U.S. patent application number 10/568324 was filed with the patent office on 2007-05-10 for composition comprising a bacteriocin and an extract from a plant of the labiatae family.
This patent application is currently assigned to Danisco A/S. Invention is credited to Bob Coyne, John Faragher, Sebastien Gouin, Carsten Bjorn Hansen, Richard Ingram, Torben Isak, Linda Valerie Thomas, Kathryn Louise Tse.
Application Number | 20070104809 10/568324 |
Document ID | / |
Family ID | 37767637 |
Filed Date | 2007-05-10 |
United States Patent
Application |
20070104809 |
Kind Code |
A1 |
Coyne; Bob ; et al. |
May 10, 2007 |
Composition comprising a bacteriocin and an extract from a plant of
the labiatae family
Abstract
The present invention provides a composition comprising (a) an
antimicrobial material; and (b) an extract obtained from or
obtainable from a plant of the Labiatae family, wherein (a) and (b)
are different; wherein the composition contains phenolic diterpenes
in an amount of greater than 1.0 wt. %, based on the composition,
and wherein when the antimicrobial material consists of nisin, the
composition comprises carvacrol in an amount of less than 0.075 wt.
% based on the composition and carvone in an amount of less than 15
wt. % based on the composition.
Inventors: |
Coyne; Bob; (Lenexa, KS)
; Faragher; John; (Richfield, WI) ; Gouin;
Sebastien; (Arhus, DK) ; Hansen; Carsten Bjorn;
(Knebel, DK) ; Ingram; Richard; (Yeovil Somerset,
GB) ; Isak; Torben; (Hinnerup, DK) ; Thomas;
Linda Valerie; (Dorset, GB) ; Tse; Kathryn
Louise; (Kobenhaven S, DK) |
Correspondence
Address: |
STEPTOE & JOHNSON LLP
1330 CONNECTICUT AVENUE, N.W.
WASHINGTON
DC
20036
US
|
Assignee: |
Danisco A/S
Langebrogade 1
Copenhagen K
DK
DK-1001
|
Family ID: |
37767637 |
Appl. No.: |
10/568324 |
Filed: |
August 6, 2004 |
PCT Filed: |
August 6, 2004 |
PCT NO: |
PCT/GB04/03423 |
371 Date: |
October 13, 2006 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10820147 |
Apr 8, 2004 |
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10568324 |
Oct 13, 2006 |
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60497409 |
Aug 22, 2003 |
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60533053 |
Dec 30, 2003 |
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60560270 |
Apr 8, 2004 |
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60497409 |
Aug 22, 2003 |
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Current U.S.
Class: |
424/745 ;
514/2.9 |
Current CPC
Class: |
A01N 37/38 20130101;
A23L 3/3472 20130101; A01N 31/08 20130101; B01J 13/043 20130101;
Y10T 428/2984 20150115; A23B 4/12 20130101; B01J 13/14 20130101;
A23L 13/72 20160801; A01N 25/28 20130101; A23B 5/06 20130101; A23B
4/20 20130101; Y02A 40/90 20180101; A23L 2/52 20130101; A23L 33/135
20160801; A23V 2002/00 20130101; A01N 63/50 20200101; B01J 13/08
20130101; A01N 35/06 20130101; B01J 13/22 20130101; A23B 4/22
20130101; A23L 3/34635 20130101; A23L 3/3571 20130101; A23B 5/16
20130101; A01N 37/36 20130101; A23P 10/30 20160801; A01N 37/46
20130101; A23C 19/11 20130101; A23C 19/084 20130101; A01N 65/22
20130101; A23L 3/3544 20130101; A21D 2/00 20130101; A23L 33/127
20160801; A23B 5/14 20130101; A23B 4/10 20130101; A23P 10/35
20160801; A23V 2002/00 20130101; A23V 2200/224 20130101; A23V
2200/10 20130101; A01N 25/28 20130101; A01N 37/36 20130101; A01N
37/46 20130101; A01N 63/50 20200101 |
Class at
Publication: |
424/745 ;
514/002; 514/010 |
International
Class: |
A61K 36/53 20060101
A61K036/53; A61K 38/12 20060101 A61K038/12 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 22, 2003 |
GB |
0319817.3 |
Oct 6, 2003 |
GB |
0323335.0 |
Claims
1. A composition comprising (a) an antimicrobial material; and (b)
an extract obtained from or obtainable from a plant of the Labiatae
family, wherein (a) and (b) are different wherein the composition
contains phenolic diterpenes in an amount of greater than 1.0 wt.
%, based on the composition, and wherein when the antimicrobial
material consists of nisin, the composition comprises carvacrol in
an amount of less than 0.075 wt. % based on the composition and
carvone in an amount of less than 15 wt. % based on the
composition.
2. A composition according to claim 1 wherein the composition
comprises carvacrol in an amount of less than 0.075 wt. % based on
the composition.
3. A composition according to claim 1 or 2 wherein the composition
comprises carvacrol in an amount of less than 0.04 wt. % based on
the composition.
4. A composition according to claim 1, 2 or 3 wherein the
composition comprises carvacrol in an amount of less than 0.02 wt.
% based on the composition.
5. A composition according to any one of the preceding claims
wherein the composition comprises carvone in an amount of less than
0.075 wt. % based on the composition.
6. A composition according to any one of the preceding claims
wherein the composition comprises carvone in an amount of less than
0.04 wt. % based on the composition.
7. A composition according to any one of the preceding claims
wherein the composition comprises carvone in an amount of less than
0.02 wt. % based on the composition.
8. A composition according to any one of the preceding claims
wherein the composition comprises thymol in an amount of less than
0.1 wt. % based on the composition.
9. A composition according to any one of the preceding claims
wherein the composition comprises thymol in an amount of less than
0.075 wt. % based on the composition.
10. A composition according to any one of the preceding claims
wherein the composition comprises thymol in an amount of less than
0.05 wt. % based on the composition.
11. A composition according to any one of the preceding claims
wherein the antimicrobial material is a bacteriocin.
12. A composition according to any one of the preceding claims
wherein the bacteriocin is selected from lanthionine containing
bacteriocins, Lactococcus-derived bacteriocins,
Streptococcus-derived bacteriocins, Pediococcus-derived
bacteriocins, Lactobacillus-derived bacteriocins,
Carnobacterium-derived bacteriocins, Leuconostoc-derived
bacteriocins, Enterococcus-derived bacteriocins and mixtures
thereof
13. A composition according to any one of the preceding claims
wherein the bacteriocin is at least nisin.
14. A composition according to any one of the preceding claims
wherein the extract is obtained from a plant of the Labiatae
family.
15. A composition according to any one of the preceding claims
wherein the plant of the Labiatae family is selected from rosemary,
sage, oregano, marjoram, mint, balm, savoury, and thyme.
16. A composition according to any one of the preceding claims
wherein the plant of the Labiatae family is selected from rosemary,
sage, oregano, marjoram, mint, balm, and savoury.
17. A composition according to any one of the preceding claims
wherein the plant of the Labiatae family is rosemary.
18. A composition according to any one of the preceding claims
wherein the composition contains phenolic diterpenes in an amount
of is greater than 2.0 wt. %, based on the composition.
19. A composition according to any one of the preceding claims
wherein the composition contains phenolic diterpenes in an amount
of is greater than 3.0 wt. %, based on the composition.
20. A composition according to any one of the preceding claims
wherein the composition contains phenolic diterpenes in an amount
of is greater than 3.5 wt. %, based on the composition.
21. A composition according to any one of the preceding claims
wherein the phenolic diterpene is selected from carnosic acid,
carnosol, methylcarnosic acid and mixtures thereof.
22. A composition according to any one of the preceding claims
wherein the extract comprises or is selected from phenolic
diterpenes, phenolic triterpenes and rosmarinic acid.
23. A composition according to any one of the preceding claims
wherein the extract comprises a phenolic triterpene.
24. A composition according to claim 17 wherein the amount of
phenolic triterpenes, based on the composition, is greater than 3.5
wt. %.
25. A composition according to claim 16, 17 or 18 wherein the
phenolic triterpene is selected from betulinic acid, oleanolic
acid, and ursolic acid.
26. A composition according to any one of the preceding claims
wherein the extract comprises a rosmarinic acid.
27. A composition according to claim 20 wherein the amount of
rosmarinic acid, based on the composition, is greater than 3.5 wt.
%.
28. A composition according to any one of the preceding claims
wherein the combined amount of phenolic diterpenes, phenolic
triterpenes and rosmarinic acid, based on the composition, is
greater than 3.5 wt. %.
29. A composition according to any one of the preceding claims
wherein the extract contains flavour-inducing compounds and/or
essential oils in an amount of less than 1 wt. % based on the
composition.
30. A composition according to any one of the preceding claims
wherein the extract contains essential oils in an amount of less
than 1 wt. % based on the composition.
31. A composition according to any one of the preceding claims
wherein the antimicrobial material and/or the extract are present
in an amount to provide a microbicidal or microbiostatic
effect.
32. A composition according to any one of the preceding claims
wherein the antimicrobial material and the extract are present in
an amount to provide a microbicidal or microbiostatic effect.
33. A composition according to any one of the preceding claims
wherein the antimicrobial material and the extract are present in
an amount to provide a microbicidal or microbiostatic synergistic
effect.
34. A composition according to any one of the preceding claims
wherein the antimicrobial material and the extract are present in
an amount to provide a microbicidal synergistic effect.
35. A composition according to any one of claims 31 to 34 wherein
the microbicidal or microbiostatic effect is a bactericidal or
bacteriostatic effect.
36. A composition according to claim 35 wherein the bactericidal or
bacteriostatic effect is in respect of Gram-positive bacteria.
37. A composition according to claim 36 wherein the bactericidal or
bacteriostatic effect is in respect of an organism selected from
species of Bacillus, species of Clostridium, Listeria
monocytogenes, lactic acid bacteria, and Lactobacillus species.
38. A composition according to any one of claims 35 to 37 wherein
the bactericidal or bacteriostatic effect is in respect of Listeria
monocytogenes.
39. A composition according to any one of the preceding claims
wherein the composition is a foodstuff.
40. A composition according to any one of the preceding claims
wherein the composition is a protectant composition suitable for
addition to a foodstuff.
41. A composition according to claim 39 or 40 the foodstuff is
selected from raw meat, cooked meat, raw poultry products, cooked
poultry products, raw seafood products, cooked seafood products,
ready to eat meals, pasta sauces, pasteurised soups, mayonnaise,
salad dressings, oil-in-water emulsions, margarines, low fat
spreads, water-in-oil emulsions, dairy products, cheese spreads,
processed cheese, dairy desserts, flavoured milks, cream, fermented
milk products, cheese, butter, condensed milk products, ice cream
mixes, soya products, pasteurised liquid egg, bakery products,
confectionery products, fruit products, and foods with fat-based or
water-containing fillings.
42. A composition according to any one of the preceding claims
wherein the composition further comprises an emulsifier.
43. A composition according to claim 42 wherein the emulsifier is
selected from polysorbates, monoglycerides, diglycerides, acetic
acid esters of mono-diglycerides, tartaric acid esters of
mono-diglycerides and citric acid esters of mono-diglycerides.
44. A composition according to any one of the preceding claims
wherein the composition further comprises a chelator.
45. A composition according to claim 44 wherein the chelator is
selected from EDTA, citric acid, monophosphates, diphosphates,
triphosphates and polyphosphates.
46. A composition according to claim 44 or 45 wherein the chelator
enhances the antimicrobial activity and/or antimicrobial spectrum
of the antimicrobial material.
47. A composition according to claim 44, 45 or 46 wherein the
chelator enhances the antimicrobial activity and/or antimicrobial
spectrum of the antimicrobial material in respect of Gram-negative
bacteria.
48. A composition according to any one of the preceding claims
wherein the composition further comprises a lytic enzyme.
49. A composition according to claim 48 wherein the lytic enzyme is
a lysozyme.
50. A composition according to claim 1 comprising (a) an
antimicrobial material, wherein the antimicrobial material is
nisin; and (b) an extract obtained from a plant of the Labiatae
family, wherein the extract is a phenolic triterpene selected from
betulinic acid, oleanolic acid or ursolic acid wherein the
antimicrobial material and the extract are present in an amount to
provide a bactericidal or bacteriostatic synergistic effect.
51. A composition according to claim 1 comprising (a) an
antimicrobial material, wherein the antimicrobial material is
nisin; and (b) an extract obtained from a plant of the Labiatae
family, wherein the extract is rosmarinic acid, wherein the
antimicrobial material and the extract are present in an amount to
provide a bactericidal or bacteriostatic synergistic effect.
52. A process for preventing and/or inhibiting the growth of,
and/or killing a microorganism in a material, the process
comprising the step of contacting the material with (a) an
antimicrobial material; and (b) an extract obtained from or
obtainable from a plant of the Labiatae family, wherein (a) and (b)
are different wherein the composition contains phenolic diterpenes
in an amount of greater than 1.0 wt. %, based on the composition,
wherein when the antimicrobial material consists of nisin, the
composition comprises carvacrol in an amount of less than 0.075 wt.
% based on the composition and carvone in an amount of less than 15
wt. % based on the composition.
53. A process according to claim 52 wherein the antimicrobial
material and the extract are added to the material together.
54. A process according to claim 52 wherein the antimicrobial
material and the extract are added to the material
sequentially.
55. A process according to any one of claims 52 to 54 wherein the
bacteriocin is added to the material.
56. A process according to claim 52 wherein the bacteriocin is
formed in situ in the material.
57. A process according to any one of claims 52 to 56 wherein the
material is a foodstuff.
58. A process according to any one of claims 52 to 57 characterised
by the features of any one of claim 2 to 51.
59. Use of (a) an antimicrobial material; and b) an extract
obtained from or obtainable from a plant of the Labiatae family,
for preventing and/or inhibiting the growth of, and/or killing a
micro-organism in a material; wherein (a) and (b) are different;
wherein the composition contains phenolic diterpenes in an amount
of greater than 1.0 wt. %, based on the composition, and wherein
when the antimicrobial material consists of nisin, the composition
comprises carvacrol in an amount of less than 0.075 wt. % based on
the composition and carvone in an amount of less than 15 wt. %
based on the composition.
60. Use according to claim 59 wherein the material is a
foodstuff.
61. Use according to claim 59 or 60 for synergistically preventing
and/or inhibiting the growth of, and/or killing a micro-organism in
a material.
62. Use according to any one of claims 59 to 61 characterised by
the features of any one of claim 2 to 51.
63. A kit for preparing a composition as defined in any one of
claims 1 to 51, the kit comprising (a) an antimicrobial material;
and (b) an extract obtained from or obtainable from a plant of the
Labiatae family, wherein (a) and (b) are different in separate
packages or containers; optionally with instructions for admixture
and/or contacting and/or use.
64. A composition as substantially hereinbefore described with
reference to any one of the Examples.
65. A process as substantially hereinbefore described with
reference to any one of the Examples.
66. A use as substantially hereinbefore described with reference to
any one of the Examples.
67. A kit as substantially hereinbefore described with reference to
any one of the Examples.
Description
[0001] The present invention relates to a composition that exhibits
a microbicidal or microbiostatic action.
BACKGROUND
[0002] Bacteriocins are antimicrobial proteins or peptides that can
be produced by certain bacteria, which can kill or inhibit the
growth of closely related bacteria. The bacteriocins produced by
lactic acid bacteria are of particular importance since they have
great potential for the preservation of food and for the control of
foodborne pathogens. (Wessels et al. 1998.)
[0003] The most well known bacteriocin is nisin, which is the only
bacteriocin currently authorised as a food additive. Nisin is
produced by fermentation of the dairy starter culture bacterium
Lactococcus lactis subsp. lactis, and is sold as the commercial
extract Nisaplin.RTM. Natural Antimicrobial (Danisco). Nisin has an
unusually broad antimicrobial spectrum for a bacteriocin, being
active against most Gram-positive bacteria (e.g. species of
Bacillus, Clostridium, Listeria, lactic acid bacteria). It is not
normally effective against Gram-negative bacteria, yeasts or
moulds. Nisin is allowed as a food preservative worldwide but its
levels of use and approved food applications are strictly
regulated, varying from country to country.
[0004] Other bacteriocins have since been discovered with potential
as food preservatives, e.g. pediocin, lactcin, sakacin,
lactococcin, enterococin, plantaricin, leucocin. These are also
active, although usually with a more narrow spectrum, against
Gram-positive bacteria. Their food use is at present restricted to
production of the bacteriocin in situ, i.e. by growth of the
producer organism within the food.
[0005] Antioxidants are widely used in food products susceptible to
oxidative degeneration. An antioxidant is defined by the Food and
Drug Administration (21 CFR 170.3) as "a substance used to preserve
food by retarding deterioration, rancidity, or discoloration due to
oxidation". Spices or plant extracts can be used in food as
antioxidants and to impart flavour. One advantage of such extracts
is that they are perceived as natural ingredients when compared to
chemical antioxidants such as butyl hydroxyanisol (BHA) and
butylated hydoxytoluene (BHT). Plants of the family Labiatae
contain several well known herbs. Extracts from these plants have
been shown to have antioxidant and, in some cases, antimicrobial
activity (Nychas & Skandamis, 2003; Smid and Gorris, 1999;
Loliger, 1989). Such extracts may be essential oils and oleoresins
(extracts with essential oil content used in flavours and
fragrances) or "deodorised", extracts that have a high phenolic
diterpene content and low level of flavour-inducing compounds.
[0006] Essential oils are extracted by simple steam distillation of
the plant material. The most effective antioxidant compounds in
rosemary and sage are reported to be carnosic acid, carnosol and
rosmarinic acid (Cuvelier et al. 1996). Carnosic acid, a phenolic
diterpene (C.sub.20H.sub.28O.sub.4), occurs naturally in leaves of
plants of the Labiatae family, particularly rosemary and sage, but
also thyme and marjoram. Dried leaves of rosemary or sage contain
1.5-2.5% carnosic acid and 0.3-0.4% carnosol (US6231896). Carnosol
is an oxidative artefact of carnosic acid (Wenkert et al. J. Org.
Chem 30:2931, 1965). The oxidation takes place in the presence of
harvesting in the leaves left to dry in the air and if the leaves
are subjected to extraction with solvents. Rosmanol may also be a
product of the oxidation of carnosic acid.
[0007] The use of extracts of plant material for inhibiting the
growth of micro-organisms has been taught in the art. Examples of
such teachings include: WO 02/069741 teaches Labiatae herb extracts
and hop extracts for extending the colour lie and inhibiting the
growth of micro-organisms in fresh meat, fish and poultry. Periago
et al. 2001. Food Science & Technology International. 7:
487-492 relates to the use of Carvacrol and thymol at 0.3
mmol/litre in combination with nisin. It is taught that synergy is
observed. JP 2001172159 relates to cosmetics comprising a range of
components including antimicrobial agent and Labiatae solvent
extract. WO 98/56395 teaches a mix of tea-tree oil and thyme
Essential oil. GB 2275 194 A discusses plant extract disinfectant.
US 6083921 discusses a combination of plant extracts including one
from Labiatae: Scutellaria, preferably root (Radix scutellariae).
U.S. Pat. No. 5,472,684 teaches an oral composition for plaque and
gingivitis containing thymol and eugenol
[0008] Food safety and prevention of food spoilage is an ever
present concern worldwide, particularly with the increasing trend
for convenience foods such as ready to eat meals, soups, sauces or
snacks. Spoilage of food is a major economic problem for the food
manufacturer. Food manufacturers need to protect the health and
safety of the public by delivering products that are safe to eat.
Such food must have a guaranteed shelf life, either at chilled or
ambient temperature storage. Consumers prefer good tasting food of
high quality--this is difficult to achieve with chemical
preservatives, harsh heating regimes and other processing measures.
Food safety and protection is best achieved with a multiple
preservation system using a combined approach of milder processing
and natural preservatives. Foodborne micro-organisms are also less
able to adapt and grow in food preserved with different
preservative measures.
[0009] There is much concern about food safety and the growth of
food pathogens such as Listeria monocytogenes. This particular
pathogen can grow at low temperatures, which are often used as an
additional preservative measure. Foodborne pathogens can sometimes
adapt to different preservatives and storage conditions, thus a
combination of preservative measures can be more successful than
individual measures.
[0010] There is an increasing need to develop economical, natural
and effective food preservative systems to meet the public demand
for convenient, natural, safe, healthy, good quality food products
with guaranteed shelf life. Bacteriocins such as nisin can be used
as preservatives in food to help meet this need. Nisin is a proven
safe, natural preservative with GRAS status. Other bacteriocins can
be used for preservation if produced in situ, by growth of the
bacteriocin producer organism in the food.
[0011] In some cases the bacteriocin levels required to ensure
preservation or food safety may prove uneconomical, or are below
effective levels due to regulatory and legislation constraints.
When bacteriocins are produced in situ, the resulting bacteriocin
levels may not be high enough to achieve the required preservative
effect.
[0012] The present invention alleviates the problems of the prior
art.
[0013] In one aspect the present invention provides a composition
comprising (a) an antimicrobial material; and (b) an extract
obtained from or obtainable from a plant of the Labiatae family,
wherein (a) and (b) are different; wherein the composition contains
phenolic diterpenes in an amount of greater than 1.0 wt. %, based
on the composition, and wherein when the antimicrobial material
consists of nisin, the composition comprises carvacrol in an amount
of less than 0.075 wt. % based on the composition and carvone in an
amount of less than 15 wt. % based on the composition.
[0014] In one aspect the present invention provides a process for
preventing and/or inhibiting the growth of, and/or killing a
microorganism in a material, the process comprising the step of
contacting the material with (a) an antimicrobial material; and (b)
an extract obtained from or obtainable from a plant of the Labiatae
family, wherein (a) and (b) are different; wherein the composition
contains phenolic diterpenes in an amount of greater than 1.0 wt.
%, based on the composition, and wherein when the antimicrobial
material consists of nisin, the composition comprises carvacrol in
an amount of less than 0.075 wt. % based on the composition and
carvone in an amount of less than 15 wt. % based on the
composition.
[0015] In one aspect the present invention provides use of (a) an
antimicrobial material; and b) an extract obtained from or
obtainable from a plant of the Labiatae family, for preventing
and/or inhibiting the growth of, and/or killing a micro-organism in
a material; wherein (a) and (b) are different; wherein the
composition contains phenolic diterpenes in an amount of greater
than 1.0 wt. %, based on the composition, and wherein when the
antimicrobial material consists of nisin, the composition comprises
carvacrol in an amount of less than 0.075 wt. % based on the
composition and carvone in an amount of less than 15 wt. % based on
the composition.
[0016] In one aspect the present invention provides kit for
preparing a composition as defined herein, the kit comprising (a)
an antimicrobial material; and (b) an extract obtained from or
obtainable from a plant of the Labiatae family, wherein (a) and (b)
are different; wherein the composition contains phenolic diterpenes
in an amount of greater than 1.0 wt. %, based on the composition,
and wherein when the antimicrobial material consists of nisin, the
composition comprises carvacrol in an amount of less than 0.075 wt.
% based on the composition and carvone in an amount of less than 15
wt. % based on the composition, in separate packages or containers;
optionally with instructions for admixture and/or contacting and/or
use.
[0017] Aspects of the invention are defined in the appended
claims.
[0018] Of the Labiatae plant family, rosemary and sage have
antioxidant activity in foods that is mainly related to phenolic
diterpenes such as carnosic acid and carnosol, as well as other
phenolic compounds, including phenolic triterpenes such as
betulinic acid, oleanolic add and ursolic acid; and rosmarinic
acid. Antimicrobial activity has been attributed to some of these
compounds, all of which can be obtained by selective extraction
from the plants. The phenolic diterpenes, phenolic triterpenes and
rosmarinic acid are distinct from the essential oils and oleoresins
that are often used in flavours and fragrances. The high flavour
and odour levels of essential oils is not conducive to their use in
food. One skilled in the art would expect a combination of an
antimicrobial material and an extract from the Labiatae plant
family to provide a simple additive bactericidal or bacteriostatic
effect. However, in vitro studies described herein have
demonstrated synergistic enhancement of bacteriocin activity by a
deodorised extract of Rosmarinus officinalis. This enhanced
activity was also observed in a food model, increasing bacteriocin
(for example nisin) kill and growth control of Gram-positive
bacteria. Enhanced bacteriocin activity was also observed with
rosemary extracts specifically prepared to contain high levels of
the phenolic diterpenes carnosol and carnosic acid, indicating
these compounds play an important role in the synergy. Enhanced
bacteriocin activity was also observed with rosmarinic acid.
[0019] The present invention provides a synergistic combination of
components for preventing and/or inhibiting the growth of, and/or
killing a micro-organism in a material, such as foodstuff. This
combination of components allows lower levels of the antimicrobial
material to be used to provide effective action and prevent the
development of tolerance to the antimicrobial material. This is
particularly important in food applications where reduction of
dosage and/or avoidance of development of tolerance is desired for
commercial and regulatory reasons.
[0020] For ease of reference, these and further aspects of the
present invention are now discussed under appropriate section
headings. However, the teachings under each section are not
necessarily limited to each particular section.
Preferred Aspects
Antimicrobial Material
[0021] In one preferred aspect the antimicrobial material is a
bacteriocin.
[0022] The antimicrobial material, such as a bacteriocin, may
typically be selected from materials (bacteriocins) that can be
used as preservatives in food
[0023] Preferably the antimicrobial material is selected from
lanthionine containing bacteriocins, Lactococcus-derived
bacteriocins, Streptococcus-derived bacteriocins,
Pediococcus-derived bacteriocins, Lactobacillus-derived
bacteriocins, Carnobacterium-derived bacteriocins,
Leuconostoc-derived bacteriocins, Enterococcus-derived bacteriocins
and mixtures thereof
[0024] Preferably the antimicrobial material is at least nisin.
[0025] Preferably the antimicrobial material consists of nisin.
[0026] Nisin is a lanthionine-containing bacteriocin (U.S. Pat. No.
5,691,301) derived from Lactococcus lactis subsp. lactis (formerly
known as Streptococcus-lactis) (U.S. Pat. No. 5,573,801). In a
preferred aspect of the present invention the bacteriocin used in
the present invention is at least nisin.
[0027] As discussed in U.S. Pat. No. 5,573,801 nisin is a
polypeptide bacteriocin produced by the lactic acid bacteria,
Lactococcus lactis subsp. lactis (formerly known as Streptococcus
lactis Group N).
[0028] Nisin is reportedly a collective name representing several
closely related substances which have been designated nisin
compounds A, B, C, D and E (De Vuyst, L. and Vandamme, E. J. 1994.
Nisin, a lantibiotic produced by Lactococcus lactis subsp. lactis:
properties, biosynthesis, fermentation and applications. In:
Bacteriocins of lactic acid bacteria. Microbiology, Genetics and
Applications. Eds.: De Vuyst and Vandamme. Blackie Academic and
Professional, London). The structure and properties of nisin are
also discussed in the article by E. Lipinska, entitled "Nisin and
Its Applications", The 25th Proceedings of the Easter School in
Agriculture Science at the University of Nottingham, 1976, pp.
103-130 (1977), which article is hereby incorporated by reference.
In 1969 the FAO/WHO Joint Expert Committee on Food Additives set
specifications for the purity and identity of nisin (FAO/WHO Joint
Expert Committee on Food Additives. 1969. Specifications for
identity and purity of some antibiotics. 12.sup.th Report. WHO
Technical Report Series No. 430). This committee recognised nisin
as a safe and legal preservative based on extensive toxicological
testing. Nisin has the food additive number E234 and is classed as
GRAS (Generally Recognised As Safe) (Food and Drug Administration.
1988. Nisin preparation: Affirmation of GRAS status as a direct
human ingredient. Federal Regulations 53: 11247). The international
activity unit (IU hereinafter) was defined as 0.001 mg of an
international nisin reference preparation. Nisaplin.RTM. Natural
Antimicrobial is the brand name for a nisin concentrate containing
1 million IU per g, which is commercially available from
Danisco.
[0029] Nisin is an acknowledged and accepted food preservative with
a long history of safe, effective food use. There have been several
reviews of nisin, e.g. Hurst 1981; 1983; Delves-Broughton, 1990; De
Vuyst and Vandamme, 1994; Thomas et al. 2000; Thomas &
Delves-Broughton, 2001). Nisin was discovered over 50 years ago and
the first commercial preparation, made in 1953, was Nisaplin.RTM..
Nisin has several characteristics that make it particularly
suitable as a food preservative. It has undergone extensive
toxicological testing to demonstrate its safety. It is heat-stable,
acid-stable and effective against a broad spectrum of Gram-positive
bacteria. It is not normally effective against Gram-negative
bacteria, yeasts or moulds but activity against Gram-negative
bacteria and yeasts has been reported in the presence of chelating
agents (PCT/US 8902625. WO 89/12399). Nisin is an effective
preservative in pasteurised and heat-treated foods (e.g. processed
cheese, cheese, pasteurised milks, dairy desserts, cream,
mascarpone and other dairy products, puddings such as semolina,
tapioca etc., pasteurised liquid egg, pasteurised potato products,
soy products, crumpets, pikelets, flapjacks, processed meat
products, beverages, soups, sauces, ready to eat meals, canned
foods, vegetable drinks) and low acid foods such as salad
dressings, sauces, mayonnaise, beer, wine and other beverages.
[0030] Although some loss of activity may be expected when used
with processed foods, this may be ameliorated e.g. by increasing
the amount of nisin applied. Effective levels of nisin to preserve
foodstuffs reportedly range from 25-500 IU/g or more. Other
effective levels would be appreciated by one skilled in the art.
For example levels of 50-400 IU/g may be utilised.
[0031] Since the discovery of the first bacteriocin, nisin, many
other bacteriocins have now been found (Hoover, 1993; Ray &
Daeschel, 1994; Axelsen, 1998; Naidu, 2000; Ray et al. 2001; Ray
& Miller, 2003). The bacteriocin pediocin, produced by
Pediococcus pentosaceus, P. acidilactici, or Lactobacillus
plantarum, may be used in the present invention. Like nisin,
different structures of pediocin have been described. At present
pediocin and other bacteriocins are not allowed as food additives
but their antibacterial activity can be achieved by production of
the bacteriocin in situ, as a consequence of the growth of the
producer organism in the food. This is the purpose of commercial s
protective cultures such as HOLDBAC.TM. Listeria (Danisco).
Pediocin has a more narrow antimicrobial spectrum compared to
nisin, but there is much interest in its food safety ability to
kill, prevent or control the growth of the food pathogen Listeria
monocytogenes (Ray & Miller, 2000). Other bacteriocins may be
used in the present invention, including those named generally as
divercin, leucocin, mesentericin, sakacin, curvacin, bavaricin,
acidocin, bifidocin, carnobacteriocin, pisicocin, piscicolin,
mundticin, enterocin, thermophilin, lacticin, plantaricin,
lactococcin, divercin, diplococcin, mesenterocin, leuconosin,
camosin, acidophilin, lactacin, brevicin, lactocin, helevticin,
reutericin, propionicin.
Extract
[0032] As discussed herein when the antimicrobial material consists
of nisin, the composition comprises carvacrol in an amount of less
than 0.075 wt. % based on the composition and carvone in an amount
of less than 15 wt. % based on the composition.
[0033] In one preferred aspect when the antimicrobial material
consists of nisin, the composition comprises carvacrol in an amount
of less than 0.05 wt. % based on the composition, preferably less
than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less
than 0.01 wt. %, preferably less than 0.004 wt. %, based on the
composition.
[0034] In one preferred aspect, the composition comprises carvacrol
in an amount of less than 0.05 wt. % based on the composition,
preferably less than 0.04 wt. %, preferably less than 0.02 wt. %,
preferably less than 0.01 wt. %, preferably less than 0.004 wt. %,
based on the composition.
[0035] In one preferred aspect when the antimicrobial material
consists of nisin, the composition comprises carvone in an amount
of less than 10 wt. % based on the composition, preferably less
than 7 wt. %, preferably less than 5 wt. %, preferably less than 2
wt. %, preferably less than 1 wt. %, preferably less than 0.75 wt.
%, preferably less than 0.5 wt. %, preferably less than 0.2 wt. %,
preferably less than 0.1 wt. %, preferably less than 0.075 wt. %,
preferably less than 0.05 wt. %, preferably less than 0.04 wt. %,
preferably less than 0.02 wt. %, preferably less than 0.01 wt. %,
preferably less than 0.004 wt. %, based on the composition.
[0036] In one preferred aspect the composition comprises carvone in
an amount of less than 10 wt. % based on the composition,
preferably less than 7 wt. %, preferably less than 5 wt. %,
preferably less than 2 wt. %, preferably less than 1 wt. %,
preferably less than 0.75 wt. %, preferably less than 0.5 wt. %,
preferably less than 0.2 wt. %, preferably less than 0.1 wt. %,
preferably less than 0.75 wt. %, preferably less than 0.05 wt. %,
preferably less than 0.04 wt. %, preferably less than 0.02 wt. %,
preferably less than 0.01 wt. %, preferably less than 0.004 wt. %,
based on the composition.
[0037] In one preferred aspect when the antimicrobial material
consists of nisin, the composition comprises thymol in an amount of
less than 1 5 wt. % based on the composition, preferably less than
10 wt. %, preferably less than 7 wt. %, preferably less than 5 wt.
%, preferably less than 2 wt. %, preferably less than 1 wt. %,
preferably less than 0.75 wt. %, preferably less than 0.5 wt. %,
preferably less than 0.2 wt. %, preferably less than 0.1 wt. %,
preferably less than 0.075 wt. %, preferably less than 0.05 wt. %,
preferably less than 0.04 wt. %, preferably less than 0.02 wt. %,
preferably less than 0.01 wt. %, preferably less than 0.004 wt. %,
based on the composition.
[0038] In one preferred aspect the composition comprises thymol in
an amount of less than 15 wt. % based on the composition preferably
less than 10 wt. %, preferably less than 7 wt. %, preferably less
than 5 wt. %, preferably less than 2 wt. %, preferably less than 1
wt. %, preferably less than 0.75 wt. %, preferably less than 0.5
wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt.
%, preferably less than 0.075 wt. %, preferably less than 0.05 wt.
%, preferably less than 0.04 wt. %, preferably less than 0.02 wt.
%, preferably less than 0.01 wt. %, preferably less than 0.004 wt.
%, preferably less than 0.004 wt. %, based on the composition.
[0039] In one preferred aspect when the antimicrobial material
consists of nisin, the composition comprises eugenol in an amount
of less than 15 wt. % based on the composition, preferably less
than 10 wt. %, preferably less than 7 wt. %, preferably less than 5
wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %,
preferably less than 0.75 wt. %, preferably less than 0.5 wt. %,
preferably less than 0.2 wt. %, preferably less than 0.1 wt. %,
preferably less than 0.075 wt. %, preferably less than 0.05 wt. %,
preferably less than 0.04 wt. %, preferably less than 0.02 wt. %,
preferably less than 0.01 wt. %, preferably less than 0.004 wt. %,
based on the composition.
[0040] In one preferred aspect the composition comprises eugenol in
an amount of less than 15 wt. % based on the composition preferably
less than 10 wt. %, preferably less than 7 wt. %, preferably less
than 5 wt. %, preferably less than 2 wt. %, preferably less than 1
wt. %, preferably less than 0.75 wt. %, preferably less than 0.5
wt. %, preferably less than 0.2 wt. %, preferably less than 0.1 wt.
%, preferably less than 0.075 wt. %, preferably less than 0.05 wt.
%, preferably less than 0.04 wt. %, preferably less than 0.02 wt.
%, preferably less than 0.01 wt. %, preferably less than 0.004 wt.
%, based on the composition.
[0041] In one preferred aspect when the antimicrobial material
consists of nisin, the composition comprises carvacrol in an amount
of less than 0.075 wt. % based on the composition and each of
carvone and thymol in amounts of less than 15 wt. % based on the
composition (preferably less than 10 wt. % based on the
composition, preferably less than 7 wt. %, preferably less than 5
wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %,
preferably less than 0.75 wt. %, preferably less than 0.5 wt. %,
preferably less than 0.2 wt. %, preferably less than 0.1 wt. %,
preferably less than 0.075 wt. %, preferably less than 0.05 wt. %,
preferably less than 0.04 wt. %, preferably less than 0.02 wt. %,
preferably less than 0.01 wt. %, preferably less than 0.004 wt. %,
based on the composition).
[0042] In one preferred aspect, the composition comprises carvacrol
in an amount of less than 0.075 wt. % based on the composition and
each of carvone and thymol in amounts of less than 15 wt. % based
on the composition (preferably less than 10 wt. % based on the
composition, preferably less than 7 wt. %, preferably less than 5
wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %,
preferably less than 0.75 wt. %, preferably less than 0.5 wt. %,
preferably less than 0.2 wt. %, preferably less than O.1 wt. %,
preferably less than 0.075 wt. %, preferably less than 0.05 wt. %,
preferably less than 0.04 wt. %, preferably less than 0.02 wt. %,
preferably less than 0.01 wt. %, preferably less than 0.004 wt. %,
based on the composition).
[0043] In one preferred aspect when the antimicrobial material
consists of nisin, the composition comprises carvacrol in an amount
of less than 0.075 wt. % based on the composition and each of
carvone, thymol and eugenol in amounts of less than 15 wt. % based
on the composition (preferably less than 10 wt. % based on the
composition, preferably less than 7 wt. %, preferably less than 5
wt. %, preferably less than 2 wt. %, preferably less than 1 wt. %,
preferably less than 0.75 wt. %, preferably less than 0.5 wt. %,
preferably less than 0.2 wt. %, preferably less than 0.1 wt. %,
preferably less than 0.075 wt. %, preferably less than 0.05 wt. %,
preferably less than 0.04 wt. %, preferably less than 0.02 wt. %,
preferably less than 0.01 wt. %, preferably less than 0.004 wt. %,
based on the composition).
[0044] In one preferred aspect, the composition comprises carvacrol
in an amount of less than 0.075 wt. % based on the composition and
each of carvone, thymol and eugenol in amounts of less than 15 wt.
% based on the composition (preferably less than 10 wt. % based on
the composition, preferably less than 7 wt. %, preferably less than
5 wt. %, preferably less than 2 wt. %, preferably less than 1 wt.
%, preferably less than 0.75 wt. %, preferably less than 0.5 wt. %,
preferably less than 0.2 wt. %, preferably less than 0.1 wt. %,
preferably less than 0.075 wt. %, preferably less than 0.05 wt. %,
preferably less than 0.04 wt. %, preferably less than 0.02 wt. %,
preferably less than 0.01 wt. %, preferably less than 0.004 wt. %,
based on the composition).
[0045] In one preferred aspect when the antimicrobial material
consists of nisin, the composition comprises each of carvacrol and
carvone in an amount of less than 1 wt. % based on the extract.
Preferably when the antimicrobial material consists of nisin the
composition comprises each of carvacrol and carvone in an amount of
less than 0.075 wt. %, preferably less than 0.05 wt. %, preferably
less than 0.04 wt. %, preferably less than 0.02 wt. %, preferably
less than 0.01 wt. %, preferably less than 0.004 wt. %, based on
the extract.
[0046] In one preferred aspect when the antimicrobial material
consists of nisin, the composition comprises each of carvacrol,
carvone and thymol in amounts of less than 1 wt. % based on the
extract. Preferably when the antimicrobial material consists of
nisin the composition comprises each of carvacrol, carvone and
thymol in an amount of less than 0.075 wt. %, preferably less than
0.05 wt. %, preferably less than 0.04 wt. %, preferably less than
0.02 wt. %, preferably less than 0.01 wt. %, preferably less than
0.004 wt. %, based on the extract.
[0047] In one preferred aspect when the antimicrobial material
consists of nisin, the composition comprises each of carvacrol,
carvone, thymol and eugenol in amounts of less than 1 wt. % based
on the extract. Preferably when the antimicrobial material consists
of nisin the composition comprises each of carvacrol, carvone,
thymol and eugenol in an amount of less than 0.075 wt. %,
preferably less than 0.05 wt. %, preferably less than 0.04 wt. %,
preferably less than 0.02 wt. %, preferably less than 0.01 wt. %,
preferably less than 0.004 wt. %, based on the extract.
[0048] In one preferred aspect the composition comprises each of
carvacrol and carvone in an amount of less than 1 wt. % based on
the extract. Preferably the composition comprises each of carvacrol
and carvone in an amount of less than 0.075 wt. %, preferably less
than 0.05 wt. %, preferably less than 0.04 wt. %, preferably less
than 0.02 wt. %, preferably less than 0.01 wt. %, preferably less
than 0.004 wt. %, based on the extract.
[0049] In one preferred aspect the composition comprises each of
carvacrol, carvone and thymol in amounts of less than 1 wt. % based
on the extract. Preferably the composition comprises each of
carvacrol, carvone and thymol in an amount of less than 0.075 wt.
%, preferably less than 0.05 wt. %, preferably less than 0.04 wt.
%, preferably less than 0.02 wt. %, preferably less than 0.01 wt.
%, preferably less than 0.004 wt. %, based on the extract.
[0050] In one preferred aspect the composition comprises each of
carvacrol, carvone, thymol and eugenol in amounts of less than 1
wt. % based on the extract. Preferably the composition comprises
each of carvacrol, carvone, thymol and eugenol in an amount of less
than 0.075 wt. %, preferably less than 0.05 wt. %, preferably less
than 0.04 wt. %, preferably less than 0.02 wt. %, preferably less
than 0.01 wt. %, preferably less than 0.004 wt. %, based on the
extract.
[0051] The extract used in the present invention is obtained from
or is obtainable from a plant of the Labiatae family.
[0052] In one aspect the extract used in the present invention is
obtained from a plant of the Labiatae family.
[0053] It will be appreciated by one skilled in the art that by the
term "extract" or "extracts" it is meant any constituent of the
plant which may be isolated from the whole plant.
[0054] In one aspect the extract used in the present invention is
obtainable from a plant of the Labiatae family. It will be
appreciated by one skilled in the art that an extract obtainable
from a plant may be obtained from a plant or may be isolated from
the plant, identified and then obtained from an alternative source,
for example by chemical synthesis or enzymatic production. For
example the extract may be produced by a eukaryotic or prokaryotic
fermentation, by a process of genetic manipulation. The present
applicant have recognised that products present in a plant of the
Labiatae family may synergistically increase the activity of a an
antimicrobial material, preferably a bacteriocin. These products
may be obtained from any source and will fall within the scope of
the present invention.
[0055] The invention comprises use of a combination of a
bacteriocin such as nisin and a of the Labiatae plant family, such
as rosemary (Rosmarinus officinalls) or sage (Salvia officinalis)
that together give enhanced control of Gram-positive bacteria in a
food system. The extracts responsible for synergy in the present
invention preferably refer to extracts of the plant family Labiatae
that have been selectively extracted ("deodorised extracts") to
increase their phenolic diterpene content (such as carnosol and
carnosic acid), phenolic tripterpene content (such as ursolic acid,
betulinic acid and oleanolic acid) or rosmarinic acid content.
These deodorised extracts can be distinguished by their high
phenolic diterpene content (for example greater than 3.5 wt. %) and
their low level (less than 1 wt. %) of flavour-inducing compounds
from plant essential oils and oleoresins that are used as flavours
or fragrances. Essential oils are typically extracted by simple
steam distillation of the plant material.
[0056] Essential oils comprise the various essential oils in plants
having the odour or the flavour of the plant from which they were
extracted. The essential oils are typically terpenoids often
comprising monoterpenes. For example an antioxidant type of
rosemary extract, which could be described as selectively extracted
or deodorised, contains >3.5% phenolic diterpenes but less than
1 wt. % essential oils. A non-selective, flavouring extract
contains 10-30 wt. % essential oils and a phenolic diterpene
content of 2->3.5 wt. %.
[0057] An essential oil is commonly described as the volatile
ethereal fraction obtained from a plant or plant part by a physical
separation process such as distillation or chromatographic
separation. Essential oils have also been described as a "group of
odorous principles, soluble in alcohol and to a limited extent in
water, consisting of a mixtures of esters, aldehydes, ketones and
terpenes. Essential oils are typically obtained by distilling
plants with water; the oil that separates from distillate usually
has highly characteristic odors identified with the plant origin.
The resulting mixture of organic compounds was thought, in the days
of alchemists, to be the essence of the plant, hence the term
"essential oil".
[0058] In one preferred aspect the extract is a deodorised extract.
Preferably the (deodorised) extract contains from 1.0 to 70 wt. %
phenolic diterpenes, preferably 3.5 to 70 wt. % phenolic diterpenes
and less than 1 wt. % essential oil.
[0059] In one preferred aspect the extract is selected from
phenolic diterpenes, phenolic triterpenes and rosmarinic acid.
[0060] In one preferred aspect the extract is or comprises a
phenolic diterpene. Preferably the phenolic diterpene is selected
from carnosic acid, carnosol and methylcarnosic acid. Preferably
the phenolic diterpene is selected from carnosic acid and
carnosol.
[0061] In one preferred aspect the combined amount of phenolic
diterpenes, and phenolic triterpenes and rosmarinic acid, based on
the extract, is greater than 1.0 wt. %. In one preferred aspect the
combined amount of phenolic diterpenes, and phenolic triterpenes
and rosmarinic acid, based on the composition, is greater than 1.0
wt. %.
[0062] In one preferred aspect the combined amount of phenolic
diterpenes, and phenolic triterpenes and rosmarinic acid, based on
the extract, is greater than 3.5 wt. %. In one preferred aspect the
combined amount of phenolic diterpenes, and phenolic triterpenes
and rosmarinic acid, based on the composition, is greater than 3.5
wt. %.
[0063] In one preferred aspect the amount of phenolic diterpenes,
based on the extract, is greater than 1.0 wt. %, for example
greater than 5.0 wt. %, greater than 10.0 wt. %, greater than 20.0
wt. %, or greater than 25.0 wt. %. In one preferred aspect the
amount of phenolic diterpenes, based on the composition, is greater
than 1.0 wt. %.
[0064] In one preferred aspect the amount of phenolic diterpenes,
based on the extract, is greater than 3.5 wt. %. In one preferred
aspect the amount of phenolic diterpenes, based on the composition,
is greater than 3.5 wt. %.
[0065] In one preferred aspect the amount of phenolic diterpenes,
based on the composition, is greater than 1.0 wt. %, preferably
greater than 2.0 wt. %, preferably greater than 3.0 wt. %,
preferably greater than 3.5 wt. %, preferably greater than 5.0 wt.
%, preferably greater than 10.0 wt. %, preferably greater than 20.0
wt. %, preferably greater than 40.0 wt. %, preferably greater than
50.0 wt. %.
[0066] In one preferred aspect the amount of phenolic diterpenes,
based on the composition, is from 2.0 to 2.5 wt. %, such as 2.3 wt.
%.
[0067] In one preferred aspect the amount of phenolic diterpenes,
based on the composition, is from 4.0 to 4.5 wt. %, such as 4.2 wt.
%.
[0068] In one preferred aspect the amount of phenolic diterpenes,
based on the extract, is greater than 1.0 wt. %, preferably greater
than 2.0 wt. %, preferably greater than 3.0 wt. %, preferably
greater than 3.5 wt. %, preferably greater than 5.0 wt. %,
preferably greater than 10.0 wt. %, preferably greater than 20.0
wt. %, preferably greater than 40.0 wt. %, preferably greater than
50.0 wt. %.
[0069] In one highly preferred aspect the extract contains one or
more phenolic triterpenes. Preferably the phenolic triterpenes are
selected from betulinic acid, oleanolic acid, and ursolic acid.
[0070] In one preferred aspect is or comprises a phenolic
triterpene. Preferably the phenolic triterpene is selected from
betulinic acid, oleanolic acid, and ursolic acid.
[0071] In one highly preferred aspect the amount of phenolic
triterpenes, based on the extract, is greater than 3.5 wt. %. In
one highly preferred aspect the amount of phenolic triterpenes,
based on the composition, is greater than 3.5 wt. %.
[0072] In one preferred aspect the extract is or comprises
rosmarinic acid.
[0073] In one preferred aspect the amount of rosmarinic acid, based
on the extract, is greater than 3.5 wt. %. In one preferred aspect
the amount of rosmarinic acid, based on the composition, is greater
than 3.5 wt. %.
[0074] In one preferred aspect the extract contains
flavour-inducing compounds and/or essential oils in an amount of
less than 1 wt. % based on the extract. In one preferred aspect the
extract contains flavour-inducing compounds and/or essential oils
in an amount of less than 1 wt. % based on the composition.
[0075] Typically flavour-inducing compounds and/or essential oils
are camphor, verbenone, borneol and alfa-terpineol.
[0076] In one preferred aspect the combined amount of camphor
present in the extract is less than 1 wt. % (preferably less than
0.2 wt. %, more preferably less than 0.15 wt. %, more preferably
less than 0.1 wt. %) based on the extract.
[0077] In one preferred aspect the combined amount of verbenone
present in the extract is less than 1 wt. % (preferably less than
0.2 wt. %, more preferably less than 0.15 wt. %, more preferably
less than 0.1 wt. %) based on the extract.
[0078] In one preferred aspect the combined amount of borneol
present in the extract is less than 1 wt. % (preferably less than
0.2 wt. %, more preferably less than 0.15 wt. %, more preferably
less than 0.1 wt. %) based on the extract.
[0079] In one preferred aspect the combined amount of
alfa-terpineol present in the extract is less than 1 wt. %
(preferably less than 0.2 wt. %, more preferably less than 0.15 wt.
%, more preferably less than 0.1 wt. %) based on the extract.
[0080] In one preferred aspect the combined amount of camphor,
verbenone, borneol and alfa-terpineol present in the extract is
less than 1 wt. % (preferably less than 0.2 wt. %, more preferably
less than 0.15 wt. %, more preferably less than 0.1 wt. %) based on
the extract.
[0081] In one preferred aspect the extract contain less than 1 wt.
% of plant essential oils and/or oleoresins based on the extract.
In one preferred aspect the extract contain less than 1 wt. % of
plant essential oils and/or oleoresins based on the
composition.
[0082] In one preferred aspect the extract contains essential oils
in an amount of less than 1 wt. % based on the extract. In one
preferred aspect the extract contains essential oils in an amount
of less than 1 wt. % based on the composition.
[0083] In one preferred aspect the plant of the Labiatae family is
selected from rosemary, sage, oregano, marjoram, mint, balm,
savoury and thyme. In one preferred aspect the plant of the
Labiatae family is selected from rosemary, sage, oregano, marjoram,
mint, balm, and savoury. It will be understood that these name
cover all species and varieties of plants known by these names.
[0084] In one preferred aspect the plant of the Labiatae family is
selected from rosemary (Rosmarinus officinalis L.), sage (Salvia
officinalis L.) oregano (Origanum vulgare L.), marjoram (Origanum
marjorana L.), mint (Mentha spp.), balm (Melissa officinalis L.),
savoury (Satureia hortensis), thyme (Thymus vulgaris L.).
[0085] In one preferred aspect the plant of the Labiatae family is
selected from rosemary (Rosmarinus officinalis L.), sage (Salvia
officinalis L.), oregano (Origanum vulgare L.), marjoram (Origanum
mailorana L.), mint (Mentha spp.), balm (Melissa officinalis L.),
and savoury (Satureia hortensis).
[0086] In one preferred aspect the plant of the Labiatae family is
selected from rosemary (Rosmarinus officinalis L.), sage (Salvia
officinalis L.), marjoram (Origanum marjorana L.), mint (Mentha
spp.), balm (Melissa officinalis L.), and savoury (Satureia
hortensis).
[0087] In one preferred aspect the plant of the Labiatae family is
rosemary.
[0088] In a further preferred aspect the phenolic diterpenes,
phenolic triterpenes and rosmarinic acid are obtained by chemical
synthesis.
[0089] Thus in highly preferred aspects the present invention
provides [0090] a composition comprising (a) an antimicrobial
material and (b) a compound selected from carnosic acid, carnosol,
methylcarnosic acid, betulinic acid, oleanolic acid, ursolic acid
and rosmarinic acid (preferably carnosic acid and carnosol). [0091]
a process for preventing and/or inhibiting the growth of, and/or
killing a microorganism in a material, the process comprising the
step of contacting the material with (a) a bacteriocin; and (b) a
compound selected from carnosic acid, carnosol, methylcarnosic
acid, betulinic acid, oleanolic acid, ursolic acid and rosmarinic
acid (preferably carnosic acid and carnosol). [0092] use of (a) an
antimicrobial material and (b) a compound selected from carnosic
acid, carnosol, methylcarnosic acid, betulinic acid, oleanolic
acid, ursolic acid and rosmarinic acid (preferably carnosic acid
and carnosol), for preventing and/or inhibiting the growth of,
and/or killing a micro-organism in a material. Microorganism
[0093] As discussed herein the present invention may prevent and/or
inhibit the growth of, and/or kill a micro-organism in a material.
This may be slowing or arresting a micro-organism, such a bacteria,
or by killing the micro-organism present on contact with the
present composition.
[0094] In one aspect the antimicrobial material and/or the extract
are present in an amount to provide a microbicidal or
microbiostatic effect.
[0095] In one aspect the bacteriocin and the extract are present in
an amount to provide a microbicidal or microbiostatic effect.
[0096] In one aspect the bacteriocin and the extract are present in
an amount to provide a microbicidal or microbiostatic synergistic
effect.
[0097] In one aspect the bacteriocin and the extract are present in
an amount to provide a microbicidal synergistic effect.
[0098] In a highly preferred aspect the microbicidal or
microbiostatic effect is a bactericidal or bacteriostatic
effect.
[0099] It is advantageous for the bactericidal or bacteriostatic
effect to be in respect of Gram-positive bacteria and Gram-negative
bacteria. Preferably the bactericidal or bacteriostatic effect is
in respect of Gram-positive bacteria.
[0100] In a preferred aspect the bactericidal or bacteriostatic
effect is in respect of an organism selected from Gram-positive
bacteria associated with food spoilage or foodborne disease
including Bacillus species, Bacillus subtilis, Bacillus cereus,
Listeria species, Listeria monocytogenes, lactic acid bacteria,
lactic acid spoilage bacteria, Lactobacillus species,
Staphylococcus aureus, Clostridium species, C. sporogenes, C.
tyrobutyricum.
[0101] In a preferred aspect the bactericidal or bacteriostatic
effect of the invention in combination with a chelating agent is in
respect of an organism selected from other micro-organisms
associated with food spoilage or foodborne disease, including
yeasts, moulds and Gram-negative bacteria including Escherichia
coli, Salmonella species, and Pseudomonas species.
[0102] In a preferred aspect the bactericidal or bacteriostatic
effect is in respect of an organism selected from Bacillus cereus
204, B. cereus Campden, B. cereus NCTC2599, B. subtilis Campden,
Clostridium sporogenes strain Campden, Clostridium sporogenes
strain 1.221, Clostridium sporogenes NCIMB1793, Listeria
monocytogenes 272, L. monocytogenes NCTC12426, L. monocytogenes
S23, Lactobacillus sake 272, Escherichia coil S15, E. coli CRA109,
Salmonella Typhimurium S29, Pseudomonas fluorescens 3756,
[0103] In a preferred aspect the bactericidal or bacteriostatic
effect is in respect of Listeria monocytogenes.
Foodstuff
[0104] The composition, process and use of the present invention
may prevent and/or inhibit the growth of, and/or kill a
micro-organism in any material. However, in view of the problems
associated with spoilage and contamination of foodstuffs and in
view of the particular effectiveness of the present invention in
foodstuffs, preferably the composition is a foodstuff or may be
added to a foodstuff. It will be appreciated by one skilled in the
art that when the present composition is a foodstuff the essential
components of (a) an antimicrobial material and (b) a extract
obtained from or obtainable from a plant of the Labiatae family are
already present in the foodstuff. They may have been provided by
one or more means. For example they may have been added in the form
of a composition containing the bacteriocin and the extract. The
two components (the bacteriocin and the afore mentioned extract)
may have been added to the foodstuff sequentially. In one further
aspect one or more of the components may have be formed in situ in
the foodstuff. For example the bacteriocin may be formed in situ in
the foodstuff by fermentation of the dairy starter culture
bacterium Lactococcus lactis subsp. lactis.
[0105] In one aspect the composition of the present invention is a
protectant composition suitable for addition to a foodstuff.
[0106] Many foodstuffs may be protected by the present invention.
Typical foodstuffs are raw meat, cooked meat, raw poultry products,
cooked poultry products, raw seafood products, cooked seafood
products, ready to eat meals, pasta sauces, pasteurised soups,
mayonnaise, salad dressings, oil-in-water emulsions, margarines,
low fat spreads, water-in-oil emulsions, dairy products, cheese
spreads, processed cheese, dairy desserts, flavoured milks, cream,
fermented milk products, cheese, butter, condensed milk products,
ice cream mixes, soya products, pasteurised liquid egg, bakery
products, confectionery products, fruit products, and foods with
fat-based or water-containing fillings.
Additional Components
[0107] The compositon of the present invention or the composition
for use in the present invention may contain one or more additional
components. However, in some aspects the protectant composition of
the present invention (suitable for addition to a foodstuff)
contains no additional components or contains no additional
components that materially affect the properties of the
composition. In these aspects the present invention provides [0108]
a composition consisting essentially of (a) a bacteriocin and (b)
an extract obtained from or obtainable from a plant of the Labiatae
family, wherein (a) and (b) are different [0109] a composition
consisting of (a) a bacteriocin and (b) an extract obtained from or
obtainable from a plant of the Labiatae family, wherein (a) and (b)
are different [0110] a composition consisting essentially of (a) a
bacteriocin and (b) an extract obtained from or obtainable from a
plant of the Labiatae family, wherein (a) and (b) are different,
wherein when the antimicrobial material consists of nisin, the
composition comprises carvacrol in an amount of less than 0.075 wt.
% based on the composition and carvone in an amount of less than 15
wt. % based on the composition. [0111] a composition consisting of
(a) a bacteriocin and (b) an extract obtained from or obtainable
from a plant of the Labiatae family, wherein (a) and (b) are
different, wherein when the antimicrobial material consists of
nisin, the composition comprises carvacrol in an amount of less
than 0.075 wt. % based on the composition and carvone in an amount
of less than 15 wt. % based on the composition.
[0112] In one preferred aspect the composition further comprises an
emulsifier. Preferably the emulsifier is selected from
polyoxy-ethylene sorbitan esters (E432-E436) otherwise known as
polysorbates (e.g. Tween 80, Tween 20), monoglycerides,
diglycerides, acetic acid esters of mono-diglycerides, tartaric
acid esters of mono-diglycerides and citric acid esters of
mono-diglycerides.
[0113] In one preferred aspect the composition further comprises a
chelator. Preferably the chelator is selected from EDTA, citric
acid, monophosphates, diphosphates, triphosphates and
polyphosphates.
[0114] Further suitable chelator are taught in U.S. Pat. No.
5,573,801 and include carboxylic acids, polycarboxylic acids, amino
acids and phosphates. In particular, the following compounds and
their salts may be useful:
[0115] Acetic acid, Adenine, Adipic acid, ADP, Alanine, B-Alanine,
Albumin, Arginine, Ascorbic acid, Asparagine, Aspartic acid, ATP,
Benzoic acid, n-Butyric acid, Casein, Citraconic acid, Citric acid,
Cysteine, Dehydracetic acid, Desferri-ferrichrysin,
Desferri-ferrichrome, Desferri-ferrioxamin E, 3,4-Dihydroxybenzoic
acid, Diethylenetriaminepentaacetic acid (DTPA), Dimethylglyoxime,
O,O-Dimethylpurpurogallin, EDTA, Formic acid, Fumaric acid,
Globulin, Gluconic acid, Glutamic acid, Glutaric acid, Glycine,
Glycolic acid, Glycylglycine, Glycylsarcosine, Guanosine,
Histamine, Histidine, 3-Hydroxyflavone, Inosine, Inosine
triphosphate, Iron-free ferrichrome, Isovaleric acid, Itaconic
acid, Kojic acid, Lactic acid, Leucine, Lysine, Maleic acid, Malic
acid, Methionine, Methylsalicylate, Nitrilotriacetic acid (NTA),
Omithine, Orthophosphate, Oxalic acid, Oxystearin, B-Phenylalanine,
Phosphoric acid, Phytate, Pimelic acid, Pivalic acid,
Polyphosphate, Proline, Propionic acid, Purine, Pyrophosphate,
Pyruvic acid, Riboflavin, Salicylaldehyde, Salicyclic acid,
Sarcosine, Serine, Sorbitol, Succinic acid, Tartaric acid,
Tetrametaphosphate, Thiosulfate, Threonine, Trimetaphosphate,
Triphosphate, Tryptophan, Uridine diphosphate, Uridine
triphosphate, n-Valeric acid, Valine, and Xanthosine
[0116] Many of the above sequestering agents are useful in food
processing in their salt forms, which are commonly alkali metal or
alkaline earth salts such as sodium, potassium or calcium or
quaternary ammonium salts. Sequestering compounds with multiple
valencies may be beneficially utilised to adjust pH or selectively
introduce or abstract metal ions e.g. in a food system coating.
Additional information chelators is disclosed in T. E. Furia (Ed.),
CRC Handbook of Food Additives, 2nd Ed., pp. 271-294 (1972,
Chemical Rubber Co.), and M. S. Peterson and A. M. Johnson (Eds.),
Encyclopaedia of Food Science, pp. 694-699 (1978, AVI Publishing
Company, Inc.) which articles are both hereby incorporated by
reference.
[0117] The terms "chelator" is defined as organic or inorganic
compounds capable of forming co-ordination complexes with metals.
Also, as the term "chelator" is used herein, it includes molecular
encapsulating compounds such as cyclodextrin. The chelator may be
inorganic or organic, but preferably is organic.
[0118] Preferred chelator are non-toxic to mammals and include
aminopolycarboxylic acids and their salts such as
ethylenediaminetetraacetic acid (EDTA) or its salts (particularly
its di- and tri-sodium salts), and hydrocarboxylic acids and their
salts such as citric acid. However, non-citric acid and non-citrate
hydrocarboxylic acid chelators are also believed useful in the
present invention such as acetic acid, formic acid, lactic acid,
tartaric acid and their salts.
[0119] As noted above, the term "chelator" is defined and used
herein as a synonym for sequestering agent and is also defined as
including molecular encapsulating compounds such as cyclodextrin.
Cyclodextrins are cyclic carbohydrate molecules having six, seven,
or eight glucose monomers arranged in a donut shaped ring, which
are denoted alpha, beta or gamma cyclodextrin, respectively. As
used herein, cyclodextrin refers to both unmodified and modified
cyclodextrin monomers and polymers. Cyclodextrin molecular
encapsulators are commercially available from American
Maize-Products of Hammond, Ind. Cyclodextrin are further described
in Chapter 11 entitled, "Industrial Applications of Cyclodextrin",
by J. Szejtli, page 331-390 of Inclusion Compounds, Vol. III
(Academic Press, 1984) which chapter is hereby incorporated by
reference.
[0120] Preferably the chelator enhances the antimicrobial activity
and/or antimicrobial spectrum of the bacteriocin. More preferably
the chelator enhances the antimicrobial activity and/or
antimicrobial spectrum of the bacteriocin in respect of
Gram-negative bacteria and other micro-organisms.
[0121] In one preferred aspect the composition further comprises a
lytic enzyme. Preferably the lytic enzyme is a lysozyme.
Process
[0122] As discussed herein in one aspect the present invention
provides process for preventing and/or inhibiting the growth of,
and/or killing a micro-organism in a material, the process
comprising the step of contacting the material with (a) an
antimicrobial material; and (b) an extract obtained from or
obtainable from a plant of the Labiatae family, wherein (a) and (b)
are different; wherein the composition contains phenolic diterpenes
in an amount of greater than 1.0 wt. %, based on the composition,
and wherein when the antimicrobial material consists of nisin, the
composition comprises carvacrol in an amount of less than 0.075 wt.
% based on the composition and carvone in an amount of less than 15
wt. % based on the composition.
[0123] In one aspect the bacteriocin and the extract are added to
the material together.
[0124] In one aspect the bacteriocin and the extract are added to
the material sequentially.
[0125] Thus the present invention provides in one aspect a
preservative/protectant composition which may be added to a range
of materials such as food systems and in another aspect a
combination of two separate products which may added sequentially
to materials such as food products.
[0126] In one aspect the extract is added to the material.
[0127] In one aspect the bacteriocin is added to the material.
[0128] In one aspect the extract is formed in situ in the
material.
[0129] In one aspect the bacteriocin is formed in situ in the
material. Preferably when the bacteriocin is nisin, the bacteriocin
may be formed in situ in the foodstuff by fermentation of the dairy
starter culture bacterium Lactococcus lactis subsp. lactis.
Highly Preferred Aspects
[0130] As discussed herein in one aspect the present invention
provides a process for preventing and/or inhibiting the growth of,
and/or killing a micro-organism in a material, the process
comprising the step of contacting the material with (a) an
antimicrobial material; and (b) an extract obtained from or
obtainable from a plant of the Labiatae family, wherein (a) and (b)
are different; wherein when the antimicrobial material consists of
nisin, the composition comprises carvacrol in an amount of less
than 0.075 wt. % based on the composition and carvone in an amount
of less than 15 wt. % based on the composition.
[0131] In one aspect the present invention provides use of (a) an
antimicrobial material; and b) an extract obtained from or
obtainable from a plant of the Labiatae family, for preventing
and/or inhibiting the growth of, and/or killing a micro-organism in
a material; wherein (a) and (b) are different; and wherein when the
antimicrobial material consists of nisin, the composition comprises
carvacrol in an amount of less than 0.075 wt. % based on the
composition and carvone in an amount of less than 15 wt. % based on
the composition.
[0132] Some highly preferred aspects of the present invention are
set out below [0133] a composition comprising (a) a bacteriocin,
wherein the bacteriocin is nisin; and (b) an extract obtained from
a plant of the Labiatae family, wherein the composition contains
phenolic diterpenes in an amount of greater than 1.0 wt. %, based
on the composition, and wherein the composition comprises carvacrol
in an amount of less than 0.075 wt. % based on the composition and
carvone in an amount of less than 15 wt. % based on the
composition. [0134] for preventing and/or inhibiting the growth of,
and/or killing a micro-organism in a material, the process
comprising the step of contacting the material with (a) a
bacteriocin; wherein the bacteriocin is nisin; and (b) an extract
obtained from or obtainable from a plant of the Labiatae family,
wherein the composition contains phenolic diterpenes in an amount
of greater than 1.0 wt. %, based on the composition, and wherein
the composition comprises carvacrol in an amount of less than 0.075
wt. % based on the composition and carvone in an amount of less
than 15 wt. % based on the composition. [0135] use of (a) a
bacteriocin, wherein the bacteriocin is nisin; and (b) an extract
obtained from or obtainable from a plant of the Labiatae family,
for preventing and/or inhibiting the growth of, and/or killing a
micro-organism in a material, wherein the composition contains
phenolic diterpenes in an amount of greater than 1.0 wt. %, based
on the composition, and the composition comprises carvacrol in an
amount of less than 0.075 wt. % based on the composition and
carvone in an amount of less than 15 wt. % based on the composition
[0136] a composition comprising (a) a bacteriocin, and (b) an
extract obtained from a plant of the Labiatae family selected from
rosemary, sage, thyme, mint, balm, savoury and oregano, wherein (a)
and (b) are different [0137] a process for preventing and/or
inhibiting the growth of, and/or killing a microorganism in a
material, the process comprising the step of contacting the
material with (a) a bacteriocin; and (b) an extract obtained from
or obtainable from a plant of the Labiatae family selected from
rosemary, sage, thyme, mint, balm, savoury and oregano, wherein (a)
and (b) are different [0138] use of (a) a bacteriocin, and (b) an
extract obtained from or obtainable from a plant of the Labiatae
family selected from rosemary, sage, thyme, mint, balm, savoury and
oregano, for preventing and/or inhibiting the growth of, and/or
killing a micro-organism in a material, wherein (a) and (b) are
different [0139] a composition comprising (a) a bacteriocin, and
(b) a compound selected from carnosic acid, carnosol,
methylcarnosic acid, betulinic acid, oleanolic acid, ursolic acid
and rosmarinic acid. [0140] a process for preventing and/or
inhibiting the growth of, and/or killing a microorganism in a
material, the process comprising the step of contacting the
material with (a) a bacteriocin, and (b) a compound selected from
carnosic acid, carnosol, methylcarnosic acid, betulinic acid,
oleanolic acid, ursolic acid and rosmarinic acid. [0141] use of (a)
a bacteriocin, and (b) a compound selected from carnosic acid,
carnosol, methylcarnosic acid, betulinic acid, oleanolic acid,
ursolic acid and rosmarinic acid, for preventing and/or inhibiting
the growth of, and/or killing a micro-organism in a material.
[0142] a composition comprising (a) a bacteriocin, and (b) carnosic
acid. [0143] a process for preventing and/or inhibiting the growth
of, and/or killing a microorganism in a material, the process
comprising the step of contacting the material with (a) a
bacteriocin, and (b) carnosic acid. [0144] use of (a) a
bacteriocin, and (b) carnosic acid, for preventing and/or
inhibiting the growth of, and/or killing a micro-organism in a
material. [0145] a composition comprising (a) a bacteriocin,
wherein the bacteriocin is nisin; and (b) an extract obtained from
or obtainable from a plant of the Labiatae family selected from
rosemary, thyme, mint, balm, savoury, sage and oregano, wherein the
composition contains phenolic diterpenes in an amount of greater
than 1.0 wt. %, based on the composition, and wherein the
composition comprises carvacrol in an amount of less than 0.075 wt.
% based on the composition and carvone in an amount of less than 15
wt. % based on the composition. [0146] a process for preventing
and/or inhibiting the growth of, and/or killing a microorganism in
a material, the process comprising the step of contacting the
material with (a) a bacteriocin; wherein the bacteriocin is nisin;
and (b) a selectively extracted extract obtained from or obtainable
from a plant of the Labiatae family selected from rosemary, sage,
thyme, mint, balm, savoury and oregano, wherein the composition
contains phenolic diterpenes in an amount of greater than 1.0 wt.
%, based on the composition, and wherein the composition comprises
carvacrol in an amount of less than 0.075 wt. % based on the
composition and carvone in an amount of less than 15 wt. % based on
the composition. [0147] use of (a) a bacteriocin, wherein the
bacteriocin is nisin; and (b) a selectively extracted extract
obtained from or obtainable from a plant of the Labiatae family
selected from rosemary, sage, thyme, mint, balm, savoury and
oregano, for preventing and/or inhibiting the growth of, and/or
killing a micro-organism in a material, wherein the composition
contains phenolic diterpenes in an amount of greater than 1.0 wt.
%, based on the composition, and wherein the composition comprises
carvacrol in an amount of less than 0.075 wt. % based on the
composition and carvone in an amount of less than 15 wt. % based on
the composition. [0148] a composition comprising (a) a bacteriocin,
wherein the bacteriocin is nisin; and (b) a compound selected from
carnosic acid, carnosol, methylcarnosic acid, betulinic acid,
oleanolic acid, ursolic acid and rosmarinic acid, wherein the
composition contains phenolic diterpenes in an amount of greater
than 1.0 wt. %, based on the composition, and wherein the
composition comprises carvacrol in an amount of less than 0.075 wt.
% based on the composition and carvone in an amount of less than 1
5 wt. % based on the composition. [0149] a process for preventing
and/or inhibiting the growth of, and/or killing a microorganism in
a material, the process comprising the step of contacting the
material with (a) a bacteriocin, wherein the bacteriocin is nisin;
and (b) a compound selected from carnosic acid, carnosol,
methylcarnosic acid, betulinic acid, oleanolic acid, ursolic acid
and rosmarinic acid, wherein the composition contains phenolic
diterpenes in an amount of greater than 1.0 wt. %, based on the
composition, and wherein the composition comprises carvacrol in an
amount of less than 0.075 wt. % based on the composition and
carvone in an amount of less than 15 wt. % based on the
composition. [0150] use of (a) a bacteriocin, wherein the
bacteriocin is nisin; and (b) a compound selected from carnosic
acid, carnosol, methylcarnosic acid, betulinic acid, oleanolic
acid, ursolic acid and rosmarinic acid, for preventing and/or
inhibiting the growth of, and/or killing a micro-organism in a
material, wherein the composition contains phenolic diterpenes in
an amount of greater than 1.0 wt. %, based on the composition, and
wherein the composition comprises carvacrol in an amount of less
than 0.075 wt. % based on the composition and carvone in an amount
of less than 15 wt. % based on the composition. [0151] a
composition comprising (a) a bacteriocin, wherein the bacteriocin
is nisin; and (b) carnosic acid. [0152] a process for preventing
and/or inhibiting the growth of, and/or killing a microorganism in
a material, the process comprising the step of contacting the
material with (a) a bacteriocin, wherein the bacteriocin is nisin;
and (b) carnosic acid. [0153] use of (a) a bacteriocin, wherein the
bacteriocin is nisin; and (b) carnosic acid, for preventing and/or
inhibiting the growth of, and/or killing a micro-organism in a
material. [0154] a composition comprising (a) a bacteriocin,
wherein the bacteriocin is nisin; and (b) an extract obtained from
a plant of the Labiatae family selected from rosemary, sage, thyme,
mint, balm, savoury and oregano, wherein the bacteriocin and the
extract are present in an amount to provide a bactericidal or
bacteriostatic synergistic effect, wherein the composition contains
phenolic diterpenes in an amount of greater than 1.0 wt. %, based
on the composition, and wherein the composition comprises carvacrol
in an amount of less than 0.075 wt. % based on the composition and
carvone in an amount of less than 15 wt. % based on the
composition. [0155] a process for preventing and/or inhibiting the
growth of, and/or killing a microorganism in a material, the
process comprising the step of contacting the material with (a) a
bacteriocin; wherein the bacteriocin is nisin; and (b) an extract
obtained from or obtainable from a plant of the Labiatae family
selected from rosemary, sage, thyme, mint, balm, savoury and
oregano, wherein the bacteriocin and the extract are present in an
amount to provide a bactericidal or bacteriostatic synergistic
effect wherein the composition contains phenolic diterpenes in an
amount of greater than 1.0 wt. %, based on the composition, and
wherein the composition comprises carvacrol in an amount of less
than 0.075 wt. % based on the composition and carvone in an amount
of less than 15 wt. % based on the composition. [0156] use of (a) a
bacteriocin, wherein the bacteriocin is nisin; and (b) an extract
obtained from or obtainable from a plant of the Labiatae family
selected from rosemary, sage, thyme, mint, balm, savoury and
oregano, for preventing and/or inhibiting the growth of, and/or
killing a micro-organism in a material, wherein the bacteriocin and
the extract are present in an amount to provide a bactericidal or
bacteriostatic synergistic effect, wherein the composition contains
phenolic diterpenes in an amount of greater than 1.0 wt. %, based
on the composition, and wherein the composition comprises carvacrol
in an amount of less than 0.075 wt. % based on the composition and
carvone in an amount of less than 15 wt. % based on the
composition. [0157] a composition comprising (a) a bacteriocin,
wherein the bacteriocin is nisin; and (b) a compound selected from
carnosic acid, carnosol, methylcarnosic acid, betulinic acid,
oleanolic acid, ursolic acid and rosmarinic acid, wherein the
bacteriocin and the compound are present in an amount to provide a
bactericidal or bacteriostatic synergistic effect wherein the
composition contains phenolic diterpenes in an amount of greater
than 1.0 wt. %, based on the composition, and wherein the
composition comprises carvacrol in an amount of less than 0.075 wt.
% based on the composition and carvone in an amount of less than 15
wt. % based on the composition. [0158] a process for preventing
and/or inhibiting the growth of, and/or killing a microorganism in
a material, the process comprising the step of contacting the
material with (a) a bacteriocin, wherein the bacteriocin is nisin;
and (b) a compound selected from carnosic acid, carnosol,
methylcarnosic acid, betulinic acid, oleanolic acid, ursolic acid
and rosmarinic acid, wherein the bacteriocin and the compound are
present in an amount to provide a bactericidal or bacteriostatic
synergistic effect wherein the composition contains phenolic
diterpenes in an amount of greater than 1.0 wt. %, based on the
composition, and wherein the composition comprises carvacrol in an
amount of less than 0.075 wt. % based on the composition and
carvone in an amount of less than 15 wt. % based on the
composition. [0159] use of (a) a bacteriocin, wherein the
bacteriocin is nisin; and (b) a compound selected from carnosic
acid, carnosol, methylcarnosic acid, betulinic acid, oleanolic
acid, ursolic acid and rosmarinic acid, for preventing and/or
inhibiting the growth of, and/or killing a micro-organism in a
material, wherein the bacteriocin and the compound are present in
an amount to provide a bactericidal or bacteriostatic synergistic
effect wherein the composition contains phenolic diterpenes in an
amount of greater than 1.0 wt. %, based on the composition, and
wherein the composition comprises carvacrol in an amount of less
than 0.075 wt. % based on the composition and carvone in an amount
of less than 15 wt. % based on the composition. [0160] a
composition comprising (a) a bacteriocin, wherein the bacteriocin
is nisin; and (b) a compound selected from carnosic acid, wherein
the bacteriocin and the compound are present in an amount to
provide a bactericidal or bacteriostatic synergistic effect [0161]
a process for preventing and/or inhibiting the growth of, and/or
killing a microorganism in a material, the process comprising the
step of contacting the material with (a) a bacteriocin, wherein the
bacteriocin is nisin; and (b) a compound selected from carnosic
acid, wherein the bacteriocin and the compound are present in an
amount to provide a bactericidal or bacteriostatic synergistic
effect use of (a) a bacteriocin, wherein the bacteriocin is nisin;
and (b) a compound selected from carnosic acid, for preventing
and/or inhibiting the growth of, and/or killing a micro-organism in
a material, wherein the bacteriocin and the compound are present in
an amount to provide a bactericidal or bacteriostatic synergistic
effect [0162] a composition comprising (a) a bacteriocin, wherein
the bacteriocin is nisin; and (b) an extract obtained from a plant
of the Labiatae family, wherein the composition contains phenolic
diterpenes in an amount of greater than 1.0 wt. %, based on the
composition, wherein the composition comprises carvacrol in an
amount of less than 0.04 wt. % based on the composition and carvone
in an amount of less than 0.04.% based on the composition. [0163] a
process for preventing and/or inhibiting the growth of, and/or
killing a microorganism in a material, the process comprising the
step of contacting the material with (a) a bacteriocin; wherein the
bacteriocin is nisin; and (b) an extract obtained from or
obtainable from a plant of the Labiatae family, wherein the
composition contains phenolic diterpenes in an amount of greater
than 1.0 wt. %, based on the composition, wherein the composition
comprises carvacrol in an amount of less than 0.04 wt. % based on
the composition and carvone in an amount of less than 0.04.% based
on the composition. [0164] use of (a) a bacteriocin, wherein the
bacteriocin is nisin; and (b) an extract obtained from or
obtainable from a plant of the Labiatae family, for preventing
and/or inhibiting the growth of, and/or killing a micro-organism in
a material, wherein the composition contains phenolic diterpenes in
an amount of greater than 1.0 wt. %, based on the composition,
wherein the composition comprises carvacrol in an amount of less
than 0.04 wt. % based on the composition and carvone in an amount
of less than 0.04.% based on the composition.
[0165] Further broad aspects of the present invention are defined
below: [0166] a composition comprising (a) an antimicrobial
material; and (b) an extract obtained from or obtainable from a
plant of the Labiatae family, wherein (a) and (b) are different;
wherein the composition contains phenolic diterpenes in an amount
of greater than 1.0 wt. %, based on the composition. [0167] a
process for preventing and/or inhibiting the growth of, and/or
killing a microorganism in a material, the process comprising the
step of contacting the material with (a) an antimicrobial material;
and (b) an extract obtained from or obtainable from a plant of the
Labiatae family, wherein (a) and (b) are different; wherein the
composition contains phenolic diterpenes in an amount of greater
than 1.0 wt. %. [0168] use of (a) an antimicrobial material; and b)
an extract obtained from or obtainable from a plant of the Labiatae
family, for preventing and/or inhibiting the growth of, and/or
killing a micro-organism in a material; wherein (a) and (b) are
different; wherein the composition contains phenolic diterpenes in
an amount of greater than 1.0 wt. %. [0169] a kit for preparing a
composition as defined herein, the kit comprising (a) an
antimicrobial material; and (b) an extract obtained from or
obtainable from a plant of the Labiatae family, wherein (a) and (b)
are different; wherein the composition contains phenolic diterpenes
in an amount of greater than 1.0 wt. %, based on the composition.
[0170] a composition comprising (a) an antimicrobial material; and
(b) an extract obtained from or obtainable from a plant of the
Labiatae family, wherein (a) and (b) are different; wherein when
the antimicrobial material consists of nisin, the composition
comprises carvacrol in an amount of less than 0.075 wt. % based on
the composition and carvone in an amount of less than 15 wt. %
based on the composition. [0171] a process for preventing and/or
inhibiting the growth of, and/or killing a microorganism in a
material, the process comprising the step of contacting the
material with (a) an antimicrobial material; and (b) an extract
obtained from or obtainable from a plant of the Labiatae family,
wherein (a) and (b) are different; wherein when the antimicrobial
material consists of nisin, the composition comprises carvacrol in
an amount of less than 0.075 wt. % based on the composition and
carvone in an amount of less than 15 wt. % based on the
composition. [0172] use of (a) an antimicrobial material; and b) an
extract obtained from or obtainable from a plant of the Labiatae
family, for preventing and/or inhibiting the growth of, and/or
killing a micro-organism in a material; wherein (a) and (b) are
different; wherein when the antimicrobial material consists of
nisin, the composition comprises carvacrol in an amount of less
than 0.075 wt. % based on the composition and carvone in an amount
of less than 1 5 wt. % based on the composition. [0173] a kit for
preparing a composition as defined herein, the kit comprising (a)
an antimicrobial material; and (b) an extract obtained from or
obtainable from a plant of the Labiatae family, wherein (a) and (b)
are different; wherein when the antimicrobial material consists of
nisin, the composition comprises carvacrol in an amount of less
than 0.075 wt. % based on the composition and carvone in an amount
of less than 15 wt. % based on the composition, in separate
packages or containers; optionally with instructions for admixture
and/or contacting and/or use.
[0174] The present invention will now be described in further
detail by way of example only with reference to the accompanying
figures in which:
[0175] FIG. 1 is a graph showing synergistic enhancement of nisin
cidal activity against Listeria monocytogenes in chicken soup at
25.degree. C. by a selectively extracted rosemary extract
[0176] FIG. 2 is a graph showing synergistic enhancement of nisin
control of Listeria monocytogenes growth in chilled chicken soup by
a selectively extracted rosemary extract (GRE09)
[0177] FIG. 3 is a graph showing synergistic enhancement by a
selectively extracted rosemary extract of nisin control of B.
cereus spore outgrowth in chilled chicken soup. Minimal detection
limit was 100 cfu/g. For the length of the testing period, the
samples containing the combination of nisin and rosemary had
Bacillus counts at or below 100 cfu/g.
[0178] FIG. 4 is a graph showing combined effect of nisin,
selectively extracted rosemary extracts and rosemary extract
components against L. monocytogenes in chicken soup at 20.degree.
C. (Minimum detection limit 100 cfu/g)
[0179] FIG. 5 is a graph showing synergistic enhancement of nisin
activity by selectively extracted extracts of rosemary or
rosmarinic acid against Listeria monocytogenes in a chicken soup at
ambient temperature.
[0180] FIG. 6 is a graph showing a demonstration of synergy between
nisin and phenolic diterpene-containing rosemary extract.
Inhibition of L. monocytogenes at 8.degree. C.
[0181] FIG. 7 is a graph showing a demonstration of synergy between
nisin and phenolic diterpene-containing rosemary extract.
Inhibition of B. cereus at 15.degree. C.
[0182] FIG. 8 is a graph showing enhanced nisin growth inhibitory
activity by a phenolic diterpene-containing rosemary extract.
Control of L. monocytogenes in carbonara sauce at 8.degree. C.
[0183] FIG. 9 is a graph showing enhanced nisin growth inhibitory
activity by a phenolic diterpene-containing rosemary extract.
Control of B. cereus spores in carbonara sauce at 15.degree. C.
[0184] FIG. 10 is a graph showing enhanced cidal effect of a nisin
and phenolic diterpene-containing rosemary extract against L.
monocytogenes in chicken soup at 20.degree. C. a) pH 4.5
[0185] FIG. 11 is a graph showing enhanced cidal effect of a nisin
and phenolic diterpene-containing rosemary extract against L.
monocytogenes in chicken soup at 20.degree. C. b) pH 6.7
[0186] The present invention will now be described in further
detail in the following examples.
EXAMPLES
Experimental Evidence of Benefit
[0187] In vitro studies described herein have shown synergy between
nisin and extracts of Rosmarinus officinalis containing >3.5%
phenolic diterpenes, increasing the efficacy of nisin
significantly. This enhanced activity was also observed in food
models, increasing nisin kill and growth control of Gram-positive
bacteria. The experimental studies also demonstrated that the
phenolic diterpenes carnosic acid and carnosol were implicated in
this synergy. The results also indicated that rosmarinic acid may
also enhance nisin activity, although this synergistic effect was
not as strong as that observed with the phenolic diterpenes.
I) In vitro Demonstration of Nisin and Deodorised Rosemary Extract
Synergy
[0188] Materials: GUARDIAN.TM. Rosemary Extract 09 (Danisco)
(GRE09). This is a water dispersible deodorised rosemary extract
containing 4% phenolic diterpenes and <1% essential oils,
extracted from rosemary leaves, combined with the carriers
polyoxyethylene sorbitan monooleate (Tween 80) and propylene
glycol. A commercial extract of nisin at potency of
1.times.10.sup.6 IU/g: Nisaplin.RTM. Natural Antimicrobial
(Danisco).
[0189] Test strains: Bacillus cereus 204, B. cereus Campden, B.
cereus NCTC2599, B. subtilis Campden, Listeria monocytogenes 272,
L. monocytogenes NCTC12426, L. monocytogenes S23, Lactobacillus
sake 272, Escherichia coli S15, E. coli CRA109, Salmonella
Typhimurium S29, Pseudomonas fluorescens 3756.
[0190] Method of microbial growth curve analysis. A 100,000 ppm
GRE09 solution was prepared in water and filter sterilised (0.2
.mu.m). Further dilutions were prepared in sterile deionised water
at 1,250-20,000 ppm. Brain Heart Infusion broth (Oxoid) was
prepared and GRE09 stock solutions were added to give the following
test solutions of GRE09; 125, 250, 500, 750, 1000, 1250, 1500, 2000
ppm. A 10,000 IU/ml nisin solution was prepared, filter sterilised
and a range of stock solutions then prepared. A range of nisin
concentrations was then prepared in Brain Heart Infusion broth. A
fully automated microbial growth analyser was used to determine
microbial growth curves (Microbiology Reader Bioscreen C analyser
linked to a PC with installed software BioLink v 5.30; Labsystem
Oy, Finland). Tests were prepared in honeycomb 2 (HC 2)
microtitre/cuvette plates with a capacity of 100 wells per plate.
The wells were loaded with 270 .mu.l of the prepared media and
inoculated at a level of 10.sup.3 CFU (colony forming units)/ml
with 30 .mu.l of microbial suspension. Incubation time and
temperature was as appropriate for the test organism. This test
allowed suitable test levels for the compounds to be determined.
The rosemary extract and nisin were then tested in combination,
using the same procedure. Nisin solutions were prepared at 50-1000
IU/ml in broth as above. GRE09 solutions were prepared at 250, 500
and 1000 ppm as above. Combinations of all these test levels were
prepared and tested in the Bioscreen as before.
[0191] Results: The minimum inhibitory concentration of nisin
alone, rosemary extract GRE09 alone and the two in combination in
the Bioscreen after 48 h at 30.degree. C. is shown in Table 1. The
minimal inhibition was taken as the lowest concentration that
caused total inhibition of the bacteria after 48 h at 30.degree. C.
Synergy was observed between nisin and the rosemary extract GRE09
against all Gram-positive bacteria but no significant effect was
observed against Gram-negative bacteria. This can be determined
from the table by comparing the column of data showing MIC levels
of nisin alone, GRE09 alone and the two combined. The latter column
gave levels much lower than the other two for Gram-positive
bacteria (Bacillus, Listeria) but not for Gram-negative bacteria
(E. coli, Salmonella). TABLE-US-00001 TABLE 1 Synergy tests of
nisin and the rosemary extract GRE09 Other test levels of the MIC
in broth after 48 h at 30.degree. C. (total combination causing
total inhibition) inhibition Nisin GRE09 MIC of nisin Test organism
(IU/ml) (ppm) (IU/ml) + GRE09 (ppm) Nisin (IU/ml) + GRE09 (ppm) B.
cereus 204 500 >1000 50 + 250 50 + 500 50 + 1000 100 + 250 100 +
500 100 + 1000 200 + 250 200 + 500 200 + 1000 B. cereus 500
>1000 50 + 250 50 + 500 NCTC2599 50 + 1000 100 + 250 100 + 500
100 + 1000 200 + 250 200 + 500 200 + 1000 B. subtilis 100 >1000
50 + 250 50 + 500 Campden 50 + 1000 100 + 250 100 + 500 100 + 1000
200 + 250 200 + 500 200 + 1000 L. >500 >1000 50 + 250 50 +
500 monocytogenes 50 + 1000 S23 100 + 250 100 + 500 100 + 1000 200
+ 250 200 + 500 200 + 1000 L. >500 >1000 50 + 250 50 + 500
monocytogenes 50 + 1000 272 100 + 250 100 + 500 100 + 1000 200 +
250 200 + 500 200 + 1000 L. >500 >1000 50 + 250 50 + 500
monocytogenes 50 + 1000 12426 100 + 250 100 + 500 100 + 1000 200 +
250 200 + 500 200 + 1000 E. coli S15 >500 >1000 >1000 +
>1000 -- E. coli CRA109 >500 >1000 >1000 + >1000 --
S. Typhimurium >500 >1000 >1000 + >1000 -- S29 Ps.
fluorescens >500 >1000 >1000 + >1000 -- 3756
II) Demonstration of Nisin and Rosemary Extract GRE09 Synergy in
Food A) Synergy Against Listeria monocytogenes
[0192] Test compounds: GRE09 at 0.1%, 0.5%, Nisaplin.RTM.
(Danisco).
[0193] Test strains: a cocktail was prepared of L. monocytogenes
strains NCTC12426, NCTC5105, NCC FSM60 and CRA3930. The Listeria
strains were grown at 30.degree. C. on Brain heart infusion agar
overnight then inoculated into broth at 30.degree. C. overnight. A
volume of each broth was mixed together to give a cocktail of
strains with a cell concentration of approximately 10.sup.9
CFU/ml.
[0194] Media: A chilled pasteurised chicken soup was used as a food
model because it was a 15 good mix of different food components
including vegetables, dairy products and poultry meat It was
comprised of a chicken stock with the addition of chicken, cream,
vegetables, flour and seasonings. The pH was 6.12. After addition
of nisin and rosemary extract GRE09, the soup was pasteurised at a
core temperature of 80.degree. C. for 2 minutes. The Listeria
cocktail was diluted to 10.sup.4 CFU/ml and inoculated into soup
tests to give a final cell count of approximately 10.sup.2 CFU/g
(growth inhibitory tests) and 10.sup.7 CFU/ml (cidal tests). The
latter test was incubated at 25.degree. C. for 2 h and then tested
by viable count enumeration to estimate the extent of cidal
activity. The growth test was incubated at 8.degree. C. with
regular sampling to estimate bacteriostatic activity.
[0195] Results. The rosemary extract GRE09 alone at 0.5% showed no
listericidal activity. Nisin at 250 IU/g caused a 1 log drop in
Listeria numbers after 2 h, but only a slight delay in growth after
24 h (FIG. 1). In comparison the combination of the two test
products at these levels caused a 2-3 log drop in Listeria numbers
after 2 h. After 24 h the cells still had not recovered to their
initial inoculum level. This was a particularly harsh test for any
preservative system, since the test medium was a rich food model,
the incubation temperature was at ambient and the bacterial numbers
high. Therefore any enhanced nisin activity was a good indication
of synergy.
[0196] Incubation for the bacteriostatic test was for 43 days:
results of this are shown in FIG. 2 and Table 2. The nisin/rosemary
synergy was again clearly demonstrated in the food model against
the Listeria cocktail. For example, Listeria growth reached
10.sup.6 CFU/ml after 13 days in the presence of 100 IU/ml nisin;
after 10 days in the presence of 0.1% GRE09 but only after a much
longer period, 34 days, in the presence of the combination of these
two ingredients. Similarly, Listeria growth reached 10.sup.6 CFU/ml
after 13 days in the presence of 100 IU/ml nisin; after 20 days in
the presence of 0.5% GRE09. The combination of the two components
resulted in no growth being observed by then end of the test
period. TABLE-US-00002 TABLE 2 Summary of growth inhibition of
Listeria in chilled chicken soup (Trial lasted 43 days) Days until
growth Test conditions reached 10.sup.6 CFU/ml Control 6 Nisin at
100 IU/ml 13 Nisin at 250 IU/ml 27 Rosemary extract GRE09 at 0.1%
10 Rosemary extract GRE09 at 0.5% 20 Nisin (100 IU/ml) + GRE09 at
0.1% 34 Nisaplin (100 IU/ml) + GRE09 at 0.5% >43 Nisaplin (250
IU/ml) + GRE09 at 0.1% >43 Nisaplin (250 IU/ml) + GRE09 at 0.5%
>43
[0197] During the test period (a) Nisaplin (100 IU/ml) +GRE09 at
0.5%, (b) Nisaplin (250 IU/ml)+GRE09 at 0.1%, and (c) Nisaplin (250
IU/ml)+GRE09 at 0.5% did not give any total aerobic viable counts
above 100 cfu/g.
B) Synergy Against Bacillus cereus
[0198] Test strains: a cocktail of Bacillus spores was prepared as
an inoculum, using Bacillus cereus strain 204, Bacillus cereus
strain 199, B. cereus strain Campden, and B. cereus strain ABC
4/9.
[0199] Additions of the test compounds were made to chicken soup,
prepared as above. The soup was pasteurised at 70.degree. C. for 2
minutes, cooled and inoculated with approximately 10.sup.3 CFU/g of
a cocktail of Bacillus cereus spores. Incubation was for 56 days.
Results are shown in FIG. 3 and summarised in Table 3.
Bacteriostatic synergy between the nisin and rosemary extract GRE09
was evident. For example, spoilage (i.e. 10.sup.6 CFU/ml) resulted
after 13 days in the presence of 25 IU/ml nisin, and after 10 days
in the presence of 300 ppm GRE09. In the presence of both these
ingredients, no spoilage had occurred by the end of the trial (56
days). TABLE-US-00003 TABLE 3 Summary of results of chilled chicken
soup trial inoculated with Bacillus cereus spores (Trial lasted 70
days). Days until growth Test conditions reached 10.sup.6 CFU/ml
Control 6 Nisin at 25 IU/ml 13 Rosemary extract GRE09 at 300 ppm 10
Rosemary extract GRE09 at 600 ppm 13 Nisin (25 IU/ml) + GRE09 at
300 ppm >70 Nisaplin (25 IU/ml) + GRE09 at 600 ppm >70
C) Synergy Against Clostridium sporogenes
[0200] Test strains: a cocktail of Clostridium spores was prepared
as an inoculum, using Clostridium sporogenes strain Campden,
Clostridium sporogenes 1.221, and Clostridium sporogenes
NCIMB1793.
[0201] Additions of the test compounds were made to chicken soup,
prepared as above. The soup was pasteurised at 70.degree. C. for 2
minutes and transferred to sterile test tubes. These were
inoculated with a cocktail of heat-shocked Clostridium sporogenes
spores, at a level of 2.2.times.10.sup.2 CFU/g, then anaerobic
conditions were created by plugging the tubes with agar. The
samples were incubated at 37.degree. C. and checked daily for gas
production (observed by blowing of the gas plug and by the
distinctive clostridial odour). Results for a 27 day incubation
period, demonstrating synergy, are shown in Table 4. For example,
synergy was clearly seen by the combined effect of 50 IU/ml nisin
and 300 ppm GRE09, which prevented growth for 27 days (the length
of the trial), whereas the individual ingredients both prevented
clostridial growth for 2 days (the same as the control).
TABLE-US-00004 TABLE 4 Summary of results of chicken soup trial
inoculated with Clostridium sporogenes spores incubated at
37.degree. C. (Trial lasted 27 days). Days until growth Test
conditions observed (gas production) Control 2 Nisin at 25 IU/ml 2
Nisin at 50 IU/ml 2 Nisin at 100 IU/ml 7 Rosemary extract GRE09 at
300 ppm 2 Rosemary extract GRE09 at 600 ppm 2 Nisin (25 IU/ml) +
GRE09 at 300 ppm 3 Nisin (50 IU/ml) + GRE09 at 300 ppm >27 Nisin
(100 IU/ml) + GRE09 at 300 ppm >27 Nisaplin (25 IU/ml) + GRE09
at 600 ppm 10 Nisaplin (100 IU/ml) + GRE09 at 600 ppm >27
III) Demonstration of in vitro Synergy with Different Deodorised,
Selectively Extracted Rosemary Extracts and Rosmarinic Acid
[0202] Growth curves of Listeria monocytogenes and B. cereus
strains in laboratory media were analysed as described above using
the Bioscreen C analyser. Minimal inhibitory concentrations (MIC)
were determined for the test compounds used singly or in
combination after 24 h at 30.degree. C. Results are shown in Table
5. The test compounds comprised nisin (as Nisaplin.RTM.; Danisco),
GRE09 (Danisco), pure rosmarinic acid (RA; Sigma) and a range of
deodorised rosemary extracts. These had been prepared by selected
extraction with either organic solvents or CO.sub.2 to obtain
extracts containing 28% phenolic diterpenes (28RE; Danisco) and a
rosemary extract containing 6% rosmarinic acid (6RA; Danisco).
Enhanced nisin activity was evident with a combination of nisin
combined with pure rosmarinic acid (RA; this may have partly been
due to low pH levels), a combination of nisin with a rosemary
extract containing 6% rosmarinic acid (6RA) and a combination of
nisin with a deodorised rosemary extract containing 28% phenolic
diterpenes and <1% essential oils (28RE). The known nisin
synergy with Tween 80 was also observed. The other carrier
propylene glycol did not enhance nisin activity. The synergies can
be observed, as before, by comparing the MIC levels for nisin
alone, the other test compound, and the two together (see Table 5).
TABLE-US-00005 TABLE 5 MIC after growth at 30.degree. C. in
laboratory medium MIC in Bioscreen after 24 h at 30.degree. C. Test
organism Individual components Combination with nisin L.
monocytogenes Nisin at 1000 IU/ml -- strain S23 0.1% GRE09 0.05%
GRE09 + 50 IU/ml nisin 1% RA 0.25% RA + 250 IU/ml nisin 0.5% RA +
100 IU/ml nisin 0.75% RA + 50 IU/ml nisin 1% 6RA <0.1% 6RA + 250
IU/ml nisin 0.5% 6RA + 50 IU/ml nisin L. monocytogenes 500 IU/ml
nisin -- strain 272 0.25% GRE09 <0.05% GRE09 + 50 IU/ml nisin 1%
of RE28 <0.05% RE28 + 50 IU/ml nisin >2% Tween 80 0.5% Tween
80 + 250 IU/ml nisin L. monocytogenes 250 IU/ml nisin -- strain
NCTC12426 0.25% GRE09 <0.05% GR 09 + 50 IU/ml nisin 1% of RE28
<0.05% RE28 + 50 IU/ml nisin >2% Tween 80 0.5% Tween 80 + 100
IU/ml nisin B. cereus 500 IU/ml nisin -- Campden spores 0.1% GRE09
0.05% GRE09 + 50 IU/ml 1% RA 0.5% RA + 250 IU/ml nisin 0.75% RA +
100 IU/ml nisin 0.75% RA + 50 IU/ml nisin 1% 6RA 0.25% 6RA + 100
IU/ml nisin 0.5% 6RA + 50 IU/ml nisin
IV) Demonstration of Synergy for Nisin Activity with Different
Deodorised Rosemary Extract Components in Food
[0203] Test strains: Listeria monocytogenes strains 272, CRA3930
and NCTC12426
[0204] The chicken soup model was used as before. The following
samples were tested: GRE09, deodorised rosemary extracts containing
28% or 70% phenolic diterpenes (RE28 and RE70; Danisco), a water
soluble rosemary extract containing 6% rosmarinic acid (6RA;
Danisco) and pure rosmarinic acid (RA; Sigma). Additions to the
soup were made as appropriate. The soup was pasteurised (70.degree.
C./2 minutes), the pH recorded and the soup was then inoculated
with a cocktail of Listeria cells prepared as described before. The
tests were incubated at 20.degree. C. and viable count enumeration
performed after 0, 2, 4 and 24 h at 20 .degree. C. Initial Listeria
levels were 1.3.times.10.sup.5 CFU/ml. The test was repeated at two
nisin levels and over different time periods. The pH of the soup
without addition was pH 6.06-6.20. Addition of rosmarinic acid at
0.1% resulted in a slight pH drop to pH 5.75. Addition of 6% RA
resulted in a soup pH of pH 5.75-5.78. Addition of 0.5% RE28
resulted in a soup pH of pH 5.98. Addition of 0.5% RE70 resulted in
a soup pH of pH 6.10. Addition of 0.5% GRE09 resulted in a soup pH
of pH 6.02-6.09.
[0205] Results, shown in FIGS. 4 and 5, indicate that all the
deodorised extracts tested and rosmarinic acid contributed to
synergy with nisin in achieving kill of Listeria cells. This could
not be attributed to the drop in pH caused by some of the
additions. The additional synergy with Tween 80 was observed in
GRE09. The results indicate that the antioxidant compounds carnosol
and carnosic acid, present at 28 and 70% in two of the extracts
tested, synergistically enhanced the cidal and growth inhibitory
activity of nisin against Listeria monocytogenes. A nisin synergy
with rosmarinic acid was evident but not as strong.
V) Demonstration of Synergistic Enhancement of Nisin's Growth
Inhibitory Activity in Different Food Systems Using a Blend of
Nisin with a Phenolic Diterpene-Containing Rosemary Extract
A) Pasteurised Chicken Soup Tests
[0206] Method: Different additions of nisin (as Nisaplin.RTM.,
Danisco), a Rosemary extract containing 28% phenolic diterpenes
(RE28), and a blend of nisin with the Rosemary extract at levels of
50 IU/mg and 4.2% phenolic diterpenes were added to commercial
chicken soup that contained no other preservatives. After addition
of the compounds the soup (pH 5.8) was pasteurised at a core
temperature of 70.degree. C. for 2 minutes. The soup was cooled to
ambient temperature and either inoculated with a cocktail of
stationary phase cells of Listeria monocytogenes strains or spores
of Bacillus cereus. The strain cocktails comprised: L.
monocytogenes strains NCIMB12426, strain 358, strain 272, strain
CRA3930. The B. cereus cocktail comprised strains 204, 199, ABC4/9
and 3.046. Initial inoculum levels were approximately
10.sup.2-10.sup.3 CFU/g. Bacillus tests were incubated at
15.degree. C., Listeria tests were incubated at 8.degree. C.
Microbiological analysis was conducted at regular intervals (Milk
Plate count Agar, Oxford Listeria Selective agar).
[0207] Results: The results, shown as the time taken for bacterial
numbers to reach 10.sup.6 CFU/g, are summarised in Table 5. The
full data are shown in FIGS. 6 and 7. The results show that the
Rosemary extract alone had no activity against Bacillus, and only
slight activity against Listeria. The Rosemary extract
significantly enhanced the growth inhibitory activity of nisin.
TABLE-US-00006 TABLE 5 Summary of results demonstrating
nisin/phenolic diterpene synergy against Listeria and Bacillus in a
pasteurised chicken soup Days until growth reached Phenolic
10.sup.6 CFU/g Nisin diterpene L. monocytogenes B. cereus Test
content content at 8.degree. C. at 15.degree. C. Control 0 3 2 RE28
at 75 ppm 0 IU/g 21 ppm 5 2 Nisaplin at 100 IU/g 0 ppm 6 3 100
mg/kg Nisaplin at 250 IU/g 0 ppm 16 6 250 mg/kg Nisin/Rosemary 100
IU/g 8.4 pm 15 >26 blend A Nisin/Rosemary 250 IU/g 21 ppm 52
>26 blend B
B) Pasteurised Meat Pasta Sauce Tests
[0208] Method: The sauce was prepared from lean minced beef (50%),
tomatoes and juice (48.9%), starch (0.5%), saKt (0.4%) and sucrose
(0.2%). The beef was fried for 5 minutes until brown, then the dry
ingredients mixed in followed by the tomatoes with juice. The sauce
was simmered for 10 minutes and allowed to cool before blending to
a smooth consistency. Final pH was 5.13. Additions were made of
nisin, rosemary extract and blends. The sauce was pasteurised to a
core temperature of 80.degree. C. for 2 minutes. A cocktail of
Listeria monocytogenes strains (as above) were inoculated after
pasteurisation and the tests incubated at 8.degree. C.
[0209] Results: The results, shown as the time taken for bacterial
numbers to reach 10.sup.6 CFU/g, are summarised in Table 6. These
show that the rosemary extract alone had no activity against
Bacillus, and only slight activity against Listeria. The rosemary
extract significantly enhanced the growth inhibitory activity of
nisin. TABLE-US-00007 TABLE 6 Summary of results demonstrating
nisin/phenolic diterpene synergy against Listeria in a pasteurised
meat sauce at 8.degree. C. Phenolic Days until 10.sup.6 Test Nisin
diterpene CFU/g Control 0 0 4 RE28 at 60 ppm 0 IU/g 16.8 ppm 5
Nisaplin at 100 mg/kg 100 IU/g 0 ppm 11 Nisin/Rosemary blend A 100
IU/g 8.4 pm >76
C) Carbonara Pasta Sauce Tests
[0210] Method: A commercial chilled pasteurised sauce was used,
containing cream, smoked bacon, cheese, mascarpone, butter, starch,
onion, garlic puree. Protein 7 g, carbohydrate 6 g, fat 17 g.
Additions of test compounds were made prior to the pasteurisation
(core temperature of 70.degree. C. for 10 minutes). Inoculations
were made once the sauce had cooled. Samples were analysed
regularly for microbial numbers.
[0211] Results: These are shown in FIGS. 8 and 9. As before, the
phenolic diterpene-containing extract (8.4 ppm) synergistically
enhanced the nisin growth inhibitory activity against Listeria
cells and Bacillus spores. The rosemary extract alone showed no
activity.
VI) Demonstration of Synergistic Enhancement of Nisin's Cidal
Activity in a Food System Using a Blend of Nisin with Phenolic
Diterpene-Containing Rosemary Extract
[0212] Method: The diluted chicken soup (pH 6.2) was prepared as
above, and split into 2 batches with one batch being adjusted to pH
4.5 with HCl. Appropriate additions of nisin, rosemary extract and
blends were made, then the soup was pasteurised. A cocktail of
Listeria strains was inoculated to give an initial inoculum of
10.sup.5 CFU/g. Viable cells were enumerated by microbiological
analysis at 0 and 2 h.
[0213] The test blends contained 1) 100 IU/g nisin+30 ppm rosemary
extract (i.e. 8.4 phenolic diterpenes), and 2) 150 IU/g nisin+45
ppm rosemary extract (i.e. 12.6 phenolic diterpenes).
[0214] Results: The results demonstrated that the presence of the
phenolic diterpene containing rosemary extract synergistically
enhanced the cidal activity of nisin (FIGS. 10 and 11),
particularly at more acidic conditions (FIG. 10). The rosemary
extract alone had no significant cidal effect.
* * * * *