CN114836343B - 一株能高产γ-氨基丁酸且拮抗有害菌的植物乳杆菌P10及其应用 - Google Patents
一株能高产γ-氨基丁酸且拮抗有害菌的植物乳杆菌P10及其应用 Download PDFInfo
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Abstract
本发明涉及微生物技术领域,尤其涉及一种植物乳杆菌P10及其应用。本发明分离得到一种广谱抑菌、产苯乳酸、γ‑氨基丁酸和胞外多糖的植物乳杆菌,将其命名为植物乳杆菌(Lactobacillus plantarum)P10,并且对其进行了保藏,保藏编号为CGMCC No.21505。本发明提供的植物乳杆菌(Lactobacillus plantarum)P10具备抑制多种腐败与致病微生物的功能,同时能够产生苯乳酸、γ‑氨基丁酸和胞外多糖等功能因子,这在功能性食品、保健品、日用品或农产品的生产领域具有重要意义。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一种植物乳杆菌P10及其应用。
背景技术
功能性食品是满足现有生活需要的可选项之一,也是食品产业高质量健康发展的需要。益生菌可通过产生多种功能性代谢产物发挥保障安全与提升营养健康的作用。因此,开发多功能性益生菌株资源具有重要的研究价值和应用前景。
苯乳酸(phenyllactic acid,PLA)是一种具有多功能性的有机酸,对常见的食源性致病菌和腐败菌具有广谱抑菌活性;细胞实验表明苯乳酸具有免疫调节作用;同时作为丹参素(3,4-二羟基苯乳酸)的结构类似物,苯乳酸在冠心病治疗方面具有重要应用。
γ-氨基丁酸(γ-aminobutyric acid,GABA)是一种抑制性神经递质,在神经系统中发挥重要作用,如与GABA-A受体结合可以扩张血管降低血压;可以增加神经元细胞膜对Cl-的通透性,引起细胞超极化,产生突触后抑制效应,达到镇静神经的效果。GABA通过促进GABA神经递质的释放,因此增加GABA能神经元细胞数目,缓解因GABA神经元细胞减少导致的小鼠抑郁。此外,GABA还可以调节β-淀粉样蛋白相关的功能活性,缓和阿尔兹海默症。
胞外多糖(exopolysaccharides,EPS)常作为增稠剂、乳化剂、稳定剂用于改善乳制品、面制品等食品的流变特性、口感,以及延缓其老化等作用。
发明内容
为了解决现有技术存在的问题,本发明提供一种植物乳杆菌P10及其应用。
第一方面,本发明提供一种植物乳杆菌(Lactobacillus plantarum)P10,所述植物乳杆菌的保藏编号为:CGMCC No.21505。
本发明分离筛选得到一种植物乳杆菌,并对其进行生物保藏,保藏信息如下:
保藏编号:CGMCC No.21505,分类命名为:植物乳杆菌Lactobacillus plantarum,保藏单位: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:北京市朝阳区北辰西路1号院3号,邮编为100101,保藏日期:2020年12月18日。
进一步地,所述植物乳杆菌(Lactobacillus plantarum)P10的16SrDNA序列如SEQID NO.1所示。
本发明进一步提供一种菌剂,包括所述的植物乳杆菌(Lactobacillusplantarum)P10或其发酵液。
本发明进一步提供所述植物乳杆菌(Lactobacillus plantarum)P10,或所述菌剂在抑制腐败与致病微生物中的应用;
所述腐败与致病微生物包括革兰氏阳性菌、革兰氏阴性菌或真菌中的一种或多种。
进一步地,所述革兰氏阳性菌包括金黄色葡萄球菌、蜡样芽孢杆菌或枯草芽孢杆菌中的一种或多种;和/或,所述革兰氏阴性菌包括大肠杆菌、荧光假单胞菌或沙门氏菌中的一种或多种;和/或,所述真菌包括黑曲霉、桔青霉、毛霉或扩展青霉中的一种或多种。
本发明进一步提供所述植物乳杆菌(Lactobacillus plantarum)P10,或所述菌剂在制备苯乳酸、γ-氨基丁酸或胞外多糖中的应用。
