Introduction:

The four food groups are things we have known about from a very young age. Daily nutritional requirements vary depending on the person, but a general system can be followed in order to ensure a healthy diet. Canada’s food guide for healthy eating has been approved by Health Canada as a good way to obtain all of the essential nutrients. It is most commonly presented in the form of a pyramid, and divided into four large food groups; namely: grain products, fruits and vegetables, milk products, and meat and alternatives. 

The meat and alternatives subcategory is often referred to as the proteins group. While protein can be found in other food groups (such as dairy), the meat and alternatives section is the food group where highest concentrations can be found. Although the least amount of daily servings come from this food group (2-3 as opposed to 5-12 for grain products, 5-10 for fruits and vegetables and 2-4 for milk products), proteins are nevertheless essential to our development and health. For these reasons, our lab is based on proteins, and their concentrations found in different sources. The integrative question we will be addressing in our project is the impact of different protein diets in different societies.

Proteins are macromolecules made up of amino acids. They are essential in growth and repair, metabolism and digestion. People can obtain protein through foods. There are many food sources that provide proteins, the most known of these being meats. Foods from animal sources are called complete proteins because they include all 20 amino acids our body requires.

The alternatives such as beans, peas and peanuts are also sources of protein, but are called incomplete proteins as they do not contain all of the essential amino acids. However, it is possible to acquire these amino acids by eating meat alternatives in combination with plant foods (grains, vegetables, etc.) Certain precautions can be taken to ensure that protein is digested properly. Our bodies digest and use animal protein more easily than plant protein; and these more easily than legumes. The protein sources that are most difficult for our bodies to digest and use are the remaining plant sources and grains. Digestion of meats is easiest when the fat content is low. Marinating meats also causes them to be easier to digest.

It is possible to receive too much protein in our diets. Our bodies recycle proteins and use them to form new ones. This is done by tissues that break them down. Our bodies require around 50 grams of protein daily; however the average Canadian consumes almost double this amount. Excess protein can lead to fat and high cholesterol. Research has also shown that an over intake of protein can lead to increased risk of osteoporosis, cardiac disease, kidney disease and artery damage.  On the other hand, a lack of protein is also detrimental to our health. Extra precautions must be taken to ensure that all nutrients are made available to those lacking meat in their diet. Vegetarians face this dilemma on a daily basis.

Differences in culture, religion, economy and climate influence the diet of a given population. Throughout the world, several different diets can be found. The Mediterranean diet is one high in fruits, vegetables, bread, cereals and olive oil. It is also low in red meats and fish. In this specific diet, poultry is ingested in moderation. An Asian diet consists mostly of rice, noodles, beans and soy products. Fish, nuts, grains and seeds are the main sources of protein. Starch, root vegetables and spices are the main focus in African meals; however, many areas incorporate fish, red meat and poultry into their diet. Most Indian diets are vegetarian ones: low in meats but high in soy products and tofu. South and Central American diets are very much like the Mediterranean diet, containing high contents of fruits, vegetables, nuts, whole grains and fish. The above demonstrates the wide range of protein content in different diets. Our project will aim to reveal some of the advantages and disadvantages of these diets.

Our lab focuses on the difference in protein concentrations found in three sources, one of which is an animal source, and the other two being plant sources. Our chosen sources were red meat, tofu and soy nuts. 126.4 g of red meat (hip French grilling steak) was used as our meat source, and our two plant sources were regular tofu (225 g) and salted soy nuts (280 g). The meat we used was a steak, which is a piece of meat from a fleshy part of a cow, cut perpendicular to the muscle fibers. Tofu is a soft substance with the consistency of cheese, made by curdling soy milk with a coagulant. It absorbs the flavors of the other ingredients. Soy nuts (glycine soja) are a member of the legume family. They are the world’s primary source of vegetable protein. The method used to determine the protein concentrations is called the Lowry-Folin method. This method is best to use when protein content is between 25 mg/mL and 500 mg/mL. This method is photosensitive, which makes it somewhat difficult to work with; however, it is more sensitive than other methods (Biuret for example), causing results to be more precise. It also requires a fair amount of preparation; it is a multi step process and is quite labor-intensive. It is the most widely used method for protein concentration determination, and can be performed at room temperature. We hypothesize that meats will be the source containing the most protein.

