Process for making chitin derivatives.

(54) Title: PROCESSES TO DEVELOP CHITINA DERIVATIVES. (54) Title: PROCESS FOR MAKING CHITIN DERIVATIVES.

(57) Summary

The present invention relates to a process characterized in that it comprises (1) forming an aqueous mixture comprising a microbial composition and solid chitin, wherein said microbial composition comprises one or more microbes that produce enzymes to digest chitin; and (2) fermenting the mixture long enough to enzymatically digest all or part of the chitin to form a fermented mixture comprising chitosan and glucosamine. In some embodiments, chitin is derived from the biodegradation of chitin-containing marine arthropods. In other modalities, chitin is obtained from fungi, filamentous fungi and yeast that contain chitin which is extracted via a chemical process. In yet another embodiment, chitin is obtained by biodegradation of chitin-containing fungi, filamentous fungi, yeast, and / or insects, preferably using HQE for digestion. In some embodiments, the process is carried out with a solution that already contains chitosan and / or glucosamine such as HYTb, the aqueous fraction is obtained from the biodegradation of organisms containing chitin.

(57) Abstract

Disclosed is a process comprising (1) forming an aqueous mixture comprising a microbial composition and solid chitin, where said microbial composition comprises one or more microbes that produces chitin digesting enzymes; and (2) fermenting the mixture for a time sufficient to enzymatically digest all or part of the chitin to form a fermented mixture comprising chitosan and glucosamine. In some embodiments, the chitin is derived from the biodegradation of chitin containing marine Arthropods. In other embodiments, the chitin is obtained from chitin containing fungí, filamentous fungí and yeast which is extracted via a Chemical process. In yet another embodiment, the chitin is obtained by the biodegradation of chitin containing fungí, filamentous fungí, yeast and / or insects, preferably using HQE for the digestion. In some embodiments, the process is carried out with a solution that already contains chitosan and / or glucosamine such as HYTb, the aqueous fraction obtained from the biodegradation of chitin containing organisms.

_SE_,: // tyRITEM 01 ECONOMY

Institute

Mexican Property

Industrial

<img file="MX337718B_D0001.tif" />

PATENT TITLE NO. 337718

Owner (s): AGRINOS AS

Address: Fornebuveien 1, N-1366, Lysaker, NORWAY

Name: PROCESSES TO PREPARE CHITIN DERIVATIVES.

Classification: IC.8: C12P19 / 04 inventor (s): JAIME LÓPEZ-CERVANTES

REQUEST

Number:

MX / a / 2013/015363

Country:

US

International filing date:

Jumo 2012

Number:

61/500,527

PRIORITY

Date:

June 2011

Validity: Twenty years

Due date! June 25, 2032 |

Yes B

The reference patent is granted based on articles 1. 2nd section V, 6 'section III, and 59 of the Lev of «Industrial property.

In accordance with article 23 of the Industrial Property Law, this patent has a validity of twenty years from the date of filing of the international application and will be subject to the payment of the fee to maintain damages.

prayable, before the

Qfjen subscribes to the preeenteatul Industrial Ropiedad (Diario 20/01/2004, 16/06/2005, 25 subsection a), 4 ° and 12 'fraction OW7 / 2002, 15/07/2004, 281 de | Instituto Mexicano de la and É subparagraph a) of the Agreement qi Autum Regionals, Subdlr I did it on the official basis of the Federation (OJ Fi 27 / C6 1/2006, '

I and III 7/2004 and property delegated to the Dlvj reformai fi stors by articles 6 fractions III and 7 ° bis 2 of amended on 08/02/1994, 2S / 10 / 19B8, 12/26/1997, 06/08 / 2010, 01/27/2012 and 04/09/2012)! Articles 1 of Industrial Property (DOF 14/12/1999,

V Clause a), 'β fractions 1 and til and 30 of the Statute, 04/08/2004 and 13/09 /; [Divisional rectors, Title of the Mexican Institute of

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I 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

/ 05 / 2009,06 / 01/2010, 1 Regulation (09/2007); Idustrial article (DOF

¿Ultades in the I lionales, Coordinators Departai

Law of 705/1999, ration V signed the Organic I7); 1st, 3rd res of the properties

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Issue Date: March 16, 2016

DIVISIONAL DIRECTOR OF PATENTS

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•*1'

NAHANNY CANAL REYES

Sand! No, 550 Floor 1,

Co!. Pueblo Santa María Tepepan, Xochunilco, CP 1G020.

Mexico City

Tel. (55) 53 34 07 03 www.irnpi.gob.rnx

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MX / 2016/21876

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OR#/.&amp;

PROCESS TO DEVELOP CHITIN DERIVATIVES

IMPI

Background of the Invention

Chitin, poly (β (1-4) -N-acetyl-D-glucosamine) is a major natural polysaccharide. This polymer is synthesized by a huge number of living organisms including crustaceans, insects, fungi, filamentous fungi, and yeasts. Considering the amount of chitin produced annually in the world, it is the most abundant polymer after cellulose.

The main commercial sources of chitin have been crabs and shrimp shells. In industrial processing, chitin is extracted from crustaceans by acid treatment to dissolve calcium carbonate followed by alkaline extraction to solubilize proteins. The most important derivative of chitin is chitosan, obtained by (partial) deacetylation of chitin in the solid state under alkaline conditions (concentrated NaOH) or by enzymatic hydrolysis in the presence of qutin deacetylase. Under controlled conditions, chitin and chitosan can be polymerized to provide water soluble derivatives such as chitin oligosaccharides (ChOS) and chitosan oligosaccharides (COS), respectively.

These oligomers are recognized for their

Ref. 245821 i ivi ri

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX337718B_D0008.tif" />

bioactivity; which include anti-tumor, bactericidal and fungicidal activity, stimulating chitinase and regulating plant growth. Chitin is involved in defending the host against bacterial invasion, has been used to prepare the affinity chromatography column, and is widely used to immobilize whole cells and enzymes.

Taking into account its biodegradability, non-toxicity, physiological inertness, antibacterial properties, hydrophilicity, gel-forming properties and protein affinity, chitin has found applications in many areas other than food such as in biosensors. Chitin-based materials are also used for the treatment of industrial contaminants. Chitin can be processed in the form of film and fiber. Chitin-derived fibers

<td>regenerated</td><td>are</td><td>used</td><td colspan="2">as binders in the process</td><td>of</td>

<td>elaboration</td><td>of</td><td>paper,</td><td>the</td><td>fiber improves endurance</td><td>to the</td>

<td>breach</td><td>of the</td><td>paper.</td><td>Without</td><td colspan="2">However, the main development</td>

of the chitin and fiber film is in medical and pharmaceutical applications as wound dressing materials.

When the degree of deacetylation of chitin reaches approximately 50%, it becomes soluble in aqueous acidic medium and is called chitosan. Chitosan is the

<img file="MX337718B_D0009.tif" />

IMPI

MEXICAN INSTITUTE

EU PROPERTY

INDUSTRIAL unique pseudo-natural cationic polymer and thus used in many applications. Being soluble in aqueous solution, it is widely used in different applications such as solutions, gels, films and fibers. The main investigations of chitosan concern its preparation weights with varied molecular and deacetylation, the dependence of its solution properties on deacetylation, the preparation of derivatives and applications.

Chitosan is much easier to process than chitin, but the stability of chitosan materials is generally lower, due to its more hydrophilic character and, especially, pH sensitivity. Chitosan and its derivatives have various functional properties that have made it possible to use them in many fields including food, cosmetic, biomedicine, agriculture, environmental protection, wastewater management. The most important fields where the specificity of chitosan must be recognized are cosmetic, pharmaceutical and biomedical applications. Drug delivery applications include oral, nasal, parenteral and transdermal administration, implants, and gene delivery.

Another point to note is its biological activity with respect to agriculture since chitosan presents

IMPI AND

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<img file="MX337718B_D0010.tif" />

antiviral and antifungal activities. It inhibits the growth of bacteria and bacterial infection, and stimulates the natural defenses in the plant. It is also used for seed coating, frost protection, release time of fertilizers and nutrients to the soil.

Even though chitosan is known to have important functional activities, the high molecular weight and high viscosity may restrict uses in some special fields, particularly in medicine and the food industry, because most animal intestines, especially the human gastrointestinal tract, it does not have enzymes such as chitinase and qutosanase, which directly degrade the β-glucosidic bond in chitin and chitosan. Unlike chitosan, its hydrolyzed products and chitosan oligosaccharides (COS) are easily soluble in water due to their shorter chain length and their free amino group in D-glucosamine units. The low viscosity and higher solubility of COS at neutral pH has attracted the interest of many researchers to use chitosan in its oligosaccharide form. Especially in areas of food and nutrition they have emphasized their ability to improve food and quality and the progress of human health.

TUTO MEXICANO Jg THE PROPERTY

INDUSTRIAL *** __

INDUSTRIAL

Chemical and enzymatic methods are widely used for COS production and among them chemical hydrolysis is most commonly used in industrial scale production. However, chemical hydrolysis has some disadvantages to being commercialized, due to the toxic compound environment, higher risk associated with environmental contamination, and low production yield. Enzymatic processes are generally carried out in baths and are preferably using chemical methods. This is due to minimized adverse chemical modifications of products during enzymatic hydrolysis.

Another product generated from chitin and glucosamine, IT can be used in agriculture, has shown that the presence of glucosamine in the composition of the soil causes an increase in absorbent trichomes, which are manifested by increasing the vigor of the plant. The first reaction that can be observed is a strengthening of the tips that take on a deep green color, with the leaf margins slightly curled. This is because, when glucosamine is applied in the soil, the plant induces a response similar to that which could result when the plant tries to defend itself from the attack of fungi, nematodes or insects without these really existing.

IMPI

M-XiVANO INSTITUTE '^N DELA PROWEDAD

INDUSTRIAL

<img file="MX337718B_D0011.tif" />

In the area of ​​medicine, glucosamine has been used for the treatment of arthritis, promotes the development of cartilage tissue, and is used in cartilage reconstruction. Glucosamine is involved in the formation of nails, tendons, skin, eyes, bones, ligaments and heart valves, it is also involved in the production of collagen and proteoglycans.

Brief Description of the Invention

Processes for increasing chitosan and / or glucosamine in HYTb are described. The process comprises (1) forming a mixture comprising HYTb, a microbial composition, and solid chitin, wherein such a microbial composition comprises one or more microbes that produce enzymes to digest chitin; and (2) fermenting the mixture long enough to enzymatically digest all or part of such chitin to form a fermented mixture. The amount of at least one of chitosan and glucosamine in the fermented mixture is greater than in such HYTb.

In an alternative embodiment, the mixture is diluted to form a dilute mixture which is fermented to digest all or part of such chitin to form a fermented mixture. The absolute amount of at least one of chitin and glucosamine (taking into account the dilution step) in the fermented mixture is greater than that in HYTb.

In some modalities the HYTc is the source of such

<img file="MX337718B_D0012.tif" />

chitin. In general, HYTc is micronized to form micronized chitin and residual chitin. The chitin used in the process can be micronized chitin. However, since this form of chitin has other commercial uses, it is preferred that the residual chitin be used in the process.

Depending on the extent of chitin digestion and the end use of the process product, solids can be conveniently separated from the fermented mixture by centrifugation or filtration. It is desired that the microbes in the microbial composition be retained, filtration is preferred although low centrifugation g can be used.

The chitin source need not be from HYTc. For example, chitin derived from filamentous fungi and / or yeast can be used. See, for example, US Patent 7,556,946 which describes a chemical process for extracting chitin from fungi, including filamentous fungi, and yeast from groups including Zygomycetes, Basiomycetes, Ascomycetes, and Deuteromycetes. Examples include Aspergillum, Penicillium, Trichoderma, Saccaromyces abd Schizosacaromyces species and edible fungi such as Agaricus, Pleurotus, Boletus and Lentinula species.

In preferred embodiments, the microbial composition comprises HQE.

IMPI &lt;\

MEXICAN INSTITUTE.

OF INDUSTRIAL PROPERTY

<img file="MX337718B_D0013.tif" />

In another embodiment, the process comprises (1) mixing a marine animal or marine animal derivative with a first microbial composition to form a first mixture, where the first microbial composition contains one or more microbes that produce enzymes that digest the marine animal or derivative in solid, aqueous and lipid fractions, where the solid fraction comprises chitin and the aqueous phase comprises amino acids, chitosan and glucosamine; (2) ferment the first mixture; (3) separating the first mixture into solid, aqueous and lipid fractions, where the solid fraction comprises chitin and the aqueous phase comprises chitosan and glucosamine; (4) forming a second mixture comprising the aqueous fraction, chitin and a second microbial composition, where the second microbial composition comprises one or more microbes that produce enzymes to digest the chitin; (5) fermenting the second mixture to form a second fermented mixture; and (6) optionally, separating the second fermented mixture into a second aqueous fraction and a second solid fraction, where the second aqueous fraction has a higher content of at least one of chitosan and glucosamine compared to the first aqueous fraction.

As with the modalities described above, the second mixture can be diluted to form a second diluted mixture which is then fermented to digest

<img file="MX337718B_D0014.tif" />

IMPI

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL all or part of the chitin to form a second fermented mixture. The absolute amount of at least one of chitin and glucosamine in the second fermented mixture is greater than that in the first aqueous fraction.

HYTc is the preferred source of chitin. It can be micronized chitin or residual chitin.

In this multiple phase fermentation process the first and second microbial compositions preferably comprise HQE.

In yet another embodiment, the process comprises (1) forming a mixture comprising a microbial composition and solid chitin, wherein such a microbial composition comprises one or more microbes that produce enzymes to digest the chitin; and (2) ferment the mixture long enough to enzymatically digest all or part of the chitin to form a fermented mixture. The source of the chitin may be HYTc. Alternatively, chitin can be derived from fungi, including filamentous fungi, and / or yeast by a non-enzymatic process. See for example, US Patent 7,556,946.

Still further, chitin and other useful products can be obtained from the biodegradation of chitin-containing biological sources such as fungi, including filamentous fungi, yeast and insects identified above. The process includes: (1)

<img file="MX337718B_D0015.tif" />

mixing a biological source containing chitin, such as fungi, including filamentous fungi, yeast and / or insects, with a first microbial composition to form a first mixture, where the first microbial composition contains one or more microbes that produce enzymes that digest the biological source containing chitin in solid, aqueous and optionally lipid fractions, where the solid fraction comprises chitin and the aqueous phase comprises amino acids, chitosan and glucosamine; (2) ferment the first mixture; (3) separating the first mixture into solid, aqueous and optionally lipid fractions, where the solid fraction comprises chitin and the aqueous phase comprises chitosan and glucosamine.

Brief Description of the Figures Figure 1 is a flow chart showing the digestion of crustaceans to form HYTb and HYTc. HYTc and HYTb are subsequently processed with HQE to form

HYTd, a solution with relatively high amounts of chitosan and glucosamine compared to HYTb.

Figure 2 depicts glucosamine formation as a function of time compared to HYTb.

Figure 3 is a flow chart showing the digestion of fungi, which include filamentous fungi, yeasts, and / or insects to form HYTb and HYTc. The HYTc and HYTb are optionally further processed with HQE to form

<img file="MX337718B_D0016.tif" />

high of

ΙΜΡΪ

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL

HYTd, a solution with relatively small amounts of chitosan and glucosamine compared to HYTb.

Figure 4 represents a process for making

HYTd.

Detailed description of the invention

A process is described comprising (1) forming an aqueous mixture comprising a microbial composition and solid chitin, wherein such a microbial composition comprises one or more microbes that produce enzymes to digest chitin; and (2) fermenting the mixture long enough to enzymatically digest all or part of the chitin to form a fermented mixture comprising chitosan and glucosamine.

In some embodiments, chitin is derived from the biodegradation of chitin-containing marine arthropods. In other modalities, chitin is obtained from fungi, filamentous fungi and yeast that contain chitin which is extracted via a chemical process. See for example the

US Patent 7,556,946. In yet another embodiment, chitin is obtained by biodegradation of chitin-containing fungi, including filamentous fungi, yeast, and / or insects as described herein.

In some embodiments, the process is carried out with a solution that already contains chitosan and / or glucosamine. The breakdown of solid chitin in the process produces more

<img file="MX337718B_D0017.tif" />

chitosan and / or glucosamine so that the final ...... solution contains higher amounts of these components. In a preferred embodiment, the starting solution containing chitosan and / or glucosamine is HYTb. The solution obtained after fermentation is referred to as HYTd. HYTd, in some embodiments, is essentially HYTb with a higher concentration of chitosan and / or glucosamine. If, for example, HYTb contains 1.2% by weight of chitosan and 1% by weight of glucosamine, the resulting HYTd will contain

<td>higher concentrations</td><td>of</td><td>one or both</td><td>of these</td>

<td>components, preferably</td><td>both of them</td><td colspan="2">of the components.</td>

<td>Chitin Sources</td><td></td><td></td><td></td>

<td>1. Biodegradation</td><td>of</td><td>Arthropods that</td><td>they contain</td>

Chitin

Figure 1 is a flow chart showing the digestion of the crustacean to form HYTb containing chitosan and glucosamine and HYTc which contains solid chitin. This figure also shows the subsequent processing of HYTc and HYTb with the microbial composition HQE to form HYTd, a solution with relatively high amounts of chitosan and glucosamine compared to HYTb.

Briefly, in the arthropod biodegradation process, a microbial composition is used to degrade the arthropod or residual components of the arthropod. It is a lactic acid fermentation process. The composition

ΪΜΡΙ γΒΖ

MEXICAN INSTITUTE

OF PROPERTY V

I kl ΓΤΙ 1 CTD 'AI · *

INDUSTRIAL microbial contains microbes that produce enzymes that can degrade the chitin-containing components of the arthropod to chitin, chitosan, N-acetyl glucosamine and glucosamine. It also contains microbes that produce enzymes that can break down proteins and fats to produce amino acids and lipids. A preferred microbial composition for arthropod degradation is referred to as HQE. HQE was filed with the American Type Culture Collection (ATCC) Manassas, VA, USA on April 27, 2010 and provided the Patent Deposit Designation PTA-10861.

In a preferred embodiment, the marine arthropod is a crustacean and the preferred crustacean is shrimp. The shrimp derivative comprises cephalothorax and / or shrimp exoskeleton.

In the biodegradation process, it is preferred that the fermentation is facultative aerobic fermentation. It is also preferred that the fermentation is carried out at a temperature of about 30 ° C to 40 ° C. The pH is preferably less than about 6, more preferably less than about 5.5. However, the pH should be kept above about 4.3. Fermentation takes place for approximately 24-96 hours. In some embodiments, fermentation takes place for approximately 24-48 hours and more preferably 24-36 hours. These fermentation times are much shorter

IMP

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MEXICAN INSTITUTE Vt - *⁸® ifo

FROM INDUSTRIAL PROPERTY than the prior art fermentation times of 10 to 15 days to achieve substantially the same amount of digestion, either without detectable formation of chitosan and glucosamine.

The separation of the mixture is preferably by centrifugation. (For example, approximately 920 g). Gravity separation can also be used but is not preferred due to the time required to achieve the separation.

The mixture is separated into three fractions: solid, aqueous and lipid. The aqueous fraction comprises hydrolyzed protein, amino acids, chitosan and the lipid fraction comprises sterols, vitamins A and E and carotenoid pigments such as astaxanthin.

As used herein, the term HYTb refers to the aqueous fraction and HYTc refers to the solid fraction obtained from the above biodegradation process. This process is described in US Patent Application Series No. 61 / 289,706, filed on 12/23/09 entitled Biodegradation of Crustacean Derivatives, US Patent Application Series No. 61 / 299,869, filed on 1/29/10 titled Biodegradation Process and Microbial Composition and US Patent Application Series No. 61 / 355,365 filed on June 16, 2010 titled Process and Composition of

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Biodegradation each of which is incorporated by reference herein in its entirety. HYTb contains amino acids (approximately 12% by weight), chitosan by weight, glucosamine by weight) and microelements (approximately

0.5-1.5 (approximately 0.5-1.5% (approximately 6% by weight) including calcium, magnesium, zinc, copper, iron and manganese. It also contains enzymes such as lactic enzymes, proteases, lipases, chitinases among others, lactic acid, polypeptides and other carbohydrates.

In addition to the uses described above for chitosan and glucosamine, HYTb alone or in combination with HYTc and the microbial composition HYTa are useful in the treatment of soil, seeds, seedlings and foliage as described in US Patent Application Series No.

61 / 355,447 presented on June 16, 2010 entitled Processes and Microbial Composition for Agricultural Use and in the

US Patent Application Series No. 13 / 160,333 filed on July 14, 2011 entitled Microbial Process and Composition, each of which is incorporated herein by reference in its entirety.

HYTd and the chitosan / glucosamine solutions obtained from the microbial digestion of chitin as described in the patent are similarly useful. See

US Patent Application 61 / 500,543 filed on

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MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

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June 2011 titled Agricultural Uses of HYTd.

It is preferred that HQE is used in the biodegradation process. In other embodiments, it is preferred that previously prepared HYTb be added to HQE or the fermentation broth. As described above, HYTb contains amino acids, chitosan, glucosamine, and micro elements including calcium, magnesium, zinc, copper, iron, and manganese.

HYTb also contains enzymes such as lactic enzymes, proteases, lipases, chitinases, lactic acid, polypeptides, and other carbohydrates. HYTb may also contain latent microorganisms from a previous biodegradation process. Such microorganisms can become reactivated and, in combination with HQE, contribute to a more robust biodegradation process compared to when HQE is used by itself as otherwise described herein.

More particularly, the process includes the following stages:

to. Activation of microbial cells in a sugar-based solution to improve their growth and biomass formation.

b. Shredding of the shrimp derivatives (cephalthorax and exoskeleton) to make a homogeneous paste.

c. Homogeneous mixing of the shrimp derivative paste with at least 10% of the activated inoculum.

<img file="MX337718B_D0022.tif" />

MPI

MEXICAN INSTITUTE Dt THE INDUSTRIAL PROPERTY

d. Adjustment of the pH values ​​to less than 6.0 in the mixture using a citric acid solution to inhibit the growth of microorganisms and promote the development of microbial cells that constitute the inoculum,

and. Fermentation of the mixture in a non-continuous stirred system at temperatures within a range of 30 to 40 ° C for at least 96 hours of pH maintenance below 5.0. The pH is periodically monitored. If the pH rises above 5.0, a citric acid buffer is added in an amount to keep the pH below 5.0.

F. Centrifugation of the ferment to separate the three main fractions: chitin, liquid hydrolyzate and pigmented paste.

g. Rinsing of the raw chitin and collection of the rinsed water to recover fine solids or minerals.

h. Chitin drying and storage.

i. Drying and storage of the liquid hydrolyzate, j. The pigmented paste (lipid fraction) is stored in closed containers for preservation.

Operational processes and foundations are best understood with reference to the following detailed description.

Microbial cell activation

Microbial compositions as described in

ΪΜΡΙ

<img file="MX337718B_D0023.tif" />

the present are used as inoculum. The HOE inoculum has a microbe concentration of approximately 2.5 to 3.0% (w / v). HQE is activated by dilution to 5% in sugarcane solution (3.75% of final concentration of sugarcane), and incubated at 37 ° C for 5 days. HYTb (10 ml per liter of culture) is preferably added to provide a source of naturally derived minerals and amino acids. The cell growth of the microorganisms was estimated by optical density measured at 540 nm. Activation is complete at an optical density of approximately 1.7. The concentration of microbes after activation is approximately 1.9 to 3.0% (w / v).

Preparation of sample

Samples of shrimp derivatives are obtained from shrimp processing plants. The slightly thawed and crushed residue (1500 g per batch) is mixed with 99 grams of sugar cane (final concentration 6.6% wt%) and 85.5 ml of 5% (v / p) activated HQE (optical cell density). = 1.7). Then the pH is adjusted to 5.5 using 2M citric acid.

Fermentation control

The mixture is incubated at 36 ° C with non-continuous shaking for 96 h. During the fermentation process, the pH is monitored using a potentiometer, and the total titratable acidity (TTA,%) is determined by titration with NaOH 0. IN

PT

<img file="MX337718B_D0024.tif" />

until a pH of 8.5 is obtained. TTA is expressed as a percentage of lactic acid. '

Separation conditions

The fermentation product is a viscous silage which has an intense orange color, due to the presence of astaxanthin. The silage is centrifuged (5 ° C) at 1250 rpm (930g) for 15 min to obtain chitin, liquid hydrolyzates, and pigment paste. The upper phase (pigment paste) is manually separated. The liquid hydrolyzates are separated by decantation, and the sediment that constitutes the pure chitin is washed with distilled water to separate the fine solids. The resulting liquid is collected and dried. Pure chitin, liquid hydrolyzates and fine solids are dried at 60 ° C. All fractions are stored to protect them from light.

2. Biodegradation of Filamentous Fungi, Yeast and Chitin-Containing Insects

The same process is used to enzymatically degrade filamentous fungi, yeasts, and / or insects containing chitin. Figure 3 is a flow chart showing the digestion of filamentous fungi, yeasts and / or insects to form HYTb and HYTc. HYTc and HYTb are optionally further processed with HQE to form HYTd, a solution with relatively high amounts of

IMPI chitosan and glucosamine compared to ΗΥΤΒ^τ.ο_Εϊ / ρ^μ/ ο? ιεοαο

Fungi, including filamentous fungi, and yeasts from groups including Zygomycetes, Basiomycetes, Ascomycetes, and Deuteromycetes can be used in the biodegradation process. Examples include Aspergillum, Penicillium, Trichoderma, Saccaromyces abd Schizosacaromyces γ edible fungi species such as Agaricus, Pleurotus, Boletus and Lentinula species.

A preferred microbial composition for digesting the fungus, which includes filamentous fungi, yeasts, and insects, is HQE.

3. Chemical Extraction of Fungi and / or Yeast The source of chitin does not need to be from HYTc. For example, fungal derived chitin, including filamentous fungi, and / or yeast can be used. For example, US Patent 7,556,946 describes a chemical process for extracting chitin from fungi, including filamentous fungi, and yeast from groups including Zygomycetes, Basiomycetes, Ascomycetes, and Deuteromycetes. Examples include Aspergillum, Penicillium, Trichoderma, Saccaromyces abd Schizosacaromyces species and edible fungi such as Agaricus, Pleurotus, Boletus and Lentinula species.

Microbial Compositions that Digest Chitin

one. HQE Consortium

HQE was deposited with ATTC on April 27, 2010,

<img file="MX337718B_D0025.tif" />

IMPI

MÜUCANO INSTITUTE

OF THE PROPERTY

INDUSTRIAL and provided the Designation of Depós-rfeo -'- ^ de—

10861.

HQE was developed, in part, by the biodegradation of marine chitin-containing arthropods such as crustaceans. However, it has been determined that HQE can also be used to enzymatically convert solid chitin to chitosan and glucosamine. It is believed that other microbial compositions as described herein can be used in the processes described herein.

The following are the microorganisms in HQE which are believed to be involved in the biodegradation process and their known properties. In some cases the strain is identified as Bioderpac, 2008. Where species are unknown, species and strains are identified as Bioderpac, 2008.

Bacillus subtilis (SILoSil® BS) is a bacterium

Gram positive which is mesophilic and grows at an optimal temperature between 25 and 35 ° C. It is aerobic and can grow in anaerobic conditions and uses a wide variety of carbon sources. It contains two nitrate reductases, one of which is used for nitrogen assimilation. It is capable of secreting amylase, proteases, pullulanases, chitinases, xylanases, and lipases.

Bacillus thuringiensis (Strains HD-1 and HD-73 (SILoSil® BT)) are facultative anaerobic bacteria Gram

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M pi

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

<img file="MX337718B_D0026.tif" />

Positive, in the form of peritrichous flagella. Strains HD and HD-73 synthesize crystals with various geometric shapes of protein and insecticidal activity during the spore period. Strains HD-1 and HD-73 secrete exochitinases when in a chitin-containing medium and can be used for the degradation of crustacean residues during chitooligosaccharide production.

Bacillus cereus (Bioderpac, 2008) is a gram-positive, aerobic, facultative bacterium that forms spores. It is mesophilic and grows at an optimal temperature between 20 and 40 ° C.

Produces the antibiotics zwittermicin A and canosamine.

Bacillus lichenifonnis (Bioderpac, 2008) is a gram-positive, mobile, spore-forming and facultative anaerobic bacterium. Produces bacitracin, alpha amylases, lactamases, proteases, and alkaline proteases. This is a non-pathogenic microorganism that is associated with plants or plant materials.

Bacillus megaterium (Bioderpac, 2008) is a Gram positive aerobic bacteria. It is considered a saprophyte. Produces glucose dehydrogenase, penicillin amidase, betaamidase, and neutral proteases.

Lactobacillus acidophilus (Bioderpac, 2008) is a member of one of eight species of lactic acid bacteria. It is Gram positive, non-sporulating and produces lactic acid during fermentation that uses lactose as a

<img file="MX337718B_D0027.tif" />

main source of carbon to produce energy. It grows with or without the presence of oxygen in an acidic medium (pH 4-5).

It produces the bactereocinas called lactacina B, organic acids, diacetyls and hydrogen peroxide.

Lactobacillua caseí (Bioderpac, 2008) is a facultative anaerobic mesophilic bacterium which is Gram positive and does not form spores. It has the ability to adapt to cold temperatures. The optimal pH for growth is 5.5. Ferments galactose, glucose, fructose, trickle, mannitol and acetylglucosamine. These species can be grown over a wide range of pH and temperature. Produces amylase enzymes. Inhibits the growth of pathogenic bacteria such as H. pylori reducing the pH through the production of (1) organic acids such as acetic, propionic or lactic acid or (2) hydrogen peroxide. This microorganism secretes bacterocins.

Pseudomonas fluorescens (Bioderpac, 2008) is a bacteria with multiple flagella, forced aerobic and its optimal temperature for growth is between 25 and 35 ° C. Produces thermoset lipases and proteases. It is antagonistic to a large number of strains of soil fungi. It produces secondary metabolites such as antibiotics, iron chelates, and cyanides. Produces endochitanase and cellulase in media with different glucose concentrations.

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Trichoderma harzianum (TRICHOSIL) is a fungus

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<img file="MX337718B_D0028.tif" />

saprophyte. It has antibiotic action and biological competition and for this reason it has biological control properties. It produces enzymes that degrade cell walls or a combination of such activities. It produces glucanases, chitinases, lipases, and cellular proteases when it interacts with some pathogenic fungi, such as Fusarium.

Rhizobium japonicum (Bioderpac, 2008) is a nitrogen-fixing bacterium. It synthesizes a hydrogenase system that participates in the recycling of hydrogen to avoid its loss during nitrogen fixation.

Azotobacter vinelandii (Bioderpac, 2008) is an aerobic bacterium. Produces nitrogenase and is capable of fixing nitrogen

Clostridium pasteurianum (Bioderpac, 2008) is a Gram positive, anaerobic obligate bacterium. Produces ferroxin (an electron-carrying protein) that acts as a direct electron donor in reducing protein iron.

Proteus vulgarís (Bioderpac, 2008) It is a facultative, anaerobic, Gram positive bacterium that grows at temperatures close to 23 ° C. Proteolytically it breaks down proteins to free amino acids by the enzymes they produce.

Streptomyces sp. (Bioderpac, 2008) is a bacterium

Gram positive soil. Produces multiple enzymes that

<img file="MX337718B_D0029.tif" />

MEXICAN INSTITUTE f AND 'OF INDUSTRIAL PROPERTY metabolize various nutrients. They can survive significant changes in temperature, humidity, and nutrient sources. The extracellular enzymes produced by these bacteria use chitin and chitosan as substrates at a pH of 4.5 to 6.5 and at 60 ° C. These are conditions generated at the beginning and at the end of the lactic fermentation stages in the biodegradation process.

Nitrobacter sp. (Bioderpac, 2008) is a bacterium

Gram negative, aerobic, which converts nitrites to nitrates. It grows at a pH between 6 and 9 and at temperatures between 10 to 34 ° C. The bacterium breaks down organic polymers such as chitin into compounds that are used by other organisms, such as Pseudomonas fluorescens and Rhizobium japonicum (Bioderpac2008).

Micrococcus sp. (Bioderpac, 2008) is a spherical Gram positive bacterium. This microorganism in association with Streptomyces sp () is capable of degrading colloidal chitin derivatives.

HQE can be used to enzymatically digest chitin. However, of these microbes, one, two, three, four, or more of the following are believed to be isolated from HQE and used to degrade chitin to chitosan and glucosamine: Bacillus subtilis ((SILoSil® BS), Bacillus thuringiensis (Strains HD-1 and HD-73 (SILoSil® BT), Pseudomonas (Bioderpac, 2008), fluorescens

Trichoderma harzianum

II í

<img file="MX337718B_D0030.tif" />

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INDUSTRIAL (TRICHOSIL), Streptorayces sp. (Bioderpac, 2008), and Micrococcus sp. (Bioderpac, 2008). In preferred embodiments, the microbial composition contains at least one of, at least two of, and preferably each of Bacillus subtilis (SILoSil® BS), Bacillus thuringiensis (Strains HD-1 and HD-73 (SILoSil® BT), and Trichoderma harzianum (TRICHOSIL) In yet another preferred embodiment, the microbial composition comprises

Trichoderma harzianum (TRICOSIL).

2. Groups and enzymatic activity of microorganisms in HQE

Biodegradation of chitin-containing arthropods, fungi, filamentous fungi, yeast and / or insect components requires hydrolytic enzymes such as proteases, lipases, and chitinases. The following groups and group combinations are also useful for the digestion of chitin in chitosan and glucosamine.

The primary group of microbes in HQE includes Lactobacillus acidophilus (Biodepac 2008), Bacillus subtilis (SILoSil® BS), Pseudomonas fluorescens (Biodepac 2008), Bacillus licheniformis (Biodepac 2008), and Trichoderma harzianum (TRICHOSIL). These microorganisms are capable of biodegrading arthropods or arthropod derivatives. One or more of the members of this primary group also have a synergistic action when

<img file="MX337718B_D0031.tif" />

combine with other HQE microorganisms. _

The first group of microorganisms includes microorganisms which cause the reduction of pH and which stabilize the fermentation due to the production of organic acids and hydrogen peroxide. This group includes Lactobacillus acidophilus (Biodepac 2008) and Lactobacillus casei (Biodepac 2008). Its activity is important at the beginning of fermentation and during the final stages of fermentation to produce the optimal pH for hydrolytic enzymes. Its activity also creates a culture environment which prevents the growth of unwanted microorganisms and favors the demineralization of chitin residues. Lactobacillus acidophilus (Biodepac 2008) is a member of the primary group.

The second group of microorganisms includes microorganisms which produce extracellular enzymes. This second group includes Bacillus subtilis (SILoSil® BS), Bacillus cereus (Biodepac 2008), Trichoderma harzianum (TRICHOSIL), Rhizobium japonicum (Biodepac 2008) and Azotobacter vinelandii (Biodepac 2008). Chitin chains in arthropods or arthropod derivatives are associated with protein molecules. The separation of such polymers requires the hydrolytic action obtained from the chitinolytic and proteolytic enzymes produced by these

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OF THE INDUSTRIAL PROPERTY microorganisms. Both types of enzymes break the chains in the inner portion of the polymer to produce oligomers of varying sizes. The action of these enzymes occurs in a successive manner within the intermediate and final phases of the fermentation process when the appropriate pH conditions are achieved. The microorganisms in this group and the environmental conditions they produce facilitate the release of pigments and the lipid fraction attached to these residues. Bacillus subtilis (SILoSil® BS) and Trichoderma harzianum (TRICHOSIL) are members of the primary group.

The third group of microorganisms includes the microorganisms Bacillus licheniformis (Biodepac 2008), Pseudomonas flourescens (Biodepac 2008), Sptreptomyces, (Biodepac 2008) and Clostridíum (Biodepac 2008). These microorganisms hydrolyze oligomers (chitooligosaccharides and peptides) to produce chitobioses, glucosamine, and free amino acids. Bacillus licheniformis (Biodepac 2008) and Pseudomonas flourescens (Biodepac 2008) are members of the primary group.

In preferred embodiments, one or two of the first, second, and third groups of microorganisms can be combined. Alternatively, all of the first, second and third groups can be combined.

A fourth group of microorganisms includes

<img file="MX337718B_D0032.tif" />

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<img file="MX337718B_D0033.tif" />

HD-73),

Bacillus thuringiensis (HD-1 and / or Streptomyces strains (Bioderpac, 2008), Micrococcus (Bioderpac, 2008), Nitrobacter (Bioderpac, 2008) and Proteus vulgaris (Bioderpac, 2008). The fourth group of microorganisms can be combined with (1 ) the primary group of microorganisms (2) any of the first, second and third groups of microorganisms (3) the combination of one or two of the first, second and third groups of microorganisms or (4) the combination of all of the first, second and third group. The addition of this fourth group results in a synergistic effect which improves the biodegradation process.

Each of these groups, which includes the primary group, is usefully separated and can be combined with prior art microbial compositions to improve their performance. In this regard, the fourth group is particularly preferred.

Table 1 sets out some of the combinations mentioned above. Column 1 is a list of known microorganisms in HQE that are believed to be active in the biodegradation process. Column 2 lists the microorganisms in column 1 without the microorganisms in the fourth group of microorganisms. Column 3 shows the combination of the primary microorganisms while columns 4, 5 and 6 identify the combination of

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<img file="MX337718B_D0034.tif" />

microorganisms of the first, second and third groups. Column 4 is the combination of groups 1 and 2; column 5 of groups 1 and 3 and column 6 of groups 2 and 3. Other useful combinations are discussed in columns 7-10.

Table 1

Crop Composition

<td>Microorganism</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>Bacillus subtilis</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td>

<td>Bacillus</td><td>X</td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td>

<td>Microorganism</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>cereus</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Bacillus megaterium</td><td>X</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Azotobacter vinelandii</td><td>X</td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td>

<td>Lactobacillus acidophilus</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td><td></td>

<td>Lactobacillus casei</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td></td><td></td><td>X</td><td>X</td><td></td>

<td>Trichoderma harzianum</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td>

<td>Rhizobium japonicum</td><td>X</td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td>

<img file="MX337718B_D0035.tif" />

<img file="MX337718B_D0036.tif" />

<td>Microorganism</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>Clostridium pasteurianum</td><td>X</td><td>X</td><td></td><td></td><td>X</td><td>X</td><td></td><td></td><td>X</td><td>X</td>

<td>Bacillus licheniformis</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td>

<td>Pseudomonas fluorescens</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td></td><td></td><td></td><td></td>

<td>Bacillus thuringiensis</td><td>X</td><td></td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Streptomyces</td><td>X</td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Nitrobacter</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Micrococcus</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Proteus vulgaris</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td>

Particularly preferred crops are 1-4, 68, and 10. I

The activity of the enzymatic extracts produced by the microorganisms with HQE is complex, but has allowed the degradation of the chitinous residues of arthropods such as crustaceans. The microorganisms in HQE are activated in a successive way in accordance with the environment generated by the organisms used.

HYTb

HYTb contains amino acids (approximately 12% / ¾ ΐ-JG

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INDUSTRIAL by weight), chitosan (approximately 0.5-1.5% by weight), glucosamine (approximately 0.5-1.5 lipases, modalities polypeptide chitinases, by weight) and micro elements (approximately 6% by weight) including calcium, magnesium, zinc, copper , iron and manganese. It also contains enzymes such as lactic enzymes, proteases, among others, lactic acid, and other carbohydrates. In some the degree of acetylation of the chitosan produced is 20% or less, preferably 15% or less, more preferably 10% or less, still more preferably 8% or less is preferred and most preferably 5% or less. The specific gravity of HYTb is typically approximately 1,050-1,054. The average amino acid content in HYTb for certain amino acids is set forth in Table 2.

Table 1

Hydrolyzates in dry powder of the amino acid profile (mg per g in dry weight)

<td>Amino acid</td><td>Hydrolyzed powder dry</td>

<td>Aspartic acid</td><td> 38</td>

<td>Glutamic acid</td><td> 39</td>

<td>Serine</td><td> 16</td>

<td>Histidine</td><td> 9</td>

<td>Glycine</td><td> 28</td>

<td>Threonine</td><td> 14</td>

<img file="MX337718B_D0037.tif" />

<img file="MX337718B_D0038.tif" />

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<td rowspan="2">Amino acid</td><td>Hydrolyzed powder</td>

<td>dry</td>

<td>To the girl</td><td> 36.1</td>

<td>Proline</td><td> 25.8</td>

<td>Tyrosine</td><td> 70</td>

<td>Arginine</td><td> 22.2</td>

<td>Valine</td><td> 20</td>

<td>Methionine</td><td> 16.4</td>

<td>Isoleucine</td><td> 18.3</td>

<td>Tryptophan</td><td> 3.1</td>

<td>Leucine</td><td> 23</td>

<td>Phenylalanine</td><td> 39</td>

<td>Lysine</td><td> 13</td>

<td>Total</td><td> 431</td>

HYTc

The primary component of HYTc is chitin. It has an average molecular weight of approximately 2300 Daltons and constitutes approximately 64% by weight of the composition. Approximately 6% HYTc contains minerals including calcium, magnesium, zinc, copper, iron, and manganese, approximately 24% by weight of protein, and 6% of water. It has a specific gravity of approximately 272 Kg / m³.

HYTd

HYTd is obtained by fermentation of chitin with a microbial composition suspended in HYTb. HYTb already contains chitosan (approximately 0.5-1.5% by weight) and glucosamine (approximately 0.5-1.5% by weight). The amount of chitosan and glucosamine in HYTd ranges from about 2% by weight to 2.5% by weight of chitosan and from about 2% by weight to 5% by weight of glucosamine. This represents an increase in the amount of chitosan and glucosamine compared to HYTb from approximately 0.5% by weight to 2.5% by weight

<img file="MX337718B_D0039.tif" />

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INDUSTRIAL chitosan and from approximately 0.5% by weight to 5% by weight of glucosamine.

As used herein the term glucosamine includes glucosamine or a mixture of glucosamine and N-acetyl glucosamine. In most modalities, HYTd contains glucosamine and N-acetyl glucosamine.

HYTd may also contain particulate chitin that has not been completely deferred. In general the fermentation mixture is filtered to remove large chitin particles. The filtrate usually contains no more than 2% by weight of chitin. HYTd when diluted is similar to HYTb in that it contains amino acids (approximately 12% by weight) and micro elements (approximately 6% by weight) that include calcium, magnesium, zinc, copper, iron, and manganese. It also contains enzymes such as lactic enzymes, proteases, lipases, chitinases, among others, lactic acid, polypeptides and other carbohydrates. In some embodiments, the degree of acetylation of the chitosan produced is 20% or less, preferably 15% or less, more preferably 10% or less, still more preferably 8% or less and most preferably 5% or less. The average amino acid content in HYTd for certain amino acids is similar to HYTb. See Table 2.

HYTd preferably comprises 12% by weight of Lamino Acids (Aspartic Acid, Glutamic Acid, Serine, Histidine,

Glycine, Threonine, Alanine, Proline, Arginine, Valine, Methionine,

<img file="MX337718B_D0040.tif" />

<img file="MX337718B_D0041.tif" />

Isoleucine, Tryptophan, Phenylalanine, Lysine and Threonine) and 5% by weight of glucosamine and chitosan. HYTd also preferably contains one or more or all of the soluble minerals (P, Ca, Mg, Zn, Fe and Cu), enzymes and lactic acid from the chitin digestion process as well as other polysaccharides.

The fermentation mixture, eg HYTb and HQE, can be diluted early in the chitin digestion process. If diluted, the ratio of chitosan and / or glucosamine to amino acids will be higher in the HYTd produced after digestion. That is, dilution before fermentation produces relatively more chitin and / or glucosamine taking into account the dilution factor than if dilution does not occur.

Chitosan / Glucosamine Products

When the starting fermentation mixture does not contain chitosan and glucosamine, for example, when activated HQE is used to digest chitin, the amount of chitosan and glucosamine in the final product ranges from about 0.5% by weight to 1.0% by weight of chitosan and from about 0.5% by weight to 1.8% by weight of glucosamine.

Example 1

Production of chitosan and glucosamine oligosaccharides

Chitosan and glucosamine oligosaccharides

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<img file="MX337718B_D0042.tif" />

they can be produced under different conditions. The hydrolysis time can be varied to find the optimal conditions to produce chitosan and / or glucosamine.

Manual agitation three times a day was used in each of the following experiments. The temperature was 35 ° C.

Table 2

<td>Treatment</td><td>Chitin (HYT-C)</td><td>Amino acid (HYT-B)</td><td>3-Day Activated Inocula (HQE)</td>

<td> 1</td><td>2% 20 g</td><td>1000 mi</td><td>0 my</td>

<td> 2</td><td>3% 30 g</td><td>1000 mi</td><td>0 my</td>

<td> 3</td><td>4% - 40 g</td><td>1000 mi</td><td>0 my</td>

<td> 4</td><td>2% - 20 g</td><td>970 mi</td><td>30 mi</td>

<td> 5</td><td>3% 30 g</td><td>970 mi</td><td>30 mi</td>

<td> 6</td><td>4% - 40 g</td><td>970 mi</td><td>30 mi</td>

<td> 7</td><td>3% , chitin micronized</td><td>1000 mi</td><td>0 my</td>

Glucosamine quantification

Glucosamine analysis was determined as previously reported in Tsuji et al. (1969), with some modifications: Specifically, it was added to a sample of

<img file="MX337718B_D0043.tif" />

<img file="MX337718B_D0044.tif" />

300 μΐ, 300 μΐ of KHSO₄ (5%), 300 μΐ NaOH₂ (5%) . The sample was then allowed to sand with occasional shaking for 15 minutes. Excess nitrous acid was removed by adding 300 μΐ NH4SO3 NH₂ (12.5%). 300 μΐ of MBTH (0.5%) were added to the mixture and incubated in a water bath for 60 minutes. Finally, 00 μΐ of FeCl3 (0.5%) was added and the absorbance at 653 was read after 30 minutes against a blank containing water.

Example 2

The following protocol can be used for the industrial production of HYTd with the high concentrations of glucosamine and chitosan. The following table shows the parameters used. The amount of activated HQE is proportional to that used in Example 1.

Table 3

Industrial Droduction Parameters

<td>Carrier solution: HYT-B</td><td>15,000 L</td>

<td>Micronized chitin (residual chitin from the grinding process)</td><td>300 kg</td>

<td>Temperature</td><td>Ambient temperature (30- 35 ° C)</td>

<td>Agitation</td><td>8 hours / daily</td>

<td>Production time</td><td>7 days</td>

Example 3

Production Kinetics

Table 4 shows the production of glucosamine in

HYTd as a function of time. Chitin (20 grams) is

<img file="MX337718B_D0045.tif" />

<img file="MX337718B_D0046.tif" />

digested with HQE activated on 970 mi of HYTb.

Table 4

<td>Days of production</td><td>% total of glucosamine (HYTd)</td><td>Glucosamine at HYTb</td><td>Glucosamine produced</td>

<td> 3</td><td> 1.11</td><td> 0.74</td><td> 0.37</td>

<td> 4</td><td> 1.54</td><td> 0.74</td><td> 0.8</td>

<td> 6</td><td> 1.78</td><td> 0.74</td><td> 1.04</td>

<td> 7</td><td> 1.93</td><td> 0.74</td><td> 1.19</td>

<td>Packaging days (approx. 20 days)</td><td> 2.29</td><td> 0.74</td><td> 1.55</td>

The results of this experiment are also represented in Figure 2.

Example 3

One kilogram of residual HYTc chitin was combined with two liters of HYTb. One liter of activated HQE was added.

After mixing, twenty liters of water were added. The resulting mixture was fermented for six days at 35 to 36 ° C. This resulted in a solution containing 6% by weight of glucosamine and 2% by weight of chitosan. See Figure 4.

It is noted that in relation to this date, the best method known by the applicant to put the aforementioned invention into practice is the one that is clear from the present description of the invention.

Α / 8 Μ Κ

<img file="MX337718B_D0047.tif" />