Separation and Identification of Glutamine Peptides from Defatted Soybean Meal by Enzymatic Hydrolysis

Grain & Oil Science and Technology 2018, 1(1): 28-31

DOI: 10.3724/SP.J.1447.GOST.2018.18006

Separation and Identification of Glutamine Peptides from Defatted Soybean Meal by Enzymatic Hydrolysis WANG Chen, XIE Yanli* , MA Chengye, XIE Dongdong College of Food Science and Technology, Henan University of Technology, Zhengzhou 450001, China Abstract: Glutamine peptides were obtained from defatted soybean meal by enzymatic hydrolysis using a combination of Protamex® and trypsinase. The results showed that the extent of hydrolysis and the concentration of Gln peptides in the hydrolysate were 22.02% and 6.05 mmol/L, respectively. The hydrolysates were fractionated by size-exclusion chromatography on a Sephadex G-15 into five major fractions (GelF1–GelF5). The peptide (GelF1) fraction with the highest glutamine peptide content (51.8%) was further evaluated to determine its molecular weight distribution. Most (92.37%) peptides were less than 1 000 Da. Glutamic acid and glutamine were the most abundant amino acids, accounting for up to 12.98% of the total amino acid content. In addition, the total amino acid content in GelF1 was higher than that in GelF2 and GelF3. Keywords: Defatted soybean meal hydrolysates; Glutamine peptides; Chromatographic separation; Amino acid analysis

1 Introduction Soybean is one of the most important oilseed crops cultivated worldwide. Defatted meal, the main by-product after oil extraction, is an interesting raw material for the production of protein hydrolysates, which possess beneficial biological activities after they are released during gastrointestinal digestion[1-3] . The transport mechanism of peptides in the intestinal mucous membrane differs from that of free amino acids, suggesting that the peptides may offer physiological advantages of efficiency over amino acids[4] . Protein hydrolysates are also used as raw materials in enteral products for hospitals, hypoallergenic infant formulas, dietetic foods, and sport drinks[5] . Glutamine plays a critical role in maintaining numerous physiological functions, including protein synthesis, gastrointestinal tract function maintenance, immune cell growth/maturation and function, and glutathione synthesis in the liver[6] , so glutamine deficiency may compromise immunosuppressive and disease recovery processes that affect patient survival[7-11] . As free glutamine is rather unstable and easily broken down into pyroglutamic acid and ammonia during autoclave sterilization, peptide-bound glutamine extracted from plant protein by enzymatic hydrolysis has the advantage of good safety and has been used to overcome this problem in enteral nutrition[12] . Plant protein hydrolysates mostly serve as protein ingredients or supplements in food and beverages[13] or as ingredients in special formulation for clinical nutrition[14] . Defatted soybean meal is rich in glutamic acid/glutamine (Glx), accounting for 20.20% of the meal content[15] . The average size distribution and amino acid compositions of the Received: 8 October 2017 /Accepted: 13 November 2017. Supported by National Key R&D Program of China (2016YFD0400203). *Corresponding author. E-mail: [email protected] ©Henan University of Technology 2018 WANG C, XIE Y L, MA C Y, et al. Separation and Identification of Glutamine Peptides from Defatted Soybean Meal by Enzymatic Hydrolysis[J]. Grain & Oil Science and Technology, 2018, 1(1): 28-31.

peptides are very important for their biological activities[5] . The objective of this study was to separate and characterize the hydrolysates obtained from defatted soybean meal using a combination of Protamex® and trypsinase and determine the molecular weight distribution and amino acid composition.

2 Materials and Methods 2.1 Materials and Reagents Defatted soybean meal (crude protein 43.5%, the crude fat 0.34%, ash 5.94%, and moisture 8.68%) was supplied by Xuchang Bangdi Protein Industry Co., Ltd. (Henan, China). Protamex® (40 000 U/g) and trypsinase (48 000 U/g) commercial enzymes were kindly provided by Novozyme (Beijing, China). L-Gly-Gln standards were purchased from Sigma Chemical Co. (St. Louis, MO, USA). [Bis(trifluoroacetoxy)iodo] benzene was purchased from Shanghai Yurlic Chemical S&T Co., Ltd. (Shanghai, China). Isothiocyanic acid phenyl ester was purchased from Acros Organics (Pittsburgh, PA, USA). Acetonitrile (chromatographically) was purchased from Tianjing Kemiou Chemreagen Co., Ltd. (Tianjing, China). All other reagents used were of analytical grade.

