Abstract This study aimed to optimize the enzyme-assisted extraction of juice from soursop (Annona muricata) using α-amylase to improve juice yield and quality. Soursop is rich in bioactive compounds and has significant market potential, but conventional extraction methods often result in limited yield and suboptimal quality. Enzymatic extraction was explored as a solution, using Response Surface Methodology with a Box-Behnken Design to optimize three processing variables: α-amylase concentration (0–1.25%), incubation temperature (40–60 °C), and incubation time (30–240 minutes). Samples with 1:1 (w/w) pulp-to-water ratio were subjected to the different combinations of processing variables. A reduced quadratic model (R² = 0.99) significantly explained the variations in juice yield. The optimized conditions, with the use of 0.41% α-amylase incubated at 51.99 °C for 63.53 minutes, resulted in a maximum yield of 80.04%. Despite enzymatic and thermal treatment, antioxidant activity (DPPH, FRAP, ABTS) was effectively retained, and total phenolic content increased significantly. HPLC analysis confirmed elevated levels of individual phenolic compounds and detected resveratrol (0.27 mg/100 mL) in the optimized juice. The optimized extract also exhibited higher total soluble solids, increased titratable acidity, and noticeable color changes. These findings demonstrate the effectiveness of α-amylase treatment in enhancing juice extractability and bioactive compound retention, offering promising implications for functional beverage development.

Keywords: Optimization, Soursop, Response surface methodology, Resveratrol

Funding: The authors are grateful for the research funding provided by the Department of Science and Technology – Philippine Council for Industry, energy and Emerging Technology Research and Development (DOST-PCIEERD).

Citation:  Estanislao, J.I.H., Evangelista-Albacea, A.R.B., Ilano, M.C.R., Flandez, L.E.L., Benedicto, J.B., Felismino, R.M., Duque, S.M.M., and Castillo-Israel, K.A.T. 2026. Process optimization for amylase-mediated juice extraction from soursop (Annona muricata). Natural and Life Sciences Communications. 25(4): e2026082.

Graphical Abstract:

INTRODUCTION

Soursop (Annona muricata) is a tropical fruit valued for its pleasantly sweet slightly sour, rich phytochemical content pulp, particularly antioxidants linked to various health benefits. The fruit is widely cultivated in equatorial regions like Mexico, Brazil and Southeast Asian Nations. Soursop contains 27.30% starch that not only contributes to its characteristic creamy, fibrous texture but it also encapsulates important bioactive compounds (Nwochoka and Williams, 2009; De Los Santos-Santos et al., 2023). These compounds, including protein, carbohydrates, non-reducing sugars, potassium, dietary fiber and vitamins B1, B2, and C, flavonoids, anthocyanins, carotenoids and water even are highly bioavailable and may be specifically contained within the starch matrix (Márquez-Cardozo et al., 2012; Afzaal et al., 2022).

These characteristics make it ideal for juice extraction and processing into shelf-stable products. However, in fruits with high starch content, such as soursop, conventional extraction techniques may not efficiently break down the pulp matrix, resulting in suboptimal yields. Enzyme-assisted extraction offers a promising alternative. This method utilizes microbial enzymes, such as α-amylase, to break down complex fruit matrices, facilitating pulp maceration, juice clarification, and improved nutrient recovery (Lanzarini and Pifferi, 1989, as cited in Shiv, 2015).

Alpha-amylases are capable of hydrolyzing and cleaving glycosidic bonds of the starch components, namely the amylose and amylopectin polymers that are present in fresh commodities like soursop. However, present studies on enzyme-assisted juice extraction of soursop have focused on cell wall degrading enzymes such as pectin lyase and cellulase enzymes. For instance, a study by Yusof and Ybrahim (1994), examined the combined effect of pectinase concentration and incubation duration on soursop juice yield, reporting a 41% increase on juice yield. Similarly, Fuzi et al. (2021) applied a cellulase–pectin lyase treatment at 50°C for 120 minutes, achieving a maximum yield of 75% at a ratio of 2.0:0.0 (% w/v).

Despite this information, there are limited studies investigating the use of amylase in soursop juice extraction. Given the starch component in the soursop pulp, amylase could be valuable in improving juice yield and quality. Furthermore, there is limited research on systematic optimization of enzyme-assisted extraction using combined effects of enzyme concentration and incubation time and temperature, emphasizing the need for rigorous process optimization.

