Trypsin Inhibitor from Soybean Whey Wastewater: Isolation, Purification and Stability

Abstract

Soybean trypsin inhibitor (STI) was obtained from simulated soybean whey wastewater through a sustainable method consisting of isoelectric precipitation, ammonium sulfate salting out, and gel filtration chromatography, and the effect of temperature, pH, and pepsin on the stability of STI was also discussed. The results showed that the recovery rate of the trypsin inhibitory activity was 89.47%, the purity and the specific activity of STI were 71.11%, and 1442.5 TIU/mg in the conditions of pH 4.0 and 40% ammonium sulfate saturation. The soybean Kunitz trypsin inhibitor (KTI) and soybean Bowman–Brik trypsin inhibitor (BBI) were obtained via gel filtration chromatography, and their specific activity levels were 1733.5 TIU/mg and 2588.3 TIU/mg, respectively. The STI displayed good stability over a wide temperature and pH range. The STI, KTI, and BBI were all resistant to pepsin hydrolysis, and their ability was ranked as BBI > STI > KTI. These findings will provide a theoretical basis for recycling STI from soybean whey wastewater and promoting better active compound utilization.

1. Introduction

As an important food ingredient, soybean protein exhibits significant potential for extensive use in food formulations due to its excellent nutritional, functional properties, and health benefits. Soybean protein isolate, a vital soybean protein product, is usually produced via alkali extraction-acid precipitation [1]. However, many soybean whey by-products are also generated during the production process. Typically, 20 tons of soybean whey liquid are generated per ton of soybean protein isolate produced [2]. Millions of tons of soybean whey are produced annually in China [3]. Soybean whey contains substantial nutrients consisting of simple sugars, oligosaccharides, and soybean whey protein. Soybean whey protein, which makes up 9~15.3% of soybean seed protein, is mainly composed of lipoxygenase, β-amylase, soybean agglutinin, and trypsin inhibitors [3,4]. However, due to the lack of economical and feasible recycling methods, most soybean whey is directly discharged without treatment after production [2], leading to serious environmental issues, such as odor, and the pollution of surface and groundwater. In addition, useful resources are wasted, reducing the economic value of soybeans [5,6]. Therefore, designing a simple, feasible method is essential for obtaining valuable compounds from soybean whey wastewater and reducing environmental pollution.

Soybean trypsin inhibitors (STI) are small molecule proteins that inhibit trypsin activity and form the main components of soybean whey protein [7]. The trypsin inhibitor activity is attributed to two polypeptides, namely the Kunitz trypsin inhibitor (KTI) and Bowman–Birk trypsin inhibitor (BBI) [8,9]. KTI has a molecular weight of about 20 kDa and consists of 181 amino acid residues, and the amino acid sequence contains two disulfide bonds and a reaction site, which can specifically inhibit trypsin activity [10]. BBI has a molecular weight of about 8 kDa and consists of 71 amino acid residues with an amino acid sequence containing seven disulfide bonds and two reactive sites, which can inhibit both trypsin and pancreatic rennet activity [11,12]. Trypsin inhibitors negatively affect the digestion of dietary proteins by blocking the activity of trypsin and chymotrypsin in the gastrointestinal system [13]. However, previous studies have shown that STI not only inhibits trypsin and chymotrypsin activity but also performs unique physiological functions, such as displaying anti-cancer, anti-inflammatory, anti-bacterial, and satiety properties [14,15,16,17,18,19]. Therefore, STI exhibits excellent application prospects in functional foods, biomedicine, and packaging materials.

