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    • 专利摘要:
    The present invention is a process for obtaining an enzymatic hydrolyzate with antihypertensive activity comprising the grinding of chicken leg claws and lyophilization to obtain a powder material with a particle size <2 mm; adjusting an aqueous solution of this powder to a different ph at a value of 0.5 relative to the ph of the starting powder and heating between 80 and 120ºC for between 10 and 120 min; cool the previous solution at a temperature between 45 and 55ºC and perform an enzymatic hydrolysis for a time between 1 and 24 h, with selected enzymes evaluating and selecting the inhibitory activity of the angiotensin converting enzyme. The invention is also the use of the hydrolyzate obtained in the treatment of hypertension, and the pharmacological utility of the peptides that the hydrolyzate contains. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2606954A1
    申请号:ES201531372
    申请日:2015-09-25
    公开日:2017-03-28
    发明作者:Francisca Isabel BRAVO VÁZQUEZ；Lluís AROLA FERRER；María Begoña MUGUERZA MARQUÍNEZ
    申请人:Universitat Rovira i Virgili URV；
    IPC主号:
    专利说明:

    PROCEDURE FOR OBTAINING A HYDROLYZE OF CLAWSCHICKEN PAW WITH ANTlHIPERTENSIVE ACTIVITY. HYDROLYZEDOBTAINED AND PEPTIDES CONTAINED
    TECHNICAL SECTOR The chicken claw hydrolyzate of the present invention is useful in the treatment of hypertension, in the field of medicine or the food industry. The peptides found may be useful in dietary as
    10 supplements of food products.
    BACKGROUND OF THE INVENTION The term antihypertensive means any substance or procedure that reduces blood pressure (BP). Several effective antihypertensive agents are known to be
    15 classified according to their mechanism of action. Diuretics, beta adrenergic blockers, calcium channel blockers, central and peripheral adrenergic inhibitors, angiotensin receptor inhibitors or angiotensin-converting enzyme (ACE) inhibitors are identified. ECA catalyzes the conversion of angiotensin 1 to angiotensin 11, which is a
    20 vasoconstrictor hormone.
    ACE inhibitors, or ACE inhibitory activity (ACEI), interfere in the production of angiotensin 1I by blocking the enzyme that produces it. Such an effect not only reduces BP but also reduces vascular damage caused by
    25 hypertension, which in turn decreases the incidence of complications in the patient, particularly renal failure or heart failure. However, experience shows that it is not possible to establish a direct a priori correlation between observing an ACLL activity in vitro and obtaining a correlative in vivo antihypertensive effect in patients.
    WO 2007004876 A2 describes the sequence of several peptides with in vitro ACE activity obtained from a milk protein hydrolyzate. The peptides are 2 to 14 amino acids in length and at least one of them has a sequence identity of 66.7% with the peptide identified by SEO.iD.NO: 1 35 of the present invention. This identity is too low to establish a
    relationship between the two peptides, logically because the two substrates are very different.
    The application WO 2007108554 A1 describes the obtaining from chicken collagen of an enzymatic hydrolyzate with ACE activity that also shows antihypertensive properties. Identify a 3000 Da peptide with such activity. However, using only collagen as a starting material excludes a series of proteins present in the skin of the claws of the animal that if incorporated into the hydrolyzate of the present invention. It also excludes proteins other than collagen present in other tissues of chicken leg claws, such as cartilage, bones or nails. The peptides obtained in the present invention will therefore be different.
    JP H42 64098 A describes obtaining a hydrolyzate of a lean chicken protein treated with thermolysin that has in vitro ACE activity. Being lean chicken meat, typical muscle tissue proteins predominate in the starting product, almost completely absent in chicken claws and therefore different from those of the present invention. Again, other major proteins are excluded from this substrate. In the hydrolyzate obtained, the document identifies the peptide identified by SEQ.ID.NO:10 as an active agent, which does not have any identity with the peptides of the invention.
    Cheng describes a hydrolyzate with antihypertensive activity obtained from the hydrolysis of the complete chicken leg with alcalase (Cheng, F. et al. "Determination of angiotensine-I converting enzyme inhibitory peptides in chicken leg bone protein hydrolysate with alcalase". 2009. Anim Sci. J. 80, 91-97). The procedure includes subjecting the liquefied starting material to a "water bath" heat treatment prior to the enzyme treatment. 10 peptides of between 5 and 10 amino acids are obtained with said in vitro ACE activity, none of which nevertheless has a significant homology with the peptides of the invention; again because the substrate used by Cheng includes bone and especially muscle tissue, and majority compounds of chicken claws are excluded. The peptides obtained by digestion of this substrate with alcalase are logically distinct, so that the procedure does not suggest the results obtained by the present invention.
    The document of the technique that is considered closest to the invention is the applicationTW 201106955 A. This document describes the preparation of a hydrolyzateEnzymatic with antihypertensive activity from chicken claws. Hemethod of obtaining comprises the homogenization of the starting material and5 a heat treatment in "water bath" followed by a digestion with protease N.After digestion by the protease the product obtained is filtered, lyophilized andselect those hydrolysates that show ACE activity. However theproduct obtained by protease N is different from that obtained by proteases of theinvention. The fundamental difference with respect to the present invention is that the
    10 thermal pretreatment in the latter is performed at modified pH. This modification in the procedure is not suggested by the prior art.
    According to the applicant's best knowledge, the peptides of the invention have not been described as such in the art. Only the one identified by SEQ.ID.NO.ID:2 15 is described in several publications, although always being part of more extensive sequences. Several of these sequences are related to the treatment of angiogenesis for example in the application US 2012101029 A 1, which contains in its SEQ.ID.NO:18 the peptide of the invention. However, these documents do not describe or suggest that fragments of the sequences can be considered more
    20 extensive, nor in particular a fragment that may correspond to the peptide of the invention.
    The problem that arises then in the art is the obtaining of an alternative protein hydrolyzate effective for the treatment of hypertension. The solution proposed by the present invention is a hydrolyzate obtained from a modified process of hydrolysis of a chicken claw substrate.
    DESCRIPTION OF THE INVENTION The present invention is a process for obtaining a hydrolyzate.
    Enzymatic with antihypertensive activity comprising the grinding of chicken leg claws and lyophilization to obtain a powder material of a particle size <2 mm, adjust an aqueous solution of this powder to a pH between 3 and 10 with the condition of The pH value is different from that corresponding to the powder material by at least 0.5, and heat between 80 and 120 ° C for between 10 and 120
    35 min, preferably between 60 and 90 min, cool the above solution to a temperature between 45 and 55 ° C and perform an enzymatic hydrolysis for a time between 1 and 24 h, preferably between 2 or 24 h, more preferably 2 or 24 h , with enzymes selected from the group consisting of:
    -proteolytic enzymes of Bacillus licheniformis and BacjIJus5 amyloliquefaciens identified as E.C. 3.4.21.62 and 3.4.24.28,-a Bacillus licheniformis serine protease identified as E.C. 3.4.21 .62,
    - a Zinc-dependent metalloprotease from Bacillus amyloliquefaciens identified as E.C. 3.4.24, 10 -a Aspergillus oryzae aminopeptidase identified as E.C. 3.4. 11 .1, and
    - a mixture between them, evaluating the inhibitory activity of the angiotensin converting enzyme according to the method described in Sentandreu, MN and Toldrá F. 2006. Food Chem. 97, 546-554, and the
    15 selection of a hydrolyzate with activity> 80% in 50% aqueous dilution v / va from the previous evaluation.
