Effect of carbonic maceration (CM) on mass transfer characteristics and quality attributes of Sanhua plum (Prunus Salicina Lindl.)

LWT - Food Science and Technology 87 (2018) 537e545

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Effect of carbonic maceration (CM) on mass transfer characteristics and quality attributes of Sanhua plum (Prunus Salicina Lindl.) Kejing An, Jijun Wu, Daobang Tang, Jing Wen, Manqin Fu, Gengsheng Xiao, Yujuan Xu* Sericulture and Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture/ Guangdong Key Laboratory of Agricultural Products Processing, Guangzhou 510610, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 June 2017 Received in revised form 17 September 2017 Accepted 18 September 2017 Available online 23 September 2017

Sanhua plum is an important tropical fruit in China with epicuticular wax and dense pulp, causing difficulties for dehydration and sucrose penetration. Carbonic maceration (CM) is an efficient and low cost technology, which can increase the mass transfer rate during candying/drying process and improve the quality indices of Sanhua plum. The results showed that CM treatment could disturb wax layer, produce porous structure and increase the permeability of cell membrane, which could significantly increase the rate of sugar intake and water loss during candying and drying processes. The sugar intake rate can be increased by 82.52 ± 5.38% and total drying time can be shortened by 39.47 ± 2.24%. In addition, CM could help to overcome the plant extraction barrier and enhance the active components content. The lower pH environment and inactivation of polyphenol oxidase (PPO) and peroxidase (POD) were also beneficial to the stability of phenol compounds, thus the increase of individual phenolics retention and antioxidant activity of Sanhua plum. The total anthocyanins, phenolics, flavonoids and the results of the ABTS assay were increased by 14.91e29.63%, 3.95e16.93%, 11.10e23.10%, and 2.10e56.71%, respectively after CM treatment. Therefore, CM is a very promising pretreatment for fruits and vegetables dehydration. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Wax layer PPO and POD activity Circular-dichroism analysis Individual phenolics Antioxidant activity Chemical compounds studied in this article: Cyanidin 3-glucoside (PubChem CID: 92131208) Cyanidin 3-rutinoside (PubChem CID: 441674) Quercetin 3-galactoside (PubChem CID: 44259103) Quercetin 3-glucoside (PubChem CID: 44259229) Neochlorogenic acid (PubChem CID: 5280633) Chlorogenic acid (PubChem CID: 1794427) Guaiacol (PubChem CID: 460) Cathecol (PubChem CID: 289) Trolox (PubChem CID: 40634) 2,20 -azinobis (3-ethylbenzo thiazoline-6sulphonic acid) diammonium salt (ABTS) (PubChem CID:9570474)

1. Introduction Sanhua plum (Prunus Salicina Lindl.) originated from Sanhua town Guangdong province, with a cultivation history for 500 years. It belongs to Rosaceae plants, which have a natural remedy against

* Corresponding author. Sericulture and Agri-Food Research Institute, Dong Guanzhuang Yiheng RD., Tianhe District, Guangzhou 510610, PR China. E-mail address: [email protected] (Y. Xu). https://doi.org/10.1016/j.lwt.2017.09.032 0023-6438/© 2017 Elsevier Ltd. All rights reserved.

various diseases due to their high level of polyphenols and antioxidant activity (Hai, Zhao, Lin, & Dong, 2015). It was reported that the major polyphenolics identified in plums were hydroxycinnamic acids, flavonols, and anthocyanins (Chun, Kim, Moon, Kang, & Lee, 2003a; Kim, Chun, Kim, Moon, & Lee, 2003). However, there is limited information on individual polyphenolic constituents and antioxidant activity of Sanhua plum. The moisture content of fresh harvested Sanhua plum is about 85e90% (w.b.) which makes it susceptible to microbial spoilage and chemical deterioration. Besides, due to the high content of organic

