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

S1350-4177(24)00286-4
10.1016/j.ultsonch.2024.107038
107038
Original Research Article
The ultrasonic-assisted enzymatic extraction, characteristics and antioxidant activities of lychee nuclear polysaccharide
Song Zongyan a
Huang Gangliang huangdoctor226@163.com
a⁎
Huang Hualiang hlhuang@wit.edu.cn
b⁎
a Key Laboratory of Carbohydrate Science and Engineering, Chongqing Normal University, Chongqing 401331, China
b School of Chemistry and Environmental Engineering, Key Laboratory of Green Chemical Process of Ministry of Education, Key Laboratory of Novel Reactor and Green Chemical Technology of Hubei Province, Wuhan Institute of Technology, Wuhan 430074, China
⁎ Corresponding authors. huangdoctor226@163.comhlhuang@wit.edu.cn
21 8 2024
11 2024
21 8 2024
110 10703812 6 2024
12 8 2024
20 8 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A response surface methodology (RSM) and one-factor method were established to investigate the optimum conditions for the extraction of lychee nuclear polysaccharides (LSP) by ultrasonic-assisted pectinase. The content of basic components in polysaccharides was determined. The antioxidant activity was well determined and compared the differences in the activities of the polysaccharides extracted by water extraction (LSP-HW) and those extracted at ultrasound-assisted enzyme (LSP-UAE). The activity of lychee nuclear polysaccharide increased with the increase of concentration. The anti-hydroxyl radical and anti-lipid peroxidation abilities of lychee nuclear polysaccharide were more excellent, and LSP-HW was slightly better than LSP-UAE in terms of activity, but the difference was not significant. In terms of solubility; LSP-HW without deproteinization was the best, followed by LSP-UAE, and the worst was LSP-UAE after deproteinization. The higher the glycoprotein content, the better the solubility of its polysaccharide. Compared with the existing extraction methods, this experiment greatly improved the extraction rate of lychee nuclear polysaccharides and further enriched the antioxidant activities of polysaccharides.

Keywords

Lychee nuclear polysaccharide
Extraction
Response surface analysis
Antioxidant activity
Solubility
==== Body
pmc1 Introduction

As an ancient Chinese treatment method, traditional Chinese medicine therapy had been benefiting people from ancient times until today [1]. Due to its friendly price and non-toxic active ingredients extracted from natural materials, it had always held a place in the modern medical world. Some plant extracts in our lives were effective in inhibiting bacteria [2], warding off toxic attacks [3], [4], displaying anti-parasitic activity [5], and lowering blood sugar levels [6], [7]. With the continuous development of the society, Chinese medicine treatment was also constantly innovated and improved. Extracts of different herbs are not only beneficial for treating diseases, but also used in beauty care and catering industry. Therefore, it had also caused a boom in researchers' study of natural Chinese herbs.

Lychee belonged to the Sapindaceae family and was a subtropical fruit. People especially like its sweet and juicy pulp. However, every year, many lychee skin and lychee nuclear were discarded. Among them, the lychee nuclear had great utilization value. The lychee nuclear was oval and dark brown, with a length of 1–3.3 cm and a width of 0.6–1.2 cm. The annual global production of lychee was about 70,000 tons, while the nuclear production was about 20,000 tons. Studies had shown that lychee nuclears can be extracted for beneficial substances such as phenols [8] and polysaccharides [9]. The lychee nuclear added nutrients needed for health and promoted energy circulation in the body. The bioactive substances in lychee nuclear also had antioxidant, anti-tyrosinase [10], [11], [12], analgesic [13], anti-tumor [14], anti-cancer [15], [16], [17], and hypoglycaemic effects [18]. Therefore, it was necessary to study the nutritional composition of lychee nuclears. Lychee nuclear polysaccharides were the main nutritional components of lychee. It had an outstanding contribution to enhancing human immunity and accelerating metabolism. Not only that lychee nuclear polysaccharide had a role in lowering blood sugar and inhibiting α-glucosidase [19], [20]. Therefore, lychee polysaccharide was widely used in the medical field. Studies on the extraction methods of lychee nuclear polysaccharides included ultrasound-assisted extraction [9], which gave an optimum extraction rate of 3.39 %. A small number of researchers had also made preliminary investigations on the structure of lychee nuclear polysaccharide. However, it was not comprehensive enough in the study of the extraction methods, biological activities, and structural diversity of lychee nuclear polysaccharides. Therefore, we needed to find a better extraction method to improve the yield of polysaccharides and to investigate their structure as well as biological activities. So, the present study was carried out to investigate the extraction, antioxidant activity, and structure of lychee nuclear polysaccharides further. Water extraction was a commonly used extraction method, but it was considered to be low-yielding and time-consuming [21]. However, when the material was cavitated by ultrasound, plant cell walls and cell membranes were easily disrupted, making it easier to extract polysaccharides. Of course, there was no absolute advantage of different methods, for example, very strong ultrasound could lead to disruption of the polysaccharide structure, ultimately leading to lower polysaccharide yields [22]. At the same time, enzymes could assist the ultrasound waves, which made it easier for the plant cells to break down, so that the active substances within the cells could more easily run into the water [23], [24]. Some studies had shown that ultrasound could also enhance enzyme activity within certain limits [25], [26]. Based on the above advantages [27], ultrasound-assisted pectinase (UAE) was used to extract lychee nuclear polysaccharides in this experiment. Therefore, a series of experiments were carried out to optimize the UAE method based on the aim of increasing polysaccharide yield.

