U.S. patent application number 11/860845 was filed with the patent office on 2008-03-13 for isomaltooligosaccharides from leuconostoc as neutraceuticals.
Invention is credited to Chang-Ho Chung, Donal F. Day.
Application Number | 20080064657 11/860845 |
Document ID | / |
Family ID | 33452458 |
Filed Date | 2008-03-13 |
United States Patent
Application |
20080064657 |
Kind Code |
A1 |
Day; Donal F. ; et
al. |
March 13, 2008 |
Isomaltooligosaccharides from Leuconostoc as Neutraceuticals
Abstract
Isomaltooligosaccharides (IMOs) produced by Leuconostoc
mesenteroides ATCC 13146 fermentation with a sucrose:maltose ratio
of 2:1 have been discovered to be effective prebiotics in mixed
cultures of microbial populations, including cultures from chicken
ceca. Surprisingly in mixed microbial cultures this IMO composition
proved as effective as FOS. Thus, these IMOs can be used as
effective prebiotics for both birds and mammals. Moreover, the IMOs
were discovered to be effective non-competitive inhibitors of
.alpha.-glucosidase. These IMOs also will be useful, as an
.alpha.-glucosidase inhibitor, in a therapeutic application for
several diseases, including obesity, diabetes mellitus,
pre-diabetes, gastritis, gastric ulcer, duodenal ulcer, caries,
cancer, viral disease such as hepatitis B and C, HIV, and AIDS. A
diet with 5-20% IMOs was also shown to reduce the abdominal fat
tissue in mammals.
Inventors: |
Day; Donal F.; (Baton Rouge,
LA) ; Chung; Chang-Ho; (Baton Rouge, LA) |
Correspondence
Address: |
PATENT DEPARTMENT;TAYLOR, PORTER, BROOKS & PHILLIPS, L.L.P
P.O. BOX 2471
BATON ROUGE
LA
70821-2471
US
|
Family ID: |
33452458 |
Appl. No.: |
11/860845 |
Filed: |
September 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10848981 |
May 19, 2004 |
7291607 |
|
|
11860845 |
Sep 25, 2007 |
|
|
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60471942 |
May 20, 2003 |
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Current U.S.
Class: |
514/54 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 31/715 20130101; A61P 3/00 20180101; A61P 31/04 20180101 |
Class at
Publication: |
514/054 |
International
Class: |
A61K 31/715 20060101
A61K031/715; A61P 43/00 20060101 A61P043/00 |
Claims
1. A method to increase the population number of the beneficial
bacteria relative to the population number of the pathogenic
bacteria in an animal's intestine, said method comprising orally
administering to the animal an effective amount of a composition
comprising one or more maltsoyl-isomaltooligosaccharides with only
.alpha.-1,4 and .alpha.-1,6 linkages and with a degree of
polymerization less than or equal to 7, such that the number of
beneficial intestinal bacteria increase substantially or that the
number of pathogenic bacteria decrease substantially.
2. A method as in claim 1, additionally comprising the step of
producing the composition by fermentation of sucrose in the
presence of maltose, in a sucrose:maltose ratio of about 2:1, by
Leuconostoc mesenteroides ATCC 13146.
3. A method as in claim 1, wherein said animal is a bird.
4. A method as in claim 1, wherein said animal is a mammal.
5. A method as in claim 1, wherein said animal is a human.
6. A method as in claim 1, wherein the number of pathogenic
bacteria substantially decrease.
7. A method as in claim 6, wherein said pathogenic bacteria is one
or more selected from the group consisting of Salmonella,
Escherichia, and Campylobacter.
8. A method as in claim 1, wherein the number of beneficial
bacteria substantially increase.
9. A method as in claim 8, wherein said beneficial bacteria is one
or more selected from the group consisting of Lactobacillus and
Bifidobacteria.
10-17. (canceled)
Description
[0001] The benefit of the May 20, 2003 filing date of provisional
application Ser. No. 60/471,942 is claimed under 35 U.S.C.
.sctn.119(e).
[0002] This invention pertains to the use of
maltosyl-isomaltooligosaccharides as a dietary supplement for birds
and mammals, specifically, to promote the growth of beneficial
intestinal microbes, inhibit the growth of pathogenic intestinal
microbes, and for therapeutic intervention in diseases such as
diabetes by inhibiting the activity of .alpha.-glucosidase to slow
the rate of glucose release from carbohydrates and thereby reduce
the uptake of glucose.
[0003] Prebiotics are nondigestible food ingredients that
selectively stimulate the growth and/or activity of beneficial
microbial strains (probiotics) residing in the host intestine. See
R. Barrangou et al., "Functional and comparative genomic analyses
of an operon involved in fructooligosaccharide utilization by
Lactobacillus acidophilus," Proc. Natl. Acad. Sci. USA, vol. 100,
pp. 8957-8962 (2003). It is believed the ability of these
probiotics to catabolize oligosaccharides (two to ten
monosaccharide units linked with glycosidic bonds) is a key factor
in bestowing beneficial health effects. Certain oligosaccharides
are used as prebiotics. They are resistant to metabolism and
adsorption in the small intestine and ultimately positively
influence the composition of microflora in the large intestine.
Oligosaccharides are also widely used in foods such as soft drinks,
cookies, cereals, candies, and dairy products. Other applications
for oligosaccharides such as an anti-cariogenic agent or a low
sweetness humectant have been explored. See S. K. Yoo, "The
production of glucooligosaccharides by Leuconostoc mesenteroides
ATCC 13146 and Lipomyces starkeyi ATCC 74054, Ph.D. Dissertation,
Louisiana State University (1997).
[0004] Oligosaccharides used as prebiotics are currently produced
either by extraction from plant sources, acid or enzymatic
hydrolysis of polysaccharides or enzymatic synthesis by
transglycosylation reactions. See P. Monsan et al.,
"Oligosaccharide feed additives," In: R. J. Wallace and A. Chesson
(eds) Biotechnology in animal feeds and animal feeding, pp.
233-245, VCH Velagsgesellshaft mbH, Weinheim, Germany (1995).
[0005] Types of Oligosaccharides
[0006] Types of oligosaccharides include fructooligosaccharides
(FOS), glucooligosaccharides (GOS), and
.alpha.-galactooligosaccharides. The differences in structures are
illustrated in FIG. 1. Fructooligosaccharides (FOS) have attracted
serious commercial interest as prebiotics. They are composed of a
D-glucopyranose unit at the non-reducing end (G) linked via an
.alpha.-1,2 linkage to two or more .beta.-2,1-linked fructosyl
units (F). This group includes 1-kestose (GF2), nystose (GF3), and
IF-fructofuranosyl nystose (GF4). Many of the oligosaccharides
marketed commercially are FOS, e.g., Raftilose and Nutraflora in
the United States.
[0007] .alpha.-Galactooligosaccharides, which are
.alpha.-galactosyl derivatives of sucrose, are present in many
legume seeds. Mono-, di-, and tri-.alpha.-galactosylsucrose, known
respectively as raffinose, stachyose, and verbascose, are produced
by extraction from plants, particularly soybeans. These
oligosaccharides are known to be, in part, responsible for the
flatulence and diarrhea that follows consumption of beans, because
of the absence of an .alpha.-galactosidase in the gastrointestinal
tracts of humans and animals.
[0008] Glucooligosaccharides (GOS) is a generic term for
poly-glucose oligomers. GOS may contain a number of different
linkages and are generally obtained from starch hydrolysates
(maltose and maltodextrins) through the action of the
.alpha.-transglucosidase (EC 2.4.1.24) from Aspergillus sp.
Glucooligosaccharides can also be produced by restricting polymer
size during the fermentation process. A subcategory of GOS is the
.alpha.-isomaltooligosaccharides (IMO) which contain .alpha.-1,6
bonds in their main chain See H. J. Koepsell et al., "Enzymatic
synthesis of dextran. acceptor specificity and chain initiation,"
J. Biol. Chem., vol. 200, pp. 793-801 (1952). Dextransucrase (EC
2.4.1.5), an enzyme produced mainly by species of Leuconostoc and
Streptococcus, catalyzes the synthesis of high molecular weight
glucans (dextrans).
[0009] Oligosaccharides as Prebiotics
[0010] Ingested oligosaccharides (prebiotics) are capable of
reaching the colon without being digested. It has been proposed
that fructooligosaccharides are preferentially utilized by
Lactobacilli and Bifidobacterial species which are considered
beneficial species of the human intestinal tract. See H. Kaplan et
al., "Fermentation of fructooligosaccharides by lactic acid
bacteria and Bifidobacteria," Appl. Environ. Microbiol., vol. 66,
pp. 2682-84 (2000). Substituting fructooligosaccharides as a carbon
source would preferentially increase the concentration of
Lactobacillus and Bifidobacteria species with a concomitant rise in
the intestinal production of lactic acid and short-chain fatty
acids (SCFA). Both these products would have the net effect of
lowering the pH in the large intestine. This appears to be one mode
by which beneficial species can out-complete and indeed help
prevent the establishment of undesirable pathogenic organisms such
as Salmonella. See B. J. Juven et al., "Antagonistic effects of
Lactobacilli and Pediococci to control intestinal colonization by
human enteropathogens in live poultry," J. Appl. Bacteriol., vol.
