Diary Microbiology (1)

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Diary Microbiology

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COMPOSITION OF MILK
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Factors affecting Milk composition

• Animal factor
• Genetic, Species, Breed, Individual cow,
  Lactation period
• Age Genetic factors
• Breed, species, feed and individuality
• Stage of lactation, pregnancy, nutritional
  balance
• Health status of the cow
• Oestrus, gestation, presence of mastitic
  infection
• Environmental factors
• Extreme climates, stress, exhaustion, housing
• Milking technique and milking frequency and stage

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Factors affecting Microbial Growth

• Intrinsic Parameters (inside the milk)
• Factors inherent to the food. They are chemical
  and physical characteristics of food.
• pH
• Moisture
• Oxidation-Reduction Potential Inside Food
• Nutrient Content
• Natural Antimicrobial Constituents
• Biological Structures Natural Microflora
• Extrinsic Parameters (environment around the
  milk)
• Storage conditions of the food i.e. properties
  of the environment in which the food is stored
• Temperature
• Relative Humidity
• Presence of Gases or Oxygen
• Antimicrobials or Added Microorganisms

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pH

• Microorganisms sensitive to changes in acidity
  because H and OH- interfere with H bonding in
  proteins and nucleic acids.
• Microbes have no mechanism for adjusting their
  internal pH.
• Therefore, pH of food significantly affects the
  microbial growth on it.

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WATER ACTIVITY

• It is a ratio of water vapour pressure of the
  food substance to the vapour pressure of pure
  water at the same temperature.
• Water activity is expressed as
• Water activity (aw) P/ Pw where P water
  vapour pressure of the food substance and Pw
  water vapour pressure of pure water (Pw 1.00).
• The growth of microorganisms is limited due to
  minimum water activity values (Table 2)
• Milk having high water activity is more
  susceptible to spoilage by micro-organisms.

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NUTRIENT CONTENT

• Microorganisms require
• Energy source such as carbohydrates, amino
  acids, proteins, organic acids and alcohol.
• Nitrogen source such as amino acids, peptides,
  nucleotides, urea, proteins and ammonia.
• Carbon source
• Minerals such as phosphorus, iron, manganese,
  magnesium, calcium and potassium.
• e. Vitamins and other growth factors
• Milk being a rich source of all the above gets
  easily spoiled by the micro-organisms.

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PRESENCE OF ANTIMICROBIALS

• Natural constituents of foods which affect
  microbial growth are
• Lactoferrin e.g. Milk
• Lactoperoxidase e.g. Cows milk
• Conglutinin e.g. Cows milk
• These antimicrobials help preserve milk for
  longer period of time.

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MICROFLORA IN MILK

• In addition to being a nutritious food for
  humans, milk provides a favourable environment
  for the growth of microorganisms.
• Yeasts, moulds and a broad spectrum of bacteria
  can grow in milk, particularly at temperatures
  above 16C.
• Microbes can enter milk via the cow, air,
  feedstuffs, milk handling equipment and the
  milker.
• Once microorganisms get into the milk their
  numbers increase rapidly.

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• It is more effective to exclude micro-organisms
  than to try to control microbial growth once they
  have entered the milk.

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Microflora of UHT milk

• Ultra-high temperature is carried out at
  135-150C coupled with aseptic packaging.
• The only microflora survive UHT treatment are
  bacterial spore of thermophlic bacilli( B.
  stearothermophilus) and sometimes to mesophilic
  bacilli and clostridia.
• Major spoilage organisms in heat processed milk
  are, B. megaterium( main cause), B. firmus, B.
  polymyxa, B. coagulans and Clostridium spp.
• Microorganisms entering through faulty packaging
  practices are usually associated with stagnant
  water on dairy floors (
  Pseudomonas, Coryneform, Micrococci etc).
• Major defects --gt coagulation, bitterness and
  gassiness.

