Riboflavin-loaded soy protein isolate cold gel treated with combination of high intensity ultrasound and high hydrostatic pressure: Gel structure, physicochemical properties and gastrointestinal digestion fate

Graphical abstract

Highlights

• •Riboflavin was encapsulated in HIU-HHP combined treated SPI cold gel.
• •HIU-HHP combination enhanced mechanical property and structural stability of gel.
• •Stronger gel network improved encapsulation and chemical stability of riboflavin.
• •HIU and HHP synergistically promoted controlled release of riboflavin by gel.
• •Protein structure change during digestion led to higher riboflavin bioaccessibility.

Abstract

Transglutaminase (TGase) was added to soy protein isolate (SPI) dispersion after the combination treatment of high intensity ultrasound (HIU) and high hydrostatic pressure (HHP) to catalyze the formation of cold gel, which was used to encapsulate riboflavin. The structure, physicochemical properties and in vitro digestion characteristics of riboflavin-loaded SPI cold gel were investigated. HIU-HHP combined treatment enhanced the strength, water retention, elastic property, thermal stability and protein denaturation degree of riboflavin-loaded SPI cold gels, and improved the gel network structure, resulting in a higher encapsulation efficiency of riboflavin and its chemical stability under heat and light treatment. HIU-HHP combined treatment reduced the erosion and swelling of SPI cold gel in simulated gastrointestinal fluid, and improved the sustained release effect of SPI gel on riboflavin by changing the digestion mode and rate of gel. In addition, HIU-HHP combined treated gels promoted the directional release of riboflavin in the simulated intestinal fluid, thereby improving its bioaccessibility, which was related to the secondary structure orderliness, tertiary conformation tightness and aggregation degree of protein during the gastrointestinal digestion. Therefore, HIU-HHP combined treatment technology had potential application value in improving the protection, sustained/controlled release and delivery of SPI cold gels for sensitive bioactive compounds.

1. Introduction

The functional nutrients or bioactive compounds in foods, such as vitamins, probiotics, and bioactive peptides, participate in many metabolic reactions and play a positive role in disease prevention and health maintenance. However, they are easily destroyed and degraded, due to their instability under processing conditions (heat, light and oxygen) and in the gastrointestinal environment (pH and digestive enzymes), resulting in a low bioavailability, which limits their application in food processing [1]. Soy protein isolate (SPI), as a high-quality protein resource, has high nutritional value and excellent gelation ability. The three-dimensional network structure formed by SPI gelation can encapsulate functional nutrients or bioactive compounds, especially the SPI cold gel formed at low temperature is more suitable as a colloidal delivery carrier for the protection and controlled release of sensitive compounds [2]. It has been found that the protein cold gels induced by calcium chloride [3] or glucono-δ-lactone [4] can resist the digestion by pepsin, which has a certain controlled release effect on vitamins. The transglutaminase (TGase)-induced protein cold gels have more covalent interactions (ε-(γ-glutaminyl) lysine isopeptide bonds) and show a greater application potential in the protection and delivery for bioactive compounds [5]. Moreover, TGase-induced cold gelation does not need to undergo the thermal denaturation of protein, which thus simplified the preparation process. However, TGase-induced SPI cold gels have low strength and weak structure, which cannot meet the application requirements for food processing and nutrient delivery.

Currently, high intensity ultrasound (HIU) and high hydrostatic pressure (HHP), as alternatives to thermal treatment, can be used for structure modification and function improvement of proteins, such as the enhancement of gelation properties. The influence of HIU (power of 10–1000 W/cm², frequency of 20–100 kHz) on the structural and functional properties of proteins is mainly related to cavitation, shear stress and turbulence [6]. The HIU treatment enhanced the viscosity and solubility of SPI dispersion and improved its fluid characteristics [7]. Zheng et al. [8] reported that HIU treatment of SPI reduced particle size and increased surface hydrophobicity, forming soluble aggregates, which resulted in a more uniform and denser gel network. Hu et al. [9] found that HIU enhanced the covalent cross-linking, hydrophobicity, and gel strength of TGase-induced SPI cold gels, which provided a sustained release effect on riboflavin. Zhao et al. [10] pointed out that ultrasound and heat treatment synergistically improved acid-induced SPI cold gels. HHP (0–1000 MPa), commonly using water as the pressure-transmitting medium, can cause protein denaturation and conformational changes, thus improving its functional properties [11]. The different degrees of protein denaturation induced by HHP resulted in structural unfolding as well as the reconstruction of secondary and tertiary structure, which formed more soluble aggregates [12]. He et al. [13] reported that HHP treatment improved the structure and heat-induced gelation properties of rapeseed protein, which might be related to higher denaturation and aggregation degree of protein. The HHP combined with salt [14] or TGase [15] enhanced the intermolecular interactions in sweet potato protein gels, which thereby improved their structural characteristics and mechanical properties. Our previous study reported the influence of combination of HIU and HHP under different conditions and sequence on TGase-induced SPI cold gel [16]. However, the reports on the effect of HIU-HHP combined treatment on the encapsulation and delivery of sensitive functional nutrients such as vitamins by SPI cold gels were scarce.

In this study, riboflavin (vitamin B₂) was used as a model for functional nutrients or bioactive compounds, which was encapsulated by TGase-induced SPI cold gels treated with HIU-HHP combination. The mechanical properties and physicochemical stability of riboflavin-loaded SPI cold gel were analyzed. The structural changes of SPI cold gel and its protection and controlled release effects on riboflavin after the simulated gastrointestinal digestion were investigated. The results provide theoretical basis and technical support for the application of high-performance SPI cold gel in the delivery of bioactive compounds and the development of innovative functional foods.

2. Materials and methods

2.1. Materials

Soy protein isolate (SPI) containing 90.6 % protein, TGase (Activa TI, 200 units of enzyme activity per gram of powdered preparation), pepsin (10,000 U/g), pancreatin (4,000 U/g) and riboflavin (purity of 98 %) were purchased from Yuanye Biotechnology Co. Ltd. (Shanghai, China). Bile salt and potassium bromide were provided from Sigma-Aldrich (St Louis, MO, USA). All reagents used were of analytical grade.

