CN112080449B - 一种屎肠球菌r40及其在降胆固醇、产胞外多糖和抗氧化中的应用 - Google Patents
一种屎肠球菌r40及其在降胆固醇、产胞外多糖和抗氧化中的应用 Download PDFInfo
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Abstract
本发明提供了一种屎肠球菌R40及其在降胆固醇、产胞外多糖和抗氧化中的应用。该屎肠球菌R40同时具有降胆固醇、产胞外多糖和抗氧化等作用,且更适合在酸性条件下生长,对胃肠道条件耐受力较强,也具有较为良好的耐胆盐能力,可以将屎肠球菌R40的活菌或代谢产物添加到食品中,在人体内起到降低胆固醇、抗氧化、助消化等作用。本发明还包括屎肠球菌R40在酸奶中的应用以及包括屎肠球菌R40的酸奶,该酸奶各项指标均优于市售发酵剂发酵酸奶。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种屎肠球菌R40及其在降胆固醇、产胞外多糖和抗氧化中的应用。
背景技术
随着人们对健康的意识和需求的强烈,益生菌、益生元、功能食品等逐渐成为人们日常饮食关注重心。2020年益生菌与健康国际研讨中,中国食品科学技术学会组织相关专家对益生菌的基本特点达成了以下共识:菌株为活菌状态、菌株数量足够、且具有有益健康的功能。近年来对益生菌的研究主要焦点在于其医学方面——如肠道功能的改善、呼吸道疾病的治疗等;畜牧养殖方面——如利用益生菌替代抗生素或用来提高肉的品质以及禽畜产品的生产能力等;食品加工——代谢产物的应用、冰淇淋产品中的应用、发酵食品、以及果汁、口服制剂等非乳制品中应用。乳酸菌(LAB)是一种普遍存在于自然界中的益生菌,其本身并非微生物分类学概念,而是对一类可发酵葡萄糖产生乳酸、无芽孢的革兰氏阳性细菌的总称。乳酸菌种类多样,至少有200多种,包括乳酸杆菌、双歧杆菌、植物乳杆菌等。乳酸菌其益生功能主要体现在调节肠道微生态、利用大分子物质以帮助人体消化、激活巨噬细胞增强人体免疫力、降低人体胆固醇浓度、预防肿瘤、延缓衰老等方面。现目前关于乳酸菌的报道中,提到了一些屎肠球菌具有降胆固醇的作用,但并没有关于同时起到降胆固醇、产胞外多糖和抗氧化作用的相关报道;因此,找到一种同时具有降胆固醇、产胞外多糖和抗氧化作用的菌株是十分有意义的。
发明内容
针对现有技术中存在的上述问题,本发明提供一种屎肠球菌(Enterococcusfaecium),其命名为屎肠球菌R40,于2020年9月14日保藏于中国典型培养物保藏中心,保藏编号为CCTCC No.M 2020496;该屎肠球菌R40具有降胆固醇、产胞外多糖和抗氧化的作用。
上述屎肠球菌R40在酸奶中的应用。
一种酸奶,包括上述屎肠球菌R40。该酸奶的口感良好,酸奶性质检验的结果表明,乳酸菌酸奶各项指标均优于市售发酵剂发酵酸奶。
一种固体或液体制剂,包括上述的屎肠球菌R40,可将其用于降胆固醇、产胞外多糖和抗氧化,以及酸奶制作。
上述屎肠球菌R40是一种具有综合性能的菌株,降胆固醇能力较强,其胆固醇降解率达到了60.99±8.82%,且更适合在酸性条件下生长,对胃肠道条件耐受力较强,也具有较为良好的耐胆盐能力;屎肠球菌R40还具有一定的产胞外多糖能力,产量最高达710.73±23.25mg/L,而屎肠球菌R40具有较强的DPPH自由基的清除能力、ABTS自由基的清除能力、氧化自由基吸收能力ORAC和FRAP铁还原能力,分别为12.04±1.05h mg/g、124.73±14.94efmg/g、91.68±5.56b mg/g和6.57±0.68bc mg/g(其中上述mg/g为维生素E mg/g)。
综上所述,本发明具备以下优点:
本发明提供了一种屎肠球菌R40,其同时具有降胆固醇、产胞外多糖和抗氧化等作用,且更适合在酸性条件下生长,对胃肠道条件耐受力较强,也具有较为良好的耐胆盐能力,可以将屎肠球菌R40的活菌或代谢产物添加到食品或保健品中,在人体内起到降低胆固醇、抗氧化、助消化等作用。还可以将屎肠球菌R40应用在酸奶中,使得酸奶口感良好,同时酸奶性质检验的结果表明,乳酸菌酸奶各项指标均优于市售发酵剂发酵酸奶。
附图说明
图1为胆固醇标准曲线;
图2为R40耐酸实验生长曲线;
图3为琼脂糖凝胶电泳图;
图4为系统发育树。
具体实施方式
以下结合实施例对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实施例1屎肠球菌R40的分离、筛选和鉴定
1、屎肠球菌R40的分离
取样品(鱼肠道内容物、泡菜汤、胃肠道药物)溶解于10mL灭菌生理盐水中,然后对其进行梯度稀释,选择合适的稀释倍数,吸取0.1mL稀释液涂布于固体MRS+CaCO3培养基上,37℃,静置培养24h。挑取培养基上产生明显透明圈、革兰氏染色阳性的菌落,分别划线纯化至菌落形态单一,观察记录菌落的基本形态特征,并挑取菌落于30%甘油管、置于超低温冰箱-70℃保存。其中,上述胃肠道药物为“妈咪爱”枯草杆菌二联活菌颗粒、金双歧(双歧杆菌乳杆菌三联活菌片)。
MRS(1000mL):蛋白胨10.0g、牛肉粉10.0g、酵母浸粉5.0g、柠檬酸氢二胺2.0g、葡萄糖20.0g、乙酸钠5.0g、磷酸氢二钾2.0g、硫酸镁0.58g、硫酸锰0.25g、吐温80 1.0mL和蒸馏水1000mL(配制固体培养基每1000mL需加入15-20g琼脂),高压蒸汽灭菌锅121℃,20min灭菌后使用;
MRS+CaCO3培养基:MRS固体培养基中加入碳酸钙,使得碳酸钙的质量浓度为0.3%,高压蒸汽灭菌锅121℃,20min灭菌后使用,倾注MRS+CaCO3培养基时应注意边摇匀边倒,以防止碳酸钙沉淀,培养基不均匀。
实验筛选出MRS+CaCO3培养基中产生透明圈且革兰氏染色阳性的乳酸菌共114株,其中从发酵食品泡菜汤中筛选出54株,依次编号为R1-R54,鱼肠道中筛选出40株,依次编号为C1-C40,胃肠道药品中筛选出20株,依次编号为Y1-Y20,其菌落形态、镜检形态等基本特征如表1所示。
表1 114株乳酸菌菌落形态及镜检形态
2、屎肠球菌R40的筛选
(1)降胆固醇能力的测定:将上述分离的所有菌种以2%(v/v)的接种量接种于MRS液体培养基中活化,37℃条件下培养48h即得发酵种子液。取发酵种子液5mL,4℃、3000r/min离心15min,弃上清液,用pH=6.5的PBS缓冲液调节菌液OD600nm=1,取调节好浓度的菌悬液,以2%(v/v)的接种量接种于MRS-CHOL培养基中,37℃培养24h后,将培养液漩涡振荡摇匀,吸取0.5mL培养液于10mL离心管中,加入4.5mL无水乙醇沉淀蛋白,静置反应10min,4℃、3000r/min离心15min后,取0.5mL上清液于洁净试管中,加入1mg/mL邻苯二甲醛溶液0.2mL,混合酸溶液4.3mL,漩涡振荡摇匀后静置反应20min以上,测定其在550nm处的光密度值,未接菌的空白MRS-CHOL培养基作对照组,未加胆固醇溶液的空白MRS培养基作为空白对照消除培养基本身对试验结果的影响。按照公式(1)计算菌株的胆固醇降解率,所有试验重复3次,计算结果求平均值。
