Note: Descriptions are shown in the official language in which they were submitted.
CA 02420095 2003-02-26
_. _2_
CRYUPROTECTIQN
'technical Field
This invention relates to the protection of microorganisms against lethal and
sub-
lethal damage caused by exposure to low temperatures. This protection is of
use in froxen
microorganisms and freeze drying microorganisms and in the preparation and
storage of
fermented and probiotic foods.
>sackgrpund Art
ro Microorganisms find application as starter cultures in the production of
fermented
foods, or as probiotic microorganisms providing health benefits to the
consumer. Starter
cultures and probiotic organisms can be- i) used for fermentation of raw
ingredieztts; or ii)
probiotic microorganisms can be added in suitable concentrations to the
finished product
prior to packaging (l3ulIimore; 1983; Gilliand, 1985). Starter and probiotic
cultures can be
15 preserved in frozen or freeze dried forms that can be either inoculated
into the bulk starter
media or directly into the product prior to fermentation or packaging.
Froaen starter cultures are the least expensive to produce but do require a
continuous cold chain from place of manufacture to the point of use. Further
to this, long
term storage of frozen blocks is best below -20°C, temperatures that
might not be easily
2o attainable in the factory. Freeze dried starter cultures are more expensive
to produce but
have the added advantage of being able to be transported without refrigeration
{Champagne et al., 1991), Consequently, freeze dried cultures are a better
alternative for
the food manufacturer that does not possess the facilities to produce and
store its own
cultures. Spray dried tactic acid bacteria cultures are still in an
experimental stage and
25 good cell viability is not yet standard (To and Etzel, 1997)_
Freezing and freeze drying microorganisms can affect their viability.
Gryoprotectants are compounds that provide some protection to biological
materials during
freezing, freeze drying and subsequent storage These compounds are used to
prevent
lethal and sub-lethal damage from occurring to the microorganisms, in order to
maintain
3o maximum viability, metabolic activity pr health benefits in subsequent
applications of the
microorganisms.
Many compounds have been trialed as cryoprotectants. Cryoprotectants including
skim milk; disaccharides: lactose, sucrose and trehalose; polyols: glycerol,
adonitol,
sorbitol; polysaccharides: pectin, dextran, resistant starch; amino acids;
polymers: gelatin,
CA 02420095 2003-02-26
-3-
gums, maltodextrin; and antiaxid$iits: ascorbic acid; have been trialed with
mixed and
often poor results (Champagne et al. 1991}.The modes of action of
cryoprotectants are not
generally well understood. Cryoprotectant action is thought to be a
combination of many
factors including stabilization of microbial cell membranes, retention of high
water
activity and prevention of ice crystal formation, preventing oxidation,
eradication of free
radicals and prevention of subsequent cell disruption (Champagne et al.,
1991).
Detrimental treatment of microorganisms during freeze drying, may result in
reduction of viability, metabolic pathway damage, reduced fermentation
activity or
tolerance of adverse conditions. Changes in metabolic activity, pathogen
inhibition, salt,
x0 bile and acid tolerance are indicative of the degree of damage sustained
during freeze
drying. The most important feature of a freeze dried culture is retention of
cell viability.
Aiirer freeze drying, probiatic cultures should preferably retain both
ma~cimum viability and
activity, or the specific attributes associated with that isolate.
For probiotic microorganisms to be of benefit to the consumer, they must be
1s present iz~ the food or tablets in a suitable concentration. When
incorporated into a
product, probiotics need to remain viable at a concentration of lOdcfulgram or
greater for
the entire shelf life of the product to be of benefit to the consumer. A
person vrould then
need to consume at least 100 grams of the product every day to ensure a
minimum daily ,
dose of probiotics of l O8cfu in total, in order to gain health benefits.
20 Probiotic viability in food products has been shown to be a significant
issue, as
initial results revealed that many products, such as yoghurt, did not maintain
adequate
probiotic survival (Rybka and Fleet, 1997}. Better survival of probiotic
bacteria in yoghurts
has been achieved by adding resistant starch to the yoghurt (CRC for Food
Industry
innovation, 1997) Probiotic microorganisms incorporated into frozen frrmented
dairy
25 yoghurt and ice cream products have shown better viability during shelf
life when
compared to chilled yoghurts. As well as being present in adequate
concentrations,
probiotics need to be in a condition suitable to cope with the adverse acid
attd bile
conditions encountered in the ,gastrointestinal tract. Product manufacture may
injure
probiotic cells. Although they remain viable, impaired cells are readily
inactivated if less
3o than ideal conditions are experienced, for exarmple, on exposure to acid
conditions of the
stomach and bile salts of the gastrointestinal tract. Sublethal injury can be
assessed by
measuring changes in normal cell activity or cell resistance, such SS 13-
galactosidase
activity or bile tolerance.
CA 02420095 2003-02-26
- !~ -
Description of the Invention
The present inventors investigated the use of various substances as
cryoprotectants
for microorganisms used in the preparation of, or incorporated into, foods.
The cryoprotectant which is currently typically used is skim milk. One problem
with skim milk is that its animal origin makes its use unacceptable to strict
vegetarians.
Trehalose has also been used but it is an expensive compound which detracts
from
its use in relation to foods.
Inulin is a plant-derived oligo/polysaccharide comprising branched glucose and
fructose chains of heterogeneous lengths with the chemical structure of a-D~-
G1u-( 1-2)-[(~-
zo D-Pru-(1~2r~° (Crittenden, 1999). Inulin, marketed as RaRiline
{Orafti, Aandorenstraat
1, 3300 Tienen, Belgium), is non-digestible to humans, but acts as a
'prebiotic',
selectively being utilised by Biftdobacterium species in the human gut
(Roberfroid, 1993).
The information supplied by the marketing company reports that the strain B,
lactic Bb-12
can utilise inulin although the test conditions 'are not mentioned. Inulin has
a low calorific
15 content and also acts as soluble dietary fibre. Inulin is already used in
foods intended as
dietary aids, with fibre, fat repla,cer and improved te~ctural qualities.
The present inventors included the prebiotac inuiin in the Substances they
tested as
cryoprotectants reasoning that if a substance reported to function as a
prebiotic proved to
have cryoprotective properties it would be particularly beneficial to the
production of
2o probiotic foods, especially, in the case of inulin, those acceptable to
vegetarians.
The present inventors surprisingly found that inulin was not only
cryoprotective but
could provide better cryoprotection than skim milk as illustrated by providing
better
survival of microorganisms during freeze drying.
The present invention provides use of an aligolpolysaccharide comprising
branched
25 glucose and fructose chains of heterogeneous lengths as a cryoprotectant
for
microorganisms. Typically the oligo/polysaccharide is of plant origin. Plants
which can
act as a source of such an oligolpolysaecharide include topinambour, chicory,
union,
asparagus and artichoke.
In particular the present invention provides use of inulin as a cryoprotectant
for
3o microorganisms.
The present invention provides a method for freeze drying a microorganism,
which
method comprises freeze drying the microorganism in the presence of an
oligolpolysaccharide comprising branched glucose and fructose chains of
heterogeneous
lengths.
CA 02420095 2003-02-26
-5-
The present invention provides a method fox freeze drying a microorganism,
which
method comprises freeze drying the microorganism in the presence of inulin.
The present invention provides a method fQr freezing a microorganism, which
method comprises freezing the microorganism in the presence of an
oligo/polysaccharide
comprising branched glucose and fructose chains of heterogeneous lengths.
The present invention provides a method for freezing a microorganism, which
method comprises freezing the microorganism in the presence of inulin.
