Biotechnological

Communication

Biosci. Biotech. Res. Comm. 10(2): 112-116 (2017)

Antimicrobial effects of grape pulp, Bakhtiari savory,

and lemon extracts on

Listeria monocytogenes

isolated

from meat

Ehsan Ghasemi, Gholamreza Banisharif and Iman Alavi*

MSc Microbiology, College of Basic Science, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

ABSTRACT

Listeria monocytogenes is a coccobacilli, gram-positive, non-spore forming bacteria. The distribution of these bacteria

in nature is high, which is a human and animal pathogen and has different complications. The main transmission

way for human infection is through the consumption of contaminated foods. This study is aimed to investigate the

antimicrobial effects of Bakhtiari savory, grape pulp and lemon extracts against Listeria monocytogenes. The pulp

extract of a kind of lemon and the aalcoholic extract of grape seed were extracted and the concentrations of 16

to 1000 mg/ml were prepared. The minced meat was infected with Listeria monocytogenes, and then the prepared

concentrations of the extracts were injected into the meats carrying the bacteria. After 3, 7 and 10 days, a micro-

bial culture was done from the meats and the colonies were counted. The study showed that the Bakhtiari savory

extract and then the grape pulp has the greatest inhibitory effect both in vitro and in food. It was also shown that

the Bakhtiari Savory could be effective as a preservative to increase meat durability and to prevent the growth of

pathogens such as Listeria monocytogenes. According to the inhibitory effect of Bakhtiari Savory extract and the

grape pulp on Listeria monocytogenes, the substances may be used for the treatment of diseases caused by these

bacteria.

KEY WORDS:

LISTERIA MONOCYTOGENES

, MEAT, BAKHTIARI SAVORY, GRAPE PULP, LEMON

112

ARTICLE INFORMATION:

*Corresponding Author: Imanalavi66@gmail.com

Received 12

March, 2017

Accepted after revision 31

May, 2017

BBRC Print ISSN: 0974-6455

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Online Contents Available at: http//www.bbrc.in/

Ehsan Ghasemi et al.

INTRODUCTION

Listeria monocytogenes (L. monocytogenes) is a gram-

positive, non-acid-fast and short rod shaped, opportun-

istic intracellular, Gram-positive, facultative anaerobic,

nonspore-forming bacterium of the genus Listeria, which

is widely spread in the environment, and can cause

severe diseases including meningoencephalitis, sep-

ticemia, mastitis and abortion in humans and animals,

primarily affecting pregnant, new-born, and immuno-

compromised individuals (Dehkordi et al. 2013, Reddy

and Lawrence 2014).

According to previous epidemiological investigations,

various types of foods and especially those with animal

origins are the main reservoirs for virulent strains of

L. monocytogenes (Dehkordi et al. 2013, Di Pinto et al.

2010, Gray et al. 2004, Tompkin 2002).The high ability

of bacterium to resistance against environmental condi-

tions such as temperature, pH, salt concentration and

also dryness is the main reason for considerable role of

the L. monocytogenes as an emerging food-borne patho-

gen (Mataragas et al. 2006, Zhang et al. 2012). Meat is

raised as one of the most important reservoirs of the

L. monocytogenes (Goh et al. 2012, Indrawattana et

al. 2011, Siriken et al. 2013). Meat of animals prepares

favorable conditions including optimum levels of nutri-

tional materials, temperature, activated water (AW), pH

and salt for persistence of L. monocytogenes (Mataragas

et al. 2006, Zhang et al. 2012).

Antibiotic therapy is the most effective way to treat-

ment the cases of food-borne listeriosis but high pres-

ence of antibiotic resistant strains of this bacterium cause

hardness and prologues in treatment period and usu-

ally occur severe economic losses (Camargo et al. 2015,

Dehkordi et al. 2013, Granier et al. 2011, Morvan et al.

2010). In keeping with this, an epidemiological research

revealed that the L. monocytogenes strains of animal ori-

gins had the ranges of 10-80 percent resistance against

commonly used antibiotics (Dehkordi et al. 2013). High

prevalence of antibiotic resistance and increasing in

the consumers’ concerns regarding food security and

consuming foods without any chemical preservative,

led the food industries to use from the traditional plant

extracts to reduce the microbial load of various types

of foods (Kang et al. 2011). Now a days, application of

medicinal plants and especially grape pulp, savory, and

lemon due to their high antimicrobial effects and also

complete contents of  avonols, antioxidants, phenolic

acids, catkins, proantocianidine, anthocyanins, catkins,

folic acids and other phenolic components have been

increased in the food industries (Mahboubi and Kazem-

pour 2011, Oliveira et al. 2013). However, using from

medicinal planst in food industries requires more exten-

sive research.

The aim of this study was to evaluate the antimicro-

bial effect of grape pulp, Bakhtiari savory, and lemon

extracts on L. monocytogenes isolated from Iranian meat

samples.

MATERIALS AND METHODS

SAMPLES COLLECTION

Grape sample was prepared from Shiraz native RISH

BABA as well as the pulp extract dewatered from a kind

of lemon, an extracted savory was also selected for the

study. To dry the samples in a large volume, followed by

the dewatering and the pulp separation, the grape cores

were kept for a week at 25 to 30 ° C in the shade with

proper ventilation.

ESSENCE EXTRACTION AND PREPARATION

The dried grape cores were kept in cool, dry place until

the time of experiments. 100 gr core was weighed in

order to take the essence and extracting (Mahboubi and

Kazempour 2011). In order to prepare the extracts, 96%

ethanol was used. For this purpose, the extraction was

performed from 100 grams of vegetable using ethanol

solvent, followed by concentrating the samples and

alcohol separation using Rubia tinctorum in a vacuum

(Siriken et al. 2013). To investigate the effective dose

which has antibacterial properties, different concentra-

tions were made from the extracts and selected doses

were applied according to the sources.

To provide the desired concentrations, 0.01 gram of

the extract was weighted and poured into the test tube,

then 96% Ethanol was added to the  nal volume of 10

ml, followed by mixing thoroughly, the solution is called

the Mother solution. Then the concentrations of 1000,

500, 250, 125, 62, 31 and 16 mg/ml were prepared.

STUDY THE ANTIMICROBIAL EFFECTS

To investigate the antimicrobial effects of desired extracts,

Hinton agar plates were used. To inoculate the bacteria,

100 μl of bacteria suspension was poured on already pre-

pared solid medium. Then, the liquid was spread over the

medium surface using a sterile cotton swab or a sterile

Pasteur L-shaped pipette. It was done identically for all

the plates. Then, plate disks impregnated with the extracts

were placed on the plates using sterile forceps,in the fol-

lowing, the antimicrobial properties were evaluated after

24 hours. To evaluate the antimicrobial properties of

extracts and to determine the Minimum Bacterial Con-

centration (MBC) and Minimum Inhibitory Concentration

(MIC) of each extract, the concentrations of MBC and

MIC were used for inoculating on food sample (meat). For

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECTS OF GRAPE PULP, BAKHTIARI SAVORY, AND LEMON ON

LISTERIA MONOCYTOGENES

113

Ehsan Ghasemi et al.

Table 1. The investigation of grape pulp, lemon and savory

extracts on Listeria monocytogenes in the minced meat.

Extract

Inoculated

bacteria

Bacterial counts in days after

inoculation

3rd 7th 10th

Grape pulp

103 102 × 2.2 102 × 9.5 102 × 8.8

106 104 × 1.4 104 × 6.6 104 × 8.6

Savory

103 101 × 8.3 101 × 4.5 102 × 2.5

106 103 × 3.6 103 × 8.5 103 × 3.4

Lemon

103 102 × 9.7 102 × 1.9 103 × 8.1

106 104 × 2.9 105 × 3.2 105 × 1.4

Table 2. The effect of grape pulp, lemon and savory extracts on Listeria monocytogenes in vitro

conditions.

Samples

Dilution

1000 500 250 125 62.5 31 15

Grape pulp

16- 18- 18

MBC

17- 17- 17

MBC

15- 15-16

MIC

14- 15- 14

Growth

12- 13- 13

Growth

12- 13- 13

Growth

9- 9- 10

Growth

Savory

Growth

MIC

Growth

Lemon

15- 15-16

MIC

14- 14- 15

MIC

13- 13- 14

MIC

14- 13- 14

Growth

8- 9- 9

Growth

7- 8- 8

Growth

this purpose, beef minced meat with about 15% fat was

selected and minced with a meat mincer which the cut-

ting blade, spiral parts and etc, were washed well and

disinfected with 96% alcohol and dried. The desired meat

was packed in 10 g plastic bags and was sterilized in the

presence of UV radiation for 3 hours. In the following, the

meats were inoculated with 10

and 10

concentrations of

L. monocytogenes in the both groups, and then the pre-

pared concentrations of the extracts were injected into

the meats carrying the bacteria. After 3, 7 and 10 days,

1 g of the desired meat was put into test tubes contain-

ing sterile distilled water and the desired concentrations

were given to the medium, then the bacteria grown were

counted after 24 hours.

STATISTICAL ANALYSIS

To analyze the data, the one-way analysis of variance

was used, as well as Duncan’s and Tukey’s mean com-

parisons with 5% probability level using SPSS statistical

software (Ver. 20).

RESULTS

In the present study, the antibacterial effect of Bakh-

tiari savory, grape pulp and lemon pulp on L. monocy-

togenes was examined in vitro and in the minced meat.

The results of 5- 10, 6- 10 and 7- 10 dilutions during 3,

7 and 10 days following the counting of bacteria sug-

gested the best inhibitory effect in the Bakhtiar savory,

grape pulp and lemon extracts, respectively.

Also, Bakhtiari savory, grape pulp and the lemon

extracts were prepared and the effect of MBC and MIC

were evaluated. The results showed that the Bakhtiari

Savory have the greatest inhibitory effect in vitro com-

pared to the Grape pulp and lemon, respectively.

DISCUSSION

In general, the amount and the distribution of L. mono-

cytogenes in nature and environment is very high.

These bacteria can be found everywhere, from human

and animal feces to the soil, water and the variety of

foods such as raw meat, fruits, milk and their products

(19 of 30). Therefore, human beings, as a member of

nature, can easily be infected with the bacteria, certainly

by exposing one of the above mentioned items and

having special conditions (8 and 30). The main trans-

mission way for human infection is through the con-

sumption of contaminated foods. The use of chemical

preservatives to increase food durability and to prevent

the growth of pathogens has resulted in the increase

of chronic and incurable diseases. Therefore, the

consumers mainly purchase and prepare foods without

114 EFFECTS OF GRAPE PULP, BAKHTIARI SAVORY, AND LEMON ON

LISTERIA MONOCYTOGENES

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Ehsan Ghasemi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECTS OF GRAPE PULP, BAKHTIARI SAVORY, AND LEMON ON

LISTERIA MONOCYTOGENES

115

additives and preservatives or with natural and safe

preservatives. In the meantime, in recent years, several

studies have been conducted on the antibacterial and the

antioxidant effects of plant seed extracts.

Ghasemi et al. (2009) showed that the essential oil of

Thymus aenensis and other species of thyme (ecotype

Elam) have antimicrobial activity against L. monocy-

togenes present in chicken. In this study, the minimum

inhibition concentration for 50% growth inhibition <MIC

against L. monocytogenes for Thymus daenensis and Thy-

mus sp. Were 70.0 and 7.1 mg / ml, respectively (Mah-

boubi and Kazempour 2011). According to the Rasouli

and colleagues (2006), the extract and the essential oil

of Thymus erioealyx and T. Porlock inhibits the growth

of L. monocytogenes. The essential oils and the extracts

of some aromatic plants (such as the mint family, Lami-

aceae) with a high percentage of Carvacrol and thymol,

have good activity against some bacteria (Chedea et al.

2013). Zdenka and colleagues (2004) showed the effect of

antibacterial activity of the ethanol extract derived from

the pulp and the seed of grapefruit against Staphylococcus

aureus, Proteus vulgaris, Klebsiella pneumoniae, Candida

albicans and other  ora and the results showed that the

extract of grapefruit seed has positive effects on the mor-

tality of microorganisms (Cvetnic and Vladimir-Knezevic

2004). Joseph et al. (2009) showed that grape skin has

the highest antimicrobial activity compared to the seeds

(Brown et al. 2009). Veronica et al. (2011) showed in a

study the effectiveness of phenolic extract against gram-

positive bacterium linens and reducing the growth of E.

coli (Chedea et al. 2013).

Jalali et al. (1385), studied the antimicrobial effects of

hydroalcoholic extract derived from eucalyptus, rosemary,

thyme, chamomile and sage on L. monocytogenes and

showed that only the eucalyptus has antibacterial effects

against L. monocytogenes. In a study by Mena and his col-

leagues in Portugal (2004) on commercial food produc-

tion, the contamination of L. monocytogenes was reported

7.17% in raw meat, 7.16% in raw milk, 6.12% in raw

 sh, 18.5% in  our and 12.9% in fresh vegetables (Mena

et al. 2004). In this study, the Bakhtiari savory showed

the greatest antimicrobial effect against L. monocytogenes

with a high percentage of carvacrol. After the Savory

Bakhtiari, the grape pulp with anthocyanins and  avo-

noids chemical compounds, and the lemon with limonene

chemical compounds had the highest antimicrobial effect

against L. monocytogenes, respectively. Their inhibitory

effects were studied and approved by the MBC and MIC

tests in vitro and in the minced meat.

CONCLUSION

The study showed that the Bakhtiari Savory extract and

then the grape pulp had the best inhibitory effect on L.

monocytogenes both in vitro and in food. On the other

hand, it was shown that the Bakhtiari Savory could be

effective as a preservative to increase meat durabil-

ity and to prevent the growth of pathogens such as L.

monocytogenes.

ACKNOWLEDGEMENTS

The authors would like to thanks from the Dr. Farhad

Safarpoor Dehkordi in the Department of Food Hygiene

of the University of Tehran for his technical support.

This work was support by the Islamic Azad University of

Shahrekord (Grant number: MSAB 220146).

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