CN113061543B - 一种植物乳杆菌及其用途 - Google Patents
一种植物乳杆菌及其用途 Download PDFInfo
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- CN113061543B CN113061543B CN202010002627.6A CN202010002627A CN113061543B CN 113061543 B CN113061543 B CN 113061543B CN 202010002627 A CN202010002627 A CN 202010002627A CN 113061543 B CN113061543 B CN 113061543B
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Abstract
本发明涉及微生物技术领域,具体涉及一种植物乳杆菌及其用途。本发明保护一种植物乳杆菌,其保藏编号为CGMCC No.18760。该植物乳杆菌可促进抗生素引起的肠道菌群失调的恢复,抑制致病菌的体外增殖,存活率高,用于食品发酵生产中,可提高食品的营养利用度,改善口感。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一种植物乳杆菌及其用途。
背景技术
益生菌是一类通过改善宿主肠道微生物菌群的平衡而发挥作用的活性微生物。2001年,世界粮农组织(FAO)和世界卫生组织(WHO)对益生菌作出了如下定义:通过摄取适当的量,对使用者的身体健康能发挥有效作用的活菌。目前普遍认为,益生菌应该具有以下条件:对宿主有益;无毒性作用和致病作用;能够在消化道存活,能耐受胃酸和胆盐;能在消化道表面定植;能够产生有用的酶类和代谢物;在加工和贮存过程中能保持活性;具有良好的感官特性等。自1899年法国Tissier博士发现第一株益生菌双歧杆菌以来,人类对益生菌的研究已有一百多年的历史。科学家们对益生菌的研究已经逐步趋向于辅助治疗各类疾病,越来越多的研究表明,益生菌可在人肠道内,通过栖生、偏生、竞争或吞噬等复杂关系,改善宿主肠道微生物菌群的平衡进而发挥促进食物有益代谢、提高免疫力、防治代谢性疾病等作用。
乳酸菌是对于人体具有有益作用的一类主要的益生菌,乳酸菌可利用葡萄糖从而产生乳酸,降低了宿主对能量的吸收和利用。乳酸的产生也能促进肠道蠕动,减少营养物质在小肠内留存的时间,提高营养物质的转运速度。
如何从传统食品中分离筛选出具有特殊功效的乳酸菌,具有非常重要的意义。
发明内容
本发明要解决的技术问题是:提供一种植物乳杆菌及其用途,该植物乳杆菌可促进抗生素引起的肠道菌群失调的恢复,抑制致病菌的体外增殖,存活率高,用于食品发酵生产中,可提高食品的营养利用度,改善口感。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种植物乳杆菌,其保藏编号为CGMCC No.18760。
本发明提供了上述植物乳杆菌在制备改善肠道菌群失调的组合物中的应用。
优选地,所述肠道菌群失调由抗生素引起。
优选地,所述组合物为食品、保健品或者药品。
本发明提供了一种活性菌剂,包括上述植物乳杆菌。
优选地,还包括辅料。
本发明还提供了一种活性菌剂的制备方法,包括以下步骤:
将上述的植物乳杆菌在液体培养基内扩大培养,收集菌体;
将扩大培养后收集的菌体加入保护剂重悬,真空冷冻干燥,然后经过粉碎,得到活性菌剂。
本发明还提供了一种发酵产物,利用上述的植物乳杆菌进行发酵获得。
