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LECTURE NOTES For B. Tech (Dairy Technology) Degree

INTRODUCTORY DAIRY MICROBIOLOGY (Course No: DM 121)

By

Dr. Velugoti Padmanabha Reddy, Ph.D

Professor 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 & 40°C with the optimum growth temperature (OGT) of 37°C 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 7°C (refrigerated) but the OGT is between 15 & 20°C 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 50°C 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 40-85°C. 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 63°C\30 min. with O.G.T of 35-37°C 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 don't 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 ­ 30°C ; 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 5°C. 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 - 42°C. 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 42°C. May grow at 41°C 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 37°C 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: 30-37°C, but grow at below 10 and above 45°C 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 37°C, 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 2-45°C 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 60°C for 1 hr and 61.7°C 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 25°C, Many species are heat resistant and survive 63°C/ 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 37°C 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 parker's 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 10°C 45°C 6.5% Nacl 9.6 pH 0.1% Methylene blue NH3 from Arginine Litmus reduction before clotting Resistance to 60°C/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 45°C but not at 10 and 53°C. OGT is 40-45°C. 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 37°C . They resist 63°C/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 10°C but not 45°C. 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 40°C 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 30°C. 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 80°C/ 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 45°C 65°C 7% Nacl Citrate utilization B. cereus Central or paracentral + + 30°C V V + B. subtilis Central or paracentral 30-40°C + + + B. stearothermophilus Subterminal to terminal + 55-65°C + + B. coagulans Subterminal to terminal + 35-45°C + V V B. megatherium Central or paracentral + 28-35°C V + +

B. cereus : Don't grow at 5°C but grows at 20-35°C. 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 20-45°C. 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 65°C 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 + 30-37°C Acetic, Butyric acids Butanol

Cl. tyrobutyricum Oval Sub-terminal Swell the cell + 37°C Butyric acid Co2, H2 from lactic acid

Cl. botulinum Oval Central to Sub-terminal + 37°C Slow increase in acidity

Cl. Perfringenes Oval Central to eccentric Large distend the cell 37°-45°C 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 45°C 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 40°C,

Used in Yoghurt, Swiss & Italian cheeses. Produces antibiotic `Bulgaricin' (stable for 1hr at 100°c) which is active against Ps.fragi and to a lesser extent to S. aureus L. acidophilus:Thermophilic, OGT 45°c; do not grow at 20°C & 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 40°c/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 ­ 35°C Antibiotic "Lactolin" is produced which is effective against G+ve organisms

BETABACTERIUM GROUP L. brevis:Serology E, Mesophilic, OGT 30°C; Do not grow at 45°C, but grow at 15°C. 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 37°C, 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:- Loeffler's 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 : 37°C 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 : 20-30°C, 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: 30-32°C 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 newborn's 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 <100-10,000/ml from normal udder, but anticipated average is 500-1000/ml in advanced countries. Micro organisms enter the udder through duct at the teat tip. The duct varies in length from 5-14mm and its surface is heavily keratinized. This keratin layer retains milk residues and exhibit antimicrobial activity. During progress of a milking, bacteria are present in the largest number at the beginning and gradually decrease. This is mainly due to mechanical dislodging of bacteria, particularly in teat canal where the number are probably highest. Discarding the first few streams of milk results in lower counts of microbes in milk. Milk from different quarters varies in numbers. Species of bacteria found in milk as it comes from udder are very limited. Micrococci Streptococci Asporogenous G+ve rods G-ve rods Bacillus spores Miscellaneous 30-99% 0-50% <10% <10% <10% <10

Micrococci are slow growing, but if allowed to grow, cause acid formation and proteolysis. They are mostly non-pathagenic. Streptococci ­ Less frequent that micrococci, St. agalactiae may be present even non clinical mastitis and thus it appears to be natural habitant of udder. Lactococcus lactis ssp lactis is never been associated with udder flora and occurs from external sources. Among G+ve rods organisms of diphtheria i.e. Corynebacterium bovis has been found in large numbers. It is non-pathogenic, but if grown causes rancidity. It has not been found from other sources such as faeces & milk products. Exterior of Udder: Number & type of organisms associated with udder vary depending on type of amount soil. Udder & teat become soiled with dung, mud, bedding material such as saw dust, straw etc. With Heavily soiled udder teats the counts may be 1 lakh cfu/ml Bedding material in winter has high number of bacteria, Main organisms .in order of maximum numbers are psychrotrophs, coliforms, Bacillus sp.In summer cows turned to pasture, the number of bacteria in bedding declines. Udder micro flora is not affected very much by washing. Sodium hypochlorite washing and accompanied by drying help in reducing in number

Teat surface predominantly micrococci and coagulase ­ve staphylococci Next predominantly faecal streptococci, but G-ve bacteria including coliforms are less. Coliforms do not survive well on teat surface. Aerobic thermoduric organisms are entirely Bacillus sp. The more frequent are B. licheniformis, B. subtilis, B. pumilis and less frequent ones are B. cereus, B. circulans Greater number of bacteria gets into milk from the surface of the cow's body and from utensils that have not been washed thoroughly. teat surface may also contain clostridia spores that are usually found in cows fodder, bedding & faeces Prevention: Prevent regular soiling of teat surface Wash with disinfectant Drying of teats before milking. Chlorine: presence of organic matter interferes & it is also irritant to hands. QAC: Satisfactory Soaps: Only detergent action. In herd with recurring mastitis chlorine between 200 to 400 ppm, & QAC 200ppm were also not effective, in preventing spread of mastitis. Cloths ­ Used for only one cow separately and Moistened in sanitizers after each use. Paper towels are preferable.

Coat of Cow: Coat serves as vehicle to contribute bacteria directly to milk. Clipping of hair around udder, flanks and tail reduces the count of bacteria in milk. Coat may indirectly contribute organisms into air of the barn, especially Bacillus sp. Coat may carry bacteria from stagnant water pools especially ropy milk organisms. Coliforms may gain from soil & manure Prevention:1. Periodic clipping of hair 2. Regular brushing of coat. 3. Machine milking Air Air is not important source of bacteria in milk. Air count in sheds rarely exceeds 200 cfu/litre. Micrococci account for >50% aerial microflora. Others are aerobic spore formers, coryneforms, streptococci and G-ve rods. Next important group is molds. Practices that increase aerial counts through milk Sweeping or short time before milking Handling hay and feed just before milking Brushing animals just before milking Having dusty bedding material Allowing dust and dirt to accumulate on wall and ceiling.

Prevention Use of small ­ top milk pails Machine milking Clean milk sheds Milker: Milker with infected wounds on hands contribute pathogenic streptococcus & micrococcus During the wet hand milking lubricant enters milk and adds bacteria from hands and teats. Pathogens causing typhoid, paratyphoid, dysentery, scarlet fever, septic sore threat. Diphtheria, cholera etc are contributed from humans. Action of milker may dislodge dust and dirt and increase air contamination. Water Water used should be potable & good in terms of bacteriological quality. Direct sources of contamination are: a) Storage tanks, not protected from rodents, birds, insects and dust. b) Hoses c) Water troughs Untreated water supplies from bore wells, lakes and rivers may be contaminated at source with faecal streptococci, Coliforms, G-ve rods, Lactic acid bacteria, Bacillus sp., and corynebacterium sp. Chlorination ­ with hypochlorites is recommended Warm water used (37°c) for udder washing is potent source of pseudomonas & Coliforms Utensils Inadequately cleaned/sanitized milk contact surfaces may contribute to the contamination.Milk cans that are not cleaned properly & lids are kept when they are still moist, result in multiplication of bacteria. Important organisms are Bacillus cereus, thermoduric bacteria Milking machines: microflora varies qualitatively & quantitatively. Predominant are thermoduric micrococci, bacillus sp. Occurring in less numbers are coliforms and streptococci while the spore formers constitute minor flora.

CLEAN MILK: Clean milk is that which comes from the healthy cows, is of good flavour; is free from dirt; contains relatively few bacteria and none of which are harmful to human health. High quality milk should be of longer keeping quality of proper nutritive value of normal taste, color, odor of excellent from health stand point of view free from extraneous matter in it. Important organisms in found in milk: 1. 2. 3. Pathogens cause mastitis and appear in milk are S. aureus, Ps. aeruginosa, St. agalactiae,. E.coli , List monocytogenes Organisms from systemic infections shedding in milk are Brucella sp., Coxiella burnetii, Mycobacterium bovis Environment i.e. soil, litter, feed, water faeces contaminating teats, udder Adjacent skin and nail ultimately entering the milk are B. cereus Cl. perfringens : from soil, litter, feed and faeces Salmonella : from feed or faeces Yersinia : from water or faeces of Infected animal E.coli, Enterobacter, Enterococci : from faeces List. monocytogenes : from feed especially silage 4. Organisms of the diseases such Typhoid, septic sore throat, Diphtheria, scarlet fever, Hepatitis A, poliomyelitis, Staphylococcal enterotoxicosis are from the infected milker or other milk handlers

Unhygienic practices are mostly present into rural areas. Practices are related to 1) Milk animal 2) Milker 3) Milking process 4) Environment Milk animals:Pathogens and Non-pathogens may enter milk if any one of the following conditions exist. A. Unhealthy animal: mastitis udder udder carrying ulcers on teats systemic diseases of animals such as tuberculosis, brucellosis

B. Unclean udder and body of the animal: Unwashed due to lack of adequate water supply Washing with unclean water before milking

Infected faces, urine & other discharges contaminating the body especially, the flanks and hind quarters Brushing of body of the animal just before milking may also contaminate milk due to spreading dust.

Milker: Unhealthy milker directly transmits disease producing microbes to milk through sneezing, coughing etc Uncleaned hands and cloths of milker

Milking processes: Incomplete milking ­ this may lead to multiplication of organisms in the left over milk, thus contaminating the next lot. Wrong method of milking ­ knuckling method of milking may cause injury to the teats and lead to udder infection Un-cleaned utensils for milk collection

Environment: 1. Poor housing:- Usually in improperly ventilated houses,Floors made up of mud without adequate drainage 2. Feeding :- Spreading of straw, silage and other feed materials on flooring, Use of hay and dusty-feed stuffs immediately before milking. These two causes may lead to increasingly polluted air, filth (manure mucus etc) and flies around the animal. 3. Unclean surroundings:-The Sewage, manure pits, stagnant water etc, cause environmental pollution. Prevention:Animals: Purchased animals are tested for Tuberculosis and brucellosis and quarantined Animals are regularly checked for mastitis and udder lesions Isolation of infected animals and following prescribed treatment Milk of infected animal should not be mixed with bulk milk. Cleanliness of udder and body: Clean the animals atleast 15min before milking. Clip the long hair around flanks, udder and tail. Udder and teats are cleaned just before milking and wiping with a cloth. use warm water if possible & usage of chlorinated water is good practice.

