Antibacterial Effect of Essential Oils against Spoilage Bacteria from

1386 Journal of Food Protection, Vol. 81, No. 8, 2018, Pages 1386–1393 doi:10.4315/0362-028X.JFP-17-474 Copyright Ó, International Association for Food Protection

Research Paper

Antibacterial Effect of Essential Oils against Spoilage Bacteria from Vacuum-Packed Cooked Cured Sausages AZITA KHORSANDI,1 ESMAEIL ZIAEE,1 EHSAN SHAD,1 MARYAM RAZMJOOEI,1 MOHAMMAD HADI ESKANDARI,1* AND MAHMOUD AMINLARI1,2 1Department

of Food Science and Technology, School of Agriculture, and 2Department of Biochemistry, School of Veterinary Medicine, Shiraz University, Shiraz 7144165186, Iran MS 17-474: Received 13 November 2017/Accepted 9 April 2018/Published Online 19 July 2018

ABSTRACT Nonfermented sausages, which have a pH of around 6.0, a low salt concentration, and high moisture with a water activity higher than 0.95, are highly perishable. In this study, culture-dependent techniques and 16S rDNA approaches were used to identify the presumptive spoilage lactic acid bacteria (LAB) in sliced vacuum-packed cooked sausage during storage at 48C. The antibacterial properties of essential oils (EOs) from the medicinal plants Carum carvi, Cinnamomum zeylanicum, Curcuma longa, Citrus medica, and Eugenia caryophyllata against isolated LAB were also investigated. A total of 106 colonies were obtained on de Man Rogosa Sharpe medium after storage of sausages samples, and 16 isolates were identified from conventional morphological analysis of the bacterial populations. DNA extraction and 16S rDNA analysis indicated that Lactobacillus curvatus, Weissella viridescens, Leuconostoc mesenteroides, Enterococcus faecium, Lactobacillus reuteri, Lactobacillus dextrinicus, Lactobacillus sakei, and Pediococcus dextrinicus were the main spoilage LAB. The antibacterial properties of EOs against isolated LAB were indicated by inhibition zones on culture plates of 7.8 to 31 mm, depending on the susceptibility of the tested LAB strain. The MICs and MBCs of five EOs were determined. The most effective EO against the LAB was C. zeylanicum followed by C. carvi and C. medica, and the least effective EO was C. longa. The EO from C. zeylanicum had the highest antimicrobial activity (lowest MICs) against LAB, with EO MICs of 4.66 to 5.33 lL/mL. The most susceptible isolate was L. mesenteroides, with a MIC of 4.66 lL/mL for the C. zeylanicum EO. These data indicate that the EO from C. zeylanicum could be used as a natural preservative for vacuum-packed emulsion-type sausage. Key words: 16S rDNA; Antibacterial properties; Essential oils; Lactic acid bacteria; Vacuum-packed cooked cured sausages

Vacuum-packed cooked cured sausages are one of the most popular meat products in Iran. In general, the shelf life can be up to 30 days at refrigerated temperatures (4 6 28C). Vacuum-packed cooked cured sausages are perishable because of their low salt concentration (~2%), a pH of around 6.0, and a water activity of 0.95 to 0.97 (23). Spoilage of cooked meat products results in sensory quality defects such as sour off-flavors, discoloration, gas production, and ropy slime formation (3, 15, 26). Lactic acid bacteria (LAB) are a major component of the microbial population found on various types of vacuumpackaged cooked sausages. These bacteria grow on the surface of the sausages and produce undesirable sensory attributes, such as sour aroma and taste. Lactobacillus sakei, Lactobacillus curvatus, and Leuconostoc mesenteroides are the common species in these products (9). Consumer preference is increasing for high quality, minimally processed meats with low concentrations of preservatives. Secondary metabolites derived from plants from various parts of the world have antibacterial activity * Author for correspondence. Tel: þ98 71 32286110; Fax: þ98 71 32286110; E-mail: [email protected].

against food spoiling microorganisms and can be used in food systems (4, 18, 28). Essential oils (EOs) and other extracts may have moderate to high antimicrobial activity against foodborne pathogens (1, 4). Although the antimicrobial effects of EOs have been well documented, few studies have been conducted to investigate the feasibility of using EOs as antimicrobial agents against LAB. The main objective of this study was to isolate and identify LAB as spoilage agents in vacuum-packed cooked cured sausages and to evaluate the antimicrobial activity of EOs from five medicinal plants against the LAB isolated on a laboratory medium.

MATERIALS AND METHODS Sampling procedure. Eighteen samples (S1 through S18) of vacuum-packed cooked cured sausages were collected from various manufacturing companies in Iran. Sausage samples from the first day of production were transferred to the food processing laboratory in the Department of Food Science and Technology (Shiraz University, Shiraz, Iran) and stored at 48C. Sausages differ by their percentages of beef, sodium chloride, soya isolate protein, potato starch, nitrite, water, sodium glutamate, sucrose, and flavoring agents. The sausage samples in the present study were

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composed of beef (40 to 60%), protein (11 to 24%), fat (15 to 27%), salt (1.4 to 2.7%), starch (3.4 to 5.1%), and ash (0.8 to 2.4%).

removed from the gels and purified with a PCR CLEAN-UP kit (GF-PC-050, Vivantis, Subang Jaya, Malaysia) following the manufacturer’s protocol.

pH values. The pH of the samples was determined according to the method described by Hu et al. (19). Two grams of sliced vacuum-packed cooked cured sausages was homogenized in 18 mL of distilled water (pH 7.00), and pH was determined with a digital pH meter (type 361, Systronics, Mumbai, India) calibrated with standard buffer solutions (Merck, Darmstadt, Germany).

