Effects of salicylic acid combined with gas atmospheric control on postharvest quality and storage stability of wolfberries: Quality attributes and interaction evaluation

Wolfberry ( Lycium barbarum ) is abundant in bioactive substances but prone to rapid deterioration after harvest, and thus wolfberry is usually dried. However, drying of wolfberry requires high energy consumption and can normally lower wolfberry bioactive contents. Cold storage, edible coating, and modified atmosphere packaging have been developed to preserve wolfberry but scarce data are available on postharvest quality and storage stability. In particular, the combined effects of salicylic acid (SA)

atmosphere (CA) on wolfberry quality have not been investigated. In the current study, SA and gas atmospheric control were applied either separately or in combination to explore changes in physiochemical properties during storage. Results showed that color quality and weight loss (WL) were the main indicators of quality change during storage, with high color quality and low WL representing excellent quality. SA and hypoxia (controlled and modified atmosphere conditions) could synergistically suppress respiration rate and antagonistically maintain ascorbic acid (AsA) and color quality, while SA could alleviate physiological disorder. Principal component analysis and cluster analysis verified that CA and SA + CA were excellent in enhancing the stability during storage and exhibited minimal quality deterioration. Thus, combining SA and gas atmospheric control could serve as a potential postharvest hurdle technology for preserving wolfberry.

Practical Applications
As a valuable and prestigious traditional Chinese medicine and food material, wolfberry (Lycium barbarum) is highly susceptible to mechanical damage after harvesting and presents a short shelf-life due to rapid softening. Wolfberry is thus usually consumed in dry form. The current study investigated the effects of salicylic acid (SA) and control atmosphere (CA) applied either separately or in combination on the changes in physiochemical properties during storage, and results showed that both CA and SA + CA storages possessed excellent abilities in preserving fresh wolfberry fruit. Therefore, the current study indicated that combining SA and gas atmospheric control could serve as a potential postharvest hurdle technology for preserving wolfberry, which should be very useful for the agriculture and food industry.
Previous studies have revealed that low-oxygen (O 2 ) and highcarbon dioxide (CO 2 ) concentration environment can significantly preserve fruit and vegetables during storage with long shelf-life by inhibiting respiration rate (RR) (Cliffe-Byrnes & O'Beirne, 2007;Wang et al., 2017). Therefore, gas atmospheric control including modified atmosphere packaging and controlled atmosphere (CA) have been widely applied in preserving perishable fresh products such as mushroom (Cliffe-Byrnes & O'Beirne, 2007), broccoli (Wang et al., 2017), peach (Girardi et al., 2005), and loquat (Ding, Chachin, Ueda, Imahori, & Wang, 2002). However, several studies have also reported that respiration is partly required for postharvest quality maintenance by energy status and cell redox state regulation (Cao, Cai, Yang, Joyce, & Zheng, 2014;. SA has shown important roles in regulating energy status to reduce bacterial infection in longan , alleviate chilling injury in plum (Luo, Chen, & Xie, 2011), and preserve nutrients in various berries (Babalar, Asghari, Talaei, & Khosroshahi, 2007;Cai et al., 2014;Valero et al., 2011;Xu, Liu, Xu, & Fu, 2019). It has also been proved that antioxidant capacity can be enhanced after SA treatment to balance the redox state of cell during storage (Cheng, Zhu, & Sun, 2021;Valero et al., 2011). Therefore, treatment with SA might be a potential strategy for compensating gas atmospheric control.
Although gas atmospheric control and SA have been applied in the agricultural industry (Valero et al., 2011;Wang et al., 2017), there are scarce reports about their effects on product quality, balancing energy status, and respiratory suppression. In particular, The combined effects of atmospheric control and SA treatment on quality and storage stability of wolfberry fruit have not been reported. Therefore, the current study aimed to investigate the impacts of atmospheric control combined with SA for the quality stability of wolfberry during storage and reveal the relationship between quality attributes and treatments. Principal components analysis (PCA) and cluster analysis were also employed to determine the main factors affecting wolfberry quality changes as well as the interactions between gas concentrations and SA and shelflife (Tian, Zhang, Zhu, & Sun, 2021). Results from the current study should provide useful guidance for using this postharvest hurdle technology to preserve other perishable agricultural products in the future.

