MILK Def It is the secretion of mammary

MILK

Def: ►It is the secretion of mammary glands in human and animals after labour. ► It is the only food for newborn for a long period till weaning time. ► Before birth the fetus receives its nutrition through the placenta.

►Milk secretion is stimulated by Prolactin. ►Milk flow is decreased by Estrogen and Progesterone while increased by Thyroxin. Also Squalene present in HELBA increase milk flow. ►Initiation of lactation may result from sudden removal of the placenta and other factors. ►Oxytocin (posterior pituitary hormone) responsible for milk ejection.

Milk is considered as a complete diet as: It contains all of the components necessary for growth, maintenance of life and reproduction. e. g: Carbohydrates, Lipid, Protein, Some minerals and Vitamins. But it is deficient in: Vitamin C, Vitamin D, Vitamin K, Iron, Copper.

Physical Properties of Milk

1 - Color: White color → due to presence of: ► Fat globules in emulsion form. ► Protein in colloidal form. ► Ca. phosphate and Ca. Casinate. * Yellowish (creamy) color → due to: ► Presence of Carotene and Xanthophyll pigments specially in cow's milk and colostrums.

2 - Reaction: Fresh milk is amphoteric in reaction as it contains acid and base. Milk is an excellent buffer as it contain: § § Protein. Phosphate. Bicarbonate. Citrate. ** PH of fresh milk: § 6. 6 – 6. 8 § 6. 8 – 7. 4 Cow's milk. Human's milk

Ø Milk p. H changed to alkaline in case of: Ø Mastitis Ø Late period of lactation. Ø If the milk left to stand for a long period specially in warm temperature, lactose is fermented to lactic acid by bacteria present normally in milk as: * Streptococcus Lactis. * Lactobacillus Lactis. Result in decrease in milk PH and milk Souring.

3 - Specific Gravity: It is the ratio between weight of a given volume of milk compared with the same volume of water at a specific temperature. q It measure total solids of milk, and determine if any constituent added or removed from milk. . Measured by Lactometer. q Normal specific gravity: q § § 1020 – 1030 – 1035 Cow's milk. Human's milk.

§ Fat is the only constituent in milk with specific gravity lower than 1000, so, when fat present in milk in high amount, specific gravity decrease than normal values. § When milk is skimmed (removal of fat content), the specific gravity increased due to: Removal of light constituents of milk. § When water is added to milk (adulteration) the specific gravity decreased due to dilution of total solids.

4 - Taste: § Normal characteristic milky taste. § Milk taste changed in case of : Ø Souring: due to increased acidity. Ø Boiling: due to certain biochemical changes and evaporation of volatile fatty acids. Ø Late stage of lactation: due to increase chloride percent. Ø Mastitis: inflammation of udder. 5 - Odor: Characteristic milky odor.

6 - Freezing point of milk: ► It is the temperature at which the liquid milk freeze or crystallize. ► The freezing point of cow or buffalo milk ranges from (-0. 53) to (-0. 57) °C with average ( -0. 55) °C ► Milk freezes at a temperature slightly lower than that of water due to the soluble constituents in milk such as lactose, minerals which lower the freezing point.

► This test is important for detection of amount of water added to milk the ► The addition of 9% water to milk will raise the freezing point by 0. 05 °C. § i. e. , if milk freezing point is -0. 45 °C, means addition of 18% water. -0. 45 – (-0. 55) = -0. 45 + 0. 55 = +0. 10 0. 05 → 9 % 0. 10 → X X= 9 x 0. 1 0. 05 = 18 % it

7 - Boiling Point: ► Milk boiling point is 100. 5 °C , more than water due to presence of dissolving substances 8 - Cream Line Formation: ► When milk left at room temperature the fat globules coalesce, clumped at surface of the container and form cream line.

Normal Chemical composition of milk

Milk is formed of: 1) Water: form 87%. 2) Solids: form 13%. A- Organic constituents of milk. 1. Protein. 2. Lipid. 3. Carbohydrate. B- Inorganic constituents of milk: 1. Minerals. 2. Vitamins.

A- Organic Constituents of Milk

1 - Protein: Milk protein less in human than in cow's milk. ► All milk protein synthesized in the mammary gland. ► Milk protein of high coefficient digestibility (85 – 95%) Amount of N 2 absorbed X 100 Total N 2 in the protein ►

Characterized by: 1 - Protein of high biological value as: ►It contains all essential amino acids. ►Easily digested. ►Easily absorbed. ►Easily metabolized. 2 - Contain moderate amount of non essential amino acids to decrease stress on body cells. 3 - Essential to keep positive nitrogen balance (nitrogen intake more than nitrogen output). 5 - Essential to maintain growth of newborn.

