Anti-oxidative and Antibacterial coating for fish steaks using composite pullulan and polyphenols of

pomegranate
Mozhiarasu S, Satish Kushwaha, Dr. Manjeet Aggarwaal
National Institute of Food Technology Entrepreneuship and Management

Abstract
In order to reduce the spoilage in foods, various technologies can be used hand in
hand, one such technology is the edible coating, carbohydrate based coating on food
can help to reduce the oxygen diffusion and if the coating contains antimicrobials, it
also helps to protect against microbial growth. Pomegranate peel having large set of
polyphenols which are reported to act both anti-oxidative and antimicrobial agents.
In this work the pomegranate polyphenols were extracted with water, purified using
Amberlite XAD 16 column obtained the yield of 70.3612.56 mg gallic acid eq /g of
peel and 52.8211.83 mg gallic acid eq /g of peel during extraction and purification respectively. The anti-oxidative and antimicrobial properties were analysed. The DPPH
IC 50 value of polyphenol was found to be 16.15 g/mL and MIC of 5 test bacterium
was determined. The effect of different formulation of polyphenols and pullulan coating solution on microbial growth and TBARS values were evaluated. Thus 9.5% pullulan, 0.5% Polyphenol with 0.5% carboxy methyl cellulose improved coating stability
and the increased the shelflife of the fish steak from 3 days to 8 days at 4oC in terms
of microbial quality. Coating with pullulan-Polyphenols has also reduced the TBARS
value approximately 4 fold and improved the fish steaks from oxidative stress.

ator. The samples were taken on 3, 6, 8, 10th day and all the parameters were
analyzed until all the sample reached the maximum permissible microbial count
107 CFU/g or 7 log units.

Results and Discussions

Anti Bacterial activity represent the polyphenols are more active against gram
positive bacteria than gram negative bacteria except Pseudomonas aeruginosa.
While all the tested bacteria can be inhibited at 2mg/mL concentration but Samonella typhimurium was quite resistant, inhibited only at 4 mg/mL.
Basic-Total Volatile Nitrogen has increased more in control samples while reduction
upto 2.5 fold in sample containing Polyphenol. TBARS determination results shown,
decreasing in TBARS value in coated sample even without polyphenols shows presence of pullulan reduced oxygen diffusion. There was 4 fold reduction in the CMCPullulan-Polyphenol coated sample.

Introduction
The safety and quality of the food depends on the nutrition, hygiene, and organoleptic
characteristic of the food [3]. But the characteristic may deteriorate during storage and
commercialization. The shelf-life of the fish depends on two factors, the first one is the
microbiological load, in which fishes are highly prone to microbial spoilage because
of its nutritious quality, water activity (0.95) and neutral pH 6.5-7 [6], apart from that,
autooxidation of lipids is another factor which converts the nutritious fatty acids into
harmful toxic components like malondialdehyde, so in order to prevent from spoilage,
various technologies can be used hand in hand, one such is edible films and coatings,
coating material will consist of biopolymers like carbohydrates, proteins, or lipids and
resins and composite material made of those. Carbohydrate coating can prevent the
diffusion of the oxygen and prevent oxidation, thus an antibacterial and antioxidative
components along with polysaccharide coating helps to achieve the goal of enhancing
shelf-life. Pomegranate are rich in phytochemicals and its peel found to have components which are antibacterial and anti-oxidative potential. Using of pomegranate peel
utilizes the fruit waste and added value to both food (fish steak) and waste (peel).

Approximately 2 fold increase of antioxidative activity of the polyphenols after purification.

An edible coating has been developed using pullulan and cmc (polysaccharide) and
polyphenol from pomegranate peel.

Scavenging property for free radicals of pomegranate polyphenols is greater

than BHT IC 50 value: 16.15 g/mL (BHT- 21.2 g/mL L (Kannat et al., 2010))
COATING EXPERIMENT RESULTS

Materials & Methods

Extraction of Polyphenols from pomegranate peel: Pomegranates (Bhaguva
variety from local market at Narela) were washed with potable water and peels
were removed. The peels blended with water in ratio 1: 10 (peel:water) (w/v)
transfered to conical flask and it was kept at 50oC for 15 minutes for maximum
extraction with occasional stirring. Then it was filtered through muslin cloth and
centrifuged at 3000g for 15 minutes to remove insoluble. The extract was stored
in brown bottle.
Purification of polyphenols: The pomegranate polyphenols were purified by
using Amberlite XAD 16 resin [7]
Total Phenolic Content: It was measured using Folin Ciocalteu method
Characterization of polyphenols in HPLC-UV: HPLC System: Waters Inc USA
Model 2707; Column: C18 Spherisorb ODS 250 column 4.6mm (5); Column
Temperature : 25oC; Mobile phase A- 1.3g potassium di hydrogen phosphate
solution in water ; Mobile phase B Acetonitrile 100%; Standards used: Gallic
acid, Ellagic Acid, Punicalgin.
Antioxidative Assay: DPPH scavenging activity was performed as described in
Sweetie Kannat, 2010
Antibacterial activity: Antibacterial well diffusion assay and Minimum inhibitory
concentration(1-10mg/mL) was performed as described in Al-Zoreky, 2009.
Preparation and Coating of Fish: The fishes were procured in local market and
brought to lab with ice pack, it was wased with potable water, beheaded, degutted and made into steaks. The fishes were placed in coating formulation in the
ratio (1:2) in a container and kept inside the vacuum chamber for the vacuum impregnation at 100mmHg at room temperature for 15 minutes (Duan, Cherian, &
Zhao, 2010). The coated fish samples were removed and air dried for 30 minutes and kept inside the LDPE zipped pouches for the storage at 4oC in refriger-

Effect on Microbial Load:

References
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coatings, Food Chem., vol. 119, no. 2, pp. 524532, Mar. 2010.
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Purified Powder of Pomegranate Polyphenol

Presence of ellagic acid and punicalgin.

The coating on to fish steaks increased the shelf life from 3 days to 8 days at
4oC.
The present project involving the use of polyphenols from pomegranate peel
helps in waste utilization from peel
Besides increasing the shelf-life of the product, the coating helps in providing the
functionality to the product.
The cost economics of the product can be further brought down by blending a
polymer from other sources (possibly from fruit waste).

Polyphenols reduced the bacteral count and enhanced the shelf life, while pullulan-polyphenol coating enhanced shelflife from 3 to 6 days and addition of CMC into pullulan and polyphenols increased the retention and stabilized the coating from
washout during drip loss.