Protein Possibilities

Protein Possibilities
October 1997 - Applications

By: Lynn A. Kuntz
Editor

  What do these groups have in common? People who follow "The Zone," a diet for optimum health espoused by Barry Sears, Ph.D. Vegetarians who shun all animal-derived food products. Food product designers. The answer: All maintain a keen interest in protein. But while the former two groups' primary interest lies in consuming the right types and levels, food technologists are more interested in using the right types and levels. They recognize that protein is not only a critical nutrient, but that it contributes functional properties in a number of applications.

The amino acid link

  Proteins consist of amino acids joined together by peptide bonds to form high-molecular-weight polymers. These chains generally appear in complex fibrous, twisted, coiled, folded or globular conformations. The number and type of amino acids and their sequence control the molecular configuration of the protein and its surface charge. Certain proteins complex with other molecules, such as carbohydrates or lipids. All of these characteristics, in turn, affect functional properties, such as solubility, water-binding, stability and emulsification.

  These bonds can be enzymatically or chemically hydrolyzed into smaller chains, polypeptides and peptides, or down to single amino acids. The conditions affect the degree of hydrolysis. Enzymatic reactions are typically more selective. Chemical reactions tend to be more random and, unless carefully controlled, will take the protein down to peptides and amino acids that can promote bitterness and off-flavors. Hydrolysis can be used to change the functional characteristics of a protein or provide proteins that are less allergenic and easier to digest.

  The amino-acid composition also determines the protein's nutritional value. Nine amino acids are considered essential because the human body cannot synthesize them in sufficient amounts for growth and health: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. In order to be considered high-quality, a protein must contain these amino acids in amounts proportional to the body's requirements.

  The industry uses two methods to designate protein quality: the older PER (Protein Efficiency Ratio) and the newer PDCAAS (Protein Digestibility-Corrected Amino Acid Score). Typically, the PER method gave animal-based proteins a higher score than that from vegetable sources, but PDCAAS ranks some forms of soy proteins with egg white and casein. Look for more information on this technique in the sidebar titled "Evaluating Proteins -- The PDCAAS Method."

  The exact process used to derive the protein ingredient might affect the quality score, according to Russ Egbert, Ph.D., manager, soy protein application, Archer Daniels Midland, Decatur, IL. "Different proteins may have certain amino acids that restrict its nutritional value. As you further process soy protein, you tend to lose some of the sulfur-containing amino acids, and those will restrict the value on a soy protein," he explains.

  From a formulation perspective, it may be preferable to use a single source of high-quality protein, such as casein. But from a nutrition standpoint, different protein sources can be combined to make up for a deficiency in one or more amino acids that brings the score down for a single protein. "What these scores don't take into account is the effect of a mixed diet," points out Ralph Knights, Ph.D., manager, development, New Zealand Milk Products, Santa Rosa, CA. It's what vegetarians refer to when they talk about "complementary" proteins, such as beans and grains. So, it turns out that Mom was right; it does all get mixed up in your stomach.

Milking the source

  One of the most common sources of protein is cow's milk. Ingredients range from nonfat dry milk to specific proteins, such as immunoglobulins.

  About 85% of the protein content of milk consists of casein, a mixture of phosphoproteins in a spherical complex known as a micelle. This consists of smaller globular particles, containing 10 to 100 casein molecules, called submicelles. These possess a hydrophobic core and a hydrophilic surface, making them insoluble in water. Caseins precipitate at pH 4.6. The casein micelles aggregate with enzymes (chymosin), acid and heat, and also can gel with age.

  "Most people think of casein in terms of fortification, but it has some functional properties, especially when making an extruded product like a snack food or pet food," says Jim Klein, vice president, technical service/sales, Erie Foods International, Inc., Erie, IL. "It has a textural effect in those applications. But most people use it as a caseinate. When you solubilize the casein, you end up with a variety of properties."

  Converting casein to a caseinate, usually calcium or sodium, but also potassium and magnesium, increases solubility. Exact characteristics depend on the specific alkaline ion. Caseinates impart a creamy character and stabilize fats in nondairy products, such as coffee whiteners and cheese-sauce powders, and bind water in processed meat and baked goods.

  "In the health-food industry, the use of calcium caseinate is much more prevalent," observes Klein. "Many of those products want to eliminate the word 'sodium' on the label. Sodium caseinate brings foaming and viscosity; sodium caseinate especially emulsifies and stabilizes fats. Calcium caseinate in comparison, doesn't do anything. If you want a product that does not contain sodium, but you want it to be functional, potassium caseinate is the product."

  In cheese-making, certain milk proteins -- ß-lactoglobulins, (-lactalbumins, bovine serum albumin (BSA), and immunoglobulins (Ig) do not coagulate. These are known as whey proteins. These globular proteins are more water-soluble and acid-stable than caseins, but denature at temperatures above 170°F. Denaturation increases their water-holding capacity. The isoelectric point of the proteins covers a wide range, depending on the compound. The major whey protein, ß-lactoglobulin has an isoelectric point at about 5.4, making whey stable at lower pHs.

  "At the isoelectric point (of whey), you don't get as characteristically insoluble curd formation as you do with casein or soy proteins," says Knight. "They're globular proteins and they act the same way in association with each other."

  Whey also contains lactoferrin and lactoperoxidase, two bioactive ingredients that can be isolated via advanced enzyme separation technology. Lactoferrin, a high affinity iron-binding protein found in mother's milk, is believed to enhance iron absorption, promote the growth of beneficial intestinal bacteria and act as an antioxidant. Lactoperoxidase also has probiotic applications and acts as a natural preservative. In addition to these, bioactive dairy peptides, such as casein phosphopeptide, may provide additional health benefits.

  Whole-milk proteins contain a combination of whey and casein proteins. These take advantage of the nutritional and functional advantages of both proteins.

  Designers looking for more than standard protein fortification might take a look at New Zealand's calcium-fortified caseinates and total-milk proteins. The patent-pending process allows the dispersion and suspension of insoluble calcium at levels of more than 5% of the dry protein rate. According to the manufacturer, the resulting product is not gritty or chalky, does not settle out in low-viscosity foods, and does not affect heat processing.

Additional animal

  Whole eggs, yolks and whites are available for ingredient use in liquid and dry forms. Most dried-egg products are produced by spray-drying. Removing the glucose from egg white prior to drying improves storage stability. Whipping aids may be added to enhance whipping properties or to increase the volume of baked goods.

  Egg protein coagulates over a wide temperature range; this is affected by pH, salts, other ingredients, and duration of heating. Egg white coagulates at 144° to 149°F, egg yolk 149° to 158°F. The presence of dissolved solids raises these temperatures.

  Egg proteins provide exceptional nutritional quality as well as unique functional, textural, visual and flavor characteristics to a finished product. However, many food manufacturers are reluctant to incorporate them into products because of cost, handling and health issues. However, egg producers have lowered the cholesterol level through feeding practices and genetic selection, so eggs have a better nutritional profile than 20 years ago.

  Meat proteins usually find their way to the plate as some sort of muscle food. However, some are used in broth or other forms as flavoring agents. Often, they undergo hydrolysis to pump up the level of flavorful peptides.

  Gelatin, a product that is generally categorized as a hydrocolloid stabilizer, comes from the hydrolysis of collagen, an insoluble animal protein. Most food gelatin is type A, an acid extraction typically from pork skins. Some type B, or alkaline-hydrolyzed product also is used.

  Process conditions, especially the number of extractions, affect the gelatin's quality. This is determined by gel strength, a property measured in Bloom, and viscosity. The higher the Bloom, the harder the set.

  Gelatin's isoelectric point falls between 7 and 9. Gelatin forms a clear, thermoreversible gel which remains stable over a wide pH range. It acts as a protective colloid by stabilizing a variety of hydrophobic materials.

Vegetarian options

  Soy is one of the most economical and abundant vegetable proteins. Soy also is enjoying a positive health image; a study published in the New England Journal of Medicine (August 3, 1995) by Dr. James Anderson, at the University of Kentucky, Lexington, and others, showed that an average consumption of 47 grams of soy protein per day may actually reduce serum cholesterol by 9.3% and LDL by 12.9%. In addition, soy contains a group of compounds, called isoflavones, that may help reduce the risk of osteoporosis, heart disease and certain cancers. They also may help alleviate some menopause symptoms.

  Soy flour and soy grits consist of ground-up whole or defatted soybeans. Flours pass through a No. 100 U.S. Standard Screen, while the screen size of grits falls between a U.S. No. 10 and 80. Toasting promotes a nutty flavor. Lecithinated and refatted forms are also manufactured to improve the water dispersability and emulsification properties.

  Flour may not be appropriate for some applications. "Flour can have flavor issues and you may have problems with the raffinose and stachyose, the difficult to digest oligosaccharides," notes Egbert.

  During processing, soy flour receives a controlled, moist-heat treatment, which determines the grade. The degree of heat affects the solubility: the higher the heat, the lower the NSI (nitrogen solubility index). This affects water dispersability, so it can be used to tailor ingredient functionality. Heating also denatures the enzymes; this reduces the beany flavor.

  Soy protein concentrates can be prepared by three processes: acid leaching, extraction with aqueous alcohol, and protein denaturation with moist heat before water extraction. Acid leaching produces a higher water-soluble content than heat-denatured or alcohol-leached ingredients. Subjecting product made by alcohol extraction to steam injection or similar processes increases solubility. Concentrates offer emulsification and adhesive properties, and bind fat and water.

  "To fortify bars or cereals or similar applications, the protein people usually look at are the traditional concentrates," Egbert says. "It's economical. You have dietary fiber present at about a 20% level. And at the cost per unit of protein, it's a very economical choice."

  Soy protein isolates consist of highly refined protein extracted from dehulled and defatted soybeans with water or mild alkali. Centrifuging removes the fiber residue. The extract is adjusted to a pH of 4.5, so that most of the protein precipitates out. The next step is centrifuging to remove soluble carbohydrates, including oligosaccharides, then washing, then spray-drying. This product is known as an isoelectric isolate. Neutralizing the isolate to a sodium or potassium salt renders it more soluble.

  Soy protein also can be used in the form of textured or structured products. Textured flours and concentrates are made through thermoplastic extrusion of soy flours or concentrates into granules, chunks or flakes. They absorb water and provide meat-like attributes when hydrated. Isolates and concentrates, formed in specific shapes, are called structured isolates and concentrates. Isolates also can be extruded into an acid/salt bath, coagulating the protein into fibers that can be joined together with edible binders into fibrous or laminar bundles.

  Other vegetables and grains also supply protein, which can provide advantages in certain applications. Rice protein has the highest quality protein of all the grains, is hypoallergenic and extremely bland. High-lysine protein has been extracted from yellow peas to produce pea flour and pea protein isolate; this is recommended for use as a stabilizer for water-in-oil emulsions. Potato protein recovered during the starch extraction process also has been developed as a commercial ingredient.

  In corn, zein accounts for about 30% of the protein. This is an excellent film former and is frequently used as such in the confectionery industry. The non-zein fraction of corn protein from high-lysine varieties has been used as a replacement or extender for traditional proteins. According to Ramu M. Rao, Ph.D., professor of food science at Louisiana State University, Baton Rouge, a commercial corn protein isolate from EnerGenetics Inc., Keokuk, IA, provides emulsification stability similar to that of milk proteins.

  The importance of wheat protein is familiar to anyone in the baking industry -- it supports the porous structure of leavened products. The major wheat protein is gluten, made up of gliadin, glutenin and other individual proteins. Currently, most gluten (vital wheat gluten) supplements the existing protein in wheat flour, or makes up the difference when using a nongluten-containing flour like rye. Gluten also serves as the basis for several meat analogs.

On the level

  One of the more important considerations when selecting a protein for fortification, and often for protein-based functionality, is the protein content. Mother Nature isn't particularly generous with the levels of protein in most foodstuffs, except for muscle foods and egg whites. For example, fluid milk is only 3% to 4% protein. Luckily, as technology progresses, it allows us to "harvest" protein from many different sources, concentrate it, purify it, and even modify it from its original form.

  What makes the protein level so important for supplementation? The first reason is easy -- economics; it only makes sense to get more of that key component for your money. Look at the cost for the equivalent level of protein. Additionally, a more purified protein source has less excess baggage to carry through into the finished product. For example, lower-protein milk and whey products also carry significant levels of lactose and minerals. Upon heating, the lactose can promote browning and reactive minerals, such as calcium, magnesium and phosphate and might affect protein stability and functionality.

  Ingredient manufacturers use various processes for protein concentration and isolation, singly and in combination, including: evaporation and spray-drying, ultrafiltration, ion exchange gel filtration, reverse osmosis, and precipitation. The process can affect functionality, composition and quality. For example, heat tends to denature protein, lowering the solubility and often reducing their emulsification and aerating properties.

Functional facts

  "(For proteins) what people first look at first is the aura of nutritional quality," says Knights. "So people usually have in mind what kind of protein they want. Secondly, they may have a flavor requirement. Thirdly, is functionality. They have to be dispersible in a dry mix or they need to provide certain textural properties in a bar or they may need emulsion properties. These functional attributes are how you go about selecting a protein."

  Solubility. A protein's solubility is the most important property in most applications. And while we tend to think of compounds as either completely soluble or insoluble, proteins actually have a certain degree of solubility, depending on their composition. Undenatured proteins are generally more soluble.

  "During the separation process, as you separate the protein from the carbohydrate sources, you're using processes that change the configuration of the protein," says Egbert. "Some of those tend to make it more insoluble. From there, you can resolubilize the protein by means such as heat or homogenization. In a beverage, you'd want the protein to stay in suspension, so in cases like these, the higher the solubility, the better."

  Other factors that affect solubility in a given formulation include pH, ionic strength and solids content. In general, proteins are least soluble at their isoelectric point, the pH at which a protein precipitates.

  Emulsification and foaming. Proteins contain both hydrophobic and hydrophilic structures that allow them to orient onto an oil/water or air/water interface. They then interact with each other to form a stabilizing film that further prevents water-droplet or air-cell coalescence.

  Protein denaturation and the resultant decrease in solubility can lower the emulsification properties, although a mild heat treatment that exposes additional hydrophobic areas in certain proteins can increase the emulsifying properties. The pH and ionic strength of the matrix also may affect the emulsification ability. The presence of fat also influences the foaming properties by acting as a competing surfactant. Generally, the ability to form finer, more uniform air cells increases with increasing concentration up to a point -- then decreases with increasing protein concentration.

  "If you look at the literature, the best protein emulsifiers are the milk proteins," says Knights. "Egg may be comparable, but it doesn't get as much use because of its other attributes, like gelation. If you look at the kinetics of emulsion formation, and the protein coating, and the protective effects, and the longevity of that stable emulsion, it turns out that what you need is, first, a soluble protein, and secondly, one that spreads out over the surface of the fat -- you can't have aggregates or proteins that coat one another instead of the fat. It turns out that casein does that extraordinarily well. Whey proteins coat the fat just about as well, but they take a while to spread over the surface."

  Gelation and viscosity. Protein forms links, and sets up an elastic network that immobilizes water and particles, such as starch granules or fat globules. In addition, heat denaturation may form linked aggregates that impact the gel, making it more brittle and less resistant to syneresis. Covalent bonds may form which straighten the gel; this helps proteins absorb and retain moisture, and can increase juiciness in meat products while decreasing shrinkage and purge. Gelation increases viscosity; inversely, solubility will decrease viscosity. Some gels, like those produced by gelatin, are thermoreversible; others, like heat-induced whey gels are irreversible.

  The viscosity of an ingredient isn't necessarily related to the protein content -- two 80% concentrates could have different viscosities, depending on how they were processed. "There are (soy) isolates that have very low viscosity, there are those that have very high viscosity," Egbert says. "It's related to processing. We have what we call 'functional concentrates' where we have resolubilized the protein, and those can have various viscosities as well." The use of a higher viscosity protein in a given application could provide a degree of viscosity that would cut the level of additional stabilizers required, and consequently provide cost savings, he points out.

  Film-forming. Sodium caseinate can form an encapsulating film on homogenized fat globules, making them into a powder for use in bakery mixes, according to Klein. Whey proteins also can form films that act as barriers to oxygen, aroma and oils. They might be used as encapsulating materials or to protect sensitive product from undesirable oxygen-induced reactions. Low levels of heat can partially denature the protein, exposing hydrophobic amino acids, and increasing a protein's ability to form and stabilize films.

  Food designers also have taken advantage of many of these characteristics to use protein as a fat replacer. Also, a number of products have been developed using dairy proteins to mimic some of fat's characteristics, including yogurtésse(, NutraSweet's Simplesse and Cultor's Dairy-Lo(. One key to fat replacement with proteins is particle size. Small particles, 0.1 to 2.0 µm, help imitate the mouthfeel of fat.

