Dynamic changes in phenolic compounds, colour and antioxidant activity of mulberry wine during alcoholic fermentation

Journal of Functional Foods 18 (2015) 254–265

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Dynamic changes in phenolic compounds, colour and antioxidant activity of mulberry wine during alcoholic fermentation Lihua Wang, Xiangyu Sun, Fan Li, Dan Yu, Xingyan Liu, Weidong Huang *, Jicheng Zhan ** College of Food Science and Nutritional Engineering, Beijing Key Laboratory of Viticulture and Enology, China Agricultural University, Beijing 100083, China

A R T I C L E

I N F O

Article history:

A B S T R A C T Dynamic changes in total phenolics (TP), total flavonoids (TF), total anthocyanins (TA), two

Received 3 May 2015

main monomer anthocyanins, cyanidin-3-O-glucoside (C3G) and cyanidin-3-O-rutinoside (C3R),

Received in revised form 10 July

colour and antioxidant activities as well as correlations among these factors were investi-

2015

gated in mulberry wine during alcoholic fermentation. TP and TF increased rapidly from

Accepted 15 July 2015

day 0 to 3 and showed unobvious changes from day 3 to 10, whereas TA, C3G and C3R in-

Available online

creased first and then decreased. C3G and C3R reached their maximum at days 1 and 2, respectively; thereafter, C3G decreased rapidly, whereas C3R was more stable. Five colour

Keywords:

parameters, L*, a*, b*, C* and H*, changed significantly from day 0 to 2 and showed unobvious

Mulberry wine

changes from day 2 to 10. For antioxidant activity, changes of the scavenging activity of 1,1-

Phenolic compounds

diphenyl-2-picrylhydrazyl (DPPH·) and reducing power were similar to changes of colour

Colour

parameters. TP, TF, TA and C3R exhibited significant correlations (P < 0.01) with antioxidant

Antioxidant activity

activity, whereas C3G exhibited weaker correlations.

Alcoholic fermentation

© 2015 Elsevier Ltd. All rights reserved.

Dynamic changes

1.

Introduction

Morus alba L., which belongs to the Morus genus and the Moraceae family, is a type of perennial woody plant (Ercisli & Orhan, 2007). This genus has 24 species with one subspecies, and 100 varieties are known to date (Ercisli & Orhan, 2007). It has the characteristics of wide adaptability and easy cultivation because it adapts to a wide area of tropical, subtropical, and temperate zones in the northern hemisphere and to the

tropics of the southern hemisphere (Ercisli & Orhan, 2007; Natic´ et al., 2015). It is also widely distributed in most areas of China. Ripe mulberry, which belongs to the fruit of Moraceae family, is oval purple-black or white jade. Mulberry belongs to the group of berries and has the characteristics of thin skin, succulence and rich nutrition. Furthermore, mulberry has a strong seasonal characteristic and a short harvest season; the harvesting period is between May and June in Beijing, China. It is unfavourable for storage and transportation because it is susceptible to spoilage at room temperature. To prolong its shelf

* Corresponding author. College of Food Science and Nutritional Engineering, Beijing Key Laboratory of Viticulture and Enology, China Agricultural University, Beijing 100083, China. Tel.: +86 010 62737535; fax: +86 010 62737553. E-mail address: [email protected] (W. Huang). ** Corresponding author. College of Food Science and Nutritional Engineering, Beijing Key Laboratory of Viticulture and Enology, China Agricultural University, Beijing 100083, China. Tel.: +86 010 62737535; fax: +86 010 62737553. E-mail address: [email protected] (J. Zhan). http://dx.doi.org/10.1016/j.jff.2015.07.013 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 18 (2015) 254–265

