04 Microbiology

8/12/2019 04 Microbiology

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Dairy Processing Handbook/Chapter 454

winner for medicine, discovered pathogenic (disease-producing) bacteriasuch as the tubercle bacillus and cholera bacterium. In addition, he devisedingeniously simple methods to enable safe study of these organisms.Alexander Fleming, 1881 1955, a British microbiologist, professor and1945 Nobel Prize winner for medicine, discovered penicillin (1929), which iseffective against many bacteria, but not tuberculosis.Selman A. Waksman, 1888 1973, an American mycologist, microbiologistand 1952 Nobel Prize winner for medicine, discovered streptomycin, whichis effective against many bacteria including tuberculosis.

Fig. 4.1Micro-organisms can be foundeverywhere ... in the air ... in the soil ...

and in water. Micro-organisms in natureMicro-organisms are found everywhere - in the atmosphere, in water, onplants, animals and in soil. Because they break down organic material, theyplay an important role in the cycle of nature.

Micro-organisms occur most abundantly where they find food, moisture,and a temperature suitable for their growth. Since the conditions that favourthe survival and growth of many micro-organisms are those under whichpeople normally live, it is inevitable that we live among a multitude ofmicrobes.

Listed below are the key characteristics of the different groups of micro-organisms, see Table 4.1.

Protozoa Unicellular, small aquatic organisms Can ingest solid food particles Ultimately become food for fish and larger animals Generally not food spoilage organisms A few protozoa are food-borne pathogens, transmitted by water Some protozoa are pathogens, transmitted by insects

Algae Uni- or multicellular organisms, frequently found in water Contain chlorophyll and are photosynthetic Used as food supplement and in pharmaceutical products

Some are source of agar for microbiological media Some produce toxic substances Generally not food spoilage organisms

Table 4.1

Micro-organisms

Groups Range of size

Protozoa 5 200 mAlgae 5 m a few metresFungie

Yeast 5 10 mMould 5 10 m by metres

Bacteria 0,5 5 mViruses 0,015 0,2 m

MicrobiologyMicrobiology is the study oforganisms that are so small,they can only be seen with the

aid of a microscope. Size ismeasured in micrometer (m).1 m = 0,001 mm


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Morphology of bacteriaMorphology is the study of the form of bacteria. This covers morphologicalfeatures such as shape, size, cell structure, motility (ability to move in aliquid), and spore and capsule formation.

Fig. 4.2Spherical bacteria (cocci)

occur in different formations.

Fig. 4.3Rod- and spiral-shaped

bacteria.

Fig. 4.4 Schematic view of a bacterialcell.

Ribosomes

Cell wall

Cytoplasm

Capsule

Chromosome

Cellmembrane

Pili

Flagellum

Reservematerial

Function of cell structuresCell wall gives the cell its

specific shapeCell membrane active transport of

nutrients andmetabolites

Cytoplasm place of productionof cell constituents

Chromosome carrier of geneticinformation

Shape of bacteriaBacteria come in many different shapes. However, three main characteristicshapes can be distinguished: spherical-, rod- and spiral-shaped.

The spherical bacteria are named cocci and when arranged in pairs theyare called diplococci (Figure 4.2 a) and when arranged in chains they arecalled streptococci (b). Cocci can also be arranged in irregular clusters likegrapes (c) and arranged in groups of four or as cubic arrangement (d).

Rod-shaped bacteria vary in both length and thickness. Some of themalso form chains in the same way as many Bacillus species (Figure 4.3).Theword bacilli means small rods.

Spiral bacteria can be of variable length and thickness, and the numberof turns also differs. Spiral bacteria with many turns are among the largestbacteria, up to 20 m are common.

Cell structure and function of bacteriaThe smallest basic unit of life, functionally and structurally, is a cell, andbacteria have the most simple cell organisation. The bacterial cell isprocaryotic. All other cells, including fungi, algae, protozoa, plant and animalcells, are eucaryotic.

The minimum number of structural components in a bacterial cell isrelatively small. The cell wall is the only rigid structural component. It givesthe cell its shape. Bacteria with a thick cell wall are Gram-positive andbacteria with a thin cell wall are Gram-negative. The cell membranedetermines what should be inside and outside of the cell. Only water-soluble substances can be transferred through the cell membrane. Thus noexchange between the cell and its environment or growth takes place ifwater is not present.

The contents of the interior of the bacterial cell are dispersed into tworecognisable parts: the cytoplasm and the chromosome. The chromosomeis the carrier of the genetic information in its DNA, deoxy-ribonucleic acid. Inprinciple, a bacterium contains only one chromosomei.e.one molecule ofDNA. Its information is transformed into enzymes in the cytoplasm. Inelectron microscope pictures, the cytoplasm most often appears granular.The actual sources of production of cell components are the ribosomes,which are big proteins, and RNA, ribonucleic acid. In addition to the

recognisable structural components (Figure 4.4) there are many solutes inthe cytoplasm: break down products, vitamins, co-factors and buildingblocks for the synthesis of cell components.