    Within the scope of the present application, when it is said that the pH of the aqueous solution of the invention is different from that corresponding to the powder material in at least one
    The value of 0.5 refers to the fact that the powder also measured in aqueous solution is different from the pH of the material. Obviously, this last measurement can be carried out on a solution for this purpose or in the solution which is then to be adjusted according to the process of the invention.
    In a preferable aspect, the enzymes used in hydrolysis are proteolytic enzymes of Bacillus Iicheniformis and Bacillus amyloliquefaciens, and identified as E.C. 3.4.21.62 and 3.4.24.28.
    In a more restrictive aspect, the invention consists in the above procedure.
    In a very preferable aspect, the invention is a process comprising grinding chicken leg claws and lyophilizing to obtain a powder material with particle size <2 mm, adjusting an aqueous solution of this powder to a pH of 7.5 in that this pH value is different from that corresponding to the powder material of step 35 above in at least 0.5, heat at 100 ° C for 90 min, cool the previous solution
    at a temperature of 50 ° C and hydrolyze for a time of 2 or 24 h with proteolytic enzymes obtained from Bacillus licheniformis and Bacillus amyloliquefaciens identified as E.C. 3.4.21.62 and 3.4.24.28, evaluation of angiotensin converting enzyme inhibitory activity according to the method described in Sentandreu, MN and Toldrá F. 2006. Food Chem. 97,546-554, and the selection of a hydrolyzate with activity> 80% in 50% aqueous dilution v / v from the previous evaluation.
    The raw material is the Paws of Polio Claws (GPP), which is a byproduct of the meat industry. The production process of the hydrolyzate is cheap and easy to industrialize.
    The inventive aspect of the process of the invention is the pretreatment of GPP powder at modified pH to achieve maximum solubilization and denaturation of the protein, which optimizes its subsequent hydrolysis. This pretreatment facilitates the access of enzymes to proteins.
    The choice of the hydrolysis enzyme in the process of the invention is decisive. Among the innumerable enzymes that can be used none was particularly suggested in the art. Randomly, the present invention chose Protamex® (Novozyme) which at the date of the present application are the proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens (EC. 3.4.21.62 and 3.4.24.28); Alcalase® 2.4 L, also called Carlsberg subtilisin, which is a Bacillus Iicheniformis serine protease (E.C. 3.4.21.62); Neutrase® 0.8l which is a Zinc-dependent metalloprotease from Bacillus amyloliquefaciens (EC. 3.4.24); and Flavourzyme® which is an aminopeptidase from Aspergillus oryzae (EC. 3.4.1 1.1) (Novozyme, Nordisk). One aspect of the invention would also comprise a mixture of all of them.
    Many of the hydrolysates obtained showed a good ACE activity and 3 of them a clear antihypertensive activity. In particular, the antihypertensive activity of the hydrolyzate obtained with Protamex®, referred to in the present application as hydrolyzate 1. was surprisingly high, which represents a definite technological advantage over the technique.
    The invention is also the hydrolysates that are obtained. So another highly preferable aspect is an enzymatic hydrolyzate comprising the peptides identified by SEQ.ID.NO:1-9, their stereoisomers and / or salts thereof. Another preferable aspect is the use of this enzymatic hydrolyzate in the preparation of a
    5 medication for the treatment of hypertension in a subject. Or also, thehydrolyzed for use in the treatment of hypertension in a subject. The subject ispreferably human.
    A more preferable aspect is a pharmaceutical composition in liquid or syrup form.
    10 comprising an enzymatic hydrolyzate containing the peptides identified by SEQ.ID.NO:1-9, their stereoisomers and / or salts thereof, and pharmaceutically acceptable excipients.
    From the hydrolysates of the invention, a series of bioactive peptides were obtained. In particular, the peptides identified by SEQ.1D.NO:2 and 4 showed lECA activity in vitro and also a surprising antihypertensive activity.
    So another preferable aspect of the invention is a peptide identified by one of the sequences SEQ.ID.NO:1-9. Another more preferable aspect is the use of a peptide
    20 identified by SEQ.ID.NO:2 or 4, its stereoisomers and / or salts thereof, or their combination in the preparation of a medicament for the treatment of hypertension. Or also, the peptides identified by SEQ.ID.NO:2 or 4, their stereoisomers and / or salts thereof, or their combination for use in the treatment of hypertension.
    Another highly preferred embodiment are the peptides identified by SEQ.ID.NO:2 or 4, their stereoisomers and / or salts thereof, or their combination for use as a medicine.
    Another highly preferable aspect is a pharmaceutical composition comprising a peptide identified by the sequence SEQ.ID.NO:2 OR 4, its stereoisomers and / or salts thereof or their combination, and pharmaceutically acceptable excipients. A more preferable aspect is a food supplement comprising a peptide identified by the sequence SEQ.ID.N0: 2 or 4, its stereoisomers and / or salts thereof.
    35 themselves or their combination, and food additives.
    The results of Example 2 show how the pretreatment in the process of obtaining affects the lECA activity of the hydrolysates, causing them to increase in most cases and often with values greater than 80%. This
    5 pretreatment is not excessively expensive since it does not use too high temperatures.
    The determination of the ICso of the hydrolysates showed that protein hydrolysis with the Protamex® enzyme produced hydrolysates with lower ICso values at all 10 hydrolysis times, that is, with more potent ACE activity and therefore with greater antihypertensive potential. The results of Tables I and 11 show that the pH used in the pretreatment, and not only the temperature, influences the ACE activity. The pH modification treatment improves protein extraction and also increases its degree of denaturation, thus conditioning the hydrolysis
    15 later.
    Furthermore, it has been ruled out that the antihypertensive hydrolyzate exhibits hypotensive activity through an in vivo study in which normotensive rats (Wistar-Kyoto) were used and to which a hydrolyzate of the invention was administered, as
    20 shown in the Example This hypotensive activity is not a desirable activity since it is the decrease in BP of a healthy person or animal, while antihypertensive activity is the decrease in BP in a hypertensive person or animal. It is of great importance that the hydrolyzate of the invention show a therapeutic effect in sick people and not in healthy people.
