EVALUATION OF MYCOLOGICAL STATUS AND DETICTION OF ITS TOXINS IN BASTERMA AND LUNCHEON IN ASSUIT CITY

Evaluation of mycological status and detiction of its toxins in Basterma and Luncheon in Assuit city

Lubna M.Ebraheem and Ghada M.Mohamed

Animal Health Research Institute, Assiut Laboratory.

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                                             ABSTRACT

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تقييم الحاله الفطريه وتحديد مدي افرازها للسموم في البسطرمة واللانشون في مدينه اسيوط

لبنى محمد ابراهيم , غادة محمد محمد

لقد تم جمع 60 عينة من اللانشون والبسطرمة بواقع 30 عينة من کل نوع جمعت عشوائيا من محلات السوبر مارکت المختلفة بمحافظة اسيوط ، وتم تحليل هذه العينات لمعرفة العدد الکلي للفطريات والخمائر وذلک باستخدام طريقة الزرع علي الوسط الغذائي داي کلوران روز بنجال وکذلک التعرف علي الفطريات المعزولة. ولقد تم معرفة السموم المفرزة باستخدام طريقة (Thin Layer Chromatographic). أظهرت النتائج ان العدد الکلي للفطريات لکل جرام في کلا من اللانشون والبسطرمة 1.86 X ²10 ، 1.29 X ²10 علي التوالي وکان العدد الکلي للخمائر لکل جرام في اللانشون 1.8 X ¹10 وفي البسطرمة 1.1 X ¹10 وکان فطر الأسبرجلس هو الأکثر إنتشاراً فکانت نسبته 96.7 % ، 53.3%  في اللانشون والبسطرمة علي التوالي وکان يمثل 30.9 % ، 23.6 % بالنسبة للعدد الکلي للفطريات المعزولة من نوعي العينات علي التوالي وکانت الأنواع السائدة لهذا الفطر في اللانشون هي أسبرجلس فلافس 56.7 % ، اسبرجلس فيوميجاتس 23.3 % ، أسبرجلس أوريزا 20% وأسبرجلس نيجر 20% بينما الأنواع التي أکتشفت في البسطرمة کانت اسبرجلس فلافس 10% ، اسبرجلس نيجر 36.7% وأسبرجلس تماري 6.6%. ويعتبر فطر البنسيليم وهو الثاني إنتشاراً موجود بنسبة 40% ، 33.3% في عينات اللانشون والبسطرمة علي التوالي وکان يمثل 8.3% ، 13.6% بالنسبة للعدد الکلي للفطريات المعزولة من نوعي العينات علي التوالي وکانت الأنواع السائدة لهذا الفطر في اللانشون هي بنسيليم أوکزاليکم 23.3% ، بنسيليم جانثينليم 10% وبنسيليم جلاسيريم 6.6% بينما الانواع التي اکتشفت في البسطرمة کانت بنسيليم أوکزاليکم 16.7% ، بنسيليم إکسبانيس 10% وبنسيليم بريقي کومباکتم 6.6 %. فطر الفيوزيريم هو الثالث إنتشاراً حيث کان موجود بنسبة 23.3% في کلاً من عينات اللانشون والبسطرمة وکان يمثل 4.9% ، 7.9% بالنسبة للعدد الکلي للفطريات المعزولة من اللانشون والبسطرمة علي التوالي. ولقد وجد نوع واحد من الفيوزيريم في عينات اللانشون وهو فيوزيريم سيلويدس 23.3% 23.3% ونوعان فى عينات البسطرمة وهما فيوزيريم نيجامى 7.6%, فيوزيريم فيرتى سيلويدس 13.3% وکان فطر الريزوبس موجود بنسبة 13.3% ، 6.7% في عينات اللانشون والبسطرمة علي التوالي وهذا يمثل 2.9% ، 2.1% من العدد الکلي للفطريات المعزولة. في هذه الدراسة تم عزل 4 أنواع من السموم الفطرية بنسبة 26.7% في اللانشون ، 13.3% في البسطرمة وکانت هذه السموم هي أفلاتوکس B, ، أوکراتوکس A ، ستريجما توسينين ، زيرالينون بواقع 13.3% ، 3.3% ، 6.7% ، 3.6 % في اللانشون علي التوالي بينما وجدت هذه السموم بنسبه 3.3% لکلاً منها في عينات البسطرمة .ولقد تم مناقشة النتائج واهمية الفطريات والخمائر التى تم عزلها للحفاظ على الصحة العامة.

