Influence of different carbohydrate sources on physicochemical properties and metabolites of fermented greengage (Prunus mume) wines

LWT - Food Science and Technology 121 (2020) 108929

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Influence of different carbohydrate sources on physicochemical properties and metabolites of fermented greengage (Prunus mume) wines

T

Xueyuan Han1, Qi Peng, Huanyi Yang, Baowei Hu, Chi Shen, Rungang Tian∗ School of Life Science, Shaoxing University, Shaoxing, Zhejiang Province, 312000, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Greengage wine Glutinous rice Sucrose Fermentation Metabolites

Sucrose and glutinous rice were used as carbohydrate sources for greengage wine fermentation. The physicochemical properties of the two greengage wines (RGW, glutinous rice greengage wine; SGW, sucrose greengage wine) were compared. The results showed differences in the contents of total sugar, total acid, alcohol, total protein and ascorbic acid between the two greengage wines. The evaluation of their sensory quality suggested that RGW was superior to SGW. However, SGW contained more total flavonoids and total phenols and showed a stronger antioxidant capacity than those of RGW. Volatile compound analysis showed that 1-hexanol and benzaldehyde were unique to RGW. Compared with SGW, RGW contained more contents of certain esters and alcohols in volatile components. Metabolomics detection indicated that RGW was richer in lipids and amino acids. Instead, SGW presented relatively high contents and a variety of organic acids and flavonoids. This study was expected to provide a new approach to improving the quality of fermented greengage wine.

1. Introduction Fruit wine with a low-alcohol content is a nutritious drink. In recent years, specific health benefits have been ascribed to the consumption of fruit wine (Careina, 2011; German & Walzem, 2000; Yoo, Saliba, MacDonald, Prenzler, & Ryan, 2013). For example, particular phenolic antioxidant compounds that are found in wine have an effect in the prevention of coronary hearth diseases (CHD) (Joshi, Sharma, & Devi, 2009; Renaud & de Lorgeril, 1992). Greengage wine as an alcoholic beverage is popular in Southeast Asia for its characteristic fruit flavor, and for its potential health benefits, such as its anticancer and antioxidant properties (Adachi et al., 2007; Jeong, Moon, Park, & Shin, 2006; Jo et al., 2006; Zheng, Zhang, Fang, & Liu, 2014). It not only contains organic acids, minerals, phenols, and amino acids, but also contains specific components that are beneficial to the human body, which makes it unique (Gil, Tomás-Barberán, Hess-Pierce, & Kader, 2002). Greengage wine can be produced by two different procedures: steeping and fermenting (Zheng et al., 2014). Although, steeped and fermented greengage wine can already be found in the market, most of it, especially fermented greengage wines, are still in the experimental stage. In practice, greengage wine is more often produced through steeping greengage fruits in rice wine for a long time (Gao, Zeng, &

Xiao, 2009; Yang, Wu, Wang, & Peng, 2005). However, freshly steeped wine is recognized as unsuitable for direct drinking due to the high concentration of fusel oils, harsh taste and pungent smell (Zheng et al., 2014). Compared with the characteristics of steeped greengage wine, fermented greengage wine exhibits not only better sensory characteristics but also increased nutritional value. Many factors affect the quality and the amounts of flavor compounds in fermented greengage wine, such as the raw materials, yeast and fermentation conditions (Bokulich, Thorngate, Richardson, & Mills, 2014; Jones & Davis, 2000). Fermentation and microbial metabolism accumulate a wide range of flavor compounds, including alcohols, esters, terpenes, aldehydes, benzene derivatives, acids and furans, which give the wine a unique character and style (Franitza, Granvogl, & Schieberle, 2016; Hoang et al., 2016; Steinhaus & Schieberle, 2007). During the fermentation of greengage wine, more bioactive components (tryptophol, melatonin, tyrosol, glutathione and hydroxytyrosol) are biosynthesized and released, making them more biologically available for absorption (Guerrini et al., 2018; Shahidi, 2009). However, greengage fruit is highly acidic and its utilization as an ingredient in the preparation of fruit wine in processing is limited because of its sour taste. The potential of greengage in wine brewing has not been fully explored. As a potential raw material for fruit wine

∗

Corresponding author. E-mail address: [email protected] (R. Tian). 1 Xueyuan Han will handle Correspondence at all stages of refereeing and publication, also post-publication. https://doi.org/10.1016/j.lwt.2019.108929 Received 18 September 2019; Received in revised form 3 December 2019; Accepted 5 December 2019 Available online 06 December 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

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prepared. The volume of NaOH solution required for titration was recorded. Alcohol content. The alcohol concentration was determined by the distillation-alcoholmeter method with rapid distillation apparatus (Super DEE, GIBERTINI, Italy) and alcohol tester (AlcoMat-2, GIBERTINI, Italy). Ascorbic acid content. The ascorbic acid content was determined according to the 2, 6-dichlorophenol-indophenol titration method described by the Association of Office Analytical Chemists (1996). L-ascorbic acid (1.0 mg/mL) was prepared as a standard solution. Total protein. The total protein content was determined according to the Bradford method (Bradford, 1976) with minor adjustments, and bovine serum albumin (BSA) was used as the standard. The Comassie Brilliant Blue G-250 dye reagent combines with the existing proteins and presents a blue–black colour with a measurable absorbance at 595 nm with a spectrophotometer.

brewing, greengage fruit is characterized by a high content of organic acids and significant amounts of flavonoids, phenols, vitamins and minerals in chemical components (Sun, Li, & Yang, 2009; Tian et al., 2018). The production of greengage wine that contains phenolic compounds would be quite attractive especially since greengage is a relatively inexpensive raw material compared to other fruits. Considering the above, this work intended to improve the taste and quality of greengage wine using two different carbohydrate sources for fermentation, namely, sucrose and glutinous rice. It is widely known that glutinous rice is one of the main ingredients for Chinese rice wine (Huangjiu) fermentation. The saccharification process of glutinous rice could produce amounts of monosaccharide and release many bioactive components into the fermentation broth (Chen & Xu, 2013). Therefore, glutinous rice was innovatively used as a substitute for pure sucrose and was fermented with greengage juice to produce greengage wine in this work. The objective of this study was to compare and evaluate the physicochemical and metabolomic properties of greengage wines fermented with sucrose or glutinous rice as a carbohydrate source. The changes in the flavor components and other substances affecting the taste of wine, such as acids, saccharides and esters, were assessed. This paper was expected to provide a new approach for producing fermented greengage wine with strong flavor, mellow taste and a high nutritional content.

2.4. Determination of total phenols, total flavonoids and total antioxidant capacity Total phenols. Based on the Folin-Ciocalteu method (Singleton & Rossi, 1965), the contents of total phenols in the wine samples were determined and gallic acid was used as the standard. The absorbance was measured at 760 nm and the results were expressed as mL gallic acid equivalent (GAE)/mL of wine. Total flavonoids. The contents of total flavonoids in the wine samples were determined according to the method of Zhishen, Mengcheng, and Jianming (1999) with minor modification. Samples of 2.0 mL were mixed with 3.0 mL of 60% ethanol and 0.3 mL of a 5% solution of sodium nitrite and incubated for 6 min. Then, 0.3 mL of 10% AlCl3 solution was added. After 6 min, 4.0 mL of 4.3% NaOH was added and mixed evenly and incubated for 15 min. The absorbance was measured at 510 nm and the content of total flavonoids was expressed as mL of rutin equivalents (RE)/mL of wine. Total antioxidant capacity. Total antioxidant capacity was analyzed according to the instructions of total antioxidant capacity (T-AOC) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The measurement of absorbance included a measuring tube and a control tube. The formula for calculating T-AOC was as follows:

2. Materials and methods 2.1. Preparation of greengage juice Greengage fruits free from pests were harvested based on the uniformity of size and shape at commercial maturity from Wangtan East Village, Shaoxing, China. Greengage juice was prepared by the freezing press method. Greengage fruits were first washed clean and frozen at −20 °C for 48 h. Then, fruits were thawed at room temperature (approximately 25 °C) until the central temperature was 0 °C. Next, the fruits were squeezed with a presser, and the resulting juice was collected. 2.2. Fermentation Glutinous rice greengage wine (RGW). Fermentations were performed in 5.0 L glass vessels containing 1.25 L of greengage juice, 1.25 L of distilled water, 1.5 kg steamed glutinous rice, 3.0 g saccharifying enzyme and 0.25 g nisin. Last, 0.6 g activated acid-reducing yeast (Saccharomyces cerevisiae, Lalvin71B) was added to the mixture, accounting for approximately 0.6 g/kg glutinous rice. Bioreactors with a total weight of approximately 4.0 kg were maintained at 22 ± 1 °C. During fermentation, the sugar content (°Brix) was measured by an Erma hand refractometer every day. The fermentation was regarded as being done on the 10th day when the sugar content was stable. After the completion of fermentation, the wines were racked and then properly clarified. Sucrose greengage wine (SGW). All fermentation conditions were the same as those of the RGW production, except that sucrose (0.85 kg) was used instead of glutinous rice.

T− AOC(U/mL)  =

ODM − ODC ×N×n 0.01 × 30

Note: ODM, the absorbance of the measuring tube; ODC, the absorbance of the control tube; N, dilution multiple of reaction system (total volume of reaction solution/sampling volume); n, dilution multiple before sample testing.

2.5. Sensory evaluation of greengage wines The sensory characteristics of the greengage wines were evaluated in terms of appearance, aroma, taste and typicality by 20 qualified and experienced panelists (10 females and 10 males). All samples were presented individually and randomly to the panelists in a sensory evaluation room at 20 ± 1 °C. Between sample evaluations, clean water was provided to rinse the mouth to avoid the lingering effect or aftertaste. According to the ISO standard, the 100-point method with minor modification was used, following the allocation of color (5 scores), clarity (5 scores), aroma (30 scores), taste (40 scores) and typicality (20 scores) (H. Yang et al., 2019; Ye, Yue, Yuan, & Wang, 2013). Detailed sensory evaluation rules are listed in Supplementary Table 1. Samples with a total score of over 85 were excellent, those over 80 were considered acceptable and good, those between 70 and 80 were generally acceptable, and those less than 70 were not acceptable.

2.3. Physicochemical analyses Total sugar. The protocol of the DNS method (Miller, 1959) was employed to determine the content of total sugar spectrophotometrically. Glucose solutions with different concentrations were prepared to generate a calibration curve with absorbance measured at 540 nm. Total acid. Total acid was determined using the method of titratable acidity (Rangana, 1979) and expressed as tartaric acid equivalents (g/ L). Phenolphthalein indicator and 0.1 mol/L NaOH solutions were 2

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2.6. Volatile compounds determination

Table 1 Physicochemical properties of greengage wines.

Instrument and software. Analyses of volatile compounds were performed on a FlavourSpec® static headspace (SHS) gas chromatographyion mobility spectrometry (GC-IMS) instrument (Gesellschaft für Analytische Sensorsysteme mbH (G.A.S.), Dortmund, Germany) with a heated splitless injector. The ionization source of the IMS was tritium (3H). In addition, a WAX-30 m × 0.53 mm ID capillary column (CSChromatographie Service GmbH, Düren, Germany) and an autosampler (PAL RSI, CTC Analytics AG, Zwingen, Switzerland) were equipped on this device. IMS instrument data were collected under positive mode and assessed with the help of LAV® software (G.A.S.) including the Reporter, Gallery Plot and Dynamic PCA plugins. Further, GC × IMS Library Search® software (G.A.S.) was employed to identify compounds. GC-IMS method. According to the method of Li et al. (2019) with several modified parameters, the volatile compounds of greengage wines were analyzed. The headspace (100 μL) was sampled and automatically injected through a heated syringe (85 °C). The carrier gas of N2 passed through the GC-IMS injector under an inlet pressure of 3 bar and introduced the sample into the capillary column. Analytes were eluted isothermally at 60 °C and driven to the ionization chamber. Compounds were ionized by the 3H source under atmospheric pressure, and ionization depended on analytes' chemical nature and concentration, yielding product ions of protonated monomers or proton-bound dimers. Next, the ions were pushed into a drift tube 9.8 cm in length through the shutter grid operating at a constant temperature (45 °C) and voltage (500 V/cm). Each spectrum was an average of 16 scans with a repetition rate of 30 ms. The flow rate of drift gas (N2) was set at 150 mL/min. The double separation obtained in the IMS drift tube and in the GC column was presented in a topographic plot. Each feature was defined by intensity value, a drift time and retention time in the plots.

Characteristics

SGW

RGW

Degree Brix (°Bx) Total sugar (g/L) Total acid (g/L) Alcohol content (%(v/v)) Total protein (mg/L) Ascorbic acid (g/L) Total phenols (mg GAE/L) Total flavonoids (mg RE/L) Total antioxidant capacity(U/mL) Score of sensory evaluation

5.5 ± 0.2 7.67 ± 0.34 13.65 ± 0.17 8.53 ± 0.25 113.31 ± 2.64 10.99 ± 0.85 33.89 ± 3.56 343.53 ± 5.07 56.40 ± 3.41 74.50 ± 3.33

