The Biophysical Properties of the Fruit Cuticles of Six Pear Cultivars during Postharvest Ripening

Abstract

Pears are abundant in bioactive compounds, which exert favorable effects on human health. The biophysical attributes of fruit cuticles are pivotal in determining fruit quality, storability, and susceptibility to mold growth. This study aimed to elucidate the biophysical properties of six pear cultivars, ‘Conference’, ‘Celina’, ‘Abate Fetel’, ‘Packham’s Triumph’, ‘Sweet Sensation’, and ‘Williams’. Two maturity stages, unripe and fully ripened, were investigated. Furthermore, the efficacy of trimethyl-chitosan-coated pear surfaces in preventing Penicillium expansum (P. expansum) growth was assessed. Basic maturity indices (CIE color, ethylene evolution, firmness, soluble solids), cuticle contact angle, roughness, and zeta potential were analyzed. Surface roughness was measured using an optical profilometer, hydrophobicity was measured via profilometry, and zeta potential was quantified using an electrokinetic analyzer. The ‘Celina’ cultivar exhibited the highest roughness, whereas ‘Williams’ had the lowest roughness. All the cultivars’ cuticles demonstrated hydrophilic characteristics, with contact angles ranging between 65° and 90°. For pH values exceeding 3.5, all pear surfaces exhibited a negative zeta potential. P. expansum growth was the slowest on the ‘Packham’s Triumph’ and ‘Conference’ cultivars. Treatment with trimethyl chitosan effectively inhibited P. expansum growth in the initial hours of incubation. In conclusion, diverse pear cultivars manifest distinct biophysical surface properties and varying susceptibility to P. expansum growth. The growth of P. expansum correlates positively with roughness, contact angle, and zeta potential. These differences can significantly impact shelf life potential and the overall postharvest quality of pears.

1. Introduction

Pears belong to a group of ‘climacteric’ fruit that, at the onset of ripening, display a burst in respiration and biosynthesis of ethylene. Endogenous ethylene promotes climacteric fruit ripening at a steady rate once the fruit reaches maturity. After reaching a climacteric peak, ethylene production decreases, terminating in senescence, physiological breakdown, and/or microbial invasion of the fruit.

At the onset of ripening, other changes, e.g., the degradation of starch into soluble sugars and an increased activity of hydrolytic enzymes responsible for fruit softening, occur. All these changes lead to fruit softening, changes in peel color, and the synthesis of volatile aroma compounds [1,2]. With regard to peel color, fruit ground color changes from green to yellow due to the degradation of chlorophyll and de novo synthesis of yellow-colored carotenoids. All the above-mentioned parameters of the fruit maturity stage are frequently used to determine the harvest date. The most important and widely utilized pear maturity parameters are firmness, starch content, and soluble solids. In the optimal harvest date for storage, the recommended firmness of pears should be in the 5–7 kg/cm² range, the starch index should be in the 4–6 range, and soluble solid content should be in the 11–13% range.

In dietary recommendations, the consumption of fruits, including pears, is universally advocated. Pears boast a rich content of dietary fiber, vitamin C, potassium, fructose, sorbitol, and antioxidants, notably anthocyanins [3]. The consumption of pears is associated with enhancing gut health, preventing constipation, and reducing the risk of type 2 diabetes and stroke [4,5]. The biophysical and chemical traits of fruits after storage and ripening significantly influence their appearance and quality for consumption.

Ethylene, a plant hormone, is a key regulator of the ripening process in pears [6]. As shown in tomatoes, ethylene acts through ethylene biosynthesis genes, namely ACC oxidase and ACC synthase, as well as ethylene-inducible genes [7]. Enzymatic actions during ripening induce cell wall composition and structure changes, influencing fruit softening [8]. Unripe fruits exhibit greater firmness, which decreases during ripening. Harvesting at the optimal maturity stage minimizes weight loss and softening [9], coinciding with color changes in the fruit. From consumers’ point of view, edible pears must have the acceptable flesh firmness set between 7.4 and 24.5 N [10].

The amounts and types of pigments in plant tissues determine the colors observed in plants. The transition from green to yellow hues in pears results from the initial masking of carotenoids by chlorophyll at harvest. Zhao et al. [11] and Naeem et al. (2019) [7] demonstrated that the shift from green to red colors in fruits is associated with chlorophyll reduction. Besides chlorophyll, carotenoids are important pigments; their synthesis correlates positively with ethylene synthesis [12]. Additionally, authors Charoenchongsuk et al. [13] observed decreasing chlorophyll levels in pear cultivars during ripening, indicating the regulation of chlorophyll degradation through ethylene-dependent and -independent pathways.

Beyond these well-studied changes, less explored variations include biophysical alterations in fruit peels [14]. The plant cuticle is an extracellular layer and acts as a protective barrier for non-lignified plant parts, significantly influencing fruit quality and post-harvest shelf life [15]. The cuticle is involved in water transpiration and fruit dehydration, as well as susceptibility to microorganisms, insects, and various pre- and post-harvest disorders [15]. The biophysical properties of fruit cuticles change during fruit ripening pre- and post-harvest and are responsive to the conditions surrounding the fruit.

Research highlights the cuticle’s role in traits like water retention, disease susceptibility, and fruit firmness, which evolves after harvest under external conditions. Fleshy fruits rely on these hydrophobic barriers to prevent water loss and microbial infection while preserving taste and aiding seed dispersal [16]. Fruit cuticles, primarily composed of the lipid polymer cutin, significantly influence biophysical properties [17] and are modulated by components like waxes and flavonoids [18]. Understanding natural cuticle changes post-harvest is vital for evaluating storage conditions [19]. Cuticles’ mechanical properties impact the appearance of agricultural produce, potentially influencing fruit disorders, fungal infections, and pest infestation [20].

The biophysical attributes of fruit cuticles are crucial for determining fruit quality, storability, and susceptibility to mold growth. A higher contact angle indicates greater hydrophobicity, meaning the surface repels water more effectively, preventing microbial proliferation and contributing to enhanced storability and reduced susceptibility to mold growth. Surface roughness and zeta potential influence microbial adhesion, with smoother cuticles and higher negative zeta potentials deterring microbial attachment, thus reducing contamination and decay risks and improving storability. Cuticle color affects the consumer perception of fruit quality and ripeness, while firmness provides structural support to the fruit, preventing physical damage during handling and storage and thus enhancing quality and prolonging shelf life. The proper management of ethylene production is essential for controlling fruit ripening and maintaining optimal quality and storability. Soluble solids, such as sugars and organic acids, contribute to fruit flavor and sweetness, impacting fruit quality and susceptibility to microbial decay, as high sugar concentrations can act as preservatives, reducing water availability and inhibiting microbial growth.

Several diseases, such as grey, blue, and pink mold, as well as brown, bull’s eye, bitter, Phytophthora, Rhizopus, and side rot, alongside conditions like black spot, flyspeck, powdery mildew, and sooty blotch, can affect pear fruit [21]. Controlling post-harvest diseases typically involves fungicide application directly onto consumable products, yet this practice raises environmental concerns and poses serious human health risks [22]. Alternative approaches, such as applying a thin edible chitosan coating on fruit surfaces, have shown promise in inhibiting fungal diseases. Research indicates that this treatment inhibits spore germination and mycelial growth of fungal species such as Penicillium expansum, Alternaria kikuchiana Tanaka, Physalospora piricola Nose, and Botryosphaeria sp. [23,24,25,26]. Moreover, the application of a chitosan layer reduces water loss and surface browning in cut fruits during storage, preserving fruit firmness without compromising nutritional quality [27]. In the case of bacterial adhesion, surface characteristics like hydrophobicity and roughness substantially contribute to their adhesion rate [28,29]. Fruit cuticle characteristics depend on apple cultivars, as shown by Bohinc et al. [14], but also, in the case of pears, on climatic conditions [30].

The primary objective of this investigation was to examine the biophysical attributes of unripe and fully ripe pear cultivars, specifically focusing on parameters such as peel contact angle, roughness, and zeta potential. These parameters provide valuable insights into the physical state of the fruit surfaces. In order to better comprehend the progression of pear maturity during storage and the subsequent attainment of optimal maturity, alterations in ground color, firmness, ethylene production, and soluble solids content were continuously monitored. Furthermore, this study sought to evaluate the influence of chitosan treatment on the growth of P. expansum on pear cultivars. In summary, the overall aim of this research was to observe changes in the biophysical characteristics of pear peels following storage that ultimately led towards an edible maturity stage.

2. Results and Discussion

2.1. Basic Maturity Parameters of Pear Cultivars

Investigated maturity parameters of pear cultivars for the unripe and fully ripe stage are presented in

Table 1. Maturity parameters of pear cultivars for the unripe and fully ripe stage.

	L*	a*	b*	Firmness (kg/cm2)	Soluble Solids (Brix)	Ethylene (µL/kg hour)
‘Conference’
Unripe	56.26 ± 3.04 a	-4.66 ± 2.81 a	35.10 ± 4.10 a	5.85 ± 0.29 a	12.02 ± 0.14 a	12.76 ± 1.13 a
Fully ripe	64.94 ± 2.80 b	3.45 ± 1.02 b	41.42 ± 2.53 b	0.45 ± 0.06 b	13.70 ± 0.66 a	11.56 ± 0.40 a
‘Celina’
Unripe	50.23 ± 6.70 a	4.62 ± 9.64 a	29.23 ± 8.70 a	7.53 ± 0.63 a	16.07 ± 0.11 a	66.38 ± 5.52 a
Fully ripe	60.36 ± 4.46 b	7.45 ± 3.00 a	41.07 ± 4.15 b	2.85 ± 0.74 b	17.86 ± 0.43 a	6.46 ± 0.27 b
‘Abate Fetel’
Unripe	60.48 ± 6.64 a	-0.07 ± 3.37 a	35.56 ± 4.06 a	5.30 ± 0.50 a	13.12 ± 0.09 a	107.55 ± 6.48 a
Fully ripe	62.88 ± 5.30 a	6.40 ± 2.22 b	43.05 ± 4.75 b	2.26 ± 0.54 b	16.32 ± 1.65 b	30.07 ± 0.00 b
‘Packham’s Triumph’
Unripe	57.56 ± 4.72 a	-4.78 ± 3.28 a	33.75 ± 2.75 a	6.30 ± 0.32 a	11.72 ± 0.17 a	1.65 ± 0.00 a
Fully ripe	61.50 ± 3.62 a	0.13 ± 1.89 b	40.70 ± 2.51 b	0.60 ± 0.10 b	13.57 ± 0.16 b	85.81 ± 3.56 b
‘Sweet Sensation’
Unripe	50.64 ± 9.27 a	4.56 ± 9.71 a	27.64 ± 10.66 a	4.80 ± 0.26 a	14.06 ± 0.18 a	7.00 ± 0.26 a
Fully ripe	57.85 ± 9.98 a	16.74 ± 10.75 a	29.09 ± 3.74 a	0.48 ± 0.15 b	15.62 ± 0.04 b	17.11 ± 0.92 b
‘Williams’
Unripe	68.33 ± 2.70 a	-1.15 ± 1.57 a	38.81 ± 1.68 a	5.97 ± 0.16 a	11.87 ± 0.39 a	97.20 ± 1.79 a
Fully ripe	64.13 ± 6.10 a	7.10 ± 2.58 b	35.81 ± 5.34 a	0.59 ± 0.24 b	12.75 ± 0.40 b	11.49 ± 1.92 b

Different letters within the same pear cultivar and the same maturity parameters indicate significant differences (p < 0.05; Paired-Samples t Test).

. The color parameter L* amounts to around 55 for unripe fruits and slightly above 60 for fully ripe fruits. The fruits thus become lighter in color, which was clearly evidenced by the increased L* values of the pear cultivars, except for ‘Williams’. During ripening, pear peels also become less green and more yellow, leading to a lighter color, evidenced here as a higher L* value, although only ‘Conference’ and ‘Celina’ showed significantly higher L* values. Due to the degradation of chlorophyll and the synthesis of carotenoids, the color parameters a* and b* also increased. Parameter a* (ratio of green/red) for unripe fruits was around 0 or slightly below, while fully ripe fruits showed values from 0.13 to 7.45. ‘Conference’, ‘Abate Fetel’, ‘Packham’s Triumph’, and ‘Williams’ had significantly higher a* values. Unripe fruits showed b* values (ratio of blue/yellow) from 27 to 38, and fully ripe fruits had b* values in ranging from 29 to 41. While ripening, fruit color becomes less green and more yellow, confirmed here by increasing a* values and a more yellow color and evidenced by significantly increased b* values for all cultivars except for ‘Sweet Sensation’ and ‘Williams’. The exception was ‘Williams’, which turned yellow with brownish tints. The results of our color parameters are in accordance with those of Hendges et al. [1], who found a greenish color for less ripe ‘Conference’ and ‘Alexander Lucas’ pears. After storage, fruit firmness amounted from 7.53 kg/cm² for ‘Celina’ to 4.80 kg/cm² for ‘Sweet Sensation’. After 7 days of shelf life, firmness decreased and ranged from 0.45 kg/cm² to 2.85 kg/cm². Most of the firmness from the pre-shelf-life and shelf-life values are in accordance with the results of Hendges et al. [1] and Dave et al. [31], except for ‘Celina’ and ‘Abate Fetel’, both of which ripened at a slower pace. The lowest degree of firmness that still enables acceptable sensory quality is 8.0 N [10,32], which amounts to 0.8 kg/cm². Most of the pear cultivars had soluble solids content ranging from 12.0 Brix to 15 Brix, with the exception of ‘Celina’, which showed higher values. Soluble solids content increased during the shelf life for all cultivars. Ethylene evolution closely mirrored the physiological stage of ripeness. Here, the ethylene production rate amounted from 1.6 µL/kg hour to 107.5 µL/kg hour and was differentiated among cultivars as well as by maturity stage. A decrease in ethylene production during shelf life was observed for ‘Conference’, ‘Abate Fetel’, ‘Celina’, and ‘Williams’, while for other cultivars, ethylene production increased. Overall, ethylene production was in accordance with the results of Hendges et al., [1], who reported a range from 1 to 32 µL/kg hour for ‘Conference’ and ‘Alexander Lucas’.

