Bio-engineered proteins - includes related articles

Prepared Foods, Oct, 1996 by Claudia D. O'Donnell

Researchers at Cornell University investigate protein properties from flavor release to how they may be altered for increased functionality.

"Mixing and matching" ingredient components in order to produce the most optimum properties is a time-honored scientific endeavor. As food manufacturers expect increasingly sophisticated ingredients, the analysis and alteration of these food components has increased in complexity as well.

At Cornell University, researchers are looking at [Beta]-lactoglobulin to determine how this unique whey protein--and bio-engineered versions of it--can work at such tasks as improving the flavor or texture of foods, or facilitating nutrient absorption.

AT THE RIGHT TIME (AND TEMPERATURE)

One of [Beta]-lactoglobulin's functions in animal physiology is to transport retinol, a vitamin A precursor. This ability to bind hydrophobic compounds extends to other molecules, such as lipid-based flavorings as well.

"The goal in one of our projects was to investigate the ability of gels made from [Beta]-lactoglobulin or dialyzed whey protein isolates (DWPI rich in [Beta]-lactoglobulin) to entrap and then release under controlled conditions lipid-based flavorings (orange and lemon oil, benzaldehdye and citral)," says Carl Batt, associate professor, Dept. of Food Science, Cornell.

The gel properties were altered by varying the gel's protein concentration, pH, ion concentration (Ca[Cl.sub.2]) and type and concentration of emulsifier.

Flavor, protein, pH and heat treatment all impacted gel strength. (See chart.) Thus, gels made from 13% DWPI with 5 mM Ca[Cl.sub.2] released encapsulated volatile flavors upon heating at 90 [degrees] C for one minute. A gel with 15% DWPI and a pH of 10 would not release the flavor upon heating even up to 130 [degrees] C.

[ILLUSTRATION OMITTED]

The gels retained about 0.5% flavoring. However, the level could be increased to 1 to 2% (depending upon the flavor) when 1% of an emulsifier was added.

ALTERED STATES

On a smaller molecular scale, a protein's amino acid sequence greatly influences its shape and thus its properties. Batt's group has looked into how altering the amino acids in [Beta]-lactoglobulin could improve its molecule-binding properties, its stability to heat, and its ability to form gels.

A potential use for the [Beta]-lactoglobulin--or new forms of it--would be to transport water-insoluble vitamins or pharmaceuticals undigested through the stomach to the small intestine. The protein would then release the molecule for its absorption into the body, says Batt. Such proteins could be made to perform a similar function in aqueous-based foods or beverages where they would entrap water-insoluble colorants or flavorings which need to be evenly dispersed.

Changing [Beta]-lactoglobulin's shape (conformation) could help in other areas as well.

For example, although as a whey protein [Beta]-lactoglobulin stays soluble under acidic (pH [is greater than] 3) conditions, it denatures when heated above 85 [degrees] C. It forms aggregates which generally precipitate from solution, but under certain conditions will form gels, says Batt.

In all these cases the goal has been to obtain more functional proteins by altering their amino acid sequence.

Milk characteristics traditionally have been improved through selective breeding techniques; however, waiting for herds to evolve into animals that produce more desirable proteins would require an enormous amount of patience, to say the least, notes Batt. A quicker alternative is to introduce new genes into the population by genetic engineering.

Initial steps in this process include understanding the location of critical amino acids in [Beta]-lactoglobulin's structure (through crystallographic studies), computer simulation of the dynamics of the alternative structures, and theoretical studies of the changed protein's functionality.

"We completed these steps and have moved on to producing desired modifications in the protein by cloning and expressing the complementary DNA (cDNA) in the microbe Escherichia coli," says Batt.

PRELIMINARY RESULTS

One of the first mysteries to be solved was determining why retinol was bound in a central location of the protein molecule--a site common to other transporter molecules.

"This will set the age for designing new lactoglobulins that can serve as molecular transports," says Batt.

In efforts to improve the protein's thermal stability, Batt's group replaced one of two amino acids (a critically located alanine or leucine) with a cysteine amino acid. This additional cysteine was then oxidized with another cysteine to form a disulfide linkage. "We found that when either amino acid was replaced with cysteine, it decreased the protein's tendency to aggregate--that is, increased their thermal stability [by 8-10 [degrees] C]," says Batt. Additionally, neither protein gelled at 65 [degrees] C. This would be a desirable property for proteins to be used in products such as beverages.

Further research has lead to the engineering of proteins with increased free thiol groups ("free" cysteine amino acids) which enhanced gel strength. Such alterations would be of benefit in foods such as pudding where firmer textures are desired.