【作者】 车建美；

【导师】 关雄； 刘波；

【作者基本信息】 福建农林大学 ， 生物防治， 2011， 博士

【摘要】 本研究通过分析龙眼果实采后腐烂过程相关酶系的变化规律,探讨龙眼褐变和腐烂的机理,并筛选了龙眼保鲜微生物,研究了该保鲜微生物的生物学特性和保鲜机理,制备了龙眼微生物保鲜菌剂。主要研究结果如下:1龙眼保鲜微生物的筛选及相关生物学特性分析从本实验室已有菌种库中筛选到6株对龙眼腐生真菌和细菌均具有较好拮抗作用的菌株,其中短短芽胞杆菌FJAT-0809-GLX(Brevibacillus breivs FJAT-0809-GLX)对龙眼腐生细菌和真菌的抑菌效果最好,抑菌圈直径为15.87 mm,抑菌带为6.27 mm。对短短芽胞杆菌FJAT-0809-GLX培养条件的研究表明,该菌株培养的最适条件为:转速180 rpm、pH值7.0的培养基、250 mL三角瓶装瓶量20-40 mL、培养48 h。对短短芽胞杆菌FJAT-0809-GLX的全基因组测序初步分析表明,该菌株的基因组规模为6 Mb,预测得到5666个基因,平均长度为933 bp,基因密度达到87.83%,基因组的GC含量为47.30%,具有177个菌株特异基因,这些基因的功能如何还有待于进一步的分析。2龙眼果实致腐机理的分析从细胞形态上看,龙眼外果皮结构未发生明显的变化,但内果皮细胞结构变化比较明显,从2级果实开始,内果皮开始发生破裂,4级和5级果实的内果皮细胞骨架完全发生瓦解,内果皮完全腐烂,有的部位还可以看到菌丝。综合龙眼果肉和果皮的腐烂特征,将龙眼果实划分为6个腐烂等级。随着腐烂程度的加深,龙眼果实的呼吸速率加快,0级龙眼果实呼吸速率为61.17 mg/kg·hr,4级的龙眼果实呼吸速率达到最大为382.71 mg/kg·hr。龙眼果实过氧化物酶(Peroxidase,POD)和多酚氧化酶(Polphenol oxidase,PPO)活性随腐烂程度加深也增高,腐烂程度达到5级的果皮POD酶活性最高,为15625 U/min·g,腐烂程度达到4级的龙眼果皮PPO酶活性最高,为687.5 U/min·g。而可溶性固形物(Total soluble solids,TSS)含量随腐烂程度加深却呈现降低的趋势。龙眼果实腐生细菌和真菌的分离和鉴定结果表明,烂果的真菌和细菌数量和种类均多于好果。腐生细菌主要包括藤黄微球菌(Micrococcus lutens)、科氏葡萄球菌(Staphylococus cohnii)、短芽胞杆菌(Brevibacillus choshinensis)和多粘类芽胞杆菌(Paenibacillus polymyxa);腐生真菌主要包括粉红聚端孢菌(Trichothecium roseum)和Neofusicoccum parvum。从龙眼果实腐生细菌和真菌的鉴定结果看,未分离到龙眼果实采后病原菌,说明采后病原菌的侵染不是龙眼果实腐烂的主要因素。3短短芽胞杆菌主要功能成分分析对短短芽孢杆菌FJAT-0809-GLX活性物质提取方法优化的结果表明,采用大孔树脂法和氯仿萃取法这2种方法提取的短短芽胞杆菌FJAT-0809-GLX活性物质的得率和抑菌效果有所不同,大孔树脂法提取的物质得率较高,为1.593 g/L,抑菌效果也最好,抑菌圈直径为27.33 mm。进一步对大孔树脂提取法各因素的优化表明,短短芽胞杆菌FJAT-0809-GLX活性物质收集的最佳方案为:将短短芽胞杆菌FJAT-0809-GLX发酵液常温3600 rpm离心30 min,取上清液,按比例与40 g/L大孔树脂AmberliteXAD16混合,28℃,160 rpm振荡4 h后,填柱,并用水洗柱后,采用3倍洗脱体积的丙酮洗脱,收集丙酮洗脱液,40℃旋转蒸发进行浓缩,所得物质即为短短芽胞杆菌FJAT-0809-GLX活性物质。短短芽胞杆菌FJAT-0809-GLX活性物质溶于二甲基亚砜、超纯水,不溶于石油醚。随着处理温度的升高,短短芽胞杆菌FJAT-0809-GLX活性物质的抑菌圈直径逐渐减小,在100℃条件下,抑菌圈直径最小,为18.17 mm。在100℃条件下处理0 h、2 h、4 h和6 h后,其抑菌效果逐渐降低,抑菌圈直径分别为20.47 mm、16.67 mm、15.42 mm和13.39 mm。随着pH值的增大,活性物质的抑菌活性有所降低,当pH=7.0时,抑菌圈直径为20.52 mm,当pH=11.0时,抑菌圈直径为18.46 mm。短短芽胞杆菌FJAT-0809-GLX活性物质对青枯雷尔氏菌、尖孢镰刀菌、串珠镰刀菌、茄形镰孢菌、苹果树腐烂病菌和苹果轮纹病菌等6种植物病原真菌都具有较强的抑制作用,对大肠杆菌K88和沙门氏菌ATCC14028这2种动物病原菌也具有一定的抑制作用,对大肠杆菌K88的抑菌效价为29.22 U/mL。短短芽胞杆菌FJAT-0809-GLX活性物质在三氯甲烷、丙酮、乙醇和甲醇这4种不同有机溶剂中的共有功能成分包括10种,分别为邻苯二甲酸单(2-乙基己基)酯、双氢麦角胺、羟苯乙酯、邻苯二甲酸二丁酯、绵马次酸、3-异丁基-六氢吡咯[1,2-a]吡嗪-1, 4-二酮、二乙基二硫代磷酸、3-苯甲基-六氢吡咯[1,2-a]吡嗪-1, 4-二酮、4-(乙酰苯基)苯甲烷和尼泊金丙酯。其中,羟苯乙酯和邻苯二甲酸单(2-乙基己基)酯的相对含量较高,对大肠杆菌K88都具有抑菌效果。羟苯乙酯的抑菌效果最好,与硫酸链霉素(5 U/mL)的抑菌效果相差不大(P﹤0.05),抑菌圈直径为17.81±0.28 mm,最低抑菌浓度为1.57 mg/mL,推测羟苯乙酯是其主要的抑菌功能成分。4短短芽胞杆菌保鲜机理的分析短短芽胞杆菌FJAT-0809-GLX活性物质对龙眼果实、鲜切苹果和台湾大青枣都具有很好的保鲜效果,对龙眼果实的保鲜率为87.50%,并且可以防止腐生菌的侵染,保持果实色泽,维持可溶性固形物的含量,保持果实的风味。短短芽胞杆菌FJAT-0809-GLX功能成分对龙眼果实腐生真菌和细菌抑菌能力有所不同。羟苯乙酯对腐生真菌和细菌的抑菌能力最大,抑菌圈直径在18.41 mm-31.06 mm之间。短短芽胞杆菌FJAT-0809-GLX活性物质对龙眼果皮POD酶活性具有一定的抑制作用,当其浓度为150 mg/mL时,抑制率最高,为9.57%。对其功能成分的抑制作用分析表明,在其中起主要作用的为羟苯乙酯,当其为50 mg/mL时,对龙眼果皮POD酶活力抑制率为25.69%,其次为麦芽酚和苯酚。不同浓度短短芽胞杆菌FJAT-0809-GLX活性物质对二苯代苦昧酰基自由基(DPPH·)均具有一定的清除作用,且在一定范围内短短芽胞杆菌FJAT-0809-GLX活性物质清除自由基的能力与其浓度呈明显的量效关系,当其浓度为100 mg/mL时,对DPPH·的清除能力最强,清除率最高为61.35%。