CN112877266B - 植物乳杆菌qs7t及其应用 - Google Patents
植物乳杆菌qs7t及其应用 Download PDFInfo
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- CN112877266B CN112877266B CN202110413337.5A CN202110413337A CN112877266B CN 112877266 B CN112877266 B CN 112877266B CN 202110413337 A CN202110413337 A CN 202110413337A CN 112877266 B CN112877266 B CN 112877266B
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Abstract
本发明提供一种植物乳杆菌QS7T及其应用,属于微生物领域。该植物乳杆菌QS7T保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为:CGMCC No.21510。该菌株是从传统四川泡菜中分离得到,其能够高效、特异性的代谢并利用菊粉。本发明还进一步提供了含有植物乳杆菌QS7T的相关产品以及植物乳杆菌QS7T的相关应用,例如一种功能性泡菜,其能够基于“益生菌+膳食纤维”,发挥提高益生菌菌株在肠道中的存活率和定殖能力的功效,以此来进一步调节改善肠道微生态,降低肠道相关疾病的发生率,促进人体健康。
Description
技术领域
本发明属于微生物领域,更具体地,涉及一种植物乳杆菌QS7T及其应用。
背景技术
益生菌是指一类能够对宿主产生有益作用的活性微生物,通过定植于宿主肠道、生殖***内,对宿主产生确切的健康功效,从而改善宿主体内微生态平衡、发挥有益作用的活性微生物总称。乳酸菌作为益生菌中最具代表性的菌属,具有帮助消化、促进营养物质的吸收、调节肠道菌群、调节机体免疫功能、降低血液总胆固醇、缓解乳糖不耐症等生理活性。
由于乳酸菌的益生活性具有菌株特异性,不同菌株之间的益生活性差别较大。因此,需要更有针对性的筛选具有特殊生理活性的菌株。
泡菜作为中国传统食品中典型的乳酸菌发酵制品,有着悠久的食用传统,除了具有独特的风味和丰富的营养,其保健功效也逐渐引起人们的关注。随着人们生活水平的提高,人们的健康意识越来越强,对食品的要求已从数量型向质量型转变,安全、营养、保健是未来食品发展的必然方向,我国传统的腌制蔬菜已难以满足人们的需求。因此,应该通过革新自身科学技术,形成具有中国特色并符合国际饮食潮流的现代功能泡菜产品,使其在人们的现代健康生活中扮演更加重要的角色。
发明内容
本发明的第一目的在于提供一种植物乳杆菌QS7T,该菌株是从传统四川泡菜中分离得到,其能够高效、特异性的代谢并利用菊粉。该菌株已保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为:CGMCC No.21510,保藏日期为2020年12月21日,并鉴定为植物乳杆菌(Lactiplantibacillus plantarum)。
本发明的第二目的在于提供一种微生物菌剂,其包括上述植物乳杆菌QS7T。
进一步地,该植物乳杆菌QS7T为活菌。
更进一步地,该植物乳杆菌QS7T为活菌,菌体浓度为107~1014CFU/mL。
更进一步地,该微生物菌剂的制备方法包括:取甘油保种菌液划线于MRS平板上活化,37℃恒温培养24h-48h,挑取单菌落接种于MRS液体培养基中培养过夜,所得菌液离心、用生理盐水洗涤,菌体沉淀分散于生理盐水得到活菌菌悬液,浓度为107~1014CFU/mL。
本发明的第三目的在于提供一种组合物,其包括上述的微生物菌剂,还可包括一些生理上可接受的赋形剂,该组合物可以是食品、保健品或药品。
进一步地,该组合物还包括益生元。益生元为益生菌的食物,可促进肠道内益生菌的增殖和存活,提供肠道定植能力,起到调节肠道菌群作用,进而发挥益生功效。
更进一步地,该益生元为菊粉。
