African Journal of Biotechnology Vol. 6 (4), pp. 384-387, 19 February, 2007 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals Full Length Research Paper Control of Aspergillus niger with garlic, onion and leek extracts Reyhan Irkin1 and Mihriban Korukluoglu2 1Balıkesir University,Susurluk Vocational School, Susurluk, Balikesir, Turkey. 2Uludag University, Agricultural Faculty, Food Engineering Department, Görükle, Bursa, Turkey. Accepted 7 February, 2007 Antifungal activity of “Allium” vegetables that is garlic (Allium sativum L.), onion (Allium cepa L.) and leek (Allium porrum L.) were investigated against Aspergillus niger. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of aqueous, ethyl alcohol and acetone extracts were determined by disc diffusion and broth dilution methods in the test tubes. Onion extract with ethyl alcohol (275 mg/mL MFC), aqueous garlic extract (325 mg/mL MFC) and aqueous leek extract (900 mg/mL MFC) found the most inhibitory against A. niger. Key words: Aspergillus niger, garlic, onion, leek, antifungal activity. INTRODUCTION Aspergillus sp. are the most common fungal species and include the powerful antioxidants, sulfur and other which are able to produce mycotoxins in food and feed- numerous phenolic compounds which arouse significant stuffs. Mycotoxins are known to be potent hepatocarcino- interests (Block, 1985; Topal, 1989; Yin and Cheng, gens in animals and humans. The presence and growth 1998; Phay et al., 1999; Harris et al., 2001; Kyung and of fungi may cause spoilage and result in a reduction in Lee, 2001; Rivlin, 2001; Griffiths et al., 2002; Benkeblia, quality and quantity of foods (Paster et al., 1995; Belt- 2004; Haciseferogullari et al., 2005). mont and Carjaval, 1998; Sahin and Korukluoglu, 2000; The aim of this study was to investigate minimal inhibi- Candlish et al., 2001; Galvano et al., 2001; Juglal et al., tory concentrations (MIC) and minimal fungicidal concen- 2002; Soliman and Badeaa, 2002; Rasooli and Abyaneh, trations (MFC) doses of garlic (Allium sativum L.), onion 2004). (Allium cepa L.) and leek (Allium porrum L.) extracts Natural plant extracts may provide an alternative to (aqueous, acetone and ethyl alcohol) against to Asper- chemical preservatives. Over the years much effort has gillus niger. been devoted to the search for new antifungal materials from natural sources for food preservation (Karapınar, 1989; Topal, 1989; Paster et al., 1995; De et al., 1999; MATERIALS AND METHODS Yin and Tsao, 1999; Nielsen and Rios, 2000; Galvano et Allium samples and water content al., 2001; Juglal et al., 2002; Soliman and Badeaa, 2002; Onyeagba et al. 2004; Boyraz and Ozcan, 2005; Hacise- Onions (A. cepa L.) and leeks (A. porrum L.) were cultivated in ferogullari et al., 2005) Yenice-Canakkale region and garlic (A. sativum L.) was obtained Allium genus has over 500 members, each differing in from Balikesir regions of Turkey during harvesting season. Samples freshly harvested were classified for homogeneity and lack of flaws maturing, color and taste, but with similar biochemical, and prepared for analysis. Water content of fresh Allium samples phytochemical and neutraceutical content. Alliums were were determined by using the official methods of AOAC (1990). revered to possess anti-bacterial and anti-fungal activities Microorganism *Corresponding author. E-mail: mihriban@uludag.edu.tr. Tel: Aspergillus niger strain was isolated from tulum-cheese in Uludag +90 2244428970 Fax:+902244428077. (*This study is part of University, Department of Food Engineering in Bursa, Turkey and R.Irkin’s PhD.thesis results). identified using standard fungi determination procedures. Irkin and Korukluoglu 385 Table 1. Water content of Allium fresh samples. incubated at 30ºC for 5 days to determine if the inhibiton was rever- sible or permanent. Minimal Fungicidal Concentration (MFC) was Allium sample Water content (%) ± SD determined as the highest dilution (lowest concentration) at which no growth occured on the plates. In control tubes 1 mL (extract and Garlic bulb (Allium sativum L.) 