The Response Surface Analysis (RSM) method used in this experiment is an integrated experimental design and mathematical modeling optimization method. The advantage of this method is that it takes into account random errors and the results are accurate and reliable. The method also systematically takes into account the interaction between the factors, avoids redundant experiments, and obtains reliability of results between experimental data and response values [28], [29]. In order to determine the optimal conditions for ultrasound-assisted enzyme extraction of lychee nuclear polysaccharide (LSP), the process was optimized using a Box-Behnken design of experiments [30]. A large number of studies had found that the influencing factors include the sonication ratio, the sonication time, the amount of pectinase added, and the interaction between these conditions [31], [32]. Based on the above-influencing factors, this experiment was carried out to investigate the improvement of polysaccharide yield.

2 Materials and methods

2.1 Pretreatment and ultrasound-assisted enzymatic extraction of polysaccharides

Lychee nuclear was provided by Sichuan Zangxi Tang Biotechnology Co. Lychee nuclear was native to Qinzhou, Guangxi. The ultrasonic device used in this experiment was produced by Xiangyi Instrument Co, Ltd, model KQ5200-DB. The centrifuge used in this experiment was a tabletop centrifuge provided by Xiangyi Instrument Co, Ltd, whose model was L420. Improvements based on existing methods [23], [33]. Lychee nuclears were dried and crushed through a sieve with a grinder. 10 g of lychee nuclear powder was taken in 50 mL of anhydrous ethanol, oil bath at 60 °C for 2 h. Defatted lychee nuclear powder and distilled H2O (pH = 7) were added to a beaker in a ratio of 1:50, followed by 250 mg of pectinase (enzyme activity was 500 u/mg), and the mixture was allowed to soak well for 5 min. The ultrasonic device was preheated to 55 °C, the extraction time was set to 55 min and the ultrasonic power was set to 360 w. The beaker was placed in the ultrasonic machine and the extraction was carried out using an ultrasonic bath. If the temperature of the water rose during this period, open the lid of the sonicator and wait until the water temperature returns to 55 °C. After extraction, the solution was cooled down on a table and then filtered under reduced pressure to obtain a supernatant. Next, the polysaccharide solution was concentrated, and when the volume of the concentrate became 1/4 of the original, 4 times the volume of anhydrous ethanol was added to the solution. Finally, the solution was placed in a refrigerator overnight. The polysaccharide precipitate was obtained by centrifugation for 6 min at a centrifuge speed of 3650 r/min. A small amount of distilled water was added to the polysaccharide, and then the polysaccharide was placed in a freeze dryer at a pressure of 0.05 pa and a temperature of −40 °C for 48 h to obtain the crude polysaccharide LSP-UAE. The yield of the polysaccharide was calculated according to the following formula.(2.1) Yield=WeightofLSPWeightoflycheekernelpowder×100

2.2 One-factor experiments

This experiment would investigate the effects of enzyme type, enzyme addition, ultrasonic extraction time, and ultrasonic feed solution on the extraction rate of polysaccharides. 10 g of defatted lychee nuclear powder was weighed and the polysaccharides were extracted in the following conditions respectively. Pectinase, papain, and cellulase were used for extraction under ultrasonic conditions at 360 W, respectively. The controlled pectinase addition was 1.5 %, 2 %, 2.5 %, 3 % and 3.5 %. The ultrasonic extraction time was varied to 25 min, 35 min, 45 min, 55 min and 65 min. The feed-liquid ratios were 1:10, 1:20, 1:30, 1:40, and 1:50. The one-way optimum conditions for each exploration were used as the basis for the next experiment.

