Breeding Of Natamycin High Yield Strain And Low PH Resistance Strain By Mutagenesis

Natamycin is a polyene macrolide antifungal antibiotic,which can effectively inhibit fungal growth and mycotoxin production.Natamycin is characterized by low dose,high efficiency and high safety.It is widely used in food raw material preservation,finished product preservative and medicine.At the same time,with people’s attention to food safety management,the market demand of natamycin increased year by year.But the low fermentation level and high production cost of natamycin limit its further development and application.In recent years,with the reports of mutagenesis breeding and various screening methods,it is possible to improve the yield of natamycin and screen strains resistant to low p H by mutagenesis breeding.In this paper,Streptomyces gilvosporeus TUST01 was used as the starting strain,and a strain with high yield and a strain with low p H resistance was screened out by mutagenesis breeding combined with appropriate screening methods,The mechanism of acid resistance of Y-10 was also studied.In order to improve the yield of natamycin,reduce the restriction of conditions of natamycin fermentation production,and reduce the consumption of alkali to reduce the cost,and provide the original starting strain with excellent characters for further genome rearrangement breeding.The main results are as follows:(1)In this paper,the fermentation performance of the starter strain S.gillvosporeus TUST01 was identified.The maximum yield of natamycin was 14.76 g/L under the condition of 5 L fermentation tank supplemented culture.It was hoped that the yield of natamycin of the high-yielding strain was increased by 20%or more than that of the starter strain.Under the condition of 5 L fermentation tank supplemented culture without controlling p H,the growth of strain was obviously limited by low p H environment during fermentation.By comparing various parameters,the target tolerance of acid-resistant strain screening was determined to be 4.4.In order to obtain the target strains,an efficient and reasonable screening method was established,and the optimal screening conditions such as the best liquid loading amount and the best sealing mode of 24-hole plate were determined.(2)In this study,S.gilvosporeus TUST01 was selected as the starting strain.High-yielding strain S.gilvosporeus Y-4-23 was screened using diethyl sulfate(DES)mutagenesis and Atmospheric and Room Temperature Plasma(ARTP)technology combined with streptomycin resistance screening and high throughput on 24 deep-well plates.The 5 L fermentation tank feed culture further improved the fermentation level of the strain,and the highest yield of natamycin reached 18.07 g/L after 120 h fermentation,which was 22.43%higher than that of the original strain.The yield of natamycin and biomass of Y-4-23 were measured every three generations after 15 successive generations of culture.The results showed that the genetic property of Y-4-23 was stable.(3)ARTP mutagenesis combined with low p H plate screening and 24 deep well plate screening was used to finally screen a strain of S.gilvosporeus Y-10.Acid-resistant strain Y-10 grew well on p H 4.0 plate,and the yield of natamycin and biomass were significantly increased under shaking flask fermentation conditions of p H 4.4 and p H 7.4,indicating that Y-10 had good acid-resistant ability.Process magnification was carried out in 5 L fermenter under different p H conditions.When p H was not controlled,strain Y-10 grew vigorously and its maximum biomass was 32.40 g/L,which was 56.22%higher than that of the original strain,but its fermentation titer was only 5.58 g/L.The biomass of Y-10 reached29.25 g/L under the condition of normal 5 L fermentation tank with p H 6.3.During the whole fermentation process,Na OH concentration of 2 mol/L was consumed 350 m L less than that of the starter,but the yield of natamycin was only 5.80 g/L.The high biomass also proved that Y-10 had a good acid resistance,while the low yield might be caused by the fact that during the fermentation process of 5 L fermenter,the bacteria had less contact with the medium due to the abnormal upwelling of the bacteria and did not make full use of the carbon and nitrogen sources.The Y-10 strain was cultured for 15 generations and the yield of natamycin and biomass were determined.The results showed that the genetic traits of the screened strain were stable.(4)The effects of low p H on S.gilvosporeus Y-10 were studied at physiological level.The acid-resistance mechanism of Y-10 was studied by comparing the differences of intracellular p H and intracellular ATP between Y-10 and starter.The results showed that Y-10 had higher levels of intracellular p H(p H_i)in the lower p H environment.The relative stability of p H_iis conducive to the growth and metabolism of Y-10 in the low p H environment.In addition,the cellular ATP level of the Y-10 is significantly higher than that of starter bacteria,and the ATP can be able to keep the H~+in the cell out of the cell to maintain p H_iand ensure Y-10 grows normally under acidic conditions.Metabolomics analysis was carried out on the selected low-p H resistant strain Y-10 and the starter bacteria,and principal component analysis and orthogonal projections to latent structures analysis were carried out on a variety of small molecular metabolites detected.Significant changes in fatty acids as biomarkers were found between S.gilvosporeus TUST01 and S.gilvosporeus Y-10.The relative contents of saturated fatty acids and unsaturated fatty acids in Y-10 were significantly lower than those in the starter,which directly affected the fluidity of cell membrane.The low yield of natamycin of Y-10 may be affected by membrane fluidity.The relative content of mannose and D-glucose in the starter was higher than that in Y-10,which was consistent with the higher yield of natamycin in the starter.