CN112972505B - 一株发酵乳杆菌在制备具有降甘油三酯功能的食品或药品中的应用 - Google Patents
一株发酵乳杆菌在制备具有降甘油三酯功能的食品或药品中的应用 Download PDFInfo
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- CN112972505B CN112972505B CN202110214751.3A CN202110214751A CN112972505B CN 112972505 B CN112972505 B CN 112972505B CN 202110214751 A CN202110214751 A CN 202110214751A CN 112972505 B CN112972505 B CN 112972505B
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
本发明属于微生物技术领域，特别涉及一株发酵乳杆菌在制备具有降甘油三酯功能的食品或药品中的应用，所述的发酵乳杆菌命名为发酵乳杆菌(Lactobacillus fermentum)HCS08‑005，该菌株已于2018年08月14日保藏在“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.16259。本发明所述的发酵乳杆菌(Lactobacillus fermentum)HCS08‑005具有降甘油三酯功能，降解率达38.1％；且具有优秀的耐消化道逆环境能力。当从口摄入进入人体时，能够耐受胃酸及胆盐环境以较高的存活率到达肠道。
Description
技术领域
本发明属于微生物技术领域，特别涉及一株具有降甘油三酯功能的发酵乳杆菌及其应用。
背景技术
如今人们的生活水平越来越高，饮食结构的改变及缺乏运动等生活习惯，出现高血脂的人群比例越来越高，据某项统计数据显示，我国成年人中，出现高血脂问题的比例大约在40％左右，而且这个比例仍然在不断扩大，不断的向低龄化的趋势发展。甘油三酯是血脂中的一种，体内甘油三酯过高会引发人体许多疾病，如高血压、动脉粥样硬化、心血管疾病等。目前防治高脂血症的主要办法有合理饮食、适量运动、适当的理疗和药物治疗，但是对于严重的高脂血症进行长期的药物治疗会出现明显的副作用，包括睡眠问题、腹泻或便秘、记忆或精神问题等。服用安全有效的具有降解甘油三酯功效的菌制剂食品或药品，可以避免或降低现有药物的副作用，但根据现有研究关于菌制剂的甘油三酯降解率普遍较低，列举如表1。
表1列举现有研究公开的菌制剂甘油三酯降解率
本发明中的发酵乳杆菌具有较强的降甘油三脂功能，为高脂血症的人群治疗提供更多的选择。
发明内容
针对现有问题，本发明目的在于提供一株耐酸耐胆盐能力强、耐胃肠液能力强、具有降甘油三酯功能的发酵乳杆菌及其应用，尤其在食品或药品组合物中的应用。
为实现上述目的，一株发酵乳杆菌在制备具有降甘油三酯功能的食品或药品中的应用，所述的发酵乳杆菌命名为发酵乳杆菌(Lactobacillus fermentum)HCS08-005，该菌株已于2018年08月14日保藏在“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.16259。
上述的应用，所述的食品为菌剂或发酵食品、饮料。
一种发酵食品，是以上述的一株具有降甘油三酯功能的发酵乳杆菌作为发酵剂生产的食品。
上述的一种发酵食品，所述的发酵食品是以上述的一株具有降甘油三酯功能的发酵乳杆菌作为发酵剂得到的酵素液。
一种药物制剂，包括有效剂量的上述的一株具有降甘油三酯功能的发酵乳杆菌和药学上可接受的辅料。
