Citric Acid Cross-Linked Gelatin/Pectin Coatings Increase Shelf Life of Ripe Grapes

Abstract

Grapes, one of the most widely consumed fruits, present commerce challenges due to their short shelf life. One promising solution is the chemical cross-linking of polymers such as gelatin and pectin, which can create stronger and more biodegradable networks. This study evaluated the cross-linking of gelatin/pectin to extend the shelf life of ripe grapes, using citric acid as a cross-linker. Three different ratios of gelatin and pectin (1:1, 2:1, and 1:2) were tested in coatings applied to grapes. The results showed that the 1:1 ratio (gelatin/pectin) was the most effective in delaying the ripening of grapes. An analysis of the characteristics of the cross-linked networks revealed the formation of covalent bonds between the polymers, confirmed by FTIR, X-ray diffraction, and electron microscopy. The 1:1 coverage was superior, maintaining the visual quality of the grapes and delaying the loss of mass and firmness during the 10-day storage period. Grapes covered with this mixture showed less reduction in firmness and a lower accumulation of sugars, demonstrating its effectiveness in maintaining the quality of the fruit.

1. Introduction

Grapes are one of the oldest and most appreciated fruits by humans, playing a fundamental role in the global agricultural economy. Their cultivation accounts for billions of dollars in exports and is an important source of income for many countries, especially those in temperate climates, such as Brazil, the United States, Italy, and Spain. In addition to being consumed fresh, grapes are also transformed into juices, raisins, and, of course, wines, which are products that are in great demand both in the domestic and foreign markets [1].

However, despite their economic and nutritional value, grapes face significant challenges in the distribution process, especially with regard to their durability on supermarket shelves [2]. As a highly perishable fruit, grapes have a short shelf life, which makes their marketing a complex task. The time spent on shelves, often accompanied by inadequate storage conditions, can result in loss of quality and waste, affecting both consumers and producers [3].

To address the challenges of the short shelf life of grapes on supermarket shelves, the use of edible films and coatings has proven to be a promising solution. These innovative technologies have the potential to significantly increase the shelf life of fruit, maintaining its sensory and nutritional properties for longer, which, in turn, reduces waste and improves the efficiency of the distribution chain [1,4].

Although edible coatings are an advantageous option, most of them are highly hydroscopic and permeable [5]. One solution to the coating problem is the synthesis of covalent structures, which are more resistant and function as biodegradable plastic. Chemical cross-linking is a process that generates stable chemical bonds, both intra and intermolecularly, connecting polymer chains and transforming them into three-dimensional polymers [6]. During this procedure, the chemical agents used must interact with the polymer through specific functional groups, creating cross-links between the molecules. To be used in the food industry, the cross-linking agent must be biocompatible, stable, and non-toxic [6,7].

The cross-linking of natural polymers is an advanced and frequently explored approach to improve their physicochemical and technological properties. Over the past ten years, there has been a significant increase in the number of academic publications and patents related to modified polymers, along with an advance in the understanding of the mechanisms and effects of this process. Among the most studied polymers are gelatin, collagen, and polysaccharides such as pectin, chitosan, and alginate [8].

Gelatin cross-linked with tannic acid improved the stability of silver nanoparticles (AgNPs) at a basic pH. It was confirmed that 5% tannic acid improved the molecular structure of gelatin through covalent bonds, which was observed through high mechanical strength and a low water barrier. The silver nanoparticles showed excellent antimicrobial activity [9].

The cross-linking of gelatin with high methyl pectin using tannic acid improved encapsulation capacity and thermal stability. This study demonstrated that intense cross-linking causes changes in the alpha helix and random coil and hardens the gelatin. In addition, cross-linking causes a considerable increase in the size of the hybrid particles [10].

The cross-linking of gelatin with polysaccharides can be further optimized through reactions using a Schiff base. However, the process requires the polysaccharide to undergo an oxidation process [11]. The process of creating Schiff bases is interesting to control the reaction mechanism and also to improve the interactions of the cross-linked complex with secondary molecules, such as alkaloids and flavonoids. For example, cross-linking between gelatin and oxidized alginate improves the stability of the controlled release of anthocyanin through Schiff bases, electrostatic interaction, and hydrogen bonds [12].

