two experiments:

In this lab, we will conduct two experiments: one to demonstrate the enzymatic hydrolysis of proteins as a function of time, and the other to. Show the effects of pH on protein solubility, have a deeper understanding of a protein’s isoelectric point, and investigate the impact of salt addition on protein solubility.
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 provided by tissues in the form of cathartic enzymes in meat or by extra-cellular enzymes contributed by microorganisms growing in high-protein food products, such as milk. In the first case the end results are desirable as there is a tenderizer of meat, while the second case, if proteolysis it too extensive, bitter peptides can form, spoiling the flavor of products such as the 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.

Introduction

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.

Materials

10 –inch pre-moistened dialysis tubing.

Skim milk.

Strings to tie the knots.

Distilled water.

Dialysis bag.

Stopwatch.

Source of heat.

0.10% ninhydrin reagent.

Test tubes.

Tripod stand.

Method

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 100 ml beaker containing 50 ml 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 3 ml of an aliquot of dialysate into a clean large test tube, and then 1 ml 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 principal group of milk proteins, are fractionated 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.

Materials

Skim milk

M HCl

Beakers, stirrer, glass rods

0.5 M NaOH

Distilled water

Solid ammonium sulfate

pH meter

Method

The weight of 50 ml 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. Excess 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 50 ml 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.

Results

Experiment 1 – Proteolysis of Milk Proteins.

During boiling of the dialysate, it was observed that the solution begins to turn transparent/clear purplish color. After boiling for 30 min, 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

Discussions

Experiment 1:

1. In this experiement, a 12,000 Dalton cutoff dialysis membrane was used. 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. All molecules of size larger than 12,000 Daltons were selectively retained inside the dialysis tube, while smaller molecules diffused out of it. As, more proteins diffused out, the absorbance of the solution increased with time. The absorbance of the blank at the beginning was 0, 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 1.367.

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, known as Ruhemann’s purple. Ninhydrin test is used to detect the presence of proteins, for example fingerprint detection. It can be used qualitatively (e.g. for chromatographic visualisation) or quantitatively (e.g. for peptide sequencing) measure protein in a sample.

Experiment 2:

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.

Weight of skimmed milk = 50.5545g

Protein yield of casein = 4.182/50.5545 *100 = 8.2 %

Or it can be expressed as 0.17 g/ml of skimmed milk

Weight of centrifuge tube #1 (WT1): 7.6523g

After adding whey proteins solution (WTS1) 🡪 26.6152g

Tube #2 (WT2): 7.6488g

After adding whey proteins solution (WTS2)🡪 26.8937g

Average weight of whey protein = ((26.6152 – 7.6523) + (26.8937 – 7.6488))/2 = 19.1039 g

Protein yield of whey = 19.1039/30 *100 = 63.67%

Or 0.6367 g/ml of skimmed milk.

Theoretically, milk has about 3.3 % protein, out of which 82% is casein and rest in majority is whey. (source: https://milkfacts.info/Milk%20Composition/Protein.htm). Our estimate here is above those reported in literature. This difference may be due to error in measurement of the weight of the casein, or precipitation of additional milk proteins at the applied pH.

Additionally, the following factors may have contributed to the error in our estimated values:

1. After centrifugation, the whey and the casein protein did not separate, contributing to the high estimated weight of the whey protein. 2. It is also possible that we heated the skimmed milk to over 60°C by accident (instead of keeping it constant 40°C), which caused casein to coagulate and make it difficult to separate with whey. As the precipitation of proteins is highly dependent on the temperature, this can contribute to a high amount of error.

3. Similar to scope of error arising due to fluctuating temperature, pH also has a great influence on protein solubility. If the pH was too low (acidic), casein can dissolve back into the solution , lowering precipitation. This however seems to not be the case in our experiments as we estimate a higher mass of casein than expected.

2. Isoelectric point is the point at which pH of a particular molecule carries no net electrical charge. Caesin has an isoelectric point of 4.6, which is the pH at which caesin precipitates in the experiement. At this pH, the solubility of the protein is at its lowest, causing its precipitation.

On increasing the pH, the caesin regains in solubility. This happenes when the pH of the solution exceeds the pI (isoelectric point) of caesin.

3.Salting out is the method due for separating proteins by the effect based on the electrolyte –nonelectrolyte interaction. It is based on the fact that solubility of the nonelectrolyte is reduced at high salt (electrolyte) concentration. This process can also be used to concentrate dilute proteins. Ammonium sulfate is commonly used for salting out of proteins, as its solubility is so high that salt solutions with high ionic strength are allowed.

Technically, whey proteins are proteins present in milk serum after precipitating out casein protein. Also, the pI of whey protein is similar to that of casein. So we need to employ a different method to precipate it out, and the salting out effect is used here.

Conclusion.

The first experiment successfully demonstrated enzymatic hydrolysis of proteins in milk, evaluated by measuring absorbance with Ninhydrin reagent. The increase in absorbance with time shows increased proteolysis, as molecules smaller than 12,000 Dalton (amino acids) were formed which diffused out of the dialysis tube.

In the second experiment, we fractionated milk proteins. Two methods were used to precipitate milk proteins – lowering the pH to that below the pI of the protein of interest, and salting out phenomena. Both these methods take advantage of the effect in solubility of protein as function of various parameters like pH, ionic strength, temperature etc.

References.

Laboratory manuals – Report and data for protein hydrolysis.

Appendices.

Class Data for a spectrophotometer (my data is group 2B):

Group

milk sample

Absorbance at 570nm

0A

0 min

0.046

1A

20 mins

0.459

1B

40 mins

0.366

2A

20 mins

0.109

2B

80 mins

0.228

3A

0 min

0.034

3B

20 mins

0.084

4A

40 mins

0.096

5A

60 mins

1.374

5B

40 mins

0.064

6A

80 mins

1.084

6B

0 min

0.438

7A

20 mins

0.218

7B

40 mins

0.517

8A

60 mins

1.026

8B

80 mins

1.367

9A

80 mins

0.367

Combined Class Data (all group’s data combined)

Time

Data

Mean

Std

0 min

0.046

0.034

0.438

0.172666667

0.229863728

20 mins

0.459

0.109

0.084

0.218

0.2175

0.209662427

40 mins

0.366

0.096

0.064

0.517

0.26075

0.165895549

60 mins

1.374

1.026

1.2

0.24607316

80 mins

0.228

1.084

1.367

0.367

0.7615

0.593035412

Graphs.

Combined Data graph