Effect of Temperature and Duration of Gasbio Heat Energy on Bee Pollen Bioactive Compounds and Bee Propolis Extraction Trigona sp
Yasri Rahmawati1), Mochammad Junus1)
1) Animal Production Department. Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
2) Animal Product Technology Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
*Corresponding Email: [email protected] Submitted 4 March 2024; Accepted 22 March 2024
ABSTRACT
Processing of dried bee pollen and propolis extraction to optimize the use of processed by- products of Trigona sp bees in the general public by utilizing biogas as heat energy. This research uses bee pollen and raw bee propolis (Trigona sp). The aim of the research was to determine the effect of temperature and length of time for drying bee pollen and propolis extraction on bioactive compounds. The research method used a completely randomized design (CRD) with 5 treatments and 3 replications, consist with (1) Bee pollen drying temperature with sunlight, 45 oC, 55 oC, 65 oC, and 75 oC, (2) Long oven time 3 days, 2,5 hours, 2 hours, 1 hour, and 15 minutes. Meanwhile, the treatment for propolis extraction with distilled water solvent is (1) Temperature 70 oC, 75 oC, 80 oC, 85 oC, and 90 oC, (2) Length of extraction time is 220 minutes. Data were analyzed using Variant ANOVA. If there is a significant difference and effect (P<0.05) or highly significant (P<0.01), then proceed with least significant difference test. Dried bee pollen research results the best bioactive compounds were obtained at a temperature of 65 oC (T3) with protein content (14.09 %), total phenol (1.314 mgGAE/g), and antioxidant content (53.97 μg/mL). The best results for propolis extraction were at a temperature of 70 oC (T0) with antioxidants (21.15μg/mL) and total phenolics (0.357 mgGAE/g). Gasbio can be used as an alternative heat for drying bee pollen and propolis extraction, but when use gasbio must determine the right temperature and time.
Key words: Activity antioxidant IC50; bee bread; propolis; proteins; total phenolics
INTRODUCTION
Trigona sp bee is a stingless bee and has the advantage of a distinctive, more sour taste of honey, a higher honey price and many of them are bred traditionally. This type of bee is used to provide benefits for breeders (Rosyidi et al., 2018). The products produced by bees are honey, bee pollen, royal jelly, propolis and beeswax. These products provide economic benefits to farmers and health benefits to the community. Bee products are widely consumed to maintain human health. Apart from honey, propolis and bee pollen are widely sold because they contain compounds that are good for the body, and are quite expensive.
Bee pollen is flower pollen collected by worker honey bees. The pollen that has been collected is used to feed bee larvae in the early stages of their development (Spulber et al., 2018). Bee pollen contains high levels of protein, vitamins and total phenols so it functions as an antimicrobial, antioxidant which can increase the body's immunity (Bogdanov, 2016). Just as honey is the energy source for bee colonies, pollen (bee pollen) is also the main source of nutrition that is important for bees. Bee bread contains protein, minerals, fat and other substances. So, an adequate supply of pollen is very important to ensure the long- term survival of the colony and maintain its productivity.
Collecting bee pollen when bees visit flowers, the bees take the stamens while their bodies are covered in pollen dust.
Honey bees use their hind legs to press pollen into the pollen nest. The bee wets the pollen with secretions and its mouthparts help the pollen stick to the bristles of the
basket. This secretion contains different enzymes, for example amylase and catalase.
Bee bread contains up to 10% nectar, for packaging. A bee has to visit around 200 flowers of different types while the pollen collected averages around 8 mg (Bogdanov, 2016).
Propolis or super glue is a sticky substance produced by bees from sap and tree sap taken from flowers, shoots, leaves and bark. Usually, bees collect these materials and mix them with saliva and special enzymes in the bee's mouth. This substance is then used by bees to wrap the nest and protect the colony from bacteria, fungi, viruses and other insects (Safitri and Purnobasuki, 2022).
