Antibacterial Activities of Phenolic Benzaldehydes and Benzoic Acids

Home , Dihydroxybenzoic acid, Gallic acid, Gentisic acid, Methylparaben, Trihydroxybenzoic acid

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Journalof Food Protection, Vol. 66, No. 10, 2003, Pages 1811– 1821

Antibacterial Activities ofPhenolic Benzaldehydes andBenzoic Acids against Campylobacter jejuni,Escherichia coli,Listeria monocytogenes, and Salmonellaenterica

MENDEL FRIEDMAN, * PHILIP R.HENIKA, AND ROBERT E.MANDRELL

WesternRegional Research Center,Agricultural Research Service, U.S.Department of Agriculture, 800 Buchanan Street, Albany,California 94710, USA

MS02-481:Received 19December 2002/Accepted 6April2003

ABSTRACT

Weevaluatedthe bactericidal activities of 35 benzaldehydes, 34 benzoic acids, and 1 benzoicacid methyl ester against Campylobacterjejuni, Escherichia coli O157:H7, Listeriamonocytogenes, and Salmonellaenterica whenthese compounds weresubstituted on the benzene ring with 0, 1, 2, or 3 hydroxy(OH) and/ ormethoxy (OCH 3)groupsin a pH7.0 buffer. Dose-responseplots were used to determine the percentage of the sample that induced a 50%decrease in CFU after60 min (BA50).Ofthe 70 compounds tested, 24 were found to be activeagainst all four pathogens, and additional 4, 10,and 12 were foundto be active against three, two, and one of the pathogens, respectively. C. jejuni was ;100times as sensitive as the otherthree pathogens. The 10 compoundsthat were most active against the four pathogens (with average BA 50 valuesranging from0.026 to 0.166)and are candidates for studies of activityin foodsor fordisinfections were 2,4,6-trihydroxyb enzaldehyde, 2,5-dihydroxybenzaldehyde,2,3,4-trihydroxyb enzaldehyde,2-hydroxy-5-metho xybenzaldehyde,2,3-dihydroxyben zaldehyde, 2-hydroxy-3-methoxybenzaldehyde,4-hydroxy-2,6-dime thoxybenzaldehyde,2,5-dihydroxybenz aldehyde,2,4-dihydroxybenzaldehyde,and 2-hydroxybenzald ehyde.Comparison of the chemical structures of the test compounds and their activities revealedthat (i) the aldehyde (CHO) groupwas more active than the carboxyl (COOH) groupwhether or notOH groupswere present;(ii) compounds were most active with trisubstituted OH . disubstitutedOH . monosubstitutedOH; (iii)for disubstitutedderivatives, 2-OH enhanced activities were exhibited by benzaldehyde but not by benzoic acid; (iv) compounds were moreactive with OH thanwith OCH 3,irrespectiveof the position of substitution on the benzene ring; (v) compounds with mixedOH andOCH 3 groupsexhibited variable results, i.e., in some cases OCH 3 groupsenhanced activity and in other cases theydid not; (vi) methoxybenzoic acids were largely inactive; and (vii) gallic acid was 20 times as active against S. enterica atpH 7.0 as it was at pH 3.7, suggesting that the ionization of its OH groupsmay enhance bactericidal activity.

Pathogenicstrains of Campylobacterjejuni, Escherich- havenumerous bene cial properties by virtueof theirabil- iacoli, Listeria monocytogenes, and Salmonellaenterica ityto act as antioxidants, anticarcinogens, antimutagens, arelinked to foodborne illnesses. Nontoxic food-compatible andantiglycemic agents (19,27, 37, 40– 42). These prop- naturalproducts are a sourceof compounds that may be ertiescan be used in the prevention of rancidity in food usefulin interventions to reduce pathogens on surfaces of andin the development of health-promoting food ingredi- fruitsand vegetables and in antimicrobialformulations add- ents.However ,phenoliccompounds can also darken pro- edto foods such as fruit and vegetable juices, meat, and tein-containingfood ingredients and reduce the nutritional poultryfor disinfectionor to minimize the contamination qualityof proteinthrough their ability to participatein poly- ofthese foods during storage and during commercial and phenol-proteinenzymatic browning reactions catalyzed by homeprocessing. polyphenoloxidases (11, 13). Phenoliccompounds are secondary plant metabolites Inaddition to their cited bene cial effects, phenolic foundin numerous plant species, and they play key roles compoundsare also reported to have antimicrobial activi- inthe biochemistry and physiology of plants. These com- tiesagainst human pathogens. The literature contains nu- pounds,especially oxidation products of phenolic com- merousstudies on theantibacterial, antifungal, and antiviral pounds,appear to be involved in the defense of plants activitiesof natural and synthetic phenolic compounds (1– againstinvading pathogens, including bacteria, fungi, and 5,7– 9, 16, 18, 20, 23– 25, 27, 29, 31– 35, 39, 44, 45), and viruses.In addition, polyphenol-oxida se-catalyzedpoly- thesestudies go back to the 19th century following the merizationof monomericphenols such as chlorogenic acid discoveryby Lister and others that carbolic acid (phenol) helpsto seal the injured plant surface and initiates the heal- canact as an antibiotic against wound infections (21). As ingprocess, analogous to the formation of brinblood clots describedin detail in the excellent reviews by Davidson (6) ininjured animals and humans (12). andFung et al. (17), thesynthetic methyl and propyl esters Dietaryphenolic compounds have also been shown to of p-hydroxybenzoicacid, the so-called parabens, as well astertiary butylhydroxyani sole(BHA) andtertiary butyl- *Authorfor correspondence. Tel: 510-559-5615; Fax: 510-559-5777; hydroquinone(TBC), are on the generally-recognized -as- E-mail: [email protected]. safe listfor usein food at a maximumconcentration of 1812 FRIEDMANET AL. J.FoodProt., Vol. 66, No. 10