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Microflora of Boiled Milk

• In India, milk is boiled to 100C for brief
  period before consumption.
• Boiling kills vegetative forms of all microbes
  except heat stable enterotoxins esp. of
  Staphylococci.
• Post pasteurization contamination can also occur
  due to improperly cleaned utensils and due to
  improperly heated portions of milk.
• Major defects--gt off-flavour (proteolytic),
  coagulation and gassiness.
• Boiled milk should be utilized within 16 hrs,
  particularly in absence of refrigeration.

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• MILK SPOILING BACTERIA

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Bacillus cereus

• B. cereus is a thick long rod shaped Gram
  positive, catalase positive aerobic spore former
  and the organism is important in food borne
  illness.
• It is quite often a cause of diarrheal illness
  due to the consumption of desserts, meat, dishes,
  dairy products, rice, pasta etc that are cooked
  and kept at room temperature as it is
  thermoduric.
• Some of the B. cereus strains are psychrotrophic
  as they grow at refrigeration temperature.
• B. cereus is spread from soil and grass to cows
  udders and into the raw milk.
• It is also capable of establishing in cans. It is
  also capable of producing proteolytic and
  amyloltic enzymes and also phoslipase C
  (lecithinase).

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• The production of these enzymes by these
  organisms can lead to the spoilage of foods.
• The diarrheal illness is caused by an enterotoxin
  produced during the vegetative growth of B.
  cereus in small intestine.
• The bacterium has a maximum growth temperature
  around 48C to 50C and pH range 4.9 to 9.3. Like
  other spores of mesophilic Bacillus species,
  spores of B. cereus are also resistant to heat
  and survive pasteurization temperature.

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Clostridium perfringens

• C. perfringens is a Gram-positive encapsulated
  anaerobic non-motile bacterium commonly found on
  meat and meat products.
• It has the ability to cause food borne disease.
  It is a toxin producing organism-produces C.
  perfringens enterotoxin and ß -toxin that are
  active on the human GI tract.
• It multiplies very rapidly in food (doubling time
  lt 10 min).
• Spores are resistant to radiation, desiccation
  and heat and thus survive in incompletely or
  inadequately cooked foods.

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• However, it tolerates moderate exposure to air.
• Vegetative cells of C. perfringens are also
  somewhat heat tolerant as they have relatively
  high growth temperature (43C -45 C ) and can
  often grow at 50C.
• They are not tolerant to refrigeration and
  freezing. No growth occurs at 6 C . C.
  perfringens is present in soil and the other
  natural environment.

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Clostridium botulinum

• C. botulinum produces the most potent toxin
  known.
• It is a Gram-positive anaerobic rod shaped
  bacterium. Oval endospores are formed in
  stationary phase cultures.
• There are seven types of C. botulinum (A to G)
  based on the serological specificity of the
  neurotoxin produced.
• Botulism is a rare but very serious disease.
• The ingestion of neurotoxin produced by the
  organism in foods can lead to death.
• However, the toxin (a protein) is easily
  inactivated by heat.
• The organism can grow at temperature ranging from
  10-48 C with optimum growth temperature at 37C.
  

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• Spores are highly heat resistant. The outgrowth
  of spores is inhibited at pH lt 4.6, NaClgt 10 or
  water activitylt 0.94.
• Botulinum spores are probably the most radiation
  resistant spores of public health concern.
• Contamination of foods is through soil and
  sediments where they are commonly present.
• The organism grows under obligate anaerobic
  conditions and produces toxin in under processed
  (improper canning) low acid foods at ambient
  temperature.

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Campylobacter

• Gram negative nonspore forming rods.
• Campyloleacter jejuni is an important food borne
  pathogen. It is one of the many species within
  the genus Campylobacter.
• Campylobacter species C. jejuni and C. coli cause
  diarrhea in humans.
• The organism is heat sensitive (destroyed by milk
  pasteurization temperature). It is also sensitive
  to freezing.
• The organisms are curved, S-shaped, or spiral
  rods that may form spherical or coccoids forms in
  old cultures or cultures exposed to air for
  prolonged periods.

