U.S. patent application number 16/676021 was filed with the patent office on 2020-05-07 for food grade bacteria for the removal of toxic compounds.
This patent application is currently assigned to LONDON HEALTH SCIENCES CENTRE RESEARCH INC.. The applicant listed for this patent is LONDON HEALTH SCIENCES CENTRE RESEARCH INC. COMPAGNIE GERVAIS DANONE. Invention is credited to Jordan BISANZ, Jeremy BURTON, Marc MONACHESE, Gregor REID, Tamara SMOKVINA, Johan VAN HYLCKAMA VLEIG.
Application Number | 20200140806 16/676021 |
Document ID | / |
Family ID | 48471060 |
Filed Date | 2020-05-07 |
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United States Patent
Application |
20200140806 |
Kind Code |
A1 |
BISANZ; Jordan ; et
al. |
May 7, 2020 |
FOOD GRADE BACTERIA FOR THE REMOVAL OF TOXIC COMPOUNDS
Abstract
The present invention relates to food-grade bacteria and methods
for removing toxic compounds, including lead, cadmium, mercury,
arsenic and pesticides, from contaminated environments or
substances.
Inventors: |
BISANZ; Jordan; (St. Thomas,
CA) ; REID; Gregor; (Komoka, CA) ; MONACHESE;
Marc; (Oakville, CA) ; VAN HYLCKAMA VLEIG; Johan;
(Marly Le Roi, FR) ; SMOKVINA; Tamara; (Orsay,
FR) ; BURTON; Jeremy; (London, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LONDON HEALTH SCIENCES CENTRE RESEARCH INC.
COMPAGNIE GERVAIS DANONE |
London
Paris |
|
CA
FR |
|
|
Assignee: |
LONDON HEALTH SCIENCES CENTRE
RESEARCH INC.
London
ON
COMPAGNIE GERVAIS DANONE
Paris
|
Family ID: |
48471060 |
Appl. No.: |
16/676021 |
Filed: |
November 6, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14390685 |
Oct 3, 2014 |
10487305 |
|
|
PCT/CA2013/000328 |
Apr 5, 2013 |
|
|
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16676021 |
|
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61620796 |
Apr 5, 2012 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 39/00 20180101;
A23K 20/00 20160501; A23Y 2220/73 20130101; A61K 35/747 20130101;
C12N 15/86 20130101; C12R 1/225 20130101; C12N 15/102 20130101;
A23C 9/1234 20130101; A61K 38/00 20130101; C12N 1/20 20130101; A61K
35/74 20130101; C12N 15/746 20130101; A23K 20/10 20160501; C12N
15/70 20130101; A62D 3/02 20130101; A23K 10/18 20160501 |
International
Class: |
C12N 1/20 20060101
C12N001/20; C12R 1/225 20060101 C12R001/225; A23C 9/123 20060101
A23C009/123; C12N 15/74 20060101 C12N015/74; A61K 35/747 20060101
A61K035/747; A62D 3/02 20060101 A62D003/02 |
Claims
1.-35. (canceled)
36. A method for reducing a subject's uptake of toxic compounds
consumed by the subject, the method comprising administering to the
subject a Lactobacillus capable of sequestering the toxic compound
consumed by the subject wherein the Lactobacillus is selected from:
Lactobacillus reuteri RC-14, Lactobacillus casei 21052,
Lactobacillus casei 393T, Lactobacillus rhamnosus GR-1,
Lactobacillus rhamnosus R3, Lactobacillus rhamnosus R37,
Lactobacillus johnsonii 20553, Lactobacillus plantarum 14917T, or
any combination thereof.
37. The method of claim 36, wherein the toxic compound is selected
from the group consisting of: lead, cadmium, arsenic, malathion and
parathion.
38. The method of claim 36, wherein the Lactobacillus is provided
in a viable form.
39. The method of claim 36, wherein the Lactobacillus is provided
in a non-viable form.
40. The method of claim 36, wherein the Lactobacillus is provided
as an extract.
41. The method of claim 36, wherein the Lactobacillus comprise a
combination of two or more different strains of Lactobacillus.
42. The method of claim 36, wherein the lactobacillus is provided
in a composition comprising the Lactobacillus and a suitable
carrier.
43. The method of claim 42, wherein the carrier is a milk-based
product.
44. The method of claim 42, wherein the composition comprises a
combination of two or more strains of Lactobacillus rhamnosus,
Lactobacillus casei, Lactobacillus crispatus, Lactobacillus
fermentum, Lactobacillus johnsonii, Lactobacillus plantarum,
Lactobacillus reuteri, and Lactobacillus amylovorus.
45. A method for reducing in a subject gastrointestinal uptake of
toxic compounds consumed by the subject through edible or drinkable
substances contaminated with the toxic compounds, the method
comprising administering to the subject a Lactobacillus, wherein
the Lactobacillus is selected from: Lactobacillus reuteri RC-14,
Lactobacillus casei 21052, Lactobacillus casei 393T, Lactobacillus
rhamnosus GR-1, Lactobacillus rhamnosus R3, Lactobacillus rhamnosus
R37, Lactobacillus johnsonii 20553, Lactobacillus plantarum 14917T,
or any combination thereof.
46. The method of claim 45, wherein the toxic compound is selected
from the group consisting of: lead, cadmium, arsenic, malathion and
parathion.
47. The method of claim 45, wherein the Lactobacillus is provided
in a viable form.
48. The method of claim 45, wherein the Lactobacillus is provided
in a non-viable form.
49. The method of claim 45, wherein the Lactobacillus is provided
as an extract.
50. The method of claim 45, wherein the Lactobacillus comprise a
combination of two or more different strains of Lactobacillus.
51. The method of claim 45, wherein the lactobacillus is provided
in a composition comprising the Lactobacillus and a suitable
carrier.
52. The method of claim 51, wherein the carrier is a milk-based
product.
53. The method of claim 51, wherein the composition comprises a
combination of two or more strains of Lactobacillus rhamnosus,
Lactobacillus casei, Lactobacillus crispatus, Lactobacillus
fermentum, Lactobacillus johnsonii, Lactobacillus plantarum,
Lactobacillus reuteri, and Lactobacillus amylovorus.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/390,685 filed Oct. 3, 2014, which in turn is a national
stage application under 35 U.S.C. 371 of International Application
No. PCT/CA2013/000328, filed 5 Apr. 2013, which in turn claims the
benefit under 35 U.S.C. 119(e) of U.S. Provisional Ser. No.
61/620,796, filed Apr. 5, 2012, the contents of each of which are
hereby incorporated by reference into the present disclosure.
FIELD OF THE INVENTION
[0002] The present invention relates to food grade bacteria for
improving detoxification. More particularly, the present invention
relates to food grade bacteria, or extracts thereof, and to methods
of using food grade bacteria or extracts thereof to reduce uptake
of ingested toxic compounds and to methods of sequestering toxic
compounds from the environment to which the food-grade bacteria is
exposed to.
BACKGROUND OF THE INVENTION
[0003] Humans and animals in general, are exposed to many toxic
compounds that contaminate the environment, food chain, water
supply and various items that are part of everyday life. These
range in number, type and exposure from ingredients in toothpaste
and shampoos to drugs and pathogens in well-water. Amongst Canadian
First Nation and Inuit populations, environmental toxins are risk
factors for other highly prevalent diseases, especially type 2
diabetes [Sharp D. Environmental toxins, a potential risk factor
for diabetes among Canadian Aboriginals. Int J Circumpolar Health.
2009; 68(4):316-26]. A large over-the-counter consumer market has
arisen under the guise of `detox`, but most of the products have no
rationale or clinical evidence to support their use. The concept of
detox has great appeal to consumers, both the health-conscious and
others concerned with the growing number of stories in the media
about pollution and diseases related to toxic substances. Thus,
there is substantial interest in this area, few effective products
and a growing need.
[0004] The replenishment or boosting of the beneficial organisms
through administration of probiotics has become feasible in Canada
relatively recently, and has led to much interest amongst consumer
and healthcare professionals. Indeed, probiotics are one of the
fastest growing food segments in North America. However, gaining
insight into the mechanisms by which indigenous microbes and
exogenous probiotics affect the subject has been limited.
[0005] Probiotic Lactobacilli and bifidobacteria have been shown to
help manage several gut pathologies. For example, U.S. Pat. No.
6,641,808 disclosing the use of Lactobacilli for the treatment of
obesity; U.S. Pat. No. 5,531,988, discloses a mixture of an
immunoglobulin and a bacterium, such as Lactobacilli or
bifidobacterium or mixtures thereof, that may be used to treat
diarrhea, constipation, and gas/cramps; U.S. Pat. No. 6,080,401
discloses a combination of probiotics having Lactobacillus
acidophilus and Bifidobacterium bifidus and herbal preparations for
aiding in weight loss, and so forth.
[0006] The ability of probiotic products to ameliorate toxins has
been much less studied, but nevertheless has some foundation. For
example, Lactobacilli and/or bifidobacteria have been found to
alter the subject's intestinal metabolic signature [Ndagijimana, M.