2.2 Preparation of Defatted Soybean Meal Hydrolysates Defatted soybean meal (50 g) was first passed through a 60 mesh sieve, resuspended in 500 mL distilled water, and heated at 80 ◦ C for 5 min to inactivate endogenous enzymes. Enzymatic hydrolysis was conducted under optimal conditions previously established for the combination of Protamex® and trypsinase, i.e., pH 7.5, 50 ◦ C, 10 h, E/S 1.90% (w of enzyme/w of substrate for Protamex® ), and E/S 4.5% (w of enzyme/w of substrate for trypsinase). The two enzymes were added at the same time. The pH of the mixture was maintained during hydrolysis using 0.5 mol/L NaOH and 0.5 mol/L HCl. Enzymatic hydrolysis was stopped by heating at 80 ◦ C for 20 min to inactivate the enzyme. Hydrolysates were clarified by filtration through 0.45 µm filters (Millipore, Bedford, MA, USA) to remove insoluble material.

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WANG C, et al. Separation and Identification of Glutamine Peptides from Defatted Soybean Meal by Enzymatic Hydrolysis

Supernatant solutions were then freeze-dried and stored at −20 ◦ C until further analysis[16] .

2.3 Determination of the Degree of Hydrolysis The degree of hydrolysis (DH) was calculated as the percentage of cleaved peptide bonds (h) of the total number of such bonds in the substrate (htot ) and calculated from the amount of base (NaOH) added to keep the pH constant during hydrolysis as described by Adler-Nissen[13] according to the following equation: DH =

h × 100% B × Nb × 100% = htot α × Mp × htot

where B is base consumption in mL; N b is normality of the base (NaOH); α is the average degree of dissociation of the α-NH2 groups; M p is mass of protein (N × 6.25) in g; h is the hydrolysis equivalents in meqv/g protein; and htot is total number of peptide bonds in the protein substrate (7.75 meqv/g soy protein).

2.4 Determination of Glutamine in Defatted Soybean Meal Hydrolysates One hundred microliters of defatted soybean meal hydrolysates (2.5 mg/mL) was mixed in an Eppendorf reaction tube containing 100 µL of a freshly prepared solution of excess BTI (10 mg/mL) in acetonitrile and with 25 µL of an aqueous pyridine solution (50 mmol/L). The mixture was reacted for 4 h at 50 ◦ C in a thermostat. Subsequently, the samples were dried by vacuum (room temperature)[17] . These samples were hydrolyzed by 200 µL of hydrochloric acid (6 mol/L) at 110 ◦ C for 23 h and freeze-dried. The hydrolysis procedure was performed under vacuum. HPLC analysis was performed using a Waters system (Waters Corporation, Milford, MA, USA) with a UV detector and Waters symmetry column C18 oven set to 40 ◦ C, flow rate 0.3 mL/min, and sample volume of 5 µL. The freeze-dried acid digested samples was loaded on a 2.1 mm × 50 mm C18 column (Waters) and eluted using an aqueous sodium acetate-acetonitrile gradient and detected at 254 nm[18] .

2.5 Fractionation of Hydrolysates by Sephadex G-15 Gel Filtration Gel filtration chromatography was applied to isolate the protein hydrolysates according to their molecular weights[19] . The freeze-dried hydrolysates (1 g) were suspended in 20 mL of distilled water and then loaded onto a Sephadex G-15 gel filtration column (1.6 cm × 100 cm), pre-equilibrated, and eluted with distilled water. Fractions (4 mL each) were collected at a flow rate of 0.3 mL/min. Elution curves were obtained by measuring absorbance at 280 nm using an online spectrophotometer. Fractions were collected and lyophilized[20] .

