Banner
Current IssueEditorial BoardOnline First ArticlesArchive
Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Isolation and the Use of Fungal-derived Chitosan as a Substrate-Free Edible Coating to Extend the Postharvest Shelf Life of Guava

R
Rekha Anantharaman1,*
1Centre for Global Health Research, Department of Obstetrics and Gyanecology, Saveetha Medical College and Hospital, Saveetha Institute of Medical and Technical Sciences, Chennai-600 001, Tamil Nadu, India.

ABSTRACT

Background: Guava (Psidium guajava) is a nutrient-rich tropical fruit with a very short postharvest shelf life due to its thin skin, high respiration rate and susceptibility to microbial spoilage. Chitosan is an edible film and a preservative, which can be developed from the fungal source and it benefiting the shelf life of perishable foods and vegetables.

Methods: The fungal species were isolated and identified by standard methods. Acid and alkali treatments were used to extract the chitosan from the chosen fungal isolates. FTIR and XRD were used to characterize the isolated chitosan. The surface of the guava fruit was coated with the fungal-based chitosan (1% and 2%). Various physiological properties such as weight loss, ripening and shelf life were evaluated at regular intervals. The DPPH assay and the Agar well diffusion method were used to assess antibacterial and antioxidant properties.

Result: Fungal isolates were selected, namely Fusarium oxysporum and Aspergillus fumigatus, based on morphological stability for the biomass yield for chitosan extraction. The yields of the chitosan for A. fumigatus (0.685 g/100 ml) and F. oxysorum (0.850 g/100 ml), respectively. Fruits that have been treated with 2% fungal-based chitosan exhibited a lower weight loss (3.2%), slow ripening and longer shelf life (9 days) and retained higher antioxidant activity (68.2%), than 1% and control group. 2% chitosan of A. fumigatus showed high antibacterial activity. The current study focusing on developing a fungal derived chitosan, which can be used as a suitable sustainable, non-toxic and safe alternative preservative to prolong the shelf life of guava.

INTRODUCTION

Guava (Psidium guajava) is a very nutritious fruit encountered in the tropics. It contains a large amounts of dietary fiber, protein, soluble carbohydrates, riboflavin, essential amino acids, vitamin C and antioxidants (Khan et al., 2025). It is easily prone to spoilage after harvest because of the cuts, growth of microbes, dehydration and enzyme degradation. The industry faces a lot of trouble mainly due to the thin delicate nature of skin in guavas, but even more so due to high demand in the market and the many benefits associated with them. Maintaining the long shelf life of guava remains a difficult aspect to the producers and retailers, particularly in regions where there is a low level of the development of the cold storage facilities (Nor et al., 2020). The conventional use of chemical preservatives as a way of extending shelf life through the incorporation of formaldehyde, BHA and BHT is also diminishing as people become more cognizant of the eventual risks to their health and safety (Hanani et al., 2023). One of the potential solutions to reducing post-harvest losses, preserving fruit quality and ensuring food safety is natural edible coating such as chitosan (Francisco et al., 2020). Edible coatings are an economically viable solution to protraction of food as well as sustainable health of customers (Kaur et al., 2024; Hazarika et al., 2023).
       
Chitosan is a linearly structured polysaccharide composed of covalently bound D-glucosamine and N-acetyl- D-glucosamine and possesses remarkable characteristics to be biodegraded, biocompatible, non-toxic and able to form a film (Muxika et al., 2017; Rajaraman et al., 2020). This has made it suitable to be used in many areas such as preservation of foods, agriculture, pharmaceuticals and bio-medical applications (Kouhi et al., 2020). It is a highly valuable source in the food industry due to being a natural non-synthetic polymer capable of serving as an edible film, benefiting the shelf life of perishable foods and vegetables by sealing a semi-permeable structure to moisture, oxygen and pathogen penetration (Wang et al., 2024; Jeyakumari et al., 2023).
       
The potential of fungal biomass as a substitute source has gained more attention for the production of chitin and chitosan by fungal mycelium. Fungal mycelia also have significantly lower than normal amounts of inorganic constituents than in traditional crustacean-based chitin, which removes the necessity of any demineralization processes to support fungal mycelia-derived products. Moreover, the occurrence of allergens in crustacean residues is a risk to vulnerable customers, which restricts its usage in foods and biomedical products (Huq et al., 2022). Classically, chitosan is produced with a two-phase procedure of treating the fungal cell walls with both acids and alkali (Manal et al., 2019). The other pathway through which chitosan is made by the fungi is through enzymatic deacetylation of chitin (Kumar et al., 2023).
       
Some filamentous fungi, especially the genus of Aspergillus, Fusarium, Mucor and Rhizopus, have chitin and chitosan in cell walls (Pochanavanich and Suntornsuk, 2002). Chitosan derived fungi can be generated in the regulated fermentation environment with readily available and easily accessible substrates available throughout the year and with little impact on environment. In addition, the produced chitosan is allergen free as well as vegetarian with cheaper as well as less polluting extraction process. Because of these benefits, fungal chitosan is a promising option to be utilized in the pharmaceutical, food and biomedical sectors (El-Sabbagh et al., 2023).
       
Dipping was one of the oldest methods of commercial coating and is commonly seen today as constant improvements are made to this in perpetuity way. This process is used to soak the fruits in a coat solution to ensure that they get the full surface coating. This technique is effective with solutions of a variety of viscosities in coating requirements (Fathi et al., 2021).
       
The objective of the research is to investigate, explore and compare the usefulness of the fungal produced chitosan as naturally occurring biodegradable and edible coating on the guava fruit shelf life. Through analysis of the fungal basis resources in terms of aspects such as physicochemical parameters of chitosan, antimicrobial activity, a possibility of fungal derived chitosan as a precious source of antimicrobial activating compounds as a replace-ment of the standard sources used as food preservatives is assessed. In addition, the innovative aspect of the study is the choice of the fungal strains as very few studies have been done on extraction of chitosan over the two strains.

MATERIALS AND METHODS

Isolation and identification ientification of fungi
 
Soil samples were collected and serially diluted to isolate fungal strains using a standard protocol. Identification was done using Lactophenol cotton blue (LPCB) stain. The characterization, antibacterial activity was done in Saveetha University, Thandalam, Chennai and the other works were performed at Valliammal College for women, Anna Nagar, Chennai. A research period of December 2019- January 2022 was established.
 
Inoculation of fungal spores
       
Aqueous preparation of fungal inoculum was prepared aseptically by directly pouring sterile distilled water on fully growing of the fungal mycelia that had been grown on Sabouraud Dextrose Agar (SDA) plates. Spores were scraped gently with a sterile inoculating wire and the suspension of spores was filtered into sterile flask. A hemocytometer was used to measure the concentration of the spores and they were diluted to 107 spores/ml. Thereafter, respective flasks were inoculated with 1 mL of fungal spore suspension. The cultures were placed under batch conditions and grown at 28oC over 5 days period and a complete growth of the fungal mycelium was observed (Bhargava and Tandon, 2015).
 
Mass cultivation
 
The culture of the selected strains of fungi was prepared in a synthetic microbial liquid growth medium, which included 20% potato extract, 2% dextrose, 1% peptone, 0.1% NaCl, 0.01% CaCl2 and 0.05% of MgSO4 7. H2O. To achieve good growth of the mycelium, the cultures were incubated at 28oC centrifuge at 120 rpm for 10 to 15 days. Produced fungal biomass was collected through filtration and then washed excessively using distilled water in the process of eliminating medium constituents; followed by air-drying at the room temperature.

 
Chitosan extraction and purification
 
Fungal mycelia were collected at the final stage of the cultivation phase using Whatman No. 1 filter paper. The harvested biomass was washed twice with distilled water and dried at 65oC until a constant weight was achieved. Chitosan extraction from the fungal biomass involved a series of chemical treatments, starting with deproteinization and deacetylation using an alkaline solution, followed by solubilization in dilute acetic acid (Nwe et al., 2010). Initially, the biomass was treated with 1 M sodium hydroxide (NaOH) at a ratio of 1:40 (w/v) and heated at 90oC for 3 hours. This process yielded a solid residue known as alkali-insoluble material (AIM). The AIM was separated by centrifugation at 6000 rpm for 15 minutes, thoroughly washed with distilled water and centrifuged repeatedly until a neutral pH was reached.
       
