Wuolah Free FoodMicrobiology2Partial

FoodMicrobiology2Partial.
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Beatriz13XO
Microbiología de los Alimentos
2º Grado en Ciencia y Tecnología de los Alimentos
Facultad de Veterinaria
Universidad de Córdoba
Reservados todos los derechos.

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Units 15 & 16: Alternative methods for
microbiological analysis of food. Conventional
methods automation and rapid-very rapid
methods.
Introduction
Nowadays, food contamination is still the major problem associated with the
consumption of food. This contamination can be made by microorganisms or microbial
metabolites.

The contamination can be from:
• Food animal origin.
• Food of vegetable origin.
• Contamination of food through unsanitary manufacturing practice.
10 years ago, “Estudio Strategic Consulting Inc’s” made a microbiological analysis carried
out a worldwide survey (encuesta):
• 58% Food industry
o 49% Food.
o 9% drinks.
o They look 80% of the times a routine microbiological analysis.
▪ It gives us a general idea, and if everything is okay, then it is not
necessary to look for pathogenic microorganisms.
o They look 20% of the times pathogenic microorganisms.
▪ Looking for pathogens is expensive and needs a long time in order
to confirm if they are present.
The food contaminated with pathogenic microorganisms can have serious

consequences:
• From a health point of view: pathogens pose a risk to human health.
• As they may have a potential impact on economic aspects:
o Destruction of stocks.
o Business closing for good.
o Work-hours losses.
o Medical treatment of the people feeling sick: hospitalization and
compensation.
o Epidemiological researches, etc.
Number of pathogen responsible of food poisoning (foodborne illnesses) have grown

within the last decades.
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Classical or traditional methods
Cuantification of Colony Forming Unit (CFU) on solid medium/culture.
Most Probable Number (MPN): colonies cannot be counted as a liquid medium is used,
but it is possible to detect the growth of microorganisms by the presence of both gas
and cloudiness.
Alternative methods
The reasons for them to be developed were:
• Implementation of Hazard Analysis and Critical Control Points (HACCP) Systems
on Food Industry in order to produce safe food products.
o Reduce to the minimum every single hazard in the food production.
• Sampling plans based on a great number of samples to be processed.
o Need to analyze hundreds of samples.
• It is necessary to get results as soon as possible.
• To set right measures to correct errors and mistakes sooner and save resources
and time.

*The characteristics are: an ideal alternative/modern/rapid method must show some
aspects to be considered a suitable method.
• Accuracy: alternative techniques must be exact and precise.
• Rapidity: results must be provided faster than conventional methods within a
few hours.
• Production: a larger number of samples can be analyzed.
• Internal validation: verification must be performed for a method to be applied.
o This verification or recognization is issued by and independent certifying
body organization or institution (AOAC, AFNOR, MicroVal).
• Simple to use: no need for highly trained staff.
• Training offered.
• Common reagents.
• Well-known brand.
• Good service.
• Economical.
• Not to big equipment.
*The classification categories are:

• Automation of classical methods → before everything was done by hand.
• Direct counting (we are able to count) techniques and living cells counting.
• Biomass detection/measurement (indirect detection).
• Miniaturized systems and analytical kits.
• Immunological methods → linked to pathogens.
• Genetic methods → linked to pathogens.
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Unit 17: Control of microbiological quality of food
Introduction
One of the most fundamental concerns in the management of the agri-food sector (from
farm to fork) refers to the quality and safety aspects of food and agricultural products.
This concern is shared equally and collectively by the producer, consumer and regulatory
authority, and they all effort to seek methods of ensuring production of high quality
food products.
Food safety definitions:

• No human being is going to be starved (people not dying from starvation).
• Food being quality (not give poison to people).
Food is fundamental to human survival. Its quality, therefore, is of paramount

importance to all of us. We must all effort to ensure that our agri-food products are:
• Wholesome.
• Nutritious.

• Safe and risk-free.
o It is impossible to guarantee 0 risk, but we are going to try to reduce it to
the minimum → this is going to be done in all the stages from the agri-
food chain.
▪ We are going to look for quality and safety.
This goal can be achieved by setting high quality-safety standards and instituting
effective total quality management systems to ensure their conformance.
Quality management/department has recently played an important role in food

production.
• Quality defines the place of food products on the world market: the more quality
my product has, the better is going to be sold, the more reputation → the better.
• Here, people are looking for quality factors, pathogens, etc.
Substantial part of quality management are:

• Food quality assessment: monitoring myself, we are going to test ourselves in
order to have the highest quality.
o HACCP system controls and prevents the critical points of the production
of the product.
• Food quality control.
Everyone involved with food, form the farmer to the consumer, shares in the
responsibilities to keep the food supply safe by taking the necessary precautions to keep
food protected from not only food spoilage but hazards that can increase human health
risks.

These actions will also prevent or reduce food losses, which is especially important in
those situations where food safety is threatened.
The increased emphasis on food safety during the past decades has decreased the
emphasis on the loss of food through spoilage, particularly in developed countries
where food is more abundant. In these countries spoilage is a commercial issue that
affects the profit or loss of producers and manufacturers.
Quality control needs: during the Second World War a major effort was given to
developing the technologies needed to ship foods to different regions of the world
without spoilage. The food was essential to the military and to populations in countries
that could not provide for themselves. Since then, progress has been made in improved
product formulations, processing, packaging and distribution systems.
Control of microbiological quality
Quality control system: Definition

HACCP (Hazard Analysis and Critical Control Point) is a system that helps food business
operators look at how they handle food and introduces procedures to make sure the
food produced is safe to eat.

• This system was created by the NASA when they needed to give food to
astronauts because if they got sick, no one would be able to help them.
• Initially it consisted of 3 designed purposes:
o Identification and assessment of hazards (biological, chemical or
physical) associated with the growing, harvesting (plants), processing-
manufacturing, marketing, preparation and use of a given raw material
or food product → all the stages.
▪ In every single step you have to keep in mind every single hazard
which that step could have.
o Determination of critical control points (CCPs) at which identified hazards
could be controlled → an estimate of the probability of a hazard being
present.
▪ A CCP is a point where you cannot do anything later, in a stage
after, to correct/prevent that CCP.
• Like not cooking the omelet sufficiently.
o Establishment of procedures to monitor the CCPs.
▪ Like using a thermometer to see if the omelet is well-cooked
(>65ºC is perfect).
Quality Management Systems (QMS) are used to control the quality and safety of
products.
• The use of a QMS will ensure that all aspects of a business are working efficiently
and cost effectively → detect possible failure during food chain processing and
avoid them to occur.
• A quality control system is a “group of statistical methods or measures which
has the ability to detect and correct a change or difference of any kind (both

predictable and unpredictable variations) all along the production process, to
finally get a product that meet stated requirements previously established”.
Quality control system: Functions

After admitting that a Quality Control system is needed, its functions will be listed:
• To set specification for raw material and final product.
o All the precautions are going to be different depending if it is the raw
material or the final product.
• To keep these specifications: take them into practice.
• To act in case of non-compliance (not following the law): write which precautions
you are going to do in order to reconduct yourself and do things right.
o These acts can be more or less dangerous.
• To design improvements (achievements): even though all the production is okay,
you have to keep moving on.

In conclusion, thanks to the Quality control, there will be:
• Product improvement.
• Improving the production.
• Improving the order and functioning of agri-food industry → have organization
so that the consumer is happy with the product.
Quality control system: Organization

It is based on:
• All ingredients and raw materials used are selected according to a priority order.
o Example: flour to bake bread.
• Once ingredients are selected they will be used to carry out quality trials or tests.
o Now and then they test the ingredient in case the product from the
supplier has lost quality.
• Products will be put on the market after analytical results are provided.
• CCPs of the process must be defined, taking special attention on them.
• Final inspection of products must be reduced to the minimum.
o Because any time I analyze myself, it costs money.
• The greater degree of integration the global organization of agri-food industry
has, the more effective the quality control will be.
o I want to be accepted/recognized as a quality product.
Quality control system: Situation

Quality control department is recommended to depend on the direction of the business.
The quality control department is recommended to hold enough authority to stop the
production chain, if necessary.
The quality control department must carry enough fundamental controls, without
ignoring their cost → insufficient vs. perfectionist.
The maintenance of the Quality Control system is convenient to everyone in the food
industry.
The quality control department is recommended to hold as great authority as other

departments.
The quality control department is recommended not to be subordinated to the

production department.
Quality control system: Cost
Quality control management often has a high cost which includes: material,
installations, equipment, working personnel salaries, etc.
• However, the lack of quality is even more expensive: loss of prestige on the
market, decay of image, etc.
• The highest cost is at the beginning. Later on, the analysis can be reduced.
The key is to reach a balanced point between quality and quantity, which allows to:
• Protect consumer’s health.
• Guarantee the quality of the product.

The benefit of quality control could be represented as the difference between the cost
of the quality control itself and the consequences of its lack (losing reputation, money,
etc.).

Unit 18: Microbiology of meat and meat products
Introduction
Broadly, the composition of meat, after rigor mortis but before post-mortem
degradative changes, can be approximated:
• Water: 75%.
• Protein: 19%.
• Soluble non-protein substances: 3,5%.
• Fat: 2,5%.
Meat is a very good source of various micronutrients:

• Low-fat pork contains 1,8 mg iron and 2,6 mg zinc.
• Pigs’ liver contains 360 mg magnesium, 20 mg iron and 60 g selenium per 100
g.
A daily intake of 100 g of meat and liver can supply up to 50% of the recommended daily
allowance for iron, zinc, selenium, vitamins B1, B2, B6, B12 and 100% of vitamin A.

The importance of meat as an essential source of some micronutrients is due to the fact
that it is either their only source, or they have a higher bioavailability.
Vitamins A and B12 (necessary for our nervous system) occur exclusively in meat and
can hardly be compensated for by plant-derived provitamins.
Microorganisms in meat
There can be an association of undesirable microorganisms with meats.
Fresh meat is a highly nutritious substrate with water activity (free for microorganisms
to be used) of about 0.99, meaning that it is suitable for the growth of most
microorganisms.
Raw meat in general may contain bacteria, including pathogenic (diseases) and spoilage
ones.
As warm-blooded animal naturally may carry bacteria such as enterobacteria in their

intestines, raw meat may be contaminated with bacteria during the slaughtering process
such as evisceration and dressing procedures.
• The slaughtering happens in a slaughter house or abattoir → matadero.

Initial biota: endogenous microbial contamination (initial microbial load of meat)
The endogenous microbiota is present in the muscles.
Muscles of healthy animals are regarded as sterile, but the slaughtering and butchering
process of animals provides bacteria with an opportunity to colonize meat surfaces.
Muscles from unhealthy animals may contain microorganisms.
Except the external or outer surface and the respiratory and the respiratory and
digestive tracts, inner tissues contain very few microorganisms.
While the animal is alive, its defense mechanism controls efficiently the infective agents.
When the animal is sacrificed, this defense mechanism fails.
Initial biota: exogenous microbial contamination

The typical biota on the surface comes from digestive and respiratory tracts, because of
something that they have eaten or inhaled.
The degree of cleanliness of the animals depends on:

• The location of the farm and the weather (temperature).

• Mean of transport (stress → they can release urine, feces, etc.).
• System of stabling (intensive/extensive).
Microbial contamination
The most important source of contamination of meat is external contamination:
bleeding and dressing of carcasses (clean, remove skin, etc.).
In the abattoir itself, even though it can be very clean, there are many potential sources
of contamination of meat by microorganisms. These include the animal hide (skin), hair,
hooves and soil adhering thereto, the contents of the gastrointestinal tract, etc.
In addition, there are some other external sources in abattoir as the equipment and
tools used in the processes, the hands and clothing of personnel as well as the
environment (air and water) may also contaminate the meat with bacteria.
At the butcher’s and at home, meat may get contaminated too.
The microorganisms causing spoilage in fresh and frozen meat and meat products
include:
• Pseudomonas spp.
• Alcaligenes spp.
• Carnobacterium spp.
• Moraxella spp.
• Micrococcus spp.
• Leuconostoc spp.

The pathogens of concern in fresh and frozen meat and meat products include:
• Salmonella spp.
• Escherichia coli O157:H7 and other enterohaemorrhagic E. coli (EHEC).
• Listeria monocytogenes.
• Staphylococcus aureus.
• Yersinia enterocolitica.
• Campylobacter spp.
• Clostridium perfringens and the potential for Cl. botulinum in cured hams and
sausages (embutido).
Microbial contamination in POULTRY

The skin of live birds may contain numbers of bacteria and could also be derived from
the feet, feathers and feces.

Contamination of the skin and the lining of the body cavity occurs during washing,
plucking and evisceration.
The process of sticking and bleeding can also introduce contamination.
Bacteria in raw meat

Meat has potential risk to carry foodborne pathogens that can cause illness and lead to
food safety problems.
• These pathogenic bacteria are able to invade our bodies or produce toxins to
cause illness.
They cannot be seen or smelled on the meat, but can generally be killed by normal
cooking conditions.
• Example: cooking to a core (inside) temperature of at least 75ºC instantaneously
or other effective time/temperature combinations.
Pathogenic bacteria may need to complete with other bacterial flora (e.g. spoilage
bacteria) for their growth on the meat.
Certain pathogenic bacteria such as Staphylococcus aureus are relatively poor

competitors and may be outgrown by other flora.
• They are not going to do anything to avoid the growth of other bacteria,
however, they are dangerous for public health because of their toxins, since they
cannot be destroyed.
Spoilage bacteria will cause food to deteriorate or lose quality by developing a bad odor
or feeling sticky on the outside of the meat, signs that consumers would normally notice.
These spoilage bacteria are normally not harmful, however, when consumed in very
large numbers, they can cause gastrointestinal disturbance.
• Consumers should throw away the mat that sows any signs of food spoilage.
Indications of spoilage in meat are:
• Surface slime (CFU >106/cm2): may be caused by species Pseudomonas,
Leuconostoc, Bacillus, etc.
o This means that the higher the number, the more possibilities there are
of being pathogens.
• Changes in the colour of meat pigments: the production of oxidizing agents
causes changes in red colour of meat to shades of green, brown and grey as
result of the production of oxidizing compound (e.g.: hydrogen peroxides and
hydrogen sulfide) by Lactobacillus.
• Changes in fats: the oxidation of unsaturated fats takes place chemically in air
and may be catalyzed by light and copper (e.g.: oxidative rancidity).
• Phosphorescence: this rather uncommon defect caused by phosphorescent or
luminous bacteria (e.g.: Photobacterium spp. grows on the surface of meat).
• Various surface colours due to the pigmented bacteria: red spots (Serratia (not
pathogenic) or Pseudomonas). Other colours are blue, yellowish, green, brown,
etc.
Bacteria in cooked meat

As mentioned before, thorough cooking can generally destroy most bacteria on raw
meat, including pathogenic ones.

Nevertheless, if there are subsequent lapses in food safety practices, food poisoning
may still occur.
• To start with, raw meat may be contaminated with spores of certain pathogenic
bacteria (e.g.: Clostridium perfringens) and spores are not readily destroyed by
normal cooking temperature.
Heat of cooking can rather activate the spores to germinate and develop into vegetative
which can multiply rapidly in foods that are placed at ambient temperature for a long
period.
In addition, pathogenic bacteria may be introduced into the ready-to-eat cooked meat
through cross-contamination and multiply to larger amount as a result of time and
temperature abuse of the food, causing foodborne illness in consumers.
• Raw products should be on the bottom part of the fridge, and if not, it should be
isolated as best as possible.
Measures to reduce risks of food poisoning caused by bacteria

To prevent food poisoning, raw meat should be cooked thoroughly before consumption.
The ready-to-eat cooked meat should be discarded if it has been held at room
temperature for more than 4 hours.
• If the cooked meat is held at room temperature for less than 2 hours, it can be
refrigerated for final use later or used before the 4 hours limit is up.

