OBJECTIVES:
1. Isolation of lipolytic microorganisms from Dairy products.
2. Screening of the isolates with high lipolytic activity.
3. Identification of the isolates with high lipolytic activity.
INTRODUCTION :
Milk contains about 87 percent water and 13 percent solids. The fat portion of the milk contains fat soluble vitamins. The solids other than fat include proteins, carbohydrates, water soluble vitamins, and minerals. These nutrients in milk help make it nature’s most nearly perfect food.
Milk products contain high quality proteins. The whey proteins constitute about 18 percent of the protein content of milk. Casein, a protein found only in milk, contains all of the essential amino acids. It accounts for 82 percent of the total proteins in milk and is used as a standard for evaluating protein of other foods. Protein is needed to build and repair body tissues and to form antibodies which circulate in the blood and help fight infection.
Milk contains the following nutrients: Calcium, phosphorus, magnesium, and potassium. The calcium found in milk is readily absorbed by the body. Phosphorus plays a role in calcium absorption and utilization. Phosphorus is needed in the proper ratio to calcium to form bone.
Milk and milk products like cheese, yogurt, and frozen dairy desserts are the main source of calcium contributing about three-quarters of the calcium in the food supply. Milk provides these two minerals in approximately the same ratio as found in bone. Milk is also a significant source of riboflavin (vitamin B2) which helps promote healthy skin and eyes, as well as vitamins A and D,B-12, and also of riboflavin, calcium, phosphorus, magnesium, potassium, and zinc.Calcium is important from a public-health perspective, because current calcium intakes by many consumers are not sufficient for them to attain optimal peak bone mass and to prevent age-related loss of bone, leading to osteoporosis. Bone mass peaks around age 30, usually remains stable in the 30's, and commonly begins a decline in the 40's that accelerates around age 50. Recent research also indicates that adequate calcium intake is one key to achieving optimal blood pressure.
Milk is one of the widely consumed nutrient food and also it is an excellent Culture medium for the growth and reproduction of micro organisms. Microbial growth can be controlled by cooling the milk. Most micro-organisms reproduce slowly in colder environments. Cooling milk also slows chemical deterioration. The temperature of freshly drawn milk is about 38°C. Bacteria multiply very rapidly in warm milk and milk sours rapidly if held at these temperatures. If the milk is not cooled and is stored in the shade at an average air temperature of 16°C, the temperature of the milk will only have fallen to 28°C after 3 hours. Cooling the milk with running water will reduce the temperature to 16°C after 1 hour. At this temperature bacterial growth will be reduced and enzyme activity retarded. Thus, milk will preserved for longer period if cooled.
Natural souring of milk may be advantageous: For example, in smallholder butter-making, the acid developed assists in the extraction of fat during churning. The low pH retards growth of lipolytic and proteolytic bacteria and therefore protects the fat and protein in the milk. The acidity of the milk also inhibits the growth of pathogens. It does not, however, retard the growth of moulds.
Naturally soured milk is used to make many products, e.G. Irgo, yoghurt, sour cream, ripened buttermilk and cheese. These products provide ways of preserving milk and are also pleasant to consume. They are produced by the action of fermentative bacteria on lactose and are more readily digested than fresh milk.
The initial microflora of raw milk reflects directly microbial contamination during production. The microflora in milk when it leaves the farm is determined by the temperature to which it has been cooled and the temperature at which it has been stored. The initial bacterial count of milk may range from less than 1000 cells/ml to 106/ml. High counts (more than 105/ml) are evidence of poor production hygiene. Rapid tests are available for estimating the bacterial quality of milk.
The first and most universal change effected in milk is its souring. So universal is this phenomenon that it is generally regarded as an inevitable change which can not be avoided,The phenomenon is well understood. It is due to the action of certain of the milk bacteria upon the milk sugar which converts it into lactic acid, and this acid gives the sour taste and curdles the milk. After this acid is produced in small quantity its presence proves deleterious to the growth of the bacteria, and further bacterial growth is checked. After souring, therefore, the milk for some time does not ordinarily undergo any further changes.
Milk souring has been commonly regarded as a single phenomenon, alike in all cases. When it was first studied by bacteriologists it was thought to be due in all cases to a single species of micro-organism which was discovered to be commonly present and named Bacillus acidilactic. Such balanced diet milk becomes contaminated with several types of micro organisms which originate form soil , water, skin and the air of animals , utensils , from the milk handlers. The number of species of bacteria which have been found to sour milk has increased until something over a hundred are known to have this power.
