Gluten Deterioration In Frozen Dough: Mechanism And Improvement Study

The advantages such as standardization and convenience of frozen dough technique have brought revolutionary development to the industrialization and the brand-chain business model of the bakery industry. Dough quality deterioration is the major problem to the shortened shelf life of frozen dough, wherein gluten deterioration is one of the main factors to the quality loss of frozen dough. Previous studies have focused on the mechanism of gluten deterioration using gluten as a whole part while the direct effects of frozen storage on its componential glutenin and gliadin remain largely unexplored. The vacancy of this knowledge remains the issue of reliability to elucidate the detailed deterioration mechanism of gluten deterioration essentially, thus weakens the effectiveness of the current protocols in regulating the dough quality. In this research, the mechanism of gluten deterioration induced by frozen storage was revealed from comparative study of the structural and physical alterations in gluten, glutenin and gliadin. This could provide a theoretical basis and technical support for improving the gluten characteristics. On the mechanism basis assessed in the hydrated gluten proteins system, the pivotal contribution of depolymerization effect of key gluten components-gluten macropolymers(GMP) on the frozen dough steamed bread quality was confirmed by the practical frozen dough system. Lastly, utilizing the water extractable arabinoxylan(WEAX) from rye bran, the frozen dough steamed bread quality was improved and the effectiveness of the mechanism was also proved simultaneously. The main research contents and conclusions were summarized as follows:During the frozen storage, depolymerization of GMP occurred in both gluten and glutenin with different depolymerization degrees. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and size exclusion high performance liquid chromatography(SE-HPLC) were combined to study the molecular weight(Mw) and subunits of gluten and glutenin. It was found that the depolymerization of GMP was conducted through breakage of interchain disulfide(SS) bonds, generating SDS soluble polymers(Mw≈91,000~688,000) and monomers(Mw≈16,000~91,000). Meanwhile, the free thiol(SH) content and subunits of gliadin were investigated using visible spectrophotometer and reversed phase high performance liquid chromatography(RP-HPLC). The results showed that the intrachain SS bonds of gliaidn were stable and the subunit composition remained constant during frozen storage. However, gliadin could further promote the depolymerization behavior of GMP, resulting in a higher depolymerization degree of gluten than that of glutenin. In addition, the promoting effects could be illustrated by the following equation: Y=0.012X1+0.609 X2-0.169, R2=0.94, where Y is the depolymerization degree of GMP, X1 is the frozen storage time and X2 is the gliadin content. The alterations in the non-covalent force decreased the spatial aggregation order for all the gluten proteins. The changes in secondary structure of gluten proteins upon frozen storage were studied by fourier transform infrared spectroscopy(FTIR). The present data suggested that the more ordered α-helix structure was partially conversed to antiparallel β-sheet and β-turn structure, indicating the non-covalent force of gluten proteins was also affected and further led to the more disordered spatial aggregation.Frozen storage weakened the interactions between gluten proteins and water. Hydration properties of gluten proteins were evaluated by combined techniques of water vapour adsorption isotherm, time domain nuclear magnetic resonance(TD-NMR) and differential scanning calorimetry(DSC). The results showed that subdued hydration properties of gluten proteins could be manifested by the reduced water absorbing capacity, enhanced water mobility and the increased level of freezable water content of gluten proteins. Furthermore, compared with the varying trends of water state in gluten proteins upon frozen storage, the weakened association between gluten and water probably originated from the increased water mobility of non-freezable rigid(Transverse relaxation time in TD-NMR spectra, T2=0.1~1 ms) and confined water(T2=1~10 ms) in gliadin. However, gliadin had the ability to stabilize glutenin network, so as to confine the water mobility of freezable free water(T2=10~100 ms) in gluten during the frozen storage. The declined hydration of gluten proteins also indicated the elevated hydrophobicity of protein structure. Using DSC and thermogravimetric analysis(TGA) techniques, the thermodynamics behavior of protein was assessed. The results showed that thermal stability at lower temperature(≈50°C) increased whist the melting enthalpy decreased, which could confirm the enhancement of hydrophobicity and more disordered structure in the frozen-stored gluten. Meanwhile, the decreased thermal stability at higher temperature(≈300°C) and the increased weight loss(600°C) illustrated the degraded gluten network structure upon frozen storage. The microstructure of gluten and glutenin network were characterized by scanning electron microscope(SEM). The presented images elucidated that the ice recrystallization could destroy the glutenin network in gluten. Finally, the viscoelastic properties of gluten proteins were tracked by the dynamic rheological avenues. The data suggested that the deterioration of network structure led to the diminished viscoelastic modolus(G’ and G’’) of gluten and glutenin. However, the viscoelastic properties of gliadin remained unaltered throughout the frozen storage. Furthermore, the viscoelastic loss of gluten and glutenin had significant positive correlations with the GMP depolymerization(r=0.96~0.98), suggesting that depolymerization of GMP was also one of the main factors to the functional loss of gluten.Frozen storage induced the degradation in the foaming properties of gliadin, which were mainly attributed to the reduction in the flexibility of molecular chain, surface hydrophobicity and absorption ability at the air-water interfaces of γ-gliadin. The surface tension, secondary structure and surface hydrophobicity were determined by the surface tensiometer, circular dichroism(CD) spectrum and fluorescence spectrophotometer, respectively. The results suggested that the α-helix structure increased at the cost of β-sheet and unordered structure, resulting in a less flexible molecular chain with the decreased surface hydrophobicity. These factors led to the weakened ability of gliadin in adsorbing at air-water interfaces, resulting in the enhanced surface tension of gliadin aqueous solutions and thus of the deteriorated foaming properties. The distribution of gliadin content after foaming was determined by biuret method and the data illustrated that the decreased gliadin content involved in the foam directly contributed to the reduced foam volume, however, the elevated foam density and protein concentration in the foam improved the foam stability. Finally, the distribution of subunits for gliadin solution and gliadin foam were analyzed by RP-HPLC and compared furtherly. The present results elucidated that γ-gliadin was the most sensitive to freezing among all the subunits and the main contributor to the weakened foaming properties during the frozen storage.GMP depolymerization induced the quality deterioration of frozen dough steamed bread by impairing the elasticity and gas retention capability of dough. According to the main characteristics of frozen dough deterioration, the GMP content, steamed bread quality, gassing power and gas retention properties of dough during freeze/thaw cycles were investigated in the yeast-(YLD) and chemical-leavened dough(CLD) system by the fractionation and reconstitution methodology. The SE-HPLC result suggested that the gluten network was strengthened in both type of dough by incorporating GMP. The strengthened network would be more resistant to the freeze/thaw effects and further resulted in the suppressed depolymerization degree of GMP. Meanwhile, the steamed bread quality made from frozen dough was recovered by the addition of GMP, indicating that the GMP depolymerization contributed greatly to the frozen dough quality loss. Rheological analysis showed that the frozen dough elasticity was improved with GMP addition. By analyzing the gassing curve and volume increment curve during dough expansion, the gassing power of YLD decreased along with the reduced yeast viability while the gassing power remained constant in CLD. Furthermore, incorporation of GMP exerted no significant effects on the gassing power in both types of dough, which could exclude the factor that the alteration in gassing power would contribute to the steamed bread quality. However, the rupture volume of frozen dough was elevated with additional GMP, illustrating the gas retention capability was improved. These results could elucidate that depolymerization of GMP contributed to the frozen dough steamed bread quality loss via inducing negeative effects on the dough elasticity and gas retention capability.Water extractable arabinoxylan(WEAX) from rye bran significantly improved the frozen dough steamed bread characteristics in terms of specific volume, texture and crumb grain structure. This was due to that WEAX could inhibit the dehydration of gluten and hinder the ice recrystallization, which further improved the yeast viability and alleviated GMP depolymerization.Combined the main deterioration mechanism of frozen gluten with the physico-chemical properties of WEAX, the water state and Mw distribution of gluten in the dough were assessed by DSC and SE-HPLC, respectively. Our results showed that WEAX reduced the freezable water content in dough and could also further inhibit the ice recrystallization to a certain degree during the frozen storage. Therefore, the yeast viability was better preserved and the enhanced gassing power was detected. Meanwhile, WEAX developed higher GMP content with lowered level of SDS-soluble monomers(Mw≈16,000~91,000) throughout the frozen storage. WEAX could induce the aggregation of GMP and soluble proteins, promoting the increased levels of GMP size and content. Thus, the GMP depolymerization was alleviated and further conductive to the improved steamed bread quality.