Journal Pre-proofs

Ultrasonic-assisted enzymatic extraction and identification of anthocyanin

components from mulberry wine residues

Lixia Zhang, Gongjian Fan, Muhammad Ammar Khan, Zheng Yan, Trust
Beta

PII: S0308-8146(20)30576-8
DOI: https://doi.org/10.1016/j.foodchem.2020.126714
Reference: FOCH 126714

To appear in: Food Chemistry

Received Date: 5 January 2019

Revised Date: 24 March 2020
Accepted Date: 29 March 2020

Please cite this article as: Zhang, L., Fan, G., Ammar Khan, M., Yan, Z., Beta, T., Ultrasonic-assisted enzymatic
extraction and identification of anthocyanin components from mulberry wine residues, Food Chemistry (2020),
doi: https://doi.org/10.1016/j.foodchem.2020.126714

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 1 Ultrasonic-assisted enzymatic extraction and identification of anthocyanin

2 components from mulberry wine residues

4 Lixia Zhang 1*, Gongjian Fan2, Muhammad Ammar Khan3, Zheng Yan1, and Trust Beta4

6 1. Research Institute of Agricultural Product Processing, Jiangsu Academy of Agricultural

7 Sciences, Nanjing 210014, Jiangsu, China

8 2. College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing

9 210037, Jiangsu, China

10 3. Department of Food Science & Technology, University College of Agriculture &

11 Environmental Sciences, The Islamia University of Bahawalpur, Pakistan

12 4. Department of Food & Human Nutritional Sciences, University of Manitoba, Winnipeg,

13 Manitoba, Canada R3T 2N2

14 Corresponding author1: Dr. Lixia Zhang, Associate Professor

15 Tel: 86-25-84391571 Fax: 86-25-84391677

16 Address: Research Institute of Agricultural Product Processing, Jiangsu Academy of Agricultural

17 Sciences, Zhong Lingjie road No. 50, Nanjing, 210014, Jiangsu, P. R. China.

18 E-mail: zlxluck@aliyun.com

19 Corresponding author2: Trust Beta, Certified Food Scientist,Canada Research Chair in

20 Functional Foods, Professor in Food Science

21 Address: Department of Food & Human Nutritional Sciences, 250 Ellis Building,Winnipeg MB

22 ,Canada R3T 2N2

23 E-mail: Trust.Beta@umanitoba.ca
1
24 Abstract

25 Mulberry wine residues produced during the wine-brewing process contain several anthocyanins

26 and other bioactive compounds. Therefore this study optimized the conditions for

27 ultrasound-assisted enzymatic extraction of anthocyanins from mulberry wine residues. A

28 three-level, four-factor Box–Behnken design was used to optimize the extraction conditions.

29 Moreover, anthocyanins were determined using an ultra-performance liquid chromatograph

30 coupled to a mass spectrometer (UPLC-MS). The mathematical model suggested a high

31 coefficient of determination (R2 = 0.9475) for the optimum conditions, namely 52°C, 315W,

32 0.22% enzyme and 94 min incubation. The yield (5.98 mg/g) was close to the predicted value

33 (5.87 mg/g). The two anthocyanins (cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside)

34 identified are consistent with those present in mulberry. The optimized conditions increased

35 anthocyanin yield, through improved utilization of mulberry wine residues. The findings will

36 potentially lead to a reduction in the environmental burden of this waste and improve the

37 efficiency and productivity of the mulberry fruit processing industry.

38

39

40 Keywords: Mulberry wine residues; Anthocyanins; Ultrasonic-assisted enzymatic extraction;

41 Box–Behnken design; UPLC-MS

42

43

44

45

2
46 1. Introduction

47 Mulberry is an excellent source of bioactive compounds, including anthocyanins, flavanols

48 and phenolic acids (Li, Bao, & Chen, 2018; Liu, Wu, Fan, Li, Ying, & Miao, 2017; Wang, Sun,

49 Li, Yu, Liu, Huang, et al., 2015). The antioxidant capabilities of these compounds have gained

50 much interest due to such functional properties (Swer, Mukhim, Bashir, & Chauhan, 2018;

51 Tomas, Toydemir, Boyacioglu, Hall, Beekwilder, & Capanoglu, 2015; Wu, Qi, Liu, Guo, Zhu,

52 Chen, et al., 2013). Consumption of foods enriched in these compounds might be associated with

53 reduced risk of ageing, cancer and cardiovascular disease and improved immune function and

54 glucose control (Huang, Chang, Wu, Hung, & Wang, 2011; Kumar & Mandal, 2017; Li, Bao, &

55 Chen, 2018; Peng, Lin, Chung, Huang, & Wang, 2018). Mulberries are suitable for processing

56 into mulberry wine, mulberry juice and mulberry jam because of their nutritious value, excellent

57 taste and characteristic flavour (Liu, Wu, Fan, Li, Ying, & Miao, 2017; Machado, Pereira,

58 Barbero, & Martínez, 2017; Wang, et al., 2015). Anthocyanins are abundantly present in the skin

59 of mulberries (Kim, Bartley, & Rimando, 2010), therefore, the wine residues produced in the

60 wine-brewing process would contain these and other bioactive compounds (He, Zhang, Yue,

61 Liang, Jiang, Gao, et al., 2016; Rubio, Maciel, da Silva, Corrêa, Peralta, & Haminiuk, 2018).

62 However, these organic residues are often discarded or used as animal feed, which results in a

63 cost increase and most likely, a negative environmental impact. This study was designed to

64 optimize the technological conditions to increase anthocyanin recovery from the residues of

65 mulberry wine so as to enhance profitability if utilized for functional food purposes.

66 Anthocyanin extraction from berry residues or wine pomace using maceration,

67 enzyme-aided (Swer, Mukhim, Bashir, & Chauhan, 2018), microwave-assisted (Koyu, Kazan,

68 Demir, Haznedaroglu, & Yesil-Celiktas, 2018) and ultrasonic-assisted (He, et al., 2016) was

3
69 previously reported. Ultrasound-assisted enzymatic extraction method was used to extract

70 polysaccharides from Chinese herb medicines, such as Corbicula fluminea (Liao, Zhong, Ye, Lu,

71 Wang, Zhang, et al., 2015). However, ultrasonic-assisted enzymatic extraction is yet to be

72 reported for mulberry residues. Therefore, this technique was investigated for optimum

73 extraction of anthocyanins from mulberry wine residues so as to achieve maximum yield.

74 The extraction, separation, purification and stability of anthocyanin from mulberry fruit

75 were previously reported (Espada-Bellido, Ferreiro-González, Carrera, Palma, Barroso, &

76 Barbero, 2017; Tchabo, Ma, Engmann, & Zhang, 2015; X. Wu, Liang, Zou, Zhao, Zhao, Li, et

77 al., 2011), but there is no literature on the extraction of anthocyanins from mulberry wine

78 residues. The separation and purification of anthocyanins using column chromatography,

79 high-performance liquid chromatography (HPLC) and high-speed counter-current

80 chromatography were previously reported (Lee & Hwang, 2017; X. Wu, et al., 2011). Fourteen

81 anthocyanins with uneven distributions and dissimilar levels were identified in the extracts of

82 mulberry (Morus alba L.) fruits grown in Vojvodina, North Serbia (Natic, Dabic, Papetti, Aksic,

83 Ognjanov, Ljubojevic, et al., 2015). Previously, the isolation of anthocyanins from black rice and

84 the elucidation of their stability (Yan, Dai, & Zheng, 2016) and blackberry juice (Zhang, Zhou,

85 Liu, Khan, Huang, & Gu, 2012) were reported. However, ultrasonic-assisted extraction and

86 characterisation of anthocyanins from mulberry wine residue have not yet been reported. Several

87 statistical designs, including response surface methodology (RSM), polynomial regression and

88 probabilistic design can enhance the output, process efficiency and simulation modelling. RSM

89 is used as a popular simulation modelling tool for the prediction of process conditions for

90 scientific studies and industrial processes. The Box–Behnken design is an experimental design

91 for RSM that can be used for optimisation of process parameters to improve yield, recovery and

4
 92 profitability. Hence, this study used the Box–Behnken design for the optimisation of anthocyanin

93 extraction from mulberry wine residues.

94 The objective of this work was to optimize the extraction process of total anthocyanins

95 from mulberry wine residue via ultrasonic-assisted pectinase and pectinase compound enzymes.

96 2. Materials and methods

97 2.1 Experimental materials

98 Pre-treatment of mulberry wine residues was performed as described in the literature

99 (Bonfigli, Godoy, Reinheimer, & Scenna, 2017) with some modifications. The squeezed

100 mulberry wine residues weighing 50kg was purchased from Jurong Dongfang Zijiu Liquor Co.,

101 Ltd. (Jurong, Jiangsu Province, China), placed into a plastic container, and then transported to

102 the laboratory within one and a half hours in November 2017. The wine residues were dried for

103 24 h at 50 °C until the moisture content was <5%. The dried residues were then stored at −18 °C

104 for 24 h before being crushed. The frozen samples were thawed, crushed into powder (include

105 screen size used or information on particle size distribution) with an electric mill (JYS-M01,

106 Jiuyang Co., Ltd, Jinan, Shandong Province, China), sealed in a brown glass container to avoid

107 light and stored at 4 °C for future use.

108 2.2 Reagents and solvents

109 Hydrochloric acid (HCl), anhydrous ethanol, disodium hydrogen phosphate, monosodium

110 phosphate, potassium chloride (KCl) and citric acid of analytical purity were purchased from

111 China Pharmaceutical Group Chemical Reagent Co., Ltd (Nanjing, Jiangsu Province, China).

112 Pectinase and pectin complex enzyme were purchased from He Shi Bi Biotechnology Co., Ltd

113 (Yinchuan, Ningxia Province, China). Pectinase was produced by Aspergillus niger, with an

114 enzyme activity of 1.0×105–6.0×105 U/g. The pectin complex enzyme was produced from the

115 three strains of Aspergillus niger, Trichoderma and Bacillus subtilis, with an enzyme activity of

116 1.0×105–6.0×105 U/g. Macro-porous resin HPD700 was obtained from the Bohong Resin

117 Technology Co., Ltd. (Tianjin, Province, China) and the Sephadex LH-20 was bought from
5
118 Sigma-Aldrich (St. Louis, Missouri, USA).

119 2.3 Ultrasound-assisted enzymatic extraction

120 Ultrasound-assisted extraction of anthocyanins was carried out following a previous report

121 with some modifications (He, et al., 2016). The stock mulberry wine residues were mixed with

122 distilled water acidified to pH 3.5 at a ratio of 1:20. The mixture was rehydrated for 30 min. The

123 pectinase and pectin complex enzyme (what levels? How much was added?) were then added

124 into the mixture. Ultrasonic-assisted extraction was performed using an ultrasonic apparatus

125 (KQ-2500E type ultrasonic apparatus, Kunshan Hachuang Instrument Co., Ltd. Kunshan,

126 Jiangsu Province, China). Thereafter, the extracted samples were centrifuged at 2680 g for 20

127 min at room temperature (XYJ-2 type high speed centrifuge, Jiangsu Jintan Hengfeng Instrument

128 Manufacturing Co. Ltd. (Jintan, Jiangsu Province, China) to obtain the anthocyanin extracts. The

129 extracts were concentrated using a vacuum rotary evaporator at 40 °C (RE-600 type rotary

130 evaporator, Shanghai Yarong Biochemistry Instrument Factory, Shanghai, China) and this was

131 followed by freeze-drying for 24 h using a vacuum freeze dryer (Shanghai Bi Lang Instrument

132 Manufacturing Co., Ltd. Shanghai, China). After drying, the crude anthocyanin powder was

133 stored at 4 °C in a brown glass container.

134 2.4 Determination of total anthocyanins content

135 Total anthocyanins content was determined based on a pH differential method as

136 previously described (Tchabo, Ma, Engmann, & Zhang, 2015) with a UV/Vis spectrophotometer

137 (Shanghai Precision Instrument Co. Ltd., Shanghai, China) using two buffers, namely pH 1.0

138 buffer and pH 4.5 buffer. The pH 1.0 buffer was prepared by mixing 0.2 M KCl and 0.2 N HCl

139 solutions (0.2 mol/L KCl:0.2 mol/L HCl=25:67 (v/v)). Buffer at pH 4.5 was prepared by mixing

140 CH3COONa and 1.0 N HCL solutions (1 mol/L NaAc:1 mol/L HCl: H2O=100:60:90 (v/v/v)),

141 and pH was adjusted using acetic acid. A 0.1 ml aliquot of the anthocyanin solution was

6
142 transferred to a 10 ml volumetric flask in duplicated flasks and the volume made up with either

143 pH 1.0 buffer or pH 4.0 buffer. Each of these flasks was stored away from light for 2 h at 25 °C.

144 The absorbance of the solutions was then detected at 520 (λmax) and 700 nm using UV-vis

145 spectrophotometer detection. Total anthocyanins were calculated as cyanidin-3-O-glucoside

146 according to the following equation:

147

148 Where A = (A520–A700) pH 1.0 – (A520–A700) pH 4.5; MW is the molecular weight of

149 cyanidin-3-O-glucoside, 449.2 g/mol; V is the volume of solution, mL; DF is the dilution factor;

150 ε is the molar extinction coefficient of cyanidin-3-O-glucoside, 26,900 L/mol*cm; L is the

151 optical path, 1cm, and m is the mass of dry residue. The results were expressed in mg/g.

152 2.5. Single-factor experiments for total anthocyanin extraction

153 To investigate the influence of extraction temperature, ultrasonic power, enzyme dosage

154 and extraction time on anthocyanin yield, single-factor experiments were performed. The

155 optimal conditions for each parameter were determined according to the highest total

156 anthocyanin content, expressed as mg cyanidin-3-O-glucoside equivalents/g of dried mulberry

157 residue weight, and were used for all the subsequent optimisation experiments. For the

158 single-factor experiments, the levels of ultrasonic power ranged from 100 W to 500 W, the

159 extraction temperature varied from 20 °C to 60 °C, the enzyme dosage ranged from 0.05% to

160 0.25% and the extraction time varied from 30 min to 150 min. Each single factor experiment was

161 repeated three times and the average values recorded.

162 2.6. Optimisation of total anthocyanin extraction

163 The relationship between the extraction conditions (extraction temperature, ultrasonic

164 power, enzyme dosage and extraction time) and the total anthocyanin yields (Y) was investigated

165 using a three-level, four-factor Box–Behnken design to determine the combined effect of various

7
166 variables for optimisation. The four independent variables and the corresponding levels were as

167 follows: X1, temperature (°C): 40 °C, 50 °C, 60 °C; X2: power of ultrasonic, (W): 200, 300, 400;

168 X3, dosage of enzyme (%): 0.15%, 0.20%, 0.25%; X4, time (min): 60, 90, 120. The four

169 independent variables were coded at three levels (1, 0, +1) (Table 1). Levels of total

170 anthocyanins (Y) were considered as the response variables, which were substituted into the

171 following second order polynomial model equation (Eq. (1)) which describes the relationship

172 between the response and the independent variables:

173

174

175 where Y is the response variable (total anthocyanins); Xi and Xj are independent variables.

176 is the constant coefficient; i is the linear coefficient; is the quadratic coefficient;

177 is the cross-product coefficient, is the number of variables and i is an integer with values

178 starting from 1.

179 2.7. Purification of anthocyanin in mulberry wine residue

180 The crude extracts were pre-purified to remove sugars and salts to obtain the anthocyanin

181 fraction according to a procedure previously reported (Xiao, Fang, Niu, & Yu, 2015). The

182 anthocyanin crude extract was dissolved in 5 mL 0.1% (v/v) acetic acid aqueous solution. The

183 total? aqueous solution (50 mL?? see above you have 5 mL) was loaded onto a column (4.5

184 cm×40 cm) of HPD700 macro-porous resin. The column was washed with 2 L of deionized

185 water at a flow rate of 1 mL/min to remove the majority of proteins, sugars, organic acids, and

186 ions. Elution of anthocyanins was then performed using 1 L of 0.1% acetic acid-acidified 80%

187 (v/v) ethanol at 1.5 mL/min. The eluate (around 500 mL) was collected on the basis of the color

188 band and UV−vis detector at 520 nm. The eluate was concentrated at 45 °C with a rotary

8
189 evaporator. The concentrate was loaded onto a column (2.5 cm×120 cm) of Sephadex LH-20 gel

190 column and eluted twice using methanol, distilled water and formic acid. The elution peaks were

191 concentrated at 45 °C and freeze-dried to obtain the purified anthocyanin samples.

192 2.8. Identification of anthocyanins using UPLC-MS

193 Sep-Pak-C18 solid phase microextraction column and Ultra Performance Liquid

194 Chromatography (UPLC) were performed according amethod reported previously (Bornsek,

195 Ziberna, Polak, Vanzo, Ulrih, Abram, et al., 2012) to obtain the major monomeric anthocyanins

196 with high purity from the Sephadex LH-20 column fractions. The anthocyanin fractions were

197 pooled, dried and resolubilised in 5 mL of 5% formic acid. They were then loaded on a new

198 Sep-Pak C18 solid phase microextraction column, previously activated with 5 mL methanol

199 containing 0.01% HCl and 5 mL acidified water containing 0.01% HCl. After washing with 10

200 mL 0.01% HCl, the anthocyanins were eluted with 10 mL 0.01% HCl in methanol. The

201 anthocyanin fraction was dried in rotavapor, resolubilised in 1 mL pure methanol, passed

202 through 0.45 μm filter membrane and stored at −18 °C until it was analysed.

203 UPLC analysis was carried out using an UPLC apparatus equipped with a Waters

204 Acquity PAD detector and a BEH C18 column (2.1  100 mm2; particle size, 1.7 m) (Waters,

205 Milford, Massachusetts, USA), according to our previous report ( Zhang, Zhou, Liu, Khan,

206 Huang, & Gu, 2012). Two solvents were used in the mobile phase: solvent A [aqueous 0.1% (v/v)

207 formic acid] and solvent B (100% HPLC-grade acetonitrile). The gradients were set as follows:

208 0–15 min with 25% B, 15–20 min with 45% B, 20–30 min with 60% B, 35 min with 90% B and

209 40 min with 5% B, at a constant flow rate of 0.5 mL/min. The peaks of the anthocyanin

210 compounds were monitored at 520 nm. UV/Vis absorption spectra were recorded from 200 nm

211 to 700 nm. Column temperature was set at 45 °C. The entire elution time was 40 min, and the

212 injected volume was 10 L.

213 Using the same method with UPLC, the purified anthocyanin samples were detected on a

214 SYNAPT Mass Spectrometry System (Waters 2690, Waters, Milford, Massachusetts, USA)

9
215 equipped with an electrospray ionisation source operating in the positive mode of polarity

216 (Mustafa, Yixiao, Re?At, Finley, & Zhimin, 2013). Data acquisition and processing were

217 performed using Esquire Control. ESI conditions include a capillary voltage of 3.0 kV for the

218 positive mode, a cone voltage of 20 V, an ion guide at 1 V and a source temperature of 100 C.

219 The nitrogen desolation gas temperature was 250 C and its flow rate was 500 L/h.

220 Collision-induced dissociation was performed with a fragmentation amplitude of 1.0 V using

221 helium as a collision gas. Mass spectra were recorded with a heat capillary voltage of 5 kV, a

222 heat capillary temperature of 275 C, a sheath gas flow rate of 80 U and an auxiliary gas flow

223 rate of 20 U. The scan range for the molecular weights was from 200 Da to 1500 Da.

224 2.9 Statistical analysis

225 All experiments were carried out in triplicates. Results were presented as means ±

226 standard deviation (n=3). The differences among factors were determined using ANOVA,

227 followed by Tukey multiple comparison tests. Statistical significance was defined at a level of

228 p<0.05. Response surface of Design-Expert 8.0.1 Trial (Stat-Ease Inc., Minneapolis, MN, USA)

229 was used for data analysis.

230 3. Results and discussion

231 3.1 Single factor analysis of total anthocyanin extraction

232 Experiments were designed to evaluate the effects of each single factor on the extraction

233 yield of the total anthocyanins (Fig. 1). In the first single-factor analysis, total anthocyanin yield

234 increased gradually with increasing extraction temperatures from 30 °C to 50 °C (Fig. 1A);

235 however, further increase in temperature decreased the yield. The decrease in yield is attributed

236 to poor heat-resistance of the anthocyanins (Mustafa, Yixiao, Apak, R, Finley, & Zhimin, 2013),

237 which underwent degradation at high temperature. Hence, the extraction temperature was set at

238 50 °C for the next single-factor experiments. In the second single-factor analysis, total

239 anthocyanin yield increased with increasing ultrasonic power (Fig. 1B) up to a maximum of 4.65

240 mg/g for 300 W; however, further increase in ultrasonic power decreased the yield. Initially, the

10
241 ultrasonic power assisted the formation of numerous gas bubbles in the solvent, which imploded

242 rapidly, accelerating the bursting of cell walls and increasing the seepage of cellular constituents

243 (Kauer, Belova-Magri, Cairós, Linka, & Mettin, 2018), and therefore improving the extraction

244 efficiency. However, excessive heat generation occurred in the reaction system when ultrasonic

245 power increased above 300 W, contributing to the degradation of the mulberry anthocyanins

246 (Espada-Bellido, Ferreiro-González, Carrera, Palma, Barroso, & Barbero, 2017) and resultant

247 low extraction yield. Based on these observations, ultrasonic power was set at 300 W for further

248 experiments. In the third single-factor experiment, increasing the extraction time initially

249 increased the extraction yield until 90 min, followed by a decrease (Fig. 1C). Theoretically,

250 increase in extraction time ensured sufficient contact between the substance and the extracting

251 agent but also contributed to oxidative degradation (Ryu & Koh, 2018). After 90 min, the

252 reaction equilibrium shifted towards oxidative degradation, thus decreasing the total anthocyanin

253 yield. The extraction time for further experiments was set at 90 min.

254 Finally, increasing the enzymatic dosage enhanced the total anthocyanin yield up to 0.2%,

255 but further increase in enzyme concentration did not change the yield (Fig. 1D). A high pectin

256 concentration contributed to high enzymolysis in the mulberry wine residues, which accelerated

257 the dissolution of anthocyanins. Thus, the target dosage of enzymes was set at 0.2% for further

258 analysis.

259 3.2 Establishment of regression equation and analysis of variance

260 In the present study, a four-factor, three-level Box–Behnken design was employed to

261 determine the optimal ultrasonic-assisted enzymatic extraction conditions to obtain the highest

262 anthocyanin yield from mulberry wine residues. The experimental design matrix is shown in

263 Table 1. Taking anthocyanin yield as a response value (Y), ANOVA was conducted in

264 Design-expert 8.0.1 software (Stat-Ease Inc., Minneapolis, MN, USA) to perform quadratic

265 regression analysis on the experimental data in Table 2. The coefficients of the equation were

266 calculated, and the variance analysis was carried out. The regression equation (model) between

11
267 the four factors and Y was obtained as follows (Eq. 3):

268
269 where, Y is the yield of anthocyanins; X1, X2, X3 and X4 are the coded variables for the

270 extraction temperature, extraction power of ultrasonic, dosage of enzyme and extraction time,

271 respectively.

272 As shown in Table 2, the F-value and p-value of the model were 24.60 and 0.0001,

273 respectively, which suggest that the model was significant. Furthermore, the determination

274 coefficient (R2 =0.9475) for this model was close to 1, which indicated that the F test was not

275 significant (p>0.05), and thus, an effective correlation existed between the predicted values and

276 the actual ones. The p-value of lack of fit was 0.5223 (p>0.05), indicating that the lack of fit was

277 insignificant compared with the pure error. The findings revealed that the regression equation

278 was fitting the model very well; hence, it could be used to analyse the experimental results.

279 The coefficients of X1, X2 and X3 (A1, A2, A3) were significant (p<0.05), indicating that the

280 effects of extraction temperature, ultrasonic power and enzyme dosage were significant to the

281 extraction rate of anthocyanin from mulberry wine residue (p<0.01). By contrast, the coefficients

282 of X4 (A4) were not significant, suggesting that the effect of extraction time was insignificant to

283 the extraction rate (p>0.05). The quadratic terms A12, A22 and A32 and the interactive terms A1A2

284 and A3A4 were significant (p<0.01), but the other coefficients were non-significant (p> 0.05).

285 These results revealed a clear, quadratic relationship of the three factors with the extraction rate

286 of anthocyanins. The “Numerical analysis” in the “optimisation program” software indicated that

287 the factor conditions corresponding to the maximum response values could be obtained. The

288 optimum extraction conditions of the total anthocyanins from mulberry wine residues were

289 found to be as follows: ultrasonic power (315 W), enzyme dosage (0.22%), temperature (52 ℃)

290 and extraction time (94 min). The predicted extraction rate of the total anthocyanins under these

291 conditions is 6.03 mg/g.

12
292 3.3 Optimisation of interactions between factors of response surface

293 The response surface plots for the interactions of the four independent variables

294 (extraction parameters) with extraction yields of total anthocyanins are shown in Fig. 2. The

295 interaction of temperature and ultrasonic power for the total anthocyanin yield was investigated (Fig.

296 2A). Enzyme dosage and extraction time were kept constant. Results revealed that at the same

297 temperatures, increasing ultrasonic power level initially increased the yield of total anthocyanins

298 in the mulberry wine residues, followed by a decline. Similarly, an increase in temperature at the

299 same ultrasonic power levels initially increased the yield slowly, followed by a decline after

300 reaching the maximum. These findings suggested an antagonistic relation between these

301 parameters and indicated a quadratic function. Extraction temperatures exceeding 52 °C and

302 ultrasonic power exceeding 315 W instigated a rapid decline in total anthocyanin yield as a result

303 of temperature-assisted degradation (He, et al., 2016). The interaction between ultrasonic power

304 and enzyme dosage for the total anthocyanin yield at constant temperature and extraction time (Fig.

305 2 B) was also significant (p<0.05). At the same enzyme dosages, raising the level of ultrasonic

306 power initially increased the yield of total anthocyanins which latter declined. The decline was

307 rapid above 315 W due to temperature-assisted degradation (López, Caleja, Prieto, Barreiro,

308 Barros, & Ferreira, 2018). Finally, the interaction of extraction time and enzyme dosage on the

309 yield of total anthocyanins at constant extraction temperature and ultrasonic power (Fig. 2C) was

310 also significant (p<0.01). At the same extraction times, increasing enzymatic dosage initially

311 increased the yield of total anthocyanins which declined latter. The maximum yield was

312 achieved at the 0.22% enzyme concentration. Furthermore, the yield declined at extraction times

313 beyond 94 min likely due to increased oxidative degradation (Chen, Zhao, & Yu, 2015).

314 3.4 Model optimisation and verification

315 The extraction conditions were optimized using Design Expert 8.0.6 (Stat-Ease Inc.,

316 Minneapolis, MN, USA) as follows: extraction temperature (52 °C), ultrasonic power (315 W),

317 enzymatic dosage (0.22%) and extraction time (94 min). The predicted yield was 6.03 mg/g.

13
318 Three parallel experiments were performed based on optimum conditions, for which the total

319 anthocyanin yield was 5.98 mg/g, with a deviation of 1.9% from the prediction. These findings

320 suggest that the Box–Behnken model successfully optimized the anthocyanin extraction from the

321 mulberry wine residues, with an accurate and reliable prediction.

322 3.5 Analysis of anthocyanin composition using UPLC–MS

323 Finally, individual anthocyanins from the mulberry wine residue extractions were

324 identified using UPLC–MS (Fig. 3). UPLC chromatographs (Fig. 3A) confirmed the presence of

325 two anthocyanins in the final extracts, with retention times of 10.26 and 10.89 min for peaks 1

326 and 2, respectively. Anthocyanins showed maximum absorption at 500–540 nm in visible light

327 and at 280 nm in UV (Escribano-Bailón, Santos-Buelga, & Rivas-Gonzalo, 2004).

328 Spectrophotometric information, including retention times, λmax, molecular ion peaks and main

329 fragment results revealed that the anthocyanins extracted from the mulberry wine residues were

330 of two types (Table 3). Mulberry anthocyanins are mainly present in the form of cyanidin

331 glucosides (Jin, Yang, Ma, Cai, & Li, 2015). MS results showed that the peak 1 displayed strong

332 signal for a molecular ion peak having a mass-to-charge ratio (m/z) of 449, combined with

333 fragment ion having an m/z of 287 with λmax of 280 nm (Alcalde-Eon, Escribano-Bailón,

334 Santos-Buelga, & Rivas-Gonzalo, 2003), which represents cyanidin. The loss of neutral fragment

335 162 is consistent with the molecular weight of hexose. Previous studies reported that the

336 glucoside of anthocyanin has a connection with hydroxyl normally at the C-3 position, at C-5 or

337 C-7 in the presence of numerous glucosidic bonds but hardly at the position of C-3′ or C-5′.

338 These results indicated that peak 1 was cyanidin-3-O-glucoside (Escribano-Bailón,

339 Santos-Buelga, & Rivas-Gonzalo, 2004). In addition, peak 2 (Fig. 3C) indicated a molecular ion

340 peak with an m/z of 595, and fragment ion having an m/z of 287. The fragment ion originated

341 from the m/z of 595 following loss of a neutral fragment of molecular weight 308. The

342 component was indicated as cyanidin taking into account m/z of 287 and wavelength of

343 maximum absorption at 518 nm. The molecular weight of 308, equivalent to the molecular

14
344 weight of cyanidin-di-glycosides was due to the loss of two glycosides. It was similar to peak 1,

345 with connection at position C-3 when only one glycoside is present. The molecular ion peak and

346 fragment ion of peak 2, in accordance with literature, confirmed the component as

347 cyanidin-3-O-rutinoside (Kumar & Mandal, 2017; Wang, et al., 2015; Zhang, Shao, Bao, &

348 Beta, 2015). The two compounds, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside in the

349 mulberry wine residue extract are consistent with the main anthocyanin structures previously

350 reported in the mulberry fruit (Liang, Wu, Zhao, Zhao, Li, Zou, et al., 2012). Isolating and

351 purifying anthocyanins using only one method is challenging as anthocyanin crude extracts

352 contain several non-anthocyanin phenolic substances. Hence, the macro-porous adsorption resin

353 and Sephadex LH-20 gel column were used to remove these impurities from mulberry wine

354 residues in this study. Initially, anthocyanins and other phenolic compounds were adsorbed on

355 macro-porous resin, through which organic acids, proteins, sugar and salts were removed. In the

356 next step, impurities, such as flavanones and phenolic acids, were further removed using

357 Sephadex LH-20 gel column. Jin et al. (2015) previously isolated cyanidin-3-O-glucoside,

358 cyanidin-3-O-rutinoside, pelargonidin-3-O-glucoside and pelargonidin 3-O-rutinoside from the

359 mulberry fruit. Espada-Bellido et al. (2017) isolated and purified cyanidin-3-O-glucoside,

360 cyanidin-3-O-rutinoside, cyanidin-3-O-(6-malonyl-glucoside) and

361 cyanidin-3-O-(6″-dioxalyl-glucoside) as the main anthocyanin components in the mulberry fruit

362 Geranidin-3-O-glucoside, geranidin-3-O-galactoside, geranidin-3-O-rutin and

363 delphinidin-3-O-rutinoside were also detected in mulberry fruit (Hosseini, Akramian, Khadivi, &

364 Salehi-Arjmand, 2018; Lee & Hwang, 2017; Liu, Wu, Fan, Li, Ying, & Miao, 2017). In this

365 research, the main components of extracts from the residue of mulberry wine were determined as

366 cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside. Variation in anthocyanin levels in

367 mulberry wine residue could results from differences mulberry species and planting area.

368 4. Conclusion

369 Anthocyanins in mulberry wine residues were extracted via ultrasonic-assisted enzymatic

15
370 extraction. A three-level, four-factor Box–Behnken design was successfully applied to optimize

371 the parameters for the extraction of total anthocyanins. The extraction yield was optimized using

372 an RSM based on four single-factor experiments, and a quadratic extraction model was

373 established. The mathematical model showed a high coefficient of determination against the

374 optimum conditions. The optimum conditions for extraction include extraction temperature of 52

375 ℃, ultrasonic power of 315 W, enzyme dosage of 0.22% and extraction time of 94 min. The

376 optimum yield (5.98 mg/g) was close to the predicted yield (6.03 mg/g). UPLC-MS indicate that

377 cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside are the two main anthocyanins in mulberry

378 wine residues, which is in agreement with the structures of anthocyanins in mulberry fruits.

379 Finally, our finding indicates that ultrasonic-assisted enzymatic extraction of the desired

380 anthocyanin components from mulberry industry residues is efficient, economical and

381 environmentally friendly.

382
383 Acknowledgements

384 We thank Professor Xueming Xu and Guanjun Tao for their contributions to the sample

385 analyses. This study was funded by the National Natural Science Foundation of China

386 (31401489) and Key R & D projects of the Jiangsu Science and Technology Department

387 (BE2018374).

16
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503

21
505
506 Fig. 1

507

508
A B
509

510

511

512

513

514

515
C D
516

517

518

519

520

521

522

523 Fig. 1 Effects of ranges in extraction temperature of 20 °C to 60 °C (A), ultrasonic power of

524 100–500 W(B), enzyme dosage of 0.05%–0.25% (C) and extraction time of 30–150 min (D)

525 on total anthocyanin yield in single-factor experiments.

526

22
528

529 Fig. 2

B
A

530

531 Fig. 2 Response surface analysis for the extraction variables on total anthocyanin content.

532 A.Total anthocyanin content versus ultrasonic power and extraction temperatures. B. Total

533 anthocyanin content versus ultrasonic power and enzyme dosage. C. Total anthocyanin

534 contents versus enzyme addition and extraction time.

23
535

536 Fig. 3 SG2

SG2
20110616-15 592 (10.302)
20110616-15 2: TOF MSArray
3: Diode ES+
287.0 10.89 1.72e4
520
537 100 A Range: 1.105
10.26

8.0e-1

6.0e-1
%
AU

4.0e-1
288.0 449.1

289.1 450.1
2.0e-1
0 SG2
0.0
630 (10.963)4.00
20110616-152.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 2: TOF MS ES+
20.00
20110616-15 592 (10.293)
20110616-15
287.0 1:1:TOF
TOF MS ES+1.48e4
MS ES+
100 B 449.1 10.29 TIC
1.29e4
100 7.98 10.92 2.53e4

287.0 595.1
15.05
8.24 9.69
%
%
%

288.0 450.1 596.1

8.96 12.12 12.53
288.0 5.98 13.37
0.81 0.890.96 289.15.34355.06.85 7.10 451.1 597.2 15.33 17.09
1.26 2.25 2.70
4.30 17.71 19.05 19.56
00 m/s m/z
8 Time
100 200
2.00 300
4.00 6.00400 8.00500 10.00600 12.00700 14.00 800 16.00 900 18.00 1000 20.00 1100
20110616-15 628 (10.919) 1: TOF MS ES+
OH
595.1 1.60e4
100 C OH

+
HO O

OH
596.1 O
O
OH
OH OH
%

O
OH
O OH
CH3

597.2
287.0
0 m/s m/z
100 200 300 400 500 600 700 800 900 1000 1100

Fig. 3 UPLC-MS- of anthocyanins in mulberry wine residue.

A. UPLC Chromatogram; B. MS-spectra of anthocyanin B. peak 1; C. peak 2.

24
539

540 Table 1

541 Table 1 Box–Behnken design for the optimisation of extraction conditions for total

542 anthocyanins
Coding Yield of total
experiments anthocyanins
X1 X2 X3 X4
（mg/g）
1 0 −1 0 1 2.40
2 −1 0 0 1 1.89
3 0 1 −1 0 4.67
4 0 0 0 0 6.02
5 1 1 0 0 4.29
6 0 −1 −1 0 2.59
7 1 −1 0 0 2.53
8 0 −1 1 0 4.35
9 0 1 0 −1 3.74
10 −1 0 1 0 3.65
11 1 0 0 1 3.90
12 1 0 0 −1 2.72
13 0 0 0 0 5.98
14 −1 0 −1 0 2.11
15 0 1 0 1 3.01
16 0 0 −1 1 2.59
17 −1 1 0 0 1.89
18 0 0 1 1 5.06
19 1 0 −1 0 3.90
20 0 0 −1 −1 2.98
21 0 −1 0 −1 2.14
22 0 0 0 0 5.41
23 −1 0 0 −1 1.86
24 0 0 1 −1 2.91
25 −1 −1 0 0 2.46
26 0 1 1 0 4.38
27 1 0 1 0 4.51
543

25
545
546 Table 2

547 Table 2 Results of ANOVA for response surface quadratic model

Source Sum of square Degree of Mean square F p

freedom

Model 39.32 11 3.57 24.60 <0.0001

A1 5.33 1 5.33 36.70 <0.0001

A2 2.52 1 2.52 17.37 0.0008

A3 3.02 1 3.02 20.76 0.0004

A4 0.52 1 0.52 3.57 0.0782

A1A2 1.36 1 1.36 9.39 0.0079

A2A3 1.05 1 1.05 7.22 0.0169

A3A4 1.60 1 1.60 11.00 0.0047

A12 14.11 1 14.11 97.12 <0.0001

A22 8.77 1 8.77 60.39 <0.0001

A32 2.09 1 2.09 14.41 0.0018

A42 15.24 1 15.24 104.91 <0.0001

residual 2.18 15 0.15

Lack of fit 1.95 13 0.15 1.28 0.5223

Error 0.23 2 0.12 R2=0.9475

Sum 41.50 26
548 notes: “p>F”<0.05, significant, “p>F”>0.05, non-significant.

26
550
551 Table 3

552 Table 3 Characterisation of anthocyanins in mulberry wine residue using UV-Vis and

553 UPLC-MS

RTa RPAb λmax MS+ MS/MS

Peak Anthocyanin
(min) (%) (nm) (m/z) (m/z)

1 10.26 41.57 281,517 449 287 cyanidin-3-O-glucoside

2 10.89 58.43 281,518 595 287 cyanidin-3-O-rutinoside

554 a RT, retention time, min.

555 b RPA, peak area relative to the total peak area, %.

556

557

558 Declaration of interest

559 The authors declare no competing financial interest.
560

561 Highlights

562 1. Ultrasonic-assisted enzymatic extraction of anthocyanins was done from

563 mulberry-wine waste.

564 2. Box-Behnken design was used to optimize the parameters for extraction of

565 anthocyanins.

566 3. Macro-porous resin and Sephadex LH-20 gel column increased anthocyanins purity.

567 4. Cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside are the main anthocyanin

568 structures.

569 5. Ultrasonic-assisted enzymatic extraction is a useful method for the extraction of

27
570 natural products from fruit wine residues materials.

571

572

573

574 Highlights

575 1. Ultrasonic-assisted enzymic method extracted anthocyanins from mulberry-wine waste

576 2. Optimum extraction parameters were successfully obtained using Box-Behnken design

577 3. Optimum extraction yield (5.98 mg/g) was close to the predicted yield (6.03 mg/g)

578 4. Macro-porous resin and Sephadex LH-20 gel column increased purity of anthocyanins

579 5. Cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside were the main anthocyanins

580

28