RO135638A2 - Protein hydrolysate with bioactive properties isolated from fish by-products and process for preparing the same - Google Patents

Abstract

The invention relates to a process for the preparation of a protein hydrolysate composition applicable in the pharmaceutical and cosmetic industries. The process consists of the extraction of bioactive peptides from fish by-products using tissue decalcification and delipidization treatments, enzymatic hydrolysis with one or two successive enzymes, namely flavourzyme and/or alkalase, followed by the extract bleaching, tangential ultrafiltration and drying by atomisation, to result in a powder composition having a content of more than 85% protein, up to 2% lipids and 1.5% ash, respectively, a molecular weight of up to 3000 Da, which has bioactive properties and antioxidant, antiproliferative and antihypertensive activity, respectively.

Description

t lbl OF STATE PENîrTî^^^ 135638 A2

Patent application

PROTEIN HYDROLYZATE WITH BIOACTIVE PROPERTIES ISOLATED FROM FISH BY-PRODUCTS AND PROCEDURE FOR OBTAINING

The present invention refers to a protein hydrolyzate, composed of low molecular weight peptides, with bioactive properties (antioxidant, antihypertensive, antiproliferative) isolated from fish by-products, bones, skin, meat, viscera, and to the process for obtaining it. The invention has applicability in the pharmaceutical, cosmetic and nutraceutical industry.

Recently, there has been an increased interest in obtaining protein hydrolysates from fish with bioactive properties by capitalizing on various residual sources (Halim et al. Trends in Food Sciences and Technology, 2016, 51:2433). The by-products resulting from fish processing are resources of negligible commercial value and are a major cause of environmental pollution. in order to capitalize on these bioresources, hydrolysis processes have been developed to convert fish proteins from unused materials (head, skin, viscera, bones, etc.) into forms accepted by the market that can be used on a large scale and in areas other than food for animals or fertilizers (Villamil et al., Food Chemistry, 2017, 224:160-171; Gildberg et al., Process Biochemistry, 1994, 28:1-15).

Fish hydrolysates are known to be a safe source of protein for nutrition due to their amino acid composition and positive effect on gastrointestinal absorption (Benjakul et al., Fish protein hydrolysates: production, bioactivities, and applications, in Antioxidant and Functional Components in Aquatic Foods, Kristinsson ed., 2014, pg. 237-272). The methods of obtaining these hydrolysates which involve the transformation of proteins into peptides, can be chemical or enzymatic and produce peptides with different functional properties and bioactivities comparable to the native counterparts (Kristinsson and Rasco, Criticai Reviews in Food Science and Nutrition, 2000, 40: 4381) . Enzymatic technologies for the recovery of proteins from fish waste are more efficient compared to chemical ones, because the hydrolysis process is easier to control, requiring milder working conditions. Also, the use of enzymes determines a high degree of hydrolysis and ensures the formation of soluble peptides, with high nutritional value, structural properties useful for nutraceutical foods and functional properties of biomedical interest. Bioactive peptides resulting from enzymatic hydrolysis of ^proteins from different biological functions $j0^^aî^^ activities of _ζ ΐϊΐίίίΐΡβ!^ inhibition

3ί angiotensin-converting enzyme (ACE), antioxidants, anticoagulants and antimicrobials (Ngo et al., International Journal of Biological Macromolecules, 2012, 51:378-383). Fish by-products can also be sources of antitumor or chemopreventive compounds (Picot et al., Process Biochemistry, 2006, 41:1217-1222).

A series of procedures for obtaining products based on peptides extracted from various fish sources with industrial applications are known. Thus, patent US6753407 B2 refers to obtaining 2 peptides, isolated from the gills of oceanic fish (hybrid sea bass Morone saxatilis x Morone chrysops), which show antimicrobial properties - e.g. bactericidal activity against E coli. Patent application CN 101294185 A protects a method for the preparation of collagen peptides from fish scales, in which fermentation is used with a mixture of strains of microorganisms belonging to different genera (Bacillus, Aspergillus, Streptomyces, Trichoderma). The resulting peptides are used to obtain products intended for nutrition and health care.

