LECTURE NOTES For B. Tech (Dairy Technology) Degree

INTRODUCTORY DAIRY MICROBIOLOGY (Course No: DM 121)

By

Dr. Velugoti Padmanabha Reddy, Ph.DProfessor and University Head Dept of Dairy Microbiology College of Dairy Technology Tirupati-517 502

COLLEGE OF DAIRY TECHNOLGY SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI- 517 502 2006

MILK MICROBIOLOGY Prokaryotes Ex. Bacteria, Blue green algae. Microbes Eukaryotes Ex. Fungi, Protozoa, Algae. Plants and Animals Binomial nomenclature --Second word is name of bacterium First word is genus & begins with capital,

PROCARYOTES: Grouped under 4 Divisions and 29 Sections 1. Gracilicutes: - Prokaryotes with thinner cell wall implying G ve type cell wall Classes: Scotobacteria, Anoxyphotobacteria, Oxyphotobacteria. 2. Firmicutes: - With thick and Strong cell wall indicative of G +ve type cell wall Classes: Firmibacteria, Thallobacteria. 3. Tenericutes: - With Pliable & soft nature indicative of lack of a rigid cell wall Classes: -Mollicutes. 4. Mendosicutes: - With faulty cell walls suggesting the lack of conventional peptidoglycon Classes: - Archaeobacteria.

DAIRY IMPORTANT MICROORGANISMS Section 1: Spirochetes Family: Leptospiraceae Genus: Leptospira Ex. Leptospira interogans Spirillaceae Aerobic, Micro-aerophillic, Motile Genus: Campylobacter Ex. Campylobacter jejuni. Gram Ve Aerobic Rods & Cocci A. Pseudomonadaceae Genus: Pseudomonas Genus: Xanthomonas B. Neissericeae Genus: Neisseria Genus: Acinetobacter C. Other Genus: Genus: Alteromonas Genus: Flavobacterium Genus: Alcaligenes Ex. Alcaligenes viscosus Genus: Brucella Ex. Brucella. abortus

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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

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Section 13:

Section 14:

Section 15: Section 16:

CLASSIFICATION OF DAIRY IMPORTANT BACTERIA I. Based on the Size & shape of arrangement of cells. 1) Cocci: cells are spherical or ellipsoidal 2) Bacilli: cylindrical or rod like cells 3) Spirilla: spiral or helical shaped cells Pleomorphic: cells appearing in different shapes or lack of uniform shape. Ex. Arthrobacter Palisade Arrangement: cells linked side by side like matchsticks Cocci: a) Diplococci: Cells divide in one plane & remain attached in pairs Ex. Neisseria b) Streptococci: Cells divide in one plane &remain attached after some divisions, in the form of chains. Ex. Lactococcus c) Tetrads: Cells divide in two planes & form 4 cells. Ex. Pediococci d) Sarcinae: Cells divide in 3 planes & form a cuboidal arrangement. e) Staphylococci: Cells divide in 3 planes in an irregular pattern producing bunches of cocci. Bacilli: a) Diplobacilli: Pairs. b) Streptobacilli: Chains Ends may be rounded as in Lactobacillus delbruckeii ssp. bulgaricus or squamosed as in Bacillus anthracis Spirilla: Single curves: Vibrio Many curves: Spirochetes Few curves: True spirilla II. Classification based on Temperature 1. Mesophillic: Microorganisms capable of growing between 20 & 40C with the optimum growth temperature (OGT) of 37C are termed as Mesophiles. All pathogenic organisms are mesophillic in nature Ex. S. aureus, E. coli 2. Psychrotrophic: Organisms capable of growing at or below 7C (refrigerated) but the OGT is between 15 & 20C are termed as psychrotrophs.These are the significant spoilage organisms of refrigerated milk & milk products. Ex. Pseudomonas sp. Alkaligenes sp. 3. Thermophilic: Organisms capable of growing over 50C with O.G.T of 55 C are termed as thermophiles. They are the important organisms causing outbreaks in heat

processed milk & milk products. Some are capable of growing between 40-85C. Organisms produce enzymes at rapid rate, so that enzymes are replaced quickly. Ex.: Bacillus stearothermophilus, Streptococcus salivarius ssp. thermophilus. 4. Thermodurics: Organisms capable of withstanding pasteurizing temperatures of 63C\30 min. with O.G.T of 35-37C are termed as thermodurics. They form important flora of pasteurized or heat processed foods. Ex. Micrococcus varians III. Classification based on Oxygen Requirement 1. Aerobic: Organisms capable of growing in the presence of oxygen are termed as aerobic organisms. They can grow in a standard air atmosphere of 21% oxygen. They are more efficient in utilization of available nutrients. Ex. Bacillus species 2. Anaerobic: Organisms which cannot grow in the presence of oxygen but can grow in the presence of CO2 are termed as anaerobic organisms. They dont use O2 for energy yielding reactions. They are ever poisoned by O2.Some tolerate low concentrations of O2.They produce catalase & peroxidase enzymes. High tolerance Clostridium perfrigens Moderate tolerance Clostridium tetani 3. Facultative: Organisms which can grow either in the presence or absence of oxygen are termed as facultative organisms. Ex. E. coli, Lactococcus lactis ssp. lactis 4. Microaerophillic: Organisms which grow best at 1-15% of O2 levels. They can use O2 for energy yielding reactions but cannot withstand high levels of O2 i.e, 21% of O2. Ex.: Campylobacter jejuni

PHYSIOLOGICAL GROUPING 1. Acid producers: Capable of fermenting lactose to form lactic acid. Lactic acid coagulates milk by producing precipitation of Casein at 4.6 pH Homofermenters: Heterofermenters: Lactococcus, some Lactobacilli SomeLactobacilli, Lueconostoc sp.,

2. Gas producers: Capable of producing CO2\&H2 from lactose fermentations. Ex. E. coli, yeasts, Clostridium species. 3. Proteolytic: Degrade milk proteins into soluble components by enzymes known as proteinases or proteases. Ex. Bacillus species, Pseudomonas species.

