CN108102959A - 人源性降胆固醇植物乳杆菌zy08及其应用 - Google Patents
人源性降胆固醇植物乳杆菌zy08及其应用 Download PDFInfo
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- CN108102959A CN108102959A CN201711410714.XA CN201711410714A CN108102959A CN 108102959 A CN108102959 A CN 108102959A CN 201711410714 A CN201711410714 A CN 201711410714A CN 108102959 A CN108102959 A CN 108102959A
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- lactobacillus plantarum
- cholesterol
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- lactobacillus
- bacterium
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- A—HUMAN NECESSITIES
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Abstract
本发明涉及食品微生物技术领域,具体而言,本发明公开了一种植物乳杆菌ZY08:为植物乳杆菌(Lactobacillus plantarum),其保藏号为CGMCC NO.14847。其具有强降胆固醇功能,以及具有较强的胆盐水解酶活力。本发明还同时公开了上述植物乳杆菌ZY08在制备具有降低胆固醇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,具体涉及到具有降胆固醇功能的植物乳杆菌ZY08及应用。
背景技术
目前,心血管疾病已经成为世界人口死亡的首要病因。血清中胆固醇含量被认为是诱发高血压、冠心病等众多心血管疾病的主要因素。因此,降低血清胆固醇水平直接关系到人体的健康。迄今为止,已有大量试验证实了服用部分乳酸菌及其相关制品具有减少人体血清胆固醇含量,降低心血管疾病发病率的功效。乳酸菌是人体肠道中重要的益生菌,其数量和组成对维持宿主的微生态平衡和提高免疫系统功能起着至关重要的作用。
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。植物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。植物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、降低血清胆固醇、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
本发明要解决的技术问题是提供一种人源性降胆固醇植物乳杆菌ZY08及其应用。
为了解决上述技术问题,本发明提供一种植物乳杆菌ZY08:为植物乳杆菌(Lactobacillus plantarum),其保藏号为CGMCC NO.14847。
作为本发明的植物乳杆菌ZY08的改进:植物乳杆菌ZY08的16S rDNA全序列为SEQID No:1所示。
本发明还同时提供了上述植物乳杆菌ZY08在制备具有降低胆固醇功能的产品中的应用。
作为本发明的应用的改进:所述具有降低胆固醇功能的产品为具有降低胆固醇功能的食品、药物、保健品及饲料。
具有降低胆固醇功能的食品包括具有降低胆固醇功能的发酵果蔬汁(例如为发酵南瓜火龙果汁);具有低胆固醇功能性发酵酸肉;
具有降低胆固醇功能的药物包括具有降低胆固醇功能的活菌制剂,该活菌制剂中植物乳杆菌ZY08的活菌数为1.0×1010~5×1010CFU/g;
具有降低胆固醇功能的饲料为具有降低胆固醇功能的青贮饲料(例如为青贮苜蓿饲料)。
本发明的植物乳杆菌ZY08单独或与其他菌株混合发酵用于制备具有降低胆固醇功能的青贮饲料。
本发明的植物乳杆菌ZY08,保藏名称:植物乳杆菌Lactobacillus plantarum;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.14847,保藏时间2017年11月1日。
本发明从健康母乳喂养婴儿的粪便中筛选出一株植物乳杆菌ZY08(Lactobacillus plantarum ZY08),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。
本发明所提供的植物乳杆菌ZY08(Lactobacillus plantarum ZY08)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。
本发明所提供的植物乳杆菌ZY08(Lactobacillus plantarum ZY08)的降胆固醇能力达到62.1~68.6%。其具有以下能力:
1、具有较强的胆盐水解酶活力;
2、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。
本发明所提供的植物乳杆菌ZY08(Lactobacillus plantarum ZY08)与植物乳杆菌CGMCC NO.14216、植物乳杆菌CGMCC NO.14217相比,其降胆固醇能力和益生特性更为突出。
综上所述,本发明从健康母乳喂养婴儿的粪便中分离获得的益生菌中筛选出来具有强降胆固醇功能的植物乳杆菌。该菌株具有较强的胆盐水解酶活力。该菌株在耐酸耐胆盐方面比其他乳酸菌有明显的优势,适合肠胃环境并具有增殖能力。无抗生素耐性,具有抗菌活性。本发明植物乳杆菌可广泛用于开发降胆固醇相关益生功能产品。
附图说明
图1为植物乳杆菌ZY08的菌落形态图。
图2为植物乳杆菌ZY08革兰氏染色的菌体形态图。
图3为植物乳杆菌ZY08的16S rDNA的电泳鉴定图;
图3中1和2均为本发明菌株,M为DNA Marker。
图4为OPA法测得胆固醇标准曲线。
图5为空白样品(未接菌)胆固醇峰图。
图6为阳性对照菌株(嗜酸乳杆菌ATCC43121)发酵上清液胆固醇峰图。
图7为植物乳杆菌ZY08发酵上清液胆固醇峰图。
图8为植物乳杆菌ZY08的胆盐水解酶活性测定图。
图9为植物乳杆菌ZY08对抗生素敏感性的平板示例。
图10为植物乳杆菌ZY08抗菌活性的平板示例。
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、植物乳杆菌ZY08的筛选及鉴定:
1.植物乳杆菌ZY08的筛选
1.1样品来源
本发明所使用的菌株均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
取大约5g新鲜粪便样品用无菌管收集,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。
1.3菌株发酵液的制备
将于甘油管中保存的菌株先在MRS琼脂平板上划线活化2~3次,然后挑取单菌落在MRS液体培养基中扩大培养18h,菌液的浓度达到108CFU/mL左右。将扩大后的菌悬液以2%(体积%)的接种量接种于胆固醇(水溶性)浓度为100μg/mL的MRS液体培养基中,于37℃培养48h后得到菌株发酵液,用于后面的测定。
每个菌种做3个平行,每组实验重复3次,同时以不接菌的含有胆固醇的MRS液体培养基作阴性对照,以嗜酸乳杆菌ATCC43121作阳性对照菌株。
1.4邻苯二甲醛比色法(OPA法)测菌株的降胆固醇能力
1.4.1标准曲线的绘制
配置胆固醇浓度为20μg/mL,40μg/mL,60μg/mL,80μg/mL,100μg/mL的胆固醇标准溶液(正己烷作为溶剂),4℃保存备用。各取0.5mL标准溶液(0.5mL正己烷为空白对照)进行氮吹,加入2mL邻苯二甲醛溶液(浓度为50mg/100mL),混匀10min静置,再加入1mL浓硫酸,混匀后静置10min,测550nm吸光度。以胆固醇浓度为横坐标,吸光值为纵坐标做标准曲线,计算出其线性回归方程为y=3.47x+0.076,相关系数为R2=0.9952。标准曲线如图4所示。
1.4.2具体操作方法
将上述1.3中所制备的菌株发酵液于8000r/min离心5min,得到菌株发酵上清液。取发酵上清液0.5mL于螺口试管,加10%氢氧化钾甲醇溶液2mL,旋紧试管,于75℃水浴皂化50min,期间每10min震荡一次,皂化完成后,冷却,加入1mL水,2mL正己烷(重复萃取一次),旋涡振荡2min,静置10min,分层,取上层正己烷层于新试管中,合并两次萃取液(3.5mL),氮气吹干(50℃),加入2mL邻苯二甲醛溶液,混匀10min静置,再加入1mL浓硫酸,混匀10min静置,测550nm吸光度。
1.4.3胆固醇降低率的计算公式:
注:Y—胆固醇降低率;
C0—为阴性对照样品中胆固醇浓度,μg/mL;
C1—为测试样品中胆固醇浓度,μg/mL。
1.5以胆固醇降低率Y为指标,筛选得到乳酸菌ZY08胆固醇降低率最高,并且优于嗜酸乳杆菌ATCC43121。
2.植物乳杆菌ZY08的鉴定
2.1菌落特征
植物乳杆菌ZY08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:
植物乳杆菌ZY08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,结果表明该菌株为植物乳杆菌。
表1、植物乳杆菌ZY08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,扩增片段长度为1.5kb左右,见图3。将PCR产物送至生工生物工程(上海)有限公司进行测序,结果如SEQ ID NO:1所示,在NCBI网站上进行BLAST序列比对,结果显示该序列与植物乳杆菌的16S rDNA序列同源性超过99%。
将菌株ZY08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ZY08为植物乳杆菌(Lactobacillus plantarum)。
