patenta de insectos

1. (19) United States
(12) Patent Application Publication (10) Pub. No.: US2014/0100361 A1
US 201401.00361A1
Le Roux et al. 43) Pub. Date: Apr. 10, 20149
(54) EXTRACTION OF CHITINS IN A SINGLE (30) Foreign Application Priority Data
STEP BY ENZYMATIC HYDROLYSIS IN AN
ACID MEDIUM May 26, 2011 (FR) ....................................... 115458O
(75) Inventors: Karine Le Roux, Milly-la-Foret (FR): O O
Jean-Pascal Berge, Saint-Ave (FR): Publication Classification
RiseBy hapeleurline (51) Int.Cl./ . s COSB 37/08 (2006.01)
Saint-Etienne-de-Montluc (FR); (52) U.S. Cl
Abdellah Arhaliass, Saint-Nazaire (FR) CPC .................................... C08B 37/003 (2013.01)
(73) Assignee: IFREMER (INSTITUT FRANCAIS USPC ............................................. 536/20: 435/274
DE RECHERCHE POUR
LEXPLOITATION DE LA MER), (57) ABSTRACT
Issy-les-Moulineaux (FR)
A method ofenzymatic extraction ofchitin is realized in a
(21) Appl. No.: 14/122,427 single step wherein the chitin is obtained by enzymatic
1-1. hydrolysis of raw material constituted by animal biomass
(22) PCT Filed: May 25, 2012 including chitin, the enzymatic hydrolysis using an enzyme
act1Ve 1n ac1d medium. Also d1SCIOSed 1S arOceSS Of Ot1m1(86). PCT No.: PCT/FR2O12/05118O ive in acid medium. Also disclosed is a p foptimi
S371 (c)(1),
(2), (4) Date: Nov. 26, 2013
Raw rateria
Washing, dying, giding
zation ofthe methodofenzymaticextractionofchitin,as well
as the chitin susceptible to be obtained by the method of
enzymatic extraction.
Reaction edium
sowent + acid
control ofpH and temperature
homogenization
2 enzyme additio:
Soisbie phase
pigments, tigids, Sigars, i.e.ai
Saits, airino acids, peptides...}
Neutralizatio aid
separatios by categories
of compouds
Enzymatic hydroEysis inan acid
nedin =
proteoysis and Scitizatio:
of the litera sats
filtration f rising
insibie phase
= CH3S
bleaching aidfor
deacetylation
foration of chitosan,
oligo-chiti chitosai,
gucosamines

2. Patent Application Publication Apr. 10, 2014 US 2014/0100361 A1
Reaction medium
{so went + acid}
contro ofpH and temperatuie
Raw materia
Washing, dying, giding
homogenization
2} enzyme addition
Enzymatic hydrolysis in an acid
medium =
proteoiysis and soiuxilization
of the irrera sats
fitration f rising
Soluble phase
pigments, tipids, Sugars, minera:
saits, aimino acids, peptides...}
insolubie phase
= CHENS
bleachingandfor
deacetylation
Neutralization aid
Separation by categories
of compounds r
iformation of chitosan,
oligo-critifchitosa,
gucosamines
F.G. 1

3. US 2014/0100361 A1
EXTRACTION OF CHTINS IN A SINGLE
STEP BY ENZYMATIC HYDROLYSIS IN AN
ACID MEDUM
FIELD OF INVENTION
0001. The present invention relates to the field of the
recovery ofbiomass, preferably animal biomass, more pref
erably marine and/or entomological biomass. In particular,
the present invention relates to a method for the enzymatic
extraction of chitin in a single step, from animal biomass
elements comprising chitin, preferably from marine and/or
entomologicalby-products,usinganactiveenzymeinanacid
medium.
BACKGROUND OF INVENTION
0002 The production and consumption of marine prod
ucts, and particularly of crustaceans, notably prawns, is
increasing each year. By-products (heads and shells) gener
ally represent more than 50% ofthe fresh weight ofcrusta
ceans. Theusethereofisthus amajorissuegiventheVolumes
involved and also the slow natural biodegradability thereof.
Chitin is the main product derived from these by-products.
0003 Moreover, entomophagy is a common dietary prac
ticeinsomecountries whichistendingtodevelop worldwide.
Indeed, insects represent an advantageous dietary resource
due to the nutritional qualities thereof. Furthermore, insect
production offers a very advantageous environmentally
friendly alternative compared to the production ofotherani
mal proteins. The by-products obtained from insect protein
production include chitin-rich shells, as for crustaceans.
0004 Chitin is the second most plentiful polysaccharide
on the surface ofthe Earth after cellulose. It does not have a
single chemical structure, but several, since it includes
polysaccharides consisting of N-acetyl-B-D-glucosamine
units and D-glucosamine units.
0005 Chitinpartiallyformstheexoskeleton ofinsectsand
crustaceans and the wall of fungi and bacteria. Chitin thus
represents 20 to 30% of the shells of crustaceans. Besides
chitin, the exoskeleton ofcrustaceans contains 20 to 40% of
proteins, 30 to 60% ofmineralsand 0to 14% offat (Waldeck
J. Daum G., Bisping B. and Meinhardt F., Appl. Env, Micro
biol., 2006, 72 (12), 7879-7885). Chitin thus represents 3 to
60% oftheshellsofinsects. Besideschitin, theexoskeletonof
insects contains 20 to 80% ofproteins, 1 to 20% ofminerals
and 10 to 50% offat (“Forest insects as food: humans bite
back. Proceedings ofa workshop onAsia-Pacific resources
andtheirpotential fordevelopment, 19-21 Feb. 2008,Chiang
Mai, Thailand FAO). Whether for crustaceans or for
insects, the proportions of the various constituents vary
according to the species, age, genus and may fluctuate
according to the seasons and environmental conditions.
Chitin extraction conditions should thus be adapted accord
ing to the raw material used (Tolaimate A. Desbrieres J.
Rhazi M.andAlaguiA., Polymer, 2003, 44(26), 7939-7952).
0006 Chitin is found incrustaceanandinsectby-products
in the form ofchitin/proteinS/minerals complexes. It is usu
ally extracted in two “chemical extraction' steps:
0007 demineralization by means ofacid hydrolysis, to
remove minerals; and
0008 deproteinization by means ofbase hydrolysis, to
remove proteins.
0009 Chitin extraction from marine by-products is cur
rently carried out on an industrial scaleby means of"chemi
Apr. 10, 2014
cal extraction'. Chitin extraction from insects has not been
very developed to date but has already been the subject of
studies, essentially using a chemical process (cicada chitin:
Sajomsang W. and Gonil P. Mat. Science Engineering C,
2010, 30(3), 357-363; silkworm pupa chitin: Paulino A.,
Simionato J. Garcia J. and Nozaki J. Carbohydrate Poly
mers, 2006, 64, 98-103; bumblebee chitin: Majtan J., Bil
ikova K. Markovic O. Grog, J., Kogan G. andSimuth J., Int.
J. Biol. Macromol., 2007, 40,237-241).
0010. A third optional bleaching step, for example using
Sodium hypochlorite, is frequently used to remove residual
pigments. Washing operations, generally with water, are
required between these various steps.
0011 Chitincan thenbereadilydeacetylated, forexample
using sodium hydroxide, to produce chitosan.
0012 Chitin is conventionally extracted for a wide range
ofapplications: medical,pharmaceutical, dietary, food,tech
nical (water filtration and depollution), etc. Indeed, chitin,
chitosan and the derivatives thereof, particularly the oligo
mers thereof, are biocompatible, biodegradable and non
toxic. Thetype ofapplication depends on the physicochemi
cal characteristics of chitin and the derivatives thereof. In
particular, chitosan may be particularly used for producing
mulching film, Stomach protection gels, but also for active
ingredient encapsulation, waste water filtration, cartilage
replacement, tissue regeneration, etc.
0013 Industrial chitin extraction from marine by-prod
ucts is essentially implemented in emerging countries. Con
ventional chemical extraction uses large quantities of
reagents (essentially hydrochloric acid, Sodium hydroxide
andbleachingagents)whichareharmful foroperators,equip
mentandthe environment. Furthermore, thebasicdeprotein
ization step is generally performed hot and thus requires a
high energy input. Moreover, the washing steps give rise to
very large volumes ofpolluted effluents, which are techni
cally difficult and expensive to recycle.
0014. Oneoftheproblems associated with currentextrac
tion methods is thepossibility ofchitin being denatured dur
ing the process (Crini G., Badot P. and Guibal E., Chitine et
Chitosan. Du polymered l'application, 2009, Presses Uni
versitaires de Franche-Comté).
0015 Studies have shown that chitin could be extracted
using biological methods, notably by means of enzymatic
extraction or microbiological fermentation, particularly for
the deproteinization step.
0016. Of the research on the fermentation process, the
studies conducted by Beaney use the exoskeleton of the
Nephrops norvegicus prawn as the study Substance (Beaney
P. Lizardi-Mendoza.J. andHealy M.,J. Chem. Tech. Biotech.,
2005,80, 145-150). In this study, chitinisextractedby means
oflactic fermentation in thepresenceofbacterialstrains for5
days at 30°C. Theacidification ofthe medium, due to lactic
acid production by the bacteria, results in partial demineral
ization while the bacteria carry out deproteinization. In this
study, the pH decreases to a value of 3.5 after 7 days of
fermentation. However, under these conditions, the chitin
extracted still contains 13% ofproteins and 14% minerals. A
purer chitin may then be obtained by performing further
chemical treatments. This typeofmethodis thus not suitable
fordirectlyobtainingahigh-quality chitin,limitingtheappli
cations thereof.
0017. A further microbial fermentation study was con
ducted to extractchitin from redcrab shellsby co-fermenting
the shells in the presence oftwo bacteria: firstly, Lactobacil

4. US 2014/0100361 A1
lus paracasei tolerans KCTC-3074, which is a lactic acid
producing bacterium, and secondly Serratia marcescens
FS-3,which isanextracellularprotease-producingbacterium
(JungW.,JoG., Kuk.J., Kim K. andPark R. Appl. Microbiol.
Biotechnol., 2006, 71,234-237). Co-fermentation was main
tained for7 days at30° C. and resulted in a demineralization
rate of97.2% and a deproteinization rate of merely 52.6%.
Thechitin obtainedwas notcharacterisedin this studybutthe
low deproteinization rate is limiting in terms of the use
thereof.
0018. A further microbial fermentation study was con
ducted by the same team, with a bacterial strain producing
proteases for deproteinizing and demineralizing marine by
products (Jo G., JungW. KukJ. Oh K. KimY. and Park R.
Carbohydratepolymers, 2008, 74, 504-508). A fermentation
testwasconductedfor7daysat30°C.,in thepresenceof10%
of bacterial strain, and resulted in a deproteinization and
demineralization rate of 84% and 47%, respectively. As
above, the demineralization is due to the pH decrease over
time (pH 5.6 after 7 days of fermentation), associated with
bacterial acid production. The low degree of purity of the
chitins obtained is limiting in terms of the applications
thereofand this method, like the previous method, involves
the drawback ofrequiring a very long reaction time.
0019. The best demineralization and deproteinization
yields by means of a two-step fermentation process were
obtained by Waldeck (Waldeck J. Daum G., Bisping B. and
Meinhardt F., Appl. Env, Microbiol., 2006, 72 (12), 7879
7885).Afterfermentingfor6daysfrom42to55°C.,followed
by a 3hour lactic acid treatment, the residual protein content
is less than 10% and the demineralization rate is equal to
98.8%. As in the study by Jo et al., the reaction time is
relatively long.
0020 Chitin extraction by means ofa fermentation pro
cess thus results in a chitin with a higher residual protein
content than in the case of chemical extraction and further
treatments are frequently required to improvethe demineral
ization. Furthermore,thereactiontimesare muchlongerthan
with the chemical process.
0021 Chitin extraction by means ofa biological process
may also be performed using enzymatic extraction.
0022. A method for extracting chitin comprising the
removalofproteinsbymeansoftheenzymaticactivity offish
viscera was proposed in the international patent application
WO 86/06082. In particular, the method described in this
patent application comprises the extraction of chitin from
prawn shells by means ofdemineralization with an acid fol
lowed by deproteinization using fish viscera, optionally pre
ensiledat pH 1.2-2.5. The raw material, i.e. the prawn shells,
is first ensiled in a Sulphuric acid solution. Ensiling makes it
possible to store the raw material before use and enables the
demineralization thereof. Secondly,thepre-ensiled shellsare
placed in contact with fish viscera for deproteinization. The
characteristics of the chitin obtained using this two-step
method are not specified.
0023. Enzymatic extraction may also be performed using
apurifiedenzyme,generallyaproteolyticenzyme.This is,for
example, the case in the study conducted by N. Gagné, with
the use ofchymotrypsin orpapain for extracting chitin from
prawn shells (Gagné N. “Production ofchitin and chitosan
from crustacean wasteandtheiruseasfoodprocessing aid',
1993 McGill University—Montreal, doctoral thesis). Aftera
conventional chemical demineralization step, the proteins
present are hydrolysed by the enzymes. The optimal depro
Apr. 10, 2014
teinization conditionsparticularly involveapH of8.0-8.7 for
chymotrypsin and papain. Under the conditions used, the
residual protein content is very low (1.3% and 2.8% forchy
motrypsin and papain, respectively).
0024. The same type ofmethod was used by Synowiecki
and Al Khateeb for conducting the enzymatic digestion of
prawn shells, previously demineralized with hydrochloric
acid, usingalcalase, at 55° C. and pH8.5 (Synowiecki J. and
Al Khateeb, Food Chemistry, 2000, 68, 147-152).
0025. A method for producing chitin involving an enzy
matic hydrolysis step has been patented (CN1715255). This
method offers a general approach for processing the raw
material sincecompounds otherthan chitinarealso extracted
from prawn shells. In particular, this method comprises an
enzymatic hydrolysis step followed by solvent extract. The
solidportionobtained is thenplacedinthepresenceofhydro
chloricacidtoperform demineralizationandfinishextracting
chitin.
0026. All the enzymatic chitin extraction methods cur
rently described involvean independentconventional chemi
cal demineralization step, before or after the enzymatic
hydrolysis step. In this way, even ifthe deproteinization step
is carriedoutusingabiological method,there is still achemi
calstep requiring washing operations andproducingpolluted
effluents and liable to affect the properties of the extracted
chitin.
0027. The current methods are thus not satisfactory and
thereisaneedforchitinextractionmethodswhicharesimple,
rapid, efficient, inexpensive and more environmentally
friendly. These methods should be suitable for producing
chitins wherein thepurity iscompatible with usein the food,
dietetic or cosmetic industries. Furthermore, the chitins pro
ducedshould meetthespecifications requiredtobeprocessed
into chitosan, oligo-chitosan orglucosamines,etc. Inparticu
lar, the degree of polymerization of the chitin should be
sufficiently high and it should not be denatured during the
process.
0028 Moreover, further compounds can potentially be
recovered using crustacean and insect by-products, particu
larly in nutraceuticals, dietetics or cosmetics. Indeed, these
marine and insect by-products contain soluble compounds
Suchas lipids,pigments, Sugars, mineral salts, aminoacids or
peptides. Targeted extractions of these soluble compounds
have been developed, such as for example the extraction of
pigmentsand inparticularofastaxanthin which is usedin the
food industry (U.S. Pat. No. 7,241,463). However, these
methods targeted on Soluble compound extraction require
further steps to conduct chitin extraction.
0029. In current chitin extraction methods, chitin is
obtained in Solid form and the liquid extraction phases, con
taining soluble compounds of potential interest, are not
recovered.Thislackofrecovery canparticularlybeexplained
by the poor quality ofthe Soluble compounds present in the
liquid phases. Indeed, in these chitin extraction methods,
soluble compounds are frequently degraded due to the rela
tively severe conditions used.
0030 Therefore, there is a need for chitin purification
usinga methodthatrespects theinitial structurethereofmore
and is also suitable forbeingassociated with soluble product
co-extraction.
0031. TheApplicantconducted research inordertoobtain
full recovery of the portions of animal biomass containing
chitin, notably full recovery of marine by-products, in par
ticular crustacean shells and entomological biomass, in par

5. US 2014/0100361 A1
ticularinsectshells. Inparticular,co-extraction methods were
studied with a view to the advantages thereof in relation to
targeted extractions.
SUMMARY
0032. The invention thus relates toa method fortheenzy
maticextraction ofchitin,characterisedin thatsaid methodis
carried out in a single step, hereinafter referred to as the
“single step', wherein chitin is obtained by the enzymatic
hydrolysis ofanimal biomass comprising chitin, said enzy
matic hydrolysis using an active enzyme in an acid medium.
0033 Accordingto oneembodiment, saidsinglestepisan
enzymatic hydrolysis intended for deproteinizing and dem
ineralizing marine by-products simultaneously.
0034. According to one embodiment, saidactive enzyme
in an acid medium is a protease having a broad spectrum of
activity inan acid medium, preferably pepsin ora stableacid
protease.
0035. Accordingto oneembodiment,theenzyme concen
trationused forhydrolysis is 0.1 to 75%,preferably5 to30%,
more preferably from approximately 23 to approximately
27% in weight relative to the weight of the protein mass
estimated in the raw material.
0036. According to one embodiment, the acid medium is
obtained by means ofthe presence of an acid, preferably a
dietary acid, morepreferably phosphoric acid orformic acid.
0037 Accordingtooneembodiment, saidanimalbiomass
comprising chitin comprises marine by-products, preferably
marine by-products obtained from crustaceans, preferably
prawns, crabs orkrill, orfrom cephalopods,preferably squid
or cuttlefish.
0038 Accordingtooneembodiment, saidanimalbiomass
comprising chitin comprises insect by-products, preferably
insect by-products obtained from beetles orhymenoptera.
0039. According to one embodiment, said methodfurther
comprises operationsforwashing,dryingand/orgrindingthe
raw material, preferably water washing, cold drying and/or
grinding operations resulting in fragments less than 1 mm in
S17C.
0040. According to one embodiment, said methodfurther
comprisesreactionmediumtreatmentoperationsattheendof
the enzymatic hydrolysis, said operations comprising opera
tionsforseparatingtheSolidandliquidphases, rinsingand/or
dryingtheinsolubleportion,preferably operations consisting
offiltration, rinsing with waterand/oroven-drying.
0041. The invention also relates to a method for optimis
ing said method forthe enzymatic extraction ofchitin, char
acterized in that said optimization method comprises at least
one ofthe following steps:
0042 a) selecting the pH oftheacid medium in the range
pH-0-2, preferably pH-0-1.5, preferably pH-0-1,82 82 82
wherepH isthepHatwhichtheenzymeexhibits maximum
activity,
0043 b) selecting the temperature ofthe acid medium in
therangeTit.0-20°C.,preferablyTit.0-15°C., preferably
T+0-10° C., where T is the temperature at which the
enzyme exhibits maximum activity,
0044 c) determining the mineral and protein content of
the raw material,
0045 d) calculating the acid concentration to be used in
the reaction medium, accordingto the mineral content ofthe
raw material, itbeing understood that, according to one pre
Apr. 10, 2014
ferred embodiment, the pH is selected such that the reaction
medium is maintained throughout the enzymatic hydrolysis
atthe pH selected in step a).
0046 e) calculating the proportion ofenzyme to be used
with respect to the protein content ofthe raw material,
0047 f)determiningthe reaction timeforobtaining chitin
orthe chitin derivatives sought.
0048. The invention further relates to chitin that can be
obtained by means ofthe method accordingto the invention.
0049. The invention also relates to chitosan that can be
obtained by deacetylating chitin according to the invention.
0050. Theinventionalso relatesto acompositioncompris
ing chitin accordingto the invention and/or chitosan accord
ing to the invention.
0051. Theinvention also relates to a pharmaceutical com
position comprising chitin according to the invention and/or
chitosan according to the invention, a cosmetic composition
comprising chitin according to the invention and/or chitosan
according to the invention, a medical device comprising
chitinaccordingtotheinventionand/orchitosanaccordingto
the invention.
0.052 Theinventionalso relatestoafoodproduct,a nutra
ceutical composition, a dietetic composition, a food Supple
ment or a functional food comprising chitin according to the
invention and/or chitosan according to the invention.
0053. Theinventionalso relatesto acompositioncompris
ing chitin accordingto the invention and/or chitosan accord
ing to the invention for the use thereofin water treatment,
filtration and/or water depollution.
0054 The invention also relates to a texturingagent com
prising chitin according to the invention and/or chitosan
according to the invention.
DEFINITIONS
0055. In the present invention, the following terms are
defined as follows:
0056 “Chitin’ refers to N-acetyl-glucosamineandglu
cosamine polysaccharides.
0057 "Chitosan” refers to chitin deacetylation prod
ucts. The borderline between chitosan and chitin con
sists ofa 50% degree ofacetylation: below, the com
pound is called chitosan, above, chitin.
0.058 Animal biomass” refers to all organic matter of
animal origin.
0059) “Marineby-products” refers to parts not used by
the food industry in marine products, particularly crus
tacean shells and heads.
0060 “Entomological by-products”or“insectby-prod
ucts” refers to parts not used by the food industry in
entomological products, particularly insect shells and
heads.
0061 “Degreeofpolymerization”referstothelengthof
apolymerchain,particularly chitin.Thedegree ofpoly
merization consists of the number of monomer units
forming the polymer chain.
0062 “Crystallinity index' refers to the proportion of
material found in the crystalline state.
0063 “Demineralization” referstoamethodforremov
ing minerals.
0.064 “Deproteinization” refers to a method for remov
ing proteins.
0065 “Depolymerization” referstothereductionofthe
length ofthe polymeric chain ofchitin.

6. US 2014/0100361 A1
0066 “Deacetylation” refers to the removal of acetyl
groups and corresponds to the transition from chitin to
chitosan.
0067 “Moisturecontent” refers to the mass percentage
ofwater contained in a sample.
0068 “Proteincontent” refers to the masspercentageof
protein contained in a sample.
0069. “Mineral content” refers to the mass percentage
ofminerals contained in a sample.
0070 "Chitin content” refers to the mass percentage of
chitin contained in a sample.
0071 "Approximately,placedbeforea number, means
more or less 10% ofthe nominal value ofthe number.
0072 Unless specified otherwise, the percentages are
mass percentages.
DETAILED DESCRIPTION
0073. The present invention relates to a method for the
enzymatic extraction of chitin in a single step, from a raw
material obtained from animal biomass and comprising
chitin,preferably a raw material made ofmarine by-products
and/orentomologicalby-products, usingan activeenzyme in
an acid medium, preferably a protease, the acid used being
preferably a dietary acid, this method also being suitable for
extracting soluble compounds such as lipids, pigments, Sug
ars, mineral salts, amino acids orpeptides.
0074. In the present invention, the two key steps of the
conventional method forextracting chitin, i.e. demineraliza
tion in an acid medium and deproteinization in an alkaline
medium, are merged into a single step. This merging into a
single step is enabled through the use ofan enzyme wherein
the optimal activity pH is acidic: the enzyme performs the
deproteinization, while the acidic pH makes it possible to
carry outthe demineralization simultaneously.
0075. The method according to the invention only com
prising a single key step, it offers the advantage ofreducing
rinsing-related material losses between the two steps ofthe
conventional method. This method also makes it possible to
decrease the reagentand solvent consumption and limit pol
luted effluent production. This method is thus both inexpen
sive and environmentally friendly.
0076. Theconditions used in the method according to the
present invention are such that the biological activity of
chitins and the native structure thereofare preserved better
than in the extraction methods existing to date.
0077. The method according to the present invention
offers the advantage ofenabling destructuration ofthe crus
tacean and/or insect by-product matrix by separating chitin,
proteins and minerals, these three major constituents being
initially strongly linked.
0078. The method according the invention comprises an
enzymatic hydrolysis step in an acid medium performing
demineralization and deproteinization simultaneously. The
minerals and proteins are detached from the solid phase and
carried in the liquid phase.
0079 According to one embodiment, the method accord
ing to the present invention may comprise, in addition to the
enzymatichydrolysisstepinanacid medium,preparationand
processing operations:
0080 preparing the raw material,
0081 preparing a reaction medium according to the
optimal conditions for enzymatic activity and compris
ing at least one acid,
Apr. 10, 2014
0082 mixing the raw material prepared in the reaction
medium, homogenizing and adding enzyme,
0.083 enzymatic hydrolysis step with controlled tem
perature, pH and stirring:
I0084 simultaneous deproteinization and demineraliza
tion reactions foran optimized time,
0085 separating the soluble and insoluble portions of
the “reaction liquor.
I0086) washing the “insoluble' portion, drying and
packaging,
0.087 optionally, characterising the extracted products.
0088 Raw Material
I0089. The term "raw material', according to the present
invention,denotestheanimalbiomasscomprisingchitinused
forextracting chitin, preferably marineby-products used for
extracting chitin and/or entomological by-products used for
extracting chitin.
0090 According to one embodiment, the raw material
used in the method according to the present invention com
prises marine by-products, preferably crustaceans, prawns,
crabs, krill, more preferably crustacean shells and heads:
accordingto oneparticularembodimentofthe invention, the
raw material is obtained from cephalopods, preferably squid
orcuttlefish.
0091. According to one embodiment, the raw material
used in the method according to the present invention com
prises entomological by-products, preferably from beetles
such as the Tenebrio molitorbeetle, hymenoptera suchasthe
Hermetia illucens fly, more preferably insect shells and
heads.
0092. The raw material preparation operation should be
suitable for retaining the qualities thereofwhile meeting the
requirements ofthe method.
0093. According to oneembodimentofthe presentinven
tion,theraw materialpreparation comprises cleaning, drying
and/orgrinding operations.
0094. According to one embodiment, the raw material is
cleaned with water.
0.095 According to one embodiment, the raw material is
driedfrom 1 hourto36hourspreferably forapproximately 18
hours,preferablyinventilatedair,preferablyata temperature
of 5 to 35° C., more preferably of approximately 12° C.
According to one embodiment, the raw material is ground to
obtain fragments having a maximum diameter equal to
approximately 10mm,preferably havingadiameterlessthan
approximately 1 mm.
0096. According to one embodiment, the raw material,
preferably prepared by cleaning, drying and grinding, is
stored prior to extraction at a temperature between -30 and
-10°C., preferably at -20°C., preferably limiting the pres
ence ofoxygen.
0097. Reaction Medium
0098. The term “reaction medium', according to the
present invention, denotes the medium wherein the enzy
matic hydrolysis reaction in an acid medium takes place.
0099. The reaction medium preparation should account
forthe enzyme activity conditions used Such as thetempera
ture, solventandpH. Theselectionoftheseconditions makes
it possible to optimize the reaction time and yields.
0100. Accordingtooneembodiment,thereactionmedium
is maintained during the enzymatic hydrolysis at a tempera
turebetween2 and80°C., preferablybetween35 and45° C.,
morepreferably fromapproximately37 toapproximately 40°
C.

7. US 2014/0100361 A1
0101. According to one embodiment, the temperature of
the reaction medium is adapted to the enzyme used so that
saidenzymehasaquasi-optimalactivitythroughouttheenzy
matic hydrolysis.
0102) Accordingtooneembodiment,thereaction medium
is maintained during the enzymatic hydrolysis at a tempera
tureintherangeTit.0to20°C., preferablyTit.0to 15°C.,
preferably T0 to 10°C., where T is the temperature at
which the enzyme exhibits maximum activity. The tempera
tureselectedshould notinducethedegradation oftheenzyme
orinhibittheactionthereof.Advantageously,thetemperature
ofthe reaction medium is less than T. So as to limitenergy
consumption.
0103) According to one embodiment, the pH ofthe reac
tion medium is 0.5 to 6.5, preferably from 1.8 to 3.8, more
preferably from approximately 1.9 to approximately 2.1. If
the enzyme is pepsin, the pH of the reaction medium is
preferably from approximately 1.9 to approximately 2.1.
0104. According to one embodiment, the pH ofthe reac
tion medium is acidic and the value thereofis adapted to the
enzyme used so that said enzyme has an optimal activity.
01.05
tion medium is in the range pH-t2, preferably pH-t1.5.
preferably pH-1, where pH is the pH at which the
enzyme exhibits maximum activity. The pH selected should
beacidic toensurethatthechitin extractionyieldis sufficient.
0106. Accordingtooneembodiment,thereaction medium
is ready for use when the temperature and pH conditions
selected for the enzymatic hydrolysis reaction are stabilized.
0107 According to a first embodiment, the reaction
medium comprises at least one acid. According to a second
embodiment, the reaction medium further comprises a sol
vent such as water or an aqueous solution.
0108. According to oneembodimentofthepresentinven
tion, the acid used is preferably a dietary acid, preferably
phosphoric acid or formic acid.
0109 Whentheacidused intheenzymatichydrolysisstep
is a dietary acid, the products extracted by means of the
methodaccordingtothepresentinventionoffertheadvantage
of being suitable for easier use in the food and cosmetic
SectOrS.
0110. According to one embodiment, the acid concentra
tioninthereaction medium is from 0.1 to 6 mol-L', prefer
ably from 0.8 to 2.8 mol-L, more preferably from 0.9 to 1
mol-L".
0111. According to one embodiment, the acid concentra
tion in the reaction medium is adaptedto the mineral content
oftheraw material usedsothatthepHofthereaction medium
is acidic and remains constant throughout the enzymatic
hydrolysis.
0112
0113. According to one embodiment, the enzyme used in
the presentinvention is an activeenzyme in an acid medium,
preferablyaproteasehavingabroadspectrum ofactivityinan
acid medium, preferably pepsin ora stable acid protease.
0114. Accordingto oneembodiment,theenzymeconcen
tration in the reaction medium is adapted to the protein con
tentofthe raw material used. Accordingto one embodiment,
theenzymeconcentration is0.1 to 75%, preferably 5 to30%,
more preferably from approximately 23 to approximately
27% by mass in relation to the protein mass estimated in the
raw material.
According to one embodiment, the pH ofthe reac
Enzyme
Apr. 10, 2014
0115 Reaction Conditions
0116. According to one embodiment, the raw material is
mixed with the reaction medium andthe resulting mixture is
optionallyhomogenized by stirring for 0to 30 minutes, pref
erably for3to 10 minutes, morepreferablyforapproximately
5 minutes.
0117. According to one embodiment, the ratio between
the weight ofraw material prepared and the volume ofreac
tion medium is 1:60 to 2:1, preferably 1:7 to 1:3, more pref
erably equal to 1:5.
0118. According to one embodiment, the ratio between
the weight ofraw material prepared and the volume ofreac
tion medium is adapted to the size ofthe fragments of raw
material prepared. In particular, account is taken ofthe fact
that,whenthesizeofthefragmentsofraw material decreases,
Solventabsorption increases andthat, consequently, it is nec
essary to increase the Volume ofreaction medium.
0119) Adding raw material into the acid reaction medium
may give rise to the formation offoam dueto theproduction
ofcarbon dioxideduetothepresenceofcalcium carbonatein
the exoskeleton ofcrustaceans and insects.Accordingto one
embodiment, the vessel used for performing enzymatic
hydrolysis has a suitable Volume for preventing the foam
liable to form from overflowing. The riskoffoam production
increases when the temperature of the acid before mixing
increases.
I0120 According to one embodiment, the temperature of
thereaction mediumbeforeaddingtheraw materialis5to 65°
C., preferably 20 to 30°C., more preferably approximately
25°C. In this embodiment, the temperature of the reaction
medium is selectedin orderto beless than the temperatureat
which the enzymatic hydrolysis is to be conducted, so as to
limit foam formation when adding raw material.
I0121 According to a first embodiment, the enzyme is
added directly into the homogenized reaction medium
optionally containing the raw material.
0122. According to a second embodiment, the enzyme is
solubilized in water, or in a solution, preferably an aqueous
Solution,andisaddedintothehomogenized reaction medium
containing the raw material.
I0123. According to one embodiment, the enzymatic
hydrolysis reaction is performed under stirring so as to opti
mize the contact between the raw material and the enzyme.
0.124. According to one embodiment, the initial pH and
temperature conditions ofthe reaction medium are kept con
stant throughout the enzymatic hydrolysis reaction. Accord
ingtoafurtherembodiment,theinitialpHand/ortemperature
conditions of the reaction medium are not kept constant
throughout the enzymatic hydrolysis reaction.
0.125. According to one embodiment, the enzymatic
hydrolysis reaction is performed in areactorequipped with a
device for regulating the temperature. According to a first
embodiment, said reactorisadouble-jacketreactorwherein a
heat transfer fluid circulates, the temperature of said fluid
beingpossibletocontrol.Accordingtoasecondembodiment,
said reactor is equipped with a heating resistor, thetempera
ture ofsaid resistor being suitable forbeing controlled.
I0126. According to a first embodiment, the pH is stable
throughoutthe enzymatic hydrolysis. According to a second
embodiment, the pH is adjusted, during the enzymatic
hydrolysis reaction, to the pKa value between the acid used
and calcium carbonate by adding a concentrated acid solu
tion, the acid being the same as that used in the reaction
medium.

8. US 2014/0100361 A1
0127. According to one embodiment, the duration ofthe
enzymatic hydrolysis is from 30 minutes to 24 hours,prefer
ably from 1 hour to 12 hours, preferably from 3 hours to 8
hours, more preferably approximately 6 hours.
0128. According to one embodiment, the duration ofthe
enzymatichydrolysis is adaptedto theactivity oftheenzyme
usedforconductingtheenzymatic hydrolysis reaction, to the
acid used and to the raw material.
0129. According to one embodiment, the duration ofthe
enzymatic hydrolysis is adapted according to the features
sought fortheendproducts, such as the degree ofpurity, the
degree ofpolymerization and the degree ofacetylation.
0130. According to one embodiment, theenzymatic reac
tion produces a reaction liquor comprising soluble and
insoluble portions.
0131 Products Separation
0.132. According to one embodiment, the soluble and
insolubleportions ofthe reaction liquorare separated by any
suitable means known to those skilled in the art.
0.133 According to a first embodiment, the soluble and
insoluble portions are separated by filtration. According to
one embodiment, the filtration is performed by a filtration
system preserving the integrity of the structures of the
extracted compounds. According to a further embodiment,
the filtration is performed by a membrane press filtration
system. According to a further embodiment, the filtration is
performed on a filter cloth, preferably on bolting cloth.
0134. Accordingtoa secondembodiment,the solubleand
insoluble portions are separated by centrifugation.
0135 According to one embodiment, the insoluble por
tion of the reaction liquor very predominantly contains
chitins and the soluble portion contains various compounds
Suchas lipids,pigments, Sugars, mineral salts, aminoacids or
peptides.
0136. According to one embodiment, the insoluble por
tion is rinsed using a solvent. According to a first embodi
ment, the solvent is water or an aqueous Solution. This
embodiment is preferred ifthechitins are subsequently used
fordietary applications. Accordingto a secondembodiment,
the insoluble portion is first rinsed with water or an aqueous
Solution and then with a bleaching agent such as hydrogen
peroxide, Sodium hypochlorite orpotassium perSulphateand
is rinsedagain with wateroranaqueous solution.Thissecond
embodiment is preferred ifbleachingofthechitins is sought.
In this embodiment, the bleaching agents used are in accor
dance with legislation.
0.137 Chitins are very hygroscopic substances, wherein
the biological activity may be degraded by an increase in
temperature.
0138 According to one embodiment, the filtered and
rinsed insoluble portion is then dried for 8 to 16 hours, pref
erably for approximately 12 hours, in an oven wherein the
temperature is preferably less than 100° C., preferably
between50and95°C.,morepreferablyapproximately90°C.
0.139. Accordingtooneembodiment,thefilteredinsoluble
portion is neutralized with sodium hydroxide. According to
oneembodiment, the insoluble portion is freeze-dried.
0140. According to one embodiment, the dried and/or
freeze-dried insoluble portion is packaged in vessels such as
glass orplastic bottles or vacuum pouches and storedprefer
ably atambienttemperature in a dry place. Accordingto one
particularembodiment, theinsolubleportion(chitin)isstored
ata temperature less than ambienttemperature, preferably at
Apr. 10, 2014
atemperaturefrom -30 to0°C., morepreferably from -20to
-10°C., morepreferably at approximately -20°C.
0.141. Accordingtoafirstembodiment, thesolubleportion
is centrifuged. According to a second embodiment, the
soluble portion is dialysed and ultrafiltered. According to a
third embodiment, the compounds from the neutralized
soluble portion are extracted using organic solvents. The
organic oraqueous solvents arethen evaporatedto beable to
obtain the compounds ofinterest.
0142. The technique for processing the soluble phase is
dependent on the nature ofthe compounds to be recovered.
0143. ExtractionYields
0144 Controlling the reaction medium (enzyme concen
tration, pH and temperature) according to the raw material
used makes itpossible to control the yieldand thebiochemi
cal and physicochemical characteristics of the chitins
obtained.Theoretically,byextendingthehydrolysistime,the
degree ofpolymerization tends to be reduced.
0145 The mass extraction yield ofthe insoluble portions
(Yd) is dependenton the nature ofthe raw material, acidand
enzyme used and is calculated using the following formula:
Yd%=100*(dried insoluble weight)/(dry raw material
weight)
0146 According to one embodiment, the insoluble por
tionpredominantly contains chitins andresidualproteinsand
minerals not removed during the enzymatic hydrolysis reac
tion.
0147 Applying conventional chemical extraction treat
ment to the insoluble portions obtained using the method
according to the present invention makes it possible to esti
mate the residual impurity content in the insoluble portion.
Indeed,this treatment is suitablefor removingthe majority of
residual proteins and minerals.
0.148. The degree ofchitin purity (D purity) is estimated
by means of gravimetry, by measuring the mass of the
insoluble sample before and after treating the insoluble por
tions with 1.25 mol-L' sodium hydroxide at 90° C. for 1
hour. As mentioned above, this treatment is suitable for
removing residual proteins and minerals. The estimated
degree ofpurity is calculated using the following formula:
D-purity=100*(mass ofinsoluble portion aftertreat
ment)/(mass ofinsoluble portion beforetreat
ment)
0149 Accordingtooneembodiment,theestimateddegree
of chitin purity (D'purity) is greater than 75%, preferably
greater than 80%, more preferably greater than 85%, more
preferably greater than 90%.
0150. According to one embodiment, the mass residual
proteincontentinthedriedinsolubleportionisless than20%,
preferably less than 15%, preferably less than 10%, more
preferably less than 5%.
0151. According to oneembodiment, the massproportion
ofproteins removed by the method according to the present
invention is greater than 80%, preferably greater than 85%,
more preferably greater than 90%, more preferably greater
than 95%.
0152. According to one embodiment, the weight amount
ofresidual mineral in the dried insoluble portion is less than
5%, preferably less than 3%, more preferably less than 1%.
0153. According to one embodiment, the weight amount
ofminerals removed by the method according to the present
invention is greater than 95%, preferably greater than 97%,
more preferably greater than 99%.

9. US 2014/0100361 A1
0154 According to one embodiment, an additional
bleachingoperation isperformedontheinsolubleportion, for
removing pigments along with a portion ofthe residual pro
teins and minerals.
0155 According to one embodiment, an additional
deacetylation operation is performed on the insoluble por
tion, for producing chitosan and removing a portion of the
residual proteins.
0156 Features ofthe Chitins Extracted
0157 Accordingtooneembodiment,thechitins extracted
using the method according to the present invention may be
usedas is or converted into chitosan, chitin oligomers, chito
san oligomers or optionally N-acetylated glucosamines.
0158. The method according to the present invention is
Suitableforobtainingawiderangeofchitin qualityinrespect
ofthedegreeofpurityandpolymerization.Theotherfeatures
(pattern distribution, C, B and Y form) are dependent on the
nature of the raw material and not on the features of the
method according to the invention.
0159. Accordingtooneembodiment,thechitins extracted
usingthe methodaccordingtothepresent inventionaresimi
lar in form to the natural form ofchitin. In other words, the
chitins extracted using the method according to the present
invention are not denatured or only slightly denatured in
relation to natural chitin.
0160 Accordingtooneembodiment,theestimateddegree
ofpurity ofthe chitins extracted using the method according
to the present invention is greater than 85%, preferably
greater than 90%, more preferably greater than 95%.
0161 Thedegreeofpurity ofthechitinsobtainedusingthe
method according to the present invention is sufficient to be
able to convert said chitin in the form of chitosan, chitin
oligomers, chitosan oligomers and glucosamines.
0162 According to one embodiment, the degree ofpoly
merization ofthe chitins isestimatedby calculatingbasedon
the average molecular mass ofsaid chitins. Accordingto one
embodiment, the average molecular mass of the chitins is
estimated by calculatingbased on the intrinsic viscosity. The
intrinsic viscosity may be determined using the method
described by Poirier et al. (Poirier, M. and Charlet, G., Car
bohydrate Polymers, 2002, 50, 363-370).
0163 According to one embodiment, the degree ofpoly
merization ofthe chitins extracted using the method accord
ing to thepresentinventionis from 1.10 to 1.10, preferably
from 1.10 to 1.107, more preferablyfrom 1.10 to 1.10.
0164. According to oneembodiment, the degreeofacety
lation ofthe chitins extracted using the method according to
the present invention is from 80% to 100%, preferably from
90% to 98%, more preferably from 95% to 97%.
0.165 According to one embodiment, the crystallinity
index ofthe chitins extracted using the method according to
the present invention is from 10% to 70%, preferably from
20% to 50%, more preferably from 30% to 40%.
0166 Soluble Compounds
0167 According to one embodiment, the soluble sub
stances extracted using the method according to the present
invention may be peptides, pigments, Sugars and mineral
salts.Theuseofdietary acidinthis methodenables the useof
these compounds in the food, dietetic and nutraceutical sec
tOrS.
0168 The present invention thus offers the advantage of
limiting the quantity ofwaste since all Substances other that
chitin extracted using the method according to the invention
can also be used or recovered.
Apr. 10, 2014
0169. According to one embodiment, the pigments
extractedusingthemethodaccordingtothepresentinvention
are astaxanthin.
BRIEF DESCRIPTION OF THE DRAWINGS
(0170 FIG. 1 represents a scheme of the method for
extracting chitin according to the invention.
EXAMPLES
0171 The invention will be understood more clearly on
readingthefollowing example, illustratingthepresentinven
tion in a non-limiting fashion
Example 1
Enzymatic Hydrolysis Using Pepsin in the Presence
ofPhosphoricAcid
0172 Materials
(0173 The raw material usedis the raw Panaeus vannamei
prawn exoskeleton. The raw material is dried at 12° C. in
ventilated air and ground to produce fragments less than 1
mm in size.The raw materialprepared is stored at -20°C. in
a WaCl.
0.174. The reagent used to maintain theacidic pH is phos
phoricacid.Theacidconcentration is calculatedaccordingto
theinitial mineralcontentintheraw materialprepared. Foran
initial mineral contentof25% in weightrelative to theweight
ofdryraw material, a 0.94 mol-L' phosphoric acid solution
is used to keep the pH ofthe reaction medium around 2.
(0175. The acid protease used is pepsin (CAS 9001-75-6,
supplier: Sigma, activity: 8112 U/mg). It is stored in powder
form at +4°C. It is solubilised in distilled water for 15 min
beforebeing introducedintothereaction medium. The quan
tity ofenzymeadded in this example is equivalent to 25% of
the estimated protein mass in the initial raw material. In this
way, for a sample of 5 g ofraw material having a moisture
content ofapproximately 15% andaprotein content ofclose
to 40%, this isequivalent to8.5% ofenzyme in relation tothe
raw material, i.e. a quantity ofpepsin of0.43g.
(0176)
0177 5g ofraw material, prepared as describedabove, is
weighed. The composition ofthe dry extract is determined
according to the analytical methods described below.
(0178 A 0.94mol-L' phosphoricacidsolution(25mL)is
preheated to 30° C. and added to the raw material. The mix
ture is stirred for 5 min so that it homogenises. The pH
measured with a pH-meter should be stable and be between
1.9 and 2.1.
0179 Pepsin (0.43 g), previously solubilised in 1 mL of
water,isadded to thereaction medium.Themixture isheated
to 40°C. on a hotplate and incubated in an oven kept at 40°
C.1° C.
0180. After6hoursofincubation,themixtureisfilteredon
bolting cloth and rinsed with plenty of distilled water. The
retentate is resuspended in distilled water, the mixture is
stirred for 10 min beforebeing filtered and rinsedagain with
water.Thesolidfractionobtainedis transferredintoacupand
dried overnight at 90° C. in an oven. The mass ofdry extract
obtained (m=1.29g) is suitable forcalculating theextraction
yield, i.e. 30.26% w/w.
Protocol

10. US 2014/0100361 A1
0181 Analyses
0182. The moisture content ofthe sample is measured by
means ofgravimetry, by measuring the mass ofthe sample
before and after being placed overnight at 105°C.
0183 The mineral content is determined by means of
gravimetry, by measuring the mass ofthe sample before and
after incineration at 600° C. for 6 hours.
0184 The protein content is estimated by means ofgas
chromatographybyassayingthetotalaminoacids. Itcan also
be measured by means ofa colorimetric assay (Lowry, BSA,
Bradford orCoomassieblue)orby meansofa Kjeldahlassay.
0185. The chitin content can be measured by means of
gravimetry, by measuring the mass ofthe sample before and
after the following treatments:
0186 for the raw material: treatment for 60 minutes
with 1NHClatambienttemperature, followedby 1.25N
NaOH at 90° C. for 120 minutes and finally bleaching
with 33% hydrogen peroxide and acetone;
0187 for the hydrolysis products, the treatment is lim
ited to a treatment with 1.25 N NaOH at 90° C. for one
hour.
0188 The molecular mass ofthe chitins is estimated by
calculatingbased on the intrinsic viscosity. The intrinsic vis
cosity can be determined using the method described by
Poirier etal., which is based on the Mark-Houwink equation
(Poirier, M. and Charlet, G., Carbohydrate Polymers, 2002,
50, 363-370). In this way, the intrinsic viscosity was deter
minedby measuring the reduced viscosity using Solutions of
variouschitinconcentrations inN,N-dimethylacetamidecon
taining 5% LiCl. The apparatus used is an Ubbelohde capil
lary viscometer. The viscometerconstant K is 0.3 cSt/s. The
measurement Volume is 15 mL.
0189 Thedegreeofpolymerizationiscalculatedusingthe
molecular mass ofthe chitins.
0190. The degree ofacetylation is estimated by means of
protein liquid NMR, according to the method described by
Einbu A. Varum K., 2008. Chitin (20 mg) is solubilized in 1
mLofDC1 (7.6N in DO, Euriso-top) with magnetic stirring
atambient temperature for5hours.The "H NMR analysis is
performed at 300 K using a Bruker ALS300 spectrometer
(300 MHz, referenceTMSP 0.00ppm). The degreeofacety
lation is then calculated basedon the intensity ofthecharac
teristic proton NMR signals, according to the formula given
by Einbu et al.
0191 The crystallinity index is determined by means of
X-raydiffraction.Thediffractometerusedisa Bruker-axS D8
Discover (Karlsruhe, Germany). Radiation is produced in a
coppertube(CuKC-1.5405A)and thebeamsproducedare
recorded every 10 min. Using the spectra obtained, the
method forcalculating the crystallinity index is based on the
ratio between theareas ofthecrystalline Zones overthe total
area (Osario-Madrazo A. David L., Trombotto S. Lucas J.
M., Peniche-Covas C. and Domard A. Carbohydrate Poly
mers, 2011, 83, 1730-1739).
(0192
0193 After6hoursofenzymatichydrolysiswithpepsinin
the presence of phosphoric acid, the extraction yield is
30.26+0.32% w/w. The composition of the dry extract
obtainedcan becomparedto thatofthe fully dried raw mate
rial orthe prepared raw material used in this example (table
1):
Results and Discussion
Apr. 10, 2014
TABLE 1
mois
ture minerals proteins chitin lipids Sugars
fully dry
8W
material:
% by mass O% 25% 40% 30% ND ND
prepared
8W
material:
% by mass 14.55% 21.25% 34% 25.5% 3.5%. 1.2%
dry
extract:
% by mass O% O.99 10.98 88.42 ND ND
(+0.03%) (+1.01%) (+1.22%)
per 100 g Og 0.30 g 3.32 g 26.76 g ND ND
ofdry raw
material
ND: not determined
0194 In view ofthecomposition ofthe dry raw material,
the quantities of minerals and proteins removed using the
method are 98.5% and91.7%, respectively.
0.195 The residual mineral and protein contents (table 1)
are those found in the unprocessed end product, without a
bleaching step. Applying a bleaching agent or washing with
Sodium hydroxide enhances the degree ofpurity.
(0196. The degree ofacetylation measured by NMR is, in
this example, in the region of95%. The molecular weight of
this sample is in the region of 10 to 10 g/mol and the
crystallinity index35%. These featuresaresimilarto those of
native chitin.
0197) Theperformances ofthis examplecan be enhanced
by increasing the quantity of pepsin used. In this way, the
experiment was conducted with a pepsin concentration of
41% with respectto thequantity ofproteinspresentintheraw
material, instead of 25% previously. The degree of chitin
purityincreases (96.78%insteadof88.42%)as thedeprotein
ization isenhanced(92.00% ofproteins removed)along with
the demineralization (99.23% ofminerals removed).
1.A methodfortheenzymaticextractionofchitin,wherein
said method is carried out in a single step, wherein chitin is
obtainedby theenzymatichydrolyzis ofraw materialconsti
tuted by animal biomass comprising chitin, said enzymatic
hydrolyzis using an active enzyme in an acid medium.
2.A methodaccordingto claim 1, wherein said single step
is an enzymatic hydrolysis fordeproteinizinganddemineral
izing said raw material simultaneously.
3. A method according to claim 1, wherein said enzyme
active in an acid medium is a protease having a broad spec
trum of activity in an acid medium, preferably pepsin or a
stable acid protease.
4. A method according to claim 1, wherein the enzyme
concentration usedforhydrolysis is 0.1 to 75%,preferably 5
to 30%, more preferably from approximately 23 to approxi
mately 27% in weight relative to the estimated weightofthe
protein in the raw material.
5.A methodaccordingtoclaim 1,whereintheacidmedium
is obtainedby means ofthe presence ofan acid, preferably a
dietary acid, morepreferably phosphoric acid orformic acid.
6.Amethodaccordingto claim 1, whereinanimalbiomass
comprising chitin comprises marine by-products, preferably

11. US 2014/0100361 A1
marine by-products obtained from crustaceans, preferably
prawns, crabs orkrill, orfrom cephalopods,preferably squid
or cuttlefish.
7. A method according to claim 1, wherein said animal
biomass comprising chitin comprises insect by-products,
preferably insect by-products obtained from beetles or
hymenoptera.
8. A method according to claim 1, further comprising
operations forwashing, drying and/orgrindingtheraw mate
rial, preferably water washing, cold drying and/or grinding
operations.
9.Amethodaccordingtoclaim 1, furthercomprising reac
tion medium treatmentoperationsattheendoftheenzymatic
hydrolyzis, said operations comprising operations for sepa
rating the Solid and liquid phases, rinsing and/or drying the
insoluble portion.
10. A method foroptimizingthe method fortheenzymatic
extraction ofchitindescribedin claim 1, wherein said method
comprises at least one ofthe following steps:
a) selecting the pH of the acid medium in the range
pHenzi-2, preferably pHenzit1.5, preferably pHenz+1,
where pHenz is the pH at which the enzyme exhibits
maximum activity,
b)electingthetemperatureoftheacid medium intherange
Tenzi-20° C., preferably Tenzi.15° C., preferably
Tenzi-10°C.,whereTenzis thetemperatureatwhich the
enzyme exhibits maximum activity,
c) determining the mineral and protein content ofthe raw
material,
d) calculating the acid concentration to be used in the
reactionmedium,accordingtothemineral contentofthe
Apr. 10, 2014
raw material. Such thatthepH is maintainedthroughout
the enzymatic hydrolysis atthe pH selected in step a),
e) calculating the proportion of enzyme to be used with
respect to the protein content ofthe raw material,
f) determining the reaction time forobtaining chitin orthe
chitin derivatives sought.
11. Chitin that can be obtained by means ofthe method
according to claim 1.
12. Chitosan that can be obtained by deacetylating chitin
according to claim 11.
13. Composition comprising chitin according to claim 11.
14. A pharmaceutical composition comprising chitin
according to claim 11.
15.Acosmetic compositioncomprisingchitinaccordingto
claim 11.
16.A medical device comprising chitin accordingto claim
11.
17. A food product, nutraceutical composition, dietetic
composition,foodSupplementorfunctional food comprising
chitin according to claim 11.
18.Acomposition comprisingchitinaccordingto claim 11
fortheuse thereofin watertreatment, filtrationand/ordepol
lution.
19.Atexturingagentcomprising chitin accordingto claim
11.
20. A method according to claim 2, wherein said enzyme
active in an acid medium is a protease having a broad spec
trum of activity in an acid medium, preferably pepsin or a
stable acid protease.