Milker: Should be free from infectious diseases Should cut his nails regularly (to avoid staphyolococci) Should clean hands with soap and water before milking Should wear clean cloths and caps

Utensils: Use utensils which have smooth surface and free from dents and crevices Should be cleaned with detergent ­ sanitizer formulae (Sod.carb+Iodophores) Do not use wider mouth utensils

Milking processes Complete milking and elimination of fore milk which contains higher bacteria. Follow full hand milking; Dry milking and Fat milking ( a flavourless fat as lubricant; not used widely but a good method). Use strip cup test Cool milk on farm preferably to <5°c

Environment Separate housing away from human dwelling, sewage, manure pits and stagnant water pools. House ­ well ventilated; normal sloppy drainage, water proof floors, hard and easy to clean. Feed manger smooth without ridges Ample sunlight ­ North & south direction. Air space 500 cft/cow Dry bedding; for tick free bedding DDT may be used to kill Periodic lime washing

Others: General quietness in the shed Speedy milking is desirable Clean and adequate water supply on the farm Use wet or pellet food stuffs during milking Keep feed and weed flavours out of milk Proper manure disposal ­ pit away from farm and remove manure periodically Control of flies and insects Incentive payment plan Educative propaganda.

EXAMINATION OF MILK FOR MICROBIOLOGICAL QUALITY There are different approaches for checking the bacteriological quality of raw milk and they are broadly classified into 1) Indirect tests such as dye reduction tests and 2) Direct tests such as direct microscopic count test and standard plate count tests. 1. INDIRECT METHODS FOR GRADING THE RAW MILK: Dye reduction tests are the indirect methods of assessing the microbiological quality of milk. They are based on the metabolic activity of the microbes and rely on the oxidationreduction potential of milk. In the tests a correlation is made between the time required for the reduction of the dye and probable bacterial population of milk. The bacteria present in the milk multiply and consume oxygen for their metabolic activity. The rate of depletion of oxygen influences the oxidation-reduction potential and depends on the number and type of bacteria. The bacterial activity is depended on the dehydrogenase enzymes, which are flavin enzymes, which can transfer hydrogen/ electron from the organic substrate to biological acceptors or in their absence to the reduction dyes. With the depletion of oxygen the redox potential of milk gets reduced to such an extent where it causes the reduction of the dye changing its colour. A. Methylene blue reduction test: The test is useful in assessing the bacteriological quality of milk by determination of the time taken for the reduction of methylene blue in milk indicated by its colour change. Milk in udder exhibits to have a very low oxidation-reduction potential which raises to + 0.3 volts during the process of milking, dumping and cooling due to the incorporation of oxygen. At this potential the methylene blue will be in oxidized form and have a blue color. The dye gets reduced when the Oxidation-Reduction potential is decreased to = 0.06 to - 0.01 volts due to the depletion of oxygen from milk as a result of metabolic activity of the organisms. The greater is the number of microorganisms in milk, the greater is the metabolic activity and the faster is the reduction of methylene blue and vice-versa. Standard solution of methylene blue: One tablet of methylene blue thiocyante or chloride (BDH or Merck) is dissolved in 200 ml of cold sterile glass distilled water by gentle heating to facilitate dissolving and then add another 600 ml distilled water. One ml of this solution when mixed with 10 ml of milk gives rise to a final concentration of 1 in 3,00,000 of the dye. The samples of the milk are mixed thoroughly. If the milk is in a bottle/ sachet it shall be inverted atleast 25 times by a rapid rotary movement of the wrist in order to mix the fat uniformly with the milk. Take 10 ml of milk into a test tube and add 1 ml of standard methylene blue solution. Invert the test tubes to mix the milk and methylene blue solution. Place the test tubes in a thermostatically maintained water bath at 37 0C+ 0.50C and note down the time of incubation. Observe the test tubes after 30 minutes for reduction of dye (Decolourization). If there is no decolourization the tubes are inverted once and transferred to the water bath for further incubation. After 30 minutes, at an interval of every one-hour continue to observe for the reduction of dye. The milk shall be regarded as decolorized when the complete column of milk is completely decolorized or is decolorized up to within 5mm of the surface.

The quality of raw milk is judged by using the following specifications MBR Time (Hrs) 5 and above 3 and 4 1 and 2 1/2 and below Quality of raw milk Very good Good Fair Poor

B. Resazurin reduction test The principle of this test is same as that of methylene blue reduction test. However, unlike methylene blue the resazurin dye undergoes reduction through a series of colour shades viz., blue, purple, lavender, pink before completely getting reduced to colourless. The reszurin dye which is blue in colour at the oxidation-reduction potential of + 0.3 volts changes into pink colour compound (resorufin) as the redox potential reduces to + 0.2 volts. This reaction is irreversible. However the colour of dye changes to colourless (dihydro resorufin) when the redox potential is reduced to +0.1 or less. Usually the degree of reduction of the dye is measured after a fixed time of incubation of the milk sample in the presence of dye. The reduction of the dye to a particular shade of colour is dependent upon the extent of depletion of oxygen by the metabolic activity of microorganisms. The colour change is measured with the help of a Lovibond comporator and a standard colour disc. Standard solution of Resazurin: One tablet of Resazurin is dissolved in 50 ml of cold sterile glass distilled water by gentle heating to facilitate dissolving. This is the bench solution for direct use and should always be used as fresh solution. Alternatively dissolve 0.05 g of resazurin powder in 100 ml of distilled water and boil the contents for 1/2 hour. This will make a standard solution of 0.05%, which should be always kept, in a cool and dark place stored in an amber colored bottle. The bench solution (0.005%) for regular use should be prepared freshly by diluting the standard solution with distilled water i.e. 1 ml of standard solution with 10 ml of distilled water. Take 10 ml of milk into a test tube and add 1 ml of working solution of resazurin solution. Invert the test tubes to mix the milk and methylene blue solution.Place the test tubes in a thermostatically maintained water bath at 37 0C+ 0.50C and note down the time of incubation. At the end of one hour of incubation match the colour of the milk with one of the colour standards of resazurin disc Resazurin disc No 4 or higher 31/2 to 1 1/2 and below Quality of milk Good Fair Poor

2. DIRECT METHODS A. Direct Microscopic Count test: Direct microscopic count test is based on the microscopic examination of stained film of a measured volume of milk spread over a specified area on glass slides. The method is useful for a rapid estimation of the total bacterial population (both live and dead cells) of a sample of milk and also in giving useful information for tracing the sources of contamination of milk with bacteria. In this test, prepared milk smear on one square centimeter area is stained with a special stain called Newman's stain and examined under microscope. Each microscopic field examined represents a quantitative aliquot of the milk sample. The number of microscopic fields occurring in one square centimeter area of the milk smear will vary as the diameter of the microscopic field varies with each microscope. This necessitates the calculation of microscopic factor. Determination of Microscopic factor (MF): Microscopic field is the area of the field observed through the microscope. It is given by r2 where the r is the radius of the field. The diameter of the microscopic field is measured with the help of stage micrometer. Each division on the stage micrometer is equivalent to 0.01 mm. The diameter of the microscopic field varies with length of the draw tube, objective and ocular tube. As the diameter of the field varies the number of microscopic fields occurring in one square centimeter area (100 sq mm) will vary and hence there is need to calculate microscopic factor. Microscopic factor(MF) is calculated as follows: MF = Area of the smearr (1 sq.cm) Area of microscopic field ( r2) = 100 sq mm x 1 r2 x 0.01 10,000 r2 Preparation of milk film or smear with the help of a sterile Breed's pipette Take 0.01 ml milk and spread very evenly on a grease free slide. Dry the smear on a warm surface at 40-45°C with in 5 minutes. Rapid drying results in cracked surfaces on the film or peels off on further processing. Immerse the slide in Newman's stain (in a staining jar) for ½ to 1 minute. Newman's stain is intended to remove the milk fat, fix the smear and stain the bacteria in a single operation. The tetrachloroethane of the stain helps to dissolve the milk fat globules, ethyl alcohol fixes the smear and methylene blue stains the smear. Microscopic examination of stained film: Examine under oil immersion and Count the number of organisms (individual cells or clumps of cells) in a number of fields of the film by examining under the oil immersion objective. The field for counting the bacterial cells is selected at random. The number of microscopic fields occurring in one square centimeter area of the smear will be very high. So only a representative number of fields depending on the concentration of bacterial cells in a microscopic field are chosen for counting the bacterial cells. = X 1 Volume of milk

The total number of fields is counted as follows Number of clumps in a field < 0.5 0.5 to 1.0 1.0 to 10 10 to 30 > 30 Number of fields to be counted 50 25 10 5 dilute the sample of milk and repeat the test

Calculate the average number of clumps per field and multiply by the microscopic factor to get the direct microscopic count per ml of milk. Direct microscopic counts per ml Bacteriological quality of milk Less than 5, 00,000 5, 00,000 to 40, 00,000 40, 00,000 to 2, 00, 00,000 Over 2, 00, 00,000 Good Fair Poor Good

Sources of contamination of milk: Type of organisms Source of contamination

Presence of cocci in clumps accompanied by Improperly cleaned milk utensils rods Excessive number of rod shaped bacteria Exposure of milk to dust and dirt especially spores Large number of cocci in pairs and short chains Large number of leucocytes cells (over 500,000 / ml) together with long chains of cocci Improper cooling of milk Mastitis infection

B. Standard plate count test: The standard plate count method is also called as pour plate technique or colony count test. It is useful in the estimation of number of viable microorganisms in the given sample of milk. The test employs the serial dilution technique for easy quantification of the organisms in view of a wide range of bacterial population that may occur in milk. The appropriate dilutions of the milk sample are mixed with a sterile nutrient medium that can support the growth of the organisms when incubated at a suitable temperature. Each bacterial colony that develops on the plate is presumed to have grown from one bacterium in the inoculum. The total number of colonies counted on the plates multiplied by the dilution factor is taken to represent number of viable organisms present in the sample. This method has been widely used with satisfactory results and is particularly suitable where low bacterial population is expected. This method is especially useful for pasteurized milk and for line testing at various stages of processing and for detecting the sources of contamination.

