BIOCONJUGATE MOLECULES WITH BIOLOGICAL AND TECHNO-FUNCTIONAL ACTIVITY, METHOD FOR THE PRODUCTION THEREOF AND USE THEREOF

Abstract

The present invention regards the synthesis of bioconjugated molecules, formed between two or more of the following functional groups: sugars, prebiotics, oligosaccharides, polysaccharides, triglycerides, fatty acids, fatty acids esters, anti-inflammatories; with its production process by biocatalyzed synthesis with hydrolases such as esterases, proteases, lipases or cutinases, and its purification with several methods that include washing and drying. In addition, its applications in foods, pharmaceuticals and cosmetics, such as: prebiotic nutraceutical, anti-inflammatory, antitumoral, intestinal vector, techno-functional ingredient for food applications (emulsifier, fat substitute) and cosmetic emollient; which are possible since these are non toxic molecules according to the Ames tests.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. A process for the production of bioconjugated molecules with biological and techno-functional activities with a weight ratio of 1:1 to 1:10 of sugars, oligosaccharides or branched polysaccharides: carboxylic acid or its derivative, the process including the steps of: a. producing a reaction by mixing on a hermetically closed recipient with agitation at 100-1000 r.p.m. and incubation at 40-80 C., two substrates according to claim 1 in a proportion of 1:1 to 1:10 w/w, in presence of an enzyme in a proportion of 1:1 to 1:10 w/w to the acylating agent; with or without solvent that if it is added is in a ratio of 1:2 to 1:100 w/v; b. filtering with Whatman filters numbers 1,3,4, or similar, or centrifuge for separation of the organic solvent and the immobilized enzyme; c. recovering the bioconjugates from the organic phase by solvent evaporation at reduced pressure (vacuum) in a rotary evaporator, heating at a temperature where the solvent boils depending on the vacuum used, or by heating at a temperature above the solvent boiling point; and d. recovering the bioconjugates from the solid phase by washing with a hydrophilic solvent in a ratio of 1:2 to 1:15 v/v and further evaporation as in step 4c.

5. The process according to claim 4, wherein the enzyme from the step 4a is a serine-hydrolase that is immobilized, the enzime selected from the group consisting of a protease, lipase, esterase, and cutinase.

6. The process according to claim 4, wherein in the step 4a molecular sieves, zeolites, clays or other water absorbents are added in a ratio of 1:1 to 1:5 w/w to the acylating agent.

7. The process according to claim 4, further including a drying step comprised by a hydrophilic solvent wash in a ratio of 1:2 to 1:15 v/w, followed by evaporation according to step 4c, with an optional drying under nitrogen, followed by freezing at 5 to 80 C. and lyophilization.

8. The process according to claim 4, further including a purification step to eliminate unreacted sugars and acylating agent, and includes using a diluted alkali in a concentration of 0.1 to 1N, or water in a ratio of 1:1 to 1:20 v/v with respect to the organic phase; recovering the purified bioconjugates in the interphase, organic phase and aqueous phase respectively, after drying according to step 4c or the process from claim 7.

9. The process according to claim 4, further including an agitation step, the agitation step is selected from the group consisting of manual, mechanic, magnetic, orbital, by vibrations, thermic or passive diffusion agitation.

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention regards the synthesis of bioconjugated molecules formed between two or more of the following functional groups: prebiotics, triglycerides, fatty acids, sugars, anti-inflammatories; with its production process (synthesis and purification); and its applications as prebiotic nutraceutical, anti-inflammatory, antitumoral, intestinal vector, techno functional ingredient for food applications as emulsifier and fat substitute, and cosmetic; which are possible since these are non toxic molecules. The characteristic details of these molecules and the production process are clearly shown in the following description and Figures, which are mentioned as examples and should not be considered to limit the scope of the present invention:

[0013] FIG. 1 shows a scheme of a general process for producing and obtaining bioconjugates according to the present invention;

[0014] FIG. 2 shows a scheme of an optional drying process for trace water and solvent elimination of the bioconjugates;

[0015] FIG. 3 shows a scheme of an optional purification process with diluted alkali for the elimination of acylating molecules form the bioconjugates;

[0016] FIG. 4 shows a scheme of an optional purification process with hot water for the separation of water soluble bioconjugates;

