WO2012057597A1

WO2012057597A1 - Method for obtaining free amino acids from protein hydrolysates

Info

Publication number: WO2012057597A1
Application number: PCT/MX2011/000016
Authority: WO
Inventors: Jaime LÓPEZ CERVANTES; Dalia Isabel SÁNCHEZ MACHADO; Karl Reiner Fick Rochin
Original assignee: Lopez Cervantes Jaime; Sanchez Machado Dalia Isabel; Karl Reiner Fick Rochin
Priority date: 2010-10-29
Filing date: 2011-02-04
Publication date: 2012-05-03
Also published as: MX2010011898A

Abstract

The present invention relates to a method for obtaining free amino acids from protein hydrolysates derived from shrimp by-products. The method was used to quantify the amino acids present in the liquor obtained as final product in lactic fermentation. It was found that the linearity, precision and recovery of the method are suitable for the quantification of the amino acids selected.

Description

Method to obtain free amino acids from protein hydrolysates

TECHNICAL FIELD

The present invention has its technical field in chemistry, since it is a novel method to obtain free amino acids from protein hydrolysates and the mixture used.

BACKGROUND OF THE INVENTION

Proteins are complex macromolecules that can represent more than 50% of the dry weight of cells, in whose structure and function they play a fundamental role. There are many that have been isolated and purified. Its molecular weight ranges from 5000 to many millions of Daltons. These biopolymers are composed of carbon, hydrogen, oxygen, nitrogen and usually, sulfur. Some also contain iron, copper, phosphorus or zinc. The total hydrolysis (acidic, alkaline or enzymatic) of the proteins yields L-shaped amino acids, which differ from each other by the nature of their side chains. The amino acids of most proteins belong to a small group, consisting of twenty of these compounds. Amino acids bind to form proteins through amide bonds called peptide bonds, forming polypeptide chains containing up to several hundred units (Cheflel, et. Al; 1993). Proteins can be classified into two groups: homoproteins consisting only of amino acids, and heteroproteins, which in addition to amino acids contain several non-protein compounds, collectively referred to as prosthetic groups. According to the chemical nature of the prosthetic group, nucleoproteins (such as those present in ribosomes and viruses), lipoproteins (such as gamma globulin), phosphoproteins (such as caseins), hemoproteins (such as hemoglobin and cytochrome C, can be distinguished, catalase and myoglobin) and metalloproteins (such as alcohol dehydrogenase and carbonic anhydrase), (Cheflel, et. al; 1993).

Amino acids share common aspects in their chemical structure. Almost all amino acids are derived from α-amino carboxylic acid, in which there is an additional substitution in carbon a. Of the amino acids from proteins, only tryptophan, tyrosine and phenylalanine absorb ultraviolet radiation and have a maximum absorbance at 278, 274.5 and 269 nm respectively. Cystine absorbs slightly at 238 nm and all amino acids do so at wavelengths close to 210 nm. Tryptophan, tyrosine and phenylalanine are the only amino acids that show natural fluorescence. Tryptophan fluorescence persists including when the amino acid is part of a protein (excitation at 287 nm; maximum fluorescence at 348 nm) (Cheflel, et. Al; 1993). According to Badui (1999), the protein content of marine products is very variable, ranges from 12 to 23% (on a wet basis) and is distributed in: 70 to 80% globulins, 10 to 20% are albumin and 2 to 4% are keratins and collagen. These contain very little amount of connective tissue and a high lysine content.

DESCRIPTION OF THE INVENTION

The characteristic details of this novel mixture and method to obtain free amino acids from protein hydrolysates are clearly described in the following description and in the accompanying figures and following the same reference signs to indicate the parts and figures shown.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Methodology followed in the experiment.

Figure 2. Amount of tryptophan and tyrosine obtained with 3 ml of 4.2M NaOH at 4, 8, 12 and 16 hours of hydrolysis.

Figure 3. Amount of tryptophan and tyrosine obtained with 6 ml of 4.2M NaOH at 4, 8, 12 and 16 hours of hydrolysis.

Figure 4. Amount of tryptophan and tyrosine obtained with 9 ml of 4.2M NaOH at 4, 8, 12 and 16 hours of hydrolysis.

Figure 5. Tryptophan and tyrosine pattern chromatograms. A) Ultraviolet detection B) Fluorescence detection.

