Improvement of edible coating properties by combining it...

Middle East Journal of Applied Sciences ISSN 2077-4613

Volume : 07| Issue :03|July-Sept.| 2017 Pages: 574-585

Corresponding Author: M.A. Youssef, Food Engineering and Packaging Dept. Food Tech. Res. Institute, Agric. Res. Center, Giza, Egypt.

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Improvement of edible coating properties by combining it with an advanced biological materials "Astaxanthin"

M.A. Youssef

Food Engineering and Packaging Dept. Food Tech. Res. Institute, Agric. Res. Center, Giza, Egypt.

ABSTRACT

In this study, the natural carotenoid "Astaxanthin" were cheaply produced from primary biological sources by a heterobasidiomycetes yeast Phaffia rhodozma species; to investigate the utilization of Astaxanthin as antioxidant and antimicrobial agent incorporated into edibe pectin-gelatin films. In order to overcome its poor water-solubility and the decomposition under light, heat and oxygen. Astaxanthin was loaded in nanostructured lipid carriers (NLC). Three concentration of Astaxanthin in NLC was evaluated (0.02, 0.05, 0.08%) for improving the microbiological safety of storage minced beef meat when incorporated with pectin-gelating film by a constant concentration, and to prevent the lipid oxidation of the coated meat storage samples. Among the concentrations tested, the formulation containing 0.08% Astaxanthin incorporated into NLC films had the maximum stability and revealed a major antioxidant activity and antimicrobial effect, which the results indicated that it afforded protection for beef meat samples against spoilage and prevented the growth of pathogenic bacteria for 5 weeks storage at 4oC. and prevent the lipid oxidation of coated meat samples. A comparative with the free Astaxanthin, the results indicated that the light, thermal, and oxidation stability of Astaxanthin in NLC was significantly enhanced. Key words: Astaxanthin, Nanostructured lipid carriers (NLC), Antimicrobial activity, edible film,

Antioxidants, stability

Introduction

More than 600 known carotenoids are widely distributed in nature, particularly in the plant kingdom, and in a wide variety of bacteria, algae and fungi.

The functions of cartenoids include the following: oxygen transporters, absorbers of light energy, colorants, provitamin A, scavengers of active oxygen and enhancers of in vitro antibody production in red blood cells (Xia et al., 2015).

In recent years, researchers found that there was a link between the consumption of food products rich in these carotenoids to the reduction in risk of developing certain types of cancer and many carotenoids act as antioxidants and have protective properties for human health (Marchiore et al., 2017). Some carotenoids have provitamin A activity, such as -carotene, - carotene and -cryptoxanthin (Gomes et al., 2017).

Carotenoids are lipophilic compounds and can be stored in a lipophilic environment, therefore, the outstanding antioxidant effects are in lipid phases by free radical scavenging or singlet oxygen quenching (Silva et al., 2016).

Astaxanthin is a diketo-dihydroxy-carotenoid (3,3-dihydroxy-B,B,-carotene-4,4-dione). It is the principal and abundant carotenoid pigment responsible for the pink to red color of the flesh of many marine animals. (Miaomiao et al., 2016). Astaxanthin possesses a higher antioxidative activity than B-carotene and -tocopherol, so that it is attractive as a powerful antioxidative reagent. Although astaxanthin does not have provitamin A activity, recent studies have demonstrated that it has considerable preventive activities against concer and may be used in medicine (Oh Yoon Ah et al., 2017).

Astaxanthin 550 times stronger than vitamin E which plays a critical role in protecting the polyunsaturated acids deposited in large amounts in the muscles reproductive organs, improved protection against oxidation and photo-oxidation (Tamjidi et al., 2014) and it was an excellent

Received: 28 July 2017 / Accepted: 28 August 2017 / Publication date: 17 Sept. 2017

Middle East J. Appl. Sci., 7(3): 574-585, 2017 ISSN 2077-4613

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quencher of O2 compared to several other carotenoids tested due to its anticarcinogenic activity. These properties have given rise to considerable our interest in using astaxanthin as food

antimicrobial. The chemical synthesis of astaxanthin is complex and costly due to the presence of chiral centers

in its molecular structure. Recently, the industrialist used microorganisms in synthesize and production of astaxanthin. The red-pigmented fermenting yeast phaffia rhodozyma synthesize it at high production rate and high purity (An et al., 2004). Molasses was shown to be an equivalent carbon source to glucose in the medium, which they reported that the high cell mass was obtained by glucose fed-batch culture. They showed that the antioxidant effect of the yeast carotenoids was found to be higher than that for the standard antioxidant (such as (+) catechin).

