解淀粉芽胞杆菌生物防治扩展青霉的机制及苹果采摘后防腐保鲜技术研究

发布时间：2019-01-14 09:34

【摘要】：扩展青霉(Pennicilum expansum)及其水溶性代谢产物展青霉素(patulin, PAT)是导致苹果采后腐烂及苹果制品污染的重要因素。为减少由感染P. expansum所致的苹果采后腐烂并提高贮藏期间的果实品质,本研究采用原位筛选、N+注入诱变技术选育出一株可高效抑制P. expansum的菌株BA-16-8,并以该菌为试材,分别对其抑菌活性、去除PAT能力、抗菌物质的鉴定、抗菌机制、作为生物活性膜对苹果采后的防腐保鲜及生物安全性进行了深入研究,得到如下结论。从苹果表面原位分离出可有效抑制P. expansum的菌株BA-16,经形态学、AP150CH鉴定和16S rDNA系统发育等分析,该菌株被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。以N+注入技术对其进行诱变选育,获得抗菌性能强且遗传性能稳定的突变株B. amyloliquefaciens BA-16-8.经检测,该菌株的菌悬液及无细胞发酵液可抑制P. expansum的生长及PAT的分泌量。为明确菌株B. amyloliquefaciens BA-16-8拮抗P. expansum的关键物质,采用PCR技术对菌株基因组进行脂肽抗生素合成相关基因检测；采用高效液相色谱对菌株发酵液中的脂肽类抗生素进行分离和纯化；采用基质辅助激光解析电离飞行时间质谱鉴定脂肽结构。鉴定结果显示该菌株可产脂肽类抗生素surfactin和fengycin。经抑菌实验和基因敲除实验,失去了fengycin合成能力的突变株B. amyloliquefaciens BA-16-8-△fen对P.expansum的拮抗效果显著降低,从而确定抑制P. expansum的关键物质为fengycin 。为探究fengycin对P. expansum的具体抑制机制,采用扫描电镜、透射电镜、核酸染色、荧光显微观察和凝胶阻滞等技术从细胞层面分析fengycin的抑菌机制；采用呼吸作用检测、线粒体复合酶活性检测等实验从生理生化层面探究fengycin处理P. expansum代谢所产生的影响。结果发现fengycin不仅可改变P. expansum细胞膜的通透性、破坏线粒体等细胞器结构,还可通过结合P. expansum线粒体复合酶II和IⅡ的相关基因,抑制其表达,使线粒体酶的活性下降,呼吸及代谢受到阻碍。为研究fengycin对P. expansum合成PAT的影响,采用实时荧光定量PCR的方法考察fengycin处理下PAT合成关键基因6-MSAS和IDH的表达情况。结果表明, fengycin处理可通过下调6-MSAS基因的转录从而抑制P. expansum对PAT的合成。为确保拮抗菌B. amyloliquefaciens BA-16-8在苹果防腐保鲜应用中的安全性,对其进行了28 d喂养实验和急性经口毒性实验等安全性评估。结果表明, B. amyloliquefaciensBA-16-8菌悬液(1×108CFU/mL)对小鼠无不良影响,具备安全性。以腐烂率为评价指标,分别确定拮抗菌菌悬液和壳聚糖的最佳浓度及配比,并将其制成防腐抗菌复合膜。结果显示：与对照组相比,2%的壳聚糖溶液与108CFU/mL的B. amyloliquefaciens BA-16-8菌悬液等比例复配制成的生物活性膜具有显著的防腐效果。以苹果多酚含量为评价指标,确定水杨酸、氯化钙、茉莉酸甲酯的最佳浓度及配比,并将其制成保鲜液。结果表明：与对照组相比,由200 μg/mL的水杨酸、2%的氯化钙和0.05 μmol/mL的茉莉酸甲酯等比例复配制成的混合液具有良好的保鲜效果。以红富士苹果为实验材料,对其分别进行涂膜处理并贮藏。结果表明：与对照组相比,由拮抗菌菌悬液、壳聚糖、水杨酸、氯化钙和茉莉酸甲酯按照等比例复配制成的生物活性膜可有效降低苹果的失重率和腐烂率,并可较好地保持果实硬度,延缓Vc、可溶性固形物和可滴定酸含量的下降以及丙二醛的上升,防腐和保鲜效果显著。综上,B. amyloliquefaciens BA-16-8产生的fengycin可通过改变胞膜通透性、破坏细胞结构、与DNA结合影响线粒体复合酶活性、抑制呼吸及代谢和下调PAT合成基因的表达等方式有效抑制P. expansum的生长及产毒,将其用于苹果采后的涂膜处理,展现出良好的防腐保鲜效果。
[Abstract]:Penicilum expansum and its water-soluble metabolite, patulin, PAT, are an important factor leading to the decay of apple and the pollution of apple products. In order to reduce the rot of apple caused by infection of P. exansum and improve the quality of the fruit during storage, in-situ screening, N + injection mutagenesis was used to select a strain BA-16-8 which could effectively inhibit the P. exansum, and the strain BA-16-8 which can effectively inhibit the P. exansum was selected and the antibacterial activity of the strain BA-16-8 was removed by using the strain as a test material, and the PAT capability was removed. The identification and antibacterial mechanism of the antibacterial substance, as the biological active membrane, made a deep study on the anti-corrosion and fresh-keeping and the biological safety of the apple, and the following conclusions were obtained. The strain BA-16, which can effectively inhibit P. exansum, was isolated from the surface of the apple, and analyzed by morphology, AP150CH identification and 16S rDNA phylogenetic analysis. The strain was identified as Bacillus amyloliquefaciens. and the mutant strain B. amyloniquefacens BA-16-8 with strong antibacterial property and stable hereditary property is obtained by using the N + injection technology to carry out mutation breeding. The bacterial suspension and the cell-free fermentation broth of the strain can inhibit the growth of P. exansum and the secretion of PAT. A high performance liquid chromatography was used to isolate and purify the lipopeptide antibiotics in the strain of the strain. The structure of lipopeptide was identified by matrix-assisted laser ionization time-of-flight mass spectrometry. The results showed that the strain can produce lipopeptide antibiotics surfac and fengyin. The antagonistic effect of the mutant B. amyloniquefaconiens BA-16-8-Difen on the synthesis ability of P. exansum was reduced by