Evaluation on simulative transportation and storage quality of sweet cherry by different varieties based on principal component analysis

Sweet cherry, which peel is thin, is rich in nutrition, and the flesh is soft and juicy. In recent years, sweet cherry has been welcomed by consumers because of its unique taste and strong antioxidant function (Gao et al., 2017). Sweet cherry is suitable for growing in tropical and subtropical areas with abundant rain, sunny and suitable temperature (Tokatlı & Demirdöven, 2020). In order to eliminate the regional limitation of sweet cherry and improve its commercial value, cold chain transportation and storage has become an inevitable development trend (Alique et al., 2005).


Introduction
Sweet cherry, which peel is thin, is rich in nutrition, and the flesh is soft and juicy. In recent years, sweet cherry has been welcomed by consumers because of its unique taste and strong antioxidant function (Gao et al., 2017). Sweet cherry is suitable for growing in tropical and subtropical areas with abundant rain, sunny and suitable temperature (Tokatlı & Demirdöven, 2020). In order to eliminate the regional limitation of sweet cherry and improve its commercial value, cold chain transportation and storage has become an inevitable development trend (Alique et al., 2005).
In the whole cold chain process, transportation and storage, interacting parts between the fruit and the box or fruits will be subjected to various pressures, such as squeezing, friction, collision and impact (Wei et al., 2019). When the relative strength of these forces that damage the fruit is relatively high, the fruit will be damaged by the shallow surface, and due to the repeated action of the external force, the connection force between the fruit cells and the strength between the cells will change, resulting in brittleness or plasticity of the fruit injury makes the fruit soft (Wei et al., 2019;Zhou & Wu, 2018). Directly damages the plasma membrane of the damaged area, softens it and reduces its oxidation resistance (Cliff & Toivonen, 2017).
At present, a lot of research has been carried out on post-harvest storage and preservation technologies of sweet cherry, such as modified atmosphere preservation method (Cozzolino et al., 2019), preservation of fungicides, chemical preservation method (Ni, 2018;Yang, 2009;Ma et al., 2019a), and physical preservation method (Michailidis et al., 2019;Tian et al., 2019). However, there are relatively few studies on the effect of different circulation and packaging conditions on fruit quality during transport.
The red cherry "Summit" has small epidermal cells, dense arrangement, thick surface wax and cell walls, and uniform internal cell size, so has good storage and transportation characteristics. The yellow cherry "Nanyang" has a loose epidermis and internal cell arrangement, and the surface layer is not waxy, which is extremely resistant to transportation. In this paper, "Summit" and "Nanyang" were used as experimental samples, studied the effect of transport vibration on the nutritional quality and antioxidant activity of sweet cherry fruit during cold chain transportation. The results will provide a reference for the development of post-harvest long-distance logistics transportation and storage technology development.

Sample preparation and treatments
The test sweet cherries were picked from orchards in Dalian, Liaoning Province, China. Then they were quickly transported to the laboratory. Sweet cherries with a uniform size (6 ± 2 g) and no obvious mechanical damage was selected as the test sample. Samples of each variety were randomly divided into two groups for processing. Each group was treated as follows: (1) Control group processing: 0 h treatments were used as the control group; (2) Vibration group processing: two layers of paper towels were glued on the bottom and around the incubator. Sweet cherries were put into the incubator gently, and the surface was covered with two layers of paper towels. The sealed insulation box was put into a constant temperature culture oscillator to simulate the mechanical vibration during road transportation. The conditions were set by the constant temperature culture oscillator: vibration frequency (120 r / min), temperature (4 °C), relative humidity (90-95%) and time (4 h). To simplify recording, the "Summit" control and vibration treatment were referred as S-CK and S-V; the "Nanyang" control and vibration treatment were referred as N-CK and N-V.

Determination of soluble solids content, titratable acid and vitamin C
The content of soluble solids (SSC) was determined according to the method described by NY/ T2637-2014T2637- (China, 2014. Titratable acid (TA) and vitamin C (VC) were determined by Hernández-Muñoz et al. (2008).

Determination of total phenol content and flavonoids
The total phenol content (TPC) was determined by the Folinphenol colorimetric method (Rodov et al., 2010). Flavonoids content was determined by aluminum nitrate colorimetric method (Jiao et al., 2017).

Determination of polyphenol oxidase
The activity of sweet cherry polyphenol oxidase (PPO) was extracted according to the method described by Cao et al. (2007) with slight modifications. Crude enzyme extract (100 μL) was incubated with a buffered substrate (1.0 mL 50 mmol/ L catechol in 4 mL sodium phosphate buffer pH 7.8), and monitored by measuring the increase in absorbance at 398 nm.

Determination of peroxidase
The activity of cherry peroxidase (POD) was determined by Cao et al. (2007).

Determination of catalase
Catalase (CAT) was extracted according to the method described by Pasquariello et al. (2015) with slight modifications. The reaction solution contained 0.2 mL enzyme solution, 1.5 mL 50 mmol/L phosphate buffers (pH 7.8) and 0.3 mL 10mmol/L H 2 O 2 the time was counted immediately and the absorbance was measured at 240 nm.

Determination of ascorbate peroxidase
The activity of ascorbate peroxidase (APX) was determined according to the previously published method (Cao et al., 2007).

Determination of superoxide anion
The content of superoxide anion radical (O 2 -) was modified according to the previously published method (Zhao et al., 2008).

Determination of DPPH free radical scavenging rate
The DPPH free radical scavenging rate (DPPH) was determined according to the method of Chavan et al. (2013) with slight modifications. The reaction solution contained supernatant (0.2 mL) and DPPH solution (2.8 mL). Thoroughly mix, and leave the water bath at 37 °C for 30 minutes, and then determination of absorbance at 517 nm, which was referred to as A i . At the same time, the absorbance A 0 of DPPH solution mixed with anhydrous ethanol (1:1) and the absorbance A j of mixed supernatant solution and anhydrous ethanol (1:1) were determined (Equation 1).

Determination of agreement of basic telecommunications services
The Agreement of Basic Telecommunications Services (ABTS + ) free radical scavenging rate was determined according to the method of Zhang et al. (2019).

Statistical analysis
Analyses of data were carried out by one-way ANOVA in SPSS 19.0 for Windows (SPSS Inc., Chicago, IL, USA). Statistic differences were considered to be significant at p < 0.05. All results were expressed as the mean ± SE. Difference significance among treatments was analyzed by Duncan's multiple range test.

SSC, TA and VC
SSC was an important nutritional indicator of fruits and had a strong relationship with the acceptability of consumers, reflecting the sugar, acid, vitamins, minerals and other nutrients in the fruits (Cliff & Toivonen, 2017). As shown in Table 1, SSC first increased slowly and then decreased during the storage and transportation period. The content of SSC in the vibration treatment group of "Summit" or "Nanyang" was lower than All means in the same row followed by different letters (A-D) are significantly (p < 0.05) different by Duncan's multiple range test. All means in the same column followed by different letters (a-d) are significantly (p < 0.05) different by Duncan's multiple range test. Values are mean ± SE of triplicate samples. Bars represent SE (p < 0.05). All samples were prepared in triplicates.
that in the control treatment groups. The vibration treatment group reached the maximum value on the 10th day, while the control treatment group on the 12th day. At the 14th day, SSC of the S-V and N-V were 2.44% and 4.92% lower than that of S-CK and N-CK respectively. SSC included soluble proteins and soluble sugars. During storage period, basic physiological activities were maintained and the substances such as soluble solids were accumulated. However, with the ripening and aging of sweet cherry, the fruit itself cannot make up for consumption, so the soluble solid content gradually decreased.
TA is also an important part of the quality of the fruits (Yang, 2009). The organic acid content has an important influence on the taste, flavor, sugar-acid ratio and pH of fruits (Fan et al., 2014). As shown in Table 1, TA content of sweet cherry in each treatment group showed a downward trend. Whether it was "Summit" or "Nanyang", the TA content of the vibration treatment group was always lower than that of the control treatment group. At the 14th day, the decline trend of the S-V was the most obvious, with a decline of 32.14%. From this, it could be seen that the simulated transport vibration treatment promoted the consumption of the TA content of the sweet cherry.
VC is an important nutrients component, but it is easily decomposed during storage. The change of VC content reflects the freshness of fruits, and it is also one of the indicators for judging the freshness preservation effect (Xu et al., 2017). As shown in Table 1, the VC content of all treatment groups gradually decreased. The simulated transport vibration treatment accelerated the decomposition of VC in sweet cherry, and the VC content of the S-V decreased more rapidly than the N-V. At the 14th day, the decline rates of S-CK, S-V, N-CK and N-V were 48.55%, 64.96%, 45.85% and 56.24%, respectively.

TPC and flavonoids content
After mechanical damage, fruits often produce a series of secondary metabolites, such as phenols, flavonoids, and alkaloids. These substances are mainly concentrated in the wound and its adjacent parts, and participate in the formation of callus and resist the invasion of insects or germ (Ma et al., 2018). As shown in Figure 1a, TPC of each treatment group increased first and then decreased, and the content of the vibration treatment group was significantly higher than that of the control treatment group. In the early storage period, the TPC of S-V was at a relatively high level, and in the later storage period, the total phenol content of N-V was significantly higher than that of other treatment groups. On the 14th day, the total phenol contents of S-CK, S-V, N-CK and N-V were 1.45, 1.75, 1.50 and 2.08, U, respectively. This indicated that simulated transport vibration accelerated the TPC of sweet cherries.
As shown in Figure 1b, the flavonoids content of sweet cherry in each treatment group first increased and then decreased, and its peak value appeared on the 8th day of storage. The peak values of S-CK, S-V, N-CK, and N-V were 30.08, 36.59, 39.32 and 42.38, mg/g, respectively. On the 14th day, the flavonoid content of S-V was 18.39% higher than that of S-CK, and the flavonoid content of N-V was 3.59% higher than that of N-CK. The results indicated that simulated transport vibration increased the secondary metabolites of sweet cherry. The increase of secondary metabolites is also a stress response of fruits to injury stress, and a self-protection effect of fruits against adversity.

PPO and POD
PPO is a copper-based enzyme that exists in the free form of plant cytoplasm or bounds to mitochondria, chloroplasts and other subcellular organelles (Ma et al., 2019a). It catalyzes the reaction of various phenols with O 2 to form hydrazine, which is also a major part of terminal oxidase. PPO is a key enzyme that promotes enzymatic browning of fruits. As shown in Table 2, the PPO activity of sweet cherry showed an upward trend. On the 12th day, the PPO activity of each treatment group increased significantly. The activity of the S-V and N-V were higher than that of the corresponding control treatment group. Among them, the activity of the S-V was always at the highest level.  This indicates that simulated transport vibration significantly increased the PPO activity of sweet cherry. Moreover, compared with "Nanyang", the simulate transport vibration treatment made the PPO activity of the "Summit" easier to rise. Studies showed that PPO activity could increase significantly when plant tissue was susceptible or causes damage under other adverse conditions, and it played a protective role.
POD is one of the important enzymes in the active oxygen free radical scavenging system (Ma et al., 2019a). It plays a role in the decomposition of H 2 O 2 in the last step of lignin biosynthesis and has a certain effect on the color change of fruits. The increase of POD activity is an important sign of the ripening and aging of fruit tissues (Chen et al., 2019). POD can be activated by H 2 O 2 , which triggers a series of toxic effects on tissues. The mechanism is that POD degrades the indoleacetic acid in fruit tissues by catalytic oxidation (Zhong & Zhu, 2004). As shown in Table 2, the POD activity showed an increasing trend. At 8 days after storage, the POD activity of each treatment group changed slowly, and the activity of POD increased rapidly on the 10th day. And during the whole storage period, the POD activity of S-V and N-V were always higher than the corresponding control treatment group. It indicated that simulated transport vibration promotes an increase in POD activity of sweet cherry.

CAT and APX
CAT is a protective enzyme in the ripening and aging process of fruits, and one of the key enzymes in the biological defense system. It can decompose H 2 O 2 into oxygen and water, remove H 2 O 2 and prevent cells from being poisoned by H 2 O 2 , so as to reduce damage to the membrane and achieve the purpose of delaying cell senescence . As shown in Table 2, the CAT activity of each treatment group showed a slow increase first, and reached the highest on the 6th day, then decreased rapidly. This may be due to the accumulation of H 2 O 2 during the early storage period of sweet cherries, which induced the increase of CAT enzyme activity. When the accumulated amount is not cleaned in time, it is possible to inhibit the CAT enzyme activity. Meanwhile, The CAT activity of S-V and N-V were always higher than S-CK and N-CK, respectively. It indicated that simulated transportation vibration promoted the expression of CAT activity of sweet cherry. APX is a highly specific peroxidase that exists in the fruit. It can catalyze the oxidation-reduction reaction of ascorbic acid with H 2 O 2 to achieve the purpose of removing H 2 O 2 and thus limit the reaction between O 2 and H 2 O 2 to produce peroxides such as ·OH . As shown in Table 2, the APX activity increased first and then decreased. The content of each treatment group reached the maximum value on the 4th day of storage. The APX activity of the S-CK, S-V, 9.78,10.60 and 9.40, U/g·min FW. The activity levels of S-V and the N-V were lower than the corresponding control treatment group, and the activity of S-CK was always at the highest level, while the activity of N-V was always at the lowest level.

O 2 production rate
During the storage and transportation of fruits, the intracellular free radical metabolism balance is often disrupted, accumulating a large amount of free radicals and triggering or aggravating lipid peroxidation, resulting in the loss of the cell plasma membrane system. Free radicals are atoms or groups of atoms with unpaired valence electrons. Oxygen molecules that enter the body through respiration receive only one electron and are converted into O 2 -. O 2 acts with other oxygen free radicals on biofilms, attacks membrane proteins, and causes the unsaturated fatty acids on the membrane undergo peroxidation, which destroys the membrane system (Nan et al., 2017). Table 3, the O 2 tended to rise as a whole. At the beginning of storage, the content of O 2 in each treatment group increased slowly. From the 6th day, the content of O 2 increased rapidly. The content of O 2 in vibration treatment groups were always higher than that in control treatment groups, and the increase of N-V was the most obvious. This indicates that simulated transport vibration increased the rise of O 2 of sweet cherry.

MDA content
Lipids exist on the cell membrane, which are related to the stress resistance of the fruit during storage. During the storage process, with the content of fatty acids decreases, the peroxidation of internal membrane lipids and the increase of active oxygen and free radicals will destroy the structure and function of the membrane, thereby destroying the structure of the cell membrane, enhancing permeability and graduality oxidation and aging of fruits (Nan et al., 2017). MDA is an important product of membrane peroxidation, which can directly produce toxic effects on cells, caused cross-linking and inactivation of enzyme proteins in biofilms, lead to membrane pores and enhance permeability. MDA can be used as membrane quality index of peroxidation.
With the extension of storage time, MDA content of sweet cherry increased gradually. At the same time, MDA contents of simulated transportation vibration treatments S-V and N-V were higher than their control treatments, and MDA content of N-V was always at the highest level during the later stage of storage (Table 3). It showed that simulated transportation vibration increased the degree of peroxidation of sweet cherry membrane lipids.

Effect of the antioxidant capacity by simulated transport vibration in sweet cherry.
According to the DPPH and ABTS + methods which determine the antioxidant capacity, the higher clearance value indicated higher antioxidant capacity (Ma et al., 2019b;Pasquariello et al., 2015). As shown in Table 3, their antioxidant capacity decreased generally, and the antioxidant capacities of S-V and N-V treatment were lower than that of its control treatment, respectively. In the early storage period, the antioxidant capacity of "Summit" was always higher than that of "Nanyang". In the later stage, the result is just the opposite.  All means in the same row followed by different letters (A-E) are significantly (p < 0.05) different by Duncan's multiple range test. All means in the same column followed by different letters (a-d) are significantly (p < 0.05) different by Duncan's multiple range test. Values are mean ± SE of triplicate samples. Bars represent SE (p < 0.05). All samples were prepared in triplicates.

