Optimizing techno-functionality of germinated whole wheat flour steamed bread via glucose oxidase (Gox) and pentosanase (Pn) enzyme innovation

Muhmmd Bill ,Yining Zhng ,Dndn Li,b ,Chong Xie,c ,Runqing Yng,c ,Zhenxin Gu,c ,Dong Jing,Pei Wng,,＊

a College of Food Science and Technology,Whole Grain Food Engineering Research Center,Nanjing Agricultural University,Nanjing,Jiangsu 210095,China

b National Technique Innovation Center for Regional Wheat Production/Key Laboratory of Crop Physiology,Ecology,and Management,Ministry of Agriculture/National Engineering and Technology Center for Information Agriculture,Nanjing Agricultural University,Nanjing,Jiangsu 210095,China

c The Sanya Institute of Nanjing Agricultural University,Sanya 572024,China

Keywords:Germinated wheat Steamed bread Enzymes Rheology Starch crystals Techno-functionality

ABSTRACT Germination,a powerful biofortification technique,holds immense potential in bolstering the micronutrient profile of essential staple grains,thereby paving the way for optimal nutritional enhancement.The primary goal of this study was to improve the technological functionality of germinated wheat flour by incorporating pentosanase (Pn) and glucose oxidase (Gox) enzymes,with particular emphasis on the evolutionary changes in its components.The inclusion of Gox did not produce any substantial impact on the volumetric characteristics of the steamed bread.The incorporation of Pn and Gox has been seen to enhance the overall excellence of steamed bread by optimizing loaf volume and textural characteristics while also improving the thermal stability of the dough.The existence of two endothermic peaks could be attributed to bound water or alterations in the granules within the starch crystallization region.Adding Pn and Gox reduced and increased the formation and stability time of the dough,respectively.A certain ratio was employed to assess alternations in the crystallinity of starch granules over a limited range.After steaming,a significant decrease in IR1047/1022 was observed,indicating that the elevated temperature partially disrupted the internal starch crystal structure,leading to a gelatinization reaction with water.The ratio of tensile resistance (R) and elongation (E) of dough increased significantly compared to the control.The results obtained from this study indicate that the simultaneous inclusion of enzymes (Pn+Gox) holds significant promise for expanding the technological functionality of germinated wheat flour dough and improving the quality attributes of steamed bread.

1.Introduction

Wheat (TriticumaestivumL.) is a significant staple crop that is commonly consumed in the form of flour.This versatile flour serves as a key ingredient in a wide range of food products,such as breads,biscuits,cookies,cakes,pasta,noodles,and more[1].The refined flour produced through advanced milling processes leads to the removal of several essential nutrients,including dietary fiber,minerals,and vitamins,which are predominantly present in the bran and germ portions of the kernel [2].Whole grain flours comprise the complete,milled,fragmented,flattened,or otherwise treated seed after eliminating nonconsumable components like the hull and husk.It is imperative that all anatomical constituents,namely the endosperm,germ,and bran,maintain their relative proportions as observed in the intact kernel[3].

In contemporary times,whole wheat products have garnered recognition as a nutritious dietary option and have experienced a notable surge in global popularity [4].Consuming whole wheat products has demonstrated the potential to lower the likelihood of various chronic conditions,such as colorectal cancer,cardiovascular issues,diabetes,and obesity.Therefore,it is advisable to partially substitute refined grain meals [5].Moreover,seed germination is pivotal in guaranteeing the continual generation of botanical organisms,constituting an indispensable prerequisite for perpetuating human beings.This is particularly relevant in terms of providing a steady supply of medicinal plants,serving as a source of feed for animals used in grazing,and ensuring food security[6].The germinated wheat grains tend to enrich useful and bioactive substances such as phenolic compounds,folates,andγ-aminobutyric acid (GABA) in under-regulated and ideal germination conditions[7,8].In addition,the process of dictated germination has demonstrated the potential to conserve or improve the technological effectiveness of germinated wheat[9].Prior research has indicated that unfettered germination leads to an excessive breakdown of starch and protein due to the activation of enzymes such asα-amylases and proteases.This subsequently leads to the degradation of flour quality and the impaired functionality of dough[10].Therefore,it is imperative to maintain nutritional quality and technological functionality,which are crucial for utilizing and advancing the novel functional product through dictated germination.In our prior investigation,we successfully augmented the concentrations of GABA,an endogenous non-protein amino acid renowned for its function as a principal inhibitory neurotransmitter within the mammalian central nervous system and its significance in promoting mental well-being.This enrichment was achieved in germinated wheat by implementing a hypoxia strategy.Additionally,we observed a reduction in the degradation of components in whole wheat flour [11].

