ES2548804B1 - PROCEDURE FOR OBTAINING AMINO ACIDS FREE OF INTEREST FROM LEGUMINOUS FLOURS - Google Patents

Abstract

Method of obtaining free amino acids of interest from legume flours. # The present invention relates to a method of obtaining free amino acids from legume flours, preferably from chickpeas, which comprises the steps of extraction, nanofiltration, purification by ion exchange resins and concentration of the final product. This method is useful for obtaining amino acids of interest such as arginine, homoarginine or canavanine, depending on the starting material.

Description

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Procedure for obtaining free amino acids of interest from flours

of legumes

DESCRIPTION

The present invention relates to a process for obtaining free amino acids from legume flours, preferably from chickpeas and other legumes very rich in free amino acids such as g. Vicia and Lathyrus, which allows you to easily obtain amino acids of interest such as arginine, homoarginine or canavanine.

STATE OF THE TECHNIQUE

Free amino acids have multiple applications today both in the food industry and in the pharmaceutical industry. They are used as nutritional supplements and also as flavorings in the case of glutamic. But many also have health promoting properties and their use has been suggested for the treatment of various diseases.

In legumes free amino acids are among the most abundant secondary compounds. They can be classified as protein and non-protein. In the first group are the twenty amino acids that are part of the proteins. In the second are the amino acids described to date (more than 240), which are not part of the proteins. Due to their nutritional and pharmacological interest, the amino acids arginine, canavanine, homoarginine and glutamic acid are in principle those of greatest interest, especially arginine, canavanine and homoarginine, due to their joint involvement in nitric oxide (NO) synthesis and their abundance and predominance in chickpea and other legumes of the genera Vicia and Lathryus.

The processes of extraction of amino acids free of interest from plant material have long been known as described in GB1377675, although it is necessary to improve the yield and minimize the use of organic solvents and strong acids such as sulfuric acid to reduce their impact about the environment and human and animal health.

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The process of purification of arginine and other amino acids that is currently carried out, is carried out from the fermentation of microorganisms as for example described in US 4,080,137. These procedures involve the manipulation of living beings and the use of fermentation reactors that imply the need for much more strict, complicated and expensive controls.

Therefore, it is of great interest to develop processes for obtaining free amino acids that are simple and efficient and with an easily obtainable raw material and in which production costs are minimized

DESCRIPTION OF THE INVENTION

In the present invention, thanks to a unique combination of various processes or stages, the various physical and chemical properties of the starting material (legume flour), of the different compounds present in these flours and of the final product (the amino acids) for the purification of these amino acids and the generation of fractions of interest in food and pharmacy. For this, a procedure of purification of compounds and fractions is described considering their different solubilities according to the pH (alkaline extraction, precipitation acid), their different solubilities according to the percentage of ethanol in the solution (precipitation or crystallization), according to the different size of their molecules (nanofiltration) and according to their chemical affinities for solid supports (ion exchange resins). Unlike other procedures, which start from much less heterogeneous and complex starting materials, the compositional complexity of legume flours from which free amino acids mentioned in the invention are extracted and purified must be considered. Complexity manifests in the diversity of compounds of chemical characteristics (solubility, etc) and physical (size, etc.) very different and where free amino acids are also a minor fraction compared to compounds such as proteins, fiber or carbohydrates. However, this complexity of the starting material makes the described procedure present another advantage added in comparison with other related ones and it is the generation during its development, thanks to the unique combination of various processes, of by-products that constitute, in addition to the pure amino acids other

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fractions with interest in food or pharmaceutical and that can be exploited because the starting material is food matrices (edible flours).

In the present invention a process for producing free amino acids from plant material is described which is economically more advantageous compared to production by fermentation. Reagent costs are negligible. The fungible material, ion exchange resins and nanomembranes, are cheap and reusable. The necessary machinery is very simple and partly already present in a plant for obtaining protein concentrates, such as extraction tanks, centrifuges, concentrators and atomizers. In conclusion, production costs are negligible compared to the added value of the products obtained.

On the other hand, the process of purification of arginine and other amino acids is comparablely much simpler to how it is carried out from the fermentation of microorganisms. This implies the manipulation of living beings and the use of fermentation reactors that imply the need for much more strict, complicated and expensive controls.

On the other hand, in addition the fractions rich in bioactive components that are generated during the purification process of the free amino acids have a value added to the process. Such would be the case of a protein concentrate rich in isoflavones, a nanoretained rich in polyphenols, a nanofiltrate rich in free amino acids and sugars, washed with resin water rich in sugars and washed with resin ammonia rich in free amino acids.

The process of making use of chickpea flour and other legumes is viable and economically profitable both in the food industry for the production of bioactive extracts, and in the pharmaceutical industry for the production and purification of amino acids such as arginine. In addition, the yields and profit margins of this process are increased by extending its application to flours of other legumes richer in free amino acids and with greater riches in some amino acids in particular, both in wild and cultivated species of the Vicia (Canavanine amino acid) genera and Lathyrus (amino acid homoarginine).

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In a first aspect, the present invention relates to a process for obtaining free amino acids from legume flour comprising the following steps:

a) extraction of legume flour;

b) nanofiltration of the aqueous extract obtained in the previous stage;

c) purification by means of ion exchange resins of the nanofiltrate obtained in the previous stage and

d) concentration and purification of the product obtained in the previous stage.

In a preferred embodiment, the legume is selected from the Vicia and Lathyrus genera.

In another preferred embodiment, the legume is selected from chickpeas, peas, beans, beans, alfalfa, lentils, lupins, peanuts, soybeans.

