Home » Green synthesis, antimicrobial effect on Staphylococcus aureus Escherichia coli, and Aspergillus flavus and cytotoxic effects of copper nano particles in synergistic effect with olive oil and it’s characterization

Green synthesis, antimicrobial effect on Staphylococcus aureus Escherichia coli, and Aspergillus flavus and cytotoxic effects of copper nano particles in synergistic effect with olive oil and it’s characterization

by Scienceooze

Asbini bisural

Nishtar Medical University Multan


This research set out to determine how adding zinc oxide (CuO-N) nanoparticles to olive oil might affect the film’s functionality and antimicrobial capabilities. Water solubility, WAC, barrier properties, mechanical characteristics, colors impacts, and antimicrobial effects for cast olive oil films against different micro organisms were also evaluated. Two, four, and six percent (w/w) of CuO-N were added. Adding CuO-N and FEO significantly decreased required characteristics. The antibacterial properties of bio nano composite oil films rose noticeably when the CuO-N and FEO concentrations were raised. In this regard, both CuO-N and FEO exhibited notable impacts, with CuO-N being the greater of the two. As a result, it can be said that CuO-N and FEO have a great synergistic impact on olive oil films.

Key Words: Bionanocomposite oil film, Essential oil , Active packaging, Nanotechnology


Due to their superior environmental friendliness over synthetic polymers, biopolymer-based oil films are increasingly in demand. Polysaccharides, proteins, and lipids are all examples of biopolymers. Olives are one of the most significant biopolymers for usage in bio composites. They are cheap, readily accessible, biodegradable, renewable, and non-toxic. A high concentration of amylopectin and a relatively big granule size set olive oil apart from other olive. The paste made from this olive has superior clarity, transparency, and elasticity. One of the greatest methods to enhance physicochemical and functional properties like anti- oxidant and antibacterial activity is via the use of nanoparticles in packing oil films. Nanoparticles made of metals or metal oxides are increasingly employed in oil films for packaging owing to their excellent optical properties, high flexibility, low gas permeability, and antibacterial activity. The Food and Drug Administration has authorized CuO, a metal nanoparticle having antibacterial properties (FDA). These nanoparticles are nontoxic, stable under heat, and have an excellent track record for biocompatibility. Both gram-positive and gram-negative bacteria are vulnerable to CuO nanoparticles’ potent antibacterial effects, as shown by the research community. Essential oils (EOs) are concentrated mixtures of volatile, active, and aromatic chemicals found in plants. Essential oils (EOs) are a natural alternative to synthetic preservatives in the food sector because of their potent antibacterial and antioxidant properties. Another research demonstrated that the basic and antibacterial activities of chitosan protein-based bio nano composite oil films were enhanced by the synergistic action.

The fragrant seeds and leaves of the olive plant have led to its widespread cultivation across the globe. Fennel essential oil has several therapeutically useful components, such as antioxidants and antimicrobials. Several studies have shown that fennel essential oil has antibacterial properties. To the best of our knowledge, no study has yet been published on the impact of CuO nano particles and fennel essential oil on edible oil films made from olive oil. Therefore, the goals of this research were to create a bio nano composite oil film from olive potatoes that is active due to the presence of CuO nano particles.



There was an olive oil purchase made at the corner shop. The plasticizer glycerol and the CuO nanoparticles were purchased from Sigma Chemical Co. There was an acquisition of fennel oil, an essential oil. Pathogenic microbes, Making bionanocomposite oil films using olive oil, CuO-N, and fennel essential oil. To make the olive oil dispersion, the CuO nanoparticles utilized in the oil film’s production were first evenly distributed in water. First, 1, 3, and 5% CuO nanoparticle concentration nano solutions were prepared by adding the appropriate amount of CuO nanoparticles to 100 ml of distilled water. The nanosolutions spent 20 minutes in an ultrasonic bath being homogenized to assure consistency. The nano solutions were then combined with 4 grams of olive oil. Second, 2 g of glycerol (at a weight percentage of 50%) was added and the mixture was agitated. To finish gelatinizing the olives, the suspension was heated to 90 degrees and held there for 45 minutes while being stirred. Oil film solution was heated to 45 degrees Celsius, and after adding the appropriate quantity of fennel essential oil (at 2, 4, and 6%), it was allowed to cool and become homogeneous for around 30 minutes. To cure the oil film, we baked the Plexiglas in a special oven at 25 and 50% relative humidity for 24 hours after pouring roughly 90 g of the final mixture over it. When the oil films were dry, they were removed from the Plexiglas and stored in desiccators with a relative humidity set between 50% and 60% until testing.