在其中起主要清除作用的功能成分为麦芽酚,当其浓度为200 mg/mL时,清除率最高为68.35%。不同浓度短短芽胞杆菌FJAT-0809-GLX活性物质对羟基自由基(·OH)的清除率也有所不同,其中以7.5 mg/mL时的清除率最高,达到98.20%,在其中起主要清除作用的成分为羟苯乙酯,当其浓度为150 mg/mL时的清除率最高,达到68.38%。5龙眼保鲜菌剂的研究将2‰琼脂和2-5%的NaCl添加到龙眼保鲜菌发酵液中制备成龙眼保鲜菌剂,其活菌含量较高,胶悬剂效果最好,质地均匀,无上下分层。龙眼保鲜菌剂对龙眼果实具有较好的保鲜效果,常温条件下(温度28-32℃,湿度50-70%)贮藏5 d,龙眼保鲜菌剂处理的腐烂率低于对照,仅为32.67±1.20%,对“早钟六号”和“解放钟”枇杷果实也具有较好的保鲜作用,保鲜率在80%-90%之间。此外,该保鲜菌剂还可以降低龙眼和枇杷果实的失重率,防止腐生菌侵染,维持可溶性固形物含量,保持果实风味。在高温高湿条件下,该保鲜菌剂还对不同鲜切水果(西瓜、皇冠梨、苹果、草莓和台湾大青枣)也具有较好的保鲜效果,防止腐生菌的滋生,保持果实色泽和口味。更多还原

【Abstract】 This paper deals with the browning and decaying mechanisms of longan (Dimocarpus longan Lour.) by analyzing the variation of enzyme activity on pericarp in the course of fruit decomposition. Meanwhile, the fresh-keeping microorganism was screened, and its biological characteristics and fresh-keeping mechanism were also studied. Finally, the fresh-keeping agent was prepared by studying the bacterial fermentation parameters. The main results were summarized as follows:1. Screening of fresh-keeping microorganism for longan fruit and its microbiological characteristicsFrom our lab strain library, six antagonistic strains with high inhibition against saprophytic fungi and bacteria of longan fruit were screened. Brevibacillus brevis FJAT-0809-GLX had the best inhibition effect, whose inhibition zone diameters were 15.87 mm to saprophytic bacteria and 6.27 mm to saprophytic fungi. The optimization experiment of cultural conditions of FJAT-0809-GLX was conducted. The results showed that the best cultural conditions for FJAT-0809-GLX were 180 rpm of rotation speed, 7.0 of pH value for cultural medium, 20 to 40 mL/250 mL of media amount and 48 h of cultural time. Genome sequencing results of FJAT-0809-GLX showed that the genome size of the strain was 6 Mb, including 5666 genes with 933 bp of average size. Its genomic density was 87.83% and GC contents were 47.30%, while 177 special genes were found to exist in the strain.2. Rotting mechanism of longan fruitAlong with fruit rotting, no significant change was observed in the structure of exocarp of longan fruit. But its structures of endocarp changed dramatically. Rupture of endocarp started at 2nd rotting grade fruit. Cell cytoskeleton of endocarp completely disintegrated at 4th and 5th rotting grade fruit. Some hyphae were observed in the ruptured endocarp. The longan fruits were divided into six rotting grades according to the rotting characteristics of pericarp and pulp.Respirational rate of longan fruit increased along with the rotting. The respirational rate was 61.17 mg/kg·hr for 0 rotting grade fruit and increased to 382.71 mg/kg·hr for 4 rotting grade fruit. The total soluble solids of longan fruit decreased along with rotting of the fruit.Saprophytic fungi and bacteria of longan fruit were isolated and identified. The amounts and species of fungi and