The effects of different extraction methods on the yield, microstructure, and antioxidant activity of polysaccharides from Rhodomyrtus tomentosa berry

The effects of different extraction methods on the yield, microstructure, and antioxidant activity of polysaccharides from Rhodomyrtus tomentosa berry | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The effects of different extraction methods on the yield, microstructure, and antioxidant activity of polysaccharides from Rhodomyrtus tomentosa berry DINGJIN LI, Wan Zunairah Wan Ibadullah, Radhiah Shukri, Qiuxia Duan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5141599/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Extraction of polysaccharides from Rhodomyrtus tomentosa berry (RTP) is essential for understanding their bioactive ingredients and developing functional foods, nutraceuticals, and pharmaceuticals. This study aimed to evaluate the effects of ultrasonic-assisted enzymatic extraction (UAE) and microwave-assisted enzymatic extraction (MAE) on the yield, physicochemical properties, and antioxidant activity of (RTP). The single-factor and orthogonal experimental design revealed that the optimal conditions for ultrasound assisted RTP extraction are at liquid to solid ratio of 35 mL/g, ultrasonic time of 11 min, ultrasonic power of 240 W, and complex enzyme dose of 2.5% with RTP extraction yield of 35.67 ± 0.32%. FT-IR spectroscopy showed that UAE caused less disruption of the polysaccharide molecular structure and better retention of functional groups than MAE. The scanning electron microscopy results demonstrated that the ultrasonically treated samples exhibited a greater degree of structural disruption, which could more effectively facilitate the release of polysaccharides. In addition, RTP obtained by the UAE has a better extraction yield and ABTS radical scavenging activity than MAE. This study demonstrated that the UAE method is a promising method for extracting high-quality R. tomentosa berry polysaccharides based on its high yield, high efficiency, and outstanding antioxidant activity. Rhodomyrtus tomentosa berry polysaccharides ultrasound microwave antioxidant activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Rhodomyrtus tomentosa (Ait.) Hassk is an evergreen shrub. This plant is native to Southeast Asia and southern China and is commonly found in forests, thickets, and coastal dunes in tropical and subtropical regions [ 1 ]. Ripe Rhodomyrtus tomentosa ( R. tomentosa ) fruit is a rich source of dietary fiber (66.56%, wb), polysaccharides (17%, wb), and lipids (4.19%, wb) [ 2 ]. Plant polysaccharides have attracted extensive attention because of their unique biological effects, such as antioxidant, hypoglycemic, immune regulation, and regulating intestinal flora [ 3 – 4 ]. Optimizing the polysaccharide extraction and purification processes can greatly facilitate the analysis of the polysaccharide structure and functional activity. Extraction techniques and conditions strongly influence the compositions and microstructures of polysaccharides, resulting in beneficial or adverse effects on their chemical properties and bioactivities [ 5 ]. Hot water extraction is a traditional method for extracting polysaccharides. However, it has several drawbacks, such as time-consuming and low extraction yield [ 6 ]. Enzymatic extraction has become a widely adopted method for extracting polysaccharides because it’s ability to degrade cell wall structure to release the polysaccharides [ 7 ]. This method typically yields better results than traditional hot-water extraction. However, the extraction efficiency of enzymatic methods is suboptimal. This limitation may be attributed to the limited number of enzyme active sites available on the surface of glycoproteins [ 8 ]. To address these limitations, researchers have combined enzymatic methods with physical techniques to enhance the extraction efficiency [ 9 ]. Microwave-assisted enzymatic extraction (MAE) has been shown to increase the polysaccharide yields. The high-energy effect of microwaves causes partial degradation and hydrolysis of cellulose, hemicellulose, and other complex polysaccharides in the plant cell wall, hence relaxing the cell wall structure and increasing cell membrane permeability [ 10 ]. When combined with enzyme catalysis, this approach accelerates cell wall destruction and increases polysaccharide release [ 11 ]. Previous authors, Le et al. (2019) [ 12 ] reported that microwave-assisted enzyme extraction significantly improved the polysaccharide yield of Ulva pertusa to 41.91%. Ultrasound-assisted enzymatic extraction (UAE) has emerged as another effective technique for enhancing polysaccharide extraction. Ultrasonic waves create cavitation bubbles that collapse, generating high local temperatures and pressures. This mechanical effect can break cell walls more effectively, facilitating polysaccharide release. The cavitation effect accelerates mass transfer, resulting in shorter extraction times than conventional methods. UAE often allows extraction at low temperatures, which helps preserve heat-sensitive compounds and reduces energy consumption. When combined with enzyme extraction, ultrasound increases enzyme-substrate contact and enhances enzyme activity, thereby improving yields [ 13 ]. Given the potential benefits of MAE and UAE extraction, there is a need to directly compare these two extraction techniques to determine their relative effects on the yield, physicochemical properties, and antioxidant activity of plant polysaccharides. Although both methods have shown promising results, comparative studies that systematically evaluate MAE against UAE for polysaccharide extraction from R. tomentosa is limited. Therefore, this study aimed to investigate the effects of ultrasonic-assisted enzyme extraction (UAE) and microwave-assisted enzyme extraction (MAE) on the extraction yield, physicochemical properties, structural properties, and surface morphology of RTP. In addition, we examined the impact of these extraction methods on the antioxidant activity of RTP. By conducting this comparative analysis, we identified the most effective method for obtaining high-quality polysaccharides with enhanced bioactivity from R. tomentosa . Our results contribute to optimizing extraction processes for plant-derived polysaccharides and provide a scientific foundation for the application of R. tomentosa polysaccharides in functional foods. 2. METHODS 2.1. Materials and chemicals Ripe R. tomentosa berry was purchased from a local market (Hezhou, Guangxi, China). The fruits (5 kg) were dried using a heat pump dryer at 50°C at a relative humidity of 35% and a speed of 1.5 m/s,. The dried fruits were stored in glass desiccator. The dried fruits were ground, passed through a 40-mesh sieve, and then used for the extraction of polysaccharides. All chemicals were purchased from Solarbio Life Sciences Co., Ltd (Beijing, China). 2.2 Extraction of RTP using different techniques 2.2.1 Ultrasonic-assisted enzymatic extraction (UAE) The RTP was extracted using an ultrasonic-microwave extraction system (Sineo Microwave Chemistry Technology Co., Ltd, Shanghai, China). RTB powder (2 g) was mixed with the extraction solvent at various liquid-to-solid ratios (20–60 mL/g). The complex enzyme consisted of papain, pectinase and cellulase at a ratio of 1:1:2 was used. The extraction parameters investigated included liquid to solid ratio (20, 30, 40, 50, 60 mL/g), ultrasonication time (10, 15, 20, 25, 30 min), ultrasonic power (140, 180, 220, 260, 300 W) and complex enzyme dose (1.0, 2.0, 3.0, 4.0, 5.0%; complex enzyme dose: solid, w/w). After adding the enzyme complex and mixing well, ultrasonic extraction was started. Ultrasonication was performed using a probe with a frequency of 25 kHz operating in pulse mode (40 seconds on, 20 seconds off). Throughout the extraction process, the extreme temperature increase was controlled by placing the beaker containing the sample in an ice bath. One variable was studied at each experiment while the other factors were kept constant. All experiments were performed in three replicates. 2.2.2. Microwave-assisted enzymatic extraction (MAE) The RTP was extracted using ultrasonic-microwave extraction system (Sineo Microwave Chemistry Technology Co., Ltd, Shanghai, China). RTB powder (2 g) were used at different liquid-to-solid ratios. The complex enzyme consisted of papain, pectinase and cellulase at a ratio of 1:1:2 was used. The liquid to solid ratio (20, 30, 40, 50, 60 mL/g), microwave time (6, 8, 10, 12, 14 min), microwave power (100, 180, 300, 450, 600 W), and complex enzyme dose (1.0, 2.0, 3.0, 4.0, 5.0%, complex enzyme dose: solid, w/w). After adding the enzyme complex and mixing well, microwave extraction was started. Throughout the extraction process, the extreme temperature increase was controlled by placing the beaker containing the sample in an ice bath. One variable was studied at each experiment while the other factors were kept constant. All experiments were performed in three replications. 2.2.3. Orthogonal experimental design An L 9 (3 4 ) orthogonal experimental design with three levels was constructed to optimize the UAE. There were four main factors: A (liquid/solid ratio), B (extraction time), C (ultrasonic power), and D (complex enzyme dose). The experimental design was shown in Table 1 . Table 1 Orthogonal experimental design of the UAE Levels A (mL/g) B (min) C (W) D (%) 1 25 9 200 2.5 2 30 10 220 3.0 3 35 11 240 3.5 (A: liquid to solid ratio, B: ultrasonic time, C: ultrasonic power, D: complex enzyme dose, Y: extraction yield.) An L 9 (3 3 ) orthogonal experimental design with three levels was constructed to optimize the MAE. There were four main factors included A (liquid to solid ratio), B (extraction time), and C (complex enzyme dose). The experimental design was shown in Table 2 . Table 2 Orthogonal experimental design of the MAE Levels A (mL/g) B (min) C (%) D (W) 1 35 7 2.5 300 2 40 8 3 300 3 45 9 3.5 300 (A: liquid to solid ratio, B: microwave time, C: complex enzyme dose, Y: extraction yield.) 2.3. The extraction yield The polysaccharide content was measured according to the method reported by Liang et al. (2024) [ 14 ] with some modifications. The absorbance was determined using a spectrophotometer at 620 nm. The sample solution (2 mL), obtained directly from the extraction process in section 2.2.2 and 2.2.3 , was mixed with anthrone-sulfuric acid (6 mL) and the mixture was heated for 15 min at 100 ℃. The absorbance was determined using spectrophotometer at 620 nm. Standard curve was plotted using glucose as standard at concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL. The polysaccharide concentration in each sample solution was calculated from the absorbance value of the sample solution, and its polysaccharide content was calculated based on the Eq. ( 1 ). Where, C is the mass concentration of the RTP solution (mg/mL), N is the dilution ratio, V 0 is the total volume of RTP solution (mL), m is the weight of powder used for the extraction process (g). 2.4. Deproteinization Deproteinization of crude RTP was performed using trichloroacetic acid (TCA) [ 14 ]. To remove proteins from the crude polysaccharides, the same volume of 3% TCA was added to different crude polysaccharide solutions (100 ml) obtained from optimal extraction conditions of UAE and MAE. The mixture was allowed to stand overnight, and then the precipitate was removed by centrifugation (10 min, 10000 rpm), and the above procedure was repeated three times to obtain a polysaccharide solution without proteins. The deproteinized water phase was collected, condensed to one fifth of its total volume using a rotary evaporator, and precipitated with ethyl alcohol at 4°C for 24 h. The precipitates were freeze-dried to obtain polysaccharide samples, named as RTP UAE and RTP MAE , respectively. The dried polysaccharides were then subjected to subsequent analyses. 2.5. Physicochemical properties 2.5.1. UV-vis spectrum and FT-IR analysis The dried samples (4 mg) of RTP UAE and RTP MAE were dissolved in distilled water (4 mL), and the UV-vis absorption spectrum of the solution was recorded in the wavelength range of 200–500 nm [ 15 ]. The dried samples (3 mg) of RTP UAE and RTP MAE were mixed with KBr (150 mg) and pressed into a disk to obtain a pellet (1.0 mm). The Fourier transform infrared spectroscopy (FT-IR) was used to determine samples between the wavelength range of 400 and 4000 cm − 1 [ 15 ]. 2.5.2. Scanning electron microscope (SEM) analysis The morphology of RTP UAE and RTP MAE were analyzed using a scanning electron microscopy. The dried samples (3 mg) were applied to the sample stage and coated with a layer of conductive gold film. The accelerated voltage was set as 15 kV [ 16 ]. 2.6. Antioxidant capacity assay 2.6.1. Scavenging DPPH radical activity assay The DPPH radical scavenging activity was measured according to a previous method with slight modifications [ 17 ]. Different concentrations of RTP UAE and RTP MAE (1 mL; 0.25, 0.5 1.0, 2.0, and 4.0 mg/mL) were mixed with DPPH ethanolic solution (4 mL, 0.1 mM). The mixture was reacted at room temperature in a dark for 30 min. The absorbance of the sample was measured at 517 nm. The positive control was ascorbic acid (VC). Eq. ( 2 ) was used to calculate the scavenging rate. Where, A 0 control solution (no sample) and A 1 is the sample solution. 2.6.2. Scavenging ABTS radical activity assay The ABTS radical scavenging activity was assessed using the method described by Mohammed et al. (2020) [ 18 ] with minor modifications. The ABTS radical solution (4 mL, 7 mM) was combined with 1.0 mL of the sample solution (0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL). After reaction at 25 ºC for 6 min, absorbance was measured at 734 nm. The positive control was ascorbic acid (VC). Eq. ( 3 ) was used to calculate the scavenging rate. Where A 0 is the control group; A 1 is the sample group. 2.7 Statistical analysis All the experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD). Prism 10 (GraphPad., Massachusetts, USA) was used for statistical analysis and figure drawing. 3. RESULTS 3.1. Effect of UAE conditions on the extraction yield of polysaccharides 3.1.1 Liquid to solid ratio The effect of the liquid to solid ratio (20, 30, 40, 50, and 60 mL/g) on the extraction yield of RTP UAE was investigated under extraction time of 10 min, ultrasonic power of 220 W, and complex enzyme dose of 3% (w/w). As shown in Fig. 1 (A), the yield of RTP UAE gradually increased as the liquid to solid ratio increased, reaching its maximum value (34.06 ± 0.65%) at 30 mL/g. Increasing the liquid to solid ratio may increase the diffusivity of the solvent in the cell and promote the desorption of polysaccharides from the material. However, a decrease in the extraction rate was observed at higher liquid to solid ratio (> 30 ml/g), which may be due to the dilution effect of the complex enzyme. This dilution reduces enzyme-substrate collision frequency, potentially reducing the reaction rate [ 19 ]. Therefore, 30 mL/g was an appropriate liquid to solid ratio for the UAE. 3.1.2 Extraction time To determine the effect of extraction time (6, 8,10, 12, and 14 min) on the RTP UAE extraction yield, the UAE was performed under the liquid to solid ratio of 30 mL/g, ultrasonic power of 220 W, and complex enzyme dose of 3%. Figure 1 (B) showed that there is an increasing trend in extraction yield from 6 to 10 min; the yield reached the maximum value at 10 min, followed by a significant reduction in yield with further increase in the extraction time. The shorter optimal time for RTP extraction may be due to the synergistic effect of ultrasound and enzymatic treatment, which accelerates the extraction process. The increase in extraction yield with extraction time is attributed to the more cell wall disruption by the ultrasonic cavitation and enzymatic activity, which enhanced the mass transfer of polysaccharide from the plant matrix into the extraction solution [ 13 ]. However, the subsequent decrease after 10 min can be attributed to polysaccharide degradation. Ultrasonic treatment has been reported to cause the cleavage of glycosidic bonds via mechanical, sonochemical, and localized heating. Collectively, these mechanisms contributed to the decrease in polysaccharide extraction with increasing ultrasound time [ 20 ]. Therefore, extraction times of 9, 10, and 11 min were selected for the orthogonal experimental design. 3.1.3. Ultrasonic power Ultrasonic power is also one of the most important factors because it can affect the enzyme activity as well as the movement and stability of polysaccharides molecules in the extraction system. RTP UAE were extracted under different ultrasonic power (140, 180, 220, 260, and 300 W). The other extraction conditions were as follows: liquid to solid ratio of 30 mL/g, extraction time of 10 min, and complex enzyme dose of 3%. Figure 1 C demonstrated that the extraction rate gradually increased with increasing ultrasonic power. The highest extraction yield (34.06 ± 0.65%.) was obtained at ultrasonic power of 220 W. However, the extraction yield decreased when the ultrasonic power exceeded 220W. Appropriate ultrasonic power promotes the release of polysaccharides through cavitation. The tiny bubbles formed during cavitation burst in the liquid, generating localized high temperature, high pressure, and strong shear force. These extreme conditions can destroy plant cell walls and enhance the extraction of polysaccharides [ 13 ]. However, excessive intensity of ultrasound power can lead to the destruction of polysaccharide structure, such as degradation of polysaccharides which reduces the extraction rate of polysaccharides [ 21 ]. Precise control of ultrasound parameters is needed to achieve optimal extraction while avoiding polysaccharide degradation. Therefore, the ultrasonic power of 220W was chosen for the UAE. 3.1.4. Complex enzyme dose The plant cell wall consists of a rigid skeleton of cellulose embedded in a gel-like matrix, the main components of which are pectic compounds, hemicellulose, and glycoprotein. To enhance the extraction speed and yield of polysaccharides, complex enzymes, including cellulose, pectinase, and papain, are used to break the plant cell wall and hydrolyze structural polysaccharides [ 8 ]. RTP UAE were extracted under different complex enzyme doses (1, 2, 3, 4, and 5%), and the other extraction conditions were as follows: liquid to solid ratio of 30 mL/g, extraction time of 10 min, and ultrasonic power of 220 W. Figure 1 (D) showed that when the complex enzyme dose increased from 1–3%, the extraction yield of RTP remarkably increased to reach the maximum value at 3%. Meanwhile, with a further increase in the enzyme dose, the extraction yield decreased. The initial increase in yield with enzyme concentration can be attributed to increased hydrolysis of cell wall components by the respective enzymes, facilitating polysaccharide release. Additionally, synergistic effects between different enzymes may occur, where the action of one enzyme exposes the enzymes to other substrates [ 11 ]. The observed decrease in extraction yield at higher complex enzyme dose (> 3%) may be due to intensified enzyme aggregation resulting from the cavitation effect of ultrasound, which reduces effective enzyme-substrate interactions [ 19 ]. Thus, the range of the complex enzyme dose for further optimization by orthogonal experiment was selected as 2.5–3.5%. 3.1.5. Orthogonal experimental design optimization results for the UAE Based on the single-factor experimental results, three levels of liquid to solid ratio, extraction time, ultrasonic power, and complex enzyme were chosen. An orthogonal experimental design L 9 (3 4 ) was performed to further optimize the UAE (Table 1 ). The results are shown in Table 3 . Using the comparison of R values obtained from the extreme difference analysis, the order of effect of experimental factors on the extraction yield was A > C > D > B, which is liquid to solid ratio > ultrasonic power > complex enzyme dose > ultrasonic time. In addition, polynomial regression analysis of the orthogonal test results (Table 4 ) showed that the model had a P-value of 0.0002, which is below the commonly used significant level of 0.05. This demonstrated that the model was highly statistically significant. Based on the‾k values, the optimal parameters would be the combination A 3 B 3 C 2 D 1 . Therefore, the optimal ultrasound-assisted extraction process was a liquid to solid ratio of 35 mL/g, ultrasonication time of 11 min, ultrasonic power of 240 W, and complex enzyme dose of 2.5%. The verification experiment performed on the basis of the condition stated resulted in an extraction yield of 35.67 ± 0.32%. Table 3 Results of orthogonal experimental design for UAE No. A (mL/g) B (min) C (W) D (%) Y (%) 1 25 9 200 2.5 24.60 ± 0.47 2 25 10 220 3.0 28.50 ± 0.08 3 25 11 240 3.5 29.27 ± 0.44 4 30 9 220 3.5 28.53 ± 0.36 5 30 10 240 2.5 25.12 ± 0.28 6 30 11 200 3.0 18.73 ± 0.82 7 35 9 240 3.0 24.77 ± 0.37 8 35 10 200 3.5 25.65 ± 0.78 9 35 11 220 2.5 35.51 ± 0.33 K 1 82.37 77.9 68.98 85.23 K 2 72.38 79.27 92.54 72.00 K 3 85.93 83.51 79.16 83.45 ‾k 1 27.46 25.97 22.99 28.41 ‾k 2 24.13 26.42 30.85 24.00 ‾k 3 28.64 27.84 26.39 27.82 Best levels A 3 B 3 C 2 D 1 R 4.52 1.87 4.46 4.41 (A: liquid to solid ratio, B: ultrasonic time, C: ultrasonic power, D: complex enzyme dose, Y: extraction yield. Where 1–3 are the levels for each condition tested. ) Table 4. Results of orthogonal experimental design for MAE No. A (mL/g) B (min) C (%) Y (%) 1 35 7 2.5 20.88±0.91 2 35 8 3.0 20.60±0.64 3 35 9 3.5 21.94±0.41 4 40 7 3.0 21.43±0.86 5 40 8 3.5 20.54±0.14 6 40 9 2.5 19.71±0.61 7 45 7 3.5 19.63±0.56 8 45 8 2.5 27.03±0.33 9 45 9 3.0 26.21±0.62 K 1 63.43 61.94 67.63 K 2 61.67 68.17 59.95 K 3 72.88 67.87 70.41 `k 1 21.14 20.65 22.54 `k 2 20.56 22.72 19.98 `k 3 24.29 22.62 23.47 Best levels A 3 B 2 D 2 R 3.73 2.08 3.49 (A: liquid to solid ratio, B: microwave time, C: complex enzyme dose, Y: extraction yield. Where 1-3 are the levels for each condition tested.) 