CHAPTER 1
LIQUID MILK PRODUCTS
1.1 Definitions
Milk is a complex biological fluid secreted in the mammary glands of mammals. Its function is to meet the nutritional needs of neonates of the species from which the milk is derived. This section of the handbook refers mainly to bovine milk, but the milk of other species, such as sheep and goats, is used for human consumption.
Typically, bovine milk is composed of approximately 87% water, 3.7 - 3.9% fat, 3.2 - 3.5% protein, 4.8 - 4.9% carbohydrate (principally lactose), and 0.7% ash. However, the exact composition of bovine milk varies with individual animals, with breed, and with the season, diet, and phase of lactation. Milk produced in the first few days post parturition is known as colostrum. Colostrum has a very high protein content, and is rich in immunoglobulin to help protect the newborn against infections. Colostrum is not generally allowed to enter the food supply in most countries.
Fresh milk products here refers to liquid milk, which accounts for about half of the total dairy market in the UK. Liquid milk is largely heat treated in developed countries, but a small quantity of raw (unpasteurised) milk is still sold in the UK. Skimmed and semi-skimmed milk, which are defined by their fat content (0.5%, and 1.5 - 1.8%, respectively), are increasingly important products in the liquid milk market.
1.2 Initial Micro flora
1.2.1 Contamination from the udder
Although milk produced from the mammary glands of healthy animals is initially sterile, microorganisms are able to enter the udder through the teat duct opening. Gram-positive cocci, streptococci, staphylococci and micrococci; lactic acid bacteria (LAB), Pseudomonas spp. and yeast are most frequently found in milk drawn aseptically from the udder; corynebacteria are also common.
Where the mammary tissue becomes infected and inflamed; a condition known as mastitis, large numbers of microorganisms and somatic cells are usually shed into the milk. Mastitis is a very common disease in dairy cows, and may be present in a subclinical form, which can only be diagnosed by examining the milk for raised somatic cell counts. Many bacterial species are able to cause mastitis infection, but the most common are Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis and Escherichia coli. These bacteria enter the udder by the teat duct, and Staph. aureus is able to colonise the duct itself. Although the organisms involved in mastitis are not usually able to grow in refrigerated milk, they are likely to survive, and their presence may be a cause of concern for health.
Diseased cows may also shed other human pathogens in their milk, including Mycobacterium bovis, Brucella abortus, Coxiella burnetii, Listeria monocytogenes and salmonellae. Recently, concerns have also been raised over the presence of Mycobacterium avium var. paratuberculosis (MAP) (the causative organism of Johne's disease in cattle) in milk from infected animals.
The outer surface of the udder is also a major source of microbial contamination in milk. The surface is likely to be contaminated with a variety of materials, including soil, bedding, faeces and residues of silage and other feeds. Many different microorganisms can be introduced by this means, notably salmonellae, Campylobacter spp., L. monocytogenes, psychrotrophic spore-formers, clostridia, and Enterobacteriaceae. Good animal husbandry and effective cleaning and disinfection of udders prior to milking are important in minimising contamination.
1.2.2 Other sources of contamination
Milking equipment and bulk storage tanks have been shown to make a significant contribution to the psychro trophic microflora of raw milk if not adequately sanitised (1). Exposure to inadequately cleaned equipment and contaminated air are also sources of contamination (2). Milk residues on surfaces, and in joints and rubber seals can support the growth of psychrotrophic Gram-negative organisms such as Pseudomonas, Flavobacterium, Enterobacter, Cronobacter, Klebsiella, Acinetobacter, Aeromonas, Achromobacter and Alcaligenes, and Gram-positive organisms such as Corynebacterium, Microbacterium, Micrococcus and spore-forming Bacillus and Clostridium (3). These organisms are readily removed by effective cleaning and disinfection, but they may build up as biofilms in poorly cleaned equipment. Milk-stone, a mineral deposit, may also accumulate on inadequately cleaned surfaces, especially in hard water areas. Gram-positive cocci, some lactobacilli, and Bacillus spores can colonise this material and are then protected from cleaning and disinfection. Some of these organisms may survive pasteurisation and eventually cause spoilage (4).
Other, less significant, sources of contamination include farm water supplies, farm workers and airborne microorganisms.
1.2.3 Natural antimicrobial factors
Raw milk contains a number of compounds that have some antimicrobial activity. Their purpose is to protect the udder from infection and also to protect neonates, but they may also have a role in the preservation of raw milk during storage and transport.