4-methoxybenzaldehyde(4-methoxysalicyla ldehyde,152); 2-hydroxy-4-methoxybenzoicacid (4-methoxysalicylic acid, 168); 2-hydroxy-5-methoxybenzaldehyde(5-methoxys alicylaldehyde, 152);2-hydroxy-5-methox ybenzoicacid (5-methoxysalicylic acid, 168);2-hydroxy-6-methox ybenzoicacid (6-methoxysalicylic acid, 168);3,4-dihydroxyben zaldehyde(protocatechualdeh yde,138); 3,4-dihydroxybenzoicacid (protocatechuic acid, 154); 3,4-dimethoxybenzaldehyde(veratraldehyde,166); 3,4-dimethoxybenzo ic acid(veratric acid, 182); 3,5-dihydroxyben zaldehyde(138); 3,5- dihydroxybenzoicacid ( a-resorcylicacid, 154); 3,5-dimethoxybenzaldehyde(168); 3,5-dimethoxybenzo icacid (182); 3-hydroxy-4-methoxybenzaldehyde(isovanillin, 152); 3-hydroxy-4- methoxybenzoicacid (isovanillic acid, 168); 4-hydroxy-3-methoxybenzaldehyde(vanillin, 152); 4-hydroxy-3-metho xybenzoic FIGURE 1. Structuresof benzaldehydes and benzoic acids eval- acid(vanillic acid, 168); 2,3,4-trihydroxybe nzaldehyde(154); uatedfor antibacterial activity. Table 1 showsthe positions of the 2,3,4-trihydroxybenzoicacid (170); 2,3,4-trimethoxybe nzaldehyde OH and OCH groupson the benzene rings. 3 (196);2,3,4-trimethoxybe nzoicacid (212); 2,4,5-trimethoxybe n- zaldehyde(196); 2,4,5-trimethoxyben zoicacid (asaronic acid, 212);2,4,6-trihydroxybe nzaldehyde(phloroglucinoca rboxaldehy- 0.02% (38). However,theyappear not to be widely used, de,154); 2,4,6-trihydroxybe nzoicacid monohydrate (188); 2,4,6- possiblybecause of their low solubility in water . trimethoxybenzaldehyde(196); 3,4,5-tr ihydroxybenzaldehyde Toassessthe antimicrobial potential of natural com- monohydrate(172); 3,4,5-trihydroxyb enzoatemethyl ester (gallic poundsand plant extracts, we previouslyevaluated the rel- acidmethyl ester, 184); 3,4,5-trihydroxyb enzoicacid (gallic acid, ativebactericidal activities of 96plantessential oils and 23 170);3,4,5-trimethoxybe nzaldehyde(196); 3,4,5-trimethoxybe n- oftheir active components by testing them separately zoicacid (212); 2-hydroxy-4,6-dime thoxybenzaldehyde(182);3- againstthe four major human pathogens (14). Inaddition, hydroxy-4,5-dimethoxybenzoicacid (182); 3,4-dihydroxy-5-me th- we havebeen evaluating other compounds in abroadeffort oxybenzaldehyde(168); 4-hydroxy-2,6-di methoxybenzaldehyde tode ne the chemical basis for theantimicrobial activities (182);4-hydroxy -3,5-dimethoxybenzaldehyde(syringal dehyde, ofdifferent classes of natural products. As partof this ef- 182);4-hydroxy-3,5-dime thoxybenzoicacid (syringic acid, 198); fort,the general objective of the present study was toscreen and5-hydroxy-3,4-dime thoxybenzaldehyde(182).Standard test compoundsincluded trans-cinnamaldehyde,gentamyc in,and avarietyof benzaldehydesand benzoic acids substituted on chloramphenicol(Sigma). All compounds were purported to be thebenzene ring with one, two, and three OH and/orOCH 3 96to 99% pure. groups(Fig. 1). The data obtained in this study indicate thatthe benzaldehydes are more active than the benzoic Testbuffers. Phosphate-bufferedsaline (PBS, pH7.0) was acidsand that bactericidal activities are in uenced by both preparedby mixing dibasic sodium phosphate (100 mM) and monobasicsodium phosphate (100 mM) ata 2:1ratio, diluting thenumber and the position of the OH andOCH 3 groups onthe benzene rings. byhalfwith H 2O,andadding NaCl (150 mM). For stronger buff- ering,the benzoic acids were prepared in 200 mM PBS (pH7.0) MATERIALS AND METHODS buffer.Forthe pH study, the PBS buffer(pH 7.0) was adjusted topH 8.4with 5 NNaOH, anda 2mM citricacid– 150 mM NaCl Testcompounds. Thefollowing compounds were purchased bufferwas adjusted to pH 3.7 or pH 5.4. fromSigma (St. Louis, Mo.) or Aldrich (Milwaukee, Wis.) (the commonnames are followed by molecular weights of the com- Testsolutions. Testsolutions or suspensions were prepared poundsin parentheses): benzaldehyde (106); benzoic acid (122); by adding 10 mlofliquid test compound or 10to 15 mg of solid acetylsalicylicacid (aspirin, 180); 2-hydroxybenzald ehyde(sali- testcompound to 1 mlof buffer and vortexing for 1 min.If they cylaldehyde,122); 2-hydroxybenzoic acid (salicylic acid, 138); 2- wereinsoluble, the solutions were microwaved for 4 to5 sonthe methoxybenzaldehyde( o-anisaldehyde,136); 2-methoxybenzoic highsetting so that condensation on the tube was observed but acid (o-anisicacid, 152); 3-hydroxybenzald ehyde(122); 3-hy- volumewas not lost. The samples were vortexed for 1 minand droxybenzoicacid (138); 3-methoxybenzaldeh yde( m-anisalde- shaken. hyde,136); 3-methoxybenzoic acid ( m-anisicacid, 152); 4-hy- Suspendedstock solutions were shaken vigorously by hand droxybenzaldehyde(122);4-hydroxybenzoic acid (138); 4-meth- in1.9-ml sterile plastic snap-cap microcentrifuge tubes. A 200- ml oxybenzaldehyde( p-anisaldehyde,136); 4-methoxybenzoic acid aliquotwas added to 400 mlofbufferand then shaken vigorously (p-anisicacid, 152); 2,3-dihydroxyben zaldehyde(138); 2,3-dihy- priorto the addition of the next aliquot. For E.coli,S. enterica, droxybenzoicacid (154); 2,3-dimethoxybenz aldehyde(166); 2,3- and L.monocytogenes, thesamples were diluted threefold, and for dimethoxybenzoicacid (182); 2,4-dihydroxyben zaldehyde( b-re- C. jejuni, thesamples were diluted fourfold for each of six dilu- sorcylaldehyde,138); 2,4-dihydroxyben zoicacid ( b-resorcylic tions,respectively. The antibiotic gentamycin was used as a pos- acid,154); 2,4-dimethoxybenz aldehyde(166); 2,4-dimethoxyben- itivecontrol for C.jejuni,E. coli, and S.enterica, andthe anti- zoicacid (182); 2,5-dihy droxybenzaldehyde(gentisa ldehyde, bioticchloramphenicol was used as apositivecontrol for L. mon- 138);2,5-dihydroxyben zoicacid (gentisic acid, 154); 2,5-dime- ocytogenes. Onthe basis of our extensive experience with cin- thoxybenzaldehyde(166);2,5-dimethoxybenzo icacid (182); 2,6- namaldehyde (14), thiscompound was also used as a positive dihydroxybenzoicacid ( g-resorcylicacid, 154); 2,6-dimethoxy- control for C.jejuni,E. coli, and S.enterica. benzaldehyde(166); 2,6-dimethoxybenzo icacid (182); 2-hydroxy-3-methoxybenzaldehyde( o-vanillin,152);2-hydroxy -3- Sourcesof bacteria. C. jejuni RM1221, E. coli O157:H7 methoxybenzoicacid (3-methoxysalicylic acid, 168); 2-hydroxy- strainRM1484, L. monocytogenes RM2199, and S. enterica ser- J.FoodProt., Vol. 66, No. 10 ANTIBACTERIAL PHENOLICCOMPOUNDS 1813 ovarHadar strain RM1309, obtained from contaminated food and wereusually small but were counted at 24h. Wealsoobserved a clinicalsources and described previously (14), wereused in this purplishblack staining of the iron-supplemented BA withsome study. ofthe compounds. The benzaldehydes and gallic acid used in the pHstudies were observed to be chromogenic at neutral and al- Bactericidalassays. Thebactericidal assay was developed kalinepHs. Experimental results were analyzed only when control bymodifying a previouslydescribed microtiter plate bactericidal countswere 60 to 200 CFU. Ineach case, at least two separate assay (14). Brie y, C. jejuni wascultured on iron-supplemented experiments,involving bacterial pellets processed from two dif- brucellaagar (BA) (Difco Laboratories, Sparks, Md.) in Hefty ferentcultures, were performed. One-ZipSlider Freezer bags (Hefty, Lake Forest, Ill.) lledwith amicroaerophilicgas mixture (5% O 2, 10% CO2, and 85% N2) Bactericidalactivity. Bactericidalactivity was de ned as the for18 to19 hat42 8C.Thebags were lledwith gas, the gas was percentageof test compound that kills 50% of the bacteria after expelledthree times, and then the bags were sealed tightly to 60 min (BA50)andwas determined as follows (14). Each com- ensurethe proper atmosphere. Streaked plates, rather than broth poundwas tested as a seriesof six dilutions from 0.00065 to cultures,were used as the source of C.jejuni.E. coli and S. en- 0.67%in the reaction mixture. The CFU countsfor all experi- terica werecultured in Luria-Bertani agar broth (Difco) for 16 to mentswere transferred to a MicrosoftExcel 8.0 Spreadsheet. The 18 h at 378C, and L.monocytogenes wascultured in iron-supple- numberof CFU fromeach dilution was matched with the average mentedbrain heart infusion broth (Difco) for 16 to 18 h at37 8C. controlvalue to determine the percentage of bacteria killed per A1-mlsample of stationary-phase bacteria ( E.coli,S. en- well.Each of the dose-response pro les (percentage of testcom- terica, or L.monocytogenes )inbroth was centrifuged, and the poundversus percentage of bactericidal activity) was examined pelletwas resuspended in 1 mlof sterile PBS. Thesample’ s op- graphically,and the BA 50 valueswere