Method for producing self-assembling dipeptides

Abstract

The invention relates to a method for producing the self-assembling dipeptides Valine-Valine (Val-Val), Isoleucine-Isoleucine (Ile-Ile), Leucine-Leucine (Leu-Leu), Phenylalanine-Phenylalanine (Phe-Phe), Isoleucine-Valine (Ile-Val / Val-Ile), Isoleucine-Leucine (Ile-Leu / Leu-Ile), Isoleucine-Phenylalanine (Ile-Phe / Phe-Ile), Phenylalanine-Valine (Phe-Val / Val-Phe), Phenylalanine-Leucine (Phe-Leu / Leu-Phe) and Leucine-Valine (Leu-Val / Val-Leu), or any combination of said dipeptides, from the yeast Metschnikowia pulcherrima.

Description

[0001] PROCEDURE FOR OBTAINING SELF-POLYMERIZING DIPEPTIDES DESCRIPTION

[0002] The present invention relates to a process for obtaining self-polymerizing dipeptides, useful as biostimulants both in direct application to crops and integrated into other fertilizers.

[0003] More specifically, the invention provides a method for obtaining the self-polymerizable dipeptides Valine-Valine (Val-Val), Isoleucine-lsoleucine (lle-lle), Leucine-Leucine (Leu-Leu), Phenylalanine-Phenylalanine (Phe-Phe), Isoleucine-Valine (He-Val, Val-lile), Isoleucine-Leucine (lle-Leu / Leu-lle), Isoleucine-Phenylalanine (lle-Phe / Phe-lle), Phenylalanine-Valine (Phe-Val / Val-Phe), Phenylalanine-Leucine (Phe-Leu / Leu-Phe) and Leucine-Valine (Leu-Val / Val-Leu), or any combination of said dipeptides, from the yeast Metschnikowia pulcherrima.

[0004] Promoting plant growth and productivity is important in agriculture. Today, these processes are primarily managed using fertilizers, plant growth agents such as hormones, physical soil modifications, and so on. However, the use of these agrochemicals has long-term environmental consequences, such as resource depletion, environmental damage, and health effects. To limit the use of environmentally hazardous chemical inputs, environmentally friendly natural products, particularly biostimulants, have been developed. Biostimulants are materials, distinct from fertilizers, that promote plant growth when applied in small quantities (Khan et al.).

[0005] 2009, Seaweed Extracts as Biostimulants of Plant Growth and Development, Journal of Plant Growth Regulation 28(4):386-399, D0l:10.1007 / s00344-009-9103-x). According to a more recent definition, plant biostimulants are substances and materials, with the exception of nutrients and pesticides, which when applied to plants, seeds, or growing media in specific formulations, have the ability to modify physiological processes in plants in a way that provides potential benefits to growth, development, and / or stress response (Du Jardin P 2012, The Science of Plant Biostimulants).

[0006] Within this range of environmentally friendly products, amino acids and / or protein hydrolysates are among the most potent and widely used biostimulants. Amino acid-based biostimulants, as well as other biostimulants based on readily assimilable carbon, which are typically applied directly to the soil, have a short half-life due to the high capacity of soil microorganisms to process and utilize them. While the effect of these biostimulants applied to the soil is rapid and can last for some time even after the biostimulant itself has disappeared, due to the changes in soil microbiology they induce, repeated application is necessary in longer-cycle crops to maintain the effects throughout the growing season and achieve maximum yields.This situation is limiting in those low value added crops where there is no automated irrigation application system.

[0007] For example, document EP2618664B1 describes the use of a composition containing individual L-amino acids to influence vital processes in plants, the individual L-amino acids being selected from the group of glutamine, asparagine, histidine and combinations thereof with each other and / or with arginine and / or with glutamic acid and / or with aspartic acid and / or with lysine, and where the total amount of the selected individual L-amino acids when the composition is applied to crops is at least 0.5 g / ha and at most 250 g / ha.

[0008] In the context of the present invention, a biostimulant is understood to be any single substance or mixture of substances whose application stimulates biological processes and, therefore, improves nutrient availability and optimizes their absorption by plants (as defined by the European Biostimulants Industry Council, EBIC). In contrast to known biostimulants, the aforementioned self-polymerizing dipeptides provide delayed biostimulation, making their use a significant advantage for crop treatment. Furthermore, pulcherhmin, an iron chelate derived from pulcherhminic acid, is naturally obtained from the yeast Metschnikowia pulcherrima. In this context, see, for example, EP3130578, from the same applicant.