Preparation of dilutions of the milk sample: Transfer 1ml of the sample with a sterile pipette to 9ml of dilution blank (1st dilution) which will make 1 in 10 dilution of the milk sample. Take 1ml from 1st dilution and transfer to second 9-ml dilution blank to get 1 in 100 dilution. Mix thoroughly and transfer 1ml from second dilution to third dilution blank to make 1 in 1000 dilution and so on till a series of required dilutions of the sample are ready. Use a fresh pipette for each successive dilution. Plating the sample and preparation of plates: Transfer 1ml of each required dilution into sterile petri dish. To each petri dish add 15 to 20 ml of sterilized standard plate count agar which was previously melted and cooled to 45°C. Allow the agar to cool and set. Invert the plates and incubate at 37C for 48 hours. After the incubation determine the average of the counts in the two plates and multiply this by the dilution factor. Selection of plates and counting the colonies: Each colony is represented to have grown from a single bacterium or clump of bacteria. Select such plates of the dilution that would contain between 30 to 300 colonies. Take always the average of the duplicate plates In case of two consecutive dilutions take the average, if the higher dilution count is less than twice than the lower dilution count. If the it is more than twice the lower count then report lower computed count. In case of spreaders, Count only if spreaders occupy less than ½ of the plate and if colonies are well distributed in spreader free area If all plates show No colonies, excess spreaders, unsatisfactory results such as contamination report as Lab accident,/ Inhibition of growth/ spreaders etc., Raw milk is graded based on the following specifications SPC/ml Not exceeding 2,00,000 2,000,00-10,00,000 10,00,000-50,00,000 Over 50,00,000 Grade Very good Good Fair poor

Pasteurized milk: A standard plate count of lower than 50,000 cfu per ml. of pasteurized milk is indicative satisfactory quality. 3. Enumeration of Coliforms in Milk: Coliforms are Gram's negative, oxidase negative, non-spore forming rods which can grow aerobically or facultatively in presence of bile salts or surface active agents with similar growth inhibitory properties and are able to ferment lactose with the production of acid and gas with in 48 hours at 37°C. Their presence in milk or milk products is indicative of possible faecal contamination and are found especially when they are handled under unsanitary conditions. The presence of these organisms is considered undesirable because they produce acid, gas and objectionable taints in the milk products.

Generally coliform organisms are destroyed during pasteurization. Their presence in pasteurized milk indicates post pasteurization contamination. The test is chiefly based on the principle that the members of this group are capable of producing acid and gas from lactose in the presence of bile salts. Presence of typical coliform colonies in petri plates is taken as evidence of coliform contamination. Interpretation: Absence of coliforms in 1: 100 dilution (less than 100 per ml) in raw milk and in 1:10 dilution (less than 10 per ml) of pasteurized milk is accepted as criterion of satisfactory quality. MPN Method: The test is based on the principle that the coliforms are capable of producing acid and gas from lactose in the presence of bile salts. However, sometimes false positives may arise due to the growth of other types of microorganisms such as Clostridium, Bacillus and certain yeasts. So the test is termed as "Presumptive test". In case of doubtful cases, the completed test is commonly employed to confirm the presence of coliforms. The Presumptive test also enables to obtain some idea of the number of organisms present in milk by means of a technique called Most Probable number method. The tubes of lactose broth or MacConkey's broth inoculated with samples of milk are being tested and a count of the number of tubes, showing acid and gas production, is made and the figure is compared to MPN (statistical) table. Differentiation of Coliforms using IMViC Tests : Indole test The test is based on the production of indole from tryptophane which is detected by using Kovac's reagent. Methyl red test Methyl red is used as pH indicator to detect a large concentration of acid as end product produced by certain organisms by utilizing glucose. This indicator turns red in the pH range of 4 and yellow in the pH range of 6 Voges-Proskaur test On oxidation, acetylmethylcarbinol the neutral or non acidic end product from glucose metabolism releases a compound known as diacetyl and this is detected in the presence of Barritt's reagent producing a deep rose color. Citrate test Citrate inside the bacterial cell undergoes enzymatic degradation and finally producing pyruvic acid and carbon dioxide. This carbon dioxide combines with sodium to form sodium carbonate and converting the reaction of the medium into alkaline. Bromothymol blue indicator previously incorporated into the medium turns the medium from green into deep Prussian blue.

4. OTHER IMPORTANT GROUPS OF BACTERIA IN MILK: A. PSYCHROTROPHILIC Psychrotrophes are capable of growing at refrigeration temperature (7-10C). The common psychrotrophs belong to the genus Pseudomonas, Flavobacterium, Alcaligenes, Acetobacter etc. Their presence in milk will result in undesirable flavours during storage of milk and milk products under refrigerator conditions due to the production of enzymes that cause proteolysis and lipolysis. The plates for psychrotrophic count are incubated in a refrigerator (7-10C) for 10 days or keeping the plates in a BOD incubator at 15C for 3 days. Suggested standards for psychrotrophes in raw milk Lees than 104 cfu per ml Above 104 cfu per ml B. THERMOPHILIC The thermophilic organisms are those organisms that are capable of growing at 55°C. Majority of these organisms are spore formers such as bacillus sp., and clostridium sp. They cause problems particularly in processing (heat) plants of milk. The plates for thermophilic count are incubated at 55°C for 24 to 48 hours Excessive number of thermophilic organisms is problematic if the milk is meant for heat processing C. THERMODURIC Thermoduric bacteria constitute a major group of undesirable bacteria that are heat resistant and capable of with standing the pastuerization temperature of milk i.e. 63°C for 30 min or 72°C for 15 sec. They belong to the genus Micrococcus, Microbacterium, Bacillus and Coryneform group. These bacteria under favourable conditions multiply rapidly and bring about different types of spoilage conditions such as acid or rennet coagulation, peptonisation and production of off flavours in milk.. Theromoduric Count : Per ml Below 10,000 10,000 to 30,000 Above 30,000 Remarks Good Fair Poor - Satisfactory - Unsatisfactory

MICROBIAL INTERACTIONS Microbial association in the same environment can be Neutralism, Antagonistic (negative) and Symbiosis (Positive) Neutralism: This type of association is most unlikely as the two organisms living in a close proximity are not affected by each other. This may exist between two organisms whose growth requirements are quite different and hence affect neither kind as there will be no competition between these two for nutrients. Antagonistic association: When an organism adversely affects the environment another organism it is said be antagonistic. Antibiosis is antagonistic association between two organisms in which one is adversely affected Ex. Production of antibiotic or inhibitory substances by one organism that affect the growth or survival of another organism Lactic acid bacteria produce lactic acid that is inhibitory to spoilage organisms Symbiosis: Symbiosis is defined in the dictionary as the relationship between two (or more) organisms that live in a close association that may but is not necessarily of benefit to each. This dictionary definition is a bit misleading. In the vast majority of symbioses one or both partners gain something positive from the association. A pair of symbionts may be able to live separately, but they almost always do better in the long run by living together. Mutualism ­ each organism benefits from the association but the manner in which benefit is derived varies a.) exchange of nutrients between two species (Syntrophism) b.) Association results in metabolic end products which are different from association as compared with sum of the products of separate species. Ex. Proteus vulgaris ferment lactose and produce acid, Staphylococcus aureus ferment lactose and produce acid but together they produce gas and acid Commensalisms: It refers to a relationship between microorganisms in which one organism benefits from the association but the other organism is not affected. Host organism by its growth affects the physical or physiological environment in such a way that the commensal species is favoured. Ex: a) Facultative organism grows and produces anaerobic conditions that favour growth of anerobes b) Growth of yeasts in sugar solutions reduce the concentration of sugar thus permitting growth of bacteria Synergism: The ability of two or more organisms to bring about an effect greater than the sum of their individual effects, (or the changes usually chemical in nature that neither can accomplish alone)

UNDESIRABLE FERMENTATIONS OF MILK: Gas Production: The production of gas mainly Co2 by certain organisms in dairy products is responsible for a defect called "gassiness". In cases, where it is associated with acid production for ex. In high fat milk there is foaming and the gas escapes the partially coagulated mass and the defect is called "Frothiness" This is due to associative action of acid producing bacteria with yeasts. The production of gas in canned dairy products causes bulging of cans and the defect is called `blowing' of cans. Causative organisms: 1) 2) Coliforms: E. coli, Enterobacter aerogenes ferment lactose of milk or cream into gas and acid. These are called `early gas producer' and produce early blowing condition. Anaerobic spore forming bacteria : ex. Cl. butyricum, Cl. sporogenes-- produce gas only in anaerobic conditions. Mostly in canned dairy products like processed cheese, concentrated milk. They are called "Late gas producers' and produce late blowing condition. Lactose fermenting yeasts ex: Candida psedutropicalis, yeasts produce Co2 and small amounts of ethyl alcohol in milk and cream, whey at or below 37°C

3)

Control: 1. Avoid excessive contamination of causative organisms 2. Holding of milk & cream in ambient temperature should be avoided 3. Adequate heat treatment of milk Ropiness/ sliminess: Ropy fermentation is brought about by the growth of bacteria leading to change in consistency of the produce that forms threads of viscous masses when poured. Ropiness develops only on storage and milk is drawn out as fine threads and may appear gel like consistency. Sometimes the change is so much pronounced that the milk can be drawn into long thread. Causative organisms: I. Gram ­ve rods: Alcaligenes viscosus II. Coli-aerogenes group. This group consists of ropy strains belonging to enterobacter, citrobacter and related genera, a. Enterobacter aerogenes, b. Ent. cloacae, c. Citrobacter freundii, d. Serratia marcescens, III. Aerobic sphere formers a. B. cereus, b. B. subtilis, c. B. circulans have also been infrequently involved in ropiness. IV. LAB: S. lactics var hollandicus, L. casei, Lactobacillus delbruckeii ssp bulgaricus show ropiness before detectable acid development. Ropiness decreases as acidity increases.

Mechanism of ropiness: 1. True gums or gum like substances which are polysaccharides. Gums are galactans produced by fermentation of lactose. 2. Mucins ­ Nitrogenous mucous like substance. Peptonizing bacteria produce mucins which are combination of proteins with carbohydrate radical 3. Exopolysaccharides: produced as capsules associated with cell (or) as slime unattached to the cell. Al. viscosus produces capsular materials Sources: The bacteria causing ropiness are generally not present in the aseptically drawn milk, but come from water, dairy equipment, dust laden air, coat of cows, cow feed etc. Significance: Affect the acceptability of the products. Less frequent in pasteurized milk. Ropiness has particularly been observed in milk held at low temperature. At higher temperature ropy bacteria are overgrown by lactic acid bacteria. Ropy bacteria are not harmful to consumers. Ropiness is higher in summer, more prevalent in spring and autumn. Desirable in Scandinavian cultured products like villi in which slime is produced by lactococci. This slime may primarily consist of glycoprotein rather than polysaccharide. Yoghurt consistency to some extent is improved lactobacilli. Prevention a. b. Proteolysis: It is the process by which casein or some insoluble casein derivatives are broken down to water soluble compounds through the action of organisms or their enzymes. It is of significance because of Loss of quality of products due to proteolytic spoilage. Desirable for the manufacture of cultured dairy products Since milk contains only small amounts of Non-proteinous Nitrogenous substances, to sustain prolonged growth or maximum growth organisms depend on enzymes that hydrolyze milk proteins. Causative organisms: Psychorotrophs are the actively proteolytic organisms and grow at 7°C or less especially Pseudomonas fluorescens, Ps. fragi, Alteromonas putrefaciens Thermoduric bacteria especially Micrococcus caseolyticus, cereus, B. subtilis B. stearothermophilus B. Clean milk production and effective heat treatment Post pasteurization contamination should be avoided. by polysaccharide production by

Pseudomonas produces heat stable proteinases. s and caseins are selectively attacked but whey proteins unaffected. P. fluorescens produces extra and endo proteinases where as Ps. fragi produces extracellular neutral endopeptidases. Proteins Proteinases Proteoses Proteases Peptones Proteases Polypeptides polypeptidases Peptides Dipeptidases Amino acids

Lipolysis: Lipolysis is the hydrolysis of milk fat by lipases resulting into the accumulation of free fatty acids. The lower chain FFA particularly butyric and caproic, are responsible for the lipolytic off flavours, also referred to as rancidity (Hydrolytic rancidity). Causes of lipolysis: 1. Intrinsic milk lipase: Present in sufficient quantities to cause hydrolysis of milk fat. However, this fat globule membrane protects the milk triglycerides from attack by the lipolytic enzymes. Hence little or no lipolysis occurs in normal milk. Lipolytic microbes or enzymes: a) Psychrotrophes Pseudomonas sp. mainly Ps.fragi. Ps.fluoresens Achromobacter lipolyticum b) other types Micrococcus frendenreichii Bacillus cereus, B. subtilis, B.coagulans c) yeasts & molds C.lipolytica Geotricum candidum Penicillium spp. and Aspergillus spp.

2.

Control:1. 2. 3. Clean milk production Cooling of milk Processing of milk prior to microbial growth reaching log phase.

Sweet curdling Curdling without pronounced acid production is called the sweet curdling. The defect is due to the production of an extracellular enzyme similar to rennin by bacteria which causes casein to precipitate in the term of small specks of curd before the development of sufficient acidity i.e between 6.2 and 6.6 pH

Common in milk and cream particularly the moderately heat treated products Causative organisms: 1. Cocci: S.liquifaciens 2. Aerobic spore formers ­ B. cereus, B. subtilis 3. Psychrophilic spore forms- B. cereus, B. licheniformis, and certain Microbacterium spp. 4. Non spore forming rods: Proteus and Escherichia Significance: Prevalent in heat treated products, particularly in summer months. refrigerated milk and boiled milk stored for longer duration. Factors affecting sweet ­ curdling: 1. High temperature 2. Age of the milk 3. Pasteurization 1. 2. 3. Control: 1. 2. 3. 4. Avoiding the Contamination Control of the Temperature Control of Ageing Pasteurization: higher temperature High temperature: summer months Age of milk ­ Milk held at ambient temperature for more than 24 hours due to production of more rennin by B. mycoides. B. mycoides does not grow rapidly as it may be inhibited by lactic acid bacteria. Pasteurization: Also observed in

Bitty Cream (or) Broken cream: It is characterized by the appearance of flakes in the cream which do not mix again when milk is shaken. If such milk is used in tea, the flakes float on the surface making it unaccepted to mainly people. Flaking normally occurs before the changes in flavour or heat stability. It is a well known example of effect of spore formers on milk (raw and pasteurized milk) Mechanical / physical origin - & cream plug; It is formed when the fat globule membrane is partly disrupted and the globules stick together. Bacteria origin ­ Produced partly by the lecithinase enzyme of B. cereus and B. cereus var mycoides attacking the phospholipids part of the fat globule membrane and partly from the coagulation of casein associated with the membrane. The bacteria flakes do not change materially when heated to 100°C where as physical flakes change at 40-45°C. Bitty cream is the cheif spoilage problem of pasteurized milk. Failure of Refregeration, Seasonal variation prolonged storage etc., are the main reasons. Control: Clean milk production to avoid spore contamination.

Development of abnormal Flavours 1. Furity Flavours : These are due to ethyl ester formation usually catalyzed by esterases from psychrotroph or lactic acid bacteria. Ester formation by Ps. fragi involves liberation of butyric & caproic acids from one and three positions of milk triglycerides and are esterified with ethanol. Predominate esters are Ethyl butyrate, Ethyl hexanoate Malty flavour: Caused by Malty strains Lactococcus lactis sp Bitty flavour : Caused by proteolytic organisms especially Bacillus sp., and Pseudomonas sp., Fishy flavour: Caused by Ps. icthyosmius Potato flavour Caused by Ps. mucidolens and Ps. graveolens Phenolic flavour: Caused by Bacillus circulans Soapy flavour: Caused by Ps. sapoticum Bitty/Musty flavour: Caused by Actinomyces and certain yeast Burnt of caramel flavour: Caused by Malty strains of Lactococcus lactis ssp.lactis Barny flavour: Caused by Aerobacter oxytocum

2. 3. 4. 5. 6. 7. 8. 9. 10.

Development of abnormal colour fermentations: 1. 2. 3. 4. 5. 6. 7. Yellow coloration: Pseudomonas synxantha Blue coloration : Pseudomonas cyanogens Green coloration: Penicillium roqueforte Black coloration : Pseudomonas nigrifaciens Red coloration : Serratia marcescens/Micrococcus resen Brown coloration: Pseudomonas fluorescens Greenish coloration: Pseudomonas fluorescens

MASTITIS MILK Mastitis milk is the milk obtained from animals suffering from the udder infection called Mastitis. IDF defines mastitis as "An inflammation of udder, almost always of microbial origin and is the outcome either of a local or of a general infection. Common symptoms are: Swollen, hot, red and painful udders Mastitic milk has i) Higher microbial count and somatic cell count ii) Altered composition iii) Reduced milk yield. Acute or clinical ­ has macroscopic changes to udder or milk, readily detectable by milker Mastitis Forms: Symptoms Chronic ­ little compositional changes with almost complete absence of pain in the udder Sub acute/ sub clinical ­ Most common form Udder & milk appear normal Diagnosed by pathogens and somatic Cells and change in composition The Changes in composition of mastitis milk are 1. Increase of ­ whey proteins i.e. bovine serum albumin,immunoglobulin; ions such as Na & Cl* and also CU, Fe, Zn; antitrypsin pH and Catalase enzymes. 2. Decrease of Lactose, Possassium and Ca, Mg and P Fat and alpha, fractions of Casein, Some whey proteins such as alpha lactalbumin & -lactoglobulin Cauastive Organisms: Staphylococcus aureus Streptococcus agalactiae are most common organisms Streptococcus dysgalatiae Streptococcus uberis and E. coli causes Environmental mastitis Corynebacterium pyogenes causes Summer mastitis E. coli causes winter mastitis

Microbes enter through teat tip into the teat duct where they get colonized. The duct varies in length from 5-14mm and the surface is heavily keratinized. The keratine layer retains

some milk residues and exhibit antimicrobial activity. Among the various species, St. aureus readily gets colonized in the duct and persist for very long time. The mechanism of penetration of teat duct by microbes may involve the growth of bacteria through the duct. But the exact mechanism is still under investigation. S. aureus and St. agalactiae organism spread between quarters and animals, during milking through milker's hands and udder cloth St. uberis and E. coli Organisms are distributed on cows and in the environment and less dependent on the milking process for dissemination of organisms with in the herd. St. uberis is isolated from lips, teats, vulva, rectum, bedding and E. coli is from bedding and faeces Cory. pyogenes ­ most prevalent in summer among unmilked cows and heifers. Very serious form and is transmitted by head fly "Hydroteae irritans" Numbers :- fluctuate but highest number are found in fore milk. Staphylococci are excreted in less numbers unlike streptococci and coliforms. Average number in subclinical form may be <10,000/ml where as in clinical form the number may be >105/ml of herd milk. Organisms survive well in raw milk cooled to 10°c or less with the exception of St. uberis. TESTS FOR DETECTION OF MASTITIS MILK Mastitis is the inflammation of the udder caused by the infection of one or more quarters of udder by certain species of bacteria ex. Streptococcus agalactiae, Streptococcus dysgalactiae, Streptociccus uberis, Staphylococcus aureus etc., When these organisms occur in milk they cause alteration in chemical composition of milk, making it unsuitable for human consumption. It is therefore very important to check the milk for mastitis to suggest suitable measures, to undertake periodical checkup of the animals, to adopt necessary prophylactic measures for controlling the spread of the infection in the herd. It will also help in rejecting the milk supplies, as otherwise the bulk milk mixed with mastitis makes it unfit for heat processing. DMC Detection:Tests based on Detection of causative org pH Compositional Change towards Blood Sampling:- done aseptically. Discard first milk into stripcup. Take sample directly into sterile sample container. For Herd test - take mixed quarter samples For Detail test ­ take individual quarter samples chloride catalase Hotis test Blood agar plating

