CN112094323B - 一种植物乳杆菌源广谱抗菌肽及其应用 - Google Patents
一种植物乳杆菌源广谱抗菌肽及其应用 Download PDFInfo
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- CN112094323B CN112094323B CN202011050636.9A CN202011050636A CN112094323B CN 112094323 B CN112094323 B CN 112094323B CN 202011050636 A CN202011050636 A CN 202011050636A CN 112094323 B CN112094323 B CN 112094323B
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- lactobacillus plantarum
- antibacterial peptide
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- plantaricin
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Abstract
本发明提供一种植物乳杆菌源广谱抗菌肽及其在肉品保鲜中的应用，属于食品微生物技术领域。本发明的植物乳杆菌FZU 122，其保藏编号为：CGMCC NO.20601。该菌株产生的抗菌肽对食品中多种致病、腐败菌具有显著的抑制作用。FZU122产生的抗菌肽Plantaricin‑FZU122的氨基酸序列为：ARLGLPVHVV。Plantaricin‑FZU122应用于肉品保鲜中，能够显著抑制肉品中微生物的生长与繁殖。此外，当Plantaricin‑FZU122与Nisin联用时对肉品在冷藏过程中展示出协同抑菌效果，可有效延缓因微生物生长繁殖导致的腐败变质。
Description
技术领域
本发明属于食品微生物领域，具体涉及一种植物乳杆菌源广谱抗菌肽及其应用。
背景技术
猪肉、牛肉、羊肉、禽肉等肉品因营养素含量丰富，能够为许多天然微生物的生长繁殖提供良好的营养条件。因此，此类食品在储藏过程中极易因微生物的滋生与代谢活动而发生腐败变质，出现变色、变味、表面发粘等不良变化。这些优势菌群主要包括革兰氏阳性（G+）的葡萄球菌属、微球菌属、芽孢杆菌属与革兰氏阴性（G–）的假单胞菌属、肠杆菌科等。
植物乳杆菌、嗜酸乳杆菌、干酪乳杆菌、棒状乳杆菌、德氏乳杆菌等多种乳酸菌可产生胞外抗菌肽，对常见食品源致病、腐败菌具有良好的抑菌效果。目前，国内外关于Plantaricin、Lactococcin、Acidocin、Lactocin等乳酸菌源抗菌肽的分离纯化、结构鉴定、抑菌活性等研究已有大量报道。乳酸菌抗菌肽具有来源广、抗菌活性强、安全性高等特点，是一类具有广阔开发前景的绿色生物源食品抗菌剂，可应用于猪肉、牛肉、羊肉、禽肉等肉品的防腐保鲜。
研究发现，大部分乳酸菌抗菌肽对G+菌具有抑菌活性，仅少数抗菌肽如Plantaricin MG、Lactocin XN8-A等可抑制G–菌。不同乳酸菌源抗菌肽的抑菌作用靶点和主要抗菌途径存在差异，对于G+菌主要通过破坏菌体细胞壁膜，使核酸、蛋白质、离子等细胞内容物泄漏或阻碍细胞壁合成等，导致细胞死亡。如Lacticin Q可快速结合到菌体细胞膜外侧，并由细胞膜外侧向内侧迁移形成类似脂质触发器结构，在细胞膜上形成巨大环孔，导致胞内蛋白质泄露。Lactococcin A等Ⅱa型抗菌肽可以与甘露糖憐酸转移酶系统结合，在细胞膜上形成孔洞，造成离子泄漏，最终导致细胞死亡。Nisin通过与肽聚糖合成过程中的重要中间物脂质Ⅱ结合从而抑制细胞壁主要组分肽聚糖的生物合成；此外，Nisin与脂质Ⅱ靶向结合，并在细胞上膜形成孔洞，导致菌体溶解死亡。也有少数乳酸菌抗菌肽以菌体DNA、生物被膜等为作用靶点，发挥抗菌作用。