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Sclerotial formation inhibition by vitamin A, C, and E in Aspergillus flavus.

INTRODUCTION

The fungi Aspergillus flavus synthesize a group of structurally related secondary metabolite named aflatoxins [1,7]. Aflatoxins are potent toxic, carcinogenic, mutagenic, immunosuppressive agents, produced as secondary metabolites by the fungus Aspergillus flavus and Aspergillus parasiticus on variety of food products. Among 18 different types of aflatoxins identified, major members are aflatoxin B1, B2, G1 and G2. Aflatoxin B1 (AFB1) is normally predominant in amount in cultures as well as in food products. Aflatoxin B1 (AFB1) is normally predominant in amount in cultures as well as in food products. Pure AFB1 is pale-white to yellow crystalline, odorless solid. Aflatoxins are soluble in methanol, chloroform, acetone, acetonitrile [3,9]. Moreover, the fungi Aspergillus flavus has this morphological structure known as sclerotium (plural sclerotia). It is a compact mass of hardened mycelium stored with reserve food material that, in some higher fungi such as ergot, becomes detached and remains dormant until a favorable opportunity for growth occurs [4]. Sclerotia (sing. Sclerotium) are important structures for most of the fungi especially for the Aspergillus flavus [5]. Spores (ascospores) formed by a sexual process in these bodies are shot into the air, and wind currents may carry them to grain heads [8]. Without these structures, the fungi would not be able to grow for these structures serve as the storage of food materials needed for survival. It has been also determined the link between aflatoxin production and sclerotial morphogenesis and that the two were interrelated [6]. In this regard, the need for protection from foods and feedstuffs against Aspergillus flavus are universally recognized and several approaches have been suggested. Thus, this study was design to evaluate the ability of Vitamins A, C, and E to inhibit the growth of the sclerotial bodies (or sclerotia) of the fungi A. flavus.

MATERIALS AND METHODS

Culture Conditions:

Stock cultures were grown in PDA slants for about 24 hours at 25 degrees Celsius. Reference stock cultures were stored at room temperature. Working stock culture was obtained by subculturing mycelia from covered slants to screw-cap vials (16 by 125 mm), each vial containing a 10-ml PDA slant. The culture used in inoculation was obtained from working stock cultures after seven (7) days of incubation at room temperature. It was then placed in the environment with total darkness because vitamin C is light sensitive and to ensure that sclerotial formation would not be affected by light.

Vitamins Used:

The vitamins used were retinol palmitate (vitamin A) having 25,000 IU; Ascorbic acid (vitamin C) which was in liquid form having formulation of 100mg/ml; and d-alpha tocopherol (vitamin E) having 400 IU. Both A and E were bought in soft gel form wherein the liquid inside the capsule was removed using a sterile syringe under aseptic conditions. It was transferred to a sterile vial and kept in the refrigerator having temperature not exceeding 30[degrees]C when not in use.

Spore Suspensions:

Spores were harvested by adding 5 ml of sterile distilled water to 14-day-old sporulated fungal culture, and then the surface of the agar was gently and carefully scraped. The resulting solution was transferred to a sterilized and clean test tube. One (1) ml of this spore suspension was pipetted to each of the five (5) centrifuge tubes after which were centrifuge for 5 minutes in 1,500 RPM under normal temperature (25 degrees Celsius). After the centrifugation, the tubes were shaked using the vortex for 30 seconds and ready for inoculation.

Preparation of the Solid Media:

The media used in this study was PDA (Potato Dextrose Agar). Each of the four (4) Erlenmeyer flask were filled with this agar then autoclaved for 15 minutes. The concentration of vitamin A E, and C were 20ml, 10ml, 5ml, and 2ml respectively were added, but as for small volumes a pippetor was used to achieve an accurate measurement of the volume. After which, the medium was dispensed on sterile plates thereby having three (3) replicates and was allowed to cool and dry for 15 minutes. The same procedure was employed but this time using the vitamin C.

A control medium was prepared of which no vitamins was added for comparison purposes between treated and non-treated culture media.

Inoculation and Growth Conditions:

After the medium was supplemented with various volumes of vitamins, the media were allowed to dry for few minutes and were inoculated with the fungal test species. 1000pl of the spore suspension was used for one-point inoculation at the center of the PDA plates mixed with different volumes of the different vitamins aforementioned. It was incubated at the room temperature for seven (7) days in total darkness.

Number of Sclerotium:

To determine the number of sclerotium present in every plate of Apergillus flavus treated with different concentrations of vitamins, manual counting of the sclerotium was done. The difference in the number of sclerotium was calculated by linear regression.

