Chitin nanowhisker production method

Abstract

The present invention relates to a method for the recovery and purification of crystalline chitin nanowhiskers comprising or consisting of the following steps: a) providing a raw material comprising chitin; b) washing, grinding and sieving the raw material of step a) to obtain a powder comprising chitin and having a uniform particle size less than 250 micrometres; c) dissolving the powder of step b) in a deep eutectic solvent (DES) comprising or consisting of an organic acid and ammonium formate (AF), so as to obtain a mixture containing DES, amorphous chitin and packed chitin nanowhiskers; d) adding water to the mixture of step c) and heating in the presence of carbon dioxide to obtain a mixture containing DES, water, amorphous chitin and disintegrated chitin nanowhiskers; e) separating the disintegrated chitin nanowhiskers from the mixture of step d) to obtain a solid powder of chitin nanowhiskers

Description

[0001] CHITIN NANOWHISKER PRODUCTION METHOD

[0002] Technical field of the invention

[0003] The present invention relates to a chitin nanowhisker production method.

[0004] The invention is applied in the field of environmentally-friendly biomaterials, usable as food and non-food film, having oxygen barrier effect, fillers for composite materials, for medical -biomedical applications, and for pharmaceutical and cosmetic products.

[0005] State of the art

[0006] The extraction of chitin from waste is a very current research topic, especially in the field of green chemistry. The specific case of crustacean exoskeletons as a source of chitin is one of the most studied areas.

[0007] The chitin content in crustacean waste ranges from 6% to 72% depending on the origin of the biomass. Crab exoskeletons have proved to be the most difficult to treat due to their rigidity; therefore, a suitable protocol for crabs will likely be suitable even for simpler biomass, such as shrimp shells, with the adjustment of experimental conditions. Furthermore, the waste / food ratio in crabs is unbalanced towards waste, which suggests a large amount of available biomass.

[0008] Nanochitin is currently produced by strong acid hydrolysis (for example by means of HC1 and H2SO4) or TEMPO-mediated oxidation of purified chitin, which in turn is obtained through a traditional chemical extraction, involving two consecutive steps with strong and hazardous acids and bases.

[0009] Alternative methods have used, for example, citric acid as a demineralizing agent for the extraction of chitin, and / or the use of liquids comprising strong acids for the deacetylation and disintegration of alpha-chitin fibres.

[0010] These methods not only require multiple steps, including acidification and subsequent neutralization, but employ risky reagents, such as strong acids and bases.

[0011] Further methods comprise the use of imidazole ionic liquid or BMIrnHSC , for the isolation of nano-chitin respectively from lobster / crab / shrimp shells.

[0012] However, imidazole ionic liquids are expensive, have toxicity proportional to alkyl chain length, are poorly biodegradable, and have potential toxicity to aquatic food. All these features do not favour their use in commercial applications.

[0013] The use of deep eutectic solvents (DES) based on choline chloride and zinc chloride in the presence of acetic acid (or acetic anhydride) or based on FeCE and betaine hydrochloride, has been introduced as an alternative to the use of ionic solvents (ILs) inchitin extraction methods.

[0014] However, choline chloride-based DESs are relatively expensive; acetic acid and acetic anhydride are highly corrosive and can lead to significant problems in the scaling of processes for chitin and nano-chitin production.

[0015] Furthermore, the use of metal-based DES (Zn or Fe) leads to both economic and environmental problems.

[0016] In view of the above, there is still a need for a method for obtaining chitin nanowhiskers, which overcomes the disadvantages of the known methods.

[0017] Summary of the invention

[0018] The invention relates to a method for the recovery and purification of crystalline chitin nanowhiskers comprising or consisting of the following steps:

[0019] a) providing a raw material comprising chitin;

[0020] b) washing, grinding and sieving the raw material of step a) to obtain a powder comprising chitin and having a uniform particle size less than 250 micrometres;

[0021] c) dissolving the powder of step b) in a deep eutectic solvent (DES) comprising or consisting of an organic acid and ammonium formate (AF), so as to obtain a mixture containing DES, amorphous chitin and packed chitin nanowhiskers;

[0022] d) adding water to the mixture of step c) and heating in the presence of carbon dioxide to obtain a mixture containing DES, water, amorphous chitin and disintegrated chitin nanowhiskers;

[0023] e) separating the disintegrated chitin nanowhiskers from the mixture of step d) to obtain a solid powder of chitin nanowhiskers; and

[0024] optionally:

[0025] f) dispersing the solid powder of chitin nanowhiskers of step e) in aqueous solution; and / or

[0026] g) (re)dispersing the solid powder of chitin nanowhiskers of step e) in an aqueous acidic solution and subsequent further treatment to obtain further disintegrated chitin nanowhiskers.

[0027] The invention further relates to crystalline chitin nanowhiskers obtainable by the method of the invention.

[0028] Finally, a further aspect of the invention is the use of the crystalline chitin nanowhiskers of the invention in the field of environmentally-friendly biomaterials, e.g. usable as food and non-food film, having oxygen barrier effect, fillers for composite materials, for medical -biomedical applications, and for pharmaceuticals and cosmetics.Brief description of the figures

[0029] Figure 1: XRD spectra of commercial chitin and sample 1 of Example 1;

[0030] Figure 2A-D: SEM images of the samples listed in Table 2 of Example 3;

[0031] Figure 3A-D: TEM analysis of the samples listed in Table 2 of Example 3;

[0032] Figure 4A-C: SEM images of the samples listed in Table 2 of Example 3;

[0033] Figure 5A-B: TGA analysis of the samples in Table 2 of Example 3;

[0034] Figure 6: FT-IR spectra of the samples in Table 2 of Example 3;

[0035] Figure 7: FT-IR spectrum of sample 5 in Table 4 of Example 6;

[0036] Figure 8: XRD spectra of the waste raw material, commercial chitin and sample 5 of Example 6 and 7;

[0037] Figure 9: FT-IR spectra of the waste raw material, commercial chitin and sample 5 of Example 6 and 7;

[0038] Figure 10: TGA analysis of the waste raw material, commercial chitin and sample 5 of Example 6 and 7;

[0039] Figure 11A-F: SEM images of samples A: ground crab shell; B, C: nanowhiskers obtained after lyophilization of the dispersion obtained by means of the procedure reported in Table 4, sample 5; E: photograph of film obtained by solvent evaporation from the dispersion obtained by means of the procedure reported in Table 4, sample 5; D, F: Film of nanowhiskers obtained after solvent evaporation of the dispersion obtained by means of the procedure shown in Table 4, sample 5;

[0040] Figure 12A-C: TEM images of nanowhiskers obtained by means of the procedure shown in Table 4, sample 5.

[0041] Figure 13: 'H-NMR spectra of the nanowhiskers obtained with the method of invention.

[0042] Figure 14: Enlarged views of the spectrum of Figure 13 showing (a) 8.75-8.25 ppm and (b) 1.65-1.15 ppm. The presence of aldehyde moieties (formylation of hydroxyl and -free amino groups) is shown by the peaks in the range 8.75 - 8.25 ppm (14a), while the peaks ~ 1.5 ppm are due to the lactate moieties (14b).

[0043] Detailed description of the invention

[0044] To address the disadvantages of the prior art, a method for the production of nanowhiskers is therefore proposed herein which, starting from raw materials comprising chitin, also from waste and / or scrap, eliminates the onerous passage of chitin isolation, reduces the amount of chemicals required and decreases the volume of the process.The invention relates to a method for the recovery and purification of crystalline chitin nanowhiskers comprising or consisting of the following steps:

[0045] a) providing a raw material comprising chitin;

[0046] b) washing, grinding and sieving the raw material of step a) to obtain a powder comprising chitin and having a uniform particle size less than 250 micrometres;

[0047] c) dissolving the powder of step b) in a deep eutectic solvent (DES) comprising or consisting of an organic acid and ammonium formate (AF), so as to obtain a mixture containing DES, amorphous chitin and packed chitin nanowhiskers;

[0048] d) adding water to the mixture of step c) and heating in the presence of carbon dioxide to obtain a mixture containing DES, water, amorphous chitin and disintegrated chitin nanowhiskers;

[0049] e) separating the disintegrated chitin nanowhiskers from the mixture of step d) to obtain a solid powder of chitin nanowhiskers; and

[0050] optionally:

[0051] f) dispersing the solid powder of chitin nanowhiskers of step e) in aqueous solution; and / or

[0052] g) (re)dispersing the solid powder of chitin nanowhiskers of step e) in an aqueous acidic solution and subsequent further treatment to obtain further disintegrated chitin nanowhiskers.

[0053] In the present invention, the following terms have the following definitions:

[0054] "raw material comprising chitin" means a raw material comprising chitin in the form of microfibrils or bundles of highly oriented fibrils, packed and held together by Van der Walls forces or hydrogen bonds, formed by chitin nanofibrils containing highly oriented chitin nanowhiskers, all incorporated within an amorphous chitin matrix that surrounds or alternates chitin crystalline nanostructures;

[0055] "nanowhiskers" means rigid crystalline nanostructures in the form of a bar or needle with a length between 100 and 500 nanometres and a width between 2 and 20 nanometres;

[0056] "packed" means highly oriented nanowhiskers that can be part of or form chitin nanofibrils closely interconnected by Van der Walls forces or hydrogen bonds, which in turn constitute more complex structures such as microfibrils or highly oriented chitin bundles.

[0057] "DES" or "deep eutectic solvent" means a solvent composed of two or more components, of which a hydrogen bond donor (HBD) and a hydrogen bond acceptor(HBA), which interact with each other by self-associating, by means of hydrogen bonding interactions and Van der Waals forces, forming a eutectic mixture with a melting temperature much lower than that of its components. In the case of the present invention, the hydrogen bond acceptor (HBA) is represented, for example, by ammonium formate (AF) and the hydrogen bond donor (HBD) is represented, for example, by organic acid.

[0058] The raw material comprising chitin of step a) preferably comprises any synthetic and / or biological source of chitin, more preferably biological, even more preferably chosen from mushrooms, algae and crustaceans.

[0059] In an embodiment, the raw material comprising chitin of step a) derives from waste, preferably agri-food waste, more preferably agri-food waste deriving from crustaceans, even more preferably deriving from the shells of crustaceans.

[0060] The process of the invention allows the use not only of synthetic chitin, but also of waste derived from different sources, including agri-food, with a view to green and circular industry.

[0061] Step b) involves washing, grinding and sieving the raw material, preferably from food waste, of step a), to obtain a powder having a uniform particle size less than 250 micrometres, preferably in a range from 250 to 10 micrometres, more preferably from 100 to 10 micrometres, even more preferably from 50 to 10 micrometres.

[0062] Preferably the washing of step b) takes place in water.

[0063] Also preferably, the grinding of step b) takes place in a mortar, mill, blender, millstone, seed grinder, ball miller, more preferably in a mortar.

[0064] The washing has the purpose of eliminating any residues adhering to the raw material comprising chitin.

[0065] Subsequently, the powder is sieved in step b) by means of a stainless steel mesh laboratory sieve, until a uniform particle size of less than 250 micrometres is obtained.

[0066] The fine and uniform particle size guarantees an effective performance of the subsequent steps of the method of the invention and promotes the dissolution of the raw material in the DES, allowing a better penetration thereof into the powder matrix, with a consequent reduction in the formation of clots and precipitation thereof.

[0067] Step c) involves dissolving the powder of step b) in a deep eutectic solvent (DES) comprising or consisting of an organic acid and ammonium formate (AF).

[0068] Preferably, the organic acid of the DES of step c) is chosen from glycolic acid, ascorbic acid and lactic acid, preferably it is lactic acid or glycolic acid, more preferably it is lactic acid.Using a DES has the advantage of not requiring the use of acids and bases to deproteinize and demineralize, in separate steps, the powder comprising chitin.

