CN110218681A - 一株发酵乳杆菌kp101及其应用 - Google Patents
一株发酵乳杆菌kp101及其应用 Download PDFInfo
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- CN110218681A CN110218681A CN201910555559.3A CN201910555559A CN110218681A CN 110218681 A CN110218681 A CN 110218681A CN 201910555559 A CN201910555559 A CN 201910555559A CN 110218681 A CN110218681 A CN 110218681A
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- lactobacillus fermenti
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- milk
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Abstract
本发明公开了一株发酵乳杆菌KP101,它的保藏编号为CCTCC No.2019085;及其在制备发酵酸奶和益生菌固体饮料方面的应用;本发明发酵乳杆菌KP101耐受酸胁迫、耐受胆盐胁迫和耐受氧化胁迫良好,抗致病菌感染、调节代谢紊乱、调节高血脂症等,同时减肥降脂;动物实验表明,能够有效降低由高脂饮食引起的血清中TG、LDL‑c的增多,分别为50.5%、67.0%;能够显著提高肝脏中HDL‑C水平,增加率为78.9%;降低肝脏中TC、TG和LDL‑C的含量,分别为36.2%、64.6%和42.8%;还具有极显著降脂作用,降低其肾周脂肪系数及腹部脂肪系数分别为97.2%和95.5%。
Description
技术领域
本发明属于微生物发酵技术领域,具体涉及一株发酵乳杆菌KP101及其应用。
背景技术
高血脂症及其并发症已成为全球发病率和死亡率最高的疾病之一,但目前的治疗手段还很局限,主要的临床处理方法有两种,一是饮食干预,二是药物治疗。饮食干预的核心为限制高脂肪及高胆固醇食物的摄入。但该方法仅在短期内有效且难以长久坚持,其治疗效果逐渐变差;药物治疗虽然较为明显,降胆固醇药物往往副作用大且价格昂贵。益生菌作为价格低廉,安全性高的一类物质,通过服用益生菌制剂调节和控制血清胆固醇水平对于高血脂症的预防及辅助治疗有很大的开发前景。
目前,通过大量动物试验和临床试验已经发现众多益生菌如部分植物乳杆菌、干酪乳杆菌、双歧杆菌等都具有调节人体血清胆固醇水平的功效。如今,已有大量研究者对益生菌在实验动物体内的降胆固醇功能进行了评价与研究。通过研究发现,益生菌主要能显著降低实验动物血清TC和LDL-C水平,还未发现益生菌能够对实验动物血清的HDL-C水平产生显著影响。由于我国居民的饮食习惯及遗传因素等与国外人种存在差,对于从国外引入的益生菌菌株可能不一定完全适合我国居民服用。我国暂无自主知识产权的降胆固醇益生菌菌株或相关产品,因此研究和开发具有自主知识产权的适合我国居民服用的益生菌菌株及其制品对于推进我国益生菌产业发展及促进国民身体健康具有重要意义。
发明内容
本发明目的是提供一株具有减肥降脂作用的发酵乳杆菌KP 101及其应用。
一株发酵乳杆菌Lactobacillus fermentum KP 101,它的保藏编号为CCTCC No.M 2019085。
一种发酵酸奶,它是由下述方法制备的:
1)菌株的活化:将保藏编号为CCTCC No. M 2019085的发酵乳杆菌KP101和嗜热链球菌的冻干菌粉接种于无菌牛奶培养基中,分别按最适温度进行培养,传代2~3次,每次接种量1~10%,活菌数达到106CFU/mL以上;