2.3 Design optimization of experimental conditions

In the one-way experiment, we found the three factors that most affected the polysaccharide yield, which were the Liquid-material ratio (A), pectinase addition (B), and extraction time (C). Later experiments would continue to investigate the combined effect of the three factors on the polysaccharide yield [34]. The related model design as well as the symbols were shown in Table 1. The 17 conditions designed by the model were used to conduct experiments, and the yield of polysaccharides obtained was used as an index to measure the advantages and disadvantages of the extraction conditions. Five of them had the same repeatability test, which could be used as an error analysis to predict the reliability of the experiment.Table 1 The factors of Box-Behnken test and its levels.

Level	Factor	
A
Liquid-material ratio (mL/g)	B
Petinase addition (%)	C
Extraction time (min)	
1	50	3	65	
0	40	2.5	55	
−1	30	2	45	

2.4 Determination of the sugar content

In this experiment, the phenol–sulfuric acid method [23] was used to determine the sugar content of the samples. The resulting linear equation was (2-2). The sugar content of the LSP was calculated by the same method as above.(2.2) y=0.0096x+0.089(R2=0.999)

y indicated the absorbance of polysaccharide at 490 nm.

x indicated glucose concentration (μg/mL)

2.5 Measurement of protein content

This experiment was based on the Caumas Brilliant Blue method with some improvements [35]. The formula for calculating the protein content of the polysaccharide in question was shown in Eq. (2-3).(2.3) y=0.8005+0.0056x(R2=0.998)

y indicated the absorbance of polysaccharide at 595 nm.

x indicated the concentration of bovine serum protein (μg/mL).

2.6 Determination of glycuronic acid content

The relevant changes were made in this experiment [36]. The absorbance at 530 nm was measured. The formula for calculating the glyoxylate of the polysaccharide in question was shown in Eq. (2-4).

y=0.0396+0.0065x(R2=0.999) (2-4)y indicated the absorbance of the polysaccharide at 530 nm.

x indicated the concentration of galacturonic acid (μg/mL).

2.7 Hydroxyl radical scavenging capacity of LSP

In this experiment, the method for the determination of hydroxyl radical scavenging capacity was improved [37]. The 4 mg/mL polysaccharide solution was first prepared by taking six identical centrifuge tubes and adding 1000 μL, 500 μL, 250 μL, 125 μL, and 62.5 μL of polysaccharide solution to the tubes and added distilled H2O to make a total volume of 1 mL. Next, 0.5 mL of H2O2 solution, 0.5 mL of salicylic acid–ethanol solution, and 0.5 mL of FeSO4 solution were mixed well and then placed in a water bath at 36 °C for 24 min. The absorbance was measured at 510 nm.(2.5) E(%)=A0-AA0

A was the sample absorbance.

A0 was the background absorbance.

2.8 Antilipid peroxidation capacity of LSP

A solution of 1 mg/mL was obtained by dissolving soybean phospholipids with phosphate buffer with a pH value of 7.2–7.4. Different concentrations of LSP-UAE solution were added to 6 centrifuge tubes, and then the configured trichloroacetic acid and thiobarbituric acid solutions were added sequentially to the above different polysaccharide solutions. The above mixture was heated in boiling water for 25 min. The supernatant from the mixture was taken. The absorption value was measured at 535 nm [38].(2.6) E(%)=A0-AA0

A was the sample absorbance.

A0 was the background absorbance.

2.9 Determination of reducing capacity

Following the above steps, the polysaccharide solution was configured to obtain 0.125–4 mg/ml. Then 1 ml of phosphate buffer (0.25 mol/L at pH = 6.6) and 1 ml of K3Fe(CN)6 (0.4 %) solution were added to the polysaccharide solution. The above solutions were placed in a water bath at 54 °C for 20 min. 1 mL of trichloroacetic acid (4 %) was added after the reaction cooled down. The above solutions were centrifuged to obtain the supernatant and 1 ml of distilled water and 0.2 ml of FeCl3 solution (0.4 %) were added respectively. The reaction was carried out at room temperature for 15 min. the supernatant was obtained by removing the precipitate from the reaction solution by centrifugation. The absorbance of solution was measured at 700 nm [37].

2.10 Testing of LSP's ability to scavenge DPPH free radicals

The experimental method was slightly modified concerning this literature [39]. A 4 mg/mL polysaccharide solution was first configured. Then different volumes of sugar solution were added to each of the six test tubes to obtain solutions with concentrations from 0.125 to 4 mg/mL. Then 4 mL of configured DPPH-ethanol (0.41 mmol/L) was added to each test tube and the reaction was carried out under dark conditions for 46 min. Finally, the absorbance value of the reaction solution was measured at 517 nm and compared with the same concentration of vitamin C (Vc) as a positive control and distilled water as a blank control. The clearance was calculated by the following formula:(2.7) E(%)=1-A1-A2A0×100

A1 indicates the absorbance of the sugar solution.