实施例2、植物乳杆菌ZY08降胆固醇能力的确认
1.气相色谱法(GC法)
1.1具体操作方法
按照实施例1中1.3的方法制备植物乳杆菌ZY08的发酵液。将其于8000r/min离心5min,得到植物乳杆菌ZY08发酵上清液。用无菌注射器取1mL发酵上清液,0.22μm滤器过滤到气相进样瓶中,将制备好的样品进行气相检测。
1.2气相色谱法的分析条件
仪器型号为Agilent 7890A;色谱柱(HP-5:30m×0.32mm×0.25μm);升温程序为100℃升至300℃(以20℃/min升温12min);进样口温度为250℃,检测口温度为300℃;载气(He)流速1.5mL/min,进样量0.5μL;分流比5:1。
2.胆固醇降低率的计算公式:
注:S0—阴性对照样品中胆固醇含量对应的峰面积;
S1—测试样品中胆固醇含量对应的峰面积。
根据气相色谱峰图中胆固醇所对应的峰面积,即可求得菌株降低胆固醇的能力。
备注:空白样品(未接菌)胆固醇峰图如图5所示,阳性对照菌株(嗜酸乳杆菌ATCC43121)发酵上清液胆固醇峰图如图6所示,植物乳杆菌ZY08发酵上清液胆固醇峰图如图7所示。
3.如表2所示,本发明筛得植物乳杆菌ZY08对MRS培养基中的胆固醇降低率达到了62.1~68.6%,比众多研究报道的对胆固醇去除效果较好的嗜酸乳杆菌ATCC43121高。说明植物乳杆菌ZY08具有高效的胆固醇去除能力。
表2、菌株的胆固醇降低率
4.本发明提供了用邻苯二甲醛比色法和气相色谱法相结合的筛选降胆固醇菌株的方法。先用邻苯二甲醛比色法初步筛选降胆固醇菌株,再用气相色谱法进一步确认菌株的降胆固醇能力。因此,本发明所得植物乳杆菌ZY08的胆固醇降低率更精确,提供了一株高效降胆固醇的菌株。
本发明的植物乳杆菌ZY08,保藏名称:植物乳杆菌Lactobacillus plantarum;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.14847,保藏时间2017年11月1日。
实施例3、植物乳杆菌ZY08的胆盐水解酶活力的确认
1.植物乳杆菌ZY08产胆盐水解酶定性测定
在新鲜配制的MRS琼脂培养基中添加0.3%(m/v)脱氧牛磺胆酸钠、0.2%(m/v)巯基乙酸钠、0.37g/L CaCl2,将其完全溶解。于121℃灭菌15min,倾倒入无菌平板中,待凝固后把无菌滤纸片均匀放入平板中,在滤纸片上滴加10μL植物乳杆菌ZY08菌悬液(约108CFU/mL),以滴加10μL的无菌磷酸盐缓冲液为空白对照。将平板于厌氧罐(OXOID)中37℃厌氧培养72h。若滤纸片周围有白色沉淀物则认为有胆盐水解酶活性。
2.植物乳杆菌ZY08胆盐水解酶活力的定量测定
取植物乳杆菌ZY08发酵液(约108CFU/mL)10mL于离心管中。在4℃、8000r/min离心10min,弃去上清液,收集菌体沉淀。用无菌生理盐水漂洗菌体2次,再用30mL 0.1mol/L的磷酸盐缓冲溶液(pH 7.0)将其悬浮。细菌悬浮液于冰浴中进行超声波破碎20min(100W,工作时间/间隙时间=3:5,保护温度为37℃)。之后,10000r/min、4℃离心10min,其上清液即为植物乳杆菌ZY08的胆盐水解酶溶液。
取0.1mL植物乳杆菌ZY08的胆盐水解酶溶液,向其中加入1.8mL 0.1mol/L的磷酸盐缓冲溶液和0.1mL的6mmol/L牛磺胆酸钠。这些混合物在37℃培养30min,之后加入0.5mL15%的三氯乙酸终止酶促反应。将其进行离心,取0.5mL上清液加入1mL的茚三酮显色液。将其旋涡震荡混匀,煮沸35min。冷却后测定其在570nm处的吸光度值。一个胆盐水解酶活力单位定义为每分钟从底物释放1μmoL牛磺酸所需要的酶的量。
3.以嗜酸乳杆菌(Lactobacillus acidophilus)ATCC43121作为阳性对照,进行上述胆盐水解酶活力的测定。蛋白质浓度的测定采用牛血清蛋白作为标准品,所有实验重复3次。
4.图8为植物乳杆菌ZY08和阳性对照菌株胆盐水解酶定性测得结果。从图中可以看出植物乳杆菌ZY08和嗜酸乳杆菌ATCC43121与空白对照相比,其滤纸片周围均有白色沉淀圈产生。表明,两株菌对胆盐具有水解作用。表3为两株菌的胆盐水解酶活性定量测定结果。从表中可以看出,这两株菌的胆盐水解酶活力均在1.0U/mg以上,植物乳杆菌ZY08的胆盐水解酶活力为1.05U/mg,与对照菌株无明显差异。实验结果表明,植物乳杆菌ZY08具有较高的胆盐水解酶活力。
表3、菌株的胆盐水解酶测定结果
注:+表示产生沉淀圈,-表示不产生沉淀圈。
5.胆盐水解酶可将体内结合型胆盐水解为游离型胆盐,而游离型胆盐不参与肝肠循环,随粪便排出体外,因而胆盐水解酶活性是降低体内胆固醇的关键因素。本发明所提供的植物乳杆菌ZY08具有较强的胆盐水解酶活力,可降低体内胆固醇。
实施例4、植物乳杆菌ZY08的耐酸性及耐胆盐性的确认
1.耐酸实验
挑取植物乳杆菌ZY08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%的量接种到MRS液体培养基,37℃培养18h后。将培养液于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。菌体悬浮于pH 3.0的MRS液体培养基中,将初始活菌数校正至大约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,测定其存活率,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
将活化扩大后的植物乳杆菌ZY08菌悬液以1%的量接种到MRS液体培养基中,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.以嗜酸乳杆菌(Lactobacillus acidophilus)ATCC43121作为对照,进行上述耐酸、耐胆盐性能测定。
4.如表4所示,植物乳杆菌ZY08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培养基中的平均存活率为93.32%。在含有0.3%的牛胆盐的环境中活菌数减少2~2.5个数量级,说明其具有较好的胆盐耐受性。实验证明,植物乳杆菌ZY08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
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。本发明提供的植物乳杆菌ZY08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,植物乳杆菌ZY08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、植物乳杆菌ZY08的抗生素感受性的确认
将培养18h的浓度约为107CFU/mL植物乳杆菌ZY08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。图9为植物乳杆菌ZY08抗生素敏感性的平板示例。
植物乳杆菌ZY08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,植物乳杆菌ZY08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、复方新诺明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,植物乳杆菌ZY08对常见的抗生素敏感。
表5、植物乳杆菌ZY08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。本发明所提供的植物乳杆菌ZY08对常见的抗生素敏感,不会对人体健康造成危害。
实施例6、植物乳杆菌ZY08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10mLLB琼脂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/孔加入植物乳杆菌ZY08发酵上清液样品,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。图10为植物乳杆菌ZY08对三种致病菌抑制作用的平板示例。
如表6所示,植物乳杆菌ZY08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ATCC43121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
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。本发明所提供的植物乳杆菌ZY08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、植物乳杆菌ZY08的对比试验
将下述表7中所述的菌株如同实施例2~实施例4所述方式进行各项性能的检测,与本发明的植物乳杆菌ZY08的结果对比如表7所述。
表7对比试验结果
从表7中可以看出,本发明所提供的植物乳杆菌ZY08的胆固醇降低率比其余两株菌高10%左右。进一步的证实,本发明的植物乳杆菌ZY08具有优秀的胆固醇降低能力。此外,植物乳杆菌ZY08的胆盐水解酶活力明显高于其余两株植物乳杆菌。其对酸及胆盐的耐受性也优于另外两株菌。本对比试验更能说明,本发明所提供的植物乳杆菌ZY08其在降胆固醇能力方面具有突出优势,同时其所具有的优良益生特性为降胆固醇益生产品的开发提供保障。
实施例8、利用植物乳杆菌ZY08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)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,0.5~1min,121℃处理,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%,再用蔗糖调节可溶性固形物含量至10°Brix,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,接入活化好的植物乳杆菌ZY08,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9、利用植物乳杆菌ZY08制备功能性发酵酸肉