2.2. Preparation of riboflavin-loaded TGase-induced SPI cold gel treated with HIU-HHP combination

The HIU-HHP combined treatment of SPI was performed according to our previous report [16]. SPI was dispersed in 100 mL of 0.01 M phosphate buffer (pH 7.0) and stirred at room temperature for 2 h to obtain 8 % (w/v) protein dispersion. The SPI dispersion was treated using an ultrasound probe (JY92-IIN, Ningbo Scientz Biotechnology Co. Ltd, Ningbo, Zhejiang, China) at 20 Hz and 480 W for 30 min (pulse durations of 2 s on and 4 s off). The procedure was carried out in an ice bath with the sample temperature controlled in the range of 20–30 °C. The ultrasonic intensity at 480 W output power measured by calorimetry was about ∼ 130 W/cm². The SPI was treated with HHP after HIU treatment. The HHP treatment was performed using a high pressure processing device (BaoTou KeFa High Pressure Technology Co., Ltd., Baotou, Inner Mongolia, China). Water was used as the pressure transfer medium; the SPI dispersion was placed in a polyethylene bag for vacuum package heat sealing and then a pressure of 400 ± 15 MPa was applied. The pressure was increased to the target value at a speed of 3.5 MPa/s, held for 5 min, and then released within 5 s.

The riboflavin powder was added to the SPI dispersion treated by the combination of HIU and HHP to achieve riboflavin concentration of 0.32 %, and magnetically stirred for 1 h. The mixture after adding riboflavin was completely shielded from light to avoid degradation of riboflavin by light. Subsequently, 40 units/mL TGase solution (prepared as 2 g Activa TI in 10 mL 0.1 M phosphate buffer, pH 7.0) was added under rapid stirring for 10 s. The final concentration of TGase was 40 units/g protein. The mixture was incubated in a water bath at 37 °C for 3 h, forming riboflavin-loaded TGase-induced SPI cold gel. The unbound water and unencapsulated riboflavin on the gel surface were removed after overnight storage at 4 °C. The SPI cold gel treated by the combined application of HIU and HHP was denoted as HIU-HHP, while those treated by individual HIU and HHP were recorded as HIU and HHP, respectively. Untreated SPI cold gel was used as the control.

2.3. Characterization of riboflavin-loaded gel

The gel strength [17], water holding capacity (WHC) [10], encapsulation efficiency (EE) [7], rheological properties [18] and differential scanning calorimetry (DSC) [11] of the riboflavin-loaded gels were measured. The specific measuring methods were shown in Table 1.

Table 1.

Measuring methods for gel strength, water holding capacity, encapsulation efficiency, rheological properties and differential scanning calorimetry of riboflavin-loaded soy protein isolate cold gels treated with combination of high intensity ultrasound and high hydrostatic pressure.

Measuring items	Measuring methods	Reference
Gel strength	The gel samples were compressed to 50 % of their original height by the probe after placing at room temperature for 30 min. The compression speed was 1 mm/s and the trigger force was 5 g. The gel strength was defined as the maximum force used during compression.	[17]
WHC	The gel samples were centrifuged at 10,000 g for 20 min. Then, the residual liquid was removed with dry filter paper. The WHC was the percentage of water retained in the gel after centrifugation to total mass of water in the gel before centrifugation.	[10]
EE	The lyophilized gel powder was mixed with water to form a dispersion of 1 mg/mL, and 1 % (w/v) pancreatin was added. The mixture was then violently shaken at 37 °C for 4 h, and centrifuged at 10,000 g for 20 min. The absorbance of the supernatant was measured at 445 nm. The EE was the percentage of riboflavin encapsulated in gel to total riboflavin addition.	[7]
Rheological properties	SPI dispersions after the addition of TGase were placed between parallel plates with a diameter of 25 mm and a gap of 1 mm. The dispersions were incubated for 3 h at 37 °C with a strain of 0.05 % and a frequency of 1 Hz.	[18]
DSC	Approximately 5 mg of the lyophilized gel sample was placed in an aluminum pan, which was hermetically sealed for analysis. The temperature was raised from 20 to 200 °C at a heating rate of 10 °C/min with the nitrogen flow rate of 50 mL/min. An empty pan was used as a reference.	[11]

2.4. Chemical stability of riboflavin

The chemical stability of riboflavin in gel samples under heat and light was evaluated according to the method of Lv et al. [19]. The fresh gel samples were placed in sealed nitrogen-filled glass bottles, and incubated in dark at 55, 75 and 95 °C or under ultraviolet light with power of 15 W and wavelength of 254 nm at 25 °C for 6 h to assess the thermal and light stability of riboflavin. The samples were collected at specific time intervals and the remained content of riboflavin was determined according to the assay method of EE in Table 1. The retention ratio of riboflavin was calculated using the following equation:

$$\mathrm{R}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\left(\%\right)=\frac{{\mathrm{C}}_{\mathrm{r}}}{{\mathrm{C}}_{\mathrm{i}}}\times 100$$

where C_r is the remained content of riboflavin after incubation and C_i is the initial content of riboflavin before incubation.

2.5. Preparation of simulated gastrointestinal fluid

The simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the method described by Caillard et al. [20]. The preparation of SGF: 2 g sodium chloride and 7 mL 37 % (v/v) hydrochloric acid were added into 1000 mL deionized water, and the pH was adjusted to 1.2. The preparation of SIF: 6.8 g potassium dihydrogen phosphate was dissolved in 250 mL deionized water, and mixed with 190 mL 0.2 M sodium hydroxide. The final volume of mixture was fixed with deionized water to 1000 mL, and the pH was adjusted to 7.5.

