行发酵所获得的产物在体内具有温和的降压作用,这种降压作用比较温和、平稳、不会产生(药物)依赖性,而且对患者体重、心跳速率、红血球、总胆固醇、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、以及其它的血清指标如总蛋白、葡萄糖、脲氮、肌酐等多项指标与对照组相比,都不会产生变化。因此,是一种将预防疾病发病风险融于日常生活的简便而有效的方法。
发明内容
本发明人针对上述课题,以天然发酵食品(干酪、马奶酒)中的乳酸菌为目标菌群,采用特定的体外筛选模型,从中筛选出发酵乳以后产生降压肽的益生性乳酸菌,并进一步研究了其在抗高血压方面的功能与应用。
本发明的目的是提供一种干酪乳杆菌LC2W的新菌株,其具有抗高血压功能。
本发明的另一目的是公开该菌株在抗高血压方面的应用。
本发明中的干酪乳杆菌(Lactobacillus casei)LC2W的分离过程如下:以天然发酵食品(干酪或马奶酒)为样品,先通过MRS液体培养基(乳杆菌选择性培养基,德国Merck公司购得)进行富集培养,然后将富集培养物用0.5%(重量/体积百分比)无菌的蛋白胨水稀释105~106倍,采用MRS-salicin[将MRS琼脂(德国Merck公司购得)中的葡萄糖以水杨素替代,水杨素的最终浓度为1%(重量/体积百分比)]或LC琼脂(干酪乳杆菌选择性琼脂,配方见Ravula,R.R.et al.,1998.Biotechnol.Tech,12:819-822.)倒平板,37℃厌氧培养72小时(H2∶CO2∶N2=10∶5∶85);待平板出现典型的菌落以后,挑取相关的菌落在LC琼脂上进一步纯化。
挑取纯化后的单菌落在MRS琼脂上37℃、厌氧条件下(H2∶CO2∶N2=10∶5∶85)进行扩增,并进行菌株冷藏保存。
将上述菌株新鲜的培养物在无菌条件下转接于10%(重量百分比)灭菌(118℃、15分钟)的脱脂奶中,37℃培养24小时;发酵产物经在4℃、4000rpm离心20分钟后,收集上清液;上清液经过分子截留极限为10,000道尔顿的膜(VIVAFLOW 200,英国Vivascience公司购得)进行超滤后,超滤透过液采用体外模型测定其对ACE的抑制作用(见N.Yamamoto et al.,1999.J.Dairy Sci.82:1388-1393.);对于在体外对ACE有明显抑制作用的菌株进行菌种鉴定,其中可获得干酪乳杆菌LC2W,并测定其菌体及代谢产物在自发性高血压大鼠(SHR)中的降压作用。
该干酪乳杆菌LC2W的微生物学特性见表1。
表1 干酪乳杆菌LC2W的微生物学特性
实验项目 | 结果 | 实验项目 | 结果 |
革兰氏染色细胞形状形成芽孢 | 阳性杆状- | 碳水化合物产酸(续)海藻糖 | + |
接触酶氧化酶在空气中生长兼性厌氧生长O/F实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葡萄糖葡萄糖酸钠纤维二糖阿拉伯糖蔗糖 | --++发酵+-++++-+ | 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甘露醇麦芽糖山梨醇蜜二糖半乳糖 | -++-+-+-+++++-+ |
本发明的干酪乳杆菌LC2W除一般乳杆菌所具有的对人体健康的有益作用外,该菌株还能用于具有抗高血压功能产品的生产,例如具有抗高血压功能的各种乳制品的生产;既可以混合的形式存在于特定的产品中,也可以纯菌剂(菌粉、胶囊、片剂)的形式生产。
通过借助以下实施例将详细说明本发明。以下实施例仅具有说明性,本发明并不受这些实施例的限制。
本发明所称的干酪乳杆菌LC2W已于2002年10月30日在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏(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,邮政编码100080),保藏登记号为CGMCC No.0828。
具体实施方式
实施例一 样品采集和分离
样品为:内蒙古自治区呼伦贝尔市等地农家自制的天然干酪或马奶酒。
步骤1、样品采集。
取上述样品25克(或25ml)加入到225ml 0.5%无菌的蛋白胨水中,充分混匀。
步骤2、样品的富集。
取步骤1中的样品5ml加入到100mL MRS肉汤中,37℃培养过夜,然后按以下步骤处理。
步骤3、LC-琼脂或MRS-Salicin琼脂分离
