Abstract
In order to effectively protect the biological activity of Sparassis latifolia β-glucan, improve its stability, and realize its high-value utilization, single-factor test and orthogonal test were carried out to optimize the microencapsulation conditions of S. latifolia β-glucan prepared using spray drying method. The β-glucan microcapsules were characterized by scanning electron microscopy, thermogravimetric analysis, Fourier-transform infrared spectroscopy, and laser particle size analyzer. The results showed that the optimal microencapsulation conditions were as follows: maltodextrin and whey protein with a mass ratio of 1:2, core and wall material with a mass ratio of 1:2, and monoglyceride and core material with a mass percentage of 0.3. Under these conditions, the powder yield and embedding rate of β-glucan microcapsules were 47.32 ± 0.58% and 86.76 ± 1.19%, respectively. The preparation technique was proved to be stable. The β-glucan microcapsules were spherical particles, with the characteristics of a smooth surface, no cracks. The particle size of microcapsules was smaller, and its Dv (50) was 8.43 µm. The distribution of microcapsules was more uniform, and its uniformity was 0.503. The good embedding performance and high thermal stability can effectively protect the biological activity of the core material.
1 Introduction
Sparassis latifolia is a delicious and nutritious mushroom with high healthcare and medical benefits [1,2]. S. latifolia is rich in nutrients such as proteins and polysaccharides in which β-glucan is strong bioactivity predominated [3]. According to the determination results of the Japan Food Analysis Laboratory, the content of S. latifolia β-glucan reached as high as 43.6% of the dry weight [4]. The content of β-glucan in the aqueous extract of S. latifolia also reached 39.3% as determined by Kim et al. [5]. According to Lian et al. [6], the content in the stalk of S. latifolia even exceeded 50%. These results showed that there was a high content of β-glucan in S. latifolia. A large number of modern studies showed that S. latifolia β-glucan had many effects such as immune regulating effects [7], antitumor [8,9], anti-inflammatory [10], and promoting hematopoietic function of organism [11]. Therefore, S. latifolia is an edible and medicinal fungus which is not only precious but also healthy. Microencapsulation is the embedding technology of dispersed solids, droplets, and gases into microcapsules using natural or synthetic polymer materials [12]. Currently, food microencapsulation has been used to microencapsulate the core material in order to effectively protect its bioactivity and improve its stability [13,14]. Spray drying is one of the most practical and widely applied physical methods. Microcapsules prepared by the spray drying method have the advantages of applicability for heat-sensitive materials, smaller particle size of microcapsules, higher thermal stability, better solubility, and dispersibility of products [15,16]. Thus, to better protect the bioactivity of S. latifolia β-glucan, improve the stability of the product, prolong the storage time, and enhance the nutritional value, in this study, the spray drying method was used for the preparation of S. latifolia β-glucan microcapsules by the microencapsulation technology. The optimum embedding rate of β-glucan microcapsule was investigated, providing theoretical evidence for the diversified development and application of S. latifolia β-glucan functional food.
2 Experiment
2.1 Materials and instruments
Materials: Dry products of S. latifolia were provided by Fujian Rongyi fungus technology R&D Co., Ltd. Maltodextrin, lactoalbumin, gum arabic, gelatin, and monoglyceride were from Shanghai Aladdin Biochemical Technology Co., Ltd. Anhydrous ethanol, hydrochloric acid, sodium hydroxide, potassium dihydrogen phosphate, and other analytical pure reagents were from Sinopharm Chemical Reagent Co., Ltd. Congo red was from Shanghai EBE Chemical Reagent Co., Ltd. Pectinase, cellulose, papain, amylase was from Shanghai Yuanye Biotechnology Co., Ltd.
Instruments: The instruments used were YC-501 spray dryer, DC800 freeze dryer, UV mini-1204 UV visible spectrophotometer, SU8000 scanning electron microscope (SEM), Fourier transform infrared spectrometer (FTIR), RE-5296 rotary evaporator, MASTERSIZER 3000 Malvin laser particle size analyzer, TGA 209 F3 thermogravimetric analyzer, and AH-NANO high pressure homogenizer.
