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Microbiology


Bacteria

Bacterial growth

Moulds
Yeasts
Viruses
Milk Microbiology

Milk pasteurisation
Milk sterilisation

Microbiology of Butter

Sources of contamination
Control of micro-organisms in butter


Micro-organism is the term applied to all microscopically small living organisms. We tend to associate micro-organisms with disease. Micro-organisms which cause disease are called pathogens. However, few micro-organisms are pathogens and micro-organisms play a crucial part in the life of our planet. For example, they provide food for fish, they occur in soil where they provide nutrients for plants and they play an important role in ruminant digestion.

In dairying some micro-organisms are harmful—e.g. spoilage organisms, pathogens—while others are beneficial—cheese and yoghurt starters, yeasts and moulds used in controlled fermentations in milk processing.

The micro-organisms principally encountered in the dairy industry are bacteria, yeasts, moulds, and viruses.

Bacteria

Bacteria are single-celled organisms. They are present in air, water and on most solid materials. Bacterial cells are very small and can only be seen with the aid of a microscope.

When observed under a microscope the cells can be seen to differ in shape and in conformation of groups of cells. Cells are either spherical or rod-shaped (Figure 7). Spherical bacteria are called cocci; those that are rod shaped are called bacilli. This is the first basis for differentiating between bacterial cells.

Figure 7. Rod-shaped (bacilli) and spherical (cocci) bacteria.

Bacteria are also classified according to cell cluster formation:

Diplococci        – two cocci cells paired
Staphylococci  – a number of cells clustered together
Streptococci    – a number of cells arranged in a chain

Some bacteria are capable of locomotion by means of flagellae—long, hair-like appendages growing out of the cell. Some rod-shaped bacteria contain spores. These are formed when the cells are faced with adverse conditions, such as high temperature: once suitable conditions are reestablished the spores germinate to form new cells.

Close examination of the simple cell reveals that it is composed of the following components (Figure 8):

Figure 8. Schematic illustration of bacterial structure.

The cell membrane is semipermeable and allows the cell to feed by osmosis, i.e. the exchange of water between the cytoplasm of a living cell and the surrounding watery material. Only small molecules can pass in and out of the cell, e.g. with a sugar solution on one side of a semipermeable membrane and water on the other, water will diffuse in, diluting the sugar solution. The sugar molecules cannot pass out so a hydrostatic pressure, known as osmotic pressure, develops.

Bacteria can feed by selective intake of nutrients dissolved in water. They can also take in nutrients against the normal osmotic flow—active transport.

Bacterial growth

Bacterial growth refers to an increase in cell numbers rather than an increase in cell size. The process by which bacterial cells divide to reproduce themselves is known as binary transverse fission. The time taken from cell formation to cell division is called the generation time. The generation time can therefore be defined as the time taken for the cell count to double.

The curve shown in Figure 9 shows the phases of bacterial growth following inoculation of bacteria into a new growth medium. The following phases can be identified:

  1. Lag phase: There is usually some delay in growth following inoculation of bacteria into a new medium, during which time the bacteria adapt to the medium and synthesise the enzymes needed to break down the substances in the growth medium.
  2. Log phase: Once the bacteria have adapted to the new medium they start to reproduce quickly and their numbers multiply evenly for each increment of time. A plot of the log number of cells against time gives a linear relationship: this is therefore called the log phase. The cells are at their greatest activity in this phase. Transferring cultures to a fresh medium at regular intervals can maintain the cells in an active state. An active culture can rapidly dominate any new environment.
  3. Stationary phase: As the bacteria dominate the growth medium, they deplete the available nutrients or toxic waste products accumulate, slowing the rate of reproduction. At the same time, cells are dying off: A state of equilibrium is reached between the death of old cells and formation of new cells, resulting in no net change in cell numbers. This phase is called the stationary phase.
  4. Death phase: In the next phase the formation of new cells ceases and the existing cells gradually die off: This is called the death phase.
  5. The log phase can be prolonged by removing toxic waste, by adding more nutrients or both.
The log phase can be prolonged by removing toxic waste, by adding more nutrients or both.

The log phase can be prolonged by removing toxic waste, by adding more nutrients or both.

Figure 9. The four phases of bacterial growth.

Factors affecting bacterial growth

Bacterial growth is affected by (1) temperature, (2) nutrient availability, (3) water supply, (4) oxygen supply, and (5) acidity of the medium.

