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Publication numberUS3775540 A
Publication typeGrant
Publication dateNov 27, 1973
Filing dateOct 31, 1972
Priority dateSep 22, 1970
Publication numberUS 3775540 A, US 3775540A, US-A-3775540, US3775540 A, US3775540A
InventorsJurd L, King A, Stanley W
Original AssigneeUs Agriculture
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Cinnamyl phenols useful as antimicrobial agents
US 3775540 A
Abstract  available in
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Claims  available in
Description  (OCR text may contain errors)

e r 3,775,540 P Ice Patented Nov. 27, 1973 the invention are given below by way of illustration and 3,775,540 not limitation: CINNAMYL PHENOLS USEFUL AS OH ANI'lMICltgIgAL IAGIIEINTSJ M Leonard Jurd Berkeley ougas 'ng r. artinez h and William L. Stanley, Richmond, calit, assignors t5 5 P m1 the United States of America as represented by the (also known as Secretary of Agriculture No Drawing. Original application Sept. 22, 1970, Ser. No. obtusastyrene) cH -cp 74,485, now Patent No. 3,745,222, dated July 10, 1973. Divided and this application Oct. 31, 1972, Ser. No.

Int. Cl. A0111 9/26 US. Cl. 424-341 1 Claim cu -ca ou ABSTRACT OF THE DISCLOSURE 1 z'cimmyr'lhem Substances which are subject to microbial spoilage are preserved by addition of a cinnamyl phenol, e.g., 2-cinnamyl-phenol, 4-cinnamyl-phenol, 2-methoxy-4 cinnamylphenol, 2-cinnamyl-5-methoxy-quinol, etc.

This is a division of our copending application, Ser. No. y h y z- W 74,485, filed Sept. 22, 1970, now US. Pat. 3,745,222 hem issued July 10, 1973. P

A non-exclusive, irrevocable, royalty-free license in the 5 invention herein described, throughout the world for all purposes of the United States Government, with the power to grant sublicenses for such purposes, is hereby granted to the Government of the United States of America.

This invention relates to the preservation of substances z-c h pawhich are normally subject to microbial spoilage. The objects of the invention include the provision of novel processes and compositions for accomplishing such preservation. Further objects of the invention will be evident 35 06113 from the following disclosure wherein parts and percentages are by weight unless otherwise specified. The abbreviation p.p.m. used herein refers to parts per million. Temperatures are given in degrees centigrade. The symbol is used herein to designate the phenyl radical.

In accordance with the invention it has been found that certain agents exhibit unexpected antimicrobial activity and are useful for preserving all kinds of materials which are normally subject to microbial spoilage. The agents in question are certain cinnamyl-phenols, and their antimicrobial activity is unusual and unexpected because it is OK not shared by closely-related phenol derivatives.

Generically, the agents in accordance with the inven- 2-Methoxy-4-cinnamy1- tion have the structurephenol.

0H Z-Me thy1-4-cinnamy1- 40 phenol.

0on i phenol CH -CK Cli-tl R a (I) CH -C Cu- R z a 0 OH 4-Cinnamy1-resorcinol. OH

where at least one but not more than three of the Rs are CH 5 CH5 hydroxy radicals, and the remainder of the Rs are each independently seledted from the group consisting of lower alkyl, lower alkoxy, and hydrogen.

The compounds of the invention are especially useful because they are active against many microorganisms in OH the categories of bacteria, yeasts, and molds. In other 2-.CinnamyI-5-methoxy- CH -Cll ctr-d words, the compounds are not just active against one or a few organisms; rather, they display broad-spectrum anti- B microbial activity. on

Examples of particular compounds within the scope of t-Cinnamyl-pyrogallol OH ca -ca era-( The compounds of the invention display activities which are equal or even superior to those of widely-used antimicrobial agents. This is illustrated by the following: In general, the compounds of the invention are superior to such known agents as phenol, resorcinol, o-phenylphenol, and the alkali metal sorbates and benzoates. For instance, compounds of the invention, at concentrations of 12 to 25 p.p.m., were found to inhibit the growth of four bacteria (Bacillus cereus, Sarcina lutea, Staphylococcus aureus, and Streptococcus laclis) whereas to achieve the same effect it required 100 to 200 p.p.m. of o-phenylphenol, more than 800 p.p.m. of potassium sorbate, and more than 1000 p.p.m. of sodium benzoate.