A2 indicates the background absorbance.

A0 indicates the blank absorbance.

3 Results and discussion

3.1 Effect of enzyme species on LSP yields

According to scientific research the cell wall was mainly composed of pectin, cellulose, and proteins [40], [41]. Papain cleaved the proteins in the cell wall into small proteins and amino acids. Cellulase hydrolyzed cellulose into glucose and small molecule fibers to release more polysaccharides. Pectinase, on the other hand, hydrolyzed the pectin chains in the cell wall to reduce the binding force of pectin on water-soluble polysaccharides. The results showed that pectinase had good results with the highest yield of polysaccharide along with good solubility. It was observed from Fig. 1 that in terms of yield papain > pectinase > cellulase. In terms of sugar content, it was pectinase > cellulase > papain. The determination of polysaccharide content revealed that pectinase extracted the highest polysaccharide content with 82 % sugar content. The least polysaccharide content of 30 % was obtained from papain. The reason may be that the cell wall was rich in pectin, which accounted for about 1/3 of the cell wall [42], and these pectins could firmly bind the active polysaccharides. When pectinase hydrolyses these pectin macromolecules, then a large amount of active polysaccharide were released. But, the protease hydrolyzed protein into a large number of small molecules, they wrapped up polysaccharide and made it difficult to dissolve in water, and a large number of pectin and vegetal color was dissolved out [43]. But in general the yield of polysaccharide extracted by cellulase was lower than that extracted by pectinase. And the protein content of polysaccharides extracted by cellulase (11.33 %) was slightly less than that of polysaccharides extracted by pectinase (13.00 %) as measured by Caumas Blue method. Therefore, overall the polysaccharide extracted by pectinase had the best yield and effect.Fig. 1 The effect of enzyme species on LSP yield.

3.2 The effect of pectinase addition on LSP yield

Most of the active polysaccharides of the plant are bound in the cell wall [43], [44], which have a relatively high pectin content that combined the active polysaccharide in it. The effect of pectinase addition on extraction rate was shown in Fig. 2. When the pectinase dosage was controlled at 1.5 %–2.5 %, the higher the pectinase addition was, the higher the polysaccharide yield and sugar content were. The sugar content reached up to 78 %. This was because the higher the pectinase dosage was, the more pectin chains were cleaved, which weakened the binding force of pectin on polysaccharides. In this way active polysaccharide was released. However, when pectinase dosage was greater than 2.5 %, the polysaccharide yield was gradually decreased again. This was because excessive pectinase will lead to hydrolysis of pectin and active polysaccharides [45], resulting in the destruction of the glycosidic bonds of active polysaccharide. Because the price of enzyme was relatively expensive, the optimal pectinase addition was 2.5 %, the sugar content was 78 %, and the crude polysaccharide yield was 2.65 %.Fig. 2 The effect of pectinase addition on LSP yield.

3.3 Effect of ultrasound time on LSP yields

Ultrasound enhances the degree of destruction of cell walls on the basis of water extraction, which was also due to the unique penetrating power of ultrasound [46]. The addition of pectinase on top of ultrasound could greatly increase the extraction rate of polysaccharides. Thus, the variation in polysaccharide yield was explored by the extraction time under the conditions that pectinase dosage was the 2.5 % and ultrasonic power was 360 w. It was found that the polysaccharide yield gradually increased in 35 min-55 min, while the sugar content slowly decreased, as shown in the Fig. 3. The highest polysaccharide sugar content of 62 % was extracted at 35 min of ultrasound, but the lowest yield was obtained. The total sugar content of polysaccharide extracted at 55 min was 56.8 % but the yield was the highest. The reason for this was that the longer the extraction time was, the higher the yield would be. At the same time, it would let more impurities such as pigment dissolve, but the overall increase in impurities was not obvious [47]. More than 55 min polysaccharide yield and sugar content decreased rapidly. Extraction time was too high will also increase the dissolution rate of impurities. Therefore, considering the extraction time, it was best to control the extraction time at 55 min, the yield was 2.00 % and the sugar content was 56.8 %.Fig. 3 Effect of ultrasound time on extraction rate.