At present, the main methods of soybean whey utilization are biotransformation and recovery of nutrients [2]. These processing treatments could effectively reduce whey wastewater. Chua et al. investigated the feasibility of converting soybean whey into soybean alcohol beverage by using the commercial Saccharomyces cerevisiae strain [20]. Li et al. used lactic acid bacteria (Leuconostoc citreum 1.2461) to ferment soybean whey to prepare extracellular polysaccharides with antioxidant activity [21]. Guan et al. used the ceramic membrane filtration, ultrafiltration, and organic solvent precipitation method to recover water-soluble polysaccharide SSPS1 with immunomodulatory function from soybean whey [22]. Minimal studies are available regarding the recovery of STI from soybean whey wastewater, while the technology to achieve a commercial level is insufficient. Li et al. purified Bowman–Birk protease inhibitor (BBI) from soybean whey based on the principle that chitosan and carrageenan can compound and condense with trypsin inhibitor in soybean whey [4]. Li et al. used the sodium sulfate precipitation method to precipitate potato trypsin inhibitor from potato wastewater and obtained the trypsin inhibitor with high activity and good stability [15]. Precipitation, membrane filtration, chromatographic techniques, and their combination are commonly used for protein recovery in the industry [23]. Salting out is a classic, scalable technique for the graded separation of proteins via precipitation. It is a simple, inexpensive process requiring no specialized equipment and is often used to separate and purify active proteins [24,25]. Gel filtration chromatography is a separation technique based on the size and shape of the protein. The protein mixture enters a porous mesh material where they are separated by molecular size. Gel filtration chromatography presents the significant advantage of an almost 100% recovery rate [26].

This study aims to design a simple, feasible method to isolate and purify valuable STI from soybean whey wastewater while discussing the impact of temperature, pH, and pepsin on its stability. First, the STI is obtained from imitated soybean whey wastewater via pH precipitation, ammonium sulfate salting out, and gel filtration chromatography. Furthermore, the impact of pH and heat treatment on the STI activity and secondary structure, as well as the influence of in vitro gastrointestinal digestion, are explored. The results are beneficial for recovering and utilizing soybean whey wastewater by-products. This work provides ways to realize zero emission and the sustainable development of soybean-processing enterprises.

2. Materials and Methods

2.1. Materials and Reagents

Cooled, defatted soybean meal was provided by the Yuwang Ecological Food Industry Co., Ltd. (Dezhou, China), which was crushed to 100 mesh and stored at 0~4 °C. The BCA Protein Assay Kit, bovine serum albumin (BSA), protein marker (11–180 kDa), and 2 × loading buffer were purchased from Beijing Solarbio Technology Co., Ltd., (Beijing, China). The benzoyl-l-arginine-p-nitroaniline (BAPA), KTI (T9128), BBI (T9777), and porcine pepsin (P7000) were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). The trypsin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China. All other reagents were of analytical grade and chromatographically pure.

2.2. Preparation of the Soybean Whey

The soybean whey was prepared as follows [4]: cooled defatted soybean meal was added to deionized water at a solid/liquid ratio of 1:10 (w/v), adjusted to pH 7.5 with 1 M NaOH, extracted in a constant temperature water bath at 50 °C for 50 min, and centrifuged at 4000 rpm for 20 min for solid/liquid separation. The supernatant was collected, after which the precipitate was added to deionized water at a solid/liquid ratio of 1:4. This process was repeated, after which the subsequent supernatant was mixed with that obtained during the first procedure. The supernatant was adjusted to pH 4.5 with 20% HCl and centrifuged to remove the protein isolate to obtain the soybean whey.

2.3. Isolation and Purification of the STI from the Soybean Whey

2.3.1. Isolation and Purification of the STI Using the Isoelectric Point Method

The molecular surface static charge was zero when the pH of the amphoteric electrolyte solution was at the isoelectric point. This weakened or destroyed the double electric layer and hydration film and increased the intermolecular attraction while decreasing the solubility, as well as the mutual aggregation and precipitation. This allowed the target protein to either be transferred to the solid phase or to remain in the liquid phase, preliminarily separating it from impurities [27,28]. The pH of soybean whey was accurately adjusted to 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5, respectively, by using 1 M HCl. The soybean whey was placed in a refrigerator at 4 °C for 120 min and then centrifuged at 4000 rpm for 20 min. The precipitate was dissolved in 0.1 M PBS buffer. The soybean whey protein concentration and trypsin inhibitory activity were determined, and the precipitated protein was analyzed via gel electrophoresis.

2.3.2. Isolation and Purification of the STI via Ammonium Sulfate Salting Out

The soybean whey was adjusted to pH 4.0 with 1 M H₂SO₄. Ground ammonium sulfate powder was slowly added to the soybean whey until reaching final ammonium sulfate saturation levels of 25%, 30%, 35%, 35%, 40%, 45%, 50%, 55%, 60%, and 65%, respectively [29]. The soybean whey was placed in a refrigerator at 4 °C for 120 min and centrifuged at 4000 rpm for 20 min. The protein precipitate was collected and dissolved in deionized water, after which the soybean whey protein concentration and trypsin inhibitory activity were determined. The precipitated protein obtained via fractional salting out was analyzed by using gel electrophoresis.

