WO2011135513A1 - Procédé d'obtention d'extraits peptidiques bioactifs par hydrolyse de protéines lactosériques avec des enzymes de cynara cardunculus, extraits ainsi obtenus et leurs utilisations - Google Patents

Procédé d'obtention d'extraits peptidiques bioactifs par hydrolyse de protéines lactosériques avec des enzymes de cynara cardunculus, extraits ainsi obtenus et leurs utilisations Download PDF

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WO2011135513A1
WO2011135513A1 PCT/IB2011/051811 IB2011051811W WO2011135513A1 WO 2011135513 A1 WO2011135513 A1 WO 2011135513A1 IB 2011051811 W IB2011051811 W IB 2011051811W WO 2011135513 A1 WO2011135513 A1 WO 2011135513A1
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seq
peptides
extracts
peptide
hydrolysis
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PCT/IB2011/051811
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English (en)
Portuguese (pt)
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Maria Manuela Estevez Pintado
Tânia Sofia GRANJA TAVARES
Maria Manuela Faria Amorim
Francisco Xavier Delgado Domingos Antunes Malcata
Rui Manuel Matos Meireles De Barros
João Ernesto DE CARVALHO
Carlos José DIAS PEREIRA
Marta Helena Fernandes Henriques
Isidra SÁNCHEZ RECIO
Mercedes GONZÁLEZ RAMOS
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Consejo Superior De Investigaciones Cientificas
Escola Superior Agrária De Coimbra
Universidade Católica Portuguesa - Ucp
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Publication of WO2011135513A1 publication Critical patent/WO2011135513A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C21/00—Whey; Whey preparations
    • A23C21/02—Whey; Whey preparations containing, or treated with, microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
    • A23J3/343—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • 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.
  • 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.
  • FUFOSE Federal Food Science in Europe
  • Whey proteins have a mixture of various secreted proteins such as ⁇ -lactalbumin (oc-La), ⁇ -lactoglobulin ( ⁇ -Lg), lactoferrin, lactoperoxidase, immunoglobulins and caseinomacropeptide (CMP), as well as a number of factors. of growth.
  • oc-La ⁇ -lactalbumin
  • ⁇ -Lg ⁇ -lactoglobulin
  • lactoferrin lactoperoxidase
  • CMP caseinomacropeptide
  • 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.
  • ACE Angiotensin-Converting Enzyme
  • 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 ⁇ - 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.
  • 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.
  • said process uses inter alia trypsin, chymotrypsin or a combination of both.
  • 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.
  • the concentration of C. candunculus flower extract should be between 1 and 5% (v / v), and preferably 1.6% (v / v).
  • the process may comprise the following steps:
  • Whey skimming (1) preferably by centrifugation
  • Microbial load reduction (2) preferably by microfiltration
  • 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.
  • 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.
  • 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,
  • 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.
  • 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,
  • Said peptides may be used:
  • 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.
  • peptides and extracts containing bioactive peptides were obtained by hydrolysis reactions of enzyme-catalyzed whey protein solutions from Cynara cardunculus.
  • CC-Lactoalbumin (OC-La), the second quantitatively higher protein. representative of bovine whey; Caseinomacropeptide (CMP), heterogeneous polypeptide fraction derived from the disruption of the ⁇ -casein (K-CN) Pheios-Met106 bond; and Bovine Whey Protein Concentrate (CPS) as substrates; and cardosins as hydrolysis enzymes.
  • CMP Caseinomacropeptide
  • K-CN ⁇ -casein
  • CPS Bovine Whey Protein Concentrate
  • the hydrolysis reaction takes place at temperatures between 50 and 60 ° C and at pH values between 4,8 and 6,2.
  • the hydrolysis reaction takes place at a temperature of 55 ° C and a pH value of 5.2 over a period of 7 hours.
  • 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%.
  • 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.
  • 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 ⁇ -La solutions with other concentration values where the system is also effective. Under preferred conditions, the degree of hydrolysis of ⁇ -La protein is 8%.
  • the hydrolysis reaction takes place at temperatures between 50 and 60 ° C and at pH values between 4,8 and 6,2.
  • the hydrolysis reaction takes place at a temperature of 55 ° C and a pH value of 5.2 over a period of 7 hours.
  • 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%.
  • Extracts obtained through hydrolysis reactions of systems 1, 2 and 3 were found to have ACE inhibitory activity, with an IC 50 index of 105.4, 47.6 and 296. , 0 ⁇ g / 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 ⁇ g / mL for systems 1, 2 and 3, respectively.
  • the fraction> 3000 Da has IC 50 values of 717.0, 539.5 and 717.0 ⁇ g / mL for the same systems.
  • 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 also show antioxidant activity of 0.96 ⁇ 0.08 1.12 ⁇ 0.13 and 0.22 ⁇ 0.002 ⁇ Trolox equivalents / mg protein. hydrolyzate, respectively.
  • 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.
