Drying curve simulation and LF-NMR online monitor of water state in ursolic acid loaded chitosan nanoparticles during microwave freeze drying

: The changes in various states of water in ursolic acid (UA) loaded chitosan nanoparticles were assessed using low-field nuclear magnetic resonance (LF-NMR) during microwave freeze drying (MFD) process, and six thin-layer models were applied to simulate the drying kinetics. UA nanoparticles were dried at different microwave power densities (1 W/g, 2 W/g and 4 W/g). The results showed that three water fractions with different transverse relaxation times ( T 2 ) were detected in fresh UA nanoparticles. The T 2 relaxation time of water decreased significantly with drying time at different microwave power densities. And the mutual migration and transformation of water in different states during the drying process of chitosan nanoparticles occurred. Furthermore, mathematical model analysis showed that the Page model provided the best description during the process of UA nanoparticle dried by MFD. The Page model can better simulate the drying kinetics of chitosan nanoparticles dried by MFD, and LF-NMR technology can monitor the changes in water status of UA nanoparticles. The results revealed that LF-NMR can monitor the changes of water in UA nanoparticles during the drying process.


Introduction
Ursolic acid (UA) is a representative pentacyclic triterpenoid carboxylic acid existing in many medicinal plants [1] .UA has multiple pharmacological effects such as anti-inflammatory, antibacterial, antitumor, antioxidant activity [2] .Although UA has various functional properties, it has poor solubility and low bioavailability [3] , which limits their use in functional foods.To improve the solubility and bioavailability of UA, UA loaded chitosan nanoparticles were prepared using the cross-linking agent sodium tripolyphosphate by ionotropic gelation method.Drying is an essential step to obtain and preserve UA nanoparticles.Therefore, it is of importance to achieve high-efficiency control of water content of the microcapsules via evaluating dry characteristics of UA nanoparticles.
In recent years, microwave freeze drying (MFD), as a new type of drying technology, has been extensively studied due to its advantages in improving food quality and reducing energy consumption [4] .Freeze drying (FD) as a common freeze drying method also can obtain dried product in high quality, but it always requires more time and energy consumption.For example, freeze dried Litsea cubeba essential oil microcapsules required 48 h [5] .The pumpkin seed oil microcapsules were dried for 48 h using freeze drier [6] .Compared with FD, MFD can greatly improve drying rate, reduce the drying time [7] .In order to avoid the disadvantages of FD, MFD can be used to produce products in high quality with less energy consumption.Duan et al. [8] reported that the time for drying sea cucumber by MFD was nearly a half of that for FD, and there were no significant differences on the quality of microwave freezeand freeze dried sea cucumber.Jiang et al. [9] reported that compared with FD, MFD can reduce drying time by 40% and provide similar quality.Moreover, MFD could realize better dry mushroom quality and drying efficiency than FD [10] .Therefore, MFD is one of the most promising technologies for accelerating drying and improving the overall quality of products [11] .To effectively improve the quality of MFD products and optimize the drying process, it is necessary to use mathematical models to describe the drying process under different drying conditions.Thin layer drying equations are of importance in the mathematical modeling in the drying process, which can better understand the drying mechanism, effectively map the trend of drying characteristics, and optimize energy efficiency [12,13] .Li et al. [14] found that the Page model could provide suitable description on the foam mat drying process of cantaloupe puree.Parveen et al. [15] predicted the drying kinetic of aonla varieties through the application of the Newton model.Kadriye et al. [16] found that the Two-term exponential model could adequately describe the drying behavior of kiwi slices.However, information on the study of the drying kinetic model and drying mathematical models of UA nanoparticles dried by MFD is still unknown.
Knowledge of water state change of materials during drying is important for prediction of dehydration.Low field nuclear magnetic resonance (LF-NMR), an excellent noninvasive tool, is an advantageous choice for analyzing dynamic water states of product during drying with high sensitivity and short detection time [17] .The change in relaxation time in LF-NMR was used to explain the water mobility and distribution inside the sample from a microscopic point of view.The 1 H proton relaxation time was measured to obtain the moisture information about free water, immobile water and bound water in materials.LF-NMR is widely used in food drying process to analyze changes in water distribution and mobility [18] .Li et al. [19] adopted LF-NMR to monitor the water state and distribution of mulberries in real-time during the drying process and the T 2 reduction trend of free water was consistent with the drying kinetic curves.Lin et al. [20] studied that the water mobility and distribution of soybean antioxidant peptide powders were monitored in real-time using LF-NMR during storage environment.Xu et al. [21] reported that LF-NMR could be used to understand the water changes of the broccoli samples during the drying process by studying the water distribution.LF-NMR technique is preponderantly used in evaluating the effect of pullulan on the water distribution of rice starch gels [22] .Therefore, LF-NMR can effectively monitor changes in the water state of material during drying.The water state and distribution of microwave freeze-dried UA loaded chitosan nanoparticles measured using LF-NMR may be more effective and intuitive.However, researches on evaluating drying characteristics of UA nanoparticles via LF-NMR cannot be available.
In this study, UA loaded chitosan nanoparticles were dried by MFD at different microwave power levels, and mathematical models were used to reveal the dehydration of UA nanoparticles dried by MFD.Meanwhile, the drying curve and LF-NMR technique were chosen to explore the water migration characteristics of UA loaded chitosan nanoparticles.

Preparation of UA loaded chitosan nanoparticles
UA loaded chitosan nanoparticles were prepared according to the method of Yan et al. [23] with some modification.Chitosan was added to 1% (w/v) acetic acid solution for magnetic stirring for 8 h at 25℃ to obtain transparent solution.The pH of chitosan solution was adjusted to 5.0 with 0.1 g/mL sodium hydroxide solution, and then 1% Tween-80 was added and magnetically stirred for 20 min at 25℃.The UA solution dissolved in ethanol was further added to the above solution at the UA-chitosan mass ratio of 4:1, and under magnetic stirring for 30 min.TPP solution of 2.0 mg/mL was slowly dropped into the mixed solution at the chitosan-TPP mass ratio of 4:1, and magnetically stirred for 45 min to obtain UA loaded chitosan nanoparticles suspension.The nanoparticles suspension was centrifuged at 12 000 r/min for 20 min, and the precipitate was washed with distilled water to remove unbound UA, and dried for further studies.

Drying experiments
The precipitate of the same weight is filled in a 60 mm petri dish and dried by a microwave freeze dryer which was developed by Duan et al. [24] The precipitate was frozen at -25℃ for at least 8 h.The pressure of the independent polypropylene drying cavity and cold trap temperature were carried out at 100 Pa and -40℃, respectively.The power density loading levels of microwave were set at 1.0 W/g, 2.0 W/g and 4.0 W/g, respectively.Remove the petri dishes every 15 min and weigh them, put the samples into NMR bottles, and then put the new petri dishes containing the sample into it to dry.All the above drying experiments were performed in triplicate.

Detection of LF-NMR
The relaxation time (T 2 ) of UA loaded chitosan nanoparticles was measured by an LF-NMR analyzer (Shanghai Nguyen Electronic Technology Co., Ltd.) with a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence.Take 5 mg of UA nanoparticles each time with forceps and place them in a NMR bottle.The main LF-NMR parameters were set as follows: main frequency (SF), 21 MHz; sampling point (TD), 89 998; offset frequency (O1), 306 350.93; sampling frequency (SW), 100 kHz; time echo (TE), 0.4 ms; time waiting (TW), 1500 ms; the number of scan (NS), 16 and the number of echoes (NECH), 2250.The T 2 inversion program was used to obtain the relaxation map of the samples.

Mathematical modeling of drying curves 2.5.1 Moisture content and moisture ratio (MR)
The moisture content in UA loaded chitosan nanoparticles was determined using an oven at 105℃ by direct drying method [25] .All the tests were performed in triplicate.During the MFD experiments, the moisture content, drying rate (DR) and moisture ratio (MR) were calculated using Equations ( 1)-( 3), respectively.
where, W t and MR are the wet basis moisture content (%) and the moisture ratio of the samples, respectively; DR is the drying rate, %/min; m t is the weight at any time, g; m g is the constant weight after drying at 105℃, g; M t+dt is moisture content at the time of t+dt, %; t is the time interval of drying, s; X t is the moisture content of the chitosan nanoparticles, %; X o is the initial moisture content (%) and X e is equilibrium moisture content (%) of the samples, which is usually ignored in engineering applications [26] .

Mathematical models
Six mathematical models (Table 1) were selected for describing the MFD process of UA loaded chitosan nanoparticles [27,28] .The fitness between the mathematical model and the experimental data could be evaluated by the highest value of the coefficient of determination (R 2 ) and the lowest values of the chi-square test (χ 2 ) and the root mean square error (RMSE) [29] .The R 2 , χ 2 and RMSE values were calculated with the following Equations ( 4)-( 6) [30] .
where, MR exp,i , MR pre,i , n and z are the experimental moisture ratios, the predicted moisture ratio, the number of observations and a constant, respectively.

Statistical analysis
Results were performed as means ± standard deviation.Origin 2017 software was used to draw result diagrams.SPSS software (version 25, SPSS Inc., Chicago, IL) was used to analyze the significance of the data.In order to determine the significant difference between the group samples, the confidence interval was selected as 95% (p<0.05).

Drying characteristics
UA-loaded chitosan nanoparticles were dehydrated at three microwave power density of 1, 2 and 4 W/g to equilibrium moisture content.The drying curve and drying rate curves of UA-loaded chitosan nanoparticles are shown in Figure 1.Figures 1a and 1b showed that during the MFD process, the drying curves displayed similar trend, but the greater the microwave power density, the shorter the drying time and the greater the maximum drying rate.Figure 1a showed that at microwave power density level of 1, 2 and 4 W/g, the time to reach the final moisture content were 150 min, 120 min and 110 min, respectively.The drying time decreased with the increase of microwave power density, similar results were also found in drying Chinese Yam (Dioscorea opposite) Tubers by MFD [31] .Compared with the drying time at 1 W/g, the drying time at 2 and 4 W/g were reduced by 20% and 26.7%, respectively.This might be attributed to that the higher microwave power density provided more energy for water molecules to evaporate.It can be seen from Figure 1b that the drying rate is mainly manifested as a deceleration stage in the whole process.When the microwave power density is 4 W/g, the maximum drying rate can reach 1.87 %/min, while the drying rate at 1 W/g is the smallest, and the maximum value at 1 W/g can reach 1.07 %/min.The reason may be that when the microwave power density is higher, the penetration effect of the microwave radiation energy is stronger, and the drying rate is also higher.Therefore, in the actual drying process, the microwave power density can be appropriately enhanced to increase the drying rate to efficiently obtain chitosan nanoparticles.

Models of drying kinetics
The data obtained during the drying process of UA nanoparticles at different microwave power densities were fitted to six mathematical models, as listed in Table 2.The constants of the drying model varied with the microwave power densities.The higher value of R 2 and the lower values of the χ 2 and RMSE indicated accuracy of mathematical tested models to describe the drying behavior.Table 2 shows that the values of R 2 are greater than 0.99 and the χ 2 and RMSE values are both less than 0.01, indicating that all six excellent models have a good fit.Among them, the highest R 2 value given by the Page model was 0.999 91, and the lowest χ 2 and RMSE values were 8.9×10 -6 and 0.002 829, respectively.More specifically, R 2 value obtained by the Page model fitting at different microwave power densities was ordered 4 W/g > 2 W/g > 1 W/g.Therefore, the Page model was selected to describe the drying kinetics of UA-loaded chitosan nanoparticles.This was also in agreement with that the Page model was the most suitable model to exhibit the drying characteristics of ripe mango cubes [32] .Herein, the constants obtained in the Page model can be better used to fit the drying processes of UA-loaded chitosan nanoparticles.

Drying mathematical model verification
To verify the accuracy of the Page model, the results of the experimental values and the predicted values of MR under 1, 2, and 4 W/g conditions were compared in Figure 2. It can be seen from Figure 2 that the Page model curve was basically consistent with the experimental value.At the same time, Figure 3 shows that the experimental values are equal to the predicted values of MR.Moreover, Figure 3 shows that the experimental value and the predicted value have a high degree of fit, and the Pearson correlation coefficient is 0.997, indicating that the Page model could better reflect MR in MFD of UA-loaded chitosan nanoparticles.

Water states in wet chitosan nanoparticles evaluated by LF-NMR
The indicator of water mobility T 2 is generally used to measure the binding force of water and matter in materials.The larger the T 2 is, the weaker the water and matter is bound [33] .Figure 4 shows that the water states in wet chitosan nanoparticles.The internal moisture in chitosan nanoparticles can be divided into three areas, and the bound water range is 0.1-10 ms (T 21 ), the immobile water range is 10-100 ms (T 22 ) and the free water range is 100-1000 ms (T 23 ) [34] .The peak area can characterize the moisture content, and the corresponding peak areas of T 21 , T 22 and T 23 are A 21 , A 22 and A 23 , respectively, which represent the content of bound water, immobile water and free water.

LF-NMR analysis of chitosan nanoparticles dried by microwave power densities
The continuous T 2 distribution curves of UA-loaded chitosan nanoparticles during drying process at different microwave power densities are shown in Figure 5.The chitosan nanoparticles were dried by MFD at different microwave power densities, and the moisture state change showed a similar tendency.Figure 5 shows that as the MFD process continue, there is a significant decrease in the peak amplitude.The T 2 distribution tended to move to the left side as a whole in Figure 5, indicating that water mobility of chitosan nanoparticles decreased.Figure 5 shows that the similar T 2 distribution of wet chitosan nanoparticles are present at the initial stage of MFD under different microwave power densities, and the free water is firstly removed and the greater the microwave power density, the faster the free water is removed.The moisture of chitosan nanoparticles changed in the later stage of drying, and the remaining water mainly existed in the form of bound and immobile  water.The results were in agreement with the spray drying characteristics of saffron bioactive components encapsulation.Figure 5 shows that a higher microwave power density could better promote the conversion of water from a high degree of freedom to a low degree of freedom, resulting in different time to reach the equilibrium moisture content of 5%.The results showed that the degree of moisture conversion was different due to the different microwave power densities.

Effect of different microwave power densities on free moisture state
Figure 6 shows that the free water peak area A 23 variation curves of UA-loaded chitosan nanoparticles at different microwave power densities.It can be seen from Figure 6 that free water peak area A 23 gradually decreases as the drying process progresses and the greater the microwave power density, the shorter the drying time for A 23 to reach the minimum value, indicating that the microwave is beneficial to the sublimation of moisture during the drying process.The order of free water removal under different microwave power densities is 4 W/g > 2 W/g > 1 W/g.The change trend of the free water peak area A 23 in chitosan nanoparticles is similar to the drying curve in Figure 1a, and the change of free water content during the drying process would directly affect the drying effect.

Effect of different microwave power densities on immobile moisture state
The immobile water A 22 variation curves of UA-loaded chitosan nanoparticles at different microwave power densities are shown in Figure 7.As shown in Figure 7, the immobile water curve in the chitosan nanoparticles presented a decrease and increase alternately as the drying process progresses at 1, 2, 4 W/g.The reason for the decrease may be attributed to that a part of the immobile water migrated to the free water and then was removed after free water was removed in the early drying stage.The second reason for the reduction may lie in that the hydrophilic group structure of polysaccharides can reduce the dynamic water flowing around them, leading in the migration of immobile water to bound water [35] .The immobile water increase may be caused by that part of the unstable bound water was irradiated by microwaves and migrated to the immobile water.In addition, with the extension of the drying time, the shrinkage of chitosan nanoparticles occurred, and the concentration difference caused part of free water to migrate to immobile water [36] .The results showed that with the increase of microwave power density during the drying process of UA-loaded chitosan nanoparticles, the degree of mutual migration and transformation of water in different states increased.

Effect of different microwave power densities on bound moisture state
The effect of different microwave power densities on the migration change of bound water A 21 is shown in Figure 8.As shown in Figure 8, the bound water of UA-loaded chitosan nanoparticles during drying process displayed an increase in the whole stage followed by a decrease at different microwave power densities.At the primary stage of drying, part of the free water was combined with chitosan material, resulting in an increase in the bound water peak area A 21 .In the later stage of drying, part of bound water is converted into immobile water and removed.The result was consistent with the change trend of Pleurotus eryngii bound water A 21 during microwave hot-air flow rolling drying [37] .

Conclusions
In this study, the MFD drying process was described by drying curves, the Page model, and the LF-NMR technology was used to characterize the water distribution and changes of UA nanoparticles.The drying curves of UA-loaded chitosan nanoparticles showed a decline trend during the drying process at different microwave power density, and higher microwave power can realize shorter time to reach equilibrium moisture.The MFD process of UA-loaded chitosan nanoparticles could best be fitted by Page model.During drying process, T 2 distribution tended to move to the left side.Free water was removed at the initial stage of drying, and the remaining water mainly existed in the form of bound and immobile water in the later stage of drying.During the MFD process of chitosan nanoparticles, free water kept decreasing, the immobile water peak area curve showed fluctuating, and the bound water peak area curve showed an increase followed by a decrease.All these findings proved the changes in the moisture content and distribution of chitosan nanoparticles during the MFD process.This study can provide a reference for exactly stimulating and optimizing MFD process of UA nanoparticles.

− 1 )Figure 1
Figure 1 Drying curves and drying rate curves of UA loaded chitosan nanoparticles at different microwave power densities

Figure 3 Figure 4 1 Figure 5 T
Figure 3 Fitting curve of drying mathematical model

Figure 6
Figure 6 The free water A 23 variation curves of UA-loaded chitosan nanoparticles during drying process at different microwave power densities

Figure 7
Figure 7 The immobile water A 22 variation curves of UA nanoparticles during drying process at different microwave power densities

Figure 8
Figure 8 The bound water A 21 variation curves of UA nanoparticles during drying process at different microwave power densities