Food additive and / or food supplement, production method, formulation of functional food and / or food supplement, use

Abstract

The present invention pertains to the field of biotechnology, nutrition and protein synthesis. More specifically, the invention discloses a food additive, the use thereof, a process for the solid-phase synthesis of a peptide providing a reduction in the generation of toxic by-products, a food and / or food supplement formulation comprising said additive and / or supplement. In one embodiment, said peptide is of SEQ ID NO: 1, Asp-Ile-Ile-Ala-Asp-Asp-Glu-Pro-Leu-Thr, or DIIADDEPLT, also known as Pep19 or WATACAP. The food additive and / or supplement of the invention comprises said peptide and provides essential amino acids that promote metabolic improvements in vertebrates, including humans and dogs.

Description

Descriptive Report of Invention Patent FOOD ADDITIVE AND / OR FOOD SUPPLEMENT, PROCESS OF OBTAINING, FORMULATION OF FUNCTIONAL FOOD AND / OR FOOD SUPPLEMENT, USE Field of Invention

[0001] The present invention is in the field of biotechnology, nutrition, and protein synthesis. More specifically, the invention discloses a food additive, its use, a process for the solid-phase synthesis of a peptide that reduces the generation of toxic byproducts, and a food and / or dietary supplement formulation comprising said additive and / or supplement. In one embodiment, said peptide is of SEQ ID NO: 1, Asp-Ile-Ile-Ala-Asp-Asp-Glu-Pro-Leu-Thr, or DIIADDEPLT, also known as Pep19 or WATACAP. The food additive and / or supplement of the invention comprises said peptide and provides essential amino acids that promote metabolic improvements in vertebrates, including humans and dogs. Background of the Invention

[0002] Dietary supplements are ways of providing nutrients in addition to a normal diet. A normal diet is often insufficient to provide all the nutrients the body needs; for example, deficiencies in certain amino acids are common. Of particular relevance in the context of this invention, essential amino acids are those not normally synthesized in the body (animal or human), despite being essential for its functioning.

[0003] Of the twenty common amino acids, the human body can synthesize only about half, making it essential to consume the remaining (essential) amino acids through food or supplements. The essential amino acids are phenylalanine, histidine, isoleucine, lysine, leucine, methionine, threonine, tryptophan and valine.

[0004] Because it is very difficult for an average human to know exactly the composition of each food, to determine whether all essential amino acids have been ingested, food additives or dietary supplements that provide some of the essential amino acids are often developed. In one aspect, the present invention provides an alternative food additive or nutritional supplement that provides some of the essential amino acids, as well as other non-essential amino acids.

[0005] On the other hand, obesity is a global epidemic. Conventional management strategies, such as diet and exercise, can be inconvenient and difficult to adhere to, and generally provide only short-term results. Bariatric surgery is another type of strategy, but it involves risk, pain, and high costs.

[0006] Some obesity control strategies involve the use of appetite-suppressing medications, such as amphetamines, but such approaches can cause undesirable side effects, such as blood pressure changes and / or depression, among others. In this context, it is highly desirable to develop an approach that is useful for obesity control but does not result in the problems known in the art. The present invention provides a solution to these problems.

[0007] The peptide used in the present invention (SEQ ID NO: 1), disclosed in document WO2014 / 008567 of the same applicant, comprises ten amino acids, in the following order: aspartic acid, isoleucine, isoleucine, alanine, aspartic acid, aspartic acid, glutamic acid, proline, leucine and threonine, that is, it contains 4 units of essential amino acid (two isoleucines, one leucine and one threonine).

[0008] The nature of the amino acids that make up the described peptide means that the conventional synthesis process produces toxic byproducts that are undesirable when used as food additives and / or dietary supplements. The present invention provides a solution to these problems.

[0009] The present invention is therefore useful in both humans and animals (notably domestic animals or pets, such as dogs), since the problem of lack of essential amino acid availability and / or excess weight, obesity, or abnormal adiposity is not yet adequately addressed with approaches known in the art. Furthermore, the peptide used requires an unconventional synthesis process to ensure that the peptide is substantially free of toxic byproducts and, therefore, particularly useful in food additives and / or dietary supplements.

[0010] In the search for the state of the art in scientific and patent literature, no documents were found anticipating or suggesting the teachings of the present invention, which has a very limited scope.

[0011] The closest prior art is document W02014008567 from the same applicant

[0012] From what can be inferred from the literature researched, no documents were found anticipating or suggesting the teachings of the present invention. Summary of the Invention

[0013] The present invention provides an improved food additive and / or food supplement that provides notable nutritional and functional advantages.

[0014] In one aspect, the functional food and / or dietary supplement of the invention provides 4 essential amino acid units, two of which are isoleucines, one leucine and one threonine.

[0015] In another aspect, the improved functional food and / or dietary supplement promotes fat loss in vertebrates, including humans and dogs. In one embodiment, administration of a functional food formulation of the invention to obese or overweight humans resulted in significant weight loss.

[0016] In another embodiment, administration of the food and / or food supplement of the invention to animals (rats and dogs) provided substantial reduction in adiposity. Consequently, the present invention also provides an alternative functional feed and / or dietary supplement for animals that provides surprising weight and / or adiposity reduction.

[0017] In one aspect, the improved functional food and / or dietary supplement of the invention comprises the peptide of SEQ ID NO: 1, or Asp-ll-ll-Ala-Asp-Asp-Glu-Pro-Leu-Thr (DIIADDEPLT), also known as WATACAP. In one embodiment, said peptide is free of or has a negligible amount of toxic byproducts, an aspect that in this embodiment results from the synthesis process described in the present invention, which has sensitive parameters modified to reduce or eliminate the toxic products.

[0018] In the process of the present invention, the reagents used are used in the synthesis of peptides and also the reagents are listed in the FDA's Food Ingredients and Packaging inventories as substances that can be added to foods.

[0019] An important technical aspect of the invention's process is the final deprotection and cleavage using a low concentration of hydrochloric acid. This approach effectively and gently removes the tert-butyl protecting groups from the peptide side chains. Furthermore, it cleaves the linker (trityl) from the sequence, releasing the peptide from the resin.

