Improved processes are provided for preparing ACE inhibitory peptide containing hydrolysates from a plant material such as a seed meal or flour. In one embodiment, the seed meal or flour is extracted with an organic solvent prior to digestion. Also provided are ACE inhibitory peptides Val-Ser-Val and Phe-Leu.

Description of the Invention

SUMMARY OF THE INVENTION

In accordance with one embodiment of the invention is a process for preparing an angiotensin converting enzyme (ACE) inhibitory peptide-containing hydrolysate comprising

contacting a substantially oil-free seed meal or a flour with an organic solvent,

separating the meal or flour from the solvent, and

treating the meal or flour with at least one proteolytic enzyme to produce an ACE inhibitory peptide-containing hydrolysate.

In accordance with a further embodiment is a process for preparing an ACE inhibitory peptide-containing hydrolysate from flax or canola comprising

treating a substantially oil-free flax seed meal or a substantially oil-free canola seed meal with at least one proteolytic enzyme to produce an ACE inhibitory peptide-containing hydrolysate.

In accordance with a further embodiment is an ACE inhibitory peptide-containing hydrolysate prepared by a process as described above.

In accordance with a further embodiment is an ACE inhibitory peptide-containing hydrolysate produced by partial proteolytic digestion of a flax meal or a canola meal.

In accordance with a further embodiment is a powder produced by drying a hydrolysate in accordance with the invention.

In accordance with a further embodiment is an edible food product comprising a hydrolysate in accordance with the invention.

In accordance with a further embodiment is a pharmaceutical composition comprising at least one of peptides Val-Ser-Val and Phe-Leu and a pharmaceutically acceptable carrier.

In accordance with a further embodiment is a peptide of the formula Val-Ser-Val.

In accordance with a further embodiment is a peptide of the formula Phe-Leu.

In accordance with a further embodiment is a method of inhibiting ACE activity in a mammal comprising administering to the mammal an effective amount of a hydrolysate or powder or edible product or composition in accordance with the invention.

Such inhibition of ACE activity can be used to produce a lowering of elevated blood pressure in the mammal. The invention thus provides a method and compositions for treating elevated blood pressure in a mammal, including a human subject.

The invention further provides use of a hydrolysate or powder or edible product or composition in accordance with the invention for preparation of a medicament for the treatment of elevated blood pressure in a mammal, including a human subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved processes and new sources for preparing an ACE inhibitory composition from a plant material such as a meal or a flour without the need to first isolate a highly enriched or purified protein fraction from the plant material.

As used herein, a "meal" means the non-oil portion of oilseeds after oil extraction, in ground form, and a "flour" means the ground seed of a non-oil producing plant such as a cereal or a legume. The processes of the invention may be applied, for example, to oilseed meals produced by conventional methods such as flaking or expelling an oil seed and defatting and solvent extraction of the resulting meal and to flours obtained from non-oil seeds, such as cereals, pseudocereals and legumes.

A "hydrolysate" as used herein means the digestion mixture obtained after proteolytic digestion or partial proteolytic digestion of a seed meal or flour.

In accordance with one embodiment of the invention, the inventors have found that extracting a seed meal or flour with an organic solvent, prior to proteolytic digestion to produce ACE inhibitory peptides, provides hydrolysates with increased ACE inhibitory activity. By using the organic solvent extraction step, the inventors have been able to overcome problems of low yield and low ACE-inhibitory activity which can be encountered when plant material is subjected to direct proteolytic digestion. Table 1 (see Original Patent) shows that for most plant materials tested, organic solvent extraction of the meal or flour prior to proteolytic digestion increased IC50 and protein content of the hydrolysate.

The plant material to be processed may be obtained from an oil-seed, such as flax, canola/rapeseed, soybean, cottonseed, sunflower, peanut or mustard, from a legume seed, such as soybeans, peas, lentils, beans or chickpeas or from a cereal grain such as wheat, oats, barley or rye or a pseudocereal such as buckwheat.

For oil seeds such as canola, flax and soybean, substantially oil-free seed meal is obtained by subjecting the seed to conventional defatting or oil-extraction methods such as expelling, expelling with solvent extraction and flaking and solvent extraction, as practiced in the commercial vegetable oil industry. The resulting material usually contains less than 1% fat. Seeds such as cereal and pseudocereal grains (eg. wheat, oats, rye, buckwheat) are naturally substantially oil free and do not require defatting.

The ground plant material can be in the form of grits obtained by coarsely grinding the whole seed or defatted meal, followed by screening to give particles classified as coarse (10-20 mesh), medium (20-40 mesh) or fine (40-80 mesh) grits according to size, or the ground plant material may be in the form of flour produced by grinding to very fine particles, so that 97% of the product passes through 100-mesh screen. The hull, husk and bran which are generally low in protein may be removed prior to production of the flour.

