Key Technology Of Green And Efficient Cycle Culture Of Spirulina And Preparation And Characterization Of Polysaccharide In And Out Of Cell

Spirulina and its products are widely used in food,biomedicine and other fields because of their high nutritional value.But spirulina culture requires a lot of water.Therefore,culture medium recycling can not only reduce water consumption,but also efficiently utilize nutrient salts and reduce culture cost.However,there were organic substances secreted by spirulina growth process in the culture medium,which would inhibit its own growth and nutrient accumulation if directly used to culture spirulina.It has been reported that the main components of endogenous growth inhibitors secreted by Spirulina spirulina are carbohydrates（IPS）.However,there are few studies on extracellular polysaccharide,and the differences between extracellular polysaccharide and intracellular polysaccharide（EPSS）in postgangliatory function are not reported.In this study,the effects of culture medium on cell growth during the cycle culture of Spirulina were studied,and activated carbon,S-8 macroporous resin and ultrafiltration membrane（10 kDa）were used to treat the culture medium,and the biomass yield,intracellular nutrient content,and polysaccharide and protein content changes in culture medium were observed.Four methods were used to extract polysaccharides from Spirulina platensis.The extracellular polysaccharides were optimized according to the yield and biological activity of polysaccharides.The structure composition and biological functional activity of IPS and EPS in Spirulina were compared.The results of this paper will provide theoretical basis and technical support for the low-cost and efficient culture of Spirulina and the preparation,processing and utilization of polysaccharides in and out of cells.The main research results are as follows:（1）the culture of spirulina separately by 0.45μm 10 kDa membrane,membrane filter,S-8 macroporous resins and activated carbon treatment,training for five consecutive cycles.The results showed that the water consumption could be greatly reduced after repeated use of culture medium,but the stem cell weight（DCW）of Spirulina decreased gradually during the cycle culture.The contents of IPS and EPS increased with the increase of cycle times and time,and the content of nutrients in biomass decreased to varying degrees.However,after the treatment of culture medium,the negative effects can be effectively reduced,the content of polysaccharide and protein in culture medium can be reduced,and the difference between the reused medium and the fresh medium for the culture of microalgae can be reduced.Treatment effect for 10 kDa ultrafiltration membrane＞S-8 macroporous resin＞activated carbon.（2）Four extraction methods,namely hot water extraction,lye extraction,high pressure homogenization and ultrasonic crushing,were used to extract spirulina intracellular polysaccharide,and the extraction method was optimized according to its structural and functional characteristics.The results showed that the extraction rate of intracellular polysaccharide was the lowest,and the content of uronic acid was less（1.12%）.The high pressure homogeneous extraction method had the highest yield of intracellular polysaccharide（6.02%）and the highest content of intracellular polysaccharide and uronic acid.The purity of intracellular polysaccharide by lye extraction was low.But for intracellular polysaccharide of spirulina functional group composition,primary structure,sugar ring type and thermal stability were no significant influence（p＜0.05）.The highest heat resistant temperature of Spirulina polysaccharide was 200℃.The intracellular polysaccharide potential of Spirulina spirulina was all negative,and the absolute value of intracellular polysaccharide negative potential was larger（-36）in high pressure homogeneous cells,and it was more acidic.At higher concentrations,high pressure homogeneous intracellular polysaccharide had the best scavenging efficiency(IC₅₀=3.65 mg/mL),and lye extraction had the worst scavenging efficiency.However,the clearance rate was much lower than Vc（88.9%）.Hot water extraction,lye extraction,high pressure homogeneous extraction and ultrasonic crushing had high（over 90%）scavenging ability at 2.5 mg/mL,but the ultrasonic crushing had poor scavenging effect at low concentration.The intracellular polysaccharide of Spirulina spirulina obtained by the four methods had inhibitory effects on the three bacteria,and the sequence of minimum inhibitory concentration was Bacillus subtilis（10 mg/mL）>Escherichia coli（5 mg/mL）>Staphylococcus aureus（2.5 mg/mL）.The four polysaccharides had inhibitory zones against Staphylococcus aureus,Escherichia coli and Bacillus subtilis.Staphylococcus aureus had the largest antibacterial zone diameter（16.38 mm）,followed by Bacillus subtilis and E.coli.Among the four kinds of polysaccharides,hot water extraction had the best bacteriostatic effect,while lye extraction had poor bacteriostatic effect.Comprehensive the above results,the best extracting method to extract the intracellular polysaccharide spirulina for high pressure homogeneous＞ultrasonic broken＞lye extraction＞hot water extraction.（3）The extraction conditions of intracellular polysaccharide from Spirulina spirulina were optimized to determine the structural properties of intracellular polysaccharide and extracellular polysaccharide.The results were as follows:The optimal extraction conditions were:solid-to-liquid ratio 1:30（g/mL）,extraction pressure 60 MPa,extraction three times.Under the optimal conditions,the extraction yield of polysaccharide was 60.61 mg/g.The composition of IIPS and EPS monosaccharides is similar,both containing mannitol,ribose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,xylose and fucose,but the proportion of each monosaccharide content is different.The surface of IPS is rough and has irregular pinstripes,which is connected by loose,fine sheet and porous network structure.The surface of EPS is smoother and flatter,with a coarser sheet structure and poor porosity.Both IPS and EPS are acidic,but EPS is more acidic and thermal stable than IPS.IPS is an excellent antioxidant with excellent DPPH and ABTS free radical scavenging ability.EPS is a strong gold ion chelating agent with strong HO^-removal ability and ferrous ion chelating ability.