[0020] The use of HCl promotes a reaction that leads to the formation of free radicals, a common occurrence in peptide synthesis, due to the use of molecular acids such as trifluoroacetic acid. The process of the invention eliminates the need for scavengers such as EDT, thioanisole, and phenol, which are traditionally used in processes known in the art. The advantages of the process of the invention are particularly useful for the peptide of SEQ ID NO: 1, although they are not limited to it.

[0021] Therefore, the objects of the present invention are a food additive and / or food supplement, its use, an improved peptide synthesis process, a food, and an improved food formulation.

[0022] These and other objects of the invention will be immediately appreciated by those skilled in the art and will be described in detail below. Brief Description of the Figures

[0023] The following figures are presented:

[0024] Figure 1 shows data on the reduction in average body weight of human volunteers measured over time (in weeks) through a normal diet with the addition of the food and / or dietary supplement proposed by the invention at a dose of 5 mg / day. The trend line plotted represents the weight reduction over a 1-year period.

[0025] Figure 2 shows the results of tests on the administration of the food and / or dietary supplement of the invention in rats, showing the evolution of body weight over time with normal feeding with the addition of the food of the invention (600 mcg / kg.day, using the FDA recommendations for transposition of doses between rats and humans, corresponds to a dose of 5 mg / day in humans). The points on the graph represent the initial weight and throughout the study in % of the initial weight.

[0026] Figures 3a and 3b show the results of tests on the administration of the food and / or food supplement of the present invention in humans, showing the evolution of body weight over time with normal feeding with the addition of the food of the invention (dose of 10 mg / day in humans). The points on the graph represent the initial weight and throughout the study in % of the initial weight.

[0027] Figures 4a, 4b, and 4c show the results of a study conducted in a group of 10 individuals with an average BMI of 33.5 who received 10 mg / day of Pep19 for 30 days. Figure 4a presents the data on body weight reduction over 3 weeks (wk). Figure 4b presents the data on hip measurement reduction over 3 weeks. Figure 4c presents the data on waist measurement reduction over 3 weeks.

[0028] Figures 4d, 4e and 4f show the results of a study conducted in a group of 10 individuals with an average BMI of 33.5 who received 10 mg / day of Pep19 for 30 days. Figure 4d presents the data on body weight reduction over 4 weeks (wk). Figure 4e presents the data on hip measurement reduction over 4 weeks. Figure 4f presents the data on waist measurement reduction over 4 weeks.

[0029] Figures 4g, 4h, and 4i show the results of a study conducted on a subgroup of 7 individuals who lost circumference after 30 days. Figure 4g presents the data on body weight reduction over 4 weeks (wk). Figure 4h presents the data on hip measurement reduction over 4 weeks. Figure 4i presents the data on waist measurement reduction over 4 weeks.

[0030] Figure 5 shows the weight loss or gain data in relation to the dose (mcg / kg) used of the additive and / or food supplement of the present invention.

[0031] Figure 6 presents weight loss data in obese individuals in relation to dose (mcg / kg).

[0032] Figure 7 presents weight loss data in individuals with high BMI in relation to dose (mcg / kg).

[0033] Figure 8 shows an embodiment of the process of the invention, illustrated by a flowchart in which: 1) step in which the first deprotection occurs: to an amino acid AA1 bound to the resin (AA1-resin) piperidine is added, with five washing steps, obtaining NH2-AA1-resin; 2) the elongation or elongation step is done by adding AAn-COOH in DMF, with stirring for 40 minutes followed by 5 washing steps, resulting in AAn-AA1-resin; 3) piperidine is added followed by five washing steps, the process being able to return to step 2 according to the number of amino acids to be added to the peptide chain, always adding five washing steps between each cycle; 4) Final washing to obtain the peptide-resin; 5) cleavage and deprotection step, using HCIM for 24h, followed by drying, crystallization in ether and filtration and final drying to obtain of Pep19.

[0034] Figure 9 shows one of the effects of administering the product of the invention: the significant reduction of the enzyme alanine aminotransferase (ALT or TGP) in U / l in the blood in a group of 10 people with an average BMI of 33.5 who received 10 mg / day of Pep19 for 30 days.

[0035] Figure 10 shows one of the effects of administering the product of the invention: the change in weight from baseline to day 28. The average weight of the seven dogs that lost weight showed a statistically significant weight reduction (p<0.01).

[0036] Figure 11 shows one of the effects of administering the product of the invention: the change in body condition score from baseline to day 28. The mean body condition score (BCS) of the seven dogs that lost weight showed a statistically significant reduction (p<0.05). Detailed Description of the Invention

[0037] The present invention provides a food additive and / or food supplement, its use, an improved process of peptide synthesis, and an improved food formulation comprising said additive and / or supplement.

[0038] The food additive and / or dietary supplement of the invention and the formulation thereof are functional foods. More specifically, dietary supplements that, in addition to providing nutrition due to their composition comprising peptides / amino acids (a source of essential amino acids), also promote metabolic improvements in vertebrates, including humans and dogs.

[0039] In one aspect, the functional food and / or dietary supplement of the invention provides 4 essential amino acid units, two of which are isoleucines, one leucine and one threonine.

[0040] In another aspect, the formulation of improved functional food provides fat loss and / or weight reduction through simple ingestion.

[0041] The functional food and / or food supplement formulation of the invention comprises the peptide of SEQ ID NO 1, or Asp-Ile-Ile-Ala-Asp-Asp-Glu-Pro-Leu-Thr (DIIADDEPLT, also known as WATACAP or Pep19) and at least one food-acceptable excipient.

[0042] Excipients acceptable as food include diluents, fillers, binders, flow agents, acidulants, disintegrants, lubricants, among others.

[0043] Acceptable excipients may be selected from starch derivatives, calcium, salts, lactose, vegetable oils, sugar, cellulose, citric acid, malic acid, tartaric acid, aspartic acid, among others, or combinations thereof.

