WO2024072249A1

WO2024072249A1 - Production process for high average molecular weight chitosan biopolymers

Info

Publication number: WO2024072249A1
Application number: PCT/PT2023/050035
Authority: WO
Inventors: Luísa Margarida DIAS RIBEIRO DE SOUSA MARTINS; Ana Paula DA COSTA RIBEIRO
Original assignee: Instituto Superior Técnico
Priority date: 2022-09-29
Filing date: 2023-09-28
Publication date: 2024-04-04
Also published as: PT118233A

Abstract

The present invention relates to a process for producing chitosan biopolymers characterised by comprising the steps of mechano-chemical extraction of chitin from biomass; and mechano- chemical deacetylation of chitin to chitosan, in which the steps take place in a planetary ball mill, at room temperature, without the addition of a solvent and in which chitosan with an average molecular weight of over 1000 kDa is obtained.

Description

DESCRIPTION

"PRODUCTION PROCESS FOR HIGH AVERAGE MOLECULAR WEIGHT CHITOSAN

BIOPOLYMERS"

FIELD OF INVENTION

The present invention relates to a process for producing high average molecular weight chitosan biopolymers. The chitosan biopolymers produced by the new process are insoluble in water and could be applied in pollution remediation, food packaging, and pharmaceutical devices.

BACKGROUND TO THE INVENTION

In recent years, biopolymers produced from renewable resources (e.g. agri-food waste) have received increasing attention due to their unique characteristics of biocompatibility, non-toxicity and biodegradability. This has boosted their application in sectors as varied as the cosmetics, medical and pharmaceutical industries (as vehicles, fillers in pharmaceutical compounds, treatment for diseases such as kidney failure, obesity or anaemia, in biomaterials, corneal dressings, surgical threads, and other medical devices (e.g. tissue regeneration) ) ; in dietetics and the food sector (such as food additives and dietary supplements, in food packaging and animal nutrition, more specifically in aquaculture and poultry farming) ; in bioremediation (such as filtering, texturizing, flocculating or adsorbing agents, in particular for water filtration and pollution control of water and aqueous effluents) ; electronic devices, the automotive industry, chemistry (e.g. as a support for metal catalysts) , the energy sector, etc.

One of the biopolymers that has generated the most interest is chitin. Chitin is a polysaccharide made up of p- (l-4) linked 2- deoxy-2-acetamido-D-glucose units and is the second most abundant naturally produced polymer (after cellulose) with annual production levels of billions of tonnes. In fact, chitin is a biopolymer synthesised by various species in the living world. It forms part of the exoskeleton of crustaceans and insects, and the side wall that surrounds and protects fungi. In insects in particular, chitin makes up between 3 and 60 per cent of their exoskeleton .

Chitin can undergo deacetylation, i.e. the conversion of amide functions into amine functions, giving rise to chitosan, a water- soluble biopolymer that has great chemical versatility as well as antibacterial and antifungal properties. In fact, chitosan already has a worldwide market that reached a value of USD 2.49 billion in 2020 and is expected to grow ca. 12% during 2021-2026.

It is known that the degree of deacetylation of chitin as well as the average molecular weight and polydispersity of the chitosan obtained play a crucial role in the properties of chitosan-based biopolymers, and consequently in their applications. For example, the use of a chitosan biopolymer with a high average molecular weight maximises its antimicrobial effect and limits trans epidermal water loss or skin irritation.

Commercial chitin is commonly extracted from crustaceans through a two-step process. The first step consists of deproteinization, i.e. removing proteins from the cuticle, using sodium hydroxide under moderate to drastic conditions, as described by Younes, I. et al. Mar. Drugs, 2015, 13, 1133-1174. A strong base and more or less drastic conditions are needed because chitin is insoluble in most common solvents (partially soluble only in extremely polar solvents) . The next step in chitin extraction consists of demineralization using dilute hydrochloric acid at room temperature, as described by Okafor, N., Biochim. Biophys. Acta, Mucoproteins Mucopolysaccharides, 1965, 101, 193- 200. Hydrochloric acid is favoured over treatments with other acids such as nitric, sulphuric, acetic or formic acid.

The commercial method for obtaining chitosan by deacetylating chitin has been described by several authors and consists of heating the chitin ( ca . 100 °C) in a highly concentrated solution (>50%) of sodium hydroxide for several hours. Multiple treatment cycles are sometimes required. Although this process produces chitosan with a molecular weight of between 80 and 800 kDa, it is a high-risk process in terms of environmental safety.

In this regard, several alternative processes have been developed to produce chitosan from chitin that are safer and more environmentally acceptable. The deacetylation of chitin is known, namely by: microwave irradiation (Sahu, A. et al., J. Mater. Sci. Mater. Med. ,2009, 20, 171-175.) ; ultrasound (Anwar, M. et al., AIP Conf. Proc., 2017, 1823) ; high pressure (No, H. K. et al., J. Agric. Food Chem., 2000, 48, 2625-2627) ; solvothermal process (Domard, A. et al. , Int. J. Biol. Macromol., 1983, 5, 49-52; Alimuniar, A. et al., In Advances in Chitin and Chitosan, C.J. Beine, P.A. Sanford and J.P. Zikakis (eds.) , Elsevier Applied Science, London and New York, pp. 627-632, 1992; Rong Huei, C., Carbohydr. Polym, 1996,29, 353-358) ; freezing/ thawing (Nemtsev, S. V. et al., Appl . Biochem. Microbiol., 2002, 38,521-526) ; planetary ball milling (Chen, X. et al., Green Chem., 2017, 19, 2783-2792) , vibrating ball milling (Di Nardo, T. et al. , Green Chem., 2019, 21, 3276-3285) , etc.

With the exception of the high-pressure process and the combined method of milling and curing in a controlled atmosphere in a vibrating ball mill, the chitosans obtained from chitin have low average molecular weights, typically less than 500 kDa. This is due to the simultaneous occurrence of depolymerisation by the attack of OH~ ions on the glycosidic bonds of chitin, which is not intended .

Thus, currently, to produce a chitosan with a high average molecular weight (> 1000 kDa) and good mechanical properties, an energy-intensive process is used, such as the high-pressure method (potentially dangerous for scaling up) or the combination of milling in a vibrating ball mill and curing in a controlled atmosphere (98% relative humidity, 22 °C) for ca. a week. The latter, although considered a low-energy mechano-chemical milling method, produces a degree of deacetylation of less than 30%. In order to increase the degree of deacetylation it is necessary to subject the material resulting from solvent-assisted mechano- chemical milling (LAG) to a curing process in a controlled atmosphere (98% relative humidity, 22 °C) for 6 days, which proves to be very time-consuming and expensive and, for this reason, not very environmentally sustainable.

Patent FR2859726 describes a method for producing chitin or chitosan derivatives which involves: (a) fine grinding of crustacean shells or cephalopod endoskeletons in the presence of a strong base in the form of granules, flakes or powder, plus 0.1- 20% by weight of water (based on the reaction mixture) so that the mixture remains as a powder ; (b ) adding an organic acid in the form of a solid or highly concentrated solution together with 0 . 1- 20% by weight of water and continuing grinding with the mixture still in powder form; and ( c ) extracting the final mixture with water or an aqueous solution . This method uses a stainless steel or ceramic vibrating ball mill (unit weight of the grinding ball greater than 200 g, occupied volume in the reactor from 30% to 60% ) . The average molecular weight of the chitosan obtained by this method is not disclosed .

This process requires prior cleaning of the biomass used as raw material ( crustacean shells , cephalopod endoskeletons ) .

As well as making the process more expens ive , pre-treating the biomass has the disadvantage of increasing the water content of the initial biomass and this directly af fects the first step of chitin extraction in which a concentrated base solution is used .

In addition, the costs of processing and puri fying water, especially when large flows have to be treated, are high and complex .

There is therefore a need to provide a process for producing chitosan biopolymers with a high average molecular weight and a high degree of deacetylation and purity that is , at the same time , environmentally favourable both in terms of energy and resource consumption and in terms of reducing waste generation .

The process of the present invention solves the above problem and surprisingly provides a high yield with a very good chitosan conversion rate . SUMMARY OF THE INVENTION

The present invention provides a process for producing high average molecular weight chitosan biopolymers, characterised by comprising the steps of: i) mechano-chemical extraction of chitin from biomass; and ii) mechano-chemical deacetylation of chitin to chitosan in which steps i) and ii) are carried out in a planetary ball mill, at room temperature, without the addition of solvent and in which chitosan with an average molecular weight of more than 1000 kDa is obtained .

In a preferred implementation, step i) of the chitosan biopolymer production process of the present invention comprises the following steps: a) crushing the biomass, for a time span of 5-10 minutes, under the conditions of mill number of grinding balls/ diameter of grinding balls/mass of biomass of 3/0.5 cm/ 100 mg ; b) adding a solid base in a mass ratio of 1:5 to 1:75, relative to the ground biomass, and dry milling under the same mill conditions as in step a) , for a time span of 3-9 hours, at a frequency of 150-500 rpm; c) adding an acid and grinding for a time span of 6-9 hours under the same conditions as the planetary ball mill in step a) ; d) washing with distilled water, whilst stirring, to neutralise and eliminate water-soluble compounds; and e ) filtering and drying the mixture obtained in step d) to isolate solid chitin .

In another preferred implementation, step ii ) of the chitosan biopolymer production process of the present invention comprises the following steps : f ) adding a solid base to the planetary ball mill in a ratio of 20 : 1 to the mass of chitin and dry milling for 6- 9 hours at a frequency of 150-500 rpm; g) washing with distilled water, whilst stirring, to neutralise and eliminate water-soluble compounds ; and h) filtering and drying of the mixture obtained in step g) to isolate solid chitosan .

In yet another implementation, in the process of the present invention the crushing of the biomass in step a ) takes place in a time span of 5 minutes .

In a more favoured implementation, the solid base in step b ) and/or step f ) , respectively in steps i ) and ii ) , is selected from the group comprising sodium hydroxide , magnesium hydroxide , potassium hydroxide , calcium hydroxide , or mixtures thereof . In a much more favoured embodiment of the present invention, the solid base is magnesium hydroxide .

In yet another more favoured implementation, the acid in step c ) of step i ) is selected from the group comprising anhydrous oxalic acid, lactic acid, and hydrochloric acid . In a much more favoured implementation the acid is anhydrous oxalic acid .

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for producing high average molecular weight chitosan biopolymers ( greater than 1000 kDa ) comprising the steps of mechano-chemical extraction of chitin from biomass and mechano-chemical deacetylation of chitin to chitosan, in which the steps are carried out in a planetary ball mill , in which the raw materials are placed at room temperature and without the addition of any type of solvent .

In the context of the present invention, " chitin" is understood to be a polymer of natural origin predominantly composed of glucosamine and N-acetyl glucosamine units ( i . e . between 55% and 98 % by weight , preferably between 75% and 90% by weight , of glucosamine and N-acetyl glucosamine units ) , in which said units can optionally be replaced by amino acids and/or peptides . In the present invention, the chitin is isolated chitin, which is understood as chitin that has been isolated or extracted from its natural environment .

In the context of the present invention, " chitosan" means the products of the deacetylation of chitin . In the present invention, chitosan is isolated chitosan, which is understood to be obtained by deacetylating isolated chitin or chitin extracted from its natural environment .

"Biomass" is understood to mean the raw material used in the process of the invention, which is obtained from the exoskeletons of fly larvae , beetle larvae , or shrimps . However, the use of raw materials obtained from di f ferent species that synthesise chitin, in particular crustaceans or insects , is not excluded .

" Insects" means insects at any stage of development , such as an adult stage , a larval stage , or a nymph stage ( intermediate stage ) . In the invention process , the exoskeletons used are the result of the end of a larval stage i f the insects are holometabolous or of a nymph stage ( intermediate stage ) i f the insects are heterometabolous .

Preferably, the insect exoskeletons used in the present invention should be those of coleoptera, diptera, lepidoptera, isoptera, orthoptera, hymenoptera, hemiptera, heteroptera, ephemeroptera and mecoptera .

The present invention uses planetary ball mills , in which the reactors are placed on a support disc and rotate around their own axis .

The preferred planetary ball mill is the Planetary Ball Mill PM 100 model (Retsch) with stainless steel or zirconium grinding balls and reactors (Retsch) or the Emax High Energy Ball Mill model (Retsch) with stainless steel or zirconium grinding balls and reactors (Retsch) .

A set of parameters are defined for the operation of the mill during the process of producing high average molecular weight chitosan biopolymers , such as : mill ing time in a range of 0 . 5 to 9 h, milling frequency in a range of 150 to 500 rpm, si ze of the milling balls in a range of 1 mm to 5 mm and quantity of milling balls in a range of 1 to 5 units. The ratio (by mass) of grinding balls : reagents is also defined in a range from 1:15 to 1:50.

It was found that controlling the milling parameters in the planetary ball mill is crucial to producing a chitosan with the desired characteristics, i.e. high average molecular weight (over 1000 kDa) , a high degree of deacetylation, and high purity. In fact, an energy-intensive process leads to the depolymerisation of chitin by breaking its glycosidic bonds, resulting in a chitosan with a low average molecular weight. The use of too few grinding balls or too short a reaction time will lead to an inefficient process, both for extracting chitin from biomass and for converting chitin into chitosan.

Surprisingly, the chitosan obtained by the process of the present invention was found to have a degree of deacetylation of ca. 95%, a high average molecular weight (> 1000 kDa) and high purity (compared to commercial chitosan) . The process of the invention is carried out under safer and more environmentally favourable conditions than currently known methods, since it does not require any added solvents, has low energy consumption and generates minimal waste.

Using this process, the final product, isolated chitosan of high average molecular weight, is obtained more efficiently and economically, in a single reactor, without requiring separation/isolation of intermediate material, thereby reducing waste production and preserving the polymer chain.

Furthermore, the biomass used in the process of the present invention does not need to be pre-treated, which is a significant advantage over known processes. Pre-treating the biomass not only makes the process more expensive, but if pieces of the crustacean are present it also increases the water content of the biomass and this would directly affect the first chitin extraction step, in which a concentrated base is used.

It has thus been found that the process of the present invention reflects an exploitation of part of the surplus biomass from the agri-food sector, for example insect exuviae, which enables it to be reintegrated into the original ecosystem, thus preventing waste and also contributing to the use of bioplastics.

The process of the present invention is described in more detail below.

The process begins with the mechano-chemical extraction of chitin from biomass. In a first step, the biomass is crushed in a planetary ball mill for 5 to 10 min under the following conditions: number of grinding balls/diameter of grinding balls/mass of biomass = 3/0.5 cm/100 mg. Biomass with a grain diameter of approximately 0.25 to 0.50 mm is obtained.

A solid base is added to the powder yielded by the previous step. The base is selected from the group comprising sodium hydroxide, magnesium hydroxide, potassium hydroxide, calcium hydroxide, or mixtures thereof, in a mass ratio of 1:5 to 1:75 in relation to the crushed biomass. The preferred base used is magnesium hydroxide.

Dry grinding is carried out with number of grinding balls/diameter of grinding balls/mass of biomass of 3/0.5 cm/100 mg, for 3 to 9 hours at a frequency of 150 to 500 rpm. Next , an acid selected from the group comprising anhydrous oxalic acid, lactic acid and hydrochloric acid is added to the reactor . Preferably, the acid is anhydrous oxalic acid . A further grinding step is carried out for 6 to 9 hours under the grinding conditions mentioned above .

After grinding, it is washed with distilled water under stirring until neutralisation is complete and the water-soluble compounds are removed .

The solid chitin is separated from the mixture by filtration and dried in a muf fle furnace at ca . 80 ° C under vacuum for up to an hour .

The yield of mechano-chemical extraction of chitin isolated from biomass is between 49% and 89% .

The chitin is then mechano-chemically deacetylated to chitosan . The isolated chitin powder, obtained by mechano-chemical extraction from biomass , is ground (number/diameter of grinding balls appropriate to the amount of chitin) using a solid base ( selected from the bases presented above ) with a base : chitin mass ratio of 20 : 1 for 6- 9 hours . The chitosan is then washed with distilled water to remove soluble substances , separated by filtration and heat-treated in a muf fle furnace at 50 ° C for 0 . 5 hours .

The degree of deacetylation of the isolated chitosan ranges from 63% and 95% . The degree of deacetylation is determined by Fourier trans form infrared spectroscopy using Sabin ' s law . Preferably, the average molecular weight is determined according to a rotational viscosity measurement method, as described by Halpern A.M., Experimental Physical Chemistry, Prentice Hall, Upper Saddle River, 1997.

EXAMPLES

Example 1 - Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia illucens) . Mechano-chemical method (Emax) - Mg (OH) 2 / oxalic acid

The process of mechano-chemically extracting chitin from the cocoons of black soldier fly larvae (Hermetia illucens) was carried out in a planetary ball mill model Emax High Energy Ball Mill (Retsch) , using stainless steel reactors and grinding balls (Retsch) .

In a first step, the cocoons were crushed for 5 min in a planetary ball mill using the following conditions: number of grinding balls/diameter of grinding balls/mass of cocoons of 3/0.5 cm/ 100 mg, at 500 rpm.

A powder with a grain diameter of 0.25 mm was obtained, to which magnesium hydroxide was added in a mass ratio in relation to the mass of fly larvae cocoons of 1:20. Dry grinding was carried out for 6 hours at 500 rpm under the following conditions: number of grinding balls/diameter of grinding balls/mass of crushed cocoons of 3/0.5 cm/100 mg. More anhydrous oxalic acid was then added to the reactor, and grinding was continued for 6 hours under the conditions mentioned above .

After grinding, distilled water was added while stirring to complete the neutralisation and eliminate the water-soluble reagents .

The solid chitin was separated from the mixture by filtration and dried in a muf fle furnace at ca . 80 ° C under vacuum for one hour .

The yield of the mechanochemical extraction of chitin from black soldier fly larvae cocoons using magnesium hydroxide and oxalic acid was 87 % .

Structural analysis by Fourier Trans form Infrared Spectroscopy - Attenuated Total Reflectance ( FTIR-ATR) revealed the following characteristic bands : 5 ( cur¹ ) 3273 , 2918 , 1540 , 1353 , 1025 .

Example 2 Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia ill ucens) Mechano-chemical method -

Mg ( OH) ₂ / HC1

The same mechano-chemical chitin extraction process as in Example 1 was used, but with an extra of hydrochloric acid ( 37 % m/v) as the acid . The yield of mechano-chemical extraction of chitin from black soldier fly larvae cocoons using magnesium hydroxide and hydrochloric acid was 82 % .

Example 3 - Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia ill ucens) . Mechano-chemical method - NaOH/HCl

The same mechano-chemical chitin extraction process as in Example 2 was used, but with sodium hydroxide as the base .

The yield of mechano-chemical extraction of chitin from black soldier fly larvae cocoons using sodium hydroxide and hydrochloric acid was 89% .

Example 4 - Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia ill ucens) . Mechano-chemical method - Mg ( OH) 2 / oxalic acid

The same mechano-chemical chitin extraction process as in Example 1 was used, but with the following grinding conditions after initial crushing : number of grinding balls/diameter of grinding balls/mass of pulverised cocoons = 3/ 0 . 1 cm/ 100 mg .

The yield of mechano-chemical extraction of chitin from black soldier fly larvae cocoons using magnesium hydroxide and oxalic acid was 83% .

Example 5 - Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia ill ucens) . Mechano-chemical method - Mg ( OH) 2 / oxalic acid The same mechano-chemical chitin extraction process as in Example 1 was used, but with the following grinding conditions after the initial crushing : number of grinding balls/diameter of grinding balls/mass of pulverised cocoons = 1 / 0 . 5 cm/ 100 mg .

The yield of the mechano-chemical extraction of chitin from black soldier fly larvae cocoons using magnesium hydroxide and oxalic acid was 79% .

Example 6 - Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia ill ucens) . Mechano-chemical method - Mg ( OH) 2 / oxalic acid

The same mechano-chemical chitin extraction process as in Example 1 was used, but the milling steps were carried out after the initial crushing at 150 rpm and for 9 hours after the addition of magnesium hydroxide and 6 hours after the addition of oxalic acid .

The yield of the mechano-chemical extraction of chitin from black soldier fly larvae cocoons using magnesium hydroxide and oxalic acid was 80% .

Example 7 - Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia ill ucens) . Mechano-chemical method - Mg ( OH) 2 / oxalic acid

The same mechano-chemical chitin extraction process as in Example 1 was used, but the milling steps were carried out after the initial crushing, for 0 . 5 hours after the addition of magnesium hydroxide and 3 hours after the addition of oxalic acid . The yield of mechano-chemical extraction of chitin from black soldier fly larvae cocoons using magnesium hydroxide and oxalic acid was 49% .

Example 8 - Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia ill ucens) . Mechano-chemical method ( PM 100 ) - Mg ( OH) 2 / oxalic acid

The process of mechano-chemical extraction of chitin from the cocoons of black soldier fly larvae (Hermetia ill ucens) was carried out in a planetary ball mill model Planetary Ball Mill PM 100 (Retsch) , using stainless steel reactors and grinding balls (Retsch) .

In a first step, the cocoons were crushed for 5 min in a planetary ball mill using the following conditions : number of grinding balls/diameter of grinding balls/mass of cocoons = 3/ 0 . 5 cm/ 100 mg .

A powder with a grain si ze of 0 . 20 mm in diameter was obtained, to which magnesium hydroxide was added in a mass ratio relative to the mass of fly larvae cocoons of 1 : 20 . Dry grinding was carried out under the following conditions : number of grinding balls/diameter of grinding balls/mass of pulverised cocoons = 3/ 0 . 5 cm/ 100 mg, for 6 hours at a frequency of 500 rpm .

Anhydrous oxalic acid was then added to the reactor, and grinding was continued for 6 hours under the conditions mentioned above . After grinding, distilled water was added while stirring to complete the neutralisation and remove the water-soluble reagents .

The yield of the mechano-chemical extraction of chitin from black soldier fly larvae cocoons using magnesium hydroxide and oxalic acid was 60% .

Example 9 - Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia ill ucens) . Mechano-chemical method - Mg ( OH) 2 / oxalic acid

The same mechano-chemical chitin extraction process was used as in Example 8 , but zirconium reactors and balls (Retsch) were used .

The yield of mechano-chemical extraction of chitin from black soldier fly larvae cocoons using magnesium hydroxide and oxalic acid was 67 % .