Laghi L, Vitali B, Placucci G, Brigidi P, Guerzoni M E. Effect of
synbiotic food consumption on human gut metabolic profiles
evaluated by 1H nuclear magnetic resonance spectroscopy. Int J Food
Microbiol. 2009; 134: 147-153]; bind to aflatoxin (Lactobacillus
strains) [Hernandez-Mendoza A, Garcia H S, Steele J L. Screening of
Lactobacillus casei strains for their ability to bind aflatoxin B1.
Food Chem Toxicol. 2009; 47(6):1064-8]; and detoxify or bind and
negate other mycotoxins (B. animalis) [Fuchs S, Sontag G, Stidl R,
Ehrlich V, Kundi M, Knasmuller S. Detoxication of patulin and
ochratoxin A, two abundant mycotoxins, by lactic acid bacteria.
Food Chem Toxicol. 2008; 46(4):1398-407].
[0007] In summary, the problem associated with toxic compounds is
real, and of growing concern to consumers.
Heavy Metals
[0008] Heavy metal toxicity is one of the largest health risks in
the 21st century. Consumption of lead and cadmium through
environmental exposure and diet has been directly responsible for
poor health outcomes including: impaired neurological function and
loss of IQ, osteoporosis, lung and kidney cancer.
[0009] Heavy metals such as lead and cadmium are present in the
natural environment, and therefore many bacteria over time have
developed mechanisms of resistance to these metals which generally
include actively precipitating and sequestering the metals
intra/extra cellular or the active efflux of metals out of the cell
cytoplasm. Non-food grade bacteria have been investigated for their
use in sequestration and detoxification of heavy metals and have
shown success (JS Singh et al. Genetically engineered bacteria: An
emerging tool for environmental remediation and future research
perspectives. Gene. July 2011. 40 (1-2):1-9); Rajkumar et al.
Potential of siderophore-producing bacteria for improving heavy
metal phytoextraction. Trends Biotechnol. March 2010. 28
(3):142-149).
Mercury
[0010] Mercury is one of the most toxic substances known to man and
its consumption by a subject is linked to poor health outcomes
including altered neurological development in children. Yet, North
Americans and Europeans are estimated to consume 6.7 .mu.g daily of
inorganic mercury and methylmercury (World Health Organization,
1991).
[0011] Mercury is present in the natural environment, and as such,
many bacteria have adopted mechanisms of resistance to it, which
generally reduce mercury levels in the surrounding environment.
Many non-food grade bacteria have been investigated for their use
in sequestration and detoxification of mercury and mercury
compounds in the environment, however the application of food grade
bacteria has not been demonstrated to date.
Arsenic
[0012] Arsenic is a metalloid element which commonly comes in two
oxidation states: arsenate (As V) and arsenite (As III). Arsenic is
found distributed globally often in the earth's crust, it is highly
soluble in water and is found in high concentrations in ground
water. Arsenic toxicity has been linked to a number of cases and is
known to cause organ failure, cancer and death. Main routes of
exposure is through ingestion via diet, often arsenic contaminated
waters are used for irrigation of farmland resulting in
accumulation of the metal in plants and food.
Pesticides
[0013] Pesticides such as malathion and parathion fall into the
class of organophosphate compounds and act as cholinesterase
inhibitors. Malathion is one of the most widely used pesticides in
the U.S., and parathion use has recently been limited and is not
used in many developed nations due to high toxicity. However,
produce imports still consistently detect levels of parathion on
produce and it is used in some rare instances in North America.
[0014] Major routes of public exposure is through consumption via
diet. Agricultural workers and industrial workers are at increased
risk of exposure through work place by absorption or inhalation if
safety protocols not properly followed.
[0015] In view of the problems associated to the exposure of any of
the above toxic compounds, it would be advantageous to provide for
food grade bacteria that can sequester toxic compounds, including
heavy metals, mercury, arsenic, pesticides, such as malathion and
parathion, or a combination thereof, from the gastrointestinal
tract of a subject to reduce the amount of the toxic compound
available to be absorbed by the subject, while detoxifying the
toxic compounds directly reduces the toxicity of toxic compounds
available to be absorbed by the subject.
SUMMARY OF THE INVENTION
[0016] It is an object of the present invention to provide for
food-grade bacteria or extracts thereof for the removal and/or
neutralization of toxic products from an environment or from a
substance to which the food-grade bacteria is exposed to, that
solve the deficiencies inherent in traditional detoxification
treatments. The present invention provides methods and uses of food
grade bacteria for removal and/or neutralization of toxic products
found in the internal environment of animals, in the environment to
which the animal is exposed or in substance ingested or to be
ingested by the animals that may avoid adverse side effects, is
reasonable in cost, and may be beneficial in reducing the risk of
diseases related to said toxic products. Further, the present
invention is relatively easy to manufacture and deliver to a
subject.
[0017] It is an object of the present invention to provide for food
grade bacteria, or extracts thereof, to detoxify and/or sequester
toxic compounds, including heavy metals, mercury, arsenic and
pesticides, with the application of reducing a subject's toxic
compounds exposure and uptake.
[0018] As such, in one embodiment, the present invention provides
food-grade bacteria or extracts thereof for removing of toxic
compounds from a substance or environment to which the food-grade
bacteria is exposed to.
[0019] In one embodiment, the present invention provides for a
composition comprising a food-grade bacteria and a suitable
carrier, whereby the composition comprises an effective dose of the
food-grade bacteria to remove a toxic compound from a substance or
environment to which the food-grade bacteria is exposed to.
[0020] In one embodiment of the composition of the present
invention, the therapeutically effective dose is at least about
1.times.10.sup.9 of the food-grade bacteria per milliliter or less
of the suitable carrier.
[0021] In another embodiment of the composition of the present
invention, the suitable carrier is a carbohydrate-containing
medium.
[0022] In another embodiment of the composition of the present
invention, the carbohydrate-containing medium is a milk-based
product.
[0023] In another embodiment of the composition of the present
invention, the toxic compound is selected from the group consisting
of lead, cadmium, mercury, arsenic, malathion and parathion.
[0024] In another embodiment of the composition of the present
invention, the food-grade bacteria are provided dead or live.
[0025] In another embodiment of the composition of the present
invention, the food-grade bacteria are provided as an extract.
[0026] In another embodiment of the composition of the present
invention, the composition comprises a combination of two or more
different species of food-grade bacteria.
[0027] In another embodiment of the composition of the present
invention, the composition comprises a combination of two or more
strains of Lactobacillus rhamnosus, Lactobacillus casei,
Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus
johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and
Lactobacillus amylovorus.
[0028] In another embodiment of the composition of the present
invention, the food-grade bacteria is selected from the group of
food-grade bacteria listed in Table 1 shown bellow. It is mentioned
that a bacteria strain of interest is the Lactobacillus rhamnosus
strain deposited, according to the Budapest Treaty, at CNCM
(Collection Nationale de Cultures de Microorganismes, 25 rue du
Docteur Roux, Paris) on Mar. 5, 2013, under the accession number
CNCM 1-4719. This strain is also referred to as "DN 116-060" or
R37.
[0029] In another embodiment of the composition of the present
invention, the environment is an aqueous environment.
[0030] In another embodiment, the present invention is a
composition, the composition including food-grade bacteria, a
carrier and an animal's feed, wherein the food-grade bacteria is
capable of removing a toxic compound from a substance or
environment to which the food-grade bacteria is exposed to and the
food-grade bacteria comprises a bacterial isolate selected from the
group consisting of the food-grade bacteria listed in Table 1 or
any combination thereof.
[0031] In one embodiment, the present invention is a method for
reducing a subject uptake of toxic compounds consumed by the
subject, the method including administering to the subject an
effective dose of a food-grade bacteria capable of sequestering the
toxic compound consumed by the subject.
[0032] In another embodiment, a method for removing a toxic
compound from a substance or environment which is contaminated or
suspected of being contaminated with the toxic compound is
provided, the method including contacting the substance or
environment with food-grade bacteria capable of removing the toxic
compound from the substance or the environment.
[0033] In one embodiment, the present invention is a method of
reducing the toxic effects of a toxic compound in a subject, the
method including: administering to the subject a therapeutically
effective amount of a food-grade bacteria capable of removing the
toxic compound from a substance or environment.
[0034] In one embodiment of the previous methods of the present
invention the toxic compound is selected from the group consisting
of lead, cadmium, mercury, arsenic, malathion and parathion.
[0035] In another embodiment of the previous methods of the present
invention the food-grade bacteria are provided dead or live.
[0036] In another embodiment of the previous methods of the present
invention the food-grade bacteria are provided as an extract.
[0037] In another embodiment of the previous methods of the present
invention the food-grade bacteria comprise a combination of two or
more different species of food-grade bacteria.
[0038] In another embodiment of the previous methods of the present
invention the composition comprises a combination of two or more
strains of Lactobacillus rhamnosus, Lactobacillus casei,
Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus
johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and
Lactobacillus amylovorus
[0039] In another embodiment of the previous methods of the present
invention the food grade bacteria are selected from the group of
food-grade bacteria listed in Table 1.
