CN113430153B - 降血压的罗伊氏乳杆菌zjuids09及其应用 - Google Patents
降血压的罗伊氏乳杆菌zjuids09及其应用 Download PDFInfo
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Abstract
本发明涉及食品微生物技术领域,具体公开了一种罗伊氏乳杆菌(Lactobacillus reuteri)ZJUIDS09,其保藏号为CGMCC NO.22843。本发明还同时提供了罗伊氏乳杆菌(Lactobacillus reuteri)ZJUIDS09在制备具有降血压功能的产品中的应用。
Description
技术领域
本发明涉及食品微生物技术领域,具体涉及到具有降血压功能的罗伊氏乳杆菌ZJUIDS09及应用。
背景技术
目前,高血压病已经成为中国及世界人口的数量很多的代谢性疾病。高血压对人体健康有严重影响,会伴有心、脑、肾等器官的功能或器质性损害的临床综合征。高血压是最常见的慢性病,也是心脑血管病最主要的危险因素。血管紧张素转化酶(Angiotensinconverting enzyme,ACE)在血压方面起着重要作用,它可将血管紧张素I转化为具有收缩血管功能的血管紧张素II,它还可以水解具有血管舒张作用的缓激肽。发酵乳中的降压肽可以通过抑制ACE活性,来引起血管扩张反应,从而降低血压。发酵乳被推荐作为高血压的非药物治疗,主要是因为它基本无不良副作用。发酵乳的降压效果主要归因于乳蛋白中的降压肽,这些肽可以在特定乳酸菌发酵或胃肠消化过程中释放。
乳酸菌发酵是产生降压肽的重要途径,乳杆菌与人类生活密切相关,是广泛应用于食品发酵、工业乳酸发酵及医疗保健领域的有益微生物之一。罗伊氏乳杆菌作为乳杆菌的一种,将其添加到食品中,可以改善食品的口感、质地和风味。罗伊氏乳杆菌也能够定植于人体内发挥益生作用,如调节肠道菌群、抑制肠道病原菌生长、降低血压、增强机体免疫力、提高乳糖消化和抗肿瘤、抗氧化等。
CN110384719A的发明《可改善高血压的罗伊氏乳杆菌菌株GMNL-263及其组合物》公开了罗伊氏乳杆菌(Lactobacillus reuteri)GMNL 263,为热灭杀死菌,透过抑制一促发炎细胞激素IL 1β与提升一双歧杆菌属的生长,而具有降血压的功效。该方案体现的是罗伊氏乳杆菌菌株GMNL-263和双歧杆菌的组合物在人群中的应用效果,且,该方案中采用的是罗伊氏乳杆菌菌株GMNL-263灭活后的产品,通过抑制一促发炎细胞激素IL 1β与提升一双歧杆菌属来起作用,但是并没有证明GMNL-263活菌有很好的降压效果。
发明内容
本发明要解决的技术问题是提供一种降血压罗伊氏乳杆菌ZJUIDS09及其应用。
为了解决上述技术问题,本发明提供一种罗伊氏乳杆菌(Lactobacillusreuteri)ZJUIDS09,其保藏号为CGMCC NO.22843。
作为本发明的罗伊氏乳杆菌(Lactobacillus reuteri)ZJUIDS09的改进:其16SrDNA全序列为SEQ ID No:1所述。
本发明还同时提供了上述罗伊氏乳杆菌(Lactobacillus reuteri)ZJUIDS09在制备具有降血压功能的产品中的应用。
所述具有降血压功能的产品例如为具有降低血压功能的食品、药物、保健品及宠物食品。
具有降低血压功能的食品包括具有降低血压功能的发酵果蔬汁(例如为发酵南瓜火龙果汁)、具有降压功能的酸奶、具有降压功能的奶酪;
具有降低血压功能的饲料为具有降低血压功能的宠物食品(例如为宠物奶粉)。
作为本发明应用的改进:
具有降血压功能的药物包括具有降低血压功能的活菌制剂,该活菌制剂中罗伊氏乳杆菌(Lactobacillus reuteri)ZJUIDS09的活菌数为1.0×1010~1×1011CFU/g。
本发明的罗伊氏乳杆菌ZJUID S09单独或与其他菌株混合发酵可用于制备具有降低血压功能的青贮饲料。
