WO2012057598A1

WO2012057598A1 - Method for quantifying amino acids using high-performance liquid chromatography (hplc) from lactic fermentation

Info

Publication number: WO2012057598A1
Application number: PCT/MX2011/000017
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: MX2010011900A

Abstract

The present invention relates to an HPLC method for quantifying free amino acids in the shrimp-head hydrolysate obtained by means of lactic fermentation. For the purpose of analysis thereof, the samples were derivatized with 9-fluorenylmethyl chloroformate (FMOC). Detection was performed using fluorescence (ex: 270 nm; em: 316 nm) in a column (SGE Hypersyl ODS C18 250 nm × 4.6 mm, 5 min) and three solutions were used as mobile phase: in phase A, 30 mm ammonium phosphate (pH 6.5) in 15:85 methanol/water; in phase B, 15:85 methanol/water; and in phase C, 90:10 acetonitrile/water. The pump flowrate was 1.20 mL/min, with a column temperature of 38°C.

Description

AMINO ACIDS QUANTIFICATION METHOD BY

HIGH RESOLUTION LIQUID CHROMATOGRAPH (HPLC) FROM

OF A LACTIC FERMENTATION.

TECHNICAL FIELD

The present invention has its technical field within chemistry, since it deals with a novel method for determining the amino acids tryptophan and tyrosine.

BACKGROUND OF THE INVENTION

The use in some marine products is only partially realized, that is, that what is not used as food is discarded as is the case of the head and shell of shrimp and other animals such as crab and fish. These products have been studied for decades, proving in various investigations that they are an important source of very good quality proteins, in addition to chitin and dyes that can be used for industrial and agricultural processes. In an investigation where lactic fermentation (with Lactobacillus plantarum) of shrimp head (Macrobrachium vollenhovenii) was carried out, the nutrient composition analysis of the hydrolyzate obtained gave a yield of 65.6% crude protein (Fagbenro and Bello-Olusoji , 1991).

In addition, to know the amount of protein that food has it is necessary to estimate its content in essential amino acids, since it indicates the quality of the protein. Amino acids are organic substances that contain a carboxyl group and an amino group attached to their R chain, which when linked by peptide bonds form long chains called peptides (up to

20 bound amino acids), which in turn form proteins together.

Some of them are essential, that is, that the organism is not able to synthesize them by itself, so it has to be obtained externally, among them it is found in tryptophan (Murray, et al., 1988). Tryptophan and tyrosine are aromatic amino acids, essential constituents of proteins, nutritionally essential for many animals and for the human body in the production of substances important to the body (Kivi, 2000).

Commonly the quantification of amino acids is performed after the breakdown of the peptide bonds by acid hydrolysis (6M HCI) followed by derivatization. However, this hydrolysis destroys tryptophan completely (Landry and Delhaye, 1988). On the other hand tryptophan does not need derivatization because it is fluorescent by nature, just like tyrosine (Chen and Barkley, 1994).

Tryptophan and tyrosine, like other amino acids, after protein hydrolysis, have been analyzed by a variety of methods in the past. High efficiency liquid chromatography (HPLC) is the most widely used analytical separation technique due to its sensitivity, easy adaptation to exact quantitative determinations and suitability for the separation of nonvolatile or thermolabile species. There are other published and patented methods such as the RU2305832 patent that describes a method for the independent determination of tryptophan and tyrosine in aqueous solution. RU2155747 describes a method of isolating the amino acids contained in a mixture for use in other processes. These methods are different from the one stated in this document.

Conventional amino acid determinations involve derivatization to facilitate identification. However, tryptophan and tyrosine due to their structure have intrinsic fluorescence, a characteristic that can be used for identification and quantification. Therefore, in this novel method of quantification of amino acids by high performance liquid chromatography (hplc) from a lactic fermentation the simultaneous determination by HPLC of tryptophan and tyrosine in hydrolysates obtained from shrimp residues without derivatizing is illustrated. DESCRIPTION OF THE INVENTION

The characteristic details of this novel HPLC amino acid quantification method from lacticide fermentation 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 . Chromatogram of the standard amino acid solution.

