Production and Evaluation of Green Soybean (Glycine max L.) Powder Fortified with Encapsulated Crude Procyanidin Extract Powder

Abstract

Green soybean (Glycine max L.), commonly known as edamame, is recognized for its rich phytochemical content and nutritional and functional benefits. However, its limited shelf life and susceptibility to quality degradation restrict its commercial potential in fresh form. To address this, green soybean seeds can be processed into extract and powder forms, which offer greater stability and added value. The preparation of crude procyanidin extract was examined in this study along with the effects of three distinct extraction techniques: enzyme incubation, ultrasonic-assisted extraction (UAE), and enzymatic hydrolysis followed by ultrasonic-assisted extraction (EUAE). Additionally, the effects of two drying methods (drum-drying and spray-drying) on the retention of bioactive compounds and antioxidant activity were assessed. Optimal conditions for each drying method were selected to enhance antioxidant properties by fortifying instant green soybean powder (GSP) with encapsulated crude procyanidin extract (ECPE). The chemical, physical, and sensory properties of ECPE-fortified GSP were analyzed. Results indicated that the EUAE method was the most effective for procyanidin extraction. Encapsulation allowed for procyanidin retention of over 83% after storage at 25 and 35 °C for 12 weeks. The optimal conditions were determined to be drum-drying at 3 rpm and spray-drying at an inlet temperature of 200 °C for the drying techniques. Fortification of GSP with 3–5% ECPE powder positively correlated with increased phytochemical content and antioxidant activity. Both drum- and spray-dried GSP maintained color integrity comparable to the control. Drum-dried GSP preserved greater concentrations of bioactive compounds and exhibited superior antioxidant activity compared to spray-dried GSP. All powdered products had acceptable water activity (≤0.60) and moisture content (≤12%), suggesting suitability for long-term storage. Although spray-dried powders exhibited greater hygroscopicity, they demonstrated lower emulsion stability and solubility compared to drum-dried powders. Drum-dried GSP retained higher levels of carbohydrate, fat, fiber, and ash compared with spray-dried powder, while protein content was similarly preserved by both methods. In conclusion, ECPE powder serves as a promising functional ingredient in instant green soybean powder. Both drum-dried and spray-dried GSP products exhibit potential for application in a variety of functional food products.

1. Introduction

Ascorbic acid, vitamin B, minerals, and dietary fiber are all abundant in edamame, also known as green soybean (Glycine max L.) [1]. Along with lowering cholesterol, it also prevents cancer and cardiovascular disease [2]. Because of their high moisture content and immaturity, fresh green soybeans have a short shelf life and are therefore perishable. By lowering the moisture content and water activity, drying is one of the best methods to increase the shelf life of pulse products because it prevents deteriorative reactions, enzyme activity, and microbial reproduction [3]. Nevertheless, the quality of the final products and their commercial value may be compromised by the loss of nutrients and unnecessary structural changes that may result from drying. Therefore, it is essential to research how drying techniques affect the end product’s quality. Since spray drying offers the benefits of cost savings, scalability, and process simplification, it can be regarded as a high-throughput process. Furthermore, due to its brief heat exposure, drum drying is a practical and cost-effective drying technique for plant material that contains heat-sensitive compounds [4]. These gentle processes can handle a wide range of substances, including heat-sensitive materials like pharmaceuticals, food nutrients, and biologic products [5].

Because of their biological properties, including their antimicrobial, anticancer, and antioxidant properties, flavonoids have attracted a lot of attention from the pharmaceutical, cosmetic, and functional food industries [6]. Proanthocyanidins, also referred to as condensed tannins, are the source of procyanidins, which are phenolic compounds that are members of the flavonoid class [7]. Nuts, grains, legumes, fruits, and vegetables are among the foods that contain procyanidins and pro-anthocyanidins in varied amounts. Our previous studies also observed the fortification of the green soybean pod extracts at 3% (v/v) in green soybean milk, which resulted in higher levels of bioactive compounds, especially procyanidins, as well as antioxidant activity [8]. Nevertheless, the thermal stability of these materials is intrinsically weak, which restricts their potential applications. Moreover, the maintenance of the active ingredients’ bioavailability, activity, and controlled release is necessary for their effectiveness [9]. One convincing way to address these problems is through the encapsulation process [10]. By encapsulating procyanidins instead of using free extracts, the product’s shelf life can be extended by shielding the core material from harmful environmental factors, including the exposure of oxygen, humidity, and light [11]. Spray drying is the most frequently employed technique in the food industry of the various methods employed for the encapsulation of bioactive compounds. Several benefits of this method include the product’s limited exposure to severe temperatures, rapid drying, and continuous operation [12].

Pasteurized green soybean milk fortified with bioactive compounds from seeds extracted from our previous studies has a short shelf life and limited stability, restricting commercial potential. Converting it into an instant powder via spray- or drum-drying with added procyanidin extract can improve stability and broaden application opportunities. This study aimed to examine the preparation of encapsulated crude procyanidin extract (ECPE) powder using different methods and the stability of procyanidin content during storage. Additionally, the total bioactive compounds of green soybean powder were assessed in relation to various drying techniques. Different concentration levels of encapsulated procyanidin extract fortified in green soybean powder were tested to select the best condition of the prototype product while maintaining the bioactive substances and antioxidant capabilities. The chemical and physical properties, along with sensory analysis, were evaluated for the instant green soybean powder that was fortified with encapsulated procyanidin extract powder. This evaluation could serve to motivate the industrial sector to expand the implementation of this technology.

2. Materials and Methods

2.1. Raw Materials

Lanna Agro Industry Co., Ltd. (LACO, Chiang Mai, Thailand) supplied the whole green soybeans involved in this study. After being cleaned and separated from the pod, the seeds were placed in vacuum-sealed polyethylene bags and then kept at 4 °C until they were needed. Analytical-grade reagents were employed throughout the course of the research. iKnowZyme PXC is an enzyme complex (2.94–4.25 U/mg) from REACH Biotechnology Co., Ltd., Pathum Thani, Thailand, consisting of pectinase, cellulase, and xylanase, obtained through deep fermentation of Aspergillus niger.

2.2. Preparation of Encapsulated Procyanidin Extract Powder

To achieve a moisture content below 10%, fresh green soybeans were soaked in tap water for 1 min and oven-dried for 48 h at 60 °C in a hot air oven (Memmert UF 110, Schwabach, Germany) [13]. The Cyclotec sample mill (HR2602, Philips, Shanghai, China) was used to grind the dried seeds into a fine powder with a 40-mesh sieve before they were extracted further.