The invention provides a method for preverning cell deactivation during freeze
drying of a microorganism which method comprises freeze drying the
microorganism in
to the presence of an oligolpolysaccharide comprising branched glucose and
fructose chains
of heterogeneous lengths.
The invention provides a method for preventing cell deactivation during freeze
drying of a microorganism which method comprises freeze drying the
microorganism in
the presence of inulin.
15 The invention provides a method for preventing cell deactivation during
freezing of
a microorganism which method comprises freezing the microorganism in the
presence of
an oligolpolysaccharide comprising branched glucose and fructose chains of
heterogeneous
lengths,
The invention provides a method for preventing cell deactivation during
freezing of
2o a microorganism which method comprises freezing the microorganism in tlae
presence of
inulin.
The invention provides a method for preventing sublethal injury during freeze
drying of a microorganism, which method comprises freeze drying the
microorganism in
the presence of an oligolpolysaccharide comprising branched glucose and
fructose chains
25 of heterogeneous lengths_
The invention provides a method for preventing sublethal injury during freeze
drying of a microorganism, which method comprises freeze drying the
microorganism in
the presence of inulin.
The invention provides a method for preventing sublethal injury during
freezing of
30 a microorganism, which method comprises freezing the microorganisrtt in the
presence of
an oligolpolysaccharide comprising branched glucose and fructose chains of
heterogeneous
lengths.
CA 02420095 2003-02-26
.. _
The invemion provides a method for preventing sublethal injury during,freezing
of
a microorganisnn, which method comprises freezing the microorganism in the
presence of
inulin.
The invention provides a method for enhancing stoiage survival of a
microorganism which method comprises freeze dt3ring the microorganism in the
presence
of an oligolpolysaccharide comprising branched glucose and fructose chains of
heterogeneous lengths,
The invention provides a method fox enhancing storage survival of a
microorganism which method comprises freeze drying the microorganism in the
presence
to of inulin.
The invention provides a method for enhancing storage survival of a
microorganism which method comprises freezing the microorganism in the
presence of an
oligo/polysaccharide comprising branched glucose and fructose chains of
heterogeneous
lengths.
15 The invention provides a method for enhancing storage survival of a
microorganism which method comprises $eezing the microorganism in the presence
of
inulin.
The invention provides a method for enhancing storage survival of a
microorganism which method comprises storing the microorganism in the presence
of an
zo oligoJpolysaecharide comprising branched glucose and fructose chains of
heterogeneous
lengths.
The invention provides a method for enhancing storage survival of a
microorganism which method comprises storing the microorganism in the
presexice of
inulin.
25 The present invention provides a culture of a microorganism which has been
prepared using one or more methods of the present invention.
The microorganism is particularly selected from a microorganism used in the
preparation of a food and a probiotic microorganism.
The present invention also provides a food incorporating one or more
3o microorganisms prepared by a method of the inve»tion.
The present invention also provides a food prepared using one or more
microorganisms prepared by a method of the invention.
In one aspect the present invention provides a cold food incorporating one or
more
microorganisms prepared by a method ofthe invention. The cold food is a food
which is
CA 02420095 2003-02-26
maintained at a temperature below room temperature but above freezing.
Typically the
storage temperature is about 4°C.
In another aspect the present invention provides a frozen food incorporating
one or
more microorganisms prepared by a method ofthe invention. The frozen food is a
food
which is maintained at a temperature below its freezing point. Typically the
storage
temperature is about -20°C.
rn another aspect the present invention provides a cold food prepared using
one or
more microorganisms prepared by a method of the invention. The cold food is a
food
which is maintained at a temperature below mom temperature but above freezing.
Typically the storage temperature is about 4°C.
In another aspect the present invention provides a frozen food prepared using
one
or more microorganisms prepared by a method of the invention. The frozen food
is a food
which is maintained at a temperature below its freezing point. Typically the
storage
temperature is about ~20°C.
13 In yet another aspect the present invention provides a vegetarian food
incorporating
one or more zx~icroorganisms prepared by a method of the invention. The
vegetarian food
may be a cold food or a frozen food.
In yet another aspect the present invention provides a vegetarian food
prepared
using one or more microorganisms prepared by a method of the invernion. The
vegetarian
2o food may be a cold food or a frozen food.
The food may be prepared by adding the one or more microorganisms to the
already prepared food.
Alternatively, the one or more microorganisms may be added to the food during
preparation. This may result in growth of the microorganisms) within the food
and
25 resultant effects on the properties of the food. The microorganisms) may
participate in
fermentation of raw materials in the preparation. The microorganisms) may
provide
partial or complete fermentation ofraw materials in the preparation. The
microorganisms
used in this way may only provide fermentative functions or may provide
fermentation and
probiotic functions.
3o The food may be prepared using one or more microorganisms and then have
further
microorganisms added.
The present invention provides a method for extending the shelf life of a food
containing one or more microorganisms which method comprises incorporating an
CA 02420095 2003-02-26
.. g _
oligolpolysaccharide comprising branched glucose and fructose chains of
heterogeneous
lengths in the food.
The present invention provides a method for extending the shelf life of a food
containing one or more microorganisms which method comprises incorporating
inulin in
the food.
The present invention provides a method for increasing the survival of one or
microorisms in a food or health supplement which method comprises
incorporating an
oligo/polysaccharide comprising branched glucose and fructose chains of
heterogeneous
lengths in the food or supplement.
14 The microorganisms) may be prepared in capsular form with the
oligolpolysaccharide comprising branched glucose and fructose chains of
heterogeneous
lengths present in the encapsulated form. The microorganism{s) in this farm
may be used
as a health supplement or may in turn be incorporated into a food.
The microorganisms) may also be prepared in tablet form with the
~5 oligolpolysaccharide comprising branched glucose and fructose chains of
heterogeneous
lengths present in the tablet. The microorganism{s) in this form may be used
as a health
supplement or may in turn be incorporated into a food.
The present invention provides a method for increasing the survival of one or
microorganisms in a food or health supplement which method comprises
incorporating
ZO inulin in the food or supplement.
The microorganisms) may be prepared in capsular form with inulin present in
the
encapsulated form. The microorganisms) in this form may be used as a health
supplement
or may in turn be incorporated into a food. The microorganisms) may be
prepared in tablet
form with inulin present in the tablet. The microorganism{s) in this form
ttaay be used as a
zs health supplement or incorporated lento a food.
The present invention encompasses the use of all fermentative and probiotic
microorganisms acceptable for food production for humans or animals.
Utilisation of commercially available isolates ensures the continuing
availability of
the strain; secondly, the strains are already guaranteed to be safe for
consumption; and for
3o probiotic microorganisms ensures they will survive the passage through the
gastrointestinal
tract, and as long as they are present in su~cient numbers, will confer their
particular
health benefits to the consu~r.
lactic acid bacteria and probiotic cultures for commercial food production,
and
starter cultures for fermentation can be purchased from international
companies such as
CA 02420095 2003-02-26
_g_
Christian Hansen Fty Ltd (Bayswater, Australia) and Gist-Brocades Australia
Pty Ltd
(Mooreban~, Australia). Other research organisations such as the CST)~O
Starter Culture
Collection (Highett, Australia) and the Australian Starter Cuhure Research
Centre
(Vflerribee, Australia), have goad lactic acid bacteria and potential
probiotic collections but
these organisms are only available on a small scale.
Fable 1 Species currently used as probiotics around the world.
Lactobacillus spp. ~~~obact~~m
spp.
