Nutritional quality of Cavendish banana (Musa acuminata, AAA) as affected by basil oil and determination of basil oil residues by GC-MS

The effectiveness of basil oil on the nutritional properties of Cavendish banana and chemical composition of basil oil and oil residue levels of treated banana fruits were evaluated in this study. Cavendish banana hands were treated with 1% alum (w/v), 1% alum (w/v) + 0.4% Ocimum basilicum (basil) oil, distilled water (control) and packaged in Low Density Polyethylene (LDPE) bags and stored at a cold room at 12-14oC. After two weeks of cold storage banana were induced ripened and nutritional contents of treated Cavendish banana were determined. Gas Chromatography Mass Spectrometry (GCMS) was instrumental in identifying the chemical constituents of basil oil as well as residues in basil oil treated Cavendish banana peel after two weeks of storage at 12-14oC. *Corresponding author Email: kris@kln.ac.lk; https://orcid.org/0000-0001-6562-3151 DOI: http:://doi.org/0.4038/josuk.v12i0.8020 This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution and reproduction in any medium provided the original work is properly credited.


INTRODUCTION
Ocimum basilicum L. which is known as Sweet Basil in the family Lamiaceae is a native herb to Asia, enriched with plenty of phytochemicals with considerable nutritional and antioxidant properties as well as ample health benefits (Paton, 1992;Shafique et al., 2011). Basil is widely cultivated and extensively used for food, perfumery, cosmetics, pesticides, and medicine due to their natural flavor and aroma and bioactive properties (Kobaet al., 2009). Sweet basil is widely used for preparation of essential oils, dried leaves as a culinary herb, condiment/spice in various dishes and food preparations (salads, sauces, pasta and Mediterranean cuisine). In medicine it is used for treating of headaches, kidney malfunctions, constipation, coughs, diarrhea, worms and warts (Ben-aliet al., 2014).
Basil essential oil has attracted attention of many scientists due to its antimicrobial and antioxidant properties which is very valuable in terms of food Industry. Utilization of essential oil in the food industry reduces the usage of synthetic fungicides / additives, and subsequently improves the freshness and sensory quality of the produce. Further, there is an increasing demand from public for natural food additives (Koba et al., 2009). Several researchers had provided evidence on the antifungal action of basil oil. According to Doube et al.(1989) basil oil at 1.5 ml/l completely inhibited the growth of 22 mold species, including 99 aflatoxigenic Aspergillus parasiticus and A. flavus. Soliman and Badeaa (2002) reported that basil oil was effective as a fungistatic agent against F. verticillioides at 2000 ppm concentration, and as a fungicidal agent at 3000 ppm concentration. Fandohan et al. (2004) demonstrated a complete inhibition of growth of F. verticillioides at basil oil concentrations higher than 2.7 μl/ml. According to Zollo et al. (1998) basil oil showed a complete inhibition of the growth of Candida albicans and A. flavus at 5000 ppm concentration, during a 7-day incubation period. Further, this oil extract showed strong antifungal activity towards Fusarium spp. (F. proliferatum, F. oxysporum, F. Verticillioides and F. subglutinans) isolated from spoiled cakes and the fungal growth was inhibited completely at 1.5 ml / 100ml concentration of basil oil (Kocić-Tanackov et al., 2011).
Crown rot disease of Cavendish banana is a serious postharvest disease caused by a range of different fungi including Colletotrichum musae, Lasiodiplodia theobromae as well as Fusarium spp., Verticillium spp. and Cephalosporium spp. (Abd-Alla et al., 2014).
In a previous study at the University of Kelaniya, efficacy of basil oil on crown rot disease control of Cavendish banana was evaluated (Siriwardana et al., 2016). During the study, basil oil at 0.4% (v/v) together with modified atmosphere packaging significantly managed crown rot disease of Cavendish banana and physicochemical and sensory properties of treated banana were not adversely affected in comparison to distilled water control (Siriwardana et al., 2016).

Preparation of Cavendish banana
Twelve (12) week mature Cavendish banana (Grande Naine cultivar) bunches were harvested from CIC banana plantation in Pelwehera, Dambulla, Sri Lanka from plants previously identified and tagged. Banana bunches were transported to the CIC banana pack house, at CIC Agri Business Centre. Bunches were dehanded and approximately 1 kg hands were selected as experimental units. All hands were washed in water to remove dirt and then with potassium aluminium sulphate (alum) (1% w/v) except the control (only washed with water). Subsequently, bananas were allowed to drip dry.

Preparation of treatments
Ocimum basilicum (sweet basil) oil was purchased from Aromatica laboratories (Pvt.) Ltd. Sri Lanka. Basil oil (400 ul) was added to 0.1 l of distilled water to prepare 0.40% (v/v) concentration with a drop of 'Tween'80 (Park Scientific Limited, Northampton, UK).
The mixture was stirred using a magnetic stirrer for 10 minutes and transferred to a handsprayer and mixed well by shaking. The control was prepared by adding one drop of 'Tween' 80 to 0.1 l of distilled water and stirring for 10 minutes (Siriwardana et al., 2016).