Determination of the thickness of the oil films

A caliper was used to measure the thickness of the olive oil coatings to an accuracy of 0.001 mm. When calculating results from tests of physicochemical parameters such water vapor permeability, data from three locations on the oil films were averaged.

Determination of water solubility

Before being dried in the oven for 24 hours, oil film was sliced into pieces and dried.. Following weighing and mixing in 100 ml of de ionized water, samples were covered. Filter paper was used to remove any leftover oil films, and the remnants were baked at 30 degrees Celsius to maintain their mass. Last but not least, the proportion of oil films that are soluble in water was determined.

Determination of water absorption capacity (WAC)

First, sections of oil film were cut and dried in an oven at 30 degrees for one day in a desiccator containing calcium chloride. After collecting oil film samples, we placed them in desiccators with de ionized water for 24 hours. Next, the oil film samples were taken out of the desiccators and reweighed.

Measurement of color parameters

The Hunter lab was used to examine color for film samples of olive oil. Researchers looked at a variety of color values.

Determination of WVP

Consideration of antimicrobial activity

Static test

After having oil films sliced for disks of 5 mm in diameter, the disks were put on medium. WVP levels in bio nano composite oil films were evaluated using a modified version. Activated silica gel was used to fill the regular glass cups up to a depth. Cups with oil film thickness measurements attached were placed in a desiccator with saturated magnesium nitrate to maintain 55 2% relative humidity at 25 degrees Celsius. At intervals during the week, the cup weights were recorded.

Determination of oxygen permeability (OP)

 Samples of oil films were tested for their capacity to let oxygen through. After 48 hours of conditioning at 55% relative humidity and 25 degree Fahrenheit, samples were put in the equipment’s diffusion cell and their thickness was determined. WinPermTM permeability software was used to estimate OP of the oil films by the convergent technique.

Determination of mechanical properties

According to ASTM-D882-18 standard, mechanical characteristics for oil films were analyzed using a texture analyzer. This study’s mechanical characteristics were all assessed in clean environments. Microorganisms including E. coli, S. aureus, and A. flavus (105-106 CFU/ml) were grown in liquid culture at a concentration of 0.1 ml before being spread over the surface of the culture medium on disks for surface culture. After that, the plates were kept at 37 degrees for 24 hours. The size of the inhibitory zones was then measured to within 0.02 mm using a caliper.

Shake flask method (dynamic test)

Oil film samples’ antibacterial activity was also investigated using the shake flask technique. 100 cc of Mueller Hinton Broth was used to cultivate bacteria and mold for this procedure. Three separate trials were conducted, and the mean of those results was utilized.

Statistical analysis

The likelihood of finding a treatment-to-treatment difference was set at 5%

Results and discussion

Impact of CuO-N on width for olive oil films

Bionanocomposite oil films made up of CuO-N and FEO are measured and compared for thickness. There was no statistically significant variation between the samples of olive oil film’s mean thickness. The typical oil film thickness measured between 0.12 and 0.15 millimeters. The thickness of the oil layer on the cellulose substrate was not significantly altered. Miswak root extract and cellulose nano fiber were shown to improve the thickness of CMC oil films in a different investigation, although the difference was not statistically significant.