Moreover, good hygienic practices should be observed. Hands, cutting boards, knives
and other utensils should be washed thoroughly after touching raw meat.
Measures should be adopted to the prevention of cross contamination between raw

meat and ready-to-eat foods including cooked meat, e.g. using one cutting board for
ready-to-eat foods and a separate one for raw meat.
Minced meat
It is easier to contaminate because they have a greater surface. They should be cooked
as soon as possible or if not, frozen.
It is more juicy than bigger pieces or cuts.
Not proper handling of the meat.
Microorganisms are spread all over the meat.
If the mincer/grinder is not properly clean:

• Rest of some other meat species may be left on the blades.

• These rests may contaminate the meat if proper cleaning and disinfection is not
carried out.
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Unit 19: Microbiology of fish, mollusks and crustaceans
Introduction
Fish: composition and nutritive value

In general, its composition is:
• 66-84% water → that is why we feel hungrier after an hour in comparison with
meat.
• 15-24% protein.
• 0,1-22% fat → that is why we classify them into white/blue fish o lean/fatty fish.
o Hake (merluza) and cod (bacalao) are lean fish.
• 1-2% carbohydrates.
• 0,8-2% minerals.
Fat fish: it has more than 5% of fat stored in the muscle, specially triglycerides.
Lean fish: it has fat stock in the liver and only 0,5-1,5% of fat in the muscular tissue.
There is different chemical composition in different parts of the fillet.

Nutritional value varies according to:
• The specie.
• The age: >age means more fat and less water.
• The season: more water and less protein and fat after spawn.
• Feeding.
• Sexual status: spawn.
• Anatomical region: the closer to the tail, the more protein content and the more
content of fat closer to the medial region.
Fish and fishery products (catch and aquaculture) shopping habits
• In Spain we eat fish at least once a week. Our consumption is higher than the
average in Europe. There are very few people that have never eaten fish.
• Mainly, we have fresh products, including alive ones.
• The shop where they sell fish or fresh fish is Fish Monger’s.

Per capita consumption of fish and fishery products (Live weight equivalent – kg
per capita/year/State member)
• We consume much more fish and fishery products than the average in Europe.
Seafood or Shellfish

Definition: invertebrate edible animals, marine or continental, fresh or conserved by
different authorized procedures (Spanish Food Code).
Shellfish are mainly categorized into crustaceans and mollusks.

• Crustaceans: they have crusts on top or a shell, which acts as an armor. Shellfish
have a pigment called “astaxanthin”, which on contact with heat turns into a
coral red colour.
o Decapods Macrura: prawn.
o Decapods Brachyuran: crab.
o Barnacles: barncacle.
• Mollusks: they are shellfish that have a hard inedible shell. They are classified
into three other subgroups: gastropods/univalves, bivalves and cephalopods.
o Bivalves: mussle.
o Univalves: bigaro.
o Cephalopods: squid.
Microorganisms in fish
Association of undesirable microorganisms with fish

Initial biota: endogenous microbial contamination (the initial microbial load of fish)
Originally internal tissues of alive and healthy fish are regarded as sterile. This is why it
is important to monitor the quality of the water.
Fish meat from unhealthy animals may contain microorganisms.
Fish and crustaceans: they are a true image or reflection of the marine environmental
biota they share their habitat with.
Mollusks live close to the coast line, which is the dirtier part of the sea.
• Like mussels (mejillones).
• We cannot consume them straight away because they have a filter which can
make them dirty from the storing of the dirt of the coast line. They must be
depurated and filtrated before considered clean for human consumption.

Factors that may influence:
• Fishing techniques: multihooks (palangre) and trawlers (arrastre).
o 90% of captured items are not fishery products.
o They destroy the ground.
• Fishing grounds: Subsaharian and Terranova. We cannot fish here because they
are trying to get recovered.
• Treatment of the captures: overcrowding. The bigger the amount of fish, the
higher possibilities for cross-contamination.
Depending on the pH, salt content, etc., different bacteria can grow:
• Fresh waters (not from the ocean): Alcaligenes and Aeromonas bacteria.
• Marine waters: Pseudomonas and Achromobacter.
Fish and crustaceans: mucus surface. We can find microorganisms here, that is why they
can cross-contaminate between fished.

Mollusks: varies according to the quality of the capture water, washing, etc.
Pathogenic microorganisms associated with fish

Microbial contamination: Fish
Microbiological Spoilage Live fish is normally considered to be sterile, but
microorganisms are found in varying numbers on all the outer surfaces (skin and gills
(branquias)) and in the digestive tract of live and newly caught fish.
When the aquatic system is contaminated with pathogenic Vibrio, these bacteria
become part of the shellfish microflora and when consumed with the fish the hazardous
pathogenic Vibrio causes life threatening food-borne infections and poses a
considerable public health threat.
Vibrios are common inhabitants of marine and estuarine environments. They are very
dangerous.
• We can see that Vibrios are there because we can see that the skin of the fish
has stains that look like blood.
Ships/boats, boxes, tools and personnel are sources of external fish contamination
(bacteria) during the processing and handling of fish and fish products.
Self-depuration of sea waters is not fast enough to destroy all pathogenic biota that
terrestrial waters vehicles to the sea. It means that microorganisms are going to live
longer enough to contaminate fish, mollusks and crustaceans.

For this reason, mollusks MUST be depurated, especially when mollusks are supposed
to be eaten raw, such and oysters (ostras), clams (almejas) or cockles (berberecho) →
Spanish legislation.
The depuration system could be:

• Passive (natural): the mollusks are kept alive during 24-48 hours in
uncontaminated marine water.
• Active (artificial): the system is more or less the same, the difference is that
water is previously treated with Cl, O3 or ultraviolet light.
o We are going to be faster because the water is sanitized/treated.
Microbial contamination: Crustaceans

They are really similar to the fish ones:
• Crabs: Achromobacter (they are psychrophilic so they can grow in refrigeration
temperatures) prevails over temporary increase of Pseudomonas (they are
everywhere).
• Prawns: Acrhomobacter and Pseudomonas. When the temperature raises,
Proteus (responsible for ammoniac smell) occurs.
• Fish and crustaceans: mucus surface.
• Mollusks: varies according to the quality of the capture water, washing, etc.

Microbial contamination: Mollusks
Determination of pH → Quality: the lower the pH, the lower quality.
• 6,5-5,9 = good/optimum.
• 5,7-5,5 = spoiled.
•  5,2 = soured and putrefied.
Oysters:
• At 0ºC or superior: if we do not want Pseudomonas and Achromobacter.
• When the temperature raises Mesophilic Aerobic Bacteria and Coliforms occur.
• Psychrophilic yeasts are present in high quantities: they make a pinky colour in
fish.
Fish is specially perishable due to:

Its chemical composition: carbohydrates content are nearly null and when the fish dies,
its post mortem changes are less evident that those on the meat.
• Crustaceans in general present a low percentage of carbohydrates, slightly
greater than in fish, but really insignificant values.
• Mollusks are different, they have greater values: fermentation.
• Even though there are nearly no carbohydrates, there are still proteins and fats,
which are going to be responsible of the fermentation (mainly in mollusks).
Psychrophilic biota: may be initially present in fish. Thus, under refrigeration conditions,
the microorganisms may be able to develop. This happens because of the TMAO present
in seawater fish.

