In Vitro Activity of Balanites aegyptiaca and Tamarindus indica Fruit Extracts on Growth and Aflatoxigenicity of Aspergillus flavus and A . parasiticus

Aflatoxin and especially aflatoxin B1 (AFB1) is a carcinogenic secondary metabolite synthesized by certain Aspergillus species. They contaminate natural and processed agricultural and animal products which render them unfit for consumption. The aim of this study was to evaluate the in vitro effects of Balanites aegyptiaca and Tamarindus indica fruit extracts on the growth and aflatoxin secretion of Aspergillus flavus (SQU21) and A. parasiticus (CBS921.7) strains. The two fruit extracts significantly (P < 0.05) reduced aflatoxin and did not inhibit mycelial dry weights of the two Aspergillus strains. At different concentrations of balanites (2.5-10%), the inhibition of total aflatoxin was 49.9-84.8% for A. flavus (SQU21) and 32.1-84.4% for A. parasiticus (CBS921.7), whereas the inhibition of aflatoxin Bwas 38.2-81.4% and 32.8-80.6% for the two strains. Tamarind fruit extract (2.5-7.5%) caused 28.8-84.2% and 40.7-85.5% reductions in total aflatoxin and 37.1-83.5% and 33.9-85.9% in aflatoxin B for the two strains, respectively. None of these extracts inhibited the fungal growth or detoxified synthetic aflatoxin B1. We have concluded that these fruits contain various inhibitors to aflatoxin biosynthesis and secretion. Therefore, they can be used in combination as safe green biopreservatives to combat aflatoxin contamination of food.

Balanites aegyptiaca (L.) Del, Balanites "Lalob, Hegleeg" in Arabic which belongs to the Zygophyllaceae is one of the most common wild plant species to dry land of Africa and South Asia (Deshmukh & Bhuyar, 2009;AlAshaal et al., 2010;Shalaby, El Namaky, Khalil, & Kandil, 2012).It is known as desert dates and used as edible fruits, fuel wood, charcoal, timber and fodder.The plant is known to be potential of medicinal value and used in herbal medicine.The leaves are edible and effective for sleeping sickness (Sheded, Pulford, & Hamed, 2006), and diabetes (Morsy, Ahmad, & Kamel, 2010).The outer rind of fruit used in the treatment the skin diseases, hypoglycemic agent, roots bark as antimalarial, Candida infection, and promising for HIV/AIDS patients (AlAshaal et al., 2010;Cook et al., 1998).On the other hand, Tamarindus indica L. of the family Caesalpinaceae and now drawn up in Fabaceae is commonly known as tamarind tree, is one of the most important multipurpose tree species in India sub-continent (Havinga, Hartl, Putscher, Prehsler, Buchmann, & Vogl, 2010;Prabhu & Teli, 2011).Almost all parts of the plant are found to be of some uses.The fruit pulps have been known for a very longtime in food, chemicals and pharmaceuticals (Dagar, Singh, & Singh, 1995).It is incorporated as seasoning or flavouring agents in food, drink, or beverages, removing off odour of fish, and as sanitizers or remedies for various ailments, management of diabetes mellitus, and immense therapeutic potential in many pathological conditions (Maiti, Jana, Das, & Ghosh, 2004;Wan Norhana, Nor Azman, Poole, Deeth, & Dakes, 2009;Dey, Swarup, Saxena, & Dan, 2011).The fruits are used as laxative or febrifuge and for wound treatment throughout the Sahel and Sudan ecological zones (Havinga et al., 2010).
From the above it is obvious that several attempts have been made to search for effective control methods to combat mould growth and aflatoxin contamination of different plant and animal products.The priority is for testing natural products of no residual effects, eco-friendly, and harmless to both humans and animals and can be used as excellent preservatives to food and feed.Therefore, the objective of the present investigations is to study the in vitro effects of fruit extracts from Balanites aegyptiaca and Tamarindus indica on the growth and aflatoxin secretion of two strains of Aspergillus namely A. flavus (SQU21) and A. parasiticus (CBS921.7).We anticipate that these results will successfully contribute in replacing toxic synthetic chemicals and provide alternative green biocontrol methods to protect food and feed against moulds invasion and aflatoxin secretion.

Fungal Strains
For this study, two aflatoxin producer strains of Aspergillus flavus (SQU21) and A. parasiticus (CBS921.7)[NRR22999]used in our previous studies were selected (El-Nagerabi et al., 2012, 2013).The strain were inoculated on Czapek Dox Agar (CDA) and identified with the help of the taxonomic manual of Raper and Fennell (1965).The growing fungi were used as inocula in the present investigations.

Inoculation of Aspergillus Strains on Media Containing Balanites and Tamarind Fruit Extracts
For inoculation of A. flavus (SQU21) and A. parasiticus (CBS921.7)two discs from each fungal inoculum were transferred aseptically to 200 ml sterile yeast malt broth in 250 ml conical flasks containing 0.0%, 2.5%, 5%, 7.5% and 10% (v/v) from balanites extract and 0.0%, 2.5%, 3.5%, 5% and 7.5% (v/v) from tamarind as described in our recent studies on Hibiscus sabdariffa, Nigella sativa, and A. digitata (El-Nagerabi et al., 2012, 2013).As a negative control, 10% balanites and 7.5% tamarind were added to yeast malt broth without any fungal inoculation.Triplicates of the inoculated flasks were incubated at ambient temperature 25-29 °C for 15 days.Similar sets were prepared and the mycelia of the fungi were filtered and the dry weight was determined using Oven method.

Effect of Balanites and Tamarind Fruit Extracts on Pure Aflatoxin B 1
Pure aflatoxin powder (Sigma Company) was added to 100 ml sterile distilled water which gave an aflatoxin B 1 concentration of 870 ppb.To this balanites concentration (10 ml/100 ml) and tamarind (7.5 ml/100 ml) were added separately to different flasks containing pure aflatoxin B 1 .The flasks were incubated at 25-29 °C for a week.The concentration of the aflatoxin B 1 was detected.

Extraction and Determination of Aflatoxin by Alfa Test-P Affinity
The extraction method used in similar studies was adopted (El-Nagerabi et al., 2012, 2013).The extraction mixture composed of 200 ml of fungal culture, 5 g of NaCl salt and 100 ml extraction solution of methanol: water (70: 30 V/V).The filtered mixture was passed through Afla-Test-P Affinity Column at a rate of 1-2 drops per second.To the elute aflatoxin, 1 ml of aflatoxin AflaTest developer was added to cuvette, and the concentration of the aflatoxin was measured using calibrated Vicam fluorometer (Series-4EX) with excitation wavelength of 360 nm and emission wavelength of 440 nm (Elshafie & Al-Shally, 1998). www.ccsen

Statisti
In this stu effect of Aspergillu of version
In the present study, inoculation of A. flavus (SQU21) and A. parasiticus (CBS921.7)strains on yeast malt broth containing different fruit extracts of balanites (2.5, 5, 7.5, and 10 g/100 ml) (Figure 1c) and tamarind (2.5, 3.5, 5, and 7.5 g/100 ml) (Figure 2c) significantly (P < 0.05) enhanced the fungal growth and mycelial dry weights of the two Aspergillus strains.On the contrary, extract of H. sabdariffa apparently retarded the vegetative growth and vigour of different fungi (Guerin & Revillere 1984).Nonetheless, calyx extract (5-12.5%)from H. sabdariffa did not show any effect on the mycelial growth of Aspergillus species (El-Nagerabi et al., 2012).Some herbal drugs and medicinal plants inhibit the mycelial growth of A. flavus and A. parasiticus while others improved mycelial growth, but retarded aflatoxin secretion (Bahk & 1983;Gowda et al., 2004;Joseph et al., 2005;Suleiman et al., 2008;Reddy et al., 2009;Da Costa et al., 2010).The fruit and oil extracts of A. digitata significantly reduced the vegetative growth and the mycelial dry weights of A. flavus and A. parasiticus (El-Nagerabi et al., 2013).Cinnamon at the concentrations of between 0.02-2.0%inhibited aflatoxin biosynthesis and the growth of A. parasiticus by 16-100% (Bullerman et al., 1977).The leaf extracts of cassia and bay enhance the mycelial growth of A. parasiticus and inhibit the mycelial growth and aflatoxin production by A. flavus (Paranagama et al., 2003;Krishnamsrthy & Shashikala, 2006;Sandosskumar, Karthikeya, Mathiyazhaga, Mohankumar, Chandrasekar, & Velazhahan, 2007).Therefore, it is evident that fruit extracts of Balanites aegyptiaca and Tamarindus indica displayed no antifungal effect on the growth of either A. flavus or A. parasiticus; however, it apparently inhibited total aflatoxins and aflatoxin B secretion by the two Aspergillus species.Similar findings were reached by using fruit extract of baobab and calyx extract of H. sabdariffa,N.sativa and A. digitataoils (Maraqa et al., 2007;El-Nagerabi et al., 2012, 2013), oil of cassia and bay leaves (Attanda et al., 2007), and anise and caraway oil (Montes-Belmont & Carvajal, 1998;Farag et al., 1989;Patkar et al., 1993;Hasan, 1994).It is most likely that different aflatoxin inhibitors may be found in both balanites and tamarind fruit extracts targeting the metabolic pathways of aflatoxin biosynthesis as we concluded in our previous studies (El-Nagerabi et al., 2012).

Detoxification of Aflatoxin B 1 by Fruit Extracts of Balanites and Tamarind
Detoxifications with biological factors offer green alternatives for aflatoxin elimination and maintaining the quality and safety of food and feed as well as human and animal health (Alberts, Gelderblom, Botha, & Van Zyl, 2009;Oguz, 2011;Prakash et al., 2011;El-Nagerabi et al., 2012, 2013).The ability of some herbal and medicinal plants as detoxifying agents was reviewed by many researchers (Sandosskumar et al., 2007;El-Nagerabi et al., 2012).This includes garlic (Allium sativum L. x) and onion (Allium cepa L.) roots extracts which cause 58.5% reduction in aflatoxin B 1 (Velazhahan, Vijayanandraj, Vijayasamundeeswari, Parandidharan, Samiyappan et al., 2010).Seed extract of Trachyspermum ammi degraded 90% of aflatoxin G 1 by altering the ring structure of lactone (Velazhahan et al., 2010) Figure 1 Staphylococcus albus, S. aureus and Sarcina lutea . The presence of inactivation factors in T. ammi seed extract a, 83,