Differences in the sugars, protein content, amino acid and lipid content among soymilk, 2% cow’s milk, 2% chocolate cow’s milk, and rice milk

By Claire Armstrong, Julia Richardson, and Ashleigh Tazzioli

Abstract

The macromolecule composition of 2% white milk, 2% chocolate milk, soymilk, and rice milk was investigated in this experiment. The sources of the various milks are very different (plant and animal), so it is expected that the composition will differ accordingly. It was hypothesized that the soymilk would be more similar in macromolecule content to the cow’s milk’s than the rice milk. It was expected that the cow’s milks have more protein and lipids than the rice milk and soymilk, but similar kinds if sugars and amino acids. The soymilk would have almost as much protein as the cow’s milks. To determine the composition differences certain tests were performed: Barfoed’s test, Selivanoff’s Test, the Bradford Assay, Thin Layer Chromatography for amino acids, and the Lipid test. Through the completion of Barfoed’s test, it was found that all four milks contain polysaccharides. Using Selivanoff’s test, we found that 2% white milk contained aldoses, while 2% chocolate milk, soymilk, and rice milk all contained ketoses. The cow milks and soymilk all contained about the same amount of protein, whereas rice milk contained less than half of the amount of protein as the others. The 2% white milk contained 2.680 mg protein/mL milk, the 2% chocolate milk contained 2.630mg/mL, the soymilk contained 2.720mg/mL and rice milk contained 0.943mg/mL. All the milks tested contained polysaccharides and all except the 2% white milk contained ketoses. For the TLC test it was predicted that the 2% white, 2% chocolate and soymilks would have similar amino acids. Even though the test was inconclusive it showed that soymilk, rice milk and 2% white milk had three amino acids in common. For the Lipid test, it was predicted that both of the 2% milks would contain lipids, while rice milk and soymilk would not. The Lipid test showed that they all contained lipids.

Discussion

The nutritional differences between the milks are related to the diversity of their sources, plant and animal. Previous studies have found that soymilk is an adequate cow’s milks substitute because it has a similar amount of proteins and similar sugars. Also soy protein has been shown to have certain health benefits. For children and infants who are allergic to cow’s milk or their mother’s milk often soy is an acceptable alternative; this is very important to their health. The protein content was expected to be similar among the 2% milks and the soymilk; the rice milk was expected to be less. It was expected that all the milks would have similar sugar content. It was expected that the 2% milks and the soymilk would have similar amino acids than the rice milk. The lipid test would show that the 2% milks have similar lipid content and that soymilk and rice milk would have less.

The Bradford Assay showed that the two percent milks and soymilk all contained about the same amount of protein, whereas rice milk contained less than half of the amount of protein compared to the others. The two percent white milk contained 2.680 mg protein/mL milk, the two percent chocolate milk contained 2.630mg/mL, the soymilk contained 2.720mg/mL and rice milk contained 0.943mg/mL. Using Barfoed’s test, it was found that all four milks contained polysaccharides. From Selivanoff’s test, it was found that two percent white milk contained aldoses, while two percent chocolate milk, soymilk, and rice milk all contained ketoses. The TLC test was inconclusive except that it showed that the soymilk, 2% milk, and rice milk had three amino acids in common: Pro, Val, and Try. The chocolate milk sample did not produce good data. The Lipid test showed that all the samples contained lipid.

The results supported the hypotheses for Bradford Assay and Barfoed’s test. The Bradford test found that, consistent with the hypothesis, the 2% white, 2% chocolate, and soymilk all had the same amount of protein while rice milk had less. Barfoed’s test showed that all the samples contained polysaccharides, supporting our hypothesis. However for Selivanoff’s test the 2% white milk contradicted the hypothesis because it showed the presence of aldoses while the other milks demonstrated the presence of ketoses. This refuted the hypothesis that all the samples would contain polysaccharides. The results for the TLC test were inconclusive and did not provide enough data to support to either the hypothesis or our null hypothesis. The Lipid test showed the presence of lipid in all the samples. This refuted the hypothesis that only the 2% white and chocolate milks would contain a detectable amount of lipid.

The two carbohydrate tests used, Barfoed’s test and Selivanoff’s test, distinguished between polysaccharides and monosaccharides, and aldoses and ketoses respectively. These tests were clear in their results and were simple to execute. The results supported the hypotheses for Barfoed’s test. Selivanoff’s test also supported the hypothesis except for the 2% white milk. This could be because the white milk is the only milk without added sugars (Giampietro, 1998). Previous research has found that with added sugars (commercial) soymilk and cow’s milk do not differ greatly in carbohydrate content (Giampietro, 1998). Also it was found that milks such as rice milk that are high in carbohydrate content but low in protein content are not good substitutes for regular milk (Giampietro, 1998). Soymilk was historically used as a good nutritional source of carbohydrates and protein for peasants. It contains a balance of carbohydrates and protein similar to milk (Anonymous, Unknown).