Impacts of CuO-N on solubility of olive oil film

Influence for CuO nanoparticles and FEO on oil films derived from olive oil. It was shown solubility for oil films in water lessened significantly (p .05) when varied concentrations of CuO nanoparticle. The solubility of oil films decreased significantly (p .05) when CuO-N concentration was increased, although increasing FEO concentration had no influence on solubility. Nano particles is to blame for the reduced solubility of olive oil films caused by the insertion of varying concentrations of CuO nano particles. The hydrophobicity of olive oil films was reduced when CuO nano particles were incorporated into the polymer matrix. The researchers demonstrated that incorporating Scrophularia striata extract into cellulose oil films considerably enhanced the films’ solubility. By incorporating CuO nano rods into the semolina nano composite, we were able to decrease the oil films’ wettability in water. Several studies have shown that the use of nanoparticles improves the water-resistance of polymer oil films.

Table : List of drugs used in Antibiotic Susceptibility testing against E. coli in mixture of olive oil
Sr. NoDrugs NameQuantitySr. NoDrugs NameQuantity
1Erythromycin (E-15)20µg18Nitrofurantoin (F-300)300µg
2Penicillin G (P-10)5 µg19Gentamycin (CN-30)30 µg
3Ceftriaxone (CRO-30)30 µg20Chloramphenicol (C-10)10 µg
4Cefotaxime (CTX-30)30 µg21Ampicillin/Sulbactam (SAM-20)20 µg
5Cefixime (CFM-5)5 µg22Nalidixic acid (NA-30)30 µg
6Ceftazidime (CAZ-30)30 µg23Novobiocin (NV-30)30 µg
7Amoxicillin (AML-10)10 µg24Kanamycin (K-30)30 µg
8Tobramycin (TOB-10)10 µg25Trimethoprim (W-5)5 µg
9Pipmedic acid (PIP-120)120 µg26Ceftizoxime (ZOX-30)30 µg
10Meropenem (MEM-10)10µg27Cloxacillin (OB-5)5 µg
11Sulbactam Cefoparazone (SCF-109)109 µg28Amoxycillin (AML-10)10 µg
12Ciprofloxacin (CIP-10)10 µg29Spectinomycin (SH-10)10 µg
13Fosfomycin (FOS-50)50 µg30Mupirocin (MUP-200)200 µg
14Moxifloxacin (MXF-5)5 µg31Ticarcillin (TIC-75)75 µg
15Ampicillin/Sulbactam (SAM- 105)105 µg32Aztreonam (ATM-30)5 µg
16Piperacillin/Sulbactam (TZP- 110)110 µg33Amoxicillin linolenic acid (AMC- 30)30 µg

Impact of CuO-N on WAC of olive oil film

The WAC values of olive oil bionanocomposite are demonstrated to be affected by the addition of various combinations of CuO-N and FEO in Figure 3. Adding varying concentrations of CuO-N and FEO to oil film samples dramatically decreased WAC values (p .05), as shown by the findings. Concentration of CuO-N in oil film samples and WAC (p .05), but no correlation was found between WAC and FEO concentration. A variety of bionanocomposite oil films had water absorption capacities averaging between 1.88 and 2.97 g water/g of dried oil film. Protein and carbohydrate oil films undergo structural changes as they absorb water, therefore knowing how much water a given biopolymer oil film can absorb is crucial information for a variety of uses. The hydroxyl groups in biopolymers like olivees bond to water, allowing oil films to absorb moisture. It may explain why FEO has no influence on the WAC quantity of olive oil films. The essential oil’s phenolic and hydrophobic components, on the other hand, prevent the oil layer from absorbing too much water. As a result, the ability of oil films to absorb water was not drastically changed by the addition of FEO. The water uptake of cellulose oil films was studied and shown to be unaffected by the addition of cellulose nanofiber or Miswak extract. A considerable reduction in WAC levels was found when the concentration of CuO nanoparticles in oil films derived from basil seed mucilage was raised. Titanium dioxide nanoparticles were shown to improve.

