FORM-2
THE PATENTS ACT, 1970
( 39 of 1970 )
PROVISIONAL/COMPLETE
SPECIFICATION SECTION 10
TITLE
AMBIENT STABLE BEVERAGE
APPLICANT
HINDUSTAN LEVER LIMITED, a Company incorporated under ths Indian Companies Act, 1913 and having its registered office at Hindustan Lever House, 165/166, Backbay Reclamation, Mumbai 400 020, Maharashtra, India
The following specification particularly describes the nature of the invention and the manner in which it is to be performed

AMBIENT STABLE BEVERAGE
Field of the invention
The present invention relates to an ambient stable beverage, particularly a tea based beverage, that is preserved by a preservative system that comprises cinnamic acid, dimethyl dicarbona.te. ..and ..at ..least one. esseatial. oil...
Background and prior art
In recent years there has been an ever increasing choice for
consumers who wish to' quench their thirst with ready made
beverages.'"" Many of those are now turning from the well known soft
drinks to' tea based beverages, be those carbonated or-still, and
the "natural" refreshment they can' providev~ ■•.-.-.
Tea contains a complex combination of enzymesV' biochemical intermediates" and structural elements normally associated with plant growth and photosynthesis. There are also many natural 'substance's'-Jt^
colour." ~"iiahy of these "are produced by the oxidation reactions that occur during the so-called fermentation stage of black tea manufacture. Tea production has long been driven by traditional processing methods with only a fundamental understanding of the chemistry that is involved. As a consequence manufacturers have discovered making ambient stable tea based beverages at the volumes required to compete with more traditional soft drinks is not simply a matter of flavouring a soft drink with tea.
The flavour of a tea based beverage and its stability rely on the stability of the beverage as a whole. The fungi including yeasts

and moulds that can grow in tea based beverages and other soft drinks can be killed by'heat treatment or at least controlled by use of preservatives. Some tea based beverages are therefore pasteurised and then bottled in glass or special heat stable PST containers. This is known as "hot filling". Unfortunately this can be an expensive operation that creates a great deal of environmentally unfriendly waste. It has therefore become more attractive for manufacturers to pack their tea based products in standard PET containers which can range from single serve units to mu it i-serve...packs, and maintain. the., stability , of., the product, using tailor made flavour and preservative systems. This is known as "cold filling". It is also useful in that one can readily use a tea concentrate or powder.
Unfortunately the use of common preservatives can affect the flavour of a tea based beverage. This is particularly true for sulphite and sorbate.. Adding a. strong flavour such as lemon can : of f set the ..preservative, taste. . However consumers are keen to experience other flavours.. . Furthermore, some of those consumers that were drawn to tea based products as a more healthy and natural alternative to soft drinks sometimes view preservatives as the sort of .synthetic additives. they would rather avoid. _.,
..Many....countries.have regulations that, prohibit the. use of certain food .additives, including some fungicides and preservatives, in foods and beverages. Regulations can vary widely but there is a clear trend for foods to contain fewer and lower levels of chemical fungicides and preservatives, particularly synthetic ones.
Accordingly there is a need for pleasantly flavoured, ambient-stable, tea based beverages that have low levels of synthetic preservatives.
/ In response to that need the present inventors have now developed an ambient stable tea based beverage that is preserved by a

preservative system that comprises cinnamic acid, dimethyl dicarbonate and at least one essential oil. Non-tea based beverages including fruit and soft drinks can be stabilised in a similar way.
Statement of the Invention
The invention can in broad terms.be said to relate to an ambient stable beverage, such as a tea based beverage, that is preserved by a preservative system that comprises cinnamic acid, dimethyl dicarbonate and at least one essential oil;
The beverage preferably contains 1 to 175 ppm cinnamic acid, 1 to 500 ppm dimethyl dicarbonate (DMDC) „and 1.: to. 100 ppm of at least one essential oil. When the beverage is tea based it preferably contains 0:01 to 3%:tea solids, especially about 0.14%.
The invention can also be said to,relate ..to a;.method for preparing an ambient-stable tea based beverage suitable for cold filing comprising' adding' cinnamic. acid;idimethyl .•:dicarbonate... and . at least one' essehtialxbil to' a" tea extract.^-: .-.•..■ ;..0M:. .:. ;. -
"Beverage"..', for; the; purposes of the present invention means -any • drink, other than water,". and ^includes; soft- drinks, fruit drinks, coffee based drinks and tea based/drinks.
"Essential oil" for the purposes of the present invention includes any of the volatile oils in plants having the odour or flavour of the plant from which they are extracted. It also includes one or more of the components of that oil that is or are responsible for or at least contributes to the odour or flavour of that plant.
"Tea" for the purposes of the present invention means leaf material from Camellia sinensis var. sinensis or Camellia sinensis