Lactoperoxidase is an enzyme found in milk. It has no inherent antimicrobial activity, but, in the presence of hydrogen peroxide (usually of microbial origin), it oxidises thiocyanate to produce inhibitors such as hypothiocyanite. This is referred to as the lactoperoxidase system, and it has bactericidal activity against many Gram-negative spoilage organisms, and some bacteriostatic action against many pathogens. For this reason it has been investigated as a possible means of extending the life of stored milk (5)
Lactoferrin is also found in milk and is a glycoprotein that binds iron so that it is not available to bacteria. The chelation of iron in the milk inhibits the growth of many bacteria. In addition to producing an iron- deficient environment, lactoferrin is thought to cause the release of anionic polysaccharide from the outer membrane of Gram-negative bacteria, thereby destabilising the membrane.
Lysozyme acts on components of the bacterial cell wall, causing cell lysis. Gram-positive organisms are much more susceptible to lysozyme than Gram-negatives, although bacterial spores are generally resistant.
Immunoglobulins of maternal origin are often present in milk, and colostrum is a particularly rich source. These proteins may inactivate pathogens in milk, but their significance in preservation is uncertain.
1.3 Processing and its Effects on the Microflora
1.3.1 Raw milk transport and storage
In developed countries, raw milk on the farm is usually cooled quickly and stored in refrigerated bulk tanks at <7 °C prior to collection. Collection by insulated tanker is often on alternate days, or sometimes less frequently, and therefore some of the milk in the tank could be 48 hours old at the time of collection. Temperature control is thus critical to minimise microbial growth, and tanker drivers are usually permitted to refuse milk stored at too high a temperature, or which has an abnormal appearance or odour. Bacterial numbers in the milk may increase during transport, either as a result of contamination from inadequately cleaned tankers or from the growth of psychrotrophic organisms, particularly Pseudomonas spp.. Milk temperature and duration of the transport stage are therefore important factors.
On arrival at the processing site, the milk is transferred to bulk storage tanks, or silos, prior to processing. The milk may be stored in the silos for 2 - 3 days, and further growth of psychrotrophic bacteria is likely during this period. The degree of growth is dependent on the initial microbial load, and the storage time and temperature. Pseudomonads are the predominant organisms present in stored raw milk, with Pseudomonas fluorescens, Pseudomonas fragi, and Pseudomonas lundensis being commonly isolated (6), but Enterobacteriaceae, Flavobacterium, Alcaligenes, and Gram-positive species can also be found. The growth of psychrotrophic bacteria may also be accompanied by the production of heat-stable, extracellular proteolytic and lipolytic enzymes. These enzymes are often capable of surviving pasteurisation and, in some cases, ultra high temperature (UHT) processing, and they may subsequently cause spoilage in the processed milk.
A number of techniques have been used to limit the growth of psychrotrophs during raw milk storage.
1.3.1.1 Thermisation
The most commonly used technique is to apply a mild heat treatment (thermisation), by heating to around 57 - 68 °C for 15 - 20 seconds and then cooling rapidly to <6 °C. This reduces the psychrotrophic population significantly and can extend the storage life of the raw milk by several days. However, thermisation cannot eliminate vegetative pathogens, and is therefore not a reliable control for the hazard. For example, L. monocytogenes can survive the process and could then grow during chilled storage (7).
1.3.1.2 Deep cooling
As the storage temperature is a key factor for the rate of growth of psychro trophic spoilage organisms, storing milk at as Iow a temperature as possible can also extend the storage life significantly. Reducing the storage temperature from 6 °C to 2 °C has been shown to give a 2-day gain in storage life for milk of good microbiological quality (8).
1.3.1.3 Carbon dioxide addition
There has been some interest in extending the storage life of raw milk by the addition of carbon dioxide at a concentration of 20-30 mM. Three mechanisms are thought to be involved in carbon dioxide inhibition of microorganisms: the first is by the displacement of oxygen; the second is a lowering of the pH of the milk due to the dissolution of carbon dioxide and formation of carbonic acid, particularly for Gram-negative psychrotrophic aerobes; and the third is a direct effect on the metabolisms such as inhibiting the production of enzymes by these organisms. It has also been suggested that the technique could be used to extend the shelf life of pasteurised milk, but concerns have been raised that the use of carbon dioxide addition could allow growth and toxin production by psychrotrophic Clostridium botulinum. However, recent work indicates that the risk of botulism is not increased by the use of this treatment (9).
Following storage, the milk then undergoes further processing.
1.3.2 Separation
If necessary, the milk is separated into skimmed milk, cream and sediment fractions, using centrifugal separators. The sediment may contain a comparatively high number of microorganisms and must be carefully discarded. The agitation involved may also break up clumps of bacteria, potentially producing an apparent increase in the number of colony-forming units. This process also allows the milk to be standardised to a specified fat content by adding back the correct quantity of cream.
1.3.3 Homogenisation
The fat globules in milk can coalesce and form a cream layer. Homogenisation reduces the size of the milk fat globules (to an average diameter of <1 (µm) by using a pump to force milk through a valve under pressure. The fat globules are then small enough to remain in suspension. This process has little microbiological effect, although clumps of bacterial cells may be broken up. Homogenisers used for pasteurised milk may be linked to the pasteuriser, and run at raised temperature in order to minimise possible microbial contamination. UHT processed milks are homogenised in sterile conditions after heat treatment and before aseptic filling. Effective cleaning and sterilising of the homogeniser are then critical to product safety.
