Effects of chitosan coatings fused with medicinal plant extracts on postharvest quality and storage stability of purple passion fruit (Passiflora edulis var. Ester)

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Advance access publication 9 March 2022 Article

Received 1 December 2021; Revised 25 February 2022; Editorial decision 3 March 2022

Article

Effects of chitosan coatings fused with medicinal plant extracts on postharvest quality and storage stability of purple passion fruit (Passiflora edulis var. Ester)

Kwanele A. Nxumalo and Olaniyi A. Fawole

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Postharvest Research Laboratory, Department of Botany and Plant Biotechnology, University of Johannesburg, Auckland Park, Johannesburg, South Africa

*Correspondence to: Olaniyi A. Fawole, Postharvest Research Laboratory, Department of Botany and Plant Biotechnology, University of Johannesburg, P.O.

Box 524, Auckland Park, Johannesburg 2006, South Africa. E-mail: [email protected]

Abstract

Chitosan edible coating (Ch; 2%, mass concentration) enriched with 2% of 0.1 mg/L Bidens pilosa (Ch+B), Lippia javanica (Ch+L), Syzygium cordatum (Ch+S), or Ximenia caffra (Ch+X) was applied as a composite edible coating in alleviating shrivel and maintaining the quality of purple passion fruit (Passiflora edulis var. Ester). Treated fruit was dipped for 3 min in the coating solution, and control fruit was dipped in distilled water.

The fruit were stored at (8±2) °C and 90%±5% relative humidity (RH) for 32 d. Sampling was done every 8 d plus 3 d ((20±2) °C and (50%±5%) RH) to simulate retail conditions. Efficacy of medicinal plant extracts in the chitosan matrix varied; lower ethylene production (82.42 µL C₂H₄/(kg·h)) was seen in fruit coated with Ch+S, and the lowest respiration rate (75 mL CO₂/(kg·h)) was observed in fruit coated with Ch+B. The control fruit showed the highest ethylene production (84.90 µL C₂H₄/(kg·h)) and respiration rate (117.98 mL CO₂/(kg·h)). Fruit coated with Ch+B had the lowest weight loss (41.67%), higher juice content (60.13%) and BrimA (3.31); while the control fruit had the highest weight loss (88.03%), lowest juice content (21.90%), and BrimA (2.49). Shrivel incidence was lowest (23.70%) on fruit coated with Ch+L and highest (83.30%) on the control fruit.

Fruit coated with Ch+X had the lowest electrolyte leakage (71.40%), while the control fruit had the highest (91.97%). Fruit coated with chitosan alone performed better than the control fruit but did not exceed the quality of composite chitosan-coated fruit. Based on the principal component analysis, it can be concluded that passion fruit coated with Ch+B was more effective in alleviating shrivel incidence, better maintained the quality of passion fruit during storage, and shows potential for commercial applications.

Keywords: Quality attributes, anthocyanins, perishability, respiration, granadilla.

Introduction

Passion fruit, known as granadilla, is a delicious purple (Passiflora edulis (P. edulis) Sims. f.) or yellow (P. edulis f.

flavicarpa) fruit (Joy, 2010). Due to its known medicinal properties, high nutritional value, and exotic taste, the de- mand for fresh and processed passion fruit products has significantly increased over the past few years (Janzantti et al., 2014; Charan et al., 2017). In South Africa, the commercially grown passion fruit (P. edulis var. Ester) is a cross between the yellow and purple passion fruit varieties (ARC-ITSC, 2021).

South Africa is one of the top five exporters of passion fruit to the European Union market, whereby 60% is sold fresh, and 40% is processed (ARC-ITSC, 2021).

Due to its high perishability and climacteric nature, the commercialization of passion fruit as a fresh product is limited (ARC-ITSC, 2021). Fruit quality loss and the subse- quent decline in the marketability of fresh passion fruit is due to moisture loss, resulting in the reduction of fruit volume, cell wall breakdown, a shriveled appearance and eventual senescence (Janzantti et al., 2014; Charan et al., 2017).

Shriveling is a major physiological disorder in locally sold and exported passion fruit, rendering the fruit unsaleable due to

their undesirable appearance (ARC-ITSC, 2021). Preservation methods are needed to enhance the shelf life of passion fruit.

Edible coatings (ECs) have been used as a positive measure to improve fruit quality, reducing the metabolism rate of horticultural crops by creating a modified atmosphere around them to maintain their freshness (Fawole et al., 2020a; Riva et al., 2020). ECs create a semi-permeable film around harvested fruits reducing moisture loss, respiration, gaseous exchange (O₂ and CO₂), and retarding ethylene production and other undesirable changes as the fruit matures or ripens (Ncama et al., 2018; Riva et al., 2020).

ECs such as chitosan are biodegradable, consumed with the coated product, and generally recognized as safe by the U.S.

Food and Drug Administration (Zhang et al., 2014; Ncama et al., 2018; Riva et al., 2020). Besides its biodegradability, other unique, albeit reportedly minor characteristics of chitosan edible coating in food preservation include its biocompatibility, antioxidant, and antimicrobial activities (Dutta et al., 2009;

Fernandez-Saiz et al., 2009).

Many studies have reported the use of plant extracts as alternatives to synthetic antioxidant and antimicrobial agents (Siripatrawan and Vitchayakitti, 2016; Nxumalo et

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al., 2021). In trying to improve the functional properties of chitosan, especially the antioxidant and antimicrobial activity, plant phenolic compounds have been introduced into the chitosan polymer matrix to formulate composite edible coatings (Siripatrawan and Vitchayakitti, 2016). For example, the enhancement of antioxidant and antimicrobial activities and the improvement of physical and mechanical properties of chitosan films have been achieved by incorporating polyphenols derived from green tea extract (Gramzaa et al., 2006;

Siripatrawan and Harte, 2010) and cinnamon essential oil (Jiarpinijnun et al., 2013). In another study by Kubheka et al.

(2020), moringa leaf extract in a gum arabic matrix success- fully maintained the physicochemical properties of ‘Maluma’

avocado fruit. Aloe vera (alone or with thymol) was also effective in reducing decay in nectarines caused by Botrytis cinerea and Penicillium digitatum by 50% and 70%, respectively (Navarro et al., 2011). Dipping banana fruits in zimmu leaf extract at 25% concentration exhibited 100% inhibition of crown-rot disease in cold storage (14 °C) and increased the shelf life to 64 d (Sangeetha et al., 2013). These studies demonstrated that ECs fused with polyphenols from botan- icals could maintain the quality and prolong the shelf life of horticultural crops (Siripatrawan and Vitchayakitti, 2016;

Nxumalo et al., 2021). These useful characteristics of plant extracts in an edible coating matrix could eliminate the use of synthetic preservatives while also being beneficial to both human health and the environment at large (Ncama et al., 2018; Nxumalo et al., 2021).

Southern African countries host a wide range of flora that contains biological activities and is mainly used in traditional medicine (Van Wyk et al., 2013; Van Wyk, 2017). After a com- prehensive literature review on medicinal plants used for food preservation, the following medicinal plants were selected and studied from the rich flora of Eswatini: water berry leaves (Syzygium cordatum (S. cordatum)), large sour plum leaves (Ximenia caffra (X. caffra)), black-jack leaves (Bidens pilosa (B. pilosa)), and fever-tea leaves (Lippia javanica (L.

javanica)). Syzygium cordatum Hochst. ex Krauss is a valuable herbal plant found in eastern and southern African countries, and it is included in the monographic guide of the most valuable herbal medicines in southern Africa (Van Wyk et al., 2013; Van Wyk, 2017). In Eswatini, S. cordatum is a multi-purpose plant species that is important for local liveli- hoods as herbal medicine and food source as its fruit is edible.

