Potato peel extracts as an antimicrobial and potential antioxidant in active edible film

Abstract Potato peel is phenolic‐rich plant byproduct that has multiple biological functions. The study explores the antimicrobial efficiency of the peel and its antioxidant potential incorporated with potato starch film. Antimicrobial sensitivity test using microdilution and diffusion test revealed positive response in Escherichia coli, Salmonella enterica, and Staphylococcus aureus with minimum inhibitory concentration, 7.5 ± 2, 5.8 ± 2, and 4.7 ± 1 mg/ml, respectively, but had a negative response for Klebsiella pneumoniae and Listeria monocytogenes. HPLC analysis revealed that caffeic, chlorogenic, and neochlorogenic acids are the main chemical compounds found in potato peel extract and responsible for its antimicrobial property. The scavenging ability and phenolic content in the active film range from 10 to 22 mg GAE/g of the dried film with 24%–55% of inhibition, respectively. The scanning electron microscope of the starch film showed homogeneously and ordered structure; however, some cracks and pores shown in the active film. The simulation experiment of phenols migration from the film into fatty and aqueous foods showed higher concentration in aqueous and less in the fatty foods. Therefore, potato peel extract has antibacterial as well as antioxidant property and incorporating with potato starch produce an active film which can be used as an alternative technology for active food packaging.

Fat oxidation is the main factors influence the value of food because this reaction results in off-flavor of processed foods (Kingston, Monahan, Buckley, & Lynch, 1998). Fat undertakes prominent oxidative changes at boiling temperature during storage; however, the addition of antioxidant delays the oxidation reaction. Potato peels (PP) is one of the phenolic-rich food processing industrial wastes and has a strong antioxidant ability which is equal to the synthetic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Generally, in food processing industries, synthetic antioxidants are the most efficient ways to decrease rancidity and toxic oxidation molecules, but they have a long-term consumer's health hazard (Gebrechristos & Chen, 2018;Ito et al., 1986;Perelman et al., 1998;Sébédioo, Kaitaranta, Grandgirarda, & Malkk, 1991). Such concerns and demand for safe food for consumers the food industry seek innovative food preservation strategies. The recent trends in food packaging technology are focusing on the development of films that play an effective role in the preservation of food quality (Galdi, Nicolais, Maio, & Incarnato, 2008). Therefore, to my knowledge, a study dealing with PP extracts incorporated with potato starch film to produce active film has not performed yet. So the objective is exploring the antimicrobial efficacy of PP extracts and its antioxidant potential incorporation with an active film made of potato starch.

| Preparation of PP extracts
Fresh potato was purchased from local Market, washed, peeled, and dried in a hot air oven (Horizontal Forced Air Drier, Proctor and Schwartz Inc.) at 55°C. The dried PP was ground into a fine powder in a mill (Tecator Cemotec 1090 samples mill). The material that passed through an 80 mesh sieve was reserved for use. About 10 g of ground PP was extracted with 100 ml of ethanol overnight in a shaker at room temperature. The extract was filtered through cheesecloth, and the residue was re-extracted three times under the same conditions. The combined filtrate was evaporated in a rotary evaporator (EVF-530-010K-GallenKamp) below 40°C temperature.
The extracted dried powder was then stored at −20°C.

| Bacterial strains
A total of five bacterial strains attained from food science and technology institution, CAAS microbiology laboratory were used to test the antimicrobial effects of the PP extract. These include three strains of gram-negative bacteria and two strains of gram-positive bacteria (Table 1). All strains of these bacteria are well-known foodborne bacterial pathogens, except E. coli which used as surrogates for E. coli O157:H7. Before used, the bacterial strains were consecutively subcultured three times in 24 hr interval. From the subcultured bacteria strain, 10μl of culture transferred into 10 ml liquid medium, and then the media incubated at 37°C for 24 hr.

