WO2011135513A1 - Process for obtaining bioactive peptide extracts by hydrolysis of whey protein by cynara cardunculus enzymes, aforementioned extracts and respective applications - Google Patents

Abstract

The present invention relates to a process for obtaining, peptide extracts from whey proteins by hydrolysis of one or more proteins or peptides containing the amino acid sequences of bioactive peptides, using for that purpose, proteolytic enzymes in particular cardosins and other similar enzymes- produced by Cynara's species. Alternatively, peptides can be obtained by chemical, synthesis or by enzymatic processes and/or fermentation. The process enclosed by the present invention allows obtaining protein concentrates and peptides with low-lactose and salts content, which have biological activities relevant to the formulation of products in food and pharmaceutical industries. Thus, the hydrolysates and their fractions or peptides resulting from this process can be used in food products acting as a preservative or after eating, strengthening the body's natural defenses. Alternatively, they can be also used in the preparation of pharmaceutical products aimed at diseases, particularly to facilitate control of blood pressure and/or bacterial infections, as well as inflammatory and ulcerogenic processes. The invention allows enlarging the applications of milk proteins, thus contributing to the recovery and in particular the valorization of whey.

Description

 DESCRIPTION

 "PROCEDURE FOR OBTAINING BIOACTIVE PEPTITIC EXTRACTS THROUGH HYDROLYSIS OF CYNARA CARDUNCULUS Whey Proteins, REFERRED TO AND

 ITS USES "

Technical field of the invention

The present invention relates to a process for producing peptide concentrates derived from whey proteins by enzymatic hydrolysis and filtration techniques. The resulting process concentrates according to the present invention comprise in their constitution peptides with various biological activities, such as antihypertensive, antiinflammatory, antiulcerative and antioxidant, which may be applied in the food and pharmaceutical industries.

Background of the Invention

These days, it is increasingly important to have a healthy diet; Most people are predisposed to do so as long as they do not alter their eating pattern too much. Consumers' barriers to changing their eating habits have led to the emergence of a wide range of foods that look similar to conventional foods but which, due to the existence of certain functional ingredients, bring additional health benefits (these foods can act preventively) (Korhonen and Pihlanto, 2006).

A food may be considered "functional" if, in addition to its nutritional effect, it has benefits over one or more functions of the body which may improve the state of health or well-being or reduce the risk of disease. This is the definition proposed by FUFOSE (Functional Food Science in Europe), and it also differentiates three important aspects: (1) the functional effect is distinct from the nutritional one; (2) the benefit presented must be scientifically substantiated; and (3) the effect may be an improvement in physiological functions (including well-being) or a reduction in the risk of developing a disease process. Today, and due to growing consumer concern about the food / health binomial, the functional food market is booming.

The possibility of obtaining a new food product that reduces or controls chronic disease is very promising, as is the potential for functional foods to mitigate disease, promote health and reduce healthcare costs (Korhonen and Pihlanto, 2006; Hartmann and Meisel, 2007).

For a long time, it was thought that food proteins were only meant to provide amino acids for the body to build new tissues. Recent advances in nutritional biochemistry and various biomedical research have helped unravel the complex relationships between nutrition and disease, suggesting that food proteins and peptides derived from them, either through the digestive process or through in vitro proteolysis, can play important roles in preventing and / or treatment of nutrition-related conditions, pathogen infections or chronic diseases. Some of these functional agents appear to be important in preserving health, promoting well-being and increasing longevity. Cheese whey is a by-product of the dairy industry, which has been considered as waste over the years, in addition to its pollutant character due to its high lactose and protein content. However, whey represents a rich and varied mixture of proteins that under certain circumstances appear to perform physiological actions in vivo and / or in vitro; Therefore, whey concentrates have been used as a protein supplement in several functional foods (Smithers, 2008).

In the case of bovine milk approximately 9 L of whey is produced from 10 L of milk during cheese production. Bearing in mind that approximately 12.6 million tonnes of cheese were produced in the world in 2002 (USDA Foreign Agricultural Service, 2003) and that production tends to increase the use of such a quantity of whey (thus avoiding its elimination) This becomes a problem as some products are close to market saturation. For environmental reasons, whey cannot be discharged into rivers due to its high biochemical and chemical oxygen demand.

For strictly economic reasons, it is difficult to use whey in animal feed or as fertilizer. In fact, although it contains c. 93% water, serum is a reservoir of high value components as it contains c. 50% of the nutrients found in milk. This composition depends on the way cheese is manufactured, but the most produced residue is lactose, in addition to protein and minerals, particularly calcium, and vitamins such as thiamine, riboflavin and pyridoxine. It is therefore crucial to find ways of valuing this by-product by obtaining high value added products. Whey proteins have a mixture of various secreted proteins such as α-lactalbumin (oc-La), β-lactoglobulin (β-Lg), lactoferrin, lactoperoxidase, immunoglobulins and caseinomacropeptide (CMP), as well as a number of factors. of growth.

The use of these proteins, after concentration or even isolation, or biologically active peptides obtained therefrom as nutraceutical supplements constitutes an economically appealing (and even desirable) use for serum. Studies to date have shown a number of biological effects of these proteins, ranging from anticancer effects to beneficial effects on digestive function, through in vitro and in vivo antimicrobial activity (Smithers, 2008).

 Most of the bioactive components of milk are also present in whey, except for caseins.

Bioactive peptides are amino acid sequences that are inactive in the proteins that originate them, but once released by enzymatic and / or fermentative processes, play a physiological role. As mentioned above, there are several types of bioactive peptides depending on their activity: ACE (Angiotensin-Converting Enzyme) inhibitor peptides and / or antihypertensive, antithrombotic, hypocholesterolemic, opioid, phosphopeptide (mineral chelating) regulators. satiety, immunomodulators, antimicrobials, antivirals, antitumor and antioxidants.

Of these bioactive peptides, ACE inhibitor peptides should be highlighted, given the incidence that hypertension has on the population of developed countries, which is a worldwide problem affecting 15-20% of also because other complications are associated with it.

Bioactive peptides derived from dairy proteins can be obtained on an industrial scale, mainly by hydrolysis with digestive-like enzymes but of microbial, plant or animal origin, as well as by fermentation with starter dairy cultures. In some studies, it has been found that the combination of these processes may be determinant for obtaining small functional peptides (Korhonen and Pihlanto, 2006). Potential applications of these hydrolyzates in fermented milks and / or their derived peptides as dietary supplements, pharmaceutical preparations or functional ingredients emerge with particular interest to the pharmaceutical and food industries - as they are foods and have a therapeutic effect, respectively. There are, therefore, some related patents - which are briefly described below.

WO1999065326 describes a process for obtaining protein products by partial hydrolysis of whey with essentially protease ^β- type, Protease A, Protease, Peptidase, Neatrase, Validase and AFP 2000 proteases for incorporation into industrial products. thus providing functional food products with improved organoleptic properties such as sweeter taste and lower levels of additives.

On the other hand, WO2008108649 describes a process for obtaining a peptide-based food product with antihypertensive activity by hydrolysis of whey proteins, and subsequent separation and / or concentration of the fraction rich in IIAEK pentapeptide, IPAVF and / or IPAVFK hexapeptide. To this end, said process uses inter alia trypsin, chymotrypsin or a combination of both.

summary

The present invention relates to a process for obtaining peptide extracts, characterized in that they are produced by hydrolysis reactions of whey proteins by the action of at least one enzyme of C. candunculus aqueous extracts.

In one process, the concentration of C. candunculus flower extract should be between 1 and 5% (v / v), and preferably 1.6% (v / v).