2.3.3. Isolation and Purification of the Trypsin Inhibitors via Gel Filtration Chromatography

The STI was purified by using a Superdex 75 Increase 10/300 gel filtration pre-loaded column. After equilibrating three column volumes with 0.1 M phosphate buffer solution, 500 μL of STI solution was slowly injected. The flow rate was set to 0.5 mL/min, the detection wavelength was set to 280 nm, and the automatic partial collector was set to 0.5 mL/tube. The protein solution of each peak was collected, dialyzed, freeze-dried, and stored at −20 °C in a refrigerator [30].

2.4. Analytical Method

2.4.1. Determination of the Protein Concentration

The soybean whey protein concentration was determined by using the disodium 2,2-biquinoline-4,4-dicarboxylate method (BCA protein quantification kit). A total of 0.5 mg/mL of BSA was used as a protein standard [3].

2.4.2. Determination of the Trypsin Inhibitory Activity [15,31]

Reagent Preparation

With regard to the BAPA solution (0.4 mg/mL), 40 mg of BAPA was dissolved in 1 mL dimethyl sulfoxide and reduced to 100 mL with Tris-HCl buffer solution. With regard to the trypsin solution (20 μg/mL), 10 mg of trypsin, with a measured specific activity of at least 10,000 benzoyl-L-arginine ethyl ester (BAEE) units/mg protein, was dissolved in 500 mL of an HCl solution. With regard to the acetic acid solution (30% v/v) to terminate the colorimetric reaction (also known as reaction stop solution), 30 mL of glacial acetic acid was mixed with 70 mL of water.

Determination of the Trypsin Inhibitory Activity

The prepared sample solution (1 mL) and Tris-HCl buffer solution (1 mL) were pipetted into separate 15-mL tubes. After incubation in a water bath at 37 °C for 10 min, 2.5 mL of a BAPA solution dissolved in Tris-HCl buffer (0.4 mg/mL) was added to each tube and mixed, followed by 1.0 mL of trypsin. The mixture was allowed to react at 37 °C for exactly 10 min, after which 0.5 mL acetic acid was added to terminate the reaction. Moreover, the trypsin was only pipetted after adding acetic acid as a negative control.

The trypsin inhibitor activity was calculated by using the following equation:

$$Trypsin inhibitor activity/\left(TIU/mL\right)=\left[\left(Ar-Abr\right)-\left(As-Abs\right)\right]\times \frac{F}{0.02} ,$$

where Ar is the absorbance of the standard solution, Abr is the absorbance of the standard blank solution, As is the absorbance of the sample solution, Abs is the absorbance of the sample blank solution, and F is the dilution multiple.

2.4.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The composition of the soybean whey protein was analyzed via SDS-PAGE. Here, 15% separation gel and 5% concentrated gel were prepared. The marker and soybean whey samples were loaded at 5 μL and 10 μL, respectively. Samples were concentrated at 80 V for 0.5 h in the concentrated gel and separated at 120 V. After electrophoresis, a Coomassie brilliant blue G-250 solution was used for staining for 60 min, and the decolorizing solution was used to decolored. The sample was scanned and analyzed by using a gel electrophoresis system imager.

2.4.4. Circular Dichroism (CD) Spectroscopy

All far-UV CD spectra (190–250) were obtained in a nitrogen atmosphere at 25 °C and recorded with a spectropolarimeter (MOS-500, BioLogic, Seyssinet-Pariset, France) by using a solution with a protein concentration of 0.5 mg/mL in a quartz cuvette with a 1-mm optical path. The spectra were analyzed via https://bestsel.elte.hu/index.php (accessed on 15 June 2022), to calculate the α-helix, β-fold, β-turn, and random curl percentages.

2.4.5. Examination of the STI Stability [15,32]

The Effect of Temperature on the STI Inhibitory Activity

The impact of temperature on the stability of STI was investigated by measuring the residual trypsin inhibitory activity. Here, 0.2 mg/mL samples were prepared by dissolving the STI powder in distilled water and treated at different temperatures (40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, and 100 °C) for 30 min in a water bath. Next, the samples were cooled to room temperature and centrifuged at 10,000 rpm for 20 min to determine the trypsin inhibitory activity of the supernatant residue.

The Effect of pH on the STI Inhibitory Activity

The effect of pH on the stability of STI was determined by measuring the residual trypsin inhibitory activity. Samples of 0.2 mg/mL were prepared at room temperature (25 °C) and treated at different pH levels (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, and 12.0) for 30 min.

The Effect of Pepsin Treatment on the STI Inhibitory Activity

The effect of pepsin treatment on the stability of STI was investigated based on earlier studies with slight modifications [15]. Here, 120 mg of STI was dissolved in 60 mL of simulated gastric juice (0.2 g/100 mL NaCl, 0.32 g/100 mL pepsin, and HCl adjusted to pH 1.5) pre-warmed at 37 °C, and incubated in a water bath shaker. Samples were taken at 10 min, 20 min, 30 min, 50 min, 70 min, 90 min, and 120 min, respectively, to determine the trypsin inhibitory activity. The KTI (Sigma T9128), BBI (Sigma T9777), blank trypsin inhibitor, and blank pepsin were used as controls.

2.5. Statistics

Microsoft Office Excel 2007 was used for data processing, and IBM SPSS Statistics 19 software (IBM, Chicago, IL, USA) was used for one-way ANOVA. The experimental data were expressed as “X ± SD” of three replicates. Significant differences between means were stated at p < 0.05. Origin 2021 was used to prepare the graphs.

3. Results and Discussion

3.1. The Analysis of the Soybean Whey

The soybean whey protein concentration was measured as 4.3 mg/mL by using the BCA method, which was higher than the 0.3~3.0 mg/mL summarized in the review by Chua [2]. The specific trypsin inhibitor activity in the soybean whey was 918.5 U/mg. The soybean whey contained mainly seven proteins. Their molecular weight was 100 kDa, 75 kDa, 32 kDa, 30 kDa, 28 kDa, 20 kDa, and 12 kDa, respectively, of which the isolated and purified KTI (21.20%) and BBI (17.40%) accounted for 38.60% of the total proteins (Figure 1). The results were generally consistent with the soybean whey prepared by Li. The electrophoretic pattern of soybean whey prepared by Li showed five bands, which were 75 kDa, 30 kDa, 24 kDa, 19 kDa, and 12 kDa, respectively [4].

3.2. Isolation and Purification of STI

3.2.1. The Effect of pH on the Isolation and Purification of STI

The pH of the solution affects the dissociation status and solubility of the proteins. When the pH approaches the isoelectric point, most of the proteins were precipitated readily.

As shown in Figure 2a, the recovery rate of the soybean whey protein was U- shaped in a range of pH 3.5 to pH 7.5. The maximal recovery rate of soybean whey protein (13.02%) was obtained at pH 3.5, followed by pH 7.5, with a recovery rate of 10.07%. The recovery rate of trypsin inhibitory activity in soybean whey gradually decreased, with the increase of pH, after which it stabilized and remained constant in a range of pH 5.0 to pH 7.5, displaying a recovery below 4.00%. As shown in Figure 2b, the STI bands were clearly visible at pH 3.5 and pH 4.0, whereas the protein band with a molecular weight of 30 kDa was also obvious. The STI bands gradually became blurred at higher pH levels in the range of pH 3.5 to pH 7.5. Although no STI band was evident at pH 7.5, the 30 kDa protein band was individually distinct. Therefore, this study improved the STI purity by adjusting the soybean whey pH to 7.5 to remove some of the heteroprotein with a molecular weight of about 30 kDa.

The results indicated that the soybean whey displayed a large amount of protein precipitation in acidic or alkaline conditions. The protein precipitated from the soybean whey at the pH < 4.5 mainly consisted of STI, whereas that obtained at pH 7.5 contained less STI and almost represented a non-trypsin inhibitor. This was consistent with the isoelectric points of STI reported in previous studies at pH 4.2 (BBI) and pH 4.5 (KTI). Therefore, pH 7.5 can be used as a condition for STI isolation and purification from soybean whey to remove the heteroprotein, and a pH < 4.5 can be used as a condition for STI precipitation. This study used pH 4.0 during the next step of isolating and purifying STI from soybean whey.

3.2.2. The Effect of Ammonium Sulfate Saturation on STI Isolation and Purification

Salting out is commonly used for protein separation and enrichment. In highly concentrated salt solutions, salt ions can bind to water molecules on the protein surface, destroying the hydration membrane and reducing the solubility of the protein, consequently separating the protein from other impurities, such as sugars [24].

This study evaluated the effect of different ammonium sulfate saturation concentrations (25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, and 65% w/v) on the STI in soybean whey. As shown in Figure 3a, a higher level of ammonium sulfate saturation gradually increased the recovery rates of the soybean whey proteins and trypsin inhibitory activity, which displayed respective values of 64.48% and 89.47% as the ammonium sulfate saturation reached 40%. As ammonium sulfate saturation increased, the recovery rate of the trypsin inhibitory activity stabilized and did not increase, whereas that of the soybean whey protein continued to rise. The results indicated that the saturation of ammonium sulfate reached 40% and STI was precipitated basically, and the increasing saturation of ammonium sulfate would reduce the purity of STI. The electrophoretic analysis results of the precipitated protein also confirmed these findings (Figure 3b). When the ammonium sulfate saturation exceeded 40%, the trypsin inhibitor bands disappeared in gel, and the protein precipitated was a non-trypsin inhibitor with a molecular weight greater than 20 kDa. The results showed that the STI was the least soluble and most readily precipitated when the ammonium sulfate saturation level reached 40%, which was the optimal saturation for salting out STI from soybean whey. Because the STI recovered via ammonium sulfate salting out still contained some non-trypsin inhibitor, the precipitated protein required purification during the next step.

3.2.3. The Effect of Gel Filtration Chromatography on STI Isolation and Purification

As shown in Figure 4a, five fractions were obtained after the STI passed through the Superdex 75 Increase 10/300 gel filtration pre-load column. Examination of the positions of KTI (SigmaT9128) and BBI (SigmaT9777) indicated that the solutions in tubes 10–13 were KTI, whereas those in tubes 14–19 were BBI. The combined total peak area of KTI and BBI was 71.11%, of which KTI accounted for 44.27% and BBI for 26.84%. The recovered soybean whey protein, after ammonium sulfate salting out, re-solubilization, and centrifugation to remove the precipitate, contained 71.11% STI, including 44.27% KTI and 26.84% BBI. The heteroprotein was further removed from the STI after gel filtration chromatography. Furthermore, the two types of STI were separated. The collected KTI and BBI were analyzed via electrophoresis (Figure 4b), and the results showed that the trypsin inhibitor displaying a single band could be obtained via gel filtration chromatography. Therefore, to obtain a purer STI, solutions were collected from tubes 10–13 and 14–19, respectively. They were then dialyzed, freeze-dried, and stored at −20 °C in a refrigerator.

3.3. Analysis of the STI Activity

As shown in Figure 5, the BBI activity was higher than that of KTI (p < 0.05). The KTI (1733.5 TIU/mg) and BBI (2588.3 TIU/mg) were obtained from the soybean whey via pH precipitation (soybean whey was precipitated at pH 7.5 to remove some of the heteroprotein), ammonium sulfate salting out (the soybean whey was treated at pH 4.0 and 40% ammonium sulfate), and gel filtration chromatography. Their inhibitory activity did not differ significantly from the KTI (Sigma T9128) (1770.8 TIU/mg) and BBI (Sigma T9777) (2602.0 TIU/mg) standards. Both the KTI and BBI obtained via column chromatography showed higher inhibitory activity than the STI obtained via ammonium sulfate salting out (1442.5 TIU/mg) (p < 0.05). The analysis of trypsin inhibitory activity showed that it was feasible to isolate and purify STI from soybean whey via ammonium sulfate salting out, whereas the STI was highly active and suitable for utilization in a follow-up study. In addition, isolating and purifying STI from soybean whey can help maximize resource utilization in terms of valuable soybean whey, whereas the subsequent reduction in sewage treatment costs and economic efficiency enhancement of enterprises present an obviously positive impact.

