Vol. 70, Nr. 9, 2005—JOURNAL OF FOOD SCIENCE M393Published on Web 11/15/2005

© 2005 Institute of Food TechnologistsFurther reproduction without permission is prohibited

M: Fo

od M

icrob

iolog

y & Sa

fety

Effectiveness of Some Natural AntimicrobialCompounds in Controlling Pathogen or SpoilageBacteria in Lightly Fermented Chinese CabbageYYYYYASUHIRASUHIRASUHIRASUHIRASUHIROOOOO I I I I INANANANANATTTTTSUSUSUSUSU, M. L. B, M. L. B, M. L. B, M. L. B, M. L. BARIARIARIARIARI, S, S, S, S, SUSUMUUSUMUUSUMUUSUMUUSUMU K K K K KAAAAAWWWWWASAKIASAKIASAKIASAKIASAKI, , , , , ANDANDANDANDAND S S S S SHINICHIHINICHIHINICHIHINICHIHINICHI K K K K KAAAAAWWWWWAMOAMOAMOAMOAMOTTTTTOOOOO

ABSTRAABSTRAABSTRAABSTRAABSTRACTCTCTCTCT: : : : : This study was designed to evThis study was designed to evThis study was designed to evThis study was designed to evThis study was designed to evaluate the bacteraluate the bacteraluate the bacteraluate the bacteraluate the bactericidal or bactericidal or bactericidal or bactericidal or bactericidal or bacteriostatic effect of chitosan, an allyliostatic effect of chitosan, an allyliostatic effect of chitosan, an allyliostatic effect of chitosan, an allyliostatic effect of chitosan, an allylisothiocyanate (AITisothiocyanate (AITisothiocyanate (AITisothiocyanate (AITisothiocyanate (AIT) pr) pr) pr) pr) product, and nisin for the aroduct, and nisin for the aroduct, and nisin for the aroduct, and nisin for the aroduct, and nisin for the artificially inoculated pathogenic bactertificially inoculated pathogenic bactertificially inoculated pathogenic bactertificially inoculated pathogenic bactertificially inoculated pathogenic bacteria (ia (ia (ia (ia (EEEEEscherichia colischerichia colischerichia colischerichia colischerichia coli, , , , , Salmo-Salmo-Salmo-Salmo-Salmo-nellanellanellanellanella Enteritidis, Enteritidis, Enteritidis, Enteritidis, Enteritidis, Staphylococcus aureusStaphylococcus aureusStaphylococcus aureusStaphylococcus aureusStaphylococcus aureus, and , and , and , and , and Listeria monocytogenesListeria monocytogenesListeria monocytogenesListeria monocytogenesListeria monocytogenes) or natural microflora of fermented Chinese) or natural microflora of fermented Chinese) or natural microflora of fermented Chinese) or natural microflora of fermented Chinese) or natural microflora of fermented Chinesecabbage. Addition of 0.1% chitosan decreased the population of pathogens from 0.7 to 1.7 log colony-forming unitscabbage. Addition of 0.1% chitosan decreased the population of pathogens from 0.7 to 1.7 log colony-forming unitscabbage. Addition of 0.1% chitosan decreased the population of pathogens from 0.7 to 1.7 log colony-forming unitscabbage. Addition of 0.1% chitosan decreased the population of pathogens from 0.7 to 1.7 log colony-forming unitscabbage. Addition of 0.1% chitosan decreased the population of pathogens from 0.7 to 1.7 log colony-forming units(CFU)/g after 4 d of storage at 10 °C. The bactericidal activity of chitosan was found to be stronger than that of nisin(CFU)/g after 4 d of storage at 10 °C. The bactericidal activity of chitosan was found to be stronger than that of nisin(CFU)/g after 4 d of storage at 10 °C. The bactericidal activity of chitosan was found to be stronger than that of nisin(CFU)/g after 4 d of storage at 10 °C. The bactericidal activity of chitosan was found to be stronger than that of nisin(CFU)/g after 4 d of storage at 10 °C. The bactericidal activity of chitosan was found to be stronger than that of nisin(0.05 mg/g). Addition of 0.2% of the AIT product (containing AIT and hop extract) exhibited a bacteriostatic effect.(0.05 mg/g). Addition of 0.2% of the AIT product (containing AIT and hop extract) exhibited a bacteriostatic effect.(0.05 mg/g). Addition of 0.2% of the AIT product (containing AIT and hop extract) exhibited a bacteriostatic effect.(0.05 mg/g). Addition of 0.2% of the AIT product (containing AIT and hop extract) exhibited a bacteriostatic effect.(0.05 mg/g). Addition of 0.2% of the AIT product (containing AIT and hop extract) exhibited a bacteriostatic effect.HHHHHooooowwwwwevevevevevererererer, a combination of AIT pr, a combination of AIT pr, a combination of AIT pr, a combination of AIT pr, a combination of AIT product and chitosan enhanced bacteroduct and chitosan enhanced bacteroduct and chitosan enhanced bacteroduct and chitosan enhanced bacteroduct and chitosan enhanced bactericidal efficacy against icidal efficacy against icidal efficacy against icidal efficacy against icidal efficacy against L.L.L.L.L. monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes. . . . . TheTheTheTheTheaddition of chitosan or AIT product was observed to suppress the populations of mesophilic and coliform bacteriaaddition of chitosan or AIT product was observed to suppress the populations of mesophilic and coliform bacteriaaddition of chitosan or AIT product was observed to suppress the populations of mesophilic and coliform bacteriaaddition of chitosan or AIT product was observed to suppress the populations of mesophilic and coliform bacteriaaddition of chitosan or AIT product was observed to suppress the populations of mesophilic and coliform bacteriadurdurdurdurduring storing storing storing storing storage at 10 °C for 4 d. Mage at 10 °C for 4 d. Mage at 10 °C for 4 d. Mage at 10 °C for 4 d. Mage at 10 °C for 4 d. Morororororeoeoeoeoeovvvvvererererer, the use of chitosan or the AIT pr, the use of chitosan or the AIT pr, the use of chitosan or the AIT pr, the use of chitosan or the AIT pr, the use of chitosan or the AIT product did not change the sensoroduct did not change the sensoroduct did not change the sensoroduct did not change the sensoroduct did not change the sensory quality ofy quality ofy quality ofy quality ofy quality ofthe lightly fermented vegetable. Therefore, these results suggest that chitosan or the AIT product could be useful tothe lightly fermented vegetable. Therefore, these results suggest that chitosan or the AIT product could be useful tothe lightly fermented vegetable. Therefore, these results suggest that chitosan or the AIT product could be useful tothe lightly fermented vegetable. Therefore, these results suggest that chitosan or the AIT product could be useful tothe lightly fermented vegetable. Therefore, these results suggest that chitosan or the AIT product could be useful toimprove the microbial safety and quality of lightly fermented vegetable.improve the microbial safety and quality of lightly fermented vegetable.improve the microbial safety and quality of lightly fermented vegetable.improve the microbial safety and quality of lightly fermented vegetable.improve the microbial safety and quality of lightly fermented vegetable.