1.3.4 Pasteurisation
Some form of heat process is commonly applied to milk to ensure microbiological safety, and to extend shelf life. In the UK, the most commonly used process is pasteurisation. Time-temperature requirements for pasteurisation vary between countries, and are often specified in legislation. In the UK, both low-temperature, long time (LTLT, 63 - 65 °C for 30 minutes), and high-temperature, short time (HTST, 71.7 - 72 °C for at least 15 seconds) minimum processes are permitted. However, in practice, the HTST process is now generally used. Recent concern about the possible survival of MAP in pasteurised milk (discussed further in section 1.7.2.8: MAP) has seen many dairies increase the length of the HTST process to 25 seconds. Higher processes (such as ultra-pasteurisation at 138 °C for at least 2 seconds) (3) may also be applied to products with high fat and solids content. Plate heat exchangers are the most common method for milk pasteurisation, but it is essential that they are designed, constructed and operated in such a way as to minimise the possibility of recontamination of the pasteurised milk by raw milk. Most commercial pasteurisers are fitted with sensors that continuously monitor the pasteurisation temperature, and are linked to automatic divert valves. If the pasteurisation temperature falls below a specified value, the valve opens and diverts the under-processed milk away from the post pasteurisation section of the plant and the filling line, into a divert tank. The correct operation of these monitoring systems is critical and should be regularly checked. It is also essential that there are no cross-connections between the raw and pasteurised sides of the process, and this should include separate clean-in-place (CIP) systems. It is also usual to maintain a higher pressure in the pasteurised milk to minimise the risk of cross contamination in the heat exchanger. Recontamination of this kind may have serious public health consequences (discussed further in section 1.7.1: Pathogen growth and survival in raw milk).
Accepted pasteurisation processes are designed to reduce the numbers of vegetative microbial pathogens to levels that are considered acceptable, although bacterial spores are not destroyed. Most of the potential psychrotrophic spoilage bacteria are also eliminated. However, certain heat-resistant mesophilic organisms, referred to as thermoduric, are able to survive pasteurisation. Thermoduric species commonly isolated from pasteurised milk include Micrococcus spp., Enterococcus faecium and Enterococcus faecalis, Bacillus subtilis, Bacillus cereus, and certain lactobacilli. Psychrotrophic strains of these organisms may be able to grow slowly in the pasteurised milk at 5 °C, and, if present initially in high numbers, could eventually cause spoilage. Effective cleaning of the cooling sections of pasteurisers is important to ensure that these organisms do not build up on surfaces.
1. 3.5 UHT or sterilisation processes
Milk may also be subjected to more severe heat processes sufficient to achieve "commercial sterility". This may be done by batch heating in closed containers, or continuously with aseptic filling into sterile containers. Both conventional retort sterilisation and UHT processes must achieve a minimum Fo of 3 minutes to ensure product safety. These processes destroy all vegetative cells in the milk, and the majority of spores, although certain very heat-resistant spores may survive. This results in a long shelf life without the need for refrigeration, but also causes organoleptic changes in the milk, such as browning.
Conventional sterilisation processes involve heating the milk in thick-walled glass bottles, closed with a crimped metal cap, at about 120 °C for approximately 30 minutes. However, modern large-scale production methods often use an initial UHT treatment prior to filling the container, followed by retorting for a reduced time (10 - 12 minutes), and then a rapid cooling process. This is said to give a product with improved organoleptic properties.
UHT processes may be direct or indirect. Direct systems inject high-pressure steam directly into the milk to obtain the desired temperature, and then employ flash cooling under vacuum to remove the resulting excess water. Indirect systems utilise heat exchangers and holding tubes. Direct systems are said to give better organoleptic properties, as the heating and cooling processes are very rapid, but they are more complex and expensive to install. UHT processed milk involves preserving milk by holding at a temperature of 140 - 150 °C for 1 - 2 seconds (minimum treatment is 130 °C for 1 sec) (3, 10). Heat treatment is usually followed by aseptic filling into sterile cartons or other containers. The maintenance of sterility in filling is vital to prevent recontamination of the treated milk. As with pasteurised milk, it is also vital to ensure that raw milk cannot recontaminate the UHT-treated milk.
Certain very heat-resistant spores of mesophilic bacilli, classified as Bacillus sporothermodurans (11) are able to survive UHT processes and may subsequently grow in the final product. However, this organism has been shown not to be pathogenic (12) and does not seem to cause any detectable changes to the product. Thermoduric Bacillus stearothermophilus are able to survive UHT processes and cause flat-sour spoilage (3).
