Milk Fermentation Lecture 4 PDF

Milk Lecture 4 “MILK FERMENTATION” Bacteria in Milk: Milk contains: water carbohydrates, proteins, carbohydrates, fats, vitamins, and minerals, and has a PH of about 6.8. Therefore, it is considered as: An excellent food for human. An excellent growth mediu...

Milk Lecture 4 “MILK FERMENTATION” Bacteria in Milk: Milk contains: water carbohydrates, proteins, carbohydrates, fats, vitamins, and minerals, and has a PH of about 6.8. Therefore, it is considered as: An excellent food for human. An excellent growth medium for microorganism. Milk is sterile at the moment of its secretion in the udder, but it is contaminated by bacteria even before leaving the udder. Most of the bacteria found in milk from healthy animals are few in numbers and harmless. Further contamination of milk by microorganisms can take place during: milking, handling, storage, transport, and other pre-processing and processing activities. Contamination of milk with disease-producing microorganisms (Pathogens) is possible from diseased animals, dairymen, polluted water, soil, equipment, and other sources. In general bacteria in milk fall into two groups: 1. Pathogenic organisms: (of public health importance) They cause diseases to man and/or animals. They have a mild effect on milk constituents. 2. Non-pathogenic organisms: (of economic importance) They cause changes on milk constituents (economic losses). Their main types are lactic acid bacteria, spore-forming organisms and those responsible for abnormal conditions or taints. Normal Fermentation of milk: Natural changes in milk. Fermentation: Is a process by which change is produced in milk due to the activity of microorganism. As milk consists of a variety of chemical compounds, which are utilizable by M.O’s, and As raw milk is not free from M.O’s, therefore it is to be expected many changes are possible to occur as a result of the growth of these M.O’s. If a sample of milk is placed immediately after milking in a shallow dish and left at room temperature (25°C), a serious of sequence changes usually take place. These changes are called Normal Fermentation of Milk. These changes could be detected by: Organoleptic exam. Microbiological exam. Chemical analysis. These changes may be divided into 4 phases or periods as follows: 1. Germicidal period. 2. Souring period. 3. Neutralization period. 4. Putrefactive period. 1. Germicidal Period (Phase): Immediately after milk is drawn from the udder there is a period during which there is no active growth of bacteria, but there may be a decrease in their numbers. This germicidal action is due to the presence of “Anti-microbial system” in milk, which is destroyed by heating milk at 60°C for 30 minutes. The length of the germicidal period varies considerably from few minutes to several hours depending upon: Species and breed of the animal. Quarter. Fraction of milk from the same quarter. Initial bacterial count.  Storage temperature (the higher the storage temp. the shorter the germicidal period and vice versa). Anti-microbial system in milk Functions of the anti-microbial system: 1. Protection of the mammary glands from infection. 2. Protection of the suckling young against diseases. 3. Protect the milk against bacterial growth. This system is not definitely known, but it may be due to Elements of the anti-microbial system in milk: Immunoglobulins (Ig). Polymorph nuclear Leucocytes. Lactoferrin. Lactoperoxidase system. Lysozyme. N Acetyl-ß-D glycosaminidase (NAGase enzyme). 1. Immunoglobulins (IG): They are antibodies against some pathogens. They may be locally produces in the udder (Ig A), or transferred to the milk from circulation (Ig G). Functions of antibodies: 1. Protection of the udder from infection. 2. Protection of the suckling young from infection (transfer of passive immunity from the mother to the neonate). 3. Reducing the severity of udder infection by neutralizing the operating toxins. 2. Polymorph nuclear Leucocytes: They protect the udder from infection by phagocyting the invading bacteria. The total somatic cell count of normal milk ranges from 100,000 to 500,000/ml milk of which 10% are polymorph nuclear leucocytes.  In case of mastitis this count is raised up to 10x106/ml milk of which 90% are polymorph nuclear leucocytes. 