Process for producing monosaccharides from chitin and / or chitosan by chemical and / or enzymatic hydrolysis

Abstract

The present invention discloses a method for obtaining monosaccharides from chitin or chitosan by chemical and / or enzymatic hydrolysis in an acid solution. By using low-cost, easily obtainable reagents, this method makes it possible to obtain industrially important sugar solutions. With this new production method, the technological sectors to which this invention pertains aim to offer the food and / or chemical industries the use of an alternative method for producing monosaccharides that is less complex and therefore more technically and financially viable.

Description

Technical Field

[0001] The present invention discloses a method for obtaining monosaccharides from chitin or chitosan in an aqueous acid solution. The method enables the obtaining of a solution of hexose using reagents that are easily obtained at low cost. The technical field related to this invention aims to provide an alternative method for producing sugars that is more technically and economically feasible by means of innovative manufacturing methods in the food industry and / or the chemical industry.

Background Art

[0002] The production of second-generation ethanol strongly depends on the efficiency of obtaining fermentable monosaccharides from biomass. The traditionally used methods rely on chemical and / or enzymatic hydrolysis of lignocellulose, the most abundant source of biomass. The main problems associated with ethanol from lignocellulosic biomass are derived from the heat-resistant structure of lignocellulose, which requires the process of biomass delignification (lignin present in biomass inhibits the activity of the cellulase enzymes used) and the fermentation of pentoses generated during the process (e.g., xylose and arabinose, which cannot be fermented by most microorganisms used to produce ethanol, especially yeasts of the genus Saccharomyces). An alternative approach being studied for producing second-generation ethanol is the use of chitin. Chitin, a natural poly-N-acetyl-glucosamine polysaccharide, is a structural component of crustaceans, insects, fungi, and other chitinous sources and is the second most abundant biopolymer in nature, inferior only to cellulose. It is estimated that 1 to 100 billion tons of chitinous waste are generated worldwide each year, mainly from the fishing industry. The hydrolysis of chitin causes its deacetylation, producing chitosan, a polymer whose monomer unit is glucosamine. The process of producing chitosan from chitin by acid or base hydrolysis is affected by several factors, such as temperature and reaction time, as recognized by REGE, P.R. and BLOCK, L.H. (1999) in a paper published in the journal Carbohydrate Research, volume 321.

[0003] The obtaining of sugars from chitosan produced by chitosan depolymerization reactions through both enzymatic and chemical pathways has been studied by several authors. Pan S. K. et al. (2011) were able to produce glucosamine (an amino sugar) from chitosan by using commercially available alpha - amylase and glucoamylase. Nitrous acid (HNO2) has been extensively studied with respect to chitosan depolymerization. This acid can react with amines, producing nitrogen gas (N2) and water. Due to the amino groups (-NH2) present in the monomer, chitosan is particularly susceptible to this Reaction .

[0004] Using a combination of acetic acid (2.5%) and mild conditions (temperature 4°C, reaction time 24 hours, dark environment and concentration 15 millimoles of NaNO2), Tommeraas et al. (2001) were able to obtain oligomers with chains having monomers with a 2,5 - D - mannose structure at their ends in a paper published in the journal Carbohydrate Research, volume 333, but were unable to obtain monomers (sugars).

[0005] By using more drastic conditions (room temperature, reaction time 24 hours, concentration 0.66M of NaNO2 and concentrated hydrochloric acid - 37%), Salim et al. (2014) were able to produce oligomers with terminal chain 2,5 - D - mannofuranoside.

[0006] A detailed study of the deacetylation and depolymerization mechanisms carried out by Knight et al. (2007) in a paper published in the Journal of Biomedical Materials Research, volume 83A demonstrated that using NaNO2 at dilute concentrations and various pH values (1.6, 2.9 and 5.1), the pH affects the products of the reaction in a decisive way, and at very low pH values, N - nitrosoamines are formed.

[0007] In other studies carried out by Valum et al. (2001) and published in the journal Carbohydrate Research, volume 46, it was shown that in a medium containing concentrated HCl, the depolymerization reaction of the glucoside bonds in partially acetylated chitosan is 10 times faster than the deacetylation reaction, while in dilute acid, the two reactions occur at equal rates.