[0069] In fact, the specific DES based on an organic acid and ammonium formate (AF), contains ammonium formate which guarantees the demineralization and de-proteinization of the raw material comprising chitin and the solubilization within the DES of amorphous chitin, promoting the purification of chitin without the use of a double step with strong acid and strong base for the elimination of minerals and proteins, and an organic acid that favours the dissolution of chitin in amorphous form, freeing the crystalline chitin nanowhiskers without therefore requiring the further use of a strong acid.

[0070] Furthermore, the specific DES of the method of the invention is readily available and inexpensive.

[0071] The method of the invention, by means of its steps and thanks to the aforesaid characteristics of DES, therefore allows the recovery and purification of chitin nanowhiskers having high crystallinity, directly from the raw material, in a single step.

[0072] Preferably in step c) the ratio of DES to powder of step b) is from 50:1 to 10:1 w / w, preferably 15:1 w / w or 20:1 w / w.

[0073] The ratio between the two components is such as to ensure the homogeneity of the final mixture, avoiding the formation of agglomerates, and at the same time ensuring good reaction kinetics without an excessive use thereof.

[0074] Preferably, the deep eutectic solvent (DES) of step c) has a molar ratio between ammonium formate (AF) and the organic acid comprised in a range from 4:1 to 1:10 preferably of 2:1.

[0075] Preferably, the ratio of ammonium formate (AF) : powder of step b) is greater than 9:1 w / w, and the ratio of acid to powder of step b) is greater than 6:1 w / w.

[0076] The above ranges ensure that the DES of the invention effectively dissolves amorphous chitin, ensures good acylation of the amine and hydroxyl groups, thus allowing subsequent separation, remaining liquid and homogeneous.

[0077] In an embodiment, the powder of step b) is dissolved in step c) in the DES by stirring, preferably at a speed of 1500 to 400 rpm, more preferably of 600 rpm.

[0078] Preferably, the stirring of step c) takes place at room temperature, for example at a temperature ranging from 30 to 20°C, preferably 25°C.

[0079] Also preferably, the stirring of step c) takes place for a time from 170 hours to 2 minutes, more preferably from 72 to 48 hours, even more preferably of 24 hours or 5 minutes.Step c) has the advantage, especially in the case of waste raw material, in the dispersion of the raw material within the DES solvent, of facilitating the demineralization with consequent separation of any calcium carbonate contained in the waste comprising chitin and the subsequent separation of the amorphous chitin from the other components of the raw material, including waste.

[0080] From step c), in fact, a mixture containing DES, water, amorphous chitin and packed chitin nanowhiskers is obtained, specifically a mixture in which the nanowhiskers are separated from the amorphous chitin.

[0081] The method comprises a step d) of adding water to the mixture of step c) and heating in the presence of carbon dioxide.

[0082] Preferably, the carbon dioxide of step d) is added in gaseous form, always preferably at a pressure from 1 bar to 50 bar, more preferably from 2 to 20 bar, even more preferably from 4 to 10 bar, even more preferably of 8 bar.

[0083] Step d) takes place by heating, preferably to a temperature of from_80°C to 150°C, more preferably from 90°C to 120°C, even more preferably of 100 °C or 150 °C and for a time ranging from 2 to 10 hours, preferably 3 hours.

[0084] Also preferably, the heating of step d) takes place under stirring, more preferably at a speed from 1500 to 400 rpm, even more preferably of 600 rpm.

[0085] Step d) takes place by adding water to the mixture of step c), preferably in an amount of 1 to 5 equivalents with respect to the DES of step c).

[0086] The water of step d) is preferably deionized or milliQ.

[0087] Still preferably, the final molar ratio used in step d) is EhCkAF: organic acid = 1-5: 2:1, more preferably 5:2:1.

[0088] The addition of water and carbon dioxide (CO2), preferably in the amounts and pressures described above, during heating, allows to better treat the chitin contained in the raw material of step b), increasing the degree of crystallinity of the resulting crystalline chitin nanowhiskers, disintegrating them from the packed form and thus improving the reaction efficiency.

[0089] The addition of water contributes to the decrease in the viscosity of DES, so as to facilitate the dispersion of the raw material powder of step b), without altering the characteristics of the DES and its ability to improve the reaction kinetics, while the concomitant use of carbon dioxide in aqueous solution guarantees modulable and reversible acidic conditions, since the aforesaid depend on the reaction between carbondioxide and water during its addition in solution, with modulable but reversible development of carbonic acid.

[0090] In fact, on the one hand the addition of carbon dioxide in aqueous solution allows the reaction to form carbonic acid which guarantees an acidic pH to the DES, on the other hand, at the end of the addition of anhydride in water, the pH returns to neutral conditions without further use of strong bases.

[0091] The method of the invention, also by means of step d) of adding water and carbon dioxide, allows the recovery and purification of crystalline chitin nanowhiskers directly from the raw material comprising chitin, for example of step b), without further steps; said nanowhiskers having a molecular weight suitable to keep them in dispersion and having a better poly-dispersity index and crystallinity.

[0092] The method involves a step e) of separating the disintegrated crystalline chitin nanowhiskers from the mixture of step d).

[0093] From step d), in fact, a mixture containing DES, water, amorphous chitin and disintegrated chitin nanowhiskers is obtained, and specifically a mixture in which the minerals and residual proteins and the amorphous chitin remain dissolved in the aqueous solution containing the DES, while the crystalline chitin nanowhiskers precipitate and are therefore separated from the aqueous DES in the next step e).

[0094] Preferably, the separation of step e) comprises a washing step el), which takes place, more preferably, with water and possibly at least one polar protic or aprotic solvent, preferably chosen from ethanol, ethanol, acetone, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF) and water, even more preferably with deionized water, even more preferably in an added amount from 10 to 50 ml.

[0095] In an advantageous form, the washing step el) takes place at a concentration of 0.1% w / w, therefore 0.5 g of nanowhiskers are suspended in 50 g of water.

[0096] When washing with water alone, this step el) has the further advantage of being an economically advantageous solvent.

[0097] Also preferably, step e) comprises a centrifugation step e2), which takes place, more preferably, in a centrifuge, even more preferably at a speed from 1000 to 9000 rpm, even more preferably 9000 rpm.

[0098] The centrifugation of step e2) preferably takes place for a time from 2 to 20 minutes, more preferably 10 minutes.

[0099] Step e) preferably comprises a separation step e3), more preferably separation by sedimentation.Step e) preferably comprises a filtration step e4).

[0100] Steps el) - e4) can be repeated several times in this sequence and preferably until neutral pH is reached, more preferably possibly up to three times.

[0101] The method involves an optional step f) of dispersing the solid powder of chitin nanowhiskers of step e) in aqueous solution, preferably in water. This step allows the storage of the nanowhiskers, keeping them stable over time.

[0102] At this step, or as an alternative thereto, there may optionally be present an optional step g) of (re)dispersing the solid powder of chitin nanowhiskers of step e) in an acidic aqueous solution and subsequent further treatment to obtain further disintegrated chitin nanowhiskers.

[0103] Preferably step g) takes place by means of ultrasonic treatment or mechanical treatments such as the use of an extruder, microfluidizer, high-pressure homogenization.

[0104] Step g) has the advantage of promoting and improving the breaking of hydrogen bonds and Van der Walls forces that keep the chitin nanowhiskers connected.

[0105] Preferably, the acidic aqueous solution has a pH of from 1 to 5, more preferably 3. Still preferably, a 5% w / w acetic acid solution is added to the aqueous solution until a pH of 1 to 5, more preferably equal to 3, is reached.

[0106] Preferably, step g) takes place by ultrasonication, more preferably by means of an ultrasonic probe, even more preferably by means of the Branson sonifier SFX 250 probe, whose maximum power is 250 Watts, at amplitude 35.

[0107] Also preferably, step g), preferably ultrasonication, takes place for a time from 2 minutes to 60 minutes, more preferably 40 minutes.

[0108] Also preferably, step g) takes place from 100 to 480 cycles, preferably 480 cycles of ultrasonication.

[0109] Step g) preferably has a duration of 5 seconds interspersed with 5 seconds of pause. Step g) allows a further stabilization and homogenization, as well as a further improvement in the separation / disintegration of the nanowhiskers obtained from step e) or f), since it favours the disintegration of any aggregates remaining from step e) or f), while having no effect on the molecular weight and poly dispersity index of the final nanowhiskers of step e) or f).

[0110] In a preferred form, the method of the invention comprises a further step h) of drying, preferably by lyophilization or evaporation of the solvent.

[0111] The invention further relates to crystalline chitin nanowhiskers obtainable by the method of the invention.In an embodiment, the crystalline chitin nanowhiskers obtained or obtainable by the method of the invention are characterized by a molecular weight of 3.5*105g / mol to 4*105g / mol, preferably 3.5*105g / mol, obtained by GPC.

[0112] The molecular weight in this range has the advantage of having nanowhiskers with high crystallinity and satisfactory dimensions, so that subsequent applications can exploit the potential of a highly homogeneous starting matrix.

[0113] Preferably, the crystalline chitin nanowhiskers obtained or obtainable by the method of the invention are characterized by a polydispersity index (PDI) between 1.79 and 2.19, preferably 2.13.

[0114] The polydispersity index in this range has the advantage of having nanowhiskers that are similar to each other in terms of size, providing a material that has homogeneous chemical -phy sical characteri sties .

[0115] In an advantageous form, the crystalline chitin nanowhiskers obtained or obtainable by the method of the invention have a degree of acetylation between 84.5% and 98.2%, preferably higher than 97%.

[0116] In fact, the method of the present invention allows to recover and purify nanowhiskers with a high degree of acetylation, and higher than commercial chitin, which is usually obtained by conventional extraction with a strong base. Furthermore, chitin with a high degree of acetylation, for example >97%, is usable in applications where the use of nanowhiskers with fully protected amino group is required.

[0117] In fact, this occurs because DES can also act in this system by REM (reactive eutectic media), acetylating the free amino groups and, partially, the secondary hydroxyl groups present in the structure. In this sense, the nanowhiskers obtainable or obtained by the method of the invention, employing the aforesaid DES, possess hydroxyl groups protected by acyl and aldehyde groups that prevent the formation of stable hydrogen bonds once the nanowhiskers are lyophilized or solid obtained by solvent evaporation. This can lead to the production of nanowhiskers that can thus be transported anhydrides and redispersed or re-solubilized in water at a later time.

[0118] Finally, a further aspect of the invention is the use of the crystalline chitin nanowhiskers of the invention in the field of environmentally-friendly biomaterials, e.g. usable as food and non-food film, having oxygen barrier effect, fillers for composite materials, for medical -biomedical applications, and for pharmaceuticals and cosmetics.

[0119] The following examples further illustrates the invention.EXAMPLES

[0120] Materials and Methods

[0121] All the chemicals (ammonium formate, DL-lactic acid) were purchased from Sigma Aldrich (Merck) and used without further treatment.

[0122] Commercial chitin was purchased from TCI and used without further purification. The shells of crustaceans, for example spider crab, were obtained from waste from restaurants using raw materials of local origin (Adriatic Sea).

[0123] SEM and TEM images of the nanowhiskers are obtained by scanning electron microscopy (SEM) and were recorded with the Gemini 1550, Zeiss AG (Oberkochen, Germany) at an acceleration voltage of 3.00 kV. Images obtained using transmission electron microscopy (TEM) were acquired with a TEM 912 Omega, Zeiss AG (Oberkochen, Germany), operating at 120 kV. The samples were negatively stained using an aqueous solution of uranium acetate.

[0124] X-ray diffraction spectra of the nanowhiskers were recorded in the 29 range of 5°-60° at a rate of 1.5° / min with the Rigaku SmartLab instrument, with a Cu-Ka radiation source ( = 1.5418 A). The crystallinity index (CrI) was calculated according to the equation CrI110= FOiio MOIxoo where Ino is the maximum intensity at 29 = 19.5° and Iamis the intensity of L Mo J

[0125] amorphous diffraction at 29 = 16.0°.

[0126] TGA and DTG of the nanowhiskers was performed by means of a TG 209 Fl Libra thermomicroscale (Netzsch, Selb, Germany) under nitrogen atmosphere with a constant flow of 100 mL / min. Each measurement was made using 10±l mg of the lyophilized sample, which was heated from 25 to 600 °C at a scanning rate of 10 °C / min in an aluminium crucible. The degradation temperature was defined as the temperature of onset of weight loss in the obtained TGA curves.

[0127] Elemental Analysis of EA3000 nanowhiskers (EuroVector Sri, Pavia, Italy) in CHNS mode.

[0128] ICP-OES of the nanowhiskers Perkin Elmer ICP-OES Optima 8000.

[0129] FT-IR / ATR spectra of the nanowhiskers were performed using Nicolet i S 5 with an ATR iD5 crystal, Thermo Fisher Scientific Inc. (Waltham, Massachusetts, USA). All spectra were recorded between 4000 cm’1and 400 cm’1with a resolution of 0.5 cm’1in absorbance mode for 16 scans at room temperature.

[0130] spectra of the nanowhiskers was performed by dissolving the samples of chitin and nanowhiskers in a solution containing 35% deuterated hydrochloric acid in deuterated water inan ultrasonic bath for 30 minutes at 50°C. 'H-NMR spectra were obtained using a Bruker Ascend 400 (AV400) NMR spectrometer, operating at 400 MHz for 'H nuclei. The DA (deacetylation) values were calculated from the NMR spectra using the equation DA% =

[0131]

[0132] GPC of the nanowhiskers GPC-Agilent / PSS 1260 Iso Pump instrument using a PSS-gram-100 column and a PSS-gram-1000 column in series, after a PSS-gram pre-column. For each analysis, 5 mg of sample were solubilized in 5% LiCl / NMP (sample concentration: 1 mg / mL) by stirring at room temperature until it completely dissolves. The solution was then filtered through a 0.45 pm syringe filter. The sample was eluted with 5% LiCl-NMP (flow rate, 0.5 mL / min, injection volume: 100 pL, temperature: 70°C, measurement time: 60 minutes) and the PSS SECcurity-UV-300 nm-1260 VWD and PSS SECcurity-RI-1260 RID detectors were used. The calibration curves were constructed using polystyrene as internal standard.

[0133] Zeta potential of nanowhiskers Zetasizer Nano ZS, Malvern Instruments (Malvern Panalytical, UK), equipped with a 632 nm HeNe laser and operating at a detection angle of 173 degrees. The colloids were diluted prior to measurements and placed in a disposable collapsible capillary cell. The measured electrophoretic mobility was converted to zeta-potential using Smoluchowski's equation.

[0134] Example 1 - synthesis of chitin nanowhiskers starting from commercial chitin A DES was formed having lactic acid (LA) and ammonium formate (AF) in a 1:2 mol / mol ratio.

[0135] The commercial chitin was washed, and possibly ground and sieved on a sieve with 250 micrometre mesh (step b)).

[0136] Subsequently 0.5 g of the chitin obtained and the DES (DES : chitin ratio = 15:1 w / w) were loaded in a Teflon reactor equipped with a magnetic stirrer (step c)).

[0137] The mixture was mixed (600 rpm) at room temperature for 5 minutes, then 2.5 equivalents of deionized water were added and the Teflon reactor was placed in a thermally controlled autoclave and subsequently carbon dioxide (CO2) was added at a pressure of 8 bar. The reactor was then heated at 100°C for 3 hours with continuous stirring (600 rpm) (step d)).

[0138] After 3 hours, the reaction was cooled to room temperature in an ice bath and 40 mL of deionized water was added to the reaction mixture.

[0139] The mixture was centrifuged (9000 rpm for 10 minutes) to separate the crystalline chitin nanowhiskers from the residual amorphous chitin. The solid powder of chitin nanowhiskers was washed with deionized water and centrifuged again: this procedure was repeated at leastthree times, or in any case until a neutral pH was obtained (step e)).

[0140] The solid powder of centrifuged chitin nanowhiskers was then re-dispersed in water, the pH of the dispersion was adjusted to about 3 with 5% v / v acetic acid and the solution was ultrasonicated using a Branson SFX 250 ultrasonic probe (amplitude 35) for a time of 40 minutes, (480 cycles of 5 seconds) (step g)).

[0141] The same synthesis under the same conditions was repeated using ascorbic acid (AA) and glycolic acid (GA) as the organic acid.

[0142] The samples were subsequently analysed and the results are reported in Table 1.

[0143] Table 1

[0144]

[0145] All the acids tested thus demonstrated the formation of a mixture comprising crystalline, disintegrated and electrostatically stabilized chitin nanowhiskers.

[0146] Example 2- synthesis of chitin nanowhiskers starting from waste chitin

[0147] A DES was formed having lactic acid (LA) and ammonium formate (AF) in a 1 :2 mol / mol ratio.

[0148] The chitin from waste, i.e. the waste of crab shells, containing chitin, was washed, ground and sieved on a sieve with a 250 micrometre mesh (step b)).

[0149] Subsequently 0.5 g of the obtained waste chitin and DES (DES : chitin ratio = 20:1 w / w) were loaded in a Teflon reactor equipped with a magnetic stirrer (step c)).

[0150] The mixture was mixed (600 rpm) at room temperature for 48 hours, then 5 equivalents of deionized water were added and the Teflon reactor was placed in a thermally controlled autoclave and subsequently carbon dioxide (CO2) was added at a pressure of 8 bar. The reactor was then heated at 150°C for 3 hours with continuous stirring (600 rpm) (step d)).

[0151] After 3 hours, the reaction was cooled to room temperature in an ice bath and 50 mL of deionized water was added to the reaction mixture.

[0152] The mixture was centrifuged (9000 rpm for 10 minutes) to separate it. The solid powder of chitin nanowhiskers was washed with fresh water and centrifuged again: this procedure was repeated three times, or in any case until a neutral pH was obtained (step e)).

[0153] The solid powder of centrifuged chitin nanowhiskers was then re-dissolved in water, thepH of the solution was adjusted to about 3 with 5% v / v acetic acid, and the solution was ultrasonicated using an ultrasonic probe (amplitude 35) for a time of 40 minutes, 480 cycles of 5 seconds) (step g)).

[0154] Example 3 - influence of step c) in the synthesis of nanowhiskers according to Example 1

[0155] Four samples were analysed starting from commercial chitin as a starting material, to analyse the variation of the molecular weight, poly-dispersity index and crystallinity index, after the treatment with DES, the addition of water and the treatment with CO2 of the invention.

[0156] The following samples were analysed:

[0157] 1) first sample (commercial chitin): the commercial chitin was washed, and possibly ground and sieved on a 250 micrometre mesh sieve (step b)), dissolved in water, the pH of the solution was adjusted to about 3 with 5% v / v acetic acid and the solution was ultrasonicated using an ultrasonic probe (amplitude 35) for a time of 40 minutes, 480 cycles of 5 seconds) (step g)). After the aforesaid treatments, the chitin was analysed, reporting its molecular weight, polydispersity index and crystallinity index. Commercial chitin is the starting material for the following samples.

[0158] 2) second sample (sample 1): commercial chitin (already subjected to step b)) was treated with DES (lactic acid (LA) and ammonium formate (AF) in a 1:2 mol / mol ratio), i.e. 0.5 g of commercial chitin and DES (DES : chitin ratio = 15:1 w / w) was loaded in a Teflon reactor equipped with a magnetic stirrer (step c)).

[0159] After stirring at 600 rpm for 5 minutes, the reactor thus loaded was then heated at 100°C for 3 hours with continuous stirring (600 rpm), without the addition of either water or carbon dioxide.

[0160] After 3 hours, the reaction was cooled to room temperature in an ice bath and 40 mL of deionized water was added to the reaction mixture.

[0161] The mixture was centrifuged (9000 rpm for 10 minutes) to separate it.

[0162] The solid powder of chitin nanowhiskers was washed with fresh water and centrifuged again: this procedure was repeated three times, until a neutral pH was obtained (step e)).

[0163] The solid powder of centrifuged chitin nanowhiskers was then re-dispersed in water, the pH of the solution was adjusted to about 3 with 5% v / v acetic acid, and the solution was ultrasonicated using an ultrasonic probe (amplitude 35) for a time of 40 minutes, 480 cycles of 5 seconds) (step g)).3) third sample (sample 2): commercial chitin (already subjected to step b)) was treated with DES (lactic acid (LA) and ammonium formate (AF) in a 1:2 mol / mol ratio), i.e. 0.5 g of commercial chitin and DES (DES : chitin ratio = 15:1 w / w) was loaded in a Teflon reactor equipped with a magnetic stirrer (step c)).

[0164] After stirring at 600 rpm for 5 minutes, 2.5 equivalents of deionized water was added. The final molar ratio of the suspension was water: HBA : HBD equal to 2.5:2:1. The reactor thus loaded was then heated at 100°C for 3 hours with continuous stirring (600 rpm).

[0165] After 3 hours, the reaction was cooled to room temperature in an ice bath and 40 mL of deionized water was added to the reaction mixture.

[0166] The mixture was centrifuged (9000 rpm for 10 minutes) to separate it. The solid powder of chitin nanowhiskers was washed with fresh water and centrifuged again: this procedure was repeated three times, until a neutral pH was obtained (step e)).

[0167] The solid powder of centrifuged chitin nanowhiskers was then re-dissolved in water, the pH of the solution was adjusted to about 3 with 5% v / v acetic acid, and the solution was ultrasonicated using an ultrasonic probe (amplitude 35) for a time of 40 minutes, 480 cycles of 5 seconds) (step g)).

[0168] 4) fourth sample (sample 3): commercial chitin (already subjected to step b)) was treated with DES (lactic acid (LA) and ammonium formate (AF) in a 1:2 mol / mol ratio), i.e. 0.5 g of commercial chitin and DES (DES : chitin ratio = 15:1 w / w) was loaded in a Teflon reactor equipped with a magnetic stirrer (step c)).

[0169] After stirring at 600 rpm for 5 minutes, 2.5 equivalents of deionized water was added. The final molar ratio of the suspension was water: HBA : HBD equal to 2.5:2: 1. Subsequently, carbon dioxide (CO2) was added at a pressure of 8 bar. The reactor thus loaded was then heated at 100°C for 3 hours with continuous stirring (600 rpm) (step d)).

[0170] After 3 hours, the reaction was cooled to room temperature in an ice bath and 40 mL of deionized water was added to the reaction mixture.

[0171] The mixture was centrifuged (9000 rpm for 10 minutes) to separate it.

[0172] The solid powder of chitin nanowhiskers was washed with fresh water and centrifuged again: this procedure was repeated three times, until a neutral pH was obtained (step e)).

[0173] The solid powder of centrifuged chitin nanowhiskers was then re-dissolved in water, the pH of the solution was adjusted to about 3 with 5% v / v acetic acid, and thesolution was ultrasonicated using an ultrasonic probe (amplitude 35) for a time of 40 minutes, 480 cycles of 5 seconds) (step g)).

[0174] Samples 4, 5 and 6 were made following the same procedure as samples 1, 2 and 3 and have as their only difference the use of glycolic acid instead of lactic acid in the formation of DES.

[0175] The results of the analyses of Z potential, molecular weight, polydispersity index and crystallinity index of the aforesaid samples are reported in Table 2.

