Quality of Soybean Seeds after Microwave Drying

Featured Application: The use of microwaves in drying has been the subject of studies in the post-harvest sector. Some authors have identiﬁed damage to the seeds because of microwaves, while others have found promising results for the technique. This study veriﬁed that the use of microwaves can be safe if the appropriate temperature and power conditions are applied. In this work, readers will also be able to verify that the use of electromagnetic energy can also be more economical and efﬁcient, as it greatly reduces the drying time, in addition to being possible to increase germination during the process, which is highly desirable. In addition to the temperature x power binomial , water content is another variable that can affect the process. This should be a stimulus for further research and studies in the area that address a broader spectrum of varieties and water content to that the technique can be safer to the point that its use reaches the public. Abstract: The use of electromagnetic energy for drying seeds has been shown to be a promising alternative. However, studies show that the technique still lacks careful evaluation due to the sensitivity of soybean seeds to damage caused using microwaves. Studies have shown that the use of microwaves during drying can be safe in seeds of soybeans, corn, and beans, among others. These studies recognize that drying using microwaves has a great potential for savings in the drying process, as it considerably reduces the drying time. This work aimed to evaluate the immediate damage caused in soybean seeds subjected to drying at temperatures of 40, 50, and 60 ◦ C, with power of 0, 0.5, and 1 W/g at a frequency of 2.45 GHz. The results of the vigor and germination tests showed that the action of microwaves on seeds was not only safe and more efﬁcient but also brought about increases in seed germination and vigor. However, the process must be judicious and obey precise indices of power and temperature to not cause damage to the seeds.


Introduction
Drying is a technology whose main function is to reduce the water content and can be applied to different materials. The procedure limits the chemical reactions of microorganisms, conserving the physical and biological structure of the stored material and its applicability of extreme contribution in sustainable processes for different segments [1][2][3][4]. The electromagnetic radiation emitted by the microwave is concentrated in the heating of The experiment was conducted with constant temperature and intermittent power. The microwave used for the study has a maximum power output of 600 W. The control circuits were modified to allow the power to be controlled. The temperature of the soybean seeds was measured by inserting an optical fiber sensor into a hole made in the soybean seed.

Constant Temperature
The temperature of the soybean samples was kept constant throughout the experiment. When the temperature crossed the desired level, the microwave energy supply was cut off automatically by the control circuit. The temperature levels used for the experiments were 40, 50, and 60 °C.

Intermittent Power
This method of microwave drying was conducted with an intermittent supply of power on a cycle of 30 s. A hundred grams of soybean seeds with an average moisture content of 21% (w.b.) were taken for microwave drying. The microwave energy generator was adjusted in such a way that the power shut off for 30 s and switched on for 30 s. The power levels used for these experiments were 0, 0.5, and 1.0 W/g. Drying was carried out until reaching final weight stability, represented by the same measurement on the scale for three consecutive readings.
Conventional drying was carried out in the same microwave equipment, without activating the power, that is, with 0 W/g, at a temperature of 40 °C.

Vigor and Germination Tests
To carry out the "first count" vigor test and the germination test, the soybean seeds subjected to drying were placed on moistened germination paper. The paper rolls were closed in plastic bags and kept in a germination chamber for 5 and 8 days at 24 °C. After the first 5 days, the germinated seeds that produced strong seedlings were counted, and the germinated percentage represented the "First Count" vigor test. The seeds that did not germinate were kept on the germination paper and returned to the germination chamber until the 8 days were completed. After this period, the paper rolls were again removed to count germination (normal seedlings) and protrusion (seeds that emitted at least 2 mm of radicle). Abnormal seedlings were those that were missing some part, such

Constant Temperature
The temperature of the soybean samples was kept constant throughout the experiment. When the temperature crossed the desired level, the microwave energy supply was cut off automatically by the control circuit. The temperature levels used for the experiments were 40, 50, and 60 • C.