Alkyl 4-hydroxybenzoates are well known to exhibit potent microbial activity. In general, the compounds of of the invention display a superior activity as compared to these benzoates wherein the alkyl group contains less than seven carbon atoms. For instance, whereas the compounds of the invention at a concentration of 12 to 25 p.p.m. will inhibit the growth of the four bacteria noted above, it requires concentrations of 100 to 400 p.p.m. for the same result to be achieved with the C -C alkyl 4-hydroxybenzoates. Moreover, it may be noted that the compounds of the invention have an advantage in that their water-solubility is higher than that of the higher alkyl (e.g., heptyl) 4-hydroxybenzoates, and that the compounds of the invention do not exhibit the strong and unpleasant odor which characterizes the said benzoates. Also, with respect to some microorganisms, the compounds of the invention inhibit growth when applied at levels less than required with the higher alkyl 4-hydroxybenzoates. This is the case, for example, with bacteria such as Acaligenes faecalis and E. coli, yeasts such as Pichia chodati, Hansenula anomala, and Saccharomyces cerevz'siae, and molds including Aspergillus flavus, A. niger, Penicillus chrysogenum, Rhizopus senti, Botrytis cinerea, Byssochlamys fulva, and Alternaria sp.

Of the various compounds included within the scope of the invention, the cinnamyl-substituted monophenols display particularly high antibiotic activity against a large variety of different microorganisms, and therefore are preferred with respect to the cinnamyl derivatives of dior tri-phenols. Coming into special consideration is 4-cinnamyl-phenol because it displays antibiotic activity over a wide range of pH. The cinnamyl derivatives of monophenols are also preferred because they are essentially colorless, whereas those derived from dior tri-pheno'ls exhibit various shades varying from red to brown.

As evident from the explanation immediately following Formula I above, the invention includes compounds wherein there may be nuclear lower alkyl or lower alkoxy substituents in addition to the hydroxy groups. In general, the compounds are preferred wherein such additional substituents are absent or, if present, are in small number, e.e., a total of one or two lower alkyl and/or lower alkoxy substituents. Taking the foregoing facts into consideration, we prefer to employ the compounds of the sub-generic category represented by the formulawherein- R is lower alkyl,

R" is lower alkoxy,

n is an integer from 0 to 2,

m is an integer from 0 to 2, and

the sum of n and m is not more than 2.

The invention encompasses not only the use of any of the above-described agents individually, but also mixtures thereof.

In preserving substances in accordance with the invention, any of the aforesaid agents or mixtures thereof are incorporated with the substance, using an amount of the agent to inhibit microbial growth. Additional conventional treatments such as dehydration, canning, refrigeration, or freezing may be applied to the substance containing the added agent. The incorporation of the agent with the substance may involve a mixing of the substance and the agent--this is especially suitable where the substance is in liquid or particulate form. Where the substance is in the form of pieces of large dimensions the agent may be incorporated therewith by coating it on the surface of the pieces. For such purpose the agent is preferably dispersed in a carrier-a liquid such as water, alcohol, water-alcohol blends, oils, or a finely-divided solid such as salt, starch, talc, or the like.

The invention is of wide versatility and can be applied for the preservation of all kinds of substances which are normally subject to microbial spoilage. Typical examples of such substances are listed below by way of example. Foodstuffs such as fruits, vegetables, juices, milk, eggs, meat, fish, grains, cereal products, cheese, etc. Animal glues and mucilages; dextrins; starch pastes and solutions; cosmetic, medicinal, and dental preparations; vitamin preparations; pastes, solutions, or other preparations of natural gums such as tragacanth, Arabic, acacia, karaya, locust bean, agar-agar, pectin, elgin, etc.; fermentation broths, mashes, and residues from fermentation processes; whey; wines and Vinegars; animal feeds and ingredients of animal feeds such as fish meals, blood meals, feather meal, meat scraps, bone meal, tankage, grains, and oil-seed meals; proteins and protein hydrolysates; textile printing pastes; paints containing proteins or other spoilable dispersing agents; solutions of bark extracts or other tanning agents; molasses; byproducts or wastes that contain potentially valuable carbohydrate, proteinous or fat ingredients such as stick liquor, corn steep liquor, fruit cannery wastes, citrus peels, cull fruit and vegetables, tops of root vegetables, distillers slops, pulp liquors, wash water from textile de-sizing operations, waste liquors from wool scouring plants, dairy and slaughter house wastes an liquors, etc.

