Studies On Protoplast Fusion And Synthetic Complete Medium Of Mortierella Alpina

Arachidonic acid plays an important role in many physiological processes.The need for ARA as an important functional oil has been growing rapidly in the fields of food,medicine and cosmetics.In recent years,the production of ARA using the method of fermentation by Mortierella alpine,has been the main method of industrial production because of its high content of ARA and reasonable composition of oil.M.alpina is also the designated strain of ARA production in "Food safety national standards for the use of food nutrition fortifier"(GB 14880-2012).Breeding is one of the key factors to improve the yield and quality of ARA.The composition of the oil from Mortierella alpina affects the quality of the final product directly.In the actual production process,it was found that when the content of tetracosanoicacid(24C,lignoceric acid)in the total oil is more than 8%,the final product of oil precipitates at low temperature.Therefore,in this study,the strains with 24C percentage less than 8%in total oil were screened by natural hybridization method.The YS-88447 strain which has a low content of 24C and a low yield of ARA,was hybridized with the strain G5(an ARA high yield strain with a relative high 24C content).A strain,ZJ-3,with a low content of 24C but relatively high percent of ARA was obtained by screening,its content of 24C was 3.39%,and the final yield of ARA was 5.21 g/L.The percentage of 24C in total oil has been reduced 170%and the percentage of ARA has been increased 6.72%compared to in the G5 strain.Furthermore,the method of Fe2+ and magnetic field joint action on Mortierella alpina to induce mutations for achieving ARA high yield strain was established in this study.high yield strain,G16,with a high total oil content of 44.85%and an ARA yield of 6.43g/L was obtained,and its content of 24C was about 8.98%.In this study,the protoplast fusion technique was applied to obtain ARA high yield strains with low 24C percentage and also to speed up the whole breeding process.ZJ-3 and C16 were fused by protoplast fusion method,and the related factors of protoplast preparation and regeneration were studied,and the optimum conditions were achieved.The screening step is crucial,to improve the screening efficiency and accuracy,and two methods were studied.They are the double inactivation method and the flow cytometry method for screening fusants.After the fusants regeneration and fermentation,a strain,YR-2,was selected,its content of total oil was as high as 43.18%,the content of 24C was 3.26%,and the yield of ARA was up to 6.32 g/L.The yield stability was confirmed by subculture.This study provides a new idea for the screening of Mortierella alpina.From lab to factory,ARA fermentation production of Mortierella alpina always use natural media(yeast).The composition of yeast is complex,and different batches have different qualities,which is not conducive to the fine experiment analysis and stability of industrial production.In this study,the addition conditions in medium of amino acid mixture,inorganic salts and other growth factors were determined by classification and single factor experiments.Although the final yield only achieved 70%of the yield from natural medium,it provides strong support for research on cell growth,metabolism,genetic breeding and so on.Furthermore,the optimised amino acid composition,vitamin concentration and several inorganic salts were added to the natural fermentation medium to verify their effect on ARA's yield.The result showed that the yield of ARA in the experimental group was 14.7%,which is higher than that in the control group.It illustrated that amino acids,vitamins and inorganic salts had a promoting effect on the fermentation of Mortierella alpina.It demonstrated a research foundation for the optimization of medium for industrial production.