Recovery of Yam Soluble Protein from Yam Starch Processing Wastewater

Over the past two decades, many studies have shown that the yam storage protein dioscorin, which is abundant in the wastewater of starch processing, exhibits many biological activities both in vitro and in vivo. In the present study, the acid-precipitation method was optimized using Box-Behnken design (BBD) combined with response surface methodology (RSM) for the recovery of yam soluble protein (YSP) from wastewater. The experimental yield of YSP reached 57.7%. According to relative quantitative proteomics (LC-MS/MS), the crude YSP was mainly composed of 15 dioscorin isoforms, which was further verified by anion-exchange and size-exclusion chromatography. YSP was found to be rich in glutamic acid and aspartic acid, and the eight essential acids made up approximately 33.7% of the YSP. Moreover, the YSP demonstrated antioxidant activity, including scavenging DPPH, hydroxyl and superoxide anion radicals, and the possible structure-activity relationships were discussed. These results indicated that YSP produced by acid precipitation may be used as a protein source with antioxidant properties.

Effect of the ratio of liquid to material on the yield of YSP. As shown in Fig. 1b, the effect of the liquid-to-material ratio (1:1, 2:1, 3:1, 4:1 and 5:1 mL/g) on the yield of YSP was studied when the extraction pH and time were 9.0 and 20 min, respectively. The yield increased to its greatest amount of 52.7% at 3:1 mL/g and then gradually increased with increasing ratio, which may have been due to enhancement in the dilution effect, where the viscosity and protein concentration of the extracts decreased. Increased protein dissolution results in increased extraction yield 21 . When the liquid-to-material ratio was greater than 3:1 mL/g, the rate of diffusion decreased, resulting in a slower yield increase. Therefore, a 3:1 mL/g liquid-to-material ratio was used for the following experiments. Fig. 1c, the effect of extraction time (0, 20, 40, 60 and 80 min) on the extraction yield of YSP was studied when the extraction pH and liquid-to-material ratio were fixed at 9.0 and 3:1 mL/g, respectively. The extraction yield reached 55.7% after 20 min and then gradually increased as the extraction continued. The results suggested that 40 min of extraction is sufficient to obtain the greatest extraction yield of YSP.

Effect of extraction time on the yield of YSP. As shown in
Response surface analysis. The  where R 1 is the yield of YSP (%) and A, B and C are the coded factors of the tested variables: extraction pH, liquid-to-material ratio and time, respectively. As shown in Table 1, the analysis of variance (ANOVA) of the response surface quadratic model indicated that the regression model obtained was highly significant (P < 0.0001) with an F value of 263.99. The lack of fit F value of 0.72 implied that the value was not significant compared to the pure error. The results showed that all investigated parameters had a notable effect on the extraction yield of YSP and could be ranked in decreasing order of importance in their effects as follows: extraction pH > liquid-to-material ratio > extraction time. The coefficient of determination (R 2 ) obtained was 0.9971, suggesting that the obtained model adequately represented the real relationship among the selected parameters. These results demonstrated that the yield of YSP was affected by three independent factors (A, B and C), two interaction terms (AB and BC), and two quadratic terms (A 2 and B 2 ).
The response surface and contour plots of the mutual interactions of the independent variables are shown in Supplementary Fig. 1. First, the effects of extraction pH and liquid-to-material ratio and their interaction on protein yield are presented in Supplementary Fig. 1a,b. The protein yield initially increased with increasing liquid-to-material ratio but decreased when the extraction pH and liquid-to-material ratio were further increased beyond a certain point. As shown in Supplementary Fig. 1a, a fractional elliptic contour was observed, suggesting the existence of significant and positive synergistic effects of extraction pH and liquid-to-material ratio on protein yield, which was in accordance with the results obtained from Supplementary Table 1. Supplementary  Fig. 1c,d present the effects of the liquid-to-material ratio and extraction time and their interaction on the protein yield. The protein yield first increased with increasing liquid-to-material ratio but then decreased when the liquid-to-material ratio and extracting time further increased beyond a certain point.