Example 10 - Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia ill ucens) . Mechano-chemical method - Mg ( OH) 2 / oxalic acid

The same mechano-chemical chitin extraction process as in Example 1 was used, but the grinding step was carried out after adding oxalic acid, for 3 hours . The yield of the mechano-chemical extraction of chitin from black soldier fly larvae cocoons using magnesium hydroxide and oxalic acid was 85%.

Comparative Example 11 (state of the art) - Extraction of chitin from the cocoons of black soldier fly larvae (Hermetia illucens) - Solvothermal method - NaOH/HCl

The process of solvothermal extraction of chitin from the cocoons of black soldier fly larvae (Hermetia illucens) was carried out in a round-bottomed glass flask.

As a first step, the cocoons were crushed in a blade mill for 15 minutes at 5000 rpm.

A powder (100 mg) with a particle diameter size of 0.25 mm was obtained, to which hydroxide (2.1 g) previously dissolved in 100 mL of distilled water was added. It was stirred (650 rpm) for 24 hours.

Hydrochloric acid (37% w/v) was then added to the flask and stirring continued for 24 hours under the conditions mentioned above .

After 48 hours, distilled water was added while stirring to complete the neutralisation and remove the water-soluble reagents.

The solid chitin was separated from the mixture by centrifugation (10000 rpm for 10 min) , followed by filtration and dried in a muffle furnace at 80 °C under vacuum for one hour. The yield of the solvothermal extraction of chitin from black soldier fly larvae cocoons using magnesium hydroxide and hydrochloric acid was 80% .

Table 1 shows a comparison of the maximum yields of mechanochemical extraction of chitin obtained from black soldier fly larvae cocoons , using di f ferent bases and acids , respectively, in the deproteini zation and deminerali zation steps . The maximum yields of the mechano-chemical extraction of chitin are also compared with that obtained by the solvothermal extraction process of the state of the art ( Comparative example 11 ) .

Table 1 - Maximum yields of mechanochemical or solvothermal extraction of chitin from black soldier fly larvae cocoons .

Example 12 - Mechano-chemical conversion of chitin, extracted as in Example 3 , into high average molecular weight chitosan

The process of mechano-chemical conversion of chitin, extracted from the cocoons of black soldier fly larvae (Hermetia ill ucens) from Example 3 , into chitosan was carried out in a planetary ball mill model Emax High Energy Ball Mill (Retsch) , using stainless steel reactors and grinding balls (Retsch) . The chitin powder samples from Example 3 were ground for 9 h with sodium hydroxide (0.35 g) with a sample mass: number of grinding balls ratio of 50:3 (0.018 g of sample) .

Then distilled water was added, the chitosans were filtered and placed in a muffle furnace at 50 °C for 0.5 hours.

The degree of deacetylation was 90%. Structural analysis by FTIR-ATR revealed the following characteristic bands: 5 (cur¹ ) 3400, 3300, 2900, 1400, 1000.

Example 13 - Mechano-chemical conversion of chitin, extracted in Example 2, into high average molecular weight chitosan

The process of mechano-chemical conversion of chitin, extracted from the cocoons of black soldier fly larvae (Hermetia illucens) from Example 2, into chitosan was carried out in a planetary ball mill, model Emax High Energy Ball Mill (Retsch) , using stainless steel reactors and grinding balls (Retsch) .

The chitin powder samples from Example 2 were ground for 9 h with magnesium hydroxide (1 g) with a sample mass: number of grinding balls ratio of 50:3 (50 mg of sample) .

The degree of deacetylation was 88%.

Example 14 - Mechano-chemical conversion of chitin, extracted in Example 1, into high average molecular weight chitosan The process of mechanochemical conversion of chitin extracted from the cocoons of black soldier fly larvae (Hermetia illucens) from Example 1 into chitosan was carried out in a planetary ball mill, model Emax High Energy Ball Mill (Retsch) , using stainless steel reactors and grinding balls (Retsch) .

The chitin powder samples from Example 1 were ground for 9 h with magnesium hydroxide (1 g) with a sample mass: number of grinding balls ratio of 50:3 (50 mg of sample) .

Distilled water was then added and the chitosans were filtered and placed in a muffle furnace at 50 °C for 0.5 hours.

The degree of deacetylation was 95%.

Example 15 - Mechano-chemical conversion of chitin, extracted in Example 1, into high average molecular weight chitosan

The process of mechano-chemical conversion of chitin extracted from the cocoons of black soldier fly larvae (Hermetia illucens) from Example 1 into chitosan was carried out in a planetary ball mill, model Planetary Ball Mill PM 100 (Retsch) , using stainless steel reactors and grinding balls (Retsch) .