life, postharvest mulberry should be processed quickly. Mulberry can be consumed either fresh or processed, and it can be processed into many forms, including syrup, jam, pulp, ice cream, vinegar, concentrate, and fruit wine (Gundogdu, Muradoglu, Sensoy, & Yilmaz, 2011). Mulberries have been reported to exhibit several biological activities, including antioxidant (Kamiloglu, Serali, Unal, & Capanoglu, 2013; Yang, Yang, & Zheng, 2010), anti-inflammatory (Liu & Lin, 2013, 2014), hypolipidaemic (Yang et al., 2010) and neuroprotective effects (Kang, Hur, Kim, Ryu, & Kim, 2006), which are linked to the presence of phenolics in mulberry. Black mulberries have cyanidin-based anthocyanins, particularly cyanidin-3-O-glucoside (C3G) and cyanidin-3-O-rutinoside (C3R), and numerous flavonoids, including rutin, quercetin and isoquercitin (Ercisli & Orhan, 2007; Özgen, Serçe, & Kaya, 2009), as well as chlorogenic acid, gallic acid and caffeic acid (Kamiloglu et al., 2013). Mulberry wine is one product of mulberries. It has the advantages of fruit wine and is consistent with the shift of Chinese alcoholic beverage industry’s policies of low consumption grain and low alcohol content. Furthermore, mulberry wine can be more adaptable to changes in the alcohol consumption market and exhibits a huge development space and market potential. Therefore, processing mulberry into fruit wine not only makes full use of sericulture resources but also greatly improves the economic benefits of sericulture and can enrich fruit wine varieties and boom the fruit wine market. Current research into mulberry wine brewing has primarily focused on analysing the volatile aromatic compounds (Butkhup et al., 2011), evaluating the colour parameters and antioxidant activity (Kalkan Yildirim, 2006) and optimising the fermentation process for preparation (Wang et al., 2013). However, research into dynamic changes in phenolic compounds, colour, and antioxidant activity of mulberry wine during alcoholic fermentation is not as common. Furthermore, because processing mulberry into fruit wine exhibits many of the advantages mentioned above, it is necessary to study the changes in phenolic compounds, colour, and antioxidant activity that occur when processing mulberry into mulberry wine. Therefore, dynamic changes in the phenolic compounds, colour and antioxidant activity of mulberry wine during alcoholic fermentation were investigated in this study. Dynamic changes in the content of total phenolics (TP), total flavonoids (TF), total anthocyanins (TA), two main monomer anthocyanins, i.e., C3G and C3R, colour, and antioxidant activity as well as the correlations between them were analysed in mulberry wine during alcoholic fermentation of four mulberry varieties, which may provide a theoretical basis for processing mulberry into mulberry wine.

2.

Materials and methods

2.1.

Materials

C3G (Purity ≥ 98.0%, HPLC) was purchased from Chengdu Must Bio-technology Co., Ltd. (Chengdu, Sichuan, China). C3R (Purity ≥ 98.0%, HPLC) was purchased from Sigma (St. Louis, MO, USA). The other reagents used were of analytical grade.

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Completely ripe mulberries were picked from Daxing, Beijing, China. Saccharomyces cerevisiae was LALVIN CY3079 (LALLEMAND, Birkerød, Denmark). Pectinase was purchased from ENARTIS (Novara, Italy).

2.2.

Vinification process

The vinification process of mulberry wine was performed according to a mulberry wine patent by Sheng and Huang (2011). Completely ripe mulberries were picked out and then crushed (added SO2 60 mg/l, pectinase 30 µl/l). Then, sucrose was added to adjust the sugar content, and the mixture was inoculated with active dry yeast at 0.25 g/l. The fermentation temperature was controlled at 20–25 °C. The cap was punched twice a day during the first seven days of fermentation. The skins were removed on day 7. Samples of 30 ml were taken every day from day 0 to day 10 during fermentation to determine the changes in the related indicators. Fermentation was done with three parallel sets for each mulberry variety. Samples were placed in a −20 °C refrigerator for subsequent experiments after centrifugation (5000 × g, 10 min).

2.3.

Determination of TP, TF and TA content

The Folin–Ciocalteu method with some modification was used for the determination of the TP content (Li et al., 2014; Ma et al., 2013, 2014). Briefly, 1 ml of sample, 60 ml distilled water, and 5 ml Folin–Ciocalteu reagent were added in a 100 ml volumetric flask successively. After reaction for 5 min, 15 ml of 20% Na2CO3 was added. The solution was diluted with water to 100 ml and mixed well. The mixture was allowed to react at room temperature in the dark for 2 h, and absorbance was measured at 765 nm. The results were expressed as gallic acid equivalents (GAE) (mg/l of GAE). The TF content was determined according to a previously described protocol (Peinado, de Lerma, Moreno, & Peinado, 2009) with some modification. Briefly, in a 10 ml centrifuge tube, 100 µl of sample was mixed with 1 ml distilled water and 100 µl NaNO2 (0.5 mol/L), and the mixture was allowed to react for 5 min. At the end of the reaction, 200 µl AlCl3 (0.3 mol/L) was added and the mixture was allowed to stand for 6 min. Finally, 1 ml NaOH (1 mol/L) and 5 ml distilled water were added to the reaction mixture, and absorbance was read at 510 nm. The results were expressed as catechin equivalents (mg/l of CTE). The amount of TA content was determined using the pH differential method (Lee, Durst, & Wrolstad, 2005) with some modification. Briefly, the absorbance of sample in 0.025 mol/L potassium chloride solution (pH 1.0 buffer) and 0.4 mol/L sodium acetate buffer (pH 4.5 buffer) was measured simultaneously at 510 and 700 nm by a UV-vis spectrophotometer and calculated using the equation A = (A 510 − A 700 ) pH1.0 − (A 510 − A 700 ) pH4.5. The TA content was calculated using the following formula: TA content = (A × MW × DF × 1000)/(ε × 1), where A is the absorbance, MW is the molecular weight of cyanidin-3-glucoside (449 g mol−1), DF is the dilution factor, and ε is the molar extinction coefficient of cyanidin-3-glucoside (29600). The results were expressed as C3G equivalents (mg/l of C3GE).

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Table 1 – Basic physical and chemical indicators of different varieties of mulberry. Indicators

Glucose (g/l) Fructose (g/l) Reducing sugar (titration method, glucose equivalents, g/l) Total acid (citric acid equivalents, g/l) TP (GAE, mg/g) TA (C3GE, mg/g)

Varieties Dashi

Hongguo

Longsang

Shani

56.35 ± 0.14b 61.10 ± 0.15b 112.17 ± 1.89b 2.67 ± 0.04d 4.81 ± 0.13b 4.16 ± 0.19a

43.03 ± 3.04c 47.35 ± 3.38c 109.77 ± 0.00b 3.87 ± 0.00a 5.30 ± 0.18a 4.34 ± 0.10a

65.72 ± 0.11a 72.23 ± 0.16a 117.12 ± 2.23a 3.38 ± 0.06b 4.71 ± 0.21b 3.62 ± 0.17b

34.40 ± 2.73d 38.70 ± 2.95d 75.04 ± 0.16c 2.78 ± 0.01c 3.23 ± 0.17c 2.32 ± 0.02c

Note: different letters in the same row indicate significant differences.

2.4.

Determination of C3G and C3R content

C3G and C3R analysis was performed on a reversed-phase HPLC (Waters 2695, photodiode array detector, Milford, MA, USA) equipped with the Waters Empower software. Chromatographic separations were performed on a Merck LiChrospher 100 RP-18e column (250 mm × 4.0 mm, I.D. 5 µm, Darmstadt, Germany) placed in a column oven set at 40 °C. The injection volume was 20 µl. The mobile phase was composed of a solvent A (water:formic acid = 100:1.5, v/v) and solvent B (water:methanol = 25:75, v/v; pH adjusted to 2.35 using formic acid). For the elution program, linear solvent gradient was applied as follows: 0–10 min 90% A, 10–50 min 90–40% A, 50– 55 min 40–10% A, 55–60 min 10–90% A (You et al., 2015). The flow rate was at 1.0 ml/min, and the runs were monitored at a 520 nm wavelength.

2.5.

Colour parameter measurements

The colour parameters were measured using the CIELab space according to a previously described method (Pérez-Caballero, Ayala, Echávarri, & Negueruela, 2003). The samples were filtered by passing through 0.45-µm filter membranes. The samples were diluted 5 times, and the measurements were carried out in a SHIMADZU UV-1800 spectrophotometer (Kyoto, Japan) using a 0.2-cm path-length quartz cuvette. Measurements of absorbance were taken at 450, 520, 570 and 630 nm wavelengths with distilled water as the blank control group. L*, a* and b* were calculated according to the formula, τ = 10−A, X = 19.717τ 450 + 1.884τ 520 + 42.539τ 570 + 32.474τ 630 − 1.841, Y = 7.950τ 450 + 34.764τ 520 + 42.736τ 570 + 15.759τ 630 − 1.180, Z = 103.518τ450 + 4.190τ520 + 0.251τ570 − 1.831τ630 + 0.818, L* = 116((Y/ Y10)1/3 − 0.1379), a* = 500((X/X10)1/3 − (Y/Y10)1/3, b* = 200((Y/Y10)1/ 1/3 3 − (Z/Z10) ), where A represents the absorbance, τ represents transmittance and tristimulus values for the blank, with D65 illuminant and CIE1964 standard observer, are X10 = 94.825,Y10 = 100, Z10 = 107.381 (Pérez-Caballero et al., 2003). The H* (H* = arctan b*/a*) and C* (C* = [(a*)2 + (b*)2]0.5) were also calculated (de Ancos, Gonzalez, & Cano, 1999).

2.6.

Determination of the antioxidant activity

2.6.1.

DPPH· scavenging activity

DPPH· scavenging activity was determined according to a previously described method (Ivanovic, Dimitrijevic-Brankovic, Misic, Ristic, & Zizovic, 2013; Li, Wang, Li, Li, & Wang, 2009). Briefly, 0.1 ml of the sample (diluted 20 times) was added to

3.9 ml of a 6 × 10−5 M solution of DPPH· in methanol. A control sample containing the same volume of solvent in place of extract was used to measure the maximum DPPH· absorbance. After the reaction was allowed to occur in the dark for 20 min, the absorbance at 515 nm was recorded to determine the concentration of remaining DPPH·. The results were expressed as Trolox equivalent antioxidant capacity. Trolox standard solutions were prepared at concentration ranging from 15.625 to 500 µM.

2.6.2. Potassium ferricyanide (K3Fe(CN)6) reducing activity (PFRA) PFRA was based on the slightly modified method of Fang et al. (2011). A volume of 20 µl of sample was mixed with 2.5 ml phosphate buffer (0.2 mol/l, pH 6.6) and 2.5 ml of 1% K3Fe(CN)6 in a 10 ml volumetric test tube. The mixture was allowed to react for 20 min at 50 °C and cooled in an ice bath for 5 min. Then, 2.5 ml of 10% trichloroacetic acid was added, and the mixture was centrifuged at 6000 × g for 10 min. The upper layer (1 ml) was mixed with 2.5 ml distilled water and 0.5 ml 0.1% ferric chloride (FeCl3) and, after 5 min, absorbance was read at 700 nm. The absorbance of the reaction mixture indicated the antioxidant activity of the sample, with a larger absorbance value indicating stronger antioxidant ability.

2.7.

Statistical analysis

Data for all of the measurements were obtained in triplicate and are expressed as the mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to analyse the differences between groups. Correlation analysis and ANOVA were conducted using IBM SPSS version 19.0 (Armonk, New York, NY, USA).

3.

Results and discussion

3.1. The basic physical and chemical indicators of mulberry fruit and mulberry wine The physical and chemical characteristics of four mulberry varieties are presented in Table 1.There were significant differences in the glucose and fructose content of four mulberry varieties, which can be ranked as Longsang > Dashi > Hongguo > Shani. The fructose content was higher than the glucose content for all four mulberry varieties. Reducing sugar content was

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Table 2 – Basic physical and chemical indicators of mulberry wine fermented from four mulberry varieties. Indicators

Alcoholic content (%) Glucose (g/l) Fructose (g/l) Total acid (tartaric acid equivalents, g/l) TP (GAE, mg/l) TA (C3GE, mg/l)

Varieties Dashi

Hongguo

Longsang

Shani

9.00 ± 0.60a 0.00 ± 0.00a 0.62 ± 0.04c 6.18 ± 0.10d 4681.33 ± 105.04a 865.01 ± 20.08a

8.44 ± 0.38a 0.00 ± 0.00a 2.36 ± 0.12b 10.27 ± 0.02b 3971.33 ± 35.12b 556.44 ± 6.66b

9.45 ± 0.00a 0.00 ± 0.00a 0.10 ± 0.00d 10.80 ± 0.01a 3554.67 ± 134.29c 510.91 ± 1.10b

8.60 ± 0.16a 0.00 ± 0.00a 8.33 ± 0.07a 7.49 ± 0.01c 3034.67 ± 119.30d 440.09 ± 5.37c

Note: different letters in the same row indicate significant differences.

determined using the titration method, and mulberry wine reached a maximum alcohol content of approximately 6.5%, as calculated by 18 g/l of sugar fermented into 1% alcohol; thus, exogenous sugar had to be added during the alcoholic fermentation process to reach the alcohol content requirements for fruit wine. There were significant differences in the total acid content of the four mulberry varieties, which can be ranked as Hongguo > Longsang > Shani > Dashi. For the TP content of the four mulberry varieties, the highest variety was Hongguo, and the lowest variety was Shani. For the TA content of the four mulberry varieties, the lowest variety was Shani. Fang et al. (2011) found that TP content of Cabernet Sauvignon grapes obtained from the northern and southern sides of the canopy was 2.793 and 3.318 mg gallic acid g−1, respectively, for grape berries without seeds, and TA content was 0.903 and 1.242 mg cyanidin3-glucoside g−1, respectively, for grape berries without seeds. The TP content of the Dashi, Hongguo and Longsang was higher than those of the grapes from the northern and southern sides of the canopy mentioned above, and that of the Shani was almost similar to those of the grapes from the southern sides of the canopy mentioned above. The TA content of the four mulberry varieties was higher than the grapes from the northern and southern sides of the canopy mentioned above. This finding may indicate that mulberry is rich in phenolics and anthocyanins and that it is both a berry with a higher nutritional value and a raw material with a high capability of developing functional food. The physical and chemical characteristics of mulberry wine fermented from four different mulberry varieties are shown in Table 2. The TP and TA content in fruit wine are primarily influenced by the berry (Meng et al., 2012) and fermenting technology used (Gao et al., 2012). The TP and TA content of the Shani variety were lower than that of the other three mulberry varieties. The fermenting technology used for the mulberry wine fermented from these four mulberry varieties was the same; thus, the different TP and TA content observed in the resultant mulberry wines may have been primarily due to the different TP and TA content in the mulberry varieties. Li et al. (2009) studied the TP content of thirty-seven China wines (24 red wines, 11 white wines and 2 rosé wines) that were produced from different geographical origins. Their results showed that the TP content (expressed as GAE) ranged from 1402 to 3130 mg/l, which an average of 2068 mg/l, for the red wines, and from 189 to 495 mg/l, with an average of 302 mg/l, for the white wines; the values for the rosé wines were 741 and 1086 mg/l. Gao et al. (2012) reported that the TP content (expressed as GAE) of blackberry wines fermented by

traditional fermentation and carbonic maceration fermentation was 1647 and 2953 mg/l, respectively, and that their TA content (expressed as C3GE) was 6 and 8 mg/l, respectively. The TP content (expressed as GAE) of the mulberry wines that were fermented from the four mulberry varieties in our study varied from 3034.67 to 4681.33 mg/l. Compared with the wines studied by Li et al. (2009), the TP content of mulberry wine in our study was higher than red wines. Compared with the blackberry wine studied by Gao et al. (2012), the TP and TA content in our mulberry wines were higher. This finding may indicate that mulberry wine is rich in phenolics and anthocyanins.

3.2. Dynamic changes in TP, TF and TA content of mulberry wine during alcoholic fermentation Changes in the TP, TF and TA content of mulberry wine during alcoholic fermentation are presented in Fig. 1A–C. From Fig. 1A, it can be observed that the TP content of the four mulberry varieties increased from day 0 to day 3 and remained basically unchanged or slightly decreased from day 3 to day 10. From Fig. 1B, the change laws for the TF content of the four mulberry varieties during alcoholic fermentation were similar to those observed for the TP content. Our results concerning the changes in TP and TF content during alcoholic fermentation were consistent with the results of Di Egidio, Sinelli, Giovanelli, Moles, and Casiraghi (2010), who examined such changes in red wine during alcoholic fermentation. The time required for the TP and TF contents to reach their maximum was different, however, which was due to a difference in the alcoholic fermentation rate. Fig. 1C shows changes in the TA content of different mulberry varieties during alcoholic fermentation. It can be observed that the change laws of four mulberry varieties were consistent during alcoholic fermentation, which increased from day 0 to day 1, decreased from day 1 to day 7 and showed basically unobvious changes from day 7 to day 10. From Fig. 1C, it is obvious that the TA content of the Dashi variety was significantly higher than the other three varieties during alcoholic fermentation. It has been reported that some of the anthocyanins were transferred into the wine via skin maceration during wine fermentation; the extraction curve obtained for anthocyanins increased rapidly at the beginning, reached its maximum after 2 or 3 days, and then decreased slowly (Nagel & Wulf, 1979). Our result is in accordance with the change laws mentioned above. One explanation for our result presented in Fig. 1C may involve the following factors: the mulberry wines

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Journal of Functional Foods 18 (2015) 254–265

A 5000

Dashi Longsang

GAE ( mg /L)

4500

Hongguo Shani

4000 3500 3000 2500 2000 1500 1000 0

1

2

3

4

5

6

7

8

9

10

Fermentation time (d)

B 1400

Dashi Longsang

Hongguo Shani

CTE (mg/ L)

1200 1000 800 600 400 200 0

1

2

3

4

5

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7

8

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Fermentation time (d)

C

C3GE (mg/l)

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Dashi

Hongguo

1600

Longsang

Shani

1400 1200 1000 800 600 400 200 0 0

1

2

3

4 5 6 7 Fermentation time (d)

8

9

10

Fig. 1 – Changes in the TP (A), TF (B), and TA (C) content of mulberry wine fermented from four mulberry varieties during alcoholic fermentation.

Journal of Functional Foods 18 (2015) 254–265

fermented with skin from day 0 to day 7; the anthocyanins from the skin dissolved continuously from day 0 to day 7; the rate of anthocyanins dissolution was greater than their degradation from day 0 to day 1; the rate of anthocyanins degradation or anthocyanins conversion into other forms of anthocyanins was greater than the rate of anthocyanins dissolution from day 1 to day 7; and the TA content showed basically unobvious changes after skin removal, which occurred on day 7.

3.3. Dynamic changes in two main monomer anthocyanins content of mulberry wine during alcoholic fermentation Anthocyanins are the main substances that influence the colour of mulberry and mulberry wine. C3G and C3R are the major anthocyanins in mulberry (Dugo, Mondello, Errante, Zappia, & Dugo, 2001; Kamiloglu et al., 2013; Pawlowska, Oleszek, & Braca, 2008; Pérez-Gregorio, Regueiro, Alonso-González, Pastrana-Castro, & Simal-Gándara, 2011; S¸tefa˘nut¸, Ca˘ta, Pop, Mos¸oarca˘, & Zamfir, 2011; Zhang, He, Pan, Han, & Duan, 2011). HPLC chromatograms of C3G and C3R during mulberry wine fermentation are presented in Fig. 2A. From Fig. 2A, it can be observed that C3G and C3R showed a good separation. The standard curve equations of C3G and C3R were y = 50819x + 202941 and y = 49733x + 128282, respectively, where x represented the concentration of C3G or C3R and y represented the chromatographic peak area of C3G or C3R. The correlation coefficients (R2) were 0.9994 and 0.9990, respectively, which showed a good linear relationship. Changes in the two main monomer anthocyanin content of mulberry wine during alcoholic fermentation are shown in Fig. 2B and C. As Fig. 2B shows, the C3G content of four mulberry varieties increased rapidly from day 0 to day 1, reached its maximum at day 1, decreased rapidly from day 1 to day 5 and showed unobvious changes from day 6 to day 10. As shown in Fig. 2C, the C3R content of four mulberry varieties increased rapidly from day 0 to day 2, reached its maximum at day 2 and decreased gradually from day 2 to day 10. Compared with the Hongguo, Longsang and Shani, Dashi, which had a higher C3R content, decreased more obviously from day 2 to day 10. The rate of decline of the C3G content was greater than that of the C3R content after they reached their maxima. Compared with the C3R content, the effect of alcoholic fermentation was greater on the C3G content, which was in accordance with the finding of Lai (2013). However, the changes in the C3G and C3R content within the first two days were different from the results of Lai (2013). One explanation for this difference was that Lai used mulberry juice in the fermentation, whereas we fermented with skin for the first seven days. Medina, Boido, Dellacassa, and Carrau (2005) found that yeast interacted with the anthocyanins during wine fermentation and that the removal rate of polar compounds was thus higher. The degradation of C3G and C3R was faster during yeast growth, which produced a great amount of ethanol. Romero-Cascales, Fernández-Fernández, López-Roca, and Gómez-Plaza (2005) hypothesised that the degradation of anthocyanins had something to do with the enzymes produced by yeast during fermentation but that this degradation only occurred during the first few days of the fermentation process because yeast

259

and polyphenol oxidase (PPO) competed for the limited oxygen; thus, the anthocyanins did not change after day 7 primarily because PPO stopped functioning. Anthocyanins were released from grape skin into grape juice in the form of monomer anthocyanins during wine fermentation; however, these anthocyanins were not stable, and they gradually transformed into a more stable structure and formed pyran anthocyanins and polymeric pigments during wine fermentation and aging (Chinnici, Sonni, Natali, Galassi, & Riponi, 2009). We speculate that the reduced content of the two main monomer anthocyanins occurred because the monomer anthocyanins transformed into the more stable form.

3.4. Dynamic changes in colour parameters of mulberry wine during alcoholic fermentation and correlation analysis According to the OIV recommendation, colour value is measured using L*, a* and b*, which are set by CIE (Pérez-Caballero et al., 2003). This measuring method is called the CIELab method, and it achieves an objective evaluation of both wine colour and any colour changes by using three specific quality attributes, as determined by visual perception: tonality, luminosity and chroma. Although this method does not present an accurate definition of colour, it can effectively track changes in wine colour during wine fermentation. The CIELab threedimensional space includes luminosity (L*; L* = 0 represents black, and L* = 100 represents colourless), the red/green colour component (a*; a* > 0 is associated with red, and a* < 0 is associated with green) and the yellow/blue colour component (b*; b* > 0 is associated with yellow, and b* < 0 is associated with blue), from which the parameters correlated with colour perception are obtained, including chroma (C), hue angle (H) and saturation (S). Saturation is defined as C*/L* (C* = [(a*)2 + (b*)2]0.5) (Ayala, Echávarri, & Negueruela, 1997; Vázquez, Segade, & Fernández, 2010). The changes in L*, a*, b*, C* and H* of four mulberry varieties during alcoholic fermentation are presented in Fig. 3A–E. L* changed significantly in the first two days, showed unobvious changes from day 2 to day 7, and decreased slightly after skin removal at day 7. L* of the Dashi and Shani showed unobvious changes from day 0 to day 10, which may mean that the luminosity of Dashi and Shani showed unobvious changes. Compared with day 0, L* of Hongguo and Longsang showed an upward tendency, which may indicate that the luminosity of Hongguo and Longsang weakened. The changes in a* observed for all four varieties during alcoholic fermentation were consistent with each other. The a* increased from day 0 to day 2, showed unobvious changes from day 2 to day 7, and decreased slightly after skin removal at day 7. Compared with day 0, the overall a* of all four varieties increased, which may mean that the red was reinforced and may be related to skin maceration. The b* of all four varieties during alcoholic fermentation changed significantly in the first two days, showed unobvious changes from day 2 to day 7, and decreased slightly after skin removal at day 7. The changes in C* that were observed during alcoholic fermentation were similar to those observed for a*. The H* changed significantly in the first two days and showed unobvious changes after day 2. For a preliminary understanding of the changes that occurred in the CIELab parameters, Pearson significant correlation

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Journal of Functional Foods 18 (2015) 254–265

A

B

900 800 700

C3G (mg/l)

600 500 400 300 200

Dashi

Hongguo

Longsang

Shani

100 0 0

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Fermentation time (d)

C 1400 1200

Dashi Longsang

Hongguo Shani

C3R (mg/l)

1000 800 600 400 200 0 0

1

2

3

4 5 6 7 Fermentation time (d)

8

9

10

Fig. 2 – HPLC chromatogram of the C3G and C3R (A) and changes in the C3G (B) and C3R(C) content of mulberry wine fermented from four mulberry varieties during alcoholic fermentation.

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A

C 60

Dashi Longsang

Hongguo Shani

50

Dashi

Honguo

40

Longsang

Shani

40

30

30

20 b*

L*

50

10

20

0

10

-10

0

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4 5 6 Fermentation time (d)

7

8

9

10

-20

0 0

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5 6 7 Fermentation time (d)

8

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D

B Dashi Longsang

60

Hongguo Shani

55

70

Dashi

Hongguo

Longsang

Shani

65 50 a*

60 C*

45

55 50 45

40

40 35

35 0

1

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3

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5

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9

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10

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Fermentation time (d)

Fermentation time (d)

H*

E 50 45 40 35 30 25 20 15 10 5 0 0

1

2

3

4

5

Dashi

Hongguo

Longsang

Shani

6

7

8

9

10

Fermentation time (d)

Fig. 3 – Changes in the L* (A), a* (B), b* (C), C* (D) and H* (E) of mulberry wine fermented from four mulberry varieties during alcoholic fermentation.

analyses between TA, C3G, C3R, TP and CIELab parameters were evaluated. The results are shown in Table 3. Significant negative correlations (P < 0.01) between L* and TA (R = −0.475) and between H* and TA (R = −0.510) were found. A significant negative correlation (P < 0.05) between C* and TA (R = −0.309) was also found. These results showed that the higher the TA content was, the smaller L* and H* were; thus, the lower the luminosity was in mulberry wine, the smaller H* was and the closer

Table 3 – Pearson correlation analysis between TA, C3G, C3R, TP and CIELab parameters. Correlative coefficient

TA C3G C3R TP

L*

a*

b*

C*

H*

−0.475** −0.094 −0.335* −0.717**

−0.185 −0.017 −0.001 −0.237

−0.263 −0.127 −0.069 −0.208

−0.309* −0.076 −0.125 −0.306*

−0.510** −0.291 −0.340* −0.333*

** Correlation is significant at the 0.01 level, *Correlation is significant at the 0.05 level.

to a purple hue the mulberry wine was. Significant negative correlations (P < 0.05) between L* and C3R (R = −0.335) and between H* and C3R (R = −0.340) were found. These results showed that the higher the C3R content was, the smaller L* and H* were; thus, the lower the luminosity was in mulberry wine, the smaller H* was and the closer to a purple hue the mulberry wine was. A significant negative correlation (P < 0.01) between L* and TP (R = −0.717) was found. Significant negative correlations (P < 0.05) between C* and TP (R = −0.306) and between H* and TP (R = −0.333) were also found, which indicates that the TP content may influence L*, C* and H*. This may be due to copigmentation, which is similar to the copigmentation that occurs in red wine during alcoholic fermentation. Phenolic acids, flavanols, flavonols and other phenolics in wine are very good copigments. In addition, studies have found that the chroma of wine was closely related to the total phenolics and anthocyanins content in both grapes and wines (González-Neves et al., 2004). Cano-López, Pardo-Minguez, López-Roca, and Gómez-Plaza (2006) showed that chroma was not correlated with monomer anthocyanins and found a low correlation with anthocyanins

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Journal of Functional Foods 18 (2015) 254–265

A 9000

Dashi Longsang

8000

Hongguo Shani

Trolox (µM)

7000 6000 5000 4000 3000 2000 1000 0

1

2

3

4

5

6

7

8

9

10

Fermentation time (d)

B 0.7

Dashi Longsang

0.6

Hongguo Shani

A700

0.5 0.4 0.3 0.2 0.1 0.0 0

1

2

3

4

5

6

7

8

9

10

Fermentation time (d) Fig. 4 – Changes in DPPH· scavenging capacity (A) and PFRA (B) of mulberry wine fermented from four mulberry varieties during alcoholic fermentation.

connected to the ethane (R2 = 0.39) but a significant correlation (R2 = 0.96) between chroma and pyran anthocyanins using regression analysis. We speculate that the changes that occur in L*, a*, b*, C* and H* during alcoholic fermentation may be related to the acylation and pyran of anthocyanins, but the effect of such changes in the content of monomeric anthocyanins (C3G and C3R) on changes in L*, a*, b*, C* and H* is not obvious.

3.5. Dynamic changes in antioxidant activity of mulberry wine during alcoholic fermentation and correlation analysis 3.5.1. Changes in antioxidant activity of mulberry wine during alcoholic fermentation According to the roles that antioxidant activity plays in inhibiting lipid oxidation and degradation, scavenging free radicals, inhibiting pro-oxidant (such as chelating transition metal), and reducing power, the methods used for the determination of antioxidant activity in vitro can be classified into five categories, according to their basis: (1) lipid oxidation and degradation, (2) scavenging free radicals, (3) chelating transition metal to prevent generating free radicals, (4) determining the reducing power of samples, or (5) other methods (Tan & Lim, 2015).

