CN113151064B - 一种戊糖片球菌及其应用 - Google Patents
一种戊糖片球菌及其应用 Download PDFInfo
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- CN113151064B CN113151064B CN202110323420.3A CN202110323420A CN113151064B CN 113151064 B CN113151064 B CN 113151064B CN 202110323420 A CN202110323420 A CN 202110323420A CN 113151064 B CN113151064 B CN 113151064B
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- pediococcus pentosaceus
- pediococcus
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Abstract
本发明涉及微生物技术领域，具体公开了一种来源于青稞的戊糖片球菌及其应用。所述戊糖片球菌具有如SEQ ID NO.1所示的16s DNA序列。本发明的戊糖片球菌具有不利用蔗糖的性质，可以降低发酵饮品后酵期酸化的影响，提高发酵饮品的适口性；并且本发明的戊糖片球菌还具有抑制金黄色葡萄球菌、大肠埃希氏菌、痢疾志贺氏菌、小肠结肠炎耶尔森菌、肠沙门氏菌肠亚种肠炎血清型增殖的能力，显著的抑制了致病菌，同时还可以很好的调节肠道菌群。
Description
技术领域
本发明涉及微生物技术领域，尤其是涉及一种来源于青稞的戊糖片球菌及其应用。
背景技术
乳酸菌作为益生菌在世界范围内被公认为是安全(GRAS)级食品微生物。随着对益生菌的研究，戊糖片球菌作为乳酸菌，备受关注与重视。戊糖片球菌微生物制剂具有广泛功能并且在动物生产、青贮饲料加工领域中都有应用。2006 年我国农业部658号公告《饲料添加剂品种目录》中就将戊糖片球菌列入了可允许使用的微生物名录。有研究表明戊糖片球菌JWS939具有杀死李斯特菌的能力(文献1)。水产方面，Munoz-Atienza等发现戊糖片球菌对嗜水气单胞菌和鳗利斯顿氏菌均表现出较强的抑制作用(文献2)。戊糖片球菌还有具有提高动物免疫力的能力(文献3)。戊糖片球菌还是青贮饲料发酵过程中的固有菌种，可以提高秸秆品质、适口性、产奶量(文献4)，还是饲养抗生素的最具发展潜力的替代品(文献5)。食品方面，从发酵食品中分离得到的戊糖片球菌能显著抑制食源性致病菌的生长，显示出作为新型抗菌剂的良好发展前景，如戊糖片球菌L1显著抑制了福氏志贺氏菌CM-CC(B)51592、大肠杆菌CMCC44825和鼠伤寒沙门氏菌CM-CC(B)50115的生长，Gurira等筛选得到的戊糖片球菌具有抑制单胞李斯特菌ATCC7644和蜡样芽孢杆菌ATCC1178增殖的能力。中华人民共和国国家卫生和计划生育委员会2014年第6号公告指出，根据《中华人民共和国食品安全法》和《新食品原料安全性审查管理办法》有关规定，批准戊糖片球菌为食用菌种，其作为原料生产的食品符合国家法律、法规、标准规定。
不同的戊糖片球菌具有不同的性能，本身其均有的碳源及氮源的利用能力，决定了其对原料的代谢差别，同时根据其自然筛选及适应性，菌种自身会有特性的功能性，包括其抑制治病菌的种类。根据益生菌株的菌株类别、理化性能及其功能性，可以针对菌株自身具有的性能及功能进行不同领域的应用。
乳制品数十年来一直是采用掺入益生菌的方法制作而成，其中益生元或共生素是最常用的食品基质。然而目前乳糖不耐受，高胆固醇水平，对牛奶蛋白和/ 或素食主义者过敏的人数不断增加，这增加了对非乳制品的需求。此外，健康意识已经增强，使消费者寻找功能性食品，并且可以用于特殊领域的非乳制品益生菌的需求也在增加。
目前市面上，一般较适口的发酵酸奶和益生菌饮品大多都含有蔗糖作为口感调节剂，然而常用于作为酸奶发酵剂的保加利亚乳杆菌和嗜热链球菌一般会在贮藏期，即后酵期利用蔗糖继续产酸，增加酸奶的酸度，进而影响酸奶的适口性。