Application of treatments
Cut surfaces of crown and fingers of banana hands were sprayed with 0.40% (v/v) basil oil emulsion, or distilled water. Another set was washed in 1% alum solution only. banana to provide protection to fruit. Each treatment comprised of five replicate boxes, each containing five hands (weighing 5.0 -5.5 kg). All treatment boxes were stored in a cold room at CIC banana packhouse, Dambulla at 12-14 °C and 85-90% relative humidity. The experimental arrangement was a completely randomized design (CRD).

Ripening of banana
After two weeks storage period banana hands were subjected to induced ripening by exposure to ethylene (thrill -480g / l ethephon, 1ml / 1 l of water) for 24-48 hours at ambient temperature (Siriwardana et al., 2016).

Moisture content
Five induced ripened fruits (randomly selected) from each treatment were used. Ten grams of pulp from each finger were placed in a dried weighed crucible. The crucible with samples were placed in a drying oven (FEB87, Astell Hearson, UK) at 105 º C and heated for 3 hours. After cooling, dried samples were reweighed. This process was repeated until a constant weight was obtained. The difference in weight was calculated as a percentage of the original sample according to formula given by AOAC (1990) and Nwosu et al. (2011).
Five replicate samples were used per treatment and mean value was taken as moisture content.

Ash content
One gram of dehydrated banana fruit sample was placed in a clean, oven dried incineration crucible of known weight. Crucible was covered with pricked aluminium foil and total weight was recorded. Incineration crucible was incinerated at 550 º C in a muffle furnace (ECF 12/6, Lenton Furnaces, UK) until it turned white and free of carbon. Weight of the cooled crucible with sample was measured and the percentage of ash was calculated.
Five replicate samples were used per treatment and mean value was taken as ash content (AOAC, 1990;Nwosu et al., 2011).
Percentage Ash = (Weight of Ash / Weight of original of sample) 100 (2)

Crude protein content
The Kjeldahl method was used in determining the crude protein content. From each of the dehydrated banana samples, 0.5 grams was transferred to the 30 ml Kjeldahl flask without allowing the sample to cling the neck of the flask. Ten (10)

Fat content
Two grams of the sample was loosely wrapped with a filter paper and put into the thimble which was fitted to a clean round bottom flask, which has been cleaned, dried and weighed. The flask contained 120 ml of petroleum ether. The sample was heated with a heating mantle and allowed to reflux for 5 hours. The heating was then stopped and the thimbles with the spent samples kept and later weighed. The difference in weight was recorded as mass of fat and is expressed as a percentage (%). contents (ppm) were determined using standard curves. Mineral content were expressed as mg/100 g of fresh weight (AOAC, 1990). Five replicate samples per treatment were used for determining each mineral and mean value were calculated and expressed as the final mineral content.

Residue analysis of Cavendish banana
Cavendish banana hands (3 hands) were treated with 0.4% basil oil and stored in a cold room at 12-14 ºC and 85-90% RH for 14 days. Each treatment was replicated three times. One banana fruit from each hand was weighed using an electric balance and peeled and chopped. Hydro distillation of peel samples was carried out using a Clevenger apparatus.
Any oil present in the peel was trapped in normal Hexane. The collected extract was passed through a nitrogen steam in order to concentrate (Cox et al., 1974). This extract was

Statistical analysis
Nutritional properties were analyzed by ANOVA, while mean separation was done using Tukey's Multiple Comparison test at P < 0.05 using Minitab.

Nutritional properties
Moisture content ranged between 76.35 -76.76% while protein content ranged between 1.37 -1.55% of alum treated, alum + basil oil treated and (distilled water treated) control Cavendish banana. Ash contents were within the range of 0.90 -0.91% while fat was not detected in all samples. However, these values were not significantly different between the treatments indicating treatments had not adversely affected the nutritional properties of banana (Table 1).

Residue analysis of basil oil treated MAP stored Cavendish banana
Gas chromatogram of authentic Sweet basil oil displayed number of peaks ( Figure 1) and relevant chemical components were identified and shown in Table 3. α -Terpineol was present at 0.1% level while α -Pinene was detected at a level of 0.02% in basil oil. Out of the identified compounds in present study, methyl chavicol, linalool, eugenol, cinnamaldehyde, α-terpineol and α-pinene has been reported as components having profound antifungal properties (Marei et al., 2012;Nurzyńska-Wierdak et al., 2013;Costa 111 98 et al., 2015;Rahemi et al., 2015). They can even be present in minute amounts and exert considerable antifungal effect on pathogenic fungi.
Similar to results of present investigation, Lis-Balchin et al. (1998)  According to Anthony et al. (2003) eugenol was the major compound in basil oil responsible for crown rot disease control. Conidial germination and appressoria formation of fungal pathogens were inhibited by eugenol and membrane permeability of fungal pathogens were affected. Conidia of Colletotrichum musae germinate after depositing on a plant surface and produce a melanised, thick walled appressoria which is essential to adhere firmly to the host surface. In a previous study, it was reported that, basil oil prevent melanisation of appressoria and cause leakage of cell contents in conidia of C. musae resulting in the death of microbial cells (Herath and Abeywickrama, 2008). However, synergistic effect of antifungal components present in basil oil such as methyl chavicol, eugenol, linalool, cinnamaldehyde, α-terpineol and α-pinene may lead to antimicrobial efficacy, which needs further investigation.