Note: Descriptions are shown in the official language in which they were submitted.
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CA 02383021 2002-04-23
Cereal 13 glucan - probiotic compositions
Background of the Invention
Technical Field
The present invention relates generally to probiotic cultures, cereal flour
(meal), cereal
bran, and B glucan. More particularly, the invention relates to methods and
compositions
for producing probiotic-supporting preparations derived from oatmeal, oat
bran, and/or
13 glucan.
Background of the Invention
The =,concept of probiotics is based ori the premise that there are
microorganisms in the
intestinal tract which have a beneficial health effect on the host. Some of
these effects
are, for example, antibiotic production, lactose digestion enhancement,
cholesterol
reduction, antimicrobial effects, duration of diarrhea limitation, and gut
mucosal immune
system stimulation. These effects are dependent on the strain of bacteria
which affect the
composition of the gastrointestinal flora. A significant number of
publications have
focused on the fact that high amounts of lactobacilli and bifidobacteria are
antagonistic
to many pathogenic and putrefactive bacteria in the human intestine, which in
turn may
reduce the risk of cancer. (See Rafter J.J. (1995) Sca_n_d. J. C*astroenterol.
135:497)
Fermentation has been used to preserve perishable foods for centuries with
milk receiving
most of the attention. "Sour milk" or "yoghurt" are often referred to in
historical literature
(Tamine A Y and Robinson. R.K. (19$5). YQghu_rt Science and Technology.
Pergamon
Press. Chapters 1. 8. & 10.). Yoghurt is milk that has peen transformed to a
solid by the
fermentative action of organisms such as Lactobacillus delbrueckii ssp.
Bulgaricus and
Streptococcus salivarus ssp. Thermophilus. These microorganisms do not have
the ability
to colonize the intestinal tract in large number because they are not
resistant to the gastric
acidity and bile salts. Therefore, it has been proposed that the inclusion of
probiotic foods
in the diet may enhance the gut population of beneficial bacteria.
Presently some yoghurt is fermented with bacterial cultures of intestinal
origin and is
. . . . . .. ..;::.,: -
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CA 02383021 2002-04-23
-2-
called Bio-Yoghurt. In Bio-Yoghurt, the microorganisms must survive
distribution and
retailing and it is imperative to functionality that the bacterial count in
the final product
is high. To provide health benefits, the suggested probiotic concentration is
3106 CFU/ml.
See RohinsonL$K (1987) Suid Afikaanse TYdskrif Vir Suiwelkunde 19:25. However,
studies have shown low viability of probiotics in current market preparations
see Shah.
NP (1995) t. Datrv J 5:515
Traditional inoculated grain based products are known in Europe. In Scotland
and Wales,
flummery is prepared from oatmeal steeped in water and kept until it becomes
sour.
Similarly, in the Scandinavian and Baltic nations, soured cereal grains have
been
described, the majority of which are.cooked after fermentation, an action that
kills the
useful microorganisms, and thus destroys probiotic benefits.
Formulations of lactobacilli grown in combination with oat bran or oatmeal are
known.
An oat bran product inoculated with defined, living microbes has been
described by
Salovaara (EP00568530). This product has an inherent granular texture due to
bran
particles. The Salovaara product contains combinations of lactobacilli but
does not
describe the use of bifidobacteria nor does it present evidence on the
protection of
bifidobacteria from competition with lactobacilli. YosaTM sold in Finland is
based on the
technology described in Salovaara (EP00568530). Other publications have
described that
oat pastes proved to be a suitable growth media for yoghurt organisms
(Salovaara. H...
Backstr¾m. K. Mantere-Ajhonen. S.. 1991. J. Dairy Sci. 49: 3 7).
~
25. In US5190755 a nutrient health drink composition is described suitable for
enteral
feeding comprising a fermented cereal based product, enzyme,, and optionally
further
nutrient components, in combination with lactobacilli. Two stages of enzyme
treatment
are used in the preparation of an oat gruel, firstly incubation with a
am,ylase and secondly
incubation with P glucanase.