Figure 6. Wavelength manipulation chromatograms. TO)

Chromatogram in the original conditions where the peaks are observed not wanted. B) Chromatogram with modified wavelengths to eliminate unwanted peaks.

The experiment was carried out in the LV-712 laboratory of the Technological Institute of Sonora, Obregón-Náinari unit in Cd. Obregón, Sonora, Mexico.

The standards used L-tryptophan and L-tyrosine were obtained from Sigma (St. Louis, MO, USA), the solutions were prepared with ultrapurified water with a NANO Diamond UV puree system (Branstead International, Dubuque, lowa, USA). HPLC grade methanol was obtained from AMD CHOMASOLV (Steinheim, Germany). Glacial acetic acid, boric acid, hydrochloric acid, sodium hydroxide and anhydrous sodium acetate, all analytical grade, were obtained from Monterrey Chemical Products (Monterrey, Nuevo León, Mexico).

The amino acid standards were diluted in 0.1 M HCI at different concentrations and calculated for calibration. The acidic water used in the free amino acid analysis consisted of acidified Milli-Q water at pH 6.3 with 1 M HCI. The 4.2M NaOH used in the total amino acid hydrolysis was prepared by dissolving 41.99 g of NaOH beads in 250 ml of Milli-Q water in a volumetric flask. The borate buffer used in the dilution of the hydrolyzed samples consisted of a 250 mM boric acid solution: 1,743 g of boric acid was diluted in 90 ml of Milli-Q water, pH 9 was adjusted with 1 M NaOH and it was adjusted 100 ml with Milli-Q water.

For the chromatographic analysis an HPLC system (GBC, Dandenong, Australia) equipped with an LC1650 autoinjector, an LC1460 solvent degasser, a WinChrom chromatographic data analysis system, an LC1 150 quaternary pump and an LC 255S fluorescence detector was used.

The chromatographic conditions for the simultaneous separation of tryptophan and tyrosine are indicated in Table 1. The fluorescence detector program started at the optimum wavelengths for the tyrosine for 5.8 min, and was subsequently automatically changed to the optimal wavelengths for Tryptophan The total time between injections was 20 minutes.

Table 1

I Parameter Conditions I 4.6 mm SS Exil ODS 250 mm, 5μιη column

Mobile phase Socratic system: Sodium acetate buffer

40mM adjusted to pH 4.5 with glacial acetic acid / water solution (1: 4), with 20% methanol

Flow rate 0.8 ml / min

Fluorescence Detection

Tryptophan: Ex: 280 nm Em: 348 nm

Tyrosine: Ex: 274 nm Em: 304 nm

Temperature 26 ° C

Sample treatment

500 g of sample are placed in a container, inoculum of Lactobacillus sp. (50% v / p), cane sugar (6.66% p / p), 25% citric acid in order to lower the pH to 4.5, and finally incubate at 30 ° C for 24 hours in an incubator (HOTPOINT ) (Figure 5). The fermented was separated in a refrigerated centrifuge (SOL-BAT SA, model C-600), at 6 ° C, 6000 rpm for 15 minutes, to obtain a fraction rich in insoluble chitin (sediment), a protein-rich liquid ( liquor) and a lipid fraction (astaxanthin). A fraction of the liquor samples were dried in a lyophilizer (LABCONCO, USA, Freezone 4.5 model) at -40 ° C and 130 bar pressure. Another part of the samples was dried in a vacuum oven (NATIONAL, USA, model 5831) at 60 ° C for 3 days. Subsequently, the samples were stored in a desiccator and in the dark until analysis. The methodology followed is shown in Figure 1.

Moisture analysis The moisture content of the dried and lyophilized shrimp waste samples was determined in duplicate directly in a vacuum oven at 60 ° C for 5 hours. This in order to express the results on a dry basis.

Tryptophan and tyrosine free extraction. The amino acids were extracted from the dried samples with acidic water. Specifically, 25 mg of dry or lyophilized finely powdered sample was dissolved in a 50 ml volumetric flask, titrated with acidified water at pH 6.3 with 0.1 M HCI, to obtain a concentration of 0.5 mg / ml. Subsequently, the samples were sonified (BRONSON, USA, model 1510) for 2 minutes for complete dissolution and subsequently analyzed by HPLC. Extraction of tryptophan and protein tyrosine. For the extraction of tryptophan and protein tyrosine an alkaline hydrolysis was carried out: 25 mg of sample were dissolved in glass tubes with 3 ml of 4.2 M NaOH and sonified for 2 minutes, the tubes were sealed under nitrogen atmosphere and incubated at 120 ° C for 4 hours. The hydrolysates were first cooled to room temperature and after about 4 ^o C in an ice bath. They were filtered through Wathman filter No. 2 and diluted to 50 ml with borate buffer pH 9.0 in a volumetric flask. Finally aliquots of this solution were filtered through 0.45 μιη Millipore membrane before chromatographic analysis.

