Microwave and ultrasound-assisted extraction of bioactive compounds from Papaya: A sustainable green process

Graphical abstract

Highlights

• •Microwave pretreated ultrasound-assisted extraction (MPUAE) was performed for bioactive compounds.
• •The Box-Behnken tool of response surface methodology was applied for optimization.
• •The findings of MPUAE were compared with the ultrasound-associated extraction (UAE).
• •MPUAE process used less energy and emitted less CO₂ to the environment.
• •MPUAE might be introduced as a viable and green extraction process.

Abstract

The demand for sustainable and eco-friendly extraction methods for bioactive compounds from natural sources has increased significantly in recent years. In this study, we investigated the effectiveness of the microwave pretreated ultrasound-assisted extraction (MPUAE) process for the extraction of antioxidants (TPC, DPPH, and FRAP) from papaya pulp and peel. The optimized variables for the MPUAE process were determined using the Box-Behnken design tool of response surface methodology. Our results showed that the optimized variables for pulp and peel were 675.76 and 669.70 W microwave power, 150 s of irradiation time, 30 °C ultrasound temperature, and 19.70 and 16.46 min of ultrasonic extraction time, respectively. Moreover, the MPUAE process was found to be more energy-efficient and environmentally friendly compared to the conventional ultrasound-associated extraction (UAE) technique. The MPUAE process emitted less CO₂ to the environment and had a shorter extraction time, resulting in a more sustainable and cost-effective extraction process. Our study suggests that the MPUAE process has the potential to be a promising and eco-friendly alternative for the industrial extraction of bioactive compounds from papaya and other natural sources.

1. Introduction

Natural products have been widely recognized as an important source of bioactive compounds with a variety of biological activities, including antioxidant, anti-inflammatory, and antimicrobial properties [1]. The use of these compounds has been increasing in various fields, such as the food and pharmaceutical industries [2]. The extraction of bioactive compounds from natural sources is a crucial step in their production and application. However, the traditional methods used for extraction, such as maceration, Soxhlet extraction, and steam distillation, have several limitations, including long extraction times, low yield, and the use of toxic organic solvents, which pose environmental and health risks [3]. Recently, there has been a growing interest in developing sustainable and eco-friendly methods for the extraction of bioactive compounds from natural sources. One such method is the use of ultrasound-assisted extraction (UAE), which has been demonstrated to be an effective and eco-friendly technique for the extraction of bioactive compounds. Ultrasound enhances mass transfer, improving the movement of solvents in and out of plant materials. UAE utilizes high-frequency sound waves to disrupt the cell wall structure and enhance the penetration of the solvent into the plant tissue, resulting in higher yields of bioactive compounds in a shorter period of time [4]. However, the energy consumption of UAE can be high, which is a significant challenge for industrial-scale applications.

To overcome this challenge, the combination of microwave pretreatment and ultrasound-assisted extraction (MPUAE) has been proposed as a promising alternative method for the extraction of bioactive compounds. The microwave pretreatment enhances the permeability of the plant cell wall and facilitates the release of bioactive compounds during subsequent ultrasound-assisted extraction [5]. The combination of microwave and ultrasound energy also allows for a shorter extraction time, which is beneficial for reducing the energy consumption and improving the cost-effectiveness of the extraction process [6].

Carica Papaya fruits are widely cultivated in the tropical and subtropical areas of the world and are well known for their high nutritional value and high concentrations of antioxidants and phytochemical compounds such as polyphenols, carotenoids, and ascorbic acid [[7], [8]]). Carotenoids, vitamins, minerals, saponins, flavonoids, alkaloids ascorbic acids, and phenols are abundant in papaya, indicating a high antioxidant activity that is beneficial to human health [9]. A huge quantity of wastes and byproducts are produced; papaya peel waste accounted for around 12 % of the fruit weight, and postharvest losses of 40–60 % are observed [10]. Papaya peel is used for a variety of uses in the food and medicine industries, including cosmetics, due to its high content of phytochemicals, bioactive compounds, vitamins, minerals, and antioxidant activity [11]. Therefore, the extraction of bioactive compounds from papaya pulp and peel using sustainable and eco-friendly methods is of considerable interest.

While previous studies have reported the extraction of phenolic compounds from various sources, including papaya leaves, the specific conditions of microwave power, irradiation time, ultrasound extraction time, and temperature have not been optimized. These variables play a crucial role in bioactive extraction [12]. The diverse composition and bioactive compounds in natural sources make developing a universally optimal extraction process challenging [13]. Furthermore, the type and quantity of polyphenols may vary based on plant species and extraction conditions. Consequently, the ideal extraction process is expected to differ depending on the fruit and its edible and non-edible components.

To address these knowledge gaps, our study introduces a systematic framework for optimizing bioactive compound extraction, focusing on microwave-dried papaya pulp and peel using the MPUAE technique. This method leverages the benefits of both microwave and ultrasound, enhancing the permeability of plant cell walls and promoting more sustainable extraction. This innovative extraction method offers a new dimension to bioactive compound extraction in papaya. However, despite the advantages of the MPUAE approach, there are considerable concerns about whether microwave irradiation and ultrasound individually would degrade the quality of the extracted bioactive. As a result, a novel method known as MPUAE was developed in this study, which uses a combination of microwave energy as pretreatment and ultrasonic waves for extraction. To extend the scope of our investigation, we also assessed the environmental consequences of the optimized MPUAE process, encompassing electrical consumption and CO₂ emissions. This eco-conscious approach adds a layer of novelty to our study, contributing to a broader understanding of the environmental implications of innovative extraction methods. The objectives of this study were to optimize the extraction of bioactive compounds from papaya pulp and peel using a combination of microwave power, irradiation time, ultrasonic temperature, and time through response surface methodology and to compare the process yield and environmental consequences of the optimized process (MPUAE) with the ultrasound-associated extraction (UAE) technique. Understanding the fundamentals of this method is crucial for its practical application in extracting bioactives from a range of plant sources, fruits, and vegetables.

2. Materials and methods

2.1. Sample collection and preparation for extracts

Fresh mature green unripe Carica papaya L. was collected from Sylhet local market with uniform size for quantitative measurement experiment. After accumulating, samples were rinsed with clean water to remove dirt. The seeds were removed and separated into the pulp and peel of papaya fruits. The pulp was cut into small pieces of slices. After that, the pulp and peel of the papaya were dried in a microwave oven (Model- MC28H5025VK/D2) at 300 W until gaining equilibrium moisture conditions. Based on a prior study, the exploratory experiment was recommended [14]. Then the dried parts were powdered by a laboratory-type hammer mill and passed through a sieve of 1 mm in size. All the samples were immediately preserved inside air-tight polythene bags. The microwave-dried pulp and peel powder from unripe papaya were extracted using 60 % concentrated ethanol, according to preliminary research and initial analysis [15]. The study design is depicted in Fig. 1 as a schematic outline.

Fig. 1.

Schematic outline of research design.

2.2. Microwave pretreated ultrasound-assisted extraction (MPUAE) process

For each extraction, 200 mg of sample (dry powder) was mixed with 25 mL of ethanol solution (60 %, v/v). For the extraction, a Box-Behnken design was utilized, with A representing the microwave power (W), B representing the microwave irradiation time (s), C representing the ultrasound temperature (^oC), and D representing the ultrasound extraction time (min) as independent variables. At first, the samples were vortexed to ensure that the samples and solvents are correctly mixed before extraction. After that, the samples were pretreated by a microwave oven (Model- MC28H5025VK/D2) with different microwave power and irradiation time. The container was covered with parafilm and cooling the sample’s container to prevent solvent evaporation during the microwave process. Then, the ultrasound was performed by ultrasonic water bath (Model- VGT – 1860QT) with different ultrasound temperatures and extraction times. To avoid evaporation losses during extraction, the samples were maintained as airtight as feasible. Afterward, the samples were centrifuged (Gyrozen-Benchtop centrifuge, Model- 416G) at 1130 g for 5 min. The specimens were filtered with Whatman filter paper at the end of each removal. After that, the filtrates' total phenolic content (TPC), DPPH radical scavenging activity, and ferric-reducing antioxidant power (FRAP) were determined.

2.3. Evaluation of total phenolic content (TPC)

The modified method of Almusallam et al. [16] was used to calculate the total phenolic content. Briefly, 0.5 mL of the ethanolic extract was placed in a 10-mL flask. The extracts were then treated with 0.5 mL 1 M Folin-Ciocalteu reagent. Then, the volume was increased to 10 mL using double-distilled water after adding 1.0 mL of Na₂CO₃ (7.5 % w/v) reagent. Subsequently, the mixture was kept at 25 °C for 1 h, the absorbance of the supernatant was measured against a reagent blank using a UV Spectrophotometer (PG Instruments Ltd, Model- T60U) at a wavelength of 725 nm. The calibration curve (y = 4.802x; R² = 0.998) for gallic acid was determined by comparing absorbance to corresponding standard gallic acid solutions, with the water blank adjusted to zero (0). The results were presented in mg gallic acid equivalent per 100 g dry matter (mg GAE/100 g DM).

2.4. DPPH radical scavenging activity

For the DPPH radical scavenging assay, Zzaman et al. [17] method was used. Firstly, 1.0 mL ethanolic extracts were vortexed with 4 mL, 0.06 mM DPPH solution in a tube. The tubes were then left in the dark for 30 min at room temperature without being handled. The absorbance of the mixtures was then measured using a UV Spectrophotometer (PG Instruments Ltd, Model- T60U) at a wavelength of 517 nm. A DPPH solution was used as a control but without the aliquot. Finally, the following Eq. (1) was used to calculate the scavenging activity.

$$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\left(\%\right)=\frac{{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$ (1)

2.5. Determination of ferric-reducing power (FRAP)

The modified method of Hossain and Hossain [12] was used to calculate the FRAP. Briefly, 0.3 mL of extracted samples were vortexed with 0.85 mL of phosphate buffer (pH 6.6; 0.2 M) and 0.85 mL of potassium ferricyanide (1 % w/v). After 20 min of incubation at 50 °C, 0.85 mL of trichloroacetic acid (10 % w/v) was added and vortexed thoroughly. Finally, 2.85 mL distilled water and 0.57 mL FeCl₃ (1 % w/v) were added, and the mixture was maintained at 25 °C for 30 min. A UV Spectrophotometer (PG Instruments Ltd, Model- T60U) was used to detect absorbance at a wavelength of 700 nm after the second incubation. In the meantime, a blank was made using distilled water instead of the extract. The standard ascorbic acid was made by diluting the stock solution in water serially. Fitting the absorbance versus its matching standard ascorbic solutions, with the water blank set to zero (0), got the standard curve (y = 2.915x; R² = 0.998). The results were expressed as mg of ascorbic acid equivalent antioxidant capacity per 100 g dry matter (mg AAE/100 g DM).

2.6. Statistical analysis and experimental design for optimization

The experiments were designed, and the statistical data was analyzed (ANOVA) using Minitab (version 20.4.0.0). The relevance of Tukey's test of significance between means was demonstrated. The BBD was used by RSM to optimize the experimental extraction procedure. Initially, the four most important factors were chosen: microwave power, microwave irradiation time, ultrasound temperature, and extraction time. The main influential independent variables were determined as 300–900 W microwave power (A), 30–150 s microwave irradiation time (B), 30–60 °C ultrasound temperature (C), and 10–30 min ultrasound extraction time (D) after trial studies. Every variable was encoded with three possible values based on trial experiments and previous studies: high (+1), medium (0), and low (-1). The most important three dependent variables (multiple responses) TPC, DPPH, and FRAP extraction yield were chosen in the experimental design.

Multiple regressions using the least-square approach were employed to analyze the data, and the BBD was used to fit the experimental model with the typical second-order quadratic equation Eq. (2):

$${\text{y}}_{\mathrm{r}}={\mathrm{\alpha }}_0+\sum _{\mathrm{i}=1}^{\mathrm{n}}{\mathrm{\alpha }}_{\mathrm{i}}{\mathrm{x}}_{\mathrm{i}}+\sum _{\mathrm{i}=1}^{\mathrm{n}}{\mathrm{\alpha }}_{\mathrm{ii}}{\mathrm{x}}_{\mathrm{i}}^2+\sum _{\mathrm{i}\ne \mathrm{j}=1}^{\mathrm{n}}{\mathrm{\alpha }}_{\mathrm{ij}}{\mathrm{x}}_{\mathrm{i}}{\mathrm{x}}_{\mathrm{j}}$$ (2)

where, ${\text{y}}_{\mathrm{r}}$ denotes the measured response variables and ${\text{x}}_{\mathrm{i}}$ and ${\text{x}}_{\mathrm{j}}$ denote the independent variable levels. ${\text{α}}_0$ are a constant (predicted response at the center) and the linear, quadratic, and two-factor interactive coefficients of the model are ${\text{α}}_{\mathrm{i}}$, ${\text{a}}_{\mathrm{ii}}$, and ${\text{α}}_{\mathrm{ij}}$, respectively. The total error criteria were used in all statistical significance tests, with a confidence level of 95 %.

2.7. Ultrasound-assisted extraction (UAE) process

Bioactive compound extraction was also performed using the UAE process, based on the MPUAE procedure's optimal conditions, in a comparison investigation. After optimization of the MPUAE process condition, the samples were extracted from both UAE and MPUAE. Triplicate extractions were carried out.

2.8. Effect of process on the environment

Electrical consumption and CO₂ emission of the applicable procedures were also assessed in the current study regarding the environmental effect. As a result, electrical consumption (E_C) was computed as the electrical power for a certain period, as follows in Eq. (3) [18]:

$${\text{E}}_C=\frac{\text{P×t}}{\text{1000}}$$ (3)

When 1 kWh of coal or fuel is burned, 0.8 kg of CO₂ is emitted into the environment [18]. As a result, CO₂ emissions may be computed using the following Eq. (4):

$${\text{E}}_{{\mathrm{CO}}_2}={\text{E}}_{\text{C}}\times 0.8$$ (4)

where, ${\text{E}}_C$ = electrical consumption (kWh); P = electrical power (W); t = process time (h); and$E_{{\mathit{CO}}_2}$ = CO₂ emission (kg).

3. Results and discussion

3.1. Fitting of regression model and statistical analysis

The RSM linear models were applied to associate with the BBD of bioactive compound extraction to determine the best process yield conditions for microwave-dried papaya pulp and peel against various trials. Bioactive componds (assessed by TPC, DPPH radical scavenging activity, and FRAP attributes) were extracted based on several independent factors found in early studies, including microwave power (W), microwave irradiation duration (s), ultrasound temperature (^oC), and ultrasound extraction time (min). According to the applicable variables for the yield of bioactives employing the MPUAE process, the numerous extraction conditions are depicted in Table 1 as obtained results. In terms of TPC, DPPH radical scavenging activity, and FRAP, peel outperformed pulp whereas peel ranged from 491.95 to 845.95 GAE mg/100 g DM, 70.03 to 84.15 %, and 2092.62 to 3023.16 AAE mg/100 g DM comapared to pulp ranged from 411.26 to 663.74 GAE mg/100 g DM, 66.58 to 82.01 %, and 1209.26 to 2204.12 AAE mg/100 g DM, respectively. ANOVA was employed to evaluate every variable's impact and the viability of the quadratic model.

Table 1.

BBD matrix and multiple responses for process yield of microwave-dried pulp and peel of papaya against different runs using the MPUAE method.

Run	Independent variables								Pulp			Peel
	Coded level				Uncoded level
	A	B	C	D	A	B	C	D	TPC (GAE mg/100 g DM)	DPPH (%)	FRAP (AAE mg/100 g DM)	TPC (GAE mg/100 g DM)	DPPH (%)	FRAP (AAE mg/100 g DM)
1	−1	−1	0	0	300	30	45	20	411.26	74.09	1946.83	491.95	74.73	2602.92
2	1	−1	0	0	900	30	45	20	515.38	75.16	1822.47	653.33	75.80	2607.20
3	−1	1	0	0	300	150	45	20	512.78	78.80	1565.18	551.82	79.87	2367.07
4	1	1	0	0	900	150	45	20	541.41	80.51	2118.35	640.32	82.23	2688.68
5	0	0	−1	−1	600	90	30	10	596.07	74.73	1955.40	689.77	76.02	2705.83
6	0	0	1	−1	600	90	60	10	603.88	74.30	1869.64	596.07	75.16	2568.61
7	0	0	−1	1	600	90	30	30	575.25	77.52	1766.72	588.26	78.59	2534.31
8	0	0	1	1	600	90	60	30	533.60	78.80	1406.52	570.04	80.09	2199.83
9	−1	0	0	−1	300	90	45	10	476.33	68.97	1938.25	510.17	68.70	2534.31
10	1	0	0	−1	900	90	45	10	510.17	66.84	1732.42	671.55	70.03	2590.05
11	−1	0	0	1	300	90	45	30	484.14	78.16	1209.26	512.78	78.80	2092.62
12	1	0	0	1	900	90	45	30	489.35	76.87	1728.13	502.36	77.94	2388.51
13	0	−1	−1	0	600	30	30	20	517.98	78.37	2195.54	640.32	79.87	2945.97
14	0	1	−1	0	600	150	30	20	663.74	82.01	2204.12	845.95	84.15	3023.16
15	0	−1	1	0	600	30	60	20	554.42	75.80	1998.28	655.94	77.73	2688.68
16	0	1	1	0	600	150	60	20	637.72	81.80	2036.88	741.83	82.66	2993.14
17	−1	0	−1	0	300	90	30	20	507.57	80.30	1565.18	512.78	80.94	2319.90
18	1	0	−1	0	900	90	30	20	554.42	77.09	1963.98	596.07	78.37	2585.76
19	−1	0	1	0	300	90	60	20	502.36	76.87	1475.13	549.22	77.52	2174.10
20	1	0	1	0	900	90	60	20	528.39	79.23	1895.37	541.41	80.73	2251.29
21	0	−1	0	−1	600	30	45	10	525.79	66.58	1796.74	674.16	69.23	2855.92
22	0	1	0	−1	600	150	45	10	570.04	76.39	2118.35	778.27	76.13	2920.24
23	0	−1	0	1	600	30	45	30	536.20	77.52	1766.72	603.88	81.80	2731.56
24	0	1	0	1	600	150	45	30	567.44	78.80	1505.15	718.41	79.87	2508.58
25	0	0	0	0	600	90	45	20	640.32	78.37	1552.32	744.43	79.66	2555.75
26	0	0	0	0	600	90	45	20	627.30	77.09	1436.54	705.39	77.73	2598.63
27	0	0	0	0	600	90	45	20	622.10	76.66	1539.45	736.63	79.66	2654.37

The results from Table 2 revealed fitting model coefficients and statistical analysis, accompanied by the lack of fit and pure error for each response, indicating that the full quadratic model along with linear terms of independent variables strongly (P < 0.05) influenced the TPC extraction for both pulp and peel, while the linear impact of ultrasound temperature (C) was insignificant (P > 0.05) for both and ultrasound time (D) for pulp. Moreover, the quadratic terms for all parameters except ultrasound temperature (C²) for pulp and irradiation time (B²) for peel had a significant (P < 0.05) effect on polyphenol extraction. However, each response was not considerably (P > 0.05) affected by the interaction of all factors.

Table 2.

ANOVA for response and regression model coefficient for bioactive extraction process yield.

Model parameter	DF	VIF	TPC (GAE mg/100 g DM)						DPPH radical scavenging activity (%)						FRAP (AAE mg/100 g DM)
			Pulp			Peel			Pulp			Peel			Pulp			Peel
			Coefficient	F-Value	P-Value	Coefficient	F-Value	P-Value	Coefficient	F-Value	P-Value	Coefficient	F-Value	P-Value	Coefficient	F-Value	P-Value	Coefficient	F-Value	P-Value
Constant			629.9		0.000	728.8		0.000	77.373		0.000	79.015		0.000	1509.4		0.000	2602.9		0.000
Model	14			11.62	0.000		9.29	0.000		11.07	0.000		10.56	0.000		12.31	0.000		9.09	0.000
Linear	4			10.48	0.001		9.68	0.001		22.76	0.000		21.04	0.000		16.02	0.000		10.19	0.001
A	1	1.00	20.39	9.70	0.009	39.7	11.49	0.005	−0.123	0.08	0.785	0.378	0.71	0.417	130.1	20.84	0.001	85.0	7.67	0.017
B	1	1.00	36.01	30.24	0.000	46.4	15.71	0.002	2.567	33.88	0.000	2.145	22.77	0.000	1.8	0.00	0.951	5.7	0.03	0.855
C	1	1.00	−4.56	0.48	0.500	−18.2	2.42	0.146	−0.268	0.37	0.555	−0.339	0.57	0.465	−80.8	8.04	0.015	−103.3	11.30	0.006
D	1	1.00	−8.03	1.50	0.244	−35.4	9.11	0.011	3.321	56.72	0.000	3.485	60.12	0.000	−169.0	35.20	0.000	−143.3	21.76	0.001
Square	4			28.54	0.000		20.42	0.000		12.94	0.000		12.73	0.000		18.06	0.000		19.60	0.000
A2	1	1.25	−98.91	101.42	0.000	−140.9	64.32	0.000	−1.055	2.54	0.137	−1.589	5.55	0.036	49.5	1.34	0.269	−229.4	24.79	0.000
B2	1	1.25	–33.84	11.87	0.005	16.9	0.93	0.354	0.563	0.72	0.411	0.712	1.11	0.312	313.8	53.90	0.000	236.9	26.44	0.000
C2	1	1.25	−6.18	0.40	0.541	−41.6	5.62	0.035	1.937	8.58	0.013	1.680	6.21	0.028	221.0	26.75	0.000	−5.9	0.02	0.900
D2	1	1.25	−44.57	20.60	0.001	−55.6	10.03	0.008	−3.232	23.87	0.000	−3.253	23.28	0.000	28.6	0.45	0.516	−50.9	1.22	0.291
2-Way Interaction	6			1.09	0.421		1.60	0.229		2.02	0.141		2.12	0.126		5.99	0.004		1.35	0.309
A*B	1	1.00	−18.9	2.77	0.122	−18.2	0.81	0.387	0.161	0.04	0.837	0.321	0.17	0.687	169.4	11.78	0.005	79.3	2.22	0.162
A*C	1	1.00	−5.2	0.21	0.654	–22.8	1.26	0.283	1.392	3.32	0.093	1.445	3.45	0.088	5.4	0.01	0.915	−47.2	0.79	0.393
A*D	1	1.00	−7.2	0.40	0.540	−42.9	4.48	0.056	0.209	0.08	0.789	−0.546	0.49	0.497	181.2	13.48	0.003	60.0	1.27	0.281
B*C	1	1.00	−15.6	1.90	0.194	−29.9	2.18	0.166	0.589	0.59	0.456	0.161	0.04	0.840	7.5	0.02	0.882	56.8	1.14	0.307
B*D	1	1.00	−3.3	0.08	0.779	2.6	0.02	0.900	−2.132	7.80	0.016	−2.206	8.03	0.015	−145.8	8.73	0.012	−71.8	1.82	0.202
C*D	1	1.00	−12.4	1.19	0.297	18.9	0.87	0.371	0.428	0.31	0.585	0.589	0.57	0.464	−68.6	1.93	0.190	−49.3	0.86	0.372
Error	12
Lack-of-Fit	10			6.81	0.135		4.43	0.198		3.32	0.253		2.15	0.359		2.70	0.300		5.36	0.167
Pure Error	2
Total	26
R-sq			93.13 %			91.55 %			92.81 %			92.49 %			93.49 %			91.38 %
R-sq(adj)			85.11 %			81.69 %			84.42 %			83.74 %			85.89 %			81.33 %

The R-sq(adj) values in Table 2, which were observed to be 85.11 % for pulp and 81.69 % for peel, imply that the models’ parameters are quite capable of describing variance in the dependent variable, the TPC extraction response. The following Eq. 5a-5b is an explanation of the regression model utilized to calculate the expected value of TPC for pulp and peel respectively. The model offered substantial practical importance since the coefficients of determination (R-sq) for pulp and peel exhibited considerable variations between actual and predicted values of 8.02 % and 9.86 %, respectively.

$$\begin{array}{c}\mathrm{TPC\_Pulp}(\mathrm{GAE}\mathrm{mg}/100\mathrm{g}\mathrm{DM})=-412+1.581\mathrm{A}+3.810\mathrm{B}+6.07\mathrm{C}+22.66\mathrm{D}-0.001099{\mathrm{A}}^2-0.00940{\mathrm{B}}^2-0.0275{\mathrm{C}}^2-\\ 0.4457{\mathrm{D}}^2-0.001048\mathrm{A}\ast \mathrm{B}-0.00116\mathrm{A}\ast \mathrm{C}-0.00239\mathrm{A}\ast \mathrm{D}-0.0174\mathrm{B}\ast \mathrm{C}-0.0054\mathrm{B}\ast \mathrm{D}-0.0824\mathrm{C}\ast \mathrm{D}\end{array}$$ (5a)

$$\begin{array}{c}\mathrm{TPC\_Peel}(\mathrm{GAE}\mathrm{mg}/100\mathrm{g}\mathrm{DM})=-794+2.616\mathrm{A}+1.94\mathrm{B}+18.96\mathrm{C}+21.3\mathrm{D}-0.001565{\mathrm{A}}^2+0.00470{\mathrm{B}}^2-0.1851{\mathrm{C}}^2\\ -0.556{\mathrm{D}}^2-0.00101\mathrm{A}\ast \mathrm{B}-0.00506\mathrm{A}\ast \mathrm{C}-0.01432\mathrm{A}\ast \mathrm{D}-0.0333\mathrm{B}\ast \mathrm{C}+0.0043\mathrm{B}\ast \mathrm{D}+0.126\mathrm{C}\ast \mathrm{D}\end{array}$$ (5b)

The variance analysis for second-order quadratic regression models was carried out on the DPPH antioxidant activity for pulp and peel response, where Table 2 expressed the model's adaptability, coefficients, and impacts of the predictor variables. According to the analysis, both pulp and peel were significantly (P < 0.05) impacted by the full quadratic model as well as linear terms of independent variables. However, the linear influence of microwave power (A) and ultrasonic temperature (C) was not significant (P > 0.05). Furthermore, the quadratic terms of ultrasound time (D²) were also significantly (P < 0.05) impacted, but other factors were not. Besides, the interaction of irradiation time to ultrasound time (B*D) had a significant (P < 0.05) effect on the extraction of DPPH antioxidant activity for pulp and peel.

The adequacy of the models is demonstrated by the coefficient of determination (R-sq) values of DPPH radical scavenging activity in Table 2, which were 92.81 % and 92.49 % for pulp and peel, respectively. Pulp and peel had adjusted R-sq(adj) values of respectively 84.42 % and 83.74 % along with the difference between R-sq and R-sq(adj) was 8.39 % and 8.75 %. The DPPH radical scavenging activity of pulp and peel is predicted employing regression equations, which are presented in Eq. 6a-6b.

$$\text{DPPH\_Pulp }\left(\%\right)=78.0-0.0025\mathrm{A}+0.0509\mathrm{B}-1.094\mathrm{C}+1.774\mathrm{D}-0.000012{\mathrm{A}}^2+0.000156{\mathrm{B}}^2+0.00861{\mathrm{C}}^2-0.03232{\mathrm{D}}^2+0.000009\mathrm{A}\ast \mathrm{B}+0.000309\mathrm{A}\ast \mathrm{C}+0.000070\mathrm{A}\ast \mathrm{D}+0.000654\mathrm{B}\ast \mathrm{C}-0.00355\mathrm{B}\ast \mathrm{D}+0.00286\mathrm{C}\ast \mathrm{D}$$ (6a)

$$\text{DPPH\_Peel }\left(\%\right)=71.5+0.0100\mathrm{A}+0.0549\mathrm{B}-0.982\mathrm{C}+1.913\mathrm{D}-0.000018{\mathrm{A}}^2+0.000198{\mathrm{B}}^2+0.00747{\mathrm{C}}^2-0.03253{\mathrm{D}}^2+0.000018\mathrm{A}\ast \mathrm{B}+0.000321\mathrm{A}\ast \mathrm{C}-0.000182\mathrm{A}\ast \mathrm{D}+0.000178\mathrm{B}\ast \mathrm{C}-0.00368\mathrm{B}\ast \mathrm{D}+0.00393\mathrm{C}\ast \mathrm{D}$$ (6b)

Table 2 demonstrated fitting model coefficients and analysis of variance, along with model reliability, implying that the full quadratic model associated with linear terms of independent factors, substantially (P < 0.05) affected FRAP extraction for both pulp and peel, although irradiation time (B) had no significant (P > 0.05) linear effect. Correspondingly, the quadratic terms for microwave power (A²) and irradiation time (B²) for peel as well as irradiation time (B²) and ultrasound temperature (C²) for pulp exhibited a significant (P < 0.05) impact on FRAP extraction. Moreover, the interaction of microwave power to irradiation time (A*B), microwave power to ultrasound time (A*D), and irradiation time to ultrasound time (B*D) had a considerable (P < 0.05) effect on the extraction of FRAP for pulp.

Since the coefficients of determination (R-sq) for the pulp and peel indicated 93.49 % and 91.38 %, respectively in Table 2, the model provided considerable important implications for FRAP extraction. The adjusted R-sq(adj) values for the pulp and peel were 85.89 % and 81.33 %, respectively, while the variance between actual and predicted values was 7.60 % and 10.05 %, demonstrating the validity of the model. The description Eq. 7a-7b of the regression equations used to evaluate the anticipated value of the FRAP for pulp and peel.

$$\text{FRAP\_Pulp (AAE mg/100g DM) = 5284-2.335A-}16.82\mathrm{B}-86.1\mathrm{C}-22.1\mathrm{D}+0.000550{\mathrm{A}}^2+0.0872{\mathrm{B}}^2+0.982{\mathrm{C}}^2+0.286{\mathrm{D}}^2+0.00941\mathrm{A}\ast \mathrm{B}+0.0012\mathrm{A}\ast \mathrm{C}+0.0604\mathrm{A}\ast \mathrm{D}+0.0083\mathrm{B}\ast \mathrm{C}-0.2430\mathrm{B}\ast \mathrm{D}-0.457\mathrm{C}\ast \mathrm{D}$$ (7a)

$$\text{FRAP\_Peel (AAE mg/100g DM) = 2319+3.017A-}14.84\mathrm{B}+2.7\mathrm{C}+19.6\mathrm{D}-0.002549{\mathrm{A}}^2+0.0658{\mathrm{B}}^2-0.026{\mathrm{C}}^2-0.509{\mathrm{D}}^2+0.00441\mathrm{A}\ast \mathrm{B}-0.0105\mathrm{A}\ast \mathrm{C}+0.0200\mathrm{A}\ast \mathrm{D}+0.0631\mathrm{B}\ast \mathrm{C}-0.1197\mathrm{B}\ast \mathrm{D}-0.329\mathrm{C}\ast \mathrm{D}$$ (7b)

The lack of fit denoted that it was insignificant (P > 0.05) in comparison to pure errors, which was desirable, and that the models were adequate for anticipating the TPC, DPPH radical scavenging activity, and FRAP responses. In addition, Variance Inflation Factor (VIF) range for all predictor variables was found to be from 1.00 to 1.25 for multiple responses, indicating a lack of multicollinearity among independent variables. Therefore, all the models demonstrated that they were suitable and effective for predicting the responses.

3.2. Impact of variables on the bioactive extraction process

The analysis revealed that the TPC extraction yield was positively correlated with the main effects of microwave power and irradiation time for pulp and peel, but those interactions among factors had a negative impact, whereas interactions of irradiation time to ultrasound extraction time and ultrasound temperature to extraction time along with quadratic terms of irradiation time had a positive influence for the peel indicated in Table 2.

According to antioxidant activity, DPPH radical scavenging properties extraction was positively correlated to the main impacts of irradiation time and ultrasound extraction time for pulp, but other factors were negatively associated. Similarly, microwave power, irradiation time, and ultrasound extraction time positively interacted with peel extraction, but other variables were adversely related shown in Table 2. Moreover, the quadratic terms of ultrasound temperature and irradiation time were positively linked for the pulp and peel. Furthermore, the terms of the variable interactions were all positively interrelated, while the relationship between the time of irradiation and the time of ultrasound extraction for the pulp and peel, as well as microwave power to ultrasound extraction time, was negatively influenced for the peel.

Accordingly, the FRAP extraction attributes were linked positively with the main effects of microwave power and irradiation time as well as the quadratic and interaction terms of all variables, whereas the interaction between the ultrasound temperature and extraction time along with the irradiation time and ultrasound extraction time impacted negatively on the pulp as illustrated in Table 2. Besides, for the peel, these qualities were positively proportional to the main effects of microwave power and irradiation time, although other parameters were negatively influenced. Subsequently, a positive correlation of the quadratic components of irradiation time as well as the interaction terms of microwave power to irradiation time, microwave power to extraction time, and irradiation time to ultrasound temperature were observed.

Ultrasound amplitude can be used to describe the beneficial effects of ultrasonic power. Owing to the sound wave and liquid droplets formed by cavitation, the plant's cell wall seems to collapse when it is exposed to ultrasound waves. The rarefaction and compression of these waves cause the liquid to cavitate, and these processes are influenced by the intensity of the waves. Consequently, as the ultrasonic amplitude increases, there are more cavities, leading to a greater extraction yield reachable [18]. In addition, ultrasound waves considerably enhanced the yields of bioactive substances throughout the recovery process and reduced extraction time [19]. In contrast, high-intensity ultrasonics might break down the bioactive molecule, which raises concerns about the importance of the process's duration [20]. Nevertheless, in comparison to the impact of microwave power, this effect is less substantial, thus of their exposure, the yield is essentially unaffected. The conversion of electromagnetic energy into heat energy occurs without physical touch in microwave operation. Nonionizing radiation generates molecular motion through the movement of ions and the rotation of dipoles, and thus energy conversion is based on these two processes [21]. The treated material absorbed energy more rapidly, which decreased heat gradients and selective heating [22]. Moreover, the microwave heating of the water molecule within the treated biomaterial results in significant stress on the cell walls, and mechanically collapses the walls. As a result, solvent absorption into the internal tissues is boosted, thus, it is possible to attain maximum extraction yields and selection [23].

Based on the study, a proper irradiation period should be selected to obtain adequate extraction since irradiation time is one of the major variables that affect bioactive extraction yield. This variable has a favorable influence on the process when associated with time. The extraction yield was observed to be proportional to the irradiation time and its quadratic impact, however, the ultrasound extraction time had a negative influence except for DPPH activity. Nevertheless, the extraction efficiency is lowered because consistent exposition to microwave radiation causes structural deterioration of the extracted substances [24]. Therefore, a reduced period needs to maximize bioactive compound extraction in terms of industrial application. Correspondingly, the MPUAE method revealed the high bioactive extraction efficiency combining microwave radiation and ultrasound wave leading to a lower processing time. The extraction of bioactive substances from various biomaterials has been accomplished effectively using ultrasound wave and microwave radiation conjunction [25]. Hence, merging these systems can result in some synergistic benefits, such as a reduction in the amount of solvent needed and faster time for extraction, an enhancement in energy efficiency, and a reduced cost [26]. Since both ultrasound and microwave treatments independently degrade bioactive molecules during their long recovery processes, pretreatment of microwave irradiation is used in the UAE procedure to minimize the period. Therefore, the MPUAE technique performed 150 s microwave run and 19.70 min and 16.46 min ultrasonic runs for the pulp and peel, respectively.

TPC, DPPH antioxidant properties, and FRAP attribute extraction were depicted by plotting three-dimensional response surface plots (Fig. 2a, Fig. 4a, and Fig. 6a of pulp and Fig. 3a, Fig. 5a, and Fig. 7a of peel) and the contour plots (Fig. 2b, Fig. 4b, and Fig. 6b of pulp and Fig. 3b, Fig. 5b, and Fig. 7b of peel) respectively. These plots illustrated the impact of the process variables, whereas response surfaces exhibit substantial curvature and contour plots display a color gradient. Accordingly, all parameters were influenced significantly regarding multiple bioactive responses. Following the investigation, the temperature has been identified as one of the most crucial variables influencing the discharge of polyphenols from the food material, and many other researchers have also noted that raising the extraction temperature enhances extraction, diffusion coefficient, and solute solubility [25,26]. Nonetheless, some samples of bioactive can break down above a certain temperature [27]. A prior study found that some polyphenols bound to proteins may result in a bioactive depletion in the tissue at a greater temperature [28]. In addition, heat may boost the extraction of antioxidants, and the improved temperature of the extraction releases the plant material and breaks the bond between the polyphenols and the proteins or polysaccharides, raising the solubility and enhancing the diffusion rate [12]. The observable FRAP activities are brought about by the denaturation of TPC [29]. This could be because polymeric phenols could bind iron ions at reduced temperatures, enhancing the FRAP and DPPH intensity [30].

Fig. 2.

Response (a) surface and (b) contour plots of total phenolic content (GAE mg/100 g DM) vs four independent variables of papaya pulp.

Fig. 4.

Response (a) surface and (b) contour plots of DPPH antioxidant activity (%) vs four independent variables of papaya pulp.

Fig. 6.

Response (a) surface and (b) contour plots of FRAP antioxidant activity (AAE mg/100 g DM) vs four independent variables of papaya pulp.

Fig. 3.

Response (a) surface and (b) contour plots of total phenolic content (GAE mg/100 g DM) vs four independent variables of papaya peel.

Fig. 5.

Response (a) surface and (b) contour plots of DPPH antioxidant activity (%) vs four independent variables of papaya peel.

Fig. 7.

Response (a) surface and (b) contour plots of FRAP antioxidant activity (AAE mg/100 g DM) vs four independent variables of papaya peel.

3.3. Optimization of MPUAE process variables

According to the information previously stated, an optimization study was conducted to identify the optimum processing conditions for recovering from the pulp and peel. Bioactive substances are often extracted utilizing several factors, yet the combined effects of these parameters have not been extensively investigated by these basic approaches. Consequently, multiple response optimizations were employed to define the optimized field, which can then assist to select the most pragmatic proportion among microwave power, irradiation time, ultrasound temperature, and ultrasound extraction time, along with promoting bioactive yields and extract possibilities. The intended function of process optimization is the simultaneous influence of the predictor variables on the three responses. The target was to maximize TPC, DPPH radical, and FRAP attributes while also considering the viability of the analysis and predicting the process condition against a variety of responses for both pulp and peel extraction.

The findings of research on the optimization of papaya pulp (Fig. 8a) and peel (Fig. 8b) depicted the following multiple bioactive responses along with the best-fitting prediction processing condition. Therefore, the optimum independent variables condition was 675.76 W microwave power, 150 s irradiation time, 30 °C ultrasound temperature, and 19.70 min ultrasound extraction time for the maximum simultaneous bioactive extraction of papaya pulp. As well as optimal predictor variable combinations were 669.70 W microwave power, 150 s irradiation time, 30 °C ultrasound temperature, and 16.46 min ultrasound extraction time for the maximum simultaneous bioactive extraction of papaya peel. The composite desirability of pulp and peel was 0.96 and 0.93, respectively, indicating the model's validity. The actual values of bioactive were relatively near to the anticipated maximum response, which was performed based on optimal conditions. In this condition of pulp, the actual value of TPC, DPPH, and FRAP were 633.38 ± 6.55 GAE mg/100 g DM, 79.01 ± 0.64 %, and 2129.79 ± 20.27 AAE mg/100 g DM, respectively. On the other hand, the actual value of TPC, DPPH, and FRAP of peel were 803.43 ± 9.14 GAE mg/100 g DM, 82.23 ± 0.43 %, and 2913.09 ± 27.90 AE mg/100 g DM, respectively. Previous researchers optimized the antioxidant activity using DPPH and FRAP assays and TPC of Baccaurea ramiflora was employed as an analogous protocol [12].

Fig. 8.

The optimal condition for bioactive extraction of papaya (a) pulp and (b) peel by MPUAE process.

3.4. Comparison between UAE and MPUAE process

The use of MPUAE had a favorable impact on the yield of bioactive obtained. Extraction was also conducted using the UAE procedure to assess the potential of the proposed MPUAE strategy. The UAE process was carried out based on a preliminary experiment; the ultrasound temperature and time were 45 °C and 40 min for pulp and 35 min for peel extraction. Correspondingly, the MPUAE technique was used based on the optimal conditions, which performed better than the UAE method. As depicted in Table 3, the TPC, DPPH, and FRAP values recovered by the MPUAE process were 2.38 %, 2.30 %, and 7.27 % for pulp and 1.31 %, 2.31 %, and 2.41 % for peel, respectively higher. Because the synergistic impacts of microwave and ultrasound waves on plant cell walls, which result in a noticeably larger extraction yield of bioactive, can be used to explain why the MPUAE is more effective [18]. This demonstrates that MPUAE might be a suitable extraction method for extracting bioactive substances from papaya since it offers the highest extraction efficiency in the least amount of time. Consequently, the MPUAE process exhibited a shorter process time than the UAE technique, which was 19.70 min and 16.46 min, respectively, for the pulp and peel. Hence, the analysis indicates that MPUAE has enhanced bioactive compound extraction compared to UAE. Thus, the MPUAE procedure does not result in any destruction of the compounds and may be adopted as a sustainable and green process for extraction.

Table 3.

Bioactive extraction of process yields by UAE and MPUAE methods.

Bioactive Compounds	UAE		MPUAE
	Pulp	Peel	Pulp	Peel
TPC (GAE mg/100 g DM)	618.63 ± 7.95b	793.02 ± 5.42a	633.38 ± 6.55b	803.43 ± 9.14a
DPPH (%)	77.23 ± 0.89c	80.37 ± 0.75b	79.01 ± 0.64bc	82.23 ± 0.43a
FRAP (AAE mg/100 g DM)	1985.42 ± 23.88d	2844.48 ± 24.38b	2129.79 ± 20.27c	2913.09 ± 27.9a

All Values in the table depict the mean ± standard deviation of three replications - the significant difference (p < 0.05) among samples in the same row with distinct superscript characters.

3.5. Effect on energy and environment

The calculation of the required electrical power and CO₂ emissions to the environment for conducting the UAE and MPUAE procedures, using microwave and ultrasound power levels of 900 W and 450 W, respectively depicted in Fig. 9. The MPUAE procedure revealed lower energy consumption and CO₂ emissions during the process, which were 36.67 % and 37.50 % for pulp and 38.46 % and 38.10 % for peel, respectively, when compared to the UAE technique. Consequently, the MPUAE technique employs much less energy and emits significantly minimal carbon dioxide gases into the environment as well. It is worth assessing that process duration directly correlates with energy usage and CO₂ emissions. The overall protocol took 40 min for pulp extraction and 35 min for peel extraction using the UAE method with ultrasound power 450 W, whereas MPUAE used a 150 s irradiation pretreatment period with 900 W level and 19.70 min for pulp16.46 min for peel ultrasound extraction time. Therefore, the total bioactive substances were observed to be considerably higher in extraction in MPUAE. The previous study observed that the single-effect extraction strategy requires more energy and generates greater greenhouse gas emissions, however, the combined microwave-ultrasound system extraction method required the reverse [18]. Thus, it can be said that the established MPUAE was a green procedure for the bioactive extraction of papaya because a considerable reduction in extraction time was noticed in this technique.

Fig. 9.

Energy consumption and environmental effect of ultrasound-assisted extraction (UAE); and microwave pretreated ultrasound-assisted extraction (MPUAE) process.

4. Conclusions

This study highlights the successful optimization of the microwave pretreated ultrasound-assisted extraction (MPUAE) process for the extraction of bioactive compounds from papaya pulp and peel using response surface methodology (RSM) with the Box-Behnken design (BBD) tool. The results of this study indicate that the MPUAE process is an effective and environmentally sustainable approach for the extraction of bioactive compounds from papaya, with shorter extraction time and observable energy efficiency compared to the traditional ultrasound-associated extraction (UAE) technique. The acceptability attributes of all anticipated models were adequate and effectively employed for bioactive optimization. The findings of this study can guide future research on the development of innovative and sustainable techniques for the extraction of antioxidant compounds from plant materials in industrial applications using a combination of microwave radiation and ultrasonic waves based on several predictor variables effects that are optimizable.

While our study has made significant contributions to the optimization of bioactive compound extraction from papaya pulp and peel using Microwave Pretreated Ultrasound-Assisted Extraction (MPUAE), it is important to recognize certain limitations. As our investigation primarily focused on optimizing the extraction process and evaluating its environmental impact, we did not conduct a detailed assessment of potential changes or modifications to bioactive compounds during the extraction process. Further research is needed to comprehensively understand the extent to which microwave irradiation and ultrasound may impact the quality of these compounds. These limitations open avenues for future investigations, which can build upon our work and provide a more comprehensive understanding of bioactive compound extraction using MPUAE.

Funding details

The research work was funded by Shahjalal University of Science and Technology Research Center (Project ID: AS/2022/1/35).

CRediT authorship contribution statement

Rahul Biswas: Conceptualization, Methodology, Validation, Investigation, Writing – review & editing, Software, Formal analysis, Writing – original draft, Visualization, Data curation, Resources. Animesh Sarkar: Conceptualization, Methodology, Validation, Writing – review & editing, Funding acquisition, Software, Writing – original draft, Visualization, Supervision, Project administration, Resources. Mahabub Alam: Conceptualization, Methodology, Validation, Investigation, Writing – review & editing, Software, Formal analysis, Writing – original draft, Visualization, Data curation. Mukta Roy: Methodology, Validation, Writing – review & editing, Software. M.M. Mahdi Hasan: Investigation, Writing – review & editing, Formal analysis, Data curation.

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.

Data availability

Data will be made available on request.