70, pp. 95-103 (1991). The fructooligosaccharides may also interact
with carbohydrate receptors present on the surface of either
microbial or epithelial cells, affecting cell adhesion and
immunomodulation. See P. J. Naughton et al., "Effects of
nondigestible oligosaccharides on Salmonella enterica Serovar
Typhimurium and nonpathogenic Escherichia coli in the pig small
intestine in vitro," Appl. Environ. Microbiol., vol. 67, pp.
3391-95 (2001).
[0011] Fructooligosaccharides, galactooligosaccharides, and soybean
oligosaccharides were found not to be digested by enzymes secreted
by small intestine, but to be fermented by certain microorganisms
found in human and livestock intestines, especially by the
Bifidobacterium sp. See. H. Tomomatsu, "Health effects of
oligosaccharides," Food Technol., vol. 48, pp. 61-65 (1994). There
are numerous reports regarding the stimulating effects of
fructooligosaccharides on the growth of probiotic strains. See P.
Monsan et al., 1995; and M. Gmeiner et al., "Influence of a
symbiotic mixture consisting of Lactobacillus acidophilus 72-4 and
a fructooligosaccharide preparation on the microbial ecology
sustained in a simulation of the human intestinal microbial
ecosystem (SHIME reactor)," Appl. Microbiol. Biot., vol. 53, pp.
219-223 (2000). Dietary FOS have been reported to be effective in
reducing the numbers of the harmful bacteria, E. coli, in the
intestine of piglets, but did not reduce numbers of Salmonella. See
P. J. Naughton et al., "Effects of nondigestible oligosaccharides
on Salmonella enterica Serovar Typhimurium and nonpathogenic
Escherichia coli in the pig small intestine in vitro," Appl.
Environ. Microbiol., vol. 67, pp. 3391-95 (2001). However, in the
same study, commercially available glucooligosaccharides (GOS),
another oligosaccharide, showed no effect on either genus of
bacteria.
[0012] In studies of in vitro fermentation characteristics using
human fecal material, small intestinal digestibility, and effects
on fecal microbial populations in dogs, GOS (containing
.alpha.-1,2, .alpha.-1,4 and .alpha.-1,6 linkages) and FOS produced
short chain fatty acids in human fecal material more rapidly than
other substrates, such as gum arabic, guar gum and guar
hydrolysate. GOS also appeared to be indigestible in the small
intestine, while supplying a carbon source for bacterial
fermentations in the large intestine of cannulated dogs. See E. A.
Flickinger et al., "Glucose-based oligosaccharides exhibit
different in vitro fermentation patterns and affect in vivo
apparent nutrient digestibility and microbial populations in dogs,"
J. Nutr., vol. 130, pp. 1267-1273 (2000). When the viable count of
Bifidobacterium infantis and B. longum, and changes in pH due to
various carbohydrate-supplemented soymilks were monitored, B.
longum showed a significantly (P<0.05) higher count on a crude
isomaltooligosaccharide (75%) supplemented soymilk than in the
control (soymilk without the added supplement) at the end of
fermentation. See C-C. Chou et al., "Growth of Bifidobacteria in
soymilk and their survival in the fermented soymilk drink during
storage," Int. J. Food Microbiol., vol. 56, pp. 113-121 (2000).
Another study showed that GOS was only 20% digested by germfree
rats. See P. Valette et al., "Bioavailability of new synthesized
glucooligosaccharides in the intestinal tract of gnotobiotic rats,"
J. Sci. Food Agric., vol. 62, pp. 121-127 (1993). Dietary
isomaltooligosaccharides (13.5g/day for 14 days) were reported to
increase fecal Bifidobacteria levels (P<0.05) in healthy adult
males. See T. Kohmoto et al., "Effect of isomalto-oligosaccharides
on human fecal flora Bifidobacteria," Microflora, vol. 7, pp. 61 69
(1988). Another study investigated the ability of several human gut
bacteria to break the .alpha.-1,2 and .alpha.-1,6 glycosidic
linkages in .alpha.-glucooligosaccharides, in vitro, in substrate
utilization tests. See Z. Djouzi et al., "Degradation and
fermentation of .alpha.-gluco-oligosaccharides by bacterial strains
from human colon: in vitro and in vivo studies in gnotobiotic
rats," J. Appl. Bact., vol. 79, pp. 117-127 (1995). Branched
oligomers were resistant to both gastrointestinal enzymes and
utilization by pathogenic microorganisms. They also reported that
.alpha.-1,2 glucosidic bonds were more resistant than .alpha.-1,6
linkages in kinetic studies of glucooligosaccharide hydrolysis in
pH-regulated fermentations. This study indicated the differences in
utilization, and thus effectiveness, of GOS based on types and
degree of branching.
[0013] Production of Glucooligosaccharides
[0014] Glucansucrases have been extensively studied because of
their role in the production of dextran and its role in the
cariogenic process. Glucansucrases (EC 2.4.5.1), usually
extracellular but in some cases cell-associated, are primarily
produced by various species of soil bacteria. Those produced by
Leuconostoc sp. are called dextransucrase. Those produced by
Streptococcus sp. and other lactic bacteria, Lactococci, are called
glucosyltransferases. Streptococcal glucansucrases synthesize
primarily .alpha.-1,3 rich polysaccharides. Leuconostoc
glucansucrases produce .alpha.-1,6 rich polysaccharides.
[0015] Glucansucrases catalyze the synthesis of high molecular
weight D-glucose polymers from sucrose. In the presence of
efficient acceptors, e.g., maltose, they may catalyze the synthesis
of low molecular weight oligosaccharides. See F. Paul, "Acceptor
reaction of a highly purified dextransucrase with maltose and
oligosaccharides: Application to the synthesis of
controlled-molecular-weight dextrans," Carbohydr. Res., vol. 149,
pp. 433-441 (1986).
[0016] Dextransucrases catalyze the synthesis of high molecular
weight glucans (dextrans) according to the reaction: ##STR1##
[0017] Dextran is a D-glucose polymer composed mainly of
.alpha.-1,6 linked backbones in a linear chain and .alpha.-1,2,
.alpha.-1,3, and/or .alpha.-1,4 branch linkages. See U.S. Pat. No.
5,229,277. The chemical structure of the dextran is specific to the
glucansucrase of the producing strain of microbes (Table 1). See J.
F. Robyt, "Dextran," In: Encyclopedia of Polymer Science and
Engineering," (H. F. Mark et al., eds.), Vol. 4, pp. 752-767, John
Wiley & Sons, New York (1986). The dextransucrase from L.
mesenteroides NRRL B-1299 can produce .alpha.-glucooligosaccharides
(GOS) containing one or more D-glucopyranosyl branch units linked
via .alpha.-1,2 glycosidic bonds if maltose supplied as an
acceptor. See F. Paul et al., "Method for the production of
.alpha.-1,2 oligodextrans using Leuconostoc mesenteroides B-1299,"
U.S. Pat. No. 5,141,858. However, dextransucrase from L.
mesenteroides B-742 (ATCC 13146)produces two dextrans; one with
.alpha.-1,6 and .alpha.-1,3 linkages, and another with .alpha.-1,6
and .alpha.-1,4 linkages. (Table 1) Usually a high molecular weight
dextran (10.sup.6-10.sup.7 Da) is produced. This is the case, for
example, of the enzyme from L. mesenteroides NRRL-512F, which is
used to produce dextran polymers of industrial interest including
chromatography supports, photographic emulsions, iron carriers, and
blood plasma substitutes (Robyt, 1986). TABLE-US-00001 TABLE 1
Linkages in different dextrans as obtained by methylation analysis
Linkages % Dextran.sup.a Solubility .alpha.-1.fwdarw.6
.alpha.-1.fwdarw.3 .alpha.-1.fwdarw.3 Br.sup.b .alpha.-1.fwdarw.2
Br.sup.b .alpha.-1.fwdarw.4 Br.sup.b L. m. B-512F Soluble 95 5 L.
m. B-742 Soluble 50 50 L. m. B-742 Less soluble 87 13 L. m. B-1299
Soluble 65 35 L. m. B-1299 Less soluble 66 1 27 L. m. B-1355
Soluble 54 35 11 L. m. B-1355 Less soluble 95 5 S. m. 6715 Soluble
64 36 S. m. 6715 Insoluble 4 94 2 .sup.aL. m., Leuconostoc
mesenteroides; S. m., Streptococcus mutans. .sup.bBr, Branch
linkage. Adapted from Robyt, 1986.
