CN110643533B - 降解油脂的干酪乳杆菌及其应用 - Google Patents
降解油脂的干酪乳杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株干酪乳杆菌F1,保藏编号为CGMCC No.18353。干酪乳杆菌F1能够降解调和油、花生油和芝麻油,对调和油的降解效果最好,降解率为21.23%。该菌株能测到明显的脂肪酶活性,酶活高达17.79U/mL。同时,干酪乳杆菌F1能够降解调和油中的多种脂肪酸,特别是亚油酸、棕榈酸、油酸及月桂酸等主要的脂肪酸,对调和油中脂肪酸的总降解率可达1.63mg/mL。干酪乳杆菌F1在人工胃液、人工肠液和胆盐的耐受实验中表现出优异的耐酸耐胆盐能力,能够耐受人体的胃液和肠液,可作为性能优良的益生菌,在肠道中存活并发挥作用。本发明的干酪乳杆菌F1能够作为食品添加剂或者直接制成食品、保健品等。
Description
技术领域
本发明属于乳酸菌技术领域,涉及一株降解油脂的干酪乳杆菌及其应用。
背景技术
膳食脂肪是人体重要的能量来源,对维持人体能量平衡起到重要作用。随着经济的发展,食物供应充足等因素,膳食中脂肪摄入量明显增加,过高的膳食脂肪摄入可以促进肥胖、冠心病、糖尿病和肿瘤等慢性疾病的发生。目前,减少脂肪摄入的方式主要是改变食物的烹饪方式、减少摄入或者采用油脂替代物等,这些方法虽然能在一定程度上降低脂肪的摄入,但是人们还是难以抵御高油脂食品诱人的口感和风味。
油脂在摄入后主要是依靠小肠内的胰脂肪酶和胃脂肪酶将脂肪分解为短链脂肪酸,而对于老年人及新生儿,脂肪酶量少、活性低,对脂肪的消化能力较弱,经常会出现脂肪消化不良性腹泻。
研究表明,微生物能水解油脂,目前已经筛选出很多能够降油脂的微生物,包括Bacillus spp.,Pseudomonas,Aeromonas,Acinetobacter,Candida等,筛选出的这些降解油脂的微生物主要用于工业废水、餐厨垃圾处理等方面,因此,筛选能用于食品或直接食用的降油脂菌具有重要意义。
乳酸菌是一大类能利用碳水化合物代谢产生大量乳酸的细菌的统称,与人类的生活密切相关。研究发现,乳酸菌具有缓解乳糖不耐受、降低胆固醇水平、调节胃肠道菌群平衡、增强机体免疫力等功效,在工业乳酸生产、食品发酵以及保健产品等领域得到了广泛应用,如发酵酸奶、干酪、乳饮料、微生态制剂、发酵香肠等。
发明内容
本发明的目的在于提供一株能够有效降解油脂的适用于食品制备或者直接食用的干酪乳杆菌。
发明人以调和油为唯一碳源,通过菌体富集及初筛、复筛培养等,从糯米酒糟中筛选出一株降解调和油、花生油和芝麻油三种油脂的菌株,且对调和油的降解效果最好,经分子生物学鉴定为干酪乳杆菌(Lactobacillus casei),命名为干酪乳杆菌F1。该菌株已于2019年8月1日在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏,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.18353。
本发明还提供上述干酪乳杆菌F1的培养方法,具体为:将干酪乳杆菌F1接种到以油脂为唯一碳源的培养基中,培养pH为7.0,培养温度为37℃,发酵培养。
干酪乳杆菌是可用于保健食品中的一种益生菌,具有降油脂功能的干酪乳杆菌因其可降低油脂含量、产生具有特殊风味的短链脂肪酸赋予食品特殊的风味等功能,可用于多种食品加工处理中,如生产奶酪、香肠、各种含油糕点及菌粉等。另外,现有含油脂污水处理均采用好氧菌,水面油层会使溶解氧被微生物利用以后得不到有效的补充,从而抑制了微生物的生长和对油脂的降解,干酪乳杆菌是兼性厌氧菌,可以和好氧菌混合用于含油脂污水的处理。
进一步地,本发明还提供上述干酪乳杆菌F1在降解油脂中的应用。
上述应用中,具体的应用方法为:将干酪乳杆菌F1接种到含有油脂的物质中,进行油脂的降解。
更进一步地,本发明提供降解油脂的食品或食品添加剂,所述的食品或食品添加剂中含有干酪乳杆菌F1。
所述的食品或食品添加剂可以为本领域中副干酪乳杆菌作为益生菌常规使用的食品或食品添加剂,例如奶酪、香肠、含油糕点及菌粉等。
与现有技术相比,本发明具有以下优点:
本发明的干酪乳杆菌F1为首株能够降解油脂的可食用菌株。干酪乳杆菌F1能够降解调和油、花生油和芝麻油,对调和油的降解效果最好,降解率为21.23%。降解曲线表明,该菌株在12h内基本进入稳定期,活菌数为7.4LogCFU·mL-1左右,对油脂的降解率随着活菌数的增加而增加。该菌株具有明显的脂肪酶活性,酶活高达17.79U/mL。同时,干酪乳杆菌F1能够降解调和油中多种脂肪酸,特别是亚油酸、棕榈酸、油酸及月桂酸等主要的脂肪酸,对调和油中脂肪酸的总降解率可达1.63mg/mL。干酪乳杆菌F1在人工胃液、人工肠液和胆盐的耐受实验中表现出优异的耐酸耐胆盐能力,能够耐受人体的胃液和肠液,可作为性能优良的益生菌,在肠道中存活并发挥作用。
附图表说明
图1为本发明菌株驯化培养结果图。
图2为本发明菌株初筛培养结果图。
图3为本发明菌株复筛培养结果图。
图4为本发明菌株镜检图。
图5为本发明PCR扩增16S rDNA的琼脂糖凝胶电泳图。
图6为本发明菌株对油脂的降解率图。
图7为本发明菌株降解调和油的降解曲线图。
图8为本发明菌株降解调和油中脂肪酸含量变化图。
表1为试验用油脂的信息。
表2为菌株驯化结果。
表3为菌株初筛培养结果。
表4为菌株复筛培养结果。
表5为菌株生理生化鉴定结果。
表6为干酪乳杆菌F1对人工胃液、人工肠液和胆盐的耐受性结果。
表7为干酪乳杆菌F1抑菌结果。
具体实施方式
下面结合实施例、附图和表对本发明作进一步详述。
本发明先从酒糟样品中按形态分离筛选乳酸菌,通过以油脂为唯一碳源的筛选培养基筛选出具有降解油脂能力菌株,进而通过16SrDNA对菌株进行鉴定。最终确定菌株对调和油、芝麻油、花生油的降解能力。
下述实施例中的相关培养基及试剂配方以及测试方法如下。
1.试验用油脂
所用油脂的信息见表1。
表1试验用油脂的信息
2.试验用培养基
(1)驯化培养基:
MRS培养基(1L):葡萄糖20.0g、胰蛋白胨10.0g、牛肉浸膏10.0g、酵母提取物5.0g、磷酸氢二钾2.0g、柠檬酸三铵2.0g、乙酸钠5.0g、吐温80 1m L、七水硫酸镁0.2g、硫酸锰0.05g,加蒸馏水至1L,pH 7.0。
M17培养基(1L):植物蛋白胨5.0g、聚蛋白胨5.0g、牛肉浸膏2.5g、酵母提取物5.0g、抗坏血酸钠0.5g、七水硫酸镁0.25g、乳糖10.0g、甘油10.0g、磷酸氢二钠5.0g,加蒸馏水至1L,pH 7.0。
(2)初筛培养基:
在MRS培养基的基础上去掉葡萄糖,添加调和油20g/L,即调和油20.0g、胰蛋白胨10.0g、牛肉浸膏10.0g、酵母提取物5.0g、磷酸氢二钾2.0g、柠檬酸三铵2.0g、乙酸钠5.0g、吐温80 1m L、七水硫酸镁0.2g、硫酸锰0.05g,加蒸馏水至1L,pH 7.0。
在M17培养基的基础上去掉乳糖,添加调和油20g/L,即调和油20.0g、植物蛋白胨5.0g、聚蛋白胨5.0g、牛肉浸膏2.5g、酵母提取物5.0g、抗坏血酸钠0.5g、七水硫酸镁0.25g、甘油10.0g、磷酸氢二钠5.0g,加蒸馏水至1L,pH 7.0。
(3)复筛培养基:
在MRS组初筛培养基的基础上去掉牛肉膏、酵母膏等碳源,添加调和油20g/L,以油脂为唯一碳源,即调和油20.0g、磷酸氢二钾2.0g、柠檬酸三铵2.0g、乙酸钠5.0g、吐温80 1mL、七水硫酸镁0.2g、硫酸锰0.05g,加蒸馏水至1L,pH 7.0。
