LECTURE NOTES For B. Tech (Dairy Technology) Degree
INTRODUCTORY DAIRY MICROBIOLOGY (Course No: DM 121)
By
Dr. Velugoti Padmanabha Reddy, Ph.DProfessor and University Head
Dept of Dairy Microbiology College of Dairy Technology Tirupati-517
502
COLLEGE OF DAIRY TECHNOLGY SRI VENKATESWARA VETERINARY
UNIVERSITY TIRUPATI- 517 502 2006
MILK MICROBIOLOGY Prokaryotes Ex. Bacteria, Blue green algae.
Microbes Eukaryotes Ex. Fungi, Protozoa, Algae. Plants and Animals
Binomial nomenclature --Second word is name of bacterium First word
is genus & begins with capital,
PROCARYOTES: Grouped under 4 Divisions and 29 Sections 1.
Gracilicutes: - Prokaryotes with thinner cell wall implying G ve
type cell wall Classes: Scotobacteria, Anoxyphotobacteria,
Oxyphotobacteria. 2. Firmicutes: - With thick and Strong cell wall
indicative of G +ve type cell wall Classes: Firmibacteria,
Thallobacteria. 3. Tenericutes: - With Pliable & soft nature
indicative of lack of a rigid cell wall Classes: -Mollicutes. 4.
Mendosicutes: - With faulty cell walls suggesting the lack of
conventional peptidoglycon Classes: - Archaeobacteria.
DAIRY IMPORTANT MICROORGANISMS Section 1: Spirochetes Family:
Leptospiraceae Genus: Leptospira Ex. Leptospira interogans
Spirillaceae Aerobic, Micro-aerophillic, Motile Genus:
Campylobacter Ex. Campylobacter jejuni. Gram Ve Aerobic Rods &
Cocci A. Pseudomonadaceae Genus: Pseudomonas Genus: Xanthomonas B.
Neissericeae Genus: Neisseria Genus: Acinetobacter C. Other Genus:
Genus: Alteromonas Genus: Flavobacterium Genus: Alcaligenes Ex.
Alcaligenes viscosus Genus: Brucella Ex. Brucella. abortus
Section 2: Section 4:
Section 5:
Facultatively Anaerobic G Ve Rods Genus: Escherichia Genus:
Enterobacter Genus: Salmonella Genus: Yersinia Genus: Citrobacter
Genus: Aeromonas Genus: Vibrio Genus: Chromobacterium
Rickettsiaceae (Rickettsias & Chlamydias) Genus: Coxiella Ex
Coxiella burnitii G +Ve Cocci Genus: Micrococcus Ex. Micrococcus
varians Genus: Staphylococcus Ex. Staphylococcus aureus Genus:
Streptococcus Genus: Lactococcus Genus: Enterococcus Genus:
Leuconostoc Genus: Pediococcus Endospore Forming G+Ve Rods Genus:
Bacillus, Genus: Clostridium Nonspore Forming G+Ve Rods, Regular
Genus: Lactobacillus, Genus: Listeria Ex. Listeria monocytogenes
Irregular, Non Sporing G +Ve Rods Mycobacteria
Section 9: Section 10:
Section 13:
Section 14:
Section 15: Section 16:
CLASSIFICATION OF DAIRY IMPORTANT BACTERIA I. Based on the Size
& shape of arrangement of cells. 1) Cocci: cells are spherical
or ellipsoidal 2) Bacilli: cylindrical or rod like cells 3)
Spirilla: spiral or helical shaped cells Pleomorphic: cells
appearing in different shapes or lack of uniform shape. Ex.
Arthrobacter Palisade Arrangement: cells linked side by side like
matchsticks Cocci: a) Diplococci: Cells divide in one plane &
remain attached in pairs Ex. Neisseria b) Streptococci: Cells
divide in one plane &remain attached after some divisions, in
the form of chains. Ex. Lactococcus c) Tetrads: Cells divide in two
planes & form 4 cells. Ex. Pediococci d) Sarcinae: Cells divide
in 3 planes & form a cuboidal arrangement. e) Staphylococci:
Cells divide in 3 planes in an irregular pattern producing bunches
of cocci. Bacilli: a) Diplobacilli: Pairs. b) Streptobacilli:
Chains Ends may be rounded as in Lactobacillus delbruckeii ssp.
bulgaricus or squamosed as in Bacillus anthracis Spirilla: Single
curves: Vibrio Many curves: Spirochetes Few curves: True spirilla
II. Classification based on Temperature 1. Mesophillic:
Microorganisms capable of growing between 20 & 40C with the
optimum growth temperature (OGT) of 37C are termed as Mesophiles.
All pathogenic organisms are mesophillic in nature Ex. S. aureus,
E. coli 2. Psychrotrophic: Organisms capable of growing at or below
7C (refrigerated) but the OGT is between 15 & 20C are termed as
psychrotrophs.These are the significant spoilage organisms of
refrigerated milk & milk products. Ex. Pseudomonas sp.
Alkaligenes sp. 3. Thermophilic: Organisms capable of growing over
50C with O.G.T of 55 C are termed as thermophiles. They are the
important organisms causing outbreaks in heat
processed milk & milk products. Some are capable of growing
between 40-85C. Organisms produce enzymes at rapid rate, so that
enzymes are replaced quickly. Ex.: Bacillus stearothermophilus,
Streptococcus salivarius ssp. thermophilus. 4. Thermodurics:
Organisms capable of withstanding pasteurizing temperatures of
63C\30 min. with O.G.T of 35-37C are termed as thermodurics. They
form important flora of pasteurized or heat processed foods. Ex.
