CN112877241B - 一株人源性发酵乳杆菌zjuids06及其应用 - Google Patents
一株人源性发酵乳杆菌zjuids06及其应用 Download PDFInfo
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- CN112877241B CN112877241B CN202110145017.6A CN202110145017A CN112877241B CN 112877241 B CN112877241 B CN 112877241B CN 202110145017 A CN202110145017 A CN 202110145017A CN 112877241 B CN112877241 B CN 112877241B
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Abstract
本发明提供一株人源性发酵乳杆菌ZJUIDS06及其应用。分类命名为发酵乳杆菌Lactobacillus fermentum,保藏编号为CGMCC NO.21339。本发明菌株具有较好的体内降血脂能力的功能性乳酸菌,在高脂血症金黄地鼠模型的八周灌胃试验中能使血清总胆固醇、甘油三酯、低密度脂蛋白胆固醇均出现显著性降低。本发明菌株还具有较强的抗氧化、降胆固醇功效,具有良好的耐酸和胆盐性、培养液无抗生素耐性、抑制肠内有害的病原菌,证明该菌株具有良好的益生特性。在生产发酵蛋酸奶及蒙特里杰克干酪中应用时均能显著降低产品中的胆固醇含量,可制备降血脂、降胆固醇及抗氧化等功能益生产品。
Description
技术领域
本发明属于食品微生物技术领域,涉及一株人源性发酵乳杆菌ZJUIDS06及其应用,所述人发酵乳杆菌具有降血脂、降胆固醇、抗氧化等功能,且具有良好的耐酸耐胆盐等益生功能及安全性。
背景技术
目前,心血管疾病逐渐成为世界人口尤其是发展中国家致死人数最多的疾病类群。高血脂作为“三高”之一,即血清胆固醇及甘油三酯的升高,与高血压、动脉粥样硬化等心血管疾病存在显著相关性。因此,降血脂成为“三高”患者亟待解决的问题。迄今为止,已有大量实验证实了服用某些益生菌及其制品的人群体内血脂含量能发生显著性降低,缓解心血管疾病的发病风险,且相比于药品而言,可食用益生菌有更好的安全性,因此开发可食用的功能益生菌产品具有良好的市场潜力和社会效益。乳酸菌作为人体肠道中重要的益生菌之一,能平衡人体肠道微生态,加强免疫系统功能,且现在已被广泛应用于食品发酵、工业乳酸发酵及医疗保健领域。发酵乳杆菌作为乳酸菌的一种,在食品中添加后可以起到改善食品性状、风味和赋予其益生性的作用。发酵乳杆菌也能够存活于人体后肠段发挥其益生性作用,如平衡肠道菌群、抑制肠道病原菌生长、降血脂、增强机体免疫力、提高乳糖消化和抗肿瘤、抗氧化等。
发明内容
本发明的目的是提供一株人源性发酵乳杆菌ZJUIDS06,所述发酵乳杆菌ZJUIDS06的分类命名为:发酵乳杆菌Lactobacillus fermentum,已于2020年12月09日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为:CGMCC NO.21339。本发明所提供的发酵乳杆菌ZJUIDS06(Lactobacillus fermentum)的16s rDNA全序列如SEQ ID No.1所示。
本发明从健康婴儿的粪便中筛选出一株发酵乳杆菌ZJUIDS06(Lactobacillusfermentum),通过细菌形态学、生理学和培养特征,结合API 50CH鉴定试剂条(法国梅里埃)和16s rDNA测序等对该菌进行了鉴定。
本发明所提供的发酵乳杆菌ZJUIDS06(Lactobacillus fermentum)的菌落形态学特征为:在MRS固体培养基上形成明显的菌落,菌落大小0.2~1.4mm。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。
本发明的另一个目的是提供所述人源性发酵乳杆菌ZJUIDS06在制备功能性产品中的应用,所述功能包括降血脂、降胆固醇、抗氧化性,且菌株具有耐酸、耐胆盐、表面疏水性、自凝集性、抗菌等益生特性及良好的安全性能。所述产品包括食品、保健品及宠物饲料。
所述产品具体包括低胆固醇功能性蒙特里杰克干酪、功能性发酵果蔬汁、低胆固醇功能性发酵蛋乳酸奶、具有降血脂功能的活菌制剂、功能性宠物饲料等。
本发明提供的人源性发酵乳杆菌ZJUIDS06具有良好的益生特性,能耐受胃肠道环境、培养液无抗生素耐性、抑制肠内有害的病原菌,具有较高的体外胆盐水解酶活性、疏水性和自凝集性。
本发明所提供的一种人源性高效降血脂发酵乳杆菌ZJUIDS06(Lactobacillusfermentum ZJUIDS06)。其冻干菌粉经高脂血症金黄地鼠模型的八周灌胃试验证明,能使血清总胆固醇、甘油三酯、低密度脂蛋白胆固醇均出现显著性降低,分别降低了18.6%、46.6%和44.8%。
本发明提供的一株具有降血脂、降胆固醇、抗氧化功能的发酵乳杆菌ZJUIDS06,是一株具有较好的体内降血脂能力的功能性乳酸菌,在高脂血症金黄地鼠模型的八周灌胃试验中能使血清总胆固醇、甘油三酯、低密度脂蛋白胆固醇均出现显著性降低,分别降低了18.6%、46.6%和44.8%。此外,本发明菌株具有较强的抗氧化、降胆固醇功效,同时具有良好的耐酸和胆盐性、培养液无抗生素耐性、抑制肠内有害的病原菌,证明该菌株具有良好的益生特性。在生产发酵蛋酸奶及蒙特里杰克干酪中应用时均能显著降低产品中的胆固醇含量,因而发酵乳杆菌ZJUIDS06可制备降血脂、降胆固醇和抗氧化功能的产品。
