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dsw (c) 2013 dept. of chemistry
Introduction
Milk normal secretion of the mammarygland for the feeding of the young
mammals,c.q.lacteal secretion, practically free fromcolostrums, obtained by the completemilking with contain not less than 8.25%milk solid-not-fat and not less than 3.25%milk fat
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Composition
Major component:Lactose (occur naturally in milk)Casein (occur naturally in milk)
Proteins (essential amino acids)Milk fat (palatable mix. of glycerides of fatty acids)
Lactose and mineral salt------ true solutionProtein and casein phosphate ------ colloidal solt.Fat ------ macroscopic dispersion of the milk plasmaOther constituents ----vitamin, enzyme, pigment, andlactic acid
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Composition (cont.)Cows milk in average gross composition: water 87%, fat 3.7%,lactose 4.9 %, proteins 3.5%, ash 0.7 %
Water87%
Solid13%
27% Protein: casein
30% Fat: combinedwith water is calledcream
37% lactose: milk
sugar6% ash/minerals:vitamins andminerals includingcalcium andphosphorus
Protein27%
Fat30%
Lactose37%
Ash/mineral6%
Milk Solids
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Variation in composition is due to breed diff., time of year, time of day,individual diff., age of mammals, period of lactation, portion of any onemilking, feeding etc.
Breed difference: greatest magnitude effect
Time of year/day:Milk in fall and early winter is richer than spring and summerMorning milk is richer in fat than evening milk
After first few days after calving, it is known as colostrums anddiffer materially from normal milk
Lactation period:In the final lactation: there is some increase in fat and casein
Portion: first portion (fore milk) of milk drawn in milking process arelower in fat than the last portion
Composition (cont.)
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Fat
Fat and associated lipids material as an emulsionof small individual globule in an aqueous phaseSurrounding each milk fat globule is a number ofmembrane composed of cholesterol, phospholipidsprotein, and other surface-active moleculesThe membrane prevent individual fat globules fromcombining with others to form a larger globule(emulsifying agent for the fat suspended in milk)Fat globule membrane:
Inner layer: phospholipids-protein complexOuter layer: enzymes originating in mammary gland
Membrane agitation 1. disruption of the membrane (rancid flavor)2. cause for globule to coalesce to form larger globuleand butter
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Milk Fat: 12.5% glycerol, 85.5% fatty acid (by weight)Glyceride tri- (98-99%), di- (0.05%), mono- (0.04%)
contains more than 16 diff. fatty acids combined with in anumber of diff. ways.
Fatty Acids (even number of carbon atom)Milk Fat is the only natural food fat that contains butyric acid in its glyceridestructure
Butyric acid, C 6, C 8, C 10 saturated fatty acids give strongcharacteristic odour60-75% saturated fatty acids
25-35% unsaturated fatty acids4% polyunsaturated fatty acidsOleic acid and palmitic acid constitute 50% of the total fatty acid inmilk fat
Minor component: FFA, cholesterol, phospholipids, Fat-soluble vitamins(A,D,E,K)
Fat (cont.)
