Protein Lab report 1

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Feb 20, 2024

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Protein Experiment 1- Soy proteins and protein solubility Food 3030-Lab 4 Sihe (Clarice) Gao 1159333 University of Guelph Lab Section 2 November 9, 2023 Lab Partner: Zoe Wu 1

Abstract This experiment explores the effect of pH on the solubility of soy protein and the application of soy protein solubility. The relationship between absorbance and different protein concentration and solution pH, isoelectric point of the soybean protein extract was determined by the Bradford colorimetric assay. Also, the bean curd is made by using a coagulant solution containing divalent ions to obtain protein precipitates. The results demonstrated that the solubility of soybean protein in solution was affected by the pH of the solution, when the pH at 7.0 which equals to the isoelectric point of the protein extract, the solubility of the protein extract would be the lowest. The protein concentration of the soybean extract at pH 2 and 10 were the two of the highest point in the whole range of pH. The formation of soy protein precipitates was affected by the overheating of the soymilk and the addition of calcium sulfate when it was not completely cooled. The final bean curd product did not reach the expected solid state, with very little solid content and an overall beige-colored, thick liquid. Thus, precipitate formation of soy protein is affected not only by pH but also by temperature. Introduction All protein-related products are associated with the utilization of their solubility. It was found that the solubility of proteins is related to the pH value of the solution in which they are dissolved. For example, cheese is made by acidifying milk and then gathering the precipitates with rennet (a coagulant), and the same principle applies to tofu (O′Flynn et al., 2021). The solubility of protein means how well the molecule can dissolved in the solution based on the thermodynamically. The structure of protein molecule is one of the intrinsic factors to affect the solubility. In general, most of the protein can dissolved naturally in water due to 2

their folded state which is hydrophilic (Marangoni, 2021). When the inner hydrophobic structure is exposed to water, the solubility of the protein is reduced, and it becomes as insoluble as oil. In addition to the hydrophobic and hydrophilic structure of the molecule, the net charge of the protein surface affects the interactions between the molecules. Some proteins interact with each other, resulting in low solubility, but protein rejection results in good solubility in the solvent. This interaction is caused by the net charge (positive or negative) on the surface of its molecules (Halligan, 2009) . In an extremely acidic or alkaline state, the solvent gives the protein a positive or negative charge, causing the molecules to reject each other. When the surface charge is zero (neutral), the lack of electrostatic repulsion between molecules makes them more tend to interact with each other towards similar proteins (Halligan, 2009) . there are many ways to denature the characteristic of protein, either by changing pH, heating or adjust the molecule surface charge. The subject of Exp.1.1 is using the Bradford colorimetric assay to determine the absorbance of BSA solution at different concentration, its standard curve is used to derive the equation for finding the concentration of an unknown protein. The Bradford colorimetric assay is a rapid and accurate method to determine the protein concentration. Exp#1.2 using the defatted soy protein flour to obtain the soy protein extract and determine the unknown concentration by the absorbance at each pH points. Which is to study the effects on solubility of soy protein by pH changing. The last Exp #1.3 is the application of protein solubility by adding the coagulant solution to obtain the solid bean curd. The hypothesis for this experiment is whether there is a significant change in the concentration or solubility of proteins at different pH values. The solubility of proteins is greatest at extremely acidic and alkaline pH values and is lower in the neutral state. The relationship between isoelectric point and pH and protein solubility is also being investigated. 3

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Materials & Methods All the materials and procedures were followed as per lab manual Proteins Experiment #1-Soy Proteins and Protein Solubility (Marangoni, 2021). 1. Materials 4 different concentration level of the BSA Reference solution (0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1.0 mg/mL), low-fat or defatted soy protein flour, 0.1N HCl&0,1N NaOH, pH meters, centrifuge; 1:4 diluted Reconstituted Bio0-Rad Reagent, micropipette, spectrophotometer(595nm) and disposable cuvettes. Raw soybean soaked overnight at the room temperature, Calcium sulfate (5%w/v, CaSO 4 ), blender, kitchenware (saucepans, basins, beaker) and filtration apparatus (cheese cloths and Whatman No.1 paper). 2. Methods Exp1.1 Used 100uL each of four-concentration level of BSA Reference solution (0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1.0 mg/mL) mixing with 900uL 1:4 Bradford reagent, dilute the BSA standards into 1:10. Transfer 1 mL from each concertation sample into a disposable cuvette and let it sit at least 20 minutes at room temperature. Put the 4 cuvettes with samples into the spectrophotometer and 1 blank to read the absorbance at 595 nm. Exp 1.2 Protein extraction made by adding 10g of soy flour to 100mL of 0.1 N NaOH and 100mL of water, then form up a 1:20 mixture of soy flour and alkali with mixing vigorously 15 minutes. Then put the 1:20 mixture into the centrifuge with 15-20 minute at 10,000rpm until the sediment settles. Decant the supernatant about from the sample after centrifuged, and transfer 20mL of the 4