    Among the peptides of the present invention, the one identified by SEO.ID.NO : it has the lowest ACE activity values (Ieso = 7.06¡Jg / mL) which could indicate a possible antihypertensive effect; however, it did not turn out that way, probably because the peptide is hydrolyzed by digestive tract enzymes and not
    30 can be absorbed as such. Similarly, it happened with the SEO.ID.NO:1 peptide, which despite having a better ACE activity than the one shown by the SEO.ID.NOA peptide did not show antihypertensive activity. The results shown in the present application demonstrate that a potent ACE activity does not directly correspond to a decrease in BP effect.
    In any case, in the antihypertensive activity of the hydrolyzate it is not ruled out that peptides that individually do not show antihypertensive activity, for example SEQ.ID.NO:1 and 7, can act synergistically when administered together with the rest of the peptides contributing to the decrease in attributable BP
    5 to the antihypertensive hydrolyzate of the invention.
    In fact, it has been described that the degree of hydrolysis produced by the physiological digestion of a particular peptide, in addition to depending on its size and nature, also depends on the presence of other peptides in the medium.
    10 (FitzGerald et al., "Hipotensive peptides from milk proteins". 2004. J. Nutr. 134 (4), 980S-9888). This could mean that peptides that do not have antihypertensive activity individually do not lose their bioactivity when co-administered with other peptides.
    15 BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a reverse phase HPLC chromatogram on a semi-preparative scale
    of the active supernatant obtained after centrifugation and ultrafiltration, through a membrane of 3,000 Da pore size, of the hydrolyzate 1 obtained by
    20 Enzymatic hydrolysis with Protamex®, 2h, at 50 ° C from GPP powder. F.1 -F.8 correspond to the eight (8) fractions collected.
    Figure 2 are two reverse phase HPLC chromatograms on a semi-preparative scale of the two (2) most active fractions (F.3 (A) and F.6 (B)) that are
    25 obtained from the first HPLC separation of the hydrolyzate 1 of GPP powder, and the fractionation performed in each of them. Specifically, six (6) sub-fractions of F.3 (F.3.1-F.3.6) and eight (8) sub-fractions of F.6 (F.6.1-F.6.8) were collected.
    Figure 3A is a graph showing the decrease in systolic blood pressure
    30 (PAS) obtained in spontaneously hypertensive rats (SHR) to which 1.5 mL of water (.), 5 mLlkg of the hydrolyzate 1 of GPP powder (Protamex ~ "2h, at 50'C) is administered (A) , or 50 mg / kg of Captopril (.), in function of the time after administration. Data represent the mean ± E8M for a minimum of six (6) animals. Unless otherwise indicated, all doses indicated herein.
    35 request refer to the body weight of the animal.
    Figure 38 is a graph showing the decrease in diastolic blood pressure (PAD) obtained in spontaneously hypertensive rats (SHR) to which 1.5 mL of water (_), 5 mUkg of GPP powder hydrolyzate 1 is administered (Protamex®,
    5 2h, at 50'C) (...), or 50 mg / kg of Caplopril (o), depending on the last time you will administer. Data represent the mean ± ESM for a minimum of six (6) animals.
    Figure 4A is a graph showing the decrease in systolic blood pressure
    10 (PAS) obtained in Wistar-Kyoto (WKY) normotensive rats that are the normotensive control of SHR rats to which 1.5 mL of water (_) or 5 mUkg of hydrolyzate 1 of GPP powder (Protamex®) is administered , 2h, at 50 ° C) (. &.), Depending on the time spent after administration. Data represent the mean ± ESM for a minimum of six (6) animals.
    15 Figure 48 is a graph showing the decrease in diastolic blood pressure (PAD) obtained in Wistar-Kyoto normotensive rats (WKY) that are the normolense control of SHR ralas to which 1.5 mL of water is administered ( o) o 5 mUkg of hydrolyzate 1 of GPP powder (Protamex®, 2h, at 50 ° C) (. &.), depending on time
    20 passed after administration. Data represent the mean ± ESM for a minimum of six (6) animals.
    Figure 5A is a graph showing the decrease in systolic blood pressure (SBP) obtained in spontaneously hypertensive rats (SHR) to which it is administered
    25 1.5 mL of water (.), 5 mUkg of hydrolyzate 2 of GPP powder (Protamex ·, 24h, at 50'C) (o), or 50 mg / kg of Caplopril (o), depending on the time You will go past the administration. Data represent the mean ± ESM for a minimum of six (6) animals.
    30 Figure 58 is a graph showing the decrease in diastolic blood pressure (PAD) obtained in spontaneously hypertensive rats (SHR) to which 1.5 mL of water (t), 5 mUkg of hydrolyzate 2 of powder is administered. GPP (Protamex®, 24h, at 50 ° C) (_), or 50 mglkg of Captopril (.), Depending on the time spent after administration. Data represent the mean ± ESM for a minimum of six (6)
    35 animals


    Figure 6A is a graph showing the decrease in systolic blood pressure
    (PAS) obtained in spontaneously hypertensive rats (SHR) to which it is administered
    1.5 mL of water (+), 5 mL / kg of hydrolyzate 3 of GPP powder (Neutrase®, 2h, at 50 ° C)
    5 (e), OR 50 mg / kg of Captopril (.), depending on the time spent after administration.
    Data represent the mean ± ESM for a minimum of six (6) animals.
    Figure 66 is a graph showing the decrease in blood pressure
    diastolic (PAD) obtained in spontaneously hypertensive rats (SHR) to which
    the administers 1.5 mL of water (o), 5 mLlkg of the GPP powder hydrolyzate 3 (NeutraseO,
    2h, at 50 ° C) (o), or 50 mg / kg of Captopril (.), Depending on the time after the
    administration. Data represent the mean ± ESM for a minimum of six (6)
    animals.
    fifteen Figure 7 is a graph showing the decrease in systolic blood pressure
    (PAS) obtained in spontaneously hypertensive SHR rats to which it is administered 1
    mL of water (0), 10 mglkg of the peptide of SEQ.ID.NO:2 (.), or 10 mglkg of the peptide of
    SEQ.ID.NO:4 (.l.), Depending on the time spent after administration. The data
    represent the mean ± ESM for a minimum of five (5) animals.
    twenty
    EXAMPLES
    With the intention of showing the present invention in an illustrative way although in
    In no way limiting, the following examples are provided.
    25 Example 1: Procedure for obtaining GPP powder.
    15 kg of chicken leg claws (GPP) gallus gallus domesticus were washed with water
    and they were crushed in a Cato industrial crusher using an 8 mm cutting plate.
    This crushing was frozen in thin layers (1-1.5 cm) and lyophilized for 5 days. He
    Freeze-dried was ground with a Fagor BV-850 model blender and the ground obtained was
    30 sieved with a 2 mm pore size sieve. A fine powder was thus obtained with
    particle size <2 mm and a moisture content <5% (GPP powder). Dust
    of GPP that presented a higher percentage of humidity than indicated,
    lyophilize or let it dry in an oven at 50 ° C for 15 h. Subsequently, it was saved
    at -20 ° C in closed boats and surrounded by silica gel to avoid an increase in the humidity of the samples.
