Efficacy of different vinegars as antifungal agents against Cryptococuss neoformans and Sarocladium kiliense

Natural sources have caught the scientist’s eye for the development of new active substances because of side effects of chemically synthetic preservatives and their ultimate hazardous effects on human health. In the present study, three types of vinegars; white distilled vinegar, grape vinegar and apple cider have been used to test the antifungal activity against Cryptococcus neoformans and Sarocladium kiliense. Rotten vegetables; cucumber and tomato were used to isolate these two fungal isolates. These isolates were identified using microscopic technique. Anti-fungal activity was observed followed by application of vinegars through agar well diffusion assay technique. All vinegars showed significant size of zone of inhibition against fungal specie. Distilled white vinegar showed the best effect against C. neoformans and S. kiliense forming zone of inhibition of 0.58±0.05 mm and 0.6±0.05 mm. Grape vinegar forms a zone of inhibition of 0.52±0.05 mm and 0.48±0.05, while 0.4±0.05 mm was observed against both fungal specie, as a result of application of apple cider. The results showed that vinegars can be used as an alternative to synthetic antifungal agents in order to overcome the drawbacks because they have active components i.e. phenolics and flavonoids etc. which have antifungal and anti-oxidant properties.


Introduction
Different antimicrobial agents have been in use for long time and these agents do not have any adverse effects. Many of the organic compounds are the antimicrobial metabolites of bacteria that are used to produce fermented foods. In fermentation process, raw materials are converted to products by the action of microorganisms and these products have acceptable qualities of food. Fermentation of food has a great economic value and these products have been contributed in improving the human health [1].
Scientific studies categorized the food borne pathogen behavior into two divisions, i.e., food science and medical field. Two different approaches are used to determine the behavior of food borne pathogens in food; commonly refers to as in vitro and in situ. In vitro studies aim at stimulating the behavior of food borne pathogen in laboratory giving a specific media for their growth, while in situ studies describe the microbial expression in real food under the conditions of storage and consumption of food [2].
C. neoformans contains two varieties namely C. neoformans var. grubii and C. neoformans var. neoformans. Colonies of C. neoformans are cream-colored and smooth, mucoid and have yeast like appearance of colonies. C. neoformans var. grubii is cosmopolitan in its distribution and this one cause 95% of all the C. neoformans infections. C. neoformans has been isolated from the menure of caged birds including canaries and parrots etc. C. neoformans var. grubii has also been isolated from the other environmental isolations including rotten or decomposed vegetables, fruits and fruit juices, wood dairy product and soil. Sarocladium can be morphologically differentiated from Acremonium by its elongated phialides rising solitary on vegetative hyphae or on conidiophores that are sparsely or repeatedly branched, the production of abundant adelophialides and elongated conidia [6]. Different natural antimicrobials from the plant origin contain secondary metabolites having ability to inhibit or reduce the growth of bacteria, yeast and molds [7]. Plant essential oils have also been used as a flavoring agent in addition to its antimicrobial activity [8]. A large number of antimicrobial agents originated from animal origin and involved in host defense mechanisms [9]. Among the different natural products, vinegars are also contributing in its antimicrobial properties [10]. Vinegars i.e. Apple cider has an important element; maleic acid with bactericidal and fungicidal activity. Maleic acid has been reported for the treatment of various infections [11].

Material and methods Isolation and culturing of fungus
The microorganisms to be isolated; were grown on the Potato dextrose agar media (containing 3.9g PDA in 100ml water). Rotten vegetables; cucumber and tomato were used to isolate the fungal specie. Small pieces of these were inoculated in the center of two different PDA plates. Then the plates were incubated at 37ᵒC for 72 hours.

Identification of fungal isolates
After incubation period, two different fungal isolates were obtained. They were identified by microscopic technique. Fungal smear of each species was made and then stained with crystal violet stain. The slides were then observed under microscope. Agar-well diffusion method Principle: The antimicrobials present in the natural antifungal (vinegars) were allowed to diffuse out into the medium and interact in a plate freshly seeded with the test organisms. The resulting zones of inhibition were mostly circular in shape as there was a confluent lawn of growth. The diameter of zone of inhibition was measured in millimeters. Method to apply vinegars PDA pla4tes were prepared under aseptic conditions. Then, by using the borer, wells were made in the center of each plate. Three of the plates were swabbed with each of the two fungal isolates, using sterile cotton swabs. Then, 100 µl of each vinegar i.e. apple cider, distilled white vinegar and grape vinegar were added in the wells. The plates were then incubated for 72 hours at 37ᵒC. The vinegars were allowed to diffuse out into the medium and interact in a plate freshly seeded with the test organisms. After incubation period, zones of inhibition were observed in each plate. The diameter of zone of inhibition was measured in millimeters with the help of Vernier caliper and diameter of each zone of inhibition was measured from three different directions. The mean values of zone of inhibition are mentioned in results.

Results
In present study, two different fungal species were isolated and identified on the basis of their colony morphological characteristics, color charts and microscopic examination. The isolated fungal species were C. neoformans and S. kiliense. Morphological examination of fungal isolates (Fig. 1).
Microscopic examination of fungal isolates (Fig. 2). Effect of distilled white vinegar on C. neoformans (Fig. 3). Effect of grape vinegar on C. neoformans (Fig. 4). Effect of apple cider on C. neoformans (Fig.  5). Effect of distilled white vinegar on S. kiliense (Fig. 6). Effect of grape vinegar on S. kiliense (Fig. 7). Effect of apple cider on S. kiliense (Fig. 8). The best effect of distilled white vinegar against C. neoformans with mean value of 0.58±0.05 mm (Fig. 9). The zones of inhibition of vinegars against S. kiliense (Fig. 10). The varying zone of inhibition against fungal species (Table 1).