3.4 Effect of ultrasonic material-liquid ratio on LSP yield

The feed-to-liquid ratio was a significant influences on the production of polysaccharides, which were very soluble in water because they contain a large number of hydroxyl polar groups [48]. The optimum dosage of pectinase was 2.5 % and the ultrasonic power was 360 w. Different material-liquid ratios were controlled to investigate the changes in yield, and the extraction was carried out for 55 min. It was found that, as shown in Fig. 4, the material-liquid ratios ranged from 1:10–1:40, the yields of polysaccharide and the sugar content increased gradually, and the highest sugar content reached 88 %. It was also found that the solubility of the extracted polysaccharides was excellent at a material-liquid ratio of 1:40. This was because as the amount of water increases, the osmotic pressure between the cells and the water gradually increased, causing more water molecules to attract the polysaccharides out [49]. However, when the material-liquid ratio was greater than 1:40, there was no significant change in the extraction rate of polysaccharides, and it even decreased slightly, which was a waste of resources. Therefore, the final choice of the material-liquid ratio of 1:40 was the best liquid–liquid ratio, and its yield was 2.61 %.Fig. 4 Effect of liquid–solid ratio on extraction rate.

3.5 Effect of temperature on LSP yields

Under the optimal extraction conditions explored above, different temperatures were controlled to explore the changes in polysaccharide yield [12]. As can be seen in Fig. 5, the yield and sugar content were gradually increased within 40 °C–55 °C. This was probably because an increasing temperature can reduce the dielectric constant of water, and at the same time the viscosity and surface tension between the cell wall and the polysaccharide were also reduced. The above led to an increase in solubility and diffusivity of polysaccharides at higher temperatures [50]. At the same time enzyme activity was activated continuously with temperature. However, with increasing temperature, the polysaccharide yield suddenly decreased, which may be due to the inactivation of the enzyme resulting in poor cleavage of the pectin chain and the release of polysaccharides [51]. Therefore in summary, the optimum temperature was 55 °C.Fig. 5 Effect of temperature on extraction rate.

Overall, it was found that the material-liquid ratio, ultrasonic extraction time, pectinase addition, and enzyme type had the greatest influence on the polysaccharide yield by one-way test, while the temperature had the second greatest influence on the extraction rate of lychee nuclear polysaccharides. Therefore, the remaining three factors with the greatest effect on yield would be selected for further study based on the determination of the enzyme type as pectinase.

3.6 Statistical analysis and model fitting

The data were analyzed and modeled by Design-Expert 13 software and its yield results under different conditions were shown in Table 2. The actual yield obtained from the test and the predicted yield obtained from the software analysis were similar, and the correlation coefficient R2 = 0.9968 could be observed through Table 3, Table 4, indicating that only 0.32 % variability could not be explained by the model. The coefficient of variation CV = 0.0113 < 0.1 indicated that the model had good confidence in the prediction of the yield. The model could then make accurate predictions of yield within the range of variation of this experiment. The influence factors A, B, and C in the Table 3 had P < 0.0001, which indicated that all three factors had a significant effect on the yield rate. The combination of the misfit term P = 0.8167 indicated that the model was not significantly misfit. The good reproducibility of the model was indicated by the signal-to-noise ratio, as the signal-to-noise ratio of the experiment was much greater than 4. in summary, the data above indicated that the modeling was successful, and the data results were accurate with little error. The information in the Table 3 could also be derived from the magnitude of the effect of the three factors on the yield, which were pectinase addition > material-liquid ratio > sonication time. The relationship between the interaction of three factors on the yield was shown below:(3.1) Yield(%)=6.32-0.5363∗A+0.6763∗B+0.1925∗C-0.3775∗AB-0.0300∗AC+0.4350∗BC-1.09A2-1.88B2-2.88C2

Table 2 Experimental design of Box-Behnken response surfaces and corresponding data results.

Run	Solid-liquid ratio (ml/g)
A	Pectinase addition (%)
B	Ultrasonic
Time (min)
C	Yield (%)
Actual	Predicted	
1	40	2.5	55	6.4	6.300	
2	50	2.5	65	1.96	1.986	
3	40	2.5	55	6.32	6.300	
4	30	2.5	45	2.68	2.584	
5	40	3	65	2.87	2.866	
6	40	3	45	1.61	1.500	
7	50	2.5	45	1.64	1.640	
8	30	2	55	2.83	2.843	
9	40	2	45	1.11	1.084	
10	40	2.5	55	6.28	6.300	
11	30	3	55	4.92	4.910	
12	40	2.5	55	6.3	6.300	
13	30	2.5	65	3.12	3.149	
14	50	2	55	2.54	2.535	
15	40	2	65	0.63	0.628	
16	40	2.5	55	6.29	6.300	
17	50	3	55	3.12	3.103	

Table 3 ANOVA regression model.