3. Lactoferrin: It is an iron-binding protein naturally present in milk and colostrum. Most micro-organisms need iron for growth. Lactoferrin has potential to inhibit their growth or even kill them by depriving them of iron. The amount of lactoferrin increases in case of mastitis. 4. Lactoperoxidase system (LPS): “Lactoperoxidase / Thiocyanate /Hydrogen Peroxide System” Peroxidase enzyme can kill bacteria by oxidative mechanisms. Lactoperoxidase (in milk) + Peroxide (from leucocytes) + Thiocyanate (In calf’s saliva derived from the ration) = Hypothiocyanate. Hypothiocyanate oxidizes the vital bacterial metabolic enzymes causing death of bacteria. 5. Lysozyme: Lysozyme is an enzyme present in the milk of some species, especially human milk. Lysozyme kills bacteria by attacking the polysaccharides of their cell walls causing lysis of the bacteria. The limited lysozyme activity in cow’s milk increases in case of mastitis due to the high somatic cell counts. Heating cow’s milk at 75°C for 15 min destroys 25% of the activity of this enzyme. However, human milk lysozyme is more heat stable than cow milk lysozyme. 6. N Acetyl-ß-D glycosaminidase (NAGase enzyme): Is an enzyme whose activity and amount increases in milk during mastitis.  It is a lysosomal enzyme (causes lysis of bacteria). 2. Souring Period (Phase): During this phase there is more active growth of many types of microorganisms, but lactic acid producing organisms dominate the field and convert the lactose into lactic acid (with other changes taking place to a lesser extent). The souring phase may continue from several hours to several days, but will reach to the end when so much acid is produced that it inhibits the growth of microorganisms producing it. Lactic acid producing streptococci (e.g., Strept. lactis) produce lactic acid up 1–1.3 % lactic acid then their growth is inhibited. Lactobacilli are more acid resistant (acid tolerant) as they are able to survive the already produced acidity and continue to produce more lactic acid up to 3.5-4 % where they can not survive the acidity of milk (casein is precipitated at this time). The evidence of acid formation is indicated by: Firstly, development of sour flavor. Then coagulation of casein to give a solid jelly like curd (or weak curd) with the release of whey. 3. Neutralization period: In the presence of considerable quantities of acid, most bacteria are inhibited to multiply, while yeasts and molds (fungi), which usually present in milk, become more active (flourish) in the acid medium. They utilize lactic acid as a source of energy (i.e., they reduce the acidity of milk) with the production of several alkaline by-products that neutralize the acidity and change the reaction of milk to the neutral or slightly alkaline side. The neutralization period may involve several days. A thick mat of molds growth often observed on the surface of milk at the end of this phase.  4. Putrefactive period: When the acid disappears due to the activity of fungi, and the reaction of milk changed from the acidic to the neutral or slightly alkaline side, proteolytic bacteria (which were remaining dormant during the last periods) enter into an active phase and become the dominating organisms. They attack milk proteins, principally casein, and in cooperation with fungi, they completely decompose the solid constituents of milk with the production of a clear fluid, not similar to the ordinary milk and has bad odor. The PH of such fluid ranges from 8-10. It is the point of complete decomposition of milk. Such fluid is very harmful and is unfit for human consumption (it is chemically poisonous). Milk is seldom kept long enough in dairy practice to undergo such extensive fermentation. “SPECIFIC FERMENTATION OF MILK” “ABNORMAL CHANGES IN MILK” “TANTS AND ABNORMAL CONDITION” “SPOILAGE OF MILK” 1. Acid Fermentation (Souring): In the field of dairy, the most important and common type of fermentation is acid fermentation (souring), in which a portion of lactose is changed into lactic acid by the action of enzymes produced by a group of microorganisms called lactic acid producing organisms (lactics). Some of these organisms are also used as starter cultures in the production of cultured dairy products such as yoghurt, butter, cream, and cheese. Lactic acid bacteria are found almost everywhere in nature, but they are found in especially large numbers in places where there is milk.  