CN114540235B - 一种诱导乳酸菌休眠态的方法及其应用 - Google Patents
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Abstract
本发明公开了一种诱导乳酸菌休眠态的方法及其应用,属于食品加工技术领域。本发明公开的一种诱导乳酸菌休眠态的方法,利用利福平作为诱导剂诱导乳酸菌休眠态,利福平的终浓度为0.16~0.64mg/mL;在不改变其他条件下,经利福平诱导的休眠乳酸菌喷雾干燥后存活率达21.66%。
Description
技术领域
本发明涉及食品加工技术领域,更具体的说是涉及一种诱导乳酸菌休眠态的方法及其应用。
背景技术
乳酸菌是食品发酵工业和功能性食品行业中的重要组成部分,由于对肠道菌群和生理代谢具备调节作用,其益生特性在近年来被不断发掘。现在普遍认为摄入足量活的益生菌将对人体健康起到推动作用。乳酸菌主要以干燥粉剂的形式应用于工业和家庭,以便于运输和加工,并保证在产品和人体消化道内的活性和稳定性。
目前,乳酸菌制剂主要通过冷冻干燥技术制备,其存活率较高,但也存在不能连续生产、设备造价高、高能耗及生产周期长等缺点,成为限制乳酸菌干燥制剂发展的重要因素。喷雾干燥技术具备低成本、高效、可连续等优点,生产成本仅为冷冻干燥技术的18%,有望替代冷冻干燥生产乳酸菌制剂。但喷雾干燥过程中菌体与热空气接触会引起细胞损伤导致干燥后乳酸菌大量死亡、活力大幅下降。
增强菌体抗性是解决喷雾干燥制剂低存活率,低活力的有效途径。当菌体处于休眠状态,细菌对热、失水等不良条件的抗性将提升。因此,休眠态乳酸菌可以成为喷雾干燥、冷冻干燥等加工过程中保障乳酸菌存活率,活力和保质期的有效途径。
现有微囊化包埋等技术可以提高喷雾干燥等加工过程中乳酸菌的存活率,但这些技术手段并不是从提高菌体抗性角度来实现高存活率,导致乳酸菌在不同加工过程中保护配方不一致。因此通过诱导乳酸菌休眠来提高乳酸菌菌体自身对不良环境的抗性,是解决加工过程中乳酸菌损失的有效途径,但目前尚无关于诱导乳酸菌休眠状态方法的报道。
因此,提供一种诱导乳酸菌休眠态的方法及其应用是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种诱导乳酸菌休眠态的方法及其应用,主要用于休眠态乳酸菌、乳酸菌制剂、乳酸菌菌粉、发酵剂的生产中。
为了实现上述目的,本发明采用如下技术方案:
一种诱导乳酸菌休眠态的方法,利用利福平作为诱导剂诱导乳酸菌休眠态。
进一步,所述的一种诱导乳酸菌休眠态的方法,具体步骤如下:
(1)将乳酸菌培养至对数末期或稳定前期,获得菌液;
(2)在步骤(1)获得的菌液中加入利福平(RFP)溶液,使利福平的终浓度达0.16~0.64mg/mL;
(3)37℃诱导3h后,离心,收集菌体;
(4)采用生理盐水或0.01M的磷酸盐缓冲液冲洗步骤(3)收集的菌体2次,再次离心,收集菌体;
(5)将步骤(4)冲洗后收集的菌体于液体保护剂中保藏,或喷雾干燥、冷冻干燥后制成菌粉。
进一步,步骤(1)所述乳酸菌在MRS液体培养基中培养。
进一步,步骤(2)在步骤(1)获得的菌液中加入4%(v/v)利福平溶液。
进一步,步骤(3)和步骤(4)所述离心为在4℃,6000r/min离心10min。
进一步,步骤(5)所述液体保护剂为葵花籽油或脱脂乳。
进一步,所述的一种诱导乳酸菌休眠态的方法在制备乳酸菌干燥制剂中的应用。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种诱导乳酸菌休眠态的方法及其应用,利用利福平作为诱导剂可以诱导乳酸菌休眠态,利福平的终浓度为0.16~0.64mg/mL;在不改变其他条件下,经利福平诱导的休眠乳酸菌喷雾干燥后存活率达21.66%。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
使用利福平(RFP)作为乳酸菌休眠诱导剂,德氏保加利亚乳杆菌亚种sp1.1(L.bulgaricus sp1.1)(CGMCC No.16586)作为模式菌株建立乳酸菌休眠模型,以2%(v/v)的接种量将活化两代后L.bulgaricus sp1.1接种于MRS液体培养基中,培养12h后,测定吸光值OD660达1.75-1.85,加入4%(v/v)RFP DMSO溶液,使RFP终浓度达0、0.16、0.32、0.48、0.64mg/mL。37℃诱导3h后,在4℃,6000r/min下离心10min,收集菌体。使用0.01M PBS冲洗菌体两次后,用生理盐水或0.1M的磷酸盐缓冲液重悬备用,其中0mg/mL的组为对照组。
