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MILK, FERMENTATION, AND

FERMENTED AND NON-FERMENTED
DAIRY PRODUCTS
Chapter 7
Ch

Food Microbiology
By
M. Majdinasab

FERMENTATION

Numerous food p
products owe their p
production
and characteristics to the fermentative activities
of microorganisms.
Ripened
Ri
d cheeses,
h
pickles,
i kl
sauerkraut,
k
t and
d fermented
f
t d
sausages.

Due to fermentation:

Shelf life is extended

A
Aroma
and
d fl
flavor is
i increased
i
d

vitamin content of the fermented food is increased

Digestibility
g
y of the raw materials is increased

Increase or decrease the toxicity of some foods

THE LACTIC ACID BACTERIA

This g
group
p is composed
p
of 13 g
genera of Grampositive bacteria at this time:

Carnobacterium
Enterococcus
Lactococcus
L t
Lactobacillus
Lactosphaera
Leuconostoc
Weissella

Oenococcus
Pediococcus
P
Paralactobacillus
l t b ill
Streptococcus
Tetragenococcus
Vagococcus
3

Generalized pathways for the production of some fermentation products from glucose
by various organisms. (A) Homofermentative lactics; (B) heterofermentative lactics; (C)
and (D) Propionibacterium; (E) Saccharomyces spp.; (F) Acetobacter spp.; and (G)
cetobacte overoxidizers.
ove oxidize s.
Acetobacter

CO2

DAIRY PRODUCTS

Milk Composition
p
Average Chemical Composition (%) of Whole Bovine Milk

Water

87.0

Protein

3.5

Fat

3.9

Carbohydrate

49
4.9

Ash

0.7

The higher structural protein content of red meats enables

these products to exist as solids.
Water content near the surface of fresh meats of ca. 75.5%
is lower than the average of 87% for milk
The aw of both products is near 1.0

CONTI
B vitamins with p
pantothenic acid and riboflavin
Vitamins A and D are added for human
consumption

It is an ideal growth medium for heterotrophic

microorganisms, including the nutritionally fastidious
Gram positi e lactic acid bacteria.
Gram-positive
bacteria

CONTI

The pH of fresh whole milk is around 6.6 but it may reach

ca. 6.8 from a cow that has mastitis.

Mastitis is most often caused by:

Streptococcus agalactiae

S. uberis

sometimes by:
Staphylococcus aureus

Streptococcus
p
dysgalactiae
y g

PROCESSING

Milk is p
processed in a number of ways
y to p
produce a
variety of products such as cream, cheese, and butter.
Skim milk (0.5% fat)
R d
Reduced
d ffatt milk
ilk (up
( tto 2
2.0%
0% ffat)
t)
Evaporated milk: removal of about 60% water from
whole milk. lactose content about 11.5%.
Sweetened condensed milk: addition of sucrose or
glucose before evaporation. sugar content about 54%
or >64%
64% in solution.

CONTI

In the United States, grade A raw milk that is to be

pasteurized should not have an:
APC that exceeds 300,000 cfu/ml for blended milk
should not exceed 100,000/ml for milk from an individual
producer.
After pasteurization, the APC should not exceed 20,000
cfu/ml and the coliform count should not exceed 10/ml.
cfu/ml,
10/ml
Raw milk should not be held longer than 5 days at 4.4C.
Chocolate milk is processed at a slightly higher
temperature than unflavored milk (75C for 15 sec rather
than 72C).

PASTEURIZATION

Destruction of all disease-causing

g microorganisms
g
Increase shelf life
Has no effect on nutritional value
Endospores of pathogens such as:
Cl t idi
Clostridium
b t li
botulinum

and spoilage organisms such as:

Clostridium tyrobutyricum

C. Sporogenes

Bacillus cereus

are not destroyed.

destroyed

10

CONTI

Low temperature-long
p
g time ((LTLT):
) 63C,, 30 min
High temperature-short time (HTST): 72C, 15 sec
The basis for the heating time and temperature is the
thermal death time (TDT) of the most heat-resistant nonspore-forming milk-borne pathogens.
Prior to 1950:

TDT of Mycobacterium tuberculosis

After the discovery

y of the Q fever agent
g

TDT of Coxiella burnetti

IIn properly
l pasteurized
t
i d milk,
ilk the
th naturally
t
ll occurring
i
enzyme alkaline phosphatase is destroyed

11

STERILIZATION

UHT ((ultra-high
g temperature):
p
)
Destroys non-sporeforming pathogens in milk, but in
addition some sporeformers are severally reduced in
numbers.
numbers
135140C for a few sec (the minimum treatment is
130 C for 1 sec)
UHT-treated milk is commercially sterile with a shelf life of
4045 days at 40F when aseptically packaged in sterile
containers.
UHT-treated whole milk is said to be more flavorful, due
apparently to formation of some Maillard products.
12

CONTI

Although pasteurized milk is free of non-sporeforming

pathogens, it is not sterile.
The efficacy of either LTLT or HTST to destroy the
mycobacterial subspecies that is associated with:

Crohns disease

13

CONTI

Most if not all Gram-negative

g
bacteria ((especially
p
y
psychrotrophs) are destroyed along with many Gram
positives.
Thermoduric Gram positives belonging to the genera
Enterococcus, Streptococcus (especially Streptococcus
salivarius
li
i subsp.
b
th
thermophilus),
hil ) Mi
Microbacterium,
b t i
Lactobacillus, Mycobacterium, Corynebacterium, and
most if not all sporeformers survive.
Among the survivors are a number of psychrotrophic
species of the genus Bacillus

14

GENERAL MICROBIOTA OF MILK

Milk constitutes an excellent medium for the growth of

microorganisms.
Freshly drawn milk from healthy animals contains a small
number of harmless microorganisms.
Nearly all the changes that take place in the flavor and
appearance of the milk after it is drawn form the cow
cow, are
the result of the activities of microorganisms.

Th
Therefore,
f
it iis very essential
ti l tto control
t l these
th
microorganisms
15

MILK-BORNE PATHOGENS

Some of the most obvious are the animal diseases

below to which humans are susceptible and which
may occur in milk of cows:
Brucellosis
Tuberculosis
Salmonellosis
Campylobacteriosis
E t h
Enterohemorrhagic
h i colitis
liti

Anthrax
Listeriosis
Q fever
Crohns disease (?)
Staph./Strep.
St h /St
Mastitis
M titi

16

SPOILAGE

As the only
y natural source of the disaccharide lactose,,
milk undergoes microbial spoilage in a way that is
unique.
Lactic acid bacteria (LAB)
The coliform bacteria are the most conspicuous utilizers
of lactose among Gram-negative bacteria.

Thus:
The bacterial spoilage of either ra
raw or pasteuri
pasteurized
ed
milk is conspicuous by the production of lactic acid
by lactose users
17

CONTI

The thermoduric Streptococcus

p
salivarius subsp.
p
thermophilus strains preferentially use the glucose
moiety of lactose.
The spoilage of UHT milk is caused by Bacillus spp.
spp that
survive the UHT process.

B cereus
B.

B. licheniformis

B. badius

B. sporothermodurans

Paenibacillus spp.

Anaerobic spores appear

not to be a problem
because of the relatively
high Eh of milk
18

CONTI

During cold storage of pasteurized milk:

Psychrotrophic Bacillus weihenstephanensis
sweet curdling
Production of p
proteases and p
peptidases
p
The spoilage of UHT-treated milk:
Heat resistant proteases & lipases that are produced by
Heat-resistant
some psychrotrophs in raw milk
Ropiness in raw milk
Alcaligenes viscolactis
low-temperature maintenance of raw milk for several days
St i
Stringy
consistency
i t

19

CONTI

Souring Lactose fermentation

LAB
Souring & gassiness coliforms acid & gas
Aroma production starter culture diacetyl
Proteolysis
y
unpleasant
p
odors,, undesirable. controlled
desirable cheese production.
Ropiness- Milk drawn into long threads
((Alkaligenes
g
viscolactis))
Sweet curdling- due to production of rennin like
enzyme which curdles without souring
Stormy
y fermentation- Rapid
p fermentation by
y
Clostridium perfringens
Color changes P. syncyanea (blue); P. synxantha (yellow);
(
)
Serratia marcescens (red)