bacteria were found to be more in rotting fruits than that in fine fruits. The saprophytic bacteria were identified as Micrococcus lutens, Staphylococus cohnii, Brevibacillus choshinensis and Paenibacillus polymyxa. The saprophytic fungi were identified as Trichothecium roseum and Neofusicoccum parvum. No pathogenic bacteria and fungi were isolated from the rotting fruits, which indicated that infection of pathogenic bacteria and fungi to longan fruit was not the main factors for its rotting. 3. Functional components of Brevibacillus brevisDifferent extracting methods for the active substance from B. brevis FJAT-0809-GLX were compared. The results showed that macroporous resin method was the best one for the extraction with 1.593 g/L of yield rate. Active substance extracted by macroporous resin method had higher antibacterial activity than that by chloroform. The factors of macroporous resin method for the active substance from FJAT-0809-GLX were compared. Active substance could be obtained according to followed method. The fermentation broth of FJAT-0809-GLX was centrifuged at 3600 rpm for 30 min. The supernatant was mixed with 40 g/L macroporous resin and vortexed for 4 h at 160 rpm. The column was filled with the mixture and eluted with 3 times given column volume acetone. The eluent was collected and concentrated by rotary evaporation at 40℃.Active substance from FJAT-0809-GLX could be dissolved in dimethylsulfoxide and ultrapure water, but not in petroleum ether. The antibacterial activity of active substance from FJAT-0809-GLX decreased as heating temperature increasing. The antibacterial circle diameter of active substance heated with 100℃was 18.17 mm. The antibacterial activities of active substance heated for 0 h, 2 h, 4 h and 6 h with 100℃were different. Their antibacterial circle diameters were 20.47, 16.67, 15.42 and 13.39 mm, respectively. Antibacterial activity of active substance decreased as the pH value increasing. The Inhibition zone diameter was 20.52 mm as the pH value was 7.0, and it was 18.46 mm as the pH value was 11.0. The active substance had antibacterial and antifungal activities to Ralstonia solanacearum, Fusarium oxysporum, Fusarium moniliforme, Fusarium solani, Valsa mali Miyabe et Yamada and Physalospora piricola. It also had inhibition activities to Escherichia coli K88 and Salmonella ATCC14028. Its antibacterial potency to E. coli K88 was 29.22 U/mL.Ten common functional components were detected in chloroform, acetone, ethanoland methanol extractions from active substance including 1,2-Benzenedicarboxylic acid,mono(2-ethylhexyl)ester, Ergotaman-3’,6’,18-trione,9,10-dihydro-12’-hydroxy-2’-methyl-5’-(phenylmethyl)-,(5’à,10à)-(dihydroergotamine), Ethylparaben, Dibutyl phthalate, 2,5-Cyclohexadien-1-one,3,5-dihydroxy-4,4-dimethyl-, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, Diethyldithiophosphinic