CN103865837A - 对鲜绿青霉有拮抗作用的链霉菌 - Google Patents
对鲜绿青霉有拮抗作用的链霉菌 Download PDFInfo
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Abstract
本发明涉及对鲜绿青霉有拮抗作用的链霉菌，本发明获得的菌株，现保存在国家知识产权局指定的保藏单位保藏，保藏单位是中国典型培养物保藏中心，保藏日期为2013年11月18日，保藏编号为CCTCCNO：M2013575。该链霉菌被鉴定为阿南德氏链霉菌，命名为阿南德氏链霉菌JXNU02，拉丁文学名为StreptomycesanandiiJXNU02。阿南德氏链霉菌JXNU02发酵液对鲜绿青霉具有较强抑制作用，且性能稳定，对开发成新型的生物防腐剂具有较好的应用价值。
Description
技术领域
本发明涉及微生物技术领域，特别是涉及一种对鲜绿青霉有拮抗作用的链霉菌。
背景技术
鲜绿青霉（Penicillium viridicatum）属于青霉属真菌，常在谷物、水果、蔬菜、坚果和肉类等食品成熟和贮藏过程中生长繁殖。鲜绿青霉在生长繁殖过程中易产生多种真菌毒素，如赭曲霉毒素A（ochratoxin A , OTA）。OTA具有强烈的致癌性和致畸性，同时对肾脏和肝脏有严重损害，是食品主要污染源之一，对人类健康及动物养殖业有重大危害。因此鲜绿青霉的防治受到学者广泛关注。
由于食品在加工、贮藏、消费的过程中易受到各种有害微生物的侵害而变质，食品防腐保鲜便成了食品工业中的重要问题。随着人们生活水平的提高，及环保意识的增强，传统的化学保鲜剂已被证实具有潜在的危害性。因此，开发高效、无毒、安全的生物保鲜剂势在必行。
发明内容
本发明的目的在于提供具有抗真菌作用，特别是对鲜绿青霉有拮抗作用的链霉菌。
实现本发明的技术方案如下：本发明所述的对鲜绿青霉有有拮抗作用的链霉菌，是从江西省南昌市郊区采集的土样经分离制备获得，其学名为阿南德氏链霉菌JXNU02（Streptomyces anandii JXNU02）。
本发明所述的对鲜绿青霉有拮抗作用的链霉菌的菌株已保存在国家知识产权局指定的保藏单位保藏，保藏单位是中国典型培养物保藏中心，保藏地址为中国武汉，武汉大学；保藏日期为2013 年11月18日，保藏编号为CCTCC NO：M 2013575。
阿南德氏链霉菌JXNU02菌株的形态特征：
基内菌丝发育良好，无横隔、不断裂；气生菌丝在ISP2 培养基（酵母提取物 4 g，麦芽提取物 10 g，葡萄糖 4 g，琼脂 20 g， 蒸馏水1 L，pH 7.3 ）上生长良好，多分枝，可形成孢子链，孢子丝呈螺旋形，孢子椭圆形、表面光滑 (见图1)。
阿南德氏链霉菌JXNU02菌株的生理生化特征：
阿南德氏链霉菌JXNU02能使淀粉水解、能产生硫化氢、能利用酪氨酸产生黑色素、能使明胶液化、能使硝酸盐还原，不能分解纤维素、不能使牛奶胨化；能够利用葡萄糖、蔗糖、D-果糖、L-阿拉伯糖、D-木糖、棉子糖、肌醇和D-甘露醇，但不能利用L-鼠李糖；细胞壁类型属于I 型，全细胞水解液中含有核糖，未检测出特征性糖。
对鲜绿青霉有拮抗作用的阿南德氏链霉菌JXNU02发酵液抑菌谱的测定。
采用琼脂糖扩散法对阿南德氏链霉菌JXNU02发酵液抑菌谱进行测定。采用的靶菌分别为鲜绿青霉、桔青霉、酵母菌、白假丝酵母、水稻稻瘟病菌、黑曲霉、毛霉、棉花枯萎病菌、尖孢镰孢菌、水稻纹枯病菌、米根霉、核盘菌、金黄色葡萄球菌和大肠杆菌。结果表明，阿南德氏链霉菌JXNU02发酵原液对上述13 种靶菌均有抑制作用。
阿南德氏链霉菌JXNU02发酵液对鲜绿青霉菌丝生长的影响
阿南德氏链霉菌JXNU02发酵液能完全抑制鲜绿青霉的菌丝生长，且抑制率达到100%，结果如图2。
本发明的有益效果：本发明涉及的一株对鲜绿青霉有拮抗作用的菌株，并对该菌株进行分类鉴定，确定其为阿南德氏链霉菌JXNU02（Streptomyces anandii JXNU02）；对阿南德氏链霉菌JXNU02发酵液的抑菌谱进行了研究，确定其对多种真菌及细菌均有抑制作用；对阿南德氏链霉菌JXNU02发酵液对鲜绿青霉菌丝生长的影响进行了研究，确定其发酵液抑制鲜绿青霉菌丝生长，且抑制率达到100%。这些数据将为今后食品生物保鲜剂的开发和应用奠定基础。
附图说明
图1 阿南德氏链霉菌JXNU02菌丝及孢子形态（X10000）。
图2阿南德氏链霉菌JXNU02发酵液对鲜绿青霉菌丝生长的影响（A为发酵液，B为空白对照）。
具体实施方式
下面结合实施实例对本发明作进一步的说明
实施例1 从江西省南昌市郊区采集的土样，按照下述方法进行分离、培养即可获得阿南德氏链霉菌JXNU02，采用摇床液体发酵培养的方法即可获得对鲜绿青霉菌有抑制作用发酵液。
（1）链霉菌的筛选、保藏
