Effects of liquid nitrogen quick freezing on polyphenol oxidase and peroxide activities, cell water states and epidermal microstructure of wolfberry

LWT - Food Science and Technology 120 (2020) 108923

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Effects of liquid nitrogen quick freezing on polyphenol oxidase and peroxide activities, cell water states and epidermal microstructure of wolfberry

T

Zhiwei Zhua,b,c, Wenhuang Luoa,b,c, Da-Wen Suna,b,c,d,∗ a

School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China Academy of Contemporary Food Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou 510006, China c Engineering and Technological Research Centre of Guangdong Province on Intelligent Sensing and Process Control of Cold Chain Foods, Guangzhou Higher Education Mega Centre, Guangzhou 510006, China d Food Refrigeration and Computerized Food Technology (FRCFT), Agriculture and Food Science Centre, University College Dublin, National University of Ireland, Belfield, Dublin 4, Ireland b

A R T I C LE I N FO

A B S T R A C T

Keywords: Wolfberry Liquid nitrogen quick freezing Water distribution Epidermal structure Freezing characteristics

Fresh wolfberry has a very short shelf-life and drying is the most common method for preserving wolfberry. For better retention of the quality and nutritional values of wolfberry, effects of liquid nitrogen spray freezing at different temperatures of −60 °C ± 2 °C (NF-60°C), −80 °C ± 2 °C (NF-80°C) and −100 °C ± 2 °C (NF-100°C) on wolfberry were investigated, as compared with air-blast freezing (BF) at −40 °C ± 1 °C and air velocity of 0.75 m/s. Results showed that the freezing time passing the maximum ice crystal formation zone were 450 s, 150 s, 100 s and 70 s for BF, NF-60°C, NF-80°C, and NF-100°C, respectively. Comparing with NF-100°C, NF-80°C samples showed better appearance, lower peroxidase activity, water distribution more similar to fresh samples and lower damage of inner epidermal cell structure. The current study suggested that the freezing characteristics of wolfberry did not become better at the ultralow freezing temperature of −100 °C, and NF-80°C was considered the most appropriate freezing process for the freezing characteristics of the wolfberry. It is hoped that the current results could be useful to the industry for better preserving wolfberry.

1. Introduction Wolfberry, also called Lycium barbarum, is an oval orange-red berry native to Asia and is mainly grown in arid and semi-arid regions. Wolfberry contains many biologically active substances such as carotenoids, polysaccharides, polyphenols, and nitrogen-containing compounds, exhibiting strong antioxidant and anti-aging functions, and thus is used as effective supplements for preventing diabetes, and cardiovascular and anti-tumor diseases (Amagase and Farnsworth, 2011; Jin, Huang, Zhao, and Shang, 2013). Fresh wolfberry can promote full metabolism effects. However, due to its high contents in water and sugar, the tender tissue of fresh wolfberry is susceptible to mechanical damage and microbial infection after harvest (Ban et al., 2015). Normally, at room temperatures, wolfberry has a very short shelf life, which changes color and taste after storage for 3–5 days. If stored at 4 °C, wolfberry will shrink due to water loss within 2–3 weeks. Therefore, drying is the most common preservation method for wolfberry. However, during drying, some

∗

functional nutrients such as amino acids, carotenoids, and polysaccharides are inevitably lost (Zhou et al., 2017; Zhu, Geng, & Sun, 2019; Zhu, Li, Sun, & Wang, 2019), and therefore effective methods for maximizing the retention of nutrients of wolfberry during storage are still highly needed for the industry. Liquid nitrogen spray freezing is a technique for rapidly freezing foods by instantaneous vaporization of liquid nitrogen. Compared with conventional freezing methods, it has the advantages of high heat transfer coefficient, fast freezing rate, short freezing time and forming small ice crystals (Lopkulkiaert, Prapatsornwattana and Rungsardthong, 2009; Zhu, Zhou, & Sun, 2019). If the freezing rate is high enough, the maximum ice crystal formation zone of the aqueous solution can be passed very quickly without crystallization (glass transition) (Slade and Levine, 1995; Mahato, Zhu, & Sun, 2019). In order to achieve a complete glass transition, a freezing rate of 106 K/s is normally required. However, for foods with high moisture contents, it is difficult to achieve such a high freezing rate. Therefore, the use of partial glass transition is a more common practice. Generally, the tissue

Corresponding author. School of Food Science and Engineering, South China University of Technology, Guangzhou, 510641, China.. E-mail address: [email protected] (D.-W. Sun). URLs: http://www.ucd.ie/refrig, http://www.ucd.ie/sun (D.-W. Sun).

https://doi.org/10.1016/j.lwt.2019.108923 Received 19 June 2019; Received in revised form 21 August 2019; Accepted 3 December 2019 Available online 04 December 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

LWT - Food Science and Technology 120 (2020) 108923

Z. Zhu, et al.

and holding for 2 min, then an increase from −70 °C to 15 °C at 10 °C/ min and holding for 2 min, followed by a decrease from 15 °C to −25 °C at 10 °C/min and holding for 30 min, then a further decrease from −25 °C to −70 °C at 5 °C/min and holding for 2min, and finally an increase from −70 °C to 15 °C at 3 °C/min.

structure of wolfberry would damage due to recrystallization of ice crystals during the process of frozen storage, causing an increase of drip loss, resulting in the loss of nutrients such as carbohydrate and vitamin, and even the deterioration of food quality (Cheng, Sun, Pu, & Wei, 2018; Li, Zhu, & Sun, 2018; Luo, Sun, Zhu, & Wang, 2018; Tian, Zhu, & Sun, 2019; Zhan, Sun, Zhu, & Wang, 2018; Zhan, Zhu, & Sun, 2019). For foods stored below the glass transition temperatures, the loss of nutrients can be greatly controlled, and liquid nitrogen freezing is a most common technique to achieve partial glass transition for food freezing (Jensen, Bo and Nielsen, 2003; Torreggiani et al., 1999). However, limited information is available for freezing wolfberry using liquid nitrogen spraying. Therefore the aim of the current study was to investigate the effects of liquid nitrogen spray freezing of wolfberry on the color, polyphenol oxidase and peroxide activity, distribution of water states and epidermal microstructure. It is hoped the results could be useful to the industry for better preserving wolfberry.

2.4. Measurement of color parameters The color of the samples was determined using a chromameter (CR400, Konica Minolta Inc., Tokyo, Japan). The color parameters recorded were luminosity value (L*), redness or greenness value (a*) and yellowness or blueness value (b*). The difference (ΔE) in color between the thawed and fresh samples was calculated by

(Lt∗ − Li∗)2 + (at∗ − ai∗)2 + (bt∗ − bi∗)2

ΔE =

(1)

where Li*, ai* and bi* indicates the color parameters of fresh wolfberry samples, and Lt*, at* and bt* refer to the color parameters of the thawed samples. Hue angle (H) and Chroma (C) (Agnelli and Mascheroni, 2002) were calculated using equations below, respectively:

2. Materials and methods 2.1. Wolfberry Wolfberry contains about 80% water, and the main chemical compositions include Lycium barbarum polysaccharides, carotenoid, alkaloids, and vitamin C. Fresh wolfberry was harvested in October 2018 from the farm of Zhongwei, Ningxia Province, China. After harvesting, samples were transported to the laboratory within two days by air courier in low temperatures. Wolfberry with no obvious crush damage and hardened flesh were selected for experiments. Prior to the experiment, all wolfberry samples were graded, and the average length and weight of the wolfberry were 3 ± 0.2 cm and 1.1 ± 0.1 g, respectively. Before the experiment, samples were rinsed with water, and the surface was dried with paper.

C=

a∗2 + b∗2

(2)

H=  

b∗ a∗

(3)

Browning index (BI) representing the intensity of brown color. BI values were calculated using Eq. (4) according to Jiang (2013).

a∗ + 1.75L∗ ⎞ − 0.31⎤ BI = 581.395 ⎡ ⎛ ∗ + a∗ − 3.012b∗ ⎥ ⎢ L 5.645 ⎠ ⎦ ⎣⎝

(4)

2.5. Measurement of PPO and POD activities The polyphenol oxidase (PPO) and peroxidase (POD) activities of the fresh and thawed wolfberry were measured using an ultraviolet spectrophotometer (L5S, INESA Analytical Instrument Co., Ltd., Shanghai, China). An amount of 1.5 g of wolfberry sample was placed in a mortar in an ice bath and ground for 10 min with 5 mL 0.1 mol/L phosphate buffer (PB) at pH 7.8 containing 5 mmol/L dichlorodiphenyltrichloroethane and 5% polyvinylpyrrolidone. The mortar was rinsed twice with a small amount of PB, and then all the slurry was transferred into a centrifuge tube. The homogenate was centrifuged at 10000 rpm using a high-speed refrigerated centrifuge (H2050R, Hunan Xiangyi Centrifuge Instrument Co., Ltd., Changsha, China) at 4 °C for 30 min. The supernatant was collected for the determination of the enzyme activities and the total volume of the supernatant was recorded. PPO activity was determined according to the method of Soliva, Elez, Sebastián and Martı́N (2000) with slight modification. An amount of 2 mL of PB and 0.5 mL 0.05 mol/L catechol were used as a reaction substrate, and 0.5 mL of enzyme extract was then added and mixed thoroughly. The solution without adding enzyme extract was used as a blank control. The mixture was quickly poured into a cuvette and the absorbance was measured immediately at 410 nm. The absorbance was measured every 30 s for 3 min after reaction for 0.5 min. The amount of enzyme causing a change of 0.01 absorbance per minute was used as one unit of PPO activity. The activity of PPO expressed as u/(g·min) was calculated by the following equation:

2.2. Freezing experiments The wolfberry was kept in a refrigerator (BCD-640WKGPZM, Hefei Midea Refrigerator Co., Ltd, Hefei, China) at 2 °C. Samples were frozen by liquid nitrogen spraying at −60 ± 2 °C, −80 ± 2 °C and −100 ± 2 °C in a liquid nitrogen freezer (QF60, Dejieli Refrigeration Technology Ltd., Shenzhen, China), which were designated as NF-60°C, NF-80°C and NF-100°C groups, respectively. For comparison, samples were also frozen in an air-blast freezer (CIE−SE7510-05F, Scicooling Science & Technology Co., Ltd., Beijing, China) with freezing air temperature of −40 ± 1 °C and air velocity of 0.75 m/s, which was designated as BF group. Temperatures of the wolfberry during freezing were monitored by using a thermocouple (T type, Omega Engineering Inc., CT, USA) inserted in the geometric center of the sample, which was connected to a datalogger (TC-08, OMEGA Engineering Inc. CT, USA). Data were recorded at an interval of 2 s. The freezing process continued until the center temperature reached the partial glass transition temperature (Tg) of wolfberry, which was measured experimentally. The frozen wolfberry was then stored below the glass transition temperature for three weeks. For index analysis, the frozen wolfberry was thawed at 2 °C for 12 h in the refrigerator. 2.3. DSC curve

Enzyme activity =

The DSC curve for determining glass transition temperature (Tg) of wolfberry was obtained based on the method described in Bell and Touma (2010) with slight modification using a differential scanning calorimeter (DSC) (DSC-214, Netzsch Scientific Instruments Co., Ltd, Selb, Germany) with nitrogen gas flow at 20 mL/min. An amount of 10–15 mg of samples was used, and the temperatures in the DSC program was set as follows: a decrease from 25 °C to −70 °C at 20 °C/min

ΔA 410 × VT W × Vs × 0.01 × t

(5)

where ΔA 410 indicates the change in absorbance within 1 min at 410 nm; VT represents the total volume of enzyme extraction solution (mL); W is the weight of the sample (g); Vs represents the volume of the enzyme extraction solution used in the test (mL); t is the total reaction time (min). 2

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POD activity was measured at 470 nm according to the method of Moerschbacher, Noll, Flott and Reisener (1988) with slight modification. The reaction solution contained 2.9 mL of PB, 0.1 mL enzyme extract, 0.05 mL 0.05 mol/L guaiacol and 10 μL of 0.04 mol/L hydrogen peroxide (H2O2). Similarly, the amount of enzyme causing a change of 0.01 absorbance per minute was used as one unit of POD activity. The solution without adding enzyme extract was used as a blank control. The POD activity was calculated using the equation below:

Enzyme activity =

ΔA 470 × VT W × Vs × 0.01 × t

(6)

where ΔA 470 indicates the change in absorbance within 1 min at 470 nm. 2.6. Distribution of water states Low field nuclear magnetic resonance (LF-NMR) can be used to determine water states and their variation and distribution in fruit from a microscopic perspective. The distribution of water states in wolfberry was determined according to the method described in Yang et al. (2015) with slight modification using an LF-NMR (mq20, Bruker Optics Ltd., Karlsruhe, Germany). Samples were placed at the bottom of the sample tube of the LF-NMR for detecting the distribution of states, with an echo time of 0.05 m s, the number of echoes of 20000, recycle delay of 2.5, a gain of 61 and the number of scans of 8 time. The area of each of the four peaks was calculated based on the obtained spectral data, and the sum of the four areas was taken as the total area. According to the total area, the percentage of each peak was calculated, respectively. All the tests were carried out at room temperature of 25 °C.

Fig. 1. DSC curve for determining the glass transition temperature of wolfberry.

Therefore, −42.1 °C was the starting temperature for the glass transition. In the temperature range from −42.1 °C to −35.2 °C, the enthalpy value increased continuously, however, further increase in the temperature to higher than −35.2 °C, the increase in enthalpy value became slower, and thus −35.2 °C was the termination temperature of the glass transition. In the glass transition temperature range from −42.1 °C to −35.2 °C, there existed an inflection point at −39.2 °C, which was therefore determined as the glass transition temperature (Tg) of wolfberry. Fig. 2 compares the changes in the center temperature of the wolfberry under different freezing processes. It was noted that the times to pass the maximum ice crystal formation zone were about 450 s, 150 s, 100 s and 70 s for BF, NF-60°C, NF-80°C, and NF-100°C, respectively. Therefore, BF had the lowest freezing efficiency while NF-100°C showed the highest, but there was no obvious difference between NF-80°C and NF-60°C. This was probably due to that some liquid nitrogen did not fully vaporize after spraying from the nozzle, and the heat transfer coefficient for liquid nitrogen freezing was higher than air-blast freezing. George (1993) reported that the heat transfer coefficient of the air-blast freezer was between 15 W/m2K and 30 W/m2K, which was less than one-third of the liquid nitrogen freezer with the transfer coefficient of more than 100 W/m2K.

2.7. Observation of epidermal microstructure The microstructure of wolfberry epidermis was observed using a microscope (DM1000 LED, Leica Microsystems GmbH, Wetzlar, Germany). Fresh and thawed samples were sliced into slices with less than 20 μm in thickness using a slicer (R136, Taiva Technology Industrial Co., Ltd., Hubei, China). Then, the epidermis was cut into 5 × 5 mm square pieces and sealed with a coverslip. The images were captured with a magnification of 40 times. All the tests were carried out at room temperature of 25 °C. 2.8. Statistical analysis All experiments were repeated at least three times and the results were expressed as mean ± standard deviation (mean ± std). The experimental data were analyzed statistically by SPSS software (SPSS Version 24.0, SPSS Inc., Chicago, USA). One-way analysis of variance (ANOVA) was performed on the data. A significant difference between the data was expressed as p < 0.05. 3. Results and discussion 3.1. Glass transition temperature and freezing curves of wolfberry In the glass state, the energy for molecular thermal motion is very low with only a small number of motion units, and the molecular chain is frozen, leading to almost no physicochemical reactions inside the food and minimum deterioration in food quality. The glass transition is a low-intensity-change process and the glass transition temperature range appears as a thermal enthalpy transition on the DSC curve. The DSC curve for determining the glass transition temperature of wolfberry is presented in Fig. 1. With an increase in temperature, the enthalpy value of the wolfberry increased gradually. At temperatures below −42.1 °C, the enthalpy value was almost unchanged, while above −42.1 °C, the sharp increase took place in the enthalpy value.

Fig. 2. Comparison of the freezing curves of wolfberry under different freezing processes. 3

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Fig. 3. Appearance of wolfberry under different freezing processes. (a) Fresh wolfberry, (b–e) wolfberry samples after BF, NF-60°C, NF-80°C and NF-100°C, respectively.

ΔE, and the values of hue angle (H) showed no obvious differences between the four groups.

3.2. Changes in color Fig. 3 shows the changes in the appearance of wolfberry under different freezing processes. The fresh wolfberry was bright in color, but the color deteriorated and became darker after freezing. With BF and NF-100°C, the epidermis of the wolfberry showed slight wrinkles. With BF, large ice crystals could form due to low freezing rate, while with NF100°C, although the formation of fine ice crystals was expected, thermal stress in the epidermis should be high due to the large difference between the freezing environment and the epidermis. In addition, the waxy layer on the wolfberry surface would suffer irreversible damage during freezing, causing a decrease in the water retention capacity of the wolfberry, and thus leading to shrinkage of the epidermis. Table 1 shows the variation of the surface color of wolfberry under different freezing processes. Compared with fresh wolfberry, L*, a* and b* values of the wolfberry indicated obvious changes after freezing with an increase of L*, and a* and b* decreasing significantly. The color difference (ΔE) for BF group was the smallest and similar to the fresh wolfberry, while the values of ΔE were no obvious differences for NF60°C, NF-80°C, and NF-100°C groups. In addition, both the chromaticity value (C) and the browning index (BI) exhibited the same tendency as

3.3. Changes in PPO and POD activities The browning of wolfberry is due to the enzymatic oxidation of phenolic compounds caused by PPO and POD enzymes, and these oxidation products generate brown substances by polymerization. PPO and POD enzymes in plant cells exist in both free and bound states (Zhu et al., 2018). The free PPO and POD enzymes mainly exist in the cell serum, and the bound PPO and POD enzymes are mainly present in organelles such as chloroplasts and vacuoles. These two states of enzymes can change after freezing, as freezing can partially inactivate the free state enzymes, while part of the bound state enzymes in the cell wall and other organelles can be released and converted into the free state due to freezing. Fig. 4 shows the effects of different freezing processes on PPO and POD enzyme activities in wolfberry, revealing that freezing temperatures significantly affected the activities of PPO and POD, which were reduced significantly after freezing. Low freezing temperatures led to low PPO activity, but the POD activity showed a tendency of decreasing 4

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Table 1 Color difference of wolfberry under different freezing processes. Samples Fresh BF NF-60°C NF-80°C NF-100°C

L* 48.27 50.20 50.07 50.67 51.28

a* ± ± ± ± ±

a

2.24 0.76b 0.71b 1.21b 1.03c

30.37 24.75 22.80 22.36 22.29

ΔE

b* ± ± ± ± ±

a

2.11 0.42b 1.97c 1.31c 1.10c

24.75 17.02 15.71 15.65 15.67

± ± ± ± ±

a

2.14 0.80b 1.27c 0.95c 0.69c

H a

0.00 ± 0.00 9.78 ± 0.81b 13.98 ± 0.34c 13.00 ± 0.51c 12.59 ± 0.43d

0.81 0.69 0.70 0.70 0.71

C ± ± ± ± ±

a

0.03 0.021b 0.023b 0.03b 0.06b

39.18 30.04 25.61 26.72 27.27

BI ± ± ± ± ±

a

112.22 ± 3.85a 74.65 ± 2.73b 62.44 ± 1.35c 65.23 ± 1.52d 66.02 ± 2.27d

0.85 0.76b 0.35c 0.73c 0.76c

Note: Superscript letters on each column represent significant differences (p < 0.05).

Fig. 5. Effects of different freezing processes on T2 relaxation time of wolfberry. Table 2 Effects of different freezing processes on the percentage of T2 relaxation peak of wolfberry. T21(%) Fresh BF NF-60°C NF-80°C NF-100°C

T22(%) a

4.37 ± 0.20 6.77 ± 0.15e 5.3 ± 0.05c 4.74 ± 0.13b 6.13 ± 0.09d

T23(%) a

5.26 ± 0.12 9.57 ± 0.19c 7.2 ± 0.29b 10.73 ± 0.51d 10.43 ± 0.32d

19.50 30.55 35.58 35.01 36.05

T24(%) ± ± ± ± ±

a

0.43 1.13b 1.15c 0.96c 0.86c

70.86 53.11 55.90 49.52 47.39

± ± ± ± ±

1.24a 2.01b 1.36b 1.03c 1.22c

Note: Superscript letters on each column represent significant differences (p < 0.05).

3.4. Changes in the distribution of water states Water in the food system exists in three states, including bound water, immobilized water, and free water. The water in wolfberry is mainly distributed in the cell wall, cell gap, cytoplasm and the vacuole, which migrates during freezing. LF-NMR technique is often used to detect the distribution of water states in a food system. In LF-NMR, the magnitude of T2 relaxation time has a strong positive correlation with the degree of freedom of protons, and the mobility of water molecules can thus be explained by the change in T2. The peak position of the T2 inversion map and the total of the signal values in each peak can represent different water states and their corresponding water contents. Fig. 5 shows the changes of T2 of wolfberry under different freezing processes, illustrating four relaxation time peaks including T21 (8.86 m s–29.14 m s), T22 (46.92 m s–105.86 m s), T23 (154.34 m s–348.25 m s) and T24 (355.23 m s–770.30 m s), corresponding to four kinds of water states in wolfberry tissue (Vicente, Nieto, Hodara, Castro and Alzamora, 2012). T21 was regarded as the bound water in the cell wall, which was tightly bound to some macromolecular substances such as cellulose and pectin and had poor fluidity. T22 and T23 were considered as the immobilized water in cell

Fig. 4. Effects of different freezing processes on the PPO and POD activities of wolfberry. (a) PPO activity, (b) POD activity. The different letters above the histogram represent the significant difference of each group (p < 0.05).

first and then increasing. Islam, Zhang, Adhikari, Cheng and Xu (2014) found that the faster the freezing rate, the more severe the decrease in PPO and POD enzyme activities. The possible reason for the results was that when the temperature of the enzyme solution was lowered more than 10 °C below its freezing point, the activity of the enzyme began to decrease, and the lower the temperature, the faster the decrease in enzyme activity. However, it was worth noting that the activity of POD enzyme in the NF-100°C group samples was increased. The possible reason was that the samples in NF-100°C group were subjected to a large thermal stress, causing great damage to the tissue structure, liberating more POD enzymes.

5

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Fig. 6. Micrographs (40X) showing the structure of inner epidermis of wolfberry under different freezing processes. (a) Fresh wolfberry, (b–e) wolfberry samples after BF, NF-60°C, NF-80°C, NF-100°C, respectively.

change in the position of the peak. Therefore, the proportion and fluidity of water in wolfberry changed after freezing as ice crystals produced by water crystallization would cause damages to the cell structure. Table 2 shows the changes in the percentage of T2 relaxation peak area of wolfberry under different freezing processes. The proportion of free water (T24) in wolfberry decreased drastically, and those of immobilized water and bound water increased to some extent. The more

gap and cytoplasm, which were combined chemically with substances such as proteins in the cytoplasm. T24 was the free water in vacuole with strong fluidity, which was loosely combined with some small molecules such as sugars in the vacuole. As shown in Fig. 5, compared with T2 relaxation peak of fresh wolfberry, the peak height and peak width of water in each component of the wolfberry changed greatly after freezing. The peak height of the water was slightly reduced, and the peak width was widened to a large degree, but there was no obvious 6

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and low POD activity, water distribution more similar to fresh samples and smaller damage of inner epidermal cell structures. BF produced wolfberry with a color close to the fresh samples, but changes in other quality indicators were not favorable. Therefore, it was suggested that NF-80°C would be the most appropriate freezing process for wolfberry freezing.

