Structure Analysis And Cryoprotective Effect Of Antifreeze Polysaccharides From Flammulina Velutipes

Antifreeze polysaccharide has three properties of thermal retarding activity,recrystallization inhibition activity and modification of ice crystal morphology,which can bind to ice crystals and prevent ice crystals from damaging cells and tissues.Flammulina velutipe has good cold resistance,and antifreeze polysaccharides can be obtained from F.velutipes mycelium through liquid fermentation and cold induction.However,there are no reports on the research of antifreeze polysaccharide in China.In order to improve the yield of F.velutipes mycelia,six kinds of exogenous additives were added into the culture medium to explore the effects on F.velutipes mycelia.In order to improve the yield of antifreeze polysaccharide and obtain high activity antifreeze polysaccharide,the mycelium was induced by low temperature,and the antifreeze polysaccharide was extracted by high temperature and high-pressure step extraction method,which improved the yield and yield of antifreeze polysaccharide.A new type of antifreeze polysaccharide was obtained by separation,purification and structure characterization of antifreeze polysaccharide.In order to explore its practical application effect,antifreeze polysaccharide was added into surimi products,and the antifreeze protection mechanism was analyzed.The main conclusions are as follows:(1)Six kinds of exogenous additives were added into solid medium and liquid medium respectively to explore their effects on the mycelia of F.velutipes.The results showed that the addition of 20 mg/L chitosan could significantly promote the growth of mycelia.Adding 20mg/L indole acetic acid and 2.0g/L calcium chloride can significantly increase the biomass of mycelium and its antifreeze polysaccharide activity in liquid fermentation,among which 2.0g/L calcium chloride has the most obvious promoting effect.The mycelial biomass,total sugar content and total antifreeze activity can be increased by 44.13%,44.02%and 48.2%,respectively.The results are also verified by the fermenter expansion experiment.(2)The mycelia of F.velutipes were induced at 4?for 5 days.The yield,antifreeze activity and structural changes of polysaccharides extracted by water,2%alkali and 25%alkali were compared before and after cold induction.The polysaccharide yield of 25%alkali extraction increased from 1.38%to 1.97%,and the polysaccharide ratio increased from 10.3%to 13.2%,indicating that cold induction changes in the composition of mycelial polysaccharides.The antifreeze activity of the first 25%alkaline-extracted polysaccharide after cold induction increased significantly,from 48.7 to 132.5.HPLC analysis showed that the molecular weight distribution of polysaccharides showed a downward trend after cold induction.It was found that the main components of each extracted component were polysaccharides through the analysis of ultraviolet.By infrared spectra analysis,structure of polysaccharides extracted with 25%alkali for the first time changed from CH connection to-CH2,and the-OH content was increased after cold induction.(3)The polysaccharides components of F.velutipes mycelium(extracted by 25%alkali the first time)were separated by DEAE anion exchange chromatography to obtain three fractions,FVP-A,FVP-B and FVP-C,of which FVP-A showed high content and high antifreeze activity.Then FVP-A was separated by gel filtration chromatography to obtain two fractions,FVP-A1 and FVP-A2,in which the content and antifreeze activity of FVP-A1 were significantly higher.FVP-A1 was freeze-dried and subjected to physicochemical analysis and structural characterization.The results showed that the sugar content of FVP-A1 was 96.75%and the molecular weight was676k Da.The monosaccharide composition analysis showed that FVP-A1 was mainly composed of three monosaccharides:xylose,mannose and glucose.Through methylation analysis and nuclear magnetic resonance,it was deduced that FVP-A1 has a glycosidic bond of?3)-?-D-Glcp-(1?3)-?-D-Manp-(1?as the main chain,and the branched chain?-D-Xylp-(1?fragment is linked to the main chain by the O-4 bond of?3,4)-?-D-Glcp-(1?.Fvp-A1 is a new kind of antifreeze polysaccharide.(4)The FVP-A1 was applied to silver carp myofibrillar protein solution and silver carp surimi gel.The protein solution with 0.1%FVP-A1 maintained high solubility,Ca²⁺-ATPase activity and total sulfhydryl content after 7 weeks in frozen storage,suggesting that antifreeze polysaccharide can inhibit the freezing denaturation of protein.After freezing and thawing for 8 times,the gel strength,texture and water holding capacity of the surimi gel incorporated with 0.1%FVP-A1 were significantly higher than those of the control group,indicating that antifreeze polysaccharide make the surimi gel network more stable.The water state of surimi gel was measured,and it was found that antifreeze polysaccharide can reduce the content of freezable water,increase the content of bound water,and inhibit the migration of water molecules,thereby reducing the amount of water in the surimi gel.In summary,2.0g/L calcium chloride significantly improved the yield of F.velutipes mycelium and the antifreeze activity of polysaccharides;Cold induction changed structure of polysaccharides of different extraction components,and improved the antifreeze activity of polysaccharide component extracted by 25%alkali the first time.The polysaccharide fraction FVP-A1 with high antifreeze activity was obtained by step-by-step purification.The structure analysis showed that it was a new antifreeze polysaccharide.0.1%FVP-A1 can significantly inhibit the freezing denaturation of silver carp myofibrillar protein and improve the freeze-thaw stability of surimi gel,which showed a good antifreeze effect.Therefore,the antifreeze polysaccharide extracted from the mycelium of F.velutipes is a new natural antifreeze protective agent,which has a broad application prospect in frozen food and provides a new idea for the antifreeze preservation of food.