In another more detailed example, the process may comprise the following steps:

 Whey skimming (1), preferably by centrifugation;

 Microbial load reduction (2), preferably by microfiltration;

 Ultrafiltration of the serum using a 10,000-20,000 Da membrane, preferably 20,000 Da due to the size of the whey proteins, thus obtaining a protein-rich concentrate (3-retained RUFi) and a peptide extract (4-filtered FUFi). ;

 Diafiltration of the protein-rich concentrate (3) by successive dilution and membrane concentration of 10,000-20,000 Da, preferably 20,000 Da due to the size of the whey proteins;

Hydrolysis of the protein-rich extract (5) resulting from d) (RUF ₂ ) with enzymes from C. flower extract candunculus, preferably for 7 h (optimal conditions);

 Ultrafiltration of the final hydrolyzate with a 10,000-20,000 Da membrane, preferably 20,000 Da due to the whey protein size;

Concentration of the RUF ₃ (6) retentate and the FUF ₃ (7) filtrate, preferably by reverse osmosis, in order to obtain a whey protein rich concentrate (unhydrolyzed protein) and a peptide rich concentrate released during said hydrolysis, respectively; and

Nanofiltration of the FUF ₃ (7) with 1000-5000 Da membrane, preferably 3000 Da.

In a practical embodiment, the <3000 Da fraction should be isolated from the remaining fraction by a membrane separation technique. More specifically, such separation should be by nanofiltration, with pore size membrane between 1000 and 5000 Da, preferably 3000 Da.

The present invention further relates to extracts obtained by the processes described above. These extracts exhibit biological activities, such as inhibition of ACE, antioxidant, antinociceptive and / or anti-inflammatory activity.

In an even larger embodiment, extracts obtained by the procedures described above further contain at least one of the following peptides, identified with the sequences: SEQ.ID.No.1, SEQ.ID.No.2, SEQ.ID.No. , SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 or SEQ ID No. 7. In further detail, see peptide SEQ.ID. No. 7. In even more preferred cases, the extracts may contain at least one of the peptides identified with the sequences SEQ.ID.No. 8, SEQ.ID.N. SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13,

SEQ.ID. No. 14 and SEQ.ID. No. 15.

These extracts may be used:

 in the preparation of an ACE inhibitor or preparations having antioxidant, antinociceptive and / or anti-inflammatory activity;

 in the preparation of a pharmaceutical composition for the prevention and / or treatment of conditions associated with hypertension, oxidative conditions and neoplasms, and / or as an analgesic; or

 in the preparation of food supplements and / or additives.

The present invention further relates to peptides obtained by the methods described above - wherein such peptides exhibit biological activity, for example as ACE activity inhibitors, antioxidants, antinociceptive, antimicrobial and / or anti-inflammatory agents.

In another preferred application, the peptides comprise one of the following amino acid sequences: SEQ.ID.No. 1, SEQ.ID.No. 2, SEQ.ID.No. 3., SEQ.ID.N. No. 5, SEQ.ID. No. 6 or SEQ.ID. No. 7; or may further comprise the amino acid sequences: SEQ.ID.No. 8, SEQ.ID.N.9, SEQ.ID.N.10,

SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14 and SEQ. ID. No. 15.

Said peptides may be used:

in the preparation of inhibitors of ACE activity, or as antioxidants, antinociceptive agents and / or antiinflammatory agents; in the preparation of pharmaceutical compositions for the prevention and / or treatment of conditions associated with hypertension, oxidative conditions, and neoplasms and / or as an analgesic;

 in the preparation of food supplements and / or additives.

General Description of the Invention

The present invention relates to a process of preparing bioactive peptides and peptide extracts from whey proteins by hydrolytic action of cardosines and similar enzymes. Another aspect of the present invention is the development and optimization of a membrane filtration process which yields low lactose and salt protein and peptide concentrates which exhibit biological activities.

 A further aspect of the present invention is the use of these peptide extracts in food and pharmaceutical products that have health benefits, particularly in the treatment and / or prevention of pathologies.

A. Bioactive Peptide Extracts

A.l. Obtaining bioactive peptide extracts

Within the scope of the present invention, peptides and extracts containing bioactive peptides were obtained by hydrolysis reactions of enzyme-catalyzed whey protein solutions from Cynara cardunculus.

The proteolytic systems were used: CC-Lactoalbumin (OC-La), the second quantitatively higher protein. representative of bovine whey; Caseinomacropeptide (CMP), heterogeneous polypeptide fraction derived from the disruption of the κ-casein (K-CN) Pheios-Met106 bond; and Bovine Whey Protein Concentrate (CPS) as substrates; and cardosins as hydrolysis enzymes.

System 1. In the hydrolysis system consisting of CPS: commercial thistle extract, the hydrolysis reaction takes place at temperatures between 50 and 60 ° C and at pH values between 4,8 and 6,2. Preferably, the hydrolysis reaction takes place at a temperature of 55 ° C and a pH value of 5.2 over a period of 7 hours.

In the preferred embodiment of the Protein Whey Concentrate (CPS): commercial thistle extract hydrolysis system, the concentration of commercial thistle extract is between 1 and 5% (v / v), and preferably will be 1.6% (v / v). The concentration of the CPS solution to be hydrolyzed is preferably 40 g / l, without limiting application to CPS solutions with other concentration values where the system is also effective. Under preferred conditions, the degree of hydrolysis of CPS proteins is 18%.

System 2. In the hydrolysis system consisting of concentrate -La: commercial thistle extract, the hydrolysis reaction takes place at temperatures between 50 and 60 ° C and at pH values between 4.8 and 6.2. Preferably, the hydrolysis reaction takes place at a temperature of 55 ° C and a pH value of 5.2 over a period of 7 hours.

In the preferred embodiment of the hydrolysis system consisting of α-La: commercial thistle extract, the Concentration of commercial thistle extract is between 1 and 5% (v / v), and preferably will be 1.6% (v / v). The concentration of the -La solution to be hydrolyzed is preferably 40 g / l, without limiting application to α-La solutions with other concentration values where the system is also effective. Under preferred conditions, the degree of hydrolysis of α-La protein is 8%.

System 3. In the hydrolysis system consisting of CMP: commercial thistle extract, the hydrolysis reaction takes place at temperatures between 50 and 60 ° C and at pH values between 4,8 and 6,2. Preferably, the hydrolysis reaction takes place at a temperature of 55 ° C and a pH value of 5.2 over a period of 7 hours.

In the preferred embodiment of the hydrolysis system consisting of CMP: commercial thistle extract, the concentration of commercial thistle extract is between 10 and 15% (v / v) and preferably is 11.5% (v / v). The concentration of the CMP solution to be hydrolyzed is preferably 40 g / l, without limiting application to CMP solutions with other concentration values where the system is also effective. Under preferred conditions, the degree of hydrolysis of CMP is 10%.

A.2. Screening of biological activity of extracts

Extracts obtained through hydrolysis reactions of systems 1, 2 and 3 (CPS, -La and CMP, respectively) were found to have ACE inhibitory activity, with an IC ₅₀ index of 105.4, 47.6 and 296. , 0 μg / mL, respectively, for the total fraction. The <3000 Da and> 3000 Da fractions of said peptide extracts have also been found to have distinct inhibitory effects on the ACE. The first presents IC ₅₀ values of 25, 6, 22, 5 and 63.0 μg / mL for systems 1, 2 and 3, respectively. On the other hand, the fraction> 3000 Da has IC ₅₀ values of 717.0, 539.5 and 717.0 μg / mL for the same systems.

Therefore, the <3000 Da fraction of the hydrolyzate exhibits very good ACE inhibitory activity relative to the> 3000 Da fraction, thus evidencing that lower molecular weight peptides are primarily responsible for the inhibitory activity of bioactive peptides on ACE.

Extracts obtained from systems 1, 2 and 3 (total fraction) also show antioxidant activity of 0.96 ± 0.08 1.12 ± 0.13 and 0.22 ± 0.002 μιηοΐ Trolox equivalents / mg protein. hydrolyzate, respectively.

B. Pilot Serum Treatment System for Peptide Concentrate Production

 Another aspect of the present invention comprises the development of a novel whey treatment process which converts the whey into derivatives of high commercial value including bioactive peptide extracts also in accordance with the present invention.

The treatment process involves the successive application of selective filtration techniques associated with hydrolysis by enzymes in the thistle extract as described in Figure 1. The experimental conditions of the hydrolysis reaction are the same as those previously defined for obtaining the peptide extracts in systems 1 and 2.