优选地,所述发酵产物为发酵乳制品、发酵燕麦制品、发酵豆制品或者发酵果蔬制品。
与现有技术相比,本发明提供一种植物乳杆菌,其保藏编号为CGMCC No.18760。该菌株具有如下效果:
(1)本发明的植物乳杆菌可显著促进抗生素导致的肠道菌群失调的恢复,肠道内双歧杆菌和乳杆菌的数量显著上升,肠球菌数量显著降低。可显著提高粪便中短链脂肪酸和乳酸的含量;可明显抑制多种致病菌的体外增殖;耐受模拟人工胃肠液,黏附人体肠上皮细胞。可用于预防、缓解或治疗因肠道菌群失调引起的便秘,腹泻和相关肠道炎症等疾病。
(2)本发明的活性菌剂生产工艺参数简单,易于控制,周期短,保证了植物乳杆菌的高存活率,所得产品能够长期储存,产品质量稳定。
(3)本发明的植物乳杆菌可发酵生产发酵牛乳,燕麦乳和胡萝卜汁等发酵物。可提高食物的营养利用度,改善口感,同时补充人体所需活性有益菌。
附图说明
图1基于UPGMA方法构建的植物乳杆菌的RAPD聚类分析图。
具体实施方式
本发明公开了菌株及其应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
生物保藏说明:
生物材料:HOM3204,分类命名:植物乳杆菌(Lactobacillus plantarum),于2019年10月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏中心地址为:北京市朝阳区北辰西路1院3号中国科学院微生物研究所;保藏编号为CGMCC No.18760。
所述植物乳杆菌分离自内蒙古东部山区家庭自制手工酸菜,采集的酸菜样品经过了2-4个月以上腌制时间,优选3个月。称取1g酸菜,用9mL 0.9%生理盐水制成酸菜汁,进行10倍稀释,稀释液涂布于改良的MRS平板(pH 5.0-6.0,并添加溴甲酚绿作为指示剂),30-40℃厌氧培养24~72h。挑选周边变黄的单菌落,染色、镜检观察形态。选取镜检为杆菌的单菌落,纯化培养,并进行16S rDNA鉴定。所述植物乳杆菌菌株细胞呈短杆状,菌体宽0.5-1.0μm,长2-4μm,两端圆形,不形成芽孢。在MRS平板上形成乳白色圆形菌落,表面湿润光滑,边缘整齐。
对分离到的植物乳杆菌HOM3204进行随机多态性DNA分析(RAPD),结果表明HOM3204与部分商业植物乳杆菌菌株均不同,具有唯一性。
对分离到的植物乳杆菌HOM3204进行放大培养,离心收集菌体,加入适当冻干保护剂重悬,再进行真空冷冻干燥制得冻干菌粉。包括以下步骤:
(1)菌种的培养
将-80℃冷冻保存的植物乳杆菌HOM3204以0.5%-3%的接种量接种于无菌的MRS液体培养基中,37℃培养16~24小时,如此传代培养两次得到活化后的种子培养液体。将种子培养液以0.5%-3%的接种量接种于发酵培养基中,培养基的配方为:20-60g/L葡萄糖,20-60g/L酵母提取物,5-20g/L三水合乙酸钠,0.1-0.3g/L硫酸镁,1-3g/L磷酸氢二钾,2-6g/L柠檬酸三胺,0.5-2g/L吐温-80。37℃恒温培养,发酵过程中自动流加氢氧化钠溶液保持恒定pH5.0-6.5致发酵至产酸停止,氢氧化钠不再流加时终止发酵,得到植物乳杆菌HOM3204高密度培养液,活菌数可达100-200亿CFU/mL。
(2)冷冻冻干保护剂的制备
使用无菌水与保护剂原料混合制备得到含有50-200g/L脱脂奶粉、20-80g/L海藻糖、1-5g/L维生素C、2-10g/L L-谷氨酸钠的保护剂。
(3)冷冻干燥