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Dairy Processing Handbook/Chapter 4 57

Motility of bacteriaSome cocci and many bacilli are capable of moving in a liquid nutrientmedium. They propel themselves with the help of flagella, which are long,stiff-thread like appendages growing out of the cytoplasmic membrane(Figure 4.5). The length and number of flagella vary from one type ofbacterium to another. The bacteria generally move at speeds of betweenone and ten times their own length per second. The cholera bacterium is

probably one of the fastest; it can travel 30 times its length per second.

Spore formationOnly a few genera of bacteria form spores: Bacillus and Clostridium are themost well-known. The spore is a form of protection against adverseconditions, e.g.heat, disinfectants, dry condition or lack of nutrients. Someof the different types of spore formations are illustrated in Figure 4.6.

When the parent cell forms a spore, it may retain its original shape, or itmay swell in the middle or at one end, depending on where the endosporeis located. During spore formation the vegetative part of the bacteria celldies. The cell eventually dissolves and the spore is released.

The spore germinates back into a vegetative cell and starts reproductionwhen conditions become favourable again.

Spores are resting cells with no metabolism and thus cannot multiply.They can survive for years in dry air, and they are more resistant thanbacteria to chemical sterilants, antibiotics, drying and ultraviolet light. Theyare also resistant to heat: 20 30 minutes in hot water or steam at 120 Cwill generally destroy spores. However, spore-forming bacteria in thevegetative state are killed in seconds by boiling at 100 C just like any otherbacteria.

Capsule formationSome bacilli and cocci are surrounded by a capsule of strongly developedmucus. This makes them rather resistant against dry conditions. Growth ofsuch bacteria in milk makes it viscous and slimy. In both cases this

phenomenon gives ropy milk.

Growth factors for bacteria

NutrientsBacteria require certain nutrients for their growth. The need for nutrientsvaries widely among different bacteria. The main sources of food areorganic compounds, e.g.proteins, fats and carbohydrates. In addition,small amounts of trace elements and vitamins are necessary for growth.

As well as material for cell formation, organic matter also contains thenecessary energy. Such matter must be soluble in water and have a low

molecular weight,i.e.it must be broken down into very small molecules inorder to be able to pass through the cytoplasmic membrane and bemetabolised by the bacterium. Consequently, bacteria need access towater.

Some micro-organisms lack the ability to release enzymes for breakingdown substances outside the cell. They have to utilise breakdown productscreated by other micro-organisms. Such a relationship is called symbiosiswhen both parties benefit from it. When one organism producessubstances, which have an inhibiting effect on other organisms, thisprocess is called antibiosis.

Water activityThe growth and metabolism of micro-organisms demand the presence ofwater in an available form. The most useful measurement of the availabilityof water is water activity, a

w. The a

win a food may be reduced by increasing

the concentration of solutes in the aqueous phase of the food, either by

Fig. 4.5Flagella may be distributed all

over the bacterium, or located at one orboth ends.

Fig. 4.6 Various types of endosporeformation in bacteria.

Round

Oval

Cylindrical

Procaryotic are all bacteria Simple cell organisation No membrane around the

genetic material, which is oneDNA molecule

Eucaryoticare fungi, algae,protozoa, plant and animal cells Cell organisation complex

Membrane around the geneticmaterial (several DNA molecules)i.e.a true nucleus exists

Symbiosis= permanent union

between organisms, each ofwhich depends for its existenceon the other

Antibiosis = an antagonisticsituation where one organismproduces substances whichinhibit the growth of otherorganisms


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removing water or by adding solutes. Some water molecules are orientedaround the solute molecules, and others become absorbed into insolublefood constituents. In both instances, the water becomes less available toenter into chemical reactions.

Dehydration is a method of food preservation that depends on thereduction of a

wby water removal. In salting and sugaring, the addition of a

solute lowers the awand preserves the food. A small reduction in a

woften

has sufficient effect to preserve a food when combined with other factors.

Definitions of water activityThe a

wof a food or solution equals the ratio of the water vapour pressure of

the food (p) to that of pure water (po) at the same temperature. When a

solution becomes more concentrated, vapour pressure decreases and thea

wdrops from a maximum value of 1 for pure water.