the reduction of free water, the greater the damage to the vacuole. During freezing, a large part of the water was transferred from the vacuole to the cytoplasm and cell gap, and only a small part of the water was transferred to the cell wall. The above results were consistent with those of Xu, Min, Bhandari, Cheng and Sun (2015), who studied the effect of ultrasound-assisted immersion freezing on water distribution of carrots, and concluded that the proportion of vacuole water in carrots decreased and the ratio of cytoplasmic and intercellular water increased after ultrasound-assisted immersion freezing. In general, the percentage of the bound water would not change during freezing as the small increase in the water percentage in the cell wall was due to the loss of water during freezing and thawing. The content of bound water might not change, but the total water content of the wolfberry would decrease after thawing, resulting in a small increase in the proportion of the bound water. Table 2 shows that the largest decrease in the proportion of free water was that from NF-100°C group, followed by NF-80°C group. The degree of cell damage was affected by both the thermal stress generated by temperature difference and the mechanical stress caused by ice crystals, while the degree of the vacuole tissue damage might be mainly controlled by the thermal stress. The samples of NF-100°C group would suffer large thermal stress during freezing, while those of NF-60°C group were similar to the fresh samples due to low mechanical and thermal stresses.

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors are grateful to the Key R&D Program of Ningxia Hui Autonomous Region (2018BCF01001) for its support. This research was also supported by the National Key R&D Program of China (2017YFD0400404), the Agricultural Development and Rural Work of Guangdong Province (2018LM2170, 2018LM2171, 2017LM4173), the Common Technical Innovation Team of Guangdong Province on Preservation and Logistics of Agricultural Products (2019KJ145), the Contemporary International Collaborative Research Centre of Guangdong Province on Food Innovative Processing and Intelligent Control (2019A050519001) and the Innovation Centre of Guangdong Province for Modern Agricultural Science and Technology on Intelligent Sensing and Precision Control of Agricultural Product Qualities.

3.5. Changes in epidermal microstructure The epidermis of wolfberry is divided into three layers. The outer layer is composed of some palisade tissue cells, while the middle one consists of some spongy tissues with large intercellular space, which accounts for most of the epidermis. Stone cells are mixed between the outer epidermis and middle epidermis. The inner layer is a thin membrane consisting of some parenchyma cells with relatively close tissue. Relative to the outer and the middle epidermis, the inner epidermis is a thin film and is in contact with the flesh, and damage due to freezing is greater with even possible breaking of the cells. Fig. 6 compares the microstructure of the inner epidermis of wolfberry under different freezing processes, indicating a great influence of the freezing temperature on the inner epidermal structure. The microscopic image of the fresh wolfberry (Fig. 6(a)) showed an integral cell structure with normal morphology. For BF samples (Fig. 6(b)), the pigment particles inside the cells had the tendency to extravasate and the hierarchical structure between cells was confusing and it was even difficult to distinguish a relatively integral cell, indicating the severe damage of the cell structure. Fig. 6(c) shows that for NF-60°C samples, the hierarchical structure between cells was slightly confusing, but the cell morphology was normal and integral cells could be identified, while for NF-80°C samples, Fig. 6(d) indicates that the hierarchical structure between cells was clear with good cell integrity and no extravasation of pigment particles existed, which was similar to the fresh sample. However, for NF-100°C samples, the cell structure was altered with slight rupture in the cell layer and only approximate morphology of the cells could be recognized. NF-80°C was considered the most appropriate freezing process for protecting the integrity of the epidermal structure of wolfberry.

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4. Conclusions This study showed that freezing temperature affected the freezing characteristics of wolfberry, however, there was no evidence to prove that the lower the temperature, the better the freezing characteristics of wolfberry. The changes in color, activities of PPO and POD, water migration distribution and inner epidermal microstructure under different freezing processes were compared. NF-100°C exhibited advantages including short freezing time and low PPO activity. However, compared with NF-100°C, NF-80°C produced wolfberry with better sensory quality 7

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