The present study aimed to investigate the optimum combination of incubation time, temperature and alpha-amylase enzyme concentration to the soursop juice yield through response surface methodology. Thereafter, the phenolic contents, physicochemical properties and antioxidant activity of the optimized juice was studied.

MATERIALS AND METHODS

Materials

Fresh soursop (Annona muricata) fruits were sourced from Barangay Talahiban, Municipality of Bay, Laguna, Philippines. The fruits were allowed to ripen until they exhibited a soft texture and a slight paling of the dark green peel, indicative of ripeness (Worrell et al., 1994). Fruits were visually inspected to exclude any signs of spoilage or physical damage. Stems were removed, and selected fruits were pretreated by steaming until the internal temperature reached 75 ± 5 °C for 5 minutes, followed by rapid cooling in an ice bath.

After pretreatment, the fruits were peeled and manually deseeded. The pulp was then ground using a kitchen blender and homogenized. The homogenized pulp was portioned into foil packs and stored at 4°C until further use.

Enzymatic juice extraction

Sample preparation and juice extraction

A commercial food-grade α-amylase enzyme (Enzyme activity: 2204 U/g; Optimum temperature range: 60–70 °C; Effective temperature range: 35–90 °C ) was procured from Enzaide CTC Far East Philippines Inc., Quezon City, Philippines. Soursop pulp was thawed and diluted with distilled water at a 1:1 (w/w) ratio and thoroughly mixed to produce a homogenized slurry, which served as the substrate for enzymatic treatment during the optimization process. After incubation, it was filtered using cheesecloth to separate the juice from the remaining pulp and insoluble residues.

Response surface methodology – Box Behnken design

The enzymatic extraction process was optimized using response surface methodology (RSM) with a Box-Behnken Design (BBD), employing Design Expert software (version 11). Three independent variables were investigated: enzyme concentration (X₁), incubation temperature (X₂), and incubation time (X₃). The levels were set as follows: enzyme concentration (0–1.25% w/w), incubation temperature (40–80 °C), and incubation time (30–240 minutes). The selection of independent variables was based on literature and preliminary trials. The enzyme concentration range (0–1.25 %) was established from preliminary experiments, which showed that juice yield increased with increasing amylase concentration up to 1.25%, beyond which no further improvement was observed.  The temperature was selected to encompass the optimal activity range of the enzyme (60–70 °C) while the incubation time was chosen based on initial trials indicating that shorter durations were insufficient for effective starch hydrolysis, whereas longer durations resulted in minimal additional yield improvement.

 A total of 15 experimental runs, including three center points, were generated to estimate the pure error and model curvature. All treatments were conducted in randomized order to minimize systematic bias. Insignificant responses were not considered in modelling. Backward elimination regression was performed to reduce and improve the model by disregarding these factors and diminish the margin of error and increase model precision.

The optimized extraction conditions predicted by the model were validated experimentally through five independent runs. The observed juice yield was compared with the predicted value to evaluate the accuracy and reliability of the model.

Juice yield

Juice yield was determined following the enzymatic extraction of soursop pulp. The total yield was calculated based on the initial weight of the homogenized slurry (including the pulp, distilled water, and added enzyme) and the weight of the juice recovered after extraction and subsequent filtration. Juice yield (%) was calculated using the following formula:

Physicochemical tests

pH

The pH of the juice extracts was measured using a pH meter (IONIX Instruments Pte. Ltd., Singapore) following the methods established by Association of Official Analytical Chemists International (1995) with minor modifications. Thirty milliliters sample was transferred in a 100 mL beaker. Then, the calibrated pH meter was immediately submerged. The pH reading was recorded when the value had stabilized in triplicate.  

Total soluble solids

A handheld refractometer (Atago Co. Ltd., Japan) was utilized to evaluate the total soluble solids of the juice extracts. The instrument was properly calibrated with distilled water prior to analysis. One to two drops of the sample were placed on the prism and was covered with the plate. The refractive index, expressed as °Brix, was recorded in triplicate (AOAC Method 932.12; 976.20; 983.17 as cited by Nielsen, 2010).

