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Publications of Palmyrah Research Institute, Sri Lanka 2012-2016

Enhancing the Capacity of Palmyrah Palm (Borassus flabellifer L) through Science

“Touch of Wonder of Palmyrah Realized through Science”

Contents

Publications in International Journals Publications in International conference

Full papers Abstracts

Publications in National conferences Full papers

Abstracts Publications in Newspapers Publications in Book

1. Publications in International Journals

IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) 9(6), pp. 51-57, 2016

Microbial, Physico- chemical and Sensory Evaluation of Preserved Palmyrah Fruit Pulp

Robika Kailayalingam, Subajini Mahilrajan*, SriThayalan SriVijeindran and Ponnuchamy Navaratnam

Palmyrah Research Institute, Jaffna, Sri Lanka

ABSTRACT: Fruits of Palmyrah palm (Borassus flabellifer) are seasonal; therefore their fibrous (mesocarp) fruit pulp (PFP) extracted with water and should be preserved with lengthened shelf life to ensure its availability in local and international market throughout the year. Therefore a study on preservation of PFP was carried out with or without various concentrations of preservatives, Sodium benzoate (SB), Sodium metabisulphite (SMS) and combinations of the both at different ratio. pH of the PFP was adjusted to 3.8 with citric acid, heated in a water bath at 90oC for 20 Sec, preservatives were added, mixed well then bottled pulp was heated at 80oC for 30 min in thermostatic water bath and kept at room temperature (30oC) for 180 days. Initial pH with stabilization has come to about 4.2. Aliquots of them were withdrawn periodically (at 30days intervals) and were analyzed for microbial, physicochemical and sensory characteristics. PFP alone (without preservatives) was spoiled with increasing pH by showing adverse characteristics (unacceptable odour) before 24hours of storage. All the treatment showed significant (p<0.001) increased in total soluble solid (10.82 – 13.10 obrix) and declined in pH (4.42 – 4.14) was observed with a proportional increase in the acidity (0.71- 0.91%) for treatments of T1 – T5 (containing SB), T6 –T10 (containing SMS) and T11 – T15 (containing both SMS & SB) up to 180 days. But no colony (Total Plate Count) was observed in the pulp treated with SMS and with combination of SMS & SB at various concentrations up to 120 days of storage. Among the all treatments the pulp treated with SB were found to be inferior in both colour and flavour characteristics. Even though it was found that PFP treated with SMS, T6 –T10 could be stored for extended period of 180 days without any major changes in chemical,

microbiological and sensory characteristics, whereas T7 (with SMS,0.4g/l ) was selected as the best treatment based on the overall acceptability.

Keywords: Palmyrah Fruit Pulp, preservatives, sensory evaluation

1.       Introduction

Palmyrah (B.flabellifer) fruit is the oldest and most important tropical fruit. It is indigenous or naturalized throughout tropical and subtropical South and Southeast Asia. Palmyrah fruit is mostly used as fresh fruit for pinattu (Dried pulp) and oil cakes, but due to its perishable nature it cannot be stored for long period of time. Pulp is yellow in colour due to the presence of carotenoids (Provitamin A). It is a good source of vitamin C and contains appreciable amount of pectin [1]. Jeyaratnam (1986) [2] said that pulp contains appreciable amount of saponin and also believed that pulp provides dermatitis relief.

During peak of harvest season (Aug- Oct) large quantity of fruits are wasted due to limited shelf life in storage. In order to make the PFP available during the off season it has to be preserved with lengthened shelf life. Sales centres of Palmyrah Development Board, Katpakams sell bottled PFP to prepare fruit base edible products. But during storage period colour of the bottled pulp turns to yellowish brown. Despite the fact it has to be developed with favourable chemical treatment for the preservation of PFP. Because of its high fermentable nature under the influence of microbes, it is dried as Pinattu for short term preservation. But it is also preserved by making panampanam (diluted drink), cordial, crush and jam with moderate shelf life.

Sodium benzoate (SB) and potassium metabisulphite (PMS) are commonly used as preservatives for long term storage of fruit pulp because of their better antimicrobial activity [3]. The maximum level for the use of these chemicals in fruit preservation including pulp and purees as described in the Codex Standards adopted in 2001 and 2006 are 1000 mg/kg SB as benzoic acid and 500 mg/kg PMS as residual SO2 [4]. Keeping in view these facts, this study was undertaken to find out the inhibitory effect of SB, SMS and both in different ratio with varying concentrations for microbial, chemical, physical and sensory quality of PFP stored at room temperature.

The aim of this study was to extend the shelf-life of the PFP by determining the best proportions of food additives like sodium benzoate and sodium metabisulphite (SB, SMS

and both in combination) to be applied for preservation of PFP at room temperature (30ºC). If storage of pulp can be improved for a long period, both PFP and its based food products will increase earnings in the Sri Lanka domestic and foreign markets.

2.       Material and methods

This research was approved by Research and Development division of Palmyrah Development Board.

2.1   Determination of Microbial Count

The method of Sri Lankan Standard: 516 Part 1: 1991 [5] was used.

Preparation of Nutrient Agar Plates

Plate Count Agar (PCA) HIMEDIA Laboratory Pvt. Ltd medium (2.35g) in a 250ml conical flask was dissolved in 40ml of distilled water by heating in a water bath, made up the volume to 100ml with the same, plugged with cotton wool well, sterilized at 121oC and 15lb in-2 pressure for 15 min and then allowed to cool to 45oC.

Serial dilution

Sample (10g) was transferred into a labeled sterile dilution bottle, made up the volume to 100ml with peptone (HIMEDIA) water (peptone 1g, NaCl (HIMEDIA) 8.5g made up to1000ml with water) under the sterile condition. Aliquots of it were taken after the thorough mixing by using vortex mixer (VELP SCIENTIFICA ZX3) and repeated the same process to obtain required dilution.

Microbial Count

Diluted sample (1ml) was transferred into each sterile plate. 20ml portion of the medium was poured to each plate in the laminar flow chamber (BIOBASE), mixed gently and allowed it to cool at room temperature. Plates of different dilutions were incubated at 37°C for 48hrs and the appeared colonies in the plates were counted and the total colonies were calculated. This experiment was repeated twice and the mean values (CFU/g) of these were calculated.

2.2   Physicochemical analysis

Total Soluble Solids (TSS)

Total soluble solids (TSS) of each sample were determined directly by using Refractometer (HSR500, Japan) at room temperature and expressed in terms of oBrix value.

Acidity

The method of SLS: 730:2010 [6] was used. The acidity of the given sample was determined as citric acid (%w/w) by titrating 10ml of sample against 0.1 N NaOH (SIGMA) using phenolphthalein (SIGMA) as an indicator.

pH

Homogenized sample (25ml) was taken in a clean beaker (25ml) and its pH was measured by using a digital pH meter (Sension PH 31-Spain) at room temperature.

2.3   Sensory evaluation

The method described by Larmond (1977) [7] was used. Selected sample was evaluated by a panel of judges from Palmyrah Research Institute staff with Oral Consent Scripts for sensory characteristics like colour, flavour, texture, mouth feel and over all acceptability. The judges were provided with prescribed questionnaires to record their observation. The information contained on the performance was 5 = Like very much; 4 = Like slightly; 3 = Neither like nor dislike; 2 = Dislike slightly; 1 = Dislike very much. The panelists expectorated the sample and rinsed mouth using distilled water between samples.

2.4   Preparation of PFP

Well ripened Palmyrah fruits available in plenty in their season at Kaithady, Northern Region of SriLanka, were washed twice with potable water and their tepals (tops) were removed then again washed with potable water and dipped in hot water for few seconds. Then ectocarp (skin) was peeled manually, the remainder (nutlets) was macerated with warm water (nutlet: water in ml = 1:100). Diluted pulp (PFP) was extracted manually using sieve after the removal of seeds and insoluble fibres.

Adjustment of pH

Initial pH of the PFP was measured with pH meter (Sension+ PH 31-Spain) and then the pH of the pulp was adjusted to proper pH 3.8 with concentrated solution of food grade commercially available citric acid and mixed well.

Blending

Acidified pulp was blended by using electric blender at low speed for 5 min.

2.5. Effect of heating at 80oC for 30min Pasteurization in the preservation of PFP PFP (pH3.8) poured into the capped clear glass bottle (100ml, without the addition of preservatives)                          was heated in a thermostatic water bath at 80oC for 30 minutes and then allowed to cool to room temperature [8,9] and stored for a period of 150 days. Aliquots of them were taken for the analysis.

2.6 Effect of preservatives in the preservation of PFP

Common preservative Sodium Benzoate and Sodium metabisulphate (Food Grade) available in local market were used. The PFP was heated in a thermostatic water bath GEMMYCO at 90oC for 20 sec., preservatives were added according to the TABLE 1, mixed well and they were transferred into clear sterile glass bottles separately and capped well. The bottled pulp was heated in a thermostatic water bath at 80oC for 30 min and then allowed to cool to room temperature [8,9]. They were stored at room temperature for a period of 180 days. Aliquots of them were taken in 30 days interval for the analysis.

Statistical analysis

Results obtained from chemical analysis (pH, brix and acidity) with three replicate were subjected to three way ANOVA. The significant difference among the treatments was tested in Least Significant Difference (LSD) at 5 % level of significance using SAS (version

9) System software.

Friedman non-parametric statistical method was used to analyze the sensory evaluation data based on 5-point hedonic scales. In this data analysis 95% confidence interval was considered, and analysis was done using Minitab 13 software.

Table 1: Concentrations of different preservatives used in preservation of PFP, alone or their combination at varying ratio

TreatmentsSB%, (w/v)SMS%, (w/v)
T10.04
T20.08
T30.12
T40.16
T50.2
T60.03
T70.04
T80.05
T90.06
T100.07
T110.02**0.015**
T120.040.02
T130.060.025
T140.080.03
T150.10.035

**Half of the concentration of each preservative used alone before was used together here.

3.       Results and Discussion

Palmyrah fruit and their products have gained considerable importance by contributing significantly to the economy of Sri Lanka. On the other hand freshly extracted pulp is highly attractive in appearance and possesses good taste and aroma, but it deteriorates rapidly in 24h. This is mainly due to fermentation caused by moulds, yeasts and bacteria. The enzymes secreted by them may affect the colour and flavour adversely. Chemicals present in the pulp may react with one another and spoil its taste and aroma. Air coming in contact with the product may react with the glucosidal substances present in it. This deterioration must be avoided by application of the food preservation principle which first involves the prevention or delay of the microbial spoilage.

The present study was carried out to identify a suitable chemical preservative/s such as sodium benzoate, sodium metabisulphate either alone or in combination for satisfactory storage of PFP at room temperature. Efficiency of preservation and storage behavior of fruit pulp is depended on physicochemical characteristics such as acidity, pH and Total Soluble Solids (TSS) and biological parameters. Period of storage had shown a pronounced effect on physicochemical attributes of chemically preserved PFP.

3.1   Microbiological evaluation

Benzoic acid inhibits the growth of mold, yeast [10] and bacteria. It is either added directly or created from reactions with its sodium, potassium or calcium salt. The mechanism starts with the absorption of benzoic acid into the cell. If the intracellular pH changes to 5 or lower, the anaerobic fermentation of glucose through phosphofructokinase is decreased by 95%. The efficiency of benzoic acid or benzoate is thus depended on the pH of the food [11]. Sodium metabisulphite releases SO2 gas when added to water, SO2 kills yeasts, fungi and some bacteria and also it acts as an antioxidant.

.

Microbial analysis of fresh PFP showed that total palate count (TPC) at the initial time was 2×106cfu/g and also heat treated PFP at 800C (without the addition of chemical preservatives) was spoiled before 15 days whereas the pulp containing preservatives (T1- T15) exhibited no microbial growth up to 120 days period of storage. At 150 days of storage, the mean TPC was significantly increased from 0 to 9cfu/g for T5 to T1. A maximum mean value was recorded in T2 while minimum value was observed in T4. Treatments T6-T15 showed growth of microorganism at 180 days of storage but counts (cfu/g) were in the acceptable range given in SLS 730: 2010. Hence chemical preservatives decreased the microbial load significantly in PFP. These results are in accordance with the findings reported by Hussain et al., (2003) [12] and Hashmi et al., (2007) [13] for mango pulp.

3.2.   Physicochemical evaluation

3.2.1.  Acidity and pH

There were interaction between preservatives, concentrations and storage period for acidity values while except preservatives for pH. Significantly higher mean pH was observed for PFP treated with SB (4.32) when compared with SMS (4.24) and both SB , SMS (4.21) and there were significant different (p<0.05) between mean pH of the pulp with the storage period while which was decreased with period of storage. PFP treated with SMS (T6-T10) and both SB, SMS (T11-T15) showed less increase in pH compared with PFP treated with SB (T1-T5).This may be due to either utilization or neutralization of acidic compounds present in the pulp otherwise compound/s secreted by organism. This condition may facilitate organism/s to prolong their growth and thereby leads to deteriorate the pulp. Abbassi et al., (2009) [14] attributed the increase in pH and the decrease in titrable acidity with increased storage period of the mangoes.

The results relating to the increase in acidity and decrease in pH (Figure 1) during the storage of PFP are in complete agreement with other researchers [15]. pH plays dual role in the fruit juices by acting as a flavour promotion and preservation. Decrease in pH of the fruit pulp samples proportional to increase in acidity has been confirmed by several researchers and may be attributed to the presence of SB in the pulp samples [16, 17].

Fig 1: Change in pH and acidity of PFP incorporated with preservatives during the period of storage

Significantly higher mean acidity was observed for PFP treated with SB (0.88%) while no significance difference between pulp with SMS (0.79%) and both SB, SMS (0.80%).Change in acidity of PFP with the period of storage has been showed in Figure 1 while there were no significant different (p<0.05) between 60, 90 and 120 days of the storage. While that acidity was significantly increased from 30-180 days. PFP treated with SMS (T6-T10) and both SB, SMS (T11-T15) showed less increase in percentage of acidity compared with PFP treated with SB (T1-T5).The increase in acidity may be ascribed to rise in the concentration of weakly ionized acid and their salts during storage and also due to formation of acid by degradation of polysaccharides and oxidation of reducing sugars or by breakdown of pectin substances and uronic acid [19, 17]

3.2.2 Total Soluble Solids (TSS)

There were interaction between all factors such as preservatives, concentrations and storage period. Amin et al., (2008) reported the effect of time of fruit harvest affects the fruit quality. The variability in TSS in the PFP might be attributed to the alteration occurring in cell wall structure during ripening process. Moreover, various hydrolytic enzymes also affect complex carbohydrates changing them into smaller compounds. Significantly higher mean obrix was observed for PFP treated with both SB, SMS (13.00) when compared with SMS (11.02) and SB (12.15). TSS was significantly increased gradually up to a storage

period of 180 days (TABLE 2). While there were no significant difference between 60 and 60 also 120 and 150 days of storage. PFP treated with both SB, SMS (T11-T15) and SB (T1-T5) showed more increase in obrix compared with PFP treated with SMS (T6-T10) while there was significance different between treatments.

Table 2: Effect of storage on TSS of the PFP (oBrix)

Treatment sStorage (days)      
 306090120150180Mea n±SD
T111.8311.8411.8911.9511.9012.0011.90.06
T212.211.9612.0111.9811.9012.0012.010.09
T312.1712.0312.1112.4812.2512.5012.260.18
T412.2612.1312.1512.512.3512.6012.330.17
T512.4312.1712.1912.3812.2512.3012.290.09
T610.7910.9310.9710.9510.9811.0010.940.07
T710.8610.9010.9310.9810.9511.0010.940.05
T810.7710.8310.910.8910.7810.8010.830.05
T910.8610.9211.2611.4511.4011.5011.230.25
T1010.7710.8311.0911.4511.4011.5011.170.29
T1112.6512.912.9112.9512.9013.2012.920.16
T1212.8412.8512.8712.9012.8513.2012.920.13
T1312.8613.0312.2913.2012.9513.6012.990.39
T1412.7712.8513.0013.4012.9913.6013.100.3
T1512.7112.7813.0113.3013.2013.6013.100.31
Mean11.92 d11.93d11.97c12.18b12.07b12.29a  
±SD0.860.850.780.880.821.00  

Each value in the table is represented as mean ± SD (n = 3). Values in the mean row followed by a different letters (a-d) are significantly different (p< 0.05).

About half of the soluble sugars of PFP are mainly composed of fructose (3.4%), with about 6.6% sucrose and 3.5% glucose. The high sugar content of pulps from ripe fruits might be attributed to the transformation of starch into soluble sugars under the action of phosphorylase enzyme during ripening [19, 20] and water soluble pectin from insoluble proto pectin in lime squash and fruit bases, respectively [21, 22].

3.3 Sensory evaluation

Every fruit is selected by its visual appearance because colour of fruit is main attribute for judging the eatable quality of fruit and the same process is applied for the colour of PFP in this research. The values for colour of all the treated samples decreased during storage at ambient temperature. The PFP from various varieties collected from different production sites were not exactly at the similar ripening stage thus they may vary in colour and other sensory characteristics. Aina & Oladunjoye (1993) [23] reported that the colour change in mangoes is primarily associated with several biochemical changes, both degradation and synthesis of various classes of molecules including carotenoids in fruit.

A number of biochemical reactions or metabolic activities are involved in the ripening process of mango fruit such as increased respiration, ethylene production, change in structural polysaccharides causing softening, degradation of chlorophyll and synthesis of carotenoids, changes in carbohydrates or starch conversion into sugars, organic acids, lipids, phenolic compounds and a number of volatile compounds. All these changes lead to ripening of fruit with softening of texture to acceptable quality. These factors predominantly contribute towards developing a total sensory profile of the mango fruit [24].

The colour of T2 and T3 was spoiled and turned yellowish brown during 90 days of storage interval. Median values of colour score for T7 and T8 is high (44.5) when compared with other treatments while T3 showed very less score of median and also this median value decreased with increase concentration of preservatives (TABLE 3). Flavour is comprised of aroma and taste. The score for flavour decreased for PFP during storage at room temperature. Flavour score of T7 and T8 were higher than that of T12 and T13during 180 days of storage while the scores noted for T2 and T3 were very less. Score of overall acceptability for T2 and T3 was less than other treatments when compared with others and median overall acceptability score at initial time of storage was highest for T7 (Figure 2).

Table 3: Effect of selected treatments on median value of sensory analysis at 180 days of Storage

 FlavourColourMouth feelTextureOverall acceptability
T214.514.51719.515
T315.5131719.513
T74244.537.531.537.5
T84244.540.536.530
T1237.534394133.5
T1337.538.5384132.45

Organic acid and sugars ratio primarily creates a sense of taste which is perceived by specialized taste buds of the tongue. Thus, sweetness due to sugar and sourness from organic acids are dominant components in the mouth feel of many fruits [25]. But in PFP mouth feel is due to bitter compounds called flabelliferins which vary with many factors such as place at which palmyrah tree is grown, type of fruit and stage of  ripening at which that fruit is tested. These factors play a major role in the assessment of its sensory qualities and acceptability [26]. In this study, T7 had highest overall acceptability at initial and 180 days of storage therefore based on the sensory characteristics the T7 was recognized as relatively better than the selected treatments (Figure 2).

Fig 2: Effect of selected treatments on overall acceptability

4.       Conclusion

From this research, it is evident that storage of PFP incorporated with preservatives showed an increase in acidity and brix values besides the decreased level of microflora with time. However, according to the organoleptic evaluation done up to 180 days of period of storage PFP containing SB was rejected by panelists, whereas among the PFP containing SMS alone and combination of SB & SMS,  PFP with SMS (0.4 g/l)  was selected as better with respect to overall acceptability. Hence it is proved that pasteurization of PFP incorporated with SMS (0.4 g/l) at pH3.8 and 80oC for 30min is needed to store PFP for 6months without any loss of acceptable characteristics.

Competing interests

The authors declare that they have no competing interests.

Authors’ contribution

PN- made consultancy and revising the manuscript; RK carried out the research activities and revising the manuscript; SM carried out the research activities, statistical analysis and drafted the manuscript; SSV- coordinated & management of research activities. All authors read and approved the final manuscript.

Acknowledgements

The authors thank Ministry of Traditional Industries and Small Enterprise Development; Sri Lanka for the financial support and also would like to express their gratitude to all the staff of Palmyrah Research Institute, Jaffna, Sri Lanka, for their kind support and assistance in the project.

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International Journal of Scientific Research in Agricultural Sciences, 3(3), pp. 062-072, 2016

Comparative Study on Phytochemical and Antimicrobial Activity of Different Solvent Extracts of Pinattu

Srikantharasa Srishankar1, Subajini Mahilrajan*2, W.A.J.P. Wijesinghe 1 and Srithayalan Srivijeindran2

1 Uva Wellassa University, Sri Lanka

2Palmyrah Research Institute, Jaffna, Sri Lanka

Abstract

Palmyrah (Borassus flabellifer) fruit is mostly used as fresh fruit, because of its perishable nature it is traditionally preserved as dried fruit pulp as pinattu and constituents of crude extracts of pinattu were evaluated. Samples were collected from three different branches of Palmyrah Development Board and extracted with different solvents as aqueous, methanol, ethyl acetate and petroleum ether then concentrated extracts were used for the study. Alkaloids and tannins were not detectable in all the extracts while aqueous and methanol extracts gave positive results for carbohydrates, proteins, phytosterols, saponins, flavonoids, phenols and amino acids and fats and fixed oils. Spectroscopic determination of total phenolic, flavonoids and saponin content were significantly (p<0.05) different among the solvents and the highest amount was identified, in aqueous extract (19.92±0.5)mg/g, methanol extract (0.18±0.0)mg/g and methanol extract (509.88±4.18)mg/g respectively. Based on their diameter of the zone of inhibition least MIC of aqueous extract for Staphylococcus (1.4±0.1cm), E.coli (1.2±0.0cm), Pseudomonas (1±0.0cm) and methanol extract for E.coli (1.4±0.1cm) and Pseudomonas (1.1±0.00cm) was 0.5mg/ml. The Salmonella and Bacillus was showed 0.75mg/ml for aqueous extract while 0.25 and 1.0mg/ml for methanolic extract respectively. Klebsiella was showed 0.25mg/ml for both aqueous extract (1.1±0.0cm) and methanol extract (1.35±0.0cm). Highest inhibition zone was observed for 1 mg/ml of aqueous extract (2.15±0.2cm) in Protease when compared with positive control (1.9±0.1cm).This finding shows that crude aqueous and methanol extract of pinattu contains high amount of phytochemicals, exhibit significant antibacterial activity with relatively lower MIC (≤ 1mg/ml) when compared with ethyl acetate and petroleum extracts.