Its leaves and bark are used in food preservation, while the whole plant has aesthetic value among other uses (Dlamini and Geldenhuys, 2009). Its stem, bark, and leaves have many useful pharmacological activities, and they are mixed with the bark of Breonadia salicina or used alone in indigenous food preservation (Dlamini and Geldenhuys, 2009; Sibandze et al., 2010). X. caffra, also known as large sour-plum, is a member of a genus of flowering plants in the Olacaceae family, which grows mainly in the southern African region (Cheikhyoussef et al., 2010; Van Wyk, 2017). As a traditional medicine, herb- alists in southern Africa have been using the leaves and roots of X. caffra to treat wounds, infections, fever, infertility, and diarroea (Mulaudzi et al., 2011; Nair et al., 2018). Recent laboratory research has confirmed that the leaf and root extracts of X. caffra have anti-gonococcal, antibacterial, and antifungal activities, which corroborate with its traditional use as food additives and in food preservation (Fabry et al., 1998; Mulaudzi et al., 2011). Bidens pilosa L. is a popular annual weed species belonging to the daisy family Asteraceae.

Previous phytochemical studies on B. pilosa recorded the oc- currence of flavonoids, polysaccharides, carotenoids, amines, lactones, mineral elements, coumarins, and volatile oil, thus proving its antioxidant potential (Chiang et al., 2004; Silva et al., 2011; Tomczykowa et al., 2011). Although the plant is an invader and generally regarded as a nuisance weed, its essential oils and dried leaves have been reported to be effective in controlling stored grain insect pests and used to preserve and flavor food (Ngamo et al., 2007; Goudoum et al., 2016).

Its antioxidant potential can prevent the formation of free radicals and oxidation of fatty acids within food (Silva et al., 2011; Goudoum et al., 2016). Lippia javanica (Burm.f.), com- monly known as fever-tea, is an invasive plant that occurs naturally in many southern African countries like Eswatini, South Africa, Zambia, Malawi, and Zimbabwe. Due to its high antioxidant capacity, this plant has been traditionally used as a herbal tea, food additive and preservative among the different ethnic groups of the southern Africa region (Shikanga et al., 2010; Van Wyk et al., 2013; Maroyi, 2017).

Although the ability of chitosan infused with agricultural waste and some plant extracts to maintain the postharvest physiology of horticultural crops has been reported in the literature, knowledgeable use of medicinal plants in postharvest preservation of passion fruit is limited. This study explored the efficacy of chitosan enriched with different medicinal plant extracts in alleviating shrivel and maintaining the quality of purple passion fruit during cold storage and shelf- life conditions.

Materials and Methods

Procurement and handling of medicinal plants Plant materials (Supplementary Figure 1) were identified with the help of the Eswatini Institute for Research in Traditional Medicine, Medicinal and Indigenous Food Plants (EIRMIP) Agronomist and Research Fellow, Mr. E. Kunene. Matured and disease-free plant leaves were collected from the EIRMIP farm at Mafutseni and deported as KN1001 for B. pilosa (black-jack), KN1002 for L. javanica (fever-tea), KN1003 for S. cordatum (water berry) and KN1004 for X. caffra (large sour-plum). Mafutseni is in the Manzini region of Eswatini, lowveld agro-ecological zone at 26°24ʹ21.9ʹʹ S, 31°35ʹ05.3ʹʹ E. The reported indigenous uses informed the investigation of the leaves of the investigated medicinal plants. The plant part selection is also in line with the sustainable use of medicinal plants. The leaves were transported to the University of Eswatini, Kwaluseni Campus, EIRMIP Laboratory for oven dehydration (Labotec (Pty) Ltd, Bavaria, South Africa) at 50 °C for 72 h and ground into powder using a blender (Kambrook, Ningbo, China).

Preparation of plant extract and edible coating The extraction technique outlined by Ramesh et al. (2014) and Hassan et al. (2019) was adopted, with slight modifications. Briefly, 20 g of finely powdered plant leaves were mixed with 100 mL 70% ethanol in a 250-mL beaker for 30 min.

The mixture was then sonicated at 20 °C high-frequency for 1 h using a sonicator (Labotec (Pty) Ltd, Bavaria, South Africa) before filtering the extracts in a separate conical flask using Whatman No.1 filter paper (Whatman, Maidstone, UK) followed by concentrating the extracts using a rotary evapor- ator (Rotavapor R-200, Buchi Laboratory Equipment, Flawil,

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Switzerland) at 45 °C. The obtained concentrates were dried under airstreams in sterile vials and stored at 4 °C for further use.

Chitosan, a polysaccharide-based edible coating (Sigma- Aldrich, St. Louis, MO, USA), was used. The chitosan-based coating was prepared according to the procedure outlined by Siripatrawan and Vitchayakitti (2016) with slight modifications. The following formulations were prepared in distilled water in the specific order and composition under magnetic stirring at 40 °C for 1 h: chitosan (2%, mass concentration), acetic acid (1%, in volume), glycerol (1%, in volume), canola oil (1%, in volume), and Tween-20 (1%, in volume).

Thereafter, reconstituted medicinal plant extracts (2% of 0.1 mg/L) were added and stirred for another 2 min. The resultant solution was then homogenized at 3000 r/min for 10 min and sonicated at 40 °C for 60 min to remove air bub- bles. Finally, composite edible coatings, 2% chitosan (Ch) with 2% S. cordatum (Ch+S), Ch+2% X. caffra (Ch+X), Ch+2% B. pilosa (Ch+B) and Ch+2% L. javanica (Ch+L) were obtained. Chitosan (2%, mass concentration) without plant extract was also formulated.

Procurement and handling of fruit

Matured purple passion fruit (P. edulis var. Ester) were harvested from Ganico Organic Farm in Johannesburg (26°02ʹ40.2ʹʹ S, 27°53ʹ05.8ʹʹ E), South Africa, during the growing seasons of 2020 and 2021. The fruit were transported to the Postharvest Laboratory in a well-ventilated vehicle. Upon arrival, fruit were sorted for blemishes, visible external damage, and uniformity of color and size. The fruit were then disinfected by immersion in 0.04% sodium hypo- chlorite for 3 min and air-dried using a fan at (20±2) °C and 50%±5% relative humidity (RH) for 30 min.

Experimental layout and coating application

A completely randomized design was used. Passion fruit were divided into six groups, representing each composite coating and the control group (distilled water). Five commercial packaging punnets (transparent polyethylene ter- ephthalate; 15 cm×10 cm) were used as replicates of each treatment per interval, and each punnet contained five ran- domly selected fruits. Coating application was carried out by fruit immersion in respective composite coatings for 3 min.

Fruit immersed in distilled water were used as the control group. Treated and control fruit were dried at (20±2) °C and 50%±5% RH for 1 h, packed and stored at (8±2) °C and 90%±5% RH for 32 d. Sampling was done at 8-d intervals. At each sampling point, a batch of fruit were placed at (20±2) °C and 50%±5% RH for a further 3-d period to simulate retail conditions.

Physiological properties Weight loss

Weight loss of passion fruit was determined by monitoring weight change in fruit at different intervals using an electronic scale (Mettler Toledo, Model ML3002E, Zurich, Switzerland;

0.0001 g accuracy). The results were expressed as the percentage weight loss of the initial weight (0 d). Ten fruits per treatment were evaluated, and results were calculated using Equation 1 (Fawole et al., 2020b):

W=Wi−Wf

Wi ×100%, (1)

where W=weight loss (%) of fruit; W_i=initial weight (g) of the fruit at the beginning of storage and W_f=final weight (g) of the fruit at the time of sampling.

Respiration rate and ethylene production

Fruit respiration rate was measured using the closed system method previously described by Fawole et al. (2020a). Briefly, in triplicates, five passion fruit were placed in 600-mL con- tainers hermetically sealed with a lid containing a rubber septum in the middle for 2 h at room temperature. After the incubation time, CO₂ production inside the container was measured using an infrared O₂/CO₂ gas analyzer (Checkmate 3, PBI Dansensor, Ringsted, Denmark). The ethylene produced was measured using an SCS56 ethylene analyzer (Storage Control Systems Ltd., Kent , UK). Results were expressed as mL CO₂/(kg‧h) for respiration and µL C₂H₄/(kg‧h) for ethylene.

Fruit shrivel incidence

Fruit shrivel incidence was visually inspected and calculated using Equation 2:

Shrivel incidence=number of af fected fruit

total number of fruit ×100%(2)

Physicochemical attributes Rind color

The fruit color attributes were measured in the CIE L* (light- ness), a* (redness), b* (yellowness) coordinates using a calibrated Minolta Chroma Meter (Model CR-400, Minolta Corp, Osaka, Japan). In five replicates, rind color was measured on op- posite sides of the equatorial region of each fruit at three marked spots. Color intensity (C*) and hue angle (h°) were calculated using Equations 3 and 4, respectively (Arendse et al., 2014):

C∗ =»

(a^∗² +b^∗²), (3)

h^◦ =tan⁻¹ Åb∗

a∗ ã

. (4)

Furthermore, total color difference (TCD) was calculated using Equation 5:

TCD=

»(L0∗+L^∗)² + (a0∗+a^∗)² + (b0∗+b^∗)² (5)

where L₀*, a₀*, and b₀* represent the color parameters of the rind at harvest.

Fruit diameter

Fruit diameter was measured on the equatorial plane of fruit using a digital Vernier caliper (Mitutoyo, Kawasaki, Japan; ± 0.01 mm).

Fruit firmness

Fruit firmness was determined using a firmness analyzer fitted with a 5-mm cylindrical probe (GÜSS-FTA, Cape Town, South Africa) and 8.9 mm penetration at a speed of 10 mm/s (Fawole et al., 2020a).

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Juice content

Juice was extracted using a juice extractor (Kambrook, Ningbo, China) and filtered using a strainer. The juice percentage was calculated using Equation 6:

Juice content(%) =juice contenti−juice content_f

juice content_i ×100%, (6) where juice content_i=initial juice content at the beginning of storage and juice content_f=final juice content at the time of sampling.

Relative electrolyte leakage (EL)

The rate of EL was determined as described by Mirdehghan et al. (2007) and Chen et al. (2008) with slight modifications. In triplicates, six disks of peel tissue were cut out using a stainless steel 10-mm cork borer. Initial conductivity (L_t) was measured after 4 h of incubation in 25 mL of 0.4 mol/L mannitol under constant shaking using a conductivity meter (Hanna Instruments, Singapore, Singapore). Afterwards, the vials were autoclaved at 121 °C for 20 min and held for 24 h at room temperature. Final conductivity (L₀) was measured for total electrolytes. The EL was computed according to Equation 7:

EL= Lt

L0×100% (7) Total soluble solids (TSS), titratable acidity (TA), TSS/TA, and BrimA index

TSS (%) was determined in triplicates using a digital refract- ometer (Palette, ATAGO PR-32, Bellevue, WA, USA) initially calibrated with distilled water. TA (%) was determined using an automated titrator. Briefly, pooled juice samples of five fruits per replicate (triplicate per treatment) were measured by diluting 2 mL of fresh juice with 90 mL of distilled water and titrated with 0.1 mol/L NaOH to an endpoint of pH 8.2 using an Orion Star T940 titrator (Thermo Fisher Scientific, Waltham, MA, USA). BrimA, a criterion for consumer accept- ance of fruit juice, was expressed as outlined by Fawole et al.

(2012) in Equation 8:

BrimA index=TSS−k×TA, (8) where k is the tongue’s sensitivity index, which was calculated to be 3 (k=3 for passion fruit). All measurements were made on 5 individual fruit juice samples for each treatment.

Phytochemical analysis Total phenolic content (TPC)

TPC of passion fruit juice (PFJ) was determined using the Folin–

Ciocalteu (Sigma-Aldrich, St. Louis, MO, USA) reagent method according to Siripatrawan and Harte (2010), using gallic acid as a standard with slight modifications. About 1 mL of PFJ was extracted with 9 mL of 50% aqueous methanol, and the resulting mixture was vortexed and sonicated in ice for 20 min in an ice bath. PFJ (50 µL) was mixed with 450 µL of 50%

methanol followed by 500 µL Folin–Ciocalteu and sodium car- bonate (2%) after 2 min. The mixture was vortexed and incubated for 40 min in a dark room. Absorbance measured was read at 725 nm using a UV–visible spectrophotometer (United Scientific, SP-UV 300, Johannesburg, South Africa). Gallic acid solution (Sigma-Aldrich, St. Louis, MO, USA) was used

to prepare a calibration curve. The concentration of TPC was expressed as gallic acid equivalents (GAE) in mg per mL of the sample. The results were expressed as milligrams of GAE per 100 mL of crude PFJ (mg GAE/100 mL PFJ).

Total flavonoid content (TFC)

The determination of TFC was measured using the colori- metric assay outlined by Fawole et al. (2020a), with a slight modification. In a test tube, distilled water (4 mL) was added to 1 mL of PFJ extract followed by 0.3 mL of 5% sodium nitrite solution and 0.3 mL of 10% aluminum chloride solution. The mixture was incubated at ambient temperature for 5 min before adding 2 mL of 1 mol/L sodium hydroxide.

Absorbance was measured using a UV-visible spectrophotometer at 510 nm, and results were expressed as catechin equivalents (CAE) mg/100 mL PFJ).

Ascorbic acid content (AAC)

AAC was determined using a method described by Fawole et al. (2020a) with slight modifications. Briefly, 0.5 mL of PFJ was extracted with 14.5 mL of 1% metaphosphoric acid. The mixture was then vortexed and sonicated (Labotec, Bavaria, South Africa) for 3 min on ice, followed by centrifugation at 4 °C for 10 min at 5000 r/min. The extracts (1 mL) were mixed with 9 mL of 2,6-dichlorophenolindophenol (dye) and incubated for 30 min in the dark. Absorbance was measured at 515 nm using a UV–vis spectrophotometer, and AAC was calculated based on the calibration curve of standard l-ascorbic acid content. Results were expressed in milligrams ascorbic acid equivalent (AAE) per 100 mL of crude PFJ (mg AAE/100 mL PFJ).