The Bradford assay was used to determine the amount of protein in each sample. The results concluded that 2% white, 2% chocolate, and soymilk all had approximately the same amount of protein while rice milk had half that amount. The research showed soymilk and cow’s milk have a similar amount of protein, and that hydrogenised formula and other milk substitutes have significantly less protein (Giampietro, 1998). It is for this reason that soymilk makes a good substitute for cow’s milk (Giampietro, 1998). The data obtained was consistent with the research.

The results for the TLC amino acid test were inconclusive because the amino acid fronts were hard to discern and for the chocolate milk sample only one amino acid front developed. The first time the experiment was run the milks were diluted to a 1/40 ratio in order to achieve a transmittance above 10%. However it was apparent that the dilution was too high because none of the amino acid fronts developed and the strips were blank. The second time the experiment was performed both a 1/20 and a 1/40 dilution was used for each sample. The 1/20 dilution strips developed faint amino acid fronts while the 1/40 samples did not. The amino acid fronts on the 1/20 strips were then measured and the Rf values were calculated. When the experimental Rf values were compared to the known Rf values to determine which amino acids were detected many of the experimental Rf values did not correlated with a known Rf value. Previous research found that the protein and carbohydrate amount is similar between soymilk and cow’s milk, but that the proteins supplied are different (Giampietro, 1998). Thus the amino acids are similar in amount but because they are from different sources their amino acids will be different (Giampietro, 1998). The data obtained was inconclusive and did not show this to be true.

The lipid test showed that all the samples contained lipids. It was expected that only the cow’s milk samples would test positive for lipids because the amounts contained in the soymilk and rice milk were expected to be trace amounts. The research indicated that soymilk and cow’s milk contain different lipids and thus have different affects on the body (Bertolami, 2004).

Each test performed had some degree of human error affecting the results. For the carbohydrate tests there were minimal problems and the results were reliable. However it is possible that there were pipetting errors. In the Bradford assay there could have been measurement errors or errors with the spectrophotometer. In addition there could have been problems with timing because of the sensitive nature of the test. The TLC amino acid test presented many problems because the dilution to get the samples, especially the chocolate milk, over 10% transmittance was too high and the strips did not develop any amino acid fronts. When the experiment was repeated a lower dilution was used and some data was obtained but only one amino acid front was found on the strip for chocolate milk. Furthermore it was difficult to see the existing amino acid fronts.

If this research were to be continued in the future, the different tests would be run with more accuracy and precision. The TLC amino acid test would be repeated to obtain more conclusive results. It would also be interesting to test the amount of lipids in each sample rather than just testing for the presence of lipids. In addition it would be interesting to test pH and calcium content.

Figures

Figure 1.1. The four milks after the completion of Barfoed’s test. Barfoed’s solution was added to all of the samples of milk, and they were then heated and observed. No color change was noted, meaning that the milks contain no monosaccharides.

2% White Milk (A 1:3), 2% Chocolate Milk (B 1:3), Soy Milk (C 1:3), Rice Milk (D 1:3)

Figure 1.2. The controls after the completion of Barfoed’s test. These samples were prepared and tested the same way as the milk samples. Glucose, the positive control, underwent a color change, meaning that it is a monosaccharide. Sucrose and the mock, dd H₂O, did not change color, indicating either polysaccharides (sucrose) or no sugars (water).

Glucose (E 1:3), Sucrose (F 1:3), Mock ddH₂O (G 1:3)

Figure 2.1. The four milks after the completion of Selivanoff’s test. For the test, Selivanoff’s solution was added to the milk samples and the tubes were then heated and observed. The 2% white milk had a weak color change, indicating that it contains a ketose. The other four milks all tested positively for aldoses.

2% White Milk (A 1:3), 2% Chocolate Milk (B1:3), Soy Milk (C 1:3), Rice Milk (D 1:3)

Figure 2.2. The controls after the completion of Selivanoff’s test. These were prepared in the same way that the milk samples were. Glucose, the negative control, demonstrated weak color change, confirming that it is a ketose. Fructose, the positive control, turned dark red, indicating that it is an aldose. The mock, ddH₂O, did not change color at all due to its lack of carbohydrates.

Glucose (E 1:3), Fructose (F 1:3), Mock ddH₂O (G 1:3)

Figure 3.1. The four milks after the completion of the Lipid test. For the test, Oil Red-O was added drop-wise until a color change was apparent. The 2% White Milk, 2% Chocolate Milk, Rice Milk and Soy milk all had apparent color change, confirming that all samples contained lipids.

2% White Milk (A 1:3), 2% Chocolate Milk (B1:3), Rice Milk (C 1:3), Soy Milk (D 1:3)

Figure 3.2. The controls after the completion of the Lipid test. These were prepared in the same way that the milk samples were. Fructose, the negative control, demonstrated no color change, confirming that it did not contain lipids. Cream, the positive control, demonstrated a color change, indicating that it does contain lipids. The mock, ddH₂O, did not change color at all due its lack of lipids.

Fructose (E 1:3), Cream (F1:3), Mock ddH₂O (G 1:3)