For chitosan extraction, the neutralized AIM was treated with 10% (v/v) acetic acid (1:40 w/v) at room temperature for 6 hours on a rotary shaker operating at 200 rpm. The resulting acid-insoluble residue was removed by vacuum filtration. Chitosan remained in the filtrate and the pH was adjusted to 9.0 using 4 M NaOH, resulting in chitosan precipitation.The precipitate was recovered by centrifugation, washed with distilled water and dried at 60oC until a constant weight was obtained (Abdel-Gawad et al., 2017). Throughout the studies, the chitosan extraction using Fusarium oxysporum indicates the Sampe 1 or S1 whereas Sample 2 or S2 denoted for Aspergillus fumigatus.
 
Characterization of chitosan
 
Fourier transform infrared spectroscopy (FTIR)
 
Using Shimadzu IR Affinity-1S Spectrometer, FTIR analysis was done for chitosan, to define group of absorptions based on functional groups. Dried chitosan was were taken in thin cut samples, crushed in a potassium bromide (KBr) and then formed  into a disk and scanned in wavenumbers 4000-400cm-1.
 
XRD
 
The Powder X-ray Diffraction (PXRD) analysis using D8 Advance Bruker (Germany) diffractometer and Cu Kα (l=1.54059) as the source of radiation.
 
Chitosan coating on guava
 
Treatment and storage condition
 
The coating solutions was prepared with 2.5 g and 5 g of chitosan powder dissolved in 900 ml of distilled water with addition of 2% of citric acid was added to facilitate the chitosan powder to dissolve and also as a plasticizer. Each one of the solutions was adjusted to pH 5.5 using 0.1M NaOH. The treatment comprised of 0.25%, 0.5% and control (an acid solution that defied the presence of chitosan). The prepared emulsion of coating solution was dipped in the form of a coating over the guava fruits and the other was untreated and kept as control. All fruits were labeled using the sample number and other information. A comparative experiment on the treated and untreated guavas was carried out to test it.
 
Observation of shelf life
 
After the post harvests treatment, guava fruits were stored in controlled environments and their shelf life was being studied at every time interval. Different quality variables were measured periodically including the color, weight loss and symptoms of spoilage of the treated.
 
Various parameters of the guava before and after treatment
 
Weight loss
 
To calculate the weight loss, the weight of the guava samples on Day 0 and the weight measured on Days 3, 6, 9 and 12 after coating were exerted. The weight of the fruit was measured using a laboratory weighing balance. The percentage of weight loss was calculated by the following equation:

 
Where,
W1 = Initial weight of the guava.
W2 = Weight at each time point following coating.
 
Antioxidant activity
 
According to the protocol of Wani et al., (2018), the DPPH radical scavenging assay and a mixture of 3.5 mL DPPH solution determined oxidative strength and 0.5 mL guava fruit extract was added. The percentage of antioxidant activity was measured at an absorbance of 615 nm using UV-Visible Spectrophotometer. The inhibition percentage of antioxidant activity was expressed as follows:

 
Where,
AC=Absorbance control
AS=Absorbance sample
 
Determination of antibacterial activity
 
The prepared fungal-based chitosan solution was used to check the antibacterial activity against Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli using Agar well diffusion method.

RESULTS AND DISCUSSION

Isolation of fungi
 
On SDA plate, a diverse growth of fungi was observed. The plate showed a range from light green to dark green, brown and white colored colonies with a cottony, powdery hyphae were shown in Fig 1 and 2.

Fig 1: Fungal colonies on SDA.



Fig 2: Fungal colonies on SDA.


 
Identification of fungal species
 
The colonies were analyzed morphologically using Lactophenol Cotton Blue (LPCB) staining and observed under high power of objective microscope (40x). Identification of the fungal species was tabulated (Table 1). Among four morphologically distinct identified fungal strains, two strains of fungi were shown in Fig 3 and 4 Fusarium oxysporum and Aspergillus fumigatus that were picked and used for the further experiment.

Table 1: Identification of isolated fungal species.



Fig 3: Fusarium oxysporum from sample 1.



Fig 4: Aspergillus fumigatus from sample 2.



Biomass production
 
A thick fungal mat was observed on the surface of the liquid medium after 10-15 days of incubation. The resultant mycelium biomass was collected, air-dried and decanted in order to extract chitosan in the next stage shown in Fig 5.

Fig 5: Mass cultivation of fungal spores.


                    
Extraction of chitosan
 
The fungal liquid biomass was used to obtain chitosan through a renowned set of chemical reactions that successively isolated the chitosan as well as purifying it. The extraction process was evidenced by the uniformity of the finished product, which was shown in Fig 6a-6e. The final yield of chitosan from Fusarium oxisoporum (S1) was 0.685 g/100 ml whereas 0.850 g/100 ml from Aspergillus fumigatus (S2). The fungal cell walls were treated with acids and alkalis in a two-step process. Proteins and soluble polysaccharides were removed successfully after being treated with NaOH and deposition of an alkali-insoluble material (AIM) as solid sediment was the evident. During the treatment, a combination of chitin and glucan dissociates and deacetylation step produced the chitosan.

Fig 6: Extraction of chitosan.



FTIR
 
The existence of distinctive functional groups in the chitosan isolated from Fusarium oxysporum was verified by FTIR was presented in Fig 7a. Extensive hydrogen bonding is indicated by a large absorption band seen in the 3368–3683 cm-1 range, which is correlated with the O-H stretching vibrations of hydroxyl groups and the N-H stretching of amino groups. Asymmetric and symmetric C-H stretching vibrations of -CH‚  groups are responsible for the peaks at 2922.16 cm-1 and 2852.72 cm-1. Overtones or lingering contaminants could be the cause of a little band at 2374.37 cm-1 and a slight peak at 2725.42 cm-1 (Sharma et al., 2024). Partial deacetylation is indicated by a band at 1375.25 cm-1 that corresponds to the C-N stretching vibrations of amino groups. The peak at 1155.36 cm-1 is linked to the chitosan backbone’s asymmetric stretching of the C-O-C bridge (Berger et al., 2018). These spectrum characteristics verify that chitosan with structural properties comparable to those of typical fungal-derived chitosan was successfully extracted.

Fig 7a: FTIR analysis of chitosan extracted from Fusarium oxysporum.


       
The FTIR spectra of the chitosan extracted by Aspergillus fumigatus have characteristic functional groups that prove its molecular composition was shown in Fig 7b. The presence of there is quantum of hydroxyl and amine due to the presence of large sum of absorption seen at 3446.79 cm-1, which is O-H and N-H stretching vibrations. The vibrations of the -CH2 groups responsible are the C-H asymmetric and the C-H symmetric stretching and the corresponding peaks are 2922.16 cm-1 and 2852.72 cm-1. The formation of inter- and intramolecular hydrogen bonds enhances the structural stability of pure chitosan (Praveen et al., 2017). The highest peak at 1554.63 cm-1 can be attributed to N-H bending, yet strong and evident peak at 1656.85 cm-1 can be connected with amide (C=O stretching of residual N-acetyl groups. The contribution of CH2 bending is assigned at 1458.18 cm-1. The C-N stretching is found to be located at a peak 1377.17 cm-1 (Street et al., 2018). These spectrum properties are evidence that the chitosan extraction is effective since it is based on the fungal source.

Fig 7b: FTIR analysis of chitosan extracted from Aspergillus fumigatus.



XRD
 
Chitosan produced by fungal strains Fusarium oxysporum and Aspergillus fumigatus exhibited certain characteristic peak angles of the diffraction obtained on the XRD plots displayed in Fig 8a, b around 2θ = 9o-10o and 19-20o demonstrating the semi-crystalline nature of chitosan. Chitosan extracted from F. oxysporum exhibited sharper and more defined peaks at 9.15o and 19.60o denoting a better degree of crystallinity. It implies that the molecular chains of this sample might be more regular and dense, which can increase its mechanical properties, thermal stability, barrier properties. Differently, chitosan obtained in A. fumigatus showed wider and weaker peaks at 9.25o and 19.55o, indicating a more amorphous structure, with low crystallinity. These distinctions can be affected by a number of factors, which include the inherent metabolic and enzymatic characteristics of fungus used as the production organism, differences in cell wall composition, change in extraction procedures like deproteinization, deacetylase activity and drying practices.

