EP1913018A1 - Biopeptides with anti-hypertensive activity from bovine beta-casein - Google Patents

Abstract

The invention relates to the characterization of a biopeptide with anti-hypertensive activity derived from bovine β-casein, to a process for the synthesis of this peptide and to the use thereof for the preparation of functional foods for the treatment of hypertension.

Description

BIQPEPTIPES WITH ANTI-HYPERTENSIVE ACTIVITY FROM BOVINE BETA-CASEIN

This invention relates to peptides with anti-hypertensive activity derived from bovine β-casein and their use for the preparation of nutraceutical foods.

Background of the invention Studies carried out by nutritionists have shown that the biological value of food proteins must be reckoned not only on the basis of their nutritional value, but also on the basis of the beneficial physiological functions possibly exerted by some of them.

The proteins contained in some foods such as milk, may be hydrolysed to peptides which, after absorption, may be beneficial to human health, for example by reducing the risk of development of some pathologies and/or by regulating some metabolic processes. These peptides are known as "BIOACTIVE PEPTIDES" (or "BIOPEPTIDES"); some of them have already been used as ingredients in functional foods or for para-pharmaceutical preparations.

Biopeptides are contained in foods such as milk and its derivatives, eggs, fish, soy seeds, rice, corn and wheat; they are part of protein sequences in the latent form and become active only upon release.

Some biopeptides have proved able to modulate arterial pressure, due to the fact that they inhibit the Angiotensin-I Converting Enzyme (ACE), an enzyme that converts angiotensin I to angiotensin II, a potent vasoconstrictor. The first discovered competitive ACE inhibitors were the peptides normally present in snake venom. Thereafter, many other inhibitors have been identified through in vitro digestion of proteins contained in milk, fish and other foods or produced by chemical synthesis, as described "for example" by Yamamoto in Biopolymers, 43: 129- 134, 1997 and by Gobbetti in Critical Reviews in Food Science Nutrition, 42: 223-239, 2002. Casein-derived ACE-inhibitors are commonly referred to as casokinins, while those derived from serum proteins are known as lactokinins. There are three processes used for obtaining physiologically active peptides:

- isolation from in vitro enzymatically digested milk proteins through hydrolysis with enzyme extracts from lactic acid bacteria; - direct purification from food products, possibly after controlled hydrolysis of the protein components; indeed, most of them are obtained from dairy products fermented with selected lactic acid bacteria. The factors that most influence the release of amino acid sequences with hypotensive activity from protein precursors are the species and the strain of the lactic acid bacteria employed;

- chemical peptide synthesis.

Both in vitro enzymatic hydrolysis and procedures for food production give rise to mixtures of peptides from which the peptide of interest must be isolated. The preferred technique for isolation is chromatography, in particular RP-HPLC (reverse phase high performance liquid chromatography), as described for example by Tauzin in FEBS Letters 531 : 369-374, 2002 and Parrot in Nahrung Food 47: 87-92, 2003.

However, the complexity of the starting hydrolysate makes the purification process quite demanding and time-consuming; in addition, each biopeptide might require a specific process protocol. Moreover, this method cannot be used on a large scale for the preparation of biopeptides to be added to functional foods, because yields are poor. Lastly, the high production costs of the biopeptides produced using this technique should be considered. The same problems affect the preparation through chemical synthesis, which is expensive and provides poor yields and therefore is suitable for chemical and physiological studies only.

