Microorganisms Associted with Milk
Microenvironment of milk:
Milk, being considers a nutritious food for human beings, also serves
as an ideal medium for the growth of different microorganisms
particularly bacteria. The growth of microbes depends on the
environment which includes both the substrate microenvironment (viz.
properties of the substrate) and the outside conditions (of which
temperature is by far the most important). Hence, in milk held at a
favourable temperature, the growth of microorganisms is due to its
congenial microenvironment. Milk consists of a variety of nutrients
(including all the vitamins) which serve as sufficient raw material and
essential factors for the growth of microorganisms under ambient
conditions. For example, bacteria requiring lactose, milk fat, milk
proteins etc.as nutrients can proliferate luxuriantly in milk. However, all
bacteria growing in milk may not need the same nutrients. For example,
some bacteria do not find lactose as a suitable energy source whereas
others require nutrients which are either present in small amounts or are
completely lacking. In such cases, an associative growth may occur, in
which growth of one group of organisms provides nutrients for the
growth of the other group. For example, those bacteria which rely on free
amino acids as nitrogen source (which are present only in minute
amounts in fresh milk) grow only after another group of bacteria
(which hydrolyze milk proteins to release amino acids) have grown.
Another such instance is the stimulation of the growth of lactobacilli due
to production of CO2 by some lactic Streptococci (the CO2 which serves
as a stimulant for lactobacilli can however, inhibit some Gram negative
bacteria). Some microorganisms need elements which are either missing
or are present in minute quantities, e.g. Leuconostoc cremoris needs
Mn+2 (which often varies with season) for fermenting citric acid for
flavor production. Besides possible lack or insufficiency of nutrients for
some groups of bacteria, as discussed above, the milk may also possess
some unfavourable conditions for multiplication of other bacterial groups.
The pH is limiting only for a few microbes but redox potential and O2
pressure are mostly unfavourable except for the growth of obligate
anaerobic bacteria. The growth of aerobic bacteria is favoured only in
such locations where O2 pressure is adequate, e.g. in cream layer. The
microbial multiplication at ambient temperatures usually lowers both the
pH and the O2 pressure in milk and a few microorganisms get killed as
they find conditions in milk to be completely unfavourable. Milk has also
been known to possess certain antimicrobial substances which inhibit or
kill certain groups of microorganisms. Milk presents an ideal nutritious
medium for microbial growth and multiplication. Milk provides all the
essential nutrients required such as nitrogen, Carbon, Oxygen, Sulfur and
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certain mineral salts and vitamins for microbial growth. Milk, in addition
to being nutritious medium, presents a favorable physical environment for
the multiplication of microorganisms. High moisture percentage in milk
provides an ideal environment for microbial growth and for nutrients to
be in solution in order to be taken into the microbial cell through the cell
wall by osmosis. An ideal osmotic pressure and neutral pH in milk are
optimum ideal factors for the multiplication of most bacteria, while yeasts
and molds prefer acidified medium as in the fermented dairy products.
Microbiological quality of milk:
Milk when secreted in udder is free from microorganisms i.e. sterile
but as it passes through ducts and reservoirs of udder, bacteria are carried
into it, especially from the walls of the teat canal. A variety of other
sources during production, handling, transport and distribution of milk
contribute various organisms to raw milk before it reaches the consumer
or a processing dairy plant. The microorganisms entering milk may be
pathogenic or nonpathogenic to human beings. The growth of
microorganisms in milk affects the quality of milk by bringing about milk
spoilages. The rate at which this spoilage occurs depends upon not only
the initial microbial load but also on conditions under which milk is held
and the length of time for which it is held. While these microorganisms
growing on milk ingredients may release certain metabolites (like lactic
and other organic acids, gases, enzymes, flavouring compounds,
pigments, toxins etc.) in the system which may be useful or harmful and
thus directly affect its quality. Generally, these metabolites lead to
various kinds of spoilages in the product and sometimes make it a
potential health hazard. Lactose serves as the principal carbon source for
majority of the spoilage causing microorganisms growing in milk.
