Microbiology and Deterioration of Microbiology and Deterioration of Milk and Milk ProductsMilk and Milk Products

• Milk constitutes an excellent medium for the Milk constitutes an excellent medium for the growth of micro-organisms.growth of micro-organisms.

• Freshly drawn milk from healthy animals Freshly drawn milk from healthy animals contains a small number of harmless micro-contains a small number of harmless micro-organisms, however, during milking process and organisms, however, during milking process and storage the contamination takes place, the extent storage the contamination takes place, the extent of which depends upon the hygienic measures of which depends upon the hygienic measures taken before, during and after milking process taken before, during and after milking process andand storage conditions observed thereafterstorage conditions observed thereafter..

Nearly all the changes that take place in Nearly all the changes that take place in the flavor and appearance of the milk after it the flavor and appearance of the milk after it is drawn form the cow, are the result of the is drawn form the cow, are the result of the activities of m.o ,therefore, it is very activities of m.o ,therefore, it is very essential to control these m.oessential to control these m.o

Significance of Significance of m.om.o. in milk:. in milk:Microbial content serves as an indicator of Microbial content serves as an indicator of

production conditions and sanitary quality of milk.production conditions and sanitary quality of milk.Prevention of spoilage.Prevention of spoilage. Prevention of milk borne illnesses.Prevention of milk borne illnesses.Production of dairy products with desired Production of dairy products with desired

characteristics imparted via m.o. introductioncharacteristics imparted via m.o. introduction

Micro-organisms in MilkMicro-organisms in MilkThe types of m.o found in milk vary The types of m.o found in milk vary

considerablyconsiderably Bacteria, yeasts, moulds and Bacteria, yeasts, moulds and

bacteriophages are commonly encountered.bacteriophages are commonly encountered. Viruses and protozoa are seldom observed Viruses and protozoa are seldom observed

in milk, except as occasional contaminants.in milk, except as occasional contaminants.

Bacteria Bacteria

Most common and most numerous of m.o Most common and most numerous of m.o found in milk and milk products .found in milk and milk products .

They belong to four main groups:They belong to four main groups:1.1. Gram +ive cocci Gram +ive cocci 2.2. Gram +ive non-spore forming rodsGram +ive non-spore forming rods3.3. Gram +ive spore-forming rodsGram +ive spore-forming rods4.4. Gram -ive non-spore forming rods.Gram -ive non-spore forming rods.

LABLAB Normally present in milk and they are Normally present in milk and they are

also used as starter culture for the also used as starter culture for the production of cultured dairy products. production of cultured dairy products. They ferment lactose and yield lactic acid.They ferment lactose and yield lactic acid.

+

++++

LAB reclassified (older names in parenthesis).LAB reclassified (older names in parenthesis).

i.i. Lactococci Lactococci

• L.delbrueckii sub sps.lactis (Str.Lactis)L.delbrueckii sub sps.lactis (Str.Lactis)

• L. Lactis sub sps. Cremoris (Str. Cremoris)L. Lactis sub sps. Cremoris (Str. Cremoris)

ii.ii. LactobaciliLactobacili

• L. CaseiL. Casei

• L. delbrueckii sub.sps.lactisL. delbrueckii sub.sps.lactis (L. Lactis).(L. Lactis).

• L. delbrueckii sub Sps. bulgaricus L. delbrueckii sub Sps. bulgaricus (L.bulgaricus)(L.bulgaricus)

iii.iii. LeuconostocLeuconostoc

ColiformsColiforms• Facultative anaerobesFacultative anaerobes• optimum growth @ 37optimum growth @ 3700c c • indicator organism and are closely indicator organism and are closely

associated the presence of pathogens associated the presence of pathogens but not necessary pathogens but not necessary pathogens themselves. themselves.

• They ferment lactose with prodThey ferment lactose with prodnn of acid of acid and gas and gas

• cause rapid spoilage of milk cause rapid spoilage of milk • they are killed by pasteurizationthey are killed by pasteurization• Their presence is an indication of PP Their presence is an indication of PP

contamination of milk. contamination of milk.

Yeast Yeast Most frequently encountered in raw cream during Most frequently encountered in raw cream during

hot weather produce acid and cohot weather produce acid and co22. They are . They are

potential contaminants throughout the year.potential contaminants throughout the year.

