Leucine-rich peptide compositions and methods for isolation

This application claims the benefit of priority of earlier-filed U.S. provisional patent application No. 61/039,426, filed Mar. 26, 2008.

The invention relates to dietary peptide compositions for stimulating protein synthesis, decreasing protein degradation, producing vasodilation, increasing nitric oxide production, and decreasing blood flow. More specifically, the invention relates to leucine-rich peptide compositions and methods for their isolation from proteins.

Loss of muscle tissue often occurs as a result of aging, malnutrition, and catabolic diseases such as burns, sepsis, and cancer. Dietary protein supplementation may be beneficial, but supplementation with the essential amino acid leucine has been shown to be especially beneficial. Dietary leucine has, for example, recently been shown to suppress the rate of myofibrillar protein degradation and muscle weight loss in rats. Leucine also stimulates muscle protein synthesis and modulates the activity of various proteins involved in the control of mRNA translation. Leucine may stimulate protein synthesis directly or through its metabolite, α-ketoisocaproic acid. Leucine may stimulate translation either independently or by interaction with the mammalian target of rapamycin (mTOR).

Leucine is one of the branched-chain amino acids and is an essential amino acid. It is the only amino acid that is converted to acetyl-coenzyme A and alpha-ketoacids and is an important source of nitrogen for synthesis of glutamine. In addition to its effects on protein synthesis and degradation, leucine also stimulates glucose uptake by protein kinase C (PKC), while insulin modulates glucose uptake via protein kinase B.

Milk-derived whey is one good source of muscle-building proteins. Whey protein isolates and whey protein concentrates are used in protein-building dietary supplements. Whey proteins are a good source of leucine, but for many individuals who need the muscle-building amino acids dietary proteins can provide, it is difficult to digest and/or absorb proteins. It is therefore important to find ways to provide leucine-containing amino acid compositions that provide amino acids in a more bioavailable form for improving muscle protein synthesis.

The present invention relates to peptides having a leucine content of from about 12% to about 40% (“leucine-rich peptides”), those peptides being isolated from a protein source by a method comprising the steps of hydrolyzing the protein in the presence of one or more leucyl aminopeptidases, optionally in combination with additional proteases, to produce a protein hydrolysate, deactivating the enzymes, and filtering the protein hydrolysate to provide a permeate comprising peptides having a molecular weight of from about 200 to about 4,000, including sub-ranges in-between, such as, for example, a molecular weight of from about 200 to about 1,000 or a molecular weight of from about 400 to about 1,000. Optionally, the step of concentrating the leucine-rich peptides by removing the aqueous fluid from the permeate may be added. Also disclosed are method of use of such peptides for a variety of beneficial effects, such as producing vasodilation, increasing nitric oxide production, decreasing blood pressure, increasing blood flow, increasing muscle tissue, improving wound healing, and improving cognitive function.

The invention also relates to a method for isolating leucine-rich peptides from a protein source, the method comprising the steps of hydrolyzing the protein in the presence of one or more leucyl aminopeptidases, optionally in combination with additional proteases, to produce a protein hydrolysate, deactivating the enzymes, filtering the protein hydrolysate to provide a permeate, and concentrating the permeate containing the leucine-rich peptides.

In various embodiments, the protein may be derived from animal or plant sources, including, for example, legumes such as soy or pea, fish, meat, milk, blood, egg, corn, wheat gluten, maize, or combinations thereof. In some embodiments, the protein source may be bovine milk, bovine whey, whey protein concentrates, and/or whey protein isolates.

In various embodiments, the leucine content of the leucine-rich peptides may be within various subranges of the about 12 to about 40 percent range, such as, for example, from about 12 to about 30 weight percent leucine, from about 15 to about 20 weight percent leucine, from about 15 to about 25 weight percent leucine, and from about 20 to about 25 weight percent leucine.