Plant material and extraction of EOs. Rhizomes of Curcuma longa (turmeric), fresh finger fruits of Citrus medica (citron), whole air-dried seeds of Carum carvi (caraway), bark of Cinnamomum zeylanicum (cinnamon), and leaves of Eugenia caryophyllata (clove) (50 g of each plant material) were hydrodistilled with 500 mL of distilled water for approximately 2 h in an all-glass Clevenger-type apparatus. The plants from which the EOs were collected in the Shiraz area and identified and authenticated by staff at the Shiraz University Herbarium. The distilled EOs were dried over anhydrous sodium sulfate and stored in tightly closed dark vials at 188C until analysis (26, 29, 39).

Microbiological analysis. To determine the total aerobic bacteria count of the samples, 25 g of each sample was aseptically added to a sterile plastic bag containing 225 mL of sterile physiological saline (9 g/L sodium chloride), homogenized in a lab blender (Stomacher 400, Seward, London, UK) at 200 rpm for 10 min, and then serially diluted in triplicate (1:10) in saline solution. One milliliter of each dilution was inoculated onto plate count agar (PCA; LuQiao Company, Beijing, People’s Republic of China) to obtain the total aerobic counts and onto de Man Rogosa Sharpe (MRS) agar (Merck) to determine LAB counts. PCA plates were incubated at 378C for 24 h, and MRS agar plates were incubated at 308C for 48 h. Colonies were counted and grouped by their macroscopic and microscopic morphological properties. Colonies were isolated, picked, and restreaked three times, and colonies of the most abundant morphotypes were finally grown in pure culture. These cultures were stored at 808C in MRS broth supplemented with glycerol (15%, v/v) for further analysis. Phenotypic characterization of isolates. Phenotypic characterization of isolated bacteria was performed as previously described by Tamang et al. (37). Presumptive LAB isolates were stained for the Gram reaction and the presence of spores, tested for catalase production, oxidase, gas production from glucose and pentose, and carbohydrate fermentation. DNA extraction. To obtain DNA from isolates, 1 mL of cultivated cells in duplicate was centrifuged at 14,000 3 g for 10 min at 48C. The pellet was resuspended in 100 lL of TAE buffer (10 mM Tris HCl, 20 mM acetic acid, and 1 mM EDTA, pH 8.0; CinnaGen, Tehran, Iran), and DNA was extracted with a DNA extraction kit (CinnaGen) following the protocol for purification of genomic DNA from gram-positive bacteria according to the manufacturer’s instructions. The DNA was then eluted with TAE buffer and stored at 208C. Molecular identification of isolated strains. Molecular identification was achieved by PCR amplification of isolated LAB 16s rRNA genes. Reactions were performed in a final volume of 25 lL in a thermal cycler (LifeTouch, Bioer, Hangzhou, People’s Republic of China). The PCR mixture contained 103 PCR buffer (2.5 lL), Taq DNA polymerase (0.5 lL), deoxynucleoside triphosphates (0.5 lL), primers (0.5 lL) 27F (5 0 AGAGTTTGATCCTGGCTCAG-3 0 ) and 1492R (5 0 -ACGG C-TTACCTTGTTACGACT T-3 0 ) (20), 2.5 lL of MgCl2, 2 lL of DNA template, and adequate sterile deionized water to obtain the final volume of 25 lL. The following temperature program was used: initial denaturation at 958C for 3 min; 35 cycles of denaturation at 948C for 40 s, annealing at 528C for 45 s, and elongation at 728C for 50 s; and a final extension at 728C for 4 min. The PCR products were separated by electrophoresis on a 1% (w/ v) agarose gel in 13 TAE buffer at 90 V (constant voltage) for 1 h. The gels were stained with ethidium bromide (1 mg/mL) and photographed under UV light. The amplification products were

GC-FID analysis. A gas chromatography (GC) apparatus (series 7890A, Agilent, Santa Clara, CA) equipped with a flame ionization detector (FID) was used for GC-FID analysis. The GC apparatus was equipped with a fused silica capillary HP-5 column (30 m by 0.32 mm inside diameter; film thickness of 0.25 lm). The injector and detector temperatures were kept at 250 and 2808C, respectively. Nitrogen was used as the carrier gas at a flow rate of 1 mL/min. The oven temperature program was 60 to 2108C at 48C/ min, which was then programmed to 2408C at 208C/min, and finally held isothermally for 8.5 min. The split ratio was 1:50 (13, 40). Agar diffusion assay. The antibacterial activity of EOs was determined using the agar diffusion method described by Ziaee et al. (40). Each LAB isolate was spread plated and cultured overnight at 37 6 28C on MRS agar. Cells were suspended in 5 mL of sterile saline solution, and turbidity was adjusted to a 0.5 McFarland standard (1.5 3 107 CFU/mL). Sterile MRS agar was prepared in 25-mL amounts. After the agar had solidified, 100 lL of bacterial suspension was spread onto the plates. A sterile filter paper disk (6.4 mm) containing 10 lL of each EO was placed on the surface of the plate. Plates were then incubated at 378C for 24 h. The antibacterial activity of the EOs was expressed based on the diameter of the inhibition zone around each EO disk. Assays were performed in triplicate, and values were recorded as the mean 6 standard deviation (SD). MIC and MBC. A microdilution broth susceptibility assay was used to determine the MICs according to the method outlined by Moosavi-Nasab et al. (28). The MIC was defined as the lowest concentration of EO that produced no visible growth. All of the tests were performed in MRS broth supplemented with Tween 80 detergent (final concentration of 0.5%, v/v) to enhance the oil solubility (5, 16, 27). Bacterial strains were cultured overnight at 378C on MRS agar. Test strains were suspended in MRS broth to a final density of 5 3 104 CFU/mL, which was confirmed by plate counts. Geometric dilutions of the EOs at 20 to 0.156 lL/mL were prepared in a 96-well microtiter plate, including one growth control (Mueller-Hinton broth [MHB] plus Tween 80) and one sterility control (MHB plus Tween 80 plus test EO). After incubation, bacterial growth was evaluated by turbidity and a white pellet on the well bottom. To evaluate the bactericidal activity of EOs, 100 lL of each well in which microbial growth was not observed was spread plated on MRS agar, and plates were incubated for 24 to 48 h at 378C. MBC was defined as the lowest concentration of EO that resulted in no surviving bacteria.

