PROJECT RESULTS

Production of Nutraceutical Peptides from Flaxseed Meal

Applicant: 

Dr. Rotimi Aluko

Department of Human Nutritional Sciences

University of Manitoba

Winnipeg, Manitoba  Canada  R3T 2N2

ARDI Project:

#01-511

Project Status:

Completed July, 2005

Achievements:

Preparation of Protein Isolate

Defatted flaxseed meal used for the project was donated by Bioriginal Foods, Saskatoon, Saskatchewan.  Work started on the project in May, 2003 with the hiring of a summer undergraduate student.  Protein isolate was prepared from flaxseed by using a standard method that involved extraction with dilute alkali, followed by precipitation of the extracted proteins with dilute acid.  The isolated protein was freeze-dried and contained 86% protein content from kjeldahl analysis.  Due to the nature of flaxseed meal, the yield of protein isolate was very low (usually about 10-15%), therefore, the first two months were used essentially to perform routine extraction in order to obtain enough quantity of protein isolate that will be suitable for the next step, i.e., enzymatic hydrolysis. 

Protein Hydrolysis

The following section deals with the first objective of the project, which was to prepare short-chain peptides that are enriched with branched chain amino acids (BCAA).  The flaxseed protein isolate was hydrolyzed sequentially with two types of enzymes that converted the long-chain proteins into short-chain peptides.  The first step involved hydrolysis with thermolysin for 5 hr at pH 7.5 followed by a second step that used papain (pH 6.5) for 3 hr.  During the 8 hr hydrolysis period, 2 ml samples were taken every 30 min and placed in a water bath of 100^oC in order to stop the enzymatic reaction.  The 2 ml aliquots were analyzed for free amino group content using the trinitro-benzene-sulfonic acid method, so that the rate of enzymatic reaction can be determined.  The results of the free amino group determination showed that the rate of flaxseed protein hydrolysis by thermolysin was slower than the rate for hydrolysis by papain.  Overall, the level of free amino groups increased, which indicates that the flaxseed proteins were broken down into smaller peptides as expected.

After 8 hr of hydrolysis with the two enzymes, the resultant protein hydrolysate was collected by centrifugation and freeze-dried.  The freeze-dried sample contained both the desirable (BCAA) and undesirable (aromatic amino acids, AAA) peptides.  Separation of the peptides is currently being performed on a column packed with activated carbon, which has a very hydrophobic character.  Since the aromatic amino acids are more hydrophobic than the branched chain amino acids, we optimized the flow-rate.  We added acid to the protein hydrolysate, which makes the AAA more hydrophobic than the BCAA.  We passed the acidic hydrolysate through the activated carbon column during which majority of the peptides that contain AAA bind to the column while most of the BCAA-containing peptides did not bind.  In this way the BCAA-containing peptides were separated from the AAA-containing peptides.  We collect flow-through samples from the column using various flow rates in order to optimize yield and quality of the BCAA-enriched peptide fraction.  Each of the flow-through samples will be analyzed for amino acid content so that we can quantify the ratio of BCAA to AAA and determine the Fischer ratio.  At this stage of the project, we have achieved approximately 50% of the first objective.

Peptide Fractionation

We successfully optimized the fractionation protocol that enabled preparation of peptide fractions that are almost devoid of aromatic amino acids as shown in the table below.  

Where A₂₆₀ and A₂₈₀ are the ultraviolet absorbance values obtained after samples were adjusted to the same protein concentration; the higher the value the more the amount of aromatic amino acids present in the sample.  The flaxseed hydrolysate is the sample obtained after enzymatic hydrolysis and prior to separation on an activated carbon column.  Fractions 1 and 2 are the peptides obtained after the protein hydrolysate was passed through the column at the optimized flow rates.

Conclusion:

Fraction 1 contains about 5% while fraction 2 contains about 2% of the amount of aromatic amino acids (AAA) in the original flaxseed protein.  The low levels of AAA mean that the fractionated peptides may be used to treat encephalopathy (brain swelling) that is associated with liver diseases.  At this stage of the project, we have achieved approximately 90% of the first objective.  To complete the first objective we need to prepare larger quantities of fractions 1 and 2 followed by amino acid analysis that will determine the amount of branched chain amino acids present in the fractions. 

Training of Highly Qualified Personnel:

Vincent Jung (undergraduate student)

Acknowledgement:

This project was made possible through funding from the Province of Manitoba and Government of Canada under the Canada-Manitoba Agri-Food Research and Development Initiative (ARDI).

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