| Wolfberry fruit
Wolfberry (Lycium barbarum L.) fruit were hand-harvested at commercial maturity stage (total soluble solids of 19.5-21.0% and titratable acidity of approximately 0.2%) from a local farm (Zhongwei, Ningxia, China). The fresh fruit were transported at 4.0 ± 3.0 C to the laboratory within 24 hr. The selected fruit were of similar size and weight without surface injury and a total of 5,504 g wolfberry fruit were used in the current study, which was randomly divided into 172 batches, with each batch containing 32.0 g wolfberry fruit as one replication.
Four batches (128 g fresh wolfberry) representing four replicates without any treatments were used as Fresh for measuring the quality attributes of the fresh fruit. The remaining 168 batches were divided into six lots with each lot containing 28 batches (a total of approximately 896 g wolfberry fruit per lot) for the following treatments: three lots were immersed in distilled water for 6 min and the other three lots were immersed in 2.0 mmol SA for 6 min. After immersion treatments with distilled water and SA, the fruit were air-dried and then stored under the following three different conditions: 1. Ambient air condition: One lot soaked with distilled water (defined as control check [CK] group) and one with 2.0 mmol SA (defined as SA + CK group) were stored without any package in a biochemical incubator (LHS-250HC-II, Shanghai Yiheng Technology Co., Ltd., Shanghai, China) at 7.0 ± 0.5 C and 93 ± 3% relative humidity (RH).
2. CA condition: One lot soaked with distilled water and one with 2.0 mM SA were stored in a 43-L acrylic sealed-chamber incubator (IQ 822,Yamato Scientific Shanghai Co.,Ltd.,Shanghai,China) without package, and the gas concentration, temperature and RH in the chamber were controlled at 5.0% O 2 and 15.0% CO 2 by a 0.13 L s À1 mixture flow of O 2 , CO 2 , and N 2 , 7.0 ± 0.5 C and 93 ± 3% RH, respectively. These two lots were defined as CA group, and salicylic acid and controlled atmosphere group (SA + CA), respectively.
3. Modified atmosphere condition: The last one lot soaked with distilled water and the last one with 2.0 mmol SA were packaged in modified atmosphere and stored the biochemical incubator at 7.0 ± 0.5 C and 93 ± 3% RH. The packaging was covered by a high barrier film (Bimes Dongguan Wonderful Packing Co., Ltd., Guangdong, China) with a thickness of 30 μm and a surface area of 0.0013 m 2 . The O 2 permeance and water vapor transmission rate of the film were 24 ml m À2 day À1 and 8.0 g m À2 day À1 , respectively. The O 2 concentration inside the package started with 21.0% and gradually decreased during storage. These last two lots were defined as modified atmosphere packaging group (MAP) and SA and modified atmosphere packaging group (SA + MAP), respectively.
A T-type thermocouple thermometer (5SRTC-TT-T-30-36, Omega Engineering Inc., Norwalk, CT) and a hygrometer (O-257, Dretec Co., Ltd., Saitama, Japan) were used to monitor the chamber temperature and RH, respectively. Quality attributes including RR, WL, TSS, AsA, F, L * , a * , b * , ΔE, and h were investigated every 4 days during the storage of 28 days, and four replicates were tested for each measurement.
After the quality determination, the samples were frozen using liquid nitrogen and grounded into powder using a tissuelyser (Tissuelyser-64, Shanghai Jingxin industrial development Co., Ltd., Shanghai, China) and stored in a biomedical freezer at À80 C (DW-40L188, Haier Co., Ltd., Tsingtao, China) for further analysis.