取步骤2中的培养液1ml,用0.5%(重量/体积百分比)无菌的蛋白胨水稀释105~106倍,吸取1ml稀释后的菌悬液加入到培养皿中,倒入融化后冷却至50℃左右的LC-琼脂或MRS-Salicin琼脂,混匀。待完全凝固后,置37℃厌氧培养72小时,观察菌落生长情况,平板4℃保存。
步骤4、菌株的纯化
用接种环挑取步骤3中所得到的菌落,在LC-琼脂平板上划线分离,置37℃厌氧培养48-72小时,挑取单菌落,进行显微镜观察,直至能确定所获得的菌株已经纯化。对于所获得的纯培养物,采用无菌的40%甘油保存和MRS斜面保存。
步骤5、采用体外模型测定分离菌株代谢产物对ACE的抑制作用
将分离的纯菌株转接到10%(重量百分比)的无菌脱脂奶中,37℃培养20-24小时。发酵产物经过4℃、4000rpm离心20分钟后,收集上清液。上清液经过分子截留极限为10,000道尔顿的膜进行超滤后,超滤透过液采用体外模型测定其对血管紧张素I转化酶(ACE)的抑制作用(见N.Yamamotoet al.,1999.J.Dairy Sci.82:1388-1393.)。以未发酵的无菌脱脂奶的超滤透过液为对照,按以下公式计算出各个菌株的发酵产物对ACE的抑制作用,从中挑选出对ACE具有显著抑制作用的菌株,结果见表2。
其中A为反应体系中加入发酵样品后,所获得的ACE酶转化产物在λ=228nm处的光吸收度;
B为反应体系中加入无菌脱脂奶的超滤透过液后,所获得的ACE酶转化产物在λ=228nm处的光吸收度。
表2 对ACE具有抑制作用的乳杆菌的初步筛选结果
对(%) | 菌株数(株) | 比例(%) | 来源 | |
天然干酪 | 马奶酒 | |||
35以上20-3410-199以下 | 2511107 | 1.64.08.885.6 | 22542 | 03665 |
其中,LC2W的抑制作用最高,可以达到42%。
步骤6、LC2W菌种鉴定
经中国科学院微生物研究所检测鉴定,LC2W的微生物学特性如上述表1所示。
应用实施例一 干酪乳杆菌LC2W(CGMCC NO.0828)的发酵产物对SHR的降血压作用
自发性高血压大鼠(SHR)是一种遗传性高血压实验动物模型,是用来研究抗高血压药物或其它降压物质最常见的动物模型。通过观察单次或多次灌胃后大鼠收缩压的变化,可观察该种物质的临时及长期降压效果。
本发明的干酪乳杆菌LC2W的发酵乳的发酵产物经过单次和多次灌胃后,对SHR有显著的降压作用。
1、实验方法
1.1实验样品的制备
干酪乳杆菌LC2W(CGMCC No.0828)对10%(重量百分比)的脱脂奶37℃发酵20-24小时后,发酵上清液对ACE的抑制作用可达35-42%。发酵乳均质后,经过喷雾干燥(进风温度165-170℃,出风温度70-72℃)获得粉状混合物,作为实验的样品。
取10g喷干粉用90ml蒸馏水溶解,充分搅拌后,4℃、4,000rmp离心20分钟,取上清液,经过分子截留极限为10,000道尔顿的膜过滤后,测定透过液对ACE的抑制作用与肽的浓度。结果表明,经过喷雾处理,发酵乳对ACE的抑制作用不受影响(透过液对ACE的抑制作用为38-41%),每克发酵乳喷干粉含有活性样品27mg。
1.2实验动物
自发性高血压大鼠(SHR),18周龄,雄性,体重318±33g,由中国科学院上海实验动物中心提供,动物合格证号:沪动合证字152号。
1.3实验方法
清醒大鼠血压测定采用SHR大鼠电子血压仪(北京中日友好医院生产)进行尾动脉间接测压法,将大鼠置于38℃温箱内加热15~20min,测定收缩压。
自发性高血压大鼠24只,随机分为三组:对照组给予蒸馏水,2.75g发酵乳喷干粉/Kg体重的高剂量组(相当于75mg活性肽/Kg体重)和0.55g发酵乳喷干粉/Kg体重的低剂量组(每克发酵乳喷干粉含有活性样品27mg,可换算成15mg活性肽/Kg体重),每组8只。大鼠在给药前一周开始测压,待血压稳定后开始实验,大鼠每天给药一次,连续十六天,并分别在给药的第1、5和16天测定大鼠血压。在预实验中,先测定大鼠给药前血压,然后灌胃给药,并分别测定2,4,6小时药物对大鼠的降压。结果显示药物的降压作用在4~6小时到达峰值。因此将实验中测压的时间定在给药前和给药后5~6小时进行。
1.4数据处理
实验数据用
x±SD表示,采用Student t test(t检验)进行显著性检验。
2、实验结果
在首次给药时,低剂量组和高剂量组,给药前大鼠的收缩压分别为192±20mmHg和195±7mmHg,给药后(5~6小时)下降至175±24mmHg和174±8mmHg,较给药前均明显下降(P<0.05或P<0.01),结果见表3。
表3 低、高剂量LC2W灌胃给药对自发性高血压大鼠血压的影响
实验分组 | 给药前血压(mmHg) | 给药后血压(mmHg) | ||
第1天 | 第5天 | 第16天 | ||
对照组低剂量组高剂量组 | 203±12192±20195±7 | 199±8175±24**174±8* | 198±8171±18*190±17 | 201±11181±18*190±19 |
注:*P<0.05,**P<0.01与给药前比较。
低剂量组大鼠在给药过程中,观察第一天、第五天、第十六天的大鼠血压,均显示本品有明显的降压作用(P<0.05)。而高剂量组大鼠给药期间观察第五天、第十六天的大鼠血压,未显示其降压作用(P>0.05)。
低剂量和高剂量组大鼠,测定第五天给药前的血压(相当于前一天给药后24小时)分别为195±18mmHg和194±13mmHg,与对照组大鼠的血压(203±7mmHg)差别无统计学意义(P>0.05)。同样,测定两组大鼠第十六天给药前的血压(相当于前一天给药后24小时)分别为200±14mmHg和202±16mmHg,与对照组大鼠的血压(206±9mmHg)差别亦无统计学意义(P>0.05),结果见表4。
表4 低、高剂量LC2W灌胃给药对自发性高血压大鼠血压的影响
实验分组 | 给药前血压(mmHg) | ||
第1天 | 第5天 | 第16天 | |
对照组低剂量组高剂量组 | 203±12192±20195±7 | 203±7195±18194±13 | 206±9200±14202±16 |
注:*P<0.05,**P<0.01与给药前比较。
另外,从表3和4还可看出,对照组大鼠在实验期间血压变化无统计学意义(P>0.05)。
3、结论
乳品活性成份LC2W对自发性高血压大鼠具有明显的降压作用。
应用实施例二 干酪乳杆菌LC2W(CGMCC NO.0828)菌体细胞对SHR的降血压作用
1、实验方法
1.1样品制备
将干酪乳杆菌LC2W(CGMCC No.0828)在MRS液体培养基中37℃培养获得的菌体细胞经过离心、无菌生理盐水洗涤以后,重新悬浮于10%(重量百分比)无菌的复原脱脂奶中,经过冷冻干燥后,获得菌体冻干粉。将样品分为两份:一份不再进行任何处理,为LC2W-VC样品(LC2W活菌含量5×1010cfu/g),直接用于SHR灌胃(LC2W-VC组);另一份样品经过100℃干热处理30分钟,称为LC2W-HKC样品(LC2W活菌含量<1×104cfu/g),再用于SHR灌胃(LC2W-HKC组)。在喂食前,样品均以蒸馏水配制成0.1g/ml悬液,临用时新鲜配制。另称取脱脂奶粉1.0g,加蒸馏水10ml,配制成0.1g/ml(对照组),临用时新鲜配制。
1.2实验动物
自发性高血压大鼠(SHR),17~18周龄,雄性,体重323±23g,由中国科学院上海实验动物中心提供,动物合格证号:沪动合证字152号。
1.3实验方法
清醒大鼠血压测定采用SHR大鼠电子血压仪(北京中日友好医院生产)进行尾动脉间接测压法,将大鼠置于38℃温箱内加热15~20min,测定收缩压。
自发性高血压大鼠30只,随机分为三组:对照组给予脱脂奶粉0.4g/Kg/d,LC2W-VC组和LC2W-HKC组剂量均为0.4g/Kg/d,每组10只。大鼠在给药前一周开始测压,待血压稳定后开始实验,大鼠每天给药一次,连续十五天,并分别在给药的第1、5、8、11和15天测定大鼠血压。在预实验中,先测定大鼠给药前血压,然后灌胃给药,并分别测定2,4,6小时药物对大鼠的降压。结果显示药物的降压作用在4小时左右到达峰值。因此将实验中测压的时间定在给药前和给药后4小时进行。
1.4数据处理
实验数据用
x±SD表示,采用t检验进行显著性检验。
2、实验结果
在首次给药时,LC2W-VC和LC2W-HKC组,给药前大鼠的收缩压分别为187±12mmHg和196±14mmHg,给药后(4小时)下降至175±12mmHg和175±8mmHg,较给药前均明显下降(P<0.01),见表5。
LC2W-VC和LC2W-HKC组大鼠在十五天的给药过程中,观察第四天、第八天、第十一天、第十五天的大鼠血压,均显示本品有显著的降压作用(P<0.01),结果见表5。
表5 LC2W-VC和LC2W-HKC灌胃给药对自发性高血压大鼠血压的影响