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TABLE 1. Changes in pH and microbial counts in samples of vacuum-packed cooked cured sausages during 30 days of storage at refrigerated temperaturesa pH during storage Sample

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 a

Day 1

6.21 6.23 6.19 6.24 6.24 6.21 6.22 6.23 6.20 6.18 6.22 6.21 6.25 6.22 6.23 6.21 6.23 6.19

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.01 0.05 0.02 0.07 0.08 0.02 0.03 0.04 0.08 0.03 0.04 0.02 0.01 0.01 0.06 0.02 0.01 0.02

Total plate count (log CFU/g) Day 30

A A A A A A A A A A A A A A A A A A

a a a a a a a a a a a a a a a a a a

4.67 4.85 5.12 4.74 5.38 4.64 5.35 5.92 4.93 5.57 5.09 4.82 5.67 4.31 5.50 5.47 4.92 5.12

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.02 0.02 0.03 0.01 0.05 0.03 0.04 0.03 0.02 0.03 0.01 0.02 0.06 0.02 0.03 0.02 0.01 0.02

Day 1 B B B B B B B B B B B B B B B B B B

Day 30

,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2

i g e h d i d a f bc e g b j c cd f e

LAB (log CFU/g)

9.44 8.66 8.57 8.76 7.96 9.13 8.07 7.51 8.59 8.22 8.38 9.14 7.53 8.81 8.28 8.18 9.01 8.49

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

Day 1

0.23 0.16 0.17 0.08 0.24 0.33 0.14 0.09 0.08 0.08 0.19 0.28 0.22 0.06 0.16 0.19 0.07 0.11

,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2 ,2

a b b b cd ab c d b c bc ab d b c c b c

Day 30

8.63 8.39 8.25 8.33 7.46 8.88 8.67 8.21 8.09 7.97 7.81 8.24 7.17 8.08 7.56 8.67 8.65 7.54

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.15 0.09 0.29 0.38 0.05 0.17 0.08 0.28 0.32 0.27 0.32 0.17 0.19 0.21 0.21 0.11 0.08 0.07

Values are the mean 6 SD of three replicates. Within each row with means with different uppercase letters are significantly different (P , 0.05). Within each column, means with different lowercase letters are significantly different (P , 0.05).

Statistical analysis. A one-way analysis of variance and Duncan’s multiple range test were used determine the significance of differences (P , 0.05) between means. SAS version 9.1 (SAS Institute, Cary, NC) was used for statistical analysis. All experiments were performed in triplicate.

RESULTS AND DISCUSSION Bacterial enumeration and pH measurements. Both pH and the microbial population changes in the sausage samples collected from the local markets were measured

during the 30-day storage period at 48C (Table 1). The mean pH values decreased significantly (P , 0.05) during storage. The greatest decrease was observed in sample S14 (pH 6.22 to 4.31), and the least decrease was observed in sample S8 (pH 6.23 to 5.92). In previous research, the pH of sliced vacuum-packed cooked ham decreased significantly during storage at 48C from 6.54 to 5.63 on day 7 to day 15 and then decreased slowly from 5.63 to 5.24 on day 15 to day 35 (19). The pH drop may be a result of the growth of LAB.

TABLE 2. Molecular and morphological analysis of LAB isolated from vacuum-packed cooked cured sausages Morphological analysisa

Molecular analysis

Isolate

LAB

ID (%)

Strain code

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 L15 L16

Enterococcus faecium Lactobacillus dextrinicus E. faecium Leuconostoc mesenteroides Lactobacillus sakei E. faecium L. sakei L. reuteri Pediococcus dextrinicus Weissella viridescens L. mesenteroides E. faecium E. faecium Lactobacillus plantarum L. mesenteroides Lactobacillus curvatus

100 98 95 96 97 98 99 96 98 100 100 98 98 99 97 96

SHU1592 SHU68 SHU1563 SHU1370 SHU1387 SHU2412 SHU9622 SHU1872 SHU1365 SHU6811 SHU1396 SHU0913 SHU071 SHU3455 SHU1368 SUCC1365

a

a b b ab c a ab b b bc bc ab d b bc ab ab c

Accession no.