Microorganisms in raw milk:
The number and types of Microorganisms present in raw milk
depend on the nature and extent of contamination which in turn varies
with the conditions of milk production and the subsequent storage
conditions. In other words, production of milk under hygienic conditions
and subsequent cooling storage at low temperature (less than 4°C) restrict
the contamination and proliferation of microorganisms.
Microbial numbers in raw milk:
The microbial counts in raw milk may usually vary from less than
one thousand per /ml to more than one million (106) per / ml. The total
microbial load in raw milk may be estimated by standard plate count
(SPC) by plating milk on tryptone dextrose agar or plate count agar and
incubating at 37oC for 48 hours. This however, is not an exact estimate of
the total bacterial content as there are other groups of microrganisms
which require different cultivation conditions (media and incubation
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temperature). Hence, suitable test conditions are provided for
enumerating the specific groups. For example psychrotrophs need
incubation at 5-7°C for 10 days; thermodurics are enumerated by
pasteurizing the milk sample before plating; the other groups, viz,
Coliforms, Lactic acid bacteria, Gram negative rods, Lipolytic and
proteolytic types, pathogenic organisms, etc. need selective media for
their growth on the test plates. Besides enumeration by plating, the
microbiological quality can be predicted by performing other routine tests
like dye reduction time tests (Methylene blue reduction and resazurin
reduction), presumptive coliform test etc. Based on the results of some of
these tests, raw milk can be appropriately graded for its microbiological
quality. The routine grading tests like standard plate count do not reflect
the sources of contamination and the production faults for which counts
of specific groups of microorganisms such as psychrotrophs,
thermodurics, coliforms, spores, streptococci etc. have to be
determined. Such tests are therefore, mainly useful for advisory
investigational and survey purposes.
Types of microflora in raw milk:
A heterogenous population of mesophilic, thermoduric,
thermophilic, psychrotrophic and pathogenic organisms constitute the
microflora of raw milk. The presence and relative proportions of the
individual groups vary with the conditions of milk production and
handling.
a- Mesophilic microflora:
The microorganisms growing optimally at 30°C to 32°C are
designated as ″mesophilic″. The different microorganisms forming the
mesophilic microflora of raw milk as detected in plate count analysis at
32°C for 2-3 days are Micrococci, Staphylococci, Streptococci (faecal
and mastitis causing Streptococci), Asporogenous Gram positive rods
(Microbacterium spp., Corynebacterium spp. Mycobacterium spp. and
Lactbacillus spp.), Sporogenous Gram positive rods (Bacillus spp.) and
Gram negative rods (Pseudomonas spp., Alcaligenes spp. and
Coliforms).
b- Thermoduric microflora in raw milk:
Thermoduric microorganisms are those which survive
pasteurization conditions but cannot grow at pasteurization
temperatures. A wide variation in the thermoduric count has been
observed in raw milk supplies although most of them do not multiply
faster at ambient temperatures. This indicates that the variations are
due to differences in the extent of contamination from potent sources.
The genera of thermoduric bacteria in fresh raw milk are the following:
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1-Microbacterium lacticum which is mostly derived from milking
equipment.
2- Bacillus spores: they are generally higher in winter than in summer
because these spores are mainly derived from the teat surfaces spoiled
with bedding materials used for housing the cows. Milk cans could also
serve as a source of Bacillus cereus spores.
3-Clostridum spores: The clostridial spore count of raw milk is also
higher in winter because these are mainly derived from silage (used in
winter feeding) and bedding materials. Their number declines when
cow starts going out for pasture feeding e.g. Cl. tyrobutyricum.
4- Micrococcus:
Microcococcus is almost exclusively derived from milking equipment
and other utensils e.g. M. freudenreichii
c- Thermophilic microflora:
The term thermophilic applies to those bacteria which can readily
grow at 55°C and most of them have an upper limit of growth at about
70°C. In dairy industry, therefore, the term is used for that group of
organisms which not only survive pasteurization but also can grow at
that temperature. The standard method of enumerating this group of
organisms is to incubate agar plate at 55°C. A small number of
thermophilic bacteria may gain access into raw milk mainly from soil,
bedding, feeds and rarely from water supplies. The number increases
due to their multiplication if raw milk is stored under warmer ambient
conditions. The common members of thermophilic microflora of raw
milk belong to aerobic or facultatively anaerobic sporeforming rods
e.g. B. stearothermophilus, B.circulans, B. thermoacidurans and
Lactobacillus thermophillus.