var. assamice. "Tea" is also intended to include the product of blending two or more of any of these teas.
For the avoidance of doubt the word "comprising" is intended to mean including but not necessarily "consisting of" or "composed of". In other words the listed steps or options need not be exhaustive.
Except in the operating and comparative examples, or where otherwise explicitly indicated, all numbers in-this -description. . indicating amounts or concentrations of material ought to be understood as modified by the word "about".
Brief description of the Figures
Figure 1 shows the results of a control experiment of growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14%■•tea. ■■.-.; -
Figure 2 shows the combined:effeet of,citral dimethyl acetal, cinnamic acid and DMDC on growth of. yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea.
Figure 3 shows the combined effect of cumic alcohol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea.
Figure 4 shows the combined effect of citral, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea.
Figure 5 shows the combined effect of 3,7-dimethyl octanol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea.

Figure 6 shows the combined effect of myrtenol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea.
Figure 7 shows the combined effect of piperonyl acetate, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea.
Figure 8 shows the combined effect of trans,trans-2,4-decadienal, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready ..to Drink tea, 0.14% tea.
Figure 9 shows the combined effect of 5-decanolactone, cinnamic ..acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea.
Figure 10.shows;the,combined.effect of citral dimethyl acetal,
curaic alcohol, cinnamic acid and DMDC on: growth of ..yeast.,::.;.- -.. ■
Saccharomyces cerevisiae X2180-1B in a matrix of-tubes .of .-.'Ready to
Drink tea, 0.14% tea. ' -;-:r :.•;>.:.■>;;.
Figure 11 gives ;the results of control.experiment of:growth of yeast Saccharomyces.cerevisiae. X2180-1B ina matrix of tubes of synthetic soft drink,- 0% tea.
Figure■■: 12 ;shows the combined effect of citral dimethyl acetal, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of synthetic soft drink, 0% tea.
Figure 13 shows the combined effect of cumic alcohol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of svnthetic soft drink, 0% tea.

Figure 14 shows...the combined effect of citral, cinnamic acid and DMDC on growth of yeast Saccharomycas cerevisiae X2180-1B in a matrix of tubes of synthetic soft drink, 0% tea.
Figure 15 shows the combined effect of 3,7-dimethyl octanol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of synthetic soft drink, 0% tea.
Figure 16 shows the combined effect of myrtenol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X218..Q.-lB..in, a. matrix of tubes of synthetic soft drink, 0% tea.
Figure 17 shows the combined effect of piperonyl acetate, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of synthetic soft drink, 0% tea.
Figure 18 shows the combined effect of trans, trans-2, 4-decadienal/"cinnamic acid and DMDC on growth of yeast' Saccharomyces'cerevisiae X2180-1B in a matrix; of tubes of -synthetic soft drink, 0% tea.
Figure 19.shows "the combined effect of 5-decanolactone, cinnamic acid and DMDCon-growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of synthetic soft drink, 0% tea'l
Figure 20 shows the combined effect of citral dimethyl acetal, cumic alcohol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of synthetic soft drink, 0% tea.
Figure 21 shows the effective concentrations of trans, trans-2,4-decadienal.
Figure 22 shows the effective concentrations of citral.

