Accepted Manuscript

Improvement of nutritional quality of soybean meal by Fe(II)-assisted acetic

acid treatment

Lu Huang, Yong Xu, Yanmin Zhou

PII: S0308-8146(19)30153-0
DOI: https://doi.org/10.1016/j.foodchem.2019.01.085
Reference: FOCH 24180

To appear in: Food Chemistry

Received Date: 17 August 2018

Revised Date: 29 November 2018
Accepted Date: 7 January 2019

Please cite this article as: Huang, L., Xu, Y., Zhou, Y., Improvement of nutritional quality of soybean meal by Fe(II)-
assisted acetic acid treatment, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.01.085

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 Improvement of nutritional quality of soybean meal by Fe(II)-assisted acetic

acid treatment

Lu Huang a, b, c, Yong Xu a, b, c, *, Yanmin Zhou d,

a
Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources,

Nanjing Forestry University, Nanjing 210037, People’s Republic of China

b
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s

Republic of China

c
Jiangsu Province Key Laboratory of Green Biomass-based Fuels and Chemicals, Nanjing

210037, People’s Republic of China

d
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095,

People’s Republic of China

* Corresponding author at: College of Chemical Engineering, Nanjing Forestry University. No.

159 Longpan Road, Nanjing 210037, People’s Republic of China.

E-mail address: xuyong@njfu.edu.cn

Tel: +86 025 85427537

Fax: +86 025 85427537

1
Abstract

We investigated the effect of Fe(II)-assisted acetic acid treatment on improvement of nutrition

quality of soybean meal (SBM) by degrading antinutritional factors (ANFs) and maintaining initial

nutrition quality. Fe(II)-assistance reduced trypsin inhibitor (TI) content significantly from 5.20 to

0.86 mg/g, and allergenic proteins were completely degraded at 55 °C, due to changes in the

conformation of soybean protein isolate (SPI) that renders proteins more prone to acetic

acid-mediated degradation. The red-shift of maximum emission wavelength indicated that

Fe(II)-assisted acid induced molecular unfolding of SPI and increased surface hydrophobicity.

Investigation of protein secondary structure revealed that Fe(II)-assisted acid treatment decreased

the β-sheet structure by 4.65% and increased the α-helical content by 7.37%. This demonstrated that

Fe(II) and acetic acid synergistically degrade ANFs by altering protein conformations in SBM.

Keywords: Soybean meal; antinutritional factors; Fe(II); acetic acid; nutrition quality; soybean

protein isolate; protein conformation

2
1. Introduction

Soybean meal (SBM) is widely used as healthy food and animal feed because of its high

protein content and well-balanced nutrient composition. However, consuming improperly processed

or untreated soybeans can be harmful because of the presence of antinutritional factors (ANFs),

including trypsin inhibitor (TI), allergenic proteins, and flatulence-producing compounds (Hong,

Lee, & Kim, 2004). Allergenic proteins affect immunoreactivity and allergic reactions, and TI can

elicit unfavorable physiological effects, including retardation of growth (Coscueta et al., 2017;

Maria John, Khan, Luthria, Garrett, & Natarajan, 2017). Hence, correct treatments are required to

degrade ANFs and improve the nutritional quality of soy proteins.

Currently, acid treatment is being widely used to change protein conformation and improve the

functional properties of plant-based sources through intermolecular electrostatic repulsion (Zhao,

Xin, Zhao, Chen, & Cai, 2014). Previously, we showed that acetic acid-catalyzed processing

effectively degraded allergens and TI in SBM (Huang & Xu, 2018). However, a significant amount

of soluble proteins was lost (potassium hydroxide protein solubility (PS) decreased from 91.81% to

78.85%), mainly due to the relatively high temperature at 70°C. PS is a significant index to evaluate

the protein quality of SBM (Parsons, Hashimoto, Wedekind, & Baker, 1991). Evans and St. John

(1945) reported that as raw SBM was autoclaved for increasing amounts of time, the proportion of

protein soluble in 0.2% potassium hydroxide decreased. An initial study by Araba et al. (1990)

showed that PS values below 65% suggested the SBM was overprocessed. It was found that the

temperature is the most important factor affecting PS value (Huang et al., 2018). Therefore, in order

to maintain optimal nutritional value, the meal must not be overheated as it will denature proteins,

rendering it less soluble or digestible. Excessive heat reduces the availability of amino acids (Del

Valle, 1981) and destroys certain amino acids (Skrede A, 1985). Therefore, lowering of temperature,
3
combined with acid treatment and other modifications that do not affect the soluble protein content

and nutritional value, while significantly reducing the ANF content, should be used to improve

SBM quality.