These different species do not affect the milk in the same way. All produce some acid, but they differ in the kind and the amount of acid, and especially in the other changes which are effected at the same time that the milk is soured, so that the resulting soured milk is quite variable. The spoilage of milk due to production of heat resistance proteolytic enzymes degrades casein.
The spoilage of milk results in the production of many off- flavours which are characterized as fruity , musty , bitter, rancid , putrid. Bacteria may be classified according to their optimum temperature for growth and heat resistance .
The bacteria encountered in milk are of the following 4 temperature types
1)Psychrophilic
2)Mesophilic
3)Thermophilic
4) Thermoduric
A) Many psychotropic bacteria are lipase producers. The main Psychrotrophic bacteria are pseudomonas florescence, alcaligens, staphylococcus, serratia, micrococcus, coliforms, enterococcus, Achromobacter, Vibrio, Flavobacterium and Alcaligenes, They arc killed in the pasteurization process, but are sometimes found in pasteurized milk.
B) The most important mesophilic bacteria are streptococci, lactobacilli and coliforms, which produce acid and gas and off flavours. They are killed in the pasteurization process.
C) Thermophilic bacteria grow well at the temperature used in pasteurization, specially when the low temperature holding method is followed. Thermophilic bacteria develop best at 55-650C with minimum and maximum of 400C and 800C respectively. Most thermophilic forms are found in two genera, Bacillus and Clostridium .
Ex: Bacillus stearothermophilus is an example of this type.
D) Thermoduric bacteria survive pasteurization in considerable numbers but do not grow at pasteurization temperatures. Since they are not killed by pasteurization, they may contaminate the containers. As a result of the faulty cleaning of the containers, the subsequent batches of milk processed through the same containers will become heavily contamined. Microbacterium lacticum, Micrococcus luteus, Streptococcus thermophiles, Bacillus subtilis exemplify this category.The main mold is Geotrichum candidum and yeast is candida species. Sachharomycetaceae is the producer of lipases. Saccharomyces cerevisiae , Debaryomyces hasenii , Debaryomyces kleockeri and lipomyces starkeyi , cryptococcaceae , candida antarctica , C.Deformans, C.Rugosa and C.Lipolytica Candida parapsilosis, candida valida, Debramyces vanriji , Dedramyces hansenii, Kluyveromyces marxianus , pichia burtonii , pichia kluyveri , Geotrichum fermentans are producers of Lipases . The strain Hansenula anomala has the highest lipolytic activity .
Copra and Cocca Butter may be spoiled by molds.During cold storage after milk collection, psychrotrophic bacterial populations dominate the microflora, and their extracellular enzymes, mainly proteases and lipases, contribute to the spoilage of dairy products .
Milk pasteurization
Pasteurization is the most common process used to destroy bacteria in milk. In pasteurization, the milk is heated to a temperature sufficient to kill pathogenic bacteria, but well below its boiling point. This also kills many non-pathogenic organisms and thereby extends the storage stability of the milk.
Numerous time/temperature combinations are recommended but the most usual is 72°C for 15 seconds followed by rapid cooling to below 10°C. This is normally referred to as High Temperature Short Time (HTST) treatment. It is carried out as a continuous process using a plate heat-exchanger to heat the milk and a holding section to ensure that the milk is completely pasteurised.
Milk is normally pasteurized prior to sale as liquid milk. Pasteurisation is used to reduce the microbial counts in milk for cheese-making, and cream is pasteurised prior to tempering for butter-making in some factories.
Batch pasteurization is used where milk quantities are too small to justify the use of a plate heat-exchanger. In batch pasteurisation, fixed quantities of milk are heated to 63°C and held at this temperature for 30 minutes. The milk is then cooled to 5°C and packed. The lower temperature used for batch pasteurisation means that a longer time is required to complete the process-30 minutes at 63°C, compared with 15 seconds a 72°C.
Effects of pasteurisation on milk
Pasteurisation reduces the cream layer, since some of the fat globule membrane constituents are denatured. This inhibits clustering of the fat globules and consequently reduces the extent of creaming. However, pasteurisation does not reduce the fat content of milk. Pasteurisation has little effect on the nutritive value of milk. The major nutrients are not altered. There is some loss of vitamin C and B group vitamins, but this is insignificant. The process kills many fermentative organisms as well as pathogens. Micro-organisms that survive pasteurisation are putrefactive. Although pasteurised milk has a storage stability of 2 to 3 days, subsequent deterioration is cause by putrefactive organisms. Thus, pasteurised milk will putrefy rather than develop acidity. In rural milk processing, many processes depend on the development of acidity, and hence pasteurisation may not be appropriate.