Sample treatment. To determine the conditions of sample treatment, tests were first carried out to specify the amount of sample to be used, then tests were performed to determine the hydrolysis conditions (alkali amount and hydrolysis time)

Determination of the amount of sample to be analyzed. For the extraction of tryptophan and total tyrosine, an assay was carried out to determine the amount of sample to be hydrolyzed. The experiment consisted, first of all, of performing a hydrolysis using 25, 50 and 75 mg of sample with 3 and 6 ml of HCI at 120 ° C, analyzed in duplicate. For chromatographic analysis the 50 and 75 mg samples were diluted 1: 2 and 1: 4, respectively, in acidified water at pH 6.3; avoiding saturation in the chromatogram. After analyzing the tryptophan and tyrosine values obtained from each of the tests, it was observed that with 25 mg of sample, better results are obtained because the quantified amount of the two amino acids is greater. Table 2 shows the results, average of two repetitions, of the analysis.

Table 2.

Determination of hydrolysis conditions. To define the amount of 4.2 M NaOH and the time used for hydrolysis, another test was performed in which three quantities of alkali (3, 6 and 9 ml) and four different times (4, 8, 12 and 16 hours) were tested. , being a total of 12 trials in duplicate. When performing the chromatographic analyzes of each of the tests, it was obtained that with 9 ml of NaOH and in a time of 16 hours, greater amounts of tryptophan and tyrosine were quantified, as observed in table 3. According to Spies and Chambers (1949) the longer the hydrolysis time, the greater the amount of amino acid recovered. A similar conclusion was reached by Wu and Tanoue (2001), who performed protein hydrolysis tests to obtain total tryptophan, where the hydrolysis time was 12 and 16 hours, having greater or equal results in the recovery of tryptophan at 16 hours (Table 7). The results obtained using 3, 6 and 9 ml of 4.2M NaOH, respectively, are shown in Figures 5, 6 and 7.

ID. When analyzing the chromatograms of the amino acid patterns, they have been more defined and larger in the fluorescence detector (Figure 7). Tryptophan and tyrosine were identified in the chromatographic column by their retention times by a comparison with the original reference standards of each separately. The wavelengths selected for fluorescence detection are for Tryptophan: Excitation 280 nm and Emission 248 nm, and for Tyrosine: Excitation 274 nm and Emission 304 nm. The retention times obtained were 4,248 min for tyrosine and 8,327 min for tryptophan, which were obtained by calculating the average retention times of 10 trials with 2 injections each.

Subsequently, tryptophan and tyrosine were extracted from the sample. Performing the HPLC analysis, it was found that the peaks of the amino acids of independent injections had the same retention time, thus determining the presence of tryptophan and tyrosine in the sample. During the optimization of the chromatographic conditions, tests with different wavelengths were made to try to eliminate the unknown peaks that interfere with the desired peaks, and that is observed between the tyrosine peak and the tryptophan peak. For this, the previously selected tryptophan and tyrosine wavelengths were not altered, but those that They present the unwanted peak. For this, wavelengths above the optimum for garbage were programmed in the time interval in which the unwanted peak is observed. Table 5 shows the time and wavelength program to remove the waste from the chromatograms.

Table 5.

Wavelength Time

6.4-6.5 min Excitation: 280 nm

Emission: 390 nm

9.7-9.8 min Excitation: 280 nm

Emission: 348 nm

Figure 8 shows the chromatogram that presents the unwanted peaks and to which the conditions to disappear were modified.

The method for the preparation of samples of protein hydrolyzate obtained from lactic fermentation of shrimp head has been determined, for the simultaneous determination of free and total tryptophan and tyrosine.

On the other hand, optimal chromatographic conditions were reached for the determination of these two amino acids. A mobile phase of 40 mM sodium acetate: methanol (80:20 v / v), at 0.8 ml / min flow rate, on a reverse phase C-ie column at 26 ° C, allowed for the most satisfactory separation of both amino acids using a fluorescence detector at different wavelengths.