L. acidophilus $. biftdum
L.,~ohnsonii .8, lon~m
L. paracasei ssp. Paracasei .~, infantis
L. rhamnosus ,8. brev~
L. plantarum B, adolescentis
L. brevis B. lactic
L. reuteri
L: salivarius
L. fermetum
L. helveticus
L. delbrueckii ssp.
Bulgaricus
Uther Species
Streptococcus salivarius ssp_ Thermophilus
.Laciococeus lactis ssp. Lactic and cremoris
Enterococcus faeciurn
Leuconostoc mesenteroides ssp. l7extranium
Propionibacterium, freudenreichii
Pediococcus acidflactici
Saccharomyces boulardii
Escherichia colt
Bacteroides spp.
Bacillus spp.
Adapted from O'Sullivan et al. (1992), Sanders (1999) and R.olfe (2000)
Selection criteria for prohiatic isolates
There is no consensus on how to define or accredit a microorganism as
probiotic
(Guarner and Schaafsma, 1998). Each strain that is considered for use in human
probiotic
preparations needs to be subjected to strict characterisation and
experimentation to ensure
CA 02420095 2003-02-26
,
the effectiveness and safety of the particular strain. Table 2 lists the
criteria for,assessing
potential probiotic strains.
Characterisation tests for assessing potential probiotic microorganisms for
human
use are designed to select the most appropriate strains and ensure the
efficacy o~the
product. As all probiotic strains do not cover the complete range of health
benefits,
specific targets should be identified when selecting a strain.
Table 2 Requirements for goad clinical studies demonstrating unique probiotic
properties for functional food use.
~ Each specimen and each strain should be documented and tested
independently, on their own merit
~ Extrapolation of data from closely related strains is not acceptable
~ Well-defined probiotic species and strains, with well-described study
preparations
~ Double-blind, placebo-controlled human studies
~ Randomised human studies
~ Results cori~rzr~ed by different independent research groups
~ Publication in respectable international peer-reviewed journals
Taken from Salminen and Saxelin (1996).
Table 3 Desirable criteria for selecting a probiotic Strain for human use.
~ Probiotic strain must be of host origin and properly identi~ ed
~ The strain must be clinically safe far use in foods, with no side effects
~ Exhibit stable characteristics in storage and in foods
~ Be industrially compatible - able to be grown to desired concentration,
suitable taste qualities, survive production
~ Survive on route to the large intestine - acid, bile and lysozyme
resistant
Colonised or adhere to the gastroimestinal tract
Exhibit demonstrable health benefits - improved nutritional value,
prevention of diarrhoea and constipation, pathogen inhibition, immune
system stimulation and modulation, cancer prevention, modulate
metabolic activities
CA 02420095 2003-02-26
Adapted ~rom Klaenhammer and Kullen (I999) and Cribson and Fuller {2000)
Host origin
Frobiotic microorganisms should be of human origin if they are intended for
human
consumption. Not only is this a safety consideration in terms of crass-species
pathogenicity, but also strains that have been repeatedly isolated from humans
are more
likely to adapted to that ecosystem and stand a better chance of survival.
Microorganisms
isolated from other ecological systems may prove to be pathogenic or at least
undesirable
when consumed by humans.
Isolates intended for use in other animal species typically originate from the
species
to in which they are intended to be used .
Safety
Safety in consuming live cultwes is one of the most important factors when
examining potential probiatic microorganisms. Lactobacillus (with the
exception ofL.
rhamnosur), Bifddbaeterium and S baulardif are not considered to pose a risk
to
consumers, however every new strain should be examined for safety aspects.
Donohue
and Salminen (1996) suggested a set of criteria that potential probiotics
should satisfy to
ensure safety when consumed by humans.
Table 4 Suitable models and methods to test the safety of potential probiotic
strains
for human consumption.
1. Detertnir~e the intrinsic properties of the strain - antibiotic resistance,
plasmid transfers etc. ,
2. Assess tlae effects of the metabolic products from the microorganism
3. Assess the acute and subacute toxicity of ingesting large amounts of the
microorganism
4. Estimate in vitro infective properties in cell culture and then in animal
models
~_ Determine the efficacy of ingested probiotic by dose response and
impact on the composition of human intestinal microflora
6. Identify and assess any side effects in human trials
7. Epidemiological surveillance of people consuming the new introduced
probiotic
8. The most rigorous safety testing for genetically modified or animal
derived strains
CA 02420095 2003-02-26
1~ -
Taken from Donohue and Salminen ( I 99~
,Survival in thegastrointestinal tract
'USl'hen consumed, probiotic microorganisms have to survive the passage from
the
mouth, through the stomach, to the intestines to exert any influence on the
host.
Bifidobacteria are reported to be predominantly located in the caecum, whereas
Lactabacillacs species preferably colonise the ileum. For best survival rates,
strains need to
be acid, bile and lysozyme tolerant to provide a competitive advantage in
viva, Bile and
acid resistance in probiotic rniraroorganisms has been trialed in vitro, using
batch and
ZO multiple chemostat techniques, with media containing appropriate levels of
bile or
buffered at low pH to mimic the gastrointestinal system. In vitro experimems
are not ideal,
but do effectively highlight inadequate species and provide a staxti~ng point
for further
experimentation.
Intes~irtal adhesion or colonisation .
1s Ideally, it is desirable that probiotic nnicroorganisms adhere to or
colonise the
intestine. The benefrts of colonisation include displacement or e~cclusion of
undesirable
bacteria or pathogens from adhering to the intestines and prolonged existence
of desirable
bacteria in the intestines. The longer probiatic microorganisms are present in
the
intestines, the greater any possibility of beneficial effect on the host.
2o Although coionisatian does not appear to be permanent, delayed residence in
the
large intestine is apparent ~uvith some strains, After feeding has ceased,
many probiotic
strains can be isolated from faeces for days or weeks afterwards before dying
out. Sanders
(Sanders, 1993) speculates that although probiotics are not permanent
residents, continuous
consumption of these transient probiotic organisms does appear to be a
requirement for
25 prolonged health benefits.
Many pathogens rely on adhesion to the intestinal mucosa as the first stage of
host
infection and probiotic microorganisms that prevent this initial step would be
beneficial.
Bernet et al. (1893) showed that several species of B~do6acterium could
inhibit cell
adhesion and invasion by E. coli and S typhimurium. Other similar in vitro
cell culture
3o experiments have been conducted, all reporting strain specific adhesion,
put of 12
Lactabacidlus strains only 4 strains, L. casei 744, L. acidophilus Lal, l..
rhamnosus r.C-
7~5 and L. rhamnasus CxCx, displayed significantly greater adherence than the
non specific
binding of E. coli.
CA 02420095 2003-02-26
-13-
Industrial exploitation
For industrial food production, a probiotic strain must be technologically
exploitable. rt is important that the bacterium be grown easily and be able to
withstand
food processing. Additional hours incubating a slow growing bacterium adds to
the cost of
production. Probiotic species need to maintain stable characteristics during
production,
short and long term storage and the shelf life of the food product. Rapid
acidification of
fermented foods by lactic acid bacteria is required to inhibit pathogens as
well as impart
organoleptic qualities. Strain stability also includes retaining the traits
associated with
health benefits for the host, such as bile and acid tolerance, and pathogen
inhibition.
to Surwfval in food systems
Probiotic strains are isolated from and selected for their ability to survive
in the
gastrointestinal tract and consequently many species show poor survival in
foods. L.
delbrrreclaii ssp. ~bulgaricus and Streptococcus therrnophilus do not survive
in the
gastrointestinal tract environment, but they do have excellent industrial
properties. Good
15 manufacturing and survival characteristics are not inherent in all lactic
acid bacteria,
indicating that strain selection is az~ irnpQrtant factor for product
manufacture.
