A STUDY ON INCIDENCE AND GROWTH HAZARDS OF BACILLUS CEREUS IN EGYPTIAN PASTEURIZED MILK DURING REFRIGERATOR STORAGE

Dept. of Food Hygiene, Port-Said Lab.

Animal Health Research Institute, Dokki, Giza.

A study on incidence and growth hazards of bacillus cereus in Egyptian pasteurized milk during refrigerator storage

(With 2 Tables and One Figure)

By

Azza A. EL-Tabiy; Zienab I. Soliman and

Hoda A. Aideia*

* Bacterial Toxin Unit, Animal Health Research Institute, Dokki, Giza, Egypt.

(Received at 6/4/2009)

دراسة عن وجود ومخاطر نمو ميکروب الباسيلس سيرس فى الألبان المبسترة المصرية أثناء حفظها مبردة

عزة على التابعي ، زينب إبراهيم سليمان ، هدى أمين عايدية

يعتبر ميکروب الباسيلس سيرس من الميکروبات التى تتسبب فى فساد الألبان المبسترة وتقلل من فترة صلاحيتها حيث انه من الميکروبات المتجرثمة التى لا تتاثر بعملية البسترة0 ويوجد منها بعض الأنواع التى لها القدرة على العيش فى درجات حرارة منخفضة0 لذلک تم استبيان مدى وجود ذلک الميکروب فى الألبان المبسترة المصرية0 تم تجميع عدد 80 عينة من ألبان مبسترة بالحرارة العالية لفترة قصيرة (72 درجة مئوية لمدة 15 ثانية), 40 عينة من کل من ألبان کاملة الدسم والمنزوعة الدسم ا0 تم فحص الألبان عند بداية فتحها وکذلک يوميا أثناء حفظها عند درجة 5 ° م حتى انتهاء صلاحيتها (15 يوم من الإنتاج) وقد أسفرت النتائج على وجود الميکروب فى 3 (5¸7%) من عينات اللبن الکامل الدسم من اجمالى عدد العينات التي تم فحصها في البداية0 ولم يتم تسجيل اى زيادة فى العينات الايجابية خلال 8 ايام من الفحص اثناء حفظها مبردة0 ولکن بعد ذلک کان العدد الکلى للعينات الأيجابية  فى الألبان المبسترة الکاملة الدسم 19 )5¸47%) وفى عينات اللبن المنزوع الدسم10 (25%) وذلک حتى انتهاء تاريخ صلاحيتها0 هذا ولم يتعدى العدد الکلى للميکروب فى العينات الايجابية الحد الذى يشکل خطرا على صحة المستهلک (510خلية/ مل )0 اثناء الحفظ عند درجة 5 ° م0 کذلک تم حفظ جزئين من عينة من اللبن التي اثبت التحاليل ايجابيتها لفحص الباسيلس سيرس وتحتوى على عدد قليل من الميکروب ، فى درجة 25 و32 درجة مئوية 0وقد أظهرت النتائج أن الحفظ الغير جيد وخاصة فى فصل الصيف قد يؤدى إلى نمو الميکروب بصورة کبيرة تؤدى الى فساد المنتج وقد يؤدى إلى حدوث تسمم غذائي بالميکروب فى اقل من 24 ساعة0 کذلک تم الکشف عن قدرة المعزولات على إفراز إنزيمات تحلل الدهون والبروتين والتي تؤدى الى فساد المنتج وأظهرت النتائج أن 96% و60% من المعزولات لها القدرة على تحلل الدهون والبروتينات على التوالى0 لذلک يمکن القول ان سلامة اللبن المبستر مسؤولية مشترکة بين المنتج والموزع والمستهلک عن طريق الحفظ الجيد0 هذا وقد تم مناقشة التأثيرات الاقتصادية والصحية لوجود ذلک الميکروب فى الألبان المبسترة0

