CN115124603B - 一种细菌素rsq01及其应用 - Google Patents
一种细菌素rsq01及其应用 Download PDFInfo
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- CN115124603B CN115124603B CN202210747899.8A CN202210747899A CN115124603B CN 115124603 B CN115124603 B CN 115124603B CN 202210747899 A CN202210747899 A CN 202210747899A CN 115124603 B CN115124603 B CN 115124603B
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- rsq01
- bacteriocin
- bacteriocins
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- salmonella
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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Abstract
本发明公开了一种细菌素RSQ01，其氨基酸序列如SEQIDNO:1所示，本发明细菌素RSQ01结构简单，安全低毒，容易制备，抑菌谱广，具有耐高温、耐酸碱等优点，本发明细菌素RSQ01可用于抗菌药物、饲料、食品等领域，具有广阔的应用前景。
Description
技术领域
本发明属于生物技术领域，具体涉及一种细菌素RSQ01及其在制备抑菌剂中的应用。
背景技术
细菌素是乳酸菌（LAB）产生的一类由核糖体合成的多肽或蛋白质，具有抗菌活性（Guo等, 2020）。细菌素具有许多优势，它们的效率高，诱导抗性或非特异性毒性的概率低，而且它们不形成残留。据报道乳酸菌中的几种细菌素对致病菌具有明显抗菌作用（Barbosa等，2015;Jamaluddin等，2017）。例如，plantaricin LPL-1和PA-1可以抑制单核增生李斯特菌（Rodríguez等，2002；Wang等，2019）以及从棒状乳杆菌中提取的细菌素对大肠杆菌和金黄色葡萄球菌有明显的抑制作用（Yi等,2016）。大多数细菌素具有广谱抗菌性能，具有广泛的耐酸、耐碱和耐高温性能（Qin等,2019）。与传统抗生素通过抑制生物合成途径的次生代谢物来杀死细菌不同，大多数细菌素是通过破坏细胞结构和诱导细胞内容物泄漏来杀死细菌（Ovchinnikov等,2017）。这说明细菌素的作用方式能有效抑制或杀灭多重耐药（MDR）细菌。此外，细菌素具有优异的热稳定性和贮存稳定性，与传统抗生素相比应用范围广泛与化学合成的传统食品防腐剂相比，天然高效的细菌素对人类宿主安全无毒（L´opez-Cuellar等,2016）。在食品、生物防治、医疗等领域显示出良好的发展和应用潜力（Aslam等,2020;Qin等,2019）。LAB生产的细菌素因其公认安全（GRAS）而在生物保藏中具有很大的潜力，但目前只有pediocin PA-1和乳链菌肽（nisin）已作为食品防腐剂进行商业化（DelvesBroughton等,2005;Simha等,2012），其余仅限于实验研究。
不同细菌素抑菌效率和抑菌特性各有不同，且同一细菌素在不同条件下抑菌活性有所不同，不同的外部环境也会影响细菌素的结构、性质和抑菌效果。因此，寻找新型细菌素并探究其抑菌稳定性，对其后续研究及应用具有极其重要的意义。
发明内容
本发明提供了一种细菌素RSQ01，其是从来源于雷氏黄萤肠道的乳酸乳球菌（Lactococcus lactis）中分离获得的，其氨基酸序列如SEQIDNO:1所示。