[0040] In one embodiment, the present invention is a method of
obtaining a strain of Lactobacillus capable of removing a toxic
compound from an environment, the method including a step of
mutagenesis or genetic transformation of the Lactobacilus.
[0041] In one embodiment, the present invention is a method for
obtaining a cell fraction capable of removing a toxic compound from
an environment, including the steps of: a) culturing a
Lactobacillus strain, and b) recovering the cell fraction from the
culture in step a).
[0042] In one embodiment of the last two methods the toxic compound
is selected from the group consisting of lead, cadmium, mercury,
arsenic, malathion and parathion. In another embodiment
Lactobacillus is provided dead or live. In another embodiment the
Lactobacillus is provided as an extract. In another embodiment the
Lactobacillus includes a combination of two or more different
strains. In another embodiment, the Lactobacillus is selected from
the group of Lactobacilli listed in Table 1.
[0043] In one embodiment, the present invention is a use of a food
grade bacteria for the removal of a toxic compound from a substance
or an environment.
[0044] In one embodiment of the use of the food grade bacteria, the
toxic compound is selected from the group consisting of lead,
cadmium, mercury, arsenic, malathion and parathion.
[0045] In another embodiment of the use of the food grade bacteria,
the food-grade bacteria are provided dead or live.
[0046] In another embodiment of the use of the food grade bacteria,
the food-grade bacteria are provided as an extract.
[0047] In another embodiment of the use of the food grade bacteria,
the food grade bacteria are provided as a combination of two or
more different species of food-grade bacteria.
[0048] In another embodiment of the use of the food grade bacteria,
the food grade bacteria are provided as two or more strains of
Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus
crispatus, Lactobacillus fermentum, Lactobacillus johnsonii,
Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus
amylovorus.
[0049] In another embodiment of the use of the food grade bacteria,
the food-grade bacteria is selected from the group of food-grade
bacteria listed in Table 1.
[0050] In another embodiment, the present invention provides for a
method for removing a toxic compound from a substance which is
suspected of being contaminated with said toxic compound comprising
contacting the substance with food-grade bacteria or extract
thereof capable of removing the toxic compound from the
substance.
[0051] In another embodiment, the present invention provides for a
method of reducing the toxic effects of a toxic compound in a
subject, the method comprising: administering to the subject a
therapeutically effective amount of food-grade bacteria of Table 1
or any combination thereof.
[0052] In one embodiment, the present invention provides for a
method of obtaining a strain of Lactobacillus capable of removing a
toxic compound from an environment, the method includes a step of
mutagenesis or genetic transformation of the Lactobacilus.
[0053] In another embodiment, the present invention is a method for
obtaining a cell fraction capable of removing a toxic compound from
an environment. The method, in one embodiment, includes the steps
of: a) culturing a Lactobacillus strain, and b) recovering the cell
fraction from the culture in step a).
[0054] In one embodiment of the methods of the present invention,
the food-grade bacteria comprise a combination of two or more
different species of food-grade bacteria.
[0055] In one embodiment of the present invention, the food grade
bacteria is a Lactobacillus.
[0056] In one aspect of the present invention the toxic compound
includes a heavy metal.
[0057] In another aspect of the present invention, the toxic
compound includes a heavy metal and the food-grade bacteria
comprise dead bacteria.
[0058] In another aspect of the present invention, the toxic
compound includes a heavy metal and the food-grade bacteria
comprise live bacteria.
[0059] In one another of the present invention, the toxic compound
includes a heavy metal and the food-grade bacteria comprise a
mixture of dead bacteria and live bacteria.
[0060] In another aspect of the present invention the heavy metal
is cadmium.
[0061] In another aspect of the present invention the heavy metal
is lead.
[0062] In another aspect of the present invention the toxic
compound includes mercury.
[0063] In another aspect of the invention the mercury is inorganic
mercury.
[0064] In another aspect of the invention the mercury is organic
mercury.
[0065] In one aspect of the present invention, the toxic compound
includes mercury and the food-grade bacteria comprise dead
bacteria.
[0066] In one aspect of the present invention, the toxic compound
includes mercury and the food-grade bacteria comprise live
bacteria.
[0067] In one aspect of the present invention, the toxic compound
includes mercury and the food-grade bacteria comprise a mixture of
dead bacteria and live bacteria.
[0068] In another aspect of the present invention the toxic
compound includes arsenic.
[0069] In one aspect of the present invention, the toxic compound
includes arsenic and the food-grade bacteria comprise dead
bacteria.
[0070] In one aspect of the present invention, the toxic compound
includes arsenic and the food-grade bacteria comprise live
bacteria.
[0071] In one aspect of the present invention, the toxic compound
includes arsenic and the food-grade bacteria comprise a mixture of
dead bacteria and live bacteria.
[0072] In another aspect of the present invention the toxic
compound includes a pesticide.
[0073] In one aspect of the present invention, the toxic compound
includes a pesticide and the food-grade bacteria comprise dead
bacteria.
[0074] In one aspect of the present invention, the toxic compound
includes a pesticide and the food-grade bacteria comprise live
bacteria.
[0075] In one aspect of the present invention, the toxic compound
includes a pesticide and the food-grade bacteria comprise a mixture
of dead bacteria and live bacteria.
[0076] In another aspect of the present invention the pesticide is
selected from malathion or parathion.
[0077] In another aspect of the present invention, the toxic
compound includes endotoxins.
[0078] In another aspect of the present invention, the toxic
compound includes heterocyclic aromatic amines.
[0079] In another aspect of the present invention, the toxic
compound includes acrylamide.
BRIEF DESCRIPTION OF THE FIGURES
[0080] The present invention will become more fully understood from
the detailed description given herein and from the accompanying
drawings, which are given by way of illustration only and do not
limit the intended scope of the invention.
[0081] FIG. 1A is a graph illustrating the ability of food grade
Lactobacilli to remove lead (Pb) from a solution (error
bars.+-.SEM).
[0082] FIG. 1B is a graph illustrating the ability of food grade
Lactobacilli to remove cadmium (Cd) from a solution (error
bars.+-.SEM).
[0083] FIG. 2A is a graph illustrating the ability of food grade
Lactobacilli to remove lead (Pb) from a solution compared to E.
coli (error bars.+-.SEM).
[0084] FIG. 2B is a graph illustrating the ability of food grade
Lactobacilli to remove cadmium (Cd) compared to E. coli (error
bars.+-.SEM).
[0085] FIG. 3A is a graph illustrating the ability of live and dead
food grade Lactobacilli to remove lead (Pb) from a solution (error
bars.+-.SEM).
[0086] FIG. 3B is a graph illustrating the ability of live and dead
food grade Lactobacilli to remove cadmium (Cd) from a solution
(error bars.+-.SEM).
[0087] FIGS. 4A-4C are TEM microphotographs of Lactobacillus
rhamnosus R37 incubated with a control buffer without the addition
of metals (FIG. 4A), lead (FIG. 4B), and mercury (FIG. 4C).
[0088] FIGS. 5A-5C are scanning electron micrographs of
Lactobacillus rhamnosus R37 incubated with a control buffer without
the addition of metals (FIG. 5A), lead (FIG. 5B), and mercury (FIG.
5C).
[0089] FIG. 6A is a scanning electron micrograph of Lactobacillus
rhamnosus R37 (top) and a corresponding energy-dispersive X-ray
spectrum of a portion of a cell not containing visible
deposits.
[0090] FIG. 6B is a scanning electron micrograph of Lactobacillus
rhamnosus R37 (top) and a corresponding energy-dispersive X-ray
spectrum of a portion of a cell containing visible deposits.
[0091] FIGS. 7A-7C are scanning electron microphotographs of
Lactobacillus rhamnosus GR-1 incubated with lead (FIG. 7A), cadmium
(FIG. 7B), and a control without the addition of metals (FIG.
7C).
[0092] FIGS. 8A-8D are flow cytometry analysis of Caco-2 cell line
comparing viability vs. mortality of untreated cells (FIG. 8A),
cells exposed to cadmium (FIG. 8B), cells exposed to Lactobacillus
plantarum 14917T (FIG. 8C), and cells exposed to Lactobacillus
plantarum 14917T and then exposed to cadmium (FIG. 8D).
[0093] FIG. 9A is a graph illustrating the growth of a number of
Lactobacilli species in Man Rogosa Sharpe (MRS) media having
lead.
[0094] FIG. 9B is a graph illustrating the growth of a number of
Lactobacilli species in MRS media having cadmium.
[0095] FIG. 10A is a graph illustrating the ability of a food grade
bacterium of the present invention to remove Hg.sup.2+ from a
solution having a 1 part per million (ppm) Hg.sup.2+ inoculum
(error bars.+-.SEM; * signifies significant (p<0.05) difference
by an unpaired T-test).
