Evaluation of Ocimum sanctum and Cymbopogon nardus Ethanol Extract as Edible Food Coatings on Jackfruit Bulbs

Received 26 Oct, 2020

Accepted 25 Feb, 2021

Published 09 Jun, 2021

ABSTRACT

Background and Objectives: Food spoilage is a major threat to food security and this has led to the development of novel and innovative food preservation techniques using natural products. This study investigated the formulation of ethanolic extracts of the leaf and stem of Ocimum Sanctum (holy basil) and Cymbopogon nardus (citronella grass) as edible food coatings on jack fruit bulbs. Materials and Methods: Treatments included plant extract+sodium alginate coated fruits, sodium alginate coated fruits and uncoated fruits. Texture analysis, water activity and mesophilic plate count were analysed over 7 days of storage. Sensory evaluation was conducted for the following parameters: colour, appearance, surface, texture and overall acceptability. Results: Total mesophilic bacterial counts increased for all treatments, with the extract coated samples showing the lowest count on the seventh day of storage. The extract coated fruits were also firmer and had lower water activity compared to the sodium alginate and uncoated controls. In overall acceptability, the citronella grass coated sample scored the highest. Conclusion: This study has thus found that a food coating using plant extracts has the potential to delay microbial spoilage and increase the shelf life of food products during storage.

INTRODUCTION

Food spoilage is a major threat to food security and is predominantly responsible for the massive losses in nutrition globally. It has become a major concern to the growing demand for food throughout the next decennia. According to a study¹, an estimated 25-80% of harvested fresh fruits are lost annually mainly due to spoilage, with the highest losses found in the tropical regions. Consequently, lessening food spoilage will dramatically increase our food supply. Microorganisms, fungi, and insects are the major cause of food spoilage and deterioration worldwide².

To overcome the constraints mentioned above, novel and innovative techniques need to be developed by the food industry. Moreover, there are high demands for natural, healthier and fresh food without using any technology that may have potential risk to human health and the environment³. This has led to a growing interest in the development of alternative preservation methods by using natural additives. Plant extracts have gained popularity since they contain substances known as phytochemicals. They are naturally occurring components present in plants, which tend to eliminate or retard the growth of food-borne pathogens⁴.

Jackfruit (Artocarpus heterophyllus Lam) comprises three important parts namely, bulb, seed, and rind. It is mainly cultivated in Asia which includes Malaysia, Indonesia, India, Thailand and the Philippines⁵. Ripe Jackfruits are very nutritious; they are rich in pectin and minerals like calcium, iron, potassium and phosphorus. Ripe jackfruits are full of carotene, ascorbic acid, and carbohydrates⁶. The unripe bulbs are usually used in the preparation of papad and chips, while the raw fruits are popularly used as vegetables in several culinary preparations. The international demand for jackfruit has increased in recent years⁷. Jackfruit is a large fruit and difficult to peel, therefore consumers prefer to buy it ready to eat. Furthermore, Jackfruit is available year-round and is a major food supply for people when there are short supplies of staple food grains.

Ocimum sanctum and Cymbopogon nardus are grown widely and thus readily available throughout Malaysia. Ocimum tenuiflorum has been utilised in various forms. Aqueous extracts from the leaves (fresh or dried in powder form) are used in natural teas or blended with different herbs or nectar to improve the restorative system^8,9. Cymbopogon nardus is a local therapeutic plant, generally utilised for a post-partum bath. Scientific studies have shown C. nardus to possess several biological activities including antiviral and antibacterial properties¹⁰.

While most research focuses on other methods which include the calcium-infusion method into jackfruit pulps¹¹, aloe vera gel coating on jackfruit bulbs¹², the effect of pretreatment with 1-methyl cyclopropane (1-MCP) and the application of edible coatings (xanthan, sodium alginate or gellan) on the ripening rate, quality parameters and shelf life of pre-cut jackfruit¹³. One particular use of plant extracts that is relatively unexplored is as addictive in the edible coatings of fruits and vegetables. The incorporation of plant antimicrobial agents into biodegradable edible coating has proven to be a highly effective tool to reduce the risk of pathogen growth and protect food products against spoilage flora^14,15.

Previous research conducted using water, 80% ethanol and 80% methanol has shown that ethanolic extracts of these plant samples possessed potent antioxidant and antimicrobial properties¹⁶.

Therefore, a study was carried out to use the ethanol extracts of the leaf and stem of Ocimum sanctum and Cymbopogon nardus to formulate an edible food coating for Jackfruit bulbs. It is postulated that the inclusion of plant extract edible coating will prolong the shelf life of the jackfruit and at the same time preserve its original odour and taste.