2)发酵酸奶的制备:将活化后的发酵乳杆菌KP101和嗜热链球菌按体积比1~2:1的比例混合,按照1%~10%的接种量接种至牛奶中,在38~45℃下发酵3~5h,在0~4℃下冷却,得到发酵酸奶;
步骤2)所述的按照3%~5%的接种量接种至牛奶中,在40~43℃下发酵3~5h;
步骤2)所述的牛奶,是由下述方法制备的:过滤、净化,加入质量分数5%~10%的糖,过滤,搅拌均匀,加热至55~65℃,在18~22MPa下均质3~10min,在95~100℃条件下灭菌5~10min后,冷却。
一种益生菌固体饮料,它包括下列重量份数的组分:发酵乳杆菌冻干粉10份,低聚异麦芽糖5~10份,大豆低聚糖5~10份,果味粉5~15份;所述的发酵乳杆菌,它的保藏编号为CCTCC No. M 2019085;
所述的果味粉为蓝莓粉、蔓越莓粉或山楂粉;
所述的发酵乳杆菌冻干粉,是由下述方法制备的:将发酵乳杆菌KP101活化,扩大培养,在3000~5000r/min下离心8~15min、用水洗涤,离心弃上清,得到菌体;将菌体和冻干保护剂按体积比1~3:1混合,冷冻干燥,得到发酵乳杆菌冻干粉;
所述的冻干保护剂,按质量百分比计包括下列组分:脱脂奶粉10%-12%、纤维素7%-9%、海藻糖3%-5%、普兰多糖6%-8%、余量为水。
本发明提供了一株发酵乳杆菌KP101,它的保藏编号为CCTCC No. M 2019085;及其在制备发酵酸奶和益生菌固体饮料方面的应用;本发明发酵乳杆菌KP101耐受酸胁迫、耐受胆盐胁迫和耐受氧化胁迫良好,抗致病菌感染、调节代谢紊乱、调节高血脂症等,同时减肥降脂;动物实验表明,能够有效降低由高脂饮食引起的血清中TG、LDL-c的增多,分别为50.5%、67.0%;能够显著提高肝脏中HDL-C水平,增加率为78.9%;降低肝脏中TC、TG和LDL-C的含量,分别为36.2%、64.6%和42.8%;还具有极显著降脂作用,降低其肾周脂肪系数及腹部脂肪系数分别为97.2%和95.5%。
附图说明
图1 邻苯二甲醛胆固醇脱除率结果;
图2 胆盐水解酶定性与定量测定结果;
图3 菌株的16S rRNA基因序列测定结果;
图4 菌株的耐酸特性结果;
图5 菌株的耐胆盐特性结果;
图6 菌株的抗氧化活性结果;
图7 小鼠血清的TC、TG、HDL、LDL测定结果;
图8 小鼠血清的ALT、AST测定结果;
图9 小鼠肝脏的TC、TG、HDL、LDL测定结果;
图10 小鼠肝脏的ALT、AST测定结果;
图11 小鼠第八周体重、腹部脂肪系数、肾周脂肪系数。
具体实施方式
实施例1 发酵乳杆菌KP101的筛选和鉴定
1、菌株的筛选
将购买于市场的泡菜样品,在实验室进行乳酸菌分离;将泡菜发酵液以10-1的梯度稀释,涂布于MRS琼脂平板上,厌氧培养24h,挑取单菌落,在平板上划线分离,反复进行纯化培养,用革兰氏染色法观察菌落形态挑选革兰氏阳性菌株;将挑选的多株菌株,再在液体MRS培养基中37℃培养24h,按照培养液3%的接种量进行接菌,连续活化传代三次,以保证菌体旺盛的生长活力;
液体MRS培养基含有蛋白胨10.0g,牛肉膏10.0g,酵母膏 5.0 g,柠檬酸氢二铵[(NH4)2HC6H5O7] 2.0 g,葡萄糖(C6H12O6·H2O) 20.0g,吐温80 1.0mL,乙酸钠(CH3COONa·3H2O)5.0g,磷酸氢二钾(K2HPO4·3H2O)2.0g,硫酸镁(MgSO4·7H2O)0.58g,硫酸锰(MnSO4·H2O)0.25g。
2、菌悬液的制备
将上述活化并扩大培养的菌液4000r/min离心10min,用0.85%的生理盐水洗涤2遍,离心弃上清,得到菌体沉淀,悬浮于等体积的生理盐水中,得到菌悬液。
3、菌株筛选培养基与试剂的制备