一种菌剂，是含有上述的具有降甘油三酯功能的发酵乳杆菌的菌剂。
本发明所述的发酵乳杆菌(Lactobacillus fermentum)HCS08-005分离自顺产健康2月龄的男婴粪便中，是一株耐消化道逆环境能力强的发酵乳杆菌，具有发酵乳杆菌的典型特征，革兰氏阳性，兼性厌氧，无芽孢，菌落呈圆形，乳白色，表面光滑，中间有凸起，边缘整齐，直径约2-4mm；细胞呈短杆状，直径长度比约为1：2～1：4，单个或成对排列。其理化特征是：接触酶阴性，氧化酶阴性，上述发酵乳杆菌CGMCC No.16259的细胞形态和理化实验结果详见表2。
表2发酵乳杆菌CGMCC No.16259的细胞形态和理化实验结果
通过在美国生物工程信息中心(National Center for BiotechnologyInformation，NCBI)查阅国际相关的基因库，将本发明的发酵乳杆菌CGMCC No.16259的16SrDNA序列与GenBank提交菌株序列相似性比较，鉴定菌株HCS08-005为发酵乳杆菌(Lactobacillus fermentum)，比较结果见表3。
表3发酵乳杆菌CGMCC No.16259的16S rDNA序列与GenBank提交菌株序列相似性比较
本发明与现有技术相比，具有以下有益效果：
1、本发明发酵乳杆菌(Lactobacillus fermentum)HCS08-005具有降甘油三酯功能，降解率达38.1％；
2、本发明发酵乳杆菌(Lactobacillus fermentum)HCS08-005具有优秀的耐消化道逆环境能力。当从口摄入进入人体时，能够耐受胃酸及胆盐环境以较高的存活率到达肠道；
3、本发明发酵乳杆菌(Lactobacillus fermentum)HCS08-005分离自健康的人源粪便中，应用时安全性高；
4、菌种纯化及发酵过程的培养基及中和剂采用的组分原料均为食品级，故菌株安全性高，可用于食品发酵或冻干菌粉等应用；
5、益生菌对致病菌的抑制特性主要表现在抑制致病菌的生长，防止细菌性腹泻，发挥益生菌功能，提高宿主免疫力，维持肠道微生态平衡。本发明的发酵乳杆菌(Lactobacillus fermentum)HCS08-005具有较好的抑菌特性。
本发明所述的具有降甘油三酯功能的发酵乳杆菌，所述的发酵乳杆菌，所述菌株命名为发酵乳杆菌(Lactobacillus fermentum)HCS08-005，该菌株已于2018年08月14日保藏在“中国微生物菌种保藏管理委员会普通微生物中心”，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.16259。
具体实施方式
实施例1发酵乳杆菌(Lactobacillus fermentum)HCS08-005 CGMCC No.16259菌株的筛选及生理生化特性
婴儿粪便5g加到5mL缓冲蛋白胨液中，以10倍稀释法稀释至10-3，每个稀释度划线到MRS改良培养基平板上。37℃培养24～48h(平板放入自封袋中)；挑取具有目的菌株典型特征(观察形态、大小、色泽、及其透明度等)、菌落较大、活性较强的单菌落，于MRS改良培养基进行划线纯化培养，如此反复2～3次，直到划线平皿中菌落特征一致；每个纯化后的平皿挑取2个以上单菌落进行涂片、革兰氏染色，并且在显微镜下观察其颜色、菌体形状是否一致，从而确定该平皿中菌落是否为纯培养物。如镜下观察结果一致，则将得到的纯培养物(平皿菌落)作为疑似菌株，将对应平皿编号，待鉴定；如镜下观察结果不一致，则继续进行上述操作。
