Optimization of ultrasound-assisted extraction of sheep abomasum protein concentrates by response surface methodology and evaluation of their properties

Large amounts of sheep abomasum (SA) are generated as a by-product of the sheep meat industry (Toldrá et al., 2012; Martínez-Alvarez et al., 2015). Efforts have been made to convert those by-products to a new source of functional ingredients or novel products due to their high nutritional value and good bioavailability (Mora et al., 2014; Queiroz et al., 2017). Several earlier studies have reported that SA is rich in proteins and numerous other nutrients (Li & Chen, 1987; Chen, 1982; Zhao, 2011). Therefore, it is assumed that sheep abomasum protein concentrates (SAPC) are valuable animal proteins that can be applied for functional or pharmaceutical products. Freeze-melting and stirring (Zhang, 2003; Zhao, 2011) are conventional extraction methods used for SAPC extraction, but these methods are time-consuming and can adversely affect bioactivity or pharmaceutical values (Jodayree et al., 2012; Tang et al., 2015). Therefore, to gain SAPC, a more environmentally friendly and highly efficient extraction process should be applied.


Introduction
Large amounts of sheep abomasum (SA) are generated as a by-product of the sheep meat industry (Toldrá et al., 2012;Martínez-Alvarez et al., 2015).Efforts have been made to convert those by-products to a new source of functional ingredients or novel products due to their high nutritional value and good bioavailability (Mora et al., 2014;Queiroz et al., 2017).Several earlier studies have reported that SA is rich in proteins and numerous other nutrients (Li & Chen, 1987;Chen, 1982;Zhao, 2011).Therefore, it is assumed that sheep abomasum protein concentrates (SAPC) are valuable animal proteins that can be applied for functional or pharmaceutical products.Freeze-melting and stirring (Zhang, 2003;Zhao, 2011) are conventional extraction methods used for SAPC extraction, but these methods are time-consuming and can adversely affect bioactivity or pharmaceutical values (Jodayree et al., 2012;Tang et al., 2015).Therefore, to gain SAPC, a more environmentally friendly and highly efficient extraction process should be applied.
Recent years, ultrasonic-assisted extraction (UAE) has been used to extract proteins from various sources including rice bran (Chittapalo & Noomhorm, 2009), rapeseed protein (Dong et al., 2011), porcine placenta (Tang et al., 2015), and chicken egg shell membrane (Jain & Anal, 2016).Compared with conventional methods, the UAE approach is time-reducing, and allows easy operation, high extraction yield, and low energy consumption (Rocha et al., 2017;Li et al., 2017).However, the application of UAE for the extraction of protein concentrates from sheep abomasum has not been reported.
On this study, response surface methodology (RSM) and a four-variable, three-level Box-Behnken design (BBD) were used for the optimization of four parameters of UAE including extraction time, ultrasound power, liquid/solid ratio, and pH for the highest water-soluble protein concentration (WSPC).Additionally, the yield, chemical composition, and functional properties of SAPC obtained by conventional extraction method (CEM) and UAE were determined and compared.This is the first study to determine the appropriate protocol to prepare SAPC, and the results should facilitate utilization of these industrial by-products.

Materials and reagents
Fresh SA was obtained from Urumqi slaughter house (Xinjiang, China), and rinsed into cold water to remove connective tissue and the residue in stomach, then cut into pieces and immediately frozen at -80 °C for 24 h.The frozen sample was lyophilized (FDU-2100, EYELA), crushed, defatted with ligarine, and stored in polyethylene bags at -20 °C until use.All reagents were of analytical grade.The WSPC was measured by Pierce  BCA Protein Assay Kit (Thermo Scientific).

Preparation of sheep abomasum protein concentrates
SAPC samples were prepared by two different methods, the CEM and UAE methods.For UAE, this method was performed in an ultrasonic generator (JY98-OOON Ningbo Scientz Biotechnology Co. Ltd., China).On short, defatted SA were extracted with alkaline solution at the established pH, ultrasound power and time.Then, the mixture was centrifuged at 12,000 r/min for 10 min at 4 °C.The pellet was discarded and the supernatant was collected, dialyzed and the WSPC was determined.Ammonium sulfate was added to the supernatant to reach 60% saturation and stirring for 3 h, then centrifuged at 10,000 r/min for 20 min.The precipitate was washed by de-ionized water, re-dissolved and dialyzed for 24 h against de-ionized water.The solution was lyophilized and kept in a polyethylene bag at -20 °C until use.
As a comparison, CEM method was performed.Defatted SA (1.0 g) were homogenized with alkaline solution (pH = 10, liquid/solid ratio = 25) and then stirred under ice bath for 240 min.Then the mixture was processed with the same protocol as UAE.