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• Most of the species are microaerophilic. It is
  oxidase and catalase positive and does not grow
  in the presence of 3.5 NaCl or at 25 C or
  below.
• The incidence reported for gastro enteritis by
  this organism are as high as in case of
  Salmonella.
• The organism is commonly present in raw milk,
  poultry products, fresh meats, pork sausages and
  ground beef. The infective dose of C.jejuni may
  be lt1,000 organisms.

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Escherichia coli

• E. coli strains are associated with food borne
  gastroenteritis.
• These are Gram-negative asprogeneous rods that
  ferment lactose and produce dark colonies with a
  metallic sheen on Endo agar.
• The organism grows well on a large number of
  media and in many foods. They grow over a wide
  range of temperature (4 to 46 C ) and pH (4.4 to
  9.0).
• However, they grow very slowly in foods held at
  refrigerator temp. (5 C ).
• They belong to the family Enterobacteriaceae.
• The organism is also an indicator of fecal
  pollution. The organism is also capable of
  producing acid and gas and off-flavours in foods.
  

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• E. coli strains involved in foodborne-illness can
  be placed into five groups enteropathogenic
  (EPEC), enterotoxigenic (ETEC), enteroinvasive
  (EIEC), enterohemorrhagic (EHEC) and
  facultatively enteropathogenic (FEEC).
• The organism also grows in the presence of bile
  salts.
• The primary habitat of E.coli is the intestinal
  tract of most warm blooded animals. E.coli 0157
  H7 strains are unusually tolerant of acidic
  environments.

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Listeria monocytogenes

• Listeria monocytogenes in foods has attracted
  worldwide attention due to the serious illness it
  causes in human beings.
• The Listeria are Gram positive non spore forming,
  nonacid-fast rods. The organism is catalase
  positive and produces lactic acid from glucose
  and other fermentable sugars.
• It is a mesophilic organism with optimal growth
  temperature 37C but it can grow at refrigerator
  temperature also. Strains grows over the
  temperature range of 1C to 45C and pH range 4.1
  to 9.6.
• Listeria monocytogenes is widely distributed in
  nature and can be isolated from decaying
  vegetation, soil, animal feces, sewage, silage
  and water. The organism has been found in raw
  milk, pork, raw poultry, ground beef and
  vegetables.
• The HTST treatment of pasteurization is good
  enough to destroy the organism in milk.

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• SPOILAGE OF
• DAIRY PRODUCTS

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SPOILAGE OF MILK AND DAIRY PRODUCTS

• Highly perishable food because
• pH b/w 6.3-6.5
• High Moisture
• Rich Nutrients (lactose sugar, butterfat, citrate
  and nitrogenous compounds)
• Composition Protein-3.2, Carbohydrate 4.8,
  Fats 3.9, minerals-0.9
• Free aa Casein and Lactalbumin-rich N-source
• Lactose sugar-simple, fermentable
• Milk fat hydrolysed by microbial lipases.

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Spoilage Of Milk And Dairy Products

• Changes in Milk Fat
• Alkali Production
• Color Changes
• Yellow milk (Ps. Synxantha, Flavobacterium)
• Red milk (Serratia marcesans, Torula glutinis)
• Brown milk (Ps. putrfaciens)
• Blue milk (Ps. syncyannea)
• Flavor Changes
• Sour or acid flavor Clean acid flavor, Aromatic
  acid flavor, Sharp acid flavor
• Bitter flavor- Proteolysis of casein (Bacillus
  sp., Clostridium)
• Burnt/Caramel flavor (burnt milk flavor-S. lactis
  var. maltigenes)

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• Gas production accompanied by acid formation-
  mainly by coliform bacteria, Clostridium and
  gas-forming Bacillus sp.- yield H2 and CO2.
• Acid formers killed at pasteurization temps.-
  however spores of clostridium and bacillus may
  survive and cause spoilage of pasteurized milk.
• Proteolysis hydrolysis of milk proteins by m/o
  accompanied by production of bitter flavor (due
  to peptide released).
• Proteolysis is favored by storage at low temps,
  destruction of lactics and other acid formers by
  heat, destruction of formed acid in milk by molds
  and yeasts
• Eg Micrococcus, Akaligenes, Pseudomonas,
  Proteus, Flavobacterium (non sporeformers)
• Bacillus and Clostridium sp (spore formers).

