CN115193350B - 乳杆菌在低pH值果汁中微胶囊化包封方法 - Google Patents
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Abstract
本发明涉及微生物技术领域,尤其涉及乳杆菌在低pH值果汁中微胶囊化包封方法。该包封方法包括:(1)果胶制备、(2)菌种活化与悬浮液制备、(3)微胶囊程序化、(4)生物膜程序化、(5)将制备完成的微胶囊与无菌果汁混合混匀。该包封方法利用菌种自身形成的生物膜,提高单次微胶囊化对菌种的保护作用,提高了益生菌在低酸碱度果汁基质中的生存能力和稳定性。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及乳杆菌在低pH值果汁中微胶囊化包封方法。
背景技术
除乳制品外,含有益生菌的替代功能性食品,尤其是果汁,具有潜在的吸引力。益生菌在储存于许多酸碱度在4.0至5.0之间的发酵乳中时会丧失生存能力。提高果汁酸碱度的一个可能方法是将其与牛奶成分混合,从而改变其感官特性,但消费者乳糖不耐受和奶制品胆固醇含量高也是与乳制品成分相关的两个现实问题。微生物的微囊化经常用于保护免受环境因素的影响,并且活性益生菌细胞以微囊化形式的递送最近受到越来越多的关注。微囊化可为细菌提供一个特别合适的微环境,使其能够在加工和储存过程中存活,直至在胃肠道的适当位置释放。然而,现有微胶囊化方法对菌种的保护功能有限,尤其当其处于低酸碱度的环境下时,其生存能力和稳定性是特别需要注意的,鉴于此,亟需研发一种更有效的微胶囊包封方法。
发明内容
本发明为了弥补现有技术的不足,提供了乳杆菌在低pH值果汁中微胶囊化包封方法,利用菌种自身形成的生物膜,提高单次微胶囊化对菌种的保护作用,提高了益生菌在低酸碱度果汁基质中的生存能力和稳定性,解决了现有技术中存在的问题。
为实现上述发明目的,本发明采用的技术方案是:
乳杆菌在低pH值果汁中微胶囊化包封方法,包括如下操作步骤:
(1)果胶制备
取干燥的鲜浆果,提取制备果胶;
(2)菌种活化与悬浮液制备
a活化乳酸菌种;
b将步骤(1)的果胶溶解于0.1%蛋白胨水溶液中,添加适量吐温-80,充分混匀后得果胶溶液,置于水浴灭菌,冷却,将乳酸菌沉淀物悬浮在果胶溶液中,混匀备用;
(3)微胶囊程序化
将步骤(2)乳酸菌悬浮液使用微胶囊成型装置挤出,挤出速率2-5mL/min;挤出到200-300mL、3.5-4.5%(W/V)氯化钙溶液中,以200-250转/分的速度搅拌;所得微胶囊在室温下与氯化钙溶液接触30-35min,以确保完全固化;倾析除去氯化钙溶液,通过玻璃过滤漏斗重力过滤回收微胶囊;然后利用无菌0.1%蛋白胨水溶液清洗数次,去除多余钙离子及未包被的细菌;最后,悬浮在Ringer平衡盐溶液中得微胶囊化乳杆菌,备用;
(4)生物膜程序化
在MRS肉汤培养基中添加3.5-4.5%(W/V)氯化钙溶液,充分灭菌混匀后,将步骤(3)制备好的微胶囊化乳杆菌转移至培养基中,在37℃下连续培养18h;倾析除去培养基后,通过玻璃过滤漏斗重力过滤回收微胶囊,并用无菌0.1%蛋白胨溶液清洗数次,制备得微胶囊;
(5)将步骤(4)制备完成的微胶囊与无菌果汁按1g/10ml进行混合混匀。
进一步地,步骤(1)果胶的制备操作为:将干燥的鲜浆果研磨成粉后,用调整至pH6的柠檬酸-柠檬酸钠溶液混合,于65℃水浴2小时提取果胶;于4000r/min条件下离心15-25min,去除上清液后,用60%乙醇反复洗涤沉淀果胶2-3次,置于55-65℃烘箱中12h,收集至干燥箱中备用。
进一步地,离心时间20min,烘箱干燥温度60℃。
进一步地,步骤(1)采用250mL柠檬酸-柠檬酸钠溶液与5g鲜浆果粉混匀。
进一步地,步骤(2)中a活化菌种过程为:挑取乳酸菌种,在MRS固体培养基预培养,挑取菌种至新鲜MRS液体培养基预活化,37℃培养18h,同条件下活化2次;然后在4000r/min条件下离心15-25min,倾去培养基后添加生理盐水继续离心2-3次,收集沉淀物备用。