Because there is no unified standard method for studying antioxidant activity, two or more methods based on different mechanisms are usually chosen to simultaneously explain the antioxidant activity of samples (Tan & Lim, 2015). The methods that are usually used to determine the antioxidant activity of wine are (2), (3) and (4), from the five categories mentioned above (Li et al., 2009; Meng et al., 2012). The methods used to determine the antioxidant activity of mulberry and mulberry products are often based on the free radical scavenging activity on DPPH· or ABTS and on the reducing power (Jiang et al., 2013; Kamiloglu et al., 2013; Shivashankara, Jalikop, & Roy, 2010; Tsai, Huang, & Huang, 2004). Because mulberry wine generally has a higher anthocyanin content and a deeper colour than wine, some methods for determining the antioxidant activity of wine are not suitable for mulberry wine. We chose methods based on the free radical scavenging activity on DPPH· and on the reducing power to study the changes in antioxidant activity of mulberry wine during the alcoholic fermentation. Studying the antioxidant activity of mulberry wine can provide a theoretical basis for a preliminary evaluation of antioxidant activities in vitro of mulberry wine. Fig. 4A and B present changes in the free radical scavenging activity on DPPH· and the reducing power of the four

Journal of Functional Foods 18 (2015) 254–265

Table 4 – Correlation between phenolic compounds and antioxidant activity. Correlative coefficient

TP TF TA C3G C3R

DPPH· scavenging capacity

PFRA

0.852** 0.821** 0.820** 0.143 0.958**

0.935** 0.941** 0.636** −0.190 0.872**

** Correlation is significant at the 0.01 level.

mulberry varieties during alcoholic fermentation. As Fig. 4 A and B show, the changes in the free radical scavenging activity on DPPH· and PFRA during alcoholic fermentation were consistent: they increased rapidly from day 0 to day 2 and showed unobvious changes from day 2 to day 10. Mulberry wine had a higher free radical scavenging activity on DPPH· and a higher reducing power, which illustrates its higher antioxidant activity in vitro, to a certain extent. The antioxidant activity in vivo of mulberry wine can be further studied to give us a more comprehensive understanding of the antioxidant activity of mulberry wine. The free radical scavenging activity on DPPH· of mulberry wine fermented with the four mulberry varieties at day 10 ranged from 4274.71 to 6543.40 µM Trolox equivalents. Sun et al. (2014) reported that the free radical scavenging activity on DPPH· of domestic red wines from Shaanxi Province ranged from 10822.73 to 19388.62 µM Trolox equivalents, those of white wine ranged from 505.28 to 603.15 µM Trolox equivalents, and rosé wine had an average of 5439.98 µM Trolox equivalents. Li et al. (2009) reported on the free radical scavenging activity on DPPH· of thirty-seven China wines that were produced from different geographical origins. For the free radical scavenging activity on DPPH·, the values ranged from 4190 to 21362 µM Trolox equivalents for the red wines, 82 to 1122 µM Trolox equivalents for the white wines and 1402 to 3410 µM Trolox equivalents for the rosé wines. Gao et al. (2012) reported the free radical scavenging activity on DPPH· of blackberry wine that was fermented by different methods. The free radical scavenging activity on DPPH· of blackberry wine fermented by traditional fermentation and carbonic maceration fermentation were 2.53 and 3.59 mM Trolox equivalents, respectively. The DPPH· scavenging activity of mulberry wine shown in our study approached the value of rosé wine from Shaanxi Province reported by Sun et al. (2014) and some of the red wines reported by Li et al. (2009). Additionally, it was higher than the blackberry wines studied by Gao et al. (2012). This result may indicate that the DPPH· scavenging activity of mulberry wine was close to that of some wines and that mulberry wine was easier to scavenge the DPPH· than blackberry wine.

3.5.2.

Correlation analysis of antioxidant activity

The results of a correlation analysis between the DPPH· scavenging activity and phenolic compounds and between PFRA and phenolic compounds are shown in Table 4. According to Table 4, there were significant correlations (P < 0.01) between DPPH· scavenging activity and TP, between DPPH· scavenging activity and TF, between DPPH· scavenging activity and TA, and between DPPH· scavenging activity and C3R (R = 0.852, R = 0.821,

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R = 0.820 and R = 0.958, respectively). There were also significant correlations (P < 0.01) between PFRA and TP (R = 0.935), between PFRA and TF (R = 0.941), between PFRA and TA (R = 0.636), and between PFRA and C3R (R = 0.872). However, the correlations between DPPH· scavenging activity and C3G and between PFRA and C3G were weak. This result may indicate that phenolics, flavonoids and C3R make a greater contribution to both the DPPH· scavenging activity and PFRA. The results of Li et al. (2009) showed that the TP and TF content of wines exhibited the strongest correlations with antioxidant properties but that TA exhibited weaker correlations. He, Sui, Du, and Lin (2013) found that hawthorn wine showed positive correlations between antioxidant activity (DPPH and FRAP) and TA, TP and TF and that the strongest correlation was between antioxidant activity and TP. Our results were basically consistent with some of the results mentioned above.

4.

Conclusions

Mulberry wine has a higher TP content, TA content and antioxidant activity in vitro, which may indicate that mulberry wine has higher biological activities. Furthermore, processing mulberry into fruit wine makes good use of mulberry, which is unfavourable for storage and transportation. Thus, mulberry wine exhibits a huge development space and market potential.

Acknowledgments We thank the National “Twelfth Five-Year” Plan for Science & Technology Support (2012BAD31B05) and Special Fund for Agroscientific Research in the Public Interest “Berries Storage & Integration and Demonstration of Processing Technology in Origin” (project number: 201303073-2) for the generous financial support provided for this work.

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