FOOD CHEMISTRY EXPERIMENT BOOK
Unit 3. PROTEINS
Teacher Activity Guide
Student Activity Guide
Figure 1
Activity/Experiment
Powerful Proteins
Puzzling Proteins
Unit 3. PROTEINS
Teacher Activity Guide
Expected Outcomes
Students will learn about the sources of proteins and their uses in the food industry.
Activity Objective
In Part 1, the students will precipitate casein from milk using an acid. This method is used to make cottage cheese. In Part 2, the students will coagulate casein from milk using an enzyme. This method is used for making cheese. In Part 3, the student will coagulate soy protein from soymilk, using magnesium sulfate. This method is used to make tofu.
Activity Length
45 minutes
Scientific Principles
Milk protein consists of 80% casein and 20% whey protein. There are four major types of casein molecules: alpha-s1, alpha-s2, beta, and kappa. Milk, in its natural state, is negatively charged. The negative charge permits the dispersion of casein in the milk. When an acid is added to milk, the H + concentration neutralizes the negatively charged casein micelles. When milk is acidified to pH 4.7, the isoelectric point (the point at which all charges are neutral) of casein, an isoelectric precipitate known as acid casein is formed. Cottage cheese and cream cheese manufacture involves an acid precipitation of casein with lactic acid or lactic acid producing microorganisms. Acid casein is used in the chemical industry and as a glazing additive in paper manufacturing.
Casein also can be coagulated with the enzyme rennin, which is found in rennet (an extract from the stomach of calves). Rennin works best at body temperature (37°C). If the milk is too cold, the reaction is very slow, and if the milk is too hot, the heat will denature the rennin, rendering it inactive. The mechanism for the coagulation of the casein by the rennin is different from the acid precipitation of casein. The coagulation of the casein by rennin is a two-stage process. In the first stage, rennin (a proteolytic enzyme) splits a specific bond in the amino acid chain of the kappa-casein macromolecule converting it into a para-kappa-casein and a glyco-macropeptide. This causes an imbalance in the intermolecular forces in the milk system, and the hydrophilic (water-loving) macropeptides are released into the whey. Unlike kappa-casein, the para-kappa- casein does not have the ability to stabilize the micellular structure to prevent the calcium-insoluble caseins from coagulation. In the second stage, colloidal calcium phosphate bridges within the casein micellular structure are formed in the presence of the soluble calcium, resulting in the three-dimensional curd structure. The rennin coagulum consists of casein, whey protein, fat, lactose, and the minerals of the milk, and has a fluffier and spongier texture than the acid precipitate. Rennet is used in the manufacture of cheese and cheese products, and rennet casein is used in the plastics industry.
Casein is solubilized with sodium hydroxide and calcium hydroxide to produce sodium caseinate and calcium caseinate, respectively. Caseinates are added to food products to increase their protein content and are key ingredients in non-dairy coffee creamers and Cool Whip ® .
Approximately 90% of soybean proteins are classified as globulins, based on their solubility in salts. More specifically, the proteins are conglycinin (a glycoprotein) and glycinin. Tofu is manufactured by coagulating proteins in soymilk with magnesium sulfate. As bonding occurs between the positively charged magnesium ions and negatively charged anionic groups of the protein molecules, the proteins coagulate.
Vocabulary
Amino acids contain carbon, hydrogen, nitrogen, and sometimes sulfur and serve as the monomers to make peptides and proteins. Amino acids have a basic structure that includes an amino group (NH2) and a carboxyl group (COOH) attached to a carbon atom. This carbon atom also has a side chain (an “R” group). There are twenty amino acids, found in the body. Eight of them are essential for adults and children, and nine are essential for infants.
Casein is a milk protein. There are four major types of casein molecules: alpha-s1, alpha-s2, beta, and kappa.
Coagulation is the transformation of a liquid into a soft semisolid or solid mass. In the coagulation of milk, it refers to the aggregation or clumping together of proteins.
