Animal Health Research Institute,

Assiut, Regional Laboratory.

Incidence of Listeria monocytogenes in pasteurized milk and some pasteurized milk products and effect of boiling

on its viability

(With 2 Tables)

By

EMAN K. AHMED and SOHAIR Z. HUSSEIN

(Received at 2/2/2005)

مدى تواجد ميکروب الليستيريا مونوسيتوجينس فى اللبن المبستر وبعض منتجاته وتأثير درجة الغليان على حيوية الميکروب

إيمان قرشى أحمد ،  سهير زين العابدين

جمعت 75 عينة عشوائية من اللبن والقشدة والرايب المبستر بواقع (25 عينة من کل نوع) وذلک من محلات البقالة والسوبر مارکت المختلفة بمدينة أسيوط. وکذلک من معمل تصنيع الألبان بکلية الزراعة جامعة أسيوط وذلک لمعرفة مدى تلوثها بميکروب الليستيريا مونوسيتوجينس. کذلک تم دراسة مدى حيوية وبقاء الميکروب فى اللبن المحقون           بـ 37×10 ⁷ خلية / مللى من هذا الميکروب وذلک بغلى اللبن وعد الميکروب على فترات زمنية مختلفة فى ثلاث محاولات. وقد أظهرت النتائج أن ميکروب الليستيريا مونوسيتوجينس قد تم عزله من اللبن المبستر بنسبة 4% بينما لم يتم عزله من القشدة والرايب المبسترين. أما بالنسبة لدراسة تأثر ميکروب الليستيريا بدرجة حرارة الغليان فقد أسفرت النتائج عن تناقص العدد من 37×10 ⁷ إلى 274×10 ⁵  و 214×10 ⁴ و 120×10 ² فى المحاولات الثلاث فى الدقائق الأولى والثانية والثالثة على التوالى بينما لم يتم عزل الميکروب بعد الدقيقة الرابعة للثلات محاولات. هذا وقد ناقش البحث الأهمية الصحية والإجراءات التى ينبغى اتباعها لمنع تلوث المنتجات سابقة الذکر بهذا الميکروب.

Summary

A total of 75 random samples of pasteurized: milk, cream and Rayeb (25 samples of each type) were collected from different localities in Assiut City and examined for the presence of L. monocytogenes. The obtained results pointed out that L. monocytogenes could be isolated from 4% of the examined pasteurized milk samples, while could not be detected from pasteurized samples of cream and Rayeb. Furthermore, three trials on sterile milk which were inoculated with 37x10⁷ CFU/ml of L. monocytogenes as an initial count to detect the thermal resistance of the organisms when exposed to boiling temperature at different times. The obtained results of the three trials revealed that the number of the organisms decreased and reached to 274x10⁵, 214x10⁴ and 120x10² at the first, second and the third minute, respectively, while at the fourth minute the organisms could not be detected. The public health importance and the sanitary measures for control of the organisms were mentioned.

Key words: Listeria monocytogenes, pasteurized: milk, cream and Rayeb.

Introduction

            Listeria monocytogenes is a Gram positive rod shaped bacterium which was discovered nearly 60 years ago and finally named Listeria according to Dr. Lister (Pirie, 1940). The organism has a psychrophillic and a mesophillic nature as it can grow at temperatures between 1 and 45 with an optimal growth occurring between 30-37°C. Several species of Listeria were recognised in the Approved Lists of Bacterial Names (Audurier et al., 1984) including L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, L. dentrificans, L. murrayi and L. grayi.

            In recent years a gradual awareness of the occurrence of Listeria organisms in both human being and animal species spread throughout the world while a considerable interest in L. monocytogenes as an agent of foodborne disease has become increasly apparant.

            L. monocytogenes is widely distributed in man and animal species including sheep, goats and many other animals that eliminate the agent in faeces as well as in the environment. Also, it has been isolated from soil, plants, mud pasteure and streams (Acha and Szyfers, 1991).  In addition it can be isolated from the stools of a large proportion of healthy people and stools of 20-30% of pregnant women and from the female genital tracts. The infectious disease associated with Listeria monocytogenes called Listeriosis and the organism is recognised by public health authorities as it plays an important role in cases of abortion in both human and animals, absces in liver, artherities, peritonities, endocarditis, conjunctivitis, pneumonia, septicaemia and meningeoenecephalitis.  Many other clinical forms have been published by different workers including mastitis (Gray and Killinger, 1966; Gitter et al., 1980 and Seeliger, 1988). Subclinical acute or chronic cases of mastitis in cows resulting in economic losses. Moreover, serious mortality rates from Listeriosis occurred in sheep herds in Australia were observed (Dennis, 1975).