4. Lipolytic: Organisms capable of attacking milk fat by enzymes known as lipases liberating glycerides & fatty acids. Ex. Pseudomonas species, Achromobacter lipolyticum Molds: Geotrichum candidum, Penicillium roqueforte. 5. Sweet curdling: Organisms capable of causing curdling of milk by the enzyme known as rennin like enzyme before the development of sufficient acidity. Ex. B. subtilis, B. cereus, Enterococcus liquifaciens. 6. Ropiness: Causing change in the viscosity of milk or forming threads when the milk is poured from one container to other, due to production of gums, mucins etc. Ex. Alcaligenes viscosus 7. Flavour producing: Fruity Pseudomonas fragi Malty Lactococcus lactis var maltigenes Fishy Proteus icthyosmius Unclean E. coli 8. Colour fermentations: Yellow coloration: Blue coloration : Green coloration: Black coloration : Red coloration : Pseudomonas synxantha Pseudomonas cyanogenes Penicillium roqueforte Pseudomonas nigrifaciens Serratia marcescens

CHRACTERISTICS OF DAIRY IMPORTANT BACTERIA Section 1: Spirochetes GENUS: LEPTOSPIRA: Characters: Flexible helicoidal rods, Gram ve, obligately aerobic, Optimum growth temperature is 28-30 C, Chemo organotrophs (heterotrophs) using fatty acids & fatty alcohol as energy & carbon sources. Leptospira interogans: causes leptospirosis in animals & man. Kidney is the natural inhabitant & organisms are shed in the urine contaminating soil & water; causes influenza type to icteric form of illness. Section 2: Spirillaceae GENUS: CAMPYLOBACTER: Characters: G-ve. Slender spirally curved rods, may be S shaped. Aerobic \ Microaerophilic (O2 requirement 3 to 15 %). Chemo-organotrophs. Respiratory metabolism. Motile cork screw like motion by means of single flagellum at one or both ends. Campylobacter jejuni: Causes fever & gastroenteritis in human beings. Along with other species causes abortions & reproductive problems in domestic animals. Sources: reproductive organs, intestinal tract & oral cavity. Raw & improperly pasteurized milks. Section 4: G-Ve Aerobic Rods & Cocci PSEUDOMONACEAE Genus: Pseudomonas Genus: Xanthomonas Genus: Zooglea Genus: Gluconobacter Characters: G ve, slightly curved rods, motile by polar flagella, oxidase +ve, catalase +ve, aerobic; respiratory metabolism & never fermentative, growth requirements are simple. Sources: soil & water. GENUS PSEUDOMONAS (Mol % G+C : 58 72) Leptospiraceae

Pseudo means false Monas means unit. i.e false species of Monas an early generic name of Protozoan Many species of Pseudomonas produces fluorescent, diffusible pigments of a greenish, yellow, or yellow green colours; black or blue Optimum growth temperature: 25 30C ; Nutritionally versatile, grow well on solid media

Presence of these organisms in milk and milk products is highly objectionable because Produces heat stable proteases and lipases even at low temperatures versatile spoilage agents attacking fats and proteins Include pathogens which cause milk borne illness. They have very little fermentative activity on carbohydrates Some of these produce phosphatase and if they grow in pasteurized milk they cause false positive tests.

Ps. fluorescens:: Produces water soluble fluorescent greenish-brown pigment known as PYOVERDIN Optimum growth temperature: 25 30 C but grows at 5C. Causes bitterness and Lipolysisin refrigerated foods by elaborating proteinases and lipases and Gelation in UHT products This also produces phospholipases and glycosidase but causes no coagulation of litmus milk Ps. fragi : Rarely produces pigments; sometimes diffusible brown pigment Grow at 5 C, Optimum growth temperature 20 25 C. It is mostly lipolytic and rarely Caseolytic. Produces fruit like odour resembling apple or strawberries compounds responsible are Ethyl butyrate and Ethyl hexanoate. Ps. aeruginosa: Produces Greenish blue pigment Pyocyanin and Pyoverdin and non caratenoid pigments. Do not grow at 5 C but brow at 41 - 42C. Optimum growth tempr: 37 C. Opportunistic pathogen; . Causes Mastitis. GENUS XANTHOMENAS: Xanthomonas maltophilia, previously known as Pseudomonas maltophilia. Differentiating characters & Pseudomonas are o produce yellow, non diffusible cell bound pigment o Inability to reduce Nitrate o Require methionine as growth factor o No growth at 4 C and 42C. May grow at 41C Optimum temperature 30 35 C o Utilization of citrate negative GENUS ALTEROMONAS: Al. putrefaciens: formerly known as Pseudomonas putrefaciens Produces a non-diffusible pink or Reddish brown pigment; Produces H2S on Kings iron agar, Rod shaped, Optimum growth temperature : 20 25 C

Major proteolytic and less lipolytic in nature, Causes surface taint in butter and also Cheesy, putrid flavour defects Source: Water and soil

GENUS ALCALIGENES Rods or cocci, Motile, Obligate aerobic, Psychrotrophs and some are thermoduric, Causes ropiness in milk and milk products Alcaligenes viscosus Alcaligenes tolerans GENUS BRUCELLA G ve rods, Short ellipsoidal, Small, circular convex colonies, Optimum growth temperature : 37 C;, Non motile, growth may favoured with Increased CO2 tension. Characteristics Brucella abortus Brucella suis + Swine Brucella melitensis + + Goats * Sheep