Figure 2. Hydrolyzate chromatogram obtained from the lactic fermentation of shrimp residue. The method of quantification of amino acids by HPLC from a lacticidal fermentation is composed of the following steps: a) Preparation of the chemical reagent. All solutions were prepared with ultra pure purified water with a Milli-Q system (Nano Diamond mash, Barnstead). FMOC (9-fluorenyl methyl chloroformate)

(Sigma, St. Louis, MO, USA) was dissolved in acetonitrile (EMD Chemicals Inc. Darmstadt, Germany, HPLC Grade); The borate buffer was prepared with boric acid and water by adjusting the pH to 8.5 with 1 M sodium hydroxide (Monterrey Chemical Products, Nuevo León, Mexico). The reagent to remove unreacted FMOC (Cleavage) was prepared using 0.85M sodium hydroxide with 0.5M hydroxylamine and 2-methylthioethanol (Sigma-Aldrich); also the solution to adjust the reaction pH (Quench) was prepared with glacial acetic acid and acetonitrile (Monterrey Chemical Products, NL, Mexico). The mobile phase was composed of a mixture of 3 solutions of different composition:

Phase A: 30 mM ammonium phosphate (pH 6.5) dissolved in a mixture of methanol-water (15:85); phase B: methanol - Water (15:85) and phase C: acetonitrile - water (90:10), all provided by EMD Chemicals Inc. b) Hydrolyzate recovery

a) Fermentation. For fermentation 500g of ground head were deposited in plastic containers of 1 kg capacity, adding 6.6% sugar (w / v), as well as inoculum (probiotic) in 50% (w / v); lowering the pH of the mixture below 6.5 with 10% citric acid to avoid rotting the sample. The containers with the sample were incubated for 24 hours at 30 ° C and the pH and titratable acidity (0.1 N NaOH) were monitored every 3 hours until a pH of less than 4.5 and a titratable acidity of 3.5% was obtained.

b) Centrifugation. Once the fermentation was finished, the sample was centrifuged at 6440 g at 8 ° C for 15 minutes to separate the phases (chitin, liquor, supernatant - pigments), collecting the protein-rich liquor or aqueous phase.

c) Preparation of the sample for quantification of amino acids

a) Lyophilization of the sample. The liquor taken from the centrifugation was frozen and lyophilized, to obtain a concentrated and easy to use product, this was done in a lyophilizer at - 40 ° C and a pressure below 133 x10 ^"3 mBar. This treatment guarantees that the final product does not contain moisture.The lyophilized powder was stored in airtight plastic bags and protected against light inside a desiccator, then 25 mg of sample were weighed and refilled to 25 ml with the borate buffer solution, the samples they were sonicated for 2 minutes to dissolve completely and stirred 30 seconds in the vortex.The solution is ready for derivatization.

b) Derivatization of amino acids with FMOC. The derivatization of amino acids is carried out according to the method of Haynes and Sheumack (1991), making some modifications. The process was started by taking 300 μΐ of the sample solution and deposited in a vial with a capacity of 1.5 mL, 300 of the FMOC reagent was added and stirred in the vortex for 90 seconds, at the end of the time, 180 μ del_ of the Cleaveage reagent was added and the resulting solution was mixed in the vortex, once this was done, the solution was allowed to stand for 3.5 minutes, then 420 μΙ_ of the Quench reagent was added after the rest time, the solution was mixed once more in the vortex and filtered using a 0.45 μιη millipore membrane; After this, the sample was ready to be analyzed by HPLC.

d) HPLC analysis (Equipment and conditions). For the analysis a liquid chromatograph equipped with a self-sampler and a software-controlled fluorescence detector (WinCrom) was used, the column temperature is controlled at 38 ° C with a column heater, all the equipment used is provided by GBC Instrumental, Australia. The separation is carried out with a 4.6 mm SGE Hypersil ODS C18 reverse phase column, for the chromatographic analysis 5 μΐ are injected. of the derivatized sample. To achieve the method, that is, that the chromatographic conditions are the most suitable for the analysis, different controllable parameters were evaluated, different pump flow rates were tested (0.8 mL / min, 1.0 mL / min, 1.2 mL / min, 1.4 mL / min), different column temperatures (34 ° C, 36 ° C and 38 ° C) this in order to make the method more precise and accurate. The amino acids are separated with an elution gradient which is shown in table 1. The flow rate used was 1.20 mL / min. The wavelength of the fluorescence detector was 270 nm excitation and 316 nm emission.

Table 1. Pump program (gradient flow)

Time (min) Phase A% Phase B% Phase C%

0 16.5 69 14.5

26 11 44 45

26.10 0 0 100

30 0 0 100

30.10 16.5 69 14.5

43 16.5 69 14.5

Claims

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:

1. A method of quantifying amino acids by HPLC from a lactic fermentation to quantify free amino acids in shrimp head hydrolyzate characterized in that it comprises the following steps:

to. Chemical reagent preparation.

b. Hydrolyzate recovery.

i. Fermentation.

ii. Centrifugation

C. Preparation of the sample for quantification of amino acids i. Lyophilization of the sample.

ii. Derivatization of amino acids.

d. HPLC analysis.

