Tracking the dynamic changes of a flavor, phenolic profile, and antioxidant properties of Lactiplantibacillus plantarum‐ and Saccharomyces cerevisiae‐fermented mulberry wine

Abstract The process of fermentation renders the superior quality of mulberry wine based on the microorganisms utilized. The present study aimed at investigating the changes and correlation between phenols and product quality of mulberry wine fermented with Lactiplantibacillus plantarum and Saccharomyces cerevisiae combinatorially. Total anthocyanins concentration (TAC), polyphenols concentration (TPC), flavonoids concentration (TFC), and antioxidant capacity decreased significantly with high correlation in the fermentation process. TAC gradually reduced with a loss rate of 47.98% from 0 to third day of fermentation. Fermented mulberry wine obtained indicated a dynamic balance due to the presence of p‐hydroxybenzoic acids as compared to the primary phenolic constituent. Chlorogenic acid usually presents in mulberry juice. The relative intensity of sourness was the most prominent and reached the maximum (10.93) on day 2 of fermentation. A total of 21 volatile esters were quantified (6621.59 μg/L), which contributed significantly to the aroma of mulberry wine. The enhanced quality of fermented mulberry wine showed contraindications with decreased constituents and escalated wine quality. Rather than usual single inoculum, fermentation combination of LAB and yeast holistically influenced the color, taste, fragrance, phenolic profiles, and antioxidant properties in mulberry wine, ensuring palatability and fit for commercialization prospects.

Malolactic fermentation is affirmative of the present perspectives, and a tackling aspect is presented here to evaluate to the alcohol fermentation to mitigate the post-harvest losses. The native preservatives in mulberry wine are incumbents of native aroma of wine brewing industrial scenario (Tchabo, Ma, Kwaw, Zhang, Xiao, et al., 2017). Resultant metabolites of monolactic fermentation yield a wide range of alcoholic secondary metabolites including alcohols, esters, acids, aldehydes, and carbonyl compounds which are produced in the process of alcoholic fermentation comprising a majority of prominence . Esterification and alcohol accumulation are an intrinsic preparations mechanism in fermentation, owing to the finalization. Economic feasibility is redressed in rendering the applicability and suitability of fermentative microbes with escalated efficiency. With a notion for aroma and flavor, wine industry necessitates the optimization protocols based on preliminary studies.
Winery researches are established based on the color, antioxidant capacity, and volatility of organic ingredients Liu et al., 2017;Tchabo, Ma, Kwaw, Zhang, Xiao, et al., 2017). Research concerned with variation profiles of the change of phenolic constituents and holistic quality of the whole mulberry wine fermentation process are scarcely available. However, the active ingredients invigorated the changes in several active compounds during the mulberry alcohol fermentation pose process deserve attention dire benefits due to because they are vital for primary implications in human health. Earlier reports concerning the changes in bioactive phenolic compounds investigated during mulberry wine fermentation are adequately examined, and changes in flavor, quality, and texture remain to be the area unexplored (Wang, Sun, et al., 2015). Therefore, the present study aimed to discover the dynamic changes of total anthocyanin concentration (TAC), total polyphenol concentration (TPC), total flavonoids concentration (TFC), phenolic acid, and sensory parameters (flavor, color, and taste) during the process of mulberry wine fermentation.
The correlation between phenolic substances, antioxidant capacity, and color. The preliminary analysis emphasizes the theoretical basis for further optimization and essential downstream processing.

| Plant material
Zhèn shēn 1 hào (Morus nigra) was picked from Jiangxinzhou, Zhenjiang, Jiangsu Province, China. The matured black fruits were harvested and selected for wine preparation. The surface microbial contamination was eliminated by treating sodium hypochlorite (0.02%, v/v) and sterile water. Subsequently, undamaged fruits were stored in the deep freezer at −20°C.

| Fermentation process
The stored frozen mulberry fruits were defrosted at room temperature (28 ± 2°C). After crushing and treatment with 0.1% pectinase, they were pretreated for temperature regulation by incubating in water bath at 40°C for 40 min duration and centrifuged (3200 g, 4°C for 10 min). The clarified juice was added with 60 mg/L SO 2 and the fermentation was initiated by inoculating yeast (5%) and LAB (0.3%) at 22°C. Periodic samples of the fermentation broth were taken for further investigation on days 0, 1, 2, 3, 4, 5, 6, 7, 9, 11, 14, and 17.

| Color measurement
Samples' color parameters were measured with a colorimeter (HunterLab, Reston, USA) and expressed as the L*, a*, and b*. Based on a* and b*, the saturation C* and hue Angle H 0 of the samples were respectively calculated according to Equations (1) and (2) (Kwaw, Ma, Tchabo, Aapaliya, Wu, et al., 2018).