Growth in presence of Methyl violet + Urease H2S production Abortion Cow

Section 5 : Facultative Anaerobic Gram negative rods. ENTEROBACTERIACEAE Genus: Genus: Genus: Genus: Genus: Escherchia Enterobacter Salmonella Yersinia Citrobacter

Characteristics: Gram Negative, Short and straight rods, Motile by peritrichous flagella or non-motile, May be capsulated, Aerobic/facultatively anaerobic Chemoorganotrophes, Ferment glucose producing acid or acid and gas, Mainly catalase positive, Oxidase negative Inhabitant of saprophytic COLIFORMS : Coliforms may be defined as Gram negative, oxidase negative, non-spore forming rods, which can grow aerobically or facultatively anaerobic in the presence of bile salts or surface active agents with similar growth inhibitory properties and which are to ferment lactose with production of acid and gas with in 48 hrs at 37C the intestines of man and animals and some may act as pathogens or

GENUS ESCHERICHIA: E. coli, E. blattae Glucose and Carbohydrates. acid, and Formic acid. Formic Acid Pyruvate Co2 and H2 Lactic acid, Acetic

Litmus milk gets coagulated with rapid acid production and gas; Citric acid and its salts are not utilized as sole source of carbon. OGT: 30-37C, but grow at below 10 and above 45C About 35% of the acid produced by E. coli is lactic acid and so they are also called pseudolactic acid bacteria. The type of acid produced by E. coli depends on sugar fermented and nature of N2 source. Significance: Their presence in the foods is indicative of faecal contamination They are potent food spoilage organisms o They produce gas in the dairy products o They produce unclean flavour in the dairy products o Some strains produce ropiness in the dairy products Their presence in pasteurized foods indicates unhygienic productions at the plant Some strains produce enterotoxins and some are enteropthogenic in nature Charcteristics Motility C02 : H2 Origin IMViC tests Indole Methyle red Voges-proskauer Citrate E. coli Motile 1:1 Faecal Positive Positive Negative Negative E. aerogenes Non-motile 2:1 Non faecal Negative Negative Positive Positive

GENUS ENTEROBACTER : E. aerogenes, E. cloacae (faecal/non faecal) Ferments glucose by means of butanediol-formic fermentation to produce acetyl methyl carbinol to give +ve Voges-proskauer test. Encapsulated variants cause ropiness. GENUS SALMONELLA: Salmonella is named after D. E. Salmon (USA). G-ve, aerobic/ facultative anaerobes with optimum growth temperature of 37C, mostly motile by peritrichous flagella, they produce acid and gas from carbohydrates such as glucose, mannitol, maltose, sorbitol etc except Salmonella typhi. It does not ferment lactose, positive for methyl red, negative for indole and Vogesproskauer but variable in utilization of citrate. Source: Direct or indirect faecal contamination, cows suffering from salmonellosis

They are designated into (by Kauffmann-White scheme) serotypes such as O (somatic), H (Flagellar) and Vi ( Virulence antigens) It is an important group of organisms because of its ability to produce a variety of food infections and illness. It produces endotoxins. 12 to 30 hrs may elapse after ingestion of food contaminated with these organisms and the onset of symptoms are due to the elaboration of endotoxins by the growth of the organisms The following illnesses are associated: Typhoid : Salmonella typhi Paratyphoid : Salmonella paratyphi A Paratyphoid : Salmonella paratyphi B Paratyphoid : Salmonella paratyphi C GENUS YERSINIA: The important organisms are Y. enterocolitica, Y. pestis, and Y. psuedotuberculosis Y. enterocolitica enters milk by contamination through faeces, urine, and insects. They grow between 2-45C with OGT of 30 C. All the organisms of this genus are pathogenic to humans or animals or opportunistic potential pathogens. GENUS SERRATIA: Serratia marcescens produces red pigment known as prodigiosin GENUS VIBRIO: Vibrio cholerae causes cholera in humans

Section 9: Rickettsiaceae (Rickettsias & Chlamydias) GENUS: COXIELLA Ex. C. burnitii G-ve short rods, pleomorphic occurring as diplococci. The organism causes Q (query) fever. Milk is contaminated directly from circulation in infected animals. Man gets infection mostly by aerosol infection and less commonly by drinking contaminated milk The organism also show high resistance to chemical and physical agents and desiccation. It is resistant to 0.5% formalin, 1.0% phenol and can withstand the heat treatment of 60C for 1 hr and 61.7C for 30 min. The complete inactivation by pasteurization may not be always possible. Ticks are the vectors of transmission.

Section 10: G +Ve Cocci Genus: Micrococcus Genus: Staphylococcus These two genus consists of Gram positive spherical cells. They are non-motile. Aerobic or facultatively anaerobic, chemoorganotrophs and catalase positive Characteristics Grow anaerobically Carbohydrate attack Lysostaphin sensitivity Ability to ferment glucose anaerobically Micrococcus -Ve Oxidative -Ve -Ve Staphylococcus + Fermentative + +

GENUS MICROCOCCUS: Micrus = small Kokkos= seed or grain Aerobic, coagulase negative, Mesophilic, forms tetrads Some of these organisms produce yellow, orange, red pigment Lactose is not fermented, OGT 25C, Many species are heat resistant and survive 63C/ 30 min but the true micrococci are not resistant to pasteurization. They are found in lactiferous ducts of mammary gland and present in milk obtained from udder under sterile conditions and hence considered as normal microflora of milk. Contaminated equipment is the main source of micrococci in milk. They are responsible for Thickening of sweetened condensed milk and causes thermodurics out breaks in pasteurizing plants Ex. M. varians, M. luteus GENUS STAPHYLOCOCCUS They are cocci and smaller than micrococci. Occurs in grape like clusters, non motile, may produce orange or yellow pigment, Mesophilic, OGT 37C Ferment a variety of carbohydrates and resistant upto 10% of salt concentration They are capable of producing several toxins like haemolysin, fibrinolysin, leucocidin, enterotoxins and thermostable nuclease etc., Haemolysins are the substances that liberate haemoglobin from RBC. haemolysis produces clear, colorless zone around colonies. Haemoglobin destroyed to produce colorless compound. , haemolysis converts haemoglobin to methamoglobin which produces greenish zone. Sources: Skin, Nasal and mucus membranes

Staphylococcus aureus: Facultative anaerobe, Grow at 15 and 45 C and is mesophilic, The colonies are with yellowish tint (lemon) or orange-yellow Heamolysins: , , gama and delta are produced. Sensitive to Novobiocin Most of the strains produce enterotoxins (Exotoxins) which are responsible for food intoxications. Five different enterotoxins are reported. They are A, B, C, D, and E but enterotoxins A are more common. They are heat stable and produce symptoms in 2 to 6 hrs after ingestion of contaminated milk. Medium: On Baird parkers medium they produce black, shiny, convex colonies surrounded by a clear zone. In the clear zone a fine black precipitate may appear i.e zone of opalescence. The pathogenic nature is confirmed with a positive coagulase and thermo nuclease test. It produces bacteriocins viz., staphylococcin / micrococcin which are bacteriostatic / cidal to bacteria and to other staphylococci. Other organisms of staphylococcus found in milk are S. epidermis, S. caprae, S. hyicus Genus Streptococcus: Streptos means flexible, a pliable length of cocci similar to necklace G +ve, spherical or ovoid and occurs in pairs or chains, Fastidious organisms, Facultative anaerobes Sherman (1937) divided streptococci into 4 groups based on serologically active group specific polysaccharide( C) substance into pyogenic, viridans, Enterococcus, Lactic groups Character Groupantigen Growth with 10C 45C 6.5% Nacl 9.6 pH 0.1% Methylene blue NH3 from Arginine Litmus reduction before clotting Resistance to 60C/30 min Haemolysis Examples Pyogenic A,B,C,D,E, F,G,H + S.pyogenes (A) S.agalactiae(B) S.disgalactiae (C) Viridans Non specific + +/, gama S.bovis (D) S.uberis S.thermophilus Enterococcus D + + + + + + + , , gama S.durans E.faecalis Lactococcus N + + +/+ gama L.lactis ssp.lactis L.lactis ssp.cremoris

PYOGENIC GROUP Streptococcus pyogenes: Belongs to Lancefield A. It is a pathogenic organism and causes septic sore throat, scarlet fever. The habitat is upper respiratory tract, skin lesions, and inflammatory exudates. It produces haemolysis and resists Phagocytosis. It produces erythrogenic toxin. It causes acute mastitis in animals. CAMP test is positive Streptococcus agalactiae: It belongs to Lancefield B and is , weakly , gama haemolysis. It is similar to S. pyogenes and causes mastitis in cows. It causes meningitis and pneumonia in humans. CAMP test is negative Streptococcus disgalactiae: Lancefield group C Causes bovine mastitis. CAMP test is negative VIRIDANS GROUP: Streptococcus uberis: This causes winter mastitis in cows. Serologically heterogenous. Non haemolytic. CAMP test is negative Streptococcus thermophilus: This is now known as Streptococcus salivarius ssp.thermophilus. It has no group specific antigen. Grows at 45C but not at 10 and 53C. OGT is 40-45C. o o o It is thermophilic starter culture used for the preparation of yoghurt, Swiss cheeses This is used for antimicrobial agents assay Ex. Sulphadiazine This can be used in microencapsulated form when grown in milk with added amino acids along with medium and also used in hypo caloric diets for treatments of obesity.

On Yoghurt lactic agar: shows small white colonies with clear zone where as Lactobacillus delbruckeii ssp bulgaricus produces large white colonies surrounded by cloudy zones. GENUS: ENTEROCOCCUS E. faecalis, S. durans These are mesophilic organisms, with OGT 37C . They resist 63C/30 min E. faecalis: The source of the organism is the intestinal tract of humans and animals. It is used as a trail starter culture in the manufacture of certain cheeses because of its salt tolerance and pH tolerance. It is dominant organism among enterococcus in India in various milk and milk products. It is non haemolytic, sometimes may be haemolytic with graying

GENUS: LACTOCOCCUS Lancefield N Group Grow at 10C but not 45C. Reduces the litmus prior coagulating it. Grow in broth with 0.1% methylene blue but not in broth with 6.5 % Nacl and 9.6 pH Character NH3 from arginine 40C 4% Nacl 9.2 pH Gas from citrate Lactococcus lactis ssp. lactis + + + + Lactococcus lactis ssp. cremoris Lactococcus lactis ssp. lactis biovar diacetylactis + + + + +

Lactococcus lactis ssp. lactis : Elliptical cocci in pairs or short chains and elongation of cells in direction of chain. The Colonies are Grey, circular, convex, glistening. OGT is 30C. It does not produce Co2 or diacetyl and does not produce ammonia from arginine. o o o o It is a important mesophilic starter It produces antibiotic NISIN which is inhibitory to Bacillus, clostridium, lactobacillus and some other Gram positive organism Some strains produce malty flavour due to metabolism of leucine to produce 3methyl butanol Lactose is fermented to lactic acid by homofermentation and produces 0.8% to 1.0% lactic acid

