U.S. patent application number 16/972541 was filed with the patent office on 2021-08-05 for methods of making chitosan.
The applicant listed for this patent is Mykito Sciences Inc. Invention is credited to Cameron Bardliving, Brent Chamberlain, Animesh Ray, Charles Taylor, Ilya Tolstorukov.
Application Number | 20210238642 16/972541 |
Document ID | / |
Family ID | 1000005566611 |
Filed Date | 2021-08-05 |
United States Patent
Application |
20210238642 |
Kind Code |
A1 |
Tolstorukov; Ilya ; et
al. |
August 5, 2021 |
METHODS OF MAKING CHITOSAN
Abstract
A method for making a chitosan product from yeast cells is
disclosed herein. Yeast cells are cultured to form a biomass of
yeast cells. The yeast cells are induced to undergo meiosis causing
the yeast cells to form asci containing ascospores wherein each
ascospore contains a chitosan protective layer in the ascospore
wall. Chitosan is extracted from the ascospores, purified to form
purified chitosan, and precipitated and dried to form a chitosan
product.
Inventors: |
Tolstorukov; Ilya;
(Claremont, CA) ; Taylor; Charles; (Claremont,
CA) ; Ray; Animesh; (Claremont, CA) ;
Bardliving; Cameron; (Blue Bell, PA) ; Chamberlain;
Brent; (Claremont, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mykito Sciences Inc |
San Marino |
CA |
US |
|
|
Family ID: |
1000005566611 |
Appl. No.: |
16/972541 |
Filed: |
June 7, 2019 |
PCT Filed: |
June 7, 2019 |
PCT NO: |
PCT/US2019/035946 |
371 Date: |
December 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62682245 |
Jun 8, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 1/16 20130101; C12P
19/26 20130101; C08B 37/003 20130101; C12R 2001/85 20210501 |
International
Class: |
C12P 19/26 20060101
C12P019/26; C12N 1/16 20060101 C12N001/16 |
Claims
1-28. (canceled)
29. A method of making a chitosan product, comprising: culturing
yeast cells, wherein the cultured yeast cells form a biomass of
yeast cells; inducing the cultured yeast cells to undergo meiosis,
wherein the induced yeast cells form asci with ascospores and
wherein each ascospore contains a chitosan protective layer in an
ascospore wall; extracting chitosan from the ascospore walls;
purifying the chitosan to form purified chitosan; and precipitating
and drying the purified chitosan to form a chitosan product.
30. The method of claim 29, wherein the method further includes an
additional step selected from the group consisting of: disrupting
asci walls or disrupting the asci walls and the ascospore walls
after inducing the cultured yeast cells to undergo meiosis;
disrupting the asci walls and separating the ascospores from debris
after inducing the cultured yeast cells to undergo meiosis; and
disrupting the ascospore walls after separating the ascospores from
the debris.
31. The method of claim 29, wherein the yeast cells are a yeast
selected from the group consisting of an Ascomycota phylum yeast
strain, a Saccharomycotina subphyla yeast strain, a class
Saccharomycetes yeast strain, a Genus Saccharomyces yeast strain, a
Genus Komagataella yeast strain, a Genus Schizosaccharomyces yeast
strain, a heterothallic haploid yeast strain, a heterothallic
diploid yeast strain, and a heterothallic polyploid yeast
strain.
32. The method of claim 31, wherein the yeast are sporulating yeast
cells that can be fused to each other to form hybrid cells.
33. The method of claim 31, wherein the yeast are heterothallic
yeast strains of opposite mating types that can mate with each
other to form heterozygous mating type hybrid cells.
34. The method of claim 29, wherein the yeast is selected from the
group consisting of a homothallic self-fertile yeast strain, a
heterothallic heterozygous for mating type diploid yeast strain, a
heterothallic heterozygous for mating type polyploid yeast strain,
and combinations thereof.
35. The method of claim 34, wherein cells of homothallic
self-fertile yeast strain, the heterothallic heterozygous for
mating type diploid yeast strain, a heterothallic heterozygous for
mating type polyploid yeast strain or combinations thereof produce
ascospores.
36. The method of claim 29, wherein inducing the cultured yeast
cells to undergo meiosis includes: incubating the yeast cells in a
presporulation medium for a time ranging from about 6 hours to
about 72 hours; and transferring the yeast cells to a sporulation
medium and incubating the yeast cells for a time ranging from about
12 hours to about 144 hours, thereby converting about 10% to about
100% of the biomass of yeast cells to asci containing at least one
ascospore.
37. The method of claim 29, wherein inducing the cultured yeast
cells to undergo meiosis includes transferring the yeast cells to a
sporulation medium and incubating the yeast cells for a time
ranging from about 12 hours to about 144 hours, thereby converting
about 10% to about 100% of the yeast cells to asci containing at
least one ascospore.
38. The method of claim 29, wherein culturing the yeast cells is
performed using a system selected from the group consisting of a
cell culture vessel, a cell retention system, and a media exchange
system.
39. The method of claim 30, wherein disrupting the asci walls or
disrupting the asci walls and ascospore walls is performed
enzymatically or mechanically.
40. The method of claim 30, wherein separating the ascospores from
the debris includes using density gradient centrifugation or
biphasic separation.
41. The method of claim 40, wherein a density gradient is created
using a density gradient medium selected from the group consisting
of polyhydric alcohols, polysaccharides, colloidal silica, and
combinations thereof.
42. The method of claim 40, wherein biphasic separation includes:
suspending the ascospores and debris into a buffer solution or an
aqueous medium, thereby forming a suspension; mixing the suspension
with a solution having a different phase from the suspension for a
time ranging from about 1 second to about 1800 seconds;
partitioning the ascospores into an ascospore containing chitosan
layer; and retaining the ascospore containing chitosan layer.
43. The method of claim 40, wherein the biphasic separation is an
aqueous two phase extraction including mixing the ascospores and
the debris with an intermediate molecular weight polyether in an
aqueous salt solution to partition the ascospores into a polyether
phase.
44. The method of claim 29, wherein extracting chitosan from the
ascospore walls includes heating the ascospores in an acid solution
to a temperature ranging from about 60.degree. C. to about
100.degree. C. for at time ranging from about 1 hour to about 12
hours, thereby solubilizing the chitosan.
45. The method of claim 44, wherein the acid solution is present in
an amount ranging from about 0.1% w/v to about 50% w/v of a total
weight of the acid solution.
46. The method of claim 29, wherein precipitating and drying the
purified chitosan includes: washing the chitosan with an alkaline
solution, thereby precipitating the chitosan; and drying the
chitosan to form the chitosan product.
47. The method of claim 46, wherein the alkaline solution is a
solution of sodium hydroxide or ammonium hydroxide present in an
amount ranging from about 3% w/v and about 30% w/v of a total
weight of the alkaline solution.
48. The method of claim 47, wherein purifying the chitosan includes
enzymatic digestion by adding an enzyme to the acid solution at a
temperature ranging from about 4.degree. C. to about 105.degree. C.
for a time ranging from about 1 hours to about 24 hours.
Description
RELATED PATENT APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 62/682,245, filed on Jun. 8, 2018, incorporated in
its entirety by reference herein.
TECHNICAL FIELD
[0002] The present disclosure relates to methods for making a
chitosan product from the ascospores of yeast cells.
BACKGROUND
[0003] Chitosan is a polysaccharide that may be used in a variety
of industries in different products. In some examples, chitosan may
be used as a flocculating agent, excipient, or anti-microbial agent
in products across multiple industries. In addition, chitosan may
be used in therapeutic applications. Currently, chitosan is
primarily sourced from crustacean shells using an extraction
process that involves treatment with harsh chemicals. Chitosan
obtained from animal sources can be contaminated with heavy metals,
induce allergic response to shellfish proteins, and have
variability material attributes, making it especially problematic
for use in biomedical industries. Chitosan obtained from a
non-animal source does not have these problems.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] Features and advantages of examples of the present
disclosure will be apparent by reference to the following detailed
description and drawings, in which like reference numerals
correspond to similar, though perhaps not identical, components.
For the sake of brevity, reference numerals or features having a
previously described function may or may not be described in
connection with other drawings in which they appear.
[0005] FIG. 1 is a schematic example of a cross-section of a
Saccharomyces cerevisiae ascospore wall.
[0006] FIG. 2 is a flow diagram illustrating an example of a method
for making a chitosan product according to the present
disclosure.
[0007] FIG. 3 is a light microscopy image of asci produced by a
Saccharomyces cerevisiae yeast strain.
[0008] FIG. 4 is another flow diagram illustrating another example
of a method for making a chitosan product according to the present
disclosure.
[0009] FIG. 5 is another flow diagram illustrating another example
of a method for making a chitosan product according to the present
disclosure.
[0010] FIG. 6 is another flow diagram illustrating another example
of a method for making a chitosan product according to the present
disclosure.
[0011] FIG. 7A is a confocal microscopy image of a Saccharomyces
cerevisiae ascus from the dit1 mutant yeast strain that is stained
with Calcofluor White. FIG. 7B is a confocal microscopy image of a
Saccharomyces cerevisiae ascus from a wild-type yeast strain that
is stained with Calcofluor White.
[0012] FIG. 8 is an overlay showing FT-IR spectra illustrating the
% transmittance (Y-axis labeled "% T") vs. wavenumber (X-axis
labeled "cm.sup.-1") of a product from a vegetative yeast cell of a
Saccharomyces cerevisiae yeast strain, a chitosan standard, and a
chitin standard.
[0013] FIG. 9 is an overlay showing FT-IR spectra illustrating the
% transmittance (Y-axis labeled "% T") vs. wavenumber (X-axis
labeled "cm.sup.-1") of a product from an ascospore of a
Saccharomyces cerevisiae yeast strain and a chitosan standard.
MODE(S) FOR CARRYING OUT THE INVENTION
[0014] It is believed that examples of the method for making a
chitosan product disclosed herein produce chitosan from a
non-animal source in fewer steps, which may produce chitosan using
less time and labor, and fewer resources when compared to other
known methods, thus providing an important advancement to the state
of the art of chitosan production. With many methods, it may be
difficult to obtain a purified product of chitosan without
chemically converting chitin to chitosan. For example, some methods
used to produce chitosan from a non-animal source may require a
deacetylation step to convert chitin to chitosan by removing acetyl
groups on chitin to achieve a purified chitosan product.
[0015] Generally, while some fungi may contain chitosan in the
vegetative cell walls, yeast only contain chitin in the vegetative
cell walls. As such, as previously mentioned, when producing
chitosan from vegetative cell walls of yeast, chitosan cannot be
produced without deacetylation of chitin to form chitosan. Without
being bound to any theory, to avoid this step, it is believed that
some methods of non-animal chitosan production focus on obtaining
chitosan from filamentous fungi mycelium that produce chitosan in
the cell walls. However, these methods are limited specifically to
species of fungi that produce chitosan in the cells walls. However,
there is no yeast species that contain chitosan within the cell
walls of vegetative yeast cells.
[0016] Accordingly, examples of the method in the present
disclosure produce chitosan by extracting a chitosan product from
yeast ascospore walls. In characterized yeast strains, yeast
ascospore walls contain chitosan, but no chitin. In an example,
FIG. 1 shows a schematic cross-sectional view of a Saccharomyces
cerevisiae yeast ascospore wall 100. The yeast ascoscpore wall 100
includes an ascospore plasma membrane 102, a mannan layer 104, a
beta-1,3-glucan layer 106, a chitosan protective layer 108, and a
dityrosine layer 110.
[0017] As shown in FIG. 1, the ascospore wall contains chitosan as
a structural polysaccharide layer. As such, examples of the method
disclosed herein avoid additional processing steps associated with
the deacetylation of chitin by extracting chitosan directly from
the ascospore wall. Without being bound to any theory, it is
believed that since examples of the present disclosure herein do
not require a deacetylation step, the method of making chitosan
from yeast ascospores may require fewer steps and less resources
and labor when compared to methods that require the deacetylation
step. Additionally, since examples of the method herein use yeast
that are generally recognized as safe (GRAS), the chitosan product
produced from the method herein may be used in therapeutic or food
applications.
[0018] Furthermore, examples of the method in the present
disclosure use yeast species that have desirable characteristics
for chitosan production. In examples of the method for making a
chitosan product, it has advantageously been found that by
culturing specific yeast so that yeast cells form a biomass,
inducing the yeast cells to undergo meiosis, thereby forming at
least one ascospore in each ascus that contains a chitosan
protective layer enclosed in an ascospore wall, chitosan may be
extracted from the ascospore walls and purified to form a chitosan
product.
[0019] In an example, a method 200 for making a chitosan product
will be described herein with reference to FIG. 2. In this example,
the first step 202 includes culturing yeast cells to form a biomass
of yeast cells. The culturing of the yeast cells may be performed
by plating yeast cells onto any medium that supports yeast cell
growth. For example, the medium may be a Yeast Peptone Dextrose
(YPD) medium. In another example, the culturing may be performed by
inoculating a liquid medium that supports yeast growth with yeast
cells. In a further example, the culturing may be performed using a
cell culture vessel, a cell retention system, and a media exchange
system. The medium may be added to the culture vessel through the
cell retention system and replenished using the media exchange
system. The yeast may be cultured at a temperature ranging from
about 15.degree. C. to about 50.degree. C. Additionally, the yeast
may be cultured for a time ranging from about 12 hours to about 120
hours with or without feeding.
[0020] Further, in this example, the yeast cells may be a yeast
strain that includes any yeast species that produce ascospores
containing chitosan and are listed as GRAS by the Food and Drug
Administration (FDA). Some examples may include any yeast strain
that is a yeast species within the Ascomycota phylum. In additional
examples, the yeast strain may be a yeast species within the
Saccharomycotina subphyla. Still further, in another example, the
yeast strain may be selected from a yeast species within the class
Saccharomycetes.
[0021] More particularly, in another example, the yeast strain may
be a yeast species within the Genus Saccharomyces, Genus
Komagataella, and Genus Schizosaccharomyces. Some examples of
suitable yeast species that may be used as the yeast strain
include, but are not limited to, Ogataea methanolica, Ogataea
angusta, Saccharomyces cerevisiae, Komagataella pastoris,
Komagataella phaffii, Schizosaccharomyces pombe, and
Schizosaccharomyces japonicus.
[0022] In addition to the above examples, any of the yeast strains
previously disclosed herein may be a homothallic self-fertile yeast
strain, a heterothallic heterozygous for mating type diploid yeast
strain, a heterothallic heterozygous for mating type polyploid
yeast strain that are capable of producing ascospores. For example,
a heterothallic haploid yeast strain, a heterothallic diploid yeast
strain, or a heterothallic polyploid yeast strain may be used. For
any heterothallic yeast strain, the sporulating yeast strain may be
obtained after fusion of yeast cells with two separate strains or
after mating with two separate strains having opposite mating types
resulting in the formation of heterozygous for mating type hybrid
cells. It is to be understood that any suitable method may be used
that fuse or cross any of the strains described herein, such as
conventional mating, protoplast fusion, or illegal mating. In an
example, the heterothallic yeast strains of opposite mating types
may mate with each other to form heterozygous for mating type
hybrid cells. In an example, cells of heterothallic haploid yeast
strains or the heterothallic homozygous for mating type diploid or
polyploid yeast strains with the opposite mating types can mate to
each other and form diploid hybrid cells or polyploid heterozygous
for mating type hybrid cells. The homothallic self-fertile yeast
strain, the heterothallic heterozygous for mating type diploid
yeast strain or the heterothallic heterozygous for mating type
polyploid yeast strain may be cultured as previously described
herein.
[0023] In another example, in particular, "a" and "alpha" mating
type cells of yeast Saccharomyces cerevisiae, can be crossed to
each other to form heterozygous for mating type diploid cells,
which can be used to produce a biomass of yeast cells. The
heterozygous for mating type diploid cells can then be cultured as
previously described herein.
[0024] In another example, the heterothallic yeast strains may be
homozygous for mating type diploid strains. It is to be understood
that heterothallic yeast strains of any ploidy with the opposite
mating types can mate with each other and form diploid or polyploid
hybrid cells that can be cultured to produce a biomass of
heterozygous for mating type yeast cells that can be induced to
undergo meiosis and form ascospores as previously described
herein.
[0025] In another example, the yeast strain is a homothallic
self-fertile yeast strain of any yeast strain previously mentioned
herein. In one example, a homothallic yeast strain of Saccharomyces
cerevisiae may be incubated in a sporulation medium to induce
meiosis and form asci with ascospores. Homothallic self-fertile
yeast strains may be cultured as previously described herein.
[0026] In another example, the yeast species may be any species
previously mentioned herein as a wild-type (WT) yeast strain. In
some examples, a wild-type yeast strain can undergo genetic
modifications, which prevent formation of non-chitosan layers of
the ascospore wall. It is to be understood that a genetic
modification may be a mutation, disruption, or deletion of a target
gene leading to alteration of biochemical pathways involved in
biosynthesis of the outer protective layer in the ascospore wall.
The genetic modification may also lead to a change in the
biosynthesis or assembly of ascospore wall components, which
prevents formation of non-chitosan layers of the ascospore
wall.
[0027] More particularly, in an example, any yeast species within
the Genus Saccharomyces may be genetically modified to prevent
formation of the outer dityrosine layer of the ascospore wall. For
example, the genes DIT1 and DIT2 of the genus Saccharomyces are
responsible for biosynthesis of factors controlling the assembly of
the dityrosine layer 110 of the ascospore wall in the schematic
example shown in FIG. 1. Therefore, the heterozygous for mating
type diploid and polyploid Saccharomyces strains containing
inactive or mutated DIT1, DIT2 genes, or a combination thereof,
form ascospores without the outer dityrosine layer, which makes the
chitosan protective layer 108 shown in the schematic example of
FIG. 1 as the outermost layer. Inactivation and mutation of the
Saccharomyces DIT1 gene, DIT2 gene, or both DIT1 and DIT2 genes can
be results of point mutations, disruptions or deletions of the
target gene or genes. As such, in examples where a Saccharomyces
strain heterozygous for mating type genes having mutated or
inactivated DIT1 or DIT2, or both DIT1 and DIT2 genes is used, the
chitosan product may be produced more efficiently using less
resources.
[0028] Referring now to FIG. 2, the second step 204 includes
inducing the yeast cells to undergo meiosis, which causes the yeast
cells to form ascospores where each ascospore contains a chitosan
protective layer in the ascospore wall. In one example, the yeast
cells may be induced into meiosis by incubating in a liquid or on
the surface of a solid sporulation medium to induce meiosis and
sporulation. In an example, the sporulation medium may be any
liquid or solid medium that provides nitrogen starvation. An
example of a sporulation medium includes a potassium acetate
containing medium.
[0029] After transferring the yeast cells to a sporulation medium,
the yeast cells may be incubated in or on the sporulation medium
for a time ranging from about 12 hours to about 144 hours. The
yeast cells may be incubated until about 10% to about 100% of the
yeast cells are converted to asci containing at least one
ascospore. The yeast cells are incubated on the sporulation medium
at a temperature ranging from about 15.degree. C. to about
50.degree. C. FIG. 3 shows a light microscopy image of an example
of asci that are produced after Saccharomyces cerevisiae diploid
cells have been incubated in the sporulation medium for 48 hours at
30.degree. C.
[0030] In another example, the biomass of yeast cells is first
transferred to a presporulation medium, incubated in or on the
presporulation medium, transferred to a sporulation medium, and
incubated in or on the sporulation medium to convert the yeast
cells to asci. Examples of presporulation media include any liquid
or solid medium that includes a non-fermentable carbon source that
is capable of being metabolized. For example, a presporulation
medium may include potassium acetate as a non-fermentable carbon
source. The yeast cells may be incubated in or on the
presporulation medium for a time ranging from about 6 hours to
about 72 hours and then transferred to the sporulation medium as
previously described herein for further incubation to induce
meiosis. The yeast cells are incubated in or on the presporulation
medium at a temperature ranging from about 15.degree. C. to about
50.degree. C.
[0031] Referring back to FIG. 2, the next step 206 of method 200
for making a chitosan product includes extracting chitosan from the
ascospore walls. In an example, the asci containing the ascospores
may be resuspended in an acid solution with a pH of less than 7.
The suspension may then be heated to a temperature ranging from
about 60.degree. C. to about 100.degree. C. for a time ranging from
about 1 hour to about 12 hours to extract the acid soluble chitosan
from the ascospore walls into the acid solution. Examples of the
acid solution include any acid solution that will solubilize the
chitosan, such as a solution of acetic acid, hydrochloric acid,
formic acid, and combinations thereof. The acid may be present in
the acid solution in an amount ranging from about 0.1% w/v to about
50% w/v of the total weight of the acid solution. It is to be
understood that the step 206 may be used on the asci after
sporulation, on the disrupted asci debris and ascospore wall
fragments after disrupting the asci and ascospore walls, on
undisrupted ascospores after separating from the disrupted asci, or
on ascospore wall fragments of ascospores that have been disrupted
after being separated from asci debris. After the chitosan is
solubilized, the acid solution is then used for the purification
step 208.
[0032] Referring to FIG. 2, in some examples of the method 200 the
next step 208 for making a chitosan product may include purifying
chitosan to form purified chitosan. In an example of step 208, an
enzyme may be added to the acid solution containing solubilized
chitosan. The enzyme is present in an amount ranging from about 1%
v/v to about 30% v/v. Examples of the enzyme include an enzyme that
may digest any .alpha. (1.fwdarw.4) glucosidic bond linkages
between chitosan and glucan, such as an .alpha.-amylase (e.g.,
TERMAMYL.RTM.). Further examples of the enzyme include an enzyme
that may digest any glucosidic bond linkages between monomers of a
glucan polysaccharide.
[0033] The enzyme may be added to the acid solution containing the
solubilized chitosan and incubated at a temperature ranging from
about 4.degree. C. to about 105.degree. C. for a time ranging from
about 1 hours to about 24 hours to digest glucosidic bond linkages.
The acid solution may have a pH of less than 7 when the enzyme is
added to the acid solution containing solubilized chitosan.
Residual glucan will precipitate from the solution after enzymatic
digestion and may be removed and discarded using solid-liquid
separation techniques (e.g., centrifugation).
[0034] After the residual glucan is removed, in the next step 210,
the purified chitosan may be precipitated and dried to form a
chitosan product. The purified chitosan may be precipitated from
the enzyme containing solution by washing with an alkaline solution
or a polar solution. Examples of the alkaline solution may be any
alkaline solution that can raise the pH and precipitate chitosan
from the solution, such as a solution of sodium hydroxide or
ammonium hydroxide. The base in the alkaline solution may be
present in the alkaline solution in an amount ranging from about 3%
w/v to about 80% w/v of the total weight of the alkaline solution.
The pH of the acid solution may be adjusted to a pH above 7 by
adding the alkaline solution. After adjusting the pH of the
solution, the chitosan precipitate may be formed by incubating the
solution for a time ranging from about 0.25 hours to about 72 hours
at temperature ranging from about 2.degree. C. to about 50.degree.
C. Examples of a polar solution may be an alcohol or acetone that
can change the polarity of the chitosan containing solution and
precipitate chitosan. The alcohol or acetone in the polar solution
may be present in an amount ranging from about 2.5% or 100% w/v of
the total weight of the polar solution. After adjusting the
polarity of the solution, the chitosan precipitate may be formed by
incubating the solution for a time ranging from about 0.25 hours to
about 72 hours at temperature ranging from about 2.degree. C. to
about 100.degree. C.
[0035] After precipitation, the precipitated chitosan may be dried
to form a chitosan product. The precipitated chitosan may be
further purified with one or more solid-liquid separation steps.
The final chitosan product may be dried and then stored.
[0036] In another example, a method 400 for making a chitosan
product will be described herein with reference to FIG. 4. In the
method 400 for making a chitosan product, the step 402 of culturing
yeast cells and step 404 of inducing the yeast cells to undergo
meiosis are the same as previously described herein in reference to
FIG. 2, steps 202 and 204.
[0037] After step 404, in one example, the third step 406 includes
disrupting the asci walls or the asci walls and the ascospore
walls. In this step 406, the asci walls and ascospore walls may be
disrupted sequentially (i.e., disrupting the asci walls and then
the ascospore walls) or simultaneously. It is to be understood that
disrupting includes enzymatic degradation, mechanically breaking
down, or any other suitable means of disrupting the asci walls or
the asci walls and ascospore walls.
[0038] In one example, the asci walls are first disrupted by
incubating the asci in conditions that cause the ascospores to be
released from the asci. In an example of incubating the asci in
conditions that cause the ascospores to be released, the asci may
be added to a buffer solution containing an enzyme to digest the
asci walls. The buffer solution may be any buffer solution that
provides suitable conditions for enzymatic activity.
[0039] After the asci are added to the buffer solution, the asci
are incubated with an enzyme that digests the asci walls to release
the ascospores. For example, any glucoside hydrolases may be used
as the enzyme that is added to the buffer solution, such as
.beta.-glucanase or a combination of .beta.-glucuronidase and
.beta.-glucuronide sulfatase, lyticase, or zymolyase. The asci may
be incubated with the enzyme for a time ranging from about 1 hour
to about 6 hours at a temperature ranging from about 4.degree. to
about 45.degree. C. to release the ascospores from the asci.
[0040] In another example of step 406, disrupting the asci by
incubating the asci in conditions that cause the ascospores to be
released from the asci may be accomplished by autolyzing the asci
to release the ascospore.
[0041] In another example of step 406, the asci walls or both the
asci walls and ascospore walls may be disrupted by mechanically
disrupting the asci walls or the asci walls and the ascospore walls
in a high pressure homogenizer. In this example, to disrupt the
asci walls only, the asci solution from step 404 is added to a high
pressure homogenizer, which has a pressure ranging from about 10
Kpsi to about 30 Kpsi. The asci solution remains in the high
pressure homogenizer for a time ranging from about 10 minutes to
about 20 minutes. To disrupt both the asci walls and the ascospore
walls, the asci solution from step 404 is added to the high
pressure homogenizer at a pressure of greater than or equal to 100
Kpsi for the same time range previously stated herein.
[0042] In another example of step 406, the asci walls or the asci
walls and the ascospore walls may be disrupted by mechanically
disrupting the asci walls or the asci walls and the ascospore
walls. For example, when disrupting the asci walls, the asci may be
added into a suspension containing zirconia beads. The zirconia
beads may be added in an amount ranging from about 33% by volume of
the total volume of the suspension. The zirconia beads may have a
size ranging from about 0.7 mm to about 2.0 mm and a density of
about 5.5 g/cc.
[0043] In another example, when disrupting the asci walls and the
ascospore walls, the asci may be added into a suspension containing
zirconia and silica beads. The zirconia and silica beads may be
added in an amount ranging from about 33% by volume of the total
volume of the suspension. The zirconia and silica beads may have a
size ranging from about 0.1 mm to about 2.3 mm and a density of
about 3.7 g/cc.
[0044] After adding the asci to the suspension with either zirconia
beads or zirconia and silica beads, the suspension may then be
shaken using a vortex mixer or a cell homogenizer. The asci may be
shaken with the suspension at a temperature ranging from about
4.degree. C. to about 42.degree. C. In an example, the suspension
may be shaken for a time ranging from about 5 minutes to about 30
minutes. Any suitable vortex mixer or cell homogenizer may be used
that is capable of shaking the suspension.
[0045] Referring now to FIG. 4, after the asci walls or the asci
walls and ascospore walls have been disrupted, the chitosan may be
extracted from the ascospore walls or the ascospore wall fragments
in step 408 as previously described herein in reference to FIG. 2,
step 206. After extracting the chitosan from the ascospore walls or
ascospore wall fragments in step 408, in some examples of method
400, the chitosan may be purified in step 410 as previously
described herein in reference to FIG. 2, step 208. Then, in step
412, the purified chitosan may be precipitated and dried as
previously described herein in reference to FIG. 2, step 210. The
chitosan product may be dried and stored.
[0046] In a third example, a method 500 for making a chitosan
product will be described herein with reference to FIG. 5. In the
method 500 for making a chitosan product, the step 502 of culturing
yeast and step 504 of inducing the yeast cells to undergo meiosis
are the same as previously described herein in reference to FIG. 2,
steps 202 and 204, respectively. Additionally, in the method 500
for making a chitosan product, the step 506 of disrupting the asci
walls is the same as previously described herein in reference to
FIG. 4, step 406. However, in this method, only the asci walls are
disrupted in step 506. As such, the methods previously described
herein that included disrupting only the asci walls are used in
step 506.
[0047] After disruption of the asci walls, the next step 508
includes separating the ascospores from the debris. It is to be
understood that debris includes any yeast cell material except the
ascospores, such as the soluble cell components, insoluble cell
components, the disrupted ascus and cell wall fragments, the
undisrupted yeast cells, and the undisrupted asci. Some examples of
separating the ascospores from the debris include density gradient
centrifugation or biphasic separation.
[0048] In one example, density gradient centrifugation is used to
separate the ascospores. First, the ascospores with the debris may
be added to a density gradient medium. Any density gradient media
may be used that will separate the ascospores from the debris. Some
examples of density gradient media include polyhydric alcohols,
polysaccharides, colloidal silica, and combinations thereof.
[0049] After the ascospores and debris are added to the density
gradient medium, the density gradient medium may be centrifuged to
separate the ascospores and the debris into separate layers of the
density gradient. In an example, centrifugation may be performed
for a time ranging from about 30 minutes to about 90 minutes with a
speed ranging from about 8,540 RCF to about 19,200 RCF. The
temperature during the centrifugation may range from about
2.degree. C. to about 42.degree. C.
[0050] After centrifugation, at least two or more layers are formed
where the bottom layer contains an ascospore enriched layer with a
high concentration of ascospores. The top layers are a debris
enriched layer, which contain the debris and a low concentration of
ascospores. The top layers of the density gradient may be
partitioned (e.g., decanting) and discarded. The bottom layer
containing the ascospores may be washed with a nonionic surfactant
at least once. Any nonionic surfactant may be used, such as
TRITON-X.RTM.. The remaining bottom layer may undergo at least one
or more solid-liquid separation steps to remove more debris. For
example, the bottom layer may be centrifuged for a time ranging
from about 3 minutes to about 7 minutes at a speed ranging from
about 1000 RCF to about 10,000 RCF. The temperature of the
centrifugation may range from about 0.degree. C. to about
42.degree. C. The centrifugation may be repeated one or more times.
The remaining bottom layer may form an ascospore pellet that is
used in the following extraction step 510.
[0051] In another example, a biphasic separation may be used to
separate the ascospores from the debris. For biphasic separation,
first, the ascospores and the debris may be suspended in an aqueous
medium or buffer solution to form a suspension or solution. Some
examples of an aqueous medium include any medium that forms a
suspension or solution, such as water or yeast culture media. Some
examples of buffer solution include any buffer solution that forms
a suspension or solution, such as potassium phosphate. Then, the
suspension or solution may be mixed with a solution of a different
phase for a time ranging from about 1 second to about 1,800
seconds. One example of a solution of different phase than the
aqueous medium or buffer solution is a lipophilic solution.
[0052] After mixing the solution or suspension with a solution of a
different phase, the mixture may be allowed to separate into two or
more layers in a separatory funnel or similar apparatus. The
ascospore containing chitosan layer can be collected from the
separatory funnel or similar apparatus and saved for extraction as
described in step 510. The suspension may be centrifuged in a
separatory funnel or similar apparatus or subjected to gravity
settling to separate the ascospores from the debris to form an
ascospore pellet. The ascospore pellet may be used in the
subsequent step 510.
[0053] In yet another example of biphasic separation, the
ascospores are separated from the debris using an aqueous two phase
extraction. In this example, the ascospores and the debris are
added to an intermediate molecular weight polyether in an aqueous
salt solution to form a suspension.
[0054] After forming the suspension, the ascospores are within the
polyether phase, which can be partitioned to form a solution. The
debris are in the aqueous salt solution, which can be partitioned
and discarded. The remaining solution containing the ascospores may
undergo at least one solid-liquid extraction step to form an
ascospore pellet. The ascospore pellet may be used in the
subsequent extraction step 510.
[0055] Referring back to FIG. 5, after the ascospores have been
separated from the debris, chitosan may be extracted from the
ascospore pellet in step 510. Step 510 may be performed as
previously described herein in reference to FIG. 2, step 206. After
extracting the chitosan from the ascospore pellet in step 510, in
some examples of method 500, the chitosan may be purified in step
512 as previously described herein in reference to FIG. 2, step
208. Then, in step 514, the purified chitosan may be precipitated
and dried as previously described herein in reference to FIG. 2,
step 210.The chitosan product may be dried and then stored.
[0056] In a fourth example, a method 600 for making a chitosan
product will be described herein with reference to FIG. 6. In the
method 600 for making a chitosan product, the step 602 of culturing
yeast and step 604 of inducing the yeast cells to undergo meiosis
are the same as previously described herein in reference to FIG. 2,
steps 202 and 204, respectively. Additionally, in the method 600
for making a chitosan product, the step 606 of disrupting the asci
walls is the same as previously described herein in reference to
FIG. 4, step 406. However, in this method 600, only the asci walls
are disrupted in step 606. As such, the methods previously
described herein that included disrupting only the asci walls are
used in step 606.
[0057] Next, in the method 600 of making a chitosan product, in
step 608 of separating the ascospores from the debris is the same
as previously described herein in reference to FIG. 5, step 508.
After step 608, step 610 of method 600 occurs where the ascospore
walls are disrupted as previously described herein in reference to
FIG. 4, step 406 using any method described to disrupt both asci
and ascospore walls. Chitosan may then be extracted from the
ascospore wall fragments in step 612. Step 612 may be performed as
previously described herein in reference to FIG. 2, step 206. After
extracting the chitosan from the ascospore pellet in step 612, in
some examples of method 600, the chitosan may be purified in step
614 as previously described herein in reference to FIG. 2, step
208. Then, in step 616, the purified chitosan may be precipitated
and dried as previously described herein in reference to FIG. 2,
step 210.The chitosan product may be dried and stored.
[0058] To further illustrate the present disclosure, examples are
given herein. It is to be understood that these examples are
provided for illustrative purposes and are not to be construed as
limiting the scope of the present disclosure.
EXAMPLES
[0059] Strain with Inactive DIT1
[0060] A staining procedure was used, which included Calcofluor
White, to validate that the DIT1 gene was deleted in the
Saccharomyces cerevisiae dit1 mutant strain. Samples were stained
with Calcofluor White (1 .mu.L in 500 .mu.L McIlvaine's buffer) and
mounted on glass slides. Samples were imaged on the same day by
confocal microscopy using a DAPI (4',6-Diamidino-2-Phenylindole,
Dihydrochloride) wavelength (358 nm and 461 nm), which excite the
Calcofluor White.
[0061] The staining method allowed identification of the yeast
ascospores with Calcofluor White. Calcofluor White can stain chitin
and chitosan only if the DIT1 gene is inactive, thereby allowing
the dye to stain the inner parts of the ascospore wall. If the
ascospores walls are stained by Calcofluor White, then the DIT1
gene was not active. FIG. 7A shows that the dit1 mutant strain does
have an inactive DIT1 gene since the ascospore walls are stained.
In comparison, a Saccharomyces cerevisiae wild-type strain that has
a functional DIT1 gene only has the asci wall stained, as shown in
FIG. 7B.
Ascospore Generation
[0062] A single colony of a Saccharomyces cerevisiae dit1 mutant
strain from a working cell plate (WCP) was suspended in 5 mL of
liquid YPD and incubated overnight at 30.degree. C. with shaking at
250 RPM. The entire working volume culture was inoculated into 200
mL liquid YPD in 1-L baffled flask and incubated at 30.degree. C.
with shaking at 250 RPM. After 24 hours of growth, the yeast cells
were spun down via centrifugation at 6730 RCF at 4.degree. C. in a
sterile 250 mL Nalgene centrifuge bottle. The medium was discarded
and the yeast cells were suspended in a 200 mL pre-sporulation
medium within a 1-L baffled flask. The culture was incubated for
another 24 hours at 30.degree. C. with shaking at 250 RPM.
[0063] After another 24 hours of incubation, the yeast cells were
spun down via centrifugation at 5640 RCF at 4.degree. C. in a
sterile 250 mL Nalgene centrifuge bottle. A pellet of yeast cells
formed from centrifugation were then transferred to a sporulation
medium. Finally, the culture was incubated in a sporulation medium
for 72 hours at 250 RPM and 30.degree. C. and then harvested. To
harvest the asci, the asci were first centrifuged at 5640 RCF at
4.degree. C. in a sterile 250 mL Nalgene centrifuge bottle. About 5
grams of wet biomass containing ascospores was harvested from the
200 mL culture. The wet biomass was washed once with MILLI-Q.RTM.
water and then centrifuged at 2,130 RCF at 4.degree. C. for 5
minutes and the wash supernatant was decanted.
Ascus Disruption and Ascospore Isolation Example 1
[0064] Asci pellets were suspended in lysis buffer containing 40 mM
Beta-mercaptoethanol (BME), 1.4M sorbitol, 50 mM potassium and
sodium phosphate (KPhos) at a pH 7.5 with lyticase containing 25
.mu.L aliquots of 2,000 U/mg lyticase. 5 mL of buffer and 2
aliquots of lyticase were added for each gram of biomass into a 50
mL conical Falcon tube. The suspension was incubated for 3 hours at
30.degree. C. and mixed using a vortex mixer at 250 RPM and then
vigorously vortexed with 0.45 .mu.M zirconia glass beads for 10
minutes where the glass beads to culture ratio was 1:1. The ascus
disruption was validated by mounting samples stained with 1%
methylene blue on a hemocytometer and imaging by light
microscopy.
[0065] Next, after being mixed with glass beads and suspended in
lyticase buffer, the pellet of ascospores and the debris was washed
three times with 0.5% TRITON-X.RTM. at 5 mL per gram of pellet. The
pellet was then purified via Percoll centrifugation gradients.
Percoll gradient was prepared by adding 9 mL of Percoll/0.5%
TRITON-X.RTM./2.5 M sucrose solutions in following proportions: 1)
8:1:1; 2) 7:2:1; 3) 6:3:1; 4) 5:4:1 into four 40 mL Sorvall high
speed centrifuge tubes, respectively. Approximately 1.5 mL of
pellet was added to the gradient and centrifuged at 10,000 RPM for
1 hour at 4.degree. C. A majority of the ascospore fraction was
concentrated in the bottom of the gradient, whereas less dense
cells and other debris remained in three upper layers of the
gradient. After centrifugation, the top 27 mL of the gradient were
removed and the bottom 9 mL layer was washed three times with 30 mL
of 0.5% TRITON-X.RTM. and centrifuged at 2,130 RCF and 4.degree. C.
for 5 minutes to obtain an ascospore pellet separated from the
asci.
Ascus Disruption Example 2
[0066] In another example, vigorous glass-bead mixing of a
Saccharomyces cerevisiae dit1 mutant yeast strain culture, which
included asci containing ascospores, led to disruption of asci
walls. 300 .mu.L of 0.45 .mu.m zirconia beads were added to 200
.mu.L of dense water suspensions of sporulation culture in 2 mL
round-bottom plastic tubes and mixed using a vortex mixer for 10
minutes. The suspension was then centrifuged at 13,000 rpm for 12
minutes. A pellet formed in the bottom layer of the suspension.
[0067] The pellet was isolated and stained with Calcofluor White,
which selectively dyes chitin and chitosan to determine if the
pellet contained chitin and chitosan. The results showed the
presence of chitin and chitosan, indicating that the mixture
contained asci walls (with chitin) and ascospore walls (with
chitosan). This data demonstrates that mechanical digestion breaks
down the asci and ascospore walls in a single mixing step, thereby
allowing chitosan to be extracted in the following step.
Chitosan Extraction
[0068] A sample of ascospores was obtained. The sample included
approximately 0.2 grams of wet ascospore weight from the bottom
Percoll gradient layer. The sample was suspended in 30 mL of 2%
acetic acid. The chitosan was extracted from the ascospores by
heating the mixture to 95.degree. C. for 5 hours in a round bottom
flask with a condenser.
[0069] The mixture was cooled to room temperature and centrifuged
at 13,300 RCF and 4.degree. C. for 10 minutes in a 50 mL Falcon
conical tube to separate the ascospore debris. The supernatant
containing the chitosan product in a solution was collected into a
50 mL Falcon conical tube. The pH of the collected solution was
raised to a pH 12 using a Thermo Fisher pH probe to track the pH
change using about 4 mL to 5 mL of 2.0M NaOH. The solution was
incubated at 4.degree. C. for about 48 hours to about 72 hours
until a chitosan product formed as a white precipitate in the
solution.
[0070] The white precipitate was centrifuged at 13,300 RCF and
4.degree. C. for 10 minutes in a 50 mL Falcon conical tube. The
supernatant was then decanted, and the white precipitate was
transferred into 2 mL Eppendorf tubes and then washed three times
with deionized water. After each wash step, a subsequent
centrifugation at 2,130 RCF and 4.degree. C. for 5 minutes was
performed. The final chitosan containing pellet was stored in the
freezer at -20.degree. C.
Spectroscopy Results
[0071] Fourier-transform infrared (FT-IR) spectroscopy was used to
analyze the final chitosan product. In order to analyze the final
product chitosan, two different samples were analyzed using FT-IR
spectroscopy. The first sample was a vegetative cell product from a
Saccharomyces cerevisiae strain. The second sample was an ascospore
layer product from Saccharomyces cerevisiae strain.
[0072] Both samples were analyzed and compared to the analytical
standard of chitin and chitosan. It is believed that if chitin is
present in a sample, there is a larger peak between 1800 cm.sup.-1
to 1600 cm.sup.-1 representing the presence of an acetyl group.
However, if chitosan is present, there is no peak or a very small
peak between 1800 cm.sup.-1 to 1600 cm.sup.-1. Both samples were
analyzed using FT-IR spectroscopy and presented as an overlay
showing FT-IR spectra with the % transmittance (Y-axis labeled "%
T") vs. wavenumber (X-axis labeled "cm.sup.-1").
[0073] The first sample was a vegetative cell product from a
Saccharomyces cerevisiae strain that was analyzed and compared to
the analytical standard of chitin and chitosan. As shown in FIG. 8,
chitin and the vegetative cell layer both contain sharp peaks in
the 1620 cm.sup.-1 region. This supports the fact that the
vegetative cell layer contains chitin in the cell wall rather than
chitosan. In comparison, the chitosan standard spectrum does not
exhibit a peak in the 1620 cm.sup.-1 region, which is expected
since it is the deacetylated form of chitin.
[0074] The second sample was an ascospore product from a
Saccharomyces cerevisiae strain. As shown in FIG. 9, the ascospore
product and the chitosan standard did not have any peaks in the
1620 cm.sup.-1 region. Therefore, this data demonstrates that
chitosan was present in the ascospore product, but not chitin.
[0075] As used herein, the term "about" is used to provide
flexibility to a numerical range endpoint by providing that a given
value may be "a little above" or "a little below" the endpoint. The
degree of flexibility of this term can be dictated by the
particular variable and would be within the knowledge of those
skilled in the art to determine based on experience and the
associated description herein.
[0076] As used herein, a plurality of items, structural elements,
compositional elements, and/or materials may be presented in a
common list for convenience. However, these lists should be
construed as though each member of the list is individually
identified as a separate and unique member. Thus, no individual
member of such list should be construed as a de facto equivalent of
any other member of the same list solely based on their
presentation in a common group without indications to the
contrary.
[0077] Unless otherwise stated, any feature described herein can be
combined with any aspect or any other feature described herein.
[0078] Reference throughout the specification to "one example",
"another example", "an example", and so forth, means that a
particular element (e.g., feature, structure, and/or
characteristic) described in connection with the example is
included in at least one example described herein, and may or may
not be present in other examples. In addition, it is to be
understood that the described elements for any example may be
combined in any suitable manner in the various examples unless the
context clearly dictates otherwise.
[0079] It is to be understood that the ranges provided herein
include the stated range and any value or sub-range within the
stated range. For example, a range from about 0.1 w/v to about 20
w/v should be interpreted to include not only the explicitly
recited limits of from about 10 w/v to about 15 w/v, but also to
include individual values, such as 3 w/v, 7 w/v, 13.5 w/v, etc.,
and sub-ranges, such as from about 5 w/v to about 15 w/v, etc.
[0080] In describing and claiming the examples disclosed herein,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise.
* * * * *