Journal of biological and health sciences http://biotecnia.unison.mx
Universidad de Sonora
ISSN: 1665-1456
Original Article
Desarrollo y caracterización de recubrimientos comestibles de pectina con el agente de biocontrol atrapado Meyerozyma guilliermondii LMA-Cp01 para el manejo de Colletotrichum gloeosporioides de papaya
1 Tecnológico Nacional de México/Instituto Tecnológico de Tepic; Laboratorio Integral de Investigación en Alimentos; Av. Tecnológico 2595, Colonia Lagos del Country, Tepic, Nayarit, México, C.P. 63175.
2 Centro de Investigación en Alimentación y Desarrollo, A.C. Carretera a La Victoria Km. 0.6, Hermosillo, Sonora, C.P. 83304, Mexico.
3 Departamento de Farmacología. Centro Universitario de Ciencias Exactas e Ingenierías. Universidad de Guadalajara, Jalisco, México. Boulevard Marcelino García Barragán #1421, Col. Olímpica, C.P. 44430. Guadalajara, Jalisco, México.
This study aimed to formulate gels using 1% (w/v) low- methoxyl pectin, 0.5% (w/v) glycerol, and 0.5 mM calcium chloride to develop films, alone or in combination with M. guilliermondii, through the casting method, to obtain an an- tifungal edible coating. The gels obtained were characterized rheologically by frequency sweep and gelation kinetics. The effect of yeast addition on thickness, morphology, differen- tial ΔE*, tensile strength, percent elongation at break, elastic modulus of films, and its antifungal activity against Colleto- trichum gloeosporioides from papaya fruit were evaluated. The film-forming dispersions proved to be true gels, showing viscoelastic behavior and narrow molecular structure typical of cross-linked polymers. These gels presented structural stability, and their texture could allow immersion or atomi- zation applications. Films alone and those added with M. guilliermondii showed thickness values of 0.034 and 0.02 mm, respectively. ESEM revealed changes in the film’s mor- phology with yeasts, demonstrating the entrapment of the biocontrol agent. The yeast’s addition to the films improved all the mechanical parameter values and achieved a com- plete inhibition of C. gloeosporioides. This research provides new pectin-based systems which are viable candidates to produce antifungal edible-coating with entrapped M. gui- lliermondii that could protect fruits and vegetables against postharvest diseases.
Este trabajo tuvo como objetivo formular geles de pectina de bajo metoxilo al 1% (p/v), glicerol al 0.5% (p/v) y cloruro de calcio 0.5 mM para producir películas solas y combinadas con
M. guilliermondii como recubrimiento comestible antifúngi-
*Author for correspondence: Montserrat Calderón-Santoyo e-mail: mcalderon@tepic.tecnm.mx
Received: November 6, 2024
Accepted: May 6, 2025
Published: May 30, 2025
co. Los geles se caracterizaron reológicamente por barrido de frecuencia y cinética de gelificación. Se evaluó el efecto de la adición de levaduras en el espesor de las películas, cambio de color ΔE*, morfología, propiedades mecánicas y actividad antifúngica contra Colletotrichum gloeosporioides aislado de papaya. Las dispersiones formadoras de película mostraron un comportamiento viscoelástico y una estructura molecular estrecha de geles verdaderos. Estos geles débiles presentaron estabilidad estructural y su textura permitiría aplicaciones de inmersión o atomización. Las películas solas y adicionadas con M. guilliermondii mostraron espesor de 0.034 y 0.02 mm, respectivamente. ESEM mostró cambios en la morfología de las películas con levadura, lo que demuestra la encapsula- ción del agente de biocontrol. La adición de levaduras a las películas mejoró todos los parámetros mecánicos y logró una inhibición completa de C. gloeosporioides. Esta investigación proporciona nuevos sistemas basados en pectina viables para la elaboración de recubrimientos comestibles antifún- gicos con M. guilliermondii que podrían proteger a las frutas contra enfermedades poscosecha.
Most fresh vegetables and fruits are highly perishable with relatively short shelf lives; they are susceptible to large losses during pre- and postharvest due to biochemical reactions, weight loss, and physical and fungal damage (Porat et al., 2018). Various approaches have been used to raise solutions to these issues. Preservation with chemical fungicides incites environmental concerns and leaves residuality involving toxicological hazards to human health. Also, fungi with che- micals acquired resistance have increased due to repeated applications (González‐Gutiérrez et al., 2024)
Volume XXVII
DOI: 10.18633/biotecnia.v27.2496
. One approach to prevent fungal spoilage is to delay the ripening process after the harvest. Low storage temperature is used to slow down chemical and enzymatic reactions and phytopathogen growth. Nonetheless, prolonged storage of fruits from warm regions under unsuitable conditions can cause chilling physiological injury (Kaur et al., 2022). Hence, it is necessary to search for cost-effective alternative methods to improve the postharvest shelf-life of fruits and vegetables. Packaging is considered a better option for fruit preservation as it provides a thin layer barrier against oxygen, carbon dio- xide, moisture, pathogens, and light, thus delaying ripening and senescence (Pillai et al., 2024). Conventional packaging materials are usually composed of petroleum-based poly- mers such as low-density polyethylene (LDPE), polyethylene terephthalate (PET), polypropylene (PP), polyamide (PA), and polycarbonate (PC), which can lead to a significant environmental problem (Chettri et al., 2023). An appropriate alternative to plastic packaging is edible coatings, which can be consumed along with the food. These coatings are made from naturally occurring biopolymers including lipids, prote- ins, carbohydrates, and composites with excellent mechani- cal and rheological characteristics (Calderón-Santoyo et al., 2022; Pandya et al., 2023; Peerzada et al., 2023). Among these biopolymers is gelatin, which is usually used in edible coa- tings applied to fresh products (Estrella-Ozuna et al., 2024), sodium alginate, a polysaccharide widely used in the food industry (Íñiguez-Moreno et al., 2021), and alginate-chitosan bilayer films (Acuña-Pacheco et al., 2024).
Additionally, acidic sugar chains derived from galactose, such as pectins, are structural constituents in plant cells that provide support and protection by being a part of the defen- se system against phytopathogens (Rohasmizah and Azizah, 2022; Patil et al., 2023; Jhanani et al., 2024). This biological polymer forms a stable hydrogel that can perform a film on fruit surfaces, acting as a support for many additives such as antioxidants, dyes, texture improvers, phytoalexins, or anta- gonists of phytopathogenic fungi, such as lactic acid bacteria and yeasts (Aguilar-Veloz et al., 2022; Bomzon, 2022; Pandya et al., 2023). In recent years, antifungal edible coatings have emerge as an effective approach to mitigate vegetable and fruit loss caused by postharvest fungal diseases by forming a semipermeable film on the surface, which reduces mechani- cal damage and protects against fungal infections (González- Estrada et al., 2017; Zhou et al., 2023).
Meyerozyma guilliermondii (anamorph Candida guillier- mondii) is an ascomycetous yeast whose biocontrol activity has been demonstrated against several fungal diseases on fruits, such as Colletotrichum gloeosporioides from mango fruit (López-Cruz et al., 2020), Penicillium digitatum and P. ita- licum from orange, P. expansum, Rhizopus stolonifer and Bo- trytis cinerea from apples and pears (López-Cruz et al., 2023), Lasiodiplodia theobromae, Rhizopus sp. and Neofusicoccum batangarum from jackfruit (Ayón-Macias et al., 2023) and, B. cinerea and Cladosporium cladosporioides from blueberries (Covarrubias-Rivera et al., 2024). M. guilliermondii exhibits multi-mode mechanisms of action, including competing
for space and nutrients, production of cell wall-degrading enzymes, volatile organic compounds, induction of several defense-related genes, and parasitism (Yan et al., 2021; Ayón- Macias et al., 2023; Cheng et al., 2023; Herrera-Balandrano et al., 2023; López-Cruz et al., 2023).
Functionalized edible coatings using biopolymers with entrapped antagonistic yeasts play an important role in the fungal disease control trends during postharvest manage- ment, improving their efficiency and viability, being a sus- tainable and eco-friendly technology (Iñiguez-Moreno et al., 2021; Vázquez-González et al., 2024). An ideal edible coating must meet certain basic characteristics such as permeability properties to water vapor and gases to avoid an anaerobic environment, breaking strength, melting above 40 °C, low viscosity and non-sticky faces, being translucent, without affecting the quality of fruits and vegetables (Chettri et al., 2023; Miranda et al., 2024). Some edible coating characteris- tics could potentially be modified by the addition of biocon- trol agents. Hence, there is still research needed to ensure the filmogenic capacity and compatibility of biopolymeric films and antimicrobials (Nunes et al., 2023). Based on these foundations, this is the first time the yeast M. guilliermondii LMA-CP01 was added to pectin-based edible-coatings. The objective of this work was to formulate pectin-base gels using calcium chloride as a cross-linking agent and glycerol as a plasticizer to produce support matrix edible films. The film-forming dispersions were characterized by low defor- mation rheology. Furthermore, the physical and mechanical properties and antifungal activity of films were studied.
Low methoxyl pectin LA 410 was purchased from Danisco (Guadalajara, México), and glycerol and calcium chloride te- trahydrate were purchased from Sigma-Aldrich (Darmstard, Germany).
M. guilliermondii LMA-Cp01 strain was isolated from the papaya surface (Carica papaya L.) (LIIA, Instituto Tecnológi- co de Tepic) and stored in glycerol at -80°C. The yeast was inoculated in Trypticase Soy Broth (TSB) (BD Bioxon, Mexico) and harvested after three days at 28° C at 150 rpm. The stock solution of M. guilliermondii was transferred to a test tube with 10 mL of sterile TSB and maintained at 28 °C, 200 rpm for 48 h. Subsequently, this solution was incubated in a flask with 400 mL of TBS for 24 h under the same conditions. The yeast solution was centrifuged at 3000 xg for 10 min, in a Z326 K universal centrifuge (HERMLE, Wehingen, Germany) and resuspended in sterilized NaCl 0.85% solution. Yeasts were adjusted at 1x108 cells/mL using a Neubauer chamber.
Dispersions of pectin were prepared at 1% (w/v), then glyce- rol was added at 50% (w/w) related to pectin dry matter basis, and finally, calcium chloride tetrahydrate as a cross-linking agent was dosed at different concentrations (3.3, 2.7, 2.0, 1.3 and 0.5 mM) (Santana and Kieckbusch, 2013). After obtaining
the ideal concentration of calcium chloride that allowed the form of a weak gel with a continuous phase (0.5 mM), the obtained film-forming solution was placed in a Petri dish and kept in a desiccator (casting method) to obtain the film of cross-linked pectin with calcium. Similarly, cross-linked gels of pectin were prepared, and once the dispersion presented a homogeneous phase, the yeast M. guilliermondii solution was added at 1x108 cell mL-1 to obtain the films of pectin + M. guilliermondii (Fig. 1).
Film-forming dispersions of pectin and pectin + M. guillier- mondii were characterized on a hybrid rheometer Discovery HR-2 (TA instruments, New Castle, USA) using a cone-plate geometry (angle 2° 1’ 1’’, 60 mm diameter). Frequency sweep (ω) was performed from 0.1 to 100 rad/s at 25°C and sweep time was performed during 180 min at 1 rad/s at 25°C (Phan et al., 2023).
Infrared spectra of pectin, pectin films, and pectin + M. gui- lliermondii films were analyzed using a spectrophotometer Nicolet FT-IR iS50 (Thermo Fisher Scientific, Waltham, USA) by a reflection technique (ATR) and recorded between 4000 to 500 cm-1.
Films were evaluated with CIE colorimeter CIE scale, using total color differences (ΔE*ab) according to equation 1:
(1)
Where ΔL*, Δa*, and Δb* are the differences between each color value of standard color plate and film samples, respec- tively. Color differences produced by films were evaluated with colorimeter CR-400 of Konica-Minolta (Tokyo, Japan), comparing surfaces with three tonalities: green (L*= 33.83, a*= -22.04, b*=11.52), yellow (L*= 82.57 a*= -4.90 b*= 32.52)
and orange (L*= 51.11 a*= 40.03 b*=28.78); readings by over-
laying films over the same surfaces, simulating color changes from physiological to maturity stage of papaya fruit surface.
The film thickness was determined as the average of 30 measurements for each sample using a Mitutoyo micrometer (293-230) of MAC (Aurora, USA) (Iñiguez-Moreno et al., 2021).
Tensile data was obtained using a Stable brand texturometer Micro Systems TA-XT2 (Godalming, UK). Film samples (wide 1 cm and 5 cm long) were cut. Initial grip separation and cross- head speed were set at 3 cm and 0.05 cm/s, respectively. Tensile strength (RT), percent elongation at break (%E) and elastic modulus (Y) were calculated according to Jantrawut et al. (2017).