从江西省南昌市郊区采集的土样，土样自然风干，研磨过筛，121℃ 干热处理1 h，称取2 g土壤于18 ml无菌生理水中，在150 r/min 条件下振荡15 min，再进行梯度稀释，将10-2、10-3土壤悬液分别涂布于含75 mg/L K2Cr2O7 高氏一号培养基（可溶性淀粉20 g，KNO3 1 g， NaCl 0.5 g，K2HPO4·3H2O 0.5 g，MgSO4·7H2O 0.5 g，FeSO4·7H2O 0.01g，琼脂粉 20g，蒸馏水 1 L，pH 7.4~7.6）平板上，28℃培养7-14 d，挑取菌落形态不同的菌落于髙氏一号培养基平板上分离纯化，将纯化培养物接种于高氏一号斜面，并于4℃保藏备用。
采用平板对峙法初筛拮抗放线菌，以马铃薯葡萄糖琼脂培养基（马铃薯200g，葡萄糖20g，琼脂20g，水1 L）平板中心为原点，划“十”，将直径为5 mm放线菌菌块放置于距平板中心2.5 cm处，28℃培养2 d 后，再将直径为5 mm的鲜绿青霉菌块接种于平板中央，28℃培养5 d后观察结果。
采用管碟法复筛拮抗放线菌，以鲜绿青霉为靶菌，能产生抑菌圈的菌株即为拮抗放线菌，并用游标卡尺采用十字交叉法测量抑菌圈直径。
（2）阿南德氏链霉菌JXNU02发酵液的制备
阿南德氏链霉菌接种至髙氏一号平板上培养7 d后，取4块直径5 mm放线菌菌饼置于种子培养基（蔗糖22.5 g，黄豆饼粉12.5 g，K2HPO4·3H2O 0.2 g，NaCl 1 g，Na2SO4 0.1 g， FeSO4·7H2O 0.01 g ，CaCO3 2 g，蒸馏水 1 L，pH 7.2），装液量50 mL/ 250 mL三角瓶，在200 r/min、28℃条件下培养48 h，即种子液。以6 %（v/v）的接种量于50 mL/250 mL发酵培养基（可溶性淀粉10 g，黄豆饼粉15 g，KH2PO4 0.3 g，MgSO4·7H2O 0.3 g，蒸馏水 1 L，pH 7.2），在200 r/min、28℃条件下培养5 d。发酵液在10000 r/min 条件下离心20 min，收集上清液，并采用0.22 μm滤膜过滤除菌，无菌发酵液置于4℃保藏备用。
（3）阿南德氏链霉菌JXNU02发酵液抗菌谱的测定
以适量无菌生理盐水将靶菌的斜面活化培养物悬浮，得细胞悬浮液，控制细胞浓度为1X106 CFU/mL。取该菌悬浮液15 μl 加入到7 mL 预冷至50℃左右的培养基中，充分混匀后，平铺在倒有20 mL 2%的琼脂底层平板( 直径15 mm) 上。凝结后放置无菌牛津杯，再加入200 μl 发酵液，37℃ 培养24 h（细菌）或28℃培养48 h（真菌）, 测抑菌圈直径即抗生素的活性。
（4）阿南德氏链霉菌JXNU02发酵液对鲜绿青霉菌丝生长的影响
分别量取1 mL阿南德氏链霉菌JXNU02发酵液和生理盐水（空白对照），加入 10 mL 预冷至50℃左右的马铃薯葡萄糖琼脂培养基中，充分混匀后，倒入培养皿中，再将直径为5 mm的鲜绿青霉菌块接种于平板中央，28℃培养5 d后观察结果。
CGCGCGGCGCGTGCTTACCATGCAGTCGAACGATGAACCACTTCGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATATGAGCCGCCCAGGCATCTGGGTGGCTGTAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAAACCCTGGAGACAGGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAATGGTGTAAGCTGCAGGTCC。
Claims (3)
1.一种对鲜绿青霉有拮抗作用的链霉菌，其特征在于，其命名为阿南德氏链霉菌JXNU02（Streptomyces anandii JXNU02），该菌株已保存在国家知识产权局指定的保藏单位保藏，保藏单位是中国典型培养物保藏中心，保藏日期为2013 年11月18日，保藏编号为CCTCC NO：M 2013575。
2. 如权利要求1 所述的对鲜绿青霉有拮抗作用的链霉菌菌株，其特征在于，其16S rRNA基因序列为：
CGCGCGGCGCGTGCTTACCATGCAGTCGAACGATGAACCACTTCGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATATGAGCCGCCCAGGCATCTGGGTGGCTGTAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAAACCCTGGAGACAGGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAATGGTGTAAGCTGCAGGTCC。
3. 如权利要求1 所述的对鲜绿青霉有拮抗作用的链霉菌菌株，其特征在于，其用于鲜绿青霉的防治。
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