将培养结束的植物乳杆菌HOM3204发酵菌液,在2~8℃离心10min,弃上清液,收集菌泥,用0.9%无菌生理盐水洗涤菌泥1~2次,将洗涤后菌泥与上述保护剂混合,于冻干机内进行冷冻干燥,待冻干完毕,用精细研磨机将菌饼进行粉碎处理,获得所述冻干菌粉,冻干菌粉活菌数为1.0-2.0×1011CFU/g。
本发明还提供一种发酵产物,利用植物乳杆菌HOM3204进行发酵获得。可将植物乳杆菌HOM3204接种于脱脂奶粉、燕麦奶、或者胡萝卜汁中,经过30~37℃发酵,获得发酵产物。所述发酵产物中含有大量植物乳杆菌,提高食物的营养利用度,改善口感,同时补充人体所需活性有益菌。
为了进一步理解本发明,下面结合实施例对本发明提供的植物乳杆菌及其用途进行详细说明,本发明的保护范围不受以下实施例的限制。
实施例1植物乳杆菌HOM3204的分离及鉴定
(1)乳酸菌筛选培养基配方
MRS固体培养基配方:蛋白胨10.0g,牛肉浸粉8.0g,酵母粉4.0g,葡萄糖20.0g,山梨醇酐单油酸1mL,磷酸氢二钾2.0g,柠檬酸三铵2.0g,三水乙酸钠5.0g,七水硫酸镁0.2g,四水硫酸锰0.05g,琼脂10.0g,双蒸水1L,英国OXIOD公司(CM1163);在成品培养基基础上添加溴甲酚绿0.005g,充分搅拌均匀。调整pH至5.5,121℃灭菌20min。备用。
(2)植物乳杆菌HOM3204菌株的分离筛选
称取1g酸菜,用9mL 0.9%生理盐水制成酸菜汁,吸取0.5mL汁液利用10倍稀释法对样品稀释,稀释度为10-3-10-5,稀释液涂布于上述MRS平板中,35℃厌氧培养48-72h。挑取周边变黄的单菌落,划线培养,纯化3-4次至菌落单一,同时进行革兰氏染色、镜检观察菌落形态。转接单菌落至MRS液体培养基中进行纯培养,甘油保种。对每株菌进行编号。
(3)植物乳杆菌HOM3204菌株的分子生物学特性分析
利用随机扩增多态性DNA标记(RAPD)方法将所得菌株与商业菌株进行基因分型对比研究,以确定所得菌株的特异性。
选取引物OPA-02(5’-TGCCGAGCTG-3’),OPA-18(5’-AGGTGACCGT-3’),OPL-07(5’-AGGCGGGAAC-3’),OPL-16(5’-AGGTTGCAGG-3’)和OPM-05(5’-GGGAACGTGT-3’)对菌株的基因组DNA进行随机扩增。扩增条件如下:首先将模板和引物95℃保持5min,然后降至56℃,加入反应混合物,按以下方式扩增45次:94℃变性1分钟,30℃退火1min,72℃延伸2min。
取10μL PCR扩增产物于2%琼脂糖凝胶电泳检测,随后在凝胶成像仪中进行成像。
利用Bionumerics version 6.6软件,基于UPGMA方法对RAPD图谱进行聚类分析。结果如图1所示。分离所得的植物乳杆菌株分别为HOM3201,HOM3202,HOM3203,HOM3204和HOM3205。商业植物乳杆菌菌株为:LP115,LP-ONLLY,P-8,CCFM8661和STIII。
结果显示,HOM3204与所选商业菌株的条带图谱均具有明显差异,差异率大于30%。一般认为,在系统发育树上相似度大于90%以上的菌株具有同株菌的可能性。因此,HOM3204与上述商业菌株相比,具有基因型特异性。
(4)植物乳杆菌HOM3204菌株的鉴定与保藏
将菌株在MRS固体平板上划线培养,35℃厌氧培养24-48h,对HOM3204抽提细菌总DNA,进行16S rDNA扩增,利用通用引物27F,1492R进行PCR扩增和琼脂糖凝胶电泳,然后切胶回收、测序,分离获得的菌株进行16S rDNA测序。用BLAST工具在NCBI数据库中进行比对,鉴定为植物乳杆菌,命名为植物乳杆菌HOM3204,其于2019年10月28日在中国微生物菌种保藏管理委员会普通微生物中心进行保藏,保藏号为CGMCC No.18760。