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Key words:

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Introduction

Meat products are the most palatable and fast food meat. They are considered the best alternative for the fresh meat due to their low price, easily preparation and palatability (Frazer and Westhoff, 1988).

Luncheon meat usually consists of finely chopped meat and fat, with or without some added cereal and using,cured with salt and nitrite and heat processed (Ranken, 1984), while basterma was prepared from fresh salted meat coated with spice pasta and stored at room temperature (Refai et al., 2003).

Althrough meat products are available sources of protein but they are also an important potential sources of serious diseases if they contaminated by different moulds which are widely distributed in nature (Mossel, 1982; Foster et al., 1983).

Mycotoxins comprise a structurally diverise family of fungal toxins,many of which have been strongly implicated as chemical progenitors of toxicity in man and animal (Ramos et al., 1996).

Futher more, afltoxins are relatively stable compounds,not destroyed by processing and even be concentrated (Carvajal et al., 2003; Honikel, 2003).

The presence of toxinogenic moulds in a meat product does not automatically mean the presence of mycotoxins, especially if growth has not occurred, but rather that a potential for mycotoxin contamination exists. On the other hand,the absence of toxinogenic moulds doesn't guarantee that the meat products are free of mycotoxins, since the toxins may persist long after the moulds have disappeared (Ismail et al., 1994).

Fungal contamination is considered as one of the important spoilage agent of meat and meat products, occurring during slaughtering of animals,transportation,processing of meat products,and using of contaminated equipment,or other additives and spices (Misra, 1983; Abd el- Rahman, 1987).

However, the spices of mycotoxins in human food my be directly from fungal contamination or indirectly through contamination of feed stuffs consumed by the animal (Hussien et al., 1997).

Mould contamination not only causes deterioration of food and feed but also can adversely affected the healthy of humans and animals since they are capable of producing toxic metabolic known as mycotoxins causing cases of food poisoning, liver cancer in human (Mossel, 1982 and Foster et al., 1983).

Under adversety of moisture, PH, temperature conditions economic losses and various degrees of food decomposition were taken place,they can cause lipolytic and proteolytic spoilage (Besancon et al., 1992; Jakobsen and Narvhus, 1996).

Since fungi influence, the biochemical character and flavour of the product and its appearance is commercially undesirable and often result in down grading of the product (Bouton and Grappin 1995; Beuvier et al., 1997).  

However, mould and yeast have no role in mould spoilage or toxin production if the garlic content in the pasta in basterma hs not less 35% from the total content (El-Khateib et al., 1987).

The main objective of this study is to study the inadence ofthe incidence of mould and yeast in meant products (luncheon and basterma).   

Materials and Methods

Collection of samples:

Sixty samples of luncheon and basterma (30 for each) were collected from different supermarkets in Assiut city. The samples were placed in a sterile plastic bags and transferred to laboratory and kept at 4C until fungal analysis.

Enumeration and isolation of fungi:

The direct plating technique using Dichloran – Ros – Bengal media

(Pitt and Hocking, 1985) was employed for isolation of fungi from luncheon and basterma.

Determination of mycotoxins produced by fungal isolates applied in the flowing steps:

1- Fungal isolates:

Seven days old culture of each isolate on PDA (Potato dextrose agar) slope was used to abtain a spore suspension in 0.2% (v/v) aqueous tween 80 (Smith and Onions, 1983).

2-Cultivation of fungal isolates and extraction of mycoloxins:

According to El-Kady and Moubasher, 1982.

3- Purification of the crude extracts:

The extract was purified by column chromatography, which described by Josefsson and Moller, 1977.

4- Thin-Layer Chromatographic determination of mycotoxins:

For qualitative analysis of mycotoxine, Thin Layer Chromatographic (TLC) technique was applied (AOAC 1975, El-Kady and Moubasher, 1982; Dorner 1998).

5- Statistical analytical results was carried by standard errier of propotion (Z).

RESULTS

Table 1: Incidence of isolated fungi in examined luncheon and basterma samples:

Table 2: Total count of isolated fungi in examined luncheon basterma samples:

and basterma samples:

* No significant difference.

