低温脱脂大豆粕的低丰度蛋白提取和高丰度蛋白去除条件的优化

Optimization of low-abundance protein extraction and abundant protein removal from defatted soybean meal 低温脱脂大豆粕的低丰度蛋白提取和高丰度蛋白去除条件的优化 1 2 first-author †E-mail:liummzhou@163.comgqzhao@yzu.edu.cn †E-mail:liummzhou@163.comgqzhao@yzu.edu.cn liummzhou@163.com 3 1 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:liummzhou@163.comgqzhao@yzu.edu.cn †E-mail:liummzhou@163.comgqzhao@yzu.edu.cn liummzhou@163.com College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China Huai’an Bioengineering Branch of Jiangsu Union Technical Institute, Huai’an 223200, China Huai’an Bioengineering Branch of Jiangsu Union Technical Institute, Huai’an 223200, China Key Laboratory of Food and Biotechnology, Changshu Institute of Technology, Suzhou 215500, China Key Laboratory of Food and Biotechnology, Changshu Institute of Technology, Suzhou 215500, China The aim of this study was to optimize the conditions for the extraction of low-abundance proteins (LAPs) and the removal of abundant proteins (APs; β-conglycinin and glycinin) from soybean meal. Single factor and orthogonal experiments were designed to determine the effects of four factors (isopropanol concentration, total extraction time, ultrasonic power, and ultrasonic time) on protein concentration in isopropanol extracts. Proteins in the isopropanol supernatant and the cold acetone precipitate of isopropanol were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). The results showed that the optimal conditions were 50% isopropanol, ultrasonic pretreatment for 15 min at 350 W, and a total extraction time of 1 h. Under these conditions, the protein concentration in the isopropanol extracts reached 0.8081 g/L. Many LAPs were detected, including β-amylase, soybean agglutinin, soybean trypsin inhibitor, fumarylacetoacetase-like, phospholipase D alpha 1-like, oleosin, and even some unknown soybean proteins. The soybean APs (β-conglycinin and glycinin) were not found. The method may be useful for discovering new soybean proteins and extracting enough LAPs of soybean to allow further studies of their physiological effects on animals without the influence of APs. 目的 2 优化低温脱脂大豆粕中的低丰度蛋白提取和高丰度蛋白去除条件，为进一步探讨大豆低丰度蛋白对动物的生理功能影响提供试验材料。 创新点 2 结合提取时间和异丙醇浓度，将超声波（超声时间、功率）用于辅助异丙醇去除大豆高丰度蛋白和富集低丰度蛋白的研究。 方法 2 在单因素试验基础上，设计了超声时间、超声功率、提取时间和异丙醇浓度等四因素三水平的正交试验。并通过测定提取液中的蛋白浓度和通过聚丙烯酰胺凝胶电泳（SDS-PAGE）、质谱分析（MS）等蛋白质组学手段鉴定提取液或丙酮沉淀物中的蛋白质含量，实现了大豆高丰度蛋白去除和低丰度蛋白提取条件的优化。 结论 2 50%异丙醇，350 W超声15 min，提取1 h是大豆低丰度蛋白提取的最佳条件。提取物含多种低丰度蛋白（油质蛋白、大豆未知蛋白、磷酸酯D和无特征大豆蛋白），且无大豆高丰度蛋白（大豆球蛋白和β-伴大豆球蛋白）。 Protein extraction Abundant protein Low-abundance protein Defatted soybean meal MALDI-TOF-MS 蛋白提取 高丰度蛋白 低丰度蛋白 低温脱脂豆粕 质谱 Introduction 1 Soybean proteins have complex compositions, which result in the diversity of their classification and function. According to their abundance in soybeans, they can be classified as abundant proteins (APs) or low-abundance proteins (LAPs). As soybeans are the main source of plant protein, the extraction and physiological functions of soybean proteins have been widely studied. Most such studies have focused on soybean meal (Tibaldi et al., 2006 2006 ; Wang et al., 2006 2006 ; Saki et al., 2012 2012 ), soybean protein isolates (SPIs) (Jankowski et al., 2009 2009 ; Cornish et al., 2011 2011 ), and the soybean APs (β-conglycinin and glycinin) (Deak et al., 2006 2006 ; Adams et al., 2008 2008 ; Sun et al., 2008 2008 ). The extraction of most LAPs is beyond the scope of traditional techniques (Deak et al., 2006 2006 ), and they are very difficult to separate from APs. New methods are needed to facilitate studies of their extraction and physiological functions on animals and humans. With the rapid development of proteomics techniques, the separation and identification of soybean LAPs have received increasing attention. Treatment with trichloroacetic acid (TCA)/acetone, verified as an efficient and reliable method for the separation and identification of soybean proteins, has been used in the characterization of soybean storage proteins (Natarajan et al., 2006a 2006a ) and for the analysis of allergen and anti-nutritional proteins in wild and cultivated soybean seeds (Natarajan et al., 2006b 2006b ). Natarajan et al. ( 2009 2009 ) found that isopropanol treatment resulted in the depletion of APs and was better than TCA for preferentially enriching soybean LAPs. On the other hand, many