d- Pscychotrophic microflora:
Pscychotrophic organisms as understood in dairy industry are those
which are able to grow at commercial refrigeration temperatures (2-7
°C) regardless of their optimum growth temperature. Usually the term
″pscychtrophilic″ is used for organisms that have an optimum
temperature below 20°C. In the psychrotrophic microflora of milk
and milk products,very few are classical psychrophiles and majority
of them are mesophilic (optimum growth temperature = 20 to 30°C) in
nature. The Pscychotrophic bacteria detected (5-7 °C in 7-10 days) in
raw milk belong to two broad categories, viz, Gram negative rods and
Gram positive rods or cocci.
1- Gram negative rods:
This group is the most commonly occurring Psychrotrophic
microflora in fresh raw milk and may comprise about 10-15% of the
initial total microbial number. The psychrotrophic Gram negative bacteria
belong to different genera namely Pseudomonas, Acinetobacter,
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Flavobacterium, Achromobacter, Alcaligenes and Enterobacter
(Coliforms). Among these, Pseudomonas constitutes about half of the
total Gram negative rods and the
following species are prevalent:
a- Pseudomonas fluorescens (predominant).
b- Pseudomonas putida
c- Pseudomonas fragi
d- Pseudomonas aeruginosa
The species of the Coliform bacteria (mainly Enterobacter spp.) have
been known to constitute 10-13 % of the Psychrotrophic microflora of
raw milk. Some of the Gram negative psychrotrophs like Pseudomonas
species can produce heat stable enzymes (lipases and proteinases) in
refrigerated milk. During subsequent pasteurization, their vegetative cells
get killed but these enzymes are not inactivated and may be responsible
for fat and casein degradation causing milk spoilages. Improperly cleaned
/sanitized milking equipment is the major source of psychrotrophic Gram
negative rods although these may also be derived from animal’s teat
surfaces.
II –Gram positive bacteria:
Some strains of the following genera / species of psychrotrophic
Gram positive rods and cocci are also encountered in raw milk:
1-Bacillus spp (spores) e.g. B. coagulans B. circulans and B. Subtilis
2-Arthrobacter spp. 3- Streptococcus spp.
e- Coliforms:
The term ″coliform bacteria″ in milk denotes the aerobic and
facultatively anaerobic, Gram negative, nonspore forming rod shaped
bacteria which ferment lactose into acid and gas at 32°C within 48 hours.
The typical genera included under this group are Escherichia,
Enterobacter
and Klebsiella. The presence of Coliforms especially Escherichia coli in
raw milk is no longer taken as an index of direct faecal contamination
(unlike that in case of water supplies) as they may also come from other
sources like improperly cleaned / sanitized milking equipment (where
moist, milky residues allow the rapid building–up of Coliforms by
multiplication). However, relating their absence to the use of properly
cleaned milking equipment is also not necessarily true. Most of the strains
of Enterobater species isolated from raw milk are psychrotrophic
microorganisms.
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f- Pathogenic microorganisms in raw milk:
Pathogens for man may gain entry into raw milk either due to their
co-secretion into milk as a result of diseased condition of the animal and /
or its udder or due to contamination of milk from infected human
handlers, faecal and non faecal sources, etc. A list of various pathogens
reported to occur in raw milk along with their principal source of entery
is presented in
the following table:
Table 8: A composite picture of pathogens transmittable through raw
milk
Pathogens Principal cause/source
Brucella abourtus, Brucellosis in lactating animal
Brucella melitensis,
Brucella suis
Mycobacterium tuberculosis Tuberculosis in lactating animal
Mycobacterium bovis
Bovine mastitis
(udder disease)
Streptococcus agalactiae
Staphylococcus aureus
Escherichia coli
Listeria monocytogenes Bovine mastitis
Bacillus cereus
Clostridium perfrigens,
Pasteurella multocida,
Actinomyes species
Coxiella burnetii
Faecal contamination of milk or Human
handlers (infected or carriers)
Salmonella typhi
Salmonella paratyphi
Escherichia coli
Vibrio cholera
Shigella flexneri
Shigella dysenterae
Streptococcus pyogens Human handlers ( infected or carries)
Cryptococcus neoformans Bovine mastitis (infrequent causative)
Adenoviruses (20 different types) Human handlers (infected or carries)
Enterovirues, Hepatitis virus. Human handlers (infected or carriers)
Tick borne encephalitis virus. Animal which get tick-bites?