| Headspace gas composition and RR analysis
About 30.0 g wolfberries were sealed in 14 Â 30 cm high barrier pouches and incubated in darkness for 3 hr. The headspace gas composition was detected using a gas analyzer (CheckMate 3, Dansensor A/S Co., Ltd., Ringsted, Denmark). The volume of headspace gas (V) was measured using a water displacement method reported by Wang et al. (2017) with some modifications. The RR was calculated using the modified equations reported by Lamikanra, Imam, and Ukuku (2005).
where n is the number of moles (mol), p represents the gas pressure (Pa), V means the volume of headspace gas (m 3 ), R indicates the ideal gas content (J mol À1 K À1 ), T is the incubation temperature (K), P represents O 2 concentration (%) in the headspace, subscripts 0 and t mean the initial and the final state, respectively, m is the mass of the wolfberry (kg), t is the time (hr), U represents the RR (mmol kg À1 hr À1 ).

| Weight loss
The mass of wolfberries before (m 0 ) and after (m t ) storage was recorded, and the weight loss (WL) during storage was determined as:

| Total soluble solids
The five randomly selected wolfberry fruits were squeezed and about 0.2 ml juice filtrate was then analyzed using a digital refractometer (PAL-1, ATAGO Co., Ltd., Tokyo, Japan) for total soluble solids (TSS) determination. After each measurement, the prism of the refractometer was calibrated using distilled water.

| Ascorbic acid content
where c means the measured value of AsA (mg L À1 ), m is the mass of the wolfberry powder (kg), v represents the total volume of metaphosphoric acid solution (L).

| Firmness
Fifteen wolfberry fruits were randomly selected from each replicate, and the firmness (F) test was carried out using a penetrometer (FT-02, Wagner Instruments, Greenwich, CT) equipped with a cylindrical metal probe (6 mm in diameter).

| Color quality
Thirty wolfberry fruits were randomly selected for color quality determination using a colorimeter (CR-400, Konica Minolta Inc., Tokyo, Japan), and parameters including L * , a * , and b * values in the CIE color system were used to demonstrate the wolfberry color in three dimensions, where L * , a * , and b * values represent lightness, red-green, and yellow-blue values, respectively. The total color difference (ΔE) between the stored wolfberry and the Fresh was calculated with the equation below: Hue angle (h ) was used to reflect the color variation and was calculated as:

| Statistical analysis
The data were analyzed using SPSS 25.0 (SPSS Inc., Chicago, IL) and were displayed as means ± standard deviations (SD). One-way analysis of variance (ANOVA) was performed using Duncan's test at a significant level of p <.05. Besides, principal component analysis (PCA) and cluster analysis were applied to visualize the comprehensive quality variations. and Zhang, Zhang, and Yang (2015) revealed that SA could reduce O 2 consumption in bananas and cucumbers, respectively. These reports supported the current results that SA treatment showed more efficiency in slowing down O 2 consumption and CO 2 production in SA + MAP group than those in MAP group, especially from 0 to 4 days and the period after 16 days.

| Respiration rate
Respiration is closely related to shelf-life of fresh products, which can influence energy status, reactive oxygen species (ROS) production, and nutrients consumption Tian, Qin, & Li, 2013). In the current study, the RR was reduced in SA treated groups and hypoxia groups ( Figure 3). Combination of SA and hypoxia had a more evident suppression on RR during the storage. Especially, SA immersion showed significant inhibition on RR when O 2 concentration was less than 4.0% and CO 2 higher than 16.0% (Figures 1-3

| Quality evaluation of the stored wolfberry
Wolfberry can be better preserved by hurdle technology (Ban et al., 2015). Table 1 illustrates that the CK wolfberries deteriorated rapidly and lost its consumer acceptability after storage for 28 days. The wolfberries treated with SA showed better color quality and consumer acceptability, especially under hypoxia conditions. To further elucidate the quality variations between the Fresh and the stored wolfberries, a comparison of quality attributes including RR, WL, TSS, AsA, F, and color quality (L * , a * , b * , h , and ΔE) are summarized in Tables 2 and 3. Wolfberries stored in different groups suffered various degrees of deterioration. CA, SA + CA, and SA + MAP groups still presented commercial value after storage for 28 days at 7.0 ± 0.5 C, which was much better than conventional treatments (Ban et al., 2015;.
The WL of fresh products can normally indicate the degree of quality deterioration of the products, which is mainly caused by respiratory transpiration, membrane permeation imbalance and tissue damage (Li, ) and CO 2 ( ) concentrations in CK (▫), CA ( ), and MAP ( ) storage. Values are means ± SD of four replicates ) and CO 2 ( ) concentrations in SA (▫), SA + CA ( ), and SA + MAP ( ) storage. Values are means ± SD of four replicates Luo, Sun, Zhu, & Wang, 2018;Zhan, Zhu, & Sun, 2019b). The highest WL was observed in CK group (42.1 ± 3.1%), which might relate to its highest RR, and suggested severe membrane and tissue damage (Cheng, Sun, Pu, & Wei, 2018a;Cheng, Sun, Pu, & Wei, 2018b;Zhang, Zhu, & Sun, 2018;. High WL is usually accompanied by high TSS, which can reflect the ripeness of fruit. The TSS was found increasing in all the groups, especially in CK and SA + CK groups (40.07 ± 2.72% and 31.75 ± 1.48%, respectively). There was a significant difference between the TSS of CK and SA + CK, while there was no significant difference between the WL of CK and SA + CK. This might be the reason that high RR and WL accelerated biochemical reactions in the cells. AsA is regarded as a significant compo-   dimensionality reduction (Chen, Zhao, Wu, He, & Yang, 2020;Wang et al., 2018). Table 4 summarizes the total variance explained and the eigenvalue of each component for the results in the current study. Eigenvalues greater than 1.0 were extracted (Lyu et al., 2017;Lyu, Zhou, Bi, Liu, & Wu, 2015;Tian, Gou, Niu, Sun, & Guo, 2018), and two principal components were finally selected, together   Figure 4a shows the loading information of the determined variables from PCA (Tian, Chen, Zhu, & Sun, 2020;Tian, Zhang, Zhu, & Sun, 2020 Although inhibiting RR in fruit can usually prolong product shelflife and increase consumer acceptability, extremely low RR plays adverse effects on fruit and vegetable preservation (Kader, 2010;Rux, Caleb, Fröhling, Herppich, & Mahajan, 2017). RR values in CA and SA + CA groups were much higher than those in MAP and SA + MAP groups ( Figure 3). However, CA and SA + CA performed better than MAP and SA + MAP as shown in Figure 4. Besides, Mi et al. (2018) revealed that the redness of the wolfberries showed a significantly 3.3.2 | Interaction analysis of gas atmosphere and SA Score plot for PC1 versus PC2 (Figure 4b) could reflect the relationships between quality indicators and treatments, and variables located closely were highly correlated (Tian et al., 2018;Tian, Zhang, Zhu, & Sun, 2020 and AsA decline (Hertog, Nicholson, & Jeffery, 2004;Saito, Rai, & Masuda, 2007), and application of SA maintained high carotenoids content for better color quality (Mi et al., 2018), alleviated oxidative stress to retard softening (Hertog et al., 2004;Valero et al., 2011), and improved energy status to inhibit AsA decline (Rasouli, Koushesh Saba, & Ramezanian, 2019;Wei, Liu, Su, Liu, & Ye, 2011). Hence, SA resulted in compensation for the adverse effects of hypoxia stress. Therefore, the prominent preservation of SA + CA and SA + MAP may be the balance of maintaining high energy status and antioxidant capacity while suppressing RR.
To further investigate the relationships among SA, gas atmospheric control and quality attributes, cluster analysis was carried out to discriminate the similarities or nearness of data (Lyu et al., 2015;Patras et al., 2011;Tian, Chen, Zhu, & Sun, 2020). As shown in Figure 4c, three big groups were clustered in the heatmap.
CK and SA + CK merged first and then combined with MAP, and SA + CA and SA + MAP integrated initially and clustered with CA, while the Fresh formed a separate group. It was also observed in Figure 5 that the impact of SA on PC1 migration distance (ΔPC1) was more evident in modified atmosphere (44.19%) than in normoxia (27.67%) and CA (38.36%). To conclude, the gas composition played a more important role on quality preservation than SA under normoxia, and SA was a stress response phytohormone, which could alleviate adverse effects of the physiological disorder, including high AsA loss and color deterioration.