CN116042469A - 一种具有抑菌功能的乳酸菌后生元复合物及其制备方法和应用 - Google Patents
一种具有抑菌功能的乳酸菌后生元复合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种具有抑菌功能的乳酸菌后生元复合物及其制备方法和应用。该乳酸菌后生元复合物的制备方法包括如下步骤:将进行两次扩增培养得到的乳杆菌种子液接种至发酵培养基中进行发酵培养,得到第一发酵液;将金属抗菌离子溶液不断滴注至第一发酵液中,边加边搅拌,40‑50℃下螯合反应2‑3小时,得到第二发酵液;将第二发酵液离心得到菌泥复合物;将菌泥复合物高温热灭活,得到灭活乳酸菌菌体复合物;将灭活的菌体复合物干燥粉碎,得到乳酸菌后生元复合物。本发明的方法可产生大量的乳酸菌素等抗菌物质,具有高效的抑菌功能,对大肠杆菌、金黄色葡萄球菌和沙门氏菌有效抑菌时间可达12h以上,且工艺简单、成本较低,适用于大规模生产使用。
Description
技术领域
本发明涉及微生物应用技术领域,具体涉及一种具有抑菌功能的乳酸菌后生元复合物及其制备方法和应用。
背景技术
益生菌指“活的”微生物,在给予一定剂量后,能对宿主起到益生的功能。后生元指灭活的益生菌菌体以及它们的相关代谢产物,包括菌体代谢产物(酶、有机酸、蛋白质/多肽、胞外多糖等)和菌体组成成分(肽聚糖、壁磷壁酸、脂磷壁酸、细胞壁多糖、细胞表面相关蛋白、蛋白丝),其可以为宿主提供健康益处。与活的益生菌相比,后生元具有清晰的化学结构,安全的剂量参数以及更长的货架期与良好的吸收、代谢、分布和***能力,同时还克服了益生菌在使用过程中存在的潜在风险,如腹胀和肠胃气胀,益生菌相关的易位,菌血症和真菌病,以及可能的抗生素抗性基因漂移等风险。后生元的制备一般通过细胞灭活、裂解技术,如热处理、酶解处理、溶剂萃取、超声处理等,再通过分离纯化获得。现有发明专利技术有“一种后生元及其精制方法”(CN114794466A)、“一种后生元及其制备方法”(CN114868914A)、“一种后生元及其制备方法与应用”(CN114480192A)、“一种改善肠道健康的后生元复合物及其制备方法”(CN114651986A)等。
现有专利技术大多存在生产工艺复杂、生产成本高等问题。此外,当前后生元产品的开发主要聚焦于工艺方式的优化,对后生元产品功效的关注较低。到目前为止,还未看到有专注于提高抑菌效果的后生元发明专利,而抑菌功能对于保护蓄禽动物的肠道健康至关重要,此外具有抑菌功能的乳酸菌后生元复合物的开发具有重要的商业价值和广阔的应用前景。
发明内容
本发明针对现有技术的不足,为了填补后生元市场的空缺,增强乳酸菌后生元的功效,为乳酸菌后生元赋能,提供了一种具有抑菌功能的乳酸菌后生元复合物及其制备方法和应用。
为实现上述目的,本发明采用如下技术方案:
本发明的第一方面是提供一种具有抑菌功能的乳酸菌后生元复合物的制备方法,包括如下步骤:
步骤一,将进行两次扩增培养得到的乳杆菌种子液接种至发酵培养基中进行发酵培养,得到第一发酵液;
步骤二,将金属抗菌离子溶液不断滴注至第一发酵液中,边加边搅拌,40-50℃下螯合反应2-3小时,得到第二发酵液;
步骤三,将上述第二发酵液离心得到菌泥复合物;
步骤四,将上述菌泥复合物高温热灭活,得到灭活乳酸菌菌体复合物;
步骤五,将灭活的菌体复合物干燥粉碎,得到乳酸菌后生元复合物。
进一步,上述乳杆菌为植物乳杆菌,优选地为植物乳杆菌YFCC1-plRY13。
进一步地,步骤一的乳杆菌种子液获得的方式为:植物乳杆菌30℃-40℃下扩增培养12-20h,得到一级种子液;将一级种子液按5%-15%的接种比例进行第二次扩增培养,培养时间为24-48h,温度为30-40℃,得到二级种子液。
进一步地,上述扩增培养所采用的培养基包括葡萄糖、牛肉浸粉、酵母浸膏和去离子水。
进一步地,上述扩增培养所采用的培养基包括如下组分:葡萄糖10-15g/L,牛肉浸粉8-15g/L,酵母浸膏8-10g/L。
进一步地,第一次发酵培养的条件为:接种量10%-15%、厌氧培养60-72h。