Temperature: Theoretically, bacteria can grow at all temperatures between the freezing point of water and the temperature at which protein or protoplasm coagulates. Somewhere between these maximum and minimum points lies the optimum temperature at which the bacteria grow best.

Temperatures below the minimum stop bacterial growth but do not kill the organism. However, if the temperature is raised above the maximum, bacteria are soon killed. Most cells die after exposure to heat treatments in the order of 70°C for 15 seconds, although spore-forming organisms require more severe heat treatment, e.g. live steam at 120°C for 30 minutes.

Bacteria can be classified according to temperature preference: Psycrophilic bacteria grow at temperatures below 16°C, mesophilic bacteria grow best at temperatures between 16 and 40°C, and thermophilic bacteria grow best at temperatures above 40°C.

Nutrients: Bacteria need nutrients for their growth and some need more nutrients than others. Lactobacilli live in milk and have lost their ability to synthesise many compounds, while Pseudomonas can synthesise nutrients from very basic ingredients.

Bacteria normally feed on organic matter; as well as material for cell formation organic matter also contains the necessary energy. Such matter must be soluble in water and of low molecular weight to be able to pass through the cell membrane. Bacteria therefore need water to transport nutrients into the cell.

If the nutrient material is not sufficiently broken down, the micro-organism can produce exo-enzymes which split the nutrients into smaller, simpler components so they can enter the cell. Inside the cell the nutrients are broken down further by other enzymes, releasing energy which is used by the cell.

Water: Bacteria cannot grow without water. Many bacteria are quickly killed by dry conditions whereas others can tolerate dry conditions for months; bacterial spores can survive dry conditions for years. Water activity (AW) is used as an indicator of the availability of water for bacterial growth. Distilled water has an AW of 1. Addition of solute, e.g. salt, reduces the availability of water to the cell and the AW drops; at AW less than 0.8 cell growth is reduced. Cells that can grow at low AW are called osmophiles.

Oxygen: Animals require oxygen to survive but bacteria differ in their requirements for, and in their ability to utilise, oxygen.

Bacteria that need oxygen for growth are called aerobic. Oxygen is toxic to some bacteria and these are called anaerobic. Anaerobic organisms are responsible for both beneficial reactions, such as methane production in biogas plants, and spoilage in canned foods and cheeses.

Some bacteria can live either with or without oxygen and are known as faculative anaerobic bacteria.

Acidity: The acidity of a nutrient substrate is most simply expressed as its pH value. Sensitivity to pH varies from one species of bacteria to another. The terms pH optimum and pH maximum are used. Most bacteria prefer a growth environment with a pH of about 7, i.e. neutrality.

Bacteria that can tolerate low pH are called aciduric. Lactic acid bacteria in milk produce acid and continue to do so until the pH of the milk falls to below 4.6, at which point they gradually die off. In canning citrus fruits, mild heat treatments are sufficient because the low pH of the fruit inhibits the growth of most bacteria.

Bacteria in milk

Milk fresh from a healthy cow contains few bacteria, but contamination during handling can rapidly increase bacterial numbers. Milk is an ideal food and many bacteria grow readily in it.

Some bacteria are useful in milk processing, causing milk to sour naturally, leading to products such as irgo. However, milk can also carry pathogenic bacteria, such as Salmonella, Tuberculosis bovis and Brucella, and can thus transmit disease. Other bacteria can cause spoilage of the milk, and spoilage and poor yields of products.

Moulds

Moulds are a heterogeneous group of multicelled organisms which reproduce asexually either by spore formation or by fragmentation. They can grow on a wide variety of substrates and are generally regarded as spoilage organisms. However, moulds are used in the production of antibiotics and in certain cheese varieties. Moulds are aerobic organisms and their growth on foods can be retarded by excluding air through careful packaging. They can be killed by relatively mild heat treatments, but mould spores are more resistant to heat. The structure of moulds is shown in Figure 10.

Figure 10. Structure of moulds.

Yeasts

Yeasts are unicellular organisms which reproduce asexually by budding. They are used industrially to ferment carbohydrates to such products as alcohol and citric acid. Yeasts are not usually used in milk processing and are normally regarded as spoilage organisms in dairy products. The structure of yeasts is shown in Figure 11.

Figure 11. Structure of a yeast cell.