Recently,there has been a notable increase in the significance attributed to matters concerning food safety.Consequently,a considerable number of consumers believe that the inclusion of the term enzyme on food labels signifies a natural and healthful attribute,particularly when contrasted with other chemical denominations.This perception has consequently led to broadening the commercial utilization of enzymes [12].Different varieties of enzymes have the potential to be incorporated into the flour and its derivatives as alternatives to chemical modifiers like hydrophilic colloids and emulsifiers.Among them,the enzymesα-amylase (AM) and hemicellulase (HC) were employed as improvers.These enzymes exhibit catalytic activity towards damaged starch and insoluble dietary fiber[13].

Furthermore,the enzyme glucose oxidase (Gox) is utilized to promote the enzymatic oxidation of glucose,resulting in the generation of gluconic acid and hydrogen peroxide.The aforementioned procedure serves to augment the gluten network by facilitating intermolecular crosslinks.Similarly,pentosanase (Pn) is an enzyme with established applications in the baking industry.Its primary role in baking is to hydrolyze pentosans,a wheat flour component that can otherwise hinder dough development and affect the final bread quality.It can hydrolyze the significant components of hemicellulose,improving dough handling,bread volume,and crumb textural properties [14].Similarly,xylanase(Xyn)is utilized to mitigate the adverse effects caused by bran.It plays a pivotal role in mitigating adverse effects through its enzymatic activity by breaking down the complex water-unextractable arabinoxylan(WUAX) into smaller,more soluble fragments.This enzymatic hydrolysis cleaves the arabinoxylan molecules,reducing their length and complexity and improving dough solubility,water absorption,and gas retention.This mechanism ultimately contributes to the enhancement of the technological functionality of the dough,leading to the production of higher-quality steamed bread when whole wheat flour with bran is involved in the process [15,16].Meanwhile,Xyn improves dough manageability and product quality by affecting flour arabinoxylan(AX)[17].However,significant degradation of AX is unfavorable;hence,Xyn dosage should be cautiously controlled [18].The objectives of combining Pn and Gox in this study are to enhance dough rheology crumb structure,improve gluten strength,and optimize nutrient availability,ultimately improving the overall consumer experience.

The influence of Pn and Gox on the formulation,rheology,and microstructure of whole wheat dough was investigated.The rheology and microstructure of the dough were notably altered by both enzymes.This effect can be attributed to the dissolution of water-insoluble arabinoxylan,an increase in glutenin macropolymer (GMP),and the formation of protein-polysaccharide complexes[19].In our latest research,we conducted an exhaustive examination into the application of Gox,Xyn,and a blend of these two enzymes.The aim was to amplify the technological capabilities of whole wheat flour enriched with GABA.Our focus centered on how these enzymes affect the attributes and potential mechanisms of arabinoxylan and gluten proteins throughout the bread-making procedure,including the liquid lamella of the dough[20].In light of the aforementioned context,this study addresses the growing global interest in whole wheat products as a nutritious dietary choice.By investigating the effects of enzymes Pn and Gox on the quality of steamed bread(stable food in many Asian countries,mainly China),the results contributes to understanding how these enzymes can enhance the nutritional and technological attributes of whole wheat-based food products.Furthermore,this work is significant in the context of food safety,as it explores the use of enzymes as natural and healthful alternatives to chemical modifiers in flour and its derivatives.This aligns with consumer preferences for natural ingredients,which can broaden the commercial utilization of enzymes in the food industry.

Additionally,this research extends beyond the nutritional and safety aspects by delving into the molecular composition of whole wheat dough,including dynamic thermal and tensile properties and the crystallinity of starch.This comprehensive analysis provides theoretical insights into the development of novel cereal-derived products and enhances our understanding of the specific impacts of enzymes on the molecular structure of dough.In conclusion,the present study is significant in promoting healthier dietary choices,improving food safety,and advancing the science of cereal-based food product development,making it a valuable contribution to academia and the food industry.

2.Materials and methods

2.1.Materials

The wheat (TriticumaestivumL.) variety Huaimai was cultivated in Jiangsu,China,and harvested in 2017.The grains were subjected to storage at a temperature of -20?C prior to their utilization for subsequent examination.The enzymes of glucose oxidase (Gox,200 U/ mg)and pentosanase(Pn,6000 U/mg)were obtained from Shanghai Yuanye Biological Co.,Ltd.in China.The water utilized in this investigation was purified using reverse osmosis and then further processed via a Millipore system (Waters,Mississauga,ON,US).All chemicals used in the experiment were of analytical quality unless otherwise indicated.

2.2.Preparation of germinated whole wheat flour

The process for preparing germinated whole wheat flour followed our prior research[20].The wheat grains were initially treated to ensure sterility using a 1% sodium hypochlorite solution.Subsequently,these sterilized grains were placed into specially designed cultivation pots(φ6.0×9.0 cm)with lids,and the setup was kept in a dark environment at a temperature of 25?C.During this period,the grains were soaked in pure water,and oxygen was supplied at a rate of 1.2 L/min to support the germination process.Following a germination period of 12 h,the wheat seeds were rinsed with pure water,subjected to freeze-drying,and then processed into fine particles using a hammer cyclone mill.The resulting flour from this hypoxia-germinated wheat was carefully preserved in a refrigerator at -20?C until it was ready for subsequent analyses.

2.3.Steamed bread manufacturing

Steamed bread was prepared according to the prior method [21]with some modifications.The conventional steamed bread production recipe comprised 200 g of flour,2 g of yeast,and 115 g of water,serving as the control benchmark.To enhance the quality of the steamed bread,precise quantities of Pn and/or Gox were included,maintaining a proportion of 0.05% (W/W) relative to the weight of the wheat flour.A thorough amalgamation of all constituents occurred through kneading in a mixer (C-100 Mixer,Hobart Corporation,Ohio,USA) operating at speeds ranging from 60 to 120 rpm,spanning a duration of 4 to 6 mins.This meticulous procedure to achieve an impeccably smooth dough consistency is essential for optimal steamed bread attributes.Subsequently,the dough was expediently transferred to a refrigerator set at 4?C and left undisturbed for a duration of 10 mins.Following this,the dough was segmented into 80 g portions,commencing the fermentation process within a proof cabinet (Model JXFD 7,Dongfu Jiuheng Instrument Technology Co.Ltd.,Beijing,China).The proof cabinet maintained a constant temperature of(35±2)?C and a relative humidity of 80%±5%,facilitating the dough's expansion to its peak volume.Upon attaining the desired dimensions,the fermented dough was subjected to steam cooking above boiling water for a duration of 25 to 30 min.Subsequent to the steaming phase,the bread was allowed to naturally cool at room temperature for a span of 1 to 2 h.All evaluations of bread quality were performed within a 12-hour window subsequent to completion of the entire process.

