Recent innovations in processing technologies for improvement of nutritional quality of soymilk

ABSTRACT Soymilk is well known for its health and nutritional benefits and is one of the best plant substitutes for cow milk. Soymilk is high in protein, low in cholesterol, lactose-free, and rich in polyunsaturated fatty acids. The bioactive compounds in soybean contribute to the beneficial effects of soymilk and are reported to exert various bioactivities. With the rising interest in health-conscious lifestyles, the development of soymilk with high nutritional quality is a critical task of the soymilk industry. Therefore, research on novel and advanced technologies is underway to develop soymilk with maximal nutritional quality. This review aims to present the recent findings on the beneficial effects of the bioactive compounds in soymilk and to introduce the latest technological advances that enhance the nutritional quality of soymilk, focusing on increasing the amount of nutrients and bioactive compounds, anti-nutrient removal, fortification with bioactive ingredients, and bio-enrichment.


Introduction
Soybean (Glycine max (L.) Merr.) is recognized for its nutritional excellence, as evidenced by its highly balanced nutrient composition (Hassan, 2013). Soymilk is one of the most widely available plant-based beverages worldwide and can be obtained from soybeans without complicated processes. It has long been acknowledged as the best alternative to the significant obstacles of dairy milk consumption: lactose intolerance, defined as the inability to digest lactose due to lack of lactose-decomposing enzymes, and milk protein allergy (Mazumder & Begum, 2016;. In recent years, with the rapid rise in popularity of plant-based diets, the demand for soymilk has continuously increased across the world (Euromonitor International, 2020;Mordor Intelligence, 2019). As consumer expectations for gaining health and nutritional benefits from plant-based foods continue to rise, the development of soymilk products that provide proper nutrition and additional beneficial effects is important.
The soymilk manufacturing process consists of dehulling, blanching, soaking, grinding, filtering of liquid from solid residues (okara), cooking, formulation and fortification, packaging, and commercial sterilization, with some modifications depending on producer (Giri & Mangaraj, 2012). The nutritional quality of soymilk is largely affected by the technologies and conditions applied in each processing step (Giri & Mangaraj, 2012). The main processing technologies that have been extensively explored in soymilk production are (1) improvement of the contents of protein and bioactive compounds in soymilk, (2) removal of anti-nutritional factors, (3) fortification with nutrients and bioactive ingredients, and (4) bio-enrichment through fermentation.
During soymilk preparation, an insoluble solid residue known as okara is produced and typically discarded as waste. However, okara (dry basis) retains substantial amounts of dietary fiber (42.4-58.1%), protein (15.2-33.4%), fat (8.3-10.9%), and a trace amount of minerals and vitamins . Approximately 355 mg/g dry matter of total isoflavones remains in okara (Jackson et al., 2002;S. Li et al., 2013). Therefore, one of the key research challenges in the soymilk industry is the development of effective solutions that increase the extractability of protein and bioactive compounds from soybeans and transferring nutritional and bioactive components of soybeans to soymilk. Innovative extraction-aiding technologies, such as microwave, ultrasound, enzymes, and high-pressure homogenization, draw interest in the soymilk industry, as improved extraction yield not only enriches the nutritional profile of soymilk, but also is directly linked to less waste generation and cost reduction (Morales-de La Peña et al., 2018;Penha et al., 2020;Preece et al., 2017c;Varghese & Pare, 2019).
Trypsin inhibitor, phytic acid, lipoxygenase, tannin, and flatulence-causing oligosaccharides present in soymilk have been recognized as anti-nutritional factors that limit absorption of essential nutrients (Arques et al., 2014;Katrolia et al., 2019;Kim et al., 2010;Shashego, 2019;Stanojević et al., 2017). In particular, trypsin inhibitors, phytic acids, and lipoxygenases have been identified as major components that negatively impact the absorption of proteins and minerals and the organoleptic quality of soymilk (Kim et al., 2010;Stanojević et al., 2017;Xiao et al., 2012), which have been evidenced by several studies. Phytase addition has been reported to improve mineral (Ca, Mg, and Fe) availability in soymilk (Kwon et al., 2014). Moreover, in vitro protein digestibility of soymilk was enhanced with the reduction in trypsin inhibitors (Vanga et al., 2020). Soymilk made from lipoxygenase-free soybean contained fewer bean-flavored compounds and was rated as less grassy/rancid and bitter than regular soymilk (Yang et al., 2016). Heat treatments are widely used to inactivate these factors in soymilk, but excess heat treatments can result in substantial loss of bioactive compounds in soymilk, such as isoflavones (Huang et al., 2006). Therefore, to balance the destruction of anti-nutritional factors and retention of nutrients, careful adjustment of timetemperature conditions in thermal processing and applications of novel non-thermal processing technologies have been studied to obtain soymilk with high nutritional value.
Fortification is one of the effective solutions that can confer nutritional density and health-promoting properties to soymilk by conveying nutrients and bioactive ingredients (Devi & Chaturvedi, 2012;Dong et al., 2016;Pathomrungsiyounggul et al., 2013). Since consumers desire to obtain nutritional benefits from plant milk and often have concerns about the nutritional adequacy of plant-based milk as a complete nutritional alternative to dairy milk (Chalupa-Krebzdak et al., 2018), producers have made an effort to develop plant milk with an improved and more complete nutritional profile. The portion of newly launched plant milk products in the global market with a "plus" claim (products fortified with vitamin/minerals or proteins or fibers) increased by 55% during Aug 2019-Jul 2020, compared to that from the same period in 2016 (Mintel, 2020). Moreover, functional soymilk has been developed by fortification with natural and bioactive ingredients, such as ginseng extracts, plant collagen, and lutein (Mintel, 2020). Therefore, a lot of research is being carried out to maximize the beneficial effects of soymilk fortified with bioactive ingredients and effective delivery tools, such as nanoemulsion that positively affects solubility and bioavailability of fortificants in the final products (Aswathanarayan & Vittal, 2019).
In addition, fermentation is an efficient and eco-friendly processing method that can enhance the nutrition of food products through biological enrichment of food compounds and the resultant health benefits (Şanlier et al., 2019). It is widely known that bioconversion of isoflavone glycosides into the more biologically active and absorbable forms occurs during soymilk fermentation. The amount of phenolic acids is often increased during soymilk fermentation as a result of degradation of large phenolic compounds by microbial enzymes (Pyo et al., 2005;Zhao & Shah, 2014). Bioactive peptides and vitamins that are produced in the process of soymilk fermentation also enrich its nutritional profile (Tsai et al., 2006;Zhao & Shah, 2014). The changes in nutritional contents by microbial bioconversion confer several improved physiological functions in fermented soymilk. The potential health benefits that have been widely studied in fermented soymilk are anti-hyperlipidemia, anti-hyperglycemia, antihypertension, anti-oxidative, anti-cancer effects, and skin protection (Ahsan et al., 2019;Kano et al., 2016;X. L Zhang et al., 2017;Qian et al., 2020).
This review aims to introduce the nutritive value of soymilk and the beneficial effects of its bioactive compounds and to highlight recent research advances in soymilk processing techniques that contribute to the nutritional improvement of soymilk. There was a particular emphasis on the enhancement of the extractability of protein and bioactive compounds into soymilk, reduction in anti-nutrients, and fortification and bio-enrichment of soymilk. This article provides an overview of the nutritional profile and health benefits of soymilk and the associated processing technologies. Furthermore, recent research findings offer meaningful information to soymilk manufacturers and industry when selecting processing techniques to improve the nutritional quality of soymilk and to expand their product portfolio with more health-and function-oriented options.

Nutritional value and health benefits of soymilk
Soymilk has been validated as one of the most nutritious beverages due to its considerably high protein level, abundant poly-unsaturated fatty acid (PUFA) content, and various bioactive compounds. The protein content in soymilk is 2.88 g/100 g, which is comparable to that in bovine milk (3.15 g/100 g) (U.S. Department of Agriculture, 2019a, 2019b. Soymilk has a well-balanced amino acid composition with high true ileal digestibility of 92.3%, producing a digestible indispensable amino acid score (DIAAS) of 117% that can be classified as an "excellent/high" quality protein source (DIAAS ≥ 100%) (Food and Agriculture Organization, 2011). The scores are greater than those of other plant-based foods, such as seitan (28%), tofu (97%), and pea emulsion (60%) (Reynaud et al., 2020).
Soymilk is cholesterol-free and has a high content of PUFAs. The total lipid content of soymilk is 1.5-2%. The fatty acids in soymilk consist of around 15% saturated fatty acids and 55-63% PUFAs, more than other plant milks, such as almond milk and rice milk (Peñalvo et al., 2004;U.S. Department of Agriculture, 2019b;. The most abundant PUFAs in soymilk are linoleic acid (54.8%), which accounts for half of the total fatty acids in soymilk, followed by α-linolenic acid (7.53%). Oleic acid (20.4%), a monounsaturated fatty acid, is the second most abundant total fatty acid in soymilk (Peñalvo et al., 2004). The n-6:n-3 ratio of soymilk is 7.28, indicating a proper balance of essential fatty acids based on the FAO's recommendation (5-10) (Food and Agriculture Organization & World Health Organization, 1995;Peñalvo et al., 2004). Also, soymilk is a good source of phospholipids, the major components of the cell membrane, with large contents of phosphatidylserine, phosphatidylglycerol, phosphatidyl ethanolamine, and phosphatidylcholine compared to cow milk .
Soymilk provides some B-group vitamins, such as thiamine, riboflavin, and folate. Although soymilk contains negligible amounts of vitamins A, D, and E, it provides a greater amount of vitamin K (3 mg/100 g) than cow milk (0.3 mg/ 100 g) (Combs & McClung, 2016). However, vitamin B 12 and certain lipid-soluble vitamins are deficient in soymilk and should be offered through fortification. Natural soymilk normally supplies 18.5 mg of calcium, 0.5 mg of iron, 60.3 mg of phosphorus, 206 mg of potassium, 2.5 mg of sodium, 0.3 mg of zinc, and 22 mg of magnesium per 100 g (Nti et al., 2016). Soymilk provides more of the daily amounts of micronutrients of iron, potassium, and magnesium and less sodium than dairy milk, which contains 0.05 mg of iron, 151 mg of potassium, 13 mg of magnesium, and 49 mg of sodium per 100 g ). Yet, soymilk possesses less calcium than dairy milk containing 119 mg/100 g. Roughly 200-300 mg of calcium per cup is generally added to commercial soymilk to achieve a level similar to that in dairy milk (Dietary Guidelines Advisory Committee, 2015; . Soymilk is noted for its beneficial effects linked to bioactive compounds in soybean, which is abundant with isoflavones, soy saponins, and soy protein. Therefore, the health benefits of soymilk are enhanced by the physiological effects of the individual functional compounds in soybean. Isoflavones, one of the main bioactive components in soybean, are a type of flavonoid structurally similar to estrogen and known to exhibit estrogen-like activity (Bolca, 2014). Isoflavones in soybean exist mainly in the form of physiologically inactive glycosides (daidzin, genistin, and glycitin). Upon digestion, they are hydrolyzed to aglycones (daidzein, genistein, and glycitein) with higher bio-activities (Setchell et al., 2002). Equol, a daidzein metabolite produced by intestinal microbes, has more potent estrogenic activity than other isoflavone derivatives (Morito et al., 2001). Commercial soymilk contains 1.10-31.03 mg of isoflavone per 100 g (Bhagwat et al., 2008). The bioavailability of isoflavone differs depending on the type of compound-carrying food matrix. The isoflavone aglycones in soymilk were more bioavailable and absorbed faster than those in a solid food matrix (textured soy protein) (Cassidy et al., 2006). Soy saponins are triterpenoid glycosides conjugated with saponin aglycones (sapogenol) and are mainly divided into groups A and B soyasaponins based on aglycone structure (Berhow et al., 2006). Soy saponins and their aglycones have various physiological functions, including anti-cancer, immunomodulatory, and hepatoprotective effects (Guang et al., 2014). Soymilk contains 393 nmol/g soy saponins and 1.21 nmol/g soyasapogenol B (Kamo et al., 2014b). Soyasapogenols have higher bioavailability than soy saponins, and the absorption rate of soyasapogenol B was greater than that of soyasapogenol A (Kamo et al., 2014a).