[0018] The synthesis of oligosaccharides using dextransucrase can
be induced at the expense of dextran synthesis. In the presence of
sucrose, the introduction into the reaction medium of molecules,
like maltose, isomaltose, and O-.alpha.-methylglucoside, shifts the
pathway of glucan synthesis towards the production of
oligosaccharides. See Paul, 1986; and M. Remaud et al.,
"Characterization of .alpha.-1,3 branched oligosaccharides
synthesized by acceptor reaction with the extracellular
glucosyltransferases from L. mesenteriodes NRRL B-742," J.
Carbohyd. Chem., vol. 11, pp. 359-378 (1992); Koepsell et al.,
1952; and J. Robyt et al., "Relative, quantitative effects of
acceptors in the reaction of Leuconostoc mesenteroides B-512F
dextransucrase," Carbohydr. Res., vol. 121, pp. 279-286 (1983). The
molecular weight and polydiversity of this oligosaccharide product
are dependent upon the sucrose to acceptor ratio, the strain of
bacteria, and on the characteristics of the intermediate
oligosaccharides in the reaction. The ratio of sucrose to maltose
affects the composition and yield of the oligosaccharides produced
by the acceptor reaction. When the maltose to sucrose ratio was 2,
a partially purified dextransucrase from L. mesenteroides NRRL
B-512F produced 85% of ththeheoretical yield of polysaccharide as
oligosaccharides, with an average degree of polymerization (DP) of
4. See U.S. Pat. No. 5,141,858; and Paul, 1986.
[0019] Leuconostoc mesteroides B-742 ATCC 13146
[0020] Leuconostoc mesenteroides ATCC 13146 was isolated from
spoiled canned-tomatoes. (Robyt, 1986) The dextran produced by this
(B-742) Leuconostoc strain is highly branched, containing as much
as 50% .alpha.-1,3 linkages. Leuconostoc mesenteroides ATCC 13146
actually produces two exocellular .alpha.-D-glucans, a fraction L,
which is precipitated at an ethanol concentration of 39%, and a
fraction S, which is precipitated at a concentration of 45% ethanol
(Robyt, 1986). Fraction L consists of an .alpha.-1,6 backbone with
.alpha.-1,4 branch-points, and fraction S consists of an
.alpha.-1,6 backbone with .alpha.-1,3 branch-points. The L fraction
from Leuconostoc mesenteroides ATCC 13146 contains 87% .alpha.-1,6
linkages and 13% .alpha.-1,4 linkages. The percentage of
.alpha.-1,3 branch-points in the fraction S glucan is variable,
dependant on the conditions under which it is synthesized from
sucrose. The .alpha.-1,3 linkages of the S fraction of L.
mesenteroides ATCC 13146 are all branched linkages. This dextran
demonstrates extreme resistance to endodextranase. This property
seems related to its structure that has the highest possible degree
of branching and exhibits a comb-like structure with main chains of
consecutive .alpha.-1,6 linked glucose residues to which single
.alpha.-1,3 linked glucosyl residues are attached. Any change in
reaction conditions that affects the rate of acceptor reaction
relative to chain elongation also affects the degree of branching
in ATCC 13146 fraction S dextran.
[0021] The acceptor reaction of L. mesenteroides ATCC 13146 was
investigated and found that branch formation in this strain, when
maltose was the acceptor, was dependant upon reaction conditions.
L. mesenteroides ATCC 13146 in the presence of maltose produced 90%
of the theoretical yield of polymer as isomaltooligosaccharides,
under optimum conditions for sucrose fermentation. See Yoo, 1997;
S. K. Yoo et al., "Co-production of dextran and mannitol by
Leuconostoc mesenteroides, J. Microbiol. Biotechnol., vol. 11, pp.
880-883 (2001); and S. K. Yoo et al., "Highly branched
glucooligosaccharide and mannitol production by mixed culture
fermentation of Leuconostoc mesenteroides and Lipomyces starkeyi,
J. Microbiol. Biotechnol., vol. 11, pp. 700-703 (2001). The
fermentation was essentially complete in 24 hours, with
oligosaccharide production being linked to growth. The production
rate was about 0.9 g/L hr. The maltose to sucrose ratio was able
not only to alter the yield of oligosaccharide but also to change
the relative proportion of different size oligosaccharides produced
by the fermentation. The highest yields of isomaltooligosaccharides
were obtained when the ratio of sucrose to maltose in the
fermentation was two. This is the same ratio reported for optimum
oligosaccharide production in vitro by the dextransucrase of L.
mesenteroides B-512F (See Paul et al., 1986). Several Leuconostoc
strains were tested to check for oligosaccharide size profiles
produced in response to maltose, because individual Leuconostoc
species synthesize different dextransucrases in response to various
acceptors. The isomaltooligosaccharides produced by L.
mesenteroides ATCC 13146 were mostly DP (degree of polymerization)
3-5 by chemical analysis. Isomaltooligosaccharides prepared by
alcohol-precipitated, cell-free culture broths had greater amounts
of higher branched isomaltooligosaccharides up to DP 7, than
commercial preparations and had no glucose and less maltose (Yoo,
1997). These isomaltooligosaccharides were found to affect
isolated, single microbial cultures by suppressing growth of
Salmonella enteritidis, Salmonella typhimurium, Staphylococcus
aureus, Staphylococcus epidermidis, and Clostridium perfringenes,
and supporting growth of two Bifidobacterium species. (Yoo,
1997).
[0022] D-mannitol is a sugar-alcohol derived from mannose or
fructose by dehydrogenation. In sucrose fermentations, mannitol is
produced as an end product, as fructose can be used as an electron
acceptor, but the levels of mannitol produced vary with the strain.
See Yoo, 1997; and C. Y. Kim et al., "Production of mannitol using
Leuconostoc mesenteroides NRRL B-1149," Biotechnol. Bioprocess
Eng., vol. 7, pp. 234-236 (2002). Mannitol was found as one of the
major end products in this Leuconostoc fermentation. It is
necessary to separate the mannitol from the oligosaccharides if
they are to be used as prebiotics, because mannitol can act as an
additional carbon source. Its presence would hinder the ability to
ascribe the essential and unique role of oligosaccharides on
intestinal microflora. (Yoo, 1997)
[0023] Oligosaccharides as Antibiotic Alternatives in Animals
[0024] Antibiotic resistance among known pathogens such as
Salmonella and Escherichia coli is expanding due to the wide use of
antibiotics in areas ranging from medicine to animal feed. Although
only specific antibiotics are used in feed preparations and are
exclusive to non-human use, their chemical similarity to
antibiotics prescribed for humans has raised concern that
resistance will spread more rapidly, since resistant mechanisms
generally affect an entire class of antibiotics (ex: penicillinases
to inhibit the Penicillins). This, coupled with public pressure to
remove antibiotics from animal feeds, has created a need for safe
alternatives that can effectively control the growth of bacterial
pathogens in the human food supply. Selected fructooligosaccharides
and glucooligosaccharides have shown potential as alternatives to
antibiotics. See P. Monsan et al., (1995); J. V. Loo et al.,
"Functional food properties of non-digestible oligosaccharides: a
consensus report from the ENDO project (DGXII AIRII-CT94-1095),"
Brit. J. Nutr., vol. 81, pp: 121-132 (1999); and P. Valette et al.,
"Bioavailability of new synthesized glucooligosaccharides in the
intestinal tract of gnotobiotic rats," J. Sci. Food Agric., vol.
62, pp. 121-127 (1993). However, not all oligosaccharides have been
found effective. Although FOS is generally considered to be
effective in regulating and reducing pathogenic microbial
populations, conflicting reports exist about the effectiveness of
GOS. See Naughton et al., 2001; and Yoo, 1997. These conflicting
reports may be due to variability in the composition of the GOS
(the degree of branching, the size, the amount of mannitol, or the
acceptor used in fermentation production), or whether the GOS was
tested on single microbial cultures, mixed microbial cultures, or
in vivo. There may also be differences depending on the animal
tested.
[0025] During recent years, poultry production and consumption have
continually increased. Since 1992, the production of broilers grew
from 9,482,000 to 14,017,000 tons in 1996 in the United States.
Poultry is a carrier of numerous bacteria, including Salmonella and
Campylobacter. Practical experience has demonstrated the difficulty
in reducing the incidence of Salmonella on chickens once they
arrive at the processing plant. Significant reduction in Salmonella
on processed carcasses requires the delivery of chickens with
reduced Salmonella to the processing plant. One of the possible
ways to control Salmonella outbreaks may be through the judicious
addition of selected carbohydrates to the diet of chickens. Mannose
and lactose in the diet of chickens have been reported to reduce
Salmonella colonization. See B. Oyofo et al., "Effect of
carbohydrates on Salmonella typhimurium colonization in broiler
chickens," Avian Dis., vol. 33, pp. 531-534 (1989).