| Total anthocyanin concentration measurement
The TAC was determined according to the method described by Jiang and Nie (2015). Briefly, 1 ml of mulberry wine was added into two separate 10 ml volumetric flask containing two various buffer solutions with 0.2 mol/L KCl: 0.2 mol/L HCl = 25:67 (pH = 1) and 1 mol/L NaAc: 1 mol/L HCl: H 2 O = 100:60:90 (pH = 4.5), correspondingly with constant volume and kept in the dark for 2 hr. The absorbance of the two samples was measured at 510 nm and 700 nm (UV-1600 spectrophotometer, Ruili Analytical Instrument, Beijing China), respectively. TAC was calculated according to Equations (3) and (4).
The results were expressed as mg C3G/L.

| Determination of the total phenolics concentration
A modified protocol of Zhang, Yang, et al. (2018) was adopted for TPC determination employing the Folin-Ciocalteu method. The absorbance of mulberry wine was measured at 760 nm with 1:10 dilution.
The TPC of the sample was calculated from the gallic acid regression equation of the standard curve. The results were expressed in milligrams gallic acid equivalents (GAE) per liter (mg GAE/L).

| Determination of the total flavonoids concentration
The TFC was determined by Sicari's method (Sicari et al., 2016) with minor modifications. Briefly, 1 ml of sample was poured into 50 ml volumetric flask containing 20 ml of 70% (v/v) ethanol. Then, 2 ml of NaNO 2 solution (5%) was added and allowed to stand for 6 min.
Consequently, the mixture was added with 2ml of Al(NO 3 ) 3 solution (10%) with continuous stirring and left to stand for 6 min. After that, 4 ml of 1.0 mol/L NaOH was added and made up with distilled water. After incubating at 25°C for 15 min, the absorbance was measured at 510 nm. The results were expressed in milligrams rutin equivalents (RE) per liter (mg RE/L). The standard curve for rutin was A = 0.0112X + 0.0008 (R 2 = 0.9998). A and X represent absorbance and concentration, respectively. 2.8.2 | 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity (ABTS-SA) measurement ABTS-SA of the mulberry fruit wine was evaluated by the ABTS cation decolorization assay with basic modifications (Tao et al., 2016).

| Determination of the antioxidant activity
The ABTS radical cation (ABTS + ) was generated by reaction of ABTS solution (7 mM) with potassium persulfate (2.45 mM). The reaction mixture was kept in the dark for 12 h at ambient temperature. The ABTS + solution was mixed with ethanol to attain an optical density of 0.70 ± 0.02 at 734 nm. Briefly, 0.2 ml of mulberry wine at different concentrations (10, 30, 60, 100, 150, and 200 mg/L) was mixed with ABTS solution and the reaction mixture after 6 min was measured at lated as per Equation (6) and expressed in mmol TE/L.
A blank : Absorbance of ABTS + and methanol; A sample : Absorbance of sample and ABTS + .

| Determination of phenolic acid content
The phenolic acid content of the mulberry wine was determined by the methods of Kwaw, Ma, Tchabo, Aapaliya, Wu, et al. (2018) using

| Determination of taste quality
Kobayashi's method was used for the taste quality determination of the samples (Kobayashi et al., 2010). Precisely, the samples (diluted 5 times) were tested on the SA 402B Electronic Tongue (Insent, Japan).
The fermented samples collected on the day 0 were taken as internal standards. The difference between the taste intensity values of other fermentation days and the internal standard was the relative intensity value. The intensity values of the internal standard were classified as 0.

| Solid-phase microextraction
Briefly, 5 ml of sample was added with 1.0 g NaCl, and internal standard was loaded into 15 ml of headspace bottle and preheated at 40°C for 10 min. Afterward, the extraction fiber (DVB/ CAR/PDMS 50/30 µm, Supelco, USA) was inserted into the headspace bottle for 30 min, and the magnetic stirring speed was set at 800 rpm/min.

| Qualitative and quantitative methods for volatile aroma components
Qualitative Methods encompass the data retrieval from spectral library retrieval, and components classification, corroboratively . The retention time of C 7 to C 40 mixtures of n-alkanes proceeded for heating procedure as reported earlier and the retention index (RI) of aroma components was calculated according to the retention index formula (7).
RI refers to the retention index, and n and n + 1 are the number of carbon atoms of n-alkanes before and after the outflow of the substance to be measured; T n and T n+1 represent the retention time of corresponding n-alkanes; T is the retention time of the substance to be measured (T n < T < T n+1 ).
Quantitative analysis was carried out by internal standard method using 0.02 μl/ml n-propanol). The concentration of the components was measured according to formula (8).
C corresponds to the concentration of the component and A refers to the peak area of the component; C i accounts for the concentration of the internal standard and the peak area of the internal standard depicts A i .

