1. Introduction

Cereals and cereal by-products constitute a major part of the daily diet of the human and animal populations. The end products of wheat processing are, other than semolina or flour. However, they may represent a source of compounds with unique physico-chemical, nutritional, and functional properties which may have a high value for human nutrition, too ^[1]. Wheat (Triticum aestivum L.) is one of the most important grains crops providing nearly 20 % of the total world food requirement ^[2]. It is grown on more acreage than any other crop. In 2010, 653.7 mln tons of wheat was produced in the world, while for two of the other main staple food crops, maize and rice (paddy), 840.3 mln and 696.3 mln tons were produced, respectively ^[3].

During storage, a change in microflora can occur due to the reduction of the content of product-typical microorganisms or due to the reproduction of the spoilage-indicating microflora, adapted to the storage conditions. The main spoilage indicating moulds are Penicillium spp., Aspergillus spp., Scopulariopsis spp., and Mucorales spp. of wheat. Microorganisms causing spoilage are also all species of yeasts ^[4]. The growth of moulds is greatly influenced by the water content of the substrate. At low moisture content (usually less than 14-16%) most storage fungi do not grow or grow very slowly ^[5]. ^[6] evaluate mycoflora and in 53 whole wheat grain samples collected in Southern Brazil during the 2012 crop. He found that for Fusarium genera, there was predominance of Fusarium verticillioides (34%) and F. graminearum (30.2%). For Aspergillus species, 37.7% of Aspergillus flavus was determined. Regarding the Penicillium species, Penicillium digitatum (49 %) was the most found species.

Flour is a fine powder made by grinding cereals or other edible starchy plant seeds suitable for grinding. It is most commonly made from wheat. Flour is the key ingredient of bread, which is the stable food in most countries. Bread is the primary food stable for the majority of the Egyptians, who consume 270 million loaves of bread daily on average of 3 loaves per capita ^[7].

Mycotoxins prevention is very important as, once developed, they become stable at environment temperature and very resistant to thermal changes ^[8]. In order quality and safety of wheat flour and wheat products to be maintained and prevention from contamination, the objectives of the investigation were to examine molds presence or contamination of wheat flour as well as to identify the isolated species and define the effect storage of wheat flour on presence molds.

Cellulose, a major polysaccharide constituent of plant cell walls, is a 1,4 linked linear polymer of 8000~12000 glucose units. Three major enzymes are involved in the degradation of cellulose to glucose are endoglucanase (endo-1,4-d-glucanasem EG), cellobiohydrolase (exo-1,4-d-glucanasem CBH) and β-glucosidase (1.4-d-glucosidase, BG). EG acts in random fashion, cleaving linked bonds with in the cellulose molecule; CBH removes cellobiose units from the non reducing ends of the cellulose chain and BG degrades cellobiose and cellooligosaccharides to glucose ^[9]. The conversion of cellulosic mass to fermentable sugars through biocatalyst cellulase derived from cellulytic organisms has been suggested as a feasible process and offers potential to reduce use of fossil fuels and reduce environmental pollution ^[10]. Researchers have strong interests in cellulases because of their applications in industries of starch processing, grain alcohol fermentation, malting and brewing, extraction of fruit and vegetable juices, pulp and paper industry, and textile industry ^{[11, 12, 13, 14]}.

Several investigations have been carried out on the numbers of organic and inorganic nitrogen, carbon materials, pH values, temperatures and incubation periods affecting on the production of cellulase enzyme complex by several fungi [15-23]^[15].

The aim of the present investigation is to study the distribution and occurrence of various groups of fungi associated with 50 samples of wheat grains and 50 samples wheat flour collected from different mills in Taif city and cellulolytic activity of some fungal isolates and the effect of some environmental and nutritional factors on cellulase production Aspergillus niger and A.flavus.

2. Material and Methods

Fifty Samples of wheat grains (Triticum vulgare Vill., Hist. Pl. Dauphinè) and fifty samples of wheat flour (from the same grains) were collected from different mills in Taif city. Each sample was collected in a sterile polyethylene bag and transferred to mycological laboratory for fungal analysis. for isolation of various groups of fungi on glucose, Cellulose Czapek’s agar, yeast starch agar ^{[24, 25]} and Sabouraud's dextrose agar medium ^[26].

The dilution plate method was used as employed by ^{[27, 28]} for the estimation of fungal flora associated with wheat grains. A known weight of of wheat grains was suspended sterilized distilled water to obtain the desired final dilution. One mL of final dilution was transferred to a sterile petridish and poured with melted but cooled agar medium.

Four wheat seeds were placed on the surface of each glucose-, cellulose-, yeast starch- Czapek's agar and sabouroud's dextrose agar media. Four plates were used for each type of media. Plates were incubated at 28°C for 7 days and the developing fungi were counted and identified. The numbers were calculated per 16 seeds for each sample. The relative importance value (RIV) for each genus and species recovered was also calculated ^[29].

Forty-six species and 1 species variety belonging to 24 genera were screened for their abilities to produce cellulase, on solid media. The most active isolates were selected for further studies dealing with the effect of different environmental and nutritional factors on enzyme production.

1- Screening of fungal isolates for cellulase production:

Forty-six species and 1 species variety belonging to 24 genera were screened for their abilities to produce exo- and endo-β-1,4-glucanase (C₁ and C_x enzymes, respectively). Isolates were cultured on medium ^[30] of the following composition (g/L): (NH₄)₂SO₄, 0.5; L-asparagin, 0.5; KH₂PO₄, 1.0; KCL, 0.5; MgSO₄.7H₂O, 0.2; CaCl₂, 0.2; yeast extract, 0.5; cellulose microcrystalline, 10; agar, 20. pH was adjusted to 5.4 using acetate buffer. Using sterile cork borer, 10 mm diameter, discs were cut to inculate 50 ml sterile liquid medium (in 250 ml Erlenmeyer flasks) of medium ^[30] for exo-glucanase production and medium ^[31] for endo-glucanase. The later medium contained the following ingredients (g/L): NH₄NO₃, 2.1; KH₂PO₄, 1.0; MgSO₄. 7H₂O, 0.5; carboxymethyle cellulose (CMC), 10.0. Cultures were incubated at 28°C for 7 days. After 7 days of incubation at 28°C the cultures were filtered and the filtrates were used to detect the activity of the enzymes as follows:

a- Detection of exo-β-1, 4-glucanas (C₁ enzyme):

Using a sterile cork borer 3 cavities (10 mm diameter) were made in plates containing solid medium ^[30]. A 0.1 ml of culture filtrate was dropped in each of these cavities followed by incubation at 28°C for 24 hours, then the plates were flooded with chloroiodide of zinc solution and the uncolored zone gave a measure of cellulolytic power of isolates.

b- Detection of endo-β-1.4-glucanase (C_x enzyme):

Ten mm cavities were cut in plates containing solid medium of ^[32] of the following composition (g/L): carboxymethyle cellulose (CMC), 10; agar, 17; pH 5.4. A 0.1ml filtrate obtained from 7 days old fungal cultures grown on medium ^[31] was dropped in each cavity. After 24 hours of incubation at 28°C plates were flooded with chloroidide of zinc solution and the clear zone around cavities were measured.

2- Factors affecting cellulase production:

The effect of different ecological and nutritional factors on production of cellulase enzymes (C₁ and C_x) by Aspergillus niger and A. flavus, respectively. Since these species were found to be highly active in cellulase production so these species were used for this study. The previous isolates were grown on medium containing (g/L): NaNO₃, 5.0; KH₂PO₄, 1.0; MgSO₄. 7H₂O, 0.5; FeCl₃, 1.0 mg; Zn SO₄. 7H₂O, 0.9 mg; MnSO₄. H₂O, 0.4 mg; thiamine, 100 mg; biotin, 10 mg and cellulose powder, 10 g ^[33]. Fifty ml of the medium were dispensed into each 250 ml Erlenmeyer flask and each flask was inoculated with an agar mycelial disc (10- mm diameter) of the mould obtained from 7 days old fungal cultures growing on the solid basal medium. Experiments were done to indicate the best conditions which produce a good deal of the of the enzyme as well as of the best expence.

a- Effect of temperature and time course:

The inoculated flasks were incubated at 20, 30 and 40°C for 14 days and harvested at 48 hours intervals. Culture fluid were filtered and centrifuged at 5000 r.p.m. for 10 min. the clear supernatants were assayed for enzyme activity.

b- Effect of pH values:

The test isolates (A. niger and A. flavus) were grown on the basal medium of ^[33]. The initial pH of the medium was adjusted with 0.1N NaOH or 0.1N HCL to different values ranging from 2 to 12. After inoculation with A. niger and A. flavus, cultures were incubated at 20°C for 8 days for C₁ and C_x, respectively. At the end of incubation period the cultures were filtered, centrifuged at 5000 r.p.m. for 10 min. and the clear supernatants were assayed for cellulase activity.

c- Effect of different carbon sources:

The basal medium ^[33] with pH 8 (The best pH for cellulase production) was supplemented with 1% of one of the following carbon sources: glucose, fructose, lactose, sucrose, cellulose, starch and carboxymethyl cellulose. The flasks were inoculated with A. niger and A. flavus and incubated at 20°C (the best temperature of C₁ and C_x enzymes production) for 8 days (the best incubation periods for C₁ and C_x enzymes, respectively) and the cultures were filtered. After centrifugation the filtrate was used to detect the cellulase activity.

d- Effect of various nitrogen sources:

To determine the effect of nitrogen source on cellulase production, sodium nitrate (2 g/L) in the basal medium was replaced by the same amount of various nitrogen compounds such as; sodium nitrate, potassium nitrate, yeast extract, ammonium sulphate, ammonium nitrate and peptone in addition to sodium nitrate as a control. Cultures were incubated at 20°C for 8 days after 8 days cultures were filtered, centrifuged and the filtrates were used for detection of cellulase activity.

3- Assay for cellulase activity (C₁ and C_x enzymes):

The method described by ^[34] and modified by ^[35] was emplyed as follows: each of 50 mg of filter paper (Watmann No. 1) and 1 ml of 1% CMC were added separately to 1 ml of acetate buffer (pH 6) and 1 ml of each culture filtrate. The mixture was incubated for 30 min. at 25°C for assaying activity of C₁ and C_x enzymes, respectively. Similar reaction mixtures using heated inactive enzyme solution were also prepared as controls and water with reagents as a blank. 3 ml of Nelson's solution were added and the reaction mixtures, were shaken and placed in a boiling water bath for 15 min. After cooling, 3 ml of the arsenomolybdate solution was added, mixed throughly and then diluted to 10 ml with distilled water. The whole mixture was centrifuged to remove any turbidity. The amount of reducing sugars produced was estimated by determining the optical density (absorption spectrum) at 700 nm wave length with a spectrophotometer model (Spectronic ® Genesys ^TM 2PC USA). A standard curve was plotted using aqueous solution of D-glucose.

3. Results and Discussion

Thirty-one species belonging to 16 genera were collected from wheat grains on plates of glucose - (16 genera and 26 species), cellulose- (16 species and 25 genera) Czapek's agar, yeast-starch agar (12 species and 19 genera) and Sabouraud's - dextrose agar media (22 species and 10 genera) at 28°C. The most common genera on four types of media were: Alternaria (2 species), Aspergillus (10), Candida and Chrysosporium (3). They were emerged in 26-88% of the samples comprising 5.4-65% of total fungi and had RIV's 34.5-105.9. From the above genera the most prevalent species were: Alternaria alternata, Aspergillus flavus, A. niger and Chrysosporium luknowense (Table 1 and Table 2).

Table 1. Total counts, (calculated per 48 seeds), number of cases of isolation (NCI, out of 50), occurrence remarks (OR) and the relative importance value (RIV) of fungal genera and species from grans of Triticum vulgare on glucose, cellulose-Czapek's agar and starch at 28°C

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Table 2. Total counts, (calculated per 48 seeds), number of cases of isolation (NCI, out of 50), occurrence remarks (OR) and the relative importance value (RIV) of fungal genera and species from grans of Triticum vulgare on sabouraud's dextrose at 28°C

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^[36] could isolated 117 different species belonging to 31 fungal genera contributed the mycobiota of stored wheat on glucose- Czapek's agar medium. The most common species isolated in decreasing orders of frequency were: Aspergillus flavus (72.5%), A. niger (71.3%), P. chrysogenum (47.5%), P. cirinum, P. oxalicum, (40% for each), A. sydowii, A. terreus (35% for each), A. flavus var. columnaris, Rhizopus stolonifer (35% for each), A. ochraceus (27.5%), A. flavipes (26.3%) and Alternaria altrnata (25%). The data obtained by ^[37] indicated that wheat grains were contaminated with many fungal and the most common genera were: Aspergillus, Fusarium (F. graminiarium and F. verticillioides) and Penicillium. ^[38] found five pathogens to be wide spread on commercial barely seeds on plates of Sabouraud's - dextrose agar medium, and these were: Drechslera teres, D. graminea, Septoria nodorum, D. sorokiniana and Fusarium nivale. ^[39] studied the mycoflora of Algerian wheat. The commonly isolated fungi on dichloran rose-bengal chlramphenicol agar medium were species of Aspergillus, Fusarium, Penicillium, Alternaria and Mucor. Aspergillus was the genus most detected at higher frequency. Among the Aspergillus species isolated A. flavus, A. niger and A. versicolor. ^[40] isolated ten fungal species belonging to seven different genera from wheat grains and these were: Aspergillus, Alternaria, Cladosporium, Fusarium and Penicillium of the Phylum Ascomycota; Mucor and Rhizopus of the Phylum Zygomycota. The frequency of Aspergillus. niger, A. fumigatus and Alternaria alternate were higher (33.76 to 40.34%) than the other fungal species identified. The frequency of A. niger was highest which is quite alarming because this strain can produce ochratoxins. Out of the Penicillium species isolated, the commonly found species were P. expansum, and P. citrinum with comparatively low percentage. ^[41] found the most common moulds contaminated the wheat storage (silos) in Golestan Provine, North of Iran were Alternaria spp. 26.7%, Aspergillus niger 21.4%, Fusarium spp.17.8%, Aspergillus flavus 10.7%, Clasdosporium spp.10.7%, Penicillium spp. 8.9% and Rhizopus spp.3.5%. ^[42] found that Fungi associated with wheat grain samples collected from five different Gaza governorates were were Aspergillus flavus 84%, Aspergillus parasiticus 72%, Fusarium oxysporum 64%, Aspergillus niger 48%, Alternaria alternate 36%, Penicillium 22%, Aspergillus ochraceus 20% and Aspergillus versicolor 4%.

Thirty-three species belonging to 17 genera were collected from wheat flour on plates of glucose- (16 genera and 32 species), cellulose- (16 genera and 32 species) Czapek's agar, yeast-starch agar medium (15 genera and 23 species) and Sabouraud's - dextrose agar media (9 genera and 23 species) at 28°C. The most common genera on four types of media were: Aspergillus (10 species), Chrysosporium (3), Cladosporium (1) and Penicillium (5); emerging in 28-100% of the samples comprising 16.2-44.5% of total fungi. From the above genera the most common species were: Aspergillus flavus, A. fumigatus, A. niger, Chrysosporium lucknowense, Cladosporium cladosporioides, Pencillium chrysogenum and P. duclauxii (Table 3 and Table 4).

Table 3. Average total counts, maximum values (calculated per g fresh weight flours in every samples), number of cases of isolation (NCI, out of 50) and occurrence remarks (OR) of fungal genera and species recovered from flour of Triticum vulgare on glucose, cellulose- Czapek's agar and starch at 28°C

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Table 4. Average total counts, maximum values (calculated per 50 g weight flours in every samples), number of cases of isolation (NCI, out of 50) and occurrence remarks (OR) of fungal genera and species recovered from flour of Triticum vulgare on sabouraud's dextrose at 28°C

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^[43] made mycological examination on two German wheat flours and collected 45 different species from whole wheat flour and 49 species from white flour on dichloran rose-bengal chloramphenicol (DRBC) agar medium. The most common genera on whole wheat flour were: Aspergillus (83.7%), Penicillium (7.6%), Eurotium (2.9%) and Alternaria (2.5%). The white flour contained Aspergillus (77.3%), fungi of the genera Penicillium (15%) and Cladosporium (4.1%) were of minor importance. The most common species from two types were: Aspergillus candidus, Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides, C. herbarum and Penicillium aurantiogriseum. ^[44] made mycological examination on wheat flours in a local super - markets in Egypt. Forty two different species were collected. The most common isolated molds on Czapek's – dextrose agar media and potato dextrose agar (PDA) medium belong to the genera Aspergillus, Alternaria, Penicillium and Cladosporium. Aspergillus flavus was the most dominating mold followed by Penicillium duclauxii, Alternaria alternata, and Cladosporium cladosporioide.