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acid, Sanhua plum is not very suitable for fresh taste. Therefore, it has often been processed to a traditional Cantonese-style candied fruit. In traditional processing, candying and drying are timeconsuming processes, especially for fruit with epicuticular wax and dense tissue, such as Sanhua plum. Many pretreatments have been reported to remove the wax layer and dense flesh barrier, such as chemical dipping (sodium hydroxide, potassium carbonate, methyl and ethyl ester emulsions) (Doymaz, 2006), thermal treatment (Tarhan, 2007) and high pulsed electric field (HPEF) (Taiwo, Angersbach, & Knorr, 2002). However, chemical dipping could only overcome the wax barrier, and has no significant effect on dense flesh (Bain & McBean, 1969). Thermal treatment could cause substantial wastewater disposal. As for HPEF treatment, the equipment is still too expensive and complicated for the moment. Therefore, a new pretreatment named carbonic maceration was introduced, which is low cost, efficient, and easy operation. Carbonic maceration (CM) was first invented by Michel Flanzy in 1934 and had been used in cabernet making (Tesniere & Flanzy, 2011). In the making process, researchers found CM process could induce structure changes of grape, such as collapse of cell wall and hydrolysis of cell membrane, and some physiochemical reactions in the vacuole. Therefore, we wondered whether the structure changes could change the mass transfer characteristics, and whether the physiochemical reactions could affect the quality attributes of products. Liu and Wang (2011) found CM treatment prior to drying could increase the total phenolic content (TPC) and ascorbic acid (Vc) content of grape by 41.28% and 12.81%. Liu et al. (2014) found CM treatment could improve the microwave drying rate of Chilli by 1.5e1.85 times, and the TPC and Vc content were increased by 40%e60% and 121%e582%, respectively. However, there was no study about the effect of CM on mass transfer characteristics and quality attributes of Sanhua plum as well as the action mechanism of CM. Therefore, we carried out our study in three aspects: (i) to study the effect of CM on mass transfer during osmotic dehydration (candying) and heat pump drying of Sanhua plum; (ii) to determine the effect of CM on quality attributes, such as individual phenolics and antioxidant activity; (iii) to explore the possible mechanism of CM on mass transfer characteristic and quality attributes. 2. Materials and methods 2.1. Material Sanhua plums were bought from a local market and selected by noticing that all samples were disease-free, basically uniform in shape and at the same ripening stage. The Sanhua plums were roughly a spheroid with radius of 2.5 ± 0.2 cm and the initial moisture was about 88.36 ± 0.61% (w.b.). 2.2. Reagent and chemicals Reagents: Guaiacol, Cathecol, PVPP, PEG 6000, Trolox, FolinCiocalteu reagent and ABTS were from National Pharmaceutical Corporation (Beijing, China). HPLC grade of phosphoric acid and acetonitrile were purchased from Honeywell (Morris, NJ, USA). Authentic standards of cyanidin 3-glucoside, cyanidin 3-rutinoside, neochlorogenic acid, chlorogenic acid, etc were purchased from Chengdu Must Bio-technology Co., LTD (Chengdu, China). 2.3. Carbonic maceration (CM) CM experiment was conducted in a laboratory set-up equipment, the schematic diagram was shown in Supplementary Fig. 1.

Sanhua plums were weighed and placed into the tank, then the tank was filled with CO2 to a desired pressure. The temperature was controlled at 30  C. When the CM duration was reached, samples were taken out, and gently blotted with tissue paper and weighed. 2.4. Osmotic dehydration (OD) Plums were immersed into 60% sucrose solution with a mass ratio of 4:1 (solution to plums) at 45  C. For vacuum treatment, OD was carried out in a vacuum chamber under 13 kPa at 45  C. For ultrasound treatment, OD was conducted in an ultrasonic chamber (DL-800B, Shanghai Zhixin Apparatus Co., China) with an operating power of 50W. 2.5. Titratable acid (TA) and soluble sugar (SS) of plums during OD TA was measured according to AOAC method (AOAC, 1995). The titratable acidity is expressed as mmol citric acid/100 g dried plum. SS was determined by the anthrone method. The total sugar was determined from the standard curve prepared using glucose and expressed as g/100 g dried plum. 2.6. Heating pump drying Heat pump drying could recover the energy from exhaust which is an environmentally friendly technology. Plums were put into the laboratory model heat pump dryer (GHRH-20, Guangdong Hongke Corporation, China) after attaining the temperature of 60  C (25% R.H.). Each aluminum wire mesh tray (containing 100 ± 2 g plums) was weighed regularly at 30 min intervals. When moisture content reached to 0.33 d.w., the drying process ended. 2.7. Moisture content (MC) and drying rate (DR) MC and DR of plums were calculated according to Doymaz (2006):