Experimental design and statistical analysis
RSM and BBD with four variables and three levels were used to optimize UAE parameters.The extraction time (X 1 ), ultrasound power (X 2 ), liquid/solid ratio (X 3 ) and pH (X 4 ) were chosen as the independent variables.The WSPC was determined as the response of the design experiments (Y).Based on the single-factor experiments (data not shown), X 1 (20, 30 and 40 min), X 2 (200, 400 and 600 W), X 3 (20, 25 and 30 mL/g) and X 4 (8, 9 and 10) were determined as critical levels with significant effect on protein extraction.Each independent variable and relative levels are given in Table 1.Twenty-four factorial points and five replicates of central point in the total 29 experiments were performed.The experimental results were analyzed using RSM algorithm and were fitted to the following predictive quadratic polynomial Equation 1: Where Y i is the response variable, β 0 is the model constant, β i is the linear terms, β ii are the squared terms, β ij is the interaction terms, and X i and X j are independent variables.

Electrophoretic analysis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of SAPC samples were carried out according to reported procedures with slight modifications (Laemmli, 1970) using 12% separating gel and 4% stacking gel.The electrophoresis was run at 75 V in the stacking gel and 150 V in the separating gel until the tracking dye reached the bottom of the gel.Then, the gels were stained with Coomassie Brilliant Blue G 250.

Yield and proximate composition
Protein, moisture, fat and ash contents of samples were determined according to the ADAC standard procedures (Association of Analytical Communities, 2007).The contents were expressed in g/100 g.Yield of samples were estimated as Equation 2: Where m d represented as the weight of extracted SAPC (g); m de represented as the weight of defatted SA (g).

Protein solubility
The assay was determined according to the reported procedures with slight modifications (Mao & Hua, 2012).Samples containing two milliliters of aqueous solution (0.1% w/v) were adjusted to different pH values ranging from 2.0 to 12.0 using 0.1mol/L HCl or 0.1mol/L NaDH, and then stirred and vortexed for 30 min.The mixtures were then centrifuged at 10000 r/min for 10 min.The supernatants were collected and the WSPC was measured.The solubility was calculated as Equation 3: Where m represented as WSPC in supernatant (mg/g); m T represented as the content of total protein in SA (mg/g).

Emulsifying properties
Evaluation of emulsifying activity index (EAO) and emulsion stability index (ESO) were performed based on the reported procedure (Pearce & Kinsella, 1978).Samples containing thirty milliliters of solution (10 g/L) were mixed with 10 mL of soybean oil, and were homogenized with an ultrasonic disperser for 2 min at 200 W.An aliquot of the emulsion (50 μL) was immediately pipetted out after homogenization from the bottom at 0 and 10 min and diluted with 5 mL 0.1% (w/v) sodium dodecyl sulfate (SDS) solution.The absorbance of the diluted emulsion was measured at 500 nm using UV/VOS spectrophotometer (China, WFZ 754, CD, LTD).EAO and ESO were calculated as Equation 4and Equation 5 respectively: Where A 0 and A 10 are the absorbance of analyzes at 0 min and 10 min respectively, dilution factor is 100, C is the initial concentration of protein (g/mL), φ is equal to 0.25 (oil volumetric fraction), A = A 0 -A 10 and t is 10 min.

Foaming properties
Foaming capacity (FC) and foam stability (FS) of samples were determined following a previous method (Turan et al., 2015) with minor modifications.Samples containing solution (10 g/L, 25 mL) were whipped for 10 min with an ultrasonic disperser for 2 min at 200 W.The whipped samples were immediately transferred into a cylinder (50 mL).The volumes before and after whipping were recorded.The total volume of foam remaining was recorded after 30 min quiescent period at room temperature.All experiments were conducted with triplicate samples.FC and FS were calculated as Equation 6and Equation 7 respectively: Where V 1 is the volume after whipping, V 2 is the volume before whipping, V 3 is the volume after standing.

Water and oil holding capacity
Water holding capacity (WHC) and oil holding capacity (DHC) of samples were determined according to the previous method (Jain & Anal, 2016).Approximately 25 mL of distilled water or refined soybean oil were added into pre-weighed centrifuge tubes containing 1 g of dry sample and subjected to stirring and mixing for 30 min.After being centrifuged at 2000 r/min for 30 min, the supernatant was discarded and the residue was drained for 15 min at room temperature and then weighted.WHC and DHC were calculated as the weight of water and oil adsorbed by 1 g sample.

Statistical analysis
Design-Expert 8.06(State-Ease Onc., Minneapolis, USA) was used for experimental design and data analyses of RSM.Dther experiments were performed with SPSS Version 19.0 (Chicago, OL, USA) using ANDVA and Tukey analysis.The statistical differences between samples were measured by the least significant difference (LSD) test.P < 0.05 was considered to be significant.All experiments were performed in triplicate and SD was calculated.

