Research 1 :Boiling Time Affecting The Protein Concentration in Milk, Measured Using Bicinchoninic Acid Assay and Calorimetric
Abstract
Heating milk can increase the rate of interactions between different molecules within the milk and hence was very important to see how increasing boiling times would affect the milk concentration. The extended essay was purposed to find the effect of boiling time on the protein concentration in milk, measured using bicinchoninic acid assay and calorimetric. The BCA assay technique was preferred in carrying in this investigation owing to its high level of accuracy. Through the BCA technique, it was believed that it would also be possible to reduce copper (II) ions into copper (I) ions within an alkaline medium. The protein concentration of the milk was hypothesized to would increase with an increase in boiling times. Two reactive A and B were to be prepared first, using different reagents. The protein concentrations would then be measured at different instances time. The findings and subsequent analysis showed that there was a direct relationship between the two research variables. The protein concentration of the milk was found to increase in boiling times, a relationship that was confirmed by a positive gradient between the two variables, answering the research question and confirming the hypothesis. The uncertainty level in the data sets was very low, and the error bars on the graph were very small, and non-overlapping, showing a high level of reliability and accuracy. Therefore, there was enough evidence to show that boiling points directly affect the concentration of milk products.
Keywords: Protein concentration, Boiling Times, Bicinchoninic Acid Assay, Pasteurization
Rationale
My whole life has always revolved around society. One of the main heating equipment that I interact most with is a microwave oven. Almost in all homesteads within the society that I have grown in, microwave ovens find many applications. However, their mode of action has triggered me a lot, having been told by my parents that these ovens have serious repercussions on the body. I also learned that a microwave oven is capable of heating food products within a few minutes, as water found within the food particles has the potentiality of gaining heat within a fraction of a minute. Thus, the question of time came to light. As I was reading across the internet, I discovered that heating food products in ovens leads to protein unfolding at a very high degree, which triggered me even further. I, therefore, decided to carry out a scientific investigation, not now on microwave ovens, but to investigate the effect of boiling point times on the concentration of proteins found within the milk.
Introduction
The composition of caseins in milk products is about 80%, in accordance with Bhat et al. (2). However, these caseins remain stable under heat treatment, provided the enzymes within the milk are not denatured. Additionally, in developing this scientific study, it is evidenced in the research work of Bezie (1), that when high temperatures are applied within a very short period of time to heating of milk products (pasteurization conditions), both the nutritional and functional properties of the milk will not be affected in any way. Instead, such treatments would increase the rate of interactions between different molecules within the milk, and hence it would be important to see how increasing boiling times would affect the milk concentration.
Hypothesis
It is hypothesized that the protein concentration of the milk will increase with an increase in boiling times. This prediction is in line with Martins et al. (67) research, supposing that if milk products are to be heated in a microwave, the waves tend to bring conversions in the compositions of proteins. This research also shows the reason why proteins happen to be very valuable in milk products. Ideally, the composition of proteins within milk is 80% casein, which is very susceptible to temperature changes, as long as the enzymes present are not denatured.
Research Variables
Independent Variable (IV)
The IV in this investigation was the boiling times which were to be varied from 20 sec to 30 sec, to 35 sec, 40 sec, and finally, 45 sec, with the aid of a stopwatch ((+/- 0.05 sec) to check corresponding changes in the protein concentration of milk.
Dependent Variable (DV)
The DV in this investigation was the corresponding changes in the protein concentration of milk.
In the variation of boiling times which were to be varied from 20 sec to 30 sec, to 35 sec, 40 sec, and finally 45 sec.
Control Variables (CV)
The main control variables in this investigation were the heating temperatures and volume of the milk. The heating temperature was controlled at 350C across all the trials using the mercury thermometer (+/- 0.010C) to ensure consistency of the results. On the other hand, the volume of pasteurized milk was maintained at 5 mL across all the trials, with aid of the graduated measuring cylinder (+/- 0.01mL) to ensure that the only dependent variable was concentration of protein across all the trials.
Development of Method, and Planning
This experimental investigation was planned to evaluate the effect of boiling times on the concentration of the proteins found in milk. The BCA assay technique was preferred in carrying in this investigation owing to its high level of accuracy. BCA in itself comprises a compatible detergent formulated on bicinchoninic acid (BCA) to detect calorimetric and process quantification of proteins in a substance (Reichelt et al. 1272). This technique makes it possible to reduce copper (II) ions into copper (I) ions within an alkaline medium. This technique is therefore based purely on the interactions of copper-based proteins. The reagent of bicinchoninic acid will then complex in a ratio of 2:1 with cuprous ions, forming a purple compound that can be easily spectrophotometrically quantified at wavelengths of 562 nm.
Methodology
Materials and Equipment
The main materials and equipment used in this investigation were:
- 100 mL of Pasteurized milk
- A graduated measuring cylinder (+/- 0.01mL)
- A mercury thermometer (+/- 0.010C)
- A stopwatch (+/- 0.05 sec)
- BioTexk Power XS
- 5 beakers
- Microwave oven
- 1050 mL of distilled water
- Reagents for preparing reactive A, and B
- 20 g of hydrated sodium carbonate
- 4 grams of sodium hydroxide
- Sodium tartrate
- 10 grams of BCA
- 2 grams of copper sulfate
- 10 grams of sodium hydrogen carbonate
- A stirring rod
Procedure
The procedure followed in carrying out the experimental investigation was comprised of the following steps:
- Both the reactive A and B were first prepared. This preparation was performed differently for the two reaction solutions:
- For reactive solution A: The 10 grams of BCA were mixed with the 20 grams of hydrated sodium carbonate, sodium tartrate, followed by the addition of sodium hydroxide solution, then sodium hydrogen carbonate. The 1000 ml distilled water was then added to complete the process of mixing.
- For reaction solution B: The copper sulfate solid was dissolved in 50 mL distilled water.
- The reactive standard was prepared by mixing 50 ml of A, and 10 Ml of B, with the aid of the graduated measuring cylinder in a beaker.
- Thereafter, 5mL of milk that had been pasteurized would be measured, using the graduated measuring cylinder, at different wavelengths of light; 90W then 180W, 300W then at 600W, and finally 900W, for five different trials.
- The next step was to heat at controlled temperatures of 350C in a microwave oven a measured volume of 0.1 mL of milk 20 seconds (for the first trial), then mixing it with a standardized reactive solution mixture.
- Step 4 was repeated for 25 different trials, five trials at 30 seconds, five at 35 seconds, five at 40 seconds, and five at 45 seconds.
- Absorbance measurement would then be performed at 562 nm in each of the trails.
- A standard curve was then plotted for each blank obtained at 562 nm absorbance measurements under BCA standards against the concentration.
- From the curve plotted, the concentration of protein in each sample would be obtained, corresponding to the boiling times and the data recorded in the processed data table.
- The solutions containing milk and the reactive solutions were allowed to cool for some time.
Safety Precautions
The main safety measure observed while carrying out this investigation was in the heating. A significant distance was maintained from the heating chemicals and handled while wearing gloves to avoid accidental burns. Additionally, considering that the experiment was being performed using a lot of glassware apparatus, safety precaution measures were taken while handling them to prevent breakages and accidental cuts.
Results
Figure 1: Calorimetric Reactions as BCA functions of Protein Values
| Name of the Curve | The formula of the Curve | A | B | R2 |
| Curve | Y=AX+B | 0.001 | 0.29 | 0.967 |
The curve plotted in figure 1 above was generated by graphing average 562 nm correlated blank measurements for the different standard concentrations of BCA, imploring Bio Tek Power XS. The importance of this curve was to specify the concentration of protein in the Casein protein/ pasteurized milk after extraction. The resulting concentration against boiling times was recorded.
Table 1: Raw Data Table
| Boiling time, t | Protein Concentration | ||||
| Trial 1 | Trial 2 | Trial 3 | Trial 4 | Trial 5 | |
| 20 | 476.412 | 474.318 | 474.216 | 475.258 | 473.023 |
| 30 | 567.089 | 565.236 | 564.216 | 563.224 | 564.468 |
| 35 | 587.118 | 585.203 | 584.126 | 586.215 | 585.620 |
| 40 | 747.782 | 746.224 | 746.125 | 747.824 | 747.129 |
| 45 | 1199.136 | 1198.124 | 1196.201 | 1199.204 | 1198.126 |
Data Analysis and Sample Calculation
The first sample calculation to be performed was for the uncertainty of the milk protein concentration. This computation was important for determining both the accuracy and reliability of the method used in collecting scientific data. The equation used to compute this uncertainty was such that;
Thus, considering the concentrations values corresponding to 20 seconds;
The second sample calculation to be performed in this investigation was on the average milk protein concentration. This computation was also very necessary in establishing the relationship existing between the boiling times and the concentration. The equation used to find this average was such that;
Thus, considering the concentrations values corresponding to 20 seconds;
The same analytical computations were extended to all the other protein concentrations corresponding to the different boiling times, recording the results obtained in the processed data table below.
Table 2: Processed Data
| Boiling time, t | ( | ( |
| 20 | 474.6454 | 1.6945 |
| 30 | 564.8466 | 1.9325 |
| 35 | 585.6564 | 1.496 |
| 40 | 747.0168 | 0.8495 |
| 45 | 1198.158 | 1.5015 |
The data presented in Table 2 above was then used to generate a graph of concentration against time to obtain the relationship between the two variables. This graphing was performed in Microsoft Excel application, then importing the graph as in figure 2 below.
Figure 2: Protein Concentration in Milk against Boiling Time, Using BCA Assay and Calorimetric
The trend line in the graphical plot above indicated that there was a direct relationship between the two variables. Ideally, the protein concentration of the milk increased with an increase in boiling times, as evidenced by a positive gradient (25.252). The relationship was not only found to be direct but it was found to be very strong, evidenced by a very high regression constant of, a value very close to 1. Additionally, the error bars on the graph were very small and non-overlapping, indicating that the data sets used to generate the graphical plot, so the methodology was reliable and accurate.
Conclusion
The main aim of this scientific investigation was to answer the research question, “Does boiling time affect the protein concentration in milk, measured using bicinchoninic acid assay and calorimetric?” It had been hypothesized that the protein concentration of the milk would increase with an increase in boiling times. The strategy adopted to answer the research question was going by the results obtained in this investigation. After carrying out a thorough experimental-based investigation, there was a direct relationship between the two research variables. In essence, the protein concentration of the milk was found to be with an increase in boiling times, a relationship that was confirmed by a positive gradient between the two variables. Thus, the research question was fully answered, and the hypothesis was confirmed. Despite the fact that the methodology used to carry this investigation ought to be improved to have more accuracy, there was enough persuasion in the results to show that boiling points directly affect the concentration of milk products.
Evaluation
The level of uncertainty in the data sets was very low, less than 2.0. Similarly, the error bars on the graph were very small and non-overlapping, indicative that the data sets used to generate the graphical plot, and hence the whole methodology was reliable and accurate. Additionally, there high regression constant, , a value very close to 1, indicating the level of correlation between the two variables in this investigation was very strong. All these indicators showed the success of this investigation. However, the level of uncertainty observed in the results would be reduced even further by using more advanced measuring instruments, such as digitalized timer, when controlling time measurements, for more improved results of milk protein concentration.
Works Cited
Bezie, Assefa. “The effect of different heat treatment on the nutritional value of milk and milk products and shelf-life of milk products. A Review.” Journal of Dairy and Veterinary Sciences 11.5 (2019): 1-8.
Bhat, Mohd Younus, Tanveer Ali Dar, and Laishram Rajendrakumar Singh. “Casein proteins: structural and functional aspects.” Milk proteins–from structure to biological properties and health aspects. InTech, Rijeka (2016): 1-17.
Martins, Carolina PC, et al. “Microwave processing: current background and effects on the physicochemical and microbiological aspects of dairy products.” Comprehensive Reviews in Food Science and Food Safety 18.1 (2019): 67-83.
Reichelt, Wieland N., et al. “Bioprocess monitoring: minimizing sample matrix effects for total protein quantification with bicinchoninic acid assay.” Journal of Industrial Microbiology and Biotechnology 43.9 (2016): 1271-1280.