解淀粉芽胞桿菌生物防治擴展青霉的機制及蘋果采摘后防腐保鮮技術(shù)研究
發(fā)布時間：2019-01-14 09:34
【摘要】：擴展青霉(Pennicilum expansum)及其水溶性代謝產(chǎn)物展青霉素(patulin, PAT)是導(dǎo)致蘋果采后腐爛及蘋果制品污染的重要因素。為減少由感染P. expansum所致的蘋果采后腐爛并提高貯藏期間的果實品質(zhì),本研究采用原位篩選、N+注入誘變技術(shù)選育出一株可高效抑制P. expansum的菌株BA-16-8,并以該菌為試材,分別對其抑菌活性、去除PAT能力、抗菌物質(zhì)的鑒定、抗菌機制、作為生物活性膜對蘋果采后的防腐保鮮及生物安全性進行了深入研究,得到如下結(jié)論。從蘋果表面原位分離出可有效抑制P. expansum的菌株BA-16,經(jīng)形態(tài)學、AP150CH鑒定和16S rDNA系統(tǒng)發(fā)育等分析,該菌株被鑒定為解淀粉芽孢桿菌(Bacillus amyloliquefaciens)。以N+注入技術(shù)對其進行誘變選育,獲得抗菌性能強且遺傳性能穩(wěn)定的突變株B. amyloliquefaciens BA-16-8.經(jīng)檢測,該菌株的菌懸液及無細胞發(fā)酵液可抑制P. expansum的生長及PAT的分泌量。為明確菌株B. amyloliquefaciens BA-16-8拮抗P. expansum的關(guān)鍵物質(zhì),采用PCR技術(shù)對菌株基因組進行脂肽抗生素合成相關(guān)基因檢測；采用高效液相色譜對菌株發(fā)酵液中的脂肽類抗生素進行分離和純化；采用基質(zhì)輔助激光解析電離飛行時間質(zhì)譜鑒定脂肽結(jié)構(gòu)。鑒定結(jié)果顯示該菌株可產(chǎn)脂肽類抗生素surfactin和fengycin。經(jīng)抑菌實驗和基因敲除實驗,失去了fengycin合成能力的突變株B. amyloliquefaciens BA-16-8-△fen對P.expansum的拮抗效果顯著降低,從而確定抑制P. expansum的關(guān)鍵物質(zhì)為fengycin 。為探究fengycin對P. expansum的具體抑制機制,采用掃描電鏡、透射電鏡、核酸染色、熒光顯微觀察和凝膠阻滯等技術(shù)從細胞層面分析fengycin的抑菌機制；采用呼吸作用檢測、線粒體復(fù)合酶活性檢測等實驗從生理生化層面探究fengycin處理P. expansum代謝所產(chǎn)生的影響。結(jié)果發(fā)現(xiàn)fengycin不僅可改變P. expansum細胞膜的通透性、破壞線粒體等細胞器結(jié)構(gòu),還可通過結(jié)合P. expansum線粒體復(fù)合酶II和IⅡ的相關(guān)基因,抑制其表達,使線粒體酶的活性下降,呼吸及代謝受到阻礙。為研究fengycin對P. expansum合成PAT的影響,采用實時熒光定量PCR的方法考察fengycin處理下PAT合成關(guān)鍵基因6-MSAS和IDH的表達情況。結(jié)果表明, fengycin處理可通過下調(diào)6-MSAS基因的轉(zhuǎn)錄從而抑制P. expansum對PAT的合成。為確保拮抗菌B. amyloliquefaciens BA-16-8在蘋果防腐保鮮應(yīng)用中的安全性,對其進行了28 d喂養(yǎng)實驗和急性經(jīng)口毒性實驗等安全性評估。結(jié)果表明, B. amyloliquefaciensBA-16-8菌懸液(1×108CFU/mL)對小鼠無不良影響,具備安全性。以腐爛率為評價指標,分別確定拮抗菌菌懸液和殼聚糖的最佳濃度及配比,并將其制成防腐抗菌復(fù)合膜。結(jié)果顯示：與對照組相比,2%的殼聚糖溶液與108CFU/mL的B. amyloliquefaciens BA-16-8菌懸液等比例復(fù)配制成的生物活性膜具有顯著的防腐效果。以蘋果多酚含量為評價指標,確定水楊酸、氯化鈣、茉莉酸甲酯的最佳濃度及配比,并將其制成保鮮液。結(jié)果表明：與對照組相比,由200 μg/mL的水楊酸、2%的氯化鈣和0.05 μmol/mL的茉莉酸甲酯等比例復(fù)配制成的混合液具有良好的保鮮效果。以紅富士蘋果為實驗材料,對其分別進行涂膜處理并貯藏。結(jié)果表明：與對照組相比,由拮抗菌菌懸液、殼聚糖、水楊酸、氯化鈣和茉莉酸甲酯按照等比例復(fù)配制成的生物活性膜可有效降低蘋果的失重率和腐爛率,并可較好地保持果實硬度,延緩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,防腐和保鮮效果顯著。綜上,B. amyloliquefaciens BA-16-8產(chǎn)生的fengycin可通過改變胞膜通透性、破壞細胞結(jié)構(gòu)、與DNA結(jié)合影響線粒體復(fù)合酶活性、抑制呼吸及代謝和下調(diào)PAT合成基因的表達等方式有效抑制P. expansum的生長及產(chǎn)毒,將其用于蘋果采后的涂膜處理,展現(xiàn)出良好的防腐保鮮效果。
[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.
【學位授予單位】：西北大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S476;S436.611.16
本文編號：2408566
[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.
【學位授予單位】：西北大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S476;S436.611.16
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