studies have confirmed that ultrasound treatment facilitates the disintegration of particles. High-power ultrasound was reported as a powerful method to enhance the extraction of intracellular compounds from plant materials, including the extraction of oil, sugar, and protein from soy flakes (Li et al., 2004 2004 ; Karki et al., 2009a 2009a ; 2009b 2009b ; Bu et al., 2012 2012 ). To obtain enough LAPs for further studies of their physiological functions on animals without the influence of APs, we carried out single factor (isopropanol concentration, ultrasonic power, ultrasonic time, and the total extraction time) and orthogonal experiments to optimize the conditions for LAP extraction and AP removal. The concentration and identity of the proteins in the isopropanol extracts were determined by Coomassie brilliant blue R-250 (CBB) kit, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). Materials and methods 1 Materials and chemicals 2 Defatted soybean meal (crude protein, 55%) was obtained from Jilin Fengzheng Soybean Food Limited Company (China). A Coomassie brilliant blue kit was purchased from the Nanjing Jiancheng Bioengineering Institute (China). Ammonium bicarbonate, acetonitrile, and trifluoroacetic acid (TFA) were all of chromatographic grade (Sigma, USA). Trypsin (Promega, USA) was of sequencing grade. A 170-kD protein molecular mass marker (Thermo, USA) was used in electrophoretic analyses. Isopropanol and other reagents were of analytical grade (Sinopharm, China). Ultrapure water was self-made (Thermo, USA) and used for making all solutions. Single factor and orthogonal experimental design 2 First, without ultrasound treatment, the effects of isopropanol concentration and total extraction time on the extraction of soybean proteins were determined. Then, under the conditions of 40% isopropanol, a 1-h total extraction time, and ultrasound treatment for 20 min, the effect of ultrasonic power was investigated. The fourth experiment was conducted using 40% isopropanol, a 1-h total extraction time and ultrasound pretreatment at 400 W, to confirm the influence of ultrasonic time on soybean protein extraction. To further determine the optimal conditions for LAP extraction and AP removal from defatted soybean meal, orthogonal experiments were designed based on the results of the above experiments (Table 1 1 ), and the proteins in extracts were identified by SDS-PAGE and MS. A : isopropanol concentration; B : ultrasonic power; C : ultrasonic time; D : total extraction time 2 Level 4 Factor A (%) B (W) C (min) D (min) 1 40 350 5 40 2 45 400 10 60 3 50 450 15 90 Protein extraction from defatted soybean meal 2 The defatted soybean meal powder (500 mg) was homogenized with 5 ml of isopropanol solutions of known concentrations (35%, 40%, 45%, 50%, and 55% (v/v)), and then extractions were carried out for certain total time (0.5, 1, 2, and 3 h; ultrasound treatment and vibrating water-bath in sequence). The ultrasound treatment included ultrasonication at variable power (0, 200, 300, 400, or 500 W) for 10 min, or for a variable time (0, 5, 10, 15, or 20 min) at 400 W. Finally, the supernatant of the extracts was obtained after centrifugation at 12 000 r/min for 15 min at 4 °C, and the protein concentration in the supernatants was determined immediately using the Coomassie brilliant blue kit. The supernatant was isolated and placed in a clean 20-ml tube, and then double the volume of cold acetone was added to the supernatant before incubation at −20 °C overnight. Next day, the samples were centrifuged for 10 min at 12 000 r/min at 4 °C and the pellet was dried using a freeze dryer. Identification of proteins in isopropanol extracts 2 SDS-PAGE 3 According to Natarajan et al. ( 2009 2009 ) and Krishnan et al. ( 2009b 2009b ), the proteins in isopropanol extracts and cold acetone precipitates can be separated by SDS-PAGE on a 12% resolving gel at 100 V using a Mini-PROTEAN Tetra System (Bio-Bad, USA) and visualized by staining for about 1 h with CBB and fading overnight. Trypsin digestion and mass spectrometry 3 Trypsin digestion and MS were performed using the method of Liu et al. ( 2016 2016 ). After being excised and washed twice with ultrapure water and three times with 25 mmol/L ammonium bicarbonate in 50% acetonitrile for destaining, the protein bands of the CBB-stained gel were dehydrated with 200 μl of