Study On Extraction,Purification And Cryoprotective Effect Of Antifreeze Polysaccharides From Cold-acclimated Flammulina Velutipes Mycelium

Flammulina velutipes is a typical low-temperature mushroom.F.velutipes mycelia from liquid fermentation can produce polysaccharides with high antifreeze activity.At present,there are few reports on antifreeze polysaccharides at home and abroad.This paper focused research work on four aspects,the screening of the best strains of F.velutipes and the optimization of the optimal fermentation conditions,the optimization of cold acclimation and the study of antifreeze polysaccharides,the extraction and isolation of antifreeze polysaccharides,and the structural properties of antifreeze polysaccharides and it’s applications.The main conclusions are as follows:（1）Screening of the best strains of F.velutipes and optimization of optimal fermentation conditionsThe optimal strain F008 suitable for liquid fermentation and high antifreeze activity was obtained from 77 strains of F.velutipes strains,and its seed liquid culture conditions were optimized.Based on the optimal seed liquid,the fermentation medium components and fermentation culture conditions were optimized.Optimized fermentation conditions are:Initial p H 6.5,temperature 18°C,rotation speed 200 r/min,culture medium components are soluble starch 55.2 g/L,yeast peptone FP103 5.6 g/L,potassium dihydrogen phosphate concentration 1 g/L,and inoculation amount 10%.Biomass increased from 12.45g/L under initial fermentation culture conditions to20.89g/L,increased by 67.7%.The optimal fermentation time was determined to be 5days under the optimal fermentation culture.（2）Optimization of cold acclimation and production of antifreeze polysaccharidesThe best temperature for cold acclimation is 4℃,and the best time for cold acclimation is 7 days.At the beginning of cold acclimation,5 mg/L abscisic acid was added.After 5 days of cold acclimation,the total antifreeze activity of 25%alkaline-extracted polysaccharide reached a maximum,which was 86.7%higher than that of the control group,and the cold treatment time was shortened.Changes in polysaccharide content and antifreeze activity of water-extracted polysaccharides,2%alkaline-extracted polysaccharides,and 25%alkaline-extracted polysaccharides were compared before and after cold acclimation.Cold acclimation did not increase the total amount of polysaccharides in F.velutipes mycelium,but increased the total polysaccharide antifreeze activity of 1g dry weight of F.velutipes mycelium from 11061.8 to 20828,and changed the antifreeze activity ratio of each polysaccharide extraction component,especially 25%The alkali-extracted polysaccharide component increased from 22%to61%of the total antifreeze activity,which is the main active part of the antifreeze polysaccharide.Therefore,cold acclimation promoted the production of 25%alkali-extracted component antifreeze polysaccharide.（3）Extraction of antifreeze polysaccharidesThe antifreeze polysaccharide extraction method was optimized.The best drying method for mycelium was 60°C.Taking the water bath step-by-step extraction method as the initial extraction condition,the ultrasonic-assisted step-by-step extraction method,the one-step extraction method,and the high-temperature and high-pressure step-by-step extraction method were compared to the anti-freezing polysaccharide extraction effect.The results showed that after ultrasonic extraction of 1 g of mycelium dry weight,the total antifreeze activity of antifreeze polysaccharides was reduced from 1110extracted by traditional water bath to 165;Although the one-step extraction simplifies the extraction step,the optimized extraction conditions are 50%alkali concentration,temperature 115°C,extraction time 30min,material-liquid ratio 1:30,1g mycelium dry weight polysaccharide content is 288.8mg,polysaccharide The total antifreeze activity was 1511;The high-temperature and high-pressure stepwise extraction conditions are as follows:at 115°C,water extraction is performed once in the autoclave,2%alkaline extraction is performed once,25%alkaline extraction is performed twice,and each extraction is 30min.The total activity of antifreeze polysaccharides obtained by 1g mycelium dry weight extraction increased from 1110 to 4817 in the initial extraction conditions.（4）Study on the isolation,purification,structural properties and application effects of antifreeze polysaccharidesThe crude polysaccharide was separated by DEAE anion exchange chromatography to obtain two peaks N and A.The N peak showed high content and high antifreeze activity.The N component was then separated by gel filtration chromatography to obtain two peaks N1 and N2.The N1 peak showed high content and high antifreeze activity,and was a purified antifreeze polysaccharide component.The structure of the antifreeze polysaccharide was determined by physical and chemical properties,UV and infrared spectral scanning,monosaccharide composition analysis,and molecular weight determination of N1 polysaccharide,which mainly consisted of xylose,mannose,and glucose.The percentages of the three were 28.82%and 44.11%,respectively.And 22.93%.Gel chromatography results showed that the polysaccharide was a homogeneous polysaccharide with a molecular weight of 4.26×10⁵ Da.Congo red experiments show that the antifreeze polysaccharide has a triple helix structure.The results of acid and alkali resistance experiments showed that the antifreeze polysaccharides maintained a high antifreeze activity within 7 days within the range of p H 5～p H12.The results of heat resistance test showed that the antifreeze activity remained unchanged after 5 hours of 100℃treatment.The polysaccharide can interact with trehalose at low temperature.Inhibits the formation of trehalose crystals at low temperatures,thereby synergistically improving the antifreeze effect.The cryoprotective effect of antifreeze polysaccharides on frozen shrimp shows that compared with the water treatment group,the antifreeze polysaccharide increased the muscle-derived fiber protein content and Ca²⁺-ATPase activity of frozen shrimp meat,reduced the freezeable water content,and improved the hardness,chewiness,and elasticity of TPA texture analysis.HE staining showed that the muscle fibers in the polysaccharide group are arranged closely and the extracellular space is reduced.Antifreeze polysaccharide has good antifreeze protection effect on frozen shrimp.In summary,the best strain of F.velutipes F008 obtained by screening has improved biomass and polysaccharide yield through optimization of seeds and fermentation.And the antifreeze activity was improved by the optimization of cold treatment.The antifreeze polysaccharide obtained by the high temperature and high-pressure extraction method after separation and purification shows good cryoprotectant effect on frozen shrimp meat,F.velutipes antifreeze polysaccharide has broad application prospects in frozen food.