2.6. Measurement of gel erosion

The erosion of riboflavin-loaded gels in SGF and SIF was measured by the amount of protein released in the dissolution medium without digestive enzymes, which can be characterized as protein loss according to the method of Hu et al. [7]. Approximately 30 mg lyophilized gel sample was immersed in 30 mL SGF and SIF without digestive enzymes, respectively. The mixture was incubated at 37 °C with constant shaking (100 rpm) for 6 h. The protein content in simulated gastrointestinal fluid was measured by the bicinchoninic acid (BCA) assay method at specific time intervals. The equation for calculating protein loss was as follows:

$$\mathrm{P}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s}\left(\%\right)=\frac{{\mathrm{C}}_{\mathrm{p}\mathrm{r}}}{{\mathrm{C}}_{\mathrm{p}\mathrm{i}}}\times 100$$

where C_pr is the content of protein released from gel and C_pi is the initial content of protein in lyophilized gel.

2.7. Measurement of swelling properties

The swelling properties of riboflavin-loaded gels in SGF and SIF were measured according to the method described by Matalanis et al. [21]. The gel swelling was measured using the same procedure as gel erosion. The gel sample after immersion was taken out from the simulated gastrointestinal fluid, and weighed accurately after removing excess liquid on the gel surface with dry filter paper. The equation for calculating swelling of gel was as follows:

$$\mathrm{S}\mathrm{w}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}(\%)=\frac{{\mathrm{M}}_{\mathrm{a}}-{\mathrm{M}}_{\mathrm{b}}}{{\mathrm{M}}_{\mathrm{b}}}\times 100$$

where M_a is the mass of gel after swelling and M_b is the mass of gel before swelling.

2.8. In vitro release of riboflavin

Separate release in SGF and SIF: The riboflavin-loaded gel samples were immersed in SGF (pH 1.2) and SIF (pH 7.5) without digestive enzyme, respectively, and incubated at 37 °C with constant shaking (100 rpm) for 6 h.

Continuous release during simulated gastrointestinal digestion: The riboflavin-loaded gel samples were immersed in SGF (pH 1.2) with 0.1 % (w/v) pepsin, and then incubated at 37 °C with constant shaking (100 rpm) for 2 h to simulate gastric digestion. Subsequently, gastric digest was mixed with SIF at volume ratio of 1:1. The pH was adjusted to 7.5 with 1 M NaOH, and 1 % (w/v) pancreatin and 0.5 % (w/v) bile salt were added. Then the mixture was incubated at the same temperature and shaking for an additional 4 h to simulate intestinal digestion.

For both separate and continuous release, the absorbance of simulated gastrointestinal fluid was measured at 445 nm at specific time intervals, and the released amount of riboflavin was obtained from the standard curve. The release ratio of riboflavin from the gel was calculated as follows:

$$\mathrm{R}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{o}\mathrm{f}\mathrm{r}\mathrm{i}\mathrm{b}\mathrm{o}\mathrm{f}\mathrm{l}\mathrm{a}\mathrm{v}\mathrm{i}\mathrm{n}(\%)=\frac{{\mathrm{A}}_{\mathrm{r}}}{{\mathrm{A}}_{\mathrm{e}}}\times 100$$

where A_r is the amount of riboflavin released and A_e is the amount of riboflavin encapsulated in gel.

2.9. Measurement of protein digestibility

The protein digestibility of riboflavin-loaded gel digests was measured according to the method reported by Zhou et al. [22] with slight modifications. The gastrointestinal digest (including undigested gel particles) obtained from section 2.8 was evenly mixed with 10 % (v/v) trichloroacetic acid (TCA) solution in equal volume to precipitate the proteins. The mixture was placed at 4 °C overnight and then centrifuged at 10,000 g for 10 min. The protein precipitate was dissolved in 1 M NaOH, and the protein content was measured by the BCA assay method. The protein digestibility was calculated as following equation:

$$\mathrm{P}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)=\frac{{{\mathrm{C}}_{\mathrm{u}}-\mathrm{C}}_{\mathrm{p}}}{{\mathrm{C}}_{\mathrm{u}}}\times 100$$

where C_u is the protein content in the undigested sample, and C_p is the protein content in the TCA-precipitate after digestion.

2.10. Measurement of riboflavin bioaccessibility

The bioactive compounds those dissolve in the micelle phase after digestion and can be absorbed by the small intestine are used to characterize their bioaccessibility. The bioaccessibility of bioactive compounds was measured according to the method of Li et al. [23] with slight modifications. The gastrointestinal digest obtained from section 2.8 was centrifuged at 10,000 g for 30 min. The supernatant (2 mL) was collected and assumed to be the micelle fraction in which the riboflavin was solubilized. The absorbance of supernatant was determined at 445 nm, and the content of riboflavin in the supernatant was obtained from the standard curve, which represented the riboflavin in the mixed micelles. The bioaccessibility of riboflavin in the gels after digestion was calculated as follows:

$$\mathrm{B}\mathrm{i}\mathrm{o}\mathrm{a}\mathrm{c}\mathrm{c}\mathrm{e}\mathrm{s}\mathrm{s}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{u}\mathrm{r}\mathrm{c}\mathrm{u}\mathrm{m}\mathrm{i}\mathrm{n}(\%)=\frac{{\mathrm{C}}_{\mathrm{m}}}{{\mathrm{C}}_{\mathrm{e}}}\times 100$$

where C_m is the content of riboflavin in the micelles after digestion and C_e is the content of riboflavin encapsulated in gel before digestion.

2.11. Structural characterization of protein in gel after digestion

The gastric and intestinal digests of riboflavin-loaded gel samples were freeze-dried. Fourier transform infrared spectroscopy (FTIR) of samples were acquired using a Vertex 70 infrared spectrometer (Bruker Company, Germany) in the wavenumber scanning range of 400–4000 cm⁻¹ at the resolution of 4 cm⁻¹ [24]. The samples were dispersed in phosphate buffer (0.01 M, pH 7.0) to obtain 0.5 mg/mL protein dispersions. An excitation wavelength of 280 nm was used, and the intrinsic fluorescence emission spectra were recorded over the wavelength range of 300–450 nm using a Hitachi F-2000 fluorescence spectrophotometer (Hitachi, Ltd, Tokyo, Japan) [25]. The particle sizes of samples were determined using a Mastersizer 2000 (Malvern Instruments Ltd., Worcestershire, UK) with 1.33 refractive index of water at 1800 rpm pump speed and 0.001 absorption parameter. The particle size distribution and volume-mean diameter (D₄₃) were obtained from the device software [26].

