CN115595286B - 一种植物乳杆菌菌剂及其制备方法和应用 - Google Patents
一种植物乳杆菌菌剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于微生物技术领域,具体涉及了一种植物乳杆菌菌剂的制备方法及新型保护剂在冷冻干燥过程中对植物乳杆菌的保护作用。制备方法如下:将植物乳杆菌LP115接种到MRS液体培养基,于37℃活化一段时间作为种子液。将种子液接种到另一MRS液体培养基扩大培养,生长至稳定期前期。取出并分装菌液至离心管,离心倒掉上清液并用无菌生理盐水冲洗,收集菌泥,随后加入一定比例的新型冻干保护剂,涡旋混匀。然后置于‑80℃低温快速预冻,冷冻干燥后得到植物乳杆菌菌剂。本发明制备的植物乳杆菌菌剂单位活菌数为2.5×1011CFU/g,冻干存活率高达85%。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种植物乳杆菌菌剂及其制备方法和应用。
背景技术
植物乳杆菌是乳酸菌中常见的一种,属于厌氧或兼性厌氧的革兰氏阳性杆菌,广泛应用于发酵蔬菜、果汁、肉制品和发酵乳制品中。根据***粮农组织(FAO)和世界卫生贸易组织(WHO)的规定,植物乳杆菌在人体胃肠道定植活菌数大于1×106CFU/mL,才能发挥其益生功能,比如调节肠道菌群平衡,增加机体免疫力。因此,从源头上制备高活性的植物乳杆菌菌剂变得尤为重要,这也成为近年来乳酸菌的研究热点之一。
真空冷冻干燥技术是将含有细胞的水溶液冷冻,在低温下进行升华干燥除冰的过程。相比于其他干燥技术,这项技术能在更高的水平上保持细胞活力和乳酸菌的稳定性,并且为产品的运输和贮存提供方便,因而受到众多的青睐。但在这个过程中也会不可避免地给细胞带来一定的伤害,比如大颗粒冰晶的形成、渗透胁迫、机械损伤、溶质效应以及DNA和蛋白质的变性等,为此在使用冷冻干燥技术时常添加抗冻保护剂以减小伤害,最大限度地提高细胞的存活率。
目前,常用的冻干保护剂主要有碳水化合物、蛋白质类和氨基酸类,其中以海藻糖、脱脂乳、蔗糖、葡萄糖和谷氨酸钠为保护剂的研究居多。特别地,相比于其它两类保护剂,碳水化合物被认为是最好的保护剂,因为它允许细胞在分子排列的空间或表面之间,形成低分子相互作用的玻璃态能力。更多地是,碳水化合物具有的多羟基结构,可以在细胞周围形成氢键,代替水分子稳定磷脂、蛋白质等生物大分子的天然结构,从而维持细胞的活力。据报道,大分子碳水化合物具有较高的玻璃化转变温度和崩解温度以及提供物理屏障的优势,小分子碳水化合物可以进入细胞内部,抑制冰晶的形成,因此,有必要研究一种由大分子和小分子复配组成新型保护剂,能够提供微生物更好的保护效果,有效维持细胞活力,提高微生物菌剂的冻干存活率。
发明内容
为了解决现有技术中存在的问题,本发明的目的在于提供一种基于海藻糖溶液和纤维素酶解产物为冻干保护剂的植物乳杆菌菌剂及其制备方法和应用。本发明提供的植物乳杆菌菌剂在制备过程中通过采用小分子和大分子糖类物质作为冻干保护剂进行混合使用,克服了单一保护剂的劣势,在整个保护体系中相辅相成,共同为制备高活性的植物乳杆菌菌剂发挥各自的作用,制得的植物乳杆菌菌剂的冻干存活率更高,能够使冻干后的细胞维持在较低的水分含量范围,有利于菌剂的后续贮藏。
本发明的技术方案是:
一种植物乳杆菌菌剂的制备方法,包括如下步骤:
(1)菌种活化:将植物乳杆菌LP115接种到MRS液体培养基,活化一段时间作为种子液;
(2)扩大培养:将步骤(1)所得的种子液接种到另一MRS液体培养基扩大培养,生长至稳定期前期;
(3)收集菌泥:取出步骤(2)扩大培养后的菌液并分装至离心管,离心后,倒掉上清液,并用无菌生理盐水冲洗,得到菌泥;