[0096] FIG. 10B is a graph illustrating the ability of a food grade
bacterium of the present invention to remove Hg.sup.2+ from a
solution having a 15 part per billion (ppb) Hg.sup.2+ inoculum
(error bars.+-.SEM; * signifies significant (p<0.05) difference
by an unpaired T-test).
[0097] FIG. 11 is a graph illustrating the ability of a food grade
bacterium of the present invention to remove organic mercury from a
solution (error bars.+-.SEM; * signifies significant (p<0.05)
difference by an unpaired T-test).
[0098] FIG. 12 is a graph illustrating the ability of live and dead
food grade bacterium of the present invention to remove inorganic
mercury from a solution (error bars.+-.SEM; * signifies significant
(p<0.05) difference by an unpaired T-test).
[0099] FIGS. 13A-13B are graphs illustrating variability of mercury
resistance within a group of food grade bacteria of the genus
Lactobacillus. FIG. 13A illustrates growth of different strains of
Lactobacillus casei in a gradient of Hg.sup.2+ and FIG. 13B
illustrates growth of different strains of Lactobacillus rhamnosus
in a gradient of Hg.sup.2+.
[0100] FIG. 14 is a graph illustrating twenty-four hour time course
of mercury removal by Lactobacillus rhamnosus R37 and GR-1 in
HEPES-NaOH supplemented with 1 .mu.g/mL HgCl.sub.2 incubated at
37.degree. C.
[0101] FIGS. 15A-15B are graphs illustrating removal of mercury
from solution by a selection of Lactobacillus rhamnosus strains of
increased resistance (R) and strains of increased sensitivity (S)
to mercury at HgCl.sub.2 concentrations of 0.5 ppm (FIG. 15A) and 1
ppb (FIG. 15B).
[0102] FIG. 16 is a graph illustrating the ability of food grade
bacteria and E. coli species to remove As (III) and As (V) from
solution at starting inoculums of 10 ppm.
[0103] FIG. 17 is a graph illustrating the ability of food grade
bacteria to remove As (III) from solution at a starting inoculums
of 1 ppm. (Error bars.+-.SEM).
[0104] FIG. 18 is a graph illustrating the ability of Lactobacilli
to remove As (III) from solution at starting inoculums of 100
ppm.
[0105] FIGS. 19A-19B are graphs depicting the ability of probiotic
bacteria to remove malathion (FIG. 19A) and parathion (FIG. 19B)
from solution. Starting inoculums for malathion and parathion are 5
.mu.g and 0.5 .mu.g respectively. (Error bars.+-.SEM).
[0106] FIG. 20 is a graph illustrating the ability of a probiotic
bacterium to remove both malathion and parathion from solution
simultaneously. Malathion original concentration was 5 .mu.g while
parathion was 0.5 .mu.g. (Error bars.+-.SEM).
[0107] FIGS. 21A-21B are graphs depicting the ability of food grade
bacteria and E. coli to remove malathion (FIG. 21A) or parathion
(FIG. 21B) from solution. Starting inoculums of pesticides for
malathion and parathion was 10 mg/L and 3 mg/L respectively. (Error
bars.+-.SEM).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0108] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Also,
unless indicated otherwise, except within the claims, the use of
"or" includes "and" and vice versa. Non-limiting terms are not to
be construed as limiting unless expressly stated or the context
clearly indicates otherwise (for example "including", "having" and
"comprising" typically indicate "including without limitation").
Singular forms including in the claims such as "a", "an" and "the"
include the plural reference unless expressly stated otherwise.
[0109] The expression "food grade bacteria" refers to any bacteria,
alive or dead, that have no harmful effect on human health or that
have a GRAS (generally recognized as safe) status. Such bacteria
maybe selected from the group consisting of Lactobacilli and
Bacilli. Non-limiting examples of food-grade bacteria particularly
suitable for the purpose of the present invention are listed in
Table 1.
[0110] The term "probiotic" as used in this document refers to
food-grade bacteria which perform beneficial functions to subject
organisms when they are present and alive in viable form in the
subject organisms.
[0111] "Food production animal" is used herein to describe any
animal that is prepared and used for human consumption. A food
production animal can be, but not limited to, a ruminant animal
such as beef and dairy cattle, pigs, lamb, chicken, turkey or any
other fowl, or aquatic animals including shrimp, lobster or fish
used for human consumption.
[0112] As used herein, the term "removing a toxic compound from a
substance or environment" refers to a removal of one or more toxic
compounds that can be tested as described in at least one of the
examples below.
[0113] "Subject" or "subjects" are used herein to describe a member
of the animal kingdom, including food production animals and
humans.
[0114] The present invention also encompasses mutant strains or
genetically transformed strains derived from a parent strain. These
mutant or genetically transformed strains can be strains wherein
one or more endogenous gene(s) of the parent strain has (have) been
mutated, for instance to modify some of its metabolic properties
(e.g., its ability to ferment sugars, its resistance to acidity,
its survival to transport in the gastrointestinal tract, its
post-acidification properties or its metabolite production). They
can also be strains resulting from the genetic transformation of
the parent strain by one or more gene(s) of interest, for instance
in order to confer to said genetically transformed strains
additional physiological features, or to allow it to express
proteins of therapeutic or vaccinal interest that one wishes to
administer through said strains. These strains can be obtained from
a strain by means of the conventional techniques for random or
site-directed mutagenesis and genetic transformation of
Lactobacilli, such as those described by Gury et al. (2004) or by
Perea Velez et al., 2007, or by means of the technique known as
"genome shuffling" (Patnaik et al., 2002 and Wang et al.,
2007).
[0115] A subject of the present invention is also cell fractions
which can be obtained from a Lactobacillus strain. They are in
particular DNA preparations or bacterial wall preparations obtained
from cultures of said strain. They may also be culture supernatants
or fractions of these supernatants. By way of example, cell-free
supernatant (CFS) of one Lactobacillus strain can be obtained using
the method for obtaining a CFS from another Lactobacillus
strain.
[0116] A subject of the present invention is also a method for
obtaining a cell fraction, comprising the steps of:
a) culturing a Lactobacillus strain, and b) obtaining and/or
recovering the cell fraction from the culture in step a).
[0117] In compositions of the invention, said strain can be used in
the form of whole bacteria which may be living or dead.
Alternatively, said strain can be used in the form of a bacterial
lysate or in the form of bacterial fractions; the bacterial
fractions suitable for this use can be chosen, for example, by
testing their properties on mercury removal from an aqueous
environment. Preferably the bacterial cells are present as living,
viable cells.
Food-Grade Bacteria for Removing Toxic Compounds
[0118] In one embodiment, the present invention relates to
food-grade bacterial or extracts thereof, including probiotics,
capable of removing or sequestering toxic compounds from an
environment to which the food-grade bacteria is exposed to, or from
a substance which may have or may be suspected of having the toxic
compound. Substances may include edible compositions, such as
vegetable-based foods or animal-based foods, and may also include
drinkable solutions, including water, milk, syrups, extracts and
other beverages. Substances may also include raw agricultural
products used to produce foods and drinkable solutions. As such,
the present invention relates also to methods of using the
food-grade bacteria of the present invention to prevent the uptake
of toxic compounds by a subject, or in methods to filter toxic
compounds out of substances prior to exposing a subject to said
substances. The environment may include an aqueous environment,
such as the gastro-intestinal tract of a subject, or the
environment in which the subject resides, such as a pond.
[0119] The food grade bacteria may be any type of bacteria that may
be capable of removing toxic compounds from foods or solutions that
may be consumed by a subject, or from ingredients used in the
manufacture of said foods or solutions. Table 1 includes food-grade
bacteria that may be used with the present invention. In a
preferred aspect, the food-grade bacteria may be aerobically,
microaerophilically or anaerobically grown and may be selected from
the group consisting of the food-grade bacteria of Table 1.
Administration of the food-grade bacteria, or extract thereof, to a
subject may be accomplished by any method likely to introduce the
organisms into the gastro-intestinal tract of the subject. The
bacteria can be mixed with a carrier and applied to liquid or solid
feed or to drinking water. The carrier material should be non-toxic
to the subject. When dealing with live food-grade bacteria, the
carrier material should also be non-toxic to the food-grade
bacteria. When dealing with live food-grade bacteria the carrier,
preferably, may include an ingredient that promotes viability of
the bacteria during storage. The food-grade bacteria may also be
formulated as an inoculant paste to be directly injected into a
subject's mouth. The formulation may include added ingredients to
improve palatability, improve shelf-life, impart nutritional
benefits, and the like. If a reproducible and measured dose is
desired, the food-grade bacteria can be administered by a cannula
or syringe. The amount of food-grade bacteria to be administered is
governed by factors affecting efficacy. When administered in feed
or drinking water the dosage can be spread over a period of days or
even weeks. The cumulative effect of lower doses administered over
several days may be greater than a single larger dose thereof. One
or more strains of food-grade bacteria may be administered
together. A combination of strains may be advantageous because
individual subjects may differ as to the strain which is most
persistent in a given individual.
[0120] The present invention is also directed to extracts or
fragments of food-grade bacterial that may be capable of removing
or sequestering toxic compounds from a substance or sample. As
shown herein, the inventors found that dead food-grade bacteria may
be used to sequester mercury from a sample. As such the present
invention is directed to food-grade bacteria fragments capable of
binding toxic compounds found in a substance of interest.
Applications
[0121] Food-grade bacteria of the present invention may be used as
a preventive measure, to prevent a subject not presently carrying a
toxic compound, from acquiring the toxic compound by exposure to
consumables or environments where the toxic compounds are present.
Food grade bacteria of the present invention may also be used to
substantially reduce or substantially eliminate toxic compounds
from a subject.
[0122] Treatment of a subject carrying the toxic compounds may be
accomplished to reduce or eliminate the amount of the toxic
compound carried by the subject, by administering the food-grade
bacteria, or extracts thereof, to the subject carrying the toxic
compound.
[0123] The methods for administering food-grade bacteria may
essentially be the same, whether for prevention or treatment. By
routinely administering an effective dose to a subject, the risk of
contamination by the undesired toxin may be substantially reduced
or substantially eliminated by a combination of prevention and
treatment.
[0124] In one embodiment, food-grade bacteria of the present
invention may be used in methods to filter toxic compounds out of a
substance. The method, in one embodiment, may comprise contacting
the substance with the food-grade bacteria for a sufficient amount
of time, and removing the food-grade bacteria and the toxin from
the substance. To accomplish this filtration of toxic compounds
from a substance, the food-grade bacteria, extracts or fragments of
said food-grade bacteria capable of binding to the toxic compounds,
may, for example, be attached to a filter, or to a solid support,
such as an affinity column, and the substance may then be run
through the filter or affinity column.
[0125] Food-grade bacteria may also be used, according to another
embodiment of the present invention, to feed aquatic animals such
as fish and shrimp. In one embodiment, food-grade bacteria of the
present invention may, for example, be added to tanks and ponds
containing the aquatic animal. Preferably the food-grade bacteria
used for aquatic animals, may be a bacteria that occurs naturally
in fresh and sea water environments.
Preparation and Administration
[0126] Although this invention is not intended to be limited to any
particular mode of application, oral administration of the
compositions are preferred. One food-grade bacterium may be
administered alone or in conjunction with a second, different
food-grade bacterium. Any number of different food-grade bacteria
may be used in conjunction. By "in conjunction with" is meant
together, substantially simultaneously or sequentially. The
compositions may be administered in the form of tablet, pill or
capsule, for example. One preferred form of application involves
the preparation of a freeze-dried capsule comprising the
composition of the present invention. Another preferred form of
application involves the preparation of a lyophilized capsule of
the present invention. Still another preferred form of application
involves the preparation of a heat dried capsule of the present
invention.
[0127] By "amount effective" as used herein is meant an amount of
food-grade bacterium or bacteria, e.g., Lactobacillus, high enough
to significantly positively modify the condition to be treated but
low enough to avoid serious side effects (at a reasonable
benefit/risk ratio), within the scope of sound medical judgment. An
effective amount of Lactobacillus will vary with the particular
goal to be achieved, the age and physical condition of the subject
being treated, the duration of treatment, the nature of concurrent
therapy and the specific Lactobacillus employed. The effective
amount of Lactobacillus will thus be the minimum amount which will
provide the desired detoxification.
[0128] A decided practical advantage is that the food-grade
bacteria, e.g. Lactobacillus, may be administered in a convenient
manner such as by the oral, intravenous (where non-viable), or
suppository (vaginal or rectal) routes. Depending on the route of
administration, the active ingredients which comprise food-grade
bacteria may be required to be coated in a material to protect said
organisms from the action of enzymes, acids and other natural
conditions which may inactivate said organisms. In order to
administer food-grade bacteria by other than parenteral
administration, they should be coated by, or administered with, a
material to prevent inactivation. For example, food-grade bacteria
may be co-administered with enzyme inhibitors or in liposomes.
Enzyme inhibitors include pancreatic trypsin inhibitor,
diisopropylfluorophosphate (DFP) and trasylol. Liposomes include
water-in-oil-in-water P40 emulsions as well as conventional and
specifically designed liposomes which transport Lactobacilli or
their by-products to an internal target of a host subject.
[0129] The food-grade organisms may also be administered
parenterally or intraperitoneally. Dispersions can also be
prepared, for example, in glycerol, liquid polyethylene glycols,
and mixtures thereof, and in oils.
[0130] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. In all cases the form must be
sterile and must be fluid to the extent that easy syringability
exists. It must be stable under the conditions of manufacture and
storage. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycol, and the
like), suitable mixtures thereof and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion. In many cases it will be preferable to
include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought
about by the use in the compositions of agents delaying absorption,
for example, aluminum monostearate and gelatin.
[0131] Sterile injectable solutions are prepared by incorporating
the food-grade bacteria in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
food-grade bacteria into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and the freeze-drying technique
which yield a powder of the active ingredient plus any additional
desired ingredient from previously sterile-filtered solution
thereof. Additional preferred methods of preparation include but
are not limited to lyophilization and heat-drying.
[0132] When the food-grade bacteria are suitably protected as
described above, the active compound may be orally administered,
for example, with an inert diluent or with an assimilable edible
carrier, or it may be enclosed in hard or soft shell gelatin
capsule, or it may be compressed into tablets designed to pass
through the stomach (i.e., enteric coated), or it may be
incorporated directly with the food of the diet. For oral
therapeutic administration, the food-grade bacteria may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like.
[0133] The tablets, troches, pills, capsules, and the like, as
described above, may also contain the following: a binder such as
gum tragacanth, acacia, corn starch or gelatin; excipients such as
dicalcium phosphate; a disintegrating agent such as corn starch,
potato starch, alginic acid, and the like; a lubricant such as
magnesium stearate; and a sweetening agent such as sucrose, lactose
or saccharin may be added or a flavoring agent such as peppermint,
oil or wintergreen or cherry flavoring. When the dosage unit form
is a capsule, it may contain, in addition to materials of the above
type, a liquid carrier. Various other materials may be present as
coatings or to otherwise modify the physical form of the dosage
unit. For instance, tablets, pills or capsules or Lactobacilli in
suspension may be coated with shellac, sugar or both.
[0134] A syrup or elixir may contain the active compound, sucrose
as a sweetening agent, methyl and propylparabens as preservatives,
a dye and flavoring such as cherry or orange flavor. Of course, any
material used in preparing any dosage unit form should be
pharmaceutically pure and substantially non-toxic in the amounts
employed. In addition, the food-grade organism may be incorporated
into sustained-release preparations and formulations.
[0135] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of the food-grade bacteria calculated to
produce the desired preventive or therapeutic effect in association
with the required pharmaceutical carrier. The specification for the
novel dosage unit forms of the invention may be dictated by and may
be directly depending on (a) the unique characteristics of the
food-grade bacteria and the particular preventive, detoxification
or therapeutic effect to be achieved, and (b) the limitations
inherent in the art of compounding such food-grade bacteria for the
establishment and maintenance of a healthy flora in the intestinal
tract.
[0136] The food-grade organism is compounded for convenient and
effective administration in effective amounts with a suitable
pharmaceutically or food acceptable carrier in dosage unit form as
hereinbefore disclosed. A unit dosage form can, for example,
contain the principal active compound in an amount approximating
10.sup.9 viable or non-viable, e.g., Lactobacilli, per ml. In the
case of compositions containing supplementary ingredients such as
prebiotics, the dosages are determined by reference to the usual
dose and manner of administration of the said ingredients.
[0137] The pharmaceutically acceptable carrier may be in the form
of milk or portions thereof including yogurt. Skim milk, skim milk
powder, non-milk or non-lactose containing products may also be
employed. The skim milk powder is conventionally suspended in
phosphate buffered saline (PBS), autoclaved or filtered to
eradicate proteinaceous and living contaminants, then freeze dried
heat dried, vacuum dried, or lyophilized.
[0138] Some other examples of substances which can serve as
pharmaceutical carriers are sugars, such as lactose, glucose and
sucrose; starches such as corn starch and potato starch; cellulose
and its derivatives such as sodium carboxymethycellulose,
ethylcellulose and cellulose acetates; powdered tragancanth; malt;
gelatin; talc; stearic acids; magnesium stearate; calcium sulfate;
calcium carbonate; vegetable oils, such as peanut oils, cotton seed
oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols
such as propylene glycol, glycerine, sorbitol, manitol, and
polyethylene glycol; agar; alginic acids; pyrogen-free water;
isotonic saline; cranberry extracts and phosphate buffer solution;
skim milk powder; as well as other non-toxic compatible substances
used in pharmaceutical formulations such as Vitamin C, estrogen and
echinacea, for example. Wetting agents and lubricants such as
sodium lauryl sulfate, as well as coloring agents, flavoring
agents, lubricants, excipients, tabletting agents, stabilizers,
anti-oxidants and preservatives, can also be present.