The optimal extraction conditions for YSP were obtained when using the following conditions: liquid-to-material ratio of 3:1 mL/g, pH 8.5, and extraction for 20 min. Under the optimum conditions, the experimental YSP extraction yield reached 57.74%, which was similar to the predicted value (57.88%). The YSP extraction regression model was highly significant, indicating that it can be used to predict protein extraction yield.
protein composition of YSp. YSP proteins were identified by LC-MS/MS and quantified using the emPAI label-free relative quantification method. There were 29 proteins identified from YSP, and 16 proteins showed emPAI values higher than 1.0 ( Table 2). There were 15 dioscorin/tuber storage related proteins among the 16 high www.nature.com/scientificreports www.nature.com/scientificreports/ abundance proteins. Sequence alignment was conducted for these 15 proteins. As shown in Fig. 2, these proteins were all isoforms of dioscorin sharing high sequence similarity, which was similar to a previous study that identified 12 isoforms of dioscorin from a 2D gel of purified dioscorin 22 . Furthermore, acidic endochitinase (emPAI value: 12.74) was also detected in YSP, which provides defense against chitin-containing fungal pathogens 23 . Compared to dioscorin and its isoforms, however, the amount of acidic endochitinase was low. In addition, trace www.nature.com/scientificreports www.nature.com/scientificreports/ amounts of other proteins were also identified in YSP, including glyceraldehyde-3-phosphate dehydrogenase, ascorbate peroxidase and mannose-specific lectin.
Purification of YSP. The anion-exchange chromatography results of YSP are shown in Fig. 3a. A major peak was observed, and no other proteins besides dioscorin were detected on the SDS-PAGE gel, which was similar to our previous result 11 . The size exclusion chromatography result of YSP is shown in Fig. 3b. The elution profile shows that YSP was composed of two fractions, namely, a high molecular weight fraction and a low molecular www.nature.com/scientificreports www.nature.com/scientificreports/ weight fraction, which was similar to a previous report demonstrating that there are two classes of dioscorin (A and B) sharing 69.6% sequence identity 8 . Table 2 also indicates that there were mainly two molecular weights for dioscorin isoforms, namely, 30 and 31 kDa, which showed no difference on the SDS-PAGE gel ( Fig. 3b; the original gels are shown in Supplementary Fig. 2). Thus, separation of the two fractions by size exclusion chromatography was not achieved.
Based on the separation results above, crude YSP without further purification, which is easily obtained by industrial scale recovery, was used for the following experiments. Table 3, the most abundant amino acids detected in YSP were Asp and Glu, accounting for 11.9% and 18.5% of the total amino acids, respectively. Collectively, the eight essential amino acids (Lys, Ile, Leu, Met, Thr, Phe, Val and Trp) of YSP made up approximately 33.7% of the total amino acids, which was slightly lower than that of sweet potato protein (40.7%) 24 . Some of these amino acids possess the following radical-scavenging activities due to their unique structure: the proton donation ability from the imidazole group of His; the hydrogen donation ability from the phenolic groups in Tyr and Phe; and the oxidization of Met to Met sulfoxide 25 . According to Table 3, the total content of the abovementioned four amino acid residues in YSP was 14.9% on a dry basis, which was higher than their combined contents in sweet potato protein (12.3%) 26 .

Amino acid composition of YSP. As shown in
In addition, the content of some essential amino acids or pairs of amino acids in YSP preceded the standard of the 'ideal protein' described by the FAO/WHO 27 . Specifically, the contents of Ile, Met + Cys, Phe + Tyr and Val of YSP were 135%, 246%, 167% and 145%, respectively, of their requirements depicted by the WHO standard (Supplementary Table 2). However, the Lys, Thr and Tyr contents of YSP were lower than the FAO/WHO recommendation for children, accounting for 90%, 98% and 48% of the amino acid requirements, respectively. free radical-scavenging activities of YSp. DPPH radical-scavenging activity. The DPPH radical-scavenging activities of YSP and ascorbic acid were investigated (Fig. 4a). The results indicated that both YSP and ascorbic acid (positive control) showed evident scavenging activities on DPPH radicals in a concentration-dependent manner. The DPPH radical-scavenging activity of YSP increased from 1.31 to 60.4% when the protein concentration increased from 0.01 to 0.5 mg/mL. The DPPH scavenging activity of antioxidants was influenced by many factors. A previous report has suggested that the unpaired electrons of DPPH can react with protons donated by antioxidants to convert the DPPH radical into its nonradical form (DPPH-H) 28 . Hou et al. hypothesized that the thiol groups of yam storage protein (dioscorin) may reduce dehydroascorbate (DHA) to regenerate ascorbate to prevent oxidative damage to yam tubers 16 . Jheng et al. observed that there are no free thiol groups in dioscorin and that its DPPH radical-scavenging activity may be related to tryptophan, hydrophobic residues, or the hydroxyls of aromatic residues, such as tyrosine 18 . Structural studies have shown that dioscorin has a www.nature.com/scientificreports www.nature.com/scientificreports/ Cys28-Cys187 disulfide bond and that its DHA reductase activity is related to its carbonic anhydrase (CA) active center (Fig. 5) 29 . Thus, the DPPH-scavenging activities of YSP may be related to the aromatic amino acids (Trp and Tyr), the CA active center (His 95, His97 and Gln114) and some surface hydrophobic amino acids (Fig. 5).