Statistical Analysis:

The classic statistical problem is to try to determine the relationship between two random variables X and Y. It analyzes the relationship between these two variables. For each subject (experimental unit), you know both X and Y and you want to find the best straight line through the data. In some situations, the slope and/or intercept have a scientific meaning as what was used for this study. In other cases, linear regression line is use as a standard curve to find new values of X from Y, or Y from X. In this study, X is equals to the concentration of vitamins while Y is equals to the number of sclerotium (plural Sclerotia). Linear regression attempts to explain this relationship with a straight line fit to the data.

Results:

One point inoculation was done and applied on the center of the PDA plates with different concentration of vitamins A, C, and E respectively. The culture was allowed to stand for about seven (7) days in total darkness at room temperature. After which, the number of sclerotium present in the plates were counted and recorded.

Figure 1 below shows the average number of sclerotia obtained from the PDA plates with different concentrations of vitamins A, C, and E.

At 5.5% of the vitamins, the number of sclerotia is smaller while relatively bigger at lower concentration. It was shown in the results that Vitamin E has a smaller number of sclerotia found at the highest concentration, followed by Vitamin C and Vitamin A respectively. It was observed that as the concentration of the vitamins in the media deacreases, the number of sclerotium formed by A. flavus increases.

Discussion:

Vitamin A reduced the number of sclerotium formed by A. flavus even if vitamin A is known to be produced naturally by the tested fungi [10]. At higher concentration, sclerotium number is smaller and the number is increasing with decreasing concentration of the vitamins. Thus, vitamin A inhibits sclerotial formation in Aspergillus flavus. Thus, vitamin A can effectively control the growth formation of the A. flavus since the sclerotium formed by the fungi was inhibited [11].In the same manner, at higher concentration of vitamin C, the number of sclerotium is minimal. It is shown in Figure 1 the decrease of the number of sclerotium with increasing concentration of vitamin C (ascorbate). Although exogenous ascorbate can inhibit sporulation in fungi [12] and can be fungicidal [13], formation of sclerotium is still present in the PDA with different concentration of vitamin C. This is possible because vitamin C is naturally produced by fungi [14,15]. The balance between the reduced and oxidized forms of vitamin C is vital for fungal differentiation, however, the exogenous ascorbate introduced at high concentrations somehow disrupted this balance pushing towards inhibition of fungal growth [11], thus leading to minimal sclerotial formation. On the other hand, vitamin E also gives the same result like Vitamin A and Vitamin C. 20 ml-volume of vitamin E has decreased the number of sclerotium compared to its lower concentration of about 2.5 ml. Reduction of fungal growth for all the concentrations of vitamin E was possibly due to interference [11]. It was reported that this vitamin is a potent biological antioxidant and its antioxidative function is mainly due its reaction with membrane phospholipid bilayers to break the chain reaction initiated by hydroxyl radical [16]. The reduction of sclerotium by vitamin E was possible since, vitamin E has a higher affinity to aflatoxin and acts by reducing its bioavialbility through the formation of stable association [11]. It implies then that vitamin E has the capability to reduce and impede aflatoxin formation and with the reduction of such, consequently, fungal and sclerotial formation will be inhibited as well as it was observed in Aspergillus parasiticus [17].

Moreover, to determine the relationship between the number of the formed sclerotia and the concentration of the vitamins used, [R.sup.2] values was determine As observed in the results, the number of sclerotia formed by A. flavus is inversely proportional to the concentration of the vitamins used. Coeffecient of Determination ([R.sup.2]) values of the different vitamins varied from each other. Vitamin A has an [R.sup.2] value of 95.4%, Vitamin C has 99.6%, and Vitamin E has an [R.sup.2] value of 88.1%. With these values, the amount of sclerotia formed depends on concentration of the vitamins.

Conclusion:

Results showed that the number of sclerotium is inversely proportional to the concentration of vitamin A, C, and E. It is observed that there is a decrease in number of sclerotium with corresponding increase of concentration of vitamin A, C, and E. Thus, there is a possible growth inhibition of Aspergillus flavus since sclerotium is the food storage of the fungi. In addition, the result also give us the idea on the amount of aflatoxin produced by Aspegillus flavus in Potato Dextrose Agar (PDA) since aflatoxin production in the fungi are interrelated with the sclerotial formation [6]. We can then assume that the smaller the number of sclerotia, there is also low level of aflatoxin that the fungi could produce.

REFERENCES

[1] Ritter, A.C., M. Hoeltz and I.B. Noll, 2011. Toxigenic potential of Aspergillus flavus tested in different culture conditions. Cienc. Tecnol. Aliment., Campinas, 31(3): 623-628.

