Influences of post-harvest melatonin treatment on preservation quality and shelf life of fresh-cut cauliflower

Xue Jianxin, Wang Kai, Li Zezhen, Zhang Shujuan, Mu Bingyu, Li Zihui, Huang Liang, Zhao Huamin, Sun Haixia

Influences of post-harvest melatonin treatment on preservation quality and shelf life of fresh-cut cauliflower

Xue Jianxin1, Wang Kai1, Li Zezhen2, Zhang Shujuan1, Mu Bingyu1, Li Zihui1, Huang Liang1, Zhao Huamin1, Sun Haixia1

(1.,030801,; 2.,030801,)

Fresh-cut cauliflower has special economic significance in post-harvest management, as a vegetable with a high perishable rate. Among them, melatonin is a kind of indoleamine that plays an important role against abiotic stress. This study aims to evaluate the effect of 0.05, 0.10, and 0.50 mmol/L of melatonin on the preservation quality of nutritionals in the fresh-cut cauliflower during 16-day storage at 4°C. Six samples of each replicate were randomly taken at 0, 4, 8, 12, and 16 d after treatment to measure weight loss, firmness, color, polygalacturonase (PG), and lipoxygenase (LOX) activities, andandexpression. The rest of the fruits were immediately frozen in liquid nitrogen and stored at −80 °C before determining the endogenous melatonin levels and DPPH radical scavenging activity, total phenolic, ascorbic acid, and total glucosinolates contents. The results showed that the nutritional quality was clearly delayed (<0.05) by the 0.10 mmol/L melatonin treatment, as disclosed by the weight loss, firmness, color, total glucosinolates content, and endogenous melatonin content of fresh-cut cauliflower samples. Furthermore, 0.10 mmol/L melatonin treatment was optimal for the enhancement (<0.05) of total phenolic content and antioxidant capacity, indicating that it delayed (<0.05) the reduction in ascorbic acid. Moreover, 0.10 mmol/L melatonin treatment enhanced (<0.05)expression, where decreased first and then increased (<0.05)geneexpression during storage, and delayed (<0.05) the activities of PG and LOX. After storage for 16 d, the changes of cellular structure in the 0.10 mmol/L treatment group and control group were observed under a Transmission Electron Microscope (TEM). The results showed the 0.10 mmol/L treatment sample cells presented obvious slight plasmolysis after storage for 16 d, where a small amount of autophagy was observed, while the slight plasmodesmata were also found between the cells. However, some variations of cells were also observed as follows: the mitochondria of cells in the ultrastructure of cauliflower florets in the absence of melatonin treatment (control) were moderately swollen, the endoplasmic reticulum was moderately expanded, autophagy appeared, and the cytoplasm and cell wall were separated. Consequently, the finding demonstrated that the melatonin treatment may be expected to serve as a useful technique to extend the postharvest life and improve quality in fresh-cut cauliflower.

storage; quality control; fresh-cut cauliflower; melatonin; cell structure

0 Introduction

Brassica vegetables, are consumed as a healthy food rich in vitamin C, phenolic compounds, dietary fiber, glucosinolates and isothiocyanates[1]. Cauliflower (L), which belongs to the genus Brassica in the Brassicaceae family, is a popular vegetable in human diet[2]. Epidemiological studies show that a diet rich in Brassica vegetables can reduce the risk of cancer incidence[3]. Fresh-cut fruits and vegetables refer to fresh fruits and vegetables that have been selected, cleaned, drained, trimmed, graded and packed and sometimes even peeled and cut as required[4]. With the accelerated pace of people's life and their increased awareness of food safety, it is of great realistic significance to carry out fresh-cut vegetables[5]. Fresh-cut cauliflower is becoming much more common than using the intact cauliflower in food services and retail markets as a convenience product, as consumer preferences for ready-to-use or ready-to-eat vegetables are increasing[6]. Cutting is an essential procedure of minimally processed fruits and vegetables. However, the wounding of tissue caused by cutting accelerates the deteriorative processes, including texture breakdown, increased respiratory rate, ethylene production, enzymatic browning, and changes in nutritional quality[7], thus shortening the shelf life of the produce.

Melatonin, or N-acetyl-5-methoxytryptamine, is a natural hormone which existed in numerous organisms[1]. Melatonin can protect cell structure, prevent DNA damage, and reduce peroxide levels by removing free radicals, enhancing oxidation resistance, and reducing lipid peroxidation[8]. Furthermore, as an endogenous plant growth regulator, melatonin plays multiple roles in plants, such as seed germination, flower development, leaf senescence, photo-protection, membrane integrity, root development, osmoregulation and the protection of plants against biotic and abiotic stresses[9]. In recent years, exogenous melatonin applying as universal biostimulator and signaling biomolecular serve a beneficial strategy for conferring chilling[10]and fungal decay tolerance[11], delaying senescence[12], preserving sensory[13], improving antioxidant defense[14]and endogenous melatonin level[15]in fruit and vegetables. Li et al.[13]suggested that exogenous melatonin treatment effectively delayed the lignification of bamboo shoots, as indicated by a significant decrease in the degrees of firmness and yellowness, and an increase in brightness, and decreased lignin and cellulose contents and PAL and POD activities. The results of these studies provide evidence regarding the involvement of melatonin treatment in delaying the senescence of postharvest fruit and vegetables. The effect of postharvest melatonin treatment on the sulforaphane production of fresh-cut broccoli at 4 ℃ during storage was investigated by Wei et al.[1]Florets treated with 0.10 mmol/L melatonin exhibited higher contents of total glucosinolates and sulforaphane. The glucoraphanin content was significantly increased after melatonin treatment, and which was explained by gene analysis. However, this study conducted a research on melatonin only with the concentration of 0.1 mmol/L and did not compare the treatment effect of broccoli with different concentrations. Meanwhile, it only measured the content of total glucosinolate and sulforaphane, so the measured indexes were unitary.

Unfortunately, the effect of melatonin treatment after harvest on the preservation quality of fresh-cut cauliflower still remains to be explored.

Therefore, this study aimed to select the optimal concentration of melatonin and explore the effects of melatonin on the nutritional quality, total phenolic and ascorbic acid contents, antioxidant capacity, endogenous melatonin, polygalacturonase (PG) and lipoxygenase (LOX) activities, andandgene expression, and cell ultrastructure of fresh-cut cauliflower during storage. Through measuring multiple indexes, this study aimed to analyze the regulating effect of melatonin on preservation quality of fresh-cut cauliflower from the three aspects of physiology, cellular structure and gene expression level. The findings may considerably assist in the development of new postharvest treatments to extend the shelf life of cauliflower.

1 Materials and treatments

1.1 Sample treatment

Before the experiment, preliminary experiment analysis was performed for cauliflower samples purchased from supermarkets. The preliminary experiment proved that the organoleptic quality of fresh-cut cauliflower began to have obvious changes after storage for 12 d, and a large area of black spot and decay occurred on the surface of cauliflower samples, their organoleptic quality was deteriorated obviously, and they lost storage and use value after storage for 16 d. Therefore, the storage deadline was selected as 16 d in this experiment.

“Xuebai” Cauliflower (L.) were obtained from a plantation in Taigu, Shanxi province of China in October 2020. The cauliflower heads were cut carefully by a sharp knife into small intact florets (approx. 20-30 g each). Then the florets dipped for 10 min into a sodium hypochlorite solution (0.10 g/L)[16], rinsed in tap water and blotted dried. For melatonin treatment, 360 samples were selected and grouped into 4 lots (90 samples per lot) for the following treatments in triplicate (30 samples per replicate). The four solutions made for treatments were: control (distilled water, 0 mmol/L), 0.05 mmol/Lmelatonin, 0.10 mmol/Lmelatonin, and 0.50 mmol/Lmelatonin[8-10]. Following immersion 15 min[11,15], the samples were dried in air at room temperature for approximately 30 min. Then, all florets were stored at 4 ℃ and 90 % Relative Humidity (RH) for 16 d. Six samples were randomly collected after 0, 4, 8, 12, and 16 d for repeated experiment. Weight loss rate, tightness, color, PG and LOX activities andandgene expression were determined. The remaining samples were then immediately frozen in liquid nitrogen and stored at −80°C, and the endogenous levels of melatonin, antioxidant activity, total phenolic content, ascorbic acid and total glucosinolates contents in the samples were determined.

1.2 Determination of quality indicators

Weight loss was measured every 4 d during storage and compared with initial weight and presented in percentage[17].

The firmness of fresh-cut cauliflower floret was measured using a TA-XT plus texture analyzer (Stable Micro Systems Ltd., UK) based on Xue et al.[18]procedure with certain modifications. The location of the cauliflower florets was adjusted such that the plunger touched the middle point of the floret between the flower head and the end of the floret[2]. The trigger force of the texture analyzer was 5 g, and the pretest speed, test speed, and post-test speed were 3.0, 1.0, and 5.0 mm/s, respectively. A probe was pushed 2 mm into the fruit at 5 mm/s, and the peak force was taken as the firmness index (in N).

The color of fresh-cut cauliflower floret was analyzed using a CM-5 Chroma Meter (Konica Minolta, Tokyo, Japan). The color analysis for* (0: dark, 100: white),* (negative value: green, positive value: red), and* (negative value: blue, positive value: yellow) values was done on day 0, 4, 8, 12, and 16.

The 2, 2-diphenyl-1-picrylidrazil (DPPH) method was applied to investigate the antioxidant activity of samples[19]. DPPH scavenging capacity, total phenolic content and ascorbic acid content were determined according to Liu et al.[12]Total glucosinolates content and endogenous melatonin concentration were detected as described by the reference[1] and reference[20], respectively.

The PG and LOX activity of the cauliflower floret was measured by the reference [21] and [22], respectively. PG is an enzyme that plays an important role in the change of cell wall structure. Recent studies have shown that, PG activity was not only related to ripening and softening of fruits, but also related to maturation and aging of fruits[23], and closely relevant to degradation of cell walls[24].LOX considered to be an enzyme closely related to the senescence of plant tissues. LOX enzyme is one of key enzymes to change in permeability of cell membranes.

1.3 RNA extraction and real-time quantitative PCR

Sample (500 mg) of cauliflower florets stored at −80 ℃were grounded into powder using liquid nitrogen, and total RNA was extracted using Plant RNA Extraction Kit (Takara, Dalian, China) according to the instruction of the manufacturer. The concentration and integrity of the total RNA were detected by NANO DROP ONE Nucleic acid quantification instrument (Thermo, Shanghai, China) and G:BOX F3 gel documentation system (Gene Co., Ltd, USA). Then, the qualified RNA was used for cDNA synthesis by PrimeScript™ RT reagent Kit (Takara, Dalian, China). Realtime quantitative PCR was performed using the cDNA. Real-time quantitative PCR was performed using SYBR®Premix Ex Taq™ (Tli RNaseH Plus; Takara, Dalian, China) in LightCycler® 480 II Real Time System (Roche，Switzerland). The PCR conditions were as follows: 95 ℃for 60 s followed by 35 cycles of 95 ℃ for 15 s , 55 ℃ for 15 s and 72 ℃ for 45s. The expression level was normalized by the internal control gene, using the 2−ΔΔCtmethod. All samples were tested in at least three biological and technical replicates. Primer sets of target genes and the βactin gene for qPCR were shown in Table 1.

Table 1 Primers used in real-time quatitatve PCR

1.4 Cell observation

A Transmission Electron Microscopy (TEM) was further used to observe the changes in cell structure and link these to texture and exogenous melatonin content. Pre-processings of samples were detected as described by the reference [21] and [25]. The samples were firstly fixed (5 mm × 1 mm × 1 mm), followed by washing (phosphate buffer of 0.1 mol/L), dehydrating (alcohol solutions at different concentrations), embedding in acetone: 812 eposy medium, curing, slicing (60-80 nm), and dyeing (uranium lead double staining). Finally images were observed and acquired by CCD camera. The experiment had three replicates, and each replicate had 5 agar blocks to be observed using TEM (HT7700, HITACHI, Japan).