estimated by a linearre- (36). ticaldensity at 620nm (OD 620)wasadjusted to ca.0.8 with PBS. gression Thelower the BA 50 valueor thehigher the 1/ BA 50 Each 10-mlsamplewas added to 990 mlofPBS (1:100),and then value,the higher the activity. A BA 50 value of .0.67%considered 80 mlofthe1:100 sample was diluted in 20 ml of PBS toanal toindicate inactivity. dilutionof 1:25,000 containing ca. 1,500 to 2,000 cells. RESULTS Studiesto optimize the bactericidal assay for the microaero- philic C. jejuni werecarried out. Speci cally, colonies on plates Table1 showsthe BA 50 valuesfor benzaldehyde,ben- from16- to 18-h cultures were sampled with a loopand suspended zoicacid, and 68 derivatives substituted with OH, OCH , in1 mlof PBS. Thebacterial suspension was centrifuged, and the 3 andboth groups in various positions of the benzene ring. pelletwas resuspended in PBS. TheOD ofthesuspension was 620 Theobserved solubilities and colors of these compounds adjustedto 1.0 to 1.3. The diluted sample (10 ml)was added to 990 mlofPBS (1:100),and then 20 mlofthe 1:100 sample was arealso given in the T able1. Four types of preparations dilutedin 20 ml of PBS toa naldilution of 1:100,000. were observed:solutions; suspensions; milky, oily suspen- Theassay mixture consisted of PBS buffer(pH 7.0), the test sions;and immiscible oils with rapid oil separation. The 5- compound,and the bacteria. For the pH studies with S.enterica, smicrowavingused to facilitate solubilization should not 20 mlofthe 1:100 sample was diluted in 5 mlof PBS (pH8.4 affectthese compounds in view of reportsthat microwaves orpH 7.0) or citric acid-saline (pH 5.4 or pH 3.7). donot adversely affect the nutrition and safety of different Sampleswere prepared with sterile 96-well tissue culture mi- categoriesof food ingredients (10). crotiterplates (Nalge, Nunc International, Rochester ,N.Y.).Each Underthe test conditions, benzoic acid was inactive dilutionwas shaken prior to its addition to thewells. The addition againstall four bacteria (BA 50 . 0.67).In contrast, benz- of 100 mlofPBS minus/plustest substance to the wells was fol- aldehydewas highlyactive against C. jejuni (BA 5 lowedby the addition of 50 mlofbacterial suspension; thus, the 50 0.045)and less active against the other three bacteria (with originalstock solution was diluted by one-third. Six wells were usedfor each control to obtain an averagecontrol value; one pos- BA50 valuesranging from 0.36to 0.48). itivecontrol (gentamycin, chloramphenicol, or trans-cinnamalde- Tofacilitatediscussion of the effects of structure on hyde)was represented in a dilutionseries across six wells, and antimicrobialactivity, Figure 2 showsplots of 1/BA 50 val- 14test compounds were represented in dilution series across the ues(calculated from theBA 50 valueslisted in T able1) for remainingwells. The wells on the plate were covered with a the10 compounds that were mostactive against each of SealPlatecover (Marsh Bioproducts, Rochester ,N.Y.),sealedon thefour pathogens. T able2 listsaverage BA 50 values for theoutside edges with Para lm, and then incubated with gentle the24 compoundsactive against all four species, and T able shakingfor 60 minat 37 8C (428C for C. jejuni).Forthe pH stud- 3presentsdata on the effects of pH on the bactericidal ies, S. enterica wasincubated for 30 and 60 min at 37 8C. activitiesof selected compounds. Followingincubation, a 20- mlsamplefrom each well was Figure3 depictsso-called box-and-whisker plots, well- spottedat the top of a squarepetri dish containing Luria-Bertani establishedstatistical tools (26, 43) thatallow the visuali- medium (for E. coli and S. enterica),brainheart infusion agar (for L.monocytogenes ),oriron-supplemented BA (for C. jejuni). Six zationof the trends and distributions of activities against 20-mlspotswere spaced across the top— one for each dilution of thefour pathogens (1/ BA 50 values)for all70 compounds testsample or six controls. The plates were placed uncovered in listedin T able1. The description of these results will be abiologicalsafety hood until the sample liquid dried (ca. 10 min) dividedinto three parts, covering monosubstituted, disub- andwere then covered. E. coli,S. enterica, and L.monocytogenes stituted,and trisubstituted compounds as listed in T able1. plateswere incubated overnight at 37 8C. C. jejuni plates were Monosubstituted benzaldehydesand benzoic acids. placedin sealable bags lledwith gas (5% O 2, 10% CO2, and 85% N2)andincubated at 42 8C. 2-Hydroxybenzaldehyde(salicylaldehyde) was quiteactive E.coli,S. enterica, and L. monocytogenes colonieswere againstall four bacteria, with BA 50 valuesranging from countedafter 18 to 24 h. C. jejuni CFU iniron-supplemented BA 0.064to 0.26.In contrast, 2-hydroxybenzoic (salicylic) acid 1814 FRIEDMANET AL. J.FoodProt., Vol. 66, No. 10 n w o w l l o e r r y b o l s s s s s s s s s s s s s s s s s s s s s s s h h o s s s s s s s s s s s s s s s s s s s s s s s s s e e e e e e e e e e e e e e e e e e e e e e e C i i l l l l l l l l l l l l l l l l l l l l l l l n w e e r r n r r n r r n r r n r r r r r r n r r r r r r r r r t t o y e y e y e y e e w y o o o o o o o o o o o o o i o o o o i o o o o o o l l l l l l l l l l l l l l l l l l l l l l l l d l a e a e a e a e e a o h h r r r r r r r r r r o o o o o o o o o o o o o o o o o o r o o o e o o e G C C G C G C G C G C G C G C G C C C C C W C G C Y C C W C B C C C R C G C s n n n n n n y o o o o o o t i i i i i e e e e i i l l l l l s s s s s s i e e e e e e e e e e e e e e e e e e e e e e e e e e e e b b b b n n n n n n l l l l l l l l l l l l l l l l l l l l l l l l l l l l b u u u u e e e e e e u b b b b b b b b b b b b b b b b b b b b b b b b b b b b l l l l l p p p p p p u u u u u u u u u u u u u u u u u u u u u u u u u u u u o o o o o s l l s l s l l l s l l l s l l l l l l l l l l l l l l l l l l s l s s s s S u o o u o u o o o u o o o u o o o o o o o o o o o o o o o o o o u o a n n n n S S S S S S S S S S S S S S S S S I S S S I S S S I S S I S S S S S S S S S c i r e t e 4 d e 1 7 n 1 0 5 3 1 1 3 7 0 2 7 3 3 0 1 3 e a 2 1 0 0 0 0 1 1 0 0 1 0 0 0 1 2 ...... c i 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S r f e 7 d t 7 7 7 7 7 7 7 7 7 7 7 7 7 0 7 7 7 7 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 n n 6 . 6 6 6 6 6 6 6 6 6 6 6 6 5 6 6 6 6 e ...... a 0 6 0 1 9 7 4 1 0 1 1 8 3 8 0 7 6 7 8 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . , 3 2 6 1 2 1 3 3 1 3 9 4 4 4 4 5 2 1 6 S ...... s ...... 0 . . . 0 ...... 0 0 0 0 0 0 0 0 0 0 . . 0 0 0 0 0 0 0 e 0 0 n e g o t s y e 3 3 c n 0 6 7 5 6 4 4 1 1 1 6 1 0 o e 1 0 0 0 0 0 0 0 0 2 0 0 0 ...... n g o 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o a t 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 y ...... m m c 6 6 6 6 6 6 6 6 6 6 6 6 6 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 u . o i r n L 6 6 6 7 0 8 9 8 5 3 0 3 9 5 ...... e o t 4 2 6 3 4 5 4 5 3 6 4 3 2 6 , ...... c m 0 0 0 7 a 0 0 0 0 0 0 . 0 0 . 0 0 0 . . b 0 0 0 H L : r 7 o f 5 1 ) d d D O 7 3 e d e e d 6 1 e d h S e i 0 0 1 3 8 3 0 5 0 0 2 5 3 l 1 0 0 2 1 0 0 2 0 0 0 1 1 0 o 6 ...... 0 ...... c 0 0 0 0 0 0 0 0 0 0 0 0 0 i n 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 0 l . 7 7 7 a o 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 e . . . . 6 . 6 . . 6 ...... E c 6 6 6 6 6 6 6 6 6 6 6 6 6 . . . 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 , m . 0 0 0 i ( 8 4 7 5 3 1 7 3 4 3 6 2 5 E 4 ...... n 4 1 3 2 5 4 3 9 1 4 6 2 1 . . . 0 ...... 5 . . . . u 5 0 . 0 j 0 0 0 0 0 0 0 . 0 . 0 0 0 0 A e 0 0 j B . C t s n i 1 6 a b d d d 3 3 e 6 d 7 b d 0 d 7 d 0 d d g 3 3 3 2 7 0 6 5 0 6 0 6 9 3 0 0 0 8 8 0 6 8 1 4 a f 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 1 2 i ...... c 8 s n 7 7 7 7 7 7 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 7 7 7 7 7 8 d u 6 6 6 6 6 6 6 i j 6 6 6 . 6 . 6 . . . . . 6 0 c e ...... 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 j 0 0 0 0 0 0 0 a 0 0 0 0 0 0 0 . 5 4 5 3 0 1 0 8 6 1 9 5 6 8 7 0 6 0 5 1 1 8 9 8 ...... c ...... C 4 6 6 2 2 9 2 6 1 3 2 6 4 3 1 2 3 4 1 3 1 4 1 2 i ...... 0 0 0 0 0 0 0 0 o . . 0 0 0 . 0 . . 0 0 . 0 0 . 