, . . . . . .. . .... . .. ..:.,..,...=,..-....: ..::..............':r>:
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CA 02383021 2002-04-23
-3-
US5591428 discloses an oatmeal-based oral nutritional supplement fermented by
Lactobacillusplantarum strain No. 299 (at a concentration of -] 010CFU/g of
freeze-dried
product) and a feeding formulation for enteral nutrition containing the same
strain of
Lactobacillus plantarum at a concentration of 1.5x10 CFU/ml of nutrition
solution.
Importantly, the method described in US5591428 requires the preparation of
oatmeal
gruel with 1%w/w 0-glucanase, which would serve to convert 13 glucan to
glucose. This
requirement is not part of the present invention.
However, typical oat yogurt preparations do not support the survival of bf
fidobacteria in
conjunction with other bacteria and as a result the number of bifidobacteria
declines
whilst the numbers of lactobacilli are sustained in steady numbers (see figure
1). Of
additional relevance is the usual decline in numbers of bifidobacteria at the
acidic pH of
-4 found in yoghurt.
13 glucans, for example, (1-3), (1-4) !3 D glucans are found in the endosperm
cell wall
of such cereals as barley and oat, appear to influence digestion, assist in
glucoregulation,
and lower serum cholesterol. Cereal 13 glucans are useful nutritional agents
and have also
been used as bulking agents in place of sucrose. !3 glucans have also been
described as
potent immune system stimulants and promote the, see Yun et al. Int. J.
Parasitol. (1997)
27=329-337= Es da et al (1997) Microbiol Immunol. 41:991-998; Williams et al.
(,19971
US 5.676.967.
Through the addition of (3 glucan to yoghurt, synergistic formulations of
probiotics and
soluble fibre are produced with both novel composition and health promoting
functionality.
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CA 02383021 2002-04-23
-4-
Disclosure of the Invention
The present invention describes the use of cereals and cereal derived
products, including
whole oat flour, or oatmeal prepared from oats or rolled oat grain,
to..,%upport the survival
,ftdobacteria in probiotic products. The products contain high numbers
of bi
(>108CFU/ml) of viable bifidobacteria, as well as functional numbers
(>106CFU/ml) of
other lactobacilli. The yoghurt was proven to have a minimum shelf-life of 30
days with
no indications of syneresis.
The products also contain significant quantities of soluble fibre. The amount
of soluble
1.0 fibre may readily be adjusted in order to comply with the 0.75g of soluble
dietary fibre
from.'oats per serving that the US FDA requires in order to make a functional
food health
claim.
The `oat milk' formulation described is novel in both its composition a.pd its
formulation
method. The formulation method differs from other oat gruels in so far as the
P glucan
derived from the cereal source is protected from enzymatic degradation in the
manufacturing process. The oat milk product is in itself of interest as a
functional health
drink or the basis of a fermented oat milk drink with probiotic benefits.
The products described, the yoghurt, flummery, drink, and dry probiotic/fibre
cereal are
non-milk containing and are lactose free making them suitable for special
dietary needs.
Brief Description of the Figures .
Figure 1 is a graph of the typical survival of bacteria in yoghurt and the
competitive loss
of bifrdobacteria.
Figure 2 is a graph of the survival of bacteria, and the enhanced survival of
bifidobacteria
in oat yoghurt cultures.
Figure 3 is a graph of the survival of bacteria, and the enhanced siuvival of
bifidobacteria
in oat yoghurt cultures containing artificial flavouring.
Figure 4 is a graph of the survival of bacteria, and the enhanced survival of
bifidobacteria in oat yoghurt cultures containing fruit preserves as
flavouring.
. ........ .... . ... . _,,,,,..
} CA 02383021 2002-04-23
-5-
Detailed Description of the Invention,
The practice of the present invention will employ, unless otherwise indicated,
conventional methods of chemistry, cereal chemistry, microbiology, and
biochemistry,
within the skill of the art. Such techniques are explained fully in the
literature. See, e.g.
Oats: Chemistrv andT~chnologX ediWebster FH (American Association of Cereal
Chemists. St. Paui_,J<N), Aledical microbioloQV edited by Samuel Baron:. 4th
ed (,The
iversity of Texas Medical Branch. Galveston. TX) and Tarnine, A.Y. and
Robinson.
R K yughurt Science and TechnologyP r mon Press.
to
All publications, patents, and patent applications cited herein, whether supra
or infra, are
incorporated by reference in their entirety.
As used in this specification and the appended claims, the singular forms "a,"
"an," and
1.5 "the" include plural references unless the content clearly indicates
otherwise. Thus, the
term "a ¾ glucan" can include more than one 13 glucan.
1. Definitions
In describing the present invention, the following terms will be employed, and
are
20 intended to be defined as indicated below.
By "cereal" is meant any of several grains such as, but not limited to,
cultivars of barley,
oat, wheat, rye, sorghum, millet, and corn.
By "B glucan" is meant a glucan with a(3(1--3)-linked glucopyranosyl backbone,
or a
P(1-4)-linked glucopyranosyl backbone, or a mixed (3(1-3) (1 -4)-linked
glucopyranosyl
25 backbone.
A "cereal B glucan" or a `cereal R glucan extract" is a B glucan or B glucan
extract,
respectively, which is derived from a cereal source.
By "probiotic" is meani a live microbial feed supplement which beneficially
affects the
host animal by improving its intestinal balance.
.; ,
CA 02383021 2002-04-23
~
-6-
By "prebiotic" is meant non-digestible food ingredients that beneficially
affect the host
by selectively stimulating the growth and/or activity of one or a limited
number of
bacteria in the colon and thus improve the host's heAlth.
By "cereal milk" is meant an aqueous suspension of a cereal treated'with a-
amylase
and optionally containing a stabiliser.
By "oat milk" is meant an aqueous suspension oatmeal, bran or oat 13 glucan
treated with
a-amylase optionally containing a stabiliser.
By "oatmeal" is meant the product of milling or reducing in particle size
kernels of whole
oats (Avena sativa).
By "oat bran" is meant the food which is produced by grinding clean oat groats
or rolled
oats ;and separating the resulting oat f)our by sieving, bolting, and/or other
similar means
into fra.ctions such that the oat bran fraction is not more than 50%> of the
starting material,
and has a total P glucan content of at least 5.5% (dry weight basis) and a
total dietary
fibre content of at least 16.0% (dry weight basis), and such that at least one-
third of the
total dietary fibre is soluble fibre.
2. General Methods
Oats and oat extracts are readily available, commercial suppliers include
Quaker Oat
Company (Chicago, USA), Ceapro Inc. (Edmonton,. Canada), Nutrinova AG
(Frankfurt,
Germany).
Oat milli.ng and particle reduction methods are known to those skilled in the
art. Milling
methods are described in Oats: Chemistrv and 1'echnaloQV ed. Webster FH
(American
Association of Cereal Chemists. St. Paul. MN and The Oat Crop: Production and
Utilisation. ed Welch. RW(Chanm & Hall. London). For oatmeal preparation oats
were ground iii'a Fitz Mill model "M" hammer mill (W.J. Fitzpatrick Co. Model
M
Comminutator Series 17 Code 80). The oats were milled until the product passed
through
a 0.5 mm screen sieve.
Oat bran containing !3 glucan was prepared for use in the present invention by
milling
through the Fitz Mill as described above with the milled product passing
through a
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CA 02383021 2002-04-23
-7-
0.5mm screen. Of particular benefit is extruded oat bran containing supra-
normal
amounts of 13 glucan such as GlucaMaxTM 16% !3 glucan (Nutrinova: Frankfurt,
Germany). The extruded nature of the GlucaMax product enhances the performance
of
a-amylase in starch digestion and the supra normal amounts of !3 glucan
enhances the
soluble fibre content of the oat yoghurt.
To enhance the funetionality of the oat yoghurt described in the present
invention, 13
glucan from any of several known cereal sources can be used in the described
process.
Such cereals include, without limitation, any of the cultivars of e.g. barley,
oat, wheat,
rye, com, sorghum, and millet, with barley and oat preferred because of their
high 13
glucan content. Oat 13 glucan is available from commercial suppliers for
example Ceapro
Inc. (Edmonton, Canada)
The !3 glucan content can be determined using a number of inethods, known to
those
skilled in the art. For example,l3 glucan content can be assessed
colorimetrically and/or
by standard analytical techniques.such as size exclusion chromatography and
HPLC. See
Wood et al Cereal Chem. (1977) 54=524LWood et al Cereal Chem (1991) 68=31 =
and
Wood et al_. Cereal Chem. (1991) 68:530.13 glucan can also be analyzed
enzymatically
using commercially available kits, such as Megazyme (Ireland) employing the
techniques
of McCle= and Glennie-Holmes J Inst. Brew. (198 ) 9
Conventional methods of drying liquids are known to those skilled in the art.
These
methods include, but are not limited to, vacuum evaporation and spray drying,
and
lyophilization. Different spray drying operating modes and configurations are
described
in Chem. Ing. Tech (1987) 59:112. Lyophilization methods are described in
,,nQwman.
JW nownstreain process= Equipment and Technig-ues (1988) 315 Alanl. Liss.
Bacterial Strains
Bacterial strains are available from a number of depositories and collections,
for example
the American Type Culture Collection (ATCC) Rockville, USA and Collection
National
de Cultures de Microorganismes, Institut Pasteur, Paris, France.
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CA 02383021 2002-04-23
-8-
Bacterial Growth Media:
MRS broth (DeMan, Rogosa and Sharpe media) for culturing Lactobacillus
bulgaricus.
Obtained from BDH Lot #V809761. Preparation: Suspend 52.2g in 1 litre of
distilled
water. Sterilized through autoclaving for 15 min. at 15 psi.
MRS++ broth (Modified De Man, Rogosa and Sharpe media) for culturing
Bifidobacteria infantis. To obtain the modified version of MRS to the basic
MRS broth
add: 0.2 g/l of NaZCO3, 0.1 g/1 CaC1Z-2HzO and adjust pH to 7.0 before
sterilization. After
autoclaving and cooling add 10 ml of filter sterilized 5% (w/v cysteine-HC1
solution).
M-17 broth for culturing Streptococcus thermophilus. Obtained from Oxoid Ltd
(Basingstoke UK). Preparation: suspend 37.25 g in 950 ml of distilled water.
Autoclave
for 15 min. at 15 psi for sterilization. After cooling add 50 ml of a 10%
(w/v) sterilized
Lactose solution.
BL-Agar (Glucose blood liver agar) for quantification of lactic acid bacteria.
BL-agar
1.5 was prepared to include defibrinated sheep blood.
Selection of bacterial strains
Lactobacillus bulgaricus (ATCC 11842) and Streptococcus thennophilus (ATCC
19258)
were selected because of their demonstrated ability to coagulate milk without
addition
of a stabilizer and their previous use as yoghurt starter culture. These two
organisms
provided a typical yoghurt aroma and flavour when used with regular milk.
Bifidobacteria selection was based on:
(i) ability to coagulate milk without stabilizer,
(ii) human origin,
(iii) resistant to an acidic envirot1rnent (pH 4.2 to 4.5), typical of a
yoghurt
product: Human Bifidobacteria strains tested were: Bffidobacterium longum
(ATCC. 15707), Bifidobacterium breve (ATCC 15770), Bifidobacterium
adolescentis (ATCC 15703), and Bifidobacterium infantis (ATCC 15697).
. . . . . . .. . '. . .'...., , .:. ... . .. . . . ......
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CA 02383021 2002-04-23
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PreDaration of bacterial inocula
Inocula were prepared by growing bacteria aerobically: 39-40'C for
Lactobacillus
bulgaricus [ATCC 11842] in MRS and Streptococcus thermophilus [ATCC 19258] in
M-17. Bifidobacterium infantis [ATGC i5697] was grown anaerobically at 39 C in
MRS++. After growth the cells were centrifuged at 7970 G's using a Sorvall
Model
RCS5 (Rotor #10 at 7000 rpm for 20 min). The concentrated cells were washed
twice and
resuspended in 0.2M sterile potassium buffer pH 6.5 for their future use as
inocula.
Inocula consisted of between 10$ and 109CFU/ml viable Bifidobacteria and
between 10'
and 10$ CFU/ml each of viable Lactobacilli and Streptococci.
