Mechanism of low-frequency and high-frequency ultrasound-induced inactivation of soy trypsin inhibitors

Highlights

• Both low- and high-frequency ultrasound can inactivate trypsin inhibitors.

• Increased ultrasonic power accelerated the inactivation process.

• Physical effects dominate over chemical effects for low-frequency ultrasound.

• Sonochemical effects dominate the initial inactivation for high-frequency ultrasound.

• Low frequency has profound performance at low power levels.

Abstract

In this study, the effect of ultrasonic frequency and power on the inactivation of soy trypsin inhibitors (TIs) was investigated to explore the ultrasound-induced inactivation mechanism. It was observed that 20 kHz and 355 kHz ultrasound have better inactivation efficiency than 1056 kHz. First-order rate constants for the inactivation process were obtained, which increased with increasing ultrasonic power at both 20 kHz and 355 kHz. For 20 kHz ultrasound, the formation of TI aggregates resulting from the physical effects of acoustic cavitation decreased the interactions between the active sites of TIs and trypsin, thus reducing the TI activity. For 355 kHz ultrasound, most of the methionine in the TIs was oxidised within 5 mins, resulting in a faster reduction of TI activity. Subsequent aggregation of TIs resulted in further TI inactivation. SDS-PAGE showed that neither disulphide bonds nor CC coupling were involved in the formation of aggregates.

Introduction

Soy products (e.g., soy protein isolate, soy concentrate, soy milk and soy flour) are rich sources of proteins, and have become popular among consumers due to their positive health effects (Chen, Xu, Zhang, Kong, & Hua, 2014). However, there are some anti-nutritional compounds in soy products, such as trypsin inhibitors (TIs), including Kunitz-type inhibitor (KTI) and Bowman-Birk-type inhibitor (BBI) (Vagadia, Vanga, & Raghavan, 2017). TIs are a group of serine (Ser) protease inhibitors that inhibit the activity of the digestive enzymes trypsin and chymotrypsin present in the gastrointestinal tract (Vagadia, et al., 2017). Trypsin hydrolyses proteins into small peptides or amino acids (<3 kDa) that can be absorbed into the bloodstream (Wagner, Gran, & Peppas, 2018; Yin et al., 2008). The inhibition of trypsin interferes with the hydrolysis of proteins, lowering the digestibility of soy protein and consequently impairing the uptake of amino acids (Vagadia, et al., 2017).

The high TI content in soy products and resulting low digestibility of soy protein is well established (Almeida et al., 2015, Vagadia et al., 2017). Almeida, et al. (2015) demonstrated that the digestibility of soy protein in soy protein isolate powder was 33% less than that of whey protein in whey protein isolate powder after in vitro gastrointestinal digestion for 3 h. A similar result was found by Santos-Hernández, et al. (2020), who compared the digestibility of different protein isolates. The protein from soybeans had the lowest digestibility (66.1%) amongst the tested isolates after in vitro gastrointestinal digestion, followed by that from lentil (73.7%), casein (74.4%), pea (74.8%) and whey protein (75.5%). Avilés‐Gaxiola, Chuck‐Hernández, and Serna Saldivar (2018) compared TI activity from 15 legumes and found that soybean had the highest TI activity (94.1 U/mg). Vagadia et al. (2017) illustrated that TI content could exceed 30 mg/g in soy products such as soy flour and soy protein isolate, indicating that soy products may have a natural disadvantage in terms of effective digestion. So far, some studies have shown that the inactivation of TIs may increase the digestibility of protein from soy products, by reducing the TIs ability to inhibit trypsin (Vagadia et al., 2018, Vanga et al., 2020). The digestibility of soy protein in raw soy milk increased by 7% when the active TI content decreased from 10% to 1% after microwaving (Vagadia, et al., 2018). Vanga, et al. (2020) also reported that TI inactivation could improve the digestibility of soy protein from 77% to 93% in soymilk.

Before considering possible modes of TI inactivation, it is important to understand their inhibitory mechanism. The KTI has 181 amino acids with a recognition site located at Arg 63-Ile 64 on a recognition loop (Sweet, Wright, Janin, Chothia, & Blow, 1974). The recognition site of a single KTI molecule can combine with a trypsin molecule via hydrogen bonding. Subsequently, other amino acid residues around the recognition site further interact with trypsin via hydrogen bonding, hydrophobic interactions or charge-transfer interactions (Sweet, et al., 1974). BBI consists of 71 amino acids with an active site at Lys 16 on the recognition loop (Koepke, Ermler, Warkentin, Wenzl, & Flecker, 2000). Similar to KTI, the active site of BBI can also interact with a trypsin molecule via hydrogen bonding with further interactions between amino acids around the active site of the trypsin molecule (Koepke, et al., 2000). These inhibitors interact with trypsin irreversibly and the inhibition of trypsin inhibitors is regarded as competitive inhibition (Green, 1953, Kunitz, 1947).