0 0,6 0,7 0,8 0,9 1,0

Mould and Yeast

Cocci

Gram +

Gram

MilkBeverages

Sweet condensed milkTomato paste

No

Growth

Dried food

MilkEggVegetable

Meat extractAged cheeseJam

aw

:

Evaporated milkTomato paste

0 0,6 0,7 0,8 0,9 1,0

Gram +

Gram

aw

:

Fig. 4.7Effect of awon growth.

awcalculation

The awcan be calculated

according to the formula:

where p = vapour pressure ofthe food at t C,and p

o= vapour pressure of

pure water at t C

po

pa

w=

Effect of water activity on growthMany micro-organisms, including pathogenic bacteria, grow most rapidly atlevels of a

win the range of 0,99 0,98. Below this a

wthe growth rate

decreases and the length of the lag phase increases (Figure 4.7).Most micro-organisms relevant to food have their optimum growth rate

at anawof 0,98 or higher. This is also the range for a

wof milk and

beverages in general. Several gram-negative bacteria are the mostcompetitive due to growth rate,i.e.if present, they will dominate themicrobial flora. In milk, for example, a gram-negative infection often hides aninfection of gram-positive bacteria.

At anawbetween 0,98 and 0,93the gram-positive bacteria dominate,

i.e.Lactobacillus, Bacillus and Micrococcus, but a few tolerant coliformsmay be present. Food-borne bacterial pathogens (Salmonella, C. botulinumand C. perfringens) are prevented from multiplication. In contrast,Staphylococcus grow at 50 % of their maximum growth rate at a

w= 0,94.

Within this range, spoilage of low-acid food by fungi is possible and theycan compete with bacterial spoilage.

At anawbetween 0,93 and 0,85possible spoilage organisms are cocci,

moulds and yeasts. The only bacterial pathogen growing within this range isS. aureus. Intermediate moisture food is designed to have an a

w< 0,85 in

order to inhibit S. aureus.At ana

wbetween 0,85 and 0,60a few moulds and yeasts can cause

spoilage. Common foods in this range are jams and jellies preserved by ahigh concentration of sugar. There is no production of mycotoxin possible inthis a

wrange.

No micro-organisms can grow at anaw< 0,60. Such food is safe from

further microbial spoilage. It should be emphasised, however, that due toprevious history, such food might contain viable micro-organisms includingpathogens and/or toxins.


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TemperatureTemperature is the greatest single factor affecting growth, multiplication andfood deterioration (Figure 4.8). Bacteria can only develop within certaintemperature limits, which vary from one species to another. In principle,bacteria can grow at temperatures between the freezing point of water andthe temperature at which the protein in the cytoplasm coagulates.Somewhere between the maximum and minimum temperatures,i.e.the

upper and lower limits, lies the optimum temperature. This is thetemperature at which the bacterial strain multiplies most vigorously.

Temperatures below the minimum cause growth to stop, but do not killthe bacteria. The life functions of bacteria cease almost completely at atemperature close to the freezing point of water. As the cells have a highcontent of water, this will freeze at this temperature. When this happens, thebacteria can no longer absorb nutrients through the cell membranes.

If the temperature is increased above the maximum, the bacteria arequickly killed by heat. Most cells die within a few seconds of being exposedto 70 C, but some bacteria survive heating to 80 C for five minutes, eventhough they do not form spores.

It takes much more heat to kill bacterial spores, and dry heat is lesseffective than moist heat. Treatment with steam at 120 C for 30 minutes

ensures the destruction of all spores, but in dry heat, the bacteria must bekept at 160 C for two hours to guarantee destruction of spores.

Classification by temperatureBacteria can be divided into the following categories (Figure 4.9) accordingto their preferred temperature range:Psychrophilic (cold-loving) bacteriagrow well at 0 C with an optimumtemperature about 12 15 C and maximum below 20 C.Psychrotrophic (cold-tolerant) bacteriaare mesophilic strains that canmultiply at commercial refrigeration temperatures with an optimumtemperature about 20 30 C.Mesophilic bacteriahas a minimum temperature about 10 oC, and generallyan optimum of 30 35 oC and maximum at about 50 oC. Without doubt,

this is the most common range for bacterial growth. Approximately 90 % ofall bacteria can grow in this temperature interval.Thermophilic (heat-loving) bacteriahave their optimum growth temperaturesat 55 65 C. Minimum temperature about 37 C and maximum around70C.

The psychrotrophic bacteria are of particular interest to the dairy industry,because microbiological activity in farm milk and market milk usually takesplace at a temperature of 7 C or below.

OxygenMany micro-organisms need free oxygen to oxidise their food in order toproduce energy and support their life processes. Complete oxidation of

organic compounds forms CO2and water. Many micro-organisms canutilise air at atmospheric pressure, and these are called aerobic micro-organisms. Other types obtain energy from their food without need of freeoxygen, and these are called anaerobic micro-organisms.