Micrococcus varians III. Classification based on Oxygen Requirement
1. Aerobic: Organisms capable of growing in the presence of oxygen
are termed as aerobic organisms. They can grow in a standard air
atmosphere of 21% oxygen. They are more efficient in utilization of
available nutrients. Ex. Bacillus species 2. Anaerobic: Organisms
which cannot grow in the presence of oxygen but can grow in the
presence of CO2 are termed as anaerobic organisms. They dont use O2
for energy yielding reactions. They are ever poisoned by O2.Some
tolerate low concentrations of O2.They produce catalase &
peroxidase enzymes. High tolerance Clostridium perfrigens Moderate
tolerance Clostridium tetani 3. Facultative: Organisms which can
grow either in the presence or absence of oxygen are termed as
facultative organisms. Ex. E. coli, Lactococcus lactis ssp. lactis
4. Microaerophillic: Organisms which grow best at 1-15% of O2
levels. They can use O2 for energy yielding reactions but cannot
withstand high levels of O2 i.e, 21% of O2. Ex.: Campylobacter
jejuni
PHYSIOLOGICAL GROUPING 1. Acid producers: Capable of fermenting
lactose to form lactic acid. Lactic acid coagulates milk by
producing precipitation of Casein at 4.6 pH Homofermenters:
Heterofermenters: Lactococcus, some Lactobacilli SomeLactobacilli,
Lueconostoc sp.,
2. Gas producers: Capable of producing CO2\&H2 from lactose
fermentations. Ex. E. coli, yeasts, Clostridium species. 3.
Proteolytic: Degrade milk proteins into soluble components by
enzymes known as proteinases or proteases. Ex. Bacillus species,
Pseudomonas species.
4. Lipolytic: Organisms capable of attacking milk fat by enzymes
known as lipases liberating glycerides & fatty acids. Ex.
Pseudomonas species, Achromobacter lipolyticum Molds: Geotrichum
candidum, Penicillium roqueforte. 5. Sweet curdling: Organisms
capable of causing curdling of milk by the enzyme known as rennin
like enzyme before the development of sufficient acidity. Ex. B.
subtilis, B. cereus, Enterococcus liquifaciens. 6. Ropiness:
Causing change in the viscosity of milk or forming threads when the
milk is poured from one container to other, due to production of
gums, mucins etc. Ex. Alcaligenes viscosus 7. Flavour producing:
Fruity Pseudomonas fragi Malty Lactococcus lactis var maltigenes
Fishy Proteus icthyosmius Unclean E. coli 8. Colour fermentations:
Yellow coloration: Blue coloration : Green coloration: Black
coloration : Red coloration : Pseudomonas synxantha Pseudomonas
cyanogenes Penicillium roqueforte Pseudomonas nigrifaciens Serratia
marcescens
CHRACTERISTICS OF DAIRY IMPORTANT BACTERIA Section 1:
Spirochetes GENUS: LEPTOSPIRA: Characters: Flexible helicoidal
rods, Gram ve, obligately aerobic, Optimum growth temperature is
28-30 C, Chemo organotrophs (heterotrophs) using fatty acids &
fatty alcohol as energy & carbon sources. Leptospira
interogans: causes leptospirosis in animals & man. Kidney is
the natural inhabitant & organisms are shed in the urine
contaminating soil & water; causes influenza type to icteric
form of illness. Section 2: Spirillaceae GENUS: CAMPYLOBACTER:
Characters: G-ve. Slender spirally curved rods, may be S shaped.
Aerobic \ Microaerophilic (O2 requirement 3 to 15 %).
Chemo-organotrophs. Respiratory metabolism. Motile cork screw like
motion by means of single flagellum at one or both ends.
Campylobacter jejuni: Causes fever & gastroenteritis in human
beings. Along with other species causes abortions &
reproductive problems in domestic animals. Sources: reproductive
organs, intestinal tract & oral cavity. Raw & improperly
pasteurized milks. Section 4: G-Ve Aerobic Rods & Cocci
PSEUDOMONACEAE Genus: Pseudomonas Genus: Xanthomonas Genus: Zooglea
Genus: Gluconobacter Characters: G ve, slightly curved rods, motile
by polar flagella, oxidase +ve, catalase +ve, aerobic; respiratory
metabolism & never fermentative, growth requirements are
simple. Sources: soil & water. GENUS PSEUDOMONAS (Mol % G+C :
58 72) Leptospiraceae
Pseudo means false Monas means unit. i.e false species of Monas
an early generic name of Protozoan Many species of Pseudomonas
produces fluorescent, diffusible pigments of a greenish, yellow, or
yellow green colours; black or blue Optimum growth temperature: 25
30C ; Nutritionally versatile, grow well on solid media
Presence of these organisms in milk and milk products is highly
objectionable because Produces heat stable proteases and lipases
even at low temperatures versatile spoilage agents attacking fats
and proteins Include pathogens which cause milk borne illness. They
have very little fermentative activity on carbohydrates Some of
these produce phosphatase and if they grow in pasteurized milk they
cause false positive tests.
Ps. fluorescens:: Produces water soluble fluorescent
greenish-brown pigment known as PYOVERDIN Optimum growth
temperature: 25 30 C but grows at 5C. Causes bitterness and
Lipolysisin refrigerated foods by elaborating proteinases and
lipases and Gelation in UHT products This also produces
phospholipases and glycosidase but causes no coagulation of litmus
milk Ps. fragi : Rarely produces pigments; sometimes diffusible
brown pigment Grow at 5 C, Optimum growth temperature 20 25 C. It
is mostly lipolytic and rarely Caseolytic. Produces fruit like
odour resembling apple or strawberries compounds responsible are
Ethyl butyrate and Ethyl hexanoate. Ps. aeruginosa: Produces
Greenish blue pigment Pyocyanin and Pyoverdin and non caratenoid
pigments. Do not grow at 5 C but brow at 41 - 42C. Optimum growth
tempr: 37 C. Opportunistic pathogen; . Causes Mastitis. GENUS
XANTHOMENAS: Xanthomonas maltophilia, previously known as
Pseudomonas maltophilia. Differentiating characters &
Pseudomonas are o produce yellow, non diffusible cell bound pigment
o Inability to reduce Nitrate o Require methionine as growth factor
o No growth at 4 C and 42C. May grow at 41C Optimum temperature 30
35 C o Utilization of citrate negative GENUS ALTEROMONAS: Al.
putrefaciens: formerly known as Pseudomonas putrefaciens Produces a
non-diffusible pink or Reddish brown pigment; Produces H2S on Kings
iron agar, Rod shaped, Optimum growth temperature : 20 25 C
Major proteolytic and less lipolytic in nature, Causes surface
taint in butter and also Cheesy, putrid flavour defects Source:
Water and soil
GENUS ALCALIGENES Rods or cocci, Motile, Obligate aerobic,
Psychrotrophs and some are thermoduric, Causes ropiness in milk and
milk products Alcaligenes viscosus Alcaligenes tolerans GENUS
BRUCELLA G ve rods, Short ellipsoidal, Small, circular convex
colonies, Optimum growth temperature : 37 C;, Non motile, growth
may favoured with Increased CO2 tension. Characteristics Brucella
abortus Brucella suis + Swine Brucella melitensis + + Goats *
Sheep
Growth in presence of Methyl violet + Urease H2S production
Abortion Cow
Section 5 : Facultative Anaerobic Gram negative rods.
ENTEROBACTERIACEAE Genus: Genus: Genus: Genus: Genus: Escherchia
Enterobacter Salmonella Yersinia Citrobacter
Characteristics: Gram Negative, Short and straight rods, Motile
by peritrichous flagella or non-motile, May be capsulated,
Aerobic/facultatively anaerobic Chemoorganotrophes, Ferment glucose
producing acid or acid and gas, Mainly catalase positive, Oxidase
negative Inhabitant of saprophytic COLIFORMS : Coliforms may be
defined as Gram negative, oxidase negative, non-spore forming rods,
which can grow aerobically or facultatively anaerobic in the
presence of bile salts or surface active agents with similar growth
inhibitory properties and which are to ferment lactose with
production of acid and gas with in 48 hrs at 37C the intestines of
man and animals and some may act as pathogens or
GENUS ESCHERICHIA: E. coli, E. blattae Glucose and
Carbohydrates. acid, and Formic acid. Formic Acid Pyruvate Co2 and
H2 Lactic acid, Acetic
Litmus milk gets coagulated with rapid acid production and gas;
Citric acid and its salts are not utilized as sole source of
carbon. OGT: 30-37C, but grow at below 10 and above 45C About 35%
of the acid produced by E. coli is lactic acid and so they are also
called pseudolactic acid bacteria. The type of acid produced by E.
coli depends on sugar fermented and nature of N2 source.
Significance: Their presence in the foods is indicative of faecal
contamination They are potent food spoilage organisms o They
produce gas in the dairy products o They produce unclean flavour in
the dairy products o Some strains produce ropiness in the dairy
products Their presence in pasteurized foods indicates unhygienic
productions at the plant Some strains produce enterotoxins and some
are enteropthogenic in nature Charcteristics Motility C02 : H2
Origin IMViC tests Indole Methyle red Voges-proskauer Citrate E.
coli Motile 1:1 Faecal Positive Positive Negative Negative E.
aerogenes Non-motile 2:1 Non faecal Negative Negative Positive
Positive
GENUS ENTEROBACTER : E. aerogenes, E. cloacae (faecal/non
faecal) Ferments glucose by means of butanediol-formic fermentation
to produce acetyl methyl carbinol to give +ve Voges-proskauer test.
Encapsulated variants cause ropiness. GENUS SALMONELLA: Salmonella
is named after D. E. Salmon (USA). G-ve, aerobic/ facultative
anaerobes with optimum growth temperature of 37C, mostly motile by
peritrichous flagella, they produce acid and gas from carbohydrates
such as glucose, mannitol, maltose, sorbitol etc except Salmonella
typhi. It does not ferment lactose, positive for methyl red,
negative for indole and Vogesproskauer but variable in utilization
of citrate. Source: Direct or indirect faecal contamination, cows
suffering from salmonellosis
They are designated into (by Kauffmann-White scheme) serotypes
such as O (somatic), H (Flagellar) and Vi ( Virulence antigens) It
is an important group of organisms because of its ability to
produce a variety of food infections and illness. It produces
endotoxins. 12 to 30 hrs may elapse after ingestion of food
contaminated with these organisms and the onset of symptoms are due
to the elaboration of endotoxins by the growth of the organisms The
following illnesses are associated: Typhoid : Salmonella typhi
Paratyphoid : Salmonella paratyphi A Paratyphoid : Salmonella
paratyphi B Paratyphoid : Salmonella paratyphi C GENUS YERSINIA:
The important organisms are Y. enterocolitica, Y. pestis, and Y.
psuedotuberculosis Y. enterocolitica enters milk by contamination
through faeces, urine, and insects. They grow between 2-45C with
OGT of 30 C. All the organisms of this genus are pathogenic to
humans or animals or opportunistic potential pathogens. GENUS
SERRATIA: Serratia marcescens produces red pigment known as
prodigiosin GENUS VIBRIO: Vibrio cholerae causes cholera in
humans
Section 9: Rickettsiaceae (Rickettsias & Chlamydias) GENUS:
COXIELLA Ex. C. burnitii G-ve short rods, pleomorphic occurring as
diplococci. The organism causes Q (query) fever. Milk is
contaminated directly from circulation in infected animals. Man
gets infection mostly by aerosol infection and less commonly by
drinking contaminated milk The organism also show high resistance
to chemical and physical agents and desiccation. It is resistant to
0.5% formalin, 1.0% phenol and can withstand the heat treatment of
60C for 1 hr and 61.7C for 30 min. The complete inactivation by
pasteurization may not be always possible. Ticks are the vectors of
transmission.
Section 10: G +Ve Cocci Genus: Micrococcus Genus: Staphylococcus
These two genus consists of Gram positive spherical cells. They are
non-motile. Aerobic or facultatively anaerobic, chemoorganotrophs
and catalase positive Characteristics Grow anaerobically
Carbohydrate attack Lysostaphin sensitivity Ability to ferment
glucose anaerobically Micrococcus -Ve Oxidative -Ve -Ve
Staphylococcus + Fermentative + +
GENUS MICROCOCCUS: Micrus = small Kokkos= seed or grain Aerobic,
coagulase negative, Mesophilic, forms tetrads Some of these
organisms produce yellow, orange, red pigment Lactose is not
fermented, OGT 25C, Many species are heat resistant and survive
63C/ 30 min but the true micrococci are not resistant to
pasteurization. They are found in lactiferous ducts of mammary
gland and present in milk obtained from udder under sterile
conditions and hence considered as normal microflora of milk.
Contaminated equipment is the main source of micrococci in milk.
They are responsible for Thickening of sweetened condensed milk and
causes thermodurics out breaks in pasteurizing plants Ex. M.
varians, M. luteus GENUS STAPHYLOCOCCUS They are cocci and smaller
than micrococci. Occurs in grape like clusters, non motile, may
produce orange or yellow pigment, Mesophilic, OGT 37C Ferment a
variety of carbohydrates and resistant upto 10% of salt
concentration They are capable of producing several toxins like
haemolysin, fibrinolysin, leucocidin, enterotoxins and thermostable
nuclease etc., Haemolysins are the substances that liberate
haemoglobin from RBC. haemolysis produces clear, colorless zone
around colonies. Haemoglobin destroyed to produce colorless
compound. , haemolysis converts haemoglobin to methamoglobin which
produces greenish zone. Sources: Skin, Nasal and mucus
membranes
Staphylococcus aureus: Facultative anaerobe, Grow at 15 and 45 C
and is mesophilic, The colonies are with yellowish tint (lemon) or
orange-yellow Heamolysins: , , gama and delta are produced.
Sensitive to Novobiocin Most of the strains produce enterotoxins
(Exotoxins) which are responsible for food intoxications. Five
different enterotoxins are reported. They are A, B, C, D, and E but
enterotoxins A are more common. They are heat stable and produce
symptoms in 2 to 6 hrs after ingestion of contaminated milk.
Medium: On Baird parkers medium they produce black, shiny, convex
colonies surrounded by a clear zone. In the clear zone a fine black
precipitate may appear i.e zone of opalescence. The pathogenic
nature is confirmed with a positive coagulase and thermo nuclease
test. It produces bacteriocins viz., staphylococcin / micrococcin
which are bacteriostatic / cidal to bacteria and to other
staphylococci. Other organisms of staphylococcus found in milk are
S. epidermis, S. caprae, S. hyicus Genus Streptococcus: Streptos
means flexible, a pliable length of cocci similar to necklace G
+ve, spherical or ovoid and occurs in pairs or chains, Fastidious
organisms, Facultative anaerobes Sherman (1937) divided
streptococci into 4 groups based on serologically active group
specific polysaccharide( C) substance into pyogenic, viridans,
Enterococcus, Lactic groups Character Groupantigen Growth with 10C
45C 6.5% Nacl 9.6 pH 0.1% Methylene blue NH3 from Arginine Litmus
reduction before clotting Resistance to 60C/30 min Haemolysis
Examples Pyogenic A,B,C,D,E, F,G,H + S.pyogenes (A) S.agalactiae(B)
S.disgalactiae (C) Viridans Non specific + +/, gama S.bovis (D)
S.uberis S.thermophilus Enterococcus D + + + + + + + , , gama
S.durans E.faecalis Lactococcus N + + +/+ gama L.lactis ssp.lactis
L.lactis ssp.cremoris
PYOGENIC GROUP Streptococcus pyogenes: Belongs to Lancefield A.
It is a pathogenic organism and causes septic sore throat, scarlet
fever. The habitat is upper respiratory tract, skin lesions, and
inflammatory exudates. It produces haemolysis and resists
Phagocytosis. It produces erythrogenic toxin. It causes acute
mastitis in animals. CAMP test is positive Streptococcus
agalactiae: It belongs to Lancefield B and is , weakly , gama
haemolysis. It is similar to S. pyogenes and causes mastitis in
cows. It causes meningitis and pneumonia in humans. CAMP test is
negative Streptococcus disgalactiae: Lancefield group C Causes
bovine mastitis. CAMP test is negative VIRIDANS GROUP:
Streptococcus uberis: This causes winter mastitis in cows.
Serologically heterogenous. Non haemolytic. CAMP test is negative
Streptococcus thermophilus: This is now known as Streptococcus
salivarius ssp.thermophilus. It has no group specific antigen.
Grows at 45C but not at 10 and 53C. OGT is 40-45C. o o o It is
thermophilic starter culture used for the preparation of yoghurt,
Swiss cheeses This is used for antimicrobial agents assay Ex.
Sulphadiazine This can be used in microencapsulated form when grown
in milk with added amino acids along with medium and also used in
hypo caloric diets for treatments of obesity.
On Yoghurt lactic agar: shows small white colonies with clear
zone where as Lactobacillus delbruckeii ssp bulgaricus produces
large white colonies surrounded by cloudy zones. GENUS:
ENTEROCOCCUS E. faecalis, S. durans These are mesophilic organisms,
with OGT 37C . They resist 63C/30 min E. faecalis: The source of
the organism is the intestinal tract of humans and animals. It is
used as a trail starter culture in the manufacture of certain
cheeses because of its salt tolerance and pH tolerance. It is
dominant organism among enterococcus in India in various milk and
milk products. It is non haemolytic, sometimes may be haemolytic
with graying
GENUS: LACTOCOCCUS Lancefield N Group Grow at 10C but not 45C.
Reduces the litmus prior coagulating it. Grow in broth with 0.1%
methylene blue but not in broth with 6.5 % Nacl and 9.6 pH
Character NH3 from arginine 40C 4% Nacl 9.2 pH Gas from citrate
Lactococcus lactis ssp. lactis + + + + Lactococcus lactis ssp.
cremoris Lactococcus lactis ssp. lactis biovar diacetylactis + + +
+ +
Lactococcus lactis ssp. lactis : Elliptical cocci in pairs or
short chains and elongation of cells in direction of chain. The
Colonies are Grey, circular, convex, glistening. OGT is 30C. It
does not produce Co2 or diacetyl and does not produce ammonia from
arginine. o o o o It is a important mesophilic starter It produces
antibiotic NISIN which is inhibitory to Bacillus, clostridium,
lactobacillus and some other Gram positive organism Some strains
produce malty flavour due to metabolism of leucine to produce
3methyl butanol Lactose is fermented to lactic acid by
homofermentation and produces 0.8% to 1.0% lactic acid
Growth requirements are very complex. It requires B- complex
vitamins such as biotin, Niacin, thiamine, pantothionic acid,
pyridoxine, folic acid and Amino acids such as Arginine, valine,
histidine, leucine, isoleucine, methionine, phenylalanine, proline,
glutamic acid It is easily inhibited by 0.15 units of penicillin,
0.5 micrograms of aureomycin and 600-1000 ppm quaternary ammonium
compounds per ml Lactococcus lactis ssp. lactis biovar
diacetylactis : It is similar to to the above organism. It utilizes
citrate and produces Co2, diacetyl, volatile acids and acetoin.
Utilization of citrate is plasmid mediated and is unstable
character.The produced acetic acid is inhibitory to pseudomonas,
coliforms, salomonella. Lactococcus lactis ssp. cremoris: It
exhibits slow fermentation of lactose. It produces diplococcin
GENUS PEDIOCOCCUS:G+ve, nonmotile spherical organisms,
Microaerophilic, showing poor surface growth Occurs as tetrads, in
pairs / short chains. Optimum growth temp: 25 30 C, Acid is
produced from glucose, galactose and maltase not from mannitol
& dextrin, Produces diacetyl apparently from oxidation of
acetyl methyl carbinol
GENUS LEUCONOSTOCS:Leucos means colorless Nostac means
encapsulated blue green algae similar to Nostoc except
photosynthesis G +ve cocci in paris or short chains,
Microaerophilic /Aerobic/Facultatively anaerobic
Hetero-fermentative i.e. production of lactic acid, ethanol and Co2
from glucose. Some produce slime in sucrose media. It is
comparatively, inactive in litmus milk and rarely coagulates milk
Growth in medium is enhanced by yeast, tomato and other vegetable
extracts. Ferment citric acid to Diacetyl, Acetoin, 2,3 butylenes
glycol acetic acid, CO2 Causes slits in cheddar cheese made with
butter cultures due to CO2
The organisms are L. mesenteroides ssp dextranicum, L.
mesenteroides ssp mesenteroides , L. paramesenteroides, , L.
mesenteroides ssp cremoris, L. lactis Section 13: ENDOSPORE FORMING
GRAM POSITIVE RODS GENUS BACILLUS: Bacillus means little stick,
Gram positive large rods (3 to 9 um in length), Aerobic,
Saprophytic soil bacteria, May occur in single, pair or chains.
Aerobic spore formers. Sources: Air, water, soil, feed and fodder
Isolated by heating raw milk to 80C/ 10 mts before plating. Large
and rough colonies are formed Acid sensitive, potent spoilage
organisms, They may be psychrotrophes, mesophilic, Thermophilic,
Catalase positive Spores: Ellipsoidal to cylindrical in shape,
occasionally bulged sporangia Organism Spore Motility Lecithinase
OGT Growth at 45C 65C 7% Nacl Citrate utilization B. cereus Central
or paracentral + + 30C V V + B. subtilis Central or paracentral
30-40C + + + B. stearothermophilus Subterminal to terminal + 55-65C
+ + B. coagulans Subterminal to terminal + 35-45C + V V B.
megatherium Central or paracentral + 28-35C V + +
B. cereus : Dont grow at 5C but grows at 20-35C. This is
important organism because Causes food poisoning by producing
enterotoxins when the number exceeds 106 per gram Produces bitty
cream defect (broken cream) by the action of lecithinase enzyme
(Extra cellular phospholipase) Produces sweet curdling by
coagulating milk at lower acidity by producing rennet like
enzyme
B. subtilis : Grows between 20-45C. This is important organism
because Cause ropiness or sliminess in raw, pasteurized milk Causes
spoilage of UHT, concentrated/ canned milk products Causes sweet
curdling by coagulating milk at lower acidity by producing rennet
like enzyme Produces levan extracellularly from sucrose
Extracellular enzymes including those that degrade pectin, casein,
polysaccharides of plant tissue are produced Polypeptide antibiotic
subtilin are produced B. licheniformis : This is important
organisms because Causes spoilage of UHT, concentrated/ canned milk
products Produces levan extracellularly from sucrose Causes
ropiness or sliminess in raw, pasteurized milk B. coagulans: This
causes spoilage of UHT, concentrated/ canned milk products B.
stearothermophilus: Grows between 45 to 65C This is important
because It is obligately thermophilic Causes flat sour spoilage of
canned/UHT milk products Used in the detection of antibiotics in
the milk i.e antibiotic assay test
GENUS CLOSTRIDIUM: These are the anaerobic organisms and spore
formers. Gram positive rods with 1-4 in length, motile, catalse
negative. They may be mesophilic or Thermophilic The important
source of these organism is soil, intestinal tract of animal. It
may become established as contaminants on equipment. They gain
entry into milk via faeces, soil, feed and especially silage Some
are pathogenic organisms such as Cl. perfringens and Cl.
botulinum
Cl. butyricum and Cl. tyrobutyricum are Thermophilic. The
fermentation end products include acetic acid, butyric acid,
butanol, iso-proyl alcohol, acetone, H2 and Co2 Cl. perfringenes :
Causes bovine mastitis in cattle and causes neurological disorders
in humans. It produces profuse gas and breaks the coagulum of milk
causing Stormy fermentation Cl. tyrobutyricum: Causes late blowing
condition in Cheeses Cl. butyricum; Causes late blowing condition
in Cheeses after 1-2 months of manufacture Cl. sporogenes :
Develops rancidity of Emmental cheese Character Spores
Cl. butyricum Oval Central to Sub-terminal Do not swell + 30-37C
Acetic, Butyric acids Butanol
Cl. tyrobutyricum Oval Sub-terminal Swell the cell + 37C Butyric
acid Co2, H2 from lactic acid
Cl. botulinum Oval Central to Sub-terminal + 37C Slow increase
in acidity
Cl. Perfringenes Oval Central to eccentric Large distend the
cell 37-45C Acetic, Butyric, Lactic acids More H2 Ammonia and
water
Motility OGT End products
SECTION 14: NON-SPORE FORMING G+ VE RODS, REGULAR GENUS:
LACTOBACILLUS, Lac milk; Bacillus staff or stick. Long, thin rods,
G+ve, asporagenous rods; Nonmotile, Microaerophilic, Aciduric,
catalase Negative ; Require complex medium like MRS medium / Rogosa
acetate agar with layering of plates Source: Feed, silage, manure 3
groups Thermobacterium Streptobacterium Betabacterium
Thermobacterium Growth at 15 45C VP Fermentation Examples + +
Homofermentation L. bulgaricus L. acidophilus L. helveticus Hexose
fermented to lactic acid by EMP, but Pentoses not fermented
Streptobacterium + + Homo /facultatively L. casei L. plantarum
Hexoses fermented to lactic acid by EMP Some sp. also produce
acetic, Formic & ethanol under glucose limitation. Pentoses
fermented to lactic acid & Acetic acid involving
phosphoketolase Serology E
Betabacterium -ve +/Heterofermentation L. brevis L. fermentum
Hexoses fermented to lactic acid & acetic acid involving
phosphoketolase pathway
THERMOBACTERIUM GROUP: L. delbrueckeii ssp.
bulgaricus:Thermophilic, OGT 40C,
Used in Yoghurt, Swiss & Italian cheeses. Produces
antibiotic Bulgaricin (stable for 1hr at 100c) which is active
against Ps.fragi and to a lesser extent to S. aureus L.
acidophilus:Thermophilic, OGT 45c; do not grow at 20C & at 4%
salt concentartion Produces lactic acid & acidity reaches 1.8%
but to keep viable the acidity should not exceed 0.6 & 0.7%
Produces antibiotics - Acidophilin, Acidolin, Lactocidin Medium:
Aesculin cellobiose Agar with incubation at 40c/48h in CO2
atmosphere. It produces colonies surrounded by dark olive green
complex o o o o Used along with other mesophilic cultures in Kefir
Used with Bifi bifidum in special ice cream Used in Paneer In
India. Used as encapsulated forms in hypocaloric diets
Therapeutic uses: Hydrolysis of lactose suitable for lactose
intolerant people production of antimicrobial substances,
antibiotics & H2O2 prevention of constipation; ulcerative
colitis Reduction of cholesterol by fermentation products
Restoration of gut microflora after antibiotic treatment because of
their capability to grow at low surface tension.
Therauptic value is based on the assumption that this milk
combats the so called autointoxications caused by accumulation in
the body, of toxic substances elaborated by toxigenic bacteria.
STREPTOBACTERIAL GROUP L. casei:Serology B & C, Mesophilic,
Produces upto 1.5%.acid on prolonged incubation o o o o Used in
Yakult It shows Inhibitory action by producing acid, peroxides
& antibiotics to Salmonella typhi salmonella typhinurium,
Shigella dysenteriae, E.coli and P.aeuruginosa Used along with
Candida lipolytica and Lac.lactis as cheese flavour additive in
processed cheese by spray/ freeze drying. Used with Pr. shermani to
produce mycostatic preservative
L. plantarium:Serology D, Used in Brine cheese, OGT - 30 35C
Antibiotic Lactolin is produced which is effective against G+ve
organisms
BETABACTERIUM GROUP L. brevis:Serology E, Mesophilic, OGT 30C;
Do not grow at 45C, but grow at 15C. Used in Kefir. Lactobrevin
antibiotic is produced.
GENUS: LISTERIA Gram +ve, Short rods with rounded ends; may be
curved; single, short chains or V forms. Catalase positive, Aerobic
/ facultatively anaerobic Colonies are Bluish grey when seen by
normal illumination and bluish green sheen by obligately
transmitted light. Sources: Wide; Water, mud, Sewage, faeces of
animals & man Listeria monocytogenes: OGT 37C, Cattle :
abortion Human: food poisoning, meningitis accompanied by
septicaemia GENUS MYCOBACTERIUM G +ve, but staining difficult due
to high wax content, Acid-fast. Non- motile, now-sporing,
non-branching rods. M. tuberculosis grows very slowly in vitro.
Requires two weeks or more to show visible growth and requires
special media for growth. Ex:- Loefflers Serum medium. Causes
tuberculosis in humans
Section 15: IRREGULAR, NON SPORING G+ve rods GENUS
CORYNEBACTERIUM:Cells are straight to slightly cured rods with
tapered ends, Club shaped appear. Non motile, Asporogenous,
Aerobic/facultatively anaerobic forms may
Recognized by their banded and beaded, clubbed appearance, meta
chromatic granules are formed. Chemo organitrophs, OGT : 37C C.
boris is not pathogenic & causes rancidity in cream. C.
pyogenes causes Supportive mastitis
GENUS BREVIBACTERIUM:Rod-coccus growth, Gram positive,
Non-motile, obligate aerobic, Chemoorganotrophic with respiratory
metabolism. OGT : 20-30C, Non-thermoduric B. lines : Produce yellow
to deep orange red carotenoid pigments Usually present on exterior
of surface ripened chesses of Limburger type.Contributes to the
surface colour of such cheeses aid in ripening by proteolysis and
improve the flavour & aroma by the production of
methanethiol.
GENUS PROPIONIBACTERIUM:Gram positive, Non-motile, Asporogenous,
Anaerobic/aerotolerant, Pleomorphic rods, club shaped with one end
rounded and the other tapered or pointed. Cells may be coccoid,
bifid, or branched, occur in single in pairs, or Short chains (in V
or Y configuration) , 5% carbon dioxide atmosphere is good for
growth. Opt. growth temp: 30-32C Lactic acid and CHOs is converted
into propionic /acetic acids +Carbon dioxide Pr. freundenrichii is
associated with Swiss cheese flavour because of proline production
and eyes due to Carbon dioxide.
FUNGI Division Sub Division Class Sub class Order Family MOLDS
Penicillium: (Class Deuteromycetes): Penicillium have septate
vegetative mycelia which penetrate the substrate and then produce
aerial hyphae on which conidiophores develop. Condiophores may be
branched and have brush like heads bearing spores clusters of
sterigmata are usually in one place and from each is formed a chain
of conidia. With the production of conidia, colonies become green,
grey green, blue green and yellow green. The colour of mature plant
is useful in helping to identify species. Asymmetric group.
Pencillium roquefort: Used in Roquefort, of blue veined cheeses.
Asymmetric group i.e. more than one branch in the conidiophore and
this branching is asymmetrical. Colonies on malt agar blue green
spreading colonies slowly change to darker green. Smooth velvety
appearance with irregular margins of radiating lines of
conidiophores Spiders Web Arachnoid , Conidiophores are rough,
conidia globose, smooth or borne in loose columns or tangled
chains. Toxins produced are Roquefortin, mycophenolic acid, PR
toxin, P.casei : Used in Swiss cheese, Asymmetrica group, No
arachnoid margins, Colonies yellow brone P.camemberti: Used in
camembert, Brie cheeses colonies / white & gradually become
pale grayish green from the centre onwards Asymetrica group,
Conidiophores slightly rough, Conidia become sub-glabose &
borne in tangled chains Produces Toxins i.e cyclopiazonic acids
Mycota Mycotina Mycetes Mycetidae ales aceae
Geotrichum candidum: Commonly found on dairy products Colonies
White, yeast like forms true mycelium, breaks to form arthrospores
are cylindrical with rounded ends. Sporendonema sebi: Causes
buttons in SCM Asperigillus (Class Duitenomycetes): Septate
branching mycelia with vegetative portions submerged in nutrient.
Conidiophores or fertile hyphae arise from thickened foot cells
which may also be submerged. At the apex, condidiophore inflates to
form a vesicle, which gives rise to sterigma which may be single
layered or double layered. Condia arise from the sterigmata and
borne in chains. Conidia are produced within the tubular sterigmata
and are extruded to form spore chains. Conidia are of various
colors and are quite characteristic of the species. Mucor:
Sporangiophores each bear terminally a single large globose
sporangium containing many spores (spherical/ellipsoidal), No
stoleniferous growth, No septate mycelium, sporangiophores never
arise from nodes on stolons, rhizoids absent. Rhizopus stolenifer:
Bread mold. Stoleniferous type of spread. Non-septate,
sporangiophores form at are quite large and Black. Heterothalic.
which
YEASTS : Unicellular - spherical/ovoid, Pseudomycelium may form
veg.rep budding Ascospores 1 to 4 per ascus. Multiply asexually by
budding where a bud has formed on a cell a raised star remains. As
many as 23 bud scars are found on a single cell. During budding
nucleus divides by construction and a portion of it enteres the bud
along with other organelles. The cytoplasmic connection is formed
by laying down of cell wall material. Under appropriate conditions
forms asci. The cytoplasm of the cell differentiates into four
thickened wall spherical spores, although the no.of spores can be
fewer. The cells from which asci develop are diploid and nuclear
divisions which precede spore formation are meiotic. Ascospores are
of two mating types. Mating type is significantly controlled by a
single gene which exists in two alletic states a & and
segregation at reduction devision preceding ascospore formation
gives rise to 2 & 2 ascospores. Fusion occurs between two
different mating types. (legitimate copulation). Such fusion
results in diploid cells which form asci containing viable
ascospores.
S.cerevisiae: Bakers yeast, isolated from kefyr. Cells globose,
subglobose, ellipsoidal/cylindrical singles, pairs, short
chains/clusters. Ascospores - globose to short ellipsoidal,
1-4/ascus, do not liberate, Lactose not fermented, Nitrate not
assimilated. Kluyveromyces marxianus var.marxianus: - Kly
fragilis/sac.fragilis: Used in Kefyt & Kumiss Cells subglobose,
ellipsoidal to cylindrical single or pairs, Ascospores one to
4/ascus cresentiform ot reniform, Lactose may be fermented by some
strains, Nitrate not assimilated. Kluy.marxianus var.lactis:
Kly.lactis/Sacch.lactis: Associated with yoghurt, isolated from
milk, gassy cheese, Italian cheese, Cream, BM Cells spherical,
ellipsoidal or occasionally cylindrical clusters, singles, Pairs
and occasionally clusters., Ascospores one to 4, spherical to
ellipsoidal, Readily released, Lactose fermented, Nitrate not
assimilated Candida kefyr: Toru.kefyr/ Can.pseudotropicalis var.
lactosa: Associated with kefyr, buttermilk and cheese,
Morphologically variable, Budding unicellular to pseudomycelium or
true mycelium, Cells Ovoid short/long, Reproduction budding or
fission, Colonies off white to cream, Lactose may be fermented.,
Nitrate not assimilated. Candida lacticondensi (Tor.
Lactiscondensi): Isolated from SCM Cells Ovoid, budding cells,
Colonies offwhite, cream, yellowish or brownish, Mycelium not
formed or rudimentary mycelium is rarely found., Lactose not
fermented, Nitrate assimilated.
BACTERIOPHAGE Bacteriophages are the viruses that infect
bacteria. Viruses are not plants, animals, or bacteria, but they
are the quintessential parasites of the living kingdoms. Phage
literally means devouring just like phagocyte (to swallow or eat up
greedily). Widely distributed in nature & most abundant in
intestinal contents of animals and first is invented by Twort in
1915. Typical bacteriophage has a structure like tadpole. Head
enclosing nucleic acids in protein sheath. Tail a hollow tube of
proteins & bearing tail plate & tail fibers. All viruses
contain nucleic acid, either DNA or RNA (but not both), and a
protein coat, which encases the nucleic acid. Some viruses are also
enclosed by an envelope of fat and protein molecules. Without a
host cell, viruses cannot carry out their life-sustaining functions
or reproduce. They cannot synthesize proteins, because they lack
ribosomes and must use the ribosomes of their host cells to
translate viral messenger RNA into viral proteins. Viruses cannot
generate or store energy in the form of adenosine triphosphate
(ATP), but have to derive their energy, and all other metabolic
functions, from the host cell. They also parasitize the cell for
basic building materials, such as amino acids, nucleotides, and
lipids (fats). They are significant from processing point of view.
Phages attack Lactic acid bacteria & lyses them by multiplying
inside the host cell leading to the release of many phages which
can re-infect the fresh cells in the culture. This results in the
failure of starters to act & bring about the changes during
preparation of fermented products. Bacteriophages are highly host
specific, a rotation of starter cultures help to control the
problem to some extent. Ca ion deficient medium for maintaining
starter cultures prevent phage attack as the ion helps in phage
adsorption to host cell. Genetic manipulations to construct phage
resistant strains.
Typical Lactococcus lactis ssp. lactis phage might have 40-90
microns head & 100-120 microns tail.Bacteriophages can persist
for a long time in dried up whey & on utensils. Phages attach
themselves to living cells & as they divide phages also
multiply. The infective material is injected (nucleic acid) through
hollow tail of the phage particle into the bacterial cell. The rate
of phage proliferation is greater than that of bacterium.
Consequently, a point is reached at which lysis begins. Lysis is
preceded by swelling of the bacteria to 6-16 times their normal
size. Phage action may cause a number of variations in the
properties of bacteria.Irreversible adsorption of phage to the cell
triggers a no. of steps which lead to the transport of viral
nucleic acids into the cytoplasm & nucleic acid gets
metabolically active transforming cellular function to one of
synthesizing viral components. As the phage assembly completed the
lysis of the host cell occur. All this occur approx. in one
generation time of the host phage multiplying to hundred in one
generation & in two generations to 10000 & so on.
GERMICIDAL PROPERTIES OF MILK Normal milk contains varying
amounts of substances which inhibit normal development of certain
bacteria and some even kill these bacteria Level of activity
depends on 1. Type of milk 2. Quarters of same animal. Functions:
1. To protect mammary gland from infections. 2. To confer
resistance to young suckling calves. IMMUNOGLOBULINS:
Immunoglobulins are antibodies against specific antigens, often to
bacteria. In the man prenatal immunity is conferred primarily
through the transmission of IG from the mother across the placenta
to fetal circulation. In cows and buffaloes IG are transferred from
colostrum into the newborns circulation postnatally when the GI
tract is permeable to intact protein molecules. Milk has two types
of Immunoglobulins, 1. 2. Functions: IgA: IgG: Produced locally
with in the udder Transferred to milk from circulation.
Reduces the severity of udder disease by Neutralizing toxins
elaborated during disease process. Aids in phagocytosis by
polymorpho nuclear leucocytes. prevents bacterial adhesion to the
cells Suppresses bacterial growth.
PHAGOCYTOSIS Phagocytosis means Invading of pathogens by
leucocytes. Protection of udder from mastitis rests primarily on
phagocytosis and killing of pathogens by PMN. Out come of an
invasion attempt by pathogens is usually decided in early hours of
infection. o The infection will be repelled, if phagocytosis occurs
faster than the multiplication of pathogens o The pathogens grow
faster in the absence of efficient phagocytosis and cause clinical
mastitis. During mastitis the high leucocytes counts are present
and it is evident that if the infection is established they cannot
efficiently and quickly dispose off the invading pathogens.
Leucocytes of uninfected udder vary between 1 to 5 lakh cells/ml of
which approximately 10% are PMN. In infected udder PMN may be upto
90%. Phagocytosis is less effective in milk than in blood. PMN
leucocytes ingest some quantities of milk fat and casein, thus
reducing the efficiency, so udder is easily affected by even a
small number of invading bacteria. Functions: Confer protection to
udder from pathogenic bacteria.
LACTOFERIN: Earlier called as Lactotransferrin. It is a red
glycoprotein and resembles Blood serum transferrin. It is a iron
binding protein. Lactoferin combines with iron and make it
unavailable for bacteria, which is an essential growth factor.
Lactoferin by virtue of the high concentration and iron binding
ability enhance the resistance of dry mammary gland to infection.
High citrate and Low bicarbonate in milk reduces iron binding
properties of Lactoferin Colostrum contains 6 mg/ml where as 1
mg/ml shows bacteriostatic action. But Colostrum also contains high
citrates which compete for iron with LF and make it available for
bacteria. Citrate in Colostrum is 3.6 mg/ml. Mature bovine milk
contain 0.42mg Lactoferin /ml. Lactoferin is bacteriostatic to B.
subtitis and B. stearothermophilus and Inhibits S. aureus and P
aeruginosa. LYSOZYME: In human milk it is present at 30 mg/100ml
i.e., 300 times that of bovine milk. This enzyme hydrolyses 1-4
linkage of peptidoylcan of bacterial cell wall i.e., linkage
between N-acetyl muranmic acid and N- acetyl glucosamine, resulting
in weakening of cell wall ultimately resulting in lysis of cell.
Lysozyme is very active against G +ve bacteria especially
thermophilic spore formers and inhibitory to Listeria
monocytogenes, Campylobacteium jejuni, Salmonella typhi, Bacillus
cereus and Pseudomonas aeurogenosa. LACTOPEROXIDASE SYSTEM This
involves three components I. Lactoperoxidase enzyme synthesized in
mammary gland in concentration of 30 mg/ml II. Thiocyanate content
in milk is governed by nutrition of the cons. Usually 1-10 g/ml
III. H202 contributed by PMN or by some udder flora ex.
Streptococci. ( catalase -ve organisms) The Lactoperoxidase enzyme
combines with H202 to oxidize thiocyanate (SCN-) yielding various
intermediate oxidation products such hypothiocyanate,
cyanosulphurous, clyanosulphuric acid which exhibit antimicrobial
activity (OSCN-) Bactericidal to Group A streptococci, E. coli, S.
typhi Pseudomonas. Bacteriostatic to group N and group B
streptococci Lactobacilli o Inhibition of O2 uptake Inhibition of
growth by interfering with oxidation of SH groups of enzymes. o
Damage to cytoplasmic membrane causing leakage and cessation of
nutrient uptake. o Inhibition of LA production. Function: i.
Protection of calf from enteritis ii. Cold sterilization of
milk
SOURCES OF BACTERIAL CONTAMINATION IN MILK Interior or Udder:
Varying number of bacteria are found in aseptically drawn milk with
the reported counts of