Viruses

Viruses are extremely small organisms comprising a spherical head containing the genetic material and a cylindrical tail. They cannot reproduce themselves, and must invade other cells in order to reproduce. Viruses that attack bacterial cells are known as bacteriophages: bacteriophages that attack acid-producing bacteria inhibit acid production in milk.

Milk Microbiology

In addition to being a nutritious food for humans, milk provides a favourable environment for the growth of microorganisms. Yeasts, moulds and a broad spectrum of bacteria can grow in milk, particularly at temperatures above 16°C.

Microbes can enter milk via the cow, air, feedstuffs, milk handling equipment and the milker. Once microorganisms get into the milk their numbers increase rapidly. It is more effective to exclude micro-organisms than to try to control microbial growth once they have entered the milk. Milking equipment should be washed thoroughly before and after use—rinsing is not enough.

Bacterial types commonly associated with milk are given in Table 4.

Table 4. Bacterial types commonly associated with milk.

Pseudomonas

Spoilage

Brucella

Pathogenic

Enterobacteriaceae

Pathogenic and spoilage

Staphylococci

Staphylococcus aureus

Pathogenic

Streptococcus

S. agalactiae

Pathogenic

S. thermophilus

Acid fermentation

S. lactis

Acid fermentation

S. lactis-diacetyllatic

Flavour production

S. cremoris

Acid fermentation

Leuconostoc lactis

Acid fermentation

Bacillus cereus

Spoilage

Lactobacillus

L. lactis

Acid production

L. bulgaricus

Acid production

L. acidophilus

Acid production

Propionibacterium

Acid production

Mycobacterium tuberculosis

Pathogenic

Microbial growth can be controlled by cooling the milk. Most micro-organisms reproduce slowly in colder environments. Cooling milk also slows chemical deterioration.

The temperature of freshly drawn milk is about 38°C. Bacteria multiply very rapidly in warm milk and milk sours rapidly if held at these temperatures. If the milk is not cooled and is stored in the shade at an average air temperature of 16°C, the temperature of the milk will only have fallen to 28°C after 3 hours. Cooling the milk with running water will reduce the temperature to 16°C after 1 hour. At this temperature bacterial growth will be reduced and enzyme activity retarded. Thus, milk will keep longer if cooled.

Natural souring of milk may be advantageous: for example, in smallholder butter-making, the acid developed assists in the extraction of fat during churning. The low pH retards growth of lipolytic and proteolytic bacteria and therefore protects the fat and protein in the milk. The acidity of the milk also inhibits the growth of pathogens. It does not, however, retard the growth of moulds.

Naturally soured milk is used to make many products, e.g. irgo, yoghurt, sour cream, ripened buttermilk and cheese. These products provide ways of preserving milk and are also pleasant to consume. They are produced by the action of fermentative bacteria on lactose and are more readily digested than fresh milk.

The initial microflora of raw milk reflects directly microbial contamination during production. The microflora in milk when it leaves the farm is determined by the temperature to which it has been cooled and the temperature at which it has been stored.

The initial bacterial count of milk may range from less than 1000 cells/ml to 106/ml. High counts (more than 105/ml) are evidence of poor production hygiene. Rapid tests are available for estimating the bacterial quality of milk.

Milk pasteurisation

Pasteurisation is the most common process used to destroy bacteria in milk. In pasteurisation, the milk is heated to a temperature sufficient to kill pathogenic bacteria, but well below its boiling point. This also kills many non-pathogenic organisms and thereby extends the storage stability of the milk.

Numerous time/temperature combinations are recommended but the most usual is 72°C for 15 seconds followed by rapid cooling to below 10°C. This is normally referred to as High Temperature Short Time (HTST) treatment. It is carried out as a continuous process using a plate heat-exchanger to heat the milk and a holding section to ensure that the milk is completely pasteurised. Milk is normally pasteurised prior to sale as liquid milk. Pasteurisation is used to reduce the microbial counts in milk for cheese-making, and cream is pasteurised prior to tempering for butter-making in some factories.

Batch pasteurisation is used where milk quantities are too small to justify the use of a plate heat-exchanger. In batch pasteurisation, fixed quantities of milk are heated to 63°C and held at this temperature for 30 minutes. The milk is then cooled to 5°C and packed.

The lower temperature used for batch pasteurisation means that a longer time is required to complete the process—30 minutes at 63°C, compared with 15 seconds a 72°C.