2.4.Quality analysis of steamed bread

The methodology for evaluating the characteristics of steamed bread was determined by our previous study [20] with minor modifications.The bread samples were weighed to assess the specific volume,and the loaf volume was gauged using the rapeseed displacement method,denoted as mL/g.The texture analysis of the steamed bread was conducted employing a TMS-pro texture analyzer (Food Technology Corporation,Virginia,USA).Each 25 mm thick slices was obtained from the steamed bread's central portion.Subsequently,a P25 cylindrical probe was employed to determine various textural parameters.The assessment was carried out at a test speed of 60 mm/min,with a compression rate of 50%and an initial force of 0.05 N.The recorded parameters encompass hardness (measured in N),cohesiveness (expressed as a ratio),springiness (measured in mm),gumminess (measured in N),and chewiness(measured in mJ).

2.5.Differential scanning calorimetry (DSC) measurements

Precisely measure the weight of the dough (10 mg) specimen,encapsulate it within a compact aluminum container,and subject it to gradual heating from 25?C to 110?C at a rate of 10?C per minute.Subsequently,evaluate the temperature at which the denaturation peak(Tp) occurs and determine the heat enthalpy (ΔH).This analysis employed the TA System Muse software (version 1.6),as referenced[22].

2.6.Mixolab test

Using Mixolab,the kneading characteristics of the dough were determined according to reference[23]with slight modifications,which allowed mixing the dough under a controlled temperature.An amount of 70–75 g of wheat flour,accounting for its inherent moisture content,was carefully introduced into the bowl of the Mixolab analyzer.The instrument automatically recognizes the requirement of adding an appropriate amount of water according to the optimal consistency,i.e.,torque C1 is 1.1 ± 0.1 Nm.The experimentation commenced with the temperature set at 30?C for an initial duration of 8 min.Subsequently,the temperature was gradually raised to 4?C per minute until the mixture reached 90?C.Following this,the mixture was maintained at 90?C for 7 min.To conclude this stage,the temperature was progressively lowered at a rate of 4?C per minute until the mixture reached 50?C.The mixture was then held steady at 50?C for 5 min.Five distinct phases (C1-C5) can be distinguished on the mixolab curves.

2.7.Starch crystallinity

The starch crystallinity of the sample was determined using Fourier transform infrared spectroscopy(FTIR)[11].The Thermo Nicolet Nexus FTIR apparatus (Thermo Scientific,Waltham,MA,US) with a singlereflection diamond attenuated total reflection (ATR) module and a mercury-cadmium-telluride (MCT) detector was employed for spectral measurements.The ATR cell's background spectrum was established by conducting 64 scans at a resolution of 4 cm-1.The experiment involved placing flour samples consistently onto the ATR crystal under the same conditions as background measurement.Subsequently,the recorded spectra were aligned with the background parameters.The absorbance of bands centered around 1047 cm-1(A1047)and 1022 cm-1(A1022)was utilized to analyze the crystalline structure of wheat starch.The evaluation of starch crystallinity was based on the ratio ofA1047toA1022.

2.8.Extension characteristics of dough

The extension features of the dough samples were determined by referring to the method described in [24] with modifications.The germinated whole wheat flour was used as the raw material to prepare the dough in the kneading machine with different enzyme preparations.The dough was removed and allowed to stand at room temperature for 40 min.Then,it was placed in the dough preparation tank of the TA.XT-2 texture analyzer (UK) SMS KIE and pressed onto a non-viscous polytetrafluoroethylene material board to make dough strips of 2 mm×60 mm.Quickly take the dough strips from the dough preparation groove and place them in the stretch position of the dough using the texture analyzer.Immediately conduct stretching experiments until the dough strips break and obtain the tensile elongation curve of the dough.Perform 10 parallel experiments on each sample.The instrument automatically analyzes the maximum tensile resistance (R) and elongation(E).The stretching characteristics parameters are as follows: the premeasurement speed was 2.00 mm/s,the test speed was 3.30 mm/s,the post-measurement speed was 10.00 mm/s,and the test distance was 50.00 mm.

2.9.Statistical analysis

All the data was replicated three times,and the outcomes were presented as the mean±standard deviation.Unless stated otherwise,all chemical and physiological markers were determined based on dry weight measurements.The experimental data was processed using SPSS 19.0 and Origin 8.6 software and subsequently subjected to analysis of variance (one-way ANOVA) and significance testing.The probability value ofP<0.05 was considered significant.

3.Results and discussion

3.1.Quality attributes of the steamed bread

The intuitive effects of adding single and combined Pn and Gox on the quality of steamed bread are shown in Table 1.Including combined Pn and Gox enzymes can considerably improve steamed bread quality compared with CK(Fig.1).Gox had no significant effect on the specific volume (SV) of the steamed bread.The SV of steamed bread was increased by 10.44% and 20.33% after Pn and Gox+Pn treatment,respectively.Hardness is a measure of the resistance to deformation of steamed bread,which refers to the force required to compress the bread slice to 50%of its original thickness,while masticatory or chewiness is the force needed to break down a solid food until it is swallowed.Compared with CK,Gox increased the stickiness of steamed bread by 27.10%,and the hardness was close to that of the control group.On the other hand,Pn has significantly reduced the firmness and cohesion of steamed bread.Compared with CK,the firmness,stickiness,and chewiness of steamed bread treated with a combined group of Pn+Gox enzymes was decreased by 36.13%,27.87%,and 38.97%,respectively,while the elasticity of steamed bread increased by 54.99%.Liu et al.[25]investigated the effects ofα-amylase,Xyn,and cellulase enzymes on the physicochemical attributes and nutritional profile of steamed bread.When comparing the single enzyme to the combination enzymes,it wasshown that the combined enzymes resulted in a higher specific volume of steamed bread and a lower hardness value,indicating a more significant effect[26].Cao et al.[27]determined that the inclusion of soy lecithin and Gox resulted in a notable augmentation in the specific volume and an enhancement in the crumb structure and softness of steamed bread.In contrast,the enzyme(xylanase)had minimal impact on the firmness reduction and springiness retention of steamed bread during storage [28].Eugenia Steffolani et al.[29] applied a variety of enzymes to improve bread quality characteristics and found that Gox had a positive linear effect on dough firmness,while Xyn andα-amylase had adverse linear effects.In this study,however,Gox failed to significantly increase the durability of steamed bread,potentially due to the impact of the dough-softening result of the enzyme and the doughmaking process.

Fig.1.Cross section images of steamed bread prepared without (control),Pn addition,Gox addition,and with Pn and Gox addition at the same time,respectively.

Table 1 Effect of different enzymes on texture properties of steamed bread.

3.2.Thermodynamic properties of dough

The DSC thermodynamic properties are shown in Fig.2.During food processing,starch gelatinization largely determines the structure and attribute of the finished product and is related to the combined moisture content.It can be seen from Fig.2 that there are two endothermic peaks(P1,P2)on the DSC spectrum,and the temperatures are in the range of 63–65?C and 87–90?C,respectively.It may be related to the thermal aggregation or heat denaturation of soluble proteins during heating.Starch gelatinization occurs when it encounters precise moisture and heat levels,resulting in a remarkable alteration in the internal composition of starch granules.This transition involves a shift from a crystalline form to an amorphous one,accompanied by notable changes in energy [30].The integration of Gox and Pn substantially increased the peak temperature of P1 of the dough.When Gox &Pn were added separately,the peak temperature of P1 increased by 0.78?C and 0.72?C compared with CK,respectively.When adding both Gox and Pn simultaneously,the peak value temperature increased by 1.44?C,and there was no significant difference in the ΔHof the treatment groups compared with CK,indicating that Gox and Pn can improve the thermal stability of the dough.There was no significant difference in the peak temperature and ΔHof the samples at P2,indicating that enzyme treatment had no significant effect on the P2 endothermic peak.The presence of two endothermic peaks could be attributed to bound water or alterations in the granules within the starch crystallization region.The increase in P1 peak temperature may be due to the hydrolysis of WUAX,which has an adverse effect on dough properties after adding Pn.Gudmundsson et al.[31] showed that water-extractable arabinoxylan(WEAX) mainly affects starch gelatinization by reducing water availability.On the one hand,WEAX can modify the starch gelatinization process by preventing water from entering the amorphous region of starch.As well as,WEAX gel can reduce the rate of water molecules entering the interior of starch granules,thus increasing the gelatinization temperature of starch granules.Although,Wang et al.[32] suggested that the presence of WEAX was seen to partially impede the gelatinization process of starch,mainly when the WEAX had a lower molecular weight and a higher degree of branching.However,including WEAX demonstrated a significant inhibitory effect on the immediate retrogradation of starch,mainly when using WEAX with greater molecular weight and reduced branching.Another possibility is that the starch granules may possess varying degrees of crystal stability.Initially,the less stable crystallites would undergo dissolution in the presence of an adequate amount of water(P1),whereas the more stable crystallites would thereafter undergo true gelation in the absence of unbound water(P2).

3.3.Thermomechanical properties of dough

The Mixolab mixing experimenter realizes the research on the thermomechanical properties of the dough by controlling the temperature and measuring the torque (expressed in Nm) generated by the continuous mixing of the dough between the two mechanical spiral arms in real time.The process begins by combining flour with water and enzymes and kneading at a consistent temperature.This kneading destabilizes and weakens the proteins within the dough.Subsequently,the starch granules in the mixture gradually begin swelling and gelatinization.This is succeeded by a phase of uninterrupted and steady gelatinization,leading to subsequent stages of recrystallization and retrogradation.Mixolab simulates the processing mode of dough during mechanical stirring and cooking,baking,and heating,which helps to evaluate the protein characteristics of dough and the overall quality of dough-based products [33,34].The results of the thermomechanical parameters of germinated whole wheat flour dough are shown in Table 2.The inclusion of Pn resulted in a notable drop in water absorption of the dough from 61.50% to 57.10%,and the formation and stability time of the dough was substantially shorter than that of the control,suggesting a decrease in the dough strength.In addition,the heat stability of starch gelatinization decreased with the reduction of peak viscosity (C3).The inclusion of Gox resulted in a substantial increase in the dough formation time(min)compared to the control,and parameters such as water absorption did not change significantly.Liu et al.[35]showed that adding Gox alone had no significant effect on the water absorption rate,dough viscosity (C3),starch gelatinization thermal stability(C3-C4),and starch retrogradation characteristics(C5-C4)of whole wheat flour.The findings of this study were consistent with them.However,after the combined addition of Pn and Gox,the water absorption rate of the dough was considerably lower than that of Pn,whileαandβwere markedly lower than of other treatments,andγwas substantially higher than the control,indicating that the weakening rate of the protein network structure in the dough during the heating stage,while the starch gelatinization rate was slowed down although,the starch retrogradation rate was accelerated during the constant temperature process at 90?C.Similarly,Liu et al.[36]investigated the impact of Xyn,α-amylase,and cellulase enzymes on the rheological characteristics of bread dough.The results indicated that adding individual enzymes did not lead to substantial changes in water absorption,dough development time,or dough stability.However,whenα-amylase,Xyn,and cellulase enzymes were simultaneously incorporated,a significant improvement in dough extensibility and tackiness was observed [37].Liu et al.[38]assessed that the insertion of an alone enzyme decreased water absorption,development time,and stability,and the enzyme compositions exhibited lower development time,water absorption,and stability while demonstrating increased softening compared to a single enzyme.Guo et al.[39] showed that in the hydrolysis of AX,significantly,when the low molecular weight AX increases,the negativeimpact of the polysaccharide polymers on the dough stability could be reduced,resulting in a more stable dough structure.The influence of Pn on the properties of starch during the formation of dough can be ascribed to the dynamic interaction between AX and starch.On the other hand,WEAX has the ability to postpone the maturation of starch gels,resulting in the formation of gels that are more unified and less springy.However,the cohesive strength of gels achieved through AX treated with Pn remains modest.Moreover,the water-holding capacity of AX experiences modifications following oxidation by Gox.Consequently,the collaborative effect of these two enzymes affects the distribution of free water in the dough,alongside the intricate processes of starch gel formation and retrogradation [19].

Table 2 Effect of enzyme on the Mix-lab parameters of germinated whole wheat flour.

3.4.Changes in starch crystallinity

The starch molecules mainly include two parts: crystalline region and amorphous region.The formation of the crystalline zone is primarily attributed to the presence of amylopectin molecules arranged in a double helix configuration,whereas the amorphous region is predominantly composed of amylose molecules arranged in a more relaxed single helix structure.The absorbance at 1047 and 1022 cm-1corresponds to the crystalline and amorphous regions of starch,respectively.This renders the band intensity ratio of 1047/1022 cm-1(IR1047/1022)a valuable metric for assessing alterations in the crystalline structure of starch granules over a limited span.The larger theIR1047/1022,the larger the crystallization area of starch granules.[40] During the dough processing,theIR1047/1022was relatively stable under the same treatment(Fig.3A),indicating that the structural arrangement of starch granules was relatively orderly in this process.When Gox and Pn were added at the same time,theIR1047/1022was lower than that of CK during the dough processing,indicating that the two enzymes may destroy the association state between starch molecules and the crystal structure of amylopectin.After steaming,theIR1047/1022decreased significantly,indicating that the high temperature destroyed the internal crystal structure of starch to a certain extent,causing starch particles to undergo a gelatinization reaction by combining with water.Cai et al.[41]pointed out thatIRof 1047/1022 cm-1was positively associated with amylopectin content and negatively associated with amylose content.The greater the amylose content,the slower the crystal structure was destroyed in the gelatinization process,further indicating that amylose could inhibit the destruction of the starch crystal structure[42].During gelatinization,the structure of the starch crystalline zone disappears quickly,and the double helix structure of amylopectin is destroyed due to the action of heat and water molecules.The absorbance ratio at 1022/995 cm-1in the amorphous region showed an irregular trend(Fig.3B).When Gox or/and Pn were added,the 1022/995 cm-1ratio was substantially higher than that of CK.After steaming,the ratio of 1022/995 cm-1increased considerably in all treatment groups,indicating that the amylose content in the amorphous region decreased due to the gelatinization reaction at this time,resulting in an increase in the 1022/995 cm-1ratio [43].After heating,both the crystalline and amorphous regions of starch change,resulting in changes in the mobility of starch molecules [44].

Fig.3.Starch crystallinity and infrared absorbance(IR)ratio of 1047/1022(A)and 1022/995 cm-1(B),respectively,for different enzymatic treatments of the dough.F,flour;M,mixed dough;R,rested dough;Fe,doughafter the fermentation stage;and C,crumb of steamed bread sample.