[0044] In one embodiment, said excipient is microcrystalline cellulose.

[0045] In another embodiment, said excipient includes wheat flour, rice flour, chickpea flour, canola oil, lemon zest, microcrystalline cellulose.

[0046] In one embodiment, said peptide is free of or has a negligible amount of toxic byproducts, which aspect in this embodiment results from the peptide synthesis process and has sensitive parameters modified to reduce or eliminate the toxic products.

[0047] The invention's process is a solid-phase synthesis process employing the well-established FMOC strategy. However, some sensitive parameters were modified in the invention, resulting in a surprising reduction or even elimination of toxic products inherent in the conventional FMPC route. This process is particularly applicable to the peptide of SEQ ID No. 1, due to the nature of its component amino acids.

[0048] Referring to Figure 8, there is an embodiment of the invention in which the process comprises the following steps: 1) piperidine is added to an amino acid AA1 bound to the resin (AA1-resin), with five washing steps, obtaining NH2-AA1-resin; 2) the elongation step is carried out by adding AAn-COOH in DMF, with stirring for 40 minutes followed by 5 washing steps, resulting in AAn-AA1-resin; 3) pyridine is added followed by five washing steps, the process being able to return to step 2 according to the number of amino acids to be added to the peptide chain, always adding five washing steps between each cycle; 4) Final washing to obtain the peptide-resin; 5) cleavage and deprotection step, using HCI for 24h, followed by drying, crystallization in ether and filtration and final drying to obtain Pep19.

[0013] In said embodiment, AA1 is threonine (Thr), AA2 is Leu, AA3 is Pro, AA4 is Glu, AA5 is Asp, AA6 is Ala, AA7 is Ile, AA8 is Ile, AA9 is Asp, so that the final peptide formed is Pep19 or SEQ 1 ID NO: 1.

[0049] The invention is also defined by the following clauses:

[0050] Process for the solid-phase synthesis of a peptide in which the final deprotection and cleavage steps employ a low concentration of hydrochloric acid.

[0051] Process as described above comprising the following steps: - piperidine is added to an amino-terminal amino acid AA1 bound to the resin (AA1-resin), followed by washing, obtaining NH2-AA1-resin; - the elongation step is carried out by adding AAn-COOH in DMF, with stirring followed by washing steps, resulting in AAn-AA1 -resin; - piperidine is added followed by washing, the process can return to step 2 according to the number of amino acids to be added to the peptide chain, always washing between each cycle; - Final wash to obtain the peptide-resin; - cleavage and deprotection step, using HCI, followed by drying, crystallization in ether and filtration and final drying to obtain the peptide of interest.

[0052] Process as described above wherein the peptide of interest is the peptide of SEQ ID NO: 1.

[0053] Process as described above in which the elongation step of the peptide chain comprises the following steps: - in a reaction vessel, suspend a resin in DCM; - stir with the contents of the reaction vessel and drain the DCM; - deprotect the amino acid on the resin by adding a piperidine solution in DMF, followed by stirring, draining and repeating this step; - wash the resin with DMF by stirring; - prepare the amino acid for coupling with molar excess in relation to the resin loading; - dissolve the amino acid in DCM until completely dissolved and add it to the dissolved amino acid DIC and stir; - add to the resin and stir; - drain the reaction medium and wash the resin with DMF while stirring; - to add the next amino acid, repeat the procedure from the deprotection step mentioned above; - when the peptide sequence is complete, remove the FMOC protecting group by performing the deprotection steps, and following them until washing with DMF, which must be repeated 3 or more times; and - wash the resin with methanol or equivalent solvent.

[0054] Process as described above in which the final cleavage and deprotection includes the following steps: - suspend the peptide-resin in a deprotection solution containing a mixture of hydrochloric acid in methanol and DCM; - Drain the liquid phase into a flask and evaporate the solution under vacuum until the final product is dry; - crystallize the solid product in diethyl ether and filter; and - vacuum dry.

[0055] In one embodiment of the process of the invention, the materials used include: Resin = Fmoc-Thr(Otbu)-2-Chlorotrimethyl resin A (polystyrene resin support with 1% divinylbenzene); D / C = N,N'-Diisopropylcarbodiimide; DCM = Dichloromethane; DMF = N,N-Dimethylformamide; HOBt = 1-Hydroxybenzotriazole hydrated; Molar HCI3 = hydrochloric acid.

[0056] Procedure for peptide chain elongation involves the following steps and conditions:

[0057] 1. Weigh an appropriate amount of resin. For example, 1 g of resin with a loading of 0.5 mmol / g;

[0058] 2. Transfer the resin to the reaction vessel and suspend it in DCM to promote swelling. For the amount of resin in the example, add 10 to 15 ml;

[0059] 3. Stir the contents of the reaction vessel with the resin for 30 minutes;

[0060] 4. Drain the DCM;

[0061] 5. Deprotect the amino acid on the resin by adding a 20% piperidine solution in DMF. In the example, you can add ml of piperidine;

[0062] 6. Stir for 7 minutes, drain and repeat the operation from the previous item;

[0063] 7. Wash the resin with DMF 5 times, shaking it for 1 minute each time. Wash with 10 to 15 ml at a time for the example;

[0064] 8. Prepare the amino acid for coupling by weighing 3 to 5 times the molar excess in relation to the resin loading;

[0065] 9. Dissolve the amino acid in enough DCM so that the concentration is between 0.3 and 0.2 mol / L. If the amino acid does not dissolve, add drops of DMF until it is completely dissolved;

[0066] 10. Add dissolved amino acid 1 equivalent of DIC and stir for about 2 minutes;

[0067] 11. Add to the resin and stir for about 40 minutes;

[0068] 12. Drain the reaction medium and wash the resin 5 times with DMF, shaking it for 1 minute each time. Wash with 10 to 15 ml at a time.

[0069] 13. To add the next amino acid, the procedure must be repeated from item 5. As soon as the amino acid aspartic acid is present in the peptide-resin, the 20% piperidine solution must be replaced in item 5 with 0.5 molar HOBt, to avoid the formation of aspartimide.