Claims

Having sufficiently described my invention, I consider as a novelty and therefore claim as my exclusive property, what is contained in the following clauses:

A method for obtaining free amino acids from protein hydrolysates in shrimp ferments characterized in that it comprises the following phases: a. Treatment of the samples: where 500 g of sample are placed in a container, adding inoculum of Lactobacillus sp. (50% v / p), cane sugar (6.66% p / p), 25% citric acid to lower the pH to 4.5, and finally incubated at 30 ° C for 24 hours in an incubator, the fermented is separated in a centrifuge refrigerated at 6 ^o C, 6000 rpm for 15 minutes, obtaining a fraction rich in insoluble chitin (sediment), a protein-rich liquid (liquor) and a lipid fraction (astaxanthin), where the fraction of the samples of Liquor is dried in a lyophilizer at -40 ° C and 130 bar pressure and another part of the samples is dried in a vacuum oven at 60 ° C for 3 days and then stored in a desiccator and in the dark until analysis. b. Moisture analysis: The moisture content of dried and lyophilized shrimp waste samples is determined in duplicate directly in a vacuum oven at 60 ° C for 5 hours for the expression of the results on a dry basis. C. Extraction of tryptophan and free tyrosine: Amino acids are extracted from dried samples with acidic water, which specifically, for every 25 mg of dry or lyophilized finely powdered sample are dissolved in a 50 ml volumetric flask, titrated with acidified water at pH 6.3 with 0.1 M HCI, to obtain a concentration of 0.5 mg / ml, to subsequently sonify the samples for 2 minutes until completely dissolved. d. Analysis: Sample identification, hydrolysis conditions and identification.

2. A method for obtaining free amino acids from protein hydrolysates in shrimp ferments as described in claim 1 wherein the phases a and b are the same and a phase c characterized in that it deals with extraction of protein tryptophan and tyrosine by an alkaline hydrolysis that It comprises dissolving, for every 25 mg of sample in glass tubes, with 3 ml of 4.2 M NaOH and sonifying for 2 minutes, to subsequently seal the tubes under nitrogen atmosphere and incubate at 120 ° C for 4 hours. The hydrolysates were first cooled to room temperature and after about 4 ^o C in an ice bath. They were filtered through Wathman filter No. 2 and diluted to 50 ml with borate buffer pH 9.0 in a volumetric flask. Finally aliquots of this solution were filtered through 0.45 μιτι Millipore membrane before chromatographic analysis.

3. A method for obtaining free amino acids from protein hydrolysates as described in the preceding claims wherein the analysis process is composed of the following steps: a. Sample treatment, stage where tests are carried out to specify the amount of sample to be used for later testing to determine hydrolysis conditions such as alkali amount and hydrolysis time.

i. Determination of the amount of sample to be analyzed, by hydrolysis using 25 mg of sample with 3 and 6 ml of HCI at 120 ° C, analyzed in duplicate diluting in acidified water at pH 6.3 avoiding saturation in the chromatogram.

ii. Determination of the hydrolysis conditions, where 9 ml 4.2 M NaOH is used in a time of 16 hours allowing to observe greater amounts of tryptophan and tyrosine. b. Identification, the analysis of the chromatograms of the amino acid patterns are observed in a fluorescence detector by means of a chromatographic column for Tryptophan: Excitation 280 nm and Emission 248 nm, and for Tyrosine: Excitation 274 nm and Emission 304 nm.

A method for obtaining free amino acids from protein hydrolysates as described in the preceding claims characterized in that the retention times are 4,248 min for tyrosine and 8,327 min for tryptophan.

A method for obtaining free amino acids from protein hydrolysates as described in claims 3 and 4 characterized in that the time program and its wavelengths used for the elimination of unwanted peaks are:

to. 4-6.5 min Excitation: 280 nm Emission: 390 nm, and

b. 7-9.8 min Excitation: 280 nm Emission: 348 nm.

A method for obtaining free amino acids from protein hydrolysates as described in the preceding claims is characterized in that in the analysis phase the optimal chromatographic conditions for the determination of tryptophan and tyrosine consist of a mobile phase of 40 mM sodium acetate: methanol (80:20 v / v), at 0.8 ml / min flow rate, in a reverse phase Ci ₈ column at 26 ° C separating both amino acids using a fluorescence detector at different wavelengths.