Keywords: lightly fermented cabbage, pathogens, chitosan, allyl isothiocyanate, beta-acidKeywords: lightly fermented cabbage, pathogens, chitosan, allyl isothiocyanate, beta-acidKeywords: lightly fermented cabbage, pathogens, chitosan, allyl isothiocyanate, beta-acidKeywords: lightly fermented cabbage, pathogens, chitosan, allyl isothiocyanate, beta-acidKeywords: lightly fermented cabbage, pathogens, chitosan, allyl isothiocyanate, beta-acid

Introduction

Fermented vegetables are an integral part of people’s diet inmany countries (Steinkraus 2000). Normally, leafy vegetables

harbor 2 to 7 log10 colony-forming units (CFU)/g of mesophilic bac-teria including coliform or spoilage bacteria such as Pseudomonas,as their normal microflora (Daeschel and others 1987; Nguyen-Theand Carlin 1994). Lactic acid bacteria (LAB) on the leaves play animportant role in fermentation. In addition, vegetables can act asa vector in transporting pathogenic bacteria from the farm (Beuchat1996; NACMCF 1999). Chinese cabbage is commonly used for theproduction of lightly fermented products, a very popular foodstuffin Japan. These products were traditionally produced in the home.Storing vegetables with salt (3% to 5%) and other ingredients underpressure produces the fermented products. It might be assumedthat growth of pathogenic or spoilage bacteria would be sup-pressed by high salt concentration and/or development of inhib-itory chemical compounds (such as organic acid and bacteriocin)produced by the coexisting LAB during fermentation (Adams andNicolaides 1997; Lee 1997). Commercially prepared fermentedcabbage has been gaining popularity with consumers because of itsconvenience, and in most cases, these foods are consumed rawwithin 2 to 4 d of preparation. Recently, several foodborne outbreakswith lightly fermented (salted) vegetables have been reported inJapan (Ozaki and others 2003; Inatsu and others 2004).