There are some bacteria that consume free oxygen if it is present, butwhich can grow in the absence of free oxygen. Such bacteria are calledfacultatively anaerobic. Anaerobic and facultatively anaerobic bacteriagenerally obtain their energy by fermentation of organic compounds.Chemically, this is an incomplete oxidation, whereby organic waste-products are formed, e.g.lactic acid from lactose, Table 4.2.

As most organisms obtain their oxygen from the air,i.e.they are aerobic,removal of oxygen/air is a means of controlling or preventing their growth.Examples of this are vacuum packing, gas packing and the use of materials

acting as an air barrier.Anaerobic bacteria die if exposed to atmospheric oxygen for any lengthof time.

45 C

20 C

7 C

Thermophilic

Mesophilic

Psychrophilic

Psychrotrophic

Fig. 4.9Classification of bacteria by

temperature preference.

Fig. 4.8Temperature conditions for

bacterial growth.

Maximum

Optimum

Minimum

Survivaltemperature

Lethaltemperature

Growthtemperature

Maximum

Optimum

Minimum

Survivaltemperature

Lethaltemperature

Growthtemperature

Maximum

Optimum

Minimum

Survivaltemperature

Lethaltemperature

Growthtemperature


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LightLight is only essential for photosynthetic cells, which capture energy fromthe light. Micro-organisms, including most bacteria, tend to be killed whenexposed to direct sunlight. The ultraviolet part of the sunlight causeschemical changes in the DNA and cell protein.

Ultraviolet light is often used to disinfect atmospheres in starter rooms.However, it is not used to disinfect food, as chemical changes may alsotake place in the food.

pH effect of acidity on growthMost natural environments have pH values between 5 and 9, and the mostcommon micro-organisms are optimised for growth in this range. Mostmoulds and yeasts grow best in slightly acidic media, around pH 5 to 6,while optimum conditions for bacteria are neutral or slightly alkalineenvironments.

Fresh milk normally has a pH between 6,5 and 6,7. Moulds and yeastsgenerally grow well at pH as low as 3, or even pH 2. Most non-processedfoods have a pH slightly below neutrali.e.they are low acid foods. Fruitjuices are generally high-acid foods (Figure 4.10).

0 2 5 7 9 14

4,6

pH:

4,6

0

Yeast

2 5 7 9 14

4,6No growth ofpathogenes

pH:

4,6

Moulds

Bacteria

Fruitjuices

Tomatoproducts

Milk

Vegetable juices

Meat, Vegetables, Soup etc.

HIGH ACID FOOD LOW ACID FOODFig. 4.10Effect of acidity on growth.

Table 4.2

Bacterial relationship to oxygen

Bacterial group Relationship to oxygen

Aerobe Micro-organisms that use O2for growth and can

tolerate O2at atmospheric level or higheri.e.21 %.Microaeraphile Micro-organisms capable of using O

2for growth

but only at lower O2levels than in atmosphere.

Anaerobe Micro-organisms incapable of O2-dependent

growth and can not grow in oxygen ofatmospheric level. They obtain energy byfermentation.

Facultative Micro-organisms that can grow well both in theanaerobe absence and presence of oxygen of atmospheric

level. Some can grow aerobically with oxygen andanaerobically with fermentation.


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Multiplication of bacteriaBacteria normally multiply by binary fission. In Figure 4.11, multipli-cation is shown graphically. Each individual cell grows and afterreaching a critical size, it divides into two identical cells. The type of cellarrangement, which results in a characteristic cell grouping, is usuallyconstant for a given species of bacteria. Cell grouping can take theforms of chains, pairs and clumps. This characteristic is therefore used

in the description of different species.

Rate of multiplicationIn favourable conditions, multiplication of bacteria can occur atintervals of 20 30 minutes. The rate of multiplication can becalculated from the formula shown to the right. With a generation time of0,5 hour, one bacterium/ml of milk will become about one million bacteria/ml within 10 hours.

Under optimal conditions in food, 100 million 1 000 million bacteria/mlcan be formed. At that stage, the growth rate will be inhibited by lack ofnutrients and accumulation of toxic metabolic waste products.Reproduction finally stops, and large numbers of bacteria die. In reality,unfavourable conditions, such as low storage temperature or low pH will

limit or delay the growth of bacteria in food.

Growth curve of bacteriaFigure 4.12 shows a curve of the growth of bacteria transferred to asubstrate by inoculation. There is usually some delay before the bacteriastart to reproduce, as they must first adapt to the new environment. Thisphase of development (a) is called the lag phase. The reason for the lagphase may also be that the culture has to be recovered. It may, forexample, have been stored at a low temperature prior to inoculation.

The length of the lag phase varies according to how much the bacteriawere inhibited at the moment of inoculation.

