Note: Descriptions are shown in the official language in which they were submitted.
CA 02942193 2016-09-09
WO 2015/136366 PCT/1B2015/000727
ORGANIC ACID ANTIMICROBIAL COMPOSITIONS
FIELD OF THE INVENTION
[0001] The present invention relates to a stable composition, consisting of
GRAS
substances, for microbial control, specifically in fermentation processes
BACKGROUND OF THE INVENTION
[0002] Organic carboxylic acids are generally known to have antimicrobial
properties.
Recent work has shown synergistic effect between organic carboxylic acids and
other types
of antimicrobial agent, such as hops acid. For example, US application
2004/0175480
described that the hops beta acids were more effective as antimicrobial agent
if they were in
combination with food grade acid, such as lactic acid, acetic acid, propionic
acid, citric acid,
a potassium ion source, and an antioxidant.
[0003] Antimicrobial organic acids are generally soluble in water at high
concentration,
but some of the organic acid antimicrobial agents are not soluble in water or
salted out in the
presence of electrolyte. As an example, hops acids are known to have limited
solubility in
water and the antimicrobial compositions of hops acids described in prior arts
are low in
active even with the use of organic solvents such as glycols or suspension
agents.
[0004] One way to combine the different antimicrobial is to spray the
liquid antimicrobial
agent onto the powdery organic acid or salt, but the resulting composition is
not easily
dispersible in water. Although wetting or/and emulsifying agents can be
formulated into the
solid composition, it is more convenient to have liquid products with high
active content for
handling and use. Products with high active content also save cost in
processing,
transportation and storage from the sustainability point of view.
[0005] It is known that surfactant can help solubilize hydrophobic material
in water.
Surfactants such as those described in US patents 5374614, 7655613 and 7846889
were
utilized to form microemulsions, a thermodynamically stable isotropic liquid,
from water-
soluble and water-insoluble ingredients. Such microemulsion compositions
containing
organic acids were also described in US patents 5294364, 6251844, 7030070 and
7642227
as acidic cleaning liquids. The organic acid content is low and surfactants
used might be
prohibited from applications in food or articles in contact with food.
[0006] It is desirable to have a single product with all synergistic
antimicrobial
components in a stable concentrated form with water as the solvent. It is much
preferred
that the antimicrobial product is of little concern in terms of flammability,
corrosivity, toxicity
and regulatory requirements.
CA 02942193 2016-09-09
WO 2015/136366 PCT/182015/000727
2
DESCRIPTION OF THE INVENTION
[0007] The invention provides for an aqueous antimicrobial composition
comprising: (i)
water soluble organic acids or their salts, (ii) antimicrobial agents with
limited water-solubility,
and (iii) surfactants.
[0008] This invention provides a non-hazardous stable aqueous antimicrobial
composition comprising water soluble organic acids and antimicrobial agents of
limited
water-solubility at high active content, preferably greater than 10%, more
preferably greater
than 20% and most preferably greater than 30% of the total weight of the
composition. The
compositions are formulated with GRAS (Generally Recognized as Safe)
ingredients that are
authorized to be used in food and beverage and the compositions are classified
as none
hazardous materials in terms of flammability and corrosively. The compositions
are stable
as homogeneous liquid for greater than 1 month, preferably greater 3 months
and most
preferably greater than 6 months at room temperature or below.
[0009] The compositions according to the present invention were designed to
function
as antimicrobial agents, preferably in reducing or controlling the
concentration of undesirable
micro-organisms in fermentation processes. Such similar compositions could
also be used
for other purposes such as for anti-parasites or as concentrating cleaning, de-
scaling and
disinfecting agents. The active components of the composition comprise one or
more
organic acids or their salts, and one or more antimicrobial agents of limited
water solubility,
with a total active content (organic acid plus antimicrobial agent) greater
than 5% by weight,
preferably greater than 15% and most preferably greater than 25% based on the
total weight
of the composition. The compositions also comprise one or more surfactant
solubilizer.
These include but not limited to anionic surfactants, non-ionic surfactants,
amphoteric
surfactants, zwitterionic surfactants and cationic surfactants. Additional
ingredients such as
neutralizing agent, solvent can also be added to increase the total active
content without
destabilizing the composition. Functional agents could also be added such as
chelating
agents, sequestrants, corrosion inhibitor, enzymes, surface modification
agents, dyes and
fragrances.
