Research Article

Antibacterial Effect of Bay Leaf (Laurusnabilis) Aqueous Extract and its Nano-Emulsion on Some Pathogenic Bacteria

Suhair Sh. Al-Siraj1, Jihan M. Badr2 and Dalia M.A. El-Masry3*

1Biology Department, Faculty of Science, Mustansiriyah University, Bagdad, Iraq; 2Poultry Diseases Department, Animal Health Research Institute (AHRI), Agricultural Research Center (ARC), Giza, Egypt; 3Nanotechnology Research unit, Animal Health Research institute, Agricultural Research center (ARC), Giza, Egypt.

Received | April 05, 2024; Accepted | July 07, 2024; Published | July 29, 2024

*Correspondence | Dalia M.A. EL-MASRY, Nanotechnology Research Unit, Animal Health Research Institute, ARC, Giza, Egypt; Email: [email protected]

Citation | Al-Siraj S.S., Badr J.M., El-Masry D.M.A. 2024. Antibacterial effect of bay leaf (laurusnabilis) aqueous extract and its nano-emulsion on some pathogenic bacteria. Adv. Anim. Vet. Sci. 12(9): 1670-1680.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.9.1670.1680

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Chemical preservatives used to prevent food damage and food poisoning bacteria have been proven to have negative consequences, including human health risks, chemical remnant in nourishment and fodder systems, and the establishment of microorganism’s impedance to the chemicals employed. As a result of these concerns, the need for alternative preservatives that are possibly effective, healthful, safe, and natural has grown.

Bay leaves belong to the family Lauraceae and are endemic to the Mediterranean region. It has been used for over a thousand years and is a key part of many cultural traditions, including cookery (Parthasarathy et al., 2008). Due to their many uses, such as flavouring agents, antioxidants, and antimicrobials, phenolic compounds found in spices, herbs, and essential oils have recently come to light. Every essential oil has different properties depending on the country of harvest, altitude, sunlight duration, and harvesting circumstances. The Bay leaves essential oil contain sabinen, α-terpinyl acetate, methyl eugenol, linalool, eucalyptol e and carvacrol. These L. nobilis essential oil has a variety of pharmacological actions, including cytotoxic, immunomodulatory properties and antifungal effects. In addition to antibacterial action against foodborne pathogens that are both Gramme positive and Gramme negative. (Esherichia coli, Staphylococcus aureus, Salmonella species, Listeria monocytogenes), spoilage bacteria (Pseudomonas aeroginosa) (Sırıke et al., 2018).

Nanoemulsions (NE) are made from droplets in nano-sized emulsifier’s stabilization. They are often more permeable and stable than traditional emulsions owing to their unique particle sizes and larger surface area. Because of their distinct antibacterial qualities, capacity to boost drug solubility, stability, and bioavailability, and potential for organ and cellular targeting, NE have been recognized as a promising mode of antimicrobial administration., ability to target biofilms, and potential to overcome antimicrobial resistance. The essential oil-based NE that may provide antimicrobial effects primarily via physical or biochemical pathways without drug-loaded. (Garcia et al., 2022). The nanoparticles have shown to be particularly effective at inhibiting antibiotic-resistant microorganisms like Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis (Gram positive bacteria) and Escherichia coli, Proteus mirabilis, Salmonella, Klebsiellaaerogenes, Pseudomonas aeruginosa, Listeria monocytogenes, Mycobacterium tuberculosis, (Gram negative bacteria) (Cowan 1999; Agarwal et al., 2018; Sırıke et al., 2018).

The phytochemical contents of nanoemulsion may have the potential to combine with the bacterial cell membrane lipids, perturbing the basic cell structures and disrupting the microbial cell’s equilibrium. These chemicals may release of cell content lead to bacterial cell death (Yazgan et al., 2019). Whereas nanoemulsion might be considered an effective method for essential oils to increase their physicochemical stability and prevent volatilization, it could also combined with microbial cells, resulting in their elimination (Matos et al., 2019 ; Yazgan et al., 2019).

NE is often used to improve the shelf life of food products owing to its antibacterial effect against pathogenic microbial because of their very small size, NE enhances the passive absorption processes of cells, lowering mass transfer resistances and increasing antibacterial capability (Siraj et al., 2023).