Source	Sum of Squares	df	Mean Square	F-value	p-value		
Model	67.73	9	7.53	4598.56	<0.0001	**	
A-Solid-liquid ratio	2.30	1	2.30	1405.81	<0.0001	**	
B-Pectinase addition	3.66	1	3.66	2235.67	<0.0001	**	
C-Ultrasonic Time	0.2965	1	0.2965	181.16	<0.0001	**	
AB	0.5700	1	0.5700	348.33	<0.0001	**	
AC	0.0036	1	0.0036	2.20	0.1816	−	
BC	0.7569	1	0.7569	462.53	<0.0001	**	
A2	4.96	1	4.96	3030.39	<0.0001	**	
B2	14.89	1	14.89	9096.42	<0.0001	**	
C2	34.99	1	34.99	21382.24	<0.0001	**	
Residual	0.0115	7	0.0016				
Lack of Fit	0.0022	3	0.0007	0.3125	0.8167	−	
Pure Error	0.0093	4	0.0023				
Cor Total	67.74	16					
** Indicates highly significant (P < 0.001); * indicates significant (P < 0.05); − indicates not significant.

Table 4 ANOVA regression model.

Std. Dev.	0.0405	R2	0.9968	
Mean	3.57	Adjusted R2	0.9956	
C.V. %	1.13	Predicted R2	0.9973	
PRESS	0.0493	Adeq Precision	183.1294	

So in a word, the model could be chosen to predict the best method of polysaccharide extraction by ultrasonic assisted pectinase method and its optimum yield.

3.7 Response surface optimization analysis

Under optimal conditions for one of the factors, each response surface and contour represents the combined effect of the other two factors on yield at the specific range. The combined effect of the three factors on yield could be visualized more clearly. Each response surface had a corresponding contour line. The more significantly the color of the contour line changed, which meant that the effect of the two factors on the yield was more significant, and the optimal conditions as well as yield was in the middle of the smallest ellipse. In Fig. 6a, Fig. 6b, Fig. 6c A, it could be seen that the surface changed greatly, and the corresponding contour plots had drastic color changes, which could lead to the conclusion that the extraction rate was greatly affected by the pectinase addition and ultrasonic extraction time under the condition of material-liquid ratio of 1:48.49. When the pectinase addition was 2.5 %, the extraction rate increased steeply within about 55 min and reached the maximum value at 55.36 min, and then began to decline gradually. It could be seen that the response surface and contour changes in Fig. 6c were also more obvious. It could be concluded that; when the pectinase addition was controlled at 2.5 %, the ultrasonication time and material-liquid ratio also had a relatively large effect on the polysaccharide yield. Between 50–60 min, the yield increased slowly with the increase of material-liquid ratio and started to decrease once it exceeded 1:40. The response surfaces in Fig. 6b didn't change significantly from the contour lines, indicating that the material-liquid and pectinase addition didn't had a significant effect on the yield. This was also consistent with the order of magnitude of the effect of different factors on yield in 3.2. So, it could be concluded that the optimal extraction conditions for this method are: meterial-liquid ratio of 1:48.49 (g/mL), pectinase addition of 2.5 %, extraction time of 55.36 min, and a predicted extraction rate of 5.1 %.Fig. 6a The combined effect of pectinase addition and utrasonic time on yield.

Fig. 6b The combined effect of pectinase addition and liquid–solid ratio on yield.

Fig. 6c The combined effect of ultrasonic time and solid–liquid ratio on yield.

3.8 Verification of optimal conditions

In order to validate the optimal conditions predicted above, additional refinement and experimentation of the conditions was required. The pectinase addition was controlled to be 2.5 %, ultrasonic power to be 360 W, material-liquid ratio to be 1:50 and extraction time to be 55 min. Three experiments were done in parallel and the three results were averaged to obtain a yield of 4.86 %, which was nearly the forecasted value. In summary, it could be concluded that the model had good accuracy and significance.