所述的改良MRS培养基：蛋白胨10g/L，牛肉粉3g/L，酵母粉4g/L，磷酸氢二钾2g/L，柠檬酸三铵2g/L，乙酸钠5g/L，葡萄糖20g/L，七水合硫酸镁0.58g/L，四水合硫酸锰0.15g/L，吐温-80 0.6g/L，碳酸钙10g/L，中性红0.05g(1％浓度5mL)，番茄汁10mL/L(v/v)，琼脂粉18g/L，调pH至6.0；115℃，30min灭菌；
实施例2发酵乳杆菌(Lactobacillus fermentum)HCS08-005 CGMCC No.16259菌株的鉴定
将实施例1中分离纯化后的纯培养物通过划线、涂片镜检，进一步确定其为纯培养物后进行菌种鉴定，包括革兰氏染色试验，接触酶试验，糖发酵鉴定，API鉴定，及16S rDNA全序列测序鉴定。最终鉴定分离得到的菌株为一株发酵乳杆菌，命名为发酵乳杆菌(Lactobacillus fermentum)HCS08-005，已于2018年08月14日保藏在“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.16259。
上述发酵乳杆菌(Lactobacillus fermentum)HCS08-005菌株为革兰氏阳性菌，兼性厌氧，无芽孢，不运动；菌落较小且大小均一，边缘整齐，表面光滑细密，白色或乳白色；菌体呈短杆状，成堆或成链排列；其生理生化特征是：其生理生化特征是：接触酶阴性，氧化酶阴性，可利用D-核糖、D-半乳糖、D-葡萄糖、D-果糖、D-甘露糖、麦芽糖、乳糖、蜜二糖、蔗糖、棉子糖。
实施例3发酵乳杆菌(Lactobacillus fermentum)HCS08-005 CGMCC No.16259菌株的耐消化道逆环境试验
发酵乳杆菌(Lactobacillus fermentum)HCS08-005耐消化道的逆环境能力是其能够以较高存活率到达肠道并在肠道存活、定植生长及发挥功效的先决条件。
1、耐酸试验
将传代三次的菌液按照10％的接种量分别接种于空白对照、pH2.0和pH3.0的MRS培养基中。37℃静置培养，于17h取样，用灭菌的生理盐水进行10倍系列稀释，分别取适宜稀释度的菌液1000μL进行活菌计数操作，每个稀释度2次重复，37℃静置培养36～48h后计数。
耐酸试验数据指标：
测得空白对照的活菌数用N0表示，其他pH条件下测得的活菌数用N′表示，其耐酸性活菌数对数比率计算公式如下：
受试菌株耐酸性试验存活率(％)＝lgcfuN′/lgcfu N0×100％；
表4 HCS08-005耐酸试验数据表
由表4可知，HCS08-005菌株在pH 3.0的条件下经过17h后，存活率达87.2％；pH2.0条件下处理17h存活率达50.1％，这个存活率可保证其通过胃酸的抑制作用仍保持较高活性，从而发挥其益生作用。
2、耐胆盐试验
将传代三次的菌液按照10％的接种量分别接种于未含有进口牛胆盐(空白对照)、含有0.3％、0.5％、1.0％、1.5％进口牛胆盐浓度的MRS液体培养基，37℃静置培养，于17h取样，测定活菌数。
耐胆盐试验数据指标：
测得空白对照的活菌数用N0表示，其他胆盐浓度条件下测得的活菌数用N′表示，其耐胆盐活菌数对数比率计算公式如下：
受试菌株耐胆盐试验存活率(％)＝lgcfuN′/lgcfu N0×100％；
表5 HCS08-005耐胆盐试验数据表
由表5可知，虽然HCS08-005菌株随胆盐浓度的升高，活菌数逐渐下降，但是在1.5％胆盐浓度处理条件下，存活率仍然达93％。
3、模拟胃液试验
将传代三次的菌液震荡摇匀，取菌悬液10mL离心(5000×g，10min，5℃)获得菌泥，用PBS缓冲液冲洗3次，获得的菌泥重悬于10mL模拟胃液中，37℃条件下消化3h，分别于0h、3h取样测活菌数。
经在模拟胃液培养基中消化0h、3h后计数测得的活菌数分别用N和N″表示，受试菌株模拟胃液试验存活率计算公式如下：