3.4. Stability Analysis of STI

Temperature and pH are factors that cannot be ignored during food processing. They have an important impact on the physicochemical properties of food, especially protein-based food. In addition, the digestion and absorption of proteins are inseparable from the influence of temperature and pH. Therefore, investigating the stability of STI in different temperature and pH conditions is essential [15].

3.4.1. The Effect of Temperature on the STI Activity

As shown in Figure 6a, the STI remained stable between 25 °C and 50 °C, and its inhibitory activity gradually decreased at higher temperatures. The inhibitory activity decreased by 15.09% after 30 min of treatment in a boiling water bath (100 °C). The results were consistent with the experimental results of Magdi, who investigated the thermal stability of STI. The results indicated that the KTI activity remained above 90% after 60-min medium boiling water bath heat treatment in neutral conditions [33]. Roychaudhuri compared fluorescence and CD spectra, revealing that the secondary structure of KTI heat-treated (90 °C) could be restored to its natural conformation after cooling without compromising the trypsin inhibitory activity [34]. In summary, STI displayed excellent thermal stability between 25 °C and 50 °C, which decreased when exceeding 50 °C. Heat treatment modified the STI structure, hydrophobic polymerization, and precipitation, affecting the inhibitory STI activity [35].

The CD spectra during thermal denaturation are a reliable indicator of the global folding and unfolding process [33]. The secondary structures of the STI treated at different temperatures were analyzed via CD. The results were shown in Figure 6b and

Table 1. The secondary structures of STI.

Temperature (°C)	α-Helix	β-Sheet	β-Turn	Random Coil
NO heating	0.00 ± 0.00	39.23 ± 0.58	15.30 ± 0.50	45.30 ± 0.98
40	0.20 ± 0.16	39.27 ± 1.25	15.53 ± 0.12	44.97 ± 1.48
50	0.27 ± 0.09	39.33 ± 1.32	15.03 ± 0.52	45.30 ±1.87
60	0.00 ± 0.00	40.17 ± 1.27	14.20 ± 1.44	45.63 ± 1.37
70	0.33 ± 0.34	40.87 ± 1.45	15.10 ± 0.78	43.70 ± 1.93
80	0.07 ± 0.09	41.33 ± 1.52	15.30 ± 0.50	43.33 ± 1.92
90	0.43 ± 0.61	41.07 ± 0.41	15.20 ± 0.22	43.33 ± 1.08
100	0.13 ± 0.19	41.73 ± 0.52	15.17 ± 0.12	42.97 ± 0.52

Results are presented as mean values ± standard deviation (n = 3). The data in each column are not significantly different (p > 0.05).

. After heating treatment at different temperatures, the STI showed strong negative peaks at 190~220 nm without positive ellipticity. This indicated that the secondary structure of the STI contained a significant number of β-structures, and higher temperatures did not lead to a complete loss of the STI ordered conformation [32]. However, increased temperatures decreased the STI ellipticity (Figure 6b), increased the β-fold content in the secondary structure, and reduced the random curl content (Table 1). This indicated that temperature had a certain influence on the secondary structure of the STI, while the STI spatial structure became more orderly. After heat treatment, the secondary structure of the STI became more stable.

3.4.2. The Effect of pH on the STI Activity

As shown in Figure 7a, no changes were evident in the residual inhibitory trypsin activity between pH 3.0 and pH 12.0. Therefore, the STI displayed excellent stability in a wide pH range. Klomklao reported that the trypsin inhibitor obtained from mung bean seeds maintained high inhibitory activity between pH 3.0 and pH 10.0 [36]. Moreover, Benjakul revealed that the trypsin inhibitor obtained from pigeon- and cowpeas also exhibited high inhibitory activity between pH 4.0 and pH 10.0 [37] The trypsin inhibitor purified from Cassia leiandra seeds also displayed high inhibitory activity between pH 2.0 and pH 10.0 [38].

