Promising Research into Proteins as Antioxidants

By Annel K. Greene, PhD, Center Director, Clemson University, Animal Co-Products Research and Education Center

Food chemist Dr. Feng Chen, a member of the Clemson University Animal Co-Products Research and Education Center (ACREC) team, is working to isolate proteins from rendered products that will have significant economic value as antioxidants and bioactive molecules. A native of Zhejiang province in China, Chen is an associate professor in Clemson’s Department of Food Science and Human Nutrition and has a strong background in food analysis, food engineering, food biochemistry, and even entomology. His current work centers on the extraction, isolation, and identification of a variety of functional substances including anticancer and antibacterial compounds from animal and plant materials. He also conducts work on a variety of rapid detection methods for numerous substances including toxins. Although still very early in his career, Chen has published more than 40 peer-reviewed journal articles and book chapters, and has served as editor for several flavor and nutraceutical books. He also holds four U.S. and international patents for methods of isolating sugars and for new pesticides against termites, fire ants, and ticks, and has three additional patents pending. Chen is considered one of the leading young food researchers in the United States and in 2002 was awarded the Leadership “Star” of the Institute of Food Technology in recognition of his achievements.

In his research for ACREC, Chen is investigating bioactive peptides from collagen as a source of potentially useful and valuable products. All proteins are a mixture of amino acids chemically bound together. Which amino acids are present and the order in which they are linked together can be extremely complex and varies among different proteins. This variability is what gives each protein its native structure and functionality. In animals, the number of different proteins in various muscles, tissues, blood, etc., is vast. It is known that many proteins from plant and animal sources have bioactive properties and that cutting proteins into smaller subunits, or peptides, can create an entirely new set of bioactive molecules. Certain peptides have activity in reducing high blood pressure and/or modulating immune systems. Others may exhibit antimicrobial activity or stimulate taste, while even others possess strong antioxidant activity. New techniques and methods of protein and peptide analysis are catalyzing the advances in this field of research.

The potential for isolating bioactive peptides from the collagen in rendered products has great potential for nutraceutical veterinary applications. However, there is also great potential for isolating antioxidant peptides from rendered products. At the request of the ACREC Research Committee, Chen has focused his work on the search for anti-oxidants from rendered products. These materials, derived from rendered protein products, may offer an alternative source of antioxidants to protect rendered fat.

Safety concerns for artificial antioxidants such as ethoxyquin, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and n-propyl gallate have caused researchers to seek natural antioxidants. However, natural antioxidants can be expensive. Tocopherols, carotenoids, catechins, and polyphenols are natural antioxidants often used in the food industry. In the rendering industry, mixed tocopherols are probably the most frequently used natural antioxidant.

Hydrolyzing (breaking) larger proteins into smaller protein subunits (peptides) for use as antioxidants is a fairly new research area. Chen’s search for antioxidants from rendering material is a completely new and unique area of study. He is utilizing porcine collagen as a source of peptides in his search for antioxidants. Collagen is a cheap and plentiful animal by-product and is a component of all rendered animal protein meals.

In his study, Chen is methodically cutting protein chains into smaller fragments. The very short chain peptides (less than 10 amino acids) that exhibit the strongest antioxidant activity must be released from the long protein chain. If attached within the larger protein, the peptides display little to no antioxidant activity. But once released after enzymatic hydrolysis, the peptides have differing activities, including some that exhibit potent antioxidant functionality. However, each peptide subunit acts differently. The key is to isolate the peptide sequence that has the highest antioxidant activity. This can be affected by utilizing different enzymatic hydrolysis techniques and conditions (pH, temperature, etc.). The overall goal of the project is to produce the greatest concentration of peptides with antioxidant activity and determine a way that these peptides can be commercially isolated and applied.

The DPPH (1,1-Diphenyl-2-Picrylhydrazyl) radical scavenging activity test is a standard method for determining antioxidant activity. In initial tests, Chen determined that enzymatic hydrolysis of porcine collagen did have increased antioxidant activity over non-hydrolyzed collagen. In this study, he also determined that antioxidant activity increased with increased hydrolysis time. This means that the shorter subunits clipped from the larger porcine collagen molecules had better antioxidant activity than larger subunits. Different crude enzymatic preparations of the hydrolyzed collagen (i.e., mixtures of smaller subunits not yet separated) were compared to the strong food industry antioxidant compounds BHT and BHA. BHT and BHA are phenolic compounds often used to prevent oxidation of fats in foods. Chen chose to compare his peptides to these two common food additives because these are two very powerful antioxidants with predictable activity and are both components in commercially available pet food antioxidant mixtures used in rendered animal fats.

Using the DPPH radical scavenging activity test, Chen determined that three of his initial four crude hydrolysate mixtures had higher antioxidant activity than BHT. The initial four crude hydrolysate mixtures had approximately 40 to 70 percent as high an antioxidant activity as BHA. Using another method of measuring antioxidant activity, the linolenic acid peroxidation system, Chen noted differences in antioxidant strength among the crude hydrolysate samples but still obtained extremely encouraging results. In the linolenic acid peroxidation test, all of Chen’s treated collagen samples had higher antioxidant activity than one milligram per milliliter BHT.

Armed with this information, Chen proceeded to study ways to get even higher antioxidant activity by breaking the collagen chain into subunits. In studies, it has been determined that just random cuts to the protein chains will not produce the desired peptides. Instead, it is necessary to use different enzymes to break the bonds of the proteins at specific points to produce peptides that have potential to be better antioxidant peptides. Chen is continuing his studies on directing the cuts to create commercially feasible peptides with the greatest antioxidant activity.

The next step in the study has been isolating/concentrating the best antioxidant peptides and then testing those mixtures for antioxidant activity. Using sophisticated separation techniques, Chen has separated three peptide fractions from the hydrolysate and is conducting work to measure activity. So far, in preliminary trials, he has obtained excellent antioxidant activity on one of the fractions. Work is continuing to verify these findings and to compare the antioxidant properties of the hydrolysates to not only BHA and BHT, but also to commercially available antioxidants used in the rendering industry. In addition, other protein fractions continue to be generated and are being evaluated for activity.

Chen’s international research team includes graduate students Huaping Zhang, Yen-hui Chen, and visiting professor Dr. Huarong Tong. This team also includes Clemson University undergraduate students Michael Sparague, Brandon Holder, Laurin Ballowe, Joy Dean, and Sarah Shelton, who are enrolled in the Clemson University Creative Inquiry program. In addition, Chen works on bioactive moieties with his wife, Dr. Xi Wang, a research assistant professor in the Department of Genetics and Biochemistry at Clemson University. This bright young married couple has considerable expertise in various areas of chemistry, biochemistry, genetics, food science, and proteomics. The Clemson University ACREC is very proud to have Chen and his team working to create new, less expensive antioxidant compounds for use in the rendering industry.

ACREC Solutions – October 2007 RENDER | back