Fresh-keeping Mechanism Of Brevibacillus Brevis For Longan (Dimocarpus Longan Lour.)

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.