U.S. patent application number 15/549075 was filed with the patent office on 2018-09-06 for antimicrobial compositions.
The applicant listed for this patent is NATUREX SA. Invention is credited to Antoine Charles BILY, Simona BIRTIC, Melanie Marie-Paule Patricia HEUDRE, Xavier Pierre-Francois MESNIER, Anne PASSEMARD, Francois-Xavier Henri PIERRE, Marc ROLLER.
Application Number | 20180249725 15/549075 |
Document ID | / |
Family ID | 55661483 |
Filed Date | 2018-09-06 |
United States Patent
Application |
20180249725 |
Kind Code |
A1 |
BIRTIC; Simona ; et
al. |
September 6, 2018 |
ANTIMICROBIAL COMPOSITIONS
Abstract
A composition includes hesperdin and/or a Lamiaceae extract
wherein a majority of the volatile components have been removed
from the Lamiaceae extract. A method for applying the composition
to a food such as meat, fish or poultry, including processed and
fresh or unprocessed meat, poultry and fish is also provided.
Inventors: |
BIRTIC; Simona; (Cavaillon,
FR) ; HEUDRE; Melanie Marie-Paule Patricia; (Vedene,
FR) ; PIERRE; Francois-Xavier Henri; (Sait Saturin
les Avignon, FR) ; MESNIER; Xavier Pierre-Francois;
(Lausanne, CH) ; PASSEMARD; Anne;
(Entraigues-sur-la-Sorgue, FR) ; BILY; Antoine
Charles; (Vedene, FR) ; ROLLER; Marc;
(Morieres Les Avignon, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATUREX SA |
AVIGNON |
|
FR |
|
|
Family ID: |
55661483 |
Appl. No.: |
15/549075 |
Filed: |
February 8, 2016 |
PCT Filed: |
February 8, 2016 |
PCT NO: |
PCT/IB2016/000206 |
371 Date: |
August 4, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14615527 |
Feb 6, 2015 |
|
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15549075 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 3/3499 20130101;
A23B 4/22 20130101; A23V 2002/00 20130101; A23L 3/34635 20130101;
A23V 2200/10 20130101; A23L 3/3472 20130101; A23L 3/3481 20130101;
A23V 2250/2132 20130101; A23V 2250/21164 20130101; A23B 4/20
20130101; A23V 2002/00 20130101; A23V 2200/10 20130101; A23V
2250/21164 20130101; A23V 2250/2132 20130101 |
International
Class: |
A23B 4/20 20060101
A23B004/20; A23L 3/3481 20060101 A23L003/3481; A23L 3/3463 20060101
A23L003/3463; A23L 3/3499 20060101 A23L003/3499; A23L 3/3472
20060101 A23L003/3472; A23B 4/22 20060101 A23B004/22 |
Claims
1-38. (canceled)
39. An antimicrobial composition comprising an effective amount of
a phenolic diterpene and of hesperidin.
40. An antibacterial composition comprising an effective amount of
a phenolic diterpene and of hesperidin.
41. The composition according to claim 40 wherein the composition
is effective against Gram positive bacteria selected from the group
consisting of Bacillus cereus, Staphylococcus aureus, Streptococcus
mutans, Listeria monocytogenes, Clostridium perfringens,
Enterococcus hirae and Mycobacterium bovis.
42. The composition according to claim 40 wherein the composition
is effective against Gram negative bacteria selected from the group
consisting of Pseudomonas aeruginosa, Escherichia coli, Salmonella
typhimurium and Enterobacter cloacae.
43. An anti-fungal/anti-yeast composition comprising an effective
amount of a phenolic diterpene and of hesperidin.
44. The composition according to claim 43 wherein the composition
is effective against yeast selected from the group consisting of
Saccharomyces cerevisiae and Candida albicans.
45. The antimicrobial composition according to claim 39 comprising
pure hesperidin and a Lamiaceae extract, wherein a majority of
volatile oil components from the lamiaceae extract having been
removed.
46. The antibacterial composition according to claim 40 comprising
pure hesperidin and a Lamiaceae extract, wherein a majority of
volatile oil components from the lamiaceae extract having been
removed.
47. The antimicrobial composition according to claim 45, wherein
the pure hesperidin contains from 80% to 99% hesperidin.
48. The antibacterial composition according to claim 46, wherein
the pure hesperidin contains from 80% to 99% hesperidin.
49. The anti-fungal/anti-yeast composition according to claim 43
comprising pure hesperidin and a Lamiaceae extract, wherein a
majority of volatile oil components from the lamiaceae extract
having been removed.
50. The anti-fungal/anti-yeast composition according to claim 49,
wherein the pure hesperidin contains from 80% to 99%
hesperidin.
51. A food product comprising a food and the composition of claim
39.
52. A food product comprising a food and the composition of claim
40.
53. The food product according to claim 51, wherein the food is
selected from the group consisting of fresh meat, fish and
poultry.
54. The food product according to claim 52, wherein the food is
selected from the group consisting of fresh meat, fish and poultry.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. patent
application Ser. No. 14/615,527, filed Feb. 6, 2015, herein
incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions for extending
color and/or microbial stability to food and in particular to
compositions which extend color and/or microbial stability to food,
including, but not limited to meat, fish or poultry
(fresh/unprocessed and processed). These compositions that are
effective in extending the color and microbiological stability of
food.
BACKGROUND OF THE INVENTION
[0003] Food safety and prevention of food spoilage is an ever
present concern worldwide, particularly within the meat industry.
Spoilage of food is a major economic problem for a 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.
[0004] There is much concern about food protection and the growth
of food spoilage organisms such as Listeria monocytogenes. This
particular species is one of the most problematic spoilage
microorganism in meat. The unusual physiological characteristics
such as exceptional resistance toward antimicrobials are largely
responsible for their ability to cause spoilage. Additionally,
spoilage organisms can sometimes adapt to different preservatives
and storage conditions, thus a combination of preservative measures
can be more successful than individual measures.
[0005] There is an increasing need to develop economical, natural
and effective preservative systems to meet the public demand for
convenient, natural, safe, healthy, good quality products with
guaranteed shelf life. Antimicrobial materials such as those
derived from plants can be used as preservatives in food to help
meet this need. Such plant extracts are considered to be desirable
because they are regarded as being natural. Moreover from a
regulatory point of view, because of long term usage, plant
extracts typically have GRAS (generally regarded as safe) status.
There is also a continuing to desire to provide microbial
protection utilizing lower amounts of antimicrobial materials. Thus
there is a need to provide new antimicrobial materials or new more
effective combinations of antimicrobial materials.
[0006] Despite their natural origins, it is desirable that
antimicrobial products from plants be used in the lowest possible
amounts. This is desirable not only for reasons of cost but also to
meet consumer desire to minimize the amount of `additives` in
foodstuffs. Moreover, many plant materials have an associated
taste. Therefore in many demanding food applications reduction of
the amount of protectant from plant origin is advantageous.
[0007] Meat manufacturers are looking for ways to enable them to
supply retail outlets from efficient, cost effective,
central-processing centers. Increased shelf life with regard to
spoilage (consumer safety) is required to make this possible as
meat makes its way through longer distribution channels from
producer to retailer to consumer.
[0008] Color shelf life is important to consumer acceptance.
Consumers judge the freshness of meat by the presence of bright red
oxymyoglobin pigment. Oxymyoglobin in fresh meat decreases with
time during storage as it changes to the stable brown pigment,
metmyoglobin. Although oxymyoglobin pigment fades during dark
storage, for example in a meat locker, pigment loss is most
pronounced in lighted, refrigerated display cases in retail
establishments. Further, pigment loss is primarily cosmetic in
nature, it has serious economic consequences. Consumers in search
of the freshest looking cuts avoid purchasing meat containing even
small amounts of brown metmyoglobin.
[0009] Shelf life associated with microbial spoilage is a serious
issue. The potential liability associated with food borne illness
outbreaks from the sale of microbe-contaminated meat is enormous.
The meat industry and associated retail outlets are seeking ways to
insure consumers' safety by preventing microbial contamination all
along the manufacturing process. Process improvements such as
carcass washing and carefully controlled low temperature processing
are now routine in the industry. One method for increasing shelf
life associated with microbial spoilage is to package the food,
e.g. meat, using modified atmosphere package (MAP).
[0010] There is a need in the industry for antimicrobial methods
and processes which are perceived by consumers as being more
natural. The antimicrobial activity of the composition comprising
Lamiaceae extract and hesperidin has been the main subject of
study. Most prior art indicate that the antimicrobial activity of
the herbs is centered in the volatile essential oil components.
[0011] P. M. Davidson and A. S. Naidu (in Natural Food
Antimicrobial Systems, A. S. Naidu, ed., 2000, CRC Press, Boca
Raton, pages 265-294) review the antimicrobial properties of
phyto-phenolic compounds from essential oils of spices, herbs,
edible grains and seeds. The authors teach that the antimicrobial
effects of spices and herbs are primarily due to the presence of
phenolic compounds in the essential oil fractions and that some
monoterpenes seem to show some activity, as well. Carvacrol,
p-cymene and thymol are identified as the major volatile components
of oregano, thyme and savory that likely account for the observed
activity. The active antimicrobial agents of rosemary have been
suggested to be borneol, camphor, 1,8-cineole, alpha pinene,
camphene, verbenone and bornyl acetate. The active constituent of
sage has been suggested to be thujone. Minimum lethal
concentrations of essential oils of thyme oil have been shown to
range from 225-900 ppm in cultures. These concentrations of
essential oils in foods would cause serious flavor problems. Since
culture experiments underestimate the concentration necessary for
effectiveness in foods, the flavor problems in foods are likely to
be more serious than even the culture numbers suggest. In another
portion of this reference, minimum inhibitory concentrations of
essential oils were stated as 1-2% for rosemary, 0.12-2% for thyme,
0.12-2% for spearmint, 0.5-2% for sage, 0.5-2% for peppermint and
0.12-2% for oregano. In the summary, the authors state that
concentrations of antimicrobial compounds in herbs and spices are
too low to be used effectively without adverse effects on the
sensory characteristics of a food.
[0012] Y. Kimura et al. in U.S. Pat. No. 4,380,506, teach a process
for producing a preservative having antioxidant and antimicrobial
activity. The process involves partitioning an extract of herb
spices between polar and non-polar solvents. Some of the
partitioned extracts showed antimicrobial activity against Gram
positive Bacillus subtilis microorganisms in culture media. The
only taste criterion tested by Kimura et al. was the bitterness.
Kimura et al. remained silent as to essential oil taste perception.
Kimura et al. did not deodorize the extract which means that the
extract contained essential oils and impacted the taste of the
meat. This impact on taste teaches away from using rosemary
extracts obtained by the process taught by Kimura et al.
[0013] D. Ninkov (WO 01/15680) teaches that pharmaceutical
compositions can be prepared by combining extracts of essential
oils from plants of the Lamiaceae family with an organic acid.
Ninkov teaches that the antimicrobial activity of the
pharmaceutical composition is due to the presence of organic
phenols such as isopropyl o-cresol in the oil extract from the
plant.
[0014] K. Shetty and R. G. Labbe, (Asia Pacific J. Clin. Nutr.
(1998), 7(3/4), pages 270-276., describe work to clone Lamiacae
plants to produce enhanced levels of essential oil components such
as carvacrol and thymol. These essential oil components have some
antimicrobial properties but their commercial use is prevented by
the strong flavors imparted to foods by these volatile
compounds.
[0015] J. Campo, M. Amiot and C. Nguyen-the (2000, Journal of Food
Protection 63, pages 1359-1368) teach that rosemary extract has
antimicrobial properties in culture studies. Minimum inhibitory
concentrations varied with the species of bacteria being tested,
but ranged from 0.06-1%. These researchers suggest that rosemary
extract may show promise in foods with low fat and low protein
content, against Gram positive organisms. No food systems were
actually studied in this reference. This reference did not study
specifically Listeria.
[0016] E. Down, et al., "Comparison of Vitamin E, Natural
Antioxidants and Antioxidant Combinations on the Lean Color and
Retail Case-Life of Ground Beef Patties" published in October,
1999, describes the effect of rosemary extract in combination with
other natural antioxidants and vitamin E diet supplementation on
the color life of non-MAP ground beef. This reference does not
teach how to extend the microbial shelf life of the meat. The
authors failed to demonstrate a red color improvement of the meat
by using rosemary as the red color preservation in meat with a
natural antioxidant containing rosemary could not statistically
differ from the control. The red color of the control alters within
commercially desirable period. The loss of as much of the red color
in the control as in meat with the rosemary from this reference
teaches away from using rosemary extract as stability agent capable
of preserving the red color of the meat.
[0017] Ahn et al. "Effects of plant extracts on microbial growth,
color change, and lipid oxidation in cooked beef", Food Microbiol.,
Vol. 24, Issue 1, (2007): 7-14 show that rosemary extract, or
rosemary oleoresin, for which the contents in phenolic diterpenes
are not known, has an antilisterial effect. In this reference,
grape seed extract and pine bark extract had a greater
antilisterial effect than rosemary which teaches away from using
rosemary extract as the lead antilisterial natural product in meat.
Further, Ahn et al. 2007 have shown that the addition of rosemary
extract to meat significantly deteriorated the red color of the
meat, as compared to the control that lost less of the red color or
as compared to grape extract that significantly improved the
preservation of the red color of the meat. Therefore, Ahn et al.
2007 teach one to not use rosemary extract as stability agent
capable of preserving the red color of the meat.
[0018] United States Patent Application Publication No. 2004/131709
studies show that rosemary extract alone, Herbalox.RTM. Seasoning,
in which the majority of the volatile oil components has been
removed shows very little, if any, antimicrobial effect. This
reference does not teach how to extend the Gram positive, more
precisely antilisterial shelf life of meat.
[0019] In addition, plant derived antimicrobials from citrus
reported in the prior art are acids not flavonoids. For example,
prior patents directed to compounds from citrus essentially relate
to acids. KR20040001441 describes orange juice as a suppressor of
germ growth. However, only less than approximately 1/50.sup.th of
the juice reported in KR20040001441 could be used in meat without
perceiving meat as sour. As meat takes up only up to 7.2% of the
solution rich in citric acid, final levels in hesperidin taken up
in meat would then correspond to less than
0.48%*1/50*7.2%=.about.0.0007%. This reference does not teach
whether hesperidin could have an antilisterial effect in meat.
[0020] Lorente, Jose et al. "Chemical guide parameters for Spanish
lemon (Citrus limon (L.) Burm.) juices." Food chemistry 162 (2014):
186-191 discloses that citrus juice has titratable acidity of 52.4
g/L, with citric acid being the main component. According to
Lorente et al. (2014) in such juice, hesperidin levels as compared
to titratable acidity are lower by more than two orders of
magnitude (257 to 484.8 mg/L), which corresponds to 0.26 to 0.48%
hesperidin w/v. Adding such an acidic composition to meat would
impact the meat taste already at low levels.
[0021] Aktas , Nesimi, and Mukerrem Kaya. "The influence of
marinating with weak organic acids and salts on the intramuscular
connective tissue and sensory properties of beef." European Food
Research and Technology 213.2 (2001): 88-94 show that adding a
solution of from 1% weak acid (including citric acid) to meat
confers to the meat the sour taste. Also they show that when
marinated in proportions 1:1 w/v (meat/marinade) the meat gains in
weight at most 7.2% following marinating in marinades containing
citric acid.
[0022] In WO 2012/112337, it is reported that flavonoids, including
hesperidin, may provide some active antimicrobial activity without
informing on the nature of microbes, whether they are bacteria,
whether they are Gram positive bacteria nor whether they are
Listeria. WO 2012/112337 teaches that active antimicrobial
compounds are acids.
[0023] Moulehi, Ikram, et al. "Variety and ripening impact on
phenolic composition and antioxidant activity of mandarin (Citrus
reticulate Blanco) and bitter orange (Citrus aurantium L.) seeds
extracts." Industrial Crops and Products 39 (2012): 74-80 report
that citrus seed extracts contain total flavonoids of 1.31 to 2.52
mg equivalent catechins/g DW. As hesperidin represents <16% of
total flavonoids of citrus seed extract, this means that hesperidin
is present at .about.0.032% in DW citrus seed extract.
[0024] Mandalari, G., et al. "Antimicrobial activity of flavonoids
extracted from bergamot (Citris bergamia Risso) peel, a byproduct
of the essential oil industry." Journal of Applied Microbiology
103.6 (2007): 2056-2064 disclose that in vitro, citrus extracts
rich in flavonoids inhibit the growth of Gram negative bacteria
only and have no effect on the growth of Gram positive bacteria,
and have no effect on the growth of Listeria. Mandalari et al.
(2007) show that neohesperidin in pure form has no effect on
Listerial growth in vitro.
[0025] Fernandez-Lopez, J., et al. "Antioxidant and antibacterial
activities of natural extracts: application in beef meatballs."
Meat science 69.3 (2005): 371-380 show that meat supplemented with
citrus extracts containing flavonoids, the main of which is
hesperidin, has no effect on the growth of Listeria monocytogenes.
For example, Fernandez-Lopez et al. (2005) show that such extracts
exert antimicrobial effects on other bacterial strains, including
Listeria innocua, but not on Listeria monocytogenes.
[0026] Teachings of Mandalari et al. and Fernandez-Lopez et al.
teach away from using hesperidin as antilisterial compound and do
not render obvious to use any or combination of flavonoids from a
citrus extract against Listeria monocytogenes in meat. For example,
Mandalari et al. (2007) teach away from using hesperidin as
antilisterial compound and do not render obvious that a purified
flavonoid could have an antilisterial effect.
[0027] Punica extracts rich in ellagic acid have no antimicrobial
effects in raw MAP meat. For example, Hayes et al. (Hayes, J. E.,
Stepanyan, V., Allen, P., O'Grady, M. N., & Kerry, J. p. 2010).
"Effect of lutein, sesamol, ellagic acid and olive leaf extract on
the quality and shelf-life stability of packaged raw minced beef
patties", Meat science, 84(4), 613-620.) (hereinafter "Hayes et
al") teach that ellagic acid (one of active compounds from the
Punica extract) has no antimicrobial effect on raw beef MAP meat
stored in cold and when applied at 300 ppm. Hayes et al. teach that
ellagic acid did not improve the preservation of the red color of
raw beef MAP meat stored in cold and when applied at 300 ppm or at
600 ppm. Hayes et al. teach away from using lower concentrations
than 300 ppm in ellagic acid for antimicrobial effect. Hayes et al.
teach away from using ellagic acid for improving the red color of
meat.
[0028] The general problem of enhancing the shelf life of fresh
meat without impacting the taste, remains in preventing the growth
of spoilage organisms and pathogens and in preserving the red color
of the meat throughout the commercially desirable storage
period.
SUMMARY OF THE INVENTION
[0029] According to one aspect in accordance with the present
invention, compositions include a Lamiaceae (rosemary) extract
comprising phenolic diterpenes wherein the extract is essentially
free of the native essential oil; and/or hesperidin extract.
Advantageously, the hesperidin extract is pure hesperidin, i.e.
having a concentration of at least 80%.
[0030] Other aspects of the present method and composition are for
use with a food which includes but is not limited to fresh meat,
poultry and fish, and comprising the components of a composition of
this disclosure, a packaged food product and a method of packaging
food.
[0031] The present invention, in one form thereof, relates to a
composition comprising hesperidin and/or a Lamiaceae extract
wherein a majority of the volatile components have been removed
from the Lamiaceae extract.
[0032] The present invention, in another form thereof relates to a
food which includes but is not limited to meat, poultry and fish,
including both unprocessed or fresh meat, poultry and fish and
processed meat, poultry and fish, containing a composition
comprising hesperidin and a Lamiacea extract wherein a majority of
the volatile oil components have been removed from the Lamiaceae
extract.
[0033] The present invention in another form thereof relates to a
method for packaging food. The method includes applying to or
incorporating into a food which includes but is not limited to
fresh or unprocessed meat, fish or poultry, as well as processed
meat, fish and poultry, a composition comprising hesperidin and a
Lamiaceae extract wherein a majority of the volatile oil components
have been removed from the Lamiaceae extract. The method,
optionally, further includes packaging the food in an atmosphere
that contains 20% or more oxygen. In alternative further
embodiments, the amount of oxygen may be as much as 70% oxygen. In
yet an alternative embodiment, a packaged food product comprises a
food, e.g. fresh/unprocessed meat, fish or poultry or processed
meat, fish or poultry, packaged in a standard atmospheric
environment.
[0034] The present invention in another form thereof relates to a
method for packaging food. The method includes applying to, or
incorporating into a food which includes but is not limited to
meat, fish or poultry (fresh/unprocessed and processed), a
composition comprising hesperidin and a Lamiaceae extract wherein a
majority of the volatile oil components have been removed from the
Lamiaceae extract and the food is packaged in an environment which
includes 20% or more oxygen.
[0035] In one form, the present invention is directed to the
presence of hesperidin having a concentration of at least 80% up to
99%, preference 95% and added 56-5380 ppm in combination or not
with rosemary extract to food to inhibit the growth of Listerial
monocytogenes.
[0036] The present invention in another form thereof relates to a
food product comprising a food and containing a composition
comprising pure hesperidin or Lamiaceae extract.
[0037] The present invention in another form thereof relates to a
method for packaging food comprising applying to or incorporating
into a food, a composition comprising pure hesperidin or Lamiaceae
extract. Optionally, the further can further comprises packaging
the food in an atmosphere that contains 20% or more oxygen.
[0038] The present invention in yet another form thereof relates to
an antimicrobial composition comprising an effective amount of a
phenolic diterpene and/or of hesperidin.
[0039] The present invention in another form thereof relates to an
antibacterial composition comprising an effective amount of a
phenolic diterpene and/or of hesperidin. In one advantageous
further form, the composition is effective against Gram positive
bacteria selected from the group consisting of Bacillus cereus,
Staphylococcus aureus, Streptococcus mutans, Listeria
monocytogenes, Clostridium perfringens, Enterococcus hirae and
Mycobacterium bovis. In an alternative further form, the
composition is effective against Gram negative bacteria selected
from the group consisting of Pseudomonas aeruginosa, Escherichia
coli, Salmonella typhimirium and Enterobacter cloacae.
[0040] The present invention in yet another form thereof relates to
an antifungal/anti-yeast composition comprising an effective amount
of a phenolic diterpene and of hesperidin. In one advantageous
form, the composition is effective against yeast including
Saccharomyces cerevisiae and Candida albicans.
[0041] Rosemary extract in accordance with the present invention
improves the preservation of the red color of meat and extends the
microbial shelf life without impacting the meat flavor. The present
inventors' studies in actual meat systems use a deodorized rosemary
extract: an extract from which the majority of the volatile
essential oil components has been removed and that does not impact
the meat taste. When combined with hesperidin or with Punica
extract, unexpectedly, synergistic antilisterial and synergistic
color preservation effects are observed, without any impact on the
food taste.
[0042] The present method and composition can provide ways for food
suppliers of any food product or beverage, including meat
manufacturers to provide retailers with products from
cost-efficient, cost-effective central processing centers. The
present method and composition can extend the shelf-life of food,
including fresh and processed meat, fish and poultry, and provide
food that has extended microbial and color shelf-life in an
atmosphere containing 70% or more oxygen and 30% or more CO.sub.2.
The method, in accordance with this disclosure that uses
combinations of extracts can be used to improve the preservation of
the meat color, block Listerial growth in food, including meats,
fish and poultry (fresh/unprocessed and processed), and allow for
the use of lower, but more effective, inhibitory concentrations of
plant extracts, without negative flavor impacts.
[0043] The present method is particularly suited for use with
modified atmosphere packaged (MAP) meats. MAP meats are packaged in
gas impermeable materials that maintain an atmosphere above the
product. Mixtures of oxygen and carbon dioxide are often used in
MAP meats. Mixtures of these gases work very well with the present
method.
[0044] In sharp contrast to the present method and composition, the
prior art teaches that citrus extracts containing hesperidin has no
effect on Listerial growth in vitro nor in a food including meat.
Pure flavonoids such as hesperitin or neohesperidin do not have any
effect on Listerial growth in vitro, nor in meat. Mandalari et al.
(2007) disclose that in vitro, citrus extracts rich in flavonoids
inhibit the growth of Gram negative bacteria only and have no
effect on the growth of Gram positive bacteria, and have no effect
on the growth of Listeria. Mandalari et al. (2007) show that
neohesperidin in pure form has no effect on Listerial growth in
vitro.
[0045] Fernandez-Lopez et al. (2005) show that meat supplemented
with citrus extracts containing flavonoids, the main of which is
hesperidin, has no effect on the growth of Listeria monocytogenes.
Fernandez-Lopez et al. (2005) show that such extracts exert
antimicrobial effects on other bacterial strains, including
Listeria innocua, but not on Listeria monocytogenes.
[0046] Teachings of Mandalari et al. and Fernandez-Lopez et al.
teach away from hesperidin as antilisterial compound and do not
render obvious to use any or combination of flavonoids from a
citrus extract against Listeria monocytogenes in food. Further, the
teachings of Mandalari et al. teach away from hesperidin as
antilisterial compound and do not render obvious that a purified
flavonoid could have an antilisterial effect.
[0047] The present discloses shows that when hesperidin is
concentrated at least 80% up to 99%, preference at 95%, it inhibits
the growth of Listeria monocytogenes in meat. The prior art teaches
or at the least, suggests that hesperidin does not have an effect
on Listerial growth in food including meat. The present inventors
found surprisingly, that hesperidin can be used in a food
product/foodstuff, such as meat (e.g. minced meat) to extend its
microbial, color and taste shelf life. When hesperidin alone is
used, hesperidin is required to be used in higher concentrations to
ensure adequate antilisterial effects. Surprisingly and
unexpectedly, the addition of plant extracts comprising phenolic
diterpenes to hesperidin, synergistically improves antilisterial
effects and allows the use of lower doses of each extract.
[0048] Also, surprisingly, hesperidin and Lamiaceae extracts have
been found to preserve color in MAP ground beef in a synergistic
manner. In samples of ground beef stored six (6) days in cold
conditions, hesperidin plus rosemary extract exceeds the color
preserving additive effect of hesperidin or rosemary alone.
[0049] The prior art is replete with statements that hesperidin
containing extracts have no inhibitory effect on Listeria
monocytogenes organisms. Surprisingly, the present inventors found
evidence that hesperidin in the presence of high oxygen
concentrations inhibit Listeria monocytogenes, Gram positive
organism. The combination of hesperidin and high oxygen atmosphere
inhibits Listeria monocytogenes, Gram positive organism isolated as
a major spoilage organism in ground beef. Even more surprisingly,
combinations of rosemary and hesperidin show synergistic inhibition
of these Listeria monocytogenes, Gram positive organisms, under
high oxygen atmospheres.
[0050] The combinations of Lamiaceae extract containing phenolic
diterpenes and hesperidin, preserve the color of meat, fish and
poultry (fresh/unprocessed and processed) in the presence of oxygen
in a synergistic manner.
[0051] Hesperidin alone at certain concentrations does not preserve
the color life of fresh red meat and results in an unacceptable
organoleptic feature. The combination of rosemary extract and
hesperidin acts synergistically to extend the color life of ground
beef in cold storage conditions. The combination is not just
additive, but is synergistic, because it exceeds the additive
effect of hesperidin alone and rosemary alone.
[0052] The addition of Lamiaceae extract comprising phenolic
diterpenes to hesperidin, yields in flavor acceptable composition
which is effective in preserving color and in inhibiting the growth
of microorganisms in meat, fish and poultry (both fresh/unprocessed
and processed).
[0053] In accordance with the present disclosure, hesperidin alone
suppresses Listerial growth in food, including meat. Surprisingly,
combinations of Lamiaceae extract, preferably, rosemary extract,
and hesperidin, are more effective in suppressing Gram positive,
preferably Listeria monocytogenes, bacterial growth than either
Lamiaceae extract or hesperidin, alone.
[0054] The combination of Lamiaceae extract comprising phenolic
diterpenes and hesperidin in the presence of oxygen, does not
impact the flavor of ground beef in a package after a commercially
desirable storage period. Neither Lamiaceae extract containing
phenolic diterpenes nor hesperidin alone, or oxygen alone, or a
combination of two of these factors alone preserves the color as
well as the combination of the three at the end of a commercially
desirable storage period, without impacting the flavor.
[0055] Surprisingly, the addition of plant extracts comprising
phenolic diterpenes, to hesperidin, synergistically improves
antilisterial effects and allows the use of lower doses of each
extract.
[0056] Also, surprisingly, hesperidin and Lamiaceae extracts have
been found to preserve color in MAP ground beef in a synergistic
manner. In samples of ground beef stored five (5) days in cold
conditions, hesperidin plus rosemary extract exceeds the color
preserving additive effect of hesperidin or rosemary alone.
[0057] The inventors found, surprisingly, that Punica extract
containing punicalagins and ellagic acid can be used in minced meat
to extend its microbial, color and taste shelf life. When Punica
extract containing punicalagins and ellagic acid are used alone
(i.e. without rosemary extract), higher concentrations of Punica
extract are required to insure adequate antilisterial effects.
Surprisingly, the addition of rosemary extracts, or extracts of
other Lamiaceae synergistically improves antilisterial effects and
allows the use of lower amounts of the Punica extract (e.g.
punicalagins and ellagic acid extracts).
[0058] Also, surprisingly, Punica extract containing punicalagins
and ellagic acid and Lamiaceae extracts have been found to preserve
color in MAP ground beef in a synergistic manner. In samples of
ground beef stored five (5) days in cold conditions, Punica extract
containing punicalagins and ellagic acid plus rosemary extract
exceeds the color preserving additive effect of Punica or rosemary
alone. This synergistic effect was observed at different
concentrations of each extract.
[0059] Surprisingly, the inventors found evidence that Punica
extract containing punicalagins and ellagic acid in the presence of
high oxygen concentrations inhibits Listeria monocytogenes, Gram
positive organism. The combination of Punica extract containing
punicalagins and ellagic acid and high oxygen atmosphere inhibits
Listeria monocytogenes, Gram positive organism isolated as a major
spoilage organism in ground beef. Even more surprisingly, the
combination of rosemary and Punica extract containing punicalagins
and ellagic acid show synergistic inhibition of these Listeria
monocytogenes, Gram positive organisms, under high oxygen
atmospheres.
[0060] The combinations of Lamiaceae extract containing phenolic
diterpenes and Punica extract containing punicalagins and ellagic
acid, extend the color shelf life of meat, fish and poultry
(fresh/unprocessed and processed) in the presence of oxygen in a
synergistic manner. Critical to this invention is the combination
of rosemary extract or other effective Lamiaceae extract and Punica
extract containing punicalagins and ellagic acid, and the presence
of oxygen.
[0061] Punica extract containing punicalagins and ellagic acid
alone at certain concentrations decreases the color life of red
meat (e.g. fresh meat) and results in an unacceptable organoleptic
feature. The combination of rosemary extract and Punica extract
containing punicalagins and ellagic acid acts synergistically to
extend the color life of ground beef in cold storage conditions.
The combination is not just additive, but is synergistic, because
it exceeds the additive effect of Punica extract containing
punicalagins and ellagic acid alone and rosemary alone.
[0062] The addition of Lamiaceae extract containing phenolic
diterpenes to Punica extract containing punicalagins and ellagic
acid, yields in flavor acceptable composition which is effective in
preserving color and in inhibiting the growth of microorganisms in
meat, fish and poultry (fresh/unprocessed and processed).
[0063] The combinations of Lamiaceae extract, preferably, rosemary
extract, and Punica extract containing punicalagins and ellagic
acid, are more effective in suppressing Gram positive, preferably
Listeria monocytogenes, bacterial growth than either Lamiaceae
extract or Punica extract containing punicalagins and ellagic acid,
alone.
[0064] The combination of Lamiaceae extract containing phenolic
diterpenes and Punica extract containing punicalagins and ellagic
acid in the presence of oxygen, does not impact the flavor of
ground beef in a package after a commercially desirable storage
period. Neither Lamiaceae extract containing phenolic diterpenes
nor Punica extract containing punicalagins and ellagic acid alone,
or oxygen alone, or a combination of two of these factors alone
preserves the color as well and in a synergistic manner as the
combination, at least up to sixth (6.sup.th) day of storage,
without impacting the flavor.
[0065] Surprisingly, the addition of rosemary extracts, or extracts
of other Lamiaceae synergistically improves antilisterial effects
and allows the use of lower doses of each extract.
[0066] Also, surprisingly, Punica extract containing punicalagins
and ellagic acid and Lamiaceae extracts have been found to preserve
color in MAP ground beef in a synergistic manner. In samples of
ground beef stored five (5) days in cold conditions, Punica extract
containing punicalagins and ellagic acid plus rosemary extract
exceeds the color preserving additive effect of hesperidin or
rosemary alone.
[0067] Surprisingly, the inventors found evidence that Punica
extract containing punicalagins and ellagic acid in the presence of
high oxygen concentrations inhibit Listeria monocytogenes, Gram
positive organism. The combination of Punica extract containing
punicalagins and ellagic acid and high oxygen atmosphere inhibits
Listeria monocytogenes, Gram positive organism isolated as a major
spoilage organism in ground beef. Even more surprisingly, the
combination of rosemary and Punica extract containing punicalagins
and ellagic acid show synergistic inhibition of these Listeria
monocytogenes, Gram positive organisms, under high oxygen
atmospheres.
[0068] The combinations of Lamiaceae extract containing phenolic
diterpenes and Punica extract containing punicalagins and ellagic
acid, preserve the color of meat, fish and poultry
(fresh/unprocessed and processed) in the presence of oxygen in a
synergistic manner. Accordingly, advantageous to some of the
present methods and compositions of this disclosure is a
combination of rosemary extract or other effective Lamiaceae
extract and Punica extract containing punicalagins and ellagic
acid, and the presence of oxygen.
[0069] Punica extract containing punicalagins and ellagic acid
alone at certain concentrations decreases the color life of red
meat (e.g. fresh meat) and results in an unacceptable organoleptic
feature. The combination of rosemary extract and Punica extract
containing punicalagins and ellagic acid acts synergistically to
extend the color life of ground beef in cold storage conditions.
The combination is not just additive, but is synergistic, because
it exceeds the additive effect of Punica extract containing
punicalagins and ellagic acid alone and rosemary alone.
[0070] The addition of Lamiaceae extract containing phenolic
diterpenes to Punica extract containing punicalagins and ellagic
acid, yields in flavor acceptable composition which is effective in
preserving color and in inhibiting the growth of microorganisms in
meat, fish and poultry (fresh/unprocessed and processed).
[0071] The combinations of Lamiaceae extract, preferably, rosemary
extract, and Punica extract containing punicalagins and ellagic
acid, are more effective in synergistic manner in suppressing Gram
positive, preferably Listeria monocytogenes, bacterial growth than
either Lamiaceae extract or Punica extract containing punicalagins
and ellagic acid, alone. Neither Lamiaceae extract containing
phenolic diterpenes nor Punica extract containing punicalagins and
ellagic acid alone, or oxygen alone, or a combination of two of
these factors alone preserves the meat against Listeria
monocytogenes as well as the combination of the three, within
commercially desirable storage period, without impacting the
flavor.
[0072] The combination of Lamiaceae extract containing phenolic
diterpenes and Punica extract containing punicalagins and ellagic
acid in the presence of oxygen, does not impact the flavor of
ground beef in a package after a commercially desirable storage
period. Neither Lamiaceae extract containing phenolic diterpenes
nor Punica extract containing punicalagins and ellagic acid alone,
or oxygen alone, or a combination of two of these factors alone
preserves the color as well as the combination of the three after
five (5) days of cold storage period, without impacting the
flavor.
[0073] To keep the number of additives within reasonable bounds
with respect to meat, fish or poultry, it is advantageous to use
botanical extracts that provide the property of inhibiting the
growth of Listeria monocytogenes, and, more particularly, it is
advantageous to combine botanical extracts that provide synergistic
antilisterial effects and that preserve the red color of the meat
without impacting the meat taste. Advantageously, formulations of
different botanical extracts, in accordance with this disclosure,
function synergistically to increase the total antilisterial
activity and to preserve the red color of the meat without
impacting the meat taste, of the combined extracts, that are
superior to the sum of their individual contributions.
[0074] The methods and compositions in accordance with the present
disclosure, exhibit a synergistic effect, as introduced above and
will be discussed in more details to follow. It is noted that in
contrast to the synergistic effect of the methods and compositions
in accordance with the present disclosure, when two compounds
elicit the same overt response, regardless of the mechanism of
action and the combined effect is the algebraic sum of their
individual effects, the compounds are said to exhibit summation
(Levine et al., 1996). However, in synergism, the joint effect of
two compounds is greater than the algebraic sum of their individual
effects.
[0075] The technique in Levine et al. (1996) has been used to
evaluate biological effects of compound combinations. Shown in FIG.
1 (identified as "Prior Art" and taken from Basic Principles of
Pharmacology, Tulane University), top graph, compound combination
effects illustrate that when two compounds with similar mechanisms
are given together, they typically produce additive effects. This
is also referred to as summation. However, if the effect of two
compounds exceeds the sum of their individual effects, this is an
unexpected effect referred to synergism.
[0076] By analogy, a synergistic response concerning half doses, as
illustrated in the bottom graph of FIG. 1, occurs if the
combination of half the dose of compound A and B produces a
response greater than A or B alone.
[0077] Those skilled in the art of antimicrobial formulations for
food matrices such as meat, are aware that antimicrobial synergy in
meat is not predictable. Not a single synergy could be disclosed
for different combinations between three natural botanical
extracts. Gutierrez, J., Barry-Ryan, C., & Bourke, P. (2008).
"The antimicrobial efficacy of plant essential oil combinations and
interactions with food ingredients." International journal of food
microbiology, 124(1), 91-97). Synergistic effects of combinations
have rarely been disclosed for combinations between synthetic and
natural extracts (see e.g. WO 2013/169231).
[0078] One advantage of some compositions and methods, in
accordance with this disclosure, is achieved by a process which
removes volatile compounds by deodorization. The deodorization
process removes volatile compounds including borneol, camphor,
1,8-cineole, alpha pinene, camphene, verbenone and bornyl
acetate.
[0079] An additional advantage, in accordance with some aspects of
the present methods, systems and compositions of this disclosure,
is achieved through a combination of hesperidin or Punica extract
with lamiaceae extract. Further, unlike the process described in
U.S. Pat. No. 4,380,506, methods, in accordance with this
disclosure, do not require the partitioning process and the methods
avoid the use of additional processing expense.
[0080] An additional advantage in accordance with one aspect of the
present method and system, is a composition which reduces the
concentration of volatile compounds to a low level so as to not
impact the taste of a food product to which a composition is
applied such as meat, thereby not affecting the taste of the
meat.
[0081] One additional advantage of some aspects of the present
invention is the presence of Punica extract with more than 60 full
lower concentration in ellagic acid (than previously reported by
Hayes et al) resulting in anti-listerial effects when combined with
rosemary extract.
BRIEF DESCRIPTION OF THE DRAWINGS
[0082] FIG. 1 is adapted from "Basic Principles of Pharmacology",
(Tulane University), in which the top portion is "Summation:
Compounds A and B Produce Equal Effects, And Their Affects Are
Additive When Combined" and the bottom portion is "Synergism: The
Combination of Half the Dose of Compound A and Compound B Produces
a Response Greater Than A or B Alone."
[0083] FIG. 2 is a graph showing Listeria monocytogenes growth in
meat in accordance with the present invention.
[0084] FIG. 3 is a graph showing antilisterial synergy effects of
combinations of hesperidin and rosemary extract in accordance with
the present invention.
[0085] FIG. 4 is a graph showing antimicrobial surface response of
different combinations of rosemary extract and hesperidin
inhibiting and decreasing Listeria monocytogenes growth, in
accordance with the present invention.
[0086] FIG. 5 is a bar chart showing red color values of the meat
in accordance with the present invention with regard to
combinations of rosemary extract and hesperidin in accordance with
the present invention.
[0087] FIG. 6 is a bar chart showing red color values of meat in
accordance with the present invention with regard to various
combinations of rosemary and Punica extract in accordance with the
present invention.
[0088] FIG. 7 is a chart showing inhibition of Listeria
monocytogenes growth by plant extracts in minced beef.
[0089] FIG. 8 is a graph showing inhibition of Listeria
monocytogenes growth by plant extracts in minced beef at 8.degree.
C. at day 9.
[0090] FIG. 9 is a chart showing inhibition of Listeria
monocytogenes growth by plant extracts in minced beef at 8.degree.
C. at day 6.
[0091] FIG. 10 is a chart showing inhibition of Listeria
monocytogenes e by plant extracts in minced beef at 8.degree. C. at
day 9 in accordance with the present invention.
[0092] FIG. 11 is a graph showing in accordance with the present
invention, antimicrobial surface response of different combinations
of rosemary and Punica extract inhibiting and decreasing Listeria
monocytogenes growth.
[0093] FIG. 12 is a graph showing Listeria monocytogenes in poultry
sausages.
[0094] FIG. 13 is a graph showing inhibition of Listeria growth in
poultry sausages by an extract combination of R/P in accordance
with the present invention.
[0095] FIG. 14 is a graph showing inhibition Listeria growth in
poultry sausage by extract combination of R/H.
[0096] FIG. 15 is a graph showing Listeria monocytogenes in pork
sausage (control).
[0097] FIG. 16 is a graph showing inhibition of Listeria growth in
pork sausages by an extract combination of R/P.
[0098] FIG. 17 is a graph showing inhibition of Listeria growth in
pork saugsage by an extract combination of R/H.
[0099] FIG. 18 is a graph showing growth of Listeria monocytogenes
in smoked salmon (control).
[0100] FIG. 19 is a graph showing inhibition of Listeria
monocytogenes in smoked salmon on the 30.sup.th day of growth.
[0101] FIG. 20 is a graph showing inhibition of Listeria
monocytogenes in smoked salmon on the 30.sup.th day of grown.
DETAILED DESCRIPTION OF THE INVENTION
[0102] Compositions in accordance with this disclosure include
Lamiaceae extract and hesperidin and methods for using compositions
for extending the shelf life of food including meat, fish and
poultry (both fresh/unprocessed and processed) without impacting
the taste.
[0103] The present invention in another form, includes compositions
comprising Lamiaceae extract and Punica extract and methods for
using these compositions for extending the shelf life of food
including meat, fish and poultry (both fresh/unprocessed and
processed) without impacting the taste.
[0104] The present methods and compositions are based on a
discovery that rosemary extracts rich in phenolic diterpenes,
alone, or in combination with hesperidin or with Punica extracts
rich in ellagic acid and in punicalagins, preserve the red color of
the meat for commercially significant period. The present inventors
discovered that treating meat with pure hesperidin, that is a
flavonoid, extracted from citrus peels and then purified, prevents
the growth of Listeria monocytogenes in meat. Lamiaceae extracts
comprising phenolic diterpenes in combination with hesperidin or
with Punica extract comprising ellagic acid and punicalagins,
synergistically provided novel solutions for suppressing the growth
of microorganisms for a commercially desirable period and for
preserving the red color of the meat without impacting the meat
taste. Compositions of this invention have been found to inhibit
the growth of Gram positive microorganisms. Compositions of this
invention, have been found to inhibit the growth of Listeria.
Compositions of this invention, have been found to inhibit the
growth of Listeria monocytogenes.
[0105] Combinations comprising: plant extracts standardized in
phenolic diterpenes carnosic acid and carnosol, and hesperidin, or
plant extracts standardized in phenolic diterpenes carnosic acid
and carnosol, and plant extracts standardized in ellagic acid and
punicalagins, that would have been used to synergistically prevent
Listeria monocytogenes growth in food including meat, fish and
poultry (both fresh/unprocessed and processed), without impacting
the food taste and that synergistically improve the preservation of
the food color (e.g. meat color), could not be retrieved from the
prior art.
[0106] None of the prior art on the antimicrobial use of the
combination of rosemary or other Lamiaceae extracts comprising
phenolic diterpenes with hesperidin or with Punica extracts
comprising punicalagins and ellagic acid, either anticipates or
renders obvious the present methods and compositions. The prior art
focuses on the use of herb essential oils or on the use of organic
acids, such as citric acid. The rosemary extracts used in the
present disclosure are processed in a manner that makes them
essentially free of the native essential oil and rich in phenolic
diterpenes. The prior art neither anticipates nor renders obvious
the synergistic combination of Lamiaceae extracts rich in phenolic
diterpenes and Punica extracts rich in punicalagins and ellagic
acid.
[0107] The prior art neither anticipates nor renders obvious the
synergistic combination of Lamiaceae extracts rich in phenolic
diterpenes and hesperidin. The prior art neither anticipates nor
renders obvious the surprisingly beneficial antimicrobial effect of
the combination of Lamiaceae extracts comprising phenolic
diterpenes with Punica extracts comprising punicalagins and ellagic
acid, or with hesperidin, on Gram positive organisms: Listeria
monocytogenes. The prior art neither anticipates nor renders
obvious the surprisingly beneficial color preservation effect of
the combination of Lamiaceae extracts comprising phenolic
diterpenes with Punica extract comprising punicalagins and ellagic
acid, or with hesperidin.
[0108] The prior art neither anticipates nor renders obvious the
absence of the impact on food taste of the combination of Lamiaceae
extracts comprising phenolic diterpenes with Punica extract
comprising punicalagins and ellagic acid, or with hesperidin.
[0109] Other flavonoids that have the same effect including but are
not limited to: narigin, isocurametin, neohesperidin, hesperidin,
poncirin, nebiletin, and tangeretin.
Definitions
[0110] The following are a list of definitions used throughout this
disclosure:
[0111] "Effective amount" is the amount necessary in order to
achieve a specific effect, in accordance with what one of ordinary
skill in the art would be readily able to determine through routine
experimentation. For example, with regard to the present
disclosure, an effective amount of a composition comprising
hesperidin and a Lamiaceae extract to be applied to a food product
or foodstuff, e.g. meat, fish and poultry (both fresh/unprocessed
and processed), to extend the longevity of the food, e.g. red color
to the fresh meat, fish and poultry, is an amount which is
determined to provide the red color longevity based on known
parameters which include, but are not limited to the concentration
of hesperidin and a Lamiaceae extract, the volume and/or surface
area of the meat, fish and poultry, and the atmospheric environment
conditions of the meat, fish and poultry. Similarly, the effective
amounts of rosemary/Punica to extend the longevity of red color to
the meat, fish and poultry are determined in a similar way.
[0112] "Food", "food product" and "foodstuff" mean products that
people or animals eat. The food, food product and foodstuff
include, but are not limited to fresh and/or unprocessed meat, fish
and poultry and processed meat, fish and poultry.
[0113] "Fresh meat, fish, and poultry" means meat fish and poultry,
entire carcasses, cut portions thereof, and ground portions
thereof. Fresh meat, fish, and poultry includes both unprocessed
meat, fish and poultry as well as meat, fish, and poultry that
includes additives such as polyphosphates, salt, water, flavors,
broths, added proteins, sugar, starches and the like which are
incorporated into the meat, fish or poultry. It is important to
distinguish fresh meat, fish or poultry which may contain these
ingredients, from "processed" meat, fish and poultry which includes
cured meat, fish and poultry, which may contain the same
ingredients, but also contain one or more of the following:
erythorbates, erythorbic acid, ascorbates, ascorbic acid, nitrites,
nitrates or cultures. Fresh meat, fish and poultry are to be
distinguished from, and as opposed to, and does not include cured
meat, fish or poultry, known as processed meat, fish and
poultry.
[0114] "Hesperidin" means a compound extracted from nature or
synthesized.
[0115] "Lamiaceae extract" means extract from a plant of the
Lamiaceae family, preferably rosemary, sage, oregano, thyme, mints,
and the following genera: Salvia, Rosmarinus, Lepechinia, Oreganum,
Thymus, Hyssopus and mixtures thereof. The most preferred is
rosemary.
[0116] "Meat, fish and poultry" means both a) processed meat, fish
and poultry and b) unprocessed meat, fish and poultry.
[0117] "Phenolic diterpenes" means carnosic acid, carnosol,
methylcarnosate, and other phenolic diterpene derivatives
(rosmanol, isorosmanol, 11,12-di-O-methylisorosmanol,
12-O-methylcarnosic acid, rosmanol-9-ethyl ether, circimaritin,
Methylated monooxidized product of carnosic acid, genkwanin,
epirosmanol, epiisorosmanol, carnosic acid derivative, epirosmanol
ethyl ether, cryptotanshinone) and mixtures thereof.
[0118] "Processed" such as "processed foodstuff" and "processed
meat, fish and poultry" are products resulting from the processing
of food, such as meat, fish or poultry or from the further
processing of such processed products, so that the cut surface
shows that the product no longer has the characteristics of fresh
meat, fish or poultry. Processing means any action that
substantially alters the initial product, including heating,
smoking, curing, maturing, drying, marinating, extraction,
extrusion or a combination of those processes. Processes include
non-heat treated and heat-treated processes.
[0119] "Punica extract" means extract from a plant of the Punica
genus, preferably Punica granatum and Punica protoPunica, and
mixtures thereof. The most preferred is Punica granatum.
[0120] "Pure hesperidin extract" means a hesperidin extract that
has a concentration of at least 80% hesperidin.
[0121] "Unprocessed" (such as meat, fish and poultry) means not
having undergone any treatment resulting in a substantial change in
the original state of the foodstuffs (e.g. meat, fish and poultry).
However, the foodstuffs may have been for example divided, parted,
severed, boned, minced, skinned, pared, peeled, ground, cut,
cleaned, trimmed, deep-frozen, frozen, chilled, milled or husked,
packed or unpacked. Unprocessed foodstuff, including meat, fish and
poultry include untreated raw meat, fish and poultry, as well as
fresh meat, fish and poultry that has been comminuted or minced,
that has had foodstuffs seasons or additives added to it or that
has undergone processing insufficient to modify the internal muscle
fiber of the meat, fish or poultry and thus eliminate the
characteristics of fresh meat, fish or poultry.
[0122] In the development of the present method and composition, it
was discovered that hesperidin has an antilisterial effect in meat
when prepared within certain ranges of concentrations.
[0123] In the development of the present method and composition, it
was discovered that rosemary extract comprising phenolic diterpenes
combined with hesperidin or with Punica extract has a superior
effect on suppressing the growth of Listeria monocytogenes in meat
than when extracts are applied alone.
[0124] In the development of the present method and composition, it
was discovered that certain mixtures of extracts of the rosemary
combined with hesperidin or with Punica extract comprising
punicalagins and ellagic acid, provide a synergistic antilisterial
effect when prepared within certain ranges of concentration
ratios.
[0125] In the development of the present method and composition, it
was discovered that rosemary extract comprising phenolic diterpenes
combined with hesperidin or with Punica extract has a superior
effect on preserving the red color in meat than when extracts are
applied alone.
[0126] In the development of the present method and composition, it
was discovered that certain mixtures of extracts of the rosemary
combined with hesperidin or with Punica extract comprising
punicalagins and ellagic acid, provide a synergistic red color
preservation effect in meat when prepared within certain ranges of
concentration ratios.
Mixtures of Extracts Rich in Phenolic Diterpenes and Hesperidin or
Punica Extract
[0127] Phenolic diterpenes such as carnosic acid or carnosol occur
specifically in Lamiaceae. To date, carnosic acid has been
identified in only a few species, all exclusive of the Lamiaceae.
To the best of the inventors' knowledge, only seven out of seventy
(70) genera of the Mentheae tribe contain carnosic acid: Salvia
(Brieskorn and Dumling, 1969), Rosmarinus (Luis and Johnson, 2005),
Lepechinia (Bruno et al., 1991), Oreganum (Hossain et al., 2010)
and Thymus (Achour et al., 2012). It may be present in Hyssopus
where one of its possible derivatives, rosmanol-9-ethyl ether (7),
was identified (Djarmati et al., 1991). Carnosic acid also occurs
as a minor compound in one genus of the Ocimeae tribe, Ocimum
(Jayasinghe et al., 2003). Brieskorn, C. H., Dumling, H. J., 1969.
Carnosolsaure, der wichtige antioxydativ wirksame Inhaltsstoff des
Rosmarin-und Salbeiblattes. Zeitschrift fur
Lebensmittel-Untersuchung and Forschung 141, 10-16; Luis, J. C.,
Johnson, C. B., 2005; Bruno, Maurizio, et al. "Abietane
diterpenoids from Lepechinia meyeni and Lepechinia hastata."
Phytochemistry 30.7 (1991): 2339-2343; Hossain, Mohammad B., et al.
"Characterization of phenolic composition in Lamiaceae spices by
LC-ESI-MS/MS." Journal of agricultural and food chemistry 58.19
(2010): 10576-10581; Achour, S., Khelifi, E., Attia, Y., Ferjani,
E., Noureddine Hellah A., 2012. Concentration of Antioxidant
Polyphenols from Thymus capitatus extracts by Membrane Process
Technology. Journal of food science 77, C703-C709; Djarmati, Z.,
Jankov, R. M., Schwirtlich, E., Djulinac, B., Djordejevic, A.,
1991. High antioxidant activity of extracts obtained from sage by
supercritical CO.sub.2 extracton. Journal of the American Oil
Chemists Society 68, 731-734; Jayasinghe, C., Gotoh, N., Aoki, T.,
Wada, S., 2003. Phenolic composition and antioxidant activity of
sweet basil (Ocimum basilicum L.). Journal of agricultural and food
chemistry 51, 4442-4449. Seasonal variations of rosmarinic and
carnosic acids in rosemary extracts. Analysis of their in vitro
antiradical activity. Spanish Journal of Agricultural Research 3,
106-112.
[0128] Here these phenolic diterpenes were extracted from rosemary
with the aim of extracting and concentrating essentially phenolic
diterpenes: 44-85%. Thus obtained extract was then deodorized in
order to get rid of essential oils and volatile compounds that
impact the food taste.
[0129] Rosemary Extract
[0130] Rosemary (Rosmarinus officinalis) leaves can be extracted
with various solvents and yield extracts that are rich in different
compounds. For instance, aqueous extracts are rather abundant in
rosmarinic acid whereas extractions using organic solvents rather
yield in extracts rich in phenolic diterpenes such as carnosic acid
and carnosol. The detailed procedure to prepare the composition of
Rosemary extract was described in the U.S. Pat. No. 5,859,293 and
WO 96/34534, both herein incorporated by reference.
[0131] The rosemary leaf was extracted with acetone at room
temperature. After the extraction was completed, the acetone
extract was filtered to separate the solution from rosemary leaf
and concentrated under reduced pressure to make concentrated native
extract. At this time, the concentrated extract can be dried
directly in a vacuum oven under mild heat to make a powdered
extract, which is a composition comprising about 15%-30% carnosic
acid and 1%-3% carnosol. Alternatively, to the concentrated native
extract, aqueous sodium carbonate (NaHCO.sub.3) was added to
dissolve carnosic acid and other organic acids, while base
insoluble substances were precipitated out.
[0132] The solution was filtered to separate from solid, and the
filtrate was further concentrated under reduced pressure. Once
finishing concentration is achieved, phosphoric acid
(H.sub.3PO.sub.4) was added and the acid insoluble substances
(including carnosic acid, carnosol, and carnosic derivatives) were
precipitated from the concentrated solution. Charcoal active is
used during the process to decolorize the rosemary extract in
solution before filtration. Through filtering, the precipitated
solid was subsequently separated from liquid and rinsed with water
to remove impurities.
[0133] Last, the solid was dried in a vacuum oven and then milled
into powder to make a composition containing about 40-65% carnosic
acid, 2-10% carnosol, and 2-10% 12-O-methylcarnosic acid. Here used
extract contained >48% carnosic acid+carnosol. A last step was
done to deodorize the rosemary extract. It corresponded to a
subsequent extraction of the previous solid with a mix of
acetone/hexane. The purpose of this step was the elimination of
fatty molecules and of volatile compounds. The filtrate was
concentrated under reduced pressure and was directly formulated on
liquid carrier.
[0134] Within the present specification and claims, this extract
standardized in phenolic diterpenes carnosic acid and carnosol,
will be referred to either as rosemary, or rosemary extract or
rosemary (powder) or rosemary (liquid).
[0135] Hesperidin Extraction
[0136] Dried immature fruits (citrus aurantium L.) were exposed to
a vapor in order to remove pectins prior to the extraction with
water. Subsequently, sodium hydroxide and calcium hydroxide were
added in the solution to stabilize the pH value. Following the
filtration step, an acidification of the filtrate was induced using
HCl. Upon this step the hesperidin precipitates, the liquid
solution is removed and the precipitate is dried. The final product
(pure hesperidin) contains 90% to 99% hesperidin, preferably more
than 95% of hesperidin as measured by HPLC.
[0137] The obtained extracts contain essentially hesperidin
(>80%) and are considered to be pure. Throughout this
disclosure, this extract standardized in hesperidin at >95%,
will be referred to as hesperidin or hesperidin (powder) or
hesperidin (liquid).
[0138] Punica Extraction
[0139] Pomegranate skin bitter (Punica granatum L.) was extracted
with ethanol/water. The extract was filtered, then concentrated.
The extract was mixed with a carrier, in this example with
maltodextrin prior to drying. Different drying technologies can be
applied. This extract was standardized in following polyphenols:
punicalagins (>7.5% by HPLC) and ellagic acid (1.5-2.5%) as
determined by HPLC.
[0140] Throughout this disclosure, this extract standardized in
punicalagins (>7.5% by HPLC) and ellagic acid (1.5-2.5%), will
be referred to either as Punica, or Punica extract or Punica
(powder) or Punica (liquid).
[0141] Preparation of Products and Mixtures of Rosemary
Extract/Hesperidin and Rosemary Extract/Punica Extract
[0142] Plant extracts and their combinations were dried into
powders. Maltodextrin was used in order to insure the suitable
drying process of combinations of extracts. Maltodextrins are
commonly used excipients or carriers for drying processes.
[0143] Maltodextrins are defined as starch hydrolysis products with
dextrose equivalent less than 20. Dextrose equivalent (DE value) is
a measure of the reducing power of starch derived oligosaccharides
expressed as percentage of D-glucose on dry matter of hydrolysate
and is inverse value of average degree of polymerisation (DP) of
anhydro glucose units. As products of starch hydrolysis,
maltodextrins contain linear amylose and branched amylopectin
degradation products, therefore they are considered as D-glucose
polymers joined by a-(1,4) and a-(1,6) linkages.
[0144] Although maltodextrins are derived from a natural compound
(starch), their structure is different from the initial structure
of the natural molecule they derive from (starch). This difference
is induced by the hydrolysis process. Thus, maltodextrin structure
does not occur in nature.
[0145] Other possible excipients or carriers include maltodextrin,
arabic gum, dextrose, salt, mono & diglycerides of fatty acids,
MPG, Polysorbate 80, vegetable oil, mono & diglycerides of
fatty acids, glucose syrup, glycerin, water and alcohol.
[0146] Compositions Were Added to the Raw Minced Beef Meat at 15%
fat.
[0147] In the course of the work leading to the present method and
composition, mixtures of rosemary extract and of hesperidin or of
Punica extract, in a number of varying concentration ratios were
tested for antilisterial effectiveness using the classical
microbiological methods. Bacterial enumeration in all here studied
samples was performed on the Aloa medium according to the
standardized method (NF EN ISO 11-290). The growth of Listeria
monocytogenes was evaluated in meat without any antilisterial agent
and without any plant extract (control). The data of listerial
growth in a control meat are represented in FIG. 2. It will be
noted that after 6 days of growth Listeria grew slightly, only by
0.29 log CFU/mL. After 9 days of growth, Listeria grew by 2.42 log
CFU/mL. Experiments on meat were conducted in modified atmosphere
packagings (MAP) that contained more than 20% O.sub.2, more
precisely 70% O.sub.2 and 30% CO.sub.2.
[0148] Following the meat manufacture, a batch of meat was sampled
straight after the mincing process and transported in refrigerated
conditions to the laboratory. In the laboratory, the meat was
sampled into 2 kg samples and conditioned in vacuum at -20.degree.
C., 24h prior to experimentation, the 2 kg meat samples were
transferred at 2-4.degree. C. and kept at this temperature for 24
h.+-.3 h until the core temperature attained -1.degree. C.
[0149] At this stage the 2 kg meat samples were inoculated with
Listeria monocytogenes in a laboratory of a biosafety of level 3 so
that the contamination by other microorganisms was avoided. Any
further supplementation to the meat was conducted in such a
laboratory. Following the homogenization of the inoculum at
4.degree. C., the inoculated 2 kg meat samples were supplemented
with plant extracts and homogenized. Plant extracts were in powder
form and were added as such to the meat. To keep them as dry
powders, plant extracts were supplemented with maltodextrin prior
to drying process.
[0150] Plant extracts could be added as lipophilic or hydrophilic
liquids, or combinations thereof, to the meat. To do so, plant
lipophilic or hydrophilic extract need to be solubilized or liquid,
undried extracts could be used directly without undergoing the
drying step.
[0151] Immediately after the supplementation of plant extracts and
homogenization, two pieces of 100 g of thus formed minced meat were
placed together in trays. Control meat pieces, without extract
treatment, followed the same procedure.
[0152] Trays were then conditioned under modified atmosphere of 20%
or more of oxygen, preferably 70% O.sub.2 and 30% CO.sub.2 at 4 or
at 8.degree. C. Packaged meat was stored in the dark for a stated
amount of time.
[0153] A series of experiments involving rosemary and hesperidin
extracts, rosemary and Punica extracts, typical antilisterial
compounds (Sodium lactate or Sodium acetate) and untreated control
were conducted. Mixtures or alone extracts of rosemary and of
hesperidin were added at 1.18% to the meat. Mixtures or alone
extracts of rosemary and of Punica were added at 0.48% to the meat.
Typical antilisterial compounds, Sodium lactate and Sodium acetate,
were added at classic concentrations 25 g/kg and 3 g/kg,
respectively, in separate experiments.
[0154] Combinations of extracts were prepared and added to the meat
according to the following proportions and doses prior to
testing:
TABLE-US-00001 Control LM 0.5R R 0.5H H 0.5R + 0.5H 0.5R + H R +
0.5H R + H Composition of Rosemary 0.00 1.28 2.56 0.00 0.00 1.28
1.28 2.56 2.56 extracts (%) extract Carnosic acid 0.00 0.56 1.13
0.00 0.00 0.56 0.56 1.13 1.13 Carnosic acid + 0.00 0.62 1.24 0.00
0.00 0.62 0.62 1.24 1.24 carnosol Hesperidin 0.00 0.00 0.00 24.00
49.00 24.00 48.00 24.00 48.00 extract Hesperidin 0.00 0.00 0.00
22.80 46.55 22.80 45.60 22.80 45.60 Composition in Rosemary 0 151
302 0 0 151 151 302 302 minced beef extract (ppm) Carnosic acid 0
66 133 0 0 66 66 133 133 Carnosic acid + 0 73 146 0 0 73 73 146 146
carnosol Hesperidin 0 0 0 2832 5782 2832 5664 2832 5664 extract
Hesperidin 0 0 0 2690 5493 2690 5381 2690 5381 Control LM 0.5R R
0.5P P 0.5R + 0.5P 0.5R + P R + 0.5P R + P Composition Rosemary
extract 0.00 3.33 6.65 0.00 0.00 3.33 3.33 6.65 6.65 of extracts
(%) Carnosic acid 0.00 1.47 2.93 0.00 0.00 1.47 1.47 2.93 2.93
Carnosic acid + 0.00 1.61 3.22 0.00 0.00 1.61 1.61 3.22 3.22
carnosol Pomegranate extract 0.00 0.00 0.00 13.50 27.00 13.50 27.00
13.50 27.00 Ellagic acid 0.00 0.00 0.00 0.27 0.54 0.27 0.54 0.27
0.54 Punicalagins 0.00 0.00 0.00 1.22 2.43 1.22 2.43 1.22 2.43
Composition Rosemary extract 0 160 319 0 0 160 160 319 319 in
minced beef Carnosic acid 0 70 140 0 0 70 70 140 140 (ppm) Carnosic
acid + 0 77 154 0 0 77 77 154 154 carnosol Pomegranate extract 0 0
0 648 1296 648 1296 648 1296 Ellagic acid 0 0 0 13 26 13 26 13 26
Punicalagins 0 0 0 58 117 58 117 58 117 R: rosemary extract; H:
hesperidin extract; 0.5R: half concentration of rosemary extract;
0.5H: half concentration of hesperidin extract R: rosemary extract;
P: Punica extract; 0.5R: half concentration of rosemary extract;
0.5P: half concentration of Punica extract
[0155] Immediately after the supplementation and the
homogenization, two pieces of 100 g minced meat in shape of
hamburgers were placed in trays. The trays were then conditioned in
a modified atmosphere containing 70% O.sub.2 and 30% CO.sub.2 and
stored at 8.degree. C. until analysis of Listerial growth and of
organoleptic features, including the red color. Such analyses were
conducted on the 0.sup.th, 6.sup.th and 9.sup.th day of
storage.
[0156] The growth of Listeria monocytogenes was evaluated in meat
in refrigerated conditions for each extract or compound and for
their combinations. The growth of Listeria monocytogenes was
measured at the beginning of the experiment, at 2/3rd of the
commercial shelf life (6 days) and at the time point corresponding
to the commercial duration of the shelf life (9 days). Logarithmic
values of Listerial growth (log CFU/mL) were calculated for each
experiment and treatment. Differences of logarithmic values of
Listerial growth (log CFU/mL) between the meat treated with plant
extracts and the untreated control were calculated to yield a final
result. The more negative value was obtained, the higher was the
antilisterial effect of the extract or of the combination of
extracts. In meat science microbiology, for a given time, values
are considered to be significant between two series when a
difference of 0.5 Log 10 CFUg.sup.-1 is observed (Chaillou et al.,
2014); (Guide pour la validation de methodes d'essais
microbiologiques et l'evaluation de leur incertitude de mesure dans
les domaines de la microbiologie alimentaire et de
l'environnement), Schweizerische Eidgenossenschaft, Confederation
suisse, Departement federal de l'economie, de la formation et de la
recherche DEFR, Document No. 328, April 2013, Rev. 03). In
microbiology, it will be noted that a treatment has a significant
antibacterial effect if its effect exceeds -0.5 log CFU/mL as
compared to the untreated control.
[0157] During the listerial growth, color of meat was monitored and
images were taken straight after the addition of the extract (on
the day 0) and on the 6.sup.th day of growth). Images were taken
under standardized light conditions of exposure and using a system
called "PackShot Creator." Indeed, this professional equipment
consists of an optimized light box containing four fluorescent
tubes diffusing homogeneous light, resulting in images always taken
under the same conditions, with minimal reflection.
[0158] Each picture representing the "sample" at a different time
scale was loaded in the open source image analysis program ImageJ.
The software is commonly used in the food industry to measure
different food parameters such as color or density (Reineke et al.
"The Influence of Sugars on Pressure Induced Starch Gelatinization,
Procedia Food Science, 1, 2011, 2040-3046; Kelkar et al.
"Developing novel 3D measurement techniques and prediction method
for food density determination , Procedia Food Science, 1, 2011,
483-491). In order to obtain representative values of the red
color, the color unit red (R) out of the three color units red (R)
green (G) and blue (B) of the RGB model was used, and the color was
measured of each pixel of a line that was drawn across the sample.
The inbuilt RGB profile plot plugging was used to determine the
different color values of each pixel along this line, notably the
values of the red color. The results are presented as variation of
the different color value as a function of the pixel number along
this line. The results were statistically analyzed for significant
differences using ANOVA test at p<0.05. Thus, per sample, more
than 1000 pixels were analyzed.
[0159] In order to evaluate the effect of plant extracts on the
color of the meat, red color of the treated meat was compared to an
untreated control. The effect was calculated by [red color in meat
with extract]-[red color in control meat (without extract)].
Negative effect means that the addition of extracts does not
preserve the red color of the meat. Positive effect means that the
addition of extracts improves the red color of the meat as compared
to the control.
Mixtures of Rosemary and Hesperidin
[0160] Growth of Listeria monocytogenes in Raw Meat
[0161] Results of such testing at 6.sup.th and 9.sup.th day are
presented in FIG. 7 and FIG. 8. Data are means of 2 to 6
replicates. Data represent log differences in L. monocytogenes
growth in treated meat as compared to inoculated controls
(non-treated meat). Data were statistically analyzed for
significance at p<0.05 using ANOVA. Different letters indicate
significant differences at p<0.05.
[0162] Results of such testing at 6.sup.th day using rosemary
extract and/or hesperidin are set forth in the following Table
1.
TABLE-US-00002 TABLE 1 Rosemary extract (%) Hesperidin (%) Expected
effect Measured effect R 0 -0.08 0 H -0.09 R H -0.08 to -0.17 0.07*
*Unexpected effect Rosemary extract and/or hesperidin: full
concentration effects on Listeria monocytogenes growth after 6 days
of growth in meat: [(log(CFU/mL) in meat treated with plant
extracts) - (log(CFU/mL) control meat (without treatment))]
[0163] It will be noted that at such short duration (six (6) days
of growth in cold conditions), the difference in listerial growth
in meat treated with plant extracts as compared with untreated
meat, expressed in log, did not attain -0.5 log, which means that
in such short time of growth, antilisterial effects could not be
appreciated. It will be noted that at such short duration (six (6)
days of growth in cold conditions), Listeria monocytogenes grew in
control meat only by 0.29 log CFU/mL (FIG. 2).
[0164] It will be noted that when combined, the measured effect of
the combination of rosemary extract and of hesperidin does not
correspond to a synergistic effect at the above concentrations
after 6 days of growth as the combinatory effect is unexpectedly
antagonistic.
[0165] When concentrations were halved, the following expected
effects calculated from the table above and measured effects were
obtained and shown in Table 2.
TABLE-US-00003 TABLE 2 Rosemary extract (%) Hesperidin (%) Expected
effect Measured effect 0.5R 0 -0.04 0.04* 0 0.5H -0.045 -0.37* 0.5R
0.5H -0.045 to -0.085 0.11* R 0.5H -0.045 to -0.125 -0.07 0.5R H
-0.04 to -0.13 0* *Unexpected effect Rosemary extract and
hesperidin: half concentrations and combinations of half and full
concentrations effects on Listeria monocytogenes growth after 6
days of growth in meat:[(log(CFU/mL) in meat treated with plant
extracts) - (log(CFU/mL) control meat (without treatment))]
[0166] It will be noted that at such short duration (6 days of
growth in cold conditions), the difference in listerial growth in
meat treated with plant extracts as compared with untreated meat,
expressed in log, did not attain -0.5 log, which means that in such
short time of growth, antilisterial effects could not be
appreciated. It will be noted that at such short duration (6 days
of growth in cold conditions), Listeria monocytogenes grew in
control meat only by 0.29 log CFU/mL (FIG. 2).
[0167] It will be noted though, that, in the above Table 2,
hesperidin applied at a half dose alone has surprisingly a greater
antilisterial effect than at the full dose. Unexpected effect is
signified by a star. On the other hand, effects of half dose of
rosemary and of combination of half dose rosemary and full dose
hesperidin were antagonistic from what was expected. Finally,
effects of combination of half dose rosemary and half dose
hesperidin and of combination of full dose rosemary and half dose
hesperidin remained within additional range, as expected.
[0168] Results of such testing at 9.sup.th day using rosemary
extract and/or hesperidin are set forth in the following Table
3.
TABLE-US-00004 TABLE 3 Rosemary extract (%) Hesperidin (%) Expected
effect Measured effect R 0 -1.12 0 H -0.64 R H -1.76 -0.85
*Unexpected effect Rosemary extract and hesperidin: full
concentration effects on Listeria monocytogenes after 9 days of
growth in meat: [(log(CFU/mL) in meat treated with plant extracts)
- (log(CFU/mL) control meat (without treatment))]
[0169] It will be noted that after nine (9) days of growth in cold
conditions, the difference in listerial growth expressed in log
CFU/mL exceeded -0.5 log CFU/mL, which means that antilisterial
effects of all extracts and their concentrations and combinations
presented in the above table could be appreciated within the
commercially desirable period.
TABLE-US-00005 TABLE 4 Rosemary extract (%) Hesperidin (%) Expected
effect Measured effect 0.5R 0 -0.56 -1.61* 0 0.5H -0.32 -0.71* 0.5R
0.5H -0.88 -1.64* R 0.5H -1.83 -1.68 0.5R H -2.28 -0.82 *Unexpected
effect Rosemary extract and hesperidin: half concentration and
combinations of half and full concentrations effects on Listeria
monocytogenes growth after 9 days of growth in meat: [(log(CFU/mL)
in meat treated with plant extracts) - (log(CFU/mL) control meat
(without treatment))]
[0170] After nine (9) days of growth in meat, extracts alone or
their combinations at all tested concentrations inhibited the
growth of Listeria monocytogenes by more than 0.5 log which means
that they had an antilisterial effect in meat.
[0171] Unexpectedly in view of the prior art and in view of data in
vitro, hesperidin had an antilisterial effect at all tested
concentrations. Further, unexpectedly, rosemary extract or
hesperidin alone had a greater antilisterial effect when used at
half concentrations as compared to full concentrations. Still
further, unexpectedly, rosemary extract combined with hesperidin at
half concentrations had a greater antilisterial effect than each
extract alone at full concentration. This is synergy (FIG. 3).
[0172] Different concentrations and their response surfaces were
analyzed using surface response methodology factorial experimental
design that was designed at three levels. These results are shown
in FIG. 4. They indicate the following concentration ranges that
provide antilisterial response in meat which is determined as:
[(log(CFU/mL) in meat treated with plant extracts)-(log(CFU/mL)
control meat (without treatment))]<0.5 as provided in Table
5.
TABLE-US-00006 TABLE 5 Extract Extract proportion in combination
(%) Hesperidin 0.5-48.0 Rosemary extract 0.2-3.0 Extract
proportions in combination that provide antilisterial response in
meat (%)
[0173] It will be noted that to insure antilisterial effect, any of
the above extract concentrations (Table 5) can be added in
combination or alone to the meat. The total percentage of the added
extract, alone or in combination, to the meat did not exceed
1.18%.
[0174] During the listerial growth, color of meat was monitored and
images were taken straight after addition of the extract (on the
day 0) and on the 6.sup.th day of growth).
[0175] Each picture representing the "sample" at a different time
scale was loaded in the open source image analysis program ImageJ.
The software is commonly used in the food industry to measure
different food parameters such as color or density (Reineke et al.
2011; Kelkar et al. 2011. In order to obtain representative values
of the three color units red (R), green (G) and blue (B) of the RGB
models, a line was drawn across the sample. The inbuilt RGB profile
plot plugin was used to determine the different color values of
each pixel along this line. The results are presented as variation
of the different color value as a function of the pixel number
along this line. The results were statistically analyzed for
significant differences using ANOVA test at p<0.05. Per sample,
more than 1000 pixels were analyzed.
[0176] Red Color of the Raw Meat
[0177] The color of the meat was appreciated by a panel of
sensorial analysis. This panel distinguished the meat color between
bright red, red, brown and green hues. All meat samples were bright
red on the day 0 of experiments.
[0178] On the 6.sup.th day, the overall panel appreciation
described the color of different meat samples subjected to
different meat treatments as following:
TABLE-US-00007 Meat color at Day 6 Control brown Sodium acetate
brown Sodium lactate brown 0.5R brown R red 0.5H green H brown 0.5R
+ 0.5H red 0.5R + H brown R + 0.5H red R + H brown
[0179] During the listerial growth, color of meat supplemented or
not with plant extracts was monitored and images were taken
straight after addition of the extract (on the day 0) and on the
6.sup.th day of growth).
[0180] Results of such monitoring at 6.sup.th day using rosemary
extract and hesperidin alone or in combination are set forth in the
following Table 6:
TABLE-US-00008 TABLE 6 Rosemary extract (%) Hesperidin (%) Expected
effect Measured effect R 0 11.05 0 H 10.69 R H 21.75 15.39
[0181] Rosemary extract and hesperidin of full concentration
effects on red meat color after 6 days of growth in meat. The
effect was calculated using: [red color of meat with extract]-[red
color of control meat (without extract)]
[0182] Contrary to the reports from the prior art, unexpectedly,
Rosemary extract better preserved the red color of the meat as
compared to the control. Hesperidin had slightly lower but similar
effect.
[0183] The combination effect remains within the additional range
and therefore was not found to be synergistic at these
concentrations.
[0184] The combination of extracts as compared to the control
significantly improves the preservation of the red color more than
each extract alone.
TABLE-US-00009 TABLE 7 Rosemary extract (%) Hesperidin (%) Expected
effect Measured effect 0.5R 0 5.53 14.143* 0 0.5H 5.35 -1.63* 0.5R
0.5H 12.51 17.16* R 0.5H 9.42 15.03* 0.5R H 16.22 13.33 *Unexpected
effect Rosemary extract and hesperidin at full and half
concentrations and combinations of half and full concentrations
effects on red meat color after 6 days of growth in meat. Each
effect was calculated using: [red color of meat with extract] -
[red color of control meat (without extract)]
[0185] FIG. 5 shows that when the concentration in hesperidin added
to meat is halved, it significantly decreased the preservation of
the red color of meat as compared to the control.
[0186] Adding rosemary significantly improved the preservation of
the meat color as compared to the untreated control meat.
Unexpectedly, halving rosemary concentration provoked a greater
effect in red color preservation of meat than the full rosemary
concentration. In addition, unexpectedly, halving hesperidin
concentration did not yield in a preservation effect of the red
color as expected but at this concentration, hesperidin
deteriorated the preservation of the red color as compared to the
control. Further, unexpectedly, the effect on the preservation of
the red color of the meat of the combination of full concentration
of rosemary and of half concentration of hesperidin, exceeded the
expected additional effects of rosemary at full concentration or of
hesperidin at halved concentration alone. This is synergy.
[0187] Still further, unexpectedly, the effect on the preservation
of the red color of the meat of the combination of half a
concentration of rosemary and of half a concentration of
hesperidin, exceeded the expected additional effects of half a
concentration of rosemary or of hesperidin at halved concentration
alone. This is synergy.
[0188] As to the rosemary extract at halved concentration and
hesperidin at full concentration, their combination effect remained
within the additional range and therefore was not found to be
synergistic.
[0189] FIG. 5 shows that all combinations between rosemary and
hesperidin at any here presented concentration significantly
improved the preservation of the red color of the meat as compared
to the control and as compared to typical antilisterial compounds
such as sodium acetate and sodium lactate.
Mixtures of Rosemary and Punica
[0190] Growth of Listeria monocytogenes in Raw Meat
[0191] Results of such testing at 6.sup.th and 9.sup.th day are
presented in FIG. 9 and FIG. 10. Data are means of 2 to 6
replicates. Data represent log differences in L. monocytogenes
growth in treated meat as compared to inoculated controls
(non-treated meat). Data were statistically analyzed for
significance at p<0.05 using ANOVA. Different letters indicate
significant differences at p<0.05.
TABLE-US-00010 TABLE 8 Rosemary Punica extract (%) extract (%)
Expected effect Measured effect R 0 0.26 0 P 0.05 R P 0.31 0.12
*Unexpected effect Rosemary extract and/or Punica extract: full
concentration effects on Listeria monocytogenes growth after 6 days
of growth in meat: [(log(CFU/mL) in meat treated with plant
extracts) - (log(CFU/mL) control meat (without treatment))]
[0192] It will be noted that at such short duration (6 days of
growth in cold conditions), the difference in listerial growth in
meat treated with plant extracts as compared with untreated meat,
expressed in log, did not attain -0.5 log, which means that in such
short time of growth, antilisterial effects could not be
appreciated.
[0193] It will be noted that when combined, the measured effect of
the combination of rosemary extract and of Punica extract did not
correspond to a synergistic effect.
[0194] When concentrations were halved, the following expected
effects calculated from the table above and measured effects were
obtained:
TABLE-US-00011 TABLE 9 Rosemary Punica extract (%) extract (%)
Expected effect Measured effect 0.5R 0 0.13 0.28 0 0.5P 0.025 0.27
0.5R 0.5P 0.55 0.11* R 0.5P 0.53 -0.03* 0.5R P 0.33 0.26*
*Unexpected effect Rosemary extract and/or Punica extract: half
concentrations and combinations of half and full concentrations
effects on Listeria monocytogenes growth after 6 days of growth in
meat: [(log(CFU/mL) in meat treated with plant extracts) -
(log(CFU/mL) control meat (without treatment))]
[0195] It will be noted that at such short duration (6 days of
growth in cold conditions), the difference in listerial growth
expressed in log did not attain -0.5 log, which means that in such
short time of growth, antilisterial effects could not be
appreciated.
[0196] Unexpected Effect is Signified by a Star
[0197] Unexpectedly, the antilisterial effect of the combination of
half a concentration of rosemary and of half concentration of
Punica, exceeded the expected additional effects of rosemary at
halved concentration or of Punica at halved concentration, alone.
This is synergy.
[0198] Further, unexpectedly, the antilisterial effect of the
combination of full concentration of rosemary and of half
concentration of Punica, exceeded the expected additional effects
of rosemary at full concentration or of Punica at halved
concentration, alone. This is synergy.
[0199] Still further, unexpectedly, the antilisterial effect of the
combination of half a concentration of rosemary and of full
concentration of Punica, exceeded the expected additional effects
of rosemary at halved concentration or of Punica at full
concentration, alone. This is synergy.
[0200] Results of such testing at 9.sup.th day using rosemary
extract and/or Punica extract are set forth in Table 10.
TABLE-US-00012 TABLE 10 Rosemary Punica extract (%) extract (%)
Expected effect Measured effect R 0 -0.39 0 P -0.53 R P -0.92 -0.63
*Unexpected effect Rosemary extract and Punica extract: full
concentration effects on Listeria monocytogenes growth after 9 days
of growth in meat: [(log(CFU/mL) in meat treated with plant
extracts) - (log(CFU/mL) control meat (without treatment))]
[0201] As mentioned above, in microbiology, it will be noted that a
treatment has an antibacterial effect if its effect exceeds -0.5
log CFU/mL as compared to the untreated control. It will be noted
that after 9 days of growth in cold conditions, compared to the
control, the difference in listerial growth expressed in log CFU/mL
exceeded -0.5 log CFU/mL when the meat was treated with full
concentrations of Punica or of the combination of full
concentration of rosemary and of full concentration of Punica.
Rosemary alone at the full concentration did not significantly
inhibit the listerial growth as compared to the untreated control
meat. However, combining rosemary at full concentration with Punica
at full concentration had a greater antilisterial effect than when
extracts were used alone and enabled to exceed the threshold of
-0.5 log CFU/mL that is required for a significant effect in
antilisterial growth.
TABLE-US-00013 TABLE 11 Rosemary Punica extract (%) extract (%)
Expected effect Measured effect 0.5R 0 -0.195 -0.35* 0 0.5P -0.265
-0.84* 0.5R 0.5P -1.19 -1.47* R 0.5P -1.23 -1.45* 0.5R P -0.88
-0.69 *Unexpected effect Rosemary extract and Punica extract: half
concentrations and combinations of half and full concentrations
effects on Listeria monocytogenes growth after 9 days of growth in
meat: [(log(CFU/mL) in meat treated with plant extracts) -
(log(CFU/mL) control meat (without treatment))]
[0202] After 9 days of growth in meat, all but one extracts alone
or their combinations at almost all tested concentrations inhibited
the growth of Listeria monocytogenes by more than 0.5 log CFU/mL as
compared to the control which means that they had an antilisterial
effect in meat. Only rosemary extract alone when tested at half a
concentration did not attain the difference of -0.5 log CFU/mL as
compared to the control.
[0203] Unexpectedly, Punica extract alone had a greater
antilisterial effect when used at half concentration as compared to
a full concentration. Further, unexpectedly, when used at a half
concentration, rosemary extract had a greater antilisterial effect
than expected. Still further, unexpectedly, the antilisterial
effect of the combination of half concentration of rosemary and of
half concentration of Punica extract, exceeded their expected
additional effects of rosemary at halved concentration or of Punica
extract at halved concentration alone. This is synergy.
[0204] In addition, unexpectedly, the antilisterial effect of the
combination of full concentration of rosemary and of half
concentration of Punica extract, exceeded the expected additional
effects of rosemary at full concentration or of hesperidin at
halved concentration, alone. This is synergy.
[0205] Further, unexpectedly, rosemary extract combined with Punica
extract at half concentrations had a greater antilisterial effect
than each extract alone at full concentration. This is synergy (See
e.g., FIG. 1).
[0206] Different concentrations and their response surfaces were
analyzed using surface response methodology factorial experimental
design that was designed at three levels. These results are shown
in FIG. 11. They indicate the following concentration ranges that
provide antilisterial response in meat which is determined as
[log(CFU/mL) in meat treated with plant extracts]-[log(CFU/mL)
control meat (without plant extract)]<-1 as shown in Table
12.
TABLE-US-00014 TABLE 12 Extract Extract (%) Punica extract 5.0-24.0
Rosemary extract 0.5-8.0 * Extract %
[0207] It will be noted that to insure antilisterial effect, any of
the above extract concentrations (Table 12) can be added in
combination or alone to the meat. The total percentage of the added
extract, alone or in combination, to the meat did not exceed
0.18%.
[0208] Red Color of the Raw Meat
[0209] The color of the meat was appreciated by a panel of
sensorial analysis. This panel distinguished the meat color between
bright red, red, brown and green hues. All meat samples were bright
red on the day 0 of experiments.
[0210] On the 6.sup.th day, the overall panel appreciation
described the color of different meat samples subjected to
different meat treatments as following:
TABLE-US-00015 Meat color at Day 6 Control brown Sodium acetate
brown Sodium lactate brown 0.5R brown R brown 0.5P red P red 0.5R +
0.5P red 0.5R + P brown R + 0.5P red R + P brown
[0211] During the listerial growth, color of meat supplemented or
not with plant extracts was monitored and images were taken
straight after addition of the extract (on the day 0) and on the
6.sup.th day of growth). Red pixels were quantified as explained
above in the Methods section.
[0212] Results of such monitoring at 6.sup.th day using rosemary
extract and Punica extract alone or in combination are set forth in
the following:
TABLE-US-00016 TABLE 13 Rosemary Punica extract (%) extract (%)
Expected effect Measured effect R 0 -3.72 0 P -5.60 R P -9.32 1.27*
*Unexpected effect Rosemary extract and Punica extract: full
concentration effects on the preservation of the red color of meat
after 6 days of growth in meat. The effect was calculated by: [red
color of meat with extract] - [red color of control meat (without
extract)]
[0213] At the above concentrations (Table 13), when added alone,
rosemary or Punica extract deteriorated the preservation of the red
color of the meat as compared to the control. It was therefore
expected that when combined, these extracts would even further
deteriorate the preservation of the red color of the meat.
Unexpectedly, when combined, rosemary and Punica extracts improved
the preservation of the red color of the meat as compared to the
control.
[0214] Unexpectedly, the effect on the preservation of the red
color of the meat of the combination of full concentration of
rosemary and of full concentration of Punica, exceeded the expected
additional effects of rosemary at full concentration or of full
concentration of Punica alone. This is synergy.
TABLE-US-00017 TABLE 14 Rosemary Punica extract (%) extract (%)
Expected effect Measured effect 0.5R 0 -1.86 1.07* 0 0.5P -2.80
-0.34* 0.5R 0.5P 0.73 4.45* R 0.5P -4.04 -2.71* 0.5R P -4.53 6.37*
*Unexpected effect
[0215] Rosemary extract and Punica extract: full and half
concentrations and combinations of half and full concentrations
effects on the preservation of the red color of the meat after 6
days of Listerial growth in meat. Each effect was calculated by:
[red color of meat with extract]-[red color of control meat
(without extract)]
[0216] As full concentrations of rosemary and of Punica extracts
deteriorated the preservation of the red color of the meat, it was
expected that halved concentrations would have also deteriorated
the preservation of the red color of the meat. Unexpectedly,
halving the added rosemary concentration significantly improved the
preservation of the red color of the meat as compared to the
untreated control.
[0217] Adding rosemary significantly improved the preservation of
the meat color as compared to the untreated control meat.
Unexpectedly, halving the concentration of the added Punica extract
did not deteriorate as much as expected the preservation of the red
color of the meat.
[0218] Further, unexpectedly, the improvement of the preservation
of the red color of the combination of half concentration of
rosemary and of half concentration of Punica extract, exceeded
their expected additional effects of rosemary at halved
concentration or of Punica extract at halved concentration alone.
This is synergy.
[0219] In addition, unexpectedly, the improvement of the
preservation of the red color of the combination of full
concentration of rosemary and of half concentration of Punica
extract, exceeded the expected additional effects of rosemary at
full concentration or of hesperidin at halved concentration, alone.
This is synergy.
[0220] Still further, unexpectedly, the improvement of the
preservation of the red color of the combination of full
concentration of Punica extract and of half concentration of
rosemary extract, exceeded the expected additional effects of
Punica extract at full concentration or of rosemary extract at
halved concentration, alone. This is synergy.
[0221] Unexpectedly, the combination of full concentration of
Punica extract and of half concentration of rosemary extract
improved the preservation of the color of the meat whereas it was
expected that the preservation of the color be deteriorated upon
application of such combination.
[0222] FIG. 6 shows that all combinations between rosemary and
Punica extract at any here presented concentration significantly
improved the preservation of the red color of the meat as compared
to the control and as compared to classic antilisterial agents such
as Sodium acetate and Sodium lactate.
[0223] One of ordinary skill in the art will recognize that
additional embodiments are also possible without departing from the
teachings of the presently-disclosed subject matter. This detailed
description, and particularly the specific details of the exemplary
embodiments disclosed herein, is given primarily for clarity of
understanding, and no unnecessary limitations are to be understood
therefrom, for modifications will become apparent to those skilled
in the art upon reading this disclosure and can be made without
departing from the spirit and scope of the presently-disclosed
subject matter.
[0224] In vitro experiments:
[0225] Antimicrobial activities of Rosemary, Punica and hesperidin
extracts alone and in combination Plant extracts of rosemary,
hesperidin, Punica and their combinations rosemary/hesperidin,
rosemary/Punica were prepared in 10% at DMSO.sub.50% final
according to the following protocol in which combinations of
extracts were prepared, completed up to 100% with maltodextrin and
added to the test solutions according to the following proportions
and doses prior to testing.
TABLE-US-00018 TABLE 15 R/H Composition of Rosemary extract 3.05
extracts (%) Carnosic acid 1.34 Carnosic acid + carnosol 1.48
Hesperidin extract 56.7 Hesperidin 53.87 R: rosemary extract; H:
hesperidin extract
TABLE-US-00019 TABLE 16 R P R/P Composition of Rosemary extract
5.33 0 5.33 extracts (%) Carnosic acid 2.35 0 2.35 Carnosic acid +
carnosol 2.58 0 2.58 Pomegranate extract 0 21.60 21.60 Ellagic acid
0 0.43 0.43 Punicalagins 0 1.94 1.94 R: Rosemary extract; P: Punica
extract
[0226] Preparation of the Working/Test Solution
[0227] 200 mg of the extract (combination) were mixed with 1 ml
100% DMSO, vortexed, sonicated for 10 min at power 100%, 45 kHz,
normal mode, well vortexed, sonicated again and diluted 1:2 in
sterile water at a final concentration of 100 mg/ml DMSO.sub.50%
final.
[0228] These preparations at 100 mg/ml were prepared in sterile 5
ml Eppendorf tube. A sample was taken prior to preparing Minimum
Bactericidal Concentration (MBC) plates. MBC, Minimal Fungicidal
Concentration (MFC) and Minimal Inhibitory Concentration (MIC).
[0229] Principle
[0230] The minimum bactericidal concentration (MBC) is the lowest
sample concentration required to kill at least 99.99% of the
inoculum (-4 log 10). The minimum bactericidal concentration (MBC)
is the lowest concentration of an antibacterial agent required to
kill a particular bacterium. It can be determined from broth
dilution minimum inhibitory concentration (MIC) tests by
subculturing to agar plates that do not contain the test agent. The
MBC is identified by determining the lowest concentration of
antibacterial agent that reduces the viability of the initial
bacterial inoculum by 99.9%. The MBC is complementary to the MIC;
whereas the MIC test demonstrates the lowest level of antimicrobial
agent that inhibits growth, the MBC demonstrates the lowest level
of antimicrobial agent that results in microbial death. This means
that even if a particular MIC shows inhibition, plating the
bacteria onto agar might still result in organism proliferation
because the antimicrobial did not cause death. Antibacterial agents
are usually regarded as bactericidal if the MBC is no more than
four times the MIC. This test was based on the count of
microorganisms in wells displaying little or no growth visually and
then plated.
[0231] Samples prepared in 10% DMSO.sub.50% .sub.final were tested
at concentrations of 2.5, 0.5 and 0.1%, against one equivalent DMSO
control (respectively 12.5, 2.5 and 0.5%).
[0232] In the case of yeast, fungicide minimum concentrations (CMF)
were performed as the WCD. In the case of the strain A.
brasiliensis, a fungus, the mere presence of growth was interpreted
as absence of fungicidal activity. The minimum inhibitory
concentration (MIC) is the lowest concentration of an antimicrobial
that will inhibit the visible growth of a microorganism after
adapted period of incubation. Minimum inhibitory concentrations are
typically used to determine the potency of new antimicrobial
agents, such as plant extracts or their combinations. The minimal
inhibitory concentration or MIC is the lowest concentration that is
sufficient to inhibit microbial growth by here tested extracts.
This test is based on visual observation of the wells that contain
microbial strain that is studied with tested extracts or without
(control). Time length and conditions of microbial growth were
conducted according to classical methods well known by one ordinary
skilled in the art.
[0233] The tests were performed on the following bacterial strains
E. coli, Pseudomonas aeruginosa, Salmonella enterica ser
typhimurium, Staphylococcus aureus, Bacillus cereus, Listeria
monocytogenes, Streptococcus mutans, Clostridium perfringens,
Enterococcus hirae, Enterobacter cloacae, Moraxella bovis and two
yeasts Saccharomyces cerevisiae and Candida albicans and mold
Aspergillus brasiliensis. The evaluation of bactericidal and
fungicidal activity was conducted according to the internal
procedure in rich nutrient media , with a calibrated inoculum of
10.sup.5-10.sup.6 CFU/ml for bacteria and yeasts and 10.sup.5
spores/ml for A. brasiliensis.
[0234] Prior to antimicrobial evaluation of extracts, the sterility
of extracts was verified by plating. The absence of any microbial
growth was compulsory. Microbial cultures were conducted
classically as summarized in Table 17.
TABLE-US-00020 TABLE 17 Growth conditions and collections of
microorganisms Organism Collection Culture conditions Bacillus
cereus ATCC 11.778 TS-30.degree. C.-24 h Staphylococcus CIP 4.83
TS-37.degree. C.-18 h aureus Listeria ATCC 19.115 TS-37.degree.
C.-24 h monocytogenes Salmonella CIP 103.799 TS-37.degree. C.-18 h
typhimurium Escherichia coli CIP 53.126 TS-37.degree. C.-18 h
Pseudomonas CIP 82.118 TS-37.degree. C.-18 h aeruginosa
Streptococcus ATCC 35.668 COS 5% sang/Broth Streptos- mutans
37.degree. C.-24 h Enterococcus ATCC 8043 Broth TS-37.degree. C.-24
h hirae Enterobacter CIP 103.475 Broth TS-30.degree. C.-24 h
cloacae Moraxella bovis CIP 70.40 T TS/Broth Col 10% horse
serum-30.degree. C.-24 h Clostridium ATCC 13.124 RCM/37.degree.
C./Anaerobiosis/48 h perfringens Candida albicans UMIP 48.72
Sabouraud/30.degree. C./48 h Saccharomyces UMIP 1181.79
Sabouraud/30.degree. C./48 h cerevisiae Aspergillus niger IP
1431.83 Sabouraud/30.degree. C./48 h
[0235] Results
TABLE-US-00021 TABLE 18 MBC and MFC concentrations (0.1; 0.5 or
2.5%) of Rosemary (R), Punica (P), Rosemary/Punica (R/P) and
Rosemary/Hesperidin (R/H) as effective against the here presented
microorganisms. R/H R/P S. aureus 0.5 0.1 S. mutans 2.5 0.5 S.
typhimirium 2.5 0.1 P. aeruginosa 2.5 0.1 E. coli 2.5 0.5 L.
monocytogenes 0.5 B. cereus 2.5 0.1 C. perfringens 0.5 0.1 E. hirae
2.5 E. cloacae 0.5 M. bovis 0.1 0.1 R/H combination of rosemary and
hesperidin extracts according to Table 15; R: rosemary extract, P:
Punica extract, R/P: combination of rosemary and Punica extracts
according to Table 16. Empty cells: minimal concentrations (if any)
would be higher than the highest concentration here measured
(2.5%).
TABLE-US-00022 TABLE 19 Unexpected/Synergistic MBC and MFC effects
of R and P (% in test solution) Expected Measured MBC/MFC MBC/MFC
MBC/MFC MBC/MFC of R (%) of P (%) of R/P (%) of R/P (%) S.
typhimirium 0.5 0.5 0.25-0.5 0.1* P. aeruginosa 0.5 0.5 0.25-0.5
0.1* C. perfringens 0.03 >0.016 >0.016 0.0078* *Unexpected
result Unexpectedly, when applied in combination, the minimal
concentration of the combination R/P to decrease microbial growth
by 4 log, S. typhimirium and P. aeruginosa is surprisingly low; it
is 2.5-5 times lower than expected. Surprisingly, when the two
extracts are combined, their activity is synergistically increased
by 2.5-5 times.
[0236] Unexpectedly, the anti-salmonella effect of the combination
of rosemary and Punica extracts, exceeded their expected additional
effects when extracts were applied alone. Indeed, it would have
been expected that to achieve the same anti-salmonella effect and
decrease microbial growth by 4 log, MBC would have been at best
halved when the extracts are combined as compared to MBC when
extracts were applied alone. Surprisingly, here, MBC were decreased
by 2.5-5 times. This is synergy.
[0237] Further unexpectedly, the anti-pseudomonas effect of the
combination of rosemary and Punica extracts, exceeded their
expected additional effects when extracts were applied alone.
Indeed, it would have been expected that to achieve the same
anti-pseudomonas effect and decrease microbial growth by 4 log, MBC
would have been at best halved when the extracts are combined as
compared to MBC when extracts were applied alone. Surprisingly,
here, MBC were decreased by 2.5-5 times. This is synergy.
[0238] Still further unexpectedly, the anti-clostridium effect of
the combination of rosemary and Punica extracts, exceeded their
expected additional effects when extracts were applied alone.
Indeed, it would have been expected that to achieve the same
anti-clostridium effect and decrease microbial growth by 4 log, MIB
would have been at best halved when the extracts are combined as
compared to MIB when extracts were applied alone. This is synergy.
Note, Punica extract had a MIC effect of 0.016%. This implies that
its MBC must be higher than MIC, meaning higher than 0.016%. Either
way, it is further unexpected that the MBC of the combination of
rosemary and Punica extracts is more than twice lower than the MIC
of an extract alone.
TABLE-US-00023 TABLE 20 Unexpected/Synergistic MIC effects of R and
P (% in test solution) Expected Measured MIC MIC MIC MIC of R (%)
of P (%) of R/P (%) of R/P (%) S. mutans 0.25 >1 0.25 0.0625* S.
aureus 0.125 0.5 0.125-0.325 0.0625* C. albicans 1 >1 1 0.25* S.
cerevisiae 0.25 >1 0.25 0.0625*
[0239] Unexpectedly, when applied in combination, the minimal
concentration of the combination R/P to inhibit microbial growth of
bacteria or of yeast, S. mutans, S. aureus, C. albicans and S.
cerevisiae is surprisingly low; it is 2-4 times lower than
expected. Surprisingly, when the two extracts are combined, their
activity is synergistically increased by 2.5-5 times than
expected.
[0240] Further unexpectedly, the anti-streptococcus effect of the
combination of rosemary and Punica extracts, exceeded their
expected additional effects when extracts were applied alone.
Indeed, it would have been expected that to achieve the same
anti-streptococcus effect and inhibit microbial growth, MIC would
have been at best at the lowest MIC as compared to MIC when
extracts were applied alone. Surprisingly, here, MIC were decreased
by 4 times than expected. This is synergy.
[0241] Still further unexpectedly, the anti-staphylococcus effect
of the combination of rosemary and Punica extracts, exceeded their
expected additional effects when extracts were applied alone.
Indeed, it would have been expected that to achieve the same
anti-staphylococcus effect and inhibit microbial growth, MIC would
have been at best halved when the extracts are combined as compared
to MIC when extracts were applied alone. Surprisingly, here, MIC
were decreased by 2-4.5 times than expected. This is synergy.
[0242] Further unexpectedly, the anti-candida effect of the
combination of rosemary and Punica extracts, exceeded their
expected additional effects when extracts were applied alone.
Indeed, it would have been expected that to achieve the same
anti-candida effect and inhibit microbial growth, MIC would have
been at best at the lowest MIC as compared to MIC when extracts
were applied alone. Surprisingly, here, MIC were decreased by 4
times than expected. This is synergy.
[0243] And, still further unexpectedly, the anti-saccharomyces
effect of the combination of rosemary and Punica extracts, exceeded
their expected additional effects when extracts were applied alone.
Indeed, it would have been expected that to achieve the same
anti-saccharomyces effect and inhibit microbial growth, MIC would
have been at best at the lowest MIC as compared to MIC when
extracts were applied alone. Surprisingly, here, MIC were decreased
by 4 times than expected. This is synergy.
[0244] It is noted that P. aeruginosa and S. typhimirium are Gram
negative bacteria. C. perfringens, S. mutans, S. aureus and L.
monocytogenes are Gram positive bacteria. C. albicans and S.
cerevisiae are yeast.
[0245] Rosemary in combination with Punica extract clearly exhibits
synergistic antimicrobial effects against the growth of Gram
positive (including L. monocytogenes (in meat), C. perfringens, S.
mutans, S. aureus), Gram negative (including S. typhimirium, P.
aeruginosa) bacteria and yeast (including C. albicans,
Saccharomyces cerevisiae).
TABLE-US-00024 TABLE 21 Unexpected/Synergistic effects of the
combination of extracts R/P on different microorganisms are here
summarized Nature of Gram Antimicrobial Microorganism microorganism
(+ or -) effect L. monocytogenes Bacteria G+ Unexpected, Synergy S.
typhimirium Bacteria G- Unexpected, Synergy P. aeruginosa Bacteria
G- Unexpected, Synergy C. perfringens Bacteria G+ Unexpected,
Synergy S. mutans Bacteria G+ Unexpected, Synergy S. aureus
Bacteria G+ Unexpected, Synergy C. albicans Yeast Unexpected,
Synergy S. cerevisiae Yeast Unexpected, Synergy
[0246] Note that hesperidin in combination with rosemary had no
bactericidal effect in vitro on Listeria monocytogenes even at
highest here tested concentrations: 2.5%.
[0247] Yet, surprisingly, such extract combination significantly
and efficiently inhibited the growth of Listeria monocytogenes in
meat products in synergistic manner.
[0248] Similar observation could be drawn when comparing hesperidin
effects in vitro, as reported in prior art against Listeria
monocytogenes growth and in meat (this study). Indeed, although
hesperidin in vitro did not have antilisterial effects, it
inhibited listerial growth in beef meat.
[0249] Use of Rosemary/Punica and Rosemary/Hesperidin Extracts in
Meat/Poultry/Fish Matrices
[0250] Processing Methods, Extract Incorporation, Listerial
Contamination and Growth Inhibition
[0251] Fresh pork sausages and poultry were produced according to
standard recipes used by industry. As far as fish and seafood are
concerned, here exampled by smoked salmon, its processing was also
done according to the industrial procedures.
[0252] Meat/Poultry/Fish Batches
[0253] Whenever possible, to overcome the variability of raw
materials in terms of pH and endogenous flora (rate and nature of
the constituent flora) for each meat/category, the processing was
carried out on 3 batches of raw materials originating from
different meat/poultry/fish suppliers/(salmon) farms. Plant
extracts or their combinations were incorporated at the beginning
of each recipe, together with basic processing ingredients. The
plant extracts or their combinations were incorporated in food
matrices at 1-3 different concentrations, as explained in Tables
23, 24, 26, 27, 29, and 30 below.
[0254] As far as every product category is concerned, controls,
i.e. products not comprising plant extracts, were tested.
[0255] Nature and Origin of Strains of Listeria
monocytogenes/Bacterial Preparation Conditions
[0256] Two strains of Listeria monocytogenes in mixture (50/50)
were studied according to the Standard Operating Procedure NF
V01-009 and concerning any meat/poultry/fish product. As far as
tests in meat products are concerned: the reference strain CIP 7838
(serovar 4b) and a so-called field strain isolated from pork (ADIV
collection) serotype 1/2a, the one that predominates in more than
50% on the fresh pork were used.
[0257] As far as poultry meat is concerned, the reference strain
7838 was coupled to a strain of Listeria monocytogenes isolated
from poultry carcass (ADIV collection; serotype 1/2b). As far as
tests in salmon are concerned: the reference strain CIP 7838 was
mixed with a strain isolated from salmon that ADIV was donated by a
partner of its network. As far as any here studied food matrix is
concerned, the two strains were prepared and inoculated according
to the guidelines of Standard Operating Procedures NF V01-009
(Version 2014).
[0258] Frozen strains, preserved as cryobilles (-80.degree. C.),
were revived and cultivated individually. Each strain was revived
by transplanting 0.1 mlin 10 ml of BHI culture medium (for 24 h at
30.degree. C.). Two successive subcultures have resulted in a
pre-culture of each strain. Subsequently, each strain was again
cultured for 24 hours at 30.degree. C. The latter culture was used
in matrix product contamination (at end of the exponential phase or
early stationary phase). After two successive centrifugations, the
pellets were suspended in 10 ml of buffered peptone water (BPW),
counts were performed on BHI medium and the bacterial solution was
kept at 0.degree. C. for 24 hours prior to inoculation. After
reading of the bacterial growth, the concentrations were then
adjusted so as to inoculate the food matrix product with the
mixture of the two strains (50/50) at 2-3 log cfu/g.
[0259] Inoculation Method
[0260] As specified in the NF V01-009, the products inoculation
mode should match the reality of industrial contamination. Thus,
sausages (pork or poultry) were infected by inoculation in the mass
of the meat mix to simulate contamination from meat As the
contamination of smoked salmon occurs during the handling of raw
materials or when slicing/packaging, here contamination of the
surface of salmon slices was applied.
[0261] The inoculation method on the surface of the products has
been developed by ADIV and provided an accurate weight of the
inoculum, calculated in a way to obtain the desired concentration
at the surface of the products. Irrespectively of the method of
inoculation (ground or surface), in order to maintain the adequate
water activity of food products, the aqueous volume to be added to
the food matrix was calculated so as not to exceed a weight/volume
ratio of 1/100 (NF V01-009).
[0262] Meat/Poultry/Fish Product Elaboration and Extract
Incorporation
[0263] Pork Sausages
[0264] Pork sausages were manufactured according to a conventional
process, in compliance with the code of practice, from lean pork
(86%) and pork fat (14%). The initial meat mix intended for sausage
manufacturing was obtained by grinding/mincing fat and lean parts
of pork meat at low-temperature trough a grid (6 mm grid). Then,
here tested plant extracts were added whenever applicable,
according to Table 24 and Table 25. After homogenization, the mix
was then stuffed into natural casings (sheep menus, 24/26
diameter).
[0265] Contamination/Packaging
[0266] For a given batch, a meat mix of 42 kg was prepared and then
divided into different sets of 5 kg each for the production of test
series. Five (5) kg of meat mix were added to a mix prepared that
did not contain any antimicrobial agent nor additive (Mix Fraiche
230 South at 23 g/kg) and 50 g water/kg. After homogenization, thus
obtained mixture was divided into parts. A portion without
artificial listerial contamination, was placed in trays (6 sausages
per tray) that were packaged in MAP conditions (70% O.sub.2/30%
CO.sub.2) with a conservation time scenario 1/3 of conservation
time at 4.degree. C. and 2/3 of conservation time at 8.degree. C.
The other part was artificially contaminated by Listeria
monocytogenes at an average rate of 3 log cfu/g (inoculation in the
ground meat mass) before being packaged and conserved as described
above. The contaminated meat samples that did not comprise plant
extracts were termed control.
[0267] Analytical Monitoring During the Conservation
[0268] Microbiological analyses were performed on D0 and JDLC (D14)
and they concerned the enumeration of Listeria monocytogenes, the
total mesophilic acidifying flora (FAM) and the lactic flora. At
each point of analysis, a single repeat batch was performed by
analysis of a tray (n=3 for the 3 batches).
[0269] Manufacturing of Poultry Sausages
[0270] Poultry sausages were produced according to the same
experimental conditions as described for pork sausages. The only
difference remains in the nature of the raw materials used and the
nature of compounds that may have been added. Indeed, in this case,
the lean was made using upper parts of chicken thighs 85% and fat
represented by the skin.
[0271] The ground meat mix did not include any classical
antimicrobial but contained a mix from which any ingredients having
antimicrobial or antioxidative activity were removed (Mix Chipo
Flight 310 to 31 g/kg). Thus prepared mixture either comprised
plant extracts, or not (controls) and followed the same
experimental conditions and listerial/bacterial analyses as
described for pork.
[0272] Preparation and Inoculation of Smoked Salmon
[0273] The manufacture of smoked salmon was conducted according to
classical procedures well known from and available to a person
skilled in the art. As for meat products, three different batches
of salmon from three different origins were made during the same
week. The products were packaged sliced on trays under vacuum
(about 200 g per tray) on the industrial site prior to
listerial/bacterial inoculation and analysis.
[0274] Tested seafood, i.e. smoked salmon, were incorporated
extract combinations of Rosemary and hesperidin extracts, and of
Rosemary and Punica extracts according to proportions and
concentrations described in Tables 29 and 30. The salmon was
weighed and then surface-inoculated by spraying, with the mixture
of the two strains (50/50) at an average rate of 3.+-.0.5 log/g.
After inoculation, the plates were again vacuum packed and put into
storage at 8.degree. C. for 30 days. Listerial growth was monitored
upon inoculation (day 0) and on the 30.sup.th day of growth.
Non-inoculated controls were kept at cold storage for all
meet/poultry/fish matrices tested here for comparison purposes.
[0275] pH, total mesophilic acidifying flora, lactic flora were
measured throughout all experiments in beef, pork, poultry meat or
fish. The presence of here tested botanical extracts in these food
matrices had no significant impact on pH, total mesophilic
acidifying flora, lactic flora.
EXAMPLE
Antimicrobial Effects of R/P and R/H in Poultry Sausages
[0276] Classical method, available to persons skilled in the art
and based on the protocol described for minced beef and above, was
applied to processed poultry sausages.
[0277] As described above, briefly, three different batches of
fresh poultry meet processed into sausages, comprising R/P and R/H
extract combinations at different concentrations were inoculated
with Listeria monocytogenes. Controls did not comprise plant
extracts. Fresh poultry meat samples were kept in cool conditions
and Listerial growth was measured on the 14.sup.th day.
TABLE-US-00025 TABLE 22 Initial Listerial load per batch of poultry
sausages Batch L. monocytogenes (cfu/g) Batch 1 1.95E+03 Batch 2
2.93E+03 Batch 3 2.75E+03
[0278] Extracts were prepared as follows: Half quantities of R (0.5
R) and half quantities of H (0.5H) or half quantities of P (0.5P)
extracts as defined above, were mixed together according to the
Table 21 and, the mix was completed up to 100% by maltodextrin.
These powdered mixes were added to the poultry fresh meat upon
processing into sausages in proportions as described in Tables 23
and 24. Tables 23 and 24 also indicate final contents in extracts
and extract compounds in % and in ppm, in extract and in the meat
matrix.
[0279] Note that 0.5% of 0.5R+0.5H would correspond to the
quantities in extract compounds termed 0.5R+0.5H that were tested
in minced beef. Further note that 0.3% of 0.5R+0.5P would
correspond to the quantities in extract compounds termed 0.5R+0.5P
that were tested in minced beef. All obtained data were analyzed
and expressed (i.e. delta log etc) according to explanations
communicated regarding minced beef.
TABLE-US-00026 TABLE 23 Control 0.5R + 0.5H Composition Rosemary
extract 0.00 3.05 of extracts Carnosic acid 0.00 1.34 (%) Carnosic
acid + carnosol 0.00 1.48 Hesperidin extract 0.00 56.70 Hesperidin
0.00 53.87 0.6% Composition Rosemary extract 0 183 in poultry
Carnosic acid 0 80 (ppm) Carnosic acid + carnosol 0 89 Hesperidin
extract 0 3402 Hesperidin 0 3232
TABLE-US-00027 TABLE 24 Control LM 0.5R + 0.5P Composition Rosemary
extract 0.00 5.33 of extracts Carnosic acid 0.00 2.35 (%) Carnosic
acid + carnosol 0.00 2.58 Pomegranate extract 0.00 21.60 Ellagic
acid 0.00 0.43 Punicalagins 0.00 1.94 0.2% 0.4% Composition
Rosemary extract 0 107 213 in poultry Carnosic acid 0 47 94 (ppm)
Carnosic acid + carnosol 0 52 103 Pomegranate extract 0 432 864
Ellagic acid 0 9 17 Punicalagins 0 39 78
[0280] Results:
[0281] L. monocytogenes has clearly grown in control meat (FIG.
12.). When combinations of extracts were added to the meat, they
efficiently inhibited Listerial growth (FIG. 13 and FIG. 14). Those
data confirm the data found in minced beef which clearly
demonstrated synergistic antilisterial effect of rosemary and
hesperidin, and of rosemary and Punica extracts.
EXAMPLE
Antimicrobial Effects of R/P and R/H in Pork Sausages
[0282] Classical method, available to people of ordinary skill in
the art and based on the protocol described for minced beef and
above, was applied to processed pork sausages.
[0283] Briefly, three different batches of fresh pork meet
processed into sausages, comprising R/P and R/H extract
combinations at different concentrations were inoculated with
Listeria monocytogenes. Controls did not comprise plant extracts.
Fresh pork meat samples were kept in cool conditions (8.degree. C.)
and Listerial growth was measured on the 14.sup.th day.
TABLE-US-00028 TABLE 25 Initial Listerial load per batch Batch L.
monocytogenes (cfu/g) Batch 1 1.55E+03 Batch 2 2.96E+03 Batch 3
2.36E+03
[0284] Extracts were prepared as follows: Half quantities of R (0.5
R) and half quantities of H (0.5H) or half quantities of P (0.5P)
extracts as defined above, were mixed together according to the
Table 24 and, the mix was completed up to 100% by maltodextrin.
These powdered mixes were added to the pork fresh meat upon
processing into sausages in proportions as described in Tables 26
and 27. Tables 26 and 27 also indicate final contents in extracts
and extract compounds in % and in ppm, in extract and in the meat
matrix.
[0285] Note that 0.5% of 0.5R+0.5H would correspond to the
quantities in extract compounds termed 0.5R+0.5H that were tested
in minced beef. Also note that 0.3% of 0.5R+0.5P would correspond
to the quantities in extract compounds termed 0.5R+0.5P that were
tested in minced beef. All obtained data were analyzed and
expressed (i.e. delta log etc) according to explanations given
above regarding minced beef.
TABLE-US-00029 TABLE 26 Control 0.5R + 0.5H Composition Rosemary
extract 0.00 3.05 of extracts Carnosic acid 0.00 1.34 (%) Carnosic
acid + carnosol 0.00 1.48 Hesperidin extract 0.00 56.70 Hesperidin
0.00 53.87 Control 0.4% 0.5% 0.6% Composition Rosemary extract 0
122 153 183 in pork Carnosic acid 0 54 67 80 (ppm) Carnosic acid +
carnosol 0 59 74 89 Hesperidin extract 0 2268 2835 3402 Hesperidin
0 2155 2694 3232 Control 0.5R + 0.5H Composition Rosemary extract
0.00 3.05 of extracts Carnosic acid 0.00 1.34 (%) Carnosic acid +
carnosol 0.00 1.48 Hesperidin extract 0.00 56.70 Hesperidin 0.00
53.87 0.4% 0.5% 0.6% Composition Rosemary extract 0 122 153 183 in
pork Carnosic acid 0 54 67 80 (ppm) Carnosic acid + carnosol 0 59
74 89 Hesperidin extract 0 2268 2835 3402 Hesperidin 0 2155 2694
3232
TABLE-US-00030 TABLE 27 Control LM 0.5R + 0.5P Composition Rosemary
0.00 5.33 of extracts extract (%) Carnosic acid 0.00 2.35 Carnosic
0.00 2.58 acid + carnosol Pomegranate 0.00 21.60 extract Ellagic
acid 0.00 0.43 Punicalagins 0.00 1.94 0.2% 0.3% 0.4% Composition
Rosemary 0 107 160 213 in pork extract (ppm) Carnosic acid 0 47 71
94 Carnosic 0 52 77 103 acid + carnosol Pomegranate 0 432 648 864
extract Ellagic acid 0 9 13 17 Punicalagins 0 39 58 78
[0286] Results:
[0287] L. monocytogenes has clearly grown in control meat (FIG.
15). When combinations of extracts were added to the meat, they
efficiently inhibited Listerial growth at all here tested
concentrations (FIG. 16 and FIG. 17). Those data confirm the data
found in minced beef which clearly demonstrated a synergistic
antilisterial effect of rosemary and hesperidin, and of rosemary
and Punica extracts.
[0288] Classical method, available to persons skilled in the art
and based on the protocol described for minced beef and above, was
applied to smoked salmons.
[0289] As described above, briefly, three different batches of
smoked salmon, comprising R/P and R/H extract combinations at
different concentrations were inoculated with Listeria
monocytogenes. Controls did not comprise plant extracts. Smoked
salmon samples were kept in cool conditions and Listerial growth
was measured on the 14.sup.th day.
[0290] Initial Listerial load per batch of poultry sausages was as
presented in Table 28:
TABLE-US-00031 TABLE 28 Initial Listerial load in smoked salmon
Batch L. monocytogenes (cfu/g) Batch 1 1.46E+03 Batch 2 4.93E+03
Batch 3 5.64E+03
TABLE-US-00032 TABLE 29 Control 0.5R + 0.5H Composition Rosemary
extract 0.00 3.05 of extracts Carnosic acid 0.00 1.34 (%) Carnosic
acid + carnosol 0.00 1.48 Hesperidin extract 0.00 56.70 Hesperidin
0.00 53.87 Control 0.4% 0.5% Composition Rosemary extract 0 122 153
in smoked Carnosic acid 0 54 67 salmon Carnosic acid + carnosol 0
59 74 (ppm) Hesperidin extract 0 2268 2835 Hesperidin 0 2155
2694
TABLE-US-00033 TABLE 30 Control LM 0.5R + 0.5P Composition Rosemary
extract 0.00 5.33 of extracts Carnosic acid 0.00 2.35 (%) Carnosic
acid + carnosol 0.00 2.58 Pomegranate extract 0.00 21.60 Ellagic
acid 0.00 0.43 Punicalagins 0.00 1.94 0.3% Composition Rosemary
extract 0 160 in smoked Carnosic acid 0 71 salmon Carnosic acid +
carnosol 0 77 (ppm) Pomegranate extract 0 648 Ellagic acid 0 13
Punicalagins 0 58
[0291] Note that 0.3% of 0.5R+0.5P corresponds to the same
proportions and dosage of extract combination added in minced beef
and termed 0.5R+0.5P.
[0292] Results:
[0293] L. monocytogenes has clearly grown in control smoked salmon
(FIG. 18). When combinations of extracts were added to the meat,
they efficiently inhibited Listerial growth (FIG. 19 and FIG. 20).
Those data confirm the data found in minced beef which clearly
demonstrated a synergistic antilisterial effect of rosemary and
hesperidin, and of rosemary and Punica extracts.
REFERENCES
[0294] Numerous references have been cited throughout this
disclosure. All references cited in this disclosure including the
three additional references listed below are incorporated by
reference.
[0295] Kai Reineke, Henning Weich, Dietrich Knorr, "The Influence
of Sugars on Pressure Induced Starch Gelatinization", Procedia Food
Science, Vol. 1, (2011), pages 2040-2046.
[0296] Shivangi Kelkar, Scott Stella, Carol Boushey, Martin Okos,
"Developing novel 3D measurement techniques and prediction method
for food density determination", Procedia Food Science, Vol. 1,
(2011), pages 483-491.
[0297] S. Chaillou, S.Christieans, M. Rivollier, I. Lucquin, M. C.
Champomier-Verges, M. Zagorec; "Quantification and efficiency of
Lactobacillus sakei strain mixtures used as protective cultures in
ground beef"; Meat Science 97, (3) (2014), pages 332-338.
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