BSH筛选培养基:液体MRS培养基的基础上添加2%的琼脂,0.3%的牛胆盐,0.2%的巯基乙酸钠,0.37g/L的氯化钙;
MRS-THIO-OX-CHOL培养基:液体MRS培养基的基础上添加0.2%的巯基乙酸钠,0.3%的牛胆盐,浓度为100μg/ml的胆固醇溶液;
胆固醇溶液配制:准确称取胆固醇粉末0.5g,在加热的条件下,用无水乙醇充分溶解并定容至50mL,此时胆固醇溶液浓度为 10.0mg/mL,在无菌环境下,用孔径为0.45µm的微孔滤膜除菌后,按照1%的量加入已经灭菌的液体MRS培养基中,此时,液体MRS培养基中胆固醇浓度为0.1mg/mL;
邻苯二甲醛工作液:准确称取邻苯二甲醛50mg,用无水乙醇定容50mL,使得浓度为1mg/mL,冷藏备用;
混合酸配制:浓H2SO4与冰乙酸1:1混匀。
4、邻苯二甲醛法筛选体外降胆固醇能力良好菌株
标准曲线的绘制:取五支洁净试管,编号为1-5,按顺序分别加入0,0.1,0.2,0.3,0.4mL的胆固醇溶液,再按1-5的顺序分别加入0.5,0.4,0.3,0.2,0.1mL的冰醋酸后,分别加入0.2mL的邻苯二甲醛试剂,使其震荡充分溶解,静置反应10min后再分别加入4.0mL的混合酸,混合均匀,室温放置10min使其显色,用移液枪将反应液置入96孔板中,用酶标仪测定其在550nm处的吸光值,以胆固醇的浓度为横坐标,吸光度OD550值为纵坐标绘制标准曲线;回归方程:y = 3.8445x + 0.0793 R2 = 0.9903
测定样品的OD值:将菌悬液以3%接种量接入到装有10mL的MRS-THIO-OX-CHOL培养基中,37°C条件下培养24h;此时,迅速利用邻苯二甲醛法测定发酵0h的培养液;将刚接完菌的培养基在9000r/min条件下离心10min,取0.25mL的上清液,加入0.1mL的邻苯二甲醛溶液,充分震荡,静置反应10min后,加入2m L的混合酸溶液,静置10min,将反应液置入96孔板中,用酶标仪在550nm处测定其OD值;在培养过程中,分别在6、12和24h时取样,利用上述邻苯二甲醛法测定发酵液6、12和24h的OD值;然后根据胆固醇的标准曲线拟合的方程确定发酵液中胆固醇的含量,并根据以下公式测得最终胆固醇的脱除率(%);
公式:胆固醇的脱除率:V=(B-A)/B×100%;其中,A为各试验菌株发酵后上清液中的胆固醇含量;B为各试验菌株发酵前上清液中的胆固醇含量;
结果:菌体培养时间的变化,菌株在体外胆固醇的脱除率也随之变化,这表明胆固醇的脱除率与菌体的生长状态有一定的关系;菌株在6h、12h和24h的胆固醇脱除率分别为44.09%、77.59%和84.58%,表明其在体外良好的降胆固醇效果,该菌株可以应用到动物体内试验,进一步测定其在体内的降血脂能力(图1)。
5、胆盐水解酶活力测定
BSH定性测定:将灭菌的BSH筛选培养基倒入无菌平板中,待培养基凝固后将无菌滤纸片均匀放于培养基上,用移液枪将活化好的待测菌液10 µL缓慢加在直径约3~4 mm的无菌滤纸片上,待菌液完全吸收后,于37℃厌氧条件下培养72 h后,观察滤纸片周围是否有白色沉淀物产生,若出现白色沉淀则可初步验证该菌含有BSH;
BSH定量测定:使用购买于上海优选生物的BSH Elisa 试剂盒进行测定;
结果:菌株周围有明显且界限清晰的乳白色沉淀,菌株具有良好的胆盐水解酶活性,其定量测定得到总酶活和比酶活分别为65.13%和9.33%;进一步表明了,菌株具有良好的降胆固醇能力,可以应有于体内实验(图2)。
6、菌种鉴定
将挑选出体外降胆固醇能力较高,以及具有较强胆盐水解酶活力的菌株送到上海生工公司进行测序,经过16SrDNA鉴定;结果为,与植物乳杆菌显示出最高的分子***学上的亲缘关系(图3);因此命名为发酵乳杆菌KP101,拉丁文学名Lactobacillus fermentum KP 101,现保存于武汉市中国典型培养物保藏中心(中国武汉大学),保藏时间为2019年1月24日,编号:CCTCC No. M 2019085。
