Ultrasonic and microwave treatment improved jujube juice yield

Abstract The methods to leaching juice from dried jujube using six treatments and some factors related with juice yield such as total soluble solids (TSS), pectinase activity, pectin contents, galacturonic acid, and the microstructure morphology of cell were investigated. Six treatments including natural leaching, ultrasonic, microwave, ultrasonic before microwave, ultrasonic after microwave, and pressing directly were applied to extract juice. The group which treating with ultrasonic before microwave displayed its total soluble solids (TSS) was 16 °Brix, which was 6.67% higher than that of natural leaching. And its total soluble solids (TSS) reached to equilibrium in 2 hr, which was faster than that of natural leaching. The mechanism of improving the production efficiency of juice yield using ultrasonic combined with microwave was explored accordingly. The content of pectin and galacturonic acid increased by 58.52% and 59.01%, respectively, which were the highest among all samples. The activity of pectinase was 9.71 μg/(h·g), which was significantly decreased 40.23% as compared to natural leaching. And the treated cells became shriveled and pitted, which led to the leakage of the contents of cell. Thus, the result showed that treating with ultrasonic before microwave displayed the best juice yield. Ultrasonic cooperate with microwave was an efficient method to leaching juice from dried jujube.

and collapsed within a medium, and then produce cell disruption, the content of cells flowed toward matter's surfaces, or the liquid medium finally (Bermúdez-Aguirre & Barbosa-Cánovas, 2012). Cell disruption made extraction and processing more efficient. The ultrasonic device is cheap, simple, and small, and its operation is easy (Chen, Tang, Chen, Wang, & Li, 2010). Compared to other techniques, ultrasound has mild extraction conditions, fast extraction speed, and a wide application range. The advantages are high extract efficiency and short reaction time (Wang, Vanga, & Raghavan, 2019).
In recent years, it has attracted widespread attention of researchers and has become a hot topic on food industry. It can be used alone or in combination with other processing technologies.
Microwaves are high-frequency electromagnetic waves with frequency variation from 300 MHz to 300 GHz (Chandrasekaran, Ramanathan, & Basak, 2013). Microwave has been extensively used to preserve or extract chemical from food products. It can produce alternating magnetic field, make the molecule in the material produce instantaneous polarization, and then do high-speed thermal movement (Chizoba Ekezie, Sun, Han, & Cheng, 2017). Although the heated food reaches a high temperature quickly, it is worth noting that nonuniform temperature distribution during microwave heating of food materials was in existence. Microwave has gained popularity in food processing due to the characteristics of good penetration, high selectivity, significant reduction in heating time, high efficiency, energy saving, ease and safe of operation, and low maintenance (Kubo, Curet, Augusto, & Boillereaux, 2019). Moreover, owing to contact with food indirectly, it is cleaner and can achieve the selective extraction of substances meanwhile. To some extent, microwave heating might change flavor and nutritional qualities of food more or less as opposed to conventional processing (Benlloch-Tinoco, Igual, Rodrigo, & Martínez-Navarrete, 2013).
Jujube (Ziziphus jujuba Mill.) is popular for its high nutritional value and medicinal uses (Reche et al., 2018). Jujube fruit provides rich contents of natural bioactive nutrients such as ascorbic acid, amino acids flavonoids, and terpenoids, which are responsible for various pharmacological activities (Siriamornpun, Weerapreeyakul, & Barusrux, 2015). Jujube juice is one of the products originating from jujube processing. Owing to the low moisture content of dried jujube, the juice of dried jujube did not respond well to pressing. To acquiring jujube juice, the whole dried jujube without any destroy was usually leached, or grinded with water to become into pulp.
However, the production efficiency of leaching is lower and its juice yield is lower as well. The method to be grinded might quickly acquire juice, but clarification of juice is difficult. Moreover, there was some bitter taste in jujube juice whether leaching or grind method, and consumers often struggle to take it in (Jiang, Li, Wang, Li, & Hong, 2016;Li, Ding, Chen, & Zeng, 2013).
A combination of ultrasound and microwaves widely used in the extraction as innovative "green" food processing technique, and both of them have an important role in promoting sustainable food industry (Su, Zhang, Adhikari, Mujumdar, & Zhang, 2018). However, there is no report on the compound method of the two technologies in the extraction of juice and the synergistic effect of ultrasound and microwave had not been discussed in juice industry. In our experiments, the dried jujubes were firstly sliced, and then were treated with microwave combined with ultrasonic. And the jujube juice yield was researched. Further, the mechanism related with jujube juice was investigated from the point of cell wall such as pectin, galacturonic acid, pectinase, and microstructure. This research might provide an efficient method to acquire juice originating dried fruit, and expand the applications of ultrasonic and microwave in food industry.