0 0 0 0 0 0 0 0 z 0 0 0 0 0 0 0 0 . n 0 e b d n a s e e d d e e d y y d i h h m d c d y d i e i e a i a h e c c n e d c d e e e e l e l d a a a c d d d d n e d a i d a d y d y d d l y i y d d c y c o z d i c u u i i d y h i i a i i h c i i h h c h n y c c l l h h l e l h c m c e a e e c a e e a a c c h y y y e d a y a d a d d l m d e e b e c c c l c c d l l c l t t c c o a i r r r l c r y c d a i c a i a i a i a a i s s l n l l a i o o o x o C s s s s i i i s s c c r r a o i i i i s s s y y s i i o r t t n n t t o o z z i e e e c c n n n n e a a h t t i i n n t n n p r r A A l l R R R R o o A A A A s - - e e e e e e e - - - a a - r r - - - - B B A S S o o m m p p b b G G g P P V V a m d n a - y x o r d y h f e e e e e e e e e e e o d d d d d d d d d d d d d d d d d d d d d d d i i i i i i i i i i i i y y y y y y y y y y y y c c c c c c c c c c c c t h h h h h h a a a a a a e e e h h h h h e e e i a a a a a a e e e e e e d d d e e e e e d d d d d d v d d d l i i i c c c c c c s i i i i d d d d d d c c c c c c d d d d d y y y i i i i i i y y y l l l l l l t a c c c i i i i i i c c c l l l l l d h h h c c a o a o a o a o a o a o h h h a a a a a a a o a o a o o a o a o i n e e e z z z z z z z z z z z z d e e e a z z z z z z z z z z z s u i c c c c d c d c d m n n n n n n n n n n n n d d d l n n n n n n n n n n n i i i d c i l i l i l o l l l e e e e e e e e e e e e e e e e e e e e e e e e a a a o a o a o n a o a o a o h p b b b b b b b b b b b b b b b b b b b b b b b z z z z z z d z z z z z z u C i y y y y y y y y y y y y c m y y y y y y y y y y y n n n n n n n n n n n n o i e c x x x x x x x x x x x x l o e e e e e e x x x x x x x x x x x e e e e e e i p d d o o o o o o o o o o o o i y c r b b b b b b o o o o o o o o o o o b b b b b b y r r h h r r h h r r h h r h h r r h h r r h h c c e m y y y y y y y y y y y y t t t t t t t t t t t t i t h d a d d d d d d d d d d d o l x x x x x x x x x x x x e e e e e e e e e e e e c e e c y y y y y y y y y y y a t o o o o o o o o o o o o c a d s h h m m h h m m h h m m h m m h h m m h h m m i r r r r r r l h h h h h h u l i i i i i i i i i i i i i i i i i i i i i i i d B t t t t t t t a o d d d d d d y i e e e e e e e z z t t D D D D D D D D D D D D D D D D D D D D D D D y y y y y y t . ------s e n n u 1 H H M M H H M M H H M M t 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 4 4 4 4 5 5 5 5 c b e e ------, , , , , , , , , , , , , , , , , , , , , , , i u t E B B A 2 2 2 2 3 3 3 3 4 4 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 s s L o b ...... n u 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 B s o 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 i A T M D J.FoodProt., Vol. 66, No. 10 ANTIBACTERIAL PHENOLICCOMPOUNDS 1815 . 6 n e 5 e r n g r h o l s s s s s s s s s h s s s s o s s s s s s s s s s s s s s . i e e e e e e e e e e e e e C l l l l l l l l l l l l l 1 n n n n n w w w w w w w e e e e e e r r r r r r r r r r r r r 1 t t t t t t o o o o o o o w w w w w o o o i o o o i o i o o i o i o i o o l l l l l l l l l l l l l l l l l l l l d d l l l l l l l o o o o o h h h h h h o r o r o r o o r o o o e o r o o e o o e e e e e e e 5 Y C B C Y B C W B C C B C W C W C R C W B Y C W C W R C Y Y C Y Y n g n n n n y o o o o . t i e i e e e e e e e e i i i l l l l l l l l l 1 l s s s s i e e e e e e e e e e e e e e e e e e e e e e b b b b b b b b b n n n n l l l l l l l l l l l l l l l l l l l l l l b u u u u u u u u u e e e e u b b b b b b b b b b b b b b b b b b b b b b 5 l l l l l l l l l l p p p p u u u u u u u u u u u u u u u u u u u u u u o o o o o o o o o o s l l s l l l l l l l l l l l l l l l s l l l s l l n s s s s s s s s s S u o o u o o o o o o o o o o o o o o o u o o o u o o f n n n n n n n n n S S I S S S S I S S S S S I S I S I S I I S S I S I S S S S S S S S S . d 3 j d d d e 2 e d e e e e 7 h 4 4 1 2 0 0 2 6 1 2 2 6 2 1 2 0 2 5 0 a 1 1 2 1 0 0 0 0 0 0 1 0 2 1 0 . 4 ...... c 0 i n 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 r f 0 e e t 7 7 7 7 1 7 7 7 7 7 7 7 7 7 7 7 7 0 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 n 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 0 e ...... 4 5 3 9 3 9 1 5 1 0 8 0 2 8 5 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 . 5 2 2 1 6 2 1 5 3 3 3 3 5 5 5 0 S ...... 0 ...... 0 . . . 0 . . 0 . . . . 0 . . 0 0 0 0 . 0 0 . 0 . 0 . 0 0 0 5 0 0 0 0 0 n d 4 i s . e 6 2 0 2 n y 0 0 2 2 4 5 0 6 0 5 1 9 0 t e 0 0 0 0 0 0 0 0 2 0 1 0 0 i ...... g v o 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 0 0 0 0 0 0 0 0 0 0 0 0 0 i a t t 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 y ...... c m c 6 6 6 6 6 6 6 6 6 6 6 6 6 a 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 u o i n r n i 1 4 0 3 0 4 4 4 9 1 7 8 5 ...... e o t 8 3 1 5 2 6 9 6 1 5 4 3 1 s ...... c m 0 0 0 0 e a . . 0 0 0 0 0 0 0 0 0 . t . 0 b 0 0 . 0 a L 0 r c i o f d n ) h i d h 2 D i e e e e 5 e 6 7 e e e d 0 7 S 1 3 7 1 3 0 0 8 0 0 6 1 2 1 0 6 . 0 1 1 0 1 0 1 2 0 0 1 0 1 0 0 . 6 ...... 0 f 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 i n 7 7 7 7 7 7 7 7 7 7 7 4 7 7 7 7 7 7 7 l . a o 6 6 6 6 6 6 6 6 6 6 6 5 6 6 6 6 6 6 6 e ...... 6 f c 6 6 6 6 6 6 6 6 6 6 6 6 6 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o m . 2 ( 0 3 0 2 5 6 2 5 7 9 6 1 8 9 E ...... 4 e 2 4 7 6 4 6 6 5 3 2 4 5 3 1 0 ...... 0 5 u 0 0 0 0 0 0 . l 0 0 . 0 . 0 0 . 0 0 . 0 . 0 A 0 a 0 0 0 0 0 0 v B . 0 A c d d e d 3 . 8 e d 6 g 1 3 3 d 0 5 5 d 2 d 0 3 d 1 3 7 4 2 0 0 7 1 1 1 2 0 0 7 0 9 0 6 8 2 0 1 3 0 0 0 0 0 0 0 0 0 2 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 i ...... n 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 n 7 7 7 7 7 7 7 7 7 u j 6 6 6 6 6 6 6 6 6 b e ...... 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 j 0 0 0 0 0 0 0 0 0 . 2 5 5 3 6 7 8 3 7 6 3 0 1 6 3 9 3 8 3 7 8 1 7 7 5 ...... C 1 4 2 5 6 5 1 7 3 4 5 8 1 2 9 4 4 3 3 4 7 3 3 2 1 d ...... e 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t . 0 0 . 0 . . 0 . 0 . . . 0 0 0 0 0 0 0 0 0 0 0 0 a 0 . 0 0 . 0 . 0 . . . 0 . 0 . 0 . 0 0 c 0 . 0 0 0 0 0 0 0 0 . i 0 0 d n i e d d d d s i i i i i c c c c . . w a a a a r d d e l l e c c c c i a i a i i h m l l l l l l l t a e y y y y y y o o n c c c c c c e d d c i i i i i i n i i s d y l l l l l l n d e c s n d a a a a a a i y h o d b i a e e s s s s s s c e n i h l a c i m h y y y y y y a d c e d r n c n a i c l n i i p a x x x x x x a d m u i c l l c a y l l o o o o o o o l l c i p a m l n c i i a i l ) h h h h h h i i i n m m C a t t t t t t n n g g l l y 2 c a a r n o l l e e e e e e a a i n n t h i i r a l n o i i t v v l n l a n n r r n e 5 V M M M M M M o o s a e i h a a y y ------s s n o 3 4 4 5 5 6 I I V V A M G S S C G C ( s t n e e e e e r e d d d d d d d e i i t y y y y y m c c i s h h h h h e e e e e a a r e e e e e e d d d d d d d d d d d e i i i i i i c c d d d d d l y y y y y p i i c c c c c c l l l l l y h h h h h x a a a a a a a a o a a o a h e e e e e e e e e e z z z z z z z t e e e c c c c c c d d d d d d d d d d d d n n n n n n n e i i i i i i d d d l l l l l d d d i i i e i i i e e e e e e e y y y y t a o a o a o o a o a o y y y c c c c c m c b b b b b b b a h h h h z z z z z z z z z z z a a a h h h a a a r e e e e y y y y y y y n n n n n n n n n n n e e e e a c c t c d d d d x x x x x x x e e e e e e e e e e e c c c d d d i i i l l l l p i i a i l l l o o o o o o o b b b b b b b b b b b a o a o a a o e l a o a o a o o h h h h h h h y y y y y y y y y y y s z z z z z z z a t t t t t t t z z z z z z z x x x x x x x x x x x c n n n n n n n e e e e e e e n n n n n n n i o o o o o o o o o o o o e e e e e e e s e e e e e e e m m m m m m m h h h h h h h h h h h m b b b b b b b d w b b b b b b b i i i i i i t t t t t t t t t t t - e t n y y y y y y y e e e e e e e e e e e y y y y y y y d d d d d d 5 h x x x x x x x u ------r d x x x x x x x C m m m m m m m m m m m o o o o o o o o 6 5 6 5 5 4 e y o o o o o o o o ------, , , , , , . f r r h h h h r r h r r r h h p u x l 3 3 4 4 5 5 6 4 4 3 3 t t t t t t t 4 4 2 3 3 3 8 e d d d d d d d o o n ------e e e e e e e e m i r d y y y y y y y c r y y y y y y y y y y y y y y y y y t s i o d y l h h m m m m h h m h h h m m a 5 x x x x x x x x x x x x x x x x x n c i i i i i i i i i i i i i i n y h o o o o o o o o o o o o r r r r r r r r r r r r r r o o o o o o o e r s e n n h r r r r r r r r r r r r r r r r r t d i T T T T T T T T T T T T T T i e h C j d d d d d d d d d d d d d d d d d d ------e n c l p u t D y y y y y y y y y y y y y y y y y 4 4 4 4 5 5 6 6 6 5 5 5 5 5 l . o a y , , , , , , , , , , , , , , - u a c m 1 t . H H H H H H H H H H H H H H H H H 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 i m m a v ------, , , , , , , , , , , , , , - , - - - - - 0 t e a a r E 2 2 2 2 2 2 2 3 3 4 4 2 2 2 2 2 2 2 2 2 3 3 3 3 3 2 3 3 4 4 4 5 s t 1 0 v n o b 5 i n l L ...... n t u e i h i 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 B A 5 s s i 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 7 C G C B A r o n T T P a i 1816 FRIEDMANET AL. J.FoodProt., Vol. 66, No. 10

TABLE 2. Bactericidalactivities of compounds that were active againstall four pathogens

Average BA50 Compounda (%)b

2,4,6-Trihydroxybenzaldehyde(56) 0.026 2,5-Dihydroxybenzaldehyde(24) 0.042 2,3-Dihydroxybenzaldehyde(16) 0.067 2,3,4-Trihydroxybenzaldehyde(50) 0.069 2-Hydroxy-5-methoxybenzaldehyde(43) 0.090 2-Hydroxy-3-methoxybenzaldehyde(39) 0.136 4-Hydroxy-2,6-dimethoxybenzaldehyde(67) 0.144 2,5-Dihydroxybenzaldehyde(35) 0.146 2,4-Dihydroxybenzaldehyde(20) 0.163 2-Hydroxybenzaldehyde(4) 0.166 2-Hydroxy-4-methoxybenzaldehyde(41) 0.174 3,4,5-Trihydroxybenzoatemethyl ester (60) 0.220 3-Hydroxybenzaldehyde(8) 0.272 3,4-Dihydroxy-5-methoxybenzaldehyde(65) 0.276 4-Hydroxybenzaldehyde(12) 0.330 4-Methoxybenzaldehyde(14) 0.330 Benzaldehyde(1) 0.336 2-Methoxybenzaldehyde(6) 0.362 2,3-Dimethoxybenzaldehyde(18) 0.430 2,6-Dimethoxybenzaldehyde(29) 0.442 5-Hydroxy-3,4-dimethoxybenzaldehyde(70) 0.452 4-Hydroxy-3-methoxybenzaldehyde(48) 0.463 2,3,4-Trimethoxybenzaldehyde(52) 0.585 FIGURE 2. Comparisonof bactericidalactivities (1/ BA 50 values) a ofthe 10 compounds that were most active against each of path- Numbersin parentheses correspond to those listed in T able1 for ogens. C. jejuni:(1)2-hydroxy-5-methox ybenzaldehyde(highest thesame compounds. b levelof activity);(2) 3,4,5-trihydroxyb enzaldehyde;(3) 2-hydroxy- Thesum of the individual BA 50 valuesfor the four pathogens 4-mthoxybenzaldehyde;(4) 3,4-dihydroxyben zaldehyde;(5) 3,4- listedin T able1 dividedby 4. dihydroxy-5-methoxybenzaldehyde;(6) 2,3,4-trihydroxybe nzaldehyde;(7) 3,4,5-trihydroxybe nzoicacid; (8) 4-hydroxy-2,6-dimethoxybenzaldehyde;(9) 2,4,6-trihydroxybe nzaldehyde;(10) 3,4,5- adecreasein, but not the elimination of, antibacterial ac- trihydroxybenzoatemethylester. E. coli:(1)3,4,5-trihydroxy tivity(with BA valuesranging from 0.19to 0.66). benzaldehyde;(2) 2,3,4-trihydroxyb enzaldehyde;(3) 4-hydroxy- 50 Withregard to the substitutions in the 3 positionsof 2,6-dimethoxybenzaldehyde;(4) 2,5-dihydroxybenz aldehyde;(5) 2,4,6-trihydroxybenzaldehyde;(6) 2-hydroxy-5-metho xybenzalde- thebenzene rings, we foundthat the BA 50 valuesfor 3- hyde;(7) 2,3-dihydroxybenz aldehyde;(8) 2,4-dihydroxyben zalde- hydroxybenzaldehyderanged from 0.20to 0.37,whereas 3- hyde;(9) 2-hydroxybenzald ehyde;(10) 3,5-dihydroxybenz alde hydroxybenzoicacid was inactive.The BA 50 valuesfor 3- hyde. L.monocytogenes :(1)2,4,6-trihydroxyb enzaldehyde;(2) 2- methoxybenzaldehyde( m-anisaldehyde)ranged from 0.091 hydroxy-4-methoxybenzaldehyde;(3) 2,3-dihydroxyben zaldehyde; to .0.67,whereas 3-methoxybenzoic acid was inactive. (4)2,5-dihydroxyben zaldehyde;(5) 3,5-dihydroxybenz aldehyde; For compoundswith substituents in the 4 position,the (6)2-hydroxy-3-metho xybenzaldehyde;(7) 2-hydroxy-5-metho xy- BA50 valuesfor 4-hydroxy-benzaldehyderanged from 0.20 benzaldehyde;(8) 3,4,5-trihydroxyb enzoatemethyl ester; (9) to0.41, and those for 4-methoxybenzaldehyde( p-anisal- 2,3,4-trihydroxybenzaldehyde;(10) 2,4-dihydr oxybenzaldehyde. dehyde)ranged from 0.068to 0.58. Both 4-hydroxy- and S. enterica:(1)2,3,4-trihydroxybe nzaldehyde;(2) 2,4,6-trihydrox- 4-methoxybenzoicacids were inactive. ybenzaldehyde;(3) 3,4,5-trihydroxybe nzoicacid; (4) 2,5-dihydroxybenzaldehyde;(5)4-hydroxy -2,6-dimethoxybenzaldehyde; Disubstituted benzaldehydesand benzoic acids. Di- (6)3,4,5-trihydroxybe nzaldehyde;(7) 2,4-dihydroxybenz aldehyde; substitutedbenzaldehydes and benzoic acids will be de- (8)2,3,4-trihydroxyb enzoicacid; (9) 2,3-dihydroxybenz aldehyde; scribedin the following order: 2,3-, 2,4-, 2,6-, 3,4-, and (10)3-methoxybenzalde hyde. 3,5-disubstitutedderivatives with either OH orOCH 3 groupsfollowed by compounds with both OH andOCH 3 substituents.T able1 showsthat 2,3-dihydroxybenz alde- showedonly borderline activity against the four bacteria. hydewas highlyactive against all four bacteria (with BA 50 Thecorresponding 2-methoxybenzoic acid was inactive. valuesranging from 0.016to 0.11).The corresponding 2,3- Acetylationof the 2-OH groupof benzoic acid forms the dihydroxybenzoicacid was activeonly against C. jejuni inactiveacetylsalicylic acid (aspirin). Surprisingly, meth- (BA50 5 0.31)and was inactiveagainst the other three ylationof the 2-OH groupof 2-hydroxybenzalde hydeto bacteria.Again, the activity level of 2,3-dimethoxybenz al- form 2-methoxybenzaldehyde( o-anisaldehyde)resulted in dehyde(with BA 50 valuesranging from 0.29to 0.58) was J.FoodProt., Vol. 66, No. 10 ANTIBACTERIAL PHENOLICCOMPOUNDS 1817 C 8 4 7 0 1 2 3 3 0 0 0 0 1 0 0 0 0 0 t . . . . . e a n 0 0 0 0 0 7 i 4 n m i 0 6 6 6 6 6 . 0 m 0 3 9 9 8 1 6 0 9 2 2 2 1 . 6 0 0 0 0 . . . 0 0 . 0 0 0 d 0 n a d 0 0 . 3 7 r o 1 f 1 1 0 3 1 a 0 0 0 0 0 c . . . . . e i n 0 0 0 0 0 r i 6 e 5 t m 0 6 6 6 6 6 n . 0 e 0 5 6 1 1 2 3 . 3 4 3 7 1 . S 0 0 0 0 . . . . 0 t 0 0 0 0 s n i a g a s e d 1 4 y 6 1 0 4 0 1 h 1 0 0 0 0 0 e ...... n d 0 0 0 0 0 0 i l a a m z 6 6 6 6 6 6 H 0 n p 6 8 2 0 3 1 6 e r 2 1 2 6 3 4 b . . o 0 0 0 0 f 0 . . . . 0 d ) 0 0 0 0 e t D u S t c i t 4 . s 6 5 b n u a s e - 5 1 2 y m 6 0 0 3 0 4 ( x 0 0 0 0 0 0 ...... o 0 n 5 0 0 0 0 0 0 h i t A e m B 6 6 6 6 6 6 m 0 4 2 2 0 3 2 3 FIGURE 3. Box-in-whiskerplots (26, 43) of the distribution of r 4 5 3 8 4 4 o . . / 0 0 0 0

0 . . . . 0 theactivities of 70 compounds for each of the four pathogens. d 0 0 0 0 n

a Thebox portion of the box-in-whisker plot delineates the median -

y andthe 25th and 75th percentiles of thedistribution (bottom and x o

r topof the box, respectively). 3 representsthe mean of thedistri- d y 4 4 4 bution.The whisker extends to the ‘ ‘upperfence,’ ’ whichis 1.5 h 3 0 0 1 0 1 0 0 0 0 0 0 d ...... theinterquartile range, i.e., 1.5 the distance from the median to e n 0 0 0 0 0 0 t i c

m the75th percentile. Values above the upper fence are identi ed e 6 6 6 6 6 6 l 0 e 9 4 7 6 4 0 6 withnumbers that correspond to those listed in Table 1. s 1 2 3 3 3 1 . . d 0 0 0 0 0 . . . . 0 n 0 0 0 0 a d b i c 7

. lowerthan that for thecorresponding dihydroxy compound. a 3 c

i 2,3-Dimethoxybenzoicacidwas inactive. l l 1

a 7 5 1 5 3 0 Thedata for 2,4-disubstitutedcompounds show that (i) g 0 1 0 0 0 0 ...... f n 0 0 0 0 0 0 i

o the BA50 valuesfor 2,4-dihydroxybenzaldehyderanged m s 6 6 6 6 6 6 e 0 from 0.065to 0.35; (ii) 2,4-dihydyroxybe nzoicacid was i 0 4 2 0 7 0 3 t i 2 1 5 8 7 1 . . . v

0 0 0 activeagainst C. jejuni (BA 5 0.46)but not against the i 50 0 0 . . . 0 t 0 0 0 c

a otherthree bacteria; and (iii) 2,4-dimethoxyben zaldehyde l

a exhibitedactivity against C. jejuni (BA50 5 0.38)but not d i c

i againstthe other three bacteria. These results show that the r e t activitiesof the2,4-disubstituted benzaldehydes are similar c . . ) a 7 e 4 . . b

d tothose of the corresponding 2,3 compounds. Both are 3 5 ) ) y e d e h h i H d H t moreactive than the monosubstituted compounds. e e c p y p d a d n l h y o o a

o Theactivity level of 2,5-dihydroxybenzaldehyde(gen- c e t t h i e s d l i . e e d l l t d d 0 d a y

a tisaldehyde)(with BA valuesranging from 0.017to n . e

m l 50 e l . i t t h g e 7 a t y d s ( s e g z c e e

u 0.063)appears higher than that described above for the2,3- ( u d i t n d j j H d d l l i o e n d e a d a p y c n b a a s z a d and2,4-disubstituted analogs. 2,5-Dihydroxybenz oicacid h a ( y o n y e e e t e x e c H h d e s n i n l i C. jejuni o (gentisicacid) was activeagainst (BA 5 0.20) b e i

p d i d 50 o a l l h y d e y w f t a z z l a t r x h s e o s s a n n and S. enterica (BA 5 0.48)but not against the other two

e 50 o e z u e e s h m h d j d d n t t b b i t i l i d

e bacteria.2,5-Dimethoxybenzaldehydewas alsoact ive c e o c y d y a c a - e b a x x z a f s m 6 y f o o n , - s S

c against C. jejuni (BA 5 0.36) and S. enterica (BA 5 c r r

x 50 50 e i e i E 2 5 B d d l r r o - - b t t r . y y n P i y y y i 0.40)but not against the other two bacteria. 2,5-Dimethox- d 3 u h h c c x x x i i . y o o o r r M 2 1 E h r r r ybenzoicacid was inactiveagainst all four bacteria. T T i M M d d d - - L m D y y y 5 4 5 m m 5 B , , -