liquid portion into the 50mL beaker. Take 100uL of the clear extract mix with 900uL of the 1:4 Bradford reagent, dilute the sample into 1:10 as Exp 1., then transfer 1mL sample into a disposable cuvette, then read the absorbance at 595nm. Then using).1 NaOH and 0.1 HCl to adjust the soy protein extract at each of the pH level from pH 2 to pH 10, mix well for 3-5 minutes at room temperature. After that measure the volume of each pH-adjusted extract, add some deionized water to increase total volume of each to 40 mL if their volume does not reach to 40 mL. Then centrifuge the mixture as previously. In the end, use the supernatant to measure the protein concentration as in Exp1.1. Exp 1.3 Grinding the overnight-soaked soybeans(150g) with water(350mL) by the blender at highest speed. Boil the mush in a pot for 5 minutes, after that filter the soy milk by a cheese cloth. Let the soy milk cool down just below boiling then mix with the 10:1 coagulant solution (CaSO 4 ) in a voluminous container. Then let sit for 15 minute to cool and coagulate. After that let the tofu stand overnight in the fridge to wait the curds form up, then press the curds by any means available to squeeze out the whey and to compact the coagulate. Results Exp 1.1 Table 1. Absorbance at 595nm of four level BSA concentration (0.025mg/mL, 0.5mg/mL, 0.75mg/mL, and 0.1mg/mL) Concentration (mg/mL) Absorbance (AU) Absorbance of blank (AU) 0.386 0.025 0.126 0.5 0.141 0.75 0.238 0.1 0.233 5

Figure 1. Standard curve with absorbance (AU) on the x-axis of four level BSA concentration (0.025mg/mL, 0.05mg/mL, 0.075mg/mL, and 0.1mg/mL) on the y-axis Exp 1.2 1. Using the standard curve from exp 1.1 is y=0.350x to calculate the protein concentration of soy extract with 1:10 dilution at each pH; y-axis is the concertation of BSA, x-axis is the absorbance of BSA at different concentration. Table 2. The corrected absorbance (AU) at 595nm and calculated protein concertation(mg/mL) of soy extract with 1:10 dilution at each pH Sample pH Dilution Corrected Absorbance (AU) Protein concentration(mg/mL) Absorbance of blank (AU) 0.392 2 1:10 0.297 0.10398 3 0.290 0.101529 6 0 0.05 0.1 0.15 0.2 0.25 0 0.02 0.04 0.06 0.08 0.1 0.12 f(x) = 0.35 x R² = 0.96 Absobance at 595nm(AU) BSA Conc.(mg/mL)

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4 0.182 0.063718 5 0.201 0.0703 6 0.186 0.0651 7 0.180 0.0630 8 0.232 0.0812 9 0.212 0.0742 10 0.303 0.1061 11 0.198 0.0693 12 0.211 0.0738 the sample calculation of pH at 6: y = 0.350 x y = 0.350 ∗ ( 0.186 ) = 0.0651 mg / mL 2. Figure 2. Calculated 1:10 soy extract protein concentration(mg/mL) at each pH 7 0 2 4 6 8 10 12 14 0 0.02 0.04 0.06 0.08 0.1 0.12 pH Protein Concetration(mg/mL)

Exp 1.3 Figure 3. The tofu placed in the fridge overnight The tofu mixed by soymilk and coagulant solution then placed in the fridge overnight. The products didn’t coagulate completely, it’s a thick liquid. The whole product is beige in color, with a layer of dark yellow solid crust on the surface. Discussion Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein solution. The “Linear Range” in this assay means that the ratio of absorbance measurement at 590nm is strictly linear with protein concentration (Ernst & Zor, 8

