Note: Descriptions are shown in the official language in which they were submitted.
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SOY KEFIR POWDER AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to kefir, and more particularly to soy kefir
powder
(SKP) used for treating fatigue, mood disorders, improving satiety and/or
vitality.
BRIEF DESCRIPTION OF THE PRIOR ART
Kefir originates from the Northern Caucasus Mountains where it has been
consumed for centuries and has been valued for numerous health promoting
properties.6 It continues to be a popular beverage in Eastern Europe,
Scandinavia, and numerous individual countries13,14 In the former Soviet
Union, kefir has been traditionally used in hospitals and sanatoria for the
treatment of numerous conditions including metabolic disorders,
atherosclerosis, allergic disease, peptic ulcers, biliary tract diseases,
chronic
enteritis, bronchitis and pneumonia. It has also been used to treat
tuberculosis, cancer, and gastrointestinal disorders when medical treatment
was unavailable6.
Controlled clinical trials have yet to confirm the utility of most of the
above
clinical uses and currently there are no regulations on the sale of kefir or
kefir
extract as natural health products.
Kefir is a cultured milk beverage made by adding kefir grains to various milk
products (i.e., cow, goat, soy, and other commonly consumed milks). The kefir
grains ferment the milk, incorporating their probiotic organisms to create the
cultured product. Kefir grains are not consumed as part of the final product;
they are removed with a strainer at the completion of fermentation and added
to a new batch of unfermented milk.
The kefir beverage has a tart, refreshing taste that is slightly acidic due to
the
presence of lactic acid. It is naturally effervescent due to the presence of
carbon dioxide and minute concentrations of alcohol (i.e., 0.08% to 2%) as a
result of yeast fermentation. Kefir also contains a variety of approximately
40
CA 02645749 2008-11-28
aromatic compounds, including diacetyl and acetaldehyde, which give it a
characteristic flavour and aroma.'
As the microbial composition varies significantly according to the kefir grain
source, the source is critical to determining the final composition of the
kefir
product.2'3 The wide variety of microorganisms used in kefir fermentation
differentiates kefir from virtually all other cultured milk products, which
typically
use only one and rarely more than three species in the culturing process.
Kefir grains are a soft, gelatinous white biomass, 3 to 20 mm in diameter,
comprised of protein, lipids, and a water-soluble polysaccharide complex
called Kefiran. Kefiran provides for a stable matrix that functions as a
natural
immobilized cell system. The grains resemble small cauliflower florets, their
structure being the result of a symbiotic relationship shared between a large
variety of specific lactic acid bacteria and yeasts. The grain matrix is
composed of a complex of 13% protein (by dry weight), 24% polysaccharide,
plus cellular debris and unknown components.2-12
Depending on the source of kefir grains, the microbial composition can vary
significantly.2-6 The dominant microflora are Saccharomyces kefir,
Lactobacillus caucasicus, Leuconnostoc species and lactic streptococci. Other
probiotic microorganisms present in the grains include lactobacilli, such as
Lb.
acidophilus, Lb. brevis, Lb. casei, Lb. casei subsp. rhamnosus, Lb. casei
subsp. Pseudoplantarum, Lb. paracasei subsp. paracase, Lb. cellobiosus, Lb.
delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis, Lb. fructivoran,
Lb.
helveticus subsp. lactis, Lb. hilgardii, Lb. kefiri, Lb. kefiranofaciens, Lb.
kefirgranum sp. nov, Lb. parakefir sp. nov, Lb. lactis, Lb. plantarum, Lb.
cellobiosus and/or Lb. helveticus. Lactococci are also present such as
subspecies of Lc. lactis, Lc. lactis var. diacetylactis and/or Lc. lactis
subsp.
Cremoris. Leuconostoc mesenteroides, Leuconostoc cremoris and L. cremoris
are also present. Other bacteria include Streptococci salivarius subsp.
thermophilus, and/or S.lactis, Enterococcus durans. Other bacteria include
Acetobacter aceti and/or A. rasens.
2"8
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Bacteriocin may also be present, especially if the appropriate strains of
lactic
acid bacteria are present in the grains.2'9
Varieties of yeasts such as Kluyveromyces, Candida, Toru/opsis, and
Saccharomyces sp. are also present in kefir grains. Candida albicans has not
been found in kefir grains. Certain yeasts of kefir include the name Candida
as
part of their nomenclature. These kefir yeasts are not opportunistic yeasts
such as C. albicans, but are classified as Generally Regarded As Safe
(GRAS). Such yeasts may have the potential to keep C. albicans under control
in the host.
The mean ranges of unit counts of microbes in gram stained kefir grains are,
a) bacilli, 62-69%, b) streptococci 11-12%, and c) yeast, 16-20%.2,x'7'10 12
Extracts of fermented soy foods have angiotensin converting enzyme (ACE)
inhibitory and blood pressure (BP) lowering properties comparable to those of
ACE inhibitor drugs.33 Soy hydrolysates and soy ACE inhibitory peptides have
been demonstrated to inhibit ACE activity in vascular tissue and to lower
systolic blood pressure (SBP) in spontaneously hypertensive rats.33-35
Moreover, anti-hypertensive effects have been obtained from milk fermented
with a combination of various lactic acid bacteria and yeast, a process
analogous to kefir fermentation, albeit that kefir grains contain a greater
variety
of bacteria and yeast.27
Chronic fatigue syndrome has been associated with higher serum
angiotensin-converting enzyme (ACE) levels, which has been suggested to
reflect damage to the vascular endothelium (Lieberman and Bell, 1993).
Hence, a part of the efficacy of SKP in chronic fatigue might be related to
its
demonstrated ACE inhibitory activity.
Yeast fermentation generates higher levels of metabolite
hydroxymethylbutyrate from the amino.acid leucine, which promotes greater
gains of muscle mass and strength in untrained men and women initiating
resistance training (Nissen et al., 1996). Since yeasts are predominant
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microbiological species involved in kefir fermentation, kefir fermentation
likely
greatly increases the production of hydroxymethylbutyrate.
Several supplementation trials have indicated that branched chain amino
acids (BCAA) can contribute to combat fatigue and to improve mental and
physical performance in athletes (De Lorenzo et al., 2003; Blomstrand et al.,
1991, 1997). Soy protein is one of the best sources of BCAA and fermentation
increases the quantity of soluble proteins. Hence, the digestibility and
bioavailability of BCAA from soy kefir would be significantly enhanced to
provide a significant enhancement in BCAA uptake. An imbalance in the ratio
of free tryptophan to BCAA with relatively low blood levels of BCAA has been
implicated as a possible cause of acute physiological and psychological
fatigue (central fatigue) during exercise (Gastmann and Lehmann, 1998) and
the chronic fatigue syndrome (Georgiades et al., 2003).
The improvement of vitality has been sparsely investigated with no reference
to fermented soy products. Green tea and products containing green tea,
which contain caffeine, have been indicated to increase vitality or have
stimulatory effects (Dulloo et al., 1999; Boon et al., 2004).
