FOODBORNE PATHOGENS AND DISEASEVolume 4, Number 4, 2007© Mary Ann Liebert, Inc.DOl: 10.1089/fpd.2007.0040

Antibacterial Activity of Guava (Psidium guajava L.)and Neem (Azadirachta id/ca A. Juss.) Extracts

Against Foodborne Pathogens and Spoilage Bacteria

M.D. Mahfuzul Hoque, 1 M.L. Bari , 2 Y. Inatsu, 2 Vijay K. Juneja, 3 and S. Kawamoto2

Abstract

The antibacterial activity of guava (Psidiuni gunJava) and neem (Azadirac/ita iiidicn) extracts against 21 strainsof foodhorne pathogens were determined—Listeria nionocittogenes ( five strains), Stapliilococcus ajireus (fourstrains), Escliericlua co/i 0157:H7 (six strains), Salmonella Enteritidis (four strains), Vibrio paraliaeniolyticus,

and Bacillus cereus, and five food spoilage bacteria: Pseudoinoiias aerogiliosa, P. putida, Alcaligenes faecalis, andAeromonas lidroplula (two strains). Guava and neem extracts showed higher antimicrobial activity againstGram-positive bacteria compared to Gram-negative bacteria except for V. para/iaeiuolyticus, P. aeroginosa,

and A. /iiidropliila. None of the extracts showed antimicrobial activity against E. co/i 0157:H7 and SalmonellaEnteritidis. The minimum inhibitory concentration (MIC) of ethanol extracts of guava showed the highestinhibition for L. mouocijtogeues JCM 7676 (0.1 mg/mL), S. aureus 1CM 2151 (0.1 mg/mL), S. aureus JCM 2179(0.1 mg/mL), and V. para/iaemolyticus IFO 12711 (0.1 mg/mL) and the lowest inhibition for Alcaligenes

faecalis IFO 12669, Aeromonas hydrophila NFRI 8282 (4.0 mg/mL), and A. hydrophila NFRI 8283 (4.0 mg/mL).The MIC of chloroform extracts of neem showed similar inhibition for L. nionocytogenes ATCC 43256(4.0 mg/mL) and L. monoci/togenes ATCC 49594 (5.0 mg/mL). However, ethanol extracts of neem showedhigher inhibition for S. aureiis JCM 2151 (4.5 mg/mL) and S. aureiis IFO 13276 (4.5 mg/mL) and the lowerinhibition for other microorganisms (6.5 mg/mL). No significant effects of temperature and pH were foundon guava and neem extracts against cocktails of L. nionocytogenes and S. aureus. The results of the presentstudy suggest that guava and neem extracts possess compounds containing antibacterial properties thatcan potentially be useful to control foodbome pathogens and spoilage organisms.

Introduction

F

O0DBORNE PATHOGENS SUCH AS diarrhea-genic serotypes of Escherichia co/i, Salmonella

spp., Listeria monocytogenes, and Aeromonas hy-drop/u/a are widely distributed in nature, caus-ing considerable mortality and morbidity in thepopulation. It has been reported that there aremore than 1.3 billion cases of human salmonel-losis annually with 3 million deaths worldwide

(Pang et al., 1995). Among the various diarrhe-agenic serotypes of F. coli, enterohemorrhagicE. co/i 0157:1-17 is implicated in a large numberof food borne outbreaks in many parts of theworld (Mead et al., 1999). L. monocytogenes hasbeen isolated from various environments and isreported to cause 25% of all the deaths resultingfrom foodborne outbreaks in the United Statesannually (CDC, 1995). Aeromonas spp. repre-sents a group of ubiquitous microorganisms in

'Department of Microbiology, University of Dhaka, Dhaka, Bangladesh.2National Food Research Institute, Tsukuba, Ibaraki, Japan.3Food Safety Intervention Technologies Research Unit, Eastern Regional Research Center, Agricultural Research Service, U.S.

Department of Agriculture, Wyndmoor, Pennsylvania.

481

482 MAHFUZUL HOQUE ET AL.

aquatic environments (Monfort and Baleux,1990). These bacteria have broad host range andhave often been isolated from humans with di-arrhea (Janda and Abbot, 1998).

Novel approaches to the development of newantimicrobials remain an important area of re-search. In recent years, multiple drug/chemicalresistance in both human and plant pathogenicmicroorganisms has developed due to indis-criminate use of commercial antibiotics com-monly applied in the treatment of infectiousdiseases (Loper et al., 1999; Davis, 1994; Service,1995). This situation has led scientists to searchfor new antimicrobials from various sources,including medicinal plants (Cordell, 2000).

Psidiuni guajava L. (guava), a fruit plant be-longing to the family Myrtaceae, is found allover the world. Guava leaves, roots, and fruitshave been used for the prevention and treat-ment of diarrhea (Lutterodt, 1989; Almeida etal.,1995), and a high level of antibacterial activitywas detected in guava leaves (Hidetoshi andDarn-to, 2002). In several studies, guava showedsignificant antibacterial activity against com-mon foodborne diarrhea-causing bacteria suchas Staphylococcus sp., Shigella sp., Salmonella sp.,Bacillus sp., E. coli, Clostridium sp., and foodspoilage bacteria such as Pseudomonas spp.(Lutterodt, 1989; Hidetoshi and Darmo, 2002;Abdelrahim et al., 2002; Jaiaij et al., 1999).

Azadirachta indica A. Juss (neem) belonging tothe family Meliaceae is an evergreen tree, culti-vated in various parts of the Indian subconti-nent. Neem has a long history of use in thetraditional medical systems of India (Ayurvedic,Unani-Tibb). Each part of the neem plant hassome medicinal property and is thus commer-cially exploitable. Extracts from neem leaves,seeds, and bark possess a wide spectrum ofantibacterial action against Gram-negative andGram-positive microorganisms, including My-cobacterium tuberculosis, Vibrio cholerae, andKlebsiella pneumoniae (Biswas et al., 2002). Pantet al. (1986) reported antifungal activity of leafextracts of neem. Recently, the antibacterial ac-tivity of neem seed oil was assessed in vitroagainst 14 strains of pathogenic bacteria (Biswaset al., 2002). However, very limited or no workhas been done evaluating the antibacterial ac-tivity of neem plant extracts against foodbornepathogens.

Therefore, this study was designed to assessthe antibacterial activity of guava and neem ex-tracts in vitro against selected foodborne path-ogens and food spoilage bacteria.

Materials and Methods

Plants

Guava and neem leaves were tested. Theseplant samples were collected from the fields ofBangladesh.

Test organisms

A total of 26 strains of foodborne pathogensand food spoilage bacteria, including Listeriamonocytogenes, Staphylococcus aureus, Vibrio para-haemolyticus, Escherichia coli 0157:H7, SalmonellaEnteritidis, Bacillus cereus, Pseudomonas spp., Al-caligenes faecalis, and Aeromonas hydrophila wereused in the study (Table 1). The stock cultures ofthe test organisms in medium containing 20%glycerol were kept at —84C. Working cultureswere kept at 4C on tryptic soy agar slants (NIS-SLIt, Tokyo, Japan) and were periodically trans-ferred to fresh slants. A loopful of culture fromthe slant was transferred to Tiyptic soy broth andgrown overnight at 37°C. The overnight grownculture was used for the subsequent study.

Preparation of plant extracts

The guava and neem leaves were collectedand washed in distilled water and air-dried, andthen cut into small pieces. The pieces of theleaves were air-dried at 37°C for 24 hours andthe dried leaves were ground using a grinder(IWATANI, Tokyo, Japan) into a fine powder.

Chloroform extracts. Thirty grams of each groundplant sample was added in 120 m of chloro-form (WAKO, Osaka, Japan) in sterile bottles(800 mL) and rotated with constant agitation(130 rpm) overnight at 20 T C in a temperature-controlled bioshaker (BR-40 LF, TAITEC, To-kyo, Japan). The chloroform fraction was sepa-rated using sterile cheesecloth and filter throughsterile Whatman filter paper (no. 2).

Ethanol extracts. The residual plant material ofeach sample after chloroform extraction wasdried at 40 C overnight in an oven. Then 120 mL

GUAVA AND NEEM EXTRACTS AND FOODBORNE PATHOGENS

483

TABLE 1. TEST ORGANISMS USED IN THIS STUDY

Code no. of test organisms Organisms No. of type culture Origin

lLm Listeria inonocytogenes2Lm L. ,nonocvto genes3Lm L. inonocytogenes4Lm L. monocvto genes5km L. Fnonocvto genes6Sa Staph vlococcus aureus7Sa S. aureus8Sa S. aureus9Sa S. aureus1 OVp Vibrio pa rahaemolvt,cusI lEc Escherhhia Co/i 0157:H712Ec E. co/i 0157:H713Ec E. co/i 0157:H714Ec E. co/i 0157:H715Ec E. Co/i 0157:H716Ec E. co/i 0157:H717 Sal Salmonella Enteritidis18 Sal S. Enieritichis19 Sal S. Enieritidis20 Sal S. Enteritidis21 B Bacillus cereu.v22 Pa Pseudonionas aeroglno.s23 Pp P. putida24 Af Alca/igenes faecahis25 Ah Aeromona.s hvdrophila26 Ah A. hvdrophila

ATCC 43256Mexican-style cheeseATCC 49594Scott AJCM 7671 Lax ham1CM 7672 Roast beefJCM 7676 Salami sausageJCM 2151 UnknownJCM 2179 UnknownJCM 2874WoundIFO 13276Human lesionIFO 12711Shirasu food poisoning, JapanMN 28 Bovine fecesCR 3 Bovine fecesDT 66 Bovine fecesMY 29 Bovine fecesE 615 Tomato juiceJCM 1649 UrineSE 1 Chicken fecesSE 2 Bovine fecesSE 3 Chicken fecesIDC 7 EggIFO 3457 UnknownPA 01 UnknownKT 2440 UnknownIFO 12669UnknownNFRT 8282UnknownNFRI 8283Unknown

of ethanol was added to each dried residue andagitated (130 rpm) overnight at 20C in a tem-perature-controlled bioshaker (BR-40 LF, TAT-TEC). The ethanol fraction was separated usingsterile cheesecloth and filter through sterileWhatman filter paper (no. 2).

Aqueous extracts. The residual plant material ofeach sample after ethanol extraction was driedat 40°C overnight in an oven. Then 120 mL ofsterile distilled water was added to each driedresidue and agitated (130 rpm) overnight at20°C in a temperature-controlled bioshaker (BR-40 LF, TAITEC). The aqueous fraction wasseparated using sterile cheesecloth and filterthrough sterile Whatman filter paper (no. 2).

All the extracts were then concentrated witha rotary vacuum evaporator (EYELA, Tokyo,Japan) at 40°C and the concentrated extractswere diluted to 10 mg/mL using 10% dimethylsulfoxide (DMSO) solvent, filter (0.45 pm) ster-ilized, and kept at —20 C until use.

Antimicrobial sensitivity testing

The antibacterial activity of all the plant ex-tracts was done according to the method of

Bauer et al. (1966). The 8-mm diameter discs(ADVANTEC; Toyo Roshi Kaisha, Ltd., Tokyo,Japan) were impregnated with 50 fLL of differentconcentrations of each plant extract before beingplaced on the inoculated agar plates. The in-ocula of the test organisms were prepared bytransferring a loopful of culture into 9 mL ofsterile Mueller Hinton broth (Difco, Sparks,MD) and incubated at 37°C for 5 to 6 hours ex-cept for Listeria monocytogenes, which wasgrown overnight. The bacterial cultures werecompared with McFarland (Jorgensen et al.1999) turbidity standard (10 8 CFU/mL) andstreaked evenly in three directions keeping at a60° angle onto the surface of the Mueller Hintonagar plate (10 x 40 mm) with sterile cotton swab.Surplus suspension was removed from theswab by rotating the swab against the side of thetube before the plate was seeded. After the in-ocula dried, the impregnated discs were placedon the agar using ethanol-dipped and flamedforceps and were gently pressed down to ensurecontact. Plates were kept at refrigeration tem-perature for 30 to 60 minutes for better absorp-tion. During this time microorganisms will notgrow but absorption of extracts would take

484 MAHFUZUL HOQUE ET AL.

place. Negative controls were prepared usingthe same solvent without the plant extract. Re-ference antibiotics (streptomycin and gentamy-cm) were used as positive controls.

The inoculated plates containing the impreg-nated discs were incubated in an upright posi-tion at 37°C overnight and/or 24 to 48 hours (ifnecessary). The results were expressed as thediameter of inhibition zone around the paperdisk (8 mm).

Determination of the minimuminhibitory concentration

The minimum inhibitory concentrations(MICs) of all the extracts were determined bymicrodilution techniques in Mueller Hintonbroth according to Sanches et al. (2005). In-oculates were prepared in the same medium ata density adjusted to 0.5 McFarland turbiditystandard (Jorgensen et al., 1999) (108 colony-forming units {CFU]/mL) and diluted 1:10 forthe broth microdilution procedure. Microtiterplates were incubated at 37 C and the MICswere recorded after 24 hours of incubation. Twosusceptibility endpoints were recorded for eachisolate. The MIC was defined as the lowest con-centration of extracts at which the microorgan-ism tested did not demonstrate visible growth.Minimum bactericidal concentration (MBC) wasdefined as the lowest concentration yieldingnegative subcultures or only one colony.

Antibacterial activity at different temperatures

The effect of temperature on antibacterial ac-tivity of plant extracts was determined by themethods as described by Lee et al. (2004). Theplant extract solutions were incubated at 37, 50,75, and 100°C, in a water bath for 30 minutes.Then, the plant extracts heated at differenttemperatures were cooled and stored at 4°Cuntil use. The antibacterial activity was assayedby the methods described by Bauer et cii. (1966).

Antibacterial activity at different pH values

The effect of pH on the antibacterial activitiesof plant extracts of guava and neem were as-sayed by using methods reported previouslywith slight modification (Shibata et al., 1995;Ohno et al., 2003). Briefly, the pH of the plant

extracts (5 mg/mL) was adjusted to the range of5.0 to 9.0 with either 50 m phosphate buffer or20 m Tris-HC1 buffer. Then the pH-adjustedmixtures were filtered through 0.45-jim mem-brane filters, stored at 4-C and used within 30minutes. The antibacterial activity against thecocktails of five strains of L. monocytogenes, fourstrains of S. aureus, six strains of E. co/i 0157:H7,and four strains of Salmonella Enteritidis wasdone by the disc diffusion method describedabove.

Statistical analysis

The inhibition zones were calculated asmeans ± SD (ci = 3). The significance amongdifferent data was evaluated by analysis ofvariance (ANOVA) using the Microsoft Excelprogram. Significant differences in the datawere established by least significant differenceat the 5% level of significance.

Results and Discussion

Ethanol extracts of guava were found to haveantimicrobial activity against all L. inonocyto-genes strains except for L. monocytogenes JCM7672, all S. aureus strains, V. parahaemoiyticus1F012711, Aicaiigenesfaeca/js IFO 12669, and allA. hydrophila strains. The aqueous extract ofguava was active against L. monocytogenes JCM7671, all S. aureus strains, V. parahaemolyticus1F012711, B. cereus, P. aeroginosa PA01, A/ca/i-genes faeca/is IFO 12669, and all A. hydro phi/astrains (Table 2). However, S. aureus JCM2894and A. hydrophila NFRI 8282 were least sensitiveto ethanol and aqueous guava extracts, respec-tively. The chloroform extracts of guava did notshow any antibacterial activity against the teststrains. The ethanol and aqueous extracts ofguava exhibited the highest antimicrobial ac-tivities and were able to inhibit 46% and 38% ofthe test organisms, respectively. Guava extractsexhibited antibacterial activity against bothGram-positive and Gram-negative bacteria.However, no antibacterial activity was foundagainst E. co/i 0157:H7, Salmonella Enteritidis,and Pseudomonas putida (Table 2). The ethanolextracts of guava showed antibacterial activityagainst other test microorganism, with zones ofinhibition very close to the zones of inhibition ofreference antibiotics, gentamycin, used in this

GUAVA AND NEEM EXTRACTS AND F000BORNE PATHOGENS

485

TABLE 2. ANTIBACTERIAL ACTIVITY OF GUAVA AND NEEM EXTRACTS AGAINSTFOODBORNE PATHOGENS AND SPOILAGE BACTERIA

Plant extracts

Zones of inhibition'

Guava Nee;n Antibiotics

tI2 OCHC13EtOH SM GMCode no. of testorganisms EtOH

lLm2Lm3Lm5Lm6Sa7Sa85a9Sa10vp21 Bc22 Pa23 Pp24 At25 Au26 Ah

19.4 ± 0.2121.9 + 0.2621.4 + 0.2621.4 ± 0.2115.3 + 0.0611.5±0.1010.7 ± 0.3012.0 ± 0.2114.8 ± 0.26

11.4±0.2112.2 ± 0.2113.0 ± 0.21

18.0 ± 0.47

12.0±0.3111.5 ± 0.2011.0 ± 0.5610.2 ± 0.2113.1 ±0.2513.5 ± 0.2012.0 ± 0.38

14.5±0.3610.0 ± 0.3814.0 ± 0.21

20.0 ± 0.4614.5 + 0.2612.0 ± 0.3513.0 ± 0.38

9.0 ± 0.4512.0 ± 0.2112.8 ± 0.44

17.2 ± 0.2019.5 ± 0.3021.5 ± 0.6018.0 ± 0.4718.0 ± 4616.5 ± 0.7016.5 ± 0.8017.4 ± 0.4024.5 ± 0.5016.2 ± 0.2015.5 ± 0.2619.3 ± 0.21

10.7+0.2611.0 ± 0.47

18.4± 0.1222.9 ± 0.5623.0+0.3022.6 ± 0.2124.5 ± 0.4922.0 ± 0.1220.4 ± 0.2118.1 ± 0.2530.0 ± 0.3122.0 ± 0.3121.5 ± 0.6621.0 ± 0.31

9.5 ± 0.5019.2 ± 0.2115.0 ± 0.47

16.0 ± 0.3814.2 ± 0.26

Represents mean ± SD mm (ii = 3); e >0.05; EtON = ethanol; H 20 = aqueous; CHCI 3 = chloroform; SM = streptomycin (30 jig);GM = gentamycin (10 pg).

study. This result is consistent with the previousreports by Jaiarj et al. (1999), Gnan and Demello(1999), Oliver-Bever (1986), and Sanches et al.(2005).

Hidetoshi and Danno (2002) reported fourflavonoid compounds extracted by 90% (v/v)aqueous methanol from guava exhibited anti-bacterial activity against B. cereus and SalmonellaEnteritidis. However, in our experiment theethanol extracts of guava did not show any ac-tivity against B. cereus IFO 3457 and SalmonellaEnteritidis.

The chloroform extracts of neem showedantibacterial activity against L. monocytogenesATCC 43256 and L. monocytogenes ATCC 49594.However, ethanol extracts of neem showed ac-tivity against L. monocytogenes ATCC 49594, L.monocytogenes JCM 7671, L. monocytogenes JCM7676, and Staphiococcus aureus JCM 2151, S. au-reus JCM 2894, S. aureus IFO 13276, and V.parahaemolyticus IFO 12711. The aqueous ex-tracts of neem did not show any antibacterialactivity against the test strains.

The chloroform and ethanol extracts of neemshowed antibacterial activity against 7% and26% of the test organisms, respectively. In ad-dition, ethanol extracts of neem showed anti-bacterial activity against V. parahaenolyticus 1170

12711, however the chloroform extracts of neemdid not show antibacterial activity againstV. parahaemolyticus IFO 12711. The ethanol ex-tract of neem showed zones of inhibition againsttest microorganism were very close to the zonesof inhibition of reference antibiotics used in thisstudy (Table 2). Preliminary studies carried outby several investigators showed significant ef-fects of neem extracts on several bacterial strains(Rao et al., 1986; Chopra et al., 1952, 1956; Cho-pra, 1958; Rojanapo, 1985). Mahmoodin, oneof the neem's medicinal compounds, showedsignificant antibacterial activity against variousGram-positive and Gram-negative microor-ganisms (Seddiqui et al., 1992). The antibacterialactivity of neem extracts against Staphylococcusaureus (Schneider, 1986), Streptococcus pyogenes,Cornebacterium, and E. coli (Thaker and An-jaria, 1986), Salmonella typhosa (Patel and Trive-di, 1962; Chopra, 1958) has been reported. Inour experiment, extract of neem did not showantimicrobial activity against any of the Gram-negative bacteria tested but were highly effec-tive in controlling Gram-positive and spoilagemicroorganism.

The MIC results are listed in Table 3. The MICof ethanol extract of guava showed the high-est inhibition for L. monocytogenes JCM 7676

Code no. of testorganisms

I Lm2Lm3Lm5Lm6Sa7Sa8Sa9Sa1 OVp21 Bc22 Pa23 Pp24 Af25 Ah26 Ah

0.3 (0.4)0.4 (0.5)0.1 (0.5)0.1 (0.2)0.1 (0.2)0.1 (0.2)0.4 (0.5)0.7 (0.8)0.1 (0.2)

4.0 (4.5)1.5 (2.0)4.0 (4.5)

7.0 (7.5)

4.5 (5.0)4.0 (4.5)3.5 (4.0)5.0 (5.5)3.0 (3.5)3.0 (3.5)

0.9 (1.0)2.0 (2.5)3.5 (4.0)

6.5 (7.0)6.5 (7.0)6.5 (7.0)4.5 (5.0)6.5 (7.0)6.5 (7.0)4.5 (5.0)6.5 (7.0)

EtOHH70CHC13a EtOH

5.0 (5.5)5.0 (5.5)

486

TABLE 3. MINIMUM INHIBITORY CONCENTRATION (MIC)AND MINIMUM BACTERICIDAL CONCENTRATION (MBC)OF GUAVA AND NEEM EXTRACTS AGAINST FOODBORNE

PATHOGENS AND SPOILAGE BACTERIA

Plant extracts

MIC (MBC)

Guava Neem

Dashes represent no positive activity found during preliminaryscreening.

Parentheses value represents minimum bactericidal concentra-tion (MBC).

aj was done by disk diffusion method.

(0.1 mg/mL), S. atireus JCM 2151 (0.1 mg/mL),S. aureus JCM 2179 (0.1 mg/mL) and V. pam-haemolyticus IFO 12711 (0.1 mg/mL). The sameextract showed the lowest inhibition for A. fae-ca/is IFO 12669 (4.0 mg/mL) and A. hydro phi/aNFRI 8283 (4.Omg/mL). The aqueous extracts

MAHFUZUL HOQUE ET AL.

of guava showed the highest inhibition forA. faecalis IFO 12669 (0.9 mg/mL) and the low-est inhibition for L. monocytogenes JCM 7671(7.0 mg/mL). Guava leaf extracts have beenreported to have MIC values ranging from150 pg/mL to 4 mg/mL for inhibition of bacte-rial pathogens (Prabu et al., 2006; Hidetoshi andDanno, 2002; Sanches et al., 2005).

The MIC of the chloroform extract of neemshowed the same inhibition concentrations forL. inouocytogenes ATCC 43256 (5.0 mg/mL) andL. monocytogenes ATCC 49594 (5.0 mg/mL).However, the ethanol extract showed the high-est inhibition for V. parahaemolyticus IFO 12711(4.5 mg/mL) and the lower inhibition againstthe other pathogens (6.5 mg/mL).

The antibacterial activity of the extracts he-ated to 50 C for 30 minutes was found almostunchanged compared to nonheated extracts,while the inhibitory effect of boiled plant ex-tracts was significantly decreased (Table 4). Theactivity of aqueous extracts of guava was lostcompletely when heated to 100 C against acocktail of S. aureus strains. The antibacterialactivity was not affected at pH 5.0, however,significant decrease of inhibitory activity wasfound at pH 9.0. The antibacterial activity of theethanol extract of guava was lost completely atpH 9.0 against a cocktail of L. monocytogenes. Theethanol extract of neem showed no activityagainst cocktail of L. inonocytogenes at pH 7.0and 9.0 (Table 5). Lisboa et al. (2006) showedsimilar findings with bacteriocin-like substances

TABLE 4. EFFECT OF TEMPERATURE ON ETHANOL AND AQUEOUS EXTRACTS OF GUAVA AND ETHANOL EXTRACTOF NEEM AGAINST COCKTAILS OF THE FOODBORNE PATHOGENS AND SPOILAGE BACTERIA

Zones of inhibition'

Cocktails of test organismsTemperature (°C)

Plant extracts(serotype code numbers)425375075100Guava

EtOHGuava

EtOHGuava

H70Neem

EtOHNeem

EtOH

L. monocvto genes(11-m-31-m, 5Lm)

S. aureus (6Sa-9Sa)

S. aureus (6Sa-9Sa)

S. aureus (6Sa-9Sa)L. monocyto genes

(2Lm, 3Lm, 5Lm)

12.0±0.212.0±0.112.0±0.311.0±0.510.5±0.210.0±0.4

12.0±0.412.0±0.512.0±0.610.5±0.410.0±0.39.0±0.1

10.0±0.311.0±0.510.0±0.610.0±0.59.0±0.20.0±0.0

14.0±0.312.0±0.310.2±0.210.0±0.89.0±0.39.0±0.6

11.5±0.311.5±0.210.0±0.510.0±0.49.0±0.49.0±0.7

'Represents mean ± SD mm (n = 3); p >0.05.

GUAVA AND NEEM EXTRACTS AND FOODBORNE PATHOGENS 487

TABLE 5. EFFECT OF PH ON ETHANOL AND AQUEOUS EXTRACTS OF GUAVA AND ETHANOL EXTRACT OF NEEMAGAINST COCKTAILS OF THE FOODBORNE PATHOGENS AND SPOILAGE BACTERIA

Zones of inhibition'

pH

Plant extractsCocktails of test organisms (serotype code no.)5.0 7.0 9.0

GuavaEtOH

GuavaEtOH

GuavaH20

NeemEtOH

L. rno,wcvto genes (lLm-3Lm, 5Lm)

S. aureus (6Sa-9Sa)

S. aureus (6Sa-9Sa)

S. aureus (6Sa-9Sa)

12.0±0.410.0±0.20.0±0.0

11.0+0.110.0+0.19.8+0.1

12.1 ± 0.610.5 + 0.110.0 ± 0.4

14.2±0.19.5±0.29.0±0.5

NeemEtOH L. ,nonocvto genes (2Lm, 3Lm, 5Lm)

Represents mean - SD mm (n .-. 3); p > 0.05.

produced by Bacillus arnyloliquefaciens isolatedfrom Brazilian Atlantic forest against L. mono-

ct/togenes.

Conclusion

In this study, guava and neem extractsshowed antibacterial activity against selectedfoodborne pathogens and spoilage microor-ganisms. The result of this study also suggeststhat guava and neem extracts possess com-pounds containing antibacterial properties thatcan be useful to control foodborne pathogensand spoilage organisms. Antibacterial extractsobtained in the present study will be applied toactual foods to assess the microbiological con-dition of the particular food or food productswith extended shelf-life.

AcknowledgmentThis work was supported by grant from

UNU-Kirin Fellowship Program. Authors aresincerely grateful to UNU-Kirin for their finan-cial support. Authors expressed their sinceregratitude to the authorities of the NFRI, Tsu-kuba for laboratories facilities and logistic sup-ports to carry out this investigation.

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