实施例2胃肠道通过能力试验
(1)菌株的活化
将供试菌株以1%的接种量接种于MRS液体培养基中,于37℃培养24h,活化两次,备用。
(2)人工胃液的配制
取稀盐酸16.4mL加水约800mL与胃蛋白酶10g,摇匀后,调节pH为3.0,加水定容至1000mL,用0.2μm微孔滤膜过滤后待用。
(3)人工肠液的配制
取磷酸二氢钾6.8g,加水500mL溶解,调节pH至6.8,另加胰蛋白酶10g,猪胆盐3g,溶解后使两溶液混合,加水定容至1000mL,用0.22um无菌滤膜在无菌环境下过滤,备用。
(4)菌株在模拟胃肠道中存活能力评价
取供试菌株活化后菌液1mL,将菌体加入至9mL人工胃液(pH 3.0)中,混匀后计活菌数,放置于37℃培养箱培养3h后计活菌数。在人工胃液中培养3h后,将全部菌体转入至等体积人工肠液(pH 6.8)中,混匀后于37℃培养。分别于3h、24h用MRS培养基进行平板活菌计数,其存活率用以下公式计算:
胃液3小时存活率(%)=[log CFU N1/log CFU N0]×100%
肠液3小时存活率(%)=[log CFU N2/log CFU N0]×100%
肠液24小时存活率(%)=[log CFU N3/log CFU N0]×100%
N0=未处理前植物乳杆菌的活菌数,N1=经过胃液处理3小时之后的植物乳杆菌活菌数,N2=经过肠液处理3小时之后的植物乳杆菌活菌数,N3=经过肠液处理24小时之后的植物乳杆菌活菌数。
从表1可以看出:植物乳杆菌HOM3204在经过3小时的模拟胃液处理后,存活可达到95%以上,菌液继续经24小时的模拟肠液处理后,其存活率仍可高达95%以上,说明植物乳杆菌HOM3204在肠道中具有较高的存活率。
表1植物乳杆菌在模拟胃肠液中的存活率
实施例3肠上皮细胞黏附能力试验
(1)细胞的复苏与培养
将Caco-2细胞冻存管快速放入37℃水浴锅中,溶化后离心去上清后用新鲜的培养液重悬细胞,使其均匀分散于培养瓶中,在5%CO2和95%空气的气体条件下37℃进行培养,复苏时每48h更换培养液1次。待细胞生长良好时(80%融合),用胰酶-EDTA溶液对Caco-2细胞在温度为37℃的条件下进行消化,将细胞消化后调整细胞浓度为1×105个/mL,接种于24孔板中培养至细胞长至融合度达80%。
(2)粘附实验
离心收集在相应培养基中生长的菌体;用DPBS洗涤菌体3次后不完全培养基重悬菌体,并调节菌体浓度为107cfu/mL;将上述菌悬液1mL加入含有长至单层的Caco-2细胞的24孔板中,于5%CO2培养箱中37℃孵育2h;孵育后用无菌DPBS洗涤3次;胰酶-EDTA溶液对Caco-2细胞在温度为37℃的条件下进行消化,统计细胞数与黏附前后活菌数,结果见表2。结果表明,植物乳杆菌HOM3204对人结肠癌细胞Caco-2的粘附指数为4.14,与对照菌株相比,具有较好的粘附肠道上皮细胞的能力。
粘附指数=粘附后细菌数/每个平皿细胞数
粘附率=粘附后细菌数/粘附前细菌数
表2植物乳杆菌对Caco-2细胞的粘附能力
实施例4抑制常见致病菌能力试验
(1)指示菌活化
将指示菌(大肠埃希菌ATCC8739;金黄色葡萄球菌ATCC6538;鼠伤寒沙门氏菌ATCC14028;铜绿假单胞菌ATCC9027;单增李斯特菌ATCC19111)以1%接种量接种于TSB培养基中,37℃,培养18h备用。
(2)植物乳杆菌活化
分别将植物乳杆菌HOM3204,STⅢ和Lp115以1%的接种量接入到已灭菌的MRS培养基中,37℃静置培养24小时,活化两次后得菌株发酵液。然后离心10min,取上清液做抑菌试验。
