U.S. patent application number 13/825985 was filed with the patent office on 2014-06-12 for compositions and methods for augmenting kidney function.
This patent application is currently assigned to Kibow Biotech ,Inc.. The applicant listed for this patent is Natarajan Ranganathan. Invention is credited to Natarajan Ranganathan.
Application Number | 20140161780 13/825985 |
Document ID | / |
Family ID | 45938609 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140161780 |
Kind Code |
A1 |
Ranganathan; Natarajan |
June 12, 2014 |
COMPOSITIONS AND METHODS FOR AUGMENTING KIDNEY FUNCTION
Abstract
A protein-rich nutritional food product composed of a probiotic
and an edible isolated protein. The probiotic component may
comprise Lactobacillus, Bacillus, Streptococcus Bifidobacteria,
Saccharomyces or Leuconostoc. The isolated protein may comprise
whey proteins, whey growth factor extract, glutamine peptide, egg
albumen, soy proteins, or caseinates. This nutritional composition
can be in the form of a food product, dietary supplement, or
medical food which, upon ingestion, will promote a healthy
intestinal microenvironment, provide a source of protein, and
assist in the elimination of nitrogenous waste products that can
build up in concentration in the circulating blood. Increased
concentrations of the wastes are known to exert a negative impact
on an individual's physiology and contribute to a decreased sense
of well-being and general malaise. Methods for removing nitrogenous
waste products from the blood and ameliorating renal failure using
the nutritional product of the invention are also provided.
Inventors: |
Ranganathan; Natarajan;
(Broomall, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ranganathan; Natarajan |
Broomall |
PA |
US |
|
|
Assignee: |
Kibow Biotech ,Inc.
Newtown
PA
|
Family ID: |
45938609 |
Appl. No.: |
13/825985 |
Filed: |
September 12, 2011 |
PCT Filed: |
September 12, 2011 |
PCT NO: |
PCT/US2011/051171 |
371 Date: |
April 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12890951 |
Sep 27, 2010 |
|
|
|
13825985 |
|
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|
Current U.S.
Class: |
424/93.44 ;
424/93.1; 424/93.4; 424/93.45; 424/93.46 |
Current CPC
Class: |
A23L 33/17 20160801;
A61K 31/702 20130101; A61P 7/00 20180101; A61K 35/74 20130101; A61K
45/06 20130101; A61K 31/702 20130101; A61P 3/02 20180101; A61K
35/747 20130101; A61K 35/744 20130101; A61K 2300/00 20130101; A61P
13/12 20180101; A23L 33/135 20160801; A61K 2300/00 20130101; A61K
35/74 20130101; A61K 35/745 20130101 |
Class at
Publication: |
424/93.44 ;
424/93.45; 424/93.46; 424/93.4; 424/93.1 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A23L 1/30 20060101 A23L001/30 |
Claims
1. A protein-rich nutritional product comprising at least one
probiotic component and at least one isolated edible protein.
2. The nutritional product of claim 1, wherein the probiotic
component comprises a Lactobacillus, Bacillus, Streptococcus
Bifidobacteria, Saccharomyces or Leuconostoc.
3. The nutritional product of claim 1, wherein the at least one
isolated protein comprises whey proteins or isolates, concentrates
or hydrolysates thereof; milk proteins or isolates, concentrates or
hydrolysates thereof whey growth factor extract; glutamine peptide;
egg albumen; soy proteins or isolates, concentrates or
hydrolysates; or caseinates.
4. A method for removing nitrogenous waste products from the blood
comprising administering to a subject in need thereof an effective
amount of a nutritional product of claim 1 thereby removing
nitrogenous waste products from the blood of the subject.
5. A method for ameliorating renal failure comprising administering
to a subject in need thereof an effective amount of the nutritional
product of claim 1 thereby ameliorating renal failure.
6. The method of claim 5, wherein the subject has a protein
deficiency.
Description
INTRODUCTION
[0001] This patent application claims the benefit of priority from
U.S. patent application Ser. No. 12/890,951, filed Sep. 27, 2010,
the content of which is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] One of the main functions of the normal, healthy kidney,
besides its regulatory, endocrine, and metabolic functions, is the
disposal of waste products. Any impairment of excretory function
can lead to the accumulation of a variety of nitrogenous waste
products including, urea, creatinine and uric acid. High
concentrations of waste products in the blood stream can exacerbate
renal failure and promote kidney stones. Moreover, nitrogenous
solutes in the circulating blood promote osmotic diffusion into the
lumen because of the concentration gradient across the intestinal
wall. This diffusion mechanism led to the concept of oral sorbents
to augment gut-based clearance of nitrogenous waste products.
Sorbents or microbes have demonstrated their ability to remove
various compounds and nitrogenous wastes within the large
bowel.
[0003] Urea-specific sorbents such as synthetic polymers and
modified polysaccharides have been evaluated for the removal of
urea and other nitrogenous wastes via the gut. Other sorbents such
as oxidized starch, activated charcoal, and carob flour have also
been investigated for the in vivo elimination of uremic toxins with
some success. Prakash & Chang ((1996) Nature Medicine 2:883-88)
demonstrated that microencapsulated, genetically-engineered E. coli
DH5 are effective in removing urea and ammonia in an in vitro
system. The same researchers obtained similar results in oral
administration of E. coli DH5 cells in a uremic rat animal model.
