1542

Journalof FoodProtection, Vol. 64, No. 10, 2001, Pages 1542– 1548 Copyright ,International Association forFood Protection

EfŽcacy ofChitosan, Carvacrol, anda Hydrogen Peroxide– Based Biocideagainst Foodborne Microorganisms in Suspensionand Adhered toStainless Steel

JAMES KNOWLES AND SIBEL ROLLER *

Schoolof Applied Science, South Bank University, LondonSE1 0AA, UK

MS01-50:Received 6February2001/ Accepted 16April 2001 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/10/1542/1671696/0362-028x-64_10_1542.pdf by guest on 25 September 2021

ABSTRACT

Theability of naturalcompounds to inactivate foodborne organisms adhered to surfaceswas investigated with the ultimate aimof replacing synthetic biocides by more environmentally friendly, natural alternatives. The antimicrobial efŽ cacy of 0.5, 1.0,and 2.0% chitosan and Spor-Klenz RTU (acommercial biocide based on hydrogen peroxide and peroxyacetic acid) and 0.5,1.25, and 2.0 mM carvacrolwas determined at 20 8C against Listeriamonocytogenes, Salmonella enterica serovarTyphi- murium, Staphylococcusaureus, and Saccharomycescerevisiae adheredto stainless steel disks. Treatment with up to 2.0% chitosanreduced the viable cell count in the microbial Ž lmsof the four test organisms by 2.4, 1.8, 2.3, and 0.9 log CFU/ test surface(t.s.), respectively. By contrast, planktonic counts of the same organisms were reduced by 0.8 to 1.7 log CFU/ mlat 2.0%chitosan. Treatment with 2 mM carvacrolreduced the viable counts of adhered listeriae, salmonellae, and yeasts by 2 to3 logCFU/ t.s.but S. aureus countswere reduced by only0.9 log CFU/ t.s.The efŽ cacy of anysingle compound was species speciŽc. In the case of microbialŽ lmsprepared using listeriae and salmonellae, Spor-Klenz RTU wasmost biocidal, followed bycarvacrol and then chitosan. However, dried Ž lmsof S. aureus weremost sensitive to chitosan and relatively resistant to carvacroland Spor-Klenz RTU. Bycontrast, yeast Ž lmswere most sensitive to carvacrol and least sensitive to chitosan. It wasconcluded that carvacrol and chitosan may have potential for use as natural biocides although optimization of conditions wouldbe necessar y.

MicrobialŽ lmson food-processing surfaces are unde- potentiallyimportant nontoxic, biodegradable and renewa- sirableas they increase the risk of cross-contaminationbe- bleresource and has been adopted in several important in- tweenfood products (17–19). Surfacecontamination can be dustrialprocesses, including wastewater puriŽ cation and controlledby the application of a regularand thorough chelationof transitionmetals (27). Chitosanhas antibacter- cleaningregime. Sanitation programs involving both clean- ialand antifungal properties and consequently has been ingand disinfection processes can be cost effective, easy studiedas apotentialnatural antimicrobial agent for foods, tomanage, and can reduce the spread of microbialcontam- cosmeticsand medicines (10,16, 36). Althoughmore active inationprovided they are undertaken rigorously (15, 21). againstspoilage yeasts, chitosan has also been shown to For example,comparative clean-in-place trials have shown inhibitgrowth of several important foodborne bacteria in- thatcorrect detergent concentrations and temperatures were cluding Salmonellaenterica serovarTyphimurium, Staph- crucialin controlling the attachment of Pseudomonasfragi ylococcusaureus, Escherichia coli, and Lactobacillusfruc- tostainless steel (40). However,thereis growing concern tivorans(30, 34, 42, 49). However,thereported minimum aboutthe disposal of waste detergents, sanitizers, and inhibitoryconcentrations for bothbacteria and yeasts vary chemicallysynthesized disinfectants that may contain toxic widelyfrom 0.01to 5.0%, depending on pH, temperature, substancesinto the environment, as well as thepotential for andthe presence of interferingsubstances such as proteins introducingtoxic residues into foods after cleaning (25, 26). and fats (11,31, 33, 35, 42, 44, 45). Thepotential for using Coupledwith increasing demands by consumers for foods chitosanin surface disinfection has not been investigated thatare only minimally processed and rely less heavily on previously. artiŽcial additives for theirshelf life, the environmental Carvacrol(C 10H14O)isa majorcomponent of the es- movementhas led to increased research efforts toidentify sentialoil fractions of oreganoand thyme (4, 22, 46). Car- andevaluate natural antimicrobial compounds as novel vacrolhas generally been regarded as safe statusand is used foodpreservatives and sanitizers (5,24, 28, 32). asa avoringagent in baked goods, candy, beverages, and Chitosan(poly- -1,4-glucosamine)is prepared com- chewing gum (13). Theantimicrobial activity of plant es- merciallyby alkaline deacetylation of chitinobtained from sentialoils in general and carvacrol in particular is well theexoskeletons of marine crustaceans (24). Inthe last 20 documentedin vitroagainst a rangeof foodbornefungi and years,chitosan has attracted much research interest as a bacteriaincluding E.coli,Listeria monocytogenes, S. en- *Authorfor correspondence. Tel: 44 20 7815 7961; Fax: 44 20 7815 terica sv.Typhimurium, S. aureus, and Bacilluscereus (5, 7999;E-mail: [email protected]. 6,12, 20, 39, 43, 46– 48). Likechitosan, carvacrol has not J.FoodProt., Vol. 64, No. 10 ANTIMICROBIAL EFFICACYOF CHITOSAN AND CARVACROL 1543 beeninvestigated previously in the inactivation of micro- Surfacetest procedure. Thesurface test was carried out organismsadhered to surfaces. essentiallyaccording to European Standard WI216028 (1). Brie y, Theobjective of this study was toassess the antimi- stainlesssteel discs (2 cm diameter; Crown Simplimatic, Worces- crobialefŽ cacy of chitosanand carvacrol solutions for sur- ter,UK) werecleaned using detergent, distilled water ,andpro- facecleaning by comparison with an industrial cleaning pane.Fresh microbial cultures were prepared on the day of each agentcontaining hydrogen peroxide and peroxyacetic acid experiment.Aliquots (100 l)of washed cells were dispensed ontodiscs, in duplicate, and dried at 37 8Cfor60 min for the (Spor-KlenzRTU). Initially,two methods (a suspensiontest bacteriaand at 25 8Cfor45 min for the yeast. The biocide solu- anda surfacetest) were usedto investigate the biocidal tionswere dispensed in 100- laliquotsonto the dried microbial propertiesof chitosan in the presence of a rangeof poten- Žlmsand allowed to incubateat 20 8Cfora contacttime of 5min. tiallyinterfering substances including sodium chloride, cal- Thediscs were immersed in 10 ml phosphate buffer (0.25 M ciumand magnesium salts, carbonates, and protein. Sub- KH2PO4,pH7.2)to neutralize the biocides. The disks were shak- sequently,only the surface test was usedto compare the enwithglass beads (5 g) for5 minto removethe organisms from