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Spoilage of Raw Milk
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Spoilage of Raw Milk

• The temperature of freshly drawn milk is about
  38C.
• Milk sours rapidly if held at these temperatures.
  
• Some inhibitory substances (lactoperoxidase and
  agglutinins) are present in freshly drawn milk
  but soon become comparatively ineffective.
• Microbial spoilage of raw milk can potentially
  occur from the metabolism of lactose,
  proteinaceous compound, fatty acids
  (unsaturated), and the hydrolysis of
  triglycerides.
• The initial bacterial count of milk may range
  from less than 1000 cells/ml to 106/ml.

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• Outbreaks of illness are due to consumption of
  raw and pasteurized milk contaminated with a
  variety of organisms, including E. coli O157H7,
  Salmonella spp., Campylobacter jejuni, Yersinia
  enterocolitica, and Listeria monocytogenes.
• Raw milk may be a vehicle for the transmission of
  Borrelia burgdorferi, the agent responsible for
  Lyme disease,and it has been shown that the
  organism can survive for at least 46 days in milk
  stored at 5?C.
• E. coli O157H7 has also been shown to be able to
  survive in yogurt.

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Spoilage of Pasteurized Milk
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Spoilage of Pasteurized Milk

• Spoilage may result from either the growth of
  psychrotrophic thermoduric organisms that survive
  pasteurisation, or post-pasteurisation
  contamination by psychrotrophs.
• Thermoduric spoilage Gram-positive
  sporeformers,mainly Bacillus spp., Clostridium
  and organisms with heat-resistant vegetative
  cells, such as Micrococcus, Lactobacillus,
  Enterococcus,Streptococcus, Corynebacterium and
  Alcaligenes.
• However, at slightly higher temperatures (7 - 8
  C), B. cereus in particular may grow quite
  rapidly, producing a type of spoilage known as
  'bitty cream' or 'sweet curdling, caused by the
  action of lecithinase on the phospholipids in fat
  globules.

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• Post-process contamination The majority of
  post-process contaminants are Gram-negative
  bacteria.
• Initially, Enterobacteriaceae, such as
  Enterobacter, Cronobacter, and Citrobacter,
  predominate, but Gram-negative psychrotrophs,
  principally pseudomonas, but also Alcaligenes,
  Klebsiella, Acinetobacter and Flavobacterium, are
  more important in terms of eventual spoilage.
• Spoilage by Gramnegative psychrotrophs usually
  takes the form of off-flavours, often described
  as unclean, fruity, rancid or putrid.

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• Ropiness and partial coagulation may also occur
  occasionally.
• Yeast and mould are also indicators of
  post-process contamination. Their presence and
  growth contribute to fruity and yeasty flavours
  in milk.

Food Food Types of Spoilage Spoilage Microorganisms
DAIRY DAIRY
MILK (pasteurized) Bitterness Sliminess (high pH) Pseudomonas spp.
MILK (pasteurized) Souring Lactobacillus thermophilus
MILK (pasteurized) Sweet curdling Bacillus cereus
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MICROORANGISM ASSOCIATED WITH PASTEURISED MILK AND PRODUCTS
Salmonella
Campylobacter spp
Listeria monocytogenes
E. coli O157H7
Yersinia enterocolitica
Staphylococcus aureus
Bacillus spp.
Mycobacterium avium subsp.

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Spoilage of Milk Powder
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Spoilage of Milk Powder

• Spoils only when moisture content gt0.8 (alarm
  water content).
• Spoilt by molds only.
• Eg. Mucor, Aspergillus, Penicillium, Rhizopus sp.
• Milk powder when spoilt becomes lumpy in texture.
  
• Recently, it has been found that contamination of
  powdered infant milk by Enterobacter sakazakii
  can lead to infant death.
• Contamination of milk powder due to S. aureus
  enterotoxin in some cases.