5.0 ± 0.1 5.72 ± 0.16 11.57 ± 0.08 12.72 ± 0.57 140.71 ± 2.23 12.30 ± 0.78 23.15 ± 1.70 293 ± 4.74 47.91 ± 3.22 84.50 ± 3.0

value of 1.0. 2.8. Statistical analysis All experiments were carried out with at least three biological replicates. Data represent the mean value plus or minus the standard deviation ( ± SD). The difference was considered to be statistically significant when the p value was less than 0.05. 3. Results and discussion 3.1. Basic physicochemical characteristics On the 10th day of fermentation, the °Brix maintained no change with approximate 5.5 °Bx in the SGW and 5.0 °Bx in the RGW. After completion of fermentation, the wines were racked and properly clarified. Certain physicochemical characteristics of the SGW and RGW are clearly outlined and compared in Table 1. Both greengage wines were fermented to semi-dryness with the residual total sugar concentrations were between 4.1 and −12 g/L. Obvious differences were observed in the content of total acid (TA), alcohol, total protein and ascorbic acid. The RGW produced from fermenting with glutinous rice was lower in total acid, higher in alcohol and ascorbic acid and contained much more total protein than those of the SGW. The content of total acids has an important influence on the flavor and aroma of fermented products, and its level can be considered an index of shelf-life (Berenguer et al., 2016; Sivertsen, Figenschou, Nicolaysen, & Risvik, 2001). It was also reported that the TA level was related to the polymerization of phenolic compounds, resulting in brown deposits (Darias-Martı́n, Rodrı́guez, Dı́;az, & Lamuela-Raventós, 2000). Therefore, the TA content reflected, to some extent, the quality of the fruit wine and was one of the most important quality indicators. In general, the TA content of steeped greengage wine is approximately 14.00–14.50 g/L (Zheng et al., 2014). In this study, both fermented greengage wines presented a low TA content, which might be attributed to the use of acid-reducing yeast (Saccharomyces cerevisiae, Lalvin71B). Furthermore, a lower TA content and a better sugar-acid balance (approximately 1:2) than those in SGW were observed in RGW, likely related to the neutralization reactions of multiple chemical compounds in RGW. A relevant study also showed that a decrease in the TA content of rice and fruit wine was highly related the utilized starting substrate (Lee, Lee, Singh, & Lee, 2018) and fermenting strains (Chen et al., 2019; Chidi, Rossouw, Buica, & Bauer, 2015; Kunicka-Styczyńska & Pogorzelski, 2009). To assess the nutritional value of greengage wine, it was necessary to examine its content of total phenols and flavonoids and to further evaluate its potential antioxidant properties. It is known that phenols and flavonoids possess high antioxidant activities (Kong & Lee, 2010). Consistent with this, the SGW fermented with sucrose presented a stronger antioxidant capacity with greater contents of total phenols and flavonoids than those of RGW. Phenols and flavonoids were reported to

2.7. Untargeted metabolomics analysis by LC-MS/MS All chromatographic separations were performed via ultra-performance liquid chromatography (UPLC) (SCIEX, UK). An ACQUITY UPLC T3 column (100 mm × 2.1 mm, 1.8 μm, Waters, UK) was used for reversed phase separation. The mobile phase consisted of solvent A (water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid). The gradient elution procedures were as follows: 0–0.5 min, 5% B; 0.5–7 min, 5%–100% B; 7–8 min, 100% B; 8–8.1 min, 100%–5% B; and 8.1–10 min, 5% B. The flow rate was 0.4 mL/min, and the column oven was maintained at 35 °C. Metabolites were detected using a high-resolution tandem mass spectrometer TripleTOF5600plus (SCIEX, UK). The operation of the QTOF was under both positive (POS) and negative (NEG) ion modes. The related parameters and technical settings referred to Yu et al. (2018). Furthermore, a quality control (QC) sample (pool of all samples) was acquired after every 5 samples to evaluate the stability of the LC-MS during the whole acquisition. Bioinformatic analysis of the untargeted metabolomics dataset referred to Yu et al. (2018). With the use of the online HMDB and KEGG databases, the accurate molecular mass data (m/z) of samples were referenced with the data in the database, and the metabolites were annotated. An in-house fragmentation spectral library of metabolites was also applied to validate metabolite identification. MetaX software was employed to further preprocess the peak intensity data according to the method of Yu et al. (2018). The differences in metabolite concentrations between the two phenotypes were detected and assessed by conducting Student's t-tests. The false discovery rate (FDR; Benjamini–Hochberg method) was also conducted to adjust the P value for multiple tests. Supervised PLS-DA was analyzed by metaX to distinguish the different variables between groups. The VIP (variable important for the projection) values were calculated, and important features were selected with a VIP cut-off 3

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listed in Table 2. It was observed that some individual compounds have produced multiple spots or signals (monomer or dimer) because of their different concentrations. A total of ten esters were quantitatively compared in GC-IMS. Esters, alcohols, and aldehydes were regarded as the major volatile compounds, but their concentrations differed obviously with the different carbon sources for fermentation. To more directly detect the differences of volatile compounds between both greengage wines, all information given by the fingerprint analysis technique was fully utilized to qualitatively characterize the wines, as shown in Fig. 3. 1-Hexanol and benzaldehyde were unique to the greengage wine fermented with glutinous rice. Yoshizaki et al. (2010) presented the effects of three types of rice koji (black, yellow and white) on volatile aroma compounds using GC−MS based metabolic profiling (Yoshizaki et al., 2010). The flavor compounds in Chinese rice wine were influenced by using Fagopyrum tataricum grain instead of glutinous rice as feedstock in the fermentation process (Ren et al., 2019). Previous studies have reported that benzaldehyde is a typical aroma substance in steeped greengage wine (Yang et al., 2005). In this study, 2-heptanol, heptanol, 1-pentanol, propan-2-one, butyl acetate, and 2-methylpropyl acetate were present at higher concentrations in RGW than in SGW. Compared with those detected in RGW, acetic acid, ethyl octanoate, propyl acetate and ethyl butanoate were found to be higher concentrations in SGW. Overall, the greengage wine fermented based on glutinous rice contained higher levels of esters and alcohols than those in the wine fermented based on sucrose. Esters are known as the most important flavor components contributing to the fruit and floral aromas (Fan & Qian, 2006). One of the sources of esters is the esterification of alcohols and fatty acids. Another source is the anabolism of microorganisms using higher alcohols under the action of acetyltransferase (Mo, Fan, & Xu, 2009). These results confirmed those of previous sensory analyses indicating that RGW had higher overall flavor coordination than that of SGW.

have a variety of health benefits (Ghasemzadeh & Ghasemzadeh, 2011; Jaganath & Crozier, 2010; Lin et al., 2016). Sensory evaluation is the basis of consumer acceptability and is an essential tool to assess its quality. The sensory evaluation results of both greengage wines showed that RGW was clear and transparent with a light-yellow color and a strong wine aroma and was full-bodied with mellow coordination and a fruity flavor, representing the specific taste of greengage wine. Therefore, RGW had a relatively high sensory evaluation score (84.50 ± 3.00 points). In contrast, SGW, with a sensory evaluation score of 74.50 ± 3.33 points, suggested slight turbidity, a lower alcohol content, slight bitterness, a slightly dull taste and a not very elegant fruity flavor. A study on metabolites and off-flavors of Japanese sake suggested that an inharmonious bitter and off-flavors were attributable to polyamines, oxidative glutathione derivatives, products of the methionine salvage cycle, or amino acid catabolites (Takahashi & Kohno, 2016). The scores from panelists suggested that RGW was superior to SGW in appearance, aroma, taste, and typicality. 3.2. Volatile compounds 3.2.1. GC–IMS topographic plots of both greengage wines The flavor is the most important characteristic of an alcoholic beverage. The volatile compounds of both greengage wines were analyzed by GC-IMS. GC-IMS combines the advantages of the high separation efficiency of gas chromatography with the fast response and high sensitivity of ion mobility spectrometry. This analytical technique has the advantages of no sample pretreatment (non-destructive), simplicity, rapidity and accuracy, and is especially suitable for trace analysis of volatile organic compounds. The data are reflected in a 3D topographical visualization in Fig. 1, where the Z-axis represents the peak height utilized for quantification, the Y-axis represents the retention time of the gas chromatographic separation and the X-axis represents the ion migration time utilized for identification. As shown in Fig. 1, the volatile compounds in both greengage wines were similar, but the signal intensity and certain compounds were slightly different.

3.3. Non-volatile metabolites identification 3.3.1. Statistics and quality control of metabolite identification The analysis of the chemical properties of a wine beverage is very important for assessing and exploring its beneficial effects and functional properties regarding human health. To explore the global metabolic diversity of the greengage wines, untargeted metabolomic technology was applied, where 17 957 annotated metabolites were identified from 34 410 ion features (Fig. 4A), including 468 MS2 annotated metabolites. An overview of the metabolite detection of RGW and SGW in POS mode is shown in Fig. 4B–D and the corresponding statistical graphs in NEG mode are shown in Supplementary Fig. 1. According to annotations, many metabolites were assigned to more than one category of primary or secondary metabolism. The top 20 largest KEGG pathways of MS2 annotated metabolites in POS mode, including the biosynthesis of secondary metabolites (39 metabolites), the biosynthesis of amino acids (14 metabolites), ABC transporters (11 metabolites), phenylpropanoid biosynthesis (8 metabolites), 2-oxocarboxylic acid metabolism, beta-alanine metabolism, purine metabolism and other metabolites are shown in Fig. 4B. To offer a deep overview of the metabolic diversity, quality control parameters for the quantification were analyzed, including the coefficient of variation (CV), hierarchical cluster analysis (Heatmap) and principal component analysis (PCA). Good repeatability was suggested by a CV value of lower than 30% (Supplementary Fig. 2). Hierarchical cluster analysis showed the intensity of each metabolite per sample and a clear separation between samples was observed (Fig. 4C). The PCA showed that PC1 and PC2 accounted for 65.62% and 8.42% of the variation, respectively, indicating a clear separation between the two wine samples (Fig. 4D).

3.2.2. Differences of volatile compounds between both greengage wines The top view of the 3D-topographic plot of the samples in GC–IMS is shown in Fig. 2. The position of the reactive ion peak (RIP) and the ion migration time were normalized. The whole spectrum represented the total headspace compounds of the samples. Most of the signals appeared in the retention time range of 100–1200 s and the relative drift time of 1.0–2.0. Each point represents a volatile compound detected in the samples. Color represents the signal intensity of the compound. The darker the color, the stronger the intensity. Red indicates a higher intensity and white indicates a lower intensity. The compounds were characterized by comparing the retention index and IMS drift time. A total of 44 volatile compounds were detected in the two wines, of which 26 volatile compounds were identified successfully from GC × IMS Library Search, as marked in Fig. 2 and

3.3.2. Differentially accumulated metabolites between both greengage wines Metabolite profiling revealed differences in metabolite abundance

Fig. 1. 3D-topographic of GC-IMS analysis of the two greengage wines. 4

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Fig. 2. Topographic plots of GC-IMS spectra with the selected compounds obtained from different greengage wines (A, SGW and B, RGW).

Table 2 GC-IMS integration parameters of volatile compounds identified in greengage wines. Count

Compound

CAS#

Formula

MW

RI

Rt [sec]

Dt [RIPrel]

Comment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

ethyl octanoate ethyl octanoate Benzaldehyde Benzaldehyde Acetic acid Acetic acid ethyl acetate ethyl acetate ethyl butanoate 2-methylpropyl acetate ethyl hexanoate isoamyl acetate 2-Methyl-1-propanol Dimethylsulfide butyl acetate butyl acetate 1-Hexanol 1-Hexanol 2-Heptanol Propan-2-one 1-Pentanol 3-Methyl-but-3-en-1-ol Propanoicac id ethyl ester propyl acetate Ethyl isobutyrate Heptanol

C106321 C106321 C100527 C100527 C64197 C64197 C141786 C141786 C105544 C110190 C123660 C123922 C78831 C75183 C123864 C123864 C111273 C111273 C543497 C67641 C71410 C763326 C105373 C109604 C97621 C53535334

C10H20O2 C10H20O2 C7H6O C7H6O C2H4O2 C2H4O2 C4H8O2 C4H8O2 C6H12O2 C6H12O2 C8H16O2 C7H14O2 C4H10O C2H6S C6H12O2 C6H12O2 C6H14O C6H14O C7H16O C3H6O C5H12O C5H10O C5H10O2 C5H10O2 C6H12O2 C7H16O

172.3 172.3 106.1 106.1 60.1 60.1 88.1 88.1 116.2 116.2 144.2 130.2 74.1 62.1 116.2 116.2 102.2 102.2 116.2 58.1 88.1 86.1 102.1 102.1 116.2 116.2

1452.9 1450.3 1531.1 1529.5 1493.5 1493.9 895.1 896.7 1044.3 1019.5 1243.7 1132.3 1102.6 808.4 1083.9 1083.7 1368.6 1368.1 1339.9 838.5 1250.2 1215.5 963.8 982.8 971.6 1481.5

927.195 919.323 1196.41 1190.112 1058.415 1059.713 226.022 226.554 288.421 274.44 477.156 351.227 325.23 199.189 312.219 312.085 704.56 703.421 641.652 208.128 486.21 439.401 249.837 256.853 252.695 1017.677

1.4868 2.0378 1.157 1.4764 1.0567 1.1526 1.0974 1.3369 1.5609 1.6171 1.8029 1.7486 1.3794 0.9615 1.2406 1.6282 1.3244 1.6464 1.3833 1.1166 1.504 1.5079 1.4564 1.4815 1.5642 1.4035

Monomer Dimer Monomer Dimer Monomer Dimer Monomer Dimer

between RGW and SGW. After filtering, differentially accumulated metabolites (DAMs) were screened from 29 491 high-quality features. As a result, 11 148 significant DAMs were identified by statistical analysis, including 7179 RGW predominantly accumulated metabolites and 3969 SGW predominantly accumulated metabolites (Supplementary Fig. 3). Multiple and statistical tests for each comparison group were assessed using univariate Student's T-tests shown in a volcano plot (Fig. 5), which displayed a significant difference at the 0.05 level. All of the DAMs (quantitative ratio of RGW to SGW) were assigned to various metabolic pathways. Glutinous rice used for greengage wine

Monomer Dimer

fermentation mainly enhanced the metabolic pathway, the biosynthesis of secondary metabolites, arachidonic acid metabolism, isoquinoline alkaloid biosynthesis, diterpenoid biosynthesis, the synthesis of plant secondary metabolites and α-linolenic acid metabolism, etc. (Fig. 6A). Compared with RGW, the fermentation in SGW mainly promoted the biosynthesis of secondary metabolites, flavonoid biosynthesis, ABC transporters, the biosynthesis of plant secondary metabolites, 2-oxocarboxylic acid metabolism, isoflavonoid biosynthesis, flavone and flavonol biosynthesis, etc. (Fig. 6B). The DAMs were annotated to various major chemical categories, including alkaloids and their derivatives, benzenoids, lipids and lipid-

5

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Fig. 3. Gallery plot of the selected signal peak areas obtained from the two greengage wines (SGW and RGW).

like molecules, nucleosides, organic nitrogen compounds, organic acids and derivatives, organoheterocyclic compounds, organic oxygen compounds, organooxygen compounds, phenylpropanoids and polyketides,

and others (Supplementary Tables 2 and 3), out of which benzenoids, lipids and lipid-like molecules, organic acids and derivatives, organoheterocyclic compounds, phenylpropanoids and polyketides were

Fig. 4. Statistics and quality control of metabolites identification. A, Statistics of identified metabolites; B, KEGG pathway analysis of MS2 annotated metabolites under POS mode; C, hierarchical cluster analysis of annotated metabolites under POS mode (Euclidean, ward. D2 cluster method); D, Score plot of principal component analysis of annotated metabolites under POS mode. 6

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Fig. 5. Significance analysis of the DAMs between the two wines samples by Student T-test.

Fig. 6. KEGG pathway analysis of the differentially accumulated metabolites under POS mode.

characteristic of SGW. It was possible that due to the relatively low contents of phenolic compounds and flavonoids that caused bitterness and astringency, RGW presented a good taste. It was interesting to note that, flavonoids, such as quercetin, procyanidin and rutin, were well maintained in SGW. As reported, flavonoids have a certain beneficial health effect and were responsible for wine color (Cheynier, 2012; Suen, Thomas, Kranz, Vun, & Miller, 2016). However, flavonoid compounds were easily oxidized to form macromolecular compounds and insoluble substances (e.g. protein haze in wine and beer) (Cheynier, 2012), resulting in turbidity and bad taste, which was in accordance with the slight turbidity highlighted in the sensory evaluation. Esters, alcohols and amino acids are important flavor compounds. There were also significant differences in the concentrations and species of esters, alcohols and amino acids between RGW and SGW. It was conjectured that the primary metabolites were affected, likely related to associated enzymes and their activities (Lee et al., 2018). For example, the levels of amino acids gradually changed with enhanced protease activity in koji (Lee et al., 2018). The analyses of related enzyme activities, including glucosidase, protease and amylases, are needed in the future. Combining the results of GC-IMS and LCMS/MS, the fermentation with glutinous rice produced more and various alcohols, esters and amino acids, which was consistent with its higher score in the sensory evaluation. Considering all of the above, it is possible to combine glutinous rice and sucrose together as carbohydrate source for fermentation to optimize greengage wine production, which is expected to impart both good flavor and healthy properties.