Growth requirements are very complex. It requires B- complex vitamins such as biotin, Niacin, thiamine, pantothionic acid, pyridoxine, folic acid and Amino acids such as Arginine, valine, histidine, leucine, isoleucine, methionine, phenylalanine, proline, glutamic acid It is easily inhibited by 0.15 units of penicillin, 0.5 micrograms of aureomycin and 600-1000 ppm quaternary ammonium compounds per ml Lactococcus lactis ssp. lactis biovar diacetylactis : It is similar to to the above organism. It utilizes citrate and produces Co2, diacetyl, volatile acids and acetoin. Utilization of citrate is plasmid mediated and is unstable character.The produced acetic acid is inhibitory to pseudomonas, coliforms, salomonella. Lactococcus lactis ssp. cremoris: It exhibits slow fermentation of lactose. It produces diplococcin GENUS PEDIOCOCCUS:G+ve, nonmotile spherical organisms, Microaerophilic, showing poor surface growth Occurs as tetrads, in pairs / short chains. Optimum growth temp: 25 30 C, Acid is produced from glucose, galactose and maltase not from mannitol & dextrin, Produces diacetyl apparently from oxidation of acetyl methyl carbinol

GENUS LEUCONOSTOCS:Leucos means colorless Nostac means encapsulated blue green algae similar to Nostoc except photosynthesis G +ve cocci in paris or short chains, Microaerophilic /Aerobic/Facultatively anaerobic Hetero-fermentative i.e. production of lactic acid, ethanol and Co2 from glucose. Some produce slime in sucrose media. It is comparatively, inactive in litmus milk and rarely coagulates milk Growth in medium is enhanced by yeast, tomato and other vegetable extracts. Ferment citric acid to Diacetyl, Acetoin, 2,3 butylenes glycol acetic acid, CO2 Causes slits in cheddar cheese made with butter cultures due to CO2

The organisms are L. mesenteroides ssp dextranicum, L. mesenteroides ssp mesenteroides , L. paramesenteroides, , L. mesenteroides ssp cremoris, L. lactis Section 13: ENDOSPORE FORMING GRAM POSITIVE RODS GENUS BACILLUS: Bacillus means little stick, Gram positive large rods (3 to 9 um in length), Aerobic, Saprophytic soil bacteria, May occur in single, pair or chains. Aerobic spore formers. Sources: Air, water, soil, feed and fodder Isolated by heating raw milk to 80C/ 10 mts before plating. Large and rough colonies are formed Acid sensitive, potent spoilage organisms, They may be psychrotrophes, mesophilic, Thermophilic, Catalase positive Spores: Ellipsoidal to cylindrical in shape, occasionally bulged sporangia Organism Spore Motility Lecithinase OGT Growth at 45C 65C 7% Nacl Citrate utilization B. cereus Central or paracentral + + 30C V V + B. subtilis Central or paracentral 30-40C + + + B. stearothermophilus Subterminal to terminal + 55-65C + + B. coagulans Subterminal to terminal + 35-45C + V V B. megatherium Central or paracentral + 28-35C V + +

B. cereus : Dont grow at 5C but grows at 20-35C. This is important organism because Causes food poisoning by producing enterotoxins when the number exceeds 106 per gram Produces bitty cream defect (broken cream) by the action of lecithinase enzyme (Extra cellular phospholipase) Produces sweet curdling by coagulating milk at lower acidity by producing rennet like enzyme

B. subtilis : Grows between 20-45C. This is important organism because Cause ropiness or sliminess in raw, pasteurized milk Causes spoilage of UHT, concentrated/ canned milk products Causes sweet curdling by coagulating milk at lower acidity by producing rennet like enzyme Produces levan extracellularly from sucrose Extracellular enzymes including those that degrade pectin, casein, polysaccharides of plant tissue are produced Polypeptide antibiotic subtilin are produced B. licheniformis : This is important organisms because Causes spoilage of UHT, concentrated/ canned milk products Produces levan extracellularly from sucrose Causes ropiness or sliminess in raw, pasteurized milk B. coagulans: This causes spoilage of UHT, concentrated/ canned milk products B. stearothermophilus: Grows between 45 to 65C This is important because It is obligately thermophilic Causes flat sour spoilage of canned/UHT milk products Used in the detection of antibiotics in the milk i.e antibiotic assay test

GENUS CLOSTRIDIUM: These are the anaerobic organisms and spore formers. Gram positive rods with 1-4 in length, motile, catalse negative. They may be mesophilic or Thermophilic The important source of these organism is soil, intestinal tract of animal. It may become established as contaminants on equipment. They gain entry into milk via faeces, soil, feed and especially silage Some are pathogenic organisms such as Cl. perfringens and Cl. botulinum

Cl. butyricum and Cl. tyrobutyricum are Thermophilic. The fermentation end products include acetic acid, butyric acid, butanol, iso-proyl alcohol, acetone, H2 and Co2 Cl. perfringenes : Causes bovine mastitis in cattle and causes neurological disorders in humans. It produces profuse gas and breaks the coagulum of milk causing Stormy fermentation Cl. tyrobutyricum: Causes late blowing condition in Cheeses Cl. butyricum; Causes late blowing condition in Cheeses after 1-2 months of manufacture Cl. sporogenes : Develops rancidity of Emmental cheese Character Spores

Cl. butyricum Oval Central to Sub-terminal Do not swell + 30-37C Acetic, Butyric acids Butanol

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

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

Cl. Perfringenes Oval Central to eccentric Large distend the cell 37-45C Acetic, Butyric, Lactic acids More H2 Ammonia and water

Motility OGT End products

SECTION 14: NON-SPORE FORMING G+ VE RODS, REGULAR GENUS: LACTOBACILLUS, Lac milk; Bacillus staff or stick. Long, thin rods, G+ve, asporagenous rods; Nonmotile, Microaerophilic, Aciduric, catalase Negative ; Require complex medium like MRS medium / Rogosa acetate agar with layering of plates Source: Feed, silage, manure 3 groups Thermobacterium Streptobacterium Betabacterium

Thermobacterium Growth at 15 45C VP Fermentation Examples + + Homofermentation L. bulgaricus L. acidophilus L. helveticus Hexose fermented to lactic acid by EMP, but Pentoses not fermented

Streptobacterium + + Homo /facultatively L. casei L. plantarum Hexoses fermented to lactic acid by EMP Some sp. also produce acetic, Formic & ethanol under glucose limitation. Pentoses fermented to lactic acid & Acetic acid involving phosphoketolase Serology E

Betabacterium -ve +/Heterofermentation L. brevis L. fermentum Hexoses fermented to lactic acid & acetic acid involving phosphoketolase pathway

THERMOBACTERIUM GROUP: L. delbrueckeii ssp. bulgaricus:Thermophilic, OGT 40C,

Used in Yoghurt, Swiss & Italian cheeses. Produces antibiotic Bulgaricin (stable for 1hr at 100c) which is active against Ps.fragi and to a lesser extent to S. aureus L. acidophilus:Thermophilic, OGT 45c; do not grow at 20C & at 4% salt concentartion Produces lactic acid & acidity reaches 1.8% but to keep viable the acidity should not exceed 0.6 & 0.7% Produces antibiotics - Acidophilin, Acidolin, Lactocidin Medium: Aesculin cellobiose Agar with incubation at 40c/48h in CO2 atmosphere. It produces colonies surrounded by dark olive green complex o o o o Used along with other mesophilic cultures in Kefir Used with Bifi bifidum in special ice cream Used in Paneer In India. Used as encapsulated forms in hypocaloric diets

Therapeutic uses: Hydrolysis of lactose suitable for lactose intolerant people production of antimicrobial substances, antibiotics & H2O2 prevention of constipation; ulcerative colitis Reduction of cholesterol by fermentation products Restoration of gut microflora after antibiotic treatment because of their capability to grow at low surface tension.

Therauptic value is based on the assumption that this milk combats the so called autointoxications caused by accumulation in the body, of toxic substances elaborated by toxigenic bacteria. STREPTOBACTERIAL GROUP L. casei:Serology B & C, Mesophilic, Produces upto 1.5%.acid on prolonged incubation o o o o Used in Yakult It shows Inhibitory action by producing acid, peroxides & antibiotics to Salmonella typhi salmonella typhinurium, Shigella dysenteriae, E.coli and P.aeuruginosa Used along with Candida lipolytica and Lac.lactis as cheese flavour additive in processed cheese by spray/ freeze drying. Used with Pr. shermani to produce mycostatic preservative

L. plantarium:Serology D, Used in Brine cheese, OGT - 30 35C Antibiotic Lactolin is produced which is effective against G+ve organisms

BETABACTERIUM GROUP L. brevis:Serology E, Mesophilic, OGT 30C; Do not grow at 45C, but grow at 15C. Used in Kefir. Lactobrevin antibiotic is produced.

GENUS: LISTERIA Gram +ve, Short rods with rounded ends; may be curved; single, short chains or V forms. Catalase positive, Aerobic / facultatively anaerobic Colonies are Bluish grey when seen by normal illumination and bluish green sheen by obligately transmitted light. Sources: Wide; Water, mud, Sewage, faeces of animals & man Listeria monocytogenes: OGT 37C, Cattle : abortion Human: food poisoning, meningitis accompanied by septicaemia GENUS MYCOBACTERIUM G +ve, but staining difficult due to high wax content, Acid-fast. Non- motile, now-sporing, non-branching rods. M. tuberculosis grows very slowly in vitro. Requires two weeks or more to show visible growth and requires special media for growth. Ex:- Loefflers Serum medium. Causes tuberculosis in humans

Section 15: IRREGULAR, NON SPORING G+ve rods GENUS CORYNEBACTERIUM:Cells are straight to slightly cured rods with tapered ends, Club shaped appear. Non motile, Asporogenous, Aerobic/facultatively anaerobic forms may

Recognized by their banded and beaded, clubbed appearance, meta chromatic granules are formed. Chemo organitrophs, OGT : 37C C. boris is not pathogenic & causes rancidity in cream. C. pyogenes causes Supportive mastitis

GENUS BREVIBACTERIUM:Rod-coccus growth, Gram positive, Non-motile, obligate aerobic, Chemoorganotrophic with respiratory metabolism. OGT : 20-30C, Non-thermoduric B. lines : Produce yellow to deep orange red carotenoid pigments Usually present on exterior of surface ripened chesses of Limburger type.Contributes to the surface colour of such cheeses aid in ripening by proteolysis and improve the flavour & aroma by the production of methanethiol.

GENUS PROPIONIBACTERIUM:Gram positive, Non-motile, Asporogenous, Anaerobic/aerotolerant, Pleomorphic rods, club shaped with one end rounded and the other tapered or pointed. Cells may be coccoid, bifid, or branched, occur in single in pairs, or Short chains (in V or Y configuration) , 5% carbon dioxide atmosphere is good for growth. Opt. growth temp: 30-32C Lactic acid and CHOs is converted into propionic /acetic acids +Carbon dioxide Pr. freundenrichii is associated with Swiss cheese flavour because of proline production and eyes due to Carbon dioxide.

