| The improvement of acid protease activity in Aspergillus oryzae has practical value for soy sauce brewing industry. Breeding of food-grade strain with high activity of acid protease is an efficient approach to enhancing the protein utilization rate and conversion efficiency of free amino acids in soy sauce fermentation. Based on the theory of asymmetric inactivation and the technique of genome recombination, the protoplasts released from A.oryzae HN3042 and A.niger CICC2377 by enzymatic hydrolysis were induced to fuse by pulse electrofield and subsequent screening of target strains was carried out. Additionaly, the performances of the screened fusant were preliminarily examined in soy sauce fermentation. The main researches and their results are as follows:Firstly, the parameters of protoplast preparation and inactivation were explored. The conditions for protoplast release from A.oryzae HN3042 and A.niger CICC2377 were as follows: cultivation in a submerged medium consisting of 1.0 % maltose, 1.0 % polytephone and 0.5 % yeast extract for 18 and 21 h respectively at 30 oC and 100 rpm, and then incubating the mycelia with an enzyme cocktail, total concentration of 15 mg/mL and consisting of lywallzyme, cellulose R-10 and snailase with the ratio of 1:1:1, for 2.5 h at 30 oC and 80 rpm, and then regeneration on the high osmotic soybean extract solid medium. Under these conditions, the release and regeneration efficiency of protoplasts from A.oryzae HN3042 and A.niger CICC2377 both reached 107 /mL and above 30 %, respectively. According to the time curse for asymmetric inactivation, the dose of UV irradiation with a 15 W ultraviolet lamp to protoplasts from A. oryzae HN3042 was 15 min under the light with a distance of 13 cm. As for the heating inactivation towards protoplasts from A.niger CICC2377, the parameter was at a water incubation of 65 oC for 10 min. After the asymmetric inactivation, the survival frequency of protoplasts on regeneration plates was nearly zero.Secondly, genome recombination between the inactivated protoplasts was iuduced by pulse electrofield. 694 strains of fusants with higher growing speed than A.oryzae HN3042 were randomly selected and the diploidization of heterokaryons was conducted with d-camphor with the content of 500 mg per liter Czapek medium. After successive subcultures for 10 generations on camphor medium, subsequent haploidization of the fused diploids was done with benomyl with the content of 1 mg per liter Czapek medium. 10 stable fusants with an enhancement of above 50 % in activity of acid protease than A.oryzae HN3042 were obtained, in which the fusant of F76 showed an improvement of 82.19 %. Furthermore, the analysis of mycelia morphology, SDS-PAGE and esterase isoenzyme profiles, ploids of the conidium nucleus and randomLy amplified polymorphic DNA (RAPD) were applied to identify the fusants through phenotypic and genetic relationship. The results revealed there were considerable differences between the fusants and their parental strains. According to these line evidences, it can be safetly concluded that genome recombination occurred in the process of protoplast fusion.Thirdly, by salting-out, ion-exchange and molecular sieve chromatography, the acid protease from F76 was purified 17.09 folds, with a yield of 33.56 % and a specific acitivity of 4015.49 U/mg. Purified acid protease moved as single band on SDS-PAGE. Native PAGE ascertained the purified protein monomeric and the molecular weight was deduced to be about 50 kDa by SDS-PAGE. Circular dichroism studies of this enzyme revealed that the contents ofα-helix,β-sheet,β-turns and aperiodic structure in the purified protein were 7.1 %, 39.4 %, 24.7 % and 32.1 %, respectively. Compared with that of A.oryzae HN3042, the content of helix decreased 14.3 % and the content ofβ-structure and aperiodic coil increased 12.8 % and 5.7 %, which indicated that conformation of acid protease from F76 was more flexible than that from A.oryzae HN3042. Acid protease from F76 was stable in the temperature range of 35 oC-45 oC with an optimum temperature for activity of 40 oC and the enzyme was active in the pH range of 3.0-6.5 with an optimum pH of 3.5. Activity of the acid protease from F76 was significantly improved by Mn2+ and inhibited by Co2+, and pepstatin A, a specific inhibitor for aspartic protease, almost completely inhibited the activity of acid protease from F76 with a concentration of 10μmol/L. The value of Km and Ea of the purified acid protease from F76 showed an decrease of 27.08 % and 0.77 % respectively than that of A.oryzae HN3042 and an increasement of 20.0 % in heat-induced attenuation index than that of A.oryzae HN3042 was responsible for the slight decrease of thermostability of the enzyme.Then, statistical optimization was applied to maximise the production of acid protease from F76. The result showed that optimum medium was the enriched bran and wheat flour medium, with an addition of 1.24 % yeast extract, 1.15 % glucose, 0.05 % MnCl2, 0.05 % MgSO4 and 0.06 % Na2B4O7. Cultured on this enriched medium, F76 produced the activity of acid protease from 1308 U/g to 1703 U/g, an increase of 30.2 %. The optimum conditions for acid protease production were as follows: inoculum volume of 8.59×105 condium per gram of koji-seed medium, incubation temperature of 31 oC, initial mositure content of 57 % and incubation time of 86 h. Under these conditions, the activity of acid protease from F76 reached 1936 U/g from 1703 U/g, a further increase of 13.68 %. By statistical optimization, the activity of acid protease from F76 in koji-seed medium was enhanced 48.01 %.Finally, the performances of F76 in soy sauce fermentation were preliminarily evaluated. The content of total titration acid, soluble non-salt solids, total soluble nitrogen and amino-type nitrogen in fermented mash at the stage of 31 days was increased by 1.8 %, 8.0 %, 8.1 % and 6.9 % than that of A.oryzae HN3042, respectively, indicative of some improvement in compositions of the fermented mash. At the fermentation stage of 80 days, the content of proteins with molecular weight of above 10 kDa and within 5-10 kDa in fermented mash by F76 was lower 5.56 % and 10.65 % than that by A.oryzae HN3042, respectively, which indicated that F76 had better degradating performance towards soybean proteins during mash fermentation than the parental strain of A.oryzae HN3042. After a 90-day aging period, the content of sweet, umami- and bitter- free amino acids in the fermented mash by F76 was 24.98 %, 10.03 % and 61.95 %, respectively, while the counterparts by A.oryzae HN3042 was 24.63 %, 9.86 % and 62.43 %, respectively. The slight increase in sweet and umami amino acids and remarkable decrease in bitter amino acids showed that the flavor of fermented mash by F76 was more pleasant than that by A.oryzae HN3042. Volatile compouds analysis showed that the content of total volatile compouds in fermentated mash by F76 was 79.87 % while the counterparts by Aoryzae HN3042 was only 73.37 %, indicating the difference in flavor of fermented soy sauce between F76 and A.oryzae HN3042.This study provides a preliminary foundation for the application of F76 in soy sauce fermentation.
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