acid, Pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-(phenylmethyl)-, (4-Acetylphenyl)phenylmethane and Propylparaben. The relative contents of 1,2-Benzenedicarboxylic acid,mono(2-ethylhexyl) ester and ethylparaben were higher than others. The inhibition zone diameter of ethylparaben was 17.81±0.28 mm, which was almost the same as that of streptomycin sulfate (5 U/mL). And its minimal inhibitory concentration (MIC) was 1.57 mg/mL. Ethylparaben may be the main functional component of the active substance from FJAT-0809-GLX.4. Fresh-keeping mechanism of Brevibacillus brevis for longan fruit The active substance from B. brevis FJAT-0809-GLX had better fresh-keeping effects for longan, apples and Taiwan green-jujubes. The fresh-keeping rate was 87.50% for longan fruit. It could also inhibit the growth of saprophytic fungi and bacteria and keep their sensory qualities and total soluble solids.Among the functional components, ethylparaben had better inhibition effects on saprophytic fungi and bacteria, with inhibition zone diameters ranged from 18.41 mm to 31.06 mm. The active substance from FJAT-0809-GLX could inhibit the POD activity from longan. The inhibit rate was 9.57% at a concentration of 150 mg/mL. Among the functional components, ethylparaben played the main role, whose inhibit rate to POD activity was 25.69% at a concentration of 50 mg/mL. The active substance from FJAT-0809-GLX had certain clear rate on DPPH·, which had a dose-effect relationship with its concentration. The clear rate was 61.35%, when its concentration was 100 mg/mL. Among the functional components, maltol played the main role with 68.35% of clear rate on DPPH·. Clear rates of active substance from FJAT-0809-GLX on·OH were different with different concentrations. Clear rate on·OH was 98.20%, when the concentration was 7.5 mg/mL. Ethylparaben played the main role; its clear rate on·OH was 68.38%, when its concentration was 150 mg/mL.5. Study on fresh-keeping microbial agent for longan fruitFresh-keeping microbial agent for longan fruit could be prepared by adding 2‰agar and 2% to 5% NaCl to fermentation broth of B. brevis FJAT-0809-GLX. The content of FJAT-0809-GLX cells in the colloidal suspension agent was high. And the agent had the characteristic of uniform texture.Fresh-keeping microbial agent for longan fruit had better fresh-keeping effect to longan fruits under the room temperature. The rotting rate, which was 32.67±1.20%, was lower than that of control. It also had better fresh-keeping effect to‘Zao Zhong Six’and‘Jie Fangzhong’loquat fruits, which were in the range of 80% to 90%. This fresh-keeping microbial agent could inhibit the growth of saprophytic fungi and bacteria on different fresh-cut fruits, including watermelon, pear, apple, strawberry and Taiwan green-jujube and keep their sensory qualities.更多还原