All seawater fish examined contains trimethylamine oxide (TMAO). Increased TMAO
levels in tissues are considered to be characteristic of marine fish spoilage because it
stimulates the grow of anaerobic bacteria.
pH = 6,2 to 6,6-6,9. It is connected with the chemical composition.
Microbiological spoilage
Fish spoilage is a complex process, in which physical (easy peeling of the skin), chemical
(fermentation) and microbiological (low pH) mechanisms are implicated.
Many spoilage producing bacteria and fungi are often isolated from fresh and spoiled
fish and other sea foods. However, they cause problems during their shelf-life.
We can see a quick degradation of lipids in fatty fish, which produces rancid odors.
In addition, marine fish and some freshwater fish contain trimethylamine oxide that is
degraded by several spoilage bacteria to trimethylamine (TMA), the compound
responsible for fishy off odors.
Microbiological criteria

Regulation (CE) nº 1441/2007:
• Chapter 1. Food safety criteria (pathogens need to be totally absent) (Salmonella
and E. coli): crustaceans and cooked mollusks, live bivalve mollusks and live
echinoderms, tunicates and gastropods.
• Chapter 2. Process hygiene criteria (good practices = hygienic) (E. coli and
Coagulase positive staphylococci): shelled and shucked products of cooked
crustaceans and molluscan shellfish.

Tema 20: Microbiology of milk and dairy products
Definitions
According to General standard for the use of dairy terms (Codex Stan 206-1999):
• Milk.- milk is the normal mammary secretion of milking animals obtained from
one or more milking without either addition to it or extraction from it, intended
for consumption as liquid milk or for further processing.
o If no further information is given, it means the produce of the milking of
one or more cows.
• Milk product.- Milk products is a product obtained by any processing of milk,
which may contain food additives, and other ingredients functionally necessary
for the processing.

Chemical composition
It is important to stir the milk while sampling, because if not the milk will start to
separate is different layers.
The milk is one of the most nutritive and balanced food. However, is lack of iron and
vitamin C.
• Proteins:
o 3.29% easily digested.
o Types: casein (82% of the total protein content) + protein from the whey
(18%).
• Fats:
o 3,34% saturated fats prevail.
o 13,6 mg/100 g of cholesterol.
• Carbohydrates:
o 4,46%.
o The most important one is lactose.
▪ Positive: it enables the absorption of calcium.
▪ Negative: intolerance, which goes hand in hand with the non-
absorption of calcium.
• Water: 88%.
• Vitamins:
o Fat-soluble vitamins:
▪ Rich in vitamin A.
▪ Poor in vitamins D, E and K.
▪ Heat treatments reduce the vitamin content.
▪ If we remove the fat we may remove these vitamins as well.
o Water-soluble vitamins:
▪ Content in vitamin B2 and B12.
▪ Poor in vitamin C.
• Minerals: all known minerals and oligoelements are present in milk. Specially
stands out its calcium, iodine, zinc and sodium content.
The species of dairy animal, its breed, age and diet, along with the stage of lactation,
parity (number of parturitions), farming system, physical environment and season
influence the colour, flavor and composition of milk and allow the production of a
variety of milk products:
• Cow milk.
• Buffalo milk.
• Sheep milk.
• Goat milk.
• Camel milk.
Milk and dairy products in the EU

In Spain 41% of the milk we consume is from drinking milk.
• Compared to the other countries from the EU, they do not drink as much milk as
a normal drink.
In the EU 31% of the milk consumed is in form of cow cheese.

Collection of cows by dairies in the EU
The biggest farm with the biggest amount of animals is Germany, followed by France.
In Spain, in comparison with the rest of Europe, we produce only 4,5% of cow milk.
Microorganisms in milk and dairy products
Initial biota: endogenous microbial contamination (initial microbial load of milk)

When animals are healthy the synthesized and secreted milk coming out from the udder
is regarded as sterile, or the number of bacteria is extremely reduced.
When lactating animals are unhealthy or affected by intra-mammary infections, the

causative agents will be present in the milk usually in high numbers, depending on the
severity of the infection:
• Brucellosis: caused by Brucella spp.
o It can cause abortion in pregnant women, as well as infertility.
• Tuberculosis: caused by Mycobacterium spp.
• Mamitis: caused by Staphylococcus spp.
o It is an inflammation of the udder (ubre).
It should be noted, however, that occasionally in mild or less severe cases of mastitis the
enumeration of pathogens in milk is not guaranteed.

Occasionally, intermittent secretion can occur form some intra-mammary infections
pathogens. Even in healthy lactating animals, unless milk is drawn aseptically from the
udder following a proper aseptic collection protocol, freshly milked raw milk contain
contaminating microorganisms at low to moderate levels.
These initial microbial concentrations can vary considerably among animals, but they
are typically in the range of several hundreds-few thousands of CFU/mL.
Initial biota: enxogenous microbial contamination (initial microbial load of milk)
There are many possible sources of microbiological contamination of the raw milk:
• Some of them are obvious sources of milk contamination at the immediate
environment of the milking parlor include the animal hide (skin) and the udder
skin surfaces.
• These skin surfaces may be contaminated with organisms from the bedding
material or dirt from the ground, the air (dust), the milker’s hands (human
contamination → Salmonella and Staphylococcus), the milking equipment, the
animal feed and the water supply.
Measures to avoid microbial contamination (initial microbial load of milk)

To avoid milk contamination is simple by adopting basic rules of clean milk production.
Both endogenous and exogenous contamination are correlated.
Some measures or actions could be taken into account:

• To prevent endogenous contamination it is recommended to monitor or control
the health status of the milking animals.
• To prevent exogenous contamination it is recommended to monitor or control
the hygiene and sanitary aspects of the equipment, personnel, facilities, etc.
Origin of microbial contamination

Many of the microbiological hazards associated with dairy products such as butter,
cheese and yoghurt are derived from the raw milk.
Initial or normal (endogenous): it comes from udder and nipples.

• If the number of bacteria is <1000 CFU/ml it means that animals are healthy.
• Most common bacteria in this case are micrococci, streptococci and lactobacilli.
Pathogens (exogenous and endogenous): the most important ones, because they can
cause serious disease, especially children, pregnant women, elderly and compromised
individuals.
Contaminants (exogenous): feces and hide (coliforms), bedding, ground, feeding (fungi
spores and Salmonella spp.), milking equipment (Algaligenes and Pseudomonas) and
miler machine (Salmonella spp. and Staphylococcus spp.) and insects.

Pathogens in milk and dairy products
Hygienic milk production practices, proper handling and storage ok milk, and mandatory
heat-treatments have decreased the threat of milk-borne 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.
The next pathogens are only involved in milk:

• Brucellosis: caused by Brucella spp.

• Tuberculosis: caused by Mycobacterium spp.
• Mamitis: caused by Staphylococcus spp.
Spoilage of milk and dairy products

The highly nutritious nature of dairy products makes them especially good media for the
growth of microorganisms. Its water content and pH also favors spoilage of milk.
The spoilage of milk products is an enormous economic problem worldwide. The

microbial load and incidence of the bacterial pathogens in foods are indicators of food
quality.
Spoilage occurs when microorganisms degrade the carbohydrates, protein, fats of milk
and produce deleterious end products.
The signals to know that it is spoiled are:

• Sour or acid formation.
• Gas production: occurs by spontaneous fermentation.
• Proteolysis.
• Ropiness.
• Changes in milk fat: rancidity.
• Alkali production.
Sour or acid formation

Sour smell and flavor and coagulation. It is because of the fermentable sugar (lactose)
present in milk, an acid formation by bacteria occurs in raw milk.

Acid formation cause 1st sour flavor of milk, then coagulation of milk gives solid jellylike
curs or a weaker curd (coágulo) that releases clear whey.
At room temperature: takes place acid lactic fermentation in raw milk.
At 10-37ºC: coliforms and micrococci causes the souring of milk.
At >37ºC: E. faecalis may produce more acid.
Gas production
The responsible microorganisms are Coliforms, Clostridium, Bacillus and yeasts.
Gas production is usually accompanied by acid formation.

