D CONTROL (raw milk for direct consumption)

Summary Signiﬁcant hazards a r Depending on the geographical region and farming and hygienic

practices, one or more of the pathogens listed in Table 16.1 can be found in raw milk.

Control measures

r Prohibition or special requirements for direct sale of raw milk. r Risk communication.

Initial level (H 0 ) r Health status of milk-producing animals through national/international eradication programs for tuberculosis/brucellosis and FMD virus and

associated animal health monitoring programs, including for clinical and sub-clinical mastitis.

r Application of Good Hygiene Practices during milking. r Adequate water supply (drinking water quality). r Use of aﬂatoxin-free feed. r Prudent use of antimicrobial drugs observing recommendations of

veterinary authorities.

Reduction (ΣR)

r No reduction process available. r Use of natural antimicrobial systems (e.g. lactoperoxidase) where

possible.

Increase (ΣI)

r Cooling of milk ≤4-6 ◦ C.

Testing

r Surveillance activities related to pathogens as indicated above. r Mastitis diagnosis through somatic cell counts and microbiological

analyses. r Determination of microbial counts in bulk tank milk for veriﬁcation of

hygiene (cleaning, disinfection, cooling). r Testing for veterinary drugs and aﬂatoxins.

Spoilage

r Determination of total viable counts using traditional or automated methods (IDF, 1990a; ISO, 2002). Determination of psychrotrophic

(IDF, 1991d) or thermoduric microorganisms (Frank et al., 1985). a In particular circumstances, other hazards may need to be considered.

Hazards to be considered Drinking raw milk containing bovine tubercle bacilli was a primary cause of alimentary tuberculosis

in infants in central Europe in the 19th and 20th century. In regions where consumption of raw milk still continues, multiple outbreaks of salmonellosis, campylobacteriosis, and infections with entero- haemorrhagic E. coli have been reported since 1980, including milk from certiﬁed herds (Sharpe, 1987; D’Aoust, 1989b). Consumption of raw milk therefore poses a signiﬁcant threat to human health. A WHO consultation recommended that risk groups should be strongly discouraged from consuming raw milk (WHO, 1995).

All the microbial hazards that may initially be present in the raw milk (see Table 16.1) are of serious concern. Their possible presence in raw milk will depend on the health status of the animals, milking hygiene, and distribution/sales systems and will therefore vary from region to region. Experience

655 Table 16.1 Diseases transmitted through milk and their most important sources

MILK AND DAIRY PRODUCTS

Causative agent

Milking animal Environment Ba. anthracis

Disease

Man

× × Cl. botulinum

Anthrax

× Br. melitensis,

Botulism

× B. abortus Campylobacter jejuni

Brucellosis

× × Vibrio cholerae

Campylobacteriosis

× E. coli spp.

Cholera

× × Cl. perfringens

Pathogenic E. coli infections

× Corynebacterium diphteriae

Cl. perfringens infections

Diphtheria

Listeria monocytogenes

× × Salmonella Paratyphi

Listeriosis

× × Salmonella Enterica serovars

Paratyphus

Salmonellosis (exclusively typhus

and paratyphus)

Shigella spp.

× Staph. aureus

Shigellosis

× Streptococcus spp.

Staph. aureus intoxication

× × Mycobacterium bovis,

Streptococcus infections

× M. tuberculosis Adenoviruses

Tuberculosis

Adenovirus-infection

Various enteric viruses

Enterovirus infection incl.

Poliomyelitis and Coxsackie Virus

Food and Mouth Disease Virus

× Hepatitis A virus

Food and Mouth Disease

Hepatitis

Tick-encephalitis virus

× Coxiella burnetti

Tick-encephalitis

× Entamoeba hystolytica

Q-fever

× Cryptosporidiae spp.

Amoebiasis

× × Toxoplasma gondii

Cryptosporidiosis

× Modiﬁed from Kaplan et al. (1962).

Toxoplasmosis

1 Not fully demonstrated.

indicates that raw milk will often be contaminated with low levels of pathogens. Therefore measures short of pasteurization cannot be relied upon in large-scale production to provide safe raw milk for human consumption.