[0008] U.S. Patent No. 3,922,260 (PENISTON, Q.P.; JOHNSON, E.L. Process for the depolymerization of chitosan - U.S. Patent No. 3,922,260, filed November 25, 1975) describes the discovery of a process for obtaining short - chain reducing molecules using the dissolution of chitosan in dilute acetic acid and a dilute solution of sodium nitrite (at a concentration of about 0.124 mol / L). SUMMARY OF THE INVENTION

[0009] In the present invention, it relates to the discovery of a combination of temperature and nitrous acid concentration conditions that enable the hydrolysis of chitosan and / or chitin to produce monosaccharides, and a combination of such chemical hydrolysis and enzymatic hydrolysis using an enzyme with amylolytic activity ("amyloglucosidase"). This process allows monomers (monosaccharides) to be directly obtained from chitosan and / or chitin, unlike previously developed processes that produce oligomers.

[0010] Mild mineral acids such as hydrochloric acid and / or short - chain carboxylic acids (such as acetic acid) cause depolymerization with the formation of oligomers, while an acid medium containing acetic acid under severe conditions (high concentration of nitrous acid) in the depolymerization reaction of chitosan in acetic acid can produce monosaccharides (it was noted that such a change in conditions, such as a change in the chemical species used to oxidize the reaction medium or a change in the nitrous acid concentration, can produce various products).

[0011] Similarly, the chemical hydrolysis by nitrous acid, i.e., the depolymerization of chitin and / or chitosan by nitrous acid, followed by enzymatic hydrolysis by an enzyme having amylolytic activity (which can promote the depolymerization of the oligomers produced by the chemical hydrolysis by nitrous acid) also produces monosaccharides.

[0012] The production of monosaccharides described herein can be exploited by the sugar industry as well as the sugar derivative industry including bioethanol and hydroxymethylfurfural (which is a precursor of several compounds and has the advantage of being used as a raw material, a widely available, renewable and abundant biomass). This process is fast and simple to implement, and the reagents used are of low cost.

Brief Description of the Drawings

[0013] [Figure 1]

Mode for Carrying Out the Invention

[0014] Manufacturing Method For acid hydrolysis, 10.00 g of chitin or chitosan was mixed with 500.0 mL of acetic acid (CH3COOH) under constant stirring. Subsequently, 100.0 mL of 0.66 mol / L sodium nitrite (NaNO2) solution was added (for in-situ formation of nitrous acid) and heated at a gentle temperature (35 °C to 55 °C) for 3 hours. The final product is a low-viscosity yellowish suspension.

[0015] For the combined hydrolysis (chemical and enzymatic), a sample of chitin (the exoskeleton of crustaceans such as shrimp - decapod crustaceans) was subjected to the chemical hydrolysis described in the previous section, followed by hydrolysis with amyloglucosidase (an enzyme having amylolytic activity) under the conditions of optimal pH and enzyme temperature (pH about 5.0 and T about 52 °C). As in the chemical hydrolysis, this product is also a yellow low-viscosity suspension containing monosaccharides (sugars).

[0016] The isolation of fermentable hexoses present in the product for characterization was carried out by gel filtration chromatography using a glass column (50 × 1.5 cm d.i.) with a total volume (V t ) of 88 mL and a void volume (V 0 ) of 60 mL containing BioGel P-2 (exclusion limit: 1800 - 100 daltons), according to Ascencio [Ascencio, S.D. Extraction, quantification and chemical characterization of low molecular weight carbohydrates from red algae (Rhodophyta). Curitiba, 2002. Thesis (Master of Biochemistry) - Biochemistry, Federal University of Paraná. 100 pages, 2002. Supervisor: Professor Dr. Miguel Daniel Noseda]. The total volume of the column was measured by adding water to the column up to the level determined to be filled with the gel, and the eluent was deionized water (pH 6.51).