Antioxidant capacity analysis Radical-scavenging antioxidant activity

Radical scavenging activity of total antioxidant was determined using the 2,2-diphenyl-1-picryl-hidrazil (DPPH) method based on quantification of free radical scavenging activity of the PFJ as described by Fawole et al. (2020a) with slight modifications. Briefly, a methanolic extract of PFJ (15 µL) was diluted with 735 µL methanolic DPPH solution (0.1 mmol/L). The mixture was vortexed for 1 min incubated for 30 min in the dark before measuring the absorbance at 517 nm using a UV–vis spectrophotometer. The free-radical capacity of PFJ was expressed as ascorbic acid (mmol/L) equivalent per mL PFJ (mmol/L AAE/ mL PFJ).

Ferric reducing antioxidant power (FRAP)

The FRAP antioxidant power of PFJ was measured according to Fawole et al. (2012) with slight modifications. A FRAP working solution of 300 mol/L acetate buffer (50 mL), 2,4,6-tripyridyl-s-triazine (5 mL) and 20 mol/L FeCl₃ (5 mL) was freshly prepared prior to the measurements. Diluted aqueous methanolic PFJ extracts (150 µL) were mixed with 2850 µL of the FRAP working solution before incubation in the dark for 30 min. Thereafter, the absorbance was measured at 593 nm using a UV–vis spectrophotometer. Results were expressed as Trolox (mmol/L) equivalents (TE) per mL PFJ (mmol/L TE/mL PFJ).

ABTS⁺ radical scavenging activity (RSA)

The ABTS⁺ (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) RSA was analyzed as described by Tagliazucchia et

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al. (2010) and Fawole and Opara (2016) with slight modifications. The ABTS⁺ working solution containing a mixture of 7.4 mmol/L ABTS⁺ and 2.6 mmol/L potassium persulfate was prepared and allowed to stand for 12 h at room temperature in the darkroom to create a stable, dark blue–green radical solution. The working solution was then diluted with methanol to an absorbance of (1.1±0.02) at 734 nm to form the test reagent. Diluted test samples (75 μL) were mixed with 1425 μL of the prepared test reagent and vortexed for 30 s before being incubated for 10 min at room temperature in the dark. Absorbance was measured using a UV–vis spectrophotometer at 734 nm.

Enzyme activity assays Polyphenol oxidase (PPO) activity

PPO activity was determined at 25 °C by measuring the initial rate of increase at 398 nm as outlined by Gonzalez et al.

(1999) and Meighani et al. (2014) with slight modifications.

Crude enzyme extraction was obtained by mixing 2 mL of PFJ with 10 mL of phosphate buffer (pH 7) containing poly- vinyl pyrrolidone (PVPP) and ethytlene diamine tetraacetic acid (EDTA) and vortexed for 30 s. The sample was then sonicated in ice at 0 °C for 10 min and then centrifuged at 10 000 r/min for 15 min at 4 °C to obtain the supernatant.

PPO activity was determined by adding 200 µL of enzyme extract to 2.5 mL of phosphate buffer with 30 µL (60 mmol/L) catechol dissolved in the buffer (pH 7) at 25 °C. Absorbance was repeatedly measured at 398 nm using a UV–vis spectrophotometer every 1 min for 3 min against blank. An increase in absorbance of 0.01 per minute represented 1 enzyme unit (Equation 9):

PPO activity(U/(mL·min)fresh weight(FW)PFJ)

=(Abs_f−Absi) × total reaction vol.

time interval × volume of enzyme (9) where Abs_f=final absorbance; Abs_i=initial absorbance; total reaction vol.=3; time interval=3; volume of enzyme=0.2 mL.

Peroxidase (POD) activity

POD activity was determined at 25 °C by measuring the initial rate of increase at 470 nm as outlined by Gonzalez et al.

(1999) and Meighani et al. (2014) with slight modifications.

Crude enzyme extraction was obtained by mixing 2 mL of PFJ with 10 mL of phosphate buffer (pH 7) containing PVPP and ETDA and vortexed for 30 s. The sample was then sonicated in ice at 0 °C for 10 min followed by centrifugation at 10 000 r/min for 15 min at 4 °C to obtain the supernatant.

POD activity was determined by adding 200 µL of enzyme extract to 2.2 mL of 0.3% guaiacol in phosphate buffer at 30 °C for 5 min and then adding 0.6 mL 0.3% hydrogen peroxide at 30 °C. Absorbance was repeatedly measured at 470 nm using a UV–vis spectrophotometer every 1 min for 3 min against blank. An increase in absorbance of 0.01 per minute represented 1 enzyme unit was calculated as shown in Equation 10:

POD activity(U/(mL^.min)FW PFJ)

= (Abs_f−Absi)×total reaction vol.

time interval×volume of enzyme (10) where Abs_f=final absorbance; Abs_i=initial absorbance; total reaction vol.=3; time interval=3; volume of enzyme=0.2 mL

Statistical analysis

Statistical analysis was carried out using GenStat Statistical Software (GenStat, 18.2 edition, VSN International, Hemel Hempstead, UK). Data were subjected to factorial analysis of variance (ANOVA) at a 95% confidence interval. The observed difference at p<0.05 was considered statistically significant according to Duncan’s multiple range test. Mean (±standard error) values of all the studied variables were presented. Principal component analysis (PCA) was carried out using XLSTAT software (version 2012.04.1; Addinsoft, Paris, France).

Results and Discussion

Physiological response Weight loss and shrivel incidence

Quality parameters like firmness, shrivel incidence, and weight loss are important quality parameters that determine the price of passion fruit and other horticultural crops. Therefore, it is essential to ensure the minimal weight loss of fresh produce during storage or marketing. The weight loss and shrivel incidence of passion fruit were affected by the combination of storage period (p<0.0001) and the treatments applied (p<0.0001; Figure 1). After 24 d of storage, fruit coated with Ch+B had the lowest weight loss (28.5%), but it was not significantly different from the other treatments (p>0.05). It was also observed that control fruit had a weight loss percentage of 64.27% during the same storage period. According to Abbott (1999), fruits with weight loss above 50% are ren- dered unmarketable. Passion fruit coated with chitosan alone showed a moderate control of weight loss percentage at the end of the storage period (54%; Figure 1A). The chitosan coating acted as a barrier to desiccation and maintained the fresh weight of passion fruit better than the control. According to Riva et al. (2020) and Duan et al. (2011), chitosan coatings serve as a semi-permeable barrier against O₂, CO₂, and moisture, thus reducing respiration, water loss, and reactive oxygen species (ROS). From our results, the application of chitosan alone on passion fruit controlled weight loss better than the control fruit. Across all the treatments, the fruit con- tinued to lose weight as the storage period increased. About 1.4- and 1.5-fold increase in weight loss was observed at 24 d and 32 d of storage, respectively. At the end of the storage period, control fruit had the highest weight loss (88.03%), followed by chitosan alone (54.00%), Ch+L (45.57%), Ch+X (42.73%), Ch+S (42.67%), and Ch+B (41.67%; Figure 1A).