Our project reflects the scientific method in several ways. To begin, a hypothesis was formulated, and an experimental procedure was designed to test this hypothesis. After designing the experiment, the overall procedure was repeated several times in order to ensure better results. Data and observations were recorded and analyzed. Based on our results and calculations, conclusions were drawn.

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Methods and Materials:

To begin, the three solid samples were blended until they were as liquid as possible, and dissolved with phosphate buffer: 160ml of phosphate buffer was added to the meat, 518ml to the nuts and 125ml to the tofu in order to protect the proteins from being denatured by the acid. Then, each sample protein solution was mixed with 9.165 g of ammonium sulfate in a beaker on ice. The beaker was put onto a stir plate in order to ensure good mixing. Ammonium sulfate was added gradually in 3 additions. This was set aside for 20 minutes to precipitate the proteins in each sample.

When a precipitate was formed, it was put into two centrifuge tubes. At this moment, one important thing was the equal amounts of ammonium sulfate saturated protein solutions in both tubes. The solution was centrifuged at the highest speed. After 15 minutes, the liquid was decanted using a Pasteur pipette. It was then resuspended with 50 ml of phosphate buffer. With the new solution, one mL was used in order to approximate concentrations of protein in the liquid using the Warburg-Christian method. The rest of the solution was frozen for later experimentation.  

Since the aborbances obtained by approximation using the UV spectrophotometer for each sample were too high, i.e. above 2 absorbance units, a 1:100 dilution was done with each sample in order to get absorbances between O and 1. After a rough estimation of the protein concentration for each sample, the Lowry-Folin method was found to be the best method for the determination of protein content in the three types of food. This method consists of mixing 1 ml of solution A (2% of Na₂CO₃ in 0.1M NaOH) with 50 ml of solution B (0.5% CuSO₄· 5 H₂O in 1% sodium) to obtain Solution C. Then, 5 ml of solution C was added to the protein solution and this new solution was well mixed. After ten minutes, solution D, commercial Folin-Ciocalteu reagent with water, was added to the solution. This was mixed once again, and was set aside for 30 minutes. After all these steps, the absorbance of each protein sample, at a wavelength of 500 nm, was found using the thermospectronic machine. Those absorbances were then compared to the absorbances of a bovin serum solution with a known concentration of 10mg/ml on which the Lowry-Folin method was also performed.

Using the absorbances and the different concentrations of this BSA solution, a calibration curve was done in order to compare the absorbances of each of the food samples (with unknown protein concentrations), to the absorbances of the BSA solution, in order to get the original protein concentration. Back to top

Results:

A calibration curve was done with a solution of serum bovin albumin with the appropriate concentration for the range of the Lowry-Folin method (25ug/mL to

500 ug/mL). The equation of the regression line for the calibration curve will serve to determine the concentration of each protein sample.

Table 1. Raw data for the calibration curve using a 2mg/ml bovin serum albumin

The following table shows the different concentrations of a solution of bovin serum albumin and their corresponding absorbency. The raw data were obtained by the Lowry-Folin method, and were used to make the calibration curve of the different concentrations of bovin serum albumin used in the experiment.

Figure 1. Calibration Curve using a 2mg/ml solution of bovin serum albumin 

The calibration curve shows the different absorbance for each concentration of bovin serum albumin. The equation of the regression line for the graph will serve to determine the concentration of different protein samples for the nuts, the meat and the tofu.

The R²  obtained for the regression line is 0.9828. This value is close enough to 1 to assume that as the BSA concentration increases, the absorbance also increases. Thus, there is a linear relationship between the concentration of proteins in a substance and its absorbance.

Raw  and calculated data for the meat sample (hip French grilling steak) (1/100 dilution)

Table 2. Concentrations of proteins in original meat sample

The table shows the number of ml of the meat solution used with its specific absorbance. This absorbance was then associated with its corresponding concentration on the calibration curve. From this information, the original concentration of proteins in the meat sample was calculated (see sample calculations).

Raw and calculated data for the tofu sample (1/100) dilution

Table 3. Concentrations of proteins in original tofu sample

The table shows the number of ml of the tofu solution used with its specific absorbance. This absorbance was then associated with its corresponding concentration on the calibration curve. From this information, the original concentration of proteins in the tofu sample was calculated (see sample calculations).

Raw and calculate data for the Soya nuts sample (1/100 dilution)

Table 4. Concentrations of proteins in original nuts sample

This table shows the number of ml of the nuts solution used with its specific absorbance. This absorbance was then associated with its corresponding concentration on the calibration curve. From this information, the original concentration of proteins in the nuts sample was calculated (see sample calculations).

Averages of the three different concentrations (see table 2, 3 and 4) for the meat, the tofu and the Soya nuts samples were done in order to determine the concentration of proteins in each original sample.

Table 5. Averages of the three original protein concentrations for each sample

Figure 2. Comparison of the original concentration of proteins in each sample

This graph visually shows the differences in the protein concentrations of each of the original sample in mg of proteins/ g of sample. 

These averages clearly show that the sample with the highest concentration of proteins is the Soya nuts sample with a concentration of 370± 30 mg of proteins/g of nuts, followed by the meat sample with a concentration of 229± 5 mg of proteins/g of meat, and lastly, the sample which has the lowest protein concentration is the tofu sample with a concentration of 186± 3 mg of proteins/g of tofu.

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Conclusion:

To conclude, the Lowry-Folin method analysis showed that the concentration of protein for the meat (hip French grilling steak) was 229 ± 5 milligrams of protein per gram of meat compared to 279 milligrams of protein in one gram of steak. The concentration of protein for the tofu was 186 ± 3 milligrams of protein per gram of tofu compared to 163 milligrams of protein in a gram of tofu. The concentration of protein in soya nuts was 370 ± 30 milligrams of protein per gram of soy nuts compared to 263 milligrams of proteins in nuts. These comparisons are based on the Nutrient Value of some Common Foods from Health Canada. Our results are not within the uncertainty of their values, but they are quite good, considering that our samples are not exactly the same as what Health Canada used. Moreover, we found more proteins in the soy nuts since soy nuts have more protein than the other types of nuts.

There was a large difference in concentrations between the Soya nuts and the other two samples. This leads us to reject our hypothesis that meat would be the source containing the most protein. Surprisingly, the concentration of proteins in the Soya nuts is much greater than the concentration of protein found in meat. Even if Soya nuts are considered as complete proteins, we need to be careful with other type of nuts, as this is not necessarily the case. Although diets containing nuts may be extremely high in protein, this does not mean they are the most favorable. Meats contain things such as iron, which cannot be found in nuts. Also, since most nuts are incomplete proteins, they must be used in combination with other foods; this is not the case when it comes to a diet containing meat.

For our experiment we used the Lowry-Folin method. We found this method long because the time needed for the solution to react was lengthy. We needed to wait 10 minutes for the first reagent and another 30 minutes for the second reagent. Then, we found out that it was difficult to be within the range of the calibration curve.

There are some sources of errors in this method. The blender could not have mashed all the cells; therefore, some proteins could have been left in the original proteins that were not used. Also, some protein could have been left in the centrifuge tube. Then, we only did one protein extraction for each of the samples due to a lack of time. We cannot forget the fact that there is more than one type of meat, nut and tofu. This could also have an impact on our results, because our samples may not represent these groups as a whole. Meats, tofu and nuts come in many forms; therefore it is difficult to make generalizations based on just one sample. We also found out afterwards that the pH could have affected our results since the Lowry-Folin method could be affected by the pH (Davidson College, 2000). Proteins are made up of amino acids held together by peptide bonds. These peptide bonds are formed by a hydration synthesis reaction. The hydrogen bonds are what keep the orientation (and secondary structure) intact. Acid entering, which include H⁺ ions, break these hydrogen bonding, causing the secondary structure to unravel, and leading the protein to be non functional. The pH must be maintained between 10 and 10.5. Unfortunately, our pH, determined by the buffer of pH 7.1 used, was lower than it should be. This could have affected our results as well.