Conformation changes play an important role in degrading ANFs and improving nutritional

quality of SBM (Zheng et al., 2017). Reagents that mediate dissociation and aggregation of peptides

and proteins have become increasingly important for several chemical and biological applications.

However, the reaction conditions of most existing chemical agents are harsh and the reagents must

be used in high molar excess (Walker, 2002). Metal ions are used to affect conformation changes of

proteins under mild temperature and pH (Kassai, Ravi, Shealy, & Grant, 2004; Shrivastava & Nair,

2001). Goldshleger et al. (1997) showed that Fe catalyzes transition in protein conformation at

20 °C. Therefore, we hypothesized that a combination of acid treatment and metal ion-mediated

modification could be a good method for effectively degrading soybean ANFs at moderate

temperatures.

The aim of the present study was to develop a moderate temperature-based process for

degrading ANFs and improving nutritional quality of SBM. We used the feed additive FeSO4·7H2O

to investigate the effect of Fe(II) on conformation changes of soybean protein isolate (SPI). The

Fe(II)-assisted degradation was explained with respect to subunit dissociation, and changes in

surface hydrophobicity and secondary structures of SPI.

2. Materials and methods

2.1. Materials and reagents

SBM (crude protein, 43.54%) was obtained from Brazil, and was ground to 60 mesh.

N-α-Benzoyl-L-arginine-4-nitroanilide (L-BAPA) and 1-anilino-8-naphthalenesulfonic acid (ANS)

were obtained from Sigma-aldrich (Shanghai, China). Pre-stained protein markers (bands 1–10: Mr
4
10, 15, 25, 35, 40, 55, 70, 100, 130, and 180 kDa) were purchased from Thermo Fisher Scientific

(China) Co., Ltd. All other chemicals used in the present study were of analytical grade.

2.2. Treatments of SBM with acid and Fe(II)

SBM were treated by acid, Fe(II), and their combination. The experiments were carried out

in 35 ml stainless-steel tube reactors with 25 ml solvent containing 5 g of SBM at a solid:liquid

ratio of 1:5 (w/v). Distilled water was used as the solvent for the control. For acid, Fe(II), and

Fe(II)-assisted acid samples, the solvent was 10% (w/v) acetic acid, 5.4 mM FeSO4·7H2O, and

their combination, respectively (Table 1). The reactors were placed in an oil bath and reacted at

55 °C for 30 min. After reaction, the acetic acid in catalyzed mashes was removed by vacuum

rotary evaporation, and then the mashes were washed with distilled water. The recovery rate of

acetic acid was 80–90% when the above operation was repeated 5–6 times. The samples were

then dried at 40 °C until the moisture content decreased below 12%, and milled to 60 mesh for

further determination.

2.3. Analysis of ANFs

TI content was analyzed using the improved L-BAPA method (Smith, Van Megen,

Twaalfhoven, & Hitchcock, 1980). The TIs were extracted from samples (1 g) with 50 ml 10 mM

NaOH at 4 °C for 15-24 h. After precipitation, the supernatants were diluted and mixed with bovine

trypsin. The activity of the remaining trypsin was then measured by addition of L-BAPA (60 mg

L-BAPA was dissolved in 1 ml dimethyl sulphoxide and diluted to 100 ml with pH 8.2 Tris buffer)

under standard trypsin solution, the p-nitroaniline released was measured spectrophotometrically at

410 nm. This provided a linear measure of the residual trypsin activity, so that the amount of pure

trypsin inhibited per unit weight of sample can be calculated. According to Laemmli et al. (1970),

allergenic protein content was determined by sodium dodecyl sulfate–polyacrylamide gel

5
electrophoresis (SDS-PAGE). Briefly, the samples (0.25 g) were extracted with 5 ml of phosphate

buffer solution (PBS, 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH = 7.3),

and centrifuged at 10,000 rpm and 4°C for 10 min. The supernatants were denatured in 4X

denaturing buffer (500 mM Tris-HCl, 25% glycerol, 10% sodium dodecyl sulfate (SDS), and 0.1%

bromophenol blue) at 100 °C for 10 min and centrifuged at 10,000 rpm at room temperature for 10

min. A 5–10 μL aliquot from the supernatant was subsequently subjected to a discontinuous

Tris-glycine buffer (pH 8.3) system with 3.75% stacking gel and 12.5% separating gel for

concentration and separation.

