Note: Descriptions are shown in the official language in which they were submitted.
COMPOSITION AND METHOD FOR GERMINATIVE COMPOUNDS
IN PROBIOTIC FOOD AND BEVERAGE PRODUCTS FOR HUMAN CONSUMPTION
[0001]
BACKGROUND
[0002] Recent scientific advances have shed light on the significance of the
relationship between human gut bacteria (the microbiome) and general health.
The
human microbiome is a product of our genetics, our diets, and our environment.
Probiotics may be used to alter the human microbiome. Probiotics for human
consumption are available as capsules and as food products (e.g. yogurt,
beverages,
etc.). Although there are examples of probiotic tea on the market, there are
none that
include an activating component to germinate probiotic spores for improved
efficacy.
Here, an invention is described that includes the provision of activating
compounds with
Bacillus bacterial spores for human probiotic consumption in tea, soups,
gravies, sauces,
hot chocolate, and other food and beverage products, particularly heated
liquids and
fluids.
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BRIEF DESCRIPTION OF THE FIGURES
[0003] The system and method of the invention are further described and
explained in relation to the following drawing wherein:
FIG. 1 shows photographs of Bacillus species in a nutrient germinant
composition according to a preferred embodiment at time zero (when hot water
is
initially added) and after steeping in hot water for 60 minutes at various
temperatures
according to preferred methods of the invention, the darker spots indicate
germinated
bacteria and the lighter/brighter spot indicate non-germinated spores;
FIG. 2 shows photographs of Bacillus species and a nutrient germinant
composition according to a preferred embodiment in steeped tea (black and
green tea)
according to a preferred method of the invention, the darker spots indicate
germinated
bacteria and the lighter/brighter spots indicate non-germinated spores.
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DETAILED DESCRIPTION
[0004] Recent scientific advances have shed light on the significance of the
relationship between human gut bacteria (the microbiome) and overall health.
The
human microbiome is a product of our genetics, our diets, and our environment.
Probiotics may be used to alter the human microbiome in order to regulate the
digestive
system and bolster the immune system. Generally, probiotics are non-pathogenic
bacteria that may include species of lactic acid bacteria (Lactobacillus),
Bifidobacterium,
and Propionibacterium. Species of Bacillus are also common as probiotics.
Probiotics
have been widely used in animals as an alternative to hormones and
antibiotics, in
plants to enhance certain growth characteristics, and in humans as a digestive
supplement. Probiotics for human consumption are available as capsules and as
food
products (e.g. yogurt, juice, etc.). Probiotics delivered in tea are also on
the market.
Probiotic tea products contain probiotic bacteria packaged with tea in a
porous bag. Tea
bags are steeped for a period of time in hot water and are consumed.
Alternatively,
Kombucha tea is a type of probiotic drink in which a symbiotic culture of
bacteria and
yeast (SCOBY) is added to tea containing sugar. The SCOBY then acts to ferment
the
sugar in the tea to produce vitamins and acids. In this case, the sugar acts
as a
substrate and the drink does not contain amino acids that are known to
activate
bacteria.
[0005] In the case of Bacillus species as probiotics (e.g. B. coagulans), the
bacteria are supplied as spores. Spores represent a unique life stage of
specific species
of bacteria in that these species have the ability to transition into a
dormant spore stage
if environmental conditions are unfavorable. Spores are naturally dehydrated,
are stable
for extended periods of time, and are resistant to environmental stresses
(e.g. heat, salt,
pH, etc.). In the case of Bacillus spores in tea products, the spores are
preferably more
resistant to the high temperatures of steeping water.
[0006] In order for spores to return to a live, vegitatively growing state,
they must
first germinate. Spore germination is a multistep process in which spores are
revived
from the dormant state. The first step is one by which spores are activated
and are
induced to germinate, typically by an environmental signal called a germinant.
This
signal can be a nutrient such as an L-amino acid, potassium ions (e.g. KCI),
and/or
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natural osmoprotectant compounds (e.g. ectoine). Nutrient germinants bind to
receptors
in the inner-membrane of the spore to initiate germination. Additionally,
sugars have
been shown to increase the binding affinity of L-amino acids for their cognate
receptors.