3.9 Effect of the two extraction methods on polysaccharide composition

Sugar content, glucuronic acid and protein had been tested for studying the composition of polysaccharides [52], and by obtaining LSP-HW (water extraction) and LSP-UAE (ultrasound-assisted enzyme assay) under optimal conditions of temperature and material-liquid ratio, the difference in composition between the two of them was compared. As shown in Table 5, the sugar content of LSP-HW was only 55.60 %, while LSP-UAE had a high sugar content of 85.00 %. It indicated that the polysaccharide obtained by ultrasound-assisted pectinase method not only had a high yield, but also had a higher sugar content. Immediately the uronic acid of the LSP-UAE was also higher than the LSP-HW, which could prove that the acidic polysaccharide of LSP-UAE was higher than the acidic polysaccharide of LSP-HW. This might be that ultrasound transmitted mechanical waves to the nearby cellular molecules through the water. It generated a higher pressure and promoted the dissolution of the sugar molecules through the bursting of the resulting bubbles [53], [54]. Through comparing the protein content, it was found that LSP-HW contained much higher protein content than LSP-UAE. This may be that under the mild aqueous extraction conditions [55], polysaccharides and proteins were extracted due to the tight bonds between them. In contrast, under ultrasonic conditions, mechanical waves broke chemical bonds such as hydrogen–hydrogen bonds between protein molecules [56], weakening the chemical bonding forces between sugar molecules and proteins. This promoted the detachment of proteins from polysaccharides. At the same time, ultrasonic waves could also make the molecular weight of polysaccharides smaller [57] and more uniform, which could promote the dissolution of polysaccharides and increase the yield of polysaccharides.Table 5 The composition of polysaccharides obtained by the two extraction methods.

Polysaccharide species	Yield	Sugar content	Glycolic acid	Protein content	
LSP-HW	2 %	55.60 %	16.84 %	22.41 %	
LSP-UAE	4.86 %	85.00 %	21.41 %	13.00 %	

Polysaccharides were extracted with a certain amount of proteins, and these proteins had an effect on the solubility as well as the activity of polysaccharides. In this experiment, the protein and sugar contents of LSP-HW, LSP-UAE and LSP-UAE-Removed protein samples were compared on the optimal bars (Fig. 7). It was found that the content of the sugar content was LSP-UAE＞LSP-HW＞LSP-UAE-Removed protein, and the size of the protein content was LSP-HW > LSP-UAE > LSP-UAE-Removed protein. In terms of solubility, it was LSP-HW > LSP-UAE > LSP-UAE-Removed protein. In terms of protein content, it was LSP-HW > LSP-UAE > LSP-UAE-Removed protein. Therefore, it could be concluded that the higher the protein content of polysaccharides, the better the polysaccharides were in terms of solubility. This may be due to the fact that polysaccharides were macromolecular compounds with inherently lower solubility. At this time, glycoproteins contain a large number of anions and cations, resulting in a higher force of interaction between the glycoproteins and water molecules. Therefore the higher the glycoprotein content, the better the solubility of the polysaccharide. As shown in Fig. 7, the Sevage method was used for de-proteinization, and the protein removal rate was high at the same time as the loss of polysaccharides was also heavy. Because this method mainly used the organic solvent trichloromethane, the protein will be denatured and precipitated, and will also carry away part of the polysaccharide. Therefore, there was an urgent need to find a new protein removal method that c maximize both protein removal and polysaccharide retention.Fig. 7 Protein content and sugar content of different types of polysaccharides.

3.10 In vitro antioxidant activities of LSP

3.10.1 Ability to scavenge hydroxyl radicals

Iron is present in the human body and occupies an important role in hemoglobin or respiratory enzymes. However, when iron ions once behave abnormally, it leads to the production of a number of toxic free radicals, which include hydroxyl radicals [58]. This free radical can seriously damage the structure of functional proteins in the human body, which can lead to diseases such as atherosclerosis [59] and cancer [60]. In this experiment, the scavenging ability of LSP for free radicals was tested by the reaction between H2O2 and Fe2+ to generate –OH with strong oxidizing ability through the colorimetric method, which was also called the Fenton reaction. It could be clearly observed through Fig. 8 that the scavenging rate of polysaccharides did not change much in the concentration range of 0.125 mg/mL–1 mg/mL, and it was maintained at about 25 %. However, when the polysaccharide concentration exceeded 1 mg/mL, free radical scavenging by polysaccharides began to increase rapidly, and the highest scavenging reached 83.43 %. But the scavenging rate was smaller than the corresponding Vc concentration. Finally, the scavenging of LSP-HW at 4 mg/mL was smaller than that of LSP-UAE (Fig. 8).Fig. 8 Hydroxyl radical scavenging activity of LSP.

3.10.2 Antilipid peroxidation capacity

Lipids also play important functions in the human body, such as energy storage and information transfer. Once the blood lipids are abnormal, it can cause diabetes and cardiovascular diseases, etc [61]. In this experiment, the anti-lipid capacity of polysaccharides was determined, and the results were shown in Fig. 9. In the concentration range of 0.125–1 mg/mL, the scavenging ability of polysaccharide was slowly increasing. When the concentration exceeded 1 mg/mL, the scavenging ability of polysaccharide increased steeply up to 80 %. This may be due to the fact that the polysaccharide needs to reach a minimum concentration in order to show activity significantly. However, all of the polysaccharide solutions had less anti-lipid capacity than the corresponding concentration of Vc. It showed that the anti-lipid capacity of LSP-HW was significantly higher than that of LSP-UAE in Fig. 9. This might be related to the glycoprotein content in it, since proteins were also biologically active due to their unique structure [62], [63]. Thus glycoproteins also assist the activity of polysaccharides to some extent.Fig. 9 Anti-lipid peroxidation ability of LSP.