胆固醇降解率(%)=(C-C0)/C×100% (1)
其中:C0为对照组胆固醇含量;C为实验组胆固醇含量。
114株乳酸菌胆固醇降解能力如表2所示。
(2)胆固醇标准曲线的绘制
1mg/mL邻苯二甲醛溶液的配制:准确称取0.050g邻苯二甲醛,用无水乙醇溶解并定容于50mL棕色容量瓶中,放置于4℃冰箱中保存备用。
混合酸(浓硫酸:冰乙酸=1:1)配制:用量筒准确量取100mL的浓硫酸沿引流玻璃棒缓慢注入100mL冰乙酸中,混合搅匀,冷却至室温后备用;
0.1mg/mL胆固醇标准溶液配制:准确称取0.050g胆固醇,用冰乙酸溶解并定容于50mL容量瓶中。吸取10mL定容后的溶液,用冰乙酸稀释定容于100mL容量瓶中,备用。
分别吸取0、0.1、0.2、0.3、0.4、0.5mL浓度为0.1mg/mL的胆固醇标准溶液于洁净试管中,加入1mg/mL邻苯二甲醛0.2mL,混合酸4.3mL,并用冰乙酸补足反应体系总体积至5mL,用漩涡振荡器振荡使之混合均匀且无气泡,静置反应20min以上,然后测定反应后液体在550nm处的光密度值OD550nm,每组3个平行。以OD550nm为纵坐标,胆固醇含量为横坐标,做标准曲线。
横坐标分别对应浓度为0、0.02、0.04、0.06、0.08和0.10mg/mL胆固醇。胆固醇标准曲线如图1所示,该方法测得的胆固醇在浓度0-0.10mg/mL范围内呈现良好的线性关系,标准曲线回归方程为Y=11.083X-0.013,R2=0.99948。
表2 114株乳酸菌胆固醇降解率的测定(x±s,n=3)
由表2可知,胆固醇降解率<20%的共38株,占比33.33%,在20-30%间的共40株,占比35.08%;在30-40%共11株,占比9.64%;40-50%间共3株,占比2.63%;>50%共4株,占比3.51%,另有18株无明显降胆固醇作用,占比15.79%。结果表明,85%左右的乳酸菌普遍都具有一定降胆固醇能力,114株乳酸菌胆固醇降解率在2.74-69.63%不等且差异性较大。胆固醇降解能力>50%的菌株共有C35、R5、R40、R43 4株,其中R40号菌株胆固醇降解率为60.99±8.82%。
3、生理生化实验
对筛选出的R40号菌株进行过氧化氢酶试验、葡萄糖发酵试验、淀粉水解试验、葡萄糖产气试验、pH=4.0、pH=9.0、15℃、45℃生长试验、耐盐性试验、甲基红试验、v-p乙酰甲基甲醇试验、酪素水解试验。其结果见表3。
表3生理生化实验结果
注:“-”表示生理生化试验阴性;“+”表示生理生化试验阳性,或菌株在该条件下能够生长但生长能力一般;“++”表示菌株在该条件下生长良好;“NG”表示该菌株在该条件下不生长。
综合生理生化试验结果,根据《常见细菌系统鉴定手册》及《伯杰氏细菌鉴定手册》,得知R40为肠球菌属。
4、胃肠道生存能力实验
综合测定R40在胃肠道内的生存能力:
(1)耐酸实验:分别取10mL活化24h的发酵种子液,4℃、3000r/min离心15min后弃上清液,加入10mL pH=3.0的MRS培养基,分别处理0h、1h、2h后,将培养液以2%(v/v)的接种量接种于正常MRS液体培养基中,37℃静置培养18h,每3h测定一次菌悬液在600nm下的OD值,以未接菌的MRS培养基作为空白对照,绘制曲线比较不同菌株对酸的耐受能力;
(2)耐胆盐实验:取活化24h的发酵种子液,以2%(v/v)的接种量分别接种于含质量分数为0、0.1%、0.2%、0.3%牛胆盐的MRS培养基中,37℃静置培养,分别测定0h及培养15h后600nm处的光密度值OD0h和OD15h,根据公式(2)计算乳酸菌的生长率:
(3)胃肠道模拟实验:考虑到耐酸实验耐胆盐实验条件单一,且以吸光度值为指标无法分辨菌悬液中的菌体是否死亡,故采用胃肠道模拟实验,采用平板计数法测定活菌数分析菌株的存活能力。
人工胃液(100mL)的配制:0.3g酵母膏,0.1g蛋白胨,0.04g葡萄糖,0.2g氯化钠,0.3g胃蛋白酶,蒸馏水溶解,盐酸调节pH=3.0,0.40μm滤膜过滤除菌后备用,切勿用灭菌锅加热灭菌;
人工肠液(100mL)的配制:0.2g牛磺胆酸钠,0.1g胰蛋白酶,0.24g碳酸氢钠,用氢氧化钠调节pH至6.8,调节最终胆固醇含量为70-100μg/mL,0.40μm滤膜过滤后备用。
取活化24h的发酵种子液,4℃、3000r/min离心15min,弃上清液,并用无菌生理盐水洗涤3次,然后用pH6.5的PBS缓冲液重悬,并调节OD600nm=1,取1mL调节好浓度的菌悬液接种到9mL的人工胃液中,混合均匀后,37℃、150r/min条件下摇床培养2h,取出后再次4℃、3000r/min离心15min,取出上清液后加入10mL无菌人工肠液,37℃、150r/min培养3h。基于预试验结果,选择三个连续的稀释倍数进行平板涂布法计数,分别测定培养前,人工胃液中培养2h后,人工肠液中再培养3h各节点的活菌数,每组试验重复3次,结果求平均值。根据公式(3)-(5)计算乳酸菌的存活率:
其中,A0h表示刚接入人工胃液时培养液的活菌数;A2h表示人工胃液培养2h后的活菌数;A5h表示转入人工肠液后培养3h的活菌数。
耐酸实验结果如图2所示,耐胆盐实验结果见表4,胃肠道模拟实验结果见表5。
表4菌株R40在不同浓度胆盐条件下的生长情况
表5菌株R40在模拟胃肠道生存率(x±s,n=3)
由图2可知,R40更适合在酸性条件下生长,酸处理1h、2h组生长能力均高于未处理组,对酸的耐受能力更强。且在pH=3.0的MRS培养基处理2h后,37℃、18h后的菌体数量与空白37℃18h后的菌体数量作比较,得存活率=140.85%。
由表4可知,胆盐的存在能够明显抑制乳酸菌的生长,随着胆盐浓度的升高,乳酸菌生长率显著下降,胆盐对乳酸菌生长抑制作用较为明显,R40菌株表现出较为良好的耐胆盐能力,且在0.3%胆盐培养基培养15h后,菌体数量/0h的菌体数量得菌体的存活率,为5.00%。
由表5可知,胃肠道模拟实验结果与耐酸实验和耐胆盐实验结果基本吻合,肠道环境对乳酸菌的胁迫力较明显。模拟胃环境条件下处理2h后,R40的生存率均能达到82.0±8.38%,活菌数保持在108-109CFU/mL,但在小肠条件下消化3h后,活菌数的数量级发生骤降,活菌数只能保持在105-106CFU/mL,但也已达到可发挥益生功能数量级。
5、16S rRNA基因序列分析法鉴定
基因组的提取:采用试剂盒(氯仿)法提取菌株R40的基因组DNA;
基因片段的获得:以提取出的基因组DNA为模板,进行16srRNA基因PCR扩增,PCR扩增正向引物为27F(5’-AGAGTTTGATCCTGGCTCAG-3’),反向引物为1492R(5’-GGTTACCTTGTTACGACTT-3’),PCR反应体系(50μL):DNA样品1μL,前序引物27F 2μL,后序引物1492R 2μL,双蒸水补充体积至50μL。PCR反应程序为:94℃预变性4min,94℃变性30s,55℃退火30s,72℃延伸120s,程序循环35次,72℃后延伸10min,16℃冷却30min。将PCR扩增产物进行凝胶电泳,称取0.35g琼脂糖,加入35mL TAE稀释液中,加热至沸腾,待琼脂糖均匀溶解,冷却至不烫手后加入1.4μL核酸染料,轻轻搅拌均匀,倒胶,注意切勿倒入气泡影响试验。胶孔中依次加入marker、及PCR扩增产物各8μL,电泳条件110kv,30min。观察电泳结果,确认PCR扩增是否成功,凝胶电泳条带明显即扩增成功后,送PCR扩增产物样品至生工生物(上海)股份有限公司委托测序。
基因序列分析:收到测序结果后,将16S rRNA基因测序结果在NCBI进行BLAST在线比对,确认菌株分类,并用MEGA6.06建立系统发育树。
琼脂糖凝胶电泳图如图3所示,其系统发育树如图4所示。
由上可知,R40的16S rRNA PCR产物长度为1.5Kbp,将测序结果进行BLAST在线分析,结果表明,R40为屎肠球菌(Enterococcus faecium ATCC 43197);基因测序结果与生理生化试验结果吻合。
实施例2屎肠球菌R40胞外多糖实验
1、将屎肠球菌R40活化后接种到胞外多糖MRS液体筛选培养基中,37℃静置培养48h,取10mL培养液于试管中,加入1/3体积的Sevag试剂(氯仿:正丁醇=4∶1),震荡30min后,4 000r/min离心15min,收集上层水相溶液,重复以上操作至两相之间无蛋白质层为止。随后加入3倍体积的冰冻无水乙醇,放置在4℃冰箱中冷藏。第2天取出,最终可以观测到多糖呈乳白色絮状沉淀析出,再于4℃、6000g下离心30min,用10mL蒸馏水溶解沉淀,可得胞外多糖提取液,胞外多糖粗提液冷冻干燥的胞外粗多糖。
2、采用苯酚-硫酸法进行测定:
(1)葡萄糖标准曲线的绘制:精密移取葡萄糖标准液0.0mL、2.0mL、4.0mL、6.0mL、8.0mL、10.0mL于试管中,加蒸馏水稀释100倍,并从中取1.0mL稀释液于25mL的具塞试管中,再各加6%苯酚溶液1.5mL振荡摇匀,沿着玻璃棒缓慢加入5mL浓硫酸,摇匀,静置10min。再放于水浴锅沸水加热20min,拿出冷却放置至室温,在490nm处,对照测定标准液的吸光度。以葡萄糖为标准品绘制标准曲线,得到标准曲线回归方程为Y=0.0054X-0.0003,R2=0.9982。根据标准曲线计算出样品提取液的EPS含量,每组至少三个平行。
(2)吸取样品1mL,加6%苯酚溶液1.5mL振荡摇匀,沿着玻璃棒缓慢加入5mL浓硫酸,摇匀,静置10min。再放于水浴锅沸水加热20min,拿出冷却放置至室温,在490nm处对照测定标准液的吸光度。
(3)由标准曲线的回归方程和稀释倍数换算出胞外多糖质量浓度。其结果见表6。
表6屎肠球菌R40胞外粗多糖产量
由表6可知,屎肠球菌R40有一定的产胞外多糖能力,产量高达710.73±23.25mg/L。
实施例3屎肠球菌R40的抗氧化实验
1、对DPPH自由基的清除作用
将100μL的1g/L的胞外粗多糖水溶液,加入100μL DPPH自由基无水乙醇溶液(0.2mmol/L),用力搅拌,在室温下黑暗中孵育30min;用去离子水和DPPH溶液作为对照,用样品和无水乙醇作为空白;测定517nm处的吸光度,每组至少3个重复。
DPPH清除率/%=[1-(A样品-A空白)/A对照]×100
以Trolox为标准品绘制标准曲线,得到标准曲线回归方程为Y=0.5673X+9.0774,R2=0.9946。根据标准曲线计算出样品提取液的EPS含量,以每g提取物中Trolox当量计(维生素E mg/g)。
2、对ABTS自由基的清除作用
准确配制7.0mmol/L ABTS溶液和2.45mmol/L过硫酸钾水溶液,二者1:1充分混匀,室温避光静置12-16h后,得ABTS+母液;取适量无水乙醇稀释ABTS+母液,使其在734nm处的吸光度为0.7±0.02,即得ABTS+工作液。在96孔板中加入50μL 0.1g/L的胞外粗多糖水溶液和150μL ABTS+工作液为样品组,记为A样品,去离子水代替ABTS+工作液反应孔为样品对照组记为A对照,去离子水代替样品组为空白组记为A空白;待各组黑暗处充分反应30min后,于734nm处检测各组吸光度并记录,每组至少三个重复。
ABTS自由基清除率(%)=[1-(A样品-A对照)/A空白]×100
以Trolox为标准品绘制标准曲线,得到标准曲线回归方程为Y=0.6058X-0.8838,R2=0.9966。根据标准曲线计算出样品提取液的胞外多糖含量,以每g提取物中Trolox当量计(维生素E mg/g)。
3、ORAC实验
(1)向96孔板各孔加入20μL不同浓度Trolox标准品溶液(或样品),以缓冲液代替样品或标品为阳性对照(+APPH),以及随后每孔加入4.0nmol/L 20μL FL荧光试剂,37℃孵育20min。测定时加入140μL 12.8mmol/LAAPH溶液启动反应,用缓冲液替代APPH为阴性对照(-APPH)将96孔板置于预热至37℃酶标仪中5min。
(2)以激发波长(485±20)nm,发射波长(530±20)nm进行连续测定荧光强度,测定时间设定在荧光衰减呈基线后为止,约60次循环(2h)。
(3)实验所得的各微孔不同时间点的绝对荧光强度数据与其初始时间的荧光强度相比,折算成相对荧光强度f,以相对荧光强度采用近似积分法计算荧光衰退曲线下面积(AUG)。其公式为:AUC=0.5×[2×(f0+f1+…+fn-1+fn)-f0-fn]×△t,其中fn标示第n个测定点的相对荧光强度,△t标示相邻两个时间点之间的时间间隔(即2min),则上述公式可简化为:AUC=2×(f0+f1+…+fn-1+fn)-f0-fn。
ORAC值=[(AUC样品-AUC空白)/(AUCTrolox-AUC空白)]×(Trolox浓度(μmol/L)/样品浓度(g/L))
每组实验至少三个重复,以Trolox为标准品绘制标准曲线,得到标准曲线回归方程为Y=0.6741X+13.284,R2=0.9971。根据标准曲线计算出样品提取液的EPS含量,以每g提取物中Trolox当量计(维生素E mg/g)。
4、FRAP实验
将300mmol/L、pH=3.6的醋酸缓冲液、10mmol/L TPTZ溶液、20mmol/L三氯化铁溶液以10:1:1配置成FRAP工作液,在96孔板中加入50μL 1g/L的胞外粗多糖水溶液和150μLFRAP工作液为样品组,以蒸馏水代替样品为对照组,室温下放置20min,在593nm处测吸光度,每组至少三个重复。以Trolox为标准品绘制标准曲线,得到标准曲线回归方程为Y=0.0054X+0.127,R2=0.9984。根据标准曲线计算出样品提取液的EPS含量,以每g提取物中Trolox当量计(维生素E mg/g)。
上述抗氧化实验结果见表7。
表7抗氧化活性实验结果
注:不同字母显著,p<0.05。小写字母表示同一种抗氧化实验不同菌株胞外多糖的显著性;大写字母表示同一种菌株胞外多糖不同抗氧化实验的显著性。
由表7可知,屎肠球菌R40具有较强的DPPH自由基清除能力、ABTS自由基清除能力、ORAC氧化自由基吸收能力和FRAP铁还原能力。从四种不同抗氧化实验中可以看出,同一种菌株胞外多糖在不同的抗氧化实验中,差异显著(P<0.05),屎肠球菌R40外多糖的抗氧化在几组实验中ABTS体系>ORAC体系>DPPH体系>FRAP体系,表明屎肠球菌R40胞外多糖对于ABTS自由基的清除率最高,而铁原子还原能力较低。
实施例4酸奶制备及评价
1、乳酸菌酸奶的制备
(1)制备乳酸菌R40冻干菌剂
将屎肠球菌R40加入增菌培养基中进行活化,作为制备菌剂的原料。将活化后的屎肠球菌R40以4%的接种量接种于增菌培养基中,37℃,静置培养18h,将培养过后的菌液振荡均匀,在4000r/min离心5min,然后将菌泥与40g/L的明胶保护剂以1:5(w:v)的比例混合,再吸取混合液2mL于5m L西林瓶中,于-40℃预冻60min,再将预冻后的玻璃瓶放入冻干机中24h,制得乳酸菌R40冻干菌剂,将菌剂放于4℃冰箱中冷藏待用。
(2)乳酸菌酸奶的制作
500mL牛奶加入40g的白砂糖后进行巴氏灭菌(70℃,20min),冷却至40℃左右时加入1g普通酸奶发酵剂(“益升优”酸奶益生菌,含嗜热链球菌、嗜酸乳杆菌和长双歧杆菌)与0.4g乳酸菌R40冻干菌剂,用保鲜膜密封好发酵容器,42℃下发酵8h后,于4℃进行后熟24h,得乳酸菌酸奶。对照组不添加乳酸菌R40冻干菌剂,其他条件均与实验组相同。
2、感官评价
随机选取12人组成感官评价小组,对乳酸菌酸奶的气味、口感、酸甜度以及组织形态打分进行感官评价,评价标准见表8,感官评价结果见表9。
表8酸奶感官评分表
表9酸奶感官评价结果
由表9可知,加有乳酸菌R40冻干菌剂的乳酸菌酸奶拥有比普通发酵酸奶更丰富的口感,乳酸菌酸奶的香气更明显,其他各项指标也优于普通发酵酸奶。
2、酸奶品质检测
(1)乳酸菌检验
采用国标GB4789.35-2010中规定的方法对两组酸奶进行乳酸菌检验。
(2)pH的测定
采用精密pH计测定酸奶的pH值。
(3)持水力的测定
使用以下方法测定4℃后熟24h的酸奶的持水力:
50mL离心管中加入30mL酸奶,称量其质量,记作A,3000r/min离心10min,弃去上清液,再次称量,其质量记作B,离心管的质量记作C,按以下公式计算酸奶的持水力:
持水力(%)=(B-C)/(A-C)×100%
(4)粘度测定
使用转子粘度计测定4℃冷藏24h的酸奶粘度:取适量酸奶,加入R180旋转型粘度计,使用2号转子,在转速条件设置为12r/min,间隔时间为10s,于室温下进行粘度测定。
上述酸奶品质检测结果见表10。
表10酸奶品质检测结果
由表10可知,乳酸菌酸奶在含菌量和粘度上较普通酸奶相比有了较高的提升,而持水力和pH与普通发酵酸奶差异不明显。
虽然结合附图对本发明的具体实施方式进行了详细地描述,但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内,本领域技术人员不经创造性劳动即可作出的各种修改和变形仍属本专利的保护范围。
Claims (5)
1.一种屎肠球菌(Enterococcus faecium),其命名为屎肠球菌R40,于2020年9月14日保藏于中国典型培养物保藏中心,保藏编号为CCTCC No.M2020496。
2.权利要求1所述的屎肠球菌R40在降胆固醇、产胞外多糖和抗氧化中的应用。
3.权利要求1所述的屎肠球菌R40在酸奶中的应用。
4.一种酸奶,其特征在于,包括权利要求1所述的屎肠球菌R40。
5.一种固体或液体制剂,其特征在于,包括权利要求1所述的屎肠球菌R40。
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