acetonitrile. The protein band pieces were then re-swollen in 10.0 ng/ml trypsin for trypsin digestion. The digestion was performed overnight at 37 °C after incubation at 4 °C for 30 min. Next day, the supernatant was extracted three times in 50% acetonitrile containing 0.1% trifluoracetic acid (TFA) and dried using a freeze dryer. All samples were analyzed using a 5800 matrix-assisted laser desorption/ionization-time of flight (MALDI TOF)/TOF analyzer (Applied Biosystems, Framingham, MA, USA). Mass spectra ( m / z 800–4000) were acquired in positive ion reflector mode, and the 20 most intense ions were selected for subsequent MS/MS sequencing analysis in 2 kV modes. Protein identification was performed by searching MS/MS spectra, and the peptide mass was obtained from National Center for Biotechnology Information (NCBI) protein databases using the search engine Matrix Science ( http://www.matrixscience.com http://www.matrixscience.com ). Statistical analyses 2 SPSS 16.0 statistical software was used for data analysis. Data were analyzed by one-way analysis of variance (ANOVA). Results and discussion 1 Effects of single factors on soybean protein extraction 2 There were significant differences in the protein concentrations of extracts obtained using five different isopropanol concentrations (35%, 40%, 45%, 50%, and 55%) ( P < 0.01; Fig. 1a 1a ). Krishnan ( 2004 2004 ) found that isopropanol could facilitate the enrichment of LAPs and the removal of APs in soybean, but Natarajan et al. ( 2009 2009 ) found that high isopropanol concentrations (exceeding 50%) resulted in a sharp decrease in both APs and LAPs identified by SDS-PAGE. This may be why in our study we found a highly significant ( P < 0.01) decrease in protein concentrations with increasing isopropanol concentrations. Data are expressed as mean±standard deviation ( n =6). Columns labeled with different capital letters indicate a significant difference at the 0.01 level ( P < 0.01), and columns labeled with different lowercase letters indicate a significant difference at the 0.05 level ( P < 0.05) In a kinetic study, the expression of dissolution was found to be analogous to the surface diffusion control of crystal growth (Tang and Nancollas, 2002 2002 ), implying that dissolution continues until equilibrium is reached. Before reaching equilibrium, the soybean protein dissolved in the extraction solution increased with increasing extraction time. In this study, the protein concentration after 1 h of extraction was significantly higher than that after 0.5, 2, or 3 h ( P < 0.01). There were no significant differences between protein concentrations after 0.5, 2, and 3 h of extraction ( P > 0.05). So, we considered 1 h as the optimal total extraction time for protein extraction from defatted soybean meal (Fig. 1b 1b ). It has been reported that while LAPs have been observed in 30%, 40%, and 50% isopropanol extracts by 1D-PAGE analysis, there was a decrease in both APs and LAPs at higher isopropanol concentrations (Natarajan et al., 2009 2009 ). So, it is impossible to separate the maximum number of LAPs (without APs) in defatted soybean meal by changing only the isopropanol concentration. Karki et al. ( 2009b 2009b ) found that ultrasound did not modify the peptide profile, regardless of the ultrasound conditions used, and after ultrasound for 120 s the SPI yield was increased by 13% at low power and 34% at high power. The particle size decreased nearly 10-fold following ultrasonic treatment at high amplitude for 120 s, resulting in the highest increase in total protein yield (46%), compared to samples with no ultrasonic treatment (Karki et al., 2009a 2009a ). In our study, ultrasound treatment improved the protein concentration in the isopropanol extracts, which showed a parabolic tendency in response to increasing ultrasonic power or duration (Figs. 1c and 1d 1c and 1d ). When the ultrasonic power exceeded 400 W, the protein concentration was higher in isopropanol extracts than in those without ultrasonic pretreatment ( P < 0.01), but there was no difference between 400 and 500 W ( P > 0.05). The protein concentration was higher in extracts with ultrasound treatment for 10 or 20 min than in those without ultrasonic pretreatment ( P < 0.01). Orthogonal experiment for the optimization of LAP extraction and AP removal 2 We confirmed that the concentration of proteins