Optimization Of Fermentation Conditions Of Sophorolipids By Genetically Engineered Strains And Its Application

In recent decades,the development of economy and sustainable biotechnology has gradually replaced existing product synthesis methods based on petrochemistry.Sophorolipids(SLs),as a biosurfactant produced by fermentation of a selected species of nonpathogenic yeasts from renewable natural materials,not only has good general properties such as foaming,wetting,and emulsification,but also has excellent properties such as low toxicity,biocompatibility,and biodegradability.Because of its excellent characteristics,it has a wide range of applications in petroleum,food,daily chemicals,medicine and environmental protection.However,compared with the relatively low production cost of petroleum-based chemical surfactants,the high cost of large-scale production of SLs is still an obstacle to its economic competitiveness,and the actual application of SLs has not been truly broken through.In this paper,the main research included two respects:optimization of SLs fermentation conditions and its applications.The main research contents and results are as follows:1.Construction of genetically engineered strains and optimization of fermentation conditionsSophorolipids,as a kind of microbial surfactants,are mainly divided into acidic-type SLs and lactonic-type SLs according to their structures.In view of the different biological characteristics of acidic and lactonic SLs,genetically engineered strains capable of producing acidic or lactonic SLs were obtained to increase the production of acidic and lactonic SLs,thereby reducing the separation cost of downstream processes and realizing the wide application of SLs.In this paper,using Starmerella bombicola as the wild-type strain,through genetic modification,the high acidic SLs-producing strain Δsble was successfully constructed,and a high lactonic-producing strain ΔrlpΔleu3 was constructed by our laboratory.The pH,temperature,and dissolved oxygen of the SLs production of △sble and △rlp△leu3 were optimized at the level of shaking flask and fermenter to obtain the most suitable fermentation parameters.According to the optimization results,the △sble strain hardly produced lactonic SLs under the optimal fermentation conditions,the production of acidic SLs was 103.3 g/L,the volumetric productivity was 10.3 g/L/d,YSLs/Glu was 1.29 g/g.The production of lactonic SLs by the ΔrlpΔleu3 strain under the optimal fermentation conditions was 79.1 g/L,accounting for 75.9%of the total SLs production.At this time,the volumetric productivity was 7.91 g/L/d,YSLs/Glu was 1.0 g/g.In addition,the effect of feeding of glucose and rapeseed oil in batch fermentation in the 7.5-L fermenter on the production of acidic SLs by △sble was also studied.From the results,the production of acidic SLs produced by the fed-feeding fermentation of △sble strain reached 189.9 g/L,the volumetric productivity was 18.99 g/L/d,which increased by 83.8%and 84.4%,respectively,compared with batch fermentation.2.The study on the antibacterial activity of lactonic sophorolipidsAcne vulgaris is a complex skin disease.At present,70～97%of young people suffer from acne vulgaris in varying degrees.Different from acidic SLs,lactonic SLs have good antibacterial,antiviral and anti-biofilm biological activities.In this paper,the inhibitory effects of lactonic SLs on Propionibacterium acnes and several common microorganisms Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa,Klebsiella pneumoniae,Candida albicans were investigated through inhibition zone and nutrient broth dilution method.The results showed that lactonic SLs at 6 mg/mL could completely inhibit the growth of gram-positive bacteria S.aureus and gram-negative bacteria E.coli,P.aeruginosa,K.pneumoniae,and the inhibition rate against the pathogenic fungus C.albicans was 68%.The significant inhibitory effect of lactonic SLs on P.acnes causing acne was first reported.When the concentration of lactonic SLs reached 0.12 mg/mL,the growth of P.acnes could be completely inhibited.In addition,the inhibition effects of plant extract including purslane and scutellaria root extract on the above pathogenic microorganisms were also investigated.The results indicated that 1 mg/mL purslane extract and 0.45 mg/mL scutellaria root extract could completely inhibit the growth of these common microorganisms,respectively.This made it possible to combine lactonic SLs and plant extracts as antibacterial ingredients into the ointment preparation to prepare a new type of anti-acne ointment,thereby reducing the troubles of young people from acne vulgaris.