3.2. Effect of MAE conditions on the yield of polysaccharides 3.2. Liquid to solid ratio The liquid to solid ratio was a key factor influencing the extraction efficiency. The effect of different liquid to solid ratio (20, 30, 40, 50, and 60 mL/g) on the yield of RTP MAE were tested at microwave time of 10 min, microwave power of 300 W, and complex enzyme dose of 3%. Figure 2 A showed that RTP MAE extraction yield increased significantly when the liquid to solid ratio increased from 20 to 40 mL/g, but decreased when the liquid to solid ratio was higher than 50 mL/g. As the liquid-to-solid ratio increased, more liquid allowed for better dispersion of the solids, increasing the surface area available for enzymatic action. This helps facilitate the release of more polysaccharides from the plant matrix [ 22 ]. However, when the liquid-to-solid ratio becomes excessively high, the extraction yield decreases because of a combination of factors. The higher amount of liquid caused dispersion of the microwave energy over a larger area, potentially reducing the intensity of localized heating. This phenomenon, coupled with the dilution of enzymes, leads to less efficient extraction. The excess liquid also altered the mass transfer dynamics and enzyme-substrate interactions, further contributing to the decreased yield [ 9 , 11 ]. Thus, a liquid to solid ratio of 40 mL/g was chosen for further experiments. 3.1.2. Microwave time Different microwave times were set at 6, 8, 10, 12, and 14 min to investigate the effect of microwave time on the extraction yields of RTP MAE . The other conditions were set as follows: liquid to solid ratio of 40 mL/g, microwave power of 300 W, and complex enzyme dose of 3%. As shown in Fig. 2 (B), the extraction yield of RTP MAE reached a maximum value of 25.41 ± 0.31% at 8 min. This extraction time was shorter than that reported for conventional heating methods, demonstrating the efficiency of microwave-assisted extraction [ 12 ]. However, the subsequent decrease in yield with extended microwave exposure time was likely due to thermal degradation of the polysaccharides. An excessive microwave exposure time can lead to thermal degradation of polysaccharides, breaking down their long chains into smaller fragments [ 11 ]. This degradation can result in the loss of functional properties, such as viscosity, gel formation ability, and bioactivity, which are crucial for their applications in the food, nutraceutical, and pharmaceutical industries [ 23 ]. It is vital to balance microwave time to maximize the extraction yield of polysaccharides while preserving their structural integrity and functional properties. Therefore, 7, 8, and 9 min were selected for further optimization experiments. 3.2.3. Microwave power The effect of microwave power (100, 180, 300, 450 and 600 W) on the extraction yield of RTP MAE was explored at a liquid-solid ratio of 40 mL/g, a microwave time of 8 min, and an enzyme complex dose of 3%. Figure 2 C showed that the extraction yield of RTP MAE increased from 17.75 ± 0.77% to 25.41 ± 0.31% when the microwave power was increased from 100 to 300 W. Increasing the microwave power during the extraction process enhanced the solubility of the sample. This is because higher microwave power produces heat more efficiently. This elevated heat promotes the breakdown of plant cell walls and facilitates the release of polysaccharides into the solution, thereby improving the extraction yield [ 9 ]. However, the extraction yield of RTP MAE decreased when the microwave power exceeded 300 W, which was due to thermal degradation. At higher microwave powers, the intense heat can break down the polysaccharide chains into smaller fragments, reducing the overall yield [ 24 ]. In this study, because there are only five fixed parameters, 100, 180, 300, 450, and 600 W, for the microwave oven used, 300 W was chosen as the optimal microwave power, and there was no need to optimize the microwave power through an orthogonal experimental design. 3.2.4. Complex enzyme dose The effects of the complex enzyme dose (1, 2, 3, 4, and 5%) on the extraction yield of RTP MAE were explored at a liquid-to-solid ratio of 40 mL/g, microwave time of 8 min, and microwave power of 300 W. Figure 2 D showed the effects of the complex enzyme dose on the yield of RTP MAE . When the complex enzyme dose was between 1% and 3%, the extraction yield was positively correlated with the enzyme concentration. A suitable complex enzyme concentration can exert a better synergistic effect and effectively promote the degradation of the cell wall, thereby promoting the release of polysaccharides [ 24 ]. However, above 3%, the extraction yield decreased slightly. This may be because the complex enzyme dose was too high; there may have been competitive inhibition between the enzymes, leading to a decrease in the extraction yield. In this context, competitive inhibition likely occurs when a high enzyme concentration leads to over-crowding of enzymes. As a result, enzyme molecules may physically obstruct each other, competing for access to limited substrate-binding sites, reducing the overall efficiency of the extraction process [ 25 ]. Therefore, a complex enzyme of 3% is considered the optimal dose for extracting RTP MAE . 3.2.5. Orthogonal experimental design optimization results for the MAE The statistical analysis of single-factor experiments showed the factors of liquid to solid ratio, microwave time, microwave power, and complex enzyme dose significantly affect the yield of RTP MAE . An orthogonal experimental design L 9 (3 3 ) was carried out to optimize the MAE (Table 2 ). In addition, the equipment used in this study has only five fixed parameters: 100, 180, 300, 450, and 600 W. Therefore, 300 W was chosen as the optimal microwave power, and there was no need to optimize the microwave power through an orthogonal experimental design. Three main factors (liquid to solid ratio, microwave time, and complex enzyme dose) and three levels for each factor were selected for further optimization. As showed in Table 5 , using the comparison of R values obtained from the extreme difference analysis, the effect order of experimental factors on the extraction yield was A > C > B (Liquid to solid ratio > complex enzyme dose > microwave time). In addition, polynomial regression analysis of the orthogonal test results (Table 6 ) showed that the model had a P-value of 0.0001, which is below the commonly used significant level of 0.05. This demonstrated that the model was highly statistically significant. Therefore, based on the‾k values, the optimal parameters would be the combination A 3 B 2 C 2 (where 1–3 are levels). However, the optimal extraction conditions were not included in the orthogonal table; the confirmatory experiment was conducted. The verification experiment showed that the extraction yield of RTP MAE reached 27.83 ± 0.21%, which was higher than that of the optimal group 8 in the orthogonal experimental design. Therefore, the optimal MAE process was as follows: microwave power 300 W, liquid to solid ratio 40 mL/g, microwave time 8 min, complex enzyme dose 2.5%. 3.3. Physicochemical properties of RTP 3.3.1. UV-vis and FT-IR analysis Fig. 3A and B show that RTPUAE and RTPMAE did not have distinct absorption peaks on the UV-vis spectrum at 260 and 280 nm wavelengths, indicating that they did not contain proteins and nucleic acids. The physicochemical analysis we analyzed only for both RTP UAE and RTP MAE obtain at optimum extraction conditions. The FT-IR spectrum of two RTPs was presented in Fig. 3A. In the 3600–3200 cm −1 region, the strong broadbands represent the O–H stretching vibration [26]. The absorption peak around 2925 cm −1 was mainly caused by the asymmetric C–H stretching vibrations of the CH, CH 2 , and CH 3 groups [26]. The peak of about 1744 cm −1 was a characteristic of the C=O stretch of carboxylic acid. The peak at 1220 cm −1 indicated the presence of C-O stretching. RTP UAE and RTP MAE showed strong absorption peak bands in the 1000–1200 cm −1 region, indicating that these polysaccharides contain C-O-C glycosidic bonds. It is noteworthy that the characteristic peaks of RTP MAE have a higher intensity than those of RTP UAE . This may be attributed to the mild physical action of ultrasound, which does not easily destroy the structures of the major functional groups of polysaccharides [27]. The lower peak intensities indicate that the extraction process causes less damage to the molecular structure of the polysaccharides and that the functional groups of the polysaccharides remain more intact, which implied that the extracted polysaccharides may retain their natural activity and functional properties (Wang et al., 2018). In addition, because of the intense heating effect of microwaves, degradation of part of the molecular structure of polysaccharides may occur, affecting the distribution and strength of functional groups. Some functional groups may be destroyed or transformed, resulting in higher intensity of characteristic peaks in FT-IR spectra [28]. 3.3.2. Microscopy analysis The SEM images of the two polysaccharides shown in Fig. 4 indicated different morphologies in RTP powder extracted by the different methods. The surface of RTP MAE possessed a compact morphology with a relatively smooth surface. While in RTP UAE, the surface was rough, wrinkled, and irregular. This indicated that the UAE disrupted the original plant structure more effectively than MAE. This was due to cavitation effects that caused cell walls to degrade, thereby enhancing the release of intracellular materials and promoting higher polysaccharide extraction efficiency [ 29 ]. 3.4. Antioxidant Activity Analysis 3.4.1. The DPPH radical scavenging activity The DPPH radical is a tool for the assay of the radical scavenging activities of antioxidants. The DPPH radical scavenging activity of RTP UAE and RTP MAE are shown in Fig. 5 A. The DPPH radical scavenging rate of RTP UAE and RTP MAE were 75.91% and 73.52%, respectively, at a sample concentration of 4.0 mg/mL. Moreover, the IC 50 values of RTP UAE and RTP MAE were 2.25 and 2.19 mg/mL, respectively. However, The DPPH radical scavenging ability of RTP UAE was not significantly different from that of RTP MAE . 3.4.2. The ABTS radical scavenging activity The ABTS + radical scavenging activity of RTP UAE and RTP MAE are shown in Fig. 4B. The results shown that the two types of RTP had a positive scavenging effect on the ABTS radical. RTP UAE had a higher ABTS radical scavenging ability than RTP MAE at the same doses. In addition, at a sample concentration of 4.0 mg/mL, the scavenging rates of RTP MAE and RTP UAE reached 63.32% and 86.12%, respectively. The IC 50 values of RTP UAE and RTP MAE were 2.21 and 3.19 mg/mL, respectively. Our results demonstrated that RTP UAE has stronger ABTS radical scavenging activity than RTP MAE . This is because ultrasound enhances enzyme activity and improves extraction efficiency by creating cavitation and micro-streaming effects, leading to better preservation of the polysaccharide structure and heat-sensitive antioxidant components compared with the uneven heating and potential degradation caused by microwave [31]. 