[0007] The germ inant signal initiates an irreversible cascade of events, also
known as commitment. Commitment is fast and is typically >90% complete in 2
minutes.
As germination progresses, the spore rapidly takes on water. As the spore
absorbs
water it consequently loses its refractivity. This loss of refractivity
towards the end of the
first phase in the germination process allows spore germination to be observed
via
phase-contrast microscopy. The initial phase is typically complete within 30
to 60
minutes depending on the temperature. The second phase of germination is an
outgrowth step in which the spore's metabolic, biosynthetic, and DNA
replication/repair
pathways initiate. The outgrowth period includes a ripening period in which no
morphological changes (such as cell growth) occur, but the spore's molecular
machinery (e.g. transcription factors, translation machinery, biosynthesis
machinery,
etc.) is activated. This period can vary in length based on the initial
resources that are
packaged with the spore during the process of sporulation.
[0008] It is known that spores can be induced to germinate via heat-
activation.
Spores of various Bacillus species have been heat-activated at strain specific
temperatures (e.g. B. subtilis spores can be heat-activated at 75 C for 30
minutes while
B. licheniformis spores can be heat-activated at 65 C for 20 minutes). The
heat-
activation is believed to cause a transient, reversible unfolding of spore
proteins. If the
spore proteins are not presented with a nutrient germ inant, such as L-
alanine, they will
refold and effectively reverse the germination process.
[0009] The present invention describes a composition and method for activating
probiotic spores in in food and beverage products, such as steeped teas,
soups,
gravies, sauces, hot chocolate, coffee, and other products, particularly
heated liquids
and fluids. The method includes the provision of nutrient germinants with
Bacillus
spores that will be heat activated and subsequently germinated with nutrient
germ inants
while the food or beverage product is being heated, such as while tea is
steeping in hot
water. According to one preferred embodiment, the two probiotic components
(bacterial
spores and nutrient germinants) may be provided as dry components in an
automatic
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coffee maker pod (e.g. Keurig , Nespresso , Tassimo , etc.) containing tea
or another
food or beverage product. According to another preferred embodiment, the
ingredients may
be provided as mixed ingredients (nutrient germinant composition and spores)
in a single
serve packet to be added to tea or another food or beverage product, mixed
components
(nutrient germinant composition and spores) in a single bag containing tea or
another food
or beverage product, or as separated components in separate tea bags (e.g. two
bags
containing any combination of the nutrient germinant composition, spores, and
tea) or other
container for other food or beverage products. According to one preferred
embodiment,
spores can be heat-activated in the presence of nutrient germinants in a
method that is
described in U.S. Patent Application No 15/479,773.
[0010] Nutrient Compositions
[0011] Described herein are preferred embodiments of nutrient compositions
that
may contain one or more L-amino acids, D-glucose, 0-fructose, a biological
buffer, a
potassium ion source, and/or a natural osmoprotectant. All components in the
nutrient
formulation must be Generally Regarded as Safe (GRAS) for human consumption by
the
United States Food and Drug Administration. At the time of this writing, most,
although not
all, of the components described herein have been deemed GRAS.
[0012] According to one preferred embodiment, the nutrient germinant
composition can include one or more L-amino acids. Preferred L-amino acid(s)
included
in the nutrient formulation are L-alanine, L-asparagine, L- valine, and/or L-
cysteine. The
choice of L-amino acids is determined by the species of Bacillus used in the
probiotic
formulation. The L-amino acids can be provided in the form of any suitable
source, such
as their pure forms and/or a hydrolysate of soy protein.
[0013] According to another preferred embodiment, the nutrient germinant
composition can optionally contain an amount of 0-glucose and/or 0-fructose.
The choice
of sugar is determined by the species of Bacillus used in the probiotic
formulation.
[0014] According to another preferred embodiment, the nutrient germinant
composition can optionally contain one or more sources of potassium ions.
Preferably,
KCI can be included as a source of potassium ions in the nutrient formulation.
The
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inclusion of potassium ions is determined by the species of Bacillus used in
the probiotic
formulation.
[0015] According to another preferred embodiment, the nutrient germinant
composition can optionally contain one or more biological buffers. The
biologic buffer is
a buffer that can buffer the nutrient formulation and/or nutrient-spore
formulation, to
maintain the formulation at the proper pH for spore germination (about pH 6-
8).
Preferred biologic buffers include, but are not limited to, a phosphate buffer
or a HEPES
sodium salt. According to another preferred embodiment, monosodium phosphate
and
disodium phosphate, preferably used together, can be included in the nutrient
formulation as buffers. According to another preferred embodiment, a HEPES
buffer
may be used.
[0016] According to another preferred embodiment, the nutrient solution can
optionally contain an osmoprotectant compound. Preferably, ectoine, a natural
osmoprotectant produced by some species of bacteria, may be included. The
inclusion
of an osmoprotectant is determined by the species of Bacillus used in the
probiotic
formulation.
[0017] According to another preferred embodiment, the nutrient composition is
any one of the nutrient compositions described in U.S. Patent Application No
15/479,773.
[0018] According to yet another preferred embodiment, a nutrient composition
may contain ingredients in one or more of the above-reference categories of
ingredients
and may contain one or more ingredients from within those categories.
[0019] Spore Formulations
[0020] Preferred embodiments of spore formulations include one or more
Bacillus species of spores, including but not limited to, Bacillus
ficheniformis, Bacillus
subtiffis, Bacillus amyloliquiefaciens, Bacillus polymyxa, Bacillus
thuringiensis, Bacillus
megaterium, Bacillus coagulans, Bacillus lentus, Bacillus clausii, Bacillus
circulans,
Bacillus firmus, Bacillus lactis, Bacillus laterosporus, Bacillus
laevolacticus, Bacillus
polymyxa, Bacillus pumilus, Bacillus simplex, Bacillus sphaericus, Bacillus
sonorensis,
Bacillus, homeckiae, Bacillus axarquiensis, Bacillus mucilaginosus, Bacillus
olivae, and
any combinations thereof. All strains in the probiotic formulation must be
Generally
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Regarded as Safe (GRAS) for human consumption by the United States Food and
Drug
Administration. At the time of filing this application, several, although not
all, of the
above mentioned strains have been deemed GRAS. Any Bacillus species
subsequently
determined to be GRAS are included here as suitable for use with embodiments
of the
invention.
[0021] Preferred spore formulations can contain 1-20 or more species of
Bacillus spores. According to one preferred embodiment, a spore formulation
can
contain 3 strains of Bacillus bacteria. According to another preferred
embodiment, 2
strains of the Bacillus bacteria can each be a different strain of the species
Bacillus
licheniformis and the third strain is a species of Bacillus subtilis.
According to another
preferred embodiment, about 80% of the formulation can be Bacillus
licheniformis (40%
of each strain) and 20% of the spores in the spore formulation can be is
Bacillus subtilis.
According to another preferred embodiment, the spores of the strain(s)
included in the
spore formulation can be mixed with water or other suitable carrier and/or
organic salts.
[0022] Most preferably, the Bacillus species that can be contained in the
spore
formulations can produce and/or be capable of producing one or more enzymes
including, but not limited to, proteases, amylases, lipases, glycosidases,
cellulases,
esterases, and xylanases. Tests and assays for determining the production of
such
enzymes from a Bacillus species are generally known in the art and to one of
ordinary
skill in the art.
[0023] According to one preferred embodiment, the spore formulation can
contain about 0.1% to 90% by weight spores, along with salt or other suitable
carrier,
such as sodium bicarbonate, or maltodextrin, or a combination thereof. In some
embodiments, the spore formulation contains about 5% by weight spores. The
spore
formulation can be and/or include a powder or other dry form (e.g. spray-dried
form of a
liquid spore concentrate, or lyophilized spore formulation) containing spores.
According
to another preferred embodiment, the total concentration of spores in the
spore
formulation can range from about 1 x 105 CFU/mL or spores/g to 1 x 1014 CFU/mL
or
spores/g or any specific concentration or range therein. The total preferred
concentration of spores in the spore formulation can be about 1, 1.125, 1.5,
1.75, 2,
2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75,
6, 6.25, 6.5, 6.75,
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7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, or 9.75 x 105, 106, 107,
108, 109, 1019,
10" , 1012, 1013, or 1014 CFU/mL or spores/g or any range or other value
therein.
According to another preferred embodiment, any one specific spore species can
be
present in the spore formulation at a concentration that can range from about
1 x 105
CFU/mL to 1 x 1014 CFU/mL or any specific range therein. The preferred
concentration
of any one specific spore species present in the preferred embodiments of
spore
formulations according to the invention can be about 1, 1.125, 1.5, 1.75, 2,
2.25, 2.5,
2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25,
6.5, 6.75, 7, 7.25,
7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, or 9.75 x 105, 106, 107, 108,
109, 1019, 1011,
1012, 1013, or 1014 CFU/mL or spores/g or any range or other value therein.
Preferably,
the spore formulation is biodegradable. According to another preferred
embodiment, the
concentrated spore formulation can contain about 1-9 x 109 or 101 CFU/mL or
spores/g.
According to another preferred embodiment, the concentrated spore formulation
can
contain about 1010 CFU/mL or spores/g.
[0024] According to one preferred embodiment of a probiotic tea composition,
the composition comprises a nutrient composition, a spore formulation, and
tea.
According to another preferred embodiment or a probiotic composition for human
consumption, the composition comprises a nutrient composition, a spore
formulation,
and a food or beverage product, such as soups, gravies, sauces, hot chocolate.
Most
preferably, the nutrient composition and spore formulation in these
embodiments are
one of the above described embodiments, both in dry powdered or other dry
solid form.
Any variety of tea or other food or beverage product may be used. Most
preferably, the
food or beverage product is one that is heated before being consumed.
[0025] According to one preferred method of providing probiotic tea for human
consumption, a nutrient composition and spore formulation are added to any
variety of
tea. Most preferably, the nutrient composition and spore formulation are one
of the
above described embodiments. According to one preferred embodiment, the
nutrient
composition, spore formulation, and tea or other food or beverage product are
combined into a mixture prior to steeping in hot water or otherwise heating
the food or
beverage product. According to other preferred embodiments, the nutrient
composition,
spore formulation, and tea may be separate components that are added together
to hot
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water to prepare the probiotic tea or added to the other food or beverage
product to be
heated or already heated, or any combination of two of the components may be
pre-
mixed together and added to the third component prior to or after adding hot
water to
prepare the probiotic tea or prior to or after heating the other food or
beverage product.
The order of addition of the three components and the hot water or hot food or
beverage
is not critical and any order may be used. When used for tea, after mixing or
adding the
three components, individually or as a pre-mixed combination, with hot water,
the
mixture is allowed to steep, preferably for 2 to 10 minutes prior to being
consumed by a
human. Two minutes is the preferred minimum amount of steeping time, but the
total
time may vary based on the type of tea used and the strength of the tea
desired by the
person who will consume the probiotic tea. For example, green tea is typically
steeped
for 2-4 minutes while Oolong tea is typically steeped for 5-8 minutes.
Consumption is
preferably by drinking or otherwise orally ingesting the probiotic tea. Most
preferably,
the hot water is at a temperature in the range of 42 to 100 C when initially
added to the
other components. The hot water temperature is preferably not below 42 C, and
the
range may vary based on the type of tea being used. For example, green tea is
typically steeped in the range of 65-80 C and black tea is usually steeped in
the range
of 80-100 C.
[0026] Various compositions according to preferred embodiments of the
invention were tested according to preferred methods of the invention. The
compositions, methods, and results are described below.
[0027] EXAMPLE 1 ¨ A probiotic blend of Bacillus spores (B. subtilis and B.
licheniformis) at a final concentration of approx. 4 x 106 CFU/mL, where CFU
stands for
colony forming unit, and a nutrient germinant composition according to a
preferred
embodiment of the invention comprising L-alanine (0.21g), monosodium phosphate
(0.14g), and disodium phosphate (0.5g), were added to approx. 8 fluid ounces
of tap
water at various temperatures.
[0028] The amount of spores added represents a "dose" of 109 CFU of probiotic
bacteria, as recommended by a Harvard Health study. The amount of nutrient
germinants used represents the minimum concentration of germinants necessary
to
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initiate germination. The temperatures used represent a range of tea steeping
temperatures.
[0029] Spores from each reaction were observed at time 0 and after 60 minutes
using phase contrast microscopy. Slides were prepared using standard
procedures.
Spores were viewed on an Olympus BX41 microscope (100X oil emersion objective)
and imaged using an Olympus UC30 camera controlled by the cellSens Dimension
software package.
[0030] FIG. 1 shows representative images from these tests at time 0 (i.e.
immediately after water addition) and after 60 minutes in the water. The
darker spots
show germinated spores, the lighter spots show non-germinated spores. The
images in
group A represent spores that had been germinated using a nutrient-germinant
composition and heated during the incubation period at 42 C according to a
preferred
composition and preferred method of the invention. The images in group B
represent
spores that had been germinated using a nutrient-germinant composition and
heated
during the incubation period at 65 C according to a preferred composition and
preferred
method of the invention. The images in group C represent spores that had been
germinated using a nutrient-germinant composition and heated during the
incubation
period at 75 C according to a preferred composition and preferred method of
the
invention. The images in group D represent spores that had been germinated
using a
nutrient-germinant composition and heated during the incubation period at 85 C
according to a preferred composition and preferred method of the invention.
The
images in group E represent represents spores that had been germinated using a
nutrient-germinant composition and heated during the incubation period at 100
C
according to a preferred composition and preferred method of the invention.
[0031] Taken together, these images show that Bacillus spores are germinated
in the compositions and conditions of preferred embodiments of the present
invention.
[0032] EXAMPLE 2 ¨ A probiotic blend of Bacillus spores (B. subtilis and B.
licheniformis) at a final concentration of approx. 4 x 106 CFU/mL and a
nutrient
germinant composition, according to a preferred embodiment of the invention,
comprising L-alanine (0.21g), monosodium phosphate (0.14g), and disodium
phosphate
(0.5g), were added to approx. 8 fluid ounces of tap water at 85 C. A tea bag
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the indicated variety of tea (black or green as shown in FIG. 2) was steeped
in the water
for approx. 3 minutes, as recommended by the tea manufacturer. After steeping,
the tea
bags were removed while the spores remained in the mixture to be ingested as a
probiotic dose.
[0033] Spores from each reaction were observed after 60 minutes using phase
contrast microscopy. Slides were prepared using standard procedures. Spores
were
viewed on an Olympus BX41 microscope (100X oil emersion objective) and imaged
using an Olympus UC30 camera controlled by the cellSens Dimension software
package.
[0034] FIG. 2 shows representative images from these tests after 60 minutes in
the water. The darker spots show germinated spores, the lighter/brighter spots
show
non-germinated spores. The top images represent spores germinated in steeped
black
tea (Lipton 1M) while the bottom images represent spores germinated in steeped
green
tea (Bigelow ).
[0035] Taken together, these images show that Bacillus spores are germinated
in the compositions and conditions of preferred embodiments of the present
invention
and that compounds that are extracted from tea during steeping do not abate
the
germination process.
[0036] Although the above examples are in reference to use of probiotic
compositions with tea, the compositions and methods of the invention may be
used with
other types of food or beverage products, such as soups, gravies, or hot
chocolate.
Most preferably, such food and beverage products are liquids or fluids and are
consumed after heating, but other types of food and beverage products may also
be
used.
[0037] All amounts for ingredients or ratios of ingredients indicated herein
as a
range include each individual amount or ratio within those ranges and any and
all
subset combinations within ranges, including subsets that overlap from one
preferred
range to a more preferred range. Those of ordinary skill in the art will
appreciate upon
reading this specification, including the examples contained herein, that
modifications
and alterations to the composition and methodology for making the composition
may be
made within the scope of the invention and it is intended that the scope of
the invention
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disclosed herein be limited only by the broadest interpretation of the
appended claims to
which the inventor is legally entitled.
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