Food Analysis – Protein Content of Foods
Table of Contents
A. Objectives
Students will:
- Understand the basic structure of proteins
- Understand the principles behind the Biuret Assay and Spectrophotometry
- Understand the process of setting up a standard curve
- Use a standard curve to determine the protein content of various protein-containing solutions
Laboratory report due:
- Data Record, Standard Curve, and Discussion Questions (Sections E, F & G)
B. Introduction
Protein Hydrolysis
There are many proteases produced by the gastrointestinal tract. Each of the various proteases acts on a specific site on the protein. The mouth does not possess digestive enzymes that initiate any digestion of protein. But in the stomach, hydrochloric acid denatures protein and provides a substrate for the enzyme pepsin to initiate protein digestion in the stomach. Once food reaches the small intestine many more proteases and peptidases are produced by both the pancreas and the small intestine to cleave peptides into their smallest units, individual amino acids.
This lab involves quantitatively determining the amount of protein in a liquid food. A protein in solution can be quantitatively assayed by several spectrophotometric methods. Most spectrophotmetric methods for determining the protein content of a food are based on the binding of a reagent to either specific amino acids or specific bonds (peptide) in the protein. The amount of color produced from this binding reaction can be detected at some wavelength of visible light and analyzed with a spectrophotometer.
Spectrophotometry
A spectrophotometer is an instrument employed to measure the amount of light absorbed by a sample. The instrument operates by passing a beam of light through a sample in a cuvette and measuring the intensity of light reaching a detector.
The beam of light consists of a stream of photons. When a photon encounters a molecule in the solution being studied, there is a chance the molecule will absorb the photon. This absorption reduces the number of photons in the beam of light leaving the solution, thereby reducing the intensity of the light beam that reaches the detector on the far side. The more molecules there are in the cuvette to absorb photons, the greater the absorbance of the solution (and the fewer photons that make it through the sample to the detector).
Color exists on an absorbance spectrum that is dependant on its interaction with light energy. Different colors absorb different wavelengths of light depending upon the amount of energy they possess. For example, red light has slightly less energy than blue light. This difference in energy results in a different absorption and thus a different color perceived by the eye.
The amount of light absorbed by a sample is directly related to its color. When a sample absorbs light of a certain wavelength, what we see is the sum of the remaining colors. For example, if a sample absorbs all wavelengths no color reaches our eyes and the sample appears black. On the other hand, if it does not absorb any light, it would appear colorless or white. Likewise, a dye that absorbed all of the colors except blue, would give a blue appearance.
Protein Assay
This lab measures the protein content of a solution using the Biuret assay. Determination of the protein content of an unknown solution requires comparison with a known or “standard” protein solution.
In the Biuret assay, absorbance measured by the spectrophotometer is a function of protein concentration. By first analyzing samples with known amounts of protein and plotting their absorbance on a graph, one can determine the protein concentrations of the unknown solutions. This graph is called the standard curve. The absorbance of the unknown solution is then measured and then plotted on the standard curve. The standard curve is used to convert the absorbance readings for the experimental samples into a protein concentration.
In this lab you will develop the protein standard curve using solutions of the protein called bovine serum albumin (BSA) that are of known concentration. The known solutions of protein are mixed with an alkaline Biuret reagent. Under alkaline conditions, substances containing two or more peptide bonds form a purple complex with the copper salts in the Biuret reagent producing a colored solution. The more protein in the sample the more color there is in the solution and the greater the absorbance indicated by the spectrophotometer.
Summary
This lab experiment is designed to acquaint you with standard lab procedures for using a spectrophotometer and for determining the amount of protein present in a solution. In this lab, you will create your own protein standard curve and then using your standard curve, estimate the relative amounts of protein in several solutions that contain protein.
C. Materials Needed
Spectrophotometer Stock solution of Bovine Serum Albumin (BSA)
Biuret reagent Protein solutions:
13 cuvettes a. skim milk (M)
Cuvette holder b. soy milk (shake gently) (S)
Wax pencil c. chicken broth (B)
Kim wipes d. protein shake (shake gently) (P)
Distilled water
1000 ml micropipet
Pipet tips
Wooden stir sticks
D. Procedure
A). Set Up
- Warm up the spectrophotometer at least 15 minutes prior to use.
- Within 2 cm from the rim of the top of 6 cuvettes, write numbers 1-6. These will be used for the known solutions.
- Label 4 cuvettes M, S, B, & P. These will have your unknown protein solutions.
- Write “BL” on another cuvette. This will serve as your blank to reset the spectrophotometer in between readings.
- Fill the “BL” cuvette with 4 ml of distilled water and set aside until you use the spectrophotometer.
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B). Sample Preparation for determination of standard curve using BSA of known concentrations:
- Prepare the standards in cuvettes as follows (add all of the BSA amounts listed below to each of the cuvettes first, then add the corresponding amount of water to each cuvette):
| Cuvette # [BSA] | BSA (ml) | H2O (ml) |
| 1 [0.0 mg/ml] | 0 | 1000 |
| 2 [1.0 mg/ml] | 100 | 900 |
| 3 [2.0 mg/ml] | 200 | 800 |
| 4 [3.0 mg/ml] | 300 | 700 |
| 5 [4.0 mg/ml] | 400 | 600 |
| 6 [5.0 mg/ml] | 500 | 500 |
- Add 2.0 ml of the Biuret reagent to each of the 6 cuvettes above. Do this by adding 1000 ml twice.
- Stir each with the clean end of a wooden stick. Record the time.
- Let the 6 cuvettes sit for 15 minutes.