Z: standard errier of propotion

Table 3: Detection of mycotoxins produced by fungi isolated from Luncheon and Basterma using (TLC) technique:

Discussion

Earlier studies reporting environment as a source of food contaminant moulds (Kure   et al., 2001; Mizakova et al., 2002; Battilani et al., 2007; Sorensen et al., 2008). In this study the results revaled that all examined samples (100%) were contaminated with moulds.

Results given in Tables (1&2) revealed that total mould count / gm of examined samples of luncheon and basterma were 1.86 x 10² and 1.29 x 10² respectively. The count in luncheon samples were lower than that reported by Abdel-Rahman et al. (1995) (9.2 x 10³), Roushdy et al. (1996) (1.5 x 10⁶), Hussien et al. (1997) (2.7 x 10⁵), Sayed et al. (2000) (2.68 x 10²); El-Tabiy (2006) (2.4 x 10³). The higher total mould count / gm of basterma were reported by Abdel-Rahman  et al. (1995) (1.4 x 10⁴), Hussien et al. (1997) (9.2 x 10⁴) and Refai et al. (2003), (the count varied from 10 (2) to 10 (5).

Results in Tables (2) indicated that there was no significance difference in count between luncheon and basterma.

Altough the total mould count of any food article is not indicataive of its safety for consumption yet it is of supreme importance in judging the hygienic condition under which it has been produced, handled and stored (Martin and Lowery, 1992). The variation in quantitative estimation of mould counts might be attributed to improper sanitation during slaugher, preparation, manufacturing, additives specially using spices of low quality or during transportation, storage and marketing of the products (Abobaker, 1986; Refai et al., 1990;  Roushdy et al., 1996).  

The mould count in both products samples may be attributed to the widespread distribution of mould in nuture. Fungi are normal inhibitant of wool of the animal, also have ability to be adopted at wide range of temperatures (Nasser et al., 1998). The most common fungal genera in both luncheon and basterma (Tables 1&2) were Aspergillus then Penicillium. This observation was relatively agree with Wu et al. (1974); Abdel-Rahman et al. (1984); Beuchat (1987); Lotfi et al. (1987), Sayed et al. (2000); Mohamed and Hussien (2004); Aideia (2005); El-Tabiy (2006), while Roushdy et al. (1996) and Hussein et al. (1997) reported that penicillium was the common fungal genera followed by Aspergillus in different meat products.

Tables (1& 2) revealed that Aspergillus was the most prevalent genus contaminating 96.7% and 53.3% of luncheon and basterma respectively and comprising 30.9% and 23.6% of the total fungi of the same samples respectively, while Azza et al. (1997) detected Aspergillus in 32.6% of luncheon samples and in 31.1% of basterma.

From Table (1, 2) The predominant species of Aspergillus recovered from luncheon samples were A. flavus ( 56.7 %), A. Fumigatus (23.3%), A.oryzae (20%) and A. niger (20%), while species of Aspergillus recovered from samples of basterma were A. flavus (10%), A. niger (36.7%) and A. tamari (6.6%). The results obtained by Hussien et al. (1997) that A. niger was 12.5 % in luncheon and 10.9% in basterma, while Sayed et al. (2000) reported that A. flavus was moderately occurred in luncheon.

A.fumigatus has a marginal xerophile nature ranges from 12°c to 55°c Ayerst, (1966), he also reported that A.niger is more prevalent in warmer climates in field satuation and stored foods Al-Doory (1980) revealed that Aspergillus spp. were incriminated in pulmonary aspergillosis, skin infection, sinusitis and otitis for food handlers.

Also Tables (1, 2) showed that Penicillum occupied the second prevalent genus. It was encountered in 40% and 33.3% of the samples of luncheon and basterma respectively and matching 8.3% and 13.6% of the total fungi on two types of samples respectively, Hussien et al. (1997) could detect penicillium in 26.4% of luncheon and in 42.7% of basterma, Dereje et al. (2010) indicated that most of the P. nalgiovense have the ability to produce toxins on the products and can become potential food safety hazards.

The predominant species of Penicillum recovered from luncheon samples were P.oxalicum (23.3%), P.janthinellum (10%) and P.glabrum (6.6%), and those recovered from basterma samples were P.oxalicum (16.7%), P.expanus (10%) and P. brevicompactum (6.6%). Some investigators could detect another spp. of penicillium in luncheon Abdel-Rahman and El-Bassiony, (1984) found P. verrucosum var cyclopium in 94.5%, Sayed et al. (2000) isolated P.viridicatum from 20%.

Penicillosis are diseases induced by pathogenic strains of Penicillium in human involving the upper respiratory tract and lungs, carcinogenic effects are also caused by mycotoxins produced by many Penicillium species (Mossel, 1982).

Fusarium was the third frequent genus contaminating 23.3% of both luncheon and basterma and constituting 4.9% and 7.9% of total fungi on luncheon and basterma respectively, one spp. of Fusarium was detected in luncheon samples it was F. verticillioides (23.3%), and two spp. were detected in basterma samples, they were F. nygamai (6.7%) and F. verticillioides (13.3%). (Tables 1&2).      

The remaining fungi was Rhizopus spp. which contaminat 13.3% and 6.7% of the samples of luncheon and basterma comprising 2.9%and 2.1%of the total fungi, respectively, (Tables 3&4). Most of these fungi had been isolated previously, but with different frequencies from meat products, Hefnawy (1980); Hegazi et al. (1992); Zaki et al. (1995); Nagat, (1997).

Yeasts derived from fungal analysis of luncheon samples revealed 7(23.3%) which comprised 8.8% in luncheon samples (Table 1) and revealed 18 (60%) in basterma samples which comprised 7.8% of the total fungi (Table 2). While the total yeast/gm in luncheon and basterma were 1.8X10 and 1.1X10 respectively.

The growth of microbes, such as bacteria, yeasts and moulds deteriorate the safety and quality of food products and cause significant economic loss (Fittenborg et al., 1996; Pitt and Hocking, 1999; Samson et al., 2004), also growth of fungal particles can be facilitated by chains of production processes that each and every process in the chain should be considered to determine where to act (FAO, 2008).

A.flavus is the most important natural source of aflatoxins in the world's food supplies of which are B₁ , B₂ , G₁ and G₂ . The risk of B₁ and B₂ aflatoxins starts when they ingested by a lactating animal and a proportion is hydoxylated to M₁ and M₂ aflatoxin derivitives of lower toxicity but their significant due to the wide spread consumption of mycotoxicated milk by infants (Frobish et al., 1986). Also Aflatoxins produced by A.flavus exposed man and animals to acute and chronic toxicity distincted in four forms: liver damage, liver cirrhosis, induction of tumours and teratognic effects (Frisvad and Samson, 1991).

As shown in Table (3) in this study 4 types of mycotoxins were detected of which aflatoxin B₁ was detected in four samples luncheon and one sample of basterma, that 23.5% and 33.3% of isolated A.flavus could produce this toxin in both luncheon and basterma samples respectively. AFB1, is highly toxic compound and its occurrence poses a threat to the health of consumers specially young children (Pierides et al., 2000). In addition to aflatoxin Stregmatocyste in toxin was found in two samples of luncheon which produced by 28.5% of isolated A. fumigatus, also this toxin was found in one sample of basterma produced by 50% of isolated A. tamari.

OchratoxinA was detected in two samples, one in luncheon and the other in basterma in which 14.3% and 20% of P.oxalicum could produce this toxin in luncheon and basterma respectively, also zearalenon toxin was detected in one sample of luncheon and one sample of basterma in which 14.3% and 25% of F.verticillioides could produce this toxin in luncheon and basterma respectively. Other species of fungi could not produce mycotoxin.

These data showed that mycotoxin was isolated from 26.7% of luncheon and from 13.3% of basterma in which Aflatoxin B₁, Ochratoxin A, Stregmatocystein and Zaeralenone were detected in 13.3%, 3.3%, 6.7% and 3.3% of luncheon samples respectively and were detected in 3.3% in all samples of basterma. (Ismail and Zaky, 1999) could detect aflatoxin B₁ in 14% of luncheon samples which is nearly simelar to our results.

In conclusion, It could be concluded that luncheon and basterma are liable to be contaminated with several fungal species as they are normal inhibtant of air and adapt themselves at a wide range of environmental conditions. strict hygienic measures must be applied during manufacturing. Application of HACCP system (Hazard Analysis Critical Control Point) along the line of production must be paralled with the end product inspection.

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