进一步地,步骤(2)b的水浴灭菌采用95℃下水浴15min灭菌;乳酸菌沉淀物悬浮在果胶溶液中后,通过微涡旋混匀备用。
进一步地,步骤(3)微胶囊成型装置的挤出孔0.4-0.5mm、氮气压力0.3-0.5bar;挤出速率通过注射泵控制。
本发明的有益效果:
1、现有技术为提高微胶囊化对菌种的保护性,多采用二次或多次包被的方式来提供更高的保护,利用生物膜进行加固的方法十分少见。本发明创新性的采用了微胶囊和生物膜相结合的方式。
2、通过电镜扫描证实了生物膜的形成,且包封率有了显著提升。本发明结合生物膜包被相较于非果胶诱导的SA微胶囊化乳杆菌效果提升11.74%,较之二重包被的包封率效果提升27.38%。
3、现有技术的多次包被会扩大底物尺寸,导致用于饮用的果汁产生负面影响,降低了饮用口感。而本发明微胶囊化以及加固附着生物膜微胶囊化对菌种在低pH果汁环境中的发酵特性不会造成影响,相比多重包被会降低发酵特性具备明显优势。
4、本发明方法保存50天后,活菌数达7.0*109cfu/mL,相较无包埋处理的效果提升27.27%,相较普通包被(SA-无果胶)效果提升11.11%。
5、本发明结合生物膜包被后的乳杆菌表现出明显的耐热性、耐寒性和耐酸性,现有技术多次包被会导致益生菌在人体肠道内的释放受阻,不利于益生菌在肠道中附着生长,模拟胃肠消化环境发现,本发明结合生物膜包被后的乳杆菌几乎免受胃液消化。
具体实施方式
为能清楚说明本方案的技术特点,下面通过具体实施方式,对本发明进行详细阐述。
实施例1
乳杆菌在低pH值果汁中微胶囊化包封方法,包括如下操作步骤:
(1)果胶制备
将干燥的鲜浆果研磨后,用调整至pH 6的柠檬酸-柠檬酸钠溶液混合,于65℃水浴2h,提取果胶;将混合物于4000r/min条件下离心20min,去除上清液后,用60%乙醇反复洗涤沉淀果胶2次,置于60℃烘箱中12h,收集至干燥箱中备用;
(2)菌种活化与悬浮液制备
a活化乳酸菌种;
挑取市售来源的乳酸菌种,在MRS固体培养基预培养,挑取菌种至新鲜MRS液体培养基预活化,37℃培养18h,同条件下活化2次;然后在4000r/min条件下离心20min,倾去培养基后添加生理盐水继续离心3次,收集沉淀物备用;
b将步骤(1)的果胶溶解于0.1%蛋白胨水溶液中,添加适量吐温-80(0.05%,w/v),充分混匀后得果胶溶液,置于95℃下水浴15min灭菌,冷却,将乳酸菌沉淀物悬浮在果胶溶液中,混匀备用;
(3)微胶囊程序化
将步骤(2)制得的乳酸菌悬浮液使用0.4-0.5mm孔和氮气压力0.3-0.5bar的微胶囊成型装置挤出,挤出速率2-5mL/min;挤出到200mL、3.5%(W/V)氯化钙溶液中,以200-250转/分的速度搅拌;所得微胶囊在室温下与氯化钙溶液接触35min,以确保完全固化;倾析除去氯化钙溶液,通过玻璃过滤漏斗重力过滤回收微胶囊;然后利用无菌0.1%蛋白胨水溶液清洗数次,去除多余钙离子及未包被的细菌;最后,悬浮在Ringer平衡盐溶液中得微胶囊化乳杆菌,备用;
(4)生物膜程序化
在MRS肉汤培养基中添加4.5%(W/V)氯化钙溶液,充分灭菌混匀后,将步骤(3)制备好的微胶囊化乳杆菌转移至培养基中,在37℃下连续培养18h;倾析除去培养基后,通过玻璃过滤漏斗重力过滤回收微胶囊,并用无菌0.1%蛋白胨溶液清洗数次,制备得微胶囊;
(5)将步骤(4)制备完成的微胶囊与无菌果汁按1g/10ml进行混合混匀,即得。
实施例2
采用普通包被(SA-无果胶)方法制备含有乳酸菌的低pH果汁。方法采用何荣军等《海藻酸钠/壳聚糖微胶囊的制备及其应用研究进展》中的两步法。
实施例3
