Fabrication, characterization and antifungal evaluation of polyphenolic extract activated keratin starch coating on infected tomato fruits

In recent times, the application of protein-based bio-composite edible films in postharvest preservation of food and agricultural products is attracting increased attention due to their biodegradability, eco-friendliness and sustainability. In this study, an avocado pear peel polyphenolic extract enriched keratin-starch composite film was fabricated, characterized and evaluated for antimicrobial activity against fungal infected tomato fruits after 6 days of storage at room (25 ± 2 °C) temperature. The SEM/EDX and FTIR results revealed the successful film formation with high degree of compatibility and homogeneity. Following a 6-day post-coating loss in weight of the coated tomato fruits decreased significantly (p < 0.05) with increasing extract concentration while titratable acidity showed a significant (p < 0.05) increase with increasing extract load. Ascorbic acid and lycopene contents were significantly (p < 0.05) higher in the avocado pear peel polyphenolic extract-loaded films. No significant effect was observed in catechol oxidase activity of the tomato extract across the different treatment groups. In addition, fungal growth inhibition showed a dose dependent increase consistent with avocado pear peel polyphenolic load in coated tomato fruits compared to control. Results obtained in this study showed that polyphenolic activated keratin-starch coating was able to reduce spoilage-induce weight loss as well as conserve the overall quality (including titratable acid levels, lycopene and ascorbic acid contents) of fungal-infected tomato fruit and reduce microbial growth. Therefore polyphenolic activated keratin-starch coating could serve as a sustainable and ecofriendly postharvest preservation method to prolong the shelf life of tomato fruits.

The global population is projected to witness an astronomical growth in the next few years. This projection is worrisome especially if not matched with proportionate food production. This is a major challenge especially in developing countries where an estimated 1.2 billion people go to bed hungry 1,2 . One major strategy to ensure food security is to reduce wastage due to food spoilage and deterioration. Thus, research focused on increasing shelf life, as well as ensuring microbial safety of fresh fruits and vegetables through edible coating technology is an effort in the right direction.
Edible coatings are solutions applied to fresh fruits and vegetable in order to maintain their freshness as well as prevent deterioration that may be due to dehydration, loss of appearance, flavor and nutritional qualities of such produce within the time taken for them to reach the consumer. Huge losses in terms of quantity and quality are recorded during postharvest handling of food and agricultural products. Majority of these losses are www.nature.com/scientificreports/ 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 tomatoes 21 .
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 × 10 6 spores/mL in sterile distilled water. The infected tomato fruits were then incubated at www.nature.com/scientificreports/ 20 °C for 24 h to resemble common fungal infections before the coating experiment 22 . 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 sample 25 . Calculated as: 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: 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 nm 26 . Values were calculated as: Polyphenoloxidase assay (Catechol assay). Polyphenol oxidase was determined spectrophotometrically by a change in colour of catechol from a colorless to colored benzoquinone solution 27 . 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 cm 3 ) of phosphate buffer (pH 7) and 0.1% catechol solution was added to 0.1 cm 3 of enzyme extract in a test tube. Sample readings were taken with a spectrophotometer (420 nm) previously zeroed with 4 cm 3 of distilled water and 0.1 cm 3 of enzyme extract. www.nature.com/scientificreports/ 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.  Water solubility, pH and weight loss. The solubility of the fabricated film was observed to decrease with increasing concentration of avocado pear peel polyphenolic extract. However, no significant (p > 0.05) difference was observed in the rate of solubility between films containing the varying concentration of the extract (Fig. 3A). Tomato fruits coated with avocado pear peel polyphenolic extract-enriched keratin-starch film had significantly (p < 0.05) lower pH values compared control. Tomato fruits coated with 0.6, 0.8 and 1.0 mL avocado pear peel polyphenolic extract-enriched film had statistically similar pH value (Fig. 3B). The loss in weight in the different treatment groups is shown in Fig. 3C. Tomato fruits coated with distilled water (control) and those coated with keratin-starch film without extract had significantly (p < 0.05) higher loss in weight compared to those coated with films containing varying concentrations of the extract. Weight loss in tomato fruits coated with extract containing films was observed decrease with increasing concentration of the extract.

Results
Titratable acidity, ascorbic acid and lycopene content. Titratable acid levels in tomato fruits subjected to the different treatment groups is presented in Fig. 4A. Tomato fruits coated with avocado pear peel polyphenolic extract-containing film had significantly (p < 0.05) higher titratable acid level compared to tomato fruits coated with keratin-starch only and distilled water (control). The ascorbic acid concentration in the different treatment groups is shown in Fig. 4B. Tomato fruits coated with distilled water (control) and those coated with keratin-starch film without extract had significantly (p < 0.05) lower ascorbic acid level compared to those coated with films containing varying concentrations of the extract. Ascorbic acid concentration in tomato fruits coated with extract containing films was observed to increase with increasing concentration of avocado pear www.nature.com/scientificreports/ peel polyphenolic extract in the film. No significant statistical difference was observed in lycopene content between infected tomato fruits coated with distilled water (control) and those coated with avocado pear peel polyphenolic extract-based keratin-starch composite film (Fig. 4C).

Catechol oxidase and antifungal activities.
Infected tomato fruits coated with avocado pear peel polyphenolic extract-based keratin-starch composite film exhibited significantly (p < 0.05) lower catechol oxidase activity, when compared with tomato fruits coated with keratin-starch composite alone and those coated with www.nature.com/scientificreports/ distilled water. Furthermore, the enzyme activity, though not significantly changed in tomato fruits coated with keratin-starch composite films containing 0.4, 0.6, 0.8 and 1.0 mL avocado pear peel extract, was significantly lower compared to those fruits coated with 0.2 mL avocado pear peel polyphenolic extract-based composite film (Fig. 5A). Incidence and severity of A. flavus and A. niger infection were significantly reduced in tomato fruits coated with keratin-starch films functionalized with avocado pear peel extract (Table 3) compared to control. Coatings containing 0.8 and 1.0 mL avocado pear peel polyphenolic extract exhibited the most significant protective effect compared to tomato fruits coated with film containing lower concentrations of the extract. Tomato fruits coated with both keratin-starch composite only and those coated with avocado pear peel polyphenolic extract-based composite film had significantly (p < 0.05) lower fungal loads, when compared with control. Keratin-starch composite containing 1.0 mL avocado pear peel extract exhibited the best antifungal activity, when compared with composite containing lower concentration of the extract (Fig. 5B).

Discussion
Climacteric, fruits such as tomato constitute a good system for evaluating metabolic changes involved in the maturation and ripening of fruits. Several factors, including physicochemical changes, such as weight loss, pH, as well as transpiration, pathogen infestation, etc. contribute to postharvest losses during storage 28,29 . Efforts aimed at regulating these factors among other things are strategic in enhancing storage life as well as postharvest losses of agricultural products. The degree of smoothness or homogeneity of the film fabricated using ginger starch, chicken feather keratin and avocado pear peel extract as revealed is indicative of a high rate of dispersion or miscibility of the individual   www.nature.com/scientificreports/ components of the film resulting in no crack or cavity within the film. It also attested to the level of cross linking or intermolecular associations that could have resulted from the amide nitrogen and oxygen with the hydroxyl groups present in both starch and the phenolic rings of the extract. The reduction in film transparency due to the incorporation of avocado pear peel polyphenolic in the film in this study is advantageous in its application as an edible coating. Film opacity could aid in the reduction in light intensity or penetration into the coated tomatoes thus preventing the potential oxidation of molecules such as ascorbic acid and phenolics antioxidant present in the tomato fruits. Oluba et al. 12 recently demonstrated biocomposites film containing keratin exhibited a reduction in film transparency. In this study, weight loss was observed to decrease with increasing concentration of avocado pear polyphenolic extract in the keratin-starch film. Keratin-starch film coating alone had no appreciable effect in preserving the weight loss. It could thus be argued that the film only served as a carrier for the extract. One of the greatest challenges in the transportation and storage of fruits is the unregulated movement of water. The loss or gain of water during storage is undesirable. The shrinkage of fruits due to excessive water loss most often results to loss in economic value of fruits. In addition, excessive loss of water measured as weight loss in this study could affect the juiciness of fruits 30 .
Generally, the titratable acidity of tomato fruits used in the study was slightly below 0.1 μeq g −1 . This is low compared to the value reported by Sree et al. 31 for chitosan coated tomato fruit coated for a period of 30 days. www.nature.com/scientificreports/ This is not unlikely because this study unlike Sree et al. 31 utilized already ripened tomato fruits with low level of metabolic activity. The rate of respiration in ripened tomatoes is low compared to green tomatoes with higher rate of respiration. Low level of titratable acidity as measured in this study signifies low concentration of citric acid being the most abundant organic acid in tomato fruits. It has been established that there is a concomitant loss of citric acid concentration with increasing maturity and ripening 32 . The ripening process in tomato fruit is accompanied by increased metabolic breakdown of citric acid to sugars. In addition, there is a further loss of acid as a consequence of high rate of ethylene production as well as increased respiratory rate during ripening 33 .
The concomitant higher concentration of ascorbic acid recorded in the avocado pear peel polyphenolic extract enriched keratin-starch film coated tomato fruits as opposed to uncoated tomato fruits in this study could be a attributable to reduce metabolic conversion of ascorbic acid to sugars as well as low respiratory rate. This observation further reinforce the potential ability of the keratin-starch-avocado peel polyphenolic extract enriched film to serve as a barrier to oxygen. The discolouration of fruits, which most often results in consumer rejection, due to unacceptable aesthetic appearance is a major challenge in fruit production. Polyphenol oxidase activity as observed in this study was not significantly different between coated and uncoated fruits. This is significant because polyphenol oxidase catalyzes the process of enzymatic browning of fruits which results in fruit discolouration. The tomato fruits used in this study were reddish and fully ripened hence no appreciable browning reaction is expected at that stage. Catechol oxidase activity was observed to be lower in tomato fruits coated with avocado pear peel polyphenolic extract enriched keratin-starch film in comparison to uncoated fruit in this study. In addition, catechol oxidase activity showed positive correlation with ascorbic acid level across the treatment groups. High ascorbic acid concentration of the avocado pear peel polyphenolic extract enriched keratin-starch film coated tomato lowers the pH level in these tomato fruits. Catechol oxidase has been shown to be catalytically active between pH 4-pH 8. Low pH due to high ascorbic acid content could interfere with the binding of the enzyme to its active site copper 34 . Enzymatic browning of fruits not only affects the appearance of fruits but also their nutritional Quality. Over fifty percent of fruit losses due to tropical fruits are attributable to enzymatic browning 35 .
The concurrent reduction in disease incidence and severity due to application of avocado pear peel polyphenolic extract functionalized keratin-starch coating in this study suggests that the antifungal activity of the coating could be fungistatic and not fungicidal as the coating does not completely hinder the growth of the pathogen. This observation is in agreement with the report of Fagundes et al. 22 as well as Palou et al. 36 . Results from this study evidently showed that avocado pear peel polyphenolic extract remarkably enhanced the antimicrobial activity of keratin-starch composite film, when applied as coating on infected tomato fruits. This observation is corroborated by reports from earlier studies 37,38 , demonstrating that the incorporation of plant extract into edible coatings significantly improves their antimicrobial activity. Kubheka et al. 38 demonstrated that the incorporation of Moringa oleofera leaf extract remarkably enhanced the antifungal activity of edible coating against   40 demonstrated the inhibition of gray mold disease in tomato fruit by the application of Vernonia amygdalina leaf extract-based emulsion. The antimicrobial action of plant extract has been attributed to the diversity of phytochemicals or secondary metabolites present in them. The fruits, leaves, seeds, roots and bark of plant are rich sources of secondary metabolites with proven antimicrobial activity. The HPLC fingerprint obtained for avocado pear peel used in this study showed the presence of flavonoids and phenolic compounds. Specifically, quercetin and kampferol were identified as the major phenolics present in avocado pear peel extract. In addition, it is pertinent to know that the lowering effect of the avocado peel enriched keratin-starch film on the pH as well its potential effect on the retention of ascorbic acid content of the infected tomato fruits may have played significant role in its antifungal action as demonstrated in this study. The high level of citric acid vis-à-vis the low pH could constitute and unfavourable environment for fungal growth.  Table 3. Disease severity (mm) and incidence (%) of fungal spoilage on tomato fruits artificially inoculated with A. flavus and A. niger spores and coated with keratin-starch (K-SC) film functionalized with avocado pear peel polyphenolic extract. Control, infected tomato fruits coated with distilled water; K-SC, infected tomato fruits coated with keratin-starch film; while K-SC-AP 0.2 , K-SC-AP 0.4 , K-SC-AP 0.6 , K-SC-AP 0.8 and K-SC-AP 1.0, infected tomato fruits coated with keratin-starch film functionalized with 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL avocado pear peel polyphenolic extract, respectively. Data on the same row carrying different alphabets are statistically significant (p < 0.05).

Conclusion
The results presented in this study demonstrated that the application of an avocado pear peel polyphenolic extract activated keratin-starch coating on fungal-infected tomato fruit remarkably inhibited the incidence of infection as well as the severity of infection and in addition conserved their organoleptic and nutritional qualities. Thus demonstrating the antimicrobial activity of avocado peel polyphenolic extract as well as the potential application of keratin-starch composite film in food packaging and post-harvesting storage of tomato fruits. However, further research on the water vapour and oxygen/carbondioxide permeability as well as sensory evaluation of the coated tomato fruits are required to further justify its application as an edible coating.