The question of the required concentration of viable probiotic organisms is
still
unresolved. Viability is assumed to be related to activity and imparting
health benefits,
although this is not always the case. The consumption of probiotics at a level
of 1 O8 - 109
2o cfu per day is a commonly quoted figure for adequate probiotic consumption,
equating to
1008 of a food product containing 106 - 10' cfu/g.
'>rhe form in which probiotic bacteria are fed, affects the minimum dose for
detection in the faeces. L. rhamnosus CrCr could be detected in host faeces
when fed at a
lower concentration of I0~ cfu in fermented or sweet milk and as
enterocapsules, rather
zs than at 10'°cfu in a freeze dried capsules. "fhe difference in
survival was attributed to the
milk buffering capacity and insoluble enterocapsute capsule coating, providing
protection
during transit through the stomach.
The final product should contain probiotic microorganisms at an adequate
concentration for the entire shelf life of the product. The Australian Food
Standard Code
o (Standard ~8) stipulates that yoghurt must have a pl:I less than 4.5 and be
prepared with S
thefmophilus and 1,. delbrueckii ssp. bulgaricus or other suitable cultures,
but does not
stipulate required levels of probiotic bacteria. Some countries have imposed
loose
standards o~ minimum allowable levels of probiotics or lactic acid bacteria in
yoghurts.
CA 02420095 2003-02-26
-. -14-
There appear to be no specifications with regard to prabiotic products that
are not of dairy
origin.
Probiotic survival in products is affected by a range of factors including pH,
post
acidif ration, hydrogen peroxide production, storage temperature, the mixture
of starter
cultures, packaging and food ingredients. Bii~xdobacteria and L. acidophilus
show better
survival when supplied with complex carbohydrates or oligosaccharides_
Probiotic survival
is generally better in mild acidic conditions, when the pH is above 4.
Tablets and cap~ule~
1o Tablets and capsules are prepared in accordance with standard technidues
used in
the health industry for their preparation.
The inulin, used by the present inventors was a high purity inulin gel which
was
sterilised with treat prior to use. The present invention relates to use of
inulin in this form
15 but also relates to use of other forms of inulin whether heat sterilised or
not. Similarly, the
present invernion relates to use of an oligolpolysaccharide comprising
branched glucose
and fructose chains oFheterogeneous lengths as a high purity gel which is
sterilised with
heat prior to use. The present invention also relates to use of other forms
ofthe
oligoJpolysaccharide whether heat sterilised or not, The desirability of
sterilising materials
20 for use in growing or storing microorganisms will be sel:Fevident to the
skilled addressee,
as will be the fact that sterilisation can be carried out in other ways.
D~nitions
Oligosaccharide: a glycoside containing between three and ten sugar moieties
Polysaccharide: a glycoside containing between three and eight' sugar
rrtoieties
Comprising: where the terms "comprise", "comprises", "comprised" or
"comprising'' are
used in this specification, they are to be interpreted as specifying the
presence of the stated
features, integers, steps or components referred to, but not to preclude the
presence or
addition of one or mare other features, integers, steps, components or groups
thereof.
Cryoprotectant: a cryoprotectant is any compound that protects biological
material or cells
from the detrimental e~'ects of cold temperatures in preparation or storage.
CA 02420095 2003-02-26
. r _ 1fJ _
Brief Description ofthe Drawings
Figure 1 shows the expected and observed cell concentrations after freeze
drying in various
cryoprotectants, for probiotic bacteria grown in SPY 2 anr! SPX S.L.
acidophilus
MrLAl grown in a) SPY 2, b) SPX 6; .1.. rhamnosus LCSH1 grown in c) SPY 2, d)
SPY 6; and B. lactic BDBB2 grown in e) SPY 2, f) SPY 6.
Figure 2 shows the angle of decline of viability and bile tolerance during
storage of various
probiotic organisms freeze dried in different cryopratectants: a) L.
acidophilus
MJLA1, b), L. rhamnorus LCSH1 and c) B. lactic BDBB2 grown in SPY 2 (solid
circles) and SPY 6 (solid triaxlgies) when recovered on agar (open symbols)
and
agar + 0.3% bile (closed syrnbols)_
Figure 3 shows the inhibition by L. acidophilus Ml"LA~ of E, coli after growth
in a) SPY 2,
or b) SPY 6, and L. monocyto~enes after growth in c) SPY 2, d) SPY 6 and
freeze
drying in various cryoprotectants.
is Figure 4 shows the inhibition by ~,, rhamnosus LCSI~1 of E. coli offer
growth in a) SPY 2,
or b) SPY 6, and ,~. monocytogenes aRer growth in c) SPY 2, d) SPY 6 and
freeze
drying in various cryoprotectants.
Figure 5 shows the inhibition by $. lactis BDBB2 of E. coli after growth in a)
SPY 2, or b)
SPY 6, and L. monocytogenes after growth in c) SPX 2, d) SPY 6 and freeze
drying
2o in various cryoprotectants.
Figure 6 shows the acidification activity of probiotic organisms after freeze
drying in
various cryoprotectants: L. acidophidus MJLAI grown in a) SPY 2, b) SPY 6; L.
rhamnasus LCSH1 grown in c) SPY 2, d) SPY' 6; and B. lactic BDBB2 grown in e)
SPY 2, f) SPY 6 and freeze dried in cryoprotectants.
Best Method of Carrying Oat the invention
The present invention provides a cryoprotectant suitable for use in freeze
drying
microorganisms and in cold or frozen foods containing probiotic
microorganisms. The
cryoprotectant does not contain any animal-derived ingredients, and produces
e~tcellertt
3o cell viability and retention of probiotic characteristics. The
cryoprotectant acts as a
CA 02420095 2003-02-26
- 16-
replacement for non-fat skim milk (NFSM), the most commonly used
cryoprotectant. The
use ofNFSM as a cryoprotectant makes subsequent use ofthe microorganisms
unsuitable
for consumption by vegetarians ox people with milk allergy ar
hypersensitivity.
According to the literature, the ideal cryoproteeta.nt needs to bind water,
prevent ice
crystal formation, protect cell membranes, enhance cell shielding, prevent
oxidation and
eradicate free radicals. No realistic replacement far skim milk as a
cryaprotectant has been
identified prior tv the present invention.
Exogenous conditions that affect suz-vival of freeze drying include method and
time
of cell harvest and correct storage of freeze dried powders. Cells should be
harvested at
1o early stationary phase. Viability declined sooner in cells harvested during
late log phase
during extended storage. Better recovery is achieved when cells had been
harvested by
frltrataon or ultrafiltratian rather than centrifugation, however, better
survival occurred in
cells obtained by centrifugation or ultrafiltration. Turing centrifuging, a
higher
temperature aids cell separation, but temperatures around 5°C is less
detrimental to cell
15 viability.
Bozoglu et al. (19$7) modelled the survival l~inetics of lactic acid bacteria
aztd
concluded that cell death is related to the area exposed to tha external
conditions. The
shielding effect can be optimised by reducing cell surface area exposed the
external
conditions by using 'small' cell variants and harvesting cells to
concentration dense
20 enough to be beneficial without causing osmotic problems, approximately 1
Or°cfu/rnL.