The chief gas formers are:
• Clostridium, gas forming Bacillus species yield H and CO2.
• Yeast, propionics and hetero-fermentative lactis produce only CO2.
The production of gas in milk is evidenced by:

• The foam at the top of liquid milk.
• Gas bubbles caught in the curd:
o By floating curd containing gas bubbles.
o By a ripping of the curs by rapid gas production, causing stormy
fermentation of milk.
Milk heated at pasteurizing temperature or above:

• The chief acid formers will be killed.
• The spores of Clostridium & Bacillus species will survive, and gas formation by
the spore formers may take place.
Proteolysis
The hydrolysis of milk proteins by organisms is produced bitter flavor in taste due to
some of the peptides released.
Actively proteolytic bacteria found in the species of: Alcaligenes, Bacillus, Clostridium
and Pseudomonas.
• Bacillus and Clostridium are spore-formers, so we have to be very cautious.
It is favored by:
• Storage at a low temperature.
• By the destruction of lactis and other acid formers by heat.
• By the destruction of formed acid in mil by molds and yeasts.
• The neutralization of acids by products of other organisms.
Ropiness
It is caused by slimy capsular material from the cells, usually gums and mucins and
develops best at low temperatures.
By providing an adequate heat-treatment of milk, kills all kind of bacteria and prevent
the ropiness.
Main types of bacterial ropiness are Leuconostoc and Micrococcus.
Changes in milk fat
Milk fat may be decomposed by various bacteria, yeasts and molds.
Changes that occur in milk fat are:

• Oxidation of the unsaturated fatty acids: yields, aldehydes, acids, ketones and
results in tallowy odors and taste.
• Hydrolysis of the butterfat to fatty acids and glycerol by the enzyme lipase.
• Combined oxidation and hydrolysis to produce rancidity.
Species of lipase forming bacteria are Proteus, Pseudomonas, molds and yeasts.

Alkali production
Alkali formers are Alcaligenes, Micrococcus and Pseudomonas.
The group of alkali formers cause alkaline reaction in milk without any evidence of
proteolysis.
• This reaction may result from the formation of ammonia, as from urea, as from
organic seeds (citric acid).
Most of these bacteria grow from moderate to low temperatures, and many can survive
pasteurization.
Heat – treatments of milk

Pasteurization: 63ºC during 30 minutes (low pasteurization) or 72-75ºC during 14
seconds (high pasteurization).
• This treatment destroys pathogens: M. tuberculosis, Coxiella burnetti,
Salmonella.
o They should be negative after doing the pasteurization.
• This treatment destroys nearly 99,5% of banal biota:
o Thermoduric biota: non-spore-forming bacteria (thermo-resistant
bacteria and Micrococcus) and spore-forming bacteria (Bacillus and
Clostridium).
o Re-contamination biota: it can get re-contaminated by Coliforms.
UHT (Ultra High Temperature) Milk: 135ºC-150ºC during 2-10 seconds.

• It destroys microorganisms and their resistance structures.
• Microbiological control: preincubation tests.

Sterilized milk: 110ºC during 20 minutes.
• It destroys microorganisms and their resistance structures.
• Microbiological control: preincubation tests.
*
Microbiological criteria
Regulation (CE) nº 1441/2007:
• Chapter 1.- Food safety criteria (Salmonella and Staphylococcal entero-toxins):
for all types of milk.
• Chapter 2.- Process hygiene criteria (Enterobacteriaceae and Coagulase positive
staphylococci): for all types of milk.

Unit 21: Microbiology of eggs and egg-products
Definitions
Eggs with shell (excluding shield, hard-boiled and incubated eggs) are the spheroid body
obtained from breeding birds which are fit for direct human consumption or for egg
products preparation.
• If no further information is given, it means eggs are obtained from hens (Gallus
gallus).
Egg products means all, or a portion of, the contents found inside eggs separated from
the shell, with or without added ingredients, intended for human consumption. An egg
product may contain food ingredients and food additives intended to give them certain
characteristics and/or to preserve their quality.
Chemical composition
It varies depending on the part of the egg:
• Whole egg: it is 100% of its weight.

o 65,6% is from water.
o 11,8% is from protein.
o 11% is from fat.
o 11,7% is from ash.
• White: it is 58% of the whole weight of the egg.
o 88% is from water.
o 11% is from protein.
o 0,2% is from fat.
o 0,8% is from ash.
• Yolk: it is 38% of the whole weight of the egg.
o 48% is from water.
o 17,5% is from protein.
o 32,5% is from fat → this is why is tastier.
o 2% is from ash.
*Eggs commercial classification

In the EU, eggs for sale on market are classified according to their quality as follows:
• Class A eggs: extra class A or fresh eggs → for human consumption.
• Class B eggs: for industrial processing.
When placed on the market, class A eggs are further classified as of their weight into
four classes and labeled with XL (73 gr or more), L (63-73 gr), M (53-63 gr) and S (<53
gr).

Microorganisms in eggs and egg-products
Initial biota: endogenous microbial contamination (initial microbial load of eggs)
When breeding birds are healthy, a freshly laid egg is usually free of bacteria (sterile)
on the inside and is well protected from bacteria by the shell, shell membranes and
several chemical substances in the egg white.
The shell of eggs is a biological protecting structure in fact, it is the egg’s first line of
defense against bacterial contamination.
• This grainy structure has approximately 17.000 tiny pores and is made almost
entirely of calcium carbonate.
• It is semipermeable, which means that air and moisture can pass through its
pores.
• It also has a thin outermost protective coating called a cuticle.

o By blocking the pores, the cuticle helps to preserve freshness and prevent
microbial contamination of the contents.
o By using UV light, the cuticle can be seen.
o When eggs are not fresh, this layer is going to start to disappear.
The viscosity of the egg white together with the presence of natural inhibitors, inhibits
the growth of microorganisms.
• It contains avidin, a biotin inhibitor.
• It also contains lysozymes which affect Gram +.
• The high egg white pH (9,1-9,7) also retards the growth of bacteria.
When breeding birds are unhealthy or affected by ovary infections, the causative
agents will be present in the milk usually in high numbers, depending on the severity of
the infection: Salmonella spp.
Initial biota: exogenous microbial contamination (initial microbial load of eggs)

There are many possible sources of microbiological contamination of the egg. Most of
them are external sources of contamination, like from fecal matter from the digestive
tracts (intestine) of hens:
• By the cage or nest, because of fecal matter.
• By wash water if the eggs are washed. It may contain fecal bacteria.
• By improper handling.
• By the material in which the eggs are packed.
The total number of microorganisms per shell of a hen’s egg has been reported to range
from 102-107 with a mean of 105.
The most common microorganisms found on the external surface of shell, among
others, are:
• Pseudomonas.
• Salmonella.
• Enterobacteriaceae.
The great variety of microorganisms depends on different factors, inter alia, the
operating conditions:
• Small farm (the more manual operations and higher risk of contamination) Vs.
semi-automatic farms of laying hens individually caged (risk of contamination
much lower).
Salmonella and eggs
Endogenous origin (Vertical transmission)
When breeding birds suffer from infection of ovary or oviduct, internal contamination

of intact shell eggs occurs during its formation. From the hen (it is unhealthy) to the egg.
Exogenous origin (Horizontal transmission)

At oviposition, the formed shell egg comes into contact with the opening of the digestive
tract at the level of the cloaca, where the propensity for fecal contamination of the
external eggshell surface is high.
Cleanliness of the environment at the time of lay impact the microbial load greatly,
higher levels on contaminated or dirty floor eggs than on eggs laid on clean nesting
material.
Initially the eggs where healthy, but by being in contact with the dirty floor, now they
are unhealthy.

Humans
Fresh eggs, even those with clean, uncracked shells, may contain Salmonella, about 0,2-
5% of eggs may be contaminated with this pathogen.
Salmonella resides in the intestinal tracts of birds and can be transmitted to humans
when they consume contaminated eggs.
Salmonella can be present on egg shells and the interior of normal-looking eggs.
If eggs are not properly handled or are eaten raw or lightly cooked, Salmonella can still
survive and may cause food-borne illnesses.
A matter of Public Health in the EU and the USA

Spain:
• Data from the Public Administration reports that 3-4% of eggs are contaminated.
• Data from other sources report that 0,2% of eggs are contaminated by
Salmonella.
• Scientific researches claim that:
o External: 1% Salmonella enteriditis and 12% other enterobacteria.
o Internal: 7% other enterobacteria.

The USA:
• 1/10.000 eggs are contaminated with endogenous contamination.
• In August of 2010, 380 millions of eggs were recalled from the market.
Spoilage of eggs and egg products: Microbial changes

The steps for microorganisms penetrating and spoiling eggs are:
• Contaminate the shell.
• Penetrate the pores of the shell to the shell membranes.
• Grow through the shell membranes to reach the white.
• Grow in the albumen (egg white).
Bacteria
Green rots: green whites or green rots are usually caused by members of the
Pseudomonas group of organisms (Pseudomonas fluorescens), which are commonly
found on the surface of the egg shell and grows at 0ºC.
• Certain species of Pseudomonas multiply in the egg white in early stages, from
which they synthesize a characteristic fluorescent green pigment (which can be
seen under UV light).
• During later stages, yolk disintegrates and blend with the white.
• When open, odor is lacking or is fruity-sweetish.
Colourless rots: they are usually caused by members of the Pseudomonas,

Acinetobacter, Alcaligenes groups of organisms and certain coliform bacteria.
• It is detected by candling. We are going to see inside shadows because of the
growing of the bacteria, as well as the membranes being different.

• Yolk in later stages, disintegrates or shows a white incrustation.
• Odor varied from a scarcely detectable to fruity to highly offensive.
Black rots: they are caused by bacteria from the Aeromonas, Proteus and Pseudomonas
groups. They are generally opaque (grey or black colour) when viewed before the
candling light and are characterized by the presence of gas.
• Since the shell membranes apparently become impermeable, the pressure
created within the egg is often sufficient to burst the shell and scatter the egg’s
contents.
• When broken, the contents have a muddy brown appearance and give off a
repulsive, putrid odor caused by SH2.
Pink rots: they are caused by bacteria from the Pseudomonas group. They are generally
rare to see.
• They resemble colourless rots except for the pinkish precipitate on the yolk and
a pink colour in the egg white.
• Often they are considered as an advanced stage of green rots.
Red rots: they are caused by bacteria from the Serratia group. They are the most
infrequent occurring one.

• They give off a mild and not offensive odor.
Molds
Spoilage of eggs by fungi goes through stages of mold growth. The stages give the
defects their names:
• Pin spots molding: it is the very early mold growth. Small, compact colonies of
molds appear in the shell and usually just inside it. Colour varies with the kind of
mold:
o Penicillium species cause yellow or blue or green spots inside the shell.
o Clodosporium species give dark green or black spots.
o Sporotrichum species give pink spots.
• Surface molding: it is supported by an storage in a very humid environment.
• Fungal rotting: it is the final stage of spoilage. Mycelium of the mold has grown
through the pores and cracks in the egg.
o Jellying of egg white is seen.
o The hyphae of the mold may weaken the yolk membrane enough to
cause its rupture.
o After this, growth is stimulated by food release from the yolk.
Commission regulation (EC) No. 1441/2007

Chapter 1.- Food safety criteria: Salmonella.

Chapter 2.- Process hygiene criteria: Enterobacteriaceae.

Unit 22: Microbiology of fruits and vegetables
Definitions
Vegetables: they refer to a plant cultivates for its edible part(s), or refer to the edible
parts of a plant. These include the stems and stalks, roots, tubers, bulbs and leaves.
Fruits: they are a subgroup of vegetables consisting of the seeds and surrounded by
pulpy tissues.
Microorganisms in fruits and vegetables

We are going to talk about mainly spoilage microorganisms that are going to decay the
product.
Initial biota: endogenous microbial contamination (initial microbial load)

When fruits and vegetables are healthy, we are going to consider that their internal
tissues are usually free of bacteria (sterile).

When fruits and vegetables are unhealthy or affected by any infection, the responsible
microorganisms, the causative agent, will be present at that moment in the fruits and
vegetables.
• It is considered as a natural status, because they can suffer diseases.

Potential exogenous sources of contamination of fruits and vegetables are boxes,
baskets, bags or means of transport. Before, the product could have been perfectly fine.
Effects of fruits and vegetables processing on microorganisms

Washing: it can eliminate up to 90% of the superficial biota. This is why it is always
important to clean them. If we can use sanitizer or chlorine, better.
Blanching/”boiling”: fruits and vegetables are immersed in water at 86-98ºC during a

few minutes. The effects of blanching are:
• Enzymatic inactivation: we will affect the enzymatic activity.
• Reduction (up to 99%) of the microbiological load.
• Colour green highlight: the product will have a brighter green.
• Softening of the leaves of vegetables: tissues will become softer and easier to
manipulate in the cooking procedure.
• Removal of the air of tissues: this air could be responsible of contamination.

Spoilage: microbiological changes
Bacteria
Bacterial soft rots (vegetables): they are usually caused by members of the Erwinia
group of organisms (specially Erwinia carotovora), which are pectin fermenting bacteria
(hydrolysis of pectins).
• Certain species of Pseudomonas, Bacillus and Clostridium cause this common
type of bacterial spoilage as well.
• Softening can also be caused by endogenous enzymes.
• The spoiled product present a wet, soft aspect with an unpleasant odor, because
there is a putrefaction happening.
Sliminess or souring (vegetables): it is caused by saprophytic bacteria in piled, wet,

heasting vegetables when temperatures are optimal.
Fungi
Grey mold rot (fruits & vegetables): it is caused by species of Botrytis. The mycelium is
grey in colour. It is favored by high humidity and a warm temperature.
• This happens a lot in strawberries.
Rhizopus soft rot (fruits & vegetables): it is caused by species of Rhizopus. The fly

Drosophila melanogaster vehicles spores and pieces of mycelium which proliferates in
cracks and produces lesions.
• The fly is going to be a carrier of the mycelium from one product to another.
Geotrichum candidum Acid rot (vegetables): widely distributed on the ground (soil).
Again Drosophila melanogaster plays a vital role on this disease by moving pieces of
mycelium from one vegetable to another.
Black mold rot (fruits): Aspergillus niger is the main responsible. It grows nearly
everywhere, even in equipment that have to do with organic matter.
Green mold rot (fruits): Cladosporium spp. is the main responsible. It is very common
to find it in oranges. Its growth is more probable if the fruit is very ripen.
Alternaria rot (fruits): caused by Alternaria spp.
Penicillium mildew: caused by Penicillium spp.
Commission regulation (EC) No. 1441/2007

Chapter 1.- Food safety criteria:
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Chapter 2.- Process hygiene criteria:

Practical case
Concept of food infection, food intoxication and food toxicoinfection
The microbial mechanisms of action are:

• Microorganisms cause their toxic effects through metabolites produced during
their growth in food before ingestion → food intoxication → disease caused by
the toxins, so you do not need to ingest the microorganism.
• Microorganisms need to be ingested with food → food infection.
• Microorganisms need to be ingested with food and then release a toxin in the
digestive tract → food toxicoinfection.
Colloquially:

• Food intoxication is a disease contracted by the food consumption after a short
period of incubation (2-10h) and gastroenteric syndrome.
• In the food infection the food is the vehicle of microorganisms that later multiply
in the human intestine. It takes several days for it to be shown.
• Food toxicoinfection is both of the above, but it also takes several days to be
shown.
Salmonella
The results from DNA relatedness studies have indicated that all Salmonella serotypes
are placed to two bacterial species that can cause illness in human and animals, they are
Salmonella enterica and Salmonella bongori.
Characteristics of Salmonella
Its growth temperature ranges from 2 to 48ºC with an optimum of 37ºC.
Salmonella spp. are generally inhibited in the presence of 3% NaCl.
They are heat sensitive and readily destroyed by pasteurization.
Its growth pH ranges from 4.1 to 9 with an optimum of 6.5.
They produce gas and acid from glucose, but they do not ferment lactose.
They can be described on the basis of their somatic (O), flagellar (H), and capsular (Vi)
antigens.
Its minimum water activity is 0.93-0.95. it is not present in food that have less water
activity than that.
Salmonellosis syndrome
They are responsible for different foodborne diseases:
• Gastroenteritis: it is generally self-limiting among health people with intact
immune system → you do not need any medicine in order to pass it.
• Systemic disease (typhoid fever): it has a long incubation period and a lower
infectious dose than the necessary for gastroenteritis.
o It is more serious and has a higher mortality rate than non typhoidal
salmonellosis.
Gastroenteritis
It is predominantly associated with enteric strains: S. enteritidis, S. typhimurium and S.
Virchow.
• Other Salmonella strains (S. Broenden, S. Eastburne, S. Napoli, S. Oranienburg,
S. Heidelberg, S. Newport and S. Dublin) serotypes can also cause gastroenteritis.
The infection begins with 12-36 hours after consumption of food containing viable cells
of Salmonella sufficiently high to pass the first-line host defense (stomach acidity) and
colonize in the gastrointestinal tract.
The typical infective dose can range from 105 to 109 viable cells per gram of food, but
illness may occur with and infective dose as low as 10-100 viable cells per gram when
bacteria are protected in high fat or protein foods.