1.5 Statistical analysis

The experiments were conducted using a completely randomized design. The experimental results of quality indicators and gene expression were analyzed using ANOVA analysis. The treatment and storage periods were the source of variation. The overall least significant differences (Fisher’s LSD procedure,<0.05) were computed to verify the significant differences among treatments and storage time. SPSS 19.0 was used for statistical analysis. Data were expressed as the mean ± Standard Deviation (SD) with three replications (each replication contain 6 samples).

2 Results and Discussion

2.1 Effects of exogenous melatonin treatment on the weight loss and firmness

Weight loss is an important factor that affects the preservation of postharvest quality of vegetables and fruit. As shown in Fig.1a, the weight loss of melatonin-treated and untreated cauliflower samples showed an increasing trend with the advancement of the storage period. No significant difference (>0.05) was found among melatonin treatment samples during the first 4 d. The reduction in weight loss was significantly higher (<0.05) in the control samples (1.142%) than in the melatonin-treated ones (0.604%-0.665%). At the end of storage, the control samples exhibited the highest weight loss (4.728%±0.010), 1.329-, 1.561- and 1.156- fold higher than that those observed in the melatonin-treated samples (0.05, 0.10, 0.50 mmol/L), respectively. However, compared with control and 0.50 mmol/L melatonin treatment, 0.05 and 0.10 mmol/L melatonin treatments significantly decreased the weight loss of cauliflower fruit from day 12 to day 16 (<0.05). 0.10 mmol/L melatonin treatment had the lowest weight loss of 3.140% after storage for 16 d.

Firmness is one of the important indices to measure the physiological changes in harvested vegetables. In this study, melatonin treatment significantly (<0.05) positive affected the firmness of cauliflower during storage at 4 ℃. As shown in Fig.1b, the firmness of the initial samples was 7.665-7.680 N, and it decreased during storage, but this decrease was effectively delayed by melatonin treatment. Compared with control, melatonin treatments significantly maintained fruit firmness after 12 and 16 d of storage (<0.05). The most remarkable (<0.05) treatment on delaying the decrease in firmness in cauliflower floret was 0.10 mmol/L, with an improvement of 40.70% compared with the control on 16 d.

It is known that melatonin treatment inhibits the water vaporization by respiration[15]. Melatonin treatment effectively maintained the higher firmness of fruit and vegetables during storage, including mango[9], strawberry fruit[12]and bamboo[13]. Physiological breakdown of postharvest cauliflower was always associated with weight loss and softening[26]. Thus firmness and weight loss were used to appraise the effect of different concentration of melatonin treatment on the sensory quality of fresh-cut cauliflower with storage duration. The lower weight loss in fresh-cut cauliflower treated with 0.10 mmol/L was attributed to more water being retained. Similar results were also found in researches on minimally processed products such as plum fruit[27], peach[28], and jujube[29]. The lower weight loss made for the higher turgor pressure in cell, which played a major role in the firmness of fruit and vegetables[13]. Accordingly, melatonin treatment was also determined to be the most effective to maintain the firmness of fresh-cut cauliflower.

The research conducted by Sarropoulou and Therios[30]showed that the change of melatonin concentration within the plant would exert an influence on root growth, namely low concentration could promote the growth of lateral root and adventitious root, while high concentration could have an opposite effect. Moreover, Posmyk et al.[31]and Hu[4]obtained similar research results in terms of seed germination and kiwifruit cold damage. Boumail et al.[5]considered that high melatonin concentration would lead to high osmotic pressure of solutions, which would generate osmotic pressure for fruit and give rise to no obvious effects, and also redundant melatonin remained in fruit, which would produce unpleasant odor and influence the taste and organoleptic evaluation of samples.

2.2 Effects of exogenous melatonin treatment on the DPPH radical scavenging activity and total phenolics content

Changes in DPPH radical scavenging capacity in fresh-cut cauliflower during storage are shown in Fig.2a. The control and melatonin-treated cauliflower florets presented an increasing trend in DPPH scavenging activity during storage at 4 ℃. The antioxidant capacity of the samples was improved following melatonin treatment (cauliflower florets treated with 0.05, 0.10 and 0.50 mmol/L melatonin demonstrated significantly higher levels of DPPH scavenging activity than the untreated samples (<0.05) from day 4 to day 16). Such capacity following 0.10 mmol/L treatment improved significantly. At the end of storage, the DPPH value reached 38.480%.

The dynamics of total phenolics content were consistent with the changes in DPPH free radical-scavenging activity (Fig.2b). Whether in the control group or the treatment group, the total phenol content gradually increased during storage. The total phenolics content was significantly elevated (<0.05) by 0.10 mmol/L melatonin treatment throughout the storage. A comparison between the total phenol content at 16 d and 0 d following melatonin treatment showed that the total phenol content in the 0.10 mmol/L melatonin treatment group increased by 3.753 times, and the content in the control group increased by 2.934 times.

In general, the antioxidant capacity of cauliflower florets increased over time, but following melatonin treatment, especially 0.10 mmol/L treatment, the antioxidant capacity of cauliflower samples considerably improved. Similar to the results of this study, it was shown in strawberry fruit[12], pear[8]and mango[9]that fruit treated with melatonin had higher antioxidant level than untreated samples at the end of the storage period. Zhang et al.[32]noted that melatonin treatment up-regulated the expression of important enzymes genes in the phenylpropanoid pathway, such as the gene of phenylalanine ammonia-lyase (), contributing to the accumulation of total phenolics in tomato and cabbage.

However, the total phenolics content in treated tissue presented a decrease at 16 day, probably due to the faster utilization rate and slower synthesis rate of total phenolics in fresh-cut cauliflower florets[33].This positive effect of melatonin in increasing phenolics was also reported in strawberry fruit[12]as well as cucumber[14]. Based on these results, the higher total phenolics content in cauliflower florets treated with melatonin was most likely associated with the increased DPPH scavenging capacity.

2.3 Effects of exogenous melatonin treatment on the ascorbic acid content

As shown in Fig.3, the ascorbic acid content presented a decreasing trend in the control and melatonin-treated samples during the storage period. However, melatonin treatment effectively delayed the decrease in ascorbic acid content in cauliflower florets treated with melatonin, and the ascorbic acid content remained similar during the storage period. The mean ascorbic acid content was significantly higher in 0.10 mmol/L melatonin-treated samples. The average ascorbic acid content in 0.10 mmol/L melatonin treated cauliflower florets (0.201 g/kg) was approximately 32.268 % higher (<0.05) than that in untreated cauliflower florets (0.152 g/kg) after 16 d of storage.