When the conditions are favorable, lactic acid bacteria attack lactose (as a source of carbon) to produce lactic acid which gives sour flavor and then causes curdling of casein. The curd formed by acid only in firm, smooth, and gelatinous without any visible signs of decomposition or rupture by gases. Usually Casein precipitates at room temperature (about 25°C) at 0.50 - 0.65 % lactic acid (PH 4.7 - 4.8). Lactic acid producing organisms (Lactics): This group includes bacilli and cocci. They are facultative anaerobes. Heating of milk at 70-80°C kills most of them. They could be classified according to: A. The products of fermentation. B. The shape of the cells. A. Classification of lactic acid producing bacteria according to the products of fermentation: 1. Homofermentative (Homolactic): They produce lactic acid as a major or sole product of fermentation. They produce also small amount of acetic acid, CO2, and other volatile products. E.g., Strept. Cremoris and Strept. Thermophilus. They are utilized in the manufacture of some types of fermented milks and cheese. 2. Heterofermentative (Heterolactic): They produce appreciable amount of both: Lactic acid. Other volatile products (e.g., acetaldehyde and diacetyl) in addition to CO2 and ethanol. E.g., Strept. diacetylactis. They are utilized for production of flavor and aromatic compounds in some products as butter and some types of fermented milks. B. Classification of lactic acid producing bacteria according to the shape of cells: 1. Streptococci (lactococci): Can survive up to 1-1.3% lactic acid.  They could be classified as follow: I. Homofermentative Streptococci: e.g., Strept. lactis, Strept. Cremoris and Strept. Thermophilus. II. Heterofermentative Streptococci: e.g., Strept. diacetylactis, Strept. citrovorus and Strept. paracitrovorous. III. Pathogenic lactic acid producing streptococci: e.g., Strept. agalactia, Strept. pyogenes, Strept. bovis and Strept. faecalis. 2. Lactobacilli: Lactobacillus casei. L. acidophilus L. lactis. L. bulgaricus. L. helveticus. L. plantarum. 3. Leuconostoc: Leuconostoc citrovorum. Leuconostoc lactis. Leuconostoc cremoris. 2. Acid and gas fermentation “Souring and Gassiness” In this type of fermentation microorganisms ferment lactose to lactic acid with the production of gases (CO2 and H2). The produced acid gives sour flavor to milk and then coagulates milk protein (casein), while gases evolved puncture the curd making it spongy. The formed curd is smooth, gelatinous, and spongy in texture due to gas bubbles. The production of gas is evidenced by foaming at the top and floating of the curd containing gas to the surface. Causative organisms: A. Coliforms: Coliforms are facultative anaerobic bacilli; they contaminate milk from manure, soil, contaminated water and from animal feed.  They ferment lactose to lactic acid and gases (CO2 and H2) and some species are proteolytic. In dairy industry, coliforms can cause serious troubles in cheese making, as they evolve large volumes of gas, resulting in a fermented cheese with a bad taste and holes. E.g., E. coli and Enterobacter aerogenes. Some strains of E. coli cause food poisoning in man and mastitis in dairy animals. Coliforms are killed by pasteurization. They are used as test (indicator) organisms for routine bacteriological quality control in dairies. B. Lactose fermenting yeasts: Some species of yeasts ferment lactose to acid, gases and alcohol. E.g., Candida pseudotropicals, Torulopsis spherica, and Saccharomyces kefir. C. Gas forming clostridia (Anaerobic spore-formers): Many species ferment lactose to acid and gases when the product is held in a limited air. E.g., Clostridium sporogenes, Cl. butyricum, and Cl. perfringenes. 3. Lipolytic fermentation (Fat degradation) (Lipolysis) The hydrolysis or splitting of milk fat (triglycerides) into fatty acids and glycerol by the action of lipase enzyme (in milk) or lipase-like enzyme (produced by lipolytic microorganisms). This is a primary concern in dairy products with high-fat content, such as whole milk, cream, butter, and ghee. Lipase enzyme: It is normally present in milk. Its amount increases in cases of late lactation period (physiologically) and cystic ovary (Pathologically). Lipase-like enzyme:  It is secreted by lipolytic microorganisms which may contaminate milk or dairy products from soil, water, air or utensils. Examples of lipolytic microorganisms: Pseudomonas species: e.g., Ps. fragi and Ps. fluorescence. Achromobacter lipolyticum. Alckaligenes faecalis. Micrococci. Serratia marcescens. Yeasts: Oidium lactis and Candida lipolyticum. Moulds: Penicillium roqueforti. 