进一步地,所述苯乳酸的产量为80-900mg/L;和/或,所述γ-氨基丁酸的产量为300-900mg/L;和/或,所述胞外多糖的产量为300-600mg/L。
本发明进一步提供所述植物乳杆菌(Lactobacillus plantarum)P10,或所述菌剂在制备功能性产品中的应用;所述功能性产品包括:食品、日用品或农产品。
第二方面,本发明提供一种提高面制品中功能因子含量的方法,包括:在面制品的制备过程中加入所述的植物乳杆菌(Lactobacillus plantarum)P10,或所述的菌剂。
进一步地,所述植物乳杆菌(Lactobacillus plantarum)P10的添加量为106-109CFU/g。
进一步地,所述面制品为馒头、面包、蛋糕或饼干中的一种或多种。
本发明具备如下有益效果:
本发明分离筛选得到一种植物乳杆菌,将其命名为植物乳杆菌(Lactobacillusplantarum)P10,该菌株具有广谱抑菌性,可以抑制多种类型的细菌和真菌。同时发酵产物中苯乳酸产量达80-900mg/L、γ-氨基丁酸的产量达300-900mg/L,胞外多糖产量为300-600mg/L。
本发明将植物乳杆菌P10用于馒头的制备中,发现在贮藏期间微生物数量显著降低,同时苯乳酸含量最高可达24mg/kg、γ-氨基丁酸含量最高可达3102mg/kg。
本发明提供了一种具有广谱抑菌性,同时具有产苯乳酸、γ-氨基丁酸和胞外多糖等功能因子能力的植物乳杆菌,为开发新型营养安全化食品、保健品、日用品或农用品等产品提供了新的菌株资源。
附图说明
图1为本发明实施例1提供的植物乳杆菌P10的菌落示意图;其中左图为固体培养基上的菌落示意图;右图为显微镜下菌落示意图。
图2为本发明实施例1提供的系统发育树示意图。
图3为本发明实施例2提供的面团流变结果示意图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
以下实施例中未特别说明的技术手段,均为现有技术的常规手段,以下实施例中所用的材料、试剂若无特别说明,均可以从商业途径购买得到。
实施例1
1、乳酸菌菌株的分离和纯化:
挑选四川省攀枝花市当地的传统泡菜食品为样品稀释后,涂布于含有CaCO3的MRS固体培养基平板,静置,于30-37℃培养36-48h,挑选出有溶钙圈的疑似乳酸菌菌株,将分离菌株于MRS固体培养基培养24-48h后,观察发现菌落形态多呈乳白色圆形、中间略微隆起、不透明或半透明、边缘整齐(如图1所示)。革兰氏染色呈阳性,菌体形态呈短杆。将初步确定的菌落在MRS固体培养基平板上反复划线几次,从而进一步进行纯化的目的,然后挑取单菌落于MRS培养基中30-37℃下培养至对数期,制作甘油管放入-20℃保藏备用。
2、具有广谱抑菌活性的菌株筛选
指示菌的活化:取保存于河北科技大学食品与生物技术实验室的大肠杆菌ATCC44752、金黄色葡萄球菌ATCC 25923、蜡样芽孢杆菌ATCC 11778、荧光假单胞菌ATCC 13525、枯草芽孢杆菌ATCC 13358、沙门氏菌ATCC 14028于营养琼脂肉汤(NB)培养基中30-37℃恒温箱中培养活化后,再将发酵液以1-3%的接种量于NB培养基中,30-37℃培养至对数期,用紫外分光光度计调至菌悬液浓度为1×108CFU/mL,备用。桔青霉ATCC9849、毛霉ATCC16457、黑曲霉ATCC 16404、扩展青霉CICC 40658接种于马铃薯琼脂培养基PDA,28-30℃培养2-7d后,挑取孢子至0.85%的生理盐水中,过滤并用血球计数法调至菌悬液浓度为105-106CFU/mL备用。
菌株抑菌特性评价:将上述细菌指示菌以3%接种量接种到NA培养基(真菌指示菌接种于PDA培养基)后倒平板。每个平板上等距离放3个无菌牛津杯。将保藏的菌种于液体MRS培养基中30-37℃活化18-24h,之后按体积百分比1-3%接种量加入到液体MRS培养基,于30-37℃培养24-48h后,将发酵液离心后取上清液注入牛津杯,将平板放于4℃冰箱中预扩散后于30-37℃恒温培养箱中培养18-24h(真菌2-4d),观察抑菌效果,测量并记录抑菌圈的直径大小。
3、产苯乳酸的菌株筛选
