Processing math: 100%
ARTICLE   Open Access    

The optimal precise temperature alleviated chilling injury and maintained post-harvest quality for 'Mengzi' pomegranate fruit

More Information
  • Chilling injury (CI) is a highly common physiological disorder in pomegranates during cold storage. Although several approaches have been investigated to mitigate the CI symptoms among some pomegranate cultivars, the fundamental and crucial environmental factor — the precise storage temperature for the 'Mengzi' cultivation remains unknown. This research evaluated the impact of storage temperatures of 0, 1, 2, 3, and 4 °C on the post-harvest quality of pomegranates. Results indicated that pomegranates stored at 2 °C exhibited the slightest color change and browning index. After storage of 130 d, pomegranates stored at 2 °C exhibited the lower CI index (82.79% reduction) and the lowest decay incidence (24.68% reduction) compared to those stored at 0 °C. The respiratory rate of pomegranates (2 °C) was also evidently suppressed (16.60%), along with a reduction in weight loss (3.46%). Furthermore, pomegranates stored at 2 °C exhibited the lowest activities of polyphenol oxidase (PPO) and peroxidase (POD), accompanied by the highest total phenolic content, which contributed to a reduction in malondialdehyde (MDA) accumulation. Relatively higher concentrations of soluble solids and titratable acid, as well as a higher sensory evaluation, were found in pomegranates stored at 2 °C. Consequently, it was inferred that the optimal temperature maintained cell membrane integrity modulated normal respiratory metabolism, and oxidative balance, and therefore alleviated CI and deterioration. This report can provide the guiding significance for the long-term storage of 'Mengzi' pomegranates under the condition of precise temperature control in phase temperature storage.
  • The genus Nymphaea, belonging to the family Nymphaeaceae and commonly known as water lily, comprises over 60 species of perennial aquatic plants[1]. This precious aquatic plant is distributed across frigid zones to the tropical regions worldwide. In horticulture, water lilies can be divided into two categories based on their ecological habits: tropical water lilies and hardy water lilies[2]. In addition to their aesthetic appeal, economically, Nymphaea species possess a substantial quantity of phytochemicals and nutrients, making them widely utilized for beverage preparation[3], essential oil extraction[4], and as a valuable source of food, nutrition, and medicinal purposes[5,6]. The incorporation of water lily in scented tea offers potential benefits in the tea beverage industry, combining ornamental value and potential health benefits[7]. Understanding the chemical composition and metabolic profiles of water lilies is important for uncovering their biological functions and applications. Metabolomics, a comprehensive study of small molecule metabolites, provides insights into the diverse range of non-volatile and volatile metabolites in water lilies[8,9].

    Non-volatile metabolites encompass a wide range of compounds, including primary metabolites such as carbohydrates, amino acids, and organic acids, as well as secondary metabolites such as flavonoids, alkaloids, and phenolic compounds[10]. These metabolites are essential for the growth, development, and defense mechanisms of plants[11]. Phytochemical screening of water lilies indicates the presence of several bioactive compounds like phenolic, flavonoids, triterpenes, glycosides, carbohydrates, and other compounds[12]. Phenols and flavonoids are the main phytochemicals found in water lilies and are responsible for their health benefits[5]. In addition, the captivating colors of water lily petals are attributed to their richanthocyanin content. For instance, delphinidin 3′-O-(2″-O-galloyl-6″-O-acetyl-β-galactopyranoside was identified as the primary blue anthocyanin in N. colorata[13]. Most detection methods for water lily metabolites are either targeted, with limited coverage, or non-targeted, with lower sensitivity and accuracy[14]. The combination of ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with widely targeted metabolomics techniques provides fast separation, high sensitivity, and broad coverage[15].

    Volatile metabolites, low molecular weight compounds that evaporate at ambient temperatures, are essential for the aroma, pollinator attraction, communication, and defense in plants. Its compositions vary among different species of water lilies. For example, day-blooming species emitting aromatic alcohols and ethers, while nocturnal species have a higher abundance of aromatic ethers, aliphatic esters, and C5-branched chain esters, which may play a role in attracting potential pollinators through olfactory cues[16]. In addition, previous studies using solid phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS) detected 79 and 71 volatile compounds in tropical water lilies and hardy water lilies, respectively, with aromatic substances being their major volatile components[17]. However, with the advancements in analytical techniques, researchers have sought to explore more advanced methods for volatile compound analysis. Currently, comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) has been widely adopted for the analysis of volatile compounds in a variety of foods due to its high resolution, high sensitivity, and peak capacity[18,19].

    In this study, we utilized UPLC-MS/MS and GC×GC-TOFMS techniques to comprehensively identify both non-volatile and volatile constituents of water lilies in five different colors. By comparing metabolite profiles across various Nymphaea species and varieties, we can uncover the discrepancies in chemical compounds that maybe linked to variations in scent, coloration, and potential biological activity. The findings of this study will enhance our understanding of the metabolic variability and chemical constitution of water lilies, thereby improving our knowledge of their distinctive attributes and potential uses.

    Methanol, acetonitrile and ethanol were purchased from Merck (Darmstadt, Germany). Ether (GC) was acquired from Tedia (Fairfield, OH, USA). Anhydrous sodium sulfate (AR) and ethyl decanoate (AR, internal standard) were purchased from Sigma-Aldrich (Shanghai, China). Distilled water was obtained from Wahaha Group Company (Hangzhou, China). The carboxen/divinylbenzene/polydimethylsiloxane (CAR-DVB-PDMS; 50/30 μm) microextraction fiber was obtained from Supelco (Bellefonte, PA, USA). The n-alkanes (C3-C9, C8-C40) were purchased from J&K Scientific (Beijing, China).

    As shown in Fig. 1, five different water lily samples were collected from Hangzhou Aquatic Plant Society, including two species — N. lotus (NL), N. rubra (NR), and three varieties — Nymphaea 'Texas Dawn' (TD), Nymphaea 'Blue Bird' (BB), and Nymphaea 'Detective Erika' (DE). Three fully expanded flowers (on the first day after flowering) were collected in the morning, three samples of each variety, and processed immediately.

    Figure 1.  Five different species and varieties of water lilies selected in this study.

    The petals of water lily samples were ground using a mixer mill (MM 400,) with a zirconia bead for 1.5 min at 30 Hz. One hundred mg of sample powder was dissolved with 0.6 mL 70% aqueous methanol; the dissolved sample was placed in the refrigerator at 4 °C for 12 h, during which the sample was vortexed six times; finally, the sample was centrifuged at 4 °C for 10 min at 12,000 r/min, and the extracts were absorbed and filtrated (SCAA-104, 0.22 μm pore size; ANPEL, Shanghai, China), and then sealed in the injection vial for subsequent UPLC-MS/MS analysis.

    The analysis was conducted by MetWare (Wuhan, China)[20]. UPLC (Shim-pack UFLC SHIMADZU CBM30A) equipped with tandem mass spectrometry (Applied Biosystems 4500 QTRAP) was used for the wide-targeted metabolomic assays of non-volatiles in water lily samples. The analytical conditions were as follows: Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100.0 mm, 1.8 μm); flow rate of 0.35 mL/min; column temperature of 40 °C; injection volume of 4 μL. The mobile phase A was ultrapure water (dissolved in 0.04% acetic acid), and the mobile phase B was acetonitrile (dissolved in 0.04% acetic acid). The mobile phase elution gradient was as follows: the proportion of phase B was 5% at 0.00 min, the proportion of phase B increased linearly to 95% at 10.00 min and maintained at 95% for 1 min, the proportion of phase B was reduced to 5% at 11.00–11.10 min and equilibrated with the proportion of phase B at 5% for 14 min. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS.

    Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), API 4500 Q TRAP UPLC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 550 °C; ion spray voltage (IS) 5,500 V (positive ion mode)/–4,500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), curtain gas (CUR) were set at 50, 60, and 30 psi, respectively; the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. Declustering potential (DP) and collision energy (CE) for individual MRM transitions was carried out with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.

    For the qualitative analysis, the metabolites were identified by matching the retention time, fragmentation patterns, and accurate m/z values to the standards in the self-constructed metabolite database (MetWare, Wuhan, China). The quantitative analysis was conducted based on the signal intensities of metabolites obtained from characteristic ions. MultiaQuant software was used to integrate and calibrate chromatographic peaks. The peak area of each chromatographic peak represented the relative content of the corresponding substance.

    The sample preparation method underwent minor modifications, as outlined in the previous study[19]. Specifically, simultaneous distillation extraction (SDE) method was used to extract the aroma components of water lily flower. The specific steps were as follows: weigh 10.00 g of the water lily flower sample to be tested, put it in a 500 mL round-bottomed flask and add 300 mL of boiling distilled water, and heat it to a slight boil with an electric heating jacket. Add 30 mL of redistilled anhydrous ether into the extraction flask and distill the extract at 50 °C for 1 h. Remove the water from the obtained material with anhydrous sodium sulfate and concentrate under nitrogen, then put into the injection bottle for sealing and storage at –20 °C for measurement.

    The analysis of aroma components in the water lily samples was conducted using a GC×GC-TOFMS system, which consisted of a gas chromatograph (Agilent 7890B; Santa Clara, CA, USA) coupled with a TOFMS instrument (LECO Pegasus 4D; LECO Corporation, St. Joseph, MI, USA). The first dimension (1st D) column was a non-polar DB-5MS column (30 m × 250 μm × 0.25 μm) and the second dimension (2nd D) column was a moderate polar DB-17HT column (1.9 m × 100 μm × 0.10 μm), and both above columns were purchased from Agilent Technologies (Santa Clara, CA, USA). Injection port temperature: 280 °C; Transfer line temperature: 270 °C; Carrier gas: helium (purity 99.999%); No split injection; Modulation time interval: 4.0 s; Sample injection volume: 1.0 μL. The 1st D column temperature program: hold at 60 °C for 3.0 min, ramp at 4.0 °C/min to 280 °C, hold for 2.5 min; 2nd D column temperature program: hold at 65 °C for 3.0 min, ramp at 4.0 °C/min to 280 °C, hold for 2.5 min; Total analysis time: 60.5 min. MS conditions: Electron ionization source; Ionization energy: –70 eV; Mass scanning range: 33 to 600 m/z; Ion source temperature: 220 °C.

    The GC×GC-TOFMS data were processed using the LECO ChromaTOF software. The calculated retention index (RI) values calculated using C8–C40 n-alkanes, and the reference RI values were obtained from the NIST 2014 semi-standard non-polar (DB-5) column. A compound was considered tentatively identified if the difference between the calculated and reference RI values was less than 20.

    Patrial least squares discriminant analysis (PLS-DA) was performed by MetaboAnalyst 5.0 (www.metaboanalyst.ca). The data was log transformed (log10) and normalization by median before PLS-DA. Significantly regulated metabolites between groups were determined by VIP ≥ 1 and absolute Log2FC (fold change) ≥ 1. To mitigate the risk of overfitting, we conducted a permutation test with 100 permutations.

    The identified non-volatile metabolites were annotated using Kyoto Encyclopedia of Genes and Genomes (KEGG) Compound database (www.kegg.jp/kegg/compound), annotated metabolites were then mapped to KEGG pathway database (www.kegg.jp/kegg/pathway.html). Pathways with significantly regulated metabolites mapped to were then fed into metabolite sets enrichment analysis, their significance was determined using p-values obtained from the hypergeometric test.

    A total of 533 non-volatile metabolites including nine categories were tentatively identified for water lilies by a comparison with tandem mass spectrum information from published databases and standards from the MetWare self-constructed metabolite database (Supplemental Fig. S1). As shown in Fig. 2a, these were 151 flavonoids (including 64 flavonoid and flavonoid carbonoside, 51 flavonols, 11 anthocyanins, 11 flavanols, nine dihydroflavone and dihydroflavonol, five isoflavones), 109 phenolic acids, 68 amino acids and derivatives, 57 lipids, 39 nucleotides and derivatives, 30 organic acids, 27 alkaloids, 23 saccharides and alcohols, and 29 other metabolites. Results showed that flavonoids, phenolic acids, lipids, amino acids and derivatives were the dominant non-volatile metabolites in the five water lilies. To better understand the differences in the content of non-volatile components between water lily samples from different species and varieties, the analysis was calculated by Log2FC (fold change) ≥ 2 or ≤ 0.5. The plots reflect the information on differential metabolite up-regulation and down-regulation (Fig. 2b). The numbers of differential metabolites identified in NL, TD, NR, and DE were 329 (237 up, 92 down), 310 (218 up, 92 down), 314 (213 up, 101 down), and 282 (177 up, 105 down), respectively.

    Figure 2.  Overview of the non-volatile components. (a) Quantitative distribution of chemical classes of volatile compounds. (b) Number of differentiated compounds with fold change ≥ 2 or ≤ 0.5. Note: NL, N. lotus; NR, N. rubra; TD, Nymphaea 'Texas Dawn'; BB, Nymphaea 'Blue Bird'; DE, Nymphaea 'Detective Erika'.

    In the present study, all differential metabolites were investigated based on fold change, and a total of 118 non-volatiles were screened in five water lilies, mainly consisted of flavonoids, phenolic acids, amino acids and derivatives, lipids, and organic acids metabolites (Supplemental Table S1 & Supplemental Fig. S2). Specifically, we screened 10 up-regulated and 10 down-regulated metabolites with the highest fold change values in different water lilies (Fig. 3). Comparing the NL samples to the BB samples, we observed higher levels of chrysoeriol-O-malonylhexoside, 6,7,8-tetrahydroxy-5-methoxyflavone, myricetin-O-glucoside-rhamnoside, and kaempferol-3-O-neohesperidoside. On the other hand, the BB samples contained higher levels of isoquercitrin and luteolin-7-O-β-D-gentiobioside. Moreover, the TD samples exhibited high levels of neochlorogenic acid, chlorogenic acid, 1-O-p-coumaroylquinic acid, and tetragallic acid, while the BB samples exhibited high levels of 2'-hydroxygenistein, phloretin 2'-O-glucoside, and 3,4-dihydroxybenzaldehyde. Comparing the NR samples to the BB samples, we found higher levels of cyanidin-3-rutinoside, cyanidin-3-O-galactoside, and myricetin-O-glucoside-rhamnoside. Conversely, the BB samples exhibited higher levels of 7-methoxycoumarin, myricitrin, and naringenin-7-O-glucoside. In comparison to the BB samples, the DE samples displayed higher levels of myricetin-O-glucoside-rhamnoside, cis-4-hydroxy-D-proline, myricetin-3-O-rhamnoside-7-O-rhamnoside, and neochlorogenic acid. On the other hand, the BB samples contained higher levels of myricitrin, isoquercitrin, and naringenin-7-O-glucoside.

    Figure 3.  The highest fold change values of non-volatile metabolites. (a) Fold change plot of NL vs BB. (b) Fold change plot of TD vs BB. (c) Fold change plot of NR vs BB. (d) Fold change plot of DE vs BB. Note: The horizontal coordinate is the log2FC of the differentially metabolized metabolite, and the vertical coordinate is the differentially metabolized metabolite. Red represents up-regulated differentially expressed metabolites, cyan represents down-regulated differentially expressed metabolites. NL, N. lotus; NR, N. rubra; TD, Nymphaea 'Texas Dawn'; BB, Nymphaea 'Blue Bird'; DE, Nymphaea 'Detective Erika'.

    We further identified the enrichment of differential metabolites in the KEGG mapping. The results of pathway enrichment analysis of the detected differential compounds using the KEGG database are shown in Supplemental Fig. S3. A total of 354, 327, 380, 299 differential compounds from BB vs NL, BB vs TD, BB vs NR, BB vs DE samples could be annotated to the relevant metabolic pathways, which were mainly significantly enriched in the pathways of biosynthesis of secondary metabolites, flavonoid biosynthesis, anthocyanin biosynthesis, flavonoids and flavonols biosynthesis, isoflavonoid biosynthesis. Furthermore, we observed significant enrichment in pathways associated with tryptophan metabolism and phenylpropanoid biosynthesis in the BB vs TD group. Additionally, caffeine metabolism exhibited significant enrichment in the BB vs NR group.

    The fragrance of water lily contains volatile compounds such as terpenes, phenylpropanoids, benzenoids, fatty acid derivatives, and amino acid derivatives. These compounds not only attract pollinators, but also play a crucial role in transmitting signals in plant-plant interactions and providing protection and defense for the plant[17]. In this study, the volatile compounds of water lily samples were analyzed by GC×GC-TOFMS (Supplemental Fig. S4). By comparing the MS of the compounds and comparing the chromatographic peaks and tested RI values with the reported RI values, 166 volatiles were identified, including 46 aromatic compounds, 34 alkynes, 22 ketones, 10 alcohols, 18 esters, 20 aldehydes, three carboxylic acids, five heterocyclics, five sulfur-containing compounds, and three other compounds (Fig. 4a). We identified 128, 141, 142, 135, and 129 volatile compounds in BB, NL, TD, NR, and DE, respectively, and 108 of the 166 metabolites were common to all water lily samples (Fig. 4b). We observed that 20 volatile compounds were exclusively detected in specific water lily samples. For instance, in BB variety, compounds such as amorpha-4,11-diene, (Z)-geranylacetone, α-bisabolol, and (E)-β-ionone were found. In NR variety, pyrocinchonic anhydride and 2-tetradecanone were exclusively detected. NL variety exhibited unique compounds including mequinol, 4-ethylresorcinol, p-xylene, and 2,5-hexanedione. TD variety showed the presence of terpilene, (E,E)-2,6-dimethyl-2,4,6-octatriene, 1-nonanol, β-bisabolene, sabinene, 3-carvomenthenone, (4E,6Z)-2,6-dimethyl-2,4,6-octatriene, ipsdienol, and 2-thujene. Lastly, DE variety exclusively contained 2-pentoxyethyl acetate.

    Figure 4.  Overview of the volatile components. (a) Quantitative distribution of chemical classes of volatile compounds. (b) Venn plot; numbers represent the identified metabolites. (c) Relative abundance of different types of volatile compounds. Note: NL, N. lotus; NR, N. rubra; TD, Nymphaea 'Texas Dawn'; BB, Nymphaea 'Blue Bird'; DE, Nymphaea 'Detective Erika'.

    The relative content of volatiles calculated from the total ion chromatograms varied in the concentration and proportion of each chemical class in different samples. Among them, the highest peak area of volatile components was found in BB, followed by NL (Fig. 4c). Alkenes were found to be the most abundant volatile components in BB, accounting for 69.58% of the total volatile components. Aromatic compounds were found to be the most abundant volatile components in NL, accounting for 64.25% of the total volatile components. In addition, alcohols and alkenes accounted for 28.05% and 26.89% of the total volatile components in DE, respectively.

    We investigated the relative amounts of the main 24 chemical compounds released that were greater than 1% in BB, NL, TD, NR, DE, with total relative contents of 90.92%, 91.45%, 83.19%, 77.29%, and 90.83%, respectively (Table 1 & Fig. 5). The concentrations of the identical chemical compounds varied across distinct samples. Within the provided BB sample, three compounds of the alkene class, namely 1,11-dodecadiene (27.30%), (E)-β-famesene (18.28%), and α-farnesene (14.44%), collectively constitute more than half of the total volatile compounds. Among the NL samples, 2,5-dimethoxytoluene (56.18%) emerged as the most abundant compound. In TD and NR samples, dimethyl sulfide was the predominant volatile compound, comprising 33.61% and 18.75% of the total volatile content, respectively. In addition, in DE sample, the concentration of benzyl alcohol (23.83%) was the most abundant, followed by dimethyl sulfide (16.67%) and α-farnesene (16.41%).

    Table 1.  Comparison of the main volatile compounds in five water lily samples.
    No.CompoundsClassRI (ref)[a]RI (cal)[b]CASIonOdor type[c]Flavor[c]Relative content (%)
    BBNLTDNRDE
    1Benzyl alcoholAlcohols1036 ± 41037100-51-679FloralFloral, rose, balsamic0.114.486.328.1323.83
    2BenzaldehydeAldehydes962 ± 3969100-52-777FruityAlmond, burntsugar, sweet8.102.133.175.223.91
    3HexanalAldehydes800 ± 280166-25-141GreenGreen, fatty, leafy0.150.690.613.840.34
    4BenzeneacetaldehydeAldehydes1045 ± 41049122-78-191GreenGreen, floral, honey0.160.141.030.090.85
    51,11-DodecadieneAlkenes1179 ± 217635876-87-967//27.303.6712.627.0411.45
    6(E)-β-FameseneAlkenes1457 ± 2145318794-84-841WoodyWoody, citrus, herbal18.280.390.840.381.27
    7α-FarneseneAlkenes1508 ± 21506502-61-441WoodyCitrus, herbal, neroli14.440.1710.380.1816.41
    8(E)-α-BergamoteneAlkenes1433 ± 3143713474-59-493WoodyWoody2.020.110.030.031.65
    9β-SesquiphellandreneAlkenes1524 ± 2152820307-83-969HerbalHerbal, fruity, woody3.430.120.150.101.37
    10(E)-β-OcimeneAlkenes1049 ± 210483779-61-193/Herbal, sweet0.010.033.890.040.01
    112,5-DimethoxytolueneAromatic compounds1251 ± 5124924599-58-4137//0.3856.180.4912.180.52
    121,4-DimethoxybenzeneAromatic compounds1168 ± 91166150-78-7123GreenGreen, hay, sweet0.053.620.026.630.02
    13PhenolAromatic compounds980 ± 4981108-95-294PhenolicPhenolic, plastic, rubbery0.170.870.561.460.62
    14Acetic acidCarboxylic acids610 ± 1058164-19-745AcidicPungent, sour3.422.012.180.771.58
    15Benzoic acid, methyl esterEsters1094 ± 3109693-58-3105PhenolicPhenolic, wintergreen, almond0.030.570.071.860.03
    16Ethyl acetateEsters612 ± 5613141-78-643EtherealEthereal fruity sweet0.380.390.140.002.06
    17Acetic acid, phenylmethyl esterEsters1164 ± 21166140-11-4108FloralFloral, fruity, jasmine1.880.010.060.020.45
    186-Methyl-5-heptene-2-oneKetones986 ± 2984110-93-043CitrusCitrus, green, musty1.292.162.153.673.05
    19(E)-3-Penten-2-oneKetones735 ± N/A7443102-33-869//0.211.010.311.580.40
    20(E)-GeranylacetoneKetones1453 ± 214483796-70-143FloralFruity, fresh, rose2.150.760.650.721.18
    212-HeptadecanoneKetones1902 ± 719002922-51-258//1.680.411.180.700.80
    22Dimethyl sulfideSulfur-containing compounds520 ± 755375-18-347SulfurousSulfurous, sweet corn4.378.7633.6118.7516.67
    23Carbon disulfideSulfur-containing compounds549 ± 1356575-15-076/Sweet0.551.171.961.291.39
    24BenzothiazoleSulfur-containing compounds1229 ± 8124095-16-9135SulfurousSulfurous, rubbery, vegetable, cooked0.290.480.561.160.53
    Total relative content (%)90.9291.4583.1977.2990.83
    RI, Retention index, Ion, Qualitative ion; [a] RI (ref): The RI values (median ± deviation) were the reference values for semi-standard non-polar (DB-5) column obtained from NIST 2014; [b] RI (cal): The RI values were calculated from C8-C40 n-alkanes; [c] odor type and flavor were obtained from website (www.thegoodscentscompany.com/search2.html). Note: NL, N. lotus; NR, N. rubra; TD, N. 'Texas Dawn'; BB, N. 'Blue Bird'; DE, N. 'Detective Erika'.
     | Show Table
    DownLoad: CSV
    Figure 5.  Molecular formulas of major volatile compounds.

    To gain a comprehensive understanding of the variations in volatile compound content among the five water lily samples, we utilized PLS-DA with the peak areas of 166 volatile compounds as input variables. As shown in Fig. 6a, the five samples were distinctly segregated from the remaining samples along the principal component 1 axis (R2X [1] = 37.6%) and principal component 2 axis (R2X [2] = 27.1%). Cross-validation was performed using the leave-one-out method, with the first two principal components explaining 99.6% of the total variance (R2X). The model exhibited good predictive ability (Q2 = 85.1%) and was not overfitted. The evident segregation and high reproducibility observed among the various sample groups substantiated the presence of significant disparities in the volatile compositions of the five water lily species and varieties (Fig. 6a).

    Figure 6.  The partial least squares discriminant analysis (PLS-DA) of the volatile compounds. (a) Score plot of PLS-DA. (b) The loading plot of PLS-DA. (c) Variable importance in the project (VIP) plot of PLS-DA. Note: NL, N. lotus; NR, N. rubra; TD, Nymphaea 'Texas Dawn'; BB, Nymphaea 'Blue Bird'; DE, Nymphaea 'Detective Erika'.

    The variable importance in projection (VIP) value is a comprehensive metric that quantifies the contribution of a variable in describing the data and indicates the significance of an independent variable for the model[21]. By utilizing the PLS-DA model, we identified 42 key volatile compounds with VIP scores of ≥ 1 across all samples (Supplemental Fig. S5). Subsequently, a one-way analysis of variance (ANOVA) was conducted on these compounds, revealing statistically significant differences among the distinct sample groups (p < 0.05). Notably, 26 metabolites of volatile compounds exhibited VIP values exceeding 1.5 (Fig. 6c), with 2,3-butanedione, octanal, 1-methyl-4-(1-hydroxy-1-methylethyl)benzene, acetic acid, phenylmethyl ester, 2,5-dimethoxytoluene, (E)-β-ocimene being among the top-ranked compounds in descending order of VIP values.

    Although previous studies have been reported on the volatile and non-volatile components of water lilies, the number of compounds reported in these studies is relatively limited[2,22]. By employing widely targeted metabolomics, we overcome the limitations associated with both targeted and non-targeted metabolite detection methods. This approach provided us with a high-throughput analysis, increased sensitivity, and wide coverage, enabling a more comprehensive understanding of the metabolomic profile of water lilies. In the present study, we comprehensively identified 533 non-volatile and 166 volatile components in five different colors of water lilies using UPLC-MS/MS and GC × GC-TOFMS techniques.

    It is widely recognized that flower color is a crucial characteristic of ornamental plants and is influenced by various factors. Specifically, the type and concentration of anthocyanins are generally considered to be the primary determinants[23]. Anthocyanins play an important role in plant physiology, serving as attractions for pollinators and herbivores, acting as deterrents against herbivores and parasites, and also influencing visual signals and mimicry of defensive structures[24]. Among the diverse range of flower colors, blue coloration primarily results from the presence of anthocyanins derived from delphinidin[25]. In the petals of Nymphaea 'King of Siam', four anthocyanins were identified: delphinidin 3-O-β-galactopyranoside, delphinidin 3′-O-(2″-O-galloyl-β-galactopyranoside, delphinidin 3-O-(6″-O-acetyl-β-glucopyranoside, delphinidin 3′-O-(2″-O-galloyl-6″-O-acetyl-β-galactopyranoside[13]. In the present study, a total of 11 anthocyanins were detected in five different-colored water lilies. Among them, delphinidin-derived anthocyanins exhibited relatively higher relative abundances in BB, TD, and DE, while its abundance was low in DE. Notably, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-rutinoside, cyanidin-O-acetylhexoside, and cyanidin-O-syringic acid, which are glycosides of the cyanidin type, were found to be the most abundant exclusively in the DE samples. In contrast, these compounds were found at very low levels or were not detected in the other four water lily samples.

    In addition, numerous studies conducted on various plant species have provided evidence that flavonoids (including flavones, flavanols, and isoflavonoids) and their glycosides play a significant role in co-pigmentation[26,27]. According to reports, N. lotus stamen extracts contain a higher concentration of flavonoids compared to perianth extracts[22]. The flavonoids identified in the stamen extracts include kaempferol 3-O-galactoside, quercetin 3′-O-xyloside, quercetin 3-O-rhamnoside, isorhamnetin 7-O-galactoside, and myricetin 3′-O-xyloside. Another study indicated that the content of flavonoids in the petals of the N. alba flower is significantly higher than in the stem and root[28]. In addition, a large number of phenolic acids, for example, caffeic acid, chlorogenic acid, p-coumaric acid have been identified in water lilies[12]. Our present study identified various flavonoid glycosides with glucose, rhamnose, galactose, and arabinose as the major sugar ligands. As a result, the number of detected flavonoids and their glycosides in water lilies has significantly expanded. Among them, quercetin, kaempferol, apigenin, myricetin, and luteolin were identified as the five major flavonols present in water lilies. In addition, many previous studies have reported the antioxidant potential of water lily extracts that are associated with the accumulation of its phytochemicals, especially flavonoids[5,7,29]. Therefore, the variations in the composition of flavonoids among distinct water lily species and varieties, as revealed in this investigation, are expected to provide valuable insights into their diverse antioxidant properties.

    Water lilies not only exhibit a wide variety of colors throughout their flowering period but also emit a captivating fragrance, which has garnered significant attention among researchers in recent studies[2,30]. Among the seven species of N. subg. Hydrocallis (Nymphaeaceae), N. lotus was only found to contain detectable levels of 2,5-dimethoxytoluene, and the content was notably high[16]. This is consistent with the results of our study. Dimethyl sulfide, a group of sulfur-containing compounds, was detected at significant concentrations in all five water lilies. Notably, it has been identified as a crucial aromatic volatile in green tea[31] and mandarin juices[32], and even in trace amounts, it contributes to the development of sulfurous notes. Benzyl alcohol, renowned for its delightful floral and sweet essence, has been extensively documented in various Nymphaea species[2]. Its relative concentration in the DE samples was 23.83% of the total volatiles. This high concentration may be the main reason for the sweet and floral aroma of the DE samples. Additionally, according to the report, 2,3-butanedione is known for its buttery, sweet, and creamy flavor, which imparts a cheese-like taste to soy milk[33]. Our research findings indicate that the BB samples had the highest concentration of 2,3-butanedione, which may be responsible for its sweet and creamy flavor, while it was not detected in the NL sample.

    In addition, previous studies reported that the flower aroma of the cold-resistant water lily is influenced by nerolidol and lilac alcohols, with orange and clove flower aromas, while the aroma of tropical water lilies is influenced by ethyl benzoate, acetic acid phenylmethyl ester, and 2-heptadecanone, which provide a fruity and sweet flower aroma[2]. Moreover, the primary volatile compounds identified in the flowers of Nymphaea hybrida were benzyl alcohol, benzyl acetate, benzaldehyde, (E)-α-bergamotene, and these may constitute its main fragrant components[34]. We observed that the NL sample releases a greater proportion of aromatic compounds than the other samples, which may account for its fruity and sweet flavor. In addition, (E)-α-Bergamotene, (E)-β-famesene and 11 other different volatile compounds were reported in N. colorata flowers, which may serve as olfactory cues for insect pollinators[35]. (E)-β-famesene and α-farnesene possess woody, citrus, and herbal aromatic attributes, was found to be more pronounced in BB samples, accounting for 18.28% and 14.44% of the total volatile composition, respectively. These compounds are likely responsible for the woody fragrance observed in BB samples. Overall, the varying concentrations and relative amounts of volatile compounds contribute to the rich and diverse fragrance qualities of these five water lily species and varieties.

    To sum up, our study utilized advanced widely targeted metabolomics to comprehensively investigate the non-volatile and volatile components in five water lily species and varieties. The results revealed significant differences in the composition and abundance of metabolites among the different colors of water lily samples. Regarding non-volatile components, we found that cyanidin-type anthocyanins were abundant in the purple-colored DE samples, while delphinidin-derived anthocyanins were prominent in BB, which exhibited a blue color. Flavonoids, phenolic acids, amino acids, lipids, and organic acids were found to be the dominant non-volatile metabolites, expanding our knowledge of their metabolic profile. Pathway enrichment analysis of the differential compounds indicated the involvement of various metabolic pathways such as flavonoid biosynthesis and anthocyanin biosynthesis in water lily metabolism. In terms of volatile components, water lilies contain diverse volatile compounds, including aromatic compounds, alkynes, ketones, alcohols, esters, and aldehydes. Among these, the concentration ofkey compounds including 1,11-dodecadiene, benzyl alcohol, benzaldehyde, α-farnesene and dimethyl sulfide, showed significant differences among samples. By investigating both non-volatile and volatile metabolites, we have obtained a comprehensive understanding of the metabolic pathways and interplay within the water lily species and varieties. Nevertheless, the intricate mechanisms underlying the synthesis and release of metabolites in water lilies warrant further investigation.

    The authors confirm contribution to the paper as follows: study conception and design: Chen Y, Lv H; data collection: Wei J, Wu Y; resources: Chen S, Yu C; analysis and interpretation of results: Lin Z, Zhu Y; draft manuscript preparation: Yang G. All authors reviewed the results and approved the final version of the manuscript.