4. Proteolytic fermentation (protein degradation) (Proteolysis) Proteolysis is the hydrolysis (breakdown) of milk protein (casein) into water- soluble compounds by the action of protein splitting enzyme (proteinase enzyme) secreted by proteolytic organisms. The initial degradation of milk protein results in the formation of peptones and peptides (bitter flavors). Subsequent degradation results in the formation of free amino acids, which can be further degraded to give free amines or even free nitrogen that cause putrid odors. Examples of proteolytic organisms: Aerobic spore formers: e.g., Bacillus subtilis, B. mycoides, B. cereus, B. coagulance and B. calidolactis. Non-spore forming rods: e.g., Pseudomonas fluorescence and Ps. Putrefaciens, and Proteus spp. and Serratia marcescens. Cocci e.g., Strept. liquefaciens and micrococci. Anaerobes e.g. Clostridium butyricum. Yeast: e.g., Torula species. Moulds: e.g., Penicillium spp. High contamination with Proteolytic organisms significantly shorten the keeping quality of dairy products.  5. Sweet curdling Most common in pasteurized milk (heated) and occasionally in raw milk of low bacterial count that is kept at a low temperature. The milk protein (Casein) coagulates at normal acidity due to the action of the extracellular enzyme called rennin-like enzyme, which is secreted by certain organisms. The organisms that cause this are often present in raw milk but don't cause many issues because they are overgrown by lactic acid producing organisms. In pasteurized milk or raw milk with a low bacterial count, these organisms can grow rapidly and dominate. Sweet curdling is usually followed by proteolysis of the curd, which results in the accumulation of alkaline by-products and/or bitterness. As most of sweet curdling bacteria are also proteolytic. Causative organisms: Aerobic spore formers: e.g., Bacillus subtilis, B. mycoides, B. cereus, B. coagulance and B. calidolactis. Pseudomonas fluorescence. Streptococcus liquefaciens. 6. Ropy (slimy) fermentation Occurs in milk and cream. Long threads of milk appear when it is poured from one container to another. Caused by microorganisms that produce gum-like or sticky substances (capsular material) of bacterial cells. Develops best at low storage temperatures (for at least 6h) and tends to decrease with increasing acidity. Causative organisms: Alkaligenous viscousus. Strept. cremoris var hollandicus. Coliforms. Micrococci.  They contaminate milk from polluted water, soils, utensils, manure, or animal feed. N.B. Non-bacterial ropiness: Ropiness should not be confused with mastitis milk. In mastitis: The ropiness is due to the fibrin masses and leucocytes. In mastitis: ropiness occurs when milk is directly drawn from the udder, while in case of ropy milk, the milk must stay for 6-12h to be evident. 7. Alkaline Fermentation (Alkali production) An abnormal change in milk. Caused by alkali-forming organisms that cause an alkaline reaction without proteolysis. These organisms attack salts of organic acids like: Citric acid to form carbonates. Urea to form ammonia. Causative organisms: Pseudomonas fluorescence. Alkaligenes viscolactis. Alkaligenes faecalis. Micrococcus ureae. Most of alkali formers bacteria grow at moderate to low temperature and many of them can survive pasteurization. 8. Alcoholic Fermentation Alcohol can be produced in milk or cream by lactose-fermenting yeasts. Yeast enzymes invertase and zymase break down lactose into alcohol and CO2. Causative organisms: Saccharomyces kefir This organism is used as starter culture in some types of fermented milks e.g., Kefir.  9. Bitty or broken cream Occurs in raw milk, pasteurized milk, and cream. When the milk is shaken, the cream layer breaks into particles of various sizes that do not mix homogenously with the milk. When cream is added to hot tea or coffee, inhomogeneous cream particles will float onto the surface. Caused by the production of the enzyme lecithinase, which breaks down lecithin in fat globule membranes, allowing the fat globules to aggregate into larger particles. Causative organisms: Bacillus cereus. Bacillus mycoides. 10. Flavor Changes (Abnormal flavor)  11. Color Change Abnormal colors in milk can be caused by: Organisms secreting colored substances Organisms secreting substances that become colored in the acidic pH of milk. Examples of color changes include: