WO2018149773A1

WO2018149773A1 - Fertilizers

Info

Publication number: WO2018149773A1
Application number: PCT/EP2018/053386
Authority: WO
Inventors: Dario Chiavacci; Eleonora DELIA; Cristina Formantici; Marco ZILIANI; Giovanni Floridi; Giuseppe Li Bassi
Original assignee: Lamberti Spa
Priority date: 2017-02-15
Filing date: 2018-02-12
Publication date: 2018-08-23
Also published as: IT201700016547A1

Abstract

A carboxymethylated protein hydrolyzate having a degree of hydrolysis (DH) comprised between 10 and 90 % and degree of carboxymethylation (DC) comprised between 60 and 100 %, chelated with an amount of metal ion such that the primary amino groups of the protein hydrolyzate to metal molar ratio is comprised between 0.8 and 3. A fertilizer composition comprising from 0.01 to 98 % by weight of the chelated carboxymethylated protein hydrolyzate.

Description

FERTILIZERS

TECHNICAL FIELD

The present invention relates to carboxymethylated protein hydrolyzates and to metal chelates obtained from said hydrolyzates.

This invention also pertains to the use of said chelates as fertilizers.

PRIOR ART

Plants require specific amounts of nutrients to germinate, grow and remain viable.

Nutrients management is crucial for optimal productivity in commercial crop production. Nutrients that are present in concentrations of < 100 parts per million (ppm) in plant tissues are defined micronutrients and include ions such as iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), cobalt (Co), boron (B), chlorine (CI), molybdenum (Mo), and nickel (Ni). Micronutrients, such as Fe, Mn, Zn, and Cu, are subject to undesirable reactions in solution and soil, for example oxidation or precipitation, and their plain utilization is, therefore, not very efficient.

In order to increase efficiency of micronutrient absorption, chelated fertilizers have been developed . Chelated fertilizer improves the bioavailability of micronutrients, such as Fe, Cu, Mn, and Zn, and in turn contributes to increase the productivity and profitability of crop production. Chelated fertilizers have a greater potential to increase commercial yield than regular micronutrients if the crop is grown in low- micronutrient stress or soils with a pH greater than 6.5. If the soil cannot meet the micronutrient requirements for the crop growth, alternative micronutrients sources, for example chelates, need to be used, in order to grow a healthy crop.

There is a number of synthetic chelating agents that can form a chelated fertilizers with one or more micronutrients. Examples are ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), cyclohexanediamine pentaacetic acid (CDTA), ethylenediamine di(o- hydroxyphenylacetic acid) (EDDHA), nitrilo triacetic acid (NTA), (hydroxyethyl)-ethylenediamine triacetic acid (HEDTA) and the like. EDTA, DTPA, and EDDHA are among the preferred chelating agents. Since these synthetic chelates are not (easily) biodegradable, the use of such compounds is being regulated in many parts of the world.

There is also a number of natural products which can be used as chelating agents. Lignosulfonates, phenols and polyflavonoid metal chelates are produced using fermented by-products, mainly from the wood pulp industry.

Among the derivatives of natural products, chelates obtained by chelation of essential micronutrient metal ions with pure amino acids or protein hydrolyzates, which contain mixtures of amino acids and peptides, are particularly interesting. Such fertilizers are also referred as "metal amino acid chelates", when pure amino acids are used as ligands, and as "metal proteinates" or "peptide chelates of metals", when protein hydrolyzate are used as ligands. One advantage of these chelates in the field of plant nutrition is attributed to the fact that they are readily absorbed into plant tissue (bioavailable) .

Examples of these fertilizers are described in US 2010/035305, WO 2009/089493, US 2008/194407, US 2005/235718, US 6,518,240, US 6,458,981, US 6,241,795, US 4,599,152, US 4,167,564 and US 4,020,158.

Unfortunately, the stability constants of these chelates are relatively low; chelates that have a low stability do not provide adequate protection to the metal ions when applied on soil and, thus, they do not ensure a sufficient solubility and bioavailability.