Soy protein (25 g or more per day) has been claimed to reduce coronary heart disease risk by the U.S. Food and Drug Administration (Food and Drug Administration, 1999). Moreover, soy protein-derived peptides have been reported to possess various health benefits, such as anti-hypertensive, immunomodulatory, anti-diabetic, and anti-cancer properties (Chatterjee et al., 2018). Therefore, soymilk is expected to offer potential health benefits due to the presence of various bioactive compounds. The recent findings on the beneficial effects of bioactive compounds in soymilk are briefly summarized in Table 1.

Improving the amounts of protein and bioactive compounds in soymilk
The extraction technique is widely accepted as a critical factor affecting the solubility and contents of protein and bioactive compounds in soymilk (Nufer et al., 2009;Preece et al., 2017c). During soymilk production, protein (15.2--33.4%, dry basis) and isoflavones (12-30% of initial contents in soybeans, dry basis) are partly preserved in okara and thus not fully incorporated into soymilk (Kamble & Rani, 2020;Vong & Liu, 2016). Therefore, a variety of novel technological methods, including microwave, ultrasound, and enzymes, have been investigated to enhance extractability into soymilk.

Enhancing the extraction yield of protein
To release soluble protein into the aqueous medium, the cytoplasmic membrane must be disrupted (De Figueiredo et al., 2018;Preece et al., 2017c).
Microwave enables exudation of the cellular components into the surrounding media by causing rapid heating and evaporation of polar water molecules within plant cells, which eventually ruptures the cell. As assumed, soymilk obtained from microwave-treated ground soybeans attained a higher extraction yield and protein content than steamheated soymilk, with increases of 23.92% (p < .05) and 44.44% (p < .05), respectively (Varghese & Pare, 2019). The application of ultrasound to extraction has drawn attraction in the food industry due to its high efficiency, costeffectiveness, and lower operating temperature (Majid et al., 2015;Vilkhu et al., 2008). The growth and implosive collapse of cavitation bubbles near a plant cell surface result in a microjet toward the plant matrix that generates transiently high localized temperature and pressure. This leads to cell wall disruption and consequent release of the contents of the cells (Majid et al., 2015;Pico, 2013). Ultrasound treatment to soy slurry for 1 min increased the total solid and protein yield by around 10%. The treatment for 0.5-15 min to slurry resulted in an increase in the number of small particles (0.3-1.1 µm). Interestingly, intact cells were detected even after 15 min of ultrasound exposure, suggesting that the improved extractability is mainly attributable to enhanced solubility rather than cell destruction (Preece et al., 2017b).
High-pressure homogenization (HPH) can induce cell disruption and extract intracellular macromolecules by causing intense mechanical stresses on the cell during homogenization of materials under high pressure (Fayaz et al., 2019). HPH (100 Mpa) increased the protein extraction yield in soy slurry by 16% and in okara solution by 26% at a single pass. Contrary to ultrasound treatment (Preece et al., 2017b), the high-pressure treatment effectively disrupted intact cell walls in soy slurry after a single pass, suggesting that HPH may provide greater extraction assistance than does ultrasound (Preece et al., 2017a) Enzymes also can aid the release of protein molecules by enzymatic hydrolysis of the polysaccharide cell wall. When  • Reduction in calculated 10-year risk for coronary heart disease by 27% and for cardiovascular disease by 24% (Sathyapalan et al., 2018) Soy protein

Muscle function
In vivo • Increase in maximum force production and muscle contraction rate without affecting muscle mass in high-fat-and carbohydrate-diet-fed mice (Khairallah et al., 2017) Liver steatosis

Improving the content and bioaccessibility of bioactive compounds
More recent studies have shown that the processing methods used during soymilk extraction have considerably different effects on the content and bioaccessibility of bioactive compounds and anti-oxidant ability in the final products. The two non-thermal heat treatments, high-intensity pulsed electric field and high-pressure processing, increased total isoflavone content by around 15-26% in soymilk-fruit juice beverage (p < .05) relative to the untreated liquid (17.78 mg/ 100 mL) (Rodríguez-Roque et al., 2020). Ultrasound treatment has emerged as a promising technique for achieving a high extraction yield of phenolic compounds from botanical sources and for enhancing biological activities, such as anti-oxidant potential (Dzah et al., 2020). Ultrasound treatment of hydrated soybeans and soy slurry at different conditions greatly increased the amount of isoflavone aglycones by 90.3-131% and 19.6-59%, respectively, in the final soymilk (p < .05). Soymilk extracted from sonicated hydrated soybeans was more effective for the recovery of isoflavones than when using slurry (Morales-de La Peña et al., 2018). Ultrasound treatment also was shown to enhance β-glucosidase activity, which could increase the amount of isoflavone aglycones. Application of ultrasound (20 kHz, 50 W, 3 min) to soy slurry enhanced βglucosidase activity in the resulting soymilk by 1.5-fold (p < .05) and increased isoflavone aglycone content by 2.4-fold (p < .05). The ultrasound treatment also enhanced the anti-oxidant capacity of the final soymilk probably due to an improvement in aglycone content (Silva et al., 2019), which has a positive correlation with anti-oxidative activity . The enhanced β-glucosidase activity can be attributed to ultrasound-induced modifications of secondary structures in the enzyme, as demonstrated by the increase in α-helix content by 50% and the decrease in β-sheet content by 18% (Sun et al., 2019). Ultrasound affects the activation or inactivation of enzymes depending on several parameters, such as intensity, pH, temperature, and exposure duration (Nadar & Rathod, 2017). The β-glucosidase activity was promoted at certain ranges of temperature (20-45°C), ultrasound intensity (<181.53 W), and exposure duration (<15 min), while the enzyme activity decreased to below that of the untreated samples when these conditions were not met (Sun et al., 2019). Therefore, it is necessary to determine an optimal condition for applying ultrasound to soymilk.