Propolis is a source of natural nutrients and nutraceuticals derived from resin substrates collected by bees (Harvi et al., 2014) which are produced by bees from tree sap, bee saliva and other materials. This compound consists of a mixture of active substances, namely polyphenols (falvonoids, phenolic acids and their esters), terpenoids, steroids and amino acids) (Safitri and Purnobasuki, 2022). Harvi et al. (2014) added that propolis contains high-value microelements, namely vitamins (A, B and C), minerals (Ca, Mg, Na, Fe, Mn, Cu and Zn) and the enzyme succinate dehydrogenase. Propolis also has important pharmacological effects, including antibacterial properties against both gram- positive and gram-negative bacteria.
Antioxidant activity IC50 is the concentration of antioxidant compounds needed to reduce free radicals by 50%.
Antioxidants display compounds that are able to capture or reduce the negative effects of oxidants in the body, working by donating one electron to compounds that are oxidants
*Corresponding author:
Yasri Rahmawati
Email: [email protected]
Animal Production Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
How to cite:
Rahmawati, Y., & Junus, M. (2024). Effect of Temperature and Duration of Gasbio Heat Energy on Bee Pollen Bioactive Compounds and Bee Propolis Extraction Trigona sp. Jurnal Ilmu dan Teknologi Hasil Ternak, 19 (1), 33-42
so that the activity of the oxidant compounds can be inhibited (Wardaniati and Taibah, 2019). Antioxidant activity is influenced by total propolis phenols. Free radical inhibitory activity of Trigona sp propolis.
derived from phenolic compounds (flavonoids and phenolic acids), as well as non-phenolics (ascorbic acid, substances such as carotenoids, organic acids and amino acids, as well as certain protein enzymes such as glucose oxidase and catalase).
The polyphenol group of compounds is the main free anti-radical compound in propolis. Polyphenol compounds are effective as free radical scavengers because they have a molecular structure in the form of an aromatic ring with a hydroxyl group which is able to reduce free radicals (Zahra et al., 2021). Bee pollen can be used as a food supplement which comes from natural extracts which have many health benefits.
According to Vassev et al. (2015), in their experiments, bee pollen contains phenolic compounds which act as antibacterial and medicinal properties.
Gasbio has been recognized as a renewable energy source available to accelerate socio-economic development.
Gasbio is a multifaceted fuel containing methane, carbon dioxide and water vapor with various utilization options (Kapoor et al., 2020). Gasbio is a form of renewable energy that comes from waste through a fermentation process. The gasbio fermentation process comes from organic materials that are fermented with the help of anaerobic bacteria, the results are methane gas (CH4), carbon dioxide (CO2), and several other gases in small amounts.
The use of biogas is not only a better use of energy, but can reduce environmental pollution caused by livestock waste (Styana et al., 2022).
Information regarding the process of drying bee pollen and propolis extraction using gasbio heat energy is still very limited.
Bee pollen and propolis as one of the largest phenolic compounds in beekeeping products are susceptible to temperature changes
during storage and processing. Pollen drying is carried out using two methods, namely natural drying depending on the weather and artificial, namely evaporation using an oven with a controlled drying temperature of 45
oC (Wibawanti et al., 2020), for 156-198 minutes so that the bioactive compounds in the pollen are not damaged (Isik et al., 2019) Meanwhile, propolis extraction uses the maceration method and then evaporation (Rismawati and Ismiyati, 2017).
In connection with the description above, research on the use of biogas as heat energy for drying bee pollen and evaporation extraction needs to be carried out. The aim is to find out the correct temperature and length of time for processing dried bee pollen and extracting propolis with gasbio as a source of heat energy so that the content of bioactive compounds contained in both materials can be maintained. The bee pollen and propolis used in this research were Trigona sp honey bee pollen and propolis.
MATERIALS AND METHODS This research used bee pollen and Trigona sp bee propolis which came from the grazing of beekeepers in Medan.
Propolis is extracted first using the maceration method by adding 1:4 distilled water solvent, then evaporated. Evaporation of propolis and drying of fresh bee pollen using gasbio heat energy. The results of the extraction of dried propolis and bee pollen were then tested for bioactive compounds.