4. CONCLUSIONS The effects of different extraction methods (UAE and MAE) on RTP polysaccharide yield, physicochemical properties, and antioxidant activity were evaluated. An orthogonal experimental design was used to optimize the extraction parameters. The results indicated that RTP extracted using the UAE method had a higher extraction yield. The optimal parameters for using UAE to extract RTP were a liquid-to-solid ratio of 35 mL/g, ultrasonic duration of 11 min, ultrasonic power of 240 W, and complex enzyme dose of 2.5%. FT-IR spectroscopy showed that UAE caused less disruption of the polysaccharide molecular structure and better retention of functional groups than MAE. Surface morphology showed that greater matrix changes occurred in the ultrasound-treated samples, allowing more polysaccharides to be extracted. This study demonstrated that the UAE method is a promising method for extracting high-quality R. tomentosa berry polysaccharides based on its high yield, high efficiency, and outstanding antioxidant activity. Declarations Credit authorship contribution statement Dingjin Li, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Writing – original draft. Wan Zunairah Wan Ibadullah, Investigation, Validation. Radhiah Shukri , Investi­gation, Validation. Qiuxia Duan, Methodology, data curation. Yipeng Gu, Investigation, Validation. Nor Afizah Mustapha, Writing – review & editing ,Investigation, Supervision, Validation. All authors read and approved the final manuscript. Funding: This study was funded by the Disciplinary Interdisciplinary and Collaborative Research Project of Hezhou University (Grant No: XKJC202401), the Agricultural Science and Technology Self-financing Funding Project of Guangxi (Grant No: Z2024049). 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 30 Sep, 2024 Reviews received at journal 28 Sep, 2024 Reviews received at journal 27 Sep, 2024 Reviewers agreed at journal 26 Sep, 2024 Reviewers agreed at journal 26 Sep, 2024 Reviewers agreed at journal 26 Sep, 2024 Reviewers agreed at journal 26 Sep, 2024 Reviewers invited by journal 26 Sep, 2024 Editor assigned by journal 25 Sep, 2024 Submission checks completed at journal 24 Sep, 2024 First submitted to journal 23 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5141599","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":360565018,"identity":"90773437-a75b-4805-b295-150ba4da1b2b","order_by":0,"name":"DINGJIN LI","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"DINGJIN","middleName":"","lastName":"LI","suffix":""},{"id":360565019,"identity":"4cc597eb-96a3-4e1a-ad2b-5ffe08d93f4a","order_by":1,"name":"Wan Zunairah Wan Ibadullah","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Wan","middleName":"Zunairah Wan","lastName":"Ibadullah","suffix":""},{"id":360565020,"identity":"57df239a-e28a-409e-8bfc-1824804c4268","order_by":2,"name":"Radhiah Shukri","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Radhiah","middleName":"","lastName":"Shukri","suffix":""},{"id":360565021,"identity":"5fcc96a3-b4c8-43ee-8cd6-c7794d6db951","order_by":3,"name":"Qiuxia Duan","email":"","orcid":"","institution":"Hezhou University","correspondingAuthor":false,"prefix":"","firstName":"Qiuxia","middleName":"","lastName":"Duan","suffix":""},{"id":360565022,"identity":"c2f489d4-8a4a-4dda-b721-7956f180da18","order_by":4,"name":"Yipeng Gu","email":"","orcid":"","institution":"Hezhou University","correspondingAuthor":false,"prefix":"","firstName":"Yipeng","middleName":"","lastName":"Gu","suffix":""},{"id":360565023,"identity":"39dfc996-9b8a-46a2-846f-f1183bee3bf3","order_by":5,"name":"Nor Afizah Mustapha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYNACGwkGBgnmAxDOAaK0pIG0sCWQpAWIJXgMiNNizt77gOFHgoU9/+yeb9KFOQxyfDcSWDfz4NFi2XPcgLEnQYJZ4s7ZbdIztzEYS95IYLuNT4vBDaCjeH9IsDHcyN0mzbuNIXEDMVoY/yRI8MjfyHkG0lJPlBZmngQJCYMbOWwgLQkGBLWcOcbALJMgYWB4I83YeuY2CcOZZx623ZyDT8vxNgbGNwl19nI3kh/eLtxmI893PPnYjTd4tAAB+w8YixkYO0CKsYEJn8NQADOMwfgDn7JRMApGwSgYaQAANqNI6t1qOI0AAAAASUVORK5CYII=","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Nor","middleName":"Afizah","lastName":"Mustapha","suffix":""}],"badges":[],"createdAt":"2024-09-24 04:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5141599/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5141599/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68256622,"identity":"c953926f-d5eb-4803-a0cc-f7a2c69d3498","added_by":"auto","created_at":"2024-11-05 11:14:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-C\u003c/strong\u003e Effects of different extraction factors on the RTP extraction yield. Figure 1 A. Effects of liquid to solid ratio on the RTP extraction yield. Figure 1 B. Effects of ultrasonic time on the RTP extraction yield. Figure 1C. Effects of ultrasonic power on the RTP extraction yield. Figure 1D. Effects of complex enzyme dose on the RTP extraction yield.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5141599/v1/73ec18a494c846b3700d4aa4.png"},{"id":68257021,"identity":"093e35a6-e359-4cbc-99c4-c65b90c99aec","added_by":"auto","created_at":"2024-11-05 11:22:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-C\u003c/strong\u003e Effects of different extraction factors on the RTP extraction yield. Figure 2 A. Effects of liquid to solid ratio on the RTP extraction yield. Figure 2 B. Effects of microwave time on the RTP extraction yield. Figure 2 C. Effects of microwave power on the RTP extraction yield. Figure 2 D. Effects of complex enzyme dose on the RTP extraction yield.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5141599/v1/c0aafb794cbc1fd722dfe2e8.png"},{"id":68256620,"identity":"86b4a9d8-f97c-4615-8fdf-ed73c9d1b588","added_by":"auto","created_at":"2024-11-05 11:14:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-B\u003c/strong\u003e UV spectra and FT-IR spectra of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e. Figure 3 A. UV spectra of RTP\u003csub\u003eUAE.\u003c/sub\u003e Figure 3 B. UV spectra of RTP\u003csub\u003eMAE.\u003c/sub\u003e Figure 3 C.\u0026nbsp; FT-IR spectra of RTP\u003csub\u003eUAE\u003c/sub\u003e. Figure 3 D. FT-IR spectra of RTP\u003csub\u003eMAE\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5141599/v1/fd87b1e3fd3e15f139e2bb1d.png"},{"id":68257022,"identity":"b91fda2d-5142-4da7-99c1-0881d8ceef61","added_by":"auto","created_at":"2024-11-05 11:22:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":170424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-B \u003c/strong\u003eThe SEM micrographs of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE \u003c/sub\u003e\u0026nbsp;at 1000-magnification. Figure 4 A. The SEM micrographs of RTP\u003csub\u003eUAE.\u003c/sub\u003e Figure 4 B. The SEM micrographs of RTP\u003csub\u003eMAE\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5141599/v1/2d583f571437198028b24cef.png"},{"id":68256623,"identity":"38e2b9ba-78fc-4808-be2b-3738ede04aed","added_by":"auto","created_at":"2024-11-05 11:14:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-B\u003c/strong\u003e\u0026nbsp; Antioxidant activity of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e. \u0026nbsp;Figure 4 A. DPPH radical scavenging activity. Figure 4 B. ABTS\u003csup\u003e \u003c/sup\u003eradical scavenging activity.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5141599/v1/086ae091ae0f8414052a75e1.png"},{"id":68257954,"identity":"06386ecc-497f-4712-adfe-2581f1ad49dc","added_by":"auto","created_at":"2024-11-05 11:30:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1200753,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5141599/v1/d254400d-d666-417b-b136-e791df798d3a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effects of different extraction methods on the yield, microstructure, and antioxidant activity of polysaccharides from Rhodomyrtus tomentosa berry","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003e \u003cem\u003eRhodomyrtus tomentosa\u003c/em\u003e (Ait.) Hassk is an evergreen shrub. This plant is native to Southeast Asia and southern China and is commonly found in forests, thickets, and coastal dunes in tropical and subtropical regions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Ripe \u003cem\u003eRhodomyrtus tomentosa\u003c/em\u003e (\u003cem\u003eR. tomentosa\u003c/em\u003e) fruit is a rich source of dietary fiber (66.56%, wb), polysaccharides (17%, wb), and lipids (4.19%, wb) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Plant polysaccharides have attracted extensive attention because of their unique biological effects, such as antioxidant, hypoglycemic, immune regulation, and regulating intestinal flora [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Optimizing the polysaccharide extraction and purification processes can greatly facilitate the analysis of the polysaccharide structure and functional activity.\u003c/p\u003e \u003cp\u003eExtraction techniques and conditions strongly influence the compositions and microstructures of polysaccharides, resulting in beneficial or adverse effects on their chemical properties and bioactivities [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Hot water extraction is a traditional method for extracting polysaccharides. However, it has several drawbacks, such as time-consuming and low extraction yield [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Enzymatic extraction has become a widely adopted method for extracting polysaccharides because it\u0026rsquo;s ability to degrade cell wall structure to release the polysaccharides [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This method typically yields better results than traditional hot-water extraction. However, the extraction efficiency of enzymatic methods is suboptimal. This limitation may be attributed to the limited number of enzyme active sites available on the surface of glycoproteins [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo address these limitations, researchers have combined enzymatic methods with physical techniques to enhance the extraction efficiency [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Microwave-assisted enzymatic extraction (MAE) has been shown to increase the polysaccharide yields. The high-energy effect of microwaves causes partial degradation and hydrolysis of cellulose, hemicellulose, and other complex polysaccharides in the plant cell wall, hence relaxing the cell wall structure and increasing cell membrane permeability [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. When combined with enzyme catalysis, this approach accelerates cell wall destruction and increases polysaccharide release [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Previous authors, \u003cem\u003eLe et al.\u003c/em\u003e (2019) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] reported that microwave-assisted enzyme extraction significantly improved the polysaccharide yield of \u003cem\u003eUlva pertusa\u003c/em\u003e to 41.91%. Ultrasound-assisted enzymatic extraction (UAE) has emerged as another effective technique for enhancing polysaccharide extraction. Ultrasonic waves create cavitation bubbles that collapse, generating high local temperatures and pressures. This mechanical effect can break cell walls more effectively, facilitating polysaccharide release. The cavitation effect accelerates mass transfer, resulting in shorter extraction times than conventional methods. UAE often allows extraction at low temperatures, which helps preserve heat-sensitive compounds and reduces energy consumption. When combined with enzyme extraction, ultrasound increases enzyme-substrate contact and enhances enzyme activity, thereby improving yields [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Given the potential benefits of MAE and UAE extraction, there is a need to directly compare these two extraction techniques to determine their relative effects on the yield, physicochemical properties, and antioxidant activity of plant polysaccharides. Although both methods have shown promising results, comparative studies that systematically evaluate MAE against UAE for polysaccharide extraction from \u003cem\u003eR. tomentosa\u003c/em\u003e is limited.