Fig 8: X-ray diffraction of chitosan extracted from fungal strain.


 
Preparation of chitosan coating solution
 
In order to measure their stability and solubility, different combinations of chitosan coating solutions were successfully prepared and displayed in Fig 9. The chitosan (S1) turned turbid and clearly there is sedimentation implying that it was not totally dispersed. Better clarity and homogeneity were noted with 1% chitosan and 2% citric acid (S1 with CA) solution showing better solubilization of chitosan in acidic solution. As an acidic environment control, the sample appeared to be translucent on the 2% citric acid alone sample leading to the observation that it lacks particle matter.

Fig 9: Preparation of chitosan coating solutions.


 
Postharvest quality of guava with chitosan coating
 
A noticeable increase in the colour change of guava fruits was observed from the third day of storage. However, by the ninth day, fruits coated with 2% chitosan (Sample 1: Fusarium oxysporum) showed significantly less colour development compared to the untreated control group, indicating a delay in the ripening process. Among all treatments, the formulation combining 1% chitosan with 2% citric acid (Sample 1) was the most effective in preserving the visual quality of the guava during storage. The postharvest application of chitosan notably extended the shelf life of the fruits was shown in Fig 10.

Fig 10: Effect of chitosan on postharvest quality of Guava.


       
In addition to delaying ripening, chitosan-treated fruits showed better retention of firmness, reduced weight loss and lower microbial decay throughout the storage period. The semipermeable nature of chitosan film likely created a modified internal atmosphere, slowing down respiration and senescence (Soares et al., 2011). Overall, chitosan coating effectively preserved the postharvest quality of guava, making it a promising treatment for extending marketable shelf life (Dutta et al., 2012).
 
Weight loss
 
Using the initial weight, the % weight loss of coated and uncoated fruits over a 12-day storage period was calculated. At the end of day 12, coated fruits showed a progressive decrease in weight, ranging from 3.2%, whereas uncoated fruits, on the other hand, demonstrated a noticeably greater reduction in weight, ranging from 0.98% at day 12. The guava weight loss results are presented in Fig 11 showing that there was a significant (p<0.05) increase in the percent weight loss. Fresh fruit loses weight through respiration and transpiration. The key reasons leading to a decrease in weight include respiration and loss of moisture between the fruits internal and environmental external conditions of air (Hernández-Muñoz, 2006; Zhang et al., 2018). Chitosan coatings delayed the process of dehydration by avoiding the loss of water and also avoided mechanical damages and overcome the cuts that occur by default on the fruits skin (Hong et al., 2012).

Fig 11: Changes in weight after post harvesting of guava.


 
Antibacterial activity
 
The antibacterial activity of chitosan formulations was evaluated against Klebsiella pneumoniae, Escherichia coli and Staphylococcus aureus, with inhibition zones compared to a standard antibiotic (Table 2) and Fig 12. When comparing, 2% chitosan showed a highest zone of inhibition than 1% chitosan sample. Among three different bacterial strains, Escherchia coli growth was inhibited higher than Klebsiella and Staphylococcus. Usually, the mechanism of the antimicrobial is based on electrostatic interactions with the cytosolic contents, cell wall and outer membrane of the bacteria (Duan et al., 2019). Antibacterial activity of chitosan is widely demonstrated to be due to its binding to the negatively charged cell wall of bacteria thus rupturing the cell wall and altering the porousness of the membrane.

Fig 12: Antibacterial Activity of chitosan solution against.



Table 2: Antibacterial efficacy of chitosan solutions.



Antioxidant activity
 
The antioxidant activity in the control (Uncoated) samples declined significantly from an initial 87% to 20%, whereas in the coated samples, it decreased only to 87 to 68.2%. The antioxidant activity in terms of DPPH showed highest with higher concentration of chitosan Table (3). The observed reduction in antioxidant capacity in the control group is most likely determined by a generation of free radicals because of the degradation of phenolic compounds due to senescence and elevated level of respiration (Ghasemnezhad et al., 2010; Rehman et al., 2020). The edible coatings affect the internal environment of fruits by slowing down metabolic reactions by a large margin leading to a significant decrease in the production of flavonoids and phenolic compounds (Gonzalez-Aguilar et al., 2010; El-Sayed et al., 2019). Conversely, the guava that coated with chitosan maintained higher antioxidant activity than the uncoated guavas. This increase is possible due to the ability of the coating to change the internal atmosphere of the fruit to reduce the oxidative stress level and slow down the degradation of the antioxidant compounds. Wang and Gao (2013) obtained a similar result demonstrating that strawberries undergoing coating of chitosan maintained a more acceptable total phenolic content and antioxidant activity when stored.

Table 3: Percentage of DPPH inhibition.

CONCLUSION

This study shows the potential of fungal-based chitosan, Fusarium oxysporum and Aspergillus fumigatus, to provide effective, environmental friendly and non toxic edible wrapping to extend the post-harvest life of guava (Psidium guajava). The functional groups of the extracted fungal chitosan were successfully characterized using FTIR. The fungal-derived chitosan showed a strongest antibacterial against both Gram positive and Gram negative bacteria. The effectiveness of antioxidant capacity of guava fruits coated with fungal extracted chitosan was clearly depicted in the DPPH antioxidant test where the antioxidant capacity of guava fruits coated with chitosan was able to withstand the antioxidant capacity throughout the storage period than the un-coated control fruits. The coating successfully retained phenolic compounds and other bioactive antioxidants and led to the extension of fruit quality and shelf life. These results show that fungal chitosan can be considered a promising candidate of natural edible coating to keep postharvest fruits in high nutritional and functional quality. Unlike the rapid decline recorded in controls, the shelf life of the fungal chitosan coated guava significantly delayed the ripening process, reduced weight loss and preserved antioxidant activity. The chitosan treated guava fruit samples maintained the quality for upto nine days in ambient conditions. The chitosan exhibited strong antibacterial activity against pathogenic strains commonly found in food such as Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli at the 2% concentration.
       
These results support the application of fungal chitosan as alternative to the utilization of conventional synthetic preservative compounds since the results satisfy consumer preference to clean-label and non-allergens food preservatives. Fungal chitosan should be considered as a best option to be employed in postharvest handling processes because it can have a combination of both antibacterial and physical barrier actions, particularly in regions where there is no advanced cold-chain structure. Further business analysis and scale-up studies are needed to inquire into its broader use in the preservation of other perishable fruits and vegetables.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

REFERENCES


  1. Abdel-Gawad, K.M., Hifney, A.F., Fawzy, M.A. and Gomaa, M. (2017). Technology optimization of chitosan production from Aspergillus niger biomass and its functional activities. Food Hydrocolloids. 63: 593-601.

  2. Berger, L.R.R., Stamford, T.C.M., de Oliveira, K.Á.R., Pessoa, A.D.M.P., de Lima, M.A.B., Pintado, M.M.E. and de Souza, E.L. (2018). Chitosan produced from Mucorales fungi using agroindustrial by-products and its efficacy to inhibit Colletotrichum species. International Journal of Biological Macromolecules. 108: 635-641.

  3. Bhargava, B. and Tandon, N. (2015). Standard operating procedures for fungal identification and detection of antifungal resistance. Indian Council of Medical Research (ICMR): Karnataka, India. 7(9): 27-44.

  4. Duan, C., Meng, X., Meng, J., Khan, M.I.H., Dai, L., Khan, A. and Ni, Y. (2019). Chitosan as a preservative for fruits and vegetables: A review on chemistry and antimicrobial properties. Journal of Bioresources and Bioproducts. 4(1): 11-21.

  5. Dutta, J., Tripathi, S. and Dutta, P.K. (2012). Progress in antimicrobial activities of chitin, chitosan and its oligosaccharides: A systematic study needs for food applications. Food Science and Technology International. 18(1): 3-34.

  6. El-Sabbagh, S.M., Hussien, S.S., Mira, H.I., Desouky, O.A. and El- gohary, D.M. (2023). Enhancement study for production, extraction and characterization of fungal species chitosan. The Egyptian Journal Of Experimental Biology (Botany). 19(1): 101-101.

  7. El-Sayed, E.M., Ali, A.M. and Elshaghabee, F.M.F. (2019). Influence of addition of egyptian grape seed extract on the antioxidant,  antibacterial activities and shelf life of traditional labneh. Asian Journal of Dairy and Food Research. 38(4): 295-300. doi: 10.18805/ajdfr.DR-138.

  8. Fathi, M., Vinceković, M., Jurić, S., Viskić, M., Režek Jambrak, A. and Donsì, F. (2021). Food-grade colloidal systems for the delivery of essential oils. Food Reviews International. 37(1): 1-45.

  9. Francisco, C.B., Pellá, M.G., Silva, O.A., Raimundo, K.F., Caetano, J., Linde, G.A. and Dragunski, D. C. (2020). Shelf-life of guavas coated with biodegradable starch and cellulose-based films. International Journal of Biological Macromolecules. 152: 272-279.

  10. Ghasemnezhad, M., Shiri, M.A. and Sanavi, M. (2010). Effect of chitosan coatings on some quality indices of apricot (Prunus armeniaca L.) during cold storage. Caspian Journal of Environmental  Sciences. 8(1): 25-33.

  11. Gonzalez-Aguilar, G.A., Villa-Rodriguez, J.A., Ayala-Zavala, J.F. and Yahia, E.M. (2010). Improvement of the antioxidant status of tropical fruits as a secondary response to some postharvest treatments. Trends in Food Science and Technology.  21(10): 475-482.

  12. Hanani, Z.N., Soo, K.L., Zunairah, W.W. and Radhiah, S. (2023). Prolonging the shelf life of fresh-cut guava (Psidium guajaya L.) by coating with chitosan and cinnamon essential oil. Heliyon. 9(12).

  13. Hazarika, T.K., Lalhriatpuia, C., Ngurthankhumi, R., Lalruatsangi, E. and Lalhmachhuani, H. (2023). Edible coatings in extending the shelf life of fruits: A review. Indian Journal of Agricultural Research. 57(5): 555-558. doi: 10.18805/IJARe.A-5725.

  14. Hernández-Muñoz, P., Almenar, E., Ocio, M.J. and Gavara, R. (2006). Effect of calcium dips and chitosan coatings on postharvest life of strawberries (Fragaria × ananassa). Postharvest Biology and Technology. 39(3): 247-253.

  15. Hong, K., Xie, J., Zhang, L., Sun, D. and Gong, D. (2012). Effects of chitosan coating on postharvest life and quality of guava (Psidium guajava L.) fruit during cold storage. Scientia Horticulturae. 144: 172-178.

  16. Huq, T., Khan, A., Brown, D., Dhayagude, N., He, Z. and Ni, Y. (2022).  Sources, production and commercial applications of fungal chitosan: A review. Journal of Bioresources and Bioproducts. 7(2): 85-98.

  17. Jeyakumari, A., Murthy, L.N., Visnuvinayagam, S. and Laly, S.J. (2023). Quality and shelf life evaluation of chitosan- coated dried bombay duck (Harpodon nehereus). Asian Journal of Dairy and Food Research. 42(4): 511-516. doi: 10.18805/ajdfr.DR-2097.

  18. Kaur, N., Somasundram, C., Razali, Z., Mourad, A.H.I., Hamed, F. and Ahmed, Z.F. (2024). Aloe vera/chitosan-based edible film with enhanced antioxidant, antimicrobial, thermal and barrier properties for sustainable food preservation. Polymers. 16(2): 242.

  19. Khan, F.I.,  Akhtar, S., Qamar, M., Ismail, T., Saeed, W., Esatbeyoglu, T. and Jafari, S.M. (2025). A comprehensive review on guava: Nutritional profile, bioactive potential and health- promoting properties of its pulp, peel, seeds, pomace and leaves. Trends in Food Science and Technology. 156: 104822.

  20. Kouhi, M., Prabhakaran, M.P. and Ramakrishna, S. (2020). Edible polymers: An insight into its application in food, biomedicine and cosmetics. Trends in Food Science and Technology. 103: 248-263.

  21. Kumar, A. S., Malik, K. and Kumar, D. (2023). Optimization of cultural conditions for chitosan production from Aspergillus flavus strain. AF2118.

  22. Manal, G., Mahmoud, E.E.K. and Mohsen, S.A. (2019). Chitin, chitosan and glucan, properties and applications. World J. Agric. Soil Sci. 3: 1-19.

  23. Muxika, A., Etxabide, A., Uranga, J., Guerrero, P. and De La Caba, K. (2017). Chitosan as a bioactive polymer: Processing, properties and applications. International Journal of Biological Macromolecules. 105: 1358-1368.

  24. Nor, S.M. and Ding, P. (2020). Trends and advances in edible biopolymer coating for tropical fruit: A review. Food Research Inter- national. 134: 109208.

  25. Nwe, N., Furuike, T. and Tamura, H. (2010). Production of fungal chitosan by enzymatic method and applications in plant tissue culture and tissue engineering: 11 years of our progress, present situation and future prospects. Biopolymers.  7(135): e162.

  26. Pochanavanich, P. and Suntornsuk, W. (2002). Fungal chitosan production and its characterization. Letters in Applied Microbiology. 35(1): 17-21. https://doi.org/10.1046/j.1472- 765X. 2002.01118.x.

  27. Praveen, E., Murugan, S. and Jayakumar, K. (2017). Investigations on the existence of piezoelectric property of a bio- polymer–chitosan and its application in vibration sensors.  RSC advances. 7(56): 35490-35495.

  28. Rajaraman, V., Rajeshkumar, S., Nallawamy, D. and Ganapathy, D. (2020). Cytotoxic effect and antimicrobial activity of chitosan nanoparticles and hafnium metal based composite: Two sides of the same coin: An in vitro study. Journal of Pharmaceutical Research Internationa. 32(19): 122-131.

  29. Rehman, M.A., Asi, M.R., Hameed, A. and Bourquin, L.D. (2020). Effect of postharvest application of Aloe vera gel on shelf life, activities of anti-oxidative enzymes and quality of ‘Gola’guava fruit. Foods. 9(10): 1361.

  30. Sharma, V., Arthanari, A. and Ramasamy, P. (2024, April). Isolation and production of chitosan phosphorylation from marine squid pen and inhibit the growth of clinical pathogens. In Obstetrics and Gynaecology Forum.  34(2).

  31. Soares, N.D.F.F., Silva, D.F.P., Camilloto, G.P., Oliveira, C.P., Pinheiro, N.M. and Medeiros, E.A.A. (2011). Antimicrobial edible coating in post-harvest conservation of guava. Revista Brasileira de Fruticultura. 33: 281.

  32. Street, R. M., Huseynova, T., Xu, X., Chandrasekaran, P., Han, L., Shih, W.Y.,  and Schauer, C.L. (2018). Variable piezoelectricity of electrospun chitin. Carbohydrate Polymers. 195: 218-224.

  33. Wang, J., Yuan, Y., Liu, Y., Li, X. and Wu, S. (2024). Application of chitosan in fruit preservation: A review. Food Chemistry: X. 23: 101589.

  34. Wang, S.Y. and Gao, H. (2013). Effect of chitosan-based edible coating on antioxidants, antioxidant enzyme system and postharvest fruit quality of strawberries (Fragaria × aranassa Duch.). LWT-Food Science and Technology. 52(2): 71-79.

  35. Wani, S.M., Masoodi, F.A., Ahmad, M. and Mir, S.A. (2018). Processing and storage of apricots: Effect on physicochemical and antioxidant properties. Journal of Food Science and Technology. 55(11): 4505-4514.

  36. Zhang, L., Chen, F., Lai, S., Wang, H. and Yang, H. (2018). Impact of soybean protein isolate-chitosan edible coating on the softening of apricot fruit during storage. Lwt. 96: 604-611.
Published In
Asian Journal of Dairy and Food Research
Article Metrics
Metrics Icon
0
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Full Research Article

    Isolation and the Use of Fungal-derived Chitosan as a Substrate-Free Edible Coating to Extend the Postharvest Shelf Life of Guava

    R
    Rekha Anantharaman1,*
    1Centre for Global Health Research, Department of Obstetrics and Gyanecology, Saveetha Medical College and Hospital, Saveetha Institute of Medical and Technical Sciences, Chennai-600 001, Tamil Nadu, India.