One of the most helpful techniques for assessing in vitro cell viability when using the broth microdilution method or microtiter plate design is the (MTT) colorimetric assay (Houdkova et al., 2017). This method has been used to investigate drug interactions against fungi (TeDorsthorst, et al., 2002) and bacteria (Rondevaldova et al., 2017), as well as EO antimicrobial susceptibility (Houdkova et al., 2017; Ye et al., 2013).

The aim of this study to evaluate the antibacterial effect of Bay leaf (Laurus nabilis) aqueous extract and nano-emulsion on selected gram positive and gram negative bacteria using thiazolyl blue tetrazolium bromide (MTT) assay and their impact on the expression of certain genes related to virulence and resistance to antibiotics in both positive and negative gram bacteria.

MATERIALS AND METHODS

Preparation of Aqueous Solution of Bay Leaf (Laurus Nobilis)

For this study bay dry leaves were acquired from Egyptian supermarkets and then they were fine grinned. Boiling 2 g of fine ground plant material in 20 ml distilled water for 20 minutes in a flask. Then The flask was taken off the fire and set aside to cool to get clean extract, the contents of the flask were filtered by filter paper and residue discarded. (Marwaha et al., 2015)

Preparation Bay Leaf (Laurus Nobilis) Nanoparticles

Aqueous extract of Bay leaf (Laurusnobilis), deionized water Tween (80) purchasing from the Sigma-Aldrich Co. Preparation of Bay leaf (laurusnobilis) micro-emulsion (40%) Tween 80 ml, 60ml water and 40 ml aqueous solution extract were done in nanotechnology Research and synthesis unit by using method according to (Rao and McClements 2011)

Characterization of Nanoparticles

With HRTEM high-resolution transmission electron microscopy, a 1400F JEM with 80 keV beam energy, () observations was made for particle size, shape and homogenous.

Test Microorganisms

Poultry clinical bacterial isolates which were formerly identified biochemically and serologically as Staphylococcus aureus, Pseudomonas aeruginosa and E. coli were gained from bacteriology unit of poultry diseases section, Animal Health Research Institute, Egypt. Escherichia coli and Pseudomonas aeruginosa strains was used to represent pathogenic gram negative bacteria while Staphylococcus aureus strain was utilized as gram-positive bacteria. A colony of each relevant bacteria was chosen using a sterile wire loop and

 

 

suspended in a 5cc sterile Brain heart infusion liquid medium and incubated for 24 hours at 37 °C. Each active culture’s optical density was adjusted to 0.1 at 625 nm for the

purpose of standardising each inoculum using fresh broth. This resulted in a standard inoculum that contained 106 colony forming units (CFU) per millilitre. (Alzoreky, and Nakahara, 2003).

Determination of Antibacterial Activity by MIC Using Thiazolyl Blue Tetrazolium Bromide (MTT) Assay

The (MTT) technique was utilized by (Raquena et al., 2019). In brief, a sterile 96-well microtitration plate received 100 μl Muller Helton broth the 100 μl of stock solution of both the aqueous extract or the nanoemulsion of Bay leaf was added to the first well and subsequent two-fold dilutions were performed in rows on the plate, yielding concentrations from 100 to 0.781 mg/ml for every extract. Added 100 μl of 1-3×105 CFU/ml concentration of bacterial inoculum at all wells. The plates were incubated to 24 hr. at (37) °C. After incubation, all wells received 10 μl from the MTT solution, and the plates were re-incubated for 4 hr. the first two rows of the plate were kept as negative (without bacterial and without plant extract inoculation inoculation) and positive control (without plant extract inoculation) rows The MIC values were found to be the lowest active chemical concentrations at which no purple colour was seen. The test were applied in three replicate for each tested plant extract against each bacteria.

PCR Analysis

DNA was extracted from S. aureus and E.coli isolates and was checked for the organization of mecA, blaZ, coa, Sea, genes and Stx2 ,iss ,Mcr1, floR , genes DNA extraction was doing using QIAamp DNA mini Kit(Qiagen Germany- GmbH) . Oligonucleotide primers were supplied from Metabion (Germany).200 μl of specimen suspension were treated at 560 °C to 10 min. with 10 μl from proteinase K and 200 μl of lysis buffer. The lysate was then given 200 μl of 100% ethanol. The samples were then centrifuged and washed. With the assistance of 100 μl of elution buffer, DNA was extracted.