[0009] Thus, the inventive process allows for the production of a large quantity of the self-polymerizing dipeptides described above while maintaining their urease-inhibiting capacity. In this way, this process generates a product capable of inhibiting urease and also containing a significant quantity of self-polymerizing dipeptides with delayed-release biostimulatory properties.

[0010] The procedure described here allows for a significant reduction in production costs because a single process generates a product with multiple activities related to fertilizer efficiency: stabilization of urea nitrogen and delayed-action biostimulation (self-polymerizing dipeptides). The combination of these two activities is essential, since a side effect of a urease inhibitor is that nitrogen applied to the soil takes time to transform into ammoniacal and nitric forms, making them available to the crop.

[0011] The procedure for obtaining the self-polymerizing dipeptides of the invention consists of the following steps:

[0012] i. Fermentation of the yeast Metschnikowia pulcherrima in a culture medium consisting of dextrose, NH4SO4, KH2PO4, MgSC, CaSC, H3PO4 and antifoaming agent, at a temperature between 26 and 28 °C, at a pH between 4.5 and 5.5, adding between 1.5 and 3% by weight of KCI, to obtain a fermentation broth;

[0013] i. Hydrolysis of the fermentation broth for 6-10 hours with sulfuric acid at a concentration of 7.5 to 15% by weight, at a temperature between 90 and 130 °C, in the presence of Cu (II) and Fe (II).

[0014] In one embodiment, the culture medium consists of dextrose 8.00%, NH4SO40.67%, KH2PO40.83%, MgSO40.73%, CaSO40.05%, H3PO4(75%) 1.33% and antifoam (100%) 0.20%.

[0015] In a preferred embodiment, the yeast fermentation in step i) is carried out at a temperature of 27 °C, a pH of 5, and a KCl concentration of 2 wt%. In another preferred embodiment, the hydrolysis of the fermentation broth from step i) is carried out with a sulfuric acid concentration of 10 wt, at a temperature between 100 and 120 °C, and a Cu concentration of 2+ between 0.5 and 2 g / liter of broth and a concentration of Fe 2+ between 1.0 and 2 g / liter of broth, for 8 hours.

[0016] As demonstrated in the following examples and figures, the procedure of the invention allows obtaining a maximum amount of the dipeptides of interest under low temperature and sulfuric acid concentration conditions, as well as good urease inhibition capacity.

[0017] In the figures:

[0018] Figure 1: graph showing the production yield of the fermentation broth of Metschnikowia pulcherrima (fresh biomass) at different temperatures;

[0019] Figure 2: Graph showing the hydrolysis yield to obtain self-assembling dipeptides at different temperatures; Figure 3: Graph showing the production yield of the fermentation broth of Metschnikowia pulcherrima (fresh biomass) at different pH values;

[0020] Figure 4: Graph showing the hydrolysis yield to obtain self-assembling dipeptides at different pH values;

[0021] Figure 5: graph showing the production yield of the fermentation broth of Metschnikowia pulcherrima (fresh biomass) at different concentrations of KCI;

[0022] Figure 6: graph showing the hydrolysis yield to obtain self-assembling dipeptides at different KCI concentrations;

[0023] Examples

[0024] Study of temperature conditions

[0025] Fermentation of the yeast Metschnikowia pulcherrima

[0026] With the aim of maximizing the production of the above self-polymerizable peptides, different fermentations of the yeast Metschnikowia pulcherrima were carried out in a 5 liter bioreactor.

[0027] For each fermentation, 3 liters of culture medium were prepared with the following composition:

[0028] % by weight

[0029] Dextrose 8.00

[0030] NH4SO4 0.67

[0031] KH2PO4 0.83

[0032] MgSO40,73

[0033] CaSCU 0.05

[0034] H3PO4 (75%) 1.33

[0035] 100% Antifoam 0.20

[0036]

[0037] In the first group of tests, a fixed pH of 5.5 was maintained by the controlled addition of ammonia. Constant aeration of 0.5 vvm and agitation of 800 rpm were maintained. Three independent fermentations were carried out with these parameters but at different temperatures (24, 27, and 30 °C). Biomass production was similar under all three conditions, as shown in Figure 1.

[0038] Hydrolysis of the fermentation broth

[0039] Once the fermentations were complete, the biomass produced underwent complete hydrolysis. The following protocol was followed to carry out the hydrolysis:

[0040] 1- Centrifugation of the culture broth

[0041] 2- Recovery of the pellet with the yeasts, this is considered fresh biomass.