2.6 Molecular Weight Distribution of Defatted Soybean Meal Hydrolysates The molecular weight distribution of peptides in different hydrolysates was determined by gel permeation chromatography (TSKgel 2000 SWXL 300 mm × 7.8 mm) using an HPLC system (Waters 600). The samples were eluted using 0.1% trifluoroacetic

29

acid (TFA) in water (A) and 100% acetonitrile containing 0.1% TFA (B) at a flow rate of 0.5 mL/min at 30 ◦ C. The hydrolysates were pre-filtered using a 0.45 µm membrane and 20 µL of sample was injected into the HPLC system. Absorbance was recorded at 220 nm. The column was calibrated with standards: Cytochrome C (12 500 Da), aprotinin (6 500 Da), bacitracin (1 450 Da), Gly-Gly-Tyr-Arg (451 Da), and Gly-Gly-Gly (189 Da). A calibration curve was prepared from the logMw of the markers and their respective elution times. The total area of the chromatogram was integrated and separated into fractions of six molecular weight (Mw) ranges (0–132 Da, 132–500 Da, 500–1 000 Da, 1 000–2 000 Da, 2 000–5 000 Da, and >5 000 Da), expressed as the percentage of the total area[21] .

2.7 Amino Acid Analysis The amino acid profile of the hydrolysates was measured as described by Wang et al.[22] . The hydrolysates of soybean meal isolated from Sephadex G-15 gel filtration and hydrolyzed in 6 mol/L HCl at 110 ◦ C under a nitrogen atmosphere for 24 h. Amino acid analysis of the acidolysis samples was performed with a Hitachi 835-50G automatic amino acid analyzer (Hitachi Ltd., Tokyo, Japan). The hydrolysates were evaporated and then the dried residue was dissolved in 0.02 mol/L HCl. The sample was filtered through a 0.45 µm nylon filter before being injected into the amino acid analyzer.

2.8 Statistical Analysis The results of DH and Gln peptides content reflected the mean of four replicates in each case. Statistical analyses were performed by Design Expert 7.0 statistical software (Stat-Ease Inc., Minneapolis, MN, USA). The statistical significance was determined by analysis of variance. P values of less than 0.05 was considered significant.

3 Results and Discussion 3.1 Isolation Glutamine Peptides Fractions from Defatted Soybean Meal Hydrolysates The lyophilized hydrolysates, obtained with a DH of 22.02%, were fractionated by Sephadex G-15 gel filtration chromatography. Gel filtration separates substances according to molecular size. This method has also been applied for group separation of protein hydrolysates and biological extracts[23] . Hydrolytic process of proteins by protease can produce molecules ranging from individual amino acids to peptides of various sizes, and peptide length is closely related to biological activities[24-25] . As shown in Fig. 1, there were five major absorbance peaks (GelF1, GelF2, GelF3, GelF4 and GelF5) at 280 nm. Each fraction was collected, lyophilized and evaluated for the content of glutamine peptides by HPLC. The percent of glutamine peptides in the five fractions was 51.8%, 14.7%, 27.4%, 2.6% and 3.5%, respectively. GelF1 exhibited the highest glutamine peptide content, and the order of glutamine peptide content for the five fractions was GelF1 > GelF3 > GelF2 > GelF5 > GelF4. The quantity of glutamine peptides in the GelF4 and GelF5 fractions was very low.

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Grain & Oil Science and Technology 2018, 1(1): 28-31

Grain & Oil Science and Technology 2018, 1(1): 28-31

2.5

3

mealusing usingaaSephadex SephadexG-15 G-15 column shown in Table 1. The meal column areare shown in Table 1. The three components were rich in glutamic acid, glutamine, aspartic three components were rich in glutamic acid, glutamine, aspartic acid,lysine lysineand andproline. proline.The The above amino acids accounted acid, above fivefive amino acids accounted for 27.61%, 27.61%,7.72% 7.72%and and14.65% 14.65% total amino acids for of of thethe total amino acids in in GelF1, GelF2 GelF2and andGelF3, GelF3,respectively. respectively.Among Among these amino GelF1, these amino acids, glutamic glutamicacid acidand andglutamine glutamine were most abundant, acids, were thethe most abundant, accountingfor forupuptoto12.98% 12.98% total amino content. accounting ofof thethe total amino acidacid content. In In addition,the thepercent percentofoftotal total amino acids in GelF1 higher addition, amino acids in GelF1 waswas higher than lower contents of of than that thatininGelF2 GelF2and andGelF3. GelF3.However, However, lower contents sulfur-containing (Met andand Cys) were noted in all sulfur-containingamino aminoacid acid (Met Cys) were noted in all components. components.