2.4. Determination of protein solubility

The PS content was measured according to Araba et al. (1990). Briefly, ground powder

samples (1.5 g) were mixed with 75 ml of 0.2% potassium hydroxide solution, placed in a 250 ml

beaker, and stirred for 20 min. The mixture was subsequently centrifuged at 2,700 rpm and 4 °C for

10 min. Fifteen milliliters supernatant were then transferred to Kjeldahl tubes to generate a 0.3 g

aliquot of the original sample. The nitrogen content of the supernatant and the sample were

determined by Kjeldahl method, and the PS was computed as the percentage of the nitrogen

extracted as to the total nitrogen of the sample.

2.5. Analysis of chemical composition

Moisture content was measured at 105 °C by an infrared moisture analyzer. Crude protein

content was assessed by the Kjeldahl method (William, 1980). The lipid in the dried samples was

gauged by Soxhlet extraction using a petroleum ether binary extracting solution (Novakofski, Park,

Bechtel, & Mckelth, 1989), while the ash was determined by combustion of the dried samples at

500 °C for 8 h (Hong et al., 2004). The pH values of samples were measured in ultrapure water via

a pH meter (MM-60R, DKK-TOA Co., Japan).

6
2.6. Preparation of SPI

SPI was produced from SBM according to Huang et al. (2017), which was based on the

difference in protein solubility under different pH conditions. Briefly, SBM powder was dispersed

in deionized water in a solid liquid ratio of 1:15 (w/v), and the pH was adjusted to 8.0 with 2 M

NaOH. The mixture was stirred for 2 h, and centrifuged at 4,000  g for 15 min. The pH of

supernatant was adjusted to 4.5 with 2 M HCl, and then centrifuged at 4,000  g for 15 min. The

precipitate was washed with deionized water and neutralized to pH 7.0 with 2 M NaOH, then

freeze-dried to obtain SPI. The protein contents of SPIs comes from SBM, control, acid, Fe(II), and

Fe(II)-assisted acid samples were 85.07%, 86.57%, 86.28%, 86.79%, and 88.11%, respectively.

2.7. Intrinsic fluorescence emission spectroscopy

The fluorescence spectrum is used to detect the changes of protein conformation. Fluorescence

intensities were obtained on a Perkin-Elmer LS 55 fluorophotometer at room temperature. The SPI

solutions (0.15 mg/ml) were excited at 282 nm and emission collected between 300 nm and 500 nm.

2.8. Determination of surface hydrophobicity (H0)

Determination of H0 was carried out according to Liu et al. (2011), using ANS as a

fluorescence probe. Briefly, 1.5% (w/v) SPI was dissolved in 0.01 M phosphate buffer (pH 7.0),

stirred at room temperature for 1 h, and centrifuged at 10,000  g for 30 min. The protein content

in the supernatant was determined, followed by dilution of the protein to 0.008-0.700 mg/ml with

phosphate buffer. Then, ANS solution (40 μL 8.0 mM) was added to successive samples

containing 4 ml sample solution of different concentrations. Fluorescence intensity (FI) was

determined with a Perkin-Elmer LS 55 fluorophotometer, at wavelengths of 390 nm (excitation)

and 470 nm (emission). The H0 was expressed as the initial slope of the FI versus protein

concentration plot.
7
2.9. FTIR spectra

Fourier transform infrared spectroscopy (FTIR) was determined following the postassium

bromide (KBr) pellet method using a Bruker VERTEX 80V spectrometer (Germany). For each

spectrum, 32 scans were taken from the 4000 to 400 cm−1 region. The analysis of secondary

structure was measured from the infrared second derivative amide I (1700–1600 cm−1) spectra by

using Peakfit v4.12.

2.10. Statistical analysis

All determinations were conducted in triplicate and all values were expressed as mean ±

standard deviation (SD). Analysis of variance was performed using the SPSS 20.0 statistical

analysis system. A probability level (p-value) of less than 0.05 was regarded as a significant

difference between samples.

