Patent Application
20250163188
The present technology relates to methods of extraction of chitin from chitin-containing biomass using mechanochemistry.
Biopolymers are triggering intense research interests for they are envisaged as renewable sources for materials and molecules. Chitin in particular is the second most abundant naturally synthesized polymer with yearly production levels in the billions of tons.
Chitin is a natural polysaccharide composed of β-(1-4)-linked 2-deoxy-2-acetamido-D-glucose units. Its amide functionality constitutes an interesting manifold for functionalization and applications. Moreover, owing to its antimicrobial activity, chitin has potential applications in food industry, pulp and paper, water treatment, cosmetics, and biomedicine.
Natural sources of chitin (or chitin-containing biomass) include crustacean shells which are produced at around 6 to 8 million tons annually. These resources however are generally discarded by seafood industries; thereby creating undesirable landfilled crustacean wastes which are expensive to dispose of, and cause environmental issues, strong odor during decomposition, and provide human health risks.
Chitin in such chitin-containing biomaterials generally exists in composite form, and in association with proteins and minerals such as calcium carbonate. To isolate chitin from such biomaterials the steps of deproteinization, demineralization, and bleaching (depigmentation) are necessary to obtain pure colorless chitin. Since the chitin-containing biomaterial, such as crustacean shells, have poor solubility in water due to their chitin-calcium carbonate-protein matrix, harsh chemicals and elevated temperatures have been traditionally employed for the extraction of chitin resulting in the release of corrosive effluents into the environment. Specifically, in the deproteinization step, proteins are removed by heating with basic solutions such as KOH and NaOH. In the demineralization step, minerals such as calcium carbonate are removed from the exoskeleton using concentrated inorganic or organic acids (
As can be seen in
Several other strategies have aimed to tackle these problems to offer alternative methods of extraction, such as by using glycerol, ionic liquids, enzymatic techniques, and bacterial fermentation methods (summarized in
Therefore, there is a need for alternative or improved methods of chitin extraction which overcome or reduce at least some of the above-described problems.
From a broad aspect, there present technology relates to methods of extraction of chitin from chitin-containing biomass using mechanochemistry.
From one aspect, the present technology relates to methods of extraction of chitin from chitin-containing biomass and comprise milling the chitin-containing biomass with an acid to obtain chitin, wherein the chitin obtained by the milling step is demineralized and deproteinized.
From another aspect the present technology relates to methods of extraction chitin from chitin-containing biomass which consist of milling the chitin-containing biomass with an acid to obtain chitin, wherein the chitin obtained by the milling step is demineralized and deproteinized.
From a further aspect, the present technology relates to methods of producing deproteinized and demineralized chitin which comprise of milling a chitin-containing biomass with an acid.
From yet another aspect, the present technology relates to methods of producing deproteinized and demineralized chitin which consist of milling a chitin-containing biomass with an acid.
In some aspects, the methods of the present technology conserve the natural environment by using natural resources such as chitin-containing wastes to extract chitin, and mitigate the environmental impact of existing methods of chitin extraction by providing methods which use minimal solvent, energy and create minimal effluents.
In some aspects, the methods of the present technology demineralize and deproteinize chitin in a single step.
In some aspects, the methods of the present technology are scalable.
In some aspects, the methods of the present technology are sustainable.
In some aspects, the methods of the present technology are performed in solid-state, wherein the milling step is performed in the absence of water.
In some aspects, the methods of the present technology produce intact chitin at high yields, and with high purity.
Reference will now be made to the accompanying drawings.
The use of “including”, “comprising”, or “having”, “containing”, “involving” and variations thereof herein, is meant to encompass the items listed thereafter as well as, optionally, additional items. In the following description, the same numerical references refer to similar elements.
It must be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term “about” in the context of a given value or range refers to a value or range that is within 20%, preferably within 10%, and more preferably within 5% of the given value or range.
As used herein, the term “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
The recitation herein of numerical ranges by endpoints is intended to include all numbers subsumed within that range (e.g., a recitation of 1 to 5 includes 1, 1.25, 1.33, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).
As used herein, the term “biomass” refers to an organic resource of material of biological origin.
As used herein, the term “chitin-containing biomass” refers to biomass rich in chitin, examples of which will be discussed further below.
As used herein, the term “milling” refers to the process of grinding, cutting, mixing, pressing or crushing a material.
As used herein, the term “sustainable” refers to a technology having a low short-term and long-term impact on the environment.
As used herein, the expression “green” refers to a technology which helps resolve or mitigate environmental impacts and/or conserves the natural environment and resources.
As used herein, the term “mechanochemistry” refers to the use of mechanical milling or shear to induce chemical reactions.
As used herein, the term “chitin” refers to a long chain polymer of N-acetylglucosamine, an amide derivative of glucose.
As used herein, the expression “intact chitin” refers to a chitin molecule having a long or preserved chain structure.
As used herein, the expression “pure chitin” refers to chitin that is substantially free of other components such as proteins and minerals with which chitin is associated in biomass materials. In most arthropods, for example, chitin is often modified, occurring largely as a component of composite materials, such as in sclerotin, a tanned proteinaceous matrix, which forms much of the exoskeleton of insects. In the shells of crustaceans and mollusks chitin is combined with calcium carbonate and proteins to form a strong composite. In its pure, unmodified form, however, chitin is translucent, pliable, resilient, and tough.
As used herein the expression “complete” in reference to demineralization and deproteinization refers to a chitin obtained which is substantially free of minerals and/or substantially free of protein.
As used herein the term “substantially” means to a great or significant extent.
The present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
The present technology stems from the discovery by the present inventors of novel methods of chitin extraction from chitin-containing biomass. The inventors have surprisingly discovered that mechanochemistry or milling of chitin-containing biomass with organic or mineral acids leads to the deproteinization and demineralization of chitin and extraction of same in a single step reaction. Methods of the present technology result in the extraction of pure high-grade chitin with ash contents of less than about 0.5% and crystallinities of more than 60%, indicating complete deproteinization and demineralization of chitin from the biomass. The yield of chitin obtained from these methods may be up to about 50% which is significantly higher than known/traditional methods. The methods of the present technology reduce the use of harsh chemicals in comparison to the traditional chemical methods, thereby offering a green and sustainable method of chitin extraction compared to traditional methods.
In one aspect the methods of the present technology comprise milling the chitin-containing biomass with an acid to obtain chitin, wherein the chitin obtained by the milling step is demineralized and deproteinized. In another aspect, the present technology relates to methods of producing deproteinized and demineralized chitin comprising milling a chitin-containing biomass with an acid.
Chitin-containing biomass suitable for the methods of the present technology include shells or cuticles of crustaceans, such as shrimps or crabs, arthropods, insects, squids, shellfish, krill, or the like, or fungi, such as mushrooms. In certain embodiments, the methods of the present technology extract chitin from crustacean shells selected from shrimp shells, crab shells, and lobster shells, and combinations thereof.
In certain embodiments, the crab shells are selected from Green Crab shells, and Snow Crab shells, and combinations thereof. In other embodiments, the crab shells are the European Green Crab shells. The European Green Crab is among the ten most unwanted species of the planet and has been recognized as a serious environmental threat. Therefore, from an environmental standpoint, the use and recycling of Green Crabs is of interest. In yet other embodiments, the crab shells are Snow Crab shells. The high content of chitin in Snow Crab shells makes them an especially valuable source of biopolymers, and well-suited for the methods of the present technology.
The chitin-containing biomass or shells may be defleshed and isolated by any method known in the art. In some embodiments, the chitin-containing biomass may be defleshed manually, for example, to isolate the shells, and thawed in boiling water for between about 2 to 15 minutes. The shells can then be dried at room temperature or other suitable temperatures overnight, or for longer durations as needed to dry the shells.
In further embodiments, the dried chitin-containing biomass may be subjected to rough homogenization into powder form prior to use in the present methods. The rough homogenization increases the ease of contact with the acid to promote deproteinization and demineralization. Homogenization of the isolated shells may be performed by using a blender, a rough pulverizer, such as a shredder, a jaw crusher, a gyratory crusher, a cone crusher, a hammer crusher, a roll crusher, a roll mill; a medium pulverizer such as a stamp mill, an edge runner, a cutting/shearing mill, a rod mill, an autogenous mill, or a roller mill. In certain embodiments, the duration of homogenization is such that the biomass is uniformly and finely powdered as a result of the treatment. In some embodiments, the size of particles of the powdered shells is between about 10 μm and about 100 μm. In other embodiments, the size of the particles may be between about 10 μm and about 20 μm, between about 20 μm and about 50 μm, between about 50 μm and about 70 μm, or between about 70 μm and about 100 μm.