本发明另一目的是将上述细菌素RSQ01应用在制备抑菌剂中，抑制菌种包括金黄色葡萄球菌（Staphylococcus aureus）、单核增生李斯特菌（Listeria monocytogenes）、肠炎沙门氏菌（Salmonella enteritidis）、猪霍乱沙门氏菌（Salmonella cholerae suis）、大肠杆菌（Escherichia coli）、坂崎肠杆菌（Enterobacter sakazakii）、铜绿假单胞菌（Pseudomonas aeruginosa）。
本发明目的通过以上技术方案实现：
1、从雷氏黄萤肠道分离的产生细菌素乳酸乳球菌；
2、采用MRS培养基培养乳酸乳球菌，离心取上清液，上清液滤膜过滤后，使用ÄKTA纯蛋白分离和纯化平台（GE, Sweden, USA）分离纯化获得小分子肽RSQ01；
3、小分子肽RSQ01分子量、氨基酸序列的确定；
4、细菌素RSQ01的抑菌活性检测、稳定性检测。
本发明具有如下有益效果：
本发明所述的细菌素RSQ01结构简单，安全低毒，容易制备，抑菌谱广，耐高温、耐酸碱等优点。本发明的细菌素RSQ01可用于抗菌药物、饲料、食品等领域，具有广阔的应用前景。
附图说明
图1为小分子肽RSQ01的ÄKTA纯化过程检测谱图；
图2为小分子肽RSQ01对肠炎沙门氏菌形态的影响结果图，其中A图为对照组，B为处理组；
图3为小分子肽RSQ01的一级质谱图；
图4为小分子肽RSQ01的二级质谱图；
图5为细菌素RSQ01对不同温度的耐受实验结果；
图6为细菌素RSQ01对不同pH的耐受实验结果；
图7为细菌素RSQ01对不同酶的耐受实验结果。
具体实施方式
下面通过实施例对本发明的技术方案作进一步详细说明，但本发明的内容并不局限于此，本实施例中方法如无特殊说明均为常规方法，所用材料、试剂等如无特殊说明均从商业途径所得到；下述实施例中所用方法如无特别说明均为常规方法，所述百分比浓度如无特别说明均为质量/体积（w/v）百分比浓度；下述实施例中的低温离心，如无注明温度，均在4℃条件下离心;
雷氏黄萤来自于中国云南省昆明理工大学生命科学学院饲养的，乳酸乳球菌采用MRS培养基37℃培养，抑菌实验中所用菌种的培养基见表1中，在37℃下培养。将所有的细菌在-80℃下储存在含有20%甘油（v/v）的培养液中。
实施例1：产生细菌素的乳酸乳球菌分离
（1）将雷氏黄萤肠道与无菌0.85% NaCl溶液（100mL）混合，然后使用无菌0.85%NaCl溶液将混合液梯度稀释至10-7，取10-1、10-3、10-5、10-7 4个梯度菌液各100μL，分别涂布到MRS培养基上，每个浓度设置3个重复，用平板涂布法涂抹均匀，然后将MRS固体培养基置于37℃中培养24h；
（2）挑取上述MRS固体培养基中的单菌落分别接种于5mL MRS液体培养基中，37℃下恒温摇床培养24h；
（3）将不同单菌落的MRS液体培养基在4℃下离心10min（8000g），取上清液，然后使用孔径为0.22μm过滤器过滤获得不含细菌菌体的无细胞上清液；取200μL上清液加入灭菌后的牛津杯（内径6mm），然后置于分别含肠炎沙门氏菌、金黄色葡萄球菌的双层平板上，37℃恒温培养箱培养24h，通过观察抑菌圈大小来判定抑菌活性，抑菌圈直径通过游标卡尺来测量；
（4）挑选抑菌圈最大的一株菌，利用细菌16S rDNA序列测序的方法对细菌进行种属鉴定，并结合形态学特征，确定该菌株为乳酸乳球菌；
（5）此外，再步骤（3）方法进一步测定乳酸乳球菌液体培养基无细胞上清液对大肠杆菌、单核增生李斯特菌、猪霍乱沙门氏菌的抑菌活性，结果显示该菌株对这3个病原菌也具有抑菌活性，且对大肠杆菌的抑菌圈为18.56±0.13mm，因此获得具有广抗菌谱和高抗菌活性的菌株乳酸乳球菌。
实施例2：小分子肽RSQ01的分离纯化和鉴定
1、实施例1分离到的乳酸乳球菌置于100mL MRS液体培养基中培养至OD595 = 0.3~0.6，然后以0.5%的接种量接种到1L MRS液体培养基中，在37℃下培养24h；
