Improvement And Mechanism Of Amylolytic Enzymes On Gluten-free Bread Quality

With the development of diagnosis methods and health awareness of individuals,the diagnostic rate of celiac disease has increased year by year.As a result,the demand for glutenfree bread is steadily increasing.However,due to the absence of gluten and abundant starch,gluten-free bread is characterized as a bad structure and easy staling.As the main component,endogenous starch is more crucial for the structure and staling properties of gluten-free bread as compared with the common one.Therefore,endogenous starch modification may be a promising way to improve the structure and anti-staling properties of gluten-free bread.Enzymatic modification of starch is an efficient biological modification method.Amylolytic enzymes in different reaction patterns cause various changes in starch structure and properties,resulting in various performances on the structure and staling of gluten-free bread added with different reaction patterns of enzymes.However,how the reaction patterns affect the structure and staling properties of gluten-free bread have not been systematically investigated.Herein,the double network of gluten-free bread was fabricated by adopting hydrocolloids and amylolytic enzymes,clarifying the strategy of improving gluten-free bread quality through amylolytic enzymes mediated gel network structure formation and enhancement.Based on the above,the structural and textural properties,as well as staling properties were explored to elucidate the regulations and mechanism of amylolytic enzymes in different reaction patterns on gluten-free bread quality,with the view of providing a theoretical basis for the application of amylolytic enzymes in the gluten-free bread industry.The main contents and results are as follows.(1)Taking rice flour as the raw material,hydrocolloids were adopted to construct the gluten-like network of gluten-free bread.Results showed that hydroxypropyl methylcellulose(HPMC)provided gluten-free bread with a gluten-like network,and the specific volume was significantly improved from 0.88 m L/g to 3.85 m L/g.But,due to the weakness of the HPMC network,the hardness and springiness of gluten-free bread were excessively decreased,resulting in structural collapse.Therefore,rice protein(RP)in collaboration with HPMC was used to further construct the gluten-free bread texture.Results showed that the addition of RP enhanced the strength and stability of the matrix due to the formation of protein gel through covalent and non-covalent forces and then spanning the gluten-like network of HPMC.And,the addition of 3% RP(g/100 g rice flour)remarkedly increased the hardness,springiness and resilience of gluten-free bread to 121%,116% and 135% of the control one,respectively.Whereas,the aggregation of RP disrupted gluten-like network integrity,and the excessive RP(excessing 3%)would greatly decrease the specific volume of gluten-free bread with an excessively weakened structure.(2)Based on the constructed gluten-like matrix,with the evaluation indicators of structural property and textural quality,four kinds of amylolytic enzymes that only catalyze the glycosyl hydrolysis(GH-Amys)and four kinds of enzymes that specifically catalyze the glycosyl transfer(GT-Amys)were employed to investigate the feasibility of improving the gluten-free bread breadmaking quality by modifying endogenous starch,respectively.As well,the regulations of them on the breadmaking quality of gluten-free bread were investigated.Results showed that the addition of amylolytic enzymes mediated the formation of the starch gel network,which further promoted the structural properties of gluten-free bread.The addition of GH-Amys significantly improved the gluten-free bread structure,and the specific volume of gluten-free bread with added exoamylase(Exo-Amy)was higher than that of endoamylase(Endo-Amy).The addition of Exo-Amy markedly increased the springiness and resilience,while the addition of Endo-Amy resulted in a significant decrease in hardness,springiness and resilience.Compared with GH-Amys,the addition of GT-Amys made a more compact and uniform starch gel network structure and hence a higher specific volume of gluten-free bread,and they significantly increased the springiness and resilience without impacting the hardness and adhesiveness of gluten-free bread.According to the results of rheo-fermentation and morphology,GH-Amys improved the structural properties of gluten-free bread by increasing the fermentation gas production and changing the starch molecular structure,while GT-Amys further enhanced the gel strength based on the formation of gel network to improve the structural properties of gluten-free bread,which decided the quality of gluten-free bread with cyclodextrin glucosyltransferase(CGTase).It was speculated that the formation of the starch gel network and the interaction between starch and HPMC double network structure were crucial for the improvement of gluten-free bread structure and texture,and which was related to the reaction patterns of amylolytic enzymes.(3)The reconstructed double network of RS-HPMC was fabricated.To elucidate the mechanism of amylolytic enzymes in improving the breadmaking quality of gluten-free bread,rheological property,starch structure and conformation of RS-HPMC were studied to investigate the correlation of breadmaking quality with the starch gel network and its interplay with the gluten-like network of HPMC.It was found that the addition of amylolytic enzymes in different reaction patterns reduced the irregular aggregation of starch segments to a different extent during the cooling by enzymatic degradation,and the enzymatic hydrolysis of starch segments formed a gel network structure with different densities and cross-linked with the HPMC network structure.The addition of GH-Amys promoted the formation of an extended fiber structure.Among them,the addition of Exo-Amy made a more cross-linked and compacted structure than that of Endo-Amy.as compared with GH-Amys,the addition of GTAmys made less irregular aggregation of starch segments,and the extended and thickened fiber bundle structure formed by the gel of enzymatically hydrolyzed starch fragments and the interaction of double networks was observed,indicating that they were more conducive to improving the density of starch gel network and the interaction between double network.At the same time,specific enzymatic products of GT-Amys further improved the uniformity of the matrix by emulsification and stabilization,which was responsible for a better structure and texture of gluten-free bread with added GT-Amys compared with the other enzymes.(4)On the premise of a better structure and texture quality of fresh gluten-free bread,taking the firming rate as the evaluation indicator,the influence of amylolytic enzymes in different reaction patterns on the staling properties of gluten-free bread was explored by analyzing the crystal structure,thermal property and water distribution and migration during storage.The results showed that the addition of amylolytic enzymes increased the proportion of segments with DP less than 13,decreased the proportion of segments with DP between 14 and 24,and significantly delayed the retrogradation of amylopectin to a different extent,resulting in the decrease in the firming rate of gluten-free bread.For GH-Amys,the effect of linear-maltooligosaccharide forming enzyme(MFAse),which executes both endo and exomode,on slowing the firming rate of gluten-free bread was higher than that of Endo-aamy andβ-amylase(Exo-bamy)which strictly executes endo or exo-mode.Compared with GH-Amys,the addition of GT-Amys had a more significant reduction in the firming rate of gluten-free bread.compared with the crystal structure,thermal properties,starch molecular structure and firming rate of gluten-free bread,it was found that the anti-staling of gluten-free bread was affected by the reaction patterns of amylolytic enzymes as well as their modification on starch fine structure and the unique properties of enzymatic products.(5)The effect of amylolytic enzymes on the different staling stages of gluten-free bread was investigated based on recrystallization and aging kinetics to elucidate the anti-staling mechanism of amylolytic enzymes on gluten-free bread.The results showed that,for GH-Amys,the samples with added Endo-aamy and Exo-bamy obeyed the instantaneous nucleation mechanism,and Endo-aamy promoted the completion of instantaneous nucleation.both Endoaamy and Exo-bamy reduced the crystal growth rate,which was related to the reduction in the length of amylopectin clusters(Endo-aamy)or the increase in the proportion of short side chains(Exo-bamy).MFAse also obeyed the instantaneous nucleation mechanism,but crystal growth was not observed during staling.Due to the reaction patterns and the unique properties of enzymatic products,the samples added with GT-Amys tend to be in random nucleation mode,completing the nucleation and then growing at a significantly reduced rate.Especially for branching enzymes derived from Rhodothermus obamensis STB05 and β-CGTase,they did not make the growth of recrystallization during the aging.Totally,MFAse executed both endo and exo-mode and GT-Amys catalyzed both hydrolysis and glycosyl transfer are more conducive for gluten-free bread anti-staling.