Study On The Technology Of Producing Hyaluronic Acid By Streptococcus Zooepidemicus Fermentation

Hyaluronic acid?HA?was a high molecular mucopolysaccharides,composed of?-D-N-acetamidoglucose and?-D-glucuronic acid two kinds of monosaccharides as the basic unit,through the?-1,3 and?-1,4 glycosidic bond repeatedly connected.Because of its unique physicochemical properties and physiological functions,was widely used in cosmetics,food,medicine and other fields.At present,China's HA market is in short supply,the fermentation production of HA has the advantages of no limitation of raw material resources,low production costs,environment-friendly,simple operation and convenient for large-scale production and so on.However the fermentation production of HA still exist technical problems.Therefore,it was very necessary to study the fermentation process of HA,especially the downstream extraction process and degradation of HA was the key to the application of fermentation process in the actual industrial production.In this paper,using Streptococcus zooepidum NQ185 as production strain to study the fermentation,extraction and degradation of HA.The main contents and conclusions were as follows:?1?The effects of medium components on the accumulation of HA metabolism were studied by single factor and orthogonal experiments.The optimal medium components were glucose 80 g/L,yeast 14 g/L,peptone 10 g/L,yeast peptone 8 g/L,MgSO ₄1.2 g/L.The experimental results showed that the yield of HA was 0.91 g/L by shake flask fermentation according to the optimized medium conditions,which was28.17%higher than that before optimization.?2?The control parameters optimization and scale-up experiment of HA fermentation were conducted by using 5 L automatic fermentation tank.The optimal culture conditions were determined by single factor experiments:pH 7,inoculation 10%,temperature of 37?,stirring speed of 300 r/min,ventilation capacity of 2 L/min.Combined with the optimization of the fermentation medium conditions,the yield of HA was 8.54 g/L and the average molecular weight was 2.55×10⁶ Da,which was obtained by using a 5 L autoclaved fermentation tank with the fermentation of 16 h.?3?HA pretreatment,enzymatic hydrolysis and diatomite post-treatment technologies were studied,finally determine the best extraction route:HA fermentation liquid adding sodium chloride to the concentration of 0.4 mol/L,adding 2 times volume of 95%ethanol precipitation.After centrifugation,the precipitate was re-dissolved in distilled water of equal volume to the fermentation liquid.The pH of the solution was adjusted to 4.5 with trichloroacetic acid.After standing for 1 h,the pH was adjusted to7.0 with sodium hydroxide.Trypsin hydrolyzed the protein at pH 8.4,enzymolysis temperature 50?,enzyme dosage 0.174 g/g for 6 h.The solution was diluted 1-fold with 95%ethanol and then vacuum filtered using a 1 cm thick diatomaceous earth pre-coating.Finally,after ethanol precipitation,the HA product was obtained by vacuum freeze drying.The yield of HA was 83.86%,the content of glucuronic acid was43.28%,the content of protein was 0.21%,protein removal rate was 98.95%,and the molecular weight was 2.15×10⁶ Da.?4?Through the research and comparison of thermal degradation,ultrasonic degradation and oxidative degradation of HA,the ultrasonic-oxidation combined degradation method was determined.HA solution was added 2 mmoL/g HA of ascorbic acid,adjusted to pH 4 with hydrochloric acid solution,and then 4 mmoL/g of hydrogen peroxide was added.Ultrasonic degradation of 200 w was performed using an ultrasonic disrupter for 20 min.The degradation of HA was 12.16 kDa.