Colloid is a suspension of finely divided particles in a continuous medium in which the particles do not settle out of the substance rapidly, and are not readily filtered.
Denatured means changed from its natural state. In a denatured protein, its characteristics or properties have been altered in some way, by heat, chemicals, or enzymatic action, resulting in the loss of its biological activity.
Digestion is the chemical breakdown of large food compounds into smaller molecules that can be absorbed by the intestines in the human and animals. The smaller food molecules travel in the blood and are used by cells to make other components or produce energy needed by the body. Digestion begins in the mouth as salivary amylase begins to break down starch into simple sugars.
Enzymes are protein catalysts, which control specific chemical reactions in living systems (plants and animals). Enzymes are active at low concentrations and are substrate specific. The enzyme rennin catalyzes the coagulation of casein in milk, but is not effective in any other chemical reaction.
Isoelectric means having equal electric potential.
Kappa-casein is one of the four major types of casein molecules. Kappa-casein self-associates into aggregates called micelles. The alpha- and beta-caseins are kept from precipitating by their interactions with kappa-casein.
Micelle is a submicroscopic aggregation of molecules, as a droplet in a colloidal system.
Peptide bonds are covalent bonds between two amino acid molecules.
Precipitation is the removal of insoluble material from solution.
Proteins are complex polymers composed of amino acid monomers, and are considered to be the primary structure of all living organisms. Some examples of protein are muscle, hair, skin, hormones, and enzymes.
Proteolysis is the hydrolysis of proteins into peptides and amino acids by cleavage of their peptide bonds. This occurs during digestion and when rennin is used to coagulate milk.
Rennet is an extract from the inner lining (membrane) of the fourth stomach (abomasum) of the calf. The abomasum is the gastric stomach of ruminant animals such as the cow. The lining is used to make cheese because it contains the enzyme rennin.
Rennin is a proteolytic enzyme that is used to coagulate milk to make cheese. Rennin is typically used in the form of rennet, a commercial preparation taken from the abomasum (fourth stomach) of young calves, but because its demand is great and supply limited, the cheese industry has been increasingly turning to microbial rennin produced from genetically engineered microorganisms. Rennin is also known as chymosin.
Substrate is the name of a reactant molecule for enzymes. A substrate is the substance on which an enzyme acts. Using the analogy of a lock and key, the lock is the substrate, and the key is the enzyme.
Materials Required
Instructions to make homemade soymilk
Ingredients: 350 grams (2 cups) of soybeans and 2.8 liters (12 cups) of water
Method: Soak 350 grams (two cups) of dry soybeans overnight. Cover with plenty of water. The beans will swell quite a bit. On the next day, drain the water off the beans and rinse the beans with fresh water in a colander. Take ½ of the beans and put them in a blender. Add 0.70 liter (three cups) of cold water and blend on high until the beans are finely ground. Pour the bean mixture into a large pot -- a 4.5-liter (1-gallon) size is good. Blend the remaining beans with 0.70 liter of water and add them to the pot. Add 1.4 liters (six cups) more of cold water to the pot. Put the pot on the stove and bring it to a boil, stirring frequently to keep the bottom from scorching. If the mixture threatens to boil over, reduce the heat. Let the beans simmer for seven minutes, then sprinkle a small amount of cold water over the mixture until boiling stops. Let the mixture come to a simmer again. Repeat the cold water/simmer treatment two more times. Take the mixture off the heat. Pour the slurry through a sieve (lined with two to four layers of fine cheesecloth) into another pot. The strained liquid is soymilk. This procedure makes about 2.8 liters (three quarts) of soymilk. Put the soymilk into containers and let cool before refrigerating. The soymilk keeps for about one week in the refrigerator and 3 to 6 months in the freezer.
SAMPLE DATA TABLE – MILK AND SOYMILK CURDS
| |
Weight of milk/soymilk |
Weight of curd |
Describe the curd (color, texture) |
| Milk + acid |
114.4 g |
19 g |
White, fine granules |
| Milk + rennet |
114.4 g |
34.1 g |
White, fluffy, spongy, thick |
| Soymilk + Epsom salt |
123.6 g* |
29.2 g* |
Light brown, fine granules* |
*Tofu results based on the use of Original Edensoy ® organic soymilk.
The weight of beaker with milk -- weight of beaker = weight of milk
SAMPLE DATA TABLE – BIURET TEST ON FOODS
| |
Biuret test – positive or negative |
| Milk + acid precipitate |
Purple, positive |
| Milk + rennet coagulum |
Purple, positive |
| Soymilk + Epsom salt coagulum |
Purple, positive |
| Potato chip |
Blue, negative |
| Raw potato |
Blue, negative |
| Bread |
Blue, negative |
Results for variations of experiments
Part 2. Rennet at low and high temperatures
Rennet has the highest activity at body temperature (37 o C). Coagulation will occur very slowly with cold milk. High temperatures will denature the rennet, so no coagulation will occur.
Part 3. Effect of rennet on soymilk and Epsom salt on milk
No coagulation should occur when rennet is added to soymilk, because the rennet is specific for casein. No coagulation should occur when Epsom salt is added to whole milk, since magnesium sulfate does not coagulate casein.
Key Questions and Answers
1. Compare the weights of the curds from the milk (acid and rennet) with that from the soymilk.
The milk + rennet curd weighed the most; the soymilk + Epsom salt curd weighed less; and the milk + acid curd weighed the least.
2. Why did the casein that was coagulated with the rennet weigh more than the casein that was precipitated with the acid?
The rennet coagulum contains milk protein and fat, while the acid precipitate contains only casein.
3. Compare the amount of acid casein precipitated from the whole milk with the amount of soy protein coagulated from the soymilk. How do your results compare with the Nutrition Facts label for each product?
Less casein precipitated from the whole milk than soy protein precipitated from the soymilk. If you look at the Nutrition Facts label on the milk and soymilk, you will see that the milk contains 8 grams of protein per 240 milliliters, while soymilk contains 10 grams of protein per 240 milliliters. Therefore, the results for the precipitates are consistent with the labels.
4. How did the biuret test indicate the presence of proteins?
There was a color change from blue to a purple-violet in the presence of proteins. There was no color change if proteins were not present.
Web site for more information on proteins
www.talksoy.com - United Soybean Board. Contains information pertaining to soy protein, phytochemicals, isoflavones (technical information), and the effects of soy products on human health.
Student Activity Guide
Proteins
Body builders and football players eat a lot of protein (eggs, cheese, and meat) to build muscle mass. You have probably seen protein-enriched drinks and protein-enriched foods (power bars) at the supermarket.
Proteins are the most complex and important group of molecules because they possess diverse functionality to support life. Every cell that makes up plants and animals requires proteins for structure and function. Your body and plants also have enzymes. These specialized proteins catalyze chemical reactions that are necessary for metabolism and cell reproduction. Your muscles are made from a variety of proteins, and these proteins allow your muscles to contract, facilitating movement. Other types of proteins in your body are the peptide hormones; insulin and glucagon are two common examples.
Proteins are complex polymers composed of amino acids. Amino acids contain carbon, hydrogen, nitrogen, and sometimes sulfur and serve as the monomers for making peptides and proteins. Amino acids have a basic structure that includes an amino group (NH2) and a carboxyl group (COOH) attached to a carbon atom (see Figure 1A). This carbon atom also has a side chain (an “R” group). This side chain can be as simple as an -H or a -CH3, or even a benzene group.
The R groups on an amino acid are analogous to an athlete's clothing and sports equipment. By changing clothing or equipment, an athlete can become more effective as a soccer, football, or baseball player. Although this person is still an athlete, the change can make the athlete more effective in a particular activity or function. The same is true with amino acids. They are still amino acids regardless of the attached R group, but different R groups produce different functions and different properties.