| Materials and reagents
The fresh jujube (Ziziphus jujuba Mill. cv. junzao) was picked in mid-September and fully mature jujube was selected to for drying. The jujubes were picked by hand and the peel of jujube became dark red, slightly wrinkled, and soft at the condition of high maturity. The dried jujubes were purchased from a local market in Yaodu District (Shanxi, China). The jujubes in the experiment possessed some features, such as dark red color, spindle shape, varied in length from 3-5 cm, the percentage of edible part exceeds a certain number (85%), and the percentage of water content was approximately 20%.
And they were stored at room temperature, cool, and dry, away from light, heat, ventilation, humidity ≤60%. Then, they were selected for absence of defects and rot.

| Sample treating and jujube juice acquirement
The dried jujubes were washed using tap water to remove the surface blemishes. And then, the date pits were discarded. Afterward, the cleaned jujubes were cut into slices with the thickness of approximately 3 mm. Based on preliminary studies, a bulk solution of the jujube pulp was prepared by mixing the pure pulp with water at a ratio of 25% w/w. Namely, the weight ration of jujube and water is 1:4. The mixture was treated by six methods immediately when the two substances mixed well. The natural leaching group was mixing without any treatment and leaching directly, while the pressing was squeezed by a high-speed blender for some seconds and then leaching. On the basis of the natural leaching group, it was retreated by ultrasonic and microwave. And the two processes were next to each other in the composite processing of ultrasonic and microwave.
The bulk solutions were differently processed as followed.
All the selected conditions were obtained from the preliminary experiments and they could cause the results to the best. There were six different treatment methods, including natural leaching (as control check, CK), ultrasonic (US), microwave (MV), ultrasonic before microwave (US-MV), microwave before ultrasonic (MV-US), and pressing (PS), of which the experiment was mentioned. The ultrasonic (JY92-IIDN, Ningbo, China) power and the ultrasonic time were maintained at 540 W, and 15 min, respectively. The microwave (G70D20CN1P-D2, Guangdong, China) treatment of mixture was carried out at following condition: the power of 560 W for 2 min. All the treatments were finished at a water-to-pulp ratio of 4:1.
After the juice soluble solids of each leached jujube sample no longer changed, the mixture with juice and pomace was filtered. The total soluble solids of the juice were measured by a handheld refractometer (ATAGO). Collecting jujube juice and pulp respectively, and stored in the freezer for further analysis.

| The total pectin and water-soluble pectin of jujube juice
Pectin of juice samples was determined using carbazole method developed with some modifications (Ningaé et al., 2018). Pectin was precipitated by adding 25 ml of 95% w/w ethanol at 75°C to a 50 ml calibrated centrifuge tube with 2 ml of juice. The obtained mixture was heated in an 85°C water bath for 10min and stirred thoroughly. 95% w/w ethanol was added to bring the total volume to 50 ml. A gel containing insoluble solids in ethanol was formed and then separated by centrifugation for 10 min at room temperature, and the supernatant was discarded. The mixture was immersed in a water bath (DZKE-D-2) at 85°C when the precipitation was washed by 63% w/w ethanol and then centrifuged. To remove the monose, the pectin precipitate was washed thrice with ethanol. The precipitate remaining in the centrifuge tubes was collected and dissolved in 1 M NaOH solution. It was dissolved in distilled water to a volume of 50 ml. The samples were then allowed to stand for 15 min to deesterify the pectin. The total pectin and soluble pectin of jujube juice were determined after filtrating. Each sample was analyzed in triplicate by mixing 0.5 ml sample with 0.25 ml of 0.15% w/v carbazole reagent.
While the floccus precipitate generated, 3 ml of H 2 SO 4 was introduced in test tubes which were held in a water bath at 85°C. The tubes were heated for 5 min, and then allowed to cool at room temperature for 15 min. The mixtures in the tubes were used for colorimetric determination. The transmittance was measured at 530 nm using a spectrophotometer (752N).

