Coconut Research Institute Repository

Fernando, S.C.

Weerakoon, L.K.

Gunathilake, T.R.

COCOS (2004), 16, 01 - 10 
Printed in Sri Lanka 
MICROPROPAGATION OF COCONUT THROUGH 
PLUMULE CULTURE 
S C Fernando, L K Weerakoon and T R Gunathilake 
Coconut Research Institute, Lunuwila, Sh Lanka 
ABSTRACT 
Plumule tissues have found to be more responsive explants for clonal 
propagation of coconut. In the present study, the feasibility of using plumule 
explants for clonal propagation of a local coconut variety (Sri Lanka Tall) 
was assessed. Plumules were cultured in three basal media (MS, Y3 and 
72) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (24 - 400 uM) 
and activated charcoal. Somatic embryogenesis and plant regeneration 
were induced by incorporating of abscisic acid (ABA) into the medium. 
Attempts were made to improve plant regeneration by applying high agar 
concentration-induced water stress, cytokinin or silver nitrate in combination 
with ABA. 
The results revealed that callus can be initiated from plumule explants 
cultured in all three basal media tested. The callus formation depended on 
the concentration of 2,4-D in the media and the best effect was observed 
with the lower level (24 uM) tested. The plant regeneration frequency 
observed with ABA treatment was 4.4 %. The combination of ABA with high 
agar concentration-induced water stress doubled the plant regeneration 
frequency. About 50 % of the regenerated plants could be successfully 
acclimatized and field planted. 
The results of the study indicate the possibility of regenerating plants from 
plumule explants of coconut. The method can be used for multiplication of 
improved seed material. However, the plant regeneration efficiency should 
be further improved for mass scale propagation of coconut using plumule 
explants. 
INTRODUCTION 
The coconut palm is a major plantation crop and the most important palm of 
the tropics. Due to its economic importance, breeding for crop improvement 
is of high priority. Coconut is a cross-pollinated and heterozygous plant, 
which is so far, propagated exclusively through seeds. Clonal propagation of 
coconut is of prime importance for rapid multiplication and distribution of the 
best genotypes obtained through conventional breeding. Furthermore, it 
1 
would also allow rapid m< Implication of selected parental palms for 
establishing seed gardens and selected individuals, exhibiting resistance to 
biotic and/ or abiotic stresses, for increasing the yield. 
Initial attempts have been made to micropropagate coconut through 
different explants such as immature inflorescence (Branton and Blake, 
1983), tender leaf (Raju et al., 1984) and immature zygotic embryo 
(Karunaratne and Periyapperuma, 1989). Later studies by Hornung (1995) 
and Chan et al. (1998) have shown that the response of plumules (embryo 
meristem and first leaves) to in vitro culture conditions is better than that of 
other tissues. Therefore, plumules could be used as a model for clonal 
propagation of coconut. As plumules are excised from zygotic embryos, they 
can only produce clones of palms with unknown performance. However, if 
the embryos are obtained by controlled pollination of selected parents, the 
clones from these embryos are likely to show many of the desired 
characteristics of the parent palms (Chan er al., 1998). 
This paper reports the progress of clonal plant regeneration of a local 
coconut variety (Sri Lanka Tall) using plumule explant. 
MATERIALS AND METHODS 
Plant material 
Mature nuts (12-14 month-old) of the variety Sri Lanka Tall coconut were 
used as the source of mature zygotic embryos. 
Callogenesis 
Mature embryos were cultured in modified Eeuwens Y3 liquid medium 
(supplemented with 0.1 uM 2,4-D, 5 uM 6-benzylaminopurine (BAP), 6 % 
(w/v) sucrose and 0.25 % (w/v) activated charcoal [Fluka]), optimized for 
mature zygotic embryo culture of coconut (Weerakoon er al., 2002). After 
incubation in dark (at 30 + 1 °C) for 15-17 days, plumules were excised from 
these embryos (Fig. 1a). They were crushed and cultured in 28 ml screw 
capped vials containing 10 ml of culture medium. The effect of three basal 
media [medium 72 supplemented with 24 uM 2,4-D ( Karunaratne and 
2 
Figure 1: Plant regeneration from plumule explants of coconut, a - Mature 
zygotic embryo (pre-cultured in germination medium for 15 days) 
ready for plumule excision; b - Callus derived from a plumule 
explant; c - Shoots regenerated from plumule-derived callus; 
d - Field-planted plumule callus-derived coconut plant. 
3 
Periyapperuma, 1989), MS (Murashige and Skoog, 1962) supplemented 
with 400 uM 2,4-D (Hornung, 1995) and Y3 (Eeuwens, 1978) supplemented 
with 100 uM 2,4-D (Chan et al., 1998)] on callogenesis was tested. The 2,4-
D concentrations used were based on previous expe^/nents. All the media 
were solidified with 0.8 % (w/v) agar (Park Scientifk-riand supplemented with 
0.25 % (w/v) activated charcoal (Pharmacos). Cultures were incubated at 30 