    ABSTRACT

    Background: Guava (Psidium guajava) is a nutrient-rich tropical fruit with a very short postharvest shelf life due to its thin skin, high respiration rate and susceptibility to microbial spoilage. Chitosan is an edible film and a preservative, which can be developed from the fungal source and it benefiting the shelf life of perishable foods and vegetables.

    Methods: The fungal species were isolated and identified by standard methods. Acid and alkali treatments were used to extract the chitosan from the chosen fungal isolates. FTIR and XRD were used to characterize the isolated chitosan. The surface of the guava fruit was coated with the fungal-based chitosan (1% and 2%). Various physiological properties such as weight loss, ripening and shelf life were evaluated at regular intervals. The DPPH assay and the Agar well diffusion method were used to assess antibacterial and antioxidant properties.

    Result: Fungal isolates were selected, namely Fusarium oxysporum and Aspergillus fumigatus, based on morphological stability for the biomass yield for chitosan extraction. The yields of the chitosan for A. fumigatus (0.685 g/100 ml) and F. oxysorum (0.850 g/100 ml), respectively. Fruits that have been treated with 2% fungal-based chitosan exhibited a lower weight loss (3.2%), slow ripening and longer shelf life (9 days) and retained higher antioxidant activity (68.2%), than 1% and control group. 2% chitosan of A. fumigatus showed high antibacterial activity. The current study focusing on developing a fungal derived chitosan, which can be used as a suitable sustainable, non-toxic and safe alternative preservative to prolong the shelf life of guava.

    INTRODUCTION

    Guava (Psidium guajava) is a very nutritious fruit encountered in the tropics. It contains a large amounts of dietary fiber, protein, soluble carbohydrates, riboflavin, essential amino acids, vitamin C and antioxidants (Khan et al., 2025). It is easily prone to spoilage after harvest because of the cuts, growth of microbes, dehydration and enzyme degradation. The industry faces a lot of trouble mainly due to the thin delicate nature of skin in guavas, but even more so due to high demand in the market and the many benefits associated with them. Maintaining the long shelf life of guava remains a difficult aspect to the producers and retailers, particularly in regions where there is a low level of the development of the cold storage facilities (Nor et al., 2020). The conventional use of chemical preservatives as a way of extending shelf life through the incorporation of formaldehyde, BHA and BHT is also diminishing as people become more cognizant of the eventual risks to their health and safety (Hanani et al., 2023). One of the potential solutions to reducing post-harvest losses, preserving fruit quality and ensuring food safety is natural edible coating such as chitosan (Francisco et al., 2020). Edible coatings are an economically viable solution to protraction of food as well as sustainable health of customers (Kaur et al., 2024; Hazarika et al., 2023).
           
    Chitosan is a linearly structured polysaccharide composed of covalently bound D-glucosamine and N-acetyl- D-glucosamine and possesses remarkable characteristics to be biodegraded, biocompatible, non-toxic and able to form a film (Muxika et al., 2017; Rajaraman et al., 2020). This has made it suitable to be used in many areas such as preservation of foods, agriculture, pharmaceuticals and bio-medical applications (Kouhi et al., 2020). It is a highly valuable source in the food industry due to being a natural non-synthetic polymer capable of serving as an edible film, benefiting the shelf life of perishable foods and vegetables by sealing a semi-permeable structure to moisture, oxygen and pathogen penetration (Wang et al., 2024; Jeyakumari et al., 2023).
           
    The potential of fungal biomass as a substitute source has gained more attention for the production of chitin and chitosan by fungal mycelium. Fungal mycelia also have significantly lower than normal amounts of inorganic constituents than in traditional crustacean-based chitin, which removes the necessity of any demineralization processes to support fungal mycelia-derived products. Moreover, the occurrence of allergens in crustacean residues is a risk to vulnerable customers, which restricts its usage in foods and biomedical products (Huq et al., 2022). Classically, chitosan is produced with a two-phase procedure of treating the fungal cell walls with both acids and alkali (Manal et al., 2019). The other pathway through which chitosan is made by the fungi is through enzymatic deacetylation of chitin (Kumar et al., 2023).
           
    Some filamentous fungi, especially the genus of Aspergillus, Fusarium, Mucor and Rhizopus, have chitin and chitosan in cell walls (Pochanavanich and Suntornsuk, 2002). Chitosan derived fungi can be generated in the regulated fermentation environment with readily available and easily accessible substrates available throughout the year and with little impact on environment. In addition, the produced chitosan is allergen free as well as vegetarian with cheaper as well as less polluting extraction process. Because of these benefits, fungal chitosan is a promising option to be utilized in the pharmaceutical, food and biomedical sectors (El-Sabbagh et al., 2023).
           
    Dipping was one of the oldest methods of commercial coating and is commonly seen today as constant improvements are made to this in perpetuity way. This process is used to soak the fruits in a coat solution to ensure that they get the full surface coating. This technique is effective with solutions of a variety of viscosities in coating requirements (Fathi et al., 2021).
           
    The objective of the research is to investigate, explore and compare the usefulness of the fungal produced chitosan as naturally occurring biodegradable and edible coating on the guava fruit shelf life. Through analysis of the fungal basis resources in terms of aspects such as physicochemical parameters of chitosan, antimicrobial activity, a possibility of fungal derived chitosan as a precious source of antimicrobial activating compounds as a replace-ment of the standard sources used as food preservatives is assessed. In addition, the innovative aspect of the study is the choice of the fungal strains as very few studies have been done on extraction of chitosan over the two strains.

    MATERIALS AND METHODS

    Isolation and identification ientification of fungi
     
    Soil samples were collected and serially diluted to isolate fungal strains using a standard protocol. Identification was done using Lactophenol cotton blue (LPCB) stain. The characterization, antibacterial activity was done in Saveetha University, Thandalam, Chennai and the other works were performed at Valliammal College for women, Anna Nagar, Chennai. A research period of December 2019- January 2022 was established.
     
    Inoculation of fungal spores
           
    Aqueous preparation of fungal inoculum was prepared aseptically by directly pouring sterile distilled water on fully growing of the fungal mycelia that had been grown on Sabouraud Dextrose Agar (SDA) plates. Spores were scraped gently with a sterile inoculating wire and the suspension of spores was filtered into sterile flask. A hemocytometer was used to measure the concentration of the spores and they were diluted to 107 spores/ml. Thereafter, respective flasks were inoculated with 1 mL of fungal spore suspension. The cultures were placed under batch conditions and grown at 28oC over 5 days period and a complete growth of the fungal mycelium was observed (Bhargava and Tandon, 2015).
     
    Mass cultivation
     
    The culture of the selected strains of fungi was prepared in a synthetic microbial liquid growth medium, which included 20% potato extract, 2% dextrose, 1% peptone, 0.1% NaCl, 0.01% CaCl2 and 0.05% of MgSO4 7. H2O. To achieve good growth of the mycelium, the cultures were incubated at 28oC centrifuge at 120 rpm for 10 to 15 days. Produced fungal biomass was collected through filtration and then washed excessively using distilled water in the process of eliminating medium constituents; followed by air-drying at the room temperature.

     
    Chitosan extraction and purification
     
    Fungal mycelia were collected at the final stage of the cultivation phase using Whatman No. 1 filter paper. The harvested biomass was washed twice with distilled water and dried at 65oC until a constant weight was achieved. Chitosan extraction from the fungal biomass involved a series of chemical treatments, starting with deproteinization and deacetylation using an alkaline solution, followed by solubilization in dilute acetic acid (Nwe et al., 2010). Initially, the biomass was treated with 1 M sodium hydroxide (NaOH) at a ratio of 1:40 (w/v) and heated at 90oC for 3 hours. This process yielded a solid residue known as alkali-insoluble material (AIM). The AIM was separated by centrifugation at 6000 rpm for 15 minutes, thoroughly washed with distilled water and centrifuged repeatedly until a neutral pH was reached.
           