2.2 Preparation of S. latifolia β-glucan
Dry products of S. latifolia were crushed and filtered using a 80 mesh to obtain the sample for preparation of S. latifolia β-glucan. Deionized water was added according to the liquid material ratio of 30:1 mL/g; 2% bioenzymes such as pectinase and cellulase were added for 2 h of enzymatic hydrolysis. Then, ultrasonic extraction was carried out at 50℃ for 1 h, centrifuged, and the supernatant was obtained. The supernatant was enzymatically hydrolyzed with impurity removal enzymes (amylase) for 20 min and then was enzymatically hydrolyzed with papain for 20 min. After centrifugation and the addition of 12 g/L ammonium sulfate, the supernatant was stirred continuously in a boiling water bath till sufficient dissolution. Then, it was concentrated. After the addition of ethanol, it was centrifuged to collect the sediment, namely, the crude extract. Subsequently, it was dissolved and vacuum freeze-dried to obtain β-glucan [17].
2.3 Determination of S. latifolia β-glucan
β-Glucan content was determined using the Congo red method [18]. The β-glucan standard solution was prepared. The standard curve was simulated with the absorbance value (y) at the wavelength of 544 nm as well as the β-glucan concentration (x). The corresponding regression equation was y = 3.7371 × −0.0104 with a good correlation of R 2 = 0.9947. The β-glucan concentration in the sample solution was calculated by the regression equation with the determined absorbance value of the sample which was dissolved in water.
2.4 Preparation of S. latifolia β-glucan microcapsules
A certain amount of β-glucan was weighed and dissolved in deionized water. After the addition of an appropriate amount of monoglyceride (emulsifier) and ultrasonication for 10 min, the solution of β-glucan was obtained. A certain amount of maltodextrin and lactoalbumin were added to deionized water and stirred, separately [19,20]. After evenly mixing by stirring, two wall material solutions were mixed with the β-glucan solution. After keeping in a water bath at a constant temperature of 50–60oC for 20 min, the emulsion was obtained by homogenization with a high-pressure homogenizer at a pressure of 1,300 Pa. S. latifolia β-glucan microcapsules were obtained using the spray drying method of the emulsion sample with a spray dryer whose parameters were set in advance [21].
2.5 Single factor test
The spray drying parameters were set as follows: the inlet temperature of 180, the outlet temperature of 80, the peristaltic speed of 8 rpm, and the fan frequency of 45 Hz. Under those constant conditions, the effects of the influencing factors on the powder yield and the embedding rate of S. latifolia β-glucan microcapsules were investigated, separately. Factors included the categories of the wall materials [lactoalbumin + gum Arabic (L + GA), maltodextrin + lactoalbumin (M + L), gum arabic + maltodextrin (GA + M), maltodextrin + gelatin (M + G), gum arabic + gelatin (GA + G)], the mass ratio between maltodextrin and lactoalbumin (1:0.5, 1:1, 1:2, 1:3, 1:4 m/m), the mass ratio of core wall (1:1, 1:2, 1:3, 1:4, 1:5 m/m), the total solid content (12, 14, 16, 18, 20%), and the amount of monoglyceride (the mass percentage of monoglyceride and core material) (0.1, 0.3, 0.5, 0.7, 0.9%) [20,22].
2.6 Orthogonal experimental design
On the basis of the single factor test, the effects of three factors, such as the wall material ratio, the core wall ratio, and the monoglyceride dosage on the powder yield and the embedding rate of S. latifolia-β-glucan were investigated using the orthogonal experiment with three factors and three levels (L9, 33).
2.7 Calculation of the powder yield and the embedding rate of Sparassis latifolia β-glucan microcapsules
About 1.0 g of finished product of S. latifolia β-glucan microcapsule was weighed. The surface of microcapsules was quickly washed with 10 mL of 0.05 mol/L phosphate buffer with pH = 7.4. The supernatant was collected after centrifugation at 4,000 rpm. The β-glucan concentration on the surface of microcapsules was calculated using the regression equation with the absorbance value of the supernatant determined using the Congo red assay [23]. Similarly, 1.0 g of finished product of S. latifolia β-glucan microcapsule was dissolved ultrasonically and centrifuged at 4,000 rpm. The supernatant was collected whose absorbance value was determined using the Congo red assay. The total content of β-glucan in microcapsules was calculated using the regression equation. The calculating equation of the powder yield and the embedding rate of microcapsules were as follows [24]:
2.8 Characterization of β-glucan microcapsules
2.8.1 SEM
The morphology of S. latifolia β-glucan microcapsules were coated with gold and then analyzed using an SEM (Hitachi SU8000) at 5.0 kV. SEM images of the microcapsules were observed, and the size of microcapsules was determined as well.
2.8.2 FTIR
β-Glucan raw materials, β-glucan microcapsules, and blank microcapsules were carried out using the FTIR instrument (Nicolet iS5). The samples were mixed with KBr from pellets and further scanned from 4,000 to 500 cm−1.
2.8.3 Thermogravimetric analysis (TGA)
The sample was placed in an alumina pan with another empty pan used as a reference. The TGA thermogram was recorded over a temperature range from 30 to 500°C at a heating rate of 10°C/min under an N2 purge.
2.8.4 Laser particle size analyzer
The particle size of samples including Dv(90), Dv(50), and Dv(10) as well as the specific surface area was determined by Mastersizer 3000 Malvin laser particle sizer.
3 Results and discussion
3.1 Single factor test results of the preparation technology of β-glucan microcapsules
3.1.1 Effects of wall material categories on the powder yield and the embedding rate of β-glucan microcapsules
The wall material ratio of 1:3, the core/wall material ratio of 1:3, the total solid content of 18%, and the amount of monoglyceride of 0.2% were fixed for this analysis. As shown in Figure 1, different combinations of wall material categories [lactoalbumin + gum Arabic (L + GA), maltodextrin + lactoalbumin (M + L), gum arabic + maltodextrin (GA + M), maltodextrin + gelatin (M + G), gum arabic + gelatin (GA + G)] led to different powder yields and embedding rates of S. latifolia β-glucan. The powder yield and the embedding rate of β-glucan microcapsules prepared by composite wall materials such as maltodextrin and gelatin as well as gum arabic and gelatin were relatively low. Besides, the stickiness to the wall during the spray process was a major issue. The formation of microcapsules was poor, possibly because the large viscosity of gelatin led to the decrease in the embedding ability of the wall materials. The powder yield of microcapsules prepared with composite wall materials of lactoalbumin and maltodextrin reached 46.15%. The corresponding embedding rate reached 78.81%. Compared with those of other composite wall materials, the powder yield and the embedding rate were high. Besides, the phenomenon of the stickiness to the wall decreased [25]. Those illustrated that composite lactoalbumin and maltodextrin could effectively encapsulate β-glucan during the spray drying process.
Effect of wall material type on powder yield and embedding rate.
3.1.2 Effects of the composite wall material ratio on the powder yield and the embedding rate of β-glucan microcapsules
The core/wall material ratio of 1:3, the total solid content of 18%, and the amount of monoglyceride of 0.2% were fixed for this analysis. The composite wall material ratio was the key factor in the microcapsule preparation. Only appropriate wall material ratio led to good powder yields and embedding effects. As shown in Figure 2, not only the powder yield but also the embedding rate of β-glucan microcapsules initially increased with the decrease of the wall material ratio between maltodextrin and lactoalbumin. When the ratio of maltodextrin to lactoalbumin reached 1:2, the powder yield of β-glucan microcapsules reached a maximum of 46.16%, and the corresponding embedding rate reached 78.83%. Subsequently, the ratio between maltodextrin and lactoalbumin continued to decrease, and the powder yield and the embedding rate of the β-glucan microcapsules all demonstrated a decreasing trend. It was possibly because the viscosity of multiple emulsions increased and the fluidity decreased due to the increase of lactoalbumin content. The too-long heating time in the nozzle of multiple emulsions led to a large loss, resulting in the continuous decrease in the powder yield and the embedding rate of microcapsules.
Effect of wall material ratio on powder yield and embedding rate.
3.1.3 Effect of the ratio between the core and the wall material on the powder yield and the embedding rate of β-glucan microcapsules
The maltodextrin to lactoalbumin ratio of 1:3, the total solid content of 18%, and the amount of monoglyceride of 0.2% were fixed for this analysis. The core wall ratio directly influenced the powder yield and the embedding rate of microcapsules. When the ratio between the core and the wall material was large, the core material could not be fully embedded, the core material was easy to leak, and a large number of microcapsules stuck to the wall. If the ratio between the core and the wall material was too low, the embedded core material by the wall material decreased. The embedding rate decreased, leading to the waste of the wall material as well as an increase in the wall material cost. As shown in Figure 3, when the core wall ratio reached 1:2, the powder yield of microcapsules reached 45.34%. The corresponding embedding rate reached 88.12%. Both reached the best. When the mass ratio between β-glucan and the composite wall material was larger than 1:3, the embedding rate decreased significantly and sharply, possibly due to the decreased β-glucan usage amount because of the increase in the composite wall material ratio. When the mass ratio of β-glucan and the composite wall material reached 1:4, the powder yield of β-glucan microcapsules decreased significantly. With the continuous increase in the wall material content, the embedded core material continued to decrease accordingly. The excessive use of wall material also resulted in material waste.