(3)平板制备
将已灭菌的TSA培养基加热到完全融化,倒入培养皿内,置于水平台面上使琼脂层形成均匀厚度,待其凝固。将指示菌加入到TSA培养基中,震荡均匀后,倒入预先制备好的TSA空白琼脂平板内,静置凝固。
(4)抑菌实验
用无菌镊子将牛津杯轻放于平板上,中间保持一定距离,向其中分别加入0.2mL待测的乳酸菌发酵液上清液,置于4℃冰箱中扩散24小时后,37℃培养箱中培养18小时,观察抑菌圈的出现。形成抑菌圈后用直尺进行测量。以MRS液体培养基为阴性对照,以植物乳杆菌LP115、STⅢ为对照菌株,每次做3个平行。
表3植物乳杆菌HOM3204对致病菌的抑制效果
注:“–”无抑菌活性;“+”11-16mm;“++”17-22mm;“+++”≥23mm
由表3可知,植物乳杆菌HOM3204对5种致病菌均有抑制作用,对大肠杆菌,沙门氏菌及单增李斯特菌抑菌效果较好。
实施例4抗氧化指标检测试验
(1)植物乳杆菌活化
分别将植物乳杆菌HOM3204,STⅢ和LP115以1%的接种量接入到已灭菌的MRS培养基中,37℃静置培养24小时,活化两次后得菌株发酵液。然后离心将菌株浓度调整到1010CFU/mL,进行平行比较。
(2)抗氧化指标的检测
指标包含总抗氧化能力测定(T-AOC)、羟自由基清除测定(·OH)、DPPH自由基清除测定。前两个指标利用南京建成公司购买的试剂盒,按操作说明书进行测定。DPPH自由基清除试验采用比色法,其原理是自由基清除剂提供一个电子与DPPH自由基的孤对电子配对,使自身紫色变为黄色,在517nm波长处的吸光度变小,其变化程度与自由基清除程度呈线性关系,即自由基清除剂的清除能力越强,吸光度越小。结果如表4所示。
表4植物乳杆菌HOM3204体外抗氧化能力的检测
由表4可知,植物乳杆菌HOM3204在总抗氧化能力、羟自由基自由基清除、DPPH自由基清除方面表现突出。
实施例5抗生素敏感性试验
药敏试验根据美国临床标准委员会(NCCLS)推荐的K-B琼脂法进行,药敏纸片购于赛默飞世尔科技公司,并严格按照其说明书操作和判断。简要说明如下:
(1)将植物乳杆菌HOM3204接种于MRS琼脂平板,37℃培养18~24小时,然后挑取纯菌落,置于无菌生理盐水中,并制成0.5麦氏浊度标准的菌悬液。
(2)将无菌棉签浸入菌悬液中,在试管壁上旋转按压,挤出多余的培养液,在平板培养基表面至少沿着三个方向涂布平板表面。接种后15分钟内将药敏纸片置于平板上。
(3)将平板37℃培养24~48h后取出,用直尺测量并记录抑菌圈直径,根据CLSI判定标准判读,结果见表5。
表5植物乳杆菌HOM3204对18种抗生素敏感性测定结果
S(susceptible)表示敏感;I(intermediate)表示中等;R(resistance)表示耐药。
实施例6植物乳杆菌HOM3204活性菌粉的制备工艺
(1)菌种的培养
将-80℃冷冻保存的植物乳杆菌HOM3204以1%的接种量接种于无菌的MRS液体培养基中,37℃培养16~24小时,如此传代培养两次得到活化后的种子培养液体。将种子培养液以3%的接种量接种于发酵培养基(如表6所示)中,37℃恒温培养,发酵过程中自动流加氢氧化钠溶液保持恒定pH5.8致发酵至产酸停止,氢氧化钠不再流加时终止发酵,得到植物乳杆菌HOM3204高密度培养液,活菌数可达200亿CFU/mL以上。
表6发酵培养基M307配方
(2)冷冻冻干保护剂的制备
使用无菌水与保护剂原料混合制备得到含有100g/L脱脂奶粉、50g/L海藻糖、3g/L维生素C、5g/L L-谷氨酸钠的保护剂。
