In-vitro Studies on Aflatoxin Contamination and Management of Copra Quality

Coconut (Cocos nucifera L.) is a significant plantation crop in India. The crop is often called ‘Kalpavriksha’ due to its multifarious uses. Copra is a source for oil, cake and other edible products (Table 1). The copra is usually dried by air-drying, forced air-drying and by kiln drying methods. However, improper handling of any of these methods may lead to contamination with mycoflora which deteriorate the quality of copra and thus trade. The most widely used and cost-effective drying method in Asia is sun-drying, where grains are spread out in the open air for extended periods. However, this process also increases the risk of aflatoxin contamination (Lakshman et al., 2022). A.flavus is the main species that is mainly responsible for aflatoxin production and crop contamination (Kakde, 2012).  The present paper reviewed the research work on post harvest spoilage of copra and the possible aflatoxin contamination and a detailed account on the non pesticidal approaches for managing the post harvest spoilage of copra with emphasis on biocontrol of A.flavus the cause of aflatoxin contamination in copra and also the possible role of chemical preservatives in checking aflatoxin inducing A.flavus mold infestation in copra.


 

Isolation of mycoflora from copra
 
Copra was cut into small bits of 0.1 cm size and surface sterilized with 0.1 per cent mercuric chloride for 1 min for isolation of mycoflora. Then copra bits were washed thoroughly in three changes of distilled water and plated again on potato dextrose agar and incubated at 28 ± 1^oC temperature for a period of 7 days at department of Microbiology laboratory of SRR and CVR Govt Degree College (A), Vijayawada, Andhra Pradesh during the year 2022-23. The culture thus obtained was observed under compound microscope for presence of associated mycoflora with copra bits and further purified by single hyphal tip isolation.
 
Isolation of Trichoderma spp.
 
Isolation studies were carried out from soil samples collected from coconut gardens revealed the presence of three Trichoderma species and were subsequently identified as T.viride, T.harzianu and T.hamatum (Rifai, 1969).
 
Screening of antagonistic effect of Trichoderma spp. (Gams et al., 1980).
 
Antagonistic effect of Trichoderma spp. on A. flavus under in vitro conditions
 
The isolated native Trichoderma spp. were screened for antagonism under in vitro conditions against A.flavus on PDA by using dual culture technique. Eight mm diameter discs of 3 day old growing culture of the pathogen and the antagonistic fungi were inoculated at opposite ends in a Petri-dish containing PDA. A control plate with only test fungus was simultaneously maintained. The Petri-plates were incubated at 28±1^oC for 7 days and the ability of the antagonist to inhibit the pathogen was recorded by periodic observations.  The per cent growth reduction was calculated by using the formulae as given by Vincent (1947).

 

Effect of volatile metabolites of Trichoderma spp. on A. flavus
 
The production and inhibitory effect of volatile antibiotics by the antagonists were tested against the test pathogen by using the procedure as given by Denis, C. and Webster. J.  1971. The antagonists were grown on PDA for a period from 0 to 25 days and its effect on growth of A.flavus was tested by exposing inverted plates of freshly inoculated test pathogens to plates containing antagonistic cultures and sealing together by cello tape. The pathogen growth was measured after 4 days after incubation at 28±1^oC and per cent inhibition was calculated.
 
Effect of non-volatile metabolites of Trichoderma spp. on A.flavus
 
The antagonists that have shown inhibition in dual culture studies were grown on potato dextrose broth to test the effect of the culture filtrates (non-volatile antibiotics) on the test pathogen by poisoned food technique (Khara and Hadwan, 1990). The culture filtrates were purified either by autoclaving at 15 PSI for 15 min.  The sterilized filtrate was incorporated in the medium for observing fungal growth and inhibition at different concentration (10%, 20%, 50% and 100%).  The PDA mixed filtrate were poured (20 ml each) into sterilized Petri-dishes and the plates were inoculated with fresh disc of the test pathogen i.e., A.flavus and per cent inhibition was calculated after 7 days of incubation.
 
Effect of chemical preservatives on A. flavus strains and Trichoderma spp.
 
The effect of commonly used chemical preservatives at 100 ppm and 500 ppm concentrations were tested against A.flavus  strains and Trichoderma spp. on PDA by poisoned food technique under in vitro conditions. Then inoculated plates with 8 mm discs of three days old cultures of A.flavus  strains and Trichoderma spp. separately and incubated at 28±1^oC temperature. The observations on mycelial growth of A.flavus strains and Trichoderma spp. were recorded at 24 hours intervals for a period of seven days. Three replicates were maintained for each treatment.

Association of mycoflora with copra
 
Isolation studies indicated that the mycoflora commonly found on copra during storage includes Aspergillus flavus, A. niger, Rhizopus spp. Drechslera spp. Botryodiplodia spp. and Penicillium spp. (Table 2 and Plate 1). Aspergillus flavus was predominant among the mycoflora with per cent colonies was ranging from 68 to 92 per each sample.  This was followed by Penicillium spp. with a range of 61 to 69 per cent colonies. Aspergillus niger was recorded to a tune of 46 to 64 per cent colonies whereas other species of Aspergillus were recorded to an extent of 47 to 57 per cent. 
 




In vitro antagonistic studies
 
Dual culture studies carried out with three species of Trichoderma isolated from soils of coconut gardens revealed that T. viride, T. harzianum and T. hamatum were found very effective in hindering the A. flavus growth under in vitro conditions (Plate 2). Of them, T. hamatum was found very effective in controlling both the isolates. This was followed by T. harzianum and T. viride with insignificant differences in per cent of inhibition (Table 3). A clear inhibition zone was noticed with all the three Trichoderma species and the inhibition zone was prevailed up to one week duration. Biological control of aflatoxins is a cost-effective and eco-friendly approach for the reducing aflatoxin contamination in food and feed and as well as minimizing the contamination throughout the food value chain (Bandyopadhyay et al., 2019). Aflatoxins can be absorbed directly by microorganisms, either by binding to their cell wall components (Motawe et al., 2014) or by being absorbed into the cells of dead microorganisms (Mwakinyali et al., 2019).




       
Further in vitro studies carried out to determine the potentiality of Trichoderma spp. against A. flavus isolates revealed that volatile metabolites of 30 day old cultures of T. viride, T. harzianum and T. hamatum were inhibitory to A. flavus. While, 0 and 15 day old cultures of all the three Trichoderma spp. were found infective in inhibiting the mycelial growth of A. flavus through volatile metabolites (Plate 3).  Among the Trichoderma spp. of 30 days old, maximum inhibition of A.flavus isolates was obtained with T. viride (66.67%), followed by T. harzianum and T. hamatum with an inhibition of 61.11% (Table 4).





For non-volatile metabolites, a rising trend in the inhibition of A. flavus was observed as the concentration of Trichoderma spp. culture filtrate was increased. Among the Trichoderma spp. maximum inhibition in mycelial growth of A.flavus was obtained by T.hamatum (57.6 per cent) (Plate 4), followed by T. harzianum (42.3 per cent) and T. viride (30.7 per cent) at 100 per cent concentration of the culture filtrate (Table 5). However, inhibition of the test fungus to a notable extent was also achieved by all the three Trichoderma spp. at culture filtrate concentrations of 75, 50 and 20 per cent respectively. Recent studies indicated that various fungal species, including Trichoderma sp., Alternaria sp., Peniophora sp., Phoma sp., Armillariella tabescens, Mucor sp., Rhizopus sp., Pleurotus ostreatus and Phanerochaete chrysosporium, can degrade aflatoxins produced by aflatoxigenic species into less or non-toxic metabolites (Wu et al., 2009; Verheecke et al., 2016; Adebo et al., 2017).




       
Studies on the inhibition effect of chemical preservatives viz., Menadione, Potassium meta bisulphite, Benzoic acid, Sodium benzoate, L-Ascorbic acid, Propionic acid and Acetic acid (glacial) on A. flavus strains (AF2 = Highly virulent and Aggressive strain; AF3 = Moderately virulent strain) showed that all the tested chemicals decreased the linear growth of aflatoxin-producing molds from moderate to significant levels at a concentration of 500 ppm and to some extent at 100 ppm as well (Table 6). A significant positive correlation was noticed among majority of the chemicals with respect to increase in dosage from 100 ppm to 500 ppm with respect to A.flavus strain inhibition in terms of linear growth (Plate 5). The inhibition of linear growth for the highly virulent strain of A. flavus (AF2) was varied from 6.67 to 100 per cent, while the moderately virulent strain AF3 was inhibited between 7.00 and 100 per cent. Since, chemicals are targeted against all the strains i.e., right from aggressive to moderately to less aggressive strains; the current discussion on the average per cent inhibition of A.flavus is apt. Among the various chemicals tested, Menadione exhibited the highest level of inhibition against A.flavus strains at 100 per cent, followed by Potassium metabisulfite and Benzoic acid which showed inhibition rates of 77.23 and 63.33 per cent respectively. The preservatives, Sodium Benzoate and Ascorbic acid also performed well in inhibiting the A.flavus strains by more than 50 per cent i.e., 57.78 and 53.89 per cent respectively. However, the efficacy of Propionic acid is also notable with an inhibition of 43.33 per cent on A.flavus strains. On the other hand, Glacial acetic acid had a mild inhibitory effect with an inhibition of 6.84 per cent on A.flavus strains. The same chemical preservative even did prove ineffective against both the aflatoxin producing molds at 100 ppm with no inhibitory effect (Table 6). Food preservatives were also found effective in preventing rot, potassium metabisulphite 0.5 per cent followed by sodium benzoate 0.5 per cent proved most effective against the rot in both pre- and post-inoculation treatments. Manjunatha et al., 2022; Kumar et al., 2019 found that there was a drastic decrease in both the morphological growth and the aflatoxin biosynthesis of A. parasiticus in anoxic state. Seyedjafarri (2021) reported that the yoghurt bacteria (S. thermophilus and L. delbrueckii  subsp. Bulgaricus) are able to reduce the levels of AFM1 in milk during the fermentation process. 




       
Compatibility studies between Trichoderma spp. that are isolated viz., T. viride, T. harzianum and T. hamatum and chemical preservatives that inhibit growth of A. flavus revealed that sodium benzoate, Ascorbic acid and Potassium meta bisulphate were safe with regard to the growth and multiplication of Trichodrerma spp. and can be used in conjunction with biocontrol management of copra spoilage.  In contrast, Menadione, Propionic acid, Benzoic Acid and acetic acid reduced the mycelial growth of Trichoderma spp. at 500 ppm concentration (Plate 6). All these chemicals were relatively safe at 100 ppm with respect to Trichoderma spp. growth inhibition (Table 7).




               
Comparison was drawn with respect to chemicals that inhibited A.flavus growth and Trichoderma spp. growth at 500 ppm concentration under in vitro conditions. The results indicated that Menadione though effective in controlling A.flavus population, is also adverse in terms of Trichoderma spp. growth. The results with respect to Potassium meta bisulphate, Benzoic acid, Sodium benzoate and Ascorbic acid were encouraging in the sense that only A.flavus growth was reduced whereas Trichoderma spp. growth is almost unaffected under in-vitro conditions (Fig 1).  However, the efficacy of Propionic acid in terms of reduction of both A.flavus strains and Trichoderma spp. is almost on par with each other. With regard to Acetic acid, a poor mold inhibitor was also doubly disadvantageous with its inhibitory effect on Trichoderma spp. The compatibility studies of chemical preservatives on Trichoderma spp. were only a study taken up keeping in view the precautions to be adopted while applying preservatives to the copra. Potassium meta bisulphate, Benzoic acid, Sodium benzoate and Ascorbic acid can be recommended to be applied on copra along with in godowns where copra is stored, the soils of which may inhabit Trichoderma spp. Whereas, in order to have a dual check of mold growth by biocontrol agents as well, the chemical Menadione has to be applied only to the copra and not to the godowns as a general. From the present studies, it can be inferred that chemical preservatives offer a feasible and an ecofriendly approach in managing the post harvest spoilage of copra especially the aflatoxin problem.

This study highlights the significant impact of fungal contamination on copra quality, with Aspergillus flavus identified as the most prevalent species. The isolation of various fungi, including A. niger, Rhizopus spp. Drechslera spp. Botryodiplodia spp. and Penicillium spp. under scores the need for effective management strategies during storage. The efficacy of Trichoderma species, particularly T. hamatum in inhibiting the growth of A. flavus demonstrates the potential for biological control measures. Additionally, chemical preservatives such as Menadione, Potassium metabisulfite and Benzoic acid showed promise in reducing fungal growth. However, further research is essential to explore these findings in practical, in-vivo studies. An integrated approach to post-harvest technology could significantly enhance copra quality and ensure better preservation methods in the industry.

None
 
Disclaimers
 
The authors are responsible for the accuracy and completeness of the information provided.

No conflicts of interest regarding the publication of this article.