[0070] 14. When the sequence is complete, remove the protecting group FMOC executing items 5 to 7.

[0071] 15. Wash the resin with DMF 3 times, shaking it for 1 minute each time. Wash with 10 to 15 ml at a time for the example.

[0072] 16. Wash the resin with methanol 3 times, shaking it for 1 minute each time. Wash with 10 to 15 ml at a time for the example.

[0073] Procedure for final cleavage and deprotection:

[0074] 1. Prepare the deprotection solution by adding 2 parts of 3 molar hydrochloric acid in ethanol with 1 part of DCM.

[0075] 2. Suspend the peptide-resin in the deprotection solution and leave it under stirring for 24 hours at room temperature (20-28oC).

[0076] 3. Drain the liquid phase into a suitable bottle.

[0077] 4. Evaporate the solution under vacuum until the final product is dry.

[0078] 5. Crystallize the solid product from diethyl ether and filter.

[0079] 6. Dry the product under vacuum.

[0080] The surprising results presented in this patent application motivated the inventors to investigate possible mechanisms of action. In this patent application, the potential mechanisms of action are not the subject of the invention nor do they limit its scope, but they deserve comment.

[0081] On the one hand, the test results seem to suggest that the action of the peptide (WATACAP) as a food is at least partially related to some of the same mechanisms as thermogenesis, which is a normal response to cold that converts brown fat to white fat and leads to remodeling and loss of body fat.

[0082] Of particular relevance to the present invention is that feeding the food formulation containing the peptide (WATACAP), unlike anorectic agents, demonstrated no effect on the brain and no effect on food consumption. The effects associated with feeding the peptide WATACAP include attenuation of postprandial glycemia, reduction in serum triglyceride levels, and liver function. These effects are conceptually associated with the reversal of phenotypes linked to insulin resistance that, in In humans, it is the most common result of chronic and excessive carbohydrate consumption. In other words, feeding the peptide WATACAP apparently increases insulin sensitivity and promotes several metabolic and morphometric changes.

[0083] It is already known that the WATACAP peptide interacts with the cannabinoid receptor 1 (in the body, but not in the brain), a receptor conserved throughout mammalian evolution. This fact suggests that the physiological effects may also be conserved among mammals. Indeed, this has been demonstrated in at least two distinct species in this patent application. In this context, the invention provides a completely new strategy for managing conditions related to insulin resistance, including obesity, in mammals such as humans, dogs, cats, rats, and horses, since all suffer from conditions related to insulin resistance.

[0084] In one embodiment of the present invention, the effects of feeding humans a food formulation containing the peptide WATACAP were preliminarily tested to determine whether such an approach could reverse obesity in humans and, thus, obtain data for designing a future, more comprehensive clinical trial. The results were completely surprising, particularly due to the large number of positive results with no side effects detected.

[0085] Also noteworthy is the unexpected reports of improved sleep in some respondents, as it is known that insulin resistance tends to be linked to insomnia. Facial pimples also disappeared, which may be due to changes in body fat content. This observation is consistent with, or corroborates, one of the likely mechanisms of action mentioned above.

[0086] The invention is also defined by the following clauses.

[0087] Food additive and / or food supplement, comprising: the peptide of SEQ ID NO 1; and one or more food-acceptable excipients.

[0045] Additive as described above wherein said excipient is selected from diluents, fillers, binders, flow agents, acidulants, disintegrants, lubricants, among others, or combinations thereof.

[0088] Acceptable excipients may be selected from starch derivatives, calcium, salts, lactose, vegetable oils, sugar, cellulose, citric acid, malic acid, tartaric acid, aspartic acid, among others, or combinations thereof.

[0089] In one embodiment, said excipient is microcrystalline cellulose.

[0090] In another embodiment, said excipient includes flour, rice flour, chickpea flour, canola oil, lemon zest.

[0091] Additive as described above in which said peptide is obtained by solid phase synthesis, comprising final deprotection and cleavage steps using a low concentration of hydrochloric acid.

[0092] Additive as described above wherein: - the step in which an amino acid AA1 bound to the resin (AA1-resin) is added with piperidine, with five washing steps, obtaining NH2-AA1-resin; the elongation step is done by adding AAn-COOH in DMF, with stirring for 40 minutes followed by 5 washing steps, resulting in AAn-AA1-resin; the piperidine addition step is followed by five washing steps; and / or - the peptide chain elongation step comprises the following steps: in a reaction vessel, suspend a resin in DCM; stir with the contents of the reaction vessel and drain the DCM; deprotect the amino acid on the resin by adding a piperidine solution in DMF, followed by stirring, draining, and repeating this step; wash the resin with DMF by stirring; prepare the amino acid for coupling with a molar excess in relation to the resin loading; dissolve the amino acid in DCM until completely dissolved and add it to the dissolved amino acid DIC and stir; add it to the resin and stir; drain the reaction medium and wash the resin with DMF while stirring; to add the next amino acid, repeat the procedure from the deprotection step mentioned above; when the peptide sequence is complete, remove the group FMOC protector, performing the deprotection steps, and following until washing with DMF, which must be repeated 3 or more times; and washing the resin with methanol or equivalent solvent / and / or - the final cleavage and deprotection step includes the following steps: suspend the peptide-resin in a deprotection solution containing a mixture of hydrochloric acid in methanol and DCM; drain the liquid phase into a flask and evaporate the solution under vacuum until the final product is dry; crystallize the solid product in diethyl ether and filter; and dry under vacuum.

[0093] Functional food formulation comprising: the peptide of SEQ ID NO 1; and one or more acceptable excipients.

[0094] Formulation as described above presented in the form of an oral capsule containing microcrystalline cellulose as an excipient.

[0095] Formulation as described above comprising: the peptide of SEQ ID NO 1; and one or more acceptable excipients, wherein said peptide is obtained by solid phase synthesis, comprising final deprotection and cleavage steps using a low concentration of hydrochloric acid.

[0096] Use of the peptide of SEQ ID NO: 1 for the preparation of a food additive or a food or dietary supplement formulation.

[0097] Use as described above in which the said peptide is obtained by solid phase synthesis, comprising final deprotection and cleavage steps using a low concentration of hydrochloric acid.

[0098] Process for preparing a food formulation comprising the steps of mixing a peptide of SEQ ID NO 1 with one or more food-acceptable excipients.

[0099] The examples described below illustrate some of the ways in which the invention can be carried out, but are not intended to limit its scope.

[0100] Example 1 - Peptide preparation process

[0101]

[0059] The peptide synthesis process is in solid phase, employing the well-established FMOC strategy.

[0102] Referring to figure 8, we have an embodiment of the invention in which the process comprises the following steps:

[0103] 1) piperidine is added to an amino acid AA1 (threonine or Thr) bound to the resin (AA1-resin), with five washing steps, obtaining NH2-AA1-resin;

[0104] 2) the elongation step is performed by adding AAn-COOH in DMF, with stirring for 40 minutes followed by 5 washing steps, resulting in AAn-AA1-resin;

[0105] 3) piperidine is added followed by five washing steps, the process being able to return to step 2 according to the number of amino acids to be added to the peptide chain, always adding five washing steps between each cycle;

[0106] 4) Final wash to obtain the peptide-resin;

[0107] 5) cleavage and deprotection step, using HCIM for 24h, followed by drying, crystallization in ether and filtration and final drying to obtain Pep19.

[0108] In said embodiment, AA1 is threonine (Thr), AA2 is Leu, AA3 is Pro, AA4 is Glu, AA5 is Asp, AA6 is Ala, AA7 is Ile, AA8 is Ile, AA9 is Asp, so that the final peptide formed is the final peptide formed is Pep19 or WATACAP or SEQ ID NO: 1.

[0109] In one embodiment of the aforementioned process: the step in which an amino acid AA1 bound to the resin (AA1-resin) is added with piperidine, with five washing steps, obtaining NH2-AA1-resin; the elongation or elongation step is done by adding AAn-COOH in DMF, with stirring for 40 minutes followed by 5 washing steps, resulting in AAn-AA1-resin; the piperidine addition step is followed by five washing steps.

[0110] The final deprotection and cleavage steps of the aforementioned process for solid-phase synthesis of a peptide employ a low concentration of hydrochloric acid.

[0111] Process as described above comprising the following steps: to an AA1 amino acid bound to the resin (AA1-resin) is added piperidine, followed by washing, obtaining NH2-AA1 -resin; the elongation step is carried out by adding AAn-COOH in DMF, with stirring followed by washing steps, resulting in AAn-AA1-resin; piperidine is added followed by washing, the process being able to return to step 2 according to the number of amino acids to be added to the peptide chain, always carrying out washing between each cycle; Final wash to obtain the peptide-resin; cleavage and deprotection step, using HCI, followed by drying, crystallization in ether and filtration and final drying to obtain Pep19.

[0112] In one embodiment of the aforementioned process: the peptide chain elongation step comprises the following steps: - in a reaction vessel, suspend a resin in DCM; - stir with the contents of the reaction vessel and drain the DCM; - deprotect the amino acid on the resin by adding a piperidine solution in DMF, followed by stirring, draining and repeating this step; wash the resin with DMF by stirring; - prepare the amino acid for coupling with molar excess in relation to the resin loading; - dissolve the amino acid in DCM until completely dissolved and add it to the dissolved amino acid DIC and stir; - add to the resin and stir; - drain the reaction medium and wash the resin with DMF while stirring; - to add the next amino acid, repeat the procedure from the deprotection step mentioned above; - when the peptide sequence is complete, remove the FMOC protecting group by performing the deprotection steps, and following them until washing with DMF, which must be repeated 3 or more times; and - wash the resin with methanol or equivalent solvent.

[0113] In one embodiment of the aforementioned process: the cleavage stage and final deprotection includes the following steps: - suspend the peptide-resin in a deprotection solution containing a mixture of hydrochloric acid in methanol and DCM; - drain the liquid phase into a flask and evaporate the solution under vacuum until the final product is dry; - crystallize the solid product in diethyl ether and filter; and dry under vacuum.

[0114] In one embodiment of the process of the invention, the materials used include: Resin = Fmoc-Thr(Otbu)-2-Chlorotrytil resin (polystyrene resin support with 1% divinylbenzene); D / C = N,N'-Diisopropylcarbodiimide; DCM = Dichloromethane; DMF = N,N-Dimethylformamide; HOBt = 1-Hydroxybenzotriazole hydrate; 3 molar HCl = hydrochloric acid.

[0115] Procedure for peptide chain elongation involves the following steps and conditions:

[0116] 1. Weigh an appropriate amount of resin. For example, 1 g of resin with a loading of 0.5 mmol / g;

[0117] 2. Transfer the resin to the reaction vessel and suspend it in DCM to promote swelling. For the amount of resin in the example, add 10 to 15 ml;

[0118] 3. Stir the contents of the reaction vessel with the resin for 30 minutes;

[0119] 4. Drain the DCM;

[0120] 5. Deprotect the amino acid on the resin by adding a 20% piperidine solution in DMF. In the example, you can add ml of piperidine;

[0121] 6. Stir for 7 minutes, drain and repeat the operation from the previous item;

[0122] 7. Wash the resin with DMF 5 times, shaking it for 1 minute each time. Wash with 10 to 15 ml at a time for the example;

[0123] 8. Prepare the amino acid for coupling by weighing 3 to 5 times the molar excess in relation to the resin loading;

[0124] 9. Dissolve the amino acid in enough DCM so that the concentration is between 0.3 and 0.2 mol / L. If the amino acid does not dissolve, add drops of DMF until it is completely dissolved;

[0125] 10. Add 1 equivalent of DIC dissolved amino acid and stir for about 2 minutes;

[0126] 11. Add to the resin and stir for about 40 minutes;

[0127] 12. Drain the reaction medium and wash the resin 5 times with DMF, shaking it for 1 minute each time. Wash with 10 to 15 ml at a time.