2、在4℃下8000 g离心10 min除去细菌细胞，取上清液；
3、使用孔径为0.22μm的滤膜过滤步骤（2）中的上清液，得到无细胞的上清液；
4、使用ÄKTA纯蛋白分离和纯化平台（GE, Sweden, USA）分离纯化获得小分子肽RSQ01
用20mmol/L磷酸盐缓冲液（pH5.2）平衡Superdex™30 Increase 10/300 GL色谱柱后，将无细胞上清液加载到柱内，采用以下运行参数：平衡体积为2柱体积（CV），洗脱液pH为5.3咪唑，洗脱体积为1.5CV，流速为0.5mL/min；UV 280 nm下监测洗脱液的吸光度值变化，收集A1、A2、A3和A4共4个主要峰下的洗脱液各0.5mL，收集于15mL管中，采用牛津杯双层平板法测定A1、A2、A3和A4峰收集物其对肠炎沙门氏菌的抑菌活性，抑菌圈结果显示，A1峰收集物的抑菌圈大小为20.15±0.42 mm，有抑菌活性（图1A）；
再次使用ÄKTA纯蛋白分离和纯化平台对A1峰收集物进行纯化，设定参数同上；对A1峰的再次纯化得到B1单峰（图1B），使用牛津杯双层平板法测定B1峰收集物对肠炎沙门氏菌的抑菌活性，抑菌圈大小为24.30±0.23 mm；结果证实其为单一物质，且具有良好的抑菌活性，将其命名为RSQ01；
5、通过二辛可宁酸（BCA）试剂盒（bisharp,Beijing, China），操作过程参照试剂盒说明书，测定RSQ01的浓度，然后通过FD-2冻干机中冷冻干燥并保存；
6、小分子肽RSQ01对肠炎沙门氏菌形态的影响
利用扫描电镜观察被小分子肽RSQ01处理后肠炎沙门氏菌的细胞膜完整性及其形态学特征，以评估指示菌浮游细胞在RSQ01处理后的影响程度
于试管中加入2mL(107 CFU/mL)的肠炎沙门氏菌悬浮液，4℃、8000g离心5min后，取EP管中沉淀的菌体，用PBS缓冲液进行重悬，重复三次；随后，添加小分子肽RSQ01（11.63μg/mL），并在37℃下孵育60min；之后加入2mL 2.5%戊二醛，在4℃下固定12h；用PBS缓冲液清洗后在乙醇浓度梯度（30%、50%、60%、70%、80%、90%、100%）下进行脱水，随后放入冷冻干燥机过夜脱水，将脱水后的细菌细胞粘附于抛光硅片上，干燥后喷金；在FlexSEM1000扫描电子显微镜下检测；结果见图2，由图可见小分子肽RSQ01能够破坏肠炎沙门氏菌的细胞膜，改变细菌的细胞形态，从而达到抑菌效果。
6、小分子肽RSQ01的分子量
小分子肽RSQ01的分子量及氨基酸序列采用纳米液相色谱结合串联质谱分析法（nanoLC-MS/MS）测定
将纯化后的小分子肽RSQ01溶解在于ddH2O中，用10mmol/L二硫苏醇糖在26℃下还原1h，然后在室温下用20mmol/L碘化乙酰胺胺基化40min后，冻干，在色谱分析前重悬于20μL的0.1%甲酸中，肽段分析是在Ultimate 3000***中进行的，该***配有QExactiveTM混合四级杆-OrbitrapTM质谱仪，并配备ESI纳米喷雾源；使用了内置150μm×15cm的反相纳米色谱柱，该色谱柱用ReproSil-PurC18-AQ 1.9 μm的树脂（100Å）填充；流动相A由含有0.1%甲酸的超纯水组成，流动相B由含有0.1%甲酸的乙腈组成。乙腈的线性梯度洗脱如下：从6%到9%的流动相B使用5min；从9%到14%的流动相B使用15min；从14%到30%的流动相B使用30min；从30%到40%的流动相B使用8min，从40%到95%的流动相B使用2min，流速均为0.6μL/min。将5μL的样品装入***；质谱分析采用单次全扫描（MS），参数如下：范围100-1,500 m/z，400m/z的分辨率为70,000；然后进行10次数据相关扫描（MS/MS）。使用Orbittrap中的Xcalibur2.1.2软件（使用2.2 kV的喷雾电压和270 ℃的毛细管温度）进行了质谱测量的数据相关扫描；蛋白质鉴定使用PeaksStudio 8.5 （BioinfirmaticsSolutionsInc., Waterloo, ONT,Canada）进行，其一级质谱见图3，二级质谱见图4。