| Data processing and analysis
All the experiments were performed in triplicate, and the data were statistically represented as Mean ± Standard Deviation. The SPSS 17.0 (IBM, USA) was used for profiling significant differences between parametric variations. Origin Pro 2016 (OriginLab, USA) was used for computing the analysis of variance and drawing graphs with statistical inferences.

| Changes of color in mulberry wine during fermentation
Wine color changes in the mulberry wine samples are pictorially illustrated in Figure 1a, b. The results demonstrated that the transition pattern of the four-color parameters (L*, a*, b*, and C*) was comparatively prominent. However, from 0 to day 1 of fermentation, the transition pattern of L* and b* was inversely proportional to that of a* and C*. The comparison of the four-color parameters and TAC ( Figure 1c) from day 1 to 9 revealed that the transition pattern of the four-color parameters (L*, a*, b*, and C*) was opposite to TAC. Table 1 clearly revealed the significant inverse relationship between the four-color parameters and TAC. From day 9 to 17, L* decreased significantly (p < .05), while a*, b*, and C* showed a slight fluctuation. This contradicted with the findings of Wang, Sun, et al. (2015) which could be attributed to the various varieties of mulberry fruits.
The four-color parameters increased gradually and then decreased during the fermentation period, and each color parameter increased dramatically after the fermentation period (p < .05). H 0 and C* increased significantly during the fermentation period (p < .05). The trend of H 0 change was consistent with that of L* from day 3 to 17, shown by a rapid rise followed by a decrease. According to earlier studies (Kwaw, Ma, Tchabo, Aapaliya, Wu, et al., 2018;Wang, Sun, et al., 2015), the change of L* and H 0 is closely related to TAC which shows negative correlation between TAC and L* and H 0 , respectively.

| Changes of TPC, TFC, and TAC in mulberry wine during fermentation and correlation analysis
The changes of TPC, TAC, and TFC in mulberry wine during fermentation are depicted in Figure 1c. The TPC increased from day 0 to 3 F I G U R E 1 Changes of color (a and b), phenol concentration (c), and antioxidant capacity (d) during mulberry wine fermentation and reached the maximum (1792.17 mg GAE/L) on day 3. From day 3 to 9, the TPC decreased significantly (p < .05), and the decreasing rate was greater than the increasing rate at the start of fermentation. After the 9 days of fermentation, there was no significant difference (p > .05) between the TPC of the samples. TFC significantly decreased (p < .05) from day 0 to 2, and the TFC and TAC transition trends were similarly observed. Anthocyanin poses as an essential component of flavonoids, and hence, the depletion of TFC is closely linked to the deterioration of anthocyanins (Garrido & Borges, 2013).
From day 2 to 4, the TFC increased, indicating the contents were still significantly lower than the initial quantity (p < .05). After 4 days, TFC decreased gradually. At the end of the fermentation period, the TPC and TFC of the samples were significantly decreased (p < .05) showing similar trends.
Anthocyanin is an important active component of mulberry, a kind of natural antioxidant, and has a positive effect on the human body with nutraceutical benefits (Mangani et al., 2011;. TAC significantly decreased (p < .05) from day 0 to 3 with a loss rating of 47.98% (Figure 1c). The decrease was great from day 1 to 2 at a loss of 32.77%. Overall, the TAC decreased rapidly and then tended to be stable during the fermentation process.

| Changes of antioxidant capacity during mulberry wine fermentation and correlation analysis
All the antioxidant activities measured by the three different meth- significantly lower than on day 0 (p < .05). Our findings differed from the report of Wang, Sun, et al. (2015) and Wang, Xie, et al. (2015), which may be attributed to differences in the fermentation mechanism, yeast species, and mulberry varieties used.
From Table 2, the antioxidant activity was highly correlated with TPC, TFC, and TAC. Among the phenolic compounds, an extremely significant correlation was observed between TPC and ABTS-SA as well as TPC and PFRA (p < .01). The correlation between TAC, TFC, and the three antioxidant abilities was extremely significant.
The strongest correlations were found between TPC and PFRA Note: ** means the correlation is extremely significant (p < .01); * means the correlation is significant (0.01 < p < .05).