在M17组初筛培养基的基础上去掉牛肉膏、酵母膏等碳源,添加调和油20g/L,以油脂为唯一碳源,即调和油20.0g、抗坏血酸钠0.5g、七水硫酸镁0.25g、甘油10.0g、磷酸氢二钠5.0g,加蒸馏水至1L,pH 7.0。
3.主要测定方法
(1)GC-MS测定脂肪酸
样品的甲酯化:称取0.5g菌液样品置于25mL具塞试管中,加入5.0mL 0.5mol/LNaOH-甲醇溶液,漩涡混匀后置于50℃水浴中进行衍生化反应30min,其间每隔10min取出摇匀1次,30min后取出置于室温下放置2h,其间每隔0.5h摇匀1次。加入5mL正己烷,漩涡提取30s,静置分层后取上层正己烷相装入GC-MS样品瓶中,样品在进样前用0.22μm孔径的尼龙膜过滤。
脂肪酸定性定量分析:进样量为1μL,不分流,进样口温度为250℃,正模式下离子源(EI源)温度为250℃,质量分析器为四级杆质量分析器,质谱裂解电压为70eV,质量扫描范围m/z为30-450。柱温箱升温条件为:50℃保持2min,然后以4℃/min的速度升到200℃,保持5min,然后以4℃/min的速度升至220℃,保持20min。载气(氦气)流速为1mL/min。根据标准品的保留时间和质谱裂解信息确定脂肪酸种类,每克种子(干重)脂肪酸含量以等物质量的脂肪酸甲酯来表示。GC-MS能够同时获取样品的化学成分信息和样品表面化学成分空间分布信息,并以图像的形式直观地反映被测物的物质与空间分布情况,目前在物质成分分析方面被广泛运用。
(2)重量法测定油脂降解率
菌株经不同油脂培养基培养48h后,静置,菌液上清液用30mL正己烷萃取2次,收集有机层(根据样品情况可适当增加萃取次数),经无水MgSO4干燥后,用旋转蒸发仪去除有机溶剂,置于60℃恒温箱干燥1h,放干燥器内冷却0.5h后称量。重复以上操作直至恒重(直至两次称量的差不超过1mg)。油脂降解率的计算:
X=(m1-m0)/m2×100%
式中:X———试样中脂肪的含量,单位为克(g);
m1———恒重后接收瓶和脂肪的含量,单位为克(g);
m0———接收瓶的质量,单位为克(g);
m2———对照样中油脂的质量,单位为克(g)。
(3)脂肪酶酶活力测定
取4个250mL锥形瓶,分别编号0、1、2、3,在0号空白瓶中加入95%乙醇15mL,样品瓶1、2、3分别加入5mL底物溶液(量取2%PVA溶液150mL,加入橄榄油50mL,用混匀器处理3min,现用现配)和pH 4.8磷酸缓冲溶液5mL,恒温水浴预热5min。在3个样品瓶中各加入待测酶液1mL,迅速混匀,30℃下反应10min后(注意准确计时),加入15mL 95%乙醇终止反应。同时向每个锥形瓶中滴加3~4滴酚酞指示剂,用0.1mol/L NaOH标准溶液滴定至溶液呈粉红色,记录NaOH标准溶液的消耗体积,计算酶活。
脂肪酶酶活力单位定义为:在30℃下,每分钟释放出1μmol脂肪酸的酶量定义为1个脂肪酶活力单位(U)。酶活计算公式:Y=(V-V0)×5×n式中:Y为样品的酶活力,单位为U/mL;V为滴定样品时消耗NaOH标准溶液的体积,单位为mL;V0为滴定空白时消耗NaOH标准溶液的体积,单位为mL;n为酶液稀释倍数。
(4)耐酸耐胆盐实验
1)基础培养液的配置:
人工胃液:分别取0.50g NaCl和0.30g胃蛋白酶溶解于去离子水中,用1.0mol/L的盐酸调pH至3.0,pH至2.0,加去离子水至100mL,0.22μm滤器过滤除菌,4℃冰箱冷藏备用。
人工肠液:取KH2PO4 6.8g,加500mL蒸馏水溶解,用质量分数为0.4%的NaOH,1%胰蛋白酶,充分溶解后,用孔径0.22μm微孔滤膜过滤除菌,4℃冰箱冷藏备用。
胆盐培养基:称取一定量的胆盐于MRS液体培养基中,使其胆盐浓度(w/v)为0.1%和0.3%,调pH至6.5±0.2,121℃灭菌15min,冷却备用。
2)耐酸耐胆盐能力测定