利用高效液相色谱仪分析乳酸菌上清液中苯乳酸的含量,以筛选高产苯乳酸的菌株。苯乳酸液相色谱分析条件为:采用C18(4.6×250mm,5μm);流动相:A:0.02%三氟乙酸甲醇溶液;B:0.02%三氟乙酸水溶液;流速:1.0mL/min;检测波长:210nm,柱温:30℃;进样量:20μL。植物乳杆菌P10苯乳酸产量为80~600mg/L。
4、产γ-氨基丁酸的菌株筛选
将乳酸菌发酵液进行2,4-二硝基氟苯衍生化,然后利用高效液相色谱仪分析发酵液中γ-氨基丁酸的含量,筛选出高产γ-氨基丁酸的菌株。γ-氨基丁酸液相色谱分析条件为:采用C18(4.6×250mm,5μm);流动相:A:CH3CN:H2O(V:V)=1:1;B:20mM的磷酸缓冲溶液(pH=7);流速:1.0mL/min;检测波长:360nm,柱温:35℃;进样量:20μL。植物乳杆菌P10γ-氨基丁酸产量为300~500mg/L。
5、产胞外多糖的菌株筛选
取上述乳酸菌上清液添加三氯乙酸至终浓度4-6%(w/v),静置过夜;离心收集上清液,然后用乙醇4℃沉淀12-18h后离心,取沉淀溶解于去离子水后再用去离子水透析,利用苯酚硫酸法测得胞外多糖产量,进而筛选高产菌株。植物乳杆菌P10胞外多糖产量为300~400mg/L。
6、功能性菌株的筛选
将上述筛选的具有广谱抑菌性且高产苯乳酸、γ-氨基丁酸、胞外多糖的乳酸菌P10进行革兰氏染色和16S rDNA鉴定,结果显示该乳酸菌16S rDNA序列长度为1504bp。将测序结果与GenBank中的16SrDNA序列进行同源性比对,然后构建系统发育树(图2),以确定菌株的种属关系。同源性分析结果表明,乳酸菌P10与Lactobacillus plantarum MH665820.1同源性达到99%以上,因此鉴定为植物乳杆菌,分类命名为植物乳杆菌P10。
表1植物乳杆菌P10的抑菌谱
实施例2:植物乳杆菌P10在馒头生产中的应用
本实施例用于说明植物乳杆菌P10在发酵制备馒头中的应用研究。
1、面团的制备
空白组:小麦粉(100%)、去离子水(50%)。
酵母菌发酵组:小麦粉(100%)、酵母(0.5%)、去离子水(50%)。
乳酸菌+酵母菌发酵组:小麦粉(100%)、酵母(0.5%)、去离子水(50%)、乳酸菌P10接种量106-109CFU/g。
将上述实验组各组分混匀,于30℃恒温恒湿培养箱中醒发,8h后取出部分面团真空冷冻干燥后测定其胞外多糖含量,部分面团测定流变特性。
2、馒头的制备
空白组:小麦粉(100%)、去离子水(50%)。
酵母菌发酵组:小麦粉(100%)、酵母(0.5%)、去离子水(50%)。
乳酸菌+酵母菌发酵组:小麦粉(100%)、酵母(0.5%)、去离子水(50%)、植物乳杆菌P10添加量106-109CFU/g;
将上述分组中的各组分混匀,于30℃恒温恒湿培养箱中醒发4-8h后取出排气,分割成30~35g的面团,滚圆成型。将不锈钢锅里添入适量水,置于电磁炉上,待水烧开后,将成型的面坯置于不锈钢锅内,沸水汽蒸制25min。自然冷却1h后装于自封袋中,一部分放置在-20℃冰箱贮藏用于分析馒头中γ-氨基丁酸含量,剩余部分放置在25℃下贮藏。
3、植物乳杆菌P10对面团发酵期间胞外多糖含量的影响
取0.5g上述冻干面团溶于10mL去离子水中,振荡1h后8000g离心15min,取上清,添加三氯乙酸至终质量分数为4%,离心(8000g,10min)取上清,加入3倍体积95%的无水乙醇,4℃过夜沉淀,再次离心后将上清装入透析袋,于去离子中透析48h,采用苯酚硫酸法测定溶液中胞外多糖的含量。
表2不同处理组面团中EPS含量
由表2可知,发酵8h时乳酸菌+酵母菌发酵的面团EPS含量为2.54mg/g,显著高于空白发酵组面团(1.71mg/g)和酵母菌发酵的面团(1.62mg/g)。面团流变结果(图3)显示,各处理组面团G`和G``随着发酵时间的增加而降低,表明发酵过程中面团逐渐酸化导致了其黏弹性的损失,发酵8h的面团中,乳酸菌+酵母菌发酵的面团G`和G``最小,其次是单一添加酵母菌实验组,表明添加乳酸菌+酵母菌共同发酵降低了面团的黏弹性,面团更柔软。表明植物乳杆菌P10的加入增加了面团中胞外多糖含量,改善了面团的流变特性,使面团具有更好的加工性能。