Cross-linking gelatin with citric acid develops antioxidant nanofibers with improved water resistance. While cross-linking, gelatin with fructose outperforms citric acid by improving nanofiber solubility. Cross-linking with citric acid is more efficient and hinders solubility and interactions with molecules and water. This effect is considered important for the development of biodegradable films and edible coatings [13].

Considering the economic importance of grapes and the negative effect of rapid ripening on shelf life, this study sought to implement an innovative strategy to extend the shelf life of highly ripe grapes that would otherwise be discarded from supermarkets. The study developed three mixed cross-linked networks of gelatin and pectin and studied their effect as an edible coating on ripe grapes.

2. Materials and Methods

2.1. Material

High-bloom gelatin was obtained from the skin of Cynoscion acoupa. High methoxyl pectin was obtained from the fruit of genipap (Genipa americana). Citric acid monohydrate was purchased from Neon (Brazil). All other reagents were of food grade.

2.2. Preparation of Gelatin/Pectin Cross-Linked with Citric Acid

A citric acid solution (0.2 mol/L⁻¹) was prepared, and the pH of the solution was adjusted to pH3. Gelatin and pectin were mixed in a beaker in the following proportions (G:P m/m) (1:1 (3 g/3 g); 2:1 (6 g/3 g); 1:2 (3 g/6 g). Then, the mixture was homogenized with 200 mL of the citric acid solution using a magnetic stirrer at 45 °C for 1 h. After complete solubilization, the solutions were filtered through PTFE membranes with a pore size of 0.22 μm to remove impurities [14].

The mixtures were then transferred to a 500 mL round-bottom flask and coupled to a continuous reflux condenser with a heating mantle and stirrer. After assembling the equipment, the gelatin/pectin mixture with citric acid was kept at 90 °C for 3 h under continuous stirring and reflux. After 3 h, the mixtures were removed and allowed to cool to room temperature until reaching 30 °C. The samples were placed in cylindrical molds, pre-frozen at −80 °C, and freeze-dried (<5 Pa, −58 °C, 24 h) in an FD-1C-50 freeze-dryer (BIOCOOL, China). The gelatin–pectin conjugates were designated as G-P1:1, G-P1:2, and G-P2:1 [14].

2.3. Characterization of Cross-Linked Gelatin/Pectin

2.3.1. Fourier Transform Infrared Spectroscopy

The samples were ground with KBr powder and then pressed into a 1 mm pellet for Fourier transform infrared (FT-IR) measurements using the transmittance mode. FTIR spectra were obtained using a spectrometer, Spectrum Two FT-IR (PerkinElmer, Waltham, MA, USA) in the range of 4000–400 cm⁻¹ with a resolution of 2 cm⁻¹ and 64 scans [9,14].

2.3.2. X-Ray Diffraction

An X-ray diffraction (XRD) analysis was performed to investigate the phase composition and crystalline behavior of the lattices. The analysis was performed on an X-ray diffractometer Panalytical model Empyrean Diffractometer (Malvern Panalytical, Almelo, NL) equipped with CuKα radiation (λ = 0.15406 nm), an accelerating voltage of 30 kV, and a current of 15 mA. The analysis was performed in the 2θ range of 10–80° in the step-scan mode with a step size of 0.02° and at a scan rate of 0.02°/min. The diffraction peak at 22.4° was chosen for the calculation of the crystallite size and crystallinity [14]. The interplanar spacing or d-spacing was calculated based on diffraction data from X-ray diffraction experiments. This calculation works based on Bragg’s Law as follows:

$$n\lambda =2dsin\theta$$

(1)

where n = the order of the diffraction (typically 1 for the first-order diffraction). λ = the wavelength of the X-ray used. d = the interplanar spacing (d-value), which is what is attempting to be calculated. θ = the angle at which the diffraction peak occurs (the Bragg angle).

2.3.3. Micromorphology

The microstructure was examined by a G300 scanning electron microscope (SEM, Zeiss, Jena, Germany). A gold–palladium mixture was sputtered onto the samples to reduce charging effects prior to imaging. The samples were analyzed at an accelerating voltage of 3 kV, under vacuum at a voltage of 500, and scanned from ×4000. Only the best images were used [15].