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Princ ipal fat ty ac ids fo un d in m i lkt r ig lycer ides
Molecular formula Chain length Melting point
Butyric CH 3(CH 2)2COOH C4 8 C
Caproic CH 3(CH 2)4COOH C6 2 C
Caprylic CH 2(CH 2)6COOH C8 16 C
Capric CH 3(CH 2)8COOH C10 31.5 C
Lauric CH 3(CH 2)10COOH C12 44 C
Myristic CH 3(CH 2)12COOH C14 58 C
Palmitic CH 3(CH 2)14COOH C16 64 C
Stearic CH 3(CH 2)16COOH C18 70 C
Arichidonic CH 3(CH 2)18COOH C20
Oleic CH 3(CH 2)7CH =CH(CH 2)7COOH C18: 1 13 C
Linoleic CH 3(CH 2)4(CH=CH.CH 2)2(CH 2)6COOH C18: 2 5 C
Linolenic CH 3.CH 2(CH=CH.CH 2)3(CH 2)6COOH C18: 3
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Proteins
Principles protein: casein, lactalbumin (0.1-0.4%), lactoglobulin (0.4%)
Casein: as a colloidal calcium caseinate calcium phosphate complex and forms about3% of the whole milkOther component: small amount (2%) of
heat-stable milk serum protein
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Milk p rotein f rac t ion s
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Casein
Casein is defined as the fraction of skim milkthat is precipitated by acidifying to pH 4.6-4.7,leaving the milk serum protein in solutionThree electrophoretic components designatedas , , and -casein in order of decreasingmobilitycomplex of -casein -casein which ishydrolyzed from casein via the action of rennin to give para- -casein
para- -casein is insoluble in the presence ofcalcium ion, but precipitate, instead, as a clot orcurd
Basis of cheese manufacture Milk of ruminant (buffalo, cow, ewe, goat, etc.)contain at least 2.5% casein give cheese-type-curd with rennet (milk of others, not)
Rennin : enzymeproduced bycellsencapsulatingdigestion trackto coalescemilk. Thisenzyme effectssoluble-milkprotein whichconvert to
insolubleensuring milk totake more timebeforehydrolysis byprotein-digesting
enzyme
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Whey proteins
After the casein proteins have been precipitated, the wheyproteins remain in solution.Whey soluble protein retained in the supernatant
include lactalbumin and lactoglobulin unaffected by diluted acid and rennin (caseinaffected) both are coagulated by heat (depend on pH,T , t , and salt concentration)
Component: -lactalbumin , lactoglobulin , protease- peptone, blood serum, (serum albumin,immunoglobulin)
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Other proteins
After removal of casein, albumin, globulin from skim milk filtrate contains a fraction that may be precipitated bysaturation with ammonium sulfateFraction protease-peptone fraction that consist of smallermolecules size than true protein
Nonprotein Nitrogen
Includes: Amino acid, vitamin, uric acid, ammonia, creatin, creatininEnd product of nitrogen metabolism and in synthesis of milk inthe udder
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Lactose (milk sugar)
Composed of D-glucose and D-galactoseContent: 4-5% (the more lactose contains, the sweeter itstaste), 1/3 as sweet as sucrose)
Reducing Fehlings solution Soluble in hot acetic acid, insoluble in alcohol and etherFermented by bacteria lactic acid (souring of milk), butodor is due to other volatile product (during fermentation)Hydrolyzed by lactase (in digestive track) intomonosaccharide as well as -galactosidase (from bacteria)Over lactose intake (exceeds the amount that it canhydrolyse) symptoms of lactose intolerance (flatulence,cramps, diarrhea)
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dsw (c) 2008 dept. of chemistry
Vitamins
Fat-soluble vitamins: A, D, E, K Ascorbic acidThiamin, riboflavin, niacin, pantotenic acid, pyridoxine, folicacid, and cyanocobalamin
Water-soluble vitamins
(CH=CH-CH=CH) 2CH2OH
CH 3 CH 3 H3CVitamin A:Uses Maintains skin and mucous membrane oforal cavity, and digestive, respiratory, reproductive,and urinary track. Critical for vision
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Vitamins (cont..)
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Vitamins (cont..)
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Vitamins (cont..)
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Enzyme
Lypase catalyze the hydrolysis of lipidsPhosphatase hydrolyze ester ofphosphoric acidLactoperoxidase, catalase, amilase,protease, xanthine oxidase
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Grade of milk
GradeGrade A raw milk
Grade A pasteurized milkCertified milk
Classes
Class 1: fluid purpose (highest price milk)Class 2:any other purpose
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Analysis of Fluid Milk Product
Sampling:Collecting milk in one container from one or more containerMixing (with no shaking) just stirStorage in a nonadsorbing, airtight container; cold (but notfreezing)
Addition a suitable preservative may allowedTablet of HgCl 2-K2Cr 2O7 not exceeding 1 g;formal dehyde 36% (0.1 % per 30 mL milk);if phosphatase test is to be made, chloroform is the onlypermisible preservative)
Bring sample to about 20 0C, mix by pouring sample into areceptacle and back again several timesWeigh or measure the test portion
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Weighing MethodPrinciples:
Transferring a weighed quantity of milk into a flatdish (porcelain, aluminum) containing a layer ofsand (to increase the drying surface)Drying the dish and content to a constant weightCooling it in desiccatorThe increasing in weight correspond to the totalsolid in the sample
Analysis of (cont.): Total Solids
Dried solidshould be
practicallywhite . Anyappreciablebrowning indicates thatsome of lactosehas beencaramelized(error in result)
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Procedure:Add 10-15 g of white sand to flat dishHeat the dish at 100 0C in drying oven until a
constant weighWeigh 5 g of prepared milk into the dishDry on a steam bath for 15 minutes, then in airoven at 98 0-100 0C for 3 hr.
Cool in a desiccator and weigh rapidlyReport the percentage of residue as total solid
Analysis of (cont.): Total Solids
dsw (c) 2013 dept. of chemistry
composition
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Lactometer MethodConstituents of normal milk are present in a fairly constantratio total solid can be calculated with a fair degree ofaccuracy if sp .gr . and fat percentage are knownProcedure:
Bring sample to 16 0 , determine the sp gr of the milk with aQuevenne lactometerTotal Solid (TS) = L + 1.2f + 0.14
with L = Lactometer readingf = percentage of fat in the milk (by the method later in
det. of crude fat bellow)
Analysis of (cont.): Total Solids
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Determination of Crude Fat
Volumetrically:Babcockmethod as arapid method forgeneral dairy
testing
Gravimetrically:Mojonniermethod
BABCOCK Method
Principle:
Milk H2SO 4 (concentrated)
Protein denaturated hydrolyzed
Fat to coalesce processes follows(centrifuging, water adding)
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dsw (c) 2013 dept. of chemistry
Babcock MethodSample of well-mixed milk in a standard test milk bottleHold the bottle at an angle then pour H 2SO 4 slowly, in small portion,while rotating the bottleMix the acid and milk until all traces of curd have disappeared. The
mixture become hot and turns uniformly dark coloredTransfer the bottle to a centrifuge, counter balance with bottlecontaining water then centrifuge for 5 min.Remove the bottle, add hot distilled water until the bulb of the bottle isfilledCentrifuge for 2 min., again. add hot water until the fat column isforced up to the neck, centrifuge 1 min., at 55-60 0CPlace the bottle into the water bath (immerse to the top of the fatcolumn)
After equilibrium, remove bottle from the bath, dry and measure theheight of the column (read directly by weight of the fat in the milk)
Determination of Crude Fat
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Babcock Method:
Well-mix milk- ----------- Mixing -----------centrifuging
----hot water adding ------------- centrifuging ---hot water
again -----------centrifuging -----------------measurement
Determination of Crude Fat
Test milk bottle
H2SO 4
Dark colored
Until bulb filled
Fat forced up Cooling in bath
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Mojonnier Method
Fat is released from other milk constituent by treatment withammonium hydroxide and ethanol then dissolved in a mixtureof ether and PEEther layer (containing fat) is decanted into a weighed dish,the ether is evaporated then the dish again weighedDifferent in weight is the amount of the fat extracted
Procedure:
Weighing approx. 10 g of well-mixed milk into a Mojonnierextraction flask and adding 1.25 mL of concentratedammonium hydroxide . Add 10 mL of 95% ethanol , stopperthe flask and holding it in horizontally with a finger on thestopper, shake thoroughly. Add 25 mL of ether and shake for1 min., add 25 mL PE and again shake for 1 min., centrifuge the flask or let stand until upper layer is clear. Remove thestopper then decant off as much as possible of the ether layerinto an aluminum dish.Repeat the extraction of the liquid remaining by addingsuccessively 5 mL ethanol, 15 mL ethyl ether and 15 mL ofPE, shaking after each addition as before. Let stand, thendecant the layer. Evaporate the ether layer and dry in an ovenat 135 )C for 5 min., Cool in desiccator then weigh.Calculate the percentage of the fat obtained
Determination of Crude Fat
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Determination of AshOrganic matter is oxidized by combustionin a muffle furnaceRemaining residue ash is weighed andpercentage of ash calculated
Ashing should be done at temperature of 500 0C in order to preventvolatilization of the chloride (sodium and potassium)Procedure:
Heat a crucible at 500 0C for 1 hr, cool and weighPipette 10 mL of well-mixed milk into crucible and weigh.
Evaporate to dryness on a steam bath.Transfer the crucible and dried sample to a muffle furnace untilthe ash is carbon free (white ash)Cool in desiccator, weigh and calculate the percentage of ash
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Determination of Total Acidity
The acidity of fresh milk ( apparent acidity )is due to the presence of casein, phosphates, albumin, CO 2, andcitrates
Any increase in acidity is due to the fermentation of lactose to lacticacid by bacteria present ( real acidity ).When lactic acid is sufficient produced, proteins coagulate (milk istermed sour )Procedure:
Weigh 20 g (about 20 mL) of well-mixed milk into beaker placed on awhite background), dilute with an equal volume of CO 2-free water
(recently boiled and cooled), add pp indicator, titrate the milk solt. with .0.1 N NaOHReport the acidity as percentage of lactic acid by weigh (1 mL NaOH isequivalent to 0.090 (cek! Benar apa salah) g lactic acid
OH
O
OH
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dsw (c) 2013 dept. of chemistry
Determination of Total Protein
Kjeldahl methodCasein and lactalbumin the major proteinconstituent in milk, are contained approx. 15.7% nitrogenconverting factor to protein = 6.38 (=100/15.7)
Procedur:Transfer accurately a weighed sample of appox. 5 g of well-mixedmilk into an 800-mL Kjeldahl flask. Add the various kjeldahlreagent, digest, distill and titrate the distillate (as previousdiscussion)
Conduct a back determinationCalculate the percentage of protein present in the milk:
% protein = sample titration (mL) - blank titration (mL) xNsolution x 0.14 (meq of N) x 6.38
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KMnO 4
Determination of Lactose (milk sugar)Polarimetric determination: based on optic activity character of thelactose
Munson- Walker method: based on reducing properties (Fehlings A andB to produce Cu 2O)Iodine-Thiosulfate method: based on oxidation of Cu 2O with nitric acidto give soluble cupric saltPermanganometric: also based on oxidation of Cu 2O with ferric sulfateand H 2SO 4, titration of the ferrous with standard potassium
permanganateCu + + Fe 3+ Cu 2+ + Fe 2+
Folin-Wu Colorimetric method:
Milk sample is freed from suspended component, then heated with analkaline copper solt. In a Folin-Wu sugar tube to prevent reoxidation.Cu 2O is oxidized by phosphomolybdic acid and bleu color is measuredin a colorimeter. Concentration of lactose is det. by comparison with asugar solution of known concentration treated in the same manner
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Residual Phosphatase Test
The test is commonly used for detection ofimproperly pasteurized milkFact: alkaline phosphatase, an enzyme present
naturally in raw milk, is progressively inactivated atthe temperature employed in pasteurizationBasis:
hydrolysis of disodium phenyl phosphate by the enzyme
Phenolate then is quanititated by any colorimetric reaction Any addition of unpasteurized milk to pasteurized milk canbe detected
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Determination of MineralsGreatest interest: Calcium, ironOther cations: sodium, potassium
AAS as an also sensitive method in detecting small amount ofcontaminant (Pb Hg, etc.)Method
Preliminary:Removing organic matter (dry ashing or wet digestion for non-Hg analysis)Dissolving the residue in dilute acid
Measurement: Aspiration to the flame
Determining absorption at spesific (a resonance line)
Preparation:Stock solution of a salt of the metal to be determined in acid
Preparation a series of standard solutiondsw (c) 2013 dept. of chemistry
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Chemical Analysis of Calcium(Volumetrically)
Calcium is precipitated at pH 4 as the oxalateThe oxalate dissolve in acid liberating oxalic acidOxalic acid then is titrated with potassium permananganatewith bromocresol green as an indicator (until pink colour )
Equation :2 KMnO 4 + 5 CaC 2O 4 + 8 H 2SO 4 2 MnSO 4 + K 2SO 4
+ 5 CaSO 4 + 10 CO 2 + 8 H 2OCalculation:Jumlah calcium (mg) = Vol. KMnO 4 (mL) x N KMnO4 x meq Ca
Follow the procedure!
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Basis: formation of coloured complex of ferrous ion and2,2-dipyridyl
Chemical Analysis of Iron(UV-Vis Spectrometrically)
sample
protein solution
acid
heating
pH adjustment
2,2-dipyridyl
Colouredsolt. Measuring A at 522 nm
Comparing with standard
data