Three different extraction methods, including enzyme incubation, ultrasonic-assisted extraction (UAE), and enzymatic hydrolysis followed by ultrasonic-assisted extraction (EUAE), were applied to extract procyanidins from the powder of green soybean seeds. For the first extraction method, approximately 10 g of powder in 100 mL of distilled water was extracted with a 1% (w/v raw material powder) enzyme complex consisting of pectinase, cellulase, and xylanase at 25 °C on an orbital water bath shaker (Hangzhou Mui Instruments Co., Ltd., Hangzhou, China) at 120 rpm for 60 min. The second extraction method, UAE, using water as a solvent, was carried out at a maximum power of 500 W at a frequency of 20 kHz for 15 min at 50% amplitude in an ultrasonic probe (VX500, Newtown, CT, USA) [14]. The last extraction method is EUAE, which combines enzymatic and ultrasonic techniques. This method involved an enzyme complex solvent followed by UAE. The mixture was centrifuged at 5000 RPM for 15 min at 4 °C (Nüve NF400R, Ankara City, Turkey) shortly after all extraction methods were completed. The residue was removed to obtain supernatant.

For the encapsulated powder, maltodextrin was chosen as the encapsulating agent [13,15,16]. Maltodextrin (dextrose equivalence of 7) was incorporated into the extract solution at a final concentration of 10% (w/v). An IKA T25 digital Ultra-Turrax homogenizer (IKA Werke GmbH & Co., Staufen, Germany) was used to further homogenize the mixture at 4000 rpm for 5 min. A freeze dryer (Lyophilization Systems Inc., Kingston, NY, USA) was used to freeze-dry the homogenized mixture.

2.3. Storage Stability Test of Green Soybeans Extract Powder

After being finely ground, the freeze-dried microencapsulate of procyanidin extract was placed in an aluminum container, and vacuum sealed before being stored at regulated temperatures of 25, 35, and 45 °C. Their individual procyanidin and percentage retention were determined for up to 8 weeks at specified intervals (weeks 0, 2, 4, 6, and 8).

2.4. Drum Drying

The green soybean slurry was produced by blending green soybeans with a water-soybean ratio of 2:1 in a commercial mill (HR2602, Philips, Ningbo, China) at high speed for 15 min. The resultant slurry was boiled at 95 °C for 20 min. The 400 mm-diameter double-drum dryer was used to process the green soybean slurry. The drum dryer gap and temperature were set at 5 mm and 135 °C, respectively. Preliminary testing was carried out at two levels of drum rotation speed (2 and 3 rpm). Prior to milling the dry flakes into flour (300 μm) using a blending machine (HR2602, Philips, Ningbo, China), they were removed as a thin film from the drum surface and permitted to cool to room temperature. For subsequent analysis, the flour was promptly sealed and kept at 4 °C in vacuum-sealed aluminum foil bags.

2.5. Spray Drying

Green soybean milk was prepared by blending the seed in water with a water-soybean ratio of 2:1 using a commercial mill (HR2602, Philips, Ningbo, China) for 15 min at high speed. The soymilk was then separated from the residue by filtering the green soybean slurry through a double-layered muslin cloth. After boiling the green soybean milk for 20 s at 95 °C, it was thoroughly homogenized with 10% (w/w) maltodextrin using a high-speed homogenizer (9000 rpm for 10 min) to obtain a colloidally stable feed prior to spray drying [17,18]. The BUCHI Mini Spray Dryer (B-290, Flawil, Switzerland) was thereafter employed to spray dry the resulting mixture. The air velocity was maintained at 120 mL/h, with an aspirator rate of 32 m³/h, and a pump rate of 7.5 mL/min. The inlet air temperature of the spray dryer was set at 180 °C and 200 °C, resulting in outlet air temperatures of 85 °C and 90 °C, respectively. A 0.7 mm-diameter dispersing nozzle was employed. The powder was collected through a high-efficiency cyclone in a glass container after spray drying, which was then transferred to a glass vial and stored in a vacuum aluminum foil bag at 4 °C until it was needed.

2.6. Preparation of Instant Green Soybean Powder Fortified with Encapsulated Procyanidin Extract Powder

Two different drying processes of instant green soybean powder fortified with 1, 3, and 5% ECPE powder were prepared by mixing the ingredients with a laboratory blender (HR2602, Philips, China) for 20 s. The optimal concentration of procyanidin in the formulation was selected based on the recommended daily intake from Murkovic [14], as reported in procyanidins, members of the proanthocyanidin class of flavonoids, at 50–53 mg per day, which was considered a reasonable supplemental level by some doctors. The mixture powder was then sealed in a vacuum aluminum foil bag and kept at 4 °C until use for chemical, physical, and sensory analysis.

2.7. Chemical Analysis

2.7.1. Total Phenolic Contents

With minor modifications, the Folin–Ciocalteu method, as outlined by Kupina et al. [19], was used to determine the samples’ total phenolic content (TPC). Folin–Ciocalteu reagent was freshly prepared as a working solution, which was diluted to 10% v/v with distilled water. After mixing 250 μL of the sample extract with 2.5 mL of the diluted Folin–Ciocalteu reagent, the mixture was incubated for 5 min at 25 °C. The mixture was then mixed with 2.5 mL of a 7.5% (w/v) sodium carbonate solution and allowed to develop color for 2 h at 25 °C in the dark. Gallic acid solutions with known concentrations were used to generate a standard calibration curve. The UV-Vis spectrophotometer (Cary 60 Bio, Agilent Technologies, Petaling Jaya, Malaysia) was used to measure absorbance at 765 nm. The gallic acid calibration curve was used to determine the TPC, which was then expressed as milligrams of gallic acid equivalents per gram of dried sample (mg GAE/g DW).

2.7.2. Total Flavonoid Contents

The colorimetric method with aluminum chloride was used to determine the total flavonoid content (TFC) [20]. 4 mL of distilled water and 0.3 mL of 5% NaNO₂ were added together with 1 mL of extract. Following a 5 min period, 0.3 mL of 10% AlCl₃·H₂O was introduced. Then 2.4 mL of distilled water and 2 mL of 1 M NaOH were introduced 1 min later. The absorbance at 510 nm was measured after the solution had been well mixed. The results are given as milligrams of catechin equivalents per gram of dried sample (mg CAE/g), with the standard curves being established using catechin (5–300 mg/kg).