文献1：SHIN M S，HAN S K，JI A R，et al.Isolation and characteristics ofbacteriocin-producing bacteria from the intestine of duck for probiotics[J].Journal of animal science and technology,2007,49(5):621-632；
文献2：Munoz Atienza，GOMEZ-SALAB，ARA UJO C，et al.Antimicrobialactivity，antibiotic susceptibility and virulence factors of lactic acidbacteria of aquatic origin intended for use as probiotics in aquaculture[J].BMC microbiology, 2013,13:15；
文献3：MASUDA T，KIMURA M，OKADA S，et al.Pediococcus pentosaceus Sn26inhibits IgE production and the occurrence of ovalbumin-induced allergicdiarrhea in mice[J].Bioscience biotechnology and biochemistry,2010,74(2):329–335；
文献4：刘艳丰，唐淑珍，巴音巴特，等.微生物青贮添加剂处理后玉米青贮对奶牛生产性能和经济效益的影响[J].中国畜牧杂志，2012，48(23):76-78；
文献5：AKHTER N，WU B，MEMON A M，et al.Probiotics and prebioticsassociated with aquaculture:A review[J].Fish&shellfish immunology，2015，45(2):733–741。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种戊糖片球菌及其应用，本发明从植物青稞筛选得到的戊糖片球菌具有不利用蔗糖的性质，可以降低发酵饮品后酸化的影响，提高发酵饮品的适口性；并且本发明的戊糖片球菌还具有抑制金黄色葡萄球菌、大肠埃希氏菌、痢疾志贺氏菌、小肠结肠炎耶尔森菌、肠沙门氏菌肠亚种肠炎血清型增殖的能力，可以很好的调节肠道菌群。
本发明的第一方面，提供了一种菌株，其特征在于所述菌株具有如SEQ ID NO.1所示的16s DNA序列。所述菌株为戊糖片球菌(Pediococcus pentosaceus) QK-1，所述戊糖片球菌(Pediococcus pentosaceus)QK-1已于2021年2月1日保藏于中国典型培养物保藏中心，保藏地址：中国武汉，保藏编号为CCTCC NO:M2021175。
本发明发现一株具有不利用蔗糖性质的菌株，该菌株具有如SEQ ID NO:1 所示的16s DNA序列，该序列长度为1509bp，经过基因比对，该菌株被鉴定为片球菌属(Pediococcus)，为戊糖片球菌(Pediococcus pentosaceus)。
本发明的戊糖片球菌(Pediococcus pentosaceus)QK-1具有不利用蔗糖性质，使得可以降低发酵饮品后酸化的影响，提高酵饮品后酵期(即贮藏期)产品酸度的稳定性，进而提高发酵饮品的适口性，对于动物源发酵酸奶、植物源发酵酸奶及含有蔗糖且涉及后酸化的食品的开发具有非常好的潜力及前景。
本发明的戊糖片球菌(Pediococcus pentosaceus)QK-1来源于植物青稞，在 MRS培养基生长其形态呈圆形菌落，乳白色，光滑，凸起，边缘整齐，不透明菌株，经革兰氏染色鉴定为革兰氏阳性菌；经抗万古霉素试验、接触酶实验和精氨酸实验，戊糖片球菌(Pediococcus pentosaceus)QK-1的接触酶阴性，符合一般乳酸菌性质；精氨酸产氨结果阳性；不具有抗万古霉素能力，符合片球菌属特性。此外，本发明的戊糖片球菌(Pediococcuspentosaceus)QK-1还具有抑制金黄色葡萄球菌、大肠埃希氏菌、痢疾志贺氏菌、小肠结肠炎耶尔森菌、肠沙门氏菌肠亚种肠炎血清型增殖的能力，对于致病菌的抑制及肠道菌群的调节具有非常好的潜力及前景。
本发明的第二方面，提供了上述菌株在制备抑菌制剂中的应用。
本发明的第三方面，提供了一种抑菌制剂，所述抑菌制剂含有上述的菌株和可接受的载体。所述可接受的载体包括脱脂奶、乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、海藻糖、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁、矿物油中的一种或多种。
本发明的第四方面，提供了上述菌株在制备食品或保健品中的应用。
本发明的第五方面，提供了上述菌株在制备乳制品中的应用。
本发明的第六方面，提供了一种乳制品，所述乳制品包括上述菌株。
作为本发明所述乳制品的优选实施方式，所述乳制品包括发酵酸奶。所述发酵酸奶可以包括动物源发酵酸奶、植物源发酵酸奶及含有蔗糖且涉及后酸化的乳制品。
本发明的第七方面，提供了一种豆乳，所述豆乳包括豆粉、蔗糖、赤藓糖醇、水和上述菌株。
与现有技术相比，本发明具有以下有益效果：
本发明提供了一种戊糖片球菌(Pediococcus pentosaceus)QK-1，其具有不利用蔗糖的性质，可以降低发酵饮品后酸化的影响，提高发酵饮品后酵期(即贮藏期)产品酸度的稳定性，进而提高发酵饮品的适口性，对于动物源发酵酸奶、植物源发酵酸奶及含有蔗糖且涉及后酸化的食品的开发具有非常好的潜力及前景；并且本发明的戊糖片球菌(Pediococcus pentosaceus)QK-1还具有抑制金黄色葡萄球菌、大肠埃希氏菌、痢疾志贺氏菌、小肠结肠炎耶尔森菌、肠沙门氏菌肠亚种肠炎血清型增殖的能力。
附图说明
图1为本发明戊糖片球菌(Pediococcus pentosaceus)QK-1筛选示意图；
图2为本发明戊糖片球菌(Pediococcus pentosaceus)QK-1革兰氏染色示意图；
图3为本发明戊糖片球菌(Pediococcus pentosaceus)QK-1万古霉素实验的结果图；
图4为本发明戊糖片球菌(Pediococcus pentosaceus)QK-1精氨酸产氨实验的结果图；
图5为本发明戊糖片球菌(Pediococcus pentosaceus)QK-1糖发酵测定结果；
图6为采用戊糖片球菌(Pediococcus pentosaceus)QK-1对发酵饮品后酵期总酸变化的影响图；
图7为采用戊糖片球菌(Pediococcus pentosaceus)QK-1对发酵饮品后酵期 pH变化的影响图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
实施例1、菌株的筛选、鉴定和培养
1、本发明的菌株来源于植物青稞，通过MRS培养基针对性筛选形态圆形菌落，乳白色，光滑，凸起，边缘整齐，不透明菌株。同时考察菌株对氧需求能力，需氧及兼性厌氧。
将筛选得到的菌株进行革兰氏染色、镜检，保存分离株，并提取菌株基因组DNA用于后续测定分析。
本发明筛选得到的菌株具有如下形态学特征：圆形菌落，乳白色，光滑，凸起，边缘整齐，不透明，革兰氏阳性菌(参考图1和图2)。
2、接触酶实验：
通过接触酶实验筛选阴性菌株，即在上述菌落上滴加3％-10％过氧化氢，无气泡产生的菌株为接触酶阴性菌株，一般乳酸菌接触酶结果为阴性。
3、万古霉素实验
通过万古霉素实验筛选具有对万古霉素无抗性的菌株，使用MRS培养基对上述菌株进行1％传代活化3代获得菌液，使用无菌生理盐水对菌液重悬，然后稀释，比照0.5标准麦氏比浊管即菌浓108CFU/mL，吸取50μL均匀涂布于25g/ 皿MRS固体培养基，含万古霉素30μg/片的药敏片贴于涂布菌液的培养基表面， 37℃培养24h，筛选不具有抑菌圈的菌株(参考图3)。
4、精氨酸产氨实验
将符合上述性能的菌株进行精氨酸产氨实验筛选，挑选精氨酸实验阳性菌株。利用精氨酸产氨评价其耐酸能力，产生的氨可以保证菌株微环境呈碱性，中和酸环境。结果阳性则其耐酸能力强，可以抵抗胃部强酸环境。
使用MRS培养基对菌株进行1％传代活化3代获得菌液，使用无菌生理盐水对菌液重悬，然后稀释，比照0.5标准麦氏比浊管即菌浓108CFU/mL，吸取 50μL均匀涂布于25g/皿精氨酸肉汤固体培养基及不含有精氨酸肉汤的固体培养基，37℃培养48h，在菌苔表面滴加Nessler试剂，挑选产生橙黄色或铁锈色的菌株，为具有利用精氨酸产氨能力的菌株(参考图4)。
5、基因学鉴定
将上述筛选的菌株进行基因组DNA提取，使用细菌通用引物进行PCR反应，1％琼脂糖凝胶电泳观察目的条带，目的条带连接至Takara
18-T Vector，将连接好的质粒转入感受态细胞中，培养过夜，蓝白斑筛选目标菌落，质粒提取及测序，所有测序均由生工生物技术(上海，中国)商业化进行。16SrDNA 序列在核糖体数据库http://rdp.cme.msu.edu/index.jsp上比对，见表1所示。
表1
进行基因分析鉴定，该菌株具有16sDNA片段(如SEQ ID NO:1所示)，该序列长度为1509bp，经过基因比对，再结合镜检及形态学观察，该菌株被鉴定为片球菌属(Pediococcus)，为戊糖片球菌(Pediococcus pentosaceus)。
通过以上实验筛选获得菌株为圆形菌落，乳白色，光滑，凸起，边缘整齐，不透明，革兰氏阳性菌；接触酶阴性，符合一般乳酸菌性质；精氨酸产氨结果阳性；不具有抗万古霉素能力，符合片球菌属特性；16sDNA鉴定，保留鉴定为片球菌属戊糖片球菌的菌株。因此，将本发明筛选得到的菌株命名为戊糖片球菌(Pediococcus pentosaceus)QK-1。
本发明的戊糖片球菌(Pediococcus pentosaceus)QK-1已于2021年2月1日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2021175。
实施例2、糖发酵试验鉴定
1、将上述经过16sDNA鉴定的菌株根据GB 4789.35—2016进行糖发酵试验鉴别。明确筛选得到的戊糖片球菌利用不同糖类碳源的能力。糖生化的能力也可以作为菌株不同亚种的身份标志。其检测结果参见图5。
通过糖发酵试验测定，明确戊糖片球菌(Pediococcus pentosaceus)QK-1具有不利用蔗糖性能，可以降低发酵饮品后酸化的影响，提高酵饮品后酵期(即贮藏期)产品酸度的稳定性，进而提高发酵饮品的适口性，对于动物源发酵酸奶、植物源发酵酸奶及含有蔗糖且涉及后酸化的食品的开发具有非常好的潜力及前景。
实施例3、抑菌能力鉴定
将加有牛津杯的双层平板置于培养箱中培养，一方面试验菌(病原菌)开始生长繁殖，另一方面抑菌物质呈球面扩散，形成递减的梯度浓度。牛津杯周围抑菌浓度范围内的细菌生长被抑制，形成透明的抑菌圈，抑菌圈的大小反映抑菌物质对指示菌的抑制程度。使用2％的水琼脂打底一层，琼脂凝固后，每块平板均匀放置2个牛津杯，轻轻按压保证位置固定。
1、牛津杯致病菌培养基配制
将致病菌活化3代后使用比浊管比浊计数108CFU/mL，使用50℃LB琼脂培养基稀释100倍，混合均匀后即菌浓为106CFU/mL，倒入平板中作为第二层，凝固后轻轻取出牛津杯。
2、益生菌发酵液处理
将活化2代的戊糖片球菌(Pediococcus pentosaceus)QK-1以1％体积量接种于MRS培养基，37℃静置培养24-48h。戊糖片球菌(Pediococcus pentosaceus) QK-1在MRS培养基中可产生大量的乳酸，乳酸具有抑菌能力，为了排除乳酸的抑菌性，本实验需要设立对照组。将接种戊糖片球菌(Pediococcus pentosaceus) QK-1发酵好的发酵液4000rpm离心20min，取上清液，测定上清液的pH值及总酸。根据每种菌的pH及总酸值在纯水中添加85％乳酸，调整其pH及总酸一致，记为该菌的对照组。
将测定好的实验组发酵液与对照组发酵液使用0.22μM滤器过滤于无菌 10mL离心管中。
3、发酵液接种
分别吸取150μL菌株发酵上清滤液、对照滤液于牛津杯孔中，4℃扩散5h 后，37℃培养12-16h，观察并测定抑菌圈大小。说明戊糖片球菌(Pediococcus pentosaceus)QK-1对该致病菌具有抑菌作用，见表2。
表2戊糖片球菌(Pediococcus pentosaceus)QK-1抑制致病菌抑菌测定
经体外抑制条件致病菌实验，明确戊糖片球菌(Pediococcus pentosaceus) QK-1具有抑制金黄色葡萄球菌、大肠埃希氏菌、痢疾志贺氏菌、小肠结肠炎耶尔森菌、肠沙门氏菌肠亚种肠炎血清型增殖能力。因此，对于致病菌的抑制及肠道菌群的调节具有非常好的潜力及前景。
实施例4、发酵饮品后酵期产酸定能测定
1、植物基豆乳的制作及一级发酵：
植物基豆乳包括以下质量百分比的原料：豆粉14.5％、蔗糖4.5％、赤藓糖醇2.5％和水78.5％。
水加热至沸腾后，倒入豆粉、蔗糖、赤藓糖等固体原料中搅匀，搅拌温度为95℃，搅拌时间为20min，然后冷却至40℃，植物基豆乳中加入科汉森商品发酵剂，搅拌均匀，37℃发酵14-16h。
2、戊糖片球菌(Pediococcus pentosaceus)QK-1的接种与二级发酵
一级发酵即前酵结束后，分别加入戊糖片球菌(Pediococcus pentosaceus) QK-1和商品菌株戊糖片球菌CICC24444，保证接种于发酵豆浆中的菌浓为 106～107CFU/mL，4℃后酵，后酵期21-28天，分别测定pH及总酸。结果见图 6和图7。
结果表明本发明的戊糖片球菌(Pediococcus pentosaceus)QK-1具有稳定发酵饮品后酵期(即贮藏期)产品酸度的性能，可以提高发酵饮品的适口性。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 中国科学院合肥物质科学研究院、恰恰食品股份有限公司
<120> 一种戊糖片球菌及其应用
<130> 2021.02.07
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1509
<212> DNA
<213> 人工合成
<400> 1
tcaggatgaa cgctggcggc gtgcctaata catgcaagtc gaacgaactt ccgttaattg 60
attatgacgt acttgtactg attgagattt taacacgaag tgagtggcga acgggtgagt 120
aacacgtggg taacctgccc agaagtaggg gataacacct ggaaacagat gctaataccg 180
tataacagag aaaaccgcat ggttttcttt taaaagatgg ctctgctatc acttctggat 240
ggacccgcgg cgtattagct agttggtgag gtaaaggctc accaaggcag tgatacgtag 300
ccgacctgag agggtaatcg gccacattgg gactgagaca cggcccagac tcctacggga 360
ggcagcagta gggaatcttc cacaatggac gcaagtctga tggagcaacg ccgcgtgagt 420
gaagaagggt ttcggctcgt aaagctctgt tgttaaagaa gaacgtgggt aagagtaact 480
gtttacccag tgacggtatt taaccagaaa gccacggcta actacgtgcc agcagccgcg 540
gtaatacgta ggtggcaagc gttatccgga tttattgggc gtaaagcgag cgcaggcggt 600
cttttaagtc taatgtgaaa gccttcggct caaccgaaga agtgcattgg aaactgggag 660
acttgagtgc agaagaggac agtggaactc catgtgtagc ggtgaaatgc gtagatatat 720
ggaagaacac cagtggcgaa ggcggctgtc tggtctgcaa ctgacgctga ggctcgaaag 780
catgggtagc gaacaggatt agataccctg gtagtccatg ccgtaaacga tgattactaa 840
gtgttggagg gtttccgccc ttcagtgctg cagctaacgc attaagtaat ccgcctgggg 900
agtacgaccg caaggttgaa actcaaaaga attgacgggg gcccgcacaa gcggtggagc 960
atgtggttta attcgaagct acgcgaagaa ccttaccagg tcttgacatc ttctgacagt 1020
ctaagagatt agaggttccc ttcggggaca gaatgacagg tggtgcatgg ttgtcgtcag 1080
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat tactagttgc 1140
cagcattaag ttgggcactc tagtgagact gccggtgaca aaccggagga aggtggggac 1200
gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggatggtac 1260
aacgagtcgc gagaccgcga ggttaagcta atctcttaaa accattctca gttcggactg 1320
taggctgcaa ctcgcctaca cgaagtcgga atcgctagta atcgcggatc agcatgccgc 1380
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgagag tttgtaacac 1440
ccaaagccgg tggggtaacc ttttaggagc tagccgtcta aggtgggaca gatgattagg 1500
gtgaagtcg 1509
Claims (7)
1.戊糖片球菌(Pediococcus pentosaceus)QK-1在制备抑菌制剂中的应用，其特征在于，戊糖片球菌(Pediococcus pentosaceus)QK-1已于2021年2月1日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2021175，戊糖片球菌(Pediococcus pentosaceus)QK-1的16s DNA序列如SEQ ID NO.1所示。
2.一种抑菌制剂，其特征在于，所述抑菌制剂含有戊糖片球菌(Pediococcus pentosaceus)QK-1和可接受的载体；所述戊糖片球菌(Pediococcus pentosaceus)QK-1已于2021年2月1日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2021175，戊糖片球菌(Pediococcus pentosaceus)QK-1的16s DNA序列如SEQ ID NO.1所示。
3.戊糖片球菌(Pediococcus pentosaceus)QK-1在制备食品或保健品中的应用，其特征在于，所述戊糖片球菌(Pediococcus pentosaceus)QK-1已于2021年2月1日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2021175，戊糖片球菌(Pediococcus pentosaceus)QK-1的16s DNA序列如SEQ ID NO.1所示。
4.戊糖片球菌(Pediococcus pentosaceus)QK-1在制备乳制品中的应用，其特征在于，所述戊糖片球菌(Pediococcus pentosaceus)QK-1已于2021年2月1日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2021175，戊糖片球菌(Pediococcus pentosaceus)QK-1的16s DNA序列如SEQ ID NO.1所示。
5.一种乳制品，其特征在于，所述乳制品包括戊糖片球菌(Pediococcus pentosaceus)QK-1，所述戊糖片球菌(Pediococcus pentosaceus)QK-1已于2021年2月1日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2021175，戊糖片球菌(Pediococcus pentosaceus)QK-1的16s DNA序列如SEQ ID NO.1所示。
6.如权利要求5所述的乳制品，其特征在于，所述乳制品包括发酵酸奶。
7.一种豆乳，其特征在于，所述豆乳包括豆粉、蔗糖、赤藓糖醇、水和戊糖片球菌(Pediococcus pentosaceus)QK-1，所述戊糖片球菌(Pediococcus pentosaceus)QK-1已于2021年2月1日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2021175，戊糖片球菌(Pediococcus pentosaceus)QK-1的16s DNA序列如SEQ ID NO.1所示。
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