Ethics approval
This study does not require any formal consent as it does not include any human participation or animal experimentation. All the experiments were carried out in accordance with relevant institutional, national, and international guidelines/legislation.
Chemicals and reagents
Analytical grade chemicals purchased from Sigma-Aldrich Ltd (UK) were used for the study.
Plant materials
Fresh ginger rhizomes, matured green avocado pear (Persea americana) and fresh tomato fruits of similar size, shape and colour were purchased from the main market in Omu-Aran, Kwara State, Nigeria and transported to the Biochemistry Laboratory, Landmark University, Omu-Ara, Kwara State, Nigeria. Identification and authentication of the plant samples were carried at the Department of Plant Science Herbarium, University of Ilorin, Ilorin, Nigeria where the respective vouchers were deposited.
Microwave assisted polyphenol extraction
Total polyphenol extract was carried out using the modified microwave assisted method of Kumar et al.19 with little modifications. Powdered avocado pear peel (50 g) was put in 500 mL Erlenmayer flask containing 250 mL ethanol (50%). The Erlenmayer flask with its content was then put inside a BP090 microwave oven (Microwave Research & Applications, Inc. Illinois, USA). The flask was securely connected to a vertical condenser. The extraction process was performed at 300 W for 5 min. Thereafter, the liquid extract was separated from the residue through vacuum filtration. The liquid extract was vacuum evaporated at 40 °C to obtain the concentrated polyphenol extract.
HPLC analysis
The characterization of the avocado peel polyphenolic extract was carried out according to Šeruga et al.20 method.
Starch extraction
The process undergone in the extraction of starch from ginger rhizome was as detailed by Oluba et al.9. Thoroughly washed peeled ginger rhizomes soaked in sodium metabisulphite solution (1%) and blended into fine paste with electric blender. The ginger starch paste was dispersed into a large volume of water, stirred vigorously and left to stand for 12 h. The water was then carefully poured off while the starch paste was scraped into petri dish, oven-dried at 30 °C to constant weight, weighed and stored at 4 °C.
Chicken feather
White coloured waste feathers were collected from the slaughter unit of Landmark Commercial Farms, Landmark University, Omu-Aran, Nigeria.
Keratin extraction
The extraction of keratin from chicken feather waste followed the procedure detailed by Oluba et al.9.
Preparation of active keratin-starch coating
The active keratin-starch edible coating was prepared according to the procedure described by Oluba et al.9 with little modifications. A 5% starch solution was prepared and gelatinized by heating on a laboratory hot plate at 70 °C with constant stirring. Keratin solution was made by dissolving 5 g of the extracted dried keratin powder in 100 mL of 0.1 M NaOH and heated on a laboratory hot plate at 70 oC with constant stirring while adding 10 mL of 3% sodium sulphite to prevent the realignment of the di-sulphide bonds. Different blends of starch, keratin and avocado peel polyphenolic extract were prepared by varying the concentration of extract added (Table 1). To each starch-keratin blend, 2 mL of glycerol was added as plasticizer after which the mixture was heated at 100 °C on a laboratory hot plate, with constant stirring. The prepared active coating was divided into two portions. The first portion was poured into a glass petri dish and oven-dried at 75 °C for 72 h, this was used for the characterization analyses. The second portion was used for the coating experiment.
Fungi isolation
One gram of spoilt tomatoes was cut with a sterile scalpel and submerged in 10 mL Sabouraud dextrose broth (SDB) for 72 h. One milliliter (1 mL) of the resulting broth containing fungal growth was plated in sterile Sabouraud dextrose agar (SDA) plates, using the standard pour plating technique. To inhibit bacterial growth, ciprofloxacin antibiotic was added to the sterile SDA (cooled to 40 °C) at concentration of 50 mg/L. Before por plating, the sterile SDA was first cooled to 40 °C. The plates were incubated at 25 °C for 72 h and observed for growth. The resulting distinct colonies were further subcultured in fresh sterile SDA plates and incubated to obtain pure cultures. The pure cultures were tentatively identified using cultural and morphological features such as colony growth pattern, and conidial morphology as A. flavus and A. niger and were then used to infect fresh tomatoes21.
Fruit inoculation and coating experiment
Two hundred and ten (210) freshly purchased matured tomato fruits of similar size and shape, free from lesions and postharvest diseases were washed with 2% hypochlorite solution (v/v) and distilled water and allowed to air dry at room temperature (25 ± 2 °C). The tomatoes were then divided into seven groups of ten tomatoes each (each treatment group was replicated three times). Tomatoes were superficially wounded once in the equator with a sterile scalpel with a probe tip 1 mm wide and 2 mm in length. The wound was then inoculated with 10 μL suspension of a mixture of A. flavus and A. niger containing about 1 × 106 spores/mL in sterile distilled water. The infected tomato fruits were then incubated at 20 °C for 24 h to resemble common fungal infections before the coating experiment22. Following the incubation period, inoculated tomato fruits were randomly assigned to seven treatment groups (Table 1) and coated with their respective coating film by immersion for 30 s. Each treatment had three replica of 10 tomato fruits each. Thereafter, the coated fruits were drained, and left to air-dry at room temperature (25 ± 2 °C) for 12 h before being placed on plastic trays on corrugated cartons to avoid contact and then stored for 6 days at room temperature (25 ± 2 °C) under laboratory conditions.