Analysis of response surface
A series of three-dimensional (3D) response surface graphs were generated and are presented in Figure 1 a-f, which shows the relationship between WSPC and extraction parameters.According to Figure 1 c, e, f, WSPC increased significantly with the increase of pH from 8 to 10.This result was consistent with previous findings that alkaline solvents are the most effective solvent to extract SAPC (Zhang, 2003).As shown in Figure 1 a, d, e, the WSPC of SA increased when ultrasonic power increased from 200 W to 400 W, which may be attributed to the further breaking of SA cells, leading to the release of more proteins into the liquid/solid system (Tang et al., 2015).However, when the ultrasonic power was further increased from 400 W to 600 W, the WSPC slowly decreased because the higher ultrasound power generated high-pressure conditions that reduced the solubility of the protein (Zhu et al., 2009).As presented in Figure 1 a, b, c, WSPC increased rapidly from 20 min to 30 min.During this period, the cell walls of SA broke gradually, which accelerated the release of proteins.However, with additional time, no obvious increase of WSPC was observed.Similarly, as shown in Figure 1 b, d, f, WSPC increased with the increase of the liquid/solid ratio, and then decreased.

Validation of the model
The optimum UAE conditions were obtained using Design Expert 8.06 software, and determined as a practical optimum: extraction time of 28 min, ultrasonic power of 450 W, liquid/solid ratio of 25 mL/g, and pH of 10.Verification experiments were performed under optimal conditions (n = 3), to further validate the reliability of the theoretical model prediction.The results showed that experimental results for WSPC were very close to the predicted values (320.5 ± 23.6 and 331 mg/g, respectively), values were not significantly different (P > 0.05).Thus, it could be concluded that the established model in this study was appropriate and valid.

Comparison of UAE method with CEM
CEM was performed and compared to UAE to evaluate the extraction efficiency.The extraction time of UAE (28 min) was less significantly than CEM (240 min).The WSPC of extract obtained by UAE was higher than for CEM (320.5 ± 23.6 mg/g, 306.2 ± 18.9 mg/g respectively).The better performance with UAE may be due to ultrasound enhanced mass transfer and particle diffusion within the liquid/solid system, as CEM agitation only enhances external mass transfer (Tang et al., 2015).
As shown in Figure 2, the protein species and the molecular weight of proteins in the SAPC samples were determined by SDS-PAGE.The SAPC samples obtained by UAE and CEM contain a variety of protein bands of similar molecular weight (MW) between 10 and 130 kDa, based on comparison to a standard composed of a mixture of proteins of known MW.
Further analysis of the composition of the proteins in SAPC and evaluation of the biological activities of SAPC is warranted.

Yield and proximate composition
The yield and proximate composition of defatted SA and SAPC samples were measured and are shown in Table 3. Significant (P < 0.05) differences were found in yield, fat, ash, protein, and moisture contents of all samples.The highest yield and protein values were observed in SAPC obtained by the UAE method, and were 24.41 ± 0.71% and 92.36 ± 3.80 g/100g, respectively.These findings indicate that UAE was the most effective method The foaming properties are also important properties in manufacturing food or medicine formulations.FS of SAPC prepared using the UAE method was higher than that using CEM, 16.44 ± 1.70 and 12.14 ± 1.45% respectively (P < 0.05).This difference might be due to the higher protein solubility of the SAPC sample prepared using the UAE method compared to that of the sample prepared by CEM (Suppavorasatit et al., 2011).However, FC of SAPC samples were not significantly different (P > 0.05), 23.19 ± 0.48 and 23.16 ± 0.08% respectively.Similarly, previous study also showed that foam properties are typically influenced by factors affecting solubility during modification processes (Hou et al., 2017;Zou et al., 2017).
WHC and DHC are important properties that determine the shelf life of products (Wu et al., 2009;Jain & Anal, 2016).The values of WHC and DHC were significantly different (P < 0.05) for the different SAPC samples.SAPC prepared by the UAE method had higher DHC values (21.20 ± 1.10 g/g), but lower WHC values (6.43 ± 0.19 g/g) than those of the SAPC prepared by CEM.These results indicate that the SAPC sample prepared by UAE retained more oil than water, and was also more lipophilic than the material prepared using CEM.

Conclusion
This is the first report of testing UAE for SAPC extraction.The effects of extraction time, ultrasonic power, liquid/solid ratio, and pH were investigated using RSM to maximize the WSPC of the extract.The results indicated that UAE technology increased the protein contents and yield but also shortened the extraction time.The SAPC obtained by UAE method showed enhanced functional properties, such as solubility, emulsifying properties, foaming properties, and oil holding capacity as compared to the properties of the extract prepared by CEM.Taken together, the UAE method can be applied as a novel procedure to prepare functional SAPC, a valuable animal protein with potential uses as a pharmaceutical or nutritional ingredients in various food or medicines.