受试菌株模拟胃液试验存活率(％)＝lgcfu N″/lgcfuN×100％；
表6 HCS08-005模拟胃液试验数据表
由表6可知，HCS08-005菌株在模拟胃液中的存活能力较强，经3h处理，存活率可达到83.6％，其在胃内停留较长时间仍保持较高活性。
4、模拟肠液试验
将传代三次的菌液震荡摇匀，取菌悬液10mL离心(5000×g，10min，5℃)获得菌泥，用PBS缓冲液冲洗3次，获得的菌泥重悬于10mL的人工肠液中，37℃条件下培养，于0h、2h、4h分别取样测活菌数。
经在模拟肠液培养液中培养0h的活菌数用N表示，经在模拟肠液培养液中培养2h、4h后计数测得的活菌数用N″表示，受试菌株模拟肠液试验存活率计算公式如下：
受试菌株模拟肠液试验存活率(％)＝lgcfuN″/lgcfuN×100％；
表7 HCS08-005模拟肠液试验数据表
由表7可知，HCS08-005菌株在模拟肠液中的存活能力强，随处理时间延长，活菌数基本没有下降，存活率高达约100％。
综上，说明该菌株具有较强的耐酸耐胆盐能力，且能够有效抵抗胃肠液的影响，从而能够在通过消化道后依然维持较高活性。
实施例4发酵乳杆菌(Lactobacillus fermentum)HCS08-005 CGMCC No.16259降甘油三酯体外试验
1、试验菌株制备
(1)一级培养：将保藏于-80℃的菌株冻存管取出，经手握解冻，混匀，取1环菌液于MRS固体培养基平板划线，37℃静置培养36～48h；
(2)二级培养：挑单菌落至5mL MRS培养基中，37℃培养20h；
(3)三级培养：5mL菌液接种至100mL MRS培养基中，37℃培养17h；
(4)四级培养：将三级培养的菌液以3％接种量接种于100mL甘油三酯培养基中，37℃培养24h。同时将未接种菌液的甘油三酯培养基37℃培养24h。
MRS培养基：酵母蛋白胨10g/L，牛肉粉3g/L，酵母粉4g/L，磷酸二氢钾2g/L，柠檬酸2g/L，乙酸钠5g/L，葡萄糖20g/L，七水合硫酸镁0.58g/L，四水合硫酸锰0.25g/L，吐温-800.6g/L，番茄汁10mL/L，琼脂20g/L，调pH至6.5；115℃，30min灭菌。
甘油三酯培养基配方的制备：将2％的聚乙烯醇水溶液与植物油按体积比3：1混合，用超声波处理(控制参数每次超声5s，间隔时间5s，共超声70次)后混合均匀制成植物油乳化液，作为甘油三酯的来源。将上述制备的植物油乳化液按5％的比例加入到MRS培养基中，调pH至6.5±0.2，115℃，30min灭菌，制成甘油三酯培养基并于4℃冰箱贮存备用。
2、单试剂法甘油三酯含量测定试验(GPO-PAP法)
将四级培养后的菌液及未接种菌液的甘油三酯培养基分别取10mL用冷冻离心机，4℃，4000r/min离心10min，取上清液，按甘油三酯试剂盒(南京建成生物试剂公司A110-1-1)说明用96孔板将无菌蒸馏水、校准品、样本各取2.5μL分别加入空白孔、校准孔及样本孔中，每孔再加250μL工作液混匀，37℃孵育10min，每组三个平行，用酶标仪测定其510nm处的OD值。
3试验结果计算方法
以如下计算公式计算乳酸菌的TG降解率。
总TG含量——未接种菌液的甘油三酯培养基中甘油三酯(TG)含量；
残留TG含量——接种菌液四级培养后的甘油三酯培养基中甘油三酯(TG)含量。
发酵乳杆菌(Lactobacillus fermentum)HCS08-005 TG降解率为38.1％。
实施例5对照菌株降甘油三酯体外试验
试验选取了现有部分商品菌株进行对照，采用相同的GPO-PAP法测定甘油三酯降解率。试验结果见表8，对照菌株中甘油三酯降解率最高的为丹麦科汉森的动物双歧杆菌乳亚种BB-12菌株，其TG降解率为39.3％；其次为丹麦科汉森的植物乳杆菌vegestar 2.0，其TG降解率为35.7％。根据实施例4测定结果，发酵乳杆菌(Lactobacillus fermentum)HCS08-005的TG降解率为38.1％，降解能力较强。
表8对照菌株甘油三酯降解率
实施例6菌株抑菌功能特性实验
指示菌株准备：取实验菌株冻存管在37℃水浴锅中使其迅速溶化，用微量移液器取100微升菌液加入10ml液体培养基中，大肠杆菌/金黄色葡萄球菌/鼠伤寒沙门氏菌/志贺氏菌37℃恒温过夜培养。
制备固体培养基，将培养至对数期的致病菌稀释至菌液浓度为105CFU/mL，取菌悬液100μL均匀涂布于固体培养基上，用镊子将无菌牛津杯竖于涂布有菌液的平皿中，每个牛津杯中加入200μL受试样品。将平皿置于适宜温度的恒温培养箱中，培养适当时间后观察拍照，并用游标卡尺计量抑菌圈直径。
实验结果见表9。表9中采用牛津杯法，牛津杯内直径6mm，外直径8mm；使用受试菌株发酵液上清，分别进行3倍、5倍浓缩以及直接取原上清进行试验。
表9抑菌谱记录表
通过试验结果可以得出，HCS08-005对胃肠道中主要致病菌有明显抑菌作用。
实施例7发酵乳杆菌(Lactobacillus fermentum)HCS08-005 CGMCC No.16259培养方法
1、冻存菌种复苏：取存于低温冰箱内的菌种冻存管，立即放入37℃水浴锅内进行菌种复苏，15～30s，至冻存管内的固体全部融化；
2、一级培养：将复苏好的1mL冻存管菌种接种至装有10mL基础培养基试管中，35℃培养箱恒温静置培养16.5±0.5h；
3、二级培养：将菌种活化所得菌悬液按5％接种量接种于100mL基础培养基中，装液量80％，35℃静置培养8±0.5h；
4、三级培养：将二级培养的菌悬液按5％接种量接种到300mL优化培养基中，装液量80％，35℃静置培养17.5±0.5h。
所述的基础培养基配方：葡萄糖30.0g/L、酵母蛋白胨22.0g/L、酵母浸出物6.0g/L、柠檬酸4.0g/L、余量为纯化水；按照配方比例称量、加热溶解，用1mol/L食品级NaOH溶液调培养基pH值至7.00，115℃下灭菌30min；
所述的优化培养基配方：酵母蛋白胨30.0g/L、葡萄糖25.0g/L、酵母浸出物10.0g/L、乳糖20.0g/L、结晶果糖5.0g/L、柠檬酸4.0g/L、L-苹果酸5.0g/L、余量为纯化水；按照配方比例称量、加热溶解，115℃下灭菌30min，用1mol/L食品级NaOH溶液调培养基pH值至6.20；
上述发酵乳杆菌(Lactobacillus fermentum)HCS08-005三级培养所得的菌悬液活菌数为5.8×109cfu/mL。
实施例8发酵乳杆菌(Lactobacillus fermentum)HCS08-005 CGMCC No.16259降甘油三酯酵素液
发酵乳杆菌(Lactobacillus fermentum)HCS08-005 CGMCC No.16259降甘油三酯酵素液，其制备方法如下：
1、菌种活化
以发酵乳杆菌HCS08-005冻干粉、鼠李糖乳杆菌HCS01-013冻干粉、动物双歧杆菌乳亚种HCS04-001冻干粉为发酵菌种，按接种量称量后用无菌水溶解，接种至优化培养基中，培养16±0.5h，待菌液pH＜4.55、OD＞4.00时发酵结束。
所述的接种量：发酵乳杆菌HCS08-0050.0294％，鼠李糖乳杆菌HCS01-0130.0003％，动物双歧杆菌乳亚种HCS04-0010.0003％，无菌水1.5％。