Other putative bioactive ingredients in soy kefir are isoflavones. Soybeans
contain the highest natural concentration of isoflavones of any food.43 The
major dietary isoflavones found in soy are genistein, daidzein, formononetin,
biochanin A and coumestrol. The biologically active isoflavones, genistein and
daidzein, are substantially increased with soy protein fermentation.44
Soy phytoestrogens have been shown to influence learning and memory
(Lund et al., 2001a), affect aggressive and social behavior (Simon et al.,
2004) and produce anxiolytic effects (Lund et at.., 2001 b).
In studies of rats, soy protein has been shown to have short-term satiating
properties when compared to other sources of protein (Semon et al., 1987).
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However, the presence of yeast protein as found in soy kefir powder and
protein fermentation likely greatly increases satiety effects. For example,
mycoprotein produced by the continuous fermentation by mushroom
microorganisms induces acute and delayed suppressive effects on food intake
(Turnbull et al., 1993). Also, rats fed a high yeast protein load reduced
their
meal and daily energy intake to a signifinicanty greater extent than rats fed
well-balanced, high protein diets from a variety of protein sources including
soy, total milk protein, or wheat gluten (Faipoux et al., 2006). The strong
satiety may have been increased by protein hydrolysis for greater availability
of absorption and/or gastrointestinal action of resulting peptides.
Fermentation of food proteins increases their digestibility and allows for
greater absorption of peptides, without changing the overall biological
value.70
In particular, proteins with high disulfide content such as soy are relatively
resistant to digestion,71 and fermentation increases their digestibility to
allow
for greater absorption of peptides.70 72 We postulate that some
physiologically
active bioactive peptides may be present in their inactive forms in the amino
acid sequences of proteins and are normally poorly absorbed from undigested
soy proteins.
Fermentation may release these "hidden" peptides and subsequently exert
health benefits. Small dipeptides and tripeptides, and even large peptides (10-
51 amino acids) can be absorbed intact through the intestines and produce
biological effects.73,74 It is noteworthy that ACE inhibitory peptides derived
from
milk fermentation have been shown to be resistant to the digestive condition
and to exert a BP lowering effects when given orally to spontaneously
hypertensive rats.75 Isoflavonoids undergo acidic and enzymatic hydrolysis in
the human gut and the isoflavones, biochanin A and formononetin, undergo
demethylation to yield the aglycones genistein and daidzein, respectively.
This
metabolism may vary among individuals, resulting in differences in the
relative
proportions of isoflavonoid metabolites produced in the gut.76
The half-lives of isoflavones are about 4-8 h, which suggests that
maintenance of high plasma concentrations of isoflavone metabolites could be
achieved with regular and frequent consumption of soy products.77
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For centuries, Asians have consumed fermented soy 'products with ACE
inhibitory activity such as soy sauce and natto,78'79 with no documented
adverse effects being noted apart from an adverse drug-food interaction noted
with monoamine oxidase inhibitor drugs.80'81 While the presence of isoflavones
with putative hormonal like activities (i.e., genistein and daidzein) may
cause
some safety concern, a review of the literature indicates that 40g of soy
powder contains 6-23.2 mg daidzein and 0.076-33.6 mg genistein. A typical
60kg person consuming 40g soy powder/day will not be exposed to more than
0.39 mg/kg/day daidzein or 0.56 mg/kg/day genistein. Animal studies, while
limited, demonstrate that adverse effects were only observed at levels of
isoflavones that are at least approximately 100 times higher than that found
in
40g of soy powder.
A comparison of soy kefir findings with other clinical trials using sf-36
health
survey scores is described in B. Stacey, B. Parsons, S. Huang, S. Peyser and
E. Dukes (2004) Gabapentin and Improved Health Status in Elderly Patients
with Postherpetic Neuralgia: A Pooled Analysis of Three Clinical Studies. P&T
29: 646-651.
The Kadian product monograph 2005 Alpharma Branded Products Division
Inc. (KAD-CLMON-01 January 2005) (KADIAN sustained-release capsules
contain Morphine Sulfate) is described in Reg Anesth Pain Med 2004;
29(2):A32.
SUMMARY
The present invention provides the use of a soy fermented product having
increased potency.
The invention further provides the use of a soy kefir fermented product useful
for treating fatigue.
The invention further provides the use of a soy kefir fermented product useful
for treating mood disorders.
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The invention further provides the use of a soy kefir fermented product useful
for improving satiety.
The invention further provides the use of a soy kefir fermented product useful
for improving vitality.
The invention further provides Soy kefir powder for use in the treatment of
fatigue in a subject in need thereof.
The invention further provides Soy kefir powder for use in the treatment of
mood disorders in a subject in need thereof.
The invention further provides Soy kefir powder for use in the improvement of
satiety in a subject in need thereof.
The invention further provides Soy kefir powder for in the improvement of
vitality in a subject in need thereof.
Accordingly, the soy kefir powder (SKP) contemplated by the present invention
is obtained by fermentating soymilk with kefir grains from the Moscow kefir
strain, and having at least one of the following characteristics:
- a protein composition of approximately of 25 - 45 % wet weight,
- a carbohydrate composition of approximately of 5 - 45 % wet weight,
- a fat composition of approximately of 25 - 45 % wet weight, or
- an ash composition of approximately of 5- 15 % wet weight.
Accordingly, the soy kefir powder (SKP) contemplated by the present invention
is obtained by fermentating soymilk with kefir grains from the Moscow kefir
strain, and having at least three of the following characteristics:
- a protein composition of approximately of 25 - 45 % wet weight,
- a carbohydrate composition of approximately of 5 - 45 % wet weight,
- a fat composition of approximately of 25 - 45 % wet weight, or
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- an ash composition of approximately of 5- 15 % wet weight.
Accordingly, the soy kefir powder (SKP) contemplated by the invention is
obtained by fermentating soymilk with kefir grains from the Moscow kefir
strain, and having the following characteristics:
- a protein composition of approximately of 25 - 45 % wet weight,
- a carbohydrate composition of approximately of 5 - 45 % wet weight,
- a fat composition of approximately of 25 - 45 % wet weight, and
- an ash composition of approximately of 5- 15 % wet weight.
The present invention has one of the following advantages: it provides a soy
kefir powder which has increased potency over related products derived from
other processes. The soy kefir powder of the invention also has the advantage
of being a natural product which may not cause side effets. The SKP of the
invention may thus be safe to use by pregnant women or subjects under other
medications. Moreover, the SKP of the invention is easily accessible to
anyone as it may be obtained without the need of a prescription.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. represents the electrophoretic profile of fermented soymilk (having
the highest peak) compared to unfermented soymilk.
Figure 2 shows the differences in improvement of mean scores of SF-36v2
subscales at the endpoint versus baseline.
