Food processing generates substantial amounts of biodegradable waste, notably fruit peels, which, if improperly managed, can lead to environmental pollution and economic losses (Singh et al., 2022). Pomegranate peel, constituting up to 50% of the fruit, is rich in bioactive compounds such as tannins, phenolic acids, flavonoids, and anthocyanins, exhibiting potent antioxidant, antimicrobial, anticancer, and anti-inflammatory properties (Jalal et al., 2018; Usanmaz et al., 2016). India, as the world's leading pomegranate producer with 234,000 hectares under cultivation, significantly contributes to global pomegranate by-product generation (Valero-Mendoza et al., 2023). Scientific studies have demonstrated that pomegranate peel extract (PPE) effectively inhibits foodborne pathogens and extends the shelf life of perishable food items, including fish and fishery products, which are particularly susceptible to spoilage due to high levels of polyunsaturated fatty acids (Barnossi et al., 2021; Charalampia et al., 2017). With growing concerns over the health risks associated with synthetic antioxidants, such as urinary bladder and stomach cancers (Ito et al., 1986), and the rise of antibiotic resistance (Lambert, 2005), there is an increasing demand for natural, plant-derived preservatives. Consequently, pomegranate peels, often discarded as agricultural waste, present a valuable, eco-friendly resource for developing natural preservatives and functional ingredients in the food, cosmetic, pharmaceutical, and aquaculture sectors (Kahramanoglu et al., 2019; Cao et al., 2002; Barnossi et al., 2021).

 

SAMPLE COLLECTION

              Pomegranate peels was collected from different juice vendors in Korampallam, Thoothukudi, Tamil Nadu, India. The pomegranate peels were manually cleaned and washed to remove dirt and unwanted materials. They were cut into 1x1 inch size, placed on a tray, and dried in a tray drier at a constant temperature of 40°C. Dried peels were ground and sieved into powder by mechanical mixer. The coarse powder was then stored in air tight sealed container and placed at 4°C. Plate 1 showed the Pomegranate peel. (PLATE 1)

 

PREPARATION OF EXTRACTS

Twenty grams of peel powder was mixed with 180 ml of polar solvent of ethanol. The conical flasks were tightly stoppered with plugs of absorbent cotton and this was wrapped with aluminium foil as a precautionary measure. The conical flasks were placed in a shaker pre-set at 30°c at 130 rpm for 72 hours for solvent extraction to complete. After that, the flasks were removed from the shaker and the filter it using Whatman No.1 filter paper and was poured into Petri plates to allow for ethanol vaporization.  After that, ethanol extract was used for further studies.

 

ISOLATION OF FOOD PATHOGENS

Contaminated food samples were collected from home and transported to our laboratory using sterile containers.  The samples were processed in our lab using the preparation of selective media such as Mannitol salt agar, SS agar, EMB agar, Campylobacter isolation agar. One gram of contaminated sample was swabbed on the selective media. The plates were incubated with 37^o C for 48 hrs. (PLATE 2)

 

IDENTIFICATION OF FOOD PATHOGENS

All isolates were subjected to Gram’s staining, motility, morphological characteristics and bio chemical tests according to Bergy’s manual. (TABLE 1) (PLATE 3)

 

ACTIVITY OF SELECTED COMMERCIAL ANTIBIOTICS AGAINST ISOLATED  FOOD PATHOGENS

Assay of antibiotic activity  against the food pathogen was performed by Kirby-Baeur disc diffusion method. The  Muller Hinton agar plates were prepared and the organism was swabbed over it using a sterile cotton swab. The antibiotic discs such as Streptomycin, Chlorampenicol, Ampicillin and Tetracyclin were placed on the surface of the agar plates and then, the plates were incubated at 37⁰C for 24hrs. After incubation, the zone of inhibition was measured in mm. (TABLE 2) (PLATE 4)

 

 EVALUATION OF ANTIBACTERIAL ACTIVITY OF ETHANOL EXTRACT OF POMEGRANATE PEEL

INOCULUM PREPARATION

Loopfull inoculum from each bacterial isolate were inoculated into nutrient broth and incubated at 37 °C for 24 hours. Adjust the turbidity with McFarland quantity  of std (0.5) to get a uniform suspension containing 1.5×108 CFU / ml.Evaluation of Antimicrobial Activity. Antimicrobial activity of plant extracts was performed using the agar-well difusion bioassay. The antimicrobial activity of ethanol extract of Pomegranate peel against food pathogens was performed by agar well diffusion method. Mueller Hinton Agar  (Hi-Media) was prepared, autoclaved, and poured into sterile petriplates. The ethanol extract of pomegranate peel was dissolved in DMSO at the ratio of 1:1. Briely, 100µL of fresh culture (approximately10⁶ CFU/mL) was uniformly spread onto Mueller-Hinton agar (MHA) plates by sterile swab dipped with food pathogen. Then, inoculated plates were allowed to dry at room temperature for 20min. Ater that, wells of 6mm in diameter were made in the agar using a sterilized well cutter and 100 µL of different concentration of ethanol extract of PE 100,200,300,400µl/well was poured in different wells. DMSO was used as negative  control. Plates were incubated at 37∘C for 24 h. Antibacterial activity was evidenced by the presence of clear inhibition zone around each well. The diameter of this zone was measured in mm and recorded. (TABLE 3) (PLATE 5)

 

MINIMAL INHIBITORY CONCENTRATION OF ETHANOL EXTRACT OF POMEGRANATE PEEL (EPP)AGAINST FOOD PATHOGENS (MIC)

Determination of minimum inhibitory concentrations (MIC’s) of the Ethanol Extract of Pomegranate peel is defined as the lowest concentration of the extract that inhibits the bacterial growth after 24 h. of incubation. The most effective extracts which exhibiting a strong antibacterial activity at 400 µl/ml was manipulated to determine their MIC using broth dilution method and evaluate their efficiency in controlling bacterial strains causing food poisoning  /food infection causing pathogens.

 In Broth dilution technique, different concentration of EPP were prepared in ranging from 100 to 500 µl/ml. From each concentration of EPE extract was mixed with one milliliter of nutrient broth. Ten microliter of each standardized broth cultures (1.5 X10⁶ CFU/ml) was cultivated on the broth containing various concentrations of the EPP extract. Control was maintained without EPP extract. The tubes were then incubated at 37⁰C for 24 hrs according to growth requirement of each organism and observed for any visible bacterial growth. And measured  the bacterial growth by take OD value at 360nm. MIC was the lowest concentration of extract that resulted in no visible growth on the broth. The broths were incubated according to growth requirement of each organism. The lowest absorbance of turbidity in the broth dilution was the evidence of bactericidal activity. (TABLE 4)

 

 DETERMINATION OF MINIMUM BACTERICIDAL CONCENTRATIONS (MBC’S) OF  EPP EXTRACT

The one µl sample was taken from  lowest concentrations of the EPP extract exhibiting invisible growth from MIC broth dilution and subcultures onto sterile Nutrient agar (NA) plates by spread plate method. The plates were incubated at 37⁰ C for 24h.After incubation, then examined for bacterial growth in corresponding to lowest concentrations of the EPP extract. MBC was taken as the lowest concentrations of the EPP extract that did not exhibit any bacterial growth on the freshly inoculated Nutrient agar plates.(TABLE 5) (PLATE 6)

 

PRELIMINARY ASSAY OF ANTIDIABETIC ACTIVITY OF ETHANOL EXTRACT OF POMEGRANATE PEEL

The antidiabetic assay was performed by Adding 1ml of 1% starch to control and test sample tubes.Then 0.5 ml of amylase enzyme was added to all the tubes. Tubes were subjected to Incubation at 37ºC for 30 min. After incubation 1ml DNSA reagent was added to all the tubes. Then allow the tubes for heating at 95 ºC for 15 min in water bath. Atlast Read the absorbance at 510 nm. (PLATE 7)

% of  Inhibition = [Absorbance Control- Absorbance Test)/Absorbance control] ×100

 

 

ANTIOXIDANT ACTIVITY OF ETHANOL EXTRACT OF POMEGRANATE PEEL

              Antioxidant activity of ethanol extract of Pomegranate peel was done by Phosphomolybdenum assay. For the conduction of the phosphomolybdenum assay, the method was followed by Prieto et al., (1999). An aliquot of 0.1mL of sample solution of different concentrations (25–400μg/mL) treated with 1mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were incubated at 95°C in a water bath for 90 min. The samples were cooled to room temperature and their absorbance was recorded at 765 nm. Ascorbic acid was used as the positive control. Antioxidant capacity was estimated by using following equation. (TABLE 6) (PLATE 8)

Antioxidant activity % = [(Absorbance control − Absorbance sample)/Absorbance control] × 100

 

ANTI BIOFILM ACTIVITY OF ETHANOL EXTRACT OF POMEGRANATE PEEL

Test tube containing 1ml of Nutrient Broth was prepared and sterilized at 121°C for 15 minutes. Then the tubes were inoculated using a test microorganisms known to form biofilms. Add the 100 µl, 200 µl, 300 µl, 400 µl of the prepared concentration of Pomegranate extracts solution to the test tubes separately, ensuring each tube receives the desired concentration of the extract. Include the control tube without the extract. Place the inoculated test tubes in an incubator set at 37°C for 48-72 hours. After the incubation, the modified crystal violet staining (CVS) assay was performed to quantify the biofilm biomass. (TABLE 7,8) (PLATE 9)

 

CRYSTAL VIOLET STAINING (CVS) ASSAY

The tubes were carefully emptied and tubes were washed at least two times with sterile distilled water to remove unattached or loosely attached cells. The tubes were air -dried for 20 mins. The adhered cells stained with 1ml of 0.1% crystal violet solution for 10 minutes at room temperature. Excess stain was rinsed off by washing the plates at least two times with water. Thereafter, the biofilm biomass was evaluated semiquantitatively by re-solubilizing the crystal violet stain bound to the adherent’s cells with 150 µl of 100% ethanol to destain the wells. The absorbance of the tubes was read at 590 nm using a Spectrophotometer after careful and gentle shaking. The OD value (OD590nm) of the sample was determined and results expressed as percentage (FIGURE 1)

 

Inhibition using the equation below

Percentage (%) inhibition = OD Negative control – OD Sample] /  OD Negative control × 100

ANTI-INFLAMMATORY ACTIVITY OF ETHANOL EXTRACT OF POMEGRANATE PEEL

PREPARATION OF REACTION MIXTURE

Bovine serum albumin solution was prepared by adding 1g of Bovine serum albumin(BSA) in a 100ml of distilled water. In a test tube add 1ml of bovine serum albumin (BSA) solution and 0.2 ml, 0.4ml, 0.6ml, 0.8ml of the sample solution separately, then make up to 2ml using the distilled water. Include control tubes without the sample extract can be added.

 

INCUBATE THE SAMPLES

Allow the mixture to incubate at 60°C for 10 minutes in a water bath to allow any potential anti-inflammatory activity to occur. (PLATE 10)

 

 PERFORM THE LOWRY METHOD

After the incubation period, use the Lowry method to measure the protein concentration in each sample. This method involves adding 0.3ml of Folin-Ciocalteau reagents to the samples, followed by colour development and measure OD value of the standard and test solution at 660 nm and plot the standard graph. (TABLE 9)

 

The percentage inhibition for anti-inflammatory activity can be calculated using the following formula:

Percentage Inhibition = (1−Absorbance of Sample/Absorbance of Control) ×100                                    

 

PRELIMINARY PHYTOCHEMICAL SCREENING OF ETHANOL EXTRACT OF POMEGRANATE PEEL

 

 The EPP extract (ethanol extract of Pomegranate peel) was subjected to preliminary phytochemical qualitative screening for the presence or absence of various primary or secondary metabolites [Harbone et al., 1998].(TABLE 10) (PLATE 11)

1. Alkaloids: To 0.5 g of each extract, was added 5 ml of 2 N HCl and filtered. Dragndoff’s reagent was added and formation of a red precipitate was used to indicate the presence of alkaloids.

                            

2. Phenols: About 3 to 4 drops of ferric chloride solution was added to the extract. The presence of phenol was indicated by the formation of bluish black colouration.

 

3. Double bond: About 2ml of each plant extracts were mixed with a 2ml of potassium permanganate. The formation of brown colour indicates the presence of double bond.

  

4. Ninhydrin test : About 2 drops of Ninhydrin reagent was added with the 2ml of Plant extract. The positive indicates the appearance of a complex with a purple colour in the test tube.

 

5. Flavonoids: Sodium hydroxide (10%) was added to the plant extract. The formation of yellow colouration indicated the presence of flavonoids.

 

6. Phenolics and Tannins: A 10% Lead acetate solution was added to little quantity of the extract already dissolved in distilled water. The presence of tannin was indicated by formation of white precipitate.

7. Carbohydrate test : Molisch’s Test: About 2ml of each extract were mixed with two drops of α-naphthol and shaken thoroughly in a test tube. Then drops of concentrated sulphuric acid was added slowly.The appearance of violet ring indicated the presence of carbohydrate.

 

 GC - MS ANALYSIS OF ETHANOL EXTRACT OF POMEGRANATE PEEL

              The 2 micro liters of ethanol extract of Pomegranate peel was injected into the GC-MS system on a 30-m glass capillary column with a film thickness of 0.25 mm using the following temperature program: initial oven temperature of 40°C for 4 min, increased to 25°C at 158°C/ min, and then held at 25°C for 10 min. GC-MS was run under computer control at 70 eV. Chemical ionization was performed using ammonia as the reagent gas at 95 eV. The solvent (dichloromethane) peak was seen at 2.4 min during the GC- MS analysis followed by compound peaks. Identification of unknown compounds was made by probability-based matching using the system such as, WILEY Registry™95, NIST05 and NIST05s. MS data library and comparing the spectrum obtained through GC-MS compounds present in the ethanol extract of Pomegranate peel  sample wase identified. (TABLE 11) (FIGURE 2)

ANTICANCER ACTIVITY OF ETHANOL EXTRACT OF POMEGRANATE PEEL

The anticancer activity of the ethanol extract was evaluated using the MTT assay, a widely used colorimetric method for assessing cell viability and cytotoxicity (Mosmann, 1983). Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and allowed to attach overnight. The cells were then treated with various concentrations of pomegranate peel ethanol extract (10–500 µg/mL) for 24 or 48 h, while untreated cells served as the control. After incubation, 20 µL of MTT solution (5 mg/mL) was added to each well and further incubated for 4 h at 37 °C. The purple formazan crystals formed were dissolved by adding 100 µL of dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed as a percentage relative to the control, and the IC₅₀ values were calculated from dose–response curves. Apoptotic activity was assessed using acridine orange/ethidium bromide (AO/EB) dual staining and Annexin V-FITC/propidium iodide (PI) assays following standard protocols (Vermes et al., 1995; Ribble et al., 2005). Treated and untreated cells were stained and analyzed using a fluorescence microscope or flow cytometer to distinguish viable, early apoptotic, late apoptotic, and necrotic cell populations based on membrane integrity and phosphatidylserine externalization.

 

 

TABLE 1 : THE BIOCHEMICAL CHARACTERISTICS OF FOOD PATHOGENIC BACTERIA

Organisms	Oxidase test	Catalase test	Indole test	Methyl red test	Voges Proskauer test	Citrate test	Gram staining
Salmonella typhi	-	+	-	+	-	+	-
Shigella flexneri	-	+	-	+	-	-	-
E.coli	-	+	+	+	-	-	-
S.aureus	-	+	-	+	+	-	+
Camphylobacter jejuni	+	+	-	+	-	+	-

 

TABLE 2: TO DETERMINE SUSCEPTIBILITY OF COMMERCIAL ANTIBIOTICS AGAINST FOOD PATHOGENS

 

TABLE 3: ANTIBACTERIAL ACTIVITY OF ETHANOL EXTRACT OF POMEGRANATE PEEL AGAINST FOOD PATHOGENS

	Food  pathogens		Concentration of ethanol extract of EPP (µl/well)
			100		200		300		400
			Zone of Inhibition in mm
	Staphylococcus aureus		18		21		23		25
	Shigella flexneri		20		22		25		28
	Escherichia coli		17		20		22		24
	Salmonella typhi		19		25		27		29
Camphylobacter jejuni		16		18		19		20

 

TABLE 4: MIC AND MBC VALUE OF ETHANOL EXTRACT OF POMEGRANATE PEEL AGAINST FOOD PATHOGENS

 

TABLE 5: MIC AND MBC VALUE OF ETHANOL EXTRACT OF POMEGRANATE PEEL

S.No	Food pathogens	MIC value of ethanol extract of Pomegranate peel (µg/ml)	MBC value of ethanol extract of Pomegranate peel(µg/ml)
1	Staphylococcus aureus	400	500
2	Salmonella typhi	400	500
3	Shigella flexneri	400	500
4	Escherichia coli	400	500
5	Campylobacter jejuni	400	500

