In the present study screening of 45 different food samples varied form milk, yogurt, cheese, spices, processed meat, and juice were screened for microbial contamination revealed 17 samples were contaminated with E. coli (37.8%) and 5 food samples were contaminated with Salmonella (11.1%) and only one food sample was contaminated with C. albicans. Both bacteria were found mainly in milk and milk goods.
Oliver et al (2009) reviewed food safety and its hazards joined to utilization of unprocessed milk and showed that E. coli and salmonella were the most common contaminants of milk and its products.
The mechanism by which essential oils can combat microbes has not been fully explained. Essential oils do not aim a specific target in the cell nevertheless they targets many goals in the cell. When essential oils enter the cell wall penetrating the cytoplasmic membrane of pathogens they cause structure disruption of their polysaccharides, phospholipids and fatty acids layers increasing their permeability (Chaieb et al. 2007; Bakkali et al. 2008). This increased permeability is accompanied by ions depletion, inhibition of the proton pump, decreased ATP synthesis, inhibition of respiration and hence cell death of the pathogen whether it is G + ve or G − ve bacteria (Nazzaro et al. 2013; Mohammed et al. 2019).
Clove is the common name of Syzygium aromaticum family Myrtaceae. Eugenol is the main component of clove's essential oil which owes its antibacterial effect by making the cell membrane more permeable and interrelate with its proteins and that was proved by many different studies to be due to increased movement of potassium and ATP out of bacterial cells (Hyldgaard et al. 2012). Moreover when clove essential oil enters the bacterial cell it inhibits DNA synthesis and proteins necessary for bacterial growth implying damage at the molecular level rather than only physical damage (Xu et al. 2016).
In the present study results of screening of the antimicrobial activity of six ready-made oils: clove (Syzygium aromaticum) oil, black seed (Nigella sativa) oil, thyme (Thymus vulgaris) oil, garlic (Allium sativum) oil, rosemary (Rosmarinus officinalis) oil and green tea (Camellia sinensis) measured by well diffusion method against six microbial isolates: Escherichia coli, Salmonella typhi, Candida albicans, Staphylococcus aureus, Klebsiella and Pseudomonas proved that all tested oils had antimicrobial tendency when facing at least one of the above mentioned six microbes. However clove oil was the only one that has antimicrobial affinity against all of the tested microbes.
Nzeako et al (2006) in their study reported that C. albicans was the most sensitive organism to the antimicrobial activity of clove oil and thyme than S. aureus, Pseudomonas aeruginosa, E. coli and Salmonella species. They also concluded that whatever the country of origin of herbs is had no effect on their antibacterial benefit. However Ncube et al. (2008) concluded that the variety of the plant parts used in extraction as well as the extraction method led to various antimicrobial effects.
The antibacterial effects of clove oil and rosemary oil were among many other oils studied by Prabuseenivasan et al (2006) against S. aureus, E. coli, K. pneumoniae, P. aeruginosa and two other bacteria. Their results showed inhibition zone rang for clove oil from 16 to 17 mm against all four bacteria and that of rosemary was 12 to 23 mm also against the four bacteria. These results of clove oil and rosemary oil were greater than the results in this study.
Regarding the antibacterial activity of black seed (Nigella sativa) oil, quite large number of studies have been established by various researchers to explore the pharmacological action of black seed oil as stated by Safhia et al. (2014) the black seed oil commercially available was selected for their study without dilution or chemical alteration, Staphylococcus aureus, Escherichia coli and Klebsiella planticola were among other tested bacteria. Inhibition zone of S. aureus was 14 mm which was the most significant result when compared to Ciprofloxacin disc (5 µg/disc) while that of E. coli was non-significant however Klebsiella showed no zone of inhibition. The result in the present study as 13 mm agrees with their results regarding S. aureus only. Approximate results to ours were reported by Mohammed et al. (2019) concerning the antibacterial action of black seed (Nigella sativa) oil towards Staphylococcus aureus where inhibition zone diameter was 11.5 mm. They explained the antimicrobial effect of black seed oil to be attributed to thymoquinone which is a volatile oil of these seeds that characteriscally has free rotation bonds in the CH3 group which qualify thymoquinone and other constituents of black seed oil to change their shapes according to the available bacteria and easily cross or enter their boundary leading to their death. While earlier Bakathir and Abbas (2011) explained the positive inhibition of black seed to S. aureus may be due to thymoquinone and melanin.