The use of enzymes can be a viable option to enhance bioactive substance extraction from soybean. The addition of hemicellulase to soy slurry greatly increased isoflavone aglycone content by 1.7-fold (p < .05), accompanied by improved β-glucosidase activity by 1.3-fold (p < .05) and enhanced anti-oxidant activity compared to the control (Silva et al., 2019). Moreover, addition of carbohydrases to slurry (6%, v/v of soy slurry) greatly increased the recovery rate of isoflavone to 93.3% in soymilk at 45 min compared to heat-only-treated soymilk (67.3%). The proportion of aglycones in total isoflavones increased with increasing enzyme concentrations (Penha et al., 2020). In terms of phenolic and flavonoid contents in soymilk, 15 soybean cultivars soaked to prepare soymilk generally presented higher total phenolics and flavonoids and antioxidant activity compared to soymilk prepared from dried soybeans . Moreover, thermal treatments of pasteurization (95°C), incontainer sterilization (121°C), and ultra-high-temperature treatment (UHT) (143°C) markedly increased the amounts of total flavonoids in soymilk by 55.7%, 56.7%, and 35.8%, respectively, compared to raw soymilk (p < .05) .
When adopting processing techniques, it is necessary to consider the bioaccessibility of bioactive compounds. To provide potential health benefits, bioactive compounds must be liberated from the food matrix and become bioaccessible in the gastrointestinal tract (Rein et al., 2013). In this sense, food processing can affect bioaccessibility through modifications of food matrices and microstructures (Ribas-Agustí et al., 2018). Thermal treatments cause alterations in the food matrix, such as disruption of plant cell walls and modification or breakdown of matrix macromolecules, which can lead to a release of bioactive substances from the food matrix and increase the concentration and bioaccessibility of phenolic compounds Ribas-Agustí et al., 2018). Thermal treatment at 95°C for 60 min markedly increased the bioaccessibility of flavonoids in soymilk to 67.19%, greater than that in raw soymilk (36.25%). Notably, UHT-treated soymilk (121°C for 9 min) retained a considerably larger amount of phenolic compounds (321.26 mg GAE/ L) in the in vitro dialyzed fraction than raw soymilk (238.67 mg GAE/L) (p < .05), resulting in improved bioaccessibility up to 125.14% compared to a smaller amount in raw (97.27%). Heat-treated soymilk has shown higher antioxidant activity after gastric digestion due to an increase in phenolic and flavonoid contents in the digested gastric fraction . Detailed information regarding the latest findings about extraction techniques of nutrients related to soymilk production are summarized in Table 2.

Removal of anti-nutritional factors
The presence of anti-nutritional components in soymilk is a significant obstacle to its high nutritional quality. Various anti-nutrients, such as trypsin inhibitor, phytate, tannin, flatulence-inducing oligosaccharides (raffinose and stachyose), and lipoxygenases, are present in soymilk (Arques et al., 2014;Katrolia et al., 2019;Kim et al., 2010;Shashego, 2019;Stanojević et al., 2017). Among those factors, trypsin inhibitors, phytates, and lipoxygenases are major antinutrients in soymilk that negatively impact protein digestion, mineral availability, and sensory attributes, respectively (Kwon et al., 2014;Vanga et al., 2020;Yang et al., 2016). Numerous studies are underway to establish effective strategies to remove these anti-nutritional factors in soymilk. Table 2 offers an overview of the recent research findings on technologies used to eliminate anti-nutrients in soymilk.

Trypsin inhibitor
Soybean trypsin inhibitors (TI) mainly consist of Kunitz trypsin inhibitor (74%) and Bowman-Birk trypsin inhibitor (26%) (Y. Y. Chen et al., 2014). These protease inhibitors disrupt the activities of human proteolytic digestive enzymes, resulting in a marked decrease in protein digestibility and loss of • 10% increase in protein extraction yield in the slurry after 1 min, but no significant changes at more than 5 min of treatment.
• Increase in the number of smaller particles (0.3-1.1 µm) in the slurry after 0.5 min (Preece et al., 2017b) High-pressure homogenization 100 MPa, 1-5 pass; Soy slurry, okara • 16% and 26% increases in protein extraction yield and reduction in particle sizes in slurry (80°C) and okara (50°C) after one pass • Increase in protein availability by 18%p and 30%p in the samples after one pass (Preece et al., 2017a) Enzyme treatments • Greater anti-radical activity than the control (Silva et al.,  • TI inactivation and increased digestibility to a similar extent to that of the conventional method (100°C for 30 min) in a shorter time (Vagadia et al., 2018) Non-thermal treatments • Time-dependent increase of in vitro protein digestibility from 77.35% (the untreated) to 84.03% at 16 min (Vanga et al., 2020) High-pressure homogenization 200 and 300 MPa; 55, 65, and 75°C Soymilk • Reduction in TI activity to around 37% of the initial activity, a similar extent to pasteurization (95°C, 30 sec) but a lesser extent than UHT treatment (142°C, 6 sec) that inhibited 68% of the initial activity • Increase in TI inhibition with elevating pressure and initial temperature (Manassero et al (Wang & Guo, 2016) (Continued ) • 40% of the initial activity remained after 7 uses, and more than 70% activity was measured after 40 days of storage  -Orozco et al., 2013). Due to their inhibitory action, protein digestibility had a high inverse association with TI activity (r = −0.815) (Zhong et al., 2015), expecting enhanced protein digestibility of soymilk through TI inactivation. It has been recommended that TI be eliminated by 90% to achieve maximum nutritional value (Kwok et al., 1993). However, 20.7-55.6% of the residual TI activity relative to that of the whole soybean was reported in commercial soymilk, implying the necessity for TI inactivation in soymilk (Xiao et al., 2012). Thermal treatments, such as UHT processing, hydrothermal cooking, and soybean blanching and roasting, are commonly used for TI inactivation (Vagadia et al., 2017). As soybean TI is relatively heat-stable due to the presence of disulfide bonds, heating at 100°C for 20 min is required to reduce its activity to 13% of that of raw soymilk (Yuan et al., 2008). However, the long processing time and high temperature needed to fully inactivate TI can degrade bioactive compounds and decrease amino acid availability (Huang et al., 2006;Parsons et al., 1992). Therefore, it is important to select appropriate processing conditions and methods for treating soymilk to balance the destruction of TI and retention of nutrients.