This indicates that all the fruit suffered excessive water loss and started to shrivel. The high weight loss of uncoated fruit could be linked to an excessive ripening rate, high ROS, and eventual senescence (Riva et al., 2020). The increase in weight loss with prolonged storage duration could be related to the continuous transpiration and direct evaporation processes through the epidermal cells of the fruit (Kumar et al., 2017;

Kritzinger et al., 2018). However, given the lower weight loss observed in fruit coated with chitosan infused with the investigated plant extracts, it could be hypothesized that the plant constituents could have improved the moisture barrier properties of the chitosan coating, thus reducing the rate of moisture loss. The high weight loss of uncoated fruit could be caused by removing natural wax on the fruit surface, especially during washing (Magwaza et al., 2013), which leads to more opened

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lenticels than the coated fruit, thus increasing the respiration rate. This agrees with our findings that the weight loss percentage of passion fruit resulted from the effectiveness of the applied chitosan coating alone and chitosan enriched with the different medicinal plants compared to control fruit. Similar results were reported by Medeiros et al. (2021), who observed that edible coatings of Chlorella sp. containing pomegranate seed oil applied on Spondias tuberosa (S. tuberosa) showed the lowest weight loss (8.97%–10.42%) compared to the control (11.56%). Megha et al. (2021) reported that pear fruit coated with chitosan (2%)+pomegranate peel extract (PPE) and chitosan (1%)+PPE coatings were effective in reducing the weight loss of pear in comparison with the pure coatings of chitosan (1%), chitosan (2%), or PPE treatment. According to Liu et al. (2011), loss of membrane in fruits is related to membrane permeability, increasing the fruit metabolic activities, which results in higher weight loss. Therefore, reducing the weight loss of fruits like passion fruit is important to improve its shelf life and ensure good economic returns.

Shrivel incidence of passion fruit occurs because of dehydration linked to fruit weight loss and high respiration rate. After 8 d of storage, shriveling was noticed in coated and uncoated fruits, with the chitosan coating fused with

medicinal plant extracts having a shrivel incidence of 2%

(Figure 1B). In contrast, chitosan coating alone had 5% and the control fruit had a shrivel incidence of 10%. A rapid increase in shrivel incidence was noticed in control fruit at 16 d and 24 d of storage, whereby 17.5% and 46.7%

shrivel incidences were observed, respectively. At the end of 32 d of storage, control fruit had higher fruit shrivel incidence (83.3%) in comparison to fruit coated with Ch+L (23.7%), Ch+S (24.4%), Ch+B (24.9%), and Ch+X (25%;

Figure 1B). It was noted that chitosan coating alone was ineffective in controlling the shrivel incidence (45%) of passion fruit after 32 d of storage, as the fruit were deemed unmarketable. The increase in shrivel incidence of untreated passion fruit could be linked to increased respiration rate and weight loss. According to Kritzinger et al. (2018), fruit shriveling is mainly connected to moisture loss, resulting from turgor loss of underlying epidermal cells. Therefore, the application of chitosan coating alone and medicinal plant extract in the chitosan matrix can be linked to improved moisture holding capacity of passion fruit by reducing the loss of turgor in the underlying epidermal cells of the fruit. This also suggests that incorporating the medicinal plant extracts improved the chitosan structure by Figure 1. Changes in (A) weight loss and (B) shrivel incidence of passion fruit during storage for 32 d at (8±2) °C and additional 3 d at (20±5) °C. Each bar represents the mean, and error bars denote the standard error (SE) of the mean. Bars followed by different letters are significantly different at p<0.05 according to Duncan’s multiple range test. Ch+B, chitosan+Bidens pilosa; Ch+L, chitosan+Lippia javanica; Ch+S, chitosan+Syzygium cordatum;

Ch+X, chitosan+Ximenia caffra.

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improving the moisture barrier properties of the fruit, thus explaining the low shrivel incidence of the medicinal plant extracts in the chitosan matrix.

Respiration rate and ethylene production

Results indicated that there was a significant interaction (p<0.0001) between the treatment and storage period for both the respiration rate and ethylene production of passion fruit (Table 1). The respiration rate and ethylene production at harvest were 40.94 mL CO₂/(kg·h) and 21.24 µL C₂H₄/(kg·h), respectively. A spontaneous increase in respiration rate and ethylene production was observed in the first 16 d of passion fruit storage; however, it began to decline, especially in the control fruit. At 16 d of storage, passion fruit coated with chitosan coating alone had a lower respiration rate (91.47 mL CO₂/(kg·h)) than the control fruit (117.98 mL CO₂/(kg·h)). This could be due to the ability of chitosan to induce atmospheric conditions (high CO₂ and low O₂) and significantly reduce transpiration rate better than uncoated fruit at this given time, thus resulting in a low respiration rate. After 24 d of storage, treated passion fruit were characterized by a 1- to 1.2-fold increase in respiration rate; however, the control fruit showed a significant decline in the respiration rate (101.00 mL CO₂/(kg‧h)) following a peak of 117.98 mL CO₂/(kg‧h)at 16 d of storage. After 32 d of storage, fruit treated with Ch+X had the highest respiration rate (68.90 mL CO₂/(kg‧h)) after attaining a peak of 85.90 mL CO₂/(kg‧h), followed by Ch+B (65.10 mL CO₂/(kg‧h)) after attaining a peak of 88.90 mL CO₂/(kg‧h), Ch+S (62.00 mL CO₂/(kg‧h)) after attaining a peak of 83.10 mL CO₂/(kg‧h), Ch+L (60.10 mL CO₂/(kg‧h)) after attaining a peak of 89.4 mL CO₂/(kg‧h), and chitosan coating alone (53.60 mL CO₂/(kg‧h)) after attaining a peak of

91.47 mL CO₂/(kg‧h). In contrast, the control fruit had the lowest respiration rate (44.30 mL CO₂/(kg‧h)) after attaining a peak of 127.98 mL CO₂/(kg‧h) (Table 1). The decrease in respiration rate of the passion fruit at 32 d of storage may be due to the observed peak in ripening rate and eventual senescence of the fruit.

Passion fruit is a climacteric fruit; therefore, a spontaneous increase in physiological response in both coated and uncoated fruit will prompt sudden ethylene accumulation that is known to be accompanied by multi-faceted biochem- ical changes resulting in fruit ripening. The control group produced the highest ethylene (84.90 µL C₂H₄/(kg‧h)) after 16 d of storage (Table 1). It was observed that the ethylene production in both the control and the treated fruit started to decline after 24 d with no significant difference (p>0.05).