Microbiology Counts
Miciobiology counts were performed by plating samples in duplicate from T=0 to
one
month shelf life (t-28-30 days) using BL agar plates, incubated anaerobically
at 39-40 C
for 48 hrs. TSA plates were incubated aerobically at 35 C for 48 hrs.
Titratable Acidity Assav
Titratable acidity is accepted as a measure of the growth of lactic acid-
producing bacteria.
The methodology was adapted from Marshall, R T 1993. Standard Methods for the
Examination of Dairy Products 16' Edition. Cap 15, ny. 435-437. Briefly, a 9 g
of
sample of product was diluted with 18 g of distilled water. Approximately 0.5
ml of
phenolphthalein solution was added and the mixture titrated with 0.1N NaOH
solution
until first persistent (30 seconds) pink colouration appeared). Acidity was
expressed as
% of lactic acid by weight (1 ml 0.1N NaOH = 0.0090 g Lactic Acid). Duplicates
of
titrated samples were averaged before final calculation. For undiluted samples
and
samples reconstituted to original solids the calculatiqn was as follows:
% acidity =(ml NaOH) (M NaOH) (9g)/weight of sample (g)
Svneresis assay
Syneresis or whey exudates that appear on the surface of yoghurt are
considered to be a
defect. Syneresis was measured by making a hole in the base of the tub with a
soldering
iron and inverting the tub gently onto a fine mesh screen held over the top of
a funnel
. .. . _ _ ... ..... . . . .. . . . . . . .....: ;;,
CA 02383021 2002-04-23
-10-
inserted into a graduated cylinder. This process was done in a cold room (5 C)
and the
measurement of syneresis was taken after 1 hotir draining time.
C. Experimental
Below are examples of specific embodiments for carrying out the present
invention. The
examples are offered for illustrat.ive purposes only, and are not intended to
limit the scope
of the present invention in any way.
Efforts have been made to ensure accuracy with respect to numbers used (e.g.
amounts,
temperatures, etc.) but some experimental error and deviation should, of
course be
allowed for.
Example 1. Production of Oat Milk
The inability of the above-mentioned bifidobacteria to coagulate the water-
based `oat
.15 milk' required the addition of stabilizers to create a set-style yoghurt.
The stabiliser was
used at a concentration of 0.5% to meet regulatory requirements for yoghurt
manufacture.
The preferred stabilizer was determined to be 0.5% Benlacta 750CM carrageenan
(Food
Specialities, Mississauga, Canada) or alternatively 0.4% Benlacta 750CM
carrageenan
with 0.1% monoglyceride. High molecular B glucan was also useful to enhance
both
viscosity and add additional fibre content to the yoghurt.
Oat milk containing 12 or 15% oatmeal, 0.5% (w/w) stabilizer, and 0.15% (w/w)
a-
amylase (Sigma No. A-2771 type VIII-A from barley malt) were resuspended in
distilled
water, with continuous agitation for 30 min. at room temperature. To obtain a
manageable oat milk consistency from the oats in solution, different
concentrations of a-
amylase were tested. Addition of a-amylase ranged from 0%-0.45% with the
optimum
being 0.15% w/w with incubation at room temperature for 30 min. based on a
minimum
of 10% oatmeal solids.
CA 02383021 2002-04-23
-11-
The oat milk obtained was autoclaved for 15 min. at 15 psi (121 C) and cooled
before
inoculation.
Example 2. Production of oat based yoghurts
The step-by-step production protocol and quality control processes are
summarized in
Table 1.
Table 1.
Step in process Quality control
Bacterial Inoculum Microbial counts on BL and TSA agar
T=0 pH, titratable acidity, microbial counts onBL and
Inoculated Oat Milk TSA agar.
T=0 Option
Addition of flavouring or fruit preserve
T=4 h pH, titratable acidity
After 4 h. incubation at 39-40 C or
when H=4.7-4.8
T=24 h. pH, titratable acidity, microbial counts on BL agar
After 24 h. in refrigeration 5 C and syneresis.
T=7 d, 14 d, 21 d, 28130 d in pH, titratable acidity, microbial counts on BL
agar
Storage under refrigeration 5 C and syneresis.