附图说明
图1为发酵乳杆菌ZJUIDS06的菌落形态图。
图2为发酵乳杆菌ZJUIDS06革兰氏染色的菌体形态图。
图3为发酵乳杆菌ZJUIDS06的16s rDNA的电泳鉴定图。图中1和2均为本发明菌株,M为maker。
图4为第二周和第八周的金黄地鼠血脂四项指标。
图5为金黄地鼠盲肠内容物门水平属水平相关微生物相对丰度。
图6为发酵乳杆菌ZJUIDS06体外疏水性和自凝集性。
图7为发酵乳杆菌ZJUIDS06体外胆盐水解酶活性。
图8为发酵乳杆菌ZJUIDS06制作的蒙特里杰克干酪中胆固醇含量测定气相图。
具体实施方式
本发明结合附图和实施例作进一步说明。
实施例1 发酵乳杆菌ZJUIDS06的筛选及鉴定
1.发酵乳杆菌ZJUIDS06的筛选
1.1 样品来源
本发明所使用的菌株是从母乳喂养健康婴儿(3~6月)粪便样品中分离获得。粪便样品共采集20份,其中所有采样对象在被采样前的至少一个月时间内未服用任何含有活菌的发酵食品和益生菌制剂、未出现腹泻等胃肠道疾病、未服用任何种类的抗生素类药物。
1.2 菌株的分离纯化
取约5g新鲜粪便样品于无菌管中收集,立即送至实验室进行菌株分离纯化。分离时取1g粪便样品放入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培养液1mL,用无菌生理盐水进行十倍梯度稀释,选取106、107、108三个稀释梯度,每个稀释梯度取菌悬液100μL涂于MRS固体培养基上,于37℃培养48h。培养结束后,从MRS固体培养基中选择生长有30~300个单菌落的平板,挑取典型菌落,在MRS琼脂平板上多次划线分离,直至整个平板上的菌落形态一致,挑取单菌落到MRS液体培养基中扩大培养。得到的菌株在含有40%(w/v)甘油的MRS液体培养基的-80℃冰箱终冷冻保存。
2.发酵乳杆菌ZJUIDS06的鉴定
2.1 菌落特征
发酵乳杆菌ZJUIDS06在MRS固体培养基培养48h后,直径在0.2~1.4mm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:
发酵乳杆菌ZJUIDS06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。
2.3 益生菌API鉴定结果
采用API 50CH(法国梅里埃公司)生理生化试纸鉴定,结果表明该菌株为发酵乳杆菌。
表1发酵乳杆菌ZJUIDS06生理生化特性
注:+表示反应为阳性,-表示反应为阴性。
2.4 16s 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,扩增片段长度约为1.5kb,见图3。将PCR产物送至北京六合华大基因(武汉)有限公司。经测序,其序列如SEQ ID NO.1所示,在NCBI网站上进行BLAST序列比对,结果显示该序列与发酵乳杆菌的16s rDNA序列同源性超过99%。将菌株ZJUIDS06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ZJUIDS06为发酵乳杆菌(Lactobacillus fermentum)。被中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏日:2020年12月09日,保藏编号为CGMCC NO.21339。
实施例2 发酵乳杆菌ZJUIDS06金黄地鼠体内降血脂能力验证
1.1 灌胃菌悬液的制备
菌株扩培后添加保护剂冻干后等到菌株冻干粉,保存在-20℃下。动物试验中每天灌胃前将冻干菌粉悬浮在生理盐水中,灌胃给药的悬浮液中的活细胞数约为109cfu/mL。每只金黄地鼠每天每100g体重给予109cfu的益生菌。试验期每周在MRS琼脂平板对冻干菌粉的每克活菌数进行计数校正。
1.2 金黄地鼠高脂血症建模
在本试验中,将胆固醇(0.1%)和猪油(10%)添加到鼠类标准繁育日粮(中国上海普路腾生物科技有限公司)中,制备高胆固醇饮食。以雄性金黄地鼠(北京维通利华实验动物科技有限公司)为实验动物,采用高胆固醇饲料,建立模拟人的混合高脂血症的混合型高脂血症模型。动物福利和实验程序按照浙江大学实验动物管理条例的指导方针进行。
1.3 金黄地鼠试验设计
动物运输到达后,共有24只雄性金黄地鼠按标准饮食喂养1周以适应新环境。环境适应期后,进行为期1周的换料期,16只金黄地鼠改喂高胆固醇饲料,其余8只金黄地鼠仍旧采用标准饲料喂养(NC组),直至试验结束。换料期结束后开始正式试验,将16只饲喂高胆固醇饲料的地鼠按体重随机分为2组:高胆固醇阳性对照组(HC组)、已授权专利菌株(专利号ZL201711410714.X)植物乳杆菌ZY08(ZY08组)和发酵乳杆菌ZJUIDS06干预组(ZJUIDS06组)。在试验期间,NC组和HC组的金黄地鼠每100g体重每天灌胃1mL生理盐水。ZY08组和ZJUIDS06组每100g体重每天分别灌胃1mL植物乳杆菌ZY08混悬液和发酵乳杆菌ZJUIDS06混悬液(活菌浓度均达到109cfu/mL)。每周测量体重和采食量,每2周用直径为0.5mm的平底毛细管进行眼眶下静脉丛采血测定血脂。将血液在4℃过夜,3000×g离心10min后,采集上层血清进行血脂分析。血清中的总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)和甘油三酯(TG)的含量由自动生化分析仪(日本东京日立公司#3100)测定。在8周的益生菌干预期后,所有金黄地鼠都进行颈椎脱臼处死及解剖,采集血液、肝脏、肾脏、肠道样本,可称量的组织经PBS漂洗后吸干水分称重,液氮速冻并在-80℃下冷冻保存直至分析。全部试验数据分析时参数检验均采用one-way ANOVA和Tukey test,非参数检验采用Kruskal-Wallis检验。