Oian et al., (2013) revealed that the natural carotenoids could be produced by yeast in high amounts and also containing new fractions (i.e. Astaxanthin, lycopene Echineone, Canthaxanthin and Lutein) having a high antioxidants power. This would reduce the cost of production and at the same time minimize the use of chemicals and solvents maintaining it more safe.

Xia et al., (2015) found that natural microbial cartenoids are successfully utilized in food processing as antioxidants for a lot of edible oils and other fats and could be used in pharmaceutical industry as it has inhibitory effects against lipid peroxidation in the body.

Nevertheless, the utilization of astaxanthin in many foods, beverages, is currently limited owing to its low bioavailability, poor water-solubility, high melting point, and instability under adverse conditions (e.g., acidic environment, heat, light, transition metals ions, singlet oxygen, and free radicals). The incorporation of it and nanostructured lipid carriers (NLC) is suggested to improve bioavailability and utilization of astaxanthin for fortification of aqueous – based foods. (Tamjidi et al., 2014).

Recently, nanostructured lipid carriers (NLC) were introduced as promising carriers for delivery of hydrophobic molecules into foods and clear beverages. They have some advantages over other colloidal delivery systems in certain circumstances. NLC have the potential to increase the chemical stability of encapsulated compounds in comparison to other lipid nanocarriers, intrinsically, because they are very effective at immobilization of active compounds within the lipid matrix (Tamjidi et al., 2013). Moreover, a major portion (about 70-99%) of their lipid phase is constituted by solid lipids.

The effectiveness of an antioxidant depends on the dispersion characteristics and the type of encapsulated active compound. (Oian et al., 2013). Therefore, a certain antioxidant may have different effects on stability of different active compounds encapsulated within different systems.

Food safety is an important global concern with health and trade implications. According to the Centers For Disease Control and Prevention (Scallan et al., 2011) foodborne diseases account for approximately 48 million illnesses, 128000 hospitalizations, and 3000 deaths each year in the United States. Four pathogens account for more than 80% of the estimated food-related deaths: Salmonella (35%), E.coli 0157:H7 (8%), listeria monocytogenes (27%) and staphylococcus aureus (10%).

Although meat products have been implicated in several foodborne outbreaks over the years, these products also are prone to spoilage. As seen with many refrigerated foods, undesirable microbial growth is responsible for most spoilage in meat products, whereas biochemical and enzymatic deteriorations also occur.

One way to control microbial growth in these food products, thereby improving safety, and delaying spoilage, is the application of antimicrobial dips or spray on the surface of the product (Kerry et al., 2006). However, in these applications, the efficiency of the antimicrobial substances is restricted due to uncontrolled migration into the food and partial inactivation of the active compounds following interactions with food components (Oh Yoon et al., 2017). One new approach to overcome these limitations is the use of antimicrobial packaging techniques, which are considered a type of active packaging (Kuswandi et al., 2011). Antimicrobial agents can be incorporated directly into the packaging materials, coated onto the surface of the packaging film, or made into a sachet that can be added directly into the package (Cerqueira et al., 2016) to control microbial growth.

Active packaging is an innovative concept in which the package, the product, and the environment interact to prolong the shelf-life, enhance safety, or improve sensory properties, while maintaining the stability and quality of the product.

This concept has benefited recently by the use of nanotechnology materials, including nanocoatings and nanoparticles (Khalil et al., 2013).

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Film formation and properties of several polysaccharide, protein, and lipid substances have been reviewed (Victor et al., 2011). Commercial applications of edible films and coatings include Pectin- and gelatin – based increased the mechanical properties and could improve the moisture barrier of the resulting films. Gelatin produces flexible tough films when cast together with pectin and glycerine or sorbitol (Lee and Min, 2013). The combination of polysaccharide and protein material showed a synergism resulting in a better mechanical property when used to prepare biodegradable film (Cheorun et al., 2005).