[0042] 3- Resuspension of 10 grams of fresh biomass in 10 grams of 40% sulfuric acid. This results in a solution with 50% fresh biomass and 20% sulfuric acid.

[0043] 4- The mixture was heated to 130 °C and maintained for a period of 8 hours.

[0044] 5- Finally, I carry out a quantification process of the following dipeptides:

[0045] Valine-Valine (Val-Val), Isoleucine-lsoleucine (lle-lle), Leucine-Leucine (Leu-Leu), Phenylalanine-Phenylalanine (Phe-Phe), Isoleucine-Valine (He-Val, Val-lile), Isoleucine-Leucine (lle-Leu / Leu-lle), Isoleucine-Phenylalanine (lle-Phe / Phe-lle), Phenylalanine-Valine (Phe-Val / Val-Phe), Phenylalanine-Leucine (Phe-Leu / Leu-Phe) and Leucine-Valine (Leu-Val / Val-Leu):

[0046] One gram of sample is taken and the aforementioned dipeptides are extracted and quantified by LC-MS. For this purpose, the 1-gram sample of hydrolysate is placed in a 1.0 L beaker and 0.5 L of organic extractant, preferably methanol, acetonitrile, or ethyl acetate, is added. The mixture is stirred for 2–4 hours at room temperature. After extraction, the mixture is filtered, and the filtrate is concentrated to dryness under reduced pressure using a rotary evaporator. The solid residue is dissolved in 25 mL of HPLC-grade acetonitrile. The solution is transferred to a 50 mL volumetric flask and diluted to the mark with HPLC-grade acetonitrile.Finally, approximately 1.0 ml of the solution, previously filtered through a syringe filter, is taken into an HPLC vial and the sample is quantified by liquid chromatography-mass spectrometry (LC-MS), UPLC Waters Acquity coupled to a Waters Acquity QDA mass detector and equipped with an ACE Excel 3 Super C18 column (3 pm, 150 x 4.6 mm), with the following chromatographic conditions:.

[0047] Quantization: SI mode

[0048] Flow rate 0.5 ml / min • Mobile phase: A = acetonitrile with 0.1% formic acid and B = water with 0.1% formic acid

[0049] • Gradient: 0 min (5% A + 95% B); 2 min (20% A + 80% B); 20 min (98% A + 2% B); 25 min (98% A + 2% B); 27 min (5% A + 95% B); 30 min (5% A + 95% B)

[0050] • Injection volume: 15 pl

[0051] • Column temperature: 40 °C

[0052] • MS range: 100-800

[0053] • Ionization mode: ES+

[0054] • Cone voltage and capillary voltage: 10 V and 1.2 kV

[0055] • Solvation temperature: 600 °C

[0056] The quantification of the compounds is carried out through external calibration. For this purpose, calibration curves are prepared individually for each compound using standards between 0.1 and 15 ppm. The peak area associated with the m / zy retention time of each known compound is measured using its standard and extrapolated to the corresponding calibration curve. Applying the correct dilution factor, the concentration in mg of dipeptide per gram of initial fresh biomass is determined using this protocol.

[0057] 6- Finally, the quantity of all the dipeptides analyzed is added together to give a cumulative value of all the dipeptides of interest.

[0058] The results are shown in Figure 2. As can be seen, the highest total quantity of dipeptides of interest is obtained at 27 °C. If, in addition to the quantity of dipeptides per unit of fresh biomass, we also consider the amount of biomass generated per liter at each temperature, we can see that 27 °C has a higher productivity (2.91 grams / L) compared to the other temperatures.

[0059] grams dipep / L initial

[0060] 24°C 2.24

[0061] 27°C 2.91

[0062] 30°C 2.48

[0063]

[0064] Study of pH conditions

[0065] Fermentation of the yeast Metschnikowia pulcherrima. In a second set of tests, further fermentations were carried out in the 5-liter bioreactor using 3 liters of the same culture medium used in the previous tests. In this second set of tests, the temperature was kept constant at 27 °C. A constant aeration of 0.5 vvm and agitation of 800 rpm were maintained. Tests were conducted at pH 4, 5, 6, and 7. Biomass production was significantly higher at the intermediate pH values ​​and slightly lower at the extreme pH values, as shown in Figure 3.

[0066] Hydrolysis of the fermentation broth

[0067] Once the fermentation process was complete, the hydrolysis and quantification of the dipeptides of interest, as described above, were carried out. As shown in Figure 4, the highest quantity of dipeptides per gram of fresh biomass was generated at pH 5.

[0068] If we assess the overall yield considering the biomass generated per liter and the amount of peptides per unit of biomass, we see that maximum productivity occurs at pH 5, with 3.14 grams / L:

[0069] grams dipep / L initial

[0070] pH 4 2.02

[0071] pH 5 3.14

[0072] pH 6 2.77

[0073] pH 7 2.01

[0074]

[0075] Study of KCl concentration

[0076] Fermentation of the yeast Metschnikowia pulcherrima

[0077] In a third set of tests, further fermentations were carried out in the 5-liter bioreactor using 3 liters of the same culture medium as in the previous tests. In this third set of tests, the temperature was kept constant at 27 °C. A constant aeration of 0.5 vvm and stirring at 800 rpm were maintained. A constant pH of 5 was maintained by the controlled addition of ammonia. Five independent fermentations were performed, incorporating increasing amounts of potassium chloride into the culture: 0%, 0.5%, 1%, 2%, and 4%. Biomass production was significantly affected by the high KCl concentrations, as shown in Figure 5.

[0078] Hydrolysis of the fermentation broth

[0079] Once the fermentation process was complete, the hydrolysis and quantification of the dipeptides of interest, as described above, were carried out. As can be seen in Figure 6, the inclusion of increasing amounts of KCl in the culture medium has a strong impact on the quantity of dipeptides of interest per gram of wet biomass.

[0080] Finally, considering both the fresh biomass per liter and the amount of dipeptides of interest per unit of biomass, we can see that even though the amount of dipeptides per unit of biomass is much higher at a 4% KCI concentration, a 2% KCI concentration is the most productive condition due to the higher amount of biomass produced per liter of fermentation:

[0081] % KCI grams dipep / L initial

[0082] 0 3.1

[0083] 0.5 3.2

[0084] 1 4.1

[0085] 2 5.9

[0086] 4 5.6

[0087]

[0088] Assessment of urease inhibition capacity

[0089] Fermentation of the yeast Metschnikowia pulcherrima

[0090] Prepare 3 liters of the culture medium with the following formulation:

[0091] % by weight

[0092] Dextrose 8.00

[0093] NH4SO4 0.67

[0094] KH2PO4 0.83

[0095] MgSO40,73

[0096] CaSCU 0.05

[0097] H3PO4 (75%) 1.33

[0098] 100% Antifoam 0.20

[0099] KCI 2%

[0100]

[0101] The temperature was maintained at 27 °C, with constant aeration at 0.5 vvm and agitation at 800 rpm. The pH was set at 5, and the fermentation process was carried out. Once fermentation was complete, the fermentation broth was centrifuged to obtain fresh biomass. Hydrolysis of the fermentation broth

[0102] Hydrolysis is carried out using 40 grams of fresh biomass mixed with 40 grams of sulfuric acid at different concentrations. The final sulfuric acid concentrations in the reactions will be 5%, 10%, 15%, and 20%. Once the different solutions are prepared, 10 grams of each will be used and subjected to the following temperatures for a period of 8 hours: 80°C, 90°C, 100°C, 110°C, 120°C, 130°C, and 140°C.

[0103] Once the extraction processes were completed, the total content of dipeptides of interest was initially assessed using the analysis protocol described above, with the following results.

[0104] mg dipep / gram biomass

[0105] 5% 10% 15% 20%

[0106] 80 °C 0.3 0.5 0.5 1.1

[0107] 90 °C 0.2 0.1 1.2 3.7

[0108] 100 °C 8.2 9.2 12.4 17.4

[0109] 110 °C 9.24 12.21 23.8 27.6

[0110] 120 °C 18.4 16.3 29.2 24.8

[0111] 130 °C 19.2 23.5 28.9 22.5

[0112] 140 °C 24.3 28.3 27.4 18.6

[0113] As can be seen in the table above (values ​​in bold), there is a range of combinations where the generation of dipeptides of interest is at its maximum.

[0114] The urease inhibition capacity of each of these solutions was then analyzed. This involved evaluating the impact of different concentrations of the hydrolysates on reducing urease activity in vitro.

[0115] To analyze urease activity and the impact of different hydrolysates, the protocol described below is followed. Reagents:

[0116] Urease from Canavalia ensiformis (Jack bean). Sigma U1500-20KU, 25 mg / ml, 1% BSA, 0.1% Tween 20.

[0117] - HEPES buffer pH 7.0, 1 M

[0118] Tween 1%

[0119] - Urea 1.25 M.

[0120] - Milli-q water

[0121] KCl-HCl solution (ammonium quantification)

[0122] Reaction conditions:

[0123] The reaction was carried out under the following conditions: 50 mM HEPES buffer, pH 7.0, 0.001% Tween, 125 mM urea, and urease at a final concentration of 5 pg / ml. The mixture was placed in an oven at 25 °C for 30 min. At the end of the reaction, a 5-second vortex was applied, and 200 pg of the product was added to 800 ml of KCl-HCl. The ammonium content was then quantified. Preparation:

[0124] Thaw the enzyme beforehand in ice or at 4 °C, at the time of making the reaction prepare a 1 / 50 dilution of the enzyme in a 10 mM HEPES buffer solution (5 l HEPES in final volume of 500 pl+ 10 pl of enzyme).

[0125] Different dilutions of the produced hydrolysates are prepared

[0126] The reaction is prepared with the following solutions:

[0127] pl

[0128] HPES 1M 50 Buffer

[0129] Tween 1% 1

[0130] Hydrolyzed / water 10

[0131] Water 830

[0132] Enzyme 1 / 50 10

[0133]

[0134] Tubes are prepared for the samples and always a positive reaction control in which water is added instead of Metshnikowia Pulcherrima hydrolysate.

[0135] Incubate for 30 min with agitation at room temperature.

[0136] After 30 minutes, 90 pl are taken and placed in a tube with KCl-HCl (400 µl): Point 0. Vortex the tubes from point 0 and incubate for 10-15 minutes. Then, add 10 pl of urea and vortex. Save the tubes for ammonia quantification.

[0137] - To the reaction tubes, 90 ml of urea are added to each tube, vortexed and incubated for 30 min at 25 °C

[0138] - At the end of the reaction, vortex the tubes and take 100 ml of the reaction and add it to 400 ml of HCl-KCl (endpoint). Incubate for 10 min.

[0139] Proceed to quantify the ammonium.

[0140] The generation of ammonia per minute is quantified in all cases through calculations.

[0141] By extrapolating from the results of the different dilutions of each hydrolysate, the concentration of hydrolysate (ppm) capable of inhibiting 50% of urease activity (IC50) is calculated. The smaller the IC50, the greater the hydrolysate's potential to inhibit urease.

[0142] As shown in the table below, there are conditions where the IC50 is minimal. The more aggressive the conditions (more sulfuric acid and higher temperature), the larger the IC50 and, therefore, the less urease inhibition capacity the hydrolysate has. In the table, the conditions considered optimal for urease inhibition are marked in bold.

[0143] IC50 (ppm hydrolyzed)

[0144] 5% 10% 15% 20%

[0145] 80 °C 98 124 149 1765

[0146] 90 °C 105 121 189 1756

[0147] 100 °C 101 125 245 1789

[0148] 110 °C 107 158 248 1845

[0149] 120 °C 121 314 321 1854

[0150] 130 °C 263 467 687 2954

[0151] 140 °C 365 623 1241 3250

[0152] Comparing the tables above, it can be observed that the most favorable conditions for obtaining dipeptides are the least favorable for obtaining potential urease-inhibiting activity. Only one small combination offers maximum yield in both cases: 15% sulfuric acid and temperatures between 110 °C and 120 °C. The hydrolysis process is optimized by incorporating metal catalysts to obtain a significant quantity of dipeptides of interest under less aggressive conditions that allow the hydrolysate to retain its urease-inhibiting capacity.

[0153] To achieve this, biomass hydrolysis processes are carried out using Metshnikowia 2 7 CL gramsu

[0154] Pulcherrima with a sulfuric acid concentration of 10% and at a temperature of 100 °C for 8 hours. Different amounts of Cu are incorporated into the reactions.2+ (0, 0.5, 1, 2 g / L), different amounts of Fe 2+ (0, 1, 2 and 4 g / L) or a mixture of the two. The results obtained are shown in the following table:

[0155] mg dipep / gram biomass

[0156]

[0157] grams Fe 2+ / L

[0158] 0 1 2 4

[0159] 0 8.6 7.5 8.6 8.21

[0160] 0.5 10.2 24.5 23.2 12.5

[0161] 1 14.1 26.7 28.7 13.4

[0162] 2 17.2 29.4 29.6 14.7

[0163]

[0164] As can be seen, copper has a positive impact on obtaining the dipeptides of interest. This effect is not observed with the incorporation of Fe. 2+ On the other hand, the combined presence of the two elements achieves a synergistic effect in enhancing the production of a greater quantity of dipeptides. Only the incorporation of more than 4 grams of Fe 2+ / L appears to have a negative effect on the reaction.

