| Objective: The problem of wine fermentation stagnation or retardation caused by insufficient assimilable nitrogen is widespread globally,and the artificial addition of nitrogen sources remains the main measure to solve this issue.However,if the amount of addition is not properly controlled,it could lead to retention of excessive assimilable nitrogen in grape juice,causing yeast cell apoptosis and affecting fermentation efficiency.It may even result in the formation of some undesirable compounds such as ethyl carbamate and biogenic amines,etc.,seriously affecting the nutritional and safety aspects of food and posing risk to human health.Proline is an abundant natural nitrogen source in grape juice.However,it cannot be effectively utilized during fermentation due to the inhibition of nitrogen metabolism.Therefore,based on the preliminary work,this research aims to screen yeast strains that can effectively utilize proline through laboratory adaptive evolution and analyze the metabolites after proline utilization.With providing theoretical and technical support for the feasibility of developing natural nitrogen proline.Methods: The study used three laboratory strains with excellent fermentation performance screened by the team as basis.adoping laboratory adaptive evolution technology as the main approach,and EMS mutagenesis as a supplementary method,to screen mutant strains that can effectively utilize proline.SNV mutation sites were identified through genomic DNA detection.The fermentation ability,hydrogen sulfide production ability,alcohol tolerance and other characteristics of the mutant strains were analyzed.In addition,metabolomics technology was employed to identify the metabolites of the mutant strains after using proline and the potential effects of differential metabolites on health were analyzed.Results:(1)After 325 generations of continuous adaptive evolution and double rounds tests in simulated fermentation systems,a total of 8 mutant strains that can effectively utilize proline were obtained.The more stressful conditions in adaptive evolution,the greater the stability and adaptability of mutant strains.(2)SNV mutation sites were detected in mutant strains WL2-1,WL2-2,WL5-1 and WL5-2,with and varying degrees of mutations occuring in the exon regions of genes related to nitrogen metabolism,proline metabolism and nitrogen repression,which could cause protein changes.Especially in WL2-2,WL5-1 and WL5-2 with more mutation sites.(3)Laboratory fermentation tests were conducted on mutant strains WL2-1,WL5-1 and WL5-2.The fermentation rates of each strain significantly better than their parent strains,WL2 and WL5.In the normal simulated grape juice(CDGJM)fermentation system,WL5-1 and WL5-2 completed fermentation 144 h and 120 h earlier than their parent strain WL5,respectively.However,in CDGJM with insufficient assimilable nitrogen and abundant proline,the strains completed fermentation 72 hours earlier.WL2-1 completed fermentation 24 hours earlier than its parental strain WL2 in both fermentation systems.All the three mutants were able to consume more than 1000mg/L of proline.(4)Metabolomics analysis showed that mutant strains in the fermentation systems with insufficient assimilable nitrogen and abundant proline could produce more beneficial metabolites with antibacterial,anti-inflammatory,and antioxidant functions.In addition,it could significantly down-regulate the contents of carcinogens 1,5-naphthalenediamine and diethanolamine.Conclusion: Laboratory adaptive evolution can obtain mutant strains that effectively utilize proline,and the metabolic substances produced by these strains in the fermentation system with insufficient assimilable nitrogen and abundant proline have more health-promoting effects.It is highly feasible to use proline,a natural nitrogen source in grape juice,as a supplement when assimilable nitrogen is insufficient. |
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