- While the known protein solution cuvettes are sitting, move to Part C. After 15 minutes, cuvettes will be ready to have their absorbance read using the spectrophotometer for determination of standard curve.
C). Unknown Sample Preparation:
- Prepare the protein solution samples in cuvettes as follows:
(Before you pipette your protein samples, be sure to mix each gently by repeatedly tipping the tube upside down. In particular, make sure the soy milk and protein shake are well mixed.)
| Cuvette | Protein Solution (ml) | H2O (ml) |
| M (skim milk) | 100 | 900 |
| S (soy milk) | 100 | 900 |
| B (chicken broth) | 100 | 900 |
| P (protein shake) | 100 | 900 |
- Add 2.0 ml of Biuret reagent to each of the cuvettes above with your pipet (remember to just add 1000 ml twice to get 2.0 ml).
- Stir each with clean end of wooden stick. Record the time.
- Let the M, B, and P cuvettes sit for at least 15 minutes. Let the S cuvette sit for at least 30 minutes. While the unknown protein solution cuvettes are sitting, you can move to Part D and begin determination of standard curve using known solutions of BSA (be sure that the standard curve samples have had at least 15 min to react.)
D). Determination of the Standard Curve
- Adjust the spectrophotometer’s zero knob until the meter reads exactly 0% absorbance.
- Wipe the blank cuvette (BL) with a Kim wipe. Place the blank in the spectrophotometer and close the cover.
- Turn the wavelength dial to 550 nanometers.
- Adjust the transmittance/absorbance dial so that it shows 0% absorbance.
- Remove the blank and insert the cuvette #1(after wiping with a Kim wipe) into the spectrophotometer and close the cover.
- Read the absorbance and record it on your data page Table 1.
- Remove cuvette #1 and replace it with the
- Adjust the transmittance/absorbance dial so that it again shows 0% absorbance.
*Click the link below to watch: How to Use a Spectrophotometer:
https://www.youtube.com/watch?v=QcCrRDYiejg
(*Source of website: ISU Gen Chem Lab Tutorials-How to use a spectrophotometer (Spectronic 200E))
- Replace the blank with cuvette #1 (again). [In order to ensure an accurate curve, the known solutions will be analyzed 2 times. When setting up the curve you will use the average of the 2 absorbance readings.]
- Read the absorbance and record it on Table 1.
- Repeat the analysis for the remaining 5 tubes. Remember to wipe the sides of the cuvettes before placing them in the spectrophotometer. ALWAYS reset the spectrophotometer with a blank between readings.
E). Determination of the Protein Content of Unknown Solutions.
- Repeat the spectrophotmetric analysis using cuvettes M, S, B, and P.
- Determine the absorbance 2 times and record on your data page in Table 2.
- Remember to reset with the blank between measurements
F). Drawing the Standard Curve
- Create a standard curve by plotting on the graph paper (Section F). Plot the protein concentrations on the X-axis vs. the absorbance of the known protein solutions (data Table 1) on the Y-axis.
- This is your standard curve for the determination of protein content of the various food protein solutions.
G). Estimating the Protein Content of Foods Using your Standard Curve
- The standard curve tells you the absorbance of various concentrations of protein.
- Plot the average absorbance of each of your protein solutions on the standard curve. Use the following letters to label your points:
- M = milk
- S = soy
- B = broth
- P = protein shake
- Read the concentration of each of your protein solutions along the x-axis of the graph and record in Table 3.
Laboratory Report for Food Analysis – Protein Content of Foods (Sections E & F Below)
Student Name: ____________________________ Date: ______________
TA Name: _________________________________
________________________________________________________________
E and F. Data Record and Lab Write-up (10 points)
Table 1: BSA protein solutions of known concentration.
| Tube # | Protein concentration | ABS 1 | ABS 2 | ABS (avg) |
| 1 | 0.0 mg/ml | 0.075 | 0.075 | |
| 2 | 1.0 mg/ml | 0.302 | 0.299 | |
| 3 | 2.0 mg/ml | 0.356 | 0.359 | |
| 4 | 3.0 mg/ml | 0.412 | 0.412 | |
| 5 | 4.0 mg/ml | 0.470 | 0.469 | |
| 6 | 5.0 mg/ml | 0.516 | 0.516 |
Table 2: Protein solutions of unknown concentration.
| Tube # | Food sample | ABS 1 | ABS 2 | ABS (avg) |
| M | skim milk | 0.262 | 0.262 | |
| S | soy milk | 0.321 | 0.320 | |
| B | chicken broth | 0.463 | 0.462 | |
| P | protein shake | 0.541 | 0.543 |
Table 3: Determine the concentration of protein solutions by plotting the absorbance of each on the standard curve and reading the value on the X-axis.
| Protein Solution | Protein Concentration (mg/ml) |
| Skim Milk | |
| Soy milk | |
| Chicken broth | |
| Protein Shake |
F. Discussion Questions (40 points)
- Give a scientific explanation of why the absorbance increases as protein concentration of a solution increases (10 points).
- Explain the principal of a standard curve (5 points).
- According to the results of your protein assay, which solution is the best source of protein? (5 points)
- What specific part of the protein in these solutions is the biuret reagent actually reacting with? (5 points)
- Would this assay be effective for solutions of free amino acids? Why or Why not? (5 points)
***Please make sure that you turn in your standard curve (section G) with the data (from table 1) plotted to show how you calculated the protein concentrations in table 3. Note: Make sure you plot known BSA concentration in mg/ml on the x-axis and corresponding average absorbance values on the y-axis. (10 points)
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