实验分组 | 给药前血压(mmHg) | 给药后血压(mmHg) | ||||
第1天 | 第4天 | 第8天 | 第11天 | 第15天 | ||
对照组LC2W-VC组LC2W-HKC组 | 200±18187±12196±14 | 195±16175±12**175±8** | 193±20171±12**178±10** | 188±14174±9**179±6** | 189±19173±11**171±8** | 194±16178±9**171±5** |
注:*P<0.05,**P<0.01,与对照组相比。
对于LC2W-VC和LC2W-HKC组大鼠,测定第四天给药前的血压(相当于前一天给药后24小时)分别为188±11mmHg和192±9mmHg,与对照组大鼠的血压(195±24mmHg)差别无统计学意义(P>0.05)。同样,测定两组大鼠第八天、第十一天、第十五天给药前血压(相当于前一天给药后24小时),与对照组大鼠的血压差别亦无统计学意义(P>0.05),结果见表6。
表6 LC2W-VC和LC2W-HKC灌胃给药对自发性高血压大鼠血压的影响
实验分组 | 给药前血压(mmHg) | ||||
第1天 | 第4天 | 第8天 | 第11天 | 第15天 | |
对照组LC2W-VC组LC2W-HKC组 | 200±18187±12196±14 | 195±24188±11192±9 | 190±16189±11191±7 | 190±16192±12192±6 | 195±17192±10192±8 |
注:*P<0.05,**P<0.01,与同一天给药前的对照组比较。
3、结论
表明干酪乳杆菌LC2W的菌体细胞及细胞组分对SHR大鼠均具有显著的降压作用。
应用实施例三 利用干酪乳杆菌LC2W制造具有降血压功能的乳酸菌饮料或活菌乳饮料
1、将原料乳(脱脂奶、鲜奶、复原奶等)经两次95℃、20分钟或140℃、2秒钟的高强度热处理后冷却至5℃,加入本发明的干酪乳杆菌LC2W(CGMCC NO.0828),使其浓度达到109cfu/ml,5℃冷藏保存即得到甜性活菌乳饮料。
2、将原料乳(脱脂奶、鲜奶、复原奶等)经两次95℃、20分钟或140℃、2秒钟的高强度热处理后冷却至37℃,按5%(体积百分比)的量接种本发明的干酪乳杆菌LC2W(CGMCC NO.0828),并在37℃培养至最终酸度为0.6-0.7%(以乳酸计),冷却至5℃并冷藏保存即得到发酵乳饮料。
Claims (2)
1、一种干酪乳杆菌(Lactobacillus casei)LC2W CGMCC NO.0828。
2、权利要求1所述的干酪乳杆菌LC2W(CGMCC NO.0828)在制备抗高血压产品中的应用。
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EP03817186A EP1642963B1 (en) | 2003-06-20 | 2003-08-25 | Lactobacillus casei lc2w strain and its use in antihypertensive aspect |
DK03817186T DK1642963T3 (da) | 2003-06-20 | 2003-08-25 | Lactobacillus casei-LC2W-stamme og dens anvendelse mod hypertension |
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CN102533868B (zh) * | 2011-12-07 | 2014-03-19 | 光明乳业股份有限公司 | 干酪乳杆菌LC2W在制备Alpha-糖苷酶抑制剂中的用途 |
CN106434786B (zh) * | 2015-08-21 | 2020-08-25 | 光明乳业股份有限公司 | 一种由干酪乳杆菌发酵制备胞外多糖的方法 |
CN105820989A (zh) * | 2016-06-15 | 2016-08-03 | 苏州健世星生物科技有限公司 | 一种直投式发酵剂益生乳酸菌的高密度培养方法 |
CN108517308B (zh) * | 2018-05-23 | 2021-06-04 | 光明乳业股份有限公司 | 一种微生态菌剂及其制备方法和应用 |
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