In a preferred embodiment, the extraction is acidic.

In another preferred embodiment, the extraction is basic, followed by an acid precipitation resulting in the obtaining of a concentrate rich in isoflavone aglycones.

In another preferred embodiment, the nanofiltration of step (b) is carried out with membranes that allow the passage of molecules with a size smaller than 200 Da.

In another preferred embodiment, the purification of step (c) is carried out by acid adsorption to a cationic resin and subsequent desorption of this at basic pH.

In another preferred embodiment, the concentration and purification of step (d) is carried out by precipitation.

In another preferred embodiment, the concentration and purification of step (d) is performed by crystallization.

Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or

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Steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1: shows the chromatogram obtained by HPLC of the crystallized chickpea Arginine.

Figure 2: shows the chromatogram obtained by HPLC of the precipitated chickpea Arginine.

EXAMPLES

The invention will be illustrated below by tests carried out by the inventors, which shows the effectiveness of the product of the invention.

A) ALKALINE EXTRACTION OF PROTEINS AND OTHER SOLUBLE COMPOUNDS OF THE GARBANZO FLOUR.

Chickpea seeds were ground in a domestic grinder and used as a starting material for the extraction of free amino acids. For this, an alkaline extraction was carried out with water brought to pH 10.5 with 1 N NaOH with stirring for 50 min at room temperature. The mixture is then centrifuged (15,000 rpm / 20 min) and the pellet is extracted at least two more times. The final product is an alkaline extract rich in proteins and other soluble compounds at basic pH.

B) ACID PRECIPITATION OF THE ALCALINE EXTRACT.

For the precipitation of the proteins of this alkaline extract, the supernatants obtained in step A) were brought to acidic pH (in this case 4.3). The mixture was then centrifuged (15,000 rpm / 20 min) and a soluble fraction (B2) rich in free amino acids and other soluble compounds and a solid fraction rich in proteins (B1) was obtained. This fraction B1 also includes a high percentage of isoflavones.

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of the seed in its hydrolyzed form (aglycone). In the case of chickpea, it is mostly the aglycone of biochanin A.

C) NANOFILTRATION.

The soluble fraction B2 obtained in step B is nanofiltered to purify free amino acids from other compounds of higher molecular weight. For this, an Amicon filtration cell was used to which a nanofiltration membrane was incorporated with a cut-off around 200 Da. As a result of nanofiltration, a nanoretaining was obtained, which is a product rich in compounds greater than 200 Da such as proteins and polyphenols. On the other hand, a nanofiltrate rich in compounds less than 200 Da was obtained as free acids and monosaccharides.

D) PURIFICATION THROUGH ION EXCHANGE RESINS.

For the purification of the free amino acids of other low molecular weight compounds a strongly acidic cation exchange resin was used, in particular Amberlite IR-120. First, the nanofiltration incubation, containing the free amino acids, was carried out with the resin at pH 2 for 30 min to allow the amino acids to bind the resin reversibly. Subsequently, several washes (in this case 4 washes) of the resin were carried out with water for 10 minutes each. In this way, nanofiltered compounds that have not bound to the resin, such as monosaccharides, are removed. The resin was subsequently washed with 0.5 N NH4 for 30 min once to desorb amino acids weakly bound to the resin. Finally the amino acids and a small proportion of other compounds are desorbed from the resin with concentrated NH4 (7.54 N).

E) PURIFICATION AND CONCENTRATION OF THE RECOVERED FREE AMINO ACIDS OF THE RESIN BY PRECIPITATION OR CRYSTALLIZATION. The final purification of the desorbed free amino acids of the resin is carried out by precipitation or crystallization, in both cases using ethanol. For the precipitation, 4 volumes (or more than 4) of ethanol are added in relation to the volume of free amino acids. The amino acids precipitate immediately recovering by centrifugation or filtration and obtaining, in the case of chickpea, mostly arginine (90.73%) with the presence of glutamic acid (6.37%) and

aspartic (2.90%) (verified by HPLC). For crystallization, ethanol is added until turbidity and left at room temperature for 24 hours. After this time the crystals are recovered by centrifugation, and in the case of chickpea 100% corresponds to pure arginine (checked by HPLC).

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Claims (9)

5 10 fifteen twenty 25 30 1. Procedure for obtaining free amino acids from legume flour comprising the following steps: a) extraction of legume flour; b) nanofiltration of the aqueous extract obtained in the previous stage; c) purification by means of ion exchange resins of the nanofiltrate obtained in the previous stage and d) concentration and purification of the product obtained in the previous stage. 2. Method according to claim 1, wherein the legume is selected from the Vicia and Lathyrus genera. 3. Procedure according to claim 1, wherein the legume is selected from chickpeas, peas, beans, beans, alfalfa, lentils, lupins, peanuts, soybeans. 4. Method according to any one of claims 1 to 3 wherein the extraction is acidic. 5. Method according to any one of claims 1 to 3 wherein the extraction is basic, followed by an acid precipitation. 6. Method according to any of claims 1 to 5 wherein the Nanofiltration of stage (b) is carried out with membranes that allow the passage of molecules with a size smaller than 200 Da. 7. Method according to any one of claims 1 to 6 wherein the purification of step (c) is carried out by acid adsorption to a cationic resin and subsequent desorption thereof at basic pH. 8. Method according to any of claims 1 to 7 wherein the Concentration and purification of step (d) is carried out by precipitation. 9. Method according to any one of claims 1 to 7 concentration and purification of step (d) is performed by crystallization. where the