Moulds Moulds Their growth is visible as a fuzzy or fluffy growth Their growth is visible as a fuzzy or fluffy growth

on the surface of milk and milk products.on the surface of milk and milk products. They may be black, green, grey, blue or white.They may be black, green, grey, blue or white. They discolor the product and often produce They discolor the product and often produce

repulsive undesirable off odors repulsive undesirable off odors Essential in production of certain varieties of Essential in production of certain varieties of

cheese.cheese.

BacteriophagesBacteriophages

Particularly obnoxious in starter cultures used for Particularly obnoxious in starter cultures used for the production of cultured dairy products.the production of cultured dairy products.

Phages kill bacterial culture and entire fermentation Phages kill bacterial culture and entire fermentation process fails (slow or dead vat).process fails (slow or dead vat).

Factors affecting growth of m.o in milkFactors affecting growth of m.o in milk

1.1. Food supplyFood supply((HH22o,energy,C,N Vit. &Mineral sourceo,energy,C,N Vit. &Mineral source))

2.2. MoistureMoisture ( ( aaww ) )

3.3. Oxygen supply Oxygen supply ((Obligate aerobes ,facultative Obligate aerobes ,facultative ,micoaerophillic , Aerotolerant anaerobes,obligate anaerobes),micoaerophillic , Aerotolerant anaerobes,obligate anaerobes)

4.4. Acidity and pAcidity and pH H (Acidophilic) (Acidophilic)

5.5. PreservativesPreservatives

6.6. Light ( phototrophic)Light ( phototrophic)

7.7. Concentration (osmophillic yeasts)Concentration (osmophillic yeasts)

8.8. Temperature Temperature (psychrotrophs- 20-30-07 , (psychrotrophs- 20-30-07 , Mesophiles-30-40,Thermophiles-55-65Mesophiles-30-40,Thermophiles-55-65oo c c

9.9. Antimicrobial constituentsAntimicrobial constituents

Products of microbial growth in milkProducts of microbial growth in milk

~Enzymes ~ Decomposition products of protein, ~Enzymes ~ Decomposition products of protein, fat & CHO etc. ~ Pigments , Toxins etc fat & CHO etc. ~ Pigments , Toxins etc

(mycotoxins & bacterial toxin ) (mycotoxins & bacterial toxin )

Result of microbial growth in milk (SpoilageResult of microbial growth in milk (Spoilage))o Principal cause are PsychrotrophsPrincipal cause are Psychrotrophso Most of these are destroyed by pasteurizationMost of these are destroyed by pasteurizationo Some may survive e.g. Some may survive e.g. Pseudomonas fluorescens , Pseudomonas fluorescens ,

Pseudomonas fragiPseudomonas fragio Other species and strains that survive pasteurization Other species and strains that survive pasteurization

and grow at Refrigeration temp. ,Produce heat stable and grow at Refrigeration temp. ,Produce heat stable proteolytic & lipolytic enzymes and cause spoilageproteolytic & lipolytic enzymes and cause spoilage : : ~~Bacillus ~ Clostridium ~ Cornebacterium Bacillus ~ Clostridium ~ Cornebacterium ~Arthrobacter ~ Lactobacillus ~ Microbacterium ~Arthrobacter ~ Lactobacillus ~ Microbacterium ~ Micococcus~ Micococcus

~ Streptococcus~ Streptococcus

Deteriorative changes:Deteriorative changes:

SouringSouring- - LLactose fermentation LA, VFAactose fermentation LA, VFA Souring & gassinessSouring & gassiness- coliforms- coliforms acid & gas acid & gas Aroma producAroma production- starter culture tion- starter culture diacetyl diacetyl ProteolysisProteolysis – unpleasant odors- undesirable. – unpleasant odors- undesirable.

controlledcontrolled desirable –cheese prod desirable –cheese prodnn.. RopinesRopiness –Milk drawn into long threads s –Milk drawn into long threads (Alkaligenes viscolactis) (Alkaligenes viscolactis) Sweet curdling Sweet curdling –due to prod–due to prodnn of rennin like of rennin like

enzyme which curdles without souring enzyme which curdles without souring Stormy fermentationStormy fermentation: Rapid fermentation by : Rapid fermentation by Clostridium perfringensClostridium perfringens