Sterilized oat milk containing 12% oatmeal and 0.4% Benlacta 750CM carrageenan
with 0.1 % monoglyceride was inoculated with 2% each of concentrated washed
cells
of Lactobacillus bulgaricus and Streptococcus thermophilus and 4% of
Bifidobacterium longum concentrated cells. Following inoculation, 100 ml
quantities
of yoghurt were dispensed in tubs, capped, and incubated aerobically at 39-40
C for
approximately 3.5-4.0 hours or until pH reached 4.7-4.8. The tubs of yoghurt
were
refrigerated at 5 C for one month.
Physico-chemical tests included measuring pH, developed acidity, and
syneresis.
Microbial counts were monitored in the inoculum (T=0) and tmtil one month of
shelf
life (T=28-30`days). The microbial counts for B.longum indicated a consistent
survival at >108CFU/ml. Bacterial counts for both L.bulgaricus
and.S.thermophilus
- remained at >106CFU/ml. A graph of the time course for bacterial cell
numbers is
given in Figure 2.
CA 02383021 2002-04-23
12-
The yoglntrts all developed a pH of <4.7 within 24 hours and optimally within
the initial
4 hour incubation period.
The titratable acidity assay indicated the rapid achievement of >0. 18% lactic
acid within
the initial 24 hours of cultivation. Over the following 28 days the titratable
lactic acid
continued to rise to a average level of 0.27%
Surprisingly, none of the cultures displayed syneresis.
Experiment 3. Production of flavoured oat yoghurts
Sterilized oat milk containing 15% oatmeal with 0.5% Benlacta 750CM
carrageenan was
prepared using flavouring of:
a. 0.5% vanilla,
b. 0.5% cinnamon,
c. 0.5% maple syrup,
d. 0.5 % cherry, or
e. Control of 0.5% sucrose.
The oat milk was inoculated with 2% each of concentrated washed cells of
Lactobacillus bulgaricus and Streptococcus thermophilus together with 4% of
Bifzdobacterium infantis concentrated cells. Following inoculation, 100 ml
quantities of yoghurt were dispensed in tubs, capped, and incubated
aerobically
at 39-40 C for approximately 3.5-4.0 hours or until pH i=eached 4.7-4.8 and
then
the tubs were refrigerated at 5 C for one month.
The microbial growth profiles were found to be as described in Experiment 2
and
are illustrated in Figure 3. The acid profile and absence of syneresis gave
the
yoghurts ideal properties for commercial products.
- . ., . . ......................... .........:.....:..Mi..l':L.:.Y..
CA 02383021 2002-04-23
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Experiment 4. Production of flavoured oat yoghurts
Sterilized oat milk was prepared containing 15% oatmeal, 0.4% Benlacta 750CM
carrageenan, and 0.1% monoglyceride. Following sterilization of the oat milk,
and before inoculation and fermentation the fruit flavouring (preserves) were
added to avoid loss of natural colours and flavours which become dull due to
the
effect of the heat treatment. This technique also avoids reduction of
viscosity and
limits syneresis (wheying-off) in the final product.
The following flavourings were used: -
a. 10% maple syrup/2.5% sucroge,
b. 10% peach jam/2.5% sucrose,
c. 10% raspberry jam/2.5% sucrose,
d. 10% blueberryjam/ 2.5% sucrose, or
1.5 e. Control of 5% sucrose.
The oat milk was inoculated with 2% each of concentrated washed cells of
Lactobacillus bulgaricus and Streptococcus thermophilus together with 4% of
Bifidobacterium longum concentrated cells. Following inoculation, 100 ml
210 quantities of yoghurt were dispensed in tubs, capped, and incubated
aerobically
at 39-40 C for approximately 3.5-4.0 hours or until pH reached 4.7-4.8 and
then
the tubs were refrigerated at 5 C for one month.
The microbial growth profiles were found to be similar as those described in
25 Experiment 2 and are illustrated in Figure 4. The acid profile and absence
of
syneresis gave the yoghurts ideal properties for commercial products.
Because an invention has been described with regard to preferred embodiments,
however it will be understood to persons skilled in the art that a number of
30 variations and modifications can be made without departing from the scope
of the
invention as described in the following claims.
