Note: Descriptions are shown in the official language in which they were submitted.
CA 02240930 1998-06-17
WO97/23136 I PCT/GB96/03173
BACTERIAL DECONTAMINATION METHOD
This invention relates to a method for reducing the
levels of bacteria, in particular food-borne human
pathogens and spoilage organisms, which is suitable for use
in food processing or elsewhere where hygiene reguirements
mean that bacterial levels should be controlled. The method
may be effective against both gram negative and gram
positive bacteria. Kits for carrying out the method are also
claimed.
PCT patent application WO 93/00822 (MAFF) discloses a
method of destroying bacteria by use of osmotic shock
treatment in combination with cold shock and/or exposure to
the enzyme lysozyme. Lysozyme is well known to be effective
against certain gram positive bacteria but the co-m-bined
treatment extends its usefulness into the class of gram
negative bacteria. However, although effective in vitro the
combined treatment is not sufficiently effective against
Salmonella when the method is used under practical food
processing conditions.
In US Patent 5,069,922 there is disclosed a process for
treating poultry carcasses to control Salmonella growth,
which comprises treating eviscerated and defeathered poultry
with a solution contA; ni n~ an alkali metal orthophosphate,
e.g. trisodium orthophosphate ~TSP), or an alkali
orthophosphate combined with a minor amount of a basic
reagent, e.g. sodium carbonate. TSP has been accepted as
safe by the US Food and ~rug A6sociation and is an ingredient
of many food products, but the TSP process as disclosed in the
above referenced US patent, and as used to date, has certain
disadvantages. Firstly it re~uires a high TSP concentration
(around 0.4M) and thus a high pH (approximately 12.5 at 0.4M),
which may reduce its acceptability for poultry and other food
treatments. Secondly, it is ineffective against gram positive
spoilage bacteria and has ~uestionable effectiveness aqainst
certain gram negative bacteria such as Campylobacter.
.
We have surprisingly found a method of treating products such
as foodstuffs, which may be contAm;n~ted with human pathogens
and/or spoilage bacteria, which is highly effective in the
.= -
CA 02240930 1998-06-17
WO97/23136 2 PCT/GB96103173
destruction of gram negative bacteria and which will also
reduce the levels of gram positive bacteria. The process is
particularly useful in the destruction of pathogenic bacteria
such as Salmonella and Campylo~acter which hitherto have been
comparatively resistant to decontAm;nAtion processes which may
be applied to foodstuffs.
Accoraing to the present invention there i8 provided a method
for reducing the levels of gram negative and gram positive
bacteria in a sample comprising treating the sample with a
0.0005 to 0.2M solution of a trialkali metal orthophosphate,
said treatment being combined with one or more of the
~ollowing further treatments:
a) subjecting the sample to osmotic shock,
b) exposing the sample to an enzyme which breaks down
peptidoglycan after said treatment with trialkali metal
orthophosphate; and
c) exposing the sample to a bacteriocin after said treatment
with trialkali metal orthophosphate.
A preferred combination of further treatments comprises step
(a) wïth either step (b) or step (c).
We have found that a combination treatment as disclosed
herein is effective in the destruction o~ gram positive
bacteria, against which trialkali metal orthophosphate on its
own is inef~ective, and for gram negative bacteria such as
Salmonella and Campylo~acter. Surprisingly the effect of the
combination of the trialkali metal orthophosphate process with
one or more of the other processes is synergistic in that not
only is the range of bacteria destroyed greater than any of
the processes separately but also it works with lower
concentrations of alkali metal orthophosphate than utilised
previously, in~ee~ ones which would be otherwise non-lethal
when used without the secondary stage of treatment. Thus
concentrations of from 0.0005 to 0.2M, suitably from 0.001 to
0.2M and preferably from 0.005 to O.OlM of trialkali metal
orthophosphate are sufficient. This ability to use lower
concentrations of trialkali metal orthophosphate reduces the
pH of the treatment solution and therefore makes the treatment
more attractive to use in food decontAm; n~ tion applications.
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W O 97/23136 3 PCT/GB96/03173
The precise minim~lm concentration o~ TSP which will be
e~ective in any particular case may vary depending upon the
nature o~ the sample. For example, as illustrated
hereina~ter, the presence o~ serum. in signi~icant quantities
may af~ect the concentration level of TSP, within the above
mentioned range, re~uired. However this can be determined
using routine methods in any particular case, i~ it is ~elt
that a minimllm amount o~ TSP is necessary.
The present invention has a ~urther advantage over the
previously disclosed osmotic shock plus lysozyme treatment
since it does not depend upon the presence o~ nutrients to
promote killing and treatment processes are not markedly
dependent on temperature being in the range 4 to 50~C. In
addition, using the present invention, organisms washed o~
chicken skin sur~aces were found to be highly susceptible to
killing treatments and the resultant treatment solutions were
relatively ~ree o~ bacteria. This could be o~ signi~icance in
reducing cross cont~m;n~tion between carcasses in poultry
processing.
Suitably the osmotic shock comprises a hypo-osmotic shock, in
which water is induced to enter a cell and particularly a
bacterial cell. Hypo-osmotic shock is pre~erably preceded by
~ministration o~ a hyper-osmotic shock (where water is
induced to leave the cell). The osmotic shock may ~e
~m;nistered using the process described in WO 93/008822, the
disclosure o~ which is incorporated herein by reference. In
particular, osmotic shock may be induced in a sample by
exposing the sample to a ~irst solution having a water
activity (aw) o~ 0.997 or less (which induces hyper-osmotic
shock) and subse~uently exposing the sample to a solution o~ a~
higher than that o~ said ~irst solution (which then results in
hypo-osmotic shock).
Advantageously, the ~irst solution has a water activitY in the
range 0.992 to 0.96, more preferably 0.974 to 0.96. It is
suitably applied ~or a time in the range o~ 5 seconds to 30
minutes, or more pre~erably between 30 seconds and 20 minutes,
or most pre~erably in the range o~ 1 to 10 minutes.
CA 02240930 l998-06-l7
WO97~3136 4 PCT/GB96/03173
The said first solution suitably contains NaCl at a
concentration sufficient to provide a water activity of 0.997
or less, for example at a concentration of about 0.8M. The aw
of the second solution is suitably of the order of 0.999. The
sample is exposed to this solution for a sufficient period of
time to produce the aesirea hypo-osmotic shock. The time may
in fact be very short, of the order of a few seconds.
