Proteins are macromolecules composed of amino acids bound together by peptide bonds. These bonds can be broken down by proteolytic enzymes including trypsin, resulting in the release of peptides and free amino acids. Proteolytic enzymes may be supplied by tissues in the form of cathartic enzymes in meat or by extra-cellular enzymes supplied by microorganisms emerging in high-protein foods such as milk. In the first example, the end result is favorable because there is a meat tenderizer, while in the second case, if proteolysis is too extensive, bitter peptides will develop, ruining the taste of products such as milk and cheese. We are going to examine the changes taking place when trypsin is to milk and monitor it as a function
Proteins used in research extensively in the food industry for their properties, such as gelation, foaming, and viscosity. Eggs, milk, and soy are common protein sources of the food industry. Protein from these sources are used as food ingredients in the form of products such as whole egg yolk, egg albumen, whey solids, on fat dry milk, whey or soy proteins concentrates (WPC, SPC), and whey or soy protein isolates (WPI, SPI). Protein isolates are about 90 – 95% protein weight, while protein concentrates are about 50 -70% proteins,
Industrial methods used in the preparation of proteins isolates and concentrates are based on the difference in the solubility of proteins as a function of the PH and ionic strength of their environment followed by further purification using size exclusions techniques. The influence of PH on proteins solubility is due to its effect on the overall charge of the protein; this overall cost depends on the expenses of the terminal amino and the carboxyl group The overall charge of a protein is positive at low PH and negative at high PH. The particular neutral PH at which a protein molecule has a net charge of zero is called the isoelectric point of that protein. Protein is less soluble if the solution is at neutral point solution is at the protein’s isoelectric point. We will examine the fractionation of milk proteins based on these principles.
Experiment 1 – Proteolysis of Milk Proteins.
This experiment was conducted to demonstrate the enzymatic hydrolysis of milk proteins to peptides and amino acids. The lower- molecule weight end products of proteolysis passed through the dialysis bag and were detected by the reaction with ninhydrin reagent after heating. The samples of skim milk were provided in bulk then re-pipette dispensers and were dialyzed. Dialysates after one hour for amino acids were tested using ninhydrin reagent. The dialysates were then measured as a fraction of exposure time to the enzyme.
Methods and material used.
10 –inch pre-moistened dialysis tubing.
Strings to tie the knots.
Source of heat.
0.10% ninhydrin reagent.
A 10- inch pre-moistened dialysis tubing was taken from the beaker and a knot was tied tight on one end to ensure no leakage occurs.
20 ml of skimmed milk was then dispensed into a dialysis bag, and at the second end, a knot was tied. The outside of the two knots of the bag was then rinsed with distilled water to remove any residual samples.
The dialysis bag was then placed into a 100ml beaker containing 50ml of distilling water and then was let to stand for one hour. A hot plate was then set up for another 250 ml beaker containing 150 ml of distilling water that was later brought for boiling to start another experiment.
After one hour, the dialysis bag was taken out then the dialysate was mixed with a glass rod to ensure the dialysate is homogenized and uniform. A pipette was then used to taken a 3ml of an aliquot of dialysate into a clean large test tube, and then 1ml of 0.10% ninhydrin reagent was added to it. Paraffin was then used to cover the test tube to prevent water loss during heating. Water was then boiled for 30 minutes and observed if there is any color change that took place after the treatment given. Violet color indicated the presence of free amines and amino acids.
The test tube was cooled with running tap water with an immediate effect then the dialysate was measured spectrometrically against a water blank at 570 nm.
A graph of class data was then plotted as a function of exposure time of the enzymes.
Experiment 2: Fractionation of Milk Proteins.
This analysis demonstrates the fractionation of milk proteins Caseins, the principle group of milk proteins, are from skim milk by adjustment to PH 4.6. The remaining soluble proteins after removal of the caseins are the whey proteins. Heat denaturation may precipitate this hydrophilic protein in the presence of an acid. Whey proteins are also subjected to further purification in the industry using ultrafiltration or electrodialysis that remove salts and lactose.
Method and Materials used.
Beakers, stirrer, glass rods
0.5 M NaOH
Solid ammonium sulfate
The weight of 50ml of skim milk was measured in 150 ml beaker; the skim milk was then heated for approximately 400C, then PH was adjusted to 4.6 by slowly adding the 1.0 M HCl as stirring was maintained continuously with a magnetic stirrer. Casein was precipitated at PH of 4.6. Casein was then separated from the formed solution using a cheese cloth as water was squeezed out from casein. Much application of pressure was added to avoid the formation of thick curd. The supernatant was then saved, and the precipitate weighed.
Casein was suspended in a 50ml of distilling water, and a glass rod was then used gently to stir it continuously until PH was constant at 7.5. The observations were then recorded, and a chemical explanation was given.
The solution saved from step 1 was taken and placed in a 250 ml beaker on a magnetic stirrer.30 g of ammonium sulfate were weighed as it was slowly added to the whey solution with constant stirring for 10 minutes. This concentration of ammonium sulfate salted out the majority of the whey proteins.
Centrifugation was used to separate the salted whey proteins from the solution. Two centrifuge screw tube were used, and the net weight of each machine measured after they were capped. The solutions were then poured in two centrifuges (30 ml) of the settlement in total and weighed as they were labeled (Wrs1, Wrs2) and balanced with accuracy od +/- 1 g difference. The weight of each sample was identical within +/- g
The samples were then centrifuged at 4000 rpm for 15 minutes, and the supernatant was removed, and the weight of the precipitate measured with caps on and labeled as (WTP1, WTP2) Net weight of each tube was subtracted from the capped tube to get the weight of the precipitate. The weight of the precipitates was summed up from the two tubes, and that was the weight of the whey protein in 30 ml. The casein in the 50 ml solution was then calculated as the centrifuge was only 30 ml of the total 50 ml.
Experiment 1 – Proteolysis of Milk Proteins.
During boiling of the dialysate, it was observed that the solution began to turn transparent/clear purplish color. After boiling for 30min, it turns into a transparent violet color
Absorbance of blank at 570nm was found to be 0
Absorbance of dialysate solution was found to be 0.228
Experiment 2: Fractionation of Milk Proteins.
Weight of skim milk was 50.5545g
Weight of casein ball was 4.182g
Casein is supposed to dissolve back to sodium caseinate after mashing in the water and adding drops of 0.5M NaOH + continuous stirring
Every time the casein particles dissolves, the solution becomes more acidic again (the pH drops, and have to drop more NaOH into the solution to make the pH constant at 7.5)
Weight of centrifuge tube #1 (WT1): 7.6523g
1.During the one-hour period of dialysis.it was observed that the dialysis tubing had the ability to allow molecules based on the size selectively. The molecules diffused out of the dialysis tubing. The absorbance of the blank at the beginning while after one hour it was found to be 0.228.
2.The rate of absorption varied from the first minute and up to the 80th minute, and it was recorded maximum by group 8B at 1.367g.
3.Ninhydrin is a chemical that detects both primary and secondary amines. It reacts with free amines to form a purple or dark blue color.To detect the presence of proteins.
1. Ionic Strength = ½ S MiZi2
Mi. = Molarity of ion
Zi = Charge of ion
1M NaCl = ½ S (1 X 12) + (1 X 12) = 1
1M CaCl2 = ½ S (1 X 22) + (2 X 12) = 3
1M (NH4)2SO4 = ½ S (2 X 12) + (1 X 22) = 3
The weight of casein ball was found to be 4.182g.
2. Isoelectric point is the point at which pH of a particular molecule carries no net electrical charge.
3.Salting out is the method due for separating proteins by the effect based on the electrolyte –nonelectrolyte interaction. It is done to concentrate dilute solutions of proteins
The experiment was successful as the casein weight was fund to be 4.182g.
Laboratory manuals – Report and data for protein hydrolysis.
Class Data for a spectrophotometer (my data is group 2B):
Absorbance at 570nm