Environmental scanning electron microscope (ESEM) Morphological examination of films was conducted by envi- ronmental scanning electron microscope ESEM (EVO LS10) of Carl Zeiss (Jena, Germany). Cross-sectional areas of films were analyzed. The cross-sectional microstructure of both films were observed after cryo-fragmentation. The samples were placed on a support at 90° and observed at 2000x and 8000x, at 40 Pa and with a voltage of 15 kV for the samples without yeast, and at 5000x and 15000x, 25 Pa, 15 kV and work distance 7.0 mm for the one with M. guilliermondii.
In vitro antifungal activity of films
The antifungal activity of M. guilliermondii and pectin-based films were evaluated in vitro against the pathogen Colleto- trichum gloeosporioides from papaya fruit. C. gloeosporioides was cultured on potato dextrose agar (PDA) (BD Bioxon, Mexico) plates for 7 days at 28 °C. Conidial suspension was adjusted to 105 spores/mL by counting in a hemocytometer using an optic microscope (BA310 Motic, USA). 50 mL of fun- gal spores were spread onto PDA disks (8 mm) and let dry for 15 min. After that, the disks were covered with films of pectin
Figure 1. Schematic representation of edible films obtention with and without M. guilliermondii.
Figura 1. Representación esquemática de la obtención de películas comestibles con y sin M. guilliermondii.
or pectin + yeast and incubated at 28 °C. The germination rate of C. gloeosporioides was determined by microscopy by measuring the percentage of germinated spores for 12 h at 28°C in samples of approximately 200 spores. A spore was considered germinated when its germ tube was longer than half the length of the spore. The inhibition percentage was calculated according to Equation 2 (González-Estrada et al., 2017; Iñiguez-Moreno et al., 2020).
The mycelial growth inhibition of films against C. gloeos- porioides was also evaluated. Plugs of 8 mm in diameter of fungal mycelia from 7 days old were placed at the center of PDA plates. Then, the plates were covered with films contai- ning or not the yeasts and incubated at 28 °C. Petri plates were used as a positive control with only the fungi. The inhibition of mycelial growth was calculated according to Equation 3. The experiment was performed with three replicates and each experiment was repeated twice.
Data analysis was carried out by Student’s t-test using the statistical software STATISTICA v. 12.0 (StatSoft Inc., Tulsa, OK, USA). The analysis of variance (ANOVA) and Tukey’s HSD test were used for comparing mean values of percentage inhibi- tion of C. gloeosporioides. Statistical differences significance was judged at the level of p < 0.05.
The main controlling parameter to achieve film-forming dis- persions was the dose concentration of calcium chloride. The gel formulated with the lowest concentration of crosslinking
Figure 2. Gelation kinetics (A) on film-forming dispersions of pectin and pectin + M. guilliermondii and damping material values (tan δ) vs film- forming dispersions time at 25 °C and 1 rad/s, and frequency sweep (B) on film-forming dispersions of pectin and pectin + M. guilliermondii with 1% (w/v) of pectin and 0.5 mM of CaCl at 25 °C and 1 rad/s.
agent (Ca2+ 0.5 mM) exhibited a more uniform phase in its
Figura 2.
2
Cinética de gelificación (A) en dispersiones formadoras de película
maturity stage, facilitating pouring into the non-stick Petri dishes. This established a CaCl2 concentration of 0.5 mM as the optimal condition for the film formation process using the casting method. At higher concentrations, the formation of agglomerates of macroscopic size was observed due to the high reactivity of the low-methoxyl pectin solution with divalent calcium cations. These agglomerates persist in the polymeric structure of the formed films, creating a disorde- red structure with a discontinuous texture.
Gel-forming solution characterization is fundamental for the development of edible films to improve film uniformity (Shivangi et al., 2021). Gelation kinetics are shown in Fig. 2A. Graph depicts a constant increment on the elastic modulus enhancing a maximum point that resulted in a flexible gel or dispersion of cross-linked polymers as a result of the continui- ty in the formation of a structure with a low force. Damping material values, or tan δ, showed values lower than 1 (Fig. 2A). This fact confirms the gel state in both solutions used for
de pectina y pectina + M. guilliermondii, y valores de material amortiguador (tan δ) vs tiempo de dispersiones formadoras de película a 25 °C y 1 rad/s, y barrido de frecuencia (B) en dispersiones formadoras de película de pectina y pectina + M. guilliermondii con 1% (p/v) de pectina y 0.5 mM de CaCl2 a 25
°C y 1 rad/s.
film formation (Abbastabar et al., 2015), indicating that the particles are strongly associated with colliding forces. When the tan δ value is around 0.01 and 0.1, the structure of the gel is more flexible and deformable and not very susceptible to syneresis because in the frequency sweep (Figure 2B), G’ and G’’ maintain a 10:1 ratio.
Fig. 2B depicts the behavior of film-forming disper- sions of pectin and pectin + M. guilliermondii subjected to a frequency sweep. It is appreciated that elastic moduli G’ predominates for both the pectin gel and the one containing the yeast. This condition implies that the analyzed solutions have both a viscoelastic behavior, typical of fluids that obey Maxwell’s model (Hütter et al., 2020; Tseng and Chang, 2024), which indicates that the gels will flow like a liquid and will be propitious, from the rheological point of view, to form films
by the casting method or spraying to form a coating, but also have viscosity and adhesion to the surfaces by contact and thus, they are optimal for application by immersion.
Fig. 2B also illustrates how the storage module is in- dependent of the shear rate during the test, while the loss module changes its behavior at frequencies close to 100 rad/s. At low frequencies and extended oscillation periods, the macromolecular chains can unravel, and the system behaves like a solution. Conversely, at high frequencies and short time interval, the macromolecular chain remains entangled, causing the system to display solid-like behavior (Okay, 2009). This phenomenon is evidenced by analyzing the behavior of the complex viscosity (η*). When at rest, the gel without yeast exhibits a significantly higher viscosity due to its more ordered structure (p < 0.05). As the shear frequency increases up to 1 rad/s, the complex viscosity of both gels fluctuates around values close to zero (Patova et al., 2017). The decrease in G’ and G’’ values in the gel containing the yeasts is attributable to the microbial cells diminishing the strength of the molecular structure by interfering with the formation of binding sites. The behavior of the studied gels at high frequencies is characteristic of true gels, and at G’ values below 10 Pa, structural stability is assumed (Vityazev et al., 2020). This finding suggests that the gel is less prone to syneresis, avoiding the formation of an aqueous phase and preventing the proliferation of microorganisms. It is noticed that G’ > G’’, a characteristic typical of cross-linked polymer gels, indicating their ability to absorb water without fully dissolving. In addition, it can be inferred that the structure is compact because there is a 10:1 ratio between the elastic and viscous modulus, respectively (Patova et al., 2017).
Fig. 3 shows IR spectra of (A) pure pectin, (B) pectin film, and
(C) pectin + M. guilliermondii film. All the IR spectra were normalized to unity, taking the most intense band as a refe- rence band, which appears around 3200 cm-1 in all spectra. The three spectra showed similar signals: (i) an intense broad band located at 3200 cm-1 caused by stretching vibrations of the hydroxyl groups (absorption in the O-H region was due to inter- and intra-molecular hydrogen bonding of the galacturonic acid backbone) (Abid et al., 2017), (ii) a shoulder around 2905 cm-1 that is related to C-H absorption, these in- clude CH, CH2, and CH3 stretching and bending vibrations, (iii) a fingerprint region located below 1500 cm-1 and (iv) a region between 1800 and 1500 cm-1, depicted in the inset of the figure. To observe the cross-linking effect, spectra A and B are depicted since this allows the observation of infrared absorp- tion by the carboxylic acid and the carboxylic ester groups of the pectin molecules. At 1726 cm-1 (esterified carboxyl groups) and 1600 cm-1 (free carboxyl groups), characteristic weak signals from bands of low methoxyl pectin are obser- ved. Ca2+ dosage in pure pectin was able to displace the band centered on 1600 cm-1 (inset: spectrum A) to 1609 cm-1 (inset: spectrum B). This shift could be due to the modification of the functional group environment, suggesting an interaction
Figure 3. FTIR spectra of: pure pectin (A), film of pectin (B), and film of pectin
+ M. guilliermondii (C). Inset: partial IR spectrum on the region from 1500 to 1800 cm-1.
Figura 3. Espectro FTIR de: pectina pura (A), película a base de pectina (B) y película a base de pectina + M. guilliermondii (C).
between free carboxyl groups from pure pectin and calcium ions that could generate sites that promote M. guilliermondii entrapment. From early studies, electrostatic interactions exist between Ca2+ and COO− in the ‘egg-box’ model (Cao et al., 2020). As an electron-withdrawing ion, calcium induces a charge deviation, which results in a change in the band vibra- tion. In addition, the ordering and arrangement of junction zones may be different under various gel conditions. Alter- natively, on the spectrum C (inset), amide bands centered at 1643 cm-1 and 1542 cm-1 (amide I and amide II absorptions, respectively) were revealed. These bands are attributable to the vibrations of the amide groups from proteins and indicate their secondary structure. These signals are attributed by the amount of M. guilliermondii entrapped into the cross-linked pectin. These characteristic absorptions of yeasts on the spectrum A and B were not observed (Mihoubi et al., 2017). Returning to section (i), the band assigned to galacturonic acids, which are sources of substrate for M. guilliermondii, no important modifications are observed in this signal ca. 3200 cm-1 (spectrum B and C). These results suggest that a good coupling between the cross-linked pectin and the yeasts could be used as edible coatings.
Both films (pectin and pectin + M. guilliermondii films) ex- hibited a uniform superficial macroscopic texture. The film of pectin is translucent (Fig. 4A) due to the carbohydrate properties and occurs because they are capable of forming crystals that allow light waves to break through; and the one that contains the biocontrol agent is opaque with a yellowish tone, as reported by Iñiguez-Moreno et al. (2021). These characteristics in films with biocontrol agents (Fig. 4B) are attributed to their lipid and protein constituents, which are not translucent.
Figure 4. Films of pectin (A) and pectin + M. guilliermondii (B).
Figura 4. Películas a base de pectina (A) y pectina + M. guilliermondii (B).
The analysis of the films formed degree influencing the differential ΔE* are reported on Table 1. The color variation is less than 5 in the yellow spectrum, as a consequence of the transparency and coloration presented by the films formed, however, ΔE* exceeds the minimum perceptible in the oran- ge and green tones. Therefore, the application of a pectin coating with M. guilliermondii will produce a change in the perceptible coloration in the zones of the papaya fruit that present these tonalities. Consequently, the application of a coating added with M. guilliermondii as a biocontrol agent will significantly modify the appearance of the fruit, except in cases where it presents a yellow tone, corresponding to an intermediate stage of maturity between physiological and consumer stages of papaya fruit.
Films formed from the pectin gel without the biocontrol agent presented an average of 0.0343 mm in its cross-section and, for those containing M. guilliermondii have an average thickness of 0.0206 mm, significantly different (p ≤ 0.05). The presence of the yeasts is presumably the cause in the decrea- se of the thickness, since these interrupt the continuity of the molecular structure in the films, decreasing the areas of cross-linking points with calcium ions between the chains of galacturonic acids, modifying the structure of the film as well as the quantity and location of the union points (Barbut and Harper, 2019).
Micrographs of film without M. guilliermondii showed smooth uniform morphology (Fig. 5A and B). The effect that yeast addition on the structure of polymer matrix on the cross- sectional area, shows irregular longitudinal zones, which evidenced the entrapment of M. guilliermondii (Fig. 5C and D). Probably, yeasts are negatively charged in their cell-wall, forming clusters that produce a different polymeric structure regarding to the original matrix with the cross-linked pectin (Casamorin et al., 2014). Nevertheless, yeast cells were homo- geneously distributed in pectin film.
Films added with M. guilliermondii proved to withstand al- most twice as much force before yielding to rupture (Fmax), increasing the tensile strength (RT) above 3x. The percent elongation at break (%E) is also increased by 30% because the yeasts decrease the crystallinity of the pectin film, and in turn, a diminish in the longitudinal elastic modulus was observed (Y). These values are shown in Table 2. The increase in tensile properties may be due to the interactions between pectin chains and yeast wall components, as was evidenced in FTIR analysis. The same pattern was reported by Iñiguez- Moreno et al. (2021).
The results of the inhibition of C. gloeosporioides are shown in Table 3. The pectin-based film without yeast did not inhibit the spore’s germination or mycelial growth. Iñiguez-Moreno et al. (2020) reported that sodium alginate films did not inhi- bit the growth of C. gloeosporioides from avocado fruit, but, the films with the yeast Meyerozyma caribbica reached more than 99% of inhibition.
The yeast M. guilliermondii completely inhibited the germination and mycelial growth of C. gloeosporioides, as the same of the pectin film added with the yeast. The biocontrol efficacy of M. guilliermondii against several phytopathogenic fungi has been previously reported. Its biocontrol effec- tiveness is due to different mechanisms of action such as competing for space and nutrients, production of hydrolytic
Table 1. Mean values of L*a*b* and differential ΔE*.
Tabla 1. Valores promedio de L*a*b* y diferencial ΔE*.
Film of pectin | Film of pectin + M. guilliermondii | |||||
Green | Yellow | Orange | Green | Yellow | Orange | |
L* | 42.52 ± 0.81b | 81.37 ± 1.37a | 54.81 ± 0.89a | 37.47 ± 0.68a | 80.31 ± 1.40a | 53.95 ± 0.99a |
a* | 13.58 ± 0.61b | -3.64 ± 0.51a | 32.68 ± 1.36a | -17.29 ± 0.85a | -3.90 ± 0.59a | 33.47 ± 0.89a |
b* | 7.56 ± 0.59b | 30.31 ± 0.95a | 22.84 ± 0.93a | 9.22 ± 0.89a | 30.69 ± 1.03a | 23.26 ± 0.78a |
ΔE* | 6.4 ± 0.5b | 3.0 ± 0.3a | 9.0 ± 1.1a | 12.7 ± 0.5a | 2.8 ± 0.3a | 10 ± 1.2a |
Values are expressed as means ± standard deviation (n = 15). Values in columns with the same name, followed by different lowercase letters, are significantly different according to Student’s t-test at p < 0.05.
Figure 5. Cross-sectional area of films obtained by environmental scanning electron microscopy (ESEM) of pectin at 2000 x (A) and negative at 8000x (B) and, pectin + M. guilliermondii at 5000 x (C) and 15000 x (D).
Figura 5. Área de sección transversal de películas obtenidas mediante microscopio electrónico de barrido ambiental (ESEM) de pectina a 2000 x (A) y 8000x (B), y de pectina + M. guilliermondii a 5000x (C) y 15000x (D).
Table 2. Films tensile data of pectin and pectin + M. guilliermondii.
Tabla 2. Datos de tracción de películas de pectina y pectina + M. guilliermondii.
Film | Fmax (N) | RT (MPa) | %E | Y (N/mm2) |
Pectin | 7.14 ± 0.55b | 7.0 ± 0.6b | 12 ± 0.9b | 0.178 ± ±0.06a |
Pectin + M. guilliermondii | 13.41 ± 0.61a | 22.35 ± 2.78a | 43 ± ±2.1a | 0.104 ± ±0.05a |
Values are the mean ± standard deviation (n=5). Different letters in the same column indicate significant differences according to Student´s t-test (p < 0.01). Fmax= Maximum force. RT= tensile strength.
Table 3. Antifungal activity of films against C. gloeosporioides
Tabla 3. Actividad antifúngica de las películas contra C. gloeosporioides
Percentage of inhibition
Treatment Spore’s germination Mycelial growth
M. guilliermondii 100 ± 0.0 a 100 ± 0.0 a
Film of pectin 0 ± 0.0 b 0 ± 0.0 b
Film of pectin + M. guilliermondii 100 ± 0.0 a 100 ± 0.0 a
Values are expressed as means ± standard deviation (n = 6). Values in columns with different lowercase letters are significantly different according to Tukey’s HSD test at p < 0.05.
enzymes and volatile organic compounds (Ayón-Macías et al., 2025). The efficacy of the film added with M. guilliermon- dii demonstrates that pectin-based edible film is a support matrix for entrapped yeast to control pathogenic fungi.
A pectin-based gel combined with glycerol, using calcium chloride as a cross-linking agent was prepared, and exhibited rheological properties that facilitate its application through spraying or fruit immersion to create edible coatings. The
resulting films can serve as a support matrix for biocontrol agents such as M. guilliermondii. The incorporation of M. guilliermondii into the pectin-based films positively influen- ced their mechanical properties and thickness, while also demonstrating antifungal activity against C. gloeosporioides in papaya fruit. Pectin-based films enriched with M. guillier- mondii present a potential alternative for controlling the phytopathogenic fungus C. gloeosporioides. Further studies on the application of pectin-based edible coatings contai- ning M. guilliermondii on fruits are necessary.
The authors declared they have no conflicts of interest.
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