FUNGI Division Sub Division Class Sub class Order Family MOLDS Penicillium: (Class Deuteromycetes): Penicillium have septate vegetative mycelia which penetrate the substrate and then produce aerial hyphae on which conidiophores develop. Condiophores may be branched and have brush like heads bearing spores clusters of sterigmata are usually in one place and from each is formed a chain of conidia. With the production of conidia, colonies become green, grey green, blue green and yellow green. The colour of mature plant is useful in helping to identify species. Asymmetric group. Pencillium roquefort: Used in Roquefort, of blue veined cheeses. Asymmetric group i.e. more than one branch in the conidiophore and this branching is asymmetrical. Colonies on malt agar blue green spreading colonies slowly change to darker green. Smooth velvety appearance with irregular margins of radiating lines of conidiophores Spiders Web Arachnoid , Conidiophores are rough, conidia globose, smooth or borne in loose columns or tangled chains. Toxins produced are Roquefortin, mycophenolic acid, PR toxin, P.casei : Used in Swiss cheese, Asymmetrica group, No arachnoid margins, Colonies yellow brone P.camemberti: Used in camembert, Brie cheeses colonies / white & gradually become pale grayish green from the centre onwards Asymetrica group, Conidiophores slightly rough, Conidia become sub-glabose & borne in tangled chains Produces Toxins i.e cyclopiazonic acids Mycota Mycotina Mycetes Mycetidae ales aceae

Geotrichum candidum: Commonly found on dairy products Colonies White, yeast like forms true mycelium, breaks to form arthrospores are cylindrical with rounded ends. Sporendonema sebi: Causes buttons in SCM Asperigillus (Class Duitenomycetes): Septate branching mycelia with vegetative portions submerged in nutrient. Conidiophores or fertile hyphae arise from thickened foot cells which may also be submerged. At the apex, condidiophore inflates to form a vesicle, which gives rise to sterigma which may be single layered or double layered. Condia arise from the sterigmata and borne in chains. Conidia are produced within the tubular sterigmata and are extruded to form spore chains. Conidia are of various colors and are quite characteristic of the species. Mucor: Sporangiophores each bear terminally a single large globose sporangium containing many spores (spherical/ellipsoidal), No stoleniferous growth, No septate mycelium, sporangiophores never arise from nodes on stolons, rhizoids absent. Rhizopus stolenifer: Bread mold. Stoleniferous type of spread. Non-septate, sporangiophores form at are quite large and Black. Heterothalic. which

YEASTS : Unicellular - spherical/ovoid, Pseudomycelium may form veg.rep budding Ascospores 1 to 4 per ascus. Multiply asexually by budding where a bud has formed on a cell a raised star remains. As many as 23 bud scars are found on a single cell. During budding nucleus divides by construction and a portion of it enteres the bud along with other organelles. The cytoplasmic connection is formed by laying down of cell wall material. Under appropriate conditions forms asci. The cytoplasm of the cell differentiates into four thickened wall spherical spores, although the no.of spores can be fewer. The cells from which asci develop are diploid and nuclear divisions which precede spore formation are meiotic. Ascospores are of two mating types. Mating type is significantly controlled by a single gene which exists in two alletic states a & and segregation at reduction devision preceding ascospore formation gives rise to 2 & 2 ascospores. Fusion occurs between two different mating types. (legitimate copulation). Such fusion results in diploid cells which form asci containing viable ascospores.

S.cerevisiae: Bakers yeast, isolated from kefyr. Cells globose, subglobose, ellipsoidal/cylindrical singles, pairs, short chains/clusters. Ascospores - globose to short ellipsoidal, 1-4/ascus, do not liberate, Lactose not fermented, Nitrate not assimilated. Kluyveromyces marxianus var.marxianus: - Kly fragilis/sac.fragilis: Used in Kefyt & Kumiss Cells subglobose, ellipsoidal to cylindrical single or pairs, Ascospores one to 4/ascus cresentiform ot reniform, Lactose may be fermented by some strains, Nitrate not assimilated. Kluy.marxianus var.lactis: Kly.lactis/Sacch.lactis: Associated with yoghurt, isolated from milk, gassy cheese, Italian cheese, Cream, BM Cells spherical, ellipsoidal or occasionally cylindrical clusters, singles, Pairs and occasionally clusters., Ascospores one to 4, spherical to ellipsoidal, Readily released, Lactose fermented, Nitrate not assimilated Candida kefyr: Toru.kefyr/ Can.pseudotropicalis var. lactosa: Associated with kefyr, buttermilk and cheese, Morphologically variable, Budding unicellular to pseudomycelium or true mycelium, Cells Ovoid short/long, Reproduction budding or fission, Colonies off white to cream, Lactose may be fermented., Nitrate not assimilated. Candida lacticondensi (Tor. Lactiscondensi): Isolated from SCM Cells Ovoid, budding cells, Colonies offwhite, cream, yellowish or brownish, Mycelium not formed or rudimentary mycelium is rarely found., Lactose not fermented, Nitrate assimilated.

BACTERIOPHAGE Bacteriophages are the viruses that infect bacteria. Viruses are not plants, animals, or bacteria, but they are the quintessential parasites of the living kingdoms. Phage literally means devouring just like phagocyte (to swallow or eat up greedily). Widely distributed in nature & most abundant in intestinal contents of animals and first is invented by Twort in 1915. Typical bacteriophage has a structure like tadpole. Head enclosing nucleic acids in protein sheath. Tail a hollow tube of proteins & bearing tail plate & tail fibers. All viruses contain nucleic acid, either DNA or RNA (but not both), and a protein coat, which encases the nucleic acid. Some viruses are also enclosed by an envelope of fat and protein molecules. Without a host cell, viruses cannot carry out their life-sustaining functions or reproduce. They cannot synthesize proteins, because they lack ribosomes and must use the ribosomes of their host cells to translate viral messenger RNA into viral proteins. Viruses cannot generate or store energy in the form of adenosine triphosphate (ATP), but have to derive their energy, and all other metabolic functions, from the host cell. They also parasitize the cell for basic building materials, such as amino acids, nucleotides, and lipids (fats). They are significant from processing point of view. Phages attack Lactic acid bacteria & lyses them by multiplying inside the host cell leading to the release of many phages which can re-infect the fresh cells in the culture. This results in the failure of starters to act & bring about the changes during preparation of fermented products. Bacteriophages are highly host specific, a rotation of starter cultures help to control the problem to some extent. Ca ion deficient medium for maintaining starter cultures prevent phage attack as the ion helps in phage adsorption to host cell. Genetic manipulations to construct phage resistant strains.