(3)冷冻干燥
将培养结束的植物乳杆菌HOM3204发酵菌液,在2~8℃,离心弃上清液,收集菌泥,用0.9%无菌生理盐水洗涤菌泥1~2次,将洗涤后菌泥与上述保护剂混合,使混合后菌液菌浓度达到1010CFU/mL以上,于冻干机内进行冷冻干燥,待冻干完毕,用精细研磨机将菌饼进行粉碎处理,获得所述冻干菌粉,冻干菌粉活菌数高于2.0×1011CFU/g。
实施例7植物乳杆菌HOM3204对抗生素导致的小鼠肠道菌群失调的恢复作用
饲养6~8周龄的健康雄性BALB/c小鼠,随机分为3组:空白对照组、抗生素模型组、植物乳杆菌HOM3204组,每组12只小鼠,全程自由饮水、进食。实验为期35天,空白对照组在实验期间每天灌胃0.3mL无菌生理盐水溶液;抗生素模型组前7天每只小鼠每天灌胃2次,每次0.3mL的0.56g/mL氨苄青霉素溶液,之后28天每天灌胃0.3mL无菌生理盐水溶液。植物乳杆菌HOM3204组前7天每只小鼠每天灌胃2次,每次0.3mL的0.56g/mL氨苄青霉素溶液,之后28天每天灌胃植物乳杆菌HOM3204活性菌粉(HOM3204活性菌粉溶解于5mL无菌生理盐水溶液中,调整活菌数为109CFU/mL)。无菌条件下取各组小鼠35天的粪便,分别用LBS,BL,EMB,叠氮钠—结晶紫—七叶苷琼脂培养基对乳杆菌,双歧杆菌,肠杆菌和肠球菌进行平板计数,结果见表7;采用气相色谱法检测粪便中乳酸和乙酸的含量,结果见表8。采用南京建成试剂盒对小鼠眼球血血清进行超氧化物歧化酶(SOD)和谷胱甘肽酶过氧化物酶(GSH-PX),结果见表9。
由表7和表8结果可知,与抗生素模型组相比,饲喂植物乳杆菌28天,可以显著地提高小鼠肠道内乳酸杆菌(P<0.01),双歧杆菌(P<0.05)的数量,显著降低小鼠肠道内肠球菌(P<0.05)的数量,显著提高粪便中乳酸(P<0.01)和乙酸(P<0.05)的含量。因此,植物乳杆菌HOM3204对抗生素导致的小鼠肠道菌群失调具有显著的恢复作用。
由表9可知,与抗生素模型组相比,饲喂植物乳杆菌28天,可以极显著地提高血液中超氧化物歧化酶的含量(P<0.01),也有助于谷胱甘肽过氧化物酶的提升,因此,植物乳杆菌HOM3204可提升由抗生素导致的菌群失调小鼠血液中抗氧化酶的活力。
表7小鼠肠道菌群的变化(log CFU/g)
注:同列肩标为不同大写字母的表示差异极显著(P<0.01);同列肩标为不同小写字母的表示差异显著(P<0.05)。
表8小鼠粪便中乳酸和乙酸含量的变化(μmol/g)
注:同列肩标为不同大写字母的表示差异极显著(P<0.01);同列肩标为不同小写字母的表示差异显著(P<0.05)。
表9小鼠眼球血血清中抗氧化指标的变化(U/mL)
注:同列肩标为不同大写字母的表示差异极显著(P<0.01);同列肩标为不同小写字母的表示差异显著(P<0.05)。
实施例8发酵牛乳试验
(1)称取12g脱脂乳粉、4g葡萄糖和3g酵母提取物,双蒸水补足至100mL,经过搅拌,充分溶解,高压均质机均质(60℃,22MPa),95℃杀菌5min,冷却至37℃待用。
(2)将植物乳杆菌HOM3204于固体MRS培养基上划线,37℃培养48h,按照此方法传代2次,挑取MRS平板上单一菌落于MRS液体培养基培养,37℃培养15h,获得高活力的菌液,4℃冰箱保藏备用。
(3)无菌条件下,将植物乳杆菌HOM3204用0.9%生理盐水洗脱2次,后以1×106CFU/mL接种量接种至原料中,搅拌,混合均匀,在35℃环境下,静置发酵至滴定酸度为70°T,得发酵乳。
(4)发酵乳通过冰浴后搅拌的方式冷却至16℃,得冷却发酵乳。灌装至包装容器中,后转入4℃的环境中进行冷藏后熟12h,即可得富含植物乳杆菌HOM3204的发酵乳。
(5)根据食品安全国家标准GB4789.35中的乳酸菌检验方法进行植物乳杆菌计数。对上述发酵乳样品进行取样,经梯度稀释后以平板计数法进行计数。所述的发酵乳中活性植物乳杆菌的含量高于2.0×108CFU/mL,pH可达4.52。
实施例9发酵燕麦奶试验
(1)称取40g裸燕麦(张家口)双蒸水补足至1000mL,浸泡过夜,破壁机中蒸煮100min,研磨10min,高压均质机均质(60℃,22MPa),经纱布过滤除渣,得到燕麦浆,95℃杀菌5min,冷却至37℃待用。
(2)将植物乳杆菌HOM3204于固体MRS培养基上划线,37℃培养48h,按此方法传代2次,挑取MRS平板上单一菌落于MRS液体培养基培养,37℃培养24h,获得高活力的菌液,4℃冰箱保藏备用。
(3)将植物乳杆菌HOM3204用0.9%生理盐水洗脱2次,后以1×106CFU/mL接种量接种至燕麦浆中,混合均匀,35℃恒温静置发酵20h,灌装至包装容器中,获得发酵燕麦乳。
(4)对上述发酵样品进行取样,经梯度稀释后以平板计数法进行计数。所述的发酵乳中活性植物乳杆菌的含量高于1.16×108CFU/mL,pH可达4.43。
实施例10发酵胡萝卜汁试验
(1)称取新鲜胡萝卜40g,清水清洗,将胡萝卜切成薄片;将胡萝卜片置于沸水中蒸煮10min,使酶失去活性,防止褐变。
(2)将冷却后的胡萝卜片加入破壁机,加适量水,研磨10min,汁液用八层纱布进行过滤,获得滤液。添加2%葡萄糖,双蒸水补足100mL,搅拌混匀,95℃杀菌8min,冷却后得胡萝卜汁,备用。
(3)将植物乳杆菌HOM3204于固体MRS培养基上划线,37℃培养48h,依照此方法传代2次,挑取MRS平板上单一菌落于MRS液体培养基培养,37℃培养24h,获得高活力菌液,4℃冰箱保藏备用。
(4)将植物乳杆菌HOM3204用0.9%生理盐水洗脱2次,后以1×106CFU/mL接种量接种至胡萝卜汁中。混合均匀,35℃恒温静置发酵24h。灌装至包装容器中,获得发酵胡萝卜汁饮品。最终产品色泽橘红、口感酸甜、香气浓郁。
(5)对上述发酵样品进行取样,经梯度稀释后以平板计数法进行计数。所述的发酵胡萝卜汁中活性植物乳杆菌的含量高于3.20×108CFU/mL,pH可达4.52。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.一种植物乳杆菌(Lactobacillus plantarum),其特征在于,其保藏编号为CGMCCNo.18760。
2.权利要求1所述的植物乳杆菌在制备改善肠道菌群失调的组合物中的用途,
所述肠道菌群失调由抗生素引起。
3.根据权利要求2所述的用途,其特征在于,所述组合物为食品、保健品或者药品。
4.一种活性菌剂,其特征在于,包括权利要求1所述的植物乳杆菌。
5.根据权利要求4所述的活性菌剂,其特征在于,还包括辅料。
6.一种活性菌剂的制备方法,其特征在于,包括以下步骤:
将权利要求1所述的植物乳杆菌在液体培养基内扩大培养,收集菌体;
将扩大培养后收集的菌体加入保护剂重悬,真空冷冻干燥,然后经过粉碎,得到活性菌剂。
7.一种发酵产物,其特征在于,利用权利要求1所述的植物乳杆菌进行发酵获得。
8.根据权利要求7所述的发酵产物,其特征在于,所述发酵产物为发酵乳制品、发酵燕麦制品、发酵豆制品或者发酵果蔬制品。
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