The research method used was an experimental method using a Completely Randomized Design (CRD) with 5 treatments and 3 replications. The research lasted for 2 months (November-December 2023), at the Animal Production and Animal Product Technology Laboratory, Faculty of Animal Science, Universitas. The data obtained was then tabulated using Ms. Excel is then analyzed by ANOVA, if there are differences, the Least Significant Difference Test is continued. The data obtained was then described descriptively to describe the
results of the research. The materials used in the research were bee pollen and Trigona sp bee propolis. distilled water, methanol, DPPH solution (1,1-diphenyl-2- picrylhydrazyl), Folinciocalteau reagent, sodium carbonate, quercetin, FeCl3 1%, NaNO2, AlCl3, and NaOH. The tools used in the research were a blender, oven, gas bio tube (compressor), incubator, UV-VIS spectrophotometer (Labomed INC), YGH YH digital scales with an accuracy of 0,1 grams, 5 mL and 10 mL measuring cups (Pyrex), analytical scales (Denver M 310 USA), Whattman No. 1 filter paper, dropper pipette (Iwaki), 100 mL and 1000 mL glass beakers (Pyrex), test tube (Pyrex), and glass funnel (Supertek).
The variables studied in the dried bee processing research were antioxidant activity IC50, total phenolics and protein.
Meanwhile, the variables observed in the propolis extraction samples were antioxidant activity IC50 and total phenolics.
Processing Dried Bee pollen Trigona sp The objects used in this research are bee bioactive compounds pollen drying temperature combination. The independent variables in the research were temperature and drying time for bee pollen. The dependent variables were protein content, IC50 valueof antioxidant activity, and total phenol. The controlled variable is the drying method.
Drying fresh bee pollen according to what is carried out by the stingless bee cultivation farm from PT Al Aqso Honey Bee using the oven evaporation method by Bogdanov (2016) which is modified from gabio heat energy at a temperature of 45-75
oC with a comparison of sunlight. In research conducted by Isik et al. (2019), drying fresh bee pollen using an oven obtained optimal results at a temperature of 45 oC for 156-198 minutes. The research design model for drying bee pollen is according to Table 1.
Table 1. Bee pollen drying time and temperature optimization design
Treatment Drying Time
T0 Sunlight 3 days
T1 45 oC 2.5 hours
T2 55 oC 2 hours
T3 65 oC 1 hours
T4 75 oC 15 minutes
Propolis Extraction
The objects used in this research are bioactive compounds from propolis extract and their solubility levels. The independent variables in the study were temperature and propolis dissolution time. The dependent variables are the IC50 value of antioxidant activity and total phenolics. The extraction method used is propolis maceration, namely
adding distilled water in a ratio of 1:4, evaporating at a temperature of 70-90 oC for 20 minutes, storing at a temperature of 4 oC for 3-4 days, centrifuged for 20 minutes at 8.000 rpm, then the filtrate was filtered again to reduce the wax content in the extract (Rismawati and Ismiyati, 2017). The research design model for propolis extraction is according to Table 2.
Table 2. Optimization design for temperature and evaporation time for propolis extraction
Treatment Temperature Time (minutes)
T0 70 oC 20
T1 75 oC 20
T2 80 oC 20
T3 85 oC 20
T4 90 oC 20
Protein Content Test
The Kjeldahl method is widely used throughout the world and is still the standard method used for determining protein levels.