3. Results and discussion

3.1. Effects of Fe(II) on acetic acid-mediated improvement in nutrition quality and chemical

composition of SBM

With the aim to investigate the effects of Fe(II) on acetic acid-mediated improvement in

nutritional quality of SBM, the experiments were designed in Table 1. The nutrition index and

chemical composition of SBM that were subjected to different treatments are also shown in Table

1. The detailed profiles of the subunit composition changes are shown in Fig. 1. Seed proteins in

soybean comprise two major fractions that account for 70% to 80% of total protein composition:

11S and 7S globulins. The 7S globulin fraction consists of β-conglycinin, which includes three

subunits: α’ subunit (72 kDa), α subunit (68 kDa), and β subunit (52 kDa). The 11S globulin

fraction is composed primarily of glycinin, including A3 protein (40 kDa), other acidic subunits

(37 kDa) and basic subunits (20 kDa) (Teng et al., 2012). All subunits of glycinin are allergens,
8
which can generate an antibody response in mice when fed soy-protein-containing diets

(Christensen, Bruun, & Frøkiær, 2003). As shown in Fig.1, the untreated SBM exhibited almost

all bands of allergens (lane 1). The control (lane 2) showed no significant changes with regard to

the raw SBM sample, indicating that only heat treatment at 55 °C hardly degraded the allergens of

SBM. We observed that only Fe(II) treatment without acetic acid cannot completely degrade

allergens (Fig.1, lane 4). In addition to the β subunit, the other bands were completely degraded

after acid treatment (Fig.1, lane 3). Huang et al. (2017) reported that acid may dissociate certain

large subunits into smaller subunits. We observed that all bands corresponding to allergens

disappeared when SBM was subjected to Fe(II)-assisted acid treatment (Fig. 1, lane 5). Results

showed that Fe(II) improved the acid-induced degradation of allergens. After control treatment, TI

was denatured by heat and the content was decreased from 5.20 mg/g to 3.18 mg/g. Moreover,

acid and Fe(II) treatments further reduced TI, indicating that acid and Fe(II) could decompose

proteins, including TI. Compared to acid-treated SBM, the content of TI in SBM treated with

Fe(II)-assisted acid decreased remarkably from 5.20 mg/g to 0.86 mg/g, suggesting that Fe(II)

facilitated TI dissociation.

PS is an important indicator for assessing protein quality in SBM (Parsons et al., 1991). Araba

et al. (1990) reported that the nutritive quality of SBM with protein solubility values above 70%

may remain unaltered, whereas those with values less than 65% were almost certainly

overprocessed. In addition, initial protein nutrition could be maintained better when PS was higher

(Căpriţă, R., Căpriţă, A., Creţescu, I., & Nicu, 2013). After Fe(II)-assisted acid treatment, PS level

decreased slightly by 7.65%, from 91.62% to 84.61%. The PS content indicated that the

Fe(II)-assisted acid treatment did not negatively affect the initial nutritional value of SBM, whereas

the slight decrease indicated that acid and Fe(II) may have induced unfolding and exposure of
9
hydrophobic groups from the protein interior. The PS content of the Fe(II)-assisted acid-treated

samples was significantly improved compared to that of SBM subjected to acetic acid-catalyzed

processing at 70 °C in our previous study (Huang et al., 2018).

3.2. Fluorescence spectroscopy

The fluorescence spectrum is used to detect the changes of protein conformation, which is

determined by the environmental polarity of tryptophan (Trp)/tyrosine (Tyr) residues and their

specific interactions. The fluorescence emission maxima undergoes a red shift when the exposure

of chromophores to the solvent increases (Pallars, Vendrell, Avilés, & Ventura, 2004). The raw

SPI showed an emission maximum at 334 nm and did not change obviously in control samples;

however, it red-shifted to 347 nm and 343 nm in the presence of acetic acid and Fe(II),

respectively (Fig. 2). These results were indicative of increased exposure of the Trp residues of

proteins to the solvent, which probably occurred due to Fe(II) and acid-mediated unfolding of SPI.

Moreover, the wavelength of the red-shift of Fe(II)-assisted acid-treated sample was remarkably

higher than that of single Fe(II) treatment, which was similar to that obtained with the single acid

treatment. However, the FI was lower than that of raw SPI after control heating treatment at 55 °C,

which can be attributed to the shielding effect of the carbohydrate bound to SPI caused by the

Maillard reaction (Corzo-Martínez, Moreno, Olano, & Villamiel, 2008; Zhang, Yang, Zhang, Hu,

& Zhao, 2017). Moreover, the decrease of FI after Fe(II) and acid treatments suggested that the

intramolecular quenching of Trp was increased, which was due to the destruction of Trp residues.