Once freeze dried, cells must be stored under the right conditions to maintain
maximum viability. Water activity between 0.1- 0.2 has been shown to be best
rwith over
drying and under drying both detrimental to cell viability. ')"he dried
cultures should be
kept under vacuum or nitrogen gas, but not air or oxygen gas, to prevent
oxidation. The
2s cells retain greater viability when stored at refrigerated temperatures of
5°C and below-
The packaging should be moisture proof, oxygen proof and opaque.
ll~aterials and R~ethods
Microorganisms
30 Probiotic cultures Lactobacillus aciclo,~lailus MJLAI, Bi~dobacterium
lacdis
B)(7BB2, and Lactobacillus rhamnosus LCSHI, were supplied by Christian Hansen
CA 02420095 2003-02-26
-17-
(Bayswater, Victoria Australia) and used in freeze drying cryoprotectant
experiments. L.
acidophilus MJLAl was used in the cryoprotectant concentration and antioxidant
trials.
NX'icl'obiological growl)t media
SPY 2 medium was prepared by dissolving 2.5% soy peptone, 2..5% yeast extract,
and 2.5% glucose monohydrate in distilled water. The pH was adjusted to 7,4
using HCl
or NaOT~, the medium dispensed into bottles and autoclaved at 121 °C
for 15 rains. SPY 6
was prepared by fortifying SPY 2 medium with 0.1% Tween 80 prior to
autoclaving. The
medium was dispensed and autoclaved at 121°C for 15 rains and then
further
1o supplemented ~avith sterile CaC12.2HZO and MnC12.4H~0, to a. final
concentration of 6.0
mM each,
MRS medium: de Man, Rogosa and Sharpe medium (de Man et al., 1960)
RCM medium: Reinforced Clostridial medium (Hirsch and Grinsted, 1954)
is TSA medium: Tryptone Soya Agar (Oxoid Australia)
NB medium: Nutrient Broth (Oxoid Australia)
PBS: Phosphate buffered saline, (lMaC18.OOg, KCl 0.208, Na2HPO4 1.44g,
KlrI2P0° 0.248,
distilled water 1 litre, pT~ 2.0)
zo Example 1
Freeze Drying Preservation of Cultures
Probiotic cell production and han~est
Bacteria were grown in of SPY 2 and SPY b (I Litre) for 24 hours at
37°C.
Lactobacilli were incubated in 8% COz and bifidobacteria incubated in aerobic
2s atmospheres. Cells were harvested by centrifugation (7000 x g at
4°C, 6min) and
resuspended in each cryoprotectant (SOmL) (Table 5), to give an approximate
concentration of 10'° cfulmL. Cell suspensions were then frozen in a
thin film, coating the
inside of sterile conical flasks by rotating the flasks in dry ice. The frozen
cell suspensions
were then hardened at ~80°C for 1 hour and then freeze dried ( -1.8
mbar, -40°C). Freeze
30 dried powders were equilibrated to a water activity of 0.1 by exposure to a
saturated
solution of lithium chloride in a sealed chamber for 48 hours. Dried cell
suspensions were
then gently aseptically ground, placed in pre-weighed specimen containers,
reweighed, and
CA 02420095 2003-02-26
-
stored in the dark, under vacuum (-0.6 mbar) at S°C. F,ach experiment
was conducted in
triplicate.
Table 5 Compounds used as cryoproteetants for preserving various probiotic
organisms daring freeze-drying.
Cryoprotectant Sterilisation treatment
Noz~-fat skim milk (Diploma) 10% pH 6.6, inspissated 100°C, l5mins
Inulin HP-Gel ((~rafti) 10% pH 6.3, inspissated 100°C, l5mins
Yeast biomass (Sigma) 10% pH 7.0, inspissated 100°C, l5mins
?rehalose (Sigrma) 10% pH 7.0, autoclaved 121°C, l5mins
Soy milk pH 7.0 (+/- 0.2), ZJH~T
(Sanitarium Health Food Co, Australia) commercial package
Soy protein isolate 5% pH 7.0, micro~uidixed at 7500 PSI,
(IPT 545, International Protein Technologies) inspissated 100°C, l5mins
All cryoprotectant solutions were suspended in distilled water and sterilised
as
stated above_ All cryoprotectants were used for microorganisms grown in $PY2,
and skim
milk, trehalose, inulin and yeast biomass were used for microorganisms grown
in SPY 6.
x0 Cell enumeration and bile sensitivity
Cell viability and bile tolerance of the freeze dried cultures were assessed
immediately prior to storage and at regular intervals over 6 months. Freeze
dried powder
(Ioomg, weighed accurately) was rehydrated with 0.1% peptone (2mL). The
rehydrated
cell slurry was serially diluted and plated on to MRS and MRS * 0.3% for
lactobacilli or
!5 RCA agar and ItCA + 0.3% bile (Oxoid) far bifidobacteria. Plates were
incubated for 48 h
at 37°C, lactobacilli at $% CO2 and bifidobacteria incubated
anaerobicaily, after which the
resulting colonies were counted. Cell concentrations were calculated as cfulg
powder.
Survival o~ freeze dtyi~ag
24 The theoretical maximum post freeze dried cell populations, the population
in the
freeze dried powder if no cells were deactivated, was calculated and compared
to the
actual viable counts in the freeze dried powder. Assuming that no losses were
incurred
during harvest or freeze drying, the theoretical maximum cell concentration
per gram of
freeze dried powder, is the number of organisms in the total volume ofgrowth
medium
2s (cfu), divided by the final weight of the freeze dried powder (g). the
total colony forming
CA 02420095 2003-02-26
- 19-
units in 1 titre of SPY 2 or SPY b medium was calculated using maximum
population data
for each organism from previous growth medium trials,
Pathogen inhibition
Bacteria were assessed far ability to inhibit .E'. coli (NCTC 11560) and
Listeria
monocytogerter (ATCC 7644) in vitro based on the methods of Chateau et al.
(1993). Two
aliquots {10 p.I,) of rehydrated cell suspension was spotted onto two MRS or
RCA agar
plates and incubated for 24h anaerobically to prevent H20z accumulation,
Plates were then
overlayed with Tryptone Soya Agar (TSA; Oxoid) containing 0.1 mL of an
overnight
1o Nutrient Broth (Oxoid) culture of either E. coli or L. rrrortocytogenes.
Plates were incubated
for 24 hours and the resulting zones of inhibition measured. The zone of
inhibition was
considered as the clear area between the edge of probiotic culture to edge of
pathogen
growth.
Acid~cation activity
Each rehydrated cell slurry (0.2 rnl,) was inoculated into soy milk (2mL) (So
Good,
Sanitarium Health food Co, Australia) that had been ternpexed to 37°C.
The pH of the
uninoculated and inoculated soy milk (pH;~;~;,, ) was measured, The inoculated
soy milk
samples were then incubated at 37°C for a hours, the lactobacilli in
8°!o COz and
bifidobacteria anaerobically, after which the pH (pH~;,r"~) was measured. CeII
activity vcras
calculated as change in pH per hour per log cfulmL, using the following
equation:
Cell Activity = dpH (pH;~;~ - pH r",u)
time {hours) x (Log,QCfu/mL)
Cryoprotectanr concentration and antioxidants
L. acidaphilus strain MTLA1 was inoculated into SPY 6 medium (IOmL) and
incubated far 20 hours at 37°C in 8% COa,. Cells were then harvested by
centrifuging
(5000 x g, 5 mitt), the supernatant discarded and the pellet resuspended in
each
3o cryoprotectant (I.OmL}. Cryoproteetants used in this experiment are listed
in An aliquot
(O.8mL} of the resuspended cell concentrate was dispensed into an eppendorf
tube, frozen
to -80°C and then freeze dried overnight (-40°C, -1.8 mbar), The
freeze dried samples
were allowed to equilibrate to a water activity of 0.1 by exposure to a
saturated lithium
CA 02420095 2003-02-26
~Za-
chloride solution for 24 hours.
Immediately after water activity
equilibration, each tube
was rehydrated to initial volume in viability and activity
for immediate use tests. Each
cryopmtectant was trialed in Table
6.
Table 6 Cryoprotectant suspension
solutions for freeze~drying L.
acidophilus
strain 11~JLA1.
Cryoprotectant Treatment
Control distilled Hz0 pH 7.0, autoclaved i21C,
l5mins
! Trehalose~(S%).._______________.__._................_.__.___~H 7.0,
autoclaved 121C,
l5mins
Trehalose (10%)
Trehalose (15%)
,..... ..___.__..__.__ -
.
_
.....
.
.
__ ~H 6.3, inspissated 100C,
_________.__ l5mins
_
.
.
Inulin~(5%) _
Inulin (10%) ,
Inulin ( I S %)
.
.
. .pH 6.7, inspissated
.... 100C, l5mins
..Trehalose (2.5%) +~inulin (2.5%)
y___________..
Trehalose (5.0%) + inulin {5.t~%)
Trehalose (7.5f ) + inulin (7.5%)
Trehalose (15%) + tacopheral~(Sigma)~(IOULIL)_pH 7.0, trehalose
solution'___________
_-_
Trehalose (15/a) + tocopheral (100u~)autoclaved 121C, XSz~nins.
Sterile
~'rehalose (15%) + ascorbic acid antioxidant added when
(Sigma) (4mgJL) cool.
Trehalose (15%) + ascorbic acid
(40mglL,)
_ pH 6.3, inulin solution
Inulin (1 S%) + tocopheral {10pL%L)inspissated
~ ~ ~
InuIin {15%) + tocopheral (100uLIL)100C, l5mins_ Sterile
antioxidant
Inulin (15%) ~- ascorbic acid (4mgli,)added when cool.
Inulin (15%) + ascorbic acid (40mg~i.)
CeII viability and bile tolerance.
Prior to freeze drying and again immediately after rehydration, the viable
cell
populations and bile tolerant populations were enumerated by plate count using
MRS and
IvIR,S + 0.3°!o bile agar.
Acid tolerance
Acid tolerance of each cell concentrate was assessed prior to freeze drying
and
immediately after rehydration. An aliquot of cell concentrate {O.1mL) was
mixed with
phosphate buffered saline (1.0 mL, PBS, pH 2.0). The cells were held for 3
hours at 37°C
and then enumerated using MRS. The results were calculated as the number of
acid
tolerant bacteria present before and after freeze drying in the original cell
concentrate.
CA 02420095 2003-02-26
-21 -
RESULTS
Probiotic survival during freeze drying
The effect of freeze drying on cell viability varied depending on the
cryoprotectant.
Figure '1 illustrates the actual viable and bile tolerant cell populations
compared to
the calculated theoretical maximum population after freeze drying.
When probiotic organisms were grown in SPY 2 medium, the least deactivation of
cells occurred during freeze drying with trehalose and inulin as
cryoprotectants (p~0.05).
Soy milk and skim milk were the next best cryoprotectants, followed by yeast
biomass.
The highest initial deactivation occurred with soy protein as the
cryoprotectant.
The degree of sub-lethal injury varied slightly between cryoprotectants,
trehalose,
yeast biomass, inulin and soy protein producing the least sub-lethal injury
(p~0.05).
When the three strains. of probiotic bacteria were grown in SPY 6, inulin gave
the
best protection to cells during freeze drying (p<0.05). Trehalose was the next
best,
15. followed by skim milk and then yeast biomass. There were no significant
di:El:'erences
betvuee~ cryoprotectants with respect to sub-lethal injury of cells.
Survival of freeze dried probiotics during storage
Freeze drying survival data were analysed by transforming the gradient of the
'line
of best fit' to angle (degrees) from the x-axis. The greater the negative
angle, the greater
2o the decline in population. The angles were compared using AN4VA. The rate
of cell
deactivation during storage depended ari the growth medium, eryoprotectant and
the
bacterium (p~0.05)
When probiotic cells were cultured izt S>fY 2 (Figure 2), the cryoprotectants
that
provided the best storage protection were trehalose and soy protein. Cell
viability in these
25 cryoprotectants and yeast biomass was better than when in skim milk.
Trehalose and soy
protein also provided the best stability of the bile tolerant population
during storage, with
the other cryoprotectants proving to be equally as good as each other.
When SPY 6 was used as a growth medium, of the four cryoprotectants used,
trehalose, inulin, and skim milk were equally good in preserving cell
viability and bile
3o tolerance. Yeast biomass was not as e~'ective in protecting cells (p~0.05).
CA 02420095 2003-02-26
. . -22-
Probiotic Xnhibition of Pathogens
The assessment of the ability of probiotic organisms to inhibit pathogens was
conducted throughout the storage trial. The degree of inhibition of the
pathogens was
related to the cryoprotectant, the growth medium and the species of probiotic.
The ability
ofprobiotic organisms to inhibit pathogens did not obviously decline during
the trial, but
some probiotic species did display an erratic tendency on a weekly basis.
The cryoprotectants that gave the greatest inhibition were inulin and
trehalose
{p<0.05). z. acidophilus MJLAI {figure 3) and ~. rhamnosus LCSI31 (Figure 4)
gave
greater inhibition when they were grown in SPY 6 prior to freeze drying.
Inhibition by B.
to lactrs BDBB2 (Figure 5) was not affected by growth medium.
fl cidiftcation activity
The acidification activity per cell of freeze dried probiatic organisms during
storage
is presented in 'Figure 6.
When probiotic organisms were gror~m in SPY 2, using multi-factor ANOVA for
15 data analysis, trehalose and inuiin maintained the best acidification
activity during storage
(p<0.05). Skim milk and soy milk were then next best, followed by soy protein,
then yeast
bioxnass. Probiotic orgaztisms cultured in SPY 6 prior to freeze drying,
retained the highest
acidification activity in trehalose and skim milk (p<0.05). Cells stored using
yeast biomass
as the cryoprotectant, had the biggest decrease in acidification ability
during storage.
20 Overall, the decline in acidification activity o~probiotic organisms grown
in SPY Z
was lower than that of the organisms grown in SPY 6 (p<0.05). I~owever, there
was na
significant difference between cryoprotectants, using the results from both
growth media.
The variations between bacteria and between media were too great.
G~ryoprotectar~t concentration
zs The e~'ect of cryoprotectant concentration and the presence of antioxidants
an cell
viability, bile tolerance and acid tolerance during freeze drying were
assessed. There were
no significant differences in cell viability or bile tolerance due to
cryoprotectant prior to
freeae drying (Table 7). There were differences in acid tolerance prior to
freeze drying,
depending on the cryoprotectant (p<0.05). The acid tolerance treatment did
decrease cell
3U viability prior to and after freeze drying (p<0.01) (Table 8).
CA 02420095 2003-02-26
- 23 -
Freeze drying caused a significant decrease in cell viability, bite tolerance
and acid
tolerance (p<0.05). Water, the control, produced the lowest cell viability,
bile and acid
tolerance after freeze drying (p<0.05)_
Inulin {15%), trehalose (IS%), trehalose (7.5%)linulin (7.5%) and trehalose +
ascorbic acid (4mg), provided the best protection to cell viability, bile and
acid tolerance
during freeze drying (p~0.05)_
Ascorbic acid and tocopheral did not aid cell survival above that of I5%
inulin and
15% trehalose. The higher levels of antioxidants were generally detrimental to
cell
viability.
IO
Table 7 Cell viability and bile tolerance of l,. acidophilus MJLAI after
freeze drying
in various concentrations of cryoprotectants and antioxidants.
Cell viability Bile tolerance
after after
freeze freeze
drying drying
Ctyoprotectant (log cfulml,) (log cfulmL)
mean s.d. mean s.d.
Water b.BI ~ 0.1 5.79 ' t 0.25
I
Ttehalose (5%) 8.03' ~ 0.057.61br ~ 0.05
Trehalose (10%) 8,20"'6' t 0.058.07'6 ~ 0.31
Treha3ose (15%) 8.30' ~ 0.048.03"~d t 0.10
Inulun (5%) 8.12 ~ 0.157.67r~' ~ 0.19
Inulin (10%) 8.18'6' ~ 0.067.876~'F t 0.08
Inulin (15%) 8.29' t 0.098.138 t 0.04
Trehalose (2.5%) + inulin 7.89' ~ 0.067.57h t 0.06
(2.5%)
Trehalose (5.0%) + inula~a 8.044 t 0.027.81'rB t O.OI
(5.0%)
Trehalose {7.5%) + inuliuu 8.25'x' t 0.088.04" ~ 0.
(7.5%) I 1
Trehalose ( I S %) + tocopheral8.21 b' ~ 0.027.90' ~ 0.
( 10~1.,I~.) 03
Trehalose (15%) + tocophera!8.16' t 0.057.83d'f t 0.1
(IOOULIr,) I
Trehalose (I5%) + ascorbic 8.24'6 f 0.057.93"t'cae~ 0.05
acid (4mgIL)
Trehalose (15%) + ascorbic $,16~ t 0.037.87~f ~ 0_07
acid (40mgIL)
Inulin (15%) + tocopheral 8.10 ~ 0.037.$4d'f t 0_
(10~,LIL) 10
hnulin (15%) + tocopheraf 8.166' ~ 0.047.90a'd' f 0.02
(100pL/L)
Inulin (15%) + ascorbio 8.20" f 0.127.95'''' t 0.
acid (4mglL) I 1
InuIin (15%) + ascorbic 8. I4~ t 0.027.85'r t 0.07
acid (40mgJL)
s.d. = standard deVildti0ri (ri=3),'' °' e'° significantly
different survive! compared to other
Cl~'O~rOteC~ntS
IS
CA 02420095 2003-02-26
_ . -24-
Combinations of trehalose and inulin were only successful at the highest
concentration that was trialed. Trehalose (5%)linulin {5%) and trehalose
(2.5%)rnuliti
{a.5%) were not effective at maintaining cell viability, bile tolerance, or
acid tolerance at
reasonable levels.
')fable $ Acid tolerant cell populations before and after freeze drying in
various
concentrations of cryoprotectants and antioxidants.
Acid tolerance Acid tolerance
after
before freeze
freeze drying
drying
Ctyoprotectant (log cfuJmL) (log cfuJmL)
mean s.d. Mean s.d
Water 8.81' ~ 0.056.278 ~ 0.08
Trehalose (5%) 8.71 t 0.047.87 ~ 0.20
Trehalose (l0%) 8 68' f 0.078.09 f 0.12
Trehalose (15%) 8.73'x' t 0.028_30 ~ 0.08
Inulin (5%) 8.76e'a' f 0.038.07 ~ 0.17
Inulin (I0%) ~ 8.79~ ~ 0.128.09 t 0.11
Inulin (15%) 8.80a~ ~ 0,058.22ab t 0.05
Trehalose (2.5%) + inulin 8.80as' t 0.067.72r ~ 0.12
(2.5%)
Trehalose (5.0%} + inulin 8.80"~ ~ 0.017.96 ~ 0,02
(5.0%)
Trehalose (7.5%) + inulin 8.$58b ~ 0.058.16~b' t 0,05
(7.5%)
?rehalose (15%) + wcophetal8:74'~e t 0.018.04 t 0.05
(iO~i,Ji,)
Trehalose (15%) +tacopheral8.79a~ ~ 0.028.04 ~ 0:03
(100uLIL)
Trehalose (15%) + ascorbic 8.85' f 0.036.14'~b ~ 0,09
acid (4mgIL)
Trehalose (15%} + ascorbic 8.'788 t 0.038.03' ~ 0.08
acid (~OmgIL)
Inulin (15%) -r tocopheral 8:67' t 0.078.04" f 0.02
(lOpLIL)
Inulin (15%) + tocopheral 8.75'd' ~ A.Ob8.03"'' t 0.03
(100~LJL)
lnulin (15%) + ascorbic 8.78 ~ 0:078.12 t 0.15
acid (4mgl~,)
Inu(in (15%) + ascorbic $.73'd' ~ 0.078.04" ~ 0.02
acid (40mg1)_.)
s.d. ~= standard deviation (n=3), '' °' "' significantly differenk
survival compared to other
cryoprotectants
.Discussion and Conclusions
Culturing cells in SPY 2 and SPY 6 influenced the survival of organisms during
freeze drying and subsequent storage, with SPY 6 producing the better results.
Greater
survival during freeze drying a~zd a smaller population of sub-lethally
injured cells was
achieved by growing cells in SPY 6. Cells grown in SPY 6 also survived better
during
storage of the freeze dried cultures and retained a higher population of bile
tolerant. cells.
~5 Adding Tween 80 and calcium to growth medium bas been observed to improve
suz'vival,
CA 02420095 2003-02-26
-25-
without preventing sub-lethal injury. frobiotic bacteria cultured in SPY ~ did
retain a
higher degree of acidification ability during storage.
Freeze drying cryoproteetnnts
As skim milk is the most commonly used eryopratectant for freeze drying
bacteria,
it was used as a reference cryoprotectant, to which other cryoprotectants were
compared in
this experiment. The compounds trialed as eryoprotectants were trehalose, soy
milk,
inulin, yeast biomass and soy protein. Trehalose has been reported as a
successful
cryoprotectant in the past (Leslie, et al., 1995). Inulin and soy protein bind
water very well
and thus may retain a higher water content for the same water activity in a
freeze dried
to state and reduce water stress. Soy milk is a dairy mimetic containing
protein, fat, calcium,
and other nutrients. Deactivated yeast biomass may provide additional physical
cell
shielding to the probiotic organism.
Both trehalose and inulin proved to be better than skim milk as
cryoprotectants.
Initit~l survival o, f~'reeze drying
Deactivation of microorganisms can occur during centrifuging, lyophilisation
and
in storage. Centrifugation had a negligible effect on cell viability of.~.
rharnnosus LCSHl
and B. lactic BDBB2, as cells showed no difference in calculated to observed
concentration after freeze drying, where any cell deactivation through
centrifugation would
have been apparent Figure 1 d) and f~. p'rom this experiment, it is impossible
to determine
if losses observed in L. acidophilus MLAJ1 were due to centrifugation or
freeze drying or
a combination of both_
The best initial survival during freeze drying was afforded by anulxn az~d
trehalose,
regardless of whether cells were cultured in SPY 2 or SPY 6. Both
cryoprotectants
produced the largest population ofviable and bile tolerant cells.
Acidtficatian activity
Acidification activity was not significantly affected by cryaprotectant,
although
cells freeze dried in trehalose, inulin and skim milk maintained the highest
acidi~catiort
activity per cell.
B. lactis BDBB2 had the greatest acidification activity per cell. It was noted
that
acidification activity per cell increased during the storage for B. lactic
BDBB2 when
cultured in SPY 2 (Figure 6). This observation remains unexplained, as cells
would not
CA 02420095 2003-02-26
.. -26-
have the opportunity to repair cell damage during freeze dried storage and
thus increase
activity. One possible mechanism is an increase in cell permeability during
storage
allowing a greater flux of acidic products into the surrounding medium. If
present, the
change in permeability did not affect cell viability.
Iflrobiotic inhibition of pathogens
The pathogen inhibition trials area only indicative of potential probiotic
action, as
the microflora in the gastrointestinal tract are subject to a range of
different exogenous
factors from the host, resident and transient bacteria. The agar plates were
incubated
anaerobically, as conditions in the gut would be anaerobic, reducing the
el~eets of -
1o hydrogen peroxide accumulation (Tagg, et al., 1976). The plate technique
employed in
this research was not buffered (although buffering would occur in the
gastrointestinal
tract), as preliminary research determined that the strains ofB. lactis did
not grow {data nat
shown} on the buffered media described by Chateau et al. (1993}.
~. coli and ~.. monocytogenes rxrere used for the pathogen inhibition trials.
E. coli is
naturally present in the gastrointestinal tract and is commonly used as an
organism for
inhibition trials.. Listeria monocytogenes, a Gram positive bacillus, is a
cold-tolerant,
foodborne pathogen that can cause illness by attacking and multiplying within
the gut
epithelium- This organism could potentially be inhibited in vivo by a suitable
probiotic.
:~isteria monacytogenes, has been examined for inhibition by probiotic
organisms, along
2o with the observation from this work that Lisreria monocytogenes was more
sensitive than
E coli, to the effects of the probiotic organisms,
Probiotic organisms freeze dried in inulin and trehalose produced the greatest
inhibition of E. cola and L. manoeytogenes.
B. lactis HDBB2 had highest acidification ability per cell and also very high
cell
concentrations, thus a greater ability to produce acid, but produced the
smallest pathogen
inhibition out of the organisms tested. Lactobacillus inhibition of pathogens
was tested
using growth on MRS, whereas B. lactis BABB2 was tested on RC.A., containing
only a
quarter o~the amount of glucose of MRS. L. acidophilus M,1L,A.1 and L.
rhamnosus
i..CST~l are able to ferment trehalose, whereas B, lactis BDB$2 can not, so
the trehalose
3o cryoproteetant can serve as an extra carbohydrate source for those strains-
None ofthe
strains are able to utilise inuliri as a carbohydrate source, despite inulin
being described as
a 'bifidogenic' substance (Roberfroid, 1993), Alternatively, pathogen
inhibition may not
be related to acid production.
30uD'llD2,uc122l5spea426
CA 02420095 2003-02-26
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This method of testing probiotic ability to inhibit pathogens has its own
limitations.
The test is sensitive to many exogenous features such as batch of growth
medium,
incubation temperature and time. Furthermore; there are no defined
specifications for
inhibition, such as would be used with antibiotic assays, preventing the
definitive
classification of probiotic organisms as 'inhibitory' or 'non-inhibitory'
towards pathogens.
The relevance of a probiotic organism inhibiting ,t~'. colt by either l Omm or
14 mm is
questionable-
Optimisation of cryoprotectant
There were differences in the ability of each cryoprotectant to prevent cell
1o deactivation and freeze injury duriztg freeze drying compared to ongoing
survival during
prolonged storage. Soy protein, while not protective during freeze drying, was
one of the
most protective during storage, with cell numbers remaining fairly constant.
The biggest .
changes in cell viability occurred during freeze drying, rather than storage,
indicating the
emphasis required on adequately preserving cells during the initial fxeeze
drying stage.
is Trehalose and inulin were selected for optimisation of initial cell
survival, as these
cryoprotectants produced the best storage results. The initial deactivation.of
cell freeze
dried in soy protein was So low it could not be considered for optimisation-
L. acidophilus
MJL.A,>l was selected as the test organism, as it suffered the biggest
decrease in cell
viability during freeze drying, compared to the other organisms, thus being
the most
2o sensitive to freeze drying.
Optimising the action of cryoprotectants by varying concentration and
including
antioxidants in the freeze drying medium, identified that the best cell
viability and least
sublethal injury was achieved with inulin (15%), trehalose (15%), inulin
(7.5%)/trehalose
(7.5%) or trehalose (15%)/ascorbic acid {4 mglL). Ascorbic acid has been shown
to
25 improve survival o~L. acidophilus but not bifidobacteria, in yoghurt
produced with mixed
cultures. Better survival of freeze drying was achieved using these
cryoprotectant
solutions, than any of the combinations used in tlae storage experiment.
The use of either trehalose or-inulin as cryoprotectant for probiotic
organisms has
proven to be better than the reference cryoprotectam skim milk (10°/a),
at preventing initial
3o deactivation and sub lethal injury, and nnaintaining cell viability during
storage. Increasing
the cryoprotectant concentration to 15% may further improve these results, by
increasing
the survival of the freeze drying process.
CA 02420095 2003-02-26
' _ .
Trehalose (a-D-glucopyranosyl-D-glucopyranose) has been reported to be an
effective cryoprotectant for yeasts and bacteria. Trehalose litre other
disaccharides, is
thought to be an effrective cryoprotectant due to its ability to form a glass
and stabilize -
phospholipids bilayers in the cell.
Inulin has not been previously reported for use as a cryoprotectant. Trehalose
is
expensive and not a commonly used food ingredient. Inulin is less expensive
and this
coupled with other desirable qualities, such as being soluble fibre, fat
replacer and
'bifidogenic' factor (Orafti Aandorenstraat l, 3300 Tienen, Belgium), make
inulin an
excellent replacement far skim milk.
to Conclusions
Using the fortified growth medium SPY 6, and inulin or trehalose as
cryoprotectants, it is possible to produce freeze dried probiotic cultures,
that retain good
cell viability and probiotic features both after freeze drying and subsequent
storage.
Example 2
A frozen soy dessert can be prepared using freeze dried nnieroorga,nisms as
set
forth in Example 1.
1~'roaen soy desserd
All product ingredients (soy beverage, sugar, oil, stabiliser and salt) are
combined
and heated to 50°C for 10 min,, then aged at 4°C, overnight.
Freeze dried probiotic strains
prepared as in Example 1 with Inulin as a cryoprotectant, are individually
added (2%
inoculum) to the soy dessert base and evenly dispersed by mixing. The product
is then .
churned and frozen (Breville Il Gelataio 1 b00). The frozen soy dessert can
then be packed
z5 into containers, sealed and hardened to -20°C: The samples axe to be
stored at -20°C.
The product pH is 7.0 +I- 0.2.
The cryoprotection ability of inulin in the product can also be increased, by
addition of extra inulir~ to the product base;
Example 3
A yoghurt can be prepared by using starter culture inoculum freeze dried as in
Example 1
for the initial fermentation of base ingredients, followed by the optional
addition of
probiotic cultures freeze dried in inulin.
CA 02420095 2003-02-26
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Yoghurt
The base ingredients of milk and mills powder (S%) are combined and heated to
85°C for
30min and tempered to 43°C. The base is then inoculated with starter
cultures,
Streptococcus thermophilus and Lactobacillus bulgaricus freeze dried in inulin
as in
Example 1, at a level of 106 cfulmL. The yoghurt base is then incubated at
43°C until a ply
of4.5 has been achieved. Freeze dried probiotic microorganisms, such as L,
acidophilus
M1LA1 freeze dried in inulin, are added to the mix at a concentration of 106
cfulrnL and
evenly dispersed. The base is then dispensed into plastic tubs, sealed and
stored at 4°C.
1~ Bxtxa inulin (up to 2%) can be included in the yoghurt base to aid
cryoprotection of
probiodc microorganisms during storage at.low temperatures.
Industrial Applicability
The present invention has application in the food industry, with respect to
the
preparation of fermented and probiotic Foods and health supplements.
CA 02420095 2003-02-26
-30-
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