菊粉存在于约36000种植物的球茎和块茎中,是蔬菜中广泛存在的一种膳食纤维,也是自然界中含量第二大的贮藏多糖。菊粉不能被人的唾液和胃肠道消化酶消化或水解,它能作为益生元,以完整结构的形式直达肠道后端,被双歧杆菌和乳杆菌等有益细菌选择性利用,从而达到明显改善肠道内微生物种群比例的目的。然而,根据现有研究报道,并不是所有乳酸菌都能利用菊粉,仅有极少部分乳酸菌菌种或菌株具有特异性代谢利用菊粉的能力。
由于植物乳杆菌QS7T能够特异性的代谢菊粉,当该组合物中同时包括植物乳杆菌QS7T和菊粉时,菊粉作为益生元在肠道内能够为植物乳杆菌QS7T提供碳源,两者能够发挥协同增效的作用,极大的提高了植物乳杆菌QS7T在肠道中的存活率和定植能力,起到了调节肠道菌群最佳平衡的作用,为宿主的消化道健康带来益处。
本发明的第三目的在于提供一种上述植物乳杆菌QS7T、微生物菌剂或组合物在生产药品、保健品或功能性食品中的应用。
进一步地,所述功能性食品包括发酵乳制品、发酵豆制品、发酵果蔬制品;优选地,所述发酵果蔬制品包括泡菜、发酵蔬菜汁和发酵水果汁。
本发明的第四目的在于提供一种功能性果蔬制品,其通过用上述植物乳杆菌QS7T发酵果蔬所得。其中,果蔬为富含菊粉的水果和蔬菜;优选地,所述蔬菜包括:菊芋、菊苣、大蒜和洋葱。
该功能性果蔬制品的制备方法包括:将所述植物乳杆菌QS7T接种、培养至菌体浓度达到1x108CFU/mL后,收集菌体制成菌悬液;再将所述果蔬进行杀菌处理后,并与盐水混合,接种所述菌悬液后一起装坛发酵。
进一步地,果蔬与盐水按1:1~3质量比混合,其中,盐水的浓度为2~6%。
再进一步地,所述菌悬液的浓度为1x108CFU/mL;所述菌悬液的接种量为2~6%。
该功能性泡菜,采用植物乳杆菌QS7T作为发酵菌种,由于植物乳杆菌QS7T能够特异性的代谢果蔬中富含的膳食纤维,尤其是菊粉,使得该功能性泡菜能够基于“益生菌+膳食纤维”,发挥提高益生菌菌株在肠道中的存活率和定植能力的功效,以此来进一步调节改善肠道微生态,降低肠道相关疾病的发生率,促进人体健康。
附图说明
图1为植物乳杆菌QS7T代谢利用菊粉能力的结果;
图2为植物乳杆菌QS7T在菊粉,葡萄糖和水中生长曲线图,图中横坐标为发酵时间,纵坐标为菌液在600nm测定的OD值;
图3为植物乳杆菌QS7T发酵葡萄糖和菊粉的pH变化,图中横坐标为发酵时间,纵坐标为发酵液pH值;
图4为薄层色谱分析植物乳杆菌QS7T在发酵菊粉过程中菊粉组成的动态变化的结果;
图5为植物乳杆菌QS7T发酵洋姜制备泡菜中去泡菜水样品测定的pH值,图中横坐标为发酵时间(天),纵坐标为泡菜水pH值;
图6为用TLC分析植物乳杆菌QS7T发酵洋姜制备泡菜中泡菜水样品的菊粉低聚糖组分;
图7为植物乳杆菌QS7T发酵洋姜制备泡菜,发酵7天后泡菜外观形态。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实施例1
菌株的筛选与保藏
本发明中的植物乳杆菌QS7T是从采集的四川传统家庭自制泡菜水中分离获得的,通过以菊粉为唯一碳源的培养基进行发酵培养,评价乳酸菌代谢利用菊粉的能力,从中筛选出能高效代谢利用菊粉的植物乳杆菌QS7T,步骤如下:
(1)取-70℃甘油保存的泡菜水样品50μL于装有5mL MRS液体培养基的试管中,震荡摇匀,37℃恒温培养16-24h。取活化后的培养液1mL加入9mL无菌生理盐水中进行10倍梯度稀释,选择合适的稀释度,取100μL稀释样品涂布于MRS-CaCO3培养基平板,37℃恒温培养24h-48h。
(2)观察并记录菌落特征,挑取有溶钙圈的并具乳酸菌典型菌落特征的菌落,进行反复划线分离、并培养,直至菌落纯化。
(3)评价乳酸菌代谢利用菊粉能力:将上述分离纯化的菌株于MRS液体培养基中过夜培养,将过夜培养物按1%(v/v)分别接种于bMRS+菊粉(bMRS中添加1%w/v菊粉)、bMRS+葡萄糖(bMRS中添加1%w/v葡萄糖)和bMRS+H2O(bMRS中添加无菌水),在37℃厌氧培养48h;其中bMRS中添加了溴钾酚紫钠盐(300mg/L)作为pH指示剂。以bMRS+H2O为阴性对照,以bMRS+葡萄糖为阳性对照,根据培养基颜色从紫色变黄的程度来判断乳酸菌对菊粉的代谢利用能力。