2.1 体重及采食量
每周记录每只金黄仓鼠的体重,并计算出每组的平均值,喂养八周后的体重结果见表2,在喂食前和干预早期,各组之间的体重平均值无显著差异。在实验开始8周,饲喂高胆固醇饮食的阳性对照组(HC)的体重平均值显著高于饲喂标准日粮的阴性对照组(NC)。益生菌组(ZY08和ZJUIDS06)的体重有所下降,表明具有一定的减轻体重效果。
表2.体重及采食量
指标 | NC | HC | ZY08 | ZJUIDS06 |
采食量/g(8w) | 8.05±1.51 | 8.87±1.32 | 8.42±1.53 | 8.08±2.32 |
体重/g(8w) | 162.22±17.83<sup>a</sup> | 193.11±13.11<sup>b</sup> | 182.71±11.37<sup>ab</sup> | 181.89±9.29<sup>ab</sup> |
w:周;a,b:P-value<0.05
2.2 血脂四项
每两周测定一次血清总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)和甘油三酯(TG)。结果如图4所示。高胆固醇饲料喂养金黄地鼠两周后血清TC和LDL-C水平升高,表明添加0.1%胆固醇和10%猪油可适用于金黄地鼠高胆固醇血症的建模。8周后,各组TC、TG、LDL-C和HDL-C含量总体上有显著差异(图4,Tukey test)。发酵乳杆菌ZJUIDS06灌胃高胆固醇血症的金黄地鼠能使TC水平降低18.6%,TG水平降低46.6%,LDL-C水平降低44.8%,均达到显著水平。而植物乳杆菌ZY08能一定程度上降低TC、TG和LDL-C,但相比于阳性对照组(HC)而言并未达到显著水平。说明发酵乳杆菌ZJUIDS06具有显著的降血脂功效。
2.3 肠道菌群
对金黄地鼠的盲肠内容物中的细菌16srRNA(V3-V4区域)进行基于MiSeq的测序分析。结果如图5所示。Firmicutes和Bacteroidetes是盲肠的优势菌门。高胆固醇饮食喂养的金黄地鼠盲肠微生物群的特征是Firmicutes和Bacteroidetes的比例(F/B ratio)增加。值得注意的是,在属的水平上,发酵乳杆菌ZJUID06干预增加了Parabacteroides和Flavonifractor相对丰度,植物乳杆菌ZY08增加了Faecalibaculum、Ruminococcus和Desulfovibrio相对丰度。而根据报道显示,Parabacteroides是人体核心菌群之一,一些菌株能在体外产生乙酸、丙酸、异丁酸和异戊酸等短链脂肪酸,在单菌饲喂的肥胖模型小鼠的体内试验中能通过宿主互作调节糖脂代谢的复杂机制,可以显著改善肥胖模型小鼠的肥胖、胰岛素抵抗、脂代谢紊乱及非酒精性脂肪肝症状。说明发酵乳杆菌ZJUID06能够调节肠道平衡,改善肠道益生菌的数量及比例,进而改善肠道菌群。
动物试验表明发酵乳杆菌ZJUID06具有降低血脂水平、调节肠道平衡的功能,且对体重控制有一定的效果。
实施例3 发酵乳杆菌ZJUIDS06的抗氧化能力验证
1.样品制备
将于甘油管中保存的菌株在MRS固体培养基上划线分离,37℃倒置培养48h。用接种环挑取单菌落接种于灭菌MRS液体培养基中,37℃静置培养18-24h,得到培养液。将培养液用蒸馏水调整至乳酸菌菌体浓度为1010CFU/mL,4℃、8000×g离心20min,收集上清液即为发酵上清液。用0.02M PBS缓冲液(pH=7.4)将离心后的菌体沉淀重悬洗涤,4℃、8000×g离心20min,重复3次。将洗涤干净的菌体细胞重悬于PBS缓冲液中,并调整菌体浓度为1010CFU/mL,即得到菌体悬液。以抗氧化标准菌株ATCC 53103作为对照。
2.总抗氧化能力测定
总抗氧化能力(FRAP法)需在酶标板中加入150μL TPTZ工作液[0.3M pH3.6醋酸-醋酸钠缓冲液、20mM氯化铁溶液、10mM TPTZ缓冲液(40Mm HCl配置),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(μmolFeSO4/mL样品)。每个样品做3个平行,计算平均值。
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(40Mm HCl配置)、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,测定液在593nm处的吸光度。以硫酸亚铁质量浓度为横坐标,以吸光度为纵坐标绘制标准曲线。
3.还原能力测定