如来源于干酪乳杆菌的抗菌肽F1可通过破坏菌体细胞膜并与DNA结合两种途径作用于金黄色葡萄球菌，导致菌体快速死亡。LactocinAL705可作为群体感应抑制剂阻碍单增李斯特菌形成生物被膜从而发挥抑菌作用。目前，关于乳酸菌抗菌肽对G–菌抗菌作用途径研究的报道较少。研究表明，此类抗菌肽主要通过破坏菌体细胞膜或干扰外膜正常生理功能等对G–菌发挥抑菌作用。如Lactocin XN8-A主要以菌体的细胞壁膜为作用靶点，形成壁膜穿孔，对大肠杆菌发挥抑菌作用。Plantaricin MG可消除鼠伤寒沙门氏菌细胞膜上的跨膜电位（ΔΨ）和pH梯度（ΔpH），并形成离子通道导致K+、磷酸盐、ATP以及紫外吸收物质等泄漏。当Nisin与EDTA联用时，EDTA螯合细胞外膜脂多糖中的Mg2+使脂多糖流失并导致细胞膜通透性增大，从而赋予Nisin对沙门氏菌等G–菌的抑菌作用。
当前，只有乳酸链球菌素（Nisin）和Pediocin PA-1两种乳酸菌源抗菌肽被批准可用作食品防腐剂，但在我国仅Nisin被允许可应用于食品。然而Nisin只对G+菌具有抑菌活性，对G–菌则无抑菌效果，限制了其在以G–菌为优势腐菌的食品保鲜中的应用。当前，国家对食品安全高度重视，因化学合成类防腐剂存在食品安全风险，开发新型、绿色、安全、广谱的乳酸菌源抗菌肽，用于肉品防腐保鲜，抑制微生物滋生与食源性致病菌污染具有重要意义。
发明内容
本发明的目的在于针对现有技术的不足，提供一种植物乳杆菌源广谱抗菌肽及其在肉品防腐保鲜中的应用。
为实现上述目的，本发明采用如下技术方案：
一种产广谱抗菌肽的植物乳杆菌，所述产广谱抗菌肽植物乳杆菌为植物乳杆菌（Lactobacillus plantarum）FZU122，其分类命名为：植物乳杆菌（Lactobacillus plantarum），于2020年9月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为：CGMCC NO.20601，保藏地址为：北京市朝阳区北辰西路1号院3号。
上述植物乳杆菌（Lactobacillus plantarum）FZU122是从吉林省榆树市农村家庭自制酸白菜中分离获得的。
植物乳杆菌（Lactobacillus plantarum）FZU122革兰氏染色为阳性，菌落呈圆形，微白色，小而凸起，边缘整齐。
植物乳杆菌（Lactobacillus plantarum）FZU122产生的抗菌物质具有广谱的抑菌活性，对金黄色葡萄球菌ATCC 29213，金黄色葡萄球菌ATCC 12600，耐甲氧西林金黄色葡萄球菌ATCC 43300，大肠埃希氏菌ATCC 25922，大肠埃希氏菌ATCC 8739，大肠埃希氏菌8099，猪霍乱沙门氏菌CICC 13312，鼠伤寒沙门氏菌CMCC 50115，肠炎沙门氏菌CICC 21527，阪崎克罗诺肠杆菌ATCC 29544，猪霍乱沙门氏菌 ATCC 13312，10株革兰氏阴性菌，均表现出良好的抑菌效果。
上述产广谱抗菌肽即植物乳杆菌（Lactobacillus plantarum）FZU122产生的抗菌肽Plantaricin-FZU122，所述抗菌肽Plantaricin-FZU122的氨基酸序列为：ARLGLPVHVV。
上述抗菌肽Plantaricin-FZU122在猪肉食品保鲜中的应用。
本发明的优点在于：本发明筛选获得一种产广谱抗菌肽的植物乳杆菌FZU122，对多种食品中常见革兰氏阳性菌和阴性腐败致病菌具有显著的抑制作用。植物乳杆菌FZU122产生的抗菌肽Plantaricin-FZU122的氨基酸序列为ARLGLPVHVV。植物乳杆菌FZU122产生的抗菌肽Plantaricin-FZU122应用于猪肉食品保鲜中，能够有效抑制微生物的生长繁殖与代谢活动，其作用效果显著优于现有技术中用于食品防腐保鲜的乳酸菌源抗菌肽乳酸链球菌素（Nisin）。并且，Plantaricin-FZU122与Nisin联合应用时可对肉品在冷藏过程中展示出协同抑菌功效。