Pentose fermentation

Gas production

Catalase

Oxidase

Gram reaction

Spores

MG515293 MG063620 MG515288 MG515291 MG515286

þ þ þ þ þ þ

þ þ þ þ þ þ

þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ

MG515289 MG515282 MG515287 MG515281 MG515292 MG515294 MG515284 MG515290 MG515295

, negative reaction to the tests; þ, positive reaction to the tests.


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TABLE 3. Chemical compositions of essential oils obtained by hydrodistillation using GC-FID Composition (%) Row

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Total (%)

Component

RI

Chemical formula

a-Thujene a-Pinene d-3-Carene Camphene b-Pinene Myrcene a-Phellandrene d-3-Carene a-Terpinene Terpinolene p-Cymene Limonene 1,8-Cineole b-Phellandrene 1,8-Cineole c-Terpinene 1,3-Cyclopentadiene Linalool Terpinolene a-Terpineol cis-Dihydrocarvone Nerol Geraniol Neral Carvone E-cinnamaldehyde Geranial Anethole Eugenol E-cynnamyl acetate b-Caryophyllene Neryl acetate a-Humulene ar-curcumene o-Methoxycinnamicaldehyde b-Sesquiphellandrene Caryophyllenyl alcohol ar-turmerol Caryophyllene oxide Viridiflorol Curzerenone Epi-a-cadinol c-Eudesmol ar-turmerone a-Turmerone Apiole b-Turmerone Equilin

929 932 934 945 969 986 1004 1008 1016 1018 1021 1025 1026 1027 1030 1041 1071 1083 1087 1143 1208 1215 1228 1232 1244 1247 1249 1288 1359 1413 1418 1432 1452 1482 1492 1523 1572 1582 1583 1590 1605 1637 1648 1662 1667 1678 1703 2049

C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H14 C10H16 C10H18O C10H16 C10H18O C10H16 C5H6 C10H18O C10H16 C10H18O C10H16O C10H18O C10H18O C10H16O C10H14O C9H8O C10H16O C10H12O C10H12O2 C11H12O2 C15H24 C12H20O2 C15H24 C15H22 C10H10O2 C15H24 C15H26O C15H22O C15H24O C15H26O C15H18O2 C15H26O C15H26O C15H20O C15H22O C12H14O4 C15H22O C18H20O2

The total aerobic bacteria and LAB counts of the sausage samples increased significantly from ,2 to .7 log CFU/g from day 1 to 30 at 48C. Hu et al. (19) reported that even though no colonies were observed in sliced vacuumpacked cooked ham on day 0, the counts on PCA and MRS had reached 6.27 and 6.04 log CFU/g, respectively,

C. carvi

C. zeylanicum

C. longa

C. medica

2.18

0.1 0.2

4.82

0.1 0.2 0.2 0.4 1.3 0.1 0.1 5.23 38.9

0.14 0.2 1.03 0.54 0.94

0.1 0.2

0.2 6.5 0.1

2.85

0.9

3.32

0.84 0.6 3.2 0.5

6.46

0.1 2.4

0.1 11.03 3.18 6.28

0.3

0.1 0.2 0.2

0.1 0.2 2.3 0.1 0.3 0.1 29.7

E. caryophyllata

1.4 0.3

60.41 0.3

21.34 2.36 0.18

0.1 0.2 0.3 0.1

1.44 0.1 0.1 0.2 0.1

1.42

0.1 0.1 75.3

0.46 0.74

14.08

1.17 0.2

0.3 0.1

0.91 1.18 1.04

0.1

3.19 2.01 3.5

2.78 2.27 35.08

0.27

1.88 1

3.63 1.4 0.24 1.1 0.34

0.47 0.2 1.1 0.8 0.5 14.3 39.07

13.8 14.9 96.13

0.91 92.32

90.58

96.51

94.47

by day 3. Significant increases in populations on both media were observed from day 7 to day 15, then populations stabilized at 8 log CFU/g until the end of storage. LAB are the major group of spoilage bacteria that develop on various types of vacuum-packed meats and meat products (2, 34).

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TABLE 4. Inhibition zones of essential oils against LAB isolated from vacuum-packed cooked cured sausagesa Inhibition zone diam (cm) C. longa

LAB

L. curvatus W. viridescens L. mesenteroides E. faecium L. reuteri L. dextrinicus L. sakei P. dextrinicus a

11.2 16 9 7.8 9.2 8.6 8.2 9

6 6 6 6 6 6 6 6

2.39 4.42 0.71 0.45 0.45 0.55 1.30 0.71

C. carvi

ab a E bc D c E bc D bc D bc C bc C C

30.4 22 27.2 14.6 24.2 24.4 12.8 18

6 6 6 6 6 6 6 6

5.41 0.71 0.84 0.55 1.92 1.14 0.84 1.22

C. medica A B B B B B C B

a b a c ab ab c b

19.8 18.4 21.6 16.4 14 22 19.6 17.6

6 6 6 6 6 6 6 6

1.92 2.07 4.04 2.30 1.58 3.32 4.34 1.67

B

E. caryophyllata

ab ab a ab b a ab ab

B C C B C B B B

11.6 11 12.6 11.8 11 12 11 10

6 6 6 6 6 6 6 6

0.55 0.00 0.55 0.45 0.00 0.00 0.71 0.00

ab c D a C ab D c C a C bc C d C

D

C. zeylanicum

29 29.8 31 30.8 30.8 29.8 29 29.6

6 6 6 6 6 6 6 6

0.00 0.45 0.00 0.45 0.45 0.45 0.00 0.55

A A A A A A A A

b ab a a a ab b ab


DNA extraction and bacteria identification. A total of 106 colonies isolated on MRS medium from the vacuumpacked cooked cured sausages samples during storage were analyzed by conventional microbiological analysis, and 16 isolates were identified representing eight species: L. curvatus, Weissella viridescens, L. mesenteroides, Enterococcus faecium, Lactobacillus reuteri, Lactobacillus dextrinicus, L. sakei, and Pediococcus dextrinicus. The 16S rRNA sequence comparisons with those in GenBank (NCBI, Bethesda, MD) are shown in Table 2. The sequences from these species were 96 to 99% similar to the sequences listed in the GenBank database. Chemical characterization and quantification of EO components. The analyses of the chemical composition of the EOs are presented in Table 3. According to GC-FID analysis, 22 compounds were identified in the EO from C. carvi (96.1% of the total C. carvi EO). The main components were limonene (38.9%), carvone (29.8%), and apiole (13.8%). These results were consistent with those of Jiang et al. (22), who reported limonene (43.5%), carvone (32.6%), and apiole (15.1%) as major components of C. carvi EO. In another study, Laribi et al. (24) determined the chemical composition of Tunisian, German, and Egyptian C. carvi EO. They identified 41 volatile compounds in the EOs from seed of the three C. carvi ecotypes. The chemical class characterization of C. carvi EO revealed the prevalence of

ketones (61.0 to 77.9%), represented by carvone as the main constituent, and the monoterpene hydrocarbons (17.5 to 33.8%) as the second-most prevalent class, with limonene as the principal constituent. The results obtained by GC and GC-FID analysis indicated 19 compounds (92.3%) in C. zeylanicum EO. The major components were E-cinnamaldehyde (60.4%) and linalool (6.5%). In previous studies, cinnamaldehyde, eugenol, and camphor have been identified as the main components of C. zeylanicum bark, leaf, and root bark oil (7, 32). Such variations in the chemical profile of C. zeylanicum may be due to geographical variation, cultivar differences, time of plant harvest, analysis and detection methods, drying methods, and plant parts used for isolation (28, 31), which may affect the bioactivity of EOs. Twenty-three constituents (90.6%) were identified in C. longa EO. The main components in the C. longa EO were aturmerone (C15H22O), b-turmerone (C15H22O), and arturmerone (C15H20O) at 39.1, 14.9, and 14.3%, respectively. Gounder and Lingamallu (14) compared the chemical composition of C. longa in three states (fresh, dried, and cured). They identified 28 compounds in the volatile oils of fresh rhizome, with the major components a-turmerone (33.5%), ar-turmerone (21%), and b-turmerone (18.9%). Fourteen compounds were identified in dried rhizome oil, with the major compounds ar-turmerone (30.3%), aturmerone (26.5%), and b-turmerone (19.1%). In cured

TABLE 5. MICs of essential oils against LAB isolated from vacuum-packed cooked cured sausagesa MICs (lL/mL) C. longa

LAB


7.50 8.33 7.50 7.50 9.00 8.33 9.66 8.66

6 6 6 6 6 6 6 6

0.54 0.57 0.00 0.54 0.00 0.57 0.57 0.57

C. carvi

b a C b C b B a A ab A a A ab AB B

6.33 6.33 5.66 6.33 6.66 5.66 5.00 6.66

6 6 6 6 6 6 6 6

057 B a 0.57 C a 0.57 D ab 0.57 D a 0.57 D a 0.57 B ab 0.00 C b 0.57 B a

C. medica

7.33 7.66 8.33 9.00 8.00 9.33 8.66 9.00

6 6 6 6 6 6 6 6

0.57 0.57 0.57 0.00 0.00 0.57 0.57 0.00

b b B ab B a C b A a A ab A a AB B

E. caryophyllata

8.5 9.00 10.00 10.00 10.33 9.50 8.00 9.33

6 6 6 6 6 6 6 6

0.54 0.00 0.00 0.00 0.57 0.54 0.00 0.57

bc b A a A a A a A a B c A ab A A

C. zeylanicum

4.66 5.33 4.66 4.66 6.33 5.66 5.00 5.00

6 6 6 6 6 6 6 6

0.57 0.57 0.57 0.57 0.57 0.57 0.00 0.00

b ab E b E b D a B ab C b C b C

D



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TABLE 6. MBCs of essential oils against LAB isolated from vacuum-packed cooked cured sausagesa MBCs (lL/mL) LAB


C. longa

11.00 12.66 11.00 11.00 13.50 12.33 14.50 12.00

6 6 6 6 6 6 6 6

0.00 0.57 0.00 0.00 0.54 0.57 0.54 0.00

C. carvi

c b C c B c B ab B b A a C b C B

7.33 7.00 7.66 8.00 8.00 7.33 6.50 8.33

6 6 6 6 6 6 6 6

0.57 0.00 0.57 0.00 0.00 0.57 0.54 0.57

C. medica

bc c D b C ab C ab C bc C c C a D C

12.00 13.50 13.33 15.00 15.00 13.66 13.00 13.00

6 6 6 6 6 6 6 6

0.00 0.54 0.57 0.00 0.00 0.57 0.00 0.00

B

E. caryophyllata

d

AB

bc

b a A a B

A

AB B B

c c

b

14.33 14.66 15.00 15.00 14.50 14.50 15.00 14.00

6 6 6 6 6 6 6 6

0.57 0.57 0.00 0.00 0.54 0.54 0.00 0.00

b ab A a A a AB ab A ab A a A b A A

C. zeylanicum

7.33 7.00 6.00 6.33 7.66 8.50 6.50 6.33

6 6 6 6 6 6 6 6

0.57 0.00 0.00 0.57 0.57 0.54 0.54 0.57

ab b E d D c C ab C a C bc D c D C


rhizome oil, only 18 compounds were identified, with the major components ar-turmerone (28.3%), a-turmerone (24.8%), and b-turmerone (21.1%). Seventeen compounds (96.5%) were identified in the C. medica EO, and the main components, constituting 78.6% of the total EO, were limonene (35.1%), c-terpinene (21.3%), 3-carene (11.0%), b-pinene (6.3%) and a-thujene (4.8%). Twenty-two compounds (94.5%) were identified in the E. caryophyllata EO, and the main components were eugenol (75.3%) and b-caryophyllene (14.1%). Dzamic et al. (10) found that eugenol was the main constituent (78.6%) of E. caryophyllata EO followed by b-caryophyllene (15.6 %). Mahboubi and Mahboubi (25) detected 26 compounds, representing 99.8% of the total E. caryophyllata EO, in which the eugenol (68.9%) was the major compound followed by b-caryophyllene (12.6%) and eugenol acetate (12.4%).

Antimicrobial susceptibility of isolated LAB: disk diffusion assay. The antimicrobial activities of EOs against LAB isolated from the vacuum-packed sausages were determined with solid diffusion assays (Table 4). The diameters of the inhibition zones were ranked as follows: 8 mm, not sensitive; 8 to 14 mm, sensitive; 14 to 20 mm, very sensitive; and 20 mm, extremely sensitive (11). The selected EOs had various levels of antimicrobial activity. The EOs inhibited the growth of bacterial strains, with zone diameters of 7.8 to 31 mm, depending on the susceptibility of the tested species. The LAB isolated from vacuumpacked sausages were extremely sensitive to C. zeylanicum EO and very sensitive to C. carvi and C. medica EOs. The C. longa EO had the least activity against the eight LAB, with zone diameters of 7.8 to 16 mm, depending on the susceptibility of the tested species. E. caryophyllata EO had moderate antibacterial activity against the LAB, with zone diameters of 10 to 12.6 mm. Moosavi-Nasab et al. (28) indicated that differences in inhibition zone diameters with respect to various EOs may be influenced by the solubility of the EO and its ability to diffuse from the disk into the agar medium. Therefore, to obtain more precise data about the antimicrobial properties of the tested EOs, the bacteriostatic activity (MIC) and

bactericidal activity (MBC) of five EOs were determined (Tables 5 and 6). The EO most effective against the tested LAB was C. zeylanicum, followed by C. carvi and C. medica, and the least effective was C. longa. The C. zeylanicum EO had significantly higher antimicrobial activity, with the lowest MICs of 4.7 to 6.3 lL/mL. The most susceptible bacterium was L. mesenteroides, with a C. zeylanicum EO MIC of 4.7 lL/mL and a C. longa EO MIC of 7.5 lL/mL (Table 5). Regarding bactericidal activity, all tested LAB were more susceptible to C. zeylanicum EO followed by C. carvi and C. medica EOs (Table 6). Results in the present study are comparable to those of Chao et al. (6), who found that C. zeylanicum EO fully inhibited the growth of some gram-positive and gramnegative bacteria, fungi, and yeasts, and the main component, cinnamaldehyde, has been shown to be particularly effective against some species of gram-positive and gramnegative bacteria (12, 21, 30, 35). Cinnamaldehyde and eugenol inhibit production of an essential bacterial enzyme and/or cause damage to the bacterial cell wall (8, 17). Therefore, the high antimicrobial activity of cinnamon oil may be due to the presence of high concentrations of cinnamaldehyde. Reported MICs of cinnamaldehyde of 0.78 to 12.5 lL/mL have prevented the growth of a broad range of microorganisms, including Aeromonas hydrophila, Bacillus cereus, Enterococcus faecalis, Escherichia coli, E. coli O157:H7, Listeria monocytogenes, Micrococcus luteus, Pseudomonas aeruginosa, Salmonella Enteritidis, Staphylococcus aureus, Candida albicans, Saccharomyces cerevisiae, and Zygosaccharomyces rouxii (33). Even though cinnamaldehyde has antimicrobial activity against E. coli O157:H7 and Salmonella Typhimurium at concentrations similar to those of carvacrol and thymol, it did not disintegrate the outer membrane or deplete the intracellular ATP pool of these microorganisms (17). Wendakoon and Sakaguchi (39) suggested that the carbonyl group of cinnamaldehyde binds to proteins, preventing the action of amino acid decarboxylases in Enterobacter aerogenes. Smid et al. (36) observed damage to the cytoplasmic membrane of S. cerevisiae treated with cinnamaldehyde, leading to excessive leakage of metabolites and enzymes from the cell and eventual loss of viability.

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Eugenol inhibits the growth of B. cereus, Campylobacter jejuni, E. coli, E. coli O157:H7, L. monocytogenes, Penicillium digitatum, Pseudomonas fluorescens, Salmonella enterica, Salmonella Enteritidis, and S. aureus (12), with MICs of 12.5 to 50 lL/mL (33). Sublethal concentrations of eugenol inhibit the production of amylase and proteases by B. cereus. Cell wall deterioration and a high degree of cell lysis were also noted (38). The hydroxyl group on eugenol is thought to bind to proteins, preventing enzyme action in E. aerogenes (39). LAB adversely affect the aroma and taste of vacuumpacked cooked cured sausage products. Some EOs have high antimicrobial activity against these spoilage organisms and may be useful for extending product shelf life. In the present study, eight LAB species were isolated from 18 spoiled vacuum-packed sausages. The extracts of the common food spices cinnamon, caraway, citron, and clove were capable inhibiting these spoilage microorganisms. These extracts are widely used in the food industry and are generally regarded as safe. Hence, they could be used as natural preservatives acceptable by the food industry.

REFERENCES 1. Bakkali, F., S. Averbeck, D. Averbeck, and M. Idaomar. 2008. Biological effects of essential oils—a review. Food Chem. Toxicol. 46:446–475. 2. Björkroth, J., and H. Korkeala. 1996. rRNA gene restriction patterns as a characterization tool for Lactobacillus sake strains producing ropy slime. Int. J. Food Microbiol. 30:293–302. 3. Borch, E., M.-L. Kant-Muermans, and Y. Blixt. 1996. Bacterial spoilage of meat products and cured meat. Int. J. Food Microbiol. 33:103–120. 4. Burt, S. 2004. Essential oils: their antibacterial properties and potential applications in foods—a review. Int. J. Food Microbiol. 94:223–253. 5. Cannas, S., D. Usai, A. Pinna, S. Benvenuti, R. Tardugno, M. Donadu, S. Zanetti, J. Kaliamurthy, and P. Molicotti. 2015. Essential oils in ocular pathology: an experimental study. J. Infect. Dev. Ctries. 9:650–654. 6. Chao, S. C., D. G. Young, and C. J. Oberg. 2000. Screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses. J. Essent. Oil Res. 12:639–649. 7. Chericoni, S., J. M. Prieto, P. Iacopini, P. Cioni, and I. Morelli. 2005. In vitro activity of the essential oil of Cinnamomum zeylanicum and eugenol in peroxynitrite-induced oxidative processes. J. Agric. Food Chem. 53:4762–4765. 8. Di Pasqua, R., G. Betts, N. Hoskins, M. Edwards, D. Ercolini, and G. Mauriello. 2007. Membrane toxicity of antimicrobial compounds from essential oils. J. Agric. Food Chem. 55:4863–4870. 9. Dykes, G., T. Cloete, and A. Von Holy. 1995. Taxonomy of lactic acid bacteria associated with vacuum-packaged processed meat spoilage by multivariate analysis of cellular fatty acids. Int. J. Food Microbiol. 28:89–100. 10. Dzamic, A., M. Soković, M. S. Ristic, S. Grijic-Jovanovic, J. Vukojevic, and P. D. Marin. 2009. Chemical composition and antifungal activity of Illicium verum and Eugenia caryophyllata essential oils. Chem. Nat. Compd. 45:259–261. 11. Elaissi, A., K. H. Salah, S. Mabrouk, K. M. Larbi, R. Chemli, and F. Harzallah-Skhiri. 2011. Antibacterial activity and chemical composition of 20 Eucalyptus species’ essential oils. Food Chem. 129:1427– 1434. 12. Friedman, M., P. R. Henika, and R. E. Mandrell. 2002. Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica. J. Food Prot. 65:1545–1560.


13. Gahruie, H. H., E. Ziaee, M. H. Eskandari, and S. M. H. Hosseini. 2017. Characterization of basil seed gum–based edible films incorporated with Zataria multiflora essential oil nanoemulsion. Carbohydr. Polym. 166:93–103. 14. Gounder, D. K., and J. Lingamallu. 2012. Comparison of chemical composition and antioxidant potential of volatile oil from fresh, dried and cured turmeric (Curcuma longa) rhizomes. Ind. Crops Prod. 38:124–131. 15. Grant, G., A. McCurdy, and A. Osborne. 1988. Bacterial greening in cured meats: a review. Can. Inst. Food Sci. Technol. J. 21:50–56. 16. Hammer, K. A., C. F. Carson, and T. V. Riley. 1998. In-vitro activity of essential oils, in particular Melaleuca alternifolia (tea tree) oil and tea tree oil products, against Candida spp. J. Antimicrob. Chemother. 42:591–595. 17. Helander, I. M., H. L. Alakomi, K. Latva-Kala, T. Mattila-Sandholm, I. Pol, E. J. Smid, L. G. Gorris, and A. von Wright. 1998. Characterization of the action of selected essential oil components on gram-negative bacteria. J. Agric. Food Chem. 46:3590–3595. 18. Holley, R. A., and D. Patel. 2005. Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol. 22:273–292. 19. Hu, P., G. Zhou, X. Xu, C. Li, and Y. Han. 2009. Characterization of the predominant spoilage bacteria in sliced vacuum-packed cooked ham based on 16S rDNA-DGGE. Food Control 20:99–104. 20. Jackson, B. M., C. M. Beck-Sague, L. A. Bland, M. J. Arduino, L. Meyer, and W. R. Jarvis. 1994. Outbreak of pyrogenic reactions and gram-negative bacteremia in a hemodialysis center. Am. J. Nephrol. 14:85–89. 21. Jantan, I. B., B. A. Karim Moharam, J. Santhanam, and J. A. Jamal. 2008. Correlation between chemical composition and antifungal activity of the essential oils of eight Cinnamomum species. Pharm. Biol. 46:406–412. 22. Jiang, Y., N. Wu, Y. J. Fu, W. Wang, M. Luo, C. J. Zhao, Y. G. Zu, and X. L. Liu. 2011. Chemical composition and antimicrobial activity of the essential oil of rosemary. Environ. Toxicol. Pharmacol. 32:63– 68. 23. Kotzekidou, P., and J. Bloukas. 1996. Effect of protective cultures and packaging film permeability on shelf-life of sliced vacuumpacked cooked ham. Meat Sci. 42:333–345. 24. Laribi, B., K. Kouki, T. Bettaieb, A. Mougou, and B. Marzouk. 2013. Essential oils and fatty acids composition of Tunisian, German and Egyptian caraway (Carum carvi L.) seed ecotypes: a comparative study. Ind. Crops Prod. 41:312–318. 25. Mahboubi, M., and M. Mahboubi. 2015. Chemical composition, antimicrobial and antioxidant activities of Eugenia caryophyllata essential oil. J. Essent. Oil Bear. Plants 18:967–975. 26. Mäkelä, P. M., H. J. Korkeala, and J. J. Laine. 1992. Survival of ropy slime–producing lactic acid bacteria in heat processes used in the meat industry. Meat Sci. 31:463–471. 27. Mondello, F., A. Girolamo, M. Scaturro, and M. L. Ricci. 2009. Determination of Legionella pneumophila susceptibility to Melaleuca alternifolia Cheel (tea tree) oil by an improved broth micro-dilution method under vapour controlled conditions. J. Microbiol. Methods 77:243–248. 28. Moosavi-Nasab, M., M. Jamal Saharkhiz, E. Ziaee, F. Moayedi, R. Koshani, and R. Azizi. 2016. Chemical compositions and antibacterial activities of five selected aromatic plants essential oils against foodborne pathogens and spoilage bacteria. J. Essent. Oil Res. 28:241– 251. 29. Moosavi-Nasab, M., E. Shad, E. Ziaee, S. H. A. Yousefabad, M. T. Golmakani, and M. Azizinia. 2016. Biodegradable chitosan coating incorporated with black pepper essential oil for shelf life extension of common carp (Cyprinus carpio) during refrigerated storage. J. Food Prot. 79:986–993. 30. Ooi, L. S., Y. Li, S. L. Kam, H. Wang, E. Y. Wong, and V. E. Ooi. 2006. Antimicrobial activities of cinnamon oil and cinnamaldehyde from the Chinese medicinal herb Cinnamomum cassia Blume. Am. J. Chin. Med. 34:511–522. 31. Prakash, B., A. Kedia, P. K. Mishra, and N. Dubey. 2015. Plant essential oils as food preservatives to control moulds, mycotoxin


32.

33.

34. 35.


contamination and oxidative deterioration of agri-food commodities—potentials and challenges. Food Control 47:381–391. Raina, V., S. Srivastava, K. Aggarwal, S. Ramesh, and S. Kumar. 2001. Essential oil composition of Cinnamomum zeylanicum Blume leaves from Little Andaman, India. Flavour Frag. J. 16:374–376. Sanla-Ead, N., A. Jangchud, V. Chonhenchob, and P. Suppakul. 2012. Antimicrobial activity of cinnamaldehyde and eugenol and their activity after incorporation into cellulose-based packaging films. Pack. Technol. Sci. 25:7–17. Schillinger, U., and F. K. Lücke. 1987. Identification of lactobacilli from meat and meat products. Food Microbiol. 4:199–208. Shan, B., Y. Z. Cai, J. D. Brooks, and H. Corke. 2007. Antibacterial properties and major bioactive components of cinnamon stick (Cinnamomum burmannii): activity against foodborne pathogenic bacteria. J. Agric. Food Chem. 55:5484–5490.

1393

36. Smid, E., L. Hendriks, H. Boerrigter, and L. Gorris. 1996. Surface disinfection of tomatoes using the natural plant compound transcinnamaldehyde. Postharvest Biol. Technol. 9:343–350. 37. Tamang, J. P., B. Tamang, U. Schillinger, C. M. Franz, M. Gores, and W. H. Holzapfel. 2005. Identification of predominant lactic acid bacteria isolated from traditionally fermented vegetable products of the Eastern Himalayas. Int. J. Food Microbiol. 105:347–356. 38. Thoroski, J., G. Blank, and C. Biliaderis. 1989. Eugenol induced inhibition of extracellular enzyme production by Bacillus subtilis. J. Food Prot. 52:399–403. 39. Wendakoon, C. N., and M. Sakaguchi. 1995. Inhibition of amino acid decarboxylase activity of Enterobacter aerogenes by active components in spices. J. Food Prot. 58:280–283. 40. Ziaee, E., M. Razmjooei, E. Shad, and M. H. Eskandari. 2018. Antibacterial mechanisms of Zataria multiflora Boiss. essential oil against Lactobacillus curvatus. LWT Food Sci. Technol. 87:406–412.