Its symptoms are:
• Foul-smelling watery diarrhea with or without blood and vomiting.
• Dehydration and electrolyte imbalance may occur as a result of diarrheal and
vomiting.
• This is followed by abdominal pain, fever, and headache, and then may follow
with nausea, chill, prostration, muscular weakness, faintness and thirst.
• In most cases, the disease is self-limiting and symptoms usually last after 2-7
days.
• Mortality rate is low (0.1-4.1%), but this can lead to death in the very young, the
elderly, and the immunocompromised people if not treated promptly.
o The immune system does not work properly in very young and elderly
people.
• Pathogenicity islands are specific regions of chromosomic DNA that include
several virulence genes.
Problems of eggs and egg products

The increase in the incidence of outbreaks by S. enteritidis is related to eggs and by-
products.
It is the most frequent serovar in eggs in Spain and the predominant one in England
and Wales.
The organism has been detected inside the egg and ovaries of layers.
The possible ways of entry into the eggs are:

• Trans-ovarial, translocation from peritoneum to yolk or oviduct.
• Penetration of the shell when the egg passes through the cloaca.
• Washing and manipulating.
Prevention of Salmonellosis
Avoid contamination of food (human and animal).
Destroy it by using heat (cooking or pasteurization).
Prevent its multiplication by cooling.
The conditions for and outbreak are:

• Food must contain salmonella.
• The number must be high.
• Viable microorganisms should be ingested.
Listeria monocitogenes
It grows from 0 to 45ºC with an optimum from 30 to 37ºC.

Growth is inhibited with pH values below 5.5 and it shows growth in pH ranging from
5.5 to 9.5.
It is able to grow in 10% NaCl, and survives in 20%.
It grows optimally at a water activity level 0.97.
It commonly associates with surface waters, decaying vegetation, soils, sewage, silage,
seafood, meat products, poultry, eggs, sausage, decaying vegetables, raw vegetables
(fresh cut vegetables, potatoes, tomatoes, and lettuce), raw and pasteurized milk, milk
products, and dairy-processing environment (like ice cream).
Contamination sources are food workers, air, food-processing environment, food

contact surfaces, chilling and cutting rooms, hands, faces and equipment.
Listeriosis symptoms
L. monocytogenes is able to invade phagocytic and non phagocytic cells, survive, and
replicate inside them → Infection
The listeriosis appears within 1-7 days following ingestion of contaminated food with
viable L. monocytogenes cells.
The symptoms are: slight fever, abdominal pain, nausea, vomiting, chills, muscle aches,
headache and sometimes diarrhea.
• The symptoms in pregnant women, very young, elderly and the
immunocompromised people can vary from mild to meningitis.

• In pregnant women, symptoms are influenza-like illness (like a flue) with fever,
headache, bacteremia, neonatal meningitis and, occasionally gastrointestinal
disorders.
o This may associate with transplacental fetal infection that may result in
abortion.
• In non pregnant adults is usually characterized by septicemia, meningitis and
endocarditis.
The duration ranges from days to a year.
The infective dose of viable cells is more than 100 cells per gram.
It has a mortality rate about 34%
Listeriosis prevention and control

Sanitizers (acid anionic detergents, quaternary ammonium compounds, iodine,
peroxide and chlorine) reduce L. monocytogenes populations by a minimum of 99,999%.
L. monocytogenes can attach to processing surfaces where they may form biofilms. Cells
present in biofilms increase resistance to sanitizers.

• Biofilm: they can produce a wall in which they can become resistant to sanitizers.
It is more or less impossible to destroy with a simple cleaning, it is needed a
mechanical cleaning and specific sanitizers.
The general recommendations to reduce the risk of listeriosis are:

• Avoid raw/unpasteurized milk.
• Do not consume milk products made from raw milk.
• Keep raw and cooked foods separate at all times.
• Wash hands, cutting boards, knives and all food contact surfaces after handling
uncooked foods.
• Thoroughly cook all raw foods of animal and fish to an internal temperature of
71ºC and poultry to 82ºC.
• Keep hot food hot (above 60ºC) and cold food cold (at or below 4ºC).
• Reheat all left over and ready-to-eat foods before eating.
• Do not keep foods at room temperature for longer than 2h.
• Refrigerate small portions of food so that they chill rapidly and evenly.
• Consume perishable and ready-to-eat foods as soon as possible.
Pathogenic Escherichia coli (enterotoxigenic or enteropathogenic)

Toxigenic responds to a toxiinfection because it releases toxins, whereas pathogenic is
an infection.
Humans are the principal reservoir of E. coli strains that cause human diarrhea.

Fecal contaminated water and food handlers are most important sources of ETEC/EPEC.
Vegetables, potato, salad, mold-ripened soft cheese and water are also sources.
Fairly high numbers of viable cells (108-1010 cells per gram) should be ingested together
with foods for developing the symptoms within 12-36h.
Diarrhea usually follows by abdominal pain, malaise and nausea. ETEC/EPEC foodborne
disease can last for 1-3 days.
• They don’t cause fevers.
Campylobacter jejuni (campylobacteriosis)

All Campylobacter spp. grow at 37ºC.
• The optimum growth temperatures of C. jejuni ranges from 42 to 45ºC.

• They cannot grow at and below 30ºC, and cannot survive at cooking or
pasteurization temperature.
They are more sensitive to adverse conditions such as drying, oxygen (in air), NaCl
(above 2.5%), low pH (below 5.5), disinfectants, freezing, gamma radiation and heat
(pasteurization).
• Campylobacter spp. are rapidly killed by gastric acids unless protected by food
materials.
• They survive well in refrigerated foods.
They require 3-15% O2 and 3-5% CO2 for growth.
Campylobacter association with foods

Surface water, vegetables, meats, poultry, eggs, clams and mushrooms easily get
contaminated by Campylobacter spp. from fecal material of animals, infected humans
and contaminated water.
Outbreak of Campylobacter most often associates with unpasteurized dairy products,

contaminated water and poultry.
Disease and symptoms

Campylobacter can cause either gastrointestinal (enteritis) or extra intestinal infections.
Most Campylobacter spp. are associated with gastrointestinal infection.
C. jejuni is responsible for 80-90% of gastrointestinal enteric infections (acute

enterocolitis).
Minimum infective dose of C. jejuni ingested together with food may be only 10 4 viable
cells per gram.
The symptoms appear within 1-7 days after consumption of a food containing viable
Campylobacter cells.
The illness starts with more severe abdominal pain (differs than salmonellosis),
sometimes mimic acute appendicitis that can result in unnecessary surgery. The other
symptoms are headache, fever, profuse diarrhea, chills, depression and weight loss.
• Vomiting is a less common feature.
Prevention and control

Control of Campylobacter contamination on the farm and from pets.
Adequate refrigeration of poultry and meat from processing to preparation and
separation of raw and cooked foods.
Individuals who have diarrhea symptoms should not work as food handlers.
Additional precautions should be exercised in kitchens preparing food for persons a high
risk of infection, such as infants, the elderly and the immunosuppressed.
Cook poultry products, meat and seafood thoroughly (no longer pink) with inside
temperature to 77 or 82ºC.

Prevent cross-contamination in the kitchen with the use of separate cutting boards for
foods of animal origin and other foods.
Avoid consuming unpasteurized milk and untreated surface water.
Make sure that persons with diarrhea, especially children, wash their hands carefully
with soap to reduce the risk of spreading the infection.
Separate raw foods from cooked foods.
Refrigerate foods, including left over cooked foods as soon as possible.
Staphylococcus aureus
Staphylococcal food poisoning (SFP) is caused by enterotoxins that are produced by S,
aureus.
• Staphylococcal enterotoxins (SEs) are heat-stable exotoxins.
S. aureus grows at a temperature of 7-48ºC, with an optimum at 35ºC.
It grows at low aw, up to 0.86, and with 7.5% NaCl.
Growth pH ranges from 4.0 to 9.8 with an optimum of 6.5.
105 viable cells per gram of food is necessary to produce enough enterotoxin to cause
illness.

Association with food
Humans are main reservoir of S. aureus involved in food poisoning. Healthy people carry
the bacterium on their skin. About 30-50% of healthy people carry enterotoxin producer
S. aureus in their mouths, throats and nasal cavity.
• Infected wounds, cuts, sinuses, pimples, furuncles and carbuncles are abound
with S. aureus.
• It can also be present in air, soil, water, sewage, plant surfaces, meats, poultry
and dairy products.
Foods associated are handled extensively and temperature abused protein-rich foods,
meat, salami, bacon, salads (such as tuna, chicken and potato), sausages, sauces, meat-
filled sandwiches, fish and fish products, custard, milk, cheese, butter, pudding,
chocolate milk, ice cream and cream-filled bakery products (especially in cakes and
sauces).

SFP is characterized by a short incubation period, typically 30 min to 6h after ingestion
of food containing enterotoxin.
The most common complication in SFP is dehydration caused by diarrhea and vomiting.

Other symptoms are diarrhea (usually watery, may contain blood), abdominal pain,
headache, muscular cramps, sweating, chills, weak pulse and prostration. High fever
may also be observed. The illness is relatively mild and usually last only a few hours to 1
day.
It is self-limiting.

Wash hands and under finger nails vigorously with soap and water before handling and
preparing food.
Do not prepare food if nose or eye is infected.
Do not prepare or serve food for others if wounds or skin infections are present on
hands.
Keep kitchens and food-serving areas clean and sanitized.
If food storage is necessary for longer than 2h, keep hot food hot (> 60ºC) and cold foods
cold ( 7ºC).
Store cooked food in a wide, shallow container and refrigerate as soon as possible.

Clostridium botulinum
It produces seven types of serologically distinct botulism toxins (neurotoxin → because
they infect the neurological system) designated by the letters A through G.
• Single strain of C. botulinum produces one type of toxin.
pH ranges from 4.7 to 8.9 with an optimum being 6.5. It is sensitive to low pH (<4.7), low
aw (0.93) and moderately high salt (5.5% NaCl). Spores do not germinate in the presence
of nitrite (250 ppm).
The neurotoxins are heat labile and can be destroyed at 80ºC in 10 min or at boiling
temperature in 5 min.
If you do not treat it you will have a high rate of mortality.
Association with food

C. botulinum vegetative cells and spores are widely distributed. They are present in soils,
fresh water, sediments of streams, lakes and coastal waters and also in the intestinal
tracts of animals and fish.

Foods commonly associated with C. botulinum are fish, meats, chicken and infant foods
(especially honey). C. botulinum spores can contaminate fruits and vegetables,
particularly those in close contact with soil.
• It only happens in infants (2-3 years) because their intestinal system is not as
complete as ours (low pH and adverse conditions for the Clostridium, so it cannot
convert from a spore into a vegetative cell), they have a higher pH and the spore
can become as a vegetative cell and produce toxins and produces infant botulism
→ case of honey.
Low-acid canned foods may associate with botulism → canned tomatoes.

Initial symptoms of foodborne botulism appear within 18-36h after consumption of food
containing preformed neurotoxin.
• Toxins are absorbed in the upper part of small intestine and reach to blood
stream.
The earlier symptoms are gastrointestinal and generally include abdominal pain, nausea,
vomiting, diarrhea and constipation.
After the earlier symptoms we can have neurological symptoms, including visual
impairments (blurred or double vision, fixed and dilated pupils), loss of normal mouth
and throat functions, lack of muscle coordination, respiratory impairment, fatigue and
paralysis of muscles that spread to lungs and hearts (asphyxia).

The botulism lasts for 7 days. In severe cases, the mortality rate is usually high, 5-10%.
The neurotoxins are the most toxic substances.
• The lethal dose for an adult human is 1 ng/1kg of bodyweight.

Because botulism toxin is destroyed under high temperatures, home canned foods
should be boiled 10 min before consuming to ensure safety.
Commercial processors use 12D concept heat treatment → death of bacteria by heat.
Salting and smoking may be insufficient to inhibit C. botulinum.
Shelf-stable canned cured foods (such as meat and fish) are preserved by a combination
of thermal processing and added salt and nitrite.

Clostridium perfringens
The number of cases/outbreak is usually very high. Syndrome is frequently associated
with the Catering industry.
It is found in the intestine (human and animal) and soil.
They are anaerobic, sporulated and demanding in nutritional needs, optimum

temperature 45ºC, pH = 5-9.
It is required to eat foods that convey great number of vegetative cells (> 10 6).
Food most frequently involved is a great variety: beef stews (bacterial sporulation is
induced, cool spores germinate, which determines multiplication of the vegetative cell).
Preventive measures are: rapid cooling, maintenance of food at > 60ºC, reheating of
surplus food and hygienic measures of the staff.

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Microbiología de los Alimentos

2º Grado en Ciencia y Tecnología de los Alimentos

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The food contaminated with pathogenic microorganisms can have serious

Number of pathogen responsible of food poisoning (foodborne illnesses) have grown

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*The classification categories are:

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Food safety definitions:

Food is fundamental to human survival. Its quality, therefore, is of paramount

Reservados todos los derechos.

Quality management/department has recently played an important role in food

Substantial part of quality management are:

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Control of microbiological quality

Quality control system: Definition

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Quality control system: Functions

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Quality control system: Organization

Quality control system: Situation

The quality control department is recommended to hold as great authority as other

The quality control department is recommended not to be subordinated to the

Reservados todos los derechos.

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Meat is a very good source of various micronutrients:

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As warm-blooded animal naturally may carry bacteria such as enterobacteria in their

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Muscles from unhealthy animals may contain microorganisms.

Initial biota: exogenous microbial contamination

The degree of cleanliness of the animals depends on:

Reservados todos los derechos.

At the butcher’s and at home, meat may get contaminated too.

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Microbial contamination in POULTRY

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The process of sticking and bleeding can also introduce contamination.

Bacteria in raw meat

Certain pathogenic bacteria such as Staphylococcus aureus are relatively poor

Bacteria in cooked meat

Reservados todos los derechos.

Measures to reduce risks of food poisoning caused by bacteria

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Measures should be adopted to the prevention of cross contamination between raw

It is more juicy than bigger pieces or cuts.

Not proper handling of the meat.

Microorganisms are spread all over the meat.

If the mincer/grinder is not properly clean:

Reservados todos los derechos.

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Fish: composition and nutritive value

There is different chemical composition in different parts of the fillet.

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Fish and fishery products (catch and aquaculture) shopping habits

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Reservados todos los derechos.

Shellfish are mainly categorized into crustaceans and mollusks.

Association of undesirable microorganisms with fish

Fish meat from unhealthy animals may contain microorganisms.

Initial biota: exogenous microbial contamination