MATERIALS AND METHODS

Preparation of plant extracts: Fresh leaves and stems of Ocimum sanctum and Cymbopogon nardus, with no apparent physical, insect or microbial damage were collected from three different locations (Nilai University, Desa Cempaka, and Desa Melati). Samples were washed with tap water, rinsed and air-dried for 7-14 days at room temperature at about 23 degrees centigrade. Upon drying, the samples were ground using a household blender, after which they were stored in Schott bottles at 4°C for further periodical use.

The preparation of crude extract was carried out as described by Ruban and Gajakhsmi¹⁷ with slight modification. The powdered leaf and stem samples were extracted with 80% ethanol. 4 g of the samples were diluted with 100 ml of solvent in a conical flask and covered with aluminium foil. The flask was placed in an orbital shaker at 120-150 rpm at room temperature. After 24 hours, the solution was filtered using Whatman filter paper No 2 and the filtrate was evaporated to dryness in the fume hood. The dried extracts were reconstituted in 80% DMSO (Dimethyl sulfoxide) to give a stock concentration of 100 mg/ml and the solutions were kept in the fridge (4) until further use.

Preparation of jackfruit bulb: Jackfruits with brownish-yellow skin colour at optimum ripening stage J33, sweet and crispy, were acquired. The outside of the fruit was washed and allowed to dry. The fruit was opened manually with a sterile knife, cutting along the main axis. The whole bulb with seed was separated from the central axis of the fruit. Damaged or bulbs with defects were removed and those with uniform size, colour and shape were selected and used for the study¹³.

Formulation of Edible food coatings: Application of edible coating was carried out with slight modification as described by¹³. First, 5 g of sodium alginate was dissolved in 500ml of distilled water and mixed with 5 ml of glycerol as a plasticiser. To this mixture was added 1ml of ethanol plant extract. The jackfruit bulbs were randomly divided into 3 treatment groups with 500 g bulbs per group; which included: 500ml sodium alginate-based edible coating + 1ml extracts, sodium alginate-based edible coating and untreated bulbs were used as the positive (+) and negative (-) controls respectively. The fruits were dipped into each of the above-mentioned solutions of different coating for 5 minutes, sealed in clean zip lock bags and stored at 4°C until analysis. The analysis was carried out on days 1, 3, 5, and 7.

Texture analysis: According to another study¹³ with slight modification texture analyser (model- TA. X Plus) by Stable Microsystem Ltd., Vienna Court, Surrey GU7 1YL UK was used to evaluate the texture of the jackfruit bulb with a setting of; Sequence Title: TPA 1, T.A. Variable No: 1: Compression, pre-test speed: 1.00 mm/sec, Test speed: 1.50 mm/sec, Post-test speed: 5.00 mm/sec, T.A. Variable No: 5: 0.0 g, Distance: 10.000 mm, Strain: 75.0%, Trigger Type: Auto (Force), Trigger Force: 5.0 g, Probe: P/10; 10mm dia cylinder Delrin (as per the texture analyser reading).

Water activity: According to Neswati et al.¹⁸ with slight modification, water activity analyser by brand-Aqua Lab, Meter Group Inc. Pullman, WA 99163 USA was used. 1g of jackfruit bulb was placed in a small weigh plate and this was then placed into the water activity analyser.

Microbiological count: For microbiological analysis, samples were taken randomly during the 1^st, 3^rd, 5^th, and 7^th day of storage. 5g of coated and control samples of jackfruit bulbs were crushed with 45 ml of 0.1% sterile peptone water in a stomacher bag. The resulting mixture was diluted by performing serial dilutions until a 10ˉ dilution factor was obtained. 100 μl of dilution sample was spread onto standard plate count agar (PCA) for enumeration. The plates were incubated at 37°C for 48 hours¹⁹. Microbial counts were expressed as log CFU/g.

Sensory evaluation: Organoleptic observation of the jackfruit bulbs was performed on day 1 and day 7. The ratings were obtained from a panel consisting of 30 students selected from the Department of Food Science, Universiti Putra Malaysia. Without making any comparison, panellists were asked to evaluate each of the samples individually. Samples were tested for different parameters like colour, appearance, surface, texture and overall acceptability. The samples were evaluated using a 9-point hedonic scale, in which “1” attributes dislike extremely and “9” attributes like extremely.

RESULTS

Plant Extract Coated Fruits
Firmness: The firmness of the jackfruit bulbs decreased with days of storage, although the decrease was not significant (Table1). For the Basil leave, the negative value and increase in firmness may be a result of the sample becoming contaminated during coating of the said fruit (exposure to a contaminated area or plate as all the samples were taken out of a zip lock bag and place in a small plate to weigh). Fruit firmness for the rest of the samples decreased upon storage.

On Day 7 the firmness levels of the Citronella extracts (both stem and leave) were higher than the Basil extracts and both the positive and negative controls. This indicates that these extracts were able to prevent water loss from the fruit, thereby retaining the firmness.