Additionally, another important limitation to the use of proteins, and in particular of protein hydrolysates and their chelates, is the extreme susceptibility to attacks by microorganisms. For their storage, it is therefore necessary to add large amounts of preservatives, substances which certainly increase the risk to the health and environmental impact. Now, we have surprisingly discovered that protein hydrolyzates, which have been carboxymethylated, can be used to chelate metal ions, suitable as micronutrients, obtaining chelates with a much higher chemical and microbiological stability than those of simple protein hydrolyzates.

These carboxymethylated protein hydrolyzates, thanks to the low molecular weight, give metal chelates with a superior mobility in the soil and an improved bioavailability compared to the large proteins. At the same time, the high anionic charge of the chelating agent provide metal chelates with a higher solubility in water and a lower viscosifying power, so highly concentrated and easily manageable compositions can be prepared.

Moreover, said chelated carboxymethylated protein hydrolyzate can be used to prepare nontoxic and biodegradable fertilizers, which will have minimal impact on worker safety, on the environment and on the ecological considerations of the farm.

As far as the Applicant knows, no one has described the carboxymethylated protein hydrolyzates of the present disclosure or their metal chelates.

US 2,788,336 describes the reaction product of proteinaceous material with carboxymethylating agents such as monochloroacetic acid. The proteinaceous material can be used as such or it can be mildly degraded (hydrolyzed). However, the Application only cites this mild degradation, the procedure to obtain a mildly degraded proteinaceous material is not described nor exemplified . Also the degree of hydrolysis is not indicated. EP 1922331 and EP 1910411 relate to compositions for the treatment of surfaces comprising guar protein extracts, optionally derivatized, characterized in that they have a protein content of greater than 65% by weight. Protein extracts can be derivatized with cationic, anionic, non- ionic hydrophilic or hydrophobic and crosslinking groups or they may have been subjected to hydrolysis. Multiple derivatizations can be carried out. Various anionizing agents with very different characteristics are mentioned, including monochloroacetic acid . But there is no description of anionization reactions, and nothing is said about the degree of anionization . Also, only a cationized protein extract from non-hydrolyzed guar is exemplified .

The definition of "degree of hydrolysis" (DH) of the protein is described in : "Improved method for determining food protein degree of hydrolysis", Nielsen P. M ., et al., Journal of Food Science, 66, 642-646 (2001).

It represents the percentage of the cleaved peptide bonds and it is calculated with the following formula :

DH = h/h_tot * 100

where h_tot is the total number of peptide bonds per protein equivalent and h is the number of hydrolyzed bonds. These two values were determined by reacting the primary amino groups, which are originated by the hydrolysis of the peptide bonds, with o-phthalaldehyde (OPA). For the determination of h_tot, the samples of proteinaceous material have to be subjected to a preliminary complete hydrolysis with HCI 6 M for 8 hours at 100 °C.

With the expression "degree of carboxymethylation" (DC), we mean the percentage of the primary amino groups which have been subjected to carboxymethylation and it is calculated with the following formula :

DC=(h_s-h_sr)/h_{s *} 100

where h_s is the serine-NH₂ value of the protein hydrolyzate, as defined at page 644 of the article of Nielsen et al. disclosed above, and h_sr represent the serine-NH₂ value after the carboxymethylation of the protein hydrolyzate.

It must be pointed out that the degree of carboxymethylation does not represents the degree of substitution . In fact, the degree of carboxymethylation only takes into account the unsubstituted primary amino groups, on the contrary the degree of substitution consider the unsubstituted primary amino groups, the amino groups substituted with one carboxymethyl group and the amino groups substituted with two carboxymethyl groups. As a consequence, the maximum possible value of DC is 100 %, while the maximum possible value of the per cent degree of substitution is 200 %. As used herein, the expression " carboxymethylated protein hydrolyzate chelated with a metal ion", is meant to include sundry compounds where a metal ion is chelated or complexed to a hydrolyzed protein which has been carboxymethylated . Coord inate bonds, covalent bonds, and/or ionic bonds may be present between the metal ion and the proteinaceous chelating agent. As used herein, chelated carboxymethylated amino acid are included in said defin ition . However, this defin ition does not include al l types of chelated carboxymethylated amino acid, but it on ly includes those coming from the partial hydrolysis of a protein .

DESCRIPTION OF THE INVENTION

It is therefore a fundamental object of the present invention a carboxymethylated protein hyd rolyzate having a deg ree of hydrolysis ( DH) comprised between 10 and 90 % and a deg ree of carboxymetylation ( DC) comprised between 60 and 100 % .

The above d isclosed carboxymethylated protein hydrolyzate chelated with an amount of metal ion such that the primary am ino g roups of the protein hyd rolyzate to the metal ion molar ratio is comprised between 0.8 and 3, is another object of the invention .

In an another aspect, the present invention relates to a fertil izer composition comprising from 0.01 to 98 % by weight (% wt) of said chelated carboxymethylated protein hydrolyzate.

In a further aspect, the present invention relates to a method of increasing the metabol ic activity in a plant comprising ad ministering the composition of the invention, wherein the composition is ad ministered to the plant in an amount sufficient to raise the n itrogen concentration within the plant and to enhance metabolic activity of the plant.

DETAILED DESCRIPTION OF TH E I NVENTION

Preferably, the carboxymethylated protein hyd rolyzate of the invention has a DH comprised between 15 and 70 % and a DC comprised between 80 and 100 % . More preferably, said carboxymethylated protein hydrolyzate has a DH comprised between 20 and 50 % and a DC comprised between 95 and 100 %.

The carboxymethylated protein hydrolyzate of the invention can be prepared according to a process comprising the following steps:

1) providing a proteinaceous material;

2) hydrolyzing the proteinaceous material to obtain a protein hydrolyzate with a degree of hydrolysis comprised between 10 and 90 %, preferably between 15 and 70 %;

3) carboxymethylating the protein hydrolyzate to obtain a carboxymethylated protein hydrolyzate with a degree of carboxymethylation comprised between 60 and 100 %, preferably between 80 and 100 %.

The proteinaceous material suitable for the realization of the present invention can be obtained from any natural or recombinant protein source. Also proteinaceous material obtained from mixtures of different protein sources can be used .

The natural protein source can be of vegetable origin including guar meal, maize meal, rice meal, tobacco meal, soybean meal, sunflower meal, wheat meal, barley meal, pea protein, tamarind kernel powder, and peanut meal and other vegetable protein meals. Also protein source which have been subjected to further purification or fractionation process or other kinds of processes can be used for the realization of the present invention. Specific examples of these alternative sources are soy protein concentrates and soy or pea protein isolates.

Alternatively, the natural protein source can be of animal origin including casein, feather meal, poultry byproduct meal, dried yeast, blood meal, fish meal, meat and bone meal, and other protein meals of animal origin. Preferably, the protein source is of vegetable origin. Preferred protein sources are guar meal, soybean meal and pea protein and the concentrates and isolates thereof. Usually, the proteinaceous material contains from 15 to 100 % by weight of proteins, preferably from 40 to 95 % wt.

In another preferred embodiment, the proteinaceous material contains from 70 to 95% by weight of proteins.

In a further preferred embodiment, the proteinaceous material contains from 40 to 60% by weight of proteins.

The hydrolysis of the proteinaceous material can be achieved by using any of the methods described in literature. Catalysts such as mineral acids, bases and, preferably, proteolytic enzymes can be used .

In a preferred embodiment, the hydrolysis is performed by placing a comminuted protein source in an aqueous solution with one or more proteolytic enzymes (proteases). In general, the solution will contain about from 10% to 30% by weight/volume of proteinaceous material to water. Suitable proteases are pepsin, pancreatin, trypsin, papain, bacterial protease and fungal protease. The enzyme is added in an amount of about 0.1 to about 10 % by weight based on the protein content. In general, the hydrolysis is carried out at between about 25 and 70 °C, over a period of about 2 hours to about 24 hours. Preferably, the hydrolysis is carried out under neutral and/or alkaline conditions and it may be necessary, during the digestion, to adjust the pH of the protein- enzyme solution by the use of an acid or base. Multistep enzymatic hydrolysis can be also applied.

Optionally, other kind of enzymes, not proteolytic, can be added to the proteinaceous material before hydrolysis in order to improve the characteristics of the hydrolyzate, for example reducing its viscosity and/or the content of solid residues.

In another embodiment, the proteinaceous material can be hydrolyzed with a base, for example sodium or potassium hydroxyde 3-5 N, and heating to a temperature between 90 and 150 °C .

