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Ebook Production, Fractionation, and Evaluation of Antioxidant Potential of Peptides Derived from Soy Protein Digests

Submitted by puput on Sat, 07/24/2010 - 07:47

Antioxidants are defined as “any substance which significantly delays or inhibits oxidation of a substrate when present at low concentrations compared to that of an oxidizable substrate” (Jun, Park, Jung, & Kim, 2004). Antioxidants are commonly used in the food industry to prevent lipid oxidation which can lead to product degradation and production of off-flavours, but they also have applications in other fields where oxidation will degrade the product quality (Gao, Miller, & Han, 2004).

Compounds such as polyphenols and carotenoids found in brightly coloured fruits and vegetables have been shown to possess beneficial health properties such as preventing cancer, diabetes, arthritis, atherosclerosis, and other age-related diseases (Kaur & Kapoor, 2001). The primary focus has been on the antioxidant properties of molecules such as flavonoids, polyphenols, and carotenoids derived from fruit and vegetable sources (Fukumoto & Mazza, 2000, Pulido, Bravo, & Saura-Calixto, 2000, Kaur et al., 2001, Sanchez-Moreno, 2002) but some groups are also investigating the antioxidant properties of proteins and peptides derived from both animal and non-animal sources (Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara, 1998, Jao & Do, 2002, Saito et al., 2003). Wayner and co-workers recognized that 10-50% of the antioxidant properties found in human blood plasma could not be explained by known antioxidants such as vitamin E, urate, or ascorbate and theorized that a number of essential amino acids possessing labile hydrogen atoms could allow amino acids and peptides to behave as antioxidants (Wayner, Burton, Ingold, Barclay, & Locke, 1987). Research currently underway covers a wide range of potential antioxidant peptide sources such as rice, soybean, fish, skeletal muscle tissue, chicken eggs, and bovine milk.

The measurement of antioxidant capacity is a challenge because antioxidants can act by many different mechanisms, such as hydrogen-donating, electron donating, free radical scavenging, and chelating metal ions which can initiate free-radical reactions. Therefore a single test for antioxidant capacity does not exist (Prior, Wu, & Schaich, 2005). In 2004, the First International Congress on Antioxidant Methods recommended the standardization of the testing of food, botanical, and nutraceutical antioxidants by two methods: the Oxygen Radical Absorbance Capacity (ORAC) fluorescence assay to measure the hydrogen donating capacity of an antioxidant to protect a fluorescing target from attack by the free-radical AAPH, and the Folin-Ciocalteu Reagent (FCR) method to quantify the electron-donating capacity of an antioxidant to protect the FCR from degradation (Prior et al., 2005).

Soybeans are a significant food crop with world-wide soybean consumption estimated at about 150 million tons in 2000 (De Meester, Kempener, & Mollee, 2000). While proteins make up approximately 40% of the total dry matter in soybeans, soy proteins are largely used as animal feed while the oil is reserved for human consumption, although interest is resurfacing for the use of soy proteins in other applications such as adhesives and polymers (Liu 1997, Kumar, Choudhary, Mishra, Varma, & Mattiason, 2002). In 1999, the US Food and Drug Administration approved the “use of health claims about the role of soy protein in reducing the risk of coronary heart disease” (Food and Drug Administration (FDA), 1999), which has lead to further interest in the soybean.

Enzymatic modification of proteins is a mild and targeted method to modify the functional properties of the proteins with high specificity under relatively mild conditions with limited effects on the nutritional properties (Kunst, 2000, Kumar et al., 2002). The enzymatic modification of soy proteins is a well-documented process, including the production of traditional Asian foods such as tempeh and miso (Liu, 1997). More recently, enzymatic digestion by pepsin and pancreatin in a digestion model similar to that found in the human digestive tract has been employed to produce bioactive peptides, such as ACE inhibitor peptides (Lo & Li-Chan, 2005). The degree of hydrolysis is monitored by the o-phthaldialdehyde (OPA) assay, where an absorbing adduct between the OPA, ?-mercaptoethanol, and the ?-amino group of the peptide is measured by spectrometry and related to the concentration of peptides in the solution (Church, Swaisgood, Porter, & Catignani, 1983).