biocidalproperties of chitosan, carvacrol, and Spor-Klenz thesurface. Viable counts in the suspension removed from the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/10/1542/1671696/0362-028x-64_10_1542.pdf by guest on 25 September 2021 RTU againstdried microbial Ž lmsprepared from three discswere determined by pour plating with tryptone soy agar or foodbornebacteria and one yeast. maltextract agar for bacteria and yeasts, respectively. The discs wererinsed with 50 ml distilled water to remove nonadhering MATERIALS AND METHODS microorganismsand placed on tr yptonesoy agar or malt extract agar,asappropriatefor the organism, to detect residual organisms Materials. Chitosanglutamate (Seacure G210, 35 to 45% remainingon the surfaces. The reduction in viable count due to acidblend, degree of deacetylation 79%, viscosity 84 mPa.S [cPs] thebiocide (log CFU/ testsurface [t.s.]) was calculated by sub- at1%, manufacturer’ s data)was obtained from Pronova Bio- tractingthe number of viable organisms remaining on the discs polymer(Drammen, Norway). Bovine serum albumin and car- aftertreatment with biocide from the number of viableorganisms vacrol(5-isopropyl-2-meth ylphenol,98%, formula weight 150.22) remainingafter treatment with control solutions (i.e., saline, hard werefrom Sigma Chemicals Company (Poole, UK). Stocksolu- water,oralbumin).The surface test was carried out on threesep- tions(0.1 M) ofcarvacrol were prepared weekly in 95% ethanol andstored at room temperature. Spor-Klenz RTU (activeingre- arateoccasions with L.monocytogenes andtwice with each of the dients:hydrogen peroxide 0.8% [wt/ wt]and peroxyacetic acid otherthree target organisms. The reproducibility of the surface 0.06%[wt/ wt],manufacturer’ s data)was supplied by Steris Ltd. testwas checked by comparing results obtained with 10 disks (Camberley,UK). Microbiologicalmedia and diluents were from fromeach of two different batches using S. enterica sv. Typhi- OxoidLtd. (Basingstoke, UK). Allother chemicals were from muriumas the test organism and distilled water instead of a bi- FisherScientiŽ c (Manchester,UK). ocide.