The main objective of this study is to produce of edible coatings having an antioxidant & antimicrobial activity for prolonging the shelf life of its coated products evaluate the antibacterial activity of edible film containing astaxanthin in nanostructured lipid carriers (NCL) and against food borne pathogens to improve the microbiological safety of foods, when associated with patties from raw minced beef during long-term refrigerated storage (4oC). Investigate the stability of free astaxanthin (ASTA) and astaxanthin in nanostructured lipid carriers (NLC) at different conditions; light, heating and oxidation (Comparative tests).

Materials and Methods Materials: Strains:

NRRLY-10922 strain of Phaffia rhodozyma which used for the production of ASTA was

provided by the Agricultural Research Service (A.R.S) Culture Collection of the Northern Regional Research Laboratory (NRRL) USDA, Peoria, IL., USA.

Corn meal:

Egyptian white corn grains were ground completely to produce the corn meal.

Methods: Production of ASTA:

ASTA was produced from NRRL-10922 strain of Phaffia rhodozyma according to Youssef et

al., (2004). The culture was maintained on production media containing only corn meal as source of carbon & nitrogen. The medium was sterilized at 121oC for 15 min. The stock cultures were incubated at 20oC and pH 4 for 48 hours then kept in the refrigerator at 4oC and subcultured monthly. Ten ml culture were chemically treated with triethyl amine (TEA) to increase the ability of strain to produce more ASTA as described by Morsy et al., (2014). Preparation of ASTA–NLC:

Three mixtures differ in the percentage of ASTA; 0.02, 0.05 and 0.08% was prepared. A volume

of 2ml of dichoromethane was added to the lipid phase (757 mg glyceryl monostearate, 218 mg oleic acid, 5 gm Lecithin and 0.2mg ASTA for the first mixture, 0.5gm ASTA for the second and 0.08 mg ASTA for the last mixture) with a capped flask and warmed to 35oC until a clear homogeneous mixture was obtained. Then, dichloromethane was removed under N2 flushing and the lipid phase was heated to 78oC. The aqueous phase was prepared by dissolving 555mg Tween 80 in 18.445 g phosphate buffer solution (pH7) containing sodium azide (0.02%) and heated to 78oC. Subsequently, the aqueous phase was added to the lipid phase and the mixture was stirred at 2000rpm for 3 min at the same temperature. The mixture was then transferred into a bath sonicator (Powersonic 505, Seoul, Korea) at 25oC for 15 min. The suspension was warmed to 78oC again and homogenized further using a probe-type sonicator (probe: TT13, Berlin) for 4 min with 2s on and off pulses periods. Finally, the hot nanoemulsion was cooled down to 10oC in an ice bath under continuous stirring, and then transferred into capped glass tubes and stored in the dark at 35oC for 15 days (Fardin et al., 2014).


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Preparation of coating films: Pectin 2.5%, w/v + gelatin 2.5%, w/v (Sigma – Aldrich Co., St. Louis. Mo, USA) + polyvinyl

alcohol 1.25%, w/v + glycerol 2.5%, w/v were suspended in distilled water (100 ml) at 90oC after stirring for 30 min. After degassing the solution a concentration of 30% (w/v) of ASTA-NLC was incorporated in the film forming solution, at 25oC with magnet stirrer. Pure pectin-gelatin (control film) was prepared by adding 3% Thyme essential oil (as reliable antioxidant ) and without the addition of ASTA-NLC. After the components of the film had been mixed, the films were formed by pouring the pectin-gelatin formulation over a Teflon covered glass surface and drying at 25oC for 24h. All films were equilibrated at 52% RH and 25oC for 48 h. before being used and /or tested. (Cheorun et al., 2005). Patty preparation:

A 20g of homogeneous ground beef meat patty samples were prepared. The patties were

separated into five groups; uncoated, coated with 0.02% ASTA-NLC film, coated with 0.05% ASTA-NLC film, coated with 0.08% ASTA-NLC film and coated with pure pectin-gelatin based materials+ Thyme oil 3 % (control film). The beef meat patties were coated by dipping them into the prepared coating solutions for 5s at room temperature, then drying for 30s. This dipping procedure was repeated three times, then the patties were dried for 2h in a laminar flow hood at 25°C and kept in refrigerator until tested. Methods: Microbiological analysis of ground beef:

At each sampling interval, duplicate packages from each treatment were aseptically opened and a

10-g-portion from the center of the patties was homogenized in sterile maximum recovery diluent (Merck) in a Seward stomacher for 1 min to make the initial dilution. Appropriate serial dilutions (10–

1–10–6) were spread plated on Plate Count Agar (PCA. Merck, Darmstadt, Germany) for total viable counts (TVC) the count methods proposed by Downes and Ito, (2001) was used to enumerated total coliforms. Hi Crome Agar is a selective medium for E.coli 0157:H7, Baird Parker agar + egg Yolk tellurite emulsion (BPA : Merck) for staphylococcus aureus, Bacillus cereus counts was enumerated on selective ajar base medium (PEMBA) and one vial from polymyxin B supplement was added (Holbrook & Anderson, 1980) and Tryptose Sulfite Cycloserine (T.S.C) agar base for Clostridium perfringens. Plate count technique FDA (1995) for Yeast and moulds counts. Determination of lipid oxidation of minced meat samples:

Lipid oxidations in patty samples were assessed using the 2-thiobarbituric acid (TBA) distillation

method of Ke et al., (1977). TBA reactive substances (TBARS) were expressed as mg malonaldehyde/kg sample. Analyses were conducted on days 0, 4, 8 and 12. Stability of free ASTA and ASTA in NLC: Light stability:

The light stability of free ASTA and ASTA–NLC were detected at a sustaining stable light

source with 178 Lux. All samples were exposed to the light for 18 days at 25oC in an incubator. At a prescribed time, the retention rates of free ASTA and ASTA–NLC were measured by HPLC (Shimadzu, Kyoto, Japan). The detection wave length were set at 476 nm with a resolution of 1.2 nm. Thermal stability:

The thermal stability of free ASTA and ASTA–NLC were investigated at 40oC and 60oC,


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respectively. All samples were heated for 15h in the dark environment. The retention rates of free ASTA and ASTA–NLC were measured by HPLC every 3h.

Oxidation stability:

The high antioxidant activity of ASTA is because of ketonic (C=O) and hydroxylic (OH) bonds

that scavenge free radicals and quench singlet oxygen. In this experiment, the oxidation stability of free ASTA and ASTA–NLC were detected in the H2O2 solution. All samples were dispersed into aqueous solutions with various amounts of H2O2 (0, 0.02, 0.04, 0.06, 0.08, 0.10 m mol). The comparative experiments were conducted at 25oC for 2 h and kept in dark for avoiding the decomposition of H2O2. The retention rates of free ASTA and ASTA–NLC were measured by HPLC.

Methods described by Miaomiao et al., (2016) for investigate the stability of ASTA film was followed in this study. Determination of antioxidant activity of film samples:

Antioxidant activity was evaluated by determining the scavenging activity of 1,1-diphenyl-2-

picrylhydrazyl radical (DPPH; Sigma-Aldrich), following the procedure described by Kim et al., (2014). 100 mg of film samples vortexed with 5ml methanol for 3 min. The mixture was left to sit for 3 h. at 25oC, and were added to a methanol solution of DPPH (100µM). The solution was left to sit for 15 min at room temperature in the dark and then absorbance was determined at 517nm. Radical-scavenging activity was calculated from the difference in absorbance between the blank and an extract sample. Results and Discussion 1. Effect of different ASTA-NLC films on the growth of different microbial flora:

Results in Table (1) explained the effect of three ASTA films incorporate with nanostructured

lipid carriers (NLC) at different concentrations after 5 weeks of storage the samples at 4oC. It has been found that the numbers of aerobic colonies was decreased with all biolife film tested. The highest decrease in colonies was appeared with 0.08% – ASTA film (14X103CFu/g) and the lowest decrease in aerobic colony counts were found in case of 0.02% – ASTA film (55X103CFu/g). This diminution represent nine fold the decreasing of the control sample (126X103CFu/g).

