CA1043251A

CA1043251A - Process for protecting organic materials

Info

Publication number: CA1043251A
Application number: CA221,892A
Authority: CA
Inventors: David Shepherd; Pierre Hirsbrunner; Theodor Beyeler
Original assignee: Societe des Produits Nestle SA
Current assignee: Societe des Produits Nestle SA
Priority date: 1974-04-08
Filing date: 1975-03-12
Publication date: 1978-11-28
Also published as: DE2425215B2; ZA751106B; DE2425215C3; AU7974575A; DE2425215A1; AR206813A1; FR2266465B1; JPS5411372B2; ES436347A1; OA04863A; GB1458795A; CH586023A5; FR2266465A1; NL7502482A; JPS50140626A

Abstract

ABSTRACT OF THE DISCLOSURE
A process for protecting organic materials such as food-grade materials with a bacteriostatic agent, wherein an extract obtained from unroasted coffee is used as the bacteriostatic agent.

Description

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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 .. . .
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 .i ,. .

1~)4L3ZSl 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. -. . .
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.

j 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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1 ~ ~ 3 Z 5 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. ;

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 ... _ ... _ .. . ........................... .. __ (control) :
inocula- . .
tion) 0~5 160 740 60 170 2 170 850 40 230 ~.
. _. . :.0 3800 3000 3000 9200 :
(control) . .
4 0.5 1600 18000 2300 1600 . .. _ , -: ,, 06500 0000870000000 12000000 290000000 .' (control)¦
. 0.5g20000 300000 300000 340000 2200 10 20 1500 :
. . _ * ' _ _ ., ', (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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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN
EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for protecting organic materials with a bacteriostatic agent, which comprises adding to said materials an extract obtained from unroasted coffee by acid-base extraction as the bacteriostatic agent in a quantity of at least 1.5 mg/g of dry materials.

2. A process as claimed in claim 1, wherein the organic materials are food-grade materials.

3. A process as claimed in claim 11 wherein the bacteriostatic agent is added in a quantity of from 1.5 to 6 mg/g of dry materials.

4. A process as claimed in claim 1, wherein an extract obtained from decaffeination residues of unroasted coffee by acid-base extraction is added as the bacteriostatic agent.

5. A process as claimed in claim 1, wherein an extract obtained by acid-base extraction, in which the basic aqueous phase has a pH-value in the range from 10 to 12, is added as the bacteriostatic agent.

6. A process as claimed in claim 1, wherein an extract obtained by acid-base extraction, in which the aqueous acid phase has a pH-value in the range from 1 to 2, is added as the bacteriostatic agent.

7. A process as claimed in claim 1, wherein an extract obtained by acid-base extraction, in which the solvent phase is diethyl ether or diisopropyl ether, is added as the bacteriostatic agent.