CN114525221B - 一种提高益生菌耐受性的方法及其应用 - Google Patents
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Abstract
本发明公开了一种提高益生菌耐受性的方法及其应用,属于食品生物技术领域。本发明方法是以乙基纤维素纳米纤维膜作为固相载体,通过将益生菌接种至含有乙基纤维素纳米纤维膜的培养基中进行培养。发酵后获得有组织的益生菌群落形成的生物膜,经过不同浓度氯化钠、胆盐、高温以及模拟胃肠液处理后,生物膜细胞存活率高达82%以上,活菌数可达到1011CFU/g。本发明解决了开发高活性、强抗逆性的益生菌菌剂的关键共性技术问题,制备的益生菌菌剂可以应用于固体饮料、功能食品、饲料、微生态制剂、发酵食品等产品形式中。
Description
技术领域
本发明属于食品生物技术领域,具体涉及一种提高益生菌耐受性的方法及其应用。
背景技术
植物乳杆菌(Lactobacillus plantarum)属于革兰氏阳性菌,是一种厌氧或兼性厌氧菌。植物乳杆菌在自然界广泛存在,也存在于人体胃肠道中,通过胃并定植于肠道发挥有益作用,是人体肠道内的有益菌之一。目前已有大量研究表明,植物乳杆菌安全性高,具有多种益生功能如调节肠道菌群平衡、改善胃肠道功能、参与免疫系统调节等,在食品发酵、工业生产、动物饲料及医疗保健等领域,都有着广泛的应用。
只有当活菌数大于107CFU/mL时,益生菌才能更好地发挥其益生功能。然而,益生菌在加工、运输以及贮藏过程中容易受到不良条件(如酸碱、渗透压以及高温等)的影响,导致其活菌数大大降低,进而影响了其功效。因此提升植物乳杆菌对不利条件的耐受性就显得尤为重要,如何最大程度的提高植物乳杆菌耐受性是其应用于实际生产的关键限制因素。
专利CN103396962A等文献公开的提高耐受性的方法多集中于包埋壁材配方的研究,通过糖、蛋白质、盐等物质的筛选来优化配方从而提高存活率。保护剂配方的优化、包埋工艺条件的改良、微胶囊化技术的应用尽管能在一定程度上提高益生菌存活率,但上述方法的提高程度有限,且操作步骤繁琐,需要借助外力。相比保护剂配方的研究,提升自身耐受性才是提高植物乳杆菌存活率的根本途径。
发明内容
为解决相关问题,本发明的首要目的在于提供一种提高益生菌耐受性的方法。
本发明的另一目的在于提供上述提高益生菌耐受性的方法的应用。
为了实现上述发明目的,本发明采用以下技术方案:
一种提高益生菌耐受性的方法,是将益生菌接种至含有乙基纤维素纳米纤维膜的培养基中,于pH5.8~6.5、28~32℃培养12~36h。
进一步地,所述的益生菌包括但不限于乳杆菌类和双歧杆菌类益生菌,其中,乳杆菌类益生菌包括植物乳杆菌、嗜酸乳杆菌、干酪乳杆菌、詹氏乳杆菌等,双歧杆菌类益生菌包括长双歧杆菌、短双歧杆菌、嗜热双歧杆菌、青春双歧杆菌、两歧双歧杆菌等;优选指植物乳杆菌;更优选指植物乳杆菌GIM1.648。
进一步地,所述的益生菌为处于对数生长期的益生菌,优选通过如下方法制备得到:取200μL甘油管保藏的益生菌,接到10mL无菌液体培养基中,37℃静置培养活化16~18h,培养结束以后,取200μL菌液于新的10mL无菌液体培养基中,同样条件活化16~18h。当所述的益生菌为植物乳杆菌时,所述的液体培养基为MRS肉汤培养基。