Contents

Author’s Declaration
Abstract
Acknowledgements
Table of Contents
List of Figures
List of Tables
List of Abbreviations
1 Introduction
1.1 Objectives of this Study
2 Literature Review
2.1 Soybeans
2.2 Peptide Production

    2.2.1 Soy Peptides
    2.2.2 Pepsin
    2.2.3 Pancreatin

2.3 Peptide Characterization

    2.3.1 Bradford Assay
    2.3.2 OPA Assay
    2.3.3 Ninhydrin Assay
    2.3.4 TNBS Assay
    2.3.5 MALDI-TOF Assay
    2.3.6 HPLC

2.4 Membrane Ultrafiltration
2.5 Antioxidant Capacity

    2.5.1 FCR
    2.5.2 ORAC
    2.5.3 Antioxidant Peptides

3 Materials and Methods
3.1 Equipment
3.2 Chemicals and Materials
3.3 Methods

    3.3.1 Stock Solutions
    3.3.2 Enzymatic Digestion
    3.3.3 Bradford Assay
    3.3.4 OPA Assay
    3.3.5 MALDI-TOF
    3.3.6 SEC-HPLC
    3.3.7 Membrane Ultrafiltration
    3.3.8 Freeze-drying of Samples and Determination of Mass Fractions
    3.3.9 DPPH Antioxidant Assay
    3.3.10 FCR Antioxidant Assay
    3.3.11 ORAC Antioxidant Assay
    3.3.12 Statistical Analysis

4 Preliminary Investigations
4.1 SPI Characterization

    4.1.1 ADM PRO-FAM
    4.1.2 Solubility

4.2 Preliminary Digestions

    4.2.1 Preliminary Digestion
    4.2.2 Individual Enzyme Effects

4.3 Fractionation by Ultrafiltration
4.4 Peptide Characterization
4.5 Antioxidant Capacity

    4.5.1 DPPH Assay
    4.5.2 FCR Assay
    4.5.3 ORAC Assay

5 Digestion Condition Factorial Design
5.1 Digestions

    5.1.1 Additional Factorial Design Digests

5.2 Fractionation by Ultrafiltration

    5.2.1 Fouling Behaviour

5.3 Characterizing the Digests and Fractions
5.4 Antioxidant Properties

    5.4.1 DPPH Assay
    5.4.2 FCR Assay
    5.4.3 ORAC Assay

6 Extensions from Factorial Design
6.1 Digestions

    6.1.1 Effect of Increasing Pancreatin Concentration
    6.1.2 Effect of Eliminating Pepsin
    6.1.3 Additional Midpoint Digest
    6.1.4 Interpretation of Digestion Results

6.2 Fractionation by Ultrafiltration

    6.2.1 Fouling Behaviour

6.3 Antioxidant Properties

    6.3.1 FCR Assay
    6.3.2 ORAC Assay

6.4 Antioxidant Model
7 Discussion
7.1 Soy Protein Source
7.2 Enzymatic Hydrolysis

    7.2.1 Quantifying Total Peptide Concentration (OPA Assay)
    7.2.2 SPI Solubility
    7.2.3 Pepsin
    7.2.4 Pancreatin
    7.2.5 Hydrolysis Conditions

7.3 Membrane Ultrafiltration

    7.3.1 Membrane Maintenance
    7.3.2 Peptide Recovery
    7.3.3 Fouling
    7.3.4 Peptide Characterization

7.4 Antioxidant Capacities

    7.4.1 DPPH Antioxidant Capacity
    7.4.2 FCR
    7.4.3 ORAC

7.5 Antioxidant Model
8 Conclusions and Recommendations
8.1 Conclusions
8.2 Recommendations
Permissions
References
Appendix A
Appendix B
Appendix C

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