Table 1: Effect of edible films with different concentration of ASTA in corporate with nanostructured lipid

carriers (NLC) on the growth of different microbial flora when used to pack meat patties in refrigerator for five weeks

Samples Aerobic colony count

Total coliforms CFU/gx10

E. Coli 0157:H7

CFU/gx10

Staph. aureus

CFU/gx10

Cl. perfringens CFU/gx10

Bacillus cereus

CFU/gx10

Yeast & Moulds

CFU/gx10 F.C.S 102X104 27 11 64 5 n/d 47 C.S 240X104 59 23 90 14 n/d 81 Control 126X103 35 16 30 5 n/d - I 55X103 20 – 14 n/d n/d – II 37X103 9 – 5 n/d n/d – III 14X103 5 – 2 n/d n/d –

* F.C.S : Fresh sample at zero time without film. * C.S : fresh sample without film after storage at 4oC for 5 weeks. * Control : Pectin – gelatin film sample with antimicrobial material (3%) Thyme oil) after storage at 4oC for 5 weeks. * I : 0.02% ASTA – NLC film. * II : 0.05% ASTA – NLC film. * III : 0.08% ASTA – NLC film. * n/d : Not detected in 25 gm. * – : Negative in 10 gm. * CFU/g : Colony forming units per gram.


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Similarly, all the tested biolife films were able to decrease the number of total coliforms, S. aureus and mold colony count. The lowest colony count was found with 0.08% ASTA in ASTA-NLC film and the other samples showed an obvious decreases if compared with blank sample (Table 1).

All three films specimens exhibited no any colony count has been found for E.Coli 0157:H7, Clo. Perfringens and B. cereus after 5 weeks of storage the samples at 4oC.

It has been concluded that 0.08% ASTA-NLC film followed by 0.05% ASTA-NLC film were found to have the best results against all tested microorganisms.

The results were agreement with many studies demonstrated that carotenoids have an antimicrobial activity when incorporated into the packaging films. 2. Determination of lipid oxidation of minced meat samples:

Lipid oxidation developed in all patty treatments as storage time increased, however, the effect

varied with concentration of ASTA in NLC (Table 2). Patties held in ASTA-NLC film of 0.08% ASTA concentration had lower TBARS compared to held in ASTA-NLC film of 0.05 and 0.02% concentrations. But all treatments groups remained well below a value at which certain off-flavors can be detected in different samples. The concentration of 0.08% ASTA in NLC was the best with respect to impeding lipid oxidation. Miaomiao et al., (2016) were found that the higher the ASTA concentration used any nanostructured lipid carriers film, the lower the TBARS determined in its coated product.

Therefore, meat coatings consisting of pectin and gelatin were reported to be effective in protecting several meat products from lipid oxidation (Cheorun et al., 2005) and in this study, active packaging by the use of nanotechnology materials including nanocoatings and nanoparticles was shown to impede lipid oxidation in minced meat products had the best protection. Table 2: Determination of lipid oxidation of coated minced meat samples:

Samples TBARS of minced meat samples with different casings

Day 0 Day 6 Day 8 Day 12 Without ASTA (Control) 0.198 0.799 1.025 1.345 With 0.02% ASTA 0.198 0.664 0.909 1.153 With 0.05% ASTA 0.198 0.488 0.734 0.966 With 0.08% ASTA 0.198 0.347 0.598 0.710

3. Characterization of the film: 3.1. Light stability of free ASTA and ASTA in NLC:

With the purpose to detect the stability of free ASTA and ASTA-NLC comparative tests: light,

thermal and oxidation stability were performed. As shown in fig. 1, After 18 days, the retention rate of free ASTA decreased rapidly to 55%

whereas ASTA-NLC was slightly decomposed with less than 10% for samples of 0.08% concentration of ASTA and a maximum decompose with less than 16% for samples of 0.02% concentration of ASTA in NLC. The results indicated that NLC were able to protect ASTA from light when its content of ASTA between 0.05 and 0.08% which, the retention rate of ASTA–NLC was greater than 86%. 3.2. Thermal stability of free ASTA and ASTA in NLC:

The thermal stability were studied at 40oC and 60oC with 15h. (fig. 2). The retention rate of free

ASTA decreased rapidly to 86 and 75% when ASTA was heated at 40oC and 60oC, respectively (fig. 2A). The maximum loss rate of ASTA-NLC was less than 10% at 40oC and 20% at 60oC (fig. 2B) in all samples. Therefore the thermal stability of ASTA was improved through NLC. Free ASTA was degraded into many products by multiple fracture patterns under heating, and with the increasing of temperature, the degradation rate of free ASTA was accelerated dramatically (Yuan and Jin, 2010)


Figure 1: The light stability of free ASTA (A) and ASTA-NLC (B), during 18 days of storage

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Figure 2: The thermal stability of free ASTA (A): at 40o and 60oC and ASTA-NLC,

(B): at 40oC and (C): ASTA-NLC at 60oC

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3.3. Oxidation stability of free ASTA and ASTA in NLC: The oxidation stability of ASTA was investigated in H2O2 aqueous solution. H2O2 with a strong

oxidability, is often used as dispersion medium to detect the oxidation stability of ASTA-NLC. The retention rates of free ASTA and ASTA-NLC along with the amounts of H2O2 are presented in fig 3. The retention rate of free ASTA had a sharp decline of 42% by the increasing amount of H2O2 "Retention rate 58%" (fig. 3 A). The retention rates of ASTA in NLC were over 69% during experiments, (fig.3B). Especially, when the content of ASTA was 0.08%, the retention rate of ASTA in NLC could reached 84%.

Based on the above experiments, it could be concluded that the stability of ASTA was enhanced through this manostructured lipid carriers technology, which protected ASTA from degradation induced by external environment. It was mainly due to that ASTA was embedded in lipid nucleation during ASTA-NLC technology (Qian et al., 2013)

Experimental data indicated that the optimized content of ASTA was 0.08% on the basis of maximum retention rate in the studies of light, thermal and oxidation stability. 4. Effect of ASTA concentration in ASTA-NLC on the antioxidant activity of the coating film during 5 weeks of storage at 4oC:

The DPPH scavenging activities of all samples ranged from 90.2 to 92.0% on day 0 (Table 3).

Film control had no radical scavenging activity. Scavenging activity of the films significantly increased as ASTA concentration increased. According to Moradi et al., (2012), antioxidant power of edible films depended on the amount and strength of added antioxidant additives in biofilms. In general, antioxidant activity did not significantly change throughout storage at 4oC in sample – 0.05% and sample – 0.08% ASTA, however, the percent of decreasing in sample – 0.02% ASTA was reached to about 34% after 5 weeks which DPPH radical scavenging activity was 59.4% for 0.02% ASTA (Table 3), indicating that the rate of antioxidant activity loss was reduced by increasing the ASTA concentration in ASTA-NLC film. The higher antioxidant activity observed in samples on day 7 might be due to a more uniform distribution of ASTA, resulting in the greater stability of pectin-gelatin emulsion coating. This could help maintain scavenging activity longer.

Table 3: Effect of ASTA concentration in ASTA-NLC on the antioxidant activity of the different films during 5 week storage at 4oC:

Storage time (week)

DPPH radical scavenging activity (%) Without ASTA 0.02% ASTA 0.05% ASTA 0.08% ASTA

0 Zero 90.2 90.5 92.0 1 Zero 92.8 94.3 95.5 2 Zero 85.8 91.0 92.6 3 Zero 72.7 86.8 88.0 4 Zero 68.9 84.4 87.2 5 Zero 59.4 83.8 85.9

Conclusion

The present study investigated the preparation of edible film containing ASTA loaded in

nanostructured lipid carriers (NLC). ASTA produced by cheap biological sources using Yeast strains and natural by-products. Through the results obtained in this study, success was observed in the production of the formulations of ASTA-NLC. The results have demonstrated that ASTA incorporated into NLC films were effective against pathogenic microorganisms such as S. aureus, E.coli 0.157:H7 and C. perfringens when associated with fresh meat, and had the stability under various conditions (light, temperature, oxidation). It also have the potential to improve the safety and quality of edible coating foods, thereby meeting the expectations of both food manufacturers and consumers who purchase them.

In addition, the results revealed that these films prevent the lipid oxidation of the coated meat samples and may be used as an alternative for food preservation due to the higher antioxidant activity.


Figure 3: The oxidation stability of free ASTA (A) and ASTA-NLC (B) in aqueous

solution with various amount of H2O2

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This study underline the importance of adding appropriate ASTA incorporate the edible film by the use of nanotechnology materials, as antimicrobial agents. References

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