
==== Front
Ultrason Sonochem
Ultrason Sonochem
Ultrasonics Sonochemistry
1350-4177
1873-2828
Elsevier

S1350-4177(24)00285-2
10.1016/j.ultsonch.2024.107037
107037
Original Research Article
Unveiling the transformative influence of sonochemistry on formation of whey protein isolate and green tea extract (WPI-GTE) conjugates
Chinarak Khanittha a
Wongnen Chantira a
Chaijan Manat a
Tamman Arlee b
Donlao Natthawuddhi c
Cheong Ling-Zhi d
Worawan Panpipat pworawan@wu.ac.th
a⁎
a Food Technology and Innovation Research Center of Excellence, Department of Food Industry, School of Agricultural Technology and Food Industry, Walailak University, Nakhon Si Thammarat 80160, Thailand
b Thailand Institute of Nuclear Technology (Public Organization), Saimoon, Ongkarak District, Nakhon Nayok 26120, Thailand
c Food Science and Technology Program, School of Agro-Industry, Mae Fah Luang University, Chiang Rai 57100, Thailand
d School of Agriculture, Food and Ecosystem, University of Melbourne, Parkville, VIC 3010, Australia
⁎ Corresponding author at: Department of Food Industry, School of Agricultural Technology and Food Industry, Walailak University, Thasala, Thailand. pworawan@wu.ac.th
19 8 2024
11 2024
19 8 2024
110 10703726 6 2024
28 7 2024
18 8 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Graphical abstract

This study investigated the formation of conjugates between whey protein isolate (WPI) and green tea extract (GTE) using three methods: redox-pair (R), ultrasound-assisted redox-pair (RU), and ultrasonication (UL). Ultrasonication significantly reduced the reaction time for synthesizing WPI-GTE conjugates compared to the standard R method (p < 0.05). The UL methods had the highest conjugate yield determined by polyphenol binding (p < 0.05). Fourier-transform infrared spectroscopy (FTIR) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the conjugate formation, indicating an increased molecular weight due to protein binding with polyphenols through covalent and non-covalent bonds. Conjugates produced via ultrasonication exhibited enhanced solubility, smaller particle size, better emulsifying capacity, and improved foaming ability compared to those formed using the traditional R method (p < 0.05). However, conjugates from the R method showed higher antioxidant activity, as evidenced by DPPH•and ABTS•+ scavenging activities (p < 0.05). In conclusion, WPI-GTE conjugates created through ultrasonic treatment demonstrate potential as dual-functional ingredients, serving as both antioxidant and emulsifier.

Keywords

Whey protein isolate (WPI)
Green tea extract (GTE)
WPI-GTE conjugates
Redox-pair
Ultrasound treatment
Functional characteristics
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pmc1 Introduction

With the rapid growth of the global population, the demand for healthcare and functional food products has surged [1]. Consequently, the food industry is increasingly focusing on developing innovative functional foods and enhancing their bioavailability [2]. Whey protein isolates (WPI), derived from the whey portion of milk, include globular proteins such as lactoferrin (LF), β-lactoglobulin (β-LG), α-lactalbumin (α-LA), and bovine serum albumin (BSA). WPI offers superior nutritional value and easier digestion compared to other animal-based proteins [1]. It is widely used in the food industry for its functional properties, serving as emulsifying, foaming, gelling, structuring, or encapsulating agents [3]. However, beneficial properties of WPI can be compromised by physical parameter changes (pH, ionic strength, and temperature) during production and storage, leading to structural alterations that reduce its biological activity and health benefits [3], [4]. Various physical and chemical modification techniques have been explored to address these challenges and enhance functionality of WPI. One promising approach is the incorporation of functional active compounds, such as polyphenols, to form active composite products [1], [5], [6], [7], [8], [9]. The binding of polyphenols like quercetin [4], [10], gallic acid, protocatechuic acid [7], rosmarinic acid [11], and rutin [12] has been shown to enhance the functional and antioxidative properties of WPI. However, forming WPI-phenolic complexes with mixed phenolics is more cost-effective and economically feasible than using pure phenolics. Green tea extract (GTE), rich in polyphenols such as catechins (epicatechin, gallocatechin gallate, epigallocatechin, and epigallocatechin gallate), is noted for its antioxidant properties, contributing to anticancer, antidiabetic, antimutagenic, antibacterial, and neuroprotective activities [13], [14]. Due to its bioactive phenolic content and exceptional biological activity, conjugating green tea extract GTE with WPI presents significant interest for enhancing the functionality of native WPI.

Traditionally, protein–polyphenol covalent complexes have been prepared using the redox-pair method [2], [4], [15], [16]. These interactions can be either covalent (conjugates) or non-covalent (complexes), with covalent conjugates forming through the oxidation of polyphenols and subsequent reaction with protein nucleophilic groups [17]. Ultrasonication, a non-thermal processing technology, is gaining attraction in the food industry for its efficiency, cost-effectiveness, and ability to enhance food processing [18]. Ultrasound-induced mechanical, cavitation, turbulence, and microstreaming effects can unfold protein structures and expose reactive groups, facilitating protein conjugation with other molecules [2], [4], [15]. Studies have shown that combining ultrasonication with free radical methods significantly increases protein-bound polyphenol content and improves antioxidant properties [15], [16]. However, research on ultrasonication's impact on protein–polyphenol covalent complexes remains limited. The employing of ultrasonication may reduce the reaction time required, therefore enhancing conjugation efficacy and potential applicability.

This study aimed at investigating the effects of traditional free radical (redox-pair), ultrasound-assisted free radical, and ultrasonic-induced grafting methods on the structure, functional properties, and antioxidant activities of WPI-GTE conjugates. The findings shed light on the potential applications of protein–polyphenol complexes as a functional food ingredient.