Water activity: As can be seen from Table 2, the water activity of all samples increased with time. On Day 1, the water activity of the negative control was 0.911 compared to the positive control which was 0.906, while samples coated with the addition of extracts had water activity values of 0.905, 0.894, 0.9, and 0.889 for basil stem, basil leaves, citronella grass stem and citronella grass leaves respectively. In these samples, the spoilage began at a_w of 0.95.

Mesophilic plate count: The total mesophilic bacterial counts for all treatments increased throughout the storage time (Table 3). A count of 7.222 log CFU/g was recorded for jackfruit bulbs treated with sodium alginate on Day 1 and the value increased to 9.141 log CFU/g on Day 7. However, a lower count was observed in jackfruit bulbs coated with sodium alginate + extracts, 7.036 log CFU/g on Day 1 and 9.073 log CFU/g on Day 7 for basil stem, 7.047 log CFU/g was obtained on Day 1 and 9.047 log CFU/g on Day 7 for basil leaves, 7.052 log CFU/g for Day 1 and 8.947 log CFU/g on Day 7 for citronella grass stem, 7.043 log CFU/g on Day 1 and 8.838 log CFU/g on Day 7 for citronella grass leaves. Compared to uncoated sample with a value of 9.164log CFU/g, jackfruit bulbs with sodium alginate coating and sodium alginate coating + extracts reduced the count of mesophilic bacterial on the last day (Day 7) of storage. Citronella grass extracts reduced the count of mesophilic bacterial more than the basil extracts but the reduction was not statistically significant.

Consumers acceptance: Sensory evaluation to compare the quality of treated and control jackfruit bulbs were carried out using a 9-point hedonic scale with 30 members comprising the panellist. The assessment included colour, surface, appearance, texture, and overall acceptability. On Day 0 (Fig. 1a) the appearance of the basil extracts was comparable to the positive control, while the Citronella grass extracts had the lowest values. The colour was the highest for the basil stem and the Citronella grass leaf samples. The surface in all extracts was higher than both the positive and negative controls. In texture, the positive control was the highest. In overall acceptability, the positive control and the basil leaves extract gave the highest values.

Nevertheless, the overall acceptability for the uncoated jackfruit (Fig. 1b) after seven days was low compared to the coated jackfruit with extracts, showing that the coatings helped improve the acceptability of the fruit.

Consumer’s acceptance of treated and untreated jackfruit bulbs

Fig. 1a-b: Sensory evaluation of jackfruit bulbs a. Comparison of sensory evaluation of treated and untreated jackfruit bulbs on Day 0, b. Comparison of sensory evaluation of treated and untreated jackfruit bulbs on Day 7

Table 1: Comparison of firmness of jackfruit bulbs treated with plant extracts
		Hardness (g)
Sample	Day 3	Day 5	Day 7
Basil Leave	-2.093±0.683	575.957±164	1326.14±630
Basil Stem	12555.81±144	6732.235±809	625.555±256
Citronella grass Leave	13721.37±941	7113.180±458	2624.700±221
Citronella grass Stem	11375.662±158	757.373±514	7113.306±978
Control (+)	21202.9±947	15214.4±567	659.63±222
Control (-)	11410.7±498	5920.65±737	402.61±119
Values with the same capital letter are not significantly different p>0.005 among the samples with the same days. Values with the same small letter are not significantly different p>0.05 among the days with the same sample.

Table 2: Variation of water activity of treated and untreated jackfruit bulbs
		Water Activity
Sample	Day 1	Day 3	Day 5	Day 7
Basil Leave	0.894±0.000	0.907±0.000	0.916±0.000	0.953±0.000
Basil Stem	0.905±0.000	0.915±0.000	0.935±0.000	0.961±0.000
Citronella grass Leave	0.889±0.000	0.907±0.000	0.918±0.000	0.951±0.000
Citronella grass Stem	0.900±0.000	0.911±0.000	0.929±0.000	0.957±0.000
Control (+)	0.906±0.000	0.917±0.000	0.926±0.000	0.973±0.000
Control (-)	0.911±0.000	0.923±0.000	0.936±0.000	0.979±0.000
Values with the same capital letter are significantly different p<0.005 among the samples with the same days. Values with the same small letter are significantly different p<0.05 among the days with the same sample