本发明的菌株ZJUIDS09,保藏名称:罗伊氏乳杆菌Lactobacillus reuteri;保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:北京市朝阳区北辰西路1号院3号,保藏编号:CGMCC NO.22843,保藏时间2021年07月07日。
本发明筛选出一株罗伊氏乳杆菌(Lactobacillus reuteri)ZJUIDS09,通过细菌形态学、生理学和培养特征,结合16S rDNA测序等对该菌进行了鉴定。
本发明所提供的罗伊氏乳杆菌ZJUIDS09(Lactobacillus reuteri ZJUIDS09)的菌落形态学特征为:在MRS琼脂培养基上形成明显的菌落,菌落大小0.3~1.5mm。菌落圆形,边缘整齐,白色,表面湿润光滑。其菌体形态特性为:革兰氏染色呈阳性,不产芽孢,圆端直杆菌,单个、成对或短链状。
本发明所提供的罗伊氏乳杆菌ZJUIDS09(Lactobacillus reuteri ZJUIDS09)的ACE抑制率能力达到90.41%。其具有以下能力:
1、具有较强的ACE抑制率和IC50,即具有较强的降血压能力。
2、该菌还具有较优异的抗氧化能力、能耐受酸和胆盐,并且具有一定的增殖能力;对常见的抗生素敏感;具有抗菌活性;
即,能耐受胃肠道环境、无抗生素耐性、能抑制肠内有害的病原菌。
综上所述,本发明从传统发酵食品中分离获得的益生菌中筛选出来具有强降血压功能的罗伊氏乳杆菌ZJUIDS09。该菌株具有较强的ACE抑制率和IC50抑制率。该菌株在抗氧化、耐酸耐胆盐方面比其他乳酸菌有明显的优势,适合肠胃肠环境并具有增殖能力,体内消化后产品的ACE抑制率也很高。无抗生素耐性,具有抗菌活性。本发明乳杆菌可广泛用于开发降血压相关益生功能产品。
附图说明
图1为罗伊氏乳杆菌ZJUIDS09的菌落形态图。
图2为罗伊氏乳杆菌ZJUIDS09革兰氏染色的菌体形态图。
图3为罗伊氏乳杆菌ZJUIDS09的16S rDNA的电泳鉴定图;
注:M从上到下依次为5000、3000、2000、1000、750、500、250、100bp;泳道1和2均为罗伊氏乳杆菌ZJUIDS09的PCR图;
图4为罗伊氏乳杆菌ZJUIDS09对抗生素敏感性的平板示例;
图4中,1-10分别代表青霉素G、氯霉素、氨苄青霉素、庆大霉素、红霉素、环丙沙星、林可霉素、甲氧苄啶、克拉霉素、四环素。
图5为罗伊氏乳杆菌ZJUIDS09抗菌活性的平板示例;
图5中,从左至右依次为沙门、大肠、金葡和单增李斯特菌的抑菌圈。
图6为罗伊氏乳杆菌ZJUIDS09发酵不同酸奶的ACE抑制率图;
从左至右依次为水牛乳、水牛乳清、普通牛乳、普通乳清。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1、罗伊氏乳杆菌ZJUIDS09的筛选及鉴定:
1.乳酸菌的筛选
1.1样品来源
本发明所使用的菌株来源于云南地区农家自制的乳扇。样品共采集15份,样品采集并立即冷藏后邮寄到实验室进行菌株的分离纯化。
1.2菌株的分离纯化
取大约5g新鲜样品用无菌管收集,立即送至实验室进行菌株分离。取1g样品放入9mL的MRS液体培养基中,涡旋混匀后于37℃下富集培养48h;然后在超净台中吸取富集液1mL,用无菌生理盐水进行十倍梯度稀释,选取10-6、10-7、10-8三个稀释梯度,每个梯度取菌液100μL涂于MRS琼脂培养基上,于37℃培养48h。培养结束后,从琼脂培养基中选择生长有50~150个单菌落的平板,挑取典型菌落,在MRS琼脂平板上多次划线纯化,直至整个平板上的菌落形态一致,挑取单菌落到MRS液体培养基增菌培养。得到的菌株均在含40%甘油的MRS液体培养基中于-80℃冷冻保存。
所获得的一个遗传稳定性良好的菌株被命名为ZJUIDS09。
2.罗伊氏乳杆菌ZJUIDS09的鉴定
2.1菌落特征
罗伊氏乳杆菌ZJUIDS09在MRS琼脂培养基培养48h后,直径在0.3~1.5mm之间,菌落圆形,边缘整齐,白色,表面湿润光滑,见图1。
2.2显微镜下形态:
罗伊氏乳杆菌ZJUIDS09菌落涂片:革兰氏染色呈阳性,不产芽孢,圆端直杆菌,单个、成对或短链状,见图2。
2.316S rDNA鉴定