[0128] 13. To add the next amino acid, the procedure must be repeated from item 5. As soon as the amino acid aspartic acid is present in the peptide-resin, the 20% piperidine solution must be replaced in item 5 with 0.5 molar HOBt, to avoid the formation of aspartimide.

[0129] 14. When the sequence is complete, remove the FMOC protecting group by performing steps 5 through 7.

[0130] 15. Wash the resin with DMF 3 times, shaking it for 1 minute each time. Wash with 10 to 15 ml at a time for the example.

[0131] 16. Wash the resin with methanol 3 times, shaking it for 1 minute each time. Wash with 10 to 15 ml at a time for the example.

[0132] Procedure for final cleavage and deprotection:

[0133] 1. Prepare the deprotection solution by adding 2 parts of 3 molar hydrochloric acid in ethanol with 1 part of DCM.

[0134] 2. Suspend the peptide-resin in the deprotection solution and leave it under stirring for 24 hours at room temperature (20-28oC).

[0135] 3. Drain the liquid phase into a suitable bottle.

[0136] 4. Evaporate the solution under vacuum until the final product is dry.

[0137] 5. Crystallize the solid product from diethyl ether and filter.

[0138] 6. Dry the product under vacuum.

[0139] Example 2 - Formulation of functional food and its preparation.

[0140] The improved functional food formulation of the invention comprises the peptide of SEQ ID NO: 1, or Asp-Ile-Ile-Ala-Asp-Asp-Glu-Pro-Leu-Thr (DIIADDEPLT, also known as WATACAP - CAS number 1536481 -46-5) and at least one acceptable excipient. In the present example, prepared oral capsules containing around 300 mg of microcrystalline cellulose as an excipient and 5 milligrams of WATACAP.

[0141] Example 3 - Use to prepare functional food and / or dietary supplement and its consumption by humans.

[0142] In this embodiment, a single-arm, open-label study was conducted with fourteen adults in the United States / Miami. None of the volunteers were pregnant. The food and / or dietary supplement formulation of the invention was administered by taking a capsule as described in Example 1, at night before bed.

[0143] Body weight was defined as an indirect marker of body fat, and each participant's weight was assessed before the study began and during weeks 1 to 4. Each participant was questioned about health status, medication use, and observation of effects. Subgroups were defined according to body mass index (BMI) classification: normal weight BMI <25 (n=3); overweight BMI between 25 and 30 (n=6); and obese BMI>30 (n=5). Volunteer participants included nine women and five men, aged 21 to 81 years (median = 53) and with a baseline BMI between 19 and 36 (mean 28). Twelve of the participants identified as White Hispanic and two identified as White Anglo. On a normalized basis, volunteers in the normal weight, overweight, and obese groups consumed, respectively, 77, 74, and 54 micrograms of peptide per kilogram per day.Statistical analyses were performed using a two-way ANOVA test with Tu key post-test (Graph Pad Software).

[0144] As a result, all participants survived and answered the questions asked, leaving no data gaps in the study. One participant reported bloating, one reported stomach pain, and two reported improved sleep.

[0145] The results of the weight measurements indicated that weights fluctuated, with overall weight loss. In the normal weight and overweight groups, the change was not significant. On the other hand, in the obese group, the reduction weight loss was significant, approximately 0.45 kilograms per week over four weeks (Figure 1). In the obese group, the differences between the initial weight and after four weeks of ingesting the functional food formulation of the invention showed a trend towards weight loss with statistical significance (p=0.06; Figure 3A, 3B).

[0146] Referring to the figures in more detail, Figure 1 shows the results of tests on the administration of the invention's food formulation to human volunteers. The evolution of body weight over time is shown with a normal diet and intake of the invention's food formulation (5 mg / day). The points on the graph represent the average initial weight and weight over the course of the study. Among the participants in the normal weight subgroup (BMI <25) and the overweight subgroup (BMI 25-30), no obvious patterns were observed, and these were not plotted on the graph. On the other hand, all in the obese group (BMI >30) lost weight, an average of 0.45 kg per week (p=0.06).These data, projecting the results (black arrow) for one year of feeding with the ingestion of the food formulation and / or food supplement of the invention in the obese group, indicate that the expected average weight reduction is approximately 48 kg per person, a very substantial value considering that in the experiment there was no restriction on the diet.

[0147] Figure 3b shows the results of a study of fourteen adults who received a fixed daily dose of 5 mg of Watacap and were followed for four weeks. In the non-obese (BMI < 25) and overweight (BMI 25-30) subgroups, there was no statistically significant difference between baseline and four-week weight. In an obese subgroup (BMI > 30), all individuals lost weight; the average weight loss was 0.45 kg per week (p=0.06). In the obese subgroup, the difference between baseline and four-week weight approached statistical significance (p=0.06). These observations demonstrate that Watacap causes fat loss in obese humans.

[0148] A significant reduction in measurements (waist and hip) and at the end of the fourth week a significant reduction in body weight (figures 4b, 4c and 4a, respectively).

[0149] In a sample of ten individuals (n=10, Israel), after administering 10 mg of the peptide, 46% reduced hip and waist measurements, 23% reduced both body weight and waist and hip measurements; 8% reduced only body weight and 8% reduced only waist measurements; and 15% showed no significant changes. A 3% reduction in waist and hip circumference was observed (n=10, Israel). There was no change in weight on average for the group (Figure 9). These observations are consistent with the observation that fat can remodel before weight loss.

[0150] The results surprised the inventors, notably because of their magnitude. The data suggest that the association between WATACAP consumption and body fat varies according to initial weight, as no statistically significant weight change was detected in normal-weight or overweight individuals, but only in obese individuals.