The process of the present invention comprises the following steps, to which further steps may be added if the serum sample requires additional treatments that do not distort the spirit of the invention:

 Serum skimming (1), for example by centrifugation.

 - Microbial load reduction (2), for example through microfiltration.

 Ultrafiltration of the serum using a 10,000-20,000 Da membrane, preferably 20,000 Da membrane, thereby obtaining a protein-rich concentrate (3-retained RUFi) and a peptide extract (4-filtered FUFi).

- Filtration of the protein-rich concentrate (3), which has been successively diluted and concentrated by a membrane of 10,000-20,000 Da, preferably 20,000 Da, in order to decrease the content of lactose and salts present (which is in the filtrate FUF 2) and a protein rich extract is retained (5 - retained RUF ₂ ).

- Hydrolysis of the protein rich extract (5 - retained RUF ₂ ) under conditions defined for hydrolysis systems 1 and 2, ie with a pH between 4.8-6.2, preferably pH of 5.2 extract concentration 1 to 5% (v / v) thistle, preferably 1.6% (v / v), at a temperature between 50-60 ° C, preferably 55 ° C, and also preferably for 7 h.

 Ultrafiltration of the final hydrolyzate with a 10,000-20,000 Da membrane, preferably 20,000 Da, in order to separate the unhydrolyzed protein (6 - retained RUF3) from the peptides obtained by hydrolysis (7 - FUF3 filtrate).

- Concentration of RUF ₃ (6) retentate and FUF ₃ (7) filtrate, for example by reverse osmosis, to obtain a whey protein concentrate (unhydrolyzed protein) and a peptide rich concentrate released during hydrolysis, respectively. Nanofiltration of the FUF3 (7) with 1,000-5,000 Da, preferably 3,000 Da, membrane to separate large peptides from small peptides.

From the process developed as described above, three value-added fractions are used: a 73% protein and low lactose peptide concentrate (7 - FUF ₃ filtrate, which is a peptide hydrolyzate considered in its total fraction), the respective fraction <3000 From the result of 87% degradation of -La and a 90% protein low-lactose unhydrolyzed protein concentrate (6 - RUF ₃ concentrate) as a high β-Lg concentrate It can be easily applied on a large scale in the food industry and at reduced cost when compared to alternative separation methods.

The protein and peptide fractions obtained in the filtration processes were characterized in physicochemical and microbiological terms.

C. Biological activity of extracts obtained Cl and C.4. ACE inhibitory activity:

 Initial studies (section A) demonstrated an in vitro ACE inhibitory action of the extracts obtained, especially in extracts containing peptides originating from α-La hydrolysis and their fraction <3000 Da.

C.2 Microbial and prebiotic activity

The hydrolysis peptide concentrate (7) generated in the section B process (CPSH) shows in vitro antimicrobial activity for Staphylococcus aureus in the <3000 Da fraction. Concerning the prebiotic action (commercial CPS, total fraction CPSH and <3000 Da) on Lactobacillus acidophilus (Ki and LAFT ^® L10) and Lactobacillus paracasei, there was a greater potentiating power for commercial CPS.

C.3 Anticancer activity

 From the studies on anti-cancer activity for CPSH in its total fraction and <3000 Da, none of the peptide extracts were found to have cytocidal activity and cytostatic activity in virtually all strains at concentrations above 1000 pg / kg. mL, except for the leukemia strain (K562) which showed growth inhibitory effects at a concentration of 25 pg / mL. Hydrolysates have no toxicity to the normal cells analyzed.

C.5 Antiulcerogenic activity

In the in vivo study of antiulcerogenic activity in the absolute ethanol-induced ulcer model of total and <3000 Da CSFH by gavage fractions, antiulcerative activity - with ILU inhibition was observed at all concentrations. At a repetitive dose of 100, 200 and 350 mg / kg bw and single dose of 30, 100, 300 and 350 mg / kg _bw , no relationship was found between ulcerative lesion rates and different concentrations. extract (dose / effect). Acute single-dose treatment had greater effects, with values of 48.5 and 71.7% for total fraction and <3000 Da, respectively. Therefore, the most appropriate treatment for this activity is acute single-dose treatment of 100 mg / kg _bw . Ç . 7 Antinociceptive Activity

 Antinociceptive activity was determined by classical models that evaluate different mechanisms of analgesia, whether central, peripheral or inflammatory. The following two nociception models were used: Thermal (Hot Plate) (neurogenic pain, direct central action) and Formaline-Induced Algesia (neurogenic and inflammatory pain).

 The CPSH did not increase the reaction time of the animals to the thermal pain stimulus (56 ± 0.1 ° C), and the studies carried out show that the extracts do not have direct central action antinociceptive activity.

Using the mouse formalin test, which is a chemical model of nociception, a dose of 1 g / kg was used to evaluate the action of CPSH. In phase I (0-5 min) of the test, it was observed that CPSH - total fraction reduced by 42%, and CPSH - <3000 Da by 32%, the reactivity of the animals; In the case of total CPSH, the efficacy is greater than the positive morphine control group (35%), demonstrating that the antinociceptive action of the extracts may be related to peripheral mechanisms. In phase II (20 min from the beginning), indomethacin was used as a positive control. Indomethacin reduced by 36%, and CPSH extracts reduced by 38 and 37% for the total fraction and <3000 Da, respectively, the reactivity of the animals, evidencing the analgesic action of the extracts on pain of inflammatory origin. The determination of antinociceptive activity through the evaluation of heat-induced algesia and the formalin-induced neurogenic and inflammatory pain model allowed us to determine that the extracts have antinociceptive action, and that it is related to the pain of neurogenic and inflammatory origin. The extracts showed no anti-edematological activity.

D Identification and characterization of peptides

 Also within the scope of the present invention are hydrolysis peptides and extracts containing the peptides having the following amino acid sequences:

SEQ.ID.N ⁰ 3: DKVGINY (97-103),

SEQ.ID.N ⁰ 4: KVGINYW (98-104),

SEQ.ID.N ⁰ 5: DKVGINYW (97-104) from degradation of -La as well as peptide SEQ.ID.N ⁰ 15: DAQSAPLRVY (33-42) from degradation of β-Lg showing residues hydrophobic at the C-terminal position and therefore potential competitive substrates or inhibitors of ACE, and the peptide with the sequence

SEQ.ID.N ⁰ 1: KGYGGVSL (16-23),

SEQ.ID.N ⁰ 2: RELKDL (10-15) from degradation of -La, as well as the peptide with the sequence SEQ.ID.N ⁰ 14: RELEEL (1-6) from degradation of β- CN, showing C-terminal Leu residues, as well as the peptide with the sequence

SEQ.ID.N ⁰ 7: KTEIPIN (116-123) from the hydrolysis of K-CN (CMP), which present a Pro residue in the penultimate position, favoring the union of the peptide with the ACE. These peptides are obtained in the pilot serum treatment system for production of peptide concentrates fraction FUF3 filtrate (peptide hydrolyzate total fraction) and its fraction <3000 Da.

In these extracts there is also the presence of peptides, or peptides analogous to those already described, with the following amino acid sequences:

SEQ.ID.N ⁰ 9: MAIPPKKNDQD (106-115); SEQ.ID. No. 8: AIPPKKNDQD (107-115), derived from the κ-CN (CMP) hydrolysis reaction, as well as the peptide with the sequence SEQ.ID. 6: KGYGGVSLPEW (16-26), derived from the degradation of -La, whose inhibitory effects on ACE are already known.