  "For fat substitution, we are typically looking to bind up water," says Egbert. "And then for the water to be released during eating, especially in meat products. Depending on the processed meat category, there are limits to the levels you can use. For example, you can typically use 2% of isolated soy protein, or 3 1/2% of a concentrate or flour as a binder."

  Beyond these functional properties, proteins can have other characteristics that influence their use. For example, according to Egbert, "With a powdered beverage, one of the most important characteristics is its density. You need to be able to get a given weight in a certain volume of package."

  Dispersability is another issue. This can be enhanced by rewetting, agglomeration, changing the particle-size distribution, or coating with a fat or phospholipid, such as lecithin.

  Functionality is dependent on not only the application, but also on what else goes into a product. "Minerals have a big effect on the functional characteristics of protein," says Knights. "They change the characteristics during heating, and they are key to understanding what will actually work in a given application. Calcium often interacts with proteins, generally to aggregate them, which generally affects their functionality. When they aggregate, they're not soluble; when they're insoluble, they don't emulsify, they settle out." The actual calcium source influences this effect.

  The fat content of the finished product also can affect how well the protein stabilizes or destabilizes the emulsion, according to Knights. Situations that cause the protein films to form aggregates or bind together can promote fat coalescence. "You can treat that by eliminating the divalent ions, or treating them with phosphates, or using a type of protein that is not affected as drastically by the presence of divalent ions." For example, whey is less susceptible to these ions than casein.

  Protein ingredients currently offer a wide range of products geared toward filling the ever-widening needs of food designers. Advanced technologies, including cutting-edge separation techniques and genetic engineering of crops, will provide even more enhanced protein ingredients in the future.

Protein Levels

Milk yields these proteins and protein ingredients with the following protein levels. These vary by processor and specific product:

  The other common protein source, the soybean, starts out with 38% to 40% protein and is further refined into:

  Other protein ingredients include dried whole egg (47%), dried egg albumen (81%), hydrolyzed gelatin (92%), corn protein isolate (91%), pea protein isolate (90%), wheat gluten (>90%) and rice protein (35% to70%).

Evaluating Proteins - The PDCAAS Method

  The quality of protein in food sources is classified according to the protein's digestibility and bioavailability, and is based on the type and variety of amino acids present.

  The Protein Digestibility-Corrected Amino Acid Scoring (PDCAAS) method is now the preferred method of protein-quality evaluation, replacing the Protein Efficiency Ratio (PER). PDCAAS accurately depicts protein quality, because it is based on the needs of humans, rather than on the PER criterion of a protein's ability to support growth in young rats. PDCAAS also balances the value of vegetable proteins with that of animal proteins; PER is thought to have placed undue emphasis on the value of the protein from animal sources.

  A protein's PDCAAS score is calculated by first analyzing the nine essential amino acid (EAA) levels in milligrams per gram of protein, using high-performance liquid chromatography (HPLC), or another analytical testing method.

  The next step is determining the uncorrected amino acid score for each of the nine essential amino acids by dividing the milligrams of EAA in 1 gram of test protein (value from HPLC procedure) by the milligrams of EAA in 1 gram of reference protein (Use the FAO/WHO 2-to-5 year old milligram per gram crude protein requirement levels: HIS=19; ILE=28; LEU=66; LYS=58; MET+CYS=25; PHE+TYR=63; THR=34; TRY=11; VAL=35).

  Finally, multiplying the lowest of the uncorrected amino acid scores by the food's true digestibility value yields the PDCAAS score. For example, if the lowest uncorrected amino acid score for a particular food element is lysine at 1.37, and the true digestibility for this element is 53%, the PDCAAS value would be 0.73.

  A "perfect" score equals 1.00 - proteins with this value provide all essential amino acids, and are considered to be complete protein sources. Higher scores are rounded down, because although excess amino acids are broken down and utilized by the body, they are generally not used for protein synthesis. Casein, egg white and certain isolated soy proteins possess scores of 1.00; kidney beans have a score of 0.68; whole wheat is valued at 0.40; and wheat gluten has a score of 0.25.

  The Percentage of Daily Value on labels is determined by multiplying the PDCAAS score by the total protein content, measured in grams. Divide the resulting number by 50 grams (the Daily Recommended Value for protein for children older than 4 and adults); and then multiply by 100 to obtain the Percentage of Daily Value.

- Heidi L. Kreuzer

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