实施例2 益生菌耐受性试验
1、耐酸性评价
分别将菌株按照3%接种量接种于液体MRS培养基,在37℃培养18h。培养液于4℃在4000rpm·min-1离心10min,得到菌泥用0.85%的生理盐水洗涤两次,然后用无菌生理盐水将菌泥悬浮,保证活化的菌株约为108CFU·mL-1,然后按照3%的接种量将菌悬液接种于pH为2.0,3.0的MRS培养基中,分别在0h、2h、4h取样,用生理盐水进行10倍稀释,稀释适当浓度后涂布于MRS固体培养基中,37℃恒温培养48h,菌落记数,每个浓度作三个平行,求其平均值,计算活菌数和存活率;
结果:在pH2.0的条件下,菌株在4h后的存活率仍能达到90%以上,但在pH3.0的条件下,菌株的存活量有明显的增多,说明其具有良好的耐酸能力(图4)。
2、耐胆盐评价
分别将大约为108 CFU·mL-1活化的菌株以3.0%接种量接种于胆盐浓度分别为0%,0.2%,0.3%的MRS培养基中,37℃恒温培养,采用梯度稀释平板计数法,分别在0、2、4 h测定其活菌数,每个浓度作三个平行,求其平均值,计算活菌数和存活率;
结果:在0.3%的胆盐条件下,菌株在4h后的存活率能达到105.07%,在0.5%的胆盐条件下,菌株在4h后的存活率达到102.24%,说明其具有良好的耐胆盐能力(图5)。
3、代谢产物抑菌性评价
利用牛津杯法进行菌株代谢产物的抑菌实验。将活化好的大肠杆菌、金黄色葡萄球菌和沙门氏菌菌液稀释至105 CFU·mL-1,取0.1mL涂布到LB平板上,将已灭菌的牛津杯放于平板中,平行放置两个,取已活化好的目标菌株的发酵上清液,分别吸取200μL放入牛津杯内,然后将平板慢慢放于37℃恒温培养箱,培养24 h,观察并测量抑菌圈直径大小。平行实验三次;
结果见表1,从表中看出发酵乳杆菌KP101对大肠杆菌、金黄色葡萄球菌、沙门氏菌的抑菌直径分别为9.34mm、15.1mm、10.39mm,表明了其了良好的抑菌特性。
4、耐药性评价
目标菌株按1%的接种量接入MRS液体培养基,37℃培养至对数生长期,无菌吸取0.1mL至MRS琼脂平板,涂布均匀,选取6种常用的药敏纸片:四环素、万古霉素、氨苄西林、卡那霉素、庆大霉素、青霉素,无菌镊子夹取放至已涂布菌液的MRS平板上,每个平板放置3种不同药敏纸片,37℃培养24 h;表2结果表明:发酵乳杆菌KP101能够抗四环素、链霉素、卡那霉素和利福平;但是对庆大霉素、氯霉素、红霉素抗生素敏感,但仍在安全浓度的范围内。
5、体外模拟人工胃肠液评价
将菌悬液1mL,转入pH3.0人工胃液9mL,37℃厌氧培养,从0h,2h,4h分别取样平板涂布计数。再从胃液培养液中吸取1mL菌液后接入pH8.0的9mL人工肠液中,得到10mL液体,37℃厌氧培养,从0h,2h,4h分别取样平板涂布计数。
实施例3 抗氧化活性试验
1、H202耐受试验
将发酵乳杆菌KP101的纯培养物接种到补充有不同浓度的过氧化氢(0.5,1.0 mmol/L)的MRS肉汤中;从0h起每隔2h测量600nm处的吸光值,计算存活率;
结果:菌株对过氧化氢的耐受呈浓度依赖关系,菌株对低浓度过氧化氢(0.5mM)具有较强的耐受能力,37℃培养4h存活率明显增多,而对高浓度过氧化氢(1mM)存活率增多较少,8h菌株的存活率也不低于90%,表明发酵乳杆菌KP101对不同浓度过氧化氢均具有较强的耐受性(图6a)。
2、清除DPPH自由基
将菌液4000r/min离心10min,分别得到发酵上清液和菌体细胞,将菌体细胞用0.85%的生理盐水洗涤2遍,悬浮于等体积的生理盐水中,得到菌体细胞液;发酵上清液、菌体细胞液、发酵液均设置对照组、样品组和空白组,分别将发酵上清液,菌体细胞液,发酵液按照如下顺序添加试剂:
1)对照组:2mL 0.04g/L DPPH 无水乙醇溶液 + 2mL无水乙醇;
2)样品组:2mL 0.04g/L DPPH 无水乙醇溶液 + 2mL样品溶液(发酵上清液、菌体细胞液或发酵液);
3)空白组:2mL样品溶液 + 2mL无水乙醇;
充分反应20min后,将各组液体加入到96孔板中,测定其OD517nm;
清除能力公式如下:
清除活性%=[1-(样品-空白)/对照]×100%;
结果(图6b)表明发酵乳杆菌KP101的无细胞提取物、发酵上清液、菌悬液的DPPH清除率分别为33.9%、88.7%、48.6%。
3、清除超氧阴离子能力评价
使用购买于南京建成的超氧阴离子自由基试剂盒进行测定;结果(图6c)表明发酵乳杆菌KP101的无细胞提取物、发酵上清液、菌悬液的超氧阴离子自由基清除能力分别为39.45U/L、136.02 U/L、110.24 U/L。
4、清除羟自由基能力评价
使用购买于南京建成的羟自由基试剂盒进行测定;结果(图6d)表明发酵乳杆菌KP101的无细胞提取物、发酵上清液、菌悬液的羟自由基清除能力分别为701.7U/L、588.7U/L、839.5U/L。综上,表明发酵乳杆菌KP101具有较好的抗氧化能力。
实施例4 动物试验
1、动物的饲养与选择
选择健康成年的C57BL/6N 雄性小鼠30只,将小鼠随机分配为3组,每组10只,饲喂不同饮食,分别为:
A 组:正常对照组(Control):普通饲料;
B 组:高脂模型组(HFD):高脂饲料;
C 组:干预组(HFD+KP101):向高脂饲料粉中加入5%的发酵乳杆菌KP101的发酵液并充分搅拌混合均匀后干燥制成固体饲料。
所述的发酵乳杆菌KP101的发酵液,是将发酵乳杆菌KP101在37℃条件下培养18h,按照培养液3%的量进行接种,连续活化三代;将上述活化并扩大培养的种子液4000r/min离心10min,用无菌水洗涤2遍,离心后,弃上清,得到菌体沉淀,悬浮于等体积的冻干保护剂中,得到1010CFU/mL的菌悬液;将菌悬液以3%接种量接入到装有10mL的MRS-THIO-OX-CHOL培养基中,37°C条件下培养24h。
干预组需要每天将高脂饲料与相应的干预物混合并搅拌均匀,干预物按照50g/kgBW/d 的剂量进行给予,保证干预物的足量摄入。小鼠自由摄食、饮水,饲养环境为12小时循环光照,温度为20±2℃,湿度50±5%。每天记录摄食量,每周称量一次小鼠体重并更换垫料。试验周期为8周,处死前禁食12小时,记录死前体重,麻醉后打开小鼠腹腔和胸腔,迅速取出小鼠各脏器及脂肪,称重并记录。
2、小鼠样本的制备
血液样本制备:采用眼球采血收集血液样本,常温放置一段时间后,以3000rpm·min-1条件下离心10 min,分离出的血清置于1.5 mL EP管中,保存于-80℃冰箱中备用。
肝匀浆液的制备:将肝脏加入9倍体积的生理盐水,充分匀浆制得10%的肝匀浆,将其用低温高速离心机以3000 rpm·min-1条件下离心10min,取上清液,置于-80℃冰箱用于分析。
3、生化指标的检测
小鼠血清及肝脏中总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)等指标均按照购自于南京建成的试剂盒说明书进行操作。
结果及分析:与对照组相比,高脂饮食小鼠的血清中TC、LDL和TG水平显著升高;而发酵乳杆菌KP101的干预能够显著降低血清TC、TG和LDL水平(图7abc)。HDL可以将过多的胆固醇从动脉粥样斑块中移走防止血管壁沉积。与正常对照组相比,高脂组小鼠血清HDL水平显著升高,这可能是由于机体为降低血清胆固醇水平而做出的应激反应;相对于高脂组而言,发酵乳杆菌KP101干预组小鼠血清HDL水平并无显著变化,这说明发酵乳杆菌KP101对血清HDL的影响不大(图7d)。因此,发酵乳杆菌KP101的干预可有效缓解由高脂饮食诱导的血脂代谢异常。
与正常对照组相比,高脂饮食导致小鼠血清中ALT和AST水平升高且均具有显著性差异。发酵乳杆菌KP101有效抑制血清中ALT和AST水平的升高,分别降低了27.09%和14.17%(图8)。
与正常组小鼠相比,高脂饮食导致小鼠肝脏TC、TG和LDL水平均出现不同程度的升高,这表明高脂饮食不仅引起血脂代谢紊乱,对肝脏代谢也产生不同程度的损伤。而经发酵乳杆菌KP101干预后,小鼠肝脏TC、TG和LDL水平均有下降,分别降低了13.4%、50.5%和67.0%(图9abc)。然而,与正常饮食组小鼠相比,高脂饮食导致小鼠肝脏HDL水平显著降低(p<0.001);与高脂饮食组小鼠相比,发酵乳杆菌KP101干预组小鼠肝脏HDL水平显著升高(p<0.01) (图9d)。
与正常对照组相比,高脂饮食导致小鼠血肝脏中ALT和AST水平升高且具有显著性差异。发酵乳杆菌KP101有效抑制肝脏中ALT和AST水平的升高,分别降低了33.34%和33.02%。结果表明,发酵乳杆菌KP101对高脂饮食引起的肝损伤及炎症反应具有一定的缓解和改善作用(图10)。
与正常组小鼠相比,高脂组小鼠体重显著增加,而经发酵乳杆菌KP101干预后,其体重却显著降低(图11a)。与正常对照组小鼠相比,高脂饮食显著地提高小鼠腹部脂肪系数和肾周脂肪系数,而经发酵乳杆菌KP101干预后,其腹部脂肪和肾周脂肪系数分别降低了95.5%和97.2%,其中肾周脂肪系数下降显著(图11bc)。
实施例5 本发明菌株与现有发酵乳杆菌性能比较
基于本发明发酵乳杆菌KP101的各项指标测定结果,与现有的发酵乳杆菌进行了对比,根据表4所列出的结果可以看出,本发明发酵乳杆菌KP101取得了良好的效果;
综合上述实施例和表格比较结果,本发明发酵乳杆菌KP101同时具有良好的耐受酸胁迫、耐受胆盐胁迫、耐受氧化胁迫、抗致病菌感染、调节代谢紊乱、调节高血脂症等多种健康功效,同时在减肥降脂方面效果显著。大量研究表明,发酵乳杆菌能够有效的调节炎症性肠病,调节肠道菌群结构,治疗高血脂症。Radha Yadav等研究发现,补充发酵乳杆菌MTCC:5898-发酵的水牛奶,具有降低胆固醇的潜力,能够降低大鼠血清中总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白(LDL-c)的含量;但是,本发明发酵乳杆菌KP101对小鼠血清TG、LDL-c的调节能力更为显著,能够有效的降低由高脂饮食引起的血清中TG、LDL-c的增多,分别为50.5%、67.0%。发酵乳杆菌KP101不仅能够降低血清中TC、TG、LDL-C的含量,并且能够显著提高肝脏中HDL-C水平,增加率为78.9%,降低肝脏中TC、TG和LDL-C的含量,分别为36.2%、64.6%和42.8%;同时,发酵乳杆菌KP101还具有极显著的降脂作用,能够显著的降低其肾周脂肪系数及腹部脂肪系数分别为97.2%和95.5%;目前,对于发酵乳杆菌对于减肥降脂方面的研究较少,大多研究发酵乳杆菌在炎症等方面的内容。本发明发酵乳杆菌KP101,表现了其良好的能够降低由高脂饮食引起的脂肪性肥胖的能力。
实施例6 发酵乳杆菌KP101发酵酸奶的制备
(1)牛奶的制备:将检验合格的新鲜牛奶过滤净化,按比例加入5%-10%的白砂糖,过滤搅拌均匀后预热至60℃,在20MPa的条件下高压均质5min,在95℃条件下灭菌5-10min后,冷却待用;
(2)菌株的活化:将发酵乳杆菌KP101和嗜热链球菌的冻干菌粉接种于无菌牛奶培养基中,分别按最适温度进行培养,传代2~3次,每次接种量3%,活菌数达到106CFU/mL以上;