However, long exposure times do not adversely affect the
~0 reaction. Typically an exposure time of from 5 secon~ to 2
hours may be convenient, more particularly from 10 minutes to
1 hour, for example about 30 minutes.
The second solution may be prepared and applied separately
from the first solution, or it may be created by introducing
an appropriate diluent such as water, to the first solution.
In a pre~erred embodiment, the said first solution contains
0.001 to 0.2 M of a tri-alkali metal orthophosphate so that
the treatment with this reagent is combined with the osmotic
shock treatment.
In addition, the second solution may contain reagents used in
steps (b) and (c) above, so as to combine these further
treatments with the osmotic shock treatment.
Suitable enzymes which break down peptidoglycan for use in the
method of the invention are those which are not harmful to
humans, and a particular example is lysozyme. Lysozyme is
suitably applied to the sample in the form of an aqueous
solution, for example having a concentration of at least
l~gmlland preferably at least 5~gmll. Such a lysozyme solution
is conveniently provided in the form o~ a solution of freeze
dried egg white which is at a concentration of at least
O.lmgml .
We have found that further treatment with lysozyme is
particularly effective. The lysozyme may be applied in a
rinse water, following treatment with trialkali metal
orthophosphate, at a concentration of at least l~g mll and
preferably at least 5~g mll. Alternatively, lysozyme treatment
. , _
CA 02240930 1998-06-17
WO97/23136 5 PCT/GB96/03173
is combined with osmotic shock treatment by adding lysozyme to
the second solution of higher aw.
Suitably bacteriocins are also those which are considered to
be fit for human consumption such as nisin and pediocin, and
pre~erably nisin is used in the method of the invention.
Suitable nisin treatment solutions contain nisin at a
concentration of at least O.l~M, preferably l~M or more. In a
preferred embodiment, the sample is rinsed with water after
treatment with the trialkali metal orthophosphate and prior to
treatment with the solution cont~in;ng nisin.
A particular advantage of the use of nisin in this combined
treatment is that nisin is also particularly effective in
killing gram positive organisms, for example, Staphylococcus
aureus giving further improved kills over the other combined
treatments.
Thus further treatment with nisin is particularly e~fective
where there is contamination by gram positive bacteria.
In the use of the process of the invention, particularly in
the treatment of poultry, control of TSP concentrations is
desirable in order to prevent the pH o~ the enzyme or
bacteriocin treatment, in particular lysozyme or nisin
treatments respectively, being adversely affected. It may be
desirable to acidi~y the lysozyme or nisin solution to
optimise the treatment.
There~ore, suitably, treatment solutions of bacteriocin or
enzyme solutions are acidified, for example to a pH of
approximately 5Ø This may be effected by inclusion of an
acid to the solution, preferably an organic acid such as
lactic acid. Lactic acid is suitably present at a
concentration of at least 0.25mM.
A suitable trialkali metal orthophosphate for use in the
method of the invention is trisodium phosphate.
The sample is suitably a portion of a foodstuff, although it
may also comprise other consumable products such as
pharmaceuticals, cosmetics and toiletries. However, it may
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W O 97/23136 6 PCT/GB96/03173
also comprise sur~aces o~ m~ch;nery~ instruments or utensils,
such as pipework, or working sur~aces, such as ~ood
preparation sur~aces or 'clean-room~ sur~aces where the
presence o~ bacteria would be problematic.
Treatment methods used will depend upon the nature of the
sample and the nature of the solution being applied. However,
treatments may suitably be effected, ~or example by immersing
the sample in a solution, or by washing and particularly spray
w~sh;ng the sample with a treatment solution, or a combination
o~ these. Where treatment reguires that the sample is exposed
to the treatment solution ~or an extended period o~ time, ~or
instance, where a solution with a water activity o~ less than
0.997 is being applied in order to induce a hyperosmotic
shock, immersion may be the most convenient option, but
suitably spray wash solutions may be prepared, ~or example by
using electrostatic spraying technigues or by including ~ilm-
~orming chemicals such as sur~actants into the solution prior
to application.
Thus in a pre~erred embodiment, a sample is treated with a
~irst solution comprising a trialkali metal orthophosphate at
a concentration in the range o~ ~rom 0.0005 to 0.2M, suitably
~rom 0.001 to 0.2M, said solution having a water activity (aw)
o~ 0.997 or less, and subsequently treating the sample with a
second solution o~ an enzyme which breaks down peptidoglycan
or a bacteriocin, said second solution having an aw higher than
that o~ said ~irst solution.
Suitably the sample is sequentially immersed in said ~irst and
second solutions but alternatives such as immersion in the
~irst solution ~ollowed by spray w~s~i ng in the second
solution may be used.
In a pre~erred process a sample of ~oodstu~ is immersed in a
solution o~ TSP at a concentration in the range 0.0005 to
0.2M, suitably ~rom 0.001 to 0.2M and pre~erably 0.005 to
O.OlM, together with a solution of sodium chloride, pre~erably
at a concentration o~ about 0.8M, at a temperature in the
range 4 to 50~, pre~erably 37~C and then spray w~s~; ng with
lysozyme solution, which is suitably at a concentration o~
CA 02240930 1998-06-17
W O 97~3136 7 PCT/GB96/03173
>lO~g mll. The lysozyme solution may be provided in the ~orm
of ~reeze dried egg white at a concentration o~ about
O.lmg ml~~. This pre~erred process is highly e~ective in the
killing o~ gram negative bacteria on vegetable material such
as lettuce.
In an alternative pre~erred process a sample o~ ~oodstu~ is
immersed in a solution o~ trisodium orthophosphate at a
concentration in the range 0.001 to 0.2M, pre~erably 0.005 to
O.OlM, at a temperature in the range 4 to 50~C, preferably 37~C
and then spray washed with nisin at a concentration o~ greater
than l~M.
Although the treatments o~ the present invention are ~airly
lS independent of temperature, it is preierred that the treatment
with trialkali metal orthophosphate and the ~urther treatment
is carried out at a temperature in the range 4 to 50~C,
pre~erably about 37~C.
Kits ~or carrying out the above-described method ~orm a
~urther aspect o~ the invention. These kits may comprise a
trialkali metal orthophosphate and one or more o~ the
~ollowing components:
a) a reagent which can be used to induce osmotic shock in a
cell;
b) an enzyme which can break down peptidoglycan; and
c) a bacteriocin.
The reagents may be supplied per se with instructions ~or
~orming suitable treatment solutions, or they may be supplied
as ready made a~ueous solutions. The components o~ the kit
will be separated in various containers, ~or example in
multipack containers.
Products treated in accordance with the above-described method
~ and particularly ~oodstu~s so treated ~orm a ~urther aspect
o~ the invention.
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W O 97/23136 8 PCT/GB96/03173
The method of the present invention is now illustrated ~urther
with re~erence to the following non-limiting examples.
In the work conducted, the ~ollowing investigations were
S carried out:
~i) the e~fect of combining TSP with osmotic shock and/or
lysozyme treatment.
(ii) the e~~ectiveness of TSP and combined osmotic
shock/lysozyme treatments on the killing of organisms attached
to food sur~aces (chicken skin and lettuce)
~iii) the effect of using nisin to supplement or replace
lysozyme in killing procedures.
The organisms used were: Escherlchia coli, Listeria
monocytogenes, Pseu~nm~nA.~ fluorescens, Salmonella
enteritidis, Campylobacter jejuni and Staphylococcus aureus.
Pre~ar~tinn of R~cteria
Campylobacter jejuni (NCTC 11626), Listeria monocytogenes
(NCTC 7973~, Pseu~mnn~s ~luorescens (NCTC 10038) Salmonella
enteritidis (NCTC 6676) and Staphylococcus aureus (NCTC 8532)
were obt~;n~ as freeze-dried cultures from the National
Collection of Type Cultures, Colindale, UK. Escherichia coli
(NCIMB 9~85) was obtained from the Division of ~ife Sciences
Collection, King's College.
To provide inocula for experiments, the cryoprotectant
glycerol (final concentration 15% v/v) was added to early
stationary phase cultures. One ml aliquots of cultures were
then dispensed into sterile cryotubes and stored at -70~C.
Except for C. jejuni, bacteria were grown aerobically at 37~C or
30~C (Pseu~mnn~.~ only) on Brain Heart Infusion broth (Oxoid)
or Nutrient agar plates (Oxoid). Broth cultures were grown in
250ml ~lasks cont~;n;ng 25 ml medium, on a shaking incubator.
C.jejunl was grown statically in 70 ml tissue culture bottles
cont~;n;ng 25ml Brain Heart Infusion broth (Oxoid) enriched
with 10% Horse serum (Oxoid) and 0.25% Yeast extract (Oxoid),
or on Blood agar plates (7% v/v Horse blood (oxoid) in Oxoid
Blood agar base No 2). Cultures were incubated at 37~C in an
CA 02240930 1998-06-17
WO97/23136 9 PCT/GB96/03173
atmosphere o~ 10%(v/v) CO2 and 10% (v/v) ~2/ obtained using a
gas jar and gas generating kit (Oxoid BR 60).
le l: Killina of sus~ended cell~
The e~ective cell kills achieved on gram negative and gram
positive organisms subjected to treatments with TSP and
lysozyme and/or osmotic shock, at various temperatures and in
the presence or absence o~ serum was tested as ~ollows.
The methodology adopted was based on the disclosure o~ WO
93/00822 incorporated herein by re~erence which indicated that
(i) early stationary phase cells were the most resistant to
osmotic shock/lysozyme treatments; (ii) 0.8 M NaCl allowed a
m~;mAl or near-m~;m~l killing e~ect in this system for all
the gram negative organisms tested; (iii) exposure to hyper
and hypo-osmotic shocks ~or l0 and 30 minutes respectively was
adequate to allow access o~ lysozyme to gram negative cells
and longer treatment times would be cc ?rcially unacceptable;
(iv) 20~gml~1 lysozyme gave close to optimal cell kills in
osmotically shocked gram negative cells.
Overnight broth cultures were diluted l/25 in ~resh medium and
similarly grown to the early stationary phase (approximately
~h incubation); growth was monitored by ~ollowing culture
optical density using an EEL colorimeter at SSO nm.
Stationary phase cultures were diluted l/l00 into appropriate
test media, which included: TSP, TSP plus sodium chloride
(NaCl), TSP plus heat inactivated newborn cal~ serum (up to
50% v/v;Cibco), and TSP plus NaCl and serum; concentrations of
TSP and NaCl were varied and are given in the results tables.
A~ter incubation in test media ~or l0 min at 4 or 37~C, cell
suspensions were ~urther diluted (l/l00) in distilled water or
distilled water cont~;n-ng 20~gml lysozyme, (Sigma L6876).
A~ter incubation ~or a ~urther 30 min, cells were diluted as
appropriate in deionised water and plated on nutrient agar.
Plates were incubated ~or up to 48h at 37~C or 30~C
(Pseudomonas only) and cell kills estimated.
The results are shown in Tables l to 8. In the ~ollowing
Tables, annotations used are as ~ollows;
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CA 02240930 1998-06-17
W O 97~3136 1~ PCT/GB96/03173
ns, no survivors detected; cell kill > 99.7%
*pH of initial treatment solutions, i.e. TSP, NaCl, TSP +
NaC1, or water.
T~hle 1. The ~mh; ned ef~ect of TSP, o~m~tic shock and
lvsozY-m~ ~n the survival of Gr~m -ve bacteri~ ~t 37~C
Organism NaCl (M) TSP(M) pH* % cell survival
-lysozyme +lysozyme
10 E. coli none none7.Q1 100 87
none 0.00210.46 100 2.3
0.4 none6.57 73 11
0.8 none6.39 33 0.59
1.2 none6.26 13 ns
~ 15 0.8 0.0029.56 ns ns
P. none none7.12 100 100
~luorescens 0.8none 6.36 100 12
none 0.0017.59 100 100
none 0.0029.07 99 66
0.8 0.0016.81 ns ns
~.
25 enteri tidis nonenone 7.01 100 99
0.8 none6.28 86 41
none 0.0018.70 91 71
none 0.00210.69 63 28
none 0.00510.87 0.2 0.1
0.8 Q.0018.42 ns ns
C. jejuni none none7.34 100 100
0.8 none6.62 10Q g5
none 0.0018.897 100 76
none 0.0029.89 100 32
none 0.00510.67 ns ns
0.8 0.0017.38 2 2
0.8 0.0029.42 ns ns
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W097/23136 1I PCT/GB96/03173
Table 2. The c~m~ined ef~ect of TSP, osmotic shock and
lvsozvme on the survival o~ Gram -ve bacteria at 4~C
5 Organism NaCl (M) TSP(M) pH* % cell survival
-lysozyme +lysozyme
. coli none none6.80 100 100
0.8 none6.36 31 ns
none 0.002 9.33 51 10
none 0.005 11.30 ns ns
0.8 0.002 9.54 ns ns
15 fluorescens none none 7.04 100 100
0.8 none6.36 100 3
none 0.001 7.54 65 85
none 0.002 9.26 99 56
0.8 0.001 7.01 ns ns
S.
enteri tidis none none 7.15 100 100
0.8 none6.32 29 17
none 0.001 7.40 80 77
none 0.002 8.42 52 34
none 0.005 11.26 1 ns
0.8 0.001 6.77 9 ns
0.8 0.002 8.80 10 ns
0.8 0.005 10.29 1 ns
.jejuni none none7.42 100 100
0.8 none6.64 100 90
none 0.0017.84 100 67
none 0.0028.68 100 89
none 0.00510.05 4 3
0.8 0.0017.66 95 33
0.8 0.0029.00 3 2
0.8 0.0059.68 ns ns
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W O 97/23136 12 PCT/GB96/03173
TAhle 3. The com~ined e~ect of TSP, o~mntic shoc~ and
lvsozvme on ~he survival o~ GrAm +ve bacteria at 37~C
Organism NaCl(M) TSP(M) pH* % Cell Survival
- lysozyme +lysozyme
L.
mono- none none6.89 100
cytogenes 0.8none 6.32 100 5
none 0.00510.8188 3
none 0.01011.5488 8
none 0.05012.22 4 ns
none 0.10012.50ns ns
0.8 0.00510.3527 ns
0.8 0.01010.80 9 ns
0.8 0.05011.87ns ns
St. aureus nonenone6.99 100 100
0.8 none6.32 64 77
none 0.0059.92100 53
none 0.0111.51 77 78
none 0.0512.23 ns ns
0.8 0.0059.66 33 17
0.8 0.019.88 ns ns
0.8 0.0511.65 ns ns
TAhle 4. The comh;ned e~ect o~ TSP, osmotic shock ~nd
lvsQzYme on the survivAl o~ GrAm +ve bacteria at 4~C
Organism NaCl(M) TSP(M)pH* % cell survival
- lysozyme +lysozyme
L.
mono- none none7.06 100 20
35 cytogenes 0.8none 6.26 100 20
none 0.00511.38100 33
none 0.01011.82100 3
none 0.05012.34100 ns
none 0.10012.46 9 ns
0.8 0.00510.6277 ns
0.8 0.01011.2453 ns
0.8 0.05011.9315 ns
45 St. aureus nonenone7.28 100 100
0.8 none6.33 100 100
none 0.0111.51100 100
none 0.0512.28 85 65
none 0.1012.44 83 40
0.8 0.0111.2g100 91
0.8 0.0511.85 55 94
0.8 0.1012.23 13 20
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W O 97/23136 13 PCT/GB96/03173
Table S. The combined effect of TSP, osmotic shock ~nd
lvsozvme on the survival of Gram -ve bacteria at 37~C in the
~resence of 50% v/v serllm
Organism NaCl (M) TSP(M) pH* % cell survival
-lysozyme+lysozyme
E. coli none none 7.54100 100
0.8 none 7.00100 15
none 0.002 8.0140 3
none 0.005 8.3556 5
none 0.01 9.2784 ns
0.8 0.002 7.88100 ns
0.8 0.005 8.561 ns
0.8 0.01 9.20ns ns
p. none none 7.20100 97
~luorescens 0.8 none6.94 46 5
none 0.001 7.4599 88
none 0.002 7.7281 72
none 0.005 8.5974 70
0.8 0.001 7.1548 2
0.8 0.002 7.4334 3
0.8 0.005 8.45ns ns
5.
enteritidis none none 7.22 100 100
0.8 none 6.00 32 4
none 0.002 7.60 100 100
none 0.005 8.35 100 71
none 0.01 9.32 90 82
0.8 0.002 7.35 35
0.8 0.005 8.10 2
0.8 0.01 8.91 ns ns
C. je~uni none none 7.13 100 75
0.8 none 6.68 75 53
none 0.005 8.59 95 88
none 0.01 9.56 21 23
0.8 0.005 7.88 15 7
0.8 0.01 9.14 ns ns
CA 02240930 1998-06-17
W O 97~3136 14 PCT/GB96/03173
Table 6. ~he com~ined e~fect o~ TSP, osmotic shock and
1YSOZYme on the surviv~1 o~ GrAm -ve bActeri~ at ~~C in the
presence of 50% v/v serl~m
s
Organism NaCl (M) TSP(M) pH* % cell survival
-lysozyme +lysozyme
E. coli none none 7.42 100 100
0.8 none 7.18 100 20
none 0.002 7.87 100 100
none 0.005 8.92 100 100
none 0.01 9.66 100 94
0.8 0.002 7.18 53 10
0.8 0.005 8.37 25 6
0.8 0.01 9.34 1 ns
P.
fluorescens none none7.18100 100
0.8 none6.93 66 1
none 0.0017.43100 100
none 0.0027.74100 91
none 0.0058.5989 88
none 0.019.60 76 89
0.8 0.0017.1539 7
0.8 0.0027.3628 7
0.8 0.0058.56ns ns
0.8 0.019.08 ns ns
30 S.
enteri tidis none none7.18100 82
0.8 none6.99100 90
none 0.0027.50100 100
none 0.0058.2497 90
none 0.019.36100 98
0.8 0.0027.2485 61
0.8 0.0058.0889 68
0.8 0.019.09 59 10
C . j ej uni none none 7.21 100 66
0.8 none 6.53 78 68
none 0.005 8.34 71 66
none 0.01 9.48 47 42
0.8 0.005 7.38 37 37
0.8 0.01 9.42 ns ns
. ,
CA 02240930 1998-06-17
W O 97/23136 15 PCT/GB96/03173
Tahle 7. The combined ef~ect of TSP, osmotiG s~ock and
lvsozvme on the survival o~ ~ram +ve bacteria at 37~C in the
~res~nce o~ 50% erum
organism NaCl(M~ TSP(M)pH* % Cell Survival
- lysozyme +lysozyme
L.
mono- none none 7.12 100 ns
10 cytogenes 0.8 none 6.99 100 2
none 0.0109.48 98 3
none 0.05011.44 52 ns
none 0.10012.01 28 ns
0.8 0.01 9.42 90 3
0.8 0.0511.21 22 ns
0.8 0.1011.60 ns ns
St. aureus none none 7.26 100 99
0.8 none 7.07 84 85
none 0.0109.57 92 80
none 0.05011.50 89 82
none 0.10011.89 5 7
none 0.15012.11 ns ns
0.8 0.0109.14 88 89
0.8 0.05010.98 92 ns
0.8 0.10011.48 1 2
0.8 0.15011.72 ns ns
T~hle 8. The combined e~ect o~ TSP, osmotic sh~ck and
lvsozvme on the survival o~ Gram +ve bacteri~ at 4~C ;n the
presence o~ 50% serll~
Organism NaCl(M) TSP(M)pH* % cell survival
- lysozyme +lysozyme
35 ~.
mono- none none7.15 100 37
cytogenes 0.8 none 6.90 100 23
none 0.0109.71 100 54
none 0.05011.38 74 5
none 0.10011.71 12 2
0.8 0.0109.36 74 27
0.8 0.05010.94 2 ns
0.8 0.1011.71 2 ns
St. aureus nonenone 7.44 100 100
0.8 none7.18 100 100
none 0.019.67 100 100
none 0.0511.53 100 100
S0 none 0.1011.82 100 100
none 0.1512.10 100 89
0.8 0.019.39 100 100
0.8 0.0511.07 100 100
0.8 0.1011.51 100 100
0.8 0.1511.69 95 82
CA 02240930 1998-06-17
W097/23136 16 PCT/GB96/03173
S Tables 1 and 3 show results at 37~C on gram negative and gram
positive organisms respectively and Tables 2 and 4 show
results at 4~C. The TSP concentrations tested ranged ~rom 0.OM
to 0.OQ5M ~or the gram negative organisms and 0.OM to 0.10M
f or gram positive.
The results (Table 1, 37~C, Table 2, 4~C) confirm that gram
negative cells are resistant to lysozyme in the absence of
osmotic shock. Osmotic shock or TSP (up to 0.002M) alone,
also give relatively low kills. The combination o~ osmotic
shock and lysozyme treatment gave high kills, as predicted by
WO 93/00822, ~or E.coli and P.fluorescens.
At 37~C, in all cases, no surviving cells were detected when a
low concentration o~ TSP (0.001 or 0.002M) was used in
~0 combination with osmotic shock, even in the absence o~ a
subse~uent lysozyme treatment. TSP treatment in the absence
o~ osmotic shock also enhanced killing o~ cells subse~uently
exposed to lysozyme. The pH o~ cell suspensions treated with
TSP (up to 0.002M) were <10. Thus, these results show marked
improvement in cell kills when TSP treatment was combined with
either lysozyme or osmotic shock treatment at 37~C.
Results at 4~C (Table 2) were essentially similar to those at
the higher temperature. However, Campylobacter proved more
resistant than the other bacteria tested; cell kills o~
Campylobacter were not increased by lysozyme treatment, and,
in combination with osmotic shock, 0.005M TSP was re~uired to
reduce the number o~ Campylobacter to an undetectable level.
Nevertheless, even ~or Campylobacter, TSP killing was clearly
promoted by osmotic shock.
Osmotic shock treatment alone had little or no e~ect on the
viability of L.monocytogenes or St.aureus at 37 and 4~C (Tables
3 and 4, respectively). However, lysozyme (20~g ml~ reduced
the viable count o~ L.monocytogenes by 84-99%; killing
CA 02240930 l998-06-l7
WO97123136 17 PCT/GB96/03173
appeared temperature dependent, being greater at 37 than 4~C.
For Listeria, combining TSP(>0.05M) treatment with a
subsequent lysozyme treatment gave significantly higher kills
than for either treatment alone. Osmotic shock also ~n~nced
~ 5 killing by TSP (c.f. killing by 0.005-0.05M TSP in presence
and absence of NaCl-treatment; Tables 3 and 4), particularly
at 37~C. Thus, in combination with osmotic shock/lysozyme
treatments, TSP gave m~;m~l killing (no survivors detected;
kills >99.7%) with very low concentrations of TSP (0.005M).
St . aureus was more susceptible to TSP when this treatment was
combined with osmotic shock. ~owever, in contrast to results
obtained with L.monocyto~enes, exposure to lysozyme did not
enhance killing following either TSP and/or osmotic shock
treatment. However, St. aureus was markedly sensitive to low
concentrations of nisin (see Example 3).
The effect o~ the presence of serum on the TSP/osmotic
shock/lysozyme combined killing treatment was det~rm; n~ by
incubating cells in 50% v/v serum plus TSP and NaCl and
subse~uently diluting the cell suspension in lysozyme
solution. At 37~C (Table 5), 100% kills of the gram negative
bacteria, C. jejuni, E. coli, P. fluorescens and S.
enteritidis were obtained. However, the TSP concentration
required (5 to 10 mM) was higher than for cells suspended
without serum. The presence of serum lowered the pH of the
TSP (5-10mM)/NaCl treatment solution to between 8 and 9. At
4~C (Table 6), a similar result was obtained, e~cept with S.
enteritidis, for which a kill o~ 90% was obt~ne~ in the
combined treatment (TSP/osmotic shock/lysozyme) with 10mM TSP.
In the presence of serum, 100% kills of the gram positive
bacterium, L. monocytogenes were achieved at 4 and 37~C (Tables
7 and 8) using S0mM TSP in the combined TSP/osmotic
shock/lysozyme procedure. Without lysozyme treatment, kills
were 78% and 98% respectively. St . aureus was again more
resistant than L. monocytogenes. At 37~C, a TSP concentration
' o~ 150mM was required to give a 100% kill and kills were not
significantly enh~nced by osmotic shock or lysozyme treatment.
At 4~C, St. aureus was resistant (<20% kill) to TSP (>150m) and
combined treatments using TSP (>150m).
-
CA 02240930 1998-06-17
WO97/23136 18 PCT/GB96/03173
~.x~mnle 2: ~; llin~ of cells adhered to food sur~aces
The e~~ective kills achieved ~or gram negative organisms (E.
5 coli and S. enteri tidis) attached to chicken skin ~or variou~
concentrations of TSP in the presence or absence of osmotic
shock and/or a subse~uent lysozyme treatment was tested as
~ollows.
Overnight cultures of test bacteria were diluted l/25 in ~resh
medium and grown to the early stationary phase (approximately
4h incubation) as described above. The experimental ~ood
surfaces used were: lg chicken skin samples (removed ~rom
wing or thighs and purchased ~rom a local supermarket); and
circular sections (diameter 3 cm) o~ lettuce leaves. Food
samples were immersed in early stationary phase cultures for
minutes at ambient temperature and subsequently dried ~or
3 minutes in a stream of cold air, or alternatively, the
sur~aces o~ the samples were inoculated with 20~1 of the test
culture and left to air dry for 30 min.
Inoculated test samples were immersed in l0 ml TSP solution,
l0 ml TSP plus 0.8M NaCl, 0.8M NaCl or distilled water, as
appropriate. A~ter incubation on a rotary shaker for l0 mins
at ambient temperature, or ~ or 37~C, the samples were shake~
to remove excess fluid and then immersed in l0ml distilled
water or distilled water containing lOO~gmll lysozyme (Sigma
L6876). Additionally, in some experiments, nisin (3.33 x
105M) or lactic acid (2.5 x 10-3 or 2.5 x l0~M) was added to
the final treatment solution, or an additional water rinse was
included prior to the ~inal treatment. A~ter incubation for
30 minutes, samples were stomached individually for 2 minutes
with 50 ml distilled water in a stomacher (Colworth 80).
Serial dilutions o~ stomached samples, and where indicated,
~35 treatment ~luids, were plated on plate count agar (~xoid) or
blood agar ~or C. jejuni only. Plates were incubated as
described above and the percentage cell kill calculated and
recorded in Tables 9 and l0.
CA 02240930 l998-06-l7
WO97/23136 19 PCT/GB96103173
TAhle 9. (A) The combine~ e~ t o~ TSP, osmoti~ shock and
lvsozvme o~ the surviva~ of E~ coli attached to chicken skin
At ro~m tem~erature. (B) ~H values ~or ~rocessin~ sQlutions
followin~ in; tial tr~Atm~ntS with or without 0.20M TSP.
A
NaC1 (M) TSP (M) TSP,TSP/NaCl % Cell Survival
pH - lysozyme ~ lysozyme
none none 6.64 100 98
0.8 none 6.39 86 20
none 0.01 9.82 100 84
15 none 0.05 11.69 93 53
none 0.10 11.99 0.1 1.8
none 0.2 12.34 0.8 0.4
0.8 0.01 9.24 45 45
20 0.8 0.05 11.04 53 39
0.8 0.10 11.92 0.2 0.1
0.8 0.2 12.27 0.7 0,4
B
NaCl (M) TSP ~M) pH of initial ~H o~ ~inal
treatment treatment
solution * solution **
- lysozyme + lysozyme
none none6.59 6.62 6.48
0.8 none6.56 6.50 6.52
none 0.2012.41 11.67 11.68
35 0.8 0.2012.32 11.56 11.56
* initial treatment solutions were: TSP, TSP + NaCl, or water
** ~inal treatment solutions were: lysozyme solutio~ or
water
CA 02240930 1998-06-17
WO97~3136 20 PCT/GB96103173
Table lO. The combined e~fect o~ TSP, o~m~ic shnck and
lYsozvme on the surviv~l o~ E . col i attached to chick~n skin
;n~llhated at 4~C
5 NaCl (M) TSP (M)pH*% Cell Survival
-lysozyme +lysozyme
none none 6.73 lO0 9l
0.8 none 6.36 lO0 41
none O.lO12.22
none 0.2012.46 0.2 O.l
0.8 O.lO11.95 4 3
15 0.8 0.2012.30 6
* pH o~ initial treatment solutions, i.e TSP, NaCl, TSP +
NaCl, or water
2~
The TSP concentrations used was relatively high, being based
on those re~uired to kill suspended organisms in the presence
o~ high organic load, such as might exist at the skin sur~ace.
The results show synergy between TSP and osmotic shock
treatments. However, TSP and TSP/osmotic shock treatments did
not, in this case, appear to signi~icantly enhance sensitivity
to lysozyme, possibly due to carry over o~ TSP to the lysozyme
solution, which became alkaline.
The experiments were repeated at ambient temperature using
lower TSP concentrations (0.002 to O.OlM). In these
experiments the skin sur~ace was directly inoculated with test
organism rather than being immersed in culture. This was to
m; n; m; se the access o~ organisms to the underside o~ skin
samples, where they might be more easily occluded by ~atty
material. The results are shown in Tables ll and 12.
CA 02240930 1998-06-17
W O 97~3136 21 PCT/GB96/03173
Table 11. The cnmhined e~ect of low TSP cQncentration,
o~motiC shock and lvsozvme nn the survival of ~. coli attached
to ~hicken skin at room tem~erature
5 NaCl (M) TSP (M) pH* % Cell Survival
-lysozyme +lysozyme
none none 6.93100 44
0.8 none 6.4532 14
none 0.002 8.8342 14
none 0.005 10.34 50 7
none 0.01 11.08 44 9
15 0.8 0.002 9.1618 3
0.8 0.005 9.9025 5
0.8 0.01 10.58 25 7
* pH o~ initial treatment solutions, i.e TSP, NaCl, TSP +
NaCl, or water
T~hle 12. The c~h; n~ effect of TSP, osmotic shock and
lvsozvme on the s-~rvival of S. enteri tidis dried onto the
surface o~ chic~en skin
NaCl (M) TSP (M) pH* % Cell Survival
-lysozyme +lysozyme
30 none none 6.82100 87
0.8 none 6.29100 30
none 0.001 7.00100 57
none 0.002 8.56 87 85
35 none 0.005 10.3111 9
none 0.01 10.9027 7
0.8 0.001 6.7397 19
40 0.8 0.002 7.7417 9
0.8 0.005 9.90823 8
0.8 0.01 10.815 6
* pH o~ initial treatment solutions, i.e TSP, NaCl, TSP +
NaCl, or water
CA 02240930 1998-06-17
WO97~3136 22 PCT/GB96103173
Results (Table ll) show that at these low TSP concentrations,
there was synergy between TSP/lysozyme, TSP/osmotic shock and
TSP/osmotic shock/lysozyme treatments. The mA~;~llm cell kill,
following treatment with 0.002M TSP (pH approximately g) was
97%, comparable to that for cells inoculated onto the skin
surface and treated for lO min with 0. 4M TSP (pH 12 . 5 ) alone.
The pH of lysozyme treatment solutions was in all cases <7.
At the TSP concentrations used (up to O.OlM), the m~; mllm
kill o~ S.enteritidis cells attached to chicken skin was 95%
(Table 12 ) and there was some indication o~ synergy between
TSP/lysozyme and TSP/osmotic shock treatments. To test the
possible effect of TSP carry over on lysozyme activity in the
~inal washing solutions, the solutions were acidified using
2.5 or 0. 25mM lactic acid. At these concentrations, the pH
of lysozyme solutions, and controls without lysozyme,
decreased to approximately 5 . 0 (2 . 5mM lactic acid) and 6. 5
(0.25mM lactic acid). However, cell kills were not
increased, except following treatment with O.OlM TSP/NaCl. In
this case, cell kill rose from 94 to 99~, in samples
subse~uently treated with lysozyme in 2 . 5mM lactic acid. In
part, this ~nhAnced killing may be due to an increased pH
shock.
Lettuce was used as a contrasting surface to that of chicken.
Kills o$ attached E. coli clearly showed a marked synergY
between TSP and subse~uent lysozyme treatments, especially at
lower TSP concentrations (less than O.OlM). At higher
concentrations the effect oi lysozyme was less marked. The
results on lettuce leaves also showed a synergy between TSP
and osmotic shock treatments, especially at O.OlM TSP (the
highest concentration tested). MA~;m~] kills of E. coli on
lettuce were ~99% ~or both TSP/lysozyme and TSP/osmotic shock
combined treatments. Thus, in contrast to the results
observed using chicken skin, data for lettuce were much closer
to those obtained for suspended cells. This implies that the
process o~ attachment to a surface does not in itself provide
protection against the treatments used, but that the nature o~
the sur~ace is critical to survival. A single experiment was
also con~llcted using S. enteritidi~ attached to lettuce.
Results were ~ualitatively similar to those for E. coli,
though cell kills were generally less.
CA 02240930 l998-06-l7
WO97~3136 23 PCT/GB96/03173
~mrle 3: Killina e~ect o~ nisin
Experiments similar to those described above ~or lysozyme were
_ S also carried out using nisin or nisin plus lysozyme. Nisin
was obtained as a ~reeze-dried powder (2.5% nisin with NaCl
and denatured milk solids; Sigma). It was dissolved in water
and stored ~rozen (-70~C) as a stock solution (3mM). After
thawing, it was sterilised by membrane ~iltration and used at
concentrations in the range 0.114~M to 34.2~M.
The results are shown in Tables 13 to 15.
T~hle 13. The e~ect of nisin ~n~ TSP treatment on the
survival o~ C. ieiuni, ~. coli and P. fluorescens
Organism Temperature TSP % cell kill.
(~C) Concentration (with log
(mM) reduction)
C. Jejuni 37 0 98.0 (1.6)
0.5>99.9 (>5.8)
4 0 61.0 (0.7)
0.5 58.2 (0.4)
1.0 54.5 (0.3)
2.0 65.8 (0.5)
5.0 95.1 (1.3)
E. coli 37 0 72.0 (0.8)
0.5 98.1 (1.7)
2.0 98.6 (1.9)
5.0~99.9 (7.4)
E. coli 45 0 42.1 (0.5)
0,594.1 (>6.5)
P. 37 0 41.5 (0.5)
fluorescens 37 0.5 >99.9 (6.8)
1.0>99.9 (5.0)
ambient0.568.8 (0.5)
1.0 99.5 (2.3)
2.0 99.9 (6.4)
St. aureus 37 0 >99.9 (4.8)
1>99.9 (>6.5)
The results shown are ~or cells incubated in TSP at various
temperatures and diluted (1:10) in nisin solution (1.14~M) at
ambient temperature.
CA 02240930 l998-06-l7
WO97~3136 24 PCT/GB96/03173
T~hlç 14. The e~ect o~ nisi~ and TSP tr~Atm~nt on the
gllrvival of C. ieiuni, E. coli and S. enteri tidis attached to
chicken ~k; n .
S Organism TSP concentration ~ cell kill
(mM) (with log reduction)
- nisin + nisin
C. jejuni 5 87.8 (0.9) 99.1 (2.0)
89.0 (1.0) 95.6 (1.4)
E. coli 5 82.6 (0.8) 99.9 (3.6)
82.9 (0.8) 96.2 (1.4)
5* 18.2 ~0.1) 97.0 (1.5)
lS 10* 56.8 (0.4) 96.6 (1.5)
S. enteritidis 569.9 (0.5) 55.1 (0.4)
72.2 (0.6) 97.1 (1.6)
20 St. aureus O O>99 . 9 (3.1)
0~g9.9 (3.9)
0~99.9 (3.5)
* water rinse omitted
25 The results shown are for cells dried on the sur~ace o~ the
skin, incubated in TSP at 37~C, rinsed ~uickly (5sec) in water
and trans~erred to nisin solution (34.2~M) or distilled water
at 37~C.
CA 02240930 l998-06-l7
WO 97/23136 25 PCT/GB96/03173
Table 15. The effect of TSP and nisin ~lus lactic acid or
nisin ~lus lvsozvme treatments on the survival of ~.
f~nteritidis at~ached to t~ ckerl sk; n .
5 A
TSP concentration % cell kill
(mM)(with log reduction)
+ nisin + nisin + lysozyme
,10
59.8 (0.4) 87.5 (O.9)
96.5 (1.5) 93.8 (1.2)
15 B
TSP concentration % cell kill
(mM)~with log reduction)
lactic acid lactic acid + nisin
(pH5)* (pH5)*
Experiment 1
96.3 (1.4) 90.0 (l.O)
96.4 (1.5) 93.9 (1.2)
Experiment 2
99.2 (2.1) 99.3 (2.2)
99.4 (2.2) 98.0 (1.7)
* a~ter addition of chicken skin sample
The results shown are for cells dried on the suri~ace of skin,
incubated in TSP at 37~C, and either (A) rinsed quickly (5
35 sec) in water and transferred to nisin (3~1M) or nisin plus
lysozyme (lOO,ug ml ) at 37~C; or (B) trans~erred to nisin in
2.5mM lactic acid.
Table 13 illustrates the effect ol~ 1.14~1M nisin on suspended
40 cells previously treated with various concentrations of TSP.
The cells were: C. jejuni (4 and 37~C) E.coli (37~C) and
P. fluorescens (room temperature and 37~C). In experiments
~ conducted at 37~C, nisin caused high kills (up to 7 logs) oE
cells previously treated with only low TSP concentrations
45 (0.5-l.OmM, depending upon the organism). ~nder the
conditions used, nisin or TSP treatment did not cause
significant cell death when applied singly. In addition, at
higher TSP concentrations, where TSP treatment alone did cause
some cell death, a marked synergy between TSP and nisin
CA 02240930 1998-06-17
WO97~3136 26 PCT/GB96/03173
treatment was evident. Under the conditions used, nisin gave
high kills of St . aureus in both TSP-treated and untreated
cells (Table 13).
S Table 14 illustrates the results of experiments where E. coli
cells were dried on the outer surface of chicken skin. It can
be seen that nisin markedly enhanced killing at 37~C of cells
previously treated with O.Ol and 0.005M TSP; at 0.05M TSP,
kills were similar in the presence and absence of nisin (data
not shown). Cell killing by nisin following exposure to low
TSP concentrations was ~urther ~nh~nced by the introduction of
a ~uick rinse in water, after TSP treatment and ~mme~; ately
prior to immersion in nisin solution. Cell kills at 0.005M
TSP were consistently >99%. The high kills of E.coli obtA;ne~
using 0.005M TSP in combination with a further nisin treatment
were comparable to those seen at very high TSP concentrations
(0.4M) without nisin. Similarly high kills (99%) were also
observed for C. jejuni tTable 14). S. enteritidis was more
resistant, nevertheless, kills of up to 97% were observed
(Table 14). (Under the conditions used, nisin was highly
effective in k;ll;ng the gram positive bacterium St. aureus.
Even in the absence of prior treatment with TSP, kills were
>99.9~ (Table 14).
Table 15(B) shows the effect on killing of Salmonella when
lactic acid was incorporated into the nisin washing solution.
The lactic acid was added to ensure an acid pH ~approximately
5.0) which was near optimal ~or nisin activity. Cell kills in
the presence o~ nisin increased to 99%; however, o~ potential
interest, kills in the absence o~ nisin were similarly high,
suggesting that an increased pH shock was a significant cause
of cell death.
Table 15(A) shows the e~fect of lysozyme plus nisin ~ollowing
TSP and combined osmotic shock/TSP treatment~ on E. col i
attached to chicken skin as the test system. In experiments
conducted at 37~C, both nisin and lysozyme reduced the viable
count of TSP treated chicken skin by >90%. However, kills
were not further improved when skin was treated with nisin
plus lysozyme.
CA 02240930 l998-06-l7
WO97/23136 27 PC~/GBg6/03173
A similar conclusion was reached in experiments using combined
osmotic shock/TSP treatment prior to ni~in and/or lysoyme
treatment at both ambient and 37~C. Kills o~ up to 95% were
obt~;ne~; however, comparison of the data with that ~or
lysozyme or nisin alone, showed no increased killing effect by
the combination. It is possible that nisin and lysozyme may
act antagonistically or that they a~fect the same population
o~ cells.
In experiments using chicken, optimal combined treatments
involving nisin gave high cell kills. In these experiments it
was possible that cell kills were underestimated due to the
presence o~ naturally-ac~uired bacteria. Therefore, some
experiments were repeated using chicken portions sterilised by
y-irradiation. In addition, the e~ectiveness o~ a combined
TSP plus osmotic shock and nisin treatment was det~rm; n~,
i.e. chicken skin was exposed to TSP in the presence o~ NaCl
and subse~uently transferred to nisin solution. The results
are shown in Table 16.
Table 16. The ef~eGt of ~is;n, osmotic shock and TSP
treatment on survival o~ ~.coli, P. fl~orescens, S.
enteritidis, ~. monocvtoaenes and St. aureus attached to
previol~lv irradiated (sterilised) chicken skin.
Or~anlsm% cell kill (log reduction)
TSP + nisin ~1 + n; S; n TSP + NaCl +
~Li s i~
E. coli 97.4 (1.59) 85.8 (0.90) 99.4 (2.20)
P. fluorescens 99.1 (2.06) 99.4 (2.24) 99.4 (2.20)
5. enteritidis 98.5 (1.84) 96.6 (1.47) 99.1 (2.05)
L. monocytogenes >99.9 (> 5.0) 99.9 (3.38) >99.9 (~5.0)
St. aureus >99.9 (3.7) 99.9(>6.0) >99.9 (4.9)
The results shown are ~or cells dried on the sur~ace o~ the
skin, incubated in TSP (5mM), NaCl (0.8M) or TSP plus NaCl at
37~C, and trans~erred to nisin (33.3~M) solution. Where TSP
was used, a brie~ water rinse (5 sec) was applied ;mme~ately
prior to transfer to nisin solution.
For gram negative bacteria, kills det~rm~ n~ ~or TSP-nisin
treatment (>97%) were generally higher than those determined
in experiments using unsterilised chicken (c.~. Tables 14 and
16). High kills (>85%) were also observed ~or osmotic shock-
CA 02240930 1998-06-17
W O 97~3136 28 PCT/GB96/03173
nisin treatment (Table 16). However, the combined TSP plus
osmotic shock and nisin treatment was most effective (cell
kills >99%). Under the conditions used, this treatment also
reduced the viable count of L. moncytogenes and St. aureus
(gram positive species) by approximately 5 log cycles (Table
16).