2.7.3. Determination of Procyanidin

The vanillin-sulfuric acid method was employed to measure the procyanidin content [21]. A stock solution containing 100 mg/L of procyanidin (PC) was produced by dissolving it in methanol. Dilutions of the stock solution (25, 50, 100,150, 200, 250, and 300 ug/mL) were made to establish a standard curve. 2.5 mL of 1% (m/v) vanillin solution in methanol, 2.5 mL of 30% (v/v) H₂SO₄ solution in methanol, and 1 mL of the standard or sample were thoroughly mixed. The mixture was subsequently preserved at 30 °C for a duration of 30 min. A UV-visible spectrophotometer (UV9600, Bobang Co., Zhengzhou, China) was employed to quantify the absorbance at 500 nm. The concentration of PC in the dry sample was measured in milligrams per gram (mg PC/g).

2.7.4. Water Activity and Moisture Content

An AquaLab water activity meter (4TE, Decagon Devices, Inc., Pullman, WA, USA) was used to measure the water activity (a_w) at 25 °C. The samples were dried in an air oven set at 70 °C for 24 h in order to determine their moisture content [22].

2.8. Antioxidant Analysis

2.8.1. DPPH Radical Scavenging Capacity Assay

A 2,2-diphenyl-1-picryl-hydrazyl (DPPH) technique described by [23] was used to test the antioxidant properties of samples. Aliquots of 150 μL of the tested sample were placed in a cuvette, and 3 mL of 0.6 mM methanolic solution of DPPH radical was added. The UV spectrophotometer (Cary 60 Bio, UV-Vis, Klang, Malaysia) was used to measure the absorbance at 515 nm. Trolox was used to develop the calibration curve, and the data collected were reported as µmol of Trolox equivalents per g of dry sample (µM Trolox eq/g). With Y standing for light absorbance and X for compound concentration, the calibration curve’s equation was Y = 10.081X − 0.016, and the determination coefficient was R² = 0.9934.

2.8.2. Ferric-Reducing Antioxidant Power Assay

As a measure of antioxidant power, outlined by Fernandes et al. [24], the ferric reducing ability of samples was used to calculate the ferric-reducing antioxidant power (FRAP) of a sample. Acetate buffer (300 mM, pH 3.6), a solution of 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl₃ were mixed together at a ratio of 10:1:1 (v/v/v) to produce the FRAP reagent. Each well was filled with the sample solutions (150 μL) and reagent (2.85 mL), which were then thoroughly mixed. After a 30 min period, the absorbance was measured at 593 nm. Trolox was used at various concentrations to develop a standard curve. On the day of the preparation, every solution was utilized. The unit of µmol of Trolox equivalents per g of sample (µM Trolox eq/g) was used to express the results. The calibration curve was represented by the equation Y = 0.021X − 0.1947, with Y standing for light absorbance and X for compound concentration. The determination coefficient was R² = 0.9963.

2.9. Flavonoid Identification and Quantification Through HPLC

High-performance liquid chromatography (HPLC) was employed to identify and quantify flavonoid compounds. The HPLC system consisted of an automatic injector, deaerator, quaternary pump, and UV-Visible detector (Agilent Technologies, HP1260, Santa Clara, CA, USA). The C18 column (Agilent Technologies, Santa Clara, CA, USA) with a 4.6 mm internal diameter, 250 mm length, and 3.5 um particle size, was used under the chromatographic conditions outlined by Dhanani et al. [25]. The solvent mixture used as the mobile phase included 0.1% (v/v) trifluoroacetic acid (Solvent A) and HPLC-grade methanol (Solvent B). The injection volume was 5 uL, and the mobile phase flow rate was 0.8 mL/min. The temperature used for the chromatographic runs was 40 °C. Procyanidins, quercetin, rutin, and kaempferol were measured in instant GSP fortified with ECPE powder using a photodiode array detector set to 254 nm. Authentic standards diluted in the mobile phase at concentrations ranging from 0.1 to 0.6 mg/mL were used to develop the calibration curve. Peaks of green soybean isoflavone aglycones were identified by matching retention times for procyanidins, quercetin, rutin, and kaempferol of 8.5–8.9, 14.5–14.8, 12.5–12.7, and 15.8–15.9 min, respectively. Samples were injected in 5 replications. In mg per 100 g of sample, the result was reported.

2.10. Physical Properties Analysis

2.10.1. Color Determination

A colorimeter (Minolta CM-3600j, Tokyo, Japan) was employed to determine the color values of the powder ingredients at room temperature. The CIELAB system scale, where L* represents brightness, a* represents green to red, and b* represents blue to yellow, was used to express the results.

2.10.2. Creaming Stability

With minor adjustments, a previously published method [26] was used to measure the creaming index (CI). A beaker filled with 50 milliliters of distilled water was filled with the powder sample (5 g). The sample was thoroughly mixed at 10,000 rpm for 5 min in order to homogenize the emulsions. In order to measure the CI, 20 mL samples of these emulsions were immediately prepared, placed in a test tube with a plastic cap, and left at room temperature (25–28 °C). During storage, the proportions and formation of the cream layer (emulsion) were noted. Using Equation (1), the creaming index (CI%)—which measures the cream layer height (H_c) as a percentage of the total height of the emulsion height (H_E) in the tube—was used to quantify the stability against creaming. Three duplicates of the procedure were performed.

$$\mathrm{Creaming} \mathrm{Index} (\%)=\left({\displaystyle \frac{H_c}{H_E}}\right)\times 100$$

(1)

2.10.3. Water Solubility

The method described by Plazzotta et al. [27] was used to specify the powder solubility values of powder samples. Using a TE-420 shaker (Tecnal, Piracicaba, Brazil), samples (W₀, 1 mg) were suspended in 100 mL of distilled water, agitated for 1 h at room temperature, and then centrifuged for 5 min at 4 °C at 5000 rpm (Nüve NF400R, Ankara City, Turkey). To dry each supernatant to constant weight (W₁), an aliquot was taken out, put into porcelain dishes, and heated to 105 °C in a hot air convection oven (FED 53, Binder, Tuttlingen, Germany). Triplicate measurements were taken. The following is the calculation of powder solubility in percentage (%) using Equation (2):

$$\mathrm{Powder} \mathrm{solubility} (\%) = {\displaystyle \frac{W_0- W_1}{W_0}} \times 100$$

(2)

2.10.4. Hygroscopicity

The method of Wang et al. [28] was employed to conduct hygroscopicity measurements. In short, samples (0.5 g) were kept for a week at 25 °C in an airtight glass container with saturated NaCl solution (78.3% relative humidity). The mass of water absorbed per 100 g of sample (%) was used to express hygroscopicity.