20

CONTI

Lactococci
L. delbrueckii sub sps. lactis (Str.Lactis)
L. Lactis sub sps. Cremoris (Str. Cremoris)
Lactobacilli
L. Casei
L. delbrueckii sub sps. lactis (L. Lactis).
L. delbrueckii sub sps. bulgaricus
(L bulgaricus)
(L.
Leuconostoc
21

CONTI
Coliform:

Facultative anaerobes
Optimum growth @ 37 C
Indicator organism and are closely associated the
presence of pathogens but not necessary pathogens
themselves.
They ferment lactose with production of acid and
gas
Cause rapid spoilage of milk
Th are kill
They
killed
db
by pasteurization
i i
Their presence is an indication of PP
contamination of milk
22

RESULT OF
(SPOILAGE)
o
o
o

o
o
o
o

MICROBIAL GROWTH IN MILK

Principal
p cause are Psychrotrophs
y
p
Most of these are destroyed by pasteurization
Some may survive e.g. Pseudomonas fluorescens,
P d
Pseudomonas
fragi
f gi
Other species and strains that survive pasteurization
and grow at refrigeration tem. produce heat stable
proteolytic & lipolytic enzymes and cause spoilage:
Bacillus
Clostridium
Cornebacterium
Arthrobacter
Lactobacillus
Microbacterium
Micococcus
Streptococcus
23

Stages of milk decay

Rancid
(on the turn)-Milk consumable

Curdling
g
Separation of curd and whey milk still
consumable

Coagulation
period of aromatic decay with mould
growth milk
milk beyond use

Dry
dehydration
d
h d ti ensues- hard
h d and
d chalky
h lk
milk beyond use

24

MICROBIAL PATHOGENS

Bacillus cereus
Listeria monocytogens
Yersinea entrocolitica
Salmonella spp.
E.coli O157:H7
Compylobacter jejuni
Coxeilla burnetii
Mold (Aspergill s Fusarium,
Molds(Aspergillus,
F sari
Penicillium)
Pe icilli ) grow
g o
in milk and milk products & produce potentially
hazardous mycotoxins.
25

STARTER CULTURES, FERMENTED

PRODUCTS

Dairy
yp
products require
q
the use of an appropriate
pp p
starter culture
Lactic starters are also used for preparing butter,
cultured buttermilk,
buttermilk cottage cheese,
cheese and cultured sour
cream and are often referred to by product.
Lactic starters always include bacteria that convert
lactose to lactic acid:
L. lactis subsp. Lactis
L. lactis subsp. Cremoris
L. lactis subsp. lactis biovar diacetylactis
26

How Does Milk Turn Into Yogurt?

Lactic acid Bacteria

Lactose -------------------------> Lactic Acid

(Milk sugar)

pH: 3.654.40

Acid causes casein (milk

protein)
t i ) to
t denature
d
t
and
d
hold water into a semi-solid
gel = yogurt

YOGURT (YOGHURT)

Starter:
A mixed culture of S. salivarius subsp. thermophilus
& Lactobacillus delbrueckii subsp. bulgaricus in a 1:1
ratio.
ratio

28

Commercial Yogurt
Contains 2 species of bacteria specialized to grow well in milk (but cant survive
inside the human body):
First,,
Streptococcus
thermophilus is
more active, then
slows down when
acidity reaches
0.5%

Next,
Next
Lactobacillus
bulgaricus is more
acid tolerant and
takes over until
acidity >1%

These bacteria work in symbiosis. Each bacterium stimulates the growth of the
other => acidifies the milk more rapidly than either partner on its own.
Acetaldehyde: the chief volatile flavor component of yogurt

CONTI

Freshly
yp
produced y
yogurt
g
typically
yp
y contains around
109 organisms/g
During storage, numbers may decrease to 106/g,
especially when stored at 5C for up to 60 days.
days
The International Dairy Federation norm for yogurt
is 107/g or above.
The antimicrobial qualities of yogurt, buttermilk, sour
cream, and cottage cheese have been examined by
inoculating Enterobacter aerogenes and Escherichia
coli.
A sharp decline of both coliforms was noted in yogurt and
buttermilk after 24 hours.

