Effect of some food preservatives on the lipolytic activity of beef luncheon fungi.

Beef luncheon meat is one of the most popular meals in several countries in the world including Egypt. Thirty one fungal species and 3 species varieties were recovered from 30 samples of beef luncheon meat collected from different supermarkets in Qena. Alternaria, Aspergillus, Emericella, Mucor, Mycosphaerella, Penicillium and Rhizopus were the most common genera on the two types of media. From the above genera, the most prevalent species were Alternaria alternate, Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Emericella nidulans, Mucor racemosus, Mycosphaerella tassiana, Penicillium chrysogenum and Rhizopus stolonifer. Screening of fungi for their abilities to produce lipase enzyme showed 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 these fungi were used in further studies. The incorporation 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 Aspergillus niger, Fusarium oxysporum and Nectria haematococca.

Meat is a rich nutrient matrix that provides a suitable environment for proliferation of meat spoilage microorganisms and common food-borne pathogens, therefore adequate preservation technologies must be applied in order to preserve its safety and quality (Aymerich et al., 2008). Microscopic filamentous fungi often contaminate vegetal and animal products, becoming a source of diseases for human and slaughter animals (Laciakova and Laciak, 1994). The conditions of the environment in the manufacturing rooms, stores, refrigerators and shops sometimes are very suitable for the development of moulds inside the products, more frequently on the surface of various sorts of meat and meat products (Jesenska, 1987). Feeds and foods are often contaminated with various moulds that have been described as pathogens. These are particularly found in products that have ripened for a long time, such as sausages and hams (Andersen, 1995). Aspergillus, Cladosporium and Penicillium species have been identified in such products. Leistner (1986) and Casado et al. (1991) supposed that fermented sausages are spontaneously colonized by domestic microflora. The composition and development of mycoflora depends on the type of products, processing time, and the conditions of ripening. The dominant mycoflora usually belongs to Aspergillus, Scopulariopsis and Penicillium species (Grazia et al., 1986; Hwang et al., 1993). Recently, Mizakova et al. (2002) studied the occurrence of mould in pork and beef used as raw materials, in salami emulsions as well as in five kinds of fermented raw meat products. Penicillium, Acremonium, Mucor, Cladosporium and Aspergillus were the most frequently isolated genera of mould.

Lipases (triacylglycerol acylhydrolases) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long chain fatty acids. Microbial lipases are commercially significant. The applications of lipases include organic synthesis, hydrolysis of fats and oils, modifications of fats, flavor enhancement in food processing, resolution of racemic mixtures and chemical analysis (Martinelle et al., 1995; Sharma et al., 2001). In eukaryotes, lipases are involved in various stages of lipid metabolism including fats digestion, absorption, reconstitution and lipoprotein metabolism (Balashev et al., 2001). Because of their wide ranging significance, lipases remain a subject of intensive study (Alberghina et al., 1991; Bornscheuer, 2000).

To prevent the development and spread of spoilage and pathogenic microorganisms via meat foodstuffs, antimicrobial packaging materials could be a potential alternative solution (Quintavalla and Vicini, 2002; Coma, 2008). The most common antimicrobials used by researchers are the various organic acids. Organic acids have characteristic sensitivities to microorganisms, sorbic acids and sorbates are very strong antifungal agents (Han, 2005). Acetic acid, lactic acid or potassium benzoate leads to a reduction of Listeria monocytogenes populations in smoked sausage (Geornaras et al., 2006; Coma, 2008). This article was conducted to determine the occurrence and distribution of moulds in beef luncheon meat as well as the effect of five food preservatives on the activity of fungal lipases and biodegradation of food.

Materials and Methods

Collection of beef luncheon samples

Thirty beef luncheon meat samples were collected randomly from different supermarkets in Qena during the period from May to July 2007. The samples were collected in sterilized polyethylene bags and transferred directly to the Mycological laboratory for fungal analysis.

Determination of beef luncheon fungi

The baiting technique was used for determination of fungi on two types of media, Glucose-Czapek's and Malt extract agar at 25℃. Beef luncheon meat was cut into equal segments (1 cm2 each). Four segments were placed on the surface of the agar medium in each plate. Five replicates were prepared for each sample and each type of cultivation media. Cultures were incubated at 25℃ for 7 days. At the end of the incubation period, the developing fungi were counted, identified and calculated/20 segments.

Media used for isolation of fungi

Glucose-Czapek's agar medium (g/l; sodium nitrate, 3.0; potassium dihydrogen phosphate, 1.0; Magnesium sulphate, 0.5; potassium chloride, 0.5; ferrous sulphate, 0.01; glucose, 10.0; agar 15.0) was used for isolation of glucophilic fungi. Malt extract agar medium is favorable for the isolation of different genera and species of zygomycetes with chemical composition: 25.0 g Malt extract and 15.0 g agar per liter. Chloramphenicol (0.5 mg/ml) was used as bacteriostatic agent (Al-Doory, 1980).

Screening of fungi for lipase production

The lipolytic activity was measured using tributyrin as a lipid substrate (Cardenas et al., 2001). The medium used contained (g/l) peptone, 10 g; Mg SO4·7H2O, 2 g; CaCl2·2H2O, 0.2 g; tributyrin emulsion, 10 (w/v); agar, 15 g 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 sterile basal medium before solidification. Using a sterile cork borer (10 mm diameter) inoculum discs bearing myclelia from a previously prepared agar culture were obtained. Discs in triplicates were placed in cavities made in the assay agar medium. The cultures were incubated for 48 hours at 25℃. The diameter of clear zone around each disc was measured and the average was calculated in mm.