Control measures Initial level of hazard (H 0 ). International recommendations and statistics on animal tuberculosis and

brucellosis are found in publications of the Office International des Epizooties (OIE). This organization defines the absence of the disease agent in a country and provides the status to be “free of”. In general national programs follow specific recommendations of this organization.

To maintain the initial level of other pathogens as low as possible, several measures have to be applied. Generalized hygiene programs to control raw-milk production are provided on an international level by IDF (1994), FAO/WHO Codex Alimentarius, or national organizations such as the National Mastitis Council in the USA (1987).

Animals must be maintained in a healthy condition and provided with feed of good hygienic quality,

e.g. free of aﬂatoxins. Implementation of adequate mastitis control programs will reduce the incidence of contaminated raw milk. Application of good hygiene practices during milking is a further important element. Personnel should be well versed in hygienic milk production practices and proper use of equipment. If milking is not performed in a separate milking parlor, air-borne and particulate contamination must be minimized during milking by restricting movement of bedding, silage, and forage materials. Walls, ceilings, and ﬂoors should be kept free from loose materials. Appropriate cleaning of the teat

MICROORGANISMS IN FOODS 6

surfaces and adjacent parts of the animal, followed by disinfection where possible. Drying of the udder is very important to avoid subsequent multiplication. Equipment, and in particular, surfaces in direct contact with the raw milk, need to be cleaned and disinfected carefully after each milking.

Reduction of hazard (ΣR). There are no processes that will reduce the bacterial load of raw milk for direct consumption. Filtration of raw milk only eliminates larger particles such as soil, hay, but will not signiﬁcantly change the bacterial load. The activation of the lactoperoxidase system should be restricted to situations

where cooling systems are not available, mainly in sub-tropical or tropical countries. Increase of hazard (ΣI). Immediately after milking, microbial growth will initiate and freshly drawn

milk should therefore be cooled immediately to 4-6 ◦

C, or preferably lower (even if some regulations are less stringent), to slow microbial growth. However, freezing should be avoided because it can induce undesirable physicochemical changes.

Testing

A number of countries have established criteria relevant to quality and hygiene, such as microbiological counts, somatic cell count, and sediment content. Published legal criteria for different types of raw milk were reviewed by Milner (1995) and Otte-S¨udi (1996a). Traditional microbiological methods are standardized internationally (ISO, 2002) or automated techniques such as the Bactoscan r are used.

Values for raw milk for the production of heat-processed dairy products in the European Union and the United States are as follows:

European Union

United States

Total viable count <1 × 10 5 /mL at delivery and

< 1 × 10 5 /mL at delivery and

3 × 10 5 /mL before further processing Somatic cells

3 × 10 5 /mL before further processing

7.5 × 10 5 /mL With respect to inhibitory substances/veterinary drugs, an integrated system has been proposed to

4.0 × 10 5 /mL

ensure a high technological quality of milk and its safety for consumers (Suhren, 1996). It is a “hurdle” system that combines different methods and the responsibility of all parties involved. It uses elements of the HACCP concept to minimize risks and to ensure safe products and comprises two different elements:

1. Application of different detection methods with different targets to detect a range of inhibitory substances.

2. Shared responsibilities of veterinarians, dairy farmers, processing establishments, and food inspection.

This integrated system has proved to be effective in many countries. For example, in Germany the percentage of “inhibitor-positive” samples raw milk at the farm is generally <0.1%, while it is a little higher when tanker milk is tested. The dominating compounds have been β-lactams and sulfonamides.

Spoilage Depending on the intended use of the raw milk, certain groups of microorganisms may be used to identify

speciﬁc bacterial contaminations relevant to further processing of the milk. Thermoduric bacteria, for example, may indicate problems with cleaning and disinfection, as they are frequently found in bioﬁlms

657 on the milking equipment. Bacteriological methods are standardized internationally (IDF, 1990a), for

MILK AND DAIRY PRODUCTS

psychrotrophic bacteria (IDF, 1991d) or used according to relevant publications, such as for thermoduric bacteria (Frank et al., 1985).

IV Processed ﬂuid milk

A Introduction Milk products are an essential part of a balanced diet and ﬂuid milks for direct consumption represent a

major proportion. The composition of various market milks (Table 16.2) is usually expressed in terms of fat content and percent non-fat milk solids (NFMS). This is commonly speciﬁed in laws and regulations of national or supranational bodies (Staal, 1981, 1986).