            Milk and its products may be subjected to listeric infection from different sources such as infected and healthy animals and people, from silage, dust, waste water and others (Acha and Szyfers, 1991 and Franco Abuin et al., 1996). Several outbreaks of Listeriosis occurred in Europe, USA and other countries due to consumption of raw and pasteurized milk, cheese, ice cream and other dairy products (Fleming et al., 1985; Linnan et al., 1988; Ryser and Marth, 1991 and Jensen et al., 1994).  Furthermore, several investigators could isolate L. monocytogenes from milk and milk products throughout the world (Farber et al., 1988, Massa et al., 1990, Loncarevic et al., 1995, Hassan Nour, 1996 and Aman and Ahmed, 1997).

            It has been reported that the studies done to evaluate the thermic resistance of L. monocytoenes have produced conflicting results. Early investigations indicated, however, that L. monocytogenes might be relatively heat resistant. Bearns and Girard (1958) isolated Listeriae from milk inoculated with over 50/000/ml after pasteurization at 61.7°C for 30 min. Doyle (1986) reported survival of L. monocytogenes in milk after pasteurization at 72°C for 16.4S while Fernandez et al., (1986) recorded survival of Listeria spp. after pasteurization at 78°C for 15 seconds.

            Because of the public health significance of L. monocytogenes as well as the economic losses caused by this organism, the present study was undertaken to study the prevalence of L. monocytogenes in pasteurized milk and some of its pasteurized products as cream and Rayeb, as well as to study the thermal resistance of the organism to boiling temperature at different times.

MaterialS and Methods

            A total of 75 random samples of pasteurized:milk; cream and Rayeb (25 samples of each) were collected from different supermarkets, groceries and the milk Technology Laboratory of Faculty of Agriculture, Assiut University. Samples were kept in refrigerator.

I-A- Preparation of samples

            Cartons or cans of collected samples were thoroughly cleaned from outside, then well mixed and aseptically opened.

B- Isolation and identification

            The technique recommended by FDA (Lovette et al., 1987) was adopted by selective enrichment in Listeria enrichment broth (LEB) followed by selective plating onto Oxford agar plates (Curtis et al., 1989). Suspected colonies of L. monocytogenes were picked up and purified before being identified according to Hitchins (1995). 

II- Effect of milk boiling on the growth and survival of L. monocytogenes

- Culture preparation

            L. monocytogenes strain was obtained from the Institute of Milk Hygiene and Technology, Vet. Med. Univ., Vienna, Austria. The strain was inoculated into Listeria enrichment broth followed by plating 0.1 ml from decimal dilutions onto Oxford agar. The incubation was done at 37°C for 24h-48h for typical colonial morphology and purity.

- Experimental procedure:

            The previous strain was inoculated in sterile milk tested to ensure its freedom from L. monocytogenes to provide 37x10⁷ cells/ml., three trials were designed to confirm the effect of time of boiling on the growth and survival of L. monocytogenes in milk. Count of L. monocytogenes was achieved by direct plating of decimal dilutions of the boiled inoculated milk (A.P.H.A. 1992) onto Oxford agar plates, which incubated at 37°C for 24-24h and typical colonies presumed to be L. monocytogenes were counted.

Results

            The obtained results were recorded in Tables 1 & 2.

Table 1: Incidence of L. monocytogenes in the examined samples of pasteurized: milk, cream and Rayeb.

Table 2: Effect of milk boiling on the growth and survival of L. monocyotgenes.

Discussion

            Thermal processing is the most widely used method to preserve food in addition to destroy harmful microorganisms, thus rendering food safe for human consumption. Early studies dealing with possible resistance of L. monocytogenes to pasteurization (Bearns and Girard, 1958) but in 1983, interest in this topic was reviewed as a result of a Listeriosis outbreak in Massachusetts that was epidemiologically linked to consumption of pasteurized milk.

            From the results recorded in Table 1 it was obvious that L. monocytogenes were isolated from 4% of the examined pasteurized milk samples, the same results were obtained by Fleming et al., 1985, and Garayzabal et al., 1986 who noticed outbreaks of Listeriosis due to consumption of improper pasteurized and flavoured pasteurized milks in the United States 1995, also Ryser and Marth (1991) recorded 49 cases of Listeriosis due to consumption of pasteurized milk in USA (1983) and this may be attributed to post pasteurization contamination or due to the explaination of Roy (1996) who concluded that Listeria organisms secrete a sticky substance called glycocalyx to attach themselves to the surfaces to resist cleaning and disinfection so, it can multiply and contaminate pasteurized milk which comes into contact with surfaces, additionally Gitter et al., (1980) suggested that pasteurization did not offer a guarantee of complete safety if the viable bacterial count is high before heat treatment. However, Bradshaw et al. (1987) and Fedio and Jackson (1989) revealed that preheating of raw milk before sterilization or pasteurization processes leads to increase heat resistance of the organisms as compared with milk control. On the other side, the obtained results were in disagreement with Rola et al. (1994) who could not detect the organism from pasteurized milk. Concerning pasteurized cream samples, Table 1. Showed that L. monocytogenes could not be detected from the examined samples, and these results were in agreement with that of El-Marrakchi et al. (1993) and Rola et al. (1994) who failed to detect the organism from fresh and pasteurized cream.  This may be due to the high acidity that affects the organisms. Huang et al. (1993) stated that the growth rate of Listeria decreased with the increase of Lactic acid and acetic acid concentrations in the medium.

            Data recorded in Table 1. showed that L. monocytogenes could not be detected in the pasteurized Rayeb samples and this is completely in agreement with the results obtained by Sabreen and Korashy (2001) who failed to isolate L. monocytogenes from plain and fruit yoghurt samples. Other investigators obtained the same results when examined other fermented dairy products (Kerr et al., 1992; Rola et al., 1994; Abou-Eleinin, 1999 and El-Prince, 2000) this may be attributed to the high content of lactic acid and the resultant lowering of its pH value and other inhibitory compounds produced by lactic acid bacteria. In addition to the free fatty acids released during storage period which aid in destruction of food borne pathogens (Wang and Johnson, 1992). In contrast to these results, El-Gazzar and Marth 1991, El-Marrakchi et al., 1993 and Gohil et al., 1995) documented that Listeria has the ability to grow or survive for extended period in fermented and non fermented dairy products at various temperatures. While, the lower incidence (2%) of L. monocytogenes obtained by Greenwood et al. (1991) attributed to the post processing contamination from the plant environment. Also growth and survival of L. monocytogenes in fermented products have been noted elsewhere (Siragusal and Johnson, 1988; Ahmed, 1989; Sing and Chander, 1990 and Zuniga Estrada et al., 1995).

            Results from the second part of the study indicated in Table 2 depicted the effect of boiling on the growth and survival of L. monocytogenes. From the tabulated data the cell count of L. monocytogenes declared to be significantly decreased by boiling from 37x10⁷ to 274x10⁵, 214x10⁴ and 120x10² in the first, second and the third minute respectively. The organisms failed to be detected and completely disappeared after the fourth minute. These results were somewhat in agreement with that of Potel (1951) and Abdel-Hakiem and Sabreen (1993) who demonstrated that L. monocytogenes died rapidly in milk heated at 80°C. White Stenberg and Hammainen (1955) and Donnelly et al. (1987) stated that L. monocytogenes was rapidly inactivated in milk at 62°C. On the other hand, the obtained results were in disagreement with Ozgen (1952), Dedie and Schulze (1957) and Ikonomov and Todorov (1957). The heat resistance of L. monocytogenes may be due to the intracellular state of the organism in naturally contaminated milk Fleming et al. (1985).

            It is noteworthy from these trials that the reduction in viable cell number of L. monocytogenes and loss in its viability may be due to the thermal processing which is considered the most widely used method to preserve food. The high temperature short time (HTST) pasteurization supplemented by Food and Drug Administration is adequate for destruction of L. monocytogenes in milk.

            In general from the public health point of view, application of good hygienic measures during production, handling and filling in final containers is essential to safe the quality of milk and its products.  Consequently prevent the risk of human hazards. In addition, it is important for good hygienists and employees working in the field of dairy production to understand the pattern of microbial growth specially those of public health concern as L. monocytogenes to safeguard human health.

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