The application of natural antimicrobial compounds for increas-ing food quality or hygiene has been increasing based on the con-sumer’s preference. For example, bacteriocins, small peptide com-pounds effective against Gram-positive bacteria, or bacteriocin-producing LAB have been applied for bacterial control in many

foods, including some vegetable products (Choi and Park 2000;O’Sullivan and Hill 2002). Chitosan, a deacetylated form of chitin,consists of polymeric 1, 4-linked 2-amino-2-deoxy-�-D-glucose andhas been shown to have antimicrobial activity against a range offoodborne bacterial pathogens, fungi, and yeast (Helander andothers 2001; Devlieghere and others 2004). Allyl isothiocyanate(AIT), one of the major compounds responsible for pungent flavorof black mustard (Brassica nigra) and Japanese horseradish (Eu-trema wasabi Maxim), exhibits antimicrobial activity against somepathogenic bacteria, yeasts, or molds (Isshiki and others 1992;Weissinger and others 2001). The alpha or beta acid containing res-in of hop (Humulus lupulus L.) inhibits the growth of some fungiand Gram-positive bacteria, except for some beer spoilage bacteria(Simpson and Smith 1992; Sakamoto and Konings 2003). Therefore,in this study we evaluated the efficacy of these natural antimicro-bial compounds for controlling the population of pathogenic orspoilage microorganisms in lightly fermented cabbage.

Materials and Methods

TTTTTest strest strest strest strest strainsainsainsainsainsThe strains studied and their sources were as follows: Enterohe-

morrhagic Escherichia coli O157:H7 strains CR-3, MN-28, and MY-29 were isolated from bovine feces. Salmonella Enteritidis strainsSE-1, SE-3, and SE-4 were from chicken feces. SE-2 from bovinefeces were provided by the Laboratory of Zoonosis, Natl. Inst. ofAnimal Health, Tsukuba, Japan. Staphylococcus aureus strains IFO13276 (human lesion), JCM 2413 (clinical isolate), JCM2874(wound), and JCM2151 (human lesion) were purchased from theInst. for Fermentation (Osaka, Japan) and the Japan Collection ofMicroorganism Riken (Saitama, Japan). Strains of Listeria monocy-togenes were ATCC 49594 (derived from L. monocytogenes strainScott A), JCM7672 (salami sausage), and JCM7676 (roast beef). Tominimize growth on enumeration media by microorganisms natu-

JFS M: Food Microbiology and Safety

MS 20050324 Submitted 5/28/05, Revised 7/25/05, Accepted 8/5/05. The au-thors are with Natl. Food Research Inst., Food Hygiene Team, Kannondai-2-1-12, Tsukuba 305-8642, Japan. Direct inquiries to author Inatsu(E-mail: inatu@nfri.affrc.go.jp).

M394 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 9, 2005 URLs and E-mail addresses are active links at www.ift.org

M: Food Microbiology & Safety

Antimicrobial compounds in controlling microorganisms . . .

rally present on cabbage leaves, rifampicin-resistant mutants werespontaneously obtained from these strains and used in this study.All test strains were adapted to grow in tryptic soy broth (TSB, pH7.3; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented withrifampicin (50 �g/mL). The mutant strains were compared withparent strains to determine that they had no phenotypic differ-ences (Inatsu and others 2003).

Preparation of inoculaPreparation of inoculaPreparation of inoculaPreparation of inoculaPreparation of inoculaEach strain of E. coli O157:H7, S. Enteritidis, S. aureus, and L.

monocytogenes was cultured at 37 °C in brain heart infusion (BHI)(Nissui Pharmaceutical Co.) medium (100 mL) containing 0.5%yeast extract supplemented with 50 �g/mL rifampicin (BHI/Rif ).Cultures were transferred to BHI/Rif by loop at 3 successive 24-hintervals immediately before they were used as inocula. A “cocktailmethod” was used for inoculation. Cells of each strain were collect-ed by centrifugation (3000 × g, 10 min, 20 °C) and resuspended in10 mL of sterile phosphate buffered saline (PBS, pH 7.2). Equalvolumes of cell suspensions (about 5.0 × 108 CFU/mL) of 3 or 4strains of each pathogen were combined to give approximatelyequal populations of each strain. The inoculum (50 mL) with finalconcentration of approximately 8.0 log CFU/mL was maintained at22 °C ± 1 °C and used within 30 min of preparation.

Preparation of lightly fermented cabbagePreparation of lightly fermented cabbagePreparation of lightly fermented cabbagePreparation of lightly fermented cabbagePreparation of lightly fermented cabbageChinese cabbages were purchased from local supermarkets and

stored at 4 °C for a maximum of 2 d before being used in the exper-iments. For laboratory-scale preparation of lightly fermented Chi-nese cabbage, 1 whole Chinese cabbage (2.0 kg) was cut into smallpieces (3 cm × 3 cm), which were washed twice with tap water for5 min. The leaf pieces were soaked with salt (30 g/kg) and 7% sodi-um chloride water (300 mL/kg) for 6 h at 4 °C under pressure(20 g/cm2).

Preparation of antimicrobialsPreparation of antimicrobialsPreparation of antimicrobialsPreparation of antimicrobialsPreparation of antimicrobialsThree antimicrobial chemicals were used for the experiment: (1)

Chitosan 10 (Wako Pure Chemical, Osaka, Japan) was dissolved indistilled water to give 5% (w/v) by adjusting the pH to 5.0 with HCl.(2) Wasaouro EXT® (designated as “AIT-Hop”) supplied by Mitsub-ishi-Kagaku Foods Co., Tokyo, Japan, was an emulsion liquid con-taining AIT and hop extract. (3) Commercial nisin (Sigma ChemicalCo., St. Louis, Md., U.S.A.) was dissolved in distilled water with HClto give 0.25 mg/mL of the product (pH 5.0). Chitosan, AIT-Hop, ornisin was added to each cup containing salted Chinese cabbage togive the final concentration of 0.1%, 0.2%, or 0.05 mg/mL, respec-tively. Five hundred microliters of each pathogen (about 5.0 × 105

to 106 CFU/mL) were inoculated into each cup containing saltedChinese cabbage, mixed well using a sterile glass rod, and stored at10 °C for 4 d under pressure (20 g/cm2). Sterile distilled water wasused as control.

Microbiological analysisMicrobiological analysisMicrobiological analysisMicrobiological analysisMicrobiological analysisTwenty-five grams of cabbage leaves was placed in a stomacher

bag, and 225 mL of PBS was added and pummeled for 90 s. Serialdecimal dilutions were prepared with PBS (pH 7.2), and the dilutedand undiluted samples were surface plated in quadruplicate onboth selective and nonselective medium supplemented with 50 �g/mL rifampicin. Tryptic soy agar (TSA) (Nissui Pharmaceutical Co.)supplemented with 50 �g/mL rifampicin was used as nonselectivemedium for viable cell determination of all the pathogens tested.In addition, E. coli O157:H7 suspensions were surface plated ontosorbitol MacConkey agar (CT-SMAC) (Nissui Pharmaceutical Co.)(CT-selective supplement, Oxoid, Hampshire, U.K.) and CT-SMAC

containing 50 �g/mL rifampicin. Samples containing Salmonellawere surface plated onto DHL agar (Nissui Pharmaceutical Co.)supplemented with 50 �g/mL rifampicin. Suspensions of S. aureuswere surface plated onto Mannitol salt agar (Eiken Chemical Co.,Tokyo, Japan) supplemented with 50 �g/mL rifampicin. Dilutedand undiluted suspensions of L. monocytogenes were surface platedonto modified oxford medium (Oxoid) supplemented with50 �g/mL rifampicin.

Standard method agar (Nissui Pharmaceutical Co.) and desox-ycholate agar (Eiken) were used for enumeration of aerobic micro-flora and coliforms, respectively, in the uninoculated samples. Lac-tic acid bacteria were enumerated on plate count agar containingbromocresol purple (Nissui Pharmaceutical Co.). Enumerationmedia were incubated at 37 °C for 24 to 28 h before presumptivecolonies were counted. In each experiment, at least 5 presumptivecolonies of E. coli O157:H7 were confirmed with an E. coli O157 di-rect immunoassay test kit (Universal Health Watch, Columbia, Md.,U.S.A.). Salmonella confirmation was carried out by testing reac-tions on triple sugar iron (Nissui Pharmaceutical Co.) slants andLIM medium (Nissui Pharmaceutical Co.) and serological tests usingSalmonella polyvalent O, O1 antisera (Denka Seiken, Tokyo, Japan).Randomly picked presumptive colonies of S. aureus and L. mono-cytogenes were confirmed with API Staph diagnostic kits and APIListeria diagnostic kits, respectively. All experiments were done4 times with duplicate samples being analyzed at each samplingtime.

Sensory evaluationSensory evaluationSensory evaluationSensory evaluationSensory evaluationDuring storage at 10 °C, the products were sensorially evaluated

for taste, odor color, texture, and overall acceptability by a panelconsisting of 13 sensory experts. The panelists, who have consider-able background knowledge in sensory evaluation, were selectedfrom the staff, researchers, food co. personnel, and teachers fromUniv. Evaluation was conducted in a well-equipped taste panelbooth at Natl. Food Research Inst. The score given by the panelvaried from 1 (dislike extremely) to 5 (like extremely). The sensorycharacteristics of odor, texture, and taste were assessed on each testsample. For each of the quality attributes, 3 packs of each treatedand untreated uninoculated samples were examined and/or testedby the judges. A sample was considered as unacceptable for a sen-sory characteristic if the score was less than 2.5 (Delaquis and oth-ers 2000).

Statistical analysesStatistical analysesStatistical analysesStatistical analysesStatistical analysesAll experiments were done 4 times with duplicate samples being

analyzed at each sampling time. Data were subjected to analysis ofvariance (ANOVA) using Microsoft Excel (Microsoft Corp., Red-mond, Wash., U.S.A.). Significant differences in plate count datawere established by least significant difference at the 5% level ofsignificance. Sensory evaluation tables represented the mean val-ues ± SD obtained from 3 individual trials.

Results and Discussion

The initial mesophilic bacterial count of Chinese cabbage justbefore fermentation was recorded as 6.38 log CFU/g. Addition

of chitosan, AIT-Hop, and other combinations did not decrease thepopulation of mesophilic bacteria, coliforms, or LAB at day 0 (Figure1). However, addition of chitosan, AIT-Hop, and other combinationsdid reduce mesophilic and coliform bacteria nearly 0.5 log CFU/gafter 1 day of storage. These counts remained constant at day 2with slight increases or decreases at days 3 and 4. On the otherhand, the population of mesophilic or coliform bacteria in controlsamples increased gradually between 10-fold to 100-fold after 4 d

Vol. 70, Nr. 9, 2005—JOURNAL OF FOOD SCIENCE M395URLs and E-mail addresses are active links at www.ift.org

M: Fo

od M

icrob

iolog

y & Sa

fety

Antimicrobial compounds in controlling microorganisms . . .

of incubation. The combination of chitosan and AIT-Hop exhibiteda slightly greater bactericidal effect against mesophilic and coliformbacteria compared with chitosan or AIT-Hop alone after 4 d of stor-age. The combination of chitosan and AIT-Hop suppressed growthof these bacteria by approximately 2.0 log CFU/g after 4 d of stor-age compared with control. There was little difference observed insamples with chitosan and AIT-Hop alone.

LAB or coliform are considered spoilage microorganisms becausethey could increase the turbidity of the seasoning liquid and reducethe quality of the fermented vegetables (Miyao 1997). The initialcounts of LAB were 2.85 log CFU/g, and the pH of the sample was6.4. The viable cell counts of LAB in the control sample increasegradually up to 5.20 log CFU/g at day 4 without any change in thefinal product pH. In the sample containing chitosan, LAB countswere lower than that of control sample after 2 d of storage, althoughthe count increased slowly up to day 4. The addition of AIT-Hopsuppressed the growth of the LAB by 2.0 log CFU/g compared withthe control sample after 4 d of storage. However, the combinationof chitosan and AIT-Hop did not enhance the efficacy. Addition ofchitosan, AIT-Hop, or a combination did not reduce the initial pH(6.4 to 6.6) at day 0, and at day 4, a slight drop of pH (6.2 to 6.4) wasobserved.

Savard and others (2002) reported that 10 g/L of chitosan did notsuppress the growth of LAB (Lactobacillus plantarum, Pediococcusacidilactici, and Leuconostoc mesenteroides) in vegetable juice. Inour experiment, we also found that chitosan did not suppress thegrowth of LAB in fermented Chinese cabbage. The population ofLAB was suppressed to its initial level by the addition of AIT-Hop.

AIT activity is believed to involve respiratory inhibition and thussuppressed the growth of aerobic or facultative anaerobic bacteria(Kojima and Ogawa 1971). However, the inhibitory activity of AITon LAB or other strictly anaerobic bacteria tends to be less than thatof other bacteria (Tokuoka and Isshiki 1994). On the other hand,alpha- or beta-acid of hop extract act as protonophores and dissi-pate the trans-membrane pH gradient, resulting in the growth in-hibition of LAB or other Gram-positive bacteria (Sakamoto andKonings 2003). Therefore, the bacteriostatic effect of AIT-Hop onLAB was believed to be derived from hop extract.

Antimicrobial activity was also analyzed in the lightly fermentedChinese cabbage with spiked samples. There was no significantchange in viable counts observed for both the Gram-negativestrains of E. coli and S. Enteritidis in the control and AIT-Hop treat-ed samples. But the addition of chitosan alone or with AIT-Hop re-duced the viable counts by 1.2 and 0.7 log CFU/g for E. coli and S.Enteritidis, respectively, by day 4. Wang (1992) reported that theaddition of 0.5% of chitosan could reduce inoculated S. aureus, E.coli, Y. enterocolitica, and L. monocytogenes by 1 to 2 log CFU/g in 4 dat pH5.5 in nutrient broth, but was less effective against S. Typh-murium. Our results (Figure 2a) are consistent, since the combina-tion of chitosan and AIT-Hop slightly reduced the bacterial count ofE. coli but not of S. Enteritidis when compared with the chitosanonly (Figure 2a).

Both Gram-positive strains grew gradually to give 0.5 (S. aureus)and 1.3 (L. monocytogenes) log CFU/g of increase in 4 d of storage incontrol (Figure 2b). The addition of AIT-Hop, however, retained theviable counts in both strains during storage. But chitosan exhibitedbactericidal activity against these strains. The larger reduction ofviable cell counts 1.3 (S. aureus) and 0.9 log CFU/g (L. monocytoge-nes) was observed on day 1 (Figure 2b). Thereafter, S. aureus re-tained its viable counts, but the growth of L. monocytogenes start-ed after 3 d of storage. The growth of L. monocytogenes could not besuppressed completely by the addition of chitosan or nisin. Theaddition of nisin exhibited similar bacteriostatic effect to the AIT-

Hop treatment for both Gram-positive strains, although the growthof L. monocytogenes started after 2 d of storage. There were no sig-nificant differences of the effectiveness of nisin and AIT-Hop forboth strains. Similarly with Gram-negative strains, bactericidal ef-ficacy of chitosan against S. aureus partially reduced in combinationwith AIT-Hop. In contrast, synergistic effect was observed againstL. monocytogenes (Figure 2b).

Lin and others (2000) showed that the population of each patho-gen (Salmonella Montevideo, Escherichia coli O157:H7, and Listeriamonocytogenes) was reduced by 4 log CFU/g on the surface of freshproduces with gaseous AIT in 4 d. However, in our study, AIT-Hop

Figure 1—The change of viable cell counts of (a) mesophilicbacteria Aerobic Plate Count [APC]), (b) coliform, and (c)lactic acid bacteria (LAB) in lightly fermented Chinese cab-bage. The samples were incubated at 10 °C with 3% saltfor fermentation for 4 d. The symbol represents the fol-lowing food additives. � = control; � = chitosan;� = Wasaouro EXT® (designated as “AIT-Hop”);� = chitosan and AIT-Hop.

M396 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 9, 2005 URLs and E-mail addresses are active links at www.ift.org

M: Food Microbiology & Safety

Antimicrobial compounds in controlling microorganisms . . .

emulsion during fermentation only suppressed the growth of patho-gens (Figure 2a, 2b). Gaseous AIT has been reported to exhibitstronger antimicrobial activity than emulsion mixed directly infoods (Inoue and others 1983). Although AIT-Hop emulsion did notexhibit bactericidal effect, it enhanced the bactericidal activity ofchitosan (Figure 2a, 2b).

The quality of uninoculated salted Chinese cabbage was evalu-ated by a panel consisting of 13 sensory experts (9 female and 4male) were selected from the staff and researchers of Natl. FoodResearch Inst., Food Co. personnel, and teachers from the univer-sity. No significant influence on the color, taste, texture, odor, andoverall acceptability was observed by the addition of chitosan orAIT-Hop (Table 1). The sensory evaluation was done after 1 d of fer-mentation because lightly fermented cabbage was a very popularfood item and representative of commercial product in Japan. How-ever, several research reports showed that there are no significantchanges of appearance, color, odor, taste, and texture after 4 d offermentation (Anonymous 1988; Kadowaki, and others 1988).

Conclusions

AIT-Hop, chitosan, or their combination could effectively suppress mesophilic or coliform bacteria, which is recognized as an

indicator of the shelf life. In addition, AIT-Hop or its combinationwith chitosan could suppress the growth of LAB (Figure 1), whichincreased the turbidity of the soaking water and consequentlycaused the loss of commercial value of the product. The addition ofthese food additives did not change the taste or other sensory pa-rameters (Table 1). Therefore, the combination of chitosan andAIT-Hop can be useful for controlling the microbial population infermented cabbage products. A large-scale series of experimentsusing this method must be done to determine the reproducibilityof the results obtained in the studies reported here.

AcknowledgmentsThis work was supported by a grant-in-aid (Integrated ResearchProgram for Functionality and Safety of Food Toward an Establish-ment of Healthy Diet) from the Ministry of Agriculture, Forestryand Fisheries of Japan.

ReferencesAdams MR, Nicolaides L. 1997. Reviews of the sensitivity of different foodborne

pathogens to fermentation. Food Control 8(5–6):227–39.Anonymous. 1988. Quality control of lightly fermented vegetable by bacterio-

logical regulation report [in Japanese]. Iwate Prefecture Res Inst Brew Food22(1):7–12.

Beuchat LR. 1996. Pathogenic microorganisms associated with fresh produce. JFood Prot 59(2):204–16.

Choi MH, Park YH. 2000. Selective control of lactobacilli in kimchi with nisin. LettAppl Microbiol 30(3):173–7.

Daeschel MA, Andersson RE, Fleming HP. 1987. Microbial ecology of fermentingplant materials. FEMS Microbiol Rev 46(3):358–67.

Delaquis PJ, Stewart S, Cliff M, Toivonen PM, Moyls AL. 2000. Sensory quality of ready-to-eat lettuce washed in warm, chlorinated water. J Food Qual 23(6):553–63.

Devlieghere F, Vermeulen A, Debevere J. 2004. Chitosan: antimicrobial activity,

Table 1—Sensory evaluation of lightly fermented cabbagea

Control Chitosan AIT Chitosan + AIT

Color 3.5 ± 0.5 3.3 ± 0.5 3.3 ± 0.6 3.5 ± 0.5Odor 3.3 ± 0.8 3.3 ± 0.6 3.2 ± 0.7 3.4 ± 0.8Taste 3.2 ± 1.1 3.2 ± 0.9 3.0 ± 1.0 3.2 ± 0.8Texture 3.5 ± 0.5 3.4 ± 0.7 3.3 ± 0.6 3.3 ± 0.6Overall 3.4 ± 1.3 3.2 ± 0.8 3.1 ± 0.8 3.4 ± 0.9aThe mean values ± SD obtained from 3 individual trials. The evaluation wasbased on a 5-point hedonic scale (1 = dislike extremely; 2 = moderatelydislike; 3 = neither like nor dislike; 4 = moderately like; 5 = like extremely).Average (n = 3) and SD were evaluated from 13 panels.

Figure 2—(a) The change of viable cell counts of Gram-negative pathogens in lightly fermented Chinese cabbage.The samples were incubated at 10 °C with 3% salt for fer-mentation for 4 d. Symbol represents the same as shownin Figure 1. (b) The change of viable cell counts of Gram-positive pathogens in lightly fermented Chinese cabbage.Experimental condition and symbol represents the sameas shown in Figure 1.

Vol. 70, Nr. 9, 2005—JOURNAL OF FOOD SCIENCE M397URLs and E-mail addresses are active links at www.ift.org

M: Fo

od M

icrob

iolog

y & Sa

fety

Antimicrobial compounds in controlling microorganisms . . .

interactions with food components and applicability as a coating on fruit andvegetables. Food Microbiol 21(6):703–14.

Helander IM, Nurmiaho-Lassila EL, Ahvenainen R, Rhoades J, Roller S. 2001.Chitosan disrupts the barrier properties of the outer membrane of Gram-neg-ative bacteria. Int J Food Microbiol 71(2–3):235–44.