Table 16.2 Typical composition of ﬂuid milks Product

NFMS (%) Whole milk

Milk fat (%)

8.25 Low-fat milk

3.25 minimum

8.25 Skim milk

8.25 Flavored low-fat milks

B Initial processing steps Homogenization. This processing step may take place before or after further treatments of the milk.

Milk fat mostly (98%) consists of triglycerides with a minor fraction of mono- and diglycerides, phospholipids, and other fats. The fat forms globules surrounded by a phospholipid membrane. In unhomogenized raw milk, globules may coalesce to form a compact layer of cream. The sweeping action of the rising fat globule clusters can carry microorganisms upward and concentrates them in the cream layer. The homog- enizer is a pump that forces milk through small orifices under high pressure. Fat globules are therefore reduced in size and remain in suspension throughout the milk for long periods of time. However, homogenization of raw milk accelerates hydrolysis of milk fat by lipases, which may result in development of rancid flavors and may be confused with bacterial spoilage. Homogenization has little effect on the microbiology of fluid milk products, except that it breaks up clumps of bacteria (Lanciotti et al., 1994).

Heat-treated, or otherwise processed, milk may be homogenized in sterile equipment after the thermal processing prior to aseptic packaging. Inadequately cleaned and disinfected or incompletely sterilized homogenization equipment can adversely affect the quality and safety of the product and lead to recontamination.

C Basic procedures to reduce the initial microﬂora Deﬁnitions of heat treatments as applied to milk and milk products have been discussed by IDF (1985).

Thermization. Growth of psychrotrophic microorganisms, such as Pseudomonas spp., to high numbers will result in the production of heat-stable proteases and lipases. High levels of these enzymes can lead to enzymatic deterioration of ﬁnished products manufactured using such milk. To increase its keeping quality during storage at low temperatures, raw milk can be submitted to a mild heat treatment or “thermization” in a continuous ﬂow system, i.e. heating at 57–68 ◦

C for a maximum of 30 s, followed by rapid cooling to <6 ◦

C. In general, a reduction of 3–4 log 10 cycles can be expected, but thermization does not control vegetative pathogens fully. It should be speciﬁcally noted that L. monocytogenes is

MICROORGANISMS IN FOODS 6

able to survive thermization (Mackey and Bratchell, 1989), and will multiply during subsequent chilled storage. Thermization may also heat-shock endospores of Bacillus spp. Subsequent storage may then permit their germination and possibly allow their inactivation when milk is subsequently pasteurized (van den Berg, 1984).

Thermization is mild enough to have minimal effects on the physical properties of raw milk. Heat inactivation of enzymes present in the milk varies depending on the enzyme and its source. Alkaline phosphatase activity is reduced by thermization but remains present.

Pasteurization. According to the IDF/WHO/FAO deﬁnition of pasteurization (IDF, 1995), milk products are pasteurized to ensure their safety by minimizing numbers of vegetative pathogens to a level considered safe for public health. Many spoilage bacteria and yeasts are also controlled. Pas- teurization is not only applied to ensure microbiological safety and to extend shelf life during refrigerated distribution (IDF, 1986), but also to meet criteria for suitability of the milk for further

processing. Pasteurization can be carried out as batch process, also called Low Temperature/Long Time (LTLT)- pasteurization, where the product is heated in an enclosed tank. In industrial milk processing, LTLT treatments between 62 ◦

C for 30–32 min are normally applied. Pasteurization can also be performed as a dynamic process, a High Temperature/Short Time (HTST) pasteurization. In this instance, the product is heated in a heat exchanger, then maintained at the desired temperature under turbulent ﬂow conditions in a holding tube to ensure the desired killing effect. Currently, the most common HTST conditions are temperatures ranging between 71 ◦

C and 65 ◦

C for at least 15 s (often 30-40 s) or very brief heating for a few seconds at 85 ◦

C and 78 ◦

C (Kessler, 1987). Time–temperature requirements vary from country to country and are frequently under regulatory control (Staal, 1986) as are the construction and operation of heating equipment.

