CN111269850A - 一株具有高黏附能力及降血脂功效的戊糖片球菌pp04 - Google Patents
一株具有高黏附能力及降血脂功效的戊糖片球菌pp04 Download PDFInfo
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- CN111269850A CN111269850A CN202010064690.2A CN202010064690A CN111269850A CN 111269850 A CN111269850 A CN 111269850A CN 202010064690 A CN202010064690 A CN 202010064690A CN 111269850 A CN111269850 A CN 111269850A
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Abstract
本发明公开了一株戊糖片球菌Pediococcus pentosaceus PP04,它的保藏编号为CCTCC No:M 2019082;一种发酵豆乳,制备方法:1)菌株的活化;2)菌种的驯化:将戊糖片球菌PP04、嗜热链球菌及保加利亚乳杆菌分别接种于脱脂乳与豆乳的混合培养液中,40~45℃下驯化;3)发酵剂的制备:将驯化后菌种按1~10%的接种量分别接种于豆乳中,40~45℃下培养,得到3种母发酵剂;4)发酵豆乳的制备:将3种母发酵剂按一定比例混合,接种于豆乳中,发酵3~7h;本发明用平板涂布计数法记录戊糖片球菌PP04对Caco‑2细胞的黏附率为79.06%,计数比革兰氏染色法更准确。
Description
技术领域
本发明属于功能性食品技术领域,具体涉及一株具有高黏附能力及降血脂功效的戊糖片球菌PP04。
背景技术
随着社会经济水平的提高,我国的饮食结构逐渐趋向于高脂肪和高动物蛋白的西方饮食模式,使得高脂血症又称高脂蛋白血症(hyperlipoproteinemia)的发病率逐年升高,继而引发多种代谢性紊乱疾病,例如,肥胖、非酒精性脂肪性肝病、动脉粥样硬化、冠心病、高血压等。高脂血症指脂肪代谢过程中,血浆中的几种脂质水平比正常值偏高,主要表现为血清胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白胆固醇(LDL-C)比正常水平偏高。同时大量研究表明高脂饮食诱导肥胖的发生过程中会加快机体代谢速率,使得细胞内还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化途径被激活,ROS增多,造成氧化应激,导致机体氧化还原平衡状态被破坏。脂肪组织中发生氧化应激会使脂肪细胞因子分泌失调,因此降低机体氧化应激程度成为治疗肥胖相关代谢性综合症的一个主要治疗靶点。目前针对高脂血症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。饮食干预指通过合理的膳食结构维持脂质代谢平衡,基本原则是低胆固醇、低脂、低糖、高膳食纤维。但该方法仅在短期内有效且难以长久坚持,治疗效果逐渐变差;药物治疗效果明显,但降血脂药物往往价格昂贵且副作用大。
益生菌经人体摄入后在一定程度上能耐受胃液,胆汁和胰脏分泌物作用而黏附于肠道上皮细胞,以活菌形式定植于肠道中是其发挥益生作用的前提条件。目前研究表明,乳酸菌对肠道上皮细胞的黏附作用有助于其在肠道定植、增强乳酸菌与肠道细胞之间的信号交流、抑制病原菌在肠道的定植。黏附性的评价方法分为直接评价法和间接评价法。一方面利用Caco-2、HT-2和Hela细胞模型进行体外试验直接研究乳酸菌的黏附性。其中,Caco-2细胞为人结肠腺癌上皮细胞,形态和功能与正常的小肠上皮细胞非常接近,比如含有肠道上皮细胞绒毛以及相应的酶。另一方面利用细菌表面的物理化学性质如表面疏水性和自聚集能力可间接反映黏附性强弱,二者均与黏附能力呈正相关。
从二十世纪七十年代时起,国外在动物模型中进行了对乳酸菌降血脂作用的诸多研究。Park 发现用L. acidophilus ATCC 43121 菌株饲喂高脂饮食诱导的高脂血症大鼠21 天后,不仅血清中的TC 水平显著性降低,而且LDL-C、IDL-C 和VLDL-C含量都有所降低;Ding Wurong 等发现植物乳杆菌Lp3 干预喂养可显著降低高胆固醇饮食诱导的大鼠血清和肝脏中胆固醇及甘油三酯水平,并抑制了大鼠肝组织细胞中的脂质沉积。通过大量动物试验和临床试验已经发现,众多乳酸菌如部分植物乳杆菌、嗜酸乳杆菌、干酪乳杆菌、鼠李糖乳杆菌等都具有调节人体血清胆固醇水平的功效,主要表现为能显著降低实验动物血清及肝脏中TC和LDL-C等脂质水平,但鲜有报道以下调机体氧化应激程度为靶点研究乳酸菌的降血脂功能。乳酸菌作为价格低廉,安全性高的一类物质,通过服用乳酸菌制剂调节和控制脂质水平对于高血脂症的预防及辅助治疗具有很大的开发前景。
发明内容
本发明目的是提供一种具有较高肠道黏附及定植能力、能够缓解高脂血症、适合我国居民服用的戊糖片球菌PP04。
一株戊糖片球菌Pediococcus pentosaceus PP04,它的保藏编号为CCTCC No: M2019082。
一种发酵豆乳,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 2019082的戊糖片球菌PP04、嗜热链球菌及保加利亚乳杆菌接种于MRS液体培养基中,40~45℃培养12~18 h,传代3次,收获菌体;将收获的戊糖片球菌PP04、嗜热链球菌及保加利亚乳杆菌按1~3%的接种量接种于脱脂乳培养基中,在40~45℃下传代2次;
2)菌种的驯化:将戊糖片球菌PP04、嗜热链球菌及保加利亚乳杆菌按1~5%的接种量,分别接种于脱脂乳与豆乳的混合培养液中,40~45℃下驯化培养;
3)发酵剂的制备:将驯化后戊糖片球菌PP04、保加利亚乳杆菌和嗜热链球菌菌种按1~10%的接种量分别接种于豆乳中,40~45℃下培养,得到3种母发酵剂;
4)发酵豆乳的制备:分别将步骤3)得到的戊糖片球菌PP04、保加利亚乳杆菌和嗜热链球菌3种母发酵剂,以体积比1~1.5:0.5~1:1~1.5混合,按接种量为1~10%接种于豆乳中,添加质量百分比3~10%的蔗糖,40~45℃下发酵3~7h;
所述的豆乳,是由下述方法制备的:大豆,水浸泡10~15 h,碱水浸泡2~3 h,磨浆,过滤除渣,加糖,煮沸,80~90℃下均质3~7 min,杀菌,冷却;
步骤3)所述的培养,是培养至豆乳凝乳结束;
步骤4)所述的以体积比1:0.5:1混合,接种量为1~5%。
一种益生菌固体饮料,它包括下列重量份数的组分:戊糖片球菌PP04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;所述的戊糖片球菌PP04,它的保藏编号为CCTCC No:M 2019082;
所述的果味粉为蓝莓粉或山楂粉。
所述的戊糖片球菌PP04冻干粉,是由下述方法制备的:将戊糖片球菌PP04活化,扩大培养,在3000~5000r/min下离心8~15 min、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,得到戊糖片球菌PP04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6%-10%、海藻糖4%-8%、菊粉6%-9%、余量为水。
本发明提供了一株戊糖片球菌Pediococcus pentosaceus PP04,它的保藏编号为CCTCC No: M 2019082;一种发酵豆乳,它是由下述方法制备的:1)菌株的活化;2)菌种的驯化:将戊糖片球菌PP04、嗜热链球菌及保加利亚乳杆菌按1~5%的接种量,分别接种于脱脂乳与豆乳的混合培养液中,40~45℃下驯化;3)发酵剂的制备:将驯化后菌种按1~10%的接种量分别接种于豆乳中,40~45℃下培养,得到3种母发酵剂;4)发酵豆乳的制备:将3种母发酵剂按一定比例混合,接种于豆乳中,添加蔗糖,在40~45℃条件下发酵3~7h;一种益生菌固体饮料,它包括下列重量份数的组分:戊糖片球菌PP04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。本发明戊糖片球菌PP04对Caco-2细胞的黏附率为79.06%。
附图说明
图1 戊糖片球菌PP04 16S rDNA序列分析鉴定结果;
图2 戊糖片球菌PP04对高脂小鼠第八周体重的影响;
图3 戊糖片球菌PP04对高脂小鼠血清中TC (a)、TG (b)、LDL(c)、HDL (d)水平的影响;
图4 戊糖片球菌PP04对高脂小鼠肝脏中TC (a)、TG (b)、LDL (c)水平的影响;
图5 戊糖片球菌PP04对高脂小鼠血清中FFA水平的影响;
图6 戊糖片球菌PP04对高脂小鼠血清中SOD(a)和GSH-Px (b)水平的影响;
图7 戊糖片球菌PP04对高脂小鼠肝脏中SOD(a)和GSH-Px (b)水平的影响;
图8 戊糖片球菌PP04对高脂小鼠血清中AST (a)和ALT (b) 以及肝脏中AST (c)和ALT(d)水平的影响;
图9 戊糖片球菌PP04对高脂小鼠血清中TNF-α(a)和肝脏中TNF-α(b)水平的影响。
具体实施方式
实施例1 戊糖片球菌PP04的分离纯化和鉴定
1、菌株的分离纯化
将市售的东北酸菜,在实验室进行乳酸菌分离;东北酸菜发酵液以10-1的梯度稀释,涂布于MRS琼脂平板上,37℃培养24 h,挑取单菌落,平板划线法反复进行纯化,用革兰氏染色法观察菌落形态挑选革兰氏阳性菌株。
液体MRS培养基组分:蛋白胨 10.0 g,牛肉膏 10.0 g,酵母膏 5.0 g,柠檬酸氢二铵[(NH4)2HC6H5O7] 2.0 g,葡萄糖(C6H12O6·H2O) 20.0 g,吐温80 1.0 mL,乙酸钠(CH3COONa·3H2O) 5.0 g,磷酸氢二钾(K2HPO4·3H2O) 2.0 g,硫酸镁(MgSO4·7H2O)0.58g,硫酸锰(MnSO4·H2O) 0.25 g。
2、菌种鉴定
将经平板划线法分离出的生长活力较好的革兰氏阳性菌株送到上海生工公司进行测序,经过16SrDNA鉴定;结果与戊糖片球菌显示出较高的分子系统学上的亲缘关系(图1)。因此命名为戊糖片球菌PP04,拉丁文学名Pediococcus pentosaceus PP04,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: M2019082。
实施例2 戊糖片球菌PP04的驯化
按2%接种量将戊糖片球菌PP04接种于含有0.05 mol/L H2O2、0.5% (w/v)胆盐及pH2.0的MRS液体培养基中,37℃驯化培养24 h。取200uL培养液涂布于MRS固体培养基上,37℃培养48 h,接种环挑取少量长出的单菌落接种到MRS液体培养基中进行复壮,并将菌液用50%(v/v)甘油-80℃保藏,备用。
结果:经过驯化培养后的戊糖片球菌PP04能够耐受0.05 mol/L H2O2、pH 2.0、0.5%(w/v)胆盐的生长环境,更有利于其在宿主肠道中的定植。
实施例3 戊糖片球菌PP04耐受能力试验
1、戊糖片球菌PP04模拟胃肠液耐受能力
1)模拟胃液或肠液实验
人工模拟胃肠液需新鲜配制。用PBS(pH 3.0)配置浓度为3 g/L的胃蛋白酶,0.22 μm滤膜过滤后制备成所需模拟胃液。用PBS(pH 8.0)配置浓度为1 g/L的胰蛋白酶,0 .22 μm滤膜过滤后制备成模拟肠液。将戊糖片球菌PP04用生理盐水重悬,在模拟胃液(pH 3.0)中将菌液充分混匀并调节其浓度至1×109CFU/mL,37℃培养3 h后检测活菌数。3 h后,取模拟胃液(pH 3.0)中的培养液1 mL加入至9 mL的模拟肠液(pH 8.0)中,充分混匀,在37℃培养8 h后检测活菌数。存活率用以下公式计算(取三次平行试验的平均值):
实施例3 戊糖片球菌PP04耐受能力试验
1、戊糖片球菌PP04模拟胃肠液耐受能力
1)模拟胃液或肠液实验
人工模拟胃肠液需新鲜配制。用PBS(pH 3.0)配置浓度为3 g/L的胃蛋白酶,0.22 μm滤膜过滤后制备成所需模拟胃液。用PBS(pH 8.0)配置浓度为1 g/L的胰蛋白酶,0 .22 μm滤膜过滤后制备成模拟肠液。将戊糖片球菌PP04用生理盐水重悬,在模拟胃液(pH 3.0)中将菌液充分混匀并调节其浓度至1×109CFU/mL,37℃培养3 h后检测活菌数。3 h后,取模拟胃液(pH 3.0)中的培养液1 mL加入至9 mL的模拟肠液(pH 8.0)中,充分混匀,在37℃培养8 h后检测活菌数。存活率用以下公式计算(取三次平行试验的平均值):
式中:N1=经过模拟胃肠液处理之后的活菌数,
N0=未经胃肠液处理前的活菌数
本发明中的戊糖片球菌PP04经过3 h人工模拟胃液(pH 3.0)和8 h模拟胃肠液(pH8.0)作用后存活率分别达到93.11%和95.18%,耐受其胃酸和胆汁的作用(表1)。
2)模拟胃肠液连续作用实验
驯化后的菌株按2%的接种量接入到MRS液体培养基,37℃培养24h,取10mL菌液在3000g条件下离心10min下,弃上清,用无菌PBS溶液洗涤两次,收集菌体沉淀,并加入10mL人工胃液(购自 Beijing Biotechnology Co.,Ltd),37℃培养1.5h。1.5h后,菌悬液3000g条件下离心10min下,弃上清,用无菌PBS溶液洗涤两次,收集菌体沉淀,并加入10mL模拟人工肠液(购自 Beijing Biotechnology Co.,Ltd),37℃培养2.5h。按如下公式计算细菌存活率(取三次平行试验的平均值):
式中:N1为人工胃肠液处理之后的活菌数
N0为人工胃肠液处理之前的活菌数
结果见表1,戊糖片球菌PP04经过人工胃肠液处理后存活率为78.06%,活菌数有所下降,但幅度不大;表明戊糖片球菌对人工胃肠环境具有较好的耐受能力,能耐受其胃酸和胆汁的作用。
2、戊糖片球菌PP04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100μL涂布于LB平板上,取100 μL菌培养液加入牛津杯中,37℃恒温培养24 h,观察小孔周围抑菌圈直径,每个指示菌做三次平行试验,游标卡尺测抑菌圈直径,求平均值。
结果见表1-1,从表中看出戊糖片球菌PP04对大肠杆菌和金黄色葡萄球菌有明显的抑制作用,且抑菌圈直径分别为21.35和16.96,而对沙门氏菌并无抑制作用。
3、戊糖片球菌PP04耐药性评价
菌株按2%的接种量接入MRS液体培养基,37℃培养至对数生长期,吸取100 μL涂布于MRS琼脂平板,选取五种常用的药敏纸片:氯霉素、红霉素、四环素、卡那霉素、链霉素,无菌镊子夹取放至已涂布菌液的MRS平板上,每种抗生素三次平行试验,37℃培养24 h,并用游标卡尺准确称量抑制圈直径。
结果见表2,从表中看出戊糖片球菌PP04对卡那霉素和链霉素无敏感性,对氯霉素、红霉素和四环素敏感,但仍在安全浓度的范围内。
实施例4. 戊糖片球菌PP04对Caco-2 细胞黏附能力
1、Caco-2 细胞的培养
将Caco-2细胞接种到细胞培养瓶中,使用含有10%胎牛血清+1%青霉素-链霉素双抗的DMEM (Dulbecco's modified eagle medium)培养基于37℃、5% CO2条件下培养5-6 d,细胞聚合度可达到90%~100%。将生长状态良好的Caco-2细胞接种于24孔板上(孔径16 mm,有效膜面积1.9 cm2),接种密度为0.5×105/孔,37℃、5% CO2条件下培养21 d,隔天换液,倒置显微镜下观察细胞生长情况约21 d后细胞形成紧密单层。
2、戊糖片球菌PP04菌悬液的制备
菌株按2%的接种量接入到MRS液体培养基,37℃培养24 h,然后在3000 g条件下离心5min下,弃上清,收集菌体沉淀,重悬于(不含青霉素和链霉素)DMEM溶液中,并调整菌体浓度至109 CFU/mL,取100 ul 菌悬液连续稀释到合适的浓度梯度,涂布计数N0。
3、平板涂板计数法
培养至21 d的Caco-2细胞,小心吸去24孔板中的培养液,用无菌PBS溶液洗涤两次,每孔加入1 mL制备好的菌悬液或等量DMEM溶液作为空白对照,37℃、5% CO2条件下共孵育2h。2 h后吸走培养液,并用PBS溶液洗两次,以除去过量的未黏附于Caco-2细胞的戊糖片球菌PP04。每孔加入0.5 mL胰酶消化1~2 min,再加入0.5 mL DEME培养基并用移液枪小心吹打均匀,取100 ul 菌悬液连续稀释到合适的浓度梯度,涂布于MRS琼脂培养基,37℃培养24h,计数N1。黏附率按如下公式计算(取三次平行试验的平均值):
结果见表3,戊糖片球菌PP04对Caco-2细胞的黏附率为79.06%,具有较好的黏附肠道细胞的能力,有利于戊糖片球菌PP04在肠道的定植。
4、显微计数法
将上述制备的戊糖片球菌PP04菌悬液加入到培养21 d的Caco-2单层细胞中,37℃培养2 h后用PBS冲洗单层细胞三次,甲醇固定30min,再进行革兰氏染色。随机找20个视野数100个细胞,以平均每个细胞上黏附的戊糖片球菌PP04菌数来评价其黏附能力。
结果表明,戊糖片球菌PP04对Caco-2细胞的黏附率为37个/cell,表现了其更好的肠道定植能力。
实施例5 戊糖片球菌PP04表面疏水性和自聚集能力
1、戊糖片球菌PP04菌悬液的制备
菌株按2%的接种量接入到MRS液体培养基,37℃培养24 h,然后在3000 g条件下离心5min下,弃上清,收集菌体沉淀。无菌PBS溶液将沉淀物洗涤两次,并重悬于PBS溶液中,使菌悬液的吸光度(OD600 nm)达到1.0±0.02(A0)。
2、表面疏水性
取3 mL菌悬液与0.6 mL二甲苯混匀后涡旋震荡2 min,室温下静置10 min,形成两相体系(水相和有机相),37℃孵育2 h。2 h后小心吸取水相,在600 nm处测定吸光度(A1),细胞表面疏水性按如下公式计算(取三次平行试验的平均值):
3、自聚集能力
按上述方法制备戊糖片球菌PP04菌悬液,调整其在OD600 nm处的吸光值为1.0±0.02(A0 h)。取3 mL菌悬液旋涡震荡1 min,于37℃条件下孵育2 h。小心吸取静置后的上清液,在600 nm处测定吸光度(A2 h),自聚集能力按如下公式计算(取三次平行试验的平均值)
乳酸菌细胞表面的物化性质如表面疏水性和自聚集能力等可间接反映其黏附性强弱。戊糖片球菌PP04表面疏水性和自聚集能力分别为54.90%和48.79%,而二者均与黏附能力成正比,所以结果表明戊糖片球菌PP04具有较好的黏附肠道上皮细胞的能力。
实施例6 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):基础饲料;
B 组:高脂模型组(HFD):高脂饲料;
C 组:干预组(HFD+PP04):高脂饲料混合PP04菌株;
菌种活化:-80℃取出保藏的已驯化的戊糖片球菌PP04,按2%接种量接种到已灭菌的MRS液体培养基中,37℃培养24 h,连续传代三次,保证菌体浓度达到108 CFU/mL,待用。
干预组需要每天将高脂饲料与戊糖片球菌PP04菌液混合并搅拌均匀,干预物按照50g/kg BW/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3000 rpm·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℃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10 min,取上清液,置于-80℃备用。
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。
4、结果及分析
与正常对照组相比,长期摄入高脂饮食导致小鼠体重显著性增加,而经戊糖片球菌PP04干预后,其体重显著性降低了12.56%,数据表明戊糖片球菌PP04对于抑制长期摄入高脂饮食引起的体重的增加具有潜在意义(图2)。
研究表明,与正常对照组相比,长期摄入高脂饮食导致小鼠血清中TC、TG和LDL水平显著升高;而戊糖片球菌PP04的干预能够显著性降低血清TC、TG和LDL水平,分别降低了32.15%、25.10%和57.98%(图3abc)。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,戊糖片球菌PP04干预组小鼠血清HDL水平并无显著变化,说明戊糖片球菌PP04对血清HDL的影响不大(图3d)。这些数据表明,戊糖片球菌PP04有效缓解由高脂饮食诱导的血清中血脂代谢异常,从而降低高脂血症等相关疾病的发病率。
与正常对照组相比,高脂饮食导致小鼠肝脏中TC、TG和LDL水平显著性升高。戊糖片球菌PP04干预后小鼠肝脏中TC、TG和LDL水平均有不同程度降低,分别降低了25.91%、58.41%和28.73%(图4abc),其中,高脂小鼠肝脏TG水平的降低效果尤为明显(P<0.001),然而各组间肝脏HDL水平没有明显变化(数据没有表明)。这些数据与小鼠血清中脂质水平变化趋势整体一致,表明高脂饮食不仅引起血清中血脂代谢紊乱,对肝脏脂质代谢也出现损伤,而戊糖片球菌PP04具有抑制高脂饮食引起的小鼠肝脏中TC、TG和LDL水平上升的潜在作用。
FFA是机体主要供能物质之一,脂肪细胞内TG在各种脂肪酶的作用下被水解为游离脂肪酸和甘油释放入血液中被组织利用,FFA既是肝脏合成TG的底物,又是富含TG的脂蛋白的水解产物,高脂饮食会加快脂肪代谢速率,造成血清中FFA水平升高。与正常对照组相比,高脂饮食导致小鼠血清中FFA水平升高且具有显著性差异,戊糖片球菌PP04干预后显著性降低了血清中FFA水平,降低率为10.77%(图5)。结果表明,戊糖片球菌PP04干预对高脂饮食引起的血清中FFA水平的变化符合FFA与TG在体内转化呈正相关的规律。
SOD 和GSH-Px是机体中两种重要的抗氧化酶,其中SOD是可以清除生物体内的超氧阴离子自由基,而GSH-Px是存在于胞浆和线粒体基质中对H2O2有专一性,能清除自由基并抑制自由基反应的酶,二者的水平反映了机体自身清除自由基的能力。与正常对照组相比,高脂饮食导致小鼠血清中SOD 和GSH-Px水平显著性降低,而戊糖片球菌PP04干预后小鼠血清中SOD 和GSH-Px水平显著性提高,分别增加了50.56%和28.57% (图6ab)。
与正常对照组相比,高脂饮食导致小鼠肝脏SOD 和GSH-Px水平显著性降低,而戊糖片球菌PP04干预后小鼠肝脏中SOD 和GSH-Px水平显著性提高,分别提高了22.95%和10.99% (图7),这些数据与小鼠血清中变化趋势整体一致,表明长期摄入高脂饮食会破坏机体氧化还原平衡状态,降低其抗氧化酶水平,而戊糖片球菌PP04具备抑制高脂饮食引起的血清及肝脏中SOD 和GSH-Px水平下降的能力,从而降低高脂血症及心脑血管疾病的风险。
AST和ALT是反映肝实质性损害的指标,其中ALT是最常用的敏感指标。与正常对照组相比,高脂饮食导致小鼠血清和肝脏中AST和ALT水平显著性升高,戊糖片球菌PP04有效抑制血清中AST及肝脏中AST和ALT水平的升高,分别降低了17.57%、43.2%和22.26%。图8a-d数据表明戊糖片球菌PP04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。
TNF-α是由巨噬细胞产生的一个关键促炎因子,与人类非酒精性脂肪性肝炎和食物诱导的小鼠脂肪性肝炎相关 。与正常饮食小鼠相比,高脂饮食导致小鼠血清和肝脏中TNF-α水平显著性升高,经过戊糖片球菌PP04的干预后,显著性抑制了小鼠血清及肝脏中TNF-α水平的升高,数据表明戊糖片球菌PP04对非酒精性脂肪性肝损伤有一定的缓解作用(图9ab)。
综上所述,本发明中的戊糖片球菌PP04经过驯化后胁迫条件为:H2O2 0.05 mol/L、pH 2.0、胆盐0.5%(w/v),具有良好的模拟人工胃肠液耐受能力、高黏附性、抗致病菌感染、调节代谢紊乱以及高血脂症等多种健康功效,同时在减肥降脂方面效果显著。
益生菌的筛选标准为:黏附性能;对胃肠液的耐受性;抑菌活性;安全性。在筛选益生菌的过程中,许多研究都把检测黏附性作为筛选的第一步,如果黏附性弱则不再进行后续的实验,而直接被淘汰。戊糖片球菌PP04表面疏水性和自聚集能力分别为54.90%和48.79%,二者均与黏附性成正比,疏水性或自聚集能力越大,黏附性越高。Yeong Ji Oh等研究发现,Pediococcus pentosaceus(SW01)对HT-29.细胞的黏附率为66.30%,但本发明中戊糖片球菌PP04对Caco-2细胞的黏附率为79.06%,表现出更高的对肠道上皮细胞的黏附能力。虽然二者使用不同的细胞模型,但Caco-2细胞因形态和功能与小肠上皮细胞非常接近,比如含有肠道上皮细胞绒毛以及相应的酶被广泛地用作细胞模型进行黏附性实验。
大量研究表明,戊糖片球菌具有降胆固醇、抑制病原菌、免疫调节等功能。陈蕾等研究中发现,戊糖片球菌JQI-7菌悬液喂养高脂小鼠28天后股静脉抽血检测,发现与对照相比小鼠血液中TG及TC分别下降23.8%和32.5%;但本发明中戊糖片球菌PP04干预后,高脂小鼠血清中TG和TC分别下降了25.10%和32.15%,与戊糖片球菌JQI-7相比戊糖片球菌PP04能更有效的抑制长期摄入高脂饮食导致小鼠血清中TG的升高,戊糖片球菌PP04不仅能够降低高脂小鼠血清和肝脏中TG和TC、HDL水平,并且显著性降低了高脂小鼠血清中FFA水平,降低率为10.77%;提高了高脂饮食导致的小鼠血清和肝脏中SOD 和GSH-Px水平的降低,分别为50.56%、28.57%、22.95%和10.99%;戊糖片球菌PP04不仅具有极显著的降血脂作用,并且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。目前,关于戊糖片球菌减肥降血脂功效的研究非常少,大多集中在戊糖片球菌对致病菌例如大肠杆菌、鼠伤寒沙门菌、李斯特菌等抑制机制,以及对宿主肠道免疫功能等方面的研究。在本研究中,具有高黏附能力的戊糖片球菌PP04,表现了其良好的改善血脂异常的潜在作用;增加机体抗氧化酶水平,进而在一定程度上缓解了高脂饮食引起的氧化应激以及对非酒精性脂肪性肝损伤的修复作用。
实施例7 戊糖片球菌PP04发酵豆乳的制备
1、豆乳的制备
选择皮薄且有光泽无腐烂变质虫害的大豆,浸泡(加入过量蒸馏水,室温条件下浸泡12h直至大豆成饱和状态质量变为衡重),碱水浸泡(用浓度为0.25%的NaHCO3溶液室温浸泡2h,消除豆腥味,软化大豆不表皮组织缩短蒸煮时间及改善均质效果),将大豆及其浸泡液同时倒入磨浆机中磨浆,120目滤网过滤除渣后,离心,煮沸(在豆乳中加入白砂糖100℃ 煮沸10 min,使脂肪酶钝化、凝血因子及抗胰蛋白酶变性进一步去除豆腥味,并达到杀菌的目的),85℃,20 MPa均质5 min, 95℃杀菌5 min,冷却至发酵温度后备用。
2、混合菌种发酵豆乳发酵剂的制备
菌种活化:取适量戊糖片球菌PP04、嗜热链球菌及保加利亚乳杆菌接种于MRS液体培养基中,42℃培养12-18 h,传代3次,在对数期结束后收获菌体。将液体MRS培养基中的戊糖片球菌PP04、嗜热链球菌及保加利亚乳杆菌按1%的接种量接种于含有2%葡萄糖的脱脂乳培养基中(脱脂乳粉与蒸馏水的体积比为1:10),在42℃条件下传代培养2次。使最终凝乳时间大致相同时即完成菌种的活化,后置于4℃冰箱中冷藏备用;
菌种的驯化及发酵剂的制备:将戊糖片球菌PP04按1%的接种量接种于脱脂乳与豆乳的混合培养液中42℃条件下进行菌种的驯化培养;嗜热链球菌及保加利亚乳杆菌的驯化过程与上述方法相同;
发酵剂的制备:将驯化后的戊糖片球菌PP04、保加利亚乳杆菌和嗜热链球菌菌种按3%的接种量依次接种于100~300 mL三个灭菌后的豆乳培养基中,42℃培养至豆乳凝乳后即为母发酵剂;
3、发酵豆乳的制备
分别将戊糖片球菌PP04,保加利亚乳杆菌和嗜热链球菌三种母发酵剂按照体积比为1:0.5:1的接种比例接种于料水比为1:8磨制的豆乳中,添加8%的蔗糖,总接种量为5%的条件下43℃发酵5 h制得的豆乳最为可口。
实施例8 益生菌固体饮料的制备
1、戊糖片球菌PP04菌体制备
将戊糖片球菌PP04在37℃条件下培养18 h,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10 min,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6%-10%、海藻糖4%-8%、菊粉6%-9%、在80℃,20min的条件下灭菌,制成冻干保护剂。
3、戊糖片球菌PP04冻干粉制备
将悬菌液与冻干保护剂按5:1~1:1的比例混合均匀,在-50℃、真空条件下干燥48h形成戊糖片球菌PP04冻干粉。
4、系列益生菌固体饮料配方
蓝莓风味固体饮料:戊糖片球菌PP04冻干粉1 g,低聚异麦芽糖0.5-1.0 g,大豆低聚糖0.5-1.0 g,蓝莓粉0.5-1.5 g;
山楂风味固体饮料:戊糖片球菌PP04冻干粉1 g,低聚异麦芽糖0.5-1.0 g,大豆低聚糖0.5-1.0 g,山楂粉0.5-1.5 g。
产品规格:固体饮料采用3 g为1包,添加的戊糖片球菌PP04的活菌数为1010CFU/mL。
Claims (9)
1. 一株戊糖片球菌Pediococcus pentosaceus PP04,它的保藏编号为CCTCC No:M2019082。
2.一种发酵豆乳,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 2019082的戊糖片球菌PP04、嗜热链球菌及保加利亚乳杆菌冻干粉分别接种于MRS液体培养基中,40~45℃培养12~18h,传代3次,收获菌体;将收获的戊糖片球菌PP04、嗜热链球菌及保加利亚乳杆菌按1~3%的接种量接种于脱脂乳培养基中,在40~45℃下传代2次;
2)菌种的驯化:将戊糖片球菌PP04、嗜热链球菌及保加利亚乳杆菌按1~5%的接种量,分别接种于脱脂乳与豆乳的混合培养液中,40~45℃下驯化;
3)发酵剂的制备:将驯化后戊糖片球菌PP04、保加利亚乳杆菌和嗜热链球菌菌种按1~10%的接种量分别接种于豆乳中,40~45℃下培养,得到3种母发酵剂;
4)发酵豆乳的制备:分别将步骤3)得到的戊糖片球菌PP04、保加利亚乳杆菌和嗜热链球菌3种母发酵剂,以体积比1~1.5:0.5~1:1~1.5混合,按接种量为1~10%接种于豆乳中,添加质量百分比3~10%的蔗糖,40~45℃下发酵3~7h。
3.根据权利要求2所述的一种发酵豆乳,其特征在于:所述的豆乳,是由下述方法制备的:大豆,水浸泡10~15h,碱水浸泡2~3h,磨浆,过滤除渣,加糖,煮沸,80~90℃下均质3~7min,杀菌,冷却。
4.根据权利要求3所述的一种发酵豆乳,其特征在于:步骤3)所述的培养,是培养至豆乳凝乳结束。
5.根据权利要求2、3或4所述的一种发酵豆乳,其特征在于:步骤4)所述的以体积比1:0.5:1混合,接种量为1~5%。
6. 一种益生菌固体饮料,它包括下列重量份数的组分:戊糖片球菌冻干粉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;所述的戊糖片球菌,它的保藏编号为CCTCC No:M 2019082。
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:所述的果味粉为蓝莓粉或山楂粉。
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:所述的戊糖片球菌冻干粉,是由下述方法制备的:将戊糖片球菌PP04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,得到戊糖片球菌冻干粉。
9.根据权利要求8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6%-10%、海藻糖4%-8%、菊粉6%-9%、余量为水。
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