# Trypsin Inhibitor from Soybean Whey Wastewater: Isolation, Purification and Stability
## 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 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, \(\beta\)-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 molecularweight 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. [PERSON] et al. investigated the feasibility of converting soybean whey into soybean alcohol beverage by using the commercial Saccharomyces cerevisiae [20]. [PERSON] et al. used lactic acid bacteria (_Leuconostoc citream_ 1.2461) to ferment soybean whey to prepare extracellular polysaccharides with antioxidant activity [21]. [PERSON] 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. [PERSON] 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]. [PERSON] 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].