取1mL样品于离心管中,加入0.2M,pH 6.6的PBS溶液及1%(w/v)铁氰化钾溶液各1mL,混匀。50℃水浴20min,冰浴下冷却。再加入10%(w/v)三氯乙酸1mL,6000×g离心5min,取上清液1mL,加入0.1%(w/v)三氯化铁1mL、蒸馏水1mL,充分混合均匀,静置待其反应10min,测定样品在700nm处的吸光度。用PBS缓冲液或MRS液体培养基代替样品为空白组。每个样品做3个平行,计算平均值。
还原能力(%)=[(As-Ab)/Ab]*100%式中:As-样品组吸光度;Ab-空白组吸光度
4.DPPH自由基清除能力测定
配制不同浓度梯度的VC溶液(0-30μg/ml)。在酶标板中加入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100μL DPPH乙醇溶液为空白组;用100μL PBS缓冲液(或MRS液体培养基)代替100μL待测样品为对照组,并以100μL PBS缓冲液(或MRS液体培养基)和无水乙醇混合液空白调零。每个样品重复做3个平行,计算平均值。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。
表3.ZJUIDS06体外抗氧化性
表3的结果显示发酵乳杆菌ZJUIDS06的总氧化能力、还原能力及DPPH自由基清除能力等抗氧化效果显著优于标准菌株ATCC 53103(鼠李糖乳杆菌),表明发酵乳杆菌ZJUIDS06具有良好的抗氧化能力,可达到清除体内自由基,改善人体的健康功效。
实施例4 发酵乳杆菌ZJUIDS06的耐酸性及耐胆盐性的确认
1.耐酸实验
挑取发酵乳杆菌ZJUIDS06单菌落在MRS液体培养基中扩大培养18h,将扩大后菌悬液以1%(v/v)的量接种到MRS肉汤培养基,37℃培养18h后。将培养液于4℃经5000×g离心5min收集菌体,用磷酸盐缓冲液(pH 6.8)洗涤2次后。菌体悬浮于pH 3.0的MRS肉汤培养基中,将初始活菌数校正至大约1×108CFU/mL,37℃培养3h。采用倾注平板法对0h和3h样品中的活菌进行计数,倾注后的平板于37℃培养48h,菌株的酸耐受能力以0h和3h时每毫升含酸培养基中活菌数的差值的对数来表示(log CFU/m L)
2.耐胆盐实验
将活化扩大后的发酵乳杆菌ZJUIDS06菌悬液以1%(v/v)的量接种到MRS肉汤培养基中,于37℃培养18h后,漩涡混匀,将初始活菌数校正至大约109CFU/mL。以10%的量接种到含0.3%(m/v)牛胆盐的MRS肉汤培养基(对照为不含牛胆盐的MRS肉汤培养基),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0h和3h时每毫升含胆盐培养基中活菌数的差值的对数来表示(logCFU/m L)。
3.以嗜酸乳杆菌(Lactobacillus acidophilus)ATCC 43121作为对照,进行上述耐酸、耐胆盐性能测定。
4.如表4所示,发酵乳杆菌ZJUIDS06的耐酸耐胆盐性能明显优于对照菌株ATCC43121。其在pH为3.0的MRS培养基中的活菌数变动仅有0.07log cfu/mL。在含有0.3%(m/v)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,发酵乳杆菌ZJUIDS06具备较高的胃肠道生存能力。
表4菌株对酸和胆盐耐受性结果
菌株 | 酸耐受能力(ΔLog CFU/mL) | 胆盐耐受能力(ΔLog CFU/mL) |
发酵乳杆菌ZJUIDS06 | 0.07±0.01 | 2.48±0.21 |
嗜酸乳杆菌ATCC 43121 | 0.09±0.01 | 3.03±0.43 |
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。本发明提供的发酵乳杆菌ZJUIDS06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,发酵乳杆菌ZJUIDS06能耐受胆盐,其能在肠道内存活,进而能有效地发挥降胆固醇作用。
实施例5 发酵乳杆菌ZJUIDS06的抗生素感受性的确认
将培养18h的浓度为108CFU/mL发酵乳杆菌ZJUIDS06菌悬液按2%(v/v)的量加入冷却至45℃左右的灭菌的MRS固体培养基中,充分混合,每个培养基定量加入15mL。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。
发酵乳杆菌ZJUIDS06对抗生素感受性抑菌圈直径如表5所示。参考CLSI(2017)药敏试验标准可得,发酵乳杆菌ZJUIDS06对青霉素G、氨苄青霉素、头孢唑林、丁胺卡那、庆大霉素、红霉素、复方新诺名和氯霉素呈现敏感性。对于环丙沙星、氟哌酸呈现中介。实验结果表明,发酵乳杆菌ZJUIDS06对常见的抗生素敏感。
表5发酵乳杆菌ZJUIDS06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。本发明所提供的发酵乳杆菌ZJUIDS06对常见的抗生素敏感,不会对人体健康造成危害。
实施例6 发酵乳杆菌ZJUIDS06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固体培养基倒入无菌培养皿中,冷却后做为下层培养基。将培养18h菌浓度为108CFU/mL指示菌菌悬液按1%(v/v)的量加入冷却至45℃左右的灭菌的LB固体培养基中,充分混合,每个培养基定量加入10mL。在上面摆放灭过菌的的牛津杯。待上层培养基冷凝后,轻轻拔掉牛津杯。定量以100μL/孔加入发酵乳杆菌ZJUIDS06上清液样品,并且以的磷酸盐缓冲液(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6所示,发酵乳杆菌ZJUIDS06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ATCC 43121的抑菌效果。可见这株菌的代谢产物具有抑菌特性。
表6.菌株对病原菌抑制能力的结果
a,b:P-value<0.05嗜酸乳杆菌ATCC 43121:阳性对照
金黄色葡萄球菌是人类化脓感染中最常见的病原菌,一些大肠杆菌可引起严重腹泻和败血症,一些沙门氏菌种也会引起人类食物中毒。乳酸菌代谢产生的细菌素、有机酸、过氧化氢等抑菌产物均能单独或共同地抑制这些致病菌的生长。本发明所提供的发酵乳杆菌ZJUIDS06的代谢产物对这三种致病菌具有一定的拮抗作用,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7 发酵乳杆菌ZJUIDS06体外疏水性和自凝集性验证
疏水性测试微生物对不同种类的有机溶剂(二甲苯、三氯甲烷和乙酸乙酯)的粘附性。步骤是将每种细菌悬液的3mL(在pH7.0的0.1M PBS中调整到约为108CFU/mL)与每种有机溶剂的1mL混合后室温下放置30分钟进行两相分离。然后,收集水相,在570nm处检测吸光度。
根据以下公式计算疏水性或带电表面的百分比:
疏水性(%)=(A-A0/A)×100(A和A0分别是与每种溶剂混合前和混合后的吸光度),分为低吸光度(0-35%)、中等吸光度(36-70%)或高吸光度(71-100%)。
自凝集性采用分光光度法对目标菌进行测定。将4mL受试菌悬液(108CFU/mL)旋涡10s,然后在37℃下放置24h。通过测量剩余吸光度确定自凝集性,并以百分比表示为自凝集能力(%)=(1-A24h/A初始值)×100。
经测量,如图6所示,已授权专利菌株(专利号ZL201711410714.X)植物乳杆菌ZY08在二甲苯、氯仿、乙酸乙酯体系中的疏水性分别为24.79%、29.92%和27.01%而发酵乳杆菌ZJUIDS06分别为40.11%、49.96%和11.85%,表明发酵乳杆菌ZJUIDS06菌株具有较强疏水性。并且ZJUIDS06具有一定的自凝集性能,自凝集能力达到28.55%,表明菌株在肠道可以通过凝聚来抵御其他微生物的攻击和干扰。
实施例8 发酵乳杆菌ZJUIDS06体外胆盐水解酶活性测定
胆盐水解酶提取:将MRS液体培养基隔夜培养后的乳酸菌离心(8000g;10min;4℃),在0.1M PBS缓冲液(pH7.0)中洗涤两次,然后在同一缓冲液中再悬浮,使细胞密度在分光光度计中570nm(1cm路径长度)处约为2-3个光学单位。为减少酶的氧化,添加10mM二硫苏糖醇(DTT)使混合液的DTT终浓度在0.5-1.5mM。之后将菌悬液(20mL)在500W,50%的占空比下,恒定4℃冷却条件下进行细胞破碎超声处理,时间为5mL/3min。破碎后离心(10000g;15min;4℃)后,上清液作为无细胞提取物迅速放入-20℃条件下保存。
胆盐水解酶活性测定:向180μL反应缓冲液(0.1M磷酸钠,pH6.0)中加入10μL适当稀释的样品、10μL(最终达到10mM)不同的胆盐(甘氨胆酸GC、甘氨脱氧胆酸GDC、牛胆酸TC、牛脱氧胆酸TDC)和10μL液体石蜡。在37℃下反应30分钟,然后立即加入200μL 15%(w/v)三氯乙酸(TCA)。之后将样品以20000g,15min,4℃离心(5424型;Eppendorf)以除去沉淀物。将100μL上清液与1.9mL茚三酮试剂[茚三酮试剂由三部分组成:0.5mL 1%(w/v)茚三酮的柠檬酸钠缓冲液(0.5M pH5.5)中;1.2mL甘油;0.2mL柠檬酸钠缓冲液(0.5M pH5.5)]充分混合,煮沸14min。将试管在自来水中冷却3min后,在570nm处测量吸光度。以甘氨酸、牛磺酸、适量(10mM)DTT和TCA制备标准曲线。BSH活性的一个单位U被定义为每分钟从底物中释放1μM氨基酸的酶的量。蛋白质浓度用BCA蛋白浓度试剂盒(beyotine)测量。
经测定,如图7所示,已授权专利菌株(专利号ZL201711410714.X)植物乳杆菌ZY08对甘氨胆酸、甘氨脱氧胆酸、牛胆酸、牛脱氧胆酸的胆盐水解酶活性分别为0.44U/mg、0.48U/mg、0.39U/mg和0.35U/mg,而发酵乳杆菌ZJUIDS06分别为0.57U/mg、0.29U/mg、1.41U/mg和0.53U/mg,证明发酵乳杆菌ZJUIDS06具有较高的胆盐水解酶活性,特别是对牛胆酸的胆盐水解酶活性。
实施例8 发酵乳杆菌ZJUIDS06制作降胆固醇蒙特里杰克干酪
1.1 发酵菌株活化与接种
充当发酵剂的商业菌株乳酸乳球菌(丹尼斯克(中国)有限公司)按3%(v/v)接种于12g/100mL脱脂乳中,30℃培养6~8h,活化3代后用作干酪的生产发酵剂,接种量为1.5%(v/v)。
1.2 试验分组及益生菌添加
益生菌添加方式试验分组如下表7所示。
表7.益生菌添加方式
益生菌干酪需向经过63℃,30min杀菌后冷却到31℃的巴氏杀菌乳中接种含有益生菌的脱脂乳,脱脂乳为12%的种子液,种子液投放前需培养至种子液活菌数达到109cfu/mL。
1.3益生菌蒙特里杰克干酪制作流程
干酪制作过程如下流程所示:原料乳→巴氏杀菌→冷却→添加发酵剂→静置培养→添加凝乳酶→保温静止→凝块切割→热烫→恒温搅拌→排出乳清→堆酿→粉碎→加盐→加压成型→包装→成熟。
对照组干酪(未添加益生菌)的制作:鲜牛乳经63℃、30min巴氏杀菌后,冷却至32℃,添加发酵液(1.5%,v/v);32℃发酵30min。添加凝乳酶,32℃静置30~45min。待乳凝固后,用干酪刀切割成约长8mm的立方块,缓慢搅拌5min以防止凝块黏连愈合。之后每5min升高1℃,使凝块升温至39℃,当pH值降到6.1~6.2时,排乳清,并将凝块切成数块于39℃堆酿。待pH值降至5.4~5.5时,揉碎凝块,添加2%(w/v)食盐,然后装入干酪模具以8kg配重压榨15min,18kg配重压榨18h。之后真空包装,并于4℃冷藏条件成熟6个月。
益生菌干酪的制作:按照上述对照组干酪的工艺流程,原料乳灭菌后添加发酵剂,同时添加益生菌。
2.1 干酪品质测定
依据GB 5009.003—2010《食品中水分的测定》中的方法测定干酪含水量。使用巴布考克法测定干酪脂肪含量。使用凯氏定氮法测定干酪蛋白质含量。使用水活度仪测定水活度。使用色度仪测定色度。使用pH计测定pH值。
使用质构仪TPA测定干酪硬度、内聚性和黏弹性等指标。去掉干酪样品上下及边缘约5mm的部分,将干酪样品切成1.5cm×1.5cm×2cm大小的圆柱体样品。将样品在室温条件下放置30min,使其温度均衡,并避免样品在测试过程中发生温度变化。质构仪参数设定如下:预下降速度2.0mm/s,测试速度0.4mm/s,返回速度0.4mm/s,循环2次,数据频率20点/秒,触发点负载5g,压缩比50%,探头型号TA11/1000。
干酪中胆固醇含量测定需准确称量0.8g干酪样品,放入试管中并加入20.0mL0.5M氢氧化钾-甲醇溶液。密闭试管并在75℃水浴中皂化50分钟,每10分钟震荡一次。自来水冲洗冷却后,加入2.0mL去离子水和5mL正己烷。混合物使用超声处理,之后震荡混匀,静置数分钟后两相分离,取出上层含有胆固醇的有机相进行稀释分析。取1mL正己烷层(上层)转移到干净试管中,氮气吹干。加5mL无水乙醇,超声溶解,过0.22μm过滤膜,8000×g离心10min,取上清液放入液相色谱棕色小瓶,进行HPLC检测。
胆固醇标准曲线:5μg/mL,7.5μg/mL,10μg/mL,20μg/mL,40μg/mL
具体参数为:色谱柱:SinoChrom ODS-BP 4.6mm*250mm*5μm;柱温:35℃;流动相:乙腈-异丙醇(4:1,v/v,1个样品需25mL左右);流速:1mL/min;波长205nm;进样量:10μL。乳酸菌活菌数由MRS平板涂布测定干酪样品中活菌数。
乳酸菌活菌数测定需准确称取11g干酪加入89mL生理盐水,匀浆处理2min,继续用生理盐水将样品稀释到适当倍数,吸取50μL稀释液均匀涂布于MRS培养基上,厌氧培养24h后在30-300cfu的培养基上统计乳酸菌的活菌数。胆固醇测定的质谱图见图8。胆固醇含量结果如表8,发酵乳杆菌ZJUIDS06作为辅发酵剂对蒙特利杰克干酪品质无显著影响;益生性方面,发酵乳杆菌ZJUIDS06和ZY08一样能显著降低干酪中的胆固醇含量,对总乳酸菌活菌数无显著影响。
表8.干酪品质表
a,b:P-value<0.05,干酪胆固醇含量:干酪中每100g脂肪所含有胆固醇毫克数(mg)
实施例9 利用发酵乳杆菌ZJUIDS06制备功能性发酵果蔬汁
1.发酵果蔬汁的加工工艺流程:
原料→清洗→闪蒸→打浆→调配→均质→灭菌→冷却→接种→密闭发酵→后熟→灌装→冷藏
2.操作要点
(1)清洗切块:先将南瓜和火龙果清洗、去皮、去切成小块;
(2)闪蒸:采用闪蒸的方法灭酶,0.5-1min,121℃处理,迅速排气;
(3)打浆:按照南瓜∶水(质量比)=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;
(4)调配、均质:按南瓜汁15%(v/v),火龙果汁30%(v/v),再用蔗糖调节可溶性固形物含量至10°Brix,加入0.2%(w/v)的稳定剂CMC-Na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2-3um;
(5)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;
(6)接种、发酵:在无菌条件下,接入活化好的发酵乳杆菌ZJUIDS06,初始菌数控制在1×107cfu/mL。在37℃条件下恒温发酵24h;
(7)后熟:发酵结束后,放入4℃冰箱中3h;
(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。
实施例10 利用发酵乳杆菌ZJUIDS06制备功能性发酵蛋乳酸奶
1.蛋乳配制
称量200g蛋黄液(巴氏杀菌)和1800g全脂UHT灭菌乳(澳大利亚Valio),倒入均质机中均质混匀(3-5Mpa),混合液倒入螺口玻璃瓶中,于63℃水浴环境杀菌30min。放置于4℃冰箱保存备用。
2.菌种活化
各菌种在无菌环境下,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%(v/v),传代2~3次以恢复活力,放置于4℃冰箱中保存。
3.蛋乳酸奶发酵及胆固醇含量测定
主发酵剂采用保加利亚乳杆菌和嗜热链球菌(丹尼斯克有限公司),接种量1%(v/v),辅助发酵剂采用发酵乳杆菌ZJUIDS06,接种量6%(v/v)的接种量,发酵前添加5%(w/v)低聚糖到蛋乳中,在37℃下培养24h。非复配发酵时,主发酵剂和辅助发酵剂仍按上述接种量添加。酸奶成品的粘度采用Brookfield粘度计进行测定。参数设置为:探头型号为LV4,测试速度为60rpm,测试时间为10s。酸奶pH值的测定釆用精密pH计测量,参照《食品安全国家标准乳和乳制品酸度的测定》进行酸度的测量:取10g蛋乳酸奶,加入20mL蒸馏水,混匀后加入0.5mL酚酞指示液,用0.1M的氢氧化钠标准溶液进行滴定,溶液微红色,并在30s内不褪色时判定结束,记录下氢氧化钠标准溶液的用量,代入公式中进行计算:X=c×V×100m×0.1其中X为样品的酸度(°T);c为氢氧化钠标准溶液的摩尔浓度,即0.1M;V为滴定时消耗氢氧化钠标准溶液体积(mL);m为滴定所用酸奶的质量(g)。乳酸菌总数测定取100μl发酵蛋乳于无菌生理盐水中,进行梯度稀释。选择105、106、107这三个梯度液,取100μl倾注法倒入平板进行计数。于37℃培养48h后计算乳酸菌菌落总数。
胆固醇含量测定应量取一定量的蛋乳酸奶发酵液于离心管中,8000×g离心10min,得到上清液。取1g上清液于试管中,加入3mL氢氧化钾-甲醇溶液(10%,w/v),在75℃水浴皂化50min,期间每10min震荡一次。皂化完成后,冷却至常温,加入5ml正己烷萃取胆固醇,涡旋振荡,静置分层。取1mL正己烷层(上层)转移到干净试管中,用氮气吹干。然后加5mL无水乙醇,超声溶解,过0.22μm过滤膜,8000×g离心10min,取上清液进行HPLC检测。配置胆固醇浓度为40μg/mL,80μg/mL,120μg/mL,160μg/mL,200μg/mL的胆固醇标准溶液(无水乙醇定容)。然后进行HPLC测定。具体参数为:色谱柱:SinoChrom ODS-BP4.6mm*250mm*5μm;柱温:35℃;流动相:乙腈-异丙醇(4:1,v/v);流速:1mL/min;波长205nm;进样量:10μL。胆固醇测定的质谱图见图8,不同处理后胆固醇的降低率见表9,从表中看出发酵乳杆菌ZJUIDS06的胆固醇降低率最高,和专利菌株ZY08略高,显著高于普通发酵剂。表明发酵乳杆菌ZJUIDS06具有良好的降牛乳和鸡蛋中胆固醇的效果。
表9.菌种对蛋乳酸奶的降胆固醇能力比较
菌种 | 胆固醇降低率(%) |
商业发酵剂 | 13.68±0.42<sup>a</sup> |
植物乳杆菌ZY08 | 43.68±1.04<sup>d</sup> |
发酵乳杆菌ZJUIDS06 | 45.23±0.58<sup>d</sup> |
植物乳杆菌ZY08+商业发酵剂 | 33.91±0.28<sup>b</sup> |
发酵乳杆菌ZJUIDS06+商业发酵剂 | 35.44±0.08<sup>c</sup> |
植物乳杆菌ZY08+发酵乳杆菌ZJUIDS06+商业发酵剂 | 31.43±0.25<sup>b</sup> |
a,b,c,d:P-value<0.05
实施例11 利用发酵乳杆菌ZJUIDS06制备降胆固醇菌粉
1.发酵乳杆菌ZJUIDS06菌泥的制备
挑取发酵乳杆菌ZJUIDS06单菌落接种于50mL MRS液体培养基中,放置37℃培养箱中培养18h。再次按5%(v/v)的接种量于250m L MRS液体培养基中活化,放置37℃培养箱中培养24h。最后将活化好的发酵乳杆菌ZJUIDS06以5%接种量于10L发酵罐中进行高密度厌氧培养,于37℃、p H 6.8的条件下培养18h。之后在8000×g、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 7.0)漂洗菌体2次。即可得到发酵乳杆菌ZJUIDS06菌泥。
2.保护剂的制备
冻干保护剂含有15%(w/v)的脱脂乳,5%(w/v)的海藻糖,3%(w/v)的谷氨酸钠,1%(w/v)的甘油,0.5%(w/v)的半胱氨酸盐酸盐。水作为溶剂。于115℃灭菌20min备用。
3.发酵乳杆菌ZJUIDS06菌粉的制备
将上述制备的发酵乳杆菌ZJUIDS06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,即可得到发酵乳杆菌ZJUIDS06菌粉。用生理盐水复水后,洗涤两次,测得发酵乳杆菌ZJUIDS06菌粉中活菌数为1.0×1010-5×1010CFU/g。
实施例12 利用发酵乳杆菌ZJUIDS06制备宠物益生菌奶粉
1.发酵乳杆菌ZJUIDS06菌粉的制备
参照实施例11制备发酵乳杆菌ZJUIDS06菌粉冻干菌粉,菌粉中活菌数为1.0×1010-5×1010CFU/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1:100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。