(Table 1) below lists the primers that were given by Metabion (Germany). A 25μl reaction including 12.5 μl of Emerald AMP Max PCR master mix (Takara, Japan), 6 μl of DNA template, 1 μl of each primer at aconcentration of 20 pmol, and 4.5 μl of water was utilised to employ the primers. The product of PCR was separated via gel electrophoresis using 1.5% agarose gel (Applichem, Germany, GmbH) stained with Ethidium bromide and photographed with a gel documentation system under ultraviolet light.

RT-PCR Analysis

RNA Extraction

To prevent RNA degradation, one volume (0.5 ml) of the harvested culture’s broth was mixed with a double volume (1 ml) of the RNAprotect Bacteria Reagent (Qiagen, Germany, GmbH). The mixture was then vortexed, allowed to sit at room temperature for five minutes, then centrifuged for ten minutes at 8000 rpm. They decanted the supernatant. Subsequently, the pellet was mixed with 200 µl of TE buffer that included 1 mg/ml of Lysozyme (Biochemica, Applichem). Additionally, 700 µl of RLT buffer was added, with 10 μl of β-mercaptoethanol per millilitre. After adding 500 μl of 100% ethanol, the procedure was finished in accordance with the QIAamp RNeasy Mini kit’s Enzymatic Lysis of Bacteria protocol (Qiagen, Germany, GmbH). RT-PCR using SYBR green.

First, 0.5 µl of each primer with a concentration of 20 pmol was used on a 25 µl reaction that also included 12.5 µl of the 2x QuantiTect SYBR Green PCR Master Mix (Qiagen, Germany, GmbH), 0.25 µl of RevertAid Reverse Transcriptase (200 U/µL) (Thermo Fisher), 8.25 µl of water, and 3 µl of RNA template. Using a Stratagene MX3005P real-time PCR equipment, the reaction was carried out as seen in (Table 2)

Data Analysis

The SYBR green rt-PCR analysis. Software from stratagene MX3005P was used to obtain ct values and amplification curves. Using the ratio (2-DDct) to compare each specimen’s CT with that of the positive control group, the “ΔΔCt” approach (Yuan et al., 2006) was utilised to estimate the variance in gene expression on the RNA of the various samples. Whereas ΔΔCt = ΔCtreference - ΔCttargetΔCt reference = Δ control - Δ treatment, . ΔCttarg

RESULTS AND DISCUSSION

Size, Shape, AND Size Distribution of Nanoemulsion

The generated image may then be used to estimate the size and shape of the nanoparticles by image analysis. The droplets size of Bay leaf (laurus nobilis) nanoparticles measured 33.3±3.5 nm, under observation of HRTEM (Figure 1a and Figure 1b). There was no aggregation. Size homogeneity and spherical nature.

The Antibacterial Effect of Bay Leaf Extracts

The results of The antibacterial effect of Bay leaf extracts revealed that nanoemulsion had a more antibacterial effect against both gram positive bacteria (S. aureus) and gram negative bacteria (E.coli and Pseudomonas aeruginosa) with MIC values of (12.5, 50and 50 μg/ml) respectively, while the aqueous extract showed MIC values of 50 μg/ml for

 

Table 4: Gene expression detected by Threshold cycles (CT) and Folding rate of resistance genes and virulence of S.aureus before and after treatment with nanoemulsion.

S.aureus							Treatment	Sample No.
blaZ		coa		mecA		16S rRNA
Fold change	CT	Fold change	CT	Fold change	CT	CT
-	21.10	-	20.54	-	20.78	19.11	-	ST1
0.2117	24.45	0.2316	23.76	0.178	24.38	20.22	1	ST2

 

gram positive bacteria (S. aureus) and 100μg/ml for gram negative bacteria (E.coli and Pseudomonas aeruginosa). (Table 4 and 5, and Figure 2 and 3)

Gene expression of virulence and resistance genes before and after treatment with nanoemulsion using the relative threshold cycle (CT) method analyze the results.

 

 

Table 3: MIC value of Bay leaf (laurus nobilis) aqueous plant extracts and nanoparticles against S. aureus, E. coli and Pseudomonas aeruginosa.

Tested Bacteria	MIC value of Laurus nobilis extracts (µg /ml)
	Aqueous extract	Nano emulsion (40%)
S.aureus	50μg/ml	12.5μg/ml
E.coli	100μg/ml	50μg/ml
Pseudomonas aeruginosa	100μg/ml	50μg/ml

 

The results revealed that the nano-emulsion extract of laurus nobilis Sub-MIC ( 25 µg/ml) highly reduced the relative expression of both virulence gene (coA gene) and antibiotic resistant genes (mecA and blaZ genes) of S. aureus with a fold change of 0.2316 , 0.178 and 0.2117 respectively compared to control (Table 3 and Figure 5).

On the other hand, the nano-emulsion extract of laurusnobilis Sub-MIC (100 µg/ml) reduced the relative expression of both virulence gene (Stx2 and issgenes ) and antibiotic resistant genes (floR and Mcr1 genes) of E.coli with a fold change of 0.5285, 0.4323 , 0.6199 and 0.3099 respectively compared to control (Table 4 and Figure 6).

 

 

 

The study was aimed to evaluate the antibacterial effect of Bay leaf (laurus nobilis) aqueous extract and nano-emulsion to compare the effect of the substance in the the normal state and nano state in terms of strength and concentration of them on both positive and negative gram bacteria. The results of this study indicated that both Bay leaf aqueous extract and nanoemulsion have antibacterial action against both gram negative (E. coli and Pseudomonas aeruginosa) and gram-positive bacteria (S. aureus). (Mostafa et al., 2018) showed antibacterial activity of Bay leaf (Laurus nobilis) on E. coli. while (Marwaha et al., 2015) scrutinized antibacterial impact of ground bay leaf and their extracts on S. aureus. On the other hand, (Fidan et al., 2019) noted that the results matched with E. coli but not with S. aureus in the case of plant extract. It was reported that every essential oil properties differ depending on the harvesting country, the length of sunlight, the harvesting conditions, the distillation’s quality, use and storage (Benoit and Saint Gir 2010). Some research have been conducted to determine the antibacterial properties of L. nobilis essential oils. (Ouibrahim et al., 2013) reported that the essential oil of L. nobilis demonstrated strong activity against most of the 22 strains tested. P. aeruginosa was the most resistant strain, while Enterobacter species exhibited the highest sensitivity, with an inhibition diameter of 22.4 mm, 16.8 mm pure oil, and 1/8 dilution.

 

They also showed that 1.8 cineole had a role in this action by exhibiting antibacterial properties against E. coli, P. aeruginosa, and Staphylococcus aureus. Laurel essential oil includes linalool, lactones, oxides (1,8 cineole), and monoterpenes (camphene and alpha-pinene). (Yılmaz et al., 2013) extracted essential oil from Laurus nobilis leaves and identified 51.8% 1,8-cineole, 11.2% α-terpinyl acetate, and 10.1% sabinene as its primary components. They also discovered that L. nobilis essential oil has a strong antibacterial, antifungal, and antioxidant potential. The hydrophobicity of essential oils and their components is one of their most important qualities, as it enables them to separate the lipids in the bacterial cell membrane and those in the mitochondria., causing cell structures to be disrupted and become more permeable. (Sikkema et al., 1994). Usually, Gram negative bacteria are more susceptible to bay leaf essential oil than Gram positive bacteria. (Loäpez et al., 2005). The nature of cellular membrane of the bacterial group is responsible for this resistance. As a result of their exterior features, they have a very hydrophobic surface. (Ouibrahim et al., 2013).

 

Previous research found that the hydrostatic size of nanoemulsion droplets ranged from 239.5 to 357.0 nm depending on oil quantity and composition (Reis et al., 2019). In general, surfactant concentration can influence nanoemulsions, which is indirectly related to droplet size external exposure and oil/water interfacial tension (Sundararajan et al., 2018). It is also influenced by shear forces and turbulence caused by the ultrasonic homogenizer (Mehmood et al., 2017).

In contrast to prior studies, the evaluated LEO nanoemulsion, showed an increase in droplet size (247.52 nm) compared to that reported in (Ozogul et al., 2017) study was (66.02 nm).

Furthermore, tween 80 concentrations decreased from 3% to 1% (w/w). Tween 80, as a small molecule surfactant and because of its hydrophilic-lipophilic balance (HLB), was proven to be an excellent nonionic surfactant for producing oil-in-water emulsions. It was more successful in adsorbing on the droplet surface than polymer-based surfactants (Chu et al., 2020).

In this investigation, it was discovered that the effect of the drug in its nano form is greater in a lower concentration than the ordinary aqueous extract. This result is in agreemrent with that obtained by Al-Hadi (2011) found

that the antibacterial effect of bay leaf extract in the nano form had stronger antibacterial effect than the aqueous extract on S. aureus. (Vijayakumar et al., 2016) reported that the aqueous leaf extract of Laurus nobilis was used in the green production of zinc oxide nanoparticles and Ln-ZnO NPs had higher antibacterial effect against Gram- positive bacteriaS. aureus than against Gram-negative bacteria Pseudomonas aeruginosa.

 

Table 5: Gene expression detected by Threshold cycles (CT) and Folding rate of resistance genes and virulence of E.coli before and after treatment with nanoemulsion.

E. coli									Treatment	Sample No.
Mcr1		floR		iss		Stx2		16S rRNA
Fold change	CT	Fold change	CT	Fold change	CT	Fold change	CT	CT
-	21.90	-	20.78	-	23.33	-	22.61	18.43	-	E1
0.3099	23.75	0.6199	21.63	0.0.4323	24.81	0.5285	23.69	18.59	1	E2

 

Concerning the effect of the plant extracts on gene expression of some antibiotic and virulence genes of the tested microorganisms ,the results in this study revealed that the nano-emulsion extract of laurus nobilis Sub-MIC highly reduced the relative expression of both virulence gene (coA gene) and antibiotic resistant genes (mecA and blaZ genes) of S. aureus. Also, it reduced the relative expression of both virulence gene (Stx2 andissgenes) and antibiotic resistant genes (floR and Mcr1 genes) of E.coli. M any authors detected the effect of different plant extracts on the bacterial virulence and antibiotic resistant genes. (Wahdan et al., 2019) found that O. marjoram and O. vulgare were highly effective at high SICs, where the toxA and exoS genes expression levels were lowered up to 12 times lower than those of the untreated control. (El-Hamid et al., 2019) observed a significant attenuation in five virulence genes of MDR Pasteurella multocida isolates, following treatment with O. marjoram extract. (Yu et al., 2021) demonstrated that treatment with the C. Camphora essential extract dramatically suppressed the expression of many antibiotic resistance genes in E. coli such as hdeD, evgs, zraS, and zraP.

CONCLUSIONs AND RECOMMENDATIONS

Research on Multidrug antibiotic-resistant bacteria and the development of natural, eco-friendly alternatives treatment is crucial. It also involves assessing the impact of these alternatives on chickens, a profitable industry that produces safe food for human consumption. The aqueous extract of Laurus nobilis leaves and its nano emulsion can be used as alternative approaches to spoilage due to pathogenic microorganisms. Furthermore, herbal use can alter the pathogenicity and antibiotic-resistance patterns of pathogenic bacteria, encouraging their usage as antibiotic surrogates in the management of bacterial contamination.

Acknowledgements

Thanks are extended to the Biology dept., Faculty of Science, Mustansiriyah University, Iraqi and AHRI Director in Egypt for cooperation and helpful.

Novelty Statement

Pathogenic microbes can cause spoiling, however alternate methods such as using a nanoemulsion of Laurus nobilis extract are possible.

AUTHOR’s CONTRIBUTION

All authors were responsible for designing the experiment.

Dalia M.A. El-Masry Conducted the synthesis and characterization of nanomaterials. Jihan M. Badr and Suhair Sh. Al-Siraj were in charge of the bacteriology part and gene expression. All author performed the experiment and collected the data. Each author contributed to their unique section of the work. All authors have reviewed and endorsed the final manuscript.

Conflict of Interest

There is no conflict of interest

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