CA2240930A1 - Bacterial decontamination method - Google Patents
Bacterial decontamination method Download PDFInfo
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Abstract
Methods for the reduction of levels of gram negative and gram positive bacteria are disclosed which involve treatment with a solution of low concentration alkali metal orthophosphate combined with either osmotic shock and/or subsequently a lysozyme in solution and/or nisin in solution. The combination process is synergistic in extending the range of effective killing of bacteria and enables the use of more desirable processing parameters than the previous techniques and is particularly suitable for food processing.
Description
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 .= -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.
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 . , _ 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 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~
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.
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 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;
-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 CA 02240930 l998-06-l7 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 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 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 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 . , 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 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 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).
-~.x~mnle 2: ~; llin~ of cells adhered to food sur~aces The e~~ective kills achieved ~or gram negative organisms (E.
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 .= -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.
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 . , _ 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 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~
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.
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 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;
-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 CA 02240930 l998-06-l7 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 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 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 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 . , 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 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 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).
-~.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 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 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.
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 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 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 .
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) 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 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-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).
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 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 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.
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 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 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 .
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) 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 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-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).
Claims (33)
1. 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 further treatment which comprises one or more of the following steps:
a) subjecting the sample to osmotic shock, b) exposing the sample to an enzyme which breaks down peptidoglycan after said treatment with trialkali metal orthophoshphate; and c) exposing the sample to a bacteriocin after said treatment with trialkali metal orthophosphate.
a) subjecting the sample to osmotic shock, b) exposing the sample to an enzyme which breaks down peptidoglycan after said treatment with trialkali metal orthophoshphate; and c) exposing the sample to a bacteriocin after said treatment with trialkali metal orthophosphate.
2. A method according to claim 1 wherein the further treatment comprises step (a) and step (b) or step (c).
3. A method according to claim 1 or claim 2 which includes subjecting said sample to osmotic shock and said osmotic shock comprises a hypo-osmotic shock.
4. A method according to claim 3 wherein said hypo-osmotic shock is preceded by administration of a hyperosmotic shock.
5. A method according to Claim 4 wherein said osmotic shock treatment includes the steps of exposing the sample to a first solution having a water activity (aw) of 0.997 or less and subsequently exposing the sample to a solution of aw higher than that of said first solution.
6. A method according to claim 5 wherein the said first solution contains NaCl at a concentration sufficient to provide a water activity of 0.997 or less.
7. A method according to Claim 5 or claim 6 wherein said first solution contains 0.0005 to 0.2 M of a tri-alkali metal orthophosphate.
8. A method according to any one of the preceding claims which includes a step of exposing the sample to an enzyme which breaks down peptidoglycan, and said enzyme comprises lysozyme.
9. A method according to any of Claim 7 wherein the sample is treated with a solution of lysozyme at a concentration of at least lµgml-1.
10. A method according to Claim 8 wherein the lysozyme is provided in the form of a solution of freeze dried egg white which is at a concentration of at least 0.1mgml-1.
11. A method according to any one of the preceding claims wherein the sample is exposed to a bacteriocin and said bacteriocin is selected from nisin or pedocin.
12. A method according to claim 11 wherein the bacteriocin is nisin.
13. A method according to claim 12 wherein the sample is treated with a solution containing nisin of concentration of at least 0.1µM.
14. A method according to Claim 13 wherein prior to treatment with the solution containing nisin, the sample is rinsed with water.
15. A method according to any one of Claims 8 to 14 wherein the bacteriocin or enzyme solutions are acidified.
16. A method according to claim 15 wherein the solutions are acidified to a pH of approximately 5Ø
17. A method according to claim 15 or claim 16 wherein the solutions are acidified by addition of lactic acid.
18. A method according to claim 17 wherein the lactic acid is present at a concentration of at least 0.25mM.
19. A method according to any one of the preceding claims wherein the trialkali metal orthophposphate is trisodium phosphate.
20. A method according to any one of the preceding claims wherein the concentration of the trialkali metal orthophosphate solution is in the range of from 0.001 to 0.02M
21. A method according to claim 20 wherein the concentration of the trialkali metal orthophosphate solution is in the range of from 0.005 to 0.01M.
22. A method according to any one of the preceding claims wherein the sample comprises a consumable product or a surface.
23. A method according to claim 22 wherein the sample comprises a portion of a foodstuff.
24. A method according to any one of the preceding claims wherein a treatment step is effected by immersing the sample into a treatment solution.
25. A method according to any one of the preceding claims wherein a treatment step is effected by spray washing the sample with a treatment solution.
26. A method according to claim 1 wherein a sample is treated with a first solution comprising a trialkali metal orthophosphate at a concentration in the range of from 0.0005 to 0.2M, said solution having a water activity (aw) of 0.997 or less, and subsequently treating the sample with a second solution of an enzyme which breaks down peptidoglycan or a bacteriocin, said second solution having an aw higher than that of said first solution.
27. A method according to claim 25 wherein the sample is sequentially immersed in said first and second solutions.
28. A method for reducing the levels of gram negative and gram positive bacteria in a portion of a foodstuff comprising immersing the portion in a solution of trisodium phosphate at a concentration in the range 0.0005 to 0.2M, and sodium chloride at a concentration of about 0.8M, at a temperature in the range 4 to 50°C, and then spray washing with lysozyme solution.
29. A method for reducing the levels of gram negative and gram positive bacteria in a portion of a foodstuff comprising immersing the portion in a solution of trisodium phosphate at a concentration in the range 0.0005 to 0.2M, at a temperature in the range 4 to 50°C, and then spray washing with nisin at a concentration of greater than 1µM.
30. A kit for carrying out a method according to any one of the preceding claims, which comprises a trialkali metal orthophosphate and one or more of the following 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.
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.
31. A kit according to claim 30 wherein the components are in the form of aqueous solutions.
32. A product which has been treated by a method according to any one of claims 1 to 29.
33. A product according to claim 32 which comprises a foodstuff.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9526174.9 | 1995-12-21 | ||
| GBGB9526174.9A GB9526174D0 (en) | 1995-12-21 | 1995-12-21 | Bacterial decontamination of foods |
| PCT/GB1996/003173 WO1997023136A1 (en) | 1995-12-21 | 1996-12-20 | Bacterial decontamination method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2240930A1 true CA2240930A1 (en) | 1997-07-03 |
Family
ID=29404386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2240930 Abandoned CA2240930A1 (en) | 1995-12-21 | 1996-12-20 | Bacterial decontamination method |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2240930A1 (en) |
-
1996
- 1996-12-20 CA CA 2240930 patent/CA2240930A1/en not_active Abandoned
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