Also within the scope of the present invention are hydrolysis-derived peptides and peptide-containing extracts having the following amino acid sequences:

SEQ.ID. ° 1: KGYGGVSL (16-23),

SEQ.ID. ° 2: RELKDL (10-15),

SEQ.ID. 3: DKVGINY (97-103),

SEQ.ID. 4: KVGINYW (98-104),

SEQ.ID. 5: DKVGINYW (97-104),

 SEQ.ID. ° 6: KGYGGVSLPEW (16-26) from degradation of -La, as well as peptides with the sequences

SEQ.ID. 7: KTEIPTIN (116-123),

SEQ.ID. 10: TVQVTSTAV (161-169),

SEQ.ID. ° 11: QVTSTAV (163-169),

SEQ.ID. ° 12: VQVTSTAV (162-169), derived from the degradation of K-CN, the peptide SEQ.ID. ° 14: RELEEL (1-6) from degradation of β-CN and peptide SEQ.ID. ° 15: DAQSAPLRVY (33-42) from β-Lg degradation, showing hydrophobic residues at one or more positions of the C-terminal tripeptide, especially peptides displaying Pro, Trp, Tyr, Phe or Leu residues at the C-terminal. , or Pro in the penultimate position, which favors the union of the peptide with ACE, thus being potential substrates or competitive inhibitors of ACE. Such peptides are obtained in the pilot serum treatment system for producing peptide concentrates fractions 7 - filtered FUF ₃ (peptide hydrolyzate total fraction), and the respective fraction <3000 Da.

The sequences, SEQ.ID.N ⁰ 9: MAIPPKKNDQD (106-115) and

 SEQ.ID. No. 8: AIPPKKNDQD (107-115), derived from the κ-CN hydrolysis reaction (CMP), show no potential ACE inhibitory activity; however, when analyzed for the C-terminal tripeptide, it appears to contain in its constitution the tripeptide IPP, endowed with potent proven antihypertensive activity.

 In these extracts there is also the presence of analog peptides already described with the following amino acid sequences:

SEQ.ID.N ⁰ 10: TVQVTSTAV (161-169),

SEQ.ID.N ⁰ 11: QVTSTAV (163-169),

SEQ.ID.N ⁰ 12: VQVTSTAV (162-169),

SEQ.ID.N ⁰ 15: DAQSAPLRVY (33-42) and

SEQ.ID.N ⁰ 6: KGYGGVSLPEW (16-26), which includes the VTSTAV, SAPLRVY and VSLPEW sequences, as well as the LKGYGGVSLPEW sequence - whose inhibitory effects on ACE are already known.

In determining the activity of ACE inhibidora, pure peptides show the CI values ₅ 99.9 ± 8.1 μΜ for the peptide with the sequence SEQ.ID.N ⁰ 3: DKVGINY (97-103) 11.3 ± 0.9 μΜ for the peptide with sequence SEQ.ID.N ⁰ 15: DAQSAPLRVY (33-42), 9.7 ± 0.4 μΜ for peptide with sequence SEQ.ID.N ⁰ 5: DKVGINYW (97-104) and 0.9 ± 0.07 μΜ for peptide with the sequence SEQ.ID.N ⁰ 6: KGYGGVSLPEW (16-26). Among said peptides are therefore 3 with potent ACE inhibitory activity.

The results obtained in the present invention demonstrate that it is mainly the peptides obtained from the hydrolysis of La that are responsible for the unexpected increase of the inhibitory activity of the extracts obtained on the ACE. Brief Description of the Figures

Figure 1. Obtainment of whey protein concentrates in pilot system and hydrolysis of the final protein concentrate under previously optimized conditions. Skimming steps were performed by centrifugation, followed by microfiltration to reduce the microbial load. Next, two ultrafiltration (diafiltration) steps were performed to concentrate the retentate proteins and reduce the lactose and mineral content. After hydrolysis, a new ultrafiltration was performed to separate the hydrolyzate (FUF ₃ ) from the unhydrolyzed (RUF ₃ ), which were subsequently reverse osmosed, and the FUF ₃ fraction was nanofiltrated.

Table 1. Identification of <3000 Da fraction peptides of hydrolyzate resulting from hydrolysis for 7 h CPS at 40 g / l with 1.6% (v / v) thistle extract at pH 5.2 and 55 ° C . Indication of amino acid sequences (SEQ. ID. No), and amino acid composition (obtained by sequencing) of the respective whey protein fraction.

SEQ IDL NO protein fraction sequence

 1 KGYGGVSL (16-23) a- La

 2 RELKDL (10-15) a- La

 3 DKVGINY (97-106) a- La

 4 KVGNYW (98-104) a- La

 5 DKVGNYW (97-104) a- La

 6 KGYGGVSLPEW (16-26) a- La

 7 Ν ΡΠ Ν (116-123) k-CN

 8 AIPPKKNQP (107-115) k-CN

 9MM PPKKNQP (106-115) k-CN

 10 TVQVTSTAV (161-169) k-CN

 11 QS TSTAV (163-169) k-CN

 12 VQV STAV (162-169) k-CN

 13 AVEST (138-142) k-CN

 14 RELEEL (1-6) β-CN

15 DAQgAPLRVY (33-42) β-Lg Detailed Description of the Invention A. Bioactive Peptide Extracts

A.l. Obtaining bioactive peptide extracts - Proteolysis reaction:

Evaluation of thistle commercial enzyme extract activity at different concentrations over time under optimal pH and temperature conditions. A CPS (Lacprodan), CMP (Davisco ^® ) and α-La (Sigma) were used as substrate to determine the optimal enzyme / substrate ratio and reaction time as a function of the following variables. : degree of hydrolysis, ACE inhibitory activity and antioxidant activity. Monitoring of the degree of hydrolysis was performed by the TNBS method as modified by Spadaro et al. (1979), and the determination of ACE inhibitory activity was performed according to the Sentandreu and Toldrá (2006) method with modifications, while the ORAC method described by Ou et al. (2001) and modified by Dávalos et al. (2004) was used to evaluate antioxidant activity.

The effect of enzyme / substrate ratio (R) and time (Γ) on the different response variables (degree of hydrolysis, ACE inhibitory activity and antioxidant activity) was studied through an experimental factorial design. A total of 10 enzyme extract experiments were performed: four points for the full factorial design, four star points (a = 1,414 away), and two central points to verify the experimental errors. Using this experimental design, the two variables were tested at 5 experimental levels: ratio enzyme / substrate at 1.6, 3.0, 6.5, 10.0 and 11.4, and time at 0, 1, 3.5, 6 and 7 h, corresponding to the following codes: 1.414, -1 , 0, +1, +1.414, respectively. The proposed polynomial quadratic model for each response variable was (1): γ ± = β ₀ + βι-R + β ₂ τ + _lrl R ² + β ₂ , ₂ τ ² + βι, ₂ £ Γ + ε (ΐ)

Where β ₀ is a constant, βι and β ₂ are linear coefficients, βι, ι and β ₂ , 2 are quadratic coefficients, βι, ₂ is the interaction coefficient, and ε is the error. The model parameters were estimated by linear multiple regression (MLR), using the program Stat-graphics Plus v.5.1. (Statistical Graphics Corporation, Manugistics Inc., MD, USA, 2000) - which allows the creation and analysis of experimental designs.

The effect of each term in the model and its statistical significance for each response variable were analyzed using the Pareto Standard graph. Terms that did not differ significantly from zero (P> 0.10) were excluded from the model, and the mathematical model was readjusted by MLR. The best fit of the model was evaluated by the coefficient of determination (R ² ) and the residual standard deviation (RSD). For the new fitted models, the estimated response surfaces and optimal conditions that maximize each response variable were obtained.

Materials:

Substrate . Commercial CPS (Lacprodan), CMP (Davisco ^® ) and -La (Sigma). Enzymes and reagents. Vegetable rennet extracts (0.25 UK (mg protein to coagulate 10 mL milk)) (C. candunculus flower extract enzyme with standard activity - Formulab, Maia, Portugal), Abz-Gly-Phe (O ₂ ) -Pro (o-aminobenzoylglycyl-p-nitrophenylalanylproline) (Bachem

Feinchemikalien, Bubendorf, Switzerland); fluorescein (Sigma Chemical, St. Louis, MO, USA); 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Aldrich Chemie, Munich, Germany); 2,2'-azo-bis- (2-methylpropionamidine) dihydrochloride AAPH (Aldrich Chemie).