Key words: Antibacterial activity, Pinattu, Phytochemicals, Saponin and Solvents

1.       INTRODUCTION

Total phenol, total saponin and the total flavonoids content determination in palmyrah dried fruit pulp leather (pinattu) extracts is interesting helpfulness for global nutritionists due to their valuable effects on human and animal health. Thus, investigation of various phytochemicals present in palmyrah based product is the noble study in order to understand their health benefits.

Phenolics are one of the key secondary metabolites existing in the plant kingdom. They are low molecular weight compounds (mol. wt. <2000 amu) universally present in all tissues of higher plants and play an important role during the development of a plant. They have multiple biological effects, including antioxidant activity (Gulcin et al. 2005), anti- inflammatory activities and metal chelation properties (Rice-Evans et al. 1997).

Flavonoids the most common group of polyphenolic that are found universally in plants. These are widely distributed in plant achieving many functions. They are important in plant for normal growth development and defense against contamination and injury (Kähkönen et al. 1999), anti-aging (Hadnick et al. 1998), antioxidant (Croteau et al. 2000), antibacterial and antifungal activities (Hassan, 2010), anticancer, anti-cardiovascular disease and anti- inflammatory (Nijveldt et al. 2001).

Saponins are among several plant compounds which have beneficial effects. Among the various biological effects of saponins are antibacterial, anticancer (Mathers, 2002) and antiprotozoal (Avato, 2006). Saponins are surface-active glycosides with detergent, wetting, emulsifying, and foaming properties (Mitra and Dungan, 1997). The palmyrah fruit pulp contains pectin, sugar, carotenoids in addition to numerous steroidal saponins (flabelliferins) (Thabrew and Jansz, 2004). This was suspected to reduce weight gain in ICR mice (Ariyasena et al. 2002) and also inhibit the increase in blood glucose after a glucose intake, while improving the level of faecal glucose, thus suggesting an inhibition of intestinal glucose uptake (Uluwaduge et al. 2005).

Nowadays antibiotics are effectiveness against serious bacterial infections. However, only one third of the infectious diseases known have been treated from these synthetic products. This is because of the emergence of resistant pathogens due to the indiscriminate use, incessant and misuse of antibiotics. One of the methods to reduce the resistance to antibiotics is by using antibiotic resistance inhibitors from plants. Plants are known to

produce a variety of compounds to defend themselves against a variety of pathogens. It is expected that plant extracts showing target sites other than those used by antibiotics will be active against drug resistant pathogens (Sen and Batra, 2012). Palmyrah palm have been used as traditional treatments for numerous human. Hence, researchers have recently paid attention on biologically active compounds, isolated from plant species used in herbal medicines for the development of novel drugs and functional foods.

Although various studies have been reported on various plant extracts, while there was little research has been conducted on palmyrah palm. Therefore, this study was conducted to determine the amount of the total phenol, flavonoid and saponin content by using spectroscopic methods and antimicrobial activity of various extracts of palmyrah dried fruit pulp leather (pinattu).

2.       MATERIALS AND METHODS

This research proposal was approved by Research and Development division of Palmyrah Development Board and research committee of Uva Wellassa University (6.9826° N, 81.0768° E), Sri Lanka.

2.1   Collection of sample

Palmyrah fruit pulp leather was obtained from the three different branches of Palmyrah Development Board (PDB) then cut into small species and pool together. After that 100 g of sample was weighted in random manner.

2.2   Preparation of plant extracts

Palmyrah fruit pulp leather (100 g) was extracted in a soxhlet extractor for 24 hours with petroleum ether (boiling point 40-60oC, polarity index: 0.1), ethyl acetate (boiling point 76.5oC, polarity index: 4.3), methanol (boiling point 65oC, polarity index: 6.6) and water (polarity index: 9) separately based on polarity index. The extracts were evaporated under reduced pressure using rotatory evaporator (IKA), then extracts stored at 4 °C (Gulcin, 2005). All the extractions were performed in duplicate.

2.3   Qualitative evaluation of phytochemicals

Petroleum ether, ethyl acetate, aqueous and methanol extracts were tested for the presence of phytochemical constituents by performing the standard methods such as Mayer’s test, Dragen-dorff’s test and Wagner’s test for Alkaloids, Molisch’s test and Fehling’s test for

carbohydrates, foam test for saponins , Libermann-Burchard test and Salkowski’s test phytosterols, ferric chloride test for phenols., alkaline reagent test and lead acetate test for flavonoids, Ninhydrin test and Xanthoproteic test for protein and aminiacids, gelatin test for tannins, modified Borntrager’s test for glycosides and test for fat and oil.

2.4   Determination of total phenolic content

The total phenolic content of the pinattu extracts was determined using the Folin- Ciocalteu reagent (Maurya and Singh, 2010). The reaction mixture contained 0.5ml of diluted extracts, 2.5ml of freshly prepared 10 % diluted Folin-Ciocalteu reagent and 2 ml of 7.5% sodium carbonate. Mixtures were kept at ambient conditions for 30 min to complete the reaction. The absorbance at 760 nm was measured. Gallic acid was used as standard and the results were expressed as mg gallic acid (GAE)/g pinattu.

2.5   Determination of total flavonoid content

Total flavonoid content was determined using aluminium chloride (AlCl3) according to a known method (Ordonez et al. 2006) using quercetin as a standard. The plant extract (3 ml) was added to 5% NaNO2 (0.3 ml). After 5 min at room temperature, AlCl3 (0.3 ml, 10%) was added. After further 5 min, the reaction mixture was treated with 2 ml of 1 M NaOH and the absorbance was measured at 510 nm. The results were expressed as mg quercetin (QE)/g pinattu.

2.6   Determination of total saponin content

Total saponins contents in pinattu extracts were estimated (Hiai et al. 1976). Different extracts of 0.25ml of solution was taken and 0.25 ml of vanillin reagent (8%, w/v) was added. Then 2.5 ml of 72% (v/v) sulphuric acid was added slowly on the inner side of the wall. After mixing the content tubes were kept in a water bath at 60oC for 10 min then cooled in ice-cold water bath for 4min. Absorbance was measured at 544 nm using spectrophotometer against the reagent blank. Quillaja saponin was used as a standard and the content of total saponins was expressed as mg Quillaja saponin(QS) equivalents /g pinattu.

Statistical analysis

The results obtained from the four extracts with three replicate were subjected to analysis of variance by complete randomized design (CRD). The significant difference among the

extracts was tested in Least Significant Difference (LSD) at 5 % level of significance using SAS software.

3.       RESULTS

  • Qualitative evaluation of phytochemicals

Phytochemical screening in pinattu (Figure: 1) showed that all the selected tests gave positive results for any of the crude solvent extracts except tannin test (Table 1).

3.2   Quantitative evaluation of phytochemicals

Aqueous, methanol, ethyl acetate, and petroleum ether extracts were prepared to examine the total phenolic, flavonoid and total saponin content of the dried palmyrah fruit pulp by using spectrophotometry.

Total phenolic content was estimated by using Folin-Ciocalteu reagent. Total phenolic content of pinattu was dependent on different solvent extracts and expressed as milligrams of gallic acid equivalents (GAE) equivalent. Table summarizes that total phenolic compounds in solvent extracts varied widely, ranging from 0.011 and 19.92±0.42 mg/g expressed as gallic acid equivalents (GAE). Aqueous extract [19.92(±0.42)] exhibited significantly (p<0.05) highest total phenolic content than methanolic extract [5.74(±0.06)mg/g]. There were no significant different between the extracts of ethyl acetate and petroleum ether. The amount of total phenol obtained from these extract was very less when compared with aqueous extract.

The content of flavonoid expressed asquercetin equivalents. Pinattu extract obtained from water not quantifiable while methanol extract showed 0.18±0.00 mg/g of flavonoid.

Total saponin contents in pinattu (% w/w) expressed as Quillaja saponin equivalents. Which was significantly different with various extracts such as water, methanol, ethyl acetate and petroleum ether was 427.08(±7.84), 509.88(±4.18), 16.62(±0.39) and 34.08(±0.86) mg/g respectively. Methanol extract gave highest saponin content among the selected extract.

3.3   Antibacterial analysis

In the trail experiment of antibacterial activity assay, ethyl acetate extract of pinattu was not exhibited inhibition zone for all tested bacteria. It may be due to the low concentration

of the extract in the solvent. Therefore extracts of aqueous, methanol and petroleum ether obtained from pinattu was selected for antibacterial assay studies.

According to the two-way ANOVA results obtained for all test organisms showed p< 0.001 for both extract and concentration. Hence the main effects were highly significant at 99.9% probability level. Since p value for the interaction effect was found as< 0.001 so there is a significant interaction effect between extracts and concentrations showed at 99.9%

level.

According to the Table 3 all the test bacteria gave highest inhibition zone diameter for aqueous extract except Staphylococcus and Salmonella while these two bacteria showed highest inhibition zone diameter for methanol extract in the meantime petroleum ether extracts showed lowest diameter for all the tests bacteria.

The antibacterial activity of both extracts with different concentration was evaluated according to their diameter of the zone of inhibition against various bacteria and the results were compared with the activity of the standard (chloramphenicol (0.1mg/ml) and solvent, serve as positive and negative control respectively.

Least MIC of Staphylococcus was 0.5 mg/ml for aqueous extract however 0.25 mg/ml for methanol extract (Table 5). Inhibition zone of Staphylococcus was showed no significant different between 1mg/ml concentration of aqueous (1.85±0.07 cm) and methanol (1.85±

  • cm) extracts. Zone of inhibition from Staphylococcus in the concentration of 0.75 and

0.5 mg/ml methanolic extract were significantly higher than that of 0.5 mg/ml of aqueous extract. E.coli was showed same least MIC (0.5 mg/ml) for both aqueous and methanol extract. Inhibition zone of E.coli was showed significant different between the concentrations (1.0 mg/ml, 0.75 mg/ml, 0.5 mg/ml, 0.25 mg/ml) of aqueous and methanol extracts. Least MIC of Pseudomonas was 0.5 mg/ml for aqueous extract though 0.5 mg/ml for methanolic extract (Figure 2). Inhibition zone of Pseudomonas was showed no significant different between the positive and 1mg/ml [2.15± (0.07)] cm concentrations of aqueous extracts and 0.75 mg/ml and 1mg/ml [1.7± (0.00)] concentrations of methanolic extracts. Zone of inhibition for Salmonella were showed no significant different between the 1mg/ml concentration of aqueous [1.5± (0.00)] cm and 0.5mg/ml methanolic [1.5± (0.00)] cm extracts. Inhibition zone of Salmonella was showed significant different between the other concentrations of both extracts. Least MIC of Salmonella was 0.75 mg/ml for aqueous extract (Figure 3), however 0.25mg/ml for methanolic extract. MIC of

Klebsiella was 0.25 mg/ml for aqueous extract and methanol extract. Inhibition zone of klebsiella showed 1.6± (0.00), 2.0 ± (0.14) cm for 1mg/ml concentrations of aqueous and methanolic extract respectively. Protease was showed highest inhibition zone for 1mg/ml of aqueous extract when compared with positive control. There were was no significant different between the zones of inhibition obtained from Protease in the concentration of 1and 0.75 mg/ml methanolic extract and 0.75 mg/ml of aqueous extract and also no significant different between the 0.5mg/ml concentration of aqueous [1.35± (0.07)] and methanol [1.3± (0.00)]cm extracts. MIC of Protease was 0.25 mg/ml for aqueous extract and methanol extract. Least MIC of Bacillus was 0.75 mg/ml for aqueous extract whereas 1mg/ml for methanol extract. Zone of inhibition obtained from Bacillus for 1mg/ml concentration of aqueous and methanol extract was 1.55± (0.07), 1.45± (0.07) cm

respectively (Table 4).

4.       DISCUSSION

Manoharan et al. 2014 also reported that phytochemicals screening in the pinattu showed positive results for steroids, triterpenoids, carbohydrates, saponin, flavonoids and proteins in varied amounts in the ethanolic and aqueous extracts while chloroform extract showed negative results for all tested compounds except for carbohydrate while glycoloids, alkaloids and tannins were not observed in any of the extracts. Most of these results agree with our qualitative analysis of pinattu.

Analysis of the total phenol and flavonoids content in plants materials is attracting thoughtfulness for pharmaceutical universal due to their beneficial effects on health of human and animal. Which are rich in phenolics are gradually being used in the food industry because these bioactive compounds prevent the lipids oxidative degradation and improve the quality and nutritive value of foods (Kähkönen et al. 1999). Phenolic compounds are one of the phytochemicals considered as secondary metabolites and these compounds derived from phenylalanine and tyrosine occurs ubiquitously in plants and is diversified (Naczk and Shahidi, 2004). In this experiment aqueous extract contained high amount of total phenolic content because which is in the plant depends on the type of extract, i.e. the polarity of solvent which are used in extraction. The solubility of the phenols is high in polar solvents and delivers high concentration of these compounds in the crude extracts during the extraction (Mohsen and Ammar, 2008).Palmyrah pinattu contained very less amount of flavonoids when compared with other tested phytochemical. These are

probably the most important natural phenols and one of the most diverse and general group of bioactive natural compounds. These compounds contained a broad spectrum of chemical and biological activities including antioxidant properties of radical scavenging properties. Several studies reported that bioactive components concentrations are affected by plant species and plant variety (Shiraiwa et al. 1991), degree of maturity, growing environment, agronomic factors such as climate and soil, cultivation year, location grown, season (Oleszek, 1996), and extraction method (Onning, 1993). For example saponins frequently are isolated by boiling in methanol (Oleszek et al. 1992), ethanol (Oleszek, 1990) and n- butanol (Massiot, 1992).

In our study methanol and aqueous extract contained better antimicrobial activity. Duddukuri et al., 2011 (Duddukuri, 1992) also reported that the antibacterial activity of methanol extract of Borassus flabellifer L. seed coat (soft outer shell) and examined against Gram positive bacteria i.e., Bacillus subtilis, Staphylococcus aureus and Gram negative bacteria i.e., Klebsiella pneumonia and Serratia marcescens. This showed consistently significant inhibitory activity. Furthermore, the minimum inhibitory concentration was ranged between 100μg to 1 mg/ml implying the significance of antibacterial activity.

5.       CONCLUSION

This study was showed that among the four extracts, most of the biologically active phytochemicals were present in the aqueous and methanol extracts. Antibacterial activities with relatively lower MIC (≤ 1mg/ml) values, confirm that methanol and aqueous extracts of pinattu of Borassus flabellifer exhibit significant antibacterial activity when compared with other extracts. Therefore it could be considered as beneficial for further investigation on the palmyrah dried fruit pulp.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

WP- Internal supervisor of the research project; SM- External supervisor; SS- Undergraduate research student; SS & SM carried out the antimicrobial activity study,

phytochemical studies and carried out statistical analysis; SS, SM &WP – Drafted the manuscript; SSV- coordinated & management of research activities. All authors read and approved the final manuscript.

Acknowledgments

Authors would like to express their gratitude to the staff of Palmyrah Research Institute, Jaffna, Sri Lanka, for their kind support in the project also kind support given by Ms.K.Robika, is greatly appreciated.

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Table 1: Phytochemical evaluation for various solvent extracts of pinattu

PhytochemicalReagents usedResults
Test(test performed)Aqueous polarity index (9)Methanol (6.6)Petroleum either (4.3)Ethyl acetate (0.1)
Detection                    of alkaloidsMayer’s TestReddish brownYellow —–  —–  —–
Wagner’s TestReddish brownReddish brownReddish brownReddish brown
Detection                    of carbohydratesMolisch’s TestPurple color ring +++Purple color ring +++Purple color ring ++Purple color ring ++
Fehling’s TestReddish black precipitate +++Reddish black precipitate +++Blue —–Blue —–
After           heated and hydrolysis—-—-GreenGreen
Detection                    of saponinsFoam Test+++++—–—–
Detection                    of phytosterolsSalkowski’s Test+++++++
Libermann Burchard’s test+++++++++++
Detection of phenolsFerric Chloride TestBlack +Black +Yellow —–Yellow —–
Flavonoids testIn            Alkaline Reagent test++++—–—–
In Lead Acetate testBrown ++Orange ++—–—–
Proteins                   and Amino Acids testIn Xanthoproteic testReddish brown ++Reddish brown ++—–yellow +
In Biuret test:—–—–—–—–
Ninhydrin Test—–—–—–—–
Tannins testGelatin test—–—–—–—–
Fats and Fixed oilsFilter paper test+++++—–—–
Glycosides testBrontrager’s Reagent test—–—–++++

Table-2: Spectroscopic determination of total phenol, total flavonoids and total saponin content of various extracts of pinattu.

  Phytochemicals SolventTotal phenol (mg/g)Total flavonoid (mg/g)Total saponin (mg/g)
Water (aqueous)19.92(±0.42)a0.00427.08(±7.84)b
Methanol5.74(±0.06)b0.18±0.00509.88(±4.18)a
Ethyl acetate0.11(±0.00)cND16.62(±0.39)c
Petroleum ether0.01(±0.00)cND34.08(±0.86)d

Each value in the table is represented as mean ± SD (n = 6). Values in the same column followed by a different letter (a-d) are significantly different (p< 0.05), ND: Not determined.

Table 3: Effect of different solvent extracts of pinattu on inhibition zone (cm) of different bacterial species.

Solvent BacteriaAqueousMethanolPetroleum Ether
Staphylococcus0.94b1.30a0.22c
E.coli0.86a0.76b0.00c
Pseudomonas sp1.08a0.88b0.24c
Salmonella0.52b1.25a0.00c
Klebsiella1.37a1.08b0.00c
Protease1.24a1.14b0.00c
Bacillus sp0.53a0.29b0.00c

Each value in the table is represented as mean ± SD (n = 3). Values in the same rows followed by a different letter (a-c) are significantly different (p< 0.05).

Table: 4: Effect of different concentrations of various extracts on inhibition zone (cm)

Each value in the table is

represented as mean ± SD (n = 3). Values in the same rows followed by a different letter (a-i) are significantly different (p< 0.05). (- control): respective solvent, (+ control): chloramphenicol (0.1mg/ml)

Table 5: MIC of different crude extracts of pinattu against bacteria

    ExtractsStaphylococcusE.coliPseudomonas spSalmonellaKlebsiellaProteaseBacillus sp
Aqueous mg/ml0.50.50.50.750.250.250.75
Methanol mg/ml0.250.50.50.250.250.251.0

Each value in the table is represented as mean ± SD (n = 3). Values in the same rows followed by a different letter (a-c) are significantly different (p< 0.05).

Figure 1: Qualitative phytochemical screening of palmyrah fruit pulp leather or pinattu

Salmonella spp                        Klebsiella spp                            Staphylococcus spp

Escherichia coli                        Pseudomonas spp                      Protease spp

Bacillus spp

gure 2: Antimicrobial activity of methanol extract of Borassus flabellifer L. dried pulp or pinattu against different bacterial species: (-) control – 1; 0.25 mg/ml – 2; 0.5 mg/ml – 3; 0.75 mg/ml – 4;

2  

1.mg/ml – 5; (+) control -6

Salmonella spp                            Klebsiella spp                        Staphylococcus spp

ia coli                      Pseudomonas spp                          Protease spp

Description: I:\results of research\anty micro pic\12.JPG

Bacillus spp

Figure 3: Antimicrobial activity of aqueous extract of Borassus flabellifer L. dried pulp or pinattu against different bacterial species: (-) control – 1; 0.25 mg/ml – 2; 0.5 mg/ml – 3; 0.75 mg/ml – 4; 1.mg/ml – 5; (+) control – 6

Scholars Research Library Journal of Natural Products Plant Resources, 5 (2), pp. 37-42, 2015

Enhance the quality of palmyrah (Borrasus flabellifer) jaggery

Abstract

K. Velauthamurty1*, S. Mary2, G. Sashikesh1 and S. Srivijeindran2

1Department of Chemistry, University of Jaffna, Sri Lanka

2 Palmyrah Research Institute, Jaffna, Sri Lanka

Palmyrah (borrasusflabellifer) jaggery industry is one of the ancient and large cottage industries in palmyrah society and it is produced by concentrating the inflorescence sap of Palmyrah palm (Sweet Toddy) to a thick consistency. The palmyrah jaggery made in household level has bitter taste due to the presence of excess amount of lime with low purity. During the period of May 2013 to June 2013 four field experiments were carried out to optimize the amount of quick lime (CaO) for a suitable Palmyrah sweet sap collection in the areas of Jaffna district, using Randomized Complete Block design. This experiment was carried out with four different concentrations of quick lime such as 5.0, 3.0, 2.5 and

2.0 grams of CaO in liter of sap. The quick lime used in this study was produced from sea shells with 96% of purity at Palmyrah Research Institute laboratory. Existing palmyrah jaggery available in the market is inferior in its quality as liquefaction and deterioration of color. Through this research attention to be made to eradicate its hydroscopic nature in the existing jaggery to upgrade it. Physicochemical quality parameters of commercially available samples and produced jaggery samples were studied according to Sri Lankan standards for jaggery. Quality of jaggery was enhanced using sweet sap with acceptable lime with higher degree of purity. For suitable jaggery production, 2.508 ± 0.411 grams of lime (96 % purity) per one liter of sweet sap

were optimized and this jaggery was scored high score than jaggery from other field experiments.