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Spoilage of Butter
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Spoilage of Butter

• Butter not easily spoilt by m/os
• Contains Min. 80 fats-spoilt only by lipophillic
  m/os.
• Stored at v. low temp-spoilt only by
  psychrophiles.
• Contains only 15 water-low aw.
• Contains antimicrobial substances like Diacetyl
  (produced naturally).
• Usually salted.
• Wrapper of butter impregnated with sodium
  diacetyl (chemical preservative).

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Spoilage of Butter

• Skunk-like flavor -Pseudomonas mephitica
• Unclean flavor -coliform bacteria
• Musty flavor -molds and actinomycetes
• Barny flavor -Enterobacter
• Fishy flavor -Aeromonas hydrophila
• Rancidity -hydrolysis of fats by lipases of m/os
  like Pseudomonas, Aeromonas
• Surface taint/Putridity - Ps. Putrefaciens
  (swetty feet like odour-due to producn of
  volatile organic acids like isovaleric acid)

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Spoilage of Butter

• Surface discoloration growth of bacteria as
  well as fungi
• Green discoloration- Penicillium sp.
• Greenish black discoloration- Cladosporium
• Bright red/pink discoloration Fusarium
• Yellow-orange discoloration Geotrichum
• Brownsih-grey discoloration Alternaria

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Beneficial Microbes in Milk

• Milk from cows, sheep, goats and humans is rich
  in microorganisms
• Commercially processed milk contains few
  beneficial bacteria.
• Lactic acid bacteria, the most abundant
  microorganisms found in milk, facilitate dairy
  fermentation and promote health.

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• Lactobacillus
• Lactobacillus is a species of lactic acid
  bacteria
• Lactobacillus casei and rhamnosus - abundant in
  raw milk and are commonly used as probiotics
• Lactobacillus acidophilus occurs in fermented
  milks, ice cream, some cheeses, frozen yogurt and
  sometimes as an added culture in unfermented
  milks.
• Lactobacillus bulgaricus, added to milk to curdle
  it used in yogurt alongwith streptococcus
  thermophillus

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• Streptococcus
• Frequently used for culturing cheese and yogurt
  as they ferment lactose
• Also produce lactase - helping people with
  lactose intolerance to digest milk more
  efficiently to lactate.
• Its a probiotic that helps improve digestion

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• Bifidobacterium
• Bifidobacterium bifidum occurs along with
  lactobacillus acidophilus in fermented milks, ice
  cream, some cheeses, frozen yogurt and sometimes
  as an added culture in unfermented milks
• have positive effects on health including
• protection against infection by pathogenic
  bacteria
• stimulation of the immune system
• decrease of cancer risk
• lowering of serum cholesterol and aiding in the
  digestion of lactose for those who are lactose
  intolerant.

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• Enterococcus
• Found in abundance in raw cow, goat, sheep and
  human milk.
•  Protect against infection and help to relieve
  diarrhoea
• particularly beneficial for animals as well as
  humans and are frequently added as probiotics

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Fermented dairy products

• Dairy foods - fermented with lactic acid
  bacteria such as Lactobacillus, Lactococcus
  and Leuconostoc.
• There are variety of fermented dairy products
  available in market
• E.g. Cheese by variety of bacteria and molds
• Yogurt streptococcus thermophilus and
  lactobacillus bulgaricus
• Kefir mixture of bacteria and yeast

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Hygienic measures

• Hygienic measures - aim at suppressing
  pathogens and inhibiting spoilage organisms
• Protection against pathogenic microorganisms
• Pasteurization generally HTST is preferred
• UHT milk is also supposed to be free from
  pathogens

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• Measures against Spoilage Organisms
• Cleaning and disinfection of the milking
  equipment is essential
• Cooling to slow down bacterial growth in milk
• Thermalization and then cooling reduce
  psychrotropes in milk

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REFERENCE

• https//www.ilri.org/InfoServ/Webpub/fulldocs/ilca
  _manual4/Microbiology.htmP122_10360
• FOOD MICROBIOLOGY by Frazier
• DAIRY SCIENCE AND TECHNOLOGY by CRC Publications