Five-point change in the SF-36v2 health status score is considered as
"clinically meaningful" change (Frost MH, Bonomi AE, Ferrams CE et al,
and the Clinical Significance Consensus Meeting Group. Patient,
clinician, and population perspectives on determining the clinical
significance of quality-of-life scores. MayoClin Proc 2002;77:488-494;
Samsa G. Edelman D, Rothman ML, et al. Determining clinically
important differences in health status measures. Pharmacoeconomics
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1999; 15:141-155; Rowbotham MC. What is a "clinically meaningful"
reduction in pain? Pain 2001; 94: 131-132.
- PF=Physical Funtioning; RP=Role Physical; GH=General Health;
SF=Social Functioning; RE=Role Emotional; MH=Mental Health
Figure 3. is a flow chart illustrating a method for preparing soy kefir powder
according to a preferred embodiment of the invention.
Figure 4 represents the clinical trial results for 20g @ 60d; n=10.
Figure 5 represents the clinical trial results for 20g @ 30d; n=10.
Figure 6 represents the clinical trial results for 30g @ 60d; n=7 (A
multivariate
statistical analysis on the separate groups was undertaken).
Figure 7 represents the clinical trial results for 30g @ 30d; n=7. (A
multivariate
statistical analysis on the separate groups was undertaken).
Figure 8 represents the clinical trial results for 30g @ 60d; n=7. (A
multivariate
statistical analysis on the combined groups was undertaken).
Figure 9 represents the clinical trial results for 30g @ 30d; n=7. (A
multivariate
statistical analysis on the combined groups was undertaken).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
By the term "Moscow Kefir grain" it is meant the kefir grain obtained under an
exclusive licence from the All-Russia Dairy Institute (ARDI), 35 Lyusinovskaya
Street, Moscow, Russia. Table 1 summarizes the composition of the microflora
of the Moscow kefir grain.
As used herein, the term "treating" refers to a process by which the symptoms
of a disorder are alleviated or completely eliminated. Thus, in the context of
disorders caused by fatigue, mood disorders, and overeating, the symptoms
are alleviated or completely eliminated.
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The term "preventing" refers to a process by which the disorder is obstructed
or delayed.
By the term "fatigue" is intended, for the purpose of this invention, a "lack
of
energy", a "lack of vitality" or "weakness", either short term or persistent,
including symptoms of the chronic fatigue syndrome that involve unrefreshing
sleep, after any exertion, weariness that lasts for more than a day, fatigue
that
is not the result of excessive work or exercise, fatigue substantially impairs
a
person's ability to function normally at home, at work, and in social
occasions.
Mild exercise often makes the symptoms, especially fatigue, much worse,
sleep or rest does not relieve fatigue. Fatigue leads to physical symptoms
that
include sore throat, swollen lymph nodes in the neck or armpits, muscle pain,
pain without redness or swelling in a number of joints, intense or changing
patterns of headaches, short-term memory loss or a severe inability to
concentrate that affects work, school, or other normal activities.
By the term "mood disorders", is intended, for the purpose of this invention,
disturbances in emotions that inhibit an individual from functioning well be
it
depression or mania.
By the term "overeating", it refers to the consumption of an energy intake
that
is inappropriately large for a given energy expenditure, thus, leading to
obesity
in a subject.
By the term "vitality", the invention refers to a healthy capacity for
vigorous
activity.
By the term "subject" is intended, for the purpose of this invention, any live
form that is subject to fatigue, depressed mood, and overeating, including
humans, farm animals, domestic animals, or zoological garden animals.
As used herein, the expression "an acceptable carrier" means a vehicle for
containing a soy kefir powder of the invention that can be administered to a
subject without adverse effects. Suitable carriers known in the art can be of
any food format that can be mixed with powder. They include, but are not
limited to, a liquid such as water, milk, juice or a solid such as cookies,
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nutrition bars, ice cream. Carriers may include a food product such as a
yogourt.
By the term "soy kefir liquid" used in the examples, it is meant the liquid
obtained by the fermentation of soymilk with the Moscow kefir grains.
In a composition, the %wet weight refers to g/1 00g wet weight. For instance,
a
protein composition of 25% wet weight means that there are 25g of protein in
a composition of 100g.
HRQOL means Health-Related Quality of Life.
Soy kefir powder contemplated by the invention and uses thereof
The present invention relates to the use of a soy kefir powder. Such a soy
kefir
powder may be obtained by fermentating soymilk with kefir grains from the
Moscow kefir strain.
The composition of the Microflora of the Moscow Kefir Grains from the All-
Russian Scientific Research Institute of Dairy Industry (ARDI) is shown in
Table 1.
Table 1 Composition of the Microflora of the Kefir Grains from the All-Russian
Scientific Research Institute of Dairy Industry (ARDI)
Microorganism CFU/g Species Total Sum
Lactobacillus 2.65 x 10 Lactobacilli 2.92 x 10
acidophilus* (87.78)a 96.82
Lactobacillus 1.96 x 10
delbrueckil lactis (0.65) Lactococci 5.12 x 106
(1.64)
Lactobacillus kefiri 2.5 x 10 (8.28) 4.78 x 106
(formerly L. brevis) Yeasts (1.53)
Lactobacillus 4.00 x 10
kefiranofaciens (0.01) Total 3.02 x 108
Leuconostoc 1.80 x 10
mesenteroides (0.06)
cermoris
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Lactococcus lactis 1.7 x 10 (0.91)
lactis
Lactococcus lactis 1.98 x 10
cremoris (0.66)
Leuconostoc 2.00 x 10
mesenteroides (0.07)
mesenteroides
Candida kefyr 2.1 x 10 (0.70)
Candida tenuis 2.4 x 10 (0.08)
Saccharomyces lactis 1.26 x 106
(0.42)
Saccharomyces
unisporus 1.18 x 106
(Saccharomyces (0.39)
delbrueckii)
aData in parenthesis represent percentage of total microflora
The soy powder of the invention has at least one of the following
characteristics:
- a protein composition of approximately of 25 - 45 % wet weight,
- a carbohydrate composition of approximately of 5 - 45 % wet weight,
- a fat composition of approximately of 25 - 45 %wet weight, or
- an ash composition of approximately of 5 - 15% wet weight.
The % refers to g/1 00g wet weight.
More specifically, the soy kefir powder contemplated by the present invention
comprises the characteristics listed in the following Table 2:
CONTROLS SPECIFICATION RESULTS METHOD
Description Fine Powder Conform Visual
Colour Beige Powder Conform Visual
Odour Slightly milk Conform Olfactive
pH 4.0-4.5 4.22 USP < 791 >
--------- - --
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CONTROLS SPECIFICATION RESULTS METHOD
Protein 29.4-44.2% 36.8 % NPPF -143T
Fat 28.6-42.8% 35.7 % NPPF - 083T
Carbohydrate 9.6-14.4% 12.0 % NPPF - 083T
Loss on drying 7.6-11.4% 9.5% USP < 731 >
Ash 4.8-7.2% 6.0% USP<281>
Total Plate Count ------ 5.7 X 106 org/g USP < 61 >
Yeast & Molds < 1000 cfu/g 50 org/g USP < 61 >
Salmonella spp. Absent Absent USP < 61 >
S. aureus Absent Absent USP < 61 >
E. Coli Absent Absent USP < 61 >
P. aeruginosa Absent Absent USP < 61 >
Enterobacter spp. < 100 cfu/g Absent MCB - 240802
IC50 2.6 - 4.0 mg/mL 3.3 mg/mL 0PA-Chromogenic
Reaction
The method for preparing a soy kefir powder contemplated by the invention
first comprises the step of fermenting soymilk with Moscow kefir grains under
suitable time and temperature conditions to obtain a fermentation culture. The
kefir grains are placed in a fermenter at a ratio, for instance, of 1 part
grains to
40 parts soymilk (2% dextrose may be added), or at a ratio, for instance, of 1
to 20, or at a ratio, for instance, of 1 to 5. Then, the grains are fermented
at
room temperature for. approximately 10 to 24 hours, preferably for 16 to 24
hours, and more preferably for 16 hours. As used herein, the term "room
temperature" refers to a temperature value of about 23 C, for instance a
temperature of 23 C 5 C.