In a further embodiment, the proteinaceous material can be hydrolyzed with an acid, for example sulfuric acid or hydrochloric acid 6-8 N, at a temperature of about 100-150 °C for 1-12 hours. Before carboxymethylation, the hydrolyzate of the invention may be filtered/centrifuged to separate undigested tissue and other insoluble residues.

At the end of the hydrolysis, the protein hydrolyzate has a value of h, defined above, ranging from 0.6 to 5.4 meq/g, preferably from 0.9 to 4.2 meq/g and more preferably from 1.2 to 3.0 meq/g, based on the weight of protein.

The process for the carboxymethylation of various protein substrates is known in the art, by way of example, in US 2,788,336.

The carboxymethylated protein hydrolyzate can be obtained by reacting the protein hydrolyzate described above with a halo-acetic acid or a salt thereof in the presence of an alkaline catalyst. The reaction can be conducted in water or in a water/water-soluble solvent mixture.

The alkaline catalysts are usually alkali metal or alkaline earth metal hydroxides, such as sodium, potassium or calcium hydroxide.

Halo-acetic acids are normally monochloroacetic acid or monobromoacetic acids, or their alkali metal salts.

Suitable water-soluble solvents for the process can be methanol, ethanol and secondary lower alkanols, such as isopropanol, sec-butanol, sec-amyl alcohol, or tertiary lower alkanols. Preferably, the water-soluble solvent is isopropanol . The possible organic solvent is distilled off at the end of the reaction.

In an exemplary carboxymethylation process, the protein hydrolyzate and sodium monochloroacetate are reacted in water in such an amount that the monochloroacetate/primary amino groups molar ratio ranges from about 0.6 to about 4, preferably from about 1.5 to 3.5, more preferably from about 2 to about 3.3. The molar concentration of primary amino groups in the protein hydrolyzate, to be used for the determination of the optimal process conditions, is calculated using h_s, the serine-NH₂ value, described in the article of Nielsen, P. M ., et al . Advantageously, the protein hydrolyzate is reacted with the sodium monochloroacetate in such an amount that almost all the primary amino groups are substituted with two carboxymethyl groups.

Since the alkali hydroxide must react with the chlorine atom of the monochloroacetate salt, to achieve complete reaction, equimolar amounts of the hydroxide and monochloroacetate salt are employed . The use of a molar excess of alkali hydroxide achieves no apparent advantage.

The carboxymethylation reaction can take place at a temperature of about 40-100 °C, preferably at 60-90 °C. It can take place for several hours (up to 6-7 h) after having reached the desired reaction temperature and the completeness of reaction can be controlled by the residual primary amino groups determination .

Preferably, at the end of the carboxymethylation reaction, the pH of the reaction mass is adjusted to from about 6 to about 10 and the solution so obtained is filtered .

The carboxymethylated protein hydrolyzate so obtained can be directly treated with a metal ion or a mixture of metal ions to form a metal chelate. Alternatively, it can be dried, ground and sieved and subsequently reacted with the metal ion.

The carboxymethylated protein hydrolyzate of the invention can be chelated with an amount of metal ion such that the primary amino groups of the protein hydrolyzate (calculated from the serine-NH₂ value as described above) to metal molar ratio is comprised between 0.8 and 3, preferably between 1 and 2, more preferably is between 1 and 1.5.

As used herein, the term "metal" refers to nutritionally relevant metals including divalent and trivalent metals that can be used as part of a nutritional supplement for plants and are substantially nontoxic when administered in traditional amounts, as is known in the art. Examples of such metals include copper, zinc, manganese, iron, chromium, calcium, magnesium, cobalt, nickel, molybdenum, vanadium, strontium, selenium, and mixture thereof. This term also includes nutritional semi-metals including, but not limited to, boron. Examples of metal ion sources include elemental metals, metal sulfates, metal oxides, metal carbonates, metal chlorides, metal borates, and combinations thereof. Examples of metal sulfates that can be used include sulfates of iron, zinc, magnesium, copper, manganese, molybdenum, selenium and cobalt. Examples of metal oxides that can be used in the invention include oxide of iron, zinc, copper, manganese, molybdenum, selenium and cobalt. With the metal oxides, however, an acid may need to be used as a processing aid . Examples of metal carbonates that can be used include calcium and magnesium carbonate. Examples of metal chlorides that can be used in the invention include calcium and magnesium chloride. Examples of metal borates that can be used include sodium tetraborate, sodium borate, calcium borate, and various hydrated forms or derivatives of these borates.

Methods for chelating protein hydrolyzates or amino acids are known in the art.

In order to chelate the carboxymethylated protein hydrolyzate of the invention, an appropriate amount of a metal salt or a mixture of metal salts can be added to an aqueous solution of the carboxymethylated protein hydrolyzate.

Usually, the chelation process is carried out for a time and under operating conditions sufficient to substantially complete the reaction. The time and temperature for the chelating reaction will depend on the desired degree of completion of the reaction. Generally, the time for the reaction can range from about 30 min to some hours and the temperature can range from about 20 to about 90 °C. The pH of the reaction mass is usually adjusted between 5.0 and 9.0.

When the chelating reaction is complete, the chelated carboxymethylated protein hydrolyzate is in a liquid form, i.e. in solution.

In one aspect of the invention, the chelated carboxymethylated protein hydrolyzate of the invention is used in liquid form.

In another aspect of the invention, the chelated carboxymethylated protein hydrolyzate of the invention is dried into a powder using any suitable drying process. Any drying process known in the art can be used, including oven drying, drum drying, fluidized bed drying, spray-drying and other commercially viable drying methods, or combinations thereof. In a preferred aspect of the invention, the drying processes used in the invention comprises spray-drying.

If desired, the powder can be converted into another solid form like a tablet, capsule, pellet, granule or the like by any known process in the art.

According to the invention, the chelated carboxymethylated protein hydrolyzate can be used to prepare a composition useful, as fertilizer, for increasing the metabolic activity in a plant. The present fertilizer composition can be provided both as dry-blended formulation and liquid formulation, typically aqueous solution/dispersion. It can be either a concentrated formulation to be diluted in the field and a diluted formulation "ready to use" which can be applied as such.

The fertilizer composition of the invention may, therefore, comprise from 0.01 to 98% by weight of the chelated carboxymethylated protein hydrolyzate described above.

In a preferred embodiment, said composition comprises from 0.01 to 5% by weight, preferably from 0.1 to 3% by weight, of chelated carboxymethylated protein hydrolyzate of the invention .

In another preferred embodiment, said composition comprises from 5 to 98% by weight, preferably from 10 to 80% by weight, more preferably from 15 to 60% by weight of chelated carboxymethylated protein hydrolyzate of the invention .

Beside the chelated carboxymethylated protein hydrolyzate, said composition can comprise other additives commonly used in the field . Examples of suitable additives are nitrate, phosphate, and potassium compounds, which are all essential for plant growth; other protein-related compounds and N-containing compounds; biostimulants; health and growth hormones; vitamins, surfactants and mixtures thereof. Common NPK sources, such as potassium nitrate, potassium phosphate, potassium chloride, potassium sulfate, potassium thiosulfate, potassium magnesium sulfate, phosphoric acid, ammonium nitrate, ammonium phosphate, monoammonium phosphate, ammonium nitrate-sulfate, ammonium phosphate-sulfate, ammonium phosphate-nitrate, diammonium phosphate, ammonium sulfate, ammonium thiosulfate, calcium ammonium nitrate solution, calcium nitrate, calcium cyanamide, sodium nitrate, urea, urea ammonium nitrate solution, and mixtures thereof, can be mixed with the chelates described herein to provide very effective plant fertilizers.

The protein-related compounds, which can be used in addition to the carboxymethylated protein hydrolyzate chelate, are proteinates, hydrolyzed animal sourced or plant sourced proteins; peptides; polypeptides; amino acid, such as arginine, asparagine, glutamine, histidine, lysine, ornithine, cystine, tryptophan etc. ; and mixtures thereof. Other N-containing compounds include betaines, polyamines and 'nonprotein amino acids'.

Examples of suitable biostimulants are humic substances, extracted from naturally humified organic matter (e.g . from peat or volcanic soils), from composts and vermicomposts, or from mineral deposits (leonardite, an oxidation form of lignite); seaweed extract/purified compounds, which include the polysaccharides laminarin, alginates and carrageenans and their breakdown products; chitosan and other biopolymers; beneficial bacteria and fungi; and the like.

They can be applied on soils, in hydroponic solutions or as foliar treatments.

Such compositions contain optimized nitrogen/nitrate or other components to metal micronutrients ratio for optimum plant growth or other metabolic activity and can be very effective in simultaneously supplying minerals and other nutrients to plants. In particular, the metabolic activity can be enhanced growth, enhanced fruit production, reduced morbidity, or enhanced fruit size. The following experimental data show the characteristics and the chelating performances of the carboxymethylated protein hydrolyzates of the invention.

EXAMPLES

Characterization Methods

The Brookfield® (BRK) viscosity (mPa*s) was measured with a DV-E Brookfield® viscometer at 20 °C and 20 rpm.

The protein content was determined according to the Kjeldhal method. The degree of hydrolysis of the proteins (DH%) was determined using the method described in: "Improved method for determining food protein degree of hydrolysis", Nielsen P. M., et al., Journal of Food Science, 66, 642-646 (2001).

The chelating power was determined with two different method: the Ferrozine test and the Fe(II) hydroxide precipitation test.

Ferrozine Test

The Ferrozine test is based on the ability of the chelating agent under test of complexing the Fe(II) ion in competition with a known complexing agent (Ferrozine).

The test was performed preparing a solution 0.6 % by weight of sample, as dry matter, in deionized water. 10 μΙ of this solution were added to 1.0 ml of a 2 ppm Fe(II) standard solution. After 5 minutes at room temperature, 100 μΙ of a 0.01 M ferrozine solution and 50 μΙ of a 5.0 M ammonium acetate buffer at pH 9.5 were added. The absorbance of the Ferrozine/Fe(II) complex was measured at 562 nm against deionized water with a Lambda25, UV-Vis spectrophotometer from Perkin Elmer.

A blank was prepared substituting the sample solution with deionized water.

The higher the absorbance of the sample solution the lower the chelating power of the sample.

The percentage chelating power based on the Ferrozine test (FCP%) was calculated with the following formula:

FCP% = [(Abs_b - Abs_s)/Abs_b] * 100 wherein

AbSb = absorbance of the blank

Abs_s = absorbance of the sample Fe(II) hydroxide precipitation test

The Fe(II) hydroxide precipitation test is based on the ability of the sample of reducing the Fe(II) hydroxide precipitation.

The test was performed by sequentially introducing in a 100 ml volumetric flask: 1.2 g, as dry matter, of sample, 30 ml of deionized water, 0.040 g of Fe(II) and 10 ml of a 0.1 M ammonia/ammonium chloride buffer at pH 8.5.

The mixture was brought to 100 ml with deionized water and leave to equilibrate at room temperature for 1 hour. The precipitate was then separated by centrifugation .

1.0 ml of the supernatant solution was mixed with 20 ml of water, 1.0 ml of HN0₃ 65 % and 2.5 ml of HCL 37 % and heated at 100 °C for 5 minute. After cooling at room temperature, 5.0 ml of a 2.0 M potassium thiocyanate solution were added and the obtained solution was brought to 100 ml with deionized water. The absorbance of the thiocyanate/Fe(II) complex was measured at 480 nm against a blank solution of the various reactants using a Lambda25, UV-Vis spectrophotometer from Perkin Elmer. The amount of non-precipitated Fe(II) in g (SI) was calculated using a calibration curve obtained with Fe(II) standard solutions.

The percentage chelating power based on Fe(II) hydroxide precipitation test (PCP%) the was calculated with the following formula :

PCP% = SI/0.04 _* 100

Examples 1 -11

According to the invention, protein materials from different sources were subjected to enzymatic hydrolysis and subsequent carboxymethylation following the procedures of preparation described below. A hydrolyzed protein, to be used for comparative purposes, was prepared following Procedure 4.

A carboxymethylated protein, to be used for comparative purposes, was prepared following Procedure 5.

The following proteolytic enzymes were used :

• Neutrase 0.8 L and Flavourzyme 500 L, commercialized by Novozymes

• Genencor Tan L plus, commercialized by DuPont.

Table 1 reports the proteinaceous materials and the procedures utilized for the preparation of Examples 1-11, and the starting protein content in % by weight.

Table 1

Comparative

Procedure 1

The proteinaceous material was subjected to two sequential enzymatic hydrolyses: first with Genencor Tan L plus, then with Neutrase 0.8 L and Flavourzyme 500 L.

In the first hydrolytic step, 200 g of proteinaceous material were added to 760 g of 50 mM sodium phosphate buffer at pH 8.0 (buffer) in a 2 I reactor under overhead stirring and heated to 85 °C. After 15 minutes, the mixture was cooled to 50 °C and was brought to pH 8 with NaOH. 3 ml of Genencor Tan L plus were added to the mixture. The enzymatic hydrolysis was performed at 50-55 °C for 3 hours, maintaining pH at value around 8 with NaOH.

In the second hydrolitic step, the pH of the solution obtained from the previous step was adjusted to 6.5 with HCI and 3 ml each of Neutrase 0.8 L and Flavourzyme 500 L were added . After 3 hours at 50 °C, the protein hydrolyzate was cooled at room temperature and, finally, diluted to a weight of 1000 g with deionized water.

For the carboxymethylation, 105 g of a 50 % wt NaOH aqueous solution were added under stirring to the hydrolyzed protein solution and the mixture was homogenized maintaining the temperature under 30 °C.

After 1 hour, 135 g of sodium monochloroacetate powder were added always under stirring . The temperature was raised to 70 °C and mainitained at this value for 6 hours. The carboxymethylated protein hydrolizate so obtained was then cooled to room temperature and its pH was adjusted to about 7 with H₃P0₄.

Procedure 2

The proteinaceous material was subjected to two sequential enzymatic hydrolyses as in Procedure 1, but with a different temperature profile. In the first hydrolytic step, 200 g of proteinaceous material were added to 760 g of buffer in a 2 I reactor under overhead stirring . 3 ml of Genencor Tan L plus were added to the mixture.

After 3 hour of hydrolysis maintaining the temperature at 50-55 °C and the pH at value around 8 with NaOH, the pH was adjusted to about 6.5 with HCI and another hydrolytic step was performed adding 3 ml each of Neutrase 0.8 L and Flavourzyme 500 L. After 3 hours at 50 °C, the protein hydrolyzate was heated at 85 °C for 30 minutes, cooled down at room temperature and, finally, diluted to a weight of 1000 g with deionized water.

For the carboxymethylation, 105 g of a 50 % wt NaOH aqueous solution were added to the hydrolyzed protein solution and the mixture was homogenized maintaining the temperature under 30 °C. After 1 hour, 135 g of sodium monochloroacetate powder were added under stirring, raising at the same time the temperature to 70 °C. The reaction mass was maintained in these conditions for 6 hours. The carboxymethylated protein hydrolyzate was then cooled to room temperature and its pH was adjusted to about 7 with H₃P0₄.

Procedure 3

The proteinaceous material was subjected to two sequential enzymatic hydrolyses: first with Neutrase 0.8 L and Flavourzyme 500 L, then with Genencor Tan L plus.

120 g of proteinaceous material were added to 465 g of water in a 1 liter reactor under overhead stirring. 1.8 ml each of Neutrase 0.8 L and Flavourzyme 500 L were added to the mixture after homogenization.

After 3 hour of hydrolysis maintaining the temperature at 30-35 °C and the pH at value around 6.5 with NaOH, the pH was adjusted to 8 with NaOH and the second hydrolytic step was performed adding 1.8 ml of Genencor Tan L plus. After for 3 hours at 30 °C, the protein hydrolyzate was cooled at room temperature and finally diluted to a weight of 600 g with deionized water.

For the carboxymethylation, the protein hydrolyzate was diluted with 350 g of water, 84 g of a 50 % wt NaOH aqueous solution were added and the mixture was homogenized maintaining the temperature under 30 °C.

After 1 hour, 108 g of sodium monochloroacetate powder were added, raising at the same time the temperature to 70 °C. The reaction mass was maintained under these conditions for 6 hours. The carboxymethylated protein hydrolyzate was then cooled down to room temperature and its pH was adjusted to about 7 with H₃P0₄.