It would therefore be desirable to provide new biopeptides with antihypertensive activity and methods for their preparation other than those described above. Detailed description of the invention

The present invention relates to the ACE-inhibitor peptide SLVYPFPGPI (SEQ ID NO. 1) and to its precursor FAQTQSLVYPFPGPIPNSLP (BP l , SEQ ID No 2), which corresponds to fragments 57-66 of bovine /3-casein. The ACE-inhibitory peptide, released from the precursor after hydrolysis with a specific proteinase, can be advantageously used for the preparation of functional foods to be used as adjuvants for the treatment of mild hypertension, either alone or in combination with the peptides NIPPLTQTPV (SEQ ID No. 3) and DKIHPF (SEQ ID No. 4), or with their corresponding precursors NSLPQNIPPLTQTPVVVPPF (BP2, SEQ ID No. 5) and EDELQDKIHPFAQTQS (BP3, SEQ ID No. 6), suitably activated so as to release the active fragment. The isolation method and the functions of BP2 and BP3 are known in the literature (Gobbetti et al., (2000) Applied and Environmental Microbiology, 66: 3898-3904).

A further aspect of the invention relates to a process for the preparation of a β-casein peptide with anti-hypertensive activity, said process comprising: a. expression of the biopeptide as a fusion protein with glutathione- thio-transferase (GST) from Schistosoma japonicum (BP-GST), in prokaryotic cells; b. purification of the fusion protein through affinity chromatography on a glutathione-sepharose resin; c. digestion of the fusion protein (BP-GST) with Lactobacillus helveticus PR4 proteinase; and d. peptide purification. This process allows the preparation of active biopeptides, in particular of the biopeptides BPl, BP2 and BP3.

The preferred expression system is the GST FUSION PROTEIN EXPRESSION SYSTEM (Amersham-Pharmacia), which contains the following structural elements: • a "tac" promoter for chemical induction and high-level expression of the fusion protein;

• a cloning site at the carboxy-terminal region of the GST gene;

• a recognition site for thrombin upstream of the cloning site, for the release of the desired protein from the fusion product; • the Amp^r gene that confers ampicillin resistance and allows the transformed cells to be selected in a suitable medium;

• the Lac I^q gene, which allows the use of the vector in any strain of host E. coli;

• the replication origin of the pBR322 plasmid. The biopeptide is released from the fusion protein (BP-GST) through enzymatic hydrolysis with a cell wall associated proteinase (T. A. F.) partially purified from Lactobacillus helveticus PR4. Lactobacillus helveticus is a lactic acid bacterium frequently used in dairy biotechnology and is labelled as a "GRAS" (Generally Recognized As Safe) micro-organism. Most strains of Lactobacillus helveticus, as widely reported in the literature (Juillard et al., 1995 Journal of Bacteriology, 177: 3472-3478; Mierau et al., 1997, Biotech. Genetic Engineering Rev., 14: 279-301 ; Gobbetti et al., 2002 Critical Reviews in Food Science Nutrition, 42: 223-239), possess high proteolytic activity towards milk caseins and are therefore among the most used micro-organisms for the preparation of physiologically active peptides from milk and derivatives thereof.

The present invention further relates to the preparation of milk drinks added with BPl , BP2 and BP3 (purified through RP-FPLC), alone or in combination with one another. These drinks, optionally fermented, are characterized by a high specific anti-hypertensive activity ascribable to their

ACE inhibitory effect, which has been demonstrated in vitro.

The invention will be illustrated in detail in the following experimental section.

Experimental section

The following oligonucleotide pairs (primers), corresponding to SEQ ID No. 2, 5 and 6:

1. BPl For (5'-AAA AGA GAG GCT GAA GCT TAC GTA CAG ACA CAG TCT TTG GTC TAT CCC TT-3') / BPl Rev (5'- AGG GAT GGG CCC

AGG GAA GGG ATA GAC CAA AG-3');

2. BP2 For (5'- AAA AGA GAG GCT GAA GCT TTG CCA CAA AAC ATT CCA CCA TTA ACT CAA AC-3') / BP2 Rev (5'- TGG AAC AAC AAC TGG AGT TTG AGT TAA TGG TG-3'); 3. BP3 For (5'- AAA AGAGAG GCTGAA GCT GAA GAT GAA TTA CAA GAT AAG ATT CAT CCA TT-3') / BP3 Rev (5'- AGA TTG AGT TTG AGC AAA TGG ATG AAT CTT AT-3') were used to amplify DNA molecules encoding for the amino acid sequences of the three biopeptides (the triplets corresponding to each amino acid able to produce the biopeptide in the correct form were selected taking into account the E. coli codon usage). The primers have a partially complementary and inverted sequence which allows amplification of a short region of 16 base pairs. The three primer pairs were hybridized and, after amplification, sufficient amounts of DNA were obtained for cloning in the pGEM-T transcription vector.