2.11. Proximate Composition Analysis of GSP

The Association of Official Analytical Chemists (AOAC) methods were used to quantify amounts of crude fat (no. 2003.06), protein (no. 990.03), fiber (no. 2002.04) and ash (no. 942.05) (AOAC, 2005). Total carbohydrate was calculated by difference as 100 − (% moisture + % ash + % crude fat + % crude protein). Results were expressed as g/100 g dry matter.

2.12. Sensory Evaluation

In the Faculty of Food Science and Technology at Chiang Mai University, 50 untrained panelists (18 males and 32 females, 18–35 years of age) were randomly selected to conduct sensory evaluations. A transparent, clear Ziploc plastic bag measuring 3 × 4 cm was used to package the powder samples. A nine-point hedonic scale, with 1 denoting “extremely dislike,” 5 denoting “neutrality,” and 9 denoting “extremely like,” was used to rate the panelists’ preferences for four characteristics: appearance, color, odor, and overall acceptability [29].

Ethical Guidelines

The statement “I am aware that my responses are confidential, and I agree to participate in this sensory test” was used to obtain informed consent from participants in the sensory test. Providing an affirmative response was necessary in order to take the test. At any time, they were permitted to withdraw from the evaluation without providing an explanation. All of the products that were evaluated were deemed safe for consumption.

2.13. Statistical Analysis

The mean ± standard deviation (SD) was used to express all data. Using SPSS for Windows version 16, a one-way analysis of variance (ANOVA) and Tukey’s post hoc test for multiple comparisons were used to analyze the data. Statistical significance was defined as a p-value of less than 0.05.

3. Results and Discussion

3.1. Extraction of Procyanidins from Green Soybean Seed

The results of the procyanidin extraction by three methods, including enzyme incubation, UAE, and EUAE, are shown in Figure 1. The results indicated that the procyanidin concentration level of green soybean extracted by EUAE accounted for 0.81 ± 0.01 mg PC/g, which was significantly higher (p < 0.05) than that extracted by other methods. By breaking down and extracting plant cell walls, enzymes can maximize the release of bioactive substances [30]. As can be seen from Figure 1, the concentration level of procyanidins from the enzyme incubation method was 0.79 ± 0.01 mg PC PC/g, which was similar to that obtained using the EUAE method. Although it was observed that UAE resulted in the lowest procyanidin content (0.56 ± 0.02 mg PC/g) compared to other extraction methods, ultrasonic-assisted extraction employs ultrasonic waves to disrupt plant cell walls, increasing the extraction efficiency and accelerating the release of intracellular components. Hence, the most optimal procedure for procyanidin extraction was enzymatic hydrolysis followed by ultrasonic extraction, which can significantly disrupt the cell wall and increase procyanidin yield. In order to increase the extraction efficiency and promote the release of intracellular components, ultrasonic-assisted extraction uses ultrasonic waves to break down plant cell walls [31]. Wang et al. [32] pointed out that proanthocyanidins were successfully extracted from Euterpe oleracea (açaí) fruit by using ultrasonic waves in conjunction with solvents.

3.2. Assessment of Encapsulated Crude Procyanidin Extract During Storage at Different Temperatures and Time

During eight weeks, at 2-day intervals, the results obtained indicated that the procyanidin content in encapsulated crude extract does not change significantly (p ≥ 0.05) throughout the entire period either at 25 or 35 °C (

Table 1. Effects of temperature and time on the stability of ECPE during storage.

Temperature (°C)	Weeks	Procyanidins (mg PC/g)	% Retention
	0 (control)	0.92 ± 0.01 a	100
	2	0.90 ± 0.01 a	97.8 ± 0.87 a
25	4	0.90 ± 0.01 a	97.6 ± 0.08 a
	6	0.81 ± 0.01 b	88.3 ± 0.43 b
	8	0.77 ± 0.01 c	83.9 ± 0.16 b
	0 (control)	0.92 ± 0.01 a	100
	2	0.92 ± 0.01 a	99.8 ± 0.10 a
35	4	0.81 ± 0.01 b	87.9 ± 1.46 b
	6	0.77 ± 0.01 c	83.9 ± 0.53 b
	8	0.78 ± 0.01 bc	84.3 ± 0.14 b
	0 (control)	0.92 ± 0.01 a	100
	2	0.73 ± 0.01 c	78.2 ± 1.31 c
45	4	0.64 ± 0.01 d	69.1 ± 0.70 d
	6	0.61 ± 0.01 d	66.9 ± 0.65 d
	8	0.65 ± 0.01 d	70.5 ± 0.44 cd

For n = 3, the data are presented as means ± standard deviation. A statistically significant difference (p < 0.05) is indicated by different letters (a–d) in the same column.

), remaining comparable to the control sample. Procyanidins degraded more quickly when stored at 45 °C than when stored at 25 °C or 35 °C. Encapsulated samples retained more than 83% of their procyanidin content after 8 weeks of storage at 25 and 35 °C, but after 4 weeks of storage at 45 °C, the content decreased to less than 65%. This finding is in accordance with Pavlović et al. [33]: the flavonoid content of chocolate decreased by 3% after 45 days of storage at 4 °C. Higher temperatures of 22 and 35 °C increased the degradation by 6 and 9%, respectively.

Procyanidins were the most abundant phytochemicals in green soybeans, which exhibit potential therapeutic applications in the treatment of chronic metabolic disorders, including cancer, diabetes, and cardiovascular disease [33]. Additionally, procyanidins present in food can be damaged by high temperatures or degraded due to prolonged storage. Hence, procyanidin encapsulation with low storage temperature overcomes the drawbacks of sensitivity to high temperature and undesired degradation due to environmental effects [34].