Total amount of protein differ according to species and affect rate of growth of young: e. g: ► Young rabbit reach double weight in 5 days as milk protein 10 gm/dl. ► Young calf reaches double weight in 50 days as milk protein 3. 7 gm/dl. ► Young child reach double weight in 5 - 8 months as milk protein 1. 5 gm/dl. 6.

Types of Milk Proteins are: 1. Casein. 2. Lactalbumin. 3. Lactglobulin. 4. Milk enzymes.

1) Casein ► It is the main and most dominant milk protein. ► It represents 25% in human's milk and 83% in cow's milk. ► It is a compound protein (Phospho-protein) of high biological value. ► The high phosphate content of casein allows it to associate with calcium and form calcium phosphate salts. § So, at normal PH of fresh milk (6. 6 PH) casein present as insoluble Ca. caseinate phosphate complex.

► Casein contains 0. 7% phosphorous and 0. 7% sulpher, but it is Phospho-protein Not Sulphoprotein. Why? ? 1) Phosphorous: § Attached to (OH) group of serine to form phospho-serine. § Attached to (OH) group of therionine to form phospho-therionine. 2) Sulpher: § Found in methionine (But masked by the methyl group) So, act as Lipotropic factor that prevent accumulation of fats on liver.

► Casein is suspended in milk in colloidal suspension complex of casein micelles that are dispersed in the water phase of milk. ► Casein micelles are spherical in shape and much smaller in diameter than fat globules, and consists of a network of casein submicelles, each one micelle contains 400 -500 submicelles, bonded by calcium phosphate bridges.

► Casein is deficient in cystiene and cystin so give negative result with sulpher test. ► Casein : (Lactalbumin - Lactglobulin) ratio is 1: 3 which facilitate its digestion. Casein Lactalbumin + Lactglobulin = 1 3 ► According to molecular weight and electrophoresis process, casein is separated in to 3 fractions, each one of them has its own amino acid composition : § α Casein → § β Casein → § γ Casein → 75%. 22%. 3%.

► Casein is the only milk protein that not coagulated on boiling. ► The net charge of milk protein is negative charge, so protein molecules remain separated from each others due to repulsion forces. Isoelectric point of casein: It is the point of absence of net electrical charge on protein molecules (So, protein PPT and not affected by the electric field).

Milk Souring (Milk Curdling – Zabadi): ► The principle of coagulation, or curd formation, at acidic p. H is the basis for Yoghurt (Zabadi) formation. How? ► Fermentation of lactose to lactic acid by: § § § Lactic acid producing bacteria present normally in milk. Addition of acid (acetic acid). Addition of starter culture (yoghurt). Lactose + lactic acid bacteria Worm temp. Fermentation Lactic acid ↓ p. H

► Leading to decrease milk p. H to 4. 6 (Isoelectric point of casein) leads to PPT of casein as: 1) By souring and lactic acid production: some protein molecules gain the hydrogen ion with its (+Ve) charge. 2) Calcium and phosphate are dissolved and separated from casein. 3) The (+Ve) charge protein attract to the (-Ve) charge protein at 4. 6 p. H, leading to PPT of casein. 4) Part of lactic acid combines of with Ca. of casein to form Ca. lactate and free casein which PPT with fat and fat soluble vitamins. Ca. Caseinate + lactic acid Casein (pure) + calcium lactate PPT ↓ (soluble)

Whey of milk curd: It is the residual clear fluid which formed after removal of casein and contain: ►Water. ►Lactic acid. ►Lactalbumin & lactglobulin (whey protein) ►Minerals. ►Water soluble vitamins. The greenish yellow color of whey is due to riboflavin (vitamin B 2)

Milk Clotting (Cheese Manufacturing) During digestion (in new born): ► Rennin enzyme acts on casein, converting it to soluble Paracaseinate which is bounded to Ca and form insoluble Ca. paracaseinate (milk clot). ► Rennin enzyme: § Derived from 4 th stomach of calves and used externally in cheese manufacture. § It is secreted inactive as pro-rennin. § Its optimum PH 4. § It is absent in adult stomach. ► Formation of milk clot: prevents rapid passage of milk from stomach to intestine so give the sense of fullness to newborn.

Whey of milk clot: Residual clear fluid after removal of casein which contain: ►Water. ►Lactose. ►Lactalbumin & lactglobulin (whey protein) ►Minerals except Ca. ►Water soluble vitamins.

B- Lactalbumin: ► Represent 87% of whey protein. ► PPT by full saturation with ammonium sulphate. ► Rich in cystein and cystin so give +ve result with sulpher test. ► Simple protein. ► Soluble protein. ► Easily digested.

► Consists of two fractions: § α Lactalbumin 32% of whey protein. § β lactglobulin 55% of whey protein. N. B: β lactglobulin called globulin because it need small amount of Na. Cl to be dissolved but it is not PPT by half saturation as albumin.

3 - Lactglobulin: ► Represent 13% of whey protein. ► PPT by half saturation with ammonium sulphate solution. ► Rich in cystein and cystin so give +ve result with sulpher test. ► Simple protein. ► Soluble protein. ► Easily digested.

► Consists of two fractions: § True globulin (Euglobulin) 50%. § False globulin (Pseudoglobulin) 50%. ► They carry antibodies causing immunity so called immunoglobulins. ► They present in higher concentration in colostrum.

4 - Milk enzymes: 1. Catalase. 2. Peroxidase. 3. Xanthin oxidase. 4. Alkaline phophatase. 5. Amylase. 6. Lipase. 7. Aldehyde oxidase.

1 - Catalase: ► Carried by fat globules. ► Act on hydrogen peroxide (H 2 O 2) producing water and oxygen. ► Increased in case of mastitis.

2 - Peroxidase: ► Carried by lactalbumin. ► Act on hydrogen peroxide (H 2 O 2). ► It is heat stable enzyme (resist destruction by heat). ► It is destroyed by milk sterilization. Milk sterilization: Heating of milk to 116◦C for 15 minutes which destroy all harmful and non harmful microorganisms. ► Milk sterilization leads to alteration in milk taste and destruction of vitamin C and vitamin B 2. N. B: Absence of peroxidase enzyme indicates milk sterilization.

3 - Xanthin oxidase: ► Carried by fat globules. ► Act on hypoxanthin and xanthin to produce uric acid.

4 - Alkaline phophatase: ► Carried by fat globules. ► Catalyze hydrolysis of phosphate ester. ► Destroyed by the same temperature that destroys the harmful bacteria. e. g: Tubercle bacilli. ► Absence of Alkaline phophatase enzyme indicates milk pasteurization. ► Milk pasteurization: Heating of milk to 60◦C for 30 minutes or 70◦C for 15 minutes followed by sudden cooling which destroy the harmful microorganisms only.

5 - Amylase: ► Carried by lactalbumin. ► Catalyze hydrolysis of α 1, 4 glycosidic linkage of starch and glycogen. ► Increased in case of mastitis.

6 - Lipase: ► Carried by casein. ► Catalyze hydrolysis of primary ester linkage in triacylglycerol. ► Act on milk fat producing free fatty acids and undesirable taste (Rancidity).

7 - Aldehyde oxidase (Schardinger's enzyme): ► Its absence indicates milk boiling. ► Schardinger's test: Milk + Methyl blue + Formaldehyde § Blue color appeared in case of boiled milk. § Colorless in case of unboiled milk.

N. B: Beside milk proteins milk also contains: A) Lactoferrin binding protein: § It contains iron which is bounded to a glycoprotein. § It facilitates iron transport and storage. § Found in high concentration in human colostrums and milk. B) Vitamin B 12 binding protein: § It carries vitamin B 12. Lactoferrin and vitamin B 12 binding protein deprive pathogenic intestinal bacteria from iron and vitamin B 12 so they have bacteriostatic action.

2 - Lipid: ► Human's and cow's milk contain the same amount 3. 5 gm/dl but buffalo's milk is a little higher 7 gm/dl. ► Easily separated on standing. ► Responsible for white color of milk ► It consists mainly of triacylglycerol distributed as coarse emulsion which contains oleic, myristic, palmitic and stearic fatty acids.

Also contain small amounts of: ►phospholipids 0. 1%. § Milk phospholipids are lecithin, cephalin, sphingomyelin (9: 5: 1). § Phospholipids in cow's milk twice that of human milk. ►Cholesterol 0. 01%. Cow's milk contains higher proportion (mainly free form) than human milk mainly (ester form) ► Milk contain fat soluble substance: e. g:

►Fat soluble vitamins (vitamin A) which is higher in human milk than cow's milk. ► Milk fat is formed mainly in lactating mammary gland from: § Active acetate. § Glycerol: derived from glucose (dihydroxyaceton phosphate). ► Milk fat may be derived from blood triacylglycerol but to a limited extent.

Cow's milk Human's milk Contain short chain fatty Contain no short chain fatty acids 10%. acids. Contain 10% essential Contain 0. 05% essential (polyunsaturated) fatty acids. specially linoleic fatty acids. Saturated fatty acids 58%. Saturated fatty acids 48%. Unsaturated fatty acids 42%. Unsaturated fatty acids 52%.

3 - Carbohydrates: ► Lactose (milk sugar) is the only carbohydrate of milk. ► It is a reducing disaccharide consists of glucose and galactose. ► Human's milk contains 7% lactose while cow's milk contains 5% lactose. ► Lactose may be excreted in urine during last third of pregnancy physiologically so it should be differentiated from glucose by osazon test.

Importance of lactose: It is less sweet than sucrose so allow the baby to take large amount of milk without causing nausea. 2. It is non fermentable carbohydrate so it doesn't produce CO 2 in GIT and the baby doesn't suffer from abdominal colic or distention. 3. Lactose help growth of lactic acid producing bacteria so help in absorption of Ca, P, Fe, Cu which prefer acidic medium for their absorption. 4. Lactose inhibits growth of putrefactive bacteria which cause abdominal distention by increasing the acidity of the intestine. 1.

Lactose synthesis: Lactose is synthesized in mammary gland from α D glucose and β D galactose. § Galactose converted to galactose -1 phosphate by galactokinase enzyme. § Galactose -1 - phosphate and UDP glucose are converted to UDP galactose and glucose -1 phosphate by uridyle tranferase enzyme. § UDP galactose bounded to glucose by lactose synthase enzyme forming lactose.

Galactosemia: ► It is a condition occurs due to deficiency of galactose -1 - phosphate uridyle transferase. ► Characterized by accumulation of galactose and galactose -1 - phosphate in blood.

Lactose synthase enzyme: Consists of two subunits: 1. Catalytic subunit: § Can't synthesize lactose. § It catalyze the attachment of galactose to protein to form glycoprotein. 2. Modifier subunit: § Alter the specificity of the catalytic subunite which make galactose bounded to glucose to form lactose. ► During pregnancy little modifier subunite is formed in the mammary gland so no lactose synthesized But at birth large amount of modifier is formed helping the synthesis of lactose in mammary gland.

Importance of lactose: It is less sweet than sucrose so allow the baby to take large amount of milk without causing nausea. 2. It is non fermentable carbohydrate so it doesn't produce CO 2 in GIT and the baby doesn't suffer from abdominal colic or distention. 3. Lactose help growth of lactic acid producing bacteria so help in absorption of Ca, P, Fe, Cu which prefer acidic medium for their absorption. 1.

Lactose inhibits growth of putrefactive bacteria which cause abdominal disturbance by increasing the acidity of the intestine. 5. On hydrolysis it produce glucose and galactose: § Glucose act as source of energy. § Galactose used for synthesis of galactolipids so help in development of nerve tissues. 4.

N. B: Lactose level in milk tend to vary directly with the weight of the adult brain: Man has the largest brain in proportion to the body weight of all animals this may be related to the galactolipids of the brain which contains galactose. 6. Lactose has a laxative effect as some of glucose is fermented by the intestinal bacteria producing CO 2 which stimulate the intestinal movement.

N. B: If the milk is taken by the adult in large amount result in diarrhea due to decrease in lactase enzyme so lactose is hydrolyzed by intestinal bacteria to glucose and galactose. § Glucose is fermented producing CO 2 and abdominal distention. § Non fermentable galactose and unhydrolyzed lactose increase osmotic pressure of the intestine leads to diarrhea.