Inatsu Y, Bari ML, Kawasaki S, Isshiki K. 2003. Construction and validation ofantibiotic resistance Escherichia coli strains for acidic foods. Jpn J Food Micro-biol 20(4):177–83.

Inatsu Y, Bari ML, Kawasaki S, Isshiki K. 2004. Survival of Escherichia coli O157:H7,Salmonella Enteritidis, Staphylococcus aureus, and Listeria monocytogenes inKimchi. J Food Prot 67(7):1497–500.

Inoue S, Goi H, Miyauchi K, Muraki S, Ogihara M, Iwanami Y. 1983. Effect of vol-atile constituents of plants on the proliferation of Bacteria. J Antibact Anti-fung Agents 11(12):609–15.

Isshiki K, Tokuoka K, Mori R, Chiba S. 1992. Preliminary examination of allylisothiocyanate vapor for food preservation. Biosci Biotechnol Biochem56(9):1476–7.

Kadowaki M, Endo T, Masuda K, Hashimoto K, Nakamura S, Suzuki T. 1988. Studyof the shelf life of lightly fermented Chinese cabbage from the aspect of con-taminated bacterial growth. IAI Center for Food Quality, Labeling and Con-sumer Services. Saitama, Japan: Research Rep. 12(1):77–82.

Kojima M, Ogawa K. 1971. Studies on the effects of isothiocyanates and theiranalogues on microorganisms. I. Effect of isothiocyanates on the oxygen up-take of yeasts. J Ferment Technol 49(9):740–6.

Lee CH. 1997. Lactic acid fermented foods and their benefits in Asia. Food Con-trol 8(5–6):259–69.

Lin CM, Kim J, Du WX, Wei C. 2000. Bactericidal activity of isothiocyanate againstpathogens on fresh produce. J Food Prot 63(1):25–30.

Miyao S. 1997. Microbiological controlling of fermented vegetables [in Japa-nese]. J Jpn Soc Food Sci Technol 44(1):1–9.

[NACMCF] Natl. Advisory Committee on Microbiological Criteria for Foods. 1999.

Microbiological safety evaluation and recommendations on fresh produce.Food Control 10(2):117–43.

Nguyen-The C, Carlin F. 1994. The microbiology of minimally processed freshfruits and vegetables. Crit Rev Food Sci Nutr 34(4):371–401.

O’Sullivan L, Ross RP, Hill C. 2002. Potential of bacteriocin-producing lactic acidbacteria for improvements in food safety and quality. Biochemie 84(5–6):593–603.

Ozaki N, Oba M, Hiwaki H, Fujishiro T, Ethou M, Uryu K, Manabe K, Miyashiro M,Wakatsuki K, Takeda A. 2003. A diffuse outbreak of enterohemorrrhagic Es-cherichia coli O157:H-infection, probably caused by pickled cucumbers. Fuku-oka, Japan: Ann Rep Fukuoka Health Env Res Inst 28(1):120–8.

Ozaki Y, Kurazono T, Saito A, Kishimoto T, Yamaguchi M. 2003. A diffused outbreakof enterohemorrhagic Escherichia coli O157:H7 related to the Japanese-stylepickles in Saitama, Japan. J Jpn Assoc Infect Dis 77(7):493–8.

Sakamoto K, Konings WN. 2003. Beer spoilage bacteria and hop resistance. Int JFood Microbiol 89(2):105–24.

Savard T, Beaulieu C, Boucher I, Champagne CP. 2002. Antimicrobial action ofhydrolyzed chitosan against spoilage yeast and lactic acid bacteria of fermentedvegetables. J Food Prot 65(5):828–833.

Simpson WJ, Smith ARW. 1992. Factors affecting antibacterial activity of hopcompounds and their derivatives. J Appl Bacteriol 72(4):327–834.

Steinkraus KH. 2000. Fermentations in world food processing. Comp Rev FoodSci Food Safety 1(1):23–32.

Tokuoka K, Isshiki K. 1994. Possibility of application of allylisothiocyanate vaporfor food preservation [in Japanese]. Jpn Soc Food Sci Technol 41(9):595–9.

Wang GH. 1992. Inhibition and inactivation of five species of foodborne patho-gens by chitosan. J Food Prot 55(11):916–9.

Weissinger WR, McWatters KH, Beuchat LR. 2001. Evaluation of volatile chemicaltreatments for lethality to Salmonella on alfalfa seed and sprouts. J Food Prot64(4):442–50.