Microorganismsand theircultivation. L.monocytogenes RESULTS NationalCollection of Type Cultures (UK) NCTC11994, S. en- terica sv.Typhimurium NCTC 74, S. aureus NCTC 10788,and Biocidalproperties of chitosanagainst microorgan- Saccharomycescerevisiae NationalCollection of Y eastCultures ismsin suspension. Aseriesof survival curves were ob- (UK)NCYC 87were selected from a groupof target organisms tainedfor thefour target organisms following exposure to recommendedin theEuropean Standards for Suspension and Sur- chitosanat 0.5, 1.0, and 2.0% at pH7.2and 20 8C in saline, faceT estsfor Disinfectants (1–3, 8, 9, 23). Bacteriaand yeasts hardwater ,andalbumin solutions. A selectionof thesere- weremaintained on tr yptonesoy broth and agar and malt extract sultsis illustrated in Figure 1. The results show that the brothand agar ,respectively.The bacteria were grown routinely in actionof chitosan was biphasicwith rapid loss of viability tryptonesoy broth at 37 8Cfor1 dayand the yeast on malt extract occurringin the Ž rst 5minof exposure,followed by much broth at 258Cfor2 days.Microbial suspensions were prepared fromfresh cultures washed twice in 0.9%saline solution and their slowerinactivation rates between 5 and30 min.Of thefour viablenumbers were determined on tryptone soy agar and malt organismstested, S.cerevisiae and L.monocytogenes were extractagar for the bacteria and yeasts, respectively. equallysensitive, showing a reductionin viable numbers of approximately1.7 to 1.8 log CFU/ mlin the presence of Suspension testprocedure. Thesuspension test was de- 2.0%chitosan after 30 min (Fig. 1A and 1D), althoughit signedbroadly according to recommendations in BritishStandard shouldbe noted that the bacterial data were morevariable Institutionprotocols with several modiŽ cations detailed below (2, 3). Chitosanglutamate solutions (0.5, 1.0, and 2.0%) were pre- thanthose obtained with the yeast. A dose-responserela- paredin 0.9% saline with or without interfering substances (2, 3). tionshipwithin the concentration range tested (0.5 to 2.0% S.cerevisiae Theinterfering substances were hard water (2.5 mM MgCl 2, 5 chitosan)was observedwith asthe target or- mM CaCl2,and3.9 mM NaHCO 3)or0.3 g/ literbovine serum ganism(Fig. 1D), butwith the other three organisms such albuminin 0.9% saline. The pH of all solutions was adjusted to arelationshipwas lessevident. S. enterica sv.Typhimurium 7.2prior to the addition of the washed organisms. While mixing was lesssensitive than the yeast or L.monocytogenes to continuously,duplicate samples were taken at 5-,15-, and 30-min theantimicrobial action of chitosan,showing a reductionin intervals,and viable numbers were determined on duplicate agar countsof about 1.2 log CFU/ mlataconcentrationof 2.0% plates.The neutralization step consisted of additionof theorgan- chitosan(Fig. 1B). S. aureus was mostresistant with viable ismsto phosphate buffer (0.25 M KH 2PO4)ordistilled water prior toserial dilution and plating out on appropriateagars as described countsvarying up to a maximumof 0.8log CFU/ mlcom- above.The suspension test was carried out on three separate oc- paredwith the control (Fig. 1C). Notably, the presence of casionswith L.monocytogenes and S. enterica sv.Typhimurium, potentiallyinterfering substances such as sodium chloride, andon two occasions with S. aureus and S.cerevisiae. hardwater (containing calcium and magnesium salts as well 1544 KNOWLESAND ROLLER J.FoodProt., Vol. 64, No. 10

ascarbonate), or protein(albumin) had no substantialin u- enceon the antimicrobial action of chitosan. Biocidalproperties of chitosan against microbial Žlmson stainlesssteel. Thebiocidal action of 0.5,1.0, and 2.0%chitosan after 5 mincontact time at 20 8Cagainstthe fourtest organisms adhered to stainless steel is shown in Table1. It was notedthat survival of some species of or- ganismson the test surfaces was poorwhether or not an- timicrobialagents were used.For example,viable numbers of S. enterica sv.Typhimurium were reducedby as much as3.5 log CFU/ t.s.due to thedrying procedure used in the preparationof the microbial Ž lms,raising concerns about thereproducibility of the test for thistarget organism. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/10/1542/1671696/0362-028x-64_10_1542.pdf by guest on 25 September 2021 Therefore,in a separateexperiment, the survival of S. en- terica sv.Typhimurium with a startingcount of 7.5 log CFU/100 linthe suspension prior to the test procedure was assessedusing two different batches of 10 stainless steeldisks each. The mean viable counts recovered were 3.8log CFU/ t.s.for onebatch of 10 disksand 3.9 log CFU/ t.s.for theother .Statisticalanalysis of the data showed standarderrors of0.025 and 0.034 log CFU/ t.s.,standard deviationsof 0.078 and 0.103, and sample variance of 0.006and 0.011, for thetwo batches, respectively. These resultsindicated good reproducibility of thetest method in spiteof a substantialloss of viable organisms due to the dryingprocedure. Notably, there was noreduction in via- bility of L.monocytogenes asaresultof the test procedure, suggestingthat this organism may be particularly resistant todesiccation. Substantial losses in viability during drying havealso been noted by others (8). TheefŽ ciency of the glass bead method for removing microbialŽ lmsfrom thesteel disks was checkedby over- layingthe spent disks with agar and incubating at the ap- propriatetime and temperature for eachtarget organism. Nomore than one to two colonies per plate were observed followingincubation, showing that the reductions in viable countspresented in T ables1 to3 were notdue to poor recoveryof the organisms from thesteel disks. Whenthe dried microbial Ž lmswere exposedto 0.5to 2.0%chitosan in saline for 5min,the bacteria were inac- tivatedat a levelof approximately 1.0 to 1.8 log CFU/ t.s. (Table1). The yeast was lesssensitive than the bacteria witha maximumlog reduction of 0.9 log CFU/ t.s.This was incontrast to the suspension test result, which had indicatedthat the yeast and L.monocytogenes were more sensitiveto chitosan than S. aureus or S. enterica sv. Ty- phimurium.The log reduction in viable counts afforded by chitosandecreased in the presence of albumin against all thetarget organisms except S. aureus. Similarly,the pres- FIGURE 1. Inactivationof L.monocytogenes (A), S. enterica sv. enceof hard water reduced chitosan efŽ cacy against some Typhimurium(B), S. aureus (C), and S.cerevisiae (D) by 0% ( ), ofthe target organisms although the results were morevari- 0.5% ( ), 1.0% ( ), and 2.0% ( )chitosanin the presence of able(T able1). The surface test results, like the suspension albumin(A andB) orhard water (C andD). Data points are testresults, revealed no distinct dose-response relationship meansof triplicate(A 6 0.71log CFU/ ml andB 6 0.54log CFU/ withchitosan. ml)or duplicate (C 6 0.08log CFU/ ml andD 6 0.24log CFU/ ml) counts. Biocidalproperties of carvacrol against microbial Žlmson stainless steel. Theresults in T able2 showthat carvacrolhad no biocidal effect on dried microbial Ž lms preparedfrom thefour target organisms when present at a J.FoodProt., Vol. 64, No. 10 ANTIMICROBIAL EFFICACYOF CHITOSAN AND CARVACROL 1545