[0176] Table 2

[0177] >

[0178] >

[0179]

[0180] Sample 1 (treated with DES but without water or carbon dioxide) contains electrostatically stabilized nanoparticles, which form a dispersion in the supernatant, stable and having a positive zeta potential between 18.8 and 31.7 mV.

[0181] Sample 1 not only reported better performance than the initial commercial chitin, but showed a preservation of a high Crystallinity Index (CrI) (Figure 1), thanks to the presence of the organic acid, which promotes the hydrolysis and dissolution of the most susceptible amorphous part, while the crystalline region is only slightly altered.

[0182] The formation of chitin nanowhiskers after treatment with DES and water (sample 2) is confirmed by the decrease in the poly dispersity index (PDI) of the lyophilized chitin samples with respect to the starting material, calculated by GPC analysis.

[0183] The simultaneous addition of water and CO2 (sample 3) maintains the same PM as the nanowhiskers together with a clear decrease in PDI, thus leading to the formation of well-defined nanowhiskers of comparable length.

[0184] The DES / Water / CCE system (sample 3) also led to an increase in the crystallinity index (CrI) with respect to the starting material, thanks to a better removal of amorphous domains dueto the acidic character of the medium promoted by the presence of CO2.

[0185] Samples 4, 5 and 6, made following the same procedure as samples 1, 2 and 3 and having as their only difference the use of glycolic acid instead of lactic acid in the formation of DES, follow the same trend as samples 1, 2, 3 noting an improvement in the chemical-physical properties of the nanowhiskers obtained by descending along the series.

[0186] Sample 4 performed better than the initial commercial chitin in terms of average molecular weight.

[0187] All values improve with the addition of water (sample 5) leading to a clear improvement in zeta potential, average molecular weight, crystallinity index.

[0188] Sample 6 using the DES / Water / CCh system shows an excellent zeta potential and average molecular weight, a crystallinity index practically comparable to that of commercial chitin and a poly dispersity index lower than it, demonstrating in an even more tangible manner how the use of the DES / Water / CCh system is extremely advantageous regardless of the DES used.

[0189] Example 4 - morphological analysis (SEM and TEM) of the samples obtained according to Example 3

[0190] The synergy obtained by the steps of the method of the invention, i.e. by means of a DESAV ater / CCE system is also clearly supported by electron microscopy images. Specifically, Figures 2A-D report the SEM analyses of the samples listed in Table 2 of Example 3 using lactic acid as organic acid, while Figures 3 A-D report the SEM analyses of the samples listed in Table 2 of Example 3 using glycolic acid as organic acid.

[0191] The SEM images revealed a refinement in the separation and disruption of the chitin fibrils. Figures 2A-D and 3A-D clearly show the formation of a more defined material and a better individualization of the nanowhiskers, passing from Figure 2A (commercial chitin) to Figure 2D (chitin treated with DES system composed of AF:LA + water + CO2). The morphology of the starting material (Figure 2A) shows a highly compact structure with closely interconnected chitin fibrils, while images of the sample treated with lactic and glycolic acidbased DES (Figures 2B and 4A), with DES and water (Figures 2C and 4B), and finally with DES, water, and CO2, (Figures 2D and 4C) clearly highlight the disaggregation of the initial structure and the release of the individual nanowhiskers.

[0192] The TEM images of the samples treated with lactic acid-based DES (Figures 3A-D) demonstrate the formation of nanoscale chitin whiskers, the length of which can be approximated at 200-400 nm and the width at about 10-20 nm (Figure 3D).Example 5 - qualitative analysis (TGA and FT- IR) of the samples obtained according to Example 3

[0193] The TGA spectra (Figures 5A-B) of samples obtained using lactic acid as organic acid (Figure 5A) and glycolic acid (Figure 5B) confirmed the maintenance of chitin structure, showing only slight differences in Tonset, Tmax and residual mass at 600°C between nanowhiskers and starting material. The data are reported, for lactic acid, in Table 3.

[0194] Table 3

[0195]

[0196] The FT-IR spectra (Figure 6) showed that the chemical structure of the nanowhiskers is substantially the same as that of the native chitin for samples 1-6 of Example 3 and Table 2, demonstrating the preservation of the chitin skeleton.

[0197] In particular, the absorption peak at 3440 cm1is assigned to the elongation O-H; the two absorption bands represent the elongation N-H and the intramolecular bond N-H, which appeared at 3260 cm1and 3100 cm

[0198] Two absorption peaks at 1650 cm1and 1620 cm1are due to the amide band I, which divided into two absorption peaks. Finally, the absorption bands of amide II, relating to C-N-H elongation and N-H bending, appeared at 1550

[0199]

[0200] While the chitin skeleton is preserved, the nanowhiskers exhibit surface modifications to functional groups that impact their physicochemical properties (see Figures 13 and 14).

[0201] Example 6 - qualitative analysis (FT-IR, XRD and TGA) of the samples obtained in Example 2 with respect to conventional extraction conditions

[0202] The results of the molecular weight (by GPC), poly dispersity index and crystallinity index (by XRD) and degree of acetylation (by NMR) obtained from the samples starting from waste comprising chitin, are reported in Table 4 and compared with the data obtained by a conventional extraction method (i.e. by treatment with HC1 (1 M), HC1 : raw material = 40:1, 50°C, 3h; followed by treatment with NaOH (IM), NaOH : raw material = 20:1, 70°C, 3h). The DES used, when indicated, was for all AF:LA 2: 1 samples and used in quantity 20: 1 w / w withrespect to the waste raw material.

[0203] Table 4

[0204]

[0205] In fact, it was possible to obtain a chitin with a degree of acetylation (DA) between 97.5% and 98.2% by performing the reaction at 150°C, higher than the DA obtained by the conventional method (DA = 92.4%, sample 6, Table 3).

[0206] An FT-IR was carried out on sample 5 of Table 4, which confirmed the purity of the chitin obtained by the method of the invention (Figure 7).