用Ezup柱式细菌基因组DNA抽提试剂盒提取目标菌株基因组DNA,将提取的乳酸菌基因组DNA作为PCR扩增的模板,采用细菌通用引物27F和1492R进行16S rDNA的PCR实验,PCR反应扩增结束后,取PCR产物进行琼脂糖凝胶检测拍照,扩增片段长度为1.5kb左右,见图3。将PCR产物送至华大基因(武汉)公司进行测序,结果如SEQ ID NO:1所示,在NCBI网站上进行BLAST序列比对,结果显示该序列与罗伊氏乳杆菌的16S rDNA序列同源性超过99%。
将菌株ZJUIDS09的序列比对结果和生理生化结果相结合,确定筛选的乳酸菌ZJUIDS09为罗伊氏乳杆菌(Lactobacillus reuteri)。
菌株ZJUID S09的保藏信息如下:保藏名称:罗伊氏乳杆菌Lactobacillusreuteri;保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:北京市朝阳区北辰西路1号院3号,保藏编号:CGMCC NO.22843,保藏时间2021年07月07日。
实施例2、罗伊氏乳杆菌ZJUIDS09降血压能力的确认
1.ACE抑制率
菌株发酵液的制备
将于甘油管中保存的菌株ZJUID S09先在MRS琼脂平板上划线活化2~3次,然后挑取单菌落在MRS液体培养基中37℃扩大培养18h,使菌液的浓度达到约108CFU/mL左右,作为培养液。将培养液以2%(体积%)的接种量接种到灭菌复原脱脂乳(11%,w/v)中,37℃培养48h后取出,4℃冰箱放置过夜。将发酵乳从冰箱取出,pH调至4.6后,8000g离心10min留上清液,再将pH调节至8.2后,8000g离心10min后,收集的上清液作为菌株ZJUID S09的发酵上清液。
所述灭菌复原脱脂乳(11%,w/v)为:将脱脂复原乳配制成11g/100ml的浓度,然后于121℃灭菌15分钟。
以FAPGG为血管紧张素Ⅰ的模拟底物,借助酶标仪测定菌株发酵上清的ACE抑制活性。测定步骤为:在96孔酶标板上按表1,利用酶标仪测定340nm处的测吸光A1(反应前),将酶标板上的反应物在37℃恒温下继续反应40min再次测量吸光度A2(反应后),平行实验3次,结果取平均值。计算ΔA(ΔA=A1-A2),以单位时间内吸光度值的变化表示ACE酶活性,同时以PBS缓冲液作阴性对照,以其他菌株作为对照菌株。
利用酶标仪测定340nm处吸光值的变化来代表ACE酶活性,抑制率可用下列公式表示:
公式中:ΔA为加入缓冲液时吸光度在40min内的变化;ΔB为加入抑制剂(发酵上清液)时吸光度在40min内的变化。平行实验3次,结果取平均值。
表1、ACE抑制率的测定试剂和添加量
IC50的测定
抑制活性达到的抑制剂浓度即为半抑制浓度,记为IC50。将菌株发酵乳冻干(即,将菌株ZJUID S09的发酵上清液于-40℃冻干24小时),加HEPES缓冲液(pH 8.3,0.08M)复配成5个浓度梯度,分别为0.3、0.5、6、15、21mg/mL,以此作为ACE抑制剂,采用如上方法测定各浓度梯度的ACE抑制率,并绘制标准曲线,然后利用Graph Pad Prism 8软件计算出样品的ACE抑制IC50值。
ACE抑制率达到50%时的抑制剂浓度为半抑制浓度,记为IC50。其余菌株的发酵液制备和ACE抑制率(%)和IC50的测定同上。
表2、菌株发酵上清液的ACE抑制率(%)和IC50
*代表差异显著,P<0.05
如表2所示,本发明筛得的罗伊氏乳杆菌ZJUIDS09在发酵上清液抑制率方面均显著优于其他菌株。罗伊氏乳杆菌ZJUIDS09的IC50显著低,表明罗伊氏乳杆菌ZJUIDS09发酵液在低浓度下(0.33g/L)就可以达到ACE抑制率50%。ACE抑制率和IC50的结果均表明罗伊氏乳杆菌ZJUIDS09发酵物具有明显的降血压功效。
罗伊氏乳杆菌(Lactobacillus reuteri)GMNL 263的发酵上清液的ACE抑制率显著低于ZJUIDS09。
2.模拟胃肠消化后的ACE抑制率