Characterization of the fabricated polyphenol extract-activated film
Surface and internal morphology as well as elemental composition of the fabricated films were determined using scanning electron microscopy/electron dispersion X-ray (SEM/EDX). Quantitative determination of surface functional groups of the film was carried out using Fourier Transform Infra-red (FTIR) Spectroscopy. The transparency of the film was measured according to Santacruz et al.23 using a UV–VIS spectrophotometer (Jenway 7305, UK) at 560 nm.
Water solubility determination of the fabricated polyphenol extract-activated film
The solubility of the fabricated avocado pear peel polyphenolic extract-based keratin-starch film solubility in water was determined according to Fakhouri et al.24 method with slight modification. A given portion of the film was cut, weighed and dried at 75 °C for 24 h after which it was immersed in a beaker containing 50 mL of water, and stirred continuously for 24 h. The biofilm sample was then removed and dried again at 75 °C for 24 h. The solubility (expressed in %) was calculated as the difference in weight before and after immersion. Determination of solubility of samples in acidic medium was carried out by the same process with a difference in the immersion solution which for acidic medium is 1 M hydrochloric acid.
Physicochemical characteristics assessment of tomato fruits after the coating experiment
Following a six-day post coating period, tomato fruits from each treatment group was homogenized and filtered using Whatman™ number 1. The filtrate was stored in plain sterile bottles and kept at 4 °C until required for further analysis.
pH determination
Ten milliliter (10 mL) filtrate was diluted with 50 mL distilled water and the pH of the resulting solution determined using an electronic pH meter (H12210 pH meter).
Titratable acidity
Five milliliter (5 mL) of filtrate was diluted with 20 mL distilled water. Three drops of phenolphthalein was added as an indicator to the solution. The solution was titrated against 0.05 M sodium hydroxide till a persistent pink colour was formed. Values were expressed as g lactic acid/100 g of sample25. Calculated as:
$$ TA = frac{M,{rm NaOH} times {rm mL;NaOH} times 0.09 times 100}{{mL;of; sample}} $$
Ascorbic acid assay
The 2, 6 dichlorophenol indophenol assay titrimetric method was used for the determination of ascorbic acid. d. The dye solution was standardized by pipetting 5 mL of standard ascorbic acid solution into a 100 mL conical flask and titrated against 2, 6 dichlorophenol indophenol dye solution till a persistent light pink color appeared. The volume of the dye used was recorded as (V_{1}). Thereafter, 5 mL of sample was diluted three-folds in a 100 mL volumetric flask with metaphosphoric acid. The diluted sample solution (10 mL) was pipetted into a conical flask and titrated against the dye till a light pink color appears which persists for 30 s ((V_{2})).
The ascorbic acid content was calculated using the equation:
$$ Ascorbic; acid; content = frac{{0.5;{text{mg}} times V2_{2} times 100;{text{mL}} times 100}}{{V_{1} times 5;{text{mL}} times weight; of;sample}} $$
Lycopene content determination
For lycopene determination, 5 mL sample was diluted with 5 mL of distilled water and agitated in a water bath at 25 °C for 1 h, after which 8.0 mL of solvent (hexane: ethanol: acetone; 2:1:1 v/v) was added. The sample solution was covered and vortexed for some minutes, after which it was incubated in the dark for 25 min. Following incubation, 1.0 mL of distilled water was added to the samples and vortexed. The sample solution was then allowed to stand for 10 min and absorbance reading taken using a spectrophotometer at wavelength of 503 nm26.
Values were calculated as:
$$ Lycopeneleft( {{text{mg}};{text{mL}}^{ – 1} } right) = A _{503 } times 137.4 $$
Polyphenoloxidase assay (Catechol assay)
Polyphenol oxidase was determined spectrophotometrically by a change in colour of catechol from a colorless to colored benzoquinone solution27. Five milliliter (5 mL) sample was diluted with equivalent volume of water and transferred into a test tube and allowed to sediment and the supernatant carefully decanted. Two cubic centimeter (2 cm3) of phosphate buffer (pH 7) and 0.1% catechol solution was added to 0.1 cm3 of enzyme extract in a test tube. Sample readings were taken with a spectrophotometer (420 nm) previously zeroed with 4 cm3 of distilled water and 0.1 cm3 of enzyme extract.
Antifungal activity evaluation
Following storage at room temperature (25 ± 2 °C) for six days, incidence of fungal infection was estimated as the percentage of decayed fruit while disease severity was determined as the diameter of the lesion (mm)22. In addition, aerobic total fungal count for tomato fruits in each group was carried out on potato dextrose agar medium using the standard pour plating technique. Tomato fruits in each treatment replicate were crushed into paste and thoroughly mixed together. For antifungal activity estimation, 1 g of crushed tomato from each of the respective fruits was placed aseptically in test tube containing 10 mL of sterile distilled water and vortexed to homogenize. One millilitre of sample was further removed to carry out series of tenfold serial dilutions in test tubes containing 9 mL of sterile distilled water. Pour plating in SDA plates was carried out using known dilutions of the respective treatments, while ensuring the maintenance of aseptic conditions. The media was mixed thoroughly with the sample and aseptically transferred on to petri dish. The plates were incubated in an inverted position at 25 °C for 48 h. Treatment was performed in five replicates while counts were in triplicates.
Statistical analysis
All experiments were done in triplicate, results are reported as mean ± SD. Data were analysed with the aid of ANOVA using a completely randomized factorial experimental design, with two factors (treatment groups and storage time) while means comparison was carried out using Turkey multiple range test (P < 0.05). GraphPad prism 8.0 software (GraphPad Software Inc., San Diego, California) was utilized in drawing charts.