Titratable acidity

Acid-base titration was carried out to determine the titratable acid content in the juice extracts. The NaOH solution was initially standardized with KHP. The concentration of the titrant was 0.098989847 N. A 5 mL sample was drawn and combined with 10 mL distilled water in a 125 mL Erlenmeyer flask. The triplicated sample was titrated until the solution reached pH 8.2 as originally described by Association of Official Analytical Chemists International (1990). It was calculated using the following formula:

where TA is titratable acidity; and [NaOH] is concentration of NaOH used.

Color

The color of the juice extracts was measured using a calibrated chromameter (Konica Minolta CR-410). Three color parameters were determined in triplicate: L* (brightness), a* (greenness or redness) and b* (yellowness or blueness). The color difference (ΔE) was calculated through the following equation as described by Wibowo et al. (2015):

where ΔE is the color difference between optimized and untreated juice extracts; and ΔL*, Δa* and Δb* are the differences between L*, a* and b* values for the optimized and untreated juice samples.

Phenolic and antioxidant assays

Extract preparation    

Phenolic contents from the soursop juice samples were extracted using the methods by Evangelista-Albacea et al. (2025) with slight modifications. The extraction solvent used was composed of absolute methanol, distilled water and glacial acetic acid (50:50:1 ratio). The soursop juice samples were added with the extraction solvent (0.45:13.0 ratio) in amber tubes and were placed in the shaker for 1 hour. For the extracts used for HPLC analysis, a 1:3 juice to solvent ratio was applied. The extracted juice samples were filtered through a coarse filter paper and were kept in an amber bottle at 4°C prior to analysis. All analyses were performed in triplicate.

Total phenolic content          

The total phenolic content of the sample was determined according to Waterhouse (2002) with minor modifications. Gallic acid was used to establish the standard curve (2.5 to 100 μg/mL) for the calculation of the total phenolic content of the samples. A 0.4 mL from the methanolic extracts of the juice samples, gallic acid standard, and extraction solvent (50:50:1 absolute methanol: distilled water: glacial acetic acid) was drawn and mixed with 2.0 mL of freshly prepared 10% Folin-Ciocalteu reagent in an amber tube. The tube was vortexed and allowed to rest for 5 minutes at room temperature. Then, 1.6 mL of 4% Na₂CO₃ was added. The solution was vortexed and allowed to stand for 90 minutes before reading the absorbance at 760 nm using Shimadzu UV-Vis spectrophotometer. The total phenolic content was calculated using the following formula:        

where c is the standard concentration (μg/mL) determined by the calibration curve, v is the extract solution volume (mL), m is the sample volume (mL).

DPPH radical scavenging activity assay

The antioxidant activity of the juice extracts was characterized by 2,2-diphenyl-1-picrylhydrazyl or DPPH radical scavenging assay according to the method described by Pisoschi et al. (2009) with minor modifications. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used to establish the standard curve (1 to 10 μg/mL) for the calculation of antioxidant activity of the samples. A 1.5 mL from the methanolic extracts of the juice samples, Trolox standard and methanol was drawn and mixed with freshly prepared 1.5 mL DPPH solution into the amber tubes. The samples were vortexed and allowed to stand for 25-30 minutes at room temperature in a dark environment. The absorbance was read using the Shimadzu UV-Vis spectrophotometer with an absorbance of 517 nm. The antioxidant activity was computed using the formula below and were expressed as micrograms Trolox equivalents per milliliter of the sample.

where c is the standard concentration (μg/mL) determined by the calibration curve, v is the extract solution volume (mL), m is the sample volume (mL).

Percent inhibition of ABTS^●+ and DPPH radicals were computed as follows.  

where A_c is the absorbance of the control and A_s is the absorbance of the sample.

ABTS^●+ radical scavenging activity assay

The procedure established by Dong et al. (2015), with minor modifications, was followed for this assay. Freshly prepared 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid or ABTS^●+ solution was mixed with potassium persulfate (1:1) ratio in an amber bottle. The mixture was incubated for 12-16 hours in a dark environment. Prior to the analysis, the absorbance of the mixture at 734 nm was adjusted to 0.70 ± 0.5.  Trolox was used to establish the standard curve (2 to 40 μg/mL) for the calculation of antioxidant activity of the samples. A 0.4 mL aliquot of the methanolic extracts of the juice samples, Trolox standard and methanol were separately drawn and mixed with 3.6 mL ABTS^●+ solution and were incubated for 15 minutes in a dark environment at room temperature. Maximum absorbance was read at 740 nm using Shimadzu UV-Vis spectrophotometer. Results were expressed as micrograms Trolox equivalents per mL of the sample. The ABTS radical scavenging activity (%) was calculated using the previously mentioned formula.