extracted by isopropanol from defatted soybean meal was higher at low isopropanol concentrations (35%, 40%, and 45%) than at the higher isopropanol concentration (50%) (Fig. 1a 1a ). This was confirmed by the lower protein concentration under conditions 3 (5 min (ultrasonic time), 450 W (ultrasonic power), 50% (isopropanol concentration), 90 min (total extraction time)), 5 (10 min, 400 W, 50%, 40 min), and 7 (15 min, 350 W, 50%, 60 min) compared to those under the other conditions (Fig. 2 2 ). In addition, the protein concentration under condition 7 increased by 9.54% and 8.93% compared with those under conditions 3 and 5, respectively, indicating that ultrasonic power, ultrasonic time, and total extraction time play important roles in soybean protein extraction. In our study, we also found that with 45% isopropanol the protein concentration increased by 12.98% under the conditions of ultrasound treatment for 15 min at 450 W with 40 min of total extraction time compared to that of ultrasound treatment for 10 min at 350 W with 90 min of total extraction time (Fig. 2 2 ). This not only confirmed that the four factors we investigated all affect soybean protein extraction, but also showed that ultrasound treatment had a greater influence on protein extraction than the total extraction time. 1: 5 min, 350 W, 40%, 40 min; 2: 5 min, 400 W, 45%, 60 min; 3: 5 min, 450 W, 50%, 90 min; 4: 10 min, 350 W, 45%, 90 min; 5: 10 min, 400 W, 50%, 40 min; 6: 10 min, 450 W, 40%, 60 min; 7: 15 min, 350 W, 50%, 60 min; 8: 15 min, 400 W, 40%, 90 min; 9: 15 min, 450 W, 45%, 40 min. Data are expressed as mean±standard deviation ( n =6). Columns labeled with different capital and lowercase letters indicate a significant difference at P < 0.01 and P < 0.05, respectively To confirm the optimal conditions for LAP extraction and AP removal, the proteins in the isopropanol extracts under nine different conditions were identified by SDS-PAGE (Fig. 3 3 ) and MS (Table 2 2 ). There were more protein bands or the staining intensity of protein bands was stronger at lower isopropanol concentrations irrespective of the ultrasonic time, ultrasonic power, and total extraction time (Fig. 3 3 ). The staining intensity of protein bands in lane 7 (condition 7) was stronger than those in lanes 3 and 5, which was in accord with their protein content in isopropanol extracts (Fig. 2 2 ). Band No. Protein accession Protein mass Protein score Protein description [species] 1 gi|62122635 56 406 1280 β-Amylase [ Glycine max ] 2 gi|356555724 46 192 141 Fumarylacetoacetase-like [ Glycine max ] 3 gi|6729836 27 555 231 Chain A, soybean agglutinin complexed with 2,6-pentasaccharide 4 gi|543177317 27 512 110 Iron-superoxide dismutase [ Phaseolus vulgaris ] 5 gi|37495455 23 720 217 Dehydrin [ Glycine max ] 6 gi|9967357 63 184 144 α Subunit of β-conglycinin [ Glycine max ] 7 gi|9967357 63 184 121 α Subunit of β-conglycinin [ Glycine max ] 8 gi|157838208 19 853 516 Chain B, complex porcine pancreatic trypsin soybean trypsin inhibitor, orthorhombic crystal form 9 gi|356515553 17 534 80 Oleosin 16 kDa-like [ Glycine max ] 10 gi|13375351 16 124 74 Truncated kunitz trypsin inhibitor [ Glycine max ] 11 gi|145495350 17 407 143 Hypothetical protein [ Paramecium tetraurelia ] 12 gi|157838208 19 853 179 Chain B, complex porcine pancreatic trypsin soybean trypsin inhibitor, orthorhombic crystal form M, marker, indicates 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10 kD; Lanes 1–9 were the conditions 1–9 as shown in Fig. 2 2 β-Amylase, the major enzyme of starch breakdown in leaves (Fulton et al., 2008 2008 ), soybean trypsin inhibitor (STI) and soybean agglutinin (SBA), accounting for 6% and 5%–7% of soybean proteins, respectively (Rackis et al., 1986 1986 ; Bajpai et al., 2005 2005 ), have been shown to be the important anti-nutritional factors of soybean (Fasina et al., 2006 2006 ; Zang et al., 2006 2006 ; Hart et al., 2010 2010 ). β-Conglycinin is composed of three subunits, α', α, and β, which are potential food allergens (Krishnan et al., 2009a 2009a ). In our study, the proteins in SDS-PAGE gels were excised and identified by MS (Table 2 2 ). β-Amylase, kunitz trypsin inhibitor, and SBA were the top three proteins in the nine isopropanol extracts. The α subunit of β-conglycinin and many other LAPs, such as fumarylacetoacetase-like, dehydrin, oleosin 16 kDa-like, and