序列表
<110> 浙江大学
<120> 一种人源性降胆固醇发酵乳杆菌ZJUIDS06及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1509
<212> DNA
<213> 发酵乳杆菌 ZJUIDS06 (Lactobacillus fermentum ZJUIDS06)
<400> 1
ctggctcagg acgaacgctg gcggcgtgcc taatacatgc aagtcgaacg aactcttgta 60
ttgattggtg cttgcatcat gatttacatt tgagtgagtg gcgaactggt gagtaacacg 120
tgggaaacct gcccagaagc gggggataac acctggaaac agatgctaat accgcataac 180
aacttggacc gcatggtccg agtttgaaag atggcttcgg ctatcacttt tggatggtcc 240
cgcggcgtat tagctagatg gtggggtaac ggctcaccat ggcaatgata cgtagccgac 300
ctgagagggt aatcggccac attgggactg agacacggcc caaactccta cgggaggcag 360
cagtagggaa tcttccacaa tggacgaaag tctgatggag caacgccgcg tgagtgaaga 420
agggtttcgg ctcgtaaaac tctgttgtta aagaagaaca tatctgagag taactgttca 480
ggtattgacg gtatttaacc agaaagccac ggctaactac gtgccagcag ccgcgctaat 540
acgtaggtgg caagcgttgt ccggatttat tgggcgtaaa gcgagcgcag gcggtttttt 600
aagtctgatg tgaaagcctt cggctcaacc gaagaagtgc atcggaaact gggaaacttg 660
agtgcagaag aggacagtgg aactccatgt gtagcggtga aatgcgtaga tatatggaag 720
aacaccagtg gcgaaggcgg ctgtctggtc tgtaactgac gctgaggctc gaaagtatgg 780
gtagcaaaca ggattagata ccctggtagt ccataccgta aacgatgaat gctaagtgtt 840
ggagggtttc cgcccttcag tgctgcagct aacgcattaa gcattccgcc tggggagtac 900
ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 960
gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatactatg caaatctaag 1020
agattagacg ttcccttcgg ggacatggat acaggtggtg catggttgtc gtcagctcgt 1080
gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattatca gttgccagca 1140
ttaagttggg cactctggtg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1200
caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga 1260
gttgcgaact cgcgagagta agctaatctc ttaaagccat tctcagttcg gattgtaggc 1320
tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga 1380
atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaa 1440
gtcggtgggg taacctttta ggaaccagcc gcctaaggtg ggacagatga ttagggtgaa 1500
gtcgtacag 1509
Claims (4)
1.一株人源性发酵乳杆菌ZJUIDS06,其特征在于,所述发酵乳杆菌(Lactobacillus fermentum)ZJUIDS06的保藏号为:CGMCC NO. 21339。
2.权利要求1所述的一株人源性发酵乳杆菌ZJUIDS06在制备功能性产品中的应用,所述功能为降血脂、降胆固醇、抗氧化性,且该菌株具良好的益生特性及安全性。
3.根据权利要求2所述的应用,其特征在于,所述产品包括食品、保健品及宠物饲料。
4.根据权利要求3所述的应用,其特征在于,所述的产品包括低胆固醇功能性蒙特里杰克干酪、功能性发酵果蔬汁、低胆固醇功能性发酵蛋乳酸奶、具有降血脂功能的活菌制剂、功能性宠物饲料。
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