Ferric reducing antioxidant power assay 

The method used by Tomasina et al. (2012) was performed with minor modifications. Trolox was used to establish the standard curve (3 to 100 μg/mL) for the calculation of antioxidant activity of the samples. A 3.6 mL freshly prepared and pre-warmed FRAP reagent composed of acetate buffer, TPTZ and FeCl₃ solution with 10:1:1 ratio was combined with 0.4 mL of methanolic extract of juice, Trolox standard with various concentrations and absolute methanol as blank in glass test tubes. The mixture was reacted at 37°C for 5 minutes using a water bath. Samples were read at 620 nm using Shimadzu UV-Vis spectrophotometer. The ABTS radical scavenging activity (%) was calculated using the previously mentioned formula. Results were expressed as micrograms Trolox equivalents per mL of the sample.

Phenolic compound profiling

To determine the phenolic compound profile of the juice samples, the method developed by Flandez et al. (2023) was employed. The analysis utilized a reverse-phase Intersil ODS-3 column with dimensions of 250 mm × 4.5 mm and a particle size of 5 µm. Juice samples were first extracted using acidified methanol with a dilution factor of three. The resulting liquid was filtered and stored in amber bottles to protect it from light degradation. Prior to HPLC analysis, approximately 500 μL of the filtered extract was transferred into an HPLC vial, which was then placed in the autosampler. The chromatographic separation was carried out using a mobile phase composed of acidified water and acetonitrile, with the specific solvent ratios defined as follows: Solvent A consisted of 2% acetonitrile, 98% water, and 1% acetic acid (v/v), while Solvent B comprised 98% acetonitrile, 2% water, and 1% acetic acid (v/v). The analysis was conducted at a flow rate of 0.8 mL per minute, with an injection volume of 20 μL, and maintained at a column temperature of 30°C. A total of 12 phenolic standards (gallic acid, catechin, epicatechin, rutin hydrate, caffeic acid, syringic acid, ellagic acid, para-coumaric acid, trans-ferulic acid, myricetin, resveratrol, and quercetin) were used to identify and quantify the compounds present in the juice samples.

RESULTS

Optimization of enzymatic juice extraction using response surface methodology

Optimization and model fitting

The experimental juice yield values obtained from the 15 experimental treatments designed using a three-factor Box-Behnken design are shown in Table 1. The influence of enzyme concentration, incubation temperature, and incubation time on the yield of juice extracted from soursop pulp were evaluated.

Table 1. Juice yield derived from each experimental treatment.

Analysis of variance was performed to evaluate the relevance of individual factors and variable interactions to the obtained response values. The lack of fit test (p-value > 0.05), coefficient of determination (R²), the predicted and adjusted R², as well as the adequate precision were considered to check the adequacy of the model. The coefficient of determination (0.9920) indicated the fit quality of the polynomial model, as well as the predicted (0.9780) and adjusted R² (0.9521). Multilinear regression resulted in second-order polynomial equations for juice yield response. Selected runs (runs 3,7 and 15) were excluded during model fitting based on the statistical outlier diagnostics in the software to improve model adequacy. The outliers were identified through the Cook’s distance values and standardized residuals which exceeded critical thresholds.

The reduced quadratic model for juice yield was significant (p-value ≤ 0.05). Similarly, the independent variables enzyme concentration (X₁) and incubation time (X₃) were both significant in the model. Quadratic term X₁² and the interaction of factors X₁X₂, X₁X₃ and X₂X₃ were also considered significant. Conversely, the lack of fit value is 0.9207 indicating insignificance.

Effects of experimental parameters on juice yield        

To illustrate the effect of each variable and factor interaction, 3D surface plots were generated with one parameter fixed at a center point. Figure 1 demonstrates the effects of these parameter interactions. It is apparent from Figure 1a that at higher incubation time and lower incubation temperatures, the juice yield was found to be increased. As the incubation time increases at the lowest incubation temperature, the juice yield was observed to be maximum. Conversely, at minimum levels for both incubation time and temperatures, the juice yield was at the lowest.

Figure 1b demonstrates the interaction of enzyme concentration and incubation time when the incubation temperature is held constant at 60°C. The juice yield was found to be at maximum when the enzyme concentration is decreased at longer incubation time. At all levels of enzyme concentration with increasing incubation time, the yield was found to be slightly decreased and has reached the minimum point.       

On the other hand, Figure 1c shows that at lower enzyme concentration with higher incubation temperatures at 135-minute incubation time, the juice yield was found to be increasing. Similarly, at the high extreme level of enzyme concentration and lowest incubation temperature, the same trend was observed. However, when the incubation temperature increased reaching 80°C, the juice yield was at the lowest despite the enzyme concentration being at the highest.

Figure 1. 3-D surface plots showing the effects of A. Enzyme Concentration at 0.625%, Incubation Temperature at 60°C, Incubation Time at 135 minutes on juice yield.

Final equation in terms of coded and actual factors

The final equation in terms of coded and actual factors are shown below, respectively. The latter can be utilized for response prediction using new factor combinations, but it shall be noted that it is unit specific. When all other factors are kept constant, the coefficient estimate shows the expected change in response for each unit change in factor value.

Model validation          

One hundred solutions were generated in the numerical optimization. Table 2 shows the optimum condition chosen for the α-amylase-assisted extraction of soursop, as well as the predicted mean value for the response.         

Table 2. Chosen solution from numerical optimization.

Optimization of the process parameters were all set in range, as well as the response. However, for incubation temperature, 50 to 55°C was targeted based on the model responses. The lower limit criterion for the response was also modified based on the results of optimization experiments. The optimization criteria were presented in Table 3.    

Table 3. Optimization criteria of the enzymatic juice extraction from soursop.

Verification of the solution

The average experimental value obtained for juice yield at the optimum condition was 80.04%, which was within the predicted interval ranging from 79.46% to 80.55%. The percentage error between the predicted and actual value for juice yield was calculated and the value was 0.05% demonstrating excellent predictive accuracy and validating the fitted model.

Validated juice yield and physicochemical properties of enzyme-extracted soursop juice

Table 4 shows the percent juice recovery from the treated and control sample, as well as other physicochemical properties and bioactive properties of the juice samples. It is evident that the enzyme-treated juice at optimum incubation temperature and time produced a significant increase, with a mean of 80.04% compared to 77.90% recovery from the control. T-test inferential statistics were utilized for pairwise comparison of the juice yield values from each juice sample. A p-value ≤0.05 was obtained, indicating that the optimized juice has a better yield than the untreated juice.

Table 4. Physicochemical characteristics and bioactive properties of the enzyme-treated and untreated soursop juice.

Note: Data is presented as mean ± standard deviation; * indicates significant difference (per row); Tx (Trolox equivalents); GAE (Gallic acid equivalents).           

The pH, total soluble solids, titratable acidity and color of the two juice samples were also investigated. These attributes mainly contribute to the sensorial properties of the juice. Results show that the mean pH value of the untreated juice was not significantly different (p-value > 0.05) to the mean pH value of the optimized juice sample. On the other hand, it was determined that enzymatic treatment had significantly affected the soluble solids and titratable acidity of the soursop juice. Both attributes were significantly higher in the treated sample than in the untreated sample. of Lastly, the color of the juice extracts was measured based on CIE L*a*b* color system and the difference in color was measured through ΔE. The average color difference (ΔE) of the optimized and control soursop juices was 2.04 which indicates that the change in color was noticeable.

Antioxidant activity of enzyme-extracted soursop juice

The effect for enzyme treatment on the bioactive and antioxidant activity of soursop juice was also evaluated and is depicted in Table 4. The overall phenolic content of the juice was measured using the Folin-Ciocalteu method. The enzyme and incubation combination significantly increased the total phenolic content of the soursop juice by 7.23% compared to the control sample.   

