Breeding Of Bacterial Cellulose-Producing Strains And Effects Of Bacterial Cellulose On NK Cell Function

Bacterial cellulose(BC)is a kind of cellulose with unique three-dimensional nano-network structure and is produced by microorganisms.It has high purity,high water absorption,excellent biocompatibility,and has potential applications in many fields,such as food,paper,cosmetics,biomedicine,etc.Static culture is the main way for industrial production of bacterial cellulose.However,static culture needs a large space and the application of bacterial cellulose membrane from static culture has certain limits.In industrial production,the shaking culture is easy to be operated and controlled.The bacterial cellulose from shaking culture has good dispersibility and easy to use,however few strains are suitable for shaking culture.In this study,in order to select bacterial cellulose-producing strains suitable for shaking cultivation,the high-yield strains were screened by natural selection and mutagenesis.The effects of bacterial cellulose on the proliferation,apoptosis and activation of NK cells were studied,and the mechanism of cytotoxic function of bacterial cellulose-activated NK cell was analyzed preliminarily,which laid a theoretical basis for bacterial cellulose to become a high value-added commodity.The main contents are as follows:(1)The method of screening strains producing bacterial cellulose from natureA method of enriching and screening strains producing bacterial cellulose from nature was established.It is favourable to enrich and screen strains when the enrichment medium included 5%nystatin and glucose as carbon source at pH 6.0.Forty bacterial cellulose-producing strains were screened from 459 samples,and the strain 1027-per1 was suitable for shaking culture and the yield of bacterial cellulose was 3.12 g/L.According to the morphological,physiological and biochemical characteristics and 16SrRNA gene sequence analysis,strain 1027-per1 was identified as Gluconacetobacter xylinum,and which was highly homologous with the strain Gluconacetobacter xylinum JCM 10150.Scanning electron microscopy showed that the average diameter of bacterial cellulose produced by the strain 1027-per1 was 27 nm.Infrared spectrum analysis showed that the fermentation product had characteristic peaks of bacterial cellulose.Thermogravimetric analysis showed that the bacterial cellulose produced by the strain1027-per1 had thermostability.(2)Mutation breeding of strain 1027-per1NTG mutagenesis system was established using strain 1027-per1 as the starting strain.NTG mutagenesis was carried out 16 h after the seed culture of strain1027-per1.The number of cells in seed suspension was 10⁸CFU/m L.The concentration of NTG was 0.025 mg/mL or 0.05 mg/mL.A method of screening strains based on colony size was established.The positive mutation rate of the larger colony was 32.18%,which was 59.94%higher than that of the random selection.The negative mutation rate was 25.40%,which was 48.14%lower than that of the random selection.A screening method by NaBr-NaBrO₃plate was established.The concentration of NaBr-NaBrO₃was 2.5-0.5 mmoL/L.The efficiency of screening of HS plate and NaBr-NaBrO₃plate were compared.The probability that the yield of screened strain is 20%and 40%higher than the yield of wild strain in HS plate was 2.7%and 0.91%,respectively.The probability that the yield of screened strain is 20%and 40%higher than the yield of wild strain in NaBr-NaBrO₃plate was 20.56%and 5.61%,respectively.The NaBr-NaBrO₃screening plate increased the screening efficiency of high-yield strains,and the probability that screened strains with an above 20%increase in yield in NaBr-NaBrO₃plate was 7.6 times as that in HS plate.A screening method by Congo red adsorption was established.The conditions for adsorption of Congo red by bacterial cellulose were as follows.The Congo red solution had the maximum adsorption at 480 nm,and the adsorption time was 40 min,the shaking speed was 160 r/min,the adsorption temperature was 30?.There was a good correlation between the wet weight of bacterial cellulose and the OD₄₈₀of Congo red solution after adsorption.The crude yield of bacterial cellulose in flask fermentation system or baffled flask fermentation system were highly correlated with the OD₄₈₀of Congo red solution after adsorption.Freundlich isothermal adsorption equation can be used to describe the adsorption process of Congo red by bacterial cellulose.The probability of getting the strain with yield above 20%than the wild strain by Congo red adsorption method was 2.1 times of that of gravimetric method.793 strains were gotten after NTG mutation and primary screening,and 97strains were screened again.The best BC producer,strain 0511-43 was selected and the yield of bacterial cellulose was 5.52 g/L,which was 76.9%higher than that of the parent strain 1027-per1.(3)Effect of bacterial cellulose on the function of NK cellsBacterial cellulose has no cytotoxic effects on human NK-92 cells and did not induce apoptosis in NK-92 cells.Stimulation of bacterial cellulose did not promote the proliferation of NK-92 cells,but promoted the proliferation of human NK cells isolated from human peripheral blood mononuclear cells(PBMC).Stimulation of bacterial cellulose promoted NK-92 cells to secrete more IFN-?and GzmB in dose-dependent manner.NK-92 cells stimulated by bacterial cellulose showed a stronger cytotoxic effect than the control NK-92 cells.To address the mechanism,the expression of Dectin-1 was detected by FACS and the results showed that the expression of Dectin-1 was significantly increased in NK-92 cells after stimulation with bacterial cellulose.Two siRNAs were designed and siRNA315 showed the best interference efficiency.RNA interference of Dectin-1 gene by siRNA315 revealed that the cytotoxic effect of NK-92 cells with siRNA315 was lower than that of NK-92cells without interference and irrelevant interference after the stimulation of bacterial cellulose,which indicated that Dectin-1 gene played an important role in the process of bacterial cellulose-activating NK-92 cells.