Optimized Expression Of Recombinant Antifreeze Proteins And Their Effect On Freeze-thaw Properties Of Plant-based Meat

Antifreeze protein（AFP）is a kind of special functional protein which can control the growth of ice crystal by adsorbing to the surface.It has the characteristics of ice surface affinity,thermal hysteresis（TH）and ice recrystallization inhibition.In this study,the expression system of P.pastoris was used to express,screen and deglycosylate AFPs from five different sources,so as to optimize the expression of AFPs with high TH activity.The antifreeze and thaw effects of AFP in plant-based meat system and fat mimetics were explored The results were as follows:（1）Heterologous recombinant expression of AFPs:The AFP gene sequences from Daucus carota（Dc AFP）,Saussurea involucrata（Sa AFP）,Ambystoma mexicanum（Am AFP）,Chlorella vulgaris strain UTEX259（Cs AFP）and its inverse protein Re Cs AFP were inserted into p PIC9K,p PICZαA and p GAPZαA,and recombinant plasmids were constructed and transformed into P.pastoris GS115,X33,SMD1168,KM71.Finally a recombinant strain P.pastoris/p PICZαA-Dc AFP-7 was obtained with high thermal hysteresis activity（1.34℃）by antibiotic screening,protein content determination and DSC thermal hysteresis activity scan screening.The antifreeze protein was modified by co-expression of molecular chaperones and deglycosylation,and a strain of P.pastoris/p PICZαA-Dc AFP-N294G with high thermal hysteresis activity was screened,and the thermal hysteresis activity of r Dc AFP-N294G was increased by 0.23℃.The fermentation process of the antifreeze protein was optimized at the shake flask as well as at the5-L fermenter level,with a final Dc AFP maximum titer of 51.6 mg·L^-1,122.0 mg·L^-1 for Re Cs AFP.（2）The effects of pretreatment of r Dc AFP and its deglycation mutant r Dc AFP-N294G on the quality of plant-based meat（PBM）after freeze-thaw were investigated.The effects of pretreatment with recombinant AFPs（rAFPs）on the microstructure,rheological properties,water mobility,and water distribution of PBM were assessed.The r Dc AFP-N294G-treated PBM samples had superior viscoelasticity and water distribution features compared to the r Dc AFP-treated group because the complex N-linked oligosaccharides may interfere with the binding of rAFPs to ice molecules.Deglycosylated Dc AFP has potential applications as a new effective cryoprotectant in meat alternatives.（3）The effect of pretreatment of r Dc AFP and its deglycosylated mutant r Dc AFP-N294G on the post-freeze-thaw quality of PBM supplemented with novel animal fat mimetics was investigated.Firstly,glycyrrhetinic acid（GA）and phytosterols were used as raw materials to stabilize and prepare high internal phase emulsions（HIPE）as animal fat mimics by modulating the self-assembled fiber network of GA in the aqueous phase and the crystallization behavior of phytosterols in the oil phase.The macroscopic and microscopic structures of HIPE were investigated,and it was found that GA as a stabilizer dominated the formation of HIPE structure,and the prepared HIPE had properties such as viscoelasticity of ideal solid animal fat,and thus could be applied as fat mimetics in PBM.Among the three fat mimetics,the konjac glucomannan-based hydrogel was more favorable for maintaining the viscoelastic characteristics of PBM.The elastic and viscous moduli of all three PBM samples showed a significant decrease after freeze-thawing,and the 1%r Dc AFP-N294G-treated group was stronger than the 1%r Dc AFP-treated group in maintaining elasticity,demonstrating that the deglycosylation mutant was beneficial in reducing freeze-thaw damage.