± 1 °C for 10 weeks in the dark. Callogenesis was repeated four times each 
time with 15 replicates. 
Somatic embryogenesis and plant regeneration 
Embryogenic callus initiated from plumules cultured in medium 72 
supplemented with 24 uM 2,4-D was used. Callus separated from the 
original explants, was subcultured in medium 72 supplemented with 16 \M 
2,4-D for 4 weeks before transferring to somatic embryoNnduction medium. 
Somatic embryogenesis was induced by subculturing of callus in medium 72 
supplemented with 5 uM abscisic acid (ABA) (filter-sterilized) and 6 % (w/v) 
sucrose for 5 weeks. Maturation of somatic embryos was achieved by 
transferring them to medium 72 (devoid of hormones) for 4 weeks. Somatic 
embryo germination was achieved in Y3 solid medium. Regenerated shoots 
were maintained in Y3 solid for 4-6 months by subculturing at 4-weekly 
intervals (Fernando and Gamage, 2000). During somatic embryo induction, 
cultures were maintained in screw-capped vials containing 10 ml of media 
supplemented with 0.8 % (w/v) agar and 0.25 % (w/v) activated charcoal 
(Pharmacos) whereas during somatic embryo maturation and germination, 
they were maintained in 100 ml flasks containing 50 ml of media 
supplemented with 0.8 % (w/v) agar and 0.25 % (w/v) activated charcoal 
(Fluka). Cultures were maintained at 30 ± 1 °C in the dark. Regenerated 
shoots were maintained at 16-h photoperiod (intensity 17 umol m"2 s'2) until 
they were ready for transferring to ex vitro. If necessary, roots were induced 
on shoots by a pulse treatment of 500 uM indole acetic acid (IAA) as 
described by Fernando and Gamage (1994-1995). 
In the present study, attempts were made to improve plant regeneration 
frequency by testing the effect of high agar concentration-induced water 
stress (2 % agar in somatic embryo induction medium during the initial 2-
week period), 5 jiM cytokinin (BAP or isopentyl adenine [2iP]) and 1-10 uM 
AgN03 in combination with ABA. The experiments were repeated at least 
three times with 30 replicates per treatment. 
Ex vitro establishment of regenerated plants 
The fully developed plants with about three leaves and well-developed root 
systems were de-flasked, rinsed well with water and drenched in a mixture 
of fungicide (0.025 % benlate) and diluted nutrient solution for a few hours 
4 
before transfer to clear polypropylene bags containing sterilized potting 
mixture (river sand and compost 1:1) moistened with a diluted nutrient 
solution. The bags were initially kept sealed (for maintaining high humidity) 
in the glass house. After one week, the cover was gradually removed to 
reduce the relative humidity. The plants were watered regularly and a liquid 
fertilizer (Maxicrop™) was sprayed every 10 days. In addition, a solution 
containing minerals of Y 3 medium (Eeuwens, 1978) was applied at two 
weekly intervals. After 6-8 months, plants were repotted in larger polybags 
containing a potting mixture of top soil, compost, river sand, dried cow dung 
and coir dust (2:1:1:1:0.5). The plants were kept in a shade-house for about 
3-4 months before transferring to direct sunlight. During this period, a liquid 
fertilizer (Maxicrop™) was sprayed on to plants at 10-day intervals and 20 g 
of young palm mixture (urea: saphos phosphate: muriate of potash, 2:3:2) 
was applied to each plant at two weekly intervals. After exposure to direct 
sunlight for about 1-2 months, they were ready for field planting. Several 
plants were transferred to field following the recommendations of the 
Coconut Research Institute of Sri Lanka (CRISL). 
Statistical analysis 
Analysis of Variance was carried out to test the differences among 
treatments. 
RESULTS AND DISCUSSION 
Callogenesis 
Coconut somatic embryogenesis usually occurs through a callus phase. 
Therefore, production of embryogenic callus is a prerequisite for successful 
plant regeneration. Among several other factors, the presence of an auxin 
in the medium is critical for successful callogenesis (Auge, 1995). The most 
commonly used auxin in coconut tissue culture media is 2,4-D and its 
concentration depends on the explant and concentration of activated 
charcoal in the culture medium (Verdeil and Buffard-Morel, 1995). In the 
present study, the effect of culture media previously used for plumule culture 
of exogenous coconut varieties [MS supplemented with 400 u.M 2,4-D 
(Hornung, 1995) and Y3 supplemented with 100 u,M 2,4-D (Chan et al., 
1998)] and immature embryo culture of Sri Lankan coconut varieties 
[medium 72 supplemented with 24 uM 2,4-D (Karunaratne and 
Periyapperuma, 1989)] on callogenesis from plumule tissues of Sri Lanka 
Tall coconut was tested. The study was extended to assess the effect of 
medium 72 supplemented with higher level (100 uM) of 2,4-D and media MS 
and Y3 supplemented with lower level (24 \iM) of 2,4-D. 
5 
The results revealed that the callusing frequencies were higher and 
comparable in all the three basal media supplemented with lower (24 uM) 