3.10.3 Reducing ability

In this experiment, the polysaccharide was made to reduce K3Fe(CN)6 to K4Fe(CN)6 (PH = 6.6), thus allowing K4Fe(CN)6 to combine with Fe3+ to produce Fe[Fe(CN)6]3. This complex has the strongest absorption peak at 700 nm. The stronger the absorbance of the polysaccharide solution, the better the antioxidant property of the polysaccharide. It showed that the reducing resistance of the polysaccharide increased with concentration but was less than the absorbance of Vc. A comparison of LSP-HW and LSP-UAE showed that the reducing ability of LSP-HW and LSP-UAE was not much different, and the water extraction was slightly stronger in Fig. 10.Fig. 10 Reducing ability of LSP.

3.10.4 DPPH radicals scavenging activity

DPPH was commonly used as an in vitro antioxidant activity assay due to its stable conjugated large π-bonding centers leading to this free radical and its stability. When the polysaccharide encountered this radical, it would scavenge the radical. The antioxidant activity of the polysaccharide was found by the difference in absorbance of the mixed solution at 517 nm [64]. It was found that LSP had a good ability to scavenge DPPH, and its scavenging rate increased with the concentration of polysaccharides, reaching a maximum of 66.67 % (Fig. 11). However, the observation of Fig. 11 revealed that the free radical scavenging activity of LSP-UAE was slightly less than that of LSP-HW under the same concentration conditions for both polysaccharides. The overall difference between the two types of polysaccharides was not significant.Fig. 11 DPPH free radicals scavenging activity of LSP.

3.10.5 Exploring the activity of different polysaccharides in high concentration

Since the activity of polysaccharides increased with the rise in concentration, and in the above discussion it was found that the polysaccharide solution at 4 mg/mL was the most active one. Therefore LSP-HW and LSP-UAE were explored under this concentration condition to find out the difference between their antioxidant activities. Observation of Fig. 12 revealed that UAE-HW was a little stronger than LSP-UAE in terms of anti-lipid, reducing ability and scavenging of DPPH radicals. LSP-HW was slightly lower than LSP-UAE in terms of anti-hydroxyl radical ability. However, LSP-UAE was stronger than LSP-HW in terms of hydroxyl radicals. The reason for this may be that ultrasound-assisted extraction increased the antioxidant activity of hydroxyl radicals of polysaccharides. However, overall, the activity of polysaccharides extracted from water was slightly higher than that of those extracted from ultrasound-assisted enzymes, which might be related to the protein content of the polysaccharides. The deeper reasons need to be further explored and refined by subsequent researchers.Fig. 12 Differences in the activity of different LSPs at high concentrations.

4 Conclusion

The present study employed ultrasound-assisted pectinase as well as RSM for the extraction of lychee nuclear polysaccharides (LSP). It showed that the extraction time was 55 min under the conditions of material-liquid ratio of 1:50, pectinase addition of 2.5 %, and ultrasonic power of 360 W and temperature of 55 °C. Under these conditions, the yield reached 4.86 % and the sugar content reached 85 %. Compared with ultrasound-assisted extraction, this condition greatly increased the sugar content, glucuronic acid content, and yield of LSP. In terms of solubility, it was found that LSP-HW > LSP-UAE > LSP-UAE-Removed protein. LSP was found to be very effective in inhibiting hydroxyl radicals and DPPH radicals. It had also been shown to inhibit reduction and inhibit lipid peroxidation. On this basis, the difference in activity between LSP-HW and LSP-UAE was also compared. It was LSP-HW > LSP-UAE in terms of anti-lipid, DPPH radical scavenging and reducing ability. It was LSP-HW < LSP-UAE in terms of anti-hydroxyl radicals. Herein, lychee nuclear waste was utilized and bioactive polysaccharides with high concentration of sugar content were extracted, which was in line with the characteristics of green chemistry. This provided a theoretical basis for subsequent people's research and product development. This also provided some reference value for the medical or cosmetic industry. Future research on lychee nuclear polysaccharides should focus on modifying the structure and functional groups of its polysaccharides to achieve better antioxidant activity.

Author contributions

Zongyan Song wrote the manuscript. Gangliang Huang and Hualiang Huang reviewed & edited the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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