Its universal nature, high precision and good
reproducibility make this method widely used for determining protein levels (Rosaini et al., 2015). Dried bee pollen is analyzed using the following formula:
% nitrogen = 14.008 𝑥 𝑚𝐿 𝑡𝑖𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑥 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦
𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 1.000 x 100%
followed by calculating the protein content using the AOAC 984.13 method (Isik et al., 2019), using the following formula:
% 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛 (𝑔/100𝑔) 𝑥 6.25%
Antioxidant Test Method DPPH
Antioxidant testing uses the DPPH method (Baliyan et al., 2022). Pipette samples of pollen and propolis 0,5 mL, 1 mL, 1,5 mL, 2 mL, and 2,5 mL from the stock solution that has been made into a test tube, then add 0.5 mL DPPH 160 ppm methanol to each. and the volume was sufficient, the volume of the pollen sample was increased to 25 mL with a blank of
0.740. Meanwhile, in the propolis sample the volume of methanol PA was up to 5 mL with a blank of 0.690. The mixture was incubated at a temperature of 25 oC ± 5 oC for 30 minutes then its absorbance was measured at a wavelength of 517nm using a spectrometer. Antioxidant activity is expressed as (%) free radical scavenger through the formula:
%𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 =𝐵𝑙𝑎𝑛𝑘 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑆𝑎𝑚𝑝𝑙𝑒𝑠 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
𝐵𝑙𝑎𝑛𝑘 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 x 100%
The DPPH radical scavenging antioxidant activity data (% inhibition) of the samples was analyzed and the IC50 value was calculated, which is a value that shows the sample concentration (ppm) which is
able to inhibit the oxidation process by 50
%. The smaller the IC50 value means the higher the antioxidant activity (Mohdaly et al., 2015). The classification of antioxidant activity is shown in Table 3.
Table 3. Classification of antioxidant activity
No IC50 value Antioxidant
1 < 500 ppm Very strong
2 50 - 100 ppm Strong
3 100 - 150 ppm Currently
4 151 - 200 ppm Weak
Total Test Phenol (TPC)
Weighed 0,1 gram of the test sample, dissolved the bee bread sample in 10 mL of ethanol, while the propolis sample in 5 mL of ethanol solution to obtain a sample solution of 1000 ppm. A total of 0.5 mL of the test sample solution was added with 5 mL of Folin-Cioceltaeu reagent (1:10) and 4
mL of 1 M sodium carbonate. The mixture was left for 15 minutes then the absorbance was measured using spectrometry at a wavelength of 750 nm. Measurements were carried out three times. Phenol is calculated using a linear regression equation from the measured gallic acid calibration curve (Figure 1), then entered into the formula:
Y = 0.0179x, with a coefficient of determination value R 2 = 0.9904
TPC = Consentration x Volume mass
Figure 1. Gallic acid regression standard curve RESULTS AND DISCUSSION
Proteins Bee pollen
The results of research regarding the effect of temperature and length of time for drying bee pollen can be seen in Table 4.
Changes in protein levels of Trigona sp bees. dried at different gasbio oven power levels are presented in the Table 4. The results of analysis of variance showed that differences in temperature levels and drying time for bee pollen did not make a
significant difference (P>0.05) in protein content.
Bee pollen samples dried with gasbio heat energy levels produced the highest protein content, namely (T3) at 14,09%, however the protein value was still low compared to the T0 treatment (sunlight) at 26,67%. This shows that the protein content of Trigona sp. bees produces much higher protein than the results of research by Anis et al. (2021) where Apis mellifera bees contain 13.77 % bee pollen.
Table 4. Average value of bee pollen protein content
Treatment Content (%)
T0 26.67±11.9
T1 13.35±5.9
T2 12.66±5.6
T3 14.09±6.3
T4 12.31±5.5
Average 15.33±6.6
The protein content in bee pollen is influenced by the processing method, temperature, heating time and storage conditions. Protein testing can represent the quality of bee pollen, because the protein from dried bee pollen produced after heat treatment, temperature and time tends to be different. In research conducted by Isik et al.
(2019) it was explained that the crude protein content of fresh bee pollen was 30,36%, but after drying at an infrared temperature of 50-88 W with A time variation from 20-180 minute decrease from
24.38-17.44 %. This is in accordance with research that the protein values are different, namely T0 (26.67 ± 11.9 %), T1 (13.35 ± 5.9
%), T2 (12.66 ± 5.6 %), T3 (12.66 ± 5.6 %) and T4 (12.31 ± 5.5 %), differences in temperature and drying time determine the protein content of bee pollen.