Experimental procedure :

Hydrolysis reaction in model system: 25 mL of CPS, 25 mL of CMP and 25 mL of -La, with 40 g / l protein content, were hydrolyzed under the above conditions to pH 4.8-6.2 with extracts. Thistle The samples were subjected to enzymatic hydrolysis by incubation in a shaking bath at 50-60 ° C with thistle extracts (Barros and Malcata, 2001) and taken at different time periods (determined by factorial design). The reaction was stopped by placing the samples in a 95 ° C bath for 30 min. After stopping the reaction, all hydrolysates were centrifuged at 10,000-20.00 Oxg for 15 min at 2-6 ° C, and the supernatants were properly collected. Hydrolyzate supernatants were subjected to an ultrafiltration process through a 1000-5000 Da pore size hydrophilic membrane (Centricon, Amicon Inc., Beverly, MA, USA).

Determination of protein content: Determinations of protein content were made by the Kjeldahl method (IDF standard 20B - IDF 1993) and by the bicinconinic acid method.

Evaluation of Enzymatic Hydrolysis: The degree of hydrolysis (DH) is understood as the percentage of the total number of peptide bonds in a protein cleaved during hydrolysis - being useful for monitoring the extent of protein degradation. DH was evaluated for all samples resulting from model hydrolysis systems obtained with the total fraction. The degree of hydrolysis was determined by the TNBS (trinitrobenzenesulfonic acid) method according to Fields (1971), with modifications by Spadaro et al. (1979).

A.2. Screening of biological activity of extracts

 CPS, CMP and -La were obtained by selective filtration techniques associated with hydrolysis by enzymes in the commercial thistle extract using the optimal enzyme / substrate ratio and the optimal reaction time obtained in section A. filtration membranes (nanofiltration) are used to separate unhydrolyzed macropeptides.

The in vitro biological activities of the most promising specific peptide extracts (separated and purified by preparative methods) were evaluated by chemical and biological assays.

Materials:

Substrate. Commercial CPS - Lacprodan, CMP - Davisco ^® , -La (Sigma). Enzymes Plant rennet extracts (0.25 UK (mg protein to coagulate 10 mL milk)) (C. candunculus flower extract enzyme with standard activity - Formulab, Maia, Portugal).

Experimental procedure :

Hydrolysis reaction under optimized conditions: 500 ml of the 40 g / l protein CPS, CMP and -La substrates were hydrolyzed to pH 5.2 with 1.6% (v / v) thistle extract to CPS and -La, and 11.5% for CMP. The samples were subjected to enzymatic hydrolysis by incubation in a shaking bath at 55 ° C for 7 h (optimal conditions determined at 1). The reaction was stopped by heating in a 95 ° C bath for 30 min. After stopping the reaction, the hydrolyzate was centrifuged at 10,000-20.00 Oxg for 15 min at 2-6 ° C, and the supernatants were properly collected.

The supernatants of the hydrolysates were subjected to a nanofiltration process through a 3000 Da pore size membrane and 50 cm ² effective filtration area (Pall Life Science, MI, USA). The ultrafiltration equipment was a Minimate TFF System (Pall Life Science, MI, USA). The obtained <3000 Da fractions were used to characterize the final hydrolysates.

A.2.1 ACE inhibitory activity:

The ACE inhibitor potential of biopeptide rich extracts was performed by the modified Sentandreu and Toldrá (2006) method, which is based on hydrolysis of the fluorescent substrate o-aminobenzoyl glycyl-p-nitrophenylalanylproline by ACE. Such inhibition was assessed for all hydrolysis extracts, and finally for the extracts of CPS, CMP and α-La thistle extract under optimal hydrolysis conditions with either the total fraction or <3000 Da. It is recalled that the enzyme ACE converts angiotensin I to angiotensin II, which increases blood pressure and aldosterone levels, and inactivates the vasodilatory action of bradykinin.

The compound used as an ACE substrate was Abz-Gly-Phe (NO ₂ ) -Pro (o-aminobenzoylglycyl-p-nitrophenylalanylproline), which was dissolved in 150 mM Tris-HCl buffer with pH 8.3. of 1125 mM sodium so as to obtain a final concentration of 0.45 mM substrate at pH 8.3. For the assay, 160 IL of substrate was mixed with 40 IL of each sample for which ACE inhibitory activity was to be determined, and 2 mU of ACE enzyme dissolved in 50% glycerol was added. and prepared in a 150 mM Tris-HCl buffer solution with 0.1 μΜ ZnCl ₂ at pH 8.3 on a black polystyrene microplate (Nunc, Denmark). The reaction was carried out at 37 ° C for 30 min. Fluorescence measurements were taken after 30 min on an OPTIMA FluoSTAR fluorimeter (BMG Labtech, Offenburg, Germany) (Fig. 2) using the excitation wavelength of 355 nm and the emission wavelength of 405 nm. The software used was Fluostar Control v. 1.32 R2.

ACE inhibitory activity was calculated as the amount of soluble protein required to inhibit 50% enzyme (IC ₅₀ ) (in μg protein / mL). To perform this calculation, a nonlinear adjustment of the data was made using PRISM v. 4.02 for Windows (GraphPad Software, Inc., San Diego, CA, USA). The activity of each sample was determined in triplicate. To calculate activity For each sample, the following formula (3) was used:

 „. (F control - White) - F sample - White sample) „„ (3)

% Inhibitory activity = x 100

 Fcontrol - Fbranco

If F _{r co nt} powdery mildew fluorescence emitted by o aminobenzoilglicina group by the action of ACE on the fluorescent substrate Abz-Gly-Phe (N0 ₂₎ -Pro without inhibitor, the fluorescence F emitted by the _white fluorescent substrate Abz-Gly-Phe (N0 ₂₎ -Pro, _Love F ra _ts fluorescence emitted by o aminobenzoilglicina group by the action of ACE on the fluorescent substrate Abz-Gly-Phe (Ν0 ₂₎ -Pro the presence of inhibitor and the Fbranco Love stra Fluorescêncίa emitted by the fluorescent substrate Abz-Gly-Phe (NO 2) -Pro in the presence of inhibitor.

The blank received the same treatment as the samples, but instead of adding the enzyme, water was added; The positive control was also subjected to the same treatment by placing 40 IL of water instead of the sample.

A.2.2 Antioxidant activity

 Screening for antioxidant activity was performed by fluorometric method (ORAC method), and evaluations were made for all samples resulting from model hydrolysis systems in their full fraction, and for extracts resulting from hydrolysis of CPS, CMP and -La with extract. thistle under optimum hydrolysis conditions, also in its total fraction.

Oxygen radical absorption capacity (ORAC Fluorescein) was determined according to the method developed by Ou et al. (2001), adapted by Dávalos et al. (2004), using a microplate fluorescence reader, with some modifications. This method allows to determine in vitro antioxidant activity based on oxidation of fluorescein by peroxyl radicals produced in situ by thermal decomposition of 2,2'-azo-bis- (2-methylpropionamidine) dihydrochloride (AAPH). Fluorescein oxidation causes a decrease in fluorescence; However, this process can be avoided or delayed in the presence of antioxidant substances.

The fluorescein solution was prepared daily at a concentration of 116.66 nM from a fluorescein 1166.1 μΜ stock solution in 75 mM phosphate buffer (pH 7.4). As a reference antioxidant, 6-hydroxy-2,5,7,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (soluble vitamin E analogue), which was prepared at 0.1 mM in phosphate buffer, was used. (mother solution). This was diluted to obtain different concentrations (0.0002, 0.0004, 0.0006, 0, 0008, 0, 0010, 0, 0012, 0, 0014 and 0.0016 μπιοΐ) to construct a curve. Reference Calibration System (Trolox) as follows. The AAPH was dissolved in phosphate buffer to a final concentration of 46.6 mM.

For the assay, 20 L of the phosphate buffer or sample (in the case of the control) was mixed with 120 [LL of fluorescein and 60 LL of AAPH in a black polystyrene microplate (Nunc) which was incubated at 40 ° C. . 104 fluorescence measurements were taken over 137 min on a FluoSTAR OPTIMA fluorimeter (BMG Labtech), the excitation wavelength being 485 nm and the emission wavelength 520 nm. The software used was Fluostar Control v. 1.32 R2. All analyzes were done in triplicate. Fluorescence measurements were normalized based on a calibration curve (no oxidant). From the normalized curves, the area was calculated as follows: (4)

 AUC = 1+ / fi / fo where fo is the initial fluorescence measured at time 0 min and fi is the fluorescence measured in a cycle i. The sample AUC was calculated according to the formula:

AUC = Antioxidant AUC - AUC Control

AUC was plotted against oxidant amount and linear regression was calculated. The final value of 0RAC-F1 was expressed as the ratio of the slope of the sample curve to that of Trolox (μιηοΐ Trolox equivalents / mg protein / peptide).

B. Pilot Serum Treatment System for Peptide Concentrate Production

 Cow serum protein and peptide concentrates were obtained in a pilot unit by selective filtration techniques associated with hydrolysis by enzymes in the commercial thistle extract, using the optimal enzyme extract ratio and the optimal reaction time. previously obtained.

Protein and peptide fractions obtained in the filtration processes were characterized in physicochemical and microbiological terms.

Knowledge of the concentration of whey proteins that are hydrolyzed under these experimental conditions is extremely importance for further characterization of the peptides. Thus, the degree of hydrolysis of the extracts was evaluated by FPLC.

Materials:

Substrate. The whey was obtained as a byproduct of the production of dish-type cow cheese, and provided by the Coimbra School of Agriculture.

Culture mediums. The PCA (Plate Count Agar), BPA (Baird Parker Agar), VRBGA (Violet Red Bile Glucose Agar), RB (Rose Bengal Agar) and egg-tellurium yolk supplement were all purchased from LabM (Bury, UK).

Enzymes Plant rennet extracts (0.25 UK (mg protein to coagulate 10 mL milk) - C. candunculus flower extract enzyme with standardized activity - Formulab, Maia, Portugal).

Experimental procedure :

Obtaining protein concentrates of cow serum. Whey was subjected to a sequence of selective filtration processes in semi-industrial equipment. Optimum conditions for obtaining concentrates are shown in Figure 1.

200-500 L of serum (1) was started, which was creamed to remove existing fat by centrifugation and microfiltration to minimize the microbial load present (2). The serum obtained was subjected to membrane ultrafiltration of 10,000-20,000 Da, preferably 20,000 Da, where the proteins of the peptides present in serum, thus obtaining a protein-rich concentrate (3-retained RUF i) and a peptide extract (4-filtered FUF i).

The protein-rich extract (5-retained RUF ₂ ) was obtained by diafiltration and was successively diluted and concentrated by a membrane of 10,000-20,000 Da, preferably 20,000 Da, in order to decrease the lactose and salt content. Gifts

Membrane-retained protein concentrate (5-retained RUF ₂ ) was subjected to hydrolysis under predefined conditions (after study of the results obtained in point 1); therefore, enzymatic hydrolysis of the retentate at pH 4.8-6.2, preferably pH 5.2, was carried out with 1.6% (v / v) extract for 7 h at 50-60 ° C, preferably at 52 ° C. The final hydrolyzate was ultrafiltered with a 10,000-20,000 Da membrane, preferably 20,000 Da, in order to separate the unhydrolyzed protein (6-retained RUF3) from the hydrolysis (7-filtered FUF3) peptides.

The extracts were further concentrated by reverse osmosis to obtain a whey protein rich concentrate (unhydrolyzed protein) and a peptide rich concentrate released during hydrolysis; The latter was subjected to membrane nanofiltration of 1000-5000 Da, preferably 3000 Da, in order to separate large peptides from small peptides, described as having greater biological activity.

Physicochemical characterization of protein and peptide fractions. The total protein content present in the fractions obtained during the selective filtration process and hydrolysis was determined by the micro-Kjeldahl technique according to NP 1986: 1991; the fat content by Gerber's technique, standard process according to NP 1923: 1987; acidity content by NP 470: 1983; the lactose content by NP 676: 1973; dry weight according to NP 477: 1983; and microbiological analysis by enumeration of viable microorganisms (in specific media) of the samples referring to the different process steps.

Microbiological analysis: Decimal dilutions were made in sterile 0.1% peptone water, which were plated in duplicate to count viable microorganisms in different media: enumeration of mesophilic total microorganisms in aerobic incubated PCA for 3 days at 30 ° C ; enumeration of Staphylococcus in BPA with tellurite egg yolk supplement, incubated under aerobic conditions for 48 h at 37 ° C; enumeration of Enterobacteriaceae in VRBGA under aerobic conditions for 24 h at 37 ° C; and enumeration of yeast in RB under aerobic conditions for 5 days at 25 ° C. All determinations used the plating technique except the VRBGA which used the incorporation method.

Evaluation of the degree of hydrolysis. The degree of hydrolysis was determined by chromatographic methods using an FPLC system, coupled with a Superose 12 HR 10/30 gel filtration column (Amersham Biosciences, Hong Kong, China), according to Lamas et al. (2004).

C. Biological activity of obtained extracts Evaluation of in vitro biological activities The evaluation of the in vitro biological activities of the obtained concentrates was carried out by chemical and biological tests.

Materials:

Substrate. Peptide concentrates obtained in section A (hydrolyzed CPS and CMP (Davisco ^® ) and hydrolysed oc-La (Sigma)) and section B (F i - CPSH - total fraction and F ₂ - CPSH - <3000 Da). The cell lines used in this study originated from human neoplasms, and were provided by the US National Cancer Institute (NCI).

Microorganisms. Staphylococcus aureus ATCC 25923, Salmonella spp. (isolated from food), Escherichia coli ATCC 25922 and Listeria inoccua (isolated from food) and Lactobacillus paracasei, Lactobacillus acidiphilus Ki and Lactobacillus acidophilus LAFT ^® L10.

Culture media, Mueller Hinton broth, Mueller Hinton agar and BHI (Brain Heart Infusion) infusion.

Enzymes and reagents. Trypsin (Sigma Chemical Co., St. Louis, MO, USA), the culture medium RPMI-1640 (Gibco, NY), fetal bovine serum (FBS) (Gibco), Hank's (Sigma Chemical Co ^', St. Louis, MO, USA), dimethyl sulfoxide (DMSO) (Sigma Chemical Co), trichloroacetic acid (TCA) (Merck, Darmstadt, Germany).

Cl. ACE inhibitory activity

The potential for ACE inhibition by biopeptide-rich extracts was performed by the modified Sentandreu and Toldrá (2006) method, which is based on the hydrolysis of the fluorescent substrate o-aminobenzoyl glycyl-β-. nitrophenylalanylproline by ACE. It was evaluated for extracts resulting from hydrolysis of CPS, CMP and -La with thistle extract under optimal hydrolysis conditions using CPSH total fraction and <3000 Da as described in A.2.1.

C.2. Antimicrobial and prebiotic activity

Determination of the antimicrobial activity of peptide rich extracts (commercial CPS and CMP, extracts resulting from the hydrolysis of CPS and CMP with thistle extract under optimal hydrolysis conditions, by total fraction and <3000 Da) was performed on pathogenic strains and food contaminants. (Escherichia coli, S. aureus, Salmonella spp. And Listeria inoccua) previously selected, and determination of stimulating activity on strains of probiotic bacteria was performed for Lactobacillus paracasei, Lactobacillus acidiphilus Ki and Lactobacillus acidophilus LAFT ^® (by intestinal flora). absence of inhibition) The activity was evaluated for the test microorganisms in specific media and in accordance with international standards.

C.2.1. Evaluation of antimicrobial activity. Antimicrobial activity was tested for the following strains; Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Innocent Listeria and Salmonella spp. (isolated from food).

Tubes containing Mueller Hinton Broth culture medium were prepared and different volumes of the peptide extract to be tested were added starting from a 100 mg / mL solution to obtain concentrations of 10.0 mg / mL (1%). 5.0 mg / mL (0.5%), 2.5 mg / mL (0.25%) and 1.0 mg / mL (0.1%). All peptide solutions were filtered through 0.22 µm filters. Yes. The different eurotubes were then inoculated with 100 μl of the test organism at different concentrations, namely 10 ³ , 10 ⁵ and 10 ⁷ cfu / mL. Inocula were prepared in BHI liquid medium with overnight growth (37 ° C / 12h); At this time, dilutions were made to obtain the desired inoculum concentrations. 300 µl of the contents of each eurotube were transferred to the microplates, which were left for 24 h in the Bio-Rad ^® reader at 37 ° C, and the absorbance readings at λ = 660 nm were recorded.

C.2.2. Prebiotic activity. Prebiotic activity was tested for L. paracasei, L. acidophilus Ki and L. acidophilus LAFT ^® L10 strains by the microplate reading method. Tubes containing MRS culture medium were prepared, to which different volumes of the peptide extract to be tested were added starting from a 100 mg / mL solution to give concentrations of 30.0 mg / mL (3%). , 0 mg / mL (2%), 10 mg / mL (1%), 5.0 mg / mL (0.5%) and 1.0 mg / mL (0.1%). All peptide solutions were filtered through 0.22 μιη filters. Each eurotube was then inoculated with 2% test organism containing a concentration of 10 ⁷ cfu / mL. Inocula were prepared in MRS broth cysteine liquid medium for each strain used for 48 h at 37 ° C under anaerobiosis. 250 μL of the contents of each eurotube were transferred to the microplates, and then 50 μL of sterile liquid paraffin was added. The microplates remained 48 h in the Bio-Rad ^® reader at 37 ° C, and absorbance readings were at λ = 660 nm.

C.3. Anticancer activity

Anticancer activity was determined for CPSH peptide concentrates - total fraction and <3000 Da, through in vitro screening using the K562 (leukemia), MCF-7 (breast), NCI-ADR (breast with multiple drug resistance phenotype), UACC-62 (melanoma), NCI460 (lung), PC03 strains (prostate), HT29 (colon), OVCAR (ovary) and 786-0 (kidney), exposed to the test peptides. The cytotoxicity of the peptide extracts on normal cell lines VERO (liver), V79 (fibroblast) and 3T3 (fibroblast) were also determined.

Determination of anticancer activity and cytotoxicity of samples:

 To test for anticancer activity, the protocol developed by the National Cancer Institute (USA) was followed, which has been using this methodology for over fifteen years (Skehan et al, 1990; Monks et al, 1991; Rubistein et al, 1990).

In vivo biological activity assessment

 Evaluation of the in vivo biological activities of CPSH peptide concentrates - total fraction and <3000 Da was performed by chemical and biological assays.

Materials:

Substrate. Peptide concentrates obtained in section B (Fi - CPSH - total fraction and F ₂ - CPSH - <3000 Da).

Enzymes and reagents. NOR, L-Name, Indomethacin, Carbenoxolone.

Animals. The animals were acquired from the Center of Bioterismo (CEMIB) of UNICAMP (Brazil), and used in the tests after a minimum period of seven days of adaptation to the vivarium, in a light-dark cycle of 12 h and room temperature of 20 ° C with water and commercial feed ad libitum; 2-week-old male SHR rats were used.

Experimental procedure :

C.4. Determination of antiulcerogenic activity. Clinical and anatomopathological evaluation of rats (with topical administration of the test peptides) with ethanol-induced ulcers (Robert, 1979) was performed, which were sacrificed by cervical dislocation, with the stomachs removed, opened along the largest curvature and washed in saline solution for counting and evaluating the lesions produced. The Ulcerative Lesion Index (ILU) was calculated according to the methodology described by Gamberini (1991).

In this study, single dose treatments of 30, 100 and 300 mg / kg _bw of the test peptide samples were performed, and treatments of 3 consecutive doses of 100, 200 and 350 mg / kg _bw .

Determination of 50% effective dose (ED ₅₀ )

The 50% effective dose (ED ₅₀ ) corresponds to the dose required to produce 50% inhibition in the observed lesions compared to the negative control.

Participation of sulfhydryl substances in gastric cytoprotection

 To study the possible participation of sulfhydryl compounds in gastric cytoprotection, the ethanol-induced ulcer model was used (Robert, 1979).

Male wistar rats weighing between 200 and 250 g were divided into groups of 6 animals. After fasting for 16 hours, with free access to water, a negative control group and the test groups were previously treated with a subcutaneous 10 mg / kg (2.5 mL / kg _bw ) aqueous solution of N-ethylmaleimide (NEM) of according to the methodology described by Szabo, Trier, Frankel (1981). The rats were sacrificed by cervical dislocation, and their stomachs were removed, opened along the greater curvature and washed in saline solution for counting and evaluation of the lesions produced. The Ulcerative Lesion Index (ILU) was calculated according to the methodology described by Gamberini (1991).

Prostaglandin participation in gastric cytoprotection

 To study the possible participation of prostaglandins in gastric cytoprotection, the absolute ethanol ulcer induction model was used (Robert, 1979).

Male wistar rats, each weighing between 200 and 250 g, were divided into groups of 6 animals. After 16 h fasting, the negative control group and the test groups received prior indomethacin treatments at a dose of 5 mg / kg _bw in aqueous solution as 10 mL / kg _bw subcutaneously according to the methodology. described by Szabo, Trier, Frankel (1981). The rats were sacrificed by cervical dislocation, and their stomachs were removed, opened along the greatest curvature and washed in saline solution for counting and evaluation of the lesions produced. The Ulcerative Lesion Index (ILU) was then calculated according to the methodology described by Gamberini (1991).

Nitric oxide (NO) participation in gastric cytoprotection To study the possible participation of nitric oxide in gastric cytoprotection, the ethanol-induced ulcer model was used (Robert, 1979).

Male wistar rats, each weighing between 200 and 250 g, were divided into groups of 6 animals. After 16 h fasting, with free access to water, a negative control group and test groups received previous treatments with a 5 mg / kg _bw aqueous L-Name solution, intraperitoneally, according to the described methodology. by Konturek and Pawlink (1986). The rats were sacrificed by cervical dislocation, and their stomachs were removed, opened along the greatest curvature and washed in saline solution for counting and evaluation of the lesions produced. The Ulcerative Lesion Index (ILU) was then calculated according to the methodology described by Gamberini (1991).

Statistical analysis

 All results of the protein fractions assays for gastric mucosal protection were subjected to analysis of variance (ANOVA) and the Duncan test, for which a 5% probability level was used as a statistical significance criterion ( p <0.05).

C.5. Determination of anti-inflammatory activity

Croton oil-induced dermatitis. Oral administration of the test peptides to mice was provided. However, a solution of croton oil was applied to the inner surface of the right ear. In the left ear, an equivalent volume of solvent (acetone) was applied. The animals were sacrificed by cervical dislocation, and portions of both ears were removed. the weight difference considered as derived from the edema produced by croton oil, according to the method of Tubaro (1985) and Schintarelli (1982).

C.6 Determination of antinociceptive activity

Evaluation of heat induced algesia. This was done according to the method of Mazella (1991).

Model of formalin-induced neurogenic and inflammatory pain. This was done according to the Hunskaar method.

(1985).

D. Identification and Characterization of Biopeptides

Determination of the peptide profile of the extract and sequencing of the major peptides were performed by LC-MS (Liquid Chromatography, coupled with Mass Spectrometry) with a view to anticipating its bioactivity based on the appearance of specific amino acid sequences as recorded in a database. , or evaluate if it was an unknown biopeptide. The identified peptides were synthesized in vitro, and their ACE inhibitory activity was evaluated. Extracts containing the most promising specific peptides and peptide concentrates obtained in section B (Fi - CPSH - total fraction and F ₂ - CPSH - <3000 Da) were analyzed.