[0017] Fractions of 10 mL of the filtrate were collected every 15 minutes for 9 hours using a Spectrum Labs CF-2 automatic fraction collector. The first method used for chromatographic screening was thin layer chromatography (TLC), using silica gel 60 plates (ALUGRAM®) as described in WAKSMUNDZKA-HAJNOS, M.; SHERMA, J.; KOWALSKA, T. "Thin Layer Chromatography in Phytochemistry" Chromatographic Science Series: 99, 2008; pre-activated at 110 °C for 30 minutes; and consisting of being used as the stationary phase. Various samples were applied by glass capillary at the bottom, at various spots, on a line 2.5 cm above the start of the sheet. Thereafter, in the chromatography chamber, the base of the sheet was immersed in the mobile phase and the run was carried out until the mobile phase reached the defined maximum height (2.5 cm below the edge of the sheet).

[0018] The mobile phase used in this analysis consisted of ethyl acetate: isopropyl alcohol: acetic acid: distilled deionized water (4:2:2:1). Development was carried out at 100 °C until the desired coloration was reached (about 5 minutes). The developing solution consisted of 250 mg of orcinol solubilized in 95 ml of ethanol and 5 ml of sulfuric acid, which was sprayed onto the plate before taking it to the furnace. The standard sample used was glucose. All reagents and standards used exhibited a high purity level.

[0019] After the initial screening by thin layer chromatography (TLC), the fractions confirmed to contain sugars were sent for HPLC analysis. Since a refractive index detector (RID) was used instead of UV-VIS detection, it was not necessary to modify the carbohydrate molecules by derivatization.

[0020] The Shimadzu high performance liquid chromatograph (LC-10 series Avp; degassing device: DGU-14A, integrated device: class LC-10) was used, and elution was carried out at a uniform concentration by pumping a mobile phase consisting of ultrapure water (distilled and deionized) containing 5 mM sulfuric acid (LC-10AD). The flow rate of the eluent was 0.6 mL / min at 30 °C (CTO-10A column oven), and the total running time was 20 minutes. Detection was carried out with a refractive index detector (Shimadzu, model RID-10A). An aliquot of 20.0 μl of the sample was manually injected (Rheodyne injector, 20 mesh) and permeated through a Phenomenex Rezex ROA-Organic Acid H+ column (300 × 7.8 mm) directly connected to a Phenomenex Carbo-H security cartridge (4 × 3 mm) filled with a material similar to that of the main column. The presence of hexose could be confirmed in the sample of flask 9 shown in Figure 1. This method enabled the visualization of the presence of monosaccharides simultaneously with glucose retention and showed the presence of sugars in the sample, as can be seen in Figure 1.

[0021] In addition, the sample was characterized by a gas chromatograph coupled to a Saturn mass spectrometer equipped with a Factor Four capillary column VF-1ms column (30 m × 0.25 mm × 0.25 μm), model 4000. The initial temperature was 50°C, and the temperature was gradually increased to 220°C, which is the analysis temperature of alditol acetate (flow rate of 40°C per minute). The temperature was maintained constant during the analysis time (25 minutes). The carrier gas was helium, with a flow rate of 1 mL / min. The area of the peak of interest was determined by integration using Varian WS software, and mass fragments by electron impact at 70 meV were obtained. These values were observed in the mass spectrum. The analysis was performed by comparing the retention times and the fragmentation profiles and patterns of the sample.

[0022] Finally, 1D and 2D nuclear magnetic resonance analyses were performed by a Bruker Avance DRX400 (Bruker Germany) spectrometer at base frequencies of 400 MHz (1H) and 100 MHz (13C), and by a Bruker Avance III 600 spectrometer (Bruker Germany) at base frequencies of 150 MHz (13C) and 600 MHz (1H). The analysis temperature was 30 to 50°C. The sample was solubilized in 99% D2O at a concentration of 80 mg / mL for 13C analysis and 20 mg / mL for 1H and 2D analyses, and placed in a 5 mm OD tube. The chemical shifts expressed in ppm were determined for both 13C (30.20 ppm) and 1H (2.224 ppm) using acetone as an internal standard, and these visualized the presence of a glucose-like hexose with a profile corresponding to anhydro mannose.

Example

[0023] In the present invention, we report the results of efficient chitin or chitosan depolymerization to fermentable monosaccharides by the combined use of nitrous acid, which has a high yield in the conversion of biomass to monosaccharides. Alternatively, this technique also shows efficiency when using a combination that combines the chemical hydrolysis described above with the enzymatic hydrolysis of chitin or chitosan by using an enzyme having amylolytic activity.

[0024] Example 1 A 1-5% (mass / volume) chitin or chitosan solution in an acetic acid solution (1-5%) is prepared at room temperature with stirring for an appropriate time (1-30 minutes). Next, a freshly prepared concentrated sodium nitrite (NaNO2) solution (0.5 to 5 mol / L) is added to the solution (5-50% of the volume of the solution) with stirring at room temperature (since nitrite tends to oxidize to nitrate, the use of an old prepared solution reduces the efficiency of this process). This reaction produces nitrogen gas, which can be observed by vigorous gas evolution.

[0025] Example 2 A chitin or chitosan solution according to Example 1 is prepared by changing the heating of the stirred mixture to gentle heating (30°C to 55°C). The temperature reduces the total time required to accelerate the reaction and obtain the product.

[0026] Example 3 A chitin or chitosan solution according to Example 1 is prepared by changing the heating of the stirred mixture to intense heating (55°C to 99°C).

[0027] Example 4 A 1-5% (mass / volume) chitin or chitosan solution in an acetic acid solution (1-5%) is prepared at room temperature by rapid stirring for an appropriate time (1 second to 59 seconds). A freshly prepared concentrated sodium nitrite (NaNO2) solution (0.5 to 5 mol / L) is added without stirring, and the mixture is stored for a period longer than 30 minutes. The reaction usually proceeds by obtaining a solution containing glucose.

[0028] Example 5 Prepare the mixture according to Example 4 by gentle heating (30 °C - 55 °C) during the preparation of the chitosan mixture with acetic acid and the addition of the sodium nitrite solution.

[0029] Example 6 Prepare the mixture according to Example 4 by intense heating (55 °C - 100 °C) during the preparation of the chitosan mixture with acetic acid and the addition of the sodium nitrite solution.

[0030] Example 7 Prepare a mixture of short-chain organic acids or mineral acids containing chitin or chitosan at a concentration in the range of 1 - 20%. Next, add a concentrated or dilute sodium nitrite solution (NaNO2) with heating (above 30 °C) and under stirring or at rest. Subsequently, add an enzyme with amylose-degrading activity for a sufficient time under stirring or at rest under suitable pH and temperature conditions (for enzyme activity) to optimize the yield.

[0031] Example 8 Prepare the chitin or chitosan mixture according to Example 7. Next, add a concentrated or dilute sodium nitrite solution with stirring or at rest at room temperature. Immediately afterwards, add an enzyme with amylose-degrading activity for a sufficient time under stirring or at rest under suitable pH and temperature conditions (for enzyme activity) to optimize the yield.

[0032] Example 9 Prepare the chitin or chitosan mixture according to Example 7. Add an enzyme with amylose-degrading activity for a sufficient time under stirring or at rest under suitable pH and temperature conditions (for the optimal activity of the enzyme) to optimize the yield. Immediately afterwards, add a concentrated or dilute sodium nitrite solution (NaNO2) with heating (temperature above 30 °C) and under stirring or at rest.

[0033] Example 10 Prepare the chitin or chitosan mixture according to Example 7. An enzyme with amylose-degrading activity EnzymeAdd it under stirring or while standing for a sufficient time under pH and temperature conditions suitable for the activity of the enzyme to optimize the yield. Next, add a concentrated or dilute sodium nitrite solution while stirring or standing at room temperature.