0 The2,6-dihydroxybenz aldehydeisomer was notavail- H H H 4 3 5 n 2 5 A 2 n , - , - , -

T 3 4 2 2 2 2 a b c d e ableto us. T able1 showsthat the corresponding 2,6-dihy- 1818 FRIEDMANET AL. J.FoodProt., Vol. 66, No. 10

droxybenzoicacid ( g-resorcylicacid) was activeagainst C. (BA50 5 0.66),and inactive against L.monocytogenes. jejuni (BA50 5 0.40) and S. enterica (BA50 5 0.47) but 2,3,4-Trimethoxybenzaldehydeexhibited a lowlevel of ac- notagainst the other two bacteria. 2,6-Dimethoxybenzal - tivity(with BA 50 valuesranging from 0.53to 0.64). 2,3,4- dehydewas activeagainst all four bacteria (with BA 50 val- Trimethoxybenzoicacid was inactiveagainst all four bac- uesranging from 0.15to 0.66),whereas 2,6-dimethoxyben- teria.Both 2,4,5-trimethoxyben zaldehydeand 2,4,5-trime- zoicacid was inactiveagainst all four bacteria. thoxybenzoicacid were inactiveagainst all four species. 3,4-Dihydroxybenzaldehyde(protocatechualdehyde) 2,4,6-Trihydroxybenzaldehydewas highlyactive was highlyactive against C. jejuni (BA50 5 0.0031) and againstall four bacteria (with BA 50 valuesranging from lessactive against the other three bacteria (with BA 50 val- 0.008to 0.055), whereas 2,4,6-trimethoxyben zaldehyde uesranging from 0.22to 0.33). 3,4-Dihydroxybenz oicacid showeda highlevel of activity against C. jejuni (BA50 5 (protocatechuicacid) was activeagainst C. jejuni (BA50 5 0.011)and low levels of activity against the other three 0.11) and S. enterica (BA50 5 0.50)and inactive against bacteria.The corresponding 2,4,6-trimethoxyben zoicacid theother two bacteria. 3,4-Dimethoxybenzal dehyde(vera- was inactiveagainst all four pathogens. traldehyde)was activeagainst L.monocytogenes (BA50 5 Thedata for the3,4,5 series show that 3,4,5-trihydrox- 0.29) and C. jejuni (BA50 5 0.48)and inactive against the ybenzaldehydewas inactiveagainst L.monocytogenes but othertwo bacteria. 3,4-Dimethoxybenz oicacid was inactive highlyactive against the other three organisms (with BA 50 againstall four bacteria. valuesranging from 0.0026to 0.054). Gallic acid (3,4,5- 3,5-Dihydroxybenaldehydeshowed high levels of ac- trihydroxybenzoicacid) was activeagainst C. jejuni (BA50 tivityagainst all four bacteria (with BA valuesranging 50 5 0.0049) and S. enterica (BA50 5 0.038)but not against from 0.065to 0.19). 3,5-Dimethoxybenza ldehydewas ac- E. coli and L.monocytogenes. Esterication of the carboxyl tiveagainst C. jejuni (BA50 5 0.088) and S. enterica (BA50 groupof gallicacid to form 3,4,5-trihydroxybenzoatemeth- 5 0.65)and inactive against the other two bacteria. 3,5- ylester (methylparaben) resulted in BA 50 valuesof 0.0093 Dihydroxybenzoicacid was inactiveagainst all four bac- for C. jejuni, 0.19 for L.monocytogenes, 0.30 for S. enter- teria,wh ereas3 ,5-dimethoxybenzoicacid was active ica, and 0.38 for E. coli. 3,4,5-Trimethoxybenzaldehyde against C. jejuni (BA50 5 0.28)and inactive against the was inactiveagainst all four bacteria. 3,4,5-Trimethoxyben- otherthree species. zoicacid was activeagainst C. jejuni (BA50 5 0.43) and Thedata for themixed hydroxy-methoxy substitutions inactiveagainst the other three species. showthe following trends in activity. 2-Hydroxy-3-meth- Withrespect to the mixed hydroxy-methoxy trisubsti- oxybenzaldehyde( o-vanillin)showed substantial activity tutedcompounds, the results obtained in this study show againstall four bacteria (with BA 50 valuesranging from that2-hydroxy-4,6-dimeth oxybenzaldehydewas activeonly 0.012to 0.25), whereas 2-hydroxy-3-methoxy benzoicacid against C. jejuni (BA50 5 0.038).3,4-Dihydroxy-5-meth- was activeonly against C. jejuni (BA50 5 0.45).2-Hy- oxybenzaldehydewas activeagainst all four bacteria (with droxy-4-methoxybenzaldehydewas activeagainst all four BA50 valuesranging from 0.0033to 0.51), and 3-hydroxy- bacteria(with BA 50 valuesranging from 0.0025to 0.43), 4,5-dimethoxybenzoicwas activeonly against C. jejuni whereas2-hydroxy-4-methoxy benzoicacid was activeonly (BA 5 0.047).4-Hydroxy-2,6-dimeth oxybenzaldehyde C. jejuni 50 against (BA50 5 0.53).The BA 50 valuesfor the was activeagainst all four pathogens (with BA values highlyactive 2-hydroxy-5-methoxy benzaldehyderanged 50 rangingfrom 0.0078to 0.47), 4-hydroxy-3,5-dimeth oxy- from 0.00066for C. jejuni to 0.070 for E. coli to 0.10 for benzaldehyde(syringaldehyde) was activeonly against C. L.monocytogenes to 0.19 for S.enterica. 2-Hydroxy-5- jejuni (BA 5 0.031) and S. enterica (BA 5 0.58), and methoxybenzoicacid was activeagainst C. jejuni (BA 5 50 50 50 4-hydroxy-3,5-dimethoxybenzoicacid was inactive.5-Hy- 0.057)and inactive against the other three bacteria, as was droxy-3,4-dimethoxybenzaldehydewas activeagainst all alsothe case for 2-hydroxy-6-methoxybenzoicacid (with a fourspecies (with BA valuesranging from 0.37to 0.55). BA valueof 0.18 for C. jejuni).4-Hydroxy-3-methoxy- 50 50 Tofacilitatethe application of thedata to foods,Figure benzaldehyde(vanillin) was activeagainst C. jejuni (BA 50 2showsthe bactericidal activity levels of the 10 com- 5 0.073)and less active against the other three bacteria poundsthat were mostactive against each of thefour path- (with BA valuesranging from 0.53to 0.63), whereas 3- 50 ogens. For C. jejuni, BA valuesranged from 0.0007for hydroxy-4-methoxybenzaldehyde(isovanillin) was slightly 50 2-hydroxy-5-methoxybenzaldehydeto 0.0093 for 3,4,5- activeagainst S. enterica (BA 5 0.61)and inactive 50 trihydroxybenzoatemethyl ester; for E. coli, BA values againstthe other three pathogens. 4-Hydroxy-3-methoxy- 50 rangedfrom 0.042for 3,4,5-trihydroxybenzaldehydeto benzoicacid (vanillic acid) and 3-hydroxy-4-methoxy ben- 0.160for 3,5-dihydroxybenzaldehyde;for L.monocytoge- zoicacid (isovanillic acid) were inactiveagainst all four nes, BA valuesranged from 0.009for 2,4,6-trihydroxy- species. 50 benzaldehydeto 0.350for 2,4-dihydroxybenzaldehyde;and Trisubstituted benzaldehydesand benzoic acids. for S.enterica, BA50 valuesranged from 0.029for 2,3,4- 2,3,4-Trihydroxybenzaldehydewas highlyactive, as evi- trihydroxybenzaldehydeto 0.140 for 3-methoxybenzalde- denced by BA50 valuesranging from 0.0037to 0.20 for the hyde.T ofurtherfacilitate the application of the data to fourbacteria. The corresponding 2,3,4-trihydroxyben zoic foodscontaminated with multiple strains of human patho- acidwas activeagainst C. jejuni (BA50 5 0.046) and S. gens,T able2 liststhe average BA 50 valuesfor the24 com- enterica (BA50 5 0.11),slightly active against E. coli poundsthat were activeagainst all four bacteria. These J.FoodProt., Vol. 66, No. 10 ANTIBACTERIAL PHENOLICCOMPOUNDS 1819