TABLE 1. Reductionin viable count (log CFU/ t.s.)of four organisms after treatment of dried Ž lmson stainless steel with 0.5, 1.0, and2.0% chitosan a

Reductionin count due to Viable count Viable count Reductionin treatment withchitosan c insuspension on steel count due to (logCFU/ t.s.),% chitosan (logCFU/ 100 disks (log dryingb (log Microorganism Solvent l) CFU/t.s.) CFU/t.s.) 0.5 1.0 2.0

L.monocytogenes Saline 6.9 6 0.23 5.7 6 0.87 1.2 6 1.06 0.5 6 0.57 0.4 6 0.59 0.8 6 0.61 Saline 1 albumin 6.9 6 0.23 5.4 6 1.11 1.5 6 1.15 0.5 6 0.26 0.6 6 0.25 1.0 6 0.32 Hard water 6.9 6 0.23 5.0 6 0.78 1.9 6 0.64 1.0 6 0.93 1.5 6 0.80 1.7 6 0.55 S. enterica sv. Saline 7.7 6 0.10 4.2 6 0.20 3.5 6 0.20 1.8 6 0.06 1.7 6 0.03 1.4 6 0.04 Typhimurium Saline 1 albumin 7.7 6 0.10 3.7 6 0.08 4.0 6 0.08 0 6 0.07 0.6 6 0.08 0.6 6 0.02

Hard water 7.7 6 0.10 2.8 6 0.03 4.9 6 0.03 0.3 6 0.03 ,0.1 6 0.08 0 6 0.02 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/10/1542/1671696/0362-028x-64_10_1542.pdf by guest on 25 September 2021 S. aureus Saline 7.0 6 0.09 6.0 6 0.10 1.0 6 0.10 1.6 6 0.12 1.8 6 0.03 1.8 6 0.10 Saline 1 albumin 7.0 6 0.09 5.5 6 0.03 1.5 6 0.03 2.3 6 0.01 1.8 6 0.09 2.2 6 0.10 Hard water 7.0 6 0.09 6.0 6 0.01 1.0 6 0.01 2.0 6 0.10 1.8 6 0.04 1.9 6 0.05 S.cerevisiae Saline 5.3 6 0.02 3.3 6 0.08 2.0 6 0.08 0.9 6 0.10 0.7 6 0.06 0.8 6 0.07 Saline 1 albumin 5.3 6 0.02 2.4 6 0.13 2.9 6 0.13 0.2 6 0.04 0.4 6 0.07 ,0.1 6 0.04 Hard water 5.3 6 0.02 2.9 6 0.10 2.4 6 0.10 0.6 6 0.02 0.7 6 0.08 0.4 6 0.02 a Contacttime was 5 minat 20 8Cinthe presence of saline, hard water ,andprotein at pH 7.2. Results are means of duplicate deter- minations. b Calculatedby subtracting the viable count on steel disks from the viable count in the initial suspension after drying (column 1 minus column 2 5 column 3). c Calculatedby subtractingthe viable count after treatment with chitosan from the viable count on steeldisks after treatment with control solutions(column 3). concentrationof 0.5 mM. However ,someevidence of in- ontheconcentration and on thetarget organism. There was activationwas observedat 1.25mM carvacrolagainst some someevidence of a dose-responserelationship to Spor- oftheorganisms. Notably, at 2 mM,the highest concentra- KlenzRTU, although the effect was notconsistent for all tionof carvacrol tested in this study, all four organisms organisms.Of thefour organisms tested, S. aureus appeared were inactivatedand the log reduction varied from 0.9for mostresistant to the biocidal action of Spor-Klenz RTU. S. aureus to2.6 log CFU/ t.s.for S. enterica sv.Typhimu- Comparisonof biocidal properties of chitosan,car- rium and S.cerevisiae. Theresults show clear evidence of vacrol,and Spor-Klenz against microbial Ž lms. The rel- adose-responserelationship to carvacrol. ativeproportions of dried Ž lmremoved or inactivated on Biocidalproperties of Spor-Klenz RTU againstmi- stainlesssteel surfaces after treatment with chitosan, car- crobialŽ lmson stainlesssteel. Theresults in Table3 show vacrol,and Spor-Klenz RTU solutionswere calculatedfrom thebiocidal action of Spor-Klenz RTU ondried microbial thedata in T ables1 to3 andsummarized in T able4. The Žlmsprepared from thefour target organisms. The log re- calculationssuggest that the efŽ cacy of any single com- ductionvaried from ,0.1to 3.7 log CFU/ t.s.,depending poundwas dependenton the species of organism present

TABLE 2. Reductionin viable count (log CFU/ t.s.)of four organisms after treatment of dried Ž lmson stainless steel with 0.5, 1.25, and2.0 mM carvacrol a

Reductionin count due to Reductionin treatment withcarvacrol c Viable count Viable counton count due to (logCFU/ t.s.),mM carvacrol insuspension steel disks dryingb Microorganism (logCFU/ 100 l) (logCFU/ t.s.) (logCFU/ t.s.) 0.5 1.25 2.0

L.monocytogenes 7.3 6 0.05 7 6 0.06 0.3 6 0.06 0 6 0.12 1.4 6 0.25 2.3 6 0.46 S. enterica sv.Typhimurium 7.6 6 0.06 4.4 6 0.43 3.2 6 0.43 0 6 0.09 0.5 6 0.32 2.6 6 0.09 S. aureus 7.2 6 0.11 6.2 6 0.05 1.0 6 0.05 0 6 0.07 0 6 0.44 0.9 6 0.02 S.cerevisiae 5.3 6 0.02 2.6 6 0.07 2.7 6 0.07 0 6 0.13 0.4 6 0.30 2.6d 6 0 a Contacttime was 5 minat 20 8C.Resultsare means of duplicate determinations. b Calculatedby subtracting the viable count on steel disks from the viable count in the initial suspension after drying (column 1 minus column 2 5 column 3). c Calculatedby subtracting the viable count after treatment with car vacrolfrom the viable count on steel disks after treatment with controlsolutions (column 3). d Entiredried microbial Ž lminactivated. 1546 KNOWLESAND ROLLER J.FoodProt., Vol. 64, No. 10

TABLE 3. Reductionin viable count (log CFU/ t.s.)of four organisms after treatment of dried Ž lmson stainless steel with 0.5, 1.0, and2.0% Spor-Klenz RTU a

Reductionin count due to Reductionin treatment withSporKlenz RTU c Viable countin Viable counton count due to (logCFU/ t.s.),% Spor-KlenzRTU suspension steel disks dryingb Microorganism (logCFU/ 100 l) (logCFU/ t.s.) (logCFU/ t.s.) 0.5 1.0 2.0

L. monocytogenes 7.3 6 0.05 3.7 6 0.31 3.6 6 0.31 1.5 6 0.14 1.4 6 0.06 3.7d 6 0 S. enterica sv.Typhimurium 7.6 6 0.30 2.9 6 0.09 4.4 6 0.09 0.2 6 0.01 2.9d 6 0 2.9d 6 0 S. aureus 7.2 6 0.25 6.0 6 0.04 1.2 6 0.04 ,0.1 6 0.04 0.5 6 0.07 0.8 6 0.06 S.cerevisiae 5.3 6 0.14 3.3 6 0.04 2.0 6 0.04 0.8 6 0.31 1.2 6 0.06 1.2 6 0.07 a Contacttime was 5 minat 20 8C.Resultsare means of duplicate determinations. b Calculatedby subtracting the viable count on steel disks from the viable count in the initial suspension after dr ying(column 1 minus Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/10/1542/1671696/0362-028x-64_10_1542.pdf by guest on 25 September 2021 column 2 5 column 3). c Calculatedby subtracting the viable count after treatment with Spor-Klenz RTU fromthe viable count on steel disks after treatment withcontrol solutions (column 3). d Entiredried microbial Ž lminactivated. inthe dried microbial Ž lm.In the case of L.monocytogenes stainlesssteel. The yeast and listeriae were moresensitive and S. enterica sv.Typhimurium Ž lms,Spor-Klenz RTU thanthe salmonellae or the staphylococci to chitosan in was mostbiocidal, followed by carvacrol and then chitosan. suspension,as expected from previouswork in this labo- However, S. aureus was mostsensitive to chitosan and rel- ratory (31, 34), butthe opposite trend was evidentwhen ativelyresistant to carvacroland Spor-Klenz RTU. By con- theorganisms were attachedto stainlesssteel. It is possible trast, S.cerevisiae was mostsensitive to carvacroland least thatthe higher recovery of viable yeast and staphylococci sensitiveto chitosan. from chitosan-treatedsurfaces was areection of greater resistanceto a combinationof stresses not present in the DISCUSSION suspensiontest. In this context, the ability of theorganisms Numerousmethods have been developed to test the toadapt to reduced water activity and desiccation, e.g., by efŽcacy of disinfectants and some have been standardized synthesizingcompatible solutes such as trehalose (14), may atthe international level (1–3, 9, 23). Althoughit is gen- beas importantas speciŽc sensitivityto the biocidal action erallyrecognized that these standardized methods require ofchitosan. furtherdevelopment to achieve good reproducibility and Overall,the biocidal activity of chitosan was lowerin repeatability,they are nevertheless valuable tools for com- thisstudy than has been reported previously (30, 42, 44, paringthe efŽ cacy of severaldisinfectants (synthetic or nat- 49) probablybecause of the near-neutral pH used in both ural)and may be usedas a screenfor assessingthe biocidal thesuspension and surface tests. The high density of amino propertiesof novelcompounds. Suspension tests, in partic- groupsin the chitosan polymer renders it highly polyca- ular,arerelatively simple to perform andare often used in tionicbelow its pKa of 6.3.This notable feature is thought theearly stages of disinfectant efŽ cacy testing and in re- tobe linked with the extent of binding of chitosan to the search (23). However,inourwork with chitosan, the results anioniccell surface and consequently with its antimicrobial obtainedin suspension did not provide a goodindication potency (30,42, 44, 49). Thus,it has been shown that the ofefŽ cacy against the same target organisms adhered to extentof inactivationof E. coli bychitosancan vary by up to6 logCFU/ mlin the pH range 5 to9, with the greatest TABLE 4. Comparativebiocidal action of chitosan, carvacrol, reductionsin viablenumbers occurring at pH 5and6 (44). andSpor-Klenz RTU after5 mincontact time at 20 8C against Otherfactors such as temperature and growth phase of the driedbacterial and yeast Ž lmson stainless steel testorganism can also play a rolein sensitivity to chitosan (44). Thereduced ionic reactivity of chitosanat neutral pH %driedmicrobial Ž lm removed/inactivated a couldalso explain the lack of interference in antimicrobial activityby hard water and in the presence of protein. Chitosan Carvacrol Spor-Klenz Inthis study, there was littleevidence of a dose-re- b c Microorganism at 2% at 2 mM at 2% sponserelationship between chitosan and the three target L. monocytogenes 22 32 51 bacteria.Higher concentrations of chitosan (2.0%) were S. enterica sv.Typhimurium 18 34 40 sometimesless biocidal than the lower concentrations (0.5 S. aureus 26 13 11 and1.0%). It has been suggested (42) thatat low concen- S.cerevisiae 15 49 23 trations,chitosan may bind to the anionic cell membrane andcause cell death due to leakage of intracellular com- a Correctedfor inactivation due to test procedure (drying). ponents,while at higher concentrations, the additional chi- b Equivalentto 0.03% carvacrol. c Equivalentto aŽnalconcentration of 0.016% hydrogen peroxide tosanmay coat the bacterial surface to preventthis leakage, and0.0012% peroxyacetic acid. sostopping further cell death. J.FoodProt., Vol. 64, No. 10 ANTIMICROBIAL EFFICACYOF CHITOSAN AND CARVACROL 1547

Theminimum inhibitory concentration values for carvacrol REFERENCES against L.monocytogenes,S. enterica sv.Typhimurium, 1.Anonymous. 1995. WI 216028:chemical disinfectantsand antisep- and S. aureus havebeen reported previously as 1.9, 0.9, tics—quantitative surface test forthe evaluation of bactericidal and/ and3.0 mM when tested in brain heart infusion broth at orfungicidal activity of chemical disinfectantsused in food, indus- 308C (46). Althoughthe experimental conditions were very trial,domestic and institutional areas. EuropeanCommittee forStan- different,our results conŽ rm theobservation that S. aureus dardization(CEN), Brussels, Belgium. was themost resistant and S. enterica sv.Typhimurium the 2.Anonymous. 1997. BS EN1276:chemical disinfectantsand antisep- mostsensitive of thethree bacteria tested to theantimicro- tics—quantitative suspension test forthe evaluation of bactericidal activityof chemical disinfectantsand antiseptics used in food, in- bialaction of carvacrol. Detailed studies on the mode of dustrial,domestic, and institutional areas. BritishStandards Institute, action on B. cereus havedemonstrated that the hydrophobic London, UK. carvacrolmolecule interacts with the phospholipid bilayer 3.Anonymous. 1998. BS EN1650:chemical disinfectantsand antisep- ofthecell membrane leading to increased permeability, loss tics—quantitative suspension test forthe evaluation of fungicidal activityof chemical disinfectantsand antiseptics used in food, in- ofcellularconstituents, decrease in membranepotential and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/10/1542/1671696/0362-028x-64_10_1542.pdf by guest on 25 September 2021 dustrial,domestic, and institutional areas. BritishStandards Institute, pHgradient, and ultimately cell death (37,46– 48). Carva- London, UK. crolmigrates more easily into bacterial membranes at high- 4.Arrebola, M. L.,M.C.Navarro,J. Jimenez, andF .A.Ocana. 1994. er temperaturesdue to increased membrane  uidity (48). Yieldand composition of the essential oilof Thymusserpylloides Unlikechitosan, carvacrol displayed a cleardose-response subsp. serpylloides. Phytochemistry36:67– 72. relationship.Studies using B. cereus insuspension have 5.Aureli, P .,A.Constantini,and S. Zolea.1992. Antimicrobial activity ofsome plantessential oilsagainst Listeriamonocytogenes. J. Food shownthat a 2-logCFU/ mlreduction in countsafter 5 min Prot.55:344 –348. couldbe doubledby extendingthe exposure time to 25 min 6.Beuchat, L. R.1994.Antimicrobial properties of apices andtheir (48). Itis conceivable that further improvements in the bi- essential oils,p. 167– 179. In V.M.Dillonand R. G.Board(ed.), ocidalefŽ cacy of carvacrolon adheredorganisms could be Natural antimicrobialsystems andfood preservation. CAB Interna- madeby optimizing conditions of use. tional,Wallingford, UK. 7.Beuchat, L. R.2000.Use ofsanitizers inraw fruitand vegetable Spor-KlenzRTU isa liquidcommercial product for- processing,p. 63–78. In S.M.Alzamora, M.S.Tapia,and A. Lopez- mulatedfor thedisinfection of hard surfaces. It is a blend Malo(ed.), Minimally processed fruits and vegetables. Aspen Pub- ofhydrogen peroxide, peracetic acid, and acetic acid. All lishers,Inc., Gaithersburg, Md. threeingredients have antimicrobial properties, although 8.BloomŽ eld, S. F.,M.Arthur,B.vanKlingeren, J. T.Holah,and R. aceticacid is unlikely to be effective at neutral pH when Elton.1994. An evaluation of the repeatability and reproducibility ofa surface test forthe activity of disinfectants.J. Appl.Bacteriol. morethan 99% of it is in the dissociated state, which is 76:86–94. inactiveagainst microorganisms (7,29, 38, 41). Our results 9.BloomŽ eld, S. F.,andE. Looney.1992. Evaluation of the repeat- showthat Spor-Klenz RTU was veryeffective in eliminat- abilityand reproducibility of European suspension test methodsfor ing L.monocytogenes and S. enterica sv.Typhimurium antimicrobialactivity of disinfectants and antiseptics. J Appl.Bac- Žlms,particularly when used at the recommended level of teriol.73:87– 93. 10.Chang, D. S.,H.R.Cho,H. Y.Goo,and W .K.Choe.1989. A 2%.However ,Spor-KlenzRTU was lesseffective against developmentof foodpreservation with the waste ofcrab processing. S. aureus and S.cerevisiae. Yeastsare able to convert the Bull.Korean Fish Soc. 22:70– 78. peroxideradical into water by constitutive response mech- 11.Chen, C.-S., W .-Y.Liau,and G.-J. Tsai. 1998. Antibacterial effects anismsinvolving reduced glutathione and catalase, and it of N-sulfonatedand N-sulfobenzoylchitosan and applications to oys- isonlyonce these systems become overloaded that peroxide ter preservation.J. FoodProt. 61:1124 –1128. 12.Davidson, P .M.,andA. S.Naidu.2000. Phytophenols, p. 265– 294. radicalscan cause damage and death (25). Itis possiblethat In A.S.Naidu(ed.), Natural foodantimicrobial systems. CRCPress, thesemechanisms protected the yeast cells from inactiva- Boca Raton,Fla. tionat the concentrations of hydrogen peroxide used in this 13.Fenaroli, G. 1995.Fenaroli’ s handbookof  avoringredients, 3rd ed. study. CRCPress, Boca Raton,Fla. Inconclusion, we haveshown that both chitosan and 14.Francois, J., and J. L.Parrou.2000. Reserve carbohydratesmetab- olismin the yeast Saccharomycescerevisiae. FEMSMicrobiol. Rev. carvacrolhave biocidal properties against microbial Ž lms 25:125–145. preparedfrom threebacteria and a yeastof importance in 15.Gibson, H., J.H.Taylor,K. E.Hall,and J. T.Holah.1999. Effec- thefood industry. In some cases, the efŽ cacy of carvacrol tivenessof cleaning techniques used in the food industry in terms was equivalentor betterthan that of acommoncommercial oftheremoval of bacterial bioŽlms. J.Appl.Microbiol. 85:985– 990. disinfectantbased on hydrogen peroxide and peroxyacetic 16.Goosen, M. F.A.(ed.).1997. Applications of chitin and chitosan. TechnomicPublishing Co., Inc., Lancaster ,Pa. acid.Therefore, both chitosan and carvacrol have potential 17.Holah, J. T.,C.Higgs,S. Robinson,D. Worthington,and H. Spen- for developmentinto novel cleaning agents for food-pro- celey. 1990.A conductance-basedsurface disinfectiontest forfood cessingsurfaces, although further work is necessaryto op- hygiene.Letters Appl.Microbiol. 11:255– 259. timizeconditions of use and applications. 18.Hood, S. K.,andE. A.Zottola.1997. Adherence tostainless steel byfoodbornemicroorganisms during growth in model food systems. ACKNOWLEDGMENTS Int.J. FoodMicrobiol. 37:145– 153. 19.Jones, M. 1994.BioŽ lms andthe food industry, p. 113– 116. In J. We thankDr. Douglas Murray for critical readingof themanuscript. Wimpenny,W .Nichols,D. Stickler,andH. Lappin-Scott(ed.), Bac- Thefollowing organizations are gratefullyacknowledged for funding this terial bioŽlms andtheir control in medicine andindustry. Bioline, work:European Commission (Contract FAIR CT96-1066), Danisco (Aplin CardiffUniversity, UK. &Barrett Ltd.UK), CPC(UK) Ltd.,DSM Specialties (Gist-brocades) 20.Knobloch, K., H. Weigand,N. Weis,H. M.Schwarm, andH. Vi- Netherlands,and Norsk Hydro Norway. genschow.1986. Acton of terpenoids on energymetabolism, p. 429– 1548 KNOWLESAND ROLLER J.FoodProt., Vol. 64, No. 10

445. In E.J.Brunke(ed.), Progress in essential oilresearch. W.de tosanin mayonnaise and mayonnaise-based shrimp salads. J.Food Gruyter,Berlin, Germany. Prot.63:202– 209. 21.Krysinski, E. P.,L.J.Brown,and T .J.Marchisello.1992. Effect of 35.Seo, H., K. Mitsuhashi,and H. Tanibe.1992. Antibacterial and an- cleaners andsanitizers on Listeriamonocytogenes attached toprod- tifungalŽ berblended by chitosan, p. 34 –40. In C.J.Brine,P .A. uctcontact surfaces. J.FoodProt. 55:246– 251. Sandford,and J. P.Zikakis(ed.), Advances in chitin and chitosan. 22.Lagouri, V .,G.Blekas,M. Tsimidou,S. Kokkini,and D. Boskou. ElsevierApplied Science, New York. 1993.Composition and antioxidant activity of essential oilsfrom 36.Shahidi, F .,J.K.M.Arachchi,and Y .Jeon.1999. Food applications oreganoplants grown wild in Greece. Z.Lebensm.Untersuch. ofchitin and chitosans. Trends Food Sci. Technol. 10:37– 51. Forsch.197:20 –23. 37.Sikkema, J., J. A.M.deBont, and B. Poolman.1994. Interactions 23.Langsrud, S., and,G. Sundheim.1998. Factors in uencing a sus- ofcyclic hydrocarbonswith biological membranes. J.Biol.Chem. pensiontest methodfor antimicrobial activity of disinfectants. J. 11:8022–8028. 38.Siragusa, G. R.,and J. S.Dickson.1992. Inhibition of Listeriamon- Appl.Microbiol. 85:1006– 1012. ocytogenes onbeef tissueby application of organic acids immobi- 24.Li, Q., E. T.Dunn,E. W.Grandmaison,and M. F.A.Goosen.1997. lisedin a calcium alginategel. J. FoodSci. 57:293– 296. Applicationsand properties of chitosan,p. 329. In M.F.A.Goosen 39.Sivropoulou, A., E. Papanikolaou,C. Nikolaou,S. Kokkini,T .Lan- (ed.),Applications of chitinand chitosan. T echnomicPublishing Co.,