C to 127 ◦

The most severe treatments are normally applied to products with higher fat or solids, the mildest for standard liquid milk. The actual treatments applied to the milk are often more severe to ensure that heating requirements are met.

Sterilization and UHT treatment. Treatments at higher temperatures are performed either in batches in closed containers such as cans or bottles, or continuously with subsequent aseptic packaging. Steriliza- tion of milk usually involves treatments of 119.5–120 ◦

C for 10–30 min, the lower range being applied when a UHT treatment is performed before ﬁlling and sterilizing in closed containers. Single UHT treatment are performed at temperatures not less than 135 ◦

C for at least 1 s; usually combinations of 135–150 ◦

C and holding times from 1 to 5 s are applied. Miscellaneous methods. Modern food processing techniques such as microﬁltration (e.g. Trouve et al.,

1991; Grandison and Glover, 1994; Corredig et al., 2003; Papadatos et al., 2003); high pressure treatment (e.g. Garcia-Graells et al., 2003; Hayes and Kelly, 2003; Harte et al., 2003), ultrasonication (e.g. Vercet et al., 2002), electromagnetic treatment (e.g. Bendicho et al., 2002) or addition of carbon dioxide (e.g. Ma et al., 2003) are currently being explored for their use in the dairy industry. One advanced technology, which is already being applied is microfiltration, where the milk is processed and sterilized by filtration, which also allows the separation of various milk components. The technique is not yet widely used, however. A combination of filtration and heat treatment can result in “fresh” milk with an extremely extended shelf life. In this procedure, separate heat treatments are applied to skimmed milk (mild) and cream, and the skimmed milk fraction is further filtered before recombination allowing the elimination of heat-resistant microorganisms, predominantly responsible for spoilage.

MILK AND DAIRY PRODUCTS

D Cleaning and disinfection Food contact surfaces are an important source of contamination, but unclean non-contact surfaces

can also contribute by generating contaminated dust particles and aerosols. Manual cleaning using detergents, acids, or alkali, followed by rinsing and disinfecting, is routinely used in milk and dairy products facilities.

To enhance the efﬁciency of cleaning and disinfecting procedures, automated cleaning-in-place (CIP) systems are frequently used in the dairy industry. Such systems are designed to provide high and repro- ducible standards of cleanliness with the minimum of manual efforts. Disassembly and manual cleaning may be required for sensitive parts such as ﬁlters or complex elements that cannot be cleaned effec- tively by CIP. Automation may also facilitate coordination between production and cleaning schedules. To ensure proper performance, equipment design, the type of soil to be removed, water quality, and detergent and disinfectant usage must be considered when installing a CIP system. The design, and use, of CIP systems in the dairy industry was reviewed by the International Dairy Federation (IDF, 1979). The conclusions are still valid. Even if not mandatory, strict separation of CIP systems for the raw and heat-treated milk is strongly recommended.

Proper operation of cleaning and disinfection programs must be documented. Documentation may be manual or by using recording charts for time, temperature, ﬂow rate, disinfectant concentration, pH, etc.

E Effects of processing on microorganisms Pasteurization aims to reduce numbers of potentially pathogenic vegetative bacteria present to levels

that do not constitute a public health concern. Additional treatments may include microﬁltration and bactofugation of the separated skimmed milk to increase the effect of subsequent heat treatment (IDF, 1995.) Such treatments are applied for products with extended shelf life. The risk of recontamination during subsequent handling and ﬁlling becomes even more crucial.

Freshly pasteurized milk usually contains less than 1000 cfu/mL, but higher levels are observed if the initial levels in the raw milk are much higher than 10 6 cfu/mL. In the case of ultra-pasteurized milk (extended shelf life, ESL) the initial levels are much lower. Hence, minimal LTLT and HTST treatments may permit the survival of thermoduric and spore-forming organisms, particularly if they are present at high levels, making it difﬁcult to meet norms for pasteurized products. Thermoduric organisms include vegetative microorganisms showing an increased heat-resistance such as Micrococcus spp., Enterococcus faecalis, and Ent. faecium, and some lactobacilli (Deibel and Hartman, 1984). Endospores of Bacillus and Clostridium spp. display a range of heat-resistance.

