Comprehensive evaluation of physicochemical properties and antioxidant activity of B. subtilis-fermented polished adlay subjected to different drying methods.

Abstract The physicochemical properties and antioxidant activity of B. subtilis‐fermented polished adlay (BPA) subjected to different drying methods (hot‐air drying, HAD; infrared‐radiation drying, IRD; vacuum drying, VD; microwave‐vacuum drying, MVD; and freeze‐vacuum drying, FVD) were evaluated in this study. Results showed FVD was ideal for maintaining the natural appearance and higher contents of proximate compositions, free fatty acids, tetramethylpyrazine (6.91 mg/g DW), coixol (0.62 mg/g DW), coixenolide (4.21% DW), coixan (35.10% DW), and triterpenoids (17.41 mg/g DW). The higher contents of total phenolics and flavonoids, stronger antioxidant activity, and higher color differences were observed in HAD and IRD samples. MVD displayed the shorter drying time, higher γ‐aminobutyric acid content, and higher retention ratios of tetramethylpyrazine (75.54%), coixol (87.10%), coixenolide (98.57%), and coixan (99.11%). Pearson's correlation coefficient exhibited that the positive correlation between the contents of phenolics and flavonoids and the antioxidant activities of all dried BPA samples was observed (R 2 > 0.881, p < .05). Principal component analysis showed that the top three categories of comprehensive quality were FVD‐, MVD‐, and VD‐treated BPA samples. In conclusion, MVD should be a potential preservation method to obtain high‐quality dried BPA for short drying time and high comprehensive quality.


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WEN Et al. and lactams (Ting et al., 2019). Several studies claimed that adlay had various beneficial functions, including antioxidant, anti-inflammation, anticancer, enhancing immunological activity, regulating endocrine functions, antidiabetes, antiobesity, and modulating gut microbiota (Zhu, 2017). Owing to its multiple nutritional and health benefits, the consumption demand for adlay has continued to increase.
Due to high health-beneficial components and water content of B. subtilis-fermented adlay, it is far more susceptible to bacterial and fungal contamination. Therefore, the major challenge is that B. subtilis-fermented adlay must be stored using dehydration methods.
Studies have shown that drying process was used to effectively restrain the growth of other microorganisms and minimize plenty of moisture-mediated degradation reactions. Hot-air (HAD), infrared-radiation (IRD), vacuum (VD), microwave-vacuum (MVD) and freeze-vacuum (FVD) drying have been commonly used in food processing and preservation. Pham, Nguyen, Vuong, Bowyer, Scalett (2017),  observed that VD maintained the higher contents of proanthocyanidins, flavonoids, and phenolic compounds, and stronger antioxidant activities in Catharanthus roseus than HAD and IRD. Sui, Mu, Sun, and Yang (2019) demonstrated that sweet potato leaves treated by FVD showed higher contents of vitamin, mineral, and phenolics, and stronger antioxidant activity than that by MVD and HAD. Thus, the appropriate method for drying B. subtilis-fermented adlay was not directly obtained according to previous studies. And until now, no or few studies were available examined the physicochemical properties and bioactivities of B. subtilis-fermented adlay treated by different drying methods.
In the present study, the nutritional and bioactive (tetramethylpyrazine, acetoin, coixol, coixenolide, coixan, γ-aminobutyric acid, triterpenoids, phenolics, and flavonoids) components and antioxidant activity of B. subtilis-fermented polished adlay (BPA) dried by five methods were detected for the first time. Furthermore, Pearson's correlation coefficient was used to evaluate the correlation between the bioactive components and antioxidant activity. Additionally, the comprehensive qualities of BPA processed by five drying methods were evaluated by principal component analysis (PCA). The results of this study will present scientific basis for choosing appropriate methods for drying BPA, which will maintain the best possible contents of nutritional and bioactive components, and promote the industrial application of BPA.

| Materials and chemicals
Polished adlay used in this study was provided by Guizhou Renxin Agricultural Development Co., Ltd., and was sealed in plastic bags and stored at 4°C until use. B. subtilis BJ3-2 was procured from Dr.

| Drying process
The BPA samples were subjected to HAD, IRD, VD, MVD, and FVD until the final moisture content was approximately 8.50 ± 1.00 g/100 g dry weight. And FVD was set as reference object due to that it maintained dried samples with high nutritional and functional components . All dried samples were placed onto the plates with 0.5 cm thickness and then followed with drying methods as displayed in Table 1. After drying, BPA samples were blended into powder and screened through a 60 mesh sieve. All dried BPA powdered samples were stored at 4°C until used.

| Color parameters
The surface color (L*, a*, and b*) of BPA dried by five methods was detected by a colorimeter. The color parameters were expressed as L* (lightness), a* (red/green), and b* (yellow/blue).Total color difference (ΔE) was calculated according to the formula as described by Aghilinategh et al. (2015).

| Proximate compositions and free fatty acids
Moisture, protein, starch, and fat contents of dried BPA were detected according to the AOAC Official Method (2012). The free (1)

| Bioactive components
Tetramethylpyrazine and acetoin were detected according to the method of Wen et al. (2019), using Agilent 1260 high-performance liquid chromatography. γ-Aminobutyric acid was analyzed using the methods of Park et al. (2017). Coixol, coixenolide, total phenolics, flavonoids, and triterpenoids were measured as suggested by Xu, Wang, et al. (2017). The contents of total phenolics and flavonoids were performed, and the results were expressed as gallic acid equivalent (mg GAE/g DW) and rutin equivalent (mg RE/g DW), respectively.

| Antioxidant activity
The ABTS + and FRAP (ferric reducing antioxidant power) assays were determined using the method suggested by Szychowski et al. (2018). The DPPH radical scavenging assay was determined as previously described by Vu, Scarlett, and Vuong (2017). Briefly, dried BPA powder (1.00 g) was mixed with 80% ethanol (15 ml). The mixture was ultrasonic extracted for 30 min at room temperature, followed by centrifugation at 2500 g for 15 min. The supernatant was collected, and these procedures were performed in triplicate. After extraction, all the supernatants were combined and concentrated into a paste by a rotary evaporation at 45°C under reduced pressure.
The pastes were reconstituted with 10 ml of methanol, and 1.0 ml of the extract was transferred to a 50-ml volumetric flask and diluted with methanol to volume. Subsequently, the extracts were stored at −18°C until used.

| Statistical analysis
All the determinations were performed in triplicate, and the results were shown as mean ± standard deviation (SD

| Final moisture content and drying time
The final moisture content and drying times for different methods were displayed in Table 1

| Chromaticity
The color parameters of BPA treated by five drying methods were revealed in Table 2, and photographs were shown in Figure

| Proximate compositions
As displayed in thus the contents of protein, starch, and fat were higher in FVD samples (Öztürk & Gündüz, 2018). In contrast, VD, HAD, and VD drying time was so long, the cell shrinkage or expansion of the cellular structure resulted in its rupture, so there resulted in plenty of physical or chemical reactions (Li, Jin-Jia, et al., 2019).

| Fatty acid composition
The fatty acid compositions of dried BPA were shown in Table 3, and eighteen fatty acids were detected in dried BPA samples.
The content of total fatty acids ranged from 60.76 g/100 g DW to 48.08 g/100 g DW, and the order of the five methods was ing that BPA could provide a potential healthy diet for human.

| Tetramethylpyrazine and acetoin
Tetramethylpyrazine naturally existed in fermented foods, such as natto, Chinese liquor, and vinegar, and had health functions especially for cardiovascular and cerebrovascular health (Li, Huang, Wang, & Qiu, 2019). Acetoin has recently been in the focus of high-value industries because of its usability in detergents, cosmetics, dairy products, and pharmaceuticals ( (Zhu, Xu, & Fan, 2010). The lower tetramethylpyrazine content in HAD, IRD, and MVD was due to that heating and moisture evaporation both promoted the volatilization of tetramethylpyrazine during drying (Dong, Hu, Chu, Zhao, & Tan, 2017). In contrast, the higher acetoin content in HAD and IRD was detected, due to that higher heating temperature and longer time disrupted the cell wall and release acetoin from B. subtilis BJ3-2.