Detailed description of the invention
The ambient stable beverage of the present invention is preserved by a preservative system comprising cinnamic acid, dimethyl dicarbonate and at least one essential oil.
Cinnamic acid
Cinnamic acid (3-phenyl-2-propenoic acid) is a well known flavouring agent for cakes, drinks, chewing gum and ice cream. Derived from cinnamon, which has;.long been added to foods, it is regarded in most countries as a useful and harmless flavouring. When dissolved in a tea based beverage cinnamic acid imparts a mild resinous odour resembling honey and flowers with a sweet and weak spicy taste. A flavouring effect is evident at • concentrations above about 10 ppra. At concentrations above 30 ppm the flavour becomes particularlyrstrong. :••. .An: additional -benefit is the suppression of unwanted preservative notes from chemicals such as sorbic and benzoic'":acids 1' Of-.the ..two stereo isomers .-that exist, the trans-isomer is more ;commonly, of interest for-'-use in flavouring.' ■• "■ -' . .. .•.-••:..•-.:-:.•.. : :.••:.■:• --.:..\-:■■;•:..• ;.•-.- :.:.; .;L'~ „-x:::. . Cinnamic acid was given GRAS : (i.e; Generally Recognised:as: Safe) status by the FEMA :(Flavouring-..Extract .Manufacturers^.Association) in 1965. While there is no:legislation in the-European"Union that prevents or limits the use of cinnamic acid in food or beverages, the normal usage maximum that has previously been agreed within the industry is 31 ppm. More recently 174.9 ppm has been allowed for non-alcoholic beverages.
A number of cinnamic acid derivatives are known and used in the food industry. These include p-dimethylamino-cinnamate, cinnamaidehyde, cinnamyl acetate, cinnamyl alcohol, cinnamyl benzoate, cinnamyl cinnamate, cinnamyl formate, cinnamyl isobutyrate, cinnamyl isovalerate and cinnamyl phenylacetate. For the purposes of this invention one could substitute or combine

cinnamic acid with one or more of its derivatives although one would need to consider' the concentrations required to achieve desired results any impact on aroma and taste.'
While not wanting to be bound by theory, the present inventors believe that cinnamic acid works as a membrane active compound that at low pH increases the concentration of the membrane soluble cinnamic acid, i.e. it does not function as a classic weak acid preservative.
The beverage of the present invention preferably contains 1 to 175 ppm cinnamic acid, more preferably 1 to 60.ppm, more especially 1 to 30 ppm.
Dimethyl dicarbonate
Dimethyl"dicarbonate is well known ..sterilant for soft .drinks.- -.:: It is also known as dimethyl.pyrocarbonate or.DMDC and is marketed by the.Bayer AG under, the trade-name VELCORIN™. . :DMDC:isa useful; sterilant; as.upon addition it:rapidly kills microbes. DMDC breaks down rapidly in aqueous environments so it poses no risk to the consumer. • However it offers no long term preservative action..; DMDC was approved for-use as :a.yeast inhibitor in wines at.the point of bottling by the United .'States Food and Drug' Administration on October 21, 1988. The EU cleared DMDC as a cold sterilant in 1989.
DMDC is known to be inefficient against mould contamination.
For the purposes of the present invention the preservative system should preferably contain between 1 and 500 ppm dimethyl dicarbonate, more preferably between 1 and 250 ppm dimethyl dicarbonate.

Essential c
The inventors tested a large number of antimicrobials and found the following to be suitable for use in the preservative system of the present invention. The minimum inhibitory concentration (MIC) is given for each compound.
TABLE I
Preferred essential oils
COMPOUND ' MIC (ppm)
Benzyl-4-hydroxybenzoate 68
4-tert-Butylcyclohexanone 4 62
Carvone . ' 300
Cinnamaldehyde 66
Citral 228
Citral dimethyl acetal 198
: Citrpnellol„ . 125.
Cumic' alcohol" ' 450
. CyclohexariebutyriC; acid .-. 68...
2-Cyclohexylethyl^ acetate. .102
trans"; trans-2','4-Decadienal 8
. Decanal. ::. 47 ..
Decanol 24
Dihydrocarveol 540
3,7-Dimethyl-l-octanol 15.8
Ethyl cyclohexanepropionate 184
Ethyl- pyruvate 1392
Ethyl-vanillin-. 24-9 ■
Jasmone 24 6
o-Methoxycinnamaldehyde 130
Methyl anthranilate 310
cc-Methyl-trans-cinnamaldehyde 58.4
Methyl eugenol 356
Methyl nonanoate 90
2-Methyl-2-pentenal 1274
5-Methyl-2-phenyl-2-hexenal 162
Methyl salicylate 1,52
4-Methyl-5-thiazoleethanol acetate 1110
Myrtenol 137
Neomenthol 156
Nonanoic acid 63
y-Nonanoic lactone 63
5-Octalactone 568
Octanoic acid (caprylic) 115

1-Octanol 24^
1-Phenyl-l,2-propanedione 222
Piperonyl acetate 24z
Propyl benzoate 6^
Pulegone 1-2
Sorbic aldehyde (2,4-hexadienal) 86
Terpinen-4-ol ^J.6
Tolualdehyde .240
y-Undecalactone 26
Undecanal 34
1-Undecanol 1^_
Vanillin I216
The preservative system preferably contains 1 to 100. ppm.of at least one essential oil- More preferably the preservative system contains 1 to 50 ppm of at least one essential oil, more especially 1 to 32.5 ppm.
Some of the aforementioned essential oils were found to.be particularly preferred in respect of their impact on.the^taste profile of tea based beverages containing • them::.,. These'i'are listed in Table II below. .In each case the respective'minimumVinhibitory concentration (MIC) and their specific preferred concentration is also given.

TABLE II
Particularly preferred essential oils

MIC (ppm) Cone (ppm)
228 1-30
198 1-30
450 1-40
8 1-20
15.8 1-20
1392 1-40
COMPOUND
Citral
Citral dimethyl acetal
Cumic alcohol
trans,trans-2,4-Decadienal
3,7-Dimethyl-l-octanol
Ethyl pyruvate
Myrtenol 137 1-20
Piperonyl- acetate-- 242 1-20-

Tea extract
The tea extract can be obtained by any suitable.means. Preferably tea leaves are extracted in hot water over a period of between 20 minutes and 5 hours. : ?The ...extract, can be dried .to form.;.a powder, reconstituted .--to form an ;.acidic beverage, or concentrated:, to form a syrup from.rwhich one-can prepare, a tea based .beverage..:; ;■•.
Teavis knpwn.-to .have certain antibacterial .and. antiviral;?.--,r.-..r..-• properties in itself. . One must exceed a concentration-qfrabqut 3% to evidence tea beginning to suppress the growth of yeasts and moulds. At concentrations;;.;lower .than this, ..which is typical for tea based beverages, tea acts as a nutrient that enhances the potential for microbial spoilage. The beverage should therefore contain 0.01 to 3% tea solids, about 0.14% being particularly preferred.
Other factors
Water quality can seriously undermine the stability of a beverage. This is an important factor when making a tea based beverage for cold filing. For that purpose it will often be important to

minimise the yeast content of water used at all stages of production. Art known methods include chlorination/dechlorination and UV irradiation.
Ambient-stable beverages of the invention'may be still or carbonated. Carbonation appears to provide a preservative effect in itself and therefore the formulation of a carbonated product need not be the same as a still one.
Tea based., be/uerages.. commonly, contain, sugar., ox some. a.th.er .sweetener to counter the sometimes astringent taste of tea. Most microbes that can typically grow in tea based beverages thrive on sugar, a source of nitrogen, oxygen, zinc, magnesium, potassium, phosphate and vitamins. It is therefore advantageous to limit the sugar content to 8 to 10 degrees brix, however one could use up to 60 degrees brix when the product is a tea mix.
Oxygen content can be minimised by pre-pasteurisation-or-some heat
treatment or nitrogen sparging. The mineral-'content-"of ^a tea
based beverage"'can be minimised using EDTA," citrate,' of a" water
softener. For example microbes can grow in tea if the
concentration "of: magnesium ions exceeds 0.2 ppm, and they only
need-trace'cleveTs of zinc' • ' -
If desired' the preservative system can also include ascorbic acid, a well known preservative for foodstuffs that is known to most as vitamin C.
The present invention also relates to a method for preparing an ambient-stable tea based beverage suitable for cold filing. The method comprises adding cinnamic acid, dimethyl dicarbonate and at least one essential oil to a tea exrract.
Cinnamic acid is freely soluble in essential oils, benzene, ether, acetone, glacial acetic acid and carbon disulphide. However cinnamic acid is not readily soluble in tea and one would not want

to contaminate a tea based beverages with any of the aforementioned chemicals. While the preservative system of the present invention includes one or more essential oils, it may be necessary to include a solubility enhancing step before adding the cinnamic acid to the tea solution. That can be achieved by spray drying the cinnamic acid.onto.a carrier powder (which may optionally be sugar based) and adding the powder to the tea, converting the acid to its. salt, or dissolving the cinnamic acid in a small quantity of organic solvent such as ethanol, or propylene glycol. One could spray dry the essential in the same way.
The ambient stable beverage of the present invention will now be described in the following examples with reference to the accompanying Figures.
EXAMPLE.1
Ready, .to. drink, .tea. experiments
Figure 1 shows.the results of a control experiment of growth of
yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of
• •.-". rr .."■■..'.'.: .•?. wo'Iii ■;■•."• 3" -C.L.J- -l" 'SO r. '■".•> ":.:"-f-_ '; :■";■■:■■' Ready to Drink tea, 0.14% tea. The matrix of 30 ml tubes each
contained 10 mi'.RTD tea, pH 3.4, containing 1-175 ppm cinnamic
aeidr Tubes^ •'were^inocui-a'ted'fwftrh -lO^-eells*' of- the -yea'si:^ ■
Saccharomyces cerevisiae.X2180-1B. Immediately following
inoculation, dimethyl dicarbonate, DMDC was added at
concentrations ranging from 1-250 ppm. Tubes were then incubated
for 14 days at 25 °C to allow surviving yeasts to grow out. At 14
days growth was measured by optical density at 600nm in xll
diluted samples, and blank values subtracted.
Figure 2 shows the combined effect of citral dimethyl acetal, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea. The matrix of 30 ml tubes each containing lOmls RTD tea pH 3.4,

all contained 100 ppm citral dimethyl acetal and 1-175 ppm cinnamic acid. Tubes were inoculated with 104 cells of the yeast Saccharomyces cerevisiae X2180-1B. Immediately following inoculation, dimethyl dicarbonate, DMDC was added at concentrations ranging from 1-250 ppm. Tubes were then incubated for 14 days at 25 °C to allow surviving yeasts to grow out. At 14 days growth was'measured by optical'density at 600 nm in xll diluted samples, and blank values subtracted.
Comparison, of..this. Figure, with....Eigure.. 1 shows., very, substantially, fewer tubes supporting yeast growth in the presence of the essential oil component, citral dimethyl acetal.
Figure 3 shows the combined effect of cumic alcohol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea. The matrix of 30 ml tubes each containing lOmls RTD tea pH 3.4, all contained 100 ppm cumic alcohol and 1-175 ppm cinnamic acid. Tubes were'"'*'""' inoculated with 104 cells of the yeas t-Sa'ccharomyce's' cerevisiae^ X2180-1B. Immediately following inoculation, dimethyl dicarbonate,
DMDC was added at concentrations ranging from.1-250 ppm. Tubes
1 . :.: ':'.■ ■.: •■•:'■.:..;•; '.: :'. -.' -'-.'. :..'-. •••: ::■:&.'. c.i ■■•■:-.-.::. ■■:. C~OV&JJ\ t: :■.'■:. ::-r-were then incubated for 14 days at 25 °C to allow surviving yeasts
to grow out. At 14 days growth was measured by optical density at
600nm in xll diluted' samples, and blank values;subtracted.
Comparison of this Figure with Figure 1 shows very substantially fewer tubes supporting yeast growth in the presence of the essential oil component, cumic alcohol.
Figure 4 shows the combined effect of citral, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea. The matrix of 30 ml tubes each containing 10 ml RTD tea pH 3.4, all contained 100 ppm citral and 1-175 ppm cinnamic acid. Tubes were inoculated with 10* cells of the yeast Saccharomyces cerevisiae X2180-1B. Immediately following inoculation, dimethyl dicarbonate, DMDC was

added at concentrations ranging from'1-250 ppm. Tubes were then incubated for 14 days at 25 °C to allow surviving yeasts to grow out. At 14 days growth was measured by optical density at 600 nm in xll diluted samples, and blank values subtracted. 5
Comparison of this Figure with Figure 1 shows very substantially fewer tubes supporting yeast growth in the presence.of the essential oil component, citfal.
10 Figure 5 shows the combined effect of 3,7-dimethyl octanol,
cinnamic acid ;and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in :a; matrix; of .tubes of Ready to Drink .tea,:. 0.14%. tea. The matrix of 30ml tubes each containing 10 ml RTD tea pH-3.4, all contained 50 ppm 3,7-dimethyl octanol and 1-175 ppm cinnamic
15 acid. Tubes were inoculated with 104 cells of the yeast ■Saccharomyces cerevisiae X2180-IB. Immediately following
.-. inoculation,:- dimethyl, dicarbonate, DMDC was•added.at ■: .
concentrations.; ranging from .1-250 ppm..._ Tubes were then incubated for 14 days at 25 °C to. allow surviving yeasts to grow out. At 14
20 da.ys_: growth,, was,.-measured, by. optical;; density . at.; 600j: nm. in-, xll:.-. diluted- samples,;and blank., values subtracted.;
Comparison of. this.Figure with Figure 1 shows very substantially _ I lew ers-tubes'; supportinga-yeast growth in ;the presence ..of the :;.'.;•;-. •/.. 25 ' '■ essential--oil^cpmponent, v.3, 7-dimethyl octanol. -
Figure 6 shows the combined effect of myrtenol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae.X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea. The matrix of 30
30 ml tubes each containing.10 ml RTD tea pH 3.4, all contained 100 ppm myrtenol and 1-175 ppm cinnamic acid. Tubes were inoculated with 10* cells of the yeast Saccharomyces cerevisiae X2180-1B. Immediately following inoculation, dimethyl dicarbonate, DMDC was added at concentrations ranging from 1-250 ppm. Tubes were then
35 incubated for 14 days at 25 °C to allow surviving yeasts to grow

out. At 14 days growth was measured by optical density at 600 run in xll diluted samples,- and blank values subtracted.
Comparison of this Figure with Figure 1 shows very substantially 5 fewer tubes supporting yeast growth in the presence of the essential oil component, myrtenol.
Figure 7 shows the. combined effect of piperonyl acetate, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B
10 in a matrix of tubes of Ready to Drink tea,. 0.14% tea. The...ma.tr.ix. of 30 ml tubes each containing 10 ml RTD tea pH 3.4,; all contained 100 ppm piperonyl'acetate'and'1-175 ppm-cinnamic acid. Tubes were inoculated with 10* cells of the yeast Saccharomyces cerevisiae X2180-1B. ' Immediately following inoculation, dimethyl dicarbonate,
15 DMDC was added at concentrations ranging from 1-250 ppm. Tubes
were then incubated for 14 days at 25 °C to allow surviving yeasts to grow out. At-14'days growth-was measured by optical density' at '-600-nm': in'-'xll diluted" samples, 'and blank values subtracted-.-;;'-
20 Comparison'Vof: this'Figure: with' Figure" 1- 'shows very'substantially fewer tubes supporting yeast-growth in'-.the presence'f;bf the essential oil component, piperonyl acetate.
Figure" 8. shows -the" combined effect of--* t'ra'ns; trans -2,4 -decadienal, 25 cinnamic acid and DMDC-on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of Ready to Drink tea, 0.14% tea. The matrix of 30 ml tubes each containing 10ml RTD tea pK 3.4, all contained 15 ppm trans,trans-2,4-decadienal and 1-175 ppm cinnamic acid. Tubes were inoculated with 10* cells of the yeast 30 Saccharomyces cerevisiae X2180-1B. Immediately following inoculation, dimethyl dicarbonate, DMDC was added at concentrations ranging from 1-250 ppm. Tubes were then incubated for 14 days at 25 °C to allow surviving yeasts to grow out. At 14 days growth was measured by optical density at 600 ran in xll 35 diluted samples, and blank values subtracted.

Comparison of this Figure with Figure 1 shows very substantially fewer tubes supporting yeast growth in the presence of the essential oil component, trans,trans-2,4-tiecadienal.
5 Figure 9 shows the combined effect of 5-decanolactone, cinnamic
acid and DMDC on growth of yeast Saccharomyces cerevisiae^ X2180-13 in a matrix of tubes of Ready to Drink tea, 0.14% tea. The matrix of 30 ml tubes each containing 10 ml RTD tea pH 3.4, all contained lOOppm 5-decanolactone and l-175ppm cinnamic acid. Tubes were
l.Q ln.QCula.te-d...with 1Q!1 cells . of . the yeast Saccharomypes cerevisiae
X2180-1B. .Immediately, following inoculation, dimethyl:, dicarbonate, DMDC was-added at concentrations ranging from 1-250; ppm.. Tubes-were then incubated for 14 days at 25 °C to allow surviving yeasts to grow out. At 14 days growth was measured by optical density at
15 600 nm in xll diluted samples, and blank values subtracted.
, ..Comparison^of ..this Figure ..with ..Figure 1 shows..yery^ubstantially , f ewer -tubes ...supporting.,, yeast. growth .....in... the presence pf^the^ ^ ^ essential .oil-.; component, ....5-decanoJLactone.

o
0
Figure 10 shows the combined effect of citral dimethyl acetal,-cumic. alcohol/;1 cinnamic^ acid and DMDC. on, growth. qf^yjeast _,,_ ,^ . Saccharomyces.Jcerevisiae..X2180-1B. in. a.'matrix.Vof jEubesi of.: Ready to 'Drink .te'a;:7;.0^14%^.te^a^:The\: matrix, of ^O.. ml. tubes^ each^cqntainirig 10
25 ,-'mi;.;RTD .teaj'pH^ 3.4, -allV'cpntained 25. ppm.citral^.dimethyl. acetal, 35 ppm cumic alcohol and .1-175.ppm cinnamic acid. Tubes.were inoculated with 104 cells of the yeast Saccharomyces cerevisiae X2180-1B. Immediately following, inoculation, dimethyl dicarbonate, DMDC was added at concentrations ranging from 1-250 ppm. Tubes
30 were then incubated for 14 days, at 25 °C to allow surviving yeasts to grow out. At 14 days growth was measured by optical density at 600 nm in xll diluted samples, and blank values subtracted.
Comparison of this Figure with Figure 1 shows very substantially 35 fewer tubes supporting yeast growth in the presence of the

essential oil components, citral dimethyl acetal and cumic alcohol.
EXAMPLE 2
Synthetic soft drink experiments
Figure 11 gives the results of a control experiment of growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of synthetic soft drink, OT'tea. Synthetic soft drink contained' glucose, 8%w/v,'citric acid 3 g/1, potassium orthophosphate 1 g/1, magnesium chloride'0.1 g/g and yeast extract 0.1 g/1. The matrix of 30 ml tubes each contained 10 ml soft drink, pH 3.4, containing 1-175 ppm cinnamic acid. Tubes were inoculated with 104 cells of the yeast Saccharomyces cerevisiae X2180-1B. Immediately following inoculation, dimethyl dicarbonate,'DMDC-was added at concentrations ranging from 1-250 ppm. Tubes were then incubated for 14 day's at 25>DC to'allow surviving yeasts "to grbw'out;.r" At- 14 days growth was measured-by optical density-at 600 run in xll -diluted samples, and blank values subtracted.
Figure 12 shows the"combined effect of citral dimethyl acetal, cinnamic acid and DMDC'on growth of yeast Saccharomyces cerevisiae X2180-iB in- a-.'mat'rix- of" tubes --of> synthetic;:so.ft- drink,- 0% tea.-Synthetic-soft drink contained glucose, 8%w/v, citric acid 3 g/1, potassium orthophosphate 1 g/1, magnesium chloride 0.1 g/g and yeast extract 0.1 g/1. The matrix of 30 ml tubes each containing 10 ml RTD tea pH 3.4, all contained 100 ppm citral dimethyl acetal and 1-175 ppm cinnamic acid. Tubes were inoculated with 10* cells of the yeast Saccharomyces cerevisiae X2180-1B. Immediately following inoculation, dimethyl dicarbonate, DMDC was added at concentrations- ranging from 1-250 ppm. Tubes were then incubated for 14 days at 25 °C to allow surviving yeasts to grow out. At 14 days growth was measured by optical density at 600 nm in xll diluted samples, and blank values subtracted.

/ Comparison of this Figure with Figure 11 shows very substantially fewer tubes supporting yeast growth in the presence of the essential oil component, citral dimethyl acetal.
Figure 13 shows the combined effect of cumic alcohol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-13 in a matrix of tubes of synthetic soft drink, 0% tea. Synthetic soft drink contained glucose, 8%w/v, citric acid 3 g/1, potassium orthophosphate 1 g/1, magnesium chloride 0.1 g/g and yeast extract 0.1 g/1. The matrix of 30 ml tubes each containing 10 ml RTD tea pH 3.4, all contained 100 ppm cumic alcohol and 1-175 ppm cinnamic acid. Tubes were inoculated .with 10* cells.of the yeast Saccharomyces cerevisiae X2180-1B. Immediately following inoculation, dimethyl dicarbonate, DMDC was added at concentrations ranging from 1-250 ppm. Tubes were then incubated for 14 days at 25 °C to.allow surviving,yeasts to,grow out. At .14 days growth was measured.by,optical, density at 600 run in xll diluted samples, and,blank values subtracted..
Comparison of this. Figure with.. Figure. 11 shows
fewer tubes supporting.yeast, growth.in the presence;of the
essential oil component, cumic alcohol.
Figure 14 shows the^cpmbined effect, of-citral, .cinnamic acid and DMDC "on''grow-th* of yeast Saccharomyces cerevisiae X2180-1B in a..-matrix of tubes of synthetic soft drink, 0% tea. Synthetic soft drink contained glucose, 8%w/v, citric acid 3 g/1, potassium orthophosphate 1 g/1, magnesium chloride 0.1 g/g and yeast extract 0.1 g/1. The matrix of 30 "ml tubes each containing 10 ml RTD tea pH 3.4, all contained 100 ppm citral and 1-175 ppm cinnamic acid. Tubes were inoculated with 10* cells of the yeast Saccharomyces cerevisiae X2180-1B. Immediately following inoculation, dimethyl dicarbonate, DMDC was added at concentrations ranging from 1-250 ppm. Tubes were then incubated for 14 days at 25 °C to allow surviving yeasts to grow out. At 14 days growth was measured by

optical density at. 600 ran in xll diluted samples, and blank values subtracted.
Comparison of this Figure with Figure 11 shows very substantially fewer tubes supporting yeast growth in the presence of the essential oil component, citral.
Figure 15 shows the combined effect of 3,7-dimethyl octanol, cinnamic acid and DMDC on growth of yeast Saccharomyces cereyisiae X2180-1B in- a'matrix- of tubes- o-f synthetic- soft drin-k, 0%- tea-. Synthetic soft drink contained glucose, 8%w/v, citric acid 3 g/1, potassium orthophosphate•• 1 g/1, magnesium chloride 0.1 g/g and yeast extract 0.1 g/1. The matrix of 30 ml tubes each containing 10 ml RTD tea pH 3.4, all contained 50 ppm 3,7-dimethyl octanol and 1-175 ppm cinnamic acid. Tubes were inoculated with 104 cells of the yeast"Saccharomyces cerevisiae X2180-1B. Immediately following"inoculation,' dimethyl dicarbonate, DMDC was added at concentrations ranging from 1-250 ppm. Tubes were then incubated for 14 days at 25 °C to allow surviving yeasts to grow out. At" 14 days growth was'fmeas'ured by'optical density'at 600 run in xll ' diluted samples,";;and-^blank values subtracted.
Comparison of this Figure with Figure 11 shows very substantially fewer tubes, "supportirig.r'yeast,. growth., in the.'presence 'of. the . essential oil - component ,;i 3, 7-dimethyl octanol.
Figure 16 shows the combined effect of myrtenol, cinnamic acid and DMDC on growth of yeast Saccharomyces cerevisiae X2180-1B in a matrix of tubes of synthetic soft drink, 0% tea. Synthetic soft drink contained glucose, 8%w/v, citric acid 3 g/1, potassium orthophosphate 1 g/1, magnesium chloride 0.1 g/g and yeast extract 0.1 g/1. The matrix, of 30 ml tubes each containing 10 ml RTD tea pH 3.4, all contained 100 ppm myrtenol and 1-175 ppm cinnamic acid. Tubes were inoculated with 10* cells of the yeast Saccharomyces cerevisiae X2180-1B. Immediately following inoculation, dimethyl dicarbonate, DMDC was added at

concentrations ranging from 1-250 ppm. Tubes were then incubated for 14 days at 25 CC to allow surviving yeasts to grow out. At 14 days growth was measured by optical density at 600 nm in xll diluted samples, and blank values subtracted.
Comparison of this Figure with Figure 11 shows very substantially fewer tubes supporting yeast growth in the presence of the essential oil component, myrtenol.
Figure 17 shows the combined effect of piperonyl acetate, cinnamic acid and DMDC on growth of .yeast Saccharomyces cerevislae X2180-1B in a matrix of tubes of synthetic soft drink, 0% tea. Synthetic soft drink contained glucose,.8%w/v, citric acid 3 g/1, potassium orthophosphate 1 g/1, magnesium chloride 0.1 g/g and yeast extract 0.1 g/1. The matrix of 30 ml tubes each containing 10 ml RTD tea pH 3.4, all contained 100 ppm piperonyl acetate and lrlJ75-ppm_,-cinnamic acid. Tubes were inoculated.with 104 cells; of the:yeast
.. Saccharomyces. cerevislae, X2180-1_B. Immediately^following - ; y. inoculation, dime thyl:di carbonate,-. DMDCowas., added^atj •. . . ::