Biol2107 Lab W 7-9:30
Protein Concentration; Utilizing Spectrophotometer to analyze commercially available food products to Justify FDA food Labels
Scott Zoebisch
10-14-2015
Abstract
Proteins help shape the physical body and the world. The consumer relies on proper FDA labeling of protein concentration. This Lab report will discuss the importance of proteins, proper food labeling and how the protein concentration is analyzed. The experimental use of the Bradford Protein Assay and Spectrophotometer will support the hypothesis that if the FDA labels are accurate on product protein availability, then the experiments will concur with the actual protein concentrations
Introduction
The word protein in this present day of such a Health
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and Fitness conscious society puts smiles on many faces. The word alone has been applied to packaging to increase profit (Nassauer 2013). But what is protein? Proteins are a polypeptide of essential amino acids necessary for muscle growth, cell function and cell maintenance (Morris, Hartl, Knoll, Lue, 2013). Proteins are found in many foods such as animal meats, dairy products and plant products. But, how much protein is needed to suggest if an individual is getting too much or too little? The FDA suggest that adults need 0.36 grams per pound of body weight (Schardt 2014).Too little protein and muscle loss has been noted, but too much protein has been questioned to cause cancer, although no direct link of evidence has been confirmed (Schardt 2014). The question is how accurate are the FDA labels concerning protein quantity. The hypothesis proposed is that if the FDA labels are accurate on product protein availability, then the experiments will concur with the actual protein concentrations. The measurements will be tested using the Bradford Assay and Spectrophotometer, which is most accurate at or above 590nm A bsorbance (Zor, Selinger 1996). Methods Testing for the presence of proteins in the unknown solutions was the first step. Utilizing the Coomassie Blue Dye confirms protein concentration with a color of intense blue. This blue color would become darker with higher protein concentrations. The negative control was the vial of PBS, where no color change would occur. The spectrophotomer was used to accurately obtain the protein concentration. The use of Micropipettors was used in conjunction with the spectrophotometer experiment. Eight cuvettes were used during the spectrophotometer experiment, each was filled with 1ml of dye reagent. The l Micropipettor was used to obtain this volume, 1ml = 1000l . Of the eight cuvettes, one was a negative control blank, containing 20l of PBS. The other seven cuvettes were filled with different concentrations of protein standards. The cuvettes were covered with parafilm, inverted to mix and allowed to incubate no less than 5 minutes. The Spectrophotometer was set to 595 absorbance reading and calibrated using the blank cuvette. Each cuvette standard was measured, and the spectrophotometer was blanked with the PBS cuvette after each standard. The standard absorbance readings were used to create the Standard Curve of the Protein Concentrations (see Table 1). The next measurements of absorbance were of the commercially available products (Protein shake, Whole Milk, and Muscle Milk). The initial absorbance and the 1:50 serial diluted solution Absorbance of each were obtained. The 1:50 dilution is a serial dilution where the initial solution is the base and each subsequent dilution from the base of 1:10 and 1:5 reflect a more precise measurement. This serial dilution is conducted when the Optical Density Absorbance rate is above 1.0. An OD reading above 1.0 will obscure concentration readings. Results The Protein Standard curve reflects the findings of the Spectrophotometer .