| The water-soluble pectin, protopectin, and total pectin of jujube pulp
The pectin was extracted according to the method reported previously by Alvarez, Alvarez, Riera, and Coca (1998) with some modifications. The pulp (2 g) ground in a mortar was suspended in 25 ml 95% w/w ethanol taken, transferred into a 50 ml graduated centrifugal tube, and kept it in a boiling-bath for 30 min. It is crucial to add 95% w/w ethanol in time during boiling. After cooling at room temperature, centrifuge for 15 min and discard the supernatant.
Then repeat the steps above mentioned for 2-3 times.
The precipitation in the original test tube dissolved with distilled water (20 ml), and hydrolysis took place at a constant 50°C for 30 min in a water bath. Centrifugal for 15 min till the tubes were cool. The precipitation washed by distilled water not only once. All the supernatant placed and dissolved into a volume of 50 ml finally.
The obtained precipitation by hydrolyzation was dissolved with the solution of 0.5 M sulfuric acid. Heating for 1 hr to dissolve protopectin in a boiling-bath and cooling, then the precipitate is removed by centrifugation and the supernatant extract is analyzed for analytics.
Pectin of samples was determined using carbazole method as was mentioned above. The experiments were performed in triplicate.
The content of total pectin is the sum of water-soluble pectin and protopectin.

| Galacturonic acid
The mixture was made by slowly adding 8 ml anhydrous ethanol to 2 ml of juice and precipitated at 4°C overnight, followed by centrifugation at 5,000 r/min, for 10 min. The precipitation was washed twice with anhydrous ethanol, acetone, and ether, respectively.
Distilled water was added to dissolve the polysaccharide obtained to 50 ml (Yang, Mu, & Ma, 2019). The pulp (2 g) tested was added to 20 ml distilled water and placed in a 50 ml centrifuge tube. The mixture water bath at 90°C for 1 hr and repeated for 2-3 times and then centrifuged while it is at room temperature. The samples (0.5 ml) with 3 ml of H 2 SO 4 were held in a water bath at 85°C for 20 min after stirring thoroughly. The test tubes were cooled in an ice bath, and 0.25 ml of 0.15% w/v carbazole reagent was added. The mixture was allowed to stand for 2 hr, and the transmittance was measured at 520 nm using a spectrophotometer.

| Pectinase activity
Diluted juice was tested directly. The pulp tissue chopped (2 g) was homogenized with a small amount of quartz sand in a mortar for a period of time with 2 ml of citric acid-phosphate buffer (pH 7.6). Then washed with buffer solution for 3 times and centrifuged at 5,000 r/min for 15 min at 4°C. The supernatant as a crude enzyme solution was kept at low temperature until used. Two 10 ml colorimetric tubes with 2.5 ml of pectin marked as a and b respectively were prepared to preheat for 5 min at 50°C in a water bath.
2 ml of citric acid-phosphate buffer (pH 5.0) and 0.5 ml of diluted enzyme liquid were added to the tubes. Tube a response accurately for 30 min in the water bath at 50°C and tube b boiled for 5 min immediately. A reaction mixture of 0.8 ml of the samples from the tubes, 0.8 ml of distilled water, and 2 ml of 3, 5-dinitrosalicylic acid reagent was stirred adequately. The reagent system was boiled for 5 min, and then immediately cooled with tap water. The rate of reaction in absorbance at 540 nm was recorded (Khurnpoon, Siriphanich, & Labavitch, 2008).

| Microstructure characterization of jujube cell
The detail of microstructure characterization testing was as followed: The collected pulp was crumbed by a sharp blade. Then, a thin layer of crumbed pulp which were putted on the slide and the other slips covered was observed with a Digital biological microscope (CX31RTSFJY) with a magnification of 400×.

| Statistical analyses
A factorial design was used to study the effect of six different processing samples. Analysis of variance (ANOVA) with the Duncan test was carried out to verify whether there is a significant difference among different treatments in the investigated traits using SPSS 20.0 (SPSS Inc.) software. And p < .05 was considered to indicate statistical significance among different treatments.