[0165] With this new information, a new set of hydrolysis conditions was prepared where a fixed amount of 1 gram of Fe was incorporated. 2+ / L and a fixed amount of 0.5 grams Cu 2+ / L. These hydrolyses were carried out under different temperature conditions (80 °C, 90 °C, 100 °C, 110 °C, 120 °C, 130 °C and 140 °C) and with a sulfuric acid concentration of 5%, 10%, 15% and 20%.

[0166] In all these extractions, the amount of dipeptides obtained was evaluated, as well as the IC50 of urease inhibition. mg dipeptides / gram biomass

[0167] 5% 10% 15% 20%

[0168] 80 °C 14.8 16.5 19.3 20.1

[0169] 90 °C 18.2 24.9 26.1 24.1

[0170] 100 °C 25.4 29.6 29.5 19.2

[0171] 110 °C 26.3 30.1 27.4 14.8

[0172] 120°C 28.5 31.1 28.7 16.3

[0173] 130 °C 28.4 29.5 23.2 12.5

[0174] 140 °C 22.1 21.3 18.5 10.2

[0175]

[0176] IC50 (ppm hydrolyzed)

[0177] 5% 10% 15% 20%

[0178] 80 °C 49 84 142 1635

[0179] 90 °C 39 96 163 1845

[0180] 100 °C 45 97 214 1743

[0181] 110 °C 59 175 209 1940

[0182] 120 °C 124 186 341 2102

[0183] 130 °C 354 210 413 1780

[0184] 140 °C 421 754 936 2136

[0185]

[0186] As can be seen in the tables above, the use of iron(II) and copper(II) catalysts favors the synthesis of the dipeptides of interest at lower temperatures and sulfuric acid concentrations. Furthermore, it also favors obtaining a smaller IC50.

[0187] Thus, with the use of iron and copper, a maximum amount of dipeptides of interest is obtained and at the same time a very low IC50 is maintained under the following hydrolysis conditions: between 10% and 15% sulfuric acid and a temperature range of between 90 °C and 130 °C.

Claims

CLAIMS 1. Procedure for obtaining the self-polymerizing dipeptides Valine-Valine (Val-Val), Isoleucine-lsoleucine (lle-lle), Leucine-Leucine (Leu-Leu), Phenylalanine-Phenylalanine (Phe-Phe), Isoleucine-Valine (lle-Val, Val-lile), Isoleucine-Leucine (lle-Leu / Leu-lle), Isoleucine-Phenylalanine (lle-Phe / Phe-lle), Phenylalanine-Valine (Phe-Val / Val-Phe), Phenylalanine-Leucine (Phe-Leu / Leu-Phe) and Leucine-Valine (Leu-Val / Val-Leu), or any combination of said dipeptides, from the yeast Metschnikowia pulcherrima, which consists of the following steps: i. Fermentation of the yeast Metschnikowia pulcherrima in a culture medium consisting of dextrose, NH4SO4, KH2PO4, MgSO4, CaSÜ4, H3PO4 and antifoaming agent, at a temperature between 26 and 28 °C, at a pH between 4.5 and 5.5, adding between 1.5 and 3% by weight of KCI, to obtain a fermentation broth; i. Hydrolysis of the fermentation broth for 6-10 hours with sulfuric acid at a concentration of 7.5 to 15% by weight, at a temperature between 90 and 130 °C, in the presence of Cu (II) and Fe (II).

2. Process for obtaining the self-polymerizable dipeptides according to claim 1, wherein the yeast fermentation in step i) is carried out at a temperature of 27 °C, a pH of 5 with an amount of KCI of 2% by weight.

3. Process for obtaining the self-polymerizing dipeptides according to claim 1, wherein the hydrolysis of the fermentation broth of step i) is carried out with a sulfuric acid concentration of 10% by weight, at a temperature between 100 and 120 °C, with a Cu concentration 2+ between 0.5 and 2 g / liter of broth and a concentration of Fe 2+ between 1.0 and 2 g / liter of broth, for 8 hours.

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