将发酵上清液(50mL)与人工胃液按2:1(v/v)混合,然后37℃恒温水浴振荡器中消化2h,沸水浴加热10min以终止反应,取样测定其ACE抑制率;将经过人工胃液消化后的剩余样品(50mL)与人工肠液按1:1(v/v)混合,然后37℃恒温水浴振荡器中消化2h,沸水浴加热10min以终止反应。取样测定ACE抑制活性,ACE抑制率测定方法同上。
人工胃液配制:取稀盐酸(浓度为2M的盐酸)1.6mL,分别加80mL水、胃蛋白酶1g,混合均匀后,用水稀释到100mL。
人工肠液配制:取磷酸二氢钾0.68g,加100mL水溶解,取0.1mol/L氢氧化钠溶液调节pH值至6.8,加入胰蛋白酶10g,混合均匀后,稀释至1 000mL。
表3、发酵上清液模拟消化后ACE抑制率(%)
abc代表差异显著,P<0.05
表3的结果表明,罗伊氏乳杆菌ZJUIDS09发酵上清液在经过人工模拟的胃液和肠液后还有76.39%的ACE抑制率,显著高于其他菌株的发酵液。表明罗伊氏乳杆菌ZJUIDS09发酵液可在人体的胃肠道发挥良好的降血压效果。
实施例3、罗伊氏乳杆菌ZJUIDS09的抗氧化能力的确认
将实施例2制备所得的培养液于4℃经8000r/min离心5min收集菌体,用磷酸盐缓冲液(pH 6.8)洗涤2次后,将菌体悬浮于MRS液体培养基中,使活菌数校正至约108CFU/mL,得菌悬液;
将菌悬液以及将实施例2制备而得的发酵上清液作为样品。同时以鼠李糖乳杆菌ATCC53103作为阳性对照菌株测定抗氧化活性。
1、总抗氧化能力(FRAP法)
总抗氧化能力的测定方法按照Giuberti等的方法稍作修改。向酶标板中加入150μL TPTZ工作液(0.3M醋酸-醋酸钠缓冲液、20mM氯化铁溶液、10mM TPTZ缓冲液,以V:V:V=10:1:1混合,现用现配)和20μL的样品,振荡混匀,37℃反应10min,测定溶液在593nm处的吸光度。将样品测得的吸光度代入硫酸亚铁标准曲线,样品抗氧化能力以硫酸亚铁当量表示(μmol FeSO4/mL样品)。每个样品重复3次,求平均值。
硫酸亚铁标准曲线:配制不同质量浓度(0μM、50μM、100μM、200μM、400μM、600μM、800μM)的硫酸亚铁溶液,将不同摩尔浓度的硫酸亚铁溶液、10mM TPTZ缓冲液、0.3M醋酸盐缓冲液以V:V:V=1:1:10混合,取170μL混合液加入酶标板,37℃反应10min,测定溶液在593nm处的吸光度。以吸光度为纵坐标,硫酸亚铁质量浓度为横坐标绘制标准曲线。
2、还原能力
还原能力的测定参考Lin等的方法并做一些修改。取1mL样品于离心管中,加入0.2M、pH6.6的PBS缓冲液及1%(w/v)铁***溶液各1mL,混匀。50℃水浴20min,冰浴冷却。再加入10%三氯乙酸1mL,6000r/min离心5min,取上清液1mL,加入0.1%(w/v)三氯化铁1mL、蒸馏水1mL,混匀,静置反应10min,于700nm处测定吸光度。用PBS缓冲液或MRS肉汤培养基代替样品为空白组。每个样品重复3次,求平均值。
还原能力(%)=[(As-Ab)/Ab]*100
式中:As——样品组吸光度;
Ab——空白组吸光度。
3、DPPH自由基清除能力
DPPH自由基清除能力的测定方法参考Shimada等的方法并作一些修改。配制VC标准溶液1000mg/ml,稀释成不同浓度梯度(0-30μg/ml)。向酶标板中加入100μL待测样品(或VC标准溶液)和100μL 0.2mM DPPH乙醇溶液(无水乙醇配制,4℃避光保存,现配现用),摇匀后于室温下避光30min,测定溶液在517nm处的吸光度;用100μL无水乙醇代替100μLDPPH乙醇溶液为空白组;用100μL PBS缓冲液(或MRS肉汤培养基)代替100μL待测样品为对照组,并以100μL PBS缓冲液(或MRS肉汤培养基)和无水乙醇混合液空白调零。每个样品重复3次,求平均值。
DPPH自由基清除能力(%)=[1-(As-Ab)/Ac]*100
式中:As——样品组吸光度;
Ab——空白组吸光度;
Ac——对照组吸光度;
所得结构如表4所示。
表4、罗伊氏乳杆菌ZJUIDS09的抗氧化活力