Intermittent Power
This method of microwave drying was conducted with an intermittent supply of power on a cycle of 30 s. A hundred grams of soybean seeds with an average moisture content of 21% (w.b.) were taken for microwave drying. The microwave energy generator was adjusted in such a way that the power shut off for 30 s and switched on for 30 s. The power levels used for these experiments were 0, 0.5, and 1.0 W/g. Drying was carried out until reaching final weight stability, represented by the same measurement on the scale for three consecutive readings.
Conventional drying was carried out in the same microwave equipment, without activating the power, that is, with 0 W/g, at a temperature of 40 • C.

Vigor and Germination Tests
To carry out the "first count" vigor test and the germination test, the soybean seeds subjected to drying were placed on moistened germination paper. The paper rolls were closed in plastic bags and kept in a germination chamber for 5 and 8 days at 24 • C. After the first 5 days, the germinated seeds that produced strong seedlings were counted, and the germinated percentage represented the "First Count" vigor test. The seeds that did not germinate were kept on the germination paper and returned to the germination chamber until the 8 days were completed. After this period, the paper rolls were again removed to count germination (normal seedlings) and protrusion (seeds that emitted at least 2 mm of radicle). Abnormal seedlings were those that were missing some part, such as hypocotyl, root, and leaves, or those that showed some abnormality in their development, such as hypocotyl and twisted roots. The germination rate was expressed as a percentage based on the initial number of seeds used. The seed germination and vigor tests were carried out in four repetitions with 50 seeds each, according to the International Seed Testing Association (ISTA) International Rules for Seed Analysis [23][24][25][26][27][28][29].
The water absorption curve [23,30], was performed by measuring the individual weight of 30 seeds, in three repetitions. The seeds were placed in Petri dishes, on two sheets of germination paper. Each seed had its weight previously measured on a precision scale of Appl. Sci. 2023, 13, 8116 4 of 14 0.0001 g. In each plate, 20 mL of distilled water were added. During the test, the seeds were kept in germination chambers at 25 • C for a period of 8 days. However, the weight of each seed was measured again at intervals ranging from 3 to 24 h during the incubation period. At each weighing, each seed was placed on absorbent paper, making sure that there was no excess free water around the seed coat. The water absorption curve was constructed from the percentage values of the weight of water absorbed by each seed. The adjustment of the water intake curve models was performed using the Curve Expert ® 2.6 software.

Experimental Design
Variation effects in germination time and temperature were analyzed using the response surface methodology (RSM). The effect of two independent variables, namely, temperature (A) and power (B), on the quality of the seed was investigated using a twofactor CCD-RSM experimental, and the results are shown in Table 1.

Statistical Analysis
The program RStudio R 3.3.0+ was used to determine the effects of the independent variables, calculate regression coefficients (R2), carry out an analysis of variance (ANOVA), and build the response surface, at a 5% significance level.
The following second-order polynomial model (Equation (1)) was fitted to the data: where Y is the response variable, X 1 and X 2 , are the coded process variables and β 0 , β 1 , β 2 , β 11 , β 22 , β 12 are the regression coefficients. A stepwise methodology was followed to determine the significant terms in Equation (1). The Minitab 21.4 program was used to analyze the average obtained by the vigor and germination test, using a box plot, with a confidence level of 95% for the applied treatments.
Principal component analysis (PCA) was used for analyzing the vigor and germination test and was performed using the software R version 4.3.0 [31], packages (devtools and ggbiplot) functions (prcomp, screeplot ggbiplot2) ( Table 2).

Results
After drying the seeds, the samples of each treatment were submitted to the vigor and germination test. Test results were subjected to response surface analysis. The contour graphs and the generated surfaces showed an unexpected behavior in the region between 40 and 50 • C and between 0 and 0.5 W/g in this region, and the total germination obtained an increment, which may indicate a biostimulant effect coming from the effect of microwave exposure, as shown in Figure 2.

Results
After drying the seeds, the samples of each treatment were submitted to the vigor and germination test. Test results were subjected to response surface analysis. The contour graphs and the generated surfaces showed an unexpected behavior in the region between 40 and 50 °C and between 0 and 0.5 W/g in this region, and the total germination obtained an increment, which may indicate a biostimulant effect coming from the effect of microwave exposure, as shown in Figure 2. The behavior of seed drying, with the increase in germination indicated on the response surface, was repeated in the first count (vigor test) and seed germination. The contour plot shows us more clearly that this region comprises, more specifically, a small area where power was applied at lower levels, that is, between 0 and 0.5 W/g. Similar behavior was found for corn seeds [32]. For temperatures above 50 °C, the damage to seeds was drastic for any power level. The behavior of seed drying, with the increase in germination indicated on the response surface, was repeated in the first count (vigor test) and seed germination. The contour plot shows us more clearly that this region comprises, more specifically, a small area where power was applied at lower levels, that is, between 0 and 0.5 W/g. Similar behavior was found for corn seeds [32]. For temperatures above 50 • C, the damage to seeds was drastic for any power level.
The seeds start to be drying with 21% (w.b.) water content. This is considered a high moisture content level for drying soybean seed. Another study using microwaves as a biostimulant effect identified that drying could safely take place at a level of 0.2 W/g, with seeds having a water content of around 10% [33,34].
The analysis of variance and the coefficients of the models generated by the response surface are described in Table 3. The protrusion model indicates as a stationary point in original units the temperature of 42.5 • C and a power of 0.31 W/g. These points represent the maximum gain in germination rates for drying using microwave energy. The averages obtained in the germination and vigor test to determine seed quality are represented in Figure 3 through box plot analysis. Through the figure, one can identify the best treatments obtained through the vigor and germination tests right after drying the soybean seeds.  Figure 3 presents the result of the germination and vigor test as a function of temperature and power. The treatments that used the temperature of 40 °C showed the best averages compared to the temperature of 50 and 60 °C. Treatments submitted to a power of 1.0 W/g had a drastic reduction in germination rates and an increase in abnormal seedlings.
The best treatment obtained in the vigor and germination test was the one that used a temperature of 40 °C, associated with a potency of 0.5 W/g. This treatment showed the highest rates of germination with protrusion of up to 91.88%, and the lowest rates of abnormal seedlings, surpassing the control treatment.
The quality of seeds can also be discussed by PCA analysis, as shown in Figure 4 below. germination test was the one that used of 0.5 W/g. This treatment showed the to 91.88%, and the lowest rates of abt. y PCA analysis, as shown in Figure 4 , respectively. Figure 3 presents the result of the germination and vigor test as a function of temperature and power. The treatments that used the temperature of 40 • C showed the best averages compared to the temperature of 50 and 60 • C. Treatments submitted to a power of 1.0 W/g had a drastic reduction in germination rates and an increase in abnormal seedlings.
The best treatment obtained in the vigor and germination test was the one that used a temperature of 40 • C, associated with a potency of 0.5 W/g. This treatment showed the highest rates of germination with protrusion of up to 91.88%, and the lowest rates of abnormal seedlings, surpassing the control treatment.
The quality of seeds can also be discussed by PCA analysis, as shown in Figure 4 below. The first two components explained the totality of approximately 99% of all variability in the data set (62.9% explained by Principal Component 1 (CP1), 37.1% by CP2) according to [35,36], this proportion of the variance explained by (CP) must correspond to at least 70%.
We can observe in Figure 5, CP1 showed great negative associations with the variables germination and first count. In CP2, it is observed that the variables and abnormal seedlings and protrusion presented negative associations. The first two components explained the totality of approximately 99% of all variability in the data set (62.9% explained by Principal Component 1 (CP1), 37.1% by CP2) according to [35,36], this proportion of the variance explained by (CP) must correspond to at least 70%.
We can observe in Figure 5, CP1 showed great negative associations with the variables germination and first count. In CP2, it is observed that the variables and abnormal seedlings and protrusion presented negative associations.
We can observe that the samples were united in agreement with the powers used as a factor within the treatments. Treatment 1, with a temperature equal to 40 • C and power 0, proved to be efficient for germination and first count. It is noteworthy that treatments 4 and 7 are closer to germination and first count, indicating better responses to the corresponding treatments; these are 40 • C and 50 • C with a power of 0.5, respectively, which suggest that the use of this power is stimulating for the germination and vigor of seedlings associated with the first count, more than just the temperature without using power. Treatments 2,5,6,8,9, and 10 indicate that there were stimuli in cellular elongation but that these seedlings were not able to become normal, a fact that can be suggested by the temperature.
Treatments 3, 11, and 12 showed a distancing from the other groups evaluated, a fact that can be observed by the use of high power, 1. As well as the treatment that most distanced itself from the others was the 13 at 60 • C and power of 1, being opposed to the protrusion, indicating very low values for all the variables observed, being unfeasible for application in seeds.