Hydroxyl radical-scavenging activity. The scavenging activities of YSP and ascorbic acid (positive control) on hydroxyl radicals are shown in Fig. 4b. Both exhibited significant hydroxyl radical-scavenging activities that increased with increasing concentration (P < 0.05). The scavenging activity of YSP at 0.5 mg/mL was 77.1%, whereas the scavenging activity of ascorbic acid at 0.5 mg/mL was 96.6%. For YSP, the scavenging activity of hydroxyl radicals increased from 2.6% to 35.7% in the concentration range from 0.01 to 0.1 mg/mL. Tryptophan in patatin and sweet potato trypsin inhibitors have been reported to be responsible for hydroxyl radical-scavenging activities 19,30 . Figure 5 shows that Trp25 of dioscorin may be involved in the hydroxyl radical scavenging activity of YSP.
Superoxide anion radical-scavenging activity. The superoxide radical-scavenging activities of different concentrations of YSP and ascorbic acid (positive control) were tested (Fig. 4c). The scavenging activity of YSP at 5 mg/ mL was 27.6%, whereas the scavenging activity of ascorbic acid at 5 mg/mL was 97.2%. For YSP, the scavenging activity increased from 3.2% to 15.4% in the concentration range from 0.01 to 1 mg/mL. conclusion YSP was successfully recovered from the wastewater of yam starch processing using the acid precipitation method. RSM was successfully performed on the extraction optimization of YSP. The results showed that YSP was mainly composed of dioscorin isoforms, which were rich in glutamic acid and aspartic acid. The eight essential amino acids made up approximately 33.7% of the YSP. Additionally, the present results showed that YSP had the function of scavenging free radicals. Further studies on the functional properties of YSP are required to expedite progress to make YSP an effective component in the nutraceutical and health food industries.

Materials and Methods
Materials. Yams (Dioscorea opposita Thunb.) were purchased from a local market (Shenyang, China) 31 . All reagents used in the present study were of reagent grade.
Sample preparation. Starch production was conducted in the laboratory by imitating the starch factory process according to the method of Daiuto et al. with some modifications 32 . Yams were washed, peeled, sliced and crushed into a homogenate by adding different amounts of 1% sodium bisulfate solution (liquid-to-material ratio) at different pH values (extraction pH). The homogenate was stirred for a set period of time (extraction time) and filtered through a 0.25 mm sieve. After centrifugation at 5000 g for 30 min, the obtained supernatant was the wastewater that contained YSP.
The pH of the wastewater was adjusted to approximately 3.5 (the pH at which the largest amount of YSP is precipitated according to a preliminary study) using 2 mol/L HCl. After magnetically stirring for 1 h at room  www.nature.com/scientificreports www.nature.com/scientificreports/ temperature, the slurry was centrifuged at 5000 g for 30 min. YSP was prepared by resolubilizing the pellet in distilled water with the addition of 1.0 mol/L NaOH to maintain a pH of 7, followed by ultrafiltration and lyophilization for other experiments. The concentrations of the proteins in the supernatants were measured according to Markwell et al. 33 . Protein extraction yield was calculated as follows: (the protein content of the supernatant)/(the protein content of the raw material) ×100.
Parameter optimization for YSP recovery. First, the extraction pH, liquid-to-material ratio and extraction time were selected as the three variables to consider during the optimization (one-factor-at-a-time test) of www.nature.com/scientificreports www.nature.com/scientificreports/ the YSP extraction. Specifically, each variable was tested individually as follows: the pH was tested in a range from 7 to 11; the liquid-to-material ratio was tested in a range from 1:1 to 5:1 mL/g; and the extraction time was tested in a range from 0 to 80 min.
Second, the influences of the three variables, including (A) extraction pH, (B) liquid-to-material ratio and (C) extraction time, on the YSP yield (Y) were studied using RSM. Seventeen BBD runs were performed at all design points in a random order 34 .
Proximate composition. The crude protein, total sugar, crude fiber, fat and ash contents of the YSP were determined according to the method of the AOAC (2005) 35 .
SDS-PAGE. SDS-PAGE was performed according to the method of Shiu et al. 36 . The electrophoresis sample was prepared by dissolving YSP (1 mg/mL) in 2× sample buffer with β-mercaptoethanol (β-ME) and heating for 5 min in boiling water. Electrophoresis was performed on a 5% acrylamide stacking gel and 12% acrylamide resolving gel. The 65 min gradients were performed at a flow rate of 300 nL/min starting from 5% to 35% solvent B (acetonitrile with 1% formic acid) over 40 min followed by a 5 min increase to 90% B, a 5 min maintenance period at 90% B and ultimately returning to 5% B. The peptides were subjected to positive ion nanoelectrospray ionization followed by MS/MS using a Thermo Scientific Q Exactive mass spectrometer. The original MS/MS data were converted to MGF formatted files using MM file conversion, and the obtained files were searched against the UniProt database using the MASCOT search engine. The following parameters were used for protein identification: carbamidomethyl (C) was the fixed modification; oxidation (M) was the potential variable modification with a maximum missed cleavage value (trypsin digestion) of 2, a peptide mass tolerance of 20 ppm and a fragment mass tolerance of 0.6 Da. The relative quantification of the identified proteins was accomplished using the exponentially modified protein abundance index (emPAI) label-free method 37 . purification of YSp. Anion-exchange chromatography. YSP was first purified on a Resource Q anion-exchange column fitted to an AKTA FPLC system (GE Healthcare, Piscataway, NJ, USA). YSP solution (5 mg/mL; 5 mL) was injected into the system and eluted with a linear gradient of 0 to 0.3 mol/L NaCl in 0.02 mol/L Tris-HCl buffer (pH 8.0). The flow rate was 6 mL/min, and the main peak was collected and dialyzed for subsequent size exclusion chromatography.

Determination of YSP protein composition by
Size-exclusion chromatography. YSP solution (10 mL) was injected into a Superdex 75 size exclusion column fitted to an AKTA FPLC system and eluted with 0.02 mol/L Tris-HCl buffer (pH 8.0). Each 3 mL fraction was collected at a 1 mL/min flow rate. the ampoule, hydrolyzation of YSP was performed at 110 °C under vacuum for 24 h. The hydrolysate was evaporated to dryness at 60 °C under vacuum. Sodium citrate buffer (pH 2.2; 3-5 mL) was added to the dried sample to obtain a 50-250 nmol/mL amino acid concentration. The solution was then filtered and loaded on a Hitachi L-8800 amino acid analyzer (Hitachi Ltd., Tokyo, Japan) 24 .
Determination of antioxidant activities. DPPH radical-scavenging assay. The DPPH radical scavenging activity was assayed using the method of Aluko and Monu 1 . In brief, 2 mL of diluted sample solution was mixed with 2 mL of DPPH solution (0.06 mg/mL). The solution was shaken vigorously and left to stand in the dark at room temperature for 20 min, and the absorbance was then measured at 517 nm. The scavenging activity was calculated by the following equation: where A 0 is the absorbance of the control group containing all reaction reagents without sample, and A 1 is the absorbance of the reaction solution mixed with the test sample.
Hydroxyl radical-scavenging assay. Hydroxyl radical (•OH) scavenging activity was assayed as described by Sun et al. 38 . The hydroxyl radical was generated by mixing 1 mL of H 2 O 2 solution (8.8 mmol/L) and 1 mL of FeSO 4 solution (9 mmol/L). Then, 1 mL of salicylic acid solution (9 mmol/L) and 1 mL of the sample solution were added sequentially to the reaction system. The mixture was then shaken vigorously and incubated at 37 °C for 20 min, and the absorbance at 510 nm was measured. The scavenging activity was calculated by the following equation: Scavenging activity (%) [1 (A A )/A ] 100 where A 0 is the absorbance of the control group containing all reaction reagents without sample; A 1 is the absorbance of reaction solution mixed with the test sample; and A 2 is the absorbance of background value without H 2 O 2 .
Superoxide anion radical-scavenging assay. Superoxide anion radical scavenging activity was measured as described by Balavigneswaran et al. 39 . Briefly, 4.5 mL of 50 mmol/L Tris-HCl buffer (pH 8.2) and 1 mL of sample solutions at different concentrations were mixed and incubated at 25 °C for 20 min. The mixture was then shaken vigorously for 5 min after the addition of 0.2 mL of 60 mmol/L pyrogallic acid. Afterwards, 1 mL of 8 mol/L HCl was added to terminate the reaction. The absorbance was measured at 325 nm. The scavenging activity was calculated by the following equation: