| Agaricus bisporus is the most widely cultivated edible fungus in the world,China is also a big producer of this mushroom.China’s reported output accounts for two-thirds of the world’s reported mushroom production in recent years.Spawn production is the primary link of mushroom cultivation,but at present,there is a lack of systematic and efficient strain quality evaluation system and quality control system for mushroom breeding in China,which seriously restricts the sustainable development of mushroom production in factories.In view of the above situation,this paper conducted a series of studies on the main varieties of A.bisporus in China,As2796 and W192,and the main results are as follows:1.Hyphae amount is one of the species quality quantitative evaluation index,in this paper,the liquid culture mycelia of mushroom species As2796 and W192 in different culture time and medium formulations were studied.The methods of extraction and determination of chitin,ergosterol and nucleic acid in mycelium were determined and optimized.It was found that there was a good linear relationship between the OD value of content of chitin,ergosterol and nucleic acid in As2796 and W192 mycelia and mycelial mass.All the determination coefficients R2 were more than 0.999.This indicated that chitin,ergosterol and nucleic acid could be used as indicators to measure the mycelia amount of A.bisporus.The concentration of the two in PD liquid medium at 24℃after 7 days used as the reference value for the determination mycelial mass of A.bisporus.2.According to the content of chitin and ergosterol in liquid culture mycelia,the relative mycelia quantity of A.bisporus was measured and calculated.The content of chitin and ergosterol in pure mycelium produced by A.bisporus strain As2796 under liquid culture was118.06 and 4.47μg·mg﹣1（the ratio of chitin:ergosterol was 26.41）,respectively,and strain W192 can reach 121.93 and 6.15μg·mg﹣1（the ratio of the two is 19.83）.On this basis,the computative mycelial mass of strain As2796 under liquid culture were 123.3 and 180.0 mg·g﹣1,respectively.And strain W192 can also reach 120.8 and 139.2 mg·g﹣1,respectively.3.Whether the spawn is aging or not is one of the evaluation indexes of spawn quality.The aging mechanism of A.bisporus was studied by high-throughput transcriptome sequencing,mycelial cell transmission electron microscopy（TEM）and the determination of chitin and ergosterol.RNA-seq analysis was performed on As2796 and the aging strain As2796Y at the transcriptome level using high-throughput sequencing technology,and taking the normal strain As2796 as the reference genome,the sequencing results showed that there were 1610 differentially expressed genes in the aging strain As2796Y.Among them,877were up-regulated and 733 were down-regulated.Function enrichment of GO and KEGG was shown in transcriptome analysis,a large number of down-regulated genes of the aging strain As2796Y were enriched in the carbon metabolic pathway,and the gene expression of enzymes related to glycolysis pathway decreased.It shows that carbon metabolism play an important role for the strain energy,foundation and lower energy metabolism disorder could be the primary cause of strain aging.TEM images also showed that the plasmolysis and vacuole enlargement of the aging strain As2796Y were more serious than that of the normal strain,and the ratio of chitin/ergosterol was higher than that of the normal strain As2796,which may be a defense mechanism for the aging strain to resist environmental damage by thickening the cell wall in the later stage.Therefore,it is concluded that the mechanism of strain aging of A.bisporus could be the inhibition of enolase synthesis in glycolysis,which results in the reduction of ergosterol content,and further degrades the cell membrane stability,and finally leads to the plasmolysis and exudation of yellow water.4.In order to improve the nutritional structure of mushroom spawn,new synthetic substrates with high yield,high quality and disease resistance were further screened.The statistical approaches（single factor and orthogonal test）were adopted and used to screen 43kinds of nitrogen-rich matrix materials,and the growth of mushroom mycelium,the mycelial mass and N content of the matrix formula were used as the index respectively.The results showed that the optimal cultivation medium is as follows:3:1 mass ratio of vermiculite to perlite,based on this weight,corn flour 10%,feather powder 15%,and bran 15%.Under these optimized conditions,the mycelial mass of matrix spawn was increased to 120%and170%than wheat spawn,respectively based on chitin and egrosterol,compared to breatheable bag wheat spawn and the small bag wheat spawn.The fruiting production was improved by3%and 17%than two controls,and the incidence of green mould was only 10%of control.5.In order to establish a perfect quality control system for production of mushroom spawn,fifteen GMP regulations for mushroom spawn-making with breathable bag were revised and twelve SSOP documents of mushroom spawn-making with breathable bag were formulated in detail.During the process of establishing of HACCP system,three hazard factors in nine processes were identified and hazard analysis（HA）was performed.On this basis we defined four critical control points（CCP）in the spawn production process were defined.Finally,the control system for the industrial production of breathable bag mushroom spawn was established in the trinity of GMP,HACCP and SSOP. |
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