| Agar test for bacteria
The inhibitory effects of PP extract tested bacteria were determined by agar well diffusion method (Oke, Aslim, Ozturk, & Altundag, 2009). The autoclave media was added to 50°C in a water bath, and an overnight culture of the bacterial strain added at a final concentration of 10 5 CFU/ml. The prepared suspension poured into a sterile Petri dish and kept for 1h at room temperature to solidify. A well (diameter = 6 mm) was prepared. PP extract was diluted to the concentration of 5 mg/ml with broth, and 50 μl of diluted extract was dispensed into each agar well. Plates were kept for at least 1 hr at room temperature to allow the extracts to diffuse into the agar before incubation at 37°C for 24 hr. After incubation, the degree

| MIC and MLC of PP extract
The MIC and MLC of PP extract performed using a broth microdilution method. Forty-five well culture plates were prepared, and serial twofold dilutions of the extracts were distributed into the plate wells. The volume of dispensed extract was 0.1 ml per well in the concentration range of 10-0.31 mg/ml. The same volume (0.1 ml) of overnight bacterial culture at a density of 10 5 CFU/ml was added to the wells, and the culture plates were placed in an incubator for 24 hr at 37°C. MIC was selected as the lowest concentration of the PP extract required to inhibit the visible growth of the tested microorganism.
The MLC was also determined by streaking the suspension in the well with concentrations greater than the MIC. After that, the subcultured agar plates were incubated overnight at 37°C. The MLC was defined as the lowest concentration of extract at which no viable microorganism was detected by subculture.

| Aqueous potato peel extracts (PPE)
Aqueous potato peel extracts were prepared with 10 g of dried and milled potato peel mixed with 100 ml of distilled water and placed in a thermostatic bath at 50°C for 60 min. After obtained the mixed potato extracts were cooled, filtered (pore size 0.45 mm), and stored at 4°C in dark flasks until used. The extraction yield, as determined gravimetrically at 80°C until constant weigh, was 1 mg of dried extract/ml of sample. The solution was used in different proportion to make active edible film.

| Active edible film preparation
Potato starch film was prepared by mixing 93 g of distil water 5 g potato starch 1.5 g of glycerol. The mixture will start out white in color and change to transparent or translucent. It will also thicken.
Once the initial white color of the starch is completely gone and the mixture has thickened remove from the heat. The thickened solution was poured into a labeled dish, the air bubbles removed by glass stir rod and then placed in a stable, labeled table for air dry. This film considered as control film (PSF) but the active films were prepared, a water mass (5, 10 or 20 g) from the formulations replaced with the same amount of aqueous PP extract so the active film referred as

| Film observation in SEM
The prepared starch film (refer Section 2.4.2) was cut in to 20 mm size manually using sharp scissors. Three pieces of each sample were immersed into 5 ml of supersaturated solution of sodium bromide. Micrographs of cross sections of the films were obtained using a Field emission scanning electron microscopy (FE-SEM). The specimens were cryofractured by immersion in liquid nitrogen. The samples were mounted on stubs and sputtered with a thin layer of gold (thickness below 50 nm) prior to SEM observations. Images at different magnifications (2,000× up to 10,000×) were obtained using a voltage of about 3 kV and a spot size of ~2 nm. The thickness of each film was measured from SEM images at six randomly selected points, using the ImageJ software. Accordingly, selected images taken for analysis.

| Phenol content of PPE and active PSF
Extract of PP dilution in cleaned water numerous concentration lev- where Ab is the blank and As is the sample.

| Phenolic migration of PPE
Polyphenol migration tests were performed considering water and ethanol 95% as food simulators for aqueous and fatty foods, respectively (Baner, Bieber, Figge, Franz, & Piringer, 1992). Samples film pieces of 2 cm 2 were immersed in 5 ml of food simulant and placed

| HPLC-DAD analysis of phenolic compounds
Phenolic acids of potato peel were purified by microporous mem-