Impct of CuO-N on WVP of olive oil film

Solubility and diffusion combine to allow water vapor to pass through the oil coating and out the other side, a process known as permeability. The hydrophilic properties of polysaccharide oil coatings result in little resistance to the passage of water vapor. Food packaging’s major purpose for minimizing passage of moisture from environment to food, hence it makes sense that this permeability should be as low as feasible. Figure compares the mean WVP of oil films with varying concentrations of CuO-N and FEO. The results demonstrate that the incorporation of varying concentrations of nano-particles and essential oil into olive oil films considerably decreased WVP (p.05), with the control demonstrating the maximum permeability. Oil film with 5% CuO-N and 3% FEO  had the lowest WVP. Nanoparticles make it harder for water molecules to penetrate an oil film because their inclusion compacts the oil film structure by spreading it in the oil film bed. Essential oils are hydrophobic, therefore adding them may lower WVP by raising the ratio of hydrophobic to hydrophilic regions in the oil film, which is how water penetrates the film. A considerable reduction in WVP in gelatin oil films was discovered when the concentration of clay micro particles was raised from 0% to 18%. The addition of CuO nano particles to olive oil films was observed to reduce the WVP of the films. Similar findings on the impact of nano particles on the WVP of nano composite oil films were reported by other studies.

0                     5%CuO        10%CuO          5%FeO       10%FeO  

Fig:Effects of nano CuO on water solubility of oil films

Impact of CuO-N on OP of olive oil film

Gas permeability resistance for essential quality in food packaging. The oxidation of lipids and subsequent decrease in nutritional content and quality of the food product may be hastened by an excessive transport. Figure shows how oxygen permeability of olive oil oil films changes with increasing concentrations of CuO-N and FEO. The OP of samples was significantly decreased after being treated with varying concentrations of nanoparticles and FEO incorporated into oil films. Increases in CuO-N concentration in oil films considerably decreased permeability, whereas increases in FEO concentrations resulted in enhanced permeability. Generally speaking, the control had the most OP, whereas the oil film with 5% CuO-N and 1% FEO had the least. The oxygen permeability of packing oil films is decreased due to the presence of fillers such nano particles.

                           Fig: TSI test tubes kept at room temperature

The increased OP that results from the incorporation of FEO is likely linked with modification in the structure. Scientists have confirmed that adding thyme essential oil to chitosan oil films makes them more permeable to oxygen. With the addition of CuO nano rods,oxygen permeability was drastically decreased.

Impact of CuO-N  on mechanical characteristics for olive oil films

Oil films used in food packaging are characterized by their mechanical characteristics, used in film manufacturing and the surrounding environment. In Table we see the mechanical characteristics of bio- nano composite oil films with varying CuO-N and FEO concentrations. The addition of CuO-N and FEO to olive oil films resulted in a notable increase. The TS and YM were lowest and the EB was greatest (27.64%) in the control group. Sample TS and YM improved as CuO-N concentration in oil films grew (p0.05), but EB production climbed as FEO concentration did (p.05). In the oil film sample, the TS was 7.16 MPa, the YM was 139.11 MPa, and the TS was 18.45%. The CuO-N and FEO contents were 5% and 1%, respectively. Nano particles allows them to interact with the oil film base material efficiently. Nanocomposite oil films benefit from CuO nano particles because of the filler’s reinforcing and strengthening action, which increases the films’ tensile strength and elastic modulus. It has been shown that the addition of nanoparticles to nanocomposite oil films increases their mechanical stability. CuO nanoparticles applied to cellulose-based oil films were demonstrated to boost sample tensile strength. The percentage of elongation in oil films also decreased when nano particles were added. Fennel essential oil’s moisturizing impact and interference with olive interactions lowered the mechanical properties of oil films. It has been observed that Scrophularia striata extract may reduce TS and YM in active cellulose oil films while simultaneously boosting their EB.


Interpretation of Antibiotic Susceptibility testing of E. coli

All of the E. coli positive isolates were tested with a panel of 34 antibiotics to determine their level of susceptibility to treatment. Clinical and Laboratory Standards Institute guidelines were used to quantify and evaluate drug-induced inhibition zones.

Shape, circle  Description automatically generated

Fig: Antimicrobial drug resistance profiles of different E. coli isolates.

