Inhibitory Effects of CaCl₂ and Pectin Methylesterase on Fruit Softening of Raspberry during Cold Storage

Abstract

Quality of raspberry fruit experiences a rapid decline after harvest due to its vulnerable texture and high moisture content. Application of calcium chloride (CaCl₂) combined with pectin methylesterase (PME) is efficient in delaying fruit softening. In this study, the effects of exogenous CaCl₂ alone or in combination with PME on the structure of the cell wall, the molecular properties of pectin, and the amount of free water of raspberry during postharvest storage were investigated. The results showed that CaCl₂ combined with PME treatment could maintain fruit firmness and inhibit weight loss. The treatment of CaCl₂+PME maintained the cell wall structure via sustaining middle lamella integrity and reducing the activities of cell wall-degrading enzymes, such as polygalacturonase, pectin methylesterase, β-galactosidase, α-L-arabinofuranosidase, and β-xylosidase. In addition, CaCl₂+PME treatment could effectively increase the content of chelate-soluble pectin (CSP) and develop a cross-linked structure between Ca²⁺ and CSP. Moreover, CaCl₂+PME treatment was of benefit in maintaining free water content. CaCl₂ in combination with PME treatment could be a promising method for inhibiting softening and maintaining the quality of postharvest raspberry during cold storage.

1. Introduction

Red raspberry (Rubus idaeus L.), known as “golden fruit”, is an important class of soft and juicy berry fruit. Raspberry fruit is rich in quantities of bioactive compounds, including anthocyanins, ellagic acids, and flavonoids, with a high repute and nutritional and medicinal value [1,2,3]. However, raspberry is highly susceptible to pathogens during storage due to its high moisture content and vulnerable texture, which leads to considerable losses in fruit quality and commercial value [2,3]. Currently, in order to reduce quality deterioration in postharvest storage and extend the shelf life of raspberry, a great deal of preservation technologies have been developed and applied in various studies, such as modified atmosphere storage [4,5], ozone storage [6], and low-temperature storage [7]. Nevertheless, these solutions still have many disadvantages, such as high investment costs, nutrient losses, and undesirable changes in aroma substances [8]. Consequently, studies on low-cost, safe, and efficient strategies for the quality maintenance of postharvest raspberry fruit are required.

Calcium is considered as a necessary mineral element in plants, which has a great influence on the permeability and integrity of the cell membrane and the fruit quality maintenance [9]. Ca²⁺ acts as a second messenger, playing an extremely significant role in interacting with other hormones and thereby regulating the growth and development of plants and responding to various biotic and abiotic stresses [10]. In addition, calcium also contributes to maintaining the cell wall components, such as the pectin and cell wall structure via inactivation of polygalacturonase (PG) and through interactions with demethylesterified pectin to form a pectin-Ca²⁺ network [11]. At present, calcium treatment alone or combined with other reagents have been widely applied in improving texture and maintaining the quality of papaya [12], persimmon [13], cherry tomato [14], and sweet cherry [15] during storage. Recently, Lv et al. (2020) mentioned that CaCl₂ treatment delayed the fruit softening of raspberry as indicated by the reduction of activities of PG, pectin methylesterase (PME), and cellulase (Cx), as well as the maintenance of protopectin content [16].

Pectin is a major component of the cell wall of plants; distributed in the primary cell wall and middle lamella, is closely associated with fruit firmness and softening process [11,17]. Fruit softening is accompanied by the solubilization and depolymerization of pectin, due to the changes in activities of PG and PME [18]. Exogenous fungal PME, which is usually extracted from Aspergillus niger strains, can inhibit the activity of endogenous PME in plants [14]. Moreover, the exogenous application of PME could strengthen the interaction between carboxylic acid and calcium, due to PME catalyzed pectin homogalacturonan de-esterification, which could stabilize pectin [9]. Yang et al. (2017) mentioned that exogenous PME combined with calcium lactate treatment was successful in improving fresh-cut papayas’ firmness under vacuum conditions [19]. In addition, exogenous PME combined with calcium treatment on jujube fruit delayed the collapse of texture and maintained fruit quality during storage [9]. However, the synergistic effects of exogenous PME and CaCl₂ on postharvest raspberry fruit firmness and quality during cold storage need to be studied. Our study aimed to investigate the effect of the postharvest application of CaCl₂ alone or combined with PME on the quality of raspberry fruit in order to reveal the underlying mechanisms regarding fruit softening in terms of cell wall metabolism, pectin molecular properties, and free water content. These will provide insights into the development of alternative techniques for the preservation of raspberry fruit.

2. Materials and Methods

2.1. Sampling

Fruits of red raspberries (Rubus idaeus L. cv R20) were hand-picked at commercial maturity from an orchard located in Zhangqiu district, Shandong province, China. The fruits with a uniform color, size, and shape and without any visible wounds or rot were transported into the laboratory immediately. Around 1000 fruits were randomly divided into three groups. Fruits of the control group were immersed in distilled water for 1 min. Fruits of the CaCl₂ treated group were dipped in the 0.2% (w/v) CaCl₂ solution (2 g of CaCl₂ was dissolved in 1000 mL of distilled water) for 1 min. And fruits of the CaCl₂+PME treated group were dipped in the solution consisting of 0.2% CaCl₂ and 0.05% (v/v) PME (DSM, Heerlen, The Netherlands) for 1 min. After being air-dried at room temperature, the fruits were stored at 4 °C and RH 90% and sampled at 3 day intervals for a period of 9 days. At each sampling time, eight fruits per replicate of each treatment were selected to determine their firmness and weight loss; the remaining fruits were packaged in tinfoil and frozen in liquid nitrogen, and finally stored in a refrigerator at −80 °C for further experiments.

2.2. Determination of Firmness and Weight Loss

Fruit firmness was determined using TA.XT. Plus texture analyzer (Stable Micro Systems Ltd., Godalming, Surrey, UK) equipped with a 50 mm probe according to the method described previously [16]. Fruit samples were compressed to a degree of 30%. The trigger force was 10 g and the pre-test, test and post-test speeds were 5, 1, and 5 mm s⁻¹, respectively. Each treatment included three replicates with 4 fruits per replicate and the results were expressed as N. Raspberry fruits of each treatment were weighted every three days during storage time.

Weight loss was calculated using to the formula below:

$$\mathrm{weight} \mathrm{loss} (\%)=\frac{{\mathrm{m}}_0-\mathrm{m}}{{\mathrm{m}}_0}\times 100\%$$

The initial fruit weight is assigned by “m₀”, and the fruit weight after storage is denoted by “m”. The weight loss was expressed as percentage (%) of loss according to the initial weight.

2.3. Observation of Cell Wall Structure

The methodology was used in this study according to the description of Silva et al. (2012) with some modifications [20]. Briefly, fresh, small pieces of flesh (2 mm in length, 2 mm in width, and 0.5 mm in thickness) of raspberry fruit from each treatment group after 9 d storage were soaked in 2.5% (v/v) glutaraldehyde at 4 °C for 2 h for fixation. Then, tissues were washed 3 times with 0.1 mol L⁻¹ phosphate buffer (pH 7.0) and post-fixed with 1% osmium tetroxide for 2 h, followed by three additional washes with 0.1 mol L⁻¹ phosphate buffer (pH 7.0). Afterwards, the tissue was dehydrated in 30, 50, 70, 80, and 100% (v/v) acetone and then was mixed with resin+acetone at 1:1 for 4 h and 1:3 for 12 h and with 100% resin at 60 °C for 48 h. Finally, it was cut into many ultrathin sections (50 nm) using an ultramicrotome (Leica EM UC6, Wetzlar, Germany), stained with uranyl acetate for 10 min and then observed via TEM equipment (JEM-1230, JEOL, Tokyo, Japan).

2.4. Measurement of Cell Wall Enzymes

The crude enzyme extractions were performed following the method of Ge et al. (2019) with minor modifications [21]. Briefly, a frozen raspberry sample was ground into fine powder in liquid N₂. One gram of the powder was homogenized with 3 mL of 95% ethanol and centrifuged at 12,000× g for 20 min at 4 °C to remove the supernatant, and the precipitate was washed three times by 3 mL pre-cooled 80% ethanol and centrifuged again. The sediment was extracted in 5 mL of pre-cooled 1.8 mol L⁻¹ NaCl (pH 5.5) for 20 min at 4 °C. After centrifugation, the supernatant was collected and stored at 4 °C for analysis of the following enzymes’ activities.

The methods of Tang et al. (2020) were employed for the determination of the activities of PG (EC 3.2.1.15) and PME (EC 3.1.1.11) [22]. The reactive substrate for PG activity determination was 10 g L⁻¹ polygalacturonic acid, and for PME activity determination it was 10 g L⁻¹ pectin. Activities of PG and PME were both measured at the wavelength of 540 nm and the results were expressed as U g⁻¹ fresh weight (FW), where one U was defined as the amount of enzymes that produced 1 μg of galacturonic acid per hour at 37 °C.

The activities of β-Gal (EC 3.2.1.23), α-L-Af (EC 3.2.1.55), and β-Xyl (EC 3.2.1.37) were determined according to the method of Villarreal et al. (2010) [23]. β-Gal, α-L-Af, and β-Xyl used ρ-nitrophenyl-β-D-galactopyranoside, ρ-nitrophenyl-α-D-arabinofuranoside, and 4-nitrophenyl-β-D-xylopyranoside as catalytic substrates, respectively. They were all assayed at the wavelength of 400 nm and the result was expressed as U g⁻¹ FW, where one unit was defined as the amount of enzymes that released 1 μmol ρ-nitrophenol per min.

2.5. Pectin Determination

The cell wall material of raspberries was extracted according to the method described by Manganaris et al. (2008) with some modifications [24]. After 9 d storage, 8 g of fruit tissue was homogenized with 40 mL of 80% (v/v) ethanol and boiled for 30 min. After cooling at 25 °C, it was filtered and the filtrate was discarded. These processes were repeated three times. The residue was then washed by ethanol, chloroform-ethanol, and acetone, successively, and denoted as alcohol insoluble residue (AIR).

The method described by Manganaris et al. (2008) with minor modifications was utilized to determine chelate-soluble pectin (CSP) [24]. An amount of 0.25 g of AIR was dissolved in 30 mL of distilled water and shaken for 4 h at room temperature, and then centrifuged at 12,000× g for 30 min at 4 °C. The residue was dissolved with 30 mL 0.05 mol L⁻¹ EDTA (containing 0.1 mol L⁻¹ NaAc) to obtain CSP. The content of CSP was assayed by the carbazole-sulfuric acid method [17]. The results were expressed as g kg⁻¹ FW.

2.6. Observation of CSP by Atomic Force Microscopy (AFM)

Observation of CSP was performed following the method described by Yang et al. (2017) with minor modifications [19]. CSP solution of each treatment after 9 d storage was extracted according to the above method and diluted to a suitable concentration (about 10 μg mL⁻¹). Then, 10 µL CSP solution was dipped on a freshly peeled mica sheet. After air-drying, the mica sheet was fixed on AFM (Bruker Mutimode 8, Karlsruhe, Germany) using double-sided tape. The AFM equipment was operated at 22–24 °C and 17–20% RH. Representative areas were scanned in the range of 10 × 10 μm and the experiment was repeated three times.

2.7. Determination of Free Water

Free water was detected according to the method previously reported by Zhang et al. (2018) using a low-field NMR instrument (NiuMag Co., Ltd., Shanghai, China) [11]. Samples stored at 9 d of each treatment were placed into the center of the radiofrequency (RF) coil at 32 °C and scanned with the multi-pulse echo sequence (Carr-Purcell-Meiboom-Gill, CPMG). The main CPMG parameters were as follows: NS = 8, NECH = 18,000, SW = 200 KHz, TD = 1,440,330, and TE = 0.400 ms.

2.8. Data Analysis

All experiments were repeated three time and data were analyzed using the SPSS 20.0 software (IBM, Chicago, IL, USA) and Microsoft Office Excel 2016. The significance differences (p < 0.05) between the control and treatment groups were compared by a one-way analysis of variance (ANOVA) and the Duncan’s test.

3. Results

3.1. Effects of Different Treatments on Firmness and Weight Loss of Raspberry Fruit

Fruit firmness of each treatment showed a decreasing trend during storage. There was no significant difference between the control and treated groups in the early stage of storage (3 d). However, on 9 d, the firmness of the PME+CaCl₂ treated group was 3.48 N, which was 6.40 and 25.18% higher when compared with the CaCl₂ treatment and control groups, respectively (Figure 1A).

Generally, fruits lose their weight during postharvest storage. In this study, the weight loss of each treatment increased gradually during storage (Figure 1B). There was no significant difference among the three groups after 3 d storage. However, during the middle and late stages of storage (6 and 9 d), the weight loss of the CaCl₂ treated group and the PME+CaCl₂ treated group was obviously lower than that of the control group. Additionally, the PME+CaCl₂ treated group showed the least weight loss, which represented an average of 13.49 and 26.21% lower than those of CaCl₂ treatment and control, respectively.

3.2. Effects of Different Treatments on the Cell Wall Structure of Raspberry Fruit

Adjacent cells adhere through the middle lamella. Loosening of the middle lamella results in fruit softening which is aroused by normal ripening. However, excessive degradation of the middle lamella causes deterioration of fruit texture [25]. Figure 2 shows the TEM images of the cell wall structure of raspberry fruit. The middle lamella of the CaCl₂+PME treated raspberry displayed as a relatively high electron-dense region, and this could be clearly observed after 9 d storage (Figure 2C). This structure was still preserved in a dense formation in the CaCl₂ treated fruit (Figure 2B). However, the cell wall structure became more swollen and the middle lamella was almost disappeared in the control group (Figure 2A). These changes indicated that cell wall structure degraded during storage, and that CaCl₂+PME treatment could remarkably retain the integrity of the cell wall structure.

3.3. Effects of Different Treatments on Cell Wall-Degrading Enzyme Activity of Raspberry Fruit

As shown in Figure 3, PG, PME, β-Gal, α-L-Af, and β-Xyl activities were detected in this study. PG, which catalyzes the hydrolytic cleavage of the α-1,4 glycosidic bonds of pectic acid, has a key role in softening during fruit ripening. Since pectin is the main component in the middle lamella of the plant cell wall, PG is considered to be responsible for the degradation of the middle lamella during fruit ripening [26]. The PG activity of the CaCl₂+PME treatment was much lower than those in the CaCl₂ alone treatment and control groups throughout the entire storage. On 9 d, the activity of PG in the combined treatment was decreased by 28.89 and 48.43% when compared with CaCl₂ alone and control treatment, respectively (Figure 3A).

PME hydrolyzes the ester linkage between methanol and galacturonic acid in esterified pectin to yield low-methoxyl pectin and polygalacturonic acid. PME functions upstream of PG, and acts as a key component in cell wall degradation during fruit ripening as well [26]. CaCl₂+PME treatment could significantly reduce endogenous PME activity during storage. The PME activity of the CaCl₂+PME treatment had been consistently lower than the control and the CaCl₂ treatment. In the CaCl₂ treated and the control fruit, the activity of PME increased sharply throughout the whole storage, while it rose relatively slowly in the fruit of the CaCl₂+PME treatment. On 9 d, the PME activity of the CaCl₂+PME treatment was 32.37 and 30.36% lower than those of the control and the CaCl₂ treatment, respectively (Figure 3B).

β-Gal can hydrolyze the cell wall polysaccharides containing galactoside and release the free galactose. β-Gal influences ripening and softening in various fruits, such as tomato, strawberry, papaya, etc. [27]. β-Gal activity showed a similar trend in all groups during storage. As expected, the CaCl₂+PME treated fruit showed the lowest activity of β-Gal when compared with the control and the CaCl₂ treatment (Figure 3C).

The cell wall component arabinoxylan is one the most abundant hemicelluloses on earth, which consists of a linear backbone of β-1,4 xylose residues with an arabinose substitution. α-L-Af and β-Xyl are involved in the degradation of arabinoxylan [28]. The activity of α-L-Af and β-Xyl were an overall upward trend in all treatments during storage. As indicated in Figure 3, the activities of the two enzymes of the CaCl₂+PME treatment group were consistently lower than the control and the CaCl₂ treatment during storage (Figure 3D,E).

3.4. Effects of Different Treatments on the CSP Content of Raspberry Fruit

The CSP content of raspberry fruit exhibited an increasing trend during storage regardless of treatments. However, different treatments had distinct effects on the CSP content. The CSP content increased from 1.02 to 1.76 g kg⁻¹ FW for the control fruit, to 1.89 g kg⁻¹ FW for the CaCl₂ treated fruit, and to 2.01 g kg⁻¹ FW for the CaCl₂+PME treated fruit at the end of the storage. When compared with the control group, either CaCl₂ or CaCl₂+PME treatment could significantly enhance the content of CSP during storage. The CaCl₂+PME treatment performed better than CaCl₂ alone. On 6 d of storage, the CSP content of CaCl₂+PME treatment was 1.2 and 1.5 times higher than those of the CaCl₂ treatment and the control, respectively (Figure 4).

3.5. Effects of Different Treatments on the CSP Nanostructure of Raspberry Fruit

Despite the variation of CSP contents being crucial for indicating fruit softening and textural change, the depolymerization of cell wall polysaccharide is also considered to be responsible for the fruit softening [29]. Thus, to better explain the mechanism of CaCl₂+PME treatment in delaying fruit softening, it was necessary to compare the nanostructures of the CSP from different treatments. The changes of the CSP nanostructure of raspberry fruit after 9 d storage were observed using AFM equipment. As shown in Figure 5C, CSP molecules in the fruit treated by CaCl₂+PME were aggregated and most of them formed large polymers. Furthermore, a cross-linking structure was also obtained in the CaCl₂+PME treatment (Figure 5D). In contrast, samples of the control or CaCl₂ alone treatment developed less polymers and no obvious structure was observed from AFM images, which indicated that pectin depolymerized in these two groups during storage (Figure 5A,B).

3.6. Effects of Different Treatments on the Free Water of Raspberry Fruit

As shown in Figure 6, T₂₁, T₂₂, and T₂₃ represent bound water, semi-bound water, and free water, respectively. T₂₁ and T₂₂ in all samples showed little variation. The highest peak of T₂₃ appeared in the CaCl₂+PME treated group when compared with the CaCl₂ treatment and the control, which demonstrated that the fruit treated with CaCl₂+PME had the highest free water during storage. A₂₁, A₂₂, and A₂₃ represent the peak areas of T₂₁, T₂₂, and T₂₃, respectively, and A_total represents the sum of the above three peaks areas. In total, the CaCl₂+PME treated fruit possessed the maximum value of A₂₃ and A_total, which indicated that the CaCl₂+PME treatment played an important role in maintaining the free water.

4. Discussion

Raspberry has high water content and vulnerable texture, which is easily infected by pathogenic fungi after harvest and further influences its market value and consumer acceptability [2]. In our study, exogenous PME combined with CaCl₂ treatment not only significantly slowed down the reduction of firmness, but also retarded weight loss when compared with the CaCl₂ treatment alone and the control fruit during storage. Moreover, CaCl₂+PME treatment could also maintain the water content of fruit, which may contribute to the retention of fruit firmness. Our results were similar to the previous studies on fresh-cut papaya [19] and jujube [9] fruit treated by exogenous calcium salts and PME.

Firmness reduction of fruit during ripening is attributed to cell wall degradation and the loss of cell structure [16]. Application of calcium could effectively maintain the structural integrity of the cell wall and thereby maintain or even improve the texture and quality of fruit during storage [10]. A previous study has mentioned that CaCl₂ treatment helps slow down the dissolution of the middle lamella and contributes to the preservation of cell wall structure during the postharvest storage of strawberry [30]. Our results showed that the middle lamella of raspberry fruit after 9 days of storage was more obvious in the CaCl₂+PME treated fruit than the CaCl₂ treatment and the control fruit, according to TEM images. It was suggested that CaCl₂+PME treatment was able to inhibit cell wall degradation and delay fruit softening. Generally, cell wall degradation is related to the increased activities of hydrolytic enzymes including PG, PME, β-Gal, α-L-Af, and β-Xyl [31]. In our study, we observed a remarkable reduction in PG, PME, β-Gal, α-L-Af, and β-Xyl activities in raspberry fruit treated by CaCl₂+PME. Based on these results, the structural integrity of the cell wall in the CaCl₂+PME treated fruit might be related to the stabilization of the middle lamella and the lower hydrolytic enzyme activities.

Fruit softening is closely related to the changes of cell wall components, including pectin, cellulose, and hemicellulose [32]. Pectin may undergo structure modifications, such as solubilization, depolymerization, and deesterification, which causes the cells to undergo cohesion reduction during fruit ripening [33]. In our experiment, CaCl₂ applied alone or in combination with PME significantly retained the CSP content during storage, indicating that both of the two treatments played key roles in retarding the solubilization of pectin in raspberry fruit. Similar to the result of a previous study [11], Ca²⁺ treatment could significantly elevate the CSP content of strawberry with prolonged storage time under vacuum conditions. It is associated with uptake of exogenous calcium ions, which promotes cellular adhesion and enhances the cell wall stability of fruit [11]. Our AFM images also supported this finding; the CSP molecules were aggregated and formed larger polymers in the fruit of CaCl₂+PME treatment when compared with the CaCl₂ treatment alone and the control fruit. In addition, the ‘eggshell’ network formed by calcium and pectin was only observed in the treatment of CaCl₂ combined with PME, which could stabilize pectin and the cell wall and contribute to fruit firmness and quality maintenance. Similar CSP pectin nanostructures were also observed in Kyoho grapes [34], apricot [35], strawberries [11], and fresh-cut papaya [19] treated by exogenous calcium alone or combined with PME. Our results suggested that application of CaCl₂ in combination with PME could retard firmness reduction and maintain raspberry fruit quality via inhibiting CSP degradation and by developing a cross-linked structure between CSP and calcium.

Water loss is another major factor influencing fruit texture changes during storage [36]. Low-field ¹H nuclear magnetic resonance (LF-NMR) was used to analyze the procedure of fruit moisture variation during storage. Zhu et al. (2017) [37] reported that the loss of total water in sweet cherry fruit was mainly due to the reduction of free water. Moreover, Zhang et al. (2018) also found that CaCl₂+PME treatment contributed to the conservation of the free water content in jujube fruit [11]. Similarly, we also found that CaCl₂+PME treatment had the highest T₂₃ peak height and largest A₂₃ relative peak areas, which indicated that the raspberry fruit of this treatment had more free water. It is indicated that the high free water content is related to the fruit firmness.

In summary, the fruit firmness of raspberry was better maintained by CaCl₂+PME application. The cell wall degradation was suppressed through sustaining middle lamella integrity, owing to a cross-linked network forming between Ca²⁺ and pectin. In addition, the pectin chain was deesterified by exogenous PME catalysis to develop low-methoxy pectin, which could react more easily with Ca²⁺ and thereby contributing to the stability of pectin and the cell wall [11,19]. Under this mechanism, the application of CaCl₂ combined with PME improved firmness and delayed softening of raspberry fruit during cold storage.

5. Conclusions

CaCl₂+PME treatment had a positive effect on inhibiting fruit softening and maintaining raspberry fruit quality, which was attributed to sustaining the structural integrity of the cell wall, inhibiting pectin degradation, as well as maintaining free water content. Therefore, CaCl₂+PME treatment is a promising method to improve the texture and quality of raspberry fruit.