Table 3: Evaluation of Mesophilic count of treated and treated jackfruit bulbs
	Total Colony Count (log CFU/g)
Sample	Day 1	Day 3	Day 5	Day 7
Basil Leave	7.047±0.014	7.508±0.006	8.362±0.013	9.047±0.015
Basil Stem	7.036±0.051	7.527±0.005	8.377±0.049	9.073±0.011
Citronella grass Leave	7.043±0.014	7.483±0.009	8.337±0.018	8.838±0.008
Citronella grass Stem	7.052±0.043	7.545±0.008	8.379±0.014	8.947±0.003
Control (+)	7.219±0.081	7.567±0.017	8.921+0.016	9.141+0.14
Control (-)	7.325±0.028	7.582±0.019	8.892+0.008	9.164+0.017
Values with the same capital letter are not significantly different p<0.05 among the samples with the same days. Values with the same small letter are not significantly different p<0.05 among the days with the same sample + Control = sodium alginate coating - Control = No coating

DISCUSSION

The current study has shown that edible food coatings formulated with plant extracts has the ability to delay fruit spoilage due to microbial growth. A preliminary study on these plants has shown that ethanol and methanol extracts of the leaves and stems of Ocimum sanctum and Cymbopogon nardus yielded more bioactive compounds than the water extracts¹⁶. The results correlate with a recent study on the stem and leaves of Ocimum sanctum conducted by Garg and Garg²⁰, who found a good yield of extracts using methanol and ethanol as extraction solvents. Therefore, this study was conducted using the ethanolic extracts of the leaves and stem of these two plants because water yielded low bioactive compound and had no antimicrobial activity while methanol is not safe for consumption.

The firmness of the jackfruit bulbs decreased with days of storage, although the decrease was not significant. For the Basil leave, the negative value and increase in firmness may be a result of the sample becoming contaminated during coating of the said fruit (exposure to a contaminated area or plate as all the samples were taken out of a zip lock bag and place in a small plate to weigh). Fruit firmness for the rest of the samples decreased upon storage. On Day 7 the firmness levels of the Citronella extracts (both stem and leave) were higher than the Basil extracts and both the positive and negative controls. This indicates that these extracts were able to prevent water loss from the fruit, thereby retaining the firmness. A study on soursop^21,22 and on tomatoes²³ found that fruits coated with chitosan were still commercially satisfactory after 30 days of storage.

All samples maintained an a_w below 0.95 up to Day 5. On Day 7, the water activity of all samples rose to above 0.95, however, the water activity of the extract coated samples (which ranged from 0.953 to 0.961) were lower (but not significantly so) compared to the positive (0.973) and negative (0.979) controls.

Phenolic compounds including chlorogenic acid, gallic acid and rutin, are known to possess many beneficial biological activities including antimicrobial properties^24,25. Since microorganisms are known to be one of the causes that leads to the deterioration of food²⁶, polyphenols due to their antimicrobial properties can reduce microbial counts and therefore protect food from spoiling. The results have shown that all extracts in the study contain bioactive compounds which possess antimicrobial activities. Although the mesophilic counts were not significantly different between the samples tested, the coatings with extracts exhibited slightly lower total mesophilic bacterial counts compared to the controls, with the Citronella extracts exhibiting lower counts than the Basil extracts. The reason that the values are not significant might be due to the low concentration of extracts incorporated in the coatings.

The data presented above has suggested that food coatings made with Basil and Citronella extracts might act as barriers to O₂ (retaining the firmness of the fruit) and water (lower a_w activity), two factors necessary for microbial growth. This could also help to explain why jackfruit bulbs coated with plant extracts possessed lower mesophilic counts.

Since the phytochemicals present in the Basil and Citronella extracts possess antimicrobial effects and retain the firmness of the jackfruit bulbs, this would contribute to the better appearance, texture and overall physical acceptance of the fruit. Jackfruit coated with citronella grass scored the highest in overall acceptability.

CONCLUSION

The findings of the present research have proven that edible food coatings using ethanolic extracts of Ocimum sanctum and Cymbopogon nardus can prevent microbial food spoilage. The coatings incorporated with the plant extracts not only possessed antibacterial properties but also acted as oxygen and water barriers. Moreover, these edible food coatings also met with consumer acceptance thereby promoting its application in the food industry.

SIGNIFICANCE STATEMENT

This study discovers the potential application of plant based edible food coatings that can be beneficial in preventing the microbial spoilage of fruits postharvest. The findings of this research will help the researcher to uncover an innovative food preservation technique using natural products. It is hoped that this will provide a solution to the critical area of food spoilage faced globally. The preparation of the extracts is easy and obtainable with the use of simple equipment. Previous studies explored by other researchers used essential oils obtained with complicated extraction methods. Thus, a new, easy and simple extraction method of preparing plant extracts that are effective in preventing microbial food spoilage may be arrived at.

ACKNOWLEDGEMENT

Southeast Asian Regional Center for Graduate Study and Research in Agriculture (SEARCA) provided funding for study under the Master of Science in Food Security and Climate Change (MS FSCC) Project, grant number GCS18-1320. The funding was for one-semester non-degree mobility at University Putra Malaysia (UPM). The above research was conducted during the time spent at UPM. No funds were allocated for publication cost.

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