[0207] Example 7 - qualitative analysis (XRD, FT-IR and TGA) of the samples obtained in Example 2 with respect to commercial chitin and waste material

[0208] Sample 5 of Table 4 (obtained according to the conditions of Example 2), starting from waste raw material chitin, was further compared with the waste starting material and with commercial chitin and the XRD spectra are reported in Figure 8.

[0209] The XRD analysis reported in Figure 8 confirmed the purity of the chitin nanowhiskers obtained according to the method of the invention and the low content of CaCCh (0.11% by weight), since all the peaks related to CaCCh disappeared while only the peaks of chitin were observable, when compared with the waste starting material and commercial chitin.

[0210] The FT-IR spectra reported in Figure 9 also demonstrated the purity of the nanowhiskers (sample 5 of Table 4) compared to the waste starting material and the commercial chitin.

[0211] The spectra of the chitin nanowhiskers obtained according to the method of the invention (sample 5 of Table 4) show the disappearance of the net peak at 870 cm1typical of calcite and of the bands between 1650 and 1450 cm1typical of proteins.

[0212] The comparison with commercial chitin (CC) confirmed the purity of the chitin of the nanowhiskers of the invention: the amide II peaks at 1310

[0213]

[0214] the frequencies of the amide I vibration bands in the region 1660-1600 cm1and the C-0 extensions at 1155

[0215]

[0216] 1070 and 1020 cm1were comparable to those of commercial chitin in both position and intensity, indicating that there were no changes in the amide-type hydrogen bonds.

[0217] The TGA analysis (Figure 10) of the nanowhiskers obtained by AF:LA + water+ CO2(sample 5 of Table 4) showed a single decomposition phase, confirming the presence of a single material with a curve comparable to that of commercial chitin (CC).

[0218] The decomposition temperature at Tso% is very similar (386°C compared to 388°C), while Tonset, Tmax and the residual mass at 600°C present only slight differences, presumably due to slight variations in chemical structure.

[0219] Example 8 - morphological analysis (SEM and TEM) of the samples obtained according to Example 2

[0220] Finally, the size, length and morphology of nanowhiskers (sample 5 of Table 4) extracted directly from crustacean biomass were investigated by SEM and TEM analysis.

[0221] The SEM images (Figures 11 A-F) of the nanowhiskers show evident differences with the starting material and the formation of filaments at the end of the treatments with the method of the invention.

[0222] By means of the solvent casting technique, the aqueous dispersion containing nanowhiskers is left in a Petri dish and the aqueous solvent is allowed to evaporate at room temperature and at 50% relative humidity, thus obtaining a transparent film (Figure 1 IE). SEM images of the film and its cross-section (Figure 11D-F) demonstrated the presence of nanostructures and filaments that come together when the film is formed.

[0223] TEM images (Figure 12A-C) showed that the nanowhiskers obtained from crustacean biomass appear slightly longer than those obtained from commercial chitin, showing a length of about 400-600 nm and a width of about 10 nm.

Claims

CLAIMS1. A method for the recovery and purification of crystalline chitin nanowhiskers comprising or consisting of the following steps:a) providing a raw material comprising chitin;b) washing, grinding and sieving the raw material of step a) to obtain a powder comprising chitin and having a uniform particle size less than 250 micrometres; c) dissolving the powder of step b) in a deep eutectic solvent (DES) comprising or consisting of an organic acid and ammonium formate (AF), so as to obtain a mixture containing DES, amorphous chitin and packed chitin nanowhiskers; d) adding water to the mixture of step c) and heating in the presence of carbon dioxide to obtain a mixture containing DES, water, amorphous chitin and disintegrated chitin nanowhiskers;e) separating the disintegrated chitin nanowhiskers from the mixture of step d) to obtain a solid powder of chitin nanowhiskers; andoptionally:f) dispersing the solid powder of chitin nanowhiskers of step e) in aqueous solution; and / org) (re)dispersing the solid powder of chitin nanowhiskers of step e) in an aqueous acidic solution and subsequent further treatment to obtain further disintegrated chitin nanowhiskers.

2. The method according to claim 1, wherein the raw material comprising chitin of step a) derives from a waste, preferably an agri-food waste, more preferably an agri-food waste deriving from crustaceans, even more preferably deriving from crustaceans.

3. The method according to any one of claims 1 or 2, wherein the organic acid of the DES of step c) is chosen from glycolic acid, ascorbic acid and lactic acid, preferably it is lactic acid or glycolic acid, more preferably it is lactic acid.

4. The method according to any one of claims 1-3, wherein in step c) the ratio of DES to powder of step b) is from 50:1 to 10:1 preferably 15:1 w / w or 20:1 w / w.

5. The method according to any one of claims 1-4, wherein step c) has a molar ratio between ammonium formate (AF) and the organic acid comprised in a range from 4:1 to 1:10 preferably 2:1.

6. The method according to any one of claims 1-5, wherein the ratio of ammonium formate (AF) : powder of step b) is greater than 9: 1 w / w, and the ratio of acid to powder of step b) is greater than 6:1 w / w.

7. The method according to any one of claims 1-6, wherein the carbon dioxide of step d) is added in gaseous form at a pressure from 1 bar to 50 bar, preferably from 2 to 20 bar, more preferably from 4 to 10, even more preferably 8 bar.

8. The method according to any one of the claims 1-7, wherein step d) takes place by heating to a temperature from 80°C to 150°C, preferably from 90°C to 120°C, more preferably 100 °C or 150 °C.

9. The method according to any one of claims 1-8, wherein step d) takes place by adding water to the mixture of step c) in an amount from 1 to 5 equivalents with respect to the DES of step c).

10. A crystalline chitin nanowhisker obtainable by the method according to any one of claims 1-9.

11. Use of the crystalline chitin nanowhiskers according to any one of claims 1-9 or 10 in the field of environmentally-friendly biomaterials, for example usable as food and non-food film, having oxygen barrier effect, fillers for composite materials, for medicalbiomedical applications, and for pharmaceutical and cosmetic products.

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