Milk sterilization
In pasteurization, milk receives mild heat treatment to reduce the number of bacteria present. In sterilisation, milk is subjected to severe heat treatment that ensures almost complete destruction of the microbial population. The product is then said to be commercially sterile. Time/temperature treatments of above 100°C for 15 to 40 minutes are used. The product has a longer shelf life than pasteurised milk. Another method of sterilisation is ultra-heat treatment, or UHT. In this system, milk is heated under pressure to about 140°C for 4 seconds. The product is virtually sterile. However, it retains more of the properties of fresh milk than conventionally sterilised milk.
Triglycerides are Tri- esters of glycerol and three fatty acids . They are charceterized as either fats ( lipids that are solid at room temperature ) or oils( lipids that are liquid at room temperature ), and are common components of foods. Other types of lipids in foods include the fattyacid mono and di esters of glycerol, termed mono glycerides and di glycerides , respectively . These are usually generated as intermediates in the break down of fats and oils Tri glycerides are Lipolytic bacteria are the heterogenous group of bacteria, which produce lipases, which catalyze the hydrolysis of fats to fatty acids and glycerol. Triglycerides have very low water solubilities , while the solubilities of mono and Di glycerides can be greater . Hydrolysis of the ester bonds of the Tri , Di , and Mono glycerides (lipolysis) Liberates free fatty acids ( FFA) . In food systems , such lipolysis is usually catalyzed by enzymes , generally by the group of enzymes known as lipases .
Lipases are defined as those enzymes capable of hydrolyzing the carboxylic acid easterbonds of water - insoluble substrates . The biological role of lipases is to initiate the metabolism of fats and oils by reducing them to readily metabolized free fatty acids and glycerol .Humans readily detect the shorter chain length fatty acids , up to about 10 carbons in length , by smell or taste . In some cases, example : Dairy products , it is often desirable that some or all sizes of these shorter free fatty acids be released by lipolysis of endogenous lipid , or the added during processing . They confer characteristic falvor or fragnence . Fermented sausausages are also have lipolytic activity . Longer chain fatty acids , particularly those containing double bonds , will oxidize their lipolytic release from a glyceride .
Some hydrolysis of the fats and oils in foods is non-microbial in origin , the result of spontaneous lipid hydrolysis and the action of lipases that are naturally present many food material. Fatty acid oxidation can also generate undesirable favours . In some cases, oxidation and or the actions of endogeneous lipases can play a larger role In spoilage than do microbial lipases . How ever , lipase production is a wide spread trait of bacteria , yeasts and molds. This ability to produce lipase does not always result in lipolytic damage . Since the synthesis or activity of the enzyme may be inhibited by components of the food or by the conditions on incubation .
Lipases can be significant contributors to product deterioration . In addition to lipases which act on triglycerides , micro organisms can produce other lipid - hydrolyzing enzymes. Chief among these are the phospholipases , which convert Phospho lipids , the primary components of cell membranes, to FFA , Lyso phosphatides , Mono and Di glycerides , Glycero phosphotides and simplar materials . The presence of a Phospho lipase can stimulate the activity of a lipase . For example, Phospholipase -C from Bacillus cereus or pseudomonas flurosence enhances the lipolytic activities of both milk lipoprotein lipase and commercial Rhizopus lipase . Micro organism that produce glycosidic enzymes can in conjunction with bacterial proteases . Degrade milk membranes and then by expose the milk lipids to lipases . Thus , the glycosidases can contribute indirectly to lipolytic activity.
The glycosidases of pseudomonas fluroscences , in contrast to the phosphorlipase-C and lipase produced by that organism are completely inactivated by Milk pasteurization temperatures and therefore , would not be expected to play a role in Protein traiting lipase activity in pasteurized or other wise similarly heated samples. By the use of special plating media micro organisms that produce lipase can be enumerated . Such enumartion as not usually performed on a protein basis. Food manufactures and processors analyse for lipolytic micro organisms only when a problem arises .
Determination of the number of lipolytic micro organisms Present in a food sample can reveal the food processor whether the particular lipid - related problem is microbial or non- microbial in origin . The fatty parts of food made up of fats and oils. The fats and oils themselves are subject more often to chemical than to microbial spoilage. Besides the fatty glycerides, natural fats and oils usually contains small amounts of fatty acids and glycerol , other liquid alcohols and sterols , Hydrocarons, proteins and Nitrogenous Compounds , phophatides , caroteinoid pigments . The chief types of spoilage result from hydrolysis , oxidation .