After the lag phase, the bacteria begin to multiply logarithmically. Thisphase (b) is called the log phase or the exponential phase.

After some time, toxic metabolic waste products accumulate in theculture. The rate of multiplication will therefore subsequentlyslow down, while at the same time bacteria are constantlydying, so that a state of equilibrium is reached between thedeath of some cells and the formation of new ones. Thisphase (c) is called the stationary phase.

In the next phase (d), formation of new cells ceasescompletely and the existing cells gradually die off. Finally theculture is almost extinct. This is called the death phase.

The shape of the curve,i.e.the length of the variousphases and the gradient of the curve in each phase, varieswith temperature, food supply and other growth parametersas well as species.

Biochemical activityDue to biochemical activity, micro-organisms can spoil foodand cause diseases in animals and plants. Some micro-organisms havebiochemical activities that are used in food processes i.e. the manufactureof cheese, yoghurt, butter, etc.

The activity of a specific micro-organism is governed by the enzymes itpossesses, as these determine what it can feed on and break down, andconsequently what end-products it produces.

There are many biochemical and enzymatic systems in micro-organisms.The following systems are the major ones concerned with milk and milk

products. They can be sub-divided into which constituent they break downand their effects.

Fig. 4.12Growth curve of bacteriaa Lag phase

b Log phasec Stationary phased Death phase

Formula for rate of reproductionof bacteria

N = number of bacteria/mlat time t

N0= number of bacteria/ml

at time 0t = the time of growth in hoursg = generation time in hours

Fig 4.11Multiplication of bacteria.

tg

N = N0x 2

a b c d

Number of bacteria (log)

Time


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sulphide and ammonia) and other compounds. Breakdown of protein nearlyalways results in ammonia, which is alkaline and has a strong odour, Figure4.13.

Three amino acids, cystine, cysteine and methionine, contain sulphurand result in hydrogen sulphide, which also gives off a strong smell of rotteneggs.

Breakdown of protein in liquid milk takes place intwo major stages called peptonisation and consists of: Curdling (sweet as opposed to sour) or clotting

of the milk by rennin-like enzymes. This fault inmilk is called sweet curdling, a defect which iscommon in pasteurised milk that is stored warm.

Proteolysis of the protein, resulting in productionof ammonia, which is alkaline.

The degree of amino-free acids and ammonia in cheese gives an indicationof its age and maturity as proteolysis progresses. Blue, or mould-ripenedcheese has rapid proteolysis, resulting in production of large amounts ofammonia.

Breakdown of fatThe process in which fat is broken down by enzymes is called lipolysis.Lipase is the main enzyme involved in this process. During lipolysis, the fatis hydrolysed to glycerol and one, two or three separate fatty acids, Figure4.14. Some of the fatty acids are volatile and give off strong smells. Oneexample is butyric acid, which gives the characteristic rancid taste.

Pure fat cannot be broken down by micro-organisms, but fat in wateremulsions, or fat in contact with water, are broken down by many micro-organisms. Water is essential for enzymatic split. Milk fat in the form ofbutter and cream is a water emulsion and contains protein, carbohydrate,minerals, etc., which sometimes makes it even moresusceptible to enzymatic breakdown.

Many bacteria and moulds that break down proteinsalso break down fat oxidatively.

Breakdown of lecithinLecithin, the phospholipid contained in the membranesround the fat globules, is a chemical combination ofglycerol, two fatty acids, phosphoric acid and choline,an organic alkali. Strains of Bacillus cereusproduceenzymes, lecithinases, which hydrolyse the lecithin intodiglyceride and phosphoryl choline. The membranes of the fat globules aresplit, resulting in an unstable fat emulsion often seen in the form of flocs orlumps floating on the surface of the milk or cream. This fault in milk orcream is called bitty or broken cream.

Further break-down of the choline into trimethyl amine will result in afishy smell and taste.

Pigment and colour productionThe process of colour production is called chromogenesis and theorganism causing the production is referred to as chromogenic.

This process of metabolism is a feature of certain micro-organisms. It isgreater in some foods than others and takes place only at ambient or lowertemperatures. Aerobic conditions are also favourable for chromogenesis.

There are two types of pigment: Endo-pigment, which stays in the cell Exo-pigment, which diffuses out of the cell into the surrounding foodThere are three basic colour groups: Carotenoids, (yellow, green, cream or golden)

Antho cyanins, (red) Melanins, (brown or black)The name of an organism often refers to the colour it produces. Example:Staphylococcus aureus = the golden Staphylococcus.

Proteolysis = breakdownof protein

Lipolysis = breakdown of fat

NH2 COOH

R

Peptides

Amino acid

PROTEIN Protease Peptidase

+

O=

O=

O=

O=

O=

O=

CH3-R-C

COOH

COOH

CH3-R-C

CH3-R-C

COOH

COOH

COOH

COOH

CH3-R-C

CH3-R-C

CH3-R-C

Freefattyacids

LIPID Lipase

GLYCEROL

GLYCEROL

Fig. 4.13Protein is broken down toamino acid by the enzymes protease

and peptidase.

Fig. 4.14Lipid is broken down to free

fatty acids and glycerol by the lipaseenzyme.

Chromogenesis = colour

production caused bychromogenic bacteria

The species of an organism isoften named after the colour itproduces, for example:Albus = whiteLuteus = yellowCitreus = citrus yellowRoseus = pink or redAureus = golden

Violaceum = violetNigra = black or brown


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Mucus productionA number of bacteria produce a mucus (or slime) of polysaccharides, whichdramatically increase viscosity, as they are highly water-soluble and dissolvein the medium. This is utilised in certain cultured products such as yoghurtand lngfil, a Swedish ropy milk.

Odour productionSome organisms produce strong odours or smells. Below is a list of someorganisms and their associated smells: Moulds musty Actinomycetes earthy Yeasts yeasty Pseudomonadaceae fruity/fishy Coliforms manure Lactococcus lactis var. maltigenes malty

Pathogens in raw milkSome micro-organisms may cause food poisoning (pathogenic micro-organisms), either by intoxication and/or infection. Intoxication implies the

production of poisons in the food prior to its consumption. Infection meansthe establishment, active growth, and multiplication of such micro-organisms in the human body. Often rather large numbers are needed tocause an infection, but sometimes, as in the case of Salmonellatyphimurium, the MID (minimum infection dose) may be as small as onebacterium.

Table 4.4

Pathogens in milk

Infectious Toxin formers

Mycobacterium bovis Bacillus cereus

Mycobacterium tuberculosis Clostridium perfringens Escherichia coli (some strains) Staphylococcus aureus Listeria monocytogenes (some strains) Salmonella Campylobacter Corynebacterium diphteriae

Study of bacteriaBacteria occur in nature in extremly large populations made up of manydifferent species. In order to study the characteristics of a particular speciesit is necessary to separate it from all other species. Laboratory procedures

exist for this purpose.The growth of a mass of cells of the same species in a laboratory vessel

(such as a test tube) is called a pure culture. To keep the culture pure,continuous precautions have to be taken to prevent entrance of otherspecies. This is done by applying sterile technique.

Bacteria are cultivated in nutrient broth or on nutrient agar. The type ofnutrients depends on the species. Typical nutrients are a mixture ofproteins, peptides, sugar, mineral salts and co-factors. To obtain nutrientagar, a jelly-like, semi-hard substance called agar is added to the broth. Micro-organisms cultivated on or in agar substrates grow as colonies.Under favourable conditions, one cell will multiply into a mass of cells calleda cfu (colony forming unit), which can be seen with the naked eye. Bymaking dilutions of the original sample, plating on agar and counting thecolonies, it is possible to enumerate the bacteria. This well-establishedtechnique for enumeration of bacteria, yeast and mould is called a Colonycount.

Study of bacteria

Pure culture Sterile technique Cfu, colony forming unit Colony count

Pathogenic bacteria

cause disease in human beings,animals and plants.


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By using selective agar media, which allows only specific groups ofbacteria to grow, the presence of various types of bacteria can bedemonstrated.

Identification and classification of bacteriaIn an attempt to classify the many different groups of bacteria that exist,they were previously divided into families, genera and species in the same

way as higher plants and animals.In zoology and botany, this is done according to the external

characteristics of the individual (appearance). The same principle wasoriginally applied to the classification of bacteria, but it was soon found thatit was not enough to group bacteria simply by size, shape, appearance andmotility. Apart from these external characteristics, it was also necessary toconsider the metabolism of the organisms (their relationship to variouscarbohydrates, proteins, fats, etc.) and their strain characteristics. Withinformation on these matters, it was possible to group similar organisms ina bacterial classification system.

The Latin names of bacteria according to this system are nowinternationally used. Every bacterium has two names. The first representsthe genus and the second describes the species, often indicating a certain

property or origin. See the Pigment and colour production section above.Identification of bacteria to the genus level is done by a combination of

various biochemical tests and morphological analysis, including gram-reaction.

A lot of new techniques based on DNA composition have beenintroduced to identify bacteria. The most important of these is PCR,polymerase chain reaction. This method can directly identify bacteria at thespecies level. Today the method is in regular use to identify pathogens. TheFTIR (Fourier Transform infrared) method compares several bacterialcomponents at a molecular level with a databank of type-species.

The most authoritative literature on identification of bacteria is BergeysDeterminative Bacteriology. The 9thedition, (1994), identifies severalthousand species. However, these represent only a fraction of those

existing in nature. Much work remains to be done and future editions ofBDB will undoubtedly become more extensive.

Bacteria in milk

From the cowMilk is virtually sterile when it is secreted in the udder. However, even beforeit leaves the udder, milk is infected by bacteria that enter through the teatcanal. These bacteria are normally harmless and few in number, up to a fewhundred per ml.

However, in cases of bacterial udder inflammation (mastitis), the milk is

heavily contaminated with bacteria and may even be unfit for consumption.This condition also causes the cow to suffer.

There are always concentrations of bacteria in the teat canal, but most ofthem are flushed out at the beginning of milking. It is advisable to collect thefirst bacteria-rich jets of milk from each teat in a separate vessel with ablack cover. Flocculated milk from diseased animals shows up readilyagainst the black background.

Infection at the farmIn the course of handling at the farm, milk is liable to be infected by variousmicro-organisms, mainly bacteria. The degree of infection and thecomposition of the bacterial population depend on the cleanliness of the

cows environment and the cleanliness of the surfaces with which the milkcomes into contact, e.g.the pail or milking machine, the strainer, thetransport churn or the tank and agitator. Milk-wetted surfaces are usually amuch greater source of infection than the udder.

Fig. 4.15Bacteria enter through the teat

canal.

Bergey's DeterminativeBacteriology, 9th edition

Number of bacteria detected Groups 35 Genera 550 Species 4 500

Fig. 4.17Collect the first bacteria-richjets of milk from each teat in a separatevessel with a black cover.

Fig. 4.16During udder inflamation themilk is heavily infected by bacteria.


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When cows are milked by hand, bacteria can get into the milk via themilker, the cow, the litter and the ambient air. The magnitude of the influxdepends largely on the skill and the hygiene-consciousness of the milkerand the way the cow is managed. Most of these sources of infection areeliminated in machine milking, but another one is added, namely the milkingmachine. A very large number of bacteria can enter the milk this way if themilking equipment is not cleaned properly.

Bacteria in raw milkMilk is very nutrious and is susceptible to contamination and hereby growthof a wide variety of bacteria.

Farm milk may contain anything from a few thousand bacteria per ml, if itcomes from a hygienic farm and up to several millions if the standard of

cleaning, disinfection and chilling is poor. Daily cleaningand disinfection of all milk equipment is therefore themost decisive factor in the bacteriological quality of milk.Under optimal condition it should be possible to achieve abacteria count of less than 20 000 CFU (Colony FormingUnits) per ml.

Rapid chilling to below 4 C contributes greatly to the

quality of the milk at the farm. This treatment slows downthe growth of the bacteria in the milk, thereby greatlyimproving its keeping qualities.

The influence of temperature on bacterial developmentin raw miilk is shown in the graph in Figure 4.18. Startingfrom 300 000 CFU/ml the speed of development athigher temperatures and the effect of cooling to 4 C isstriking. Cooling to 4 C or even lower to 2 C, in

conjunction with milking makes it possible to deliver milk at two-dayintervals provided that the milk container/tanker is insulated.

There are principally three sources of infection: inside the cow, theudders and everything the milk comes into contact with. The most commonmicro-organisms in raw milk of good quality (


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FungiFungi are a group of micro-organisms that are frequently found in natureamong plants, animals and human beings. Different species of fungi vary agreat deal in structure and method of reproduction. Fungi may be round,oval or threadlike. The threads may form a network, visible to the nakedeye, in the form of mould on food, for example. Fungi are divided into

yeasts and moulds.

YeastsYeasts are single-cell organisms of spherical, elliptical or cylindrical shape.The size of yeast cells varies considerably. Brewers yeast, Saccharomycescerevisiae, has a diameter of 2 8 m and a length of 3 15 m. Someyeast cells of other species may be as large as 100 m.

Yeasts, like moulds, have a more complex internal structure thanbacteria. They contain cytoplasm and a clearly discernible nucleussurrounded by nuclear membrane, Figure 4.19. The cell is enclosed by awall and a cell membrane, which is permeable to nutrients from the

outside of the cell and waste products from the inside.The cell contains a vacuole that serves as storage space for reservenutrition and for waste products before they are released from the cell.Fat globules and carbohydrate particles are embedded in thecytoplasm. In the cytoplasm there is also a fine network of membranesnamed endoplasmic reticulum, mitochondria (where energy for cell growthis generated), as well as ribosomes.