SUMMARY

A total of 80 random samples, fortyof each of pasteurized full cream and skimmed milk were collected from local Egyptian markets. Samples were screened for the occurrence of Bacillus cereus. The incidence and total count of B. cereus in the different samples at the sterile opening were evaluated. 3 (7.5%) B. cereus positive samples were detected when the samples analyzed early. All milk samples were stored at 5 °C and evaluated for B. cereus up to their expiry date (at 72 °C for 15 s). No increase in incidence and count of B. cereus were observed during the first 8 days of storage at 50C but thereafter, an increase in the incidence and population of the microorganism with storage time was observed.   B. cereus was detected in 19 (47.5%) and 10 (25%) of the investigated pasteurized full cream and skimmed milk samples, respectively at the end of refrigerator storage. Quantitive analysis of growth of B. cereus in refrigerated pasteurized milk confirms that no samples achieved hygienically undesirable limit 105 cfu/ml. Two portion of milk, proved to be positive for B. cereus were kept at ambient (25°C) and optimum (32°C) temperatures and evaluated for the growth of B. cereus during 24 hours. The data indicate that, if storage temperature is abused, less than 24 h are required for low initial levels of B. cereus cells to increase to levels that are sufficient to cause foodborne illness. Out of 50 B. cereus isolates tested, 48 (96 %) possessed lipolytic activity, while 30 (60%) showed proteolytic activity. B. cereus ability to elaborate protease and lipase may give an indication of their active role in the spoilage of pasteurized milk in abuse temperature. In spite of the fact that the producer is responsi­ble for the safety of pasteurized milk, it is evident that this responsibility has to be shared with the retailer and consumer by applying proper cooling, especially in the summer period. B. cereus role in the keeping quality and hazards of pasteurized milk were discussed.

Key words: Milk, pasteurized milk, Bacillus cereus.

introduction

The gradual transition of food retailing patterns from small stores to large supermarkets has been associated with an extension of the shelf life of the perishable foods. This applies also to pasteurized milk. Improving microbial safety and extending the shelf life of pasteurized milk have always been an important concern to the dairy industry. A major factor limiting realization of these goals is microorganisms surviving the pasteurization process and/or contributing to post pasteurization contamination. B.cereus is a common bacterium that is often present in raw milk (Griffiths, 1992). Possible sources of contamination are the soil, faeces, bedding, feed, air, the milker, and the milking equipment (Van Heddeghem and Vlaemynck, 1992)           .           Under stress conditions, the B. cereus cells sporulate. The presence of spores in raw milk, their high resistance to the pasteurization temperature (Lin et al., 1998 and Janstova and Lukasova, 2001), and also the adherence of spores and vegetative cells to metal surfaces (Faille et al., 2001 and Peng et al., 2001) make B. cereus an unwelcome, but very frequent contaminant of dairy products (Huang et al., 1999).

Milk simply labeled "pasteurization" is usually treated with High Temperature/Short Time (HTST). In the HTST process, milk is forced between metal plates or through pipes heated on the outside by hot water, and is heated to 72 °C for 15-20 seconds. Pasteurized milk has a shelf life from only a couple of days in some countries to up to over 20 days in, e.g., USA (Rysstad and Kolstad, 2006). Pasteurization will actually induce spore germination, and in the absence of competing flora, B. cereus grows well (Granum and Lund, 1997). B. cereus species may be subdivided according to their thermotolerance: the psychrotrophes that can grow at temperatures as low as 4ºC, but fail to grow at temperatures above 37ºC, and the mesophiles, able to grow at temperatures ranging from 10ºC to, sometimes even, 50ºC (Kramer and Gilbert, 1989). Psychrotrophic strains of B. cereus are some of the most important organisms limiting the shelf life of pasteurized milk stored above 6°C (Griffiths, 1992). B. cereus is frequently found in pasteurized milk, causing spoilage because of the production of lipases and proteases. They can also exhibit a health risk to the consumer since they produce enterotoxins (Champagne et al., 1994).

B. cereus play a very important role in the keeping quality of pasteurized milk. Therefore, the present study was undertaken in order to assess the existence and growth of B. cereus during refrigerator storage of pasteurized milk. The effect of abuse storage temperature on growth of B. cereus in milk and the proteolytic and lipolytic potentials of isolates were also investigated.

Materials and methods

1. Milk samples and sampling:

A total of eighty, freshly produced (day 2 of production) pasteurized milk samples (at 72. °C for 15 s), fortyof each of pasteurized full fat milk and skimmed milk, packaged in plastic bottles (15 days shelf life) were collected from local Egyptian markets over a period of 6 months. The samples were transported in cooler boxes with ice to the laboratory and analyzed within 1h after collection.

Milk samples were aseptically opened under laminar flow. The incidence of B. cereus in the milk samples was investigated. All milk samples were tested for B. cereus at the sterile opening and daily during keeping the samples at 5ºC, the recommended temperature for cold storage of pasteurized milk until their expiry date.

In addition, 2 portion of milk, proved to be positive were stored in incubators at ambient (25°C) and optimum (32°C) temperatures and then tested for growth of B. cereus at intervals of 0, 3, 7, 10, 15, and    24 h. The average number of colonies on the duplicate plates was recorded for each sample at each time to study the effect of abuse storage temperature on growth of B. cereus in milk.

2. Isolation and enumeration of B. cereus:

For each milk sample, 50 ml were added to 450 ml Butterfield’s phosphate-buffered and blended for 2 min at 18,000-20,000 rpm. Serial dilutions from 10-1 to 10-6 were prepared. Isolation procedures followed the methods described by Health Protection Agency (2004), using        B. cereus selective agar (PEMBA) supplemented with polymyxin B (Oxoid) and egg yolk emulsion (Oxoid) as selective plating medium. Plates were inoculated with each dilution of sample (including 1:10) by spreading 0.1 ml evenly onto surface of each plate with sterile glass spreading rod.The inoculated Petri dishes were incubated at 30°C for  24 h. Typical 2 to 5 mm in diameter, turquoise to peacock blue in color with flat ground glass surface and surrounded by a grey to turquoise halo of dense precipitate (egg yolk reaction) were counted.

3. Confirmation of B. cereus

Turquoise to peacock blue colonies were isolated and confirmed as B. cereus. 5 or more presumptive positive colonies plates were Picked and transferred to nutrient agar (NA) slants and incubated at 30°C or for 24h for biochemical confirmation as B. cereus. All isolates from NA were examined by Gram stain for morphologic characterization. The identities of all suspected colonies were confirmed by testing for glucose fermenta­tion, Voges-Proskauer reaction, nitrate reduction, and              β-hemolytic activity. B. cereus strains should be positive for all four reaction.Motility test, rhizoid growth on NA plates were applied to differentiate typical strains of B. cereus from other members of the cereus group (FDA, 1998).

4. Determination of proteolytic and lipolytic activities of isolates

The proteolytic activity was tested by inoculating the strains on 50 percent skim milk agar. Then plates were incubated at 5°C for 10 days. The presence of clear zone of hydrolysis around the colonies confirmed as positive (APHA, 1978).

The lipolytic activity was tested by culturing the strains on tributyrin agar. The plates were incubated at 25°C for 3 days and screened for the presence of clear zone of hydrolysis (Harrigan and Mc Cance, 1976).

RESULTS

Table 1: Statistical analytical results of B. cereus incidence and counts in examined full cream and skimmed pasteurizedmilk samples during storage at 50C.

0 Time: Second day of production.

ND: Non detectable (<102).

NB: % was calculated according to the total number of each sample (40).

Fig. 1: Growth of Bacillus cereus in pasteurized milk during storage at ambient (25°C) and optimum (32°C).

Table 2: Lipolytic and proteolytic activities of B. cereus isolated from the examined milk samples.

DISCUSSION

The prevalence and total count of B. cereus in examined samples during refrigerator storage up to their expiry date were presented in Table (1).

On examination on the day of opening (day 2 of production) only   3 (7.5%) of examined full cream milk samples were found to be positive for B. cereus cr. However, no positive samples could be detected in examined skimmed milk samples on the day of opening. The initial count of B. cereus during the first 8 days of storage at 50C was low, numbers ranged from 2.5x102 to 5.0x102 with a mean count of 3.5x102 ± 7.6x101 cfu/ml were observed in the positive samples recorded. Improvedtemperature control of stored milk during sale may be reflected in the lower reportedprevalence.

For the first 8 days of storage at 50C, there was no increase in number of positive samples and count of B. cereus but thereafter, an increase in the incidence and population of the microorganism with storage time was observed. The achieved results in Table (1) showed that higher incidence of B. cereus positive samples was recorded from pasteurized full eam milk samples during observation period. B .cereus positive samples were detected in 19(47.5%) and 10 (25%) of the investigated pasteurized full cream and skimmed milk samples, respectively at the end of refrigerator storage. In this concern, Larsen and Jorgensen (1996)reported that vegetative cells and spores of          B. cereus tend to adhere to fat drop­lets of the milk. The presented results indirectly corroborate with that findings by Schiegelova et al. (2003) they reported that products made from skimmed milk (curd cheese) were contaminated in one case only (3.2% of products), while products with higher fat content (even if made with different production technolo­gies) in 10–16% of cases.

The obtained results regarding the presence of B. cereus in pasteurized milk were supported by other reports. Isolation of B. cereus from samples of pasteurized milk (35%) have been reportedby Ahmed et al. (1983).          B. cereus was detected in 2% of Chinese pasteurized milk samples ataverage counts of 280/ml (Wong et al., 1988).                       Counts of B. cereus in 38 samples of pasteurized milk in the Netherlands showed that all contained spores with a mean of around 100/100 ml (Notermans et al., 1997), with counts highest in the summer. B. cereus could be isolated from 133 (40%) from pasteurized milk in household refrigerators in The Netherlands, they found low B. cereus counts, less than five per ml in the samples that were stored below 7°C. (Te Giffel et al., 1997). Eneroth et al. (2001) conducted a study on pasteurized milk samples from Sweden they reported that counts of      B. cereus ranging from 10 to 1200 CFU/ml in the samples taken from consumer packages. however, counts of up to 104 cfu/ml have also been recovered from bottled pasteurized milk (86.7%) by Ionescu et al.(1966).        Moreover,Larsen and Jorgensen (1996) analyzed 458 samples of pasteurized milk and cream from 3 Danish dairies and found the total viable count of B. cereus ranging from 103 to 3 X 105 cfu / ml. The high prevalence of B. cereus in milk reported by these researcher may bedue to survival of spores during pasteurization and subsequent re-growth.

Many dairy scientists believe that contamination of pasteurized milk with Bacillus spp. results from spores that were present in the raw milk and survived the pasteurization process (Griffiths and Phillips, 1990 and Sutherland and Murdoch, 1994). However, other data in the literature suggest that post pasteurization contamination might significantly contribute to Bacillus counts in pasteurized milk (Crielly   et al., 1994).

               The results recorded in Table (1) pinpoint that B. cereus was found to be present in two-day pasteurized milk samples in relatively small numbers and the count did not increase during first 8 days of storage, but after this time an increase in the incidence and population     of the microorganism with storage time were observed. At 9- 11 days         of storage, the milk samples had B. cereus count ranged from 4.5x102 to           2.5x103 in examined pasteurized full cream milk and 3.5x102 to 9.0x102 in skimmed milk samples. The count increased up to 8.2 x103and to 7.2x103 cfu/ml at the end of observation period in examined pasteurized full cream and skimmed milk samples, respectively. The present findings suggest that the growth of B. cereus naturally present in pasteurized milk as regarded as a thermoduric psychrotrophic species, can survive pasteurization and grow at refrigeration temperatures.Several strains of B. cereus are psychrotrophic and are capable of growth in milk at temperatures as low as 4-6°C (Andersson et al., 1995). Possibly mesophilic B. cereus may be facultative in the sense that they develop psychrophilic activity after adaptation to prolonged low temperature storage. 

            Since psychrotrophs have a significant impact on the keeping quality of pasteurized milk the findings in this study suggest that the ability of B. cereus to grow at low temperature occurs during storage. In this concern, Kevin et al. (1998) reported that the detection of psychrotrophic strains of the B. cereus group in dairy products is at present extremely slow (5 to 10 days), due to the inability to discriminate psychrotrophic and mesophilic strains by means other than growth at low temperature. Psychrotrophic bacteria have been recognized as a recurring problem in the refrigerated storage and distribution of perishable food products, which is particularly pertinent to the dairy industry (Sorhaug and Stepaniak, 1997). It has been estimated that 25% of all shelf life problems associated with conventionally pasteurized milk and cream products in the United States may be linked to this class of thermoduric bacteria, with a large number of the contaminants being psychrotrophic B. cereus and B. mycoides (Meer et al., 1991).

In evaluating these data in term of the practical keeping problem in home refrigerators, good quality commercially pasteurized milk should maintain desirable quality for at least 8 days when stored at 50C. It has been considered that a number of 105 B. cereus present in pasteurized milk should be regarded as hazardous to the consumer (Notermans et al., 1997). Quantitive analysis of growth of B. cereus in refrigerated pasteurized milk confirms that no samples achieved hygienically undesirable limit 105 cfu/ml.

The gross abuse trials were presented in Fig (1). Based on the worst case situation, we considered the possibility that consumers might left portions of milk not use it immediately, or that unused might be left un refrigerated, and outgrowth of any B. cereus present in milk could occur. An increase in the population of B. cereus with storage time was observed in both samples and its level had reached 7 and 8.1 log cfu/ml after 24 hours in samples stored at 25 and 32 °C respectively. Approximately 3 to 7 hours were required to reach 5 log cfu/ml during storage at 32ºC, and 10 to almost 15 h during storage at 25ºC. Indicating a possibility of larger mesophilic bacterial activity at 25 and 32°C than psychrotrophic activity at 5°C. Chandler and McMeein (1985) consider the storage temperature as the basic factor influencing the growth of the microorganisms in the spoilage of milk. Several reports shown that the organism can proliferate in pasteurized milk depending on the storage temperature (Crielly et al., 1994). According to Adams and Moss (2000), these microorganisms can grow and spoil a product quite rapidly at ambient temperature, so refrigerated storage is often a requirement for an acceptable shelf-life. In this respect,Feijoo et al. (1997) studied the outgrowth of B. cereus in whipping cream stored at 23, and 32°C,from aninitial inoculum of 1 x102 cfu /ml, within 9 h at 32°C and 11 h at 23°C, numbersreached 105 to 106 cfu/ml. In this concern, Bean and Griffin (1990) reported that 94% of outbreaks involving B. cereus were attributed to improper holding temperature; therefore, the importance of maintaining adequate refrigerated storage of dairy products is obvious. There are two forms of B. cereus food poisoning. The rapid-onset emetic illness occurs one to five hours after the consumption of food contaminated with a heat stable enterotoxin produced by B. cereus during growth in the food. The second form is a diarrhoeal illness, which becomes evident eight to sixteen hours after ingestion of contaminated food (Helgason et al., 2000).

During storage under low temperature the milk undergoes spoilage due to proteinases and lipases released by psychrotrophic bacteria (Sorhaug and Stepaniak, 1997).

The number of Bacillus isolates positive for proteolysis and lipolysis is given in Table (2). Out of a total 50 isolates, 48 (96%) and 30 (60%) of total B. cereus isolated were found to be lipolytic and proteolytic respectively. Meer et al. (1993) concluded that 57% of the Bacillus isolates obtained from 59 Grade A milk samples in Oregon, US were lipolytic. Almeida et al. (2000) detected that 92% of the Bacillus isolates were found to have proteolytic and or lipolytic activity. Matta and Punj (1999) obtained 59 lipolytic Bacillus isolates and showed that   B. cereus (32.2%) was the predominant lipolytic organism. It has been estimated that 25% of all shelf life problems associated with conventionally pasteurized milk and cream products may be linked to      a large number of the contaminants being psychrotrophic B. cereus, the organism is associated with defects such as off flavours, sweet curdling and bitty cream caused by proteinase, lipase and phospholipase enzymes (Meer et al., 1991). Brown (2000) described Bacillus spp. as microorganisms that cause significant economic losses. Low concentrations of B. cereus can cause defects such as coagulation and off-odors due to proteolysis and lipolysis (Andersson et al., 1995). This defects are detected at the moment of reaching the concentration of microorganisms of 5x105 - 107 cfu/ml (Marth and Steele, 1998).

Based on the results obtained, B. cereus levels on examined Egyptian pasteurized milk samples were lessthan that considered harmful. The data indicate that, if storage temperature is abused, less than 24 h are required for low initial levels of B. cereus cells to increase to levels that are sufficient to cause foodborne illness. Pasteurized milk stored under proper conditions, at 5 ºC, and consumed within the expiration date in general cause no problems. The producer is responsi­ble for the safety of pasteurized milk, it is evident that this responsibility has to be shared with the retailer and consumer by applying proper cooling, especially in the summer period. It is evident from these studies that there might be a possibility of alteration of milk by the proteolytic and lipolytic activities of the B. cereus isolates in the present study, their activity may result in the degradation of milk during transport and storage, hence care is to be taken while handling and storage of pasteurized  milk to avoid spoilage.

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