Absorbance

2.0 1.5 1

1.0

2

0.5

5

4

0.0

Table of of GelF1, GelF2 andand GelF3 Table11Amino Aminoacid acidcomposition composition GelF1, GelF2 GelF3 0

20

40

80

60

100

120

Fraction number

Fig. 1 Elution profile of defatted soybean meal protein 259

The numbers indicate five different fractions. 500

0.20 0.18 0.16

hydrolysates separated by size-exclusion chromatography on Sephadex G-15 at 280 nm

AU

3.20.14 Molecular Weight Distribution of Defatted Soybean Meal Hydrolysates 0.12

87

997

4 998 4 405

19.850

0.08 After gel filtration, GelF1, which showed the highest 2.5 0.06 3 content of glutamine peptides among the fractions, was further 0.04

Absorbance

evaluated to determine its molecular weight. The profile of 0.02 2.0 molecular size of peptides from hydrolysates obtained using 0.00 the TSKgel SWXL column is presented in 22.00 Fig.24.00 2. 26.00 The 4.00 6.00 2.00 2000 16.00 18.00 20.00 12.00 14.00 8.00 10.00 1.5 proportion of groups with molecular weight <1 000 Da was Min 92.37%. In contrast, the group of peptides with molecular weight 1.0 Da was1 lower. Molecular weights <1 000 Da were more >1 000 common, and the distribution was as follows: 22.97% percent 2 0.5 5 56.31% percent of peptides ranging from 500 to 14000 Da, of peptides ranging from 132 to 500 Da, and 5.9% percent of peptides less than 132 Da. The above results demonstrate 0.0 that extensive degradation occurred using the combination of 0 ® 40 80 100 60 in the formation 120 Protamex and20 trypsinase, resulting small-sized Fraction number peptides with major peaks corresponding to 500–1 000 Da, 132–500 Da and <132 Da. 259

0.20

500

500

0.200.16 0.180.14 0.16

259

0.18

0.12

AU

GelF2

GelF2

GelF3

GelF3

Asp 6.38 1.81 3.38 Asp 6.38 1.81 3.38 Thr 2.16 0.61 1.12 Thr 2.16 0.61 1.12 Ser 2.32 0.58 1.27 Ser 2.32 0.58 1.27 Glu+Gln 12.98 3.68 6.87 Glu+Gln 12.98 0.67 3.68 2.686.87 Gly 2.41 Gly 2.41 0.67 1.212.68 Ala 2.35 0.65 Ala Grain & Oil0.64 2.35 0.65 1.211(1): 28-31 Cys 0.33 Science and Technology0.23 2018, Cys 0.64 0.33 1.310.23 Val 2.52 0.64 Val 2.52 0.64 0.291.31 Met 0.56 0.16 Met G-15 2.04 0.56 are 0.16 in 0.29 1. The Ile 0.53 1.15 meal using a Sephadex column shown Table Leu 0.71 1.341.15 aspartic Ile 2.04 0.53glutamine, three components were rich in2.50 glutamic acid, 0.161.34 Leu 2.50 five0.36 0.71 acids acid, lysine andTyr proline. The0.70 above amino accounted Phe 1.16 0.67 0.310.16 Tyr 0.70 0.36 for 27.61%, 7.72% and 14.65% of the total amino acids in Lys 4.30 1.12 2.290.31 PheGelF3, respectively. 1.16 0.67 GelF1, GelF2 and Among these amino His 1.54 0.43 Lys 4.30 1.12 0.832.29 acids, glutamicArg acid and glutamine were the most abundant, 2.34 0.61 His 1.54 0.43 1.260.83 accounting for up to 12.98% 3.95 of the total1.11 amino acid content. In Pro Arg 2.34 0.61 2.111.26 addition, the percent amino acids Total of total50.85 14.67in GelF1 27.81was higher

