CN113057218B - 褪黑素在抑制芽孢杆菌类食源性致病菌中的应用 - Google Patents
褪黑素在抑制芽孢杆菌类食源性致病菌中的应用 Download PDFInfo
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Abstract
本发明公开了褪黑素在抑制芽孢杆菌类食源性致病菌中的应用,属于农业技术领域。本发明经过研究发现,褪黑素可有效抑制枯草芽孢杆菌(Bacillus subtilis)、蜡状芽孢杆菌(Bacillus cereus)或地衣芽孢杆菌(Bacillus licheniformis)的生长,用于制备芽孢杆菌类食源性致病菌生长抑制剂。
Description
技术领域
本发明属于农业技术领域,具体涉及褪黑素在抑制芽孢杆菌类食源性致病菌中的应用。
背景技术
很多芽孢杆菌都是致病菌,能引起果蔬、粮食、乳制品、鱼类、肉类、蛋类及罐头食品的变质,产生的微生物毒素被人食用后可造成食物中毒。此外,芽孢杆菌产生的芽孢对热、紫外线、电磁辐射、极端温度、pH等具有很强的抗性,通常的杀菌方法很难彻底将其杀灭。因此,目前普遍使用防腐剂以阻碍致病菌的生长和微生物毒素的产生,从而起到延缓食物变质或腐烂进程的目的。
然而,过量摄入化学防腐剂会干扰人体新陈代谢、引起皮肤过敏等问题。目前使用较多的化学防腐剂苯甲酸,欧共体儿童保护集团认为它不宜用于儿童食品,日本也对其使用做了严格限制。随着绿色消费意识的增强,天然防腐剂的开发及应用符合国家政策、市场和环境需求。
褪黑素是人体的一种内源性激素,具有调整昼夜节律、抗衰老,调节神经系统、免疫系统及心血管系统等作用。近几年研究发现褪黑素能削弱致病疫霉(Phytophthora infestans)对马铃薯的危害,还可诱导植物产生抗病性。然而,褪黑素对芽孢杆菌等食源性致病菌的研究还尚未有报道。
发明内容
本发明的目的在于提供褪黑素在抑制芽孢杆菌类食源性致病菌中的应用。
褪黑素是一种天然化合物,别名N-[2-(5-甲氧基-3-吲哚)乙基]乙酰胺,分子式为C13H16N2O2。
本发明经过研究发现,褪黑素可抑制芽孢杆菌类食源性致病菌枯草芽孢杆菌(Bacillus subtilis, BS)、蜡状芽孢杆菌(Bacillus cereus, BC)、地衣芽孢杆菌(Bacillus licheniformis, BL)的生长,故提出了褪黑素在制备芽孢杆菌类食源性致病菌生长抑制剂中的应用。
根据应用,本发明提供了一种抑制水果、蔬菜中的芽孢杆菌类食源性致病菌生长的制剂,该制剂的活性成分为对褪黑素。
作为优选,所述制剂为褪黑素水溶液。
更优选地,所述褪黑素水溶液的浓度为15 mM。
与现有技术相比,本发明将褪黑素应用于预防采后小番茄等水果、蔬菜的食源性致病菌感染的制剂中,通过降低芽孢杆菌鞭毛游动性、胞外蛋白酶活性及生物膜形成的方式达到抑菌的目的。本发明无需复杂分子操作,制剂中主要活性物质褪黑素普遍存在于动物、植物、微生物体内,是一种内源性的活性物质,对人体友好。除此之外,本发明提供了一种预防采后小番茄等水果、蔬菜的食源性致病菌感染的制剂,该制剂为浓度为15 mM的褪黑素水溶液,该制剂相对于其他浓度制剂而言,预防效果最佳,且制备方法简单。
附图说明
图1为采用不同浓度褪黑素处理后的枯草芽孢杆菌菌落生长情况柱状图。ns 没有显著差异,* p < 0.05,具有显著差异。
图2为含有0 mM、1 mM褪黑素的LB培养基培养后,枯草芽孢杆菌的生长情况图。A为枯草芽孢杆菌形态图,B为枯草芽孢杆菌的细胞尺寸图。
图3为不同浓度的褪黑素对枯草芽孢杆菌鞭毛游动性的影响。Ns 没有显著差异,*** p < 0.001,**** p < 0.0001。
图4为不同浓度的褪黑素对枯草芽孢杆菌胞外蛋白酶活性的影响。* p <0.05,*** p < 0.001,**** p < 0.0001。
图5为不同浓度的褪黑素对枯草芽孢杆菌生物膜形成的影响。** p < 0.01,*** p< 0.001。
图6为不同浓度的褪黑素对番茄果实上的枯草芽孢杆菌、蜡状芽孢杆菌、地衣芽孢杆菌的作用的示意图。ns 没有显著差异,* p < 0.05 , ** p < 0.01, *** p < 0.001,**** p < 0.0001。
具体实施方式
本发明公开了褪黑素在抑制水果、蔬菜中芽孢杆菌类食源性致病菌生长方面的应用。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明中。本发明的微生物菌株和相关应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的产品及方法进行改动或适当变更与组合,来实现和应用本发明技术。
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合具体实施例进行详细描述。
实施例1
褪黑素对枯草芽孢杆菌生长影响的测定
枯草芽孢杆菌菌株活化:从-80℃冰箱中取出少量甘油保存的枯草芽孢杆菌接种于10 mL LB液体培养基中(蛋白胨10 g/L,酵母提取物5 g/L,NaCl 10 g/L),于28℃,200rpm摇床中过夜培养。
按1:1000比例将活化后的菌种转接至新的LB液体培养基中,于37℃,200 rpm培养至OD600=1.0后,4℃,8,000 rpm离心6 min收集菌体,将菌体重悬于等体积的无菌去离子水中。取4 µL重悬菌液转移至4 mL分别含有0 mM(对照组)、0.1 mM、0.5 mM、1 mM、5 mM、10 mM褪黑素的LB液体培养基中,200 rpm避光培养6 h后检测菌体浊度(OD600)。
测定结果如图1所示,随着褪黑素浓度的增高,枯草芽孢杆菌的OD600值越来越小,说明褪黑素的浓度越高,对枯草芽孢杆菌的抑制效果越明显
实施例2
褪黑素对枯草芽孢杆菌细胞形态的影响
枯草芽孢杆菌菌株活化方法同实施例1。
在含有0 mM(对照组)、1 mM褪黑素的LB液体培养基中培养枯草芽孢杆菌。分别取1mL菌液于4℃,8,000 rpm离心6 min收集菌体。用PBS溶液洗涤2次后用少量PBS重悬菌体。取一滴上述细胞重悬液转移至干净的载玻片上,利用4%戊二醛对细胞进行固定。1 h后,依次用65%、75%、85%、95%、100%的酒精浸泡载玻片15 min进行梯度脱水。待干燥机干燥后,利用扫描电子显微镜观察枯草芽孢杆菌的形态。
测定结果如图2所示。根据图2中A图可知,在含有1 mM褪黑素的LB液体培养基中枯草芽孢杆菌细胞数量明显少于对照组。在10000倍视野下可以发现,褪黑素处理的枯草芽孢杆菌细胞的长度和宽度明显小于对照组中的细胞(图2B)。这些结果表明,褪黑素处理后,枯草芽孢杆菌细胞生长受到明显抑制。
实施例3
褪黑素对枯草芽孢杆菌泳动性的影响
枯草芽孢杆菌菌株活化方法同实施例1。
制备含有0 mM、0.1 μM、0.2 mM、0.5 mM、1 mM褪黑素的LB固体培养基,向培养基正中央点入3 μL OD600=1.0的枯草芽孢杆菌菌液,将培养皿置于28℃恒温箱正置培养72 h后,测量菌落直径。
测定结果如图3所示,褪黑素的浓度越高,LB平板上枯草芽孢杆菌菌落的直径越小;含有1 mM褪黑素的LB平板上枯草芽孢杆菌菌落的直径最小,说明褪黑素对枯草芽孢杆菌的泳动能力具有抑制作用,且褪黑素的浓度越高,抑制效果越强。
实施例4
褪黑素对枯草芽孢杆菌蛋白酶活性的影响
在应对外界压力是,枯草芽孢杆菌会产生防御响应,蛋白酶活性增加。枯草芽孢杆菌菌株活化方法同实施例1。
向含有1%奶粉的LB固体培养基中加入含有0 mM、0.1 mM、0.2 mM、0.5 mM、1 mM的褪黑素,向培养基正中央点入3 μL OD600=1.0的枯草芽孢杆菌菌液,将培养皿置于28℃恒温箱培养48 h后,测量水解圈直径。