4、植物乳杆菌P10对馒头贮藏期间菌落总数的影响
每日随机取不同处理组的馒头各3个,分别用0.85%的生理盐水稀释10倍进行拍打均质,并分别于平板计数培养基(PCA)、孟加拉红平板进行涂布。细菌于30-37℃培养48h后进行菌落计数,真菌于28℃培养72h后进行计数。
表3不同处理组馒头贮藏期间菌落总数
结果(表3)显示加入乳酸菌的馒头在贮藏期间菌落总数显著低于只添加酵母组,贮藏第10d时细菌和真菌菌落总数分别为5.77Lg CFU/g、5.36Lg CFU/g,较酵母组低3.61LgCFU/g和2.65Lg CFU/g,表明植物乳杆菌P10能够起到抑制多种微生物的生长,可发挥对馒头防腐保鲜的作用。
5、植物乳杆菌P10对馒头PLA、GABA含量的影响
将上述醒发好的面团取出排气,分割成30~35g的面团,滚圆成型。将不锈钢锅里添入适量水,置于电磁炉上,待水烧开后,将成型的面坯置于不锈钢锅内沸水汽蒸制25min。自然冷却1h后装于自封袋中,放置在-20℃贮藏。将馒头样品用超纯水稀释溶解,用均质机拍打均匀后离心取上清液,采用HPLC法测定PLA含量、比色法测定GABA含量。
表4不同处理组馒头中PLA和GABA含量
结果(表4)显示酵母菌和乳酸菌共同发酵的馒头PLA、GABA含量最高,分别为24mg/kg和3120mg/kg,显著高于其他组馒头。其次是乳酸菌发酵的馒头。PLA和GABA含量分别为17mg/kg和2150mg/kg。表明乳酸菌P10的加入能显著提高馒头中PLA和GABA含量。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 河北科技大学
<120> 一株能高产γ-氨基丁酸且拮抗有害菌的植物乳杆菌P10及其应用
<130> KHP221112756.7
<141> 2022-04-19
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1504
<212> DNA
<213> Artificial Sequence
<400> 1
ggctcaggac gaacgctggc ggcgtgccta atacatgcaa gtcgaacgaa ctctggtatt 60
gattggtgct tgcatcatga tttacatttg agtgagtggc gaactggtga gtaacacgtg 120
ggaaacctgc ccagaagcgg gggataacac ctggaaacag atgctaatac cgcataacaa 180
cttggaccgc atggtccgag cttgaaagat ggcttcggct atcacttttg gatggtcccg 240
cggcgtatta gctagatggt ggggtaacgg ctcaccatgg caatgatacg tagccgacct 300
gagagggtaa tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca 360
gtagggaatc ttccacaatg gacgaaagtc tgatggagca acgccgcgtg agtgaagaag 420
ggtttcggct cgtaaaactc tgttgttaaa gaagaacata tctgagagta actgttcagg 480
tattgacggt atttaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 540
gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggttttttaa 600
gtctgatgtg aaagccttcg gctcaaccga agaagtgcat cggaaactgg gaaacttgag 660
tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa 720
caccagtggc gaaggcggct gtctggtctg taactgacgc tgaggctcga aagtatgggt 780
agcaaacagg attagatacc ctggtagtcc ataccgtaaa cgatgaatgc taagtgttgg 840
agggtttccg cccttcagtg ctgcagctaa cgcattaagc attccgcctg gggagtacgg 900
ccgcaaggct gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 960
ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atactatgca aatctaagag 1020
attagacgtt cccttcgggg acatggatac aggtggtgca tggttgtcgt cagctcgtgt 1080
cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tattatcagt tgccagcatt 1140
aagttgggca ctctggtgag actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1200
aatcatcatg ccccttatga cctgggctac acacgtgcta caatggatgg tacaacgagt 1260
tgcgaactcg cgagagtaag ctaatctctt aaagccattc tcagttcgga ttgtaggctg 1320
caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat 1380
acgttcccgg gccttgtaca caccgcccgt cacaccatga gagtttgtaa cacccaaagt 1440
cggtggggta accttttagg aaccagccgc ctaaggtggg acagatgatt agggtgaagt 1500
cgta 1504
Claims (7)
1.一种植物乳杆菌(Lactobacillus plantarum)P10,其特征在于,所述植物乳杆菌的保藏编号为:CGMCC No.21505。
2.一种菌剂,其特征在于,包括权利要求1所述的植物乳杆菌(Lactobacillus plantarum)P10或其发酵液。
3.权利要求1所述的植物乳杆菌(Lactobacillus plantarum)P10,或权利要求2所述菌剂在制备抑制腐败与致病微生物的产品中的应用;
所述腐败与致病微生物为革兰氏阳性菌、革兰氏阴性菌或真菌中的一种或多种;
所述革兰氏阳性菌为金黄色葡萄球菌、蜡样芽孢杆菌或枯草芽孢杆菌中的一种或多种;
所述革兰氏阴性菌为大肠杆菌、荧光假单胞菌或沙门氏菌中的一种或多种;
所述真菌为黑曲霉、桔青霉、毛霉或扩展青霉中的一种或多种。
4.权利要求1所述的植物乳杆菌(Lactobacillus plantarum)P10,或权利要求2所述菌剂在制备苯乳酸、γ-氨基丁酸和胞外多糖中的应用。
5.根据权利要求4所述的应用,其特征在于,所述苯乳酸的产量为80-900 mg/L;和/或,所述γ-氨基丁酸的产量为300-900 mg/L;和/或,所述胞外多糖的产量为300-600 mg/L。
6.一种提高面制品中功能因子含量的方法,其特征在于,包括:在面制品的制备过程中加入权利要求1所述的植物乳杆菌(Lactobacillus plantarum)P10,或权利要求2所述的菌剂;
所述功能因子为苯乳酸、γ-氨基丁酸和胞外多糖。
7.根据权利要求6所述的方法,其特征在于,所述植物乳杆菌(Lactobacillus plantarum)P10的添加量为106-109 CFU/g。
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