Ascorbic acid as an effective antioxidant agent is one of the most important indicators of the nutrient value of fruit and vegetables[9]. Previous studies have found that melatonin delays the degradation of ascorbic acid in fresh produces during storage[27]. Melatonin treatment could increase the resistance of the fruit to oxidative stress during ripening by improving bioactive compounds, such as ascorbic acid[34]. A previous study by Gao et al.[34]has found that melatonin treatment could maintain fruit firmness and cell-wall integrity during storage. The ascorbic acid in strawberry fruit could be synthesized from D-galacturonic acid, a principal component of cell wall pectins[12]. Therefore, reduced pectin solubilization in cell walls usually resulted in decreased ascorbic acid.

2.4 Effects of exogenous melatonin treatment on the color parameters

Postharvest decay of cauliflower include a number of visual alterations, including the colour change of the floret[3]. The total color difference*,*, and* value were selected as the most suitable indicator to express the color change of the surface of fresh-cut cauliflower (Table 2). During storage, melatonin- treated florets displayed a marked tendency to retain the color reached at harvest; on the contrary a faster tendency of color alteration was found in control florets. During storage, all samples were remained almost unchanged in terms of* values during storage, and no significant differences were observed between the control and melatonin-treated fruit. A slight increase was noted in the* value of the cauliflower floret during storage. At the end of storage, the 0.10 mmol/L melatonin-treated florets showed higher* values than the controls 0.05 and 0.50 mmol/L florets. The untreated and melatonin-treated florets exhibited a gradual decrease in* values as the storage progressed, indicating the darkening of the florets. However, the melatonin-treated fruits, except for 0.50 mmol/L melatonin-treated fruits, were shinier (higher* value) than the control fruits after 16 d of storage (<0.05).

Consistent with our results, Liu et al.[12]found that 0.10or 1 mmol/Lpostharvest melatonin treatment was most effective in delaying the color dvelopment, Wang et al.[15]found that exogenous melatonin treatment improved the levels of the*,*, and° of cherry skin.

Table 2 Effects of melatonin treatment on L*, a* and b* of fresh-cut cauliflower florets

Note: Data correspond to the means ± standard error of the independent replicates. Different small letters in the same column show significant difference (<0.05) within the same storage period.

2.5 Effects of exogenous melatonin treatment on the total glucosinolates content

Glucosinolates are natural bioactive components in cauliflower and can be hydrolyzed in to health-benefit isothiocyanates such as sulforaphane, erucin and so on[1]. Several experts believed that retaining glucosinolates in postharvest cauliflower is as important as maintaining its commercial quality. During the storage of fresh-cut cauliflower florets, the total glucosinolates increased first and then decreased. Melatonin treatment delayed the degradation of total glucosinolates in fresh-cut cauliflower tissues (Fig.4). The total glucosinolates content of cauliflower in the 0.10 mmol/L treatment group was higher than the one in the control group and other melatonin treatment groups (<0.05). In the first 8 days of the storage period, no significant difference was found between the 0.10 mmol/L treatment group and the 0.05 mmol/L treatment group (>0.05). On 16 day of storage, the content of cauliflower glucosinolates in the control group and 0.05, 0.10 and 0.50 mmol/L treatment groups decreased by 113.977 %, 63.468 %, 20.150 %, and 77.097 %, respectively, compared with the content at 0 d.

The chemical properties of glucosinolates are relatively stable. Most of them are distributed in the vacuoles of plants in the form of salts. The total glucosinolate content increased in the first 8 day of the storage period according to Jahangir et al.[35]The postharvest increase in glucosinolates and polyphenols in cold-stored Brassica is due to the fact that hydroxycinnamic acids are thought to provide cell wall rigidity, which could protect plant tissues from chilling injury. However, the total glucosinolate content decreased at the later stage of storage, which could be attributed to degradation of glucosinolate. When the plant is damaged by external attacks, myrosinase are released from the plant to catalyze glucosinolates and hydrolyze glucosinolates, thereby producing many of biologically active substances, such as isothiocyanates, thiocyanates, and nitrile compounds[36]. Wei et al.[1]found that myrosinase activity andgene expression were promoted by melatonin treatment. These works indicated that melatonin was beneficial for glucoraphanin hydrolysis and enhanced sulforaphane production in fresh-cut broccoli during cold storage. Meanwhile, the results of the present study failed to support the correlation between ascorbic acid content and total glucosinolate content[37].

2.6 Effects of exogenous melatonin treatment on the PG activity

As shown in Fig.5a, during the storage, the polygalacturonase (PG) activity of the control group increased first and then decreased slightly, and the peak of PG activity appeared. The PG activity of the melatonin treatment group was significantly lower than that of the control group, but the activity value continued to increase with the extension of the storage period. Following the storage, the PG activity of samples in the 0.10 mmol/L treatment group was 15.103 U/(g·min), 26.246 % lower than the value in the control group. The difference in PG activity between the 0.10 mmol/L treatment group and the 0.50 mmol/L treatment group (18.430 U/(g·min)) reached a significant level (<0.05). These results showed that melatonin treatment could significantly inhibit the PG activity of cauliflower floret during post-harvest storage, delay the decomposition of pectin in the cell wall, and prolong the storage time.

PG has the capacity to hydrolyze polygalacturonic acid in the cell wall, thereby participating in the degradation of pectin, and it has a close relation to the ripening and softening of fruit[21]. Tang et al.[29]also confirmed that PG is an enzyme closely related to the softening and senescence of fruits and vegetables. PG mainly acts on the-1, 4 glycosidic bond in the polygalacturonic acid molecule in pectin, it degrades the pectin material in the cell and makes the fruit soften[38]. However, Ghiani and Citterio[24]believe that PG may be related to the ripening and senescence of fruit, but it is not the only determinant. In the late storage period, the PG activity of the control samples decreased slightly, possibly due to the decline in the metabolic capacity of the cauliflower in the late storage period, which also reduced the PG activity of the fruit[23].

2.7 Effects of exogenous melatonin treatment on the LOX activity

The results of lipoxygenases (LOX) activity changes in cauliflower treated with different melatonin levels are shown in Fig.5b. As shown in Fig.5b, the freshly harvested cauliflower samples exhibited higher LOX activity than the other samples, which may be related to the higher atmospheric temperature during the harvest. The enzyme activity decreased as the storage temperature dropped. Then, the enzyme activity gradually increased, and the control group and 0.50 mmol/L melatonin treatment group reached their peaks on the 8 and 12 day, respectively. At this time, the samples began to enter the stage of rapid softening and senescence. In the late storage period, the enzyme activity of the samples in the 0.05 mmol/L melatonin treatment group was active, and an upward trend was observed. However, it was still significantly different from that of the control group at this stage (<0.05). After 0.10 mmol/L melatonin treatment, the LOX activity of cauliflower floret was significantly inhibited, with no activity peak appearing during storage, and it essentially maintained at a constant level. This finding indicated that melatonin treatment could inhibit the enzyme activity.

LOX destroys the integrity of the cell membrane and changes the permeability of the membrane by oxidizing polyunsaturated fatty acids, leading to softening and senescence of vegetables and fruit[22]. Scholars also hold the view that fat peroxidation increases the accumulation of free radicals, especially reactive oxygen species, and this increase further damages membrane lipoproteins and accelerates the senescence process[39]. Kong et al.[40]found that melatonin treatment may have inhibited the transcription ofas well as the LOX protein content in pepper fruit during cold storage. Many studies recently showed that during the ripening and senescence of fruits, the enzyme activity and expression of LOX, which were negatively correlated with firmness[41], increased as the firmness decreased. This finding is inconsistent with the conclusion of the present study.

2.8 Effects of exogenous melatonin treatment on the genes expressions

The expression levels of genes encodingandwere evaluated in cauliflower florets.expression, which was upregulated during 4 ℃ storage, showed a similar pattern.expression decreased first and then increased during storage. Theandgene expression levels were significantly inhibited in the melatonin-treated cauliflower florets, compared with the controls. According to Fig.5c, Fig.5d, the expression pattern ofandwere consistent with the PG and LOX activities, respectively. Compared with the control, melatonin treatment inhibitedexpression, and this speculation was supported by Zheng et al[8].

Wang et al.[15]believed that melatonin may increase enzyme activity by increasing the gene transcription level of protective enzymes and promote the formation of antioxidant substances. In this manner, melatonin plays a role in effectively eliminating the self-synthesizing free radicals in plants. Zhai et al.[42]reported that exogenous melatonin retarded the degradation of cell wall by inhibiting PG activity and cellulase gene expression in three pear cultivars. Tang et al.[29]thought that melatonin inhibited the activity of cell-degrading enzymes by suppressing the expression of related genes, ultimately maintaining jujubes fruit firmness. A similar result was noted in strawberry treatment with melatonin[12]. A recent study has established that melatonin inhibited the transcription of thegene and the enzymatic acitivity ofby increasing the percentages of linoleic and linolenic acids[41].

2.9 Effects of exogenous melatonin treatment on the endogenous melatonin content

Given that 0.10 mmol/L melatonin treatment seemed to be the most effective in extending the postharvest life and improving the quality of strawberry fruit, it was selected to further study the endogenous melatonin content by using UPLC-MS analysis. The multiple reaction monitoring (MRM) chromate grams data showed that the final extraction from cauliflower floret had a similar retention time and/233.15>174.17 to that of standard melatonin, demonstrating that the final extraction from the samples contained endogenous melatonin. As shown in Fig.6, the endogenous melatonin content in the control sample increased for a short time on the 4 day, and then rapidly decreased. By contrast, in the 0.10 mmol/L treatment group, the endogenous melatonin content continued to rise as the storage period went on and reached the maximum value on the 12 day and then began to decline. Exogenous melatonin treatment significantly increased the endogenous melatonin content compared with the control during the whole storage (<0.05). These results indicated that endogenous melatonin may act not only as a signaling molecule for attenuating decay but also as a powerful antioxidant for delaying the senescence of cauliflower floret.

Considering the scavenger role of the melatonin, it might be warking in coordination with its delaying senescence functionality and increasing the endogenous level indirectly[21]. A recent study has established that exogenous melatonin treatment increases the endogenous melatonin content, which not only act as a signaling molecule for reducing decay, but also act as a strong antioxidant, delaying the ripening of fruit[32]. Wang et al.[15]found that the delayed senescence in sweet cherries after exogenous melatonin treatment may be associated with high endogenous melatonin levels and increased antioxidant activity and content. Zhang et al.[43]showed that exogenous melatonin not only increased the endogenous levels of melatonin and cytokinin but also decreased the concentration of abscisic acid under heat stress inL. Ahammed et al[44]. studied tomato thermotolerance and found that the exogenous melatonin-induced enhancement in endogenous melatonin levels stimulated the antioxidant defense system in-silenced plants and alleviated heat-induced oxidative stress. A report about tomato shows that melatonin might alleviate fruit CI symptom by promoting endogenous melatonin accumulation and stimulating GABA shunt pathway activity[45].

2.10 Effects of exogenous melatonin treatment on the cell ultrastructure changes

Based on morpho-physiological and biochemical traits analysis, 0.10 mmol/L melatonin treatment seemed to be more effective than the other treatments. Therefore, 0.10 mmol/L melatonin treatment and control were selected for TEM analysis.

The changes in the ultrastructure of cells in the control and 0.10 mmol/L melatonin treatment groups after 16 d of storage are shown in Fig.7. The two groups of cells showed damages and plasmolysis to different degrees. However, the damage to the cell structure was obviously alleviated after melatonin treatment. The ultrastructure of cauliflower floret treated with melatonin had a normal electron-dense cell wall and mostly uniform cell mitochondria. The endoplasmic reticulum was slightly expanded, and the cytoplasm and cell wall in a small area were severely separated. However, the mitochondria of the cells in the ultrastructure of cauliflower florets in the absence of melatonin treatment (control) were moderately swollen, the endoplasmic reticulum was moderately expanded, autophagy appeared, and the cytoplasm and cell wall were separated.

Note: Cell wall (CW), plasmolysis (▲), nucleus (N), mitochondria (M), cytoplasm (P), vesicles (VE), rough endoplasmic reticulum (RER) and autophagy (AP)

Experimental group: The cells were moderately edematous, the cell membrane and cell wall (CW) structure were completed, most of the cell membranes had obvious slight plasmolysis (▲), and the partial separation was severe. Many flocculent materials were present in the cytoplasm. The nucleus (N) was obvious and dominated by euchromatin. Mitochondria (M) could be seen in the cytoplasm most of the structure was relatively normal, a small part was slightly swollen, and the cristae was significantly reduced and missed. Slight plastid globules could be seen in the cytoplasm (P) and vesicles (VE), the rough endoplasmic reticulum (RER) was slightly expanded, and no apparent degranulation was found.

Control group: The cells had obvious slight plasmolysis (▲). A certain amount of flocculent substances was found in the cell cytoplasm. The number of mitochondria (M) was reduced, and their volume significantly increased. The swelling and deformation were serious, the cristae were significantly reduced and missed, the matrix lightened, and the inner and outer membranes were obviously damaged. The rough endoplasmic reticulum (RER) was moderately expanded without significant degranulation. A small amount of vesicles (VE) and plastid globules (P) could be seen in the cytoplasm. A small amount of autophagy (AP) could also be seen. Slight plasmodesmata (at the arrow) were observed between the cells.

Fig.7 Effects of melatonin priming on ultrastructure of caulifower florets cells and cell wall after 16 days of storage

Many studies have confirmed that changes in the quality of vegetables and fruit after harvest and changes in cell structure are closely related to senescence[40]. Plant cell walls have the functions of stabilizing cell morphology; controlling cell growth and expansion; and participating in the transportation of intracellular and extracellular substances, information transmission and defense. Kong et al.[40]used advanced and convenient Scanning Electron Microscope (SEM) to observe the changes in pulp tissues and cell structure to evaluate the effect of melatonin treatment on pepper fruit CI during low-temperature storage. The more complete and plump cell SEM structure also showed that melatonin may strengthen the ability of pepper cells to resist oxidative stress. Recent studies have found that the application of melatonin thickened the cell walls of Arabidopsis and increased the amount of callose deposits, thereby increasing the resistance of Arabidopsis[46]. Plant melatonin-enhanced the overall antioxidant capacity of organelles and regulated the expression of stress response genes. Melatonin could enhance plant resistance when subjected to different abiotic stresses[46]. Khan et al.[47]found that the stomatal traits, such as number and stomatal length and width, were greatly improved in melatonin-primed treatment, and melatonin-priming preserved the chloroplast structure, maintained cell expansion, and strengthened the cell wall in response to drought stress. The findings in the present study suggested that melatonin treatment reduced the activity of PG enzyme and prevented degradation of cell-wall components. Similarly, melatonin treatment could reduce the activity of LOX enzymes, thus preventing changes in cell membrane permeability and helping in the formation of firmed cell wall and well-established cell expansion.

3 Conclusion

To sum up, 0.10 mmol/Lpostharvest melatonin treatment was the most effective in delaying senescence of fresh-cut cauliflower by reducing weight loss, delaying color development, maintaining fruit firmness, increasing the content of endogenous melatonin, and inhibiting the decline in total glucosinolates and ascorbic acid contents. In addition, melatonin treatmentinhibited the decline in total phenolics, resulting in increased antioxidant capacity. The increased cell wall and cell-membrane relative integrity in fresh-cut cauliflower in response to melatonin treatment may have resulted from the activities of lower cell wall and cell membrane degrading enzymes PG and LOX activities, respectively. Therefore, it can be seen that preservation performance after 0.10 mmol/L treatment is obviously better than 0.05 and 0.50 mmol/L. It could be known that the melatonin treatment of fresh-cut cauliflower with various concentrations played a promoting role in its preservation quality to a certain extent, but too low or too high concentrations would restrain the preservation effect. Thus, this study revealed that the selection of concentration was very important in the preservation experiment of fresh-cut cauliflower with melatonin. It effectively prolongs shelf life of fresh-cut cauliflower. Thus, as a safe biostimulator and signaling biomolecule, melatonin is beneficial for delaying senescence in fresh-cut cauliflower.

[1] Wei L, Liu C, Zheng H, et al. Melatonin treatment affects the glucoraphanin-sulforaphane system in postharvest fresh-cut broccoli (L.)[J]. Food Chemistry, 2020, 307: 125562.

[2] Vaishnav J, Adiani V, S Variyar P. Radiation processing for enhancing shelf life and quality characteristics of minimally processed ready-to-cook (RTC) cauliflower (L.)[J]. Food Packaging and Shelf Life, 2015, 5: 50-55.

[3] Giuffrida F, Agnello M, Mauro R P, et al. Cultivation under salt stress conditions influences postharvest quality and glucosinolates content of fresh-cut cauliflower[J]. Scientia Horticulturae, 2018, 236: 166-174.

[4] Hu M. Effect of Melatonin Treatment on Postharvest Fruit Chilling Injury and Senescence in “HUA YOU” Kiwifruit[D]. Yangling: Northwest A&F University, 2018. (in Chinese with English abstract)

[5] Boumail A, Salmieri S, St-Yves F, et al. Effect of antimicrobial coatings on microbiological, sensorial and physico-chemical properties of pre-cut cauliflowers[J]. Postharvest Biology and Technology, 2016, 116: 1-7.

[6] Mashabela S, Mahajan P V, Sivakumar D. Influence of different types of modified atmosphere packaging films and storage time on quality and bioactive compounds in fresh-cut cauliflower[J]. Food Packaging and Shelf Life, 2019, 22: 100374.

[7] Ma L, Zhang M, Bhandari B,et al. Recent developments in novel shelf life extension technologies of fresh-cut fruits and vegetables[J]. Trends in Food Science & Technology, 2017, 64: 23-38.

[8] Zheng H, Liu W, Liu S, et al. Effects of melatonin treatment on the enzymatic browning and nutritional quality of fresh-cut pear fruit[J]. Food Chemistry, 2019, 299: 125116.

[9] Rastegar S, Khankahdani H H, Rahimzadeh M. Effects of meltonin treatment on the biochemical changes and antioxidant enzyme activity of mango fruit during storage[J]. Scientia Horticulturae, 2020, 259: 108835.

[10] Jannatizadeh A. Exogenous melatonin applying confers chilling tolerance in pomegranate fruit during cold storage[J]. Scientia Horticulturae, 2019, 246: 544-549.

[11] Lin Y, Fan L, Xia X, et al. Melatonin decreases resistance to postharvest green mold on citrus fruit by scavenging defense-related reactive oxygen species[J]. Postharvest Biology and Technology, 2019, 153: 21-30.

[12] Liu C, Zheng H, Sheng K, et al. Effects of melatonin treatment on the postharvest quality of strawberry fruit[J]. Postharvest Biology and Technology, 2018, 139: 47-55.

[13] Li C, Suo J, Xuan L, et al. Bamboo shoot-lignification delay by melatonin during low temperature storage[J]. Postharvest Biology and Technology, 2019, 156: 110933.

[14] Ahammed G J, Wu M, Wang Y, et al. Melatonin alleviates iron stress by improving iron homeostasis, antioxidant defense and secondary metabolism in cucumber[J]. Scientia Horticulturae, 2020, 265: 109205.

[15] Wang F, Zhang X, Yang Q, et al. Exogenous melatonin delays postharvest fruit senescence and maintains the quality of sweet cherries[J]. Food Chemistry, 2019, 301: 125311.

[16] Hou Q. Research of Factors Influencing the Quality of Fresh-cut Cucumber[D]. Handan: Hebei University of Engineering, 2016. (in Chinese with English abstract)

[17] Tao T, Ding C, Han N, et al. Evaluation of pulsed light ofr inactivation of foodborne pathogens on fresh-cut lettuce: Effects on quality attributes during storage[J]. Food Packaging and Shelf Life, 2019, 21: 100358.

<

[18] Xue J, Zhang S, Sun H, et al. Study of Malus Asiatica Nakai’s firmness during different shelf lives based on visible/near-infrared spectroscopy[J]. Mathematical and Computer Modelling, 2013, 58(11/12): 1829-1836.

[19] Brand-Williams W, Cuvelier M E, Berset C L W T. Use of a free radical method to evaluate antioxidant activity[J]. LWT-Food Science and Technology, 1995, 28(1): 25-30.

[20] Pang L, Wu Y, Pan Y, et al. Insights into exogenous melatonin associated with phenylalanine metabolism in postharvest strawberry[J]. Postharvest Biology and Technology, 2020, 168: 111244.

[21] Zhang P, Chen F, Yang H, et al. Research advances on cell wall disassembly in fruit ripening and softening[J]. Food Science and Technology, 2010, 35(11): 62-66.

[22] Tian S, Zhou J. Relationship between different storage temperature and changes of protective enzyme or LOX in fresh Chineses jujube[J]. Journal of Agricultural University of Hebei, 2009, 29(1): 46-49. (in Chinese with English abstract)

[23] Zhou R, Li Y, Yan L, et al. Effect of edible coatings on enzymes, cell-membrane integrity, and cell-wall constituents in relation to brittleness and firmness of Huanghua pears (Pyrus pyrifolia Nakai. cv. Huanghua) during storage[J]. Food Chemistry, 2011, 124(2): 569-575.

[24] Ghiani A, Citterio S. A comparative study of melting and non-melting flesh peach cultivars reveals that during fruit ripening endo-polygalacturonase (endo-PG) is mainly involved in pericarp textural changes, not in firmness reduction[J]. Journal of Experimental Botany, 2011, 62: 4043-4054.

[25] Khan M N, Zhang J, Luo T, et al. Seed priming with melatonin coping drought stress in rapeseed by regulating reactive oxygen species detoxification: Antioxidant defense system, osmotic adjustment, stomatal traits and chloroplast ultrastructure perserveration[J]. Industrial Crops & Products, 2019, 140: 111597.

[26] Li M, Li X, Li J, et al. Responses of fresh-cut strawverries to ethanol vapor pretreatment: Improved quality maintenance and associated antioxidant metabolism in gene expression and enzyme activity levels[J]. Journal of Agricultural and Food Chemistry, 2018, 66: 8382-8390.

[27] Feng X, Dong X, Zhu S, et al. Effects of melatonin treatments on preservation of fengtang plum fruits[J]. Science and Technology and Food Industry, 2020, 41(6): 265-271.

[28] Qian C, Zhu Q, Gao S, et al. Effects of exogenous melatonin treatment on cold storage quality and chilling injury of postharvest peach fruit[J]. Jiangsu Journal of Agricultural Sciences, 2020, 36(3): 702-708. (in Chinese with English abstract)

[29] Tang Q, Li C, Ge Y, et al. Exogenous application of melatonin maintains storage quality of jujubes by enhancing anti-oxidative ability and suppressing the activity of cell wall-degrading enzymes[J]. LWT-Food Science and Technology, 2020, 127: 109431.

[30] Sarropoulou V N, Therios I N. Melatonin promotes adventitious root regeneration in in vitro shoot tip explants of the commercial sweet cherry rootstocks CAB-6P, Gisela 6, and MxM 60[J]. Journal of Pineal Research, 2012, 52(1): 38-46.

[31] Posmyk, Kuran H, Marciniak K, et al. Presowing seed treatment with melatonin protects red cabbage seedlings against toxic copper ion concentrations[J]. Journal of Pineal Research, 2008, 45(1): 24-31.

[32] Zhang N, Sun Q, Li H, et al. Melatonin improved antiocyanin accumulation by regulating gene expressions and resulted in high reactive oxygen species scavenging capacity in cabbage[J]. Frontiers in Plant Science, 2016, 7: 00197.

[33] Aghdam M S, Fard J R. Melatonin treatment attenuates postharvest decay and maintains nutritional quality of strawberry fruits (Fragaria × anannasa cv. Selva) by enhancing GABA shunt activity[J]. Food Chemistry, 2017, 221: 1650-1657.

[34] Gao H, Zhang Z K, Chai H K, et al. Melatonin treatment delays postharvest senescence and regulates reactive oxygen species metabolism in peach fruit[J]. Postharvest Biology and Technology,2016,118:103-110.

[35] Jahangir M, Abdel-Farid I B, Mahmood Z, et al. Metabolomic approach: postharvest storage stability of red radish (L.)[J]. Journal-Chemical Society of Pakistan, 2014,36(5): 931-938.

[36] Peng P, Tian Y, Deng F, et al. Identification of glucosinolate in different varieties of broccoli leaves based on HPLC-Q-TOF-MS technology[J]. Food & Machinery, 2019, 35(4): 42-47. (in Chinese with English abstract)

[37] Albrecht J A, Schafer H W, Zottola E A. Relationship of total sulfur to initial and retained ascorbic acid in selected cruciferous and noncruciferous vegetables[J]. Journal of Food Science, 1990, 55: 181-183.

[38] Liang J, Zhu D, Lv J, et al. Effect of cell wall enzyme activity on the degredation of pectin during postharvest softening of plum fruit[J]. Science and Technology of Food Industry, 2015, 36(10): 344-348. (in Chinese with English abstract)

[39] Marangoni A G, Palma T, Stanley D W. Membrane effects in postharvest physiology[J]. Postharvest Biology and Technology, 1996, 7: 193-217.

[40] Kong X, Ge W, Wei B, et al. Melatonin ameliorates chilling injury in green bell peppers during storage by regulating membrane lipid metabolism and antioxidant capacity[J]. Postharvest Biology and Technology, 2020, 170: 111315.

[41] Kou X, Yan S, Wu C, et al. Effects of lipoxygenase on ripening and softening of jujube fruit[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE),2002, 18(2): 127-130. (in Chinese with English abstract)

[42] Zhai R, Liu J, Liu F, et al. Melatonin limited ethylene production, softening and reduced physiology disorder in pear (L.) fruit during senescence[J]. Postharvest Biology and Technology, 2018, 139: 38-46.

[43] Zhang J, Shi Y, Zhang X, et al. Melatonin suppression of heat-induced leaf senescence involves changes in abscisic acid and cytokinin biosynthesis and signaling pathways in perennial ryegrass (L.)[J]. Environmental and Experimental Botany, 2017, 138: 36-45.

[44] Ahammed G J, Xu W, Liu A, et al. Endogenous melatonin deficiency aggravates high temperature-induced oxidative stress inL[J]. Environmental and Experimental Botany, 2019, 161: 303-311.

[45] Sharafi Y, Aghdam M S, Luo Z S, et al. Melatonin treatment promotes endogenous melatonin accumulation and triggers GABA shunt pathway activity in tomato fruits during cold storage[J]. Scientia Horticulturae, 2020, 254: 222-227.

[46] Shi H, Chen Y, Tan D X, et al. Melatonin induces nitric oxide and the potential mechanisms relate to innate immunity against bacterial pathogen infection in Arabidopsis[J]. Journal of Pineal Research, 2015, 59(1): 102-108.

[47] Khan M N, Zhang J, Luo T, et al. Seed priming with melatonin coping drought stress in rapeseed by regulating reactive oxygen species detoxification: Antioxidant defense system, osmotic adjustment, stomatal traits and chloroplast ultrastructure perserveration[J]. Industrial Crops & Products, 2019, 140: 111597.

采后褪黑素处理对鲜切花椰菜保鲜品质及货架期的影响

薛建新1，王 凯1，李泽珍2，张淑娟1，穆炳宇1，李紫辉1，黄 亮1，赵华民1，孙海霞1

（1. 山西农业大学农业工程学院，太谷 030801； 2. 山西农业大学食品科学与工程学院，太谷 030801）

为研究采后外源性褪黑素处理对鲜切花椰菜货架期间品质及生理的影响，试验以“雪白”花椰菜为材料，拟从中筛选出有效的使用浓度，进而从生理、细胞和基因表达水平解析褪黑素对鲜切花椰菜保鲜品质的调节作用，以期为探索鲜切花椰菜保鲜和衰老调控的有效途径提供科学依据。采用0.05、0.10、0.50 mmol/L褪黑素溶液浸泡鲜切花椰菜样本15 min，分析货架期间（0、4、8、12、16 d）样本的失重率、硬度、1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl, DPPH）值、总酚含量、抗坏血酸含量、色泽、总硫代葡萄糖苷含量、内源性褪黑素含量、多聚半乳糖醛酸酶（polygalacturonase, PG）和脂氧合酶（lipoxygenases, LOX）活性及基因表达水平、细胞超微结构的变化。结果表明：0.10 mmol/L褪黑素处理明显延缓（<0.05）了失重率、硬度、色泽和总硫代葡萄糖苷含量的下降，提升（<0.05）了样本中内源性褪黑素的含量。同时，0.10 mmol/L褪黑素处理最有利于提高总酚含量和抗氧化能力，延缓抗坏血酸的降低。此外，0.10 mmol/L可抑制细胞结构的损伤，降低和的基因表达，延缓PG和LOX的活性。由此可知，对鲜切花椰菜进行外源褪黑素处理可能是延长其采后寿命和提高品质的有效技术。

贮藏；品质控制；鲜切花椰菜；褪黑素；细胞结构

Xue Jianxin, Wang Kai, Li Zezhen, et al. Influences of post-harvest melatonin treatment on preservation quality and shelf life of fresh-cut cauliflower[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2021, 37(13):273-283.

10.11975/j.issn.1002-6819.2021.13.031 http://www.tcsae.org

薛建新，王凯，李泽珍，等. 采后褪黑素处理对鲜切花椰菜保鲜品质及货架期的影响[J]. 农业工程学报，2021，37(13)：273-283. (in English with Chinese abstract) doi：10.11975/j.issn.1002-6819.2021.13.031 http://www.tcsae.org

date：2021-03-20

date：2021-06-30

National Natural Science Foundation of China (31801632), Science and Technology Innovation Foundation of Shanxi (2019L0396) and Shanxi Agricultural of University Research Grant (2016YJ04).

Xue Jianxin, associate professor, doctor, research interest: storage, processing and non-destructivetesting of agricultural products. Email: vickyxjx@126.com

10.11975/j.issn.1002-6819.2021.13.031

S635.3; TS255.3

A

1002-6819(2021)-13-0273-11