[0139] Accordingly, the subject may be orally administered a
therapeutically effective amount of at least one food-grade
bacteria and a pharmaceutically acceptable carrier in accordance
with the present invention. The food-grade bacteria may be a
Lactobacillus. The Lactobacillus may be selected from the group
comprising the bacteria listed in Table 1.
TABLE-US-00001 TABLE 1 Strains Tested For Ability to Degrade or
Sequester Toxic Compounds Species Strain Code 1 Strain Code 2
Lactobacillus casei Shirota YIT 9029 FERM BP-1366 Lactobacillus
casei ATCC 393 Lactobacillus crispatus ATCC 33323 Lactobacillus
fermentum ATCC 11739 Lactobacillus johnsonii DSM 20553
Lactobacillus plantarum ATCC 14917 Lactobacillus rhamnosus ATCC
27773 Lactobacillus reuteri RC-14 ATCC 55845 Lactobacillus
amylovorus LAB Lactobacillus rhamnosus GG ATCC 53013 Lactobacillus
rhamnosus GR-1 ATCC 55826 Lactobacillus rhamnosus HN001
Lactobacillus rhamnosus R37 DN 116-0060 Lactobacillus rhamnosus R38
DN 116-0063 Lactobacillus rhamnosus R22 DN 116-0009 Lactobacillus
rhamnosus R17 DN 116-0136 Lactobacillus rhamnosus R29 DN 116-0064
Lactobacillus rhamnosus R3 DN 116-0061 Lactobacillus rhamnosus R10
DN 116-0032 Lactobacillus rhamnosus R11 DN 116-0141 Lactobacillus
casei C3 DN 114-0017 Lactobacillus casei C8 DN 114-0022
Lactobacillus casei C11 DN 114-0125 Lactobacillus casei C26 DN
114-0074 Lactobacillus casei C6 DN 114-0226 Lactobacillus casei C20
DN 114-0037 Lactobacillus casei C29 DN 114-0230 Lactobacillus casei
C13 DN 114-0126 Lactobacillus casei C28 DN 114-0189 Lactobacillus
casei C31 DN 114-0227 Lactobacillus casei C10 DN 114-0223
Lactobacillus casei C1 DN 114-0001
[0140] The above disclosure generally describes the present
invention. Changes in form and substitution of equivalents are
contemplated as circumstances may suggest or render expedient.
Although specific terms have been employed herein, such terms are
intended in a descriptive sense and not for purposes of
limitation.
EXAMPLES
[0141] The examples are described for the purposes of illustration
and are not intended to limit the scope of the invention.
Example 1--Demonstration of Removal of Inorganic Lead and Cadmium
from an Aqueous Environment
[0142] 1 mL inoculums of 24 hour cultures of Lactobacillus
rhamnosus GR-1, Lactobacillus casei 393T, Lactobacillus johnosonii
20553 and Lactobacillus plantarum 14917T at cell concentrations of
approx. 1.times.10.sup.9 CFU/mL were added to a 50 mM HEPES buffer
containing Pb or Cd and incubated for 5 hours at 37.degree. C.
Following incubation, cells were removed by centrifugation at 5,
000 G. The total metal concentration in the supernatant was
analyzed via Inductively Coupled Plasma-Mass Spectrometry
(ICP-MS).
[0143] FIGS. 1A-1B illustrate the ability of food grade
Lactobacilli to remove Pb (FIG. 1A) and Cd (FIG. 1B) from a
solution at starting inoculums of 2 ppm and 2.5 ppm for lead and
cadmium respectively. Depending on the species/strain of
Lactobacilli examined and the metal environment there was variation
in removal. As illustrated in FIG. 1A 45-50% of Pb was removed from
solution while as illustrated in FIG. 1B 40-80% of Cd was removed.
Removals of both Pb and Cd were deemed significant (p<0.05) by
an ANOVA one-way analysis of variance.
Example 2--Demonstration of Specificity of Lead and Cadmium Removal
by Food Grade Lactobacilli from an Aqueous Solution
[0144] 1 mL inoculums of 24 hour cultures of Lactobacillus
rhamnosus GR-1, Lactobacillus rhamnosus GG, E. coli Col and E. coli
25922 at cell concentrations of approx. 1.times.10.sup.9 CFU/mL
were added to a 50 mM HEPES buffer containing Pb or Cd and
incubated for 5 hours at 37.degree. C. Following incubation, cells
were removed by centrifugation at 5, 000 G. The total metal
concentration in the supernatant was analyzed via Inductively
Coupled Plasma-Mass Spectrometry (ICP-MS). As illustrated in FIGS.
2A-2B, for both Pb (FIG. 2A) and Cd (FIG. 2B), Lactobacilli removed
70-80% of metal in solution while E. coli removal was only 30-50%.
The amount removed by Lactobacilli compared to E. coli strains and
uninoculated control were shown to be significant (P<0.05) by an
ANOVA one-way analysis of variance.
Example 3--Removal of Lead and Cadmium by Live and Dead
Lactobacilli
[0145] In this example, the ability of live and dead Lactobacilli
to remove lead (FIG. 3A) cadmium and (FIG. 3B) from solution at a
starting inoculums of 3 ppm was tested.
[0146] The assay was carried out as previously described in
Examples 1 and 2. Viable cells of all Lactobacilli were compared to
cells that were killed by gamma irradiation at 5.5 Kilo Grays (KG)
for 1 hr. Gamma irradiation was used as it kills the cells without
destroying cell wall/membrane integrity. Equal inoculums of viable
and dead cells were used. With reference to FIG. 3B, live and cells
irradiated with gamma rays were able to remove roughly equal
amounts of cadmium. However, as illustrated in FIG. 3A, there was a
split between the ability of viable or dead cells to bind more
lead. The results obtained herein show that binding of metals may
likely be a surface associated action not requiring actively
metabolic cells. As such, the present invention is also directed to
the parts of food-grade bacteria capable of binding heavy
metals.
Example 4--Demonstration of Passive Sequestration Activity
[0147] FIGS. 4A-4C illustrate TEM micrographs of Lactobacillus
rhamnosus R37 incubated in 50 mM HEPES-NaOH buffer (FIG. 4A) with 1
mM Pb (FIG. 4B) and 1 mM HgCl.sub.2 (FIG. 4C) added. Numerous
deposits are observed throughout the cells incubated with heavy
metals (FIGS. 4B-4C) however; some smaller deposits are also
visible when no metal is added (FIG. 4A). The nature of the
deposits was confirmed using SEM and EDX analysis.
[0148] FIGS. 5A-5C are SEM micrographs of Lactobacillus rhamnousus
R37 incubated in 50 mM HEPES-NaOH buffer (FIG. 5A) with 1 mM Pb
(FIG. 5B) and 1 mM HgCl.sub.2 (FIG. 5C) added. Numerous deposits
are observed throughout the cells incubated with heavy metals
(FIGS. 5B-5C) however; some smaller deposits are also visible when
no metal is added (FIG. 5A).
[0149] FIGS. 6A-6C illustrate energy-dispersive X-ray spectroscopy
(EDX) analysis of putative metal deposits in Lactobacillus
rhamnosus R37. Osmium coated samples being imaged with SEM were
analyzed with EDX to determine the elemental composition of
putative metal deposits within the cell. FIG. 6A demonstrates the
spectrum (bottom) of a portion of cell not containing any visible
deposits and mercury was not detected. FIG. 6B shows analysis of a
large deposit which was determined to contain 36.62% mercury by
mass proving cellular sequestration of mercury (see Table 2).
[0150] Similar results were also obtained for GR-1, R3, R39,
Lactobacillus casei C3 showing mercury in the cell.
TABLE-US-00002 TABLE 2 Control Suspected Hg deposit Element Weight
% Atomic % Element Weight % Atomic % Carbon 74.44 87.03 Carbon
39.67 70.99 Oxygen 11.75 10.32 Nitrogen 7.51 11.52 Sulfur 4.50 1.97
Oxygen 8.51 11.44 Osmium 9.30 0.69 Phosphorus 1.23 0.85 Totals
100.00 100.00 Sulfur 0.97 0.65 Osmium 5.50 0.62 Mercury 36.62 3.92
Totals 100.00 100.00
Example 5--Confirmation of Precipitation and Binding of Metals on
and within Food Grade Bacteria
[0151] Lactobacilli were incubated in a 50 mM HEPES buffer for 2
hrs at 37.degree. C. in the presence of metals at a final
concentration of 10 mM. The assay was carried out by incubating
bacteria (Lactobacillus rhamnousus GR-1) for 2 hrs in a 10 mM metal
solution at 37.degree. C. Following incubation the bacteria were
diluted 100-fold and filtered through a 0.2 .mu.m filter to trap
bacteria and allow passage of solution. The filters were dried at
room temperature for 2 hrs and then coated with 5 nm of osmium
tetra oxide. The identification of the metals was confirmed by EDAX
X-ray analysis which showed that the metal precipitates were the
heavy metals added to solution.