  • the protein and peptide fractions obtained in the filtration processes were characterized in physicochemical and microbiological terms.
  • Section A demonstrated an in vitro ACE inhibitory action of the extracts obtained, especially in extracts containing peptides originating from ⁇ -La hydrolysis and their fraction ⁇ 3000 Da.
  • the hydrolysis peptide concentrate (7) generated in the section B process 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.
  • 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.
  • 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 ⁇ -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 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 ⁇ - 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.
  • CMP K-CN
  • SEQ.ID.N 0 9 MAIPPKKNDQD (106-115); SEQ.ID. No. 8: AIPPKKNDQD (107-115), derived from the ⁇ -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.
  • hydrolysis-derived peptides and peptide-containing extracts having the following amino acid sequences:
  • SEQ.ID. ° 12 VQVTSTAV (162-169), derived from the degradation of K-CN, the peptide SEQ.ID. ° 14: RELEEL (1-6) from degradation of ⁇ -CN and peptide SEQ.ID. ° 15: DAQSAPLRVY (33-42) from ⁇ -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.
  • SEQ.ID. No. 8 AIPPKKNDQD (107-115), derived from the ⁇ -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.
  • 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.
  • FIG. 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.
  • 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 osmos
  • ⁇ 0 is a constant
  • ⁇ and ⁇ 2 are linear coefficients
  • ⁇ , ⁇ and ⁇ 2 , 2 are quadratic coefficients
  • ⁇ , 2 is the interaction coefficient
  • ⁇ 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.
  • 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.
  • the degree of hydrolysis 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).
  • 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 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.
  • 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 ⁇ -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.
  • 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.
  • ACE inhibitory activity was calculated as the amount of soluble protein required to inhibit 50% enzyme (IC 50 ) (in ⁇ g 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:
  • 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.
  • 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 ⁇ stock solution in 75 mM phosphate buffer (pH 7.4).
  • phosphate buffer 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 ⁇ ) 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.
  • 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 ( ⁇ Trolox equivalents / mg protein / peptide).
  • 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.
  • the whey was obtained as a byproduct of the production of dish-type cow cheese, and provided by the Coimbra School of Agriculture.
  • PCA Plate Count Agar
  • BPA Baird Parker Agar
  • VRBGA Van Oil Red Bile Glucose Agar
  • RB Rose Bengal Agar
  • egg-tellurium yolk supplement purchased from LabM (Bury, UK).
  • 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.
  • 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.
  • 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.
  • 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).
  • Peptide concentrates obtained in section A hydrolyzed CPS and CMP (Davisco ® ) and hydrolysed oc-La (Sigma)
  • 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.
  • 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).
  • 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 ⁇ m filters. Yes. The different eurotubes were then inoculated with 100 ⁇ l of the test organism at different concentrations, namely 10 3 , 10 5 and 10 7 cfu / mL.
  • 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 ⁇ filters.
  • 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.
  • Peptide concentrates obtained in section B (Fi - CPSH - total fraction and F 2 - CPSH - ⁇ 3000 Da).
  • 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.
  • CEMIB Center of Bioterismo
  • UNICAMP UNICAMP
  • the 50% effective dose corresponds to the dose required to produce 50% inhibition in the observed lesions compared to the negative control.
  • mice 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).
  • ILU Ulcerative Lesion Index
  • mice 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).
  • ILU Ulcerative Lesion Index
  • 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).
  • mice 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).
  • ILU Ulcerative Lesion Index
  • Croton oil-induced dermatitis 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).
  • 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 ⁇ m pore size filter.
  • the sample was eluted with a 0% to 35% solvent gradient B in 70 min at 35 ° C.
  • the injected sample volume was 500 ⁇ l, 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.
  • 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).
  • 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 ⁇ m particle size) reverse phase column Bio-Rad Laboratories, Richmond, CA, USA) was used.
  • 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)) .
  • solvent A was a mixture of water and trifluoroacetic acid (1000: 0.37 (v / v))
  • 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.
  • 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 TM 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.