根据乳酸菌在菊粉为唯一碳源的培养基上发酵产酸的能力导致的发酵液颜色变黄的程度,从而筛选出一株能高效、特异性代谢利用菊粉的菌株。根据上述菌落、细胞形态鉴定及16S rDNA基因序列分析的结果,经中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)鉴定为(Lactiplantibacillus plantarum)QS7T。
表1四川传统泡菜中分离乳酸菌的发酵程度
菌株编号 | bMRS+H<sub>2</sub>O | bMRS+1%葡萄糖 | bMRS+1%菊粉 |
YT040 | - | ++++ | - |
QS7T | - | ++++ | ++++ |
YT044 | - | ++++ | - |
YT048 | - | ++++ | - |
YT049 | - | ++++ | - |
YT051 | - | ++++ | - |
YT052 | - | ++++ | - |
YT053 | - | ++++ | - |
YT054 | - | ++++ | - |
YT059 | - | ++++ | - |
YT061 | - | ++++ | - |
YT063 | - | ++++ | - |
YT065 | - | ++++ | - |
YT066 | - | ++++ | - |
YT068 | - | ++++ | - |
YT069 | - | ++++ | - |
YT071 | - | ++++ | - |
YT072 | - | ++++ | - |
YT073 | - | ++++ | - |
注:‘-’表示发酵液未变色,仍为紫色;‘+’表示发酵液pH降低后变黄的程度,其中‘+’表示轻微变黄;‘++’表示淡黄色;‘+++’表示黄色;‘++++’表示深黄色。
结果分析如下:共从四川传统泡菜中分离纯化获得19株乳酸菌,以bMRS+H2O为阴性对照,以bMRS+葡萄糖为阳性对照,以菊粉为唯一碳源设置bMRS+菊粉作为实验组,根据培养基中pH指示剂颜色从紫色变黄的程度来判断乳酸菌对菊粉的代谢利用能力,如表1所示,从结果可知仅有QS7T对菊粉表现出明显的发酵能力。植物乳杆菌QS7T在阴性对照(bMRS+H2O)中发酵48h后为紫色,由于无任何碳源,因此无生长和发酵产生;植物乳杆菌QS7T在阳性对照(bMRS+葡萄糖)中发酵48h大量产酸,降低发酵液pH,因此发酵液变成深黄色;植物乳杆菌QS7T在实验组(bMRS+菊粉)中发酵48h后,可以观察到发酵液也变成深黄色,说明QS7T可以菊粉为唯一碳源生长良好并代谢产酸、降低体系pH;综上可以知道植物乳杆菌QS7T是一株能够高效代谢并利用菊粉的乳酸菌。
图1为植物乳杆菌QS7T代谢利用菊粉能力的结果。
实施例2
植物乳杆菌QS7T的菊粉代谢利用特性,测定如下:
(1)生长曲线和pH测定:将保存的植物乳杆菌QS7T在MRS平板上划线、37℃培养24-48h活化,挑取单菌落于MRS液体培养基中,培养液(1%,v/v)接种于分别含50mL bMRS+菊粉、bMRS+葡萄糖和bMRS+H2O的无菌离心管中,于37℃培养,分别在1、2、3、4、6、8、10、12、14、15、20、24、48和72h等14个不同时间点取发酵液用分光光度计通过在波长600nm(OD600nm)的光密度来测量乳酸菌生长情况、用酸度计来测定发酵液pH值。
(2)代谢产物的薄层色谱(TLC)分析:利用TLC分析乳酸菌在发酵菊粉过程中菊粉组成的动态变化,将上述取样的发酵液12000rpm离心2min取上清液,用毛细管吸取6μL上清液点样于GF254薄层层析板上,并以6μL 1%菊粉和3μL 1%葡萄糖作为对照。展开剂按正丁醇:乙酸:水=2:2:1(v/v)的比例配制,点样完成的薄层层析板置于该溶剂中进行展开。显色剂配置:100mL丙酮中加入2g二苯胺,2mL苯胺和10mL磷酸(wt%=85%)混合得到。最后将展开的薄层层析板用该显色剂喷雾,在105℃下加热10min显色。
结果分析如下:植物乳杆菌QS7T在以1%菊粉(inulin)为唯一碳源的培养基中能够在10h后进入稳定期,OD值达到最大值,并与在葡萄糖为碳源的培养基中生长情况接近;同时测定发酵液不同时间点的pH值,发现植物乳杆菌QS7T在以菊粉为唯一碳源的培养基中,发酵时间10h后,pH就降低至4.20,对比之下,与在含葡萄糖的培养基中发酵pH降低速度和最终值都接近;采用TLC分析在菊粉为唯一碳源的培养基发酵48h后的发酵液的组分变化,发现48h后菊粉完全被植物乳杆菌QS7T降解并代谢利用。综述可知植物乳杆菌QS7T表现出非常高效的代谢能力。