Materials:

Samples. Peptide concentrates obtained in section B (Fi - CPSH - total fraction and F ₂ - CPSH - <3000 Da), and synthetic peptides. Experimental procedure :

Semi-Preparative RP-HPLC: The equipment used (Waters Series 600 HPLC from Waters Corp., Mildford, MA, USA) consisted of a pump (delta 600 model), a gradient controller (model 600), an injector (model 717 plus), a variable wavelength ultraviolet detector (model 996), a fraction collector and data acquisition and processing software (Millenium 3.2). The column used was a Prep Nova Pak® HR Cig (300 x 7.8 mm d.i., 6 µm particle size and 60 µm pore size) (Waters). Solvent A is a mixture of water and trifluoracetic acid (1000: 1) and solvent B is a mixture of acetonitrile and trifluoracetic acid (1000: 0, 8). The flow rate was 4 mL / min, and solvent absorbance was measured at 214 nm.

 To isolate peptide fractions, the <3000 Da lyophilized permeate from the ultrafiltration process was dissolved in water at a concentration of 100 mg / ml, and was forced through a 0.45 µm pore size filter. The sample was eluted with a 0% to 35% solvent gradient B in 70 min at 35 ° C. In each chromatographic analysis, the injected sample volume was 500 µl, and the separated fractions were collected a total of 12 times; Once acetonitrile was removed by spraying, they were lyophilized and stored at -20 ° C. Thereafter, ACE inhibitory activity was determined following the method described in section A.2.1.

Identification of peptides by mass spectrometry:

Mass spectrometric analyzes were performed on an HP Agilent 1100 System (Agilent Technologies, Waldbronn, Germany), connected inline to an Esquire-LC, Ion Trap mass detector (Bruker Daltonik GmbH, Bremen, Germany). The HPLC system was equipped with a pump Quaternary, an internal degasser and an automatic injector (all from the 1100 Series, Agilent Technologies). The ChemStation (Agilent Technologies) program was used as a data acquisition system. A Wide-Pore C18 (250 x 4.6 mm di, 5 µm particle size) reverse phase column (Bio-Rad Laboratories, Richmond, CA, USA) was used.

The injection volume used was 50 L. Solvent A was a mixture of water and trifluoroacetic acid (1000: 0.37 (v / v)), and as solvent B a mixture of acetonitrile and trifluoroacetic acid (1000: 0.27 (v / v)) . The peptides were eluted at a flow rate of 0.8 mL / min under a variable gradient depending on the fraction analyzed.

The absorbance of the solvent was monitored at 214 nm and at the detector output the flow rate of 0.8 mL / min was split at a ratio of 1:40 - thus giving a final flow of approximately 20 L / min to the MS nebulizer. , through a T-piece detector (Valco, Houston, TX, USA), which transported it to the mass spectrometer via the electrospray interface. The MS used nitrogen gas and helium drying at an estimated pressure of 5x10 ^-3 bar, and mass spectra were acquired with a maximum range of 100-1500 m / z. The capillary was kept at a voltage of 4 kV. About 5 spectra were analyzed on the MS and MS ⁿ . The intensity limit for MS / MS analysis was 10,000 (ie 5% of the total signal). Precursor ions were isolated with a range of 4 m / z, and fragmented with a voltage ramp of 0.35 to 1.4 V.

Spectral data were processed and transformed into mass values using the Data Analysis ™ v. 3.0 (from Bruker Daltonik). The BioTools v. 2.1 (of Bruker Daltonik) was used to process the MS / MS spectra, and to carry out peptide sequencing.

 Finally, the identified peptides were produced according to the Fmoc method to be greater than 90% pure, and then the ACE inhibitory activity was determined following the method described in section A.2.1.

Claims

Process for the production of peptide extracts, characterized in that they are produced from the whey protein hydrolysis reaction by the action of at least one C. candunculus flower extract enzyme.

Process according to Claim 1, characterized in that the concentration of C. candunculus flower extract is between 1 and 5% (v / v), preferably 1.6% (v / v).

Process according to either of Claims 1 and 2, characterized in that it comprises the following steps: a) Whey skimming (1), preferably by centrifugation;

 (b) microbial load reduction (2), preferably by microfiltration;

 c) Ultrafiltration of the serum using a membrane of

 10,000-20,000 Da, preferably 20,000 Da, thereby obtaining a protein rich extract and a peptide rich extract;

 (d) diafiltration of the protein-rich extract (3) by successive dilution and membrane concentration of 10,000-20,000 Da, preferably 20,000 Da; e) Hydrolysis of the protein rich extract (5) resulting from d), with enzymes from C. candunculus flower extract, preferably for 7 h;

f) Ultrafiltration of the final hydrolyzate with a membrane of 10,000-20,000 Da, preferably 20,000 Da; (g) concentration of the protein rich extract retentate (6) and the peptide rich extract filtrate (7), preferably by reverse osmosis for a whey protein concentrate - unhydrolyzed proteins - and a peptide rich concentrate released during hydrolysis, respectively; and

h) Nanofiltration of the peptide rich extract (7) with 1000-5000 Da membrane, preferably 3000 Da.

Process according to any one of the preceding claims, characterized in that the <3000 Da fraction is isolated from the remaining fraction by a membrane separation technique.

Process according to the preceding claim, characterized in that it is performed by nanofiltration through a pore size membrane between 1000 and 5000 Da, preferably 3000 Da.

Extracts characterized by being obtained by the process described in any one of the preceding claims.

Extracts according to the preceding claim, characterized in that they have biological activity.

Extracts according to the preceding claim, characterized in that the biological activity is selected from: ACE activity inhibitor, antioxidant, antinociceptive agent and / or anti-inflammatory agent.

Extracts according to any of claims 6 to 8, characterized by comprising at least one of the following peptides of SEQ ID NO: SEQ.ID.N ^0, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5,

SEQ.ID. # 6 or SEQ.ID. # 7.

Extracts according to the preceding claim, characterized in that they contain in their constitution the peptide SEQ.ID. No. 7.

Extracts according to claim 9, characterized in that they contain in their constitution the peptides identified with the sequences SEQ.ID.No.1, SEQ.ID.No.2, SEQ.ID.No.3, SEQ.ID No. 4, SEQ.ID. No. 5 and SEQ. ID No. 6.

Extracts according to any one of claims 9 to 11, characterized in that they further comprise at least one of the peptides identified with the sequences SEQ.ID.No. 8, SEQ.ID.No. 9, SEQ.ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ. ID No. 15.

Peptides, characterized in that they are obtained by the process described in any one of claims 1 to 5.

Peptides according to the preceding claim, characterized in that they have biological activity.

Peptides according to the preceding claim, characterized in that the biological activity is selected from: ACE activity inhibitor, antioxidant, antinociceptive agent and / or anti-inflammatory agent.

16. Peptide according to any one of claims 13 to 15, characterized by comprising the following amino acid sequences: SEQ.ID.N ⁰ ° SEQ.ID.N 2 SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 or SEQ ID No. 7.

Peptides according to any one of claims 13 to 16, characterized in that they comprise the following amino acid sequences: SEQ.ID.No. 8, SEQ.ID.No. 9, SEQ.ID.N.10, SEQ. ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14 or SEQ ID No. 15.

Use of the extracts defined in any of claims 6 to 11, characterized in that they are applied in the preparation of an ACE activity inhibitor, and / or as an antioxidant, antinociceptive agent and / or anti-inflammatory agent.

Use of the extracts according to claims 16 and 17, characterized in that they are applied in the preparation of a pharmaceutical composition for the prevention and / or treatment of conditions associated with hypertension, oxidative conditions, and / or neoplasms, and / or as a painkiller.

Use of the extracts according to claim 16, characterized in that they are applied in the preparation of supplements and / or food additives.

Use of the peptides defined in any one of claims 13 to 15, or their salts, characterized in that they are applied in the preparation of an ACE activity inhibitor and / or as an antioxidant, antinociceptive agent and / or anti-inflammatory agent.

Use of the peptides according to the preceding claim, characterized in that they are applied in the preparation of a pharmaceutical composition for the prevention and / or treatment of conditions associated with hypertension, oxidative conditions, and / or neoplasms, and / or as an analgesic.

Use of the peptides according to the preceding claim, characterized in that they are applied in the preparation of supplements and / or food additives.