Foot and mouth disease (FMD) virus Animal with FMD.
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Sources of microorganisms in raw milk:
The initial number and types of microorganism in fresh raw milk
depend on a number of sources, the primary ones being
1- The udder and teats (The interior and the exterior conditions).
2- The milker and milk handlers.
3- Water supplies.
4- Air.
5-Milking equipment.
6- The subsequent storage and transport conditions may further increase
the microbial number if proper cooling (refrigeration) is not practiced.
I- Internal contamination:
The initial number of microorganisms in aseptically drawn fresh raw
milk is almost high.
A- Interior of the udder:
1- The interior of the normal udder: milk when secreted in the healthy
udder is almost sterile but as it passes through ducts and reservoirs of the
udder, organisms are carried into it especially from the walls of the teat
canal, these bacteria enter through teat opening and spread in the interior
of the udder (lactiferous ducts) by their physical movement and
multiplication, majority of these bacteria come down in the foremilk.
These microorganisms may range from a few hundred to a few thousands
per ml of milk. They are washed out by the first few streams of milk with
drawn from the udder; therefore, fore-milk usually contains the largest
counts or number and strippings usually have a lower bacterial count.
Types of microorganisms contributed by this source are the following few
genera:
-Micrococci (predominate), Streptococci, and Corynebacterium.
2- Interior of infected udder (mastitis):
The mastitis causing bacteria get excreted in milk (most of them in
foremilk fraction)
a- Subclinical form of mastitis contributes about 10000 bacteria /ml of
milk.
b- Clinical form of mastitis contributes over 10 million bacteria / ml
(such high numbers are generally excreted in streptococcal and coliform
mastitis). In Staphylococcal mastitis the number excreted is not so high.
Mastitis may lead to the development of millions of the infectious
organisms per ml of milk from diseased quarters.
Types of microorganisms contributed by this source:
The most common microorganisms are:
1- Staphyloccus aureus.
2-Streptococcus agalactiae.
3-Streptococcus dysagalactiae.
4-Streptococcus uberis.
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5- Escherichia coli.
6-Corynebacterium pyogenes.
-Other infrequent microorganisms are:
-Leptospira, Listeria, Bacillus cereus, Pasteurella multocida, Clostridium
perfringens, Actinomycetes, Rickettsia (Coxiella burnetii), and Yeasts
(Cryptococcus neoformans).
B- Blood (Diseased animal):
Circulation of microorganisms in the blood of diseased animal as in:
1- Bacteraemia.
2- Viraemia.
3- Rickettisaemia.
4- Toxoplasma.
The infected animals excrete or shed organisms in their milk which
sometimes continues without the manifestation of diseases
(Asymptomatically).
The causative organisms of these diseases are:
1- Brucella abortus and Brucella melitensis as in (Brucellosis).
2- Mycobacterium tuberculosis as in tuberculosis (TB).
3-Clostridium botulinum as in (Botulism ).
4-Coxiella burnetti as in Q-fever.
5- Leptospira as in leptospirosis.
6- Listeria as in Listeriosis.
II - External contamination:
The secreted milk is almost sterile and contamination may occur
during production, handling, transport and distribution of milk.