1.       Introduction

Palmyrah (Borassus flabellifer) palms are mainly distributed in north and east provinces of Sri lanka and it is known to be a valuable economic plant [1]. There are varieties of palmyrah products available in the local and international market. Palmyrah leaves are used for thatching, mats, hats and different handicrafts, stalks are used to make fence and black timber is used in constructions.

The young, either male or female inflorescences of palmyrah palm produces the fresh sap. The fresh sap is a sweet clear watery liquid and it is a good source of vitamins: riboflavin, vitamin B12 vitamin C, thiamine and nicotinic acid and minerals: calcium, iron, zinc, copper and phosphorous [2]. Fresh sap plays a role in the indigenous medicine as anti- diabetic, anti-hyperglycemic and anti-hyperlipidemic agent [3]. This unfermented fresh sap is called as sweet sap or ‘pathaneer’ [4], it can be consumed freshly or prepare the sap based products. The sweet sap is also allowed to ferment to yield a mild alcoholic beverage which is called “toddy” [5] [6] [7].

The sweet sap based products as jaggery, treacle and sugar candy are made out by concentrating the sweet sap to the suitable consistency in open pans. Palmyrah palm jaggery is the main marketable product and which is more nutritious sap based product, containing 1.04 % protein, 0.19 % fat, 76.86% sucrose, 1.66% glucose, 3.15% total

minerals, 0.861 % calcium, 0.052 % phosphorus; also 11.01 mg iron per 100 g and 0.767 mg of copper per 100 g [2]. Jaggery, a sugar rich food product is produced all over the world under different names, such as Gur (India), Desi (Pakistan), Panela (Mexico and South America), Jaggery (Burma and African countries), Jaggery (Sri Lanka), Htanyet (Myanmar), Panocha (Philippines), Rapadura (Brazil), and Naam Taan Oi (Thailand) [8]. It is consumed directly or used for preparation of sweet confectionery items and ayurvedic/traditional medicines [9].

Fermentation of sweet sap is caused by micro organism and occurs during the period of collection right up to processing. The fermenting organisms are dominated by yeasts, particularly Saccharomyces cerevisiae [10]. Traditionally quick lime is used to prevent the

fermentation of sweet sap in the preparation of jaggery at the cottage industries in Jaffna peninsula [1, 10]. It is generally obtained from furnacing oysters (mostly sea shell used in Jaffna peninsula) using coconut husk charcoal and the temperature is not up to the optimum level to produce pure lime [REF].Therefore, the quick lime shows a high possible for adulteration by impurities such as ash, sand, unheated sea shells. Another major issue is the local tappers are using excess amount lime which present in sweet  toddy.  However, there are no studies carried out until now about the composition and purity of quick lime used in local production. Hence this study is to initiate the study on preserve quality for extends the shelf life of palm jaggery and to standardize and improve the techniques of production of jaggery. This leads to increase the local and export market demand for palmyrah jaggery.

2.       MATERIALS AND METHODS

This research work mainly focuses on the physicochemical properties of jaggery samples and enhance their quality to the international grade via purified and optimized the quick lime

Determine the physicochemical properties of Sweet sap and Jaggery samples Physicochemical properties of commercially available sweet sap and jaggery samples, which are collected from different areas in Jaffna district, were analyzed for this study and the physicochemical properties of the jaggery samples were compared with Sri Lankan Standards for jaggery [11].

Evaluate the purity of quick lime obtained from the traditional kiln

The quick lime samples  were  randomly collected  from two  different traditional kilns  in Anaikoddai area and Pandaitharippu area. These collected quick lime samples were cooled down to the room temperature and packed in a moisture proof container [12]. Moisture free quick lime samples were taken on a clean surface to make cone and it was quartered to get a representative sample of small fragments. 100 − 200 g of this moisture free quick lime sample fragment was ground well via using the pestle and mortar and it passes through a No. 7 mesh sieve. 0.50 g of sieved quick lime sample was transfer into the 300 mL Erlenmeyer flask containing 20 mL of CO free distilled water and plugged the flask immediately. The mixture was swirled and boiled for 2 minutes. Then 150

mL of distilled water and 15 g of sucrose were added to the above mixture. The flask was shaken well for 5 minutes and allowed to stand for 30 – 60  minutes.  Then  it  was titrated with the standard HCl  solution  by  using  the phenolphthalein as an indicator  and finally the purity of quick lime samples were estimated.

Prepare the optimum amount of purified quick lime

Naturally occurring sea shell were randomly collected in the coastal area. Two sets of 10 g of sea shell samples were taken into the crucible plates and kept into the furnace at 750

⁰C for 10 minutes and 20 minutes. The above steps were repeated again for the various temperatures such as 850 ⁰C, 950 ⁰C and 1000 ⁰C. Then the purity of the

produced lime samples was analyzed and find out the optimum purity of quick lime

Produce the quality improved sweet sap

Matured and healthy female palmyah palms were selected near jaggery producing areas of Jaffna peninsula in the period of May to June. The well prepared and healthy inflorescences were identified for the sap collection. The selected inflorescences were tapped in the evening between 5 to 6 p.m to ooze out sweet sap  and  these  were  collected in to the well hygienic quick lime applied earthen pots. The known amount of 96

% of purified quick lime was applied into the earthen pots to prevent the fermentation under the previous experience of the local tappers. The different amount of quick lime such as 5

/ , 3 g/L, 2.5 g/L and 2 g/L were applied into the sweet sap collection which are indicate as a field trial such as T1, T2, T3 and T4 respectively

The nylon covering nets were used on pots to avoid the fall/entry of ants, insects and spiders during the collection period of sweet sap. The sixteen hours later, the collected sweet sap samples in earthen pots were quickly transferred into the separate high molecular high density polyethylene (HMHDPE) bottles. Initially, these bottles were washed thoroughly with boiled distilled water, drain out completely and capped immediately to avoid microbial contamination. The sample bottles were placed into a cooler with ice to maintain the temperature below 5 ⁰C and this was immediately transferred to the laboratory in aseptic and sterile condition.

The pH of the sweet sap in each sample bottles was measured directly by using the pH meter (HATH) and Brix was measured by using the hand refractometer. The collected sweet sap samples from various earthen pots in same field trials were pooled together and measured the pH value of this pool sample. Then this pooled sweet sap was transferred into aluminum pan and heated to 120 C to prepare jaggery. These prepared jaggery samples were packed in high density polyethylene (HDPE) bags under vacuumized condition and jaggery samples were labeled as A, B, C and D which are produced from the field trials T1, T2, T3 and T4 respectively. The produced jaggery samples from various field trials were carried out to select best jaggery samples on the basis of sensory attributes as the colour, odor, appearance, flavor and texture. For this sensory evaluation, the five points hedonic scale was used to select the total acceptability of the samples and it were statistically analyzed using the MINITAB statistical analysis package according to the Friedman nonparametric test at 5 % confidence level of significance [12].

3.       RESULTS AND DISCUSSION

The study of quality characteristic of marketable sweet sap

The most significant quality characteristic of the commercially available sweet sap samples, which is obtained from different areas, were analyzed and tabulated in the Table

  1. This research study clearly indicates that the marketable

available sweet saps have shown higher pH, pale yellow in colour and bitter taste due to the presence of excess amount of quick lime. This unfavorable colour and taste have given unpleasant condition to produce jaggery without deliming treatment. Personal interviews taken from local tappers during our field visits is clearly notified that they haven’t any scientifically knowledge about the amount of lime which is adequate to prevent the microbial activities during the collection of sweet sap.

The minimum amount of quick lime addition at higher degree of purity will ensure that the optimum alkaline pH of the sweet toddy.

Table 1: pH, brix and colour of the commercially available samples

PlacepHBrixColourTaste
Pandaiththarippu11.2011.5WhiteBitter
Pandaiththarippu11.1411.0WhiteBitter
Chavakacheri11.7113.5Pale yellowBitter
Chavakacheri11.7012.5WhiteBitter
Atchuveli11.2011.4WhiteBitter
Atchuveli11.6212.5Pale yellowBitter
Atchuveli11.2311.0WhiteBitter
Kondavil11.6813.2Pale yellowBitter
Kondavil11.5512.6Pale yellowBitter
Allarai11.1212.4WhiteBitter
Allarai11.5913.0Pale yellowBitter

Study the chemical constituents of marketable Jaggery

The moisture content, total ash, acid soluble ash, matter insoluble in water and reducing sugars of the marketable jaggery samples obey the limits of specification for jaggery according to the Sri Lankan standards but sugars and non reducing sugar content is exceed the SLS standard level [12]. The most significant chemical constituents for the jaggery samples are listed down in table 2.

Table 2: Chemical constituents of the market available Jaggery

  Sample NoMoisture content percentTotal             ash percent by massAcid insoluble ash percent byMatter insoluble in water percent by mass (W/W)Reducing sugars percent by                  massSugars,    non                – reducing sugars percent by mass
16.78 %1.83 %0.11 %1.23 %6.10 %76.80 %
23.39 %1.45 %0.12 %1.65 %2.32 %91.09 %
36.79 %2.65 %0.20 %1.43 %2.16 %88.04 %
47.48 %2.62 %0.30 %1.65 %2.11 %81.20 %
57.07 %2.45 %0.13 %1.65 %5.79 %82.65 %
67.73 %1.80 %0.11 %1.23 %2.25 %85.61 %
SLS10 % (max)3.5                %0.5% (max)2 % (max)13% (max)70 % (max)

The moisture in the products exceeded the SLS limits at ambient temperature before their expiry period, is tabulated in table 3. Therefore, jaggery samples start to melt before the expired.

Table 3: The moisture content in the commercially available jaggery samples

SampleMoisture content percent byMoisture content percent by mass
17.7223.16
28.4321.82
38.1922.89

Study the purity of commercially available quick lime

This research study clearly point out that the commercially available quick lime which is collected from Anaikoddai kilns shown slightly higher purity rather than the Pandaitharippu  kilns.  However  these  two.kilns are not produce high purified quick lime, which is, tabulated in table 4. This impurity arises from the traditional kiln due to the following drawbacks such as (a) the kiln totally opens to environment and using coconut husk to heat sea shells. The important reaction occurs in the lime producing kiln take place at the optimum temperature (900 oC) which is the calcining of limestone.

CaCO3(s) →CaO(s) + CO2(g)

However, this heating process cannot be reached the actual temperature of above 900 0C [14]. (b) The sea shells are packed in several layers and between these layers coconut husk are spreaded into the traditional kiln. Therefore, there are the possibilities for adulteration by impurities as soil and coal present in quick lime. According to the results from the research work and the above drawbacks, traditional kiln should be modified in the future to produce a highly purified quick lime.

Table 4: The purity of commercially available quick lime

PlacePurity of lime expressed as CaO (%)
Anaikoddai59.99 ± 0.97
Pandaitharippu42.34 ± 0.74

Study the purity of quick lime in the laboratory scale

A various yield percentage of quick lime was produced as a function of temperature and time. In this study, 800 ⁰C is not enough to produce the lime above 90 % of purity. Other temperature treatments are selected to optimize the time. We can get lime with 96 % of purity in above the 800 ⁰C. Even the purity of lime is increasing with increased the time period and the temperature. Finally, 850 0C for 30 minutes treatment was selected as a best consumable method to produce lime with minimal amount of energy required.

Figure 1: The percentage of quick lime as a function of the various temperature for the treatment of 30 minutes (…………………………………………….. line), 20 minutes (—– line) and 10 minutes (………………………………………………………….. line).

Collection of sweet sap

Different field visits were carry out to optimize the lime for the processing of palm jaggery and all the summarized results were recorded in the table 10. In field visit 1 (T/ ), field visit 2 (T ), field visit 3 (T( ) and field visit 4 (T0 ) amount of applied quick lime were 4.668 ± 0.847 g/L, 3.188 ± 0.242 g/L, 2.508 ± 0.411 g/L and 2.051 ± 0.833 g/L and pH of them were 11.51,  10.62, 9.43 and 8.23 respectively. According to Sri Lankan Standard [1] fresh jaggery samples from T/ , T    and T( )  field visits at the time of packaging moisture content of all was below 10

%. Sweet sap samples with pH between 7 and 8 quickly changed into acidic medium with time and this indicates the microbial activity is present considerably. Jaggery from field visits T0 shows melting property and moisture content was below12.5 %. It was identified that fermented sweet sap also course melting of jaggery. Therefore pH of sweet sap needs to be maintained above pH 9 in large scale application. For the suitable jaggery production without deliming step

2.508 ± 0.411 g of lime (96 % purity) per one liter of sweet sap was optimized.

Table 10: Summary of all field visits

Filed visitName of the inflorescenceAmount of quick lime (g/L)pH of PooledBrix of
T1F1, F2, F4 & F54.668 ± 0.84711.5112.8
T2F6, F7, F8 & F93.188 ± 0.24210.6211.72
T3F11, F12, F13, F14 & F152.508 ± 0.4119.4311.63
T4F16, F17, F18, F19, F20, F21, F222.051 ± 0.8338.2311.00

Sensory evaluation of produced jaggery

Sensory data obtained through the five point hedonic evaluating test, revealed that there were significance difference in colour (p = 0.012), appearance (p = 0.003) and texture (p = 0.001) characteristic among the jaggery samples produced in the laboratory. However there were no significant difference in flavour (p = 0.296) and mouth feel (p = 0.145) among the samples. The sample C gained the highest rank for the colour, appearance, and texture. Therefore the sample C was selected as the best sample.

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Table 11: sensory attributes of the jaggery

Sensory attributesP valueSum of the rankBest sample
ABCD
Colour0.01219.527.529.513.5C
Appearance0.00317.529.030.013.5C
Texture0.00117.028.531.513.0C
Flavour0.29618.027.524.520.0B
Mouth feel0.14515.024.027.024.0B

Evaluation of moisture content of prepared Jaggery samples

Moisture content of prepared jaggery samples were determined in different time periods as in first day, after first month and after sixth month. Jaggery from fourth field trial melted before one month and this indicates even the pH 8 is suitable to produce jaggery fermented sap causes the melting of jaggery easily. Jaggery produced from other field trials can be stored for six months.

Table 12: Moisture content of jaggery with average of triplicate readings after 6 months

Field trialsMoisture             content percent by massMoisture content percent by mass afterMoisture content percent by mass after
T13.523.728.69
T23.984.018.94
T34.124.879.45
T44.4325.65

Conclusion

Palmyrah jaggery is one of the most popular sweeteners in north and east provinces of Sri Lanka. The color of the sweet sap was improved with using the proper amounts of applied lime per liter.

Sweet sap should be maintain at pH 9 by using a purified quick lime to stop the fermentation and it can be useful to get quality improved jaggery as high consumer acceptance in the market level. At this optimum pH range jaggery can be produced without deliming step from fresh, unfermented and filtered sweet sap of palmyrah palm.

Acknowledgements

Authors like to thank the Department of Chemistry, University of Jaffna, and Palmyrah Research Institute for providing necessary facilities.

REFERENCES

  • K. Theivendirarajah, Palmyrah Palm, A Monograph, K. Theivendirarajah, Roxanne Crescent, scarborough, Ontario, Canada, 2008, 1, 27.
  • D. Barh, B.C. Mazumdar, Research Journal of Medicine and Medical Sciences, 2008, 3, 2, 173-176.
  • P. Goyal, A.K. Agarwal, Lakashminarasimhaiah, G.K. Singh, world journal of pharmacy and pharmaceutical sciences, 2014, 4, 1, 1172-1184.
  • J.F Morton, Economic Botany, 1988, 42(3): 420-441.
  • T.A. Davis, D.V. Johnson, Economic Botany, 1987, 41, 2, 247-266.
  • A, Kovoor, The palmyrah palm: potential and perspectives. FAO Plant Production and Protection Paper No 52. FAO, Rome, 1983, 77.
  • K.K, Bajpayee, Ethnobotany of Phoenix, Journal of Economic and Taxonomic Botany, 1997, 21, 1, 155-157.
  • A.K. Thakur, Potential of jaggery (Gur) manufacturing in Punjab state. In: Proceedings of the National Seminar on Status, Problems and Prospects of Jaggery and Khandsari Industry in India. Indian Institute of Sugarcane Research, Lucknow, 1999, 70–76.
  • P.K. Pattnayak, M.K. Misra, Biomass & Bioenergy, 2004, 26, 79–88.
  • P.C. Vengaiah, D. Ravindrababu, G.N. Murthy, K.R. Prasad, Indian Journal of Traditional Knowledge, 2013, 12, 4, 714- 717.
  • SLS 521: 1981, Specification for jaggery, Sri Lanka Standards Institution, Colombo 3, Sri Lanka, 1981, 8-20. [12] R.S. Boynton, Chemistry and Technology of Lime and Limestone, Second Edition, Interscience Publishers, New York, London, 1980.

[13] David J. Sheski, Handbook of Parametric and Nonparametric Statistical Procedures, CRC Press, third Edition, Aug 27, 2003.

[13] P. Atkins, T. Overton, J. Rourke, M. weller, F. Armstrong, Inorganic Chemistry, W. H. freeman and company, New York, Fifth Edition, 2010, 314.

International Journal of Multidisciplinary Studies (IJMS) 1 (1): 91-103,

2014

Optimization of Palmyrah (Borrasus flabellifer) Fruit Pulp in Different Varieties of Fruit Yogurts

S. Sangheetha1, M. A. J. Wansapala2, A. Gnanasharmala1, S. Srivijeindran1

1Palmyrah Research Institute, Kandy Road, Kaithady, Sri Lanka

2 Department of Food Science and Technology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka

ABSTRACT

The research focused on processing palmyrah fruit pulp into a value added product to broaden the utilization of palmyrah fruit pulp. Set, swiss style, jelly and pulp preserve yoghurts incorporated with palmyrah fruit pulp were developed. Control yoghurt and plain yoghurt for other preparations were prepared with 13 % sugar, 1% gelatin, 12 % skim milk powder and

lactic acid bacteria culture following household method. The fresh pulp was kept at – 25 oC for 48 hours and heated up to 85 oC for 30 minutes with 5 % cane sugar, 0.6 % tartaric acid, 1 % ascorbic acid and 0.13 % sodium chloride. The treated pulp was added at 5 %, 7.5 %, 10 %,

12.5 % and 15 % into both set and swiss style yoghurts. Fruit jelly was prepared with sugar, gelatin, sodium citrate, citric acid and 20 % pulp. It was then incorporated into yoghurt at 5 % and 6 %. Palmyrah fruit  pulp preserve was  prepared by  heating sugar, pectin  and  pulp (45. 8

%) until its brix reached 68.5 o and it was topped on set yoghurt at 5 %, 7.5 % and 10 %. Sensory evaluation for colour, odour, appearance, mouth feel and texture was conducted with 21 untrained panelists using 5 point hedonic scale and the optimized pulp concentrations for set, swiss style, jelly and preserve yoghurt were 5 %, 7.5 %, 6 % and 10 % respectively (p < 0.05). The overall sensory qualities of all palmyrah fruit yoghurts were rated as good to very good. There were no significant changes in sensory attributes, brix and pH in storage at 4 oC. Shelf life was 18 days for all products at 4 oC without any preservatives except set yoghurt as it showed separation of water from the third day of the preparation. Nutritional and microbiological qualities of the products were investigated and compared with control yoghurt. Protein was higher in swiss style (6.12 %) and jelly (7.77 %) yoghurts. Carbohydrate was higher in swiss style yoghurt (36.87 %) and preserve yoghurt contained highest fat content (2.57

%).

Keywords: Carbohydrate, fruit yoghurt, palmyrah fruit, pulp, protein

1.  INTRODUCTION

Palmyrah (Borrasus flabellifer) possess a great capacity to yield several products of economic importance and hence it is  called  “Wishing tree” which means a palm that yields anything  and everything. Almost every part of the palm is utilized but most of the products from the palm are made by traditional methods known from the time immemorial.

It is estimated from the statistical data of existing female palms in Sri Lanka  that,  about 20,000 tons of palmyrah fruit pulp is available annually  during  fruit  season  but  around 10000 tons  of pulp is thrown in to nature or used for animal feeding (Jansz et al

2002) every year because its uses are limited mainly due to the presence  of  a  bitter  compound and lack of trials are done to process into various consumer attractive value added products.

Moreover, palmyrah fruit pulp is nutritious and has a yellow colour due to carotenoids which are precursors of vitamin A and therefore it has potential of being as  a source  of vitamin A and giving attractive yellow colour to foods. In addition to that, it is revealed that pulp is rich in vitamin C (ascorbic acid) and a good source of pectin which could be used to process the fruits into various products (Theivendrarajah, 2008)

With regard to palmyrah plantation, it is reported that currently 24.260 hectares equivalent with 11 million palms are available of which 3.5 million trees are in Jaffna, 3.5 million in Kilinochchi, 3 million in Mannar and balance scattered all over the country (Palmyrah development board 2010). Government’s Mahinda Chintana 10 – year plan aims to

increase the current level of palms from 11 million to 16 million by 2016 and the vision of it for the future strongly emphasizes on the need to promote palmyrah based products including its fruits. Also the government has given high priority for dairy development (Ministry of livestock and rural community development, 2010). Currently major part of milk production is done by farmers domestically and their life is as same as that of palmyrah dependents.

The above facts generated the idea to produce a value added consumer attractive product using palmyrah fruit pulp and milk. As a result the research was conducted with the aims of developing formulae for different kinds of palmyrah fruit yoghurts, analyzing nutrient contents of the yoghurts and determining shelf lives of them. The research, when industrialized will give the benefits to the palmyrah dependents of approximately 1/3rd of the population of Northern and Eastern provinces where palmyrah occurs extensively by increasing the marketing and utilization of palmyrah fruit pulp, minimizing the wastage of a nutritive resource and under exploitation of the health aspects of pulp and employment opportunities to produce novel product for consumers.

2.  BACKGROUND

Yoghurt is a healthy and delicious fermented milk product due to its nutritive and therapeutic value with a custard-like consistency which differentiates it from other fermented milk products (Perdigon et al 2002). The Codex Alimentarious Commission (2008) defines it as a coagulated milk product obtained by lactic acid fermentation through the action of Lactobacillus bulgaricus and Streptococcus

thermophilus from milk with or without additions and the microorganisms in the final product must be viable and abundant.

Yoghurt is commonly considered as safer product and nutritionists are being considering incorporating inexpensive source of nutrient to make it an almost complete food (Boghra and Mathur 2002). Yoghurt is easily digestible and palatable than milk as it contains less lactose. Further, it is treasured for controlling the growth of bacteria and in curing of intestinal disease, for instance, constipation and diarrhea, lowering of blood cholesterol and anti-carcinogenic effect (Kamruzzaman et al

2002). Yoghurt is a great source of B vitamins, phosphorus and calcium. Organoleptic properties such as flavour, texture and aroma of yoghurt depend upon so many factors including the raw materials, manufacturing process and the bacterial strains used (Kumar and Mishra 2004).

According to the Sri Lankan Standard for yoghurts (SLS 824 Part 2:1989) a standard yoghurt should be smooth, glossy surface, no crack or holes on the top, no whey synersis, no off flavor or odor and clean layer on the surface of the yoghurt. It also says that yoghurt must contain minimum of 3.0 % fat whilst it categorizes the yoghurt that contains between

0.5 % and 3.0 % fat as low fat yoghurt and that with less than 0.5 % fat is non-fat yoghurt.

Palmyrah fruit pulp has bitter taste due to flabelliferins and it gives a negative impact on utilization of pulp. Despite that from the point of health, palmyrah fruit pulp has numerous benefits to us. It is now proved that flabelliferins lowers the glucose absorption into blood,

increase the cardiac anti oxidant activity and decrease the blood cholesterol. Recent studies show that pulp is rich in provitamin A (32 ppm), vitamin C (285  ppm)  (Jeyaratnam 1986)  and contains lycopene as well (Pathberiya and Chandrika 2003). Lycopene is an anti oxidant which is beneficial in cardiovascular ailments and cancer.

3.  MATERIALS AND METHODOLOGY

  • Materials

Fresh milk was procured from Yarlco, the only dairy production unit in Jaffna peninsula and cream separation was done there using their cream separator. Other ingredients were collected from Jaffna market. Palmyrah fruits were collected from Kopay and Navatkuli.

Reagents used for chemical and microbiological analysis of the developed products were purchased from Sigma Aldrich. All glassware were obtained from ISO certified companies in United Kingdom and Germany and the  equipment  used  for  the  studies  were caliberated  and made in United Stated of America and Germany.

3.2   Quality tests and preparation of raw materials

Selection and quality of raw materials are the major determinants of the quality of end  products in a processing. Therefore raw materials are analyzed prior to be subjected to the processing to ensure the production of good quality end product. In the study, raw milk was subjected to clot boiling test with 68 % alcohol according to COMESA/East African Standards. Also the pH, titrable acidity as lactic acid, total soluble solid, fat and solid non fat of milk were determined. It was boiled up to 80 oC for 15 minutes.

Pest attack free, fresh, black skinned, well ripe fruits were selected to the study. The nutlets were dipped in warm water (45 – 50 oC) for few seconds and then macerated. The ratio of pulp: added water was 1:0.5 (v/v). Pulp was then strained through a muslin cloth. It was kept in deep freezer at -25 oC for about 48 hours.

Palmyrah fruit pulp was treated with 5 % sugar, 0.6 % tartaric acid, 1 % ascorbic acid,

0.2 % citric acid, 0.13 % sodium chloride and 1 % gelatin and the mixture was heated up  to  85 oC for 30 minutes to incorparate into set and swiss style palmyrah fruit pulp yoghurts.

For jelly yoghurt, palmyrah fruit pulp jelly was prepared with sugar (84 %), gelatin (11.5 %), tartaric acid (1.06 %), sodium citrate (0.18 %), water and 20 % pulp following the house hold method. Sugar (53 %) and pectin (0.35 %) were heated together with the pulp (45. 8 %) were heated until brix reached 68.5 o to prepare fruit preserve for fruit preserve yoghurt. The preserve was prepared freshy in each preparation, so that it can eaisly be poured on the yogurt easily before it gets set.

3.3 Preparation of yoghurts

For the plain yoghurt for all four products, 13 % sugar, 1 % gelatin and 12 % skim milk powder were added and the amounts of them were same in all products. The house hold method for yogurt production was used to prepare yoghurt using lacitic acid bacteira culture procured from Jamma. The preparation of yoghurt, its processing conditions and ingredients were kept same in the preparation of all products.

Optimization of pulp in set and swiss style yoghurts Five recipes in each category of yoghurts were developed for palmyrah fruit pulp incorporated set fruit yoghurt and swiss style yoghurt (Table 1).

TABLE 1: Optimization of fruit pulp concentrations  in  palmyrah  fruit  pulp  set yoghurt   and swiss style yoghurt

 Treatments
12345
Treated     fruit              pulp amounts (g 100 ml-1)        5.00        7.50        10.00        12.50        15.00

Optimization of pulp as jelly in fruit jelly yoghurt Two recipes  of  palmyrah  fruit  pulp jelly yogurts were prepared by blending the jelly in different percentages (Table 2). All ingredients except jelly were kept constant in all blends.

TABLE 2: Optimization of palmyrah fruit pulp jelly in swiss style yoghurt

  TreatmentPulp in the jelly added to the product (g 100 ml-1)
15.0
26.0

Optimization of pulp as preserve in fruit preserve yoghurt

Three recipes of palmyrah fruit pulp preserve incorporated set yoghurts were prepared. Table 3 shows the percentage of palmyrah fruit pulp preserve added in each treatment. The preserve was poured on the set yogurt. All ingredients except fruit pulp preserve were kept constant in all blends.

TABLE 3: Optimization of palmyrah fruit preserve in palmyrah fruit preserve yoghurt

  TreatmentPulp in the product (g 100ml-1)
15.0
27.5
310.0

Selection of the formula of each variety of prepared yoghurts

The formula in each variety of yoghurts which gained most consumer preference was selected via sensory evaluation conducted by 21 untrained panelists using 5 point hedonic scale method and analysis of variance was conducted on the sample means for flavor, colour, mouth feel, appearance,  texture  and   over   all acceptability. Statistically significant attributes were further analyzed to see where mean difference existed using Minitab (Friedman test) at 95 % confidence interval (P < 0.05).

3.3 Analysis of selected yoghurts

All selected products were subjected to the following tests and the results were then compared with that of control yoghurt.

Chemical analysis

pH, total soluble solids, and titrable  acidity were analyzed according to SLS 824: Part 2,  1989. Crude protein, moisture, crude fat, crude fibre, ash and total carbohydrate were determined in accordance with Association of Analytical Chemists Standard (2002). Same was done for commercially available plain yoghurt as control and compared with developed palmyrah fruit yoghurts.

Microbiological analysis

Total plate count and yeast and  mold  were tested for skim milk powder, sugar, milk, water and milk solution prior to the addition of culture. E. coli, yeast and mold in the selected products were assessed according to SLS 824

Part 2: 1989. Same was done for commercially available plain yoghurt.

Shelf life study

For the shelf life  evaluation  pH,  brix  and titrable acidity were measured once in in three  days for 21 days. At the same time, texture, appearance, colour, smell and air bubble  formation on the surface of the product were observed by experienced persons at the station where the research was carried out.

Experimental design and statistical analysis Friedman non parametric statistical method was used to analyze the results of sensory evaluation data at 95 % confidence level was considered.

4.   RESULTS EVALUATION AND DATA ANALYSIS

  • Quality tests and preparation of raw materials

The analysis showed that the raw milk met the quality requirements specified in SLS 824: Part 2: 1989 for yogurt preparation. In Table 4 the fat, solid non fat, density, pH and acidity of the purcehased milk are given in ranges because the milk was procured three times throughout the studies and the tests for fat, solid non fat and density of raw milk were carried out for each batch.

TABLE 4: Quality parameters (fat, solid  non fat, density, pH and acidity) of raw milk used for the study

Constituent of  Range
Fat0.02 – 0.03 g 100g-1
Solid non fat7.5 – 7.6 g 100 g-1
Density28.6 – 29.1
pH6.6 – 6.7
acidity0.14 – 0.17 g 100g-1

Clot boiling test is carried out to ensure the quality of milk. In the test poor quality milk will coagulate and fine particles of curd will be visible whereas the quality milk will not give so. When the acidity of milk exceeds 0.21% the milk get spoiled and is not good for the production. The raw milk used in the study did not clot in the alcohol test and the pH and

acidity were ranged 6.6 – 6.7 and 0.14 – 0.17 % respectively which fell into the acceptable ranges of pH and acidity for consumption.

Fruit pulp preparation

It would be better if some parameters such as penetration ability, pH and total soluble solids are standardized for the selection of good quality fruits as it will be more beneficial and easy to  the industries to select super grade fruits for the production. Palmyrah fruit pulp was collected and kept in at -25 oC till used in order to preserve and minimize the bitterness of pulp. Therefore the pH, brix and acidity were determined soon after the extraction only. The pH, total soluble solids and the acidity by means of ascorbic acid were 4.06, 15.2 % and 0.66 %. Acidity was expressed in terms of ascorbic acid as this is the dominant acid in the fruit. The results showed that the pulp was not spoiled.

Pulp has bitter principle which hinders its exploitation in food industries. However Jansz (2006) stated that the bitterness can be reduced by freezing beyond -20 oC. Therefore pulp was kept in deep freezer at -25 oC for about 48 hours to minimize its  bitter  taste  and the  bitterness of fresh pulp and frozen pulp was compared. It was experienced via tasting that the frozen pulp was less bitter than the fresh.

After the preparation of pulp for set and swiss style yoghurts, its texture, aroma and sweetness were improved and the colour remained as that of fresh pulp. Tartaric acid was added to reduce the pH closer to that of yoghurt. Citric acid and ascorbic acid were added as anti oxidants to prevent discolouration in the preparation. Sodium chloride was added as taste enhancer.

Palmyrah fruit pulp jelly prepared for the development of fruit jelly yoghurt was smooth and soft without any bubbles or foam on the top. The final pH of solution was 3.1 which is the most suitable pH for jelly to settlle off.

Palmyrah fFruit preserve was darker than the natural colour of  the  pulp  but  the texture was as same as that of jam. No air bubbles or sugar crystals formed and it was smooth and transparency.

4.2   Sensory evaluation of yoghurts

Friedman test in Minitab was used to analyze the results of sensory evaluation  to  select the most preferred blend of each type of palmyrah fruit yoghurt.

Palmyrah fruit pulp set yoghurt

The preference of each sensory attributes of all palmyrah set yoghurts developed in the study were rated from 5 to 1. Point 5 was given for like very much whilst point 1 was dislike very much.

TABLE 5: Sensory evaluation of set palmhyrah fruit yoghurt

Sensory attribut eMedian score for the products
175101115142107
Flavour  4a(45.5)  4a(44.0)3b (31.5)3b (29.0)3b (30.0)
Color4a (47.5)4a (49.0)3b(33.5)3b(25.5)3b (24.5)
Appeara nce4a (47.0)4a (52.5)3b(29.0)3b(26.5)3b (25.0)
Mouth feel4a (50.0)4a(49.0)3b(27.5)3b(26.5)3b (25.0)
Texture4a (52.5)4a (47.5)3b(31.0)3b(26.0)3b (23.0)

Results (Table 5) shows that the products 175 and 101 scored same median 4 for all analyzed sensory attributes and no significant difference existed in-between the products for each attributes. Products 115, 142 and 107 showed significant different between the other two and among them, and scored less than 175 and 101. To select the best product between 175 and 101 the sum of rank had to be considered as the medians were same for each attribute. Product 175  has  scored  higher  than the other for flavor, mouth feel and texture whereas  the   product

101    scored    higher    for  colour  and  appearance.  It  would  be  rather  good  if  the overall

acceptability of the product was also analyzed. However as 175 scored higher for flavor,  mouth feel and texture which indeed play in the success of product product 175 which contained 5% fruit pulp was selected as best.

Palmyrah fruit pulp swiss style yoghurt

Colour, flavour, appearance, mouthfeel and texture of each blend was rated from 5 to 1 where 5 was assigned to like very much and 1 was to dislike very much.

TABLE 6: Sensory evaluation of palmyrah fruit swiss style yoghurt

Senso ryMedian score for the products
2126525242227
Flavour4a4a4a4a4 (37.5)
Color4a4a4a4a4 (37.5)
Appeara nc3b.854a.153bc.853c.653.75 (36.0)
Mouth feel3.9 (39.04a3.9 (35.03.9 (36.53 (30.0)
Texture4b (34.4.3 (44.4b (36.3 (31.3.9 (34.9)

Same alphabets on means in a row show non-significant differences at the confidence level    of 95%. Sum of ranks are given along with the medians in the brackets.

175– Set yoghurt with 5% fruit pulp
101– Set yoghurt with 7.5% fruit pulp
115– Set yoghurt with 10% fruit pulp
142– Set yoghurt with 12.5% fruit pulp
107– Set yoghurt with 15% fruit pulp

Same alphabets on means in a row show non-significant differences at the confidence level of 95%. Sum of ranks are given along with the medians in the brackets.

215 – Swiss style yoghurt with 5% fruit pulp

265 – Swiss style yoghurt with 7.5% fruit pulp

253 – Swiss style yoghurt with 10% fruit pulp

242  – Swiss style    yoghurt with 12.5% fruit pulp

227 – Swiss style yoghurt with 15% fruit pulp

The results of sensory evaluation (Table 6) revealed that the all products have scored the same median 4 for flavor, colour and mouth feel and no significant difference existed among them for each of those attributes. Product 265 scored the highest median and sum rank for appearance, mouth feel and texture. Also it was clear that product 265 scored the best for all sensory attributes. Therefore product 265 containing 7.5% fruit pulp was selected.

From the above results of both types of yoghurts, it could be noted that the selected swiss style yoghurt contained higher percent of pulp (7.5%) than set yoghurt (5%). Also the comments given by the panelists brought out that the consistency and appearance of  swiss style yoghurt were better than that of set yoghurt and bitterness could not be sensed in swiss style yoghurt although the pulp was higher in that. About two or three days after the production set yoghurt showed water separation and  the  appearance  became  down. Therefore  among the selected set and swiss style yoghurts, swiss style yoghurt with 7.5 % fruit pulp was selected for further studies.

Palmyrah fruit pulp jelly yoghurt

Sensory evaluation revealed that both products scored 4.5 median to flavor, colour and texture and product 307 scored less than product 321 for appearance and mouth feel. Also the sum of ranks of product 321 were higher than those of

307 for all attributes. Therefore product 321 was selected finally (Table 7).

TABLE 7: Sensory evaluation of palmyrah fruit jelly yoghurt

SensoryMedian score for the products
attributes321(5 % of pulp)307 (6 % of pulp)
Flavour4.5a (20.0)4.5a (19.0)
Coloraa
Appearanceab
Mouth feel4.5a (23.5)3.5b (15.5)
Texture4.5a (20.0)4.5a (19.0)

Means in the each row followed by the same letters are not significantly different at p < 0.05. Sum of ranks are given along with the medians in the brackets.

Palmyrah fruit pulp preserve yoghurt

The results of sensory evaluation brought out that product with 10 % of fruit pulp scored 4.5 median to flavor, colour and texture and mouth feel and the sum of ranks of product was higher than the other products with 5 % and 7.5 % fruit pulp and thus it (product with 10 % of fruit pulp) was selected.

4.3   Analysis of selected products

Chemical analysis – pH, Brix and acidity pH, acidity and total soluble solids of the all types  of yoghurts selected in the sensory evaluation were analyzed to determine the  chemical  quality of them (Table 8).

TABLE 8: Analysis of pH, acidity and total soluble solids of palmyrah  fruit  yoghurts selected in sensory evaluation

  Yoghurt  pH  soluble solidsAcidity
Palmyrah fruit set yoghurt4.4316.90.8 ± 0.01
Palmyrah fruit swiss style yoghurt4.3618.668 0.9 ± 0.03
Palmyrah fruit jelly yoghurt4.6021.30.97 ± 0.01

Time interval

SLS 824 Part 2: 1989 says that the acidity by means of lactic acid of standard yoghurt should be between 0.8 % and 1.25 %. Acidity of all products selected in the studiy was in between the limits specified in the standard. In contrast, the pH of the products were slightly higher than the optimum pH of

4.2 for coagulation. It may be due to the acidity of fruit pulp and the organic acids (citric acid, tartaric acid etc) added in preparation of pulp for the product developments.

pH and brix of yoghurts with storage

The results show that the pH and brix did not change much and they were in acceptable limits even after 18 days. Fruit jelly yoghurt showed the pH above 4.2. The change in pH and total soluble solids of each type of yoghurt were observed for 21 days (Figures 1, 2, 3 and 4).

Values are means of triplicates. Vertical bars indicate the standard errors.

FIGURE 1: Study on change of pH and brix of palmyrah fruit pulp swiss style yoghurt for 21 days

Values are means of triplicates. Vertical bars indicate the standard errors.

The milk solids content (including the fat content) for yoghurt ranges from around 9 % for low fat yoghurt to more than 20 % for certain types of concentrated yoghurt. Many commercial yoghurt products have milk solids contents of 14 – 15 % (Tamime and Robinson, 1999). The total solids content of milk can be increased by concentration processes, such as, evaporation under vacuum, and membrane processing (i.e., reverse osmosis and ultra filtration).

Values are means of triplicates. Vertical bars indicate the standard errors.

FIGURE 4: Study on change of pH and brix of palmyrah fruit set yoghurt for 21 days

Although the characterisitcs of palmyrah fruit pulp set yoghurt were in the limits, separation of whey and water was observed on the third day from the development .

Nutrient analysis of selected palmyrh fruit yoghurts in the study

Codex Alimentarious Regulations for yoghurt (2008) indicates that the minimum milk protein content is 2.7 % (except for concentrated yoghurt where the minimum protein content is

5.6 % after concentration) and the maximum fat content is 15 %. The results (Table 9) show that the fat content of all products did not exceed 15 % and were between 0.71 % and 2.47 %. Therefore, all palmyrah fruit pulp yoghurts can be categorized as low fat fruit yoghurts. The protein content of preserve yoghurt was lower than the limit as the preserve added for yoghurt was about 40 % and  thus  the  milk  in  the yoghurt  was  reduced  drastically  and  the preserve contained almost 60 % of sugar. It would be more informative about the nutrient value and medicinal benefits of the products if the analysis of crude fibre, dietary fibre and vitamin A is carried out.

TABLE 9: Nutrient contents of developed palmyrah fruit pulp yoghurts

    NutrientAmount of nutrients (g 100g-1)
Swiss     style  Jelly yoghurtPreserv     e        fruitCommerci     al     yoghurt
  Protein6.12± 0.057.77±0.0 62.48±0.0 3  5.26±0.01
  Crude fat0.71± 0.010.90±0.0 52.47±0.0 3  0.80±0.05
  Moisture55.59± 0.366.48±0. 0261.38±0. 02  65.00±0.15
  Ash0.56 ±0.030.53±0.0 10.92±0.0 2  0.81±0.01
Acid insoluble ash  0.15±0. 02  0.46±0.0  0.29± 0.01  0.29±0.01
Carbohydra te + fibre36.87± 0.3723.86±0. 0932.46±0. 08  27.84±0.12

Palmyrah fruit pulp contains provitamin A (32 ppm), vitamin C (285 ppm) and lycopene as well (Mohanajayelauxmy

1986). Apart from the nutritional benefits of pulp, it was discovered that Flabelliferin II was preventing the absorption of glucose into the blood stream with no adverse reactions (Janz 2006). Further it has anti glycemic activities, anti microbial activities and hypocholestremic acivities which are benefits for our health. 10 – 12 % pectin in pulp decrease cholesterol  by  25 – 35 % by binding bile salts and cholesterol (Jansz 2006).

Microbiological analysis

According to SLS824 Part 2: 1989 E.coli, yeast and mold, lactic acid  bacteria,  and  Salmonella  should  be analyzed howbeit since the media was not available at the time when the research was carried Salmonella will be checked in future works of the research. Total plate count test was done for skim milk powder, sugar, milk and potable water according to

SLS 516 part 1, 1991. But bacterial growth was in acceptable range in potable water and bacterial growth was not observed in other ingredients. E. coli was absent in all products including control yoghurt. Yeast count of all products were not more than 1000 per gram and Mold did not exist in the selected products. Mostly the microbial contamination occurs during the process. Therefore implementation of hygienic practices during process will help reduce microbial contamination.

Organoleptic analysis.

Texture, flavor and appearance were observed one in three days to determine the shelf life. They remained as fresh throughout the period of 21 days. But since the pH and acidity went beyond accepted level after

18 days observations were not taken as it will not contribute to determine shelf life further.

Shelf life study

pH and brix were measured once in three days to determine the shelf life of products for 21 days. At the same time, appearance, colour, smell and texture were observed by experienced persons. As discussed above in pH and brix change, the shelf life period was determined as 18 days without any preservatives at 4 oC.

5.  CONCLUSION AND FURTHER WORK

The optimized palmyrah fruit pulp concentrations for its set yoghurt, swiss style yoghurt, jelly yoghurt and preserve yoghurt were 5 %, 7.5 %, 6 % and 10 % (v/v) respectively. All four products complied with Sri Lankan Standard 824:1989 and the pH, brix and acidity  of  the final products were acceptable ranges. The shelf life of the  product was 18 days at 4oC without any preservatives. However a  complete  analysis  of  nutrients including vitamin A and C, phyto nutrients and dietary and crude fibres can give a complete nutrient profile and nutritional  value  of  the developed  products.  Therefore,  in  the   next season   of   fruits these constituents will be analysed. Also, the study on development of yoghurt with  chemically preserved pulp will be studied.   Consumer   preference   test   and marketing survey should be done to improve its quality.

REFERENCES

AOAC International (2002). AOAC Official Methods of Analysis. (2002), 17th Edition, ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS Washington, USA.

BOGHRA, V. R. AND MATHUR O. N., (2000). Physico-chemical status of major milk constituents and minerals at various stages of shrikhand preparation. Journal of Food Science and Technology, 37: 111-115

FOOD AGRICULTURE ORGANIZATION AND WORLD HEALTH ORGANIZATION, CODEX ALIMENTARIOUS COMMISSION, (2003). Codex standard no 243 – 2003 – Codex

standard for fermented milk, United States of America

JANSZ,  E.  R., WICKREMASEKARA,  N. T. AND SUMUDUNI, K. A. V. (2002). A Review

of the Chemistry and Biochemistry of seed shoot flour and fruit pulp of the Palmyrah palm (Borassus flabellifer L.). Journal of National Science Foundation of Sri Lanka. 30(1&2): 61-87.

JANSZ, E. R., (2006). Bioactivity of Palmyrah (Borrasus flabellifer) biomolecules, Nugegoda, Sri Lanka

JEYARATNAM, M. (1986). Studies on the Chemistry and Biochemistry of palmyrah products. M.Phil. Thesis. University of Jaffna, Sri Lanka. pp. 1-200

KAMRUZZAMAN M., ISLAM M .N, RAHMAN M. M., PARVIN S. AND. RAHMAN M. F,

(2002). Evaporation rate of moisture from dahi (yoghurt) during storage at refrigerated condition. Pakistan Journal of Nutrition., 1: 209-211.

KUMAR,  P.  AND   MISHRA   H.N., (2004). Mango fortified set yoghurt: Effect of  stabilizer addition of physicochemical, sensory and textural properties. Food Chemistry., 87: 501-507

MINISTRY OF LIVESTOCK AND RURAL COMMUNITY DEVELOPMENT, (2010).

online                        available                         from http://www.livestock.gov.lk/site/index.php?l ang=en,

PALMYRAH DEVELOPMENT BOARD, (2010). Annual Report of palmyrah, Jaffna PATHBERIYA L. G, CHANDRIKA U. G (2003). Variation of bioactivity of fruit pulp of different cultivars of palmyrah research plan – National seminar on palmyrah research and development, palmyrah development board, Colombo, Sri Lanka

PERDIGON, G., A.M. DE LEBLANC, J. VALDEZ AND M. RACHID, (2002). Role of

yoghurt in the prevention of colon cancer. Eur. J. Clin. Nutr., 56: S65-S68

SIVAGNANASOTHY V., (2010). Accelerated palmyrah development work, Article  on  Daily news, (on line) 21st of September, 2010 available from http://archives.dailynews.lk/2010/09/21/fea1

  • asp. (Accessed on 20. 01. 2014)

SRI LANKA STANDARD INSTITUTE, (1991). Sri Lankan Standard for microbiological analysis, SLS 516:Part 1, Total plate count, Sri Lanka

SRI LANKA STANDARD INSTITUTE, (2013). Sri Lankan Standard for microbiological analysis, SLS 516:Part 2-2, 2013 for Yeast and mold, Sri Lanka

TAMIME A.Y. AND. ROBINSON. R. K (1999). Yoghurt, Science and Technology – International Journal of Dairy Technology, 61: 112–113.

THEIVENDRARAJAH. K. (2008). Palmyrah palm (Borrasus flabellifer) – A Monograph, Scarborough, Ontario, Canada.

BioMed Central Biological Research, 47:35, 2014

Screening the antifungal activity of essential oils against decay fungi from palmyrah leaf handicrafts

Subajini Mahilrajan1, Jeyarani Nandakumar2*, Robika Kailayalingam1, Nilushiny Aloysius Manoharan1 and SriThayalan SriVijeindran1

1Palmyrah Research Institute, Kaithady, Jaffna, Sri Lanka. 2Department of Botany, University of Jaffna, Jaffna, Sri Lanka.

Abstract Background

The palmyrah leaves handicrafts are affected by fungal attack on rainy season; this could be prevented by some protective agents. Instead of using expensive and harmful chemicals, it was decided to test the activity of natural plant extracts on fungal attack. Fungi were isolated, purified from affected palmyrah leaves then characterized as Aspergillus niger, Aspergillus flavus and Penicillium sp. and used for this study. Different concentrations of extract prepared from thyme (Thymus vulgaris L) flower and commercially available thymol has been evaluated for their antifungal activity against isolated fungi. Minimum Inhibitory Concentration (MIC) and Percentage of Growth Inhibition (GI) of thyme extract and thymol were determined to screen the antifungal activity.

Results

There was no significant different on growth inhibition among the fungi species for all the concentrations of thyme and thymol. While growth inhibitions for all concentrations of hot water extract of thyme showed significantly different (p<0.05) and increases with concentrations. The extract of 15ml/dl thyme and 0.5, 1.0, 2.0 ml/dl of thymol showed significantly higher GI (100%) for all fungi species, while 0.1 ml/dl of thymol showed significantly lesser activity when compared with 10ml/dl of thyme extract. Minimal Inhibitory Concentration of thymol was shown to be 0.5 and thyme extract was 15ml/dl respectively.

Conclusion

Based on the results thymol in the concentration of 0.5ml/dl could inhibit the fungal growth besides thyme extract of 15ml/dl could be used instead of commercially available thymol. Therefore thyme could be used for the preparation of ecofriendly fungal agent to protect the palmyrah leaves handicrafts in addition field testing is essential to achieve this in industrial level. Key words: Extracts, Fungi, Growth Inhibition and Preservation

1.       Background

Macroscopic filamentous fungi are ubiquitous micro organisms with a great capacity to colonize many kinds of substrates and to develop under humid environmental conditions. Fungi and their airborne spores have been recognized as possible causative agents of various diseases in human

  • in addition they cause discolourations on Palmyrah leaf handicrafts.

Nowadays, application of chemical compounds is considered as the common method and most inexpensive to fungal control. However, their adverse effects on human health and the environment, promoted to produce natural fungicide [2]. Biologically active compounds found in plants appear to be more acceptable and safer than synthetic compounds and exhibit a wealthy source of potential for control the fungal agents [3].

Several studies have shown that thyme oils, particularly those of Thymus vulgaris and Thymus zygis [4, 5] possess significant antifungal, insecticidal, and antimicrobial activities, it may vary based on the variation on the chemical composition.

The essential oils of more than one hundred species of the genus Thymus have been chemically investigated; reveal about 360 different volatile components in total. Among these, the monoterpenes were the most prominent group while sesquiterpenes represent a lower percentage of the volatiles. Generally, plants of the genus Thymus are considered as the most common source of the monoterpenoid phenols, thymol and carvacrol [6].

Several Thymus species are locally known as “Omum” and the dried herbal parts are used in herbal tea, condiment and folk medicine. The essential oils of some Thymus spp. are characterized by the presence of high concentration of the isomeric phenolic monoterpenes thymol and/or carvacrol [7]. Traditionally this has been used as a medicinal plant

in the treatment of headaches, coughs, diarrhea, constipation, warts, worms and kidney malfunction [8]. Thyme also possesses various beneficial effects as antiseptic, carminative, antimicrobial and antioxidative properties [9].

The main objective of this work was to characterize the in vitro antifungal activities of hot water extract of thyme and pure thymol, as comparative substance, on different fungus species isolated from Palmyrah leaf handicrafts.

2.       Results

The antifungal activity of the different concentrations thyme extracts and thymol with different treatments, in terms of percentage of growth inhibition of mycelia were calculated and tabulated in Table 1. Results showed that the thymol have great potential of antifungal activity against all of the three fungi such as Aspergillus niger, Aspergillus flavus and Penicillium sp.

Table 1: Percentage of growth inhibition with different treatments

Fungus speciesT1T2T3T4T5T6T7T8
A.niger40.68e50.13d76.98b100a59.4c100a100a100a
A.flavus2.01e2.26d76.44b100a59.4c100a100a100a
Penicillium7.02e7.27d75.38b100a66.92c100a100a100a

Activity of hot water extracts of thyme (T1-T4) was compared with that of pure thymol (T5-T8). All the four different concentrations of pure thymol (T5-T8) showed the 100% of GI for A.niger. A.flavus and Penicillium at 4th day of incubation and minimal inhibitory concentration of thymol was 0.5ml/dl (T6). The hot water extract of (T4) 15ml/dl thyme and 0.5 (T6), 1.0 (T7), 1.5 (T8) ml/dl of commercially available thymol were showed significantly (p<0.05) higher GI (100%) for all fungi species, while 0.1 ml/dl (T5) of thymol showed lesser activity when compared with 10ml/dl (T3) of thyme extract, significantly.

GI of thyme extract at the concentration of 10 ml/dl (T3) was 76.9, 76.4 and 75.3% and 1 ml/dl (T1) was 40.6, 2.0 and 7.0 for A.niger. A.flavus and Penicillium respectively at 4 th days of incubation (Figure 1) while thymol with the concentration of 0.1ml/dl showed 59.4, 59.4 and 66.9% GI for above stated fungi respectively at 7th days of incubation.

3.       Discussion

The Palmyrah leaves are long, fan-shaped, 2 to 3 m in length. The first two tender unexpanded whitish leaves and the next 12 young green leaves are used in making various handicrafts. The whitish tender leaves are used for making soft fine handicrafts while the young green leaves are used for making stronger, but coarse textured utility items like mats, baskets, packaging material, inner lining of heavy duty fibre baskets etc. The mid rib known as eekils can be used to make implements for sweeping if gardens, wall hangers, baskets and a type of carrying cases. Therefore the dried palmyrah tender leaf was selected and cut into small pieces and inoculated into the PDA plate then incubated at room temperature for 4 days. After incubation colonies were purified by repeated streaking then purified fungi were maintained in agar slants at 4°C throughout the study and used as stock cultures [10].

Antifungal activity assay

All species of fungi, using of any concentrations caused significant differences (p<0.05) on inhibitory effect of extracts. It could be observed that as extract concentration increases, the inhibitory effect also increased. In other words, the inhibitory effect of the extract was proportionate to its concentration. As stated by Rasooli et al., 2006 [11] and Amini et al., 2012 [12], with the increase of concentrations, the susceptibility of fungi increased.

According to the results, we can consider that strong antifungal activity of thyme hot water extract could be attributed to thymol itself. Rasooli and Owlia (2005) [13] showed that thyme oils were affect cell wall and cell membrane of A. parasiticus. The plasma membrane was irregular, dissociated from cell wall, invaginated and associated with the formation of lomasomes, which were found in fungi treated with imidazole components. At low concentrations, phenolic lipophilic compounds such as thymol alter cell permeability of microbes, permitting the loss of macromolecules. Exact cause effect relation for the mode of action of phenols has not been determined, but they may inactivate the essential enzymes, react with cell membrane proteins or disturb genetic material functionality [14]. From this point of view, thyme, which is rich in thymol and other antifungal components, could be used for preparation of disinfecting agent for fungi present in the palmyrah leaf handicrafts in low concentration.

Rakotonirainy and Lave´- drine (2005) [15] reported on fungi static but not fungicidal activity of linalool (295 and 415 ppm) after 21 days of exposure of mouldy books in glass chamber,

volume 10Æ2 l. Comparing with these results, vaporous phase of essential oil of thyme happens to be more effective.

4.  Conclusion

Hot water extract of thyme in the concentration of 15ml/dl, which is rich in thymol besides other antifungal components, could be used for disinfect fungi and also comparable to pure thymol. Considering the results, we recommend the use of thyme selected for development of new and safe fungicides. Further formulation and field experiments are necessary to achieve this target.

5.   Materials and methods 5.1Microbial culture and condition

The thyme extract and thymol were assayed for antifungal activity against the fungal strains A.niger, A.flavus and Penicellium sp.isolated from palmyrah leaf handicrafts which are affected by fungi. These fungi were grown on PDA plate at room temperature and maintained with periodic sub-culturing at 4oC.

5.2   Thymol and Thyme

Thyme flowers obtained from domestic market and Thymol from Sigma was used for this study.

5.3   Preparation of thyme powder

Thyme flowers were dried in oven at 40oC for 24h, ground into fine powder in an electric grinder and sieved to obtain a fine sample. Then sealed in a polythene bag and stored at room temperature until extraction.

5.4   Preparation of extract

Dried powered of thyme (20g) was weighed and macerated with 100 mL of water (boiled for 1/2h – hot water extract) and filtered through Whatman No 1 filter paper. This crude extract was used to screen the antifungal activity of extract.

5.5   Screening of thymol and thyme extract for antifungal activity by dilution method

PDA medium with different concentrations of thyme extracts and thymol were prepared as Table

2. About 15 mL of the medium with above treatments were poured into each petridish and

allowed to settle. Nine mm discs of 5 days old culture of the test fungi from the margin of the plates were incised, placed at the center of the petridishes, and incubated at room temperature for 4-7 days. After incubation the colony diameter was measured in millimetre. For each treatment three replicates were maintained. PDA medium without the extract served as control. Growth zones were measured at 4th and 7th days of incubation.

After incubation the colony diameter was measured in millimetre. For each treatment three replicates were maintained. PDA medium without the extract served as control. Growth zones were measured at 4th and 7th days of incubation. The fungi toxicity of the extract in terms of percentage of growth inhibition of mycelia was calculated by using the formula

Growth inhibition (%) = dc – dt/dc x 100

Where dc = Average increase in mycelial growth in control,

dt = Average increase in mycelial growth in treatment [16].

The antifungal agent nystatin added to the agar plates (final concentration of 1.0 mg/L) served as a positive control of Aspergillus niger, A. flavus and penicillium sp. Each experiment was repeated to confirm the results.

Table 1: Different concentrations of thyme and thymol extracts with different treatments

5.6                                                                                                                                                                      Treatments   Extracts Concentrations (ml/dl) T1 Thyme 1 T2 Thyme 5 T3 Thyme 10 T4 Thyme 15 T5 Thymol 0.1 T6 Thymol 0.5 T7 Thymol 1.0 T8 Thymol 1.5     Statistical Analysis:

The                                                                                results (percentage of inhibition) obtained

from the eight treatments with three replicate were subjected to analysis of variance by complete randomized design (CRD). The significant difference among the treatments was tested in Least Significant Difference (LSD) at 5 % level of significance using SAS software.

6.   Competing interests

The authors declare that they have no competing interests.

7.     Authors’ contribution

SM & RK participated in the conception and design of the study, carried out the antimicrobial activity study, SM- Drafted the manuscript. NAM- carried out statistical analysis and SSV- coordinated & management of research activities. All authors read and approved the final manuscript.

8.  Acknowledgements

Financial assistance given by Ministry of Traditional Industries & Small Enterprise Development is acknowledged.

9.   References

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  4. Bruneton J. Partie 3, Terpenes steroids, huiles essentielles. In Pharmacognosie. Paris: E ditions Tec & Doc & E ´ ditions Me´dicales Internationals, 1999.
  5. Pina-Vaz C Rodrigues AG Pinto E Costa-de-Oliveira S Tavares C Salgueiro LR Cavaleiro C Gonc alves M J and Martinez-de-Oliveira J. Antifungal activity of Thymus oils and their major compounds. J European Academy of Dermatology. 2004, 18, 73–78.
  6. Bruneton J. Pharmacognosy, Phytochemistry. Medicinal Plants, Lavoisier Publishing, Paris, 1995 p. 287
  7. Baser KHC. Essential oils from aromatic plants which are used as herbal tea in Turkey. In: Fla- vours, Fragrance and Essential Oils. Proceedings of the 13th International Congress of Flavours, Fragrnce and Essential Oils (Baser K. H. C., ed.). AREP Publications, Istanbul, Turkey, 1995 pp. 67D79.
  8. Simon JE Morales MR Phippen WB Vieira RF. and Hao Z. A source aroma compounds & a popular culinary & ornamental herb. 1999 p. 499 – 505. In: Perspectives on new crops and new uses (J. Janick eds.).
  9. Baranauskiene R Venskutonis PR Viskelis P and Dambrauskiene E. Influence of nitrogen fertilizers on the yield and composition of thyme (Thymus vulgaris). Journal of Agricultural and food chemistry, 2003, 51: 7751-58.
  10. Mahilrajan S Nandakumar J Kailayalingam R Manoharan NA and SriVijeindran S. Screening the antifungal activity of essential oils 3 against decay fungi from palmyrah leaf 4 handicrafts. Biological Research, 2014, 47:35.
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IOSR Journal Of Environmental Science, Toxicology And Food Technology 8, (1), PP 47-52, Jan. 2014

Optimization of dyeing condition and its dyeing on Palmyrah (Borassus flabellifer) leaves

Subajini Mahilrajan1*, Jeyarani Nandakumar2, Robika Kailayalingam1 and SriThayalan SriVijeindran1

1 (Palmyrah Research Institute, Kaithady, Jaffna, Sri Lanka

2(Department of Botany, University of Jaffna, Sri Lanka)

Abstract

Palmyrah leaf based articles have been dyed mostly with direct dyes till today, which always have problems to dyers is that complications in dye reproducibility. Therefore this study was concluded about standardize the dyeing variables of alkaline dye. The optimum wave length was 600 nm, out of a set of wave length ranging from 400-700 nm on the basis of highest absorbance. Five dye concentrations (0.1-0.5g/l) were tried and 0.3g/l dye concentrations were selected on the basis of dye absorption. Likewise for the good dye absorption optimum temperature, optimum time and optimum leaf: liquor ratio was 100oC, 30 min and 1:20 respectively. Significantly higher dye absorption and wash fastness was observed leaves with beaching than without bleaching while there was no significant different among the property of light fastness. Effect of auxiliary was studied with sodium chloride, sodium carbonate and naphthalene among the auxiliaries naphthalene was selected as the best source for the maximum dye absorption based on the light fatness properties.

Key words: Palmyrah leaf, dye absorption, naphthalene and fastness

1.  INTRODUCTION

  1. History

Dyeing is a complex process, where numbers of variables are involved. Dyeing process is broadly governed by fabric, dye and time, temperature, pH of the fabric and liquor, type of auxiliary used etc. Any minor variation in any of these variables causes problems in dye reproducibility, though it is possible to achieve reproducibility in dyeing results. By standardizing each and every variable consistent reproducible results can be achieved.

1.2  Literature Review

The temperature of the dye bath affects the affinity of the dye molecules towards fibre, rate of hydrolysis, migration and covalent bond formation, therefore the dyeing temperature selected must be as per the dye type. Percentage of dye absorption increased with increase in temperature and after that, the dye absorption decreased (Taylor, 2001; Bae, et. al., 1998).

Auxiliaries play an important role in dyeing of reactive dyes. They help in better exhaustion of the dyes. Alam, et al., 2008; Farha, et al., 2010 reported that the addition of electrolyte to the dye liquor of anionic dye increased the uptake of dye by the fabric. The electrolyte used in dyeing dissociates completely in aqueous dye liquor. For entering into the fabric, the charge on surface (negative in fabric) will have to be neutralized since both anionic dyes and fabric have the same charge. Sodium ion (Na+) from sodium chloride is cationic and in the dye liquor is attracted by the negatively charged fabric. By bonding the sodium cations neutralize the anionic surface charge of the fabric. Now the neutralized fabric can attract the organic dye molecules which have a greater affinity for the fabric than the aqueous solution.

Dyes molecules permanently bind with cellulose based fibers (cotton, rayon, hemp, linen) as well as leaves, when the pH is raised. Soda ash (sodium carbonate) is generally used to raise the pH and is either added directly to the dye or in a solution of water in which garments are soaked before dyeing. (Anonymous, 2007). Because of this reason alkaline dyes were used for this study.

1.3  Problems identified

Leaf based articles have been dyed mostly with direct dyes till today, which always have problems to dyers is that complications in dye reproducibility as well as consumers as these articles fades very easily so an attempt has been made to standardize the dyeing process for leaves using alkaline dye which have good colour fastness.

1.4  Objective

Study the dyeing process to reduce the fade of dyed Palmyrah Leaves

1.4.1    Specific objective

Optimization of alkaline dye and its dyeing on Palmyrah leaves

1.5  Scope of the study

Increases the uses of dyed palmyrah leaves when compared with leaves without dye due to improve technique. If dyeing process can be improved palmyrah leaf based products will increase the earning in domestic and foreign market.

2.  DESCRIPTION OF RESEARCH

Standardization of dyeing process for palmyrah leaves with alkaline dye: Experiments were carried out to optimize dye concentration, dyeing time, dyeing temperature and leaf liquor ratio for dyeing of palmyrah leaf with alkaline dye.

i.   Determination of optimum wave length

For determining the optimum wavelength 1 ml of dye was diluted to 200 times and absorbance of the solution was taken on a spectrophotometer at different wave lengths from 400 to 700 nm. The wavelength reflecting the highest optical density was selected.

ii.   Optimization of dye concentration

For determining the optimum dye concentration, five different concentrations of reactive dye

i.e. 0.1, 0.3, 0.5, 0.75 and 1g/L were taken and samples were dyed at 95-100°C for 30 minutes. Absorbance of dye solutions before and after dyeing was recorded at optimum wavelength. The dye solution giving the maximum dye absorption was taken as optimum dye concentration.

iii.   Optimization of dyeing temperature

To optimize dyeing temperature, dyeing was carried out using optimum concentration of dye at five different temperatures i.e. 70, 80, 90 and 100oC. The temperature giving maximum dye absorption was taken as the optimum dyeing temperature.

iv.   Optimization of dyeing time

The leaves samples were dyed using optimum dye concentration for five different time durations

i.e. 10, 20, 30 and 40 minutes. The optimum dyeing time was selected on the basis of maximum dye absorption.

v.   Optimization of leaf liquor ratio

To ascertain the optimum dyeing liquor leaf (L: R) ratio, five samples were dyed at the optimum concentration, temperature and time at 1:10, 1:15, 1:20, 1:25 and 1: 30. Optimum dyeing leaf liquor (L: R) ratio was decided on the basis of maximum dye absorption.

vi.   Effect of bleaching agent

This palmyrah leaf contained high amount of lignin therefore the leaves samples were heated at 100oC for 10min with hydrogen peroxide (4ml/l) and then dyed using optimum dyeing condition. For control treatment was done without pre heating and hydrogen peroxide. The optimum dyeing was selected on the basis of maximum dye absorption.

vii.   Effect of auxiliaries

Different auxiliaries such as sodium chloride, sodium carbonate and naphthalene (10g/l) were added in dye bath separately. Best auxiliary was decided on the basis of maximum dye absorption.

viii.   Fastness testing

Wash fastness of the leaves dyed without bleaching, dyed with bleaching, dyed with naphthalene, dyed with NaCl, dyed with Na2CO3 and dyed with bleaching and Naphthalene under optimized condition was tested according to ISO 105 –CO3 method. The above dyed leaves were washed in soap solution (Na salted) for 30 min at room temperature. Half of the dyed leaf was draped with black paper and covered with glass slide then placed on direct sun light for 25h. Then light fastness was tested by colour scale is for assessing changes in colour of leaf in colour fastness tests, for example the leaf consists of five number of colour each representing a visual difference and contrast. The fastness rating goes step-wise from:

Note 5 = no visual change (best rating)

Note 1 = a large visual change (worst rating).

The colour scale has the 5 possible values: 5, 4, 3, 2 and 1

3.  RESULTS AND DISCUSSION

Basic or cationic dyes on ionization give coloured cations and form an electrovalent bond with the –COOH group of wool and silk. These dyes are applied from neutral to mildly acidic pH. These dyes have poor light fastness.

i. Determination of wave length: Optimum wave length is the wave length at which maximum absorbance was observed. The absorbance was recorded from 400 to 700 nm. The maximum absorbance was observed at 600 nm hence this wave length was selected for further studies (Figure: 1).

0.4

0.35

Text Box: Absorbance0.3

0.25

0.2

0.15

0.1

0.05

0

400    450    500    550    600    650    700

Wave length (nm)

Figure 1: Wave length of alkaline dye (Malachite green).

ii.   Optimization of dye concentration

On the basis of dye absorption to optimize the dye concentration 0.1, 0.2, 0.3, 0.4 and 0.5 g/L dye concentration were taken (Figure 2). There was no significant difference (P > 0.05) between treatments with higher absorbance of dye with the concentration of 0.3, 0.4 and 0.5 g/L. Hence

0.3 g/L dye concentration was selected because mean difference between 0.5 and 0.3g/L is greater than mean difference between 0.3 and 0.4 g/L and also 0.3 g/L is cost effective concentration when compared with higher absorbance concentrations. It is found that dye absorption by cotton fabric increased with the increase in dye concentration in the dye bath because the absolute quantity of the absorbed dye increases while the relative quantity diminishes (Singla et al., 2012).

40

35

Text Box: Absorbance (%)30

25

20

15

10

5

0

0.1           0.2           0.3           0.4           0.5

Concentration (g/L)

Figure 2: Optimization of dye concentration on the basis of dye absorption

iii.   Optimization of dyeing temperature

Dyeing temperature is the temperature that is suitable for dye absorption and fixation of dye on the leaf material. For optimizing dyeing temperature, dyeing was carried out at four different temperatures i.e. 70, 80, 90 and 100oC. The percent dye absorption at different temperatures is given in Table: 3. Mean absorption value for temperature 90 and 100oC is significantly higher than that of temperature 70 and 80oC, while there was no significant difference between the absorptions of temperature for 90 and 100oC. Therefore 90oC was selected as the optimum dyeing temperature. Ali et al., (2008) reported that this increase in dye uptake can be attributed to better dye exhaustion at higher temperature.

Table 3: Optimization of dyeing temperature on the basis of dye absorption

Temperature (oC)Absorbance (%)
7017
8021
9064
10086

iv.   Optimization of dyeing time

Dyeing time is the time required to get the dye fixed on palmyrah leaves. The effect of dyeing time on absorbance is shown in Table 4. Mean absorption value for time at 30 and 40min is significantly higher than that of time at 10 and 20min, while there was no significant difference between the absorptions of time for 30 and 40min. Therefore 30min was selected as the optimum dyeing time for better exhaustion. Longer the dyeing time create higher absorbance until dye exhaustion attains equilibrium (Ali et al., 2008).

Table 4: Optimization of dyeing temperature on the basis of dye absorption

Time (min)Absorbance (%)
1070
2075
3086
4091

v.   Optimization of Leaf liquor Ratio

The effect of leaf liquor ratio on absorbance is shown in figure 2. Mean absorption value for L: R for1:10, 1:15 and 1:20 is significantly higher than that of L: R for 1:25 and 1:30, while there was no significant difference between the absorptions of L: R for for1:10, 1:15 and 1:20 (Figure 3). Therefore 1:20 was selected as the optimum dyeing L: R for better exhaustion. Higher absorbance value at lower L: R may be explained by crowding of dye molecule at lower L: R resulting in increased dye exhaustion of the leaf (Ali et al., 2008).

vi.   Effect of bleaching agent

The leaves samples were heated at 100oC for 10min with hydrogen peroxide (4ml/l) and then dyed using optimum dyeing condition. For control treatment dyeing was done without preheating (Figure 4). Mean percentage of dye absorption was significantly higher for dyed with bleaching than dyed with non-bleaching method.

120

100

Text Box: Absorbance80

60

40

20

0

1:10            1:15            1:20            1:25            1:30

L:R

Figure 3: Optimization of leaf liquor ratio on the basis of dye absorption

Text Box: Dyeing methodWithout bleaching

Bleaching

0        20       40       60       80      100

Dye absorption (%)

Figure 4: Effect of bleaching on dye absorption

vii.   Effect of auxiliaries

A mordant is more important than the dye itself. Moreover, the ideal mordant for bulk use should produce appreciable colour yield in practicable dyeing conditions at low cost, without seriously affecting physical properties of fibre or fastness properties of the dyes. Different auxiliaries such as sodium chloride, sodium carbonate and naphthalene (10g/l) were added in dye bath separately.

Percentage of dye absorption with Na2CO3was significantly higher than other auxiliary this may be due to de-colouration of dye on alkaline medium. Among the NaCl and naphthalene there was no significant different between on dye absorption while de-colouration to direct sunlight for 24h was higher for NaCl than naphthalene (Figure 5). Therefore Naphthalene was selected for further study.

90

80

Text Box: Dye absorption (%)70

60

50

40

30

20

10

0

Dye                 NaCl             Na2CO3                    Napththalene Type of Auxiliary

Figure 5: Effect of auxiliary on dye absorption

viii.   Fastness testing

Table 5: Colour score for fastness test of different dyed method

  Method of dyeingWash fastnessLight fastness
Dyed without bleaching43
Dyed with bleaching53
Dyed with naphthalene44
Dyed with NaCl33
Dyed with Na2CO322
Dyed   with    bleaching  and Naphthalene53

Wash fastness for colour scale for dyed with bleaching and dyed with bleaching and Naphthalene was showed higher than other treatments (Table 5) while those treatments showed less light fastness colour scale although dyed with naphthalene showed good light fastness (Figure 4) property.

Figure 4: Dyed without bleaching (B), dyed with bleaching (A24), dyed with naphthalene (A10), dyed with NaCl (C), dyed with Na2CO3 (D) and dyed with bleaching and Naphthalene (A25).

4.  CONCLUSION

This study could be conclude for optimum dyeing with good dye absorption accordingly leaf materials have to dyed with the concentration of 0.3g/l, Temperature 100oC, Time of dyeing 30 min and leaf: Liquor ratio 1: 20. Besides dyed with bleaching was showed best wash fastness and dyed with naphthalene showed good light fastness.

5.  REFERENCES

J. Singla, S. S. J.Singh, and N. M. Rose (2012). Standardization of Dyeing Variables of Reactive Dye for Tie and Dye on Cotton, Volume : 1: 5, ISSN No 2277 – 8160.

Submitted to IOSR-JBB

Glycemic Index and Insulin Index of Palmyrah Based Edible Products Commonly Consumed in Jaffna

S. Mahilrajan1, S. Balakumar2, V. Arasaratnam2, T. Kumanan3 and

R. Kailayalinkam1

1Palmyrah Research Institute, Kandy Road, Kaithady, Jaffna, Sri Lanka

2 Department of Biochemistry, Faculty of Medicine, University of Jaffna, Sri Lanka

3Department of Medicine, Faculty of Medicine, Sri Lanka

Abstract: The objective of this study was to determine the insulin index and glucose index to isoenergetic (1000- kJ) portions of palmyrah based foods commonly consumed in Jaffna and their correlations between nutrient and phytochemical contents. Subjects were selected those having fasting blood sugar less than 100 mg/dl. Glucose (reference standard) and four test foods such as pinnatu, Jaggery, odiyalpittu and pullukodiyal flour snak were administrated to four groups of 24 subjects and serving weight were calculated based on the energy content. Subjects were fasted for12h before the administration of foods then venous blood samples were collected at 0 time and every 30 min for two hours after either feeding with glucose and test foods. An insulin score and glycemic score were calculated from the area under the insulin/glucose response curve for each food with the use of reference food. Among the test foods pinattu has low glycemic (52.9%) and low insulin index (36.47%).Carbohydrate, ash and Total Dietary Fiber (TDF) present in the those products were negatively correlated with glycemic index. Insulin index was negatively correlated with TDF, protein and ash contents. Not only the nutrient contents but also the phytochemicals such as total phenols and flavonoids showed negative correlation. All the tested products showed less plasma insulin, although high plasma insulin causes coronary heart disease. Therefore palmyrah products could be used in order to develop new value added foods, which could have more beneficial health properties regarding glucose and insulin metabolism.

Keywords – Insulin, glycemic index, carbohydrate and total dietary fiber

I.INTRODUCTION

Palmyrah palm has great economic potential and every part of the palm has economic value [1]. The edible palm products are classified into sap, fruit and tuber based products. Among the sap based products ‘neera’ is obtained by tapping the inflorescences, which is consumed as such after deliming process or converted in to jaggery, sugar or candy [2]. Ripped fruit pulp used in different food preparations at domestic level. Fresh palmyrah pulp could be extracted manually or mechanically [3]. The fibers are removed by straining through muslin cloths sunlightdried by called ‘pinnatu’. Tuber based products such as palmyrah ‘odiyal flour’ (dried tuber flour) and ‘plukodiyal flour’ (boiled, dried tuber flour) are used for traditional food preparations.

Palmyrahfruit pulp has shown antidiabetic [4], anti-inflammatory [5], wound healing, anthelmintic activity [6], analgesic and antipyretic activity [5]. Methanolic extract of the male flowers of Borassus flabelliferinhibited the serum glucose levels in sucrose-loaded rats, which may be due to presence of spirostane-type steroid saponins [7]. It also has been documented that palmyrah flour possesses immunosuppressant property [8]. Even though the palmyrah has innumerable medical importance [9] it has not received due importance.

Among the palmyrah-based products, fruit pulp showed caloric value (energy) is 102.83 kcal/100g [10] and also contained more than 5% of soluble fiber [11], which may influence on glycemic and insulinemic response [4]. Lu et al., (2004) [12] have shown that ingestion of arabinoxylan rich fibers decreased postprandial insulin and glucose responses in healthy subjects. Chandalia et al., (2000) [13] have shown that a high dose intake of fibers

over six weeks had beneficial effects on type 2 diabetes patients by improving glycaemic control, attenuating hyper insulinaemia. Even though the palmyrah food based edible products are the traditional foods of Jaffna inhabitants, their nutritive values have not been studied so far.

The aim of this study was to determine the postprandial insulin responses and glucose response to isoenergetic portions of palmyrah based products such as Jaggery, pinattu, odiyalpittu and pullukodiyalflour snak in non-diabetic subjects.

  1. MATERIAL AND METHODS
    1. Materials

Glucose (Glucolin pure glucose) was from SmithKline Beecham (Pvt) Ltd, Colombo-01. ‘Pinattu’, ‘odiyal’ flour and ‘pullukodiyalflour were from ‘Katpaham’ Sale Center, Palmyrah Development Board, Jaffna, Sri Lanka and the ‘Jaggerywas from Pandatharippu Palm Development Society Jaffna, Sri Lanka.

  • Preparation of test foods

The foods were selected to represent palmyrah based natural and processed foods commonly consumed in Jaffna peninsula populates. Foods were prepared in bulk to minimize variations in composition. Each food was served as a 1000kJ portion with 250 mL water.

Pinattu’

Pinattu cut in to 2.0× 1.0×0.3 cm3 slabs was used. ‘Odiyal pittu’

The odiyal flour (250g) was soaked in 2500ml water for 60 minutes, strained through a muslin cloth and squeezed to wet dry. After mixing with wheat flour (50g) and salt the mixture which was made into fine granules, mixed with scrapped coconut (75g) and steamed for 20minutes.

‘Pullukodiyal’ floursnak

‘Pullukodiyal’ flour (200g) was mixed with sugar (20g), scrapped coconut (50g) and water (100ml) and made into small balls.

  • Analytical method

Determination of energy contents of the Palmyra based foods

The energy content of each of the food preparations were measured with Bomb Calorimeter (e2k) and serving weight was calculated for each food is have a total energy of 1000kJ [14].

Nutrient analysis

All the food samples were analyzed for moisture, fat, protein, ash and total dietary fiber contents [15]. Phytochemical analysis

Food samples (10g) were extracted withaqueous (polarity index: 9) in a soxhlet extractor for 24 hours. The extracts were concentrated using rotator evaporator (IKA), and stored at 4°C. Phytochemicals such as total phenolic content

[16] and total flavonoid content [17] were estimated and the results were expressed as mg gallic acid equivalents (GAE)/g at 760nm and quercetin equivalents (QE)/g at 510nm, respectively.

Estimation of blood glucose level

Plasma glucose concentration was analyzed with glucose hexokinase [18] in a semi-automated biochemical analyzer (TC 3300).

Estimation of serum insulin level

Serum insulin concentration was measured with insulin ELISA kit (BIOTINA).

Calculation of Glycemic index and insulin index

After the administration of the reference or test foods blood glucose and insulin levels measured for 2 hrs with 30min interval. Area Under Curves (AUC) for glucose and insulin (gAUC and iAUC) were calculated, based on Simpson’s Rule, where fasting concentrations were used as the baseline and truncated at zero. Descriptive data are expressed as mean ± standard error mean [14].

Insulin score IS (%) was calculated for each test food by dividing the insulin AUC value for the test food by the insulin AUC value for glucose (reference food), and expressed as a percentage.

IS (%) =Area under the 120 min insulin response curve for 1000 kJ test food *100 Area under the 120 min insulin response curve for 1000 kJ glucose

  • Selection of subjects

Four separate groups of 24 healthy adults [19], [20] age between 18- 40 years were selected. Informed consent was obtained from all of the subjects. The subjects taking prescribed medications and non-diabetic subjects those have fasting blood sugar greater than 100 mg/dl were excluded.

  • Administration of test and reference food

A fasting (12h overnight fast) venous blood sample (2.5ml) of each subject was collected. The subjects were administrated with either 62.5g (1000kJ) glucose in 250ml of water or with test food and where glucose was used as reference food. Venous blood samples (2.5ml) were collected at 30, 60, 90 and 120 min after the administration of glucose and test foods and dispensed into two tubes where one contained KF-Na EDTA (for the plasma glucose analysis) and the next tube without clotting agent (for serum insulin analysis). Blood samples were centrifuged at 2500 rpm for 10min and plasma, serum were separated into blood collection tubes and stored immediately at -80°C for analysis.

  • Ethical clearance

The ethical clearance (J/ERC/13/43/NDR/0063) for this study was obtained from the ‘Ethical Review Committee’, Faculty of Medicine, University of Jaffna.

  • Statistical methods and data analysis

Glycemix index (GI) for each food was also calculated by using the same equation with the corresponding plasma glucose concentration. Glycemic and insulinemic indexes of different types of foods were analyzed by Randomized Complete Block Design (RCBD) using SAS software version 9.1.

The results obtained from nutritional and phytochemical analysis with triplicate were subjected to analysis of variance by Complete Randomized Design (CRD). The significant difference among the extracts was tested in Least Significant Difference (LSD) at 5 % level of significance using SAS software.

Linear-regression analysis was used to test the associations between glucose and insulin indexes, correlation between GI and nutritional, phytochemical content, that of between SI and nutritional, phytochemical content using SAS software version 9.1.

  1. RESULTS

Freshly prepared palmyrah based foods samples were tested for their energy content with bomb colorimeter and then serving weights were calculated. Since this study contained four different types palmyrah based food items, it was decided to select six subjects in a group. Their mean height and weight were 53.8(±7.7) Kg and 164.3(±6.7) cm respectively (TABLE 1).

TABLE 1: Energy content, serving weights of different palmyrah based foods and means age, Body Mass Index (BMI) of the subjects

Test foodsEnergy (mJ/ kg)Weight of 1000kJ energy test food (g)Total Subjects (No.)Males (No.)Female (No.)Mean Age (Years)Average BMI (kg/m2)
Pinattu13.092 (± 0.05)76.364230 (±4.11)19.90 (±2.32)
Jaggery14.577 (± 0.19)68.665133 (±3.30)19.46 (±2.4)
Odiyal piitu8.670 (± 0.12)115.364225 (±1.86)21.04 (±1.77)
Pullukodiyal snak10.658 (± 0.12)93.863333 (±7.21)19.47 (±1.82)
  • Nutritional analysis of test foods

Moisture content of theodiyalpittu and pullukodiyal flour snak was higher than jaggery and pinattu. Carbohydrate content of the jaggery [57.75 (±6.8)g/100g] was significantly higher than other test foods while there were no significant difference between the carbohydrate content of odiyalpittu and pullukodiyal flour snak). Protein contents of pullukodiyal flour snak was the highest [3.90(± 0.2)] and it significantly higher than odiyalpittu [3.25(±0.2) g/100g]. There were no significant difference between fat contents of pullukodiyal flour snak [4.11(±1.3)g/100g], odiyalpittu [5.94(±1.4)g/100g] and very little amount of fat content found in jaggery and pinattu. Pinattu [11.98(±1.9)g/100g] contained highest amount of TDF and it was significantly higher than that in odiyalpittu [10.12(±1.5)g/100g], pullukodiyal flour snak [7.87(±2.7)g/100g] and jaggery [2.15(±2.0)g/100g] (TABLE 2).

  • Phytochemicals analysis of test foods

Total flavonoid and phenol contents of all the test foods significantly (p<0.05) different from each other. Both total flavonoid and phenol contents were highest in pinattu (TABLE 2).

TABLE 2: Nutrient, phytochemical contents and GI and IS of test

Nutrients/ PhytochemicalsJaggeryPinattuOdiyal pittuPullukodiyal flour snak
Nutrients (g/ serving weight )Moisture5.71(±0.01 )d12.86 (± 0.79)c56.86 (±0.01 )a35.02 (± 0.53)b
Carbohydrate57.75 (±6.8)a45.82 (±1.5)b38.66 (±8.4)c41.68 (±8.2)c
 Protein0.91 (± 0.0)c1.80 (±0.2 )b3.25 (±0.2 )b3.90 (± 0.2)a
 Fat0.03 (± 0.0)b0.05 (±0.0 )b5.94 (±1.4 )a4.11 (±1.3 )a
 Total dietary fiber2.15 (±2.0 )c11.98 (±1.9 )a10.12 (±1.5 )b7.87 (±2.7 )b
 Ash2.05 (±0.0 )b3.15 (± 0.1)a0.46 (± 0.0)d1.24 (± 0.0)c
Phytochemicals (mg/serving weight)Total Phenol content176.5 (±0.03)b315.6 (±0.02)a43.7 (±0.01)d126.0 (±0.01)c
Total Flavoniod content28.7 (±0.06)c188.1(±0.06)a0.066.6 (±0.02)b
GI (%) 53.7752.9052.2760.33
IS(%) 62.5036.4768.9040.17

Each value in the table is represented as mean ± SD (n = 3). Values in the same rows followed with a different letter (a-d) are significantly different (p< 0.05).

  • Fasting glucose and insulin concentrations

Fasting plasma glucose and serum insulin levels of it four different groups of subjects showed no significant difference between reference and test foods (TABLE 3).

  • Relationship between BMI and fasting glucose, insulin concentrations

Mean fasting insulin concentration of each test foods such as jaggery, pinattu, odiyalpittu and pullukodiyal flour snak was negatively correlated with mean BMI values of respective group that variability was 52, 91, 83, and 13% respectively while there were significant different (p>0.05) between fasting insulin concentration and BMI. Likewise there were positive correlation between mean fasting glucose concentrations of groups consuming Jaggery and pullukodiyal flour snak and mean BMI values respective group, while there were negative correlation for mean fasting glucose concentrations of groups consuming pinattu, odiyalpittu BMI values respective group therefore coefficient determination values accounted for 2, 29, 1, and 18% variability in BMI respectively and no significant different between fasting glucose concentrations and mean BMI.

  • AUC of glucose and insulin after the consumption of test and reference foods

There was a no significant different in both insulin and glucose AUCs of reference standards of all foods (TABLE 3). Although while there were significant differences between individual AUCs of both test foods and reference standards. Mean insulin AUCs of test foods were showed significant different, that of jaggery (5340.75uIU/ml/min) was showed significantly higher than other three test foods. However; Mean glucose AUC of each test foods showed not significant different (p<0.05).

TABLE 3: Glucose and insulin concentration and AUC for all the group of test and reference foods

Time (min)0 30 60 90 120 AUC
Glucose (mg/dl)/ Insulin level (uIU/ml)Glucose levelInsulinGlucose levelInsulin levelGlucose levelInsulin levelGlucose levelInsulin levelGlucose levelInsulin levelGlucos eInsulin
 Reference food78.31 (±4.92)14.82 (±8.87)114.30 (±13.26)105.21 (±28.45)109.97 (±13.48)101.47 (±46.13)99.99 (±4.12)88.77 (±40.96)91.39 (±16.71)38.61 (±21.79)2381.88544.6
Group 1  Jaggery  77.27 (±6.79)  13.02 (±12.36)  102.41 (±5.11)  92.07 (±42.68)  97.06 (±4.83)  63.85 (±33.72)  91.69 (±9.24)  39.46 (±11.87)  92.10 (±5.43)  23.47 (±6.40)  1280.6  5340.7
 Reference food77.67 (±9.42)11.10 (±11.84)132.00 (±5.41)75.64 (±42.21)117.48 (±8.41)85.38 (±49.97)98.92 (±8.07)57.10 (±42.45)86.40 (±4.57)29.00 (±28.05)3907.06085.7
Group 2  Pinattu  83.25 (±12.73)  16.00 (±14.65)  115.55 (±5.56)  41.47 (±25.65)  102.45 (±8.85)  42.07 (±29.31)  92.30 (±6.55)  30.24 (±22.06)  86.12 (±8.66)  26.91 (±23.01)  2066.6  2219.2
 Reference food90.52 (±5.66)12.74 (±2.49)127.55 (±3.34)61.21 (±14.04)111.57 (±4.63)64.68 (±14.79)98.27 (±5.62)44.68 (±9.28)87.33 (±9.14)24.45 (±12.19)2180.51139.8
Group 3  Odiyal pittu  93.72 (±1.88)  14.387 (±4.87)  115.55 (±2.99)  64.21 (±24.15)  103.53 (±7.05)  34.24 (±14.99)  96.37 (±4.68)  26.94 (±10.51)  90.13 (±7.81)  26.44 (±10.10)  4372.3  3012.6
 Reference food83.40 (±6.13)12.08 (±6.32)129.17 (±6.41)77.54 (±27.38)109.15 (±6.27)88.10 (±21.44)100.58 (±6.34)66.77 (±26.43)88.32 (±6.85)58.35 (±34.44)2968.56692.0
Group 4  pullukodiyal flour snak  85.23 (±10.70)  12.31 (±8.09)  118.05 (±8.44)  52.68 (±39.36)  97.27 (±13.39)  42.65 (±14.92)  94.75 (±6.27)  24.38 (±5.73)  85.40 (±6.21)  10.77 (±7.81)  1791.0  2688.4

Insulin AUC values were divided by glucose AUC values which were 8.59, 1.26, 3.37 and 1.28 for Jaggery, pinattu, odiyalpittu and pullukodiyal flour respectively while there were no significant different between pinattu, odiyalpittu and pullukodiyal flour. Therefore Jaggery was a markedly insulinogenic relative to their glycemic effect (Fig: 1). Consequently jaggery was a greatest product to promote the production or release of insulin to their glycemic response followed by other palmyrah products such as odiyalpittu, pullukodiyal flour and pinattu.

Text Box: Mean InsulinAUC/ Glucose AUCJaggery

Pinattu               Odiyal pittu,

Plukkodiyal flour

Test foods

Fig 1: Ratio of insulin AUC to glucose AUC responses.

  • Effects of different nutrient on GI values

Linear-regression analysis of carbohydrate was not significant different while other nutritional factors such as fat, TDF, ash and protein showed significant different (p<0.05) among the test foods. Correlation coefficient values of TDF (r=-0.02, p=0.008), carbohydrate (r=-0.03, p=0.055) and ash(r=-0.04, p=0.006) showed negatively correlated with GI values of test foods while positively correlated with protein (r=0.42, p=0.007) and fat (r=0.04, 0.003).

  • Effects of different nutrient on IS values

Linear-regression model showed not significant different between SI values of test foods and nutrients such as carbohydrate, TDF, protein, fat and ash. Mean IS values were negatively correlated with TDF (r=-0.18), protein (r=-0.02) and ash (r=-0.39) except fat (r=0.12) and carbohydrate (r=-0.01). The above same correlation pattern was found between nutritional content of composite meals and insulin response by Bao et al., 2009 [21].

  • Effects of different phytochemicals on GI and IS value

Linear-regression model was not showed significant different between GI values and phytochemical contents of test foods. There were negativelyweek correlation between total flavonoid (r=0.01), total phenol (r=0.01) and GI values of test food. As well there were negatively correlation between total flavonoid (r=0.65), phenol (r=-0.35) content and IS values of test foods therefore65 and 35% of variability in IS values of test foods respectively.

  • Glycemic and insulin indexes

The raise in blood glucose level and insulin level was less than that of reference food (glucose) (TABLE 2). Among the palmyrah based products pinattu was low GI (52.90) and low IS (36.46) while jaggery (53.77) and odiyalpittu (52.27) were low GI and medium IS and both GI and IS was medium for pullukodiyal flour snak (TABLE 2).

  1. DISCUSSION

The glycemic index and insulin index of palmyrah foods are an important tools used in treat people with diabetes and in weight control. Food with a low GI makes full lengthier and stay satisfied longer, less likely to overeat. The GI helps to know the type of foods which are useful to control the blood glucose level. Insulin index which is less well-known index is more important than the measures of glycemic response [14]. Therefore this study on glycemic index and insulin index was under taken to measure it GI and IS of the palmyrah based edible products. Nevertheless, no studies have been carried out and reports do not available on GI and IS of palmyrah based edible products.

In fact, the GI of a carbohydrate-rich food can vary greatly depending on a number of factors including the variety, origin, processing, and preparation of the food, the other nutrients that are consumed with the food and even the time of day in which the GI is measured [22].

Wolever & Bolognesi, 1996 [23] reported that glycaemic response data obtained from a test at lunch time, after a standard breakfast, differ significantly from those obtained after an overnight fast. In various in vivo studies test food servings have been determined based on calculated data or data received from food tables and those do not resemble to the actual nutrient composition of the food product. In the present study energy of the consumed test foods were determined by using bomb colorimeter. The energy obtained from pinattu, jaggery, odiyalpittu and pullukodiyal flour snak were used for calculate the serving weight of each test foods.

Gaesser, 2007 [24] actually showed that diets with higher GLs were associated with lower BMIs. In this study there were significantly negative correlation was found between mean fasting insulin concentrations and mean BMI values, same result was reported by Holt et al., 1997 [14] among the six groups of subjects. Jaggery and pullukodiyal flour showed positive correlation while pinattu and odiyal pittu showed negative correlation between mean fasting glucose concentration and mean BMI hence BMI was increased that pinattu and odiyalpittu decreased the mean fasting glucose concentration, this could be due to the amount of dietary fiber content of the test foods because order of the TDF content was pinattu>odiyalpittu>pullukodiyal flour > jaggery (TABLE 2).

Incremental AUC is the area beneath the curve only down to fasting level. If the blood glucose level falls below the baseline, the area below fasting is ignored. Thus, the incremental AUC can never be less than zero [25]. All the test foods showed insulinogenic relative to their glycemic effect base on the ratio obtained from the insulin AUC values were divided by glucose AUC values (Figure 1). In fact, the earlier study on palmyrah pinattu as an anti-diabetic food component by Uluwaduge et al., (2007) [4] showed in insulin-dependent diabetic patients (IDDM) revealed a lower glucose response to pinattu and there was a significant reduction (p< 0.01, by 15-48%) in blood glucose concentration after a glucose challenge.

Ahmed et al., 1976 found that addition fat to carbohydrate meal enhances insulin secretion even though the plasma glucose response actually decreases and also Holt et al., 1997 [14] found no relation between the postprandial insulin response and the fiber content of a food. Whereas Albrink et al (1979) [26] found that lower insulin response in healthy subjects when consume meals that have high-fiber content than low-fiber content [27]. Palmyrah pinattu contained appropriate amount of dietary fiber. Therefore it should influence the GI and IS of palmyrah pinattu and has low GI and IS (TABLE 2). Glucose and insulin responses decreased after with meals containing soluble fibers, including pectin, Oatrim (oat fiber extract), guar gum, and methyl cellulose fibers, when compared with meals without soluble fiber (Behall et al., 2002). Morgan (1992) [28] reported that three macronutrients stimulate the release of several gut peptides, but to different degrees. Protein and fat are particularly effective in stimulating gut peptide release despite a small glucose effect. Thus, the insulin response to a carbohydrate food varies with the amount of fat, protein, or both.

Palmyrah products generate lower postprandial insulin response than reference standard glucose. This response is not only due to the fiber content of products, but also due to the bioactive components such as total phenol as saponin etc. (TABLE 2) and the physical structure of palmyrah products. There were significant correlation were observed between phytochemicals and GI values of test foods.

  • CONCLUSION

This present study showed that among palmyrah based products pinattu showed low glycemic and insulin index. Specifically these products decrease postprandial insulin response compared to reference standard glucose. In other words, less insulin is required for regulation of postprandial plasma glucose concentrations in healthy subjects after intake of all tested palmyrah food products than glucose. High plasma insulin has been shown to be an independent risk factor for coronary heart disease. Therefore palmyrah based food products could be used in order to develop new value added foods, which could have more beneficial health properties regarding glucose and insulin metabolism.

Acknowledgements

The authors like to thank Palmyrah Research Institute, Palmyrah Development Board, Jaffna, Sri Lanka and Department of Biochemistry Faculty of Medicine, University of Jaffna, for providing the necessary facilities.

REFERENCES

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2.     Publications in International conference

Full papers

Proceedings of Jaffna University International Research Conference (JUICE 2014)

Antifungal Activity of Some Plant Extracts Against Decay Fungi From Palmyrah Leaf Handicrafts

Subajini Mahilrajan, Jeyarany Nandakumar, Robika Kailayalingam and SriThayalan SriVijeindran.

a Palmyrah Research Institute, Kaithady, Jaffna, Sri Lanka bDepartment of Botany, University of Jaffna, Sri Lanka

Abstract

The palmyrah leaves handicrafts are affected by fungal attack; this could be prevented by some protective agents. Instead of using expensive and harmful chemicals, it was decided to test the activity of natural plant extracts on fungal attack. Fungi were isolated from affected palmyrah leaves by repeated streaking on PDA plates, isolated fungi were purified and characterized as Aspergillus niger, Aspergillus flavus and Penicillium sp. Extract of thyme (Thymus vulgaris L) flower obtained in local market and thymol have been evaluated for their Antifungal Activity against isolated fungi by using different concentrations. To screen the antifungal activity, MIC (Minimum Inhibitory Concentration) and Percentage of Growth Inhibition (GI) of thyme extract and thymol were determined. Minimal Inhibitory Concentration of thymol was shown to be 0.5ml/dl. The hot water extract of 15ml/dl thyme and 0.5, 1.0, 2.0 ml/dl of commercially available thymol showed significantly (p<0.05) higher GI (100%) for all fungi species, while

0.1 ml/dl of thymol showed significantly lesser activity when compared with 10ml/dl of thyme extract. Therefore thyme could be used for the preparation of ecofriendly fungal agent to protect the palmyrah leaf handicrafts also field testing is essential to achieve this in industrial level.

Key words: Extracts, Fungi, Growth Inhibition and Preservation

3.      Introduction

Microscopic filamentous fungi are ubiquitous micro-organisms with a great capacity to colonize many kinds of substrates and to develop under extreme environmental conditions. Fungi and their airborne spores in the indoor and outdoor environment have been recognized as possible causative agents of various diseases in human (Nielsen 2003) and spoilage of Palmyrah leaf handicrafts.

The essential oils of more than one hundred species of the genus Thymus have been chemically investigated; reveal about 360 different volatile components in total. Among these, the monoterpenes were the most prominent group while sesquiterpenes represent a lower percentage of the volatiles. Generally, plants of the genus Thymus are considered as the most common source of the monoterpenoid phenols, thymol and carvacrol (Bruneton 1995).

Several Thymus species are locally known as “Omum” and the dried herbal parts are used in herbal tea, condiment and folk medicine. The essential oils of some Thymus spp. are characterized by the presence of high concentration of the isomeric phenolic monoterpenes thymol and/or carvacrol (Baser, 1995). Traditionally this has been used as a medicinal plant in the treatment of headaches, coughs, diarrhea, constipation, warts, worms and kidney malfunction (Simon et al., 1999). Thyme also possesses various beneficial effects as antiseptic, carminative, antimicrobial and antioxidative properties (Baranauskiene et al, 2003).

Several studies have shown that thyme oils, particularly those of Thymus vulgaris and Thymus zygis (Bruneton, 1999; Pina-Vaz et al., 2004; Stahl-Biskup & Sa´ ez, 2002), possess significant antifungal, insecticidal, and antimicrobial activities, it may vary based on the variation on the chemical composition.

The main objective of this work was to characterize the in vitro antifungal activities of hot water extract of thyme and pure thymol, as comparative substance, on different fungus species isolated from Palmyrah leaf handicrafts.

4.          Materials and methods

  • Collection of sample

Palmyrah leaf based handicrafts are generally affected by fungus during rainy season. So affected tender leaf of Palmyrah was collected from design center of Palmyrah Development Board during rainy season and used for the isolation of Palmyrah leaf born fungus.

2.2   Isolation and detection of Palmyrah leaf born fungi

  • Preparation of Potato Dextrose Agar media PDA plates and slants

Potato Dextrose Agar (3.9g) was dissolved in 40mL of distilled water in a boiling water bath and total volume was made up to 100mL then sterilized at 121oC and 15lb/in2 for 15 minutes. After sterilization, the medium was allowed to cool to 50oC and poured in to sterile petridishes (20mL/ Petridish) under aseptic condition. The PDA was prepared as above stated procedure and 7 mL of the medium was poured into boiling tubes. The tubes were plugged with cotton wool and sterilized at 121oC and 15lb/in2 for 15 minutes. The tubes were then cooled in an inclined position and used for storage of the fungus.

2.2.2    Isolation and Purification of fungal strains

Palmyrah leaf was cut into small pieces and inoculated into the PDA plate then incubated at room temperature for 4 days (Robert Koch and Friedrich Loeffler 1884). After 4 days three different colours of the colonies were selected, purified by repeated streaking and transferred to PDA slants and kept at 4oC.

2.2.3    Identification of isolated fungal strains

Selected fungal colonies were characterized to species level based on macroscopic morphology and microscopic features.

2.5   Thymol and Thyme

Thyme obtained from domestic market and Thymol from Sigma was used for this study.

2.6   Preparation of thyme powder

Thyme flowers were dried in oven at 40oC for 24h, ground into fine powder in an electric grinder and sieved to obtain a fine sample. Then sealed in a polythene bag and stored at room temperature until extraction.

2.7   Preparation of extract

Dried powered of thyme (20g) was weighed and macerated with 100 mL of water (boiled for 1/2h – hot water extract) and filtered through Whatman No 1 filter paper. This crude extract was used to screen the antifungal activity of extract.

  • Screening of thymol and thyme extract for antifungal activity by dilution method PDA medium with different concentrations of thyme extracts and thymol were prepared as Table 1. About 15 mL of the medium with above treatments were poured into each petridish and allowed to settle. Nine mm discs of 5 days old culture of the test fungi from the margin of the plates were incised, placed at the center of the petridishes, and incubated at room temperature for 4-7 days. After incubation the colony diameter was measured in millimetre. For each treatment three replicates were maintained. PDA medium without the extract served as control. Growth zones were measured at 4th and 7th days of incubation.

After incubation the colony diameter was measured in millimetre. For each treatment three replicates were maintained. PDA medium without the extract served as control. Growth zones were measured at 4th and 7th days of incubation. The fungi toxicity of the extract in terms of percentage of growth inhibition of mycelia was calculated by using the formula

Growth inhibition (%) = dc – dt/dc x 100

Where dc = Average increase in mycelial growth in control,

dt = Average increase in mycelial growth in treatment (Singh et al., 1999).

The antifungal agent nystatin added to the agar plates (final concentration of 1.0 mg/L) served as a positive control of Aspergillus niger, A. flavus and penicillium sp. Each experiment was repeated to confirm the results.

Statistical Analysis:

The results (percentage of inhibition) obtained from the eight treatments with three replicate were subjected to analysis of variance by complete randomized design (CRD). The significant difference among the treatments was tested in Least Significant Difference (LSD) at 5 % level of significance using SAS software.

3.       Results

Isolated fungi B, G & A were identified as Aspergillus niger, Aspergillus flavus and Penicillium sp respectively at species level in based on macroscopic and microscopic features (Table 2).

The antifungal activity of the extracts with different treatments, in terms of percentage of growth inhibition of mycelia were calculated and tabulated in Table 3. Results showed that the thymol have great potential of antifungal activity against all of the three fungi tested.

Activity of hot water extracts of thyme (T1-T4) was compared with that of pure thymol (T5-T8). All the four different concentrations of pure thymol (T5-T8) showed the 100% of GI for A.niger. A.flavus and Penicillium at 4th day of incubation and minimal inhibitory concentration of thymol was 0.5ml/dl (T6). The hot water extract of (T4) 15ml/dl thyme and 0.5 (T6), 1.0 (T7),

1.5 (T8) ml/dl of commercially available thymol were showed significantly (p<0.05) higher GI (100%) for all fungi species, while 0.1 ml/dl (T5) of thymol showed lesser activity when compared with 10ml/dl (T3) of thyme extract, significantly.

120.00

Text Box: Growth Inhibition (%)100.00

80.00

60.00

40.00

20.00

0.00

T1              T2              T3             T4

Different treatments with different concentrations of thyme extracts

A.flavus A.niger Penicillium

Figure 1: Average growth inhibition of thyme extract for different fungi at 4th days of Incubation

GI of thyme extract at the concentration of 10 ml/dl (T3) was 76.9, 76.4 and 75.3% and 1 ml/dl (T1) was 40.6, 2.7 and 7.0 for A.niger. A.flavus and Penicillium respectively at 4 th days of incubation (Figure 1) while thymol with the concentration of 0.1ml/dl showed 59.4, 59.4 and 66.9% GI for above stated fungi respectively at 7th days of incubation.

4.       Discussion

  • Isolation of Palmyrah leaf borne fungi

The Palmyrah leaves are long, fan-shaped, 2 to 3 m in length. The first two tender unexpanded whitish leaves and the next 12 young green leaves are used in making various handicrafts. The

whitish tender leaves are used for making soft fine handicrafts while the young green leaves are used for making stronger, but coarse textured utility items like mats, baskets, packaging material, inner lining of heavy duty fibre baskets etc. The mid rib known as eekils can be used to make implements for sweeping if gardens, wall hangers, baskets and a type of carrying cases. Therefore the dried palmyrah tender leaf was selected and cut into small pieces and inoculated into the PDA plate then incubated at room temperature for 4 days. After incubation black (B), green (G) and bluish green (A) coloured colonies were selected for further study. Selected colonies were purified by repeated streaking then purified fungi were maintained in agar slants at 4°C throughout the study and used as stock cultures.

4.2   Antifungal activity assay

All species of fungi, using of any concentrations caused significant differences (p<0.05) on inhibitory effect of extracts. It could be observed that as extract concentration increases, the inhibitory effect also increased. In other words, the inhibitory effect of the extract was proportionate to its concentration. As stated by Rasooli et al., 2006 and Amini et al., 2012, with the increase of concentrations, the susceptibility of fungi increased.

At low concentrations, phenolic lipophilic compounds such as thymol alter cell permeability of microbes, permitting the loss of macromolecules. Exact cause effect relation for the mode of action of phenols has not been determined, but they may inactivate the essential enzymes, react with cell membrane proteins or disturb genetic material functionality (Lopez-Malo et al., 2005).

5.       Conclusion

Hot water extract of thyme in the concentration of 15ml/dl, which is rich in thymol besides other antifungal components, could be used for disinfect fungi and also comparable to pure thymol. Considering the results, we recommend the use of thyme selected for development of new and safe fungicides. Further formulation and field experiments are necessary to achieve this target.

Acknowledgements

Financial assistance given by Ministry of Traditional Industries & Small Enterprise Development is acknowledged.

References

  • Amini, M., Safaie, N., Salmani, M. J. and Shams-Bakhsh, M. (2012). Antifungal activity of three medicinal plant essential oils against some phytopathogenic fungi. Trakia Journal of Sciences, 10: 1-8,
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Table 1: Different concentrations of thyme and thymol extracts with different treatments

  Treatments   Extracts Concentrations (ml/dl) T1 Thyme 1 T2 Thyme 5 T3 Thyme 10 T4 Thyme 15 T5 Thymol 0.1 T6 Thymol 0.5 T7 Thymol 1.0 T8 Thymol 1.5    

Table 2: Macroscopic and microscopic features

of isolated fungus

CharacteristicsBGA
Colony colourBlack  with    white reverseGreen with cream reverseBluish green with white revers
HyphaeSeptate hyalineSeptate hyalineSeptate hyaline
Conidial headRadiateRadiateRadiate
ConidiophoreSmooth hyalineSmooth hyalineSimple
VesicleGloboseSubglobose
ConidiaBlack globoseGloboseRound,Unicellular Unbranch
PhialideUnisereateBisereate…………..