    Example 2. Procedure for obtaining hydrolysates from the powder of
    5 GPP, without pretreatment. In a round bottom plastic tube 0.1 g of GPP powder was weighed, 4.5 mL of distilled water was added and stirred in the vortex for 20 sec. The initial pH of these samples was between 6 and 9. Subsequently, the enzyme was added in a volume of 0.5 mL at an enzyme / protein concentration of 0.4 AU (Units
    10 Anson), and 80 LAPU in the case of Flavourzyme®. Four types of enzymes Alcalase® 2.4 L, Neutrase® 0.8 L, Protamex® and Flavourzyme® 1000 L (Novozyme) were tested separately. These enzymes have different activities (2.4; 0.8; 1.5 AU / g and 1000 LAPU / g, respectively) so the added volume of enzyme varied depending on the enzyme and the enzyme / protein concentration tested. Be
    Thus, it prepared a disolution in distilled water with an enzyme concentration such that taking 0.5 mL of this solution would have the enzyme / protein concentration necessary to perform the hydrolysis. In this way the final volume to be added of all of them (0.5 mL) was unified. The Protamex® enzyme, sold in solid state, was reconstituted in distilled water at the desired concentration before adding it to the
    20 GPP powder. Once the enzyme was added, the pH of the mixtures was adjusted to 7. Hydrolysis was carried out at 25 or 50 ° C under constant stirring at 250 rpm in orbital, for 2, 4 or 24 h. As a control samples were taken in which the hydrolysis time was O h. After the hydrolysis, the enzymes were heat inactivated in a water bath at 85 ° C for 10 min. The samples were cooled on ice for 10 minutes.
    25 min and centrifuged at 10,000 x g, 20 min, 4 ° C. The supernatants obtained are the hydrolysates, which were filtered with 0.45 µm filters and frozen at -20 ° C.
    Example 3. Procedure for obtaining hydrolysates from GPP powder, with pretreatment.
    30 In other samples, once the GPP powder was obtained, the protein was pretreated with changes in pH and temperature. In round bottom plastic tubes 0.1 9 of GPP powder was weighed, a volume of distilled water was added and the pH was adjusted to 3, 7.5 or 10 with NaOH (0.1 M) or Hel (0.1 M) for a final volume of 4 mL, with the proviso that the final pH of the sample should be at
    35 minus a value of 0.5 other than the initial value determined in Example 2.
    Subsequently, the tubes were placed in a water bath at a temperature of 50 or 100 ° C, and left under stirring for 90 min. After this time, the samples pretreated at 100 ° C were allowed to cool to 50 ° C, the temperature was adjusted. pH of the broth obtained at 7 with 0.1 M HCI or 1 M NaOH, in all cases and then the enzymatic hydrolysis was carried out. After this pretreatment, 0.5 mL of a solution of Alcalase® 2.4 L enzyme, Neutrase® 0.8 L, Protamex® or Flavourzyme® 1000 L were added to the GPP samples at an enzyme concentration / 0.4 AU protein, and 80 LAPU in the case of Flavourzyme®. The preparation of the enzyme solution was carried out in the same way as in the previous example. The final volume of the reaction in all cases was also 5 ml, so the volumes were adjusted with distilled water. Once the enzyme was added, the pH of the mixtures was adjusted to 7. Hydrolysis was carried out at 25 or 50 ° C under constant stirring at 250 rpm in orbital, for 2, 4 or 24 h. As a control samples were taken in which the hydrolysis time was O h. After the hydrolysis, the enzymes were heat inactivated in
    15 a water bath at 85 ° C for 10 min. The samples were cooled on ice for 10 min and centrifuged at 10,000 x g, 20 min, 4 ° C. The supernatants obtained are the hydrolysates, which were filtered with 0.45 Jlm filters and frozen at -20 ° C.
    Example 4: Selection of the hydrollzados obtained from the GPP powder of
    20 according to their lECA activity In order to select the GPP powder hydrolysates (filtered supernatants) with lECA activity, the said activity was determined as described in Example 5, discarding those hydrolysates with activity less than 80% when the diluted hydrolyzate was tested in half. Only the
    25 hydrolysates whose supernatant diluted in half showed a percentage of inhibition greater than 80%, which turned out to be forty (40) hydrolyzed. As a sample of the variability in percentages of ACE activities shown by the hydrolysates obtained, Table I shows the AC activity of twenty-eight (28) of them. Specifically, the results corresponding to hydrolysis with a dose are shown
    30 enzyme set of 0.4 AU for Alcalase®, Neutrase®, and Protamex® and 80 LAPU for Flavourzyme® and a fixed hydrolysis time and temperature of 24 hours and in which only the pretreatment varies, comparing samples without pretreatment regarding samples to which different pretreatments have been applied for 90 min (different pH: 3, 7.5 and 10 and different temperatures: 50 and 100 ° C). Hydrolysates
    35 obtained at a hydrolysis temperature of 25 ° C were not interesting
    regardless of the pretreatment conditions, as shown by the IC values obtained in each case (Table 1). Table 1. ACE activity (%) of GPP powder hydrolysates obtained with
    5 Alcalase®, Neutrase®, Protamex® (0.4 AU) and Flavourzyme® (80 LAPU) at 25'C
    for 24 h without and with pre-hydrolysis treatment for 90 min at 50 ° C (A) or
    100'C (8).
    TO Treatment ~ revio hydrolysis (90 min, 500 CI
    Without
    Enzyme TECA ('lo) IC "Il1ll pretreatment
    pH 3 pH 7.5 pH 10 pH 3 pH 7.5 pH 10
    Alcalase® 19.5170, 6129.5763.86NONONO
    Neutrase® 37, 1088.8285.4379.531.621.822.40
    Protamex® 65.7689.7457.3374.342.79NO2.38
    Flavourzyme® 20.2070.2432.7326.49NONONO n-6 for each sample
    N.D: Not determined
    Treatment ~ revio hydrolysis {90 min, 100 ° CI Enzyme lECA ('lo) IC "(I1L)
    pH 3 pH 7.5 pH 10 pH 3 pH 7.5 pH 10
    Alcalase® 83.5283.6686.921.881.451.86
    Neutrase® 87.9391.4991.461.491.401.69
    Protamex® 92.3190.0596.371.131.101.01
    Flavourzyme® 71, 2468.1067.44Do notDo notDo not
    n = 6 for each sample 15 N.D: Not determined
    Table 11 shows some of the hydrolysates with the best lECA activities obtained and the necessary amount of supernatant necessary to produce a 50% enzymatic decrease (ICso) expressed in ~ LJmL.