图2为植物乳杆菌QS7T在菊粉,葡萄糖和水中生长曲线图,图中横坐标为发酵时间,纵坐标为菌液在600nm测定的OD值;
图3为植物乳杆菌QS7T发酵葡萄糖和菊粉的pH变化。图中横坐标为发酵时间,纵坐标为发酵液pH值;
图4为薄层色谱分析植物乳杆菌QS7T在发酵菊粉过程中菊粉组成的动态变化的结果。
实施例3
利用植物乳杆菌QS7T发酵制备功能性泡菜,步骤如下:
(1)植物乳杆菌QS7T接种于MRS液体培养基中,37℃培养至菌体浓度达到约1x108CFU/mL,4500r/min离心10min,收集菌体后用无菌生理盐水洗涤2次,并加入无菌生理盐水制成菌悬液备用。
(2)选择无病害的新鲜洋姜原料(500g)经整理、清洗、沥干、切分后,用75%食用酒精喷洒杀菌。无菌水清洗残留食用酒精后,在超净工作台紫外线下沥干,装入2.0L玻璃泡菜坛。
(3)蔬菜与6.0%食盐水质量体积比(g/mL)为1∶2,并接入5.0mL植物乳杆菌QS7T菌悬液。坛沿封水,25℃发酵7d,4得发酵成熟的洋姜泡菜。
(4)每坛每隔1d取样,用于测定酸度和植物乳杆菌QS7T数量。
结果分析如下:从接种当天开始直至发酵的第7天每天进行取样,去除发酵泡菜水样品测得pH值,发现发酵1天以后泡菜水pH即迅速降低至4左右,7天后下降至3.5左右,通过菌落计数知道泡菜水中植物乳杆菌QS7T的数量为1x108CFU/mL;同时对发酵液进行菊粉低聚糖的分析,发现接种当天和发酵1天后的发酵液中几乎没有菊粉低聚糖,随着发酵的进行从第2天开始后续泡菜样品中测定发现存在大量的菊粉低聚糖组分,说明由植物乳杆菌QS7T发酵富含菊粉的洋姜所制备的泡菜中既富含活的植物乳乳杆菌,也富含大量的菊粉益生元,是一种功能性泡菜,同时制备出的洋姜泡菜具有质地脆嫩、鲜香爽口、酸味宜人的独特风味,且可以进行工业化推广生产。
图5为植物乳杆菌QS7T发酵洋姜制备泡菜中去泡菜水样品测定的pH值。图中横坐标为发酵时间(天),纵坐标为泡菜水pH值;
图6为用TLC分析植物乳杆菌QS7T发酵洋姜制备泡菜中泡菜水样品的菊粉低聚糖组分;
图7为植物乳杆菌QS7T发酵洋姜制备泡菜,发酵7天后泡菜外观形态。
以上所述的实施例仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (10)
1.一种植物乳杆菌(Lactiplantibacillus plantarum)QS7T,其保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为:CGMCC No.21510,保藏日期为 2020年 12月21 日。
2.一种微生物菌剂,其特征在于,其包含如权利要求1所述的植物乳杆菌QS7T。
3.一种组合物,其特征在于,其包括如权利要求2所述的微生物菌剂。
4.根据权利要求3所述的组合物,其特征在于,还包括益生元。
5.根据权利要求4所述的组合物,其特征在于,所述益生元包括菊粉;所述微生物菌剂的含量为107~1014CFU/mL,所述益生元的含量为质量浓度0.1~5%。
6.权利要求1所述的植物乳杆菌QS7T、权利要求2所述的微生物菌剂或权利要求3所述的组合物在保健品或功能性食品中的应用。
7.根据权利要求6所述的应用,其特征在于,所述功能性食品包括发酵乳、乳酸菌饮料、发酵果蔬制品。
8.一种功能性果蔬制品,其特征在于,其通过用如权利要求1所述的植物乳杆菌QS7T发酵果蔬所得。
9.根据权利要求8所述的功能性果蔬制品,其特征在于,所述果蔬为富含菊粉的水果和蔬菜。
10.一种如权利要求8所述的功能性果蔬制品的制备方法,其特征在于,其包括:
将所述植物乳杆菌QS7T接种、培养至菌体浓度达到1x108CFU/mL,收集菌体,制成菌悬液;
再将所述果蔬进行杀菌处理,并与盐水混合,接种所述菌悬液后一起装坛发酵。
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