2010). This “Linear Range” reduce the limitation when unknown sample protein concentration does not fall within the limits range in the calibration graph. Which is simplifies the assay since this range does not require the protein concentration must fall in the limits range, at the same time increased the accuracy of the measurement (Ernst & Zor, 2010). However, the absorbance of BSA (Table 1.) at the 0.1 mg/mL concentration is lower than the one at 0.075 mg/mL. also, there were two lowest protein concentration in the curve (Figure 2), it might be the experimental error due to the standard curve equation of the BSA absorbance. The isoelectric point of the protein is equal to pH value at which the total charge on the protein is zero , therefore the protein at its isoelectric point is usually the least soluble (Halligan, 2009) . Poor solubility along with the low concertation of the solution. According to the protein concentration curve (Figure 2.), the concentration at pH 7 is the lowest. So, the isoelectric point is 7 of the protein isolated from soy flour by deduced roughly. Solubility of SPI is affected by the temperature and pH value, low solubility means all the protein molecules are congregated in the solution. So, for assuming the optimal pH to isolate soy protein should locate the pH with the low protein concentration. According to the protein concentration curve, the optimal pH would be 4 or 7, since this two pH have a low protein solubility. Also, there is research stated the solubility of SPI would be the lowest when pH(p I ) at 4.6 (O′Flynn et al., 2021). Compared to the pH stated by literature, the experiment result (Table 2 and Figure 1) of normal soy protein extracted from soy flour has two pH point at the low protein concertation/solubility, it may be due to their protein structure differences (O′Flynn et al., 2021). In the literature also stated the optimal pH for good solubility of SPI would be at 2.0 and 9.0, the experiment shows that at these two-pH point, the protein concentration is the two of the highest. 9

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In the experiment 1.3, the hypothesis product of tofu-making should be a curd (“solid soy milk”). The soy milk has boiled for over 5 minutes and did not cool down the soy milk completely then to adding the Calcium sulfate, it might be the reason of the final product failure. Because temperature is important to determine the texture of the bean curd. The tofu texture would firm and rough if it has only boiled 5 minutes at 95 o C, the texture of tofu would become more and more soft along with increasing the boiled time (Rekha & Vijayalakshmi, 2011). Since the solid content of tofu is affected by the boiled temperature and duration. The divalent cations are the key fact to provide insoluble precipitates of making bean curd, since the interaction of those cations in coagulant solution with the surface’s carboxyl groups of soy protein, which would cause the salting out then form the insoluble precipitates (Arii & Nishizawa, 2018) . So, the soymilk may still be able to congregate without boiling but only adding the coagulant solution. Conclusion The solubility of proteins is affected not only by pH but also by temperature. The solubility of soy proteins is more favorable at extremely acidic or alkaline pH levels because the electrostatic charge of the protein is not zero in either state. The molecules resist each other and do not aggregate and are well dissolved in the solution. On the other hand, the solubility of soy protein is lowest when the net charge is zero/neutral which is the p I value is equal to the pH value at around 7. According to the literature, the lowest solubility of isolated soy protein is at pH 6.9 (O′Flynn et al., 2021), but the experimental results show that there is lower solubility at both pH 4 and pH 7. Therefore, low solubility can help the formation of tofu. This test deviation is due to the possible use of the equation derived from the BSA standard curve. It was unable to find a reason for the 0.1 mg/mL BSA concentration to have an absorbance lower than 0.075 10

mg/mL. At the same time, the condition and texture of finished beans curd did not meet the expected hypothesis. Failure of the experimental results may be caused by the uncritical preparation of soymilk boiling. Reference Arii, Y., & Nishizawa, K. (2018). Divalent magnesium cation concentrations determine the formation of tofu-like precipitates with differing urea solubilities. Heliyon , 4 (9). Ernst, O., & Zor, T. (2010). Linearization of the Bradford Protein Assay. Journal of Visualized Experiments , (38). Halligan, B. D. (2009). ProMoST: A tool for calculating the pi and molecular mass of phosphorylated and modified proteins on two-dimensional gels. Phospho-Proteomics , 283–298. O′Flynn, T. D., Hogan, S. A., Daly, D. F., O′Mahony, J. A., & McCarthy, N. A. (2021). Rheological and solubility properties of soy protein isolate. Molecules , 26 (10), 3015. Rekha, C. R., & Vijayalakshmi, G. (2011). Influence of processing parameters on the quality of soycurd (tofu). Journal of Food Science and Technology , 50 (1), 176–180. Marangoni, A.G. (2021). Food chemistry (FOOD*3030) laboratory manual. Guelph, Ontario: University of Guelph. 11