aras, andM. Arsenakis.1996. Antimicrobial and cytotoxic activities Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/10/1542/1671696/0362-028x-64_10_1542.pdf by guest on 25 September 2021 Inc.,Lancaster ,Pa. oforiganum essential oils.J Agri.Food Chem. 44:1202– 1205. 25.Moradas-Ferreira, P .,andV .Costa.2000. Adaptive response of the 40.Stone, L. S.,andE. A.Zottola.1985. Effect ofcleaning and sani- yeast Saccharomycescerevisiae toreactive oxygenspecies: defens- tizingon the attachment of Pseudomonasfragi tostainless steel. J. es, damage anddeath. Redox Rep. 5:277– 285. FoodSci. 50:957– 960. 26.Mullerat, J., B.W.Sheldon,and N. A.Klapes. 1995.Inactivation of 41.Stratford, M. 1999.Traditional preservatives— organic acids, p. Salmonella species andother foodborne pathogens with Salmide, a 1729–1735. In R.K.Robinson,C. A.Batt,and P .D.Patel (ed.), sodiumchlorite-based oxyhalogen disinfectant. J. FoodProt. 58: Encyclopediaof food microbiology. Academic Press, SanDiego. 535–540. 42.Sudarshan, N. R.,D. G.Hoover,and D. Knorr.1992. Antibacterial 27.Muzzarelli, R. A.A.1997.Chitin. Permagon Press, Oxford,UK. actionof chitosan. Food Biotechnol. 6:257– 272. 28.Naidu, A. S.2000.Natural foodantimicrobial systems. CRCPress, 43.Thompson, D. P.1990.In uence ofpHon the fungitoxic activity of Boca Raton,Fla. naturallyoccurring compounds. J. FoodProt. 53:412– 415. 29.Oh, D., andD. L.Marshall.1995. Monolaurin and acetic acid in- 44.Tsai, G. J.,and W .-H.Su. 1999. Antibacterial activity of shrimp activationof Listeriamonocytogenes attached tostainless steel. J. chitosanagainst Escherichiacoli. J.FoodProt. 62:239 –243. FoodProt. 59:249– 252. 45.Tsai, G. J.,Z.-Y.Wu,and W .-H.Su. 2000. Antibacterial activity of 30.Papineau, A. M.,D.G.Hoover,D.Knorr,and D. F.Farkas.1991. achitooligosaccharidemixture prepared by cellulase digestionof Antimicrobialeffect ofwater-soluble chitosans with high hydrostatic shrimpchitosan and its application to milk preservation. J. Food pressure.Food Biotechnol. 5:45– 57. Prot.63:747– 752. 46.Ultee, A.2000.Bactericidal actionof carvacrol towardsthe food 31.Rhoades, J., andS. Roller.2000. Antimicrobial actions of degraded pathogen Bacilluscereus. Ph.D.Thesis. Wageningen University, andnative chitosan against spoilage organisms in laboratory media Netherlands.ISBN 90-5808-219-9. andfoods. Appl. Environ. Microbiol. 66:80 –86. 47.Ultee, A.,L.G.M.Gorris,and E. JSmid.1998. Bactericidal activity 32.Roller ,S.1995.The quest for natural antimicrobials as novelmeans ofcarvacrol towardsthe foodborne pathogen Bacilluscereus. J. offood preservation: status report on a Europeanresearch project. Appl.Microbiol. 85:211– 218. Int.Biodeterioration Biodegrad. 36:333– 345. 48.Ultee, A.,E. P.W.Kets, andE. J.Smid.1999. Mechanisms ofaction 33.Roller ,S.,andN. Covill.1999. The antifungal properties of chitosan ofcarvacrol onthe foodborne pathogen Bacilluscereus. Appl. En- inlaboratory media andapple juice. Int. J. FoodMicrobiol. 47:67– viron.Microbiol. 65:4606– 4610. 77. 49.W ang,G. 1992.Inhibition and inactivation of Ž vespecies offood- 34.Roller ,S.,andN. Covill.2000. The antimicrobial properties of chi- bornepathogens by chitosan. J. FoodProt. 55:916– 919.