30

KEFIR
Kefir
f is p
prepared
p
by
y the use of kefir g
grains.
Bacterial species of the genera
Acetobacter
Lactobacillus
Lactococcus
Leuconostoc
yeast species of the genera
Candida
Kluyveromyces
Saccharomyces

These symbionts are held together

by coagulated protein

31

CONTI
The important
p
Lactobacillus spp.
pp in kefir
f are:
L. kefiri
L. parakefiri
L. kefiranofaciens subsp. kefiranofaciens
L. kefiranofaciens subsp. kefirgranum

32

BUTTER
Composition:
p
15% water
81% fat
Less than 0.5% carbohydrate and protein

Although it is not a highly perishable product,

product it does
undergo spoilage by bacteria and molds.
The main source of microorganisms
for butter is cream

Salted butter may contain up to 2% salt

33

CONTI
Bacteria cause two p
principal
p types
yp of spoilage
p
g in
butter:
1. Surface taint or putridity: Pseudomonas
putrefaciens

47C within 710 days

The odor of this condition is apparently due to

certain organic acids, especially isovaleric acid.
Surface taint along with an apple odor is caused
also by Chryseobacterium joostei.
34

CONTI
2. Rancidity
y
hydrolysis of butterfat with the liberation of free
fatty acids.

Lipase

The causative
Th
ti organism
i
is
i Pseudomonas
P
d
f
fragi,
i
although P. fluorescens is sometimes found.

35

CONTI
3. Malty
y flavor : Lactococcus lactis var. maltigenes.
g
4. Skunklike odor: Pseudomonas mephitica
5. Black discolorations: P. nigrifaciens
Fungal spoilage of butter:
Cladosporium
Alternaria
Aspergillus
Mucor
Rhizopus
Penicillium
G t i h
Geotrichum,
especially
i ll G.
G candidum
did

36

CONTI

Discolorations on butter: Black y

yeasts of the
genus Torula
The generally high lipid content and low water
content make butter more susceptible to spoilage by
molds than by bacteria.

37

CHEESE

Cheeses result from a lactic fermentation of milk.

Milk is prepared and inoculated with an appropriate
lactic starter.
Th starter
The
t t for
f cheese
h
production:
d ti
S. salivarius subsp. thermophilus is employed for acid
production in cooked curds (up to 60C)
A combination of S. salivarius subsp. thermophilus &
L lactis subsp.
L.
subsp lactis for curds that receive an
intermediate cook
38

CONTI

The low moisture content of hard and semihard

ripened cheeses makes them insusceptible to spoilage
by most organisms
Molds
Some ripened cheeses have sufficiently low oxidationreduction potentials to support the growth of
anaerobes.
Anaerobic bacteria sometimes cause the spoilage of these
products when aw permits growth to occur.
39

CONTI

Gassiness of cheeses
Clostridium spp., especially C. pasteurianum
C. butyricum
C. sporogenes
C. tyrobutyricum
C. tyrobutyricum is well established as the cause of a
butyric acid fermentation or the late-blowing defect in
cheeses.
h
An aerobic sporeformer, Paenibacillus polymyxa:
Gassiness
40

DISEASES CAUSED BY LACTIC

ACID BACTERIA

Although
g the beneficial aspects
p
of the lactic acid
bacteria to human and animal health are
unquestioned, some of these bacteria are associated
with human illness.

Lactobacilli

Several species of leuconostocs

P di
Pediococci
i

Enterococci: nosocomial (hospital acquired) infections, with

E. faecalis and E. faecium
LAB are opportunists that are not capable of initiating
y individuals.
infection in normal healthy
41

SOME FERMENTED MILK PRODUCTS

42