[0151] On the other hand, it should also be considered that the experimental design used, and the dosage of the WATACAP peptide, varied between the different groups: on a normalized basis, volunteers in the normal-weight, overweight, and obese groups consumed, respectively, 77, 74, and 54 micrograms of peptide per kilogram per day. The lower normalized dose in the obese group could be surprisingly responsible for the greater effect—this model could be explained by the fact that some peptides exhibit an inverted-U dose-response curve, in which higher doses paradoxically produce less effect. In this hypothesis, reducing the dose of the WATACAP peptide could lead to weight loss effects in the overweight group, which will be studied in future trials.

[0152] Example 4

[0153] In this embodiment, a study was conducted on a group of 10 people with an average BMI of 33.5 who received 10 mg / day of Pep19 for 30 days.

[0154] Table i shows the reduction in body weight over 4 weeks, represented in a graph in figure 4a.

[0155] Table 1 - Weight in kg before and after starting product administration. This study was conducted with the population of Israel who received 10 mg per day for 30 days.

[0156] Table 2 shows the reduction in waist measurement over 4 weeks, represented in a graph in figure 4b.

[0157] Table 2 - Waist measurement in cm before and after starting product administration. This study was conducted with the Israeli population who received 10 mg per day for 30 days.

[0158] Table 3 shows the reduction in hip measurement over 4 weeks, represented in a graph in figure 4c.

[0159] Table 3 - Hip measurement in cm before and after starting product administration. This study was conducted with the Israeli population who received 10 mg daily for 30 days.

[0160] The results presented show a significant reduction in body weight and measurements (waist and hip) at the end of the fourth week. These surprising results suggest that the association between WATACAP consumption and body fat varies according to initial weight, with obese individuals experiencing more significant reductions in body weight and measurements.

[0161] In a subgroup of 7 participants who lost circumference, it was observed that weight loss occurred after circumference loss (figures 4g, 4h and 4i). Example 5 - Administration of feed additive to animals.

[0162] Figure 2 shows the results of tests on the administration of the food additive and / or food supplement of the invention to rats, showing the evolution of body weight over time with normal feeding plus the addition of the food additive of the invention (600 mcg / kg day). The points on the graph represent the initial weight (black) and the weight throughout the study (gray). Data analysis indicates that feeding the peptide WATACAP at a dose of 600 micrograms per kilogram per day reduces adiposity (Table 1), with a statistically significant difference in weight in the group of heavy animals (panel A; p<0.05). On the other hand, no statistically significant difference in weight was observed in the groups of light or medium-weight animals.

[0163] The results are consistent when comparing data from humans and rats, which allows generalization to other vertebrates, particularly mammals.

[0164] However, adjustments to the experimental model in future clinical trials are recommended to overcome the limitations of the study (number of participants, ethnicity, low number of doses, duration of only four weeks).

[0165] It should also be noted that even though there was no placebo group in this preliminary experimental design, there was significant weight loss in all individuals in the obese group, while in the normal-weight and overweight groups, the reduction was not statistically significant. Taken together, the results in the obese group are most likely real and not placebo-like, since if it were a placebo effect, it would affect participants in all groups, which was not the case.

[0166] Example 6 - Formulation of cookies with the peptide.

[0167] The functional food and / or dietary supplement formulation may be prepared in the form of biscuits comprising the peptide of SEQ ID NO: 1, or Asp-Ile-Ile-Ala-Asp-Asp-Glu-Pro-Leu-Thr (DIIADDEPLT, also known such as WATACAP - CAS number 1536481-46-5) and at least one acceptable excipient. In the present example, cookies were prepared containing 5 mg of WATACAP, 50% wheat flour or rice flour mixed with chickpea flour, 25% sugar, 25% canola oil and lemon zest.

[0168] The product showed stability and pleasant flavor, and can be used as a functional food.

[0169] Example 7 - Reduction of the ALT enzyme present in the blood.

[0170] In this embodiment, a study was conducted on a group of 10 people with an average BMI of 33.5 who received 10 mg / day of Pep19 for 30 days. Figure 9 demonstrates a significant reduction in the enzyme alanine aminotransferase (ALT or SGPT) in U / L in the blood in a group of 10 people with an average BMI of 33.5 who received 10 mg / day of Pep19 for 30 days. When liver cells are damaged, they release ALT into the bloodstream. Elevated ALT levels in the blood can be a sign of liver damage or disease. Some types of liver disease cause elevated ALT levels before the individual shows symptoms of the disease. Therefore, an ALT blood test can help diagnose certain liver diseases early. The ALT reduction was surprising.

[0171] Example 8 - Consumption of functional food and / or dietary supplement by dogs.

[0172] In this embodiment, a study was conducted on a group of eight healthy, neutered, university-owned adult Beagles (4 females and 4 males) who were individually housed indoors and fed an extruded kibble at a rate historically associated with weight stability. Each puppy was administered Pep19 (5 mg / puppy / day, 2.06–3.14 mg / kg / day) as a single oral dose daily for 28 days. Health screening, including physical examination, body weight, body condition score (BCS), complete blood count, chemistry profiles, total thyroxine, thyroid-stimulating hormone, and urinalysis, were collected at baseline, day 14, and day 28. Fecal score, The animal's appetite, temperament, and general condition were assessed daily. At baseline, the mean age, weight, and BCS were 3.8 ± 0.3 years, 12.7 ± 2.1 kg, and 6.4 ± 0.7 / 9, respectively, as can be seen in Table 4 below:

[0173] Table 4: Age, sex, weight and body condition score (BCS) of dogs on days 0, 14 and 28. NM, castrated male. SF, spayed female.

[0174] The intervention consisted of 5 milligrams of Pep19 (Proteimax Bio Technology Israel) formulated as a microcrystalline cellulose excipient in cellulose-based vegetarian capsules. Each dog was fed its The animals were fed a daily dose of feed and received Pep19 as an orally administered capsule, covered by a small portion of a canned diet in the morning. Weight was assessed on days 0, 14, and 28 at the same time of day by the same technician (JF); BCS was assessed on days 0, 14, and 28 by a senior veterinarian (MM, AWR). Data were collected and analyzed using two-way ANOVA with Tukey's multiple comparisons test using GraphPad Prism (GraphPad Software, www.graphpad.com).