测定结果如图4所示,浓度为1 mM的褪黑素制备的含蛋白质的LB培养基得到的枯草芽孢杆菌的水解圈直径最大,说明褪黑素的浓度越高,蛋白酶活性越强,对枯草芽孢杆菌的防御响应的诱导越强。
实施例5
褪黑素对枯草芽孢杆菌生物膜形成的影响
枯草芽孢杆菌菌液活化方法同实施例1。
于4℃,5,000 rpm离心8 min收集菌体,并将其重悬于等体积的灭菌去离子水中。向3 mL含有0 mM、0.1 μM、0.2 mM、0.5 mM、1 mM褪黑素的LB液体培养基中加入30 μL菌液,将玻璃试管置于28℃恒温培养箱中静置培养5 d。去除菌液,用去离子水轻轻冲洗2遍后,向玻璃试管中加入6 mL结晶紫溶液,室温静置1 h后弃去溶液,用去离子水洗3遍,并于空气中干燥1 h。向玻璃试管中加入3 mL 40%的甲醇和10%的冰醋酸混合液,充分溶解后,在575 nm波长下测定吸光值,评估褪黑素对枯草芽孢杆菌生物膜形成能力的影响。
测定结果如图5所示,褪黑素的浓度越高,枯草芽孢杆菌的生物膜形成能力越弱;浓度为1 mM的褪黑素处理后的枯草芽孢杆菌的生物膜形成能力最弱,说明褪黑素的浓度越高,对枯草芽孢杆菌生物膜形成的能力的抑制效果越强。
实施例6
褪黑素对番茄果实上的芽孢杆菌的抑菌作用
枯草芽孢杆菌(Bacillus subtilis, BS)、蜡状芽孢杆菌(Bacillus cereus, BC)、地衣芽孢杆菌(Bacillus licheniformis, BL)的菌株活化方法同实施例1中枯草芽孢杆菌的活化方法。
随机选取27个小番茄果实,对小番茄进行无菌处理。具体步骤为:使用5%的84消毒液浸泡30 min后,在超净台中紫外照射15 min。然后在超净台中倒掉84消毒液并用无菌水将小番茄洗净,控干水分。向果实表面分别喷洒0 μM(对照组)、0.005 mM、0.01 mM、0.05mM、0.1 mM、0.5 mM、1 mM、5 mM、10 mM、15mM的褪黑素溶液,自然晾干后备用。于4℃,8,000rpm离心6 min收集BS、BL、BC菌体,用等体积的无菌去离子水重悬菌体后,将菌液均匀喷洒到果实表面上,自然晾干。喷洒BS的小番茄于28℃培养12 h,喷洒BL、BC的小番茄于室温静置3 h。然后,将小番茄转移至含10 mL无菌水的培养瓶中,充分洗涤。取5 μL培养瓶中的洗液,转移至995 μL无菌水中,充分吹打混匀后,从中取5 μL溶液,转移至新的995 μL无菌水中,此为40000倍稀释液。取100 μL该稀释液涂布于LB固体培养基上。将涂布后的平板置于培养箱中培养12 h。BS、BL、BC的培养温度分别为28℃、37℃、30℃。对平板上生长的细菌进行计数。
测定结果如图6所示,褪黑素的浓度越高,对BS、BL、BC细胞群落的抑制能力越强。10 mM的褪黑素能完全抑制小番茄上BL细胞的生长;15 mM的褪黑素能完全抑制小番茄上BS、BC细胞的生长。
Claims (3)
1. 褪黑素在抑制芽孢杆菌类食源性致病菌中的应用,其特征在于,所述芽孢杆菌类食源性致病菌为枯草芽孢杆菌(Bacillus subtilis)或地衣芽孢杆菌(Bacillus licheniformis)。
2. 褪黑素在制备芽孢杆菌类食源性致病菌生长抑制剂中的应用,其特征在于:所述芽孢杆菌类食源性致病菌为枯草芽孢杆菌(Bacillus subtilis)或地衣芽孢杆菌(Bacillus licheniformis)。
3. 一种芽孢杆菌类食源性致病菌生长抑制剂,所述芽孢杆菌类食源性致病菌为枯草芽孢杆菌(Bacillus subtilis)或地衣芽孢杆菌(Bacillus licheniformis),其特征在于:所述制剂的活性成分为褪黑素水溶液,褪黑素水溶液的浓度为15 mM。
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