Color changes Color changes Pseud.syncyaneaPseud.syncyanea(blue)(blue);;Pseud.synxanthaPseud.synxantha(yellow)(yellow);; Serratia marcescens Serratia marcescens (red)(red)

Pathogenic micro-organisms in mPathogenic micro-organisms in milkilk• Food borne illnesses occur as a result of :Food borne illnesses occur as a result of : ~Ingestion of raw milk~Ingestion of raw milk ~Improper pasteurization~Improper pasteurization ~Poor handling / storage leading to PP ~Poor handling / storage leading to PP

contaminationcontamination• Measures to decrease the threat :Measures to decrease the threat : ~Hygienic production practices~Hygienic production practices

~~Proper handling and storageProper handling and storage

~Pasteurization~Pasteurization

Bacterial Pathogens of current concernBacterial Pathogens of current concern

Bacillus cereusBacillus cereus

Listeria monocytogensListeria monocytogens

Yersinea entrocoliticaYersinea entrocolitica

Salmonella spp.Salmonella spp.

E.coli O 157:HE.coli O 157:H77

Compylobacter jejuniCompylobacter jejuni

Coxeilla burnetiiCoxeilla burnetii

MouldsMoulds(Aspergillus,Fusarium,Penicillium(Aspergillus,Fusarium,Penicillium) ) grow grow in milk in milk and milk products & produce potentially hazardous and milk products & produce potentially hazardous mycotoxinsmycotoxins

Means of destruction of micro-organismsMeans of destruction of micro-organisms1.1. Heat - pasteurization , sterilization etc.Heat - pasteurization , sterilization etc.2.2. Ionizing Radiations- UV., gamma raysIonizing Radiations- UV., gamma rays3.3. High frequency sound waves- super & High frequency sound waves- super &

ultrasonicultrasonic4.4. Electricity - by the heat generated.Electricity - by the heat generated.

5.5. Pressure - 600 x > atmospheric pressurePressure - 600 x > atmospheric pressure

6.6. Chemicals Acids ,alkalis, halogens HChemicals Acids ,alkalis, halogens H22 0 022 etc. etc.

Microbiological Standards for milk & Milk Microbiological Standards for milk & Milk Products Products

( BIS & other related monographs) ( BIS & other related monographs)

Native Inhibitory substances in milk:Native Inhibitory substances in milk:

a)a) Immune system.Immune system.

b)b) Phagocytosis.Phagocytosis.

c)c) Bacteriostatic & bacteriacidal protein Bacteriostatic & bacteriacidal protein

HACCPHACCP Raw and end-products may be tested for Raw and end-products may be tested for

the presence, level, or absence of the presence, level, or absence of microorganisms. microorganisms.

Traditionally these practices were used Traditionally these practices were used to reduce manufacturing defects in dairy to reduce manufacturing defects in dairy products and ensure compliance with products and ensure compliance with specifications and regulations, however, specifications and regulations, however, they have many drawbacks:they have many drawbacks:

1.1. Destructive and time consuming Destructive and time consuming

2.2. Slow response Slow response

3.3. Small sample sizeSmall sample size4.4. Delays in the release of foodDelays in the release of food In 1960’s the Pillsbury company, the U.S. In 1960’s the Pillsbury company, the U.S.

Army, and NASA introduced a system for Army, and NASA introduced a system for assuring pathogen free foods for the space assuring pathogen free foods for the space programme. programme.

This system, called This system, called Hazard Analysis and Hazard Analysis and Critical Control Points Critical Control Points (HACCP), is a focus on (HACCP), is a focus on critical food safety areas as part of TQM .critical food safety areas as part of TQM .

Involves a critical examination of entire food Involves a critical examination of entire food manufacturing process to determine every manufacturing process to determine every step where there is a possibility of physical, step where there is a possibility of physical, chemical, or microbiological contamination chemical, or microbiological contamination which would render food unsafe or which would render food unsafe or unacceptable for human consumption .unacceptable for human consumption .

These identified points are critical control These identified points are critical control points (CCP). points (CCP).

Seven principles to HACCPSeven principles to HACCP::1.1. analyze hazardsanalyze hazards2.2. determine CCPsdetermine CCPs3.3. establish critical limitsestablish critical limits4.4. establish monitoring proceduresestablish monitoring procedures5.5. establish deviation proceduresestablish deviation procedures6.6. establish verification proceduresestablish verification procedures7.7. establish record keeping proceduresestablish record keeping proceduresBefore these principles can be put into place, a Before these principles can be put into place, a

prerequisite programmeprerequisite programme and and preliminary setuppreliminary setup is necessary.is necessary.