[0175] The Pep19 product was well accepted by all dogs. No adverse effects were detected, and all blood and urine tests remained normal. Despite no changes in diet or calorie intake, seven of the eight dogs lost between 0.7% and 3.8% of their initial body weight by day 28, with a weekly weight loss rate of 0.2% to 1.0%. The decrease in mean weight was statistically significant (p<0.01; Figure 10). This was accompanied by a statistically significant reduction in mean BCS (p<0.05; Figure 11).

[0176] In this embodiment, the Pep19-containing product was demonstrated to be well-tolerated and safe for dogs. The Pep19-containing product was administered in a manner associated with historical weight stability, and surprisingly, Pep19 was associated with a measurable reduction in weight and BCS within 28 days. Typical energy-restricted weight-loss programs in dogs aim to maintain a weight loss rate of 1-2% per week (Mariana YH Porsani et al., 2020). Dogs in this trial that lost weight while fed their typical diet at a maintenance calorie level achieved a weekly weight loss rate of 0.2-1%. In this embodiment, the weight and BCS data demonstrated an association between Pep19 and a reduction in weight and fat turnover, similar to the effects observed in rodents and humans.Thus, this essay suggests a new approach to canine weight management using Pep19 as a functional supplement or feed additive. The experts. in the art will value the knowledge presented here and will be able to reproduce the invention in the modalities presented and in other variants and alternatives, covered by the scope of the following claims.

Claims

1. Food additive and / or dietary supplement characterized in that it comprises:- peptide of SEQ ID NO: 1; and- at least one food-acceptable excipient.

2. Additive, according to claim 1, characterized in that the peptide is in a concentration of around 1%.

3. Additive, according to claim 1, characterized in that the excipient is selected from diluents, fillers, binders, flow agents, acidulants, disintegrants, lubricants, or combinations thereof.

4. Additive, according to any of claims 1 or 3, characterized in that the excipient is microcrystalline cellulose.

5. Process for obtaining a peptide via solid-phase synthesis, characterized in that it comprises final deprotection and cleavage steps using a low concentration of hydrochloric acid.

6. Process, according to claim 5, characterized in that it comprises the following steps:a) add piperidine to the amino-terminal amino acid attached to the resin (AA1-resin), followed by washing, obtaining NH2-AA1-resin;b) perform an elongation step by adding AAn-COOH in DMF, with stirring followed by washing steps, resulting in AAn-AA1-resin;c) add piperidine, followed by washing, the process may return to step b depending on the number of amino acids to be added to the peptide chain, always performing washes between each cycle;d) perform a final washing step to obtain the peptide-resin; ande) perform a cleavage and deprotection step using HCl, followed by drying, crystallization in ether, filtration, and final drying to obtain the peptide of interest.

7. Process, according to claim 5 or 6, characterized in that said peptide is the peptide of SEQ ID NO: 1.

8. Process, according to claim 7, characterized in that it comprises the steps of:a) adding piperidine to the amino acid AA1 linked to the resin (AA1-resin), with five washing steps, obtaining NH2-AA1-resin;b) performing an elongation step by adding AAn-COOH in DMF, stirring for 40 minutes followed by 5 washing steps, resulting in AAn-AA1-resin;c) adding piperidine, followed by five washing steps, the process can return to step b depending on the number of amino acids to be added to the peptide chain, always adding five washing steps between each cycle.d) performing a final washing step to obtain the peptide-resin; ande) performing a cleavage and deprotection step using 4M HCl for 24 hours, followed by drying, crystallization in ether, filtration, and final drying to obtain the peptide of SEQ ID NO: 1.

9. Process, according to any of claims 5 to 8, characterized in that the elongation step of the peptide chain comprises the following steps:- in a reaction vessel, suspend a resin in DCM;- stir with the contents of the reaction vessel and drain the DCM;- deprotect the amino acid from the resin by adding a piperidine in DMF solution, followed by stirring, draining, and repeating this step;- wash the resin with DMF while stirring;- prepare the amino acid for coupling with molar excess relative to the resin loading;- dissolve the amino acid in DCM until total dissolution, add it to the dissolved amino acid DIC and stir.- add to the resin and stir;- drain the reaction medium and wash the resin with DMF while stirring;- to add the next amino acid, repeat the procedure starting from the deprotection step mentioned above;- when the peptide sequence is complete, remove the FMOC protective group by performing the deprotection steps, and subsequent steps up to washing with DMF, which should be repeated 3 or more times; and- wash the resin with methanol or an equivalent solvent.

10. Process, according to any of claims 5 to 9, characterized in that the final cleavage and deprotection step includes the following steps:- suspend the peptide-resin in a deprotection solution containing a mixture of hydrochloric acid in methanol and DCM;- drain the liquid phase into a flask and evaporate the solution under vacuum until the final product is dry;- crystallize the solid product in diethyl ether and filter it; and- vacuum drying.

11. Food additive comprising:- peptide of SEQ ID NO: 1; and- at least one acceptable excipient,characterized in that said peptide is obtained by a solid-phase synthesis process, comprising final deprotection and cleavage steps using a low concentration of hydrochloric acid.

12. Additive, according to claim 11, characterized in that it is obtained by a process as described in any of the claims 5 to 10.

13. Formulation of functional food and / or dietary supplement, characterized in that it comprises:- peptide of SEQ ID NO: 1; and- at least one acceptable excipient.

14. Formulation, according to claim 13, characterized in that it is presented in the form of an oral capsule containing microcrystalline cellulose as an excipient.

15. Formulation of functional food, characterized in that it comprises:- peptide of SEQ ID NO: 1; and- at least one acceptable excipient,wherein said peptide is obtained by the process described in one of claims 5 to 10.

16. Use of the peptide of SEQ ID NO: 1, characterized in that it is for preparing a food additive and / or dietary supplement or a food formulation.

17. Use, according to claim 16, characterized in that said peptide is obtained by solidphase synthesis, comprising final deprotection and cleavage steps using a low concentration of hydrochloric acid.

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