Introduction

Lipases induce the hydrolysis and the creation of esters formed from glycerol and long chain fatty acids. Microbial lipases are commercially important as they are more steady if it is put in compare with plant and animal lipases and they can be got an inexpensive way. The implementations of lipases include organic synthesis, hydrolysis of fats and oils, modification of fats, flavor improvement in food processing and chemical tests (Martinelle et al., 1995; Sharma et al., 2001).  When lipolytic fungi contaminating meat products, they may be responsible for the unpleasant or undesirable flavors rendering them unmarketable or even not suitable for consumption. Also, protein hydrolysis by fungi in foods may produce a variety of odor and flavor defects. Thus, selected sterilization temperature may confer microbial sterility, but not inactivate considerable portion of microbial lipase leading to later loss of product. This substantiates the hypothesis that reported by (Smith and Hass 1992). Mycotoxins are low molecular weight yields of the fungal secondary metabolite, created mainly by Aspergillus, Penicillium, Fusarium and Alternaria genera. These metabolites are virulent for humans and animals if they expend or respire, and exposition to mycotoxins through the contaminated feed is one of the main hazards affecting ruminant health (Bennet and Klich, 2003; Kalac and Woolford, 1982). The contamination of meat with mold leads either to food poisoning or spoilage and render it to be in less quality (Rieth, 1973; Mossel, 1977 and Waffia and Hassan, 2000). Also, the consuming of the contaminated meat by mold and their mycotoxins induces hemorrhages with hepatotoxic, nephrotoxic, neurotoxic, dermatotoxic, genotoxic, teratotoxic, carcinogenic or hormonal effects and immunosuppression (Cheo, 1991 , Hassan et al., 2004 and Abd – Elghany & Sallam 2015 ).

Materials and Methods

Screening of Fungal Isolates for Lipase Production

The lipolytic action was scaled using tributyrin as a lipid substrate (Cardenas et al., 2001). The medium used contained (g/l) peptone, 10g; MgSO₄.7H₂O, 2g; CaCl₂.2H₂O, 0.2g; tributryin emulsion, 10 (w/v); agar, 15g with pH 6. Tributyrin was sterilized separately by autoclaving for 15 minutes at 1.5 atm. and 1 ml was added to 100 ml of the sterile basal medium before solidification. Using a sterile crock borer (10 mm diameter) inoculum discs bearing mycelia from a previously prepared agar culture were obtained. Disc in triplicates was placed in cavities made in the assay agar medium. The cultures were incubated for 48 hours at 25⁰C. The diameter of the clear zone around each disc was measured and the average was calculated.

Assay for Lipase Activity

The method described by Mateos Diaz et al. (2006) was employed. This method was designed to estimate the amount of free fatty acids (FFA) in m mole lose by the enzyme action. 10 ml of olive oil emulsion solution (0.1% olive oil) was diluted by 20 ml distilled water and added to 0.25 ml of sodium taurocholate and 5 ml of the crude enzyme (fungal filtrate), pH was adjusted to 8.8. After 20 minutes of incubation at 30⁰C with shaking, the mixture titrated with 0.05 M NaOH until pH 8.8 (the original pH). The consumed volume of NaOH was estimated and the volume activity calculated. Samples with 5 ml distilled water (replaced the enzyme solution) was used as a blank.

Factors affecting on lipase production.

The effect of some ecological and nutritional factors on the production of lipase enzyme by two fungal species ) Aspergillus flavus and Scopulariopsis fusca) were studied. Since these species were found to be the most active in lipase production.

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. Cultures fluids were filtered and assayed for lipase production.

Effect of pH Values

The test organisms (Aspergillus flavus and Scopulariopsis fusca) were grown on the medium described earlier. The initial pH values of the medium were regulated with citrate buffer to various values from 2 to 14. Inoculated flasks were incubated for 6 days at 30°C (the best temperature and time course for lipase production), then filtered and assayed for lipase production.

Effect of Carbon Sources

To study the effect of oils as carbon sources on lipase production by chosen fungal species, the basal medium previously mentioned was supplemented sporadically with 0.1% emulsion of various oils and pH of the medium was adjusted to 6 (the best pH for lipase production). These oils were: sunflower oil, flax oil, olive oil, peanuts oil, hsalban oil. Inoculated flasks were incubated at 30°C for 6 days, then filtered and the supernatant was utilized for the assay of the enzyme activity.

Effect of Nitrogen Sources

To estimate the effect of nitrogen sources on the production of lipase enzyme the 3 g/L of sodium nitrate as inorganic nitrogen source in the basal medium was substituted by an equivalent amount of different nitrogen compounds for example yeast extract, peptone, ammonium molybdate, ammonium sulfate, and potassium nitrate as well as sodium nitrate as control. the pH of the medium was adjusted to 6. Inoculated flasks were incubated at 30°C for 6 days. After the incubation period was ended, the enzyme activity was assayed in the supernatant filtrate.

Screening of Fungal Isolates for Mycotoxins Production:

Fungal Isolates

A total of 4 isolates of prevalent fungi, from fresh cow meat belonging to Aspergillus fumigatus, A. niger and Penicillium chrysogenum (2 isolates) were examined for their ability for production of mycotoxins.

Cultivation and Inoculation of Fungal Cultures

Sucrose yeast extract liquid medium was used for mycotoxins screening. The composition of sucrose yeast extract liquid medium was sucrose, 40 g, and yeast extract, 20 g per liter of distilled water (pH 6±0.2). Erlenmeyer flasks of 250 ml capacity were used. Each flask contained 50 ml of the desired medium. Flasks were sterilized at 1.5 atmospheres for 20 min. and inoculated after cooling with 2 discs taken from 7 day-old culture. Cultures were incubated for 15 days at 28°C (Gabal et al., 1994).

Detection of Mycotoxins

The levels of mycotoxins were measured by a fluorometric method using aflatoxins or ochratoxins standards for adjustment of fluorometer before reading the toxins content of the tested samples (Hansen, 1993; VICAM, chemists. Inc., U.S. America).

Results and Discussion

Lipolysis Activity

Four isolates (5.4 % of total isolates) exhibited high lipolytic activity     (clear zones 14-17 mm) and these isolates were: Aspergillus fumigatus, A. flavus, Cladosporium spongiosum, and Scopulariopsis fusca. Thirty-five isolates (47.3% of total isolates) were found to be of moderate lipolytic activity and the weakly lipolytic (clear zone less than 10) isolates were represented by 35 isolates (47.3% of total isolates) as shown in the table (1). These results similar to those obtained by Abdel-Rahman and Saad (1989) and Banwart (1989) found that fungi isolated from meat and meat products e.g. Aspergillus, Fusarium, Rhizopus, Penicillium and other had lipolytic and proteolytic activity. Godtfredsen (1990) who reported that lipolytic microorganisms are the widest spread such as Aspergillus niger, A. flavus and A. fumigatus that produce lipases it is to note that, microbial lipases are often heat resistant. In Egypt, Tawakkol and Khafaga (2007) found that all the examined species of Aspergillus (A. niger, A. flavus, A. ochraceus, A. terreus, A. parasiticus and A. candidus), Penicillium (P. chrysogenum, P. citrinum, P. expansum and P. oxalicum), Scopulariopsis spp, Mucor spp, Rhizopus spp, Candida albicans and Fusarium spp isolated from cattle and buffalo meat had lipolytic activity. In Qena city, Saleem (2008) screened a total of thirty-one fungal species and 3 species varieties isolated from beef luncheon meat for their abilities to produce lipase enzyme. He found that ten isolates represented 32.26% of total isolates appeared high lipase production, while sixteen isolates (51.61%) were moderate and 5 isolates (16.13%) were low producers. Aspergillus niger, Fusarium oxysporum, and Nectria haematococca produced the highest amount of lipase enzyme so he used these isolates in further studies. The integration of five food preservatives (Disodium phosphate, sodium benzoate, citric acid, potassium sorbate, and sodium citrate) individually in the culture medium of lipase production exhibited an inhibitive effect on the mycelial growth and enzyme production by the three tested isolates. Ouf et al. (2010) reported that most isolates of Aspergillus flavus, A. niger, Cladosporium spp., Mucor spp. and Penicillium spp. Isolated from meat and meat products have the ability to produce lipase and protease enzymes.

Table 1: Degree of lipolytic activity (calculated as the average diameter of the clear zone in mm) of the tested fungal isolates.

Table 1: Continue

Table 1: Continue

*Activity Remarks: High activity, H= from 14 – 17 mm; Moderate activity, M= from 11 – 13 mm; Weak activity, W= less than 10 mm.

Effect of Environmental and Nutritional Factors on Lipase Production

Effect of Temperature and Time Course

Maximum lipase production by both of Scopulariopsis fusca and Aspergillus flavus was achieved  6 days after incubation at 30⁰C. Cultures incubated at 40⁰C produced the lowest quantity of the enzyme whereas those incubated at 20⁰C yielded moderate amounts of lipase enzyme (Figs. 1 and 2).

Figure 1: Effect of time course and temperature (A), pH values (B), different carbon sources (C) Click here to view figure

Figure 2: Effect of time course and temperature (A), pH values (B), different carbon sources (C) Click here to view figure

Effect of pH Values

The data represented in Figs. (1 and 2) shows that considerable amounts of lipase enzyme were detected by Scopulariopsis fusca and Aspergillus flavus if the culture medium was initially adjusted at a pH range of 4-8 and the maximum production could be achieved at pH 6. Synthesis of lipase enzyme was greatly inhibited at pH lower than 4 or higher than 10.

Effect of Different Carbon Sources

Among the 5 carbon sources incorporated separately in culture medium olive oil and sunflower oil yielded the highest amounts of lipase enzyme by Scopulariopsis fusca and Aspergillus flavus, respectively. On the other hand considerable amounts of lipase enzyme were produced in case of using hsalban oil and equal amounts of lipase enzyme were produced in case of using sunflower oil, flax oil and peanuts oil by Scopulariopsis fusca but in case of Aspergillus flavus; moderate amounts of lipase enzyme were produced by using hsalban oil, flax oil, peanuts oil and olive oil (Figs. 1 and 2).

Effect of Different Nitrogen Sources

As presented in Figs. (1 and 2)ammonium molybdate and potassium nitrate were the best nitrogen sources required to induce the highest yield of lipase enzyme by Scopulariopsis fusca and Aspergillus flavus, respectively. In case of Scopulariopsis fusca moderate amount of lipase enzyme was produced by the incorporation of yeast extract and similar amounts were produced by using peptone and potassium nitrate and the lowest amount of lipase enzyme was yielded by using ammonium sulfate. On the other hand, the moderate amount of lipase enzyme was produced by Aspergillus flavus in the presence of ammonium molybdate and equal amounts were produced by using ammonium sulfate, peptone and yeast extract.

These findings are almost in agreement with those reported by Rapp (1995) found that the maximum production of lipase enzyme by Fusarium oxysporum f. sp. vasinfectum required peptone. Additions of trimyristin, olive oil, span 85, and oleic acid to growing shake-flask cultures, and the addition of oleic acid to suspensions of washed mycelium. Synthesis of lipase activity was repressed by glucose and glycerol. Lipase activity was inhibited by oleic acid and its sodium salt, but not by glycerol. The lipase activity remained constant during 1h incubation at pH 4 to 10.5 and 30⁰C. It was stable in 25% (v/v) acetone, ethanol, and n-propanol and was catalyzed remarkably in n-hexane.  Kamini et al., (2000) inspected a number of factors affecting the production of extracellular lipase by Cryptococcus sp. S-2. Sequential optimization of nitrogen, carbon sources and inducers promoted lipase activity and under optimum conditions, the lipase activity was 65.7 U/ml of the culture medium in 120 h at 25⁰C and at pH 5.6. Sardine oil, soybean oil, and triolein oil were active inducers of lipase production. Abbas et al. (2002) isolated Mucor sp strain during a screening of lipolytic fungus which may play a role in the acidification of palm oil. He found that the highest lipase production amounting to 57 U/ml when culture conditions were optimized was achieved after 6 days of cultivation. ul-Haq et al. (2002) screened ten mold cultures for the production of lipases. He found that Rhizopus oligosporous GCBR-3 supported maximum production of lipase (48.0U/g substrate) under optimum environmental and cultural conditions such as wheat bran/flask and incubation at 30⁰C for 48 h. Tan et al. (2004) optimized the culture medium including nitrogen source, carbon source, and metal ions, for lipase production from Penicillium camembertii Thom PG-3 and the optimal medium consisted of soybean meal (fat free) 4%, Jojoba oil 0.5%, (NH₄)₂HPO₄, 0.1% Tween 60, initial pH 6.4 and the inoculation was at 28⁰C for 96 h. The optimal pH and temperature for lipase activity were 6.4 and 48⁰C, respectively. Liu et al. (2009) studied the effects of temperature, pH, and various components in detergent on the activity and stability of a lipase produced by a soda lake fungus strain Fusarium solani N4-2. The lipase showed the maximal activity at pH 9.0 in a glycine-NaOH buffer at 30⁰C. At 0⁰C and 10⁰C, 52.3 % and 82.6% its maximum activities were detained, respectively.

The Occurrence of Mycotoxins in the Culture of Most Common Fungi

The results in table (2) revealed that the four tested isolates had ability for aflatoxins and ochratoxins production and the results were confirmed by HPLC analysis as shown in tables (3, 4, 5 and 6) and these results in agreement with Glinsukon et al., (1979) found that A. niger produces low level of aflatoxin.  Mahmoud et al. (2001) found that many of isolated fungi from local meat products (luncheon and minced meat) and livers of poultry and imported bulls have mycotoxin producing potential. Results of mycotoxins analysis revealed that 45 % of the examined samples were positive. Aflatoxins (B1, B2, G1, and G2), ochratoxin A, citrinin and sterigmatocystin were detected. Samples of livers imported bulls contained the highest level of aflatoxins and ochratoxin A. Varga et al. (2001) reported that Aspergillus auricomus, A. fumigatus, A. glaucus, A. melleus, A. ostianus, Penicillium aurantiogriseum, P. canescens, P. chrysogenum, P. commune and others had ability to produce ochratoxin A. Czerwiecki et al. (2002 a, b) found that Penicillium chrysogenum, P. cyclopium, P. griseofulvum, P. oslitum, Aspergillus flavus, A. versicolor and Eurotium glaucum were ochratoxin A producers. Frisvad et al. (2006) found that Aspergillus flavo-fucus, A. glaucus, A. niger, A. oryzae, A. ostianus, A. sulphureus, A. tamarii, Penicillium citrinum, P. citromyces, P. digitatum, P. frequentans, P. expansum, P. puberulum, and others could produce aflatoxins. Tawakkol and Khafaga (2007) tested 27 strains of Aspergillus flavus isolated from 200 samples of meat and 120 environmental samples for the production of aflatoxins, 21 isolates (77.7%) were found toxigenic. They could produce aflatoxins B1, B2 or G1 either singly or combined. Also, they found that 15 out of 26 isolates of Aspergillus ochraceus produced ochratoxins. Abdel- Kareem (2010) revealed that four isolates out of nine isolates of A. niger and 3 isolates of A. ochraceus could produce ochratoxin A. De Curtis et al., (2012) reported ochratoxin A production is mainly associated with Aspergillus  carbonrius and A. niger or nigris section species. Abd – Elghany & Sallam (2015) studied total aflatoxins (AFT) and ochratoxin A (OTA) levels were evaluated using the VICAM AflaTest and OchraTest immunoaffinity fluorometric method in a total of 50 meat products (25 each of beef luncheon and beef burger) bought from different supermarkets in Mansoura city, Egypt. All the meat samples analyzed were contaminated with both AFT and OTA with mean values of 1.1 μg/kg and 5.23 μg/kg, respectively, for beef luncheon and mean values of 3.22 μg/kg and 4.55 μg/kg, respectively, for the beef burger.

Table 2: Mycotoxigencity of the tested fungal isolates

Table 3: HPLC analysis of aflatoxins and ochratoxins produced by Aspergillus fumigatus

Table 4: HPLC analysis of aflatoxins and ochratoxins produced by Penicillium chrysogenum (1)

Table 5: HPLC analysis of aflatoxins and ochratoxins produced by Penicillium chrysogenum (2)

Table 6: HPLC analysis of aflatoxins and ochratoxins produced by Aspergillus niger

Acknowledgments 

The authors are thankful to all college in Botany and Microbiology Department, South Valley University. 

Conclusion 

The results confirmed that all the fungal isolates can produce lipase enzyme with variable degrees. The high producer’s isolates were Aspergillus flavus and Scopulariopsis fusca so they were chosen for moreover studies for optimization of environmental and nutritional conditions for output of lipase. The results affirmed that ideal temperature, incubation period and pH for maximum production by the two strains were; 30⁰C, 6 days  and pH 6 but the difference in the used carbon and nitrogen sources as follow olive oil and ammonium molybdate and sunflower oil and potassium nitrate as a sole carbon and nitrogen sources in basal medium which is initially adjusted to pH 6 for maximum lipase production by Scopulariopsis fusca and Aspergillus flavus, respectively. Four isolates were used as a proof for production of aflatoxins and ochratoxins by the fluorometric method the results confirmed that all the tested isolates had the ability for aflatoxins and ochratoxins production and HPLC analysis confirmed these results.

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