| Juice yield
The equilibrium time and juice yield were used as the index of production efficient. Figure 1 showed the equilibrium time of total soluble solids (TSS) after processing with different methods. The TSS of juice processed with US-WV reached 16 °Brix quickly and steady.
It took 2 hr to reach the equilibrium time. Additionally, the time of WV-US was 3 hr and its highest point was 15 °Brix that was 6.67% lower than US-WV. Compared to the latter, the time of the former reached the equilibrium in advance. Likewise, it had a time reduced about 1.2-fold compared to the CK. According to the changes of the equilibrium time of the TSS, the effects of ultrasound and microwave in the independent form were revealed that the intensity of microwave was higher in comparison. No obvious difference was observed between ultrasound and microwave (p > .05). The equilibrium time was 3.5 hr and the TSS was 16 °Brix which processed by PS and it reached the top slowly. Thus, the synergetic form of ultrasound and microwave accelerated the yield of juice. Further, the juice acquired with synergetic form method was clearer than PS method.

| The water-soluble pectin
When the cell wall was destroyed, the pectin of cell wall in which the liquid medium located was beneficial to the output of juice. The con-

| Protopectin
Protopectin as an insoluble substance is generally associated with plant polymers and cell debris having fiber-like molecular structure in immature fruits. Therefore, protopectin presented only in

| Galacturonic acid
Pectin is present in the cell wall and middle lamellae of fruit and veg-

| Pectinase
Pectinases are a heterogeneous group of related enzymes of cell wall that hydrolyze pectin polysaccharides of plant tissues into small molecules and are mainly responsible for the variation in pectin properties. As a compound enzyme, pectinase is widely used for clarification of fruit juice (Rajdeo, Harini, Lavanya, & Fadnavis, 2016).
Pectin was used as the basal material which digested to reflect the enzyme activity of juice and pulp during the extraction of jujube at the same time. And it was worth noted that microwave mainly passivated pectinase activity by heating, while ultrasound affected pectinase activity due to cavitation effect which caused the structure matrix to change (Arjmandi et al., 2016;Ma et al., 2016). The enzyme activity of juice of processing decreased significantly as Figure 5a showed. A half of the activity of control was that of pressing. Compared with the control, the enzyme activities of microwave and ultrasound were damaged tremendously by 37.25% and 50.41%.
In addition, unexpectedly, we found the synaptic forms of microwave and ultrasound decreased a little in our study. Similar to the changes trend of the activity of pectinase of the juice, the activity of enzyme of pulp ( Figure 5b) decreased with the treatments of microwave combination with ultrasound.

| The total pectin
The total pectin content of juice was shown in Figure 6a. There was a significant increase in the total pectin content after processing (p < .05). In this test system, the total pectin content highest was in US-WV (2,509.52 mg/L), and the second was in PS (2,402.85 mg/L).
The pectin content in the other combined treatment (WV-US) was only 1,872.62 mg/L, which was much lower (25.38%) than that in US-WV. The total pectin content in pulp was calculated as a percentage (Figure 6b). The results indicated that there were significant differences between the control and other groups (p < .05). The content of pectin of pulp by pressing was the highest, followed by the microwave after ultrasonic treatment, which were 0.0356% and 0.0339%, respectively. Besides, the total pectin content of the microwave before ultrasonic was decreased by 16.22% when it compared to MV-US.

| D ISCUSS I ON
The effect of treatments on juice quality was determined by meas- Microwave and ultrasonic might be used in juice prior to juice extraction in order to increase the extraction efficiency in the final product. In summary, microwaves in combination with ultrasound technologies presented an alternative in the processing of fruit juices with high yield, as demanded by consumers and industry. However, in-depth researches on the compositional mechanisms, such as browning, and the application on jujube juices are still needed to be carried out.

| CON CLUS ION
Treating dried jujube with ultrasonic before microwave might improve the production efficient of dried jujube juice yield. The total soluble solids (TSS) was 16 °Brix, which was 6.67% higher than that of natural leaching. And its TSS reached equilibrium in 2 hr, which was faster than that of natural leaching, accelerating the ratio of the juice output at the same time. In summary, microwaves in combination with ultrasound technologies presented an alternative in the processing of fruit juices with high yield, which was demanded by juice industry.

CO N FLI C T O F I NTE R E S T
None.