| Antimicrobial nature of PP
The antimicrobial activity of PP extracts was determined by agar well diffusion method, and the detail results are available in (Table 2).  The difference between gram-positive and gram-negative bacteria to antimicrobial response differs in cell wall structure. The cell wall of gram-positive contains single-coat, whereas gram-negative has multilayered structure bounded by external cell membrane (French, Whistler, Bemiller, & Paschall, 1984). Therefore, intra-cellular spaces of bacterial can easily hyperacidified, triggering functional disorder of bacterial energy metabolism. However, the exterior lipopolysaccharide coating gives more buffering capacity to gram-negative bacteria, functioning as a defensive fence for hydrophobic compounds (Haahn, Jones, Akha, & Rockland, 1977). Accordingly, the bacteria show less sensitivity to the antimicrobial activities of polyphenols.
Thus, most plant sources antimicrobial have harmless to use in food.
Therefore, PP extracts could be an alternative antimicrobial and promote the reduction in the use of synthetic preservatives in food. This could be because starch film has comparatively smooth surface impermeable for large molecules (French et al., 1984). When granules put in warm water, it initiates to hydrate then swell (Haahn et al., 1977). The multipart nature of the heated granule structure and the starch chains become more mobile as hydrogen bonding between adjacent glucose units is interrupted and water enters the matrix (Rodríguez-rojo, Visentin, Maestri, & Cocero, 2012). The increase in chain mobility allows the starch film to become more flexible and swell. At the same time, amylose and small amounts of amylopectin begin to leach from the granule matrix into the aqueous medium (Shamekh, Forssell, Suortti, Autio, & Poutanen, 1999). Granule hydration further accelerated by the presence of small pores that reportedly span from the granule surface to the core region (Kim & Huber, 2008). Therefore, the change in structural showed in PSF and active PSF is the difference in the amount of aqueous PP with the starch film. So, farther research should focus on determining the optimal level of aqueous potato peel to minimize cracked and massive pore in the surface of the edible plastic film for better performance to be used as food packaging.

| TPC and antioxidant activity of active film
The TPC, DPPH inhibition %, and migration of polyphenols of the active film are shown in Table 3; the phenol content and DPPHscavenging activity of active film showed that increasing amount of PPE to the formulations will produce higher polyphenol amount  In Figure 3, the TPC of PP extracts of three solvents showed significant variation with (p < 0.05) value. The highest TPC attained from ethanol extracts (39 ± 0 mg of GAE/g) followed by Methanol (49 ± 2mg of GAE/g) and Petroleum (22 ± 1 mg of GAE/g of dried PP). The DPPH-scavenging ability in between the solvents does not show a significant difference with % inhibition obtained 65 ± 1, 60 ± 2 and 51 ± 2 for Methanol Ethanol and Petroleum, respectively.
On the contrary, TPC reported by Arun et al. (2015). The higher TPC content obtained from ethyl-acetate extract (83.2 GAE/g of dried PP weight of extracts) and showed higher DPPH-scavenging activity.

| Migration test of PPE from active film to food simulants
The polyphenol migration test using food simulant of water and 95% ethanol migrated from the active film to stimulants measured after seven days of the active film exposure inside the simulant (Table 3). The highest amounts and fast flow of polyphenols revealed in the active film the aqueous medium. In contrast to this, in the ethanol medium, the polyphenol amount was low. Active agents of a film release to any type of food from aqueous to fatty products, so the film could be used in active packaging (Yang et al., 2016). The migration of chemical compounds from active film packaging to food simulants can be affected by temperature, concentration, and the affinity of the compounds to the food simulant (Santos, Silva, & Andrade, 2017). In addition several factors influence migration of polyphenols such as nature of the bioactive compound, the chemical composition the reaction in-between film and bioactive compound, the structural matrix, and the medium condition (Baner et al., 1992).

| CON CLUS IONS
Potato peel extract has antibacterial and antioxidant properties and active starch film through incorporating potato peel extracts was successfully developed. The phenolic compound in the extract has an ability to hinder fat oxidation and bacterial inhibition. The harmless nature of most plant extracts such as potato peel has more acceptability by food processing companies to use as a food preservative. Potato peel extracts can be used in different mechanisms for food preservative but incorporating with the starch film is the best alternative and run with the current global technology demanded environmentally friendly packaging product. Therefore, further studies addressing how to apply as a packaging system for food industries should be done.

ACK N OWLED G M ENTS
The authors wish to thank the Collaborative Innovation Task of CAAS (CAAS-XTCX2016005) for financial support. Tigray Agricultural Research Institute, Mekelle university college of Veterinary Medicine.

CO N FLI C T O F I NTE R E S T
The authors declare that we do not have any conflict of interest.

D ECL A R ATI O N FO R H U M A N S U BJ EC T S
This study does not involve any human or animal testing.

E TH I C A L A PPROVA L
The study's protocols and procedures were ethically reviewed and approved by Institute of Food and Science Technology, CAAS.