Bliss et al. ((1996) Am. J. Clin. Nutr. 63:392-398) have
demonstrated that supplemental gum arabic fiber increases fecal
nitrogen excretion and lowers urea nitrogen concentration in
chronic renal failure patients consuming a low protein diet.
Reinhart et al. ((1998) Rec. Adv. In Canine and Feline Nutr. Lams
Nutrition Symposium Proceedings. Vol. II:395-404) found that canine
renal patients fed a diet containing a fermentable fiber blend
improved clinical end-stage renal disease status, suggesting that
specific nutritional alteration allows repartitioning of nitrogen
excretion away from the kidney and into the feces by colonic
fermentation or additional bacterial growth.
[0004] Prebiotic components are food ingredients that enhance the
actions of probiotic components in the digestive tract. In this
synergistic or relationship a probiotic component, such as
Bifidobacteria, metabolizes undigested carbohydrates, such as
dietary fibers, oligosaccharides, etc., to produce short-chain
fatty acids such as acetate, propionate and butyrate. These
short-chain fatty acids may promote intestinal cell growth, enhance
water and mineral absorption, and prevent yeast, mold, and
pathogenic bacterial growth. In addition, probiotic components may
antagonize pathogens directly through production of antimicrobial
and antibacterial compounds such as cytokines and butyric acid (De
Vuyst and Vandamme. Antimicrobial potential of lactic acid
bacteria. In: De Vuyst L, Vandamme E L, eds. Bacteriocins of lactic
acid bacteria. Glasgow, United Kingdom: Blackie Academic and
Professional; 1994:91-142; Dodd and Gasson. Bacteriocins of lactic
acid bacteria. In: Gasson M J, de Vos W M, eds. Genetics and
biotechnology of lactic acid bacteria. Glasgow, United Kingdom:
Blackie Academic and Professional; 1994:211-51; Kailasapathy and
Chin (2000) Immunol. Cell. Biol. 78(1):80-8), reduce gut pH by
stimulating the lactic acid-producing microflora (Langhendries et
al. (1995) J. Pediatr. Gastroenterol. Nutr. 21:177-81), compete for
binding and receptor sites that pathogens occupy (Kailasapathy and
Chin (2000) Immunol. Cell. Biol. 78(1):80-8; Fujiwara et al.,
(1997) Appl. Environ. Microbiol. 63:506-12), improve immune
function and stimulate immunomodulatory cells (Isolauri et al.
(1991) Pediatrics 88:90-97; Isolauri et al. (1995) Vaccine
13:310-312; Rolfe (2000) J. Nufr. 130 (2S):396S-402S), compete with
pathogens for available nutrients and other growth factors (Rolfe
(2000) J. Nufr. 130 (2S):396S-402S), or produce lactase which aids
in lactose digestion.
[0005] U.S. Pat. No. 4,022,883 discloses a method for alleviating
uremic symptoms in persons suffering from renal failure comprising
administering orally thereto an effective dosage of a cell mass of
a non-pathogenic soil bacteria selected from the group consisting
of an urea degrading bacterium, a creatine degrading bacterium, a
creatinine degrading bacterium and an uric acid degrading bacterium
wherein the urea degrading bacterium is a species of Serratia; the
creatinine degrading bacterium is a non-fluorescent Pseudomonas,
Rhizobium, Agrobacterium, Corynebacterium ureafaciens, Arthrobacter
ureafaciens, E. coli, or Pseudomonas aeruginosa; and a uric acid
degrading bacterium is Bacillus subtilis, a non-fluorescent
Pseudomonas, Bacillus fastidosus, Micrococcus dentrificans,
Mycobacterium phlei, Aerobacter aerogenes.
[0006] U.S. Pat. No. 4,218,541 teaches a method for converting urea
to inocuous products. The method involves obtaining a culture of at
least one microorganism selected from the group of Enterobacter
agglomerans, Group D Streptococcus, Bacilli, and Pseudomonad or
mixtures of said microorganisms and adding the culture to a
composition containing urea.
[0007] U.S. Pat. No. 4,970,153 discloses a method of producing
urease and more particularly a method of producing acid urease by
the cultivation of Lactobacillus fermentum TK 1214. This patent
further teaches the use of such acid urease or the decomposition of
urea contained in fermentation food products.
[0008] U.S. Pat. No. 5,116,737 teaches a method for growing
acid-producing bacterial cultures, such as diary cultures, wherein
the culture is selected to contain a urease-producing strain of
bacteria and the medium used for the culturing contains added urea.
Urease-producing strains of Streptococcus thermophilus and
Bifidobacterium are also disclosed.
[0009] U.S. Pat. No. 5,716,615 discloses a pharmaceutical
composition containing several different bacteria including
Streptococcus thermophilus, Lactobacilli and Bifidobacteria wherein
the bacteria are present in the composition at a total
concentration of 1.times.10.sup.11 to 1.times.10.sup.13 per gram.
An excipient consisting of maltodextrin, microcrystalline
cellulose, maize starch, levulose, lactose or dextrose is further
taught. Methods of using the pharmaceutical composition are also
disclosed which include treatment of a gastrointestinal disorder
and hypercholesteremia or modulating a host's immune response.
SUMMARY OF THE INVENTION
[0010] The present invention is a protein-rich nutritional product
composed of at least one probiotic and at least one isolated edible
protein. In one embodiment, the probiotic component is selected
from the group of a Lactobacillus, Bacillus, Streptococcus
Bifidobacteria, Saccharomyces or Leuconostoc. In other embodiment,
the at least one isolated protein is selected from the group of
whey proteins or isolates, concentrates or hydrolysates thereof;
milk proteins or isolates, concentrates or hydrolysates thereof
whey growth factor extract; glutamine peptide; egg albumen; soy
proteins or isolates, concentrates or hydrolysates; or
caseinates.
[0011] This nutritional composition can be in the form of a food
product, dietary supplement, or medical food which, upon ingestion,
will promote a healthy intestinal microenvironment, provide a
source of protein, and assist in the elimination of nitrogenous
waste products that can build up in concentration in the
circulating blood.
[0012] Increased concentrations of the wastes are known to exert a
negative impact on an individual's physiology and contribute to a
decreased sense of well-being and general malaise. Methods for
removing nitrogenous waste products from the blood and ameliorating
renal failure using the nutritional product of the invention are
also provided.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Nitrogenous waste products accumulating in the blood stream
have detrimental affects on health. Removal of nitrogenous wastes
by diverting them into the colon is a viable approach to decrease
the negative impact that waste product accumulation has on an
individual's physiology. The present invention combines the
properties of a probiotic and edible protein into a product to both
provide a source of protein and effectively reduce the blood
concentration of nitrogenous waste products.
[0014] A probiotic component of the present invention refers to a
mono or mixed culture of live or freeze-dried microorganisms which,
when applied to man or animal, beneficially affects the host by
improving the properties of the indigenous microflora, such as
bifidobacterium organisms that metabolize undigested carbohydrates
and are beneficial to an individual. Probiotic components of the
present invention are selected for their ability to exert a
beneficial effect on the host, survive transit through the
intestinal tract, to adhere to intestinal epithelial cell lining,
to produce of anti-microbial substances towards pathogens and/or to
stabilize the intestinal microflora. Furthermore, a probiotic
component should have a good shelf-life. Products of the present
invention generally contain a large number of viable cells at the
time of consumption, and are non-pathogenic and nontoxic. Examples
of probiotic components include, but are not limited to,
Bifidobacterium spp. (e.g., bifidum, longum, infantis),
Lactobacillus spp. (e.g., bulgaricus, acidophilus, lactis,
helveticus, casei, plantarum, reuteri, delbrueckii, chamnosus,
johnsonii, paracasei), Streptococcus spp. (e.g., thermophilus,
diacetilactis, cremoris, durans, faecalis), Saccharomyces spp.
(e.g., pombe, boulardii), Leuconostoc spp. (e.g., citrovorum,
dextranicum) and Bacillus sp. (e.g., pasteurii). In one embodiment,
the probiotic component is composed of at least one, at least two,
or at least three microorganisms from the genera Bifidobacterium,
Lactobacillus, Streptococcus, Saccharomyces, Leuconostoc and
Bacillus. In another embodiment, the probiotic component is
composed of at least one microorganism from the genera
Bifidobacterium, Streptococcus or Lactobacillus. In a further
embodiment, the probiotic component is composed of at least two
microorganisms from the genera Bifidobacterium, Streptococcus or
Lactobacillus. In a particular embodiment, the probiotic component
is composed of Bifidobacterium longum, Streptococcus thermophilus
and Lactobacillus acidophilus.
[0015] Microorganisms also useful in the invention are those that
have the ability, either through natural selection or by genetic
manipulation, to catabolize various nitrogenous compounds (e.g.,
urea, creatinine, uric acid and ammonia) by expressing or
overexpressing one or more cognate catabolic enzymes. Exemplary
microorganisms are those having an elevated level of urease or
creatininase secretion.
[0016] A microorganism exhibiting elevated levels of catabolic
enzyme secretion can be selected or trained by exposing a selected
microorganism on increasing amounts of the metabolite of interest
(e.g., urea, creatinine, uric acid and ammonia). For example, it
has been found that a standard strain of Streptococcus thermophilus
can be trained to express elevated levels of urease by sequential
passage of the strain on increasing amounts of urea, e.g., a single
colony growing on 0.5% urea is selected and applied to medium
containing 1.0% urea, a single colony growing on 1.0% urea is
selected and applied to medium containing 2.0% urea, etc. Using
such a method, a S. thermophilus strain having the ability to grow
on 5% urea was isolated. This strain proliferated in artificial
intestinal fluid (AIF, US Pharmacopeia) in the pH range of 5.5 to
7.5, characteristic of the colon environment; used urea as a sole
nitrogen source; and catabolized urea in the presence of other
nitrogen sources. It was found that urea hydrolysis was growth- and
pH-dependent and that urea concentrations could be reduced by this
strain from 300 mg/dL to 20 mg/dL within 24 hours at pH 6.3 when
inoculated in AIF at an initial density of 10.sup.9 cfu/mL.
Moreover, this strain survived 3 hours in acidic pH 3.0 with only a
one-log loss in cfu and was able to pass through bile. In addition,
this strain did not appear to exhibit any resistance to eight
commonly used antibiotics. Therefore, these data indicate that a
specifically selected or trained bacterial isolate can be used as a
urea-targeted component in a product of the present invention.
[0017] Elevated levels of secretion can also be obtained by
overexpressing the gene of interest (e.g., via multiple copies or a
promoter driving high levels of expression) in a prokaryotic
microorganism of interest such as Bifidobacterium, Lactobacillus,
Streptococcus, Leuconostoc or Bacillus, or a eukaryotic
microorganism such as Saccharomyces. The gene of interest can be
under the regulatory control of an inducible or constitutive
promoter. Promoters for use in recombinant prokaryotic expression
vectors are well-established in the art and can include the
beta-lactamase (penicillinase) and lactose promoter systems (Chang
et al. (1978) Nature 275:615; Goeddel et al. (1979), Nature
281:544), a tryptophan (trp) promoter system (Goeddel et al. (1980)
Nucleic Acids Res. 8:4057; EPO App. Publ. No. 36, 776) and the tac
promoter (De Boer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21).
While these are commonly used promoters which are commercially
available, one of skill in the art can appreciate that any other
suitable microbial promoter can be used as well. Nucleic acid
sequences encoding suitable prokaryotic promoters have been
published thereby enabling one of skill in the art to readily
isolate these promoters (e.g., by standard cloning or PCR
methodologies) for cloning into plasmid or viral vectors
(Siebenlist et al. (1980) Cell 20:269). The promoter and
Shine-Dalgarno sequence (for prokaryotic host expression) are
operably-linked to the DNA encoding the gene of interest, i.e.,
they are positioned so as to promote transcription of the messenger
RNA from the DNA, and subsequently introduced into a suitable host
cell.
[0018] Eukaryotic microbes such as yeast cultures can also be
transformed with suitable protein-encoding vectors. See e.g., U.S.
Pat. No. 4,745,057. Saccharomyces cerevisiae is the most commonly
used among lower eukaryotic host microorganisms, although a number
of other strains are commonly available. Yeast vectors can contain
an origin of replication from the 2 micron yeast plasmid or an
autonomously replicating sequence (ARS), a promoter, DNA encoding
the desired protein, sequences for polyadenylation and
transcription termination, and a gene encoding for a selectable
marker. An exemplary plasmid is YRp7, (Stinchcomb et al. (1979)
Nature 282:39; Kingsman et al. (1979) Gene 7:141; Tschemper et al.
(1980) Gene 10:157). This plasmid contains the trp1 gene, which
provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, for example ATCC No. 44076 or
PEP4-1 (Jones (1977) Genetics 85:12). The presence of the trp1
lesion in the yeast host cell genome then provides an effective
environment for detecting transformation by growth in the absence
of tryptophan.
[0019] Suitable promoting sequences in yeast vectors include the
promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman
et al. (1980) J. Biol. Chem. 255:2073) or other glycolytic enzymes
(Hess et al. (1968) J. Adv. Enzyme Reg. 7:149; Holland et al.
(1978) Biochemistry 17:4900), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase. Suitable
vectors and promoters for use in yeast expression are commercially
available and further described in Hitzeman et al., EP 73,657.
[0020] As will be understood by those of skill in the art,
expression vectors containing polynucleotides which encode a
degradative enzyme of interest, e.g., a urease or creatininase, can
be designed to contain signal sequences which direct secretion of
enzyme of interest through a prokaryotic or eukaryotic cell
membrane. Such signal sequences are well-established in the art and
can be taken from other enzymes/proteins known to be secreted into
the extracellular environment.
[0021] Transforming the microorganisms as defined herein, describes
a process by which exogenous DNA is introduced into and changes a
recipient cell. It can occur under natural or artificial conditions
using various methods well-known in the art. Transformation can
rely on any known method for the insertion of foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method is
selected based on the type of host cell being transformed and can
include, but is not limited to, viral infection, electroporation,
heat shock, lipofection, and particle bombardment. Such
"transformed" cells include stably-transformed cells in which the
inserted DNA is capable of replication either as an autonomously
replicating plasmid or as part of the host chromosome. This also
includes cells which transiently express the inserted DNA or RNA
for limited periods of time.
[0022] As will be appreciated by the skilled artisan, a
microorganism can also be exposed to a mutagen to cause changes in
the genetic structure of the organism so that it expresses elevated
levels of a catabolic enzyme of interest.
[0023] Transformed or mutagenized strains are subsequently selected
for the ability to grow in the presence of the metabolite which is
degraded by the catabolic enzyme of interest. By way of example, a
strain transformed with nucleic acid sequences encoding a urease is
selected for high levels of urease secretion by growing said strain
on high levels of urea. Levels of urease secretion can also be
detected using standard enzymatic assays. As disclosed herein, the
strain can be sequentially subcultured on increasing levels of urea
to further enhance urease secretion. One embodiment of the present
invention provides a urease-secreting strain of Bacillus pasteurii,
Streptococcus thermophilus or Saccharomyces pombe. In another
embodiment, a urease-secreting strain is at least one of the
microorganisms of a probiotic component composed of at least two or
at least three microorganisms. In a further embodiment, a
urease-secreting strain is at least two of the microorganisms of a
probiotic component composed of at least three microorganisms.
[0024] The probiotics according to the invention can be obtained by
fermentation and can be stored after fermentation and before
addition to the composition of the present invention for a time and
at a temperature that prevents substantial loss of probiotic cfu.
For example, the probiotic component can be fermented until a final
concentration of 10.sup.6 to 5.times.10.sup.10, or 10.sup.7 to
10.sup.10, or 10.sup.8 to 10.sup.9 cfu per mL of fermented medium
is achieved.
[0025] When the probiotic component is a mono culture, said mono
culture is 100% of the probiotic component. When the probiotic
component is composed of at least two microorganisms, each
microorganism can be 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90% of
the probiotic component, wherein the total of all microorganisms is
100%. An exemplary probiotic component is composed of about 10-15%
L. acidophilus, about 10-15% B. longum and about 70-80% S.
thermophilus (e.g., a ratio of approximately 1:1:8,
respectively).
[0026] The probiotic component or urease-secreting microorganism is
included at a concentration of 10.sup.8 cfu/mL, 10.sup.9 cfu/mL,
10.sup.10 cfu/mL, 10.sup.11 cfu/mL, or 10.sup.12 cfu/mL when added
as a liquid or 10.sup.8 cfu/g, 10.sup.9 cfu/g, 10.sup.10 cfu/g,
10.sup.11 cfu/g, or 10.sup.12 cfu/g when added as a freeze-dried
powder. In one embodiment, the probiotic component is about 20% to
about 70% of the total product weight, In particular embodiments,
the probiotic component is about 50% of the total product
weight.
[0027] Edible nutritional proteins and their derivatives provide
specific benefits, in particular in subjects with stage 5 renal
failure who exhibit significant protein loss. For example, whey
protein isolate (WPI) and milk protein isolate (MPI) have been
shown to effectively provide a gain in lean muscle mass. WPI is
high in branched-chain amino acids. MPI is primarily casein, shown
effective in promoting muscle growth. Egg protein (albumen) also is
high in amino acid content. Whey protein hydrosylate (WPH) has been
linked to improved nitrogen retention and growth in rats.
Accordingly, in particular embodiments, the edible protein of the
invention is a whey protein or isolate, concentrate or hydrolysate
thereof; milk protein or isolate, concentrate or hydrolysate
thereof; whey growth factor extract; glutamine peptide; egg
albumen; soy protein or isolate, concentrate or hydrolysate; or
caseinate. In the instant composition, the protein is added as an
ingredient per se and is not sourced from other ingredients such as
peanut pieces. In this respect, the protein is isolated, i.e.,
obtained from its natural source as an extract or purified protein
(e.g., greater than 90% homogenous to the protein with fats and
carbohydrates removed).
[0028] When the protein is a single edible protein, said protein is
100% of the protein component. When the protein component is
composed of two or more protein, each protein can be 5, 10, 15, 20,
30, 40, 50, 60, 70, 80, or 90% of the protein component, wherein
the protein component total is 100%.
[0029] The amount of protein component added to the composition of
the invention is provided in the range of 10 to 50 grams per
serving. In one embodiment, the protein component is not less than
10 grams and not more than 100 grams per serving. In one
embodiment, the protein component is about 20% to about 70% of the
total product weight, In particular embodiments, the protein
component is about 50% of the total product weight. When the
product further includes addition additives, the percent of the
protein can be decreased to accommodate the additional
additive.
[0030] In some embodiments, the composition of the invention
further includes a prebiotic component. A prebiotic refers to a
non-digestive food that beneficially affects the host by
selectively stimulating the growth and/or activity of one or more
non-pathogenic bacteria in the colon. Prebiotic components of the
present invention are considered to have anti-carcinogenic,
anti-microbial, hypolipidemic and glucose modulatory activities.
They can also improve mineral absorption and balance. Furthermore,
bacteria belonging to the Bifidobacterium and Lactobacillus
families are stimulated by the presence of the prebiotic component
and proliferate. Pharmacokinetically, the prebiotic components
reach the colon largely intact. An exemplary prebiotic component
includes, but is not limited to, an oligosaccharide such as
fructo-oligosaccharide or inulin, isomaltose oligosaccharide,
trans-galacto-oligosaccharide, xylo-oligosaccharide, or
soy-oligosaccharide; a pyrodextrin such as arabinogalactan,
lactilol, lactosucrose, or lactulose; or a fiber source such as oat
gum, pea fiber, apple fiber, pectin, guar gum, psyllium husks,
glucomannan or guar gum hydrolysate (BENEFIBER, Novartis
Pharmaceuticals). In one embodiment, the prebiotic component is
composed of at least one, at least two, or at least three
non-digestive foods (e.g., oligosaccharides, pyrodextrins or a
fiber source). In another embodiment, an oligosaccharide is at
least one of the non-digestive foods of a prebiotic component
composed of at least two or at least three non-digestive foods. In
yet another embodiment, a fiber source is at least one of the
non-digestive foods of a prebiotic component composed of at least
two or at least three non-digestive foods. In a further embodiment,
a fiber source is at least two of the non-digestive foods of a
prebiotic component composed of at least three non-digestive foods.
In a still further embodiment, the prebiotic component is composed
of at least one of the following non-digestive foods of lactulose,
psyllium husks and guar gum hydrolysate. In particular embodiments,
the prebiotic component is composed of lactulose, psyllium husks
and guar gum hydrolysate.
[0031] A nutritional product combining the beneficial properties of
a probiotic and edible food protein can include a food product,
dietary supplement, comestible medical food or pharmaceutical
product. The ingestion of said product provides both a protein
source and reduces the blood concentration of nitrogenous waste
products that accumulate in the circulating blood stream. These
waste products of the present invention can be of an endogenous
origin such as normal or abnormal metabolic routes or bacterial
putrefaction. Furthermore, the waste products can be of an
exogenous origin as in dietary intake of proteins and amino acids.
Furthermore, repeated ingestion of the product will have a highly
beneficial effect upon the intestinal microflora by localization
and colonization in the large intestine of microbes known to
promote a healthy intestinal microenvironment.
[0032] A product of the present invention can take the form of a
food product including, but is not limited to, a health bar, health
drink, yogurt, dahi, ice cream, frozen yogurt or other frozen food
product. In addition to containing protein and probiotic
components, the product of the present invention can further
containing various fillers or additives.
[0033] Optional additives of the present composition include,
without limitation, pharmaceutical excipients such as magnesium
stearate, talc, starch, sugars, fats, antioxidants, amino acids,
proteins, nucleic acids, electrolytes, vitamins, derivatives
thereof or combinations thereof. In one embodiment, an additive of
the product is carob flour, for example, locust bean gum. In
another embodiment, an additive is a mushroom extract from Agaricus
bisporus. In particular embodiments, a gel cap contains fillers
such as magnesium stearate, talc and starch.
[0034] Further, to increase the palatability of a food product
containing a protein and probiotic, it may be desirable to add
flavors, sweetening agents, binders or bulking agents.
[0035] Flavors which can optionally be added to the present
compositions are those well-known in the art. Examples include, but
are not limited to, synthetic flavor oils, and/or oils from plants
leaves, flowers, fruits and so forth, and combinations thereof are
useful. Examples of flavor oils include, but are not limited to,
spearmint oil, peppermint oil, cinnamon oil, and oil of wintergreen
(methylsalicylate). Also useful are artificial, natural or
synthetic fruit flavors such as citrus oils including lemon,
orange, grape, lime, and grapefruit, and fruit essences including
apple, strawberry, cherry, pineapple and so forth.
[0036] Sweetening agents can be selected from a wide range of
materials such as water-soluble sweetening agents, water-soluble
artificial sweeteners, and dipeptide-based sweeteners, including
salts thereof and mixtures thereof, without limitation.
[0037] Binders can be selected from a wide range of materials such
as hydroxypropylmethylcellulose, ethylcellulose, or other suitable
cellulose derivatives, povidone, acrylic and methacrylic acid
co-polymers, pharmaceutical glaze, gums (e.g., gum tragacanth),
milk derivatives (e.g., whey), starches (e.g., corn starch) or
gelatin, and derivatives, as well as other conventional binders
well-known to persons skilled in the art. Examples of bulking
substances include, but are not limited to, sugar, lactose,
gelatin, starch, and silicon dioxide.
[0038] When the above-mentioned additives are included in the
product of the present invention, they are generally less than 15%
of the total product weight. In particular embodiments, they are
less than 5 to 10% of the total product weight.
[0039] Depending on whether the product is to be consumed by an
adult human, child or animal (e.g., companion animal or livestock),
it can be produced in various sizes and with various ingredients
suitable for the intended recipient. Further, because the probiotic
and protein components of the present invention are generally
recognized as safe, they can be consumed one, two or three times
daily or more.
[0040] The present invention also relates to a method for removing
nitrogenous waste products from the blood of an individual with
elevated levels of nitrogen-containing waste products. The method
involves administering an effective amount of a product of the
present invention so that the levels of nitrogenous waste products
in the blood are decreased or reduced, desirably to a normal range.
For example, normal levels of creatinine in the blood are in the
range of 0.6-1.2 mg/dL, whereas normal blood urea nitrogen (BUN)
levels range from 7-18 mg/dL and normal uric acid levels in males
and females is in the range of 2.1 to 8.5 mg/dL and 2.0 to 7.0
mg/dL, respectively. Further, a BUN/creatinine ratio of 5-35 is
indicative of normal levels of nitrogenous waste products in the
blood. As one of skill in the art can appreciate, means for
determining the levels of nitrogenous wastes are well-known to the
skilled laboratory clinician.
[0041] As products of the present invention can reduce the levels
of nitrogenous waste products in the blood of a mammal with chronic
renal failure, these compositions are useful in a method for
ameliorating renal failure. The method involves administering a
product of the present invention to a subject having or at risk of
having renal failure. Subjects having or at risk of having renal
failure include those with diabetic nephropathy, hypertensive
nephrosclerosis, glomerulonephritis, interstitial nephritis, or
polycystic kidney disease wherein nephron function is impaired
thereby decreasing glomerular filtration rate. Desirably, an
effective amount of a product for ameliorating renal failure is an
amount sufficient to effect beneficial or desired results,
including clinical results, and, as such, an effective amount of a
product is one which results in the alleviation or amelioration of
one or more symptoms associated with renal failure (e.g., a build
up of uremic solutes), diminishment of extent of disease,
stabilized (i.e., not worsening) state of disease by supporting
healthy bowel function, delay or slowing of disease progression, or
amelioration or palliation of the disease state. Amelioration can
also mean prolonging survival as compared to expected survival if
not receiving treatment. In particular embodiments, the instant
composition is administered to a subject with renal failure and a
protein deficiency.
[0042] It is further contemplated that compositions of the present
invention containing proteins and probiotics may have further
utility in the providing energy and overall health and well-being
in subjects undergoing cancer therapy.
Example 1
Yogurt Food Product
[0043] Yogurt or frozen yogurt can be prepared from one gallon of
commercially available whole, homogenized, pasteurized milk which
is heated to boiling and quickly allowed to cool to approximately
45.degree. C. To this is added approximately one ounce of yogurt
starter culture containing lactic acid bacteria of the genus
Lactobacillus, Streptococcus, Bacillus or Bifidobacteria. The
mixture is mixed well and allowed to ferment at 37.degree. C. for
10 to 12 hours. One or more proteins, prebiotics, whole fruit
additives, flavoring, sweetening agents, binders, or other
additives can be combined and added to the yogurt to obtain a
product of desired consistency or to suit the palette of the
prospective consumer. In one embodiment of the present invention, a
food product comprises components to meet the special dietary needs
of individuals with renal insufficiency.
Example 2
Health Bar
[0044] Health bars can be prepared by combining various excipients,
such as binders, additives, flavorings, colorants and the like,
along with the probiotic (e.g., Lactobacillus, Streptococcus,
Bacillus or Bifidobacteria) and protein such as soy protein
isolate, and mixing to a plastic mass consistency. The mass is then
either extruded or molded to form "candy bar" shapes that are then
dried or allowed to solidify to form the final product.
Example 3
Medical Food
[0045] A medical food can be prepared by combining rolled oats,
dehydrated apples, honey, inulin, carob flour, cinnamon, sugar,
vanilla extract, and protein such as whey protein isolate, and
lyophilized cultures of L. acidophilus and or L. fermentum, a
Bifidobacteria, and Streptococcus thermophilus (10.sup.8-10.sup.10
cfu each). These ingredients are mixed in appropriate proportions
with a prebiotic and formed into a rectangular bar approximately
12.5 to 15 centimeters in length, 3 to 4 centimeters in width and 1
centimeter in height and placed into a sterile vacuum oven for 12
to 24 hours to obtain an edible food product of the desired
consistency.
Example 4
Minipig Model of Chronic Uremia
[0046] The effects of a composition of the present invention in the
form of a gel cap product or formulation admixed with the pig chow
were tested using an established model of chronic uremia (mild to
moderate) in the Gottingen strain of miniature swine (Willis, et
al. (1997) J. Endourol. 11(1):27-32). At sexual maturity (3 months
of age) these pigs weigh 8-11 kg, and are about half the size of
all other strains of minipigs. The pigs grow slowly and double
their body weight in about 6 months.
[0047] In this uremic model, of the renal mass is removed through
bilateral flank incisions (McKenna, et al. (1992) J. Urol. 148 (2
Pt 2):756-9). One kidney is entirely removed, and both poles of the
contra lateral kidney are removed with the aid of electro-cautery
(for scoring the renal capsule and about 2 mm of parenchyma),
surgical staples, and Gel foam sutured over the exposed parenchyma
of the remnant. The contra lateral kidney is removed during the
same surgical procedure by gross dissection and ligation and
section of the renal artery and vein and ureter. The pigs used in
this analysis characteristically experienced a large, acute
elevation of creatinine and Blood Urea Nitrogen (BUN)
concentrations in plasma; followed by a decline over 1-2 weeks to
values that stabilized well above baseline. These pigs maintained
their appetite and stable uremia for 3-6 months, maintained or
gained body weight, and behaved no differently than normal control
pigs. Hematocrits declined slowly as the uremia progressed and
hemoglobin concentrations also declined over time. After the
surgery, the pigs were allowed to recover for 3-4 weeks prior to
administering the compositions.
[0048] Using this model, several different blinded formulations in
the form of gel caps or admixed with the pig chow were tested as
gut-based therapy for uremia. Formulations administered with the
pig chow did not demonstrate any significant differences either in
BUN or creatinine values. However, .sup.th nephrectomized mini pigs
(n=6) given a formulation containing four microbial strains (L.
acidophilus, L. bulgaricus, B. longum and S. thermophilus;
10.times.10.sup.9 CFU/gel cap) exhibited continued body weight
gains of approximately 31% and decreased BUN and creatinine levels
of approximately 13% each. Similarly, two .sup.th nephrectomized
mini pigs given a formulation containing three microbial strains
(L. acidophilus, B. longum and S. thermophilus; 10.times.10.sup.10
CFU/gel cap) also showed a decrease in BUN (average 21%) and
creatinine (average 29%) levels although the body weight of one
mini pig increased (6%) and the other decreased (20%). Additional
formulations were tested in the form of gel caps or admixed with
food and in general the results indicated that oral treatment with
a probiotic formulation effectively and significantly reduced
plasma creatinine concentrations by 22.5.+-.8.5% in a stable
porcine model of chronic renal failure. Small sample size may have
prevented detection of corresponding reductions in BUN and
elevation of hematocrit.
Example 5
Animal Dosing
[0049] Table 1 provides a suitable approximate serving dosage of a
nutritional product for administration to an animal such as a
companion animal.
TABLE-US-00001 TABLE 1 Weight pounds (Kg) Morning Dose Evening Dose
Less than 2.2 lbs 1 0 (<1 Kg) 2.2-4.4 lbs 1 1 (1-2 Kg) 4.4-8.8
lbs 2 1 (2-4 Kg) 8.8-17.6 lbs 2 1-2 (4-8 Kg) 17.6-35.2 lbs 2 2
(8-16 Kg) 35.2-70.4 lbs 2-3 2-3 (16-32 Kg) More than 70.4 lbs 3 3
(>32 Kg)
Example 6
Urease-Secreting Strains of Streptococcus thermophilus
[0050] This example discloses the isolation and selection of a high
level urease-secreting strain of Streptococcus thermophilus. Three
isolates of gram-positive, lactic acid-producing non-pathogenic
cocci of Streptococcus thermophilus were isolated from various
sources and designated KB4, KB19, and KB25. KB4 was isolated from a
probiotic product, KB19 was isolated from a commercial yogurt
product and KB25 from Dahi yogurt (from India).
[0051] Growth rates and urea hydrolysis of these bacteria in the
intestinal pH range (pH 5.5, 6.3 and 7.5) were determined by
transferring exponentially growing cultures of KB19, KB4 and KB25
into modified Artificial Intestinal Fluid M2 (AIF, US Pharmacopeia)
supplemented with 100 mg/dL filter-sterilized urea, 100 .mu.M
NiCl.sub.2, 10% MRS broth, dextrose to final concentration of 1%,
and 0.3% yeast extract, wherein the initial cell density was
10.sup.9 cfu/mL. Pancreatin was omitted from the recipe to allow
the evaluation of bacterial growth by direct OD600 nm measurement.
Urea concentration in the supernatants (% of control) and growth
(OD600 nm) were measured every 4 hours.
[0052] Concentration of urea in the supernatants of bacterial
cultures was measured using the protocol and standards supplied
with the Blood Urea Nitrogen Reagent Kit (535, SIGMA, St. Louis,
Mo.). Urea hydrolysis was monitored by comparing urea-nitrogen
concentrations in bacterial supernatants to appropriate control
medium incubated in the same conditions and expressed as percent of
control. Four to nine independent experiments were conducted and
Student t-test was used for statistical analysis.
[0053] Under similar assay conditions, exponentially growing
cultures of KB19, KB4 and KB25 were inoculated into AIF M2, pH 6.3,
supplemented with 100 mg/dL urea and with or without 100 .mu.M
NiCl.sub.2 at initial cell density of 10.sup.9 cfu/mL to determine
whether the growth and rates of urea hydrolysis by these strains
was dependent on the additional Ni++. Urea concentration in the
supernatants as a % of control and growth (OD600 nm) were measured
every 4 hours. Four to nine independent experiments were conducted
and Student t-test was used for statistical analysis. Similarly, it
was determined whether the growth and rate of urea hydrolysis of
these strains was dependent on urea concentration. Under similar
growth conditions exponentially growing cultures of KB19, KB4 and
KB25 were inoculated into AIF M2, pH 6.3, supplemented with 100
.mu.M NiCl.sub.2 and 100, 200, or 300 mg/dL urea. Urea
concentration in the supernatants as a % of the control and growth
(OD600 nm) were measured every 4 hours. Four to nine independent
experiments were conducted and Student t-test was used for
statistical analysis. The survivability of these KB19, KB4 and KB25
was determined in artificial gastric juice in the presence and
absence of urea and dextrose. The average loss in viable cell count
after exposure to artificial gastric juice (logs cfu/mL) is shown
in Table 2.
TABLE-US-00002 TABLE 2 PH/Additive KB19 KB4 KB25 1.4 7 7 7 2.0 7 7
7 2.5 3 4 4 2.5/Urea 3 4 4 2.5/Dextrose 3 3 3 2.5/Urea + Dextrose 3
3 3 3.0 2 2 3 3.0/Urea 2 3 3 3.0/Dextrose 1 2 3 3.0/Urea + Dextrose
1 2 2 Initial cell density was 10.sup.7 cfu/mL. Urea and dextrose
concentrations were 10 mg/mL and 1%, respectively.
[0054] Further it was determined whether the nutrient composition
and availability had an affect on growth and urea hydrolysis by
KB19, KB4, and KB25. Each strain was grown for 24 hours at
37.degree. C. and pH 6.0 in the presence or absence of 100 mg/dL of
urea and combinations of nitrogen and carbon sources.
[0055] Further analysis of S. thermophilus KB19 indicated that this
strain could survive a 3 hour exposure to gastric juice, pH 3.0,
followed by a 3 hour exposure to 0.3% oxgal, pH 6.0, with only 1
log loss in viability. Remaining viable cells were able to
proliferate in AIF M2, pH 6.0, supplemented with 230 mg/dL urea and
completely hydrolyzed the urea within less than 18 hours (n=4). All
test solutions were supplemented with 230 mg/dL urea and 1%
dextrose. Collectively, these analyses indicated that all three
strains studied proliferated in the fed state AIF medium in the pH
range from 5.5 to 7.5, characteristic of colon environment; they
could all use urea as a sole nitrogen source; and they each
catabolized urea in the presence of other nitrogen sources. Urea
hydrolysis was growth and pH dependent. Under the conditions
tested, the rate of urea hydrolysis was strain-dependent in tests
of pH stability: KB19=KB25>KB4; Ni requirement:
KB25>KB19>KB4; urea hydrolysis for over 300 mg/dL:
KB19=KB25>KB4; and specific nutrients: KB19>KB25>KB4.
Further, there was strain-dependent results relating to
survivability, wherein in tests of gastric juice stability:
KB19>KB4>KB25; and bile stability: KB19>KB4>KB25.
[0056] In view of the desirable traits exhibited by S. thermophilus
KB19 it was further determined, using a disc-diffusion test, that
K19 was sensitive to Spectinomycin (100 .mu.g), Kanamycin (30
.mu.g), Chloramphenicol (30 .mu.g), Spectinomycin (100 .mu.g),
Penicillin (10 IU), Carbenicillin (100 .mu.g), Doxycycline (30
.mu.g), and neomycin (30 .mu.g). To further enhance the levels of
urease secreted by strain K19, this strain was trained on
increasing levels of urea as described herein.
* * * * *