附图说明
图1植物乳杆菌FZU122的乳酸菌的生化检测反应结果，注：1七叶苷，2纤维二糖，3麦芽糖，4甘露醇，5水杨苷，6山梨醇，7蔗糖。
图2 PCR扩增产物凝胶电泳图。从左到右依次是FZU122、Marker。
图3基于16S rDNA基因序列构建FZU122菌株的系统发育树。
图4 植物乳杆菌FZU122发酵液的抑菌效果。
图5样品的Sephadex LH-20凝胶层析图谱。
图6植物乳杆菌FZU122发酵液粗提物反相HPLC层析不同分离组分的抑菌活性。图中：流动相（20%甲醇）；1、2、3、4为图7中洗脱峰1、2、3、4的收集产物，及不同分离组分的抑菌活性。
图7抗菌肽Plantaricin-FZU122中氨基酸序列的二级质谱图。
图8 抗菌肽Plantaricin-FZU122在猪肉冷藏保鲜中微生物生长繁殖的抑制作用。
具体实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是下述的实例仅仅是本发明其中的例子而已，并不代表本发明所限定的权利保护范围，本发明的权利保护范围以权利要求书为准。
实施例1
本发明中的棒状乳杆菌FZU122是从吉林省榆树市农村家庭自制酸白菜中分离获得的。
具体分离方法为：取10 g泡菜经无菌破碎，使用无菌生理盐水重悬，10倍梯度稀释后，分别涂布于MRS固体培养基平板，37 ℃条件下静置培养18 h；挑取单菌落置于MRS液态培养基内，37 ℃条件下静置培养24 h，离心取上清，在排出有机酸、过氧化氢等物质干扰的情况下，分别以金黄色葡萄球菌和鼠伤寒沙门氏菌为指示菌通过固体琼脂打孔抑菌法，筛选出产抑菌物质的菌株。
筛选所得产抑菌物质的菌株革兰氏染色为阳性，菌落呈圆形，微白色，小而凸起，边缘整齐，这些特点与典型乳酸菌菌落特征相同。从中挑选出抑菌圈直径在10 mm以上的1株菌将其命名为FZU122，通过GB4789.35-2016鉴定这株菌，将菌株培养液分别涂布于MRS琼脂平板上，置于36 ℃恒温培养箱中静置培养48 h，用灼烧灭菌后的接种针从平板上挑取单一菌落分别接种于七叶苷（用无菌液体石蜡覆盖液面）、纤维二糖、麦芽糖、甘露醇、水杨苷、山梨醇、蔗糖的生化管中，用酒精消毒后的封口膜封口，放入36 ℃恒温培养箱中，静置培养24 h后观察各生化管中颜色变化。鉴定结果见图1和表1，初步判定为乳酸菌，并命名为FZU122。
表1植物乳杆菌FZU122的生化检测结果
注：+表示菌株呈阳性
菌株FZU122接种于MRS液体培养基，37℃条件下经活化、转接培养至对数生长期。取1~2 mL培养液10000 g/min离心1 min，弃上清，菌体细胞使用细菌总DNA提取试剂盒，参照说明书提取利用总DNA。采用16S rRNA基因引物[F（5’- AGTTTGATCMTGGCTCAG-3’），R（5’-GGTTACCTTGTTACGACTT-3’）]进行PCR扩增，取5μL PCR产物进行1%琼脂糖凝胶电泳。
用试剂盒纯化回收PCR产物，由生工生物工程（上海）股份有限公司进行16S rRNA全序列测序。菌株FZU122的16S rDNA序列共1530bp，测序结果如SEQ ID NO.1所示。
AGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCG。
菌株FZU 122在MRS固体培养基上菌落呈边缘整齐的圆形、微白色、小而凸起；菌体细胞经革兰氏染色后，在显微镜下观察呈蓝紫色、长杆状、无芽孢，判断为革兰氏阳性杆菌。如表1所示，菌株FZU 122在所测试碳的水化合物中经发酵后均呈阳性。综上结果，可初步判定菌株FZU 122属于乳杆菌属。菌株FZU 122总DNA经16 S rDNA基因引物进行PCR扩增，取5μL PCR产物进行1％琼脂糖凝胶电泳，电泳结果显示PCR产物在1500bp处有一条明显条带（见图2）。菌株FZU 122的PCR产物测序结果显示为一段由1503 bp核苷酸组成的碱基序列。经同源性比对，并采用MEGA X 10.1构建菌株FZU 122的系统发育树（见图3），结果表明菌株FZU 122为一株植物乳杆菌（Lactobacillus plantarum）。