取菌株活化2代后的液体培养液6mL于10mL的离心管中,10000r/min离心10分钟去除上清取沉淀,加入等体积0.85%的生理盐水溶液,相同条件离心,去上清取沉淀,继续加入等体积的0.85%的生理盐水溶液,制备乳酸菌菌悬液。取1mL菌悬液加入9mL的人工胃液(pH=3.0,pH=2.0)、人工肠液和9mL的胆盐浓度为0.1%和0.3%(w/v)的MRS培养基中,37℃培养,依次在0,3h时进行取样,计算乳酸菌存活率。
(5)抑菌实验
将菌株接种于MRS培养基,37℃培养24h。收集培养液,4℃,8 000r/min离心10min,得发酵上清液。将指示菌稀释后均匀涂在LB平板上,用直径6mm打孔器在打孔,取150μL发酵上清液注入小孔中,室温下扩散4h后,37℃培养18h,检测抑菌圈的大小。
实施例1
1.菌株筛选
(1)菌株的驯化
无菌条件下,用移液枪吸取适量采集的五种酒糟样品,分别标注为“S1、S2、S3、S4、S5”,将这五种酒糟样品接种于MRS、M17两种培养基,分别放置于37℃、42℃恒温培养箱中培养,共计20个平行样,再将初步培养的菌株进行划线分离驯化。
经过MRS和M17两种培养基48h的驯化培养后,酒糟中的菌株培养结果如图1、表2所示,只有16个培养皿中菌落从外观上较为符合乳酸菌的特质,进一步进行初筛培养。
表2菌株驯化结果
(2)初筛结果
将驯化获得的菌株通过初筛培养基进一步进行筛选。将驯化得到的菌株按照3%的量接种于初筛培养基上,并将培养基放置于37℃、42℃这两种恒温条件下培养24-48h,进行分离初筛,并观察菌株生长情况。结果如图2及表2所示,2、3、7、8、10、13、14、16号这8株菌在初筛培养基中无法生存,无法利用油脂生长,不具有降解油脂的功能。
用接种环在长有菌株的培养皿上每个挑取两个菌落,利用革兰氏染色法初筛菌株进行镜检,判断菌株属性。由图2和表3可知,初筛得到的菌株皆为革兰氏阳性菌,符合实验要求。
表3菌株初筛培养结果
(3)复筛结果
在初筛培养的基础上再去掉牛肉膏、酵母膏等碳源,以油脂为唯一碳源进行复筛培养。由图3和表4可知,经复筛培养基的筛选,只有1、4、18号三株菌株能够在以油脂为唯一碳源的培养基中生存,判定其能够降解油脂,且从其外观形态上看符合乳酸菌的特征。
表4菌株复筛培养结果
2.乳酸菌菌株鉴定
(1)生理生化鉴定
将1号菌进行染色电镜观察,其结果为图4所示,该菌株经革兰氏染色为阳性,并呈现均匀杆状分布。
根据菌落形态及革兰氏染色结果,参照《伯杰氏细菌鉴定手册》对菌株进行生理生化实验,包括过氧化氢酶测定实验、葡萄糖发酵实验、淀粉水解实验、明胶水解实验、甲基红实验、硝酸盐还原实验、硫化氢实验。结果为表5所示,该菌株皆符合和《伯杰氏细菌鉴定手册》中关于乳酸杆菌的描述,初步判定为乳酸杆菌。
表5菌株生理生化鉴定结果
注:“﹢”为阳性,“–”为阴性
(2)16SrDNA扩增、克隆及测序
采用Ezup柱式细菌基因组DNA抽提试剂盒提取菌株的DNA,进行PCR扩增,在1500bp处条带清晰(图5)。PCR扩增后将产物切胶、纯化、回收后,送至生工生物工程(上海)有限公司进行测序,核苷酸序列见序列表SEQ NO.1所示。
测序结果经EZBioCloud中16S-based ID在线序列比对:菌株1的16SrDNA与干酪乳杆菌Lactobacillus casei strain NWAFU1575同源性为99%,相差4个碱基,具有较高的同源性,但尚有部分序列存在一定差异,属新菌株,命名为干酪乳杆菌F1。
3.降油脂能力测定
(1)恒重法测定菌株对油脂的降解率
为了研究干酪乳杆菌F1对于不同油脂的降解效果,将筛选得到的干酪乳杆菌F1接种于分别以芝麻油、花生油、调和油为唯一碳源的培养基中,恒温培养48h,通过恒重法测定菌株对油脂的降解率。
结果由图6可知,干酪乳杆菌F1能够降解调和油、花生油及芝麻油,对调和油的降解效果最好,达到21.23%,分别是花生油和芝麻油降解率的1.3和2.55倍。
(2)菌株的油脂降解曲线
将干酪乳杆菌F1按照3%的量分别接种于含20g/L的调和油为唯一碳源的油脂培养基中,每4个小时取一次样,进行活菌计数和油脂降解率的测定。由图7可知,该菌在12h内基本进入稳定期,活菌数在7.4LogCFU·mL-1。该菌对油脂的降解率随着活菌数的增加而增加,在培养后期仍呈增加的趋势。
(3)菌株的脂肪酶活力
对干酪乳杆菌F1进行脂肪酶活性测定,酶活为17.79U/mL。
(4)GC-MS法分析菌株降解油脂脂肪酸的能力
利用GC-MS测定干酪乳杆菌F1降解调和油中脂肪酸含量的变化(图8)。干酪乳杆菌F1能够降解调和油中的长链脂肪酸,对调和油中脂肪酸的总降解率可达1.63mg/mL。其中,降解率超过10%的脂肪酸有6种。降解结果如下:对亚油酸的降解能力和降解率分别为1.23mg/mL,49.1%;对月桂酸的降解能力和降解率分别为0.097mg/mL,40.89%;对棕榈酸的降解能力和降解率分别为0.083mg/mL,23.88%;对二十碳烯酸的降解能力和降解率分别为0.029mg/mL,18.2%;对油酸的降解能力和降解率分别为0.15mg/mL,16.7%;对反油酸的降解能力和降解率分别为0.023mg/mL,10.3%。
(5)耐酸耐胆盐能力
作为性能优良的益生菌,要在人体发挥作用,必须要能够耐受人体的胃液和肠液,并在通过胃肠道后仍能保证活菌数在1×106cfu/mL以上时才能发挥生物活性。胃液pH值通常为3.0左右,正常人体的小肠胆汁盐含量在0.03%-0.30%之间;食物通过胃肠道的时间一般为2h左右。干酪乳杆菌F1对人工胃液、人工肠液和胆盐的耐受性结果分别见表6。
表6干酪乳杆菌F1对人工胃液、人工肠液和胆盐的耐受性
由表6可知,干酪乳杆菌F1对人工胃液、人工肠液都有较好的耐受性。人工肠液培养3h后,活菌数仍在108cfu/mL以上。人工胃液培养3h后,pH 2胃液中活菌数仍106cfu/mL以上。另外,干酪乳杆菌F1对胆盐有一定的耐受性,而正常人体小肠中的胆汁酸盐含量在0.03%-0.3%范围内波动,由结果推测干酪乳杆菌F1在高胆盐浓度的人体小肠中应该也具有较好的耐受性和存活率。
(6)抑菌能力
采用打孔的方法,测定干酪乳杆菌F1的抑菌能力(表7)。结果表明,该菌株的发酵上清液对六种指示菌的抑菌效果明显。
表7干酪乳杆菌F1抑菌结果
序列表
<110> 扬州大学
<120> 降解油脂的干酪乳杆菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1478
<212> DNA
<213> 干酪乳杆菌(Lactobacillus casei)
<400> 1
gggcgggggg gggtgcctat acatgcagtc gaacgagttc tcgttgatga tcggtgcttg 60
caccgagatt caacatggaa cgagtggcgg acgggtgagt aacacgtggg taacctgccc 120
ttaagtgggg gataacattt ggaaacagat gctaataccg catagatcca agaaccgcat 180
ggttcttggc tgaaagatgg cgtaagctat cgcttttgga tggacccgcg gcgtattagc 240
tagttggtga ggtaatggct caccaaggcg atgatacgta gccgaactga gaggttgatc 300
ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt 360
ccacaatgga cgcaagtctg atggagcaac gccgcgtgag tgaagaaggc tttcgggtcg 420