The compounds of the invention may be synthesized by known procedures-for example, those disclosed by Hurd et al., Jour. Am. Chem. Soc., vol. 59', pp. 107-109; Barnes et al., Tetrahedron, vol. 21, pp. 2707-2715; Jurd, Experientia, vol. 24, pp. 858-860; Jurd, Tetrahedron, vol. 25, pp. 1407-1416; and Jurd, Tetrahedron Letters No. 33, pp. 2863-2866. Typically, these syntheses involve condensing a selected phenol (phenol itself, a cresol, resorcinol, guaiacol, hydroquinone monomethyl ether, etc.) with cinnamyl bromide or cinnamyl alcohol.

The invention is further demonstrated by the following illustrative examples. For comparative purposes, various compounds, including some of known antimicrobial activity, are included in the reported experiments.

EXAMPLE 1 A series of compounds were assayed for effectiveness against bacteria, molds, and yeasts, using the following test procedure.

All compounds were tested at a concentration of 500 p.p.m. (w./v.) Plates were prepared by adding a measured amount of the candidate compound (in an appropriate solvent, i.e., acetone, ethanol, or Water) to 10 ml. of sterilized medium, mixing thoroughly, pouring into 60 x mm. Petri dishes, and allowing the gel to set. The plates were then inoculated with' the test organisms. In the case of bacteria and yeasts the inoculation was done by Leder- 6 The plates were incubated at 28 C. for one to five days and evaluated by comparison with the controls.

The results are expressed on the basis:

+ for effective to inhibit growth bergs replica plating technique, applying nine bacteria or 5 for not completely effective;

seven yeasts on each plate. In the case of molds, drops of faint growth occurs homogenized culture were placed on the surface of the for ineffective; growth occurs plates, applying three to four molds per plate. The results are summarized in the following tables.

TABLE I Activity of Various Compounds (at concentration of 500 p.p.m.) Against Bacteria 4-cini-cin- 4-cin- Z-cinnamyl- 2cinnamyl- Cinnamylnamylnamylfi-methoxy- B-methoxynamyl- Phe- Resor- Pyro- Methoxyphenol resorcinol pyrogallol quinol quinone eugenol nol cinol gallol quinol Bacillus cereus Sorcino leutea Staphylococcus aure Streptococcus lactic- Acalioeues faecolis. Eacherichia coli :l: i d:

TABLE II Activity of Various Compounds (at concentration of 500 p.p.m.) Against Yeasts 4-cin- 4-cin- 4-cin- 2cinnamy 2-cinnamy Cinnamylnamylnamylfi-methoxyfi-methoxynamy Phe- Resor- Pyro- Methoxyphenol resorcinol pyrogallol quinol quinone eugenol nol cinol gallol quinol Zygosacchomyces japouicus Candida tropicalis Pichia chodati Hammula anomala Succharomyces cereoisi Saccharomyces mellis Torula uiilis Hauseuula melliqui Candida chalmersi Saccharomyces rosei Zyaosaccharomyecs barlce TAB LE III Activity of Various Compounds (at concentration of 500 p.p.m.) Against Molds 4-cin- 4-cin- 4-cin- 2-cinnamyl- Z-cinnamyl- Cinnamylnamylnamyl- 5-methoxyfi-methoxynamyl- Phe- Resor- Pyro- Methoxyphenol resoreinol pyrogallol quinol quinone eugenol nol cinol gailol quinol Botrytis cinerea- Byssochlamys fuloaa Alternana sp The media used were: plate count agar (Difco) for EXAMPLE 2 bacteria; potato dextrose agar (Difco) for yeasts and 5 molds.

Control plates were also prepared containing the media plus the same solvent used for the candidate agents and inoculated with the same organisms.

TABLE IV Minimal Inhibitory Concentrations (in ppm.) of Various Compounds Against Molds 4- 2- 2-cinnamyl- 2-methyl-4- 2-cinnamyl- Ortho- Potasclnnamylcinnamyl- 4-methoxycinnamyl- 4-n1ethyl- Phenylsium Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Bacillus cereus- 12 25 12 12 100-200 800 1, 000 Sarciua latch.-. 25 25 25 12 12 200 800 1, 000 Staphylococcus aureus 25 25 25 12 12 200 800 1, 000 Streptococcus lactis- 25 12 25 12 12 100-200 800 1 000 Acaligcues jaecalis -100 200 200 200 200 100-200 800 1,000 Escherichia coli--. 50-100 200 200 200 200 100-200 800 1, 000

TABLE V Minimal Inhibitory Concentrations (in pp. 111.) of Various Compounds Against Molds 4- 2- 2-cinnamyl- 2-methyl-4- Z-cinnamyl- 0 Potascinnamylcinnamyl- 4-methoxycinnamyl- 4-methyl- Phenylsium Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Zg gosaccharomyces japonicus 12 12 25 12 12 100-200 800 1,000 Candida tropicalis 12 12 50 25 25 100 200400 250-500 Pichia chodati. 26 50 200 200 200 100 800 1, 000 Hamenula auomala 50 100 50 100-200 800 1, 000 Saccharomyces cerevisiae. 25 25 50 25 25 100-200 800 1, 000 Torula uiilis 50 60 200 100 200 100-200 800 1, 000

TABLE VI Minimal Inhibitory Concentrations (in p.p.m.) of Various Compounds Against Molds 4- 2- Zeinnamyl- 2-methyl-4- 2-cinnamylo- Potaseinnamylcinnamyl- 4-methoxycinnamyl- 4-methyl- Phenylsium Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Aspergillus flavus 100 100 200 200 200 100 800 1, 000 Aspergillus niger 50 100 200 200 200 100 800 1, 000 Penicillium chrysoae'aum 50 25-50 200 50 100 50 800 1,000 Rhizopus senti 6 50 200 50 100 100 200 600 Boirytis cinerea 25-50 12-25 200 50 25 12-25 200 500 Byssochlamys fulva 25 25 50 25 25 50 800 1,000 Altcrnaria sp 25 25 100 50 100 50 400 500 TABLE VII Minimal Inhibitory Concentrations Yeasts, and Molds (in p.p.m.) of 4-cinnarnyl-phenol and Several Alkyl 4-hydroxybenzoates Against Bacteria,

4-cin- Heptyl Amyl I1-Butyl n-Propyl Ethyl Methyl nam yl- 4-hydroxy- 4-hydroxy- 4-hydroxy- 4-hydroxy- 4-hydroxyi-hydroxyphenol benzoate benzoate benzoate benzoate benzoate benzoate Bacillus cereus 25 12 100 200 400 200 200 Sarci'aa Zutea. 25 12 100 200 400 200 200 Staphylococcus au rcus. 25 12 100 200 400 200 200 Streptococcus lactia. 25 12 100 200 400 200 200 A caligenes faecalis- 50-100 200 200 200 400 200 200 Escherichia coli 50-100 200 200 200 400 200 200 Z yqosaccharomyccs japo nicus 12 12-25 50 100 100-200 200 200 Candida tropicalis- 12 12-25 50 100 200 200 200 Pichia chodali 25 200 100 100 200-400 200 200 Hansenula anomala. 50 200 100 100 200-400 200 200 Saccharomyces cerevisiae 25 100 50 100 200 200 200 Torula utilis 50 25 100 100 200 200 200 Aspergillus flaws 100 200 100 200 200 200 200 Aspergillus niger- 50 200 100 200 200 200 200 Penicillium chrysogen 50 200 100 100 200 200 200 Rhizopus semi 6 25 50 100 200 200 200 Botrylia cinerea 25-50 50-100 50 100 100-200 100 100 Byssoch lamys fulva 200 100 100 200 200 Alternaria 5p 50-100 50 50 100-200 100 200 EXAMPLE 3 EXAMPLE 4 Two compounds (4-cinnamyl-phenol and 4-cinnamylresorcinol) were assayed for effectiveness against bacteria, molds, and yeasts, except that in this case the assays were conducted at different concentrations of each compound and at difierent pI-ls in order to determine the minimum concentration of each compound required to inhibit growth at the particular pH. The results are tabulated below.