Microwave heating is an effective thermal method to inactivate TI in soymilk. Microwave treatment (2,450 MHz, 1,000 W) of soymilk at 100°C for 8 min reduced TI activity from 10% to 3% and enhanced in vitro protein digestibility from 80.5% to 87% in a much shorter time (8 min) than that of the conventional boiling method (30 min) (Vagadia et al., 2018). Microwave exposure for 2 min almost entirely inhibited TI activity in both soaked (99.1%) and dry beans (94.6%). Irradiation for 1 min showed potentiated inhibitory actions on soaked soybeans (86.6%) compared to dry beans (51.8%) (Kumar et al., 2019). Similarly, treatment with microwave (2,450 MHz) to soymilk decreased TI by 84% at 100°C for 10 min relative to untreated soymilk (p < .05). The protein digestibility of microwaved soymilk increased up to 93.03% at 85°C for 10 min compared to that of untreated soymilk (77.35%) (p < .05) (Vanga et al., 2020). In addition, with increasing microwave power (540-810 W) and temperature (70-90°C), the TI activity in soymilk decreased from 18% to 3%, while protein digestibility increased from 76.43% to 87.4% (Varghese & Pare, 2019). Ultrasound treatment to soymilk effectively decreased TI activity in a time-dependent manner (1-16 min) at 25 kHz and 400 W. A 52% reduction of TI activity was achieved with 16 min of treatment compared to that of the untreated (43.44 mg/g dry weight) (p < .05). The protein digestibility of soymilk treated with ultrasound improved with time, reaching 84.03% after 16 min of treatment compared to the lower value of the untreated (77.35%) (Vanga et al., 2020).
To achieve greater inactivation efficiency, heat treatment combined with high pressure or ultrasonic waves has been proposed. High-pressure homogenization (200 and 300 MPa) decreased TI activity to about 37% of the initial level under different initial temperatures (55, 65, and 75°C) (p < .05). However, the inactivation rate was lower than that of UHT (142°C for 6 s)-treated samples (p < .05) (Poliseli-Scopel et al., 2012). Recent molecular simulations have revealed that an increase in pressure from 300 Mpa to 600 Mpa for 5 ns induces compaction of soybean TI molecules, which will lead to complete unfolding under prolonged high pressure . In agreement with this, 96.7% of TI in CaCl 2 -fortified soymilk was inactivated at 600 MPa and 55°C (Manassero et al., 2016). The manothermosonication (MTS) technique employs ultrasonic waves (20 kHz) along with mild heat and pressure to synergistically inactivate TI. MTS treatment (65°C and 400 kPa) on soymilk had a 14.92 min D-value (time needed to inactivate 90% of TI), while the D-value of thermosonication (65°C and 100 kPa) was 30.16 min. Moreover, the TI inactivation rate of continuous MTS without temperature control (400 kPa) was greater than that of thermal treatment alone at 70°C, obtaining an overall D-value of 3.28 min (Chantapakul et al., 2020).
The dielectric-barrier discharge (DBD) plasma method has emerged as a novel non-thermal technology that minimizes heat-catalyzed alterations of food. DBD plasma is generated under atmospheric pressure through the electric discharging process between two electrodes separated by a dielectric layer (Sakudo et al., 2019). This method was reported to inactivate undesirable microorganisms and enzymes in food products, possibly due to reactive species in the plasma (Pankaj et al., 2013). The DBD plasma decreased soybean TI level by decreasing surface hydrophobicity and inducing breakage of disulfide bonds in a time-and inputdependent manner. Plasma treatment at 51.4 W for 210 s inactivated 95.6% of soybean Kunitz TI (p < .05). Similarly, soymilk exposure to plasma at 51.4 W for 21 min reduced TI activities by 86.1% and led to almost complete destruction of TI at 27 min (p < .05) (J. . Based on current findings, the application of DBD plasma to soymilk is an interesting topic for future investigation. Some natural compounds such as tea polyphenols and stevioside have recently been explored as candidate natural inhibitors of soymilk TI. Tea polyphenols and stevioside were reported to interact with amino acids near TI's reactive sites, impeding specific binding of trypsin to TI (Z. Chen et al., 2020;Liu et al., 2017. Epigallocatechin gallate (EGCG), the principal component of tea polyphenols, suppressed 75.3% of Kunitz TI at an EGCG/Kunitz TI ratio (w/w) of 40/1. Molecular docking analysis found that EGCG binding to Kunitz TI was driven mainly through both hydrophilic and hydrophobic interactions and hydrogen bonds with the amino acids near TI reactive sites, displaying a pattern of competitive inhibition . Also, EGCG and epigallocatechin (EGC) inhibited Bowman-Birk TI by 51% and 21%, respectively, at a TI ratio (w/w) of 20/1. The interaction involved hydrophobic contacts and hydrogen bonds between TI and the polyphenols (Z. Chen et al., 2020). Stevioside is a natural high-intensity sweetener used as a sugar alternative. Stevioside remarkably reduced the activity of Bowman-Birk TI by 81.6% at a TI ratio (w/w) of 20/1. However, it did not affect the action of Kunitz TI. Notably, replacing 30 g/L sucrose with 159 mg/L stevioside in soymilk successfully reduced TI activity to 49.9% without affecting the sweetness .

Phytate
Phytate is the principal storage form of phosphate and inositol in plants such as legumes. Raw soymilk extracted from different varieties contains 0.76-2.18 mg/g (dry basis) of phytate (Kim et al., 2010). The primary concern for dietary phytate is its interference with intestinal mineral absorption due to interactions with particular metal ions (Konietzny & Greiner, 2003). It can adversely affect protein digestibility and amino acid availability, presumably by interacting with proteins or mineral cofactors required for digestive enzyme activities (Gilani et al., 2005;Konietzny & Greiner, 2003). Reduction of phytate in soymilk improved protein digestibility and availability of calcium, iron, and zinc (I. K. Hwang & Lee, 1995). For these reasons, numerous studies are underway to establish effective strategies to remove phytates from soymilk.