Three antioxidant assays, with different mechanisms, were used to assess the antioxidant activity of the soursop juice extracted with the aid of α-amylase. The antioxidant activity of the treated juice was 330.05 μg Tx/mL, 859.74 μg Tx/mL and 479.64 μg Tx/mL for DPPH radical scavenging activity, ABTS radical scavenging activity and Ferric reducing antioxidant power, respectively. T-test inferential statistics revealed that there is no significant difference between the bioactivity of the enzyme-treated soursop juice and the untreated juice. Nevertheless, the overall antioxidant activity indicates that enzymatic treatment did not pose any negative effects to the antioxidant activity of soursop juice.

The percentage inhibition using ABTS^●+ and DPPH assay was also examined in this study. Both assays are not significantly different in terms of inhibitory activity against the free radicals in treated and untreated juice. At a dilution factor of 59.78, the percent inhibition for the ABTS assay was 12.69% and 12.55% for optimized and control samples, respectively. On the other hand, there is a 63.71% and 63.60% inhibition exhibited by the optimized and control sample, respectively. The results show that the soursop juice in general, whether untreated or treated with enzyme, was better in scavenging DPPH free radical than the ABTS^●+ radical.

Phenolic compound profile of enzyme-extracted soursop juice 

High performance liquid chromatography was performed to characterize the specific phenolic compounds present in the α-amylase-extracted soursop juice. After the analysis, 8 phenolic compounds were present for both treated and untreated soursop juice, while 1 phenolic compound was observed only from the enzyme-treated soursop juice as shown in Table 5.

Table 5. Concentration of phenolic compounds from treated and untreated soursop juice.

Note: Data is presented as mean ± standard deviation; * indicates significant difference (per row); nd - not detected.

Among the 8 phenolic compounds observed from both soursop juice samples, gallic acid, catechin, rutin hydrate and trans-ferulic acid increased in concentration while epicatechin, syringic acid, ellagic acid and para-coumaric acid declined. T-test statistics revealed that the rise and decline in concentration of these phenolic compounds were indeed significant with p-value ≤ 0.05. On the other hand, the compound resveratrol has only been found in optimized soursop juice with a concentration of 0.27 mg/100 mL. Catechin from the optimized sample has the largest concentration of 2.80 mg/100 mL which improved compared to 2.72 mg/100 mL of the control sample. This was followed by epicatechin, gallic acid and rutin hydrate with 1.16 mg/100 mL, 0.72 mg/100 mL and 0.55 mg/100 mL concentrations, respectively.

DISCUSSION

Optimization and effects of variable parameter to juice yield

A reduced quadratic model with a coefficient of determination (R²) value of 0.9920 was obtained, indicating excellent fit. All fit statistics, as well as p-value and lack-of-fit values, were within acceptable levels to consider the model adequate and significant. On the contrary, the lack of fit was insignificant which implies that the model fits and effectively captures the experimental data variation. (Corzo et al., 2008 as cited in Handique et al., 2019).

Three 3D surface plots were presented in this paper. It shows the interaction between the independent variables and how it affects the response. The analysis of variance for factors A and B (p-value ≤0.05) corresponds with the displayed surface plots, suggesting that enzyme concentration and incubation time were directly associated with the variation in juice yield. In contrast, the incubation temperature solely did not show significant correlation. Regardless, the interaction of incubation temperature with other factors caused a significant effect on the response.

The design yielded response values ranging from 76.40% to 83.90%. Different factor combinations resulted in varying response behavior, which may be attributed to the α-amylase enzyme activity that varies at different temperatures. Alpha-amylases have different classes such as regular, medium-temperature and high-temperature—functioning from room temperature to as high as boiling temperature (Paul and Genesca, 2013). This diversity may explain the α-amylase remained active and positively influenced the yield even at elevated temperatures.

Nevertheless, the optimum extraction condition for soursop pulp established in this study was 0.41% enzyme concentration at 51.99°C for 63.53 minutes to consider a more economical extraction. This parameter yielded a percentage error between the predicted and experimental mean of 0.05% which is within the acceptable range. Thus, the corresponding model for soursop juice yield was acceptable and adequate in predicting responses using new levels of experimental parameters (Amid et al., 2014; Handique et al., 2019).

As compared to the present study, Yusof and Ibrahim (1994) established that higher enzyme concentration and longer time combination were found to produce greater soursop juice yield of 67.20% using pectin lyase enzyme. The present study shows that at low enzyme concentration at longer incubation time, a higher juice yield can be attained. The effect of the addition of both pectinase and cellulase to soursop juice yield was also analyzed by Fuzi et al. (2021). They obtained a yield of 75.0% using 0.0:2.0 (%v/%w) of the two enzymes, incubated at 50°C for 120 minutes. Accordingly, the current study obtained 83.09% as the highest response during optimization and 80.04% for the model validation, significantly higher than the previous studies. This implies that the use of α-amylase enzymes at the optimum condition is more efficient.   

Juice yield and physicochemical properties of enzyme-treated and untreated soursop juice

The present study also compared the yield and various physicochemical characteristics of α-amylase-treated soursop juice to its untreated counterpart. From 77.90%, the enzyme treatment significantly increased the yield to 80.04%. A similar trend is observed for the total soluble solids and titratable acidity of the enzyme-extracted juice sample. This increasing trend is attributed to the combined effects of the factors used in the study. The study highlights a statistically significant increase in percent yield which, at the laboratory scale, seems small. The percent increase in yield is more appreciable when it is applied on a large industrial scale. As shown by the study, amylase has the potential to increase the quantity of the juice without using additional raw materials, increasing its profitability. It is therefore highly suggested that the percent yield increase be validated on a large scale extraction process.

 Alpha-amylase is capable of hydrolyzing water-insoluble solids such as starch molecules. It causes the juice and other bound compounds like sugars and organic acids to be released through the degraded amylose and amylopectin polymer (Joshi et. al, 2011; Eissa and Salama, 2015). Both heating and enzyme application contributed to the degradation of starch, converting insoluble sugars to soluble sugars. Meanwhile, the titratable acidity compared to pH has significantly increased. Nevertheless, pH and titratable acidity are two properties that are interrelated but not relative to each other. Titratable acidity is equivalent to the total acidity content while pH explains the hydrogen ion concentration (Sadler and Murphy, 2010). For juice color, there is a noticeable difference, with ΔE of 2.04, between the enzyme-treated and the untreated soursop juice. The lightness attribute was higher, indicating a lighter color for the treated juice. According to Wei et al. (2012), darker juice was deemed bitter by the consumers; hence, the lighter ones are perceived to taste better.

Parallel studies using α-amylase for enzymatic extraction shows the same increasing trend for the juice yield of apple, orange and grape juices (Sondhi et al., 2021). Alpha-amylase-treated pumpkin juice also increased in terms of total soluble solids, but its pH remained unchanged in a study by Hesham and Manal (2015).      

Bioactivity and antioxidant capacity of enzyme-treated and untreated soursop juice

Results showed a 7.23% increase in the total phenolic content of the α-amylase-treated juice. Soursop pulp is known to be rich in polyphenols and phenolic acids (Agu et al., 2017), which may exist either in free form or bound within complex plant structures such as the cell wall and starch matrix (Wang et al., 2020). Phenolic compounds can interact with starch through non-covalent bonds (Zhu, 2015), limiting their extractability. The application of α-amylase facilitates the hydrolysis of starch into simpler molecules, thereby releasing bound bioactive compounds, including phenolics. This mechanism likely explains the observed increase in total phenolic content in the enzyme-treated soursop juice.

Alpha-amylase has also been reported to be an effective enzyme for enhancing the total extractable phenolic compounds in oat flour. Chen et al. (2015) worked on examining the phenolic compounds of oat flour before and after the application of α-amylase. It was found out that there is a significant increase to the total phenolic content of the sample (0.46 to 1.35 μmol GAE/g). The study concluded that the application of α-amylase can yield better phenolics than no treatment.

The antioxidant activity of the two soursop juice samples, as evaluated in terms of scavenging DPPH and ABTS radicals, as well as the ferric reducing power, did not differ significantly (P > 0.05). The optimized soursop juice exhibited a comparable antioxidant activity to that of the untreated sample. The results suggest that despite the optimization process of extraction, the inherent antioxidant potential of the soursop juice was preserved. Hence, enzyme-assisted extraction caused no degradation of the bioactive compounds. The absence of significant differences on the antioxidant activity of the two soursop juice samples despite the significant variation in the total phenolic content of the juice may be attributed to the different mechanisms of the assays
(Opitz et al., 2014 as cited in Bibi Sadeer et al., 2020).

The observed results contribute a new perspective when compared to several studies reporting enzyme-assisted juice extraction. In general, enzymatic treatments have shown to enhance functional properties, including increased antioxidant activity, by improving the release of bioactive compounds.  In a study by Nguyen and Nguyen (2018), the application of pectinex and viscozyme increased the antioxidant capacity of mulberry juice with 82.60% percent inhibition. Similarly, pectinase-assisted extraction of apricot juice showed a significant increase in antioxidant activity (DPPH and FRAP), indicating the degradation of cell walls liberating bioactive compounds responsible for antioxidant activity (Bashir et al., 2021). While the present study did not show significant increase in the antioxidant activity of soursop juice, it still provided a valuable insight into the fruit-, enzyme- and processing parameter-dependent nature of enzyme extraction. This may also suggest that enzymes used in juice extraction not only functions to enhance the antioxidant activity of the juice but also to preserve its functional properties.

Through HPLC analysis, gallic acid, catechin, epicatechin, rutin hydrate, syringic acid, ellagic acid, para-coumaric acid, trans-ferulic acid and resveratrol were detected in the enzyme-treated soursop juice. The phenolic compounds detected and quantified in this study were parallel to the data previously identified by other researchers in soursop pulp. In a separate experiment by Jimenez et al. (2014), various caffeic acid derivatives were found at wavelength 328 and 323 nm unlike in the present study. This suggests that the variation in the methods and standards used for HPLC analysis has an impact on the extracted compounds present in the sample.

Resveratrol, a phenolic compound from the stilbene family and a potent antioxidant was only observed from the optimized soursop juice. Control juice extract showed no peak for the similar phenolic compound. This finding is consistent with the study of Da Silva et al. (2014), where resveratrol was detected only guava and surinam cherry and was absent from other fruit pulps tested including soursop. Although there was no significant improvement in the overall antioxidant activity of the optimized juice, it can be inferred that the presence of resveratrol indicates qualitative improvement of the juice. These findings imply that enzymatic juice extraction may enhance the bioaccessibility and extractability of certain phenolic compounds present in soursop pulp.

CONCLUSION

The optimum condition for the enzymatic extraction of soursop using α-amylase was performed in the present study to determine its effect on juice yield (%). The optimized condition was attained at 0.41% enzyme concentration, 51.99°C incubation temperature and 63.53 minutes incubation time. Enzyme-treated soursop juice resulted in a significant increase in juice yield and has shown changes in the physicochemical properties as compared to untreated juice. Concurrently, the enzymatic juice extraction has no negative impact on the antioxidant properties of the soursop juice but has significantly improved its total phenolic content. The study also revealed that the process improves the extractability and bioaccessibility of certain phenolic compounds like resveratrol, which have a potential health implication.

ACKNOWLEDGEMENTS

The authors would like to thank the Department of Science and Technology - Philippine Council for Industry, Energy and Emerging Technology Research and Development (DOST-PCIEERD) and the Collaborative Research and Development to Leverage Philippine Economy (CRADLE) program for funding this study. The authors would also like to extend their appreciation to the Institute of Food Science and Technology at the University of the Philippines Los Baños, for allowing the conduct of experiments.

AUTHOR CONTRIBUTIONS

Jan Irish H. Estanislao: Investigation (Lead), Formal Analysis (Lead), Writing Original Draft (Lead); Anna Rina B. Evangelista-Albacea: Investigation (Supporting), Formal Analysis (Supporting), Writing - Review and Editing (Supporting); Ma. Cristina R. Ilano: Investigation (Supporting), Formal Analysis (Supporting); Lloyd Earl L. Flandez: Investigation (Supporting); Joshua B. Benedicto: Investigation (Supporting); Romel M. Felismino: Conceptualization (Lead), Funding Acquisition, Supervision (Lead); Katherine Ann T. Castillo-Israel: Conceptualization (Equal), Funding Acquisition (Equal), Writing - Review and Editing (Supporting). Sheba Mae M. Duque: Conceptualization (Equal), Funding Acquisition (Equal), Writing - Review and Editing (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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