Effects of pasteurisation on milk

Pasteurisation reduces the cream layer, since some of the fat globule membrane constituents are denatured. This inhibits clustering of the fat globules and consequently reduces the extent of creaming. However, pasteurisation does not reduce the fat content of milk.

Pasteurisation has little effect on the nutritive value of milk. The major nutrients are not altered. There is some loss of vitamin C and B group vitamins, but this is insignificant.

The process kills many fermentative organisms as well as pathogens. Micro-organisms that survive pasteurisation are putrefactive. Although pasteurised milk has a storage stability of 2 to 3 days, subsequent deterioration is cause by putrefactive organisms. Thus, pasteurised milk will putrefy rather than develop acidity.

In rural milk processing, many processes depend on the development of acidity, and hence pasteurisation may not be appropriate.

Milk sterilisation

In pasteurisation, milk receives mild heat treatment to reduce the number of bacteria present. In sterilisation, milk is subjected to severe heat treatment that ensures almost complete destruction of the microbial population. The product is then said to be commercially sterile. Time/temperature treatments of above 100°C for 15 to 40 minutes are used. The product has a longer shelf life than pasteurised milk.

Another method of sterilisation is ultra-heat treatment, or UHT. In this system, milk is heated under pressure to about 140°C for 4 seconds. The product is virtually sterile. However, it retains more of the properties of fresh milk than conventionally sterilised milk.

Microbiology of Butter

Butter is made as a means of extracting and preserving milk fat. It can be made directly from milk or by separation of milk and subsequent churning of the cream.

Sources of contamination

In addition to bacteria present in the milk other sources of bacteria in butter are (1) equipment, (2) wash water, (3) air contamination, (4) packing materials, and (5) personnel.

Equipment

In smallholder butter-making, bacterial contamination can come from unclean surfaces, the butter maker and wash water. Packaging materials, cups and leaves are also sources of contaminants. Washing and smoking the churn reduces bacterial numbers. But traditional equipment is often porous and is therefore a reservoir for many organisms.

When butter is made on a larger processing scale, bacterial contamination can come from holding-tank surfaces, the churn and butter-handling equipment.

A wooden churn can be a source of serious bacterial, yeast and mould contamination since these organisms can penetrate the wood, where they can be destroyed only by extreme heat. If a wooden churn has loose bands, cream can enter the crevices between the staves, where it provides a growth medium for bacteria which contaminate subsequent batches of butter. However, if care is taken in cleaning a wooden churn this source of contamination can be controlled. Similar care is required with scotch hands and butter-working equipment.

Wash water

Wash water can be a source of contamination with both coliform bacteria and bacteria associated with defects in butter. Polluted water supplies can also be a source of pathogens.

Air

Contamination from the air can introduce spoilage organisms: mould spores, bacteria and yeasts can fall on the butter if it is left exposed to the air. Moulds grow rapidly on butter exposed to air.

Packaging

Care is required in the storage and preparation of packaging material. Careless handling of packaging material can be a source of mould contamination.

Personnel

A high standard of personal hygiene is required from people engaged in butter-making. For example, in New Zealand the 1938 dairy produce regulations stated "no person shall permit his bare hands to be brought in contact with any butter at any time immediately following manufacture or during the wrapping, packaging, storage and transport of such butter".

Personnel pass organisms to butter via the hands, mouth, nasal passage and clothing. Suitable arrangements for disinfecting hands should be provided, and clean working garments should not have contact with other clothes.

Control of micro-organisms in butter

Salting effectively controls bacterial growth in butter. The salt must be evenly dispersed and worked in well. Salt concentration of 2% adequately dispersed in butter of 16% moisture will result in a 12.5% salt solution throughout the water-in-oil emulsion.

Washing butter does little to reduce microbiological counts. It may be desirable not to wash butter, since washing reduces yield. The acid pH of serum in butter made from ripened cream or sour milk may control the growth of acid-sensitive organisms.

Microbiological analysis of butter usually includes some of the following tests: total bacterial count, yeasts and moulds, coliform estimation and estimation of lipolytic bacteria.

Yeast, mould and coliform estimations are useful for evaluating sanitary practices. The presence of defect producing types can be indicated by estimating the presence of lipolytic organisms.

All butter contains some micro-organisms. However, proper control at every stage of the process can minimise the harmful effects of these organisms.

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