Suitable organic solvents for extraction of the meal or flour include the lower alcohols such as methanol, ethanol, propanol and butanol, or acetone or ethyl acetate. Ethanol is preferred. The alcohols may be used alone or as an aqueous mixture containing at least 50% alcohol. 65% to 75% ethanol is preferred.

The organic solvent may be mixed with the seed meal or flour at a liquid:solid ratio in the range of about 8:1 to about 25:1, about 10:1 to about 20:1 being preferred.

The seed meal or flour is kept in contact with the organic solvent for about 1 hour up to about 24 hours, at a temperature from about 20.degree. C. to the boiling point of the organic solvent. In one embodiment, solvent treatment is for 24 hours at room temperature (25.degree. C.), or a shorter time at a higher temperature, eg. 3 hours at 50.degree. C. The meal/solvent slurry may be stirred or otherwise agitated during the contact period.

The treated meal or flour is then separated from the liquid solvent phase by any suitable conventional method, such as centrifugation, screening, filtration or decantation and may be washed with water to reduce the level of residual organic solvent. The material is then ready for proteolytic digestion to generate ACE inhibitory peptides. Alternatively, the residual organic solvent may be removed by hot air drying to produce a meal or flour that can be stored for future rewetting and proteolytic digestion.

Proteolytic digestion of the seed meal or flour to yield ACE inhibitory peptides may be carried out generally by methods known to those of skill in the art, and as further described herein. Suitable proteolytic enzymes include acid, neutral and alkaline proteases, and peptidases, including serine endopeptidases and metallo endopeptidases, or mixtures thereof. Many commercially available proteolytic enzymes can be used, such as those listed in Table 2 (see Original Patent). One of skill in the art can readily determine the appropriate digestion conditions for the particular proteolytic enzyme employed.

It is also within the skill of one in the art to determine which proteolytic enzyme gives the lowest ACE inhibitory IC50 in the hydrolysate for a particular plant material, as described in the examples and in FIG. 1 (see Original Patent). It has been found, for example, that thermolysin gives the best inhibitory activity of the enzymes tested when used for digestion of flax seed meal and soybean meal. Proteolysis may be carried out for a period of time which yields the lowest ACE inhibitory IC50 in the hydrolysate. Generally, a digestion period of about 3 hours provides maximum inhibitory activity.

If the hydrolysate is to be used as an edible product such as a food or a food supplement, it may be desirable to stop the proteolytic digestion before maximum ACE inhibitory activity is achieved, in order to retain some of the food value of the plant protein. With some plant materials, proteolytic digestion may be accompanied by the production of bitter-tasting digestion products. In such a situation, it may also be desirable to stop proteolytic digestion before maximum ACE inhibitory activity is reached to control the bitterness.

In one embodiment, the hydrolysates of the invention have an ACE inhibitory IC50 of less than 200 .mu.g powder/ml. In further embodiments, the hydrolysates have an IC50 less than 100, or less than 60 or less than 50.

Typically, the proteolytic enzyme is used at a concentration from about 0.25% to about 8.0% w/w (enzyme:protein content).

In a further embodiment, the proteolytic enzyme is used at of concentration from about 0.5% to about 4.0%.

The proteolytic digestion is terminated by any suitable method, for example heat inactivation of the enzyme or adjustment of the pH of the digestion mixture away from the pH range of enzyme activity. Such methods are well known to those in the art. The resulting hydrolysate is filtered or centrifuged, for example in a decanter centrifuge, to remove residual meal or flour.

In accordance with a further embodiment of the invention is provided a process for preparing an ACE inhibitory peptide-containing hydrolysate from flax or canola meal by digesting the meal with a proteolytic enzyme to generate ACE inhibitory peptide-containing hydrolysates of high specific activity without further purification. Flax and canola have not previously been shown to be sources of ACE-inhibitory peptides. The proteolytic enzyme is selected, and the digestion carried out, as described above. The flax or canola meal may optionally be subjected to extraction with an organic solvent, as described above, before proteolytic digestion.

After digestion of a seed meal or flour to give ACE-inhibitory peptides, and separation of the hydrolysate from the residual meal or flour, the hydrolysate can be used as an edible product or may be spray dried to give a water-soluble powder suitable for use as an edible product or as a pharmaceutical. The use of protein and hydrolysed protein preparations as edible products and their incorporation into food products or use as food supplements is well known to those of skill in the food processing art, for example as described in Clemente, A (2000) "Enzymatic Hydrolysates in Human Nutrition", in Trends in Food Science & Technology v. 11, pp. 254-262

In one embodiment, the hydrolysates are used as edible products without further processing other than standard procedures to control bacterial contamination, such as flash pasteurisation or microfiltration.

The hydrolysates of the invention in liquid or powder form may be used to supplement beverages such as soft drinks, carbonated beverages, ready to mix beverages, milk and milk beverages and their derivates, and foods such as sauces, condiments, salad dressings, fruit juices, syrups, desserts (e.g, puddings, gelatin, icings, and fillings, baked goods and frozen desserts such as ice creams and sherbets), soft frozen products (e.g, soft frozen creams, soft frozen ice creams and yogurts, soft frozen toppings such as dairy or non dairy whipped toppings), oils and emulsified products (e.g., shortening, margarine, mayonnaise, butter and salad dressings), candy and bar confections, cereal foods, and chewing gum tablet.

It has long been realised that extremely raised blood pressure is a life threatening condition. It has more recently been suggested that even modest increases in blood pressure above normal may have deleterious effects.

A systolic blood pressure of 140 mm of mercury or greater, (occurring in approximately 20% of the adult population over the age of 30) is the current clinical definition of hypertension requiring medical intervention. A number of epidemiologists are, however, now defining a second or "preventative model" of hypertension which would define a systolic blood pressure of more than 120 mm of mercury as being the point at which people should undertake steps to reduce their blood pressure and associated risk of cardiovascular disease. People who have blood pressure within the range 120-140 mm mercury generally do not consider themselves to be sick and therefore are less likely to receive pharmaceutical based therapies. An increasing percentage of people in this category are, however, seeking non-pharmaceutical options to reduce this moderately elevated blood pressure (Potential benefits of functional foods and nutraceuticals to reduce the Risk and Costs of Diseases in Canada. B. J. Holub. Report to AAFC 2002).

The hydrolysates and powders of the invention provide anti-hypertensive agents which may be ideal for such cases of modestly raised blood pressure and can be consumed along with or as part of a food.

By comparison with the blood lowering effects previously observed in humans for ACE inhibitory products derived from milk or whey, the hydrolysates of the invention with IC50 values around 40-60 .mu.g/ml may be given to human subjects at an initial dose from about 2 to about 5 gm per day. This dose may be adjusted as required once its blood pressure lowering effect is observed. The dosage would be adjusted accordingly for hydrolysates of higher or lower IC50 value, as would be understood by one of skill in the art.

The hydrolysates may also be formulated as tablets, capsules, granules, powders, syrups, suspensions or injectable solutions.

The hydrolysates produced by the methods described herein have equivalent or greater ACE inhibitory activity than the commercially available whey hydrolysates. When analysed by the ACE inhibitory assay described herein, the commercial whey product BioZate (Davisco Foods International) had an ACE inhibitory IC50 of 137 .mu.g powder/ml. As shown herein, several plant materials yielded protein hydrolysates with ACE inhibitory IC50 values considerably less than that of the whey product, and therefore greater inhibitory activity, without the need to employ any subsequent purification strategy.

Hydrolysates obtained by proteolytic digestion of canola meal were further purified, as described in Example 12, to give two fractions, each containing a single purified ACE-inhibitory peptide. These peptides, not previously described as ACE inhibitors, have the amino acid sequences Val-Ser-Val and Phe-Leu.

The ACE inhibitory peptide-containing hydrolysates of the invention may be further processed to obtain fractions with further enhanced specific activity or the purified single peptides described above, for use as edible products or as pharmaceuticals.

The ACE inhibitory activity of the hydrolysates of the invention may be further enhanced, if desired, by ultrafiltration as described in the Examples herein.

It has been found that seeds with a high polysaccharide content, such as flax and barley, give hydrolysates of high viscosity. Where the viscosity of the hydrolysate is greater than about 10.times.10.sup.-3 PaS (based on the viscosity of a 5% solution in water at 25.degree. C. and a shear rate of 200 (l/s), ultrafiltration membranes up to pore size 100,000 MWCO can be used, but membranes with pore size up to 10000 are preferred.

For hydrolysates of lower viscosity, such as those obtained from canola, membranes of pore size up to 10000 MWCO may be used and membranes with pore size up to 3000 MWCO are preferred.
 

Claim 1 of 20 Claims

1. A process for preparing an angiotensin converting enzyme (ACE) inhibitory peptide-containing hydrolysate comprising: a) contacting a substantially oil-free seed meal or a flour with an organic solvent selected from the group consisting of methanol, ethanol, propanol, butanol, acetone, ethyl acetate, and mixtures thereof; b) separating the meal or flour of step (a) from the solvent; and c) treating the separated meal or flour of step (b) with at least one proteolytic enzyme to produce an ACE inhibitory peptide-containing hydrolysate.

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