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.

Scale showing the size of an animal cell, animal nucleus, yeast cell, virus, bacteria cell (rod) and bacteria cell (coccus)

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 desiccation. 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.

Hypothetical bacterial growth curveBacterial 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 (shown on the right) varies with temperature, nutrient supply, and other growth factors. This exponential death curve is also used in modeling the heating destruction of microorganisms.

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:

  1. budding: called Fungi Imperfecti or false yeasts
  2. 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

 Hypothetical bacterial growth curveThere 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:

  1. lactoperoxidase
  2. lactoferrin
  3. lysozyme
  4. xanthine

More information on these antimicrobials can be found in the dairy microbiology textbook by Marth and Steele. See also the discussion on lactoperoxidase in this series at https://www.uoguelph.ca/foodscience/book-page/effects-milk-handling-quality-and-hygiene

Where the intrinsic factors are related to the food properties, the extrinsic factors are related to the storage environment. These would include temperature, relative humidity, and gases that surround the food.

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 (see below), acid production, electrical impedance 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.

Dye Reduction Tests: Methylene Blue and Resazurin

From: Atherton, H. V. and Newlander, J. A. 1977. Chemistry and Testing of Dairy Products. 4th Edn. AVI, Westport, CT.

Methylene Blue Reduction Test

The methylene blue reduction test is based on the fact that the color imparted to milk by the addition of a dye such as methylene blue will disappear more or less quickly. The removal of the oxygen from milk and the formation of reducing substances during bacterial metabolism causes the color to disappear. The agencies responsible for the oxygen consumption are the bacteria. Though certain species of bacteria have considerably more influence than others, it is generally assumed that the greater the number of bacteria in milk, the quicker will the oxygen be consumed, and in turn the sooner will the color disappear. Thus, the time of reduction is taken as a measure of the number of organisms in milk although actually it is likely that it is more truly a measure of the total metabolic reactions proceeding at the cell surface of the bacteria.

The methylene blue reduction test has lost much of its popularity because of its low correlation with other bacterial procedures. This is true particularly in those samples which show extensive multiplication of the psychrotropic species.

Apparatus.–The necessary equipment consists of test tubes with rubber stoppers, a pipette or dipper graduated to deliver 10 ml of milk and a water bath for maintaining the samples at 35o to 37oC. The bath should contain a volume of water sufficient to heat the samples to 35o C within 10 minutes after the tubes enter the water and should have some means of protecting the samples from light during the incubation period. If a hot-air chamber is used, the samples should be heated to 35o C in a water bath since warm air would heat the milk too slowly.

The dry tablets contain methylene blue thiocyanate and may be obtained from any of the usual laboratory supply houses. They should be certified by the Commission on Standardization of Biological Stains. The solution is prepared by autoclaving or momentarily boiling 200 ml of distilled water in a light resistant (amber) stoppered flask and then adding one methylene blue tablet to the flask of hot water. The tablet should be completely dissolved before the solution is cooled. The solution may be stored in the stoppered, amber flask or an amber bottle in the dark. Fresh solution should be prepared weekly.

Procedure in Testing.–The following procedures are recommended.

  1. Sterilize all glassware and rubber stoppers either in an autoclave or in boiling water. Be sure all glassware is chemically clean.
  2. Measure 1 ml of the methylene blue thiocyanate solution into a test tube.
  3. Add 10 ml of milk and stopper.
  4. Tubes may be placed in the water bath immediately or may be stored in the refrigerator at 0o to 4o C for a more convenient time of incubation. When ready to perform the test, the temperature of the samples should be brought to 35o C within 10 minutes.
  5. When temperature reaches 36o C, slowly invert tubes a few times to assure uniform creaming. Do not shake tubes. Record this time as the beginning of the incubation period. Cover to keep out light.
  6. Check samples for decolorization after 30 minutes of incubation. Make subsequent readings at hourly intervals thereafter.
  7. After each reading, remove decolorized tubes and then slowly make one complete inversion of remaining tubes.
  8. Record reduction time in whole hours between last inversion and decolorization. For example, if the sample were still blue after L 5 hours but was decolorized (white) at the 2.5 hour reading, the methylene blue reduction time would be recorded as 2 hours. Decolorization is considered complete when four-fifths of the color has disappeared.

Classification.–The suggested classification is listed.

Class 1. Excellent, not decolorized in 8 hours.

Class 2. Good, decolorized in less than 8 hours but not less than 6 hours.

Class 3. Fair, decolorized in less than 6 hours but not less than 2 hours.

Class 4. Poor, decolorized in less than 2 hours.

Factors Affecting the Test.–Many factors affect the methylene blue reduction test and therefore the steps of operation should be uniform.

Since the oxygen content must be used up before the color disappears, any manipulation that increases the oxygen affects the test. Cold milk holds more oxygen than warm milk; pouring milk back and forth from one container to another increases the amount, and at milking time much oxygen may be absorbed.

The kind of organisms affect the rate of reduction. The coliforms appear to be the most rapidly reducing organisms, closely followed by Streptococcus lactis, some of the faecal Streptococci, and certain micrococci. Thermoduric and psychrotrophic bacteria reduce methylene blue very slowly if at all. A large number of leucocytes affect the reduction time materially.

Light hastens reduction and therefore the tests should be kept covered. The concentration of the dye should be uniform as an increased concentration lengthens the time of reduction. Increasing the incubation temperature augments the activity of the bacteria and therefore shortens the reduction time.

The creaming of the test samples causes a number of organisms to be removed from the body of the milk and brought to the surface with the rising fat. This factor causes variations in the reduction time, since the bacteria are not evenly distributed. The accuracy of the test i s increased, reduction time shortened and decolorization more uniform if the samples are periodically inverted during incubation.

The Resazurin Test

The resazurin test is conducted similar to the methylene blue reduction test with the judgement of quality based either on the color produced after a stated period of incubation or on the time required to reduce the dye to a given end-point. Numerous modifications have been proposed. The two most commonly used are the "one-hour test" and the "triple-reading test" taken after one, two, and three hours of incubation. Other modifications have value in specific applications.

The procedure for making the resazurin test is as follows: Prepare resazurin solution by dissolving one resazurin tablet (dye content/ tablet, approximately 11 mg, certified by Biological Stain Commission) in 200 ml of hot distilled water as was done in the methylene blue test. Place one ml of dye solution in a sterile test tube, then add 10 ml of sample. Stopper the tube, place in the incubator and, when the temperature reaches 36o C, invert to mix the milk and dye. Incubate at 36o C. Tubes are examined and classified at the end of an hour in the "one-hour test" or at the end of three successive hourly intervals in the "triplereading test." The following relationships of color and quality are generally accepted:

Color of Sample: Quality of Milk

  1. Blue (no color change): Excellent
  2. Blue to deep mauve: Good
  3. Deep mauve to deep pink: Fair
  4. Deep pink to whitish pink: Poor
  5. White: Bad

The resazurin test may be a valuable time saving tool if properly conducted and intelligently interpreted, but should be supplemented by microscopic examination.

Results on the reliability of the resazurin tests are conflicting. One study in comparing the resazurin test with the Breed microscopic method on 235 samples found the test reliable. Other reports state that the resazurin test is an unreliable index of bacteriological quality in milk. A major criticism of the method is that the resazurin reduction time of refrigerated bottled milk at either 20o or 37o C is much too long to be of any value in evaluating bacteriological spoilage of stored milk.

Standard Methods notes that under no circumstances should results of either methylene blue or resazurin tests be reported in terms of bacterial numbers. The two dye reduction procedures are described in more detail in Chapter 15 of the Thirteenth Edition of Standard Methods compiled by the American Public Health Association.

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:

  1. destructive and time consuming
  2. slow response
  3. small sample size
  4. 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:

  1. analyze hazards
  2. determine CCPs
  3. establish critical limits
  4. establish monitoring procedures
  5. establish deviation procedures
  6. establish verification procedures
  7. 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 (see -> food -> safe food production systems). 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:

  1. simple or defined: single strain, or more than one in which the number is known
  2. mixed or compound: more than one strain each providing its own specific characteristics

Starter cultures may be categorized as mesophilic, for example:

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

or 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.

Please see further details in our cheese technology section.

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:

  1. Commercial culture
  2. Mother culture - first inoculation; all cultures will originate from this preparation
  3. Intermediate culture - in preparation of larger volumes of prepared starter
  4. Bulk starter culture - this stage is used in dairy product production

Please see further details in our cheese technology section.