Strip cup test : This is a simple field test used for finding out the presence of fibrin, mucous and clots of milk in foremilk, which is an indication of mastitis infection. Most cases of acute mastitis and 10 percent of chronic infections are detected by this test. A negative test cannot be taken to indicate absence of any infection. Test first few streams of milk pH:The pH of the normal milk varies between 6.6 - 6.8. The milk from infected udders is usually alkaline in reaction having >6.8 pH (pH 7.0 to 7.4) and this condition can be detected by observing the colour change shown by a suitable pH indicator, bromothymol blue, added to milk. 5 ml milk is mixed with 1ml of 0.04% bromothymol blue. Blue green to green colour is indicative of mastitis where as normal milk shows yellow colour Milk from cows in advanced lactation also has an alkaline reaction and therefore gives a positive test and thus a negative test cannot be taken as evidence of absence of infection. The test detects about 70 per cent of infected cases. Chloride test:: The chloride content increases in milk of animals suffering from mastitis. Abnormal milk has more than 0.14% chloride content. o Accurately measure 5 ml of 0.l34 per cent silver nitrate solution and transfer into a test tube. o Add two drops of 10 percent potassium chromate indicator and this gives red colour. o Add exactly 1 ml of milk to the contents of the tube and mix. o Observe the colour of the mixture. If the sample contains an abnormally high percentage of chloride, the red colour will change to yellow to indicate a positive test. A brownish-red colour indicates a negative test Animals in early or in late lactation may give false positive reaction to chloride test. Catalase Test: Living cells including leucocytes contain the enzyme catalase. The number of leucocytes in the milk increases during the infection of udder and the catalase test is used to measure the increase in the content of catalase depending on the ability of the enzyme to break down hydrogen peroxide. o o o 15 of milk in a test tube and add 5ml of 1% H2O2 solution. Invert tube and incubate at 37°c for 3hrs O2 collects at upper portion. In normal milk the amount of oxygen liberated will be about 2.0 ml. More than 2.5 ml of gas collected in the top of the tube is presumed to be due to an abnormal infected udder. Milk from chronic cases may produce as much as 10 ml of gas. Milk of animals in early or late lactation may give positive tests to catalase and may still be free of infection

o

Somatic cells:A. Leucocytes : DMC test using Newman"s stain. Leucocytes > 5x105 /ml is indicative of mastitis. B.SLST:Sodium lauryl sulphate test ­ based on the increase in viscosity of milk by adding 4% SLS in 10% teepal solution with pH 12.0 To 2ml of milk add 2 ml of test solution shaken for 20 sec gently in tube. No viscous layer ­ No mastitis (<1x105 leucocytes). Slight " " - Sub-acute (1x105 " ). Central viscous core ­ Acute (>5x105 " ) May disappear after stopping retation Hotis Test: Amongst the tests described, Hotis test gives the most accurate information about mastitis infection. It is based on the fact that Streptococcus agalactiae, when growing in milk, produces a characteristic colony like mass of cells adhering to the sides of the test tube. By the introduction of an acid indicator (bromocresol purple), the identification of these colonies or 'buttons' is facilitated by their characteristic yellow colour. The appearance of yellow colonies of organisms along the sides of the tube or on the bottom indicates infection with Streptococcus agalactiae. o o o o 9.5ml of milk is mixed with 0.5ml of 0.5% aqueous bromocresolpurple and incubated at 37°c for 24-48 in a test tube. Yellow colonies or cell mass/ indicative of streptococcus. Flocculent on side of test tubes especially St.agalactiae.. Rusty brown colour colonies ­ S.aureus.

Blood agar test After preincubation of milk sample at 37°c overnight sample is streaked or pour plated. Small colonies; & alpha or haemolysis indicates St.agalactiae (some time no haemolysis); Alpha haemolysis (small zone around colonies with green discolouration) is indicative of St,dysgalactise and No reaction is due to St. uberis. Large colonies than streptococci with haemolysis (wide zone of clearance around colonies) is indicative of S.aureus CAMP (Christie, Atlkin, Munch & Peterson) test The CAMP test is very specific for the detection of mastitis caused by Streptococcus agalactiae. o Take the pre-poured Blood agar plates and streak across the center of the agar a culture of beta toxin producing strain of Staphylococcus aureus o Suspected milk samples for mastitis due to Streptococcus agalactiae are streaked at an angle to the first streak but avoiding contact with Staphylococcus aureus. o Incubate the plates over night at 37°C and observe for the zones. o Clear zone between the streaks of Staphylococcus aureus and milk sample indicates that the milk is positive for mastitis of Streptococcus agalactiae

MILKBORNE- DISEASES Milk serves as a potential vehicle for transmission of diseases under certain circumstances Milk by virtue of possessing almost all the essential nutritional factors, can serves as an excellent culture and protective medium for most of microbes which includes pathogens. Pathogens grow & multiply to produce certain toxic metabolites and make itself an extremely vulnerable commodity from public health point of view Out break:--- it is an incident in which two or more persons experience a similar illness usually a gastro intestinal after ingestion of a common food and termed as outbreak of food borne disease Food Intoxication --- food borne intoxication Food Infection Food Toxi--Infection }----food borne infections Food Intoxication------Ingestion of toxins already synthesized by microbes in food (pre-formed) brings about poisoning syndrome in the consumers. Toxins affecting GI tract are enterotoxins Food Infection----Ingestion of viable pathogenic bacteria along with the food leads to their lodgment and establishment in consumers organs. This is termed as food infection. Food Toxi- Infection----- some organisms can produce toxins `in situ' after getting ingested with food and infect intestine Examples Food intoxication staphylococcal food poisoning Botulism E.coli diarrohea Cholera Mycotoxicosis Other Milk Borne Diseases: Bacterial: Rickettsial: Viral : 1. Tuberculosis 2. Brucellosis 3. Diphtheria 1.Q fever 1.Entero virus, 2. Infectious hepatitis 3. F&M 4. Tick borne encephalitis 4. Anthrax Food Infection typhoid Shigellosis Septic sore throat scarlet fever Food toxi- infection B. cereus food poisoning Cl. perfringen's gangrene

MILK BORNE INFECTIONS Salmonellosis:-G- ve, motile, asporogenous, facultative rods. It results from ingestion of salmonella organisms with contaminated food. This is mainly milk born illness as large number of bacteria are needed to cause the disease & no toxic product is involved. Causative organisms: Salmonella typhi -- typhoid Salmonella paratyphi A, B ,C -----paratyphoid Sal. entetidis Sal. typhimurium }---- food poisoning Sources: o In most cases infection is derived from the faeces of carriers or sick animals contaminating water which in turn contaminate milk through utensils and equipment, human handlers, milkers who are either carriers or active cases. Occasionally cow suffering from clinical salmonellosis (diarrohea, anorexia etc.,) may excrete the org., in milk. External agencies like flies. IP: 7-14 days IP:1-7 days

o o o

Symptoms: 3types 1. Typhoid fever: continuous fever Inflammation of intestines Formation of intestinal ulcers Enlargement of spleen Characteristic rose spot eruptions on abdomen toxaemia 2. Paratyphoid fever: milder than typhoid and 105-107 organisms/ml are required for infection 3. Food poisoning: nausea, vomiting, abdominal pain, greenish& foul stools. Diarrhea, headache, muscular weakness, Moderate fever, restlessness Control: Rigid sanitary conditions in handling, processing, storage and distribution of milk Health control of handlers ­periodic examination, treatment, off from duties. Educating milkers about personnel hygiene. Pasteurization of milk or other adequate heat treatments. Control of flies. Treatment of water supplies. Diagnosis:faeces ,urine &blood are subjected for Microscopic examination, Cultural Serological examination, Widal test for typhoid examination,

Shigellosis: Known as Bacillary dysentry Organisms: Shigella dysenteriae, S. flexneri, S. sonnei Multiply rapidly in milk at or > 15Oc & more so in pasteurized milk. IP: 1 ­7 days 4 days Sources: 1. Milk infected by contamination from utensils, water, flies 2. milk handlers may cause contamination. S. dysenteriae produces endotoxins, Acute intestinal disease characterized with blood, pus or mucus, Fever, Vomiting, abdominal cramps Control: control of flies rigid sanitary conditions of dairy workers working in pasteurization plants and retail shops by Diarrhoea usually

Streptococcal infections: I. II. III. St. pyogenes---------------Scarlet fever, septic sore throat, Tonsillitis, septicaemia St. agalactiae---------------mastitis; opportunistic pathogen Ent. faecalis var. faecalis Ent. faecium }------ food poisoning symptoms are Ent. faecalis var. zymogens similar to staphylococci but milder

Sources: Animals suffering from S. agalactiae mastitis Milkers act as vectors of mastitis Enterococci are chiefly of faecal origin contaminate water, milk¸ human carriers of S. pyogenes. contaminate milk & milk products

Septic sore throat: High& irregular fever sudden onset of fever Throat Some times abscesses around tonsils Scarlet fever: Acute febrile (feverish) disease of throat accompanied by scarlet rash due to elaboration of toxins Control: Rejection of milk from infected udders Health check of dairy workers Hygienic control of handling & processing of milk Holding of milk at low temperature ( 7o C). Adequate heat treatment of milk Inflammation & Swelling of lymph glands,

MILK BORNE INTOXICATIONS Staphylococcal poisoning: G +ve, non motile, cocci in clusters of irregular shapes Sources: Lactating animal i.e bovine udder human handlers as the organism is frequently present in the nose, skin, wounds, pimples& boils

Toxins of Staphylococci: Haemolysins---alpha, beta, gama &delta, Enterotoxin Enterotoxin is produced when organisms reach (106) organisms/ml. Organisms grow well & faster in low count milk or pasteurized milk as in poor quality milk competition with other organisms & changes brought about by them i.e acidity, depletion of nutrients hamper their growth. Enterotoxins A, B, C, D, E, and F; A,B are responsible for food poisoning out breaks & of human origin; C,D are mammalian origin& predominate in milk; F is recent origin & responsible for toxic shock syndrome TSS Enterotoxins are protein in nature, Heat stable & are not destroyed even after boiling for 15 min, do not alter taste of food, Exotoxins and they are elaborated into milk & milk products.

Symptoms: Nausea, vomiting, Abdominal pain, Abdominal cramps, diarrohea, Sweating, headache Toxicity depends on the amount of toxin & susceptibility of individual IP: 1-16 hours and recovery is often followed in 24 h ­ 48 Hr. Diagnosis: On Baird -parker agar medium black, shiny colonies. Thermonuclease detection - Thermo Deoxyribo Nuclease coagulase production Serological methods ­detection of enterotoxin --Precipitation of enterotoxin ­ Antibody complex --RIA --ELISA Control: Isolation of infected animals with staphylococcal udder. &mastitis control program Infected handlers should not be allowed to hand milk Adequate cooling of milk to prevent multiplication Heating of milk before enterotoxin production Avoiding post-processing contamination

Botulism: Organism: Clostridium botulinum Milk products are rarely involved because it requires an anaerobiosis and can not survive at low pH Milk products sometimes implicated are Condensed milks Organism is heat resistant. . Produces toxins which are heat labile, Toxins are A,B,C,D,E F and G Severe & very fatal ; as it affects nervous system A,B,E and F are harmful to humans.Very lethal in small doses. 1mg is sufficient to kill 1million people Source: soil, water

Symptoms: Nausea, vomiting, fatigue, dizziness, headache, dryness of mouth, skin & throat, paralysis of muscles, double vision, respiratory failure & death IP ­ 12- 96hr Duration- 1-10 days Control: Hygienic conditions at production of milk adequate heating of milk products 100°c / 10-20 min destroys toxin acidity inhibits organism Rejection of bulged cans & spoiled foods chilling after production of milk

E. coli poisoning: Enteropathogenic E. coli can be defined as any stains of E. coli having the potential to cause diarrhea. Two types:- More than 106 cells /g are required to produce toxins I. Toxigenic EEC:-- produce enterotoxins & cause cholera like symptoms. Usually referred as "Infantile diarrhea or Traveler's diarrhea" characterized by watery diarrhea leading to dehydration& shock Invasive EEC:-- produces no enterotoxins. Organisms invade epithelial cells of colon. The diarrhea is characterized with blood & mucus Toxin ----1. Heat labile toxin LT 2. Heat stable toxin HT LT is inactivated at 65°C/30minutes; 100000 daltons Mw

II.