[0152] FIGS. 7A-7C are scanning electron micrographs (SEM) of
Lactobacillus rhamnosus GR-1 incubated with (FIG. 7A) lead or (FIG.
7B) cadmium. The bright spots observable in the images represent
the precipitation of heavy metal particles on the surface and
inside the cell. FIG. 7C displays the non metal control which is
the Lactobacilli without addition of metals, note the absence of
precipitate metal particles.
Example 6--Preliminary Evidence of Protective Effect of Food Grade
Lactobacilli on a Caco-2 Cell Line as a Model of the Gut Epithelial
Barrier
[0153] Caco-2 cells were grown in 12 or 24 well plates for two
weeks using supplemented Eagles Minimum Essential Medium
(ATCC.RTM.) as described above. At two weeks, media was aspirated
and cells were washed lightly 2.times. with warm 50 mM HEPES
buffer. Bacterial cultures of interest were also grown in 5 mL
broth cultures for 22 hrs and washed 2.times. with 50 mM HEPES.
Bacterial cells were resuspended to 10 mL in Eagles Minimum
Essential Medium (ATCC.RTM.) without any Pen/Strep in solution, 400
.mu.L of media was added to wells in 24 well plates and 900 .mu.L
of media was used in 12 well plates. Bacteria were allowed to
incubate with cell line for 2 hr at 37.degree. C. During incubation
period metal spiked solutions of Eagles Minimum Essential Medium
(ATCC.RTM.) was made by adding stock concentrations of Pb, Cd or As
(Sigma Aldrich.RTM.) to the media at desired concentrations.
Following incubation period the bacterial metal solution was
aspirated so that only cells adhering to the Caco-2 cell monolayer
remained, the media was replaced with the metal spiked media in
addition control wells were set up that either did not have metal
in the media and were not incubated with bacterial species. Cells
were incubated for 5 hrs in metal spiked media at 37.degree. C.
[0154] Following this incubation, media was removed by aspiration
and discarded. Cells were washed once gently with warm HEPES buffer
and then removed from the wells using 500 ul of 0.25% (w/v) trypsin
until cells detached from flask. 500 .mu.L of cell media was added
to stop trypsin reaction and total volume of each well was
transferred into separate sterile 1.5 mL centrifuge tubes
(Diamed.RTM.). The cell suspension was mixed by pipetting to avoid
formation of bubbles. Cells were centrifuged in a bench top
microcentrifuge for 2 mins at 120 RPM, supernatant was discarded
and cells were suspended in 1.times.PBS. Cells were diluted by a
factor of 10 by suspending 50 .mu.L of cells with 450 .mu.L of
Guava Viacount.RTM. Reagent (Cat No. 4000-0041) in a clean sample
tube, cells were stained for at least 5 min. Stained cells were
then analyzed for viability using the Guava ViaCount Assay on the
Guava EasyCyte Mini bench top flowcytometer. Cells were separated
based on viability forming two distinct populations: live and dead.
Populations were analyzed and statistically compared using FlowJo
(TreeStar.TM.) analysis software for flow cytometry data. Cells
were analyzed to see differences in viability after exposure to
metals in the presence or absence of Lactobacilli.
[0155] FIG. 8 illustrates a flow cytometry analysis of the Caco-2
cell line comparing viability vs. mortality for un treated cells
(FIG. 8A), Caco-2 cells exposed to cadmium (FIG. 8B), Caco-2 cells
exposed to Lactobacillus plantarum 14917T (FIG. 8C) and Caco-2
cells pretreated with Lactobacillus plantarum 14917T and then
exposed to cadmium (FIG. 8D). As shown by FIG. 8D addition of
Lactobacillus plantarum 14917T before cadmium exposure contributed
to increased survival of the cell line then when just exposed to
cadmium (FIG. 8B).
Example 7--Viability of Lead and Cadmium Resistant Food Grade
Bacteria of the Genus Lactobacillus
[0156] The assay was carried out by inoculating a 200 .mu.L well of
Man Rogosa Sharpe (MRS) medium containing lead or cadmium at a
concentration of 100 ppm with an inoculum of 10.sup.7 bacteria from
a fresh 24 hrs broth cultures of the Lactobacilli species
Lactobacillus rhamnosus GR-1 and Lactobacillus plantarum 14917T.
Growth was measured by OD600 for 24 hrs. incubation at 37.degree.
C. Growth was measured for 24 hours with readings taken every 30
minutes by optical density measurements at a wavelength of 600 nm.
Following the growth assay all species were diluted and drop plated
on MRS agar to determine colony forming units (CFU) in
solution.
[0157] FIGS. 9A-9B show growth of all tested Lactobacilli species
in the MRS media with lead (FIG. 9A) or cadmium (FIG. 9B) at a
concentration of 100 ppm.
Example 8--Demonstration of Removal of Inorganic Mercury from an
Aqueous Environment
[0158] A 1% inoculum of a 24 hour culture of Lactobacillus
rhamnosus DN116-060 was added to de Man Rogosa Sharpe (MRS) broth
containing HgCl.sub.2 and incubated for 24 hours at 37.degree. C.
Following incubation, cells were removed by centrifugation at 5,000
g. The total mercury concentration in the supernatant was analyzed
via cold vapor atomic absorption spectroscopy (CVAAS). As
illustrated in FIGS. 10A-10B, the Lactobacilli removed 94.4% of a 1
part per million (ppm) mercury inoculum (FIG. 10A) and 85% of a 15
part per billion (ppb) inoculum (FIG. 10B). Both removals were
deemed significant (p<0.05) by an unpaired T-test.
Example 9--Demonstration of Removal of Organic Mercury Form an
Aqueous Environment
[0159] A 1% inoculum of a 24 hour culture of Lactobacillus
rhamnosus DN116-060 was added to de Man Rogosa Sharpe (MRS) broth
containing MeHgCl2 and incubated for 24 hours at 37.degree. C.
Following incubation, cells were removed by centrifugation at 5,000
g. The total mercury concentration in the supernatant was analyzed
via cold vapor atomic absorption spectroscopy (CVAAS).
[0160] FIG. 11 shows the ability of a food grade bacterium to
remove MeHg.sup.2+ from solution at a starting inoculum of 1 ppm
MeHgCl2. (Error bars.+-.SEM). As illustrated in FIG. 11, the
Lactobacilli removed 23.2% of a 1 ppm mercury inoculum (p<0.05
by an unpaired t-test).
Example 10--Inorganic Mercury Removal by Live and Dead
Lactobacillus rhamnosus DN116-060
[0161] The assay was carried out as previously described in Example
9 at a concentration of 500 ppb HgCl.sub.2. Viable cells of
Lactobacillus rhamnosus DN116-010 were compared to cells that were
killed by heating at 80.degree. C. for 10 minutes at an inoculum
equivalent to the final cell density of viable cells.
[0162] FIG. 12 illustrates the ability of live and dead
Lactobacillus rhamnosus DN116-060 to remove Hg.sup.2+ from solution
at a starting inoculum of 500 ppb HgCl.sub.2. As shown in FIG. 12,
viable cells were capable of removing significantly more mercury
than heat killed cells (p<0.05 by unpaired t-test) suggesting
that there is a passive sequestering of mercury as well as
potential metabolic detoxification.
Example 11--Variability of Mercury Resistance within Food Grade
Bacteria of the Genus Lactobacillus
[0163] Assay was carried out as previously described in Example 9
across a spectrum of HgCl.sub.2 concentrations. Growth was measured
after 24 hours at 37.degree. C. by the optical density of cultures
at a wavelength of 600 nm. A spectrum of resistances to mercury
were observed in both species demonstrating that resistance to
mercury is a variable trait among food grade bacteria.
[0164] FIGS. 13A-13B illustrate the growth of Lactobacillus casei
(n=38) (FIG. 13A) and Lactobacillus rhamnosus (n=40) (FIG. 13B) in
a gradient of Hg.sup.2+ measured by OD600 after 24 hours incubation
at 37.degree. C. Each set of connected points represents one
strain. Resistance is a strain variable trait resulting in a
spectrum of resistance profiles in both species. FIG. 13B
illustrates three Lactobacillus rhamnosus strains showing a
distinctly higher resistance as compared to the rest of the
strains.
Example 12
[0165] Twenty-four hour time course of mercury removal by
Lactobacillus rhamnosus R37 (in viable and heat killed form) and
GR-1 in HEPES-NaOH supplemented with 1 .mu.g/mL HgCl.sub.2
incubated at 37.degree. C. With reference to FIG. 14, sequestration
activity is not instantaneous and reaches a maximum after 12 h in
Lactobacillus rhamnosus R37 while maximal removal was observed at
24 hours in the case of Lactobacillus rhamnosus GR-1.
Example 13--Resistant Strains of Food Grade Bacteria Remove More
Mercury than Mercury Sensitive Strains
[0166] The assay described in Example 1 was carried out using a
selection of Lactobacillus rhamnosus strains of increased
resistance and increased sensitivity to mercury.
[0167] FIGS. 15A-15B illustrate removal of mercury from solution by
a selection of Lactobacillus rhamnosus strains of increased
resistance (R) and strains of increased sensitivity (S) to mercury
at HgCl.sub.2 concentrations of 0.5 ppm (FIG. 15A) and 1 ppb (FIG.
15B). Resistant strains removed significantly more mercury from
solution than their sensitive counterparts (p<0.05 as determined
by ANOVA with Bonferroni post test [FIG. 15A] and un-paired t-test
[FIG. 15B]). (Error bars.+-.SEM)
Example 14--Removal of Arsenite and Arsenate from an Aqueous
Environment
[0168] Bacterial cultures were grown for 24 hrs in preferential
media; Man Rogoas Sharpe (MRS) broth for Lactobacilli and
Luria-Bertani (LB) broth for E. coli. Cells were centrifuged,
washed and re-suspended in PBS. 1 mL aliquouts were distributed
between sample tubes containing 9 mL of PBS buffer spiked with
arsenic, 1 mL of MRS or LB broth was added to sample tubes. Cells
were incubated for 5 hrs at 37.degree. C.; following incubation
cells were removed by centrifugation at 5, 000 g. The total arsenic
remaining in solution was analyzed via inductively coupled
plasma-mass spectrometry (ICP-MS). As illustrated in FIG. 16
Lactobacilli were able to remove 50-60% of As (III) and As (V)
while E. coli DH5a was less effective.
Example 15--Demonstration of Removal of Arsenite (as III) by a
Panel of Lactobacilli
[0169] The assay was carried out by inoculating a 1 ppm
(9.08.times.1018 free atoms) arsenite solution (HEPES buffer) with
1.times.10.sup.9 CFU/mL of selected Lactobacilli. The solutions
were incubated for 5 hrs at 37.degree. C.; following incubation
cells were removed by centrifugation at 5, 000 g. The total arsenic
remaining in solution was analyzed via inductively coupled
plasma-mass spectrometry (ICP-MS).
[0170] As shown in FIG. 17 and Table 3, Lactobacilli removed 11-13%
of the total arsenic which was determined by looking at differences
in concentrations in total free atoms in solution vs. bound to each
species.
TABLE-US-00003 TABLE 3 Species % Removed L. rhamnosus GR-1 13 L.
johnsonii 20553 11 L. casei 393T 11 L. plantarum 14917T 11
Example 16--Demonstration of Removal of Arsenic (III) at High
Concentrations by Lactobacilli
[0171] The assay was carried out by inoculating a 100 ppm arsenite
solution of HEPES buffer with 1.times.10.sup.9 CFU/mL of the
selected Lactobacilli. The solutions were incubated for 5 hrs at
37.degree. C.; following incubation cells were removed by
centrifugation at 5,000 g. The total arsenic remaining in solution
was analyzed via inductively coupled plasma-mass spectrometery
(ICP-MS).
[0172] As shown in FIG. 18, all Lactobacilli showed ability to
remove near 70% of arsenic from solution compared to the untreated
control sample. Species to species variation in amount of arsenic
able to remove was low and not significant.
Example 17--Demonstration of Removal of Malathion and Parathion
from Aqueous Environment by Probiotic Bacteria
[0173] Bacterial broth cultures of Lactobacillus rhamnosus GR-1
were grown for 24 hrs in Man Rogosa Sharpe (MRS) broth. Cells were
collected, washed and re-suspended in a 1.times.PBS buffer. 1 mL of
cell suspension was transferred into sample tube containing a 50:50
mixture of HEPES buffer having the pesticides and MRS. Starting
inoculums of pesticides for malathion and parathion was 5 .mu.g/L
of HEPES buffer and 0.5 .mu.g/L of HEPES buffer respectively.
Samples were incubated for 5 hrs at 37.degree. C. Following
incubation cells were removed by centrifugation at 5, 000 g. The
remaining pesticide in solution was analyzed via gas
chromatography-mass spec (GC-MS) and values were compared to
untreated controls.
[0174] With reference to FIGS. 19A-19B, Lactobacillus rhamnosus
GR-1 was able to remove 20% of the malathion from solution (FIG.
19A) and 50% of the parathion (FIG. 19B).
Example 18--Demonstration of Removal of Malathion and Parathion
Simultaneously by a Probiotic Bacterium
[0175] Bacterial broth cultures of Lactobacillus rhamnosus GR-1
were grown for 24 hrs in Man Rogosa Sharpe (MRS) broth. Cells were
collected, washed and re-suspended in a 1.times.PBS buffer. 1 mL of
cell suspension was transferred into sample tube containing a 50:50
mixture of HEPES buffer having the pesticides and MRS. Starting
inoculums of pesticides for malathion and parathion was 5 .mu.g/L
of HEPES buffer and 0.5 .mu.g/L of HEPES buffer respectively.
Samples were incubated for 5 hrs at 37.degree. C. Following
incubation cells were removed by centrifugation at 5, 000 g. The
remaining pesticide in solution was analyzed via gas
chromatography-mass spec (GC-MS) and values were compared to
untreated controls.
[0176] As shown in FIG. 20, Lactobacillus rhamnosus GR-1 was able
to remove 50% of the malathion from solution and 50% of the
parathion.
Example 19--Demonstration of Removal of Pesticides by a Panel of
Food Grade Bacteria and Some E. coli Species
[0177] Bacterial broth cultures of Lactobacilli were grown for 24
hrs in Man Rogosa Sharpe (MRS) broth, E. coli species were grown
for 24 hours in Lucella Broth (LB). Cells were collected, washed
and re-suspended in a 1.times.PBS buffer. 1 mL of cell suspension
was transferred into sample tubes containing a 50:50 mixture of
HEPES buffer having the pesticide and MRS or LB. Starting inoculums
of pesticides for malathion and parathion was 10 mg/L of HEPES
buffer and 3 mg/L of HEPES buffer respectively. Samples were
incubated for 5 hrs at 37.degree. C. Following incubation cells
were removed by centrifugation at 5, 000 g. The remaining pesticide
in solution was analyzed via gas chromatography-mass spec (GC-MS)
and values were compared to untreated controls.
[0178] FIG. 21A illustrates that the Lactobacilli were able to
remove 35-60% of malathion, while E. coli was able to remove 10-25%
of malathion. FIG. 21B illustrates that the Lactobacilli and E.
coli were able to remove 55-70% of parathion.
Example 20--Demonstration of Removal of Endotoxins by a Panel of
Food Grade Bacteria
[0179] Endotoxins are well known toxins responsible for sepsis and
death. They are produced by a number of Gram negative bacteria and
to date few effective treatments have been developed. Other potent
toxins produced by bacteria include the fatal Shiga toxin produced
by E. coli 0157:H5, and TcdA and TcdB toxins from Clostridium
difficile both of which damage the human colonic mucosa and are
potent cytotoxic enzymes. Deaths from C. difficile toxins have
become a major concern in North American hospitals and care homes.
Probiotic therapy has shown great promise in preventing infections
caused by E. coli 0157:H7 and C. difficile.
[0180] Alkaline phosphatase levels (activity and protein) can be
measured in feces and blood as it has been shown that up-regulation
of this enzyme can detoxify endotoxins in the gut and improve gut
permeability. A pig model is used for this assay. C. difficile
toxins will be detected from stool by a commercially available
enzyme-linked fluorescence immunoassay.
Example 21--Demonstration of Removal of Aflatoxin by a Panel of
Food Grade Bacteria
[0181] Aflatoxin (a hepatic carcinogen) is important contributors
to disease, albeit risk of exposure to the mainstay population in
N. America is low. Aflatoxin B1 has been included because
probiotics can have an effect against it, and such results have
implications for many sub-populations in the US (eg large farming
communities) and beyond (eg Middle East, Argentina).
[0182] The aflatoxin will be measured from blood by affinity column
cleanup and LC-MS/MS fluorescence.
Example 22--Demonstration of Removal of Heterocyclic Aromatic
Amines (HAA) by a Panel of Food Grade Bacteria
[0183] Heterocyclic aromatic amines (HAA) are found in food (eg
processed meat) and cause diet-related mutagenesis which plays an
etiologic role in chronic diseases, including cardiovascular
disease and cancer. Their direct association with cancer is low,
but the potential for them to be inhibited by probiotics makes them
worth studying, as a positive detox effect provides a good consumer
message.
[0184] They will be measured from urine and blood samples using
HPLC.
Example 23--Demonstration of Removal of Acrylamide by a Panel of
Food Grade Bacteria
[0185] Acrylamide is made industrially but is highly regulated due
to its neurotoxicity. It naturally forms in certain foods,
particularly plant-based foods that are rich in carbohydrates and
low in protein, during processing or cooking at high temperatures
(French fries, potato chips). Also found heavily in cigarette
smoke. Acrylamide is monitored and studied by Health Canada, but no
levels have been established on what is toxic/safe, so it's tough
to set a `limit` or even tell in a study what would be considered
dangerous. It has a link to causing cancer and information on how
much will cause this effect is not known.
[0186] Acrylamide will be detected by HPLC.
* * * * *