Procedure 4

A comparative sample of hydrolyzed proteic material was prepared with two sequential enzymatic hydrolyses as described in Procedure 1. The carboxymethylation step was not carried out.

Procedure 5 A comparative sample of carboxymethylated non-hydrolyzed protein was prepared using the following procedure.

200 g of proteinaceous material, 1500 g of water and 105 g of 50 % wt NaOH aqueous solution were loaded in a 3 liter reactor under overhead stirring . The mixture was homogenized maintaining the temperature under 30 °C. After 1 hour, 135 g of sodium monochloroacetate powder were then added, raising at the same time the temperature to 70 °C. The reaction mass was maintained in these conditions for 6 hours. The carboxymethylated protein was then cooled down to room temperature and its pH was adjusted to about 7 with H₃P0₄.

Procedures 6-8

In order to evaluate the effect of the degree of carboxymethylation, the same protein hydrolyzate was reacted with different amounts of sodium monochloroacetate.

3 aliquots of 200 g of protein hydrolyzate were prepared according to Procedure 1.

Different amounts of 50 % wt NaOH aqueous solution (70, 35, and 16.5 g, respectively) were added under stirring to each aliquot and the mixtures were homogenized keeping the temperature below 30 °C. After 1 hour, sodium monochloroacetate powder (90, 45 and 20,5 g respectively) was added to the mixtures always under stirring . The temperature of the three solutions was raised to 70 °C and maintained at this value for 6 hours. The carboxymethylated protein hydrolyzates so obtained were then cooled to room temperature and the pH was brought to about 7 with H₃P0₄.

Table 2 reports the values of h in meq/g of protein after hydrolysis, the monochloroacetate/primary amine (as serine-NH2) molar ratio (MCA/PA), the characteristics of the carboxymethylated protein hydrolyzates of the invention and the comparative samples. Table 2

* Comparative

ND = Not Determined

NA = Not Applicable

Table 3 reports the performances as chelating agents of the Examples 1-

11.

Table 3

* Comparative

NA = Not Applicable As can be seen from the results, the carboxymethylated protein hydroiyzates of the invention show an high chelating power, superior to those of the comparative Examples. Better performances were obtained with the carboxymethylated protein hydroiyzates prepared with an higher monochloroacetate/primary amine molar ratio, because of the presence of an higher number of amino groups substituted with two carboxymethyl groups.

Claims

1) A carboxymethylated protein hydrolyzate having a degree of hydrolysis (DH) comprised between 10 and 90 % and degree of carboxymethylation (DC) comprised between 60 and 100 %, chelated with an amount of metal ion such that the primary amino groups of the protein hydrolyzate to metal molar ratio is comprised between 0.8 and 3.

2) The carboxymethylated protein hydrolyzate of claim 1), having a DH comprised between 15 and 70 % and a DC comprised between 80 and 100 %.

3) The carboxymethylated protein hydrolyzate of claim 2), having a DH comprised between 20 and 50 % and a DC comprised between 95 and 100 %.

4) The carboxymethylated protein hydrolyzate of claim 1), chelated with an amount of metal ion is such that said molar ratio is comprised between 1 and 2.

5) The carboxymethylated protein hydrolyzate of claim 1), wherein said metal ion is chosen in the group consisting of copper, zinc, manganese, iron, chromium, calcium, magnesium, cobalt, nickel, molybdenum, vanadium, strontium, selenium, boron ions and mixture thereof.

6) A fertilizer composition comprising from 0.01 to 98 % by weight of the chelated carboxymethylated protein hydrolyzate of claim 1).

7) The fertilizer composition of claim 6) comprising from 0,01 to 5 % by weight of said chelated carboxymethylated protein hydrolyzate.

8) The fertilizer composition of claim 6) comprising more than 5 and up to 98 % by weight of said chelated carboxymethylated protein hydrolyzate.

9) The fertilizer composition of claim 6) further comprising an additive chosen in the group consisting of nitrate, phosphate and potassium compounds, which are all essential for plant growth; other protein- related compounds and N-containing compounds; biostimulants; health and growth hormones; vitamins; surfactants and mixtures thereof.