WRIST ACTION SHAKER: A NOVEL METHOD FOR COLLECTING POLLEN GRAINS FROM THE INFLORESCENCE OF COCONUT (COCOS NUCIFERA L.) FOR ITS STABLE NUTRITIONAL COMPONENTS

WRIST ACTION SHAKER: A NOVEL METHOD FOR COLLECTING POLLEN GRAINS FROM THE INFLORESCENCE OF COCONUT (COCOS NUCIFERA L.) FOR ITS STABLE NUTRITIONAL COMPONENTS

Kavitha, V. Gopal *, K. Joshna and N. Balachandran

Department of Pharmacognosy, College of Pharmacy, Mother Theresa Post-Graduate and Research Institute of Health Sciences (A Government of Puducherry Institution), Puducherry, India.

ABSTRACT: To predict the cocos anther dehiscence, a novel method was devised to compile the colossal amount of pollen grains. The matured inflorescence enveloped by the spathe was collected and the anther dehiscence were noticed day by day until the shedding of entire pollen. This study was experimented for a period of eight months between (February) 2018 to 2018 (September). After an experimental trails, sieve and a new tool wrist action shaker methods were used to collect the healthy pollen grains. Proximate analysis was determined by food analysis-FSSAI and association of analytical chemicals method. The complete process of anther dehiscence took 12 -18 days and after an experimental trials the high amount of pollen grains were obtained on the twelfth day. During the month of June amount of pollen i.e. 16.21 ± 0.05 g per 100 g of the fresh flower weight was recorded in wrist action shaker whereas 2.36 ± 0.018 g per 100 g of the flower weight found in sieve method. The average dry weight of pollen grains was (9.759 g) and 0.11 ± 0.03% of moisture content was found in wrist action shaker. In addition the quality of pollen was stable at – 4 ℃ (freeze drying). The p value (p = 0.002 <0.05) is lesser than 0.05, which indicates that weight of the pollen grains is significant for wrist action shake. The total carbohydrate and protein were higher and zinc and iron were also abundant. The conclusive proof out of the two methods wrist action shaker was more efficient, reliable and cost-effective for collection and storage to use the pollen grains in the fields of pollination biology, nutraceuticals, modern herbal medicine and genetic resource conservation.

Keywords: Inflorescence, Proximate analysis, Sieve, Wrist action shake, Nutraceuticals

INTRODUCTION: The coconut palm Cocos nucifera L., (Fam: Arecaceae) popularly known as kalpa virshu, provides numerous benefits to our lives ¹.

Coconut inflorescence is monoecious, consisting both male and female flowers on the same inflorescence known as spadix, which developed from a woody sheathe or spathe.

During the flowering stage, the spathe splits lengthwise to expose the spadix ². Each spadix measures 1–1.5 m in length and contains 40–60 spikelets that bear the flowers. The male flowers are always more in number than the female flowers in the same spadix and this variation may be somewhere between a few hundreds and thousands depending upon the number of ramification in the spadix ³. The male flower has six yellow floral leaves arranged in two circles. The centre of each flower is an abortive pistal which forms the teeth at the apex, each bearing a nectar gland and six hammer-shaped stamens that yield large quantities of powdery yellow pollen ⁴.

Coconut pollen grains are monocolpate, spherical and smooth in nature when fresh, and they become ellipsoidal when dry ⁵. When hydrated a pollen tends to gain its original shape. Each pollen measures about 65 µm to 69 µm in length and 0.028 to 0.30 mm in diameter ⁶. In a male flower, the number of pollen grains present per inflorescence have been estimated about 88,000 to 1,21,000 ^{7, 8}.

Early method of pollen collection and isolation was carried out by different technology using shed microspore method by applying a force using magnetic stirrer, but this has only possible to view the image. Another  method was done by placing the spikelets in water so that the pollens will fall ⁹ were collected by using a camel hair brush, but this method had two disadvantages – very small amount of pollen was collected and also the content was reduced and the stability were loss during storage of the pollen ¹⁰.

Availability of large quantities of pollen production for controlled pollinations are possible by means of storing the pollen at freeze dry condition for germination and the viability also increased, by means of fluid bed drying method. This method of collection was efficient only for pollination and does not comprise any chemical constituents due to drying at 40 ℃ all chemical components are deteriorated ¹¹.

A matured spathe of coconut palm (Cocos nucifera) which shoots 12–14 inflorescences per year ¹². The coconut palm in general, is allogamous (out-breeding), because even though the male and female flowers are located close to each other in each inflorescence, the pollen is shed before the female flowers are ready for it. Pollens were shed during the bursting of anthers when the spikelets gets split automatically. The major phytochemicals present in C. nucifera are lignin, tannin, furfural, pentose and cellulose many researches are on-going to produce these compounds that can be used in various biological and pharmacotherapeutic applications ^13-15. Coconut pollen grains contain enormous amount of carbohydrate, proteins, lipids and secondary metabolites such has phenols, flavonoids, anthocyanins, phospholipids, minerals and vitamins and generally it is very difficult to separate these components from pollen extract ¹⁶. Recently, increasing evidence of coconut pollen possess potential therapeutic benefits including antioxidant, antibacterial, anti-inflammatory, chemotherapeutic activity, and they are also good source of food for foraging bees. Collecting large amount of pollen grain is a challenging task though. To overcome the defects observed in the earlier methods of pollen collection, this research focuses to design a new method for collecting a healthy amount of pollen grains directly from fresh spikelets.

MATERIALS AND METHODS:

Study Site: The research work was conducted at the Eco-restoration located at Pondicherry – near Ousteri Lake, about 10 km west of Pondicherry city. The matured spathe of coconut flower- Cocos nucifera was collected during the period of February 2018 to September 2018 – timing is between 8.00 am to 10.00 am.

Protection of the Inflorescence: The split spathe was kept in a clean, dry butter paper. When the matured inflorescence starts dehiscence by longitudinally splitting between spikelet, the whole process hardly took about 3 to 8 days. During these days, the pollen quantity was less due to the premature inflorescence ¹⁶.

The collected flowers were batched for a period of 8 months – the collection was done from 1^st to 19^th day after opening of spathe, i.e. the period between the opening of first female flower, which generally coincides with the bursting of the spathe and abscission of the male flowers (termed as male phase). The spadices are ivory in colour and turn brownish orange at mature stages. The mature male flowers break open the bracts to release the yellow colour pollen grains from the anthers.

Collection of Spikelets: The split spikelets were separated and collected at regular intervals in order to avoid the deviation of environmental parameters, which in turn affect the pollen release. During the period of anthers’ dehiscence, the pollen grains get released in the form of a fine dust of yellow-coloured powder. It takes 3 to 12 days to release the entire pollen from the anthers’ bract ¹⁷.

To separate the pollen grains, we devised two experimental methods that optimize the standard protocol for convenient and continuous collection of a healthy amount of pollen grains.

Isolation of Pollen Grains:

Method-1:

Sieve Method: Exactly 100 g of dried coconut flowers was taken and then transferred into a pharmaceutical sieve mesh size no. 40. The flowers were sieved for 5 min using a butter paper to collect the pollen grains separately. The yellow-coloured powder obtained after this was then weighed and the percentage of the yield was calculated. To avoid moisture, the pollen grains were immediately transferred in to an Eppendorf tube and stored at 4 ℃ to prevent contamination and germination, to improve shelf life and to prevent the viability of the pollens ⁹.

Method-2:

Wrist Action Shake Method: Exactly 100 g of the dried flowers was taken in a sterilized round bottom flask and the flask was connected to a wrist action shaker that was run for 15 min at 250 oscillations in order to separate the powdery pollens from its stamen. On continuous shaking, pollen grain got released from the woody flowers and this mixture was then transferred into a 50-ml conical tube and centrifuge at 3000 rpm for 15 min. The supernatant was discarded and the pollen grains settled at the bottom were collected and then finally sieved. The sieved pollen grains are kept in a desiccator for 1 h. to remove the moisture and then stored at freezing -4 ⁰C in air tight container.

Determination of Proximate Composition: The proximate composition (i.e. fibre, moisture, protein, carbohydrates, lipid, ash and dry matter) of the coconut pollen grains was determined according to the method of AOAC ¹⁷.

Total Proteins: The total protein content in the coconut pollen grains sample was measured using Kjeldhal method (Kelplus- Supra LX). Take 0.4 g of the sample add 5 ml of Conc. Sulphuric acid and subjected to digestion and further distilled after adding 40% Sodium hydroxide. It was collected in a flask containing 4% of boric acid and titrated with 0.1N Hydrochloric acid. The percentage of nitrogen quantified was converted into nitrogen by multiplying with 6.25 as conversion factor 9 ¹⁸.

Total Carbohydrates: The difference between100 and sum of crude protein, fat, moisture, ash and fibre is called total carbohydrate. This is referred to as total carbohydrate by difference and is calculated by the following formula ¹⁹.

100 - (Weight in grams [protein + fat + water + ash + alcohol] in 100 g of food)

Ash Content: Ash content was determined by using a muffle furnace by taking 2 g of coconut pollen grain in a crucible and kept inside a muffle furnace (Technico) heated to 550 ℃ for 4 h. The resulting ash was measured in an electronic weighing balance. The ash content of coconut pollen grain greatly depends on the variety and climatic condition ²⁰.

Total Lipids: Total lipid determined by extraction using organic solvents. Extraction of Crude fat by treating with diethyl ether and petroleum ether. Evaporate and calculate the residual weight ²¹.

Fat % (w/w) = Weight of Extracted Fat × 100

Weight of Dample:

Moisture Content: One gram of pollen grains was accurately weighed in a porcelain evaporating dish and was heated at 60 ℃ in an oven for 1 h. The percentage of moisture content of the pollen grain was calculated for the sample using the below formula ¹⁹.

Moisture content % = M₂-M₁ / M₂

Where M₁ is weight of the sample after drying and M₂ is weight of the sample before drying

Determination of Mineral Components: The mineral components were determined by using atomic absorption spectroscopy ²².

Statistical Analysis: The statistical analysis was performed using SPSS tool and the result obtained as mean ± standard deviation. Comparisons were carried out by Student’s t-test and Levene’s Test for equality of variances; tests were with p ˂ 0.05 level of significance.

Storage: Under atmospheric condition, pollen grains tend to loss their potency for germination as well as denaturation of protein happens due to the temperature of above 30 ℃ and humidity ²³. Some researchers proved pollen grains are normally stored at room temperature ²⁴. Some researchers reported that the pollen grains are stored according to freeze drying method in a closed natural atmosphere (sealed capsules) corresponding to relative humidity and rotary pump vacuum at 6 × 10 mm of Hg.

Storage could be possible at different temperatures like 4 ℃ (refrigerator), –20 ℃ and –80 ℃ (freezer). On the basis of different periods of pollen collection, the moisture content and the percentage of yield and storage temperature will be recorded. The process of drying has been referred to as auto freezing ^{25, 26},

Pollen grains belonging to a wide variety of species are best stored at temperatures 4 °C and –20 °C for short-term. Dry pollens kept at between 4 °C and –20 °C remain viable for a few days to a year, which may be adequate for use in breeding programs ^{27, 28}.

RESULT:

Mean weight of Pollen Obtained from the Inflorescence of Cocos nucifera at Different Period of Anther Dehiscence: The effective time period for pollen grains to get released from the anther of an inflorescence was 1 to 18 days – this may depend on the climatic conditions and the species. To demonstrate the developmental difference of pollen shed day by day were observed and revealed the mean weight of the pollen grains obtained by wrist action handshake method at different time periods (i.e. number of days), after the release of pollen grains in Table 1.

TABLE 1: MEAN WEIGHT OF POLLEN OBTAINED FROM THE INFLORESCENCE OF COCOS NUCIFERA AT DIFFERENT PERIOD OF ANTHER DEHISCENCE

FIG. 1: MEAN WEIGHT OF THE POLLEN GRAINS                       

FIG. 2: PERCENTAGE OF POLLEN GRAINS OBTAINED BY TWO DIFFERENT METHODS

Pollen collection was carried out over a period of 8 months (from February 2018 to September 2018) by two different methods as shown in Fig. 2. Results indicate that the maximum amount of pollen grains obtained using sieve method was found to be 2.36 ± 0.018g/100 g of flower and in wrist action shake method it is about 17.89 ± 0.97 g/100 g of flower. The moisture content also differed between these two methods: a low moisture content of 0.11 ± 0.03% was observed in wrist action shake method and in sieve method the moisture content was found to be 0.29 ± 0.07% represented in Table 2. The maximum amount of pollen grains obtained by wrist action method was during the months of June and July and they had less moisture content as shown in Fig 3.

Proximate Analysis: The proximate analysis of coconut pollen grains with various parameters – total carbohydrate: 15.903 ± 0.105 mg/g, total protein: 18.56 ± 0.115 mg/g, lipid: 11.833 ± 0.075 mg/g, moisture content: 0.970 ± 0.008% and ash: 0.896 ± 0.020%. Summarized in Table 4 and shown in Fig. 3 are the estimated values of the mineral components of the dried powder of coconut pollen grains – magnesium: 3.54 ± 0.034 ppm, calcium: 3.066 ± 0.049 ppm, manganese: 0.273 ± 0.020 ppm, copper: 38.58 ± 0.549 ppm, iron: 53.43 ± 0.445 ppm, zinc: 131.7 ± 0.359ppm, potassium: 4.733 ± 0.208 ppm and sodium: 4.233 ± 0.283ppm were summarized in Table 3.

The mean weight of the pollen grain are analysed statistically by means of Independent Sample t-test and Levene’s test for Equality of Variances were observed that mean weight of pollen grain for the Seive method is 1.5975 and Wrist action shake method is 9.7262. The p value (p = 0.002<0.05) is lesser than 0.05, which indicates that the weight of the pollen grains is not same for sieve method and wrist action shake method in Fig. 2

Accordingly by the two methods, the collected pollens were stored in Eppendorf tube and wrapped with a cellophane sheath and kept at –4 ºC and it is maintained viable for up to 3 months of storage. During this storage period, the protein and other nutrient values has no changes observed and remains same when compared those with their initial values.

TABLE 2: COLLECTION OF POLLEN GRAINS OBTAINED BY TWO DIFFERENT METHODS

Values are the Mean ± Standard deviation (n=3)

FIG. 3: MINERAL COMPONENTS OF COCONUT POLLEN GRAINS

TABLE 3: PROXIMATE COMPOSITION OF COCONUT POLLEN GRAINS

TABLE 4: MINERAL COMPOSITION OF COCONUT POLLEN GRAINS

Values are the Mean ± Standard deviation (n=3)

DISCUSSION: The study suggest that step by step spikelet’s splits starting from bursting of spadix to anther dehiscence, which probably could facilitate pollen bank. The result demonstrate that the evolutionary steps to increase the anther dehiscence by interfering the pollination as an important selective force for anther dehiscence. Each stage was monitored and recorded from day-1 morning to evening, for inspection on several changes from bursting of spadix to shedding the pollen. The removal of coconut pollen grains are characterized by several phenomenon of day by day variations of anther dehiscence were noticed on 3 to 18 days to shed their entire pollen. Overall our data revealed that step by step and day to day studies may promote the release of pollen grains increased gradually and shed their entire pollen at fourteenth day. We thus provide a direct experimental evidence for anther dehiscence. Collecting healthy amount of pollen grains is of critical task for pollen bank since coconut palm is monoecious and final step of anther dehiscence may leads to self-pollination by means of stomium and elongated pollen filaments. To prevent pollination the split spikelets were kept at sterile and controlled atmospheric room temperature for drying. Meanwhile for successful anther dehiscence a series of developmental events that leads to breakage of stomium and release of pollen grains must be synchronized with improved technique by an external force using a spontaneous shaking (wrist action shaker) and centrifugal force to separate the pollen grains. About 100 grams of dried flowers contains 17.5 to 19.8 gms/dry weight of pollen grams and it depends on the variety, weather, season and climatic condition.

Based on our observations and findings of the two methods Sieve and wrist action shaker, we suggest wrist action shake method was very effective in order to get enormous amount of pollen grains without any contamination and stability also maintained during storage (–4˚C) ²⁷. This is because pollen grains are surrounded by a pollen kit, so it cannot be released automatically by beating. Hence it needs some extra force to shed the pollen grains by shaking continuously for 15 minutes by means of wrist action shaker machine in which the pollen grains are released from the anthers and centrifuge at 3000 rpm for 15 min in order to separate and the entire pollen grains, despite being finer, less dense and damp, it is very difficult to collect the pollen grains conventionally; so we used the sieve technique by using a sieve which was the final  step to remove the anthers and stored at air tight containers. Enormous amount of copper, potassium, magnesium and zinc are found in Coconut pollen grains. But the collection of pollen grains and its processing are of critical importance for establishing the pollen bank. However the moisture content of pollen grains plays a critical role to maintain its quality as well as the viability. If it is not stored properly contamination may occur and if it is dried at above 40 ºC the thermo labile compounds will be denatured, since it rich in protein the choice of drying is only freeze to restore the phyto-compounds. Abundant of zinc and iron consumption of coconut pollen grains could aid in various biochemical processes in our body and also act as certain enzyme catalysis ^29-31. Enormous amount of copper, potassium and magnesium of coconut pollen grains has improve osmoregulation in our body and regulate homeostatic balance. Hence it supports the proper functioning of body cells, nerves, bones and muscles ³².

CONCLUSION: Our result concludes that the manual method for collecting the matured spathe and complete detachment of flowers from spikelets and better understanding the development of anther dehiscence and pollen release mechanism helps to optimize the collection and harvesting the pollen to restore the pollen grains for future research. Moreover coconut inflorescence contains immense quantity of pollen and enriched with fortified nutrients especially proteins, minearls as zinc and steroids and flavonoids as phytocompounds are abundant. After a period of an extensive experimental trials we observed that the result findings conclude a successful methods for establishing the release of pollen grains without any contaminants depends on time and climatic conditions. Thus this research work would results in successful collection of pollen grains brought out by two novel methods: sieve method and wrist action action shake method, both of which enable us to collect viable, matured coconut pollen grains but the quantity and the stability are varied. Despite being finer, less dense and damp, it is very difficult to collect the pollen grains, and that too they quickly get rehydrated upon release from anthers. For this reason Wrist action is the wright choice for collection, desiccation and efficient to obtain quality and quantity of pollen grains. Meanwhile this study also found that even though coconut inflorescence flowers throughout the year, the maximum amount of pollen grains were obtained only during the months of May, June and July, which had the most favourable climatic conditions and during this period the moisture content was also found to be less.

Our investigation of proximate analysis revealed that the coconut pollen grains are enriched with protein, lipids, fats and lipid. Mineral components like zinc are present abundantly. These findings conclude that future this would be the choice for immune booster and best neutraceuticals for women health as it contains steroid and flavonoids.

ACKNOWLEDGEMENT: We thank the Department of Food and Drug Testing Mrs. Sundarambal S, Indirangar, Gorimedu, Pondicherry, India, for allowing us to use their lab facilities.

CONFLICTS OF INTEREST: Nil

REFERENCES:

1. Karun A, Aparna V and Rajesh MK: Coconut 2017; 14(120): 139-158:,
2. Hedström I: Pollen carriers of Cocos nucifera (Palmae) in Costa Rica and Ecuador (neotropical region). Revista de Biologia Tropical 1986; 34(2): 297-01.
3. Sajini K: Cryopreservation of coconut zygotic embryos and pollen. Central Plantation Crops Research Institute. Technical Bulletin 2010; 17: 63.
4. Ranieri A, Benelli G, Castagna A, Sgherri C, Signorini F, Bientinesi M, Nicolella C and Canale A: Freeze-drying duration influences the amino acid and rutin content in honeybee-collected chestnut pollen. Saudi J Biol Sci 2019; 26: 252-55
5. Whitehead RA: The processing of coconut pollen. Euphytica 1963; 12(2): 167-77.
6. Manthriratna MA: Coconut Research Institute of Ceylon, Ceylon Cocon. Quart 1965; 16: 102-10.
7. Gangolly SR, Kamalakaran AK, Balakrishnan TK and Pandalai KM: Studies on pollen in the coconut. Ind Coconut J 1961; 14(2): 49-66.
8. Varkey T and Davis TA: Studies on coconut pollen with reference to the leaf and root (wilt) diseases. Indian Coconut Journal 1960; 14(1).
9. Whitehead RA: The processing of coconut pollen. Euphytica. 1963; 12(2): 167-77.
10. Liyanage DV: Controlled pollination of coconut palms botanist, coconut research institute Ceylon cocon. Quart 1965; 16: 102-10.
11. Harries HC: Pollen collection from coconut flowers by means of a fluid-bed dryer. Euphytica. 1973; 22(1): 164-71
12. Patel JS. The Coconut, a monograph, Govt. Press. Madras. Rican Natural History. Chicago: University of Chicago Press 1938; 8: 16-33.
13. Canale A, Benelli G, Castagna A, Sgherri C, Poli P, Serra A, Mele M, Ranieri A, Signorini F and Bientinesi M: Microwave-assisted drying for the conservation of honeybee pollen. Materials 2016; 9: 363.
14. Roopan SM: An overview of phytoconstituents, bio-technological applications, and nutritive aspects of coconut (Cocos nucifera). Applied Biochemistry and Bio-technology 2016; 179(8): 1309-24.
15. Agashe SN: Palynology and its applications. Oxford and IBH 2006; 257.
16. Whitehead RA: The flowering of Cocos nucifera in Jamaica. Trop Agric Trin 1965; 42: 19-29.
17. Barnabás B and Kovács G: Storage of pollen. Pollen Biotechnology for Crop Production and Improvement. Cambridge: Cambridge University Press 1996; 293-14.
18. Food C and Kjeldahl TM: Changes in AOAC® Official Methods of Analysis. International. Methods 934.09.
19. Kenneth Helrich: A.O.A.C. Methods 955.04 C, 979.09 (modified). Official Methods of Analysis. 1990:15.
20. Kenneth Helrich: A.O.A.C, Official Methods of Analysis, International. Methods 922.06 and 954.02;1990:15:199
21. Kenneth Helrich: A.O.A.C, Official method 905.02; 2000:17 Fat in milk.
22. Food Safety and Standards Authority of India, Ministry of Health and Family Welfare, Government of India, New Delhi. FSSAI Manual of Methods of Analysis of Foods. Beverages (coffee, tea, cocoa, chicory) sugar and sugar products and confectionery products. 2015. Available from https://old.fssai.gov.in
23. Knowlton HE. Studies in Pollen with Special Reference to Longevity. Ithaca: Cornell University 1922; 52: 751-793.
24. Connor KF and Towill LE: Pollen-handling protocol and hydration/dehydration characteristics of pollen for application to long-term storage. Euphytica 1993; 68(1-2): 77-84.
25. Kieliszek M, Piwowarek K, Kot AM, Bła˙zejak S, Chlebowska-Smigiel A and Wolska I: Pollen and bee ´ bread as new health-oriented products: A review. Trends Food Sci Technol 2018; 71: 170-80
26. Towill LE and Walters C: Cryopreservation of pollen. Cryopreservation of Tropical Plant Germpl 2000; 115-29.
27. Harries HC: The evolution, dissemination and classification of Cocos nucifera The Botanical Review 1978; 44(3): 265-19.
28. Liyanage DV: Preliminary studies on the floral biology of the coconut palm. Tropical Agric 1949; 105: 171-75.
29. Yang Y, Iqbal A and Qadri R: Breeding of Coconut (Cocos nucifera ): The Tree of Life. In Advances in Plant Breeding Strategies: Fruits 2018; 673-25.
30. Renjith RS and Rajamohan T: Young inflorescence of Cocos nucifera contributes to improvement of glucose homeostasis and antioxidant status in diabetic rats. Int J of Diabetes in Developing Countries 2012; 32(4): 193-8.
31. Thomas RJ and Rajkumar JA: Flowering and pollination biology in coconut. J of Plant Crops 2013; 41(2): 109-17.
32. Obasi NA, Ukadilonu J, Eze E, Akubugwo EI and Okorie UC: Proximate composition, extraction, characterization and comparative assessment of coconut (Cocos nucifera) and melon (Colocynthis citrullus) seeds and seed oils. Pakistan Journal of Biological Sciences 2012; 15(1): 1.
    

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    How to cite this article:

    Kavitha B, Gopal V, Joshna K and Balachandran N: Wrist action shaker: a novel method for collecting pollen grains from the inflorescence of coconut (Cocos nucifera L.) for its stable nutritional components. Int J Pharm Sci & Res 2021; 12(12): 6622-28. doi: 10. 13040/IJPSR.0975-8232.12(12).6622-28.

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