An important source of microorganisms in pasteurized milk is post-process contamination. Pseu- domonads are typical post-process contaminants (Eneroth et al., 2000) but Bacillus spp. may also be reintroduced after the heat treatment (Schraft et al., 1996). Since surviving thermoduric bacteria may adhere and multiply in the cooling section of the pasteurizer, care must be taken that plants are regularly evaluated for the prevalence of bioﬁlms, and HTST systems not operated too long without cleaning (Bouman et al., 1982; Sharma and Anand, 2002). Cooling, ﬁlling, and low-temperature storage of pasteurized milk do not inactivate microorganisms, but must be controlled to minimize growth of those surviving, to minimize recontamination and limit growth of possible post-process contaminants.

F Spoilage Pasteurized milk from modern well-operated plants can have a shelf life of well over 10 days under

refrigeration (Otte-S¨udi, 1996b). Commercially manufactured ultra-pasteurized milk is much more stable and remains stable for periods as long as 10 weeks under refrigeration (Boor, 2001).

MICROORGANISMS IN FOODS 6

As in the case of raw milk, pasteurized and ultra-pasteurized ﬂuid milk products will support abundant growth of microorganisms if contaminated. Spoilage may involve: r Growth of surviving spore-forming bacteria (Bacillus and Clostridium spp.); r Growth of thermoduric bacteria; r Growth of contaminating psychrotrophic (Gram-negative) bacteria; and r Activity of heat-stable enzymes produced pre-pasteurization.

Microorganisms causing spoilage are usually spore-forming or thermoduric bacteria surviving the pasteurization process (Meers et al., 1991), from the contamination of equipment, e.g. as bioﬁlms (Bouman et al., 1982; Carpentier and Cerf, 1993; Wong, 1997) or from post-process contamination by environmental microorganisms such as Enterobacteriaceae (Varnam and Sutherland, 1994). Further details on spoilage ﬂora such as species of Pseudomonas, Flavobacterium, Chromobacterium, Alcali- genes, Bacillus, and coliforms commonly found have been reviewed (Cousin, 1982; Meers et al., 1991; Ternstrom et al., 1993; Deeth et al., 2002). Psychrotrophic strains of both thermoduric or spore-forming organisms grow at temperatures as low as 5 ◦

C and can cause spoilage or be hazardous to health (Crielly et al., 1994). If initially present in high numbers, sufﬁcient growth may occur to cause spoilage within 10–14 days of refrigerated storage.

Microbial spoilage of refrigerated market milk products is recognized primarily by development of off-flavors, often described as unclean, putrid, and fruity, while physical changes such as ropiness and partial coagulation are less common defects. The intensity of flavor defects depends on the extent of microbial enzymatic decomposition of milk proteins, fat, and to some extent lactose. A particular aspect of spoilage is that due to preformed enzymes, such as heat-resistant lipases or proteases of psychrotrophic microorganisms, principally Pseudomonas spp. (Champagne et al., 1994; Muir, 1996a,b,c; Stevenson et al., 2003), commonly causing bitterness of the product. Bacillus cereus is particularly troublesome because it produces lecithinase that acts on the phospholipids of the milk fat globule, forming small proteinaceous fat particles that adhere to the surfaces of glasses (IDF, 1992). The defect due to the development of B. cereus is referred to as “sweet curdling” and is particularly a problem in the warm summer months (Christiansson et al., 1999). The time required for changes to occur depends on the initial numbers and type of microorganisms present, the pasteurization conditions and the storage temperature (Schröder et al., 1982; Mourgues et al., 1983; Schröder and Bland, 1984). For example, the average time required for spoilage of HTST pasteurized milk stored at 1.7, 5.6, and 10 ◦

C was 17, 12, and 6.9 days, respectively (Hankin et al., 1977). Pasteurized milks from modern well-operated plants have >10 days of shelf life under refrigeration, and even longer if the milk is aseptically packed (Otte-S¨udi, 1996b).

G Pathogens Minimum pasteurization treatments speciﬁed by law or regulation generally allow destruction of

pathogens likely to be present initially in raw milk with a sufﬁcient margin of safety. Although pasteurized market milk products have a remarkable safety record and present little health hazard, several outbreaks (campylobacteriosis, salmonellosis, yersiniosis, etc.) have been linked to pasteurized milk. Such outbreaks are usually due to inadequate pasteurization, post-pasteurization contamination, or temperature abuse during use (Snyder et al., 1978; Sharpe, 1987; Doyle, 1989).