Various techniques for encapsulation were used in food industry to deliver some active compounds such as additives, colors, enzymes and preservatives. However their incorporation into liposomes as nanoparticles is distinguished by the unique liposomal characteristic which is the capacity to entrap both hydrophobic and hydrophilic particles. Liposomes also have the advantages of being biocompatible, biodegradable, non toxic and non immunogenic. For all these reasons liposome nanocapsules have considered as one of the tempting persuasive package material in pharmaceutical and food manufacturing (Ghorbanzade et al. 2017; Corrêa et al. 2019).
A comparative study was done by Anderson and Omri (2004) between three types of formulated liposomes considering encapsulation efficacy as a crucial factor in liposome parameters they reported that DSPC liposomes showed the greatest encapsulation efficiency between the three formulations which might be due to their shorter hydrocarbon chain (C14) DSPC liposomes had also shown the most drug retention.
Regarding measurement of zeta potentials which is one of the standard characterization techniques to evaluate the final conclusion of nanoparticle surface charge character whether cationic, anionic or neutral character (Smith et al. 2017). That was clearly explained by Paolino et al. (2006) and Elmeshad et al. (2013) who stated that the magnitude of zeta potential is the clue for the potential stability of nanoliposomes composition and estimation of in-vivo fate of these nanocarriers. Because when zeta potential increases it leads to more repulsion between particles resulting in more stable colloidal dispersion. In other words when all particles in suspension have a large negative or positive zeta potential, then the particles have a propensity to repel each other leaving no chance for their aggregation together.
In the present study blank DSPC liposomes showed negative zeta potential as (− 18.6 ± 25.0 mV) in accordance with Makino et al (1991).
Results reported by Elmeshad et al. (2013) stated that the nanoliposomal formulations had average zeta potential with a range from 23.3 ± 1.2 to 35.8 ± 0.3 mV.
In the present study clove loaded liposomes had higher negative zeta potential (− 117.5 ± 85.2 mV) which may be explained by the presence of hydroxyl (OH−) group on the clove surface liposomes that might be physically associated by the surface without upsetting the packing membrane suggesting that clove oil might be caught in the hydrophobic fraction of the liposomal bilayer. The presence of Hydroxyl (OH−) group on the clove surface with more negative zeta potential acted as stabilizing material.
In the present study TEM images showed well dispersed and not aggregated round shaped empty and encapsulated vesicles had a diameter around 232.33 ± 10.63 nm and 979 ± 27 nm respectively. The large size of clove oil encapsulated liposome might be explained considering the existence of phospholipid PO2−group in the polar head of DSPC liposomes might prevent the clove hydroxyl (OH−) group from getting close to the PO2− group because of the electrostatic repulsive force between the DSPC PO2− group and the clove hydroxyl (OH−) group thus weakening the interactions between phospholipid PO2− and clove hydroxyl (OH−) group. The presence of hydroxyl (OH−) group on the clove surface liposomes that might be physically associated by the surface without upsetting the packing membrane suggesting that clove oil might be caught in the hydrophobic fraction of the liposomal bilayer. These findings show that the presence of clove oil in liposomes increased the spacing between the adjacent bilayer, resulting in larger-sized liposomes compared to the control empty liposomes.
While results reported by Elmeshad et al. (2013) showed the opposite as the loaded nanoliposomes was possessing the minimum vesicle size (532 ± 26 nm) which was a surprising finding and was explained by the hydrophilic nature of the entrapped material in their study which lead to its segregation inside the aqueous phase rather than being trapped within the hydrophobic part of the liposomes.
FTIR studies were done to examine the effect of clove oil on the DSPC liposomes. The spectrum of the DSPC liposomes (Fig. 5) exhibited the major characteristic bands demonstrating the symmetric and anti-symmetric stretching vibrations of the CH2 inside the acyl chain (2850 and 2920 cm−1 respectively) the OH stretching and bending vibrations (3470 and 1640 cm−1 respectively) the carbonyl stretching vibration C=O (1734 cm−1) the CH2 bending vibration CH2 (1470 cm−1) and the symmetric and anti-symmetric PO2− stretching vibrations (1090 and 1220 cm−1 respectively) same results were reported and published in a previous study by one of our authors with Mady et al. (2012) also these results are in accordance with Mady and Elshemey (2011).