iron-superoxide dismutase, were also detected in defatted soybean meal under some conditions (Table 2 2 ). The α subunit of β-conglycinin was not detected under conditions 3, 5, or 7. Determination of LAP extraction and AP removal 2 The preferable orthogonal experimental conditions (conditions 3, 5, and 7) were inconsistent with those identified in our previous study (Liu et al., 2016 2016 ) of the effects of ultrasonic treatment on LAP extraction (10 min, 400 W, 50%, 60 min). So, to determine the optimal conditions for LAP extraction and AP removal, a comparative analysis was carried out between the orthogonal experiment (under conditions 3, 5, and 7) and the ultrasonic treatment experiment (under optimal conditions). The results are shown in Figs. 4 4 and 5 5 , and Table 3 3 . The protein concentration of up to 0.8081 g/L under condition 7 was higher than that under the optimal conditions from the ultrasonic treatment experiment (10 min, 400 W, 50%, 60 min) ( P < 0.05), and condition 5 ( P < 0.05) and condition 3 ( P < 0.01) from the orthogonal experiment. This result confirmed the important role of the ultrasonic treatment and total extraction time in LAP extraction (Fig. 4 4 ). Band No. Protein accession Protein mass Protein score Best protein description [species] 1 gi|571503799 92 477 144 Phospholipase D alpha 1-like isoform X2 [ Glycine max ] 2 gi|62122633 56 391 267 β-Amylase [ Glycine max ] 3 gi|62122635 56 406 725 β-Amylase [ Glycine max ] 4 gi|63259123 56 457 353 β-Amylase [ Glycine max ] 5 gi|363806904 29 349 153 Uncharacterized protein LOC100799849 [ Glycine max ] 6 gi|255638626 17 463 131 16.5 kDa oleosin [ Glycine max ] 7 gi|255638626 36 632 147 Unknown [ Glycine max ] 8 gi|351720944 22 718 290 Uncharacterized protein LOC100305855 precursor [ Glycine max ] 9 gi|157838209 19 853 450 Chain B, complex porcine pancreatic soybean trypsin inhibitor, tetragonal crystal form 10 gi|571459448 22 698 285 Oleosin 1-like [ Glycine max ] 11 gi|571459448 22 698 132 Oleosin 1-like [ Glycine max ] 12 gi|356556690 15 180 98 Uncharacterized protein LOC100800510 [ Glycine max ] 13 gi|351726299 17 463 176 16.5 kDa oleosin [ Glycine max ] 1: 10 min, 400 W, 50%, 60 min; 2: 15 min, 350 W, 50%, 60 min (condition 7); 3: 10 min, 400 W, 50%, 40 min (condition 5); 4: 5 min, 450 W, 50%, 90 min (condition 3). Data are expressed as mean±standard deviation ( n =6). Columns labeled with different capital and lowercase letters indicate a significant difference at P < 0.01 and P < 0.05, respectively Cold acetone precipitation of isopropanol extracts was performed by SDS-PAGE and the proteins were identified by MS (Fig. 5 5 , Table 3 3 ). There were no differences in the number of protein bands but some differences in the intensity of their staining among the four conditions, indicating different protein contents but the presence of the same protein varieties. This result was consistent with the protein concentrations of isopropanol extracts (Fig. 4 4 ). Some LAPs were detected in the cold acetone precipitates, such as phospholipase D, β-amylase, oleosin 1-like, uncharacterized and even an unknown protein (Table 3 3 ). M, marker, indicates 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10 kD; Lanes 1–4 were the conditions as shown in Fig. 4 4 Conclusions 1 The results of this study show that: (1) isopropanol contributed to the removal of APs, and ultrasonic treatment and total extraction time played an important role in enriching the LAPs of soybean; (2) the effect of ultrasonic treatment on LAP enrichment was higher than that of total extraction time; (3) the optimal conditions were 50% isopropanol, ultrasonic treatment for 15 min at 350 W for a total extraction time of 1 h. This method may be useful for preparing sufficient soybean LAPs to study their physiological effects on animals without the influence of APs. References MR Adams , MS Anthony , H Chen , 等 . Replacement of dietary soy protein isolate with concentrates of soy 7S or 11S globulin has minimal or no effects on plasma lipoprotein profiles and biomarkers of coronary risk in monkeys . 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1600293 Project supported by the China Agriculture Research System (No. CARS-36) and the National Natural Science Foundation of China (No. 31572430) 2016-06-27 2016-11-28 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 18