Dept. of Food Hygiene,

Animal Health Research Institute, Assiut, Regional Laboratory

Detection of aflatoxins released

by some Aspergillus species isolated from processed cheese together

with some aerobic and anaerobic

spore former organisms

(With 4 Tables)

By

Nahed M. Wahba and Eman Korashy Ahmed

(Received at18/11/2008)

الکشف عن السموم الفطرية المفرزة من بعض أنواع الأسبريجيلس المعزولة من الجبن المطبوخ وبعض الميکروبات المتحوصلة الهوائية واللاهوائية

ناهد محمد وهبه ، إيمان قرشى أحمد

فى السنوات الأخيرة زاد الإقبال على استهلاک الجبن المطبوخ وذلک لقناعة المستهلک بسلامة هذا النوع من الجبن نظراً لطريقة تصنيعه وتعرضه للحرارة أثناء الطبخ. لذا هدفت الدراسة إلى معرفة مدى تلوث الجبن المطبوخ بالفطريات وخصوصاً المفرزة منها لسموم الأفلاتوکسين وکذلک تواجد الميکروبات المتحوصلة الهوائية واللاهوائية. لذا تم تحليل 120 عينة من الجبن المطبوخ وأظهرت النتائج أن 18 (15%) من العينات کانت تحتوى على فطريات وخمائر وکان العدد الکلى لها 2.7×10 ⁴ لکل جرام وأعلى نسبة بين العينات الموجبة (55.6%) کانت لفطر الأسبرجلس فلافس والذى أثبتت النتائج أن 15% منه کانت مفرزة لجميع أنواع الأفلاتوکسين. أما بالنسبة للميکوتوکسين فقد تبين أن 5 عزلات من بين 20 عزلة کانت مفرزة للميکوتوکسين والتى تسبب في موت أکثر من 50% من اليرقات المختبرة . و تناولت الدراسة أيضاً تواجد الميکروبات المتحوصلة الهوائية واللاهوائية وقد أمکن عزل الباسيلس سيرس بنسبة 14.2% بمتوسط عددى 7×10 ² ميکروب / جرام کما أمکن عزل کل من الباسيلس ماسيرنس ، الباسيلس ليشنيفورم والباسيلس ميجاتيرم بالنسب الآتية 6.7 ، 16.7 ، 11.7% على التوالي، وکذلک الکلوستردينم بيرفنجنز بنسبة 13.3% من العينات المفحوصة. ولقد خلصت الدراسة إلى ضرورة إتباع بعض الخطوات لإنتاج جبن مطبوخ سليم وآمن تتلخص في الخطوة الأولى: منع تلوث غذاء الحيوان بالفطريات حتى نمنع إفراز الأفلاتوکسين ومنع تکاثرها داخل المزرعة. الخطوة الثانية: تشمل بعض الاحتياطات اللازمة لمنع نمو وتکاثر الميکروبات في الجبن وذلک بالتبريد السليم فى کل مرحلة من مراحل التصنيع وحتى يصل إلى المستهلک.

SUMMARY

Processed cheese has an excellent history of safety; however, it is difficult to eliminate completely fungi and bacterial growth, activity and toxin production that threaten consumer's health. Methods: 120 processed cheese samples were analyzed for determination of total fungi and yeast count, as well as identification of the isolated fungi. Toxicity and aflatoxin produced by the isolated Asp. flavus using thin layer chromatographic technique. Bacillus species and C. perfringens count as spore former organisms. Results: Fungi and yeasts were present in 18 (15%) of processed cheese samples with total count of 2.7x10⁴/g. Asp. flavus was the first prevalent species (55.6%) of the positive samples. 5 (25%) out of 20 isolates of Asp. flavus proved to produce mycotoxins, three of them were able to produce all types of aflatoxins. Mycotoxins producing isolates cause more than 50% mortality of the larvae tested. Some aerobic and anaerobic sporeformers were isolated and counted. B. cereus was isolated from 17 (14.2%) of processed cheese samples with an average count of 7x10²/g.                B. macerans, B. lechniformis and B. megaterium were detected in 8 (6.7%), 20 (16.7%) and 14 (11.7%) of samples, respectively.  Moreover, C. perfringens was present in 16 (13.3%) of samples.  Conclusion: Two important points must be regarded to safe production of processed cheese; first one: is the measures to minimize the presence of spores. The second is to prevent spore germination and vegetative proliferation by adequate cooling during all steps of production till dispensation to the consumers.

Key words: Processed cheese, Aspergillus ssp., aflatoxins

Introduction

Processed cheese has an excellent history of safety. Production and consumption of processed cheese increased steadily. Today, large tons of natural cheeses are converted to processed cheese, the global production of processed cheese products is estimated at ~1.5-1.8 million ton/year (Guinee, 2003). Heat processing, packaging and cheese composition normally limit bacterial growth however, it is difficult to eliminate completely bacterial growth and activity in this type of cheese (Hamed et al., 1997). Airborne mold spores tend to grow on cheese where pockets of air exist between the packaging material and cheese surface (Hocking and Faedo, 1992).

Aflatoxins (Afs) are hepatotoxic, mutagenic, carcinogenic and immuno suppressive toxins (Bennett and Klich, 2003 and Honikel, 2003). They are secondary metabolites produced by many strains of Aspergillus flavus and Asp. parasiticus. The major aflatoxins of concern are designated Af B₁, B₂, G₁, G₂ and M₁. Af B₁ is predominant in amount and toxicity (Mountney and Gould, 1988). Outbreaks of mycotoxicosis from Af B₁ have been reported (Peraica et al., 1999). Cattle consume Af B₁ in feedstuff and transform it into Af M₁ so, milk and milk products can serve as indirect source of aflatoxins (Kisza and Domagala, 1994). Af B₁ and M₁ are highly toxic compound and its occurrence poses a threat to the health of consumers specially young children (Pierides et al., 2000).

Furthermore, aflatoxins are relatively stable compounds, not destroyed by processing and may even be concentrated. Their thermal stability disqualifies pasteurization and ultra pasteurization as methods of control (Carvajal et al., 2003 and Honikel, 2003).

Endo spore forming species of Bacillus and Clostridia are of great concern to the dairy industry causing both food spoilage and food poisoning.  B. cereus and related species cause problems to the dairy industry by deteriorating the product (Eneroth et al., 2001) and by endangering people's health up consuming contaminated food (Ghelardi et al., 2002). B. cereus is considered to be the most important species however, more recently attention has been drawn to other species of bacillus that may occur in milk products more often and in higher concentrations than B. cereus (Mansour et al., 1999).

Likewise, C. perfringens is a classical agent of foodborne disease characterized by mildness and self limiting nature (Sanz et al., 2002).  C. perfringens type A is considered the third leading cause of foodborne disease in the USA (Bos et al., 2005). Species of clostridia producing butyric acid like C. butyricum, C. perfringens and C. tertium cause sever defects in cheese as a consequence of late blowing or butyric blowing (Galle le Bourhis et al., 2005).

Therefore, and due to the dangerous role of molds in cheese, prevalence and count of mold are considered as standard test for checking the general sanitary measures adopted, together with toxin production and toxicity of Asp. flavus isolates. Moreover, the incidence and count of Bacillus species and C. perfringens are essential.

Materials and Methods

            A total of 120 samples of processed cheese were collected and subjected to the following:

1- Preparation of the samples according to APHA (1992).

2- Enumeration and Isolation of molds and yeasts were done as described by Oxoid (1990).

3- Identification of isolated fungi was done in the Mycological Center, Fac. of Science, Assiut Univ.

4- Cultivation and extraction of the fungal toxins:

            Asp. flavus isolates were collected and cultivated on Czapek's liquid medium fortified by 0.2% yeast extract and 1% peptone, incubated at 28°C for 10 days. The content of each flask (medium and mycelium) were homogenized for 5 min. in high speed blender (1600 rpm) with 100 ml chloroform. The extract procedure was repeated 3 times, washed with equal volume of dist. water, dried over anhydrous sodium sulfate, filtered then, concentrated under vacuum to near dryness and diluted to 1 ml with chloroform.

5- Thin-layer chromatographic separation of mycotoxins:

           Thin-layer chromatographic technique was used for qualitative identification of mycotoxins by comparison with appropriate reference standards (Gimeno, 1979 and Van Egmond et al., 1980).

6- Bioassay method for mycotoxins:

           The immature brine shrimp (Artemia salina L.) was used for detection of aflatoxins and for other mycotoxins toxicity as described by Scott et al. (1980).

7- Enumeration, isolation and identification of Bacillus spp. using Mannitol egg yolk polymyxin agar (MYP) according to Lancette and Harmon (1980) and Cowan and Steel (1974).

8- Enumeration of C. Perfringens using lactose sulphite broth (Beerens et al., 1982) by applying most probable number (MPN) technique.

Results

           The results were manifested and tabulated in Tables (1-4).

Table 1: Incidence and count of different molds and yeasts isolated from processed cheese samples.

Table 2: Aflatoxin production and toxicity of Asp. flavus isolated from processed cheese samples.

A: indicates more than 50% mortality of larvae tested.

B: indicates less than 50% mortality of larvae tested.

Table 3: Incidence and count of some aerobic spore former organisms in processed cheese samples.

Table 4: Incidence, count and frequency distribution of C. perfringens in processed cheese samples.

Discussion

            Consumer demand for processed food challenges the food industry to produce tasty-nutritious and microbially safe products (Leistner and Gorris, 1995). Mycobiota growing in food is often beneficial for the ripening and development of specific flavor characteristics of the product, but it can be harmful due to the production of mycotoxins (Federico et al., 2002).

            Mold and yeasts were present in 18 (15%) of processed cheese samples with total count of 2.7x10⁴ (Table 1). Higher incidence and count were reported by Wafy (2006) and Amin (2007). 100% incidence was detected by Ismail and Sabreen (2001) and Sabreen and Zaky (2001). On the other hand, no molds could be isolated from processed cheese examined by Taniwaki and Dender (1992). Processed cheese may serve as a substrate to Asp. flavus and other mycotoxins producing fungi (Sabreen and Zaky, 2001 and Amin, 2007).

            Asp. flavus was the first prevalent species encountered in processed cheese samples. It was found in 10 (55.6%) of the positive samples in counts ranged from 100-1400 cells/g, other species were isolated in different percentages and counts (Table 1). The considerable high incidence and count of Asp. flavus is of great importance for its production of Aflatoxins. Several researches detected aflatoxins in processed cheese (Sabreen and Zaky, 2001; Sarimehmetoglu et al., 2004 and Yaroglu et al., 2005). Af B₁ and M₁ are highly toxic compound and its occurrence poses a threat to the health of consumers specially young children (Pierides et al., 2000).

            In the present study, 5(25%) out of 20 isolates of Asp. flavus proved to produce mycotoxins, three of them were able to produce all types of aflatoxins (B₁, B₂, G₁, G₂ and M₁). All mycotoxins producing isolates (5 isolates) cause more than 50% mortality of the larvae tested (Table 2). The mycotoxin production and toxicity by Asp. flavus wes previously studied by El-Maraghy and Zohri (1988) and Abdel-Malek et al. (1993).

            Because mycotoxins are natural contaminants of food and their exposure is more likely to occur in world where poor methods of food handling and storage are common, their formation is unavoidable and methods of controlling are usually preventive.

            Aerobic spore formers like Bacillus species naturally existed in numerous foods owing to their wide distribution in the environment.  Growth of B. cereus often limits the shelf life of dairy products kept at refrigerator temperatures, if conditions are right, they multiply rapidly and produce sufficient toxins to induce symptoms of food poisoning (Borge et al., 2001 and Vissers et al., 2007).

            B. cereus was previously isolated from processed cheese by Wahba (1997); Sabry (2001) and Abdel-Hameed (2004). In this work, B. cereus was isolated from 17 (14.2%) of processed cheese samples with counts ranged from 1x10² to 3.8x10⁴, with the average count was 7x10² organisms/g (Table 3). Concentration of <10³/g may considered innocuous since the minimal level required to cause illness has been estimated to be >10⁵/g (ICMSF, 1996).

            B. macerans, B. lechniformis and B. megaterium were detected in 8 (6.7%), 20 (16.7%) and 14 (11.7%) of samples, respectively (Table 3). Although some foodborne gastroenteritis incidents attributed to B. lechniformis have been reported (Turnbull, 1997 and Salkinoja-Salonen et al., 1999), B. lechniformis secreted protease enzyme which produce milk curds and may be useful as a new source of milk coagulants for cheese making industry (Ageitos et al., 2007).

            The clostridium species present in cheese originate from milk which is contaminated by these spores during milking, even >0.2 clostridium spores/ml in raw milk can affect the cheese during processing (Kalzendorf, 1995).

            C. perfringes was present in 16 (13.3%) of processed cheese samples.  Most of samples lines within the range of 3-20 cfu/g (Table 4). Lower incidences of C. perfringens and other anaerobic organisms were observed by Wahba (1997) and Amin (2007).

            In most instances the actual cause of poisoning of C. perfringens is temperature abuse, small numbers of organisms are present, multiply to food poisoning levels during cool down and storage (Crouch and Golden, 2005).

            So, two important points must be regarded to safe production of processed cheese, first one is the measures to minimize the presence of spores including low initial contamination levels in the feed and preventing their growth in the farm environment. The second is emphasizing the requirement for precautions that prevent spore germination and vegetative proliferation by adequate cooling during all steps of production till dispensation to the consumers.

Acknowledgment

            The authors thanks Prof. Dr. S.S. El-Maraghy, Physiology of Fungi Lab., Fac. of Sci., Assiut Univ., for extraction, separation and bioassay of mycotoxins.

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