3.3. Effect of Drying Methods on Bioactive Compounds and Antioxidant Activity from GSP

The total bioactive compounds and antioxidant activity of GSP were influenced by various drying methods (

Table 2. Effects of drying methods on bioactive compounds and antioxidant activities of GSP.

Bioactive Compounds	Drying Process
	Drum-Drying		Spray-Drying
	2 rpm	3 rpm	180 °C	200 °C
TPC (mg GAE/g)	0.81 ± 0.01 b	0.95 ± 0.02 a	0.32 ± 0.01 b	0.66 ± 0.01 a
TFC (mg CAE/g)	0.31 ± 0.02 a	0.32 ± 0.01 a	0.15 ± 0.01 b	0.17 ± 0.01 a
Procyanidins (mg PC/g)	0.21 ± 0.01 b	0.43 ± 0.01 a	0.14 ± 0.01 b	0.28 ± 0.01 a
DPPH (µM Trolox eq/g)	1412 ± 8.31 b	1477 ± 7.80 a	341.1 ± 4.87 b	565.5 ± 8.71 a
FRAP (µM Trolox eq/g)	1617 ± 24.9 b	1902 ± 2.26 a	1064 ± 4.67 b	1248 ± 11.5 a

For n = 3, the data are presented as means ± standard deviation. A statistically significant difference (p < 0.05) is indicated by different letters (a,b) in the same row.

). In the dried powder, the content of TPC, TFC, procyanidins, and antioxidant activities (DPPH and FRAP) was assessed in relation to the drum rotation speed (2 and 3 rpm). The retention time of the feed on the heated drum surface may have decreased, resulting in a reduction in the degradation of bioactive compounds in GSP. Consequently, the TPC, procyanidins, and antioxidant activities increased significantly (p < 0.05) with an increase in drum rotation speed. The drum’s rotation speed influences the heat transfer and drying time, impacting the retention of bioactive compounds. The feed layer on the drum was renewed more frequently with higher rotation speed, which could help avoid exposure to overheating. This could promote more uniform drying and less local thermal damage [35]. Suthiluk et al. [36] also demonstrated that a drum rotation speed of 2 rpm is optimal for achieving higher DPPH and FRAP contents in the pineapple pomaces than 1 rpm. Increasing rotation speed tends to enhance moisture removal rates because of more frequent renewal of the feed film on the drum and thinner layer thickness. In addition, faster moisture removal reduces the time moisture remains in the sample under high heat, which reduces internal temperature and thereby reduces degradation of heat-sensitive phenolics and procyanidins [36].

In the case of the spray-drying process, only the effect of inlet air temperature was investigated as a spray-drying variable in order to isolate its specific influence on the retention of bioactive compounds and antioxidant activity, while other parameters such as feed flow rate, atomization air flow, and nozzle pressure were kept constant to minimize confounding effects. The bioactive compounds and antioxidant activities of GSP were significantly increased (p < 0.05) by increasing the inlet temperature of spray-drying. TPC and procyanidins were twice increased from 0.32 ± 0.01 to 0.66 ± 0.01mg GAE/g and 0.14 ± 0.01 to 0.28 ± 0.01 mg PC/g, respectively, when temperature increased from 180 to 200 °C. The reason was that the heat-sensitive phenolic compounds degraded less because of shorter exposure times brought on by the quicker drying process at higher temperatures [37]. Furthermore, rapid evaporation helped form a protective dried shell around the droplet more quickly, which insulated the interior and slowed further heat penetration, further protecting sensitive compounds [38]. Also, unwanted reactions (Maillard reactions or browning) could be avoided by reducing the time when the droplet is wet and hot, which could transform phenolics into less bioactive forms [39]. Some phenolic compounds were bound in conjugated form. A higher temperature might help break these bonds, releasing more free phenolics, which were more active in typical antioxidant assays. Thus, the measured TPC and procyanidin content can increase because more becomes extractable or detectable [40]. Spray-dried sapodilla powder showed comparable results. Upon raising the temperature from 140 °C to 220 °C, the TPC of spray-dried sapodilla powder rose from 1.07 to 1.27 mg GAE/100 g [41]. In addition, comparable findings regarding TFC and DPPH scavenging activity in the black garlic extracted powder were reported by Wijayanti et al. [42]. When the temperature was elevated from 165 to 225 °C, TCF and DPPH scavenging activity increased from 1.50 to 1.66 mg/g and 0.30 to 0.34 mg TE/g. 225 °C produced the highest total flavonoid content, while 165 °C produced the lowest.

Through a drum-drying process at a rotation speed of 3 rpm, the highest concentrations of TPC (0.95 ± 0.02 mg GAE/g), TFC (0.32 ± 0.01 mg CAE/g), procyanidins (0.43 ± 0.01 mg PC/g), and antioxidant activities DPPH (1477 ± 7.80 µM Trolox eq/g) and FRAP (1902 ± 2.26 µM Trolox eq/g) were extracted from GSP. Spray-drying the samples at an inlet temperature of 180 °C produced the minimal concentrations of TPC (0.32 ± 0.01mg GAE/g), TFC (0.15 ± 0.01 mg CAE/g), and procyanidins (0.14 ± 0.01 mg PC/g), along with antioxidant activities measured by DPPH (341.1 ± 4.87 µM Trolox eq/g) and FRAP (1064 ± 4.67 µM Trolox eq/g). According to Table 2, the bioactive compounds of the sample powder were significantly higher than those of the spray-drying process when the drum-drying process was conducted at a rotation speed of 3 rpm (p < 0.05). This might be due to the drying temperature of drum-drying (135 °C), which was lower than spray-drying (200 °C). Consequently, the optimal condition from each drying process (3 rpm rotation speed of drum-drying and 200 °C inlet temperature of spray-drying) was selected for further studies.

3.4. Development of Antioxidant-Rich Green Soybean Powder Fortified with ECPE

The effect of GSP fortification with ECPE on the bioactive content was presented in

Table 3. Changes in bioactive content and antioxidant activities of GSP after fortification with ECPE powder.