TABLE VIII Samples of fruit juice (grape and apple) with and without added 4-cinnamyl-phenol were inoculated with cultures of wild yeast or pure wine yeast (S. cerevisiae) and held at room temperature. The inoculated juices were observed at intervals to detect the time at which fermentation (gas production) began. This is a measure of microbial activity--where the organisms multiply freely, the time for initiation of fermentation is short; conversely,

Effect of pH on Minimal Inhibitory Concentration (p.p.m.) of 4-cinnamyl-phenol and 4-cinnamyl-resorcinol 4-einnarnyl-phenol pH7 pH6 pH5 pH4 pHB pH7 pHfi pH5 pH4 pH3 Bacillus cereus 25 25 25 12. 5 200 100 6. 25 Sarcina lutea 25 25 25 12. 5 100 25 Staphylococcus aureus- 25 25 25 25 50 25 Streptococcus laciis. 25 25 25 25 50 25 Acaligenes faecalis. 50 25 25 25 100 25 Escherichia coli 25 50 50 25 100 50 Zygosaccharomyces japo'nicus 12. 5 12. 5 12.5 50 50 50 Candida tropicalia. 25 25 25 50 50 50 Pichia chodaii. 50 50 25 150 100 50 Hansenula anomala. 50 50 50 100 100 Saccharomyces cerevisiae 25 25 25 50 50 Geotriehum o Torula utilis 50 50 37. 5 50 75 50 Aspergillus flaoas 100 100 100 200 200 100 Aspergillus niger.. 100 100 75 200 100 100 Penicilliam chrysogenumn 50 50 50 100 50 50 Rhizopus sentL 25 25 25 100 100 50 Boirytis cinerea" 25 25 25 100 75 50 Byssochlamus ful 25 25 25 100 75 50 50 75 50 Alternaria sp where microbial growth is inhibited the time for initiation of fermentation is long or does not occur at all.

The results are tabulated below.

nisms selected from the group consisting of bacteria, yeasts, and molds in a substance normally subject to spoilage by said microorganisms, which comprises apply- TABLE IX Influence of -cinnamyl-phenol on Fermentation of Fruit Juice Inoculated with Yeasts Time for initiation of fermentation, hours Amount of inoeulum, Zero 12.5 p.p.m. 25 ppm. 50 p.p.m. 100 p.p.m. 200 p.p.m Run Substrate Inoculum cells/ml. 4-CP of 4-0]? of 4-CP of 4-01 4-GP 01 4-01 1 Grape juice..- Wild yeast--..- 10 7 N .D. 12 ND. 44 N .D. 2 do do 1O 17 17 3O 62 96 ND. 3 Wine yeast... 10* 12 22 45 146 00 oo 4 do do 10 41 75 oo 00 oo oo 5 Apple jnim do 10 46 42 76 co co co Explanation of symbols:

4-CP designates 4-cinnamy1-phenol.

N .D. means not determined no means that fermentation did not occur; the organisms had been destroyed.

Having thus described the invention, what is claimed is: 1. A process for inhibiting the growth of microorgaing to said substance an effective microbial-growth-inhibiting amount of 2-cinnamyl-S-methoxy-quinol.

References Cited JEROME D. GOLDBERG, Primary Examiner A. I. ROBINSON, Assistant Examiner US. Cl. X.R.

Tetrahedron, vol. 21, pp. 2707-2715

Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US4044160 *Jun 15, 1976Aug 23, 1977Swift & CompanyTert-butyl hydroquinone, bacteriostat, whole milk
US20100119646 *Jan 31, 2008May 13, 2010Dsm Ip Assets B.V.Dihydroeugenol as Additive for feed
U.S. Classification514/720, 514/733, 252/404
International ClassificationA23L3/349, C07C39/00, A23L3/3463, C07C39/21
Cooperative ClassificationA23L3/3463, C07C39/21, A23L3/349
European ClassificationA23L3/349, A23L3/3463, C07C39/21