[0017] FIG. 5 shows a scheme of an optional purification process with water at room temperature for the separation of water soluble bioconjugates;

[0018] FIG. 6 shows a scheme of an optional drying process of bioconjugates with acylating traces;

[0019] FIG. 7 shows an scheme of an optional drying process of bioconjugates in aqueous solution;

[0020] FIG. 8A shows a HPLC chromatogram of bioconjugates synthetized using example 1(A);

[0021] FIG. 8B shows a HPLC chromatogram of bioconjugates synthetized using example 2(B);

[0022] FIG. 9A shows a graphic showing toxicological evaluation of bioconjugates;

[0023] FIG. 9B shows a graphic showing toxicological evaluation of bioconjugates;

[0024] FIG. 10 shows a graphic showing a prebiotic effect of the new bioconjugates molecules;

[0025] FIG. 11A shows a graph showing an Anti-inflammatory activity of the new bioconjugated molecules in vitro;

[0026] FIG. 11B shows a graph showing an Anti-inflammatory activity of the new bioconjugated molecules in vivo;

[0027] FIG. 12 shows a graph showing an antitumoral activity of the new bioconjugated molecules;

[0028] FIG. 13 shows a hydrolysis kinetic of the new bioconjugated molecules in an ex vivo digestive tract simulator;

[0029] FIG. 14A shows an example of the techno functional application in food of the new bioconjugated molecules as emulsifier; and

[0030] FIG. 14B shows an example of the techno functional application in food of the new bioconjugated molecules as fat substitute.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The process of the present invention is shown in FIGS. 1 to 4. In FIG. 1 sugars, oligosaccharides or polysaccharides are FOS, GOS or other branched oligosaccharides with (2.fwdarw.1) and (2.fwdarw.6) links, such as agave fructans, raw or purified; when purified oligosaccharides with short (DP<10) or long (DP>10) degree of polymerization can be used. As it is a mixture of sugars, not a single bioconjugate is obtained but a mixture of bioconjugated molecules depending on the sugar used as substrate and the acylating agent.

[0032] The acylating agent can be a a) carboxylic acid (free fatty acid) of medium chain (8-12 carbons) or long chain (>12 carbons); or b) one of its esters (methyl, ethyl, vinyl, etc.); or c) an oil with different carboxylic acids, saturated or unsaturated such as omega-3; or d) a mixture of esters oils such as commercially available ethyl esters of omega-3. When the acylating agents are mixtures of fatty acids or their esters the complexity of the produced molecules increases.

[0033] The enzyme used in this invention is part of the serine-hydrolases, such as proteases, lipases, esterases, cutinases or any other that act or synthetizes an ester link. For better productivity the enzyme can be immobilized making its recovery and reuse easier. If the bioconjugated molecules will be used in foods, the enzyme must be food grade, if they will be used in pharmaceuticals or cosmetics, the enzyme should comply with the required standards.

[0034] Regarding solvent, the reaction can be carried out in organic solvents, including hydrophobic solvents such as hexane, heptane, isooctane, decane, etc.; hydrophilic solvents such as 2-methyl-2-propanol, 2-metyl-2-butanol, acetone, etc.; as well as biphasic systems with hydrophobic solvents/water; or in the absence of solvents, being the acylating agent the solvent. The molecular sieve (porous silica, zeolite or clay with pore size of 3-4 , or any other water absorbent) is optional when the acylating agent produces water as reaction sub-product.

[0035] The synthesis process of a mixture of bioconjugated molecules, also denoted as bioconjugates in the present invention, includes the following steps:

[0036] 1. Synthesis reaction. In this step sugars, oligo- or polysaccharides, are conjugated (esterified) with the acylating agent in a ratio of 1:1 to 1:10 w/w, in a reaction biocatalyzed by the enzyme which is added in a ratio of 1:1 to 1:10 w/w regarding the acylating agent; in a solvent with a ratio of 1:2 to 1:100 w/v regarding the acylating agent; in an hermetically sealed container, heated to a temperature that ensures the acylating agent is in liquid state when is a solvent free reaction or at a temperature below the boiling point of the solvent (generally between 40 and 80 C.), under agitation (manual, mechanic, magnetic, orbital, vibrations, thermic or passive diffusion) that allows good mass transfer (100-1000 r.p.m.). Optionally molecular sieve or other absorbent is added, when the acylating agent produces water during the reaction, in a ratio of 1:1 to 1:5 w/w. The reaction time is between 48 and 120h. At the end of the reaction the mixture is called crude reaction mixture (2) and is composed by the bioconjugates, unreacted acylating agent and sugars, and the enzyme (also the molecular sieve it was added).