A CD analysis was performed of the STI treated at different pH levels. The results were shown in Figure 7b and

Table 2. Secondary structures of STI.

pH	α-Helix	β-Sheet	β-Turn	Random Coil
3.0	0.00 ± 0.00	40.17 ± 0.95 a	15.03 ± 0.31 ab	44.67 ± 0.84 b
4.0	0.00 ± 0.00	40.13 ± 1.67 a	15.03 ± 0.42 ab	44.83 ± 2.07 ab
5.0	0.00 ± 0.00	39.57 ± 0.94 a	14.60 ± 0.29 b	45.83 ± 1.91 ab
6.0	0.00 ± 0.00	39.67 ± 0.46 a	14.93 ± 0.17 ab	45.40 ± 0.62 ab
7.0	0.00 ± 0.00	39.76 ± 0.59 a	15.10 ± 0.22 ab	45.10 ± 0.85 ab
8.0	0.00 ± 0.00	38.33 ± 1.43 ab	14.77 ± 0.58 ab	46.87 ± 2.04 ab
9.0	0.00 ± 0.00	38.60 ± 1.53 ab	15.00 ± 0.14 ab	46.40 ± 1.67 ab
10.0	0.00 ± 0.00	37.23 ± 0.21 b	14.90 ± 0.36 ab	47.90 ± 0.45 a
11.0	0.00 ± 0.00	36.90 ± 1.19 b	15.50 ± 0.49 ab	48.60 ± 1.43 a
12.0	0.00 ± 0.00	35.50 ± 0.92 b	15.60 ± 0.64 a	48.90 ± 1.56 a

Results are presented as mean values ± standard deviation (n = 3). Different letters on the same column indicate significant differences.

. Strong negative peaks were evident at 190–220 nm, indicating that the STI mainly contained β-fold structures regardless of the pH conditions. In addition, no α-helical structures were present in the STI secondary structure in different pH conditions. The β-sheet content decreased, and the random curl content increased at higher pH levels, indicating gradual destabilization of the STI structure as the pH increased. In acidic conditions, the STI was more stable than in alkaline conditions, which could be due to the de-ionization of hydrogen bonds in the β-fold structure, resulting in structural de-folding [15].

3.4.3. The Effect of Pepsin on the STI Activity

The gastric environment substantially affects the digestion and absorption of food or oral drugs. After reaching the intestine, STI must maintain sufficient inhibitory activity to realize its functional weight control, anti-cancer, and anti-inflammatory effect. Because recent research has suggested that not all inhibitors are resistant to digestion in mammals, the digestibility of any given inhibitor must be assessed accurately. To investigate the hydrolytic inactivation of STI in a simulated gastric environment, experiments were set up to examine the effect of pepsin treatment on inhibitory STI viability [15].

As shown in Figure 8a, the STI and KTI activity retention gradually decreased with extended pepsin treatment time. After 120 min, the STI and BBI inhibition activity retention rates were 44.75% and 33.13%, respectively. The BBI activity retention was 84.93%, which was higher than STI and KTI (p < 0.05) (Figure 8b). In the presence of inactivated pepsin and the absence of pepsin, the extension of time did not affect the inhibitory trypsin activity. The results indicated that pepsin significantly affected STI and KTI, but not BBI, which could be because pepsin more likely hydrolyzed large KTI molecules rather than small BBI molecules. Therefore, the trypsin inhibitor displayed excellent resistance to pepsin hydrolysis.

4. Conclusions

This study recovers STI from simulated industrial soybean whey wastewater by using various techniques, including the isoelectric point method, ammonium sulfate salting out, and gel filtration chromatography, from the perspective of industrial production. The results showed a maximum STI recovery of 89.47% and a purity of 71.11% in pH 4.0 and 40% ammonium sulfate saturation conditions. After further purification, the KTI (1733.5 TIU/mg) and BBI (2588.3 TIU/mg) recovered from soybean whey wastewater exhibit excellent inhibitory activity, which is not significantly different from the KTI (Sigma T9128) and BBI (Sigma T9777) standards. The STI purified from the soybean whey display excellent stability in a broad temperature and pH range. Most of its trypsin inhibitory activity is retained when the STI enters the gastrointestinal tract. These results suggest that STI with high purity and activity can be obtained from soybean whey by using a simple method consisting of pH precipitation, and ammonium sulfate salting out. The soybean trypsin inhibitor from the soybean whey colloidal wastewater exhibited excellent purity (71.11%) and stability.