Milk microbiology standards for milk and milk product Shivnam Rana Msc. Microbiology (PAU) L BS -251 – M

Introduction Complete food – Carbohydrates(Lactase), Protein(Casein), Fats, Minerals due to: Complex biochemical composition High water activity Excellent culture medium for the growth and multiplication of microorganisms

Factors that influence survival and growth of microorganisms Intrinsic Nutrient availability pH Water availability Extrinsic Temperature Atmosphere

Origin of microorganism in milk Commensal micro flora- teat skin, epithelial lining of the teat canal, duct that conveys the milk from the mammary gland to the teat orifice. Staphylococcus, Streptococcus,Bacillus, Micrococcus, Corynebacterium, coliforms Environmental contamination- soil, water equipment, dairy farm area are reservoir for many food borne pathogens Salmonella species, L. monocytogenes

Diseased animal Mastitis- Staphylococcus (S.agalactiae) and Streptococcus species

Lactobacillus Obligate homofermentative lactobacillus – hexoses ferment lactic acid Growth at 45°C but not at 15°C Lactobacillus delbrueckii, lactobacillus helveticus, lactobacillus salivarius and L. Acidophilus

Facultative homofermentative lactobacillus- hexoses ferment lactic acid or glucose limitation acetic acid, ethanol, formic acid Grow at 15°C and show variable growth at 45°C Lactobacillus casei, lactobacillus curvatus, lactobacillus sakei and lactobacillus plantarum.

Obligate heterofermentative lactobacillus- Hexoses ferment lactic acid+ acetic acid+ ethanol+ CO2 pentoses ferment lactic acid + acetic acid Lactobacillus fermentum, lactobacillus brevis and lactobacillus keferi.

Benefit Resist weak acids of ph 3.5 to 4.5 resulting to a yield of 90% lactic acid. Starter culture. Preservative for the production of cheese and yoghurt.

Lactobacillus acidophilus Absorb B vitamins, vitamin K, fatty acids and calcium. Reduce lactose intolerance. Increases the production of anti-allergy cytokines, chemical messengers released by cells of the immune system.

Lactobacillus bulgaricus Helps reduce symptoms of lactose intolerance. Provides the stomach and intestine with the enzyme bacterial lactase that helps digest the lactose in milk.

Enterococci Ubiquitous gram-positive, catalase-negative Ability to survive extreme ph, temperatures, and salinity. Psychrotrophic nature, heat resistance and adaptability to different substrates and growth conditions

Enterococci also occur in natural milk (or whey) starter cultures Made by incubating it at 42–44 °C for 12–15 h Used for manufacturing of cheese ( artisan)

Strains E. Faecalis and E. Faecium producing enterocins Activity against listeria monocytogenes, staphylococcus aureus, clostridium spp., Including clostridium botulinum and clostridium perfringens, and vibrio cholerae

Bifidobacterium bifidum Optimum growth ph Optimum growth temperature °C, maximum °C, minimum °C Produce acetic acid, lactic acid- 3:2 Helping in digestion Strengthens the immune system To eliminate harmful bacteria

Lactobacillus rhamnosus Lactose-intolerant people to reduce the inflammatory response that occurs when they consume milk. Helps the immune system by stimulating the production of antibodies and combating pathogenic bacteria.

Lactose and galactose utilization by different lactic acid bacteria

Lactobacillus curd Milk is heated to a temperature of °C Complex and heterogenous flora - L. Lactis, L. Lactis subsp cremoris, S. Thermophilus, L. Debrueckii subsp bulgaricus, L. Plantarum and lactose fermenting yeast. Anaerobic respiration

Special attribute of Curd Dysentery and other gastrointestinal disorders. Improve appetite, vitality and increase digestibility.

yogurt Mixed starter culture – S. Thermophilus and lactobacillus delbrueckii or lactobacillus bulgaricus. Ratio 1:1 Fermentation :- lactose content of milk to yield lactic acid, CO2, acetic acid, diacetyl, acetaldehyde Ph reduces :- around 6.5 to 4.5 due to the production of organic acids Initially streptococcus thermophilus ferments the lactose Lactobacillus bulgaricus, which is more acid tolerant, continues to ferment the remaining lactose. Lactase Lactose glucose + galactose Hydrolyzes

Benefit Easier digestibility, The ingested organisms enhance bioavailability of nutrients Ensure gastrointestinal balance, Promoting colon health Accelerates the healing of gastrointestinal tract disorder Reduction in cholesterol level.

Cheese

Different cheese using different strains Swiss cheese formation involves a late propionic acid fermentation with ripening done by Propioni bacteria shermanii. Blue cheeses are produced by Penicillium spp. Roquefort cheese is produced by using P. roqueforti Camembert and Brie by using P. camemberti and P. candidum.