Prerequisite ProgrammePrerequisite Programmeo Premise control Premise control o receiving and storage control receiving and storage control o equipment performance and maintenance equipment performance and maintenance

controlcontrolo Personnel training Personnel training o sanitation sanitation o recall procedurerecall procedure§ Preliminary setupPreliminary setupassemble teamassemble teamdescribe the product describe the product identify intended use identify intended use construct flow diagram and plant schematic construct flow diagram and plant schematic verify the diagram on siteverify the diagram on site

Starter CulturesStarter Cultures

Starter cultures are those microorganisms Starter cultures are those microorganisms that are used in the production of that are used in the production of cultured dairy products such as yogurt cultured dairy products such as yogurt and cheese. and cheese.

The natural micro flora of milk is either The natural micro flora of milk is either inefficient, uncontrollable and inefficient, uncontrollable and unpredictable, or is destroyed altogether unpredictable, or is destroyed altogether by the heat treatments given to the milk. by the heat treatments given to the milk.

A starter culture can provide desired A starter culture can provide desired characteristics in a more controlled and characteristics in a more controlled and predictable fermentation.predictable fermentation.

The primary function of lactic starters is The primary function of lactic starters is the production of lactic acid from lactose. the production of lactic acid from lactose. Other functions of starter culture may Other functions of starter culture may include the following :include the following :

Flavour ,aroma, and alcohol productionFlavour ,aroma, and alcohol production proteolytic and lipolytic activitiesproteolytic and lipolytic activities inhibitioninhibition of undesirable organismsof undesirable organisms

There are two groups of lactic starter cultures:There are two groups of lactic starter cultures:

1.1. Simple or definedSimple or defined: single strain, or more than : single strain, or more than one in which the number is known.one in which the number is known.

2.2. Mixed or compoundMixed or compound: more than one strain : more than one strain each providing its own specific each providing its own specific characteristics characteristics

Starter cultures may be categorized as Starter cultures may be categorized as mesophillic or thermophillic:mesophillic or thermophillic:

MesophilicMesophilico Lactococcus lactis subsp. CremorisLactococcus lactis subsp. Cremoriso L. delbrueckii subsp. LactisL. delbrueckii subsp. Lactis

o L. lactis subsp. Lactis biovar diacetylactis L. lactis subsp. Lactis biovar diacetylactis

o Leuconostoc mesenteroides subsp. Leuconostoc mesenteroides subsp. CreamorisCreamoris

ThermophilicThermophilic

o Streptococcus salivarius subsp. Streptococcus salivarius subsp. Thermophilus (S. thermophilus)Thermophilus (S. thermophilus)

o Lactobcillus delbrueckii subsp. bulgaricus Lactobcillus delbrueckii subsp. bulgaricus

o L. delbruekii subsp. Lactis L. delbruekii subsp. Lactis

o L. helveticusL. helveticus

o L. plantarum L. plantarum

o L.caseiL.casei

Mixtures of mesophillic and Mixtures of mesophillic and thermophillic microorganisms can also thermophillic microorganisms can also be used as in the production of some be used as in the production of some cheeses.cheeses.

Starter culture preparationStarter culture preparation Commercial manufacturers provide starter Commercial manufacturers provide starter

cultures in lyophilized (freez-dryed), frozen or cultures in lyophilized (freez-dryed), frozen or spary- dried forms.spary- dried forms.

The dairy product manufacturers need to The dairy product manufacturers need to inoculate the culture into milk or other inoculate the culture into milk or other suitable substrate. suitable substrate.

There are a number of steps necessary for There are a number of steps necessary for the propagation of starter culture ready for the propagation of starter culture ready for production:production:

1.1. Commercial cultureCommercial culture

2.2. Mother culture-Mother culture- first inoculation; all cultures first inoculation; all cultures will originate from this preparation will originate from this preparation

3.3. Intermediate cultureIntermediate culture - in preparation of - in preparation of large volumes of prepared starterlarge volumes of prepared starter

4.4. Bulk starter culture-Bulk starter culture- this stage is used this stage is used in dairy product production in dairy product production