It is known that the binding of metal ions to Trp residues and proteins can cause the destruction of

Trp (Tabak, Sartor, & Cavatorta, 1989). Binding of Fe(II) and SPIs might also catalyze cleavages

of peptide bonds close to the Fe(II) (Goldshleger & Karlish, 1997). Additionally, the dissociation

of SPI could also lead to the quenching of fluorescence, which was associated with the ionization
10
of carboxylic acid groups from acetic acid (Renard, Lefebvre, Griffin, & Griffin, 1998). Overall,

the changes in fluorescence emission spectra confirmed that Fe(II)-assisted acid treatment

significantly affected the protein structure and induced unfolding of SPI, which promoted the

degradation of ANFs and improvement of nutritional quality of SBM.

3.3. Surface hydrophobicity of SPI

Surface hydrophobicity is one of the most important surface-related properties of proteins

(Wagner, Sorgentini, & Anon, 2000), which can detect changes in the distribution of hydrophobic

groups on the surface, as a result of changes in the molecular structure of SPI. We observed that the

H0 values of all pretreated materials ranged from 71.08 to 210.35. The increase in H0 values

indicated significant loss in the ordered structure of SPI after the treatments, followed by exposure

of hydrophobic groups. Compared to untreated SBM, a slight decrease in H0 was observed in the

control sample. The H0 values increased after acid and Fe(II) treatments, indicative of the exposure

of the previously hidden hydrophobic clusters in the native protein, and showing that acid-induced

exposure was more effective than those of Fe(II) (Fig. 3). Furthermore, the H0 value of samples

processed by Fe(II)-assisted acid increased considerably compared to that of single acid processed

samples. Dissociation of protein subunits is known to expose hydrophobic groups, which increases

surface hydrophobicity (Laligant, Dumay, Casas Valencia, Cuq, & Cheftel, 1991). Therefore, our

results showed that Fe(II) improved the acid-mediated dissociation of SPI.

In this study, the relationship between H0 of all treated materials was compared to each

corresponding PS (Fig.4). Generally, the PS of pretreated materials decreased, albeit unequally,

with increasing H0 values. For example, when the H0 increased from 71.08 to 210.35, the

corresponding PS decreased from 92.79% to 84.61%. Furthermore, a linear correlation (R2 = 0.993)

between H0 and PS was observed. Negative correlation of ANS hydrophobicity to protein solubility
11
(Fig. 4) indicated that the exposed aromatic amino acids may play an important role in protein

solubility. These results indicated that the decrease of PS in SBM processed by Fe(II)-assisted acid

treatment may be attributed to the exposure of surface hydrophobicity, which was consistent with

our previous hypothesis.

3.4. FTIR spectroscopy

FTIR spectroscopy is widely used to investigate the secondary structure of protein, and peak

identification using amide I bands is a powerful technique for quantitative analysis of secondary

structures (Zhao, Xiong, & McNear, 2013). In the present study, a Gaussian curve was used to

analyze the amide I bands, and secondary structure of proteins was revealed by the following bands:

1610–1640 cm-1 and 1670–1680 cm-1 for β-sheet; 1640–1650 cm-1 for random coils; 1650–1660

cm-1 for α-helix; 1660–1670 cm-1 and 1680–1700 cm-1 for β-turn. The results of quantification

analysis are shown in Table 2. The untreated SPI contained 15.33% α-helix, 44.70% β-sheet, 24.28%

β-turn and 15.68% random coils. The results showed that the predominant conformation in SPI is

β-sheet, which confirmed the results of previous studies (Rampon, Robert, Nicolas, & Dufour, 1999;

Yu, Ma, Yuen, & Phillips, 2004). We observed that different treatments gently changed the

secondary structural of SPI. No significant difference was observed in the α-helical structure

content after control and Fe(II) treatment. The content of α-helix increased after acid and

Fe(II)-assisted acid treatments, indicating the formation of acid-induced α-helical structure. The

result is in agreement with that of previous study (Zhao, 2011). In addition, acid and Fe(II)

treatments decreased the content of β-sheet. We noticed that the contents of secondary structure

treated by Fe(II)-assisted acid changed considerably compared to that processed by single acid and

Fe(II), suggesting that Fe(II) and acid act synergistically. Compared to SPI of untreated SBM, the

α-helical content of SBM treated with Fe(II)-assisted acid increased by 7.37%, that of β-sheet
12
decreased by 4.65%, β-turn increased by 1.69%, and random coil increased by 3.44% (P < 0.05).

This suggested that Fe (II)-assisted acid treatment induced a self-reassembly from β-sheet to α-helix

and β-turn structure, as the β-sheet structure was always found inside a folded molecule. Reports

show that the loss of β-sheet structure exposes hydrophobic sites of the protein that may increase

surface hydrophobicity (Jiang et al., 2014), which was consistent with our results. In addition,

decrease in β-sheet and increase in α-helical content lead to dissociation of SPI (Huang et al., 2017),

which might explain the degradation of allergenic proteins and TI.

4. Conclusions

In this study, we investigated the effects of Fe(II)-assisted acid treatment on nutritional quality

of SBM. After processing by Fe(II)-assisted acid, the levels of ANFs such as allergenic protein and

TI were significantly reduced, and assessment of protein solubility showed that the initial nutritional

quality of SBM was maintained. Fluorescence spectra analysis of SPI after acid and Fe(II) treatment

indicated unfolding of the molecular structure and exposure of surface hydrophobicity. SBM treated

with Fe(II)-assisted acid had higher α-helical and lower β-sheet content than SBM obtained using

other methods. These results indicated the Fe(II) facilitated the acid-induced dissociation of SPI,

which provided clues regarding ANF degradation. In conclusion, Fe(II)-assisted acid treatment is an

effective method for improving the nutritional quality of SBM via changes in SPI conformation.

This study contributes to clarifying the mechanisms of the effect of treatment on SPI structure,

further moving towards implementing Fe(II) and acid in the processing chain of SBM.

Acknowledgements

This work was supported by the National Key Research and Development Program of China

(2017YFD0601001).

Conflict of interest
13
There is no conflict of interest.

14
References

Araba, M., Dale, N. M., & Division, P. S. (1990). Evaluation of protein solubility as an indicator of

overprocessing soybean meal. Poultry Science, 69(1), 76–83.

Căpriţă, R., Căpriţă, A., Creţescu, I., & Nicu, V. (2013). Refractometric method for evaluation of

soybean protein solubility. Lucrari Stiintifice Zootehnie Si Biotehnologii, 42(1).

Christensen, H. R., Bruun, S. W., & Frøkiær, H. (2003). Antigenic specificity of serum antibodies

in mice fed soy protein. International Archives of Allergy and Immunology, 132(1), 58–67.

Corzo-Martínez, M., Moreno, F. J., Olano, A., & Villamiel, M. (2008). Structural characterization

of bovine β-lactoglobulin-galactose / tagatose maillard complexes by electrophoretic,

chromatographic, and spectroscopic methods. Journal of Agricultural and Food Chemistry,

56(11), 4244–4252.

Coscueta, E. R., Pintado, M. E., Picó, G. A., Knobel, G., Boschetti, C. E., Malpiedi, L. P., & Nerli,

B. B. (2017). Continuous method to determine the trypsin inhibitor activity in soybean flour.

Food Chemistry, 214, 156–161.

Del Valle, F. R. (1981). Nutritional qualities of soya protein as affected by processing. Journal of

the American Oil Chemists’ Society, 58(3), 419–429.

Evans, R. J., & St John, J. L. (1945). Estimation of the relative nutritive value of vegetable proteins

by two chemical methods. Journal of Nutrition, 3.

Goldshleger, R., & Karlish, S. J. (1997). Fe-catalyzed cleavage of the alpha subunit of

Na/K-ATPase: evidence for conformation-sensitive interactions between cytoplasmic domains.

Proceedings of the National Academy of Sciences of the United States of America, 94(18),

9596–9601.

Hong, K. J., Lee, C. H., & Kim, S. W. (2004). Aspergillus oryzae GB-107 fermentation improves
15
 nutritional quality of food soybeans and feed soybean meals. Journal of Medicinal Food, 7(4),

430–435.

Huang L., & Xu. Y. (2018). Effective reduction of antinutritional factors in soybean meal by acetic

acid-catalyzed processing. Journal of Food Processing and Preservation, 1–8.

https://doi.org/10.1016/j.tifs.2016.01.010

Huang, L., Ding, X., Dai, C., & Ma, H. (2017). Changes in the structure and dissociation of soybean

protein isolate induced by ultrasound-assisted acid pretreatment. Food Chemistry, 232,

727–732.

Jiang, L., Wang, Z., Li, Y., Meng, X., Sui, X., Qi, B., & Zhou, L. (2014). Relationship between

secondary structure and surface hydrophobicity of soybean protein isolate subjected to heat

treatment. Journal of Chemistry, 5, 1–10.

Kassai, M., Ravi, R. G., Shealy, S. J., & Grant, K. B. (2004). Unprecedented acceleration of

zirconium(IV)-assisted peptide hydrolysis at neutral pH. Inorganic Chemistry, 43(20),

6130–6132.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature, 227(5259), 680–685.