Pro

3.95

1.11

2.11

than that in GelF2 However, lower 27.81 contents of Totaland GelF3. 50.85 14.67 sulfur-containing amino acid (Met and Cys) were noted in all 4 Conclusions components. 4 Conclusions The DH and Gln peptides obtained from the combination Table 1 Amino acidtrypsinase composition of GelF1, and mmol/L. GelF3 were 22.02%GelF2 and 6.05 of Protamex® and The DH and Gln peptides obtained from the combination Concentration in each fraction Using gel permeation chromatography on Sephadex G-15, five ® and Amino acidtrypsinase were 22.02% and 6.05 mmol/L. of Protamex fractions were purified. Among them, the highestGelF3 content of Gln GelF1 GelF2 Using gel peptides permeation chromatography on Sephadex G-15, five glutamine was in the GelF1 fraction. The molecular 6.38 them,1.81 3.38 content of Gln fractions wereAsp purified. Among the highest weight distribution was investigated and a high quantity (92.37%) Thr 2.16 0.61 1.12 glutamine peptides was in2.32 the fraction. The molecular of oligopeptides DaGelF1 was0.58 observed. Serbelow 1 000 1.27Amino acid weight distribution wasGlx investigated and a high6.87 quantity (92.37%) analysis showed that was mainly in the GelF1 Glu+Gln 12.98present 3.68 of oligopeptides Gly below 1 000 2.41 Da was 0.67observed. 2.68 Amino acid fraction.

6.00

8.00

87

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 Minutes

Fig. 2 Molecular 22.00 14.00 distribution 6.00 18.00 10.00 weight

of glutamine peptides in GelF1 Min

26.00

The results of amino acid analysis of GelF1, GelF2 and GelF3 obtained from the chromatography of defatted soybean

259

0.56 0.16 0.29 Ile 2.04 0.53 1.15 The authors conflict 0.71 of interest. Leu declare no2.50 1.34 [1] MARSMANA D J V, et Tyr G J P, GRUPPEN 0.70 H, VAN 0.36 ZUILICHEM 0.16 al. The influence of screw configuration on the in vitro digestibility References Phe 1.16 0.67 0.31 and protein solubility of soybean and rapeseed meals[J]. Journal Lys G J P,1995, 4.30 2.29 Food Engineering, 26(1): 13-28. [1] ofMARSMANA GRUPPEN H,1.12 VAN ZUILICHEM D J V, et His S J,ofHETTIARACHCHY 0.43 on 0.83 al. The influence screw1.54 configuration theCHEN in vitroP digestibility [2] RAYAPROLU N S, Y, et al. Argsolubility 0.61rapeseed 1.26meals[J]. and protein of2.34 soybean Journal Peptides derived from high oleic acid and soybean meals inhibit colon, liver and lung growth[J]. Research of Food Engineering, 13-28. Procancer cell1995, 3.95 26(1):Food 1.11 2.11International, 50(1): 282-288. Total 50.85 14.67 N27.81 [2] 2013, RAYAPROLU S J, HETTIARACHCHY S, CHEN P Y, et al. [3] HUANG R, DING N, oleic DAI acid C H,soybean et al. Changes in the Peptides L derived from X high meals inhibit colon, structure dissociation soybean protein isolate induced by liver andand lung cancer cellofgrowth[J]. Food Research International,

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87

997

Conflict of Met Interest

2 Molecular weight distribution 3.3 AminoFig. Acid Composition Analysis of glutamine peptides in GelF1 The results of amino acid analysis of GelF1, GelF2 and obtained from the chromatography 3.3GelF3 Amino Acid Composition Analysis of defatted soybean

0.20

GelF1

Valdeclare no conflict 2.52 The authors of 0.64 interest. 1.31

997

2.00

4.00

4 998 4 998 4 4054 405

2.00

19.85019 850

AU

0.00

0.18

GelF1

Ala that Glx2.35 0.65 mainly 1.21 in the GelF1 analysis showed was present 0.64 0.33 0.23 fraction.

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%

WANG C, et al. Separation and Identification of Glutamine Peptides from Defatted Soybean Meal by Enzymatic Hydrolysis

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