concentration of 2,4-D. Increase in the concentration of 2,4-D up to 100 or 
400 u.M resulted in a decrease in callusing (Table 1). Despite the statistically 
non-significant decrease in callusing frequency in Y3 supplemented with 
100 uM 2,4-D, callus initiated in high 2,4-D containing media showed a poor 
growth compared to that of lower 2,4-D media. 
Table 1: The effect of different culture media on callogenesis in plumule 
explants of coconut 
Basal medium 2,4-D concentration Callusing frequency 
(uM) (%) 
72 24 55.2 
100 40.9 
MS 24 49.4 
400 35.5 
Y3 24 54.3 
100 46.7 
Significance p<0.05 
CV (%) 17.3 
LSD 12.1 
In this study, callusing frequencies in plumule explants cultured in previously 
published plumule culture media (MS+ 400 uM 2,4-D and Y3+ 100 uM 2,4-
D) were lower (35.5 and 46.7 % respectively) than those reported by 
Hornung (1995) (75 %) and Chan et al. (1998) (60 %) . In the present study, 
the published media were tested with high levels of 2,4-D but without the 
specific type of activated charcoal, which had been used by the relevant 
research groups. Different types of charcoal have different adsorption 
capacities, which results in variations in active auxin concentration in the 
medium. Therefore, the use of high levels of 2,4-D in the presence of 
unspecific type of charcoal could be the reason for the low callusing 
frequencies observed. This was further confirmed by the increase of callus 
production in those media by reducing the concentration of 2,4-D. With 
agreement of the results of this study, Saenz et al. (1999) reported that 
callusing in plumules cultured in medium described by Chan et al. (1998) 
could be increased up to 100 % by using a different type of charcoal. 
Furthermore, the use of different coconut genotypes for studies of different 
research groups might also have contributed to the variable results. 
Somatic embryogenesis and plant regeneration 
Generally, somatic embryogenesis in coconut callus has been induced by 
gradual reduction of auxin concentration with a corresponding increase in 
the cytokinin level in the medium (Buffard-Morel et al., 1995). In contrast, 
6 
somatic embryogenesis in coconut has also been induced by an early 
increase in 2,4-D concentration followed by a gradual reduction (Verdeil et 
al., 1994) or a rapid decrease in 2,4-D concentration and application of high 
concentration of cytokinin (Chan era/., 1998). However, research conducted 
at the CRISL revealed that an alternate treatment, removal of auxin and 
incorporating ABA to the culture medium, give rise to more consistent plant 
regeneration from coconut callus (Fernando and Gamage, 2000). Similarly, 
Samosir et al. (1999) reported improved somatic embryogenesis in coconut 
with the use of ABA. 
In this study, embryogenic callus (Fig. 1b) treated with 5 uM ABA, became 
more compact. After 5 weeks in the medium with ABA, whitish globular 
structures were visible. Upon transfer to the medium devoid of hormones for 
four weeks, some of the embryogenic structures elongated whereas the 
others developed into haustorial tissues or fused shoot-like structures. On 
repeated subculture to the Y 3 medium, the somatic embryos produced 
shoots (Fig. 1c). 
Incorporating ABA to the culture medium resulted in normal development of 
somatic embryos that can develop into complete plants. However, the plant 
regeneration frequency was low (4.4 %) . Among the treatments tested, only 
the application of high agar-induced water stress in combination with ABA 
had a positive effect and it doubled (9.0 %) the plant regeneration frequency 
(Table 2). The use of ABA in combination with osmotically active substances 
has resulted in improved somatic embryogenesis and plant regeneration in 
coconut (Samosir et al., 1999), rubber (Linossier et al., 1997) and several 
other crops. The moisture stress, induced by application of high agar 
concentration in the medium has also shown encouraging results in plant 
regeneration of rice (Jain et al., 1995). The effect of such a stress has not 
been tested for coconut previously. The enhanced results observed in this 
study might be due to promotion of the expression of embryo-specific 
genes, improved embryo development and increased production of storage 
reserves caused by moisture stress. 
Table 2: Effect of high agar concentration - induced water stress on plant 
regeneration 
Treatment Plant regeneration 
(%) 
ABA 4.4 
ABA + high agar-induced water stress 9.0 
Significance P= 0.01 
CV (%) 31.8 
LSD 0.3 
7 
An embryogenic callus clump that regenerated plants produced an average 
number of 1-2 plants. The clonal plants were maintained in Y3 medium until 
they produced 2-3 leaves and a good root system. When shoots did not 
produce roots spontaneously, the application of an IAA pulse resulted in 
successful rhyzogenesis in more than 90 % of the treated shoots. When the 
complete plants were transferred to the potting medium for acclimatization, 
about 50 % of plants survived and they could be transferred to field 
successfully (fig. Id). 
The results of.this study indicated the possibility of regenerating clonal 
plants of the local variety Sri Lanka Tall using plumule explants. The 
concentration of 2,4-D in the callusing medium was found to be the critical 
factor determining success in callogenesis. Application of ABA in 
combination with high agar level-induced water stress improved plant 
regeneration frequency. The resulting plants could be acclimatized and field 
planted successfully. This method can be applied for multiplication of 
improved seed material. However, the plant regeneration efficiency of the 
current protocol should be further improved for mass scale propagation of 
coconut using plumule explants. 
ACKNOWLEDGEMENTS 
The authors wish to thank the staff of the Tissue Culture Division for their 
assistance in carrying out the experiments. 
REFERENCES 
Auge, R. (1995). The physiological phenomena related to the realization of 
culture in vitro. In: Razdan, M.K. (Ed.), In vitro culture and its 
application in horticulture, Science Publishers Inc., USA, p. 7-31. 
Branton, R.L. and Blake, J. (1983). Development of organized structures in 
callus derived from explants of Cocos nucifera L. Ann. Bot. 52:673-
678. 
Buffard-Morel, J. Verdeil, J.L. Dussert, S. Magnaval, C. Huet, C. and 
Grosdemange, F. (1995). Initiation of somatic embryogenesis in 
coconut (Cocos nucifera L ) . In: Oropeza, C. Howard, F.W. and 
Ashburner, G.R. (Eds.), Lethal Yellowing: Research and practical 
aspects, Kluwer Academic Publishers, Dordrecht, pp.217-223. 
Chan, J.L. Saenz, L. Talavera, C. Hornung, R. Robert, M. and Oropeza, C. 
(1998). Regeneration of coconut (Cocos nucifera L.) from plumule 
8 
explants through somatic embryogenesis. Plant Cell Rep. 17:515-
521. 
Eeuwens, C.J. (1978). Effect of organic nutrients and hormones on growth 
and development of tissue explants from coconut (Cocos nucifera) 
and date (Phoenix dactylifera) palms cultured in vitro. Physiol. Plant. 
42:173-178. 
Fernando, S.C. and Gamage, CK. (1994-1995). Clonal propagation of 
coconut: Improved culture conditions for rhizogenesis. Cocos 10:20-
25. 
Fernando, S.C. and Gamage, C.K.A. (2000). Abscisic acid induced somatic 
embryogenesis in immature embryo explants of coconut (Cocos 
nucifera L ) . Plant Sci. 151:193-198. 
Hornung, R. (1995). Micropropagation of Cocos nucifera L. from plumular 
tissues excised from mature zygotic embryos. Plantations, 
Recherche, Developpment 212:38-41. 
Jain, R.K. Khehra, G.S. Lee, S.H. Blackhall, N.W. Marchant, R. Davey, M.R. 
Power, J.B. Cocking, E.C. and Gosal, S.S. (1995). An improved 
procedure for plant regeneration from indica and japonica 
protoplasts. Plant Cell Rep. 14:515-519. 
Karunaratne, S. and Periyapperuma, K. (1989). Culture of immature 
embryos of coconut, coconut, Cocos nucifera L.: callus proliferation 
and somatic embryogenesis. Plant Sci. 62:247-253. 
Linossier, L. Veisseire, P. Cailloux, F. and Coudret, A. (1997). Effects of 
abscisic acid and high concentrations of PEG on Hevea brasiliensis 
somatic embryo development. Plant Sci.124:183-191. 
Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and 
bioassay with tobacco tissue cultures. Physiol. Plant. 15:473-497. 
Raju, C.R. Prakash Kumar, P. Chandramohan, M. and Iyer, R.D. (1984). 
Coconut plantlets from leaf tissue cultures. Plantation Crops 12: 5-
81. 
Saenz, L. Chan, J.L. Souza, R. Hornung, R. Rillo, E. Verdeii, J.L. and 
Oropeza, C. (1999). Somatic embryogenesis and regeneration in 
coconut from plumular.explants. Countries. In: Oropeza, C , Verdeii, 
J.L., Ashburner, G.R., Cardena, R. and Santamaria, J.M. (Eds.), 
Current Advances in Coconut Biotechnology, Kluwer Academic 
Publishers, Dordrecht, pp.309-319. 
9 
Samosir, Y.M.S. Godwin, I.D. and Adkins, S.W. (1999). The use of 
osmotically active agents and abscisic acid can optimize the 
maturation of coconut somatic embryos. In: Oropeza, C , Verdeil, 
J.L., Ashburner, G.R., Cardena, R. and Santamaria, J.M. (Eds.), 
Current Advances in Coconut Biotechnology, Kluwer Academic 
Publishers, Dordrecht, pp.341-354. 
Verdeil, J.L. and Buffard-Morel, J. (1995). Somatic embryogenesis in 
coconut (Cocos nucifera L). In: Bajaj, Y.P.S.(Ed.), Somatic 
Embryogenesis and Synthetic Seeds I, Biotechnology in Agriculture 
and Forestry, vol. 30 Spring-Verlag, Berlin, pp. 299-317. 
Verdeil, J.L. Huet, C. Grosdemange, F. and Buffard-Morel, J. (1994). Plant 
regeneration from cultured immature inflorescences of coconut 
(Cocos nucifera L.): evidence for somatic embryogenesis. Plant Cell 
Rep. 13:218-221. 
Weerakoon, L.K, Vidhanaarachchi, V.R.M, Feranando, S.C , Fernando, A 
and Gamage, C.K.A (2002). Increasing the efficiency of embryo 
culture technology to promote coconut germplasm collecting and 
exchange in Sri Lanka. In: Coconut Embryo In Vitro Culture: Part II. 
Florent Engelmann, Pons Batugal and Jeffrey Oliver (Eds), IPGRI. 
27 - 40. 
10 


Micropropagation of coconut through plumule culture

Fernando, L.C.P.

Aratchige, N.S.

Kumari, S.L.M.L.

Appuhamy, P.A.L.D.

Hapuarachchi, S.L.M.L.

Development of a method for mass rearing of neoseiulus baraki, a mite predatory on the coconut mite, aceria guerreronis

Chandrasir, K.A.S.

Effectiveness of used engine oil in the management of coconut mite, Aceria guerreronis (Acari: Eriophyidae)

Wijebandara, D.M.D.I.

Ranasinghe, C.S.

Response of rapid decline affected coconut (cocos nucifera L,) palms to micro-nutrients and common salt

Aratchige N.S.

Lesna, I.

Sabelis, M.W.

Below-ground plant parts emit herbivore-induced volatiles: Offactory responses of a predatory mite to tulip bulbs infested by rust mite

Coconut Research Institute

Jayasekera, C.

Everard, J.M.D.T.

Appuhamy, P.A.H.N.

Idirisinghe, I.M.S.K.

Report of the coconut research institute for 2004

Peiris, S.

Distribution patterns of coconut mite Aceria guerreronis Keifer and its predator Neoseiulus aff. paspalivorus in coconut palms.

Bandaranayake, C.K.

Kearsey, M.J.

Genome mapping. QTL analysis and MAS: Importance, principle, constraints and application in coconut

Premasiri, R.D.N.

Silva, L.R.S.

Effect of mulches and cover crops on water status and gas exchange of coconut (Cocos nucifera L.) palms in gravelly soils

Gunathilake, K.D.P.P.

COCOS (2003), 15 31 - 37 
Printed in Sri Lanka 
STUDIES ON DEVELOPMENT AND STORAGE 
OF BOTTLED PENIPOL 
K.D.P.P. Gunathilake and C. Jayasekera 
Coconut Research Institute.Lunuwila, Sri Lanka 
ABSTRACT 
Penipol or "candied coconut" made from coconut and sugar, is used as a 
sweet filler in traditional food preparations in Sri Lanka. A ready-to-serve, 
preserved Penipol is likely to have a good demand among both local urban 
consumers and Sri Lankan communities overseas. The main objective of 
this study was to develop a simple technique to produce a bottled Penipol 
with an extended shelf life, in order to exploit this perceived demand. 
In a preliminary trial it was established that a Penipol product, acceptable to 
consumer in terms of colour, taste and texture, was derived from a blend of 
coconut and sugar in the ratio of 2.5:2.0. This blend with added Vitamin E 
(80 ppm), salt, spices was used to prepare Penipol for the studies on 
Pasteurization and storage, and the physico-chemical analyses. This 
product contained 3.58% protein, 20.78% fat, 7.00% fiber and 0.83% 
minerals and its water activity, pH and total soluble solids were 0.8, 5.5 and 
65°, respectively. 
Penipol, bottled in jars, was pasteurized in boiling water. Five heat 
treatments (boiled for 15, 30, 45, 60 minutes and no heat treatment) were 
tested. Microbial analysis of the Penipol, stored for one month, showed that 
pasteurizing for 30 minutes was adequate to prevent microbial growth and 
achieve commercial sterility. 
The keeping quality of bottled Penipol, pasteurized for 30 min., was studied 
by conducting sensory evaluations, at 3-monthly intervals, over a 12 months 
storage period. Statistical analysis showed that there is no significant 
difference in sensory properties between freshly prepared and preserved 
Penipol throughout the studied period. The free fatty acid levels remained 
unchanged at 0.05+/-0.02 (as % of lauric acid). Results indicated that 
Penipol can be preserved for at least 12 months by applying a simple 
bottling technique with 30 min. pasteurization in boiling water. 
INTRODUCTION 
"Penipof (Candied coconut) is a coconut based product which consists of 
grated coconut, caramelized sugar (or treacle), salt and a few selected 
spices. It is used as an ingredient or "sweet filler" in the preparation of sweet 
31 
foods like "Laveria", "Imbul kiribath", "Halapa", pancake etc. However, 
Penipol cannot be kept for more than two days without refrigeration because 
it develops rancidity and is subject to microbial spoilage. Rancidity 
development in food is mainly due to the lipids undergoing complex 
chemical changes and reacting with other food constituents during 
processing and storage. Addition of antioxidants such as Tocopherol during 
processing delays or suppresses rancidity development. Tocopherol exerts 
its maximum effectiveness at relatively low concentrations of approximately 
80ppm (Bockish, 1998). 
The shelf stability of food products are prolonged by the application of 
bottling or canning technology where food and its container are rendered 
commercially sterile by application of heat alone or in combination with pH 
adjustment or water activity (Cruess, 1958). There is an increasing demand 
for convenience foods to save time devoted to preparing food. Ready-to-
serve, bottled Penipol is a product which has a potential in the local market 
and may also have export potential especially for expatriate Sri Lankan 
communities in Europe and Middle East. It is in this context that the present 
study set out to develop "bottled Penipot' with an extended shelf life. 
MATERIALS AND METHOD 
Ratio of coconut to sugar in Penipol: 
Different quantities of good quality scraped coconut were blended with a 
standard hot caramelized sugar solution (2 kg sugar in 800 ml water-Total 
Soluble Solids (TSS) 85°) to determine the best proportion of these 
ingredients. Ten samples from each blend were observed for taste, colour 
and texture. Based on these observations, the best formulation was 
selected for preparing Penipol used in this studies. 
Preparation of Penipol: 
Penipol was prepared as follows 
1. Sugar (3.6 kg) was heated in a cooking pan with 1.44 liter water until the 
total soluble solid level reached to 85°. 
2. Vitamin E (400 mg) and salt (15 g) were dissolved in the caramelized 
sugar solution prior to mixing of coconut. 
3. Scraped coconut (4.5 kg) was added to this sugar solution and was 
mixed. 
32 
4. This mixture was heated further period of 10 min. with continue mixing. In 
the final stage of cooking, 5g of crushed mixture of cardomom and 
pepper (2:1) was added and mixed thoroughly. 
Studies on bottling technology: 
i. Pasteurization studies: 
Freshly prepared Penipol was filled into pre-sterilized jars, while it was still 
hot, leaving a headspace of 1cm. The unsealed jars were placed in a boiling 
water bath for 10 min. The jars were then sealed with caps, manually. The 
sealed Penipol jars were subjected to the following heat treatments. Three 
jars were used per treatment. 
T1- Control-No heat treatment 
T2-15 min. in boiling water 
T3- 30 min. in boiling water 
T4- 45 min. in boiling water 
T5- 60 min. in boiling water 
After a storage period of one month, microbial analyses were done by serial 
dilution of the pour plate method described in AOAC (1995). Microbial 
counts were used to determine the optimum heat treatment to achieve 
commercial sterility. 
Storage studies: 
A batch of bottled Penipol pasteurized for 30 min. in a boiling water bath 
was prepared for the shelf life study. The bottles were stored for 12 months 
at ambient condition (30° +1-2 C) and samples drawn at three monthly 
intervals were evaluated by 30 untrained judges. At each sampling Laveria 
with freshly prepared Penipol and bottled Penipol as the filler, were 
presented to the panel for grading their colour, flavour and overall 
acceptability. A five point Hedonic scale with 5 for excellent and 1 for highly 
disliked was employed for scoring. Free fatty acids level of the samples was 
also determined. 
Measurement of physico-chemical properties: 
Total soluble solid levels and pH of Penipol were measured by using a 
refractometer (ATAGO NIE) and a pH meter (Cyb er Scan 510), 
respectively. Water activity of Penipol was determined by measuring the 
Equilibrium Relative Humidity (ERH) using an electric humidity sensor 
(CHS-UPS TC 2, TDK). 
33 
Chemical analysis: 
Moisture, protein, fats, fiber and total ash contents were determined by 
standard AOAC method (1990) and the carbohydrate % was determined by 
difference. The free fatty acids content of the stored samples was 
determined employing the method prescribed by Pearson (1973) 
RESULTS AND DISCUSSION 
The results of evaluation of Penipol formulations with various ratios of sugar 
to coconut are shown in Table 1. All the formulations tested gave products 
with a good taste. Coconut to caramel sugar (85°) at 1:1 ratio gave a 
product with a juicy texture because of an excess of sugar. Coconut to 
sugar at ratios of 3:2 and 3.5:2 gave products with a light colour indicating 
insufficient caramel sugar. Coconut to caramel at the ratio of 2.5 to 2.0 was 
the only formulation that gave an acceptable product with good taste, 
texture and colour. 
Table 1: Sensory evaluation of Penipol formulations 
Formulation Blends Observations Consumer 
acceptability 
of the product 
1 Coconut 2.0 kg 
Caramel sugar 2.0 kg 
Taste-good 
Colour-good 
Texture-juicy 
Unacceptable 
2 Coconut 2.5 kg 
Caramel sugar 2.0 kg 
Taste-good 
Colour-good 
Texture-good 
Acceptable 
3 Coconut 3.0 kg 
Caramel sugar 2.0 kg 
Taste-good 
Colour-light 
Texture-good 
Unacceptable 
4 Coconut 3.5 kg 
Caramel sugar 2.0 kg 
Taste-good 
Colour-too light 
Texture-good 
Unacceptable 
Chemical analysis: 
The composition of Penipol (coconut 2.5:sugar 2.0) is presented in Table 2. 
34 
Table 2: The composition of Penipol 
Constituent Level (dry weight 
basis) 
Moisture 29.68% 
Crude protein 3.58% 
Crude fat 20.78% 
Crude fiber 7.00% 
Total ash 0.83% 
PH 5.50 
Water activity 0.80 
Total soluble solidsfTSS) 65° 
Each value represents mean of three samples. 
The composition is given as a percentage on 
dry weight basis per 100g 
Based on the proximate composition, the total energy value of Penipol was 
391 kcal/100g. Since the product contains whole coconut kernel it is rich in 
dietary fiber and coconut protein. As Penipol contains about 20.78% fat, the 
addition of an antioxident into the mixture is important to avoid rancidity 
development. Penipol is a low acid product, but has 65° brix (total soluble 
solids) and 0.8 water activity, indicating high osmotic pressure in the food. 
Pasteurization studies: 
Penipol jars stored without any heat treatment or after heating for 15 min in 
boiling water were spoiled within one month. Microbial analysis of samples 
heated for 30 min. or longer showed no evidence of microbial growth. The 
low water activity (0.8) of the product and 30 min. heat processing appeared 
to be adequate to prevent microbial activity. Most bacteria do not grow 
below a water activity of 0.91, whereas spoilage moulds can grow even at 
water activity as low as 0.80. Osmophilic yeast has been reported to grow at 
water activity value of 0.61 (Jay 1996). However, most spoilage yeasts do 
not grow below a water activity of 0.88. At any given temperature, the ability 
of microorganisms to grow is reduced as the water activity is lowered (Jay 
1996). Therefore with, lower than conventional heat treatment in 
combination with a low water activity of 0.8, it is possible to obtain a bottled 
Penipol with sterility up to required commercial standards. 
Shelf life study: 
Mean sensory scores for colour, appearance, taste, smell and overall 
acceptance of preserved Penipol samples drawn during the 12 months 
storage are shown in Table 3.0. 
35 
Table 3: Median values of sensory scores at three months intervals over a 
period of one year storage 
Sensory 
attributes 
Fresh 
sample 
Stored for 
3 months 
Stored for 
6 months 
Stored for 
9 months 
Stored for 
12 months 
F ratio 
Colour 4.6+/-0.3 4.6+/-0.1 4.6+/-0.1 4.6+/-0.3 4.6+/-0.2 0.250N!' 
Appearance 4.7+/-0.3 4.7+/-0.2 4.7+/-0.1 4.7+/-0.3 4.7+/-0.2 0.690NS 
Taste 4.5+/-0.4 4.5+/-0.3 4.5+/-0.2 4.4+/-0.5 4.4+/-0.5 0.607NS 
Smell 4.3+/-0.3 4.3+/-0.1 4.3+/-0.4 4.3+/-0.3 4.3+/-0.1 0.550NS 
Overall 4.5+/-0.1 4.5+/-0.3 4.5+/-0.3 4.5+/-0.4 4.5+/-0.2 0.250NS 
accept. 
It is evident that there is no significant difference in the sensory attributes 
tested between freshly prepared and preserved Penipol. This indicates 
bottled Penipol, pasteurized 30 min in a boiling water bath can be stored for 
at least 12 months without any deterioration or loss of consumer 
acceptance. 
Hydrolysis of ester bonds in lipids mainly occur by enzyme activity and by 
heat with moisture, resulting in the liberation of free fatty acids (Fennema, 
1995). Tocopherol is the antioxident vitamin widely used in the food industry 
to control the development of rancidity and it exerts its maximum 
effectiveness at relatively low concentrations, approximately 80ppm. 
(Blockish 1998). Although coconut contains about 40% fat, changes in free 
fatty acids content of stored samples of the Penipol were insignificant 
(p<0.05) during 12 months of storage, indicating vitamin E (80 ppm) 
together with heat processing of bottled Penipol control the rancidity 
development during storage. Throughout the study period the free fatty 
acids level was maintained at 0.05 +/-0.02 (as % of lauric acid). 
CONCLUSION 
Penipol can be preserved for at least twelve months by applying a simple 
bottling technique with 30 min. pasteurization in boiling water. 
36 
REFERENCES 
AOAC (1990) Official Methods of Analysis. Vol.2. Association of Official Analytical 
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Bockisch, M. (1998). Fats and Oils Handbook, second edition, AOCS Press, 
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Dhaliwal, M. and Hira, C.K.(2001). Effect of storage on physico-chemical and 
nutritional characteristics of carrot beet-root and carrot-black carrot juice, 
Journal of Food Science and Technology, Vol.38, No.4. 
Fennema, O.R. (1995). Food Chemistry, third edition, Marcel Dekker Inc., USA 
Jawahir, K. Badrie, N. Donawa, A. (2001). Effect of Blanching on Pickled Bitter 
Gourd (Momordica charantia). Journal of Food Science & Technology, Vol.38, 
No.2. 
Jay, J.M.J. (1996). Modern Food Microbiology, fifth edition, Chapman and Hall, 
Newyork. 
Linda, B.M. (1979). Protein Enrichment of Coco Spread with Legume Flours, 
Philippine Journal of Coconut Studies, Vol. 1V. 
Pearson, D. (1973). Laboratory Techniques in Food Analysis. Butterworth and Co. 
Ltd, England, Australia, Canada. 
Semwal, A.D. Sharma, G.K. Patki, P.E. Padmashree, A. Arya, S S. (2001). Studies 
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37 


Studies on development and storage of bottled penipol

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