    Table 11. ACE activity (percentage of inhibition and lesion) of GPP powder hydrolysates with higher inhibitions, obtained with Neutrase® and Protamex® (0.4 AU) at 50'C for O, 2, 4 and 24 h of hydrolysis.
    Weather Treatment ~ revio hydrolysis (90 min, 1000Cl
    Enzyme HydrolysisACEI (%)IC "(~ l)
    (h) pH3pH 7.5pH 10pH3pH 7.5pH 10
    OR 82.9670.9980.842.723.422.01
    Neutrase® 293.7793.7794.040.900.891.01
    4 101.5791.4091.790.991.261.44
    24 91.0393.0293.130.980.831.10
    Protamex® OR81.6382.5885.214.072.603.14
    2 94.8193.8994.790.750.630.65
    4 94.6595.8196.450.690.580.59
    24 93.1295.7797.430.500.510.54
    Three hydrolysates were selected as representatives of all those hydrolysates that showed an ICso <1 IJ L.
    • The hydrolyzate obtained with Protamex® for 2 h at 50 ° C pH 7 with
    10 pretreatment prior to hydrolysis of 100 ° C for 90 min at pH 7.5 (ICso = O, 63 IJL of hydrolyzate or 29 IJg of protein / mL of hydrolyzate), which we call hydrolyzate 1.
    • The hydrolyzate obtained with Protamex® for 24 h at 50 ° C pH 7 with pretreatment prior to hydrolysis of 100 ° C for 90 min at pH 7.5 (ICso
    15 = 0.51 IJL of hydrolyzate or 9.56 IJg of protein / mL of hydrolyzate), which we call hydrolyzate 2.
    • The hydrolyzate obtained with Neutrase® for 2 h at 50 ° C pH 7 with pretreatment prior to 100 ° C hydrolysis for 90 min at pH 7.5 (ICso = 0.89 IJL of hydrolyzate or 46.27 IJg of protein / mL of hydrolyzate), to which
    20 we call hydrolyzate 3.
    Example 5: Measurement of the lECA activity of hydrolysates and peptides The measurement of the lECA activity was performed according to the method described by Sentrandreu and Toldrá (Sentrandreu, MN, and Toldrá F. "A rapid simple and sensitive fluorescence
    25 method for the assay of angiotensin-I converting enzyme ". 2006. Food Chem. 97, 546
    554) and subsequently modified by Quiros et al. (Quirós, A. et al. UStability to gastrointestinal enzymes and structure-activity relationship of beta-casein-peptides with antihypertensive properties "2009. Peptides, 30, 1848-1853). It is a fluorimetric method in which it is used as a substrate for the RCT o-aminobenzoylglycyl-pnitrophenylalanylproline (0-Abz-Gly-p-Phe (N02) -Pro-OH) The fluorescence generated by the release of the aminobenzoylglycine group was measured using an excitation wavelength of 350 nm and a of emission of 420 nm. The percentage of inhibition (% Inhibition) was calculated by applying the following formula:
    (White) - (Sample White)% Inhibition = 1-x 100
    (Control) - (Substrate blank)
    The protein concentration of the hydrolysates to determine the leso value was determined by the Kjeldahl method (FIL-IDF. UMilk. Determination of nitrogen content (Kjeldahl method). Standard 20B ". 1993. Inl. Dairy Fed. Brussels, Belgium) multiplying the percentage of nitrogen in the sample by 6.25.
    Example 6: Isolation, identification and synthesis of peptides with ACE activity Analytical and semi-preparative high efficiency liquid chromatography (HPLC) equipment was used, as well as a tandem mass spectrometry (MS) equipment that allowed the sequencing of the peptides. The steps mentioned below were performed.
    6.A. Obtaining the soluble fraction of the hydrolyzate 1 of GPP powder Following the procedure described in Example 4, 250 ml of the protein hydrolyzate called hydrolyzate 1 were obtained, whose supernatant was centrifuged again in filtering devices (Centripep, Amicon Inc) with hydrophilic membrane of 3000 gives pore size. The permeate (fraction less than 3000 Da) obtained was collected, lyophilized and stored at -20 ° C until its subsequent fractionation.
    6.8 Fractionation by reverse phase HPlC at semi-preparatory scale The semi-preparative HPlC used was from the Agilent 1260 series (Agilent Technologies) consisting of a quaternary pump, a gradient controller, a
    35 injector, a diode array detector, a fraction collector and
    data acquisition and processing software (Agilent OpenLab CDS ChemStation
    Edition for LC & LC / MS systems A.01.04).
    The lyophilized permeate obtained in section 6A was dissolved in water at5 concentration of 10 mg / mL and the peptides were separated byreverse phase chromatography using a C18 column (Europa peptide, 120 AO, 25x 1.0 mm, 5 IJm; Teknokroma). The solvents used were a mixture of water:trifluoroacetic acid (1000: 1) and acetonitrile: trifluoroacetic acid (1000: 0.8),solvents A and B respectively, and elution was performed at a flow of 4 m Umin
    10 using the following gradients in the order of appearance: Oa 40% of solvent B in 50 min), 40-45% of B in 1 min, 45-90% of B in 4 min, 90-0% of B in 1 The volume of injected sample was 750 IJL and the absorbance of the solvent was monitored at 214 nm.
    15 8 different fractions (Figure 1) called F.1 -F.8 were collected from the protein hydrolyzate called hydrolyzate 1, which were lyophilized and reconstituted in water at different volumes. The protein content of these reconstituted fractions was determined by the bicinconinic acid method (BCA ™ Protein Assay Kit, Thermo Scientific) following the manufacturer's instructions. TO
    20 said fractions were determined lECA activity according to the protocol described in Example 5 (Table IV). Those fractions with a percentage of ACE activity greater than 80% were determined the ICso, observing that the fractions with the lowest values were F.3 and F.6, with 1.99 and 1.24 IJg / mL of protein respectively.
    25 Table IV. Protein content, percentage of ACE activity in the fractions obtained by reverse phase HPLC from the permeate of size <3000 Da obtained from the protein hydrolyzate called hydrolyzate 1.