B-Inorganic constituents of milk

1 - Minerals: Human milk contain less mineral elements (0. 4%) than cow's milk (0. 8%). ► Milk rich in Ca and P which are present in their proper ratio for absorption (2: 1) in human milk while in cow's milk (1: 2) which is not suitable for their maximum absorption. ► Ca and P are essential for: 1. Growth of bone and teeth. 2. Stability of casein. ►

► Milk is deficient in Fe and Cu which are supplied by their storage in liver during prenatal life (this store is sufficient till weaning time). N. B: Milk is deficient in Iron but it is more in human milk than cow's milk Thus anaemia in breast feeding is less common. ► Milk contain adequate amount of Na, K, Mg. ► Human milk contains Na: K (1: 2) which is suitable for the optimal growth of newborn.

2 - Vitamins: Milk is deficient in: ► Vitamin C. ► Vitamin D. ► Vitamin K. ► Milk contain adequate amount of vitamin B complex which are sufficient for first week of life e. g: ► Pantothenic acid. ► Riboflavin (gives the whey the greenish tint in sunlight). ►

N. B: ► Vitamin C must be supplied to the growing baby in the form of fruit juices to withstand infection. ► Fortified vitamin D milk is used in order to supply the baby with vitamin D requirement which is added from cod liver oil. ► Exposure to sunlight in the early morning or before sunset help in formation of active vitamin D from cholesterol.

Milk is an adequate diet for children Although milk is deficient in Fe, Cu, vitamin C, vitamin D, and vitamin K it is complete natural food for the following reasons:

1. 2. 3. 4. Easily digested absorbed and metabolized. Contain all the nutrients required for the newborn at early stage of life. Balanced ratio between carbohydrates, lipid and protein. Milk protein: Protein of high biological value as: § § It contains all essential amino acids. Easily digested. Easily absorbed. Easily metabolized.

5. Milk lipid: Milk contain essential fatty acids e. g: § Linoleic acid. § Linolenic acid. 6. Milk sugar (lactose): A. It is less sweet than sucrose so allow the baby to take large amount of milk without causing nausea. B. It is non fermentable carbohydrate so it doesn't produce CO 2 in GIT and the baby doesn't suffer from abdominal colic or distention. C. Lactose help growth of lactic acid producing bacteria so help in absorption of Ca, P, Fe, Cu which prefer acidic medium for their absorption.

D. Lactose inhibits growth of putrefactive bacteria which cause abdominal distention. E. On hydrolysis it produce glucose and galactose. F. Lactose has a laxative effect. 7. Provide Ca and P in proper ratio for absorption (2: 1) which are essential for: 1. Growth of bone and teeth. 2. Stability of casein. 8. Good source of vitamins. 9. Contain antibodies specially in colostrums which provide immunity to the newborn. 10. Cheep in price and of good caloric value.

N. B: ►Certain foreign substance in the blood plasma may enter the milk as: ►Volatile oils of certain foods (onions – garlic). ►Drugs (sulphonamids – salicylate – morphin – alcohol). ►Inorganic elements (iorn – iodin – lead – mercury).

Factors affecting milk composition

1. Species: ►Milk protein: Rabbit's milk 10% while cow' milk 3. 7%. ►Milk fat: Cow's milk 3. 5% while buffaloe's milk 7%. 2. Age: ►Old age decrease fat contents of milk. 3. Seasonal variation: ►In winter increase in fat, protein and mineral contents of milk.

4. Nutritional variation: ► ► Overfeeding has no effect on milk composition. Underfeeding decrease in milk volume and protein and lactose contents of milk while fat contents of milk increased. 5. Infection of mammary gland: ► Mastitis decrease fat, lactose and casein contents of milk while whey protein and chloride ions increased.

Stage of lactation: Colostrum secreted during 1 st week of lactation. Intermediate milk secreted during 1 st month of lactation except 1 st week. ► Mature milk secreted during 1 st year of lactation except 1 st month. ► Late milk secreted after 1 st year of lactation. Ø Watery in consistancy. Ø Contains less protein, lipid, lactose and vitamins. Ø Contains more minerals than mature milk (less sweet to help weaning). 6. ► ►

N. B: Witch's milk: Fluid secreted by mammary gland of infants during first few days of life due to the effect of placental hormones on mammary gland of infants.

A- Colostrum: Yellowish fluid secreted by mammary gland during 1 st week of lactation. ► Volume of colostrum in human (150 – 300 ml/day). ► Specific gravity (1. 040 – 1. 060) Colostrum is very important for survival of new born due to: 1. High level of protein (20%): q 40 – 55% globulin specially γ globulin (immunoglobulin) which provide immunity for newborn up to 6 months. q Globulin is a coagulable protein by heat. ►

2. 3. 4. q Absorbed without digestion due to its low molecular weight. q Globulin contain prolin which enter in Hb synthesis. Low fat (3 g%) and carbohydrate (4 g%) content. ► Easily digested. ► Contains higher content of cholesterol and lecithin. High mineral content. ► Iron found in higher concentration than mature milk. High fat soluble vitamins (E, D, A, K) and water soluble vitamins (riboflavin, nicotinic acid).

5. Trypsin inhibitor: q Inhibit action of trypsin enzyme so: immunoglobulins absorbed as such. 6. Yellowish color of colostrum is due to high level of β carotene which is present 50 – 100 times as much as in mature milk. ► Colostrum contains large granular bodies (colostrum corpuscles): Alveolar cells of mammary gland leukocytes baded with fat.

Function: 1. Has a laxative effect so help excretion of meconium. 2. Contain high level of immunoglobulins which provide immunity for newborn. 3. Easily digested, absorbed and metabolized. 4. Has high nutritive value (rich in protein, minerals and vitamins).

Humanization of cow's milk

Def: It is a process by which cow's milk is made to be as near as human's milk. Aim: q Protein of human's milk (casein) form 25% of milk protein while casein in cow's milk form 5/6 of milk protein so it form dense clot in newborn stomach which can't be digested leading to vomition. q Lactose in human's milk higher than that in cow's milk (7: 5).

Steps: 1. Pasteurization of milk: § Heating of milk to 60 ◦C for 30 minutes followed by sudden cooling then the pasteurized milk left in cool place for 4 hours to allow separation of cream. 2. Separate the cream and the residual milk called Skimmed milk. 3. Skimmed milk is divided to two halves. § To one half add: Ø Separated cream. Ø Equal amount of water. Ø Complete lactose to 7%. Ø Iron, vitamin C, vitamin D may be added. 4. Mix well then sterilize the milk: § Heating of milk to 116 ◦C for 15 minutes.

N. B: 1. The humanized milk still differs from human milk in containing less iron and copper. 2. The protein in diluted cow's milk is usually still higher than that of human milk so: Ø Bottle fed babies store greater quantities of nitrogen than do breast fed babies. This nitrogen retention result in a muscle mass 25% greater in bottle fed babies. Ø Cow's milk digested more slowly than human milk. Ø The faeces of bottle fed babies are more abundant and of a harder consistency than those of breast fed babies.

3. High mineral content of cow's milk may be a load to cardiovascular system due to high Na+ content. 4. High phosphate content of cow's milk may produce hyperphosphatemia and hypocalcemia with convulsions in the infants.

Advantages of human milk (Breast Feeding): 1. Psychological effect on both child and mother. 2. Breast milk is supplied in suitable temperature. 3. Sterile and not liable to be contaminated. 4. Cheaper than animal milk. 5. Not liable to adulteration. 6. Lactation may act as a contraceptive measure. 7. Lactation minimize the risk of breast tumors.

ESTIMATION OF LACTOSE IN MILK

Principle: Lactose in milk is estimated in the clear filtrate after precipitating proteins. Tungestic acid, and Trichloroacetic acid can be used to precipitate milk proteins.

Instrument: 1. 2. 3. 4. 5. 6. 7. Estimated pipette. Volumetric flask. Funnel. Beaker. Burette holder. Porcelain dish.

Procedures: a) PPT of casein: ►In a clean dry volumetric flask add: § 5 ml of milk § 5 ml of 10% sodium tungstate solution § 5 ml of 2/3 N sulphuric acid drop by drop. ►Shake gently after each addition. ►Complete to the 100 -ml mark by distilled water. ►Left for sometime (1/4 – 1/2 hr) until the precipitate of casein settles down.

b) Filteration: ► Filter into a dry clean beaker. N. B: The filtrate must be clear, if not refilter again.

c) Reduction: ► When about half the volume is filtered, wash the burette with little of the filtrate. ► Then fill the burette with the filtrate. ► In a porcelain dish place: q 10 ml of Fehling’s solution. q 20 ml of distilled water. § Heat the Fehling’s solution until it gently boils and ► Begin the reduction adding the filtrate slowly and regularly in order to keep Fehling’s solution boiling all the time. ► Continue the reduction until the last trace of blue color is just discharged.

► Using an external indicator near the end point (Potassium Ferrocyanide solution acidified with Glacial Acetic acid) to ensure the complete reduction of Fehling's solution: ØThe end point is reached when no brown color or precipitate is formed on the addition of one drop of the solution to the external indicator. 10 -ml Fehling’s solution are reduced by 0. 0678 -gram lactose. ØIf brown color or PPT appeared it indicates: v Incomplete reduction of fehling’s solution. Complete reduction till the yellow color of the indicator doesn’t change. v

d) Calculation: R= number of mls of the filterate used for complete reduction of fehling’s solution. ► 10 ml Fehling’s solution are completely reduced by 0. 0678 gm lactose. 100 ► Total dilution of milk (T. D): 20 = 5 ► ► Lactose in milk (g%): Lactose in milk = 0. 0678 x 20 x 100 R

Estimation of glucose or lactose in solution:

Principle of the test: The quantitative determination of glucose or lactose in gm/dl in the solution depends upon their reducing property to Fehling’s solution. Instruments used in the estimation: ► Pipette 5 and 10 -ml capacity. ► Conical flask of 100 cc capacity. ► Burette 50 ml capacity ► Burette holder. ► Porcelain dish. ► Beaker.

Reagents and solutions required: ► Glucose or lactose, solution under estimation. ► Fehling quantitative reagent. ► Potassium ferrocyanide acidified with glacial acetic acid (external indicator). Significance of the test: ► This test considered the bases of tests used for the determination of blood glucose. ► Used for estimation of lactose in milk, for detection of adulteration of milk by the addition of water.

procedures: (A) Rough estimation: ► Fill the burette with glucose or lactose solution after its careful rinsing with water. ► Put in the porcelain dish q 10 -ml of Fehling quantitative reagent. q 20 ml of distilled. Ø Heat until the Fehling boils in the dish. ► Begin the reduction by slow addition of solution in order to keep the Fehling boiling all the time. ► Continue reduction till the last trace of the blue color of the Fehling’s reagent turn to red brown PPT of Cu 2 O. ► Record the rough reading R (number of mls of glucose or lactose descended from the burette).

►If the (R) rough reading is below 5 ml make dilution to the glucose or the lactose solution under the estimation. rough reading is above 5 ml calculate the percentage of glucose or the lactose solution under the estimation. ►If the (R)

(B) Preparation of diluted sugar solution: ► Mutiply the (R) by 4. ► The result is equaled to 5, 10 or 20. ► In 100 ml volumetric flask add: q? ml of the glucose or lactose sugar solution. q. Completed with distilled water to 100 ml mark and well mixed. ► In this case the sugar solution is diluted 5, 10 or 20 times.

(C) Accurate estimation: ► Wash the burette with distilled water and rinse it with little of diluted sugar, ► fill it again with the diluted sugar. ► In the porcelain dish add: q 10 ml of Fehling’s quantitative solution. q 20 ml of distilled water. ØHeat till gentile boiling. ► Begin the reduction by slow addition of the diluted sugar solution drop by drop in order to keep the Fehling’s solution boiling all the time. ► Continue reduction till the last trace of the blue color disappeared and changed to red brown PPT of Cu 2 O

► Using an external indicator near the end point (Potassium Ferrocyanide solution acidified with Glacial Acetic acid) to ensure the complete reduction of Fehling's solution: ØIf no brown color or PPT is formed on the addition of one drop of the solution to the external indicator it indicates: v complete reduction of Fehling's solution. ØIf brown color or PPT appeared it indicates: v Incomplete reduction of Fehling's solution. v Complete reduction till the yellow color of the indicator doesn’t change. ► Record the number of mls used for reduction of 10 ml Fehling’s quantitative solution and give it symbol (V) ml.

D) Calculation: ► 10 ml Fehling’s solution are completely reduced by 0. 0678 gm lactose. Lactose in solution = 0. 0678 x 100 R 0. 0678 x T. D x 100 V g/dl

► 10 ml Fehling’s solution are completely reduced by 0. 05 gm glucose. Glucose in solution = 0. 05 x 100 R 0. 05 x T. D x 100 V g/dl

N. B. : ► Fehling’s solution should boil gently all the time. ► Never stir by glass rod. ► The rate of addition of sugar solution from the burette should be equal to the rate of evaporation of Fehling in the dish. ► Do not use the external indicator as long as there is blue coloration seen by naked eye. ► The external indicator should be present in white, clean porcelain slab.