As an answer to our integrative question, we must look at the different diets mentioned in our introduction. Of these, both Asian and North American diets seem to contain the most proteins. The main difference in these diets is the source of the proteins. North American diets include more protein from animal sources, whereas Asian diets derive most of their protein from plant sources. Advantages can be found in both of these. Diets rich in meat are also high in fats, which can be seen as a large disadvantage. Fats can leads to a range of diseases, including diabetes and cardiovascular diseases. Diets high in nuts may be lower in fat, but they may be lacking in other things such as vitamins and minerals. Both advantages and disadvantages must be considered when planning a well-balanced diet. Whether your protein consumption comes from plant or animal sources, it is important to ensure that your diet contains the suggested requirements for this food group (50 g per day). One must also remember that a good diet must be combined with exercise in order to ensure a healthy lifestyle; and this proves to be true regardless of cultural, economical and religious influences on a diet. Foods with high concentrations of protein can also lack vitamin and minerals. They can also have a high amount of things that our body doesn’t need, such as fat. Therefore, a balanced diet which includes sources of proteins, but is also rich in nutrients, is essential to our growth and development.

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Appendix:

Sample Calculations

To calculate the final concentration of BSA:

The original solution of BSA that was used had the following concentration: 10mg /ml.

A 1:5 dilution was performed on this solution so that the final BSA solution used to make the calibration curve was 2mg/ml.

Sample calculation:

To calculate the corresponding concentration on the calibration curve:

The trend line of the calibration curve has the equation:  y = 0.6746x + 0.0598

Where y is the absorbance and x is the concentration of BSA corresponding to that absorbance.

Sample calculation for 1 ml of the meat solution with absorbance of 0.415:

Mg/ml

To calculate the concentration in the original meat sample:

Originally 126.4g of meat was put in 160 ml of buffer (solution 1).

Then 15 ml of this solution was taken and added to 50ml of phosphate buffer (solution 2).

Then a 1:100 dilution was done with this solution, and a different amount of this final solution was mixed with the buffer and was put under analysis each time (for more detail see materials and methods).

Sample calculation for 1ml of meat solution with corresponding concentration of 0.527 mg/ml:

To calculate the concentration in the original tofu sample:

The same thing as with the meat was done for the tofu. However, the original solution of 225g of tofu and 125 ml phosphate buffer (solution1) was diluted taking 35ml of the solution 1 and adding 35 ml of buffer because the original solution was took thick to be analyzed.(solution 2).

Sample calculation for 1ml of tofu solution with corresponding concentration of 0.515 mg/ml:

To calculate the concentration in the original nuts sample:

The same thing as with the tofu was done for the nuts. However, the original solution of 193.487g of tofu and 518 ml phosphate buffer (solution1) was diluted taking 50ml of the solution 1 and adding 25 ml of buffer because the original solution was took thick to be analyzed.(solution 2).

Sample calculation for 0.5ml of tofu solution with corresponding concentration of 0.083 mg/ml:

To calculate the uncertainties of the final concentrations of each sample

To determine the uncertainty for each sample, the standard deviation from the mean method was used. Here is an example of this method using the meat sample.

Standard deviation from the mean:  where d is the deviation from the mean and N the number of data points. The average concentration of proteins for the meat is 229 mg of proteins/g of meat.

Example: = 5 mg proteins/ g meat is the uncertainty on the concentration of proteins in the original meat sample. The same thing was done for the nuts and tofu samples.

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