Fructooligosaccharides (FOS) have been shown to influence
intestinal bacterial populations by enhancing the growth of lactic
acid bacteria such as Lactobacillus species and Bifidobacteria, and
to inhibit Salmonella colonization of chicks. See J. S. Bailey et
al., "Effect of fructooligosaccharide on Salmonella colonization of
the chicken intestine," Poultry Sci., vol. 70, pp. 2433-2438
(1991); and T. Fukata et al., "Inhibitory effects of competitive
exclusion and fructooligosaccharide, singly and in combination, on
Salmonella colonization of chicks," J. Food. Prot., vol. 62, pp.
229-233 (1999). The mean number of Salmonella enteritidis in the
chicks of the fructooligosaccharide group was significantly
(P<0.05) decreased compared with the control group. There are no
reports that a glucooligosaccharide is effective in modifying the
gut microflora in poultry.
[0026] Importance of .alpha.-Glucosidase Inhibition
[0027] Starch is one of the most readily available fermentable
sources of energy for organisms and makes up 60-70% of the dietary
carbohydrate consumption in humans. Humans secrete a pancreatic
.alpha.-amylase that cleaves starch to a di-(maltose),
tri-(maltotriose), and branched .alpha.-dextrins in the duodenal
cavity. Because there is no integral transport process in the
intestinal enterocyte that can accommodate anything larger than
free glucose, these oligosaccharides are further processed to
glucose in the intestinal surface membrane by .alpha.-glucosyl
saccharidases, including .alpha.-glucosidase. These enzymes form
part of a large glycoprotein component of the intestinal surface
brush border membrane. Once formed, glucose then may be
cotransported into the enterocyte, along with Na.sup.+, either by a
75 kDa specific integral brush border glucose carrier or by a
transporter expressed in the small intestine. Inhibitors of
.alpha.-glucosidase are know to delay the digestion of starch, of
starch-derived oligosaccharides, and sucrose such that the rise in
blood sugar levels is slowed and insulin secretion is decreased
after a meal. These inhibitors have been proposed to be used
therapeutically for obesity, gastritis, gastric ulcer, duodenal
ulcer, caries, hyperglycemia, hyperinsulinemia, diabetes mellitis,
cancer, viral infection, hepatitis B and C, HIV and AIDS. See U.S.
Pat. Nos. 5,840,705; and 4,013,510; and U.S. Patent Application No.
2004/0081711. At least two commercial oral .alpha.-glucosidase
inhibitors, Miglitol and acarbose, are currently prescribed for use
in managing non-insulin-dependent diabetes mellitus by slowing the
appearance of glucose in the blood after eating.
[0028] We have discovered that the isomaltooligosaccharides (IMOs)
produced by Leuconostoc mesenteroides ATCC 13146 fermentation with
a sucrose to maltose ratio of 2:1 are effective prebiotics in mixed
cultures of microbial populations, including cultures from chicken
ceca. Surprisingly in mixed microbial cultures, this IMO
composition proved as effective as FOS as a potential prebiotic.
This IMO composition could be an effective alternative to
antibiotics for chickens and other poultry. Thus, these IMOs can be
used as effective prebiotics for both birds and mammals. Moreover,
the IMOs were discovered to be effective non-competitive inhibitors
of .alpha.-glucosidase. These IMOs also will be useful, as an
.alpha.-glucosidase inhibitor, in a therapeutic application for
several diseases, including obesity, diabetes mellitus,
pre-diabetes, gastritis, gastric ulcer, duodenal ulcer, caries,
cancer, viral disease such as hepatitis B and C, HIV, and AIDS. A
diet with 5-20% IMOs was also shown to reduce the abdominal fat
tissue in mammals.
BRIEF DESCRIPTION OF DRAWINGS
[0029] FIG. 1 illustrates the structures of various
oligosaccharides.
[0030] FIG. 2 illustrates the production of various
isomaltooligosaccharides by L. mesenteroides ATCC 13146 from
sucrose (10% w/v) and maltose (5% w/v) as a function of time.
[0031] FIG. 3 illustrates the flow chart for the production of
isomaltooligosaccharides as used in this study.
[0032] FIG. 4 illustrates the results of thin layer chromatography
indicating the types of branched .alpha.-isomaltooligosaccharides
of L. mesenteroides (ATCC 13146). The abbreviations used are as
follows: S, Isomaltodextrins; P, Isomaltooligosaccharide product;
C, Commercial isomaltooligosaccharides (Wako Pure Chemical Industry
Ltd., Osaka, Japan); Glc, glucose; IM.sub.3, Isomaltotriose;
IM.sub.5, Isomaltopentaose; and IM.sub.7, Isomaltoheptaose.
[0033] FIG. 5 illustrates the results of .sup.13C NMR of the
branched .alpha.-isomaltooligosaccharides of L. mesenteroides (ATCC
13146).
[0034] FIG. 6 illustrates the anaerobic growth of mixed cultures of
Salmonella typhimurium and Lactobacilli johnsonii on the branched
.alpha.-isomaltooligosaccharides of L. mesenteroides (ATCC 13146)
preparation at 37.degree. C.
[0035] FIG. 7 illustrates the .alpha.-glucosidase (maltase)
activity inhibition with increasing concentrations of the branched
.alpha.-isomaltooligosaccharides of L. mesenteroides (ATCC
13146).
[0036] FIG. 8 illustrates a double reciprocal plot of
.alpha.-glucosidase (maltase) activity inhibition as the
concentration of the branched .alpha.-isomaltooligosaccharides of
L. mesenteroides (ATCC 13146) increased.
[0037] FIG. 9A illustrates the .alpha.-glucosidase (maltase)
activity in the presence of different concentrations of panose
(branched; .alpha.-1,4 and .alpha.-1,6).
[0038] FIG. 9B illustrates the .alpha.-glucosidase (maltase)
activity in the presence of different concentrations of
isomaltotriose (linear; two .alpha.-1,6).
[0039] FIG. 10A illustrates a double reciprocal plot of
.alpha.-glucosidase (maltase) activity inhibition as the
concentration of panose (branched; .alpha.-1,4 and .alpha.-1,6)
increased.
[0040] FIG. 10B illustrates a double reciprocal plot of
.alpha.-glucosidase (maltase) activity inhibition as the
concentration of isomaltotriose (linear; two .alpha.-1,6)
increased.
[0041] FIG. 11A illustrates the anaerobic growth of Salmonella
typhimurium and Bifidobacterium longum on different combinations of
panose (branched; .alpha.-1,4 and .alpha.-1,6) at 37.degree. C.
[0042] FIG. 11B illustrates the anaerobic growth of Salmonella
typhimurium and Bifidobacterium longum on different combinations of
maltooligosaccharides (d.p. 4-10) at 37.degree. C.
[0043] FIG. 12 illustrates a comparison of growth rates in log
phase between Salmonella typhimurium and Bifidobacterium longum at
different concentrations of panose (branched; .alpha.-1,4 and
.alpha.-1,6) and maltooligosaccharides (M.O.; d.p. 4-10).
[0044] The present invention describes the production and
application of mixtures of isomaltooligosaccharides (IMOs) ranging
in size from DP (degree of polymerization) 3 to 7 units and
incorporating a maltosyl group at the reducing end of each
oligomer. The said mixtures were produced by fermentation with
Leuconostoc mesenteroides ATCC 13146 by restricting the polymer
size through the addition of maltose to the carbon source. A
specific ratio of maltose acts to limit the chain length produced
by the enzyme dextransucrase acting on sucrose. The IMOs in this
work were produced by a sucrose to maltose ratio of 2:1. Syrup
containing said fermentation products was obtained after ion
exchange and chromatographic separation of the fermentation broth.
Mannitol was then removed to produce isolated IMOs. The said
mixture produced by this process was found to be readily
catabolized by Bifidobacteria and lactobacillus but not readily
utilized by either Salmonella sp., or E. coli, pointing towards its
use in intestinal microflora modification. The said mixtures were
non-competitive inhibitors of .alpha.-glucosidase (maltase), an
enzyme required for starch or maltodextrin utilization, and
decreased the abdominal fat in mammals.
EXAMPLE 1
[0045] Materials and Methods
[0046] Organism, Culture Medium, and Inoculum Preparation
[0047] All strains of bacteria used in this study were obtained
from the American Type Culture Collection (ATCC, Manassas, Va.).
They were maintained on agar slants, at 4.degree. C. and
transferred monthly. Anaerobes were subcultured weekly. Salmonella
typhimurium (ATCC 14028) and Escherichia coli B (ATCC 23226) were
maintained on tryptic soy agar (Difco, Detroit, Mich.).
Bifidobacterium bifidum (ATCC 35914), Bifidobacterium longum (ATCC
15708), Lactobacillus johnsonii (ATCC 33200), and Leuconostoc
mesenteroides (ATCC 13146) were maintained anaerobically on
Lactobaccilli MRS slants (Difco, Detroit, Mich.) containing 0.05%
(w/v) cysteine. Chicken ceca were kindly supplied by the Russell
Research Center (USDA ARS, Russell Research Center, Athens, Ga.).
Screening and isolation for chicken ceca bacteria were conducted
following the method described by R. Hartemink et al., "Comparison
of media for the detection of Bifidobacteria, lactobacilli and
total anaerobes from fecal samples," J. Mircrobiol. Meth., vol. 36,
pp. 181-192 (1999). Basically, ceca (from 6 weeks to 8 weeks
broilers) in a plastic bag were homogenized by kneading the bag,
and a subsample of about 10 g was transferred to a preweighed glass
container containing 90 ml anaerobic buffered peptone water (Oxoid)
with 0.5 g/L L-cysteine-HCl. The container was then closed and
weighed to determine the actual sample size. Mixed samples were
diluted further with reduced physiological salt solution ("Rps,"
peptone 1 g/L, L-cysteine-HCl 0.5g/L and NaCl 8g/L) or test media
(MRSB; Difco). Finally, the samples were plated on the media and
incubated at 37.degree. C. for 48 h. Unless otherwise stated,
mixing, diluting, plating and incubation were carried out
anaerobically. Six colonies out of the hundreds were selected
randomly and designated as chicken ceca isolates #1 to #6.
[0048] Preparation of Oligosaccharides
[0049] Batch fermentations were conducted in a 2-L BioFlo II
fermentor (New Brunswick Scientific Co.) with a working volume of
1.0 L. The media had the following composition: sucrose (100 g/L);
maltose (50 g/L); yeast extract (5 g/L); MgSO.sub.4.7H.sub.2O (0.2
g/L); FeSO.sub.4.7H.sub.2O (0.01 g/L); NaCl (0.01 g/L);
MnSO.sub.4.7H.sub.2O (0.01 g/L); CaCl.sub.2 (0.05 g/L);
KH.sub.2PO.sub.4 (3 g/L) with pH 7.2. Fermentors were inoculated
from late log phase flask cultures at 1.0% of working volume.
Fermentations were conducted at pH 6.5, 28.degree. C., and 200 rpm.
After harvesting, cells were removed by centrifugation at
10,400.times.g for 20 min (Dupont Sorvall RC5C, Newtown, Conn.).
Activated charcoal (5 g/L, Sigma Chem. Co., St. Louis, Mo.) and
Celite 545 (1 g/L, Fisher Scientific, Fair Lawn, N.J.) were added
to cell-free culture broth and mixed at 50.degree. C. for 20 min.
The broths were then filtered through No. 6 filter paper (Whatman
International Ltd., Maidstone, England) to remove the carbon. The
filtered broths were desalted using ion-exchange columns filled
with an anion-exchange resin in the hydroxide form and a
cation-exchange resin in the hydrogen form (Rohm and Haas,
Philadelphia, Pa.). The eluents were concentrated by vacuum
evaporation (Brinkmann Instrument Inc., Westbury, N.Y.) to 65%
solids. Mannitol crystallized upon cooling the concentrates, and
was removed by decantation. Isomaltooligosaccharides were separated
from the mannitol free concentrates using a cation exchange column
(in calcium form); the isomaltooligosaccharide fractions were
concentrated by vacuum evaporation.
[0050] Analytical Methods
[0051] Bacterial growth was measured by turbidimetry at 660 nm,
calibrated against cell dry weight. Cells from a known volume were
harvested by centrifugation at 10,400.times.g for 2 min (Dupont
Sovall 24S, Newtown, Conn.), washed with deionized water,
resuspended in a minimum volume of water, and dried (initially
overnight at 95.degree. C. and then at 105.degree. C.) to constant
weight. An absorbance of 1.0 at 660 nm was equivalent to 0.51 g of
dry matterliter.sup.-1.
[0052] Thin Layer Chromatography (TLC)
[0053] Separation and qualitative identification of
oligosaccharides was conducted using TLC. Whatman K6F silica gel
plates of sizes (10.times.20 cm) were obtained from Fisher
Scientific (Chicago, Ill.). A homologous series of isomaltodextrins
(DP 1-10) was donated by Chonnam National Univ. (Kwangju, Korea).
Maltopentaose, maltohexaose, maltoheptaose, panose, glucose, and
isomaltotriose (Sigma Chem. Co., St. Louis, Mo.) and a commercial
mixture of isomaltooligosaccharides (Wako Pure Chemical Industry
Ltd., Osaka, Japan) were used as standards. Aliquots (1-2 .mu.L) of
the solutions to be analyzed were applied 20 mm from the bottom of
the TLC plates with 10 .mu.L micro syringe pipettes. The plates
were developed at ambient temperature, using a mixture of solvents
(acetontrile, ethyl acetate, propanol, and water in volume (ml) at
proportions of 85:20:50:70, respectively). After development was
complete, the plates were dried, and the carbohydrates visualized
using a spray of an ethanol solution containing 0.3% (w/v)
.alpha.-naphthol and 5% (v/v) H.sub.2SO.sub.4. After air-drying,
spots were developed by heating in an oven for 10 to 20 min at
100.degree. C. Isomaltooligosaccharides were identified by
comparing their chromatographic behavior with those of the
standards.
[0054] Cation Column Chromatography
[0055] Different types of cation resins (Na, K, Ca form) were
tested for separation of isomaltooligosaccharides from the end
fermentation products. Resins (Duolite CR-1320, Rohm and Haas,
Philadelphia, Pa.) in glass-jacketed columns (10 mm (Inner
diameter).times.100 mm (Length); working volume 70 ml ) were
regenerated using 5% solutions of NaCl, KCl, or CaCl. The
temperature of the water eluent and the circulating water for glass
jacket were 92 and 80.degree. C., respectively. No pressure on the
column was applied. Injection volume was 1 ml of solution (15
Brix.degree. IMO). The detector was a differential refractometer
(Waters).
[0056] High Performance Ion Chromatography
[0057] High-performance ion chromatography using a CarboPac MA1
column (Dionex, Sunnyvale, Calif.) and a pulsed amperometric
detector (PAD, Dionex) was used for quantitative analysis of
glucose, fructose, sucrose, mannitol, and maltose concentrations in
solution. The samples were eluted at 0.4 mlmin.sup.-1 with a 0.48 M
NaOH solution. Oligosaccharide concentrations were calculated from
peak areas of high-performance liquid chromatography on an
Aminex-HPX-87K Bio-Rad column (Bio-Rad Lab. Hercules, Calif.) run
at 85 .degree. C. with K.sub.2HPO.sub.4 as eluent, at a constant
flow rate of 0.5ml.min.sup.-1, using glucose as a standard.
[0058] .sup.13C Nuclear Magnetic Resonance
[0059] The isomaltooligosaccharides (DP 1 to DP 8) were analyzed
using a DPX 250 (63 MHz .sup.13C) system with help of the
Department of Chemistry, Louisiana State University (Baton Rouge,
La.). The chemical shifts were expressed in ppm relative to the
methyl signal of acetone in deuterium oxide solvent which was used
as an internal standard at .delta.=29.92 ppm. The various signals
were identified as described by F. Seymour et al., "Structural
analysis of dextrans containing 4-O-.alpha.-D-glucosylated
.alpha.-D-glucopyranosyl residues at the branch points, by use of
13C-nuclear magnetic resonance spectroscopy and gas-liquid
chromatography-mass spectrometry," Carbohydr. Res., vol. 75, pp.
275 (1979); and M. Remaud et al., "Characterization of .alpha.-1,3
branched oligosaccharides synthesized by acceptor reaction with the
extracellular glucosyltransferases from L. mesenteriodes NRRL
B-742," J. Carbohyd. Chem., vol. 11, pp. 359-378 (1992).
[0060] Kinetic Assay for .alpha.-Glucosidase
[0061] .alpha.-Glucosidase (maltase; EC 3.2.1.20), .beta.-NAD,
glucose dehydrogenase (EC 1.1.147) and other reagent chemicals were
obtained from the Sigma Chemical Co. (St. Louis, Mo.). The kinetic
assays were based on the following reaction; ##STR2##
[0062] The kinetic assays were all performed in 96-well plates and
read at wavelength 320 nm in a SPECTRAmax Plus microtiter plate
reader (Molecular Devices Corp., Sunnyvale, Calif.) at 37.degree.
C. The software package Softmax.TM. was used for data analysis.
.alpha.-Glucosidase (maltase; EC 3.2.1.20), .beta.-NAD, and glucose
dehydrogenase (EC 1.1.147) solutions were prepared with 0.1M
K.sub.2HPO.sub.4 (pH 7) buffer. Each well contained 25 .mu.l of
0.13 IU/ml glucose dehydrogenase, 25 .mu.l of 1.65 IU/ml of
.alpha.-glucosidase, and 20 .mu.l of 12 mM of .beta.-NAD in a total
volume of 200 .mu.l with different combinations of sugars and 0.1M
K.sub.2HPO.sub.4 (pH 7) buffer. Absorbance change with time was
measured at 320 nm.
[0063] Oligosaccharide Utilization by Selected Microorganisms
[0064] The growth of selected bacteria in the presence of
isomaltooligosaccharides was compared by measuring absorbance over
time at 660 nm. The media used for both the Bifidobacteria sp. and
L. johnsonii was of the same composition as Lactobacillus MRS broth
with 0.05% (w/v) cysteine, except the carbon source was replaced by
various oligosaccharide preparations. The growth media for S.
typhimurium and E. coli was tryptic soy broth, with the carbon
source replaced by purified isomaltooligosaccharides. Carbon
sources were supplied at a final concentration of 0.5% (w/v). All
carbon sources were filter sterilized (0.2 .mu.m). The following
carbon sources were compared: glucose (Sigma Chem Co., St. Louis,
Mo.), commercial fructooligosaccharides (FOS; >97.5%, Samyang
Genex Co., Seoul, Korea), and isomaltooligosaccharide preparations.
Individual culture, anaerobic growth tests were conducted in sealed
glass test tubes. Each tube was inoculated from an overnight
culture with either S. typhimurium or E. coli and a 24 to 48 hr
culture of a Bifidobacteria sp. or L. johnsonii. The experiments
with Bifidobacteria sp. and L. johnsonii were conducted under
anaerobic conditions using anaerobic jars (BBL Microbiology Sys.,
Cockeysville, Md.) or the Oxyrase plate system (Oxyrase, Inc.,
Mansfield, Ohio). MRS broth containing 0.05% (w/v) cysteine with
oligosaccharides as a carbon source was used for mixed cultures of
S. typhimurium and L. johnsonii. Total viable counts were conducted
on MRS agar and the cell numbers of S. typhimurium were determined
from growth on MacConkey agar plates (Difco, Detroit, Mich.). The
cell numbers for L. johnsonii were obtained as the difference
between total viable count and S. typhimurium numbers. TLC was used
to determine oligosaccharide consumption patterns of various
strains. The media was MRSB (Difco) for Bifidobacteria and ceca
bacteria, and TSB (Difco) for S. typhimurium and E. coli containing
0.5% (w/v) of Leuconostoc isomaltooligosaccharides instead of
glucose as the carbon source. Media pH was adjusted to 6.0 and 0.1%
(v/v) inoculum grown overnight in MRSB was used. During the growth,
samples were taken at various times. Samples (2 .mu.l) were applied
on TLC plates.
EXAMPLE 2
[0065] Oligosaccharide Production
[0066] Isomaltooligosaccharide (IMO) production by acceptor
reaction
[0067] IMO production by L. mesenteroides ATCC 13146 from sucrose
(10% w/v) and maltose (5% w/v) was followed over time up to about
30 hr. As shown in FIG. 2, IMO production was complete by late log
phase, about 10 hr post-inoculation, and levels did not drop
thereafter. Sucrose disappeared rapidly during log phase of growth,
with sucrose depletion corresponding to the transition point to
stationary phase. Once sucrose was depleted, the accumulated
fructose was metabolized to mannitol with a decrease in growth rate
compared to growth on sucrose. Fructose concentration peaked about
the end of log phase then decreased slowly. Mannitol production
occurred through the lag phase to the stationary phase and was
linked to the fructose concentration where the rate of fructose
disappearance was the inverse of the rate of mannitol formation.
Oligosaccharide production was associated with cell growth. The
conversion of fructose to mannitol was associated with the
accumulation of fructose. Upon completion of fermentation, the cell
mass was 3.2 g/L. The weight % yield of oligosaccharide (product
produced.times.100/[(160.times. mole of sucrose
consumed)+(342.times. mole of maltose consumed)]) was 82% of
theoretical, the number 160 in the equation from 342 (sucrose
M.W.)-((180 (fructose M.W.)+2 (hydrogen M.W.)) and the conversion
of fructose to mannitol was 71% of theoretical.
[0068] Thin layer chromatography (TLC) clearly showed the course of
IMO production. (Data not shown). As fermentation proceeded, mono-
and disaccharides disappeared as the higher DP (degree of
polymerization) polysaccharides were formed. By 24 hr, all mono-
and disaccharides had been converted to higher oligosaccharide
polymers. Four main isomaltooligosaccharides were found. The sizes
of these oligomers were compared with a commercial oligosaccharide
product of known composition. The Leuconostoc
isomaltooligosaccharides were branched polymers with a size range
of DP 2 to 7.
[0069] The oligosaccharides, based on their linkages, showed
different Rf values (Data not shown). The migration of branched
isomaltodextrins containing single .alpha.-1,3 or .alpha.-1,4
linkages, was faster than equivalent dextrins containing only
.alpha.-1,6 linkages, as indicated by J. F. Robyt et al.,
"Separation and quantitative determination of nanogram quantities
of maltodextrins and isomaltodextrins by thin-layer
chromatography," Carbohydr. Res., vol. 251, pp. 187-202 (1994). The
migration of the Leuconostoc oligosaccharides was faster than
equivalent isomaltodextrins (.alpha.-1,6 linkages), but slower than
equivalent maltodextrins (.alpha.-1,4 linkages).
[0070] Oligosaccharide Separation
[0071] The fermentation broth, after cell separation, contained
oligosaccharides, mannitol and some organic acids. Because the
oligosaccharides are neutral polymers, and the other components
(acids, color compounds and salts) are charged, cation resins were
used for separation of oligosaccharides. Neither K.sup.+ nor
H.sup.+ cation columns clearly separated the oligosaccharides from
the mannitol and other products, whereas Ca.sup.2+ cation columns
produced two well separated peaks (Data not shown). When analyzed
by HPLC as described above, the first peak contained all the
isomaltooligosaccharides, and the second peak contained mannitol
and organic acids, e.g., acetic acid and lactic acid (Data not
shown). Based on these results, the process for producing an
oligosaccharide product was developed as shown in FIG. 3.
[0072] Mannitol Separation
[0073] Crystallization at 4.degree. C. separated most of the
mannitol from oligosaccharides as shown by HPLC analysis of the
product at different stages: the broth after deionization, the
broth after mannitol crystallization (86.4% recovered); the
mannitol product (>99.0% purity, calculated by the HPLC peak
area); and the oligosaccharide product after the cation exchange
(Ca.sup.2+ form) column (>98.8% purity). (Data not shown) Pure
oligosaccharide solution (14.5 Brix.degree.) was concentrated to 60
Brix.degree. by evaporation. The concentrated pure
isomaltooligosaccharides (ca. 60% w/v) were used for the further
testing. Table 2 shows the product yields at various stages of the
production.
[0074] Mannitol was a major end product in the Leuconostoc
fermentation. However, mannitol must be separated from the
oligosaccharides if they are to be used as prebiotics, as mannitol
can also be a carbon source for microorganisms. Most of the
mannitol (86.4%) was recovered without further processing by
crystallization at 4.degree. C. To obtain highly purified
oligosaccharides (>98.8%), a cation exchange column was used. A
Ca.sup.2+ resin has high ionic strength and divalent properties,
which may account for the increased resolution seen when it was
used. On a Ca.sup.2+ resin, the oligosaccharides eluted first
followed by a mixture of mannitol and organic acids (lactic acid
and acetic acid). The smaller mannitol molecule eluted after the
oligosaccharides in part because of partial ionization of the
mannitol at the 6.5 pH. Divalent cations such as Ca.sup.2+ bind
strongly to the organic acids. At pH 6.5, which is above the pK
value of lactic and acetic acids, they exist in dissociated forms.
The stronger organic acid that is lactic (pK of 3.79) eluted later
because it interacts more strongly with Ca.sup.2+ than acetic acid,
pK value of 4.7. TABLE-US-00002 TABLE 2 Product yields and process
for production of glucooligosaccharides Process Components in
process Fermentation Input Sucrose 100 g/L (10% w/v) Maltose (5%
w/v) 50 g/L Yeast extract 8.28 g/L and salts ##STR3## Output
Oligosaccharides 80.1 g/L (82.3%.sup.a) Mannitol 37.6 g/L
(70.1%.sup.b) Acids, ethanol 5.75 g/L and cell mass Decolorization
and deionization ##STR4## Removal of color pigments and salts
Evaporation Concentrated to ca. 60% (w/v) Crystallization at
4.degree. C. Oligosaccharides, Mannitol (86.4% residual mannitol
recovered, 99.0% purity) Ca .sup.2+Ion exchange ##STR5## Purified
oligosaccharides 98.8% purity .sup.aWeight % yield of
oligosaccharide (product produced .times. 100/[(160 .times. mole of
sucrose consumed) + (342 .times. mole of maltose consumed)])
.sup.b% fructose conversion to mannitol
EXAMPLE 3
[0075] Composition and Structure of Isomaltooligosaccharide
Products
[0076] Thin layer chromatography (as described in Example 1) showed
that IMO were branched polymers ranging in size from DP 2 to 7
(FIG. 4). By HPLC peak area, there was 6.9% DP 2, 28.4% panose,
36.7% branched DP 4, 19.1% branched DP5, 7.4% branched DP6, and
1.2% branched DP7. In the pure form, there was only a trace amount
of monosaccharides (<0.2%) present, and no polysaccharides
larger than DP 7.
[0077] Structural analysis of IMOs by C.sup.13 NMR (as described in
Example 1) showed that the IMOs are linked mainly by .alpha.-1,4
and .alpha.-1,6 linkages (FIG. 5). These oligosaccharides were
analyzed using a DPX 250 (63 MHz .sup.13C) system. The chemical
shifts in FIG. 5 are expressed in ppm relative to the methyl signal
of acetone in a deuterium oxide solvent, which was used as an
internal standard at .delta.=29.92 ppm. The various signals were
assigned as described by Seymour et al. (1976) and Remaud et al.
(1992): 85-105 ppm, the anomeric region (mainly 97-103 ppm, as
there is only an infinitesimal proportion of reducing sugar in any
of the polymers); 70-75 ppm, C-2,3,4 and 5; 60-70 ppm, bonded and
non-bonded C-6 atoms; 75-85 ppm, signals of bonded C-2, C-3, C-4,
C-5.
[0078] Two closely separated peaks at 100.44 ppm were also
encountered in the spectrum of maltose, and both correspond to a
glucose molecule linked to a reducing residue of maltose by an
.alpha.-1,4 linkage (See Remaud et al., 1992). This also implied
that the .alpha.-1,4 linkage is located at the reducing end of
isomaltosyl residues containing .alpha.-1,6 linkages. The peaks
corresponding to the region of 98.0-99.0 ppm showed .alpha.-1,6
linked residues. However, the intensity of the resonances for
.alpha.-1,3 bonds around 100.0 and 80.6-81.2 ppm were not present
(Dols et al., 1998; Remaud et al., 1992).
[0079] The isomaltooligosaccharides produced were branched polymers
between DP 2 and 7 in size. Prior researchers had reported that the
oligosaccharides synthesized by the dextransucrase from this
bacterium had .alpha.-1,6 backbones with .alpha.-1,3, and/or
.alpha.-1,4-branched side chains when maltose was used as an
acceptor. See Remaud et al. (1992). However, under the current
conditions, the IMOs produced contained mainly .alpha.-1,4 and
.alpha.-1,6 linkages and maltose was linked to the reducing end of
the isomaltosyl residues.
EXAMPLE 4
[0080] Isomaltooligosaccharides as Microbial Growth Modifiers
[0081] Individual Cultures
[0082] Growth of selected bacteria on L. mesenteroides ATCC 13146
isomaltooligosaccharides as a carbon source was compared with
growth on a commercial fructooligosaccharide (FOS) mixture. Both
types of oligosaccharides produced significantly reduced growth of
S. typhimurium and E. coli compared with growth on glucose (Table
3). Based on the TLC analysis of the medium at different times,
these organisms could not use the IMOs efficiently (Data not
shown). There was no significant difference between growth rates on
either of the oligosaccharide preparations. The growth rate
suppression of E. coli in the presence of IMOs was marginally
greater than that of S. typhimurium (Table 3). The growth of
selected probiotic strains on IMOs was also compared. Leuconostoc
IMO supported the growth of Bifidobacterium longum and L. johnsonii
and showed no significant difference when compared to glucose as
carbon source. B. longum degraded almost all components of the IMO
within 24 hrs as shown by TLC (Data not shown). Utilization of the
IMO product by B. bifidium was less rapid (74.9% relative to growth
rate of FOS) than utilization of a commercial FOS and glucose. This
indicates that the growth of probiotic strains was also dependent
on the type of oligosaccharides. TABLE-US-00003 TABLE 3 Growth
comparison on isomaltooligosaccharide preparations: IMO,
Leuconostoc isomaltooligosaccharides; FOS, Commercial
fructooligosaccharides (Samyang Genex Co., Seoul, Korea) Growth
rate in exponential growth phase ([Absorbance Growth rate unit
.times. 100] hr.sup.-1) (on IMO/ Organism glucose IMO FOS glucose)
S. typhimurium 9.89 3.64 3.48 36.8 E. coli 9.35 2.68 2.44 28.7 B.
bifidium 13.30 9.81 13.10 73.8 L. johnsonii 11.06 10.74 10.70 97.1
B. longum 11.72 11.69 11.70 99.7
[0083] Mixed Cultures
[0084] To test for prebiotic effects of the IMO, mixed cultures of
S. typhimurium and L. johnsonii were grown on the oligosaccharides.
FIG. 6 shows the anaerobic growth of the two mixed cultures over 30
hr, as a function of time and medium pH. When the medium pH was
above 5.0, both organisms grew; however, S. typhimurium grew more
slowly than L. johnsonii. As the population of L. johnsonii
increased, the pH dropped. When the pH dropped below 5.0, S.
typhimurium populations decreased until they were below detection
level (<1).
[0085] When S. typhimurium or E. coli was grown on ATCC 13146 IMO
preparations, there was less than 37% of the equivalent growth on
glucose, similar to growth on commercial fructooligosaccharides
(less than 35%). The fact that these bacteria showed similar growth
on IMO and on FOS is surprising based on the literature.
Lactobacillus johnsonii and B. longum showed no differences in
growth rate on glucose or the IMO preparations. When L. johnsonii
and S. typhimurium were grown together on oligosaccharide
preparations, the oligomers stimulated the growth of the
Lactobacillus, but were not readily utilized by the Salmonella. TLC
showed clearly that the IMOs preferentially stimulated the growth
of Bifidobacterium, but were not readily utilized by Salmonella and
E. coli. It appears that these IMOs are selectively favored by some
probiotic strains.
EXAMPLE 5
[0086] Utilization of IMOs by Bacteria Isolated From Chicken
Ceca
[0087] Utilization of Leuconostoc IMOs by six bacterial isolates
(all showed Gram positive, catalase negative and lactic acid
formation from glucose) from chicken ceca was compared to
utilization of a commercial fructooligosaccharide (FOS). Three of
the six bacteria showed more growth after 24 hr on IMOs than on FOS
(Table 4). In mixed cultures of bacteria from chicken ceca, the
cecal bacterial isolates #5 and #6 showed the same use pattern of
IMOs. Only the DP 3 polymer (panose) was utilized in the first 24
hours as shown by TLC (Data not shown). TABLE-US-00004 TABLE 4
Growth comparison of chicken cecal bacteria on various substrates:
glucose, IMO, Leuconostoc isomaltooligosaccharide; and FOS,
Commercial fructooligosaccharides (Samyang Genex Co., Seoul, Korea)
Relative growth to glucose as a carbon source at 24 hr incubation
([Absorbance unit of Glc at 24 hr/ Absorbance unit of .times. 100]
hr.sup.-1) Organism glucose IMO FOS C.B. # 1 100.00 22.77 61.20
C.B. # 2 100.00 99.34 79.29 C.B. # 3 100.00 75.85 48.01 C.B. # 4
100.00 36.72 61.40 C.B. # 5 100.00 87.23 50.25 C.B. # 6 100.00
87.17 87.66 .sup.aGrowth level at stationary phase (at 24 hr) on
glucose was calculated as 100. C.B.; Cecal Bacterium
[0088] To test the potential of this IMO as a prebiotic in poultry,
six different microbial strains were isolated from chicken ceca.
These isolates were identified as lactic acid bacteria by colonial
morphology and chemical reaction (Gram positive, catalase negative,
and lactic acid formation from glucose, data not shown). When
utilization of the Leuconostoc isomaltooligosaccharides by these
isolates was compared with utilization of a commercially available
fructooligosaccharide (FOS), surprisingly three of the six isolates
showed better growth after 24 hr on IMO than on FOS. In tests of
mixed cultures of these lactic acid bacteria and Salmonella on the
IMOs, five of the six cecal isolates showed higher growth rates and
inhibited the growth of Salmonella. Similar results were seen with
Lactobacillus and Bifidobacterium strains. Two isolates showed
identical patterns of consumption of IMO. They only degraded the DP
3 component of the IMO mixture. Similar effects have been seen in
studies on the effect of fructooligosaccharides in feed trials with
broilers where FOS reduced susceptibility of poultry to Salmonella
colonization, increased Bifidobacterium levels, and reduced the
level of Salmonella present in the caecum. See Bailey et al.
(1991); and Chamber et al. (1997).
[0089] The low pH produced by the chicken cecal bacteria is likely
responsible for the observed suppression of S. typhimurium growth
in mixed cultures. Although other antagonistic substances such as
bacteroicins and hydrogen peroxide could be produced that can
inhibit S. typhimurium, significant levels of lactic acid bacteria
must be generated first. These studies did not directly measure in
vivo effects of IMO as produced in this study, but indicated that
this IMO composition can be effective as an avian prebiotic.
EXAMPLE 6
[0090] Inhibition of .alpha.-Glucosidase by IMOs
[0091] .alpha.-Glucosidase and IMO
[0092] The Leuconostoc IMO was found to inhibit the activity of
.alpha.-glucosidase (maltase). FIG. 7 shows the inhibition of
.alpha.-glucosidase activity with increasing concentrations of IMO
(0%, 0.25%, and 0.5%) as measured at various concentrations of
maltose (0, 50, 100, 250 mM). A double reciprocal plot of this data
indicated this was a non-competitive inhibition (FIG. 8).
[0093] In order to determine the role of branching in the
inhibition, the Ki values for panose and isomaltotriose were also
determined. FIGS. 9A and 9B shows the inhibition of
.alpha.-glucosidase activity with increasing concentration of
panose and isomaltotriose, respectively (0%, 0.25%, and 0.5%), as
measured at various concentrations of maltose (0, 50, 100, 250 mM).
A double reciprocal plot of this data is shown in FIGS. 10A and
10B. Only panose, containing .alpha.-1,4 and .alpha.-1,6 linkages,
showed an inhibition on .alpha.-glucosidase. Isomaltotriose, a
linear glucose polymer linked by two .alpha.-(1.fwdarw.6) linkages,
was not inhibitory.
[0094] Growth test of B. longum and S. typhimurium With Panose and
Maltodextrins
[0095] To further determine whether Leuconostoc IMO acts as a
starch-metabolism inhibitor, B. longum and S. typhimurium were
grown in different combinations of panose and maltooligosaccharides
(from DP 4 to DP 10). As the concentration of panose in the growth
medium increased, the growth of S. typhimurium slowed but B. longum
growth increased. (FIGS. 11A and 11B, respectively). A comparison
of growth rates at log phase clearly showed the growth inhibition
of S. typhimurium by panose (FIG. 12). In the case of B. longum,
50% panose+50% maltooligosaccharides and 75% panose+25%
maltooligosaccharides combinations showed better growth than other
combinations.
[0096] L. mesenteroides ATCC 13146 IMOs were found be a
non-competitive inhibitor of .alpha.-glucosidase (maltase). To
verify inhibition of .alpha.-glycosidase by branched oligomers,
panose and isomaltotriose were tested for inhibition. Panose
contains .alpha.-1,4 and .alpha.-1,6 linkages and is one of the
components in the Leuconostoc IMOs. Panose inhibited
.alpha.-glucosidase, whereas isomaltotriose, containing two
.alpha.-1,6 linkages in a linear structure, did not. Panose also
suppressed growth of S. typhimurium but not B. longum. B. longum
showed increased growth when panose and maltodextrins were supplied
in the medium together compared with maltodextrin alone. When
growth rates at early log phase were compared, growth inhibition of
S. typhimurium by panose was clearly evident.
[0097] Isomaltooligosaccharides (branched or partially branched)
most likely inhibit some of those enzymes required for utilization
of starch in other genera, such as Escherichia and Salmonella.
Panose and the Leuconostoc isomaltooligosaccharides reduced the
activity of .alpha.-glucosidase that degrades .alpha.-1,4 linkages
in a maltose or maltodextrin. Panose alone did not produce a higher
growth rate than maltooligosaccharides and panose together for B.
longum. It is likely that high concentrations of panose also can
inhibit enzymes seen in maltooligosaccharide inhibition. There
seems to be a synergistic effect for the probiotic strains on
carbon source utilization when maltodextrin and prebiotic sugars
are present together.
EXAMPLE 7
[0098] Isomaltooligosaccharides as Dietary Supplement for Chicks
Inoculated with Salmonella
[0099] Young chickens were used to test the effectiveness of these
branched isomaltooligosaccharides (IMO) produced as described in
Example 2 as a dietary supplement to reduce Salmonella intestinal
infections. Young chickens (commercial Leghorn broiler chickens on
day of hatch) were orally inoculated with Salmonella using a round
tip cannula attached to a syringe containing nalidixic
acid-resistant Salmonella. This unique strain was used to be able
to distinguish these bacteria from the general population. The
chickens were then divided into four groups to be fed standard
chicken feed (prepared and milled by the poultry department of the
University of Georgia) with added concentrations of IMOs of 0, 1, 2
and 4% (w/w). On day 21, the chickens were sacrificed to examine
the ceca (large intestine) and count the bacterial populations of
Salmonella, Bifidobacteria, Lactobacillus, and total anaerobic
bacteria. In addition, the weight gain efficiency was determined,
and the general condition of the birds noted. There was no
significant difference in weight gain efficiency between chickens
fed IMO in the feed and control. There was a 0. 1 pH unit drop in
cecal pH in the birds receiving IMO at all concentrations.
[0100] Based on the above data (Examples 4 and 5) and the drop in
pH, it is predicted that the IMO-supplemented food will be
effective as a prebiotic, i.e., will increase the numbers of
beneficial bacteria (Bifidobacteria and Lactobacillus) and decrease
the numbers of pathogenic bacteria (Salmonella). Thus
IMO-supplemented food would be useful as an antibiotic for
poultry.
EXAMPLE 8
[0101] Toxicity Study of Isomaltooligosaccharides
[0102] To test whether the isomaltooligosaccharide composition
produced as described in Example 2 is toxic to mammals, young rats
were used and various body organs assayed after several weeks of
feeding IMO-supplemented food. Young male Sprague-Dawley rats
(about 2 months old, mean weight of 270 g) were used. The rats were
divided into four groups of 5 to 6 rats per group. One group (the
control) was fed standard rat chow (Purina rat chow). The other
three groups were fed IMO-supplemented rat chow at a concentration
of 5%, 10%, and 20%, respectively. The food intake and weight gain
was measured twice a week for six weeks. At the end of six weeks,
the rats were sacrificed to examine the weights of the major
organs.
[0103] There were no significant differences in food intake
(although a trend toward an increase in the IMO food intake was
seen; p<0.058). Weight gain, heart weight, spleen weight, kidney
weight, lung weight, brown adipose tissue weight, and white adipose
tissue weight were determined. (Data not shown) There were
significant differences in the weight of the caecum with an
increased weight measured especially in the 10% and 20% IMO groups.
This probably indicates an increase in the population of
fermentation bacteria. Blood was also taken for future
analysis.
[0104] There was also a significant effect of the IMO concentration
on the abdominal fat gain when normalized for food intake. A
significant decrease was seen in abdominal fat with increasing
levels of IMO in the feed. (Table 5) TABLE-US-00005 TABLE 5
Accumulation of abdominal fat in rats after 6 weeks at various
concentration of isomaltooligosaccharides Concentration of
Isomaltooligosaccharides Abdominal fat (gm)/ (gm IMO/gm food) Food
Intake (gm) 0 0.0012 0.05 0.00075 0.10 0.00063 0.20 0.000316
[0105] These data indicate that IMO-supplemented food is non-toxic.
More importantly, this indicates that IMO-supplemented food can
reduce either the formation or deposition of fat. It is also
predicted that the blood glucose level will be less in rats fed the
IMO-supplemented food.
EXAMPLE 9
[0106] Effect of Isomaltooligosaccharides-Supplemented Food on
Blood Glucose
[0107] To determine the effectiveness of IMOs produced as described
in Example 2 on blood sugar levels after eating, rats will be used.
The rats will be fed IMO-supplemented food as described above in
Example 8, and the blood glucose levels monitored overtime after
feeding. It is expected that the blood sugar levels in the IMO-fed
rats will rise at a slower rate than the controls based on the data
above showing that these IMOs are effective .alpha.-glucosidase
inhibitors (Example 6). It is also predicted that the insulin level
will be decreased. This indicates that these IMOs would be
effective therapeutically for diabetes or pre-diabetes.
[0108] The complete disclosures of all references cited in this
application are hereby incorporated by reference. Also,
incorporated by reference is the complete disclosure of the
following documents: Chang-Ho Chung, "A potential Nutriceutical
from Leuconostoc mesenteroides B-742 (ATCC 13146); Production and
Properties," A dissertation submitted to the Department of Food
Science, Louisiana State University, May 2002; and D. F. Day and
Chang-Ho Chung, "Probiotics from Sucrose," a slide presentation at
the May 22, 2002 meeting of the American Society of
Microbiologists. In the event of an otherwise irreconcilable
conflict, however, the present specification shall control.
* * * * *