Md;iþ1  Md;i dM ¼ DR ¼  dt tiþ1  ti

(1)

Where M is the instantaneous mass, M0 is the initial moisture content, Me is equilibrium moisture content, which can be assumed as zero when compared with M0. Md,I, Md,iþ1 is the moisture content at time ti, tiþ1, dry basis. 2.8. Total Phenolics (TP), flavonoids(TF), anthocyanins (TA) content and ABTS assay Total phenolics were prepared according to the procedure described by Chun, Kim, and Lee (2003b) with modifications. 5 g plums pulp were ultrasonically extracted with 30 ml 1% HCl-80% methanol for 30min each time until the extract became colorless. After centrifugation (1000 g, 10min), the supernatants were combined and evaporated at 40  C. The extract was stored at 4  C. TPC was determined using Folin-Ciocalteu phenol reagent as described by An et al. (2016). Briefly, diluted extract (400 mL) were added into test tubes, followed by 2.0 mL of Folin-Ciocalteu reagents and allowed to stay at room temperature (25  C) for 5min, then 3.0 mL of sodium carbonate (7.5% w/v) solution was introduced into the mixture. After 2 h reaction in darkness, absorbance was measured at 765 nm. Results were expressed as gallic acid equivalents. TFC was measured according to the colorimetric assay described by Dewanto, Wu, Adom, and Liu (2002). At the beginning, 0.3 mL of 5% NaNO2 was added to a 10 mL flask with 2 mL diluted extracts.

K. An et al. / LWT - Food Science and Technology 87 (2018) 537e545

After 6 min, 0.3 mL of 10% AlCl3 was added, and after 6 min, 2 mL of 4% NaOH was added. 15 min later, the reaction flask was filled up to the volume with distilled water and thoroughly mixed. Absorbance of the mixture was determined at 510 nm. Results were determined as rutin equivalents. TAC was evaluated by the pH differential method (Kalt, Forney, Martin, & Prior, 1999). Phenolic extracts of plums in 0.025 M potassium chloride buffer (pH 1.0) and 0.4 M sodium acetate buffer (pH 4.5) were measured at 510 and 700 nm after 15 min of incubation at 23  C. The content of total anthocyanins was expressed as cyanidin 3-glucoside equivalent. A molar absorptivity of 26900 was used for cyanidin 3-glucoside (molecular weight 449.2 g/mol). ABTS*þ working solution was prepared by mixing 7 mM ABTS solution and 2.45 mM potassium persulfate equally and kept in darkness overnight. The concentration of the resulting blue-green ABTS*þ working solution was adjusted to an absorbance of 0.70 ± 0.02 at 734 nm. 0.2 mL sample solution was added to 2 mL ABTS*þ working solution. The mixture was incubated in darkness for 30 min and measured at 734 nm. A standard curve was prepared using Trolox solution and the results were reported as mg Trolox/g d.w.

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2.12. Scanning electron microscopy Morphologies of the waxy layer and flesh of Sanhua plums were observed under a field emission scanning electron microscope (SEM) using a JSM-6360LV machine (JEOL Co, Japan). The specimens of wax layer for SEM observation were prepared by cutting the peel near the equator of plum with central area of 0.5 cm  0.5 cm. For morphologies of flesh, 0.5  0.5  0.1 cm3 small pieces were taken from flesh tissue. The cut sections were carefully affixed on copper stubs. Each specimen was gold-coated prior to SEM observation. 2.13. Statistical analysis The results were analyzed using one-way analysis of variance (ANOVA) and Duncan's multiple-range test (P < 0.05), carried out with SPSS 18.0 (Chicago, IL, USA). All experiments were run in triplicate. 3. Results and discussion 3.1. The effect of CM on mass transfer during OD

