CN113373085A - 一株降低牛乳β-乳球蛋白抗原性的植物乳杆菌及其应用 - Google Patents
一株降低牛乳β-乳球蛋白抗原性的植物乳杆菌及其应用 Download PDFInfo
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- CN113373085A CN113373085A CN202110571986.8A CN202110571986A CN113373085A CN 113373085 A CN113373085 A CN 113373085A CN 202110571986 A CN202110571986 A CN 202110571986A CN 113373085 A CN113373085 A CN 113373085A
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- lactobacillus plantarum
- ahq
- lactoglobulin
- beta
- antigenicity
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- A23C19/00—Cheese; Cheese preparations; Making thereof
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Abstract
本发明公开了一株降低牛乳β‑乳球蛋白抗原性的植物乳杆菌及其应用,属于生物应用技术领域。该菌株已于2020年10月10日在武汉市武昌区八一路武汉大学中国典型培养物保藏中心保藏,保藏编号为CCTCC NO:M2020544。植物乳杆菌AHQ‑14具有胆盐水解酶活性、对肠细胞粘附能力高、在胃肠液中存活率高等益生性和生物胺产量低、无溶血性、对常用抗生素敏感等安全性。植物乳杆菌AHQ‑14应用于含β‑乳球蛋白食品中,可有效降解牛乳中β‑乳球蛋白,进而可降低牛乳蛋白的抗原性,减少过敏反应的发生。
Description
技术领域
本发明属于生物应用技术领域,涉及一种具有降低β-乳球蛋白致敏性的植物乳杆菌(Lactobacillus plantarum)AHQ-14,还涉及所述植物乳杆菌AHQ-14的用途。
背景技术
牛奶蛋白通常会引起儿童过敏反应,约有2-3%的婴幼儿表现牛奶蛋白过敏的症状。牛乳中β-乳球蛋白被认为是引起小儿牛乳过敏的主要过敏原。降低β-乳球蛋白致敏性的方法有很多,例如加热、糖基化、酶解等。但热处理可能会生成更多的抗原表位,使过敏性增强。糖基化会使部分营养成分损失,酶种类繁多,不同条件下酶的作用方式和程度也有差异,导致分解产物和抗原性也不同,同时还会产生苦味肽,影响口感和风味。乳酸菌发酵则是对产品品质影响较小、降低致敏性效果较好的方法之一。
乳酸菌的许多菌株已被广泛用于益生菌制剂和发酵食品的生产过程中,在维持胃肠道正常功能、抑制病原菌入侵、预防腹泻以及食物过敏等方面具有独特的生理作用。由于不同菌株的特性与能力不同,乳酸菌的效果是可变的,因此有必要发展有效降低β-乳球蛋白致敏性的乳酸菌菌株,这对于开发低致敏乳制品具有重要意义。而植物乳杆菌AHQ-14用于降解牛乳致敏蛋白的研究未见公开报道。
发明内容
本发明的目的提供一株降低牛乳β-乳球蛋白抗原性的植物乳杆菌及其应用。该菌株可以降解牛奶中的主要致敏蛋白β-乳球蛋白。
一株降低牛乳β-乳球蛋白抗原性的植物乳杆菌(Lactobacillus plantarum),所述植物乳杆菌(Lactobacillus plantarum)命名为AHQ-14,保藏编号为CCTCC NO:M2020544,保藏日期为2020年9月25日。
进一步地,所述植物乳杆菌(Lactobacillus plantarum)具有胆盐水解酶活性、对肠细胞粘附能力高、在胃肠液中存活率高等的益生性和生物胺产量在安全范围内、无溶血性、对常用抗生素敏感等的安全性。
一株降低牛乳β-乳球蛋白抗原性的植物乳杆菌的应用,所述应用为利用植物乳杆菌AHQ-14制备具有降解β-乳球蛋白,降低β-乳球蛋白抗原性的能力的发酵乳制品中的应用。
进一步地,所述发酵乳制品包括发酵乳、干酪、乳饮料或乳粉等。
进一步地,利用植物乳杆菌AHQ-14制备发酵乳制品的方法,包括如下步骤:将植物乳杆菌AHQ-14接种到发酵乳制品中,恒温培养后即得。
进一步地,植物乳杆菌AHQ-14的接种量为体积计1%~10%,优选为体积计2%接种量;培养的温度为20~50℃,优选为37℃;培养的时间为16-30h,优选为20h。
本发明的有益效果如下:植物乳杆菌AHQ-14具有胆盐水解酶活性、对肠细胞粘附能力高、在胃肠液中存活率高等益生性和生物胺产量低、无溶血性、对常用抗生素敏感等安全特性。植物乳杆菌AHQ-14应用于含β-乳球蛋白食品中,具有较强降低β- 乳球蛋白抗原性的能力。
附图说明
图1是本发明植物乳杆菌AHQ-14菌落形态图。
图2是本发明植物乳杆菌AHQ-14菌体显微镜图(X1000)。
图3是本发明植物乳杆菌AHQ-14系统发育树状图。
图4是本发明植物乳杆菌AHQ-14的胆盐水解酶活力,(a)LGG,(b)L.plantarumAHQ-14。
图5是本发明植物乳杆菌AHQ-14对HT-29细胞的粘附能力。
图6是本发明植物乳杆菌AHQ-14对模拟胃肠液的存活率。
图7是本发明植物乳杆菌AHQ-14溶血活性;(a)Listeria monocytogenes,(b)LGG,(c)L.plantarum AHQ-14。
具体实施方式
本发明涉及一种植物乳杆菌AHQ-14,该菌株已于2020年9月25日在武汉市武昌区八一路武汉大学中国典型培养物保藏中心保藏,其保藏号为CCTCC NO:M 2020544。
本发明的植物乳杆菌AHQ-14是从新疆传统发酵酸牛乳中分离获得的,具有降低牛乳中β-乳球蛋白致敏性的功能,可用于发酵乳制品中。
实施例1:植物乳杆菌AHQ-14筛选
I.菌株分离方法:制备MRS培养基:按照20g/L葡萄糖、10g/L蛋白胨、5g/L酵母浸粉、10g/L牛肉膏、5g/L乙酸钠、2g/L柠檬酸氢二胺、2g/L磷酸氢二钾、0.58g/L MgSO4·7H2O、0.25g/L MnSO4·5H2O与1mL/L Tween-80称取原料,混合均匀,溶于去离子水中,得到的溶液在温度121℃下灭菌15min,得到所述的MRS培养基;培养基中添加15g/L琼脂粉,在温度121℃下灭菌15min,得到MRS琼脂培养基。
将采集的新疆传统发酵酸牛乳样品用涡旋振荡器混匀,称取0.5g或0.5ml样品放入4.5ml浓度为0.85%的灭菌生理盐水中,对样品进行十倍梯度稀释(10-4,10-5,10-6),然后涂布在MRS琼脂培养基或者M17琼脂平板培养基上,在37℃下厌氧培养48h,挑取单菌落,观察菌落形态和革兰氏染色细胞形态特征,同时进行过氧化氢酶试验。将革兰氏染色阳性、过氧化氢酶试验阴性的菌暂定名为乳酸菌。将菌株接种于MRS液体培养基中,在37℃下培养48h,得到纯乳酸菌菌株,再进行鉴定、保存。
II.降低β-乳球蛋白抗原性能力菌株的筛选:将分离菌种其在牛乳琼脂平板培养基上培养,如果菌落周围形成透明圈,被认为这株菌是是产蛋白酶的,通过透明圈法进行初筛。利用ELISA试剂盒检测发酵β-Lg样品中β-Lg的抗原性降低情况进行复筛,获得其蛋白质水解活力与β-乳球蛋白抗原性降低率较强的本发明菌株。
实施例2:植物乳杆菌AHQ-14的形态学特征与菌种鉴定
I.菌落形态特性:本发明植物乳杆菌AHQ-14在MRS琼脂培养基上形成乳白色菌落、不透明、圆形、边缘整齐、中央凸起,直径约1.3~1.6mm,具体见附图1。
II.菌体形态:本发明植物乳杆菌AHQ-14具有如下形态学特征:细胞呈长杆状、末端钝圆,长4.0~8.0μm、宽0.9~1.1μm,呈单个或链状,具体见附图2。
III.分子生物学鉴定:采用通用引物,正向引物是27f:5′-AGAGTTTGATCCTGGCTCAG-3′;反向引物是1492r:5′-GGTTACCTTGTTACGACTT -3′,以菌体DNA为扩增模板PCR扩增16S rRNA基因区域,扩增产物纯化,再进行 16S rRNA基因序列测定,然后通过基因序列比对和系统发育关系研究进行种属鉴定。本发明菌株显示出了与植物乳杆菌最高的分子系统学上的亲缘关系,鉴定为植物乳杆菌,并命名为植物乳杆菌AHQ-14,其系统发育树状图见附图3。
所述菌株保藏于中国典型培养物保藏中心(简称CCTCC,地址:武汉市武昌珞珈山,中国科学院微生物研究所,邮编430072)。植物乳杆菌(Lactobacillus plantarum)AHQ-14的菌种保藏号为CCTCC NO:M 2020544,保藏日期为2020年9月25日,分类命名为植物乳杆菌(Lactobacillus plantarum),菌株名称为AHQ-14。
实施例3:植物乳杆菌AHQ-14的胆盐水解酶活性