二重包被制备含有乳酸菌的低pH果汁。方法采用Annan等Effect of processvariables on particlesize and viability of Bifidobacterium lactis Bb-12ingenipin-gelatin microspheres中的方法。
一、不同包被方式包封率的测定
对本方明生物膜包被方法的实施例1,现有包被方法实施例2、实施例3进行包封率测定,测定结果如下表1:
表1不同包被方式包封率
由表1结果可知,本发明方法的包封率显著提升,相较于实施例2的非果胶诱导的SA微胶囊化乳杆菌效果提升11.74%,较之实施例3的二重包被的包封率效果提升27.38%。
二、对乳酸菌种在低pH果汁环境中的发酵特性影响
发酵特性指标以发酵液总酸(以乳酸含量计)、活菌数和感官评分进行评价。
2.1发酵液总酸(以乳酸含量计)的测定及计算方法,采用GB 12456-2021《食品中总酸的测定》自动电位滴定法
2.2活菌数计算,采用GB 4789.2-2016《菌落总数测定》计数。
2.3感官评价
采用四分法量化,以优100分、良75分、中50分、差25分进行赋分,确立评分标准,建立评价分析标准表。分别取200mL样品置于白色透明玻璃杯中,挑选15名感官评价小组成员,在自然光线下用目测法观察样品的色泽和组织形态,嗅其气味,品尝滋味,对样品的色泽、口感、滋味、组织形态四个方面进行综合评判及打分,得到感官评价得分。
表2感官评价评分标准参考
设置正常发酵组,正常发酵采用乳酸菌种直接发酵低pH果汁,不作其他处理,发酵周期及发酵条件同其他进行了包封处理的实施例组。
结果表明,正常发酵组的发酵液总酸为5.25g/mL、活菌数7.8*109cfu/mL、感官79;实施例2的SA包被总酸为5.289g/mL、活菌数7.5*109cfu/mL、感官78;实施例1的生物膜包被总酸5.361g/mL、活菌数7.6*109cfu/mL、感官82;实施例3的二重包被总酸4.221g/mL、活菌数5.9*109cfu/mL、感官70.75。由此可见,本发明生物膜包被较正常发酵,总酸提升2.11%,活菌数降低-2.63%,感官提升3.80%;较SA包被总酸提升1.36%,活菌数提升1.33%,感官提升5.13%;较二重包被总酸提升27.01%,活菌数提升28.09%,感官提升15.90%。
上述不同包埋方式发酵特性的测定结果如下表2:
表2不同包埋方式发酵特性
三、稳定性考察
5℃条件下保存50天后,测定活菌数,进行稳定性考察。
结果表明,本发明方法的实施例1的活菌数、现有方法的实施例2及实施例3的活菌数分别为7.0*109、5.5、6.3cfu/mL。本发明结合生物膜的方法相较于未包埋处理效果提升27.27%;较之SA包被效果提升11.11%。
四、耐热性、耐寒性和耐酸性考察
4.1模拟95℃高温环境,将附着生物膜微胶囊化的乳杆菌放置1min,未包埋处理、SA包被、生物膜包被、二重包被活菌数分别为3.267*109、4.667*109、5.967*109、5.8*109cfu/mL,本发明实施例1的生物膜包被的效果较未包埋处理、SA包被和二重包被,耐热效果分别提升82.65%、27.86%和2.87%。
4.2在真空冷冻干燥环境下,未包埋处理、SA包被、生物膜包被、二重包被活菌数分别为5.633*109、6.067*109、7.567*109、7.7*109cfu/mL,本发明实施例1的生物膜包被的效果较未包埋处理和SA包被,耐寒效果分别提升34.32%和24.73%,但较之二重包被效果降低了-1.73%。
4.3模拟胃肠道消化环境2小时后发现,未包埋处理、SA包被、生物膜包被、二重包被的活菌数分别为2.4*109、7.067*109、7.9*109、7.567*109cfu/mL。即生物膜相较于未包埋处理效果提升229.17%、较之SA包被效果提升11.79%、较之二重包被效果提升4.41%。模拟4小时后,活菌数依次为2.167*109、6.933*109、8.733*109、8.1*109cfu/mL,即本发明生物膜相较于未包埋处理效果提升效果提升303.00%,较之SA包被效果提升25.96%,较之二重包被效果提升7.81%。已知二重包被会导致益生菌在人体肠道内的释放受阻,这是因为二重包被使得益生菌有了更强的保护,但由于粒径扩大等问题,使得益生菌在肠道中释放缓慢,且不易附着在肠道中,导致活菌数量要低于生物膜包被。且附着生物膜的乳杆菌与体外正常环境下MRS肉汤培养的活菌数几乎没有变化,即表明生物膜包被后几乎免受胃液消化。
上述不同包埋方式的耐热性、耐寒性和耐酸性活菌测定结果如下表3
表3不同包埋方式耐热性、耐寒性、耐酸性的活菌测定结果
本发明方法制得的果汁产品消费者普遍接受度高,在减少胃肠阻力消耗的同时,能降低奶制品引发的胆固醇增高和消除消费者乳糖不耐受,从而提高了益生菌的功能价值,为消费者提供适量益生菌的有效载体。
上述具体实施方式不能作为对本发明保护范围的限制,对于本技术领域的技术人员来说,对本发明实施方式所做出的任何替代改进或变换均落在本发明的保护范围内。
本发明未详述之处,均为本技术领域技术人员的公知技术。
Claims (6)
1.乳杆菌在低pH值果汁中微胶囊化包封方法,其特征在于,包括如下操作步骤:
(1)果胶制备
取干燥的鲜浆果,提取制备果胶;
(2)菌种活化与悬浮液制备
a活化乳酸菌种;
b将步骤(1)的果胶溶解于0.1%蛋白胨水溶液中,添加适量吐温-80,充分混匀后得果胶溶液,置于水浴灭菌,冷却,将乳酸菌沉淀物悬浮在果胶溶液中,混匀备用;
(3)微胶囊程序化
将步骤(2)乳酸菌悬浮液使用微胶囊成型装置挤出至3.5%(W/V)的氯化钙溶液中,以200-250转/分的速度搅拌;所得微胶囊在室温下与氯化钙溶液接触30-35min,以确保完全固化;倾析除去氯化钙溶液,过滤回收微胶囊;然后利用无菌0.1%蛋白胨水溶液清洗数次,去除多余钙离子及未包被的细菌;最后,悬浮在Ringer平衡盐溶液中得微胶囊化乳杆菌,备用;
(4)生物膜程序化
在MRS肉汤培养基中添加3.5-4.5%(W/V)氯化钙溶液,充分灭菌混匀后,将步骤(3)制备好的微胶囊化乳杆菌转移至培养基中,在37℃下连续培养18h;倾析除去培养基后,过滤回收微胶囊,并用无菌0.1%蛋白胨溶液清洗数次,制备得微胶囊;
(5)将步骤(4)制备完成的微胶囊与无菌果汁按1g/10mL进行混合混匀。
2.根据权利要求1所述的乳杆菌在低pH值果汁中微胶囊化包封方法,其特征在于,步骤(1)果胶的制备操作为:将干燥的鲜浆果研磨后,用调整至pH 6的柠檬酸-柠檬酸钠溶液混合,于65℃水浴2小时提取果胶;于4000r/min条件下离心15-25min,去除上清液后,用60%乙醇反复洗涤沉淀果胶2-3次,置于55-65℃烘箱中12h,收集至干燥箱中备用。
3.根据权利要求2所述的乳杆菌在低pH值果汁中微胶囊化包封方法,其特征在于,柠檬酸-柠檬酸钠溶液与鲜浆果配比:50:1(V:W)。
4.根据权利要求1所述的乳杆菌在低pH值果汁中微胶囊化包封方法,其特征在于,步骤(2)中a活化菌种过程为:挑取乳酸菌种,在MRS固体培养基预培养,挑取菌种至新鲜MRS液体培养基预活化,37℃培养18h,同条件下活化2次;然后在4000r/min条件下离心15-25min,倾去培养基后添加生理盐水继续离心2-3次,收集沉淀物备用。
5.根据权利要求1所述的乳杆菌在低pH值果汁中微胶囊化包封方法,其特征在于,步骤(2)b的水浴灭菌采用95℃下水浴15min灭菌;乳酸菌沉淀物悬浮在果胶溶液中后,通过微涡旋混匀备用。
6.根据权利要求1所述的乳杆菌在低pH值果汁中微胶囊化包封方法,其特征在于,步骤(3)微胶囊成型装置的挤出孔0.4-0.5mm、氮气压力0.3-0.5bar;挤出速率通过注射泵控制。
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