芽孢杆菌抗菌肽具有广谱的抗霉活性，是开发新型大米生物防霉剂的潜在资源。然而，关于芽孢杆菌抗菌肽对大米霉变防控作用的研究目前为止未见有报道。本项目拟在前期发现的Bacillus natto CSna-05菌株所产抗菌肽粗提物能有效防止大米霉变的基础上，开展以下研究：（1）用高效液相色谱（HPLC）等方法对抗菌肽粗提物进行分离纯化，确定纯化后各抗菌肽组分对大米霉变及积累黄曲霉毒素的防控效果，并对受试大米进行质量测评；（2）以圆二色谱（CD）等技术探讨非水相体系条件下，湿度、温度、稳定剂等因素对抗菌肽活性构型的影响；（3）利用荧光标记、超微分析、紫外光谱、流式细胞术(FCM)等方法研究抗菌肽抑制大米霉菌孢子萌发的作用位点与作用模式，阐明抗菌肽对大米霉变的防控机理。为开发高效、安全的大米生物防霉保鲜剂提供新途径和理论依据。

Because of their broad-spectrum antifungal activity, the antifungal peptides from Bacillus are potential resources for new biological anti-mildew products for rice preservation, but the study on their using for preventing rice from mildew is still blank. In our previous studies, the crude extraction of antifungal peptides from Bacillus natto CSna-05 was found effective in keeping rice from mildew. Based on these results, the following researches would be carried out: (1) each antifungal peptide group which can effectively prevent rice mildew will be isolated and purified from the crude extracts of fermentation broth, using high performance liquid chromatography (HPLC). The influences of these purified groups on treated rice will be evaluated to ensure rice quality. (2) The influences of humidity, temperature and stabilizer on the activities and conformation of the purified anti-mold peptides, under the non aqueous phase condition of rice storage, will be investigated using circular dichroism (CD); (3) The fluorescence labeled anti-mold peptides binding sites in mold spores and its germination mycelium will be located, and their interaction pattern will be determined through methods of ultra micro analysis, UV spectrophotometry and flow cytometry system. This program will illuminate the prevention mechanisms of the antifungal peptides from Bacillus natto on rice mildew, and is expected to provide a new way and theory basis for developing novel anti-mildew agent for rice preservation.

在前期研究优化后的发酵工艺基础上，制得纳豆菌（Bacillus natto）高抗霉活性发酵产物。采用超滤分离、固相萃取、反相高效液相、质谱分析等技术对发酵产物进行分离、纯化、鉴定的结果表明：B. natto液体发酵产物中高抗霉活性成分主要是C原子数为13-15的典型surfactin同系物（L7-surfactin）；而在固态发酵条件下，B. natto可大量合成含14个C原子，7位氨基酸为缬氨酸V的V7-surfactin。对几种霉菌的最小抑菌浓度（MIC）测定结果表明，V7-surfactin比L7-surfactin对霉菌的MIC值更低，抗霉活性更强。建立和完善了测定样品抑霉率的新方法：刃天青比色法。该方法能准确比较抗霉活性相近的样品的抑霉率大小，较抑菌圈法操作容易、灵敏度高、结果重现性好。通过显微染色、扫描电镜等方法初步探讨了surfactin对霉菌细胞作用机理。结果显示：L7-surfactin、V7-surfactin对霉菌的作用都表现在可抑制霉菌孢子萌发和菌丝生长，显微染色观察时可看到受surfactin作用的霉菌出现胞内物质外流现象，高倍扫描电镜图片显示surfactin可引起霉菌细胞壁最外层溶解，使细胞表面易出现凹陷和变形。稳定剂筛选试验中，发现EDTA 可使surfactin抗霉活性明显增强，表现在引起菌丝严重畸形、自溶和断裂。比较了纳豆菌液体发酵、固体发酵所得抗菌肽粗提物对大米霉变的防控作用。结果表明：两种条件下所得抗菌肽粗提物对大米霉变均有一定抑制作用，固态发酵所得抗菌肽粗提物对大米霉变的防控效果优于液态发酵抗菌肽粗提物，同时在颜色气味方面对受试大米无不良影响。试验条件下，每100g大米中添加1-3mL纳豆菌固态发酵抗菌肽提取物，对大米中霉菌的抑制率可达到79.1-92.5%。