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Full Research Article
Evaluate the Capability of Some Local Fungal Isolates to Produce Pectinase Enzyme using Some Fruit Peels
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First Online 02-02-2023|
Methods: The current study included the isolation and identification of some fungi from fruits and vegetables in the local markets of Al-Diwaniyah City/Iraq, the detection of their capability to produce pectinase enzyme using the standard medium and the possibility of using the peels of some fruits as basic materials.
Result: The results indicated the isolation and identification of 178 fungal isolates belonging to 11 genera at different frequency rates and it was found that the isolate P. expansum isolated from Apples was the most efficient isolate in producing pectinase enzyme and the use of Apple peels gave the highest production of the enzyme from the fungal isolate understudy compared to other fruit peels used.
MATERIALS AND METHODS
A group of fruits and vegetables (Apples, Oranges, Strawberries, Cucumbers, Potatoes, Tomatoes and Onions) were used to isolate the fungi in the local markets of Al-Diwaniyah City/Iraq, which showed symptoms of fungal infection in August 2022. The samples were brought to the laboratory, washed with distilled water and 1 cm2 from the fungus-infected area superficially sterilized for three minutes with 1% Sodium hypochlorite solution, then washed with sterile distilled water and dried between two sterile filter papers, then cultured in Petri dishes containing sterile (PDA) medium with the antibiotic streptomycin at a concentration of 50 mg/liter of three pieces for each type of fruits and vegetables, the dishes were incubated in the incubator for 7 days at 28°C, with three replications for each treatment (Saleh and Al-Thani, 2019).
Identification of fungi
After the growth of fungi on the PDA medium, small parts were taken using a culture needle from the edge of each fungal colony and were planted individually on Petri dishes containing a sterile PDA medium to obtain pure cultures of the fungi and their identification and the dishes were incubated in the incubator for 3-4 days at 28°C and after the appearance of growth was diagnosed morphologically and microscopically based on the taxonomic keys of fungi (Robinson, 2011; Watanabe, 2018). Then the diagnosed and isolated fungi were stored purely in test tubes containing sterile tilted PDA medium in the fridge at 4°C until use, the percentage of fungi frequency was then calculated using the following equation:
Preparation of suspended spores
The spores of the isolated and most frequent fungi that were diagnosed in the previous step were prepared by preparing the sterile PDA culture medium in sterile Petri dishes and then inoculating by streak plate method from the pure isolates under study. After 3 days of incubation at 30°C, 5 mL of sterile distilled water containing 1% Tween 80 was added to the dishes under sterile conditions and the spores were washed and transferred to sterile tubes, which were stored in the fridge at 4°C until use.
Pectinase enzyme production test from isolated fungi using standard medium
To test the capability of the isolated and most frequent fungi to produce pectinase enzyme, a modified Czapek-Dox agar medium was used, replacing the carbon source with citrus pectin, after sterilizing the medium in an autoclave for 20 minutes at 121°C, it was poured into sterile Petri dishes and allowed to cool before drilling holes of equal diameters in each dish, three duplicates of each isolate were inoculated with 1 ml of suspended spores at a concentration of 106 spores/ml in each hole, then for 5 days, the dishes were incubated at 30°C and the clear zones formed were determined using a regular ruler (average of two perpendicular diameters) by adding Lugol’s Iodine solution, as the appearance of a clear zones halo around the fungal colony indicates the production of the enzyme and the greater the diameter of the halo indicates an increase in the activity of the isolate in the production of pectinase enzyme (Aboaba, 2009). Then the isolate with the highest enzyme production was determined to carry out other experiments.
Pectinase production test using peels of some fruits
After determining the most productive isolate of the pectinase enzyme on the standard medium, which is Penicillium expansum (Apple), it is the capability to produce the enzyme was tested using the peels of some fruits. Rangarajan et al. (2010) methods were used to prepare the peels of fruits (Apples, Bananas, Oranges and Tangerines) a sufficient amount of peels were collected and cut into roughly equal sizes before drying in an oven at 55°C until the weight was stabilized, then the samples were ground with an electric mill and stored in glass containers away from moisture. The ground peels were added to the pectinase production medium, which is the modified Czapek-Dox agar medium by replacing the carbon source with Apple peels, the medium was then sterilized and poured into sterilized Petri dishes, which were then allowed to cool before the holes of equal diameters were drilled in each dish, each hole in the dishes was inoculated with 1 ml of suspended spores at a concentration of 106 spores/ml for the fungal isolate under study and with three replicates, then for 5 days the dishes were incubated at 30°C, the same experiment was repeated by replacing the Apple peels with peels (banana, orange and tangerine) separately. The clear zones formed were determined using Lugol’s Iodine Solution, as the appearance of a clear zone halo around the fungal colony was evidence of the enzyme production, determining the peels of fruits that gave the highest production of the enzyme to carry out other experiments.
Assay for pectinase enzyme production using solid-state and submerged fermentation methods
Apple peels were used, as they produced the most enzymes in the previous experiment using the fungal isolate P. expansum (Apple). The method of Martin et al., 2004 was used to produce the enzyme by solid-state fermentation (SSF) method, with a weight of 10 g of pre-prepared Apple peels, placed in 250 ml glass flasks and sterilized with an autoclave for 40 minutes at 121°C, then, 10 ml of the mineral nutrient solution was added to the peel solution and in sterile conditions, containing (0.1% NH4NO3, 0.1% NH4H2PO4 and 0.1% Mg SO4.7H2O), the medium was sterilized in an autoclave for 15 minutes at 121°C before being inoculated with 10 ml of spore suspension at a concentration of 106 spores/ml in three replicates, then the flasks were incubated at 30°C in a shaker incubator at 200 rpm for 5 days. The enzyme was also produced by submerged fermentation (SmF) method by preparing Apple peel extract after weighing 100 gm of Apple peels in flasks and adding 1600 ml of distilled water, placing it on a hot plate at 100°C for 3 hours and keeping the extract in the fridge at 4°C until use, 100 ml of the extract was taken and 10 ml of the mineral nutrient solution mentioned above was added to it under sterile conditions, then the medium was inoculated with 10 ml of suspended spores at a concentration of 106 spores/ml and in three replications. The flasks were then shaken for 5 days at 30°C at 200 rpm in a shaker incubator.
Crude pectinase enzyme extraction
Crude pectinase enzyme was extracted from solid cultures by adding 100 ml of buffer solution sodium acetate with pH 5.5 and a concentration of 0.05 M to the components of the fermented flasks. The medium was mixed well, then a filtration process was carried out under a vacuum and the centrifugation process was then carried out at 10,000 rpm for 20 minutes, with the filtrate representing the crude enzyme being collected. In the submerged cultures, the centrifugation process was conducted directly to the liquid culture and the filtrate was collected and the enzymatic activity of all samples was estimated (Garg et al., 2016).
Estimation of pectinase activity
The effectiveness of pectinase was estimated using citrus pectin as a base material, as the mixture contained equal amounts of 1% pectin solution prepared in a buffer solution of sodium acetate with pH 5.5, the concentration of 0.05 M and a suitable dilution of the crude enzyme, after 30 minutes in a water bath at 50°C, 1 ml of 3,5-Dinitrosalicylic acid (DNS) was added and the mixture was heated for 10 minutes before being cooled, the optical absorption at a wavelength of 540 nm was determined using a Spectrophotometer, the efficacy was estimated based on the standard curve of pre-prepared galacturonic acid and was found the unit of activity, which is the amount of enzyme required in experimental conditions to release 1 micromole of galacturonic acid per minute (Sengupta et al., 2000).
Significant differences between the means were determined using Duncan’s polynomial test at a probability level of 5% using an analysis of variance (ANOVA) (Armitage et al., 2008).