Table 3: Percentage of growth inhibition with different treatments

ungus species  T1  T2  T3  T4  T5  T6  T7  T8
A.niger40.68e50.13d76.98b100a59.4c100a100a100a
A.flavus2.01e2.26d76.44b100a59.4c100a100a100a
Penicillium7.02e7.27d75.38b100a66.92c100a100a100a

Proceedings of Jaffna University International Research Conference (JUICE 2014)

FORMULATION OF INSTANT SOUP MIX POWDER USING

UNBOILED PALMYRAH (Borassus flabellifer) TUBER FLOUR AND LOCALLY AVAILABLE VEGETABLES

N.Niththiya1 , S.Vasantharuba1, M. Subajini2, S Srivijeindran2

1Department of Agricultural Chemistry, University of Jaffna, Jaffna, Sri Lanka

2 Palmyrah Research Institute, Jaffna, Sri Lanka

Abstract

Vegetable and prawn added soup prepared with incorporation of palmyrah tuber flour known as Odiyal Kool is one of the popular and traditional food of Northern Sri Lankan population. This study was to develop dehydrated instant soup mix to reduce the difficulty in the preparation of soup. An instant dehydrated vegetable and prawn added soup mix were developed using unboiled palmyrah tuber flour as thickening agent and dried vegetables, salt, spice and tamarind paste as other ingredients. Initially the saponin content of the flour was removed by a pretreatment before adding to the soup. The ingredients such as manioc, long bean, carrot, moringa leaves, onion and garlic were dehydrated in an oven using established procedures. The optimum levels of palmyrah tuber flour, tamarind paste and garlic powder were found by conducting preliminary trials using five point hedonic scale sensory evaluation tests. Then optimum level of prawn powder was incorporated into prawn added soup mix in addition to other ingredients used in the vegetable soup mix using similar trials. Soup mix: water ratio of 1: 12.5 was selected as the most preferable level for the reconstitution of the soup mix. Proximate analysis revealed that the final vegetable and prawn added soup mix possessed 3.24 %, 4.76 % moisture 1.16%, 1.50% fat, 5.9%, 7.15% protein, 5.58%, 8,66% ash,

4.8%, 5.1% fibre, 79.32%,72.8 carbohydrate and 5-56, 6.52 pH. The total plate count of fresh vegetable and prawn added soup mix was 2.1×103 and 2.9 x 103 cfu/g respectively, which was

within the safe range. The result of the shelf life evaluation studies namely moisture content, sensory evaluation and microbial analysis revealed that both soup mixes could be stored for two months without affecting their quality. The results of the cost of production analysis reveals that the unit price for vegetable and prawn added soup mix were 30.11and 51.06 rupees respectively.

Key words: Dehydrated vegetables, Palmyrah tuber flour, Soup mix

INTRODUCTION

Soup is the one of the traditional food can be classified as an appetizer, warm food during cold and sick. In the modern world commercially prepared instant soup such as canned, dehydrated, and frozen soups have replaced homemade soup as preparing a soup is a time consuming process. Instant soup can become an alternative food for breakfast because it could fulfill the adequacy of energy and nutrient required by the body, very practical in preparation and taking only short time to serve [1].

Dry soup mix contained vegetables in many forms have several advantages over canned soups [2].These soup mixes are popular among the people because of easy to make. Among all dehydrated products instant mixes gained popularity in the recent years, by way to providing convenience, hygienic, extensible shelf life and easy to carry. So that it reduces the transportation cost and available in various packages and it requires very less time to prepare food [3].

Palmyrah has an existing potential of producing 3000 metric tons of tuber flour annually [4]. Palmyrah flour is considered a good source of carbohydrates, fiber and some micronutrients such as calcium, magnesium and ferrous ions [5].The consumption of the odiyal flour can be increased by the introduction of more value added products with good palatability. Odiyal flour was used up to certain level in the instant soup mix. The nutritive and health beneficial components found in the tuber might improve the quality of soup mix.

Vegetables are low in calories and high in fiber content. They are also best source of antioxidants and other phytonutrients. An important advantage in using dehydrated Vegetables are that they are available year round rather than just seasonally [6].

This study was carried out to develop consumer preferable nutritious precooked soup mix from locally available resources and evaluates the nutritional as well as sensory attributes of the developed mix. The objective of this study is to develop a dehydrated instant vegetable soup mix and prawn added soup mix using odiyal flour and prawn added soup mix using odiyal flour and and a mix of compatible ingredients namely vegetables (manioc, long bean, carrot, moringa leaves and onions),rice grits, spices (garlic),salt, chilli powder, tamarind and prawn. MATERIALS AND METHODS

This study was carried out in the laboratory of Palmyrah Research Institute (PRI), Kaithady, Jaffna. Raw materials were procured from the sales centre of Palmyrah Development Board (‘Katpaham’) and local market. Analytical test were conducted in PRI Laboratory and laboratory of Dept. of Agricultural Chemistry, University of Jaffna. All the chemicals used in the analysis were analytical grade and each experiment was done in triplicates.

Preparation of unboiled palmyrah tuber flour (Odiyal flour)

Odiyal flour was sieved using 60-mesh size sieve and soaked in excess water for about two hours to remove soluble bitter compounds, which are present in Odiyal flour. After drained out the excess water wet odiyal flour was mixed with the required amount of tamarind paste according to the soup composition. Then the odiyal flour tamarind paste mix was spread as thin layers and dried in an oven at the temperature of 80°C for 3 hours. The dried flour paste mix was ground by using a mechanical grinder, sieved off, packed and kept in room temperature conditions (28±2°C) until further use.

Preparation of vegetables

Selected vegetable samples such as long bean, carrot, moringa leaves were blanched for predetermined times and immersed in 0.2% sodium metabisulphite (SMS) solution and dried in an oven for standard time using established procedures Boiled manioc samples also dried using similar procedures. The dried samples were ground and sieved off to a thickness of

0.5mm using NO.35 sieves. Similarly selected onion and garlic samples also dried using established procedures, ground and sieved off to a thickness of 0.25mm using No.60 sieves. These samples were packed and kept in room temperature conditions (28±2°C) until further use [6, 7].

Preparation of prawns

Fresh and good quality prawn was selected sand their skin was peeled off and cooked with excess 2% salt solution in pan. The cooked prawns were sliced into small pieces and dried in an oven for 10 hours at 65°C and the dried product was ground, packed and kept in room temperature conditions (28±2°C) until further use.

Optimization of ingredients

All the ingredients were mixed according to the specified ratio for the development of soup mix. In sensory evaluation, each sample was subjected to five-point hedonic scale test (5-like very much, 1- dislike very much) and acceptability of sample was judged by 30 untrained members. They determine the consumer preference of each sample. The panelists judged the sensory characteristic such as appearance, colour, aroma, texture, taste mouth feel and overall acceptability of the samples of the samples.

In the first trial amount of palmyrah tuber flour was changed (10, 20, 30, 35 and 40 g) and all other ingredients were kept at the same level. Then a panel of judges tested the developed soup mix samples. The most preferable amount of odiyal flour used for the soup mix (35 g) was selected from results of the above panel.

In the second trial the soup mix was prepared by using different amount of tamarind paste (5, 10, 15 and 20 g) The most preferable amount of tamarind paste used for the soup mix (10 g) was selected from the results of the sensory panel. Similarly the most preferable amount of garlic (0.82 g) was selected in the third trial based on the evaluation of sensory panel. Based on the results of the sensory panel the ingredients needed for the vegetable soup mix was finalized.

Then in the fourth trial prawn added soup mix was prepared by using different amount of prawn powder (6.3, 12.6 and 18.9 g) and maintaining all other ingredients at the same level similar to

vegetable soup mix. The most preferable amount (12.6 g) of prawn powder was selected from the results of the sensory panel.

Table 1 gives the optimized amount of ingredients used for the preparation of both vegetable and prawn added soup mix.

Proximate analysis

Proximate analysis was done for both vegetable and prawn added soup mix. Proximate analysis including moisture, ash, fat and fibre content were analyzed by standard AOAC methods [8]. Protein content was analyzed by Kjeldhal method [9] and carbohydrate content was estimated by difference method. Calorific value was estimated by multiplying the percentages of protein, fat and carbohydrate with the recommended factors. pH value was determined by pH meter.

Reconstitution of the formulated soup mix

The optimum condition for the reconstitution of formulated soup mix was evaluated by conducting preliminary trials. Initially known weights of soup mix samples (40g) were taken and mixed with different amount water (400, 500 and 600ml) separately and heated for 5 minutes in a hot plate. The appearance and consistency of soup was observed by a panel. The optimum amount of water required for reconstitution was selected based on the evaluation of the panelists.

Shelf life study

The developed vegetable and prawn added soup mix were packed in high density poly ethylene bags and kept in ambient conditions (28±2°C) for a period of 2 months. The changes in moisture content, microbial load (total plate count and yeast and mould count) and sensory characters were evaluated periodically at monthly interval.

Cost of production

Cost of production was estimated separately for both type of soup mixes and it was given as cost for unit (40 g) weight.

Statistical analysis

Friedman non-parametric statistical method was used to analyze the sensory evaluation data based on 5-point hedonic scales using Minitab software. The triplicate data of proximate contents were statistically analyzed by Completely Randomized Design (CRD) using analysis of variance (ANOVA) in SAS statistical software (Version 9.1). The significant differences were compared at 95% confidence interval (p<0.05) using Duncan’s New Multiple Range Test (DNMRT).

RESULTS AND DISCUSSION

Proximate analysis of vegetable and prawn added soup mix powder was given in Table 2. The average moisture content of the vegetables and prawn added soup mix powder were 3.24%, 4.72% respectively. According to the U.S standard, the moisture content of the dry product must not greater than 5%. Prawn added soup mix (7.15%) had higher total protein than that of vegetable soup mix (5.09%). The two soup mix samples had lower fat content (1.16%, 1.50%) than that of results of Abeysinghe and Illeperuma [2] for formulation of an MSG free instant vegetable soup mix (4.1%). The ash content of the prawn added soup mix (8.66%) differs from the vegetables soup mix (5.58%).This difference probably exists because that the prawns are good source of minerals and pre drying treatments of the prawns (cooking with 2% salt solution). Singh et al,, [10] also observed higher percentage of ash (13.5%) content during the development of mushroom-whey soup powder. The fiber content of vegetable soup mix (4.8%) and prawn added soup mix (5.10%).The present result of soup mix, fiber content higher than other soup mix which are prepared without using odiyal flour. The reason of this increasing, odiyal flour and long bean consist higher fiber content.

Reconstitution of the formulated soup mix

Among the different ratio of reconstitution (1:10, 1:12.5. 1:15) the ratio of 1:12.5 was widely accepted by the panel of judges as the optimum level of reconstitution for both vegetable and prawn added soup mixes.

Shelf life study

There was no difference in the sensory scores of both soup powder samples stored at room temperature (28±20C) in high density polyethylene packaging materials for two months. Therefore the developed soup mix samples have acceptable sensory character up to 2 months.

Estimation of moisture content during storage

The initial moisture content of the vegetable soup mix (3.24%) increased to 3.25% after one month and next month not the change in the moisture content. The initial moisture content of the prawn added soup mix (3.21%) increased to 3.24% after the one month and the next month it was increased to 3.25 percentages. The moisture percentage not exceed 5% within 2 months, this percentage is acceptable level dehydrated vegetables products without spoilage.

Microbial analysis

Both of soup mix samples showed negative result for the yeast and mold 10-1 dilution. No growth molds and yeast detected in two samples of soup mix in fresh form. Heat treatment may be preventing the growth of yeast and mold. The growth of yeast and mold was not observed due to the two months period. Aerobic plate count of the develop fresh vegetable soup mix was 2.1×103cfu/g and prawn added soup mix was 2.9×103 cfu/g .A slight increase in aerobic plate count was observed in both samples up to 2 months.

Jey [11] reported that the product is microbiologically safe since total microbial count of dehydrated soups should generally be less than 1×104cfu/g. The ITI [12] report point out that total bacterial count of their formulated soup is 3800/g. Therefore, the developed soup mixes were within the acceptable limit for safe use for a period of 2 months.

Cost of production

The unit (40 g) price for vegetable soup mix and prawn added soup mix were 30.11 and 51.06 rupees respectively. This new product will help to open door in palmyrah related industry in the island through the creation of an opportunity tom offer comparatively low cost instant soup mix powder with higher nutritional and sensory qualities.

CONCLUSION

Vegetable and prawn added soup prepared with the incorporation of palmyrah tuber flour is one of the popular traditional foods of Northern Sri Lankan people. Nevertheless, the popularity of this soup is decreasing due to difficulty in the preparation of quality product. The developed vegetable and prawn added instant dried soup mix have an acceptable sensory, nutritional and microbial quality and it can be stored under ambient condition without affecting the quality characters. The cost of production also found in acceptable level. The developed soup mix is more convenient than traditional product and this will improve its popularity among the younger generation.

REFERENCES

[1] Sunyoto, M., Futiawati, R., and Rahimah, S. 2012.The Influence of Full Cream Milk Powder Concentration to the Characteristics of “Rasi” Instant Cream Soup. Journal of s Agricultural Science and Technology A 2 (2012) 1218-1231.

[2]. Abeysinghe, C. P., and Illeperuma, C. K.2006.Formulation of an MSG (Monosodium Glutamate) free Instant vegetable soup mix. J Natn Sci Foundation Sri Lanka, 34(2), 91-95. [3]. Premavalli, K. S.,Madhura, C. V. and Kumar, M. A.2005.Studies on traditional Indian foods-IV Development and storage stability of green leafy vegetables based instant mixes. Indian Food Packer, 59(4), and 68.

[4].Naguleswaran,S.,Vasanthan,T., hoover, R., and Liu, Q. 2010. Structure and physicochemical properties of palmyrah seed-shoot starch grown in Sri Lanka. Food Chemistry, 118(3), 634-640.

[5]. Mason, D., and Henry, C. J. K. 1994.Chemical composition of palmyrah (Borassus flabellifer) seed shoots-odiyal. International Journal of Food Sciences and Nutrition, 45(4), 287-290.

[6]. Hung, P. V. and Duy, T. L. 2012. Effects of drying methods on bioactive compounds of vegetables and correlation between bioactive compounds and their antioxidants, International Food Research Journal 19(1): 327-332.

[7]. Price, M. L. 2007. The Moringa tree. ECHO technical note.

[8]. AOAC .2000. Official methods of analysis. Association of Analytical Chemists, 15th edition Virginia, U.S.A. pp 125-39.

[9]. Pearson, D. 1976. The chemical analysis of foods, Churchill Livingstone , Edinburgh, London.

[10]. Singh,S., Ghosh.S and Patil.G.R. 2003. Development of a mushroom whey soup Powder, International Journal of Food Science and Technology 38, 217–224.

[11]. Jay, J.M. 1992.Incidence and types of microorganisms in foods. In Modern Food Microbiology (pp.63-93).Springer Netherlands.

[12].Ceylon Institute of Scientific and Industrial Research, 1992. Method of preparation of odiyal soup mix, CR-0067 Part I.

Table1: Amount of ingredients present in the soup mix

IngredientsVegetable soup mixPrawn added soup mix
Odiyal flour (g)3535
Rice grits (g)22
Manioc(g)44
Long beans(g)44
Moringa leaves(g)0.250.25
Carrots(g)0.150.15
Tamarind(g)1010
Chilli powder(g)11
Salt(g)55
Onion(g)0.750.75
Garlic(g)0.820.82
Prawn powder (g)012.6

Table 2: Proximate analysis of vegetable and prawn added soup mix powder

Components %Vegetables soup mix powder (Mean± SD)Prawn added soup mix powder (Mean ± SD)
Moisture3.24 (0.75).76(0.35)
Fat1.16(0.03)).5(0.06)
Protein5.9(0.15).15(0.13)
Ash5.58(0.140).66(0.33)
Fiber4.8(0.62).1(0.26)
Carbohydrate79.322.83
Calorific value (kcal/g)351.3233.42
pH5.56(0.00).52(0.01)

(The values in the parentheses are standard deviation)

Abstracts

  1. Optimization of Palmyrah (Borrasus flabellifer) Fruit Pulp in Different Varieties of Fruit Yogurts

Sangheetha S., Wansapala M. A. J., Gnanasharmala A. and Srivijeindran S.

International Conference on Multidisciplinary Approaches 2014, University of Sri Jayewardenepura, 2014, 129.

2.               Improving the quality of palmyarh bottled toddy and its partial clarification

Subajini Mahilrajan, Robika Kailayalingam, Srithayalan Srivijeindran and Ponnuchamy Navaratnam (July 2014)

Proceedings of the Peradeniya Univ. International Research Sessions, Sri Lanka, Vol. 18, p 520.

  • Preliminary phytochemical screening of extracts of palmyrah fruit pulp Nilushiny Aloysius Manoharan, Subajini Mahilrajan and SriThayalan SriVijeindran (July 2014) Peradeniya University International Research Session, Sri Lanka, Volume 18, pp545.

4.               Going local: Exploring consumer behavior and motivations for palmyrah based products

Shaliny S., and Sri Thayalan S.

Proceedings of International Conference on contemporary Management -2014 (ICCM- 2014), pp 779-789.

  • Standardization and enhancement of quality of Palmyrah (Borrasus flabellifer) jaggery Mary  S.,    Velauthamurty K. , Srivijeindran S. , and sashikesh G. (2014) International Conference on Multidisciplinary Approaches 2014, University of Sri Jayewardenepura, pp 123.

6.               Assessment of water purification capabilities of activated charcoal prepared from palmyrah (Borassus flabellifer) kernel shell by different carbonization and chemical activation processes

Mary S., Sashikesh G., Nilushiny A.M., and Srivijeindran S.

Proceeding of International Conference on Multidisciplinary Approaches 2015, pp 201.

7.               Determination of nutritional facts of palmyrah (Borassus flabellifer) sap based products existing in the market of Jaffna peninsula.

Nilushiny A.M., Arachige J.W.M., Mary S., and Srivijendran S.

Proceeding of International Conference on Multidisciplinary Approaches 2015, pp 227.

8.               Comparative studies of functional properties of palmyrah shoot flour with wheat and rice flour

Subajini Mahilrajan, Robika Kailayalingam, Nilushiny Aloysius Manoharan and SriThayalan SriVijeindran.

9.               Formulation and quality assessment of compost prepared with different compositions of palmyrah resources

A.M. Nilushiny, T. Karunaithan, S.Mary, S.SriVijeindran and A.Jeyakanth International Conference on Dry Zone Agriculture– ICDA 2015, 60.

10.         Effect of Furnacing Time Periods and Temperature on Purity of Quick Lime for the Production of palmyrah (Borrasus flabellifer) jaggery, (2016)

Mary S and Srivijeindran S

International Symposium on Agriculture and Environment. pp 241.

11.         Effect of different heat treatments and additives on sustaining ethanol content of palmyrah toddy

S.Mary, M. Senthilnanthanan, A.M. Nilushiny and S.SriVijeindran Proceedings of the 4 th International conferences of Eastern University, 2016.

  1. Potential of Palmyrah (Borassus flabellifer) leaf powder as ruminant feed and its chemical and physicochemical properties, (2016).

Vasanthakumar Sethurajah, Sangheetha Sriskandarajah, Karunainathan Thirunavukkarasu and Srivijeindran Srithayalan.

Accepted for Peradeniya University International Research Session, Sri Lanka

13.         Consistency of the quality of palmyrah jaggery produced from palmyrah treacle during offseason at a production facility in Jaffna, (2016).

Shanmugam Mary, Rajaratnam Shanthini and Srithayalan Srivijeindran. Accepted for Peradeniya University International Research Session, Sri Lanka.

14.         Effect of heat treatment on keeping quality of palmyrah sweet Sap

Surenther S., Mahilrajan S., Chandrasena G., Robika K. and SriVijeindran

Accepted for International Conference on Dry Zone Agriculture– ICDA 2016, Sri Lanka

1. Publication in National conference

1.       Comparative Study of Proximate Composition of Palmyrah Pinattu and Flour (Odiyal, Boiled odiyal)

Srikantharasa S., Mahilrajan S., Wijesinghe P. J. A. W, G. Bandra and S SriThayalan Proceedings of the 6th Research symposium of Uwa wellasa University, Sri Lanka, January 28- 29, 2016.

2.       Analysis of Biochemical properties of palmyrah leaf flour: Utilize as a commercial cattle feed.

Vasanthakumar S., Sangheetha S, Srivijeindran S. and Sashikesh G. Proceedings of the 23 rd

Annual Session of Jaffna Science Association, 2016.

  • Root extent and canopy coverage of palmyrah palm in the Jaffna District. Vasanthakumar S., Karunainathan S. and Srivijeindran S.

Proceeding on National Symposium Agriculture 2015 pp 25-26.

4.       Development of palmyrah (Borrasus flabellifer) tuber based precooked supplementary food – “Palmyrah nutrimix”

Piratheepan S., Sangheetha S., Perera O. D. A. N., Srivijeindran S., Jayasinghe C.V. L.

Peer reviewed proceedings of Wayamba International Conference 2014, Wayamba University of Sri Lanka, 2014, 219

5.       Optimization  of  sponge  cake  formulation     with    palmyrah     food    resources                                                                               and evaluation of its quality characteristics

KasthaniK., Sangheetha S., Vasantharoopa S., and Srivijeindran S. Peer reviewed proceedings of the annual conference – 2014,

Jaffna Science Association, 2014, 70.

6.       Effect of packaging materials on shelf life of palmyrah food resource cake

Kasthani K., Sangheetha S., Vasantharoopa S. and Srivijeindran S.,

Peer reviewed proceedings of the annual conference – 2014, Jaffna Science Association, 2014, 71.

7.       Effect of palmyrah coir dust on physicochemical properties of potting media and on growth and yield of chilli.

Puthisigamani S., Jayasinghe H.A.S.L,. Gunadasa H.K.S.G, Nilushiny A.M. and Sri Vijeindran S.

5th Research Symposium on Value addition for sustainable development, UwaWellasa University of Sri Lanka, 2014, 40-42

2. Publication in news papers

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