Drying Process	Compounds	Concentration Level of Procyanidins Fortification in GSP (%)
		0 (Control)	1	3	5
Drum-drying	TPC (mg GAE/g)	0.95 ± 0.02 c	2.62 ± 0.01 b	2.69 ± 0.02 a	2.71 ± 0.05 a
	TFC (mg CAE/g)	0.27 ± 0.01 c	0.33 ± 0.01 b	0.34 ± 0.012 a	0.35 ± 0.01 a
	Procyanidins (mg PC/g)	0.42 ± 0.01 c	0.54 ± 0.01 b	0.62 ± 0.01 a	0.63 ± 0.01 a
	Quercetin (mg/g)	0.04 ± 0.01 c	0.05 ± 0.01 b	0.07 ± 0.01 a	0.07 ± 0.01 a
	Rutin (mg/g)	0.03 ± 0.01 c	0.21 ± 0.01 b	0.22 ± 0.01 a	0.22 ± 0.01 a
	Kaempferol (mg/g)	0.04 ± 0.01 c	0.08 ± 0.01 b	0.08 ± 0.01 ab	0.09 ± 0.01 a
	DPPH (µM Trolox eq/g)	1067 ± 2.80 c	1305 ± 3.51 b	1325 ± 3.31 a	1383 ± 6.10 a
	FRAP (µM Trolox eq/g)	1502 ± 1.26 c	2114 ± 1.20 b	2129 ± 6.30 ab	2151 ± 2.56 a
Spray-drying	TPC (mg GAE/g)	0.46 ± 0.01 c	0.65 ± 0.01 b	0.65 ± 0.01 ab	0.66 ± 0.02 a
	TFC (mg CAE/g)	0.13 ± 0.01 c	0.21 ± 0.01 b	0.21 ± 0.01 ab	0.23 ± 0.01 a
	Procyanidins (mg PC/g)	0.13 ± 0.01 c	0.14 ± 0.01 b	0.14 ± 0.01 a	0.15 ± 0.01 a
	Quercetin (mg/g)	0.02 ± 0.01 c	0.03 ± 0.01 b	0.03 ± 0.01 b	0.03 ± 0.01 a
	Rutin (mg/g)	0.02 ± 0.01 c	0.11 ± 0.01 b	0.12 ± 0.01 ab	0.13 ± 0.01 a
	Kaempferol (mg/g)	0.07 ± 0.01 c	0.08 ± 0.01 a	0.08 ± 0.01 a	0.08 ± 0.01 a
	DPPH (µM Trolox eq/g)	165.48 ± 3.21 c	299.2 ± 4.83 b	387.6 ± 1.55 ab	417.5 ± 3.23 a
	FRAP (µM Trolox eq/g)	1216 ± 19.5 c	1345 ± 10.8 b	1387 ± 5.02 b	1438 ± 5.93 a

For n = 3, the data are presented as means ± standard deviation. A statistically significant difference (p < 0.05) is indicated by different letters (a–c) in the same row.

. The addition of the ECPE to GSP positively affected the phytochemical contents and antioxidant activities in samples. An increase in bioactive compounds of drum-dried powder and spray-dried powder has been found to be up to 2.7 and 2.0 times higher than that of the control, respectively. In the flavonoid group, procyanidins had the highest phytochemical content; at 5% fortification, their content was more than 1.1 times that of the control. However, the procyanidin concentration from the drum-drying process (0.63 ± 0.01 mg PC/g) was higher than the spray-drying method (0.15 ± 0.01 mg PC/g). This difference resulted from a relatively high drying temperature (200 °C) during the spray-drying process used in the present research. The health benefits of procyanidins, a significant class of bioactive molecules, are well established. Because these substances minimize oxidative stress-induced cell damage, they hold promise for treating chronic metabolic diseases like diabetes, cancer, and cardiovascular disease [15].

Furthermore, another major flavonoid in the powder sample was kaempferol, which was found in high content in the powder from the drum-dried (0.07 ± 0.01 mg/g) and spray-dried (0.08 ± 0.01 mg/g) processes. It has been discovered that kaempferol and its glycosylated derivatives have anti-inflammatory, antidiabetic, cardioprotective, neuroprotective, antioxidant, antimicrobial, and anti-cancer properties [43]. The potential of kaempferol in cancer therapy is evident in its high cytotoxicity, which is a characteristic of polyphenolic nutraceutical compounds. Numerous mechanisms have been shown to be triggered in the control of cancer cells [21].

The fortification improved the DPPH and FRAP antioxidant potential of GSP products, as displayed in Table 2. The highest values of DPPH and FRAP capacity were obtained for 5% fortified with ECPE in drum-dried powder at 1383 ± 6.10 and 2151 ± 2.56 µM Trolox eq/g, respectively. Furthermore, the fortification strongly improved antioxidant power for the spray-dried samples, which were about 2.5 (DPPH) and 1.2 (FRAP) times higher than those of the control. The bioactive content that was lost during the heat processing could therefore be improved by fortification with the extract. The content of TCF and TFC, particularly the fortification with ECPE following the drying process, has been in a positive correlation with antioxidant activity. The addition of 3% ECPE to GSP fortification in the food sector can therefore have encouraging health effects. The total phenolic content and total procyanidins of the coffee leaf teas were significantly correlated with their DPPH radical scavenging activity, as observed by Ngamsuk et al. [44].

3.5. Physical Properties of GSP After Fortified with ECPE Powder

The color measurement results of GSP from various drying processes were shown in

Table 4. Physicochemical characteristics of GSP after fortified with ECPE powder.

Drying Process	Compounds	Concentration Level of Procyanidins Fortification in GSP (%)
		0 (Control)	1	3	5
Drum-drying	L	74.0 ± 0.21 b	74.1 ± 0.24 b	74.8 ± 0.09 ab	75.2 ± 0.48 a
	a*	−9.81 ± 0.11 a	−9.76 ± 0.07 a	−9.65 ± 0.06 a	−9.80 ± 0.15 a
	b*	32.5 ± 0.22 a	32.3 ± 0.26 a	31.6 ± 0.16 a	31.8 ± 0.66 a
	aw	0.22 ± 0.01 a	0.22 ± 0.01 a	0.22 ± 0.01 a	0.22 ± 0.01 a
	Moisture content (%)	3.25 ± 0.12 a	3.28 ± 0.14 a	3.43 ± 0.21 a	3.44 ± 1.20 a
	Creaming index (%)	66.2 ± 0.23 a	66.0 ± 0.23 a	64.3 ± 0.37 b	62.6 ± 2.75 c
	Water solubility index (%)	2.02 ± 0.31 a	2.03 ± 0.31 a	2.02 ± 0.36 a	2.05 ± 0.08 a
	Hygroscopicity (%)	6.41 ± 1.65 d	7.21 ± 1.45 c	8.46 ± 1.70 b	9.09 ± 1.84 a
Spray-drying	L	91.1 ± 0.22 a	91.4 ± 0.15 a	91.2 ± 0.14 a	91.6 ± 0.11 a
	a*	−5.81 ± 0.03 b	−5.92 ± 0.05 ab	−6.02 ± 0.21 a	−6.00 ± 0.09 a
	b*	14.8 ± 0.10 a	14.9 ± 0.14 a	15.1 ± 0.63 a	15.1 ± 0.24 a
	aw	0.17 ± 0.04 a	0.16 ± 0.07 a	0.14 ± 0.03 b	0.15 ± 0.02 ab
	Moisture content (%)	2.19 ± 0.06 a	2.49 ± 0.02 a	2.54 ± 0.28 a	2.98 ± 0.29 a
	Creaming index (%)	59.4 ± 0.28 a	57.4 ± 0.38 b	54.4 ± 3.26 b	53.7 ± 4.88 b
	Water solubility index (%)	6.67 ± 0.88 b	6.67 ± 0.17 b	7.95 ± 1.11 ab	8.71 ± 0.03 a
	Hygroscopicity (%)	10.3 ± 0.47 b	11.3 ± 0.67 b	13.2 ± 0.26 a	13.5 ± 1.67 a

For n = 3, the data are presented as means ± standard deviation. A statistically significant difference (p < 0.05) is indicated by different letters (a–d) in the same row.

as L (lightness), a* (greenness), and b* (yellowness). Initial values for the spray-dried and drum-dried powders were L (74.0 ± 0.21, 91.1 ± 0.22), a (−9.81 ± 0.11, −5.81 ± 0.03), and b (32.5 ± 0.22, 14.8 ± 0.10), respectively. The drum-dried powders showed a slightly increasing trend in the values of L* when the powder was fortified by the highest concentration level of procyanidins (5%). This change could be as a result of the procyanidin extract being mixed with the maltodextrin carrier (white powder), which was required to enhance the drying process and powder qualities [45]. The color of the product may be influenced by the levels of maltodextrin, a white filler that can add brightness to the powder [46]. There were no significant differences (p < 0.05) between the a*-value and b*-value of procyanidins from various drying processes at each fortification level.

Additionally, the drum-dried powder was greener (average of 9.76) and more yellowish (average of 32.1) than spray-dried powder (average of 5.94 and 15.0, respectively). This may be due to the use of whole green soybean slurry as the initial material in the drum-drying process. Hence, most of the pigments in the group of chlorophyll and carotenoids are still retained in the flake powder. For the spray-drying process, the green soybean slurry was filtered through a double-layered muslin cloth to separate the green soybean milk from the residue. Hence, spray-dried powder was brighter (average of 91.33) than drum-dried powder (average of 74.53).

Different drying techniques resulted in GSP with a_w and moisture contents ranging from 0.14 to 0.22 and 2.19 to 3.44%, respectively. The a_w and moisture content were unaffected by the variation in procyanidin fortification levels (0–5%) in GSP. The moisture content of the prepared spray-dried powder ranged from 2.19 to 2.98%, which was lower than that of the drum-dried powder, which ranged from 3.25 to 3.44% (Table 4). The result of moisture was also consistent with the a_w value. These findings might be because a greater inlet temperature of the spray-drying process elevated the frequency of heat transmission into dry particles, providing a stronger driving force for moisture dissipation [47]. On the other hand, GSP from the drum drying process showed rather high moisture content due to the high rotation speed that reduces the retention time of feed on the heated drum surface, which increases the moisture content of the final product [35]. Moisture content and residual a_w were the two most important factors to be examined in dried products in order to guarantee food safety and product quality [48]. Overall, the GSP produced by the various drying methods had an a_w and moisture content of less than 0.60 and 12%, respectively, which could be regarded as appropriate and secure for long-term storage.

Table 4 showed the creaming index of drum-dried and spray-dried powder, which was an average of 64.8 and 56.3%, respectively. The result obtained was in the range between 53.7% and 66.2%. Furthermore, competition for water between the encapsulating materials seemed to have an important role in two-phase stability [49]. The lower CI of drum-dried powder was due to nonpolar groups of fiber and other compounds that slightly interact with the water, therefore decreasing the stability of emulsion. One of the most prevalent mechanisms of emulsion instability is creaming, which results in macroscopic phase separation into layers of cream and serum. The degree of droplet aggregation in an emulsion and its viscosity was indicated by the creaming index. High emulsion stability is achieved by emulsions with a low creaming index and low creaming rate, which exhibit satisfactory creaming behavior [50].

The GSP from spray drying showed water solubility in the range of 6.67–8.71%, which was higher than drum-dried powder (2.02–2.05%). This advantage may be attributed to the fact that the porous nature of the powder was enhanced by the small particles from the spray-drying process, which consequently provided a greater surface area for the powder to interact with water. Lee et al. [51] indicated that water transfer is facilitated by the larger surface area of the small particles. Prasetyaningrum et al. [52] have observed comparable outcomes in their research on Gac powder from the spray-drying process, which exhibited a high water solubility and a small particle size, as opposed to Gac powder from the drum-drying process, which exhibits a low water solubility and a large particle size. Furthermore, there was no significant difference in the water solubility of GSP from varying concentrations of procyanidin fortification (p ≥ 0.05).

Hygroscopicity of the drum-dried and spray-dried powders ranges from 6.41 to 9.09% and 10.3 to 13.5% (Table 4). The findings suggested that the moisture absorption was improved by increasing the quantity of encapsulated crude procyanidin extracted powder. This was attributed to the inherent hygroscopicity of the encapsulated material and the increased surface area. Prasetyaningrum et al. [52] stated that maltodextrin-encapsulated powder exhibits rather high hygroscopicity, indicating its ability to absorb moisture. Moreover, we found in our experiment that spray-dried powder was more susceptible to moisture absorption than drum-dried powder due to the smaller size and higher surface area, which allows for greater water penetration. According to Chaves [53], the moisture content of powders decreased as the inlet air temperature increased, resulting in the powder absorbing moisture from the surrounding air. An amorphous product in a metastable non-equilibrium state with a high degree of hygroscopicity is produced by the rapid removal of moisture during spray drying at higher drying temperatures [54]. The observed trends in solubility and hygroscopicity were consistent with recent reports on spray-dried soymilk powders. Scanning electron microscope (SEM) investigations of spray-dried soy beverages and soymilk powders had shown predominantly spherical particles with varying degrees of surface wrinkling, collapse, or surface convexities; such surface features increase surface area and could reduce wettability, leading to slower reconstitution and higher hygroscopicity. Conversely, better-encapsulated particles rehydrate more readily and tend to be less hygroscopic. These morphology–function relationships had been demonstrated in recent soybean studies and reviews [41,55].

3.6. Nutritional Composition of GSP Fortified with ECPE Powder

The proximate composition of GSP fortified with ECPE powder was presented in

Table 5. Nutritional composition of GSP fortified with ECPE powder.

Nutritional Value (%)	Drying Method
	Drum-Drying	Spray-Drying
Carbohydrate	20.0 ± 0.03 a	12.5 ± 0.01 b
Protein	36.9 ± 0.02 a	35.8 ± 0.02 a
Fat	18.4 ± 0.01 a	15.5 ± 0.02 b
Fiber	14.5 ± 0.01 a	11.7 ± 0.01 b
Ash	4.26 ± 0.01 a	2.23 ± 0.01 b

For n = 3, the data are presented as means ± standard deviation. A statistically significant difference (p < 0.05) is indicated by different letters (a,b) in the same row.

. Significant differences (p < 0.05) were observed between drying methods. Drum-dried powder contained higher carbohydrate (20.0 ± 0.03%), fat (18.4 ± 0.01%), fiber (14.5 ± 0.01%), and ash (4.26 ± 0.01%) compared to spray-dried powder, which showed lower values of 12.5 ± 0.01, 15.5 ± 0.02, 11.7 ± 0.01, and 2.23 ± 0.01%, respectively. Protein content was similar between drum drying and spray drying methods (36.9 ± 0.02% and 35.8 ± 0.02%, respectively), indicating that both techniques preserved the major protein fraction in GSP. The observed differences aligned with the raw material preparation of the two drying methods discussed in Section 2.4 and Section 2.5. Drum drying disseminates green soybean slurry into a thin layer on a heated surface, yielding concentrated sheets that preserve a greater proportion of non-volatile solids, including carbohydrates, dietary fiber, minerals, and surface-associated lipids. Conversely, spray drying atomizes the filtered green soybean slurry into fine droplets, yielding powders with reduced apparent fat, fiber, and ash content.

Singh et al. [56] reported that spray-dried soymilk powders-maintained protein quality but showed reduced carbohydrate and fiber content compared with drum-dried counterparts. Similarly, Taşoyan et al. [57] demonstrated that drying temperature and method significantly affect the ash and fiber composition of soymilk powder, with drum drying favoring higher mineral retention. The current findings suggest that drum drying generally improves the retention of non-protein nutrients (carbohydrates, fiber, fat, ash), whereas spray drying maintains protein levels comparably but may produce powders with diminished mineral and fiber content. The selection of the drying technique should be determined by the desired nutritional and functional use of GSP powders.

3.7. Sensory Analysis of Fortified GSP

The sensory evaluation of procyanidin-encapsulated GSP powder from drum-dried and spray-dried sources was conducted over a 12-week period of storage at 25 °C, as illustrated in Figure 2a–d. Sensorial ratings were acquired for sensory aspects, including appearance, color, odor, and overall acceptability for fortified GSP samples. The drum-dried powder and spray-dried powder showed modest differences in all the sensory attributes. The color and odor criteria depict a slight variation between the drying methods, with average scores of 7.28 ± 0.13, 7.40 ± 0.18, and 6.43 ± 0.26, 6.35 ± 0.26, respectively, as the powder gave a greenish-yellow color. Similar trends were also observed by Mazumder and Ranganathan [58]; during the storage period, the isoflavone-encapsulated milk powder showed minimal yellowing. According to the study, the sensory qualities of milk powder encapsulated with isoflavone were essentially unaffected. The outcomes of the present study’s sensory evaluation indicated that the overall acceptability for fortified spray-dried GSP samples showed a higher score with an average of 7.27 ± 0.38 than drum-dried GSP (7.03 ± 0.28) throughout 12 weeks of storage. Statistically, there was no significant difference (p > 0.05) in terms of drying method and storage time. Overall, the results revealed that the volunteers’ satisfaction with the GSP ranged from like slightly to like moderately, exhibiting a satisfactory level of more than 70% for all topics.

4. Conclusions

The extraction of procyanidins from green soybean seed was enhanced using 1% (w/v) enzymatic hydrolysis followed by ultrasonication, achieving a maximum procyanidin content of 0.81 mg/g. Additionally, this study found that storage at 25–35 °C was suitable for maintaining procyanidin content, with retention levels higher than 83%. The different conditions of drying processes, including drum drying and spray drying, affected the total bioactive compounds and antioxidant activity of GSP. Hence, the most suitable from each drying process (3 rpm rotation speed of drum-drying and 200 °C inlet temperature of spray-drying) was selected as a desirable method to preserve the maximum bioactive compounds and antioxidant properties of GSP. For the development of GSP fortified with ECPE, the phytochemical content and antioxidant activity of GSP obtained from both drying methods demonstrated a positive correlation with the increasing concentrations of ECPE fortification. Furthermore, spray-dried powder was brighter than drum-dried powder. The water activity and moisture content of all dried powders fell into an acceptable range for inhibiting the growth of microorganisms. Despite spray-dried powder being slightly higher in the degree of hygroscopicity, it also had more emulsion instability and water solubility than drum-dried powder. Drum drying enhanced the retention of carbohydrates, fats, fibers, and minerals, whereas spray drying maintained protein content at a comparable level but resulted in lower non-protein nutrient recovery. These findings indicate that the chosen drying method significantly affects the nutritional composition of GSP powders. Lastly, all the powder samples are deemed acceptable based on their appearance, color, odor, and overall acceptability. In conclusion, it could be said that the GSP powder fortified with ECPE from this study was appropriate for the production of antioxidant-rich powder with antioxidant properties that are useful in the functional food industry. Moreover, the extraction technology, stability, and evaluation of the antioxidant capacity of procyanidin extract have the potential to be employed as functional additives. However, the extract powder obtained from the freeze-drying process would be recommended for further studies on the release of encapsulated crude procyanidins in a gastrointestinal system model.