\u003c/p\u003e \u003cp\u003eTherefore, this study aimed to investigate the effects of ultrasonic-assisted enzyme extraction (UAE) and microwave-assisted enzyme extraction (MAE) on the extraction yield, physicochemical properties, structural properties, and surface morphology of RTP. In addition, we examined the impact of these extraction methods on the antioxidant activity of RTP. By conducting this comparative analysis, we identified the most effective method for obtaining high-quality polysaccharides with enhanced bioactivity from \u003cem\u003eR. tomentosa\u003c/em\u003e. Our results contribute to optimizing extraction processes for plant-derived polysaccharides and provide a scientific foundation for the application of \u003cem\u003eR. tomentosa\u003c/em\u003e polysaccharides in functional foods.\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Materials and chemicals\u003c/h2\u003e\n \u003cp\u003eRipe \u003cem\u003eR. tomentosa\u003c/em\u003e berry was purchased from a local market (Hezhou, Guangxi, China). The fruits (5 kg) were dried using a heat pump dryer at 50\u0026deg;C at a relative humidity of 35% and a speed of 1.5 m/s,. The dried fruits were stored in glass desiccator. The dried fruits were ground, passed through a 40-mesh sieve, and then used for the extraction of polysaccharides.\u003c/p\u003e\n \u003cp\u003eAll chemicals were purchased from Solarbio Life Sciences Co., Ltd (Beijing, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Extraction of RTP using different techniques\u003c/h2\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.1 Ultrasonic-assisted enzymatic extraction (UAE)\u003c/h2\u003e\n \u003cp\u003eThe RTP was extracted using an ultrasonic-microwave extraction system (Sineo Microwave Chemistry Technology Co., Ltd, Shanghai, China). RTB powder (2 g) was mixed with the extraction solvent at various liquid-to-solid ratios (20\u0026ndash;60 mL/g). The complex enzyme consisted of papain, pectinase and cellulase at a ratio of 1:1:2 was used. The extraction parameters investigated included liquid to solid ratio (20, 30, 40, 50, 60 mL/g), ultrasonication time (10, 15, 20, 25, 30 min), ultrasonic power (140, 180, 220, 260, 300 W) and complex enzyme dose (1.0, 2.0, 3.0, 4.0, 5.0%; complex enzyme dose: solid, w/w). After adding the enzyme complex and mixing well, ultrasonic extraction was started. Ultrasonication was performed using a probe with a frequency of 25 kHz operating in pulse mode (40 seconds on, 20 seconds off). Throughout the extraction process, the extreme temperature increase was controlled by placing the beaker containing the sample in an ice bath. One variable was studied at each experiment while the other factors were kept constant. All experiments were performed in three replicates.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.2. Microwave-assisted enzymatic extraction (MAE)\u003c/h2\u003e\n \u003cp\u003eThe RTP was extracted using ultrasonic-microwave extraction system (Sineo Microwave Chemistry Technology Co., Ltd, Shanghai, China). RTB powder (2 g) were used at different liquid-to-solid ratios. The complex enzyme consisted of papain, pectinase and cellulase at a ratio of 1:1:2 was used. The liquid to solid ratio (20, 30, 40, 50, 60 mL/g), microwave time (6, 8, 10, 12, 14 min), microwave power (100, 180, 300, 450, 600 W), and complex enzyme dose (1.0, 2.0, 3.0, 4.0, 5.0%, complex enzyme dose: solid, w/w). After adding the enzyme complex and mixing well, microwave extraction was started. Throughout the extraction process, the extreme temperature increase was controlled by placing the beaker containing the sample in an ice bath. One variable was studied at each experiment while the other factors were kept constant. All experiments were performed in three replications.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.3. Orthogonal experimental design\u003c/h2\u003e\n \u003cp\u003eAn L\u003csub\u003e9\u003c/sub\u003e (3\u003csup\u003e4\u003c/sup\u003e) orthogonal experimental design with three levels was constructed to optimize the UAE. There were four main factors: A (liquid/solid ratio), B (extraction time), C (ultrasonic power), and D (complex enzyme dose). The experimental design was shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eOrthogonal experimental design of the UAE\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLevels\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA (mL/g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB (min)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC (W)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e(A: liquid to solid ratio, B: ultrasonic time, C: ultrasonic power, D: complex enzyme dose, Y: extraction yield.)\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAn L\u003csub\u003e9\u003c/sub\u003e (3\u003csup\u003e3\u003c/sup\u003e) orthogonal experimental design with three levels was constructed to optimize the MAE. There were four main factors included A (liquid to solid ratio), B (extraction time), and C (complex enzyme dose). The experimental design was shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eOrthogonal experimental design of the MAE\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLevels\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA (mL/g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB (min)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD (W)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e(A: liquid to solid ratio, B: microwave time, C: complex enzyme dose, Y: extraction yield.)\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. The extraction yield\u003c/h2\u003e\n \u003cp\u003eThe polysaccharide content was measured according to the method reported by \u003cem\u003eLiang et al.\u003c/em\u003e (2024) [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e] with some modifications. The absorbance was determined using a spectrophotometer at 620 nm. The sample solution (2 mL), obtained directly from the extraction process in section \u003cspan class=\"InternalRef\"\u003e2.2.2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2.2.3\u003c/span\u003e, was mixed with anthrone-sulfuric acid (6 mL) and the mixture was heated for 15 min at 100 ℃. The absorbance was determined using spectrophotometer at 620 nm. Standard curve was plotted using glucose as standard at concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL. The polysaccharide concentration in each sample solution was calculated from the absorbance value of the sample solution, and its polysaccharide content was calculated based on the Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eWhere, C is the mass concentration of the RTP solution (mg/mL), N is the dilution ratio, V\u003csub\u003e0\u003c/sub\u003e is the total volume of RTP solution (mL), m is the weight of powder used for the extraction process (g).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Deproteinization\u003c/h2\u003e\n \u003cp\u003eDeproteinization of crude RTP was performed using trichloroacetic acid (TCA) [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. To remove proteins from the crude polysaccharides, the same volume of 3% TCA was added to different crude polysaccharide solutions (100 ml) obtained from optimal extraction conditions of UAE and MAE. The mixture was allowed to stand overnight, and then the precipitate was removed by centrifugation (10 min, 10000 rpm), and the above procedure was repeated three times to obtain a polysaccharide solution without proteins. The deproteinized water phase was collected, condensed to one fifth of its total volume using a rotary evaporator, and precipitated with ethyl alcohol at 4\u0026deg;C for 24 h. The precipitates were freeze-dried to obtain polysaccharide samples, named as RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e, respectively. The dried polysaccharides were then subjected to subsequent analyses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Physicochemical properties\u003c/h2\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.1. UV-vis spectrum and FT-IR analysis\u003c/h2\u003e\n \u003cp\u003eThe dried samples (4 mg) of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e were dissolved in distilled water (4 mL), and the UV-vis absorption spectrum of the solution was recorded in the wavelength range of 200\u0026ndash;500 nm [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe dried samples (3 mg) of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e were mixed with KBr (150 mg) and pressed into a disk to obtain a pellet (1.0 mm). The Fourier transform infrared spectroscopy (FT-IR) was used to determine samples between the wavelength range of 400 and 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.2. Scanning electron microscope (SEM) analysis\u003c/h2\u003e\n \u003cp\u003eThe morphology of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e were analyzed using a scanning electron microscopy. The dried samples (3 mg) were applied to the sample stage and coated with a layer of conductive gold film. The accelerated voltage was set as 15 kV [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003e2.6. Antioxidant capacity assay\u003c/strong\u003e\u003c/h2\u003e\n \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.1. Scavenging DPPH radical activity assay\u003c/h2\u003e\n \u003cp\u003eThe DPPH radical scavenging activity was measured according to a previous method with slight modifications [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. Different concentrations of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e (1 mL; 0.25, 0.5 1.0, 2.0, and 4.0 mg/mL) were mixed with DPPH ethanolic solution (4 mL, 0.1 mM). The mixture was reacted at room temperature in a dark for 30 min. The absorbance of the sample was measured at 517 nm. The positive control was ascorbic acid (VC). Eq. (\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) was used to calculate the scavenging rate.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n \u003cp\u003eWhere, A\u003csub\u003e0\u003c/sub\u003e control solution (no sample) and A\u003csub\u003e1\u003c/sub\u003e is the sample solution.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.2. Scavenging ABTS radical activity assay\u003c/h2\u003e\n \u003cp\u003eThe ABTS radical scavenging activity was assessed using the method described by \u003cem\u003eMohammed et al.\u003c/em\u003e (2020) [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e] with minor modifications. The ABTS radical solution (4 mL, 7 mM) was combined with 1.0 mL of the sample solution (0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL). After reaction at 25 \u0026ordm;C for 6 min, absorbance was measured at 734 nm. The positive control was ascorbic acid (VC). Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) was used to calculate the scavenging rate.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n \u003cp\u003eWhere A\u003csub\u003e0\u003c/sub\u003e is the control group; A\u003csub\u003e1\u003c/sub\u003e is the sample group.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eAll the experiments were performed in triplicate, and the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Prism 10 (GraphPad., Massachusetts, USA) was used for statistical analysis and figure drawing.