实施例2
植物乳杆菌FZU122接种于MRS液态培养基内，37 ℃条件下静置培养发酵24 h，离心取上清，分别以金黄色葡萄球菌ATCC 29213，金黄色葡萄球菌ATCC 12600，耐甲氧西林金黄色葡萄球菌ATCC 43300，大肠埃希氏菌ATCC 25922，大肠埃希氏菌ATCC 8739，大肠埃希氏菌8099，猪霍乱沙门氏菌CICC 13312，鼠伤寒沙门氏菌CMCC 50115，肠炎沙门氏菌CICC21527，阪崎克罗诺肠杆菌ATCC 29544，猪霍乱沙门氏菌 ATCC 13312为指示菌，接种于LB固体培养基中，采用打孔抑菌法评价抗菌剂对指示菌的抑菌效果。结果如图4所示，L. plantarum FZU 122的发酵液上清对金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌，大肠杆菌、猪霍乱沙门氏菌、鼠伤寒沙门氏菌、肠炎沙门氏菌、阪崎克罗诺肠杆菌等食源性致病菌均具有抑菌活性。表明，L. plantarum FZU 122在发酵液中产生具有广谱抑菌活性的物质。
实施例3
植物乳杆菌FZU122接种于MRS液态培养基内，37 ℃条件下静置培养发酵24 h，将发酵液在4 ℃，10000 rpm条件下离心10 min，得到原始发酵液上清，然后用旋转蒸发仪在45 ℃条件下将其体积浓缩8倍，置于4 ℃冷藏备用。在浓缩发酵液中缓慢加入95%无水乙醇，并搅拌均匀，最终使无水乙醇的体积浓度为75%，于4 ℃静置醇沉12 h。醇沉结束，在4℃，10000 rpm条件下离心10 min，弃去沉淀，保留上清。在45 ℃条件下旋蒸至不再有液体蒸出，回收乙醇反复利用。将样品置于4 ℃冷藏保存备用。取50 g Sephadex LH-20干粉在65%乙醇中浸泡24 h，充分溶胀，装填入层析柱。以超纯水（UP水）作为流动相，流速设定为2mL/10min，开启蠕动泵，平衡2~3个柱体积后，将流动相改为20%色谱级甲醇，其他条件不变，将紫外检测器检测波长调节至280 nm，平衡至少2个柱体积至吸光值不变并调零。样品稀释2倍，并过0.45 μm滤头。沿柱壁小心缓慢上样5 mL。用20%色谱级甲醇作为流动相，以0.2mL/min等度洗脱，每管收集10 min，并检测各管的抑菌活性。由图5可见，第58管到第62管中的分离组分具有抑菌活性。将抑菌活性较强的第60管分离组分合并收集，并采用旋转蒸发仪浓缩后置于4 ℃保存备用。
实施例4
将经Sephadex LH-20过滤层析且有抑菌活性的组分收集合并，在50 ℃条件下旋蒸浓缩至初始体积二分之一左右，去除溶液中有机相甲醇，取1 mL浓缩液用0.22 μm滤头过滤，存放在–20 ℃冰箱备用。流动相A设定为UP水，流动相B选用色谱级甲醇，梯度洗脱条件为：0~30 min，甲醇5~100％；流速为1 mL/min；进样量为5 μL；柱温为室温。收集各个洗脱峰，浓缩后用微量核酸蛋白测定仪其中抗菌肽浓度，并以鼠伤寒沙门氏菌为测试菌，用琼脂扩散法测各个活性峰抑菌活性。由图6所示，将经Sephadex LH-20凝胶柱分离获得的分离组分，采用反相HPLC进一步分离并获得不同组分的图谱。收集不同保留时间洗脱峰靠近峰尖部分的分离组分，经真空旋转蒸发仪干燥后采用UP水复溶，经抑菌试验结果显示，其中3号峰对应的分离组分具有抑菌活性（见图6）。收集3号峰分离组分备用。
实施例5
