Microbiology of milk

Describes about various microbes present in Milk.Good and Bad effects of microbes.Spoilage of Dairy products.

1. MILK PROCESSING TECHNOLOGY LIBRARY PAPER PRESENTATION PRESENTED TO:- PRESENTED BY:- ER.NITIN KUMAR YASH GAJWANI 114172 VIBHUTI MITTAL 1141 YUKTII BAHL 114174 SUMIT KUMAR 1141 SALONI GOYAL 1141

2. Topics to be covered

3. COMPOSITION OF MILK

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

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

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

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

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

9. PRESENCE OF ANTIMICROBIALS Natural constituents of foods which affect microbial growth are: Lactoferrin e.g. Milk Lactoperoxidase e.g. Cow’s milk Conglutinin e.g. Cow’s milk These antimicrobials help preserve milk for longer period of time.

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

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

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

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

14. MILK SPOILING BACTERIA

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

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

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

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

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

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

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

22. Most of the species are microaerophilic . It is oxidase and catalase positive and does not grow in the presence of 3.5% NaCl or at 25 °C or below. The incidence reported for gastro enteritis by this organism are as high as in case of Salmonella . The organism is commonly present in raw milk, poultry products, fresh meats, pork sausages and ground beef. The infective dose of C.jejuni may be <1,000 organisms.

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

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

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

26. SPOILAGE OF DAIRY PRODUCTS

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

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

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

30. Spoilage of Raw Milk

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

32. Outbreaks of illness are due to consumption of raw and pasteurized milk contaminated with a variety of organisms, including E. coli O157:H7, Salmonella spp., Campylobacter jejuni , Yersinia enterocolitica , and Listeria monocytogenes . Raw milk may be a vehicle for the transmission of Borrelia burgdorferi , the agent responsible for Lyme disease, and it has been shown that the organism can survive for at least 46 days in milk stored at 5◦C. E. coli O157:H7 has also been shown to be able to survive in yogurt.

33. Spoilage of Pasteurized Milk

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

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

36. Ropiness and partial coagulation may also occur occasionally. Yeast and mould are also indicators of post-process contamination. Their presence and growth contribute to fruity and yeasty flavours in milk. Food Types of Spoilage Spoilage Microorganisms DAIRY MILK (pasteurized) Bitterness & Sliminess (high pH) Pseudomonas spp. Souring Lactobacillus thermophilus Sweet curdling Bacillus cereus

37. MICROORANGISM ASSOCIATED WITH PASTEURISED MILK AND PRODUCTS Salmonella Campylobacter spp Listeria monocytogenes E. coli O157:H7 Yersinia enterocolitica Staphylococcus aureus Bacillus spp. Mycobacterium avium subsp.

38. Spoilage of Milk Powder

39. Spoilage of Milk Powder Spoils only when moisture content >0.8% (alarm water content). Spoilt by molds only. Eg . Mucor , Aspergillus , Penicillium , Rhizopus sp. Milk powder when spoilt becomes lumpy in texture. Recently, it has been found that contamination of powdered infant milk by Enterobacter sakazakii can lead to infant death . Contamination of milk powder due to S. aureus enterotoxin in some cases .

40. Spoilage of Butter

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

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

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

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

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

46. Streptococcus Frequently used for culturing cheese and yogurt as they ferment lactose A lso produce lactase - helping people with lactose intolerance to digest milk more efficiently to lactate . It’s a probiotic that helps improve digestion

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

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

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

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

51. Measures against S poilage Organisms Cleaning and disinfection of the milking equipment is essential Cooling – to slow down bacterial growth in milk Thermalization and then cooling – reduce psychrotropes in milk

52. REFERENCE https://www.ilri.org/InfoServ/Webpub/fulldocs/ilca_manual4/Microbiology.htm#P122_10360 FOOD MICROBIOLOGY by Frazier DAIRY SCIENCE AND TECHNOLOGY by CRC Publications