Effect of core/wall material ratio on powder yield and embedding rate.
3.1.4 Effect of monoglyceride dosage on the powder yield and the embedding rate of β-glucan microcapsules
The maltodextrin-to-lactoalbumin ratio of 1:3, the core/wall material ratio of 1:3, and the total solid content of 18% were fixed for this analysis. Monoglyceride as the emulsifier also influenced the embedding rate of β-glucan microcapsules to some extent. As shown in Figure 4, when the amount of monoglyceride continued to increase, the powder yield and the embedding rate demonstrated similar trends. When the dosage of monoglyceride was 0.5%, the powder yield and the embedding rate were the highest namely, 45.12 and 82.64%, respectively. When the dosage of monoglyceride was 0.9%, the powder yield and the embedding rate were the lowest, namely, 30.18 and 75.19%, respectively.
Effect of monoglyceride dosage on powder yield and embedding rate.
3.1.5 Effect of solid content on the powder yield and the embedding rate of β-glucan microcapsules
The maltodextrin-to-lactoalbumin ratio of 1:3, the core/wall material ratio of 1:3, and the amount of monoglyceride of 0.2% were fixed for this analysis. As shown in Figure 5, when the total solid content was in the range of 12–18%, the powder yield of β-glucan microcapsule products demonstrated a decreasing trend which was not significant. When the total solid content was 20%, the powder yield was only 30.18%. When the total solid content was 14%, the embedding rate of β-glucan microcapsules was the highest and reached 81.84%. With the increase of total solid content, the embedding rate demonstrated a trend of decreasing. When the total solid content exceeded 18%, the water content in the multiple emulsion decreased, the solution viscosity increased, and the fluidity was poor. Thus, most of the solution stuck to the wall and blocked the hose. When the content of the solidified product was too small, a large amount of heat was used for water evaporation, microcapsules were difficult to dry completely, and may adhere to each other. Considering the powder production and embedding rate of microcapsules, the total solid content of 14–18% is more suitable.
Effect of solid content on powder yield and embedding rate.
3.2 Optimization of the preparation technology of β-glucan microcapsules by the orthogonal test with two indexes
On the basis of single factor test, the L9 orthogonal test (33) was designed using three factors that had a great influence on the powder yield and the embedding rate such as the wall material ratio (A), the core wall ratio (B), and the monoglyceride dosage (C). Two indexes were investigated including the powder yield and the embedding rate of β-glucan microcapsules. Data were processed by the comprehensive score method [19]. The weight coefficients of the powder yield and the embedding rate were all 0.5. The orthogonal test results are shown in Table 1. The variance analysis is shown in Table 2.
Design and results of L9 (33) orthogonal test
| No. | Factor | Powder yield (%) | Embedding rate (%) | Comprehensive score | ||
|---|---|---|---|---|---|---|
| A | B | C | ||||
| Wall material ratio (m/m) | Core/wall material ratio (m/m) | Monoglyceride dosage (%) | ||||
| 1 | 1 | 1 | 1 | 33.46 | 74.22 | 79.34 |
| 2 | 1 | 2 | 2 | 39.50 | 73.23 | 85.48 |
| 3 | 1 | 3 | 3 | 30.70 | 74.06 | 76.19 |
| 4 | 2 | 1 | 2 | 33.58 | 75.63 | 80.27 |
| 5 | 2 | 2 | 3 | 45.07 | 87.90 | 100 |
| 6 | 2 | 3 | 1 | 42.15 | 80.81 | 92.73 |
| 7 | 3 | 1 | 3 | 27.16 | 75.08 | 72.84 |
| 8 | 3 | 2 | 1 | 42.01 | 81.69 | 93.07 |
| 9 | 3 | 3 | 2 | 30.03 | 76.35 | 76.74 |
| k 1 | 80.33 | 91.00 | 80.88 | |||
| k 2 | 77.48 | 92.85 | 81.89 | |||
| k 3 | 88.38 | 80.83 | 83.01 | |||
| R | 10.66 | 15.37 | 7.55 | |||
Note: Comprehensive score = (Powder yield/Maximum powder yield) × 0.5 + (Embedding rate/Maximum embedding rate) × 0.5; k 1, k 2, k 3 were the average value of the comprehensive score under each level of each factor; R was (Range = maxiMum average yield − Minimum average yield) for the comprehensive score.
The analysis of variance
| Source | Sum of squares | df | Mean square | F value | P value | Significance |
|---|---|---|---|---|---|---|
| A | 216.353 | 2 | 108.176 | 27.614 | 0.035 | * |
| B | 375.719 | 2 | 187.859 | 47.955 | 0.020 | * |
| C | 90.592 | 2 | 45.296 | 11.563 | 0.079 | |
| Error | 7.835 | 2 | 3.917 | — | — | — |
| Total | 690.498 | — | — | — |
Note: * shows significant differences (P < 0.05).
According to the range analysis in Table 1 and the variance analysis in Table 2, the influencing extent of three factors on the comprehensive score was in a decreasing order of the core wall ratio (B) > the wall material ratio (A), wherein the core wall ratio and the wall material ratio had a significant influence on the powder yield and the embedding rate of β-glucan microcapsules (P < 0.05). However, the influence of the monoglyceride dosage was not significant. Thus, the optimum technological condition of β-glucan microcapsules was finally confirmed as A2B2C1, namely, the composite wall material ratio between maltodextrin and lactoalbumin(A2) of 1:2, the core wall ratio (B2) of 1:2, and the monoglyceride dosage (C1) of 0.3%. Under this process condition, three replicates were conducted to obtain the powder yield of β-glucan microcapsules of 47.32 ± 0.58% as well as the embedding rate of 86.76 ± 1.19%. The technology stability was good.
3.3 Structural characterization of β-glucan microcapsules
3.3.1 Morphological characterization analysis of β-glucan microcapsules
The morphology of β-glucan microcapsules(A2B2C1) was directly shown by SEM (Figure 6). The surface of microcapsules was a structure of a smooth sphere. There was no depression, crack, or fold. The particle size was relatively uniform. The surface of microcapsules was continuous without adhesion, illustrating that the structure of the wall material could effectively protect the core material by the isolation of the core material from the outside. The good integrity of the appearance illustrated the already formed β-glucan microcapsules. β-Glucan was embedded successfully by the wall material. The microencapsulation guaranteed the good dispersion of β-glucan, which was not easy to adhere to each other.
SEM micrographs of β-glucan microcapsules by spray drying.
3.3.2 Particle size analysis of β-glucan microcapsules
Particle size and its distribution were important parameters to characterize the properties of microcapsules, which usually determined the physical, mechanical, and chemical properties of microcapsules. Therefore, particle size and its distribution were very important to the quality evaluation of β-glucan microcapsules. As shown in Figure 7, for the raw material of β-glucan, Dv (50), Dv (10), and Dv (90) were 102, 48.0, and 201 µm, respectively. The corresponding specific surface area was 74.26 m2/kg, and the uniformity was 0.503. For the microcapsulated β-glucan, Dv (50), Dv (10), and Dv (90) were 8.43, 4.21, and 17.5 µm, respectively. The corresponding specific surface area was 770.6 m2/kg, illustrating that microcapsule particles were smaller and more uniform with a more concentrated distribution and a larger surface area which was about 10 folds larger than the raw material. The emulsification before the product spray drying utilized the high-pressure homogenization treatment, which had a great influence on the size and the distribution of particles.
Particle size distribution curve of (a) β-glucan and (b) β-glucan microcapsules.
3.3.3 Infrared spectrum analysis of β-glucan microcapsules
According to the literature, the characteristic absorption peaks of β-glucan were 884, 894, and 930 cm−1 [26]. According to infrared spectrum analysis as shown in Figure 8, the wide and strong absorption peak near 3,273 cm−1 should be caused by the characteristic absorption peaks of free hydroxyl and amino functional groups between polysaccharide molecules, namely, the stretching vibration of O–H and N–H [27], which existed in empty microcapsules, β-glucan and β-glucan microcapsules. The small peak near the wavelength number of 1,633 and 2,922 cm−1 was mainly caused by the stretching vibration of C–H of –CH2– in the saccharide chain and combined water, respectively [28]. The characteristic absorption peaks of 884, 894, and 930 cm−1 occurred in not only the raw material of β-glucan but also β-glucan microcapsules. However, there were no such two peaks in the empty microcapsule, illustrating the successful embedding of β-glucan. There were two peaks of 1,532 and 1,632 cm−1 in the empty microcapsules formed by two wall materials. Such two peaks also occurred in β-glucan microcapsules, illustrating that there were not only the characteristic absorption peak of the β-glucan core material but also that of the wall material in the infrared spectra of microcapsules, further illustrating the embedding of β-glucan by the wall material.
The analysis of FT-IR spectra of different samples.
3.3.4 Thermal stability analysis of β-glucan microcapsules
According to the thermal stability analysis in Figure 9, the mass loss of β-glucan microcapsules was only 2.2% in the range of 30.0–93.3°C, possibly due to the water evaporation process on the surface of microcapsules. At 93.3–208.2°C, the microcapsules were stable. At 208.2–242.0°C, the mass loss of microcapsules increased with a great amplitude and reached 16.5%, possibly due to the thermal decomposition process of the easily decomposed organic substance of microcapsules. The thermal decomposition rate of microcapsules has slowed down since 242.0°C. But it continued to decompose. The thermal decomposition process of blank microcapsules was similar to that of β-glucan microcapsules. At 30.0–95.0°C, the mass loss of β-glucan raw material was 8.5%, corresponding to the process of water evaporation. Since 95°C, β-glucan raw materials have begun to enter the process of thermal decomposition. At 95.0–207.2°C, the mass loss was small, namely, 7.0%. Since 207.2°C, it has entered the main thermal decomposition process of the material. The above data analysis showed that maltodextrin and lactoalbumin as composite wall materials could form macromolecular polymers with a network structure through cross-linking, which embedded β-glucan [29]. Thus, microcapsules could protect the water in the interior of β-glucan in a lower temperature range. Meanwhile, the thermal decomposition temperature of β-glucan was increased. Thus, the microencapsulation technology could effectively improve the stability of β-glucan.
The analysis of TG of different samples.
4 Conclusions
In this study, lactoalbumin and maltodextrin were used as the composite embedding wall materials. The optimum technological conditions of microencapsulation of S. latifolia β-glucan microcapsules were confirmed using the single-factor test and the orthogonal test, namely, the composite wall material ratio between maltodextrin and lactoalbumin of 1:2, the core wall ratio of 1:2, and the monoglyceride usage of 0.3%. Under these technological conditions, the powder yield and the embedding rate of β-glucan microcapsules were 47.32 ± 0.58 and 86.76 ± 1.19%, respectively. The stability of the technology was good. The variance analysis showed that the influence of three factors on the comprehensive score of microcapsules was in decreasing order of the core wall ratio > the wall material ratio. The Infrared spectrum analysis, electron microscope scanning, particle size analysis, and thermal stability analysis of β-glucan microcapsules showed that the β-glucan microcapsules with good quality could be obtained by the spray drying of the multiple emulsion with the composite wall material of maltodextrin and lactoalbumin as well as the core material of β-glucan. The prepared microcapsules were spherical with characteristics of smooth surface, no crack, small particle sizes, and a uniform distribution. Besides, microcapsules required a higher temperature for decomposition. In addition, the good embedding capacity could effectively protect the bioactivity of the core material.
Acknowledgements
The authors are very grateful for the support of Research and Testing Center of Pharmaceuticals, College of Materials and Chemical Engineering, Minjiang University, and Fujian Provincial University Engineering Research Center of Green Materials and Chemical Engineering for laboratory facilities and encouragement.
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Funding information: The study was supported by the Natural Science Foundation of Fujian Province (2021Y0101, 2018J01434) and the Planned Project of Fujian Municipal Science and Technology Bureau (2020-GX-6).
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Author contributions: M.Z. and C.W. planned the experiments; M.Z.,Y.Z., and M.S. interpreted the results; M.Z. and B.L. made the write up and analyzed the data; and G.L. made illustrations.
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Conflict of interest: The authors declare there is no conflict of interest.
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Ethical approval: The conducted research is not related to either human or animal use.
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Data availability statement: The data used to support the findings of this study are available from the corresponding author upon request.
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