将菌株在添加0.2%(w/v)巯基乙酸钠、0.3%(w/v)牛磺脱氧胆酸钠水合物和0.2%(w/v)CaCl2的MRS琼脂平板上培养。在无菌培养皿中加入灭菌的上述MRS培养基,将凝固好的培养基上均匀放置直径约为3-4mm的无菌滤纸片。吸附10μL活化菌液,缓慢加入滤纸,直到液体不再流动,在37℃厌氧条件下培养3天。菌落周围的沉淀区域显示了菌株的胆盐水解酶活性。
以MRS培养液用作阴性对照,LGG作阳性对照,进行胆盐水解酶活性的定性测定。图4中大量的脱氧胆酸沉淀并扩散到AHQ-14周围的介质中,这证明了植物乳杆菌AHQ-14中存在胆盐水解酶活性。
实施例5:植物乳杆菌AHQ-14对人肠上皮细胞的粘附能力应用人结肠癌细胞HT-29(HTB-38)模拟植物乳杆菌AHQ-14对人肠上皮细胞的粘附能力研究。在添加10%(v/v)胎牛血清的RPMI-1640培养基中培养,培养温度37℃,相对湿度为90%, CO2浓度为5%,空气浓度为95%。将HT-29细胞接种(105个细胞/孔)到12孔的组织培养板中,并用磷酸盐缓冲盐水冲洗两次。过夜生长的细胞用PBS洗涤两次,并在4℃离心2min(10,000×g)。将浓度为1×108cfu/ml的乳酸菌悬浮液(100μL)加入到每个细胞培养孔内,并在每个细胞培养孔中加入1mL RPMI1640培养基,并且特点是不含抗生素。然后在含5%CO2的环境下,于37℃孵育2h。去除细菌悬浮液和非粘附菌株,使用0.5mL 0.5%Triton X-100溶液分离粘附细菌。细胞裂解物被连续稀释并铺在琼脂平板上。使用MRS琼脂培养基的平板计数法用于检测最终活细菌细胞计数(粘附细菌),并在37℃培养48小时后进行计数。粘附试验检测一式三份,粘附特性定义为粘附百分比。
植物乳杆菌AHQ-14对HT-29细胞的粘附能力如图5所示,发现植物乳杆菌 AHQ-14具有与LGG相当的粘附能力,约为78.0%。
实施例6:植物乳杆菌AHQ-14在模拟胃肠液中的耐受能力
使用1mol/L HCl液将灭菌的磷酸缓冲盐溶液(PBS)的pH值调节至2.5,加入胃蛋白酶,使其浓度达到3.0mg/ml,然后用0.22μm微孔滤膜过滤除菌,制成人工模拟胃液。使用0.1mol/L NaOH溶液将灭菌PBS的pH值调节至8.0,加入胰蛋白酶使其浓度达到以重量计0.1%,同时加入牛胆汁使其浓度达到以重量计1.8%,混合均匀,用 0.22μm微孔滤膜过滤除菌,制成人工模拟肠液。
模拟胃肠液中耐受性研究:将LGG(对照)和植物乳杆菌AHQ-14在MRS培养基中活化培养两代,用灭菌生理盐水离心洗涤3次,然后悬浮于5ml灭菌生理盐水中制成菌悬液。将0.50ml上述菌悬液加入到4.5ml上述制备的人工模拟胃液中,在37℃下培养3h,于0、3h分别取样,采用MRS琼脂培养基倾注法测定其活菌数。然后,把0.50ml已消化3h的含菌人工模拟胃液加到4.5ml上述制备的人工模拟肠液中,继续在37℃下培养,并分别于0、8h采用MRS琼脂培养基倾注法测定活菌数。
研究结果如图6所示,植物乳杆菌AHQ-14在胃肠道消化中显示出与LGG相当的存活率。
实施例7:植物乳杆菌AHQ-14产生物胺能力
菌株在含有7种0.2%前体氨基酸(L-酪氨酸,L-组氨酸,L-精氨酸,L-鸟氨酸, L-赖氨酸,L-色氨酸和L-苯丙氨酸)和0.005%吡哆醛-5-磷酸的MRS培养基中被活化两次以诱导脱羧酶,将样品通过0.22μm过滤器过滤,然后进行生物胺的衍生,用HPLC 测定生物胺的水平。
生物胺产量测定结果如表1所示,植物乳杆菌AHQ-14产生0.07μg/mL组胺,0.031 μg/mL腐胺,产生微量的色胺、苯乙胺、尸胺和酪胺,该产量低于经常食用的食物中的含量水平,低于危害人体健康的有毒水平。
表1 植物乳杆菌AHQ-14的生物胺产量
实施例8:植物乳杆菌AHQ-14的溶血性
以单增李斯特菌(Listeria.Monocytogenes)作阳性对照。将阴性对照菌LGG及植物乳杆菌AHQ-14划线接种在Columbia琼脂培养基(含5%羊血)表面,37℃厌氧培养 48h,若菌落周围产生透明的溶血圈,则具有溶血性,若没有观察到溶血圈则不具有溶血性。
图7显示L.monocytogenes周围的透明无色区作为阳性对照。而LGG和L.plantarumAHQ-14在培养基上未产生清晰的区域,表明它们是非溶血性的。因此,初步认为植物乳杆菌AHQ-14是可用作益生菌的安全菌株。
实施例9:植物乳杆菌AHQ-14的抗生素敏感性