1.发酵酸肉的加工工艺流程:
原料→切片→拌糯米粉→接种→发酵→腌制→包装→成品
2.操作要点
(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,接入活化好的植物乳杆菌ZY08,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10、利用植物乳杆菌ZY08制备降胆固醇菌粉
1.植物乳杆菌ZY08菌泥的制备
挑取植物乳杆菌ZY08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%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。最后将活化好的植物乳杆菌ZY08以5%接种量于10L发酵罐中进行高密度厌氧培养,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 7.0)漂洗菌体2次。即可得到植物乳杆菌ZY08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.植物乳杆菌ZY08菌粉的制备
将上述制备的植物乳杆菌ZY08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,即可得到植物乳杆菌ZY08菌粉。用生理盐水复水后,洗涤两次,测得植物乳杆菌ZY08菌粉中活菌数为1.0×1010~5×1010CFU/g。
实施例11、利用植物乳杆菌ZY08制备青贮饲料
植物乳杆菌ZY08制备苜蓿青贮饲料的步骤如下:
(1)原料准备:苜蓿草收割后,保证清洁,无腐败变质,将其切断至1~2cm。
(2)青贮的调制:苜蓿草青贮时水分含量控制在60~65%,称取200g苜蓿草原料装入真空包装袋,将实施例10所制备的植物乳杆菌ZY08菌粉按0.02‰接种量接种到原料中,采用真空包装机真空包装后,至于储藏室进行发酵。
(3)储藏条件:温度为15~45℃,时间为30~60d。
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> 人源性降胆固醇植物乳杆菌ZY08及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1509
<212> DNA
<213> 植物乳杆菌(Lactobacillus plantarum)
<400> 1
ctggctcagg acgaacgctg gcggcgtgcc taatacatgc aagtcgaacg aactctggta 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 ccgcggtaat 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 (5)
1.植物乳杆菌ZY08,其特征是:为植物乳杆菌(Lactobacillus plantarum),其保藏号为CGMCC NO.14847。
2.根据权利要求1所述的植物乳杆菌ZY08,其特征是:植物乳杆菌ZY08的16S rDNA全序列为SEQ ID No:1所示。
3.如权利要求1或2所述的植物乳杆菌ZY08在制备具有降低胆固醇功能的产品中的应用。
4.根据权利要求3所述的应用,其特征是:所述具有降低胆固醇功能的产品为具有降低胆固醇功能的食品、药物、保健品及饲料。
5.根据权利要求4所述的应用,其特征是:
具有降低胆固醇功能的食品包括具有降低胆固醇功能的发酵果蔬汁;具有低胆固醇功能性发酵酸肉;
具有降低胆固醇功能的药物包括具有降低胆固醇功能的活菌制剂,该活菌制剂中植物乳杆菌ZY08的活菌数为1.0×1010~5×1010CFU/g;
具有降低胆固醇功能的饲料为具有降低胆固醇功能的青贮饲料。
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Application publication date: 20180601 Assignee: Siuite (Hangzhou) Food Technology Co.,Ltd. Assignor: ZHEJIANG University Contract record no.: X2021330000751 Denomination of invention: Human cholesterol lowering Lactobacillus plantarum zy08 and its application Granted publication date: 20200602 License type: Common License Record date: 20211116 |