Pathway for Citrate – positive strain of lactococcus and leuconostoc species

Changes in milk by microorganism

Gas production: Fermentation occurs at faster rate, then raw milk present a foamy layer on the upper surface Air bubbles becomes entrapped and gas becomes saturated throughout the body of the milk Colliforms, clostridium and bacillus species.

Proteolysis Acid Proteolysis: Milk whey separated, Milk taste will be soured, Micrococcus sp. Alkaline Proteolysis: Milk whey separated and pH >6.9 (towards neutral and alkaline). Milk taste will be bitter Sweet curdling: Bacillus cerus – it release enzymes “protease” which targets the casein. Bacteria use lactose and convert into acids and aldehyde components. Milk is sweet in taste Slow Proteolysis: Release of endogenous proteases in the milk, cause slow proteolysis of the milk. Proteolysis due to Anaerobic Bacteria: Bacillus and clostridial species are heat resistant. result into specific kind of smell names as butyrine smell

Roppiness or sliminess : Milk viscosity is increased, rope like structure is formed Alcaligenes viscolactis Change in the color of milk: Blue color pseudomonas synciani Red color brubibacterium erythrogenes, sarcinia marcense Yellow color pseudomonas synxantha Brown color pseudomonas putrificians Green color pseudomonas aurogenosa

Changes in Milk Fat Oxidation of unsaturated fatty acids: Milk fat oxidation aldehydes + ketones + acids Oxidation imparts tallowy odors Hydrolysis of overall milk fat: lipase Fatty acids glycerols + free fatty acids Hydrolysis Gives rise to putrefied odor (rotten egg like smell) Combined hydrolysis and oxidation eg:- Proteus, Alcaligenes, Micrococcus

Change in the Flavor of Milk Sour Flavour: It is due to acidic changes in the milks: Clean: Low contents of acids, Streptococcus lactis Aromatic: streptococci and aroma- forming Leuconostoc sp., moderated type of acidic components. Sharp: coliform bacteria, clostridium species, volatile fatty acids, high acidic contents Bitter Flavour: It is due to alkaline changes in the milk. Potato-like Flavour: Pseudomonas mucidolense Fishiness: Acromian hydrophila, It is due to formation of tri-methyl amine

Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE)

Fluorescent in situ hybridization(FISH)

ELISA

Prevention method Pasteurization Ultra-High Temperature- 138 °C to 150 °C for 4 to 15 seconds Steam under pressure- 115 to 118°C for 14 to 18 min, caned milk Radiation- gamma, UV rays Preservatives- sorbic acid, propionic acid, sugar, salt, hydrogen peroxide Modified atmosphere packaging Refrigeration - 10 °C or low temperature Freezing to -18 °C

HACCP Principle 1: Conduct a hazard analysis. Principle 2: Determine the Critical Control Points (CCPs). Principle 3: Establish critical limit(s). Principle 4: Establish a system to monitor control of the CCP.

Principle 5: Establish the corrective action to be taken when monitoring indicates that a particular CCP is not under control. Principle 6: Establish procedures for verification to confirm that the HACCP system is working effectively. Principle 7: Establish documentation concerning all procedures and records appropriate to these principles and their application.

New bacteria discovered in raw milk Chryseobacterium oranimense, which can grow at cold temperatures(7°C) and secretes enzymes that have the potential to spoil milk.“ C. haifense and C. bovis

laws Milk and milk product order, 1992 Milk and milk product amendment regulation, 2009

Precautions The animal should be healthy and free from diseases. A healthy person should milk the animals. He should avoid sneezing, coughing, etc., and must wear clean clothes. Milking vessels should be cleaned properly with chemicals or detergents that are not injurious to health. Arrangements must be made in advance to immediately cool the milk to 4 ºC within an hour of milking. The ingredients and cleaning agents used must be of the desired quality. There should be a provision for checking the quality, sampling and testing of milk.

Reference Beresford T P, Fitzsimons N A, Brennan N L and Cogan T M (2001) Recent advances in cheese microbiology. International Dairy Journal –74. Ledenbach L H and Marshall R T 2009 Microbiological Spoilage of Dairy Products. Compendium of the Microbiological Spoilage of Foods and Beverages Nwamaka N T and Chike A E (2010) Bacteria population of some commercially prepared Yoghurt sold in Enugu State, Eastern Nigeria. African Journal of Microbiology Research 4 (10) Papademas P and Bintsis T (2010) Food safety management systems (FSMS) in the dairy industry: A review International Journal of Dairy Technology Varga L (2007) Microbiological quality of commercial dairy products. Communicating Current Research and Educational Topics and Trends in Applied Microbiology

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