Figure 1
A. Lysine is an essential amino acid (notice the blue nitrogen atom and the carboxyl group).
B. Glycylglycine is a dipeptide containing two glycine molecules connected by a peptide bond.
C. Aspartame is a dipeptide containing aspartic acid and phenylalanine.
There are twenty amino acids found in the body. Eight of these amino acids are essential for adults and children, and nine are essential for infants. Essential means that we cannot synthesize them in adequate quantities for growth and repair of our bodies, and therefore, must be included in the diet.
Amino acids are linked together by a peptide bond in which the carboxyl carbon of one amino acid forms a covalent bond with the amino nitrogen of the other amino acid (see Figure 1B). Short chains of amino acids are called peptides. Longer chains of amino acids are called polypeptides. Although the term polypeptides should include proteins, chains with less than 100 amino acid residues are considered to be polypeptides, while those with 100 or more amino acid residues are considered to be proteins.
Many of the major hormones in the body are peptides. These hormones can influence enzyme action, metabolism, and physiology. Insulin, which is given to a person with a specific type of diabetes, is an example of a peptide hormone. Certain antibiotics and a few anti-tumor agents are also peptides. The artificial sweetener aspartame (Equal ® ) is a dipeptide composed of aspartic acid and phenylalanine with a methyl group attached at the carboxyl terminal group (L-aspartyl-L-phenylalanine methyl ester) (see Figure 1C).
The sequence of amino acid residues in a polypeptide chain is critical for biological function. For example, a genetic disease (mutation of a single base pair in DNA) called sickle cell anemia is caused by the substitution of one amino acid (glutamate) with another (valine) in a structural protein called beta-globulin, which is a part of hemoglobin in red blood cells. Hence, a single structural change resulted in a dramatic alteration in physiological function. The ability of an enzyme to catalyze a particular reaction depends on its specific shape. It’s a lot like a key and lock; if the key is broken or in a different shape, it won’t open the lock. The receptor sites on cell surfaces must be in a specific shape for polypeptide hormones to interact with the cell. With twenty different amino acids and each polypeptide consisting of hundreds of amino acids, it is no wonder that proteins play such a variety of roles in the human body.
Chemistry of Proteins
The protein backbone is formed from the peptide bonds created from the amino and carboxyl groups of each monomer that repeat the pattern -N-C-C- or C-C-N-. The number and sequence of amino acids in a polypeptide chain is referred to as the primary structure of a protein. The free amino group and carboxyl group on opposite ends of a polypeptide chain allow proteins to act as pH buffers (resist changes in pH) inside the cell. The amino group (NH2) accepts a proton and becomes (NH3+ ), and the carboxyl group (COOH) donates a proton and becomes dissociated (COO-).
As noted previously, each amino acid residue in the polymer may have a different side chain or chemical group attached to it, such as hydroxyl (OH), amino (NH2), aromatic ring (conjugate rings such as the phenol ring in phenylalanine), sulfhydryl (SH), carboxyl (COOH), or various alkyl (CHn). This variety of side chain groups on the polymer backbone gives proteins remarkable chemical and physical properties. For example, carboxylate groups can function as carboxylic acids (COO-), or amino groups can behave as bases (NH3+). This allows protein polymers to be multifunctional molecules, with both acidic and basic behavior at the same time! Additionally, the presence of hydroxyls, carboxylates, sulfhydryls, and amino groups allows hydrogen bonding, and the alkyl groups provide hydrophobic interactions, both within the protein polymer itself and between separate protein molecules.