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Abstract

La présente invention concerne un procédé d'obtention d'extraits peptidiques de protéines lactosériques par hydrolyse d'un(e) ou de plusieurs protéines ou peptides contenant les séquences d'acides aminés de peptides bioactifs, consistant à utiliser, à cet effet, des enzymes protéolytiques, et notamment des cardosines et d'autres enzymes similaires produites par l'espèce Cynara. En variante, les peptides peuvent être obtenus par synthèse chimique ou par des processus enzymatiques et/ou fermentatifs. Le procédé couvert par la présente invention permet d'obtenir des concentrés protéiques et peptidiques à faible teneur en lactose et en sels, qui présentent des activités biologiques utiles pour la formulation de produits dans les industries alimentaire et pharmaceutique. Ainsi, les hydrolysats et leurs fractions ou peptides résultant dudit procédé peuvent être utilisés dans des produits alimentaires, agissant comme conservateurs pour ceux-ci ou, après leur ingestion, renforçant les défenses naturelles de l'organisme. Ils peuvent en outre être utilisés dans l'élaboration de produits pharmaceutiques contre des maladies, notamment pour faciliter la régulation de la pression artérielle et/ou la lutte contre les infections bactériennes, et des processus inflammatoires et ulcérogéniques. L'invention élargit le champ d'application des protéines lactiques, contribuant ainsi à l'amélioration et à la revalorisation du lactosérum, notamment.
PCT/IB2011/051811 2010-04-26 2011-04-26 Procédé d'obtention d'extraits peptidiques bioactifs par hydrolyse de protéines lactosériques avec des enzymes de cynara cardunculus, extraits ainsi obtenus et leurs utilisations WO2011135513A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108546725A (zh) * 2018-01-23 2018-09-18 北京联合大学 一种利用马血制备的生物活性肽及其制备方法
US11912788B2 (en) 2017-03-16 2024-02-27 Microsintesis Inc. Probiotic molecules for reducing pathogen virulence

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999065326A1 (fr) 1998-06-17 1999-12-23 New Zealand Dairy Board Hydrolysat de proteine bioactive du petit-lait
WO2005081628A2 (fr) * 2004-03-01 2005-09-09 Peptera Pharmaceutical Ltd. Peptides derives de la caseine et leurs utilisations therapeutiques
WO2008108649A2 (fr) 2007-03-06 2008-09-12 Friesland Brands B.V. Peptides inhibiteurs de l'ace issus du lactosérum et procédés pour obtenir ces peptides.

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999065326A1 (fr) 1998-06-17 1999-12-23 New Zealand Dairy Board Hydrolysat de proteine bioactive du petit-lait
US20040086958A1 (en) * 1998-06-17 2004-05-06 Ralf-Christian Scholthauer Bioactive whey protein hydrolysate
WO2005081628A2 (fr) * 2004-03-01 2005-09-09 Peptera Pharmaceutical Ltd. Peptides derives de la caseine et leurs utilisations therapeutiques
WO2008108649A2 (fr) 2007-03-06 2008-09-12 Friesland Brands B.V. Peptides inhibiteurs de l'ace issus du lactosérum et procédés pour obtenir ces peptides.

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BARROS R M ET AL: "Molecular Characterization of Peptides Released from beta-Lactoglobulin and alpha-Lactalbumin via Cardosins A and B", JOURNAL OF DAIRY SCIENCE, AMERICAN DAIRY SCIENCE ASSOCIATION, US, vol. 89, no. 2, 1 February 2006 (2006-02-01), pages 483 - 494, XP026956870, ISSN: 0022-0302, [retrieved on 20060201] *
DATABASE Geneseq [online] 17 November 2005 (2005-11-17), "Casein peptide SEQ ID NO 1886.", XP002658792, retrieved from EBI accession no. GSP:AEC18170 Database accession no. AEC18170 *
DATABASE Geneseq [online] 17 November 2005 (2005-11-17), "Casein peptide SEQ ID NO 1897.", XP002658788, retrieved from EBI accession no. GSP:AEC18181 Database accession no. AEC18181 *
DATABASE Geneseq [online] 17 November 2005 (2005-11-17), "Casein peptide SEQ ID NO 1943.", XP002658793, retrieved from EBI accession no. GSP:AEC18227 Database accession no. AEC18227 *
DATABASE Geneseq [online] 17 November 2005 (2005-11-17), "Casein peptide SEQ ID NO 1944.", XP002658790, retrieved from EBI accession no. GSP:AEC18228 Database accession no. AEC18228 *
DATABASE Geneseq [online] 17 November 2005 (2005-11-17), "Casein peptide SEQ ID NO 1945.", XP002658789, retrieved from EBI accession no. GSP:AEC18229 Database accession no. AEC18229 *
DATABASE Geneseq [online] 17 November 2005 (2005-11-17), "Casein peptide SEQ ID NO 1999.", XP002658791, retrieved from EBI accession no. GSP:AEC18283 Database accession no. AEC18283 *
PIHLANTO-LEPPALA A: "Bioactive peptides derived from bovine whey proteins: opioid and ace-inhibitory peptides", TRENDS IN FOOD SCIENCE AND TECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, GB, vol. 11, 1 January 2001 (2001-01-01), pages 347 - 356, XP002256621, ISSN: 0924-2244, DOI: 10.1016/S0924-2244(01)00003-6 *
SILVA S V ET AL: "Partial Identification of Water-Soluble Peptides Released at Early Stages of Proteolysis in Sterilized Ovine Cheese-Like Systems: Influence of Type of Coagulant and Starter", JOURNAL OF DAIRY SCIENCE, AMERICAN DAIRY SCIENCE ASSOCIATION, US, vol. 88, no. 6, 1 June 2005 (2005-06-01), pages 1947 - 1954, XP026954494, ISSN: 0022-0302, [retrieved on 20050601] *
TAVARES TANIA ET AL: "Novel whey-derived peptides with inhibitory effect against angiotensin-converting enzyme: In vitro effect and stability to gastrointestinal enzymes", PEPTIDES (NEW YORK), vol. 32, no. 5, May 2011 (2011-05-01), pages 1013 - 1019, XP002658738, ISSN: 0196-9781 *
ZYDNEY ANDREW L: "Protein separations using membrane filtration: New opportunities for whey fractionation", INTERNATIONAL DAIRY JOURNAL, vol. 8, no. 3, March 1998 (1998-03-01), pages 243 - 250, XP002658737, ISSN: 0958-6946 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11912788B2 (en) 2017-03-16 2024-02-27 Microsintesis Inc. Probiotic molecules for reducing pathogen virulence
CN108546725A (zh) * 2018-01-23 2018-09-18 北京联合大学 一种利用马血制备的生物活性肽及其制备方法
CN108546725B (zh) * 2018-01-23 2021-08-27 北京联合大学 一种利用马血制备的生物活性肽及其制备方法

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