2.9. High performance liquid chromatography (HPLC) analysis of individual phenolics HPLC analysis was performed according to the method of Lee (2002) with modifications. HPLC system (LC-20AT, Shimadzu, Japan) with a C18 reversed-phase symmetry analytical column (5 mm  250 mm  4.6 mm; Waters Corp., Milford, Mass., U.S.A.) was used. Linear solvent gradient of binary mobile phases (solvent A, 4% H3PO4 in HPLC grade water; solvent B, 100% acetonitrile) during HPLC analysis was applied as follows: 0e5 min, 6% B; 5e55 min, 6e25% B; 55e56 min, 25e75% B; 56e61 min, 75% B; 61e62 min, 75%e6% B; 62e70 min, 6% B. The flow rate was 1.0 mL/min and the injection volume was 10 mL. Hydroxycinnamic acids were monitored at 320 nm, flavonols at 360 nm and anthocyanins were at 520 nm. 2.10. PPO and POD activity measurement The enzyme extracts were made by homogenization of 5 g of each sample with 5 mL of 0.1 M acetate buffer (pH 5.5) [containing 4% (w/v) PVPP and 340% (w/w) PEG 6000]. The PPO activity was assayed using 0.1 mL extract and 4.0 mL of a solution with 0.15 M cathecol in 0.05 M acetate buffer (pH 5.5). The reaction was measured at 420 nm and 25  C. The POD activity was assayed using 0.5 mL extract and a reaction mixture composed of 25 mmol of guaiacol (50 mmol/L acetate buffer, pH 5.5) with 200 mL 0.5 mol/L H2O2. The oxidation of guaiacol was measured at 470 nm and 25  C. Enzyme activity unit was defined as an increase of 0.1 in absorbance per minute. Residual activity (RA) of enzyme was defined as:

RA ¼

enzyme activity after CM treatment; At initial enzyme activity; A0

(2)

3.1.1. The kinetics of water loss (WL) and solute gain (SG) of Sanhua plum From the kinetics of water loss (Fig. 1A), the plums presented a higher WL after CM treatment and WL was increased with the rise of CM pressure, especially at 5Mpa for 3 h. However, SG did not show corresponding increase as CM pressure increased (Fig. 1B). This is because CM could cause certain intracellular solid loss of Sanhua plum (Supplementary Fig. 2) due to the depolymerisation of cell wall and cell membrane. SG presented was the intake of solute minus the outflow of intracellular solute. We found CM at 5 MPa had higher SG than other pressure. Hence, CM condition of 5 MPa for 3 h was chosen for further experiment. 3.1.2. The kinetics of soluble sugar and titrable acid of Sanhua plum Soluble sugar and titrable acid are another indices to measure the mass transfer during OD, which can reflect the actual sugar intake and intracellular solute loss. As shown in Fig. 2 (A), with the increase of OD time, soluble sugar presented a gradual increase, when reached to 3 h, the sugar content approached the peak value. The peak value of sugar content was highest in CM þ vacuum osmosis sample, followed by CM þ ultrasound osmosis, CM þ osmosis, vacuum osmosis, ultrasound osmosis and osmosis samples. As to titratable acid (TA), it presented a gradual decline as OD proceeded (Fig. 2B). The fast decline of TA also occurred in CM treated samples. The results indicated that CM pretreatment could significantly increase the intake of soluble sugar and loss of titrable acid. The rate of sugar intake was increased by 82.52 ± 5.38% after CM treatment. According to the product of prune (Sunsweet, USA, California) whose sugar content is 42.84 g/100 g prune, acid content is 28.44 mmol/100 g prune. Therefore, we believe 3 h of OD could keep the sugar and acid content of Sanhua plum in appropriate range.

2.11. Circular dichroism (CD) analysis of PPO and POD

3.2. The effect of CM on moisture transfer during heat pump drying

CD spectra were recorded with a Chirascan™ systems (Applied Photophysics Ltd., United-Kingdom), using quartz cuvette of 1-mm optical path length at room temperature (25±1  C). CD spectra were scanned at the far UV range (260-200 nm) with four replicates at 50 nm/min, bandwidth ¼ 1 nm.The content of a-helix (%), b-sheet (%), b-turn (%) and Rndm.coil (%) were analyzed by CDNN software (Leatherhead, UK).

Moisture content (MC) curves were observed to be reduced exponentially with drying time (Fig. 3A), whose curves exhibited a steeper slope, indicating who had higher drying rate. The curve of CM þ vacuum osmosis showed the steepest slope, followed by CM þ ultrasound osmosis, CM þ osmosis, osmosis, ultrasound osmosis and vacuum osmosis. It indicated that CM treatment could significantly increase the drying rate of Sanhua plum.