| Changes of phenolic acid content in mulberry wine during fermentation
The content of phenolic acid in the mulberry wine at different fermentation times is illustrated in Figure 2a

| Taste and quality changes of mulberry wine during fermentation
Wine taste was detected using the detection sensor of the electronic tongue which comprise an artificial lipid membranous cast in an electrode widely used for the routine assessment (Lu et al., 2016). Twelve  on day 2. However, from day 9 to day 17, stability in taste was established. The sourness was reflected by the pH of the wine rendered through fermentation efficacy. During the early stages of fermentation, the yeast grew and generated many acidic metabolites and CO 2 , subsequently making the pH to drop dramatically and the fermentation broth sour. The relative intensity of bitterness reached the minimum (−2.07) on day 2. However, astringency first increased, then decreased significantly (p < .05), and reached the maximum (3.90) on day 3. The bitterness and astringency of wine are mostly due to phenolic compounds (Cai et al., 2020). The phenolic compounds in mulberry wine steadily decreased as fermentation continues, resulting Note: ** means the correlation is extremely significant (p < .01); * means the correlation is significant (0.01 < p < .05).

| Changes of aroma components in mulberry wine during fermentation
The mulberry wine samples were harvested and analyzed on days 0, 2, 4, 7, and 17. It showed the quantity and content of aroma components in Figure 3d and Table 3. In mulberry wine, 51 volatile substances were detected by using SPME-GC-MS method, including 13 alcohols, 24 esters, seven aldehydes, three ketones, three acids, and one aromatics component. The results indicated that the total volatile components content first increased and then decreased, and reached the maximum (11,808.48 μg/L) on the 7th day. Furthermore, after fermentation, the total volatile compounds of mulberry wine were significantly (p < .05) higher than mulberry juice (fermentation 0th day). As a whole fermentation process, alcohols and esters accounted for the largest proportion of the total volatile components constituents.
According to Belda et al. (2017), alcohols are essential flavor compounds in fruit wine, and they are mostly generated through glycolysis. Furthermore, amino acids precursors in resultant higher alcoholic compositions play a pivotal role. Among the identified volatile compounds, alcohols played an important role in volatile substances, accounting for 20.62% to 42.37% of the total volatile substances. Ethanol, isobutanol, isoamyl alcohol, and phenylethanol were the principal components of alcohols, and their levels F I G U R E 2 Changes of p-hydroxybenzoic acids (a), p-hydroxycinnamic acids (b), and total phenolic acid (c) during mulberry wine fermentation reached the maximum on the 7th day. Isoamyl alcohol presented the greatest concentration of higher alcohols in mulberry wine.
Additionally, phenylethanol was found to impart a distinct floral fragrance to mulberry wine (Wang, Xie, et al., 2015). Compared with mulberry juice (fermentation 0th day), the amount of alcohol in mulberry wine after day 17 of fermentation decreased. However, the overall content was significantly higher than that in mulberry juice (p < .05). The content of alcohol group increased from 80.53 μg/L to 3333.85 μg/L after fermentation. Hexanol (16.42 μg/L) was the most important alcohol volatile substance in mulberry juice, which has a grassy and woody fragrance, decreased rapidly during the fermentation period. However, it was not detected on the 17th day of fermentation. After fermentation, isoamyl alcohol was the main alcohol volatile in mulberry wine which was produced during the fermented process.
As the largest family of mulberry wine volatile compounds, esters accounted for 56.07% to 71.15% of the total identified compounds. It has been proposed that esters are formed by the reaction of alcohols and organic acids during the fermentation process, which has a significant impact on the aroma of wine (Belda et al., 2017).
Furthermore, the presence of yeast and other microbes might result in the production of esters (Wang, Xie, et al., 2015).  Abbreviations: RIExp, Retention Index Experiment; RIL, Retention Index Library;"-": Not detected.
The superscript alphabetical letters a, b, c, d, and e denote statistically significant differences between the data (p < 0.05).

| CON CLUS IONS
The strains, Lactiplantibacillus plantarum and Saccharomyces cerevisiae, used for the fermentation influenced the phenolic compounds (TPC, TAC, and TFC) of the mulberry wine samples. During the fermentation period, the total anthocyanin, phenolic, and flavonoid content decreased significantly in the mulberry wine. Furthermore, mulberry wine exhibited better free radical quenching ability due to the presence of microbially biotransformed phytochemical compounds after L. plantarum and S. cerevisiae fermentation. These compounds influenced the quality of mulberry wine. Fermentation with LAB and yeast significantly influenced color, taste, aroma, phenolic profiles, and antioxidant properties of the mulberry wine samples, which could focus the application prospects for nutraceutical and healthcare potentials. Besides, mulberry wine production makes maximum use of mulberry fruits, prevents post-harvest losses, and demonstrates significant economic potential.

ACK N OWLED G M ENTS
We appreciate Dr. Xin Wang for his guidance on the experiment and the provision of the experimental equipment. This work is supported by the Zhenjiang Science and Technology Bureau fund (NY2020020) and financial support from DANYANG YIHE FOOD CO., LTD.

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

E TH I C A L S TATEM ENT
This research article does not contain any studies with human participants or animals performed by any of the authors.