At the end of the storage period, fruit coated with Ch+X had the highest ethylene production (59.20 µL C₂H₄/(kg‧h)) after attaining a peak of 83.85 µL C₂H₄/(kg‧h), followed by Ch+S (49.50 µL C₂H₄/(kg‧h)) after attaining a peak of 84.42 µL C₂H₄/(kg‧h), Ch+B (42.50 µL C₂H₄/(kg‧h)) after attaining a peak of 84.10 µL C₂H₄/(kg‧h), Ch+L (42.4 µL C₂H₄/(kg‧h)) after attaining a peak of 83.34 µL C₂H₄/(kg‧h) chitosan only (40.90 µL C₂H₄/(kg‧h)) after attaining a peak of (83.61 µL C₂H₄/(kg‧h)), and control fruit had the lowest ethylene production (27.30 µL C₂H₄/(kg‧h)) after attaining a peak of 84.9 µL C₂H₄/(kg‧h) (Table 1). This indicates that the control fruit reached its ripening and senescence stage faster than the coated fruit, explaining the high ethylene production peak at 16 d of storage and low ethylene production at the end of the storage period. No significant difference (p>0.05) was observed between fruit coated with only chitosan and fruit coated with chitosan enriched with B. pilosa (Ch+B) and L.

javanica (Ch+L) extracts from the beginning to the end of the Table 1. Respiration and ethylene rate of passion fruit treated with chitosan coating fused with medicinal plant extracts during storage for 32 d at (8±2) °C and additional 3 d at (20±5) °C

Parameter Treatment Harvest Storage period (d) Significance level

8 16 24 32 Treatment

(A)

Storage period (B)

A×B Respiration

(mL CO₂/(kg·h)) 40.94±0.45

Ch+B 58.25±0.48^d 75.00±0.09^c 88.9±0.45^b 65.1±3.63^cd <0.0001 <0.0001 <0.0001

Ch+L 53.30±1.54^d 86.00±0.90^b 89.4±0.65^b 60.1±1.59^cd

Ch+S 54.17±1.19^d 77.00±4.58^c 83.1±1.92^bc 62.0±4.53^cd

Ch+X 54.14±1.20^d 78.87±3.81^c 85.9±0.78^b 68.9±2.45^c

Chitosan 55.71±0.56^d 91.47±2.75^b 88.8±0.41âb 53.6±0.16^d Control 66.89±4.00^cd 127.98±5.15â 101.0±2.39â 44.3±4.20ê Ethylene

(µL C₂H₄/(kg·h)) 21.24±0.56

Ch+B 33.25±0.16ê 84.10±0.16â 78.32±0.13â 42.5±2.65^c <0.0001 0.001 0.001

Ch+L 32.33±0.13ê 83.34±0.15â 77.68±0.39â 42.4±2.69^c

Ch+S 31.62±0.42ê 82.42±0.52â 77.93±0.29â 49.5±3.88^b

Ch+X 32.80±0.07ê 83.85±0.06â 78.12±0.21â 59.2±4.16^b

Chitosan 32.49±0.06ê 83.61±0.04â 78.44±0.08â 40.9±1.59^c Control 33.31±0.27ê 84.90±0.49â 81.32±1.10â 27.3±1.47^f

Data presented as mean±standard error (SE). Different letters across treatments and storage duration for each attribute differ significantly (p<0.05) according to Duncan’s multiple range test. Ch+B, chitosan+Bidens pilosa; Ch+L, chitosan+Lippia javanica; Ch+S, chitosan+Syzygium cordatum; Ch+X, chitosan+Ximenia caffra.

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storage period, suggesting that chitosan could be implicated in this observation. According to Fawole et al. (2020a), ECs reduce gaseous exchange by sealing lenticels and covering the epicarp, consequently reducing fruit respiration rate and ethylene production. The suppression of the respiration rate and ethylene production in the coated passion fruit could be associated with coating gas barrier properties, as the results indicate in our study, leading to delayed climacteric ripening, which extended the shelf life of passion fruit by 8 d.

Physical parameters Color attributes

Fruit color is an important parameter that influences the buyer’s choice and acceptability to purchase (Pathare et al., 2013). Results revealed that the applied treatments and storage period significantly (p<0.0001) increased the C* and hue angle of passion fruit, respectively (Table 2). The initial C* of passion fruit was 22.44. Generally, the C* of passion fruit decreased as the hue angle increased during the storage period. This indicates that regardless of the treatment applied, passion fruit color changed during the storage period (Table 2). Treatments and storage period significantly (p<0.0001) influenced the C* of passion fruit. At the end of the storage period, the lowest C* (5.43) was observed in the control fruit.

Coating passion fruit with chitosan only resulted in lower C* (8.49) compared to chitosan enriched with the different

medicinal plant extracts. However, coating the passion with Ch+X had no significant (p>0.05) difference in color intensity compared to passion fruit treated with chitosan only. Among the different treatments applied, passion fruit coated with Ch+L (11.1) had higher C*, while fruit treated with Ch+X (9.16) exhibited lower C*, and they were significantly different (p<0.05) from each other.

Peel browning and other rind disorders of passion fruit can contribute to a decrease in passion fruit color during storage. As a result, the passion fruit hue angle significantly (p=0.001) increased with an increased storage period (Table 2). The hue angle increased with a decrease in the purple color of passion fruit as it can measure the fruit color purity and its deviation from the initial hue angle of 15.27. After 16 d of storage, fruit coated with Ch+B had the highest hue angle (40.58), and at the end of the storage period, passion fruit coated with Ch+B had the highest hue angle (33.33), followed by Ch+L (23.33), Ch+S (21.86), Ch+X (21.52), chitosan only (20.51), and control fruit had the lowest hue angle (15.61). The TCD was significantly (p=0.034) influenced by an interaction of treatments applied and the storage period (Table 2). Generally, the TCD significantly (p<0.0001) increased during the storage of passion fruit. At the end of the storage period, control fruit (17.10) had the highest TCD, followed by fruit coated with chitosan (14.1), Ch+L (13.50), Ch+X (13.20), Ch+B (12.90), and Ch+S (12.10) had the lowest TCD. It is noteworthy that even Table 2. Color attributes of passion fruit treated with chitosan coating fused with medicinal plant extracts during storage for 32 d at (8±2) °C and additional 3 d at (20±5) °C

Parameter Treatment Harvest Storage period (d) Significance level

8 16 24 32 Treatment

(A)

Storage period (B)

A×B

C* 22.44±0.467

Ch+B 21.42±0.580â 21.03±0.580â 12.19±0.583^c 9.7±0.058^d <0.0001 <0.0001 0.1387 Ch+L 18.06±0.580âb 16.47±0.587^b 12.4±0.583^c 11.1±0.578^cd

Ch+S 14.07±0.603^bc 15.39±0.739^b 13.67±0.583^c 10.2±0.606^cd

Ch+X 16.75±0.607^b 13.12±0.572^c 12.05±0.586^c 9.16±0.574^d

Chitosan 17.95±0.583^b 17.24±0.600^b 10.05±0.583^d 8.49±0.57d7^d Control 18.78±0.508^ab 17.17±0.586^b 9.19±0.571^d 5.43±0.577^e

Hue angle 15.27±0.792

Ch+B 26.01±0.633^c 40.58±0.572^a 37.38±0.572^a 33.33±0.589^b <0.0001 0.001 0.1891

Ch+L 26.5±0.520^c 31.75±0.572^b 29.3±0.664b^c 23.33±0.589^cd

Ch+S 20.86±0.580^d 31.2±0.588^b 26.62±0.586^c 21.86±0.589^d

Ch+X 24.67±0.569^cd 31.2±0.580^b 29.45±0.592^bc 21.52±0.583^d Chitosan 21.55±0.580^d 30.86±0.575^bc 25.62±0.574^cd 20.51±0.580^d Control 22.77±0.586^d 27.08±0.577^c 22.42±0.583^d 15.61±0.595^e TCD

Ch+B 8.9±0.088^b 9.3±0.058^b 9.5±0.058^b 12.9±0.058^ab 0.0317 <0.0001 0.034

Ch+L 6±0.333^c 6.9±0.058^c 9.1±0.058^b 13.5±0.058^a

Ch+S 5.6±0.058^cd 7.4±0.058^bc 7.9±0.058^bc 10.1±0.058^b

Ch+X 6.10±0.384^c 8.4±0.058^b 8.7±0.058^b 13.2±0.058^a

Chitosan 5.5±0.058^cd 8.8±0.058^b 9±0.058^b 14.1±0.058^a

Control 3.8±0.058^d 9.4±0.058^b 13.2±0.088^a 15.1±0.058^a

Data presented as mean±standard error (SE). Different letters across treatments and storage duration for each attribute differ significantly (p<0.05) according to Duncan’s multiple range test. C*, color intensity; Ch+B, chitosan+Bidens pilosa; Ch+L, chitosan+Lippia javanica; Ch+S, chitosan+Syzygium cordatum; Ch+X, chitosan+Ximenia caffra; TCD, total color difference.

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though enriching chitosan coating with medicinal plant extracts resulted in lower TCD compared to fruit coated with chitosan alone, there was no significant difference (p>0.05) at the end of the storage, suggesting delayed ripening and probably suppressed anthocyanin synthesis in the treated fruit (Valero et al., 2013).

Fruit size reduction and juice yield

The fruit size reduction was significantly (p<0.0001) influenced by the interaction of the applied coating and the storage period, while the juice yield percentage was significantly (p<0.0001) influenced by the storage period and the treatments applied (Figure 2). The initial fruit diameter was 38.60 mm, and it generally declined as the storage period increased. Control fruit exhibited 48.26% reduced fruit diameter after 24 d of storage, rendering them unmarketable. At the end of the storage period, fruit coated with Ch+S exhibited the largest fruit diameter (32.00 mm), equivalent to 15.79% reduced fruit size (Figure 2A). It was followed by passion fruit coated with Ch+B (20.98%), Ch+L (23.13%), Ch+X (25.70%), chitosan only (41.29%), and control fruit (61.99%). The reduced fruit size could result from increased weight loss in the coated and uncoated passion fruit storage.

The application of medicinal plant extracts in the chitosan matrix reduced the weight loss in passion fruit; thus, it had lower fruit size reduction. According to Mota et al. (2003), passion fruit size and rind thickness contribute to the fruit weight because the rind is the major component of the fruit.

The decrease in fruit size could be due to water flow from the rind to the pulp, since, in the same way, the fresh matter

in the rind is rapidly reduced than the flesh in the fruit, thus affecting the fruit size and rind thickness over a long storage period.

Generally, the juice yield percentage of passion fruit declined as the storage period increased (Figure 2B). The initial juice content was 91.00%. Coated passion fruits showed a steady decline in juice yield percentage compared to the control. Control fruit exhibited a 1.9-fold decrease in juice yield percentage after 24 d of storage, and it was significantly different (p<0.05) to the coated fruit. At the end of the storage period, the control fruit had the lowest juice yield percentage of 21.90%. Among the treated fruit, the lowest juice yield percentage was observed in fruit treated with Ch+S (55%);

however, it was not significantly different (p>0.05) from fruit treated with Ch+L that obtained a juice yield of 55.47%.

The highest juice percentage was obtained from passion fruit treated with Ch+B (60.13%); however, it was not significant (p>0.05) from fruit treated with Ch+X, which obtained a juice yield of 59.20% (Figure 2B). Coating passion fruit with chitosan coating enhanced juice yield percentage retention, but it was lower when compared to the fruit coated with chitosan enriched with different medicinal plant extracts.

The observed retention of a higher juice yield percentage in the coated fruit may be related to reduced ROS, respiration, weight, and fruit size reduction (Maftoonazad et al., 2008).

Fruit firmness

The advancement of ripening and senescence changes the texture profile of horticultural crops. Firmness is one of the important quality attributes that determines the quality and Figure 2. (A) Fruit size and (B) juice yield of passion fruit during storage for 32 d at (8±2) °C and additional 3 d at (20±5) °C. Each bar represents the mean, and error bars denote the standard error (SE) of the mean. Bars followed by different letters are significantly different at p<0.05 according to Duncan’s multiple range test. Ch+B, chitosan+Bidens pilosa; Ch+L, chitosan+Lippia javanica; Ch+S, chitosan+Syzygium cordatum; Ch+X, chitosan+Ximenia caffra.

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acceptability of fruits. Table 3 illustrates the effect of chitosan composite coating during storage on the firmness of passion fruit. The initial firmness of passion fruit was 14.45 N, and it decreased over time with significant interaction between the treatment and storage period (p=0.022; Table 3). At the end of the storage period, coating passion fruit with Ch+L resulted in higher fruit firmness retention (7.80 N), followed by Ch+S (7.70 N), Ch+B (7.50 N), chitosan (5.80 N), and control fruit (2.60 N). As evidenced by firmness retention, it could be suggested that the application of the medicinal plant extracts in the chitosan matrix delayed loss of fruit membrane integrity, probably by preventing the generation of ROS on the fruit surface that can increase membrane permeability thus resulting in low fruit firmness during storage. According to Khaliq et al. (2017), fruit softness could be due to the waning of cell wall structure, loss of membrane integrity, hydrolysis of cellulose and hemicellulose, and depolymeriza- tion of pectin and starch. Coating delays ripening by reducing cell wall hydrolyzing enzymes and respiration in fruit, thus retaining the fruit firmness (Maftoonazad et al., 2008).

A similar trend was also observed in weight loss, in which the minimal change was observed in fruit coated with chitosan enriched with different medicinal plant extracts. Similar results were reported by Khaliq et al. (2019), who observed that sapodilla fruit treated with Aloe vera (100%)+Fagonia indica plant extract (1%) significantly maintained higher firmness (6.67 N) than the control (3.65 N) after 12 d of storage.

Chemical analysis Electrolyte leakage

Free radicals in the cell accumulate and harm the cell membrane in stored fruits as they are released. This affects the cell membrane and firmness of horticultural crops like passion fruit (Shi et al., 2013). Once the cell membrane is dam- aged, its permeability increases, causing an increase in the EL rate (Antunes et al., 2010). Significant interactions were observed between the treatment and storage period (p<0.030) on the EL of passion fruit (Figure 3). The initial EL was (8.90%) and significantly (p<0.05) increased in all the treatments during the storage period. Compared to the control fruit, coating passion fruit with chitosan alone controlled the leakage rate to some extent; however, incorporating the medicinal plant extracts into the chitosan matrix reduced the loss

of fruit firmness, thus resulting in lower EL of passion fruit during the storage period (Figure 3). At the end of the storage period, coating passion fruit with Ch+X resulted in lower EL (71.40%), followed by Ch+L (73.17%), Ch+B (73.77%), Ch+S (75.63%), chitosan only (83.73%), while control fruit had the highest EL (91.97%). The application of chitosan edible coating alone delayed loss in cell membrane permeability, whereas incorporating the medicinal plant extracts in the chitosan matrix improved the resistance of the cell membrane to degradation, probably due to ROS, thus reducing the EL of passion fruit. Similar findings were reported by Tesfay and Magwaza (2017), who reported that avocado fruit coated with carboxymethyl cellulose and chitosan containing moringa leaf extract showed EL of 170 μЅ/m and 210 μЅ/m respectively, which were significantly lower than that of the control fruit (292 μЅ/m). According to Sayyari et al. (2010), ultrastructural alterations in fruit membrane results in unset- tled ion balance and EL of the mesocarp. It must be noted that throughout the storage, there was a gradual increase in EL of passion fruit, indicating loss of the fruit cell membrane integrity.

