Note: Descriptions are shown in the official language in which they were submitted.
~W095/08275 2 1 72244 PCT~P9~/02891
SHELF STABLE PRODUCT
Technical Field of the Invention
The invention relates to stabilisation of products which
are vulnerable to spoilage or poisoning by microbial
spores, especially ambient stable and extended chill shelf
life products, in particular food products, and a process
for the preparation thereof.
Background to the Invention
A problem with edible products is that often they are
susceptible to spore outgrowth on storage and hence need to
undergo a preservation system to extend their shelf life.
Typical preservation systems are;
For a-mbient stable products
(i)Neutral pH plus sterilisation. Sterilisation typically
involves a m;nimllm heat treatment equivalent to 121C for 3
minutes (Fo = 3);
(ii)Low equilibrium pH (pH c 4.6) plus pasteurisation in
order to prolong shelf-life and inactivate vegetative
pathogens.
Both products produced using methods (i) and (ii) may have
a high water activity (Aw o~ above 0.93); or
(iii) Low water activity (Aw) products (Aw c 0.93) produced
by introducing a relatively high level of salt and/or sugar
into the product followed by pasteurisation, in order to
prolong shelf-life and inactivate vegetative pathogens.
F 7196 (C)
/ ~ 21 ~2244
For exten~e~ chl1l shelf li~e products
It is generally accepted from a safety standpoint, neutral
pH, high Aw products that are intended for extended shelf
life at chill temperature require a process equivalent to
90C for 10 minutes in order to inactivate non-proteolytic
Clostr;~;um botul;nl~m. From a spoilage standpoint, in
order to prevent the outgrowth of heat-resistant spoilage
Bacilli, then a process equivalent to 95C for 25 min is
required.
All such preservation systems are often detrimental to
product quality and thus for some time a novel preservation
system has been sought which provides a shelf stable
product with minimum loss of quality, for example product
texture, colour and/or taste, which can-be applied to
edible products having a pH of more than 4.6 and a water
activity of more than 0.93.
Surprisingly, it has been found that this can be achieved
if the product is first subjected to high pressure
conditions, a cell membrane destructive agent being present
either before or after the high pressure treatment; then
subjected to a treatment to inactivate any remaining
vegetative cells which may be present due to germination of
the spores and/or non-spore forming micro-organisms.
EP-A-0480422 describes a method for treating fruit juice
with high pressure, wherein a proteolytic enzyme is added
prior to the high pressure process. The proteolytic enzyme
is employed to deactivate pectin-decomposing-enzymes such
as pectin esterase or polygalacturonase. It does this by
breaking peptide bonds in the target enzyme, not by
destroying membranes.
JP-A-04075574 teaches sterilisation using high pressure in
the presence of a lytic enzyme such as lysozyme. As stated
AMENDED SH~T
~ F 7196 (C) ~ -
2 1 7 2 2 ~ 4
in Comparative Example A (see page 10 of the present
specification), lysozyme ls not a membrane destructive
agent; lytic enzymes cleave cell walls, they do not destroy
cell membranes.
GB-A-1188753 discloses the use of pressure treatment and
compounds, such as tyrosine and 1-arginine, to initiate
germination. These compounds are not membrane-destructive
agents; they are simply amino acids which make up pseudo-
random peptides such as polyarginine (which is an effectivemembrane-destructive agent). Indeed, membrane-destructive
agents do not initiate germination; they prevent the
~ormation of active micro-organisms from germinated spores.
D;sclosl~re of the Invent1on
According to the present invention, there is provided a
process for producing a shelf stable product comprising
(a) subjecting the product to a pressure of 10 to 1000
Mega-Pascal at a temperature of less than or equal to
60C for a period of 1 minute to 10 hours; and
~b) inactivating any remaining vegetative cells in th
~iiE?I~ SH~
~WO95/08275 ~ 7 ~ 2 4 4 PCT~P9~/02891
product;
wherein a membrane destructive agent is added to the
product prior to step (a) or step (b) or immediately after
5 step (b).
