Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS FOR THE PRODUCTION OF ISOMALTOOLIGOSACCHARIDES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application claims the benefit of U.S. Provisional Application
Serial No.
62/0421,006, filed August 22, 2014, and of U.S. Provisional Application Serial
No.
62/151,404, filed April 22, 2015, the contents of which are incorporated
herein in their
entirety.
BACKGROUND OF THE INVENTION
[0002] Dietary supplements, sometimes also called nutraceuticals, are either
food or food
constituents that purportedly provide medical or health benefits which can
include prevention
of disease (Stephen, D.F.L., Trends in Food ScL Tech, 1995, 6:59-61). The term
typically
includes the following representative classes: probiotics, prebiotics, dietary
fiber, omega-3
fatty acids and antioxidants (Pandey, M. et al., Asian J. Pharm. Clin. Res.,
2010, 3:11-15).
Due to increasing numbers of health conscious consumers in Asia, the United
States and
Europe, the dietary supplement market, specifically in the areas of
oligosaccharides and
prebiotics, has demonstrated significant growth over the last three decades
(Goffin, D. et al.,
Crit. Rev. Food. ScL Nutr., 2011, 51:394-409; Roberfroid, M.B., Br. J. Nutr.,
2002, 88 Suppl
2:S133-8) and new, economical methods for their production are in demand.
[0003]0f interest here are the pre- and probiotic classes of dietary
supplements. Broadly
defined, probiotics are made up of living cultures of bacteria, such as those
in yogurt, that
promote the growth of healthy gut flora by means of population support
(Gilliland, S.E. et al.,
"Health and Nutritional Properties of Probiotics in Food including Powder Milk
with Live
Lactic Acid Bacteria", 2001, p. 1-34, World Health Organization). Prebiotics,
however, are
materials, either physical (e.g. dietary fiber) or chemical (e.g. butyrate)
which can promote
the growth of selected beneficial flora (Chung, C.H., et al., Poult. ScL,
2004, 83:1302-6)
and/or exert some beneficial effect directly on intestinal epithelial cells
(thus improving
uptake of nutritive calories, vitamins, minerals, etc.). Because many
prebiotics can
overcome the resistance of the digestive barrier facilitating the
proliferation and/or activity of
desired populations of bacteria in situ (Gibson G. R. et al., J. Nutr., 1995,
125:1401-12; Van
Loo, J. et al., Br. J. Nutr., 1999, 81:121-32), research and development in
this area has
boomed. Additionally, prebiotics are often found naturally in the food supply,
especially
fermented foods and are generally compatible with most food formulations
(Macfarlane, S. et
al., Aliment PharmacoL Ther., 2006, 24:701-14; Manning, T.S. et al., Best
Pract. Res. Clin.
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Gastroenterot, 2004, 18:287-98). By definition, glucooligosaccharides are
prebiotic agents,
and many forms are commercially available.
[0004]Glucooligosaccharides are a class of carbohydrate oligomers that include
isomaltooligosaccharides (IMO). IMOs are glucosyl saccharides with a core
structure based
on an a-(1¨>6) linked backbone that may include a-(1-4), a-(1¨>3)
(nigerooligosaccharides)
and\or a-(1,2) (kojioligosaccharides) linked branches (Yun, J. et at,
Biotechnot Lett., 1994,
16:1145-1150). These glucosidic linkages are found in commercial IMO syrups
(Goffin, D. et
al., Crit. Rev. Food Sci. Nutr., 2011, 51:394-409.)
[0005]Chung and Day have produced glucooligosaccharides, specifically IM0s,
via the
action of dextransucrase generated in situ upon sucrose in the presence of a
maltose
acceptor (U.S. Patent No. 7,291,607). The IMO was an extracellular product of
the
fermentation of sucrose by Leuconostoc mesenteroides ATCC 13146Tm. Chung and
Day
demonstrated that these glucooligosaccharides (branched IM0s) are readily
utilized by
bifidobacterium sp. and lactobacifius sp., but not by Escherichia coli or
Salmonella sp. in
pure-culture studies (Chung, C.H. and Day, D. F., J. Ind. Microbiot
Biotechnot, 2002,
29:196-9).
SUMMARY OF THE INVENTION
[0006] In one aspect, this invention provides a method for the preparation of
oligosaccharides comprising the steps of (a) contacting a feedstock comprising
a fixed ratio
of sucrose to maltose with a dextransucrase-producing microorganism in an
aqueous culture
medium; (b) fermenting the feedstock with the bacteria cells at a pH between 4
and 8; (c)
removing the bacteria cells; and (d) polishing, wherein the final composition
of the
oligosaccharides produced is varied according to the pH selected and the
initial feedstock
ratio employed. In one embodiment, performing steps (a) to (c) is continuous.
In another
embodiment, the method is conducted as an immobilized enzyme or an immobilized
cell
process. In a further embodiment, the method is conducted as a batch operation
or as a
fed-batch operation.
[0007] In another embodiment, the fixed ratio of sucrose to maltose ranges
between the
ratios 1.5:1 to 7:1. In one embodiment, the fixed ratio is maintained at 2:1
or at 2.33:1 or at
2.75:1. In a further embodiment, the fixed ratio of sucrose to maltose is
adjusted during the
fermentation process by the addition of either more sucrose or more maltose.
[0008] In another embodiment, the dextransucrase-producing microorganism is
Leuconostoc mesenteroides [in particular Leuconostoc mesenteroides ATCC 13146
or
Leuconostoc mesenteroides NRRL B-742 or Leuconostoc mesenteroides subsp.
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mesenteroides (Tsenkovskii) van Tieghem (ATCC 11449T), or NRRL B-1299],
Leuconostoc citreum, Leuconostoc gasicomitatum, or Leuconostoc kimchfi. In a
further
embodiment, the dextransucrase-producing microorganism is WeiseIla confusa,
Weissella
cibaria, Lactococcus spp Penicifiium aculeatum, Pediococcus spp (pentosaceus),
Streptococcus mutans, Streptococcus oralis, Streptococcus sanguinis, or
Lactobacilfis spp
(reuteri).
[0009] In another embodiment, the pH is controlled by adding an acid or a base
to the
culture medium. In a further embodiment, the base comprises an alkali earth
metal
hydroxide or carbonate. In one embodiment, the alkali earth metal is calcium.
In another
embodiment, the base comprises sodium hydroxide.
[0010] In one embodiment, the bacteria cells are removed by centrifugation,
filtration or
clarification. In another embodiment, the polishing removes insoluble
impurities. In a further
embodiment, the polishing comprises decolorization. In one embodiment, the
decolorization
utilizes activated charcoal or activated carbon. In a further embodiment, the
decolorization
comprises using a weak base anion resin. In yet another embodiment, the
polishing
comprises de-ashing. In one embodiment, the de-ashing comprises using a strong
acid
cation resin to remove metal ions. In another embodiment, the de-ashing
comprises a two-
step process using a strong acid followed by a weak base. In a further
embodiment, the
polishing comprises removing protein. In another embodiment, the removing
protein
comprises heating, then evaporating the aqueous culture medium followed by
centrifugation
or filtration. In one embodiment, the removing protein comprises using a weak
base anion
resin. In another embodiment, the polishing comprises removing organic acids.
In one
embodiment, the removing organic acids comprises utilizing a weak base anion
resin. In
another embodiment, the removing organic acids comprises liquid chromatography
using a
chromatographic grade gel-type strong acid cation exchange resin in calcium
form (SAC-
Ca++).
[0011] In another embodiment, the polishing comprises removing mannitol. In
another
embodiment, the removing the mannitol utilizes continuous or pulsed liquid
chromatography.
In a further embodiment the removing the mannitol utilizes evaporation
followed by one or
two stages of cooling to initiate crystallization and precipitation.
[0012] In another embodiment, the final composition of the oligosaccharides
produced
comprises isomaltooligosaccharides with one or more a-(1-4) at the reducing
end and a-
(1¨>6) linkages with a degree of polymerization between 3 and 9 or between 3
and 10. In a
further embodiment, the isomaltooligosaccharides further comprise a-(1¨>4), a-
(1¨>3) and/or
a-(1¨>2) branching.
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[0013] Another embodiment further comprises providing the oligosaccharides as
a
concentrated solution, optionally prepared under suitable conditions for human
consumption.
A separate embodiment further comprises providing the oligosaccharides as a
powder,
optionally prepared under conditions suitable for human consumption. In one
embodiment,
the powder is produced by drying, spray drying or by freeze drying.
[0014] In another aspect of the invention, a composition is produced by the
method
described above. Optionally, the composition is suitable for human
consumption.
[0015] Another aspect of the invention provides for the preparation of
oligosaccharides
comprising the steps of contacting a feedstock comprising a fixed ratio of
sucrose to maltose
with a dextransucrase-producing microorganism in a culture medium; (b)
fermenting the
feedstock with the bacteria cells at a pH between 4 and 8; (c) removing the
bacteria cells;
and (d) polishing; wherein the final oligosaccharides produced are essentially
free of ash,
mineral acids, residual proteins, sugar alcohols, and organic acids. In one
embodiment,
performing steps (a) to (c) is continuous. In another embodiment, the method
is conducted
as an immobilized enzyme or an immobilized cell process. In a further
embodiment, the
method is conducted as a batch operation or as a fed-batch operation.
[0016] In another embodiment, the fixed ratio of sucrose to maltose ranges
between the
ratios 1.5:1 to 7:1. In one embodiment, the fixed ratio is maintained at 2:1
or at 2.33:1 or at
2.75:1. In a further embodiment, the fixed ratio of sucrose to maltose is
adjusted during the
fermentation process by the addition of either more sucrose or more maltose.
[0017] In another embodiment, the dextransucrase-producing microorganism is
Leuconostoc mesenteroides [in particular Leuconostoc mesenteroides ATCC 13146
or
Leuconostoc mesenteroides NRRL B-742 or Leuconostoc mesenteroides subsp.
mesenteroides (Tsenkovskii) van Tieghem (ATCC 11449Tm), or NRRL B-12991,
Leuconostoc citreum, Leuconostoc gasicomitatum, or Leuconostoc kimchii. In a
further
embodiment, the dextransucrase-producing microorganism is WeiseIla confusa,
Weissella
cibaria, Lactococcus spp, Penicillium aculeatum, Pediococcus spp
(pentosaceus),
Streptococcus mutans, Streptococcus oralis, Streptococcus sanguinis, or
Lactobacillis spp
(reuteri).
[0018] In another embodiment, the pH is controlled by adding an acid or a base
to the
culture medium. In a further embodiment, the base comprises an alkali earth
metal
hydroxide or carbonate. In one embodiment, the alkali earth metal is calcium.
In another
embodiment, the base comprises sodium hydroxide.
