| Frozen dough technology,one of the most promising technologies in Chinese food industry,is the key to realize the industrial production of flour products.However,the quality deterioration of frozen dough during the whole frozen process limits its large-scale application.The quality deterioration of wheat gluten is one of the most important factors affecting the quality of frozen dough.Antifreeze proteins(AFPs),which can bind with ice crystals,modify ice crystals' morphology and inhibit ice recrystallization,have broad application prospects in the field of frozen dough.Natural AFPs have some disadvantages such as difficult extraction and purification procedures,high cost and low yield.Therefore,in this research,three recombinant antifreeze proteins(rAFPs)exhibiting different characteristics were heterologously expressed by Pichia pastoris;the effects of these three rAFPs on the quality of dough,as well as the structure and physical properties of gluten proteins during freezing and frozen storage were systematically investigated;and on this basis,the formulation of compound rAFPs with better protective effects on frozen dough throughout the frozen process was determined.This research is of great significance for systematically understanding the effects of different rAFPs on gluten quality deterioration during different stages of the frozen process,improving the quality of frozen dough and its bakery products,and developing the application of rAFPs in frozen foods.The main research contents and conclusions were summarized as follows:Based on the amino acids sequences of carrot AFP(Genbank accession no.AAC62932.1),type II fish AFP from Epinephelus coioides(Genbank accession no.AGM15882.1)and Tenebrio molitor AFP(Genbank accession no.ABB03885.1)in the National Center for Biotechnology Information(NCBI)database,the gene sequences of three rAFPs were synthesized by codon optimization.After the construction of yeast expression vector and strains screening by G418 and 24-well plate,three recombinant Pichia pastoris strains,which could produce recombinant carrot AFP(rCaAFP),recombinant type II fish AFP from Epinephelus coioides(rFiAFP)and recombinant Tenebrio molitor AFP(rTmAFP)with high thermal hysteresis activities,were obtained,respectively.The fermentation process was preliminarily optimized,and the three rAFPs were all purified to electrophoretic purity after a series of purification procedures.The thermal hysteresis activities of rCaAFP,rFiAFP and rTmAFP were 1.62°C,4.23°C and 7.57°C(10 mg/mL),respectively.All the three rAFPs had the ability to modify ice crystals' morphology and inhibit ice recrystallization.The rTmAFP exhibited the strongest modification ability of ice crystals' morphology,while rCaAFP had the strongest ability to inhibit ice recrystallization.The effects of three rAFPs on the quality of frozen dough were investigated.The three rAFPs were proved to be efficient cryoprotectants for frozen dough.The addition of rAFPs weakened the damage of ice cystals' formation and recrystallization on yeast cells and gluten network by modifying ice crystals' morphology and inhibiting ice recrystallization,protected the fermentation performance of dough,shortened its proofing time,inhibited the increment of freezable water content,thereby improving the specific volume and texture properties of bread.The rTmAFP was most effective in protecting frozen dough quality during freezing,while rCaAFP was most effective during frozen storage.This was related to the differences of abilities to modify ice crystals' morphology and inhibit ice recrystallization.The effects of three rAFPs on the structural properties of gluten and its components(glutenin and gliadin)during freezing and frozen storage were investigated.During freezing and frozen storage,some disulfide bonds of gluten and glutenin were ruptured,resulting in depolymerization at different levels,while the intramolecular disulfide bonds of gliadin were relatively stable;for both gluten and glutenin,the rTmAFP was most effective in preventing the breakage of disulfide bonds and glutenin macropolymer(GMP)depolymerization during freezing,while rCaAFP was most effective during frozen storage.During freezing and frozen storage,some ?-helical structures of gluten,glutenin and gliadin were transformed into ?-turn and anti-parallel ?-sheet structures,while ?-sheet and intermolecular ?-sheet structures were not obviously altered,indicating the ordered degree of their spatial structure aggregation reduced;the rAFPs prevented the transformation of ?-helical structures to other secondary structures by reducing the damage of freezing and frozen storage on the non-covalent bonds system of glutenin and gliadin,thus contributing to keep the secondary structure of gluten relatively stable.In addition,the rAFPs weakened the damage of freezing and frozen storage on gluten network by modifying ice crystals' morphology and inhibiting ice recrystallization,making the gluten network more continuous and less disrupted.The rTmAFP was most effective in protecting the secondary structure and microstructure of gluten during freezing,while rCaAFP was most effective during frozen storage.The effects of three rAFPs on the physical properties of gluten and its components(glutenin and gliadin)during freezing and frozen storage were investigated.The three rAFPs,especially rTmAFP,caused freezing hysteresis by reducing the freezing temperature of gluten,glutenin and gliadin.During freezing,for gluten and glutenin,the freezable water content and water mobility increased,and the water migration and redistribution were also observed;while the interaction between gliadin and water was not significantly altered;for gluten and glutenin,all the three rAFPs,especially rTmAFP,could inhibit the increment of freezable water content and water migration;both rFiAFP and rTmAFP could inhibit the enhancement in water mobility of gluten,and only rTmAFP could inhibit the enhancement in water mobility of glutenin.During frozen storage,for gluten,glutenin and gliadin,the interaction between gluten proteins and water was weakened;all the three rAFPs,especially rCaAFP,inhibited the increment of freezable water content,water mobility and water migration,improving water-holding capacity of gluten proteins.The thermal stability of gluten and glutenin reduced during freezing and frozen storage,while the thermal stability of gliadin only reduced during frozen storage;the three rAFPs could improve the thermal stability of gluten proteins;the rTmAFP was most effective in improving the thermal stability of gluten and glutenin during freezing,while rCaAFP was most effective in improving the thermal stability of gluten,glutenin and gliadin during frozen storage.During freezing,the cryoprotective effects of rAFPs on the water-holding capacity and thermal stability of gluten were mainly achieved by protecting these corresponding properties of glutenin;during frozen storage,the cryoprotective effects of rAFPs on the water-holding capacity and thermal stability of gluten were achieved by protecting these corresponding properties of both glutenin and gliadin.During freezing and frozen storage,the rheological properties of gluten and glutenin deteriorated,while the viscoelastic properties of gliadin did not significantly change;during freezing and frozen storage,the rAFPs may weaken the adverse effect of ice crystals' formation and recrystallization on the rheological properties of gluten and glutenin by reducing the degree of GMP depolymerization,protecting network and enhancing water-holding capacities;the rTmAFP was most effective in protecting rheological properties of gluten and glutenin during freezing,while rCaAFP was most effective during frozen storage.The effects of compound rAFPs on frozen dough properties and bread quality were investigated.The results showed that compound rAFPs(the mass ratio of rTmAFP and rCaAFP was 1:4)could improve frozen dough properties and bread quality to the full extent.The compounding of rAFPs with different characteristics had better cryoprotective effects on frozen dough properties. |
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