Effects of Ultrasonic-Microwave-Assisted Technology on Hordein Extraction from Barley and Optimization of Process Parameters Using Response Surface Methodology

We investigated the process intensication of ultrasonic-microwave-assisted technology for hordein extraction from barley. Response surface methodology was utilized to optimize the extraction conditions and to analyze the interaction between four selected variables: temperature, microwave power, ultrasonic power, and extraction time. e validated extraction yield of hordein reached 8.84% at 78°C, microwave power 298W, and ultrasonic power 690W after 20min as optimum conditions. Compared with traditional water-bath extraction (4.7%), the ultrasonic-microwave-assisted technology eectively increased the hordein extraction yield and shortened the extraction time. According to the obtained quadratic model (R 0.9457), ultrasonic power and extraction time were the rst two signicant factors. However, temperature limited the eects of other factors during extraction. SDS-PAGE and scanning electron microscopy were used to identify the hordein extract and to clarify the dierence between the two hordein fractions extracted with new and traditional methods, respectively. Ultrasonic-microwave-assisted technology provided a new way to improve hordein extraction yield from barley and could be a good candidate for industrial application of process intensication.


Introduction
Hordein, as the major storage protein in barley, has attracted wide attention due to its hydrophobicity, antioxidant activity, and electrospinnability [1].Compared with other prolamins (gliadin, secalin, and avenin), hordein is distinctive for high contents of nonpolar and hydrophobic residues and low levels of charged amino acids [2,3]. is typical amino acid composition of hordein leads to multifunctional properties, which have been investigated in various applications.For example, continuous ultrathinbers with nanoscale diameters were successfully prepared from hordein in acetic acid with the unfolded molecular structure and exible conformation.In order to enhance the mechanical strength of the nano bers, a novel assembled prolamin nanofabric which was prepared by incorporating compact zein nanoparticles into electrospun hordein networks had been obtained [4,5].Hordeins are commonly subdivided by molecular weights into B-hordein (70%-80%), C-hordein (10%-20%), D-hordein, and c-hordein (1%-5%) [6].Numerous studies have revealed that the unique sequence partitioning of hordein may produce peptides with ferrous ion-chelating capacity or powerful antioxidant bioactivities [7].Barley has widely been used for beer brewage, whereas considerable B-hordein and C-hordein, as the main composition of hordein protein, would remain in the spent grain after malting and brewing at high temperature [8,9].erefore, it is possible to introduce the processing intensi cation method to improve hordein extraction yield without breaking protein molecular conformation.To our knowledge, ethanol extraction is a traditional method for extracting hordein from barley.e majority of hordein can be extracted in the first 2 h, and the protein yield increases by 2-3% between 2 and 4 h [10].Wang et al. have investigated several parameters, such as water to material ratio, pH, and temperature.ey also found that, compared with the effects of different protein isolation methods, cold precipitation gave a higher hordein content than rotary evaporation.Up to now, highly efficient extraction method for hordein is still needed.
Microwave-and ultrasonic-assisted extractions are new process intensification technologies [11].Microwave can effectively shorten the extraction time by intensifying heat transfer in liquid-solid system and accelerate substance dissolution from solid materials.Nevertheless, because of the heterogeneous mixture system, microwave radiation may give rise to spot heating exceedingly.Ultrasonic wave can clean the surface of solid particles in solvents by producing impact forces and lead to solid particles break or collapse as a result of instant high temperature and pressure produced by the cavity effect [12].Barley hordein has no changes after boiling in the brewing process owing to some hordein subunits are more resistant to proteolysis and heating [13].erefore, heat resistance properties of hordein provide a microwave-assisted method a chance to improve protein extraction yield without irreversible degradation and denaturation.
e ultrasonic-microwave-assisted technology (UMAT) takes full advantage of the high-energy effect of microwave and ultrasonic cavitation to overcome some shortcomings of conventional extraction processes [14,15].Operating power and time of UMAT play significant roles in enhanced extraction process.In this study, a response surface methodology (RSM) considering ceiling temperature, microwave power, ultrasonic power, and extraction time was employed to evaluate the effects of these parameters on hordein extraction.A 5-level-4-factor central composite experimental design (CCD) was obtained.Besides, we analyzed the characteristics of two hordein fractions under different extraction conditions.

Materials and Methods
Barley flour (Shangdong Province, China) was purchased from a local supermarket.After defatting with hexane (10 : 1 v/w) at 25 °C, barley flour was centrifuged at 7,000 ×g for 30 min and then air-dried.
e defatted barley flour was treated with 1 M NaCl solution (10 : 1 v/w) for 1 h to remove albumin, globulin, and glutelin.is step was repeated twice and then washed by deionized water 3 times (1 h each time) to eliminate the disturbance of reagents and small molecule materials, and centrifuged at 7,000 ×g for 30 min at room temperature.Finally, the resulting precipitate was dried in a vacuum oven at 45 °C for over 36 h, and the moisture was 13.37% of material weight.e weight losses of barley flour during defatting, salt dissolution, and water washing were 0.6%, 2.7%, and 2.2%, respectively.Anhydrous ethanol (>99%) and hexane (>97%) were provided by Sinopharm Chemical Reagent Co., Ltd.(Shanghai, China).All the other relevant reagents were of analytic grade.
e response surface and contour plots showed in this paper were produced by Design Expert version 8.0.5b software without any change.
e scanning electron microscope images and electrophoresis of two hordein fractions were marked by photoshop software.e diameters distribution of hordein protein was conducted by Oringin 8.0 software.

Ultrasonic-Microwave-Assisted Extraction of Hordein.
Hordein was extracted in an ultrasonic-microwave extractor.Briefly, 20 g of dried powder was transferred into a 250 ml beaker, and 200 ml of 55% (v/v) ethanol solution was then added.To decrease solvent volatilization during extraction, each beaker was sealed with parafilm.e extractor was self-assembled with a (2450 ± 50) MHz microwave oven and a 25 kHz ultrasonic processor.Microwave passed through the beaker to irradiate the pretreated sample.Once the extraction temperature exceeded the settled ceiling temperature, a platinum temperature sensor sent feedback to the temperature control system, thereby temporarily stopping microwave irradiation.Ultrasonic wave was transferred from the conical transducer assembled at the top of the oven shell via a hole.e microwave power was set from 1 to 1000 W, and the ultrasonic power was set by percentage of maximum output power (800 W) for each 10 s within 6 s pause.e experiment was duplicated twice, and the average extraction yield of hordein was taken as the response.After treatment with UMAT, resulting mixture was centrifuged (8,000 ×g for 30 min) to collect the supernatant.e yield of hordein was determined by UV-visible spectroscopy at 226 nm.Hordein fraction with 92.3% purity was used as the standard protein.e yield of barley hordein based on the dry weight of barley, defined in (1), was a valuable index for evaluating the extraction process.
where C H is the concentration of hordein in the supernatant detected by UV-visible spectroscopy at 226 nm, V is the volume of supernatant in each experiment, and M o is the dry weight of barley flour calculated according to the weight loss.

Traditional Extraction of Hordein.
Hordein was extracted in a thermostatic water bath, 20 g of dried powder was transferred into a 250 ml beaker, and then 200 ml of 55% (v/v) ethanol solution was added into the beaker with stirring for 2 h at 65 °C.To decrease solvent volatilization during extraction, each beaker was sealed with parafilm.
After treatment with water-bath method, resulting mixture was centrifuged (8,000 ×g for 30 min) to collect the supernatant.e follow-up processing was same with the UMAT method mentioned before.

Experimental Design.
A 5-level-4-factor CCD was performed to optimize the hordein extraction process, and the selected variables with coded/uncoded levels are presented in Table 1.All 31 experimental conditions and results are shown in Table 2. Seven axial points were used to estimate the pure error sum of squares.e fitted polynomial 2 Journal of Food Quality equation was expressed as surface and contour plots to visualize the relationship between the response and experimental levels of each factor and also to optimize the experimental conditions.Design Expert version 8.0.5b was used to fit the experimental data to a quadratic polynomial equation to obtain coefficients of the following equation: where Y is the response, X i and X j are the coded independent variables, β 0 is the constant, β i is the linear-term coefficient, β ii is the quadratic-term coefficient, and β ij is the cross-term coefficient.Analysis of variance (ANOVA) was performed (Table 3), and P < 0.05 was considered statistically significant.

Morphological Analysis.
e ethanol supernatants obtained from UMAT extraction under optimum conditions and traditional extraction were collected, respectively.Afterwards, the hordein ethanol extracts were concentrated to one-fifth of initial volume using a votary evaporator at 60 °C under vacuum.
en, the precipitates were isolated by centrifugation (8,000 ×g for 30 min).Both of the separated hordein fractions from two methods were redissolved and dispersed equally in 55% isopropanol-water solution and freeze-dried for 18 h under the same conditions.Both hordein fractions were glued on specimen stubs by coating with gold.Each sample was observed at an accelerating potential of 20 kV under high vacuum during micrography.

Electrophoresis of Hordein
Fractions.SDS-PAGE was performed to evaluate the differences between subunits in the two hordein fractions.Extracted hordein fractions were mixed with loading buffer (urea solution) and heated at 100 °C for 5 min.After cooling, precipitates were removed by centrifugation, 60 µg protein sample was loaded into 12% SDS-PAGE gel, and subjected to electrophoresis at a constant voltage of 80 V. en, the gel was stained with 0.1% (w/v) Coomassie brilliant blue-R-250 and destained with the solution containing water, methanol, and acetic acid (4 : 5 : 1, v/v).

Results and Discussion
3.1.Model Fitting.RSM is an effective statistical technique for process optimization, which was herein utilized to design a series of experiments to establish a predicting model for optimizing the UMAT extraction conditions for hordein.
e tted equation had a determination coe cient (R 2 ) of 0.9457, revealing that 94.57% of variation in the response could be explained by the model [16].e points in Figure 1 form a nearly linear pattern, indicating that normal distribution was a suitable model for this data set.e optimal extraction conditions were obtained from the tted model as follows: 78.2 °C, 298.9 W microwave power, 690.2 W ultrasonic power, and 20.4 min extraction time.According to the model, the predicted yield of hordein under the optimal conditions was 8.97%, and the mean experimental value reached 8.84% at 78 °C temperature, 290 W microwave power, and 690 W ultrasonic power after 20 min.
Statistical signi cance of the regression model was checked by the F and P values, and the ANOVA results for response surface quadratic model are shown in Table 3. Corresponding variables became more signi cant with the increased absolute F value and decreased P value.e quadratic model was extremely signi cant (P < 0.0001), so it was suitable for describing the UMAT extraction process.Clearly, the four variables were extremely signi cant, indicating the optimization of these factors was imperative.All the quadratic terms of X 2  1 , X 2 4 , X 2 3 , and X 2 2 were signi cant to the extraction yield of hordein, and the rst three were extremely signi cant.Additionally, two interaction coe cients (X 1 X 3 and X 3 X 4 ) were signi cant (P < 0.05), and the "lack of t" of the obtained model was statistically insigni cant (P 0.3869).[17].However, this method is rather complicated, so it is necessary to gure out the interaction between independent variables during UMAT extraction.