Salmonellae do not survive pasteurization and their presence in pasteurized milk is the consequence of either improper heat treatments or post-pasteurization contamination. The outbreaks in the United Kingdom (Salmonella Braenderup; Rampling et al., 1987) and in the United States (Adams et al., 1984) are examples of inappropriate heat treatments. In the latter example, temperatures as low as 54.5 ◦

C for

30 min were recorded.

661 The largest outbreak of salmonellosis in the history of the United States occurred in Northern Illinois.

MILK AND DAIRY PRODUCTS

Pasteurized low-fat milk (2%) contaminated with S. Typhimurium was the cause of more than 16 000 cases (Anonymous, 1985a,b; Ryan et al., 1987). Inspections of the incriminated dairy plant revealed no evidence of improper pasteurization and Bradshaw et al. (1987) conﬁrmed that the outbreak strain possessed no abnormal heat resistance. Although the cause was never completely elucidated, a thorough investigation of the plant and production lines revealed a cross-connection between the pipes used for raw and pasteurized milk that may have been at the origin of the contamination (Lecos, 1986).

It is generally accepted that current minimum pasteurization standards (71.7 ◦

C, 15 s or 62.8 ◦ C,

30 min) are sufﬁcient to inactivate L. monocytogenes in milk (Donnelly et al., 1987; Bunning et al., 1988). Nevertheless, viable Listeria spp. including L. monocytogenes have been isolated from pasteurized milk in different countries at frequencies ranging from 0.9% to about 5% (Harvey and Gilmour, 1992; Moura et al., 1993; Ahrabi et al., 1998). Fernandez-Garayzabal et al. (1986) attributed the presence of L. monocytogenes in 21.4% of the samples of pasteurized milk treated at 78 ◦

C for 15 s to the protective effect provided by type A leukocytes. A similar explanation was given for the reasons of the outbreak of L. monocytogenes 4b in Massachusetts affecting 49 consumers (Fleming et al., 1985). These explanations were, however, questioned by other researchers (Donnelly, 1990; Lovett et al., 1990).

L. monocytogenes is frequently found in the wet environments of milk processing plants (Jung and Busse, 1988; Jeong and Frank, 1994a,b; Pritchard et al., 1995) and may cause post-processing contamination.

Campylobacter jejuni has been the cause of an outbreak involving 110 patients after the consumption of inadequately pasteurized milk (Fahey et al., 1995). Additional sporadic cases of campylobacteriosis in the United Kingdom were traced to birds pecking through the caps of milk bottles. Campylobacter was isolated from the beaks of jackdaws and magpies as well as from the contaminated milk (Hudson et al., 1990; Southern et al., 1990; Stuart et al., 1997).

Although swine are recognized as the major reservoir of Y. enterocolitica in nature (Doyle et al., 1981), several studies have demonstrated the presence of this species in pasteurized milk and outbreaks have been reported (CSPI, 2004). Schiemann and Toma (1978) recovered Y. enterocolitica only from 1 of 165 samples of pasteurized dairy products produced in Ontario, Canada. Similarly, Moustafa et al. (1983) recovered Y. enterocolitica from 1% of pasteurized milk samples examined, while Tibana et al. (1987) recovered the pathogen from 13.7% of pasteurized milk samples produced in Rio de Janeiro, Brazil. Twenty-two of the 41 isolates were capable of producing heat-stable toxin in culture media but not in sterile whole milk.

Since Y. enterocolitica is rapidly inactivated at pasteurization temperatures (Francis et al., 1980; Lovett et al., 1982), its presence in ﬁnished product is most likely to be the result of post-pasteurization contamination. This is certainly true for reported outbreaks (Ackers et al., 2000). In 1976, consumption of contaminated chocolate milk at a school resulted in the illness of 36 children from Y. enterocolitica serotype O8. The same serotype was isolated from the milk and was probably introduced during manual mixing of pasteurized milk with chocolate syrup without subsequent heat treatment (Black et al., 1978). In another outbreak, pasteurized milk was implicated as the vehicle of infection of 148 persons in three states (Tacket et al., 1984) and of 19 people in Nevada in 1996 (CSPI, 2004). A very large outbreak was probably caused by the use of packaging material contaminated with pig faeces, although the implicated serovar could never be isolated from swine (Schiemann, 1989). Greenwood et al., (1990) indicated in the investigation of the incriminated dairy bottling plant the possibility of a contaminated ﬁller valve causing the outbreak.