[0010] The water soluble organic acids preferred for use in the invention
are selected
from water-soluble organic mono- and polycarboxylic acids with two to eight
carbon atoms in
the molecule and optionally substituted by one or more hydroxy groups.
Suitable classes
are alkanoic acids, hydroxyalkanioc acids, alkyl polycarboxylic acids and
hydroxyalkyl
polycarboxylic acids. Preferred herein are mono- and polycarboxylic acids
which have a
pKa value, related to the first dissociation stage of less than about 6. These
include, for
example, acetic acid, adipic acid, benzenesulfonic acid, benzoic acid, citric
acid, gluconic
acid, glutaric acid, hydroxyacetic acid, lactic acid, malic acid,
methanesulphonic acid, oxalic
CA 02942193 2016-09-09
WO 2015/136366 PCI71B2015/000727
3
acid, propionic acid, salicylic acid, succinic acid, tartaric acid, and
mixtures thereof as well as
their salts. A preferred acid is citric acid or its salt. For purposes of this
invention hops acids
is not an organic acid.
[0011] The water soluble organic acid content is from 5 to 70%, from 5 to
60%, from 5 to
50%, preferably 15 to 60%, most preferably 25¨ 50% by weight within the total
composition.
[0012] Suitable antimicrobial agents of limited solubility include hops
acids or nisin. The
term "hops acids" as used herein includes hops beta acids, hops alpha acids,
their
isomerized or derivatized products and their salts. It also includes hops
extracts such as
those described in US 5286506. The chemical identities of alpha and beta hops
acids
include cohumulones, humulones, adhumulones, colupulones, lupulones and
adlupulones.
These all are included in the term "hops acids" as use herein. Hops acids can
be prepared
by extraction and purification from natural hops or by chemical synthesis. A
preferred
antimicrobial agent is hops acids.
[0013] The hydrophobic antimicrobial component, preferably hops acids,
active contents
are from 0.05 to 15%, preferably 0.1 to 10%, most preferably 0.5 ¨ 5% by
weight within the
total composition.
[0014] The surfactant solubilizing or emulsifying agents for antimicrobial
components of
low water-solubility include nonionic, anionic, cationic, amphoteric and
zwitterionic
surfactants. Surfactants described in US patents 7846889, 7030070, 6251844 and
5294364
are herein incorporated by reference. Preferred surfactants are those
authorized to be used
as food and pharmaceutical ingredients or in articles with potential food
contact. These
surfactants include polysorbates (polyoxyethylene sorbitan fatty acid esters)
such as the
Tweens (Croda Inc), mono or dialkyl sulfosuccinates such as Aerosol OT
(Cytec), alkyl
polyglucosides such as Glucopons (BASF) and alkyl sulfates. Preferred
surfactants for use
in the invention include polyoxyethylene (20) sorbitan monooleate and alkyl
polyglucosides.
[0015] The composition can contain one or more surfactants from one single
group or
from different groups of surfactants. The amount of one or more surfactants in
the
compositions is from 0.1 to 30%, preferably 1 to 20%, most preferably 2 ¨ 15%
by weight
within the total composition.
[0016] A limited amount of organic solvent or co-surfactants can also be
used so that the
amount of surfactant can be reduced. Examples are fatty alcohols such as
methanol,
ethanol, propanol, butanol, glycols or polyhydric alcohol of ethylene glycol,
propylene glycol,
glycerol; fatty acids or diacids such as butanoic acid, adipic acid, octanoic
acid. Preferred
co-surfactants include ethanol, propylene glycol and adipic acid.
CA 02942193 2016-09-09
WO 2015/136366 PC171B2015/000727
4
[0017] The composition may contain one or more co-surfactants or organic
solvents
from one single group or from different types. The amount of the one or more
co-surfactants
or organic solvents in the composition is from 0.1 to 30%, preferably 1 to
20%, most
preferably 2 ¨ 10% by weight within the composition.
[0018] The organic acid can be neutralized totally or partially with alkali
or alkali earth
hydroxide, ammonium hydroxide, or organic amines. Examples are sodium
hydroxide,
potassium hydroxide and ammonia. The degree of neutralization depends on the
type and
the amount of organic acids. Typically, the composition should be neutralized
to pH above 2
to reduce the corrosiveness of the organic acid, but no more than pH 4 to
retain the highest
active content possible. However, for high stability of the composition,
neutralization to
alkaline pH is preferred.
[0019] The composition is preferred to be formulated as a micro-emulsion, a
thermodynamically stable, isotropic liquid mixture of oil and water containing
surfactant
preferably with a co-surfactant or solvent. The organic acid is first
dissolved in water and
neutralized to the desired pH, and then mixed with at least one surfactant, at
least one co-
surfactant and at least one antimicrobial agent of limited water-solubility.
It is preferred that
the hydrophobic antimicrobial agent is first dissolved in at least one
surfactant and at least
one co-surfactant or at least one solvent to make a pre-blend, which is then
diluted with
neutralized organic acid aqueous solution.
[0020] It is preferred all components of the antimicrobial composition are
authorized as
food ingredients or granted GRAS (Generally Recognized as Safe) status by FDA.
It is also
preferred the composition is none hazardous in terms of corrosiveness and
flammability.
[0021] The compositions provide a powerful, non-antibiotic, antimicrobial
treatment. The
composition can be used for reducing undesirable microorganism concentration,
promoting
desirable microorganism propagation, and increasing desirable microorganism
efficiency in
an aqueous system.
[0022] In the foregoing method, the "undesirable" microorganisms intended
to be
reduced are those that compete for nutrients with the desirable microorganisms
that promote
the desired fermentation processes. Unwanted or undesirable microbes in
fermentation
include the lactic acid producing bacteria (LAB) and the acetic acid producing
bacteria of
which Lactobacillus and Acetobacter are prominent representatives. Any microbe
that
competes for the fermentable substrate, denying it to the intended fermenting
organism and
thus reducing yields can be considered undesirable. In this regard, the hops
acid extract
and organic acid employed in the present method do not detrimentally affect
the growth and
viability of desirable, fermentation-promoting microorganisms, but does
eliminate or
CA 02942193 2016-09-09
WO 2015/136366 PCT/1B2015/000727
suppress the growth of undesirable microorganisms that interfere with the
fermentation
process. Moreover, the elimination or suppression of undesirable
microorganisms has a
favorable effect on the growth and viability of desirable microorganisms.
[0023] The method of controlling undesirable microorganism concentration in
an
aqueous solution employed in a fermentation process comprising the steps of:
introducing a
fermentable carbohydrate to an aqueous solution, introducing at least one
yeast to the
aqueous solution, and introducing the composition containing (a) the water
soluble organic
acids or their salts, (b) the antimicrobial agents with limited water-
solubility, and (c) the
surfactants to the aqueous solution.
[0024] The composition is added to the fermentation system in order to
control the
undesirable microorganism concentration such that the amount of the organic
acid in the
fermentation system is from 12500 ppm down to 100 ppm, from 6250 down to 100
ppm, or
from 4000 down to 100 ppm, or from 4000 down to 200 ppm. Generally at least
100 ppm or
at least 200 ppm or at least 300 ppm of organic acid is used. The composition
is added to
the fermentation system such that the amount of the antimicrobial agent with
limited water-
solubility in the fermentation system is from 0.5 ppm to 200 ppm, or from 0.5
ppm to 150
ppm, or from 2 to 150 ppm, or from 2 to 100 ppm. Generally the amount of hops
acid used
in is at least 2 ppm or at least 5 ppm.
EXAMPLES
Example #1
[0025] Mixing Tween 80 (polyoxyethylene (20) sorbitan monooleate), 10.82
parts,
absolute ethanol, 3.74 parts and hops acid, potassium salt (30% active acid),
3.15 parts in
the order described to give a homogeneous liquid. In a separate vessel,
dissolve citric acid,
anhydrous 34.49 parts in demineralized water 34.49 part, and the cold solution
was then
neutralized by adding ammonium hydroxide of 28-30% ammonia content, 6.02
parts. The
partially neutralized citric acid was then added to the organic phase
containing hops acid,
resulting in an isotropic liquid of about 35% active combined antimicrobial
agents. The
composition was found stable at 4 C without crystallization of citrate salt
and stayed as
homogeneous for 1 month at 50 C. No negative effect was observed as an
antimicrobial
agent with the formulation.
CA 02942193 2016-09-09
WO 2015/136366 PCT/1B2015/000727
6
Table 1. Compositions of Citric and Hops Acid (percent by weight)
Example # 1 2
Citric Acid, anhydrous 34.49 37.84
D. Water 41.78 45.84
Ethanol, 99.5%, absolute 3.74 2.05
Tween 80 10.82 5.94
Ammonium hydroxide, 28-30% NH3 6.02 6.60
HOPS Acid, 30% 3.15 1.73
Stability:
F/T (- 20 C) Stable as liquid Stable as liquid
4 C (crystallization) >46 days >46 days
22 C >76 days >76 days
32 C >70days >70 days
50 C (liquid phase separation) 30 days 37 days
Example #2
[0026] The same procedure as in Example #1 was applied with the charges
listed in
Table 1 to yield a composition of 38% active combined active antimicrobial
agents. The
composition was found stable for at 4 C and room temperature without
crystallization of
citrate salt and stayed as homogeneous for 37 days at 50 C (Table 1). Again,
no adverse
effects were observed in antimicrobial properties with the formulation.
Examples #3 to 11
[0027] Three GRAS status surfactants, that is, Tween 80, Aerosol OT-70PG
(Cytec),
and alkyl polyglucoside of Triton BG-10 (Dow) or Glucopon 215 UP (BASF), were
formulated
in these Examples according to charges listed in Table 2. Citric acid was
first dissolved in
water and then partially neutralized with ammonia. The surfactants, Tween 80,
Aerosol OT-
70PG, Triton BG-10 or Glucopon 215 UP were added as listed, followed by hops
acids and
ethanol. After thorough mixing, a homogeneous liquid was always obtained with
shelf life
listed in Table 2.
CA 02942193 2016-09-09
WO 2015/136366 PCT/1132015/000727
7
Table 2. Compositions of Citric and Hops Acid (percent by weight)
Example # 3 4 5 6 7 8 9 10 11
Citric Acid, anhydrous 34.49 33.77 32.33 33.37 34.18 33.56
33.35 34.49 32.03
D. Water 41.78 40.90 39.15 40.41 41.38 40.64 40.40
41.78 38.99
Ethanol, 99.5%, absolute 4.15 4.19 3.86 3.98 4.13 4.07
4.16 4.30 3.34
Tween 80 11.62 10.16 2.72 10.25
11.82
Aerosol OT-70PG 4.94 1.48 10.86 1.75 1.47
Triton BG-10 5.48 10.68 5.20 1.08 10.01
Glucopon 215 UP 11.70 4.57
Aq. NH3, 28-30% 6.02 5.90 5.65 5.83 5.97 5.86 5.83
6.02 5.62
HOPS Acid, 30% 3.14 3.08 2.96 3.04 3.11 3.14 3.10
3.16 3.63
Stability:
FIT (- 20 C) liquid liquid liquid liquid liquid liquid stable stable
stable
4 C (crystallization), days >43 >70 >43 >70 >70 >70 >56
>56 >36
22 C, days 20d >70d 20d >70d >70d >70d >56d >56d >36
50 C (phase separation), days 2 36 2 54 70 30 34 56
>36
Examples 12- 13
[0028] In these
examples potassium hydroxide was used as neutralizing agent. The
same procedure as Examples #3-10 was followed with charges by weight listed in
Table 3.
The active contents of antimicrobial agents were increased to above 40%, but
the shelf life
was limited. The instability was thought to be caused by the acid-liable
surfactant
solubilizers.
Examples 14-16
[0029] In these
examples potassium hydroxide was used to neutralize the acids toward
alkaline pH for high stability. Alternative co-surfactants of non-flammable
propylene and/or
adipic acid were also used as shown in Table 4 of these compositions. The same
procedure
as Examples #3-10 was followed with charges by weight listed in Table 4. No
hydrolysis of
the surfactants used was expected, and no destabilization and little
discoloration of hops
acid were observed upon aging for more than 6 weeks at 4, 22 and 50 C.
CA 02942193 2016-09-09
WO 2015/136366 PCT/1B2015/000727
8
Table 3. Compositions of Citric and Hops Acid (percent by weight)
Example # 12 13
D. Water 39.47 41.96
Aerosol OT-75 (in ethanol) 3.78 2.87
Tween 80 5.27 2.81
Citric acid, anhydrous 39.39 41.47
45% KOH 8.50 9.00
30% Isoalpha Acid, Extract 3.59 1.89
Stability:
F/T (- 20 C) Stay as liquid Stay as liquid
4 C >2 month >2 month
22 C 10 weeks 4 weeks
50 C < 24 hr < 24 hr
Table 4. Alkaline Compositions of Citric and Hops Acid (percent by weight)
Example # 14 15 16
Citric Acid 25.30 25.76 25.76
Adipic Acid 3.22 3.23 3.20
Potasium Hydroxide, 45% 53.90 54.78 54.80
Glucopon 215UP, 62% 11.80 10.40 10.40
Hops acid, 30% 2.87 2.92 2.92
Ethanol, 99.5% 2.91 2.91
Propylene glycol 2.92
pH 8.80 8.88 9.13
Examples 17-18
[0030] In these examples, ethanol was used as solvent to dissolve both hops
acid and
citric acid in water (Table 6). However, this composition is considered
flammable due to the
presence of large amount of flammable solvent.
CA 02942193 2016-09-09
WO 2015/136366 PCT/1B2015/000727
9
Table 6. Hops/Citric Acid Compositions in Ethanol/Water
(percent by weight)
Example # 17 18
Ethanol, 99.5% absolute 35.08 32.79
Citric acid, anhydrous 39.50 40.82
30% lsoalpha Acid, Extract 3.60 1.66
D. Water 21.82 24.53
Examples 19-20
[0031] In these samples, propylene glycol was added to mitigate the
flammability of the
compositions (Table 7). But propylene glycol is not a good solvent for citric
acid, the active
content is limited.
Table 7. Hops/Citric Acid Compositions in Propylene Glycol/Ethanol/VVater
(percent by weight)
Example # 19 20
Ethanol, 99.5% absolute 10.90 10.90
Propylene glycol 45.18 42.50
Citric acid, anhydrous 22.93 24.80
30% lsoalpha Acid, Extract 2.09 1.13
D. Water 18.90 20.67
[0032] Example 21: MIC Determinations of Citric Acid/Hops Acid Compositions
[0033] In the following examples, the endpoint used to measure levels of
antimicrobial
activity is known as the Minimal Inhibitory Concentration, or MIC. This is the
lowest
concentration of a substance or substances which can achieve complete
inhibition of growth.
[0034] Five milliliters of growth medium were inoculated with the
appropriate test
microbe and were incubated with shaking overnight. These overnight cultures
(ON) were
diluted 1:100 (100 pl of ON into 10 mL PBS). Corning 96-well microtiter plates
were used
CA 02942193 2016-09-09
WO 2015/136366
PCTi1B2015/000727
and prepared by dispensing 252 pl of media into column 1 and 140 pl into
columns 2-12.
Next, 28 pl of the formulation was dispensed into column 1 to achieve a 1:10
dilution and the
highest concentration of the composition to be tested. Column 1 was mixed by
pipetting and
twofold serial dilutions were carried out across the plate (140 pl of media +
composition
transferred each time). Column 12 was used as a composition-free control,
therefore 140 pl
of media + formulation from column 11 was removed to waste. Finally, 5 pl of
bacteria was
added to each well, yielding ¨5 x 105 cfu/ml. The 96 well plates were covered,
sealed with
parafilm and incubated at the appropriate temperature for 18-24 hours. The
wells were
scored as positive or negative for growth based on a visual examination for
turbid wells, with
turbidity being an indicator of growth. The lowest concentration of
antimicrobial which
completely inhibits growth (e.g., a clear well) is designated the Minimal
Inhibitory
Concentration.
Table 8. MIC Values
Formulation Citric Acid: Hops Acid Test Microbe Growth Media MIC
(ppm product)
Example 1 40:1 L. plantarum MRS Broth 781
M9YG 781-3125
Example 1 40:1 S. aureus
TSB ___________________________________________________________ 391-1563
M9YG 781-1563
Example 1 40:1 B. subtilis t-
TSB 781-3125
Example 2 80:1 L. plantarum MRS Broth 781-1563
Example 11 30:1 L. plantarum MRS Broth 1722
Example 14 30:1 L. plantarum MRS Broth 2365
Example 16 30:1 L. plantarum MRS Broth 781-1563