*代表差异显著,P<0.05;**代表差异显著,P<0.01。
本发明筛得的罗伊氏乳杆菌ZJUIDS09发酵上清液的总抗氧化能力、还原能力以及DPPH自由基均显著高于标准菌株鼠李糖乳杆菌ATCC53103。从而说明罗伊氏乳杆菌ZJUIDS09发酵上清液和菌悬液具有高效的抗氧化能力。
罗伊氏乳杆菌(Lactobacillus reuteri)GMNL 263菌悬液和发酵上清液的总抗氧化能力均远不如鼠李糖乳杆菌ATCC53103。
实施例4、罗伊氏乳杆菌ZJUIDS09的耐酸性及耐胆盐性的确认
1.耐酸实验
将实施例2制备所得的培养液于4℃经8000r/min离心5min收集菌体,用磷酸盐缓冲液(pH 6.8)洗涤2次后,将菌体悬浮于事先调节pH至3.0的MRS液体培养基中,使活菌数校正至约108CFU/mL,得耐酸实验用菌悬液;
将耐酸实验用菌悬液直接(37℃下培养0h)采用倾注平板法(37℃培养48h)进行菌落计数,所得记为N0;
将耐酸实验用菌悬液先于37℃下培养3h,然后再采用倾注平板法(37℃培养48h)进行菌落计数,所得记为Nt;
耐酸存活率计算公式如下:
上式中,N0为测试菌株0h的活菌数(CFU/mL);Nt为测试菌株3h的活菌数(CFU/mL)。
2、耐胆盐实验
将菌悬液(实施例3所得)以10%的量接种到含0.3%(m/v)牛胆盐的MRS液体培养基(对照为不含牛胆盐的MRS液体培养基),于37℃培养3h。然后采用倾注平板法对样品中的活菌数进行计数。倾注后的平板于37℃培养48h。菌株的胆盐耐受能力以3h时每毫升含胆盐培养基中活菌数与不含胆盐培养基中活菌数的差值的对数来表示(log CFU/mL)。
3、采用不同菌株进行上述耐酸、耐胆盐性能测定。
4、如表4所示,罗伊氏乳杆菌ZJUIDS09的耐酸耐胆盐性能明显优于对照菌株。其在pH为3.0的MRS培养基中的存活率高达98%。在含有0.3%的牛胆盐的环境中活菌数仍达到106CFU/mL以上,说明其具有较好的胆盐耐受性。实验证明,罗伊氏乳杆菌ZJUIDS09具备较高的胃肠道生存能力。
表5、菌株对酸和胆盐耐受性结果
5、益生菌必须能够耐受胃肠道中胃酸和胆汁等一系列不良环境而活着才能发挥其益生作用。本发明提供的罗伊氏乳杆菌ZJUIDS09在pH 3.0的条件下能够生长增殖,其能顺利通过胃中的酸性环境到达小肠。同时,罗伊氏乳杆菌ZJUIDS09能耐受胆盐,其能在肠道内存活,进而能有效地发挥降血压作用。
罗伊氏乳杆菌(Lactobacillus reuteri)GMNL 263胆盐耐受性远远不如本发明的罗伊氏乳杆菌ZJUIDS09。
实施例5、罗伊氏乳杆菌ZJUIDS09疏水能力的确认
1.疏水性的测量
用干净的PBS缓冲液(0.2M,pH6.8)先洗涤罗伊氏乳杆菌ZJUIDS09菌体(实施例3步骤1制备所得菌体)沉淀两次,再重悬使OD610的吸光度约为0.5左右,得罗伊氏乳杆菌ZJUIDS09菌悬液;其他菌的菌悬液制备同上。
取2ml罗伊氏乳杆菌ZJUIDS09悬液和2ml二甲苯彻底混合,37℃水浴充分震荡5min后,并于0h、2h后分别测量水相的OD610吸光值。
A0=0h的吸光值,At=th的吸光值。
所得结果如下表所示。
表6、不同菌株的表面疏水性(%)
2.结果分析
经测量,罗伊氏乳杆菌ZJUIDS09的疏水性在51.43%,显著高于其他菌株。该表明该菌株具有较强的粘附能力,可粘附到人的肠道,改善肠道菌群的健康。
实施例6、罗伊氏乳杆菌ZJUIDS09的抗生素感受性的确认
将浓度约为108CFU/mL罗伊氏乳杆菌ZJUIDS09菌悬液按1%的量加入冷却至45℃左右的灭过菌的MRS琼脂培养基中,充分混合,定量加入15mL/皿。待凝固后,用镊子取药敏纸片放于培养基上。将培养皿正面朝上放于37℃培养箱中培养24h。以无抗生素的纸片为空白对照。测量抑菌圈直径。每个重复三次。
罗伊氏乳杆菌ZJUIDS09对抗生素感受性抑菌圈直径如表7所示。参考CLSI(2017)药敏试验标准可得,罗伊氏乳杆菌ZJUIDS09对青霉素、克拉霉素、四环素、红霉素、林可霉素和氯霉素呈现敏感性。对于庆大霉素呈现中介。实验结果表明,罗伊氏乳杆菌ZJUIDS09对常见的抗生素敏感。
表7、罗伊氏乳杆菌ZJUIDS09对抗生素敏感性结果
注:S,敏感;I,中介;R,耐药
随着抗生素在临床治疗的广泛应用,乳酸菌的耐药性也越来越严重,长期摄入耐药性的乳酸菌会给临床治疗带来很大的困难。本发明所提供的罗伊氏乳杆菌ZJUIDS09对常见的抗生素敏感,不会对人体健康造成危害。
实施例7、罗伊氏乳杆菌ZJUIDS09的病原菌抑制能力的确认
采用国际通用琼脂扩散法测定乳酸菌抗菌活性。将10mL LB琼脂培养基倒入无菌培养皿中,冷却后做为下层培养基。将菌浓度约为107CFU/mL指示菌菌悬液按1%的量加入冷却至45℃左右的灭过菌的LB琼脂培养基中,充分混合,定量加入10ml/皿。在上面摆放灭过菌的牛津杯。待上层培养基冷凝后,轻轻拔掉牛津杯。定量以100μL/孔加入罗伊氏乳杆菌ZJUIDS09发酵上清液样品,并且以磷酸盐缓冲液(0.2M,pH 6.8)作为对照。挑选出小孔周围有明显抑菌圈的菌株,测量抑菌圈直径,每个重复三次。
如表8所示,罗伊氏乳杆菌ZJUIDS09的代谢产物对金黄色葡萄球菌、大肠杆菌和肠炎沙门氏菌均有一定的抑制作用,并优于对照林可霉素的抑菌效果。可见这株菌的代谢产物具有抑菌特性。
表8、菌株对病原菌抑制能力的结果
金黄色葡萄球菌是人类化脓感染中最常见的病原菌,大肠杆菌可引起严重腹泻和败血症,沙门氏菌种也会引起人类食物中毒。本发明的罗伊氏乳杆菌ZJUIDS09代谢产生的细菌素、有机酸、过氧化氢等抑菌物质均能单独或共同地抑制这些致病菌的生长。本发明所提供的罗伊氏乳杆菌ZJUIDS09的代谢产物对金黄色葡萄球菌、大肠杆菌和单增李斯特的抑菌效果优于对照,表明其对这三种菌的抑菌效果明显,这对其维持肠道微生态平衡发挥重要作用,并起到健康促进效果。
实施例8、利用罗伊氏乳杆菌ZJUIDS09制备功能性发酵酸奶和乳清
(1)原料:2L全脂UHT灭菌乳或鲜牛乳、乳清、水牛乳、水牛乳清;
(2)预热:放入容器中加热至63℃;
(3)均质、杀菌:倒入均质机中均质,压力为15-25MPa,混合液倒入铁罐中,同时加入100g白糖,于90℃水浴环境杀菌10min;
(4)冷却:杀菌后的牛乳,冷却至40-50℃备用;
(5)制备发酵剂:罗伊氏乳杆菌ZJUIDS09菌种在无菌环境下,接种于装有灭菌脱脂乳(12%,w/v)的试管中,于37℃条件下培养20小时。每次传代接种量为2-4%(v/v),传代2~3次以恢复活力,放置于4℃冰箱中保存;
(6)接种、发酵:在无菌条件下,牛乳或乳清中接入活化好的罗伊氏乳杆菌ZJUIDS09,接种量为2-4%(v/v);在42℃条件下恒温发酵6-10h;
(7)后熟:发酵结束后,放入4℃冰箱中后熟12-24h;
(8)灌装、冷藏:后熟完成后,灌装到250mL的灭菌玻璃瓶中,送至冷库冷藏。
制备好的酸奶或酸奶乳清按照实施例2所述方法测定ACE抑制率,结果见图6。图6的结果显示罗伊氏乳杆菌ZJUIDS09制备的普通牛乳和水牛乳均有近90%的ACE抑制率,而乳清也有35%以上的ACE抑制率,表明罗伊氏乳杆菌ZJUIDS09适合用于降压酸奶的制备。
实施例9、利用罗伊氏乳杆菌ZJUIDS09制备功能性发酵果蔬汁
(1)原料:选取新鲜的南瓜和火龙果;
(2)清洗切块:清洗、去皮(南瓜去瓤)、切成小块;
(3)闪蒸:采用闪蒸的方法灭酶,121℃处理0.5~1min,迅速排气;
(4)打浆:按照南瓜∶水(重量比)=1∶1的比例,将南瓜和水适量的逐渐放入胶体磨中研磨,进行粗磨和细磨各一次;将火龙果用打浆机打浆至果肉均匀无块状;
(5)调配、均质:按南瓜汁15%,火龙果汁30%,3%蔗糖,余量为水;加入0.2%的稳定剂CMC混合均匀,采用两段均质法,先低压(15MPa),后高压(25MPa),使瓜肉颗粒直径粒径为2~3μm。
(6)灭菌、冷却:调配好的复合果蔬汁在100℃下保温10min,冷却至40℃左右。
(7)接种、发酵:在无菌条件下,接入活化好的罗伊氏乳杆菌ZJUIDS09,初始菌数控制在107CFU/mL。在37℃条件下恒温发酵24h。