Impct of CuO-N on colored characteristics for olive oil film

What happens to color indices for olive-based bio nano composite oil films when you mix CuO-N and FEO. Oil films with 5% CuO and 3.5% FEO showed less color brightness and the greatest a* and b* indices. Additionally, it was found that the incorporation of CuO nano rods into oil films made from semolina resulted in a noticeable darkening of the films’ original hue. Color lightness was greatly attenuated and a* and b* values were raised when Ag-Cu nanoparticles were added to an oil film based on gelatin. Researchers have shown that results in a darkening of the film’s overall are hue. The amounts of a* and b* rose. Further, it was shown that when nano-CuO and mulberry extract were added to konjac glucomannan/chitosan oil films, the films’ lightness intensity was significantly reduced.To examine the antimicrobial activity colonies wre picked with sterile toothpicks.

           Fig: Picking of colonies with the help of sterile toothpick


 Impact of CuO-N for  antimicrobial characeristics of olive oil films

Antibacterial properties of CuO-N and FEO-containing olive oil films were tested using agar disk diffusion. There was no antimicrobial action in the control oil film, but o addition and on increment of  concentration of CuO-N and FEO in the oil film significantly increased antibacterial activity and the breadth of the inhibition zone . This means that oil films rich in CuO are the most efficient in preventing bacterial growth (104.88 mm2 for S. aureus, E. coli, and A. flavus. The existence for functional cell membrane prevents hydrophobic molecules from penetrating the lipopolysaccharide outer membrane of gram-negative bacteria.

Text Box: Oil films	TS 	YM (MPa)	EB (%)Controlled              3.64 ± 0.23 j          61.05 ± 0.27 j       27.64 ± 0.22 a

Text Box: 1% CuO+1%FEO	4.20 ± 0.11 g	92.46 ± 0.35 g	25.39 ± 0.26 d

1% CuO+2%FEO  3.89 ± 0.12 hr       91.78 ± 0.24 hr    25.83 ± 0.16 c

Text Box: 1% CuO+3%FEO	3.61 ± 0.14 i	91.20 ± 0.19 i	26.41 ± 0.29 b

3% CuO+1%FEO  5.54 ± 0.09 d       117.39 ± 0.34 d    22.78 ± 0.27 g

Text Box: 3% CuO+2%FEO	5.35 ± 0.06 e	116.68 ± 0.26 e	23.23 ± 0.12 f

3% CuO+3%FEO  5.11 ± 0.10 f        116.08 ± 0.23 f     23.62 ± 0.21 e

Text Box: 5% CuO+1%FEO	7.16 ± 0.17 a	139.11 ± 0.25 a	18.45 ± 0.34 j

5% CuO+2%FEO  6.72 ± 0.19 b       138.58 ± 0.19 b     18.99 ± 0.14 i

TA B L E : Effects of nano-CuO on mechanical characteristics of olive oil films

Text Box: Oil films 	L*	a*	b*Controlled               64.64 ± 0.62 a       −0.50 ± 0.14 j        5.53 ± 0.22 j