Escherichia coli O157:H7 was responsible for a large outbreak (more than 100 persons) due to the consumption of pasteurized milk. During inspection of the processing plant, the same phage-type was isolated from pipes to the bottling machine and from rubber seals from the same machine, suggesting post-process contamination (Upton and Coia, 1994). The reasons for another outbreak involving 114

MICROORGANISMS IN FOODS 6

persons are not known and could have been either a failure in the heat treatment or post-process contamination, since the milk was processed at the farm (Goh et al., 2002).

Overall, the risk of B. cereus enterotoxin caused gastroenteritis from pasteurized milk is very low, although it has been implicated in an outbreak in the Netherlands involving more than 250 consumers (van Netten et al., 1990). Taking into account the quantity of pasteurized milk sold, the number of cases due to B. cereus worldwide is very low, perhaps because spoilage of the products deters consumers, and the quantity of toxin required to cause illness seems high (IDF, 1992; Langeveld et al., 1996). The prevalence of B. cereus in milk varies greatly. In some countries, a prevalence of 2% has been reported (Wong et al., 1988), whilst in other areas every sample is positive (Notermans et al., 1997). Some strains of B. cereus strains are capable of growing and producing enterotoxin at chill temperatures (<7 ◦

C, spoilage occurred before high levels of B. cereus were present, however, signiﬁcant growth to levels above 10 5 cfu/mL was seen at

C) (Dufrenne et al., 1995; Notermans et al., 1997). When stored at 6 ◦

C (Notermans et al., 1997). In studies with human volunteers, only very weak symptoms were noticed when milk containing >10 7 cfu B. cereus per mL was ingested (Langeveld et al., 1996). Investi- gations to determine the origin of this spore-forming species have shown that post-process contamination is a frequent cause of its presence in the ﬁnished products (Lin et al., 1998; Eneroth et al., 2001).

In the mid 90s, PCR-based detection demonstrated the presence of Mycobacterium avium subsp. paratuberculosis (MAP) in retail pasteurized milk in the UK (Millar et al., 1996; Grant et al., 2002a). This has caused much debate since MAP in cattle and sheep causes an inflammatory chronic infection of the gut called Johne’s disease. In humans, a disease with similar symptoms, Crohn’s Disease (CD), is becoming more common. No firm link has been made between occurrence of MAP and CD, and several factors (genetic predisposition, abnormal immune response) appear to influence development of the disease. However, studies have failed to demonstrate that MAP is not involved in CD (European Commission, 2000; Harris and Lammerding, 2001; Chamberlin et al., 2001; Bernstein et al., 2004). Mycobacterium avium subsp. paratuberculosis is very difficult to culture because it grows very slowly, with incubation for up to 1 year recommended. Studies have demonstrated that despite a 4-6 log 10 reduction during commercial pasteurization, some individual cells of MAP may survive the milk pasteurization process (Grant et al., 2002b; Hammer et al., 2002). Increasing the pasteurization time, e.g. from 15 to 25 s at 73 ◦

C, appear to have no increased lethal effect (Lund et al., 2002; Hammer et al., 2002). Although there is the potential for other pathogens to be present in pasteurized milk as a result of post-processing contamination, there are very few documented cases of illness resulting from such contamination. Staph. aureus intoxications were reported from pasteurized milk, and recontamination during ﬁlling of containers was assumed to be the reason (Geringer, 1983). A large outbreak of staphylococcal enterotoxin poisoning was caused by chocolate milk containing between 94 and 184 ng of type A

C, or temperature up to 90 ◦

toxin per carton (Evenson et al., 1988). If staphylococcal enterotoxins or aﬂatoxin M 1 are present in raw milk, they also will be present in pasteurized milk.

D CONTROL (raw milk for direct consumption)