2.12. Statistical analysis

All measurements were performed in triplicate, and the data in figures and tables are expressed as mean ± standard deviation. Significant differences were defined by p < 0.05, which was determined by analysis of variance using SPSS software v. 25.0 (SPSS Inc., Chicago, IL). Graphs were plotted using Origin 2018 software (OriginLab Corporation, Northampton, USA).

3. Results and discussion

3.1. Gel strength and water holding capacity (WHC)

The gel strength and WHC of riboflavin-loaded SPI cold gels treated by HIIU-HHP combination are shown in Fig. 1A. Both individual HIU and HHP treatment enhanced the gel strength of riboflavin-loaded SPI cold gels, and HIU was more effective than HHP. The combination of HIU and HHP further increased the gel strength from 6.19 g to 40.03 g, which was increased by approximately 5.5 times. This might be related to the fact that HIU-HHP combined treatment promoted TGase-induced protein intermolecular covalent cross-linking to form a more compact three-dimensional network structure [7]. The individual HIU and HHP or combined treatment improved WHC of riboflavin-loaded SPI cold gels. The HIU-HHP treated sample had the highest WHC, which was not significantly different from the HIU treated sample. It was possible that HIU-HHP treatment promoted the protein-water interactions, which contributed to the binding of water to the gel matrix [27]. Moreover, higher gel strength and more compact network structure also facilitated the entrapment and retention of water in the gel.

Fig. 1.

Gel strength and water holding capacity (A), encapsulation efficiency (B), rheological properties (C) and thermal properties (D) of riboflavin-loaded soy protein isolate cold gels treated with combination of high intensity ultrasound and high hydrostatic pressure. Different lowercase or uppercase letters indicate that the results are significantly different (p < 0.05).

3.2. Encapsulation efficiency (EE) of riboflavin

The EE of riboflavin in HIU-HHP treated SPI cold gels is shown in Fig. 1B. The samples with different treatments all exhibited excellent encapsulation capacity for riboflavin. The EE of riboflavin in SPI cold gels approximately increased from 82 % to 89 % after individual HIU and HHP treatment. HIU-HHP combined treatment further increased EE to 92.51 %, indicating the synergistic effect of HIU and HHP in improving the encapsulation of SPI cold gels on riboflavin. It could be observed an obvious outflow of liquid for the inverted untreated gel, while a small amount of liquid can be observed for the individual HIU and HHP treated gel. However, there was almost no fluid exudation for HIU-HHP combined treated gel, suggesting more retention of liquid, prevention of dissolved riboflavin loss, and improvement of EE, which was also supported by the highest gel strength and WHC of gel treated by HIU-HHP combination (Fig. 1A).

3.3. Rheological properties of gel

The strength of gel network can be characterized by the rheological parameters such as storage modulus (G'), which reflected the elastic properties of gel [28]. Fig. 1C shows the effect of HIU-HHP treatment on G' of riboflavin-loaded SPI cold gels. The initial G' values of samples treated by individual HIU and HHP or their combination were lower than that of control (untreated sample). However, the G' of all treated samples rapidly increased with the prolongation of TGase cross-linking time, and the increasing rate of G' decreased over time, eventually reaching equilibrium, which suggested the continuous strengthening of network structure and gradual formation of stable and firm gels [24]. The final G' values of all treated samples were higher than that of control, which reached the maximum for HIU-HHP combined treated sample. This indicated that HIU-HHP synergistic effect could improve the elasticity and strength of gel as well as the compactness of network structure to the greatest extent, which was in agreement with the results of gel strength (Fig. 1A).

3.4. DSC of gel

The thermal properties of riboflavin-loaded SPI cold gels treated with HIU-HHP combination analyzed using DSC are shown in Fig. 1D. All samples exhibited similar DSC curves with a single endothermic peak. The peak temperature (T_p) represents the denaturation temperature of protein, reflecting its thermal stability [29]. Compared with the control (105.17 °C), the T_p of the samples treated with HIU, HHP, and their combination all increased, which reached the highest for the HIU-HHP combined treated gel (111.75 °C). This indicated HIU-HHP combined treatment enhanced denaturation temperature of protein, leading to the improvement of thermal and structural stability of gel, which might be attributed to a more compact tertiary conformation of protein [11]. The enthalpy change (ΔH) reflects the denaturation degree of protein during heating [30]. Compared with the control, the ΔH of all treated SPI cold gels decreased, which implied more stretched and disordered structure of SPI, leading to protein denaturation and a reduction in ΔH [22]. The ΔH of sample treated with HIU-HHP combination was the lowest, indicating the highest denaturation degree of protein. In addition, HIU-HHP combined treated sample might lose partial secondary structure such as α-helix, which also resulted in reduced ΔH [31]. Proteins with a higher denaturation degree exposed more intramolecular groups, promoting the intermolecular interactions such as hydrophobic interactions, disulfide bonds and TGase-induced covalent cross-linking, which thus improved the gel network structure.

3.5. Chemical stability of riboflavin in gel

The chemical stability of riboflavin in SPI cold gels treated with HIU-HHP combination under different heat treatment conditions is shown in Fig. 2A-C. The retention ratio of riboflavin showed a decreasing trend whether encapsulated or not with the extension of heat treatment time. After heat treatment at 55, 75 and 95 °C for 6 h, the retention ratios of free riboflavin were 41.82 %, 28.15 % and 9.99 %, respectively. This indicated that high temperature promoted the chemical degradation of riboflavin, reducing its chemical stability. The thermal stability of riboflavin encapsulated in SPI cold gels also showed similar changes, but their retention ratios increased to different degrees compared with free riboflavin. After heat treatment at all temperatures for 6 h, the riboflavin retention ratio of HIU-HHP combined treated sample was the highest, which reached 57.49 %, 39.49 % and 18.11 % at 55, 75 and 95 °C, respectively. This suggested that the HIU-HHP combined treatment improved the thermochemical stability of riboflavin in gels. A similar change in the light stability of riboflavin in SPI cold gels treated with HIU-HHP combination was also found in Fig. 2D. The degradation of free riboflavin was obvious with the extension of light time, but the riboflavin encapsulated in gel showed higher retention ratio. The retention ratio of free riboflavin decreased to 43.69 % after 6 h of light exposure, and the retention ratios of riboflavin encapsulated in gels were 60.78 % (control), 63.30 % (HIU), 59.89 % (HHP) and 68.09 % (HIU-HHP), respectively. The results showed that the HIU-HHP combined treated gel had the strongest ability to protect riboflavin from degradation by light, thereby improving its photochemical stability.

Fig. 2.

Chemical stability of riboflavin in SPI cold gels treated with combination of high intensity ultrasound and high hydrostatic pressure. (A) at 55 °C; (B) at 75 °C; (C) at 95 °C; (D) under light.

The higher chemical stability of riboflavin in HIU-HHP combined treated gel under heat and light was related to the higher strength and more stable structure of the gel network (Fig. 1). These gel matrices formed a thick and firm physical barrier, preventing the encapsulated riboflavin from contact with external environmental stresses, which effectively protected the riboflavin [32]. Lu et al. [33] reported similar result that the carriers with high stability could reduce the chemical degradation of bioactive compounds under adverse environments such as high temperature and light exposure. Overall, high temperature had a greater impact on the chemical stability of riboflavin than light exposure. The encapsulation effect of HIU-HHP combined treated gel provided a stronger protection for riboflavin, effectively slowing down the chemical degradation of riboflavin caused by environmental stresses such as heat and light, which thus improved its chemical stability.

3.6. Erosion and swelling of gel

The immersion of the gel matrix in a solvent can cause protein loss, leading to the dissolution of gel matrix, which reflects the erosion of the gel matrix by solvent. The erosion of riboflavin-loaded SPI cold gels treated with HIU-HHP combination in SGF (Fig. 3A) and SIF (Fig. 3B) without digestive enzymes is expressed as protein loss. In both SGF and SIF, the control (untreated gel sample) had the highest protein loss (approximately 9 %), indicating severe damage of the gel matrix, while all treatments reduced protein loss and gel erosion. HIU-HHP combined treated sample had the lowest protein loss (approximately 5 %) in SGF and SIF, suggesting the strongest resistance to destruction. HIU-HHP combined treatment synergistically improved physical stability of SPI cold gel by enhancing its resistance to the erosion by simulated gastrointestinal fluids, which protected the integrity of gel structure. These physical treatments promoted TGase-induced covalent cross-linking of protein, and caused protein to aggregate to form a stronger and more stable gel network structure, reducing protein loss due to the erosion of gel matrix [17], which was also corroborated by the analysis results of rheology (Fig. 1C) and DSC (Fig. 1D).

Fig. 3.

Erosion (A: SGF, B: SIF), swelling (C: SGF, D: SIF) and riboflavin release (E: SGF, F: SIF) of riboflavin-loaded SPI cold gels treated with combination of high intensity ultrasound and high hydrostatic pressure in simulated gastrointestinal fluids without digestive enzymes. SGF: simulated gastric fluid; SIF: simulated intestinal fluid.

Swelling is an important parameter of release carrier for bioactive compounds, reflecting the process of spontaneously absorbing solvent by carrier matrix, which has a significant influence and regulation on the diffusion and release of bioactive compounds in the matrix [34]. The spontaneous swelling of riboflavin-loaded SPI cold gels in SGF (Fig. 3C) and SIF (Fig. 3D) without addition of digestive enzymes might be related to the osmotic pressure and electrostatic repulsion within the gel [35]. The swelling of the control was rapid, and its network structure was easily damaged, thus the swelling data of the control was no longer collected after 30 min. All treated samples showed a lower swelling than the control. However, swelling data of treated samples was only collected within 240 min due to the collapse of the gel structure caused by prolonged immersion. All treated samples rapidly absorbed water and swelled within the first 60 min of SGF and the first 30 min of SIF, and the swelling rates decreased with the extension of immersion time, subsequently slowly reached equilibrium. The HIU treated sample exhibited a lower swelling compared with HHP treated sample, and the lowest swelling was observed in HIU-HHP combined treated sample. It was possible that an increase in TGase-induced covalent cross-linking of proteins resulted in a highly ordered and tight arrangement of protein aggregates within the gel network, thus reducing the rate of water molecules entering the gel network structure [36]. In addition, the higher non-polarity or hydrophobicity of TGase-induced SPI cold gel also decreased water absorption, which thereby lowered its swelling [7]. The high mechanical properties and structural stability (Fig. 1) of HIU-HHP combined treated riboflavin-loaded SPI cold gel could also hinder its water absorption and swelling, and delay its disintegration during prolonged swelling.

3.7. In vitro release of riboflavin in SGF and SIF without digestive enzymes

In the controlled release systems of protein gels, the change of environmental pH has a great influence on the release rate of molecules. The acidic and basic groups on the polypeptide chains will accept and release protons according to the change of environmental pH, leading to the water absorption and swelling or dehydration and contraction of the gel matrix, which effectively controlled the diffusion and release of bioactive compounds within the gel matrix [21]. The in vitro release ratio of riboflavin in SGF and SIF without digestive enzymes is shown in Fig. 3E-F. The riboflavin release from individual HIU and HHP treated gel decreased compared with the control, which was the lowest for HIU-HHP combined treated gel. Lower erosion (Fig. 3A-B) and swelling (Fig. 3C-D) of gel could delay the release of encapsulated riboflavin. Compared with the control, the release ratio of riboflavin from HIU-HHP combined treated sample after immersion in SGF and SIF for 6 h reduced from 66.92 % and 57.00 % to 30.21 % and 33.02 %, respectively. The HIU-HHP combined treated gel had a stronger and more stable network structure, enhancing its resistance to the erosion of SGF and SIF, which resulted in more retention of riboflavin in the gel [3]. Furthermore, a lower gel swelling reduced the substance exchange between digestive liquid and gel, which was more conducive to retention of riboflavin in the gel and delayed its release [7].

3.8. Protein digestibility of gel during gastrointestinal continuous digestion

The protein digestibility of the riboflavin-loaded SPI cold gels treated with HIU-HHP combination during simulated gastrointestinal continuous digestion is shown in Fig. 4A. The individual HIU and HHP or their combination all resulted in a reduced protein digestibility throughout the digestion process, which might be attributed to the formation of a more stable and compact gel network structure via TGase-induced covalent cross-linking of SPI enhanced by HIU-HHP treatment, hindering the permeation and diffusion of digestive enzymes in the gel [1]. On the other hand, the network structure could provide protection for the enzymolysis sites in the protein, which delayed the hydrolysis and digestion of the gel matrix by proteases [22]. At the initial stage of simulated gastric and intestinal digestion, the protein digestibility increased rapidly. After gastric digestion (2 h) and gastric + intestinal digestion (6 h), the protein digestibility for control, HIU, HHP and HIU-HHP treated samples were 48.71 %, 41.62 %, 47.55 %, and 39.22 %, as well as 97.32 %, 91.40 %, 96.37 %, and 89.82 %, respectively. It could be calculated that the net protein digestibility of different treated samples at the intestinal digestion phase were 48.61 %, 49.78 %, 48.82 %, and 50.60 %, respectively. The HIU-HHP combined treated gel had the strongest resistance to gastric digestion, reducing the protein digestibility in SGF,which provided a certain protection for riboflavin during the gastric digestion phase. However, it increased the protein digestibility at the intestinal digestion stage, promoting gel degradation in SIF, which was beneficial for the effective release of riboflavin in the intestine and its bio-utilization.

Fig. 4.

Protein digestibility (A), riboflavin release (B) and bioaccessibility (C) of riboflavin-loaded SPI cold gels treated with combination of high intensity ultrasound and high hydrostatic pressure during simulated gastrointestinal continuous digestion. The symbols SGF and SIF are the same as the legend of Fig. 3. The different letters indicate that the results are significantly different (p < 0.05).

3.9. In vitro release of riboflavin during gastrointestinal continuous digestion

Digestive enzymes can break down the protein matrix in the gels, thereby affecting the release of nutrients or bioactive compounds encapsulated in the gel network [14]. The release ratio of riboflavin from HIU-HHP combined treated SPI cold gels during simulated gastrointestinal continuous digestion is shown in Fig. 4B. The release of riboflavin from the gel gradually increased with the extension of digestion time. Whether at the gastric or intestinal digestion stage, the riboflavin release from sample treated with individual HIU and HHP was lower than the control throughout the digestion process. HIU-HHP combined treated sample had the lowest release amount and rate of riboflavin, showing a noticeable sustained release effect. HIU-HHP combined treatment reduced the erosion and swelling of SPI cold gel (Fig. 3A-D), decreasing the infiltrating amount of digestive fluids and the enzymolysis of digestive enzymes to the gel matrix (Fig. 4A), which consequently slowed down the release of riboflavin. Furthermore, the dense network structure formed by HIU-HHP combined treatment created a more tortuous pathway for riboflavin release [37], which also resulted in the slow release of riboflavin.

After gastric digestion (2 h) and gastric + intestinal digestion (6 h), the riboflavin release ratios for control, HIU, HHP and HIU-HHP treated samples were 74.42 %, 57.61 %, 61.45 % and 48.71 %, as well as 87.39 %, 79.56 %, 79.47 %, and 72.26 %, respectively. It could be calculated that the net release ratios of riboflavin for different treated samples at the intestinal digestion phase were 12.97 %, 21.95 %, 18.02 % and 23.55 %, respectively. Although the net release ratio of riboflavin at gastric digestion stage was higher than that at intestinal digestion stage, the HIU-HHP combined treated sample reduced the net release of riboflavin in SGF and increased its net release in SIF, suggesting the controlled directional release of encapsulated riboflavin in the intestine, which was related to the different digestion mode and protein digestibility of the gel during the gastrointestinal digestion.

3.10. Bioaccessibility of riboflavin

The protein gel matrix is destroyed by protease hydrolysis in the process of simulated gastrointestinal digestion, leading to the constant release of encapsulated bioactive compounds from the gel. The peptides generated from proteolysis, bile salts, and other substances can interact with released bioactive compounds, facilitating their micellization and the absorption by the small intestine [38], which can characterize their bioaccessibility. The bioaccessibility of riboflavin in HIU-HHP combined treated SPI cold gels after simulated gastrointestinal continuous digestion is shown in Fig. 4C. All treated gels significantly enhanced the bioaccessibility of riboflavin, especially the riboflavin in HIU-HHP combined treated sample exhibited the highest bioaccessibility, which was increased by 21.30 % compared with the control. This was mainly related to the protection for riboflavin by the gel structure and its higher directional release in the intestine (Fig. 4A-B). A higher bioaccessibility could be expected to result in a higher bioavailability, suggesting a higher absorption of nutrients or bioactive compounds by body [39]. In addition, the structural changes of protein in the gel during simulated gastrointestinal digestion might also affect the release and bioaccessibility of encapsulated riboflavin. Therefore, the protein structure in the samples after gastric and intestinal digestion will be analyzed next.

3.11. Protein structure in gel after gastrointestinal digestion

3.11.1. FTIR

The FTIR spectra of riboflavin-loaded SPI cold gels treated with HIU-HHP combination after gastrointestinal digestion are shown in Fig. 5A. In the wavenumber range of 3200–3700 cm⁻¹, gastric digests showed multiple small peaks (at 3551, 3474, and 3414 cm⁻¹), which might be related to hydrogen bonds due to the stretching vibrations of –OH [40]. The HIU-HHP combined treatment caused partial small peaks of the gastric digest to disappear, only leaving the absorption peak at 3414 cm⁻¹. All intestinal digests exhibited a broad peak in the –OH vibrational region, and the HIU-HHP combined treatment shifted this peak from 3414 cm⁻¹ to 3418 cm⁻¹, which suggested the possible disruption of hydrogen bonds in the gel. This was in agreement with the findings of Liu et al. [41], who found that a redshift in the position of –OH stretching vibrations correlated with a decrease in hydrogen bonds. The absorption peaks of 2800–3000 cm⁻¹ (at 2926 and 2854 cm⁻¹) were attributed to the stretching vibrations of C-H in the CH₃ and CH₂ groups of the polypeptide chains [42], which were moved to 2930 and 2866 cm⁻¹ by HIU-HHP combined treatment in the intestinal digestion phase, indicating an obvious change in the side chain structure of intestinal digests.

Fig. 5.

Fourier transform infrared spectroscopy (A), intrinsic fluorescence spectroscopy (B), particle size distribution (C) and average particle size (D) of riboflavin-loaded soy protein isolate cold gels treated with combination of high intensity ultrasound and high hydrostatic pressure after gastrointestinal digestion. The different letters indicate that the results are significantly different (p < 0.05).

The amide I band (1600–1700 cm⁻¹, C = O stretching vibration), amide II band (1480–1575 cm⁻¹, N-H bending vibration), and amide III band (1200–1300 cm⁻¹, C-N stretching vibration) are the characteristic amide absorption bands of protein in FTIR [43]. The gastrointestinal digestion did not change the absorption peak position of the amide I band (1639 cm⁻¹) except for the absorption peak of the intestinal digest treated with HIU-HHP combination shifting to 1641 cm⁻¹. The absorption peaks of amide II bands for all gastric digests were at 1543 cm⁻¹, shifting to 1547 or 1549 cm⁻¹ for intestinal digests. There were weak absorption peaks of amide III bands at 1234 cm⁻¹ for the gastric digests, which were disappeared after intestinal digestion (Red box in Fig. 5A). The changes in the characteristic amide bands suggested that the protein structures in the gels subjected to different treatments were relatively stable in the gastric digestion phase, while the intestinal digestion damaged the amide structures, especially for HIU-HHP combined treated sample. This view could be also supported by the appearance of small peak at 1377 cm⁻¹ in the intestinal digestion phase as well as the disappearance of absorption peak at 1742 cm⁻¹ and the shift of absorption peak position from 1022 cm⁻¹ to 1014 cm⁻¹ for intestinal digest treated with HIU-HHP combination. Moreover, the absorption peaks at 617 and 496 cm⁻¹ were shifted to 611 and 492 cm⁻¹, respectively, along with changes in peak intensities during the gastrointestinal digestion, suggesting an increase in the number of amino groups, which was also the result of protein digestion.

The amide I band in FTIR mainly reflects the secondary structure of proteins. Deconvolution and second-derivative fitting of the amide I band were performed to quantify the proportion of each secondary structure via peak area integration; the results are shown in Table 2. In the gastric digestion phase, all treated samples showed lower α-helix, β-turn, and random coil as well as higher β-sheet compared with the control. Notably, the most significant change in secondary structure was observed in HIU-HHP combined treated sample. The increase in β-sheet content enhanced the ordered structure of the proteins in the gastric digests, resulting in a more stable molecular structure [44]. Our previous study also found that a higher β-sheet structure contributed to the formation of a stronger and more stable gel network [45]. This might also be the reason for the reduction in protein digestibility (Fig. 4A) and riboflavin release ratio (Fig. 4B) of HIU-HHP combined treated sample in the gastric digestion phase. However, the influence of different treatments on the protein secondary structure of intestinal digests was opposite to that of gastric digests, which might be related to the different pH of simulated gastrointestinal fluid and different effect of gel network matrix structure on protein digestion. The reduction in β-sheet and the increase in random coil for the intestinal digests of HIU-HHP combined treated sample suggested that the protein hydrolysis caused structural unfolding, leading to more flexible and disordered protein molecules [28], which also confirmed higher protein digestibility (Fig. 4A), riboflavin release ratio (Fig. 4B) and bioaccessibility (Fig. 4C) of HIU-HHP combined treated sample at the stage of intestinal digestion.

Table 2.

Secondary structure contents of riboflavin-loaded soy protein isolate cold gels treated with combination of high intensity ultrasound and high hydrostatic pressure after gastrointestinal digestion.

Samples		α-helix (%)	β-sheet (%)	β-turn (%)	Random coil (%)
Gastric digest	Control	22.14 ± 0.09e	36.28 ± 0.48b	19.42 ± 0.46e	22.16 ± 0.39e
	HIU	18.73 ± 0.12b	49.50 ± 1.32ef	13.31 ± 0.30b	18.46 ± 0.46b
	HHP	20.04 ± 0.35c	40.55 ± 0.68c	18.39 ± 0.38d	21.02 ± 0.23d
	HIU-HHP	18.26 ± 1.10b	51.77 ± 1.09f	12.38 ± 0.44a	17.59 ± 0.19a
Intestinal digest	Control	13.39 ± 0.50a	55.42 ± 0.98 g	13.27 ± 0.83ab	17.92 ± 0.45ab
	HIU	18.11 ± 1.88b	42.08 ± 0.42d	16.16 ± 0.69c	23.65 ± 0.67f
	HHP	17.14 ± 1.45b	49.23 ± 0.36e	14.41 ± 0.99b	19.22 ± 0.08c
	HIU-HHP	21.25 ± 0.36d	32.29 ± 0.57a	19.30 ± 0.40e	27.16 ± 0.58 g

The values with different letters in the same column are significantly different (p < 0.05).

3.11.2. Intrinsic fluorescence spectroscopy

The intrinsic fluorescence absorption of proteins is primarily attributed to tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) residues, and Trp residues are more sensitive to solvent polarity. Hence, the fluorescence quantum yield of Trp can be used to identify conformational changes in proteins [8]. The intrinsic fluorescence spectra of riboflavin-loaded SPI cold gels subjected to HIU-HHP combined treatment after gastrointestinal digestion are shown in Fig. 5B. The fluorescence emission maximum wavelength (λ_max) for all samples was near 349 nm, which was not shifted significantly. The λ_max above 330 nm indicated that Trp residues were distributed in a polar microenvironment close to the protein molecular surface [46]. The fluorescence intensities of all gastric digests were higher than those of intestinal digests, which was in agreement with the findings of Liao et al. [47]. All treatments reduced the fluorescence intensity of gastric digests, but increased that of intestinal digests, especially HIU-HHP combined treated samples showed the most obvious changes. The decrease/increase in fluorescence intensity could be related to the embedding/exposure of the chromophorous amino acid residues as well as the hydrophobic/hydrophilic and non-polar/polar microenvironments in which the Trp residues were located, which indicated a tight/loose tertiary conformation of the protein [48]. Moreover, the increase in fluorescence intensity at the intestinal digestion stage might also be associated with the structural unfolding and exposure of protein molecules due to the reduction of hydrogen bonds (Fig. 5A). The HIU-HHP combined treated gel exhibited a more compact structure in the gastric digestion phase and enhanced its resistance to gastric digestion, allowing more riboflavin to retain within the gel, which provided the effective protection for encapsulated riboflavin. However, the gel structure became more relaxed and exposed in the intestinal digestion phase, ultimately leading to more protein degradation and directional release of riboflavin, which improved bioaccessibility (Fig. 4).

3.11.3. Particle size distribution

The particle size distribution and average particle size of riboflavin-loaded SPI cold gels treated with HIU-HHP combination after gastrointestinal digestion are shown in Fig. 5C-D. All the gastric digests exhibited the bimodal particle size distribution in the range of 100–10000 nm, while the intestinal digests showed the unimodal particle size distribution. At the intestinal digestion stage, the particle size distribution of the control did not change. However, the particle sizes of samples treated with HIU, HHP and their combination were distributed near 10 nm, 1000 nm and 1–10 nm, respectively, and the particle size distribution peaks became narrow obviously. This suggested that the further protein degradation due to gastrointestinal continuous digestion resulted in a more uniform particle size distribution of intestinal digests than gastric digests [49]. In the gastric digestion phase, large average particle size (D₄₃) of the samples indicated a high aggregation degree of protein in SGF, which prevented riboflavin from escaping and degrading. Compared with the gastric digests, the intestinal digests had lower D₄₃ with the particle size distribution shifting towards smaller particle sizes, suggesting the gradual decomposition of large particles into small particles by protease during gastrointestinal continuous digestion, which facilitated the release of riboflavin in SIF. Furthermore, the difference in D₄₃ between the gastric and intestinal digests of all treated samples increased compared with the control, especially the HIU-HHP combined treated sample. In the process of gastrointestinal digestion, the D₄₃ of the control and HIU-HHP combined treated sample decreased from 1408 to 1250 nm and from 2472 to 703 nm, respectively. The higher particle size of gastric digests indicated that the protein particles formed larger aggregates and had a stronger ability to resist gastric digestion. On the contrary, the reduction in particle size of intestinal digests enhanced the interactions between protein and solvent in SIF, promoting the depolymerization and enzymolysis of protein as well as the directional release of riboflavin [2], which also supported the sustained/controlled release function of HIU-HHP combined treated gel and higher bioaccessibility of riboflavin (Fig. 4).

4. Conclusions

The individual HIU and HHP treatment increased the gel strength, WHC and G' of riboflavin-loaded TGase-induced SPI cold gels, which indicated stronger mechanical properties of gels. The HIU-HHP combined treated gel displayed the highest mechanical properties, resulting in the maximum EE of riboflavin (92.51 %). The thermal and structural stability of gel were enhanced by HIU-HHP combined treatment, preventing riboflavin against partial chemical degradation caused by heat and light, which thus improved its chemical stability. Furthermore, HIU-HHP combined treatment reduced the erosion and swelling of gel, leading to the lowest protein digestibility and slowest riboflavin release rate during the simulated digestion process; but it increased the net release of riboflavin at the intestinal digestion stage. The sustained/controlled release effect of the gel on riboflavin was also closely related to the structural changes of protein during the gastrointestinal digestion. The HIU-HHP combined treated gel exhibited a more ordered secondary structure, tighter tertiary conformation and greater aggregation degree of protein in the gastric digestion phase, resulting in a stronger ability to resist gastric digestion, which provided more effective protection for encapsulated riboflavin. However, at the intestinal digestion stage, protein showed a more disordered and flexible secondary structure, less compact and more exposed tertiary conformation, and lower aggregation degree, enhancing the digestion of protein and the disintegration of gel, which promoted the directional release of riboflavin. The HIU-HHP combined treatment improved the sustained/controlled release of riboflavin by gel, resulting in the highest bioaccessibility of riboflavin, which was increased by 21.30 % compared with the control. The results indicated that the synergistic effect of HIU and HHP could strengthen the structure of SPI cold gel, which improved its functions of encapsulation, protection, sustained/controlled release and delivery for riboflavin.

CRediT authorship contribution statement

Yuxuan Mao: Writing – original draft. Xinqi Li: Methodology, Conceptualization. Qi Qi: Visualization, Software. Fang Wang: Investigation. Hao Zhang: Resources. Yuzhu Wu: Supervision. Jingsheng Liu: Validation, Funding acquisition. Chengbin Zhao: Writing – review & editing, Formal analysis. Xiuying Xu: Data curation, Project administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.