[0034] Example 11 By using an alternative form of nitrous acid, for example, saturating the reaction medium with nitrogen oxides such as dinitrogen tetroxide, N2O4, dinitrogen pentoxide, N2O5, etc., a reaction solvent containing nitrous acid is obtained, and then, by the variations described in Examples 2, 3, 4, 5, 6, 7, 8, 9 and 10, a solution of chitin or chitosan according to Example 1 is prepared. The invention described in the claims of the present application at the time of filing is appended below. [1] A method for producing monosaccharides from chitin and / or chitosan by chemical and / or enzymatic hydrolysis and its use for producing monosaccharides in solution, which comprises dissolving chitin or chitosan in an acetic acid solution and / or an inorganic acid, and subsequently reacting this mixture with concentrated nitric acid in the following steps: Step 1: Stirring a solution of acetic acid and / or an inorganic acid (1 to 5%) containing 1 to 5% (mass / volume) of chitin and / or chitosan at room temperature for an appropriate time (1 to 60 minutes, preferably 5 minutes); Step 2: Adding 5 to 50% by volume of nitrous acid freshly prepared and produced in various forms in various concentration ranges under stirring and heating. [2] A method for producing monosaccharides from chitin and / or chitosan by chemical and / or enzymatic hydrolysis and its use for producing monosaccharides, which comprises using the solution described in [1] under stirring and gentle heating in the temperature range of 30°C to 50°C. [3] A method for producing monosaccharides from chitin and / or chitosan by chemical and / or enzymatic hydrolysis and its use for producing monosaccharides, which comprises using the solution described in [1] and [2] obtained under various reaction time conditions in the range of 1 minute to 72 hours. [4] A method for producing monosaccharides from chitin and / or chitosan by chemical and / or enzymatic hydrolysis and its use for producing monosaccharides in solution, which comprises using the solution described in [1], [2] and [3] heated under intense heating in the temperature range of 55°C to 200°C under changing pressure while stirring. [5] A method for producing monosaccharides from chitin and / or chitosan by chemical and / or enzymatic hydrolysis and its use for producing monosaccharides in solution, which comprises using the solution described in [1], [2], [3] and [4], and an enzyme having amylose-degrading activity, under suitable pH and temperature conditions (for optimal enzyme activity), with stirring or standing for a suitable time, including the use of an additional step of enzymatic hydrolysis. [6] Dissolve chitin and / or chitosan in an acid solution (mineral acid or organic acid), and carry out enzymatic hydrolysis using an enzyme having amylolytic activity under suitable pH and temperature conditions (for optimal enzyme activity) with stirring or for a certain period of time while standing, and subsequently react the product with nitrous acid under various time, temperature and pH conditions. A method for producing monosaccharides from chitin and / or chitosan by chemical and / or enzymatic hydrolysis and its use for producing monosaccharides in solution.

Claims

1. A method for producing monosaccharides from chitin and / or chitosan by chemical and enzymatic hydrolysis, comprising the following steps of dissolving chitin or chitosan in an aqueous acetic acid solution and then reacting this mixture with concentrated nitrous acid (a) Stirring an aqueous acetic acid solution (1 to 5%) containing 1 to 5% (mass / volume) of chitosan and / or chitin at room temperature for an appropriate time of 1 to 30 minutes, (b) Adding an aqueous sodium nitrite solution freshly prepared at a concentration of 0.5 to 5 mol / L in an amount of 5 to 50% of the volume to the aqueous solution described in step (a) under stirring and heating, or saturating the aqueous solution of step (a) with gaseous dinitrogen tetroxide and dinitrogen pentoxide, and comprising The method further comprising an additional step of enzymatic hydrolysis using an enzyme having amylose-degrading activity under stirring or stationary conditions at a pH of 5.0 and a temperature of 52 °C, and the enzyme having amylose-degrading activity is amyloglucosidase.

2. The method according to claim 1, wherein step (b) occurs under stirring and gentle heating in a temperature range of 30 °C to 55 °C.

3. Optionally, step (b) is carried out at various reaction times in the range of 1 minute to 72 hours, the method according to claim 1 or 2.

4. The method according to claim 1 or 3, wherein step (b) is carried out with intense heating in a temperature range of 55 °C to 100 °C.

5. The method according to any one of claims 1 to 4, wherein the additional step of performing enzymatic hydrolysis is carried out between step (a) and step (b).

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