The inventors have discovered that peptides having a leucine content of from between about 12% and about 40% (“leucine-rich peptides”) may be isolated from a protein source by a method comprising the steps of hydrolyzing the protein in the presence of one or more (i.e., at least one) leucyl aminopeptidases, optionally in combination with additional proteases, to form a protein hydrolysate, deactivating the enzymes, filtering the protein hydrolysate to isolate leucine-rich peptides, and concentrating the leucine-rich peptides. In one embodiment, the method comprises hydrolyzing the protein in the presence of one or more aminopeptidases which may act as leucyl aminopeptidases (leucine aminopeptidases), optionally supplemented with additional proteases, to form a protein hydrolysate, deactivating the enzymes, filtering the protein hydrolysate using ultrafiltration, nano-filtration, or another suitable filtration method to give a permeate containing the leucine-rich peptides, and concentrating the leucine-rich peptides in the permeate using reverse osmosis, evaporation, or another suitable method for concentrating the peptides by removing the aqueous portion of the permeate from the filtration step. The invention also provides compositions comprising leucine-rich peptides prepared by the method.

Dietary leucine appears to have a number of beneficial effects, including stimulation of muscle protein synthesis and suppression of the rate of myofibrillar protein degradation and muscle weight loss. Leucine also increases hypothalamic mTOR signaling and decreases food intake and body weight (Cota, D. et al. Science (2006) 312: 927-930). Leucine administration increases leptin levels, and leptin is known to promote lipolysis in adipose tissue, but has no apparent effect on lean tissue. Increased leptin levels result in decreased hunger, decreased food consumption, and increased cellular energy expenditure.

Protein sources such as standard whey protein hydrolysates may contain as much as 10% leucine, but given the variety of beneficial effects of this amino acid and the fact that it is not synthesized in the body from other amino acids, it has been a goal of the food and nutritional supplement industry to develop ways to increase leucine levels in order to promote its beneficial effects, especially its effects on muscle building and inhibition of muscle wasting. The present invention provides amino acid compositions that are rich in leucine and such compositions may be isolated from readily-available protein sources using the method of the invention.

The inventors proposed to use leucine aminopeptidase to process whey proteins to produce bioactive/bioavailable leucine-containing peptides which they have demonstrated to have a variety of desirable effects. Leucine residues appear to play an important role in the distinction between the effects of remarkably similar proteins. For example, Angiotensin I differs from Angiotensin II by two additional carboxyl-terminal amino acids—histidine and leucine. Met-enkephalin, a neuropeptide, has many similar effects to those of Leu-enkephalin, which appear to be mediated by the aromatic side-chains of the amino acids in the peptides. Both peptides consist of 5 amino acid residues. Met-enkephalin and Leu-enkephalin differ, however, in the carboxyl-terminal fifth amino acid residue. In Met-enkephalin, it is methionine. In Leu-enkephalin it is leucine. Both appear to have effects on arterial blood pressure and vasodilation, but in this area the amino acid difference appears to be more significant, with Leu-enkephalin appearing to have a stronger effect (Moore, R. H. and D. Dowling, “Effects on Intravenously Administered Leu- or Met-Enkephalin on Arterial Blood Pressure,” Regulatory Peptides (1980) 1(2): 77-87). While others have provided hydrolysates containing a significant percentage of leucine present as a free amino acid (see, for example, U.S. Pat. No. 6,875,456 to Delest, V. et al.), the present invention provides small peptides (e.g., a hydrolysate containing a significant percentage of di- and tri-peptides) to deliver leucine in a more bioavailable/bioactive form. These peptides have demonstrated surprising beneficial results, such as potent inhibition of angiotensin converting enzyme (ACE) inhibition, vasodilation, and stimulation of increased nitric oxide production. Experiments demonstrate that the peptides of the invention provide bioavailable/bioactive leucine, supplementation with the peptides demonstrating many of the same beneficial effects as those provided by leucine supplementation, but with additional benefits not previously described as the result of leucine supplementation.

Leucine-rich peptide compositions of the present invention provide leucine in combination with other amino acids, but provide increased amounts of leucine in relation to the other amino acids. These compositions may readily be added as a powder to a drink formulation, to a food product, to a nutritional supplement composition such as a tablet, capsule, or other formulation, or to a pharmaceutical preparation, for example. Additional flavorings, carbohydrates, fats, proteins, vitamins, minerals, and other suitable food or supplement ingredients may be included in such compositions.