U.S. patent application number 15/762659 was filed with the patent office on 2018-09-27 for acid composition comprising a phycocyanin.
This patent application is currently assigned to FERMENTALG. The applicant listed for this patent is FERMENTALG. Invention is credited to Olivier CAGNAC.
Application Number | 20180271119 15/762659 |
Document ID | / |
Family ID | 55178111 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180271119 |
Kind Code |
A1 |
CAGNAC; Olivier |
September 27, 2018 |
ACID COMPOSITION COMPRISING A PHYCOCYANIN
Abstract
The invention relates to an acid composition, particularly an
acid food composition, comprising at least one phycocyanin
resistant to pH acid.
Inventors: |
CAGNAC; Olivier; (Libourne,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FERMENTALG |
Libourne |
|
FR |
|
|
Assignee: |
FERMENTALG
Libourne
FR
|
Family ID: |
55178111 |
Appl. No.: |
15/762659 |
Filed: |
September 22, 2016 |
PCT Filed: |
September 22, 2016 |
PCT NO: |
PCT/EP2016/072583 |
371 Date: |
March 23, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 1/12 20130101; A61P
19/00 20180101; A23L 2/58 20130101; C07K 1/145 20130101; A23L 5/46
20160801; A23L 5/43 20160801; C12P 23/00 20130101; C07K 14/405
20130101; C12P 17/165 20130101; C12P 21/00 20130101 |
International
Class: |
A23L 2/58 20060101
A23L002/58; C07K 14/405 20060101 C07K014/405; C07K 1/14 20060101
C07K001/14; A23L 5/46 20060101 A23L005/46 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 25, 2015 |
FR |
1559072 |
Apr 21, 2016 |
FR |
1653525 |
Claims
1. An acidic composition, comprising at least one mineral or
organic acid and at least one acidic-pH-resistant phycocyanin,
wherein.
2. The acidic composition according to claim 1, wherein said
acidic-pH-resistant phycocyanin is a phycobiliprotein the
apoprotein of which comprises the protein of SEQ ID NO 1 or SEQ ID
2 or a variant thereof.
3. The acidic composition according to claim 2, wherein the
.alpha.-subunit apoprotein of said phycocyanin comprises SEQ ID NO
1 and the .beta.-subunit apoprotein of said phycocyanin comprises
SEQ ID 2 or variants thereof.
4. The acidic composition according to claim 3, wherein said
.alpha.-subunit apoprotein consists of SEQ ID NO 1 and the
.beta.-subunit apoprotein consists of SEQ ID or variants
thereof.
5. The acidic composition according to claim 1, wherein it further
comprises an allophycocyanin combined with the phycocyanin.
6. The acidic composition according to claim 5, wherein the
.alpha.-subunit apoprotein of said allophycocyanin comprises SEQ ID
NO 3 or variants thereof and the .beta.-subunit apoprotein of said
allophycocyanin comprises SEQ ID NO 4 or variants thereof.
7. The acidic composition according to claim 6, wherein the
allophycocyanin .alpha.-subunit apoprotein consists of SEQ ID NO 3
or variants thereof and the .beta.-subunit apoprotein consists of
SEQ ID NO 4 or variants thereof.
8. The acidic composition according to claim 1, wherein the
acidic-pH-resistant phycocyanin is a phycocyanin extracted from an
alga (or microalga) of the order Cyanidiales.
9. The acidic composition according to claim 8, wherein the
phycocyanin is a phycocyanin extracted from an alga (or microalga)
of the family Cyanidiaceae or the family Galdieriaceae.
10. The acidic composition according to claim 9, wherein the
phycocyanin is a phycocyanin extracted from an alga (or microalga)
of the genera Cyanidioschyzon, Cyanidium or Galdieria.
11. The acidic composition according to claim 10, wherein the
phycocyanin is a phycocyanin extracted from an alga (or microalga)
selected among the group consisting in the species Cyanidioschyzon
merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium,
Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum,
Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria
partita, Galdieria sulphuraria, preferentially the species
Galdieria sulphuraria, Cyanidium caldarium, and Cyanidioschyzon
merolae.
12. The acidic composition according to claim 1, wherein it has a
pH of 4 or less.
13. The acidic composition according to claim 1, wherein it has a
pH of 2 to 4.
14. The acidic composition according to claim 1, wherein it has a
pH of about 2.5 to 3.5.
15. The acidic composition according to claim 1, wherein the
.alpha.-subunit apoprotein of said phycocyanin has an isoelectric
point below 3.
16. The acidic composition according to claim 1, wherein the
.alpha.-subunit apoprotein of said phycocyanin has an isoelectric
point below 2.5.
17. The acidic composition according to claim 1, wherein the
.alpha.-subunit apoprotein of said phycocyanin has an isoelectric
point below 2.2.
18. The acidic composition according to claim 1, wherein it is
solid.
19. The acidic composition according to claim 18, wherein the
phycocyanin content is from 0.25 mg/g to 2.5 mg/g.
20. The acidic composition according to claim 1, wherein it is
liquid.
21. The acidic composition according to claim 20, wherein the
phycocyanin content is from 2.5 mg/L to 2,500 mg/L.
22. (canceled)
23. The acidic composition according to claim 1, wherein it is an
acidic food composition.
24. The acidic composition according to claim 20, wherein it is a
carbonated or non-carbonated beverage.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns an acidic composition,
particularly an acidic food composition, comprising at least one
acidic-pH-stable phycocyanin.
STATE OF THE ART
[0002] Phycocyanins are food colourings which give a blue colour to
the products to which they are added. Phycocyanin extracted from
spirulina is today the only natural blue pigment approved by the
US-FDA (FR Doc No: 2013-19550). It is sold in liquid form, or in
powder form for use as blue pigment in foods.
[0003] That spirulina-derived phycocyanin, however, has the
disadvantage of being unstable at acidic pH, below 5, leading to
loss of colouring and to precipitation, which limit its use. In the
best case, the loss of stability occurs around pH 4 (cf. technical
specifications of the spirulina-derived phycocyanin Linablue.RTM.;
http://www.dlt-spl.co.jp/business/en/spirulina/linablue.html).
[0004] Consequently, there are many acidic food compositions,
notably carbonated or noncarbonated beverages, for which
spirulina-derived phycocyanin cannot be used as food colouring or
for its antioxidant properties.
[0005] A need exists to identify new phycocyanins stable at acidic
pH, particularly stable at acidic pH below 4. pH resistance or
stability is measured herein as a less than 10% loss of colouring,
after a minimum exposure of 10 minutes at acidic pH. pH stability
can be measured by other methods, such as physical characterization
of the phycocyanins within the acidic compositions as a function of
time.
DISCLOSURE OF THE INVENTION
[0006] Thus, the present invention concerns an acidic composition,
particularly an acidic food composition, which can comprise at
least one acidic-pH-resistant phycocyanin. Advantageously, said
phycocyanin can be a phycobiliprotein whose apoprotein can comprise
at least the protein of SEQ ID NO 1 or SEQ ID 2 or a variant
thereof.
[0007] According to the invention, the phycocyanin can in
particular be a phycocyanin extracted from a Galdieriaceae, more
particularly extracted from Galdieria.
[0008] The acidic composition according to the invention,
particularly the acidic food composition, can be either solid or
pasty, or liquid, in particular an optionally carbonated
beverage.
[0009] By acidic composition, particularly by acidic food
composition, is meant, according to the invention, a composition
having a pH of 4 or less, advantageously having a pH of 2 to 4,
more advantageously of 2.5 to 3.5.
DETAILED DESCRIPTION OF THE INVENTION
[0010] Phycocyanins and allophycocyanins are phycobiliproteins
comprising alpha and beta subunits composed of an apoprotein
covalently bound to a chromophore. The different phycocyanins are
distinguished essentially by the sequence of their alpha- and
beta-subunit apoproteins.
[0011] According to a particular embodiment of the invention, the
acidic composition, particularly the acidic food composition,
comprises an acidic-pH-resistant phycocyanin whose .alpha.-subunit
apoprotein comprises SEQ ID NO 1 (accession number YP_009051179.1)
and whose .beta.-subunit apoprotein comprises SEQ ID 2 (accession
number YP_009051180.1) or variants thereof.
[0012] According to a preferred embodiment of the invention, the
acidic composition, particularly the acidic food composition,
comprises an acidic-pH-resistant phycocyanin whose .alpha.-subunit
apoprotein consists of SEQ ID NO 1 (accession number
YP_009051179.1) and whose .beta.-subunit apoprotein consists of SEQ
ID 2 (accession number YP_009051180.1) or variants thereof.
[0013] According to another embodiment of the invention, the acidic
composition, particularly the acidic food composition, can further
comprise an allophycocyanin combined with the phycocyanin.
[0014] Advantageously, the alpha-subunit apoprotein of said
allophycocyanin comprises SEQ ID NO 3 (accession number
YP_009051103.1) and the .beta.-subunit apoprotein comprises SEQ ID
NO 4 (YP_009051104.1) or variants thereof.
[0015] According to another preferred embodiment of the invention,
the alpha-subunit apoprotein of said allophycocyanin consists of
SEQ ID NO 3 (accession number YP_009051103.1) and the
.beta.-subunit apoprotein consists of SEQ ID NO 4 (YP_009051104.1)
or variants thereof.
[0016] The amino acid composition of a protein can give said
protein different properties according to said amino acid
composition. The characteristics of a protein depend, among other
things, on its amino acid composition and its isoelectric point
(pI). The isoelectric point is the pH of the solution at which the
protein carries no net charge or, in other words, the pH at which
the molecule is electrically neutral and the proteins tend to
attract one another, aggregate and precipitate. At a pH above their
isoelectric point, the proteins tend to be negatively charged and
to repel one another.
[0017] Comparative analysis of the isoelectric points of various
proteins using the computational procedure described by Patrickios
and Yamasaki (Polypeptide Amino Acid Composition and Isoelectric
Point. II. Comparison between Experiment and Theory. Analytical
Biochemistry. 231, 1, 1995: 82-91.1995) shows a certain correlation
between the theoretical calculations and the acidic pH resistance
observed experimentally.
[0018] Studies carried out by the Applicant indicate that the
acidic pH resistance of a phycocyanin may be linked to the amino
acid sequence of the a subunit of said phycocyanin. Furthermore, it
is noted that within the amino acid sequence of the a subunit of
the phycocyanin, the identity of the first 26 amino acids seem
particularly important. It is particularly the case of phycocyanin
obtained by culture of microalgae strains of the genera
Cyanidioschyzon, Cyanidium or Galdieria, more particularly of
strains Galdieria sulphuraria, Cyanidium caldarium and
Cyanidioschyzon merolae.
[0019] Thus, advantageously, the composition according to the
invention can comprise at least one phycocyanin of which at least
one apoprotein, particularly that of the a subunit, can have a low
isoelectric point allowing better stability at acidic pH.
[0020] By low isoelectric point is meant an isoelectric point of 3
or less, preferentially of 2.5 or less, more preferentially of 2.2
or less.
[0021] Thus, more advantageously, the composition according to the
invention can comprise at least one phycocyanin of which at least
one apoprotein, particularly that of the .alpha. subunit, can have
an isoelectric point of 3 or less, preferentially of 2.5 or less,
more preferentially of 2.2 or less.
[0022] Thus, according to a particular embodiment of the invention,
the acidic composition, particularly the acidic food composition,
can comprise at least one phycocyanin whose .alpha.-subunit
apoprotein can have a low isoelectric point, more particularly at
least one phycocyanin whose .alpha.-subunit apoprotein can comprise
SEQ ID NO 1, or a variant.
[0023] Thus, according to another particular embodiment of the
invention, the acidic composition, particularly the acidic food
composition, comprises at least one phycocyanin whose
.alpha.-subunit apoprotein has a low isoelectric point, more
particularly at least one phycocyanin whose .alpha.-subunit
apoprotein consists of SEQ ID NO 1, or a variant.
[0024] By variant is meant, according to the invention, a protein
sequence corresponding to a reference sequence, in this case the
protein represented by SEQ ID NO 1 or SEQ ID NO 2 or SEQ NO 3 or
SEQ NO 4, modified by one or more substitutions, insertions or
deletions of one or more amino acids of the reference sequence and
which has the same functional properties as said reference
sequence.
[0025] Advantageously, the variants according to the invention have
at least 83% sequence identity with the .alpha. subunits of the
phycocyanin, and at least 82% with the beta subunits of the
phycocyanin.
[0026] Preferentially, the variants according to the invention have
at least 90% identity with the .alpha. (SEQ ID NO 1) and .beta.
(SEQ ID NO 2) subunits.
[0027] Similarly, for the allophycocyanins, the variants
advantageously have at least 89% sequence identity with the .alpha.
subunits of the allophycocyanin, and at least 90% with the .beta.
subunits of the allophycocyanin.
[0028] Those skilled in the art know how to measure protein
sequence identity using the common methods at their disposal,
notably the BLASTP program
(http://blast.ncbi.nlm.nih.gov/Blast.cgi).
[0029] Similarly, those skilled in the art know how to identify
variants of said sequences and to verify that they retain the same
structural properties by a simple stability test in acidic pH, for
example by performing a test such as the test presented in example
3.
[0030] Those skilled in the art know that a polypeptide can be
modified by substitution, insertion and/or deletion of at least one
amino acid without substantially modifying the function
thereof.
[0031] For example, the substitution of an amino acid at a given
position by another chemically equivalent amino acid is a known
example of sequence variation which does not substantially affect
the properties of the protein.
[0032] These "conservative" substitutions can be defined as
exchanges within the following groups of amino acids [0033] Ala,
Ser, Thr, Pro, Gly [0034] Asp, Asn, Glu, Gln [0035] His, Arg, Lys
[0036] Met, Leu, Ile, Val, Cys and [0037] Phe, Tyr, Trp
[0038] Thus, the variants of the apoproteins of the phycocyanins
and/or allophycocyanins according to the invention can comprise a
difference of 1 to 30 amino acids in relation to the corresponding
reference sequence, particularly concerning the .alpha. and/or
.beta. subunits of the phycocyanin, insofar as the variant obtained
retains the properties of the reference protein and the percent
homologies/identities stated above.
[0039] More precisely according to the invention, [0040] the
.alpha.-subunit apoprotein variants of the phycocyanins useful in
the acidic compositions according to the invention, deriving from
substitutions, insertions and/or deletions, can comprise a
difference of 1 to 27 amino acids in relation to the corresponding
reference sequence, insofar as the variant obtained retains the
properties of the reference protein and the percent identities
stated above; [0041] the .beta.-subunit apoprotein variants of the
phycocyanins useful in the acidic compositions according to the
invention, deriving from substitutions, insertions and/or
deletions, can comprise a difference of 1 to 30 amino acids in
relation to the corresponding reference sequence, insofar as the
variant obtained retains the properties of the reference protein
and the percent identities stated above; [0042] the .alpha.-subunit
apoprotein variants of the allophycocyanins useful in the acidic
compositions according to the invention, deriving from
substitutions, insertions or deletions, can comprise a difference
of 1 to 24 amino acids in relation to the corresponding reference
sequence, insofar as the variant obtained retains the properties of
the reference protein and the percent identities stated above;
[0043] the .beta.-subunit apoprotein variants of the
allophycocyanins useful in the acidic compositions according to the
invention, deriving from substitutions, insertions and/or
deletions, can comprise a difference of 1 to 20 amino acids in
relation to the corresponding reference sequence, insofar as the
variant obtained retains the properties of the reference protein
and the percent identities stated above.
[0044] More particularly according to the invention, and regardless
of the reference sequence considered (phycocyanin .alpha. and/or
.beta. subunit and/or allophycocyanin .alpha. and/or .beta.
subunit), the variants of said subunits can advantageously comprise
a difference of 1 to 15 amino acids, preferably a difference of 1
to 10 amino acids, in particular a difference of 1 or 2 or 3 or 4
or 5 or 6 or 7 or 8 or 9 or 10 amino acids in relation to the
corresponding reference sequence, insofar as the variant obtained
retains the properties of the reference protein and the percent
identities stated above.
[0045] According to the invention, the phycocyanin or variants
thereof useful, alone or mixed with an allophycocyanin or variants
thereof, in acidic compositions, particularly in acidic food
compositions, can be obtained by culture of a natural organism
naturally expressing the phycocyanin or the variant thereof of
interest or by culture of an organism genetically transformed to
express the phycocyanin or the variant thereof of interest selected
for its capacity to produce said phycocyanin or variants
thereof.
[0046] Exemplary natural organisms naturally expressing a
phycocyanin useful in the compositions according to the invention
or the variant thereof of interest include algae (or microalgae) of
the order Cyanidiales.
[0047] The order Cyanidiales includes the families Cyanidiaceae and
Galdieriaceae, themselves subdivided in the genera Cyanidioschyzon,
Cyanidium and Galdieria, members of which include, among others,
the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae
DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum,
Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria
maxima, Galdieria partita and Galdieria sulphuraria. Particular
mention may be made of strain Galdieria sulphuraria (also called
Cyanidium caldarium) UTEX#2919.
[0048] Thus, according to an embodiment of the invention, the
acidic composition, particularly the acidic food composition,
comprises an acidic-pH-resistant phycocyanin from natural organisms
such as algae or microalgae of the order Cyanidiales, in particular
from natural organisms of the families Cyanidiaceae or
Galdieriaceae.
[0049] More particularly according to the invention, the acidic
composition, particularly the acidic food composition, comprises an
acidic-pH-resistant phycocyanin from natural organisms which belong
to the genera Cyanidioschyzon, Cyanidium, Galdieria, advantageously
selected from the species of the genera Cyanidium and Galdieria
[0050] Even more particularly according to the invention, the
acidic composition, particularly the acidic food composition,
comprises an acidic-pH-resistant phycocyanin from natural organisms
selected from the species Cyanidioschyzon merolae 10D,
Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium
daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens,
Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria
sulphuraria.
[0051] The preferred form of the acidic composition, particularly
the acidic food composition, according to the invention comprises
an acidic-pH-resistant phycocyanin from a natural microalga such as
Galdieria sulphuraria, Cyanidium caldarium or Cyanidioschyzon
merolae. More preferentially, the acidic-pH-resistant phycocyanin
comes from a natural microalga selected from Galdieria sulphuraria
and Cyanidium caldarium.
[0052] By way of example of an organism transformed to express the
phycocyanin or the variant thereof of interest selected for its
capacity to produce said phycocyanin or variants thereof, mention
may be made of a microorganism transformed so as to express the
apoprotein of SEQ ID NO 1 and/or SEQ ID NO 2 and/or SEQ ID NO 3
and/or SEQ ID NO 4, said microorganism also comprising the
biosynthetic pathways necessary for the production of the
chromophore and for the binding thereof to the apoprotein. Yeasts
in particular may be mentioned as microorganisms that can be
modified to produce the phycocyanin and/or allophycocyanin used in
the food compositions according to the invention.
[0053] Those skilled in the art will have no difficulty in finding
in the prior art the description of the methods for culturing
natural and/or modified organisms that can produce a phycocyanin
useful in the compositions according to the invention.
[0054] For example, the culture of Cyanidiaceae or Galdieriaceae,
of the order Cyanidiales, well-known to those skilled in the art,
can advantageously be carried out in mixotrophic mode, light
usually being necessary for the biosynthesis of pigments.
[0055] Such an industrial culture can advantageously be carried out
in large-volume (i.e., 1,000-litre, 10,000-litre, 20,000-litre,
100,000-litre) fermenters. The culture can be carried out under the
conditions known to those skilled in the art. It can be carried out
in batch mode, in fed-batch mode or in continuous mode.
[0056] The phycocyanin useful in the compositions according to the
invention can more particularly be extracted from biomass obtained
by culture of an alga of the order Cyanidiales, as defined above,
cultivated in mixotrophic mode with light having a wavelength of
400 nm to 550 nm, advantageously of 420 nm to 500 nm,
preferentially of 430 to 480 nm, more preferentially of about 455
nm. It can be "white" light having a broad spectrum comprising
light of said wavelength. It can also advantageously be narrow
spectrum light consisting of said wavelength.
[0057] Such a method for industrial preparation of Cyanidiales
biomass in mixotrophic mode, and the biomass thus obtained, are in
particular described in patent application FR 15 59072 filed on 25
Sep. 2015, the contents of which are incorporated herein by
reference.
[0058] The object of the invention is to provide a composition in
which the phycocyanin is stable at acidic pH. By acidic composition
is meant, according to the invention, any composition comprising a
mineral or organic acid and a phycocyanin. Said composition can be
liquid, fluid or viscous, pasty or solid, which has an acidic pH
and into which an acidic-pH-resistant phycocyanin is
incorporated.
[0059] For the aqueous liquid compositions, the pH is measured in
the usual manner. For the non-aqueous liquid compositions or for
the pasty or solid compositions, the pH is measured after
dissolution of the composition in a sufficient amount of water to
dissolve the soluble compounds contained therein, including the
mineral or organic acids and the phycocyanin.
[0060] Advantageously, the composition according to the invention
is an aqueous liquid composition, optionally in gel form, or a
pasty or solid composition designed to be dissolved in an aqueous
solution or in a solid or pasty composition comprising water.
According to another advantageous embodiment of the invention, the
acidic composition pasty or solid composition intended to be
employed and/or stored in a humid environment.
[0061] The mineral or organic acids useful in the compositions
according to the invention are well-known to those skilled in the
art. Exemplary mineral acids include in particular carbonic,
phosphoric, hydrochloric, sulphuric, perchloric, sulphonic and
nitric acids. Exemplary organic acids include in particular citric,
lactic, malic, tartaric, succinic acids, advantageously citric
acid.
[0062] By acidic food composition is meant, according to the
invention, any composition designed to be ingested by humans or
animals and which falls within the preceding definition.
Nutraceutical acidic compositions must be regarded as falling
within the definition of the acidic food compositions within the
context of the invention.
[0063] The acidic food compositions according to the invention are
well-known to those skilled in the art. They can comprise a carrier
which can comprise structural components associated with active
compounds identified for their nutritive supply or for their health
properties which benefit humans or animals. The acidic food
composition according to the invention can also comprise food
additives such as texturing agents, flavouring agents,
preservatives, or any components well-known to those skilled in the
art. The carrier can comprise water and/or proteins and/or fats
and/or fibre and/or sugars. The components of the carrier may have
only structural properties, but they are generally known for their
nutritive supply.
[0064] The acidic food composition according to the invention can
be ready-to-use or in the form of a food additive to be added to a
solid, pasty or liquid preparation in order to prepare the edible
food.
[0065] For the food compositions, the acid will preferably be
selected from the list of acidifiers authorized for foods, in
particular carbonic, phosphoric, citric, malic, tartaric and lactic
acids, more particularly citric acid.
[0066] Concerning the non-food acidic compositions according to the
invention, they can be, among other things, pharmaceutical,
veterinary or cosmetic compositions and further comprise any
additives and/or active agents known and used in such
compositions.
[0067] In a solid, liquid or pasty acidic composition according to
the invention, the phycocyanin can be incorporated, for example, in
powder form. Said acidic composition, particularly said acidic food
composition, may thus be in any known conventional form such as
creams, gels, foams, pastes, etc. Exemplary solid food compositions
include cakes or biscuits, dry food for cooking, soluble powders,
gelatinous solid compositions (jelly), foams etc.
[0068] According to the invention, said liquid acidic composition
can be an aqueous composition into which the phycocyanin is
dissolved. It can be in the form of a ready-to-use composition or a
liquid concentrate for dilution, notably to be ingested or to be
added to a solid food either for its preparation or for its
ingestion, for example a concentrated liquid "topping" composition
to be applied to a cake to give it colour. Among these concentrated
compositions, mention may be made of syrups, optionally containing
alcohol.
[0069] The liquid acidic composition according to the invention can
be of varying viscosity and optionally comprise additives such as
viscosity agents, gelling agents, and other structuring additives
known to those skilled in the art and typical for the preparation
of liquid food compositions.
[0070] According to a particular embodiment of the invention, the
liquid food composition can be an optionally carbonated acidic
beverage. Particular mention may be made of sodas, juices, sports
drinks, energy drinks, recovery drinks, etc. The compositions of
these beverages are well-known to those skilled in the art and can
notably comprise sugars, mineral salts, food additives, dissolved
gas, etc. The beverage according to the invention is a conventional
acidic beverage in which the colouring usually employed has been
wholly or partly replaced by an acidic-pH-resistant phycocyanin
according to the invention.
[0071] According to the invention, the phycocyanin content in the
compositions according to the invention can be consistent with the
practices of those skilled in the art.
[0072] For example, when the phycocyanin will be used to colour the
acidic composition, then the phycocyanin content in said
composition can be consistent with the practice of those skilled in
the art as regards colouring.
[0073] In a liquid acidic composition within the context of the
invention, the phycocyanin content can be from 2.5 mg/L to 2,500
mg/L, preferentially from 25 mg/L to 300 mg/L.
[0074] In a liquid composition of the ready-to-use beverage type,
the phycocyanin content can generally be from 25 mg/L to 300 mg/L,
preferentially from 50 mg/L to 100 mg/L.
[0075] In a concentrated liquid composition for dilution before
use, such as a syrup, the phycocyanin content can generally be from
250 mg/L to 2,500 mg/L, preferentially from 500 mg/L to 1,000
mg/L.
[0076] In a solid composition, the phycocyanin content can
generally be from 0.01 mg/g to 10 mg/g, preferentially from 0.1
mg/g to 5.0 mg/g, more preferentially from 0.25 mg/g to 2.5
mg/g.
[0077] One of the advantages of the invention resides, as can be
seen in the following examples, in the fact that the colouring
provided by the acidic-pH-resistant phycocyanins is more stable
over time.
[0078] Other aspects and features of the invention will become
apparent from reading the examples and figures.
DESCRIPTION OF THE FIGURES
[0079] FIG. 1 describes the amino acid sequences of the Galdieria
sulphuraria phycocyanin and allophycocyanin apoproteins with:
[0080] SEQ ID NO 1: YP_009051179.1: Phycocyanin .alpha. subunit;
[0081] SEQ ID NO 2: YP_009051180.1: Phycocyanin .beta. subunit;
[0082] SEQ ID NO 3: YP_009051103.1: Allophycocyanin .alpha.
subunit; [0083] SEQ ID NO 4: YP_009051104.1: Allophycocyanin .beta.
subunit;
[0084] FIG. 2 presents the stability curve as a function of pH of
the phycocyanins extracted from Galdieria sulphuraria (UTEX2919)
and Cyanidioschyzon merolae (ACUF 199) whose apoprotein sequence
consists of SEQ ID NO 1 or a variant, in relation to the stability
curve of the spirulina-derived phycocyanin Linablue.RTM.. [0085]
(-.box-solid.-): Phycocyanin from Galdieria sulphuraria (UTEX#2919)
[0086] (.cndot..cndot..cndot.X.cndot..cndot..cndot.): Phycocyanin
from Cyanidioschyzon merolae (ACUF 199) [0087] (-.smallcircle.*-):
Lina Blue.RTM.: Phycocyanin from Spirulina platensis (Arthrospira
platensis) (data obtained from the website
http://www.dlt-spl.co.jp/business/en/spirulina/linablue.html),
showing the pH values at which precipitation of the phycocyanin
appears.
[0088] FIG. 3 represents colour change over time of an acidic
beverage comprising phycocyanin. W0, W2, W4 and W6: week 0, 2, 4
and 6.
EXAMPLES
Example 1: Production and Extraction of Phycocyanin from Galdieria
sulphuraria UTEX#2919
Materials and Methods
[0089] Strain: Galdieria sulphuraria (Also Called Cyanidium
caldarium) UTEX#2919
Culture Medium
[0090] Mixotrophy: 30 g/L glycerol, 8 g/L (NH.sub.4).sub.2SO.sub.4,
1 g/L KH.sub.2PO.sub.4, 716 mg/L MgSO.sub.4, 44 mg/L CaCl.sub.2, 3
mL/L of Fe-EDTA stock solution (6.9 g/L FeSO.sub.4 and 9.3 g/L
EDTA-Na.sub.2) and 4 mL/L of trace metal solution (3.09 g/L
EDTA-Na.sub.2; 0.080 g/L CuSO.sub.4, 5H.sub.2O; 2.860 g/L
H.sub.3BO.sub.3; 0.040 g/L NaVO.sub.3, 4H.sub.2O; 1.820 g/L
MnCl.sub.2; 0.040 g/L CoCl.sub.2, 6H.sub.2O; 0.220 g/L ZnSO.sub.4,
7H.sub.2O; 0.017 g/L Na.sub.2SeO.sub.3; 0.030 g/L
(NH.sub.4).sub.6Mo.sub.7O.sub.24, 4H.sub.2O).
Culture Conditions:
[0091] The cultures are carried out in 1- to 2-L-useful-volume
reactors with computer-controlled automated systems. Culture pH is
regulated by adding base (14% ammonia solution (wNH.sub.3/w))
and/or acid (4 N sulphuric acid solution). Culture temperature is
set to 37.degree. C. The culture is illuminated by baffles equipped
with a system of white LEDs or blue LEDs (455 nm) in a way similar
to that described in patent WO 2014/174182. Tracking of cell growth
is carried out at different times by measuring absorbance at 800
nm. And a measurement of the dry mass is carried out by
filtration.
[0092] The performance characteristics of the culture at the end of
growth are summarized in the following Table 1:
TABLE-US-00001 TABLE 1 Mixotrophy Time (h) 160 Dry biomass (g/L) 20
Phycocyanin content (mg/g dry biomass) 40
[0093] Measurements of intracellular phycocyanin content per gram
of dry matter were carried out using the extraction and assay
method described by Moon and colleagues [Moon et al., Korean J.
Chem. Eng., 2014, 1-6] while replacing the phosphate buffer with
water.
Example 2: Extraction of Phycocyanins
[0094] Strains Galdieria sulphuraria (UTEX#2919) and/or
Cyanidioscyizon merolae (ACUF199) were cultivated under the
conditions of example 1.
[0095] Phycocyanin is then extracted according to a modification of
the protocol described by Moon et al., 2014 (op. cit.). Said
modification consists in replacing the phosphate buffer used to
solubilize phycocyanin with demineralized water.
[0096] An extract (also called "phycocyanin extract" or "crude
extract") which comprises, in addition to the phycocyanin of
interest, other water-soluble proteins, is thus obtained. The
phycocyanin extract can have several possible qualities depending
on the method of extraction and/or purification used. For example,
a crude extract will contain a higher amount of water-soluble
proteins, other than phycocyanin, than that found in a purified
extract. By purified extract is meant a crude extract of which a
portion of the water-soluble proteins have been removed by
ultrafiltration, hollow-fibre filtration, or ion-exchange
chromatography, methods known to those skilled in the art, while
retaining the phycocyanin.
[0097] Purity index is traditionally expressed by calculating the
ratio of the absorbance of the solution at 618 nm (specific
absorbance of phycocyanin) to that at 280 nm, the specific
absorbance of aromatic amino acids giving an idea of the total
protein level. The lower this ratio, the higher the amount of
proteins other than phycocyanin in the solution.
[0098] The crude extract was purified using the KrosFlo.RTM.
tangential flow filtration system from Spectrum.RTM. Labs.
TABLE-US-00002 TABLE 2 Purity index measurement of a phycocyanin
extract before and after purification. Purity index Abs 618 nm/Abs
280 nm Crude phycocyanin extract 0.54 Purified extract 2.12
Example 3: Stability Study of Phycocyanin Extracted from Strains
Galdieria sulphuraria (UTEX#2919) and Cyanidioschyzon merolae
(ACUF199) and Spirulina (Arthrospira) platensis as a Function of
pH
[0099] Strains Galdieria sulphuraria (UTEX#2919) and
Cyanidioschyzon merolae (ACUF199) were cultivated under the
conditions of example 1.
[0100] The tests are carried out by taking as reference the data of
the commercial product LineBlue.RTM.
(http://www.dlt-spl.co.jp/business/en/spirulina/linablue.html) from
the company DIC Lifetec Co., Ltd. (Tokyo, Japan), which is a
phycocyanin extracted from Spirulina (Arthrospira) platensis.
[0101] These tests were carried out with the phycocyanins prepared
in example 2 and having a purity index of 2.12. Measurement of blue
colour is done by measuring absorbance at 618 nm with an ultraspec
2100 pro spectrophotometer (Amersham). Percent colour loss is
calculated relative to the absorbance measurement of the sample
under the reference conditions (pH 6).
[0102] For the resistance test under acidic conditions, the pH is
gradually lowered by adding 5% citric acid solution (Sigma 251275)
to the phycocyanin preparation. For each pH value, a sample of the
phycocyanin solution is collected and its absorbance measured at
618 nm, 10 minutes after lowering the pH to the desired value.
[0103] The results of these tests are presented in FIG. 2:
[0104] Phycocyanin extracted from Galdieria sulphuraria (UTEX#2919)
[(-.box-solid.-)] exhibits very good pH-resistance compared with
that of Spirulina, with less than 10% loss of pigmentation up to pH
2.75 (98.25% of its colouring at pH 3, 92.4% at pH 2.75), the loss
increasing as of pH 2.5 (79% at pH 2.5; 75% at pH 2.25; 46% at pH
2).
[0105] Phycocyanin extracted from strain Cyanidioschyzon merolae
(ACUF 199) [(.cndot..cndot..cndot.X.cndot..cndot..cndot.)] exhibits
good pH-resistance compared with that of Spirulina, with more than
90% of its colouring at pH 3, 70% at pH 2.75; 40% at pH 2.5; 23% at
pH 2.25; 20% at pH 2.
[0106] Phycocyanin extracted from strain Spirulina platensis
[(-.smallcircle.*-)] exhibits only 90% of its colouring at pH 4,
80% at pH 3.8; 60% at pH 3.6; 38% at pH 3.4. Phycocyanin extracted
from Spirulina platensis starts to precipitate as of pH 3.8.
[0107] In conclusion, Galdieria sulphuraria or Cyanidioschyzon
merolae phycocyanins are more resistant to acidic pH than that
extracted from Spirulina.
Example 4--Comparative Analysis of Apoprotein Sequences of
Phycocyanin and Allophycocyanin from Different Microalgae and
Measurement of the Isoelectric Points Thereof
[0108] In order to identify a possible cause for the increase in
resistance to acidic pH, a comparison of the sequences of the
.alpha. and .beta. subunits of phycocyanins and allophycocyanins
produced by various microorganisms, particularly microalgae, was
performed using the BLASTP program, the use of which is well-known
to those skilled in the art, on the basis of published sequences
accessible in databases (see accession number). The comparison is
made in relation to the apoprotein sequence of the corresponding
subunit of strain Galdieria sulphuraria.
[0109] At the same time, the isoelectric point of the .alpha.- and
.beta.-subunit apoproteins of phycocyanins and allophycocyanins
whose sequence comparison was carried out was determined by the
computational procedure described by Patrickios and Yamasaki
(1995).
[0110] Phycocyanin and allophycocyanin produced by strain Galdieria
sulphuraria serve as the reference in these studies.
[0111] Results
[0112] The results of the sequence comparisons and the pI
calculations are presented in tables 3 and 4 below:
TABLE-US-00003 TABLE 3 Comparisons of the alpha and beta apoprotein
sequences of phycocyanin from various organisms. The isoelectric
point of each protein is shown (Patrickios and Yamasaki, 1995).
aa/aa (% Identity) pI .alpha. subunit of Phycocyanin from
(accession #) Galdieria sulphuraria (YP_009051179.1) 162/162 (100%)
2.115 Cyanidium caldarium (P00306.3) 159/162 (98%) 2.1
Cyanidioschyzon merolae (NP_848986.1) 135/162 (83%) 2.129
Arthrospira maxima CS328 (EDZ96896.1) 119/162 (73%) 3.657 Spirulina
platensis (P72509.2) 119/162 (73%) 3.657 Arthrospira jenneri fz
(AEV40872.1) 118/162 (73%) 3.657 Cyanobacterium stanieri PCC 7202
123/162 (76%) 3.327 (AFZ46322.1) Halothece sp. PCC 7418
(WP_015227201.1) 130/162 (80%) 3.786 Geitlerinema sp. PCC 7407
120/162 (74%) 3.936 (WP_015173541.1) Nostoc sp. PCC 7120
(WP_010994705.1) 130/162 (80%) 3.978 .beta. subunit of Phycocyanin
from (# accession) Galdieria sulphuraria (YP_009051180.1) 171/171
(100%) 4.112 Cyanidium caldarium (AAB34027.2) 169/171 (99%) 4.112
Cyanidioschyzon merolae (NP 848987.1) 141/171 (82%) 4.112
Arthrospira maxima CS328 (EDZ96897.1) 130/171 (76%) 3.966 Spirulina
platensis (1HA7 B) 130/170 (76%) 3.966 Arthrospira jenneri fz
(AEV40871.1) 130/171 (76%) 4.062 Cyanobacterium stanieri PCC 7202
135/171 (79%) 3.966 (AFZ46321.1) Halothece sp. PCC 7418 (WP
015227202.1) 133/171 (78%) 4.074 Geitlerinema sp. PCC 7407 138/171
(81%) 4.12 (WP 015173542.1) Nostoc sp. PCC 7120 (BAB72486.1)
141/170 (83%) 4.159
TABLE-US-00004 TABLE 4 Comparisons of the alpha and beta apoprotein
sequences of allophycocyanin from various organisms. The
isoelectric point of each protein is shown (Patrickios and
Yamasaki, 1995). aa/aa (% Identity) pI .alpha. subunit of
Allophycocyanin from (accession #) Galdieria sulphuraria
(YP_009051103.1) 161/161 (100%) 3.9 Cyanidium caldarium
(AAA20110.1) 158/161 (98%) 3.932 Cyanidioschyzon merolae
(NP_849064.1) 144/161 (89%) 3.989 Arthrospira jenneri (AEV40869.1)
131/161 (81%) 4.008 Arthrospira platensis (CAA65141.1) 131/161
(81%) 4.054 Cyanobacterium stanieri PCC 7202 125/161 (78%) 3.997
(AFZ46138.1) Halothece sp. PCC 7418 133/161 (83%) 3.724
(WP_015226049.1) Geitlerinema sp. PCC 7407 137/161 (85%) 4.054
(WP_015172402.1) Nostoc sp. PCC 7120 (WP_010994198.1) 130/161 (81%)
4.041 .beta. subunit of Allophycocyanin from (accession #)
Galdieria sulphuraria (YP_009051104.1) 161/161 (100%) 3.502
Cyanidium caldarium (AAB01577.1) 158/161 (98%) 3.657
Cyanidioschyzon merolae (NP_849065.1) 145/161 (90%) 3.298
Arthrospira jenneri (AEV40870.1) 138/161 (86%) 3.502 Arthrospira
platensis (BAA19986.1) 141/161 (88%) 3.532 Cyanobacterium stanieri
PCC 7202 141/161 (88%) 3.532 (AFZ46566.1) Halothece sp. PCC 7418
138/161 (86%) 3.502 (WP_015226048.1) Geitlerinema sp. PCC 7407
140/161 (87%) 3.502 (WP_015172403.1) Nostoc sp. PCC 7120
(WP_010994199.1) 138/160 (86%) 3.657
[0113] The sequence and isoelectric point comparisons show that the
.alpha. subunits of the phycocyanins most resistant to acidic pH
have an isoelectric point below 3 and a higher percent identity
with the sequence of Galdieria sulphuraria.
Example 5: Stability Over Time in a Beverage
[0114] The stability test in acid medium over time for the
phycocyanin extracted from strain UTEX#2919 was carried out by
adding the phycocyanin to a lemonade beverage (pH 2.95; as
described in example 6: beverage 1). After adding 0.025.Salinity.
of phycocyanin the apoprotein sequence of which consists of variant
of SEQ ID NO 1, the beverage was exposed to a day/night cycle (16
h/8 h) with artificial light for 6 weeks, at room temperature.
[0115] Regular samples are taken over time in order to measure the
absorbance at 618 nm. An aliquot is taken at weeks 0 (W0), 2 (W2),
4 (W4) and 6 (W6), and the absorbance at 618 nm measured with the
ultraspec 2100 pro spectrophotometer (Amersham) in order to define
the 100% colour point. Residual colour is expressed as a percentage
of the initial reference value.
[0116] FIG. 3 shows the results of this experiment. Steady colour
loss over time during long-term exposure to light is noted.
However, after 6 weeks of exposure more than 60% of the colour
remains.
Example 6: Examples of Acidic Beverages in Liquid Form Comprising
Phycocyanin
[0117] Beverages containing phycocyanin can have the following
composition:
Beverage 1--Soda Beverage:
TABLE-US-00005 [0118] Carbonated water qs 1 L Sugars 70 g Citric
acid 1.5 g Natural organic flavour 4 to 8 g Phycocyanin extract
obtained in example 2 qs 100 mg of Phycocyanin
The pH of this beverage is 2.95
Beverage 2--Health Drink for Athletes:
TABLE-US-00006 [0119] Spring water qs 1 L Glucose 20 g Fructose 10
g Citric acid 2.7 g Sodium citrate 1.87 g Potassium citrate 0.327 g
Magnesium chloride 2.5 mg Calcium chloride 3 mg Natural organic
flavour 4 g Phycocyanin extract obtained in example 2 qs 150 mg of
Phycocyanin
The pH of this beverage is 3.5
Example 6: Acidic Beverage in Soluble Powder Form Comprising
Phycocyanin
TABLE-US-00007 [0120] Sugars 70 to 100 g Citric acid 1 to 1.5 g
Natural organic flavour 4 to 8 g Phycocyanin extract obtained in qs
250 mg of Phycocyanin example 2
The powder (75 to 110 g) thus prepared can be dissolved in 1 L of
water to obtain a blue-coloured acidic beverage.
Example 7: Solid Acidic Composition Comprising Phycocyanin: Acidic
Sweet
TABLE-US-00008 [0121] Water: qs 1 kg Sugars: 800 g Citric acid: 1
to 2 g Phycocyanin extract obtained in qs 2,500 mg of Phycocyanin
example 2
REFERENCES
[0122] Moon et al., Korean J. Chem. Eng., 2014, 1-6 [0123]
Patrickios et Yamasaki, Polypeptide Amino Acid Composition and
Isoelectric Point. II. Comparison between Experiment and Theory.
Analytical Biochemistry. 231, 1, 1995: 82-91 [0124] FR Doc No:
2013-19550 [0125] WO 2014/174182 [0126] FR 15 59072 filed on 25
Sep. 2015 [0127]
http://www.dlt-spl.co.jp/business/en/spirulina/linablue.html [0128]
http://blast.ncbi.nlm.nih.gov/Blast.cgi
Sequence CWU 1
1
41459PRTGaldieria sulphuraria 1Met Glu Thr Leu Tyr Ser Thr His Arg
Pro Arg Ile Leu Glu Thr His 1 5 10 15 Arg Gly Leu Ala Leu Ala Ile
Leu Glu Ala Leu Ala Ala Leu Ala Ala 20 25 30 Leu Ala Ala Ser Pro
Ala Ser Asn Gly Leu Asn Gly Leu Tyr Ala Arg 35 40 45 Gly Pro His
Glu Leu Glu Ser Glu Arg Ala Ser Asn Thr His Arg Gly 50 55 60 Leu
Leu Glu Gly Leu Asn Ala Leu Ala Val Ala Leu Ala Ser Asn Gly 65 70
75 80 Leu Tyr Ala Arg Gly Thr Tyr Arg Gly Leu Asn Ala Arg Gly Ala
Leu 85 90 95 Ala Ala Leu Ala Ala Leu Ala Ser Glu Arg Leu Glu Gly
Leu Ala Leu 100 105 110 Ala Ala Leu Ala Ala Arg Gly Ser Glu Arg Leu
Glu Thr His Arg Ser 115 120 125 Glu Arg Ala Ser Asn Ala Leu Ala Gly
Leu Asn Ala Arg Gly Leu Glu 130 135 140 Ile Leu Glu Ala Ser Asn Gly
Leu Tyr Ala Leu Ala Ala Leu Ala Gly 145 150 155 160 Leu Asn Ala Leu
Ala Val Ala Leu Thr Tyr Arg Ser Glu Arg Leu Tyr 165 170 175 Ser Pro
His Glu Pro Arg Thr Tyr Arg Thr His Arg Ser Glu Arg Gly 180 185 190
Leu Asn Met Glu Thr Pro Arg Gly Leu Tyr Pro Arg Gly Leu Asn Thr 195
200 205 Tyr Arg Ala Leu Ala Ser Glu Arg Ser Glu Arg Ala Leu Ala Val
Ala 210 215 220 Leu Gly Leu Tyr Leu Tyr Ser Ala Leu Ala Leu Tyr Ser
Cys Tyr Ser 225 230 235 240 Ala Leu Ala Ala Arg Gly Ala Ser Pro Ile
Leu Glu Gly Leu Tyr Thr 245 250 255 Tyr Arg Thr Tyr Arg Leu Glu Ala
Arg Gly Met Glu Thr Val Ala Leu 260 265 270 Thr His Arg Thr Tyr Arg
Cys Tyr Ser Leu Glu Val Ala Leu Val Ala 275 280 285 Leu Gly Leu Tyr
Gly Leu Tyr Thr His Arg Gly Leu Tyr Pro Arg Met 290 295 300 Glu Thr
Ala Ser Pro Gly Leu Thr Tyr Arg Leu Glu Ile Leu Glu Ala 305 310 315
320 Leu Ala Gly Leu Tyr Leu Glu Gly Leu Gly Leu Ile Leu Glu Ala Ser
325 330 335 Asn Ala Arg Gly Thr His Arg Pro His Glu Ala Ser Pro Leu
Glu Ser 340 345 350 Glu Arg Pro Arg Ser Glu Arg Thr Arg Pro Thr Tyr
Arg Val Ala Leu 355 360 365 Gly Leu Ala Leu Ala Leu Glu Ala Ser Asn
Thr Tyr Arg Val Ala Leu 370 375 380 Leu Tyr Ser Ser Glu Arg Ala Ser
Asn His Ile Ser Gly Leu Tyr Leu 385 390 395 400 Glu Ser Glu Arg Gly
Leu Tyr Gly Leu Asn Ala Leu Ala Ala Leu Ala 405 410 415 Ala Ser Asn
Gly Leu Ala Leu Ala Ala Ser Asn Thr His Arg Thr Tyr 420 425 430 Arg
Ile Leu Glu Ala Ser Pro Thr Tyr Arg Ala Leu Ala Ile Leu Glu 435 440
445 Ala Ser Asn Ala Leu Ala Leu Glu Ser Glu Arg 450 455
2172PRTGaldieria sulphuraria 2Met Leu Asp Ala Phe Ala Lys Val Val
Ala Gln Ala Asp Ala Arg Gly 1 5 10 15 Glu Phe Leu Ser Asn Thr Gln
Leu Asp Ala Leu Ser Lys Met Val Ser 20 25 30 Glu Gly Asn Lys Arg
Leu Asp Val Val Asn Arg Ile Thr Ser Asn Ala 35 40 45 Ser Ala Ile
Val Thr Asn Ala Ala Arg Ala Leu Phe Ser Glu Gln Pro 50 55 60 Gln
Leu Ile Gln Pro Gly Gly Asn Ala Tyr Thr Asn Arg Arg Met Ala 65 70
75 80 Ala Cys Leu Arg Asp Met Glu Ile Ile Leu Arg Tyr Val Ser Tyr
Ala 85 90 95 Ile Ile Ala Gly Asp Ser Ser Val Leu Asp Asp Arg Cys
Leu Asn Gly 100 105 110 Leu Arg Glu Thr Tyr Gln Ala Leu Gly Val Pro
Gly Ala Ser Val Ala 115 120 125 Val Gly Val Glu Lys Met Lys Asp Ser
Ala Ile Ala Ile Ala Asn Asp 130 135 140 Pro Ser Gly Ile Thr Thr Gly
Asp Cys Ser Ala Leu Met Ala Glu Val 145 150 155 160 Gly Thr Tyr Phe
Asp Arg Ala Ala Thr Ala Val Gln 165 170 3161PRTGaldieria
sulphuraria 3Met Ser Ile Val Thr Lys Ser Ile Val Asn Ala Asp Ala
Glu Ala Arg 1 5 10 15 Tyr Leu Ser Pro Gly Glu Leu Asp Arg Ile Lys
Ser Phe Val Leu Ser 20 25 30 Gly Gln Arg Arg Leu Arg Ile Ala Gln
Ile Leu Thr Asp Asn Arg Glu 35 40 45 Arg Ile Val Lys Gln Ala Gly
Gln Gln Leu Phe Gln Gln Arg Pro Asp 50 55 60 Ile Val Ser Pro Gly
Gly Asn Ala Tyr Gly Glu Glu Met Thr Ala Thr 65 70 75 80 Cys Leu Arg
Asp Leu Asp Tyr Tyr Leu Arg Leu Val Thr Tyr Gly Val 85 90 95 Val
Ala Gly Asp Ile Ser Pro Ile Glu Glu Ile Gly Leu Val Gly Val 100 105
110 Lys Glu Met Tyr Asn Ser Leu Gly Thr Pro Ile Ser Ala Val Ala Glu
115 120 125 Gly Ile Lys Ala Met Lys Asn Val Ala Cys Ser Leu Leu Ser
Gly Asp 130 135 140 Asp Ser Ala Glu Ala Gly Phe Tyr Phe Asp Tyr Thr
Ile Gly Ala Met 145 150 155 160 Gln 4161PRTGaldieria sulphuraria
4Met Gln Asp Ala Ile Thr Ala Val Ile Asn Thr Ala Asp Val Gln Gly 1
5 10 15 Lys Tyr Leu Asp Asn Ser Ser Ile Glu Lys Leu Lys Gly Tyr Phe
Gln 20 25 30 Thr Gly Glu Leu Arg Val Arg Ala Ala Ala Thr Ile Ala
Ala Asn Ala 35 40 45 Ala Gly Ile Ile Lys Asp Ala Val Ala Lys Ser
Leu Leu Tyr Ser Asp 50 55 60 Ile Thr Arg Pro Gly Gly Asn Met Tyr
Thr Thr Arg Arg Tyr Ala Ala 65 70 75 80 Cys Ile Arg Asp Leu Asp Tyr
Tyr Leu Arg Tyr Ala Thr Tyr Ser Met 85 90 95 Leu Ala Gly Asp Pro
Ser Ile Leu Asp Glu Arg Val Leu Asn Gly Leu 100 105 110 Lys Glu Thr
Tyr Asn Ser Leu Gly Val Pro Ile Gly Ala Thr Ile Gln 115 120 125 Ser
Ile Gln Ala Met Lys Glu Val Thr Ser Ser Leu Val Gly Ser Glu 130 135
140 Ala Gly Lys Glu Met Gly Ile Tyr Phe Asp Tyr Ile Cys Ser Gly Leu
145 150 155 160 Ser
* * * * *
References