Isolation And Screening For Limonin-producing Endophytic Bacteria And Characteristics Research

Limonin is a compound of highly oxidized triterpenoids that has many human disease-preventing biological activities,including anti-cancer,antibacterial,antinociceptive and anti-inflammatory activities and anti-HIV.At present,limonin is mainly extracted from plant,with low yield and complicated process.However,microbial fermentation,which has been used for the large-scale production of various bioactive substances owing to its short hours,low cost,fine control,small occupied land.What's more,it is conducive to the protection of plant resources.In this paper,the characteristics of endophytic bacteria were as the starting point,and the Citrus maxima(Burm.)Merr.cv.Shatian Yu.as the research object.Endophytes were isolated and identified in the different tissues of Citrus maxima(Burm.)Merr.cv.Shatian Yu.The aim of this study was to screen out microorganisms that could produce limonin and conduct related research.The research content and the results obtained are as follows:(1)Isolation,identification and antibacterial properties of endophytes in Citrus maxima(Burm.)Merr.cv.Shatian Yu.The endophytes in the peel and seeds were isolated from Citrus maxima(Burm.)Merr.cv.Shatian Yu.A total of five isolates were obtained from the tested plants: three(P,P1,P9)from the peel and two(Z1,Z2)from the seeds.According to morphological characteristics and 16 S r RNA gene sequencing identified them as Bacillus spp.;The fermentation broth which were dealt with different ways(stock solution,concentrate,ethyl acetate extract)on the antibacterial activity that against Salmonella,Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Pseudomonas aeruginosam was determined by the Oxford Cup method.The results showed that the fermentation broth concentrate and ethyl acetate extract of strains P and P9 inhibited the five pathogens to different extents,among which the antibacterial effect against Bacillus subtilis was the most obvious.while different treated samples of P1,Z1 and Z2,the bacteriostatic effect was not significant.Through the separation of protein and non-protein components,the results showed that the effective antibacterial components of P,P1 and Z1 were mainly concentrated in the non-protein component;both of the crude protein and non-protein component of the fermentation broth of strain P9 might play a bacteriostatic effect;the crude protein of strain Z2 has a weak inhibitory effect on Staphylococcus aureus,and its non-protein component extract has a significant inhibitory effect on Pseudomonas aeruginosa.(2)Screening of limonin-producing endophytes.Five endophytes were cultured in three different mediums: inorganic salt medium,natural medium 1 and natural medium 2.The high-performance liquid chromatography and mass spectrometry were used to detect limonin in the fermentation broth.Two strains of limonin-producing bacteria were screened out,and limonin was produced in all three mediums.Among them,in the inorganic salt medium,the production of the P((2.961 ± 0.12)mg/L)was larger,and the production of the P9 was small,being(0.151 ± 0.08)mg/L;after adding the pomelo peel powder In natural medium 1,the production of strains P and P9 were significantly increased,which were(4.377 ± 0.11)mg/L and(0.299 ± 0.02)mg/L,respectively.It is about two times than the inorganic salt medium.The mass spectrometric analysis of the extract of the fermentation broth of strain P with high yield of limonin was carried out,and the ion peak of m/z = 471.20([M+H]+),493.18([M+Na]+)was detected,and its characteristic ions were detected.It is m/z = 161.06,425.20,which is consistent with the characteristic ion under the same conditions as the limonin standard,and it can be judged that this substance is limonin.It indicates that the endophytic bacteria from the Citrus maxima(Burm.)Merr.cv Shatian Yu contain natural limonin-producing microorganisms,and the presence of the precursor can significantly increase the yield.(3)Optimization of conditions required by Bacillus sp.P to produce limonin.The growth curve and the curve of limonin production of P were analyzed.It was found that the growth of P-cell and the production of limonin showed a consistent trend,and the growth amount reached the maximum at 16 h,while the yield of limonin reached a maximum of(3.176 ± 0.13)mg/L.The optimal conditions for the production of limonin by strain P were determined by single factor test(bacteria age,glucose content and inoculum size)and response surface analysis: the bacterial age was 15 h,the glucose concentration was 3.4 g/L,and the inoculum was 16%.Under this condition,the maximum yield of limonin was predicted to be 3.51 mg/L,and the verified value was(3.49 ± 0.23)mg/L.