Ultrafiltration methods also have been investigated to reduce the amount of phytate in soymilk. Soymilk circulated through an ultrafiltration membrane (<10 kDa) had 67.3% of the phytate of that of original soymilk (p < .05). The phytate concentration in soymilk decreased with the increasing extent of ultrafiltration (Wang & Guo, 2016). However, ultrafiltration was capable of removing only free phytate (<10 kDa) in soymilk, even though about one-third of phytic acids in heated soymilk are bound to the protein (R. Wang et al., 2018).
The addition of phytase catalyzes the hydrolysis of phytates. Treatment with 300 U/L of phytase to soymilk for 60 min completely removed all types of phytates (p < .05) (myo-inositol tris-, pentakis-, and hexakisphosphate) in commercial soymilk. Soymilk with added phytase had increased calcium, iron, and zinc solubility by up to 20.8%, 37.1%, and 67.4%, respectively, under in vitro digestion (p < .05) (Theodoropoulos et al., 2018). However, single use of free phytase can result in relatively high production costs and thus not be practical for commercial applications. Of late, immobilized enzymatic systems have been investigated, providing the possibility of continuous processing and multiple reuses of the enzymes. Application of immobilized phytase on glass microspheres to soymilk for 120 min significantly reduced phytate level from 11.5 mg to 0 mg (p < .05). On the contrary, the boiling method reduced about 25% of phytate, comparable to the free phytase system, which took 100 min for complete removal of phytate in soymilk. Additionally, the immobilized system can be reused for seven consecutive cycles and had relatively stable catalytic activity after 40 days of storage (K. I. Chen et al., 2018). The hydrolytic activity of chitosan-and calcium-alginate immobilized phytase systems was evaluated as 24% and 33%, separately, at 4 h of incubation in soymilk. The systems could be used repetitively for about eight cycles with chitosan immobilizer and six cycles with a calcium-alginate immobilizer (Sirin et al., 2017).

Lipoxygenase
Lipoxygenase (LOX), one of the causative enzymes of offflavor, catalyzes the oxidation of PUFAs in soybean (Baysal & Demirdöven, 2007). The unique flavors of soymilk, described as "beany, green/grassy, meaty/brothy, and astringent," are critical drivers deterring people from purchasing soymilk (Lawrence et al., 2016). It was found that LOX was present in steamed soymilk in a low amount of 1.15-1.75% of soymilk protein extracts (Stanojević et al., 2017).
Heat treatments, such as blanching, roasting, and hotgrinding (Lv et al., 2011;Navicha et al., 2017), and pH adjustment of soaking water (Ashraf & Snyder, 1981) are commonly used for LOX inactivation. However, overheating for complete inactivation of LOX can reduce protein solubility in soymilk (Araba & Dale, 1990). In addition, blanching and hot grinding at 80-100°C significantly affect non-beany flavor compounds (p < .05) (Lv et al., 2011). Accordingly, non-thermal processing methods for the inactivation of soymilk LOX are topics of great interest.
Several emerging non-thermal technologies, such as pulsed electric field (PEF) and pulsed light (PL), are reported to inactivate LOX. Previously, PEF that employs high-voltage pulses in a short period was reported to irreversibly inactivate LOX, presumably by changing its secondary and local tertiary structural conformation (Luo et al., 2010). The inhibition rate for LOX in soymilk reached about 88% under PEF exposure at 42 kV/cm and 25°C for 1,036 μs (p < .05) (Y. Q. Li et al., 2008). When combined with preheating (50°C), an 84.5% inactivation rate was attained with much shorter treatment time (100 μs) at 40 kV/cm (p < .05) (Riener et al., 2008). The degree of LOX inactivation in soymilk increased as PEF strength, time, and pulse frequency and width increased (Y. Q. Li et al., 2008;Riener et al., 2008). Additionally, PL, which utilizes intense pulses of light over a broad spectrum (1-1,100 nm) in a fraction of seconds (Oms-Oliu et al., 2010), completely inactivated LOX at a distance of 7 cm under 340.2 J/cm 2 for 90 s (p < .05). However, LOX inactivation by PL was mainly caused by PL-induced photo-thermal effects (temperature increment up to 71.7°C) and was not noticeably induced during a temperature-controlled PL treatment. It was thus suggested that PL might not provide significant advantages for soymilk LOX inactivation over conventional thermal treatment (Alhendi et al., 2017).
Decrease of residual off-flavor compounds jointly with partial LOX inactivation could be another approach to increase sensory palatability while minimizing heat-induced deterioration. The addition of β-cyclodextrin (0.5-1.0%) to soymilk with mild heating at 40 or 60°C significantly decreased beany flavor compounds, including hexanal, hexanol, and 1-octen-3-ol (p < .05). The inclusion of 0.75% βcyclodextrin in soymilk under heat treatment at 60°C obtained a lower score on beany flavor indices than that of the soymilk added with lower β-cyclodextrin concentration (0.25-0.5%) and unheated sample (p < .05) (Shi et al., 2017).

Fortification of soymilk with micronutrients and bioactive ingredients
Fortification, defined as deliberately adding one or more components to a particular food, is commonly used to improve nutritional quality and health-enhancing properties (Dary & Hurrell, 2006). Soymilk is generally fortified with certain minerals and vitamins (A, D, and B 12 ) (Combs & McClung, 2016). Thermal processing conditions during soymilk production can result in severe degradation or loss of micronutrients (Arcot et al., 2002;Huang et al., 2006). Prior to commercial fortification, the appropriate form of fortificant, its absorption efficacy, and its effects on final product stability should be considered (Dary & Hurrell, 2006). Calcium, a micronutrient of great nutritional importance, has a critical role in bone metabolism. Commonly used calcium fortificants for soymilk are calcium carbonate and tricalcium phosphate, containing relatively high calcium content (about 40%). Although calcium carbonate exhibited about one-sixth lower solubility in soymilk than did tricalcium phosphate, its bioavailability in soymilk was higher than that of tri-calcium phosphate (p < .05) and comparable to that of cow milk (Chaiwanon et al., 2000;Zhao et al., 2005). However, utilization of these agents as fortificants sometimes causes calcium precipitation, decrease in dispersion stability, and chalky mouthfeel in the final product (Chaiwanon et al., 2000;Pathomrungsiyounggul et al., 2013). Therefore, it is suggested to add chelating agents (e.g. potassium citrate and trisodium citrate) and stabilizing compounds (e.g. carrageenan) to avoid these undesirable changes (Pathomrungsiyounggul et al., 2010).