收集RP-HPLC活性峰采用纳升液相色谱-四极杆飞行时间串联质谱联用仪（Nano-Liquid Chromatography）表征抗菌肽分子量、氨基酸序列。如图7所示，为用纳升液相色谱-质谱联用仪分析上述3号峰活性物质进行质谱分析获得多肽氨基酸序列为ARLGLPVHVV，其分子量为1059.6553，且带正电，带2个正电荷，由10个氨基酸组成，将其命名为Plantaricin- FZU 122。经在线抗菌肽数据库（Antimicrobial peptide database:http://aps.unmc.edu/AP/main.php）比对，未发现相同序列。表明，鉴定出1条全新的植物乳杆菌源抗菌肽。
实施例6
采用二倍等度稀释96孔板法，测定Plantaricin- FZU 122对实施例2中各菌株的最小抑菌浓度（Minimal inhibitory concentration, MIC）。简而言之，各指示菌株接种至LB液体培养基37 ℃、180 rpm条件下培养至对数期，以新鲜的LB液体培养基将菌体细胞数值调至108（OD600=0.4~0.5），进一步稀释至105待用；抗菌肽Plantaricin- FZU 122经LB液体培养基稀释成不同浓度后，以50 μL菌液与50 μL不同浓度抗菌肽溶液于96孔板板孔内混合均匀后，37 ℃条件下培养24 h，通过酶标仪测定菌液OD600值，以没有菌体生长所对应的抗菌肽浓度即为抗菌肽对于该菌株的MIC。由表2所示，Plantaricin- FZU 122对12株指示菌株均表现出不同程度的抑菌活性；其中对鼠伤寒沙门氏菌的最小抑菌浓度为8 μg/mL，对于大肠埃希氏菌 ATCC25922、甲型副伤寒沙门氏菌CMCC(B) 50093以及金黄色葡萄球菌ATCC29213的最小抑菌浓度为32 μg/mL，对于其他指示菌的最小抑菌浓度则均为16 μg/mL。结果表明，Plantaricin- FZU 122对食品中常见致病、腐败菌均具有较强的抑菌活性。
表2 Plantaricin- FZU 122的最小抑菌浓度
实施例7
取新鲜猪肉（精肉）分割称重后置于无菌密封袋，根据猪肉重量分别以500 μg/g加入抗菌肽Plantaricin- FZU 122，500 μg/g加入Nisin，以250 μg/g Plantaricin- FZU122+250 μg/g Nisin（联合处理）加入混合抗菌剂，并经混合均匀后密封，置于4 ℃条件下储藏，同时设置无菌生理盐水处理作为空白对照组，并分别于0、1、3、7、14、21天取样。猪肉经取样后称取重量2~3 g，并以1:10比例加入无菌生理盐水，均质混合后经梯度稀释，涂布于LB固体培养基，置于37℃条件下培养18h后进行菌落计数。由图8所示，经联合处理组与Plantaricin-FZU122处理组的猪肉在冷藏0~7天，微生物的繁殖生长速度得到抑制，且菌落总数显著低于生理盐水与Nisin处理组。其中，联合处理组对猪肉在冷藏过程中展示出协同抑菌效果，可有效延缓猪肉因微生物生长繁殖导致的腐败变质。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福州大学
<120> 一种植物乳杆菌源广谱抗菌肽及其应用
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 10
<212> PRT
<213> 人工序列
<400> 1
Ala Arg Leu Gly Leu Pro Val His Val Val
1 5 10
Claims (4)
1.一种植物乳杆菌源广谱抗菌肽，其特征在于：所述抗菌肽氨基酸序列为：ARLGLPVHVV。
2.一种产权利要求1所述广谱抗菌肽的植物乳杆菌，其特征在于：所述植物乳杆菌为植物乳杆菌（Lactobacillus plantarum）FZU 122，于2020年9月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为：CGMCC NO.20601。
3.如权利要求1所述的一种产广谱抗菌肽在肉品保鲜中的应用。
4.如权利要求2所述的一种植物乳杆菌在肉品保鲜中的应用。
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