通过使用MRS琼脂扩散法测试植物乳杆菌AHQ-14对所选抗生素的敏感性,将植物乳杆菌AHQ-14在MRS琼脂平板上培养,将抗生素纸片放在合并的MRS琼脂培养基上,给予半小时以进行抗生素扩散,然后在37℃孵育48小时。通过测量抑制区直径大小来区分抗生素敏感性。
表2 植物乳杆菌AHQ-14对常规抗生素的敏感性
注:S为敏感,I为中度敏感,R为抵抗性。
如表2所示植物乳杆菌AHQ-14对氯霉素,红霉素和氨苄青霉素敏感,对克林霉素和四环素均表现出中等敏感性,而对万古霉素具有抗性。植物乳杆菌AHQ-14对常用的抗生素具有敏感性,表明它们应用于食品和治疗中是可行的。
实施例10:植物乳杆菌AHQ-14应用于发酵牛乳
制备浓度为12%(w/v)的复原乳:将12重量份乳粉溶解于100重量份超纯水中,在105℃下进行灭菌15min,然后冷却至37℃以下,得到所述的复原乳。
将植物乳杆菌AHQ-14按照2%接种量(以牛乳体积计)接种到上述复原乳中,在 37℃下恒温培养16-30h,连续活化两代。另一组试验分别接种2%(v/v)商品发酵剂(保加利亚乳杆菌和嗜热链球菌以体积比1:1混合物。)和植物乳杆菌AHQ-14的组合物,在恒温培养箱中于37℃培养16-30h后,进行β-乳球蛋白抗原性测定。
利用β-乳球蛋白过敏原检测试剂盒(双抗体夹心法)检测发酵乳样品中的β-乳球蛋白抗原性。发酵的复原乳样品在10,000×g和4℃下离心15分钟,取上清液备用。按照酶联免疫吸附测定试剂盒的步骤,严格检测样品中β-乳球蛋白的抗原性,使用未发酵的复原乳作为空白对照。从β-乳球蛋白的标准曲线计算抗原降低率。
结果如表3所示,植物乳杆菌AHQ-14发酵乳中β-乳球蛋白抗原性降低64.08%,商业发酵剂发酵脱脂乳中β-乳球蛋白抗原性降低70.06%,而植物乳杆菌AHQ-14与商业发酵剂复配使用后发酵乳中的抗原降低了81.20%。表明植物乳杆菌AHQ-14适于与商品发酵剂复配使用,用于发酵牛乳的生产中。
表3 发酵牛乳中β-乳球蛋白抗原性的降低率
SEQUENCE LISTING
<110> 大连工业大学
<120> 一株降低牛乳β-乳球蛋白抗原性的植物乳杆菌及其应用
<130> 2021
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1471
<212> DNA
<213> 植物乳杆菌(Lactobacillus plantarum)
<400> 1
ccgttcggat tctatacatg cagtcgaacg aactctggta ttgattggtg cttgcatcat 60
gatttacatt tgagtgagtg gcgaactggt gagtaacacg tgggaaacct gcccagaagc 120
gggggataac acctggaaac agatgctaat accgcataac aacttggacc gcatggtccg 180
agtttgaaag atggcttcgg ctatcacttt tggatggtcc cgcggcgtat tagctagatg 240
gtggggtaac ggctcaccat ggcaatgata cgtagccgac ctgagagggt aatcggccac 300
attgggactg agacacggcc caaactccta cgggaggcag cagtagggaa tcttccacaa 360
tggacgaaag tctgatggag caacgccgcg tgagtgaaga agggtttcgg ctcgtaaaac 420
tctgttgtta aagaagaaca tatctgagag taaactgttc aggtattgac ggtatttaaa 480
ccagaaagcc acggctaaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 540
tgtccggatt tattgggcgt aaagcgagcg caggcggttt ttttaagtct gatgtgaaag 600
ccttcggctc aaccgaagaa gtgcatcgga aactgggaaa cttgagtgca gaagaggaca 660
gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaag 720
gcggctgtct ggtctgtaac tgacgctgag gctcgaaagt atgggtagca aacaggatta 780
gataccctgg tagtccatac cgtaaacgat gaatgctaag tgttggaggg tttccgccct 840
tcagtgctgc agctaacgca ttaagcattc cgcctggggg agtacggccg caaggctgaa 900
actcaaagga attgacgggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960
tacgcgaaga acccttacca ggtcttgaca tactatgcaa atctaagaga ttagacgttc 1020
ccttcgggga catggataca ggtgctgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1080
gggttaagtc ccgcaacgag cgcaaccctt tattatcagt tgccagcatt aagttgggca 1140
ctctggtgag actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg 1200
ccccttatga cctgggctac acacgtgcta caatggatgg tacaacgagt tgcgaactcg 1260
cgagagtaag ctaatctctt aaagccattc tcagttcgga ttgtaggctg caactcgcct 1320
acatgaagtc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1380
gccttgtaca caccgcccgt cacaccatga gagtttgtaa cacccaaagt cggtggggta 1440
acctctagga accagccgct aagtgacagt t 1471
Claims (6)
1.一株降低牛乳β-乳球蛋白抗原性的植物乳杆菌(Lactobacillus plantarum),其特征在于:所述植物乳杆菌(Lactobacillus plantarum)命名为AHQ-14,保藏编号为CCTCCNO:M 2020544,保藏日期为2020年10月10日。
2.根据权利要求1所述的一株降低牛乳β-乳球蛋白抗原性的植物乳杆菌(Lactobacillus plantarum),其特征在于:所述植物乳杆菌(Lactobacillus plantarum)具有胆盐水解酶活性、对肠细胞粘附能力高、在胃肠液中存活率高的益生性和生物胺产量低、无溶血性、对常用抗生素敏感的安全性。
3.一株降低牛乳β-乳球蛋白抗原性的植物乳杆菌的应用,其特征在于:所述应用为利用植物乳杆菌AHQ-14制备具有降解β-乳球蛋白,降低β-乳球蛋白抗原性的能力的发酵乳制品中的应用。
4.根据权利要求3所述的应用,其特征在:所述发酵乳制品包括发酵乳、干酪、乳饮料或乳粉。
5.根据权利要求3所述的应用,其特征在于:利用植物乳杆菌AHQ-14制备发酵乳制品的方法,包括如下步骤:将植物乳杆菌AHQ-14接种到发酵乳制品中,恒温培养后即得。
6.根据权利要求5所述的应用,其特征在于:植物乳杆菌AHQ-14的接种量为体积计1%~10%,培养的温度为20~50℃,培养的时间为16-30h。
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CN112111433A (zh) * | 2020-09-30 | 2020-12-22 | 兰州大学 | 一株具有耐酸耐胆盐活性的植物乳杆菌lzu-j-qa85及其应用 |
CN112195123A (zh) * | 2020-10-12 | 2021-01-08 | 中科美大(福建)生物科技有限公司 | 一种植物乳杆菌及其制剂和应用 |
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CN112195123A (zh) * | 2020-10-12 | 2021-01-08 | 中科美大(福建)生物科技有限公司 | 一种植物乳杆菌及其制剂和应用 |
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