The higher the protein concentration, the higher the optical density. With Absorbance rates over 1.0 there are outliers that do not stay in the linear sequence. The Optical density will begin to level off close to 1.0 OD, and this is the reason to use a serial dilution to get the most accurate OD reading . The line of best fit equation for the standard curve is y=0.5394x ensuring to go through the point of origin. (See Table 1)
Table 2 shows the product and the FDA Food Label with the mg/ml of each Dairy product . By this table it shows the Muscle Milk having the highest protein concentration (see Table 2)
Before the 1:50 dilution, the 1:1 dilution shows the Whole Milk having the highest protein concentration. The Serial dilution is mentioned previously contributes to a more accurate reading of exactly which product has the highest concentration of Protein available and is needed when the 1:1 dilution is over 1.0 OD Absorbance. Table 3 outlines the A bsorbance of the product before dilution (1:1 ratio) after serial Dilution (1:50 ratio) the calculated concentration of mg/ml divided by the standard curve and the actual FDA food label mg/ml (see table
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3) Tables (Table 1) Standard Curve (Table 2) FDA Labeling Product FDA Label (g/ml) Conversion (mg/ml) Protein Shake 5 g/8 0z 5000 mg/ 236 ml Whole Milk 8 g/236 ml 8000 mg/ 236 ml Muscle Milk 25 g/ 414 ml 25,000 mg/ 414 ml (Table 3) Dilution Factor Concentrations Product OD absorbance (1:1) OD absorbance (1:50) Calculated Concentration (mg/ml) FDA Food Label (mg/ml) Protein Shake 1.912 .164 15.20 5000mg/236ml Whole Milk 2.391 .431 39.95 8000mg/236ml Muscle Milk 2.176 .700 64.89 25,000mg/414ml Discussion The results from the experiment show no falter of accuracy to the FDA labeling. This experiment supports the H ypothesis that if the FDA labels are accurate on product protein availability, then the experiments will concur with the actual protein concentrations. With so many additives and preservatives being processed in the world’s food, effecting the Health of the consumer, it was mandatory that the FDA outline the ingredients and nutritional value of all manufactured food (FDA.gov). This is done not only to inform the consumer of healthy options but also negate any legal liability from health issues that may arise from consumption of commercial products. The research performed was to evaluate the accuracy of the FDA’s labeling and information disseminated to the consumer. The need and want to know by the consumer reflects consumer spending (Nassauer 2013). The more the consumer can trust a product the more faithful the consumer will be to said product. And all of this entails the bottom line of everything – money, boosting sales by appealing to the consumer (Nassauer 2013). Citations 1. Nassauer, S. (2013) When the Box says ‘Protein’, the Shoppers say ‘I’ll take It’ WSJ 2. Schardt, D. (2014) Protein is more better? Nutrition Action Healthletter Vol. 41 issue 9 pg. 1 3. Zor, T. , Selinger, Z. (1996) Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies 4. Morris, J., Hartl, D., Knoll, A., Lue, R. (2013) Biology How Life Works 5. FDA.gov Lab Report Scoring Guide Mechanics, Format, and Style Possible Points Points Earned An appropriate writing style is used throughout the paper (clear, concise and easy to read) and the paper is in the proper format (typed, double-spaced, 1-inch margins) 2 1 Paper is free of grammatical, spelling and typing errors 4 2 Sources of ideas and statements are always cited. NO Quotes, it must be in your own words. 4 3 All tables and graphs are appropriately labeled (appropriate units), numbered and titled. The table or graph should be able to stand alone from the paper. 4 2 Literature cited has proper format (at least 3 citations, 2 must be journal articles, the only website accepted is FDA.gov) 4 1.5 Mechanics, Format, and Style Total 18 9.5 Content Possible Points Points Earned Title is appropriate and lets the reader know what the central problem or question is 2 2 Abstract An abstract is a short summary placed prior to the introduction, used to help readers determine the purpose of the paper.
4 1
Introduction
Introductory paragraph includes clear statements of the following:
-Background and significance of the problem,
-statement of the question driving the research,
-statement of your hypothesis and prediction 6 3
Methods
Description of methods is a concise narrative of pertinent information in paragraph form. Not a bullet point list. 4 2.5
Results
Results are summarized (in writing and table or graph format) and include a statement about the results of controls used. 8 2
Discussion
Interpretation and discussion of results follows logically from the data
Conclusion is related back to the original question and hypothesis and follows logically from the data
Discussion of inconsistencies in the data, and potential biases in reference to the methods are included
8 2
Content Total 32 12.5
PAPER TOTAL: 22 (out of 50 possible points)
Any type of PLAGIARISM will result in a
ZERO.
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...at keep organisms alive. “Proteins are the most structurally sophisticated molecules known” (Campbell, 1999) which is reason enough to study them. The techniques we learned in this lab form a basis from which a detailed study of proteins is possible. Following our procedure we were successfully able to set up a quantifying assay to determine the amount of protein within a milk sample, although our yield percentage was rather low. However, errors in this lab (in the form of a low yield percentage) may have an origin from our last lab. In the process of extracting proteins from the milk sample, we may have inadvertently lost some of the protein through erroneous measurements, or perhaps through poor handling of either ammonium sulfate or the dialysis tubing. While not sufficient enough (at this point) to invalidate our results, they do explain the major difference between the expected and the actual amount of protein extracted.
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...(Complete protein. (2014, March 24). “Complete proteins could be gain from meat, fish, poultry, cheese, eggs, yogurt, and milk” (Incomplete vs. Complete Proteins. (n.d.).