Sources: contaminated water supplies with faecal matter unhygienic practices by handlers also introduce organisms gross contamination from infected animals

Cholera:Caused by vibrio cholerae Sources: Soiled hands of convalescent carrier or patient, infected raw water for dairy purposes, Adulteration of milk with contaminated water Organisms adheres to epithelial cells lining mucosa in small intestines where it produces enterotoxins causing loss of fluid and electrolytes from body followed by dehydration. IP: 3 days (few hrs to 5 days) Control: cholera vaccination control of flies sanitary production , handling, processing, distribution of milk 5. Aflatoxins: The important organisms are Aspergillus flavus A. parasiticus

Sources: milch animals upon ingesting of feed contaminated with aflatoxins .These toxins are metabolized and are secreted into milk in the from of M1&M2. Produces B1& B2 toxins in mil. B1 is most toxic aflatoxins. G1,G2 and G2a extremely heat stable & potent, Highly toxigenic & carcinogenic Aerial contamination of mold spores is one of the important one soil contamination with mold spores poor storage conditions of milk products. Symptoms: Liver hyperplasia, tissue haemorrages, Anorexia , Hepatitis , Spleen , pancreas& kidney also be involved MILK BORNE TOXI ­ INFECTIONS B. cereus poisoning: Occurs due to ingestion of contaminated food with B. cereus or its spores Sources: from mastitis udder spores from animal's teat, skin, equipment especially of cans soil ­ directly or indirectly Produces 3 toxins --------- Haemolysin, lecithinase, enterotoxin but Enterotoxin is responsible for food poisoning

Symptoms: Two types 1) Diarrohea: Abdominal pain & cramps, Profuse watery diarrhea Rectal tenesmus (spasms) Moderate nausea with rare vomiting. Lecithinase releases phosphoryl choline, a toxic sub from lecithin 2) Vomiting: Acute attack of nausea & vomiting diarrhea is not common Control: cooling of milk Environmental hygiene& air quality General hygiene in production, processing etc.. Cl. perfringens poisoning: Cl. perfringens--- causes gas gangrene as it is anaerobic conditions inside canned foods are favourable for growth. 5 Enterotoxins --- A,B,C,D & E; A, C causes food poisoning Sources: from soil & faeces ­ dust, fodder --- milk faecal contamination of water

Symptoms: Diarrhoea, vomiting, abdominal pain, IP ­ 8-22 h duration: 1day some times very fatal Control:-- same as B. cereus OTHER MILK BORNE DISEASES Tuberculosis: It is one of the most important infectious diseases. The causative organisms are Mycobacterium tuberculosis & M. bovis. M. tuberculosis causes pulmonary type infection, where as M. bovis non-pulmonary type Sources: primary source is infected udder- the bacilli can pass from blood to milk through circulation Environment- dust, fodder, manure milk handlers infected with tuberculosis

Symptoms: Parenchymal pulmonary infiltration Recognizable by X-ray---onset Next advanced stage ---- cough, fever, Fatigue, loss in weight Susceptible: Children < 3 years & > 12 years IP: variable, usually 4-6weeks

Control: isolation treatment of infected ones vaccination BCG; Bacille Calmette Guerin Boiling of milk care of milk handlers improved living conditions Environmental hygiene

Brucellosis:-Undulant fever or Malta fever B.abortus (cattle), B.melitensus(goats)--more virulent for man, virulent Sources: Diseased animals excrete organisms in milk Environment infected discharges Inhalation of dry infected materials Symptoms: Headache, sweating IP: 5 ­ 21 days Immunity is developed after first attack Control: isolation ,treatment of animals vaccination of herd heat treatment of milk Diphtheria:-Corynebacterium diphtheriae infections very rare , human sneeze or cough over milk or contaminate From fingers soiled from nasal discharges. Cows may suffer from mastitis--from udder lesions it infects milk Symptoms: Febrile infections of nose , throat, tonsils Inflammation of throat, Diphtheria toxin may affect kidney & heat muscles leading to death. Develops life immunity. Anthrax :-Caused by B. anthracis Milk born anthrax is not prevalent as meat borne. Excreted in milk but require very large no.. in blood a condition met just before death Infected material contaminate surroundings as spores survive for many years

B.suis(swines)--next

Two infections: cutaneous & pulmonary (pneumonia)

Rickettsial Q fever: Coxiella burnitti ­ etiology is questionable, so the name ` Qery' Q fever. It Survives pasteurization 72 0 C/ 15sec & freezing. Resists 0.5% formalin, 1%phenol

Sources: inhalation of infected dust of vaginal or faecal matter infected animals excrete in milk ingestion of contaminated milk Symptoms: High fever, head ache, weakness, serve sweating, virus like pneumonia IP: 2-3wks mastitis in animals. Control: Animal vaccination Heating of milk avoid post processing contamination Environmental hygiene NEW EMERGING DISEASES: Listeriosis:-Caused by L. monocytogenes which is a G + ve , non- sporing rod , grows at 1- 45°C. Very serious disease; more out breaks in recent years Sources: infected is imp source ( mastitis udder ) milk handlers faecal contamination of milk & water unhygienic practices Symptoms:-Acute meningitis with or without septicemia, Onset is with sudden fever, nausea , head ache, delirium, coma, & shock Organisms are killed by pasteurization; some reports deny as leucocytes ingesting these organisms may protect them from heat effect. Produces extra cellular alfa, beta haemolysins Campylobacterioris: Caused by C.jejuni, relatively new bacterium. Source: faecal contamination is chief source infected handlers contaminated water

Symptoms: acute gastro enteritis with severe abdominal pain, diarrhea Yersiniosis: Caused by Y. enterocolitica Source: water is the chief source faecal contamination infected handlers; contaminated raw materials

Symptoms: Acute syndrome with appendicitis. Abdominal pain , fever, vomiting , diarrhea due to enterotoxins & as well as invasion Vibriosis V.parahaemolyticus causes gastroenteritis. It is also recent illness. Milk rarely implicated in the poisoning. Mechanism of infection is not known Source:-- contaminated water is main source Symptoms:- Diarrohea is main symptom; others abdominal pain &cramps, Nausea, vomiting, headache, chills, fever. IP : 12 ­ 24 h.

VIRAL DISEASES Foot & Mouth disease: Man slightly susceptible Sources: from fluid of vesicles, saliva, to faeces, urine, &milk udder lesions ­ rupture of vesicles & contaminate milk Symptoms: Fever, difficulty in swallowing, Heat & dryness in mouth followed by eruption of blisters in mouth. Control:- Adequate heating of milk vaccination of animals discard milk from suspected animals

Entero viruses- polio virus & coxsackie virus: Polio virus killed by pasteurization of milk

Sources:-1. Faecal contamination of milk & water. 2. Flies Symptoms:-first gastro intestinal disturbances, after 1-3 days major disease causes headache, fever, muscle stiffness, paralysis associated with cell destruction in CNS Infectious Hepatitis: One of the serious diseases transmitted through milk. Hepatitis A is transmitted through milk Sources: Human handlers are imp source contaminated water contaminated environment Symptoms: -- Nausea, vomition, abdominal pain, diarrhea, fever, chills, in urine & jaundice Bile

Tick borne encephalitis Caused by group B arboviruses . Causes meningoencephalitis Source: Ticks & mites is prime source followed by contaminated milk.

GENERAL MEASURES TO PREVENT FOOD POISONING: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Isolation of infected animals Environmental hygiene of milk production Hygiene of milker Proper feeding practices Proper cleaning of utensils Hygiene of milk handler Proper cooling of milk. Hygienic condition at plant Proper processing temperature of milk Hygiene of packaging materials. Quality of water in dairy plant Environmental hygiene at dairy plant Proper drainage & sewage systems Avoid sneezing/ coughing at product manufacture.. Prevent manual handling of product during marketing Avoid eating products after expiry date Clean eating habits.

EFFECT OF DIFFERENT STAGES OF PROCESSING ON THE MICROBIAL QUALITY OF MILK: COOLING OF MILK: Cooling of milk is the process of bringing the temperature of the milk below ambient temperature using a cooling medium. The purpose of the cooling is to reduce the spoilage of milk by preventing or retarding the growth of microorganisms. Milk contains various microorganisms derived from different sources. The most important spoilage and pathogenic organisms found in milk are mesophilies with a growth temperature ranging from 20°C to 40°C. Cooling reduces the metabolic activity of the organisms by reducing the activity of the enzymes necessary for the metabolism there by reducing the growth and reproduction of the bacteria especially mesophilic organisms. The effect of reducing the temperature on the growth of microbes Storage of milk at Temperature (°C) for 18 hrs 0 5 10 15 20 Growth rate 1.00 1.05 1.80 10.00 200.00

From the above data it is evident that the critical temperature is 10°C. Hence in practice milk is cooled to below 5°C to increase the shelf life of raw milk. BACTOFUGATION: Centrifugal force provides an efficient means of improving the bacteriological quality. The principle is known as Bacterial super centrifugation. But when it is carried out under pasteurization heat conditions it is called Bactofugation. There is a tendency to use the term irrespective of process temperature. First industrial scale application in Belgium. Bactofugation removes bacteria, both living and dead, from treated substances whereas traditional heat treatment kills bacteria and leaves them in food. Bactofugation is important in food stuffs infected with bacteria containing thermo-stable endotoxins. Purposes: To improve hygienical quality. When it is necessary to avoid heat resistant bacteria without resorting to excessive heating. Where exceptionally high degree of bacteriological purity is sought. The process is based on stoke's laws V = r (d1-d2) g ------------9v

V: d1: d2: r: g: v:

Velocity or solid or semi solid particle density of particle to be separated density of fluid radius of the particle centrifugal force viscosity of fluid

Bacterial dimension i.e. 1.2 Bacterial density is 1.070 - 1.120. So it requires high centrifugal force. Based on size and density certain species are removed more easily than others. This is notably true of sporulated bacteria. Therefore 90-95% of bacteria can be removed by application of 800012000 g of gravitational field for 8 sec at 50oC. With two bactofuges in a series 99% of bacteria can be removed at 72°C this treatment has two fold effect. In this condition a cell count reduction of 99% and colony count reduction of 99.9% is obtained. Industrial machines have a capacity of 6000-7000 lts/hr. Double effect process has 1/100 of bacterial content equivalent in pasteurized milk. Bactofugation is not a substitute for pasteurization or sterilization but used in conjunction with them to improve their efficiency. Advantages: In cheese milk 1. Prevents swelling in certain cheeses by butyric acid bacteria which are heat resistant 2. Remove bacteria without pasteurization which enables cheddar cheese production with more typical cheese flavour. Powders: 1. To reduce the count of microbes and significant removal of heat resistant bacteria. Sterilized milks - the severity of heat treatment can be reduced. Cream - the condition floc caused by heat resistant Bacillus cereus avoided.