A- The exterior of normal udder and teats and animal body (belly,
Flanks and Tail): These surfaces soiled with feed, manure, mud and
bedding materials (like sand, straw and saw dusts) such dirts may fall into
the milk pail and carrying a considerable number of bacteria into the milk
during milking. The bacterial count in milk obtained from cows with
unwashed teats (soiled heavily with manure) might reach approximately
105 cfu / ml, and the bacterial count associated with bedding material
increased during winter housing (when teats are more soiled with bedding
materials containing counts as high as 1010 cfu / ml) than during summer
(when cows are sent to pastures and hence lesser exposure to such
bedding). The aerobic spore counts on teat surfaces were 102 - 105 / teat
depending on environmental conditions. The cows fed on ″bad silage″
excrete Clostridial spores in their feces which may contaminate teat
surfaces and gain entry into milk. Organisms associated with such dirt are
often of the gas–forming group as Coliform and Clostridial spores also
Bacillus spores and Micrococci, Staphylococci and faecal Streptococci.
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Under current milk production procedures, the body of the dairy animal
has become a minor source of bacteria in milk. Flanks and udders are
clipped, cows are washed, udders dried and the milk is taken with milking
machines.
B-Personnel (Milkers and handlers) unclean or unhealthy:
The number of microorganisms derived from milkers or handlers is
more when hand milking is practiced. The milker´s hands may be a
source of pathogenic bacteria of human origin. Moreover, a disease can
be easily being carried from one animal to another on the milker´s hands.
Organisms may drop directly into the milk pail from the nose and mouth
during sneezing or coughing, wet-hand milking is especially
objectionable because the liquid washes dirt and bacteria from teats,
udder and hands into milk. Several diseases caused by bacteria and spread
through milk may be transmitted, either directly or indirectly from the
milker or handler to the milk, such as:
1-Typhoid fever.
2- Diphtheriae.
3-Scarlet fever.
4- Septic sore throat.
These are sometimes transmitted from a diseased person or carrier
through milk to susceptible individuals who consume the contaminated
milk. Therefore, persons suffering from an infectious or contagious
disease or a carrier of such, should not allowed to handle milk or its
products or have a part in the cleaning or care of utensils used for them.
Filthy habits and wet-hand milking or similar practices are to be
condemned. The cloths of the milker should be clean, as must be the
milking–stool and the cord that is used to tie the animal or its legs. If
these precautions are not observed, the milker may contaminate his hands
every time he touches stool and cord. Types of microorganisms
contributed by this source are mainly Staphylococci (Staphylococcus
aureus) and Coliform from unclean personnel. Also any of the human
pathogens may come in the milk from unhealthy milker.
C- Milking utensils: The term ″utensils″ is entended to include all
containers or equipment in which or by which milk or its products are
handled, processed, stored, transported or merchandised. They include
pails, cans, milking machine (teat cup, clusters, buckets or cans, milk
pipeline, recorder, jars and pipeline receiver, milk cans or bulk tanks and
ancillary equipment namely strainer, filter, a cooler and milk flow
indicators and meters). As soon as the fresh milk comes into contact with
the inadequately cleaned and sanitized utensils, the virulent
microorganisms will contaminate it. These include thermoduric,
Thermophilic and mesophilic microorganisms. After leaving the udder,
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milk is mainly contaminated from improperly sanitized milking
equipment .The residual milk solids (milk residues / milk stones) on such
equipment support the growth of microorganisms and formation of
bacterial clusters which contaminate the subsequent supplies.
Types of microorganisms contributed by this source are:
1-Mastitis pathogens: such as Streptococcus agalactiae and
Staphylococcus auresus when hot cleaning / disinfecting solutions are
used thermodurics predominate (Asporogenous Gram-positive rods). In
bulk tanks the thermoduric count is lower (˂105 cfu/m2) but the
psychrotrophic count is higher due to prevalence of cold temperatures. In
the milk cans which are improperly cleaned and covered with lid while
still moist, the bacteria may
Types of microorganisms contributed by this source are:
1-Mastitis pathogens: such as Streptococcus agalactiae and
Staphylococcus
auresus when hot cleaning / disinfecting solutions are used thermodurics
predominate (Asporogenous Gram-positive rods). In bulk tanks the
thermoduric count is lower (˂105 cfu/m2) but the psychrotrophic count is
higher due to prevalence of cold temperatures. In the milk cans which are
improperly cleaned and covered with lid while still moist, the bacteria
may
proliferate and contaminate the incoming milk. Spores of Bacillus cereus
and other thermodurics in milk are generally contributed by milk cans.
D- Aerial contamination or Atmosphere: The feeding of dusty hay and
grains just before milking has been shown to contaminate the milk to
some
extent. Although brushing dairy animals just before milking was also
found
to increase the bacterial content because of the air borne contamination.
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Small numbers may gain entry both during hand milking (by falling in the
milk pail) and machine–milking (by getting sucked with air in the
machine
during its operation).
Types of microorganisms contributed by this source are:
1- Micrococci (more than 50% of the aerial microflora).
2- Bacillus spores.
3- Moulds spores.
E- Farm water supplies:
If polluted untreated water (from wells, lakes, springs and rivers)
gains access to milk or is used for washing udders and teats or for rinsing
milking equipment and other utensils or is used for washing milker´s
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hands. Any microorganisms present in the water will contaminate the
milk. However multiplication of some of the water-borne bacteria in any
residual water in the equipment will result in more serious contamination.
Many farms rely on untreated water supplies from wells, lakes, springs
and rivers. Bacterial contamination can be introduced from storage tanks
which are not properly protected from Rodents, Birds, Insects and dust.
So water used in the process of milk production should be of good
bacteriological quality (i.e. chlorinated). Chlorination is frequently
recommended for water of unsatisfactory bacteriological quality.
Types of microorganisms contributed by this source are:
1- Fecal types like: Coliform, faecal Streptococci and Clostridia.
2- Saprophytic derived from soil and vegetation.
3- Pseudomonas (Psychrotrophic microorganisms) and other gram
negative rods.
4- Bacillus spores.
F- Flies and Rodents:
The contamination of milk from flies is very common, as they carry
several types of microorganisms on their wings and legs from sewage or
infected sputum, uterine discharges and abscesses to utensils, milker’s
hands and water supplies. In this manner pathogenic organisms of various
types may be carried into liquid milk. The principal organisms
transmitted
in this way are those causing:
1-Typhoid.
2-Paratyphoid.
3-Tuberculosis.
4-Diphtheria.
5-Anthrax.
Such organisms can exist on flies for long periods.
G- Storage and transport: The improperly cleaned and sanitized
equipment used for storage (milk cans or bulk tank) and transport (cans or
insulated tankers) of raw milk might increase the bacterial count
depending on their hygienic conditions. Some increase in the microbial
count may also result due to pumping of milk which breaks up the
bacterial clumps and chains. Secondly, the improper cooling /
refrigeration of raw milk during storage and transport leads to an increase
in the bacterial number particularly psychrotrophic count. Hence, in the
stored milk psychrotrophs predominate. The psychrotrophic Bacillus
spores are detected but they usually do not germinate and outgrow.The
thermoduric flora also remains unchanged in stored milk. Among
psychrotrophic genera in stored milk Pseudomonas species mainly
Pseudomonas fluorescens. The type of predominant microflora depends
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on the temperature of storage as follow:
0- 5 ° C e.g. Pseudomonas fluorescens.
5-10 ° C e.g. 1- Pseudomonas fluorescens.
2- Proteus vulgaris
3- Micrococcus species
4- Alcaligenes viscolactis
5- Alcaligenes marshallii
10-15 ° C e.g.1- Streptococcus acidominimus
2- Streptococcus agalactiae
3- Streptococcus cremoris
4- Streptococcus durans
5-Streptococcus dysgalactiae
6-Streptococcus faecalis
7- Streptococcus lactis
8-Streptococcus uberis
15 -30 °C:
All Streptococci especially Streptococcus lactis
30- 40 °C:
1- Enterobacter aerogenes
2- Escherichia .coli
3- Lactobacillus brevis
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4- Lactobacillus bulgaricus
5- Lactobacillus casei
6- Lactobacillus fermenti
7- Lactobacillus helveticus
8- Lactobacillus lactis
9- Lactobacillus plantarum
10- Lactobacillus leichmanii
11- A few Streptococci
40- 50 °C:
1-Lactobacillus bulgaricus
2-Lactobacillus fermenti
3-Lactobacillus lactis
4-Lactobacillus helveticus
5- Lactobacillus thermophiles
6- Streptococcus faecalis
7-Streptococcus thermophilus
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