Effects Of Additives On Aging Of Auricularia Auricula Liquid Spawn

The phenomenon of aging of liquid spawn occurs from time to time during the production of liquid spawn of Auricularia auricular,which has an adverse effect on the yield and quality of A.auricular.Adding additive agent to the culture medium can affect the growth of the A.auricular mycelium.However,previous studies focused on the effects of additive agent on the yield of mycelium,and there have been few reports on the effects of additives on the aging of liquid spawn.In this study,the A.auricular variety"Heiwei 15"was used as the test material to explore the effects of additive agent on the growth and aging of liquid spawn of A.auricular.We screen anti-aging and easy-aging media in which we analyze the mycelial antioxidant enzymes activity,substrate degrading enzyme activity,antioxidant substances,active oxygen concentration and membrane peroxidation degree change pattern.By transcriptome technology,we detect the different expressed genes of mycelium and determine the key gene and metabolism pathways playing a role in different kinds of culture media,which has theoretical and application value for in-depth understanding of the mechanism of A.auricula aging and improving the fermentation process of liquid spawn.The main findings are as follows:?1?Result of the effects of four additive agent on the aging of liquid spawn of A.auricular,showed that 25 mg/ml VB₁ has most significant inhibitory effect on mycelial aging.The growth rate was still 3.7mm/d on the 16th day of culture,48%higher than that of the control group,followed by potassium dihydrogen phosphate.The addition of magnesium sulfate can inhibit the mycelium aging,while was adverse to the growth of mycelium,and the maximum growth of mycelium was only 6.63 mg/ml.Adding excessive peptone source can promote mycelial growth but accelerate mycelial aging.The growth rate was reduced to 1.9 mm/d on the 16th day of culture,which was24%lower than that of the control group.Therefore,the result of screening the medium was that the medium added with VB₁ and potassium dihydrogen phosphate is anti-aging medium,and the medium added with excess peptone is an easy-aging medium.?2?With the increase of fungus age,the physiological and biochemical indexes of mycelium in the selection medium and the control medium showed regularity variation.Soluble protein content,amylase activity,and ascorbate peroxidase activity increased first and then decreased,indicating that the mycelial metabolic activity and substrate degradation activity significantly decreased and the mycelium activity weakened significantly after aging.Peroxidase activity change trend was not obvious,indicating that peroxidase does not play a major role in the aging process of liquid spawn of A.auricular.Catalase activity gradually increased during culture,and increased by 9.48 times,10.28 times,5.10 times,and 6.56 times in control group,kv group,kp group,and aging group,respectively at the end of the culture,which indicated that catalase played a major role in the active oxygen removal at the late stage of mycelium growth.The activity of superoxide dismutase gradually decreased,and decreased by 87.2%,65.7%,53.3%,and 91.5%in control group,kv group,kp groupand aging group respectively,on the 16th day,indicating that the superoxide dismutase was major enzyme removing active oxygen in the early stage of mycelial growth.?3?Comparison of the mycelial physiological and biochemical indicators between the screening medium and the control medium showed that adding VB₁ and potassium dihydrogen phosphate could increase the soluble protein content,activity of peroxidase,superoxide dismutase,ascorbate peroxidase and amylase significantly,however,decrease the active oxygen species H₂O₂concentrations and O^2-production rate.Moreover,VB₁ and potassium dihydrogen phosphate inhibited membrane lipid peroxidation effectively.Additionally,the addition of excess peptone significantly increased the concentration of H₂O₂ and O^2-production rate and inhibited the activities of antioxidant enzymes and substrate degradation enzymes.And the degree of cell membrane peroxidation of easy-aging medium was significantly higher than the control and anti-aging groups.It showed that the delay of mycelial aging by additives may be related to the active oxygen scavenging system.However,the activity of antioxidant enzymes decreased and the active oxygen was not removed effectively,which resulted in the damage of cell membrane.It is one of the important reasons for the aging of agaric liquid bacteria.?4?Based on transcriptome sequencing results,1350 and 2247 differential genes were obtained from anti-aging medium and easy-aging medium,respectively.The correlation coefficient of gene expression in the same medium was more than 0.9,indicating that the sample had good repeatability.At the same time,the correlation coefficient between different media was less than 0.9,which indicated that there was significant difference in the gene expression of mycelium in different additive media at the RNA molecular level.It was concluded that the additive significantly affected the gene expression of mycelium.?5?Functional annotation and enrichment analysis showed that the additive agent may affect the aging of A.auricular liquid spawn by influencing the mycelial nutrient metabolism and transport activity.The most differentially expressed genes in the anti-aging and easy-aging groups were the genes related to membrane and membrane component functions,indicating that the synthesis,repair and damage of cells and organelle membranes may be the main reason that different additive agent affect the aging of liquid spawn.KEGG analysis showed that different additives could change the mycelial activity and affect the aging of agaric liquid strain by mediating the metabolism of nutrients and energy.KEGG enrichment analysis showed that in addition to amino acid metabolism,the expression of some secondary metabolic pathways in sphingomyelin synthesis and aging medium was significantly up-regulated.According to this description,the additives in the anti-aging medium mediate cell membrane receptor activity,thereby increasing the mycelial proliferation activity and delaying the aging reaction.The secondary metabolism of A.auricula mycelia in easy-aging medium,especially the synthesis of terpenoids,may be the main reason to promote occurrence of the aging phenomenon.