休眠乳酸菌比例由流式细胞仪测定,具体流程如下:向上述重悬后的休眠诱导后的菌液中,加入CFDASE染液使其终浓度达10μM,温和混匀后,37℃水浴15min,标记细胞后,6000r/min离心5min,加入新鲜灭菌的MRS液体培养基洗菌体两次并用MRS液体培养基重悬,37℃孵育30分钟清除胞内未标记染料,离心收集标记后的细胞并加入MRS肉汤重悬至原体积,取1mL原始菌液(未经利福平诱导的菌液)为阴性对照,取1mL染色后的菌液为CFDASE单染,4℃冷藏备用。将CFDASE染色后的菌液分装到2mL离心管中,加入不同浓度的利福平溶液,37℃诱导3h。诱导完成后,6000r/min离心5min收集菌体(取衰老后期的菌液制备PI单染管),加入PBS洗菌两次,所有样品加入含4%多聚甲醛的PBS固定液,4℃固定15min,获得固定菌液样品。所有固定菌液样品梯度稀释至终浓度达106-107CFU/mL,过200目细胞筛后备用。仪器预热完成后,设置激发通道488nm(CFDASE)保留FSC和SSC通道,测定休眠乳酸菌比例,结果见表1。
表1不同浓度利福平处理后休眠乳酸菌比例
利福平浓度(mg/mL) | 0 | 0.16 | 0.32 | 0.48 | 0.64 |
休眠乳酸菌比例(%) | 48.89 | 79.14 | 84.53 | 88.41 | 93.76 |
根据表1可知,利福平浓度为0时,只有48.89%的菌体处于休眠状态;而利福平浓度为0.16mg/mL时,休眠状态菌体比例为79.14%;而利福平浓度为0.64mg/mL时,休眠乳酸菌比例为93.76%。由此可见,当利福平浓度大于0.16mg/mL时,即可大幅度提高休眠乳酸菌比例。
实施例2休眠乳酸菌喷雾干燥
使用利福平(RFP)作为乳酸菌休眠诱导剂,德氏保加利亚乳杆菌亚种sp1.1(L.bulgaricus sp1.1)作为模式菌株建立乳酸菌休眠模型,以2%(v/v)的接种量将活化两代后L.bulgaricus sp1.1接种于100mL MRS中,培养12h后,测定吸光值OD660达1.75-1.85,加入4%RFP终浓度达0.64mg/mL。37℃诱导3h后,在4℃,6000r/min下离心10min收集菌体。使用1mL 0.01M PBS洗菌两次后重悬备用,将未经3h诱导的L.bulgaricus sp1.1菌液设为对照组。
采用实验室级喷雾干燥装置制备休眠态L.bulgaricus sp1.1菌粉,喷雾干燥条件如下:将上述3h诱导后的重悬的1mL L.bulgaricus sp1.1菌液或未经3h诱导的1mLL.bulgaricus sp1.1菌液加入到100mL30%(w/v)脱脂乳为进料液,进风风量:380m3/h,进风/出风温度:120/60℃,雾化压力0.9m3/h,调整进料速率使进出风温度满足预设值。喷雾干燥后存活率和水分含量的结果见表2。
表2利福平浓度达0.64mg/mL干预后的喷雾干燥存活率和水分含量
表2显示,在不改变其他条件下,经利福平诱导的休眠乳酸菌喷雾干燥后存活率达21.66%,远远高于未经利福平诱导的乳酸菌喷雾干燥后的存活率8.17%。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (5)
1.一种诱导乳酸菌休眠态的方法,其特征在于,利用利福平作为诱导剂诱导乳酸菌休眠态;
具体步骤如下:
(1)将乳酸菌培养至对数末期或稳定前期,获得菌液;
(2)在步骤(1)获得的菌液中加入利福平溶液,使利福平的终浓度达0.64mg/mL;
(3)37℃诱导3h后,离心,收集菌体;
(4)采用生理盐水或0.01M的磷酸盐缓冲液冲洗步骤(3)收集的菌体2次,再次离心,收集菌体;
(5)将步骤(4)冲洗后收集的菌体经喷雾干燥后制成菌粉。
2.根据权利要求1所述的一种诱导乳酸菌休眠态的方法,其特征在于,步骤(1)所述乳酸菌在MRS液体培养基中培养。
3.根据权利要求1所述的一种诱导乳酸菌休眠态的方法,其特征在于,步骤(2)在步骤(1)获得的菌液中加入4%(v/v)利福平溶液。
4.根据权利要求1所述的一种诱导乳酸菌休眠态的方法,其特征在于,步骤(3)和步骤(4)所述离心为在4℃,6000r/min离心10min。
5.权利要求1-4任一项所述的一种诱导乳酸菌休眠态的方法在制备乳酸菌干燥制剂中的应用。
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