The bee pollen protein of Trigona sp bees produced during the research was 15.33% lower than research conducted by Vassev et al. (2015), the bee pollen protein reached 22.7 % protein. These protein elements are essential for life and organisms
y = 0.0179x R 2 = 0.9904
0 0,2 0,4 0,6 0,8 1
0 10 20 30 40 50 60
Regression of total phenol pollen and propolis
cannot synthesize them themselves. This proves that bee pollen contains protein that can be consumed. Drying bee pollen with gasbio heat energy at a temperature of 65 oC for 1 hour gave the best results with an average of 14.09 % in dry bee pollen yield.
Antioxidant Activity Bee pollen and Propolis
Antioxidant activity uses the DPPH method because of its many advantages, namely: easy, simple, fast, good for certain polarities, sensitive and only requires a small sample (Khairunnisa et al., 2020). The results of the antioxidant activity test research are presented in Table 5.
Based on on Table 5, that the results of the ANOVA analysis of variance showed
that differences in temperature levels and drying time of bee pollen did not make a significant difference (P>0.05) in antioxidant activity. Based on Table 5, it describes the antioxidant activity of dried bee pollen with the lowest IC50 valueat a temperature of 45
oC (T3) of 53.97 (μg/mL), meaning that bee pollen is able to reduce 50 % DPPH at a concentration of 53.97 (μg/mL) is better compared to sunlight heat (T0), namely 70.93 (μg/mL). Table 5 also shows the results of the ANOVA analysis of variance of propolis antioxidant activity which were not significantly different (P<0.05), where the lowest mean was shown to be T0 of 21.15(μg/mL). The lower the IC50 value, the higher the antioxidant activity of the extract against DPPH (Khairunnisa et al., 2020).
Table 5. Average value of antioxidant activity of bee pollen and propolis
Treatment Content Antioxidant IC50 (μg/mL)
Bee pollen Propolis
T0 70.93±18.7 21.15±17.8
T1 73.52±20.2 37.54±20.0
T2 60.50±18.4 38.28±19.7
T3 53.97±15.8 25.27±17.1
T4 66.16±18.5 32.29±19.0
Average 65.02 ±16.8 30.91±17.1
Changes in antioxidant values occur as a result of being influenced by temperature and heating time. As in research conducted by Handayani et al. (2022), secondary metabolite compounds can usually be damaged by heating treatment, this damage can be indicated by a decrease in antioxidant activity, an increase in the antioxidant IC50
value. The antioxidant activity value of the propolis produced is higher than research by Djakaria et al. (2020) on Trigona sp propolis. from North Lombok has very weak antioxidant activity as seen from the high IC50 value (>200 μg/ml).
This high or low IC50 value can also be caused by Trigona sp food, as explained by Rosyidi et al. (2018), Trigona sp bees have the ability to fly short distances so they only intensively search for food in their nest area, which is different from bees. The results of research regarding the effect of differences
in temperature and length of drying of bee pollen and evaporation of propolis extraction can be seen in Table 5.
The antioxidant activity of IC50 in bee pollen was produced at a temperature of 45
oC for 2,5 hours with an average value of 70.93 µg/g and the highest was temperature of 65 oC for 1 hour with an average value of 53.97 µg/g, while the lowest IC50 propolis antioxidant activity was 70 oC for 20 minutes with an average value of 21,15 µg/g and the highest temperature was 80 oC with an average value 38,28 µg/g. This indicates that to reduce free radicals by 50%, 70.93 µg/g is needed for bee pollen dried at a temperature of 45 oC, and 70.93 µg/g for bee pollen dried at a temperature of 65 oC.
Analysis test ANOVA on the antioxidant content of bee pollen showed that the antioxidant activity of Trigona sp bee pollen for each heat drying treatment in sunlight
and biogas heat energy did not have a significantly different effect (P>0.05) and the data obtained showed an up-down graph.