    ICso Activity Concentration
    Fraction
    prolein (~ g / mL) LECA (%) (~ g / mL) F.1 59.38 66.88 ± 1.91
    F.2 126.18 93.07 ± 1.18 12.08
    F.3 30.56 80.45 ± 1.36 1.99
    F.4 31.43 69.64 ± 1.34
    F.5 37.61 80.33 ± 2.81 3.59
    F.6 45.2289.55 ± 0.801.24
    F.7 13.5936.27 ± 4.05
    F.8 32.25OR
    n-3 per fraction
    Fractions F.3 and F.6 with greater ACE activity were subjected to a second fractionation by reverse phase HPLC on a semi-preparative scale using the same equipment, column and solvents but eluting the sample with a linear gradient of 10-20% of the solvent B in A in 40 minutes and 20 to 30% of solvent B in A in 40 minutes for F.3 and F.6, respectively. 6 sub-fractions were collected from the F.3 (F.3.1, F.3.2, F.3.3, F.3.4, F.3.5, F.3.6) and 8 subraction F.6
    (F.6.1, F.6.2, F.6.3, F.6.4, F.6.5, F.6.6, F.6.7, F.6.8) (Figure 2A and 2B). To all these
    10 sub-fractions were determined the ACL activity following the method already described in Example 5 and the protein concentration by the BCA method (BCA ™ Protein Assay Kit, Thermo Scientific) following the manufacturer's instructions (Tables V and VI). Those fractions with a percentage of ACE activity greater than 75% were also determined the IC5 (j. From these results, the
    15 sub-fractions with a lower ICso, specifically the F .3.3 and the F .6.6 with ICso values of 0.83 and 0.86 ~ g / mL respectively. Both sub-fractions eluted approximately between 12 and 16 minutes, using the separation method described above (12.5-15.5 min and 13-14 min for F.3.3 and F.6.6, respectively). The two subfractions, which contained peptides with ACE activity, were collected, lyophilized and
    20 were kept at -20 ° C until subsequent identification of the peptides responsible for said activity.
    Table V. Protein content, ACE activity and ICso of the subfractions obtained by reverse phase HPLC from
    25 of fraction F.3.
    Subfraction Protein concentration (~ g / mL)ACE activity (%)IC "(~ g / mL)
    F.3.1 36.0667.68 ± 4.4111, 23
    F.3.2 42, 2582.37 ± 1.225.64
    F.3.3 20.9691.75 ± 4.720.83
    F.3.4 19.9381.45 ± 3.831.97
    F.3.5 65.0781.56 ± 0.4411, 77
    F.3.6 38.0372.99 ± 0.3712.01
    n 3 per fraction
    Table VI Protein content, ACE activity and ICso of the subfractions obtained by reverse phase HPLC from fraction F.6.
    Concentration
    ICso activity
    Protein subfraction
    LECA (%) (~ g / mL)
    (~ g / mL)
    F.6.1 26.6744.74 ± 2.15
    F.6.2 26.6712.75 ± 0.05
    F.6.3 26.6780.71 ± 4.296.24
    F.6.4 26.6784.03 ± 1.994.73
    F.6.S 26.6788.28 ± 2.813.90
    F.6.6 26.6791.66 ± 0.860.86
    F.6.7 26.6775.17 ± 6, 184.62
    F.6.8 26.6776.93 ± 1.555.01
    5 n-3 per fraction
    6C. Identification of peptides with ACL activity by tandem mass spectrometry (MS / MS)
    10 For the identification of the peptides responsible for the ACE activity of subfractions F.3.3 and F.6.6 obtained by semi-preparative HPLC from hydrolyzate 1 and subsequently from fractions 3 and 5, respectively, a L TQ mass spectrometer was used Orbitrap Velos (ThermoFisher Scientific) equipped with a nano-ionization source (Proxeon) connected online to a nano-HPLC Easy-LC
    15 (Proxeon, ThermoFisher Scientific). For which, they were reconstituted in water and the fractions were diluted with 0.1% TFA to a protein concentration of 0.1 ~ g / ~ 1. The separation of the peptides from the fractions was performed using a C18 EASY-Column pre-column, 2 cm, ID 100 ~ m, 5 ~ m and a C18 EASYColumn column, 10 cm, ID 75 ~ m, 3 ~ m, (Thermo Scientific) in which 10 IJL of were injected
    20 the diluted fractions. The mobile phases used were 0.1% formic acid: 2% acetonitrile (phase A) and 0.1% formic acid in 100% acetonitrile (solvent B) and the flow rate was 400 nUmin. For fraction F.3.3. The gradients used were: 0-45% B in 80 min, 45-100% B in 20 min and 100% B for 10 min. For fraction F.5.6 a gradient of 0-35% B in 40 min was used, 35-100% B in 10 min
    25 and 100% B for 10 min. All mass spectra were acquired in positive ion mode. The scanner (miz 50-2000) was acquired with a targeted value of 10,000,000 at a resolution of 30,000 to 400 mL and the 20 most intense ions were selected for collision-induced dissociation fragmentation in the L TQ with a targeted value of 10,000 Y a
    5 collision energy of 35%. For the identification of the peptide sequences, the Proteome Discoverer 1.4.288 (Thermo) program with MASCOT 2.4.1.0 was used and the sequences were searched in a chicken protein database in which 25992 sequences were included (IPI_chicken_3 .81). The peptides that are collected in Table VII were identified.
    Table VII Peptides identified in subsections F.3.3 and F.6.6
    Mass
    Mass
    Subfraction
    I KNOW THAT. ID. NO:
    Theoretical
    experimental
    F.3.3 1146 .3811 62.60
    F.3.3 21162.381162.60
    F.3.3 31211.391211.62
    F.3.3 41075.171075.47
    F.6.6 5647647.36
    F.6.6 6746.91747.44
    F.6.6 7622640.44
    F.6.6 81106.581107 .59
    F.6.6 910681068.53
    6.0 6.0 Synthesis of peptides with ACE activity The peptides of the invention were sent to Chemically synthesized to Casio ApS (Lyngby, Denmark) and were prepared with the following purity values: SEO. ID. NO: 1 = 99.68%. SEO.ID.NO:2 = 90.19%. SEO.ID. NO: 3 = 98.09%. SEO ID.NO:4 = 99.10%. SEO.ID.NO:5 = 99.33%. SEO.ID.NO:6 = 99.71%.
    20 SEO ID.NO:7 = 99.71%. SEO.ID.NO:8 = 99.64% AND SEO.ID.NO:9 = 98.39%.
    Once synthesized, the ACL activity was determined in order to know which
    which of the peptides identified in subsections F.3.3 and F.6.6 were the one or more
    responsible for such activity in the fractions. Table VII shows the results obtained in the determination of said activity represented as ie ", (~ M).
    Table VIII ICso values of the identified peptides of
    5 subsections F.3.3 and F.6.6 with better ACE activity obtained from the antihypertensive hydrolyzate called hydrolyzate 1
    Peptide subfraction le "(~ M)
    F.3.3 SEO.iD.NO: 129.71
    F.3.3 SEO.iD.NO: 211, 01
    F.3.3 SEO.iD.NO: 3> 137.6
    F.3.3 SEO.iD.NO: 444, 75
    F.6.6 SEO.iD.NO: 5> 515.4
    F.6.6 SEO.iD.NO: 680.91
    F.6.6 SEO.iD.NO: 77.06
    F.6.6 SEO.iD.NO: 8> 150
    F.6.6 SEO.iD.NO: 9> 150
    iO n "'' 'by JW ptido
    fifteen The measurement of the lECA activity of the synthesized peptides showed that of the 9 peptides tested, 5 of them presented the aforementioned activity, observing ICso concentrations below 100 IJM. Specifically, the peptides identified as SEQ.ID.NO:2 and 7 were those with the highest ACL activity (lowest value ICso), which were present in sub-sections F.3.3 and F.6.6. respectively. Both were selected to determine their antihypertensive activity in spontaneously hypertensive rats (SHR) and in Wistar-Kyoto (WHY) rats.
    twenty Example 7: Measurement of antihypertensive activity in rats of GPP goat hydrolyzate 1. The hydrolyzate obtained with Protamex® 2h at 50 ° C was selected with pretreatment of 100 ° C, pH 7.5 for 90 min, since it had a good ACE activity, to study the effect of this hydrolyzate on BP in spontaneously hypertensive rats (SHR ) and in Wistar-Kyoto (WKY) rats, which are the normotensive control of
    SHR rats. The GPP protein powder hydrolyzate called hydrolyzate 1 is
    prepared according to the procedure described in Example 4.
    To perform these studies, 17-20 week SHR and WKY male rats were used
    5 life and weight between 300 and 360 g, from Charles RiverLaboratories España S.A. The rats remained at an ambient temperaturestable of 23 () C, and with 12-hour light-dark cycles, ingesting water and food tofreely available. The measurement of BP in these animals was carried out with amodification of the tail cuff technique, originally described by
    10 Buñag (Buñag RD. "Validation in awake rats of a tail-cuff method for measuring systolic pressure". 1973. J. Appl. Physiol. 34, 279-282). In the present example, the modification of systolic blood pressure (PAS) and diastolic blood pressure (PAD) produced by the acute administration of the protein hydrolyzate obtained from GPP powder was evaluated. Before placing the cuff on the tail of the rats, they
    15 exposed at a temperature close to 37 ° C to facilitate dilation of the caudal artery. To ensure the reliability of the measurement, the animals were accustomed to the procedure 2 weeks before carrying out the test.
    The administration of hydrolyzate 1 to be tested was performed by intragastric tube in a
    20 time range between 9 am and 10 am in the morning. The SHR rats used for the study at that time had PAS and PAD values, respectively between 192 and 212 mm Hg, and between 140 and 180 mm Hg. The WKY rats used for the study had at that time PAS and PAD values respectively between 110 and 140 mm Hg, and between 90 and 110 mm Hg. Be
    25 took measures of the PAS and the PAD in the animals periodically, every 2 hours, up to 48 hours post-administration of the products to be tested using a PA meter for rats model Le50001 (Letica). The amount of hydrolyzate administered was 5 mUkg of body weight of the animal. The procedure performed on animals was approved by the Ethical Committee of the Rovira i Virgili University.
    As a negative control to establish the circadian variation of the PAS and the PAD in probed rats, the measures of the PAS and the PAD obtained in rats that were administered by intragastric tube 1.5 mL of water were used. As a positive control to establish the effect on the PAS and on the PAD of a prototype lECA drug, such as Captopril, the PAS and PAD measurements were used
    obtained in rats given by intragastric tube 50 mg Ikg of Captopril. This dose of Captopril was administered to each rat in a volume of 1.5 mL.
    5 The results obtained were grouped and the mean ± the standard error of themean (ESM) for a minimum of 6 homogeneous trials. The data of the three groupsof animals were compared in a 2-way analysis of variance (ANOVA)using the Tukey test in each post-administration time with the programSPSS statistic (18M SPSS Statistics software version 20.0) and was considered
    10 significant difference for p values <0.05.
    Figures 3A and 38 show, respectively, the decrease in SBP and PAD obtained in SH R rats at different times, after administration of GPP powder hydrolyzate with Protamex®, 2h, at 50 ° C (hydrolyzate 1) . These figures also include the decrease in SBP and PAD observed after the administration of Captopril. As expected, Captopril produced a pronounced decrease in SBP and PAD in SHR rats. The decrease in SBP and PAD were maximum at 6 hours after drug administration. This GPP powder hydrolyzate produced a significant decrease in SBP and PAD in the 20 animals at 6 hours after administration, which was significantly equal to that produced by Captopril at the same time. After 2 hours of consumption of the hydrolyzate, the SBP and PAD of the treated animals begin to decrease but this pressure drop was not significantly different from that produced by untreated rats until reaching 6 hours. The values of the PAS and the PAD
    25 observed 24 hours after the different administrations were similar to those that the animals had before them.
    Figures 4A and 48 show respectively the changes of the PAS and the PAD
    obtained in WKY rats at different times, after administration of hydrolyzate 1 of
    30 GPP powder. It can be seen that the hydrolyzate did not modify either the PAS or the PAD of the treated animals. These results allow us to rule out possible undesirable effects in the hydrolyzate tested on BP of normotensive subjects.
    Example 8: Measurement of antihypertensive activity in rats of hydrolyzate 2 of GPP powder
    The determination of antihypertensive activity in vivo in SHR rats of the hydrolyzate obtained from GPP powder called hydrolyzate 2 (Hydrolysis with Protamex® 24h at SO ° C with pretreatment of 100 ° C, pH 7.S for 90 min) was performed following the method and procedure described in example 7. The tested concentration of
    5 hydrolyzate was 5 mUkg and water (1.5 mL) and captopril (5 mg / kg) were also testedas negative and positive controls, respectively.
    Figures 5A and 58 show the variation of the PAS and the PAD, respectively, of SHR rats after intragastric administration of hydrolyzate 2, water and 10 captopril at the aforementioned doses. These PA curves show how, as the time elapses since the animal ingested the hydrolyzate 2, both the SBP and the PAD decreased until reaching the maximum descent at the post-administration ah (26 and 34 mm HG, respectively). Subsequently, the antihypertensive effect begins to reverse. The effects observed on BP after
    Water administration or captopril are as expected as described in example 7.
    Example 9: Measurement of antihypertensive activity in rats of GPP powder hydrolyzate 3.
    20 The determination of the antihypertensive activity in vivo in SHR rats of the hydrolyzate obtained from GPP powder called hydrolyzate 3 (Hydrolysis with Neutrase® 24h at 50 ° C with pretreatment of 100 ° C, pH 7.5 for 90 min) was performed following the method and procedure described in Example 7. The tested concentration of hydrolyzate 3 was 5 mUkg and water (1.5 mL) and captopril (5 mg / kg) were also tested.
    25 as negative and positive controls, respectively.
    Figures 6A and 68 show, respectively, the decrease in SBP and PAD obtained in SH R rats at different times, after administration of GPP powder hydrolyzate with Neutrase®, 2h, at 50 ° C (hydrolyzate 3) . Hydrolyzate intake
    30 3 showed an antihypertensive effect after 2 hours of oral administration, being maximum at 6 hours. PAS and PAD lowering values were observed similar to those produced by the drug captopril at that time. The PAS and PAD curves of the SHR rats treated with water and with captopril showed the expected effect as described in example 7.