3.5.Dough tensile properties

The tensile properties of dough treated with different enzyme preparations are shown in Table 3.These properties are measured by the TA.XT2 texture analyzer includes the maximum tensile resistance (R) and elongation (E),where the tensile resistance indicates the elasticity and strength of the gluten network structure,which can be used to describe the strength of gluten.The elongation indicates the fluidity and plasticity of the dough and reflects the expansion capacity of the dough,whereas the tensile ratio (R/E),which is an important indicator to measure the balance between the doughRandE,and if its value is too large or too small then it will have a negative impact on the final product.The incorporation of Pn resulted in a decrease in theRvalue,a rise in theEvalue,and a considerable reduction in theR/Eratio of the dough.On the other hand,the addition of Gox greatly enhances theRandR/Eratio of the dough,and after simultaneous treatment with two enzymes,theR,E,andR/Eratio of the samples considerably rise as compared to the CK.Liu et al.[37] findings demonstrated that the inclusion of individual enzymes as well as combined (cellulase,Xyn,andα-amylase)can enhance the extensibility and softness of the dough while simultaneously reducing its resistance to extension.Notably,the combination of enzymes(at concentrations of 6,120,and 60 ppm)resulted in a substantial increase in the extensibility of wheat flour dough,reaching up to a 42% improvement.Yang et al.[45] suggested that incorporating Xyn-Gox and Xyn-Gox &cellulase-modified dough can mitigate the adverse impacts of using a single enzyme on dough properties,specifically extensibility,tenacity,and stability.Consequently,this leads to the development of a more tender gluten matrix.Guo et al.[46] examined that the incorporation of transglutaminase (TG) at a concentration of 1%(W/W)and Gox at a concentration of 0.006%(W/W) into the dough simultaneously resulted in a significant increase in both the extensibility and maximum resistance of the dough by 9.6 mm and 14.7 g,respectively.Pn can decrease the high absorption of bran and weaken water combination competition so that water is evenly distributed in the dough,thereby improving the local aggregation of gluten proteins,effectively reducing the strength of the dough,and enhancing its extensibility[19].The oxidation of Gox causes the dough to become compact and hardened,manifested by increased maximum tensile resistance and reduced elongation.The rough tissue structure of steamed bread may be related to the rupture of the dough.The air volume will expand during the fermentation,and the film between the air chambers will extend.When the elongation is too significant,the air storage capacity of the thin film ruptures,and the dough decreases rapidly.Therefore,to a certain extent,the stretching of dough is not as significant as possible,Pn+Gox dough was well balanced compared with R and E values of other groups,and dough prepared with Pn and Gox has good flexibility due to its synergistic effect on gluten stress and strain hardening [47].

Table 3 The stretching properties of dough.

4.Conclusion

The overall quality and techno-functionality of whole germinated wheat flour were modified by Pn and Gox to enhance the specific volume,textural,and thermal stability of the steamed bread dough.Both of enzymes jointly affect the distribution of free water in the dough and the gelatinization and retrogradation behavior of starch.After steaming,the ratio of 1047/1022 cm-1decreased significantly,indicating that the high temperature destroyed the internal crystal structure of starch to a certain extent,causing starch particles to undergo a gelatinization reaction by combining with water as well as the ratio of 1022/995 cm-1increased considerably in all treatment groups.Additionally,the combination of Pn and Gox exhibited a synergistic effect,resulting in increased dough viscoelasticity and improved quality of the steamed bread.This collaborative enzymatic intervention has not only improved the physical characteristics of the final product but has also advanced our understanding of the complex interactions governing the behavior of key constituents during the processing of germinated wheat flour.As a result,these findings contribute valuable insights to optimizing processes to enhance the quality and functional properties of cereal-based products.

CRediT Authorship Contribution Statement

Muhammad Bilal:Visualization,Data Curation,Writing-Original draft.Yining Zhang:Visualization,Writing-Original draft.Dandan Li:Investigation,Review.Chong Xie:Investigation,Software.Runqiang Yang:Supervision,Review,Editing.Zhenxin Gu:Supervision,Review,Editing.Dong Jiang:Supervision,Review,Editing.Pei Wang:Conceptualization,Writing-Reviewing and Editing,Funding Acquisition.

Declaration of Competing Interest

The authors declared that they have no conflicts of interest to this work.We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

This article does not contain any studies with human or animal subjects.

Acknowledgements

This research was supported by the Young Elite Scientists Sponsorship Program by CAST (2022QNRC001),National Key Research and Development Plan Project (2022YFD2301401),Outstanding Youth Science Fund Project of Natural Science Foundation of Jiangsu Province(BK20211576),the Central Government Guides Local Funds(ZYYD2023A13),Key Technology Research and Development Program of Hainan Province (ZDYF2022XDNY233),the China Postdoctoral Science Foundation(2018 M630564),and a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.