进一步地,所述的益生菌的接种量为1%~2%(v/v)。
进一步地,所述的乙基纤维素纳米纤维的直径为841±408nm,可以通过静电纺丝法制备得到;优选如下方法制备得到:取乙基纤维素,按质量体积比1g:2~3mL加入冰乙酸,充分搅拌,得到均一透明的纺丝液,进行纺丝;纺丝的条件如下:电压15KV,针头距离接收板的距离18cm,进液速率0.5mL/h,纺丝时间2h。乙基纤维素纳米纤维膜具有良好的生物相容性。
更进一步地,获得乙基纤维素纳米纤维膜后,于50℃真空干燥12h,去除残留的溶剂。
进一步地,所述的培养基的配方为:蛋白胨5~10g/L,牛肉膏5~10g/L,酵母粉5~8g/L,柠檬酸三胺2~3g/L,乙酸钠4~5g/L,葡萄糖15~20g/L,三水合磷酸氢二钾2~3g/L,七水合硫酸镁0.05~0.1g/L,一水合硫酸锰0.05~0.2g/L,水余量;优选为:蛋白胨10g/L,牛肉膏10g/L,酵母粉5g/L,柠檬酸三胺2g/L,乙酸钠5g/L,葡萄糖20g/L,三水合磷酸氢二钾2g/L,七水合硫酸镁0.1g/L,一水合硫酸锰0.058g/L,水余量。
进一步地,所述的培养的条件为:于pH6.5、30℃培养36h。
一种利用上述方法制备的益生菌菌剂,所述的益生菌菌剂是以乙基纤维素纳米纤维膜作为固相载体,被细胞外基质紧密包裹的有组织的益生菌群落形成的生物膜。
进一步地,获得所述的益生菌菌剂后,倒掉培养基,清洗纤维膜,去除浮游菌。
更进一步地,所述的清洗所用的试剂为无菌的0.85%(w/v)生理盐水。
上述益生菌菌剂在食品领域中的应用。
进一步地,所述的应用为用于生产发酵乳制品、发酵果蔬、功能食品、饲料、微生态制剂和/或固体饮料。
本发明相对于现有技术具有如下的优点及效果:
本发明以具有较高比表面积的静电纺纳米纤维膜作为固相载体,使益生菌在其表面形成生物膜,达到提高益生菌自身抗逆性的效果。经过不同浓度氯化钠、胆盐、高温以及模拟胃肠液处理后,益生菌细胞存活率达到82%以上,活菌数可达到1011CFU/g,解决了在开发高活性、强耐受性的益生菌菌剂的关键技术问题,制备的高活性益生菌菌剂可用于固体饮料、功能食品、饲料、微生态制剂、发酵食品等产品中。
附图说明
图1为不同培养时间下生物膜的扫描电镜图。
图2为生物膜与游离细胞对氯化钠的耐受性分析图。
图3为胆盐处理2h后生物膜与游离细胞存活率的比较图。
图4为胆盐处理4h后生物膜与游离细胞存活率的比较图。
图5为生物膜与游离细胞对温度的耐受性分析图。
图6为生物膜与游离细胞对模拟胃肠液的耐受性分析图。
具体实施方式
下面结合实施例和附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1培养时间对植物乳杆菌生物膜形成能力的影响
(1)静电纺纳米纤维膜制备:称量5g乙基纤维素(EC,6-9mPa.s)于血清瓶中,加入10mL冰乙酸,放入一个2.5cm的磁力搅拌子,用磁力搅拌器在1000rpm搅拌2h,得到均一透明的纺丝液。利用自制的纺丝设备纺丝,接收板表面覆盖一层铝箔。纺丝条件如下:电压15KV,针头距离接收板的距离18cm,进液速率0.5mL/h,纺丝时间2h。最后将制备得到的乙基纤维素纳米纤维膜放于50℃的真空干燥箱中干燥12h,去除残留的溶剂,得到乙基纤维素纳米纤维膜;