2 Materials and methods

2.1 Materials and chemical reagents

Whey protein isolate (WPI) was purchased from Krungthepchemi Co., Ltd (Bangkok, Thailand). According to the product data sheet, WPI is made up of 88.9 % crude protein, 3.4 % crude carbohydrate, and 0.8 % crude fat, as well as minerals such as calcium (415 mg/100 g), sodium (249 mg/100 g), potassium (551 mg/100 g), phosphorus (238 mg/100 g), and magnesium (80 mg/100 g). Green tea leaves were provided from Royal Project Shop (Chiang Mai, Thailand). Dialysis bag (14 kDa molecular weight cut off) was purchased from Yuanye Reagent Co. (Shanghai, China). Precision Plus Protein™ Dual Color Standards was purchased from Bio-Rad Laboratories Ltd. (California, USA). Chemicals required for SDS-PAGE, Folin & Ciocalteu′s phenol reagent and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS) were purchased from Sigma Aldrich (St. Louis, Mo, USA). All other chemicals were of analysis grade and purchased from Merck Ltd, (Darmstadt, Germany).

2.2 Preparation of green tea extract (GTE)

The hot water extraction of green tea was utilized to make an aqueous extract, which was connected to general consumption of green tea with safety concerns due to the absence of an organic solvent. Ultrasonication was also used to improve extraction efficiency [14]. The ground green tea leaves were extracted using a slightly modified method described by Mujtaba et al. [13]. To prepare the extract, 10 g of ground green tea leaves were mixed with 150 mL of 80 °C distilled water. The suspension was ultrasonically sonicated for 20 min at 45 °C using a 400 W probe (Sonics & Materials Inc., USA) with an amplitude of 40 % and 5 on/off cycles. GTE was centrifuged at 3,600 rpm for 10 min at 25 °C, then filtered through Whatman filter paper No. 1. The total extractable phenolic content of crude GTE was 82.19 mg/mL, as assessed by the Folin-Ciocalteu test [12]. The extract was stored in sterilized brown bottles and refrigerated at 4 °C until use.

2.3 Fabrication of the WPI-GTE conjugates

The WPI-GTE conjugates were prepared using ultrasound-assisted free radical treatment as described by Baba et al. [4] with minor modifications. WPI powder was dissolved in deionized water and hydrated overnight at 4 °C with continuous stirring at 200 rpm. WPI-GTE conjugates were produced using three different methods: the redox-pair (R) method, the ultrasound-assisted redox-pair (RU) method, and the ultrasound method, as detailed.

2.3.1 Redox-pair (R) WPI-GTE conjugates

The R system was created by combining WPI solution at a final concentration of 4 % (w/v) with ascorbic acid (1 g) and H2O2 (4 mL, 50 mM), as a radical initiator. The mixture was stirred for 2 h at 25 °C before GTE was added to reach a final concentration of 60 mg/mL. The reaction mixture was continuously stirred at room temperature (27–29 °C) for 24 h, forming a redox-pair WPI-GTE conjugate (R).

2.3.2 Ultrasound-assisted redox-pair (RU) WPI-GTE conjugates

The reaction mixture was prepared in the same way as the R method described above, but instead of stirring for 24 h, it was immediately ultrasonicated for 60 (RU60), 90 (RU90), and 120 (RU120) min. Ultrasonication (Sonics & Materials Inc., USA) was performed using a tapered Microtip titanium probe (13 mm) in pulsed mode with a 5 s off cycle and an amplitude of 40 %, resulting in a power of 400 W. During the ultrasonication process, the solution (100 mL) was immersed in an ice bath to maintain a temperature below 30 °C.

2.3.3 Ultrasonication (UL) WPI-GTE conjugates

Ultrasonication was used to synthesize WPI-GTE conjugates (without redox pair). The GTE was added to the WPI solution at the specified concentration and ultrasonically treated for 60 (UL60), 90 (UL90), and 120 (UL120) min, using the same ultrasound conditions as described above for RU method. Untreated WPI solution (4 %, w/v) was used as a control.

To remove unreacted GTE, all WPI-GTE conjugates (R, RU60, RU90, RU120, UL60, UL90, and UL120) were dialyzed in a dialysis bag (14 kDa molecular weight cut off) for 48 h at 4 °C with 8 changes in deionized water. The WPI-GTE conjugates were then lyophilized and stored for further examination.

2.4 Characterization of WPI-GTE conjugates

2.4.1 Determination of degree of polyphenol binding

Polyphenol binding capacity was determined using the method of Guo et al. [12]. The WPI-GTE conjugate sample was dialyzed in purified water for 12 h at 4 °C. The Folin-Ciocalteu assay was carried out with extra fluid from dialysis. In brief, 300 µL of each sample was added to 1.5 mL of 10 % Folin-Ciocalteu reagent. Next, 1.2 mL of sodium carbonate solution (7 %, w/v) was added and thoroughly mixed. The mixtures were incubated in the dark for 30 min before being measured at 760 nm with a UV spectrophotometer (UV1800; Shimadzu, Japan). To determine the phenolic content of a sample, a standard curve (y = 0.0029x-0.0234, R2 = 0.9988) was created by varying the concentration of green tea standard. The total GTE content was calculated as milligrams of GTE per gram (mg GTE/g). The binding capacity of phenols was determined as follows:(1) Degreeofphenolicbinding%=totalphenoliccontent-phenoliccontentoutsidethedialysatetotalphenoliccontent×100

2.4.2 Determination of free sulfhydryl (SH) content

The free sulfhydryl content of proteins was determined using Ellman's reagent (DTNB). The samples (4 mg/mL) were mixed into 2.5 mL of Tris-glycine buffer (0.086 M Tris, 0.09 M glycine, 0.004 M EDTA, pH 8.0). Then, 0.02 mL of DTNB (4 mg/mL dissolved in Tris-glycine buffer) was added to the mixture and incubated at room temperature for 15 min before measuring absorbance at 412 nm. The free sulfhydryl content (μmol SH/g) was calculated as follows:(2) δ(μmol/g)=106×A412×D1.36×104×C

where δ is the free sulfhydryl content (μmol/g protein), A412 is the measured absorbance value, C is the protein concentration (mg/mL), and D is the dilution multiple and 1.36 × 104 is the molar absorbance coefficient [12].

2.4.3 Determination of particle size and zeta potential

The average particle size of native WPI and WPI-GTE conjugates were measured at 25 °C using dynamic light scattering (DLS) (NANO-ZS ZEN 3600, Malvern, UK) with a He/Ne laser (λ = 633 nm) and scattering angle 90°. To avoid multiple scattering, the sample (0.01 mL) was diluted with distilled water (0.99 mL). The zeta potential was also measured with the same device. Each measurement was taken four times.