Correlation ability
In order to determine the effects of antioxidant components and simulated transportation vibration on the antioxidant capacity of sweet cherry, the correlation of between antioxidant indexes and antioxidant capacity were analyzed (see Figure 2). DPPH free radical scavenging ability was positively related to TA, flavonoids, PPO, POD, CAT and APX, but negatively correlated with O 2 -, MDA and TPC. ABTS + free radical scavenging ability was positively correlated with TA, VC, flavonoids and O 2 -and MDA.
The correlation results of 13 indicators showed that TA, VC, TPC, flavonoids, PPO, POD, CAT, APX, O 2 and MDA of sweet cherry were important antioxidants. They can directly affect the antioxidant capacity of sweet cherry. The antioxidant capacity of vibration groups was become weaker, which may be due to their lower retention of antioxidant components.

Principal component analysis
The principal component analysis (PCA) showed that the cumulative variance contribution rate of the first three principal components reached 88.97% (see Table 4), which indicated that these three principal components could accurately and objectively reflect the similarity between samples, so a total of three principal components were extracted (Nascimento et al. 2020;Fernandes et al.. 2019).   Table 5 and Table 6)

PCA analysis of shelf quality of sweet cherries with different treatments
According to the results of PCA, the contribution rate of the first, second and third principal component was 42.896%, 35.027% and 11.047%, respectively (Table 4), and the cumulative contribution rate was 88.970%. Three principal components were used to evaluate the different treatments of sweet cherry, and the comprehensive score (F) was calculated (Equation 2). where: F 1 , F 2 , F 3 and F were the score of the first, second, third principal component and a comprehensive score. The higher the F score, the better the quality (Ma et al., 2018).
As shown in Table 7, the F of each group showed a downward trend with the prolongation of storage time. After 14 days storage, the comprehensive score relationship of each group was: S-CK> N-CK> S-V> N-V. This showed that the simulated transport vibration accelerated the quality degradation of sweet cherry during simulated transport storage. Meanwhile, transportation vibration stress had a greater impact on the nutritional quality and antioxidant activity of "Nanyang", a sweet cherry with poor storage resistance.

Conclusions
Transport vibration often leads to the damage of fruit surface tissue and physiological abnormalities inside the fruit, thus accelerating the softening and aging of fruit. Among them, apparent damage of the fruit which is visible to the naked eye is easy to find and deal with in time. The damage of internal tissue caused by transportation vibration is most easily overlooked and the damage of fruit is more serious. The results of this study indicated that transportation vibration has a great impact on the nutritional quality of sweet cherry, and that there is a strong correlation between antioxidant activity and nutritional quality. In order to maintain the original quality of the sweet cherry fruit, the logistics time should be shorten, and it is necessary that vibration reduction packaging measures should be taken to reduce the damage of vibration to the fruit. 1.18 ± 0.08Ha 1.03 ± 0.09Ga 0.98 ± 0.05 Fa 0.85 ± 0.09Ea 0.67 ± 0.07Da 0.23 ± 0.05Ca -0.01 ± 0.09Ba -0.15 ± 0.08Aa S-V 1.09 ± 0.09Ha 0.95 ± 0.08Gb 0.88 ± 0.08Fc 0.62 ± 0.05Ec 0.43 ± 0.09Dc 0.15 ± 0.08Cb -0.12 ± 0.06Bc -0.24 ± 0.09Ac N-CK 1.12 ± 0.03Hb 1.01 ± 0.02Ga 0.92 ± 0.06Fb 0.81 ± 0.06Eb 0.59 ± 0.06Db 0.21 ± 0.07Ca -0.06 ± 0.07Bb -0.18 ± 0.05Ab N-V 1.01 ± 0.08Hb 0.89 ± 0.06Gc 0.76 ± 0.04Fd 0.59 ± 0.09Ed 0.38 ± 0.07Dd 0.11 ± 0.06Cc -0.15 ± 0.08Bd -0.31 ± 0.08Ad All means in the same row followed by different letters (A-H) are significantly (p < 0.05) different by Duncan's multiple range test. All means in the same column followed by different letters (a-d) are significantly (p < 0.05) different by Duncan's multiple range test. Values are mean ± SE of triplicate samples. Bars represent SE (p < 0.05). All samples were prepared in triplicates.