所述的优化培养基配方为：葡萄糖25g/L，酵母蛋白胨25g/L，酵母浸出物20g/L，乙酸钠10g/L，柠檬酸2g/L，磷酸二氢钾2g/L，七水合硫酸镁0.06g/L，余量用纯化水，pH调至6.60，115℃灭菌30min。
2、菌种发酵
将菌种活化后的发酵液以5％的接种量接种至发酵原液中，装液量为60～75％，搅动培养，搅拌速度为60rpm，通气量为0，初始气压为0.02～0.03MPa，37℃培养40±4h；发酵进行36h开始，每小时监控发酵液pH值，当接连两次pH值相差小于0.1，且pH＜3.25，发酵结束，得酵素液，此时酵素液状态为深褐色液体，流动性较好。
所述的发酵原液配方及制备：决明子50g/L，菊花10g/L，葡萄糖10g/L，低聚异麦芽糖10g/L，枸杞5g/L，生姜5g/L，黄精1g/L，余量为纯化水。将决明子、菊花、枸杞、生姜、黄精原料预处理后，投入到多功能提取罐中，加入一定量的水(超过物料表面)浸泡5～10h后开始蒸煮，同时搅拌(60～100rpm)，温度达到100℃时开始计时，煮沸40～50min，冷却至50～70℃，过滤，收集滤液，按比例添加葡萄糖和低聚异麦芽糖，加水定容，混合均匀后将其冷却至室温，调节pH值至6.60，115℃灭菌30min。
实施例9发酵乳杆菌(Lactobacillus fermentum)HCS08-005 CGMCC No.16259降甘油三酯酵素液功效试验
试验选用辽宁长生生物技术有限公司提供的清洁级昆明种(ICR)小鼠40只，18-22g，雄性。无菌块状鼠料由辽宁长生生物技术有限公司提供，检验条件为清洁级动物实验室，温度22±1.5℃，湿度50％±10％，工作照度160-280lx，噪音<60dB。受试物即发酵乳杆菌(Lactobacillus fermentum)HCS08-005降甘油三酯酵素液。
1、高脂动物模型的建立
将40只ICR小鼠随机分为3组，分别为正常对照组、高脂模型组、酵素液干预组，每组10只，给予不同的饲料喂养6周。对照组给予标准饲料及生理盐水灌胃；高脂模型组给予高脂饲料及生理盐水灌胃；酵素液干预组给予高脂饲料同时给予试验酵素液灌胃。
2、观察指标及检测方法
(1)血清TG含量的测定
各组分别饲养6周后，小鼠禁食不禁水12小时，眼底静脉丛取血，5000r/min，4℃离心15min取血清，采用酶比色法按照试剂盒操作说明测定各组小鼠血清TG的含量。
(2)脏器指数测定
各组试验小鼠分别饲养6周后，各组小鼠禁食不禁水12小时，取血处死后，腹部解剖，获取肝脏、心脏、肾3个主要脏器，用生理盐水洗净血迹，滤纸吸干，分析天平称取重量，并计算脏器指数。
脏器指数＝脏器质量(mg)/体重(g)。
3、试验结果
(1)血清中TG含量测定结果
表10各组小鼠TG含量结果
根据表10TG含量测定结果，酵素液干预组小鼠血清TG含量明显低于高脂模型组，说明试验酵素液可以有效的降低高脂血症小鼠的甘油三酯水平。
(2)脏器指数测定结果
表11各组小鼠脏器指数结果
各组小鼠脏器指数测定结果见表11，高脂模型组的肝脏指数较正常对照组明显升高，酵素液干预组的肝脏指数较高脂模型组下降明显，说明试验酵素液能够降低脂肪在小鼠肝脏的聚集。
Claims (2)
1.一株具有降甘油三酯功能的发酵乳杆菌在制备具有降甘油三酯功能的食品或药品中的应用，所述的发酵乳杆菌命名为发酵乳杆菌(Lactobacillus fermentum)HCS08-005，该菌株已于2018年08月14日保藏在“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.16259。
2.根据权利要求1所述的应用，其特征在于，所述的食品为菌剂或发酵食品、饮料。
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