| Tempeh is a traditional Indonesia soy-based food which contains a high level of protein.It has been widely spread and accepted as a folk tonic and a good substitute of meat products for vegetarians in Asian countries and also around the world.The preparation of tempeh was traditionally made by natural fermentation of de-hulled and steamed soybeans covered with banana leaf.Currently,in some western counties,such as USA,monoxenie solid-state fermentation with purified strain has been realized.Commercial starter cultures(mainly composed of Rhizopus oligosporus)are already available.The nutritional values of tempeh have been sufficiently studied.However,soybean is regarded as one of the eight food allergens by Food and Agriculture Organization of the United Nations.The effect of solid-state fermentation of on protein allergens during the process of tempeh preparation is rarely investigated.In addition,several studies reported that the fermentation generated a kind of non-protein amino acid with many physiological functions,which is called gamma-aminobutyric acid(GABA).But the influencing factors and optimal conditions for the accumulation of GABA are still not clear.In this study,the effects of solid state fermentation with four filamentous fungi(Rhizopus oligosporus RT-3,Actinomucor elegans DCY-1,Neurospora crassa and Rhizopus oryzae)and their combination for the preparation of tempeh on the degradation of anti-nutritional factors and reduction of soybean allergens were evaluated.The mixted fermentation of R.oligosporus RT-3 and A.elegans DCY-1 was selected for the preparation of tempeh.On the basis of strains screening,the accumulation of GABA was carried out through after-ripening process.The after-ripening process was therefore developed.Dynamic simulation of gastrointestinal digestion model was used to investigate the stability and immunogenicity of after-ripened tempeh milk(GABA tempeh milk).Furthermore,the effect of after-ripened tempeh flour on dough quality and bread staling of wheat bread was studied.Results of the study will be helpful for the development of hypoallergenic soy-based functional foods containing a high level of GABA.The main results are listed as follows:1.Selection of the strain combination for mixed fermentationTrypsin inhibitor,oligosaccharide and protein allergen were significantly degraded by fermentation of R.oligosporus RT-3,A.elegans DCY-1,N.crassa and R.oryzae.Soluble protein and peptide(<10 kD)contents were increased during fermentation.The degradation of trypsin inhibitor(protein-based anti-nutritional factor)reached 90%for R.oligosporus RT-3,which was the highest among all strains.Except for basic subunit,the α’,α,βsubunits of β-conglycinin and acidic subunit of glycinin were 100%hydrolyzed.Tempeh fermented with R.oligosporus RT-3 displayed highest soluble protein and peptide content(<10 kD),which were 31.17 mg/g and 256.80 mg/g,respectively.R.oligosporus RT-3 showed the weakest capacity to hydrolyze soybean oligosaccharides(carbohydrate-based anti-nutritional factor).The content of stachyose and raffinose reached 9.11 mg/g and 1.17 mg/g after fermentation,while R.oryzae exhibited the strongest capacity to degrade soybean oligosaccharides.In addition,A.elegans DCY-1 exhibited potent degradation on both protein-based and carbohydrate-based anti-nutritional factors.The antioxidant abilities of all strains were further normalized and expressed as EC50 value,N.crassa and R.oryzae showed significantly lower antioxidant abilities compared with R.oligosporus RT-3 and A.elegans DCY-1.Tempeh fermented with R.oligosporus RT-3 displayed highest DPPH radical scavenging activity,chelating ability and hydroxyl radicals scavenging effect,whereas A.elegans DCY-1 treated tempeh exhibited highest ABTS-+cation radical scavenging activity and reducing power.The mixed fermentation of R.oligosporus and A.elegans showed higher soluble protein(32.54 mg/g)and peptide content(268.03 mg/g)than that of single strain.The mixed fermentation of R.oligosporus and A.elegans exerted potent ability to degrade preotein-based and carbohydrate-based ANFs.The formation of aglycones from glycosides was observed after the mixed fermentation,which was reached 69.33%for the glycosides proportion.2.Effect of mixed fermentation on the antigenicity and structure of proteinThe reduction in the antigen content was 90.75%for tempeh fermented with R.oligosporus RT-3 and A.elegans DCY-1.Sera from soy sensitive individuals were used to evaluate IgE immunoreactivity.Results revealed that the mixed fermentation of R.oligosporus RT-3 and A.elegans DCY-1 significantly decreased the binding activities of IgE with the percentage ranging from 64.60%to 78.57%,depending on the sera used,which possessed potent degradation on allergenicity compared with monoxenie solid state fermentation.The increase in α-helix,reduction in β-sheet and random coil structure was observed through FTIR.The results of exogenous and intrinsic