(4)添加冻干保护剂:向步骤(3)得到的菌泥中添加冻干保护剂,涡旋混匀,得混匀溶液;
(5)冷冻干燥:先将步骤(4)所得混合溶液进行预冻,接着冷冻干燥后得到植物乳杆菌菌剂。
进一步地,所述步骤(4)中,冻干保护剂为海藻糖溶液和纤维素酶解产物按重量比(6~10)∶(0~4)组成,所述海藻糖溶液的质量分数为8~12%。
更进一步地,所述步骤(4)中,冻干保护剂为海藻糖溶液和纤维素酶解产物按重量比8∶2组成,所述海藻糖溶液的质量分数为10%。
在本发明提供的植物乳杆菌菌剂的制备过程中,本申请发明人创造性的添加了由海藻糖和纤维素酶解产物按照特定的重量比组成复合冻干保护剂,发现二者结合能够产生明显的协同作用,海藻糖和纤维素酶解过程中产生的一些可溶性低聚糖可以借助其含有的多羟基基团在冷冻干燥阶段代替水分子,维持细胞生物大分子原先水合状态下的天然结构,结合大分子纤维物质在细胞周围形成的玻璃化基质,类似于一层物理屏障,抵抗外界环境对细胞的伤害,两者的共同作用使得细胞承受较小的活力损失。
进一步地,所述冻干保护剂在121℃、0.1MPa条件下灭菌17min后冷却使用。
进一步地,所述步骤(4)中菌泥和冻干保护剂的重量比为1∶10。
更进一步地,所述步骤(4)的冻干保护剂中的纤维素酶解产物的制备方法如下:
将纤维素溶解于pH为4.8的乙酸-乙酸钠缓冲液中,配置成浓度为0.02g/mL的纤维素悬浮液,经磁力搅拌器搅拌12~24h,然后加入1.75U/mL的纤维素酶溶液,混合均匀,在50℃和150rpm条件下水解0.5~2.5h,反应时间结束便将混合溶液浸入沸水中加热5min,使酶失活进而停止酶促反应。待其冷却至室温,利用质量浓度为4%的NaOH溶液调节酶解液pH为6.7~6.9,即得纤维素酶解产物。
进一步地,所述纤维素悬浮液和纤维素酶溶液体积比为4∶1。
进一步地,所述纤维素来源于柚子表皮,所述纤维素的制备方法如下:
将柚子表皮于50℃干燥24h,粉碎,过40目筛;利用质量浓度为15%的NaOH溶液进行碱处理,料液比为1∶30,蒸馏水和95%乙醇冲洗至中性,50℃干燥12h;利用质量分数为6%的NaClO2溶液进行漂白处理,所述NaClO2溶液采用盐酸溶液调节pH至3.8~4.0,然后用蒸馏水冲洗至中性;于冷阱温度-70℃、真空度0Pa下冷冻干燥48h,得到纤维素。
进一步地,所述步骤(1)中植物乳杆菌LP115的接种量为3×107CUF/mL,活化时间为10h,活化温度为37℃;所述步骤(2)中种子液的接种量为MRS液体培养基质量的4.4%,培养时间为9.5h。
进一步地,所述步骤(3)中的离心条件为:14136×g,4℃,离心10min;离心后,倒掉上清液,并用质量浓度为0.85%的无菌生理盐水冲洗2次。
进一步地,所述步骤(5)中冷冻干燥条件为:冷阱温度-70℃、真空度0Pa,冷冻干燥48h。
本发明还提供了一种采用上述制备方法制得的植物乳杆菌菌剂。
本发明制得的植物乳杆菌菌剂可以应用于食品中。
本发明通过植物乳杆菌LP115活化、扩大培养,离心收集菌泥,添加一定比例的新型冻干保护剂,得到植物乳杆菌LP115和新型保护剂的混合物,于-80℃预冻,随后冷冻干燥获得植物乳杆菌菌剂。
与现有技术相比,本发明提供的植物乳杆菌菌剂具有以下优势:
(1)本发明制备的植物乳杆菌菌剂冻干存活率达到88.86%。相比于传统单一的海藻糖保护剂,由海藻糖溶液和纤维素酶解产物质量比为8∶2组成的新型冻干保护剂,将其冻干存活率提高了30.23%,保护效果显著增强。而且原料价廉易得,具有较强的生产推广价值。
(2)采用扫描电镜分析发现,本发明制备的植物乳杆菌菌剂表现为短杆状,分布紧凑紧密,大量聚集在表面粗糙多褶皱的结构上,有利于维持细胞活力,保护效果相对更好。
(3)采用低场核磁共振氢谱分析发现,本发明制备的植物乳杆菌菌剂主要存在结合水和不易流动水,其中结合水含量在85%以上,维持在较低的水分含量范围,有利于菌剂的后续贮藏。
附图说明
图1为为植物乳杆菌活化前后在MRS培养基的生长曲线示意图。
图2为为植物乳杆菌在新型保护剂不同复配比例的影响下存活率的示意图。
图3为植物乳杆菌在纤维素不同酶解时间的影响下存活率的示意图。
图4为植物乳杆菌在不同保护剂组成的影响下扫描电镜(SEM)的示意图。
图5为植物乳杆菌在不同保护剂组成的影响下T2弛豫图谱的示意图。
图6为植物乳杆菌在不同保护剂组成的影响下荧光光谱的示意图。
图4~6中:a代表10%海藻糖溶液;b代表10%海藻糖溶液和纤维素酶解0.5h的产物;c代表10%海藻糖溶液和纤维素酶解1h的产物;d代表10%海藻糖溶液和纤维素酶解1.5h的产物;e代表10%海藻糖溶液和纤维素酶解2h的产物;f代表10%海藻糖溶液和纤维素酶解2.5h的产物。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明,但这并非是对本发明的限制,本领域技术人员根据本发明的基本思想,可以做出各种修改或改进,但是只要不脱离本发明的基本思想,均在本发明的保护范围之内。
以下实施例中,未作特别说明的试剂为常规试剂,均可在常规试剂生产销售公司购买。
实施例1一种植物乳杆菌菌剂的制备方法
所述的植物乳杆菌菌剂的制备方法,包括如下步骤:
(1)菌种活化:将植物乳杆菌LP115接种到MRS液体培养基,接种量为3×107CUF/mL,于37℃活化10h作为种子液;
(2)扩大培养:将步骤(1)所得的种子液接种到另一MRS液体培养基扩大培养,所述种子液的接种量为MRS液体培养基质量的4.4%,生长9.5h至稳定期前期;
(3)收集菌泥:取出步骤(2)扩大培养后的菌液并分装至离心管,在14136×g,4℃的条件下离心10min后,倒掉上清液,并用质量浓度为0.85%无菌生理盐水冲洗2次,得到菌泥;
(4)添加冻干保护剂:向步骤(3)得到的菌泥中添加冻干保护剂,所述菌泥和冻干保护剂的重量比为1∶10,涡旋混匀,得混合溶液;
(5)冷冻干燥:在-80℃条件下快速预冻步骤(4)所得混合溶液,冷冻干燥后得到植物乳杆菌菌剂;所述冷冻干燥的条件为:冷阱温度-70℃、真空度0Pa,冷冻干燥48h。
所述MRS液体培养基配方(以重量份数计):细菌学蛋白胨10份,无水葡萄糖20份,牛肉浸膏5份,酵母提取粉4份,吐温-80 1份,无水乙酸钠5份,柠檬酸三铵2份,七水合磷酸氢二钾2份,七水合硫酸镁0.2份,四水合硫酸锰0.05份,蒸馏水1000份。
所述步骤(4)中的冻干保护剂为海藻糖溶液和纤维素酶解产物按重量比8∶2组成,所述海藻糖溶液的质量分数为10%。所述冻干保护剂在121℃、0.1MPa条件下灭菌17min后冷却使用。
所述纤维素酶解产物的制备方法如下:
将纤维素溶解于pH为4.8的乙酸-乙酸钠缓冲液中,配置成浓度为0.02g/mL的纤维素悬浮液,经磁力搅拌器搅拌20h,然后加入1.75U/mL的纤维素酶溶液,所述纤维素悬浮液和纤维素酶溶液体积比为4∶1,混合均匀,在50℃和150rpm条件下水解2h,反应时间结束便将混合溶液浸入沸水中加热5min,使酶失活进而停止酶促反应。待其冷却至室温,利用质量浓度为4%的NaOH溶液调节酶解液pH为6.7~6.9,即得纤维素酶解产物。
所述纤维素来源于柚子表皮,所述纤维素的制备方法如下:
将柚子表皮于50℃干燥24h,而后高速粉碎,过40目筛;利用质量浓度为15%的NaOH溶液进行碱处理,料液比为1∶30,蒸馏水和95%乙醇冲洗至中性,50℃干燥12h;利用质量分数为6%的NaClO2溶液进行漂白处理,所述NaClO2溶液采用盐酸溶液调节pH至3.8~4.0,然后用蒸馏水冲洗至中性;于冷阱温度-70℃、真空度0Pa下冷冻干燥48h,得到纤维素。