Laligant, A., Dumay, E., Casas Valencia, C., Cuq, J. L., & Cheftel, J. C. (1991). Surface

hydrophobicity and aggregation of. beta.-lactoglobulin heated near neutral pH. Journal of

Agricultural and Food Chemistry, 39(12), 2147–2155.

Liu, Y., Zhao, G., Ren, J., Zhao, M., & Yang, B. (2011). Effect of denaturation during extraction on

the conformational and functional properties of peanut protein isolate. Innovative Food Science

and Emerging Technologies, 12(3), 375–380.

Maria John, K. M., Khan, F., Luthria, D. L., Garrett, W., & Natarajan, S. (2017). Proteomic analysis
16
 of anti-nutritional factors (ANF’s) in soybean seeds as affected by environmental and genetic

factors. Food Chemistry, 218, 321–329.

Novakofski, J., Park, S., Bechtel, P. J., & Mckelth, F. K. (1989). Composition of cooked pork chops:

effect of removing subcutaneous fat before cooking. Journal of Food Science, 54(1), 15–17.

Pallars, I., Vendrell, J., Avilés, F. X., & Ventura, S. (2004). Amyloid fibril formation by a partially

structured intermediate state of α-chymotrypsin. Journal of Molecular Biology, 342(1),

321–331.

Parsons, C. M., Hashimoto, K., Wedekind, K. J., & Baker, D. H. (1991). Soybean protein solubility

in potassium hydroxide: an in vitro test of in vivo protein quality. Journal of Animal Science,

69(7), 2918–2924.

Rampon, V., Robert, P., Nicolas, N., & Dufour, E. (1999). Protein structure and network orientation

in edible films prepared by spinning process. Journal of Food Science, 64(2), 313–316.

Renard, D., Lefebvre, J., Griffin, M. C., & Griffin, W. G. (1998). Effects of pH and salt

environment on the association of β-lactoglobulin revealed by intrinsic fluorescence studies.

International Journal of Biological Macromolecules, 147(2-3), 41–49.

Shrivastava, H. Y., & Nair, B. U. (2001). Chromium(III)-mediated structural modification of

glycoprotein: Impact of the ligand and the oxidants. Biochemical and Biophysical Research

Communications, 285(4), 915–920.

Skrede A, K. A. (1985). Heat affects nutritional characteristics of soybean meal and excretion of

proteinases in mink and chicks. Nutrition Reports International, 32(2), 479–489.

Smith, C., Van Megen, W., Twaalfhoven, L., & Hitchcock, C. (1980). The determination of trypsin

inhibitor levels in foodstuffs. Journal of the Science of Food and Agriculture, 31(4), 341–350.

Tabak, M., Sartor, G., & Cavatorta, P. (1989). On the interactions of metal ions with tryptophan and
17
 glyciltryptophan: A fluorescence study. Journal of Luminescence, 43(6), 355–361.

Teng, D., Gao, M., Yang, Y., Liu, B., Tian, Z., & Wang, J. (2012). Bio-modification of soybean

meal with Bacillus subtilis or Aspergillus oryzae. Biocatalysis and Agricultural Biotechnology,

1(1), 32–38.

Wagner, J. R., Sorgentini, D. A., & Anon, M. C. (2000). Relation between solubility and surface

hydrophobicity as an indicator of modifications during preparation processes of commercial

and laboratory-prepared soy protein isolates. Journal of Agricultural and Food Chemistry,

48(8), 3159–3165.

Walker M., J. (2002). The protein protocols handbook. Humana Press: New Jersey.

William, H. (1980). Official methods of analysis of the association of official analytical chemists

(13th ed.). Arlington, TX: Association of Official Analytical Chemists Inc.

Yu, Z., Ma, C. Y., Yuen, S. N., & Phillips, D. L. (2004). Raman spectroscopic determination of

extent of O-esterification in acetylated soy protein isolates. Food Chemistry, 87(3), 477–481.

Zhang, Y., Yang, R., Zhang, W., Hu, Z., & Zhao, W. (2017). Structural characterization and

physicochemical properties of protein extracted from soybean meal assisted by steam

flash-explosion with dilute acid soaking. Food Chemistry, 219, 48–53.

Zhao, J., Xiong, Y. L., & McNear, D. H. (2013). Changes in structural characteristics of

antioxidative soy protein hydrolysates resulting from scavenging of hydroxyl radicals. Journal

of Food Science, 78(2), 152–159.

Zhao, M. M. (2011). Subunit dissociation of soybean protein isolates in acid conditions. Journal of

South China University of Technology, 39(9), 22–27.

Zhao, M. M., Xin, P. X., Zhao, Q. Z., Chen, N. N., & Cai, M. S. (2014). Structural variations in the

subunits of peanut protein isolates under acidic conditions. Modern Food Science and
18
 Technology, 30(12), 37–42.

Zheng, L., Li, D., Li, Z. L., Kang, L. N., Jiang, Y. Y., Liu, X. Y., & Wang, J. H. (2017). Effects of

Bacillus fermentation on the protein microstructure and anti-nutritional factors of soybean

meal. Letters in Applied Microbiology, 65(6), 520–526.

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Figure captions

Fig. 1 SDS-PAGE of peptides in SBM subjected to different treatments. M: marker, Lanes 1–5

were untreated SBM, control, acid-, Fe(II)-, and Fe(II)-assisted acid-treated SBM, respectively.

Marker (from top to bottom): 180, 130, 100, 70, 55, 40, 35, 25, 15, and 10 kDa.

Fig. 2 Effects of Fe(II)-assisted acid treatment on the fluorescence spectra of SPI.

Fig. 3. Surface hydrophobicities of SPI that were subjected to different treatments. Different

letters represent significant differences between treatments (P < 0.05).

Fig. 4. Relationship between PS and surface hydrophobicity.

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Table 1 Nutritional index and chemical composition of SBM subjected to different treatments.

Treatments SBM Control Acid Fe(II) Fe(II)-assisted acid

Untreated Distilled water 10% acetic acid 5.4 mM FeSO4 10% acetic acid + 5.4 mM FeSO4

Nutritional quality

TI (mg/g) 5.20 ± 0.18a 3.18 ± 0.07b 2.16 ± 0.05d 2.49 ± 0.06c 0.86 ±0.05e

PS (%) 91.62 ± 0.19a 91.98 ± 0.64a 88.24 ±0.60c 89.67 ± 0.14b 84.61 ± 0.31d

Chemical composition

Moisture (%) 12.90 ± 0.02a 10.34 ± 0.04b,c 10.04 ± 0.01c 10.18 ± 0.02c 10.43 ± 0.01b

Crude protein (%) 44.72 ± 0.74a 44.88 ± 0.35a 44.41 ± 0.29a 45.28 ± 0.40a 45.68 ± 0.58a

Lipid (%) 1.35 ± 0.05a 1.17 ± 0.05b 1.23 ± 0.03a,b 1.20 ± 0.03a 1.09 ± 0.09b

Ash (%) 6.75 ± 0.05b 6.69 ± 0.07b 6.26 ± 0.15c 7.38 ± 0.08a 7.19 ± 0.21a

pH 6.61 ± 0.01a 5.83 ± 0.04c 5.44 ± 0.02d 6.01 ± 0.01b 5.35 ± 0.02e

Means in a row with different letters were significantly different (P < 0.05).

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Table 2 Secondary structure content of SPI subjected to different treatments.

Method α-Helix (%) β-Sheet (%) β-Turn (%) Random coils (%)

SBM 15.33 ± 0.04c,d 44.70 ± 0.08b 24.28 ± 0.05d 15.68 ± 0.05b

Control 15.22 ± 0.09d 44.95 ± 0.17a 24.17 ± 0.04e 15.66 ± 0.10b

Acid 15.68 ± 0.14b 44.21 ± 0.04c 24.50 ± 0.04c 15.61 ± 0.04b

Fe(II) 15.47 ± 0.10b,c 44.11 ± 0.05c 24.80 ± 0.03a 15.57 ± 0.06b

Fe(II)-assisted acid 16.46 ± 0.05a 42.62 ± 0.04d 24.69 ± 0.03b 16.22 ± 0.04a

Means in a column with different letter were significantly different (p < 0.05).

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Fig. 1:

23
Fig. 2:

24
Fig. 3:

25
Fig. 4:

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Highlights:

 Fe(II) and acid can synergistically degrade antinutritional factors in soybean meal

 Initial nutrition was maintained after Fe(II)-acid treatment under mild temperature

 Fe(II)-acid treatment promoted the molecular unfolding of soybean protein isolate

 Fe(II) improved the acid-mediated dissociation of soybean protein

 Fe(II)-acid treatment fostered the quality improvement of soybean meal

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