Typical Lactococcus lactis ssp. lactis phage might have 40-90 microns head & 100-120 microns tail.Bacteriophages can persist for a long time in dried up whey & on utensils. Phages attach themselves to living cells & as they divide phages also multiply. The infective material is injected (nucleic acid) through hollow tail of the phage particle into the bacterial cell. The rate of phage proliferation is greater than that of bacterium. Consequently, a point is reached at which lysis begins. Lysis is preceded by swelling of the bacteria to 6-16 times their normal size. Phage action may cause a number of variations in the properties of bacteria.Irreversible adsorption of phage to the cell triggers a no. of steps which lead to the transport of viral nucleic acids into the cytoplasm & nucleic acid gets metabolically active transforming cellular function to one of synthesizing viral components. As the phage assembly completed the lysis of the host cell occur. All this occur approx. in one generation time of the host phage multiplying to hundred in one generation & in two generations to 10000 & so on.

GERMICIDAL PROPERTIES OF MILK Normal milk contains varying amounts of substances which inhibit normal development of certain bacteria and some even kill these bacteria Level of activity depends on 1. Type of milk 2. Quarters of same animal. Functions: 1. To protect mammary gland from infections. 2. To confer resistance to young suckling calves. IMMUNOGLOBULINS: Immunoglobulins are antibodies against specific antigens, often to bacteria. In the man prenatal immunity is conferred primarily through the transmission of IG from the mother across the placenta to fetal circulation. In cows and buffaloes IG are transferred from colostrum into the newborns circulation postnatally when the GI tract is permeable to intact protein molecules. Milk has two types of Immunoglobulins, 1. 2. Functions: IgA: IgG: Produced locally with in the udder Transferred to milk from circulation.

Reduces the severity of udder disease by Neutralizing toxins elaborated during disease process. Aids in phagocytosis by polymorpho nuclear leucocytes. prevents bacterial adhesion to the cells Suppresses bacterial growth.

PHAGOCYTOSIS Phagocytosis means Invading of pathogens by leucocytes. Protection of udder from mastitis rests primarily on phagocytosis and killing of pathogens by PMN. Out come of an invasion attempt by pathogens is usually decided in early hours of infection. o The infection will be repelled, if phagocytosis occurs faster than the multiplication of pathogens o The pathogens grow faster in the absence of efficient phagocytosis and cause clinical mastitis. During mastitis the high leucocytes counts are present and it is evident that if the infection is established they cannot efficiently and quickly dispose off the invading pathogens. Leucocytes of uninfected udder vary between 1 to 5 lakh cells/ml of which approximately 10% are PMN. In infected udder PMN may be upto 90%. Phagocytosis is less effective in milk than in blood. PMN leucocytes ingest some quantities of milk fat and casein, thus reducing the efficiency, so udder is easily affected by even a small number of invading bacteria. Functions: Confer protection to udder from pathogenic bacteria.

LACTOFERIN: Earlier called as Lactotransferrin. It is a red glycoprotein and resembles Blood serum transferrin. It is a iron binding protein. Lactoferin combines with iron and make it unavailable for bacteria, which is an essential growth factor. Lactoferin by virtue of the high concentration and iron binding ability enhance the resistance of dry mammary gland to infection. High citrate and Low bicarbonate in milk reduces iron binding properties of Lactoferin Colostrum contains 6 mg/ml where as 1 mg/ml shows bacteriostatic action. But Colostrum also contains high citrates which compete for iron with LF and make it available for bacteria. Citrate in Colostrum is 3.6 mg/ml. Mature bovine milk contain 0.42mg Lactoferin /ml. Lactoferin is bacteriostatic to B. subtitis and B. stearothermophilus and Inhibits S. aureus and P aeruginosa. LYSOZYME: In human milk it is present at 30 mg/100ml i.e., 300 times that of bovine milk. This enzyme hydrolyses 1-4 linkage of peptidoylcan of bacterial cell wall i.e., linkage between N-acetyl muranmic acid and N- acetyl glucosamine, resulting in weakening of cell wall ultimately resulting in lysis of cell. Lysozyme is very active against G +ve bacteria especially thermophilic spore formers and inhibitory to Listeria monocytogenes, Campylobacteium jejuni, Salmonella typhi, Bacillus cereus and Pseudomonas aeurogenosa. LACTOPEROXIDASE SYSTEM This involves three components I. Lactoperoxidase enzyme synthesized in mammary gland in concentration of 30 mg/ml II. Thiocyanate content in milk is governed by nutrition of the cons. Usually 1-10 g/ml III. H202 contributed by PMN or by some udder flora ex. Streptococci. ( catalase -ve organisms) The Lactoperoxidase enzyme combines with H202 to oxidize thiocyanate (SCN-) yielding various intermediate oxidation products such hypothiocyanate, cyanosulphurous, clyanosulphuric acid which exhibit antimicrobial activity (OSCN-) Bactericidal to Group A streptococci, E. coli, S. typhi Pseudomonas. Bacteriostatic to group N and group B streptococci Lactobacilli o Inhibition of O2 uptake Inhibition of growth by interfering with oxidation of SH groups of enzymes. o Damage to cytoplasmic membrane causing leakage and cessation of nutrient uptake. o Inhibition of LA production. Function: i. Protection of calf from enteritis ii. Cold sterilization of milk

SOURCES OF BACTERIAL CONTAMINATION IN MILK Interior or Udder: Varying number of bacteria are found in aseptically drawn milk with the reported counts of