Received 19 June 2001 »CROBIALLY RESISTART COMPOSITIONS
This invention relates to microbially resistant compositions and methods for their preparation. It further relates to methods of use of such compositions and to products which are made microbially resistant due to the addition or application of such compositions, BACKGROUaD
Lactoperoxidase (LP) is an enzyme that is part of the natural non-immune defence systems in milk and mucous secretions (such as saliva, tears and intestinal secretions).
LP, together with various naturally occurring cofactors, foams the Lactoperoxidase system (LPS) that has pronounced anti-microbial activity.
The LP5 incorporates LP (extracted from bovine milk), a source of peroxide and a cofactor (generally thiocyanate). In many situations, glucose oxidase (from a microbial source) and glucose are incorporated to provide a source of hydrogen peroxide.
The use of an enzyme system is often preferred as this ensures that the delivery of peroxide is sustained. However this requires aerobic conditions so that in anaerobic conditions a direct source of peroxide may be required.
Reactions catalysed by LP and where the cofactor is thiocyanate yield short-lived intermediary oxidation products of thiocyanate that show the anti-microbial activity. LP
utilises peroxide to catalyse the oxidation of the thiocyanate ion in the presence of water to generate the hypothiocyanite ion (OSCN-). The hypothiocyanite then is believed to react with the sulphydryl groups on the bacterial membranes with catastrophic effects on the bacteria. Much of the thiocyanate is regenerated in the process.
The LPS is regarded as being bactericidal against Gram-negatives (eg. E. cola, Yersinia entercolitica, Pseudomnnas spp., Salmonella spp, Campylobacter spp) and bacteriostatic against Gram-positive bacteria (Listeria monocytogenes, 5'taphylococcus aureus, Streptococcus spp). The LPS is also suggested to have anti-viral activity in some situations.
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CA 02373485 2001-11-09 PCTlNZ00/00074 Received 19 June 2001 WO 00/69267 ---.
The anti-microbial effects of a number of fatty acids are also well-documented. The most active are the medium chain fatty acids lauric (dodecanoic) acid and myristic (tetradecanoicj acid. The fatty acids are regarded as especially effective against the Gram-positive bacteria and the fatty acid derivative monolaurin (1-monododecanyol rac-glycerol) is generally regarded as the most active. In addition, anti-viral activity (against the enveloped viruses] has been claimed for the fatty acid derivatives, including sodium dodecyl sulphate.
Monolaurin has GRAS status as an emulsifying agent and is used mostly in vegetable shortenings and to some extent in ice creams and baked goods.
Monolaurin is marketed as Lauricidin tE for use as an anti-microbial in food systems.
Howevez, it has not found wide acceptance as an anti-microbial because of the concentrations required and the resultant e$'ects on organoleptic quality of the treated food products.
The applicants have now found that the LPS and C,z fatty acid derivatives and salts thereof, such as monolaurin, can be used in combination and that, when combined, t~.he anti-microbial effect exceeds that which could have been preaicted based upon the known properties of the components. It is therefore broadly upon this unexpected finding of enhanced anti-microbial effect or syne:gistic interaction which the present invention is based.
SUMMARY OF THE INVENTION
Accordingly, in a first aspect, the present invention provides a method of preparing a microbially resistant composition which comprises forming a mixture of the following components:
(a] a lactoperoxidase system comprising:
(ij a lactoperoxidase (ii] a source of peroxide; and 2 _ ~Y~vtriv,::~:~.,n:~' ;,j~"~9 ~~~.~a.~U
Received 19 June 2001 (iii) a cofactor which is capable of yielding anti_microbial oxidation products; and (b) a Ciz fatty acid derivative or a salt thereof, wherein the Ci2 fatty acid derivative or the salt thereof is present in an amount effective to interact with the lactoperoxidase system to product an enhanced anti-microbial effect.
The term "enhanced anti-microbial effectp means an anti-microbial e$'ect yP~~
is more ZO microbiocidal against at least one type of microorganism than would be predicted from the known properties of the individual components.
As used herein, the term microorganism" means microbial pathogens, ineffective particles and spoilage organisms, including those of bacterial, viral, fungal or protozoal origin.
Conveniently, the peroxide is hydrogen peroxide.
preferably, the cofactor is selected from thiocyanate or iodide, and is most preferably thiocvanate.
Preferably, component (b) is or includes monolaurin (1-monododecanoyl-rac-glycerol) or 2~ sodium dodecyl sulphate.
In the currently most preferred embodiment, component (b) is monolaurin.
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Received 19 June 2001 WO OO1b9267 Conveniently, the Ci2 fatty acid derivative or the salt thereof is present in an amount which is at least 5% by weight of the total lipid present in the composition.
Preferably, said composition is formed by the addition of one or more of the components to a food product which already contains the remaining component(s).
The food product may be a dietary supplement or nutraceutical. It may also be a dairy product, meat product or fish product.
The food product may also be an animal feed, which in its simplest form may be water.
I5 Alternatively, said composition, when formed, consists of said components in admixture.
In a further aspect, the invention provides a preparative composition suitable for use in preparing a microbially resistant composition which comprises at least two components selected from:
(i) a lactope~-oxidase;
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(iii) a cofactor which. is capable of yielding anti-microbial oxidation products; and (iv) a C12 fatty acid derivative or a salt thereof, wherein the lactoperoxidase and peroxide source, if both present, are kept separate.
In yet a further aspect, the invention provides a preparative pack suitable for use in preparing a microbially resistant composition which comprises, in separate containers, a lactoperoxidase and a Cm fatty acid derivative or a salt thereof.
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Preferably, said pack further includes a source of peroxide and/or a cofactor avhich is capable of yielding anti-microbial oxidation products. Where provided, the source of peroxide and/or said cofactor are in separate containers.
In yet a further embodiment, the invention provides an anti-microbial compositic.;n which comprises a lactoperoxidase, a cofactor which is capable of yielding anti-microbial oxidation products and a Ciz fatty acid derivative or a salt thereof, wherein the Ci2 fatted acid derivative or the salt thereof is present in an amount effective to synergistically interact with said lactoperoxidase and said cofactor, in the presence of peroxide, to produce an enhanced anti-microbial effect.
Preferably, said anti-microbial composition further includes a source of peroxide.
The composition may also include a further anti-microbial agent or a chelating agent.
Pr eferably, the further anti-microbial agent is selected from phenols, organic acids, bacteriocins, derivatives of these and mixtures of these, or anti-microbial components or mixtures of components extracted from or found in milk, such as lactoferrin.
Preferably, the cheiating agent is EDTA.
In yet a further embodiment, the invention provides a microbially resistant food product which is prepared by a method as defined above.
In still a further aspect, the invention provides a food product which includes the following components:
(i) a lactoperoxidase;
(iij a source of peroxide;
(iii) a cofactor tvhich is capable ofyielding anti-microbial oxidation products; and (iv; . watt ~w:,~ :~r-:vative or a salt thereo.
which product is resistant to the growth of microorganisms.
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Received 19 June 2001 Preferably, said product is resistant to the growth of both Gram positive and Gram negative bacteria.
In one form, said food product is a dietary supplement, nutraceutical dairy product. meat product, fish product or animal feed.
.r a final as~eet. she invention provides a method of treating a food product for the purp«sr _.:. -.-v ceT.~_~ ~°,.;: produce microbially resistant which comprises the step o~," adri°~~~- t>
said product an effective amount of an anti-microbial composition as defined aoove.
I3EStrRIP~ION OF SAE IaR~IWIN~S
lahiie the invention is broadly as defined above, it will also be appreciated that it i~-~cludes embodiments of which the following description provides examples.
Furt:~ermore, a better understanding of the invention will be gained through reference to the accompanying drawings in which:
Figure t is a graph showing the e$'ect of monolaurin concentration on the growth of bacteria in a broth culture inoculated with S. aureus at a rate of 8 x 106 cfu per ml ;equivalent to a 1°'o inoculum of the spec~:ally prepared stock culture;. The Optical ~eraity f,OD) measured at o00 nm was used as an index of bacterial cell numbers.
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1 d ~;:,at,-_ WO 00/69267 PC~~ June 2001 Figure 2 is a graph showing the inlubition of the growth of bacteria by various compositions in a broth culture inoculated with E. coti at a rate of 1.5 x I06 cfu per ml (equivalent to a 0.05% inoculum of the specially prepared stock culture).
S Figure 3 is a graph showing the inhibition of the growth of bacteria by various compositions in a broth culture inoculated with S. aureus at a rate of 4 x 105 cfu per ml (equivalent to a 0.05% inoculum of the specially prepared stock culture).
Figure 4 is a graph showing the inhibition of the growth of bacteria by various compositions in a broth culture inoculated with S. aureus at a rate of 8 x 106 cfu per ml (equivalent to a I% inoculum of the specially prepared stock culture).
Figure S is a graph showing the inhibition of the growth of bacteria by various compositions in a broth culture inoculated with L. monocytogenes at a rate of 1.5 x i06 cfu per ml (equivalent to a 0.05% inoculum of the specially prepared stock culture).
Figure 6 is a graph showing the inhibition of the growth of bacteria by various compositions in a broth culture inoculated with L. monocytogenes at a rate of 3 x I0~
ciu per ml (equivalent to a 1% inocuium of the specially prepared stock culture).
L1ESCItIPTiON OF THE INVENTION
As broadly defined above, the primary focus of the present invention is on microbially _wsistant compositions. Such compositions exert an anti-mic:~obiai epee:
*..harough the 2~ syergistic combination of the LPS and a Cm fatty acid derivative or a salt thereof.
The $i.rprising finding made by the applicants is that the anti-microbial efficacy of the LPS can be supplemented markedly by addition of a Cm fatty acid derivative or a salt thPrPnf 3p :~ :rzprovement in anti-microbial efficacy is enhanced or synergistic in character. '1'luis synergism is particularly evident against Gram-negative microorganisms such as E. coll.
The LPS requires three components. These are in turn a lactoperoxidase, a source of peroxide 3~ and a cofactor. 'The cofactor is one which yields intermediary anti-microbial oxidation P~~~°.,,t;~,; E
Received 19 June 2001 WO Ua/69Z67 products. Examples of suitable cofactors include thiocyanate and halides (particularly iodide).
The lactoperoxidase itself can be any of those which are commercially available. GRAS status lactoperoxidases being particularly preferred.
The peroxide can be directly added (for example as hydrogen peroxide) or can be the product of enzymic digestion of an appropriate substrate. For example, a combination of glucose oxidase and glucose can provide the source of hydrogen peroxide.
Sodium percarbonate-based systems can also be used.
The components of the LPS will be provided in art standard amounts. For example, where the peroxide source is glucose/glucose oxidase and the cofactor is thiocyanate, the components can be included in a liquid medium in the following amounts (mg per litre):
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LP 6.85 Glucose oxidase 3.17 Glucose 31.7 SCN- 24.0 For a solid substrate, the same components can be included, for example, in the follouring amounts (mg per kg):
Glucose oxidase 9.5 Glucose 31.7 SCN- 200.
3f The use of Ci2 fatty acid derivatives includes the use of esters of fatty acids or their salts.
Cre particular ester which the applicants found to be used is monolaurin (Lauricidin l~).
Sodium dodecyl sulphate can also be used as a suitable derivative.
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~'u~ .':-' Received 19 June 2001 It is also to be emphasised that the fatty acid derivative or the salt thereof need not be in S pure form. The useful fatty acid derivative or salt thereof can be included in a mixture such as an extract of bovine milk fat or coconut oil in which the lipid has been treated to ensure that the requisite proportion of anti-microbial components are present.
In order for the fatty acid derivative or salt thereof to induce synergism with the LPS; the applicants have found that a synergistically effective amount of the fatty acid derivative or the salt thereof must be present. This amount is greater than the levels at which anti-microbial fatty acids are present in standard bovine milk (in which the lipid is predominantly in the form of triglycerides), and reflects the applicants finding that when the LPS
plus fatty acid derivative or salt thereof is applied to milk, significantly enhanced and effective anti-microbial result is achieved.
Specifically, the applicants have determined that where the synergistic anti-microbial effect is to be produced in a composition which contains lipid, the fatty acid derivative or the salt thereof must be present in an amount which is at least 5% by weight of the total lipid in the composition.
The invention will now be illustrated with reference to the following non-limiting experimental section.
EXPER.TMENTAL
SECTION A
Materials Bacterial strains, media ar~.d chemicals L. monocytogenes strain L45 and S. aureus strain R3? were obtained from Dr Roger Cook, the Meat Industries Research Institute of New Zealand strain culture collection and E. coli 0157:57 strain NCTC 12900 was obtained from Dr Heather Brooks, the Department of Microbiology, University of Otago strain culture collection.
Stock cultures of all strains were stored in skim milk at -70~C and when required were E'~" ~;i . ~' - .....;.
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subcultured onto Plate Count Agar (PCA) (Difco Laboratories, Detroit, Michigan, USA) or blood agar (BA) (Columbia Agar Base (GIBCO BRL, Life Tech Ltd, Paisly UK) supplemented with 5% whole human blood (Dunedin Public Hospital, Dunedin, NZ)).
Strains in regular use were maintained as plate cultures and subcultured every two weeks. All commercial media were prepared according to the manufacturer's spec~cations. Monolaurin (1-monolauroyl-rac-glycerol, Sigma Chemical Co., St Louis, MO, USA) was prepared by dissolving 1g in 10 mL ethanol, dispensing in 1 mL
volumes and storing at -20~C until required. All LPS components were filter sterilized. Glucose (Sigma) was prepared by dissolving 18.016 g glucose (Sigma) in 100 ml of MilliQ water LO dispensing in 3 mL volumes and stored at room temperature (RT) until required.
Glucose oxidase (GOX) and glucose oxidase were sourced from Sigma;
lactoperoxidase was sourced from Tatua Biologics, Morrinsville, NZ; and the sodium or potassium thiocyanate was sourced from Bio Serae SA Limited, Montolieu, France.
Methods Test system Growth experiments were conducted in 100 x 15 mm screw capped glass test tubes containing 8 mL Todd-Hewitt broth (THB). As required, monolaurin was added to each tube prior to autoclaving of tubes at 121~C for 15 min. As required, components were added to autoclaved tubes in the following order, cell inoculum, 16 LPX stock, glucose stock, thiocyanate stock and glucose oxidase stock. In all cases tubes were inoculated with 0.05% and 1.0% (v/v) of an overnight THB culture of the appropriate bacterial strain. The tubes were incubated at 37~C for 48 hours and their OD6ooam read by use of a spectrophotometer (Spectronic 20D+, Milton Roy Company, USA) at intervals as appropriate. Viable counts of each bacterial strain were determined by dilution in saline of five overnight cultures of each strain and plating of each dilution onto PCA by use of a spiral plating machine (Spiral Systems, Cincinnati, USA).
The concentration (mg/litre) of the components of the LPS in the culture solution was as follows: 6.85 for lactoperoxidase, 3.17 for glucose oxidase, 31.7 for glucose and 29.0 for thiocyanate.
Results The test system The viable count of overnight broths for each test strain used in the experiments was 3 x 109, 8 x 108 and 3 x 109 cfu per ml-for E coli 0157:57 strain NCTC 12900 ("E. coh"), S
aureus strain R37 ("S. aureus") and L monocytogenes strain L45 ("L.
monocytogenes") respectively.
The degree of inhibition of monolaurin against both Gram-positive strains appeared to be proportional to the concentration of monolaurin used, as is illustrated by the results shown for a 1% inoculum of S. aureus grown in the presence of 50 - 150 ppm monolaurin (Figure 1). For both monolaurin and LPS, the degree of inhibition observed was inversely proportional to the bacterial load imposed on the system; that is, the greater the starting inoculum the lesser the degree of inhibition observed.
For both monolaurin and LPS, L. monocytogenes was the strain most sensitive to inhibition and E. coli the strain least sensitive to inhibition.
Effectiveness against E. coli Monolaurin did not inhibit the growth of E. coli when tubes were inoculated at either 0.05% or 1.0%. The LPS slightly inhibited the growth of E. coli when tubes were inoculated at 0.05% but not when inoculated at 1.0%. Growth of the E. coli was strongly inhibited by combinations of the monolaurin and LPS (Figure 2).
Effectiveness against S. aureus Growth of S. aureus inoculated at 0.05% (Figure 3) was completely inhibited by monolaurin at concentrations of 100 and 500 ppm. These cultures were also strongly inhibited by LPS, with the time of culture stationary phase being extended for approximately 20 hours beyond cultures containing no LPS. No growth was observed in tubes containing both monolaurin and LPS. Growth of S. aureus inoculated at 1.0%
(Figure 4) was completely inhibited by monolaurin at a concentration of 500 ppm but was only partially inhibited by monolaurin at a concentration of 100 ppm. In the case of the 1% inoculum with 100 ppm monolaurin cultures, the stationary phase was extended for approximately 10 hours beyond that of the control tubes and these cultures never reached densities comparable to those of the control tubes.
These cultures were strongly inhibited by LPS, with the stationary phase being extended for approximately 13 hours beyond that of the tubes containing no LPS.
Significantly, the only growth seen in tubes containing both monolaurin and LPS was in the 100 ppm monolaurin + LPS system where the last time point at 48 hours showed a slight but statistically sign~cant increase in turbidity.
Effectiveness against L. monocytogenes L. monocytogenes was completely inhibited at all inoculum densities by all systems containing monolaurin at either 100 or 500 ppm (Figures 5 & 6). In the case of the 1%
inoculum with LPS cultures, the stationary phase was extended for approximately 22 hours beyond that of the uninhibited control tubes. In the case of the 0.05%
inoculum with LPS cultures, stationary phase was extended for approximately 27 hours beyond that of the uninhibited control tubes and these cultures never reached densities comparable to those of the control tubes.
Discussion The three strains of bacteria used in this study were chosen because of their status as food-borne pathogens and the range of their reported sensitivities to monolaurin. It was not surprising that growth of the E. coli 0157:H7 strain used in this study was not affected by monolaurin at concentrations of up to 500 ppm, as Kabara et al reported growth of E. coli to be unaffected by monolaurin concentrations of greater than 1000 ppm (Kabara et al, 1977). By contrast, the growth of L. monocytogenes has been reported to be inhibited by relatively modest concentrations of monolaurin, with concentrations as low as 5 ppm having been reported to delay lag phase growth in broth cultures by 8 hours (Wang et al, 1977). Although growth of the strain of L.
monocytogenes used in this study was not affected by monolaurin at a concentration of 10 ppm, it was completely inhibited by monolaurin at concentrations of 100 and ppm. S. aureus is a bacterium with an intermediate sensitivity to inhibition of growth by monolaurin as it has been reported to be sensitive to inhibition at concentrations of about 200 ppm (Kabara et al, 1977), a result which compares well with the sensitivity of the strain used in this study.
The LPS has been reported to inhibit the growth of a wide range of bacteria including E.
coli, S. aureus and L. monocytogenes (Wolfson et al, 1993). Unfortunately, the wide range of component (glucose, H202, GOX, LPX & SCN-) concentrations, incubation media, and incubation temperatures used in these studies, make direct comparisons with the present study difficult. Generally though, it appears that the degree of relative sensitivity of the strains to LPS alone seen in this study is consistent with that reported by others (Gaya et al, 1991; Kamau et al, 1990; Bjork et al, 1975; Siragu et al, 1989).
Despite the bactericidal nature of the action of LPS against E. coli, the degree of inhibition of growth of the 0157:H7 strain used in this study was considerably less than that seen against the Gram-positive species. It was also considerably less than that seen against E. coli DHSoc, a common laboratory strain (Simmonds &
Kennedy, unpublished data). Large variations in the sensitivity of E. coli strains to LPS has been noted by others, with Grieve et al reporting 6 hour reductions in viable count for different enterotoxigenic strains ranging between 3.6 and 7.3 log units (Grieve et al, 1992).
The results reported above provide strong evidence that the combination of monolaurin and LPS, has great potential for use as a preservation system. There was no inhibition of E. coli 0157:H7 by monolaurin used alone and we are not aware of any reported degree of sensitivity of any E. coli strain to monolaurin alone used at any concentration.
Thus, the synergistic effect of the monolaurin + LPS combinations against E.
coli 0157:H7 (inhibition far in excess of that expected from LPS alone) was unexpected. By contrast, the inhibitory effect of the monolaurin + LPS combinations against S. aureus was not as surprising on the basis of its significant inhibition by each agent used alone.
One unexpected result however, was the degree of inhibition of S. aureus obtained in the combined systems. One problem with the use of monolaurin as a food preservative has always been that the quantity of monolaurin required to obtain the desired level of inhibition is such that it may make the process uneconomic, or result in the development of undesirable organoleptic properties (texture, flavour) in the food. The results reported above indicate that monolaurin + LPS combinations will be effective at concentrations of monolaurin much lower than those required to achieve an equivalent inhibitory effect by use of monolaurin alone.
SECTION B
This section further illustrates aspects of the invention.
A broth culture system (Todd-Hewitt Broth, THB) inoculated with either S.
aureus R37 or E. coli 0157:H7 (-vt) was used as a screening system to evaluate different lipid components. The strains were cultured in THB overnight at 37 °C and dispensed to tubes. The initial loadings were 1 x 105 per ml for both the S. aureus and the E. coli.
The components of the lactoperoxidase system, LPS, were present at concentrations of:
20 mg/litre of lactoperoxidase (c.3000 Units activity/litre), glucose oxidase (c.300 Units activity/1), 290 mg/1 of thiocyanate ion (496 mg/1 of NaSCN) and 12000 mg/1 of glucose. The growth of the organisms was then followed by measuring the increase in absorbance (at 600nm) of the broth regularly over 48 hours using a spectrophotometer (Spectronic 20D+) and attached data logger.
A number of lipid components (fatty acids and monoesters) exhibit anti-microbial effects when combined with the lactoperoxidase system as evidenced in the experiments summarised in Table 1. Monolaurin was the most effective lipid component against S. aureus and in this simple system, it was just as effective whether or not the LPS was included. The Table 1 data indicate that synergy is apparent for both the Gram positive S. aureus and the Gram negative E. coli but the effect is greater with the S. aureus. The effect of monopalmitoleate was very similar to that of monolaurin against E. coli alone or in the presence of the LPS. However sodium lauryl sulphate was very effective against E. coli, whether or not the LPS was present.
Table 1: Inhibition of bacterial growth in broth culture by a combination of a lipid component and the lactoperoxidase system. The data are expressed as the concentration of the lipid component when bacterial growth is inhibited by about 50%
or 100% after 48 hours; eg. 100/250 means that growth is about 50% inhibited at 100ppm and completely inhibited at 250ppm of the lipid component; the *
indicates that the organism was not 100% inhibited at any concentration up to 1000ppm.
S. aureus E, cola R37 0157:H7 Lipid component Lipid only Lipid + Lipid onlyLipid +
LPS LPS
Monolaurin <50/50 <50/50 100/* 50/*
Lauric acid 250/ 500 50/ 100 500/* 50/ 1000 Sodium lauryl sulphate500/* 250/* 100/250 100/250 Caprylic acid (C8:0)500/* 100/ 1000 500/* 500/ 1000 Palmitoleic acid 50/250 <50/50 250/* 50/*
(C16:1) Monopalmitoleate 500/* <50/50 50/* 50/*
Tables 2 and 3 present the results of experiments in which milk and mince were inoculated with S. aureus R37. The synergistic effects of the LPS/monolaurin combination are apparent.
Table 2: Evaluation of the monolaurin ( 1000ppm) + lactoperoxidase (LPS with 20mg per litre of lactoperoxidase) system in milk (S. aureus R37 at 37~C; cfu per ml).
Milk with Experiment Experiment Treatment effect S. aureus R3 O 5 h 24 h 5 h 24 h compared with hours Control Control 7 x 1 x 1 x 5 x 1 x Control 104 108 108 10~ 108 Monolaurin 7 x 2 x 9 x 1 x 9 x 3 log @ 5h;
104 105 108 104 108 nil @
24h LPS 7 x 5 x nil nil 2 x 4 log to complete kill @ 5h &
24h Monolaurin 7 x Nil nil nil nil Complete kill + LPS 104 @ 5h & 24h Table 3: Evaluation of the monolaurin ( 1000ppm) + lactoperoxidase (LPS with 200mg per kg of lactoperoxidase) system in mince (S. aureus R37 at 37~C; cfu per g).
Mince with Experiment Treatment effect S. aureus R37 O hours 5 hours 24 hours compared with Control Control 2 x 105 3 x 108 7 x 108 Control Monolaurin 8 x 104 2 x 108 9 x 108 Nil @ 5h & 24h LPS 9 x 104 3 x 105 1 x 103 3 log @ 5h & >5 log @ 24h Monolaurin + 9 x 104 4 x 103 2 x 102 5 log @ 5h & >6 log LPS ~ @ 24h Tables 4 and 5 present the results of experiments in which milk was inoculated with E.
coli 0157:H7 (a Gram negative organism) or S. aureus R37 (a Gram positive organism).
Table 4: Evaluation of the monolaurin + lactoperoxidase system in milk against a Gram negative organism (E. coli 0157:H7, at 12~C; cfu per ml). The ratio of LPX to GOX
(Units of enzyme activity) was 9:1, with the thiocyanate ion and glucose each present at l2mg/1.
Milk with O 1 day 2 days 3 days Treatment effect E. coli 0157:H7 compared with Control Control 2 x 1 x 1 x 1 x Control 105 10~ 109 109 Monolaunn (500 2 x 2 x 3 x 2 x 1 log @ 1 & 2d;
ppmJ 105 106 108 109 nil @ 3d LPS (50 mg LPX/litre)2 x 4 x 7 x 7 x 2-3 log @ 1d; 1-2 105 104 10~ 10~ log @ 2 & 3d Monolaurin (500)+2 x 3 x 2 x 3 x 2-3 log @ 1d; 5 LPS (50) 105 104 104 106 log @ 2d;
2-3 log @ 3d Table 5: Evaluation of the monolaurin + lactoperoxidase system in milk against a Gram positive organism (S. aureus R37 at 37°C; cfu per ml; component concentrations as for Table 4).
Milk with O 5 hours 24 hours Treatment effect S. aureus R37 hours compared with Control Control 9 x 1 x 108 3 x 109 Control Monolaurin (1000 ppm)1 x 4 x 106 2 x 109 1-2 log @ 5h; nil 105 @ 24h LPS (5 mg LPX) 1 x 6 x 10~ 2 x 109 nil @ 5 & 24h LPS (50 mg LPXJ 1 x 1 x 108 1 x 109 nil @ 5 & 24h Monolaurin (1000)+ 9 x 4 x 104 1 x 109 3-4 log @ 5h; nil LPS (5J 104 @ 24h Monolaurin (1000)+ 9 x 9 x 104 2 x 109 3 log @ 5h; nil LPS (50) 104 @ 24h Again, the efficacy of the LPS/monolaurin combination is apparent.
Table 6 presents the results of two experiments that show in certain circumstances, the presence of the milk itself actually has an inhibitory effect on the efficacy of the monolaurin treatment. That is, the anti-microbial effect of the monolaurin is adversely affected by the normal levels of lipid (generally in the form of triglycerides) naturally present in milk. Similarly, the efficacy of the combination of monolaurin plus the LPS
is also affected by the concentration of milk (and hence the lipid) present in the culture medium (Table 7). However the effect of the (competing) lipid content is less with the combination of monolaurin + LPS than with monolaurin alone; in other words, the synergistic effect is greater.
Table 6: Comparison of different levels of milk (re lipid) on the efficacy of the monolaurin system against S. aureus R37 (cfu per ml) in a Todd-Hewitt Broth (THB)/milk mixture at 37°C.
Treatment Microbial Effect of monolaurin count O hours6 hours treatment on S.
aureus 100% THB [Control, lOml THB 1 x 3 x 10~ Control only) 105 100% THB + 500ppm Monolaurin 1 x nil 7 log @ 6h 25% milk/75% THB 1 x 5 x 10~ Control 25% milk/75% THB + Monolaurin 1 x 5 x 103 4 log @ 6h Treatment Microbial Effect of monolaurin count O hours6 hours treatment oa S.
aureus 50% milk/ 50% THB 1 x 7 x 10~ Control 50% milk/50% THB + Monolaurin 1 x 2 x 103 4-5 log @ 6h 75% milk/25% THB 1 x 8 x 10~ Control 75% milk/25% THB + Monolaurin 1 x 3 x 106 1-2 log @ 6h 100% milk 1 x 4 x 10~ Control 100% milk + Monolaurin 1 x 1 x 10~ nil @ 6h Table 7: Comparison of different levels of milk (ie lipid) on the efficacy of the monolaurin + lactoperoxidase system (with 5 ppm of LPX) against S. aureus R37 (cfu per ml) in a THB/milk mixture at 37°C.
Treatment Microbial Effect of count O hours 6 hours monolaurin + LPS
treatment 100% THB (Control, lOml THB 1 x 105 2 x 10~ Control only) 100% THB + 500ppm Monolaurin 1 x 105 nil 7 log @ 6h + LPS
25% milk/75% THB 1 x 105 3 x 10~ Control 25% milk/75% THB + Monolaurin 1 x 105 nil 7 log @ 6h + LPS
50% milk/ 50% THB 1 x 105 5 x 10~ Control 50% milk/50% THB + Monolaurin 1 x 105 3 x 103 4 log @ 6h + LPS
75% milk/25% THB 1 x 105 8 x 10~ Control ?5% milk/25% THB + Monolaurin 1 x 105 8 x 103 4 log @ 6h + LPS
100% milk 1 x 105 6 x 10~ Control 100% milk + Monolaurin + LPS 1 x 105 4 x 104 3 log @ 6h The experimental results reported in Tables 8 and 9 further demonstrate the synergistic efficacy of the LPS/monolaurin combination.
Table 8: The synergistic effect of the components through comparison of the effect of the efficacy of the LPS or monolaurin systems alone and the monolaurin + LPS
against the Gram-positive bacteria, S. aureus R37 in milk at 37°C.
Treatment Effect Monolaurin @ 1000 of treatment @
5h (reduction in microbial count, cfu per ml) ppm & LPS (with Synergistic LPX LPS Monola Monolaurin effect @ + 5 or 50 ppm) alone urin + of the LPS the combination compared with alone LPS Monolaurin Expt 67 (from Table alone alone 5) 1. 100% milk + Nil 1-2 log 3-4 log 3-4 2 log LPX log 1. 100% milk + Nil 1-2 log 3 log 3 log 1-2 log Table 9: The synergistic effect of the components through comparison of the effect of different levels of milk (ie fat) on the efficacy of the monolaurin system alone and the monolaurin + LPS against the Gram-positive bacteria, S. aureus R37 at 37°C.
Treatment Effect of treatment @ 6h (reduction in microbial count, cfu per ml) Monolaurin @ 500 ppm Monolaurin Monolaurin Synergistic (& LPX @ + 5 ppm) alone + the LPS effect Expt 72 & 77 (from Tables compared 6 & 7) with monolauria alone 2. 100% THB (Control, lOml Control Control NA
THB
only) 3. 100% THB + Monolaurin 7 log 7 log Nil (+LPS) 4. 25% milk/75% THB + Monolaurin4log 7log 3 log (+ LPS) 5. 50% milk/50% THB + Monolaurin4log 4 log Nil (+ LPS) 6. 75% milk/25% THB + Monolaurin1-2 log 4 log 2-3 log (+ LPS) 7. 100% milk + Monolaurin Nil 3 log 3 log (+ LPS) As summarised in Tables 8 and 9, LPS alone had no inhibitory effect on against S. aureus R37. Monolaurin alone at 1000 ppm had a small effect ( 1-2 log) as reported in Table 8 but had no effect at the lower level of 500 ppm as reported in Table 9. The synergistic effect of the combination of monolaurin and the LPS (compared with monolaurin alone or the LPS alone) however is clearly evident. The extent of the synergy is affected by both the presence of and by the level of lipid. In the presence of lipid, the anti-microbial efficacy of the combined composition is much enhanced compared with the monolaurin alone. However, the efficacy of the anti-microbial composition is influenced by the proportion of lipid present.
Table 10 presents the results of an experiment in which the effect of the concentration of monolaurin alone on S. aureus in THB was evaluated. In this experiment, a concentration of only 25 or 50ppm was required to have a significant effect on the population of S. aureus. The efficacy of the 50ppm monolaurin treatment was equivalent to that achieved with 500ppm in Table 6. As the experiment reported in Table 10 was conducted in a fat-free medium, the comparison provides further evidence of the compromising effect of the presence of other lipids on the efficacy of the monolaurin.
Table 10: Comparison of different levels of monolaurin on the population of S.
aureus R37 in THB (cfu per ml) at 37~C.
Treatment O hours 6 hours 24 hours Effect of monolaurin treatment 8. Control THB only 3 x 104 4 x 10~ 2 x 109 Control 9. THB + lppm 3 x 104 2 x 10~ 2 x 109 Nil @ 6 & 24h M onolaurin 10. THB + 5ppm 3 x 104 1 x 10~ 2 x 109 Nil @ 6 & 24h Monolaurin 11. THB + lOppm 3 x 104 8 x 106 1 x 109 Nil @ 6 & 24h M onolaurin 12. THB + 25ppm 3 x 104 5 x 101 2 x 106 6 log @ 6h & 3 log@
Monolaurin 24h 13. THB + 50ppm 3 x 104 nil 1 x 106 Complete kill @ 6h &
Monolaurin 3 log@ 24h With reference to Tables 6 to 10, a solution of 100% milk contains around 3%
milk lipid. Monolaurin at a concentration of 500 ppm represents 0.05 grams per 100m1 (0.05%). From the above it is clear that a certain threshold concentration of selected anti-microbial lipid components (expressed as a percentage of the total lipid) must be exceeded in order to ensure a sign~cant anti-microbial effect of the total composition.
Table 11 provides a summary of the relevant data classed according to the level of added anti-microbial lipid (in this case monolaurin) both in the actual amount present and as a proportion of the total lipid. The data in Table 11 indicate that 3.2%
monolaurin was marginal in that the effect was variable and ranged from a 1 log to a 4 log reduction in microbial count at 5 or 6 hours for monolaurin alone and a 3 log to 8 log reduction for monolaurin + LPS.
Table 11, Summary of data from Tables 2, 8, 9 & 10: Evidence for the effectiveness of, and the synergistic effect of, the components of the anti-microbial composition as affected by the quantity of monolaurin present and the proportion of the total lipid present as monolaurin (reduction in population of S. aureus after 5 or 6 hours at 37°C).
Treatment Monolaurin as a Effect of Effect of Synergistic No ex Tableproportion and as monolaurin monolaurin effect a + LPS
percentage of total lipid 11, Table 0.001/0.001 100% Nil Not done ND
10 of (ND) total lipid 13, Table 0.005/0.005 100% 7 log ND ND
3, Table 0.05/0.05 100% 7 log 7 log Nil 4, Table 0.05/0.8 6.3% 4 log 7 log 3 log ex Table 0.10 / 3.1 3.2% 3 log 8 log 5 log 1, Table 0.10/3.1 3.2% >1 log 3 log 2 log 5, Table 0.05/ 1.6 3.2% 4 log 4 log Nil 6, Table 0.05/2.3 2.2% > 1 log 4 log 3 log It is therefore the applicants view that the concentration of the selected anti-microbial lipid components must be equal to or greater than 5% of the total lipid present in order to include the synergistic anti-microbial effect.
In the case of bovine milk the free fatty acids with anti-microbial properties (or their derivatives) must therefore constitute more than 5% of the total lipids present for the milk to be transformed into anti-microbial composition to achieve a substantial and consistent anti-microbial effect. Such a composition will be effective against both Gram positive (as exemplified by S. aureus) and Gram negative organisms (as exemplified by E. coli, see Table 5).
Such an amount of anti-microbial lipid can only be achieved by addition of the selected anti-microbial lipid or derivative in accordance with the invention.
The importance of the presence of all components of the system in ensuring an effective anti-microbial composition was tested in the experiment summarised in Table 12.
A simple medium of the following composition was prepared:
Phosphate Buffered Saline (pH 7) with bactotryptone and yeast extract (PBS/T/Y): 195 ml of 0.2 M NaH2P04, 305 ml of 0.2 M Na2HP04 and 8.994 g NaCl, 10 g bactotryptone, 5 g yeast extract made up to 1 litre with MilliQ
water and autoclaved in screw-capped tubes at 121 °C for 15 min. The tubes were held at incubation temperature (37 °C) until used.
Two strains S. aureus R37 or E. coli 0157:H7 (-vt) were used in the experiments (as for the experiments in Table 1), with the following base combinations of monolaurin and the LPS selected for the two strains based on titrations of the organisms against monolaurin + LPS to define the sensitivity: S. aureus ( 100mg LPX
(lactoperoxidase) per litre in the LPS and 20mg/litre of monolaurin) and E. coli (100mg LPX per litre in the LPS and 50mg/litre of monolaurin). The LPX to glucose oxidase (GOX) ratio was 9:1 and the thiocyanate and glucose were incorporated at l2mg/litre.
Table 12: The importance of the individual components in ensuring the efficacy of the anti-microbial system. The degree of inhibition is defined by the time (hours of incubation 37 °C) at initiation of the logarithmic growth phase of the organism and the time at plateau (maximum absorbance).
E. coZi 0157:H7 No inhibition Partial inhibition Maximal inhibition (O hours and 12 (4 hours and 16 hours)(12 hours and 20 or hours) >20 hours) Monolaurin (ML) ML + LPX + GOX ML + LPX + GOX + SCN
alone ML + LPX ML + GOX + glucose ML + the complete LPS
ML + glucose ML + GOX + SCN
ML + thiocyanate ML + GOX + glucose (SCN) + SCN
No inhibition Partial inhibition Maximal inhibition (O hours and 12 (4 hours and 16 hours)(12 hours and 20 or >20 hours) horns) ML + LPX + glucose ML + LPX + GOX + glucoseThe above may be interpreted ML + LPX + thiocyanateAll- of the above as the effects of monolaurin may be ML + glucose + interpreted as the plus the complete LPS
effects of as some thiocyanate monolaurin plus peroxideglucose would have been ML + LPX + glucose as the GOX would generatepresent in the medium.
+
SCN peroxide with glucose as the substrate.
S. aureus R3?
No inhibition Partial inhibition Maximal inhibition (8-12 hours and (12-16 hours and 24 (16 hours and 24-28 hours) 16- hours) 20 hours) ML + glucose Monolaurin (ML) alone ML + LPX + GOX + SCN
ML + thiocyanate ML + LPX ML + the complete LPS
(SCN) ML + LPX + GOX ML + LPX + glucose ML + LPX + ML + SCN + glucose thiocyanate ML + LPX + glucose +
SCN
ML + GOX The above may be interpreted ML + GOX + glucose as the effects of monolaurin ML + GOX + SCN plus the complete LPS
as some ML + GOX + glucose glucose would have been + SCN
ML + LPX + GOX + glucosepresent in the medium.
The above group of treatments may be interpreted as the effects of monolaurin plus peroxide as the GOX would generate peroxide with glucose as the substrate.
w0 oo~69z67 _ _$.eceived 19 June 2001 Again, the efficacy of the applicants approach is demonstrated.
INDUSTRIAL APPLICATIOf~
S
The applicants findings in respect of synergism between the LPS and monolaurin (which exemplify the interaction between LPS and anti-microbial fatty acid / fatty acid de::vazines~ °aas a number of applications. Principal amongst these is that this synergistic anti-microbial effect can be reproduced in food products which are prone to contamination or spoilage.
The present invention has particular benefit where the fatty acid component is monolaurin.
This reflects the fact that monoiaurin has GRAS status and is already inclwded in some food products as a emulsifying agent.
Food products in which the components can be included are any foodstu$s subject to spoilage as well as dietary supplements and nutraceuticals. The invention has particular application to dairy products (such as yoghurts), fish products (particularly shellfish) and meat products (including both ground meats and carcasses), as well as to animal feeds. It should however be appreciated that "feed" is intended in its most general sense, and can include water which is fed to farmed animals including but not limited to bovines, ovines, pigs, caprines, equines and avians (such as poultry).
To such products, the individual components can be added together or independently.
In some instances, where a product may inherently contain one or more of the components in appropriate amounts, the remaining components only need be added.
The components can also be added to farm a mixture as part of the product (such as a dairy product), or can be applied to at least partially coat the products (such as shellfish).
Wher a the ingredients are to be added individually, a preparative pack can be provided with at least the lactoperoxidase and the fatty acid derivative or salt thereof in different containers.
~=a.a';~.,~..:r~.:. ,.rd:::
E::x~.P _,e',~ri WO OOI69267 I'~'~g'1~9 June 2001 The anti-microbial compositions of the invention can also be users to supplement the action of other agents. 1n such circumstances, the additional agent can be used separately or, more usually, as part of a mixture with the present components.
Other optional components which can be included where desirable include further anti-microbial agents, chelating agents, enzymes and nutritional nutraceutical components.
Specifically, the composition may also include any of the following:
- a chelating agent, such as EDTA;
- a phenol, such as the esters if pares-hydroxybenzoic acid (the parabens) including the methyl, ethyl, propyl, butyl or heptyl esters of tert-butyl hydroxyanisole (BHA);
- an organic acid, (which is recognised as a preservative), such as formic acid, acetic acid, propionic acid, lactic acid, sorbic acid, benzoic acid, citric acid or derivatives of any of these acids;
- a bacteiiocin, such as raisin;
- lyzozyme;
- extracts of milk.
It will be appreciated by those persons skilled in the art that the above description is provided by way of example only and that modifications and/or variations thereto can be made without departing from the scope of the invention, which is limited only by the lawful scope of the appended claims.
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Siragusa, G. R. and M. G. Johnson. 1989. Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanate-H202 antimicrobial system. Appl. Environ.
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