进一步地,上述发酵培养基包括如下组分:葡萄糖10-15g/L,牛肉浸粉20-40g/L,磷酸缓冲液1-10g/L、乙酸钠1-10g/L、硫酸镁0.1-1g/L、吐温80 0.5-1g/L。
进一步地,上述金属抗菌离子溶液为可溶性锌盐溶液,其配制方法为:将锌盐加入至去离子水中,使终浓度达到20%-30%。
进一步地,可溶性锌盐溶液的添加比例为5%-15%,滴注速度为10-15L/h。
本发明的第二方面是提供上述制备方法制备的乳酸菌后生元复合物。
本发明的第三方面是提供上述乳酸菌后生元复合物制备增强免疫力的膳食补充剂、保健品、药品或食品中的应用。
本发明采用以上技术方案,与现有技术相比,具有如下技术效果:
(1)本发明提供的方法实现了乳酸菌高密度发酵,灭活后的菌体数量高达10亿以上;
(2)本发明提供的方法将无机抗菌金属离子和菌体特异性吸附结合,提升了后生元的抑菌功能;
(3)本发明提供的方法工艺简单、成本较低,适用于大规模生产使用;
(4)本发明提供的方法可产生大量的乳酸菌素等抗菌物质,具有高效的抑菌功能,对大肠杆菌、金黄色葡萄球菌和沙门氏菌有效抑菌时间可达12h以上。
附图说明
图1是本发明制备乳酸菌后生元复合物的工艺流程图;
图2显示了乳酸菌后生元复合物对大肠杆菌的抑菌效果;
图3显示了乳酸菌后生元复合物对沙门氏菌的抑菌效果;
图4显示了乳酸菌后生元复合物对金黄色葡萄球菌的抑菌效果。
具体实施方式
本发明提供了一种具有抑菌功能的乳酸菌后生元复合物及其制备方法和应用。下面通过具体实施例和附图对本发明进行详细和具体的介绍,以使更好的理解本发明,但是下述实施例并不限制本发明范围。
实施例中方法如无特殊说明的采用常规方法,使用的试剂如无特殊说明的使用常规市售试剂或按常规方法配制的试剂。
实施例1
本实施例筛选乳酸用于制备乳酸菌后生元复合物,具体的步骤如下:
1、以实验室保藏的6株乳酸菌为对象,采用牛津杯法筛选高抑菌性能的乳酸菌。
2、6株乳酸菌包括:嗜酸乳杆菌(YFCC1-acJD)、保加利亚乳杆菌(YFCC1-bu16)、植物乳杆菌(YFCC1-p1RY13)、干酪乳杆菌(YFCC1-caJD17)、副干酪乳杆菌(YFCC1-paJD17)、罗伊氏乳杆菌(YFCC1-reJD17)。
3、抑菌筛选步骤:(1)致病菌菌悬液制备:将大肠杆菌ATCC25922、沙门氏菌H9812、金黄色葡萄球菌ATCC43300甘油管接种于LB液体培养基内,37C振荡培养12h,置于4℃冰箱备用。(2)乳酸菌发酵液制备:将6种乳酸菌按照2%的接种量接种到MRS液体培养基中,活化2代,取第3代发酵培养基,离心取上清,再用0.22μm的细菌过滤器过滤,即为乳酸菌的无细胞发酵液。(3)采用牛津杯琼脂扩散法筛选,以大肠杆菌ATCC25922、沙门氏菌H9812、金黄色葡萄球菌ATCC43300为指示菌,测定抑菌圈直径大小,平行测定三次取平均值。
4、筛选结果:
如下表1可知,植物乳杆菌(YFCC1-plRY13)的抑菌性能最强,对大肠杆菌、沙门氏菌和金黄色葡萄球菌都具有很强的生长抑制效果。
表16株乳酸菌的抑菌活性
注:“+++”、“++”、“+”表示不同的抑菌强度;“-”表示无抑菌。
实施例2
本实施例提供一种具有抑菌功能的乳酸菌后生元复合物,由以下步骤制得(参考图1):
步骤一:将1g植物乳杆菌菌粉接种至100m1种子培养基(含1g葡萄糖、牛肉浸粉1g、酵母浸膏0.8g、余量为去离子水),38℃培养下培养12h得到一级种子液;
步骤二:按照10%的接种量,将一级种子液接种至2L种子培养基中,38℃培养30h得到二级种子液;
步骤三:按照10%的接种量,将二级种子液接种至5L发酵培养基中(50g葡萄糖、100g牛肉浸粉、5g磷酸缓冲液、5g乙酸钠、0.5g硫酸镁,2.5g吐温80、余量为去离子水),厌氧培养72h,得到第一发酵液;
步骤四:将第一发酵液升温至40℃,将浓度25%的可溶性锌盐溶液不断滴注至第一发酵液中,滴注比例10%,滴注速度10L/h,滴注结束后螯合3小时,得到螯合液;
步骤五:对螯合液进行离心取沉淀、高温灭活、干燥、粉碎,得到乳酸菌后生元复合物。
实施例3
本实施例提供一种具有抑菌功能的乳酸菌后生元复合物,由以下步骤制得(参考图1):
步骤一:将5g植物乳杆菌菌粉接种至600m1种子培养基(含8g葡萄糖、牛肉浸粉8g、酵母浸膏5g、余量为去离子水),40℃培养下培养15h得到一级种子液;
步骤二:按照15%的接种量,将一级种子液接种至10L种子培养基中,40℃培养35h得到二级种子液;
步骤三:按照15%的接种量,将二级种子液接种至30L发酵培养基中(320g葡萄糖、640g牛肉浸粉、30g磷酸缓冲液、30g乙酸钠、3g硫酸镁,14g吐温80、余量为去离子水),厌氧培养72h,得到第一发酵液;
步骤四:将第一发酵液升温至45℃,将浓度20%的可溶性锌盐溶液不断滴注至第一发酵液中,滴注比例15%,滴注速度15L/h,滴注结束后螯合2小时,得到螯合液;
步骤五:对螯合液进行离心取沉淀、高温灭活、干燥、粉碎,得到乳酸菌后生元复合物。
验证实施例
1、抑菌性能分析
将实施例2制备的后生元复合样品按照固液比1∶3比例浸提30min,离心取上清:上清液经灭菌20min后,再次离心将沉淀离去,即得到后生元样品浸提液,静置于4℃冰箱中备用;取浸提液5ml加至45mL LB培养基内,分别加入50μl对数生长期的大肠杆菌ATCC25922、沙门氏菌H9812、金黄色葡萄球菌ATCC43300,38℃、200rpm摇床培养12h,每2h取样测OD600吸光值,并与对照(不加浸提液组)对比,结果如图2、图3、图4所示。
图2、图3和图4显示的是乳酸菌后生元复合物对大肠杆菌ATCC25922、沙门氏菌H9812、金黄色葡萄球菌ATCC43300的抑菌性能,由结果可知,对照组大肠杆菌、沙门氏菌和金黄色葡萄球菌均长势良好,本次试验结果稳定可信;乳酸菌后生元复合物组12h内均无致病菌生长情况,抑菌效果显著。
以上对本发明的具体实施例进行了详细描述,但其只作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
Claims (10)
1.一种具有抑菌功能的乳酸菌后生元复合物的制备方法,其特征在于,包括如下步骤:
步骤一,将进行两次扩增培养得到的乳杆菌种子液接种至发酵培养基中进行发酵培养,得到第一发酵液;
步骤二,将金属抗菌离子溶液不断滴注至第一发酵液中,边加边搅拌,40-50℃下螯合反应2-3小时,得到第二发酵液;
步骤三,将所述第二发酵液离心得到菌泥复合物;
步骤四,将所述菌泥复合物高温热灭活,得到灭活乳酸菌菌体复合物;
步骤五,将灭活的菌体复合物干燥粉碎,得到乳酸菌后生元复合物。
2.根据权利要求1所述的制备方法,其特征在于,所述乳杆菌为植物乳杆菌,优选地为植物乳杆菌YFCC1-plRY13。
3.根据权利要求2所述的制备方法,其特征在于,步骤一的乳杆菌种子液获得的方式为:植物乳杆菌30℃-40℃下扩增培养12-20h,得到一级种子液;将所述一级种子液按5%-15%的接种比例进行第二次扩增培养,培养时间为24-48h,温度为30-40℃,得到二级种子液。
4.根据权利要求3所述的制备方法,其特征在于,所述扩增培养所采用的培养基包括葡萄糖、牛肉浸粉、酵母浸膏和去离子水。
5.根据权利要求3所述的制备方法,其特征在于,所述扩增培养所采用的培养基包括如下组分:葡萄糖10-15g/L,牛肉浸粉8-15g/L,酵母浸膏8-10g/L。
6.根据权利要求1所述的制备方法,其特征在于,所述第一次发酵培养的条件为:接种量10%-15%、厌氧培养60-72h。
7.根据权利要求1所述的制备方法,其特征在于,所述发酵培养基包括如下组分:葡萄糖10-15g/L,牛肉浸粉20-40g/L,磷酸缓冲液1-10g/L、乙酸钠1-10g/L、硫酸镁0.1-1g/L、吐温800.5-1g/L。
8.根据权利要求1所述的制备方法,其特征在于,所述金属抗菌离子溶液为可溶性锌盐溶液,其配制方法为:将锌盐加入至去离子水中,使终浓度达到20%-30%;优选地,所述可溶性锌盐溶液的添加比例为5%-15%,滴注速度为10-15L/h。
9.如权利要求1-8任一项所述制备方法制备的乳酸菌后生元复合物。
10.如权利要求9所述的乳酸菌后生元复合物制备增强免疫力的膳食补充剂、保健品、药品或食品中的应用。
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