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[0019] In one embodiment, the bacteria cells are removed by centrifugation,
filtration or
clarification. In another embodiment, the polishing removes insoluble
impurities. In a further
embodiment, the polishing comprises decolorization. In one embodiment, the
decolorization
utilizes activated charcoal or activated carbon. In a further embodiment, the
decolorization
comprises using a weak base anion resin. In yet another embodiment, the
polishing
comprises de-ashing. In one embodiment, the de-ashing comprises using a strong
acid
cation resin to remove metal ions. In another embodiment, the de-ashing
comprises a two-
step process using a strong acid followed by a weak base. In a further
embodiment, the
polishing comprises removing protein. In another embodiment, the removing
protein
comprises heating, then evaporating the aqueous culture medium followed by
centrifugation
or filtration. In one embodiment, the removing protein comprises using a weak
base anion
resin. In another embodiment, the polishing comprises removing organic acids.
In one
embodiment, the removing organic acids comprises utilizing a weak base anion
resin. In
another embodiment, the removing organic acids comprises liquid chromatography
using a
chromatographic grade gel-type strong acid cation exchange resin in calcium
form (SAC-
Ca++).
[0020] In another embodiment, the polishing comprises removing mannitol. In
another
embodiment, the removing the mannitol utilizes continuous or pulsed liquid
chromatography.
In a further embodiment the removing the mannitol utilizes evaporation
followed by one or
two stages of cooling to initiate crystallization and precipitation.
[0021]1n another embodiment, the final composition of the oligosaccharides
produced
comprises isomaltooligosaccharides with one or more a-(1-4) at the reducing
end and a-
(1¨>6) linkages with a degree of polymerization between 3 and 9 or between 3
and 10. In a
further embodiment, the isomaltooligosaccharides further comprise a-(1-4), a-
(1¨>3) and/or
a-(1¨>2) branching.
[0022]Another embodiment further comprises providing the oligosaccharides as a
concentrated solution, optionally prepared under suitable conditions for human
consumption.
A separate embodiment further comprises providing the oligosaccharides as a
powder,
optionally prepared under conditions suitable for human consumption. In one
embodiment,
the powder is produced by drying, spray drying or by freeze drying.
In another aspect of the invention, a composition is produced by the method
described
above. Optionally, the composition is prepared under suitable conditions for
human
consumption.
[0023]Other objects of the invention may be apparent to one skilled in the art
upon reading
the following specification and claims.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0024]The novel features of the invention are set forth with particularity in
the appended
claims. A better understanding of the features and advantages of the present
invention will
be obtained by reference to the following detailed description that sets forth
illustrative
embodiments, in which the principles of the invention are utilized, and the
accompanying
drawings of which:
[0025] FIG 1 shows the taxonomy of oligosaccharides.
[0026] FIG 2A depicts a thin-layer chromatograph (TLC) of
glucooligosaccharides of L.
mesenteroides ATCC 13146 fermentations with three pH control methods wherein
Lane 1
shows a 5% NaOH fermentation batch; Lane 2, a 5% lime fermentation batch; and
Lane 3, a
5% lime sucrate fermentation batch. The following abbreviations are shown in
the figure:
Gluc., glucose; Fruc., fructose; Suc., sucrose; IM2, isomaltose; IM3,
isomaltotriose; IM4,
isomaltotetraose; M2, maltose; M3, maltotriose; M4, maltotetraose; M5,
maltopentaose. FIG
2B depicts a typical HPAEC chromatogram, for comparison. The blue line
represents a
typical product, and the red line represents a standard (1,4-DPx = maltosyl
oligosaccharide
or DPx).
[0027] FIG 3 illustrates the production patterns of glucooligosaccharides and
mannitol by L.
mesenteroides ATCC 13146 from sucrose and maltose as a function of time.
Fermentation
of L. mesenteroides ATCC 13146 with (A) 5% NaOH pH control; (B) 5% Lime pH
control;
and (C) 5% Lime sucrate pH control. Fermentation samples (carbohydrates)
harvested
according to the times shown were analyzed by high performance liquid
chromatography
(Agilent 1200 HPLC with a differential refractive index detector at 45 C,
BioRad Aminex
HPX-87K at 85 C eluted with 0.01 M K2SO4 at 0.8 mUmin).
[0028] FIG 4 shows an HPLC analysis where cation resin chromatography (60 mL
loading
with 57 Brix sample) was carried out with 10 mL/min flow rate at 50 C, where
fractions
(each 15 mL) were collected. For carbohydrates (GlcOS (marked as GOS), panose,
maltose, and mannitol), Agilent 1200 HPLC was used with a differential
refractive index
detector at 45 C, BioRad Aminex HPX-87K at 85 C eluted with 0.01 M K250.4at
0.8 mL/min.
For organic acids (acetic acid and lactic acid), Agilent 1100 HPLC was used
with an Aminex
HPX-87H column at 65 C eluted with 1.0 mL/min of 0.005 N H2SO4 with detection
via
absorbance at 210 nm.
[0029] FIG 5 shows a bar graph illustrating product patterns of maltosyl-
isomaltooligo-
saccharides produced at different pHs as determined by HPLC. The bar graph
gives the
relative percentages. Samples were analyzed by high performance liquid
chromatography
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(Agilent 1200 HPLC with a differential refractive index detector at 45 C,
BioRad Aminex
HPX-87K at 85 C eluted with 0.01 M K2SO4 at 0.6 mL/min).
[0030] FIG 6 shows the effect the sucrose:maltose (S/M) ratio has on the
molecular weight
distribution at a fixed pH of 5.5. As S/M increases, so does the molecular
weight distribution.
[0031] FIG 7 shows the effect that pH has on the molecular weight distribution
at fixed
sucrose:maltose (S/M) ratio. As pH decreases toward 5.5, the molecular weight
distribution
increases.
[0032] FIG 8 shows pH values needed for optimization of the yields of each
oligomer.
Maltose and panose should be minimized, and higher oligomers maximized.
[0033] FIG 9 shows a comparison of fermentations using Leuconostoc spp. NRRL
B742,
1299, and kimchi as the dextransucrase-producing microorganism; trace A is
from sample
MIMO 070814 using a species found in sourdough wild starter, which produced a
low
product concentration that appears to be both linear and branched; trace B is
from sample
MIMO 070314 using a mixed species of Leuconostoc derived from kimchi, which
produced a
high linear product concentration; trace C is from sample ISOT 071714 using
PWSA-L.
citreum B742, which produced a high product concentration that appears to be
linear (DP3-
8), with branching at greater DP; trace D is from sample ISOT 070914 using L.
mesenteroides B1299, which produced a medium product concentration with
branching at
DP>4; and trace E is from sample ISOT Classic 2012 using L. citreum B742,
which
produced a high product concentration that appears to be linear (DP3-8) with
branching at
greater DP.
DETAILED DESCRIPTION OF THE INVENTION
[0034] This application is not limited to particular methodologies or the
specific compositions
described, as such may vary. It is also to be understood that the terminology
used herein is
for the purpose of describing particular embodiments only, and is not intended
to be limiting,
since the scope of the present application will be limited only by the
appended claims and
their equivalents.
[0035] Unless defined otherwise, all technical and scientific terms used
herein have the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although any methods and materials similar or equivalent to
those
described herein can be used in the practice or testing of the present
application, the
preferred methods and materials are now described. All publications mentioned
herein are
incorporated herein by reference to disclose and describe the methods and/or
materials in
connection with which the publications are cited.
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Definitions
[0036] As utilized in accordance with the present disclosure, the following
terms, unless
otherwise indicated, shall be understood to have the following meanings:
[0037]"BI-ix", also known as degrees Brix (symbol Bx), refers to the sugar
content of an
aqueous solution. One degree Brix is 1 gram of sucrose in 100 grams of
solution and
represents the strength of the solution as percentage by weight (% w/w). Brix
also accounts
for dissolved salts, organic acids, and other solutes that increase the
refractive index of the
solution. As such, it is less useful as a quantitative measure of saccharide
content in
complex broth (fermentation mixtures), but is quite accurate with respect to
the refined
product. Thus, 1 degree brix = 1 g refractive dry solids per 100g of material.
If the solution
contains dissolved solids other than pure sucrose, then the Bx only
approximates the
dissolved solid content.
[0038] "Degree of polymerization", or "DP", refers to the number of sugar
units in a given
oligosaccharide.
[0039] "Oligosaccharides" refers to glycans of all kinds with DP>2 and <10.
[0040] "Glucoolioosaccharide", or "GlcOS", refers to an oligosaccharide
comprised of
glucose in any structural arrangement. An example of a GlcOS is
maltooligosaccharide [-0-
a-(1,4)-], maltopentaose, which has the following chemical structure:
J4D, HO
( OHa4 54) (a.-1,4)
DP 5 or
cm- (a-1,4)
= -s-s,
[0041] "Isomaltoolioosaccharide", or "IMO", refers to glucosyl saccharides
with a core
structure based on an a-(1¨>6) linked backbone that may include a-(1-4), a-
(1¨>3)
(nigerooligosaccharides) and/or (kojioligosaccharides) linked branches. An
example of an
IMO is a GlcOS assembled with [-O-a-(1,6)-] linkages, isomaltopentaose, which
has the
following chemical structure:
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- NO CP
:ic pii 1,, t
............................... I
HO_
/OH HOibw-c .µ)--.41101-t
HO lOw-t,
õ)-+Ell 0 . C (
DP = \._..0, RA-1,6) ---- F
H= .............
0
o. 4, tm.s. ,k.) .4,400F;
H 8 .= (i. =,,,4 it)
\ ____________________________________
HD 'OH
[0042] "Maltosyksomaltooligosaccharides," or MIM0s, refers to an
oligosaccharide,
specifically glucan, of less than 10 degrees of polymerization comprised of a-
(1¨>6) linkages
terminated by an a-(1¨>4) linkage. The a-(1¨>4) terminal group is maltose,
therefore
maltosyl-isomaltooligosaccharide or MIMO is produced by an acceptor reaction
by maltose
or other maltooligosaccharide. An example of an MIMO with a single maltosyl
linkage [-O-a-
(1,4)-] linkage at the reducing end is maltosyl-isomaltotriose, which has the
following
chemical structure:
Rd.
Rd 44.1 i =-
lli.____
I' ¨. / ¨
iltili
Ai---1. A . ( :\-mr
, OH
' ' -Si
DP õ:õ, 5 No....-c ),...,:,`.si,
,
................... d
-c = . .
, .....4,,,
______________________________________________ õ). N
[0043] "Branched MIMO" refers to an oligosaccharide, specifically glucan, of
less than 10
degrees of polymerization comprised of a-(1¨>6) linkages terminated by an a-(1
¨>4) linkage
and a-(1,2), a-(1¨>3) and/or a-(1,4) branches. Examples of a branched MIMO
with
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glucose branching linkages at positions 1,2 and 1,3 and/or 1,4 have the
following structures:
¨ OR
,..3
7.---'\ (0'..-/$4)
\-..ovaaõ, OH
\ 'I ';',.. SP
; ________________________________ am 0 7----\,.....i
l Ori
\ ________________________________________ /
/ %11
\
H 0
(
=.., a\ i
\ ________________________
ti0; O _______ = 0,,
\
isfOliew-1 )000/
\\,.._0 01÷L i Hoe ai 7
\
\ON i (,
\\OH OH
MIM.0^1-0-a-(1,2)]
,----0H
----;\
HO K ii ....001Q'...
\ ' __ *'.e'.. . . 'ail $4-
;'=
\
'CH
- \
OA 4) C
HIO, DH 0 __
/:,...,
.,ti,
SNP.- \___
/
, -0
.. i
...i
,
\H P----,
ik),,,..-== \)--",
i_.....,"
HO '.........
laH
lie
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.i.,`
.
Mit..S ii
., i---="*ail '-',.;_;., ;1.1
1.-- 1 'µõ__..
HO 161 ,-......OH
\ /\ ..,
(a.4,6) 9
. ,3
s
¨01Ft..-N-1
\----(µ
;:-
\ /\----'k
o.t Killiimo.õ( \
MEN104-041-(1,4)1
[0044]"Dietary supplement" refers to a food, food ingredient or food additive
that produces a
health benefit, including carbohydrates such as oligosaccharides.
[0045] "SAC" means a Strong Acid Cation exchange resin, typically one with
sulfonic acid
groups, i.e., a sulfuric acid equivalent.
[0046] "SBA" means a Strong Base Anion exchange resin, typically one with free
amine
groups, that can be made equivalent to hydroxyl groups.
[0047] "WAC" means a Weak Acid Cation exchange resin, typically one with free
carboxylic
acid groups (pKa -4.2).
[0048] "WBA" means a Weak Base Anion exchange resin, typically one with
tertiary amine
groups which are not stronger than the corresponding free base (pKa -9.8).
[0049] Reference will now be made in detail to certain preferred methods of
treatment,
compounds and methods of administering these compounds. The invention is not
limited to
those preferred compounds and methods but rather is defined by the claim(s)
issuing
herefrom.
Overview of the Invention
[0050]The glucooligosaccharides of the present disclosure are produced by the
growth of L.
mesenteroides (ATCC 13146) or any other dextransucrase-producing microorganism
that
produces the enzyme dextransucrase on sucrose in the presence of maltose or
other
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receptor. The pH is adjusted prior to inoculation to 6.5-6.8 depending to the
optimal pH to
achieve maximum cell growth at the start of fermentation. It is also possible
to adjust the
starting pH to any level in the range of 4 to 8. The pH of the fermentation
naturally drops
from its starting pH as the organism grows in culture in the presence of an
appropriate
feedstock. The optimum pH for cell growth is 6.5 to 6.8. Optimum enzyme
production and
activity is in the range of 5.5 to 6Ø When pH 5.5 is reached, pH control is
maintained by the
addition of an alkaline material such as sodium hydroxide. The organism ATCC
13146
performs at an optimum pH of 5.5. In a preferred embodiment of the process of
the
invention, the pH of the fermentation broth is adjusted to pH 6.5 with 50%
aqueous sodium
hydroxide and is maintained around pH 5.5 during the desired length of the
fermentation
step.
[0051] Other dextransucrase-producing enzymes may prefer a different pH for
optimum
performance. Thus, in the process of the present invention, the initial pH is
set to the
preferred pH for the cell growth of the organism utilized. The pH is then
allowed to drop
naturally due to the production of organic acids during the process of the
present invention to
the pH that is the preferred for the specific dextransucrase-producing enzyme
being utilized.
If the pH is not controlled by external means, the pH of the fermentation
broth will continue to
drop until bacterial growth stops and the production of the desired maltosyl-
isomaltooligosaccharides (IM0s) ceases.
[0052] The most desirable mixture of IMOs is produced during fermentation by
bacterial
enzyme(s) that carry out the fermentation process at an optimal pH of 5.5.
Shifts in pH to
values above or below 5.5 can alter both the yield and the mix of
oligosaccharide sizes
present in the product IMOs. Alternatively, this mix can also be changed by
varying the
starting sucrose to maltose ratio as well as varying the calcium content.
[0053] It is possible to increase the overall production of IMOs in a given
fermentation by a
continuous addition of a sucrose/maltose feed in the desired ratio of sucrose
to maltose.
This increase in product output and composition can be done independently of
pH control if
pH control is accomplished by the addition of an alkaline material such as
sodium or
potassium hydroxide, or can be done in conjunction with pH control if a
sucrose containing
material such as lime sucrate, supplemented with maltose, is used. Lime
sucrate, also
known as saccharate of lime, is a form of calcium hydroxide in which the
calcium is
complexed with sucrose.
[0054] The disclosed process also comprises an improvement on known commercial-
scale
methods for the fermentative production of GlcOS using L. mesenteroides (ATCC
13146) by
reducing the cost of production. Costs associated with alkali can be reduced
by a factor of
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-2.4 by using Ca(OH)2 rather than NaOH. Replacing two Na + ions with one Ca2+
ion also
negates the need for de-ashing the chromatographic ion exchange column, which
eliminates
two potentially expensive chromatographic steps. The use of lime sucrate
solves the issue of
solubility of calcium in water, and the sucrose-base is used as supplemental
feedstock for
the fermentation.
Disadvantades of Known Production Methods
[0055] Mixtures of isomaltooligosaccharides are generally produced by the
action of
immobilized enzymes on mono- or disaccharide feedstocks and can also be
produced by
transglycosylation of starch hydrolysates followed by chromatographic
separation. In an
early process, Chludzinski et al. produced branched isomaltooligosaccharides
using
dextransucrase (EC 2.4.1.5) expressed from bacterial cultures such as
Leuconostoc spp.
and Streptococcus ssp. (Chludzinski, A. M., et al., J. Bacteriol., 1974, B:1-
7). Roper and
Koch later disclosed the production of isomaltooligosaccharide mixtures from
starch
hydrolysates (maltose and maltodextrins) through the action of the a-
transglucosidase (EC
2.4.1.24) from Aspergillus sp. (Starch, 1988, 40:453-459).
[0056] More recently, use of the enzyme glucosyltransferase isolated from
Leuconostoc
mesenteroides has been reported. Remaud et al. disclosed the production of
linear and
short-branched oligosaccharides with a-(1¨>6) linkages and a maltose at the
reducing end
using a feedstock starting ratio of 7:1 sucrose:maltose from a reaction
catalyzed by extra-
cellular Leuconostoc mesenteroides ATCC 13146 glucosyltransferase (Remaud, M.,
et al., J.
Carbohydrate Chem., 1992, 11(3):359-378) producing branched dextrans with 1-3
linkages.
The same group has reported the production of branched a-(1¨>2)
isomaltooligosaccharide
mixtures from sucrose with an acceptor reaction catalyzed by dextransucrase
(Remaud-
Simeon, M. et al., Appl. Biochem. Biotechnol., 1994, 44:101-17). Paul et al.
disclosed the
synthesis and purification of branched isomaltooligosaccharide mixtures
containing an a-
(1,2) bond by the action of soluble and insoluble glucosyltransferase isolated
from
Leuconostoc mesenteroides B-1299 on sucrose and a glucosyl acceptor such as
maltose (or
a material rich in maltose, such as a starch hydrolysis product), isomaltose,
methyl a-
glucoside, isomaltotriose or glucose (or a material rich in glucose, such as a
starch
hydrolysis product) (Paul, F. et al., U.S. Patent No. 5,141,858).
[0057] Chung and Day produced glucooligosaccharides (MIMO) generated in situ
during
fermentation via the action of dextransucrase generated in situ during live
fermentation upon
sucrose in the presence of a maltose acceptor (U.S. Patent No. 7,291,607). The
isomaltooligosaccharides produced were an extracellular product of the
fermentation of
sucrose by Leuconostoc mesenteroides ATCC 13146. Chung and Day demonstrated
that
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these glucooligosaccharides (MIMO) are readily utilized by bifidobacterium sp.
and
lactobacillus sp., but not by Escherichia coli or Salmonella sp. in pure-
culture studies
(Chung, C.H. and Day, D. F., J. Ind. MicrobioL BiotechnoL, 2002, 29:196-9).
[0058] Immobilized enzymatic synthesis of isomaltooligosaccharides has been
used to
produce these compounds, but is a costly, complex process requiring enzyme
isolation,
immobilization and separation and likely requires more costly steps in
purification of the end
product. The method of the present invention is a step change improvement in
how to make
isomaltooligosaccharides in terms of efficiency of production and purity of
the end product.
The immobilized enzyme production approach also yields a different mix of
molecules
compared with the natural in situ fermentation process. Hence, the in situ
approach is
preferred for producing a natural product that may be more desirable as a
dietary
supplement than products produced by other methods.
Improved Method for the Production of Isomaltoolicosaccharides
[0059]Conventional fermentation is a more practical approach for industrial
manufacture of
glucooligosaccharides, and in particular isomaltooligosaccharides, than the
use of
immobilized enzymes. Live cultures that produce dextransucrase in situ also
metabolically
convert the D-fructose to D-mannitol which can be economically separated. D-
fructose is a
difficult compound to separate from isomaltooligosaccharides and can be a
detriment to use
of this product in human nutrition, given the current information about the
negative effects of
high fructose syrups. (See: Ouyang, X., et al., J. Hepatol., 2008, 48(6):993-
9. doi:
10.1016/j.jhep.2008.02.011. Epub 2008 Mar 10; Dhingra, R. et al., Circulation,
2007,
116:480-488; Swanson, J. E., et J. Clin. Nu/r., 1992, 55(4):851-856; and
Vartanian,
L. R., et al., Am. J. Public Health, 2007, 97(4):667-75. Epub 2007 Feb 28.)
[0060] By utilizing the method of the present invention, the size and
composition of the
product MIMOs may be closely controlled. The sucrose:maltose (S/M) ratio
provides the
primary control of product composition, in that it determines the general DP
distribution.
Close control of the pH of the fermentation mixture allows refinement of the
product
composition. Specifically, within the range of pH 6.5 to 5.5, the product
composition bell
curve shifts to higher DP as the pH decreases and vice versa. Introduction of
specific
amounts of Ca" also determines the degree of branching of the product MIMO by
promoting
one or more isoforms of dextransucrase (Chae, et al., J. Microbiol.
Biotechnol., 2009,
19(12):1644-1649).
[0061]The composition of the MIMO produced is primarily determined by the
bacteria
utilized in the fermentation step. The action of bacteria genus species
Leuconostoc
mesenteroides ATCC 13146 on a sucrose/maltose mixture under the conditions of
the
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present invention produces IMOs with a-(1-4) and a-(1¨>6) linkages with or
without a-
(1-4), a-(1¨>3) and a-(1¨>2) branching. The enzyme utilized by Leuconostoc
mesenteroides to catalyze the linkage reaction, dextransucrase, has previously
been
isolated from various strains of the bacteria and used to produce similar
IMOs. IMO
synthesized by dextransucrase isolated from Leuconostoc mesenteroides ATCC
13146 had
a-(1¨>6) backbones with a-(1¨>2), a-(1¨>3), and/or a-(1-4)-side chains when
maltose was
used as an acceptor (Remaud, M. et al., J. Carbohyd. Chem., 1992, 1 1 :359 -
378). It is
reasonable to assume that MIMOs containing a-(1¨>2), a-(1¨>3), and a-(1-4)
side chains
will be effective prebiotics.
[0 06 2] Any organism that produces dextransucrase is applicable for use in
the process
described herein. For example, as shown in Figure 1, the GlcOS produced by the
method of
the present invention is a family of isomaltooligosaccharides that include,
but are not limited
to, branched isomaltooligosaccharides. The final mixture contains IMOs with
DP2 to DP10,
which are considered to be desirable prebiotics (Van Loo, J. et al., Br. J.
Nutr., 1999, 81 :121-
32). Dextransucrase from Leuconostoc mesenteroides strain ATCC 13146 reliably
prefers
to synthesize GlcOS with a-(1¨>6) linkages when maltose is used as the
acceptor. The
larger GlcOS oligomers (DP4 to DP8) may have continuous a-(1¨>6) linkages to
maltose,
that is, MIMOs. Many species of Leuconostocacea also produce branched MIMOs.
[0063] Any microorganism species capable of producing dextransucrase,
including
Leuconostoc mesenteroides, may be utilized in the process of the present
invention. For
example, L. mesenteroides ATCC 13146 may be used. This bacterium is known by
other
designations by those skilled in the art, including the designation
Leuconostoc citreum ATCC
13146, the designation NRRL B-742, and the designation PWSA-L. citreum B742,
the
designation Leuconostoc citreum Farrow, and the designation L. amelibiosum.
The
bacterium Leuconostoc mesenteroides subsp. mesenteroides (Tsenkovskii) van
Tieghem
(ATCC 11449T"), NRRL B-1299, may also be employed. Other useful
dextransucrase-
producing microorganisms include, but not limited to, Leuconostoc spp
(specifically
mesenteroides, citreum, gasicomitatum and kimchii), Weisella spp (specifically
confusa,
such as NRRL # B 1064), Lactococcus spp., Streptococcus spp. (specifically
mutans),
Lactobacillis spp. (reuten), Pediococcus pentosaceus spp., especially
Pediococcus
pentosaceus (ATCC #33316), and certain mutant E. coli. Useful microorganisms
may also
be isolated from natural sources including, but not limited to, sourdough wild
starter (the
bioorganism mixture used in the production of sourdough bread) and kimchi (a
traditional
fermented Korean dish made of vegetables and seasonings).
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[0064]The feedstock ratio of sucrose to maltose (S:M) utilized in the process
of the present
invention is a determiner of the chemical composition of the product oligomer.
In one
embodiment, under batch conditions, the bacteria are grown in a nutrient
mixture (culture
medium) suitable to support growth of the bacteria and a fixed ratio of
sucrose:maltose and
fermentation is allowed to continue until all of the fructose generated during
the reaction is
converted to mannitol. The oligosaccharide production is complete when the
sucrose is
exhausted. Additional fermentation time may result in the reorganization of
the MIMOs by
chemical recombination that changes the DP distribution. In some cases, longer
chains are
formed, possibly from continued residual enzyme activity. Continuation until
the fructose is
converted to mannitol also simplifies purification of the final product. The
further steps of the
process, removing the spent bacteria cells, decolorizing the product and
separating the
mannitol and organic acids from the product oligosaccharides, are then carried
out.
[0065] On a commercial scale, the method of the present invention is carried
out in
equipment known to those skilled in the art. Upon start-up of the fermentation
process, the
entire equipment system is flushed, cleaned and sterilized. A fermentation
tank is charged
with the requisite media components (typical vitamins, sulfates, phosphates,
salts and other
materials used for bacterial growth such as those media recommended by ATCC
for use in
growing the microorganism being cultured, including DIFC0 dehydrated culture
media and
ingredients) and sucrose and maltose in a defined ratio. Separately, the
innoculum (in the
preferred approach, ATCC 13146) is grown until achieving to OD-1 (Optical
Density or
absorbance at 660 nm via UV-VIS spectrophotometer) and added to the
fermentation in a
volume in the range of about 1% to about 10% of the amount contained in the
fermentation.
The fermentation takes place at a temperature around about 28 C. The
fermentation is
continued until no fructose is present, for a period of approximately 25 to 60
hours. The cells
are separated from the broth by microfiltration, centrifugation or
clarification and then
discarded. The broth is decolorized through the use of granular activated
charcoal at about
70 to about 80 C. Alternatively, powdered activated charcoal may be used and
removed by
filtration. The product is separated from the decolorized broth using pulsed
or simulated
moving bed chromatography at about 60 to about 65 C. The extract is then
concentrated as
desired and polished to remove the insoluble impurities, which can be
performed through
centrifugation or microfiltration. It can then be spray dried or freeze dried
if the intent is to
yield a powdered product.
[0066]1n another embodiment, under fed-batch conditions, the process of the
invention may
run on a continuous, or semi-continuous basis. Additional feedstock may be
introduced to
keep the fermentation going and to manage the sucrose:maltose ratio. The pH
may be
controlled during the extended fermentation at the same time by the addition
of sodium
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hydroxide, lime, lime sucrate or another suitable base as described below. As
the initial
feedstock is consumed, additional feedstock, either sucrose or maltose, may be
added to the
culture medium. Such feedstock additions may be used (a) to maintain the
initial fixed
sucrose:maltose ratio or (b) to change the sucrose:maltose ratio as the
fermentation
proceeds. In one embodiment, sucrose is added separately from the maltose to
achieve a
specific sucrose:maltose ratio. In another embodiment, maltose is added
separately from
the sucrose as a means to adjust the sucrose:maltose ratio. Such maltose-only
addition
may also prevent its degradation from continuous contact with the strong base
prior to
addition to the culture. Either approach may be used to produce the first-
order MIMO end
product with the desired chemical composition.
[0067] At any time during the extended fermentation, pH control may take place
utilizing
sodium hydroxide, lime, lime sucrate or other base. When lime sucrate is used,
adjustments
in feedstock addition, sucrose:maltose ratio, and pH control may take place
simultaneously.
Such fed-batch process may involve an input pump for each feedstock component
and each
pH control component, which allows for close control of the sucrose:maltose
ratio and pH
control. The product-containing broth is siphoned off and the other steps of
the process
carried out. In another embodiment, lime sucrate is added to the culture to
control pH, in
particular, to maintain a specific pH in the range of 5.5 to 6.8. It is also
possible to add at the
same time additional maltose so as to introduce a specific sucrose:maltose
ratio with each
dose of pH control. In another embodiment, the sucrose (or lime sucrate) and
maltose may
be mixed just prior to use, kept cool, and used immediately. In these ways, it
is possible to
control for sucrose:maltose ratio as well as pH at the same time whether using
sodium
hydroxide, lime, lime sucrate, or another base in order to achieve the desired
MIMO
chemical composition.
[0068] Under any of the embodiments disclosed herein, the sucrose:maltose
ratio may
range from 1.5:1 to 7:1. The sucrose:maltose ratio may range from 2:1 to
3.5:1. Preferably,
the sucrose:maltose ratio ranges from 2:1 to 3.2:1. Additionally, the
following
sucrose:maltose ratios have utility: 2:1, 2.33:1; 3.17:1, and 3.2:1. All of
these
sucrose:maltose ratios can be utilized in the process of the invention to
provide commercially
desirable product MIMOs for mammalian or avian cosumption, preferably for
human
consumption. For example, a sucrose:maltose ratio of 2:1 produces a DP range
of 4 to 7
using the process of the invention.
[0069] Varying the S:M ratio changes the degree of polymerization and the
average
molecular weight of the isomaltooligosaccharide distributions that are
produced, shifting
them to smaller average values as the amount of maltose increases relative to
the sucrose.
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This appears to be a common effect for any dextransucrase producing organism,
independent of strain (Day, D.F. and Yoo, S. K., "Natural Glucans: Production
and
Prospects," in R. A. Gross and C. Scholz (eds.), Biopolymers from
Polysaccharides and
Agroproteins, ACS Symposium Series, American Chemical Society, Washington
D.C., 2001,
786:292-300). The degree of branching of MIMOs may be determined by the
presence of
appropriate amount of a divalent metal cation in the culture medium. While not
wishing to be
held by any particular theory, the divalent metal cation may influence the
final IMO
composition by forming a complex with the feedstock sugars, the active enzyme,
or both.
Appropriate divalent metal cations include, but are not limited to, calcium
(Ca"), magnesium
(Mg"), strontium (Sr), zinc (Zn"), manganese (Mn"), and iron (Fe").
pH Control
[0070] Sodium hydroxide (NaOH) is routinely used as a pH control reagent in
fermentation.
For instance, the lactic acid bacteria, including Leuconostoc spp., need a
significant amount
of NaOH to maintain the optimum pH for their active growth due to the
production organic
acids during fermentation. Without pH control, the pH of Leuconostoc
mesenteroides ATCC
13146 in fermentation rapidly drop to pH 3.5 within 10 hours (Dissertation,
Yoo, Sun Kyun,
"The Production of Glucooligosaccharides by Leuconostoc mesenteroides ATCC
13146 and
Lipomyces Starkeyi ATCC 74054," 1997, Louisiana State University).
[0071] In the method of the present invention, the base utilized may be any
hydroxide
selected from the alkali or alkaline earth metals, including but not limited
to, MgO, Mg0H2,
CaO, Ca0H2, Sr0, Sr(OH)2, NaOH, Li0H, and KOH. In one embodiment, calcium
hydroxide
saccharates (sucrates) are preferred. The use of a calcium hydroxide sucrate
permits
addition of feedstock sucrose simultaneously with the base required to
regulate pH at the
desired level. For example, approximately a solution of 5% Ca0H2 in 25%
sucrose is one
desirable combination. Ca0H2 may be provided as slaked lime, lime or calcium
oxide. In
another embodiment, the base utilized may be an alkali earth metal carbonate.
[0072] The use of Ca0H2 obtained from lime has economic advantages in the
present
invention. As shown in Example 5 below, a two-liter fermentation required 16.2
g of NaOH
to maintain a pH of 5.5. Using 2013 price information (see Example 5, Table 2,
below), on a
bulk basis the cost of NaOH would be $44.55 for a 10,000 L fermentation, while
the cost of
equivalent lime would be $18.15. On an industrial scale, lime ($0.14 per 10 kg
product) can
be a low cost alternative of NaOH, even if it is necessary to increase the
amount of lime two-
fold or more to maintain an appropriate pH. Lime sucrate method ($0.07 per 1
kg product) is
more attractive because the solubility of lime is increased up to 5% in 22.5%
sucrose
solution (Dissertation, Madsen, L. R. "Iron Mediated Precipitation of Phenol:
Protein
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Aggregates from Sugar Cane Juice", Louisiana State University, Baton Rouge,
2009) and
feeding in maltose at the same time increased the observed yield of product
over time.
[0073]The use of calcium as the basic counter-ion has process advantages. A
cation
(calcium, Ca"form) chromatographic exchange resin in this embodiment is used
to separate
the MIMO's from organic acids and mannitol. Due to the high concentration of
sodium ions
(Na) present in the fermentation broth, the sodium ions replace the calcium
ions (Ca") in
the resin used during separations. The presence of the calcium ions mitigates
the need to
regenerate the separation resins during processing. Thus, in the present
method, the use of
a calcium-ion-based alkali reduces the overall cost of the production of
MIMOs.
Product Purification
[0074] Under the method of the present invention, the MIMOs are separated from
the
fermentation mixture after fermentation is complete as determined by the
conversion of the
fructose to mannitol. Alternatively, the fermentation is continued for a
certain amount of time
to allow the MIMOs produced to spontaneously rearrange themselves into longer
chains, if
desired in the end product. The amount of time needed for this rearrangement
may be
determined by conducting experiments as described herein in order to optimize
the desired
MIMO chain lengths.
[0075]The bacteria cells are then removed from the fermentation mixture, which
is then
subject to a de-ashing process in two steps (strong acid/weak base) and then
decolorized.
Mannitol is then removed through a crystallization process and the product
MIMOs optionally
then may be separated from the by-products via chromatography
[0076]Alternatively, the MIMOs are separated from the fermentation mixture
after
fermentation is complete as determined by the conversion of the fructose to
mannitol. In a
further embodiment, the fermentation is continued for a certain amount of time
to allow the
MIMOs produced to spontaneously rearrange themselves into longer chains. The
amount of
time needed for the rearrangement may be determined by conducting experiments
as
described herein in order to optimize the desired MIMO chain lengths.
[0077]The bacterial cells are removed from the fermentation mixture, which is
then
decolorized, and subject to a de-ashing process in two steps: a strong acid
followed by a
weak base. Mannitol is then removed by using a single or a compound
crystallization
process. The product MIMOs may optionally be separated from the remaining by-
products
via chromatography.
[0078]Suitable methods for removing the bacterial cells include
centrifugation, filtration or
chemical clarification. In one embodiment, centrifugation is employed. Types
of separation
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include continuous liquid or batch centrifugation, using a horizontal
decanter, cream
separator/disc centrifuge, and/or chemical clarification followed by
decantation and/or
filtration. Types of filtration include, but are not limited to,
ultrafiltration, microfiltration or gel
filtration.
[0079]Suitable methods for decolorization include, but are not limited to, the
use of
activated carbon in powder or granular form, with or without pH buffering
(e.g. magnesite),
and may be performed in either batch or continuous (e.g. column) mode.
Activated charcoal
may also be used. A suitable powered carbon is Carbochem CA-50 (Carbochem
Inc.,
Wynnewood, PA) or an equivalent activated carbon. In batch mode, decolorizing
carbon is
added at about 60 to about 70 C, the bulk mixture is allowed to cool to a
temperature of
about 40 C, and after agitation, a filter aid is added. The bulk mixture is
filtered to yield
decolorized liquor.
[0080] In continuous mode, the bulk liquor is passed through a column charged
with
granulated activated carbon at about 65 to about 70 C. Once saturated, the
carbon can be
kilned or regenerated in-place via treatment with alkaline ethanol or
equivalent (Bento).
Examples of suitable filter aids include, but are not limited to, silicon
dioxide, diatomaceous
earth, diatomite, and kieselguhr. Suitable brand names include Celite 545
(Sigma-Aldrich,
St. Louis, MO) and Celatom (Sigma-Aldrich). The grade of filter is selected
according to
the desired time for filtration to occur at an optimum rate, as finer grades
will slow down the
filtration significantly.
[0081] In a preferred embodiment, the bulk of the side-product mannitol can be
removed by
concentrating the mixture and cooling it until crystallization occurs. The
crystals may then be
separated via decantation, filtration or use of a basket centrifuge.
Alternatively, fractional
precipitation of products can be done using organic solvents such as ethanol.
[0082] In another embodiment, the mannitol and the organic acids may be
further removed
by continuous or pulsed chromatography. (See Figure 5.) For example, a
chromatographic
grade gel-type strong acid exchange (SAC) resin in calcium form (SAC-Ca) kept
at 45-70 C
may be utilized. Two main fractions result from the chromatography: 1) MIMO
plus acetic
acid; and 2) mannitol plus lactic acid. It is possible that some lactic acid
and some acetic
acid may remain in the MIMO fraction after the completion of the
chromatography without
interfering with further processing.
[0083] If de-ashing is desirable, an anion exchange resin in partial free-base
form may be
used as described by Saska and Chen (U.S. Patent No. 6,451,123).
Alternatively, the
oligosaccharide product-containing fraction may be further purified by removal
of any heavy
metal ions present utilizing an acid/base combination of ion exchange resins.
Example
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combinations include, but are not limited to, SAC/SBA, SAC/WBA, WAC/SBA, and
WAC/WBA in series, or used as mixed-resin beds.
[0084] In a preferred embodiment, decolorization occurs prior to de-ashing so
as to protect
the de-ashing resins from contamination of the color bodies. Also, the use of
WBA vs SBA
offers three key improvements: (1) the WBA removes all of the organic acids
that would
otherwise only be partially removed by liquid chromatography, (2) removes any
residual
proteins in the fermentation broth and (3) removes any residual color that was
not removed
via the activated carbon step. Removal of the residual color is important
because the resin
can be irreversibly fouled without this step. By using this specific de-ashing
set, additional
downstream options for final product polishing such as mannitol
crystallization become
available in addition to liquid chromatography or both in combination.
[0085] In a further embodiment, the MIMO-containing liquor is concentrated by
evaporation
to give a solution with a brix range between about 20 and about 70 (gram
refractive dry
solids/100g). Other possible brix ranges are between about 30 to about 65,
between about
50 to about 60, between about 55 to about 65, and between about 55 to about
59. Other
brix ranges may be preferred for specific end-product uses.
[0086] In another embodiment, the mannitol may be further removed by
continuous or
pulsed chromatography. See Figure 5. For example, a chromatographic grade gel-
type
strong acid exchange (SAC) resin in calcium form (SAC-Ca) kept at 45-70 C may
be utilized.
Two main fractions result from the chromatography, MIMO and mannitol, with
some residual
mannitol remaining in the MIMO fraction.
[0087] In a further embodiment, the MIMO-containing liquor is concentrated by
evaporation
to give a solution with a brix range between 20 and 70 (gram refractive dry
solids/100g).
Other possible brix ranges are between about 30 to about 65, between about 50
to about 60,
between about 55 to about 65, and between about 55 to about 59. Other brix
ranges may
be preferred for specific end-product uses.
[0088] In yet another embodiment, the MIMO is made into a powder by spray
drying or by
freeze drying or other forms of vacuum evaporation. Alternatively, the
purified liquor may be
precipitated with ethanol or a similar solvent to yield a solid product that
is easily dried.
[0089] The following examples are offered by way of illustration and not by
way of limitation.
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EXAMPLES
Example 1
Preparation of Bacterial Strain and Culture Medium
[0090] L. citreum was purchased from the American Type Culture Collection
(ATCC 13146,
Manassas, VA). After re-isolation, the strain was stored in a -74 C freezer
in 20% glycerol.
This two-liter culture was grown at 28 C in a medium composed of sucrose (100
g/L),
maltose (50 g/L), yeast extract (5 g/L), MgSO4=7H20 (0.2 g/L), FeSO4=7H20
(0.01 g/L), NaCI
(0.01 g/L), MnSO4=7H20 (0.01 g/L), CaCl2 (0.05 g/L), KH2PO4 (3 g/L at pH 6.5).
For two-liter
fermentations, yeast extract (10 g), MgSO4=7H20 (400 mg), FeSO4=7H20 (20 mg),
NaCI (20
mg), Mn504=7H20 (20 mg), CaCl2 (100 mg), and KH2PO4 (6 g) were dissolved in
distilled
water (1250 mL) and adjusted to pH 6.5 using 6 M NaOH prior to innoculation.
The mixture
was autoclaved for 15 min at 120 C. Solutions of maltose (100 g/ 250 mL) and
sucrose
(200 g/500mL) were sterilized prior to transfer to the fermentor.
Example 2
pH Control-materials and Preparations
[0091] The pH control capacity of lime was compared with sodium hydroxide
(NaOH).
Sodium hydroxide pellets were purchased from Fisher Scientific (Hanover Park,
IL) and
hydrated lime powder was purchased from Batesville Marble Hydrated Lime
(Arkansas Lime
Company, Batesville, AR). NaOH (5% w/v, 1.25M, 1.25M eq. [OH]) and lime (5%
w/v,
0.68M, 1.35M eq. [OH]) solutions were prepared by dissolving 50 g of each in
1L distilled
water. To prepare a 5% lime sucrate solution, lime powder (50 g in 1L bottle)
and sucrose
(250 g in 805 mL distilled water) were autoclaved separately. After
autoclaving, sucrose
solution was transferred into the 50 g of lime to give a final solution
concentration of 5% lime
in 25% sucrose, and given the identification "5% lime sucrate". A solution of
maltose (1L of
12.5%) was also prepared separately.
Example 3
Fermentation and pH Control
[0092] Batch fermentations were conducted using 2L BioFlo II fermentors (New
Brunswick
Scientific, New Brunswick, NJ). The fermentors were inoculated from late log-
phase flask
seed cultures at 1.0% (20 mL) of working volume. Fermentations were conducted
at 28 C
with stirring at 200 rpm.
[0093] The pH of the cultures decreased from a starting pH of 6.5 as the cells
produced
organic acid and continued to drop until automatic control began, when pH
reached 5.5
(optimal for the dextransucrase used). This took about 5.5 hrs. The pH was
maintained at
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5.5 until completion of the fermentation(-30 hrs) using 5% NaOH (w/v), 5% lime
(w/v), or 5%
lime sucrate (together with 12.5% w/v maltose solution). A feed of 5% lime
sucrate and
12.5% maltose were used to adjust pH and maintain a sucrose to maltose ratio
at 2:1. For
the lime sucrate method, the lime sucrate and maltose were fed separately with
same flow
rate for the initial 18 hours to control the pH and then replaced with a lime
solution for 12
hours until the end (30 hours) to avoid the residual fructose in the final
fermentation broths.
Example 4
Purification of MIM0s, Mannitol, and Organic Acids
[0094] After harvesting, cells were removed by centrifugation at 10,400g for
20 min.
Activated charcoal (5g/L, Sigma, St. Louis, MO; 100-400 mesh) and Celite 545
(1 g/L,
Fisher Scientific, Hanover Park, IL) was added to cell-free culture broth and
mixed at 50 C
for 20 min. The broth was filtered through No. 3 filter paper (Whatman,
Maidstone, England)
to remove the carbon. The filtered broth was concentrated using a Yamato
rotary evaporator
RE71 (Yamato, Santa Clara, CA) at 65 C to 85 C to 57 g/100g ( brix).
[0095] Cation-exchange chromatography (6.0 x 70 cm column) with pre-swelled
Dowex
Monosphere 99 320 resin (sulfonated styrene-DVB, 300-330 pm, gel, 1.5 eq/L [H
], Ca"
form; Dow, Midland, MI) was used to separate the MIMOs. (See Figure 5.)
Elution was
monitored in real-time by periodically measuring the refractometer brix (Atago
Pallet).
During elution, the void volume was discarded and then fractions were
collected and
analyzed for both carbohydrates and acids by HPLC.
[0096] Based on the results of HPLC analysis (or brix), MIMO fractions
containing either
MIMO:acetic acid or mannitol:lactic acid were combined. After concentration to
30 'brix, the
mannitol was separated from the lactic acid via ethanolic precipitation (70%
ethanol). The
precipitated solid fraction (mannitol) was washed again with 100% ethanol and
air-dried at
55 C. The MIMO fraction and lactic acid fractions were freeze-dried. The
acetic acid is
volatile and the bulk of it was removed during lyophilization. (See Figure 3.)
Example 5
Purification of MIM0s, Mannitol, and Organic Acids
[0097] After harvesting, cells were removed by centrifugation at 9-14k*g for
20-30 min.
Activated charcoal (0.5-3.0 % w/w, Carbochem DC-50, or equivalent) was added
to cell-free
culture broth and mixed at 40-70 C for 20-60 min. The broth was filtered
through No. 3 filter
paper (Whatman, Maidstone, England) topped with diatomite filter aid (fast-
flow, Celite 545
or equivalent, 1.0-6.0 % w/w) to remove the carbon. The filtered broth was
concentrated
using a Buchi R-220 rotary evaporator at 65 C to 85 C to 57 g/100g ( brix).
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Example 6
Comparing MIMO Production with Lime, Lime-Sucrate or Sodium Hydroxide
[0098] Using lime rather than NaOH for pH control, GlcOS was produced from
Leuconostoc
mesenteroides ATCC 13146 according to the method of Chung and Day (2004, Poult
ScL,
83:1302-6). Progress was monitored via TLC. The GlcOS products (indicated by
arrows in
Figure 2) were primarily DP3 (Degree of Polymerization, panose) through DP6
polysaccharides with Rf values corresponding to neither maltooligosaccharides
(M2-M5) nor
isomaltooligosaccharides (IM2-1M4).
[0099] Once the fermentations were complete, as assessed by TLC, the yields of
GlcOS
(DP3), mannitol, and maltose produced using pH control with 5% NaOH or 5% lime
or 5%
lime sucrate, with 12.5% maltose were compared in terms of total GlcOS as
determined by
HPLC. Table 1 shows the GlcOS production by weight percent of the carbohydrate
feed.
Table 1
Product NaOH Lime Lime sucrate
GlcOS (DG>3) 42.40 + 1.50 41.40 + 0.51 40.00 + 1.35
Mannitol 32.45 + 1.49 32.42 + 0.82 31.15 + 1.26
Maltose 12.85 + 1.61 12.45 + 0.31 12.96 + 0.80
[00100] Using lime, the
yields (% of GlcOS (DP?3) per total carbohydrate amount
input) were similar (42.4 0.51 %) with the NaOH (41.1 1.50 %) control (Table
1). In all
fermentations, the production of GOS (D1=3) and mannitol were complete
approximately 15
h and 21 h post-inoculation depending on the type of pH control used (Figure
4). With 5%
lime sucrate, the final GlcOS production (387.7 g) was greater than with NaOH
(Table 2) and
the 40.0 1.35 % yield (GlcOS (D1=3) per total carbohydrate amount input) was
slightly
lower than the 42.4 1.50 % observed using 5% NaOH (Table 1).
Table 2
Total
Fermentations NaOW Limea carbohydrates Productb Cost of
NacOH or
Lime pH control cost per
(g per 2L) (g per 2L) (g per 2 L) (g per 2L) 10 kg
of GlcOS
(10 kL ferment)
(sucrose/maltose)
NaOH 16.2 g 300 g 263.5 g $ 44.55 $0.33
Lime 26.3 g 300 g 257.9 g $18.15 $0.14
Lime sucrate 21.5 g 461.25 g 387.7 g $14.84 $0.07
aAmounts of NaOH and lime used for 2L fermentations.
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bThe final products are GlcOS (D13), mannitol, and maltose.
C Price of NaOH ($550 per metric ton) per global chemical market intelligence
service ICIS
pricing on May, 2013 (at world wide web URL
icis.com/Articles/2013/05/02/9664807/three-
us-producers-announce-price-initiatives-for-caustic.html). Price of hydrated
lime ($138 per
metric ton) in the United States (personal communication, M. Michael Miller,
Lime Specialist,
U.S. Geological Survey, Dec. 3, 2013).
[00101] A two-liter fermentation required 16.2 g of NaOH, 26.3 g of lime, and
21.5 g of lime
sucrate to maintain the optimum pH. The costs of either NaOH or lime relative
to the
respective product yield were calculated and are given in Table 2.
Example 7
pH Control of Maltosyksomaltoolidosaccharides Product Patterns
[00102] Maltosyl-isomaltooligosaccharides are produced by fermentation of
sucrose in the
presence of maltose by the organism L. mesenteroides ATCC 13146. Generally,
the pH of
the fermentation medium drops from its preferred starting pH of 6.5-6.8 to 5.5-
4.5 as the
organism grows. In this example, maltosyl-isomaltooligosaccharides were
produced from
fermentation at three different pHs (pH 6.5, pH 6.0, and pH 5.5).
[00103] Batch fermentations were conducted in 10L BioFlo fermentors (New
Brunswick
Scientific, New Brunswick, NJ). The fermentors were inoculated from late log-
phase flask
seed cultures at 1.0% (100 mL) of working volume. Fermentations were conducted
at 28 C
with stirring at 200 rpm. To maintain the fermentation medium was initially
set at a pH of
6.5, and allowed to drop during fermentation to pH 5.5, and thereafter
maintained at pH 5.5
using 10 N NaOH fed by automatic pH control. If different process pH values
were required,
the same process was used, except that the automatic control was set to start
at the desired
fermentation pH.
[00104] High performance liquid chromatography (Agilent 1200 HPLC with a
differential refractive index detector at 45 C, BioRad Aminex HPX-87K at 85 C
eluted with
100% water at 0.6 mUmin) was used for quantitative analysis of carbohydrates.
A three-
point curve made of maltose, panose, mannitol, glucose, and fructose was used
to
standardize the instrument. The final patterns of maltosyl-
isomaltooligosaccharides
produced differed according to the pH maintained during the fermentation
process. As
shown in Figure 6, a bar graph of the data from Table 3, a lower pH produced
longer
oligomers (DP 4-7) of maltosyl-isomaltooligosaccharides, while higher pH
yielded the shorter
oligomers maltose or panose (DP 2-3).
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Table 3
Maltose Panose
pH DP4 DP5 DP 6-7
(DP2) (DP3)
5.5 5.58 21.37 37.75 25.45 9.86
6.0 9.41 27.02 36.44 19.88 7.25
6.5 9.07 31.98 35.39 17.33 6.24
[00105] Figure 7 shows the effect the sucrose:maltose (S/M) ratio has on
the
molecular weight distribution at a fixed pH of 5.5. As S/M increases, so does
the molecular
weight distribution. Additional experiments were conducted at three different
fermentation
pHs. Profiles of each carbohydrate component during these fermentations at
three different
pHs were compiled and included monitoring of cell growth density, sucrose,
maltose,
mannitol, monomers (glucose, fructose), and maltosyl-isomaltooligosaccharides
polymers
(DP 3-7).
Example 8
Effect of Sucrose:Maltose Ratio on Product Composition
[00106] Oligosaccharides were produced according to the method previously
described.
However, the sucrose to maltose ratio (S/M) was varied in order to determine
the optimum
ratios for the production of maltosyl-isomaltooligosaccharides in various
ranges of degree of
polymerization (DP). Fermentation samples were analyzed via HPAEC-PAD (high
pressure
ion exchange chromatography using a Thermo Dionex ICS-5000 with a Carbopac PA-
100
column and a pulsed amperometric detector).
[00107] Figure 8 demonstrates the effect of pH when the S/M is fixed at 2:1.
It is clear
that the closer the pH approaches the enzyme's optimum, the greater the
proportion of
higher molecular weight oligosaccharides. It is also evident that maltose
conversion
becomes more efficient at pH <6.8 and that panose is glycated at a greater
rate.
Example 9
Use of Different Dextransucrase-Producinq Microoraanisms
[00108] Leuconostoc spp. ATCC 13146 and Kimchi were evaluated through the
same
process as that set forth for the present invention. The Kimchi juice was
sequenced by PCR
to review 16SrRNA to determine the bacterial species in the Kimchi juice and
confirmed the
presence of Leuconostoc spp. (comprising 72.5% of the bacteria present in the
juice)
capable of making dextransucrase. See Table 4 below. In the case of NRRL 1299,
the
species was cultured from revived freeze dried isolates provided by ATCC. With
Kimchi, the
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active fermenting juice was used as the innoculum. In each case, after the
sugar and
nutrient media were prepared, it was sterilized in place in the fermenter and
then inoculated.
The inoculum was prepared by growing the bacteria to OD-1 (Optical density or
absorbance
at 660nm via UV-VIS spectrophotometer). The pH in the fermenter was adjusted
with
sodium hydroxide to 6.5 at initial fermentation. The fermentation of the media
was carried
out for 25 to 65 hours until complete as described in the present invention.
During
fermentation the pH was allowed to drop to 5.5 and then maintained at 5.5 by
controlled
addition of sodium hydroxide. The cells were then removed using centrifugation
and the
resulting clear liquor was decolorized with powdered carbon. The resulting
broth was then
purified by chromatography. The resulting purified liquor was concentrated to
55 to 65 brix
and the resulting syrup was centrifuged to remove proteins and other solids.
Table 4
Sample ID Species Kimchi Juice
NR 102781 Leuconostoc camosum JB16 6.8%
NR 074997 Leuconostoc gasicomitatum LMG 42.1%
NR 102984 Leuconostoc gelidum JB7 12.2%
NR 025204 Leuconostoc inhae strain IH003 2.6%
[00109] The evaluation of Kimchi by PCR (16ssRNA) as described above
demonstrates that fermentation with mixed species of Leuconostoc is capable of
producing
dextransucrase and the desired MIMO product. In addition, species of
Pediococcus, such
as Pediococcus pentosaceus, can also produce desirable MIMO product. The data
shown
in Figure 9 demonstrates that dextransucrase-producing microorganisms other
than L.
mesenteroides both individually and in mixed cultures may be used in the
method of the
present invention.
Example 10
Commercial Scale Production
[00110] L. citreum was purchased from the American Type Culture Collection
(ATCC
13146, Manassas, VA). After re-isolation, the strain was stored in a -74 C
freezer in 20%
glycerol. This two-liter culture was grown at 27 C in a medium composed of
sucrose (100
g/L), maltose (50 g/L), yeast extract (5 g/L), MgSO4=7H20 (0.2 g/L),
FeSO4=7H20 (0.01 g/L),
NaCI (0.01 g/L), Mn504=7H20 (1.5 g), CaCl2 (0.05 g/L), KH2PO4 (3 g/L at pH
6.5). Yeast
extract (0.075 kg), MgSO4 (1.46 g, anhydrous), FeSO4=7H20 (1.5 g), NaCI (0.15
g),
MnSO4.H20 (20 mg), CaCl2(0.8 g), and KH2PO4(40.00 g) were dissolved in
distilled water
(12.6 kg) and adjusted to pH 7 using with NaOH (3.2 g, 50%) prior to
innoculation. The
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mixture was autoclaved for 15 min at 120 C. Solutions of maltose. H20 (0.864
kg) and
sucrose (1.910 kg) were sterilized prior to transfer to the fermentor.
[00111] At startup, the entire system was flushed, cleaned and sterilized. The
fermenter
was then charged with maltose, sucrose and nutrients in the quantities given
above. The
materials were thoroughly mixed. The inoculum is grown to OD-1 (Optical
density or
absorbance at 660 nm via UV-VIS spectrophotometer).
[00112] The fermentation was continued until complete. The inocula cells
were
removed from contents of the fermenter and discarded. The broth was
decolorized as
described above.
Example 11
Commercial Scale Production Using Optional De-Ashina and Crystallization Steps
[00113] After harvesting, cells are removed by centrifugation at 9-14k*g
for 20 to 30
min. After removal of the cells, the liquor is concentrated to about 35-40
brix prior to
treatment with activated charcoal (0.5-3.0 % w/w, Carbochem DC-50, or
equivalent) to
decolorize the liquor. After filtration and washing, the decolorized liquor is
about 35 brix.
This liquor is put through SAC and WBA chromatography as described above for
further
purification.
[00114] Alternatively, following decolorization, the broth is either put
through de-ashing or
concentrated to 56-59 brix for crystallization. Crystallization occurs
overnight at room
temperature. The crystallized mannitol is spun off (-400g/15L batch) and the
liquor is
refrigerated at 5 C overnight. A second crop of mannitol may then be spun off,
producing,
for example, about 100g/15L per batch). The liquor is then concentrated to 63-
65 brix to
yield the final IMMO product.
[00115] Alternatively, MIMOs may be concentrated to 30-40 brix, then spray
dried to
yield a powdered product.
Example 12
Production of Maltosvl-isomaltooliaosaccharides (Ml MOs)
3,000-Liter Fermenter Scale
[00116] The MIMO product prepared in this example was prepared under cGMP and
kosher parve conditions. Details of the process that are not specifically
mentioned herein
are the same as those for Examples 10 and 11. Unless otherwise specified, all
equipment
was cleaned in place before being utilized or re-utilized in the process
steps. Care was
taken to sanitize all equipment and transfer lines and hoses in accordance
with cGMP
standards in the biofermentation industry for products for human consumption.
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[00117] Samples were taken throughout the fermentation and purification
process for
continuous QC monitoring as summarized in Table 5.
Table 5
Shake Formulation
Formulation
Analysis Performed Fl Seed Fermenter Batch
Phase; Hold Phase;
asks
T=0-24 hrs T=24-55 hrs
0D600 Final Every 4 hours Every 4 hours Every 4
hours
pH Final Every 2 hours Every 2 hours Every 2
hours
Micro exam Final Final Every 8 hours Every 8
hours
Every 8 hours +
Brix measurement** X Final Every 8 hours
Final
Every 2 hours +
Retained QA samples Final Final Every 2 hours
Final
**Brix measurements were taken by a hand-held device.
[00118] The following reagents and starting materials were dissolved in 3.7
liters RO
water (3.7 liters): potassium phosphate monobasic (0.0118 Kg), anhydrous
magnesium
sulfate (0.000431 Kg), ferrous sulfate heptahydrate (0.000045 Kg), manganese
sulfate
monohydrate (0.000045 Kg), sodium chloride (0.000045 Kg), and calcium chloride
dihydrate
USP (0.00024 Kg), yeast extract (bacteriological grade; 0.0221 Kg), sucrose
(Pure Cane
Extra Fine Granulated Sugar; 0.53021 Kg); maltose (Sunmalt-S[N] maltose
monohydrate;
0.2935 Kg). The initial pH was adjusted to 7.0 using 50% sodium hydroxide FCC
(0.005
Kg). This medium was divided into six Fernbach flasks, plugged, autoclaved at
12100 for 15
minutes, and then cooled to room temperature.
[00119] L. citreum (B-742; ATCC 13146), previously preserved as 1.0 ml
aliquots of a
1:1 w/w broth/glycerol mixture), were thawed at room temperature. Five flasks
were
aseptically inoculated with 1.0 ml of the prepared L. citreum. The sixth flask
served as an
uninoculated control. All flasks were shaken at 27 C at 150 rpm overnight (16
hours) until
the optical density (OD) was > 1. The 0D600of a sample from each flask was
measured on a
Shimadzu spectrophotometer. A microscopic examination of a sample from each
flask was
also conducted to look for growth of the L. citreum and to rule out
contamination. The
healthy cultures from the five flasks were aseptically transferred to a
sterile, 2-gallon
pressure vessel.
[00120] To produce the sugar and salt stock solution, a 1200-gallon
fermenter was
charged with 1440 Kg RO water, sucrose (Pure Cane Extra Fine Granulated Sugar;
498.96
Kg); maltose (Sunmalt-SIN1 maltose monohydrate; 200.0 Kq), sodium chloride
(0.037 Kg),
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and calcium chloride hydrate USP (0.204 Kg). The mixture was thoroughly
stirred before
transferring by sterile filtration to seed and stock production tanks.
[00121] A 300-gallon seed fermenter fitted with two pH probes and an air
sparger was
prepared for fermentation by mixing 238 Kg RO water, 2.76 Kg yeast extract
(bacteriological
grade), 1.48 Kg potassium phosphate monobasic, 54.2 grams anhydrous magnesium
sulfate, 5.7 grams ferrous sulfate heptahydrate, and 5.7 grams manganese
sulfate
monohydrate.
[00122] The sugar and salt stock solution (309.2 Kg) was aseptically
transferred to the
seed fermenter via filter sterilization. Immediately after the transfer, 10 Kg
RO water was
added to a 15-gallon pressure vessel, the vessel pressurized and the water
aseptically filter
sterilized into the seed tank. An additional 20 Kg RO water was added to
adjust the brix of
the fermentation mixture.
[00123] The seed tank was inoculated with the healthy combined cultures via
aseptic
lines. The pH of the tank was recorded. Fermentation was allowed to occur
without pH
adjustment for 16 hours at 27.0 C + 1.0 and aeration. The pH was monitored
every 2 hours
and the 0D600 measured every four hours.
[00124] The 1200-gallon fermentation tank was then charged with 1332 Kg RO
water,
15.01 Kg yeast extract (bacteriological grade), 8.01 Kg potassium phosphate
monobasic,
292.2 grams anhydrous magnesium sulfate, 30.0 grams ferrous sulfate
heptahydrate, and
30.0 grams manganese sulfate monohydrate. The mixture was stirred at 50 rpm to
dissolve
the dry ingredients. The pH was recorded at 5.70, but not adjusted. The tank
was held at
37 C for two additional hours while making certain that the air sparger
continued to function
properly.
[00125] The media was then sterilized at 121 C for 60 minutes. The tank was
cooled
to 27 C via slight aeration through the sparger line. The previously prepared
sugar and salt
stock solution was aseptically transferred (1676.1 Kg) via filter
sterilization. The initial pH
was 5.71. The previously prepared seed solution (31.0 Kg) was aseptically
transferred to
the production fermenter. The air supply to the headspace was turned on. The
pH was
again measured and adjusted to approximately 7.0 using 50% sodium hydroxide
solution.
[00126] Fermentation was allowed to continue in the tank at 27 C + 1.0,
with head
space aeration and agitation. The pH was maintained at 5.5 + 0.05 using 50%
sodium
hydroxide solution. The 0D600, brix, and microbiological inspection were
monitored as given
in Table 5. The fermentation was allowed to continue until complete and
fructose was not
detectable. The fermentation tank was then cooled to 10 C.
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[00127] The spent L. citreum bacterial cells were removed from the
fermentation broth
via microfiltration, followed by six stages of diafiltration at an inlet
pressure of up to 20 psi.
Briefly, the fermentation broth was pressure filtered through microfiltration
membranes to
ensure removal of as much cellular debris as possible. The cellular material
retained by the
microfilters was washed with 750 Kg RO water six times to enhance product
recovery and
yield. The aqueous permeates were combined for concentration and de-
colorization.
[00128] The product mixture was concentrated using a heated wiped film
evaporator.
Periodically, the concentrated product mixture was sampled and the brix level
determined
via a hand-held meter. Concentration was discontinued when the brix level
reached 40.
[00129] The concentrated fermentation broth was de-colorized using powdered
activated carbon (26.25 Kg, CA-50S). The product mixture was heated to 65 C
and the
carbon was added along with Celite 545 (21.0 Kg). The resulting slurry was
stirred for
about 30 minutes before reducing the temperature to 50 C. The activated carbon
was
removed by pressing the product mixture through a Sperry filter press that was
pre-coated
with more Celite 545 (21.0 kg) suspended in 300 Kg RO water. The product
mixture was
then recirculated through the pre-coated filter press until the filtrate was
clear. Finally, it was
pumped through a 1 micron polishing filter. Additional MIMO product was
recovered by
washing the Sperry filter press by adding 250 Kg RO water, agitating the tank
contents and
then recirculating through the filter press until the filtrate was clear.
Again, this filtrate was
pumped through a 1 micron polishing filter.
[00130] In appropriately sized batches (1350 L at 40 brix total, 6 stages
of 225 L feed
each for a total of six feed/regeneration cycles), ion exchange chromatography
(IEX) is
utilized to remove ash (comprised of minerals not consumed during the
fermentation
process), residual colored organic compounds, organic acids, proteins and
amino acids.
The resulting combined product is evaporated to a concentration of 56 brix.
The product is
then cooled to room temperature while agitating to facilitate crystallization
of any remaining
mannitol. The mannitol crystals are removed by basket centrifugation or by
filtering out the
crystalline mannitol crystals. The crystals are washed with cold water to
remove the product,
which is retained for future use. The product is evaporated to a concentration
of 65 brix.
[00131] The product mixture is again cooled to room temperature while
agitating to
facilitate crystallization of any remaining mannitol. The product mixture is
further cooled to
4 C with agitation to facilitate crystallization of any remaining mannitol.
Any resulting
crystalline mannitol is removed by basket centrifuge or by filtration of the
final product and
the crystals again washed with cold RO water to recover any remaining MIMO
product. The
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wash water is retained for future use. The final product is a clear,
colorless, slightly viscous
liquid.
Example 13
Improvements in Yield by Fermentation at Hidher Total &mars
[00132] Typically, fermentation production processes for MIMOs are not run at
high total
solids in order to avoid issues related to osmotic shock of the working
organism. Previous
work (12 full batches at 15.5 kg) indicated that fermentation at approximately
17.6 % total
sugar gives the proper product distribution, yield, and is reproducible. Thus,
increasing the
total sugar (TS) content should increase product yield. Here, 44% is given as
overall product
recovered at pilot scale, not yield in the production-scale fermenter, which
is typically 56-
58%. The TS can be thus increased until the organism either loses productivity
or begins to
reject its condition, metabolically, in order to protect itself, e.g. by the
production glycerol or
ethanol, which are undesirable contaminants in a final product. A second
limitation on
osmotic strength is the resistance of the enzyme to deformation from its
preferentially active
conformation due to insufficient hydration and/or co-substrate (e.g. water, in
the case of
hydrolytic enzymes).
[00133] In order to attempt to span a reasonable range of working
concentrations, a
two-kilogram fermentation was run at a sucrose:maltose ratio of 2.75:1 and at
a TS of 30 %.
The fermentation behaved identically at 30% TS to the established method
fermenting
17.6%. The yield, purity, and product distribution were identical once
normalized over the
total refractive dry solids (RDS). For yield calculations, the % w/w and
derived values are
given below in Table 6 and Table 7.
Table 6
Analytics 3000 kg 2 kg
% w/w 37 Hr A 30Bx #1
Brix 18.3 29.2
mannitol 4.885 7.400
glucose 0.007 0.057
fructose 0.011 0.002
sucrose 0.825 0.317
maltose 0.315 0.806
panose 1.370 2.383
MIMO-DP4 3.469 5.470
MIMO-DP5 3.431 5.423
MIMO-DP6 1.488 2.573
MIMO-DP7 0.435 0.777
MIMO-DP8 0.186 0.324
MIMO-DP9 0.000 0.000
MIMO-DP10 0.000 0.000
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Lactate 2.137 3.061
Glycerol 0.027 0.060
Formate 0.000 0.000
Acetate 0.899 1.420
Ethanol 0.097 0.153
Table 7
Fermented
amount 3000 kg 2.005 kg
Sucrose, kg 388.8 0.420
Maltose, kg 156.5 0.169
Total, kg 3072.3 2.005
Totals
MIMO, % 10.38 16.95
TOTAL, % 19.58 30.23
Purity, % 53.00 56.08
Yields 3000 kg 2.005 kg
TS, % 17.7 29.4
TS, kg 545.2 0.589
ISOT, kg 318.9 0.340
yield, % 58.48 57.71
MWD, Da 763.77 759.06
ISOT
kg@3000 318.85 542.50
@44% 239.90 397.05
[00134] These data show that the yields of a 3000-Kg batch at 17.7% TS are
not
significantly different from the 2-Kg batch fermented at 29.4% TS, 58.48 vs.
57.71%,
respectively. Mass-average molecular weight distribution (MWD) was likewise
similar at
763.77 vs 759.06 Da (target 760 Da) for TS fermented at 17.7% and 29.4%,
respectively.
Further, the increase in TS did increase yield from 318.85 kg to a calculated
542.5 Kg/batch
improving the recoverable yield by, in the worst case 49.28 % and in the best
case, 70.14%.
Example 14
Removal of Mannitol via Crystallization from the MIMOs
[00135] As described in Example 12, the MIMO product can be further processed
at the
end of the described process to remove any remaining mannitol to further
improve the purity
of the final product. This example describes improvements to the process of
removing
mannitol via crystallization.
[00136] In one method, IEX product ("IEX out" in Table 8) was evaporated to
56 brix
and was allowed to crystallize ("XL #1") at room temperature. The mannitol
crystals were
removed (cake #1) via either a basket centrifuge fitted with a polypropylene
filter bag with 10
pm pores or a nutsch pressure filter with 10 pm equivalent plate/screen. The
cake was
washed with 500 mL cold water (appx. 125% cake w/w) and the washings were
collected for
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recycle ("wash #1"). The resulting liquor ("liquor #1") was cooled to 2-5 C
and was allowed to
crystallize ("XL #2"). The mannitol crystals were removed ("cake #2") via
either a basket
centrifuge fitted with a polypropylene filter bag with 10 pm pores or a nutsch
pressure filter
with 10 pm equivalent plate/screen. The cake was washed with 500 mL cold water
(approximately125% cake w/w) and the washings were collected for recycle
("wash #2").
The crystals are removed as before, and the resulting liquor is sent to final
concentration to a
product brix of 63-64 . Table 8 shows a typical mass balance, given as the
averages from 11
batches.
Table 8
Stage !EX XL #1 XL #2
Evap Liquor Wash Cake Liquor Wash Cake
Values
!EX in !EX out 2 #1 #1 #1 #2 #2 #2 Recycle
kg 7.290 26.839 3.265 2.548 0.647 0.402 2.425 0.612 0.114 1.259
Brix 35.46 8.03 57.36 52.82 19.79 89.59 50.51
13.64 89.26 16.72
RDS, kg 2.48 1.81 1.87 1.346 0.13 0.317 1.225
0.08 0.087 0.21
Ash, kg 0.032 0.000 0.000 0.000 0.000 0.000 0.000
0.000 0.000 0.000
ISOT, kg 1.097 1.064 1.064 0.988 0.09 0.031 0.939
0.07 0.010 0.16
Acids, kg 0.517 0.000 0.000 0.000 0.000 0.000 0.000
0.000 0.000 0.000
Mannitol,
kg 0.698 0.677 0.677 0.360 0.03 0.317 0.230
0.02 0.087 0.05
Solids,
kg 2.34 1.74 1.74 1.35 0.13 0.35 1.17 0.08
0.10 0.21
Purity, % 46.77 61.10 61.10 73.28 73.28 8.82 80.33
80.33 10.16 76.06
[00137] In an improved method, IEX product ("IEX out") was evaporated to 56
brix
and was allowed to crystallize (XL #1) at room temperature. The mannitol
crystals were
removed ("cake #1") via either a basket centrifuge fitted with a polypropylene
filter bag with
pm pores or a nutsch pressure filter with 10 pm equivalent plate/screen. The
cake is
washed with 500 mL cold water (appx. 125% cake w/w) and the washings collected
for
recycle ("wash #1"). The resulting liquor ("liquor #1") was allowed to
crystallize ("XL #2") at
room temperature. Once cooled and crystallization was observed, the entire
mixture was
cooled to 2-5 C and was allowed to crystallize until complete. The mannitol
crystals were
removed ("cake #2") via either a basket centrifuge fitted with a polypropylene
filter bag with
10 pm pores or a nutsch pressure filter with 10 pm equivalent plate/screen.
The cake was
washed with 500 mL cold water (approximately 125% cake w/w) and the washings
were
collected for recycle ("wash #2"). The data from this experiment are shown
below in Table 8.
The product has a brix of 63-64 brix and was ready for pasteurization and
packaging as
commercial material.
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Table 8
Stage !EX XL #1 XL #2
!EX !EX Evap Liquor Wash Cake Evap Liquor Wash Cake
Recycle
Values
in out 2 #1 #1 #1 3 #2 #2 #2
kg 7.027
29.747 2.908 2.46 0.632 0.354 1.916 1.545 0.481 0.120 1.113
Brix 32.20 5.86 57.01 51.29 14.90 93.58 65.85 63.45 21.40 92.16 17.71
RDS, kg 2.26 1.74 1.73 1.26 0.09 0.33 1.26 0.98
0.10 0.11 0.200
Ash, kg 0.157 0.000 0.000 0.000 0.000 0.000 0.000
0.000 0.000 0.000 0.000
ISOT, kg 1.199 1.137 1.137 1.058 0.040 0.003 1.058
0.892 0.067 0.002 0.107
Acids, kg 0.363 0.000 0.000 0.000 0.000 0.000 0.000
0.000 0.000 0.000 0.000
Mannitol' 0.595 0.569 0.569 0.218 0.049 0.259 0.218 0.066 0.032 0.081 0.081
kg
Solids, kg 2.31 1.71 1.71 1.28 0.09 0.26 1.28 0.96
0.10 0.08 0.19
Purity, % 53.70 64.26 64.26 78.63 43.74 1.14 78.63
87.59 64.85 2.53 57.07
[00138] Alternatively, after purifying the product mixture using IEX as
described
above, the product mixture is evaporated to 56 brix, then cooled to room
temperature while
stirring to encourage crystal formation of the mannitol present. After about
12 to about 16
hours, the crystals are removed via basket centrifugation, or by utilizing a
vibrating screen
filter, or by using a nutsch filter, or by using combinations of these
techniques which are well
known to one skilled in the art. The remaining liquid is evaporated to 65-66
brix, then cooled
to room temperature while stirring to encourage crystal formation for 12 to 16
hours. The
mixture is cooled to 4 C to encourage crystallization of any remaining
mannitol. A two-stage
crystallization process, i.e., one using two different temperatures, is
necessary in order to
allow the crystals to form properly. After about 12 to about 16 hours, the
crystals are
removed via basket centrifugation, or by utilizing a vibrating screen filter,
or by using a
nutsch filter, or by using combinations of these techniques. The product is
now ready for
pasteurization and packaging as commercial material.
[00139] All publications and patent applications mentioned in this
specification are herein
incorporated by reference to the same extent as if each individual publication
or patent
application were specifically and individually indicated to be incorporated by
reference.
[00140] From the foregoing it will be appreciated that, although specific
embodiments of the
invention have been described herein for purposes of illustration, various
modifications may
be made without deviating from the spirit and scope of the invention.
Accordingly, the
invention is not limited except as by the appended claims.
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