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Fig. 1. The effect of CM pressure on water loss (A) and solute gain (B) of Sanhua plums during OD.

Fig. 2. The effect of CM on soluble sugar (A) and titrable acid (B) content of Sanhua plums.

Fig. 3. The curves of moisture content (A) and drying rate (B) of Sanhua plums after CM treatments.

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Table 1 The content of individual hydroxycinnamic acids, flavonols and anthocyanins of fresh and dried Sanhua plum. Fresh Sanhua plum

Anthocyanins Cy-3-glu (mg/100 g d.w) Cy-3-rut (mg/100 g d.w) Hydroxycinnamic 5-CQA (mg/100 g d.w) 3-CQA (mg/100 g d.w) Flavonols Qu-3-gala (mg/100 g d.w) Qu-3-glu (mg/100 g d.w)

Dried Sanhua plum Without osmosis

osmosis

Vacuum osmosis

Ultrasound osmosis

Without CM

CM treatment

Without CM

CM treatment

Without CM

CM treatment

Without CM

CM treatment

288 ± 10a

29.8 ± 4.6f

39.2 ± 7.1e

19.7 ± 3.7g

55.8 ± 4.0c

48.0 ± 6.3d

79.2 ± 7.0b

27.9 ± 6.9f

50.4 ± 2.9d

349 ± 21a

34.2 ± 4.3f

53.9 ± 9.8e

27.4 ± 4.4g

81.1 ± 2.6bc

66.4 ± 13.4d

89.9 ± 9.6b

30.4 ± 8.2f

78.1 ± 1.0c

acid 2.82 ± 0.76d

4.01 ± 0.55c

7.34 ± 0.91a

3.39 ± 0.02d

4.85 ± 0.78b

2.97 ± 0.88d

4.58 ± 0.82b

2.01 ± 0.78e

3.92 ± 0.58c

3.21 ± 0.62a

1.25 ± 0.22d

2.55 ± 0.53b

0.55 ± 0.02e

1.52 ± 0.49c

1.27 ± 0.43c

1.55 ± 0.58c

0.60 ± 0.02e

1.34 ± 0.11d

3.62 ± 0.47b

3.83 ± 0.58b

5.97 ± 0.21a

1.42 ± 0.21e

3.52 ± 0.14b

2.81 ± 0.13c

3.65 ± 0.59b

1.94 ± 0.42d

3.99 ± 0.50b

b

b

a

d

1.89 ± 0.59c

e

2.65 ± 0.43

2.47 ± 0.26

3.66 ± 0.67

0.88 ± 0.11

1.50 ± 0.64

2.33 ± 0.76b

1.35 ± 0.55

cd

2.01 ± 0.28bc

Cy-3-glu, cyanidin 3-glucoside; Cy-3-rut, cyanidin 3-rutinoside; 5-CQA, chlorogenic acid; 3-CQA, neochlorogenic acid; Qu-3-gala, quercetin 3-galactoside; Qu-3-glu, quercetin 3-glucoside. Values are means ± SD (n ¼ 3). For each row, values followed by the same small superscript letter did not share significant differences at p < 0.05 (Duncan's test).

The curves of drying rate were identified in three distinct periods: a warming-up, constant rate and falling rate period (Fig. 3B). This is attributed to the different roles of capillary diffusion and moisture evaporation playing in moisture transfer during drying (Wang & Chen, 2000). According to Azzouz, Guizani, Jomaa, and Belghith (2002), the drying efficiency depends on the nature of the material and difficulty of capillary diffusion. As shown in Fig. 3B, CM treatment could significantly increase the drying rate of plum, especially in constant rate and falling rate period, which indicated CM could change the structure of material and overcome the difficulty of capillary diffusion. Samples pretreated by CM combined with vacuum osmosis had the shortest drying time, which was 39.47 ± 2.24% less than untreated samples. Wang et al. (2014) and Liu et al. (2014) also reported CM pretreatment could shorten the drying time and increase the drying rate. This phenomenon could be ascribed to the structure modifications of plant tissue such as depolymerisation of pectin and other biopolymers. According to Krall and McFeeters (1998), when a plant material was put in a low pH environment, depolymerisation of pectin and other connecting biopolymers occurred. Femenia, Sanchez, Simal, and Rossello (1998) also found the low pH not only hydrolyzes glycoside bond, but also hydrolyzes ester linkages. In our study, we found the pH value of Sanhua plum was decreased from 3.46 to 3.2 after CM treatment (Supplementary Fig. 3).

Besides, when plant tissue was subjected to a variety of adversely environmental conditions, cell membrane structure was destroyed, showing intracellular electrolyte leakage and increase in cell membrane permeability. We found the cell membrane permeability of Sanhua plum was increased significantly after CM treatment (Supplementary Fig. 4). The hydrolysis of cell wall and increase in cell membrane permeability could considerably reduce the mass transfer resistance and increase the drying rate. 3.3. The effect of CM on quality attributes of Sanhua plum 3.3.1. Individual polyphenols According to previous studies (Chun et al., 2003a; Kim et al., 2003), the predominant phenolic compounds in plums were hydroxycinnamic acids, such as neochlorogenic acid (3-O-caffeoylquinic acid, 3-CQA) and chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA). However, in Sanhua plum, anthocyanins were the principal polyphenols, especially cyanidin 3-rutinoside (Cy-3-rut) and cyanidin 3-glucoside (Cy-3-glu). Flavonols such as quercetin 3galactoside (Qu-3-gala) and quercetin 3-glucoside (Qu-3-glu) also made contributions to polyphenols of Sanhua plum. As shown in Table 1, the content of Cy-3-glu and Cy-3-rut were decreased significantly after drying, while 5-CQA, 3-CQA, Qu-3-gala and Qu-3glu did not show significant decrease. The changes in the content of

Table 2 The content of total phenolics, flavonoids, anthocyanins and antioxidant capacity assay of fresh and dried Sanhua plum. Fresh Sanhua plum

TPC (mg GAE/g d.w) TFC (mg RE/g d.w) TAC (mg CGE/g d.w) ABTS (mg Trolox/g d.w)

Dried Sanhua plum Without osmosis

Osmosis

Vacuum osmosis

Ultrasound osmosis

Without CM

With CM

Without CM

With CM

Without CM

With CM

Without CM

With CM

30.4 ± 0.2a

15.3 ± 0.4c

17.0 ± 0.3b

11.3 ± 0.1f

13.3 ± 0.4d

13.0 ± 0.4d

10.6 ± 0.2g

11.0 ± 0.2f

57.0 ± 2.5a

36.0 ± 0.9c

44.1 ± 0.3b

26.0 ± 0.8e

31.6 ± 1.0d

30.7 ± 0.4d

14.4± 0.1e 37.8 ± 0.6c

23.5 ± 0.8f

26.1 ± 0.5e

1.20 ± 0.06e

1.46 ± 0.06cd

1.08 ± 0.1f

1.40 ± 0.03d

1.60 ± 0.12c

2.04 ± 0.06b

1.14 ± 0.1e

1.31 ± 0.02d

38.5 ± 0.4e

60.3 ± 1.0b

33.3 ± 0.2f

37.8 ± 0.3e

51.8 ± 0.9d

56.7 ± 0.7c

32.4 ± 1.3e

33.1 ± 0.1f

8.79 ± 0.04 107±1a

a

TAC: Total anthocyanins content; TPC: Total phenolics content; TFC: Total flavonoids content; ABTS: Antioxidant activity with ABTS assay. Values are means ± SD (n ¼ 3). For each row, values followed by the same small superscript letter did not share significant differences at p < 0.05 (Duncan's test).

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Fig. 4. The residual activity of POD (A) and PPO (B) of Sanhua plum after CM treatments.

Fig. 5. Far-UV CD spectra of POD(A) and PPO (B) after CM treatments.

phenolics, flavonoids and anthocyanins during drying could be ascribed to many simultaneous effects. According to Raynal and Moutounet (1989), anthocyanins could have been degraded by the joint action of temperature/enzymatic activity during drying process. Hydroxycinnamic acids could have been influenced by polyphenoloxidase (PPO) enzymatic activity (Piga, Caro, & Corda, 2003). While the degradation of flavonoids is not directly associated with the PPO activity, since they are not direct substrates of the oxidases. Mileti c et al., (2013) and Piga et al. (2003) found the content of flavonoids lowers more rapidly with the rise in temperature. Therefore, the high temperatures and high oxygen concentrations involved in the heat pump drying could lead to rapid degradation of the anthocyanins. Flavonoids (Qu-3-gala, Qu-3-glu) are more stable than anthocyanins at mild temperatures (60  C). The content of chlorogenic acid remained unaffected or even increased after drying could be ascribed to the inactivation of oxidase and the production of chlorogenic acid during drying. According to Piga et al. (2003), the chlorogenic acid can act as an intermediary in PPO enzymatic degradation of the anthocyanins (the compounds deprived of glycosides). The individual polyphenols all showed increase after CM treatment. Compared with non-CM samples, the content of Cy-3-glu, Cy-3-rut, 5-CQA, 3-CQA, Qu-3-gala and Qu-3-glu were increased by 31.49e182.80%, 35.43e196.31%, 43.07e95.02%, 22.05e176.36%, 29.89e147.89% and 38.11e114.77%, respectively.

3.3.2. Total phenolics, flavonoids, anthocyanins and antioxidant capacity assay with ABTS As shown in Table 2, the TPC, TFC, TAC and antioxidant capacity assay with ABTS of fresh Sanhua plum were 30.4 mg GAE/g d.w, 57.0 mg RE/g d.w, 8.79 mg CGE/g d.w and 107 mg Trolox/g d.w., respectively. After drying, they were decreased by 44.09e65.03%, 22.59e58.76%, 76.79e87.71% and 43.88e69.83%, respectively. It indicated that drying process caused more damage to anthocyanins, which was correlated well with degradation of Cy-3-rut and Cy-3-glu. However, after CM treatment, the retention of TAC, TPC, TFC and results of ABTS assay of plums were increased by 14.91e29.63%, 3.95e16.93%, 11.10e23.10%, and 2.10e56.71%

Table 3 The secondary structure changes of PPO and POD of Sanhua plum after CM treatments.

PPO

POD

CM (MPa)

a-helix (%)

b-sheet (%)

b-turn (%)

Rndm. coil (%)

0 1 3 5 0 1 3 5

24% 20.10% 17.50% 15.20% 32.80% 29% 26.80% 27.80%

23.70% 27.70% 31.10% 34.70% 17.4% 19.7% 21.3% 20.5%

18.80% 19.80% 20.60% 21.40% 17.10% 17.80% 18.30% 18.10%

39.40% 43.10% 46.30% 49.50% 30.80% 33.90% 35.90% 35.00%

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Fig. 6. The microstructure of epicuticular wax and inner tissue of Sanhua plum: A, C: epicuticular wax of untreated samples (x 5000) (x10000); B, D: epicuticular wax of samples treated by CM (x 5000) (x10000); E: inner tissue of untreated samples (x 200); F: inner tissue of samples treated by CM under 3 MPa (x150); G: inner tissue of samples treated by CM under 5 MPa (x 50); H: inner tissue of samples treated by CM under 7 MPa (x 50).

respectively. Wang et al. (2014) and Liu et al. (2014) also found the similar phenomenon. The increased individual phenolics and antioxidant activity could be attributed to many reasons. According to Chen (2015) the increased cell permeability could help to overcome the plant extraction barrier and enhance the extraction performance. In the CM process, cell membrane permeability was increased as more active components such as phenolics can be extracted. Besides, acid environment is believed to be beneficial to the stability of polyphenols (Ruenroengkin, Zhong, & Jiang, 2008). Therefore, the

increased cell permeability and lower pH environment caused by CM could account for the increased phenolic compounds of Sanhua plum. 3.4. The effect of CM on activity and conformational changes of PPO and POD 3.4.1. The effect of CM on activity of PPO and POD PPO and POD are the main enzymes responsible for quality loss due to phenolic degradation. As shown in Fig. 4, the activity of PPO

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and POD were decreased significantly after CM treatment. As CM pressure increased from 1 to 7 MPa, the residual activity of PPO was decreased from 88.89% to 47.26% and POD was decreased from 10.59% to 5.56%. The phenolic compounds are the substrates of oxidative enzymes. Therefore, the inactivation of PPO and POD could reduce the loss of polyphenols.