The chitin-containing biomass in powder form contains impurities such as protein, phosphoric acid, iron, copper, zinc, molybdenum, silicon, aluminum, calcium, magnesium, potassium, sodium, calcium carbonate and other minerals derived from such raw material.
Mechanochemistry is currently the topic of intense research effort, in particular for biomass conversion. Mechanochemical methods tackle the issues related to solubility in common solvents, separation or selectivity, while cutting overall effluents and energy demands. Previous methods such as those disclosed in PCT/CA2019/051048 (incorporated herein by reference) use mechanochemistry together with aging to deacetylate chitin and to thereby produce chitosan. Such methods for example include amorphizing chitin-containing powdered shells for 30 min in ZrO2 jar with ZrO2 ball, immediately mixing and milling the amorphized shells with NaOH, and aging to yield deacetylated chitosan. Other methods, such as those disclosed in EP 3 450 462 (incorporated herein by reference) use mechanochemistry to produce chitin oligomers typically containing about two to seven N-acetylglucosamine (NAG) molecules (monomers), NAG, and a 1-O-alkyl-N-acetylglucosamine (methanolysis product and NAG derivative) from chitin-containing biomass through a hydrolysis reaction of chitin by pulverizing the chitin-containing biomass with a pulverization apparatus in the co-presence of water and an acid catalyst selected from phosphoric acid, nitrous acid, and an organic acid. To date, however, the use of mechanochemistry in the extraction of intact chitin having a long or preserved chain structure from chitin-containing biomass has not been explored.
In certain embodiments, mechanochemistry is performed by milling the chitin-containing biomass with an acid. Surprisingly, compared to the traditional methods of extraction, as discussed above, the methods of the present technology result in the extraction/production of deproteinized and demineralized chitin by milling with an acid only, thereby reducing the time needed to extract chitin, the number of chemicals and/or solvents used in the reaction and the resultant release of corrosive effluents into the environment. Therefore, in certain embodiments, the methods of the present technology are said to comprise a single step.
In certain embodiments, the milling step is performed in solid state, using a solid acid. In other words, the milling is performed in the absence of solvents, such as water.
In other embodiments, the milling step may be performed in the presence of a liquid. This technique is also known as liquid-assisted grinding (LAG). According to the definition of LAG, mechanochemical reactivity is affected by the ratio (η) of the liquid additive relative to the weight of solids. In this definition, LAG lies in the range of η≈0-1 μL/mg, while η>10 μL/mg corresponds to a typical solution reaction, and 1<η<10 μL/mg indicates slurry reactions. In certain implementations of these embodiments n may be between about 0.2 μL/mg and about 5 μL/mg. In other embodiments, n may be between about 0.2 μL/mg and about 1.0 μL/mg, between about 0.5 L/mg and about 1.5 μL/mg, between about 1.0 μL/mg and about 2.0 μL/mg, between about 2.0 μL/mg and about 3.0 μL/mg, between about 3.0 μL/mg and about 4.0 μL/mg, or between about 4.0 μL/mg and about 5.0 μL/mg. In one embodiment, n is between about 0.42 μL/mg and about 2.12 μL/mg. In other embodiments, η may be about 0.2 μL/mg, about 0.4 μL/mg, about 0.6 μL/mg, about 0.8 μL/mg, about 1.0 μL/mg, about 1.5 μL/mg, about 2.0 μL/mg, about 2.5 L/mg, about 3.0 μL/mg, about 3.5 μL/mg, about 4.0 μL/mg, about 4.5 μL/mg, or about 5.0 μL/mg.
In certain embodiments, the acid used in the milling step may be an organic acid. Organic acids are especially suited in the methods of the present technology as they can be produced from low-cost biomass, are less harmful to the environment, and the resulting organic salts derived by their use from the present methods have the potential to be reused as environmentally friendly de-icing agents or preservatives. Organic acids used in the methods of the present technology may be selected from citric acid, ascorbic acid, acetic acid, L-malic acid, succinic acid, salicylic acid, L-lactic acid, formic acid, benzoic acid, and glutaric acid and combinations thereof. In certain embodiments, the organic acid is selected from citric acid, ascorbic acid, acetic acid, L-malic acid, succinic acid, and salicylic acid, and combinations thereof.
In further embodiments, the organic acid may be selected from L-malic acid, succinic acid, and salicylic acid, and combinations thereof. Advantageously, L-malic acid, succinic acid, and salicylic acid are available in nature, can be obtained at low costs, and are categorized as green chemicals according to the GSK's acid and base guide (Henderson, R. K. et al., “Development of GSK's acid and base selection guides”, Green Chem. 2015, 17 (2), 945-949, incorporated herein by reference). Similarly, succinic acid is a valuable building block found in nature that can be applied as a precursor for surfactants, solvents, synthetic resins, and pharmaceuticals. Currently, much of its global production relies on a fossil-based route, using non-renewable feedstocks. However, efficient, sustainable, bio-based, and cost-competitive alternative processes for the production of succinic acid are being developed and optimized. Salicylic acid is also naturally synthesized by plants and it is an important hormone for their growth and development. Industrially, salicylic acid is used as a food preservative, bactericide, antiseptic, and starting material for the synthesis of important pharmaceuticals.
In other embodiments, the acid used in the milling step may be a mineral acid. Mineral acids suitable for the methods of the present technology are mild mineral acids such as hydrochloric acid, nitric acid, perchloric acid, sulfuric acid, and phosphoric acid and combinations thereof. In certain embodiments, the mineral acid is hydrochloric acid.
In certain embodiments, the amount or ratio of acid used in the milling step is calculated with respect to a mineral content, and more specifically, the calcium carbonate (CaCO3) content, in the chitin-containing biomass. In some embodiments, the ratio of acid to the mineral content of the chitin-containing biomass is between about 2:1 and about 10:1. In other embodiments, the ratio of acid with respect to the mineral content in the chitin-containing biomass is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.
In any of the embodiments of the present technology milling may be performed by using any one or more of a mixer mill, a ball mill, a planetary mill, a jar with at least one ball, a food processor, a blender, a vortex, a rapid mixer, an extruder, and an acoustic mixer. In certain embodiments, milling comprises using a mixer mill and a jar with at least one ball. In such embodiments, the jar may be made of one or more steel, zirconia and polytetrafluoroethylene (PTFE). The at least one ball may be a zirconia ball, or a steel ball. A combination of balls may also be used, such that at least two balls are used in milling with a jar, wherein the first ball is made of a first material and the second ball is made of a second material. For example, a first zirconia ball and a second steel ball may be used for milling. In other embodiments, the ball may be made of the same material as the jar, such that, for example, a zirconia ball is used in zirconia jar for milling. In other embodiments, the ball may be made of a different material than the jar.
In certain embodiments, milling is performed for about 5 minutes to about 60 minutes. In other embodiments, milling may be performed for about 10 to about 30 minutes, such as for about 15, about 20 or about 25 minutes. In one embodiment, milling is performed for about 30 minutes. In another embodiment, the milling is performed for about 10 minutes. Advantageously, the methods of the present technology use short milling times and thereby decreasing the energy input required to extract chitin from chitin-containing biomass compared to known methods, while still providing high yields and high purities of chitin.
In certain embodiments, the milling is performed at room temperature. Advantageously, this feature also contributes to the green and sustainable characteristics of the methods of the present technology as this limits the energy input required to extract chitin from chitin-containing biomass compared to known methods, as the mixture of chitin-containing biomass and acid does not need to be heated to high temperatures.
The methods of the present technology may further comprise an additional step of aging after the step of milling. Accelerated aging by the methods disclosed herewith is considered to be a low energy, solvent-free alternative to solvothermal methods yielding organic and inorganic materials. The inventors of the present technology have found that aging in addition to milling may result in further demineralization of chitin when liquid acids such as hydrochloric acid and acetic acid are used for milling. Aging, however, was not required when milling was conducted with solid acids (i.e., in solid-state). However, an aging step may optionally be added to the methods of the present technology when a solid acid is used.
In certain embodiments, aging may be performed under controlled humidity, with optional heating. In such embodiments, aging may be performed in a humidified chamber, with a relative humidity (RH) of between about 43% to about 98%. In other embodiments, aging may be performed at a RH of about 98%.
Heating may also be performed during the aging step. In certain embodiments heating is performed at temperatures ranging between about 20° C. and about 100° C., such as about 30° C., about 40° C., about 50° C., about 60° C., about 70° C., about 80° C., or about 90° C. In one embodiment, heating is performed at about 50° C. during the aging step.
The duration of aging may range from about 0 to about 6 days depending on the conditions used. In certain embodiments, aging may be performed for 0 days (i.e., no aging may be required). In other embodiments, aging may be performed for about 12 hours to about 6 days. In further embodiments, aging may be performed for about 1 day. In yet further embodiments, aging may be performed for about 3 days.
In certain embodiments, the methods of the present technology comprise milling a chitin-containing biomass selected from any one or more of shells and cuticles of crustaceans, arthropods, insects, squids, shellfish, krill, and fungi with an organic acid selected from citric acid, ascorbic acid, acetic acid, L-malic acid, succinic acid, and salicylic acid, and combinations thereof; wherein the ratio of organic acid to a mineral content in the chitin-containing biomass is between about 2:1 and about 10:1. In certain implementations of this embodiment, the method comprises a single-step. In other implementations of this embodiment, the method further comprises aging. In certain other implementations of this embodiment, the milling is performed in solid state. In other implementations of this embodiment, the milling is performed in the presence of liquid; wherein n is between about 0.2 μL/mg and about 5 μL/mg.
In other embodiments, the methods of the present technology comprise milling a chitin-containing biomass selected from any one or more of shells and cuticles of crustaceans, arthropods, insects, squids, shellfish, krill, and fungi with a mineral acid selected from hydrochloric acid and combinations thereof, wherein the ratio of mineral acid to a mineral content in the chitin-containing biomass is between about 2:1 and about 10:1. In certain implementations of this embodiment, the method comprises a single-step. In other implementations of this embodiment, the method further comprises aging. In certain other implementations of this embodiment, the milling is performed in the presence of liquid, wherein n is between about 0.2 μL/mg and about 5 μL/mg.
In other embodiments, the method of the present technology may further comprise a step of washing and filtering the chitin obtained to remove byproducts and wastes from the reaction and to neutralize the pH. Washing may be performed with water, acetone, or other known suitable solvents. The chitin obtained may further be dried under vacuum or without vacuum after washing to remove the residual solvents. The duration of drying may be any duration necessary to dry the chitin obtained. Drying may be performed at room temperatures or at any other temperature suited for drying chitin without affecting its chemical and physical properties. In one embodiment, the chitin obtained is dried at 50° C. overnight.
In certain embodiments the mass yield of chitin obtained by the methods of the present technology is at least about 2%. In other embodiments the mass yield of chitin obtained is between about 2% and about 50%. In yet other embodiments, the mass yield of chitin obtained is more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, or more than about 40%. In further embodiments, the mass yield of chitin obtained is about 2%, about 6%, about 8%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%, about 40% or about 50%.
Crystallinity is related to the degree of order and the crystal size of a given crystalline substance. The crystallinity index is a quantitative indication of the purity and crystalline structure of the chitin polymer obtained. Various techniques, such as X-ray diffraction (XRD), powder X-ray diffraction (pXRD), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy may be used to measure the crystallinity index of a substance. In certain embodiments of the present technology, the crystallinity index of chitin was measured by pXRD. Generally, crystallinity indices of more than 60% are considered to indicate high grade pure chitin wherein the chitin is substantially free of minerals and proteins.
In certain embodiments, the chitin obtained by the methods of the present technology have a crystallinity index of at least about 60%. In other embodiments, the chitin obtained by the methods of the present technology have a crystallinity index of at least about 70%.
Ash content is another important parameter to be considered during the analysis of chitin. Ash is the inorganic residue remaining after water and organic matter have been removed by heating in the presence of oxidizing agents and provides a measure of total amounts of mineral within a sample. In some embodiments of the present technology, the ash content of chitin was determined by degradation of chitin samples in the presence of air and was measured by Thermogravimetric analysis (TGA). The ash content thus representing the amount of mineral oxide present in the chitin framework is an indicator of the efficiency of demineralization. A high content of ash present in chitin can negatively affect certain properties of the chitin polymer, including solubility, viscosity, and purity. Approximately 30% of ash can be generally removed from crustacean shells after demineralization, and because of its influence in the properties of chitin polymer, the ash content of high-quality grade chitin should be less than about 1%.