 

 

Table 6: ANTIOXIDANT ACTIVITY OF ETHANOL EXTRACT OF POMEGRANATE PEEL

Sample	Concentration µg/ml	OD @ 695 nm	% Antioxidant activity
Ascorbic acid	1000	1.96	100
EPP	400	0.32	83.67

 

Table 7: ANTI-BIOFILM ACTIVITY OF EPP AGAINST STAPHYLOCOCCUS AUREUS

Sample concentration	OD Value @ 620nm
Control	0.28
100µl	0.25
200µl	0.22
300µl	0.20
400µl	0.19

 

Table 8: ANTI-BIOFILM ACTIVITY TEST AGAINST SALMONELLA TYPHI

Sample concentration	OD Value @ 620nm
Control	0.29
100µl	0.22
200µl	0.20
300µl	0.18
400µl	0.16

 

 

 

 

 

 

 

FIGURE 1:  INHIBITION PERCENTAGE OF BIO-FILM ACTIVITY OF ETHANOL EXTRACT OF POMEGRANATE PEEL

 

TABLE 9: ANTI-INFLAMMATORY TEST OF ETHANOL EXTRACT OF POMEGRANATE PEEL

Sample concentration	OD Value @ 620nm	% inhibition
Control	1.96	-
1st Tube (200µl)	1.23	37.56
2nd Tube (400µl)	1.19	39.59
3rd Tube (600µl)	1.14	42.13
4th Tube (800µl)	1.11	43.65

 

TABLE 10: PHYTOCHEMICAL ANALYSIS OF ETHANOL EXTRACT OF POMEGRANATE PEEL

 

FIGURE 2: GCMS ANALYSIS OF ETHANOL EXTRACTION OF POMEGRANATE

 

 

TABLE 11: GC MS ANALYSIS OF ETHANOL EXTRACT OF POMEGRANATE PEEL

 

 

 

 

 

PLATE 1 : PREPARATION OF   POMEGRANATE PEEL POWDER

 

Pomegranate peel

Dried Pomegranate peel

Powder of Pomegranate peel

 

 

PLATE 2 : ISOLATION FOOD PATHOGENIC BACTERIA FROM CONTAMINATED FOODS

 

EMB agar - E.coli                                           Mannitol salt agar - Staphylococcus aureus

Camphylobacter isolation agar - Camphylobacter jejuni

 

SS agar - Salmonella typhi                                                                      SS agar - Shigella flexneri

 

 

PLATE 3: BIOCHEMICAL TEST FOR FOOD PATHOGENIC BACTERIA

Biochemical test for Salmonella typhi

 

PLATE 4 : SUSCEPTIBILITY OF COMMERCIAL ANTIBIOTICS AGAINST FOOD PATHOGENS

 

Plate 5: ANTIBACTERIAL ACTIVITY OF ETHANOL EXTRACT OF POMEGRANATE PEEL AGAINST FOOD PATHOGENS

 

 

Camphylobacter jejuni                                                                  shigella fexneri

 

Salmonella typhi

 

Staphylococcus aureus                                                             Escherichia coli

PLATE 6: MBC OF ETHANOL EXTRACT OF POMEGRANATE PEEL

 

a)     Salmonella typhi

b) Staphylococcus aureus

 

PLATE 6: ANTI-DIABETICS ACTIVITY FOR ETHANOL EXTRACT OF POMEGRANATE PEEL

 

 

PLATE 7: ANTIOXIDANT ACTIVITY FOR ETHANOL EXTRACT OF POMEGRANATE PEEL

PLATE 8 : ANTI-BIOFILM ACTIVITY OF ETHANOL EXTRACTION OF POMEGRANATE PEEL

a) Salmonella typhi

b) Salmonella typhi

 

PLATE 9 : ANTI-INFLAMMATORY ACTIVITY FOR ETHANOL EXTRACT OF POMEGRANATE PEEL

 

 

 

 

 

 

PLATE 10: PHYTOCHEMICAL ANALYSIS OF ETHANOL EXTRACT OF  POMEGRANATE PEEL

 

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