(8)后熟:发酵结束后,放入4℃冰箱中3h。
(9)灌装、冷藏:后熟完成后,灌装到250mL的灭菌玻璃瓶中,送至冷库冷藏。
实施例10、利用罗伊氏乳杆菌ZJUIDS09制备功能性发酵酸肉
(1)切片:将市售新鲜五花肉,切成3cm见方薄片。
(2)拌糯米粉:1000g原料肉与250g糯米粉混匀,加入葡萄糖1%。
(3)接种、发酵:在无菌条件下,接入活化好的罗伊氏乳杆菌ZJUIDS09,初始菌数控制在107CFU/mL。在37℃条件下恒温发酵18h。
(4)腌制:加入2%的盐,于25℃下腌制20d。
实施例11、利用罗伊氏乳杆菌ZJUIDS09制备降血压菌粉
1.罗伊氏乳杆菌ZJUIDS09菌泥的制备
挑取罗伊氏乳杆菌ZJUIDS09单菌落接种于50mL MRS液体培养基中,放置37℃培养箱中培养18h。再按5%的接种量于250mL MRS液体培养基中活化,放置37℃培养箱中培养24h。最后将活化好的罗伊氏乳杆菌ZJUIDS09以5%接种量于10L发酵罐中进行高密度培养,于37℃、pH 6.8的条件下培养18h。之后在8000r/min、4℃条件下离心15min,弃去上清液,收集菌体沉淀,用无菌磷酸盐缓冲液(pH 6.8)漂洗菌体2次。即可得到罗伊氏乳杆菌ZJUIDS09菌泥。
2.保护剂的制备
冻干保护剂含有15%的脱脂乳粉,5%的海藻糖,3%的谷氨酸钠,1%的甘油,0.5%的半胱氨酸盐酸盐。水作为溶剂。于110℃灭菌备用。
3.罗伊氏乳杆菌ZJUIDS09菌粉的制备
将上述制备的罗伊氏乳杆菌ZJUIDS09菌体沉淀按1:5的比例与保护剂溶液充分混匀。于-40℃条件下预冻5h,使其均匀冻结在容器内壁上,然后进行真空冷冻干燥,干燥18~20h后,即可得到罗伊氏乳杆菌ZJUIDS09菌粉。用生理盐水复水后,洗涤两次,测得罗伊氏乳杆菌ZJUIDS09菌粉中活菌数为1.0×1010~1×1011CFU/g。
实施例12、利用罗伊氏乳杆菌ZJUIDS09制备宠物益生菌奶粉
1.罗伊氏乳杆菌ZJUIDS09菌粉的制备
参照实施例11制备罗伊氏乳杆菌ZJUIDS09菌粉冻干菌粉,菌粉中活菌数为1.0×1010-1×1011CFU/g。
2.宠物配方粉的制备
原料初选:奶粉、鱼粉、骨粉、谷物、植物油,添加剂:维生素、微量元素、功能因子、其他;
自动配料:按照配方将得到的物料原料投入物料仓;
粉碎:将称量好的物料通过粉碎机粉碎;
混合:粉碎的物料再添加植物油和微量元素,加入到混合机混合均匀;
膨化:混合好的物料通过膨化机制成颗粒物料;
烘干:混合好的物料通过烘干机烘干,温度控制在65-70度;
分级筛选:将物流通过分级筛,颗粒控制在2.5-5毫米;
3.宠物益生菌配方粉的制备
将1制备的菌粉和2制备的宠物饲料按照1:1000的比例混合均匀,最终产品中的出厂的活菌在108CFU/g以上。产品灌装后,入库保存销售。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
序列表
<110> 浙江大学
<120> 降血压的罗伊氏乳杆菌ZJUIDS09及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1402
<212> DNA
<213> 罗伊氏乳杆菌(Lactobacillus reuteri)
<400> 1
tggctcagga tgaacgccgg cggtgtgcct aatacatgca agtcgtacgc actggcccaa 60
ctgattgatg gtgcttgcac ctgattgacg atggatcacc agtgagtggc ggacgggtga 120
gtaacacgta ggtaacctgc cccggagcgg gggataacat ttggaaacag atgctaatac 180
cgcataacaa caaaagccac atggcttttg cttgaaagat ggctttggct atcactctgg 240