76.1±2.2 A.niger culture) were added in to the 5 mL of solvent; ethyl alcohol Onion bulb (Allium cepa L.) 89.3±0.65 or acetone or water seperately All the tests were done in three replicates (Abbasoglu, 1996; Yin and Tsao, 1999; Flörl et al., 2003; Whole Leek (Allium porrum L.) 87.9±0.36 Rasooli and Abyaneh, 2004). RESULTS AND DISCUSSION Preparation of material extracts 270 g of fresh onion, leek and garlic were peeled and then was Water contents of fresh Allium samples are listed in Table chopped with 300 mL distilled water, ethanol (Panreac 1. The MIC and MFC (mg/mL) concentrations of Allium 121086.1612, 99.5%) and acetone (Merck 1.00013.2500, 99.5%) plant extracts and inhibitory zones (mean ± SD) against (w/v) by using a domestic blender (Braun model 4259, Germany) to A. niger are presented in Table 2. It is seen that Allium for 1 min at average speed. The mixture were macerated during 24 plants have antifungal effects to A. niger. The most inhi- h at the + 4ºC. After that, resulting extracts of materials were filtered and sterilized using a 0.45 µm pore size cellulose acetate bitory plant was garlic, followed by onion and leek. Ethyl membrane filter (Cole-Parmer-47 mm) under nitrogen gas pressure. alcohol extracts of the garlic and onion significantly show The extracts were used directly. Dilutions were prepared from 900 inhibitor effect against A. niger. Also, inhibitor activities to 100 mg/mL (by 25 mg/mL intervals). These dilutions were used in were observed for aqueous extracts of garlic and leek. antifungal analysis. Ethyl alcohol, acetone and distilled water Acetone extracts of onion and leek did not show any served as control. effect on A. niger. Ethyl alcohol extract of the onion has the highest inhibitory activity when their MIC and MFC values are compared. Preparation of inoculum The inhibitory activity of Allium vegetables extracts A. niger was cultured on Sabouraud Dextrose Broth-SDB-(Oxoid against mould have been reported by numerous authors. CM0147) at 30ºC for 22 h. Test fungi in SDB were enumerated by It has also been observed that alliicin, thiosulfonates and using serial dilution method. Final cell concentration of culture was 4 5 other compounds show fungistatic activities against A. 10 - 10 cfu/mL. niger, Rhodotorula nigricans, Penicillium italicum, Penici- llium cyclopium, Aspergillus flavus, Cladosporium macro- Antifungal activity tests carpum, Aspergillus fumigatus, Aspergillus alutaceus, As- pergillus terreus and Penicillium chryogenum (Wei et al., Disc diffusion method was used as an antimicrobial method (Yin and Tsao, 1999; Karaman et al., 2003; Benkeblia, 2004). Sterile 1967; Graham and Graham, 1987; Topal, 1989; Hafez Sabouraud Dextrose Agar-SDA-(Oxoid CM0041) at 43-45ºC and and Said, 1997; Ankri and Mirelman, 1999; Harris et al., poured into the petri plates (9 cm diameter). Then the agar was 2001). Similarly, ajoene compound which is a deri-vative allowed to solidify at + 4ºC for 1 h. 0.2 mL of A. niger culture of alliicin and obtained from garlic with ethyl alcohol ext- inoculum applied of each plate. Inoculum was evenly spread on raction is very inhibitory against A. niger, Candida albi- agar using a glass L- rod spreader. The petri dishes were left at + cans and Paracoccidioides brasiliensis (Naganawa et al., 4ºC for 1 h to allow agar surface to dry. Sterile filter papers (Schleicher and Schüll 2668, Germany, 6 mm diameter) were 1996). Yoshida et al. (1987) reported that ajoene comp- placed on the culture mediums and were impregnated with 50 µL ound from garlic have stronger antifungal activity than material extracts between 100 - 900 mg/mL concentrations and alliicin. They are determined that ajoene damages the placed on the inoculated plates. Distilled water and other solvents cell walls of fungi. Yin and Tsao (1999) studied antifungal were added at the same concentrations on the discs to provide a effects of various Allium plants. They found 35 ± 3 control. After 30 min, plates were turned upside down and incu- bated at 30ºC for 24 h. At the end of the period, inhibition zones, µg/mL MFC for garlic bulb, 748 ± 15 µg/mL MFC for on- formed in the medium were measured in millimeters (mm). All ion bulb and 91 ± 10 µg/mL MFC for chinese leek against experiments were done in three replicates. A. niger. These authors observe that garlic shows the highest antifungal activity against three Aspergillus species. Determination of minimal ınhibitory concentration (MIC) and Antifungal effects of onion and garlic essential oils minimal fungicidal concentration (MFC) against some fungi have also been investigate. A. niger is 5 mL of sterile extracts at different concentrations were taken in to less inhibited by low concentrations of essential oils of the sterile empty tubes and 1 mL of A. niger culture was added in to green and yellow onions but red onion and garlic essen- the extracts and mixed. After that 1 mL of (extract + A. niger) tial oils show strong inhibitory effects against A. niger culture was added in to the 5 mL of sterile SDB in the tubes. Then (Benkeblia, 2004). Fistulosin, an antifungal compound all the tubes were incubated at 30ºC for 15 days. Observations isolates from onions, shows antifungal activities against were made for visible growth of fungi. The highest dilution (lowest concentration) showing no visible growth was regarded as Minimal several fungal species (Phay et al.,1999). Combined extr- Inhibitory Concentrations (MIC) during 15 days. Cells from the act of corni fructus, cinnamon and chinese chieve (1:6:6, tubes showing no growth were subcultured on SDA plates and v/v/v) also exhibit high inhibitor activity against A. niger 386 Afr. J. Biotechnol. Table 2. The minimal inhibitory and minimal fungicidal concentrations (MIC and MFC) and inhibitory zones diameters of Allium plant extracts against A. niger. Materıals Aqueous MIC MFC Ethyl alcohol MIC MFC Acetone MIC MFC (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) Garlic (Allium sativum L.) MIC/MFC 325 325 425 450 800 875 Inhibition zones* 15.5±0.9 15.5±0.9 15±1.0 16.8±0.8 13.7±1.6 16.5±0.5 Onion (Allium cepa L.) MIC/MFC > 900 > 900 250 275 > 900 > 900 Inhibition zones* - - 11.8±0.3 16.7±0.6 - - Leek (Allium porrum L.) MIC/MFC 900 > 900 >900 >900 >900 >900 Inhibition zones* 12.3±1.1 - - - - - *Inhibition zones (mm, mean ± SD). (Mau et al., 2001). Ankri S, Mirelman D (1999).Antimicrobial properties of allicin from A. niger, P. italicum, Tryptophyton gypseum and Micro- garlic. Microb. Infec.2: 125-129. sporon audouini are inhibited by thiosulfonates compo- AOAC (1990).Official methods of analysis.Association of Official Analytical Chemists. Washington. unds in onions. However, antifungal studies about onions Belmont RM, Carjaval M (1998).Contol of Aspergillus flavus in maize are very limited. Researchers have observed that ether with plant essential oils and their components. J. Food Prot. 61: 616- extracts of onion show inhibitor effect against A. flavus 619. and A. parasiticus (Wei et al., 1967; Sharma et al., 1979; Benkeblia N (2004).Antimicrobial activity of essential oil extracts of various onions (Allium cepa) and garlic (Allium sativum).Lebensm.- Pruthi, 1980). Topal (1989) reported that onion exhibits Wiss.u-Technol. 37: 263-268. more inhibitor activity against bacteria than yeast and Block E (1985). The chemistry of garlic and onion. Sci. Am. J. 3: 94-99. fungi. Researches on antifungal effects of leek are also Block E, Naganathan S, Putman D, Zhao SH (1992). Allium Chemistry: very limited. In Kivanc and Kunduhoglu (1997) study, leek HPLC analysis of thiosulfinates from Onion, Garlic, Wild Garlic (Ramsoms), Leek, Scallion,Shallot, Elephant Garlic, Chieve and was found to have the lowest inhibitory vegetable against Chinese Chieve. Uniquely high allyl methyl ratios in some garlic yeasts compared to onion, cabbage, radish and garlic. samples. J. Agric. Food Chem. 40: 2418-2430. Tsao and Yin (2001) obtained 32 mg/L MIC for chinese Boyraz N, Özcan M (2005). Antifungal effect of some spice hydrosols. leek oil and 20 mg/L MIC for garlic oil against A. niger. Fitoterapia. 76: 661- 665. 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