TSS, TA, TSS/TA, and BrimA index

The accumulation of TSS content is one of the important fruit quality indicators of maturity and ripening. There was significant interaction (p<0.0001) observed between the treatment and storage period on the TSS of passion fruit (Table 4). In all the treatments, the TSS levels increased during the first 16 d of storage and gradually declined. At harvest, the TSS level was 15.10%, and the highest increase in TSS was observed in the control fruit (16.4%) at 16 d of storage. This is a result of the faster ripening of the control passion fruit. Among the coated passion fruit, fruit treated with Ch+B had a higher TSS (15.77%), and it was significantly different (p<0.05) from the other treatments (Table 3). At the end of the storage period, the TSS content had declined that the control fruit having the lowest TSS (14.73%), followed by chitosan alone and Ch+L (15.00% for both treatments), Ch+S and Ch+X (15.10% for both treatments), and the highest TSS content was observed from passion fruit treated with Ch+B (15.40%). Coating passion fruit with a chitosan coating delayed senescence and controlled the fast conversion of starch to sugar. Incorporating the medicinal plant extract in the chitosan matrix further Table 3. Fruit firmness of passion fruit treated with chitosan coating fused with medicinal plant extracts during storage for 32 d at (8±2) °C and

additional 3 d at (20±5) °C

Parameter Treatment Harvest Storage period (d) Significance level

8 16 24 32 Treatment (A) Storage period (B) A×B

Fruit firm-

ness (N) 14.45±0.302

Ch+B 13.20±2.78^b 12.26±2.39^bc 9.41±1.23^de 7.5±0.45^g <0.0001 0.0006 0.022

Ch+L 13.92±3.07â 12.11±2.33^bc 9.07±1.06ê 7.8±0.57^g Ch+S 13.06±2.72^b 12.11±2.33^bc 9.96±1.45^d 7.7±0.53^g Ch+X 13.68±2.97âb 11.32±2.01^c 8.89±1.02êf 7.0±0.24^h Chitosan 12.79±2.61^bc 10.14±1.53^cd 7.50±0.45^g 5.8±0.24ⁱ Control 11.40±2.04^c 8.67±0.93^f 4.18±0.91îj 2.6±1.55^j

Data presented as mean±standard error (SE). Different letters across treatments and storage duration for each attribute differ significantly (p<0.05) according to Duncan’s multiple range test. Ch+B, chitosan+Bidens pilosa; Ch+L, chitosan+Lippia javanica; Ch+S, chitosan+Syzygium cordatum; Ch+X, chitosan+Ximenia caffra.

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decreased the fast conversion of starch to sugar of passion fruit, indicating a slow ripening rate; therefore, the TSS increased at a reduced rate in comparison to the control fruit.

According to Parven et al. (2020), generally, climacteric fruits exhibit a cumulative increase of TSS during postharvest storage. This increase could be due to the conversion of sucrose to sugars, thus having free soluble sugar accumulation (Ali et al., 2010), primarily leading to an increase in TSS concentration during metabolic processes at storage (Cheour et al., 1990; Parven et al., 2020). Yang et al. (2014) and Gull et al. (2021) reported similar results in apricot fruit coated with chitosan enriched with pomegranate peel extract and blueberry coated with chitosan and blueberry leaf extract, respectively.

The initial TA of the fruit at harvest was 4.15% citric acid (CA). The TA decreased with storage, but it eventually increased after 24 d of storage in almost all the treatments (Table 4). There was a significant interaction (p=0.023) observed between the treatment and storage period on passion fruit TA, with the storage period significantly (p<0.0001) influencing the changes in TA during storage (Table 4). At the end of the storage period, fruit coated with control had higher TA (4.15% CA), followed by chitosan coating (4.08%

CA), Ch+B (4.03% CA), Ch+L (4.02% CA), Ch+S (4.00%

CA), and passion fruit coated with Ch+X showed the lowest TA (3.92% CA). Reduction of TA is attributed to oxidation of organic acids (Ali et al., 2010); therefore, the application of chitosan coating and incorporating the medicinal plant extracts into the chitosan matrix enhanced the capacity of chitosan coating in restricting O₂ availability and subse- quently decreasing organic acids oxidation on the passion fruit. Similar results were reported by Bahmani et al. (2015), who reported that the TA levels of sapodilla fruit decreased with an application of thyme essential oil. Several other au- thors have also reported decreases in TA during the storage of different fruit. However, Medeiros et al. (2021) reported an increase in TA for S. tuberosa fruits with or without coatings during storage.

The taste of passion fruit juice is primarily determined by juice TSS level and the ratio between the TSS and TA.

According to Fawole and Opara (2016), the TSS/TA ratio influences the taste of products, and it measures the balance between the acids and sugars that it contains. Metabolic processes such as respiration influence the TSS and TA differences during fruit ripening (Zhang et al., 2017). The initial TSS/TA ratio at harvest was 3.97, and the coated fruit showed a significant (p<0.0001) increase in TSS/TA during the first 16 d of storage, which declined afterwards. Passion fruit coated with Ch+S had the highest increase with a 4.19 TSS/TA ratio (Table 4), and at the end of the storage period, fruit coated with Ch+B had a significantly (p<0.05) higher TSS/TA (3.92), followed by Ch+X (3.83), Ch+S (3.78), Ch+L and chitosan only (3.73 for both), while control fruit had the lowest TSS/

TA (3.61). Changes in TSS/TA were significantly (p<0.0001) influenced by the different treatments and storage period. The increase in TSS/TA ratio could be due to the observed increase in TSS and decrease in TA values during storage, resulting in a higher TSS/TA ratio. According to Zafari et al. (2015), starch degradation into water, soluble sugars, sucrose, and glucose are the main attributes of TSS/TA ratio levels. This might also be due to the breakdown of organic matter into sugars and their involvement in the respiration cycle (Fawole et al. 2020a).

BrimA index is based on the TSS/TA ratio and tongue’s sensitivity index (k) to determine the acceptability of juices (Jordan et al., 2001). The storage period had a significant (p<0.0001) effect on the BrimA index of passion fruit. The initial BrimA index was 3.67, and the results from this study indicate that except for control fruit, the BrimA initially increased with Ch+S having the highest BrimA index of 4.61 after 16 d of storage with the control fruit having the lowest of 3.37 (Table 4). Afterwards, the BrimA index declined across all treatments, and at the end of the storage period, the highest BrimA index was obtained from passion fruit treated with Ch+B (3.31), followed by Ch+X (3.14), Ch+S (3.10), Ch+L (2.94), chitosan only (2.55), and control fruit (2.49) that exhibited the lowest BrimA index. The capacity of chitosan coating and medicinal plant extracts in the chitosan matrix created a modified atmosphere around the passion fruit, which slowed down the metabolic rate, Figure 3. Electrolyte leakage of passion fruit during storage for 32 d at (8±2) °C and additional 3 d at (20±5) °C. Each bar represents the mean, and error bars denote the standard error (SE) of the mean. Bars followed by different letters are significantly different at p<0.05 according to Duncan’s multiple range test. --- represents investigated parameter at harvest. Ch+B, chitosan+Bidens pilosa; Ch+L, chitosan+Lippia javanica; Ch+S, chitosan+Syzygium cordatum; Ch+X, chitosan+Ximenia caffra.