BA50 valuesrange from 0.026for 2,4,6-trihydroxybenzal- monosubstitutedphenolic derivatives of benzaldehyde are dehydeto 0.585 for 2,3,4-trimethoxybenzaldehyde. muchmore active than the parent unsubstituted compounds. Theresults of the present study also revealed the following pH effects. SincepH can affect both the susceptibility orderof bactericidalactivity intensities elicited by thephe- of S. enterica toinactivation (22) andthe ionization of the noliccompounds (with some exceptions): trisubstituted OH OHgroupsof the test compounds (15), additionalexplor- . disubstitutedOH . monosubstitutedOH. For disubsti- atorystudies were carriedout to ascertain to what extent, tutedderivatives, the 2-OH and4-OH groupsenhance the ifany,the pH of themedium would in uence antibacterial antimicrobialactivities of benzaldehyde but not those of activitiesagainst this pathogen. The limited data shown in benzoicacid. Both the 2-OCH 3 andthe 4-OCH 3 substitu- Table2 suggestthat pH has a variableeffect on the anti- tionsalso increased activity levels for benzaldehyde.How- microbialactivities of thesix compounds evaluated at pHs ever,theincrease for themethoxy groups was smallerthan rangingfrom 3.7to 7.0. Gallic acid was anexception. Its thatfor thecorresponding hydroxy groups. Compounds activityincreased about 20-fold after 60 min when the pH withthree methoxy groups were inactive.Compounds with was increasedfrom 3.7to 7.0. Attempts to determine ac- mixedOH andOCH 3 groupsexhibited variable results, i.e., tivitiesat pH 8.4 were unsuccessful.The results were not insome cases the added methoxy groups enhanced activity reproducible,presumably because of the sensitivity of the andin others they had no effect. microorganismsin the control samples to inactivation at the Theesteri cation of gallicacid to 3,4,5-trihydroxyben- higher pH. zoatemethyl ester results in a signicant increase in activ- DISCUSSION ity.This increase is presumably the basis for theextensive studieson theantimicrobial properties of thenumerous syn- Structuraland mechanistic aspects. Thestructure- theticgallic acid esters mentioned below. Antibacterial ac- functionanalysis of thecompounds tested in this study con- tivitiesof gallic acid esters of different chain lengths are tributesto efforts designedto elucidate the effects of struc- relatedto the length of the alkyl group of the ester side turesand action mechanisms of antibacterial activities for chain (16). Theinhibition of oxygen use by methyl esters thisclass of compounds. The requirement of the phenolic ofhydroxycinnamates(alkacins) was foundto be correlated OHgroupfor antimicrobialactivity is strikingly demon- withgrowth inhibition when hydroxycinnamates were test- stratedby the observation (44) thatthe phenolic OH group ed against Pseudomonas uorescens(2). Theseresults sug- ofcarvacrolis essential for activityagainst Bacilluscereus, gestthat bacteriostatic activities of phenolic compounds are sincecymene, a biologicalprecursor of carvacrol lacking basedon cellular energy depletion and not on membrane- theOH group,had a muchless extensive effect on the disruptingeffects. membranepotential associated with bactericidal activity Anexamination of the trends in activities shown in thandid carvacrol. In relevant studies, Ramos-Nino et al. Tables1 and2 andFigures 2 and3 indicatessimilar dis- (31) describequantitative structure-activity relationships for tributionsand clusterings of activities for E.coli,S. enter- severalbenzaldehydes with regard to the activities of these ica, and L.monocytogenes. Incontrast, levels of activity compoundsagainst L.monocytogenes,Salmonella Enteri- against C. jejuni were approximately100 times as high as tidis, and Lactobacillusplantarum. Multiregressionanalysis theobserved levels of activity against the other three spe- ofthedata and arti cial neural network models showed that cies.The plots also show a muchbroader distribution of stericand polar parameters of the benzaldehydes were im- activitiesagainst C. jejuni. Wedonotknow the reasons for portantbut a lipophilicparameter was not.These ndings thesedifferences. The summary of theactivities for the24 suggestthat rather than disrupting cell membranes, the compoundsthat were activeagainst all four species, pre- compoundsacted outside the cell. Additional chemical sentedin T able2, will be helpful in devising antibacterial studiesindicated that the benzaldehydes became covalently formulationswith which to protect food and possibly also attachedto surface SH groupsof the bacterial cells (31). nonfoodsubstrates against multiple pathogens. Ramos-Ninoet al. (30) alsoused mathematical rela- tionshipsto describe the inhibition of a cocktailof 18 Effect ofpH onbactericidal activities of phenolic strains of L.monocytogenes intermsof stericand electronic compounds. Theionization of a phenolicgroup to a phen- 2 1 effectsassociated with substituted benzoic and cinnamic ac- oxideion (ROH ® RO 1 H )isgoverned by its pK a idsand benzaldehydes. It is noteworthythat the mathemat- valuesas well as by the pH ofthemedium. The higher the icalmodel used to predict the activities of the pure com- pH,the more extensive the ionization. Since pK values of poundscould not predict their activities in milk, meat, or phenolicOH groupsare usually .8 (15), theconcentration soups.Activities against Listeria inthese foods were gen- ofphenoxide ions is low at pH values of ,7. Thus, if erallylower than those observed in vitro. Payne et al. (28) phenoxideions rather than the protonated forms arethe alsoevaluated the antimicrobial activities of phenoliccom- activeantibacterial species, expectations are that activities poundsagainst L.monocytogenes in milk. ofthe phenolic compounds should increase with pH. It is Our resultsshow that the aldehyde (CHO) groupof thereforeof inherent interest to ndout whether the anti- benzaldehyde,but not the carboxyl (COOH) groupof ben- bacterialactivity levels of phenoxideions (RO 2) are higher zoicacid, is a structuralfeature that can contribute to an- thanthose of the protonated forms (ROH). If theyare, the timicrobialactivity in the absence of any phenolic OH enhancementof activity when the medium’ s pHis higher groupson the benzene ring. Our dataalso show that the probablyarises from thenegative charge of the phenoxide 1820 FRIEDMANET AL. J.FoodProt., Vol. 66, No. 10 ions.These charges may alter the electrochemical balance G.Cristinzio.1995. Antibacterial polyphenols from olive oil mill inproximity to the microenvironment of the bacteria, fa- waste waters. J.Appl.Bacteriol. 79:393–398. 5.Cho, J. Y.,J.H.Moon,K. Y.Seong,and K. H.Park.1998. Anti- cilitatingcell death. microbialactivity of 4-hydroxybenzoic acid andtrans 4-hydroxycin- Exploratorystudies summarized in T able3 showthat namic acid isolatedand identi ed from rice hull. Biosci.Biotechnol. activityappears to increaseas thepH increasesfrom 3.7to Biochem. 62:2273–2276. 7.0.However ,exceptfor gallicacid, this increase appears 6.Davidson, P .M.1993.Parabens and phenolic compounds, p. 263– notto be signi cant in the narrow pH range studied. For 306. In P.M.Davidsonand A. L.Branen(ed.), Antimicrobials in gallicacid, the BA valuedecreased from 0.20at pH 3.7 foods.Marcel Dekker,New York. 50 7.Domenico, P .,D.C.Straus,D. E.Woods,and B. A.Cunha.1993. to0.035 at pH 7.0 after 30 min (a sixfoldincrease in ac- Salicylatepotentiates amikacin therapyin rodent models of Klebsi- tivity).The corresponding change after 60 min was from ellapneumoniae infection. J.Infect.Dis. 168:766–769. 0.19to 0.0093, a highlysigni cant 20-fold increase in ac- 8.Eklund, T .1985.The effect ofsorbicacid andesters of p-hydroxy- tivity.Expectations were thatthis trend would accelerate at benzoicacid onthe protonmotive force in Escherichiacoli mem- higherpH values. However ,ourefforts tomeasure anti- branevesicles. J.Gen.Microbiol. 131:73–76. microbialactivity at pH 8.4 were unsuccessfulbecause the 9.Eklund, T .1985.Inhibition of microbialgrowth at differentpH levels bybenzoicand propionic acids andesters ofp-hydroxybenzoic acid. highpH killed signi cant amounts of the bacteria in the Int.J. FoodMicrobiol. 2:159–167. controlsample. In related studies, we foundthat certain 10.Finot, P .A.,and M. Merabet.1993. Nutritional and safety aspects antibacterialphenolic compounds such as gallic acid are ofmicrowaves. Historyand critical evaluationof reported studies. themselvesunstable at high pHs (15). Sincemany foods, Int.J. FoodSci. Nutr. 44(Suppl.1):S65– S75. suchas fruit and vegetable juices, vinegar salad dressings, 11.Friedman, M. 1996.Food browning and its prevention. J. Agric. Food Chem. 43:6–29. andsweet and sour sauces, are on the acid side of the pH 12.Friedman, M. 1997.Chemistry, biochemistry, and dietary role of scale,the effect of pHontheantibacterial activities of phe- potatopolyphenols. J.Agric.Food Chem. 45:1523–1540. noliccompounds merits further study (9, 22, 32). 13.Friedman, M., andF .F.Bautista.1995. Inhibition of polyphenol ox- idase bythiols in the absence andpresence ofpotato tissue suspen- CONCLUSIONS sions. J.Agric.Food Chem. 43:69–76. 14.Friedman, M., P.R.Henika,and R. E.Mandrell.2002. 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