    Example 10: Measurement of antihypertensive activity in rats of peptides with ACE activity identified in antihypertensive hydrolyzate 1 Once the peptides present in the antihypertensive hydrolyzate have been identified (Table VII) and to determine which of them had ACE activity (Table VIII)
    5 proceeded to determine the in vivo antihypertensive activity of the most active peptides. During digestion, ingested peptides can be hydrolyzed by digestive tract proteases and lose their activity or not be able to cross the intestinal barrier and therefore not be absorbed. That is why it is necessary to conduct in vivo studies to evaluate their antihypertensive activity once they are
    10 digested The degree of hydrolysis produced by the physiological digestion of a particular peptide will depend on its size, its nature, and the presence of other peptides in the medium (FitzGerald et al., "Hipotensive peptides from milk proteins". 2004. J. Nutr. 134 (4) .9805-9885).
    15 The antihypertensive activity of the peptides that had the highest ACE activity (SEO.ID.NO:1, 2, 4, 6 and 7) was performed in male SHR rats aged 17-20 weeks following the same method described in the Example 5. The conditions of maintenance and feeding of the animals were also the same as those described in Example 7. Similarly, they were used as a negative control and
    20 positive, water and captopril (50 mg / kg), respectively and in the case of the peptides, were tested individually, administering to each animal a volume of 1.5 ml of the peptide dissolved in water at a concentration of 10 mg / kg, since this volume was comparable to the volume of hydrolyzate 1 that each animal received. These peptides were chemically synthesized as described in section 40.
    The results obtained were grouped and the mean ± the ESM was obtained for a minimum of 5 homogeneous trials. The most promising results were those obtained with the peptides denominated as SEO.ID.NO: 2 and 4. The data of the three groups of animals (treated with water, with the peptide SEO.ID.N0: 2 and with the peptide 30 SEO.ID.NOA) were compared in a 2-way and 1-way analysis of variance (ANOVA), in the latter case, to verify the effect of peptides on BP at study hours (O, 2, 4, 8 and 24 h) individually. In both cases, the Tukey test was used and the analyzes were performed using the SPSS statistical program (18M SPSS Statistics software version 20.0). The difference was considered significant for
    35 values of p <0.05.
    According to the results shown in Figure 7, the intake of peptides SEQ.ID.NO:2 and 4 produced a decrease in SBP, at 4 or 6 h after administration, respectively. In both cases, the maximum drop occurred at
    5 6 h after administration, a significantly higher antihypertensive effect (P <0.01) was observed with the peptide of SEQ.ID.NO:4. The PAS recovered its initial values at 8 or 24 hours after its intake.
    Individual administrations of peptides SEQ.ID.NO:1, 6 and 7 to SHR 10 rats did not produce a significant decrease in SBP (data not shown).
    权利要求:
    Claims (8)
    [1]
    1. Procedure for obtaining an enzymatic hydrolyzate with antihypertensive activity, characterized in that it comprises the following steps: a) crush chicken leg claws and lyophilize to obtain a powder material 5 with particle size <2 mm;
    b) adjust an aqueous solution of the above powder to a pH between 3 and 10, and heat between 80 and 120 ° C for between 60 and 90 min, with the proviso that said pH value is different from that corresponding to the powder material of the previous stage in at least 0.5;
    10 c) cool the previous solution to a temperature between 45 and 55 ° C and carry out an enzymatic hydrolysis for a time between 1 and 24 h with proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens identified as E.C. 3.4.21.62 and 3.4.24.28;
    d) evaluation of the inhibitory activity of the angiotensin-converting enzyme according to the method described in Sentandreu, MN and Toldrá F. 2006. Food Chem. 97, 546-554; And e) selection of a hydrolyzate with activity> 80% in 50% aqueous solution v / va from the previous evaluation.
    A method according to claim 1, characterized in that the hydrolysis time of step c) is between 2 and 24 h.
    [3]
    3. A method according to claim 1, characterized in that it comprises the following steps: a) grind chicken leg claws and lyophilize to obtain a powder material with particle size <2 mm;
    b) adjust an aqueous solution of the powder to a pH of 7.5 and heat at 100 ° C for 90 min, in which said pH of 7.5 is different from that corresponding to the powder material of the previous stage by at least one value of 0.5;
    c) cooling the above solution to a temperature of 50 ° C and hydrolyzing for a period of 2 or 24 h with proteolytic enzymes obtained from Bacillus licheniformis and Bacillus amyloliquefaciens identified as E.C.
    [3]
    3.4.21.62 and 3.4.24.28; d) evaluation of the converting enzyme inhibitory activity of
    angiotensin according to the method described in Sentandreu, MN and Toldrá F. 35 2006. Food Chem. 97,546-554; Y
    e) selection of a hydrolyzate with activity> aO% in 50% aqueous dilution v / v from the previous evaluation.
    [4]
    4. Enzymatic hydrolyzate characterized in that it comprises the peptides identified by SEO.ID .NO: 1-9 or salts thereof.
    5. Use of the enzymatic hydrolyzate of claim 4, in the preparation of a medicament for the treatment of hypertension in a subject.
    [6]
    6. Use according to claim 5, characterized in that said subject is human.
    [7]
    7. Pharmaceutical composition comprising an enzyme hydrolyzate containing
    10 the peptides identified by SEO.ID.NO:1-9 or salts thereof, and pharmaceutically acceptable excipients, in which said composition is in liquid or syrup form.
    to. Peptide identified by one of the SEO.ID.NO:2 or 4 sequences.
    [9]
    9. Use of a peptide identified by SEO.ID.NO:2 or 4 or salts thereof, or
    15 the combination of them in the preparation of a medication for the treatment of hypertension.
    [10]
    10. Pharmaceutical composition comprising a peptide identified by the sequence SEQ.ID.NO:2 or 4 or salts thereof or the combination thereof, and pharmaceutically acceptable excipients.
    20 11. Food supplement comprising a peptide identified by the sequence SEO.ID.NO:2 or 4 or salts thereof or the combination thereof, and food additives.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US6767990B1|1999-12-01|2004-07-27|Food Industry Research And Development Institute|Peptides used as angiotensin converting enzyme inhibitor and preparation process thereof|ES2702617A1|2017-09-04|2019-03-04|Univ Rovira I Virgili|Hydrolyzed claws of chicken legs, their peptides and uses thereof |
    ES2804649A1|2019-08-06|2021-02-08|Univ Rovira I Virgili|Hypoleptinémic composition and its use |TW201106955A|2009-08-26|2011-03-01|Nat Univ Chung Hsing|The method for preparation bioactive hydrolysate which have antihypertensive activity from chicken feet|CN110272934A|2019-07-08|2019-09-24|山西原生肽科技有限公司|The extracting method and its application of endothelium corneum gigeriae galli small-molecular peptides|
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