Table 5. Shows that dry bee pollen and Trigona sp bee propolis extract with distilled water as a solvent can inhibit DPPH very well. The parameter used as a DPPH reduction test is the IC50 (half maximal inhibitory concentration) value, namely the concentration of the extract that can inhibit free radicals by 50 %. The IC50 value is obtained from the regression equation. The results of dried bee pollen and Trigona sp propolis extract have an average IC50 value of 65.02 μg/mL and 30.91 μg/mL which means the average value antioxidant activity bee pollen is classified as a strong antioxidant and propolis is classified as a very strong antioxidant. Kus and Jerkovic (2018) also stated that the composition and antioxidant capacity depend on the processing method. Apart from that, the intensity of the color can affect the biological activity of the sample, the darker the color of the propolis, the higher the total phenolic content and antioxidant activity.
The positive effect of antioxidant bioactive compounds on bee pollen and propolis is proven by the results of this study, namely that the antioxidants of bee pollen and propolis are classified as good.
This provides a scientific basis for the use of bee pollen and propolis for the prevention and treatment of diseases caused by oxidative stress. Liaudanskas (2021) states that good antioxidants can be used as a treatment for anti-inflation, anti-allergies, anti-bacterial and antiviral.
Total Phenolic Bee Pollen and Propolis Phenol is calculated using a linear regression equation from the measured gallic acid calibration curve (Figure 1), then entered into the TPC formula. The research results can be seen in Table 6. Based on statistics analysis of variance ANOVA dried bee pollen and propolis mathematically it is not significantly different (P<0.05). In the Table 6, the highest score total phenolics Dried bee pollen obtained T3 treatment with a value of 1.314 mgGAE/g while propolis obtained T0 with an average of 0.357 mgGAE/g. The research results show that heating temperature and time can damage secondary metabolite compounds in bee pollen. This is proven by the drying results bee pollen use gasbio shows different results. The antioxidant content found in bee pollen and propolis is influenced by the phenolic content, where the higher the phenolic content value, the lower the IC50
value(Rosyidi et al., 2018).
Table 6. Average total phenolic value of bee pollen and propolis
The difference in total phenolic bee pollen and propolis in each treatment was according to the temperature and drying and evaporation time used. Some phenolic components are heat sensitive and easily oxidized. This is supported by Kunarto and Sani (2020) that the increase in total phenolics is adjusted to the increase in
extraction temperature, extraction time and solvent concentration. High temperatures are able to release phenolic compounds from cell walls and extraction time can cause the accumulation of compounds that can increase oxidation.
Phenolic levels are also influenced by the type of feed consumed by Trigona sp
Treatment Total Phenolics (mg GAE/g)
Bee pollen Propolis
T0 1.314±0.75 0.357±0.21
T1 1.160±0.66 0.301±0.17
T2 1.137±0.65 0.346±0.19
T3 1.314±0.76 0.355±0.20
T4 1.148±0.66 0.340±0.19
Average 1.257±0.61 0.339±0.02
bees. This is supported by Zahra et al.
(2021) that plant origin can influence physicochemical parameters, the higher the total phenolic content, the stronger the free radical activity produced. This is also supported by Agus et al. (2019) in their research who stated that geographical location can significantly influence the phenolic and antioxidant content values of Trigona sp propolis. The components in bee pollen and propolis are also influenced by several factors, such as the type of bee, climatic and geographical conditions, type of stup, availability of natural food sources and the strength of the bee colony (Safitri and Purnobasuki, 2022).
CONCLUSION
Optimal temperature and duration of drying bee pollen using gasbio heat energy is 65 oC for 1 hour producing protein, antioxidant activity IC50 and the best total phenolics (14.09±6.3 %, 53.97±15.8 μg/mL, and 1.314±0.76 mgGAE/g), while the best propolis extraction temperature and evaporation time was 70 oC resulting in the best IC50 and total phenolic antioxidant activity (21.15±17.8 μg/mL and 0.357±0.21 mgGAE/g).
ACKNOWLEDGMENT
The researcher would like to thank the supervisors who have conducted research and this research is funding for the 2023 Brawijaya University Professor Grant.
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