The positive clones (recognized by colony PCR, DNA extraction, restriction analysis and sequence analysis) were used as a template for further amplification with primer pairs specific for cloning in pGEX-4T-l .

The insertion of the fragment in the pGEX vector was carried out by digestion of both the fragment and the vector with EcoRI and Xhol, followed by ligation. The fragments were inserted in the vector and used to transform E. coli competent cells (BL21-DE3). Colony screening was carried out with colony PCR and the isolated plasmid DNA was sequenced.

After inoculum and induction of the bacterial culture, transformed E. coli cells were sonicated in order to disrupt the cell wall and allow the release of the fusion protein in the soluble phase.

Glutathione-thio-transferase fusion proteins were purified from the bacterial lysate through affinity chromatography on the Glutathione Sepharose 4B resin (glutathione molecules bound to a sepharose matrix).

Purification was first tested on a small amount of cells (2 ml of cell culture). After verifying the presence of the fusion protein through SDS Gel Electrophoresis (SDS-PAGE) a protein fraction was prepared from 500 ml of cell debris and purified through column chromatography.

Starting from 500 ml of induced bacterial culture, as determined by quantitative (Bradford assay) and densitometric analyses, an average amount of about 15 mg of recombinant fusion protein of each of the three biopeptides was obtained from 45 mg of purified fusion protein present in the sonicate.

In order to verify the presence of the GST-BP fusion protein, 50 μg of each GST-BP was digested with thrombin. After SDS-PAGE, the analysis showed the presence of a peptide of about 3 kDa compatible with the dimensions of each biopeptide.

As further confirmation of the expressed sequences, the fusion proteins were subjected to ESI/MS (Electro Spray Ionization/Mass Spectrometry) analysis, which confirms the presence of the cloned biopeptide downstream of the GST protein.

In order to exert their ACE-inhibitory activity, the three biopeptides need to be released in the active form from the GST-fusion proteins through digestion with an enzyme fraction isolated from Lactobacillus helveticus PR.4. The cell wall Tightly Associated Fraction (TAF) containing a serine- proteinase was extracted from one litre of a 24-hour culture of this microorganism.

The protease activity of the TAF fraction was preliminary assayed on bovine β-casein, after suitable incubation times (4h-8h-24h). SDS-PAGE of the incubated β-casein showed the proteinase activity of the TAF.

This fraction was then used for digesting the BP-GST fusion proteins, under the same conditions as β-casein hydrolysis. SDS-PAGE confirmed the release of the biopeptide after excision of the BP-GST fusion protein with the TAF of L. helveticus PR4. The subsequent step was the evaluation of the ACE-inhibitory activity of GST-BP proteins hydrolysed with TAF for 24h at 37⁰C. Suitable concentrations of these samples were used in the in vitro ACE-inhibition assay described by Nakamura (Nakamura et al., 1995 Journal of Dairy Science, 78: 777-783), suitably modified. This analysis allows the measurement of the percentage of inhibitory activity of GST-BPl, GST-BP2 and GST-BP3 using the uncleaved fusion proteins and the native GST proteins as negative controls.

The results showed the high ACE inhibitory activity of TAF-hydrolysed GST-BP l , GST-BP2 and GST-BP3. In detail, 80 μg of hydrolysed fusion protein inhibit the ACE activity by 50%, whereas the GST native protein, TAF-hydrolized GST protein and uncleaved fusion proteins do not show any inhibitory activity. The peptides thereby released were then purified through RP-HPLC and their IC₅₀ was determined (the peptide concentration causing 50% ACE inhibition). A 3.2 ml RPC Resource column equipped with an AKTA-prime instrument with detector at a wavelength of 214 nm for peptide detection was used (both from Amersham-Pharmacia). The fractions containing the biopeptides were suitably collected and lyophilized after removal of the solvents. The peptide concentration was evaluated using the orto- phthaldialdehyde (OPA) method. The BPl IC_5O, determined according to Nakamura, was 467 μM, the BP2 IC₅₀ was 807 μM and the BP3 IC₅₀ was 1729 μM. These data, compared with those from experiments carried out on biopeptides containing the BP l, BP2 and BP3 sequences, allow us to conclude that the ACE-inhibitory activity of biopeptides obtained using the process of the invention is comparable to that of peptides purified by RP-HPLC from hydrolysed casein. Uses of the biopeptides

The ACE-inhibitory biopeptides of the invention can be formulated as a milk drink, either fermented or not; if taken regularly, they can contribute to the maintenance of acceptable pressure levels in subjects with moderate hypertension. For this purpose, BPl, BP2 and BP3 were singularly added to reconstituted skimmed milk powder, at concentrations of 2, 2.2 and 4.4 mg/ml respectively. The ACE inhibition value was determined in vitro, applying the method described by Nakamura et al. (1995), suitably modified, to the pH 4.6 soluble fractions of these lactic drinks. The assay was also carried out on a control, i.e. reconstituted skimmed milk powder without the addition of biopeptides, in order to exclude any ACE-inhibitory activity of substances naturally occurring in the product under study. The results showed that milks added with the single biopeptides have much greater ACE-inhibitory activity than the control.

In order to evaluate the possibility of developing a fermented milk product added with ACE-inhibiting biopeptides to be used as a the diet- supplement for patients suffering from moderate hypertension, milk added with BPl , BP2 and BP3 at the above-mentioned concentrations is inoculated with lyophilized Lactobacillus acidophilus LA 14 (initial cell density of about 10⁷ ufc/ml); fermentation was carried out at 37°C for 22 hours. After fermentation, in vitro ACE-inhibition assays on pH 4.6 soluble fractions added with each biopeptide showed that their antihypertensive activity remained essentially unchanged after 22-hour fermentation in comparison with the activity of non-fermented milk drinks. This result suggests that the added peptides are not degraded by the proteolytic system of the starter used and that therefore their bioactivity remains unchanged.

The described application is only an example of the possible uses of the three ACE-inibitors biopeptides; in fact, it is also in principle possible to add the tested peptides to different foods, such as fruit juices or other drinks.

Claims

1. A bovine β-casein peptide with ACE-inhibiting activity selected from SEQ ID No. 1 and SEQ ID No. 2.

2. Nutraceutical foods containing the peptides of claim 1.

3. Nutraceutical foods according to claim 2, further comprising a peptide selected from SEQ ID No. 3, 4, 5 and 6.

4. Nutraceutical foods according to claim 3, added with lactobacilli.

5. Nutraceutical foods according to claim 3 or 4, in the form of an optionally- fermented milk drink.

6. Use of the peptides of claim 1 for the preparation of nutraceutical foods, optionally in combination with the peptides of claim 3.

7. A process for the preparation of a β-casein peptide, which comprises the following steps: a. expression of the peptide in bacterial cells in the form of fusion protein with glutathione-thio-transferase (GST); b. purification of the fusion protein through affinity chromatography on a glutathione-sepharose resin; c. digestion of the fusion protein by Lactobacillus helveticus PR4 proteinase preparation; d. purification of the peptide.

8. The process according to claim 7 wherein the β-casein peptide is selected from:

SEQ ID No. 1 ; SEQ ID No. 3;

SEQ ID No. 4.

9. The process according to claims 7 or 8 wherein the Lactobacillus helveticus PR4 proteinase preparation is the cell wall tightly associated fraction containing a serine-proteinase.