2.4. Preparation and Application of Cross-Linked Coatings on Ripe Grapes

Gelatin–pectin conjugates, designated as G-P1:1, G-P1:2, and G-P2:1, were dissolved in distilled water at 5% shrinkage. In each experiment, 3% sorbitol was added under continuous stirring at 50 °C for 1h. Sorbitol was used as a plasticizing agent, with the aim of inducing bonds between water molecules and the gel network of gelatin–pectin conjugates and increasing solubilization [16].

In this study, seedless green grapes (Thompson variety) purchased from a supermarket were used. The grapes were selected from batches with an expired shelf life. The grapes were purchased in a non-refrigerated environment, with the following characteristics: 25 °C and a relative unit of 80–90% kept in sealed plastic containers. The grapes with a high degree of ripeness were selected and separated from the box with the help of scissors, maintaining the grape stems. The grapes were then sanitized with a moist solution of sodium hypochlorite (2.5% active chlorine) for 20 min. The properly sanitized grapes were divided into groups for the application of the coating [14].

After separating the batches, the grapes were submerged for 1 minute in the film-forming solutions and then placed on a support to drain the excess solution. After drying at room temperature, the procedure was repeated twice to ensure efficient coverage. After drying (2 h/± 25 °C), the fruits were stored at 25 ± 2 °C and 85 ± 2% relative humidity in a BOD incubator (Quimis, Q315M, São Paulo, Brazil) with air circulation for 10 days. The experiments were performed in duplicate and analyzed every 2 days [16,17].

2.5. Shelf-Life Study of Ripe Grapes

2.5.1. Titratable Acidity and pH

To determine titratable acidity, 10 mL of the sample was titrated with 0.1 N sodium hydroxide until pH 8.1, and the result was expressed in citric acid (g/100 g). The pH of the fruits was determined using a bench pH meter (Pro Linelab model, São Paulo, Brazil). For each treatment, solutions were prepared (10 g of grapes and 100 mL of distilled water), and three readings were taken [17].

2.5.2. Soluble Solids and Maturation Index

Soluble solids were measured using a refractometer (Instrutemp, ITREF95, São Paulo, Brazil). The maturation index was determined by the relationship between the soluble solids content and the titratable acidity [17].

2.5.3. Loss of Mass and Firmness

The mass loss was calculated according to Equation 1. The firmness of the grapes (N) was checked using a digital penetrometer model FR-5120 (Lutron Electronic Enterprise Co., Ltd., Taipei, Taiwan) supplied with an 8 mm plunger [17]. Three readings were taken for each fruit.

$$Lossofmass\left(g\right)={\displaystyle \frac{Mi-Mf}{Mi}}$$

(2)

where Mi = initial mass. Mf = final mass.

2.5.4. Thickness

The thickness was measured with a digital micrometer with a resolution of 0.001 mm (Insi-ze, model IP 54) at 10 random points of the coating, 20 mm from the edge. The analysis was performed on grape samples separated in triplicate for the gelatinous coating and for the reticulated networks. The coating was applied to the grapes, and after drying, it was separated from the grapes, and the reading was taken [15].

2.6. Statistical Analysis

Analysis of variance and Tukey’s test were applied with a significance level of 5% (p < 0.05) using the Statistica version 7.0 program STATSOFT Inc., 2004 (STATSOFT Inc., Tusa, OK, USA).

3. Results and Discussion

3.1. Reticulation Analysis

The cross-linking process between gelatin and pectin was observed through spectroscopic techniques and complementary analyses to verify the diffraction patterns and micromorphology. The FTIR spectra for reference are those observed in (Figure 1A). For gelatin, we observed an increase in the spectroscopic peaks (3272 cm⁻¹) corresponding to the functional groups OH and NH (amide A) [10]. The spectroscopic peak corresponding to the vibrations of the =C-H groups and charged amino groups -NH₃⁺ (amide B) can be seen at 2363 cm⁻¹. The spectrometric peaks between 1645 cm⁻¹ and 1540 cm⁻¹ are characteristic of the double bond stretching vibrations of the C=O and C=N groups (amide I and amide II). Finally, the spectrometric peaks between 1462 cm⁻¹ and 970 cm⁻¹ correspond to the stretching vibrations of C=O and C=N (amide III) [12].