The degree of deacetylation was 87%. Example 16 - Mechano-chemical conversion of chitin, extracted in Example 1 , into high average molecular weight chitosan

The process of mechano-chemical conversion of chitin extracted from the cocoons of black soldier fly larvae ( Hermetia ill ucens) from Example 1 into chitosan was carried out in a planetary ball mill , model Planetary Ball Mill PM 100 (Retsch) , using zirconium reactors and grinding balls (Retsch) .

The chitin powder samples from Example 1 were ground for 9 h with magnesium hydroxide ( 1 g) with a sample mass : number of grinding balls ratio of 50 : 3 ( 50 mg of sample ) .

Distilled water was then added and the chitosans were filtered and placed in a muf fle furnace at 50 ° C for 0 . 5 hours .

The degree of deacetylation was 63% .

Example 17 - Mechano-chemical conversion of chitin extracted in Example 1 into high average molecular weight chitosan

The process of mechano-chemical conversion of chitin extracted from the cocoons of black soldier fly larvae ( Hermetia ill ucens) from Example 1 into chitosan was carried out in a planetary ball mill , model Emax High Energy Ball Mill (Retsch) , using zirconium reactors and grinding balls (Retsch) .

The chitin powder samples from Example 1 were ground for 9 h with magnesium hydroxide ( 1 g) with a sample mass : number of grinding balls ratio of 50 : 3 ( 50 mg of sample ) . Distilled water was then added and the chitosans were filtered and placed in a muf fle furnace at 50 ° C for 0 . 5 hours .

The degree of deacetylation was 78 % .

Example 18 - Mechano-chemical conversion of chitin extracted in Example 1 into high average molecular weight chitosan

The process of mechano-chemical conversion of chitin extracted from the cocoons of black soldier fly larvae ( Hermetia ill ucens) from Example 1 into chitosan was carried out in a planetary ball mill , model Emax High Energy Ball Mill (Retsch) , using stainless steel reactors and grinding balls (Retsch) .

The chitin powder samples from Example 1 were ground for 6 h with magnesium hydroxide ( 1 g) with a sample mass : number of grinding balls ratio of 50 : 3 ( 50 mg of sample ) .

The degree of deacetylation was 83% .

Example 19 - Mechano-chemical conversion of chitin extracted in Example 1 into high average molecular weight chitosan

The process of mechano-chemical conversion of chitin extracted from the cocoons of black soldier fly larvae ( Hermetia ill ucens) from Example 1 into chitosan was carried out in a planetary ball mill , model Emax High Energy Ball Mill (Retsch) , using zirconium reactors and grinding balls (Retsch) . The chitin powder samples from Example 1 were ground for 6 h with magnesium hydroxide (1 g) with a sample mass: number of grinding balls ratio of 50:3 (50 mg of sample) .

The degree of deacetylation was 74%.

Example 20 - Mechano-chemical conversion of chitin extracted in Example 1 into high average molecular weight chitosan

The process of mechano-chemical conversion of chitin extracted from the cocoons of black soldier fly larvae (Hermetia illucens) from Example 1 into chitosan was carried out in a planetary ball mill, model Emax High Energy Ball Mill (Retsch) , using stainless steel reactors and grinding balls (Retsch) .

The chitin powder samples from Example 1 were ground for 9 h with magnesium hydroxide (1 g) with a sample mass: number of grinding balls ratio of 25:1 (50 mg of sample) .

The degree of deacetylation was 79%.

Example 21 - Mechano-chemical conversion of chitin extracted in Example 1 into high average molecular weight chitosan

The chitin powder samples from Example 1 were ground for 9 h with magnesium hydroxide (1 g) with a sample mass: number of grinding balls ratio of 50:1 (50 mg of sample) .

The degree of deacetylation was 67%.

Comparative example 22 (state of the art) - Solvothermal conversion into chitosan of chitin extracted in Example 11

In a first step, the chitin obtained in Example 11 (80 mg) was crushed in a blade mill for 15 min at 5000 rpm.

A powder with a grain diameter of 0.05 mm and a mass of 80 mg was obtained, to which sodium hydroxide (60%) previously dissolved in 100 mL of distilled water was added. It was then stirred at 650 rpm for 72 hours.

The chitosans were then filtered and placed in a muffle furnace at 50 °C for 0.5 hours.

The degree of deacetylation was 92%.

Table 2 shows a comparison of the degrees of deacetylation of chitosans obtained from black soldier fly larvae cocoons using the mechanochemical method (MBP) and different bases. The degrees of deacetylation of chitosans obtained mechano-chemically are also compared with those obtained through the solvothermal conversion process ( S ) .

Table 2 - Degree of deacetylation of chitosan .

Example 23 - Extraction of chitin from cocoons of beetle larvae ( Tenebri o moli tor) . Mechano-chemical method - Mg ( OH) 2 / oxalic acid

The same process as that used in Example 1 was used for the mechano-chemical extraction of chitin from the cocoons of beetle larvae ( Tenebri o moli tor) .

The yield of the mechano-chemical extraction of chitin from beetle larvae cocoons using magnesium hydroxide and oxalic acid was 70% .

Example 24 - Mechano-chemical conversion of chitin extracted in Example 23 into high average molecular weight chitosan The process described in Example 14 was used for the mechanochemical conversion of chitin extracted from the cocoons of the beetle larvae ( Tenebri o moli tor) of Example 23 into chitosan .

The degree of deacetylation was 85% .

Example 25 - Extraction of chitin from shrimp exoskeletons ( Li topenaeus vannamei , Boone 1931 ) . Mechano-chemical method - Mg ( OH) 2 / oxalic acid

The same process as that used in Example 1 was used for the mechano-chemical extraction of chitin from shrimp exoskeletons ( Li topenaeus vannamei , Boone 1931 ) , except that , after initial crushing, the powder obtained had a particle diameter of 0 . 30 mm .

The yield of the mechano-chemical extraction of chitin from shrimp exoskeletons using magnesium hydroxide and oxalic acid was 73% .

Example 26 - Mechano-chemical conversion of chitin extracted in Example 25 into high average molecular weight chitosan

The process of mechano-chemical conversion of chitin extracted from shrimp exoskeletons ( Li topenaeus vannamei , Boone 1931 ) from Example 25 into chitosan was used as described in Example 14 .

The degree of deacetylation was 78 % . The average molecular weight , estimated by viscosimetry, of the chitosan obtained by the process of the present invention is greater than 1000 kDa . This high average molecular weight is in line with the biopolymer being practically insoluble in water, which also indicates the absence of depolymerisation reactions (which would lead to a lower average molecular weight ) . The fact that chitosan is able to resist dissolving makes it a great product to use in new applications , such as pollution remediation, packaging, etc .

Claims

CLAIMS Process for producing high average molecular weight chitosan biopolymers characterised by comprising the following steps i) mechano-chemical extraction of chitin from biomass; and ii) mechano-chemical deacetylation of chitin to chitosan in which steps i) and ii) take place in a planetary ball mill, at room temperature, without the addition of solvent, and in which chitosan with an average molecular weight of over 1000 kDa is obtained. Process according to claim 1 characterised in that step i) comprises the steps of: a) crushing of biomass, for a time span of 5-10 minutes, under mill conditions: number of grinding balls/diameter of grinding balls/mass of biomass of 3/0.5 cm/100 mg; b) adding a solid base in a mass ratio of 1:5 to 1:75, relative to the crushed biomass, and dry milling under the same mill conditions as in step a) , for a time span of 3-9 hours, at a frequency of 150-500 rpm; c) adding an acid and grinding for a time span of 6-9 hours under the same conditions as the planetary ball mill in step a) ; d) washing with distilled water, under stirring, to neutralise and eliminate water-soluble compounds; and e) filtering and drying of the mixture obtained in step d) to isolate solid chitin. Process according to claim 1 characterised in that step ii) comprises the steps of: f) adding a solid base to the planetary ball mill in a ratio of 20:1 relative to the mass of chitin, and dry milling for 6-9 hours at a frequency of 150-500 rpm; g) washing with distilled water, under stirring, to neutralise and eliminate water-soluble compounds; and h) filtering and drying of the mixture obtained in step g) to isolate solid chitosan. Process according to claim 2 characterised in that the crushing of biomass in step a) lasts for 5 minutes. Process according to claim 2 characterised in that the solid base, in step b) of step i) , is selected from the group comprising sodium hydroxide, magnesium hydroxide, potassium hydroxide, calcium hydroxide, or mixtures thereof. Process according to claim 3 characterised in that the solid base, in step f) of step ii) , is selected from the group comprising sodium hydroxide, magnesium hydroxide, potassium hydroxide, calcium hydroxide, or mixtures thereof.

7. Process according to claim 5 and/or claim 6 characterised in that the solid base is magnesium hydroxide. 8. Process according to claim 2 characterised in that the acid in step c) of step i) is selected from the group comprising anhydrous oxalic acid, lactic acid, and hydrochloric acid.

9. Process according to claim 8 characterised in that the acid is anhydrous oxalic acid.