CN110106121A - 一种产胞外多糖的植物乳杆菌 - Google Patents
一种产胞外多糖的植物乳杆菌 Download PDFInfo
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- CN110106121A CN110106121A CN201910461706.0A CN201910461706A CN110106121A CN 110106121 A CN110106121 A CN 110106121A CN 201910461706 A CN201910461706 A CN 201910461706A CN 110106121 A CN110106121 A CN 110106121A
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- Prior art keywords
- whey
- jnulcc001
- lactobacillus plantarum
- product
- polysaccharide
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Abstract
本发明公开了一种产胞外多糖的植物乳杆菌,属于微生物技术领域。本发明提供的植物乳杆菌JNULCC001具有生物安全性,不产生毒素,以该菌株为发酵菌株,以乳清粉为原料可以生产功能性发酵产品。所生产的发酵乳清制品质地均一、感官状态佳、流变学特性较好、产生特征风味物、且后熟稳定性好,活菌数高。此外,该方法生产发酵制品无需外源添加任何食品添加剂,实现了乳清的再利用,改善了环境污染以及资源浪费的问题,具有环境友好性和可持续发展性。
Description
技术领域
本发明涉及一种产胞外多糖的植物乳杆菌,属于微生物技术领域。
背景技术
每年全球都会产生大量的食物废弃物以及食物残渣,不仅造成环境污染也带来了巨大的经济损失。随着人们对发酵乳品喜爱程度的增加以及乳品企业的日益发展,随之产生了大量的乳清以及其他液体残渣(Food&Function,2018)。乳清占整个乳体积的85-95%(Bioresource technology,1996,57:1-11),被认为是一种营养价值丰富的物质,因为它含有55%的乳品营养成分。乳清含有93-94%水和乳糖、蛋白质、矿物质、乳酸和脂肪(Journalof environmental management,2012,110:48-68.)。
传统的有氧过程处理乳清的方法成本很高(Enzyme and Microbial Technology,2007,41:876-880),并且大部分乳制品工厂没有相应的处理系统,因此,大约47%的乳清被排放到河流或陆地造成严重的污染问题(Biotechnology Progress,2004,20:1091-1095),这同时也造成了营养物质的浪费。因此,提供一种低成本的、操作简便的乳清处理方法,对于环境保护有着重要的意义。
植物乳杆菌是一种多功能乳酸菌,广泛存在于发酵的乳、肉、蔬菜制品中,同时在人体肠道内也常有发现,其在食品领域有很长的使用历史,对人类健康有益。随着L.plantarum WCFS1,和L.plantarum NCIMB 8826基因组测序的完成,人们发现部分植物乳杆菌可以利用多种糖类以及蛋白质产生氨基酸。另外,植物乳杆菌的大量表面蛋白使得它们更容易接近不同的表面以及底物。这些都使得植物乳杆菌可以适应不同的环境条件。
乳酸菌胞外多糖是由乳酸菌产生的高分子多糖聚合物,可分为荚膜多糖和胞外多糖。荚膜多糖一般分布在菌体的细胞壁上,而胞外多糖通常被释放到培养液中。乳酸菌胞外多糖在提高发酵乳制品的流变学特性、质构以及适口性方面有着极好的应用(FEMSmicrobiology reviews,1999,23(2):153-177)。乳酸菌胞外多糖可以在肠道内长时间存在进而增加益生菌在肠道内的定植(International dairy journal,2002,12(2):163-171)。此外,人们发现乳酸菌胞外多糖对重金属离子和染料分子也有一定的吸附效果(Bioresource technology,2014,160:15-23;Plos one,2016,11(2):e0148430)。
乳酸菌胞外多糖根据其组成和结构可以分为同多糖和异多糖。同多糖主要有葡聚糖和果聚糖。相比之下,异多糖则由多个重复的低聚糖组成,每个重复单元通常含有两个或两个以上单糖并具有不同的连接方式(Biotechnology advances,2001,19(8):597-625)。乳酸菌胞外多糖带有一些带电基团,如羧基、磷酸根以及羟基。这些基团有助于胞外多糖吸附重金属。
发明内容
本发明的第一个目的是提供一株植物乳杆菌(Lactobacillus plantarum)JNULCC001,已于2019年4月25日保藏于中国典型培养物保藏中心,保藏编号为CCTCC M2019296,保藏地址为中国,武汉,武汉大学。
本发明的第二个目的是提供含有上述植物乳杆菌JNULCC001的微生物制剂。
在本发明的一种实施方式中,所述微生物制剂中含有活菌数≥107CFU/g的植物乳杆菌细胞。
本发明的第三个目的是提供上述的植物乳杆菌JNULCC001在生产发酵制品中的应用。
本发明的第四个目的是提供上述的植物乳杆菌JNULCC001在制备胞外多糖中的应用。
本发明的第五个目的是提供一种生产乳清制品的方法,以乳清为底物,应用植物乳杆菌JNULCC001进行发酵。
在本发明的一种实施方式中,是将在MRS培养基中培养12-24h后的植物乳杆菌JNULCC001按照5%-10%的接种量接种于含乳清的物质中,置于25-42℃下,发酵12-72h。
在本发明的一种实施方式中,包括以下步骤:
a)将乳清粉加水配成3%-12%(m/V)的悬浊液,105℃杀菌15min,优选10%;
b)将植物乳杆菌JNULCC001在MRS培养基中培养12-24h,优选18h;
c)按5%-10%的接种量取b)中得到的菌液(OD600=1.5-3),离心,用无菌生理盐水清洗后,接种于a)中已灭菌的乳清中,置于25-42℃下,发酵12-72h,优选31℃,优选48h。
本发明的第六个目的是提供上述的植物乳杆菌JNULCC001在制备食品、保健品、饲料或生物医药材料中的应用。
本发明的第七个目的是提供上述的微生物菌剂在制备食品、保健品、饲料或生物医药材料中的应用。
本发明筛选获得了一株高产胞外多糖的植物乳杆菌,该植物乳杆菌可以用来发酵乳清生产功能性产品。相对于现有技术,该发明主要具有以下优势:
(1)该菌株具有生物安全性,不产生毒素,具有环境友好性和可持续发展性;
(2)该植物乳杆菌产生胞外多糖,避免在乳清发酵中加入外源添加剂;
(3)发酵所获得的产品性状良好,流变学特性,风味物质都有所提高,同时产生多种氨基酸。
生物材料保藏
一株植物乳杆菌(Lactobacillus plantarum),分类命名为Lactobacillusplantarum JNULCC001,已于2019年4月25日保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 2019296,保藏地址为中国,武汉,武汉大学。
附图说明
图1为发酵液的拉丝(A)及纯化后涂板(B)结果图。
图2为植物乳杆菌JNULCC001的革兰氏染色结果图。
图3为植物乳杆菌JNULCC001在不同碳源条件下多糖的产量情况。
图4为单糖组成分析结果。
图5为植物乳杆菌JNULCC001发酵乳清乳清粉浓度的优化结果示意图,A为乳清浓度对pH的影响,B为乳清浓度对活菌数的影响,C为乳清浓度对多糖产量的影响。
图6为植物乳杆菌JNULCC001发酵乳清温度的优化结果示意图,A为发酵温度对pH的影响,B为发酵温度对活菌数的影响,C为发酵温度对多糖产量的影响。
图7为植物乳杆菌JNULCC001发酵乳清发酵时间的优化结果示意图,A为发酵时间对pH的影响,B为发酵时间对活菌数的影响,C为发酵时间对多糖产生的影响。
图8为植物乳杆菌JNULCC001发酵乳清接种量的优化结果示意图,A为接种量对pH的影响,B为接种量对活菌数的影响,C为接种量对多糖产生的影响。
图9为植物乳杆菌JNULCC001发酵的乳清与未发酵乳清的状态示意图。
图10为植物乳杆菌JNULCC001与商业用酸奶发酵剂发酵的乳清后的流变学特性变化结果。
图11为植物乳杆菌JNULCC001发酵的乳清后的氨基酸产生情况。
图12为植物乳杆菌JNULCC001发酵的乳清后的后熟稳定性。
具体实施方式
胞外多糖提取和含量的测定方法:胞外多糖含量的提取采用醇沉法进行,具体如下:在液体MRS或乳清中发酵18-96h的发酵液经离心(1000g,4℃,30min)去除菌体,之后向发酵液上清加入终浓度为4%的TCA(三氯乙酸)放于4℃冰箱过夜处理,离心(1000g,4℃,30min)去除蛋白,之后在上清液中加入3倍体积95%的乙醇,放于4℃冰箱醇沉过夜,离心(1000g,4℃,30min)取沉淀,之后用8000-14000Da的透析袋于4℃条件下流动透析48h。冻干称重。
单糖组成分析方法:5mg EPS-JNULCC001加入4mL,2.5M的三氟乙酸(TFA),将其水解成单糖,于121℃水解2h。之后,水解产物用N2吹干,加入甲醇用N2吹干,重复三吃以去除残余TFA,最后加入去离子水溶解干燥的水解产物至色谱瓶中用于检测。采用离子色谱法对样品进行检测,所用色谱柱为DionexTMCarboPacTMPA20(赛默飞),所用检测器为电化学检测器。流动相:A相:ddH2O;B相:NaOH;C相:NaOH/NaAC,流速:0.5mL/min。
上机程序如下:
选取果糖,阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖、核糖、半乳糖醛酸和葡萄糖醛酸作为标准品。数据分析采用变色龙软件(7.2)对数据进行分析。
GC-MS分析方法:选择配有1310-ISQ LT的GC-MS对制品的风味物质进行分析,柱子选用TG-WAXMS柱(60m×0.25mm×0.25μm)。将6mL发酵制品转移至20mL的螺口瓶中,测试前加入氯化钠至饱和状态,此操作有助于风味物质的挥发,便于检测。操作条件如下:氦气作为载体,流速为1.2mL/min,分流比为50:1,程序设定为:(a)40℃维持1min;(b)以3℃/min的速率将温度从40℃升至160℃,维持0min,(c)再以10℃/min的速率将温度从160℃升至230℃维持8min。进样温度维持在250℃。然后,将测试结果与NIST17数据库进行比对。
活菌数的测定方法:活菌数的测定采用稀释涂平板法进行,具体如下:用移液枪吸取0.1mL样品液于无菌的的离心管中用无菌生理盐水稀释成稀释度为10-1,10-2,10-3,10-4,10-5,10-6,10-7,10-8,10-9,10-10,10-11,10-12。选择稀释度为10-4至10-12涂布于MRS培养基,每个稀释度做两个重复,置于31℃的恒温箱中培养48h后计数。
实施例1产胞外多糖乳杆菌的分离筛选
(1)样品液的稀释:用无菌的1mL移液管吸取1mL样品液于无菌的的小试管中用无菌生理盐水稀释成稀释度为10-1,10-2,10-3,10-4,10-5,10-6,10-7,10-8,10-9,10-10,10-11,10-12。选择稀释度为10-4至10-12的稀释液备用。
(2)平板分离:首先在超净台上在无菌操作的条件下,将已经灭菌的含有碳酸钙的MRS固体培养基倒平板,待温度为室温后用移液枪吸取以上稀释度为10-4至10-12的发酵液0.1mL。分别用倾注法和涂布法倾注和涂布到含有碳酸钙的MRS固体培养基的培养皿上,每个稀释度做两个重复,并用保鲜膜装好至于37℃的恒温箱中培养48h。筛选出有透明圈的菌落,如图1所示,并进行液体培养。
(3)拉丝实验:对获得的乳酸菌进行拉丝实验确定产多糖。并对这些菌株进行多糖的提取及测定,如图1所示。
(4)菌株在不同条件下产糖情况测试:将活化好的菌株分别接种到含有乳糖、蔗糖、葡萄糖、甘露糖的MRS以及10%乳清液中发酵18h,提取多糖并测定其含量,结果如图3所示。
实施例2单糖组成分析
按照具体实施方式中“单糖组成分析方法”对单糖组成进行分析,结果如图4所示,该提取的多糖主要由葡萄糖、甘露糖、半乳糖和半乳糖醛酸组成,含量分别260、27、82和20μmol/g。
实施例3菌株的鉴定
(1)菌体形态观察及革兰氏染色
在超净台中用接种环挑去少量菌体样本均匀涂抹在载玻片上,再用酒精灯加热杀死细菌,滴加草酸铵结晶紫,染色1min。用水冲掉洗载玻片上多余的染色液,再用吸水纸吸干。滴加少量碘-碘化钾溶液,静置1min后水洗,用吸水纸吸干,连续滴加乙醇进行脱色,直至流出的液体无色,然后水洗。最后用蕃红染液复染30s。染色结束后,用显微镜进行观察,革兰氏阳性菌呈紫色,革兰氏阴性菌呈红色。鉴定结果,分离的乳酸菌为革兰氏阳性菌(如图2所示)。
(2)菌株的分子生物学鉴定
对所获得的菌株提取基因组DNA,采用通用引物对16s rDNA进行扩增,之后进行测序,对测序结果在NCBI上进行比对,确定其为植物乳杆菌。
实施例4植物乳杆菌发酵乳清时发酵条件的优化
(1)乳清浓度的优化:
植物乳杆菌JNULCC001按照3%比例接种于MRS培养基,于31℃静置培养18h后用生理盐水清洗两遍,按照5%的接种量分别接种于3%、5%、8%、10%和12%的灭菌乳清粉中,发酵24h后测定产品的pH,统计产品中的活菌数,并提取胞外多糖并测定其含量。
如图5所示,乳清浓度对pH影响较小,当乳清浓度为10%时,pH最低为4.6(图5A)。不同乳清浓度对EPS产量影响较大,当乳清浓度为12%时,EPS产量为2.1g/L(图5C),但此时活菌数为4.3×108CFU/mL,当乳清浓度为8%时,活菌数为3.1×109CFU/mL(图5B)。
(2)发酵温度的优化:
植物乳杆菌JN001按照3%比例接种于MRS培养基于31℃静置培养18h,用生理盐水清洗两遍,按照5%的接种量接种于12%的乳清粉中,分别于25℃、28℃、31℃、37℃和42℃静置培养24h后测定产品的pH,统计产品中的活菌数,并提取胞外多糖并测定其含量。
如图6所示,温度对pH的影响不是很明显(图6A),当发酵温度为31℃时,乳酸菌菌落数最高,达到1.1×109CFU/mL(图6B)。EPS产量随着温度的升高逐渐降低。当温度升至42℃时,EPS产量仅为85mg/L(图6C),此时的活菌数为1.8×108CFU/mL。
(3)发酵时间的优化:
植物乳杆菌JNULCC001按照3%比例接种于MRS培养基于31℃静置培养18h,用生理盐水清洗两遍,按照5%的接种量接种于12%的乳清粉中,于31℃条件下分别静置培养4、8、12、24、36、48、72、96h后测定产品的pH,统计产品中的活菌数,并提取胞外多糖并测定其含量。
如图7所示,随着时间的延长,pH逐渐降低,活菌数变化不大,维持在5.4×108CFU/mL,发酵48h多糖产量最高达到最高为2.0g/L。
(4)接种量的优化:
植物乳杆菌JNULCC001按照3%比例接种于MRS培养基于31℃静置培养18h,用生理盐水清洗两遍,之后分别按照1%、3%、5%、7%和9%的接种量接种于12%的乳清粉中,于31℃条件下分别静置培养486h后测定产品的pH,统计产品中的活菌数,并提取胞外多糖并测定其含量。
如图8所示,不同的接种量(1%-9%)对pH的影响不大(图8A)。活菌数在接种量为1%-7%范围内也比较稳定(图8B),当接种比率增加至9%时,活菌数显著升高(图8B)。当接种量为5%时,EPS产量达到最高2.1g/L(图8C)。
实施例5产品的性状观察
经MRS培养18h的JNULCC001用生理盐水清洗两次后接种于10%的乳清,置于31℃发酵48h后用玻璃棒搅拌并挑取做粘度观察,同时按照“胞外多糖提取和含量的测定方法”中的方法对多糖进行醇沉观察多糖产生情况。同时对该样品进行粘度测试测试方法如下。
图9为对发酵后的乳清制品进行粘稠度观察的结果及发酵后乳清中多糖醇沉结果,如图8所示,发酵后的乳清制品具有良好的粘稠度,且发酵产品中多糖含量丰富。
图10为三种制品的粘度测定结果,三种制品分别为:对照(未发酵乳清);常用酸奶发酵剂发酵乳清所获得制品;JNULCC001发酵乳清所得发酵制品。粘度的测定选用HAAKEMARSⅢ流变仪进行测定,该流变仪配有平行板几何形状(P35Ti-L,35-mm直径,1-mm间隙),测试条件为25±0.5℃。The HAAKE Rheo Win 3软件用于数据采集和分析。样品在分析之前首先在样品台上于25±0.5℃平衡2min。所有测试做三个平行。黏度曲线为剪切率γ=0.1-1000s-1条件下测试结果。
如图9所示,当剪切应力从30到1000s-1变化时,三种制品都表现出剪切变稀的现象反映出,制品从水相向凝胶相的转变。使用低剪切应力30s-1时,JNULCC001发酵后的乳清制品黏度为市售酸奶发酵剂发酵制品的4.3倍,是未发酵乳清的11.1倍。
图11为氨基酸含量分析结果。采用HITACHI的L8900氨基酸分析仪进行测定,测试采用柱后茚三酮衍生法。
如图11所示,JNULCC001发酵后的乳清制品中含有丰富的氨基酸,包括磷酸乙醇胺、苏氨酸、丝氨酸、缬氨酸、胍氨酸、半胱氨酸、亮氨酸、苯丙氨酸、β-丙氨酸、γ-氨基正丁酸、鸟氨酸、赖氨酸、精氨酸、羟脯氨酸和脯氨酸。植物乳杆菌JNULCC001发酵乳清还能产生风味物质(如表1所示)。
图12为发酵制品的稳定性检测。活菌数检测同具体实施方案。pH测定采用pH计测定,酸度检测采用SBA-40C生物传感分析仪进行检测。
如图12所示,发酵制品在17天内活菌数都维持在106CFU/mL以上。pH和乳酸变化不明显,说明产品稳定性良好。
实施例6利用扫描电镜对所发酵前后的乳清产品进行观察
上述所获得的乳清发酵产品经10000rpm,4℃,离心20min后取上清液冻干,冻干后的产品配成1mg/mL的溶液,用扫描电子显微镜观察其结构。结果显示,采用JNULCC001发酵的乳清制品更加平滑细腻。
表1 GC-MS分析菌株JNULCC001发酵乳清产生的风味物质
序号 | 保留时间 | 峰面积(%) | 化合物名称 | 分类 |
1 | 4.99 | 0.59 | 辛烷 | 碳氢化合物 |
2 | 5.26 | 0.27 | 丙酮 | 酮类 |
3 | 6.35 | 0.33 | 乙酸乙酯 | 酯类 |
4 | 6.84 | 1.01 | 异丙醇 | 醇类 |
5 | 7.33 | 3.89 | 乙醇 | 醇类 |
6 | 8.58 | 5.49 | 丁二酮 | 酮类 |
7 | 13.77 | 0.99 | 丙醇 | 醇类 |
8 | 16.24 | 0.62 | 2-庚酮 | 酮类 |
9 | 18.37 | 7.00 | 2-正戊基呋喃 | 杂环类 |
10 | 19.60 | 0.13 | 苯乙烯 | 碳氢化合物 |
11 | 20.78 | 19.85 | 乙偶姻 | 酮类 |
12 | 21.51 | 0.26 | 二丙酮 | 酮类 |
13 | 21.41 | 12.08 | 脱氢芳樟醇 | 醇类 |
14 | 23.60 | 0.17 | 1-己醇 | 醇类 |
15 | 25.36 | 0.84 | 壬酮 | 酮类 |
16 | 27.84 | 0.06 | 1-辛烯-3-醇 | 醇类 |
17 | 28.02 | 0.08 | 正庚醇 | 醇类 |
18 | 28.77 | 8.61 | 乙酸 | 酸类 |
19 | 29.50 | 0.51 | 乙基己醇 | 醇类 |
20 | 29.95 | 0.45 | 十五烷 | 碳氢化合物 |
21 | 31.25 | 0.41 | 异丁基苯乙酮 | 酮类 |
22 | 33.04 | 1.68 | 十一酮 | 酮类 |
23 | 34.04 | 0.41 | 十六烷 | 碳氢化合物 |
24 | 34.11 | 0.49 | 十一酮 | 酮类 |
25 | 35.57 | 0.29 | 萘 | 碳氢化合物 |
26 | 36.03 | 1.99 | 丁酸 | 酸类 |
27 | 36.35 | 0.22 | 丙烯酸,辛酯 | 酯类 |
28 | 36.47 | 1.21 | 糠醇 | 醇类 |
29 | 37.89 | 0.21 | 萘 | 碳氢化合物 |
30 | 39.81 | 0.16 | 萘 | 碳氢化合物 |
31 | 40.82 | 0.30 | 4-氢-1,3-苯并二氧烷 | 碳氢化合物 |
32 | 42.10 | 0.24 | 十三烷酮 | 酮类 |
32 | 42.47 | 0.01 | 苯甲醛 | 醛类 |
33 | 44.00 | 5.14 | 己酸 | 酸类 |
34 | 47.41 | 5.05 | 苯并噻唑 | 杂环类 |
35 | 51.23 | 3.58 | 辛酸 | 酸类 |
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一株植物乳杆菌(Lactobacillus plantarum)JNULCC001,已于2019年4月25日保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 2019296,保藏地址为中国,武汉,武汉大学。
2.含有权利要求1所述植物乳杆菌JNULCC001的微生物制剂。
3.如权利要求2所述的微生物菌剂,其特征在于,所述微生物制剂中含有活菌数≥107CFU/g的植物乳杆菌细胞。
4.权利要求1所述的植物乳杆菌JNULCC001在生产发酵制品中的应用。
5.权利要求1所述的植物乳杆菌JNULCC001在制备胞外多糖中的应用。
6.一种生产乳清制品的方法,其特征在于,以乳清为底物,应用权利要求1所述的植物乳杆菌JNULCC001进行发酵。
7.如权利要求6所述的方法,其特征在于,是将在MRS培养基中培养12-24h后的植物乳杆菌JNULCC001按照5%-10%的接种量接种于含乳清的物质中,置于25-42℃下,发酵12-72h。
8.如权利要求7所述的方法,其特征在于,包括以下步骤:
a)将乳清粉加水配成3%-12%的悬浊液,灭菌;
b)将植物乳杆菌JNULCC001在MRS培养基中培养12-24h;
c)按5%-10%的接种量取b)中得到的菌液,离心,用无菌生理盐水清洗后,接种于a)中已灭菌的乳清中,置于25-42℃下,发酵12-72h。
9.权利要求1所述的植物乳杆菌JNULCC001在制备食品、保健品、饲料或生物医药材料中的应用。
10.权利要求2或3所述的微生物菌剂在制备食品、保健品、饲料或生物医药材料中的应用。
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