    For chitosan extraction, the neutralized AIM was treated with 10% (v/v) acetic acid (1:40 w/v) at room temperature for 6 hours on a rotary shaker operating at 200 rpm. The resulting acid-insoluble residue was removed by vacuum filtration. Chitosan remained in the filtrate and the pH was adjusted to 9.0 using 4 M NaOH, resulting in chitosan precipitation.The precipitate was recovered by centrifugation, washed with distilled water and dried at 60oC until a constant weight was obtained (Abdel-Gawad et al., 2017). Throughout the studies, the chitosan extraction using Fusarium oxysporum indicates the Sampe 1 or S1 whereas Sample 2 or S2 denoted for Aspergillus fumigatus.
     
    Characterization of chitosan
     
    Fourier transform infrared spectroscopy (FTIR)
     
    Using Shimadzu IR Affinity-1S Spectrometer, FTIR analysis was done for chitosan, to define group of absorptions based on functional groups. Dried chitosan was were taken in thin cut samples, crushed in a potassium bromide (KBr) and then formed  into a disk and scanned in wavenumbers 4000-400cm-1.
     
    XRD
     
    The Powder X-ray Diffraction (PXRD) analysis using D8 Advance Bruker (Germany) diffractometer and Cu Kα (l=1.54059) as the source of radiation.
     
    Chitosan coating on guava
     
    Treatment and storage condition
     
    The coating solutions was prepared with 2.5 g and 5 g of chitosan powder dissolved in 900 ml of distilled water with addition of 2% of citric acid was added to facilitate the chitosan powder to dissolve and also as a plasticizer. Each one of the solutions was adjusted to pH 5.5 using 0.1M NaOH. The treatment comprised of 0.25%, 0.5% and control (an acid solution that defied the presence of chitosan). The prepared emulsion of coating solution was dipped in the form of a coating over the guava fruits and the other was untreated and kept as control. All fruits were labeled using the sample number and other information. A comparative experiment on the treated and untreated guavas was carried out to test it.
     
    Observation of shelf life
     
    After the post harvests treatment, guava fruits were stored in controlled environments and their shelf life was being studied at every time interval. Different quality variables were measured periodically including the color, weight loss and symptoms of spoilage of the treated.
     
    Various parameters of the guava before and after treatment
     
    Weight loss
     
    To calculate the weight loss, the weight of the guava samples on Day 0 and the weight measured on Days 3, 6, 9 and 12 after coating were exerted. The weight of the fruit was measured using a laboratory weighing balance. The percentage of weight loss was calculated by the following equation:

     
    Where,
    W1 = Initial weight of the guava.
    W2 = Weight at each time point following coating.
     
    Antioxidant activity
     
    According to the protocol of Wani et al., (2018), the DPPH radical scavenging assay and a mixture of 3.5 mL DPPH solution determined oxidative strength and 0.5 mL guava fruit extract was added. The percentage of antioxidant activity was measured at an absorbance of 615 nm using UV-Visible Spectrophotometer. The inhibition percentage of antioxidant activity was expressed as follows:

     
    Where,
    AC=Absorbance control
    AS=Absorbance sample
     
    Determination of antibacterial activity
     
    The prepared fungal-based chitosan solution was used to check the antibacterial activity against Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli using Agar well diffusion method.

    RESULTS AND DISCUSSION

    Isolation of fungi
     
    On SDA plate, a diverse growth of fungi was observed. The plate showed a range from light green to dark green, brown and white colored colonies with a cottony, powdery hyphae were shown in Fig 1 and 2.

    Fig 1: Fungal colonies on SDA.



    Fig 2: Fungal colonies on SDA.


     
    Identification of fungal species
     
    The colonies were analyzed morphologically using Lactophenol Cotton Blue (LPCB) staining and observed under high power of objective microscope (40x). Identification of the fungal species was tabulated (Table 1). Among four morphologically distinct identified fungal strains, two strains of fungi were shown in Fig 3 and 4 Fusarium oxysporum and Aspergillus fumigatus that were picked and used for the further experiment.

    Table 1: Identification of isolated fungal species.



    Fig 3: Fusarium oxysporum from sample 1.



    Fig 4: Aspergillus fumigatus from sample 2.



    Biomass production
     
    A thick fungal mat was observed on the surface of the liquid medium after 10-15 days of incubation. The resultant mycelium biomass was collected, air-dried and decanted in order to extract chitosan in the next stage shown in Fig 5.

    Fig 5: Mass cultivation of fungal spores.


                        
    Extraction of chitosan
     
    The fungal liquid biomass was used to obtain chitosan through a renowned set of chemical reactions that successively isolated the chitosan as well as purifying it. The extraction process was evidenced by the uniformity of the finished product, which was shown in Fig 6a-6e. The final yield of chitosan from Fusarium oxisoporum (S1) was 0.685 g/100 ml whereas 0.850 g/100 ml from Aspergillus fumigatus (S2). The fungal cell walls were treated with acids and alkalis in a two-step process. Proteins and soluble polysaccharides were removed successfully after being treated with NaOH and deposition of an alkali-insoluble material (AIM) as solid sediment was the evident. During the treatment, a combination of chitin and glucan dissociates and deacetylation step produced the chitosan.

    Fig 6: Extraction of chitosan.



    FTIR
     
    The existence of distinctive functional groups in the chitosan isolated from Fusarium oxysporum was verified by FTIR was presented in Fig 7a. Extensive hydrogen bonding is indicated by a large absorption band seen in the 3368–3683 cm-1 range, which is correlated with the O-H stretching vibrations of hydroxyl groups and the N-H stretching of amino groups. Asymmetric and symmetric C-H stretching vibrations of -CH‚  groups are responsible for the peaks at 2922.16 cm-1 and 2852.72 cm-1. Overtones or lingering contaminants could be the cause of a little band at 2374.37 cm-1 and a slight peak at 2725.42 cm-1 (Sharma et al., 2024). Partial deacetylation is indicated by a band at 1375.25 cm-1 that corresponds to the C-N stretching vibrations of amino groups. The peak at 1155.36 cm-1 is linked to the chitosan backbone’s asymmetric stretching of the C-O-C bridge (Berger et al., 2018). These spectrum characteristics verify that chitosan with structural properties comparable to those of typical fungal-derived chitosan was successfully extracted.

    Fig 7a: FTIR analysis of chitosan extracted from Fusarium oxysporum.


           
    The FTIR spectra of the chitosan extracted by Aspergillus fumigatus have characteristic functional groups that prove its molecular composition was shown in Fig 7b. The presence of there is quantum of hydroxyl and amine due to the presence of large sum of absorption seen at 3446.79 cm-1, which is O-H and N-H stretching vibrations. The vibrations of the -CH2 groups responsible are the C-H asymmetric and the C-H symmetric stretching and the corresponding peaks are 2922.16 cm-1 and 2852.72 cm-1. The formation of inter- and intramolecular hydrogen bonds enhances the structural stability of pure chitosan (Praveen et al., 2017). The highest peak at 1554.63 cm-1 can be attributed to N-H bending, yet strong and evident peak at 1656.85 cm-1 can be connected with amide (C=O stretching of residual N-acetyl groups. The contribution of CH2 bending is assigned at 1458.18 cm-1. The C-N stretching is found to be located at a peak 1377.17 cm-1 (Street et al., 2018). These spectrum properties are evidence that the chitosan extraction is effective since it is based on the fungal source.

    Fig 7b: FTIR analysis of chitosan extracted from Aspergillus fumigatus.



    XRD
     
    Chitosan produced by fungal strains Fusarium oxysporum and Aspergillus fumigatus exhibited certain characteristic peak angles of the diffraction obtained on the XRD plots displayed in Fig 8a, b around 2θ = 9o-10o and 19-20o demonstrating the semi-crystalline nature of chitosan. Chitosan extracted from F. oxysporum exhibited sharper and more defined peaks at 9.15o and 19.60o denoting a better degree of crystallinity. It implies that the molecular chains of this sample might be more regular and dense, which can increase its mechanical properties, thermal stability, barrier properties. Differently, chitosan obtained in A. fumigatus showed wider and weaker peaks at 9.25o and 19.55o, indicating a more amorphous structure, with low crystallinity. These distinctions can be affected by a number of factors, which include the inherent metabolic and enzymatic characteristics of fungus used as the production organism, differences in cell wall composition, change in extraction procedures like deproteinization, deacetylase activity and drying practices.

    Fig 8: X-ray diffraction of chitosan extracted from fungal strain.


     
    Preparation of chitosan coating solution
     
    In order to measure their stability and solubility, different combinations of chitosan coating solutions were successfully prepared and displayed in Fig 9. The chitosan (S1) turned turbid and clearly there is sedimentation implying that it was not totally dispersed. Better clarity and homogeneity were noted with 1% chitosan and 2% citric acid (S1 with CA) solution showing better solubilization of chitosan in acidic solution. As an acidic environment control, the sample appeared to be translucent on the 2% citric acid alone sample leading to the observation that it lacks particle matter.

    Fig 9: Preparation of chitosan coating solutions.


     
    Postharvest quality of guava with chitosan coating
     
    A noticeable increase in the colour change of guava fruits was observed from the third day of storage. However, by the ninth day, fruits coated with 2% chitosan (Sample 1: Fusarium oxysporum) showed significantly less colour development compared to the untreated control group, indicating a delay in the ripening process. Among all treatments, the formulation combining 1% chitosan with 2% citric acid (Sample 1) was the most effective in preserving the visual quality of the guava during storage. The postharvest application of chitosan notably extended the shelf life of the fruits was shown in Fig 10.

    Fig 10: Effect of chitosan on postharvest quality of Guava.


           
    In addition to delaying ripening, chitosan-treated fruits showed better retention of firmness, reduced weight loss and lower microbial decay throughout the storage period. The semipermeable nature of chitosan film likely created a modified internal atmosphere, slowing down respiration and senescence (Soares et al., 2011). Overall, chitosan coating effectively preserved the postharvest quality of guava, making it a promising treatment for extending marketable shelf life (Dutta et al., 2012).
     
    Weight loss
     
    Using the initial weight, the % weight loss of coated and uncoated fruits over a 12-day storage period was calculated. At the end of day 12, coated fruits showed a progressive decrease in weight, ranging from 3.2%, whereas uncoated fruits, on the other hand, demonstrated a noticeably greater reduction in weight, ranging from 0.98% at day 12. The guava weight loss results are presented in Fig 11 showing that there was a significant (p<0.05) increase in the percent weight loss. Fresh fruit loses weight through respiration and transpiration. The key reasons leading to a decrease in weight include respiration and loss of moisture between the fruits internal and environmental external conditions of air (Hernández-Muñoz, 2006; Zhang et al., 2018). Chitosan coatings delayed the process of dehydration by avoiding the loss of water and also avoided mechanical damages and overcome the cuts that occur by default on the fruits skin (Hong et al., 2012).

    Fig 11: Changes in weight after post harvesting of guava.


     
    Antibacterial activity
     
    The antibacterial activity of chitosan formulations was evaluated against Klebsiella pneumoniae, Escherichia coli and Staphylococcus aureus, with inhibition zones compared to a standard antibiotic (Table 2) and Fig 12. When comparing, 2% chitosan showed a highest zone of inhibition than 1% chitosan sample. Among three different bacterial strains, Escherchia coli growth was inhibited higher than Klebsiella and Staphylococcus. Usually, the mechanism of the antimicrobial is based on electrostatic interactions with the cytosolic contents, cell wall and outer membrane of the bacteria (Duan et al., 2019). Antibacterial activity of chitosan is widely demonstrated to be due to its binding to the negatively charged cell wall of bacteria thus rupturing the cell wall and altering the porousness of the membrane.

    Fig 12: Antibacterial Activity of chitosan solution against.



    Table 2: Antibacterial efficacy of chitosan solutions.



    Antioxidant activity
     
    The antioxidant activity in the control (Uncoated) samples declined significantly from an initial 87% to 20%, whereas in the coated samples, it decreased only to 87 to 68.2%. The antioxidant activity in terms of DPPH showed highest with higher concentration of chitosan Table (3). The observed reduction in antioxidant capacity in the control group is most likely determined by a generation of free radicals because of the degradation of phenolic compounds due to senescence and elevated level of respiration (Ghasemnezhad et al., 2010; Rehman et al., 2020). The edible coatings affect the internal environment of fruits by slowing down metabolic reactions by a large margin leading to a significant decrease in the production of flavonoids and phenolic compounds (Gonzalez-Aguilar et al., 2010; El-Sayed et al., 2019). Conversely, the guava that coated with chitosan maintained higher antioxidant activity than the uncoated guavas. This increase is possible due to the ability of the coating to change the internal atmosphere of the fruit to reduce the oxidative stress level and slow down the degradation of the antioxidant compounds. Wang and Gao (2013) obtained a similar result demonstrating that strawberries undergoing coating of chitosan maintained a more acceptable total phenolic content and antioxidant activity when stored.

    Table 3: Percentage of DPPH inhibition.

    CONCLUSION

    This study shows the potential of fungal-based chitosan, Fusarium oxysporum and Aspergillus fumigatus, to provide effective, environmental friendly and non toxic edible wrapping to extend the post-harvest life of guava (Psidium guajava). The functional groups of the extracted fungal chitosan were successfully characterized using FTIR. The fungal-derived chitosan showed a strongest antibacterial against both Gram positive and Gram negative bacteria. The effectiveness of antioxidant capacity of guava fruits coated with fungal extracted chitosan was clearly depicted in the DPPH antioxidant test where the antioxidant capacity of guava fruits coated with chitosan was able to withstand the antioxidant capacity throughout the storage period than the un-coated control fruits. The coating successfully retained phenolic compounds and other bioactive antioxidants and led to the extension of fruit quality and shelf life. These results show that fungal chitosan can be considered a promising candidate of natural edible coating to keep postharvest fruits in high nutritional and functional quality. Unlike the rapid decline recorded in controls, the shelf life of the fungal chitosan coated guava significantly delayed the ripening process, reduced weight loss and preserved antioxidant activity. The chitosan treated guava fruit samples maintained the quality for upto nine days in ambient conditions. The chitosan exhibited strong antibacterial activity against pathogenic strains commonly found in food such as Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli at the 2% concentration.
           
    These results support the application of fungal chitosan as alternative to the utilization of conventional synthetic preservative compounds since the results satisfy consumer preference to clean-label and non-allergens food preservatives. Fungal chitosan should be considered as a best option to be employed in postharvest handling processes because it can have a combination of both antibacterial and physical barrier actions, particularly in regions where there is no advanced cold-chain structure. Further business analysis and scale-up studies are needed to inquire into its broader use in the preservation of other perishable fruits and vegetables.

    CONFLICT OF INTEREST

    All authors declare that they have no conflict of interest.

    REFERENCES


    1. Abdel-Gawad, K.M., Hifney, A.F., Fawzy, M.A. and Gomaa, M. (2017). Technology optimization of chitosan production from Aspergillus niger biomass and its functional activities. Food Hydrocolloids. 63: 593-601.

    2. Berger, L.R.R., Stamford, T.C.M., de Oliveira, K.Á.R., Pessoa, A.D.M.P., de Lima, M.A.B., Pintado, M.M.E. and de Souza, E.L. (2018). Chitosan produced from Mucorales fungi using agroindustrial by-products and its efficacy to inhibit Colletotrichum species. International Journal of Biological Macromolecules. 108: 635-641.

    3. Bhargava, B. and Tandon, N. (2015). Standard operating procedures for fungal identification and detection of antifungal resistance. Indian Council of Medical Research (ICMR): Karnataka, India. 7(9): 27-44.

    4. Duan, C., Meng, X., Meng, J., Khan, M.I.H., Dai, L., Khan, A. and Ni, Y. (2019). Chitosan as a preservative for fruits and vegetables: A review on chemistry and antimicrobial properties. Journal of Bioresources and Bioproducts. 4(1): 11-21.

    5. Dutta, J., Tripathi, S. and Dutta, P.K. (2012). Progress in antimicrobial activities of chitin, chitosan and its oligosaccharides: A systematic study needs for food applications. Food Science and Technology International. 18(1): 3-34.

    6. El-Sabbagh, S.M., Hussien, S.S., Mira, H.I., Desouky, O.A. and El- gohary, D.M. (2023). Enhancement study for production, extraction and characterization of fungal species chitosan. The Egyptian Journal Of Experimental Biology (Botany). 19(1): 101-101.

    7. El-Sayed, E.M., Ali, A.M. and Elshaghabee, F.M.F. (2019). Influence of addition of egyptian grape seed extract on the antioxidant,  antibacterial activities and shelf life of traditional labneh. Asian Journal of Dairy and Food Research. 38(4): 295-300. doi: 10.18805/ajdfr.DR-138.

    8. Fathi, M., Vinceković, M., Jurić, S., Viskić, M., Režek Jambrak, A. and Donsì, F. (2021). Food-grade colloidal systems for the delivery of essential oils. Food Reviews International. 37(1): 1-45.

    9. Francisco, C.B., Pellá, M.G., Silva, O.A., Raimundo, K.F., Caetano, J., Linde, G.A. and Dragunski, D. C. (2020). Shelf-life of guavas coated with biodegradable starch and cellulose-based films. International Journal of Biological Macromolecules. 152: 272-279.

    10. Ghasemnezhad, M., Shiri, M.A. and Sanavi, M. (2010). Effect of chitosan coatings on some quality indices of apricot (Prunus armeniaca L.) during cold storage. Caspian Journal of Environmental  Sciences. 8(1): 25-33.

    11. Gonzalez-Aguilar, G.A., Villa-Rodriguez, J.A., Ayala-Zavala, J.F. and Yahia, E.M. (2010). Improvement of the antioxidant status of tropical fruits as a secondary response to some postharvest treatments. Trends in Food Science and Technology.  21(10): 475-482.

    12. Hanani, Z.N., Soo, K.L., Zunairah, W.W. and Radhiah, S. (2023). Prolonging the shelf life of fresh-cut guava (Psidium guajaya L.) by coating with chitosan and cinnamon essential oil. Heliyon. 9(12).

    13. Hazarika, T.K., Lalhriatpuia, C., Ngurthankhumi, R., Lalruatsangi, E. and Lalhmachhuani, H. (2023). Edible coatings in extending the shelf life of fruits: A review. Indian Journal of Agricultural Research. 57(5): 555-558. doi: 10.18805/IJARe.A-5725.

    14. Hernández-Muñoz, P., Almenar, E., Ocio, M.J. and Gavara, R. (2006). Effect of calcium dips and chitosan coatings on postharvest life of strawberries (Fragaria × ananassa). Postharvest Biology and Technology. 39(3): 247-253.

    15. Hong, K., Xie, J., Zhang, L., Sun, D. and Gong, D. (2012). Effects of chitosan coating on postharvest life and quality of guava (Psidium guajava L.) fruit during cold storage. Scientia Horticulturae. 144: 172-178.

    16. Huq, T., Khan, A., Brown, D., Dhayagude, N., He, Z. and Ni, Y. (2022).  Sources, production and commercial applications of fungal chitosan: A review. Journal of Bioresources and Bioproducts. 7(2): 85-98.

    17. Jeyakumari, A., Murthy, L.N., Visnuvinayagam, S. and Laly, S.J. (2023). Quality and shelf life evaluation of chitosan- coated dried bombay duck (Harpodon nehereus). Asian Journal of Dairy and Food Research. 42(4): 511-516. doi: 10.18805/ajdfr.DR-2097.

    18. Kaur, N., Somasundram, C., Razali, Z., Mourad, A.H.I., Hamed, F. and Ahmed, Z.F. (2024). Aloe vera/chitosan-based edible film with enhanced antioxidant, antimicrobial, thermal and barrier properties for sustainable food preservation. Polymers. 16(2): 242.

    19. Khan, F.I.,  Akhtar, S., Qamar, M., Ismail, T., Saeed, W., Esatbeyoglu, T. and Jafari, S.M. (2025). A comprehensive review on guava: Nutritional profile, bioactive potential and health- promoting properties of its pulp, peel, seeds, pomace and leaves. Trends in Food Science and Technology. 156: 104822.

    20. Kouhi, M., Prabhakaran, M.P. and Ramakrishna, S. (2020). Edible polymers: An insight into its application in food, biomedicine and cosmetics. Trends in Food Science and Technology. 103: 248-263.

    21. Kumar, A. S., Malik, K. and Kumar, D. (2023). Optimization of cultural conditions for chitosan production from Aspergillus flavus strain. AF2118.

    22. Manal, G., Mahmoud, E.E.K. and Mohsen, S.A. (2019). Chitin, chitosan and glucan, properties and applications. World J. Agric. Soil Sci. 3: 1-19.

    23. Muxika, A., Etxabide, A., Uranga, J., Guerrero, P. and De La Caba, K. (2017). Chitosan as a bioactive polymer: Processing, properties and applications. International Journal of Biological Macromolecules. 105: 1358-1368.

    24. Nor, S.M. and Ding, P. (2020). Trends and advances in edible biopolymer coating for tropical fruit: A review. Food Research Inter- national. 134: 109208.

    25. Nwe, N., Furuike, T. and Tamura, H. (2010). Production of fungal chitosan by enzymatic method and applications in plant tissue culture and tissue engineering: 11 years of our progress, present situation and future prospects. Biopolymers.  7(135): e162.

    26. Pochanavanich, P. and Suntornsuk, W. (2002). Fungal chitosan production and its characterization. Letters in Applied Microbiology. 35(1): 17-21. https://doi.org/10.1046/j.1472- 765X. 2002.01118.x.

    27. Praveen, E., Murugan, S. and Jayakumar, K. (2017). Investigations on the existence of piezoelectric property of a bio- polymer–chitosan and its application in vibration sensors.  RSC advances. 7(56): 35490-35495.

    28. Rajaraman, V., Rajeshkumar, S., Nallawamy, D. and Ganapathy, D. (2020). Cytotoxic effect and antimicrobial activity of chitosan nanoparticles and hafnium metal based composite: Two sides of the same coin: An in vitro study. Journal of Pharmaceutical Research Internationa. 32(19): 122-131.

    29. Rehman, M.A., Asi, M.R., Hameed, A. and Bourquin, L.D. (2020). Effect of postharvest application of Aloe vera gel on shelf life, activities of anti-oxidative enzymes and quality of ‘Gola’guava fruit. Foods. 9(10): 1361.

    30. Sharma, V., Arthanari, A. and Ramasamy, P. (2024, April). Isolation and production of chitosan phosphorylation from marine squid pen and inhibit the growth of clinical pathogens. In Obstetrics and Gynaecology Forum.  34(2).

    31. Soares, N.D.F.F., Silva, D.F.P., Camilloto, G.P., Oliveira, C.P., Pinheiro, N.M. and Medeiros, E.A.A. (2011). Antimicrobial edible coating in post-harvest conservation of guava. Revista Brasileira de Fruticultura. 33: 281.

    32. Street, R. M., Huseynova, T., Xu, X., Chandrasekaran, P., Han, L., Shih, W.Y.,  and Schauer, C.L. (2018). Variable piezoelectricity of electrospun chitin. Carbohydrate Polymers. 195: 218-224.

    33. Wang, J., Yuan, Y., Liu, Y., Li, X. and Wu, S. (2024). Application of chitosan in fruit preservation: A review. Food Chemistry: X. 23: 101589.

    34. Wang, S.Y. and Gao, H. (2013). Effect of chitosan-based edible coating on antioxidants, antioxidant enzyme system and postharvest fruit quality of strawberries (Fragaria × aranassa Duch.). LWT-Food Science and Technology. 52(2): 71-79.

    35. Wani, S.M., Masoodi, F.A., Ahmad, M. and Mir, S.A. (2018). Processing and storage of apricots: Effect on physicochemical and antioxidant properties. Journal of Food Science and Technology. 55(11): 4505-4514.

    36. Zhang, L., Chen, F., Lai, S., Wang, H. and Yang, H. (2018). Impact of soybean protein isolate-chitosan edible coating on the softening of apricot fruit during storage. Lwt. 96: 604-611.
    In this Article
      Contact us
      Follow us
      Published In
      Asian Journal of Dairy and Food Research

      Editorial Board

      View all (0)