| γ-Aminobutyric acid
γ-Aminobutyric acid, a four-carbon nonprotein amino acid, is a major inhibitory neurotransmitter in the sympathetic nervous system . As displayed in Figure 1c, the highest γ-aminobutyric acid content was observed in MVD-prepared samples (22.71 mg/g DW), followed by FVD (21.70 mg/g DW), but the TA B L E 3 Contents of fatty acids in BDA dried by five drying methods (g/100 g DW)  (2015) showed that the γ-aminobutyric acid content changed insignificantly in high temperature (90-150°C).

| Phenolics, flavonoids, and triterpenoids
Phenolics, flavonoids, and triterpenoids are an essential group of plant metabolites during development and in response to various conditions, and exhibit pharmacological effects on treating many diseases such as inflammation, cardiovascular disease, hypertension, arteriosclerosis, and cancer .
As noted in Figure 1d, different dehydrated treatments displayed variable effects on the contents of total phenolics, flavonoids, and triterpenoids in BPA samples. The contents of total phenolics and flavonoids of dried BPA samples were 9.45-10.69 mg GAE/g DW and 5.68-6.82 mg RE/g DW, respectively. The highest phenolic contents were presented in HAD samples, approximately 1.13 times higher than that of FVD. And the highest flavonoid content was found in IRD samples, approximately 1.60 times higher than that of FVD. It was supported by Cheng et al (2019), who also found that FVD resulted in less phenolic and flavonoid contents of green coffee beans compared with MVD and HAD, which was consistent with our results. Heating created more destruction of the tissue, which in turn led to the thermal degradation or transformation into simpler phenolic compounds, and polymerization or oxidation of phenolic and flavonoid compounds (Si et al., 2016). During the freezing step prior to FVD, the cellular structure of samples was damaged by ice crystals formation and FVD processing was treated under lower exposure to oxygen. After the end of FVD, enzymatic oxidation of phenolics was more easily to happen when exposed to air (Duodu, 2011). Additionally, lower flavonoid and phenolic contents in MVD samples were due to that heat generation from microwave radiation
Meanwhile, high temperature could promote the conversion of flavone glycoside into flavone aglycone compounds and low molecular weight phenolic compounds with stronger antioxidant activity (Ming et al., 2019). Additionally, phenolic compounds were bound to the skeleton of melanoidins of Maillard reaction products in the form of noncovalent bond and synthesized melanoidin compounds with strong antioxidant activity (Wang, Qian, & Yao, 2011). Therefore, the antioxidant activities of BPA treated by HAD and IRD were significantly higher than other drying methods.
Pearson's correlation was used to evaluate the relationship between bioactive components and antioxidant activities of dried BPA as shown in Table 4

| Principal component analysis
Principal component analysis (PCA) was used to investigate the comprehensive quality of dried BPA samples processed by the different drying methods. Figure 3a,  quality, but high cost of FVD might limit its application. Therefore, MVD could be an alternative preservation method when considering the economy and drying time.

| CON CLUS ION
The nutritional and bioactive components, and antioxidant activity of BPA processed by five drying methods were evaluated. The higher contents of tetramethylpyrazine, coixol, coixenolide, coixan, and triterpenoids were detected in FVD samples. The higher contents of total phenolics and flavonoids, and antioxidant activity were exhibited in HAD and IRD samples. The shorter time, higher γ-aminobutyric acid content, and higher retention rate of tetramethylpyrazine, coixol, coixenolide, and coixan were obtained in MVD samples. Through principal component analysis, BPA subjected to FVD, MVD, and VD had higher comprehensive quality. MVD was a promising technology for preserving BPA for short drying time and high bioactive components. For further work, it is certainly worth examined that the change rules and kinetic models of bioactive components loss in BPA.

CO N FLI C T O F I NTE R E S T
The authors declare no financial or commercial conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
This study does not require informed consent because it does not use humans as research subjects.