Flavour reversion :
Flavor reversion is defined as the appearance of object is on able flavors from less oxidation than is needed to produce rancidity .Oils that contain lenolenic acid , fish oils , vegetable oils . Butter fat and Meat fats become “ tallowy” as the result of oxidation but butter fat is called rancid well only hydrolysis of fatty acids and glycerol has taken place.
Some of the pigments produced by micro organisms are fat soluble and therefore, can diffuse into fat , producing discoloration , ranging through yellow, red, purple and brown . Stamping-Ink discoloration of MEAT fat caused by Yellow pigment micro cocci and bacilli . The fat- soluble pigment is an Oxidation-Reduction indicator that changes from Yellow- green - blue and finally to purple as Fat becomes more oxidized by the peroxides formed bythe bacteria , yellow , pink , red fat-soluble pigments may be produced by various bacteria, yeasts and molds .Many of the proteolytic bacteria are also lipolytic. The main source for isolation of lipolytic microorganisms is butter and other dairy products.Yeasts and yeast like fungi are specific group of micro organisms on various substrates . It is assumed that yeasts and yeast like fungi can adopt to substrates rich in fat under conditions of anthropogenic impact . This characteristic has become urgent due to utilization of industrial waste. The main index of their activity is excreted lipolytic enzymes. Microorganisms, may be involved in the oxidation of fats, auto oxidation is common. The Microbiological quality of butter depends upon the quality of cream and sanitary conditions used in the processing.
BIOCHEMICAL ACTIVITIES :
If allowed to stand under condition that permit bacterial growth, raw milk of a good sanitary quality will rapidly undergo a series of chemical changes. The principal change is lactose fermentation to lactic acid. This change is brought about by acid uric lactic organisms, especially Strepotococcus lactis and certain lactobacilli. These include two distinct biochemical types, homo-and heterofermentative. In homofermentation lactic acid is the major product of lactose fermentation. Hetero fermentative organisms, however, produce lactic, acetic, propionic, and some other acids, and some alcohols and gases such as CO2 and H2 Organisms continue to form lactic acid until the concentration of acid is itself too great for the organisms to remain live.
Microbacteria, micrococci, coliforn18, etc. Also ferment lactose to lactic acid and other products. Many Clostriifiul1J species and, some yeasts such as Torula lactic, and Torula cremoris ferment lactose with acid and gas production.
As the acidity continues to increase and reaches a pH of 4.7, it eventually causes a precipitation of casein. Organisms capable of metabolizing lactic and other acids develop especially acid uric, yeasts and moulds. The acidity of milk is diminished and the alkaline products of protein decomposition such as amines, ammonia and the like are produced.
This is accomplished by many species of the genera Bacillus, Clostridium, Pseudomonas,Proteus and numerous other forms.
The action of microorganisms does not involve fat as readily as it does lactose and protein. Lipolysis results from the action of lipase produced by bacteria such as Pseudomonas, Achromobacter and by some yeasts and moulds. Fat is hydrolysed to glycerol and fatty acids. Some of the fatty acids, for example, butyric and caproic acid give milk products, distinctive and usually rancid, odours and flavours.
Several microorganisms also bring about certain objection able changes in the milk which may not be deleterious to health. Rapines in milk is sometimes encountered. The milk become ropy or slimy and may be pulled out into long threads. It is produced by several organisms but the most important species is Alcaligenes viscolactis. A rapid fermentation of lactose in milk is sometimes observed and is known as stormy fermentation. This is brought about by Clostridium perfringens. The curd become torn to shreds by the vigorous fermentation and gas production. Several organisms have been isolated from milk which impart brilliant colours. Pseudomonas syncyanea imparts blue colour, pseudomonas synxantha yellow colour and Serratia marcescens red colour to the milk. From the review of the literature it was Observed the importance of the analysis. In day to day human.
Bacterial types commonly associated with milk.
Pseudomonas | Spoilage |
Brucella | Pathogenic |
Enterobacteriaceae | Pathogenic and spoilage |
Staphylococci | |
Staphylococcus aureus | Pathogenic |
Streptococcus | |
S. agalactiae | Pathogenic |
S. thermophilus | Acid fermentation |
S. lactis | Acid fermentation |
S. lactis-diacetyllatic | Flavour production |
S. cremoris | Acid fermentation |
Leuconostoc lactis | Acid fermentation |
Bacillus cereus | Spoilage |
Lactobacillus | |
L. lactis | Acid production |
L. bulgaricus | Acid production |
L. acidophilus | Acid production |
Propionibacterium | Acid production |
Mycobacterium tuberculosis | Pathogenic |
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