2.4.4 Determination of ultraviolet absorption (UV) spectra

The UV absorption spectra of native WPI and WPI-GTE conjugates were recorded using a UV spectrophotometer (UV1800, Shimadzu, Japan) with a diluted sample solution in a 1.0 cm quartz cuvette. The wavelength range was 200 to 500 nm at 25 ℃.

2.4.5 Determination of Fourier-transform infrared (FTIR) spectra

The FTIR spectra of WPI and WPI-GTE conjugates were recorded using an ATR-FTIR spectrophotometer (Bruker Co., Ettlingen, Germany), as described by Guo et al. [12]. Freeze-dried samples were placed on the ATR crystal and the spectra was collected in the wave number ranges of 530–4,000 cm−1 with 32 scans and a resolution of 4 cm−1. The data were examined with the program OPUS 8.5 (Bruker Optik GmbH 2020, Ettlingen, Germany).

2.4.6 Determination of protein pattern by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to investigate the protein patterns in all samples [19]. A 5 % SDS solution (90 °C) was used to solubilize the samples. Protein content was analyzed by the Biuret method and then adjusted to 20 mg/mL before being mixed with sample buffer under reducing condition. The samples were boiled at 85 °C for 5 min before being loaded into stacking and separating gels made of 4 % (w/v) and 12 % (w/v) acrylamide, respectively. Then electrophoresis was performed (Mini Protein II unit; Bio-Rad Laboratories, Inc., Richmond, CA, USA). Precision Plus Protein™ Dual Color Standards were used as standard markers. The gel was stained for 30 min with Coomassie Brilliant Blue R250, then discolored for 24 h with a methanol and acetic acid solution.

2.4.7 Determination of surface hydrophobicity

The surface hydrophobicity was determined using the Baba et al. [4] method. In brief, 1 mL of each sample (1 mg/mL) was mixed with 200 μL of bromophenol blue (BPB; 1 mg/mL) and vortexed. The control sample was similar in volume to the original sample, but it was replaced with tris-HCl buffer (20 mM, pH 8). The reaction mixture was shaken for 10 min at room temperature before centrifugation for 15 min at 2,000 × g. The supernatant was diluted with deionized water (1:10), and the absorbance was measured at 595 nm. The amount of BPB bound was determined using the following equation. The greater the BPB content, the higher the surface hydrophobicity.(3) AmountofBPBboundμg=absorbanceofcontrol-absorbanceofsampleabsorbanceofcontrol×200μg

2.5 Functional properties measurement

2.5.1 Determination of protein solubility

Freeze dried samples were suspended in water (1 mg/mL) and centrifuged at 12,000 rpm for 30 min. The supernatant was analyzed for protein content using the Biuret method. A 500 μL aliquot of each sample's supernatant was added to 2 mL of Biuret reagent. The reaction mixture was incubated for 30 min in the dark and analyzed at 540 nm. Bovine serum albumin was used as a benchmark to calculate protein content.

2.5.2 Determination of foaming property

The foaming properties of WPI and WPI-GTE conjugates were determined in the manner described by Baba et al. [4], with minor modifications. Approximately 20 mL of the liquid sample was homogenized (Ultra Turax, T25, Germany) in a 50 mL graduated cylinder at 12,000 rpm for 90 s, with a high shear mixer to produce foam. The formula used to calculate the foaming capacity and stability of samples was:(5) Foamingcapacity%=V2V1×100

(6) Foamingstability%=VfV2×100

where V1 is the initial volume before shearing, V2 is the volume of the foam after shearing and Vf volume of the foam 60 min after shearing.

2.5.3 Determination of emulsifying property

The emulsion activity index (EAI) and emulsion stability index (ESI) of the WPI and WPI-GTE conjugates were determined using the method described by Chaijan et al. [20], with minor modifications. One milliliter of soybean oil was homogenized with three milliliters of WPI or WPI-GTE conjugates solution (5 mg/mL) at 12,000 rpm for three min (IKA Labortechnik homogenizer, Selangor, Malaysia). After 0 and 15 min, 50 µL emulsions were collected and diluted with 5 mL of SDS (0.1 g/100 mL). The absorbance was then read at 500 nm (UV1800, Shimadzu, Tokyo, Japan). The formulas below were used to calculate EAI and ESI:(7) EAIm2/g=2×2.303×A×DFlφC

(8) ESImin=A0ΔA×Δt

where A is absorbance at 500 nm, DF is dilution factor, l is path length (m), φ is oil volume fraction, C is protein concentration (g/m3), A0 is absorbance at 500 nm, ΔA is A0 – absorbance at 500 nm for 15 min, and Δt is 15 min.

2.6 Antioxidant activity analysis

2.6.1 Determination of DPPH radical scavenging activity

The DPPH• scavenging activity was measured using the method of Rodsamai et al. [21], with minor modifications. The WPI or WPI-GTE conjugates (80 μL) were mixed with 320 µL of deionized water and 2 mL of a 0.06 mM methanolic DPPH solution. The absorbance at 517 nm was measured following a 30-min incubation at room temperature. A calibration curve was created with Trolox (0–1 mM) as a standard (y = − 0.7867x + 0.6604, R2 = 0.9954). The DPPH• scavenging activity was measured in milligrams of Trolox equivalent antioxidant capacity (TEAC) per gram of sample.

2.6.2 Determination of ABTS radical scavenging activity

The ABTS•+ scavenging assay was performed using the method described by Zheng et al. [15] with minor modifications. The original ABTS solution was made by combining ABTS (7 mM) and potassium persulfate (2.45 mM) solutions in a 1:1 ratio and storing it in the dark for 12 to 16 h. The original ABTS solution was diluted with phosphate buffer (10 mM, pH 7.2) until the absorption value reached 0.7 ± 0.02. The samples (30 µL) were mixed with 3 mL of diluted ABTS solution, and the absorbance was measured at 734 nm after 5 min of incubation in the dark. The original ABTS solution was diluted with phosphate buffer solution (10 mM, pH 7.2) until the absorption value reached 0.7 ± 0.02. The samples (30 µL) were mixed with 3 mL of diluted ABTS solution, and the absorbance at 734 nm was measured after 5 min of incubation in the dark.

2.7 Statistical analysis

Throughout the entire study, including the fabrication of the WPI-GTE conjugates and their techno-functionality, a completely randomized design (CRD) was used. All experiments were performed in triplicate, and results are presented as mean ± standard deviation. ANOVA and Duncan test were used to analyze all data (p < 0.05). Data analysis was carried out using IBM SPSS Statistics 25 statistical software, and graphing was done using Origin 2019b software. Significant differences were indicated by different letters in the chart below (p < 0.05).

3 Results and discussion

3.1 Degree of polyphenols binding

The phenolic binding of WPI-GTE conjugates prepared using the traditional redox- pair (R) method was 56.35 %, which was lower than ultrasound-assisted redox-pair (RU), and ultrasonication (UL) methods (Table 1). The R method produced hydroxyl radicals through redox reactions to attack the hydrogen atoms at hydroxyl, amino, and sulfhydryl sites on WPI to form intermediates, which were then bound to polyphenol [2]. For RU, the degree of phenolic binding slightly increased (58.07–60.71 %, Table 1) as the reaction time increased from 60 to 120 min. The RU treatment may accelerate the interaction between WPI and GTE by promoting free radical formation caused by both oxidation and cavitation. This resulted in reducing the time required for conjugation. The RU group then facilitated the formation of WPI-GTE interaction, resulting in higher bound phenolic equivalents than the R and UL samples (Table 1). Furthermore, ultrasonics can denature the protein by exposing hydrophobic sites, which allows for both covalent and non-covalent binding of a hydrophobic phenolic compound to whey protein [4]. The binding capacity of conjugates subjected to UL increased at 60–90 min (73.25–74.25 %) and then decreased at 120 min (67.51 %), owing to the prolonged ultrasonic exposure. The decrease in binding capacity could be attributed to the excess energy generated by ultrasound via long-term treatment, which enhanced protein aggregation with fewer reaction sites for phenolic compounds [15]. This was consistent with the findings of Zheng et al. [15], who noticed that the total EGCG content of ovalbumin-EGCG conjugates increased at first and then decreased over ultrasonic time during ultrasound-assisted treatment. The results indicated that excessive ultrasound treatment causes protein denaturation and aggregation, as well as polyphenol decomposition, reducing polyphenols' ability to bind to proteins. It appeared that UL enabled higher phenolic bound content than RU at the same reaction time. When compared to UL alone, the ultrasound and oxidation system combination in RU may increase protein–protein interaction and restrict protein–phenolic binding. All by itself, UL might make it easier for proteins' active groups to get readily bound to phenolic groups. The ultrasound treatment for 90 min (UL90) could significantly produce WPI-GTE conjugates with a higher degree of phenolic binding while also shortening the time required.Table 1 Degree of polyphenols binding and free sulfhydryl group in whey protein isolate (WPI) and WPI-green tea extract (GTE) conjugates.

Sample	Degree of polyphenols binding (%)	Free sulfhydryl group (µmol/g)	
WPI	−	3.00 ± 0.11f	
R	56.35 ± 0.19e	3.57 ± 0.07d	
RU60	58.52 ± 1.28d	3.76 ± 0.08c	
RU90	58.07 ± 0.40d	4.49 ± 0.08a	
RU120	60.71 ± 0.32c	4.50 ± 0.09a	
UL60	73.25 ± 0.96a	4.05 ± 0.14b	
UL90	74.25 ± 0.09a	3.28 ± 0.07e	
UL120	67.51 ± 0.46b	3.33 ± 0.11e	
Note: Values are given as mean ± standard deviation from triplicate determinations. Different letters in the same column indicate significant differences (p < 0.05). WPI=whey protein isolate. R=WPI-GTE conjugates prepared by traditional free radical treatment. RU60, RU90 and RU120 = WPI-GTE conjugates prepared by ultrasound-assisted free radical treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively. UL60, UL90 and UL120 = WPI-GTE conjugates prepared by ultrasound treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively.

3.2 Free sulfhydryl (SH) content

The free SH groups are highly chemically active and participate in interaction with phenolic compounds, which affects the functional characterization of proteins [22]. They also play an important role in stabilizing the tertiary structure of proteins and indicating microstructural changes [10], [12]. As shown in Table 1, the free sulfhydryl content of all WPI-GTE conjugates increased significantly compared to native WPI, indicating that polyphenols in GTE interact with WPI. This result was consistent with the findings of Guo et al. [12], who observed an increase in free sulfhydryl content when rutin interacts with WPI. The interaction of polyphenols with nucleophilic protein groups such as histidine, cysteine, tryptophan, and amino resulted in the formation of C-N or C-S bonds, exposing the free sulfhydryl groups within the WPI. The ultrasound-assisted treated samples (RU and UL) had higher SH content (3.76–4.50 µmol/g) compared to the R sample (3.57 µmol/g). This was most likely due to the unfolding of the protein structure and dissociation of WPI aggregates, resulting in the exposure of its interior SH groups after subjected to ultrasound treatment [16]. Yan et al [16] reported a similar finding, observing a higher SH content in soy protein isolate (SPI)-EGCG conjugates prepared using the ultrasound-assisted method. This suggests that the ultrasonication process not only facilitates the formation of GTE-WP complex but also destroys the native structure of protein, as indicated by exposing more SH groups [16]. Long-term ultrasound treatment led to a slight decrease (3.28–4.05 µmol/g) of free SH groups in the WPI-GTE complex compared to the R sample (Table 1). The result could be due to the oxidation of exposed free SH groups by hydroxyl radical generated during ultrasonication, resulting in a decrease in free SH content [12]. Compared with native WPI, ultrasound treated samples showed higher free sulfhydryl content. Similar results were observed after the introduction of 500 W of ultrasonication to myofibrillar protein-spices flavor compounds. In contrast with the control group, the ultrasonication group exhibited greater free SH content. This suggests that ultrasonication could increase free SH content by breaking of the disulfide bond or release of the thiol group embedded in the molecules [18]. It should be noted that combining redox-pair and ultrasound treatments resulted in a greater increase in free SH content than either alone (Table 1). This could be attributed to the intensive exposure of thiol groups following a combination of redox-pair and ultrasound treatments, which resulted in a higher rate of SH group exposure than polyphenol conjugation. According to the results, all synthesized methods resulted in a structural change of WPI, as evidenced by higher SH content. The net SH content in each sample was determined by the degree of WPI unfolding after different methods were applied, as well as the interaction with free SH groups via GTE [23].

3.3 Surface hydrophobicity of the WPI-GTE conjugates

Surface hydrophobicity characterizes the changes of hydrophobic groups inside proteins and impacts the functional properties of protein such as protein emulsification, foaming, and gelation ability because it is an essential indicator of protein tertiary structure [10], [12]. The surface hydrophobicity of the WPI-GTE conjugates prepared via the R, RU, and UL methods exhibits different trends (Fig. 1). All conjugates showed markedly increased surface hydrophobicity compared to native WPI (5.22 µg BPB bound). However, a decrease in the surface hydrophobicity of WPI-GTE conjugates produced using RU (14.44–16.15 µg BPB bound) and UL (9.06–11.92 µg BPB bound) methods was observed (Fig. 1). The enhanced binding of WPI to polyphenols via hydrophobic interaction and other bonds may be responsible for the decreased hydrophobic surface caused by ultrasonic treatment. The results obtained showed that, in comparison to R and RU treatment, the surface of WPI-GTE conjugates treated with UL became more hydrophilic. This alteration could be attributed to the introduction of polar groups from phenolic compounds and the exposure of some buried hydrophilic regions [22]. However, the surface hydrophobicity of conjugates increased with the longer ultrasonication time (UL90 < UL120), which can be explained by the exposure of different hydrophobic regions induced by the breakdown of non-covalent bonds during larger denaturation [4].Fig.1 Surface hydrophobicity of whey protein isolate (WPI) and WPI-green tea extract (GTE) conjugates. Note: Different letters indicate significant differences (p < 0.05). WPI = whey protein isolate. R = WPI-GTE conjugates prepared by traditional free radical treatment. RU60, RU90 and RU120 = WPI-GTE conjugates prepared by ultrasound-assisted free radical treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively. UL60, UL90 and UL120 = WPI-GTE conjugates prepared by ultrasound treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively.

3.4 Particle size and zeta potential of the WPI-GTE conjugates

The redox-pair (R) produced WPI-GTE conjugates with the largest mean particle size of 1,300 nm (p < 0.05, Fig. 2a). All WPI-GTE conjugates made with the R and RU methods had larger average particle sizes than native WPI (Fig. 2a). The opaque solutions were obtained in all R- and RU-made complex groups (Fig. 2b), corresponding to the large particle size (Fig. 2a). Turbid solution is caused primarily by the formation of aggregates between protein molecules as a result of electrostatic interaction, hydrophobic interaction, and disulfide bonds [12]. The precipitation was detected after 10 min of incubation in all R- and RU- made complex-solutions, indicating low stability (Fig. 2c). Ultrasound-assisted WPI-GTE conjugates (UL) had smaller particle diameters compared to native WPI (p < 0.05). As the ultrasonic time increased, the particle size diameter of sample prepared using the UL method decreased significantly (Fig. 2a). The cavitation and shearing effects of high-intensity ultrasound resulted in a strong intermolecular interaction and the formation of a compact particle. As a result, smaller particle sizes were observed [12]. WPI-GTE treated with UL for 120 min resulted in the smallest particles (p < 0.05). All WPI-GTE treated with UL yielded clear solutions (Fig. 2b), which correlated well with the smaller particle size (Fig. 2a). Stable dispersions were also produced, as evidenced by no phase separation after 10 min of storage (Fig. 2c). This could be because cavitation produced a stable colloidal substance, such as liposomes, that dispersed easily in the water phase. The binding of WPI and GTE polyphenols may also alter the charge distribution on the protein surface, disrupting intermolecular protein aggregation and increasing solubility.Fig.2 Average particle size diameter and zeta potential of native whey protein isolate (WPI) and WPI-green tea extract (GTE) conjugates (a), appearances of freshly prepared WPI and WPI-GTE conjugates (b), appearances of WPI and WPI-GTE conjugates after standing for 10 min (c). Note: Different letters indicate significant differences (p < 0.05). WPI = whey protein isolate. R = WPI-GTE conjugates prepared by traditional free radical treatment. RU60, RU90 and RU120 = WPI-GTE conjugates prepared by ultrasound-assisted free radical treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively. UL60, UL90 and UL120 = WPI-GTE conjugates prepared by ultrasound treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively.

The zeta potential is useful for analyzing intermolecular electrostatic interactions. High absolute zeta potential values indicate non-aggregation of proteins and improved protein dispersion [18]. Fig. 2a shows the zeta potential of the samples. All the samples, including WPI and WPI-GTE conjugates, had a negative zeta potential, indicating the presence of anionic groups on the surface. All R- and RU-made samples showed a significant decrease in negative zeta potential values in comparison to WPI solution (p < 0.05), indicating aggregation of unfolded protein molecules via electrostatic interaction, hydrophobic interaction, and disulfide bond, and thus decreased intermolecular repulsive forces. This was consistent with the formation of larger mean particle sizes in these solutions via flocculation and coalescence (Fig. 2a). The higher the free SH group (Table 1) and surface hydrophobicity (Fig. 1) of these complexes, the more intermolecular interactions between conjugate molecules occurred. This contributed to the reduced absolute zeta potential of these complex groups, facilitating protein precipitation as shown in Fig. 2b and 2c. The ultrasound treatment alone (UL) resulted in a significant increase in absolute zeta potential, indicating that cavitation caused by ultrasonication aided WPI-GTE conjugate dispersion (Fig. 2a). Interestingly, the highest absolute zeta potential was found after 60 and 90 min of ultrasonic time (Fig. 2a), resulting in the smallest mean particle size caused by strongly electrostatic repulsion stabilized particles as seen in transparent solution (Fig. 2b and 2c).