The bioavailability of minerals is primarily affected by individual nutritional status, nutrient interaction, and dietary inhibitors/enhancers in foods. Calcium is often co-fortified with vitamin D, since calcium absorption is mediated by a vitamin D-dependent mechanism and is affected by individual vitamin D status (Heaney et al., 2003;Pansu et al., 1983). Also, vitamins C and A are potent enhancers for iron absorption (García-Casal et al., 2003). Thus, the WHO/FAO guideline on fortification recommends adding vitamin C and iron at a molar ratio of 4:1 to high-phytate foods such as soymilk (Dary & Hurrell, 2006).
As an increasing number of consumers are becoming interested in food products that help them achieve wellbeing, fortification of soymilk with bioactive ingredients has gained attention throughout the soymilk industry. Bioactive ingredients are compounds that confer some physiological benefits when added to foods, such as phytochemicals, carotenoids, peptides, fibers, PUFAs, and plant sterols (Fernandes et al., 2019). Addition of bioactive ingredients to soymilk is expected to provide certain health benefits beyond the basic nutritive value of the soymilk itself.
Plants are abundant and contain many natural bioactive compounds. A wide array of botanical extracts and their bioactive compounds that impart health benefits has been identified, some of which have been further applied for soymilk enrichment. In a recent study, glutamate decarboxylase (GAD) isolated from germinated brown rice was incorporated into raw soymilk. GAD is an enzyme that converts L-glutamic acid to γ-aminobutyric acid (GABA), which functions as an inhibitory neurotransmitter in the nervous system and has a potent anti-hypertensive effect (Diana et al., 2014). Soymilk with added GAD (1.0%, v/v) had 1.23 times higher GABA content relative to the control soymilk (Pramai et al., 2019). Additionally, soymilk containing green coffee extracts indicated significantly high total phenolic content and antioxidative capacity with increasing concentration of phenolic contents in coffee extracts (0.25-1 mg/mL) (p < .05). When fortified with 1 mg of phenolics per 1 mL of soymilk, the in vitro digestibility of starch and protein of soymilk was increased by 19.8% and 17.9%, respectively, relative to unfortified soymilk (p < .05) (Sęczyk et al., 2017).
Soymilk served as a highly effective vehicle for phytosterol fortification for positively altering lipid profiles. Phytosterol, mainly present in plant oil, is reported to lower cholesterol levels because it interferes with intestinal absorption of cholesterols by competing for micellar uptake (Nissinen et al., 2002). Consumption of soymilk enriched with 2% plant sterol powder markedly lowered total cholesterol (TC), triglycerides, and LDL-c level in modestly hypercholesterolemic subjects (p < .05). Fortified soymilk effectively lowered TC by 8.7% and LDL-c by 12.6% to a greater extent than other fortified fruit bars, yogurt, and flavored milk (Penchalaraju et al., 2018). Similarly, 6-month administration of soymilk fortified with 2 g of plant sterols decreased TC, LDL-c, and non-HDL-c levels compared to those of the control group (p < .05) in Chinese elderly with moderate hypercholesterolemia (Dong et al., 2016).
However, some technical constraints remain to be solved when incorporating nutrients and bioactive ingredients into soymilk. Poor stability during processing and storage, low solubility, and bioavailability need to be addressed for optimal delivery. Nanoemulsion has been proposed as a novel delivery system for its properties in entrapping compounds in nano-sized lipid droplets (<200 nm) (Aswathanarayan & Vittal, 2019). Recently, corn oil-based nanoemulsion containing vitamin D3 significantly increased in vitro bioaccessibility by 3.94-fold relative to that of vitamin D3-containing coarse conventional emulsion (p < .05). It also upregulated in vivo serum 25-(OH)D3 level by 73% compared to that of the vehicle nanoemulsion (p < .05) (Kadappan et al., 2018). Also, vitamin D3-encapsulated nanoemulsion (<200 nm) was developed based on soybean oil, soy lecithin, Tween 20, and water was successfully incorporated into whole-fat milk. The vitamin D3 nanoemulsion remained stable in the milk for 10 days without phase separation (Golfomitsou et al., 2018). Not only for nutrients, nanoemulsion can be utilized as an effective carrier for bioactive ingredients in functional foods. Incorporation of turmeric extractcontaining nanoemulsion into milk protected curcuminoids from degradation by gastric acids and effectively released them during intestinal digestion, ensuring the controlled release of the bioactive compounds in the intestine (Park et al., 2019). Although it was pointed out that food-grade nanoemulsions made of edible ingredients can effectively be employed to fortify typical O/W emulsion food matrices, there are a limited number of studies regarding their application to soymilk. Therefore, nanoemulsion-based fortification of soymilk is a promising field for future research and industrial applications.

Bio-enrichment of soymilk with functional compounds and nutrients
Bio-enrichment with bioactive compounds and micronutrients occurs during soymilk fermentation. The suitability of soymilk as a culture media for the growth and biochemical activities of various microorganisms has been reported in earlier studies (Li et al., 2012). Certain lactic acid bacteria species grew well on raw soymilk without nutrient supplementation (Li et al., 2012). Lactic acid bacteria not only confer various physiological benefits to soymilk by degrading different substrates into more bioactive forms, but also increase nutritive values of fermented soymilk by producing micronutrients.