In certain embodiments, the ash content of the chitin obtained by the methods of the present technology is at least about 0.5%. In other embodiments, the ash content of the chitin obtained by the methods of the present technology is between about 0.5% and about 10%. In one embodiment, the ash content of chitin obtained is between about 0.5% and about 1.0%. In further embodiments, the ash content is about 0.6%, about 1.0%, about 1.5%, about 2.0%, about 3%, about 4%, or about 9%. Advantageously this further indicates that chitin obtained by the methods of the present technology maintains its chemical and physical properties even when exposed to extreme environments.
Solid char residues in TGA may also be measured and represent the amount of residual carbonaceous materials that cannot be dissociated into volatile fragments. In certain embodiments, the char content is between about 10% and about 70%.
The examples below are given to illustrate the practice of various embodiments of the present disclosure. They are not intended to limit or define the entire scope of this disclosure.
It should be appreciated that the subject matters of this disclosure are not limited to the particular embodiments described and illustrated herein but includes all modifications and variations falling within the scope of the subject matters of this disclosure as defined in the appended claims.
Frozen GC were received from Parks Canada and stored in the freezer at −20° C. until use. Acetic acid, hydrochloric acid, and acetone were obtained from Fisher Scientific. Citric acid and L-Ascorbic acid were purchased from Sigma-Aldrich. KOH was obtained from ACP.
The GC shells were defleshed manually by thawing in boiling water for 5 minutes. The shells were then washed by deionized (DI) water, dried overnight at room temperature and homogenized into powder with a blender. The resulting powder containing chitin, minerals and proteins was stored at −18° C. until further treatment.
Mechanochemistry was used thereafter to remove proteins and minerals from GC shells. Milling was performed using different acids (citric acid, ascorbic acid, hydrochloric acid, or acetic acid) at different ratios (2:1 to 10:1 of acid with respect to the mineral content in the shells). For example, for a ratio of 2:1 of acetic acid to mineral content of the shells, 1.0 g GC shell powder and 0.80 ml acetic acid were combined in a zirconia jar with one zirconia ball (10 mm) using a Retsch MM 400 mixer. The mixture of GC shell powder and acetic acid was then milled for 30 minutes at 30 Hz. The procedure was similar for other acids. The total reagent mass of solid in all experiments was kept to around 1.0 g.
In certain embodiments, wherein aging was used after milling, the mixture was transferred to a 10 ml beaker in a standard Tupperware glass container with a petri dish of super-saturated K2SO4 solution to achieve a RH of 98% in the container.
The sample was worked up by washing with 100 ml of water and filtering using a Whatman filter paper until neutral pH, followed by washing with 50 ml acetone and vacuum drying at 50° C. overnight.
Powder X-ray diffraction (pXRD) spectra was acquired using a Bruker D8 Advance X-ray diffractometer equipped with a CuKα filament, scanned with a 20 range between 5-55° with an increment of 0.02°. Chitin crystallinity was obtained by comparing the area of the peaks (reduced area) and the area of the peaks (reduced area), where crystallinity %=(reduced area)×100/(global area).
Thermogravimetric analysis (TGA) was carried out by a Q50TM from TA Instruments under nitrogen flow at a temperature range of 30 to 800° C. and a ramp rate of 10° C. min-1 in aluminum pans.
Solid-state nuclear magnetic resonance (NMR) spectra were collected on a Varian VNMRS operating at 400 MHz for the 13C acquisition using a 4 mm double-resonance Varian Chemagnetics T3 probe. The number of scans was 640 for each sample for a total time of 1.5 h. Each data point in the spectra was acquired by a 3 s recycle delay, 512 co-added transients and contact times between 0.06-8 ms.
Power consumption was measured using a RioRand Plug Power Meter. The power consumption was measured for the mill, hot plate and the oven while in experimental use.
Materials were purchased, and GC shell powder was prepared as described in Example 1.
The legacy method was carried out as described in Naczk M. et al., “Compositional characteristics of green crab (Carcinus maenas)”, Food Chemistry 2004, 88 (3), 429-434, and Fulton, B. A. & Fairchild, E. A., “Nutritional analysis of whole green crab, Carcinus maenas, for application as a forage fish replacement in agrifeeds”, Sustainable Agriculture Research 2013, 2 (3), 126 (the contents of which are incorporated herein by reference). Briefly, 20 g of powdered shell was deproteinized with 400 ml of 5% KOH solution for 2 h at 100° C. with occasional mixing. After the deproteinization, the biomass was isolated using Whatman filter paper (90 mm) and washed with water until neutral pH. Subsequently, the sample was treated with 5% HCl at a ratio of 1/25 (w/v) at room temperature for 2 h with constant mixing. Finally, the product was washed by DI water to pH 7, and then with 100 ml acetone, followed by drying under vacuum at 50° C. overnight.
In order to analyze and quantify the residual minerals and proteins, TGA analyses of GC shells before and after treatment and that of the intermediate deproteinized product were performed (
13C SS-NMR is one of the important analysis techniques to assess the purity of chitin.
GC shell powder prepared as described in Example 1 was milled using a Retsch MM 400 mixer mill with a 20 ml zirconia jar and one 10 mm zirconia ball for 30 minutes. Three different organic acids (citric acid, ascorbic acid, and acetic acid) and one mineral acid (hydrochloric acid) were investigated at different concentrations to find the optimal reaction conditions. The purity of the chitin obtained from each reaction was ascertained by pXRD, TGA, and 13C SS-NMR.
Although mechanochemical treatment of GC shells with acid was initially expected to result in the demineralization of the chitin, analysis by 13C SS-NMR and pXRD surprisingly showed that proteins were also removed from the GC shells during the mechanochemical treatment of GC shells with the acids used. In other words, the production of chitin from GC shells was achieved in a single step without using a basic (e.g., KOH) deproteinization step as used in the legacy method.
Furthermore, as will be described in further detail below, unlike other existing extraction methods (e.g., Zhang, J. et al., “Base-free preparation of low molecular weight chitin from crab shell”, Carbohydrate Polymers, 2018, 190, 148-155; incorporated herein by reference), the methods of the present technology were carried out with milder acids in a shorter amount of time with less energy. Since the methods of the present technology do not involve any harsh chemical environments, the chitin produced by the methods of the present technology remains intact after the separation step.
Citric acid has been used for the treatment of biomass such as chitin, cellulose and lignin as a green modifier. Since citric acid contains 3 acidic functionalities, one mole of calcium carbonate can theoretically react with 2/3 moles citric acid. However, the pKa of the third proton is ˜6.4, indicating a weak acid. Moreover, since there are proteins and other minerals in the crab shells which must be removed, the amount citric acid used was selected to be in excess to completely remove the minerals and proteins.
Initially, GC shell powder was subjected to 2 equivalents citric acid (ratio 2:1 acid with respect to the mineral content in the shells) in order to obtain pure chitin. The process was carried out by milling the GC shells and citric acid for 30 minutes without aging or any other post-reaction modifications. After the isolation of the product, the residual minerals were determined by pXRD and TGA. The TGA of deproteinized GC shell and the final chitin by the legacy method is shown for comparison in
The pXRD of chitin separated from GC shells by 4 equivalents of citric acid (
Initial attempts at milling the shells with 2 equivalents HCl (ratio 2:1) resulted in minor demineralization (57% mineral content) when the samples were milled 30 minutes without aging (
To further improve the demineralization, increasing the aging time combined with increasing HCl ratio was attempted. Complete demineralization was achieved by using 10 equivalences of HCl (ratio 10:1) and 3 days of aging, which was confirmed by the lack of mineral peaks between 30° to 55° in pXRD spectra and the one-step decomposition in TGA (
In these embodiments liquid HCl was used to control demineralization in solid-state. About 2-10 equivalents of HCl was used in for liquid assisted grinding, which equated to an n ranged from 0.42 to 2.12 μL/mg. As best seen in
The effect of acid to shells ratio (2:1, 6:1, 10:1 acid to minerals) was investigated by milling the GC shells with acetic acid for 30 minutes. The method used liquid assisted grinding.
The effect of aging at 50° C. and 98% RH for 1 day was further explored. As seen in
To further optimize the methods of the present technology, other organic acids were tested. Specifically, L-malic, succinic, and salicylic acids were investigated for the extraction of chitin.
Chitin extraction was conducted by mechanochemistry as described above. Specifically, chitin was extracted form GC shells employing 2 equivalents of L-malic, succinic, and salicylic acids. The results shown in
aCalculated in respect to the calcium carbonate (CaCO3) content in the shells (65%);
bCalculated considering the mass of GC shells used during extraction (250 mg);
cDetermined via Power X-Ray Diffraction (PXRD);
dDetermined via Thermogravimetric Analysis (TGA).
In comparison to the values obtained using citric and ascorbic acids (Table 1, Entries 1 and 2), a slightly higher char content (19%) and lower crystallinity index values (65%) were observed for chitin extracted after milling with L-malic acid (Table 1, Entry 3). Regarding reaction efficiency, yields of chitin after extraction with malic acid (16.1%) are about 2 and 7 times higher than the ones obtained using citric and ascorbic acid, respectively. Chitin after extraction with succinic acid shows an increased crystallinity index (69%), but higher char content (20%) in comparison to the analogue extracted using L-malic acid (Table 1, Entry 4). Nevertheless, chitin yields of 10.7% are still obtained using only 400 mg of organic acid. Crystallinity index values remain similar (68%), but yields are increased to 13.5% and char values are decreased to 16% after reaction of GC shells with salicylic acid (Table 1, Entry 5). Although the material obtained is slightly less crystalline, the extraction employing L-malic, succinic, and salicylic acids uses about 70% less reagents than the methods using citric and ascorbic acids, and also allows the achievement of higher yields of chitin (11-16%) directly from GC shells.
As seen in Table 1, about 38% of ash could be removed from the GC shells and the highest ash content observed for the chitin polymer extracted from GC shells was 2.1% (Table 1, Entry 2). Therefore, the methods disclosed herein produces high-quality chitin, which maintains its chemical and physical properties even when exposed to extreme environments.
To further decrease the quantity of reagents required and reduce costs related to the neutralization of acidic waste generated, chitin extraction was attempted using only 1 equivalent of the proposed organic acids (
With the objective of using even milder reaction conditions and reducing energy consumption in the method of the present technology, lower milling times (10 min) were explored for the extraction of chitin. As best seen in Table 2, using citric and ascorbic acid (Table 2, Entries 1 and 2), a decrease in the milling time slightly decreases polymer crystallinity but increases the yields of chitin extracted, thus reaching a value of 25.3% for salicylic acid (Table 1, Entry 5). The amount of char and ash present in the samples milled for 10 min (Table 2) is also higher in comparison to the chitin extracted after 30 min (Table 1), which suggests a more effective demineralization of GC shell samples after longer processing times.
aCalculated in respect to CaCO3 content in the shells (65%).
bCalculated considering the mass of GC shells used during extraction (250 mg).
cDetermined via PXRD.
dDetermined via TGA.
Using 400 mg of L-malic, succinic, or salicylic acids, high-quality grade chitin with low ash content (<2%), good crystallinity (>60%), and yields higher than 10% can be obtained from GC shells after 30 min of ball milling. Optimum reaction conditions include the use 2 equivalents of salicylic acid and 30 min processing time, in which the lowest ash content (0.9%) is obtained with good yields (13.5%). Lower processing times could further improve the yields of chitin extracted, at the cost of polymer quality (i.e. increased ash content).
The methods of the present technology were modified such as to start with 40 g of green crab shells instead of 1 g. Milling was performed using a large-scale blender. High yields and a high purity of chitin were obtained as disclosed above.
Variations and modifications will occur to those of skill in the art after reviewing this disclosure. The disclosed features may be implemented, in any combination and subcombinations (including multiple dependent combinations and subcombinations), with one or more other features described herein. The various features described or illustrated above, including any components thereof, may be combined or integrated in other systems. Moreover, certain features may be omitted or not implemented. Examples of changes, substitutions, and alterations are ascertainable by one skilled in the art and could be made without departing from the scope of the information disclosed herein.
It should be appreciated that the present technology is not limited to the particular embodiments described and illustrated herein but includes all modifications and variations falling within the scope of the present technology as defined in the appended claims.
All references cited in this specification, and their references, are incorporated by reference herein in their entirety where appropriate for teachings of additional or alternative details, features, and/or technical background.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2023/050321 | 3/10/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63319111 | Mar 2022 | US |