Effect of food preservatives on lipase production

Aspergillus niger, Fusarium oxysporum and Nectria haematococca were selected since these species were found to be active lipase producers. Fifty ml aliquots of modified Czapek's liquid medium (g/l: NaNO3, 3.0; KH2PO4, 1.0; MgSO4·7H2O, 0.5; KCl, 0.5; FeSO4 0.01 and Olive oil emulsion, 10 ml) were individually dispensed in 250 ml Erlenmeyer conical flasks. PH of the medium was adjusted to 6.5 using citrate buffer. The flasks were autoclaved for 15 minutes at 1.5 atm. Five types of food preservatives (sodium citrate, potassium sorbate, disodium phosphate, citric acid and sodium benzoate) which are most common in food industries in Egypt, were added under aseptic conditions to the sterilized liquid medium at rates of 100, 200, 400 and 800 ppm. Media without preservatives served as control. Each flask was inoculated with two agar mycelial discs (10 mm diameter) obtained from 7 days old cultures. Inoculated flasks were incubated for 7 days at 25℃, then filtered. The filtrate was centrifuged and the clear supernatents were used for assaying of lipase activity. Mycelial dry weights of the tested fungi were also determined.

Assay for lipase activity

The method described by Mateos Diaz et al. (2006) was employed. This method was designed to calculate the amount of free fatty acids (FFA) in m mole liberated 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 30℃ with shaking, the mixture titrated with 0.05 M NaOH until pH 8.8 (the original pH). The consumed volume of NaOH was determined and the volume activity calculated. Samples with 5 ml distilled water (replaced the enzyme solution) was used as a blank.

Results and Discussion

Thirty one fungal species and 3 species varieties were recovered from 30 samples of beef luncheon meat on glucose-Czapek's (18 Genera + 30 species + 3 variety) and Malt extract (19 Genera + 31 species + 3 variety) agar at 25℃. Alternaria (1 species), Aspergillus (8+2 var.), Emericella (1 + 1 var.), Mucor (2), Mycosphaerella (1), Penicillium (5) and Rhizopus (1) were the most common genera on the two types of media. From the above genera, the most prevalent species were Alternaria alternate, Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Emericella nidulans, Mucor racemosus, Mycosphaerella tassiana, Penicillium chrysogenum and Rhizopus stolonifer (Table 1). These species were isolated with different numbers and frequencies from various types of meat products in different places of the world by several workers (Grazia et al., 1986; Casado et al., 1991; Hwang et al., 1993; Mizakova et al., 2002). Recently, Mizakova et al. (2002) isolated 78 species of moulds from meat and various meat products. The most frequently isolated genera were Penicillium, Acremonium, Mucor, Cladosporium and Aspergillus. Most of them are reported to be toxicogenic (Zaky et al., 1995; Ostry, 2001; Mizakova et al., 2002).

Table 1

Total counts (TC, calculated per 20 segments in all samples), number of cases of isolation (NCI, out of 30), occurrence remarks (OR) and the relative importance values (RIV) of fungal genera and species recovered from beef luncheon meat on glucose-Czapek's and Malt extract agar at 25℃

H = High occurrence, 15~30, M =Moderate occurrence, 8~14, L = Low occurrence, 4~7, R = Rare occurrence, 1~3.

Screening of fungal isolates for their abilities to produce lipase enzyme

Thirty one fungal isolates representing 16 genera, 28 species and 3 varieties were tested for their ability to produce lipase enzyme. The result obtained indicated that, all fungi tested are able to produce lipase enzyme, but with variable degrees (Table 2). In this respect, ten isolates represented 32.26% of total isolates appeared high lipase production, while sixteen isolates (51.61%) were found to be moderate and 5 isolates (16.13%) were low producers. Aspergillus niger, Fusarium oxysporum and Nectria haematococca were on the top for enzyme production, so these fungi were selected for further studies. Nagy et al. (2006) screened thirty-eight filamentous fungi cultivated under solid state fermentation conditions for lipase production. Many of these fungi yielded good activities of lipase enzyme. Gliocladium roseum and Trichoderma harizianum produced the highest amount of lipase.

Table 2

Screening of 31 fungal isolates recovered from beef luncheon meat for their abilities to produce lipase enzyme

High > 24 mm Moderate = 19~24 mm Low < 19 mm

Effect of food preservatives on growth and lipase production by fungi

The incorporation 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 Aspergillus niger, Fusarium oxysporum and Nectria haematococca, except in few cases there is no effect by the low dose. Generally, the inhibition degree was correlated with the type and the concentration of preservatives in the culture medium for lipase (Figs. 1~6). Beuchat (1981) reported that potassium sorbate and sodium benzoate acted synergistically with heat to inactivate ascospores or vegetative cells of four molds including Aspergillus flavus, Penicillium puberulum, Byssochlamys nivea and Geotrichum candidum. The two preservatives had varied degrees of effectiveness on molds. Potassium sorbate was clearly more inhibitory than sodium benzoate to colony formation by A. flavus, and the presence of sucrose and sodium chloride enhanced this inhibition. Organic acids have characteristic sensitivities to microorganisms, sorbic acids and sorbates are very strong antifungal agents (Han, 2005). Recently, Geornaras et al. (2006) and Coma (2008) reported that acetic acid, lactic acid or potassium benzoate leads to a reduction of Listeria monocytogenes populations in smoked sausage.

Fig. 1

Effect of food preservatives on lipase production by Aspergillus niger.

Fig. 6

Effect of food preservatives on mycelial growth of Nectria haematococca.