For pectin, FTIR spectra analysis shows the carbohydrate fingerprint in the region between 900 and 1200 cm⁻¹. The peaks associated with the degree of esterification of pectin correspond to the bands between 1610 and 1630 cm⁻¹ [11]. This region is characteristic of the symmetric stretching vibrations of the carboxylic group. The marked presence of the stretching absorption of the OH groups is located in the range of 3000 to 3500 cm⁻¹. This region is directly associated with the inter and intramolecular hydrogen bonds of the galacturonic acid (GalA) molecule of pectin [11].

Finally, the typical absorption bands of citric acid are located at 1690 cm⁻¹ and 1743 cm⁻¹ and correspond, respectively, to the free carboxylic acid group and the hydrogen-bonded carboxylic acid group [13].

The FTIR spectra of the cross-linked networks are shown in (Figure 1B). All spectra of the cross-linked networks are similar and indicate that cross-linking between gelatin and pectin has been achieved. We observed that the relative intensity of the spectra of the amide I and amide II bands changed. The most evident result was observed for the G-P 1:1 cross-linked network. Similar results were observed for gelatin/agar cross-linked films with citric acid [18].

The amide I and amide II bands came together to form a single absorption band at 1390 cm⁻¹. Conversely, the absorption bands above 3000 cm⁻¹ split into three. The amide A bands underwent a large reduction, and the amide B bands disappeared. Another relevant observation is that the absorption bands in the region of 1700 cm⁻¹ for both gelatin and pectin disappeared in all formulations [19].

The disappearance of the peak can be attributed to the formation of a new chemical bond between gelatin and pectin. Furthermore, the chemical shift in the low-intensity absorption bands in the region of 1800 cm⁻¹ can be attributed to the formation of diester bridges between the gelatin and pectin chains. Finally, we observed the disappearance of the 1743 cm⁻¹ peak, characteristic of the carboxylic group, indicating the reaction between the carboxylic group and the amino group of gelatins [20].

X-ray diffraction patterns were used to investigate the atomic arrangements in the crystal unit cells (Figure 1C). All lattices exhibited scattering at 2θ = 22°, indicating that crystallinity increased systematically in the composites. The XRD peak at 2θ = 22° is commonly associated with the hexagonal packing of alkyl groups in the side chain. The increase in intensity of these peaks at 2θ = 22° is consistent with chemical cross-linking responses [21].

The observation of inflections in the diffraction patterns suggests the formation of lamellar arrangements, or porous ribs, after the mixing and reaction between gelatin and pectin. According to previous studies, cross-linking reactions in semicrystalline polymer networks cause the packing and formation of lamellar domains of the most varied types [22].

The d-value of 0.40 nm suggests that the average distance between the molecular units or the cross-linked structures is organized with strong molecular interactions. This result suggests organization on a nanometric scale, with regular distances between the structural units and crystals. This is typically observed in materials with some structural order but not perfect crystallinity. In addition, the broad scattering of the peak shows that the structure is of the semicrystalline type. These results reinforce how the cross-linking was not complete and that there are many sites with amorphous proportions and weak bonds [21,22].

Cross-linking occurs between the two polymers by means of a gelatin chain cross-linked with partially oxidized pectin using free diester chains. The process requires that a portion of the gelatin and pectin molecules be cross-linked. During the process, the temperature favors the formation of covalent bonds randomly, with priority given to the most reactive groups in the following order: carboxylic acid>amide>diester. After cross-linking part of the gelatin and pectin, intramolecular cross-linking begins through the diester groups and the already cross-linked gelatin networks [11,20].

The gelatin molecule undergoes stepwise cross-linking with citric acid to form amide bonds (Figure 2). The amino group (-NH₂) of gelatin reacts with the carboxylic group (-COOH) of citric acid at an acidic pH. The mechanism involves a nucleophilic acyl substitution reaction, which results in an unstable tetrahedral intermediate. The intermediate can rearrange to form a variety of compounds. The main mechanism, favored by the reaction medium, releases water and leads to the formation of amide bonds and cross-linked gelatin.

Reaction mechanism 2 involves the formation of ester bonds between pectin and citric acid in an acidic medium (Figure 3).

The hydroxyl group (-OH) of citric acid reacts with the carboxylic group of pectin (-COOH), forming an ester bond (-COO-). The mechanism occurs as follows: the carboxylic group of pectin acts as an acid, donating a proton (H⁺). The hydroxyl group of citric acid acts as a nucleophile, attacking the carbon of the carboxylic group. In the end, the nucleophilic reaction process stabilizes by forming an ester bond and releasing water.

Finally, reaction mechanism 3 involves the formation of a multi-cross-linked complex of gelatin and pectin (Figure 3). This process involves multiple rearrangements and by-products, which are difficult to express in the reaction mechanism. Therefore, for simplicity, we show only the rearrangement and cross-linked formation.

The amide and ester bonds form a hybrid network between gelatin and pectin chains, with citric acid connecting both. The amide and ester bonds occur simultaneously at different points in the gelatin and pectin molecules. Citric acid acts as a cross-linking agent, forming a three-dimensional network with mixed properties. Depending on the extent of cross-linking, the three-dimensional cross-linked network has semicrystalline characteristics, with regions of high crystallinity and others with amorphous characteristics.

The cross-sectional images taken with the scanning electron microscope corroborate the presence of crystalline domains dispersed within the amorphous regions (Figure 4).

Notably, all the cross-linked networks present patterns very different from those observed for gelatin and pectin. The mapping images show extensive networks with crystalline domains, both duly and homogeneously distributed in the amorphous regions. In addition to the crystallinity patterns, we observed that the entire extension of the surface is porous. The extensive cross-linking of the polymers is noted by observing how much the polymer network has restructured itself, changing the basic characteristics from amorphous to crystalline [23].

3.2. Shelf Life Study Analysis

The shelf life of ripe grapes was studied for a period of 10 days under controlled conditions (Figure 5). We observed that the grapes in the control group showed deterioration from the second day onwards and drastic changes in visual quality from the fourth day onwards. The grapes in the control group (gelatin) showed visual changes from the sixth day onwards and drastic changes in visual quality from the eighth day onwards.

In the test group, we observed that the reticulated net (G-P1:1) presented the best results, managing to maintain the visual quality of the grapes throughout the study period. Similarly, the reticulated net (G-P1:2) presented satisfactory results until the sixth day. By contrast, the reticulated net (G-P2:1) presented satisfactory results until the eighth day.

Acidity is one of the most relevant parameters in the study of shelf life, and it naturally decreases as fruits ripen. Organic acids, especially citric acid, are consumed in the process of cellular respiration [24]. Although there were fluctuations in acid values, a constant reduction pattern could be observed for all treatments (Figure 6A). The control treatment and the gelatin coating were the ones that presented the greatest reduction in acidity as a function of time. The result shows that these isolated treatments (control and gelatin) are not sufficient to maintain the quality of ripe grapes for a long time.

Treatments using reticulated nets showed the best results, delaying the reduction in acidity and preventing the accelerated ripening of the grapes. Both treatments (G-P1:2 and G-P2:1) showed similar behavior, with small fluctuations in acidity results up to the eighth day. After this time, we observed a sharp reduction, decreasing as the metabolic responses intensified. The most efficient treatment was with (G-P1:1), resulting in a controlled acidity reduction with few fluctuations.

The pH parameters for all treatments followed the same pattern observed in acidity (Figure 6B). The control treatment showed that the grapes were very ripe, and the pH increased considerably faster compared to the other treatments. The oscillations in pH values were due to the metabolic reactions that occur during fruit ripening [25]. Although the treatments (gelatin, G-P1:2, and G-P2:1) slowed an increase in pH, this was not efficient enough to extend the shelf life for 10 days. The results obtained in this study for the pH prove that the coating (G-P1:1) acted in reducing fruit ripening due to its lower pH value.

The soluble solids content gradually increased in the grapes during storage time (Figure 6C). The treatments (control and gelatin) presented the highest soluble solids content each day during storage. At the end of 10 days, except for treatment (G-P1:1), all other treatments showed high soluble solids content. This result indicates that an increase in the concentration of free sugars and organic acids is responsible for grape ripening [24,26].

The soluble solids content was already considered high on day 0 since the grapes were already very ripe, as indicated by the ripeness index results (Figure 6D). For all treatments, we observed that the ripeness index increased progressively. In the end, the grapes from the treatment (G-P1:1) showed a lower ripening index with a difference of (p ≤ 0.05). This result proves that the use of (G-P1:1) prevents gas exchange, thus delaying the ripening process.

Degradation and mass exchange during grape ripening is a natural and inevitable process (Figure 6E). The loss of mass and reduction in firmness are a direct result of gas exchange and the loss of water and organic acids during biological processes such as cellular respiration and transpiration [27]. Mass loss was constant throughout the storage period and was greater in the control treatment. Although the other treatments showed mass loss, after 10 days, we did not observe significant differences, and all grapes were rotten. Only the treatment (G-P1:1) showed reduced and controlled mass loss. This result shows that the ideal cross-linking is in the gelatin–pectin ratio (1:1). Although the other samples were cross-linked, these results show that complete cross-linking was achieved only in the G-P1:1 treatment.

Finally, grape firmness decreased as mass loss and extracellular fluids increased (Figure 6F). As previously reported, we observed a considerable increase in soluble solids during storage days, which reduced the structural firmness of the cells. Although all treatments followed a similar pattern of firmness loss, the best treatment was still (G-P1:1).

The thickness of the coatings increased according to the intensity of the cross-linking reaction between gelatin and pectin (

Table 1. Thickness of gelatin coatings and cross-linked gelatin/pectin networks.

Sample	Thickness (mm)
Control	0.0
Gelatin	0.141 ± 0.002 a
G-P1:1	0.201 ± 0.003 b
G-P1:2	0.312 ± 0.004 c
G-P2:1	0.311 ± 0.012 c

Equal letters in the same column do not differ from each other according to Tukey’s test at 5% significance (p ≤ 0.05).

). The thickness of the gelatin coating is in agreement with previous studies [16,17]. We noticed that the coating (G-P1:1) presented a statistically significant difference in thickness between the cross-linked networks. The increase in the thickness of the coatings was already expected, considering the mass balance in the cross-linking reaction in comparison to the gelatin coating.

The cross-linked network (G-P1:1) presented the greatest cross-linking since it was the one with the smallest thickness in relation to the other experiments. The decrease in coating thickness occurred because the cross-linked network prevented the movement of the individual polymer chains of gelatin and pectin, making the coating more compact and less flexible. The cross-linked network tends to form a more rigid structure, which can reduce the volume of gel that forms during drying [24].

The increased thickness observed in the cross-linked networks (G-P1:2 and G-P2:1) indicates light-to-moderate cross-linking. Moderate cross-linking may result in an increase in film thickness due to the formation of a denser three-dimensional network between the gelatin and pectin molecules. This increase in density may cause the film layers to expand more when formed since the cross-linked network may attract more water or other solutes during preparation [24].

Although all tests were performed with cross-linked samples, it was evident that only the (G-P1:1) treatment was the most efficient. These results show that the extent of cross-linking between treatments is different [28]. The ideal ratio for cross-linking between high-bloom gelatin and high-methoxyl pectin should be the ratio (1:1). This ratio should be the one that has the most effective cross-linking reactions and, therefore, was the one that best protected the grapes during storage [29].

4. Conclusions

The results presented confirm that the cross-linking process between gelatin and pectin is an effective strategy for the preservation of ripe grapes, especially when used in the ideal proportion of 1:1 (G-P1:1). This treatment demonstrated superiority over the others in preserving visual quality, controlling acidity, maintaining pH, structural firmness, and reducing mass loss during the storage period. The formation of reticulated networks provided more efficient control of gas exchange and grape ripening, significantly delaying natural degradation and extending their shelf life. Detailed analysis of the diffraction patterns and microstructure reinforces the fact that the G-P1:1 treatment promoted more complete cross-linking, resulting in well-structured and homogeneous crystal networks. These findings highlight the importance of precise control of the proportions and conditions of the cross-linking process to optimize the results. The use of cross-linked polymeric networks represents a promising solution for the storage and preservation of fruits, offering a sustainable and innovative approach for the food industry.