(3)发酵酸奶的制备:分别将发酵乳杆菌KP101和嗜热链球菌按体积比1:1的比例混合,制得发酵剂,按照3%-5%的接种量接种至已经冷却好的牛奶中,在42℃条件下培养发酵4h后,放置在4℃条件下冷却储藏。发酵乳杆菌KP101活菌数达到106CFU/mL以上。
实施例7 益生菌固体饮料的制备
(1)发酵乳杆菌KP101菌体制备:将发酵乳杆菌KP101在37℃条件下培养18h,按照培养液3%的量进行接种,连续活化三代。将上述活化并扩大培养的种子液4000r/min离心10min,用无菌水洗涤2遍,离心后,弃上清,得到菌体沉淀,悬浮于等体积的冻干保护剂中,得到1010CFU/mL的菌悬液;
(2)保护剂制备:在水溶液中分别添加脱脂奶粉10%-12%,纤维素7%-9%,海藻糖3%-5%,普兰多糖6%-8%,在80℃,20min的条件下灭菌,制成冻干保护剂;
(3)冷冻干燥制备发酵乳杆菌冻干粉;
(4)系列益生菌固体饮料(3g)配方:蓝莓风味固体饮料:发酵乳杆菌冻干粉1 g,低聚异麦芽糖0.5-1.0g,大豆低聚糖0.5-1.0g,蓝莓粉0.5-1.5g;蔓越莓风味固体饮料:发酵乳杆菌冻干粉1 g,低聚异麦芽糖0.5-1.0g,大豆低聚糖0.5-1.0g,蔓越莓粉0.5-1.5g;山楂风味固体饮料:发酵乳杆菌冻干粉1 g,低聚异麦芽糖0.5-1.0g,大豆低聚糖0.5-1.0g,山楂粉0.5-1.5g,制得不同口味的益生菌固体饮料;
(5)产品规格:固体饮料采用3g为1包,添加的发酵乳杆菌KP101的活菌数为1010CFU/mL。
Claims (8)
1. 一株发酵乳杆菌Lactobacillus fermentum KP 101,它的保藏编号为CCTCC No. M2019085。
2.一种发酵酸奶,它是由下述方法制备的:
1)菌株的活化:将保藏编号为CCTCC No. M 2019085的发酵乳杆菌KP101和嗜热链球菌的冻干菌粉接种于无菌牛奶培养基中,分别按最适温度进行培养,传代2~3次,每次接种量1~10%,活菌数达到106CFU/mL以上;
2)发酵酸奶的制备:将活化后的发酵乳杆菌KP101和嗜热链球菌按体积比1~2:1的比例混合,按照1%~10%的接种量接种至牛奶中,在38~45℃下发酵3~5h,在0~4℃下冷却,得到发酵酸奶。
3.根据权利要求2所述的一种发酵酸奶,其特征在于:步骤2)所述的按照3%~5%的接种量接种至牛奶中,在40~43℃下发酵3~5h。
4.根据权利要求3所述的一种发酵酸奶,其特征在于:步骤2)所述的牛奶,是由下述方法制备的:过滤、净化,加入质量分数5%~10%的糖,过滤,搅拌均匀,加热至55~65℃,在18~22MPa下均质3~10min,在95~100℃条件下灭菌5~10min后,冷却。
5.一种益生菌固体饮料,它包括下列重量份数的组分:发酵乳杆菌冻干粉10份,低聚异麦芽糖5~10份,大豆低聚糖5~10份,果味粉5~15份;所述的发酵乳杆菌,它的保藏编号为CCTCC No. M 2019085。
6.根据权利要求5所述的一种益生菌固体饮料,其特征在于:所述的果味粉为蓝莓粉、蔓越莓粉或山楂粉。
7.根据权利要求6所述的一种益生菌固体饮料,其特征在于:所述的发酵乳杆菌冻干粉,是由下述方法制备的:将发酵乳杆菌KP101活化,扩大培养,在3000~5000r/min下离心8~15min、用水洗涤,离心弃上清,得到菌体;将菌体和冻干保护剂按体积比1~3:1混合,冷冻干燥,得到发酵乳杆菌冻干粉。
8.根据权利要求7所述的一种益生菌固体饮料,其特征在于:所述的冻干保护剂,按质量百分比计包括下列组分:脱脂奶粉10%-12%、纤维素7%-9%、海藻糖3%-5%、普兰多糖6%-8%、余量为水。
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