The behavior of water loss during seed drying in the two main treatments observed by the germination and vigor test is shown in Figure 6. The seed drying rate was higher at the beginning of the process, and after a period, it stabilized until the end. We can observe that the samples were united in agreement with the powers used as a factor within the treatments. Treatment 1, with a temperature equal to 40 °C and power 0, proved to be efficient for germination and first count. It is noteworthy that treatments 4 and 7 are closer to germination and first count, indicating better responses to the corresponding treatments; these are 40 °C and 50 °C with a power of 0.5, respectively, which suggest that the use of this power is stimulating for the germination and vigor of seedlings associated with the first count, more than just the temperature without using power. Treatments 2,5,6,8,9, and 10 indicate that there were stimuli in cellular elongation but that these seedlings were not able to become normal, a fact that can be suggested by the temperature.
Treatments 3, 11, and 12 showed a distancing from the other groups evaluated, a fact that can be observed by the use of high power, 1. As well as the treatment that most distanced itself from the others was the 13 at 60 °C and power of 1, being opposed to the protrusion, indicating very low values for all the variables observed, being unfeasible for application in seeds.
The behavior of water loss during seed drying in the two main treatments observed by the germination and vigor test is shown in Figure 6. The seed drying rate was higher at the beginning of the process, and after a period, it stabilized until the end. The values of the water content of the seeds at the end of the drying process in each treatment were 6 and 4% for conventional drying and with the use of microwaves, respectively. At the end of the drying process, the water content for conventional drying at 40 °C and 0 W/g was 15.15% (w.b.), and for drying using microwaves at a temperature of The values of the water content of the seeds at the end of the drying process in each treatment were 6 and 4% for conventional drying and with the use of microwaves, respectively. At the end of the drying process, the water content for conventional drying at 40 • C and 0 W/g was 15.15% (w.b.), and for drying using microwaves at a temperature of 40 • C and power of 0.5 W/g was 17.29%. For storage purposes, both processes were inefficient since drying should occur up to a content of 12% (w.b.) of water. However, the conditions of the drying air did not allow the removal of a higher value, which could be reached at a temperature of 42 • C, with 0.3 W/g, as identified by the RSM model, as the ideal condition for maximizing gains in biostimulation of applied energy. Table 4 shows the parameters adjusted for the models applied for fit in the drying behavior. The behavior of the temperature of the soybean seeds and the insufflated air during microwave drying can be seen in Figure 7. In the image, we can identify that there is a slight increase in the internal temperature of the seeds during conventional drying, which did not happen in the same intensity for dry seeds using microwaves.  Figure 7 may have the key to begin to understand the reasons why seed damage was considerably higher in conventional drying. In the microwave drying process, the temperature did not exceed the recommended index for most soybean seeds of 42 °C. In addition, the seeds had an exposure time to temperature a little more than eight times longer in the conventional process, which was approximately 8 h 37 min against 46 min for the microwave process.
In Figure 8, the water absorption test was performed, showing a similar behavior between the two main treatments studied. However, in the first 10 h, the seeds that were treated by microwave drying showed greater water absorption.  Figure 7 may have the key to begin to understand the reasons why seed damage was considerably higher in conventional drying. In the microwave drying process, the temperature did not exceed the recommended index for most soybean seeds of 42 • C. In addition, the seeds had an exposure time to temperature a little more than eight times longer in the conventional process, which was approximately 8 h 37 min against 46 min for the microwave process.
In Figure 8, the water absorption test was performed, showing a similar behavior between the two main treatments studied. However, in the first 10 h, the seeds that were treated by microwave drying showed greater water absorption. Figure 7 may have the key to begin to understand the reasons why seed damage was considerably higher in conventional drying. In the microwave drying process, the temperature did not exceed the recommended index for most soybean seeds of 42 °C. In addition, the seeds had an exposure time to temperature a little more than eight times longer in the conventional process, which was approximately 8 h 37 min against 46 min for the microwave process.
In Figure 8, the water absorption test was performed, showing a similar behavior between the two main treatments studied. However, in the first 10 h, the seeds that were treated by microwave drying showed greater water absorption.  The ability to absorb more water, especially in the first hours of germination, is an indication of good quality in seeds [33,37,38]. The result of this test corroborates the performance of the microwave treatment vigor test using a power of 0.5 W/g, which surpassed conventional drying in its germination percentage. In a combined study of microwaves and ultrasound (US-MW) in slices of green olives, the authors noticed that the power of 180 W provided a higher rate of rehydration for dry olives compared to higher powers, determining that the US pre-treatment -MW is promising [39]. Table 5 are presented the adjusted models for the water up take curve. Table 5. Parameters of the exponential plus linear model given by y = a + br x + cx and adjusted for the water absorption curve in soybean seeds for conventional drying and microwave drying. In okra samples, the rehydration rate had a better performance at 540 W of microwave power; the pre-treatment maintained the characteristics of the product compared to the fresh product [40]. Again, we have a result that indicates the biostimulant effect of microwave drying, which has an advantage over conventional drying.