Prior to the step of fermenting soymilk with the kefir grain, kefir grains are
activated in soymilk (2% dextrose may be added) for instance at a ratio of 1-
part grains to 2-parts milk and maintained at room temperature for
approximately 24 hours.
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The method of the invention also comprises the step of separating the kefir
grains from the fermentation culture. Indeed, as kefir grains increase in
volume
during fermentation, a portion of grains are removed to maintain constant
grain-to-milk ratio. For instance, the grains are removed by coarse sieving
and
can be advantageously used as the inoculum for fermenting a subsequent
batch of soymilk. Alternatively, grains may be lyophilized for long-term
storage.
In order to remove any significant amount of alcohol, the method may further
comprise a step of spray-drying the fermentation culture so as to obtain a soy
kefir powder. The fermentation culture thus obtained may be spray-dried at a
temperature of 65 C 13 C until a significant amount of alcohol is removed.
Alternatively, the fermentation culture may be spray-dried at 180-250 C
instantaneously (< 1 sec.). The kefir powder is then processed to separate
from the same agglomerated kefir powder called "chunks". In such a case, the
chunks may be crushed and then added and mixed to the kefir powder.
Current Status
Soy Kefir Powder is developed by the Applicant and contains live organisms, it
is a probiotic; however, it is currently developed for its other properties.
Using
the Applicant's fermentation process, different peptide products are produced
from soymilk vs. cow's milk, leading to important differences in the
bioactivities
of each product in cell culture; a greater bioactivity has been associated
with
soymilk based kefir. Therefore, the Applicant is focusing development efforts
on fermented soymilk. The Applicant does not produce an actual kefir
beverage, rather fermented soymilk is spray-dried and provided as a powder.
The spray-drying process produces an alcohol free product. To consume, dry
powder is preferably mixed with a suitable amount of liquid (i.e., water,
juice,
milk, etc.).
Physical And Chemical Properties
Natural Health Product Substance
Appearance: Beige powder, similar in consistency to powdered milk.
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Solubility: Soluble in water.
Manufacturer: The Applicant
Storage: Store refrigerated at 2 - 8 C.
Constituents:
Protein (approximate) : 35 40%
Carbohydrate: 10 14%
Fat: 35 - 40%
Moisture: 8 - 10%
Ash: 5 - 7%
The % refers to g/1 00g wet weight.
pH: 4.2
Stability: 12 months refrigerated at 2 - 8 C.
Active Ingredients
The therapeutically active anti-hypertensive agents in soymilk fermented with
kefir grains are postulated to be isoflavones.
Two main isoflavones in soybeans are genistein, and daidzen with minor
amounts of glycetein and glycetin. Isoflavones can exist in several different
forms (glucoside, rnalonyl, acetyl, etc.) and these forms are interconvertible
by
enzymatic hydrolysis during fermentation or heat treatment. However, the total
isoflavone content remains approximately the same. The presence of
bacterial glucosidases during fermentation produces the aglycone form,
genistein and daidzen, as the main products. Tempeh, miso and other
fermented soy foods have lower isoflavone levels (0.36 to 1.38 mg/g) and
higher aglycone levels than tofu due to the fermentation.
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Soy Kefir Powder contains the following isoflavone content:
0.130 to 0.150 mg diazidin/g soy kefir powder = 4.55 to 5.25 mg diazidin/35g
soy kefir powder
0.450 to 0.550 mg genistin/g soy kefir powder = 15.75 to 19.25 mg genistin/35
g soy kefir powder
0.120 to 0.140 mg daidzein/g soy kefir powder = 4.2 to 4.9 mg diazidin/35 g
soy kefir powder
0.225 to 0.245 mg genistein/g soy kefir powder = 7.875 to 8.575 mg
genistein/35 g soy kefir powder.
Total isoflavone content for 35 mg = 32.375 to 37.975 mg/35 g soy kefir
powder. This amount of isoflavones corresponds to between 0.925 and 1.085
mg isoflavones/g soy kefir powder.
In another embodiment, Soy Kefir Powder contains the following isoflavone
content:
0.149 mg diazidin/g soy kefir powder = 5.215 mg diazidin/35g soy kefir
powder
0.492 mg genistin/g soy kefir powder = 19.441 mg genistin/35 g soy kefir
powder
0.129 mg daidzein/g soy kefir powder = 4.52 mg diazidin/35 g soy kefir
powder
0.235 mg genistein/g soy kefir powder = 8.22 mg genistein/35 g soy kefir
powder.
Total isoflavone content for 35 mg = 37.40 mg/35 g soy kefir powder. This
amount of isoflavones corresponds to 1.07 mg isoflavones/g soy kefir powder.
c
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Isoflavone concentrations in foods are usually expressed on a per gram
protein basis, since we previously have assessed the protein content of SKP
to be 43%, the isoflavone concentration would be 2.49 mg/g protein, which is
slightly above the range of isoflavones seen in the richest sources of
isoflavones in soy products such as soy flour (approximately 2 mg/g protein).
A study has shown that intake of 45g soy flour leads to 50 to 100-fold
increase
in blood and urinary concentrations of isoflavones, respectively.
Studies using isoflavones have suggested that effective doses start about 30
mg isoflavones/day; however, it should be noted that the content of the more
bioactive aglycone isoflavones genistein and diazidein is important
clinically. It
is interesting that there is a high proportion of daizidein and genistein,
which is
the most important biologically active isoflavone, in soy kefir powder.
Investigations have shown that the isoflavone absorption is greater in humans
consuming fermented soy products since fermented soy contains a high
proportion of the better absorbed aglycone isoflavones (daidzein and
genistein) than the glucose-conjugated forms (daizidin and genistin).
S-equol is a product of daidzin or daidzein biotransformation by intestinal
bacteria. There is greater efficacy of soy protein or soy food diets in people
that have an active bacterial flora capable of converting daidzein into S-
equol.
Using an in vitro model of the gastrointestinal tract, recent work has
indicated
that supplementation of equol-producing bacteria on daidzein metabolism can
increase equol production and so it is possible that the probiotic effects of
SKP bacteria may increase the potency of isoflavones in the product.
There is increasing evidence that isoflavones are ineffective when provided
alone but require the presence of soy protein and other soy components to be
effective such as lignans, proteins, etc..
Assay Methodologies
Method for Determining Isoflavone Content
CA 02645749 2008-11-28
Analysis for isoflavone content of soy kefir powder was carried out as
following:
Standard samples of the following isoflavones were used to make standard
mixtures as following:
1- Daidzin 1.0 mg dissolved in 1.2mL acetonitrile:H20:acetone (4:1:1)
2- Genistin 1.0 mg dissolved in 1.25 mL acetonitrile:H20 (4:1)
3- Daidzein 1.0 mg dissolved in 2.0 mL acetonitrile:H20 (9:1)
4- Genistein 1.0 mg dissolved in 1.25 mL acetonitrile:H20 (4:1)
5- Biochanin A 1.0 mg dissolved in 1.0 mL acetonitrile
6- In an Eppendorf vial, the following amounts of each standard solution were
mixed to obtain a mixture which contains 100pg of each isoflavone:
a. 120pL Daidzin
b. 125pL Genistin
c. 200pL Daidzein
d. 100pl- Genistein
e. 100pL Biochanin A
f. 350pL acetonitrile
Using the 100pg/mL solution mixture, a serial dilution was carried out to make
standards mixtures of the following concentrations: 50, 25, and 12.5pg/mL.
The standards mixtures were used to build the calibration curves.
The samples were extracted and analyzed as following:
1. 400 mg of each sample powder was suspended in 4 ml of
acetonitrile: methanol (1:1) solution in 10 mL screwed cap tube. Each tube was
subjected to sonication for 1 hour in ice cold water bath, with interval
vortexing
every 15 minutes.
2. Samples were allowed to settle down on bench for 15 min. Then the clear
upper solution of each sample was transferred into 1.5mL Eppendorf vial, and
then centrifuged at 20000G for 5 minutes.
CA 02645749 2008-11-28
The clear supernatant was further filtered through nylon filters (0.2pm pores)
and was analyzed by HPLC-UV.
Use of the soy kefir powder of the invention for improving mood
An object of the invention relates to the use of the soy kefir powder as
defined
above for improving mood, and for preventing and/or treating mood disorders
in a subject.
The Soy Kefir Powder of the invention is a Natural Health Product currently
being developed for improving energy, mood and satiety. It is produced by the
fermentation of soymilk using genuine Russian Kefir grains sourced from the
All-Russia Dairy Institute. Milk is spray-dried following fermentation,
resulting
in a probiotic powder with a protein composition of approximately 35 - 40%.
Isoflavones, which- are also found in more bioavailable forms in fermented
soymilk, have been demonstrated to possess aniolytic properties, decrease
aggressive behaviour, diminish the stress response and to lower incidence of
depressive mood and improve cognitive performance in postmenopause.
Therefore, the Soy Kefir Powder of the invention may provide multiple
therapeutic modalities and be of potential benefit in numerous stress
disorders.
In summary, the Soy Kefir Powder of the invention is a unique fermentation
product that provides therapeutic benefits and improves the mood of subjects.
Use of the soy kefir powder of the invention for treating fatigue
In another object, the present invention relates to the use of the soy kefir
powder contemplated by the invention for preventing and/or treating fatigue.
The mechanisms of action for energy improvement with SKP are unclear but
are likely multi-factorial. Chronic fatigue syndrome has been associated with
higher serum angiotensin-converting enzyme (ACE) levels, which has been
suggested to reflect damage to the vascular endothelium (Lieberman J and
CA 02645749 2008-11-28
Bell, 1993). Hence, a part of the efficacy of SKP in chronic fatigue might be
related to its demonstrated ACE inhibitory activity.
Yeast fermentation generates higher levels of metabolite
hydroxymethylbutyrate from the amino acid leucine, which promotes greater
gains of muscle mass and strength in untrained men and women initiating
resistance training (Nissen et al., 1996). Since yeasts are predominant
microbiological species involved in kefir fermentation, kefir fermentation
likely
greatly increases the production of hydroxymethylbutyrate.
Several supplementation trials have indicated. that branched chain amino
acids (BCAA) can contribute to combat fatigue and to improve mental and
physical performance in athletes (De Lorenzo et al., 2003; Blomstrand et
al.,1997). Soy protein is one of the best sources of BCAA and fermentation
increases the quantity of soluble proteins. Hence, the digestibility and
bioavailability of BCAA from soy kefir would be significantly enhanced to
provide a significant enhancement in BCAA uptake. An imbalance in the ratio
of free tryptophan to BCAA with relatively low blood levels of BCAA has been
implicated as a possible cause of acute physiological and psychological
fatigue (central fatigue) during exercise (Gastmann and Lehmann, 1998) and
the chronic fatigue syndrome (Georgiades et al., 2003).
Use of the soy kefir powder of the invention for increasing vitality
In another object, the present invention relates to the use of the soy kefir
powder contemplated by the invention for increasing vitality.
The improvement of vitality has been sparsely investigated with no reference
to any fermented soy products. Green tea and products containing green tea,
which contain caffeine, have been indicated to increase vitality or have
stimulatory effects.
The findings shown in Example 2 thus are unexpected showing that healthy
individuals showed improvement in energy levels and vitality despite low daily
intakes of soy kefir liquid (only 200 mL) or 25-35 g soy kefir powder, major
a
CA 02645749 2008-11-28
improvements were noted in relatively quickly (i.e., within 1-7 days). As
shown
in Figure 2, vitality was greatly improved in the clinical trial involving soy
kefir
powder, as a significant increase in the sub-scale measure of vitality in the
SF-
32v2 questionnaire scores was demonstrated with a 12.8 point increase over
baseline measures. A five point increase in considered to be clinically
meaningful. (Rowbotham, 2001).
Use of the soy kefir powder of the invention for improving satiety
In a further object, the present invention relates to the use of the soy kefir
powder contemplated by the invention improving satiety.
In studies of rats, soy protein has been shown to have short-term satiating
properties when compared to other sources of protein (Semon et al., 1987).
However, the presence of yeast protein as found in soy kefir powder and
protein fermentation increases greatly satiety effects. For example,
mycoprotein produced by the continuous fermentation by mushroom
microorganisms induces acute and delayed suppressive effects on food
intake. Also, rats fed a high yeast protein load reduced their meal and daily
energy intake to a signifinicanty greater extent than rats fed well-balanced,
high protein diets from a variety of protein sources including soy, total milk
protein, or wheat gluten (Faipoux et al., 2006). The stong satiety may have
been increased by protein hydrolysis for greater availability of absorption
and/or gastrointestinal action of resulting peptides.
Fermentation of food proteins increases their digestibility and allows for
greater absorption of peptides, without changing the overall biological
value.70
In particular, proteins with high disulfide content such as soy are relatively
resistant to digestion,71 and fermentation increases their digestibility to
allow
for greater absorption of peptides.70-72 We postulate that some
physiologically
active bioactive peptides may be present in their inactive forms in the amino
acid sequences of proteins and are normally poorly absorbed from undigested
soy proteins.
oL~
CA 02645749 2008-11-28
The findings shown in Example 2 thus are unexpected showing that despite
low daily intakes of soy kefir liquid (only 200 mL) or 25-35 g soy kefir
powder,
major satiety was noted in relatively quickly (i.e., within 1-7 days).
Methods of use
In related objects, the present invention provides methods for preventing
and/or treating mood disorders, for treating fatigue, for increasing vitality,
for
improving satiety and/or preventing and/or treating fibromyalgia. Such
methods comprise the step of administering to said subject an effective
amount of the soy kefir powder of the invention.
The soy kefir powder may be associated with an acceptable carrier.
The amount of soy kefir powder of the invention administered is preferably an
effective amount. An effective amount of soy kefir powder of the invention is
that amount necessary to allow the same to perform its desired role without
causing, overly negative effects in the subject to which the soy kefir powder
is
administered. The exact amount of soy kefir powder to be administered will
vary according to factors such as the type of condition being treated, as well
as the mode of administration. In the context of the present invention, is
useful
to administer an amount that will treat fatigue, mood disorders, improve
satiety, improve energy and improve vitality.
The soy kefir powder of the invention may be given to a subject through
various routes of administration, but it is however preferably administered
per
os. Suitable dosages will vary, depending upon factors such as the desired
effect (short or long term), the route of administration, the age and the
weight
of the subject to be treated.
The present invention will be more readily understood by referring to the
following examples. These examples are illustrative of the wide range of
applicability of the present invention and is not intended to limit its scope.
Modifications and variations can be made therein without departing from the
2 21
CA 02645749 2008-11-28
spirit and scope of the invention. Although any methods and materials similar
or equivalent to those described herein can be used in the practice for
testing
of the present invention, the preferred methods and materials are described.
EXAMPLES
EXAMPLE 1 : PREPARATION OF SOY KEFIR POWDER ACCORDING TO
A PREFERRED METHOD OF PREPARATION OF THE INVENTION.
Hundred (100) cases of sterile (UHT) SO NICE Natural soy milk (Soyaworld
Inc.), each case consisting of twelve 946 mL Tetrapaks, were obtained (three
production lots). Milk was stored at 4 C in a walk-in cold room.
A 150 L Chemap fermenter situated in the Biotechnology Research Institute
(BRI, Montreal, Quebec) pilot plant was used for all production fermentations.
Prior to the first fermentation, the fermenter was cleaned using standard BRI
protocols and then steam sterilized at 1210C using the computer controlled
sterilization cycle. The fermenter was equipped for on-line control of
temperature and continuous monitoring of pH. The fermentations were run
without air addition (anaerobic) and without agitation, except for brief
periods
during startup and harvesting.
The fermentation substrate consisted of soy milk and dextrose. Except for
batches designated K0830A and K0830B, each batch used 9 cases of milk
(102 L) plus 2 kg of dextrose. Batches K0830A and K0830B used 90 L of milk
plus 1.78 kg of glucose. The temperature controller was set to a constant
23 C. The milk and glucose were added to the fermenter manually and then
agitated for 2 minutes at 250 rpm prior to addition of the grains (starter
culture). The initial starter culture consisted of 5 kg of wet grains plus 16
L of
fermented kefir. In subsequent fermentations the grains consisted of the
solids
filtered from the previous batch of harvested kefir. After addition of the
grains,
the agitation was continued at 250 rpm for an additional 2 minutes. At this
time
the fermentation was considered as started and the pH and temperature were
noted.
22,
CA 02645749 2008-11-28
The fermentation was continued at constant temperature with no agitation for
a specified length of time based on prior experience and ongoing analysis of
the IC50 from previous batches. Except for the first batch, fermentation times
ranged between 16 and 24 hours.
At the end of the batch and prior to harvesting, the kefir was agitated at 250
rpm for 2 min and the harvest line was flushed with steam. Since a pump was
not used for harvesting, the kefir was removed from the fermenter by gravity
flow, aided by 0.5 barr of air pressure introduced into the fermenter
headspace.
The harvested kefir was filtered using a custom-made 316 SS cone sieve with
3.2 mm openings. The filtered liquid was collected in a 200 L SS tank and the
collected solids retained for addition into the subsequent batch. From the SS
tank, the filtered liquid was placed into 19 L plastic pails, sealed airtight
with
gasketed lids and placed in a walk-in cold room at 4 C (note: this was a
different cold room from that used for storage of the soy milk substrate).
The above-mentioned process can be repeated several times, for instance for
a total of 11 fermentation batches.
After completion of all the fermentation batches, the liquid kefir was stored
in a
walk-in cold room at 4 C. Spray drying of the kefir was performed using a Niro
Atomizer Spray Dryer Model HT-10-530. Each fermentation batch was spray
dried separately. The spray drying conditions for each batch were maintained
constant by controlling outlet air temperature to between 60-70 C by
adjustment of throughput rate. The time required and solids yield from each
spray dried batch were recorded and, after obtaining a sample for analysis,
each batch of powder was hermetically sealed in a plastic bag. After all
batches had been spray dried, the powder from all batches (except K0817,
first fermentation batch) was sieved using a Kason vibrating screen with 2.1
mm hole size. The total mass of large chunks collected by the sieving
operation was 11.66 kg or about 23% of the total product yield. The large
chunks were crushed using an Urschel high speed chopper and then added to
the powder. All sieved and crushed powder was blended together for 30
CA 02645749 2008-11-28
minutes using a double-action ribbon blender. A 500 g sample was obtained
for analysis.
The soy kefir powde was packaged in ten hermetically sealed plastic bags.
Each bag was weighed and placed. in an airtight plastic pail and stored at 4 C
until use.
EXAMPLE 2: USE OF THE SOY KEFIR POWDER OF THE INVENTION
FOR TREATING FATIGUE, MOOD ELEVATION, AND INCREASING
SATIETY
RAW MATERIALS USED IN MANUFACTURING
Source of Soymilk
Soyaworld Inc., Burnaby, BC, Canada.
Source of Kefir Grains
The All-Russia Dairy Institute (ARDI), 35 Lyusinovskaya Street, Moscow,
Russia.
FERMENTATION AND PROCESSING
Kefir grains are activated in unsweetened soymilk (2% dextrose added) at a
ratio of 1-part grains to 2-parts milk and maintained at room temperature for
approximately 24 hours. Grains are then removed by coarse sieving, placed in
the fermenter at a ratio of 1 part grains to 5 parts soymilk (2% dextrose
added), then fermented at room temperature for approximately 24 hours.
Fermentation produces carbon dioxide gas and lowers the pH to 3.5 to 4,
producing a thick foaming liquid, creamy in texture and consistency, with an
approximate alcohol content of 0.5% to 1 % by volume.