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Effect of UAE conditions on the extraction yield of polysaccharides\u003c/h2\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1 Liquid to solid ratio\u003c/h2\u003e\n \u003cp\u003eThe effect of the liquid to solid ratio (20, 30, 40, 50, and 60 mL/g) on the extraction yield of RTP\u003csub\u003eUAE\u003c/sub\u003e was investigated under extraction time of 10 min, ultrasonic power of 220 W, and complex enzyme dose of 3% (w/w). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(A), the yield of RTP\u003csub\u003eUAE\u003c/sub\u003e gradually increased as the liquid to solid ratio increased, reaching its maximum value (34.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65%) at 30 mL/g. Increasing the liquid to solid ratio may increase the diffusivity of the solvent in the cell and promote the desorption of polysaccharides from the material. However, a decrease in the extraction rate was observed at higher liquid to solid ratio (\u0026gt;\u0026thinsp;30 ml/g), which may be due to the dilution effect of the complex enzyme. This dilution reduces enzyme-substrate collision frequency, potentially reducing the reaction rate [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, 30 mL/g was an appropriate liquid to solid ratio for the UAE.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.2 Extraction time\u003c/h2\u003e\n \u003cp\u003eTo determine the effect of extraction time (6, 8,10, 12, and 14 min) on the RTP\u003csub\u003eUAE\u003c/sub\u003e extraction yield, the UAE was performed under the liquid to solid ratio of 30 mL/g, ultrasonic power of 220 W, and complex enzyme dose of 3%. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(B) showed that there is an increasing trend in extraction yield from 6 to 10 min; the yield reached the maximum value at 10 min, followed by a significant reduction in yield with further increase in the extraction time. The shorter optimal time for RTP extraction may be due to the synergistic effect of ultrasound and enzymatic treatment, which accelerates the extraction process. The increase in extraction yield with extraction time is attributed to the more cell wall disruption by the ultrasonic cavitation and enzymatic activity, which enhanced the mass transfer of polysaccharide from the plant matrix into the extraction solution [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the subsequent decrease after 10 min can be attributed to polysaccharide degradation. Ultrasonic treatment has been reported to cause the cleavage of glycosidic bonds via mechanical, sonochemical, and localized heating. Collectively, these mechanisms contributed to the decrease in polysaccharide extraction with increasing ultrasound time [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, extraction times of 9, 10, and 11 min were selected for the orthogonal experimental design.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.3. Ultrasonic power\u003c/h2\u003e\n \u003cp\u003eUltrasonic power is also one of the most important factors because it can affect the enzyme activity as well as the movement and stability of polysaccharides molecules in the extraction system. RTP\u003csub\u003eUAE\u003c/sub\u003e were extracted under different ultrasonic power (140, 180, 220, 260, and 300 W). The other extraction conditions were as follows: liquid to solid ratio of 30 mL/g, extraction time of 10 min, and complex enzyme dose of 3%.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC demonstrated that the extraction rate gradually increased with increasing ultrasonic power. The highest extraction yield (34.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65%.) was obtained at ultrasonic power of 220 W. However, the extraction yield decreased when the ultrasonic power exceeded 220W. Appropriate ultrasonic power promotes the release of polysaccharides through cavitation. The tiny bubbles formed during cavitation burst in the liquid, generating localized high temperature, high pressure, and strong shear force. These extreme conditions can destroy plant cell walls and enhance the extraction of polysaccharides [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, excessive intensity of ultrasound power can lead to the destruction of polysaccharide structure, such as degradation of polysaccharides which reduces the extraction rate of polysaccharides [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. Precise control of ultrasound parameters is needed to achieve optimal extraction while avoiding polysaccharide degradation. Therefore, the ultrasonic power of 220W was chosen for the UAE.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.4. Complex enzyme dose\u003c/h2\u003e\n \u003cp\u003eThe plant cell wall consists of a rigid skeleton of cellulose embedded in a gel-like matrix, the main components of which are pectic compounds, hemicellulose, and glycoprotein. To enhance the extraction speed and yield of polysaccharides, complex enzymes, including cellulose, pectinase, and papain, are used to break the plant cell wall and hydrolyze structural polysaccharides [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. RTP\u003csub\u003eUAE\u003c/sub\u003e were extracted under different complex enzyme doses (1, 2, 3, 4, and 5%), and the other extraction conditions were as follows: liquid to solid ratio of 30 mL/g, extraction time of 10 min, and ultrasonic power of 220 W. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(D) showed that when the complex enzyme dose increased from 1\u0026ndash;3%, the extraction yield of RTP remarkably increased to reach the maximum value at 3%. Meanwhile, with a further increase in the enzyme dose, the extraction yield decreased. The initial increase in yield with enzyme concentration can be attributed to increased hydrolysis of cell wall components by the respective enzymes, facilitating polysaccharide release. Additionally, synergistic effects between different enzymes may occur, where the action of one enzyme exposes the enzymes to other substrates [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. The observed decrease in extraction yield at higher complex enzyme dose (\u0026gt;\u0026thinsp;3%) may be due to intensified enzyme aggregation resulting from the cavitation effect of ultrasound, which reduces effective enzyme-substrate interactions [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, the range of the complex enzyme dose for further optimization by orthogonal experiment was selected as 2.5\u0026ndash;3.5%.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.5. Orthogonal experimental design optimization results for the UAE\u003c/h2\u003e\n \u003cp\u003eBased on the single-factor experimental results, three levels of liquid to solid ratio, extraction time, ultrasonic power, and complex enzyme were chosen. An orthogonal experimental design L\u003csub\u003e9\u003c/sub\u003e(3\u003csup\u003e4\u003c/sup\u003e) was performed to further optimize the UAE (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The results are shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Using the comparison of R values obtained from the extreme difference analysis, the order of effect of experimental factors on the extraction yield was A\u0026thinsp;\u0026gt;\u0026thinsp;C\u0026thinsp;\u0026gt;\u0026thinsp;D\u0026thinsp;\u0026gt;\u0026thinsp;B, which is liquid to solid ratio\u0026thinsp;\u0026gt;\u0026thinsp;ultrasonic power\u0026thinsp;\u0026gt;\u0026thinsp;complex enzyme dose\u0026thinsp;\u0026gt;\u0026thinsp;ultrasonic time. In addition, polynomial regression analysis of the orthogonal test results (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) showed that the model had a P-value of 0.0002, which is below the commonly used significant level of 0.05. This demonstrated that the model was highly statistically significant. Based on the\u0026oline;k values, the optimal parameters would be the combination A\u003csub\u003e3\u003c/sub\u003eB\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e1\u003c/sub\u003e. Therefore, the optimal ultrasound-assisted extraction process was a liquid to solid ratio of 35 mL/g, ultrasonication time of 11 min, ultrasonic power of 240 W, and complex enzyme dose of 2.5%. The verification experiment performed on the basis of the condition stated resulted in an extraction yield of 35.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32%.\u003c/p\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of orthogonal experimental design for UAE\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA (mL/g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB (min)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC (W)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eY (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026oline;k\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026oline;k\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026oline;k\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBest levels\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e(A: liquid to solid ratio, B: ultrasonic time, C: ultrasonic power, D: complex enzyme dose, Y: extraction yield. Where 1\u0026ndash;3 are the levels for each condition tested. )\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u003cstrong\u003eTable 4. Results of orthogonal experimental design for MAE\u003c/strong\u003e\n \u003c/div\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eA (mL/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eB (min)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eC (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eY (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e20.88\u0026plusmn;0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e20.60\u0026plusmn;0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e21.94\u0026plusmn;0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e21.43\u0026plusmn;0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e20.54\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e19.71\u0026plusmn;0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e19.63\u0026plusmn;0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e27.03\u0026plusmn;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e26.21\u0026plusmn;0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eK\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e63.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e61.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e67.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eK\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e61.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e68.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e59.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eK\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e72.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e67.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e70.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e`k\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e21.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e20.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e22.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e`k\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e20.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e22.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e19.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e`k\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e24.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e22.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e23.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eBest levels\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eA\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eB\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eD\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e3.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e2.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e3.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e(A: liquid to solid ratio, B: microwave time, C: complex enzyme dose, Y: extraction yield. Where 1-3 are the levels for each condition tested.)\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Effect of MAE conditions on the yield of polysaccharides\u003c/h2\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Liquid to solid ratio\u003c/h2\u003e\n \u003cp\u003eThe liquid to solid ratio was a key factor influencing the extraction efficiency. The effect of different liquid to solid ratio (20, 30, 40, 50, and 60 mL/g) on the yield of RTP\u003csub\u003eMAE\u003c/sub\u003e were tested at microwave time of 10 min, microwave power of 300 W, and complex enzyme dose of 3%. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA showed that RTP\u003csub\u003eMAE\u003c/sub\u003e extraction yield increased significantly when the liquid to solid ratio increased from 20 to 40 mL/g, but decreased when the liquid to solid ratio was higher than 50 mL/g. As the liquid-to-solid ratio increased, more liquid allowed for better dispersion of the solids, increasing the surface area available for enzymatic action. This helps facilitate the release of more polysaccharides from the plant matrix [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, when the liquid-to-solid ratio becomes excessively high, the extraction yield decreases because of a combination of factors. The higher amount of liquid caused dispersion of the microwave energy over a larger area, potentially reducing the intensity of localized heating. This phenomenon, coupled with the dilution of enzymes, leads to less efficient extraction. The excess liquid also altered the mass transfer dynamics and enzyme-substrate interactions, further contributing to the decreased yield [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, a liquid to solid ratio of 40 mL/g was chosen for further experiments.\u003c/p\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.2. Microwave time\u003c/h2\u003e\n \u003cp\u003eDifferent microwave times were set at 6, 8, 10, 12, and 14 min to investigate the effect of microwave time on the extraction yields of RTP\u003csub\u003eMAE\u003c/sub\u003e. The other conditions were set as follows: liquid to solid ratio of 40 mL/g, microwave power of 300 W, and complex enzyme dose of 3%. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(B), the extraction yield of RTP\u003csub\u003eMAE\u003c/sub\u003e reached a maximum value of 25.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31% at 8 min. This extraction time was shorter than that reported for conventional heating methods, demonstrating the efficiency of microwave-assisted extraction [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the subsequent decrease in yield with extended microwave exposure time was likely due to thermal degradation of the polysaccharides. An excessive microwave exposure time can lead to thermal degradation of polysaccharides, breaking down their long chains into smaller fragments [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. This degradation can result in the loss of functional properties, such as viscosity, gel formation ability, and bioactivity, which are crucial for their applications in the food, nutraceutical, and pharmaceutical industries [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. It is vital to balance microwave time to maximize the extraction yield of polysaccharides while preserving their structural integrity and functional properties. Therefore, 7, 8, and 9 min were selected for further optimization experiments.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.3. Microwave power\u003c/h2\u003e\n \u003cp\u003eThe effect of microwave power (100, 180, 300, 450 and 600 W) on the extraction yield of RTP\u003csub\u003eMAE\u003c/sub\u003e was explored at a liquid-solid ratio of 40 mL/g, a microwave time of 8 min, and an enzyme complex dose of 3%. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC showed that the extraction yield of RTP\u003csub\u003eMAE\u003c/sub\u003e increased from 17.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77% to 25.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31% when the microwave power was increased from 100 to 300 W. Increasing the microwave power during the extraction process enhanced the solubility of the sample. This is because higher microwave power produces heat more efficiently. This elevated heat promotes the breakdown of plant cell walls and facilitates the release of polysaccharides into the solution, thereby improving the extraction yield [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the extraction yield of RTP\u003csub\u003eMAE\u003c/sub\u003e decreased when the microwave power exceeded 300 W, which was due to thermal degradation. At higher microwave powers, the intense heat can break down the polysaccharide chains into smaller fragments, reducing the overall yield [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, because there are only five fixed parameters, 100, 180, 300, 450, and 600 W, for the microwave oven used, 300 W was chosen as the optimal microwave power, and there was no need to optimize the microwave power through an orthogonal experimental design.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.4. Complex enzyme dose\u003c/h2\u003e\n \u003cp\u003eThe effects of the complex enzyme dose (1, 2, 3, 4, and 5%) on the extraction yield of RTP\u003csub\u003eMAE\u003c/sub\u003e were explored at a liquid-to-solid ratio of 40 mL/g, microwave time of 8 min, and microwave power of 300 W. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD showed the effects of the complex enzyme dose on the yield of RTP\u003csub\u003eMAE\u003c/sub\u003e. When the complex enzyme dose was between 1% and 3%, the extraction yield was positively correlated with the enzyme concentration. A suitable complex enzyme concentration can exert a better synergistic effect and effectively promote the degradation of the cell wall, thereby promoting the release of polysaccharides [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, above 3%, the extraction yield decreased slightly. This may be because the complex enzyme dose was too high; there may have been competitive inhibition between the enzymes, leading to a decrease in the extraction yield. In this context, competitive inhibition likely occurs when a high enzyme concentration leads to over-crowding of enzymes. As a result, enzyme molecules may physically obstruct each other, competing for access to limited substrate-binding sites, reducing the overall efficiency of the extraction process [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, a complex enzyme of 3% is considered the optimal dose for extracting RTP\u003csub\u003eMAE\u003c/sub\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.5. Orthogonal experimental design optimization results for the MAE\u003c/h2\u003e\n \u003cp\u003eThe statistical analysis of single-factor experiments showed the factors of liquid to solid ratio, microwave time, microwave power, and complex enzyme dose significantly affect the yield of RTP\u003csub\u003eMAE\u003c/sub\u003e. An orthogonal experimental design L\u003csub\u003e9\u003c/sub\u003e(3\u003csup\u003e3\u003c/sup\u003e) was carried out to optimize the MAE (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, the equipment used in this study has only five fixed parameters: 100, 180, 300, 450, and 600 W. Therefore, 300 W was chosen as the optimal microwave power, and there was no need to optimize the microwave power through an orthogonal experimental design. Three main factors (liquid to solid ratio, microwave time, and complex enzyme dose) and three levels for each factor were selected for further optimization.\u003c/p\u003e\n \u003cp\u003eAs showed in Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, using the comparison of R values obtained from the extreme difference analysis, the effect order of experimental factors on the extraction yield was A\u0026thinsp;\u0026gt;\u0026thinsp;C\u0026thinsp;\u0026gt;\u0026thinsp;B (Liquid to solid ratio\u0026thinsp;\u0026gt;\u0026thinsp;complex enzyme dose\u0026thinsp;\u0026gt;\u0026thinsp;microwave time). In addition, polynomial regression analysis of the orthogonal test results (Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e) showed that the model had a P-value of 0.0001, which is below the commonly used significant level of 0.05. This demonstrated that the model was highly statistically significant. Therefore, based on the\u0026oline;k values, the optimal parameters would be the combination A\u003csub\u003e3\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e (where 1\u0026ndash;3 are levels). However, the optimal extraction conditions were not included in the orthogonal table; the confirmatory experiment was conducted. The verification experiment showed that the extraction yield of RTP\u003csub\u003eMAE\u003c/sub\u003e reached 27.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21%, which was higher than that of the optimal group 8 in the orthogonal experimental design. Therefore, the optimal MAE process was as follows: microwave power 300 W, liquid to solid ratio 40 mL/g, microwave time 8 min, complex enzyme dose 2.5%.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Physicochemical properties of RTP\u003c/h2\u003e\n \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.1. UV-vis and FT-IR analysis\u003c/h2\u003e\n \u003cp\u003eFig. 3A and B show that RTPUAE and RTPMAE did not have distinct absorption peaks on the UV-vis spectrum at 260 and 280 nm wavelengths, indicating that they did not contain proteins and nucleic acids.