试验例一、植物乳杆菌活化前后在MRS培养基生长曲线分析
(1)植物乳杆菌活化前生长曲线的测定及分析
将植物乳杆菌LP115按照3×107CUF/mL的接种量接种到液体MRS培养基,于37℃隔水式培养箱培养,分别在发酵0、2、4、6、8、10、12和24h分别取样测定活菌数。以发酵时间为横坐标,植物乳杆菌活菌数的常用对数为纵坐标,绘制生长曲线,确定活化终点。
(2)植物乳杆菌活化后生长曲线的测定及分析
将植物乳杆菌LP115按照3×107CUF/mL的接种量接种到液体MRS培养基,培养至活化终点,作为种子液。将种子液以4.4%(w/w)的接种量接到另一液体MRS培养基,于37℃扩大培养,同样在发酵0、2、4、6、8、10、12和24h分别取样测定活菌数。以发酵时间为横坐标,植物乳杆菌活菌数的常用对数为纵坐标,绘制生长曲线,确定菌泥收获期。
(3)乳酸菌活菌数测定方法参照GB 4789.35-2016食品安全国家标准乳酸菌检验。
(4)试验结果:由图1可知,植物乳杆菌活化前在液体MRS培养基的生长情况为:0~2h,处于延滞期,植物乳杆菌活菌数变化较小,生长缓慢;2~12h,处于对数期,植物乳杆菌活菌数迅速升高,呈现指数级增长趋势;12~24h,处于稳定期,植物乳杆菌活菌数基本保持不变,生长达到动态平衡。因此,选择植物乳杆菌生长的对数期后期10h作为活化终点。而植物乳杆菌活化后在液体MRS培养基的生长情况为:没有延滞期,0h直接进入对数期,到8h对数期结束,植物乳杆菌活菌数迅速增加;8~24h,处于稳定期,植物乳杆菌活菌数基本维持不变。据报道,稳定期的细胞是冷冻干燥过程的最佳选择,因此选择稳定期前期9.5h作为菌泥的收获期。
试验例二、植物乳杆菌冻干保护剂复配比例和纤维素酶解时间优化分析
(1)冻干保护剂复配比例的优化
在制备植物乳杆菌菌剂过程中,根据菌泥质量,添加其10倍质量的冻干保护剂。其中,对照组添加10倍的10%海藻糖溶液,实验组则分别按照10%海藻糖溶液和纤维素酶解产物(酶解2h)9∶1、8∶2、7∶3和6∶4(m/m)的比例添加保护剂,分别记为10∶0、9∶l、8∶2、7∶3、6∶4,其它操作步骤不变。然后对冻干样品进行活菌数测定,计算冻干存活率,进而优化保护剂的复配比例。
(2)纤维素酶解时间的优化
在制备植物乳杆菌菌剂过程中,根据菌泥质量,实验组保持海藻糖溶液和纤维素酶解产物复配比例8∶2,改变纤维素酶解时长,分别设置0.5、1、1.5、2和2.5h共5个梯度,对照组则保持不变,仍添加10倍菌泥质量的10%海藻糖溶液,其它操作步骤不变。然后对冻干样品进行活菌数测定,计算冻干存活率,进而筛选出最佳纤维素酶解时长。
所述冻干存活率的计算公式如下:
(3)试验结果:(1)冻干保护剂复配比例的优化的实验数据如表1和图2所示,(2)纤维素酶解时间的优化的实验数据如表2和图3所示;图2和图3中,在p<0.05水平下进行显著性差异分析,a、b、c表示数据存在显著性差异(Duncan'stest)。
表1
保护剂不同比例 | 冻干存活率(%) |
10∶0 | 62.17 |
9∶l | 75.78 |
8∶2 | 85.37 |
7∶3 | 61.17 |
6∶4 | 54.83 |
表2
酶解时间(h) | 冻干存活率(%) |
0 | 58.63 |
0.5 | 75.35 |
1 | 77.65 |
1.5 | 81.94 |
2 | 88.86 |
2.5 | 59.61 |
由表1和图2可知,当10%海藻糖溶液和纤维素酶解产物复配比例为8∶2(m/m)时,由新型冻干保护剂制备的植物乳杆菌菌剂冻干存活率达到85.37%,比传统单一的海藻糖保护剂的冻干存活率提高了23.2%。由表2和图3可知,当纤维素酶解时间为2h时,由该对应产物组成的新型冻干保护剂制备的植物乳杆菌菌剂冻干存活率达到88.86%,比传统单一的海藻糖保护剂的冻干存活率提高了30.23%,显著提高了菌剂的存活率。
试验例三、植物乳杆菌菌剂的微观形貌和T2弛豫图谱分析
(1)微观形貌分析
将植物乳杆菌经过不同的冻干保护剂冻干后,采用扫描电子显微镜进行微观形貌的观察分析,扫描电镜图揭示了植物乳杆菌菌剂的形貌特征,直观反映了植物乳杆菌在不同保护剂组成的分布状态。其中,a代表10%海藻糖溶液;b代表10%海藻糖溶液和纤维素酶解0.5h的产物;c代表10%海藻糖溶液和纤维素酶解1h的产物;d代表10%海藻糖溶液和纤维素酶解1.5h的产物;e代表10%海藻糖溶液和纤维素酶解2h的产物;f代表10%海藻糖溶液和纤维素酶解2.5h的产物。
由图4可知,图a-f展现的植物乳杆菌形状为短杆状,长度均保持在2μm左右,形态相似。然而,图a表现出相对光滑的表面结构,植物乳杆菌内嵌在海藻糖物质表面,大量被包裹在海藻糖内部,不易被发现。图b-f呈现出相类似的结构,表面粗糙多褶皱,有效栖息面积较大,细胞排列紧密。此外,植物乳杆菌多粘附在纤维基质片状结构的凹陷处,暴露完整的细胞形态,分布紧凑致密,有利于菌株的复水,复苏细胞活力。
(2)T2弛豫图谱分析
利用核磁共振成像仪测定植物乳杆菌添加不同保护剂冻干后的水分分布情况。称取约0.7g植物乳杆菌菌粉于透明带塞试管中,选择磁体探头40mm,在Q-FID序列下进行水模校正和样品参数的确定,参数为:SF=20MHz,SW=200kHz,RFD=0.002ms,P1=9.52μs,RG1=20db,DRG1=3,PRG=2,NS=8,TD=60002,TW=6000ms,P2=19.04μs,然后在Q-CPMG序列下设置TE=0.150ms,NECH=2000,进行样品的测定,并进行反演,得到T2弛豫图谱(如图5所示)。不同保护剂组成的植物乳杆菌冻干后的水分分布状态如表3所示。
表3
注:T2i,A2i和W2i(i=1,2,3)分别是指图5植物乳杆菌冻干后T2弛豫图谱中的三个峰及其峰面积以及所占的比例。
由图5和表3可知,所有样品的T21峰的面积所占比例W21最大,说明植物乳杆菌冻干后的水分主要以强结合水的状态存在,代表了植物乳杆菌的脂质和蛋白质等大分子物质紧密结合的水。考虑到弱结合水T22的所占比例W22,可以知道植物乳杆菌冻干后结合水的含量超过85%,说明菌粉中大部分自由水已经去除,维持在较低的水分含量范围,有利于在后续贮藏过程中保持良好的细胞活力。而不易流动水T23相比于T21和T22,其弛豫时间最长,说明该部分水与植物乳杆菌结合相对松散,在后期贮藏过程中变化波动较大。
试验例四、植物乳杆菌菌剂的荧光光谱分析
取0.1g植物乳杆菌冻干后的菌剂,充分溶解在9.9mL生理盐水中,梯度稀释(不同样品间稀释倍数保持相同),直到OD600nm为0.2左右。取3mL菌液,加入0.3mL 1mg/mL的二乙酸荧光素(FDA)染液,混合均匀,然后加入0.3mL200μg/mL的碘化丙啶(PI)染液,涡旋混匀,4℃避光静置10~15min。每个样品吸取150μL液体滴加至酶标板,设定激发波长480nm,步长为2nm,在发射波长500~670nm范围内进行荧光光谱扫描。
FDA染液配制方法:50mg FDA溶于10mL丙酮中,配制终浓度为5mg/mL的母液,4℃避光保存备用。
PI染液配制方法:10mg PI溶于10mL超纯水,配制终浓度为1mg/mL的PI母液,4℃避光保存备用。
不同保护剂组成的植物乳杆菌菌剂细胞膜完整性的表征如表4所示。
表4
由图6和表4可知,对照组a和实验组b-f的荧光光谱曲线表现出相似的走向,在520nm和630nm附近有两个明显的峰,其峰高分别反映植物乳杆菌细胞膜正常和受损的程度。经计算,对照组a和实验组b-f在520nm和630nm附近两个峰峰高的比值分别为0.92,1.40,1.45,1.73,2.21和0.83。其比值越大,植物乳杆菌细胞膜受损程度越小,完整性更高。因此,与对照组a相比,除了实验组f,其余b-e植物乳杆菌细胞膜的完整性更高,且逐渐升高,并在以纤维素酶解2h的产物和海藻糖溶液为新型保护剂制备的植物乳杆菌菌剂细胞膜完整性达到最高。
上述实施例仅例示性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
Claims (5)
1.一种植物乳杆菌菌剂的制备方法,其特征在于,包括如下步骤:
(1)菌种活化:将植物乳杆菌接种到MRS液体培养基,活化一段时间作为种子液;
(2)扩大培养:将步骤(1)所得的种子液接种到另一MRS液体培养基扩大培养,生长至稳定期前期;
(3)收集菌泥:取出步骤(2)扩大培养后的菌液并分装至离心管,离心后,倒掉上清液,并用无菌生理盐水冲洗,得到菌泥;
(4)添加冻干保护剂:向步骤(3)得到的菌泥中添加冻干保护剂,涡旋混匀,得混合溶液;
(5)冷冻干燥:先将步骤(4)所得混合溶液进行预冻,接着冷冻干燥后得到植物乳杆菌菌剂;所述步骤(1)中植物乳杆菌的接种量为3×107CFU/mL,活化时间为10h,活化温度为37℃;所述步骤(2)中种子液的接种量为MRS液体培养基质量的4.4%,培养时间为9.5h;所述步骤(4)中,冻干保护剂为海藻糖溶液和纤维素酶解产物按重量比8∶2组成,所述海藻糖溶液的质量分数为8~12%;所述冻干保护剂在121℃、0.1MPa条件下灭菌17min后冷却使用;所述步骤(4)的冻干保护剂中的纤维素酶解产物的制备方法如下:
将纤维素溶解于pH为4.8的乙酸-乙酸钠缓冲液中,配置成浓度为0.02g/mL的纤维素悬浮液,搅拌12~24h,然后加入1.75U/mL的纤维素酶溶液,混合均匀,在50℃和150rpm条件下水解0.5~2.5h,反应时间结束便将混合溶液浸入沸水中加热5min,使酶失活进而停止酶促反应,待其冷却至室温,利用质量浓度为4%的NaOH溶液调节酶解液pH为6.7~6.9,即得纤维素酶解产物;所述纤维素来源于柚子表皮,所述纤维素的制备方法如下:
将柚子表皮于50℃干燥24h,粉碎,过40目筛;利用质量浓度为15%的NaOH溶液进行碱处理,料液比为1∶30,蒸馏水和95%乙醇冲洗至中性,50℃干燥12h;利用质量分数为6%的NaClO2溶液进行漂白处理,所述NaClO2溶液采用盐酸溶液调节pH至3.8~4.0,然后用蒸馏水冲洗至中性;于冷阱温度-70℃、真空度0Pa下冷冻干燥48h,得到纤维素。
2.根据权利要求1所述的植物乳杆菌菌剂的制备方法,其特征在于,所述步骤(4)中菌泥和冻干保护剂的重量比为1∶10。
3.根据权利要求1所述的植物乳杆菌菌剂的制备方法,其特征在于,所述纤维素悬浮液和纤维素酶溶液体积比为4∶1。
4.根据权利要求1所述植物乳杆菌菌剂的制备方法,其特征在于,所述步骤(3)中的离心条件为:14136×g,4℃,离心10min;离心后,倒掉上清液,并用质量浓度为0.85%的无菌生理盐水冲洗2次;
所述步骤(5)中,预冻温度为-80℃,所述冷冻干燥的条件为:冷阱温度-70℃、真空度0Pa,冷冻干燥48h。
5.一种根据权利要求1~4任一项所述的制备方法制得的植物乳杆菌菌剂。
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