^[45] found that the most prevalent genera recovered from white wheat flour on plates of malt extract agar and Czapek's -dextrose agar media were Aspergillus (73.7%), Penicillium (5.6%), Eurotium (3.9%) and Alternaria (1.5%). ^[46] were isolated Absidia, Alternaria, Aspergillus, Cladosporium Epicoccum, Eurotium, Fusarium, Mucor, Penicillium and Rhizopus from wheat flour mill in Argentina. ^[47] were isolated Aspergillus, Acremonium, Alternaria, Fusarium, Mucor, Penicillium and Cladosporium spp. from consumed flour in the bakeries of Tabriz city. ^[48] were isolated Aspergillus, Penicillium, Fusarium, Cladosporium, Alternaria, Mucor, Rhizoctonia, Trichoderma, Rhizopus, Nigrospora, Bipolaris, Macrophomina from wheat flour samples by using PDA (Potato dextrose Agar) medium according to decreasing frequency.

Table 5. Activity of exo-B-1,4glucanase (C₁) (calculated as average diameter of clear zone in mm) of the fungal isolates

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Forty-six species and 1 species variety belonging to 24 genera were screened for their abilities to produce C₁ and C_x enzyme on solid media proved to be active to utilize cellulose, but with different degrees. Two isolates (4.2% of total isolates) showed high cellulolytic activity in both exo- and endo-β-1,4- glucanases and these were: Aspergillus flavus and A. flavus var. columnaris. Ten isolates (20% of total isolate) showed high cellulolytic activities in production of C₁ enzyme only and these were: Acremonium strictum, Aspergillus flavus, A. flavus var. columnaris, A. fumigatus, A. niger, Cochliobolus lunatus, Fusarium oxysporum, Penicillium steckii, Trichoderma hamatum and T. viride. On the other hand five fungal isolates (10.4% of total isolates) showed high cellulolytic activities for C_x enzyme only, and these were: Aspergillus flavo-furcatis, A. flavus, A. flavus var. columnaris, Nectria haematococca and Penicillium citrinum. Twenty-six and thirty-eight isolates (55% and 79.2% of total isolates) were found to be moderate production of C₁ and C_x enzymes, respectively, while 11 and 5 isolates (25% and 10.4% of total isolates) were of weak cellulolytic activity (Table 5 and Table 6). Most of the above fungal isolates were reported as cellulase producers, but with variable capabilities by several workers [15,17,18,19,22,23,49-57].

Table 6. Activity of endo-B-1,4glucanase (C_x) ( calculated as average diameter of clear zone in mm) of the fungal isolates

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Figure 1. Effect of time course and temperature, pH values, carbon sources and nitrogen sources on production of exo-ß-1,4-glucanase(C₁) by Aspergillus niger

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Figure 2. Effect of time course and temperature,pH values, carbon sources and nitrogen sources on production of endo-ß-1,4-glucanase(C_x) by Aspergillus flavus

Aspergillus niger and A. flavus headed the list of the most active cellulase producers (for C₁ and C_x, respectively), so they were chosen for further studies to achieve the most favourable environmental and nutritional conditions for C₁ and C_x enzymes production. Maximum production of exo and endo-β-1,4-glucanase by A. niger and A. flavus were obtained after 8 days of incubation at 30°C with culture media contain cellulose and glucose as a carbon sources and sodium nitrite as nitrogen source and the culture medium was initially adjusted to pH 8 (Figure 1 and Figure 2). These findings are almost in agreement with those reported by ^[54] studied the effect of environmental factors on production of cellulase enzyme by Aspergillus fumigatus and A. niger. They reported that the optimum pH for cellulase production 6 to 7 and optimum temperature around 40°C. Also, ^[18] found that maximum production of endo -ß- 1,4 glucanase by Cheatomium globosum was achieved 6 days after incubation at 30°C with incorporation of maltose as carbon source and NH₄NO₃ as nitrogen source in the culture medium which is initially adjusted to pH6. Recently ^[23] found that maximum production of exo- and endo-ß-1,4 glucanase by Mucor circinelloides and Aspergillus flavus was achieved 6 days after incubation at 30°C with incorporation of fructose or sucrose as a sole carbon source and potassium nitrate or sodium nitrate as a sole nitrogen source, respectively in the basal medium initially adjusted to pH 6.

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