    The datasets generated during and/or analyzed during the current study are not publicly available due to management requests, but are available from the corresponding author on reasonable request.

    This work was supported by the Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2014-TRICAAS) for financial support.

  • The authors declare that they have no conflict of interest.

  • [1]

    El Hosry L, Bou-Mitri C, Bou Dargham M, Abou Jaoudeh M, Farhat A, et al. 2023. Phytochemical composition, biological activities and antioxidant potential of pomegranate fruit, juice and molasses: a review. Food Bioscience 55:103034

    doi: 10.1016/j.fbio.2023.103034

    CrossRef   Google Scholar

    [2]

    Limongelli R, Minervini F, Calasso M. 2023. Fermentation of pomegranate matrices with Hanseniaspora valbyensis to produce a novel food ingredient. LWT 180:114687

    doi: 10.1016/j.lwt.2023.114687

    CrossRef   Google Scholar

    [3]

    Yu K, Huang X, Yu Z, Chen C, Li P, et al. 2023. Application of steam explosion pretreatment for accelerating the phenolics extraction from pomegranate peel: Mechanism and modeling. Journal of Food Engineering 357:111629

    doi: 10.1016/j.jfoodeng.2023.111629

    CrossRef   Google Scholar

    [4]

    Zheng Y, Jia X, Zhao Z, Ran Y, Du M, et al. 2023. Innovative natural antimicrobial natamycin incorporated titanium dioxide (nano-TiO2)/poly (butylene adipate-co-terephthalate) (PBAT)/poly (lactic acid) (PLA) biodegradable active film (NTP@PLA) and application in grape preservation. Food Chemistry 400:134100

    doi: 10.1016/j.foodchem.2022.134100

    CrossRef   Google Scholar

    [5]

    Wang S, Liu S, Liu C, Tang S, Gu D, et al. 2023. Affinity screening of potential anti-obesity and anti-diabetic component from pomegranate peel by co-immobilization of lipase and α-amylase using carbon nanotube and hydrogel. Process Biochemistry 126:51−60

    doi: 10.1016/j.procbio.2023.01.002

    CrossRef   Google Scholar

    [6]

    Ordaz-Rodríguez SB, López-Hernández LH, Mendoza-Sánchez MdJ, Escobar-Ortiz A, Abadía-García L, et al. 2023. Green extract of pomegranate peel (Punica granatum L.) obtained by ultrasound assisted extraction and its preservative properties on raw chicken burgers. Food and Humanity 1:1046−54

    doi: 10.1016/j.foohum.2023.08.020

    CrossRef   Google Scholar

    [7]

    Mishra V, Kaplan Y, Ginzberg I. 2022. Mitigating chilling injury of pomegranate fruit skin. Scientia Horticulturae 304:111329

    doi: 10.1016/j.scienta.2022.111329

    CrossRef   Google Scholar

    [8]

    Molla SMH, Rastegar S, Omran VG, Khademi O. 2022. Ameliorative effect of melatonin against storage chilling injury in pomegranate husk and arils through promoting the antioxidant system. Scientia Horticulturae 295:110889

    doi: 10.1016/j.scienta.2022.110889

    CrossRef   Google Scholar

    [9]

    Jia X, Li J, Du M, Zhao Z, Song J, et al. 2020. Combination of Low Fluctuation of Temperature with TiO2 Photocatalytic/Ozone for the Quality Maintenance of Postharvest Peach. Foods 9:234

    doi: 10.3390/foods9020234

    CrossRef   Google Scholar

    [10]

    Li W, Liu Z, Wang H, Yuan J, Zheng Y, et al. 2024. Heat shock pretreatment and low temperature fluctuation cold storage maintains flesh quality and retards watercore dissipation of watercored 'Fuji' apples. Scientia Horticulturae 323:112492

    doi: 10.1016/j.scienta.2023.112492

    CrossRef   Google Scholar

    [11]

    Kuang X, Chen Y, Lin H, Lin H, Chen G, et al. 2023. Comprehensive analyses of membrane lipids and phenolics metabolisms reveal the developments of chilling injury and browning in Chinese olives during cold storage. Food Chemistry 416:135754

    doi: 10.1016/j.foodchem.2023.135754

    CrossRef   Google Scholar

    [12]

    Gao H, Lu Z, Yang Y, Wang D, Yang T, et al. 2018. Melatonin treatment reduces chilling injury in peach fruit through its regulation of membrane fatty acid contents and phenolic metabolism. Food Chemistry 245:659−66

    doi: 10.1016/j.foodchem.2017.10.008

    CrossRef   Google Scholar

    [13]

    Yao M, Ge W, Zhou Q, Zhou X, Luo M, et al. 2021. Exogenous glutathione alleviates chilling injury in postharvest bell pepper by modulating the ascorbate-glutathione (AsA-GSH) cycle. Food Chemistry 352:129548

    doi: 10.1016/j.foodchem.2021.129458

    CrossRef   Google Scholar

    [14]

    Kong X, Wei B, Gao Z, Zhou Y, Shi F, et al. 2018. Changes in Membrane Lipid Composition and Function Accompanying Chilling Injury in Bell Peppers. Plant and Cell Physiology 59:167−78

    doi: 10.1093/pcp/pcx171

    CrossRef   Google Scholar

    [15]

    Wang L, Huang X, Liu C, Zhang C, Shi K, et al. 2023. Hydrogen sulfide alleviates chilling injury by modulating respiration and energy metabolisms in cold-stored peach fruit. Postharvest Biology and Technology 199:112291

    doi: 10.1016/j.postharvbio.2023.112291

    CrossRef   Google Scholar

    [16]

    Kong XM, Ge WY, Wei BD, Zhou Q, Zhou X, et al. 2020. Melatonin ameliorates chilling injury in green bell peppers during storage by regulating membrane lipid metabolism and antioxidant capacity. Postharvest Biology and Technology 170:111315

    doi: 10.1016/j.postharvbio.2020.111315

    CrossRef   Google Scholar

    [17]

    Ge W, Kong X, Zhao Y, Wei B, Zhou Q, et al. 2019. Insights into the metabolism of membrane lipid fatty acids associated with chilling injury in post-harvest bell peppers. Food Chemistry 295:26−35

    doi: 10.1016/j.foodchem.2019.05.117

    CrossRef   Google Scholar

    [18]

    Niu Y, Ye L, Wang Y, Shi Y, Liu Y, et al. 2023. Transcriptome analysis reveals salicylic acid treatment mitigates chilling injury in kiwifruit by enhancing phenolic synthesis and regulating phytohormone signaling pathways. Postharvest Biology and Technology 205:112483

    doi: 10.1016/j.postharvbio.2023.112483

    CrossRef   Google Scholar

    [19]

    Rehman RNU, Malik AU, Khan AS, Hasan MU, Anwar R, et al. 2021. Combined application of hot water treatment and methyl salicylate mitigates chilling injury in sweet pepper (Capsicum annuum L.) fruits. Scientia Horticulturae 283:110113

    doi: 10.1016/j.scienta.2021.110113

    CrossRef   Google Scholar

    [20]

    Kashash Y, Mayuoni-Kirshenbaum L, Goldenberg L, Choi HJ, Porat R. 2016. Effects of harvest date and low-temperature conditioning on chilling tolerance of 'Wonderful' pomegranate fruit. Scientia Horticulturae 209:286−92

    doi: 10.1016/j.scienta.2016.06.038

    CrossRef   Google Scholar

    [21]

    Li X, Yang H, Lu G. 2018. Low-temperature conditioning combined with cold storage inducing rapid sweetening of sweetpotato tuberous roots (Ipomoea batatas (L.) Lam) while inhibiting chilling injury. Postharvest Biology and Technology 142:1−9

    doi: 10.1016/j.postharvbio.2018.04.002

    CrossRef   Google Scholar

    [22]

    Fan X, Xi Y, Zhao H, Liu B, Cao J, et al. 2018. Improving fresh apricot (Prunus armeniaca L.) quality and antioxidant capacity by storage at near freezing temperature. Scientia Horticulturae 231:1−10

    doi: 10.1016/j.scienta.2017.12.015

    CrossRef   Google Scholar

    [23]

    Zhang W, Jiang H, Cao J, Jiang W. 2021. Advances in biochemical mechanisms and control technologies to treat chilling injury in postharvest fruits and vegetables. Trends in Food Science & Technology 113:355−65

    doi: 10.1016/j.jpgs.2021.05.009

    CrossRef   Google Scholar

    [24]

    Qiao L, Deng X, Yu X, Feng M, Jiao Y, et al. 2024. Appropriate sodium nitroprusside dose contributes to the quality maintenance of fresh walnuts. Food Innovation and Advances 3:42−51

    doi: 10.48130/fia-0024-0006

    CrossRef   Google Scholar

    [25]

    Ban Z, Niu C, Li L, Gao Y, Liu L, et al. 2024. Exogenous brassinolides and calcium chloride synergically maintain quality attributes of jujube fruit (Ziziphus jujuba Mill.). Postharvest Biology and Technology 216:113039

    doi: 10.1016/j.postharvbio.2024.113039

    CrossRef   Google Scholar

    [26]

    Li J, Han Y, Hu M, Jin M, Rao J. 2018. Oxalic acid and 1-methylcyclopropene alleviate chilling injury of 'Youhou' sweet persimmon during cold storage. Postharvest Biology and Technology 137:134−41

    doi: 10.1016/j.postharvbio.2017.11.021

    CrossRef   Google Scholar

    [27]

    Caleb OJ, Opara UL, Mahajan PV, Manley M, Mokwena L, et al. 2013. Effect of modified atmosphere packaging and storage temperature on volatile composition and postharvest life of minimally-processed pomegranate arils (cvs. 'Acco' and 'Herskawitz'). Postharvest Biology and Technology 79:54−61

    doi: 10.1016/j.postharvbio.2013.01.006

    CrossRef   Google Scholar

    [28]

    O'Grady L, Sigge G, Caleb OJ, Opara UL. 2014. Effects of storage temperature and duration on chemical properties, proximate composition and selected bioactive components of pomegranate (Punica granatum L.) arils. LWT - Food Science and Technology 57:508−15

    doi: 10.1016/j.lwt.2014.02.030

    CrossRef   Google Scholar

    [29]

    Lorente-Mento JM, Guillén F, Valverde JM, Valero D, Badiche F, et al. 2024. Chilling injury control in pomegranate fruit with compostable stretchable skin film. Scientia Horticulturae 323:112480

    doi: 10.1016/j.scienta.2023.112480

    CrossRef   Google Scholar

    [30]

    Luo Y, Wang R, Lei X, Ren Y, Yuan C. 2023. Melatonin treatment delays senescence and alleviates chilling injury in spaghetti squash during low-temperature storage. Scientia Horticulturae 310:111778

    doi: 10.1016/j.scienta.2022.111778

    CrossRef   Google Scholar

    [31]

    Andrade KS, Aguiar GPS, Rebelatto EA, Lanza M, Oliveira JV, et al. 2020. Encapsulation of pink pepper extract by SEDS technique: Phase behavior data and process parameters. The Journal of Supercritical Fluids 161:104822

    doi: 10.1016/j.supflu.2020.104822

    CrossRef   Google Scholar

    [32]

    Kato M, Guan S, Zhao X. 2021. In-situ observation of graphene using an optical microscope. Applied Surface Science Advances 6:100138

    doi: 10.1016/j.apsadv.2021.100138

    CrossRef   Google Scholar

    [33]

    Guo Y, Liang P, Tang Y, Zhang M, Li B. 2022. Effects of postharvest deastringency and 1-methylcyclopropene treatments on membrane permeability, membrane-degrading enzymes and their encoding genes in persimmon (Diospyros kaki, cv Mopanshi) fruit. Scientia Horticulturae 297:110941

    doi: 10.1016/j.scienta.2022.110941

    CrossRef   Google Scholar

    [34]

    Si J, Ye BB, Liu ZL, Xiao XM, Yang YY, et al. 2022. Transcriptional repression of MaRBOHs by MaHsf26 is associated with heat shock-alleviated chilling injury in banana fruit. Postharvest Biology and Technology 193:112056

    doi: 10.1016/j.postharvbio.2022.112056

    CrossRef   Google Scholar

    [35]

    Chen LL, Shan W, Cai DL, Chen JY, Lu WJ, et al. 2021. Postharvest application of glycine betaine ameliorates chilling injury in cold-stored banana fruit by enhancing antioxidant system. Scientia Horticulturae 287:110264

    doi: 10.1016/j.scienta.2021.110264

    CrossRef   Google Scholar

    [36]

    Wei K, Ma C, Sun K, Liu Q, Zhao N, et al. 2020. Relationship between optical properties and soluble sugar contents of apple flesh during storage. Postharvest Biology and Technology 159:111021

    doi: 10.1016/j.postharvbio.2019.111021

    CrossRef   Google Scholar

    [37]

    Moon P, Fu Y, Bai J, Plotto A, Crane J, et al. 2018. Assessment of fruit aroma for twenty-seven guava (Psidium guajava) accessions through three fruit developmental stages. Scientia Horticulturae 238:375−83

    doi: 10.1016/j.scienta.2018.04.067

    CrossRef   Google Scholar

    [38]

    Li W, Liu Z, Wang H, Zheng Y, Zhou Q, et al. 2024. Harvest maturity stage affects watercore dissipation and postharvest quality deterioration of watercore 'Fuji' apples. Postharvest Biology and Technology 210:112736

    doi: 10.1016/j.postharvbio.2023.112736

    CrossRef   Google Scholar

    [39]

    Islam M, Ali S, Nawaz A, Naz S, Ejaz S, et al. 2022. Postharvest 24-epibrassinolide treatment alleviates pomegranate fruit chilling injury by regulating proline metabolism and antioxidant activities. Postharvest Biology and Technology 188:111906

    doi: 10.1016/j.postharvbio.2022.111906

    CrossRef   Google Scholar

    [40]

    Qiao J, Guo L, Huo J, Huang D, Zhang Y. 2024. Controlled atmosphere effects on postharvest quality and antioxidant capacity of blue honeysuckle (Lonicera caerulea L.). Food Innovation and Advances 3:155−66

    doi: 10.48130/fia-0024-0015

    CrossRef   Google Scholar

    [41]

    Rai A, Kumari K, Vashistha P. 2022. Umbrella review on chilling injuries: Post-harvest issue, cause, and treatment in tomato. Scientia Horticulturae 293:110710

    doi: 10.1016/j.scienta.2021.110710

    CrossRef   Google Scholar

    [42]

    Kou X, He Y, Li Y, Chen X, Feng Y, et al. 2019. Effect of abscisic acid (ABA) and chitosan/nano-silica/sodium alginate composite film on the color development and quality of postharvest Chinese winter jujube (Zizyphus jujuba Mill. cv. Dongzao). Food Chemistry 270:385−94

    doi: 10.1016/j.foodchem.2018.06.151

    CrossRef   Google Scholar

    [43]

    Lufu R, Ambaw A, Opara UL. 2021. Functional characterisation of lenticels, micro-cracks, wax patterns, peel tissue fractions and water loss of pomegranate fruit (cv. Wonderful) during storage. Postharvest Biology and Technology 178:111539

    doi: 10.1016/j.postharvbio.2021.111539

    CrossRef   Google Scholar

    [44]

    Ginzberg I, Stern RA. 2016. Strengthening fruit-skin resistance to growth strain by application of plant growth regulators. Scientia Horticulturae 198:150−53

    doi: 10.1016/j.scienta.2015.11.016

    CrossRef   Google Scholar

    [45]

    Carvajal F, Palma F, Jamilena M, Garrido D. 2015. Cell wall metabolism and chilling injury during postharvest cold storage in zucchini fruit. Postharvest Biology and Technology 108:68−77

    doi: 10.1016/j.postharvbio.2015.05.013

    CrossRef   Google Scholar

    [46]

    Barman K, Asrey R, Pal RK. 2011. Putrescine and carnauba wax pretreatments alleviate chilling injury, enhance shelf life and preserve pomegranate fruit quality during cold storage. Scientia Horticulturae 130:795−800

    doi: 10.1016/j.scienta.2011.09.005

    CrossRef   Google Scholar

    [47]

    Ali S, Khan AS, Nawaz A, Naz S, Ejaz S, et al. 2023. The combined application of Arabic gum coating and γ-aminobutyric acid mitigates chilling injury and maintains eating quality of 'Kinnow' mandarin fruits. International Journal of Biological Macromolecules 236:123966

    doi: 10.1016/j.ijbiomac.2023.123966

    CrossRef   Google Scholar

    [48]

    Liu Q, Guo X, Du J, Guo Y, Guo X, et al. 2023. Comparative analysis of husk microstructure, fruit quality and concentrations of bioactive compounds of different pomegranate cultivars during low temperature storage. Food Bioscience 52:102400

    doi: 10.1016/j.fbio.2023.102400

    CrossRef   Google Scholar

    [49]

    Babalar M, Pirzad F, Sarcheshmeh MAA, Talaei A, Lessani H. 2018. Arginine treatment attenuates chilling injury of pomegranate fruit during cold storage by enhancing antioxidant system activity. Postharvest Biology and Technology 137:31−37

    doi: 10.1016/j.postharvbio.2017.11.012

    CrossRef   Google Scholar

    [50]

    Acosta-Ramírez CI, Lares-Carrillo ID, Ayón-Reyna LE, López-López ME, Vega-García MO, et al. 2024. A comprehensive study from the micro- to the nanometric scale: Evaluation of chilling injury in tomato fruit (Solanum lycopersicum). Food Research International 176:113822

    doi: 10.1016/j.foodres.2023.113822

    CrossRef   Google Scholar

    [51]

    Zhang Z, Huber DJ, Qu H, Yun Z, Wang H, et al. 2015. Enzymatic browning and antioxidant activities in harvested litchi fruit as influenced by apple polyphenols. Food Chemistry 171:191−99

    doi: 10.1016/j.foodchem.2014.09.001

    CrossRef   Google Scholar

    [52]

    Du M, Jia X, Li J, Li X, Jiang J, et al. 2020. Regulation effects of 1-MCP combined with flow microcirculation of sterilizing medium on peach shelf quality. Scientia Horticulturae 260:108867

    doi: 10.1016/j.scienta.2019.108867

    CrossRef   Google Scholar

    [53]

    Zhao Y, Song C, Brummell DA, Qi S, Lin Q, Duan Y. 2021. Jasmonic acid treatment alleviates chilling injury in peach fruit by promoting sugar and ethylene metabolism. Food Chemistry 338:128005

    doi: 10.1016/j.foodchem.2020.128005

    CrossRef   Google Scholar

  • Cite this article

    Li L, Luo J, Li X, Pang L, Jia X, et al. 2024. The optimal precise temperature alleviated chilling injury and maintained post-harvest quality for 'Mengzi' pomegranate fruit. Food Innovation and Advances 3(4): 385−395 doi: 10.48130/fia-0024-0036
    Li L, Luo J, Li X, Pang L, Jia X, et al. 2024. The optimal precise temperature alleviated chilling injury and maintained post-harvest quality for 'Mengzi' pomegranate fruit. Food Innovation and Advances 3(4): 385−395 doi: 10.48130/fia-0024-0036

Figures(7)  /  Tables(1)

Article Metrics

Article views(1295) PDF downloads(205)

ARTICLE   Open Access    

The optimal precise temperature alleviated chilling injury and maintained post-harvest quality for 'Mengzi' pomegranate fruit

Food Innovation and Advances  3 2024, 3(4): 385−395  |  Cite this article

Abstract: Chilling injury (CI) is a highly common physiological disorder in pomegranates during cold storage. Although several approaches have been investigated to mitigate the CI symptoms among some pomegranate cultivars, the fundamental and crucial environmental factor — the precise storage temperature for the 'Mengzi' cultivation remains unknown. This research evaluated the impact of storage temperatures of 0, 1, 2, 3, and 4 °C on the post-harvest quality of pomegranates. Results indicated that pomegranates stored at 2 °C exhibited the slightest color change and browning index. After storage of 130 d, pomegranates stored at 2 °C exhibited the lower CI index (82.79% reduction) and the lowest decay incidence (24.68% reduction) compared to those stored at 0 °C. The respiratory rate of pomegranates (2 °C) was also evidently suppressed (16.60%), along with a reduction in weight loss (3.46%). Furthermore, pomegranates stored at 2 °C exhibited the lowest activities of polyphenol oxidase (PPO) and peroxidase (POD), accompanied by the highest total phenolic content, which contributed to a reduction in malondialdehyde (MDA) accumulation. Relatively higher concentrations of soluble solids and titratable acid, as well as a higher sensory evaluation, were found in pomegranates stored at 2 °C. Consequently, it was inferred that the optimal temperature maintained cell membrane integrity modulated normal respiratory metabolism, and oxidative balance, and therefore alleviated CI and deterioration. This report can provide the guiding significance for the long-term storage of 'Mengzi' pomegranates under the condition of precise temperature control in phase temperature storage.

    • The pomegranate (Punica granatum L.), a berry of the pomegranate family (Punicaceae), is a medicinal and edible fruit[1]. In addition to numerous vitamins and minerals[2], pomegranates are also rich in valuable bioactive substances, including anthocyanins, carotenoids, tannins, polyphenols[3], and flavonoids[4]. It is proven that these substances have anti-diabetic, anti-inflammatory, antioxidant[5], anti-tumour, anti-hyperglycaemia, and anti-hypertensive properties in vivo and in vitro[6].

      Pomegranates are highly sensitive to temperature and are typically stored in cold storage at a range of 0−5 °C[7]. Higher temperatures lead to obvious water loss, browning, and decay. While, under unfavorable low temperatures condition, the fruits are vulnerable to chilling injury (CI), thus leading to metabolic disorder and decay during post-harvest storage[8]. Besides, low temperature fluctuation (LTF) ranging from ± 0.1 to ± 0.2 °C was discovered to significantly alleviate the CI in peach during 60 d of storage, as opposed to a temperature fluctuation ranging from ± 0.5 to ± 1 °C[9]. Similarly, LTF storage was demonstrated to be an effective approach to maintaining flesh quality and retarding water core dissipation in 'Fuji' apples[10]. There are many publications revealing that the occurrence of CI is accompanied by an increase in oxidative enzyme levels, including polyphenol oxidase (PPO) and peroxidase (POD) activity, as well as total phenolics. Specifically, the chilling temperatures exerted a stimulating effect on the activities of PPO and POD and resulted in a decrease in the content of phenolic substances in olives[11] and peaches[12].

      It is well known that the membranes guarantee the biochemical and physiological reactions are carried out orderly and efficiently[13]. There is a hypothesis that CI begins with internal cell membrane injury. Kong et al.[14] proposed that the increased incidence of CI in bell peppers is accompanied by enhanced membrane permeability, and it was further demonstrated that the incidence of CI is closely related to the integrity of the cell membrane. Likewise, Wang et al.[15] found more malondialdehyde (MDA) accumulated along with CI occurrence in peaches. Also, the impaired function of cell membranes led to an insufficient supply of metabolic energy, which was closely associated with respiratory abnormalities. This led to the excessive buildup of MDA and reactive oxygen species (ROS)[16]. The accumulation of these harmful substances disrupted a series of physiological and metabolic disturbances, ultimately resulting in further exacerbation of the development of CI symptoms[17].

      To date, a diversity of methods have been explored to mitigate the CI among pomegranates and other fruits. Physical treatment for controlling CI is primarily achieved through temperature conditioning[18], near-freezing temperature storage[19], and heat treatment[20]. A study in which sweet potato tuberous roots were pre-cooled at 10 °C for 5 d before cold storage demonstrated that low-temperature conditioning could inhibit the CI progression[21]. Fan et al.[22] proved that the near-freezing point temperature of 0 °C extended the storage time of apricot from 45 to 90 d without CI symptoms. Furthermore, it was demonstrated that pomegranates were soaked with hot water at 45 °C for 4 min, and subsequently stored at 2 °C for 3 months, leading to a significantly reduced incidence of CI symptoms[23]. Additionally, chemical treatments, such as sodium nitroprusside[24], hydrogen sulfide[15], brassinolides[25], and 1-MCP[26] are also the research hotspots for mitigating CI among fruits and vegetables.

      Although many techniques have been proposed to reduce CI and increase storage-ability, the temperature always remains the key environmental factor that governs the extent of CI. An appropriate and precise preservation temperature prominently maintains pomegranate physiological quality[27,28]. Further, it has been observed that the chilling injury of pomegranates was remarkably different in different cultivars and different harvest periods[20]. While there are few researches on the influence of precise storage temperature for 'Mengzi' pomegranate. This research investigated the optimal temperature for 'Mengzi' pomegranate during 130 d of storage. Not only their storage parameters were determined, but also the indicators related to micro-structure and ROS-redox balance in all storage temperatures were evaluated. The aim is to provide a reference for the long-term commercial storage of 'Mengzi' pomegranates.

    • The 'Mengzi' pomegranates (Punica granatum L.) were harvested at 80% maturation (the color of pomegranate surface was green-red, and the single fruit weight was 330−350 g) in October from the Heyuan Ten Thousand Acres Pomegranate Manor of Honghezhou Heyuan Agricultural Development Co., Ltd located at Liwu Village, Jianshui County, Honghezhou City, Yunnan Province, China (23°37′32″ N, 102°49′16″ E). Then they were wrapped with single-fruit net bags, placed in foam boxes with ice packs (the ice packs were covered with a foaming net), and airlifted to the Agricultural Products Processing and Preservation Laboratory of Tianjin University of Science and Technology (Tianjin, China). To ensure the transportation environment was as uniform as possible, the same ice packs, and the same quantity of pomegranates were placed in each box during cold chain transportation. With a bright red color, uniform size, smooth surface, no mechanical lesions, no cracks and no disease spots, pomegranates were selected for the experiment. After pre-cooling at 3 °C for 12 h, the samples were packaged into 0.02 mm PE bags, containing 20 samples per bag. Each treatment comprised 10 bags and were subsequently stored at 0 ± 0.2 °C, 1 ± 0.2 °C, 2 ± 0.2 °C, 3 ± 0.2 °C, and 4 ± 0.2 °C, respectively.

    • The CI indicator was evaluated in light of the proportion and severity of visible symptoms area[29]. Fifteen individual fruits were taken and calculated using five grades and the following formula: 0 = no CI, 1 = slight (0 < CI ≤ 5%), 2 = regular (5% < CI ≤ 15%), 3 = moderate (15% < CI ≤ 25%); 4 = severe (CI > 25%).

      CIindex(CI)=NumberoffruitswithCIsymptom×CIscoreTotalnumberofeachtreatment×4
    • The BI indicator was evaluated, in light of the browning area. The extent of BI was categorized into these five levels given the approach of Luo et al.[30]: 0 = no BI, 1 = slight (0 < BI ≤ 10%), 2 = regular (10% < BI ≤ 25%), 3 = moderate (25% < BI ≤ 50%), 4 = severe (> 50%). The BI indicator was computed as below:

      BIindex(BI)=NumberoffruitswithBIsymptom×BIscoreTotalnumberofeachtreatment×4
    • L*, a*, b* and ΔE*, as peel colour parameters, were assessed through a color meter (HP-200, China). Decay incidence was the ratio of decay fruits to the total fruits denoted as per
      Gao et al.[12].

    • Microscopic morphology of pomegranate peel was observed based on the approach of Andrade et al.[31]. Several pieces of pomegranate husk (5 mm × 5 mm) were randomly cut and fixed in 4% glutaraldehyde solution (pH 6.8) prepared with 0.1 mol·L−1 phosphate buffer. After 3 h, they were rinsed with 0.1 mol·L−1 phosphate buffer three times and subsequently dehydrated using isoamyl acetate for 20 min. Finally, the specimens were freeze-dried using one vacuum freezing dryer (FD-1A-50, China) for 1 d. The slices were sprayed with gold sputtering coater for 60 s and then were observed with SEM (LEO, Germany).

      Similarly, the microstructure of pomegranate samples was observed in accordance with the approach of Kato et al.[32] with some adjustments. Pomegranate peel tissue samples were cut into cubes with the size of 1 cm3 and fixed in FAA solution (5% acetic acid, 40% formaldehyde, and 70% ethanol) at low temperatures (4 °C). After 48 h, the samples were successively put into 70%, 80%, 90%, 95%, and 100% ethanol solutions (2 h per solution) for dehydration. Subsequently, the samples were successively put into 100% alcohol: 100% xylene (1:1) solution, 100% xylene, and 100% xylene solution (1.5 h per solution) for transparency. After that, the samples were placed in xylene: paraffin (1:1) solution, and subsequently embedded in pure paraffin. Once solidified, the paraffin wax was cut into pieces with 10 μm in thickness. Finally, the slices were stained with eosin solution and micro-photographed using an inverted fluorescence microscope (TE2-PS, Japan).

    • The permeability of the cell membrane was assessed according to the method of Guo et al.[33] with some adjustments. Ten cylindrical tissue samples (10 mm in diameter) were extracted from pomegranate peel using one punch and rinsed three times using distilled water. Then, tissues were placed in a triangular bottle containing distilled water (20 mL) at 25 °C for 1 h. The initial conductivity P1 was determined through one digital conductometer (DDS-11A, China). Subsequently, the specimen was boiled for 10 min and then cooled at 25 °C. The conductivity of P2 was determined. The conductivity of distilled water was P0. The cell membrane permeability (%) = (P1 - P0) × 100 / (P2 - P0). Throughout the entire process, all utensils were rinsed with distilled water, and the slice samples were not touched directly by hand. Before measurements, the electrode was calibrated using a blank solution. Additionally, before each measurement, the electrode was rinsed with distilled water and then washed with the liquid to be measured three times.

    • Respiratory rate was measured through one fruit and vegetable respiration detector (FS-GH100, China). The results were represented in CO2 mg·kg−1·h−1. Specifically, after weighted, one pomegranate fruit was placed into an air-tight jar (1 L) and detected at room temperature (25 °C) for 3 min. Then the respiration rate was calculated automatically by inputting the weight of the pomegranate. Weight loss was measured as the distinction between the initial weight and the weight of each storage time, then divided by initial weight.

    • MDA concentration of pomegranates was measured according to the method of Si et al.[34]. As well as pomegranate peel (5 g), 10% trichloroacetic acid (TCA) solution (10 mL) was taken into pre-cooled mortar and ground into homogenizing pulp under the condition of ice bath. After grinding, the homogenizing pulp was centrifuged at 5,000× g for 10 min under 4 °C. Two mL supernatant was blended with 0.6% of 2 mL thiobarbituric acid (TBA) solution and bathed in water for 5 min under 100 °C. After that, it was cooled to room temperature and then centrifuged at 5,000× g at 4 °C for 10 min. Absorbance of samplers was determined at 600, 532, and 450 nm, respectively. The MDA content (μmol·g−1·FW) = [(OD532 − OD600) − 0.56 × OD450] × V/(Vs × m × 100), where, V represented the volume of the extract (mL); Vs indicated the volume of the extract utilized for the measurement (mL); and m represented the weight of the sample (g).

    • The total phenolic content of pomegranates was measured as described by Chen et al.[35]. Pomegranate tissues (0.2 g) and 70% methanol (4 mL) were ground into a homogenate in a mortar and then bathed at 70 °C for 15 min. Then, the homogenate were centrifuged at 5,000× g for 20 min after cooled to room temperature. The supernatant was added up to 25 mL using distilled water. After that, the above supernatant (1 mL) was taken and blended with Folin-Ciocalteus (1 mL) and 7.5% sodium carbonate solution (1 mL). After the blend was incubated at 25 °C for 1 h, the phenolic content was determined at an absorbance of 760 nm and denoted as mg·g−1.

    • The peroxidase (POD) activity and polyphenol oxidases (PPO) activity were measured according to the method of Si et al.[34]. The pomegranate peel (3 g) and 10 mL PBS solution (1 mM PEG 6000, 4% PVPP, 1% Triton X-100) were taken into pre-cooled mortar and ground into homogenate under the condition of ice bath. Afterward, the mixture was centrifuged at 5,000× g at 4 °C for 30 min. The supernatant was collected as enzyme extracts and stored at 4 °C for later tests. As for POD activity, 0.5 mL enzyme extracts were mixed with 1.0 mL of 5 mM guaiacol solution and 0.5 mL 2% H2O2. At 15 s of the reaction, the absorbance was measured at 470 nm six times (once per 10 s). POD activity (U·g−1·FW) = ΔA470 × Vt/0.01 × m × Vs × t, here in, ΔA470 represented value change in absorbance, t denoted the reaction time, m signified the mass of the sample, Vt indicated the total volume of supernatant, and Vs stood for the volume of supernatant utilized for the measurement. As for PPO activity, 0.1 mL enzyme extracts were blended with 2.9 mL phosphate buffer (pH 5.5) and 1mL of 1 mM catechol solution, and then the mixture was incubated at 37 °C for 10 min. At 15 s of the reaction, the absorbance was read at 420 nm for 6 min (once per 10 s). PPO activity (U·g−1·FW) = ΔA420 × d/0.01 × t, where, ΔA420 represented the value change in absorbance, t denoted the reaction time, and d signified the dilution multiple. In the control group, 0.5 mL distilled water was added to replace the enzyme extracts.

    • As claimed by Wei et al.[36], SSC was determined through one pocket refractometer (PAL-3, Japan). TA was evaluated in accordance with the approach of Moon et al.[37].

    • Sensory evaluation was conducted in line with the approach of Li et al.[38] with minor adjustments. Pomegranates were eliminated from low-temperature storage after 130 d and stored at ambient conditions (25 °C). The 20 panelists were invited to evaluate the pomegranate samples that were stored at 25 °C on day 1, day 4, day 7, and day 10. The evaluation panelists (10 males and 10 females) were members of the Agricultural Products Logistics Preservation Laboratory. They were between the ages of 20 and 30, and had extensive experience in food sensory evaluation. The members have all received systematic training in the specialized course of food sensory evaluation. Meanwhile, the processing methods of the samples were confidential, and the samples were randomly numbered for sensory evaluation. The sensory evaluated attributes of pomegranates included color, flavor, firmness, texture, and overall acceptability. The preference scale is as follows: very low intensity (scores ≤ 3); low intensity (3 < scores ≤ 5); moderate intensity (5 < scores ≤ 7); elevated intensity (7 < scores ≤ 9); excessively elevated intensity (9 < scores ≤ 10).

    • Both technical replications (repeated measurements of the same sample) and biological replications (different samples from the same treatment group) experiments were conducted in triplicate for analysis of each treatment. The SPSS 27 (SPSS Inc., USA) was used to analyze the significance of differences. Moreover, mean values with standard errors were represented. Further, digraph analysis was implemented through Origin 2021 (Microcal Software, MA, USA). Duncan's multiple range tests were applied for analysis of variance (ANOVA). In addition, p < 0.05 is significant.

    • The appearance of pomegranates was diverse after 130 days cold storage. Apparent CI symptoms of pomegranates at 0 °C were observed as follows: depression, browning of the husk, pitting, and scald (Fig. 1a). As depicted in Fig. 1b, the CI gradually rose as storage temperature decreased and storage time extended. The results demonstrated that the CI index exhibited a distinctly elevated trend among the pomegranate samples stored at 0 and 1 °C, as compared to the other treatment groups. On day 130, the values reached 0.523 and 0.278, respectively. Similar results have been published by Zhang et al.[23]. The pomegranates stored at 2 °C resulted in a 30 d postponement of the CI occurrence, accompanied by a markedly reduced CI indicator on day 130 in comparison to the group stored at 0 °C. Similar publications have been released in persimmon, where the alleviation of CI was achieved at 4 °C but aggravated at lower storage temperatures[26].

      Figure 1. 

      Effects of storage temperatures on (a) appearance, (b) CI, (c) decay incidence, along with (d) BI. Values of the figures were exhibited as means ± standard error (n = 3). Vertical bars denoted the standard errors of the means. Disparate small letters denoted significant differences in the treatments for each sampling period at p ≤ 0.05.

      The surface decay on the pomegranate was another pervasive CI symptom[39]. The variation of decay incidence is shown in Fig. 1c. Similar to the trend of the BI index, significantly lower decay incidence was noted from 90 to 130 d for pomegranates stored at 2 °C as compared with other groups. On day 130, the pomegranates stored at 0 °C exhibited a decay incidence of 10.09%, while those stored at 2 °C had the lowest decay incidence at 7.65%. In CI fruit, the amino acids, sugars, and some minerals were released from chilling-injured cells, which could offer the substrate to microorganisms, particularly fungi. The pathogens then infected the fruit and led to decay[27].

      Colouration parameters L*, a*, b*, and ∆E* for pomegranates during storage as displayed in Fig. 2. The L* values of the fruit peel exhibited a decline as the storage time increased. The L* value of pomegranates stored at 0 °C showed a dramatic decline of 37.50% on day 130, while those stored at 4 °C showed a reduction of 26.49% (Fig. 2a). The results suggested that the storage of pomegranates at 0 and 4 °C resulted in the CI and browning, respectively, which in turn led to a reduction in fruit surface luster. The occurrence of CI and browning both led to the decrease of gloss of the pomegranate surface[40], which may be the reason for the higher L* value of samples stored at 2 °C than those stored at other temperatures. Furthermore, there was a similar study in which the decreasing L* values of water core 'Fuji' apples indicated increasing browning[38].

      Figure 2. 

      Effects of storage temperatures on the (a) L* value, (b) a* value, (c) b* value, and (d) ∆E* value of pomegranates appearance. Values of the figures were displayed as means ± standard error (n = 3). Vertical bars denoted the standard errors of the means. Disparate letters denoted prominent differences in the treatments for each sampling period at p ≤ 0.05.

      The a* values raised constantly in all treatments during the initial 20 d, followed by a gradual decline (Fig. 2b). The a* values of pomegranates stored at 2 °C exhibited a higher level during 130 d of storage than those of other treatments. The a* values of pomegranates stored at 2 °C exhibited the greatest increase, while those stored at 0 °C had the smallest increase during the initial 20 d. It might be that chilling temperature could significantly inhibit the ripening of fresh fruits[20]. Failure to maturity was another ordinary CI symptom among fruits like bananas, tomatoes, and melons[41]. Besides, the a* values of pomegranate samples stored at 3 and 4 °C were found to be lower than those at 2 °C, suggesting that higher storage temperature (3 and 4 °C) may contributed to a higher degree of browning. This was consistent with the results of BI.

      Moreover, the b* values (Fig. 2c) and ∆E* (Fig. 2d) values showed an increasing trend with time. The b* and ∆E* values of pomegranate stored at 0 °C increased the most, while those stored at 2 °C was the least. These changes might correlate to maturity and browning[42]. The results demonstrated that the optimal storage temperature (2 °C) was beneficial to maintain the appearance of pomegranate and delay the browning and senescence.

    • SEM observations showed varying degrees of cracking on the surface of the peel at different temperatures (Fig. 3a). By comparison, pomegranates stored at 0 °C exhibited considerable cracks, a rough surface, and severely damaged wax layer. Crack extension might result in further water loss and microbial aggression, which might accelerate fruit decay[43]. Pomegranates stored at 2 °C exhibited no epidermal cracks and retained a complete waxy layer compared to other treatments. Generally, the significant decrease in waxy cuticle thickness might result from the wax falling off the peel surface during fruit storage[43]. The waxy cuticle is a momentous barrier for fruits, which contributes to anti-mechanical damage, resistance to moisture loss, and pathogen penetration[44].

      Figure 3. 

      Effects of storage temperatures on micro-structure ((a) SEM images, (b) paraffin section images), (c) cell membrane permeability, (d) respiratory rate, and (e) weight loss. Values in the figures were exhibited as means ± standard error (n = 3). Vertical bars stood for the standard errors of the means. Disparate letters denoted prominent differences in treatments for per sampling period at p ≤ 0.05.

      It was noteworthy that cells with CI typically exhibited an irregular and collapsed shape, as illustrated in Fig. 3b. The vascular bundle cells of pomegranates stored at 0 °C were damaged, and the surrounding stone cells were irregularly arranged. Besides, no apparent boundaries emerged between epidermal cells (Fig. 3b.b-1), suggesting that the cell membranes were degraded[17]. Similar findings were observed in CI samples of zucchini, which could be ascribed to dissolution and degradation of cell walls[45]. During 130 d of storage at 2 °C, the majority of cells retained their integrity and original shape (Fig. 3b.b-3).

      The changing patterns of cell membrane permeability are represented in Fig. 3c. Similar to the trends of CI, the cell membrane permeability of pomegranates stored at 0 °C exhibited a notable increase in comparison to the other treatments, particularly within the final 30 d. Notably, cell membrane permeability of pomegranates stored at 0 °C exhibited the greatest increase, reaching 57.46%, while those stored at 2 °C displayed the smallest increase, reaching only 33.68% on day 130. Reportedly, cell membrane lipids experienced a physical variation from a liquid-crystalline to a solid gel state at chilling temperature, resulting in an elevation of membrane permeability and ions leakage[46].

      Respiration, as a basic metabolic activity, was a crucial factor in maintaining the fruit quality[15]. The respiratory rate was presented in Fig. 3c. On day 40, the respiratory rate of pomegranates stored at 0 °C increased significantly and reached a respiratory peak of 34.85 mg∙kg−1∙h−1, which was roughly 1.17 times as high as that of the samples on day 20. Subsequently, respiratory rate slowly declined over times, yet remained higher than other groups. The respiratory rate of fruit stored at 2 °C was decreased steadily and was always lower than other groups throughout the storage time. Thereby, it was indicated that the CI occurrence might trigger a prominent burst in respiratory rate by combining the CI index. CI induced the abrasion of cell membrane and other cell organelles, especially the mitochondrial membrane, resulting in abnormally high respiration rate[46]. The aforementioned findings consisted with previous reports on cucumber and persimmon[46].

      Weight loss is chiefly attributed to the water evaporation from stored pomegranates[46]. Weight loss of pomegranates significantly increased during 130 d of storage (Fig. 3d). On day 130, the pomegranates stored at 4 °C demonstrated the highest weight loss at 6.05%. Following that, the fruit stored at 0 °C exhibited a weight loss of 5.48%. In contrast, the pomegranates stored at 2 °C exhibited the lowest weight loss, at approximately 3.46%. A robust relationship between weight loss and pomegranates respiration rate has been established and the natural barrier of the waxy layer in reducing the fruit weight loss has also been confirmed by Ali et al.[47]. Therefore, less weight loss of pomegranates at 2 °C might be ascribed to stable respiratory rate and slight CI. Pomegranates stored at 0 °C exhibited an abnormal respiratory rate and a destructed waxy layer of peel, which might lead to excessive water loss[48].

    • MDA and cell membrane permeability are important indicators of fruit senescence and chilling injury caused by low temperatures[17]. The MDA content raised continuously during the storage of 130 d, as depicted in Fig. 4a. On day 20, the MDA content of pomegranates stored at 0 °C rose to 6.56 μmol·g−1·FW, exhibiting a rise of 3.08 μmol·g−1·FW compared to the initial value, while the samples stored at 2 °C increased by only 1.29 μmol·g−1·FW. The increased MDA content indicated that mild chilling injury had occurred in the pomegranates under the storage condition of 0 °C. It was also reported that both loquat and banana fruits with chilling injury exhibited higher levels of MDA accumulation and electrolyte leakage[49]. The MDA content of pomegranates stored at 2 °C was relatively lower compared to other groups on day 130, reaching only 13.23 μmol·g−1·FW. Notably, the MDA content of pomegranates stored at 0 °C was almost twice as much as that stored at 2 °C on day 130. Accordingly, it was indicated that the formation of MDA in the 'Mengzi' pomegranate was aggravated by the cold stress. The present findings are in agreement with reports on bell peppers and peaches[12].

      Figure 4. 

      Effects of storage temperatures on (a) MDA, (b) total phenolic content, (c) POD, and (d) PPO. Values of the figures were displayed as means ± standard error (n = 3). Vertical bars denoted the standard errors of the means. Disparate letters represented remarkable differences in the treatments for each sampling period at p ≤ 0.05.

      All treatments exhibited a declined tendency in the total phenolic content during a storage of 130 d (Fig. 4b). The total phenolic content of pomegranates stored at 2 °C declined slower than others, accumulating 35.56 mg·g−1·FW on day 130. In particular, the total phenolic content of pomegranates stored at 0 °C declined dramatically, decreasing by 38.53% on day 130. The total phenolic content of pomegranates stored at 3 and 4 °C were also lower than those stored at 2 °C, which may be due to the increased tissue aging and browning (Fig. 1d) of pomegranates with increasing storage temperature[50]. These results suggested that storage temperature (2 °C) improved the retention of phenolic content in pomegranates. In turn, the high phenolic content increased the resistance to senescence and CI during the post-harvest storage in pomegranates. This may be because the total phenolics can prevent the membrane lipid peroxidation by inhibiting the occurrence and propagation of oxidative chain reactions[12].

      The pattern of POD activity is presented in Fig. 4c. The POD activity of pomegranates stored at 0 °C increased significantly, peaking at 6.44 U·g−1·FW on day 40, and then decreased steadily until day 130. The POD activity of pomegranates stored at 2 °C, by contrast, increased steadily and changed minimally during the storage of 130 d. The changes might be attributed to the accumulated toxic substances (free radicals and MDA) binding to proteins such as defense-related enzymes, destroying structure and function[51]. Consistently, the PPO activity of pomegranates in these groups showed a similar rising tendency with POD activity during storage of 130 d (Fig. 4d). The PPO activity of pomegranates at 0 °C increased to a maximum peak (24.45 U·g−1·FW) on day 70, while other groups peaked on day 100. Notably, the changes in PPO activity of samples stored at 2 °C was always the smallest compared to other groups. Our results corresponded to the former report on phenolase metabolism of Chinese olives under CI storage temperature[11].

      POD and PPO, two key defense-related enzymes, were highly associated with alleviating enzymatic browning. They were also considered as important factors causing browning in post-harvest agricultural products[52]. It was considered that PPO and POD exerted an antagonistic influence over phenolic anabolism in the case of CI. The phenols were oxidized to the quinones by the action of two enzymes (PPO and POD), resulting in typical CI in peaches[51].

    • The differences in TA, SSC, and SSC/TA of pomegranates during the storage of 130 d are shown in Table 1. Compared to the initial value, all values of pomegranates declined to varying degrees. Specifically, the SSC content of pomegranates stored at 0 °C was 12.50%, while those stored at 2 °C was 14.34% on day 130. A few soluble sugars could be adopted as signal molecules, adjusting progression and growth, which were strongly associated with maturity and senescence of horticultural products[53]. The possible reason is that the storage temperature of 2 °C could reduce the metabolic intensity and the consumption of the organic matrix, while the reduced SSC in CI pomegranates was possibly attributed to sugars as an energetic response to cold stress[50]. Pomegranates stored at 2 °C exhibited remarkably higher TA values than those at 0 °C. The higher TA loss in pomegranates could be related to increased respiration intensity, in which the organic acid was severely depleted during the high respiration[41]. The solid-acid ratio increased on day 130, however, there were no significant differences among treatments.

      Table 1.  Effects of storage temperatures on soluble solids content (SSC), titratable acids (TA), and SSC/TA of pomegranates.

      Parameter Time (d) Storage temperature
      0 °C 1 °C 2 °C 3 °C 4 °C
      SSC content (%) 0 16.80 ± 0.32
      20 17.31 ± 0.48a 17.43 ± 0.40a 17.97 ± 0.40a 17.65 ± 0.47a 17.01 ± 0.54a
      40 17.54 ± 0.70ab 17.74 ± 0.27ab 18.32 ± 0.31b 17.89 ± 0.44ab 17.34 ± 0.31a
      70 16.01 ± 0.30c 16.31 ± 0.50bc 17.21 ± 0.49a 17.11 ± 0.40ab 15.67 ± 0.56c
      100 14.89 ± 0.40c 15.26 ± 0.40bc 16.46 ± 0.50a 15.66 ± 0.30b 13.57 ± 0.40d
      130 12.50 ± 0.36c 12.75 ± 0.26c 14.34 ± 0.29a 13.40 ± 0.33b 11.83 ± 0.41d
      TA content (%) 0 2.01 ± 0.23
      20 1.66 ± 0.05b 1.75 ± 0.12ab 1.97 ± 0.28a 1.85 ± 0.04ab 1.83 ± 0.04ab
      40 1.54 ± 0.15b 1.64 ± 0.08b 1.93 ± 0.06a 1.71 ± 0.15b 1.67 ± 0.07b
      70 1.43 ± 0.07c 1.55 ± 0.06bc 1.87 ± 0.09a 1.62 ± 0.11b 1.57 ± 0.09b
      100 1.26 ± 0.07d 1.38 ± 0.06c 1.71 ± 0.04a 1.51 ± 0.08b 1.48 ± 0.05bc
      130 1.16 ± 0.04c 1.24 ± 0.05c 1.57 ± 0.07a 1.45 ± 0.05ab 1.39 ± 0.04b
      SSC/TA ratio (%) 0 8.46 ± 1.32
      20 10.44 ± 0.60a 10.02 ± 0.89a 9.28 ± 1.52a 9.55 ± 0.46a 9.30 ± 0.50a
      40 11.43 ± 0.66a 10.86 ± 0.67ab 9.49 ± 0.14ab 10.55 ± 1.16ab 10.39 ± 0.25b
      70 11.23 ± 0.30a 10.55 ± 0.16ab 9.19 ± 0.16c 10.60 ± 0.44ab 9.99 ± 0.90bc
      100 11.85 ± 0.98a 11.06 ± 0.19ab 9.61 ± 0.10cd 10.37 ± 0.71bc 9.17 ± 0.04d
      130 10.81 ± 0.05a 10.32 ± 0.53a 9.13 ± 0.54b 9.25 ± 0.11b 8.54 ± 0.51b
      The results denoted mean ± standard deviations, n = 3. Different letters denoted prominent differences in the treatments for each sampling period at p ≤ 0.05.

      The sensory evaluation is presented in Fig. 5. Results demonstrated that pomegranates stored at 2 °C exhibited the highest scores, which were higher than those stored at 0 °C during room temperature storage. Loss of flavor and softening were typically macroscopic CI symptoms, which were usually not easily detected until the fruits were subsequently transferred from cold storage to ambient temperatures[17]. A similar result was found in tomatoes, and the potential mechanisms leading to the specific CI phenomenon were also elucidated. To be specific, the water was absorbed by the middle lamellar when its movement from the symplast to the apoplast occurred, which subsequently reduced the cell turgor pressure and exacerbated the softening induced by CI[23].

      Figure 5. 

      Pomegranates were stored at low temperatures for 130 d and further stored at 25 °C for 10 d. The pomegranates sensory scores were performed on (a) day 1, (b) day 4, (c) day 7, and (d) day 10 when stored at 25 °C.

    • The associations among different quality attributes for pomegranates (Fig. 6) were evaluated using Pearson's correlation analysis. It was found that the rising CI index was positively correlated with BI, b* value, ∆E* value, decay incidence, cell membrane permeability, MDA, POD, PPO, and weight loss, while negatively related to L* value, a* value, SSC, and TA, respiratory rate, and total phenolic content.

      Figure 6. 

      Pearson's correlation coefficients on the quality attributes of pomegranates stored at 2 °C. The red dots stood for positive correlation; and the blue dots stood for negative correlation. The mark * on dots represented a significant correlation at the level of p ≤ 0.05.

      Consequently, our study corroborated that CI symptoms first appeared around the vascular bundle cells and epidermic cells. The cell membranes were degraded due to the cold stress, and then the total phenolics were oxidized to the quinones by the action of PPO and POD, leading to browning and aging[41]. For another, cell membranes and mitochondrial membranes were degraded, which resulted in the leakage of metabolites and salt ions. Thus, these metabolic disorder induced by the accumulation of toxic substances like ROS and MDA, which accelerated the senescence of fruit and the infection of pathogenic bacteria (Fig. 7)[23].

      Figure 7. 

      The possible mechanism of precise storage temperature (2 °C) alleviated CI occurrence in 'Mengzi' pomegranate fruit through moderating the antioxidant-oxidant balance and respiration metabolism, which contributed to maintaining membrane integrity and pomegranate quality.

    • In this study, the optimal temperature for the long-term cold storage of the 'Mengzi' pomegranates was determined to be 2 °C among the range of 0 to 4 °C. This temperature storage maintained the best appearance quality, with minimal color change and browning index. Not only was the CI index minimized, but the decay rate was also the lowest after 130 d of storage. Meanwhile, the respiratory rate of pomegranates was suppressed, along with a reduction in weight loss when stored at 2 °C. Additionally, the levels of oxidants, including PPO, POD, and MDA, were reduced, while the levels of antioxidants (the total phenolics) were increased. SEM observation and membrane permeability examination revealed the cell integrity of pomegranates stored at 2 °C were the highest. Furthermore, pomegranates stored at 2 °C produced a relatively higher content of soluble solids and titratable acid, as well as better sensory scores. Overall, the results indicated that storage at 2 °C had a significant effect on moderating the antioxidant-oxidant balance and respiration metabolism, contributing to the maintenance of cell integrity and thus alleviating chilling injury. This report may provide the guiding significance for long-term storage of 'Mengzi' pomegranates under precise temperature.

      • This research was funded by the National Natural Science Foundation of China (3200161737), Science and Technology Action Project of Rural Revitalization Industry Development of Xinjiang Uygur Autonomous Region (2022NC119), Postdoctoral Research Foundation of China (2022M712375), and Open Project Program of State Key Laboratory of Food Nutrition and Safety (SKLFNS-KF-202316).

      • The authors contributed the article as below: Li L: compiling the first draft & software. Luo J: validation. Li X: resources & methodology. Pang L: editing. Jia X: visualization. Liu L: software & editing. Kačániováe M: review. Song J: Supervision. Qiao L: editing & review. All authors checked the results and approved the final version.

      • All data generated or analyzed during this study are included in this published article.

      • The authors declare that they have no conflict of interest.

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press on behalf of China Agricultural University, Zhejiang University and Shenyang Agricultural University. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (7)  Table (1) References (53)
  • About this article
    Cite this article
    Li L, Luo J, Li X, Pang L, Jia X, et al. 2024. The optimal precise temperature alleviated chilling injury and maintained post-harvest quality for 'Mengzi' pomegranate fruit. Food Innovation and Advances 3(4): 385−395 doi: 10.48130/fia-0024-0036
    Li L, Luo J, Li X, Pang L, Jia X, et al. 2024. The optimal precise temperature alleviated chilling injury and maintained post-harvest quality for 'Mengzi' pomegranate fruit. Food Innovation and Advances 3(4): 385−395 doi: 10.48130/fia-0024-0036

Catalog

  • About this article

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return