the experiments of bacteriostasis and gene knock-out, and the key material of the inhibition of P. exansum was determined to be fengyin. In order to investigate the specific inhibition mechanism of fengysin for P. exansum, the inhibition mechanism of fengysin was analyzed by scanning electron microscope, transmission electron microscope, nucleic acid staining, fluorescence microscopic observation and gel block technique. The effects of the activity of the mitochondrial complex enzyme on the metabolism of P. exansum were investigated from the physiological and biochemical level. The results showed that fengysin can not only change the permeability of the cell membrane of P. exansum, destroy the structure of the organelle such as mitochondria, but also inhibit the expression of the mitochondrial complex enzyme II and I II of P. exansum, so that the activity of the mitochondrial enzyme is reduced, and the respiration and metabolism are hindered. In order to study the effect of fengysin on the synthesis of PAT, the expression of the key gene 6-MSAS and IDH was investigated by real-time fluorescence quantitative PCR. The results showed that the fengyin treatment could reduce the P. exansum's synthesis of PAT by down-regulating the transcription of the 6-MSAS gene. In order to ensure the safety of the antagonistic bacteria B. amyloniquefacens BA-16-8 in the application of the anti-corrosion and fresh-keeping of the apple, the safety assessment of the 28-day feeding experiment and the acute oral toxicity experiment was carried out. The results showed that the suspension of B. amylliqiensBA-16-8 (1-108CFU/ mL) had no adverse effect on the mice, and it was safe. The optimum concentration and ratio of antagonistic bacteria suspension and chitosan were determined by the decay rate as the evaluation index, and the anti-corrosion and antibacterial composite membrane was prepared. The results showed that, compared with the control group, 2% of the chitosan solution and 108CFU/ mL of B. amyloniquefacens BA-16-8 suspension liquid were compounded to prepare the bioactive film with a significant anti-corrosion effect. The optimum concentration and proportion of salicylic acid, calcium chloride and methyl jasmonate were determined with the content of polyphenol of apple as the evaluation index, and it was made into fresh-keeping liquid. The results showed that the mixed solution prepared by the ratio of 200. m u.g/ mL of salicylic acid, 2% of calcium chloride and 0. 050.mu. mol/ mL of methyl jasmonate in the control group had good fresh-keeping effect. The red Fuji apple was used as the experimental material, and the film was treated and stored separately. Compared with the control group, the bioactive film prepared by compounding the antagonistic bacteria suspension liquid, the chitosan, the salicylic acid, the calcium chloride and the jasmonic acid methyl ester can effectively reduce the weight loss rate and the decay rate of the apple, and can better maintain the fruit hardness and delay the vitamin C, the content of soluble solid and titratable acid is reduced and the content of malondialdehyde is increased, and the anti-corrosion and fresh-keeping effect is remarkable. In general, the fengyin produced by B. amyloniquefacens BA-16-8 can effectively inhibit the growth and the production of P. exansum by changing the permeability of the cell membrane, destroying the cell structure, binding to the DNA to influence the activity of the mitochondrial complex enzyme, inhibiting the respiration and the metabolism, and reducing the expression of the PAT synthetic gene. It is used for the film treatment after apple production, and has good anti-corrosion and fresh-keeping effect.
【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S476;S436.611.16

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