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Dairy Microbiology

Basic Microbiology
Microorganisms
Microorganisms are living organisms that are individually too small to see with the naked eye. The unit of measurement used for microorganisms is the micrometer (µ m); 1 µ m = 0.001 millimeter; 1 nanometer (nm) = 0.001 µ m. Microorganisms are found everywhere (ubiquitous) and are essential to many of our planets life processes. With regards to the food industry, they can cause spoilage, prevent spoilage through fermentation, or can be the cause of human illness.

There are several classes of microorganisms, of which bacteria and fungi (yeasts and moulds) will be discussed in some detail. Another type of microorganism, the bacterial viruses or bacteriophage, will be examined in a later section.

Bacteria
Bacteria are relatively simple single-celled organisms. One method of classification is by shape or morphology:
Cocci:
- spherical shape
- 0.4 - 1.5 µ m
Examples: staphylococci - form grape-like clusters; streptococci - form bead-like chains

Rods:
- 0.25 - 1.0 µ m width by 0.5 - 6.0 µ m long
Examples: bacilli - straight rod; spirilla - spiral rod
There exists a bacterial system of taxonomy, or classification system, that is internationally recognized with family, genera and species divisions based on genetics.
Some bacteria have the ability to form resting cells known as endospores. The spore forms in times of environmental stress, such as lack of nutrients and moisture needed for growth, and thus is a survival strategy. Spores have no metabolism and can withstand adverse conditions such as heat, disinfectants, and ultraviolet light. When the environment becomes favourable, the spore germinates and giving rise to a single vegetative bacterial cell. Some examples of spore-formers important to the food industry are members of Bacillus and Clostridium generas.

Bacteria reproduce asexually by fission or simple division of the cell and its contents. The doubling time, or generation time, can be as short as 20-20 min. Since each cell grows and divides at the same rate as the parent cell, this could under favourable conditions translate to an increase from one to 10 million cells in 11 hours! However, bacterial growth in reality is limited by lack of nutrients, accumulation of toxins and metabolic wastes, unfavourable temperatures and dessication. The maximum number of bacteria is approximately 1 X 10e9 CFU/g or ml.
Note: Bacterial populations are expressed as colony forming units (CFU) per gram or millilitre.

Bacterial growth generally proceeds through a series of phases:

Lag phase: time for microorganisms to become accustomed to their new environment. There is little or no growth during this phase.
Log phase: bacteria logarithmic, or exponential, growth begins; the rate of multiplication is the most rapid and constant.
Stationary phase: the rate of multiplication slows down due to lack of nutrients and build-up of toxins. At the same time, bacteria are constantly dying so the numbers actually remain constant.
Death phase: cell numbers decrease as growth stops and existing cells die off.
The shape of the curve varies with temperature, nutrient supply, and other growth factors.

Yeasts
Yeasts are members of a higher group of microorganisms called fungi . They are single-cell organisms of spherical, elliptical or cylindrical shape. Their size varies greatly but are generally larger than bacterial cells. Yeasts may be divided into two groups according to their method of reproduction:
budding: called Fungi Imperfecti or false yeasts
budding and spore formation: called Ascomycetes or true yeasts
Unlike bacterial spores, yeast form spores as a method of reproduction.

Moulds
Moulds are filamentous, multi-celled fungi with an average size larger than both bacteria and yeasts (10 X 40 µ m). Each filament is referred to as a hypha. The mass of hyphae that can quickly spread over a food substrate is called the mycelium. Moulds may reproduce either asexually or sexually, sometimes both within the same species.
Asexual Reproduction:
fragmentation - hyphae separate into individual cells called arthropsores
spore production - formed in the tip of a fruiting hyphae, called conidia, or in swollen structures called sporangium
Sexual Reproduction: sexual spores are produced by nuclear fission in times of unfavourable conditions to ensure survival.

Microbial Growth
There are a number of factors that affect the survival and growth of microorganisms in food. The parameters that are inherent to the food, or intrinsic factors, include the following:
nutrient content
moisture content
pH
available oxygen
biological structures
antimicrobial constituents
Nutrient Requirements: While the nutrient requirements are quite organism specific, the microorganisms of importance in foods require the following:
water
energy source
carbon/nitrogen source
vitamins
minerals
Milk and dairy products are generally very rich in nutrients which provides an ideal growth environment for many microorganisms.
Moisture Content: All microorganisms require water but the amount necessary for growth varies between species. The amount of water that is available in food is expressed in terms of water activity (aw), where the aw of pure water is 1.0. Each microorganism has a maximum, optimum, and minimum aw for growth and survival. Generally bacteria dominate in foods with high aw (minimum approximately 0.90 aw) while yeasts and moulds, which require less moisture, dominate in low aw foods ( minimum 0.70 aw). The water activity of fluid milk is approximately 0.98 aw.

pH: Most microorganisms have approximately a neutral pH optimum (pH 6-7.5). Yeasts are able to grow in a more acid environment compared to bacteria. Moulds can grow over a wide pH range but prefer only slightly acid conditions. Milk has a pH of 6.6 which is ideal for the growth of many microoorganisms.