By shelf-stable product is meant a product that has
extended shelf-life either under ambient storage conditions
(stable for ~ 3 months) or under chill conditions (stable
for > lO days).
By vegetative cells is meant both germinated spores and
non-spore forming micro-organisms.
Preferably the membrane destructive agent is added prior to
step (a):
In order to obtain the required shelf stable product it is
necessary to either prevent any spores present in the
product from germinating, or to encourage the spores to
germinate and then kill the germinated spores.
Although the applicants do not wish to be bound by any
theory, it is believed that the following occurs in the
above steps. In step (a) the pressure conditions at
moderate temperatures for l minute to lO hours will allow
the germination of the spores to occur. Preferably this is
done in the presence of a membrane destructive agent.
Surprisingly, it has been found that the combined
application of the pressure conditions and the membrane
destructive agent leads to a very high degree of spores to
be germinated and a surprisingly low level of super-dormant
spores. It is believed that this very effective
germination and removal of super-dormant spores to a low
level is unique for the combination of the pressure
conditions and presence of membrane destructive agent and
will not be found under other conditions, for example, the
W095/08275 PcT~ps~m28sl ~
21 72244
combined use o~ temperature and membrane destructive agent.
Furthermore, the presence of the membrane destructive agent
will effectively prevent the formation of active micro-
organisms from the germinated spores.
In step (b) the vegetative cells are inactivated by, for
example, heat treatment (conventional pasteurisation),
repetitive heat treatments, pressure treatment, repetitive
pressure treatment, or by a combination thereo~. This step
will not only kill the non-spore forming micro-organisms,
but will also kill the germinated spores. The net result
is that spores are very effectively removed and no active
micro-organisms are formed from the spores.
The conditions in step (a) are 10-1000 Mega-Pascal at a
temperature of less than or equal to 60C for a time between
1 minute to 10 hours.
Preferably the pressure in step (a) is from 50 to 400
Megapascal, more preferred 50 to 300 Megapascal, most
preferred 50 to 250 Megapascal. Pressure may be generated
by any method ~or high pressure generation, for example
liquid pressure may be applied whereby the product is
liquid in itself or is emersed in water after which the
pressure of the water is raised. High pressure technology
in general and its potential use is for example described
by R.G. Earnshaw (Food Technology International Europe '92
pages 85-88).
The temperature is pre~erably from 10 to 60C, more
preferred 20 to 60C, most preferred 35 to 60C. The time
for step (a) is preferably from 10 minutes to 8 hours, more
preferred 20 minutes to 5 hours, most preferred 20 minutes
to 4 hours.
For the purpose of this speci~ication the term cell
membrane destructive agent has the meaning as known in the
~ Wo95/08275 2 1 7 ~ 2 4 4 PCT~P9~/02891
art: ie. any agent capable of affecting microbial
membranes for example, lantibiotics, pseudorandom peptides,
magA;nins, attacins, cecropins, defensin, eugenol, allecin,
subtilin, Pep 5, epidermin, cinnamycin, RoO9-0918,
duramycin and ancovenin. The amount of cell membrane
destructive agent is preferably from 10 to 1000 ppm, more
preferred 15 to 300 ppm, most preferred 25 to 150 ppm.
Especially preferred cell membrane destructive agents are
so-called lantibiotics such as described in EP 427 912.
Members of this group include nisin, and pediocin.
Within this class nisin is particularly preferred.
Another class of membrane destructive agents are pseudo-
random peptides. For the purposes of the present
specification the term 'pseudo-random' will be used to
refer to both completely random peptides and to those in
which no steps have been taken to inhibit or restrict
peptide growth to single step elongation of the peptide
ChA; n.~ SO as to produce an ordered sequence of residues.
These peptides, having far less structure than those ~ound
in nature, are known and have found applications as drug
carriers or as reagents in immunological assay techniques.