In the present study incorporation of clove oil into the DSPC liposomes showed no significant variation in the frequency of the symmetric and anti-symmetric CH2 stretching bands within the acyl chain observed implying that clove oil does not alter the number of gauche conformers. While FTIR measurements reported by Mady et al (2011) revealed that gold nanoparticles made significant alterations in the frequency of the CH2 stretching bands suggesting that gold nanoparticles increased the number of gauche conformers and generate a conformational change within the acyl chains of phospholipids.
An important finding that could be observed was that the CH2 bending vibration modes that were located at 1469.49 cm−1 were affected by the encapsulation of clove oil into DSPC liposomal preparation as wave number was lowered to 1468.53 cm−1. This suggested the presence of ordering outcome within acyl chain packing in the gel forms of phospholipids.
The interaction between clove oil and DSPC liposomal head group was monitored by means of the PO2− antisymmetric stretching band, which is positioned at 1247.72 cm−1. The wave number was moved to higher values from 1247.72 to 1249.65 cm−1 after the encapsulation of clove oil in DSPC liposomes. This suggested the absence of hydrogen bonding between clove oil and liposome head group. According to previous investigational observations lowered frequency values are exhibited an enhancement of existing hydrogen bond strengthening or in creation of new hydrogen bonding between the constituents Severcan et al. (2005). These results suggesting that clove oil was located in the interfacial region of the membrane.
In the present study analyzing data of clove oil interaction with glycerol backbone close to phospholipids head group within the interfacial region the C=O stretching band and the wave number deviation of this band revealed shift of wave number value of C=O group towards higher value from 1738.51 to 1739.48 cm−1 regarding liposome sample containing clove oil without any proof of hydrogen bonding formation where the amount of hydrogen-bond formation was observed in the glycerol backbone region of the DSPC molecule by alteration in the contours of the ester C=O stretching. Analyzing these data suggesting dryness concerning these functional groups in the interfacial area of the lipid membranes. Consequently any effects in the spectra of this region can be assigned to an interaction between clove oil and the polar/a polar interfacial region of the membrane. Clove oil tends to decrease hydrogen bonding formation in the interfacial region of DSPC liposome suggesting the existence of the free carbonyl groups in the arrangement. In other words clove oil is likely to substitute some H2O molecules from the interfacial region and lead to an increase in the number of free carbonyl groups.
When using liposomes for drug encapsulation the preparations is a mixture of entrapped and unentrapped drug fractions and science Entrapment Efficiency (EE) is the percentage of the drug that is successfully entrapped into liposomes so the first step for EE determination was to separate the entrapped drug inside the liposomes from the free one. However the process of separation and measurement of concentrations are heavy, tiring and taking long time moreover the separation procedure itself might be unsuccessfully completed. (Laouini 2012; Bano 2000). Differential scanning calorimetry (DSC) is very efficient method that does not need a separation step meanwhile it is able to access more than one parameter of liposomal encapsulation as EE, size, drug–lipid interactions and partition coefficient so that achieving quality control of liposomal encapsulation (Bakonyi et al 2017).
In the present study results of encapsulation efficiency (EE%) of clove was 22%. This may be attributed to the lipo-solublized state of the drug owing to its entrapment within multiple lipoidal domains of liposomal vesicles which enables a sustainable release of clove oil as slowly leaking out of the clove compared to its free form in the vicinity of the bacteria in culture which might explain the enhancement of the antibacterial activity by 10 times.
Differential Scanning Calorimetry (DSC) was mainly applied to determine the thermodynamics of biological macromolecules measuring phase transitions, conformational changes and temperature dependence (Chiu and Prenner 2011).
In the present study incorporation of clove oil into liposomes exhibited noticeable expansion and move to lower temperature 72 °C of the main characteristic endothermic peak of unloaded DSPC that was existed at 79 °C which suggested that clove oil had a significant consequence on the acyl chains of DSPC bilayer and that its presence lowered the mutual transition of the lipid acyl chains. The decreased temperature of the major DSPC transition process showed that clove oil assimilation was more likely due to the configuration of acyl chains in a disorganized and free state. The pre-transition temperature was found to be shifted from 64 to 54 °C indicating that liposomes encapsulating clove oil had a membrane fluidization effect.