In the process of fermentation, various organic compounds are degraded into smaller and more bioactive forms by microbial enzymes, positively altering the nutritional profile of the food (Fukuda et al., 2017). β-Glucosidase, broadly distributed in microorganisms, hydrolyzes sugar-bound forms of isoflavones and other phenolic compounds into isoflavone aglycones and free phenolics (Madeira Junior et al., 2015;Pyo et al., 2005). The conversion of isoflavone glucosides into aglycones (daidzein, genistein, and glycitein) is required to obtain higher bioavailability and bioactivity of isoflavones in soymilk (Izumi et al., 2000;Kano et al., 2006). Since the levels of total phenolic compounds and isoflavone aglycones are positively correlated with the free radical scavenging capacity of soymilk, increasing the contents of these components through fermentation would provide the resultant soymilk with enhanced antioxidative and health-promoting effects . Therefore, application of β-glucosidase-producing bacteria (e.g. certain Lactobacillus species and Bacillus subtilis) and fungi (e.g. Rhizopus oligosporus) as a functional starter culture has been studied to enrich fermented soymilk with bioactive isoflavone aglycones and other free phenolic compounds (Table 3).
In addition to bioconversion of isoflavones and release of free phenolics, it has been shown that several lactic acid bacteria strains can produce vitamins. The major fermentative microbe, Streptococcus thermophilus, as well as the most common probiotic species, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus rhamnosus, are reported to generate several B-group vitamins during soymilk fermentation (Albuquerque et al., 2017;LeBlanc et al., 2011). Several reports indicate that vitamin-producing bacteria produce relatively Table 3. Recent findings on microorganisms that increase the contents of isoflavone derivatives, vitamins, and phenolic acids in soymilk during fermentation.
Tabla 3. Hallazgos recientes relacionados con microorganismos que aumentan el contenido de derivados de isoflavonas, vitaminas y ácidos fenólicos en la leche de soya durante la fermentación. A co-culture of the two bacterial strains was used for fermentation in natural soymilk and soymilk supplemented either with passion fruit by-products, fructooligosaccharides, or both as prebiotics. 4 The culture is mainly composed of Acetobacter and Lactobacillus bacterial species and yeast species (predominantly Pichia and Dekkera) (Tu et al., 2019) 1 Se utilizó como sustrato leche de soya suplementada con 2% de concentrado de proteína de suero.
high levels of riboflavin, folate, and cobalamin (2.332 mg/L (Ge et al., 2020), 837 μg/L (Albuquerque et al., 2017), and 3.951 μg/L (Bhushan et al., 2017), respectively) in soymilk. These levels are comparable or higher to those of whole milk (1.62 mg/L of riboflavin, 50 μg/L of folate, and 3.57 μg/L of cobalamin) (Combs & McClung, 2016). The exploitation of microbial functionality appears to be a more natural approach to produce soymilk with enhanced nutritive values, without undesirable side effects. However, the ability of microbial cultures to release and produce functional components varies considerably by strain. Good fermentation conditions and the selection of appropriate strains are necessary to maximize the nutritional quality of soymilk. The recent findings on fermenting microbes that are successfully employed to enrich soymilk with bioactive compounds (isoflavones, vitamins, and phenolic acids) are detailed in Table 3.

Future perspectives and conclusion
Soymilk is well recognized as a nutritional milk substitute for people with lactose digestion disorders and milk protein allergies. As consumers have become interested in plantbased diets and want to promote a healthy lifestyle through foods, soymilk expands and thrives in its own market in the plant-based food category, beyond the perception as a simple milk substitute. This review highlighted the recent research regarding various processing technologies that can improve the nutritional quality of soymilk, primarily focusing on increase in the contents of protein and bioactive compounds, inactivation of anti-nutritional factors, fortification with nutrients and functional compounds, and bio-enrichment. When producing soymilk, insoluble fiber, protein, and bioactive compounds remain in okara and fail to be incorporated into soymilk. Extraction is an essential process in preparing soymilk that delivers beneficial soluble components in soybean to soymilk, contributing to its high nutritional density and enhanced bioactivities, such as antioxidative capacity. Innovative technologies that aid the extraction process have been utilized to provide as high amounts as possible of nutrients and bioactive compounds into soymilk. These processes, including microwave, ultrasound, high-pressure homogenization, and enzymes, effectively increased the amounts of protein, isoflavones, and flavonoids in soymilk and improved their bioaccessibility and antioxidant activity.
Removal of anti-nutritional components, represented by TI, phytates, and LOX, is essential to maintain and improve nutritional quality. It enables soymilk to be consumed to its full potential as a nutritious drink. Furthermore, research on non-thermal processing techniques is currently underway to remove anti-nutritional factors and improve the overall nutritional quality of soymilk while minimizing thermal degradation of nutrients.
Fortification is a practical solution that compensates for deficient nutrients in soymilk and replenishes nutritional loss during processing. Functional soymilk that provides specific health benefits can be created by adding various bioactive ingredients such as phytosterols. With the development of a nano-sized delivery platform, such as nanoemulsion, nutrients and bioactive compounds can effectively be incorporated into the diverse food matrix. However, few pieces of research have been undertaken for its application to soymilk. Therefore, further investigation is necessary to assess the fortifying efficacy of nanoemulsion in soymilk.
As the last area of interest, bio-enrichment through fermentation has been investigated as a more natural and sustainable tool to improve the nutritional quality of soymilk. Since the nutritional profile of fermented soymilk varies greatly depending on the type of microorganisms and fermentative conditions, bio-enrichment of soymilk is a promising area of interest in terms of its versatility for improving organoleptic and nutritional attributes. Thus, progressive research to find novel nutrient-producing strains and optimal conditions for soymilk fermentation is required to provide more diverse and healthy options for soymilk consumers.
Although these technological advances seem to possess excellent potential prospects in future industrial applications, most of them, at present, are at a laboratory scale suitable for controllable experimental conditions rather than commercial use. Further research needs to be performed to develop cost-effective technologies that meet commercial objectives, to simplify the processing procedures suited for industrial operation, and to establish optimal processing conditions that yield consistent and desired outcomes at industrially relevant scales. This progress will allow consumers to purchase more cost-effective and nutritious soymilk, which ultimately leads to the growth of the global soymilk industry.