Typically, the peptide comprises a co-polymer of at least
one amino acid having an isoelectric point above 7 and at
least one amino acid having a bulky functional group.
For the purposes of the present speci~ication, bulky groups
are those having a total of five or more carbons and
heteroatoms. These include the aromatic groups derived
from toluyl rings (as in phenylalanine and tyrosine) and
indoles (as in tryptophan), as well as sufficiently long
side rhA; ns such as the guanidino group of arginine, and
the amino group of lysine.
W095/08275 ~1 ~ 2 2 4 4 PCT~P94/02891
Typically the amino acid having an isoelectric point above
7 is selected from the group comprising arginine, lysine,
histidine and mixtures thereof.
Typically, the amino acid having an aromatic or other bulky
functional group is selected from the group comprising
arginine, tryptophan, tyrosine, phenylalanine and mixtures
thereof.
It should be noted that arginine, and ornithine have a
sufficiently high isoelectric point and sufficiently bulky
functional group in the side chain that poly-arginine and
poly-ornithine are effective peptides.
Particularly preferred peptides are those which comprise
homopolymers of arginine and copolymers of lysine and
phenylalanine, arginine and tryptophan and/or lysine and
tryptophan. Mixed systems are also envisaged.
All of the above-mentioned amino acids are naturally
occurring in the L chirality and consequently it is
preferred that this form of the amino acid is used.
The molar ratio of amino acids of the two types is
preferably in the range l0:l-l:l0, more preferably 5:l-l:2
with an equal or pre~om;n~nt molar quantity of the basic
amino acid being preferred.
Other amino acid residues can be present in the peptide,
including non-standard amino acids such as ornithine, which
is not an essential amino acid but does have an lsoelectric
point close to l0.
Accordingly it is preferred that the membrane destructive
agent is selected from lantibiotics, pseudo-random
synthetic peptides and mixtures thereof. Preferably the
membrane destructive agent is nisin.
~ W095/08275 2 1 722 4 4 PCT~P94/02891
The inactivation of the veyetative cells (step (b)) can be
carried out in any suitable way, for example by heat
treatment to a temperature of 60 to 100C for 1 to 100
minutes. Particularly preferred, however is that the
inactivation is a high pressure sterilisation at a pressure
of 300 - 1500 Megapascal, a temperature of 60C or below and
a time of 1 minute to 10 hours. If high pressure
conditions are used in step (a) and in step (b) this will
provide a surprisingly good product quality.
In an especially preferred embodiment of the invention the
pressure during step (b) is at least 50 megapascal higher
than the pressure during step (a), more preferred more than
100 Megapascal higher e.g. 100 to 400 megapascal higher
than the pressure in step (a). Preferably the pressure in
step (b) is between 350 and 500 Megapascal. The
temperature during the high pressure inactivation in step
(b) is preferably from 5 to 50C, more preferred 10 to 45C,
most preferred 15 to 40C, for example ambient temperature.
The time for high pressure inactivation in step (b) is
preferably from 5 minutes to 8 hours, more preferred 10
minutes to 5 hours, most preferred 10 minutes to 4 hours.
Where appropriate step (a) and (b) may be repeated for even
further reducing the number of spores in the products. For
example the product may be subjected to 2 to 10
preservation cycles as described above. Preferably the
number of cycles is from 2 to 4.
The preservation process of the invention may
advantageously be applied to all products which tend to
suffer from problems with spores. Examples of suitable
products are ~ood products, personal products and
detergency products.
Advantageously the preservation process is applied to
products, having a pH of more than 4.6, more preferred more
woss/08275 ~ 2 2 4 4 PcT~P9~m289l ~
than 4.6 and less than 10, most pre~erred more than 4 7 and
less than 8. Also preferably the water-activity aw of the
food is more than 0.93, more preferred aw is from 0.96 to
1.00, most preferred 0.97 to 1.00.
Since the process of the invention allows for the
production of shelf stable products whereby only moderate
temperatures are applied, the process of the invention is
especially suitable for products to which a high
temperature is detrimental to its quality e.y. products
which are unstable or undergo undesired structural changes
or form off-flavours when heated at temperatures normally
used in preservation.
Examples of cosmetic products which may be subjected to the
process of the invention are creams, lotions, tonics,
toothpaste, lipstick, gels, shampoo and other hair
products.
Examples of detergent products are liquid systems like
liquid fabric washing detergents, household cleaners,
abrasives, fabric conditioners and (semi-) solid detergents
e.g. pastes and soap bars.
Preferably the process is used to provide shelf stable
foodstuffs. Examples of suitable food products are
spreads, in particular zero or extremely low fat spreads,
dressings, dairy and non-dairy creams, toppings, processed
cheese, pates, semi-hard cheese, sauces, sweet spreads,
margarines, ice-cream, meat and fish products, bavarois,
bakery and dough products, vegetables, fruit, soups, dairy
products, beverages. In particular this process may be
used to provide ambient stable sauces, soups and dressings.
Before, during or after steps (a) and (b) the edible
products are preferably filled into a suitable package for
further use. If the filling takes place before steps (a)
~Wo 95/08275 ~ 1 7 ~ 2 ~ 4 PcT/EP94/n289l
and (b) it is not necessary to use aseptic filling since
the package will be sterilised during steps (a) and (b).
.,
If, however the product is filled during or after steps (a)
? 5 and (b), then preferably aseptic filling conditions are
applied.
The invention will now be illustrated by means of the
following examples:
Example 1
Shows the effect of nisin added prior to pressure treatment
on B.subtilis.
Preparation of B.Subtilis Spores
An overnight culture of Bacillus subtilis was made in BHI
at 30C. Appropriate aliquots were streaked on agar plates
(plate count Agar (Difco)) supplemented with 0.04 mg/L
MgCl2. The agar was incubated at 30C for 3 to 5 days.
Well sporulated cultures (>30~ spores) were harvested by
washing once in sterile distilled water.
Pressure Treatment
A spore concentration of from 106 to 107/ml in 5ml distilled
water was used. The spores were subjected to a high
pressure treatment of 200 mPa at 35C for 180 min, followed
by pasteurisation at 85C for 5 min. A number o~ different
concentrations (0-100 ppm) of nisin were added prior to
high pressure treatment. After treatment the spores were
disseminated into agar and incubated for 5 days at 30C
before counting. Results are expressed as a log reduction
factor.
w095/08275 ~ 4 4 PCT~P94/02891 ~
Log Reduction Factor =
log (Startinq Inoculum Colony forminq units (cfu)
finishing c~u in inoculum
Results were compared with the log reduction factor
achieved when;
(a) no pressure treatment and no pasteurisation occurred
(b) no pressure treatment occurred ie. only pasteurisation
(c) pressure treatment only ie. no pasteurisation.
Results are shown in Table 1.
Comparative Example A
Shows the effect of lysozyme (not a membrane destructive
agent) added prior to pressure treatment of B.subtilis.
Example 1 was repeated except a number of different
concentrations (0-200 ppm) of lysozyme were added prior to
high pressure treatment instead of nisin. Results given in
Table 2 are expressed as a log reduction factor.
~x~le 2
Shows the effect of nisin or pediocin addition at different
stages in the process on C.Botulinum.
Preparation of C.botulinum spores
Clostridium botulinum type A strains ZK3, 62A, VII, type B
strains 2345, bolus alba, 6 were used. A cocktail was made
by mixing equal amounts o~ the spores together.
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Woss/08Z75 2 1 72244 PCT~Pg~m289l ~
Sporulation of proteolytic C.botulinum
Approximately O.lml of cooked meat stock (Difco) culture
was inoculated into lOml of liquid TPGS medium and
incubated for 24 hour at 30C.
TPGS medium
Tryptone (Difco) 5
bactopeptone (Difco) 0.5
glucose 0.2
soluble starch 0.2
cysteine 0.05
pH 6.8
sterilised 15 min at 120C.
The entire culture was inoculated into 1000 ml TPGS. This
TPGS was then poured over the agar phase. The biphasic
culture was incubated anaerobically for 5 to 7 days at 30C.
Aqar Phase
Meat Extract (Liebig) 1.67
bactopeptone (Difco) 1
tryptone (Difco) 1
gelatin (Gelatine Delft) 1
agar 2
sterilised 15 min at 120C.
When sufficient spores were present (10-70~ as observed by
microscopic ~m; n~tion) spores were harvested by
collecting the liquid phase.
The spore suspension was washed 3 times with distilled
water by repeated centrifugation at about 5C at 12000g for
~ WO95/08275 21 72244 PcT~Pg4m289l
10 min each time.
The spores were then treated in an ultrasonic bath after
washing to ensure loose spores. The treated suspension was
heated for 10 min at 80C to ensure elimination of botulinum
toxin and/or vegetative cells and enumerated.
Sporulation of non-proteolytic C.botulinum
Sporulation was carried out as described for proteolytic
C.botulinum except that the treated suspension was heated
for 30 min at 60C to ensure elimination of botulinum toxin
and/or vegetative cells before enumeration.
Enumeration of Spores
Dilutions were done in a peptone (0.1~) and physiological
salt (0.85~) solution. Proteolytic spores were enumerated
in pour plates cont~;n;ng TSA, cystein and egg yolk
(0.05g/ml). Incubation was at 30C for 3 to 5 days. Non-
proteolytic spores were enumerated in TPG and egg yolk
containing pour plates.
Treatment
35 ppm nisin or 300 ppm Pediocin were added either prior to
high pressure treatment (200 MPa at 45C for 180 min), prior
to pasteurisation (protocol (i) - 85C for 5 min; protocol
(ii) - 95C for 10 min) or after both pressure and
pasteurisation treatments.
The C.botulinum was inoculated into CMM (cooked meat
medium, Difco) to a final concentration of 107/ml for the
high pressure treatment and pasteurisation treatment.
Recovery was carried out in Tryticase peptone glucose (TPG)
(to enumerate C.botulinum). The TPG agar plates were
incubated anaerobically for 5 days at 30C.
W095/08275 ~l 7 -2- ~ 4 ~ PCT~P9~/02891 ~
Results, expressed as a log reduction factor are shown in
Table 3.
Table 3
Treatment Nisin Pediocin
Membrane 4.5 4.4
destructive
p agent added
R prior to
o pressure
T treatment
C Membrane 4 2 4.2
0 destructive
L agent added
(i) prior to
pasteurisation
Membrane 5.3
destructive
agent added
after
pasteurisation
Membrane 5.4 4.8
p destructive
R agent added
o prior to
T pressure
o treatment
C
0 Membrane 5.2 4.5
L destructive
(ii) agent added
prior to
pasteurisation
Membrane 5.8
destructive
agent added
after
pasteurisation
Exam~le 3
Shows the effect of nisin and high pressure on C.botulinum
~ W095/0827~ 2 1 72 2 4 4 PCT~P94/02891
in cheese.
C.botulinum cocktail was prepared as detailed in Example 2.
Treatment
Spores of the cocktail of C.botulinum were mixed into a
cheese having the following formulation;
Cheese formulation
~ by weight
Skim milk powder 12.2
Milk protein concentrate4.0
Butter 39.0
Salt 1.1
Na pyrophosphate 0.2
Na citrate dihydrate 0.4
Water 43.1
The final spore concentration was 107/ml. Nisaplin was
added to the cheese at a level of 0.05~ by weight
(corresponds to 10 mg/kg pure nisin).
The cheese was subjected to a number of different pressure
treatments for 180 min as detailed in Table 4, followed by
a pasteurisation treatment of 80C for 10 minutes.
Results expressed as log reduction factor are given in
Table 4.
W09510827s ~ ~ 7 ~2 4 ~ PCT/EP91/02891
Table 4
Pressure Temperature Log Reduction
Factor
O.lMPa 35C 0.0
45C 0.0
60MPa 35C 0.4
45C 1.0
200MPa 35C 1.3
45C 2.4
Example 4
Shows the effect of pressure and nisin on C.botulinum.
A C.botulinum cocktail was prepared as detailed in Example
2.
Treatment
Spores of the cocktail of C.botulinum were mixed into
cooked meat medium (CMM, Difco) to a final concentration
107/ml. The CMM contained 0, 10 or 35 ppm nisin. The 3
inoculated CMM varieties were subjected to different
pressure treatments as detailed in Table 5. After pressure
treatment the inoculated CMM varieties were subjected to
one of the following pasteurisation protocols:
(np) no pasteurisation
(i) 85C for 5 min
(ii) 95C for 10 min
Recovery was done in TPG with the same level of nisin
present as in the CMM. Results expressed as log Reduction
factor are shown in Table 5.
~WO 95/08275 2 1 7 2 2 4 4 PcT/EPg~m289l
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W095/08275 2 ~ ~ 2 2~4 4 pcT~ps~loz89l ~
18
Example 6
Shows effect of nisin on B.subtilis in 2 stages of
pressure/post pasteurisation treatment.
B.subtilis spores were prepared as for Example 1.
A spore concentration of from 106 to 107/ml in 5ml distilled
water was used.
Nisin at 35 ppm concentration was added prior to pressure
treatment and/or pasteurisation treatment. The pressure
treatment was 200MPa, at 35C for 180 min. The
pasteurisation treatment was 80C for 5 min.
Results, expressed as a log reduction factor, are given in
Table 7.
Table 7
nisin present nisin present log reduction
pre-pressure post- factor
treatment pasteurisation
treatment
~ X 5.2
X ~ 5.3
~ ~ 5.3
Example 7
Shows a comparison of pasteurisation and pressure treatment
to inactivate the vegetative cells.
B.subtilis spores were prepared as for Example 1.
A spore concentration of from 106 to 107/ml in 5 ml
distilled water was used. The spores were subjected to a
~W095108275 2 ~ 7 22 4 4 PCT~P94/02891
19
high pressure treatment of 60MPa at 35C for 30 min followed
by either
J
(i) Pasteurisation of 5 min at 85C or
(ii) Pressure treatment at 400 MPa at 35C for 10 min.
r
35 ppm nisin was added prior to high pressure treatment
(step (a)). After treatment the spores were disseminated
into agar and incubated for 5 days at 30C before counting.
Results are expressed as a log reduction factor and are
shown in Table 8.
Table 8
Treatmentlog reduction factor
(i) High pressure plus ~5.9
pasteurisation
(ii) Repetitive High ~5.9
Pressure
Example 8
Shows a comparison of pasteurisation and pressure treatment
to inactivate the vegetative cells.
C.Botulinum cocktail was prepared as for Example 2.
Spores of the cocktail of C.botulinum were mixed into
cooked meat medium (CMM, Difco) to a final concentration of
107/ml. The CMM contained 35 ppm nisin. The spores were
subjected to a high pressure treatment of 60MPa-at 35C for
30 min followed by either
(i) Pasteurisation of 5 min at 85C or
(ii) Pressure treatment at 400 MPa at 35C for 10 min.
Recovery was done in TPG with 35 ppm nisin added. Results
WO9S/08275 2 t 7 ~ 2 4 4 PCT~P9~/02891 ~
expressed as log reduction ~actor are shown in Table 9.
Table ~ ,
Treatment log reduction ~actor
(i) High pressure plus 2.5
pasteurisation
(ii) Repetitive high 2.4
pressure