As kefir grains increase in volume during fermentation, a portion of grains
are
removed to maintain constant grain-to-milk ratio. When fermentation is
completed, grains are removed by coarse sieving and used as the inoculum
CA 02645749 2008-11-28
for fermenting a subsequent batch of soymilk.5 Alternatively, grains can be
lyophilized for long-term storage.'
Following removal of the grains, liquid kefir is approximately 8% total
solids. It
is then converted to powder by spray-drying, thus removing any significant
amount of alcohol.
There are no significant variations of vitamin and mineral content following
kefir fermentation from the original sourced soymilk; however, a small
increase
in proteolysis leads to an increase in free amino acids.23 Indeed, the
capillary
electrophoretic profile of the fermented soymilk demonstrates a
protein/peptide
profile unique from that of unfermented soymilk (Fig. 1).
PACKAGING
The soy kefir powder is packadged in 4W' x 5W' paper/foil pouches, each
containing 35 grams of powder.
CASE REPORTS
Human data with Soy Kefir Powder or Soy Kefir Liquid shows significant
effects in terms of mood elevation (i.e., stress reduction), relief of fatigue
including the treatment of chronic fatigue syndrome in 10 subjects with and
without chronic fatigue syndrome. Satiety effects were also specifically noted
by some subjects.
Some subjects initially received the Soy Kefir Liquid only whereas others
received Soy Kefir Liquid and SKP or SKP only. Subjects received initially the
original batch of SKP (Trial 1) and at a later time point, most of the same
subjects received the batch of SKP to be used in the clinical trial (Trial 2).
The first batch used generic Oriental store soy milk using a ratio of grains
to
milk of 1:5 whereas the second batch used SoNice soy milk using a ratio of
grains to milk of 1:20.
CA 02645749 2008-11-28
For the batch of SKP produced for the clinical trial (Trial 2), symptoms
regarding fatigue, stress/depression, sleep disturbances or other disturbances
were rated on a scale 0-5 with a 0 being symptom-free and 5 meaning
symptom at its greatest intensity. Any adverse event was also noted.
OPEN LABEL CLINICAL TRIAL WITH PATIENTS WITH CHRONIC
FATIGUE SYNDROME
As anecdotal evidence indicated that the Soy Kefir Powder may improve
several features of the chronic fatigue syndrome, i.e., weakness, lack of
energy and depressed mood, an open label pilot study was carried out to test
the tolerance and effects of the product on a small group of chronic fatigue
patients. Eleven patients with chronic fatigue syndrome received 56 pouches
of 37.5 grams of product, to be taken as I pouch twice a day for 4 weeks.
Patients answered the SF-36v2 Health Survey quality of life questionnaire
before, and after the 4 week treatment period.
The SF-36v2 Health Survey, is a highly validated, widely-used health status
assessment tool that measures eight concepts: physical functioning (PF), role
limitations due to physical health (RP), bodily pain (BP), general health
perceptions (GH), vitality (VT), social functioning (SF), role limitations due
to
emotional problems (RE), and general mental health (MH). Scores for people
at the top or bottom of a scale can be interpreted by looking at the items and
response choices that must be chosen to earn those scores. For example,
someone at the top score of the SF-36 Physical Functioning (PF) scale does
not have limitations in any of the SF-36 activities due to health. A person
scoring at the bottom of the PF scale is very limited in all activities,
including
bathing and dressing. Physical Functiohing, Role Physical, and Bodily Pain
are primarily measures of physical health, while the other three scales are
primarily measures of mental health. Research has demonstrated that scales
associated with the physical health construct are sensitive to detecting the
impact of physical health interventions. Similarly, scales that are the
strongest
measures of mental health are sensitive in detecting the impact of mental
health interventions. SF-36 is a FDA approved tool that is used in a wide
CA 02645749 2008-11-28
variety of clinical areas ranging from cardiac rehabilitation programs and hip
replacement surgery to the impact of medications on pain relief.
In the open label efficacy trial, two patients had to discontinue the
treatment:
one because of gastric pain after 3 days even though she said she had never
felt so energetic from the time that she had the disease. She suggested that
she wanted to try to take the product 1 or 2 days a week as her improvements
were so remarkable. The second patient had gastric discomfort and vomited
at her first ingestion of the product. Increased satiety effects were also
noted
after the ingestion of the product. All other patients took the product for 4
weeks. For the statistical analysis, a two-tailed Wilcoxon test was used. The
results of the questionnaire are assembled into 8 scales and the average
score for each scale before and after,the treatment were compared. The alpha
risk was 5%.
The results of this pilot study show that the product had significant
beneficial
effects on. the subjects in terms of pain, energy and mood (Figure 2). Two
scales, Bodily Pain and Vitality show differences with an alpha risk < 5% the
accepted threshold in sciences and 4 other with an alpha risk _< 12% which
qualify them for "trend" because they are compatible with a true effect (and
consequently have a good chance to be shown in a larger study). Those
additional scales that showed strong trends included Role Emotional, Role
Physical, Social Functioning and General Health. A satiety effect was also
noted in one subject in the pilot study, which was also observed in the case
study among some subjects.
The clinical trial was extended from a two week to a four week intervention as
there is evidence that the placebo effects typically fade after a two-week
time
frame. Hence, the placebo effect was therefore less likely as positive results
were seen over the more extended period of four weeks. Moreover, literature
indicates that the chronic fatigue patient population is quite resistant to
placebo effects due to their high utilization of a wide variety of other
products
that show no efficacy. Also, improvement in the energy, emotional health and
social functioning scores is concordant with the case study results showing
CA 02645749 2008-11-28
consistent improvements in energy, mood elevation and satiety among
subjects either with or without chronic fatigue syndrome.
EXAMPLE 3 CASE STUDIES OF INDIVIDUAL PATIENTS
Eleven patients experiencing different symptoms, some having chronic fatigue
syndrome and the others suffering from other conditions were given soy kefir
product individually at different times.
Table 3. The results of the clinicat study with patients having chronic
fatigue
syndrome:
CA 02645749 2008-11-28
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EXAMPLE 4: PROTOCOL OUTLINE
((EFFECTS OF SOY KEFIR POWDER IN MILD TO MODERATE ON THE
QUALITY OF LIFE OF PATIENTS WITH CHRONIC FATIGUE SYNDROME"
Introduction:
From anecdotic evidence, it was hypothesized that a fermented soy milk
product may improve several features of the chronic fatigue syndrome:
asthenia, pain, and mood. A pilot study was planned to test the tolerance and
effects of the product on a small group of patients and with an open design.
Patients and procedures:
Eleven patients with chronic fatigue syndrome received 56 pouches of 37, 5 gr
of product, to be taken as 1 pouch twice a day for 4 weeks. Patients answered
a quality of life questionnaire before and after the 4 week treatment period.
This questionnaire, the SF36v2 is a validated tool that is widely used in this
area of research.
Results:
Tolerance: 2 patients had to discontinue the treatment: one because of gastric
pain after 3 days even though she said she had not felt so energetic since she
had the disease. She suggested trying to take the product 1 or 2 days a week.