3.4.2. CD spectra analysis of PPO and POD The CD spectra of PPO and POD treated by CM are shown in Fig. 5. PPO has double negative peaks in far UV CD spectra at around 208 and 222 nm and POD has one positive peak at 195 nm, and two negative peaks at 208 nm and 222 nm. Those peaks are feature characteristic of the a-helix secondary conformation of protein, and their intensity reflects the amount of helical structure. The intensity of peaks at 195, 208 and 222 nm were decreased significantly after the CM treatment, indicating losses of a-helix conformation in PPO and POD. As shown in Table 3, with the CM pressure increased, the relative contents of a-helix of PPO and POD were decreased from 24% to 15.2% and 32.80%e26.80%, respectively. However, the content of b-sheet, b-turn and random coil were increased correspondingly. This was consistent with many studies (Guo et al., 2017; Wimmer & Zarevúcka, 2010). In the carbonic maceration process, CO2 might penetrate into the folding site of enzyme structure, and interact with their protein strands and binding site to form a carbonated complex, which could cause the unfolding of a-helix occur simultaneously with the folding of b-sheet, b-turn and random coil, implying a conversion of a-helix to b-sheet. Therefore, the changes in enzyme conformation lead to the active site loss in their functions.

3.6. The mechanism of effect of CM on Sanhua plum The results can be interpreted as three reasons for CM to increase the mass transfer characteristic as well as three reasons for CM to increase the quality attributes of Sanhua plum. Firstly, the lower pH environment created by CM treatment could induce depolymerisation of cell wall and cell membrane, which might considerably reduce the mass transfer resistance. Secondly, the increased cell permeability caused by CM treatment could lead to the increase of drying rate. Thirdly, certain damage to wax layer and enlarged porous structures caused by CM are beneficial to internal moisture diffusion and evaporation. The reasons for increased quality attributes. Firstly, the increased cell permeability could help to overcome the plant extraction barrier and enhance the extraction of active components. Secondly, lower pH was beneficial to the stability of phenol compounds. Thirdly, the inactivation of PPO and POD caused by conformational changes could reduce the loss of polyphenolics. 4. Conclusion CM technique presented in our study is a favorable pretreatment for osmotic dehydration and drying of fruits and vegetables. It could help to reduce the mass transfer resistance, and increase internal moisture diffusion and evaporation of plant material. It can also enhance the extraction of active components, inactivated oxidase enzymes and improve the stability of phenolic compounds. Therefore, CM is a promising pretreatment for fruits and vegetables, especially for fruits like plums with wax layer and dense flesh. Acknowledgements

3.5. The effect of CM on microstructure of wax layer and internal tissue Waxes are a complex mixture of alcohols, alkanes, aldehydes, ketones, and esters made from long-chain fatty acids. The structure of waxy layer is important since it influences the rate of water loss during natural and artificial drying. The wax crystals structure depending on wax composition present different shapes among different cultivars. The crystalline microstructure of epicuticular wax on Sanhua plum can be described as curved fibrils and small thin platelets (Fig. 6A, C). After CM treatment, the crystalline structure was changed to a packed granular structure protruding from the surface. The increased complexity of surface structure was evident due to the increased size and number of protuberances after CM treatment. (Fig. 6 B, D). According to Bain and McBean (1967), the regeneration of the waxy layer occurs soon after plant surfaces have been disturbed. Skene (1963) found new wax forming on the surface of polished apples, not in the form of original platelets. Therefore, we speculated that CM treatment could cause damage to the wax layer of Sanhua plum. According to Bain and McBean (1969), the change of wax structure could reduce the water barrier efficiently, but the rate of water diffusion through the flesh is the major limiting factor for fleshy fruits like plum. The cell membrane permeability suggested that CM treatment not only took effect on peel, but also had greater effect on inner tissue (Supplementary Fig. 4). The microstructure of flesh tissue also proved this. As shown in Fig. 6E, parenchyma cells of dried plums showed ambiguous contours of cell wall, turgor loss and degradation of middle lamella. After CM pretreatment, the contours of cell wall became clear and the porous structures were produced (Fig. 6FeH). In addition, with the increase of CM pressure, the pore structures were increased and enlarged, which is beneficial to internal moisture diffusion and evaporation.

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