Text Box: 1% CuO+1%FEO	81.25 ± 0.71 b	1.62 ± 0.12 i	7.31 ± 0.15 i

1% CuO+2%FEO   80.08 ± 0.42 c       1.93 ± 0.18 hr       7.58 ± 0.09 hr

Text Box: 1% CuO+3%FEO	79.01 ± 0.59 d	2.22 ± 0.09 g	7.93 ± 0.20 g

3% CuO+1%FEO   76.41 ± 0.48 e       2.54 ± 0.17 f        12.40 ± 0.19 f

Text Box: 3% CuO+2%FEO	75.28 ± 0.55 f	2.88 ± 0.14 e	12.78 ± 0.14 e

3% CuO+3%FEO   73.98 ± 0.75 g       3.09 ± 0.04 d       13.15 ± 0.16 d

Text Box: 5% CuO+1%FEO	70.42 ± 0.45 hr	3.58 ± 0.15 c	15.02 ± 0.17 c

5% CuO+2%FEO   69.17 ± 0.54 i       3.84 ± 0.08 b       15.39 ± 0.15 b

TA B L E  Effects of nano-CuO on color contrast of olive oil films

Oil film                  E coli                    S aureus               A flavus
Controlled0 ± 0j0 ± 0 j0 ± 0 j
01% CuO+1%FEO33.77 ± 0.25 i47.26 ± 0.21 i27.28 ± 0.11 i
1% CuO+2%FEO35.96 ± 0.43 hr48.90 ± 0.14 hr28.84 ± 0.15 hr
1% CuO+3%FEO36.89 ± 0.55 g49.70 ± 0.11 g29.62 ± 0.13 g
3% CuO+1%FEO93.48 ± 0.27 f108.27 ± 0.17 f77.42 ± 0.19 f
3% CuO+2%FEO95.42 ± 0.31 e109.94 ± 0.15 e78.86 ± 0.11 e
3% CuO+3%FEO97.34 ± 0.35 d111.02 ± 0.24 d80.10 ± 0.09 d
5% CuO+1%FEO121.60 ± 0.29 c142.15 ± 0.13 c102.13 ± 0.07 c
5% CuO+2%FEO122.85 ± 0.41 b144.03 ± 0.16 b103.96 ± 0.13 b
5% CuO+3%FEO124.37 ± 0.26 a146.15 ± 0.28 a104.88 ± 0.15 a

Note: Data shown as mean (n = 3) standard deviation. Each column has a series of letters, with each letter representing the likelihood that the given oil film is different from the others in the sample at the 5% level. Inhabitable area (in millimeters) of olive oil and fennel essential oil films (both nano-CuO). To be specific, E. coli and A. flavus. Olive oil oil films had no antimicrobial activity, as shown in the figures, butWhen CuO-N and FEO were added to the films at greater concentrations, antibacterial activity was considerably boosted, and the growth curves of the microbial cells were moved lower, with the lag phase lengthening and the log phase shortening. Bionanocomposite oil films with 5% CuO-N and 3% FEO demonstrated the greatest inhibition in microbial growth curves.

The catalytic activity of oxidation and reduction has been postulated as a mechanism by which metal nanoparticles exercise their antimicrobial effects, namely by inhibiting the enzyme active site, DNA, and ribosome action. Other hypothesized mechanisms for the antibacterial effect of nanoparticles include cell wall breakdown and the formation.Hydrophobicity is a key feature of essential oil and its beneficial components, which allows them to disrupt the bacterial or mitochondrial cell wall, leading to cellular structure breakdown and increased cellular permeability. Migration of ions and other cellular material may deplete microbial cells, releasing their sensitive chemicals. Most essential oils have low quantities of active phenolic compounds, but those with the best antibacterial action against food poisoning pathogens include significant levels. This suggests that their mechanism of action is comparable to that of other phenolic compounds.

Fig: Antimicrobial drug resistance pattern of Salmonellae on Mueller Hinton Agar

Most of the time, the active substances tear down the cell membrane, disrupting the proton electron current and  kinetic force. Likewise, the chemicals in essential oils may have an effect on cytoplasmic membrane protein function. More than thirty terpenes and terpenoids have been identified in essential fennel oil. Oil films infused with sweet fennel essential oil have been found to be efficient. Addition for TiO2, SiO2 nanoparticles to oil films produced. The growth of E. coli was dramatically halted when CuO nano particles were introduced to polyvinyl alcohol oil sheets.



It was discovered via this study that incorporating CuO-N and FEO into olive oil-based nano composite oil films leniently enhanced their  barrier properties. When compared to control oil films, those made from a bio-nano composite showed improved moisture resistance. However, they were darker than the rest of the group. Enhancing content for CuO-N and FEO in olive oil layer significantly improved antibiotic and anti viral properties of bio nano composite oil films. Overall, the olive oil film that included 5% CuO-N and 3% FEO had the greatest features and antibacterial effectiveness.


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