Patent application CN 1383732 A describes a technology for preparing a mixture of fish peptides by using 2 enzymes, namely pancreatin and pepsin. The peptides obtained have antibacterial and immunological regulatory properties and are easy to digest and assimilate. Patent application EP2766383 B1 presents an enzymatic process for obtaining peptides of low molecular mass and low ash content, starting from gelatin made from fish skin and bones. Gelatin is broken down into low molecular weight peptides using enzymes from Bacillus sp. The gelatin solution is clarified by filtration, after adjusting the pH to 7, with sodium or ammonium hydroxide. The filtered solution is desalinated at a temperature of 20 to 55°C in a diafiltration mode, until the salt content drops below 0.05% (mass/volume). The product thus obtained is hydrolyzed with a bacterial protease, preferably from Bacillus licheniformis, maintaining the pH in the range from 7 to 10 and the temperature in the range from 25 to 75°C. The hydrolyzed product is filtered through an ultrafiltration membrane at a temperature between 15 and 55°C, and the permeate is concentrated at a temperature between 50 and 100°C, under vacuum, to obtain the peptide solution according to the invention.

A main disadvantage of fish hydrolysates is the bitter taste, which

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10201806926Y describes masking the bitter taste of peptides produced by enzymatic hydrolysis of fish proteins using glycerophospholipids. Patent JP 3483875 B2 presents a process for extracting with 10% ethanol the peptides resulting from hydrolysis with an alkaline prosthesis of fish proteins. Patent KR 101415225 refers to a process in which a combination of proteins, a bacterial and a fungal protease, followed by column chromatography, are used to hydrolyze collagen. The patent US6905704 protects a process by which peptides containing the valine (Val) - tyrosine (Tyr) sequence, with ACE inhibitory activity, are mainly purified. The process involves purifying the peptides by reverse phase liquid chromatography using a stationary phase with octadecylsilyl (ODS) groups.

All these procedures known to remove the bitter taste also affect the biological activity of the peptides (Idowu and Benjakul, Journal of food biochemistry, 2019, 43(9), e12978).

The technical problem that the invention solves is to create a composition of peptides from fish proteins, with a high degree of purity, molecular weight less than 3000 Da, reduced bitter taste and bioactive properties, namely antioxidant, antiproliferative and antihypertensive activity, reproducible.

The protein hydrolyzate obtained, according to the invention, is a yellowish-white powder, which has a low bitter taste and a content in peptides with an average molecular mass of less than 3000 Da of more than 85% and which exhibits the following bioactive properties: the antioxidant activity of neutralizing free radicals 1,1-diphenyl-2picryl-hydrazyl (DPPH) of at least 60%, antihypertensive - by inhibiting the angiotensin-converting enzyme - by at least 50% and antitumor activity, by inhibiting the growth of Hep 2 cells of at least 60%.

The process of obtaining fish protein hydrolyzate, according to the invention, consists of the following stages:

intensive washing of a quantity of 100...1000 g of fish by-products, with tap water, in continuous flow, for 1...3 hours, then with distilled water, for 30...50 min;

mechanical shredding of the washed tissue using an electric meat mincer with a 3 mm sieve, followed by decalcification of the tissue by milling with

wet: decalcification solution of 1:10...1:40, at room temperature, with continuous gentle stirring;

separation by filtration of decalcified tissue, followed by intensive washing with water for 1...3 hours, drainage of excess water and delipidation by treatment with 4...10 volumes of acetone/distilled water mixture 1:1, for 4 ...20 h, at temp. room, or with 1...4 volumes of isopropanol, at temp. of 3O...7O°C, for 1...10 hours, with stirring;

washing with water in a continuous flow of delipidated tissue, to remove traces of fat, mixing with cold distilled water, in a ratio of 1:2...1:5 (g/vol), homogenization 2...5 min at 500.. .2000 rpm and treatment with 3...5 volumes of 0.1 M sodium hydroxide solution, for 1...4 hours, with cold stirring;

separating the tissue sediment from the homogenate by centrifugation at 2000...5000 rpm, for 20...40 min and removing the supernatant;

intensive washing of the tissue obtained after alkaline treatment;

enzymatic hydrolysis by treatment with microbial proteases, previously dissolved in 6...10 volumes of 0.2M phosphate buffer, pH 7.0...8.5, in an enzyme:substrate weight ratio of 1:20...1 :60, at 5O...7O°C, for 1...8 hours;

enzyme inactivation by heating the extract at 90...100°C for 10...30 min, followed by cooling and separation of non-extracted tissue by centrifugation at 3000...5000 rpm for 30...50 min , adjusting the pH to 7.0 with concentrated solutions of sodium hydroxide or hydrochloric acid and treating with activated charcoal 0.5...2.0%, by mixing and gentle stirring, for 2...15 hours, at room temperature;

decantation of activated carbon, followed by tangential ultrafiltration, using a filter membrane with polysulfone fibers, with the exclusion limit of molecular masses of 3000 Da;

concentration of the resulting solution after filtration to 10...20 % dry matter and spray drying at 130-145 °C inlet temperature and 80°C outlet temperature. »

Fish by-products can be scraps of skin, bones, meat and viscera, separated or in the original mixture, and preferably come from farmed cyprinids, carp

52 The enzymes used are subtilisin A, a serine endopeptidase produced by Bacillus licheniformis, and/or a mixture of aminopeptidases, dipeptylpeptidases and endo-peptidases produced by Aspergillus oryzae.

The invention presents the following advantages:

- the process described in this proposal is an optimal method for obtaining peptides with low molecular weight, high degree of purity from fish by-products (skin, bone, meat, viscera) and without bitter taste;

- the reduction of bitter taste formation is due to the use in the first stage of enzymatic hydrolysis of a mixture of peptidases, aminopeptidases, dipeptylpeptidases and endo-peptidases, which limit the formation of bitter peptides in fish;

- the obtaining procedure is feasible and does not require the use of aggressive substances and equipment with a high degree of complexity;

- the use of two enzymes for hydrolysis of fish waste allows the cleavage of the protein chain at specific sites of these enzymes under moderate and controlled working conditions (temperature, pH, extraction times, etc.);

- the fish peptides obtained and tested in vitro present defined and reproducible bioactive properties, respectively antioxidant capacity, antiproliferative and antihypertensive activity;

- the proposed process achieves a superior and efficient utilization of the by-products resulting from the fish processing processes and reduces environmental pollution;

- the conditioned product in the form of soluble powder, of high purity, can be used for the development of new formulations with applications in the pharmaceutical and nutraceutical industry;

- the proposed process reduces environmental pollution with waste from the fish processing industry.

The present invention proposal is highlighted by the following examples:

Example 1

A quantity of 200 g of mixed meat and bones of carp, Cyprinus carpio, is washed with tap water, in a continuous flow, for 3 hours, then with distilled water, for 30 min, after which the washed tissue is minced the help of an electric meat mincer with a Je^^rri sieve. Penw'da^^Bfefe;.the shredded tissue is entered into the laboratory prevaatt with the

J C MEDICA yVft pT “ / ?· 1-ARMlMPEy J V add 2000 ml of 0.2 M HCI solution for 24 hours at room temperature with gentle agitation. The decalcification solution is removed by filtration and the resulting tissue is washed intensively with water in a continuous flow for 2 hours and then 30 min with distilled water. After washing, add 1200 ml acetone/distilled water 1:1 mixture over the decalcified and washed tissue for delipidation and shake for 12 hours. The tissue obtained after filtering through a textile filter, is washed with water in a continuous flow, to remove traces of fat, and then it is placed in a stainless steel vessel equipped with a stirrer, in which 200 ml of cold distilled water is added and homogenized for 5 min at a speed of 500 rpm.

Separately, prepare 1000 ml of 0.1M NaOH solution, which is introduced over the homogenate prepared earlier and stirred in the cold for 3 hours to remove the carbohydrate components from the tissue structure. After the alkaline treatment, the mixture is centrifuged at 4000 rpm for 20 min and the supernatant is removed. The tissue separated by centrifugation is washed extensively with water for 2 hours and then with distilled water for 30 min, after which it is processed by enzymatic hydrolysis.

the washed tissue is placed in a stainless steel vessel over which 200 ml of Phosphate buffer 0.2 M pH 7.0, in which 0.006 g of Flavourzyme® (Novozymes, Bagsvaerd, Denmark) were previously dissolved, a mixture of peptidases, aminopeptidases, dipeptylpeptidases and endo-peptidases produced by Aspergillus oryzae (Merz et al., Journal of Agriculture! and Food Chemistry, 63(23), 5682-5693). The activity of the enzyme mixture used is 500 LAPU/g. One LAPU unit is that amount of enzyme that hydrolyzes 1 pmol of L-leucine-p-nitroanilide per minute. Any mixture of enzymes with the same characteristics can be used.

The enzymatic hydrolysis takes place at a temperature of 55°C, for 6 hours, after which the obtained extract is heated to 90°C, for 20 min, to stop the enzymatic reaction. Then, the hydrolyzate is cooled, centrifuged at 5000 rpm for 30 min and the obtained supernatant is recovered and mixed with 20 g of activated animal charcoal with which it is left in contact for 3 hours. After settling, the decolorized solution is ultrafiltered by tangential filtration using a membrane with polysulfone fibers, with a molecular weight cutoff of 3000 Da. The permeate is recovered, represented by the solution with a molecular weight of less than 3000 Da, which is concentrated under vacuum to 12% dry matter^i^'lis^ spraying, at the end of which 1°C inlet temperature and 80°C are obtained protein tempeți in the form of ^f^ere of cu

3ΰ average molecular weight below 3000 Da, with a content of 85% protein, 1.8% lipids and 1.2% ash.