Response Surface and Contour Plots. UMAT is a typical technique that intensi es bioactive extraction from raw materials
e three-dimensional response surface and contour plots generated from the predicting model (3) are shown in Figures 2-5.Fixing other two variables at zero level, the three-dimensional response surface showed that temperature and microwave power had quadratic e ects on the yield of hordein (Figure 2).In addition, circular contour plot indicates that the interactions between corresponding variables are negligible [18].e yield of hordein was elevated with increasing ceiling temperature and microwave power.Microwave is a rapid heating process that conducts heat from internal and external material particles simultaneously.e main disadvantage of applying microwave during bioactive extraction is inhomogeneous heating, thereby decreasing the extraction   yield due to partial overheating [19].erefore, we set ceiling temperature as a variable.According to the contour plot in Figure 2, the e ect of microwave power on hordein extraction yield is limited by temperature.Once temperature was xed, the variation of microwave power did not change the yield evidently as a result of temporary stop of irradiation.
e elliptical shape of contour plot in Figure 3 indicates that the interactions between temperature and ultrasonic power signi cantly a ect the hordein yield.e impact of ultrasonic power at 640 W on the hordein yield was elevated from 7% to over 8% with increasing ceiling temperature.Ultrasonic wave can cause solid particles break or collapse due to the cavity e ect.Subhedar and Gogate [20] studied the ultrasonic-intensi ed e ect at a xed microwave power and found that raising the ultrasound power hardly a ected the transfer process.Table 3 shows that ultrasonic power played a more important role than microwave power, suggesting that microwave might augment the extraction yield mainly through rapid heating.Once the ceiling temperature was xed, the e ects of microwave were weakened.However, compared with traditional water-bath extraction, microwave shortened the heating time.
Fixing microwave power and ultrasonic power at zero level, the three-dimensional response surface in Figure 4 shows the e ect of extraction time on hordein yield was nearly linear.Although the contour plot was similar to that in Figure 3, there was no signi cant interaction between temperature and extraction time (Table 3).Additionally, the yield of hordein from barley our increased with rising ceiling temperature, which directly re ected the in uence of microwave.To increase the extraction yield within a short time, microwave power should be elevated.A at plain three-dimensional surface plot was showed in Figure 5, and the interaction (X 3 X 4 ) between ultrasonic power and extraction time was the most signi cant one (Table 3).Probably, both of the two factors strongly stimulated the hordein yield at the same ceiling temperature.As shown in Figures 2-5, temperature in each experiment was the foremost limitation during the extraction of hordein from barley, and ultrasonic power interacts with suitable temperature.Both of the factors were positively correlated with the hordein yield.In addition, ultrasonic power and extraction time were interactive parameters for the extraction process, managing to augment the extraction yield.In short, UMAT immensely shortened the extraction time and increased the extraction yield.

Morphology and Preliminary Characterization of
Fractions.As shown in Figure 6, the hordein fraction from UMAT had accumulation block appearance (Figure 6(a))  ).e global protein diameters ranged from 400 to 1800 nm (Figure 6(d)), and the average diameter was 931 nm.As a substitute for mechanical stirring or other intensi cation technologies, ultrasonic waves could markedly a ect the physical morphology of bioactive material and has been widely applied to some physicochemical processes for bioactive crushing [20].In this study, the morphological di erence indicated that UMAT decreased the hordein molecule size and promoted protein aggregation because of the intense vibration and mechanical function of ultrasound.SDS-PAGE (Figure 7) showed that the hordein fractions from two methods had identical subunits.e main storage subunits were B-hordein (36-43 kDa) and C-hordein (45-60 kDa).

Conclusion
In summary, we herein optimized the conditions of UMAT for hordein extraction from barley.e optimal extraction yield was 8.84% at 78 °C temperature, 298 W microwave power, and 690 W ultrasonic power after 20 min, being consistent with the quadratic model (8.97%).e selected variables remarkably increased the hordein extraction yield from 4.7% (traditional ethanol solution extraction) to 8.84% in the extraction process.In addition, the subunit fractions of hordein extracted from UMAT and traditional method had no obvious di erence, although the particle diameters were slightly di erent due to the ultrasonic vibration e ect.
i c r o w a v e p o w e r A : t e m p e r a t u r e Yield of barley hordein

Figure 2 :
Figure 2: Response surface plot and contour plot showing the e ect of temperature and microwave power on the yield of barley hordein at ultrasonic power 640 W and extraction time 18 min.

Figure 3 :
Figure 3: Response surface plot and contour plot showing the e ect of temperature and ultrasonic power on the yield of barley hordein at microwave power 255 W and extraction time 18 min.

Figure 4 :
Figure 4: Response surface plot and contour plot showing the e ect of temperature and extraction time on the yield of barley microwave power 255 W and hordein at ultrasonic power 640 W.

Figure 5 :Figure 6 :
Figure 5: Contour plot showing the e ect of ultrasonic power and extraction time on the yield of barley hordein at temperature of 65 °C and microwave power 255 W.

Table 2 :
Experimental design and results of the yield of barley hordein.

Table 1 :
Independent variables and their levels used for the CCD.

Table 3 :
Variance analysis of the regression model.
According to CCD, 31 experimental points were run regularly.Response values in Table2showed that the yields of hordein ranging from 6.7% to 8.8%.e regression equation obtained for hordein extraction is presented below: