Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to a process for protecting
organic materials, more especially food-grade materials, by
means of a bacteriostatic agent extracted from unroasted
coffee.
It is known that the growth of bacteria on or in
organic materials can be ret~rded and even completely
I inhibited by the addition of a bacteriostatic agent. There
I are a large number of active substances which are suitable
! f4r this purpose, including for example phenyl mercuricborate, hexachlorophene, sulphur dioxide, benzoates. --
Unfortunately, problems are involved in the use of substances
of this kind in the food industry (toxicology, laws, etc.).
It is for this reason that the food industry has turned
towards natural substances or extracts. Various bacteriostatic
agents hav~ been prepared from plants, above all from spices,
for example from celery oil~ caraway oll, clove oil, etc.
Unfortunately, spices being by de~hition materials with
distinctive flavours and odours, it is almost impossible ~;
to prepare by extraction bacteriostatic agents completely
free of any organoleptic component. In addition~ these
l agents are not very artive or, more preclsely, if they are
very active, their activity is highly specific and they have
~,~ to be used in considerable quantities. As a result, they
impart their flavour and odour to the food-grade products
to which they are added.
The present invention relates to a process for protecting
organic materials, especially food-grade materi~ls, by means
of a bacteriostatic agent of remarkable activity which,
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organoleptically, does not betray its origins. The process
according to the invention is distinguished by the fact that
an extract obtained from unroasted ~ffee is used as the
bacteriostatic agent.
This bacteriostatic agent is obtained by treating
coffee waxes, that is to say the film of fat surrounding -
the grain of unroasted coffee. The waxes themselves do not -
have any significant bacteriostatic effect because the
bacteriostatic agent present in them is too diluted. It is
for this reason that the bacteriostatic agent is isolated
by the treatment referred to above which comprises at least
one so-called acid-base extraction of the type normally
applied in the field of chemistry, for example a distribution ,
between a solvent phase and a basic aqueous phase, followed
after decantation and separation by acidification of the
basic aqueous phase and then by distribution between a ;
solvent phase and the aqueous phase thus acidlfied,
separation of the solvent phase and evaporation of the
solvent in cases where it is desired to recover the
bacteriostatic agent in dry form. In one advantageous ;
method of extraction, the caffeine present in the unroasted ;
coffee is simultaneously extracted by any known
decaffeination process using a solvent, after which the
caffeine extraction residue is treated by a process
~5 comprising various purifications to eliminate the caffeine, ~;
and an acid-base extraction such as, for example, the acid-;;;
base extraction defined above.
The residue obtained in this way represents the
bacteriostatic agent. It has an oily consistency, is
yellowish to brownish in colour, has a moderate inherent
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taste and odour and is perfectly edible. It shows remarkable
bacteriostatic activity which is governed to some extent by
the method ~ extraction used and~ in particular, ~y the
type of solvent(s) used. It is possible to use a wide
variety of soIvents, such as methylene chloride, hexane, -
ethyl acetate, although the best effects are achieved with `~
agents obtained by extraction with diethyl and diisopropyl
ethers.
The results of preliminary chromatographic tests show
that this bacteriostatic agent is in the form of a complex
mixture of several substances of unknown type. In addition,
it would seem that most of the bacteriostatic power derives `
from one or two substances and not from all the constituent
substances of this mixture. Owing to its method of preparation,
the whole has an acld character, but is non-phenolic in ~ I
nature, as shown by the negative results of ferric chloride
tests, so that the substance!s in question are not tannins.
Providing it is not stored in dry form, the agent is stable
at temperatures of up to approximately 60C and keeps well
in air.
The bacteriostatic agent may be added to finished organic
! materials, more especially to food-grade materials ready for
consumption, in quantitles by weight of at least 0.15 7O~
i i.e. 1.5 mg of dry agent per g of dry materials. In the case ~;
I ~ 25 of-a processed material, addition of the agent may thus form
¦ the last stage in the manufacturing process or may be made
at any stage during manufacture, so that the agent is unable
¦~ to undergo any changes as a result. It may be added for
example in solution or emulsion in a suitable carrier or
~,~ 30 solvent. The quantities normally used are sllghtly greater ~ ;
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than the minimum dose by which bacterial growth is inhibited,
for example 2 times greater, and have to be adapted according
to the particular type of material to be protected and to the
environmental conditions, especially the climatic conditions. -~
In addition, the bacteriostatic agent may be used as a
bactericide, the doses required in that case being approximately
3 times greater. When used in doses of this order,the
bacteriostatic agent, which has only a slight odour and
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flavour, is too dilute to impart its odour and flavour to ;~
the food-grade material.
This agent may of course be added in admixture with
other substances, such as antioxidants, flavourings,
colorants, etc. Finally, where practical requirements
dict~e, it is possible to treat only part of the organic
material with the bacteriostatic agent in one form or
another and then to mix the material thus treated with the ;~
rest of the u~treated material. It is then advisable to
sub~ect the mixture to careful homogenisation.
In one preferred embodiment of the process according to ;~
the invention, the bacteriostatic agent is added in such a
way that its concentration in the end food product is in
the range from 0.15 to 0.6 %, i.e. in the range rom1.5 to
6 mg of dry agent per g of dry materials.
In a first modification of this embodiment, the
bacteriostatic agent is prepared by directly treating
unroasted caffee with methylene chloride in a quantity of
;~ approximately 10p~rts by weight of methylene chloride to
1 part by weight of unroasted coffee. Evaporation of the
methylene chloride leaves a greasy residue which is taken ;
up in an aqueous alkaline soda solution with a pH-value of
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from 10 to 12. After vigorous stirring at a temperature in ;
the range from 20 to 30C, the product is decanted and the
supernatant fat phase is separated off. The aqueous phase -
is then æ~ified with a 1 N to 5 N sulphuric acid solution
until a pH-value in the range from 1 to 2 is obtained.
This acidified aqueous phase is then extracted with diethyl
ether or diisopropyl ether, for example with 3 times the
same volume of diisopropyl ether, after which the bacteriostatlic ~
agent is recovered by evaporating the ether, optionally :
after drying with an anhydrous salt. It is preferred to
keep the agent in solution, even in concentrated solution,
rather than in dry form. In this case, the ether does not
have to be evaporated to dryness, or the bacteriostatic
agent ls redissolved, for example in alcohol. It is also
possible before evaporation to change the solvent, for example
by adding alcohol to the ethereal phase, removing the ether
and optionally concentrating the alcoholic phase containing
the bacteriostatic agent.
In a second modification of this embodiment, the
decaffeination residues obtained for example by treating
previously moistened unroasted coffee with methylene -~
chloride are used as starti~g material. It is known that
the water thus applied to the coffee beans makes them swell
and, at the same time, causes the caffeine~chlorogenic acid
complex to dissociate. Accordingly, the caffeine accompanies
the waxes into the methylene chloride, so that evaporation
of the methylene chloride leaves a fatty residue rich both in
crystallised caffeine and in water. It is then possible as
required either to filter the undissolved caffeine, to
separate the waxes from the aqueous phase and then to treat ~
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, the waxes thus separated with an alkaline solution, or to
take up the residue in an acid solution of pH 1 - 2, to separate
~1 the waxes from the aqueous acid phase, in which the caffeine
¦ is dissolved, and then to treat the waxes with an alkaline
solution, or even to combine the purification processes
described above. In both cases, the treatment by which the
~` bacteriostatic agent is isolated is with advantage continued
in the same way as described earlier on in reference to the
first embodiment. These operations involving crystallisation
or dissolution in acid medium m~y of course be repeated as
many times as necessary in order suitably to remove the
~ caffeine before the alkaline treatment of the waxes.
;j The process according to the invention is illustrated
by the following Examples. Examples 1 and 2 relate to
the extraction of the bacteriostatic agent, Example 3 describes
the tests demonstrating the bacteriostatic power of the agent,
~ while Example 4 demonstrate~:J the efectiveness of the
! bacteriostatic ~gent in prolecting food-grade materials
i against microbial growth.
j 20 EXAMPLE 1
240 kg of unroasted coffee beans are treated with
vigorous stirring at 20C with 3 separate 800 litre batches
of methylene chloride. These 3 volumes of liquid phase are
combined and the methylene chloride removed in vacuo, leaving
; 25 2.5 kg of a greasy resîdue greenish in colour with an odour -
j;~ of unroasted coffee which is immediately treated with
10 litres, and then 8 litres and then another 8 litres of a
0.05 N aqueous soda solution at a temperature of 25C.
After each operation, the mixture is left to settle, after
which the supernatant fatty phase is separated from the
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aqueous phase. These 3 volumes of aqueous alkaline phase are
then com~ined and then acidified to pH 1 with 0.3 litre of a
5 N sulphuric acid solution. This aqueous acidified phase
is then extracted with 3 volumes of diethyl ether measuring
~5 litres, 20 litres and 20 litres, respectively, after
which the 3 volumes of ethereal phase are combined. 10 litres
of ethanol are then added to the ethereal phase, after which
the ether is removed in vacuo at a temperature kept below
30C. Finally, most of the ethanol is evaporated in vacuo
at a temperature of approximately 35C, leaving approximately -~
1 litre of an alcoholic solution containing 18 g of bacterio- ~;
static agent which is maintained in this form. -
EXAMPLE 2
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60 kg of water are added to 240 kg of unroasted coffee
beans at a temperature of 60C and under a pressure of 1.5 ~
atms. After a contact time of 1 hour, the pressure is ~ ;
reduced to 1 atm and the swollen beans are treated with
6 times320 litres of methylerle chloride. These 6 volumes of
liquid phase are combined, after which the methylene chloride
is removed in vacuo, leaving 10~2 kg of a non-homogeneous
brown-green mixture, with the odour of unroasted coffee,
consisting af a fatty phaseand an aqueous phase containing
crystals of caffeine. The caffeine is removed by filtration :
(2.5 kg). The two liquid phases are then separated, followed
by the successive addition to the fattyFhase of 3 separate -~
12.5 litre batches of a 0.05 N sulphuric acid solution.
After each operation, the mixture is left to seffle~ the
two phases are separated and the fatty phase recovered.
On completion of these purify:ing operations, the fatty phase
no longer contains caffeine. It is then treated with 10 litres
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of a 0.05 N aqueous soda solution, and separation of the
bacteriostatic agent is continued in the same way as described
in Example 1, except that diisopropyl ether is used as the
extraction solvent. The proportions of solvent used are
the same or equivalent. 1 litre of an alcoholic solution
containing 20 g of bacteriostatic agent is thus obtained,
the bacteriostatic agent being maintained in that form.
EXAMPLE 3
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The bacteriostatic activity of the agent was assessed
by the minimum inhibition concentration method ~ the - -
Public Health Laboratory Service Committee described in
British Med. J. 408 (1965).
Five cultures of the following microorganisms are
prepared:
bacteria Escherichia coli ATCC 8739
Staphylt~lcoccus aureus ATCC 155
~ ynas aeru~osa ATCC 10145
Bacillus cereus ATCC 14579
yeast Candida utilis CBS 567
containing approximately 2.108 cells/ml of nutrient medium
(culture time approximately 24 hours).
At the same time, samples of the bacteriostatic agent
to be tested are prepared by dilution from a mother
suspension (2 g/100 ml, i.e. 2/100) in ethanol. 1 ml of
water is added to a first 1 ml sample of this mother
suspension, 2 ml of wate~ are added to a second sample
~; and so on, so as to obtain a series ranging in dilution
from 1/100 to 1/300, after which this intermediate series
and the initial mother suspension are diluted 10 times with
the nutrient medium of m~roorganisms. A series of test
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samples ranging in dilution from 1/500 to 1/3000 is
prepared in this way.
0.2 ml of each of the aforementioned microorganism
cultures are then added to 10 ml of each of the diluted
samples thus prepared. This is followed by incubation for
24 hours at 30C for the bacteria and at 35C for the yeast,
after which the optical density of the mixture is measured -~
at 600 nm and compared with the optical density of reference I -
samples. ~; -
Here now are the minimum inhibition concentrations
found for the "diisopropylic" agent prepared in Example 2:
1/2500 against Ps. aeru~inosa
.
1/1000 against E coli, Staph. aureus, B cereus
no bacteriostatic activity against C utilis
1 15 The minimum inhibition concentrations for the
"diethylic" agent prepared in Example 1 are approximately
2 times higher. ;
EXAMPLE 4
4 groups of 4 sterile samples af reconstituted skimmed
1 20 milk, i.e. a total of 16 samples, are prepared and then
inoculated with the bacteria mentioned in Example 3 and ~:
protected against them by variable doses of the bacteriostatic
agent prepared in Example 2. Evolution of the colonies of
bacteria in the samples incubated at 30C is then measured
~; 25 as a function of time by a conventional counting method.
¦ The results are set out in the following Table:
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number of bacteria present per ml of
skimmed milk :
~ of _
incubation ba~rio- Staph. E. coli B. cereus Ps. ~
time in h static aureus aeru~inosa .
addned
... _ ... _ .. . ........................... .. __
0 0 210 860 60 220
(control) :
inocula- . .
tion) 0~5 160 740 60 170
1 210 870 50 190
2 170 850 40 230 ~.
. _. . :.0 3800 3000 3000 9200 :
(control) . .
4 0.5 1600 18000 2300 1600
1 800 1000 60 500
2 100 20 30 75
. .. _ , -: ,,
06500 0000870000000 12000000 290000000 .'
(control)¦
. 0.5g20000 300000 300000 340000
8 18000 110000 350 47000
2200 10 20 1500 :
. . _ * ' _ _ ., ',
0230000000 780000000 12000000 175000000
(control)¦ :
0.5140000000460000000 5800000 300000000
24 11 oooboooo300000000 6500000 140000000
, ~_ . 2¦ 150 10 10 120000000
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* coagulated samples
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In the above Table, the percentages of bacteriostatic ~.
agent added are percent of a 3 % ethanolic solution of
that agent in skimmed milk which itself has a solids content :. :
of approximately 10 %. Accordingly, these percentages
express the following quantities by weight:
0.5 % = O.S ml/100 ml = 1.5 mg/g of solids
1 % = 1 ml/100 ml = 3 mg/g of solids
2 % = 2 ml/100 ml = 6 mg/g of solids
Naturally the figures quoted in the above Table are
-by no means precise and are to be considered above all as
orders of magnitude. Nevertheless it can be seen that :
the bacteriostatic agent is extremely effective in inhibiting ;:
¦ bacterial growth, except perhaps in the long term with
¦ respect ~ Ps. aeruginosa, for this particular substrate of
¦ 15 skimmed milk.
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