3.5 Ultraviolet absorption (UV) spectroscopy of the WPI-GTE conjugates

Changes in the conformation of proteins and the microenvironment of hydrophobic amino acid residues caused by phenolic insertion could be reflected in a shift in UV absorption spectra [4], [12], [15], [16]. Fig. 3 depicts the UV absorption spectra of WPI-GTE conjugates synthesized using various methods as compared to native WPI. Native WPI possessed a maximum absorption peak wavelength (λmax) of 280 nm, attributed to the presence of aromatic amino acids like phenylalanine, tryptophan, and tyrosine. However, the WPI-GTE conjugates had a blue shifted maximum absorption peak at 268–277 nm, indicating the presence of covalent bonds. In comparison to traditional R and RU treatments, UL treatment significantly increased the intensity of UV absorption in WPI-GTE conjugates. This indicated that ultrasonic cavitation altered the aromatic amino acid microenvironment of the conjugates. The favorable ultrasonic conditions changed the molecular structure of WPI, resulting in unfolding and realignment. The blue shift with a larger absorption peak in the conjugate obtained using the UL method suggested that the WPI and GTE conjugated more efficiently. This result was consistent with the findings of Zheng et al. [15], who produced WPI and rutin complex.Fig.3 UV absorption spectra of whey protein isolate (WPI)-green tea extract (GTE) conjugates. Note: WPI = whey protein isolate. R = WPI-GTE conjugates prepared by traditional free radical treatment. RU60, RU90 and RU120 = WPI-GTE conjugates prepared by ultrasound-assisted free radical treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively. UL60, UL90 and UL120 = WPI-GTE conjugates prepared by ultrasound treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively.

3.6 Fourier-transform infrared (FTIR) spectra of the WPI-GTE conjugates

FTIR spectra identify the various types of molecules present in a sample and how they interact with one another [3]. The stretching and bending vibrations of the peptide backbone in the amide I (C=O stretching vibration) and II (N-H, C-N) bands of the FTIR spectrum can be used to represent various secondary structures. Amide I and Amide II groups are protein absorption bands located at 1600–1700 cm−1 and 1450–1550 cm−1, respectively [12]. Fig. 4 shows the FTIR spectra of different WPI-GTE conjugates. Native WPI showed four characteristic peaks: amide I (1,632.03 cm−1, C=O stretching), amide II (1,524.08 cm−1, N-H bending), amide III (1,233.21 cm−1, C-N stretching and N-H deformation), and amide A (3,273.35 cm−1, N-H stretching with hydrogen bonding). The amide I band (C=O stretching vibration) of the WPI-GTE conjugates shifted from 1,632 cm− 1 to 1,630 cm− 1, and the amide II band (N-H, C-N) changed from 1,524 cm− 1 to 1,518 cm− 1 (Fig. 4), indicating that GTE caused a change in the secondary structure of WPI. The WPI-GTE conjugates changed slightly in the amide I band before and after ultrasound. The amide II band changed from 1518 cm− 1 to 1515 and 1526 cm− 1 for RU and UL treatments, respectively. These findings suggested that ultrasound influenced hydrogen bonding and other interactions within WPI-GTE conjugates. Peak shifting in the amide I and amide II regions was also observed during the covalent and non-covalent interactions of quercetin [4]. and rutin [12] with WPI, respectively. This was attributed to structural changes induced in the protein following binding with polyphenols [4]. The increased peak area of amide A could be due to the non-covalent incorporation (hydrogen bond) of polyphenol. Clearly, both RU and UL conjugates showed a significantly larger increase in area, indicating increased hydrogen bonding due to polyphenol-protein binding [4]. Thus, UL and RU method can successfully be used for conjugation of GTE with WPI in comparable or even better than the traditional R method.Fig.4 Fourier-transform infrared (FTIR) spectra of whey protein isolate (WPI)-green tea extract (GTE) conjugates. Note: WPI = whey protein isolate. R = WPI-GTE conjugates prepared by traditional free radical treatment. RU60, RU90 and RU120 = WPI-GTE conjugates prepared by ultrasound-assisted free radical treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively. UL60, UL90 and UL120 = WPI-GTE conjugates prepared by ultrasound treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively.

3.7 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the WPI-GTE conjugates

SDS-PAGE was used to monitor the molecular weight distribution of WPI and WPI-GTE conjugates prepared using different methods. Fig. 5 shows the protein pattern of WPI and its conjugates. The bands contained major whey proteins like β-lactoglobulin (β-Lg) and α-lactalbumin (α-La) [24]. Original β-Lg of WPI had a molecular weight of 15 kDa. The presence of conjugated WPI with polyphenol bands varies according to the conjugation process, resulting in shifting and less intense protein bands. Because of the covalent binding of polyphenols and proteins, the treated WPI caused a slight shift in the protein bands toward high-molecular-weight protein mass. All UL groups had protein bands with higher molecular weights than the R and RU groups. As a result, high molecular mass bands on SDS-PAGE indicated the formation of WPI-GTE conjugates. These results demonstrated that the polyphenols in the sample conjugates were covalently bound, although SDS-PAGE was unable to detect minor differences in their molecular weights [2]. Shi et al. [25] reported that the bands of SPI-chlorogenic acid conjugates migrated upwards compared to the unmodified SPI bands, likely due to the formation of complexes from the covalent binding between SPI and chlorogenic acid. A similar trend was observed by Baba et al. [4], where whey protein bands shifted slightly towards higher molecular weights as a result of traditional and ultrasonic-assisted free radical treatments, indicating that polyphenol binding increased the protein mass. In this study, the formation of protein–polyphenol covalent conjugates using ultrasound and ultrasonic-assisted methods produced a band on SDS-PAGE with higher molecular mass positions than native WPI.Fig.5 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of whey protein isolate (WPI) and WPI-green tea extract (GTE) conjugates. Note: WPI; whey protein isolate (Lane 1), R; WPI-GTE conjugates prepared by traditional free radical treatment (Lane 2), RU60 (Lane 3), RU90 (Lane 4) and RU120 (Lane 5); WPI-GTE conjugates prepared by ultrasound-assisted free radical treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively, UL60 (Lane 6), UL90 (Lane 7) and UL120 (Lane 8); WPI-GTE conjugates prepared by ultrasound treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively.