Example 2

The preliminary stages of washing the tissue: shredding, decalcification, delipidization and alkaline treatment are similar to those presented in example 1, with the difference that delipidization is done with 800 ml of isopropanol at a temperature of 40°C, for 5 hours. The enzyme treatment is carried out with Alcalase® (Novozymes, Bagsvaerd, Denmark) which is prepared by dissolving 0.005 g of enzyme in 250 ml of 0.2 M phosphate buffer pH 8.0 and is added over the tissue resulting from the alkaline treatment step. Alcalase is a subtilisin A-type serine endo-peptidase produced by Bacillus licheniformis. The enzyme used had an activity of 0.75 Anson units per gram. One Anson unit is defined as that amount of enzyme that liberates 1.0 pmol of L-tyrosine from hemoglobin per minute at 25°C, pH 7.5.

The hydrolysis reaction takes place at a temperature of 60°C for 5 hours. The steps of stopping the enzymatic reaction, centrifugation, treatment with activated carbon and ultrafiltration are carried out under the conditions shown in example 1. The resulting solution after tangential filtration with a molecular weight below 3000 Da is concentrated to 17% by vacuum centrifugation and spray dried. After drying, a peptide powder with a molecular weight of less than 3000 Da is obtained with a content of 87% protein, 1.5% lipids and 1.5% ash.

Example 3

The procedure for obtaining peptides from fish is similar to that described in example 1 with the difference that the raw material was a mixture of skin, bones and fish meat and that the enzymatic treatment is done with both enzymes, successively, as follows: - in the first stage enzymatic hydrolysis is carried out with 0.005 g of Flavourzime®, previously dissolved in 150 ml of 0.2 M phosphate buffer, pH 7.0, at a temperature of 50 °C, for 5 hours, followed by inactivation of the enzyme by heating the solution to 100° C, for 15 min;

- the resulting solution is cooled, brought to pH 8.0 with 10 M sodium hydroxide and enzymatically hydrolyzed with a solution of 0.007 g of Alcalase®, dissolved in phosphate buffer

decolorize the solution and filter according to example 1. The solution recovered after ultrafiltration is concentrated to 12% and dried as a powder using an atomizer. The protein hydrolyzate obtained has a molecular weight of less than 3000 Da, a protein content of 90%, lipids of 1.5% and ash of 1.5%.

Protein hydrolyzate samples, obtained according to the above examples, were analyzed physicochemically and biologically. The results of the physicochemical and biochemical analyzes demonstrated that the analyzed peptide samples had a high content in protein (over 85%) and low in lipids (under 2%) and in ash (under 1.5%).

Example 4

The procedure is as in example 3, except that by-products of the novac, Arystlchtys nobilis, are used.

Example 5.

Work as in example 3, except that by-products of cteno, Ctenopharyngodon idella are used.

Example 6.

Work as in example 3, except that blood byproducts / phytophagous, Hypophthalmichthys molitrix are used.

The evaluation of the antioxidant activity of the protein hydrolysates prepared by controlled enzymatic hydrolysis was carried out by the DPPH method (W Wang et al., Czech J Food, 2013, 31/1:1-4) which consists in the quantitative determination of the ability to inhibit/neutralize free radicals DPPH (1,1-diphenyl-2-pyri hydrazyl), using as a standard a sample of Trolox, a substance with known antioxidant activity. The results expressed as DPPH radical inhibition percentages demonstrated that all the hydrolysates obtained according to the above examples showed antioxidant activity. The DPPH radical inhibition effect of the samples at the concentration of 10 mg/ml, expressed as a percentage, varied between 50% and 62%; the highest value (61.5%) was presented by the protein hydrolyzate sample prepared by successive treatment with the two enzymes, flavorzyme and alcalase.

The antihypertensive activity of the obtained protein hydrolyzate variants was evaluated by determining the inhibition potential of the converting enzyme

angiotensin (ACE), according to the method described by Păpadii Chemistry, 2007, 1Q5j647-656). Ab inhibition degree values. (Food xprinted

2/ proteins. The highest antihypertensive activity, respectively 78.5%, was presented by the protein hydrolyzate variant obtained by hydrolysis with alcalase.

Testing of the antiproliferative effect of protein hydrolysates obtained from fish by-products, according to the examples presented, was carried out on a stabilized Hep 2 tumor cell line originating from human epithelial carcinoma, in accordance with the international standard ISO/EN 10993-5/2009. in this context, cell viability and proliferation were investigated by the quantitative MTT method (reduction of tetrazolium bromide to insoluble formazan in viable cells) in the presence of sample variants, during 48 hours of cultivation. The obtained results showed that all the hydrolysates show a significant antiproliferative activity, at concentrations higher than 8 mg/ml. Our data revealed that the hydrolyzate variants tested at this concentration induced an inhibition of Hep2 tumor cell growth of 38.6% for the flavorzyme-extracted variant, 42.5% for the alcalase-extracted variant and 61.7 % for the variant combined with the two enzymes.

Claims (4)

demand 1. Protein hydrolyzate according to the invention, characterized in that it has a low bitter taste, a peptide content with an average molecular weight of less than 3000 Da of more than 85% and the following bioactive properties: antioxidant activity to neutralize free radicals 1,1- diphenyl-2-picryl-hydrazyl (DPPH) of at least 60%, antihypertensive, inhibiting angiotensin converting enzyme of at least 50% and antitumor activity, inhibiting the growth of Hep 2 cells of at least 60%. 2. The process for obtaining the fish protein hydrolyzate according to the invention, characterized in that it consists of the following steps: intensive washing of a quantity of 100 ... 1000 g of fish by-products, with tap water, in continuous flow, time for 1 ... 3 hours, then with distilled water, for 30 ... 50 min; mechanical crushing of the washed tissue by means of an electric meat grinder with a 3 mm sieve, followed by decalcification of the shredded tissue by treatment with a solution of hydrochloric acid 0,2 ... 1,5 M, for 4 ... 24 hours, wet tissue weight ratio: decalcification solution of 1:10 ... 1:40, at room temperature, with continuous gentle shaking; filtration separation of decalcified tissue, followed by intense washing with water for 1 ... 3 hours, draining excess water and delipidization by treatment with 4 ... 10 volumes of acetone / distilled water mixture 1: 1, for 4 ... 8 p.m., temp. chamber, or with 1 ... 4 volumes of isopropanol, at temp. of 3O ... 7O ° C, for 1 ... 10 hours, with stirring; washing with continuous flow of delipidized tissue with water, to remove traces of fat, mixing with cold distilled water, in a ratio of 1: 2 ... 1: 5 (g / vol), homogenization 2 ... 5 min at 500 .. .2000 rpm and treatment with 3 ... 5 volumes of 0.1 M sodium hydroxide solution, for 1 ... 4 hours, with cold stirring; separation of the tissue sediment from the homogenate by centrifugation at 2000 ... 5000 rpm, for 20 ... 40 min and removal of the supernatant; intensive washing of the tissue obtained after alkaline treatment, followed by enzymatic hydrolysis by treatment with microbial proteases, previously dissolved in 6 ... 10 volumes of 0.2M phosphate buffer, pH 7.0 ... 8.5, by weight enzyme: substrate of 1:20 ... 1:60, at 5O ... 7O ° C, for 1 ... 8 hours; inactivation of enzymes by heating the extract to 90 ... 100 ° C for 10 ... 30 min, followed by cooling and separating the non-extracted tissue by centrifugation at 3000 ... 5000 rpm, for 30 ... 50 min , adjust the pH to 7.0 with concentrated sodium hydroxide solutions and / or -To be . · Stir and stir the treatment with activated charcoal 0.5 ... 2, Q% ^ tin IOI8 Ά ^ INIUS z / 1, u _UV r < J // pm ^Λ you Λ 2 ... 15 hours, at room temperature; decanting of activated carbon, followed by tangential ultrafiltration, by using a filter membrane with polysulfone fibers, with a molecular weight exclusion limit of 3000 Da; concentration of the resulting solution after filtration at 10 ... 20% dry matter and spray drying at 130-145 ° C inlet temperature and 80 ° C outlet temperature. The process for obtaining the fish protein hydrolyzate according to claim 2, characterized in that the fish by-products may be skin, bone, meat and viscera residues, separated or in an initial mixture, and preferably come from farmed cyprinids, crap Cyprinus carpio, novac, Arystichtys nobilis, cteno, Ctenopharingodon idella, sânger / fitofag, Hypophthalmichthys molitrix. Process for the production of fish protein hydrolysate according to claim 2, characterized in that the enzymes used are a mixture of aminopeptidases, dipeptylpeptidases and endopeptidases produced by Aspergillus oryzae, and / or subtilizin A, a serine endopeptidase produced by Bacillus licheniformis.