2.2. Surface Topography and Roughness

Figure 1 shows photos of six pear cultivars (‘Conference’, ‘Celina’, ‘Abate Fetel’, ‘Packham’s Triumph, ‘Sweet Sensation’, and ‘Williams’), with corresponding optical micrographs showing the 2D topography of a representative area of the pear surface. Inside these areas, the root mean square (RMS) values for surface roughness were calculated on smaller 30 µm × 30 µm areas.

Statistical analysis of the average surface roughness demonstrated a significant difference among pear cultivars (p < 0.0001). The average surface roughness of pear cultivars (Figure 2) measured on the equatorial region of the unripe pears ranged from (0.94) µm for ‘Williams’ to (3.62) µm for ‘Celina’ pears. The surface roughness of the fully ripe pears ranged from (0.74) µm for ‘Sweet Sensation’ to (3.01) µm for ‘Celina’ pears. There was no statistically significant difference between the surface roughness of unripe and fully ripe pears (p = 0.8046). However, the results showed differences in average roughness among pear cultivars. The ‘Celina’ cultivar exhibited the highest average surface roughness. A two-way analysis of variance (ANOVA) demonstrated significantly lower average surface roughness for all pear cultivars before (p < 0.0001) and after (p < 0.0001) the ripening process in comparison to the ‘Celina’ cultivar. Among the cultivars exhibiting the lowest surface roughness (‘Conference’, ‘Abate Fetel’, ‘Sweet Sensation’, ‘Packham’s’, and ‘Williams’) additional differences were observed. Among both unripe and fully ripe cultivars ‘Conference’ and ‘Abate Fetel’, a statistical difference was not observed (p > 0.0771). However, compared to ‘Abate Fetel’, the ‘Packham’s’ (p < 0.0038), ‘Sweet Sensation’ (p < 0.0001), and ‘Williams’ (p < 0.0215) cultivars exhibited statistically lower surface roughness. Overall, the results demonstrate that the surface roughness decreases in the following order: ‘Celina’ >> ‘Conference’ ≈ ‘Abate Fetel’ > ‘Packham’s’ ≈ ‘Sweet Sensation’ ≈ ‘Williams’. According to Heredia-Guerrero et al. (2018) [33], roughness depends on wax crystals that change during maturation.

Measurements show very low standard deviation for ‘Williams’ both before and after ripening. No outliers in the roughness values were detected in our 2 × 54 datapoints. Besides having very low surface roughness, the ‘Williams’ cultivar also appeared to have a very homogeneous surface. The ‘Packham’s Triumph’ and ‘Sweet Sensation’ cultivars had a less homogeneous surface. The ‘Conference’, ‘Celina’, and ‘Abate Fetel’ cultivars had the most heterogeneous surface topography compared to ‘Williams’.

2.3. Static Contact Angle

The contact angles of unripe pear cultivars ranged from 64.6° to 77.4°, and no significant differences were found between cultivars (Figure 3). The lowest contact angle was recorded for ‘Sweet Sensation’ and ‘Williams’, whereas the highest contact angle was observed for ‘Celina’. A two-way analysis of variance (ANOVA) demonstrated that statistical significance in the contact angle was obtained for ‘Williams’ between the unripe cultivars (p < 0.0167). Meanwhile, the contact angle of fully ripe pear cultivars ranged from 71.2° to 84.2°, with the lowest contact angle being shown for ‘Sweet Sensation’ and the highest being shown for ‘Conference’ and ‘Packham’s Triumph’. The statistical analysis demonstrated a significant difference in the contact angle of the fully ripe cultivars, especially for the ‘Sweet Sensation’ cultivar (p < 0.0276). Considering the differences between unripe and fully ripe cultivars, it can be concluded that the overall contact angle is higher for the fully ripe samples. A statistically significant change was obtained in the case of the ‘Conference’ (p < 0.0129) and ‘Williams’ (p < 0.0004) cultivars. The results of contact angles show that the surface of pears becomes more hydrophobic during ripening. The contact angles obtained are very close to the contact angles of different apple cultivars, as shown by Bohinc et al. [14]. In blueberry (Vaccinium corymbosum) skin, the contact angle was found to slightly increase during storage [34].

2.4. Zeta Potential

The measured zeta potentials as a function of pH are shown in Figure 4. Before ripening, the isoelectric point was between pH = 2.7 and 3.3, whereas after ripening, the isoelectric point was below pH = 3 for most of the pears. At lower pH values, the surfaces are positively charged, whereas for higher pH values, the surfaces are negatively charged. At a neutral pH, the potential reaches strongly negative values. Before ripening, ‘Abate Fetel’ reached the most negative values of the potential. Generally, before ripening, the potential was more negative than after ripening. In this research, the pear zeta potentials were in accordance with the values obtained for apples [14].

2.5. P. expansum Growth

Figure 5 shows the lesion diameters of P. expansum inoculated on these different pear cultivars: ‘Abate Fetel’, ‘Williams’, ‘Conference’, ‘Packham’s Triumph’, and ‘Celina’, before and after chitosan treatment, observed during fourteen days of incubation. The influence of chitosan treatment on P. expansum growth on pear cultivars was evaluated. An examination of P. expansum lesions (Figure 5) indicated a statistically significant alteration in lesion size over an extended incubation period, irrespective of the application of the chitosan layer. An analysis of P. expansum growth (Figure 6) revealed statistically significant differences among the cultivars investigated. Between day 4 and 8, ‘Conference’ showed a significantly lower lesion diameter as compared to other cultivars, except for ‘Packham’s Triumph’. Conversely, P. expansum growth was slower on the ‘Packham’s Triumph’ and ‘Conference’ cultivars. During the initial eight days of incubation, greater lesion growth was observed on the ‘Packham’s Triumph’ cultivar compared to the ‘Conference’ cultivar. However, with prolonged incubation (ten days or more), lesion growth notably accelerated on the ‘Conference’ cultivar, which was particularly evident after fourteen days. Overall, these results suggest that P. expansum growth is slowest on the ‘Packham’s Triumph’ cultivar and indicate that it could be the most resistant to mold growth. In the first days of incubation, a statistically significant impact of chitosan on P. expansum growth was obtained. With regard to mold attachment on the surface, it is important to mention the biophysical characteristics of P. expansum spores. Fernandes et al. [35] report that P. expansum spores have a contact angle of 51.3° and a zeta potential of −34 mV. Biophysical characteristics of the cuticle, as well as biophysical characteristics of microorganisms, interplay in attachment process. To our knowledge, there is no evidence of such a study on different cultivars of pears.

Chitosan quaternary salt has demonstrated effective antifungal properties in on-site experiments, mostly involving apples. De Britto and Assis [36] assessed the application of chitosan and several of its quaternary derivatives on sliced apples as active coatings. The findings revealed that these coatings exhibited reduced browning on the cut apple surface compared to uncoated samples, and they exhibited notable antifungal activity against P. expansum and a moderate inhibitory effect on Botrytis cinerea.

2.6. Statistical Analysis

PCA (Figure 7) was used to explore differences between various pear varieties and their ripening stages, according to ten measured variables. A total of 58.1% of the data variation was explained by the relation between principal component 1 (PC1) and principal component 2 (PC2).

Plotting the data in a two-dimensional coordinate system (Figure 7) shows that PC1 provides a good distinction between the unripe and fully ripe pear varieties. The fully ripe pears were associated with higher values of contact angle, soluble solids, a*, b*, L*, mold diameter, and zeta potential, while the unripe pears were associated with higher values of firmness and ethylene production. Unripe ‘Abate Fetel’ and ‘Williams’ pears had significantly higher ethylene production. Interestingly, the ‘Celina’ pears stood out in both groups (ripe, unripe), as they had the highest values for roughness and soluble solids among the pears of different varieties. Among the fully ripe pears, the ‘Sweet Sensation’ pear differed the most; it had the lowest levels of L* and b* values, mold diameter, contact angle, roughness, and zeta potential, while its a* value level was the highest.

The PCA diagram (Figure 7) reveals that the samples were not grouped according to pear variety but according to the degree of ripeness; the degree of pear ripeness had a greater influence on the variables examined than the pear variety. In Figure 8a, a total of 72.1% of the data variation was explained by the relation between PC1 and PC2. Three varieties (‘Williams’, ‘Packham’s Triumph’, and ‘Conference’) with similar characteristics were gathered in one group. These pears had higher b* and L* values and lower levels of soluble solids and a* value. ‘Sweet Sensation’ pears had significantly lower levels of b* value and firmness than other varieties, while ‘Celina’ pears had the highest level of a* value, firmness, soluble solids, contact angle, roughness, and zeta potential. Among the pear varieties analyzed, ‘Abate Fetel’ pears distinguished themselves by exhibiting notably elevated levels of ethylene production, a higher L* value, and the lowest recorded level of zeta potential. A second PCA diagram (Figure 8b) was performed to evaluate the differences between various varieties of fully ripe pears. PC1 explained up to 43.8%, and PC2 explained 32.8%. The samples were separated along the first PC by differences observed in b* value, mold diameter, roughness, and zeta potential. The second PC separated the samples based on L* and a* values, as well as firmness, soluble solids, ethylene production, and contact angle. From Figure 8b, it is clear that there are two groups of samples, indicating some similarities among the samples within each group. In one group, there are ‘Celina’ and ‘Abate Fetel’ pears, while in the other group, there are ‘Williams’, ‘Packham’s Triumph’, and ‘Conference’ pears. It is evident that one sample (‘Sweet Sensation’), which cannot be assigned to any of the groups, is positioned in the upper left quadrant. This sample exhibits the highest a* value, while other parameters such as ethylene production, L* and b* values, contact angle, zeta potential, mold diameter, and roughness are at their lowest. The position of ‘Celina’ and ‘Abate Fetel’ pears indicate that they had the highest levels of firmness, soluble solids, roughness, and mold diameter. ‘Williams’, ‘Packham’s Triumph’, and ‘Conference’ pears had higher level of ethylene production, L* value, and contact angle, as well as a lower level of firmness, soluble solids, and mold diameter.

Table 2. Correlation matrix based on all measurements, i.e., unripe and fully ripe samples.

	L	a	b	Firmness	Soluble Solids	Ethylene	Mold Diameter	Contact Angle	Roughness	Zeta Potential
L		−0.057	0.78	−0.464	−0.238	0.235	0.329	0.226	−0.453	−0.05
a			−0.202	−0.575	0.694	−0.268	0.344	0.229	−0.08	−0.012
b				−0.398	0.072	0.14	0.548	0.506	−0.064	0.223
Firmness					−0.241	0.258	−0.41	−0.611	0.501	−0.017
Soluble solids						−0.173	0.524	0.393	0.475	0.228
Ethylene							−0.274	−0.235	0.032	−0.546
Mold diameter								0.461	0.172	0.396
Contact angle									0.216	0.449
Roughness										0.433

unfolds the correlations between the parameters studied, based on measurements for unripe and fully ripe samples. Firmness is negatively correlated with all color parameters; a decrease in firmness corresponds to an increase in lightness (L*) and yellowness (b*) and a decrease in green color (a*). Firmness is also negatively correlated with the mold diameter (−0.41) and contact angle (−0.61) and is positively correlated with roughness (0.50). Ethylene, as the most important maturity indicator, is negatively correlated with the contact angle (−0.23) and negatively correlated with zeta potential (−0.55). Correlations confirm that contact angle, roughness, and zeta potential increase with the progress of maturity.

We also conducted a correlation analysis between the mold diameter and biophysical attributes (roughness, contact angle, and zeta potential) for each individual pear cultivar. The strongest correlations were observed between mold diameter and roughness, with strong positive correlations in the ‘Abate Fetel’ cultivar (r = 0.77) and moderate positive correlations in the ‘Celina’ cultivar (r = 0.51). The correlation between mold diameter and contact angle is strongest in the ‘Conference’ cultivar (r = 0.77), followed by moderate positive correlations in the ‘Williams’ (r = 0.41) and ‘Packham’s Triumph’ (r = 0.23) cultivars. Zeta potential shows the strongest negative correlations with the mold diameter in the ‘Abate Fetel’ (r = −0.84) and ‘Williams’ (r = −0.79) cultivars, indicating a very strong association between these factors. Overall, these correlations suggest that roughness and zeta potential may have the most significant impact on mold growth in the pear cultivars studied, with varying strengths of association observed across different cultivars.

3. Materials and Methods

3.1. Plant Material

The pear cultivars (‘Conference’, ‘Celina’, ‘Abate Fetel’, ‘Packham’s Triumph’, ‘Sweet Sensation’, ‘Williams’) were provided by Evrosad d.o.o. Krško, Slovenia. These are common cultivars grown and consumed in Slovenia as fresh pears. All the pear cultivars were grafted on quince MA and trained as slender spindles; winter pruning was conducted to maintain tree canopies. The pear trees were 7 years old; the agricultural practice in the orchard included ‘integrated’ pest management, i.e., the application of environmentally friendly phytopharmaceuticals. Fertilization was carried out according to the soil analyzed, i.e., 90 kg of N/ha and 60 kg of K were administered. Administration of Ca was carried out 3 times with maxflow Ca, along with other treatments. Each cultivar was harvested at the optimal maturity stage, as defined by firmness value, starch index, and soluble solids content. Meteorological data from the nearest meteorological station are presented in

Table 3. Meteorological data from the nearest station, Novo Mesto.

Meteorological Station NOVO MESTO lon = 15.1773 la t = 45.8018 Altitude = 220 m	Average T (°C)	Precipitation (mm)	Sunshine Duration (h)
2023/04	9.8	92.4	151.6
2023/05	15.4	142	148.6
2023/06	20.6	61.4	259.6
2023/07	22.6	153.1	262.9
2023/08	21	178.2	260.6
2023/09	18.2	46.7	215.3

. The data of harvest dates and maturity parameters are presented in

Table 4. Harvest date and maturity parameters of the pear cultivars at harvest.

	Date of Harvest	Firmness (kg/cm2)	Starch Index (1–10)	Soluble Solids (Brix)
‘Conference’	31 August	6.80 ± 0.26	4.35 ± 0.40	11.92 ± 0.26
‘Celina’	8 August	6.72 ± 0.32	4.68 ± 0.52	15.40 ± 0.62
‘Abate Fetel’	31 August	5.82 ± 0.38	6.10 ± 0.39	13.20 ± 0.28
‘Packham’s Triumph’	25 August	7.50 ± 0.28	4.86 ± 0.42	11.60 ± 0.34
‘Sweet Sensation’	7 September	5.30 ± 0.35	6.10 ± 0.58	13.90 ± 0.32
‘Williams’	20 August	6.10 ± 0.41	5.86 ± 0.52	11.52 ± 0.42

.

After harvest, all the fruits were stored in cool storage conditions (+1 °C) until the beginning of the experiment. On the day of the experiment (10 October 2023), sound fruits of uniform size, with no visible damage, were transported to the laboratory at the Biotechnical faculty, Ljubljana, where the study was conducted. Analyses of these unripe fruits were carried out the next day, while analyses of fully ripe fruit (edible maturity) were carried out after 10 days of storage at room temperature (20 °C).

3.2. Roughness

The surface topography was imaged by a Zygo Zegage Pro HR non-contact optical profilometer (Zygo Corporation, Middlefield, CT, USA). The optical profilometer was based on the principle of coherence scanning interferometry. The light from an LED illuminator was split into a path directing the light onto the pear surface and a path directing the light to an internal reference. Reflections from both surfaces were recombined and projected onto an array detector, which produced interference fringes for every pixel. From these interference patterns, the topography of the surface could be modeled.

For every cultivar, 3 different representative areas—sized 1000 µm × 1000 µm—in the equatorial region of the pear surface were selected, using the 10× large field of view objective. In each of these zones, 3 profile images—sized 170 µm × 170 µm—were taken using the 50× objective with 0.52 µm optical lateral resolution. Inside these detailed 170 µm × 170 µm images, RMS values were calculated on 30 µm × 30 µm squares. The relative coordinates of the 30 µm × 30 µm squares were kept fixed for every measurement. This procedure resulted in a dataset of 54 RMS values for every pear cultivar.

3.3. Static Contact Angle

The static contact angle between the pear surface and a sterile water droplet was measured. A drop shape on the pear surface was analyzed with Theta Attension Optical Tensiometer (Biolin Scientific, Gothenburg, Sweden). The water droplet was placed on the pear surface with a steel syringe needle, and photos of droplets were taken with a charge-coupled device (CCD) camera of a goniometer. After that, the photos were saved to a personal computer. The droplet’s contour on the pear surface was mathematically evaluated by using the solution of the Young–Laplace equation Δp = −γ(1/R_1 + 1/R_2), where Δp is the Laplace pressure, and gamma is the surface tension. R1 and R2 are the principle radii of curvature. The contact angle between the droplet and the pear surface was as the slope of the contour line at the phase contact point between the pear surface, the water, and air.

3.4. Zeta Potential

The zeta potential of the pear surfaces was measured with an electrokinetic analyzer (SurPASS, Anton Paar GmbH, Graz, Austria). A 0.001 M KCl electrolyte solution in ultrapure water was used as a starting point, followed by titration with 0.05 M hydrochloric acid into an acidic range. The zeta potential measurements at each pH point value were repeated four times. From the measured streaming potential, the zeta potential was calculated from the Smoluchowski equation.

3.5. Color Measurements

Pear skin color was measured according to CIE. L*, a*, and b* color parameters were measured by using the Konica Minolta colorimeter CM-5 reflectance mode, with a measured area of 3 mm on 5 fruits at the point of the largest diameter. Each pear was measured 3 times.

3.6. Ethylene Evolution

The ethylene evolution rate was measured in 1 mL air samples withdrawn from a hermetic glass jar (2.8 L), extracted with a 1 mL syringe. Three fruits were closed for 1 h at 20 °C. The 1 mL withdrawn samples were injected into a gas chromatograph (Agilent Technologies 6890 N) equipped with a Carbon plot column (60 m × 0.32 mm × 1.5 µm) and flame ionization detector (FID). The column temperature was set at 60 °C; the injector and detector temperature was set at 250 °C. The ethylene production rate was expressed as µL ethylene/(kg fruit h).

3.7. Soluble Solids Content and Starch Content

Soluble solids content was measured from the squeezed juice of 10 fruits on an Atago RX-5000CX digital refractometer and expressed in Brix. Starch content was determined by a starch iodine test. Horizontally around its equator, cut pears were dipped in an iodine solution consisting of 10 g of KI and 2.5 g I per liter of water. After 2 min, the starch pattern on the cut pear surface was recorded as index 1 = 100% starch or 10 = 0% starch.

3.8. Flash Firmness

Flesh firmness was determined using a penetrometer (Fruit Texture Analyser GÜSS GS; South Africa) with a plunger of 0.5 cm² on the largest diameter area. Firmness was measured on 5 pears, four times on each pear, and expressed in kg/cm². Ethylene evolution, soluble solids content, and firmness are the most common maturity parameters widely used to assess the maturity stage of climacteric fruit. These parameters are employed to determine either the harvest date of pears or simply check their maturity during shelf life.

3.9. Chitosan Coating and P. expansum Inoculation

Pear surfaces were coated with a chitosan film before inoculation with P. expansum. Trimethyl chitosan (CAS Number: 52349-26-5) was prepared as 1% solution in 1% of acetic acid.

For pear fruit inoculation, seven-day old culture of P. expansum grown on MEA (Malt Extract Agar; Sigma Aldrich, Darmstadt, Germany) was used for inoculum preparation. The mold surface was scrapped with a sterile loop and transferred to a physiological solution containing 0.05% Tween 80. The conidia were counted with a Bürker–Türk counting chamber, and the concentration was adjusted to 10⁸ mL⁻¹. An ‘X’-shaped wound (approx. 2 mm × 2 mm) was made with a sterile scalpel three times on the same pear, and 10 µL of the inoculum was added to each wound and left to dry. The effects of chitosan against P. expansum growth were monitored at 25 °C for 14 days. Lesion diameter measurement was carried out every two days.

3.10. Statistical Analyses

A two-way analysis of variance (ANOVA) was employed to examine statistical differences among pear cultivars and incubation time in relation to P. expansum lesion growth. All the experiments were repeated 6 times. Statistical analysis was performed using the Tukey test (GraphPad Prism 8, La Jolla, CA, USA) and two-way analysis of variance (ANOVA) to evaluate the significance of the cultivar type and incubation time, with significance levels set at * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. The difference in maturity parameters among unripe and fully ripe pears cultivars was analyzed with the paired-samples t-test (SPSS program, version 22). Statistical significance was considered for a p-value below 0.05, i.e., 95% of the confidence interval.

Also, the statistical distinctions between untreated and chitosan-treated pear cultivars were considered. Principal component analysis (PCA) and correlation coefficients was performed using the OriginPro 2024 program.

4. Conclusions

Various factors affect the longevity of pears after storage. The cuticle, as the outermost fruit tissue, is considered crucial in preventing microbial attack and consequent fruit deterioration. Cuticle characteristics such as contact angle, roughness, and zeta potential have a role mostly in the first stage, i.e., the attachment of microorganisms to fruit. In non-fruit surfaces, microbial attachment substantially depends on surface roughness and, to a lesser extent, on the contact angle and zeta potential. We investigated six pear cultivars; all showed hydrophilic characteristics that tended to decrease during maturation, from unripe to fully ripe maturity. Roughness differed between cultivars, ranging from 1 to 3 µm, and tended to increase for 2 cultivars but did not change significantly in most of the cultivars. All the pear cultivars had negative zeta potential; fully ripe fruits showed fewer negative values.

Although, in most cases, no significant parameters were observed in the biophysical parameters between unripe and fully ripe pear cultivars, interesting trends were elucidated from PCA and correlation analyses, i.e., fully ripe fruits tend to have a higher contact angle, roughness, and zeta potential and are more prone to P. expansum growth. The results of the cuticle characteristic analysis found here might further broaden the knowledge regarding the interplay of fruit surface and microbial spoilage.