3.8 Functional properties

3.8.1 Protein solubility

Solubility is regarded as an important index for assessing the functional properties of proteins because it is a prerequisite for other functionalities such as gelling and interfacial properties. The solubility of WPI-GTE complexes produced by the redox-pair method alone (R) or in combination with ultrasound (RU) was significantly lower than that of native WPI (Fig. 6). These findings were primarily explained by the higher surface hydrophobicity of these complexes compared to control WPI (Fig. 1). It should be noted that increased sonication time in RU treatment increased the solubility of the protein–phenolic complex, owing to the realignment of hydrophilic moieties at the surface caused by cavitation. Ultrasonication can produce complexes with comparable or better solubility than native WPI (p < 0.05). This result showed that ultrasound treatment increased protein solubility and hydration by facilitating the formation of hydrogen bonds. Furthermore, the cavitation effect of ultrasonication can break non-covalent interactions such as hydrophobic protein connections, exposing more hydrophilic amino acid residues and increasing solubility [25]. In addition, increased solubility can be explained by more negative zeta potential (Fig. 2a) in WPI-GTE conjugates, leading to enhanced protein solubilization [4]. The covalent conjugation of WPI with rutin [12] and quercetin [4] using ultrasonication resulted in a significant increase in protein solubility. However, increased ultrasonic time had no significant effect or trended toward lower solubility (p > 0.05, Fig. 6). This could be because prolonged sonication disrupts the hydrophilic and hydrophobic interactions within the protein, reducing its solubility. This result demonstrated that implementing UL alone for 60 min increased the solubility of the final complexes, while combining it with R resulted in lower solubility than the native WPI.Fig.6 Protein solubility of whey protein isolate (WPI) and WPI-green tea extract (GTE) conjugates. Note: WPI = whey protein isolate. R = WPI-GTE conjugates prepared by traditional free radical treatment. RU60, RU90 and RU120 = WPI-GTE conjugates prepared by ultrasound-assisted free radical treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively. UL60, UL90 and UL120 = WPI-GTE conjugates prepared by ultrasound treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively.

3.8.2 Foaming property

As shown in Fig. 7, conjugation of WPI with GTE had a significant effect on the foaming properties of WPI. WPI-GTE conjugates treated with RU and UL had significantly higher foaming capacity (p < 0.05) compared to redox-pair (R), indicating that ultrasonication improved the formation of complexes with superior foaming capacity. The improvement in foaming capacity could be due to the increased protein solubility of the resulting complexes (Fig. 6), leading to more protein molecules being efficiently transferred to the air–water interface [25]. The addition of chlorogenic acid to SPI via ultrasonic process can improve conjugate foaming capacity [25]. The EGCG-induced modification in the secondary structure of WPI improved its emulsifying and foaming properties [6]. Baba et al. [4] stated that the increased foaming capacity of whey conjugated with quercetin was due to increased molecular flexibility caused by polyphenol conjugation. Longer ultrasonication times resulted in significantly higher foaming capacity (Fig. 7), which is explained by the sonication action on proteins, which denatures and reorganizes the hydrophilic balance of the protein molecule to improve foaming capacity [4].Fig.7 Foaming properties of whey protein isolate (WPI) and WPI-green tea extract (GTE) conjugates. Note: WPI = whey protein isolate. R = WPI-GTE conjugates prepared by traditional free radical treatment. RU60, RU90 and RU120 = WPI-GTE conjugates prepared by ultrasound-assisted free radical treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively. UL60, UL90 and UL120 = WPI-GTE conjugates prepared by ultrasound treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively.

Foam stability of conjugates appears to be influenced by the nature and structure of the polyphenol, protein, and conjugation method used. Complexes treated with RU had the highest foam stability, whereas complexes treated with UL possessed the lowest (p < 0.05; Fig. 7). The high surface hydrophobicity of RU-treated conjugates may explain their high foaming stability by flavoring proteins' adsorption to the air–water interface, reducing surface tension and forming a viscoelastic film linked by hydrophobic interaction. It should be noted that the conjugates made by UL possessed high foaming capacity, but had the lowest foaming stability (p < 0.05; Fig. 7). High phenolic compound conjugation may reduce the foaming stability of all UL-made complexes, resulting in reduced protein molecular flexibility and increased steric hindrances at the air-gas interface. This resulted in a looser film formation at the interface, causing low foam stability. Furthermore, the high repulsion of an excessively negative zeta potential of the UL conjugates makes it more difficult for the proteins to maintain the stability of the air-gas interface [4]. To summarize, the molecular structure of protein–phenolic complexes significantly altered the realignment at the air-gas interface.

3.8.3 Emulsifying property

Emulsion activity index (EAI) and emulsion stability index (ESI) are commonly used to evaluate the emulsifying properties of protein derivatives. EAI typically indicates the ability of proteins to adsorb stably at the oil–water interface during emulsion preparation, whereas ESI indicates the ability of an emulsion to resist strain after the formation of small droplets [16], [26]. Fig. 8 depicts the effects of R, RU, and UL methods on the EAI and ESI of WPI-GTE conjugates. The UL method produced WPI-GTE conjugates with significantly higher EAI (p < 0.05) compared to native WPI and other methods of complex production. This correlated well with foaming capacity (Fig. 7). The UL complex after 90 min of sonication had the highest EAI and ESI, 1.5 and 3.5 times higher than the native WPI, respectively (p < 0.05). These findings suggested that conjugation with polyphenols could improve the emulsifying properties of WPI. Conjugates such as WPI-quercetin [4], ovalbumin-EGCG [15], and SPI-chlorogenic acid [25] have previously been shown to improve their emulsifying properties. Furthermore, the application of ultrasonic treatment may have caused a response in conjugate particle size, accelerating the diffusion rate of protein molecules at the oil/water interface, and improving the EAI and ESI of WPI-GTE conjugates [29]. Since flexible proteins principally have greater emulsifying properties than rigid proteins, the enhancement of emulsification could also due to the increase of protein flexibility caused by structural changes of protein [25]. This behavior could be caused by some internal hydrophilic group exposure and the partial unfolding of protein structures, which results in the improved solubility (Fig. 6) of the conjugates and the transport of protein to the interfacial regions [18], [25]. Clearly, when compared to the control WPI and RU groups, the use of UL produced complexes with significantly higher emulsifying capacity and stability, indicating that ultrasonication was a superior strategy for producing conjugates with improved oil–water interface properties. According to the findings, both traditional R and ultrasound-assisted free radical (RU) treatments significantly reduced emulsifying properties of conjugates (Fig. 8), which was strongly associated with low solubility (Fig. 6). In summary, the WPI-GTE complexes prepared using the UL method demonstrated superior emulsifying property, emulsifying stability, and foaming ability with low foaming stability.Fig.8 Emulsifying properties of whey protein isolate (WPI) and WPI-green tea extract (GTE) conjugates. Note: WPI = whey protein isolate. R = WPI-GTE conjugates prepared by traditional free radical treatment. RU60, RU90 and RU120 = WPI-GTE conjugates prepared by ultrasound-assisted free radical treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively. UL60, UL90 and UL120 = WPI-GTE conjugates prepared by ultrasound treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively.

3.9 Antioxidant activity

The antioxidant properties of WPI-GTE conjugates produced using various methods were evaluated by examining the scavenging abilities of DPPH and ABTS free radicals (Fig. 9). All conjugates showed a significant increase in DPPH and ABTS radical scavenging activities when compared to native WPI (p < 0.05; Fig. 9). This could be due to the introduction of polyphenols, which improve antioxidant capacity. The presence of OH groups of phenolic molecules in the complexes could be attributed to the increased free radical scavenging activity by donating hydrogen atoms [27]. The primary active polyphenols of green tea are catechin (C), gallocatechin gallate (GCG), gallocatechin (GC), epicatechin gallate (EGC), epicatechin gallate (EGC), epicatechin (EC), and catechin gallate (CG), which strongly exhibit free radical scavenger [14]. Our results were consistent with the SPI-EGCG conjugates, which had significantly greater free radical scavenging ability than the native SPI [16]. The antioxidant capacity of lentil protein increased following the formation of lentil protein and onion skin phenolic complexes [28]. Zheng et al [15] found that binding ovalbumin to EGCG increased its antioxidant capacity.Fig.9 Antioxidant activity of whey protein isolate (WPI) and WPI-green tea extract (GTE) conjugates. Note: WPI = whey protein isolate. R = WPI-GTE conjugates prepared by traditional free radical treatment. RU60, RU90 and RU120 = WPI-GTE conjugates prepared by ultrasound-assisted free radical treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively. UL60, UL90 and UL120 = WPI-GTE conjugates prepared by ultrasound treatment under different ultrasonic response times of 60 min, 90 min, and 120 min, respectively. TEAC = Trolox equivalent antioxidant capacity.

The WPI-GTE conjugates prepared using the traditional R method (70 mg TEAC/g) demonstrated significantly higher DPPH free radical scavenging ability (p < 0.05) compared to other approaches (Fig. 9). The DPPH free radical scavenging activity of complexes prepared using the combined RU method ranged from 52.15 to 61.93 mg TEAC/g, increasing by approximately 1.98–2.36 folds when compared to native WPI (26.22 mg TEAC/g). Furthermore, the DPPH free radical scavenging activity of complexes prepared using the UL method ranged from 54.22 to 56.83 mg TEAC/g, representing a 2.07–2.67-fold increase over native WPI.

The ABTS radical-scavenging activity of conjugates was comparable to their DPPH radical scavenging activity (Fig. 9). Conjugates outperformed native WPI for ABTS radical scavenging activity (p < 0.05) (Fig. 9). ABTS free radical scavenging capability was equivalent to 191.16 and 174.65–186.37 mg TEAC/g after R and RU treatments, respectively. UL treatment showed lower scavenged ABTS radicals (84.80 to 95.63 mg TEAC/g) compared to the free radical method. However, similarly to the DPPH radical-scavenging assay, increasing scavenging ABTS radicals in conjugates were found in the increasing ultrasonic time of each group. This is explained by the cavitation force produced during long ultrasonication, which encourages the partial unfolding of the protein structure and reveals more aromatic amino acids, like tryptophan, which are involved in direct electron transfer and have strong antioxidant activity [2], [16]. This suggested that polyphenol addition and conjugate treatment could both successfully improve the antioxidant properties of WPI.

4 Conclusion

The newly synthesized method for preparing WPI-GTE conjugates was successfully developed using ultrasound-assisted free radical (RU) and ultrasonic (UL) treatments, resulting in a higher conjugate yield and functionality than the traditional free radical (R) method. In comparison to the standard R method, the RU and UL treatments could significantly reduce the reaction time from 24 h to 60–120 min to produce WPI-GTE conjugates. The UL treatment produced the highest conjugate yields, the smallest particle size, and the highest absolute zeta potential. The covalent and non-covalent bonds between WPI and polyphenols altered the secondary structure of the native WPI, as evidenced by the SDS-PAGE pattern, UV spectra, and FTIR spectra. WPI-GTE conjugates produced using the UL method demonstrated superior solubility, foaming properties, emulsifying properties, and antioxidant properties. Ultrasonication (UL) for 60–90 min is an effective method for producing WPI-GTE conjugates with high functional and antioxidant properties. As a result, UL-produced WPI-GTE conjugates have the potential to be employed as a functional food emulsifier/foaming agent with antioxidant properties.

CRediT authorship contribution statement

Khanittha Chinarak: Writing – review & editing, Writing – original draft, Software, Resources, Methodology, Investigation, Data curation, Conceptualization. Chantira Wongnen: Writing – review & editing, Writing – original draft, Methodology, Conceptualization. Manat Chaijan: Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Methodology, Conceptualization. Arlee Tamman: Writing – review & editing, Writing – original draft, Methodology, Conceptualization. Natthawuddhi Donlao: Writing – review & editing, Writing – original draft, Methodology. Ling-Zhi Cheong: Writing – review & editing, Writing – original draft, Methodology, Conceptualization. Worawan Panpipat: Conceptualization, Methodology, Visualization, Validation, Resources, Supervision, Writing – review & editing, Funding acquisition.

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.

Acknowledgement

We would like to thank Walailak University for providing partial funding through Research Assistant Grant No. WU03. The Thailand Institute of Nuclear Technology (Public Organization) was also given financial assistance.
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