THERMIZATION: It is the least heat treatment followed in Dairy industry. Heat treatment used is in the range of 63-65oC/15-20 sec and cooling to below 6°C. Optimum is 66 - 70oC/15 sec. Widely used in Europe. Used as a routine in Netherlands in cheese milk. Purpose: 1) 2) 3) To destroy enzyme `lipase' because milk produced a farm is not taken daily to dairy i.e., in western countries after 2 to 3 days it is transported to diaries. To extend storage of raw milk at refrigeration. To maintain daily production of products when supply is reduced.

Designed to lower the number of psychotrophs of raw milk to take care of lipolysis and proteolysis Factors: 1) 2) Raw milk - Initial load should be 5,00,000 (cfu/ml). As load increase the corresponding count after thermization in 3 days storage at 6oC also increases because of elimination of only a portion of psychotrophs. Heat treatment above 65oC is effective. But increasing the temperature at constant holding time promoted greater extension of shelf life than increasing holding time at constant temperature. As temperature increases more number of flora of enterobacteriaceae are destroyed. Some bacteria may increase in number Ex. S. thermophilus ( at 68°C/10 sec)

3)

DESTRUCTION OF MICROORGANISMS BY HEAT Microbes play important role in milk and milk products because they act as Spoilage organisms Disease causing organisms Index organisms of sanitary conditions at the production and processing sites of products

Therefore it is essential to study The conditions which destroy or remove these microorganisms Conditions that destroy all microorganism of a particular type while destruction of others being incidental Ex. Pasteurization

Destruction by heat: Microbes are destroyed by heat when microbial proteins are coagulated and enzymes required for their metabolism are inactivated. The death of microbes is also due th thermal denaturation of the secondary and tertiary structure of macromolecular cellular organizations such as DNA, proteins and membranes. Thermal inactivation is of first order kinetics -dN KN = ---------- K is the temperature dependent reaction constant dt Decline of viable count (dN) in a given interval (dt) is proportional to the initial concentration of living cells. The effect of heat depend on the intensity of temperature and duration of exposure The effect varies with Kind of organism, state of organism and environment during heating. Terms used to denote heat destruction Decimal reduction time ( D value) Z value Q 10 Value Sterilizing effect F0 value

D- Value D value is defined as the time in minutes taken to destroy 90% of the viable micro-organisms ( or to reduce the number to 1/10th of its original population) at some specific temperature. D-value is the index of the time-temperature needed to reduce microbial number in a system by one log cycle. Eg: (106) one million organism ­ reduced to 100(102) by heating the milk for the time equivalent to four D values. Z-value It designates the slope of a thermal death curve. It is the number of degree of temperature in 0 F required for a specific death curve to reduce the counts by 1 log cycle. In other words the temperature difference which results in a ten fold change in D value. Z value for sporeformers is 18 and for nonsporeformers is 10-14

Q 10 Value : It is defined as a factor by which reaction rate is increased by increasing the the by 10 0C temperature

Q 10 Value = Rate of reaction (T+ 10 0C) divided by rate of reaction at T 0C Q 10 Value for chemical reactions is 2 to 4; for microbial inactivation it is 10 to 30 and for spore destruction it is 8 to 12 10 Z= log Q 10 F0 value: It is a total integrated lethal effect and it is used to measure microbial severity of a thermal process. It is expressed as minutes at a specified reference temperature of 121 oC when Z equaled 18. F0 value for low acid foods is 3 and milk puddings is 4 to 10 F is thermal death time of an organism at 121oC and F0 value indicates F value when Z equaled 18. Ex: F0 value is 10 minutes at 2500F (1210C) which is equivalent to 1 minute at 268 0F Clostridium botulinum cook: The minimum heat treatment recommended for low acid products (>4.5 pH) is 121oC for 3 min or equivalent. This results in 12 decimal reductions for Clostridium botulinum. It is selected because it is most heat resistant pathogen and so the minimum heat treatment recommended to achieve 12 decimal reductions is known as minimum Clostridium botulinum cook

PASTEURIZATION OF MILK: Louis Pasteur (1860-64) a France scientist heated wine to 50-55°c to increase the keeping quality. IDF Definition: LTLT --- 63°C/30 mt or HTST --- 72°C/15sec & cooled to 5°c or below. Objective: To destroy all the pathogenic organisms present in milk To reduce the load of non-pathogenic organisms

The important pathogen is Mycobacterium tuberculosis. So the minimum heat treatment was established to guarantee the inactivation of Mycobacterium tuberculosis. The pasteurization deals effectively with almost all pathogens satisfactorily except for Bacillus cereus. Milk contains alkaline phosphotase enzyme which is inactivated at the temperature-time combination similar to pasteurization. Alkaline phosphotase can be measured by a simple chemical fest known as phosphotase test. Particulars M. tuberculosis Phosphatase Pasteurization Cream line reduced Milk:Raw milk contains a mixed micro flora arising from several sources. Holding temperature prior to Pasteurization is very important. >10°c­all the organisms in raw milk multiply actively. This includes heat resistant nonspore formers and spore formers. If this number of heat resistant organisms increases the total count of milk after pasteurization will also increase. <10°c ­ Growth of contaminating strains rapidly falls. <5°c- G-ve rods grow significantly such as Pseudomonas which are capable of producing heat resistant enzymes. B.cereus may grow and produce toxins. It is not theoretically possible to destroy all the microbes in milk by heat treatment but the purpose of pasteurization is to reduce probability of pathogens surviving the process to such a level that the public health risk from drinking such milk is negligible. Heat treatment Survivors% Bacillus Coryneforms G+ve G-ve 63°c/30min 54% 46% 0 0 80°c/10min 61% 37% 2 0 30min 58.9°c 61.1°c 61.7°c 62.2°c Time 15sec 70°c 71.1°c 71.7°c 72.2°c

Thermoduric organisms in Market milk I. Source of thermodurics from milk: 1. G-ve organisms:- Only Alcaligenes tolerans survives pasteurization 2. G+ve cocci a) b) Micrococci ­ large number in raw milk but outgrown by others at 7°c. They are unable to grow due to lactenin, a natural inhibitor in milk. They also occur on dairy equipments. St. thermophilus, Ent. faecalis and St. uberis : A number of strains of these (b),(c),(d) are thermoduric but grow very slowly at refrigeration temperature between pasteurization and consumption.. Milk containing high thermoduric counts also contain appreciable number of haemolytic streptococci.

3. Anaerobic spore formers ­ Unable to grow because of high redox potential of milk but sometimes isolated from milk. 4. Aerobic spore formers ­ Survive pasteurization Mesophilic ­ B.licheniformis most important followed by B.pumilus, B.subtilis Thermophilic ­ B.stearothermophilus Psychrotrophic ­ B.coagulans, B.circulans, B.mycoides 5. Coryneform groups ­ Survive & form substantial portion of microflora of pasteurized milk. But do not grow at low temperature. II. Inadequately cleaned utensils. III. Accumulated in pasteurization plant due to improper cleaning methods. IV.Re-pasteurization of returned milk : because of growth of thermodurics on storage after first pasteurization. V. Use of pasteurized skim milk or cream for standardization. VI. Summer months due to poor cooling. Psychrotrophs in market milk: Pseudomonas, flavobacterium, Acaligenes, Achromobacter Part of normal flora of milk and grow fast as temperature increases upto 25°c. Proper pasteurization destroys almost all psychrotroph or at least to the extent that survivors do not a factor in flavour deterioration over extended period of storage. But post pasteurization contamination is the major problem. Psychrotroph cause the problems in pasteurized milk like Unclean flavour, Putrid, fruity, Rancid, sour, ropy, Greenish yellow discolorations.

Thermophilic in market milk: They are non-pathogens and usually associated with high acidity and off-flavours Facultative thermophiles grow at 37°C & at 55°C and Obligatory thermophiles do not grow at 37°C but grow at 55°C and even upto 70°C

(i)

Raw milk ­ contains few number but may increase on storage at higher temperature and gain access through soil beddings, feeds.

(ii) Pasteurizer ­ inside surface & holder tubes, pre heaters, fitter cloths HTST ­ no problem of thermophiles because of too short residence time of milk (total 70-80 sec), but fitter cloths used between regeneration and heating sections are trouble some. (iii) Re-pasteurization & returned milk:a) addition of returned milk b) Dripping from bottle fillers to raw milk contain very high thermophiles c) Skim milk or cream from returned milk for standardization. Milk foam left in equipment is an excellent source Dead ends where hot milk is allowed to stand for any appreciable time.

(iv) (v)

Coliforms in market milk: Coliforms are undesirable in pasteurized milk a) Raw milk ­ improper sanitation of production i.e faecal contamination through water, exterior of animal and improperly cleaned utensils. b) Pasteurizing plants: i. ii. iii. iv. Improperly cleaned pipelines, pumps, fillers, bottles etc. condensate drippings at various places personnel unsanitary practices detective or worn-out equipment having pits and pockets which favour accumulation of milk solids

The presence of coliforms in pasteurized milk can be tackled as o o o As heat resistant species, is not a major problem Phosphotase test is useful in case of improper pasteurization, Major problems only due to Post-pasteurization contamination.

Defects - Gassiness, Ropiness, Unclean flavour, Medicinal & Bitter flavour Pathogens in market milk: Their presence is due to improper pasteurization, post processing contamination. No recorded cases of pathogens if pasteurization is done as per IDF. Incidents of staphylococci, Salmonella, Campylobacter, Yersinia are recorded. B. cereus was not recorded though it survives pasteurization. Possible sources of post-processing contamination. (a) Human carriers (b) Thermophiles in final regeneration section are shed into pasteurized milk (c) Pin holes in PHE of regeneration section allow raw milk mixes with pasteurized milk which can be avoided by using high pressure on the processed milk

Keeping Quality of market milk: KQ of pasteurized milk is 5-7 days at refrigeration. Spoilage depends on type & number of organisms and also on temperature of storage Unclean & bitter flavours B.cereus at 106 cfu/ml. are due to G-ve rods and Bitter off flavours are due to

Acid production, Coagulation, Protein destabilization are brought by acid producers such as Lactococcus lactis ssp lactis Enzymes : Psychrotrophs are destroyed but enzymes are "thermostable" present and if number is 106 ­ 107 cfu/ml. Proteases, Lipases and Phospholipase C have less time to react at low temperature storage because of KQ 5-7 days. Enzymes of Bacillus species are not heat resistant

UHT PROCESSING OF MILK: The purpose of UHT treatment of milk is to produce a sterilized milk which is meant to 1. Keep without deterioration i.e remain stable and of good commercial value for a sufficient period 2. be free of microorganisms and toxins harmful to the health of consumes 3. be free of microorganisms liable to proliferate during storage IDF recommended 135-150°c/1-4sec for UHT treatment. Microbes in milk and their response to heat treatment: Class I: Microbes killed by conventional pasteurization temp of 71-72°c for 15-30 sec. This eliminates most vegetative cells of bacteria like S. aureus, haemotylic streptococci, G-ve enterococci (E. coli, Salmonella sp.) pseudomonas, B. abortus, M. tuberculosis, all yeasts and molds. Class II: Resistant to HTST, but sensitive to UHT i.e. 135-150°c/1-4sec HTST is tolerated by some thermoduric vegetative cocci like entenococci some micrococci, Microbacteria, thermophilic bacilli (L. delbrueckii ssp. bulgaricus Lactobacillus lactis), S. salivarius ssp. thermophilus, thermoduric aerobic & anaerobic spores.

Class III: Obligate thermophilic soil bacterium B.Stearothermophilus are known to withstand UHT treatment of milk. Same spores of mesophilic bacilli and clostridia may survive, if milk is heavily contaminated. ( Growth temp. of 40 ­ 45°C of B. Stearothermophilus is observed during storage)

The thermal effect is due to denaturation of secondary & tertiary structure of macromolecular cellular organizations like DNA, Proteins & membranes. Heat resistance of common spore formers (sterilized milks) Decimal reduction time at 121°C (sec) Cl. botulinum 3 B. cereus 2-4 B. coagulase 18 B. subtilis 3-20 B.stearothermophilus 200-500 B.stearothermophilus is most resistant and constitute greatest hazard to spoilage of sterilized product. Determining the lethal effect of or system:Equal exposure times and temperature do not produce the same lethal effect when commercial UHT systems are compared to the conventional oil bath and vial procedure. Similarly a comparison between direct and indirect UHT systems does not result in same total lethality at equivalent times and temp. So sterilizing effect / sporicidal efficiency (SE) is calculated. Gales loot defined SE = log (initial spore concentration) Final spore concentration. SE of mesophilic spore formers = 8 SE of Thermophilic spore formers = 1-2-1-6 Quality of raw milk: No. of bacteria in raw milk is influenced by contamination from milk utensils Bacterial growth between production & delivery at processing dairies. Quality of milk used for UHT process should be good with low spore count and psychrotrophic count and their enzymes. Spoilage of UHT milk:- Either due to (a). Enzymes which are heat resistant (b) Post processing contamination either from packaging operation or improper cleaning of system. Spoilage is characterized by bitter off flavour, gelation and coagulation of milk proteins. Psychrotrophes and aerobic spore formers do produce proteases and lipases at a storage temperature of as low as 4°C. Some of these enzymes are most heat resistant. Enough enzymes are produced at 5°C by as few as 103-104 pseudomonas/ml to cause significant loss of native milk proteins and bitter flavour. Most of UHT spoilage can be traced to packaging failures. Aseptic filling of UHT milk is of utmost importance. Since contamination with one viable bacterium able to reproduce in milk will inevitably spoil the product during storage with in few days. Contamination risk with sterility of packaging material and contamination is mainly by class I and II organisms.

This is mainly due to o o o o At the time of change of paper rolls Worn out gaskets in sterile microbes Condensed water at the filling pipes faulty sealing of packages. portions of equipment which harbour

If contamination by insufficient sterility of packaging material due to faulty sealing or corroded plates of plate heat exchanger, then the contamination flora is most variable and varies between package to package. If contamination is due to gaskets or condensed water, then all contaminated packages contain uniform microflora. Only one microbe is found as pure culture. Microbes which will enter the product more probably are those found in water stagnant on dairy floors ­ pseudomonas, micrococci, enterococci Bacillus and some yeasts. But these do modify milk visibly (coagulation, proteolysis, staining & flocculation) or produce off flavours or a measurable property (PH, acidity, redox potential) but UHT milk cannot attain cell concentration of >105/ml so these must be detected by direct counts on nutritive medium. Quality control of UHI milk: one per 1000 containers Satisfactory process shows spoilage level not higher than

How many packs to test ­ 50 to 100 units daily/line if line gives 4000 units per hour. This sums upto 0.1 ­ 0.2% of whole production. Incubation ­ pre incubation of packs for sufficient period. The temperature should be between 25-30°C/7-9 days and incubated at 55°c for thermophilic spores. The appearance, pH, taste and blowing of packages are recorded. The defect packages are tested by streaking on agar plate and incubated at 30°C/2 days or 55°C/4days. Streaking is gone to know the nature of micro organism by going morphological tests, oxidase and catalase tests. Interpretation:Thermophitic spores ­ insufficient heating of the product Mesaphilic spores - survived heat treatment or recontamination. Methods to improve the quality of UHT milk:1. 2. 3. 4. 5. 6. 7. 8. Use raw milk of low SPC and Psychrotrophs Milk collected from hygienic milk shed areas Immediately after collection, milk is subjected to pasteurization (or) thermization Test sample of milk in sealed ample in oil both at 130 ­ 140°c Better use recombined or reconstituted milk with very low spore count LP system preservation of farm milk Bactofugation with 1000 litre/hr.flow rate Nisin ­ active against spore formers (Aerobic/anaerobic) but not approved by PFA.

STERILIZATION OF MILK Sterilization means exposing the milk to 118 to 120°C for 15 to 20 minute. Sterilized milk should contain neither bacteria nor bacterial spores. Spore flora of raw milk: Raw milk contains a considerable number of bacterial spores although this number may be low in comparison with total bacterial count. The bacterial number in raw milk is mainly influenced by the a) b) Degree of contamination from the milking utensils. Bacterial growth rate that has taken place between time of production and delivery at the daily.

Bacterial spores gain entry into milk mainly by other means for ex. - Contamination with dust particles - Contamination with soil - Contamination with manure. Thus the density of spores in the milk is thus higher in the milk when the animal is milked in stable than in open air. It is generally assumed that their number will not increase during lapse of time between milking and delivery, therefore chiefly governed by the cleanliness of methods in milk production. Heating of 10mts at 80-85°c gives the spore count. Heating 30 mt at 100°c often used for the enumeration of more thermo-resistant spores. More than half of the spores in raw milk belong to the species of Bacillus licheniformis. Other spores of considerable importance are B.pumillus, B.subtitis. But lesser number of spores of B.cereus, B.circulance, B.megatherium B.stearothermophiulus (thermophilic) and members of genus clostridium are encountered. Among the mesophilic spores present in raw milk those of "B.subtilis" are most thermoresistant.

Sterility control: Mesophilic spores less than one per litre. Incubation at 30°C ­ 37°C for 5-7 days is generally used with unopened bottles for mesophilic spore count but at 37°C for 3-5 days facultative thermophiles may also develop. Sub lethally heat treated mesophilic spores cannot grow at 37°C but grow only at 30°C. Obligate thermophiles grown at 55-65°C for 5 days. Survival of bacteria in sterilized milks: Because of deleterious effects of sterilization upon certain physico chemical properties of milk, there is often tendency to use the minimum amount of heat treatment, with the result occasionally bacteria are developed in sterilized product. When "B. subtilis" is present, it gives extremely "bitter taste" and "proteolysis" occurs. "B.circulans" presence in sterilized milk produces "carbolic taint". When sterilized milk is incubated at higher temperature it is possible that more species may be found. The presence of B. coagulance and of facultative thermophilic bacilli can be demonstrated by

tests at 37°C. Test at 55°C can demonstrate the `presence of bacteria of B. coagulance and groups of obligate & facultative thermophilic bacilli. Presence of B. cereus and B.subtilis indicates the lower intensity of heat treatment. The spores of B. cereus are less thermo-resistant than those of B.subtitis. Hence the presence of B.cereus in greater no. than B.subtitis indicates insufficient degree of heat treatment. The presence of B. circulans which produces carbolic taint is often associated with improperly washed bottles. These bacterial grow even at rather low temperature quickly and abundantly form spores in milk returned bottles" contaminate the bottle washing machine. When the temperature and alkalinity of detergent solutions are not sufficiently efficient, large number of B. circulans spores may survive the treatment and infect clean bottles. The inbottle-treatment fails to kill the spores; hence more infected bottles are returened and contaminate the bottle washing machine. Thus a vicious circle is set up which is fatal to the product unless measures are taken to improve the standards for bottle sterilization. The presence of "B. coagulance" group and thermophilic bacilli is usually associated with the filler. For reasons of economy and quality milk is mostly bottled at higher temperature. These bacteria develop in filler, forms spores there and contaminate milk at bottling stage, often surviving later in bottle treatment. Development of B. coagulance group can be fairly easily controlled by using filling temperature and their growth is retarded at 60°c and prevented at 65°. The development of thermophilic bacilli can be controlled only by through and frequent cleaning and disinfection of filler. At a bottling temperature of 80°c which is the maximum, thermophilic organisms develop much lesser that at frequently used bottling temperature of 65-70°c. Anaerobic spore forming bacteria are practically never present unless milk has been strongly under sterilized, as anaerobic spore forms are less resistant then aerobic spores.

HOMOGENIZATION Theories: Shearing and grinding - Subjects the globule to unequal forces as it moves at different rates in a fluid stream which reform the globule beyond its yield point. Explosion - occur as the high pressure is suddenly released. Shattering -- Shatters as fat globules impinge against a retaining wall or impact ring. Attentuation -- or unstable fat thread formation due to stretching or fat globules beyond elastic limits by phenomenal rapid changes in velocity of liquid. Cavitation -- In which vapour bubbles are formed and collapsed at subsequent pressure point releasing energy in the form of shock waves which break up fat globules.

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