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eThe physicochemical analysis we analyzed only for both RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e obtain at optimum extraction conditions. The FT-IR spectrum of two RTPs was presented in Fig. 3A. In the 3600\u0026ndash;3200 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e region, the strong broadbands represent the O\u0026ndash;H stretching vibration [26]. The absorption peak around 2925 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e was mainly caused by the asymmetric C\u0026ndash;H stretching vibrations of the CH, CH\u003csub\u003e2\u003c/sub\u003e, and CH\u003csub\u003e3\u003c/sub\u003e groups [26]. The peak of about 1744 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e was a characteristic of the C=O stretch of carboxylic acid. The peak at 1220 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e indicated the presence of C-O stretching. RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e showed strong absorption peak bands in the 1000\u0026ndash;1200 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e region, indicating that these polysaccharides contain C-O-C glycosidic bonds. It is noteworthy that the characteristic peaks of RTP\u003csub\u003eMAE\u003c/sub\u003e have a higher intensity than those of RTP\u003csub\u003eUAE\u003c/sub\u003e. This may be attributed to the mild physical action of ultrasound, which does not easily destroy the structures of the major functional groups of polysaccharides [27]. The lower peak intensities indicate that the extraction process causes less damage to the molecular structure of the polysaccharides and that the functional groups of the polysaccharides remain more intact, which implied that the extracted polysaccharides may retain their natural activity and functional properties (Wang et al., 2018). In addition, because of the intense heating effect of microwaves, degradation of part of the molecular structure of polysaccharides may occur, affecting the distribution and strength of functional groups. Some functional groups may be destroyed or transformed, resulting in higher intensity of characteristic peaks in FT-IR spectra [28].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.2. Microscopy analysis\u003c/h2\u003e\n \u003cp\u003eThe SEM images of the two polysaccharides shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e indicated different morphologies in RTP powder extracted by the different methods. The surface of RTP\u003csub\u003eMAE\u003c/sub\u003e possessed a compact morphology with a relatively smooth surface. While in RTP\u003csub\u003eUAE,\u003c/sub\u003e the surface was rough, wrinkled, and irregular. This indicated that the UAE disrupted the original plant structure more effectively than MAE. This was due to cavitation effects that caused cell walls to degrade, thereby enhancing the release of intracellular materials and promoting higher polysaccharide extraction efficiency [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Antioxidant Activity Analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.1. The DPPH radical scavenging activity\u003c/h2\u003e\n \u003cp\u003eThe DPPH radical is a tool for the assay of the radical scavenging activities of antioxidants. The DPPH radical scavenging activity of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e are shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA. The DPPH radical scavenging rate of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e were 75.91% and 73.52%, respectively, at a sample concentration of 4.0 mg/mL. Moreover, the IC\u003csub\u003e50\u003c/sub\u003e values of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e were 2.25 and 2.19 mg/mL, respectively. However, The DPPH radical scavenging ability of RTP\u003csub\u003eUAE\u003c/sub\u003e was not significantly different from that of RTP\u003csub\u003eMAE\u003c/sub\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec35\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.2. The ABTS radical scavenging activity\u003c/h2\u003e\n \u003cp\u003eThe ABTS\u003csup\u003e+\u003c/sup\u003e radical scavenging activity of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e are shown in Fig. 4B. The results shown that the two types of RTP had a positive scavenging effect on the ABTS radical. RTP\u003csub\u003eUAE\u003c/sub\u003e had a higher ABTS radical scavenging ability than RTP\u003csub\u003eMAE\u003c/sub\u003e at the same doses. In addition, at a sample concentration of 4.0 mg/mL, the scavenging rates of RTP\u003csub\u003eMAE\u003c/sub\u003e and RTP\u003csub\u003eUAE\u003c/sub\u003e reached 63.32% and 86.12%, respectively. The IC\u003csub\u003e50\u003c/sub\u003e values of RTP\u003csub\u003eUAE\u003c/sub\u003e and RTP\u003csub\u003eMAE\u003c/sub\u003e were 2.21 and 3.19 mg/mL, respectively. Our results demonstrated that RTP\u003csub\u003eUAE\u003c/sub\u003e has stronger ABTS radical scavenging activity than RTP\u003csub\u003eMAE\u003c/sub\u003e. This is because ultrasound enhances enzyme activity and improves extraction efficiency by creating cavitation and micro-streaming effects, leading to better preservation of the polysaccharide structure and heat-sensitive antioxidant components compared with the uneven heating and potential degradation caused by microwave [31].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eThe effects of different extraction methods (UAE and MAE) on RTP polysaccharide yield, physicochemical properties, and antioxidant activity were evaluated. \u0026nbsp; An orthogonal experimental design was used to optimize the extraction parameters. The results indicated that RTP extracted using the UAE method had a higher extraction yield. The optimal parameters for using UAE to extract RTP were a liquid-to-solid ratio of 35 mL/g, ultrasonic duration of 11 min, ultrasonic power of 240 W, and complex enzyme dose of 2.5%. FT-IR spectroscopy showed that UAE caused less disruption of the polysaccharide molecular structure and better retention of functional groups than MAE.\u0026nbsp; Surface morphology showed that greater matrix changes occurred in the ultrasound-treated samples, allowing more polysaccharides to be extracted. This study demonstrated that the UAE method is a promising method for extracting high-quality\u003cem\u003e\u0026nbsp;R. tomentosa\u003c/em\u003e berry polysaccharides based on its high yield, high efficiency, and outstanding antioxidant activity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCredit authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDingjin Li,\u003c/strong\u003e Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Writing – original draft. \u003cstrong\u003eWan Zunairah Wan Ibadullah,\u003c/strong\u003e Investigation, Validation.\u0026nbsp;\u003cstrong\u003eRadhiah Shukri\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e Investi­gation, Validation. \u003cstrong\u003eQiuxia Duan,\u0026nbsp;\u003c/strong\u003eMethodology, data curation. \u003cstrong\u003eYipeng Gu,\u003c/strong\u003e Investigation, Validation. \u003cstrong\u003eNor Afizah Mustapha,\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; editing ,Investigation, Supervision, Validation. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was funded by the Disciplinary Interdisciplinary and Collaborative Research Project of Hezhou University (Grant No: XKJC202401), the Agricultural Science and Technology Self-financing Funding Project of Guangxi (Grant No: Z2024049).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWang, R., Yao, L., Lin, X., Hu, X., \u0026amp; Wang, L. (2022). Exploring the potential mechanism of Rhodomyrtus tomentosa (Ait.) Hassk fruit phenolic rich extract on ameliorating nonalcoholic fatty liver disease by integration of transcriptomics and metabolomics profiling. 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Degradation Method, Structural Characteristics, Biological Activity and Structure-Activity Relationship of Degraded Polysaccharides. Food Reviews International, 0(0), 1\u0026ndash;30. https://doi.org/10.1080/87559129.2023.2273933\u003c/li\u003e\n \u003cli\u003eChen, X., Chen, G., Wang, Z., \u0026amp; Kan, J. (2020). A comparison of a polysaccharide extracted from ginger (Zingiber officinale) stems and leaves using different methods: Preparation, structure characteristics, and biological activities. International Journal of Biological Macromolecules, 151, 635\u0026ndash;649. https://doi.org/10.1016/j.ijbiomac.2020.02.222\u003c/li\u003e\n \u003cli\u003eGharibzahedi, S. M. T., Marti-Quijal, F. J., Barba, F. J., \u0026amp; Altintas, Z. (2022). Current emerging trends in antitumor activities of polysaccharides extracted by microwave- and ultrasound-assisted methods. International Journal of Biological Macromolecules, 202, 494\u0026ndash;507. https://doi.org/10.1016/j.ijbiomac.2022.01.088\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Rhodomyrtus tomentosa berry, polysaccharides, ultrasound, microwave, antioxidant activity","lastPublishedDoi":"10.21203/rs.3.rs-5141599/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5141599/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtraction of polysaccharides from \u003cem\u003eRhodomyrtus tomentosa\u003c/em\u003e berry (RTP) is essential for understanding their bioactive ingredients and developing functional foods, nutraceuticals, and pharmaceuticals. This study aimed to evaluate the effects of ultrasonic-assisted enzymatic extraction (UAE) and microwave-assisted enzymatic extraction (MAE) on the yield, physicochemical properties, and antioxidant activity of (RTP). The single-factor and orthogonal experimental design revealed that the optimal conditions for ultrasound assisted RTP extraction are at liquid to solid ratio of 35 mL/g, ultrasonic time of 11 min, ultrasonic power of 240 W, and complex enzyme dose of 2.5% with RTP extraction yield of 35.67 ± 0.32%. FT-IR spectroscopy showed that UAE caused less disruption of the polysaccharide molecular structure and better retention of functional groups than MAE. The scanning electron microscopy results demonstrated that the ultrasonically treated samples exhibited a greater degree of structural disruption, which could more effectively facilitate the release of polysaccharides. In addition, RTP obtained by the UAE has a better extraction yield and ABTS radical scavenging activity than MAE. This study demonstrated that the UAE method is a promising method for extracting high-quality\u003cem\u003e R. tomentosa\u003c/em\u003e berry polysaccharides based on its high yield, high efficiency, and outstanding antioxidant activity.\u003c/p\u003e","manuscriptTitle":"The effects of different extraction methods on the yield, microstructure, and antioxidant activity of polysaccharides from Rhodomyrtus tomentosa berry","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-05 11:14:29","doi":"10.21203/rs.3.rs-5141599/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-30T09:56:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-28T19:46:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-27T14:33:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250533376877141831555863941705452790683","date":"2024-09-27T00:23:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78627368963403872994687645058292662171","date":"2024-09-26T22:00:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257248894050060527711254804933948638596","date":"2024-09-26T16:56:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168768974538483579167750670033370934980","date":"2024-09-26T14:55:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-26T11:34:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-26T01:56:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-24T05:22:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2024-09-24T03:55:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f129c32b-c912-4af6-a6cc-5a7d5b40b8c4","owner":[],"postedDate":"November 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-11-05T11:14:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-05 11:14:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5141599","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5141599","identity":"rs-5141599","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}