RESULTS AND DISCUSSION
The results in Table (1) indicated the isolation and identification of 178 fungal isolates from some different fruits and vegetables belonging to 11 fungal genera and these genera are: (Alternaria sp., Aspergillus sp., Fusarium sp., Penicillium sp., Rhizoctonia sp., Cladosporium sp., Botrytis sp., Stemphylium sp., Cephalosporium sp., Rhizopus sp. and Mucor sp.) and the results indicated that the most frequent fungi, according to the isolated source, were P. expansum and Al. alternata isolated from Apples with a frequency of 34.78% and 21.73%, respectively, P. italicum and P. digitatum isolated from oranges with 30.76% and 26.92%, respectively, Al. alternata and Botrytis cinerea isolated from Strawberries with 36.0% and 24.0%, respectively, Al. alternata and F. solani isolated from Cucumbers with 30.30% and 21.21%, respectively, A. niger and F. oxysporum isolated from Potatoes with 30.0% and 25.0%, respectively, Al. alternata and F. solani isolated from Tomatoes at 34.61% and 19.23%, respectively, then A. niger and P. expansum isolated from Onions at 44.0% and 24.0%, respectively. These findings support prior studies that show it is possible to isolate various fungi from fruits and vegetables after harvesting, as A. niger and F. oxysporum were isolated from Apples (Oelofse et al., 2006; Chatanta et al., 2008). As well as A. niger from Tomatoes and Oranges (Yildiz and Baysal, 2006; Reddy et al., 2008). Fusarium sp. is a moisture-loving fungus found on fruits high in moisture content such as Oranges (Tournas et al., 2005). As well as Alternaria sp., Penicillium sp. and Rhizopus sp (Bukar et al., 2009). Alternaria sp. and Rhizopus sp. from cucumber (Hoque and Shamsi, 2011). The high moisture content of fruits and vegetables and the different transportation and storage conditions that may increase the humidity and temperature of fruits and vegetables are among the most important factors that encourage the growth of fungi on them, as well as external environmental conditions such as insect intrusion and wounds that facilitate the entry and spread of fungi as well as the presence of foreign materials such as sand and dust and the remnants of other damaged fruits and vegetables among them (Rawat, 2015).
Pectinase enzyme production test from isolated fungi using standard medium
The results indicated that the fungal isolates of Penicillium spp were one of the most efficient isolates in producing pectinase enzyme using the standard medium, the isolate of P. expansum isolated from Apples was the most efficient isolate in producing the enzyme, reaching a clear zones diameter of 5.9 cm, followed by isolating P. expansum isolated from Onions with 4.7 cm, then the two isolates P. italicum and P. digitatum with 4.2 cm and 4.0 cm, respectively, with significant differences compared to the other fungal isolates under study (Table 2). These findings are consistent with previous studies that found that the species of Penicillium spp. it can produce pectinase enzymes, including P. italicum, P. viridicatum, P. roqueforti, P. expansum and P. griseoroseum (Alaña et al., 1990; Silva et al., 2002; Perièin et al., 2007; Cardoso et al., 2007). Therefore, the fungal isolate P. expansum isolated from Apples was chosen to carry out other experiments in this study.
Pectinase production test using peels of some fruits
The results in Fig (1) indicated that the use of Apple peels gave the highest production of pectinase enzyme, as it reached a clear zones diameter of 7.2 cm, followed by orange peels with 6.3 cm, tangerine peels with 5.8, then banana peels with 5.6. The majority of developed countries use agricultural and industrial waste to produce high-value raw materials, juice factories have used waste products such as citrus peels and Apple waste and these wastes contain percentages of pectic substances that reach 15-25% in citrus peels and 15-30% in Apple peels, which helps the growth of fungi that can produce pectinase enzyme (Ravindran and Jaiswal, 2016; Maric et al., 2018). So Apple peels were selected to carry out other experiments in this study.
Pectinase enzyme production assay using SSF method and SmF method
The results in Fig (2) indicated the superiority of the solid-state fermentation method over the submerged fermentation method in enzyme production, as the efficiency reached 760 and 425 units/mg, respectively, which could be due to a variety of factors, including lower catabolic repression in SSF compared to SmF and lower microorganism diffusion in SSF, as well as increased growth rates due to higher oxygen levels in the solid-air phase (Doriya et al., 2016).
Conflict of interest
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