[2] Frisvad, J.C., T.O. Larsen, R. de Vries, M. Meijer, J. Houbraken, F.J. Cabanes, K. Ehrlich and R.A. Samson, 2007. Secondary metabolite profiling, growth profiles and other tools for species recognition and important Aspergillus mycotoxins. Studies in Mycology, 59(1): 31-37.

[3] Reddy, S.V. and F. Waliyar, 2012. Properties of Aflatoxin and it producing fungi. Aflatoxins http://www.icrisat.org/aflatoxin/aflatoxin.asp (Accessed on March 2015)

[4] Duca, R.C., 2009. Food quality monitoring and analytical techniques optimization of some aliments within plant-animal correlation Contaminated aliments effects on the detoxication enzymes. Medication. Universit'e Paris Sud--Paris XI

[5] McAlpin, C.E.. 2001. An Aspergillus flavus mutant producing stipitate sclerotia and synnemata. Mycologia, 93: 552-565.

[6] Cotty, PJ., 1988. Aflatoxin and sclerotial production by Aspergillus flavus: Influence of pH. Phytopathology, 78: 1250-1253.

[7] Mahoney, N.E. and S.B. Rodriguez, 1993. Inhibition of aflatoxin production of surfactants. Appl. Environ. Microbiol, 60: 106-110.

[8] McMullen, M. and C. Stoltenow, 2002. Ergot PP-551 Revised Edition. Retrieved from: http://www.ag.ndsu.edu/pubs/plantsci/crops/pp551w.htm

[9] Onilude, A.A., O.E. Fagade, M.M. Bello and I.F. Fadahunsi, 2005. Inhibition of aflatoxin-producing aspergilla by lactic acid bacteria isolates from indigenously fermented cereal gruels, Biotechnology, 4: 1404-1408

[10] Georgiou, C.D. and A. Zees, 2001. Lipofuscins and Sclerotial Differentiation in Phytopathogenic fungi, Mycopathologia, 153: 203-208.

[11] Daud, H.F., J. Bautista and F.G. Teves, 2014. Effects of Vitamins A, C and E on Growth and Colonial Morphology of Aspergillus flavus. International Research Journal of Biological Sciences. ISSN 2278-3202 3(10): 52-59.

[12] Hansberg, W. and J. Aguirre, 1990. Hyperoxidant states microbial cell differentiation by cell isolation from dioxygen. J. Theor. Biol, 142: 201-221.

[13] Elad, Y., 1992. The use of antioxidants (free radical scavengers) to control gray mold (Botrytis cinerea) and white mould (Sclerotinia sclerotiorum) in various crops. Plant. Pathol, 41: 417-426.

[14] Petrescu, S.A., A.S. Hulea, R. Stan, D. Avram and V. Herlea, 1992. A yeast strain that uses D-galacturonic acid as a substrate for L-ascorbic acid biosynthesis. Biotech Lett, 14: 1-6.

[15] Spickett, M.C., N. Smirnoff and R.A. Pitt, 2000. The biosynthesis of erythroascorbate in Saccharomyces cerevisiae and its role as an antioxidant. Free Rad. Biol. Med, 28: 183-292.

[16] Nair, A. and R.J. Verma, 2001. Vitamin E ameliorates aflatoxin-induced biochemical changes in testis of mice, Asian J Androl, 3: 305-309.

[17] Cleveland, T.E., D. Bhatnagar, C.J. Foell and S.P. McCormick, 1987. Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus, Appl Environ Microbiol, 53: 2804-2807.

(1) Mark Anthony I. Jose and (1) Franco G. Teves

(1) Department of Biological Sciences, College of Science and Mathematics, Mindanao State University-Iligan Institute of Technology, Andres Bonifacio Avenue, Tibanga Highway, 9200 Iligan City, Lanao del Norte, Philippines

ARTICLE INFO

Article history:

Received 23 June 2015

Accepted 25 July 2015

Available online 30 August 2015

Corresponding Author: M.A.I. Jose, Department of Biological Sciences, College of Science and Mathematics, Mindanao State University-Iligan Institute of Technology, Tibanga Highway, Iligan City, 9200 Philippines. Tel: 221-4050 137 E-mail: jose.markanthony@gmail.com
Fig. 1: The mean number of sclerotia in the media with
different concentrations of vitamins A, C, and E after
seven (7) days of incubation in total darkness.

Vitamins Used (ml)           Number of Sclerotia

                     20ml   10ml   5ml   2.5ml   Control

Vitamin A             19     25     31     35       80
Vitamin C              9     27     39     43       80
Vitamin E              3     22     26     46       80

Note: Table made from bar graph.
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Author:Jose, Mark Anthony I.; Teves, Franco G.
Publication:Advances in Environmental Biology
Article Type:Report
Date:Aug 1, 2015
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