Mycological Evaluation of Serving Some Dairy Products with Special Reference to Mycotoxins Production in Azhar University Student Hostels

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1 World Journal of Dairy & Food Sciences 8 (): , 01 ISSN X IDOSI Publications, 01 DOI: /idosi.wjdfs Mycological Evaluation of Serving Some Dairy Products with Special Reference to Mycotoxins Production in Azhar University Student Hostels Marwa I. Khalifa, Maha.A. Al-Ashmawy, A. Abdel-Khalik and M. El-Sherbini Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Mansoura University, 5516 Mansoura, Egypt Abstract: Milk and milk products especially cheeses and yoghurt are essential constituents in daily meals in Student hostels in Azhar University, Egypt. This dairy products may be contaminated with moulds and/or mycotoxins, among mycotoxins aflatoxins are the most dangerous especially aflatoxin B1 which is classified as the potent human carcinogen. The objectives of this study were (i) evaluate dairy products distributed in Azhar University mycologically for incidence, isolation and identification of mould species, (ii) determination the occurrence of aflatoxins in this dairy products and (iii) to compare between Sabouraud dextrose (SDA) and Aspergillus Differentiation Media (ADM) for isolation and counting of Aspergillus flavus as a one of most important species which has a public health significant in toxin production. Mycological evaluation was done for 00 random samples of soft cheeses (Feta and Istanboli, 65 each) and 70 plain yoghurt samples distributed in student hostels. About 64.5% of all different samples were positive for mould; 70.7%, 56.9% and 65% of Feta cheese, Istanboli and yoghurt samples respectively. The highest incidences (70.7%) as well as the highest mean (1.50 x 10 cfu) were detected in Feta cheese; all the samples were aflatoxin free by HPLC. According to these values, we considered the students in Azhar University are in safe side away from aflatoxicosis. Key words: Dairy products Mould Aflatoxin Aspergillus Differentiation Medium INTRODUCTION Mycotoxins are secondary toxic by-products (not essential for survive) of some moulds produced Milk has been used by humans since the beginning under certain conditions to achieve a competitive of recorded as the most popular and nutritious human advantage for mould over other mould species and food, this value put the food hygienists in a real bacteria. It is a heat stable, cytotoxic toxin for all living challenge, to provide milk to consumer safe and at the cells including higher organisms cells [4]. same time keep its nutritional value high as much as Mycotoxins produced by certain moulds as a toxic possible. In some countries including Egypt most milk substances can be found in dairy products from two is manufactured into more stable dairy products of origins: indirect contamination, which results when worldwide commerce, such as cheese and yoghurt. dairy cows ingest feed that contains mycotoxins Dairy products manufactured from milk have the same which pass into the milk such as aflatoxin M 1 and direct properties of original milk moreover; several stages of contamination, which occurs because of the intentional or processing may be unfavorable and add more points of accidental growth of moulds which secrete aflatoxins B1, hazard and weakness allowing entrance of moulds. B, G1 and G [5], therefore, feeding on a low quality Unfortunately, these products support mould growth and 6 ration contaminated with moulds more than 10 /g and kept toxin production due to its contents of protein, low ph under humid conditions cause mycotoxin intoxication to and storage under unfavorable conditions [1, ]. both animals and human whom consumed this animal Moulds are microscopic organisms that live on plant products. or animal matter. No one knows how many species of Therefore our work was adapted to evaluate dairy fungi exist, but estimates range from tens of thousands to products distributed in Azhar University mycologically perhaps 00,000 or more []. for incidence, isolation and identification of mould Corresponding Author: Marwa I. Khalifa, Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Mansoura University, 5516 Mansoura, Egypt. 165

2 World J. Dairy & Food Sci., 8 (): , 01 species, to determine the occurrence of aflatoxins in this samples percent were detected in feta cheese while, the dairy products and to compare between Sabouraud and lowest mean counts were detected in yoghurt and the aspergillus differentiation media for isolation and lowest incidence of contaminated samples were in counting of Aspergillus flavus as a one of most important istanboli cheese samples (56.9%). About 6% of cheese species which has a public health significant in toxin and 4% of yoghurt samples were complying with production. Egyptian standards for mould count while 100% for aflatoxins. Aspergillus, Penicillium and mucor genera MATERIALS AND METHODS were more frequently detected than other genera of fungi. Amoung the media used the ADM recovered more This study included examination of two hundreds isolates of A. flavus as shown in Table and Fig.1. All random samples of soft cheeses (Feta and Istanboli) (65) samples of cheese and yoghurt that contaminated by each and yoghurt (70) samples which were collected from Aspergillus species were free of aflatoxins. different student s hostels in Azhar University for mycological evaluation. Each cheese sample was represented by tetra bag packet of 65 g, while sample of DISCUSSION yoghurt by cup of 80 g weight. All samples in this study were collected in the period Dairy products can be contaminated by mycotoxins between October 01 and May 01. from two ways first way; indirectly by contamination of The collected samples were transferred directly to the animal feed with subsequent passage of the mycotoxins laboratory with a minimum of delay under aseptic to milk (AFM1) [11] or directly by contaminating dairy condition to be examined mycologically. Each sample was products with moulds which multiply during processing, divided into two portions. One for mycological manufacturing and storage and produce aflatoxins examination and the second for mycotoxins testing for (B, B, G1 and G); that direct contamination is our samples shown mould count. research target. Fungal contamination of cheese and The samples were prepared according the technique yoghurt is a serious threat in the students' hostels reported by APHA [6]. especially with the lack of awareness of healthy nutrition Sabouraud Dextrose agar Chloramphenicol medium and the absence of refrigerator in student rooms. (SDA) was used. While, Aspergillus Differentiation agar Total mould count is considered an indicator for hygienic medium (ADM) was prepared for isolation and giving an condition in dairy plants in many countries [1]. indication about Aspergillus flavus toxicity [7]. The prevalence and count of mould in Feta and Determination of the total number of mould in cheese Istanboli cheese and yoghurt samples collected from and yoghurt samples was conducted in duplicate plates student hostels of Azhar University had been of SDA and ADM according to APHA [6]. The isolated demonstrated in Table 1. However, it declared that species were identified on the basis of investigation 70.7, 56.9 and 65.7% of Feta cheese, Istanboli and yoghurt of the macro morphological properties of conidial and samples, respectively are contaminated with moulds. other structures according to the key described by It meant that a total of 64.5% of all different samples were Samson et al. [8]. positive for mould. These findings agree with those reported by El-Shrief [1] who detected mould in Aflatoxin Detection: Thin layer chromatography (TLC) for 7 of 50 samples (54%) fresh Damietta cheese. detection of Aflatoxins preparation and development of Montagna et al. [14] found Moulds in 54 of the 1 TLC plates according to method described in AOAC [9]. analyzed cheese samples (44.%). Sabreen and Zaky [15] After that the quantitative analysis of Aflatoxins B1, B, could detect moulds in 55% of examined soft cheese. G1 and G were determined by HPLC after the method of El- Asuoty [16] examined 5 yoghurt samples, 9 (6%) of Slubblefield and Shotwell [10]. them were mould positive. We obtained a mould count ranged from 1x10 and 7.6x10 cfu/g with a mean of RESULTS 1.5x10 cfu/g in feta cheese and in Istanboli cheese samples from 1x10 and 5x10 cfu/g with a mean value of Fungal Contamination and Aflatoxins Production: Mould 9.9x10 cfu/g. Ghazal [17] analyzed 0 cheese samples was detected in more than 64.5% of all different samples. (10 of soft fresh and 10 soft brined cheeses) mycologically The highest mean counts as well as the highest positive and found that contamination with moulds in counts 166

3 World J. Dairy & Food Sci., 8 (): , 01 Table 1: Prevalence and statistical analytical results of moulds examination for soft cheese and yoghurt samples +ve samples count cfu/g Examined Samples samples No. % Minimum Maximum Mean ±SE Feta cheese x 10² 7.6 x x10 ±0.48x10 Istanboli cheese x 10² 5 x x10 ±.54x10 Yoghurt x10 x10 1.5x10 ±0.0791x10 Table : Comparison between the results obtained from mould examination for examined samples and Egyptian standards (005) Samples comply with Egyptian standard Count (cfu/g) Egyptian standard Soft cheeses yoghurt Mould count (6%) 4(4%) Table : Frequency distribution of isolated moulds from examined soft cheeses and yoghurt samples Feta Istanboli Yoghurt Mould spp. No % No % No % A. flavus A. niger A. ochraceous A. nomius A. terres Acremonium spp Alternaria Cladosporium spp Fusarium spp Geotrichum Mucor spp P. albocormium P. cammberti P. chrysogenium P. citrinum P. funiculosum P. oxalicum P. roquforti P. rubrum P. species Trichophyton Total Table 4: Prevalence of Aspergillus flavus in examined white soft cheese and yoghurt samples on SDA and ADM Media A. flavus samples No. of Type Medium +ve samples No. % isolates cheese SDA ADM yoghurt SDA ADM *SDA= Sabouraud Dextrose Agar *ADM= Aspergillus Differentiation Medium 4 ranges from 10 to cfu /g and to The results in Table indicated that only 47 (6%) of cfu/g, respectively. Bullerman [18] found similar results in examined white soft cheese and 4 (4%) of plain yoghurt cheese (10-7.5x10 cfu/g). Arizcun et al. [19] declared total comply with Egyptian standard [0, 1]. Presence of mould count less than 10 cfu/g while, yoghurt maximum moulds in cheese and yoghurt reflect unsanitary hygienic count was x10 cfu/g and mean value 1.5x10 cfu/g. measures in manufacturing and packaging [, ]. 167

4 World J. Dairy & Food Sci., 8 (): , 01 5 Isolation of A.flavus on media 0 No. of isolets Sabouraud ADM 5 0 Feta Istanboli Yoghurt Fig. 1: Prevalence of Aspergillus flavus in examined white soft cheese and yoghurt samples on SDA and ADM media. From the aforementioned results it is obvious that the mycotoxicosis. This can be achieved through, avoid mould contamination levels in Feta cheese were higher 4 contaminated feeds by more than 10 / g from animal feed, than that found in Istanboli cheese and yoghurt. This may application of restricted hygienic measures in dairy plants be due to the low salt concentration (5%) in Feta cheese and preserving opened milk products in refrigerator. associated with long shelf life which expanded to 1 In comparing the efficiency of SDA and ADM, months comparing to 14 days for yoghurt which are ADM showed high recovery rate of A. flavus as declared processed from sterile milk which destroy huge in Table and Fig. 1 as reported by Cotty [0]. proportions of mould populations. Also high salt Fourteen (0.04%) of examined Feta cheese samples were contents (6%) of Istanboli cheese may act as mould contaminated with A. flavus and 15 isolates were suppressor. recovered on SDA, while 16 (.5%) samples and 0 Identification of isolated mould species revealed isolates were recovered on ADM. also Istanboli that Aspergillus, Acremonium, Alternaria, cheese samples reflected 4 (10%) with 5 isolates and 6 Cladosporium, Fusarium, Geotrichum, Mucore and (16.%) with 6 isolates and yoghurt samples contained Penicillium ontaminate the samples. El-Diasty and 5 and 8 isolates of A. flavus on SDA and ADM, El-Kaseh [4] also isolated Aspergillus spp., respectively. Cladosporium spp., Mucor spp., Curvularia spp., Samples contaminated by Aspergillus flavus were Penicillium spp., Geotricum spp. from milk and yoghurt. screened by TLC and HPLC and all of samples were free In this study Aspergillus, Mucor and Penicillium were of aflatoxin. It reflects that the presence of Aspergillus the most dominant in the contaminated samples. flavus in food does not mean necessarily the presence of These results are in agreement with that reported by mycotoxins and vice versa [1]. Cano-Sancho et al. [] El-Asuoty [16] Ghazal [17] and Berikten. and Kivanc [5] analyzed cheese and yoghurt samples for aflatoxin and while, in some studies [6, 14] Penicillium species were could not detect any occurrence of aflatoxin in cheese the most frequently isolated followed by Geotrichum spp. samples and only of 7 yoghurt samples were (7.%), Aspergillus spp. (4.%) and Mucor spp. (4.%). contaminated but in acceptable limit according to legal EU Otherwise, Allaraj et al. [7] recorded Aspergillus spp, limits. Positive cheese samples for AFM1 were recorded Penicillium spp, Fusarium and Mucor as the most in the study of Montagna et al. [], 16.6%, in medium common moulds found in analyzed cheeses. term (19.%) and long term ripened cheeses (17.1%), Among isolated Aspergillus spp. A. niger and A. flavus they returned this to the high affinity of AFM 1 for milk were the major isolates (.59,.5and 17.%) for A. niger proteins and therefore during the cheese-making process and (4.5, 1.5 and 9.6%) in Feta, Istanboli and yoghurt ripening does not reduce the levels of AFM 1 but on the samples, respectively, as shown in Table. These contrary increases them, due to water loss. Higher results findings are similar to those reported by El- Asuoty [16], were recorded by Kokkonen et al. [4]. Gazal [17] and Abo Donia [8]. In conclusion, the obtained results showed high Mould contamination may represent a great hazard contamination of white soft cheese and yoghurt with even in small counts if the package is opened and kept different types of moulds which constitute a public health under uncontrolled temperature and moisture conditions hazard. Storage of food under condition which prevents which activate mould to produce toxins [9]. Moulds must mould growth and strict hygienic measures and be prevented from reach to food and food commodities regulations should be imposed during processing, especially milk as a vital step in prevention of packaging and transportation. 168

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