gatggacctg cggtgcatta gctagttggt aaggtaacgg cttaccaagg cgatgatgca 300
tagccgagtt gagagactga tcggccacaa tggaactgag acacggtcca tactcctacg 360
ggaggcagca gtagggaatc ttccacaatg ggcgcaagcc tgatggagca acaccgcgtg 420
agtgaagaag ggtttcggct cgtaaagctc tgttgttgga gaagaacgtg cgtgagagta 480
actgttcacg cagtgacggt atccaaccag aaagtcacgg ctaactacgt gccagcagcc 540
gcggtaatac gtaggtggca agcgttatcc ggatttattg ggcgtaaagc gagcgcaggc 600
ggttgcttag gtctgatgtg aaagccttcg gcttaaccga agaagtgcat cggaaaccgg 660
gcgacttgag tgcagaagag gacagtggaa ctccatgtgt agcggtggaa tgcgtagata 720
tatggaagaa caccagtggc gaaggcggct gtctggtctg caactgacgc tgaggctcga 780
aagcatgggt agcgaacagg attagatacc ctggtagtcc atgccgtaaa cgatgagtgc 840
taggtgttgg agggtttccg cccttcagtg ccggagctaa cgcattaagc actccgcctg 900
gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 960
agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atcttgcgct 1020
aaccttagag ataaggcgtt cccttcgggg acgcaatgac aggtggtgca tggtcgtcgt 1080
cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgttactagt 1140
tgccagcatt aagttgggca ctctagtgag actgccggtg acaaaccgga ggaaggtggg 1200
gacgacgtca gatcatcatg ccccttatga cctgggctac acacgtgcta caatggacgg 1260
tacaacgagt cgcaagctcg cgagagtaag ctaatctctt aagccgttct cagttcggac 1320
tgtaggctgc aactcgccta cacgaagtcg gaatcgctag taatcgcgga tcagcatgcc 1380
gcggtgaata cgttcccggg cc 1402
Claims (4)
1.罗伊氏乳杆菌(Lactobacillus reuteri)ZJUIDS09,其特征在于:保藏号为CGMCCNO.22843。
2.根据权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)ZJUIDS09,其特征在于: 16S rDNA全序列为SEQ ID No:1所述。
3. 如权利要求1或2所述的罗伊氏乳杆菌(Lactobacillus reuteri)ZJUIDS09在制备具有降血压功能的药物中的应用。
4.根据权利要求3所述的应用,其特征在于:
具有降血压功能的药物包括具有降低血压功能的活菌制剂,该活菌制剂中罗伊氏乳杆菌(Lactobacillus reuteri)ZJUIDS09的活菌数为1.0×1010~1.0×1011 CFU/g。
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