The second patient had gastric discomfort and vomited at her first ingestion
of
the product. All other patients took the product for 4 weeks.
Anecdotic findings: one patient noticed that after the ingestion of the
product
at around 10 am and 3 pm, she felt "full' and did not experience hunger at the
time of the next meal. Several patients had problems with the taste and did
not find a perfect vehicle for the product. One of them did a thorough
investigation of different beverages and found the brand of fruit juices CERES
was the best.
One patient said that she had suffered from chronic muscle pain in here thighs
and that it disappeared soon after the beginning of the treatment.
2
CA 02645749 2008-11-28
III Statistical analysis: the questionnaire is divided into 11 questions, with
several sub questions in most of them, for a total of 36 questions. For the
analysis, a 2 tailed Wilcoxon test was used. The results of the questionnaire
are assembled into 8 scales and the average score for each scale before and
after the treatment are compared. The alpha risk is 5%. The results are
summarized on table 4 and the statistics on table 5.
Mean Std. Deviation
PREPF Physical health 47,78 25,51
POSPF 54,44 29,94
PRERP Role physical 25,00 12,50
POSRP 43,06 26,04
PRERE Role emotional 60,19 30,84
POSRE 81,48 20,32
PRESF Social functioning 25,93 16,67
POSSF 44,44 36,00
PREBP Bodily pain 27,00 17,68
POSBP 45,78 13,41
. PREGH General health 43,22 14,10
POSGH 51,44 20,89
PREVT Vitality 21,11 11,40
POSVT 33,89 16,91
PREMH Mental health 56,44 7,33
POSMH 58,67 8,72
PREHTI Health transition 3,11 ,33
POSHTI 3,44. 1,01
Table 4: Means and standard deviations pre and post treatment for each scale
Asymp. Sig. (2-
Z tailed)
POSPF - PREPF Physical health -,666(a) ,506
POSRP - PRERP Role physical -1,690(a) ,091
POSRE - PRERE Role emotional -1,859(a) ,063
POSSF - PRESF Social
functioning -1,552(a) 121
POSBP - PREBP Bodily pain -1,997(a) ,046
POSGH - PREGH General health -1,548(a) ,122
POSVT - PREVT Vitality -2,198(a) ,028
POSMH - PREMH Mental health -,108(a) ,914
POSHT1 - PREHT1 Health -1,134(a) 257
transition
b Wilcoxon Signed Ranks Test
Table 5: Z scores for comparisons of the means and p values
CA 02645749 2008-11-28
Interpretation: The results of this pilot study show that the product had
significant beneficial effects on the subjects. Two scales, Bodily Pain and
Vitality show differences with an alpha risk < 5% the accepted threshold in
sciences and 4 other with an alpha risk _< 12% which qualify them for "trend"
because they are compatible with a true effect (and consequently have a good
chance to be shown in a larger study).
It is noteworthy that among the 9 subjects tested, only 3 reported spectacular
results such as the disappearance of a pain or more vitality or more hours of
functioning. This suggests that the questionnaire can identify small
improvements. The results of the comparison with the general population
should be interpreted with caution because it is not a population matched for
gender and age. They show however that each scale has shown a significant
change for the better.
Conclusions and future direction: The results clearly support the hypothesis
that the product has important beneficial effects on fatigue, pain and
vitality. It
also may have positive effects on mood. Finally, an observation on a possible
satiety effect was made.
In the future, larger controlled and randomized trial should be designed to
test
the following hypothesis:
The product has beneficial effects on pain. Patients suffering from pain that
are not adequately treated by conventional medicine, such as fibromyalgia
may be a good population. Alternatively, patients who suffer from chronic pain
which treatment may be harmful, such as arthritis could be considered.
The product has beneficial effects on fatigue. This could be tested on
patients
with chronic fatigue again.
In addition, pilot studies should test the following:
The product has beneficial effect on mood. An exploratory investigation with
patients suffering from mood disorders or in women at menopause, who
present with such problems, is indicated.
3J
CA 02645749 2008-11-28
The product has satiety effects. This deserves a pilot study because if it is
true, a huge portion of the population will be interested.
On a practical standpoint, the vehicle of the product should be worked on in
order to obtain a palatable solution.
EXAMPLE 5 Clinical trial
Product Summary
SKP is a new product for patents that suffer from Chronic Fatigue Syndrome
(CFS) and Fibromyalgia (FMS) and that are coping with limitations due to
pain, fatigue, and lack of energy and vitality.
SKP is a dried powder made of soy milk fermented with Kefir grains. The
product has shown its efficacy in separate clinical trials on quality of life
parameters such as bodily pain, role physical and vitality.
This presentation will summarize the results obtained from one of those
clinical trials.
Clinical trial design
The clinical trial was designed to confirm the results previously obtained by
the Applicant by use of the SF-36v2 questionnaire.
Patients were to be recruited, and divided in three treatment arms of 10g, 20g
and 30g per day of flavoured kefir powder.
Patients suffering from chronic fatigue syndrome and fibromyalgia of
moderate intensity were selected. Symptoms of the disease include prominent
fatigue as well as chronic widespread muscle and joint pain. These symptoms
CA 02645749 2008-11-28
greatly limit a person's ability to work, entertain a social life, enter or
maintain
affective relationships, and practice any kind of physical activities.
Patients were followed for 60 days and SF36v2 questionnaires were filled at
baseline, and after 30 and 60 days of treatment.
In the absence of a placebo group, statistical analysis aimed at comparing
treatment groups at any time point to their baseline values by use of a two
tailed paired t-test.
Clinical trial results
A total of 18 patients have been completed so far (10 at 20g, 8 at 30g).
Approximately 9 patients are currently ongoing and should complete by mid-
December.
There has been some drop-outs during the course of the study. Most patients
dropped out of lack of compliance or lack of motivation (typical of CFS and
FMS patients). Two patients stopped using the product for reasons of side
effects in the 20g group but still managed to finish the study. Those results
were included in the analysis. Two patients also stopped the product on the
30g group because of side effects. One of those two patients did not complete
any follow-up questionnaire and was thus eliminated from the analysis.
Mean compliance was 55.6 days in the 20g group Vs 49.8 days in the 30g
group.
The statistical analysis of the results of this clinical analysis is shown in
Figures 4 to 9.
The results show a consistent and dose-dependent effect on many HRQOL
parameters as summarized below in Table 6:
HRQOL Clinical meaningful Statistical significance
CA 02645749 2008-11-28
parameter
Bodily Pain All doses, all time points 20g @ 60d, 30g @ 30, 60d
Physical
Functionning 20g @30, 60d, 30g @ 30d J20g @ 60d
Role Physical 20g @ 60d, 30g @ 30, 60d 20g @ 60d
Social Function 20g @ 60d, 30g @ 30, 60d 20g @ 60d => p=0.064
Vitality 20g @ 60d, 30g @ 30,60d ' None
Role Emotional 30g @ 30, 60d INone
Mental Health 30g @ 30, 60d None
General Health None None
L(2
CA 02645749 2008-11-28
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