结果显示，MALDI-TOF-MS显示小分子肽RSQ01的分子量为1901.87Da，根据完整的基因组序列和分子量分析，小分子肽RSQ01的氨基酸序列如SEQ ID NO:1 所示。
使用针对GenBank的蛋白组BLAST（www.ncbi.nlm.nih.gov/BLAST），细菌素RSQ01与已报道的细菌素没有表现出同源性。此外，其他成熟的Ⅱ类细菌素的序列比对结果显示细菌素RSQ01是新的细菌素。因此，来自乳酸乳球菌的细菌素RSQ01是一种新的Ⅱa类细菌素。
实验例3、细菌素RSQ01的功能鉴定
1、细菌素RSQ01的抗菌谱
将实施例2中纯化的细菌素RSQ01用于测定抗菌谱，测试了针对含有食品腐败细菌和食源性病原体的指示菌株的抗菌谱，结果见表1：
表1
；
表中：b抗菌圈直径（mm）：+++：>20mm； ++：11-20mm；+：5-10mm。
2、细菌素RSQ01的稳定性
为了评估细菌素RSQ01的稳定性及蛋白性质，检测了其热稳定性、酸碱耐受性、储存稳定性以及酶敏感性。
细菌素RSQ01的热稳定性评估是将细菌素冻干粉溶于PBS缓冲液（pH7.4）中至终浓度为11.63μg/mL，分别在60℃、80℃和100℃下处理30min，同时以室温（25℃）下的细菌素作为对照；使用牛津杯双层平板法检测不同温度处理后细菌素对金黄色葡萄球菌的抑菌活性，结果见图5；
将细菌素冻干粉溶于不同pH值（2、4、6、8、10）的PBS缓冲液中，并在37培养1h，以细菌素原始pH为4.5作对照，使用牛津杯双层平板法检测不同温度处理后细菌素对金黄色葡萄球菌的抑菌活性，结果见图6；
细菌素（11.63μg/mL）对各种酶的敏感性检测，在细菌素溶液中添加酶至终浓度为1mg/mL，在37℃下孵育2h，然后在80℃下灭活10 min，未添加酶的细菌素溶液作为阳性对照，酶分别为胰蛋白酶、α-淀粉酶、β-淀粉酶、蛋白酶K、胃蛋白酶、脂肪酶、过氧化氢酶；使用牛津杯双层平板法检测不同温度处理后细菌素对金黄色葡萄球菌的抑菌活性，结果见图7。
上述结果表明细菌素RSQ01对于高温、酸碱、酶都表现出良好的耐受性。
序列表
<110> 昆明理工大学
<120> 一种细菌素RSQ01及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> PRT
<213> 乳酸乳球菌( Lactococcus lactis)
<400> 1
Gly Thr Pro Ser Gly Gly Ala Asp Gly Ile Gly Phe Ala Phe His Pro
1 5 10 15
Glu Glu Val Gly
20
Claims (2)
1.一种细菌素RSQ01，其氨基酸序列如SEQIDNO:1所示。
2.权利要求1所述的细菌素RSQ01在制备抑菌剂中的应用；
所述抑菌剂抑制金黄色葡萄球菌（Staphylococcus aureus）、单核增生李斯特菌（Listeria monocytogenes）、肠炎沙门氏菌（Salmonella enteritidis）、猪霍乱沙门氏菌（Salmonella choleraesuis）、大肠杆菌（Escherichia coli）、坂崎肠杆菌（Enterobacter sakazakii）、铜绿假单胞菌（Pseudomonas aeruginosa）。
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| CN106591174A (zh) * | 2016-11-10 | 2017-04-26 | 江南大学 | 一株产细菌素的弯曲乳杆菌及其应用 |
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