Available Oxygen: Microorganisms can be classified according to their oxygen requirements necessary for growth and survival:

Obligate Aerobes: oxygen required
Facultative: grow in the presence or absence of oxygen
Microaerophilic: grow best at very low levels of oxygen
Aerotolerant Anaerobes: oxygen not required for growth but not harmful if present
Obligate Anaerobes: grow only in complete absence of oxygen; if present it can be lethal
Biological Structures: Physical barriers such as skin, rinds, feathers, etc. have provided protection to plants and animals against the invasion of microorganisms. Milk, however, is a fluid product with no barriers to the spreading of microorganisms throughout the product.
Antimicrobial Constituents: As part of the natural protection against microorganisms, many foods have antimicrobial factors. Milk has several nonimmunological proteins which inhibit the growth and metabolism of many microorganisms including the following most common:

Temperature: As a group, microorganisms are capable of growth over an extremely wide temperature range. However, in any particular environment, the types and numbers of microorganisms will depend greatly on the temperature. According to temperature, microorganisms can be placed into one of three broad groups:

Psychrotrophs: optimum growth temperatures 20 to 30° capable of growth at temperatures less than 7° C. Psychrotrophic organisms are specifically important in the spoilage of refrigerated dairy products.
Mesophiles: optimum growth temperatures 30 to 40° C; do not grow at refrigeration temperatures
Thermophiles: optimum growth between 55 and 65° C
It is important to note that for each group, the growth rate increases as the temperature increases only up to an optimum, afterwhich it rapidly declines.

Detection and Enumeration of Microorganisms
There are several methods for detection and enumeration of microorganisms in food. The method that is used depends on the purpose of the testing.
Direct Enumeration:

Using direct microscopic counts (DMC), Coulter counter etc. allows a rapid estimation of all viable and nonviable cells. Identification through staining and observation of morphology also possible with DMC.

Viable Enumeration:

The use of standard plate counts, most probable number (MPN), membrane filtration, plate loop methos, spiral plating etc., allows the estimation of only viable cells. As with direct enumeration, these methods can be used in the food industry to enumerate fermentation, spoilage, pathogenic, and indicator organisms.

Metabolic Activity Measurement:

An estimation of metabolic activity of the total cell population is possible using dye reduction tests such as resazurin or methylene blue dye reduction, acid production, electrical impedence etc. The level of bacterial activity can be used to assess the keeping quality and freshness of milk. Toxin levels can also be measured, indicating the presence of toxin producing pathogens.

Cellular Constituents Measurement:

Using the luciferase test to measure ATP is one example of the rapid and sensitive tests available that will indicate the presence of even one pathogenic bacterial cell.
Isolation of microorganisms is an important preliminary step in the identification of most food spoilage and pathogenic organisms. This can be done using a simple streak plate method.

Microorganisms in Milk
Milk is sterile at secretion in the udder but is contaminated by bacteria even before it leaves the udder. Except in the case of mastisis, the bacteria at this point are harmless and few in number. Further infection of the milk by microorganisms can take place during milking, handling, storage, and other pre-processing activities.
Lactic acid bacteria: this group of bacteria are able to ferment lactose to lactic acid. They are normally present in the milk and are also used as starter cultures in the production of cultured dairy products such as yogurt. Note: many lactic acid bacteria have recently been reclassified; the older names will appear in brackets as you will still find the older names used for convenience sake in a lot of literature. Some examples in milk are:

lactococci
L. delbrueckii subsp. lactis (Streptococcus lactis )
Lactococcus lactis subsp. cremoris (Streptococcus cremoris )
lactobacilli
Lactobacillus casei
L.delbrueckii subsp. lactis (L. lactis )
L. delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus )
Leuconostoc
Coliforms: coliforms are facultative anaerobes with an optimum growth at 37° C. Coliforms are indicator organisms; they are closely associated with the presence of pathogens but not necessarily pathogenic themselves. They also can cause rapid spoilage of milk because they are able to ferment lactose with the production of acid and gas, and are able to degrade milk proteins. They are killed by HTST treatment, therefore, their presence after treatment is indicative of contamination.Escherichia coli is an example belonging to this group.

Significance of microorganisms in milk:

Information on the microbial content of milk can be used to judge its sanitary quality and the conditions of production
If permitted to multiply, bacteria in milk can cause spoilage of the product
Milk is potentially susceptible to contamination with pathogenic microorganisms. Precautions must be taken to minimize this possibility and to destroy pathogens that may gain entrance
Certain microorganisms produce chemical changes that are desirable in the production of dairy products such as cheese, yogurt.
Spoilage Microorganisms in Milk
The microbial quality of raw milk is crucial for the production of quality dairy foods. Spoilage is a term used to describe the deterioration of a foods' texture, colour, odour or flavour to the point where it is unappetizing or unsuitable for human consumption. Microbial spoilage of food often involves the degradation of protein, carbohydrates, and fats by the microorganisms or their enzymes.
In milk, the microorganisms that are principally involved in spoilage are psychrotrophic organisms. Most psychrotrophs are destroyed by pasteurization temperatures, however, some like Pseudomonas fluorescens, Pseudomonas fragi can produce proteolytic and lipolytic extracellular enzymes which are heat stable and capable of causing spoilage.