2. The method of quantification of amino acids by HPLC from a lactic fermentation described in claim 1, characterized in that all the solutions were prepared with ultra pure water purified with a Milli-Q system (Nano puree Diamond, Bamstead), FMOC ( 9-Fluorenyl methyl chloroformate) (Sigma, St. Louis, MO, USA) is dissolved in acetonitrile (EMD Chemicals Inc. Darmstadt, Germany, HPLC Grade), the borate buffer is prepared with boric acid and water by adjusting the pH to 8.5 with 1M sodium hydroxide (Monterrey Chemical Products, Nuevo León, Mexico), the reagent to remove unreacted FMOC (Cleavage) is prepared using 0.85M sodium hydroxide with 0.5M hydroxylamine and 2-methylthioethanol (Sigma-Aldrich), The solution to adjust the pH of the reaction (Quench) is prepared with glacial acetic acid and acetonitrile (Monterrey Chemical Products, NL, Mexico), where the mobile phase is composed of a mixture of 3 solutions of different compos Ion: Phase A: 30 mM ammonium phosphate (pH 6.5) dissolved in a mixture of methanol - water (15:85); phase B: methanol - Water (15:85) and phase C: acetonitrile - water (90:10).

The method of quantification of amino acids by HPLC from a lactic fermentation described in claims 1 and 2, characterized in that the recovery of the hydrolyzate is carried out by the steps: i. Fermentation, depositing 500g of ground head in plastic containers of 1 kg capacity, or equivalent, adding 6.6% sugar (w / v), as well as inoculum (probiotic) in 50% (w / v); lowering the pH of the mixture below 6.5 with 10% citric acid to avoid rotting the sample, incubating the containers for 24 hours at 30 ° C and monitoring the pH and titratable acidity (0.1 N NaOH) each period of time, constant, until a pH of less than

4.5 and a titratable acidity of 3.5%,

ii. Centrifugation, at 6440 g at 8 ° C for 15 minutes to separate the phases (chitin, liquor, supernatant - pigments), to collect the protein-rich liquor or aqueous phase.

The method of quantification of amino acids by HPLC from a lactic fermentation described in claim 1, 2 and 3, characterized in that the preparation of the sample for quantification of amino acids is carried out by the steps:

i. Lyophilization of the sample, where the liquor taken from the centrifugation is frozen and lyophilized, to obtain a concentrated and easy-to-handle product, performing it in a lyophilizer at - 40 ° C and a pressure below 133 x10 ^"3 mBar, guaranteeing that the final product does not contain moisture, storing the lyophilized powder in airtight plastic bags and protected against light inside a desiccator, to subsequently weigh 25 mg, or equivalent, of the sample and have a volume of 25 ml, or equivalent, with the borate buffer solution, the samples are sonified for 2 minutes so that they dissolve completely and shake for 30 seconds in the vortex,

Derivatization of the amino acids, with FMOC, where the derivatization of the amino acids is carried out according to the method of Haynes and Sheumack (1991), where the process begins by taking 300 μΙ_, or equivalent, of the sample solution and deposited in a vial with a capacity of 1.5 ml_, or equivalent, 300 μί, or equivalent, of the FMOC reagent is added and stirred in the vortex for 90 seconds, then 180 μΐ_, or equivalent, of the Cleaveage reagent is added and the resulting solution is added Mix in the vortex, then let the solution stand for 3.5 minutes, then 420 μΙ_, or equivalent, of the Quench reagent is added, the solution is mixed once more in the vortex and filtered using a 0.45 μιτι millipore membrane.

5. The method of quantification of amino acids by HPLC from a lactic fermentation described in claim 1, 2, 3 and 4, characterized in that HPLC analysis is performed using a liquid chromatograph equipped with a self-sampler and a fluorescence detector software controlled (WinCrom), where the column temperature is controlled at 38 ° C with a column heater, the separation is carried out with a 4.6 mm SGE Hypersil ODS C18 reverse phase column, for chromatographic analysis they inject 5 μί of the derivatized sample, and the flow rate used in the process was 1.20 mL / min with a fluorescence detector wavelength of 270 nm excitation and 316 nm emission.

6. The method of quantifying amino acids by HPLC from a lactic fermentation described in claim 5, characterized because the amino acids are separated with the following elution gradient (Time in minutes, Phase A%, Phase B%, Phase C%) corresponding:

i. 0 16.5 69 14.5

ii. 26 1 1 44 45

iii. 26.10 0 0 100

iv. 30 0 0 100

V. 30.10 16.5 69 14.5

saw. 43 16.5 69 14.5