(2)植物乳杆菌活化液准备:取200μL甘油管保藏的植物乳杆菌GIM1.648,接到10mL无菌MRS肉汤培养基中,在37℃恒温培养箱中静置培养活化16~18h,培养结束以后,取200μL菌液于新的10mLMRS肉汤培养基中,同样条件活化16~18h,得到菌种活化液;
(3)用于生物膜形成的培养基制备:蛋白胨10g/L,牛肉膏10g/L,酵母粉5g/L,柠檬酸三胺2g/L,乙酸钠5g/L,葡萄糖20g/L,三水合磷酸氢二钾2g/L,七水合硫酸镁0.1g/L,一水合硫酸锰0.058g/L,水余量;
(4)生物膜形成:把制备得到的纳米纤维膜放于培养皿内,然后加入3mL步骤(3)制备的液体培养基,接种0.5%(v/v)菌种活化液,37℃静置培养12~72h后,倒掉培养基,用无菌的0.85%(w/v)生理盐水清洗纤维膜三次,去除浮游菌,得到生物膜。
(5)生物膜活菌计数:培养结束后将长有生物膜的纤维膜用无菌镊子夹入无菌的3cm培养皿中,用3mL的0.85%无菌生理盐水将其浸没,将培养皿放置在超声清洗装置中超声(150w,14min,25℃),保证生物膜从纤维膜上洗脱下来,同时混悬于生理盐水中。梯度稀释生物膜悬液,取100微升稀释液涂布在固体平板,培养48小时,计算活菌数,单位CFU/g。一式三个平行,重复三次实验。
结果如表1所示,培养时间对生物膜形成有一定的影响,当培养时间从12h增加到36h,生物膜活菌数也有所增加,36h时达到最大值,即11.53Log CFU/g。
表1培养时间对生物膜活菌数的影响
实施例2扫描电子显微镜观察不同培养时间对植物乳杆菌生物膜形成能力的影响
按照实施例1相同步骤进行操作形成生物膜,唯一不同的是培养时间为4h、8h、12h、16h、20h、24h和36h,纤维膜片在室温条件下干燥以后,用手术刀剪成1cm×0.5cm的长条后通过导电胶贴在电镜专用样品台上,离子溅射仪喷金后用场发射扫描电子显微镜观察生物膜。放大倍数6.0k倍。
电镜观察结果分析:如图1所示,当培养时间为4h时,仅有少量的游离细胞粘附在单根纤维上,培养时间增加到12h时,开始出现细胞聚集现象,且有的细胞团块聚集在纤维与纤维之间的空隙处,24h时,细胞相互聚集在一起,几乎填满了纤维原有的孔隙结构,另外,出现了孔洞,是细菌生物膜的特征结构。当培养时间延长到36h,大量的菌体聚集,可以看到菌体之间紧密排列在纤维膜表面,甚至覆盖了纤维,说明培养36h后,纤维膜上形成了成熟的生物膜。
实施例3植物乳杆菌在不同培养温度下形成生物膜的能力
按照实施例1相同步骤进行操作形成生物膜,区别在于,设置培养温度为25℃、30℃和37℃。
结果如表2所示,随着培养温度的增加,生物膜活菌数呈现先增加后降低的趋势,当培养温度为30℃时,菌落数为11.44Log CFU/g,达到最大值,所以30℃是植物乳杆菌生物膜形成的最佳培养温度。
表2培养温度对生物膜活菌数的影响
实施例4植物乳杆菌在不同接种浓度下形成生物膜的能力
按照实施例1相同步骤进行操作,区别在于,设置接种浓度为0.1%、0.5%、1%、1.5%、2%、2.5%和3%(v/v)。
结果如表3所示,可以看出,接种浓度对生物膜活菌数有较大的影响,当接种浓度从0.1%增加到3%时,活菌数呈现先增加后降低的趋势,在接种浓度为1%时,活菌数达到最大,为11.29Log CFU/g,说明最佳的接种浓度是1%。
表3接种浓度对生物膜活菌数的影响