fluorescence spectrum demonstrated that the fluorescence intensity decreased and the absorption peak was red shifted after fermentation,indicating the rearrangement of protein conformation.Ultraviolet spectrum showed that chromophoric group of protein was aggregated with the increase of polarity.The results obtained revealed that the mixed fermentation changed secondary and tertiary structure of protein.The average particle size of protein was 272.07 nm for non-fermented soybeans.The mixed fermentation of R.oligosporus RT-3 and A.elegans DCY-1 resulted in a significant decrease of particle size,which was 166.27 nm.The bimodal distribution of particle size distribution was observed.Fermentation resulted in the decrease in particle size of protein and increased the proportion of protein particles with the size ranged from 10-100 nm.The mixed fermentation increased the absolute value of Zeta potential,which was-20.6,indicating the increase of protein stability.3.Effect of after-ripening process on the formation of y-aminobutyric acidGABA accumulation through after-ripening process of tempeh was optimized by single-factor experiment and response surface methodology.The optimal condition was listed as follow:170 μM of PLP,0.63%of MSG and 8.1 h of after-ripening time.GABA content reached to 1642.25 mg/100g under such condition.After the after-ripening process,soluble protein content decreased which indicated the hydrolysis of soluble protein.Results of SDS-PAGE showed that all protein bands were almost 100%decomposed including some small proteins existing during the fermentation process with the molecular mass of 15 kDa.RP-HPLC profiles of peptides indicated that the category of peptide decreased after the after-ripening process,but the genetation of smaller peptides were observed which leaded to the increase of peptide content.The antioxidant activities of water extracts in a order from strong to weak was:after-ripened tempeh>overripe tempeh>tempeh>soybean.The linear correlation coefficient(R2)between the peptide content and hydroxyl radicals scavenging effect,DPPH radical scavenging activity and chelating ability were 0.9245,0.9171 and 0.9075,respectively.The increase of antioxidant activity of tempeh through after-ripening process was closely related to the production of peptides.4.Effect of dynamic simulation of biomimetic digestion on protein stability of after-ripened tempeh milkResults of dynamic simulation of gastrointestinal digestion showed that less protein bands were detected in GABA tempeh milk(after-ripened tempeh milk)compared with soybean milk and conventional tempeh milk.Proteins were degraded further during the 3 h of gastrointestinal digestion.All protein bands could not be detected by electrophoresis for GABA tempeh milk after 60 min of intestinal digestion.Soluble protein content decreased during the dynamic simulation of gastrointestinal digestion.The contents of soybean milk,conventional tempeh milk and GABA tempeh milk were 13.00 mg/mL,2.63 mg/mL and 1.78mg/mL before digestion.After the gastrointestinal digestion,soluble protein contents were decreased to 1.31 mg/mL,0.54 mg/mL and 0.42 mg/mL,respectively.The initial peptide content in GABA tempeh milk was 18.98 mg/mL,significantly(P<0.05)higher than soybean milk(1.22 mg/mL)and tempeh milk(11.33 mg/mL).The level of peptide in conventional tempeh milk and GABA tempeh milk decreased during the gastrointestinal digestion,indicating the degradation to smaller peptides and amino acids.However,the increase in peptide content was observed for soybean milk.Great reduction of IgE binding activity and allergenicity was detected in GABA tempeh milk.GABA tempeh milk showed the lowest allergenicity compared with other samples.5.Effect of after-ripened tempeh on the quality of wheat flour dough and breadAddition of tempeh flour and GABA tempeh flour(after-ripened tempeh flour)in wheat bread preparation significantly improved the fermentation performance and rheological properties of dough,increasing G’,G" and the elasticity of fermented dough and specific volume of fresh bread.During the storage,tempeh bread displayed the lowest moisture migration rate,lowest values of hardness and chewiness but highest values of springiness and resilience.Three different proton populations were obtained through LF-NMR measurement(population A,B,C),which were attributed to both rigid and exchangeable protons of starch and gluten in contact with water molecules,exchangeable protons of water,starch and gluten,and the protons of lipids.Bread treated with tempeh flour and GABA tempeh flour had small changes in proton distribution and only a small amount of protons shifted to population with shorter relaxation time and formed amylopectin crystals during the storage that could delay bread staling.Addition of GABA tempeh flour significantly increased the content of GABA in bread,which reached 58.31 mg/100g. |
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