Overall, TI inactivation usually involves a change at the binding site of the TI, including structural break-down, redox reactions, or aggregation (chemical/physical) (Chen, et al., 2014; Koide & Ikenaka, 1973). For the structural break-down, the recognition sites of TIs can be destroyed by enzymatic hydrolysis (Kanekar, Joshi, Sarnaik, & Kelkar, 1992). As a consequence, the mechanism of binding between trypsin and the TI is prevented (Kanekar, et al., 1992). Redox-induced structural changes can alter the spatial positions of amino acids or cause the reduction of disulphide bonds, such as methionine oxidation of bovine trypsin inhibitor (Ripoll, Piela, Váasquez, Scheraga, & Bioinformatics, 1991). For the former, the distance and angle between the amino acids of TIs and those of trypsin are altered. For the latter, the breakage of SS bonds can accelerate the dissociation of the TI-trypsin complex, which decreases the inhibitory activity of the inhibitors (de Veer et al., 2015, Koide and Ikenaka 1973). Apart from the aforementioned inactivation mechanisms, the formation of protein aggregates can also be considered. The active sites can be buried inside aggregates, hence reducing the possibility of TI binding with trypsin (Koepke, et al., 2000).

Based on these mechanisms, some methods have been developed to inactivate TIs, including heat treatment (Chen, et al., 2014), microwaves (Vagadia, et al., 2017), fermentation (Song, Frías, Martinez-Villaluenga, Vidal-Valdeverde, & de Mejia, 2008), irradiation (Siddhuraju, Makkar, & Becker, 2002) and addition of an oxidant (Griffiths & Cooney, 2002) or reductant (Creighton, 1974). The most common method used is heat treatment. However, the high temperature may also destroy some essential nutrients (proteins, vitamins, etc.) in soy products (Koshiyama, Hamano, & Fukushima, 1981). Other technologies such as fermentation and microwave irradiation are either time-consuming, high-cost or high-energy processes (Vagadia, et al., 2017). In addition, fermentation can also cause unwanted loss of functional properties, such as gelling, viscoelasticity and water holding capacity (Nüchter, Ondruschka, Bonrath, & Gum, 2004). Therefore, it is vital to find a technology to inactivate TIs which is low-cost, energy-efficient and causes minimal negative effects on soy products.

As a non-thermal technology (low processing temperature with a short treatment time), ultrasound is an eco-friendly and easy-to-operate method that has been used to inhibit the activity of enzymes in foods such as lipoxygenase, peroxidase and pectin esterase (Zhang, Wang, Zeng, Han, & Brennan, 2019). Additionally, it has also been successfully used to improve the physico-chemical properties of soybean protein (Martínez-Velasco et al., 2018). Therefore, ultrasonic technology may be considered for inactivating TIs in soy products.

Based on the frequency, ultrasound can be classified into three types: low-frequency (power) ultrasound (20–100 kHz) (LFUS), high-frequency ultrasound (100 kHz-1 MHz) (HFUS) and diagnostic ultrasound (1–500 MHz) (Wu, Guo, Teh, & Hay, 2012). Up to now, low-frequency ultrasound has been successfully applied to inactivate TIs in soy milk (Rude et al., 1998, Vanga et al., 2020). Rude, et al. (1998) investigated the effect of parameters including temperature, ultrasonic amplitude and ultrasonic time via response surface methodology and found that the maximum decrease of TI activity in soy milk was achieved when treated at 80 °C and 18 W/mL (20 kHz) for 7 min. Vanga, et al. (2020) reported that the TI content in soymilk decreased by 52% after ultrasound treatment at 25 kHz and 13 W/mL for 16 min. However, ultrasound-induced inactivation of TIs is not well understood, especially with respect to the effect of ultrasound frequency.

It is known that low-frequency ultrasound (e.g. 20 kHz) generates strong physical forces, while higher frequency ultrasound (200 kHz–800 kHz) creates high radical yields by acoustic cavitation (Kentish & Ashokkumar, 2011). In comparison, acoustic cavitation and the associated chemical effects are less in the MHz range, with acoustic streaming effects dominant instead (Kentish & Ashokkumar, 2011). It has been reported that strong physical effects, such as shear forces and microstreaming, could alter the conformation of enzymes by disrupting van der Waals’ interactions or hydrogen bonding (Nadar, Rathod, & Biotechnology, 2017). In addition, the hydrogen and hydroxyl radicals produced by acoustic cavitation can chemically damage proteins, resulting in the loss of enzyme activity (Nadar, et al., 2017). Based on the fundamentals of acoustic cavitation and sonochemistry, it is reasonable to speculate different inactivation pathways, and therefore process efficiency, for low-frequency (LF) and high-frequency (HF) ultrasonication. However, until now a systematic investigation of the effect of ultrasonic frequency on the inactivation mechanism of TIs has not been reported.

The current study was focused on the effect of ultrasound frequency on the mechanism and efficiency of inactivation of soybean TIs. For this, three acoustic frequencies of ultrasound (20 kHz, 355 kHz and 1056 kHz) were applied to purified soybean TIs, with the extent of ultrasonicTI inactivation determined using trypsin digestion assays. A simple kinetic model was used to compare the inactivation efficiency at the above-mentioned frequencies and at varying acoustic power levels and sonication times. In addition, the tertiary structure, particle size, molecular weight and amino acid composition were further analysed in order to probe the TI inactivation mechanisms as a function of ultrasound frequency. Detailed mechanisms of ultrasound-induced TI inactivation have been developed for both low-frequency and high-frequency ultrasonication.