综上,本专利提出,通过静电纺丝技术制备得到的纳米纤维膜具有较高的比表面积和孔隙率,适宜植物乳杆菌粘附进而形成生物膜。
效果实施例1植物乳杆菌生物膜对不同浓度氯化钠的耐受能力研究
(1)静电纺纳米纤维膜制备:称量5g乙基纤维素(EC,6-9mPa.s)于血清瓶中,加入10mL冰乙酸,放入一个2.5cm的磁力搅拌子,用磁力搅拌器在1000rpm搅拌2h,得到均一透明的纺丝液。利用自制的纺丝设备纺丝,接收板表面覆盖一层铝箔。纺丝条件如下:电压15KV,针头距离接收板的距离18cm,进液速率0.5mL/h,纺丝时间2h。最后将制备得到的乙基纤维素纳米纤维膜放于50℃的真空干燥箱中干燥12h,去除残留的溶剂,得到乙基纤维素纳米纤维膜;
(2)植物乳杆菌活化液准备:取200μL甘油管保藏的植物乳杆菌,接到10mL无菌MRS肉汤培养基中,在37℃培养箱中静置培养活化16~18h,培养结束以后,取200μL菌液于新的10mL无菌MRS肉汤培养基中,同样条件活化16~18h,得到菌种活化液;
(3)用于生物膜形成的培养基制备:蛋白胨10g/L,牛肉膏10g/L,酵母粉5g/L,柠檬酸三胺2g/L,乙酸钠5g/L,葡萄糖20g/L,三水合磷酸氢二钾2g/L,七水合硫酸镁0.1g/L,一水合硫酸锰0.058g/L,水余量;
(4)生物膜形成:把制备得到的纳米纤维膜放于培养皿内,然后加入3mL步骤(3)制备的无菌肉汤培养基,接种1%(v/v)菌种活化液,30℃静置培养36h后,倒掉培养基,用无菌的0.85%(w/v)生理盐水清洗纤维膜三次,去除浮游菌,得到生物膜。
(5)生物膜活菌计数:培养结束后将长有生物膜的纤维膜用无菌镊子夹入无菌的3cm培养皿中,以3mL的0.85%无菌生理盐水将其浸没,将培养皿放置在超声清洗装置中超声(150w,14min,25℃),保证生物膜从纤维膜上洗脱下来,同时混悬于生理盐水中。梯度稀释生物膜悬液,取100微升稀释液涂布在固体平板,培养48小时,计算活菌数,单位CFU/g。一式三个平行,重复三次实验。
(6)生物膜对氯化钠的耐受能力实验:在无菌MRS肉汤培养基中加入不同浓度的氯化钠,制备得到含有不同浓度氯化钠(2%、4%、6%、8%、10%、12%、14%(w/v))的培养基,115℃下灭菌30min,用无菌镊子将通过(4)得到的生物膜转移到含有3mL不同浓度氯化钠的培养基中,37℃静置培养24h,倒掉培养基,用无菌生理盐水洗涤三次去除浮游菌,按照(5)进行活菌计数。生物膜细胞存活率计算公式:存活率(%)=(N/N0)×100,式中,N为氯化钠处理后(Log CFU/g)的生物膜细胞活菌数;N0是氯化钠处理之前生物膜细胞活菌数(LogCFU/g)。
对比例1游离植物乳杆菌对不同浓度氯化钠的耐受能力研究
(1)MRS肉汤培养基制备:蛋白胨10g/L,牛肉膏10g/L,酵母粉5g/L,柠檬酸三胺2g/L,乙酸钠5g/L,葡萄糖20g/L,三水合磷酸氢二钾2g/L,七水合硫酸镁0.1g/L,一水合硫酸锰0.058g/L,吐温80 1g/L,水余量;
(2)植物乳杆菌活化液准备:取200μL甘油管保藏的植物乳杆菌,接到10mL无菌MRS肉汤培养基中,在37℃培养箱中静置培养活化16~18h,培养结束以后,取200μL菌液于新的10mL无菌MRS肉汤培养基中,同样条件活化16~18h,得到菌种活化液;
(3)在3mL无菌MRS肉汤培养基中接种1%(v/v)菌种活化液,37℃静置培养36h后,将菌液离心(6000rpm,5min,4℃)得到菌泥,用无菌盐水洗涤三次去除培养基,加入3mL含有不同浓度氯化钠的培养基中,37℃静置培养24h后,离心(6000rpm,5min,4℃),倒掉上清后,在离心管中加入3mL无菌生理盐水重悬菌泥,梯度稀释菌悬液,取100微升稀释液涂布在固体平板上,培养48小时,计算活菌数,单位CFU/g。一式三个平行,重复三次实验。游离细胞存活率计算公式如效果实施例1生物膜细胞存活率计算公式。
游离细胞和生物膜存活率如图2,可以看出,无论氯化钠浓度多少,生物膜细胞的存活率均要高于游离细胞。当氯化钠浓度为6%时,生物膜细胞的存活率为96%,而游离细胞的存活率为83%。说明形成生物膜以后,植物乳杆菌对氯化钠的耐受能力得到了明显提高。
效果实施例2植物乳杆菌生物膜对不同浓度胆盐的耐受能力研究
按照效果实施例1相同步骤进行操作,唯一不同就是所用的培养基是含有不同浓度胆盐(0.05%、0.1%、0.2%、0.3%(w/v))的无菌MRS肉汤培养基,37℃静置培养2h或4h。游离细胞对氯化钠的耐受能力研究按照对比例1进行操作。
由图3和图4所示结果可知,与游离细胞相比,植物乳杆菌在形成生物膜后,其存活率较高,在胆盐浓度为0.3%的条件下处理2h后,生物膜细胞存活率高达95%,而游离细胞存活率仅为77%,同浓度下处理4h后,生物膜细胞存活率高达89%,而游离细胞存活率仅为72%,虽然增加处理时间后,二者的存活率均有所降低,但是生物膜细胞的存活率依然要远高于游离细胞,以上说明植物乳杆菌生物膜具有较强的胆盐耐受能力。
效果实施例3植物乳杆菌生物膜耐热性研究
按照效果实施例1相同步骤进行操作,唯一不同就是将长有成熟生物膜的纤维膜放于3cm培养皿中,加入3mL无菌肉汤培养基,然后将培养皿放入水浴锅(55℃、60℃、65℃、70℃)中加热30min。游离细胞对高温的耐受能力研究按照对比例1进行操作。
由图5所示结果可知,与游离细胞相比,植物乳杆菌在形成生物膜后,其存活率较高,70℃加热30min后,生物膜细胞存活率高达82%,而游离细胞存活率仅有43%,说明植物乳杆菌形成生物膜以后具有较强的耐热能力。
效果实施例4植物乳杆菌生物膜对模拟胃肠液耐受能力研究
按照效果实施例1相同步骤进行操作,唯一不同就是将长有成熟生物膜的纤维膜放于3cm培养皿中,加入3mL模拟胃液,37℃,100rpm,温育2h后倒掉模拟胃液,用无菌的生理盐水洗涤三次,按照实施例1(5)进行活菌计数,按照实施例1(6)计算存活率,然后将经模拟胃液消化后的生物膜转移到无菌培养皿中,加入3mL模拟肠液,37℃,100rpm,温育3h后倒掉模拟肠液,用无菌生理盐水洗涤三次,按照实施例1(5)进行活菌计数,按照实施例1(6)计算存活率。游离细胞对模拟胃肠液耐受能力研究按照对比例1进行操作。
模拟胃肠液的配制如下:
模拟胃液:用1M盐酸调节0.85%(w/v)生理盐水的pH至3.0,加入一定量的胃蛋白酶,使其终浓度为3g/L,用0.22μm滤膜过滤除菌得到模拟胃液。
模拟肠液:在0.85%(w/v)生理盐水中加入一定量的胆盐和胰蛋白酶,使得胆盐的终浓度为0.3%(w/v),胰蛋白酶的浓度为1g/L,调节pH至6.8,用0.22μm滤膜过滤除菌得到模拟肠液。
由图6所示结果可知,与游离细胞相比,植物乳杆菌在形成生物膜后,其存活率较高,经过2h的模拟胃液消化后,生物膜细胞存活率高达92%,而游离细胞存活率为83%,再经过3h的模拟肠液消化后,生物膜细胞存活率高达82%,而游离细胞存活率为57%,以上说明植物乳杆菌形成生物膜以后具有较强的耐受模拟胃肠液能力。
综合以上实验结果,形成生物膜以后,植物乳杆菌对氯化钠、胆盐、高温以及模拟胃肠液均表现出良好的耐受能力,说明植物乳杆菌生物膜具有良好的耐受性。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受所述的实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (8)
1.一种提高益生菌耐受性的方法,其特征在于:将益生菌接种至含有乙基纤维素纳米纤维膜的培养基中,于pH5.8~6.5、28~32℃培养12~36 h;
所述的乙基纤维素纳米纤维膜通过如下方法制备得到:取乙基纤维素,按质量体积比1g:2~3 mL加入冰乙酸,充分搅拌,得到均一透明的纺丝液,进行纺丝;纺丝的条件如下:电压15 KV,针头距离接收板的距离18 cm,进液速率0.5 mL/h,纺丝时间2 h;获得乙基纤维素纳米纤维膜后,于50℃真空干燥12 h,去除残留的溶剂;
所述的益生菌为植物乳杆菌;
所述的益生菌的接种量为1%~2%v/v。
2.根据权利要求1所述的提高益生菌耐受性的方法,其特征在于:
所述的益生菌通过如下方法制备得到:取200μL甘油管保藏的益生菌,接到10 mL无菌液体培养基中,37℃静置培养活化16~18 h,培养结束以后,取200μL菌液于新的10mL无菌液体培养基中,同样条件活化16~18 h。
3.根据权利要求1所述的提高益生菌耐受性的方法,其特征在于:
所述的培养基的配方为:蛋白胨5~10 g/L,牛肉膏5~10 g/L,酵母粉5~8g/L,柠檬酸三胺2~3g/L,乙酸钠4~5 g/L,葡萄糖15~20 g/L,三水合磷酸氢二钾2~3g/L,七水合硫酸镁0.05~0.1g/L,一水合硫酸锰0.05~0.2 g/L,水余量。
4.根据权利要求1~3任一项所述的提高益生菌耐受性的方法,其特征在于:
所述的培养基的配方为:蛋白胨10 g/L,牛肉膏10 g/L,酵母粉5 g/L,柠檬酸三胺2 g/L,乙酸钠5 g/L,葡萄糖20 g/L,三水合磷酸氢二钾2 g/L,七水合硫酸镁0.1g/L,一水合硫酸锰0.058 g/L,水余量;
所述的培养的条件为:于pH6.5、30℃培养36 h。
5.一种利用权利要求1~4任一项中所述的方法制备的益生菌菌剂,其特征在于:所述的益生菌菌剂是以乙基纤维素纳米纤维膜作为固相载体,被细胞外基质紧密包裹的有组织的益生菌群落形成的生物膜。
6.根据权利要求5所述的益生菌菌剂,其特征在于:
获得所述的益生菌菌剂后,倒掉培养基,清洗纤维膜,去除浮游菌;
所述的清洗所用的试剂为无菌的0.85%w/v生理盐水。
7.权利要求5或6所述的益生菌菌剂在食品领域中的应用。
8.根据权利要求7所述的应用,其特征在于:所述的应用为用于生产发酵乳制品、发酵果蔬、功能食品、饲料、微生态制剂和/或固体饮料。
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