Discussion
Soybean seeds, when subjected to microwave drying at temperatures of 40 to 50 • C and microwave power above 0 to 0.5 W/g, may have their drying time reduced by up to eight times. The process is safe for water contents below 21%, as shown by the results of this experiment.
Several studies report gains in reducing drying time or other advantages using microwaves and other electromagnetic energies [2,3,41,42]. However, studies that report gains in seed quality are still incipient. Recently, it was proved in a study on the use of magneto priming in soybean seeds they can promote biostimulation and carry out the removal of water from the seeds [33].
In this study, soybean seeds were submitted to different drying conditions, which led to a region of exploration of potential gains in bio stimulus by microwaves. The gain in seed vigor observed in the physiological tests indicates a large area of study to be explored as new post-harvest technologies for seeds. Similar results have already been shown in previous studies [43][44][45][46][47], but little is known about the biological or biophysical reasons related to this phenomenon.
In other types of food products, the MW power can minimize the nutritional degradation influencing the oxidation effect of different products; in samples of sugarcane juice, it was verified that the lower power of the lesser antioxidant activity verified through the elimination of free radicals (DPPH), being the device an efficient technique for concentrate juice [48]. The same antioxidant effect was observed in peas treated at a power of 100 W; there was a decrease in DPPH and an increase in flavonoid levels [49]. In jelly processing, the microwave had an advantage, such as higher DPPH, phenolic content, and ascorbic acid content over the conventional use of the open pan [50]. Despite this, these products are no longer like soybean seeds-the antioxidant effect shown by these studies could explain a possible similar effect on seeds during drying.
This study corroborates the data presented in [51], which using a power of 0.13 W/G, identified that it is possible to dry soybean seeds without damage to germination with greater speed and efficiency of the process. Microwave drying is still a field of study that needs many exploratory studies to certify, over a wide range of drying conditions, the safety and benefits of this process. This study demonstrated that we have a reach region for drying, considering the binomial temperature and power that can not only be safe but also promising for the performance of seeds in the field.

Conclusions
The results showed that microwave drying could not only safely dry soybean seeds but also increase their quality level. There is a germinal biostimulation zone for soybean seeds located between the temperature of 40 to 45 • C and the potency of 0 to 0.5 W/g.
The power of 1 W/g is not recommended for drying seeds, according to this study, at any of the temperatures studied. The use of a temperature of 40 • C is indicated for drying; when associated with a power of 0.5 W/g, it becomes a biostimulant to increase seed quality and increase water absorption during the first moments of germination.
The drying carried out on soybean seeds with 21% water content at 40 • C and a power of 0.5 W/g is safe and drastically reduces drying time.