further annotated with secondary mass spectral data (Supplementary Fig. 4). For most of these categories, amino acids, lipids and certain phenols were predominantly detected in RGW. For instance, the quantitative ratio of amino acids in RGW to those in SGW, including isoleucine, lysine, glutamic acid, histidine, etc., and that of fatty acyls including valeric acid, senecioic acid, lineolic acids, azelaic acid, sebacic acid, hydroxyoctadecanoate, etc. and dihydroxyphenyl-gamma-valerolactone were significantly high (Supplementary Tables 2 and 3). In contrast, the quantitative ratio of organooxygen compounds and flavonoids in RGW to those in SGW, including allose, quinic acid, coumaroylquinic acid, caffeoylquinic acid, procyanidin B1, procyanidin B2, gambiriin A1, kaempferol- 7-O-neohesperidoside, chlorogenic acid, rutin, etc., were significantly low. Lee et al. (2018) observed and determined 67 primary and secondary metabolites that were significantly different in koji produced with varying degrees of milling (Lee et al., 2018). In detail, the koji made with varying degrees of milling contained different abundances of carbohydrate and lipid derived metabolites, and some koji had a relatively high content of antioxidant secondary metabolites (flavonoids and phenolic acid) (Lee et al., 2018; Liu et al., 2015). Previous research also correlated structural contours and metabolic compositions of different rice varieties in koji (Lee et al., 2017). Therefore, appropriate carbohydrate sources or starting substrates are important for improving the quality and nutrient function of fermented wine (Lee et al., 2017, 2018; Liu, Zheng, & Chen, 2017; Yoshizaki et al., 2010). In this study, the higher contents of organic oxygen compounds and flavonoids were the main chemical 7

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4. Conclusion

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Changes in the basic physicochemical characteristics, volatile and involatile metabolites and sensory quality of fermented greengage wine fermented with different carbohydrate sources were studied in this work. The results definitely demonstrated significant differences in the metabolite levels of RGW and SGW. RGW, with a higher alcohol content, showed good sensory quality, but few antioxidant substances and slightly lower antioxidant capacity than that of SGW. Through GC-IMS analysis, it was found that 1-hexanol and benzaldehyde were unique to RGW, and esters, alcohols, and aldehydes were regarded as the major components of the volatile compounds present in greengage wines. By LC-MS/MS analysis, more and various lipids and amino acids were detected in RGW, while relatively high content and diverse organic acids and flavonoids were present in SGW. These results revealed that the difference in sensory characteristics of the two types of greengage wines could be explained by the significantly different levels of volatile and nonvolatile metabolites due to the two different fermentation. The carbohydrate source in greengage wine fermentation affected the accumulation and diversity of metabolites. These results could be regarded as an exploration to improve the quality of greengage wine. Funding This research was supported by the Scientific Research Start-up Fund of Shaoxing University (20195006) and the National Natural Science Foundation of China (31772365). Declaration of competing interest The authors declare no competing financial interest or other conflict of interest. Acknowledgements All authors whose names appear in the paper have contributed sufficiently to this work. Conception and design: RG Tian and XY Han. Analysis and interpretation of the data: XY Han, Q Peng and HY Yang. Drafting of the article: XY Han. Critical revision of the article: RG Tian, BW Hu and C Shen. Final approval of the article: RG Tian, XY Han, Q Peng, HY Yang, BW Hu and C Shen. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.108929. References Adachi, M., Suzuki, Y., Mizuta, T., Osawa, T., Adachi, T., Osaka, K., et al. (2007). The “Prunus mume Sieb. et Zucc”(Ume) is a rich natural source of novel anti-cancer substance. International Journal of Food Properties, 10(2), 375–384. Association of Office Analytical Chemists (1996). Official methods of analysis (15th ed.). Washington, DC: George Banta. Berenguer, M., Vegara, S., Barrajón, E., Saura, D., Valero, M., & Martí, N. (2016). Physicochemical characterization of pomegranate wines fermented with three different Saccharomyces cerevisiae yeast strains. Food Chemistry, 190, 848–855. Bokulich, N. A., Thorngate, J. H., Richardson, P. M., & Mills, D. A. (2014). Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. Proceedings of the National Academy of Sciences, 111(1), E139–E148. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254. Careina, S. S. (2011). Therapeutic value of wine: A clinical and scientific perspective: A perspective. Handbook of Enology, 1, 146–208. Chen, A. J., Fu, Y. Y., Jiang, C., Zhao, J. L., Liu, X. P., Liu, L., et al. (2019). Effect of mixed fermentation (Jiuqu and Saccharomyces cerevisiae EC1118) on the quality improvement of kiwi wine. CyTA - Journal of Food, 17(1), 967–975. Chen, S., & Xu, Y. (2013). Effect of ‘wheat Qu’on the fermentation processes and volatile flavor‐active compounds of Chinese rice wine (Huangjiu). Journal of the Institute of Brewing, 119(1–2), 71–77.

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