Some species and strains of Bacillus, Clostridium, Cornebacterium, Arthrobacter, Lactobacillus, Microbacterium, Micrococcus , and Streptococcus can survive pasteurization and grow at refrigeration temperatures which can cause spoilage problems.

Pathogenic Microorganisms in Milk
Hygienic milk production practices, proper handling and storage of milk, and mandatory pasteurization has decreased the threat of milkborne diseases such as tuberculosis, brucellosis, and typhoid fever. There have been a number of foodborne illnesses resulting from the ingestion of raw milk, or dairy products made with milk that was not properly pasteurized or was poorly handled causing post-processing contamination. The following bacterial pathogens are still of concern today in raw milk and other dairy products:
Bacillus cereus
Listeria monocytogenes
Yersinia enterocolitica
Salmonella spp.
Escherichia coli O157:H7
Campylobacter jejuni
It should also be noted that moulds, mainly of species of Aspergillus , Fusarium , and Penicillium can grow in milk and dairy products. If the conditions permit, these moulds may produce mycotoxins which can be a health hazard.

HACCP
Raw and end-products may be tested for the presence, level, or absence of microorganisms. Traditionally these practices were used to reduce manufacturing defects in dairy products and ensure compliance with specifications and regulations, however, they have many drawbacks:
destructive and time consuming
slow response
small sample size
delays in the release of the food
In the 1960's, the Pillsbury Company, the U.S. Army, and NASA introduced a system for assuring pathogen-free foods for the space program. This system, called Hazard Analysis and Critical Control Points (HACCP), is a focus on critical food safety areas as part of total quality programs. It involves a critical examination of the entire food manufacturing process to determine every step where there is a possibility of physical, chemical, or microbiological contamination of the food which would render it unsafe or unacceptable for human consumption. These identified points are the critical control points (CCP). There are seven prinicples to HACCP:
analyze hazards
determine CCPs
establish critical limits
establish monitoring procedures
establish deviation procedures
establish verification procedures
establish record keeping procedures
Before these principles can be put into place, a prerequisite program and preliminary setup is necessary.
Prerequisite Program:
premise control
receiving and storage control
equipment performance and maintenance control
personnel training
sanitation
recall procedure
Preliminary Setup:
assemble team
describe the product
identify intended use
construct flow diagram and plant schematic
verify the diagram on-site
Food Safety Enhancement Program-FSEP is The Canadian Food Inspection Agency's HACCP initiative. There is extensive information at their Web site regarding FSEP, including implementation manuals, HACCP curriculum guidelines, and generic models.

Starter Cultures
Starter cultures are those microorganisms that are used in the production of cultured dairy products such as yogurt and cheese. The natural microflora of the milk is either inefficient, uncontrollable, and unpredictable, or is destroyed altogether by the heat treatments given to the milk. A starter culture can provide particular characteristics in a more controlled and predictable fermentation. The primary function of lactic starters is the production of lactic acid from lactose. Other functions of starter cultures may include the following:
flavour, aroma, and alcohol production
proteolytic and lipolytic activities
inhibition of undesirable organisms

There are two groups of lactic starter cultures:
simple or defined: single strain, or more than one in which the number is known
mixed or compound: more than one strain each providing its own specific characteristics
Starter cultures may be categorized as mesophilic or thermophilic:
Mesophilic

Lactococcus lactis subsp. cremoris
L. delbrueckii subsp. lactis
L. lactis subsp. lactis biovar diacetylactis
Leuconostoc mesenteroides subsp. cremoris
Thermophilic

Streptococcus salivarius subsp. thermophilus (S.thermophilus)
Lactobacillus delbrueckii subsp. bulgaricus
L. delbrueckii subsp. lactis
L. casei
L. helveticus
L. plantarum
Mixtures of mesophilic and thermophilic microorganisms can also be used as in the production of some cheeses.

Bacteriophage
Bacteriophages are viruses that require bacteria host cells for growth and reproduction. Initially, the bacteriophage attaches itself to the bacteria cell wall and injects nuclear substance into the cell. Inside the cell, the nuclear substance produces shells, or phage coats, for the new bacteriophage which are quickly filled with nucleic acid. The bacterial cell ruptures and dies as the new bacteriophage are released.
Bacteriophages are ubiquitous but generally enter the milk processing plant with the farm milk. They can be inactivated heat treatments of 30 min at 63 to 88° C, or by the use of chemical disinfectants.
Bacteriophages are of most concern in cheese making. They attack and destroy most of the lactic acid bacteria which prevents normal ripening known as slow or dead vat.
Starter Culture Preparation
Commercial manufacturers provide starter cultures in lyophilized (freeze-dryed), frozen or spray-dried forms. The dairy product manufacturers need to inoculate the culture into milk or other suitable substrate. There are a number of steps necessary for the propagation of starter culture ready for production:
Commercial culture
Mother culture - first inoculation; all cultures will originate from this preparation
Intermediate culture - in preparation of larger volumes of prepared starter
Bulk starter culture - this stage is used in dairy product production

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