In the case of macromolecules, such as proteins, the polymeric structure of the macromolecule allows it to simultaneously carry many different charges (on different amino acid residues). However, unlike the small single molecules, the amino acid residues are constrained by linear peptide linkages and thus cannot move freely to randomly associate with other charged molecules. Assuming that charged residues will seek to bond with the nearest convenient counter ion, it is most likely that oppositely charged amino acid residues located at different points within a single protein chain will bond. These structural differences result in the folding of proteins into a three-dimensional structure, which is, in part, responsible for their functional properties as biocatalysts, structural materials, muscles, and chemical receptors. Proteins can be shaped as long flat sheets or in globular spheres. This leads to the names fibrous or globular for protein shapes. Most enzymes are globular proteins.
In standard acid base chemistry, students learn that molecules carry electrostatic charges based on the type of atoms that make up a molecule and the environment of the molecule. Given that opposite charges attract, cationic and anionic atoms can combine to form covalent bonds, in which electrons are shared between atomic orbitals, or form ionic bonds, in which only electrostatic attractions exist. In solution with smaller molecules, such as HCl (an acid) or NaOH (a base), protein molecules can freely move around and associate with each other on a more-or-less random basis.
Protein polymers extend the simple acid base charged chemical species concepts to explain how biological systems have greater levels of complexity and can utilize simple, monomeric chemical structures (like amino acids) to create exquisitely complex biological structures like antibodies, muscle, and skin. Protein polymers have physical structure, even when dissolved in liquids. The charged and hydrophobic residues within a protein tend to associate, causing the protein to fold up. When you unfold the protein molecule (called denaturation), its charged residues can reassociate with other charged molecules (precipitation or coagulation). Protein precipitation is widely used to recover recombinant protein products, enzymes, or in the production of many common foods. Cheeses and soybean tofu are examples of coagulated protein food products.
Enzymes
Enzymes are protein polymers that possess the ability to specifically “recognize” biological molecules, bind to them, and catalyze a chemical reaction. In contrast to non-protein catalysts, enzymes are specific catalysts -- they usually react with only one substrate. Since all biochemical reactions are enzyme catalyzed, many different enzymes must exist. An Escherichia coli bacterium, one of the simplest biological organisms, has more than 1,000 different enzymes working at various times to catalyze the reactions necessary to sustain life of the bacterium.
The complex molecules that are contained in food provide the energy needed by living organisms to carry out all life functions. These molecules are not useful to the organism unless they are first broken down into smaller, simpler forms through digestion. Digestion involves the hydrolysis (breakdown) of proteins to amino acids, starches to monosaccharides, and fats to fatty acids and glycerol. Unfortunately, hydrolysis at body temperature occurs at a rate that is too slow to be useful to the organism. To speed up (catalyze) the hydrolysis reaction, living organisms produce and use enzymes.
Carbohydrate digestion begins in the mouth with an enzyme called salivary amylase. This enzyme is an alpha-amylase whose function is to reduce starch, a complex carbohydrate, to simple sugars. Starch is initially reduced to maltose and then to glucose. The glucose is absorbed by the intestines and used to supply energy for the body.
Food Uses of Proteins
Proteins also serve important roles in the processing of food products. They are used for their thickening, gelling, emulsifying, and water-binding properties in meats (sausages), bakery products, cheese, desserts, and salad dressings. Proteins are used for their cohesive and adhesive properties in sausage making, pasta, and baked goods. Egg proteins are used for their foaming properties in desserts, cakes, and whipped toppings. Milk, egg, and cereal proteins are used as fat and flavor binders in low-fat bakery products. Proteins are used for texture and palatability in bakery products (breads, cakes, crackers, and pizza crust) and sausages.
Milk protein consists of 80% casein and 20% whey proteins. There are four major types of casein molecules: alpha-s1, alpha-s2, beta, and kappa. Milk, in its natural state, is negatively charged. The negative charge permits the dispersion of casein in the milk. When an acid is added to milk, the H+ concentration neutralizes the negatively charged casein micelles. When milk is acidified to pH 4.7, the isoelectric point (the point at which all charges are neutral) of casein, an isoelectric precipitate known as acid casein is formed. Cottage cheese and cream cheese manufacture involves an acid precipitation of casein with lactic acid or lactic acid producing microorganisms. Acid casein is used in the chemical industry and as a glazing additive in paper manufacturing.
Casein also can be coagulated with the enzyme rennin, which is found in rennet (an extract from the stomach of calves). Rennin works best at body temperature (37°C). If the milk is too cold, the reaction is very slow, and if the milk is too hot, the heat will denature the rennin, rendering it inactive. The mechanism for the coagulation of the casein by the rennin is different from the acid precipitation of casein. The coagulation of the casein by rennin is a two-stage process. In the first stage, rennin (a proteolytic enzyme) splits a specific bond in the amino acid chain of the kappa-casein macromolecule converting it into a para-kappa-casein and a glyco-macropeptide. This causes an imbalance in the intermolecular forces in the milk system, and the hydrophilic (water-loving) macropeptides are released into the whey. Unlike the kappa-casein, the para-kappa-casein does not have the ability to stabilize the micellular structure to prevent the calcium-insoluble caseins from coagulation. In the second stage, colloidal calcium phosphate bridges within the casein micellular structure are formed in the presence of the soluble calcium, resulting in the three-dimensional curd structure. The rennin coagulum consists of casein, whey protein, fat, lactose, and the minerals of the milk, and has a fluffier and spongier texture than the acid precipitate. Rennet is used in the manufacture of cheese and cheese products, and rennet casein is used in the plastics industry. Casein is solubilized with sodium hydroxide and calcium hydroxide to produce sodium caseinate and calcium caseinate, respectively. Caseinates are added to food products to increase their protein content and are key ingredients in non-dairy coffee creamers and Cool Whip®.
Approximately 90% of soybean proteins are classified as globulins, based on their solubility in salts. More specifically, the proteins are conglycinin (a glycoprotein) and glycinin. Tofu is manufactured by coagulating the proteins in soymilk with magnesium sulfate. As bonding occurs between the positively charged magnesium ions and negatively charged anionic groups of the protein molecules, the proteins coagulate.
Activity/Experiment: Download PDF Version (bottom of page)
Activity Objective
In Part 1, you will precipitate casein from milk using an acid. This method is used to make cottage cheese. In Part 2, you will coagulate casein from milk using the enzyme rennin. This method is used for manufacturing cheese. In Part 3, you will coagulate soy protein from soymilk, using magnesium sulfate. This method is used to make tofu.
Materials Required
HIDDEN MESSAGE: ELMER’S GLUE
NAME______________________________CLASS_________________PERIOD____
Puzzling Proteins: Download PDF Version
Solution to Puzzling Proteins
S T I A N Y P H Y S P D I C C
A L N L O Y A C T D R N I H V
E T S I I H R O E U E O R E G
H O U U T T Y N P O C B E E U
R L L I A R A F R E I E N S +
+ + I + L T O + O + P D N E +
+ + N + U + + G T + I I I + +
+ + + R G + + + E + T T N + +
+ + E + A + + + I N A P + + +
H O R M O N E S N N T E + + E
+ M U S C L E + + + I P + N +
A M I N O A C I D T O E Z + +
E N I S Y L + + O + N Y S + +
+ + + + + + + F + + M + + A +
+ + + + + + U + + E + + + + C
(Over, Down, Direction)
AMINO ACID (1, 12, E)
CASEIN (15, 15, NW)
CHEESE (14, 1, S)
COAGULATION (5, 11, N)
DENATURE (10, 2, SW)
ENZYME (15, 10, SW)
HORMONES (1, 10, E)
INSULIN (3, 1, S)
LYSINE (6, 13, W)
MUSCLE (2, 11, E)
NITROGEN (3, 2, SE)
PEPTIDE BOND (12, 11, N)
PRECIPITATION (11, 1, S)
PROTEIN (9, 4, S)
RENNIN (13, 3, S)
TOFU (10, 12, SW)
HIDDEN MESSAGE:
You should stay physically active throughout your life!
Continue to Appendices 