taaaactctg ttgttggaga agaatggtcg gcagagtaac tgttgtcggc gtgacggtat 480
ccaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540
cgttatccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa 600
agccctcggc ttaaccgagg aagcgcatcg gaaactggga aacttgagtg cagaagagga 660
cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 720
aggcggctgt ctggtctgta actgacgctg aggctcgaaa gcatgggtag cgaacaggat 780
tagataccct ggtagtccat gccgtaaacg atgaatgcta ggtgttggag ggtttccgcc 840
cttcagtgcc gcagctaacg cattaagcat tccgcctggg gagtacgacc gcaaggttga 900
aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960
aacgcgaaga accttaccag gtcttgacat cttttgatca cctgagagat caggtttccc 1020
cttcgggggc aaaatgacag gtggtgcatg attgtcgtca gctcgtgtcg tgagatgttg 1080
ggttaagtcc cgcaacgagc gcaaccctta tgactagttg ccagcattta gttgggcact 1140
ctagtaagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc 1200
ccttatgacc tgggctacac acgtgctaca atggatggta caacgagttg cgagaccgcg 1260
aggtcaagct aatctcttaa agccattctc agttcggact gtaggctgca actcgcctac 1320
acgaagtcgg aatcgctagt aatcgcggat cagcacgccg cggtgaatac gttcccgggc 1380
cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccgaagccg gtggcgtaac 1440
ccttttaggg agcgagccgt ctaaggggga caaaagtg 1478
Claims (6)
1.干酪乳杆菌(Lactobacillus casei)菌株F1,保藏编号为CGMCC No.18353。
2.根据权利要求1所述的干酪乳杆菌菌株F1的培养方法,其特征在于,具体为:将干酪乳杆菌菌株F1接种到以油脂为唯一碳源的培养基中,培养pH为7.0,培养温度为37℃,发酵培养。
3.根据权利要求1所述的干酪乳杆菌菌株F1在降解油脂中的应用。
4.根据权利要求3所述的应用,其特征在于,具体的应用方法为:将干酪乳杆菌菌株F1接种到含有油脂的物质中,进行油脂的降解。
5.降解油脂的食品或食品添加剂,其特征在于,所述的食品或食品添加剂中含有权利要求1所述的干酪乳杆菌菌株F1。
6.根据权利要求5所述的食品或食品添加剂,其特征在于,所述的食品或食品添加剂为奶酪、香肠、含油糕点或菌粉。
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