Reproduction of yeastYeast cells normally reproduce by budding, as shown in Figure 4.20,although other methods of reproduction can also be found. Budding is anasexual process. A small bud develops on the cell wall of the parent cell.The cytoplasm is shared for a while by parent and offspring. Eventually

the bud is sealed off from the parent cell by a double wall.The new cell does not always separate from its parent and may

remain attached to it while the latter continues to form new buds. Theoffspring cell may also form fresh buds of its own. This can result inlarge clusters of cells attached to each other.

Some types of yeast reproduce sexually,as in Figure 4.21, by formingspores, ascospores and basidiospores (not to be confused with bacterialspores). Two cells fuse together and the two nuclei also fuse. Followingdivision of the nuclear material, eight ascospores are formed within thecells, each containing a similar set of DNA. When the spores aremature, they are released and germinate, forming new cells, whichthen reproduce asexually by budding.

Conditions for the growth of yeast

Environment and nutrientsYeasts have the same need for nutrients as other living organisms.They usually flourish in habitats where sugars are present, such asfruits, flowers, and the bark of trees. A few species are pathogenic toanimals and humans. Yeasts are the predominant spoilage organism of fruitjuices. Like bacteria, they have a system of intracellular and extracellularenzymes capable of breaking down large molecules in the substrate tomanageable size for the metabolism of the cells. In the laboratory, yeastsare cultivated in sugar-rich substrates with pH 5 to 6.

MoistureLike bacteria, yeasts must have access to water in order to live, but yeastsrequire less water than bacteria. Some species can grow in media with verylow water content, such as honey or jam.

Fungi are divided into: yeasts moulds

Vacuole

Nucleus

Cell wall

Ribosomes

Mitochondrion Endoplasmicreticulum

Cellmembrane

Fig. 4.19The structure of a yeast cell.

Fig. 4.20Budding yeast cells.

Yeast can cause defects incheese and butter.


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supplied by companies that specialise in developing and propagating themunder strictly-controlled hygienic conditions. The micro-organisms used inthe dairy industry are called starter cultures. A starter culture is, for example,a mixture of organisms that form lactic acid by fermenting the lactose inmilk. However, it is important that the quality of the starter cultures ispreserved after arrival at the dairy by maintaining high standards of hygiene,and that when used, sterile technique is applied in critical steps of theprocessing chain.

In this context, it should be mentioned that the milk may contain residuesof antibiotics emanating from treatment of cows suffering from mastitis; themost commonly occurring one is penicillin. In spite of regulations saying thatmilk from cows treated with antibiotics must not be sent to the dairy, youmay find sufficiently high levels of antibiotics in bulk tank milk to stop or

Fig. 4.28 Effect of penicillin in milk on acid production.

*/ I.U = International unit. (Penicillin causeslysis of bacterial cells.)

Levels of penicillin that inhibitsome micro-organisms

Lactococcus lactisLactococcus cremoris

Streptococcus thermophilus

Lactobacillas bulgaricus

Starter (mixed culture)

0,1 - 0,250,05 - 0,1

0,01 - 0,05

0,25 - 0,5

0,25 - 0,5

Micro-organism Penicillin (I.U) */ml

Time

Acid

Starter A

Starter A inpenicillin milk

Fig. 4.29Growth of starter bacteria andphages and influence on infected starter

culture.

10

9

8

7

6

5

4

3

2

1

5 10 15 20 25 30

Log No.

Non-infected single-strain culture

Number of phages

Phage-infected single-strain cultureRef: Nordisk Mejeriindustri 4/82

Hours

retard growth of the starter cultures to be used. Figure 4.28 illustrates theinfluence of even small residues of penicillin on the most commonly usedstarter cultures.

As raw milk is usually contaminated with bacteriophages, it is important

that the milk used for starter cultures, generally skim milk, is heated to atleast 90 C for 30 minutes to inactivate the phages. Figure 4.29 showswhat will happen if this is not done or if the milk is re-contaminated byphages afterwards. The time it takes for one non-infected bacterium toproduce four new bacteria is two generations. In the same period, onephage infects one bacterium, which releases 150 new phages in onegenerations time. These phages infect 150 new bacteria and in a furtherone generations time, 22 500 phages are released. This is the reasonwhy a phage-infected starter culture suddenly collapses after a while.

It would be a false idealisation of micro-organisms to omit to mentionthat some of them the pathogenic micro-organisms are regarded asmankinds worst enemies. The threat against Man is becoming more andmore serious due to the use of antibiotics. Some pathogens are todayresistant against all antibiotics. It is also true that without the manyharmless micro-organisms, life would not be possible for Man and manyother living organisms.

In most countries, governments have passed laws requiringpasteurisation of milk produced at a dairy and intended for consumption.A typical temperature/time combination for pasteurisation is 72 C for15 20 seconds, which kills all pathogens. In order to avoidrecontamination it is of utmost importance that good hygiene practiseprevails.


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04 Microbiology