[0037] 2. Filtering. As observed in FIG. 1, in the process of this invention, unlike other previously described inventions, the bioconjugates are also recovered from the solid phase (10) besides the liquid phase (6), by filtration or centrifugation (3), using Whatman filters number 1, 3, 4, or similar, and/or by centrifugation. From this step the liquid organic phase (OP, 4) contains the bioconjugates, solvent and the unreacted acylating agent, and in the retained solid phase (SP, 7), the bioconjugate, enzyme, molecular sieves (if added), unreacted sugars and acylating agent.

[0038] 3. Bioconjugate recovery from organic phase is carried out by solvent evaporation (omitted if the acylating agent was the solvent). This is achieved by heating above the boiling point of the solvent used or using a rotary evaporator with reduced pressure and appropriate temperature. The dried product is called organic phase bioconjugate (OPB, 6) and although it might have unreacted acylating agent, they can be used in the applications described.

[0039] 4. Bioconjugate recovery from solid phase is carried out by washing with an alcohol, such as methanol, ethanol, t-butanol, isopropanol or other hydrophilic solvent, in a ratio of 1:2 to 1:5 w/v. In the solid phase of this step (8), the enzyme, molecular sieve (if added) and unreacted sugars are recovered; while in the liquid phase, after alcohol evaporation as described in step 3, the solid phase bioconjugates (SPB, 10) are recovered, and although it might have unreacted acylating agent, they can be used in the applications described

[0040] 5. Drying (optional). FIG. 2 shows the optional process to completely drying the bioconjugates. The OPB and SPB are washed with a hydrophilic solvent such as an alcohol or acetone. Washing (11) removes the residual reaction solvent. The washing solvent is removed as described in step 3, so it can be recovered and recycled (12). The solvent free bioconjugates (13) can be optionally dried using nitrogen when the reaction solvent is retained in the bioconjugates for complete elimination of the solvents and recovery of the bioconjugates (14). However once the organic solvent is removed, the bioconjugates can contain water due to the hydrophobicity of the sugar component in the bioconjugate, therefore they can be frozen (15) at a temperature of 5 to 80 C. and lyophilized (16) to remove residual water and recovery of washed and dried bioconjugates (17).

[0041] 6. Purification (optional): If the reaction did not reached 100% yield or if the acylating agent was in excess, the acylating agent can be removed by several purification methods that will be described:

[0042] a. With diluted alkali (FIG. 3). The SPB (10) or OPB (6) are dissolved in a hydrophobic solvent in a ratio of 1:1 to 1:10 w/v (this is not necessary if the reaction was carried out in an hydrophobic solvent for OPB). An aqueous solution of diluted alkali (19), in a concentration of 0.1 to 1N, is added to the solution 18 in a ratio of 1:1 to 1:5 v/v. After agitation (20) (manual, mechanic, magnetic, orbital, by vibration, thermic or passive diffusion), is decanted (21) and three phases separated; 1) organic phase (22) with bioconjugates with retention times larger than 3.5 min (24) (FIG. 8-A) and can be evaporated following step 3 (23) for solvent recovery; 2) the interphase (25) containing soap and 3) an aqueous phase (26) containing the bioconjugates with retention times smaller than 3.5 min (FIG. 8-A). The aqueous phase bioconjugates (26) are dried with an air flow or oven (27) to obtain bioconjugates (28) with retention times smaller than 3.5 min (FIG. 8-A).

[0043] b. With water: It can be hot (FIG. 4) or room temperature water (FIG. 5). With this purification method the bioconjugated molecules soluble in water can be separated. The hot water procedure is: the OPB (6) or SPB (10) are heated to 40 to 70 C. (29) and are washed with water at a temperature of 40 to 70 C. (30). The mixture is agitated manually or mechanically (31) and centrifuged at 1000-10000 r.p.m. (32). The upper phase (33) has the bioconjugates with unreacted acylating agent (if it was the case) and in the inferior phase the bioconjugate in aqueous phase with sugar traces (34). Alternatively the water-soluble product can be obtained from the reaction solid phase (SP, 7) washing with water at room temperature (35). The mixture is manually or mechanically agitated (36) and centrifuged at 1000-10000 r.p.m. (37). The upper phase (38) has the bioconjugate in solution with traces of unreacted sugar (if it was the case), while the lower phase (39) has a non water soluble bioconjugate with the enzyme, molecular sieve (if is was added) and the unreacted acylating agent (of it was the case). The upper phase bioconjugate with unreacted acylating agent (39) can be dried through freezing (40) and lyophilization (40) or oven drying (42), obtaining a non water soluble dried bioconjugate (43) with traces of acylating agent (if it was the case). The water soluble bioconjugates (34 or 38), with or without sugar traces, can be directly dried from the aqueous solution (FIG. 7) through freezing (44), followed by lyophilization (45) or spray dry (46) to obtain dried water soluble bioconjugates (47).

[0044] Depending on the purification method employed the final state of the product can be a water-soluble gel or a powder of molecules with longer retention times. The present invention has human and veterinarian applications.

EXAMPLES OF APPLICATIONS

[0045] Example 1. Bioconjugated molecules agave FOS+lauric acid. 16 g, agave FOS 50 g, vinyl laurate 500 ml, hexane 50 g, immobilized lipase (C. antarctica B) 50 g, molecular sieve of 3 The mixture is agitated at 60 C. for 96h, is filtrated and purified following one of the methods mentioned in step 6. The chromatogram of the bioconjugates synthetized this way is presented in FIG. 8-A.

[0046] Example 2. Bioconjugated molecules agave FOS+omega-3. 16 g, agave FOS 50 g, fish oil 500 ml, hexane 50 g, immobilized lipase (C. antarctica B) 50 g, molecular sieve of 3 The mixture is agitated at 60 C. for 96h, is filtrated and purified following one of the methods mentioned in step 6. The chromatogram of the bioconjugates synthetized this way is presented in FIG. 8-B.

[0047] Example 3. Application of bioconjugates in foods and pharmaceuticals.

[0048] The bioconjugated molecules synthetized according to example 1 and purified by one of the methods in FIGS. 2 and 3, were evaluated for toxicity to confirm their applicability in foods and pharmaceuticals. FIG. 9 shows the absence of toxicity in the two methods testes: (A) evaluation of mutagenicity in the TA98 strain by the generation of alkylating compounds; (B) evaluation of mutagenicity in the TA102 strain by the generation of oxidizing compounds. Both according to the method of Marron and Ames (Marron and Ames, 1983), that has been postulated as an acceptable test to detect as non-mutagenic the non-carcinogenic compounds (Dobo et al., 2006). The treatments were: (A): 1-Espontaneous reversion, 2-DMSO (solvent), 3-TWEN (solvent), 4-Picrolonic acid, 5-Control1, 6-Contorl2, 7-Bioconjugates. (B): 1-Espontaneous reversion, 2-DMSO (solvent), 3-TWEN (solvent), 4-4-nitroquinalone, 5-Control1, 6-Contorl2, 7-Bioconjugates.

[0049] Example 4. Application of bioconjugates as prebiotics.

[0050] The bioconjugated molecules synthetized according to example 1 (purified by one of the methods in FIGS. 2 to 7), were used as carbon source for the growth of probiotic microorganisms, that showed acceptance in the consumption of the bioconjugates (FIG. 10). Lactobacillus casei and L. rhamnosus had the highest growth rates without statistical differences, concluding that the bioconjugates of branched oligosaccharides have prebiotic functions.

[0051] Example 5. Application of bioconjugates as prebiotics anti-inflammatories.

[0052] The bioconjugated molecules synthetized according to example 1 (purified by one of the methods in FIGS. 2 to 7), can be used as anti-inflammatories as shown in FIG. 11. FIG. 11-A shows the in-vitro anti-inflammatory activity measured as COX-2 inhibition using a previously reported (Szymczak et al., 2008). The bioconjugates are compared to a known anti-inflammatory Diclofenac. Concentrations 1, 2 and 3 for Diclofenac were 50, 100 y 200 g/ml respectively; for the bioconjugates were 400, 800 y 1600 g/ml. (Mean values are presented for duplicates. Mean standard deviation was of 4.5%. FIG. 11-B shows the in-vivo anti-inflammatory activity of the bioconjugates measured using the plantar edema induction methodology (Xu et al., 2012). Samples were evaluated at a dosis of 100 mg/kg. Results show the plantar edema induction of the animals two hours after inflammation induction with carrageenan. The smaller the size of edema translates as a higher anti-inflammatory activity. In this FIG. Bioconjugates 1 were purified according to the method in FIG. 2 and Bioconjugates 2 were purified according to the method in FIG. 3.

[0053] For the concentrations tested, FIG. 11-A shows that the bioconjugates have between 60% and 70% of the in vitro anti-inflammatory activity found for Diclofenac, without presenting its secondary and hepatotoxic effects (Aithal, 2011). FIG. 11-B shows the in vivo anti-inflammatory activity. Bioconjugates 1 had an anti-inflammatory activity similar to Diclofenac while Bioconjugates 2 presented a lower activity.

[0054] Example 6. Application of bioconjugates as antitumorals.

[0055] The bioconjugates synthetized according to example 1, purified according to the method in FIG. 3, were used as cytotoxic agents in the HeLa cervical cancer cell line. As show in FIG. 12, the bioconjugates are cytotoxic from a concentration of 500 g/ml.

[0056] Example 7. Application of bioconjugates as intestinal vector.

[0057] The bioconjugates synthetized according to example 1, purified according to one of the methods in step, were used as intestinal vector. In this case the prebiotic non-digestible part of the bioconjugates vectorizes the acylating part of the molecules. For this it was verified that the bioconjugates were not hydrolyzed before reaching the intestine in an intestinal tract simulator. The simulator was described by Gonzalez-Avila et al., (Gonzalez-Avila et al., 2012). FIG. 13 shows a thin layer plate with the samples taken from the digestive tract simulator: 1-Laurie acid (standard), 2-Bioconjugates (control), 3-Food with bioconjugates in stomach, 4-Food with bioconjugates in small intestine, 5-Food with bioconjugates in ascendant colon, 6-Food with bioconjugates in transverse colon, 7-Food with bioconjugates in descending colon. Since no presence of lauric acid was observed in stomach and small intestine, it is concluded that the bioconjugates work as vectors to carry molecules of interest to the colon.

[0058] Example 8. Application of bioconjugates as emulsifiers.

[0059] The bioconjugated molecules synthetized according to example 1 (purified by one of the methods in FIGS. 2 to 7), were used as emulsifiers in the preparation of a strawberry mousse (FIG. 14-A), according to the following formula (%):

TABLE-US-00001 Bioconjugates 0.5 Gelatin 0.5 Strawberry 47.6 Plain yogurt 37.55 Sour cream 11.85 Sugar 4.0

[0060] Example 9. Application of bioconjugates as fat substitute.

[0061] The bioconjugated molecules synthetized according to example 1 (purified by one of the methods in FIGS. 2 to 7), were used as fat substitute in the preparation of a coconut shake (FIG. 14-B), according to the following formula (%):

TABLE-US-00002 Bioconjugates 0.43 Gelatin 0.43 Evaporated milk 31.56 Condensed milk 24.5 Coconut-vanilla flavor 0.16 Hot water 10.22

[0062] Example 10. Cosmetic formulation using bioconjugates.

[0063] The bioconjugated molecules synthetized according to examples 1 or 2 (purified by one of the methods in FIGS. 2 to 7), were used in the preparation of a cosmetic emollient, according to the following formula (%):

TABLE-US-00003 Bioconjugates 6.0 Liquid silicone f350 cts 10.0 Sorbitan 5.0 Stearic acid 4.0 Mineral oil 2.0 Lanoline 1.0 Cetyl alcohol 1.0 Triethanolamine 0.9 Antioxidant 0.1 Purified water c.s.p. 100

[0064] Having sufficiently described the invention, it is a novelty and what is claim is: