Published online May 26, 2022
Journal of Ecology and Environment (2022) 46:13
1School of Agriculture and Natural Resources, University of Phayao, Phayao 56000, Thailand
2Meliponini and Apini Research Laboratory, Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 52000, Thailand
3Department of Plant Medicals, Andong National University, Andong 36729, Republic of Korea
Correspondence to:Bajaree Chuttong
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Background: In northern Thailand, the longan flower is the principal nectar source for honey production. Microorganisms play a critical function in the agricultural ecology. The morphological characteristics of fungal species found in longan pollen were studied. Aspergillus spp. were found to be invertase-producing strains and were employed in the longan syrup production process. The purpose of this study was to evaluate the effects of invertase-added longan syrup on the adult honey bee population numbers that were fed by this syrup for 16 weeks.
Results: Different fungal species were found in longan pollen samples. Aspergillus was the main genus, with three predominant sections: Nigri, Flavi, and Terrei. Other isolated species were Trichoderma spp., Rhizopus spp., Neurospora spp., Chaetomium spp., Fusarium spp. and Penicillium spp. However, Aspergillus spp. is the only fungal species that produces the enzyme invertase. The invertase-producing strains belonging to the Aspergillus section Nigri were found to be A. niger LP5 with an optimum activity at pH 6.0 and 60°C. When A. niger LP5 invertase was used for longan syrup processing, the highest levels of glucose (3.45%) and fructose (2.08%) were found in invertase added longan syrup (C), while fresh (A) and boiled longan syrup (B) had lower contents of both sugars. The sucrose content was detected in (A) at 4.25%, while (B) and (C) were at 4.02% and 3.08%, respectively. An appropriate amount of sugar to feed and maintain the honey bee population was considered. The data showed no statistically significant differences between the two selected forms of longan syrup compared to the sugar syrup examined by the adult honey bee population.
Conclusions: The main species of isolated fungi from longan pollen were Aspergillus spp. The discovery of an invertase-producing strain of A. niger LP5 has enabled its application for enzyme utilization in the invert sugar preparation process. The adult worker bee populations fed by longan syrup from both boiled and invertase-added sources showed an increasing trend. Artificial syrup made from longan fruit to feed honey bees when natural food sources are limited can be applied.
Keywords: Aspergillus niger, honey bee, invertase, longan pollen, longan syrup
Nutrition is important for the survival of strong and healthy honey bee colonies. Honey bees obtain energy from carbohydrates, proteins for growth and development, and lipids for energy reserves, while minerals, vitamins, and water are required for optimal survival (Frizzera et al. 2020). However, when natural resources are insufficient and do not meet the requirements of honey bee colony, supplemental feeding of honey bee colonies compensates for natural pollen and nectar shortages (Gemeda 2014; Semkiw and Skubida 2016). Beekeepers feed an artificial supplementary diet when honey bees suffer from low nectar flow during the rainy season and unusual weather conditions. An artificial diet that is high in carbohydrate can also be used as an alternative energy source to support colony development and prevent starvation of honey bees (Frizzera et al. 2020). Longan (
Honey bees consume the sucrose or invert sugar syrups (constituted mostly of fructose and glucose), add enzymes during this process, and store them in the cells (Bugarova et al. 2021). Somerville (2000) revealed that in an experiment involving sucrose, glucose, fructose and invert sugar, fed to colonies of the same strength and under similar conditions, sucrose and invert sugar were found to be the more attractive sugars for honey bees. The invertase enzyme is responsible for the hydrolysis of sucrose to produce invert sugar. Invertase can break down sucrose into glucose and fructose at low temperatures of 30°C–60°C, thus reducing the occurrence of hydroxymethylfurfural (HMF) (Amaya-Delgado et al. 2006; Chaira et al. 2011; Kotwal and Shankar 2009). In Thailand, biological processes involving the invertase enzyme to produce invert syrup have been studied but are limited to
Ten samples of longan pollen (LP1–LP10) were obtained from longan trees in the area near the honey bee apiary in Phayao province, Thailand (19°02’44.4”N, 99°52’38.2”E) in 2020. The samples were preserved in a sterile plastic bag and transferred to the laboratory. The samples were dried at 30°C in a hot air oven for 3 days before being used as a substrate for fungal investigation. The modified
Longan pollen was used as a resource to isolate different strains of
An Erlenmeyer flask containing 100 mL of the SS medium with 2.0% (w/v) sucrose was prepared as the sole carbon source. The culture was inoculated with 2.0 mL of each spore suspension (106 spores/mL) of invertase-producing strains and incubated at 30°C for 7 days. Crude invertase was determined by estimating the reducing sugar liberated using dinitrosalicylic acid reagent (DNS reagent) (Miller 1959) in international units. Under the assay condition of 30 minutes and 60°C, one unit of invertase was defined as the amount of enzyme that liberates 1.0
The optimal pH for enzyme activity was determined by changing the assay reaction mixture using several buffers in the range of 3.0 to 10.0. The optimum temperature for the enzyme activities was examined by measuring the activity at different temperatures (30°C to 80°C) in the optimal buffer. The effects of metal ions, which included Mg (MgSO4), Ca (CaCl2), Mn (MnSO4), Fe (FeSO4), Co (CoCl2), Cu (CuSO4), Zn (ZnSO4), and ethylenediaminetetraacetic acid (EDTA), were investigated by incorporating them into a 0.1 M final concentration mixture prior to determination of enzyme residual activities.
Fresh longan fruit was taken to the laboratory; first, the longan fruit was water-rinsed and washed. Extraction involves the use of a pressing machine with their material being filtered through a food strainer to produce a freshly prepared extract. Fresh longan syrup was prepared to 50 Brix and pH 5.5. The syrup was sterilized by adding a sodium metabisulfite solution at a concentration of 200 ppm and setting it aside for 24 hours. Sugar content was determined for fresh longan syrup (A), boiled longan syrup at 100°C for 15 minutes (B), and enzymatically treated invert syrup (C) incubated at 60°C for 60 minutes after adding ten units of invertase per liter of longan syrup.
High-performance liquid chromatography (HPLC) coupled to a refractive index detector (RID) was used to determine the sugar contents (fructose, glucose, maltose, and sucrose) of 3 forms of longan syrup according to Association of Official Analytical Chemists (AOAC) method 977.20. A 5.0% (w/v) honey solution in distilled water was injected into a HPLC system (Shimadzu, Kyoto, Japan) equipped with an LC-10AD pump, CBM-10A system controller, and RID-10A refractive index detector and connected to a computer running class LC10 controller software. An Inertsil NH2 column (5 m, 250 × 4.6 mm) (GL Science Inc., Tokyo, Japan) was used to determine sugars, with a mobile phase of HPLC acetonitrile/water (72:25) was used at a flow rate of 1.0 mL/min, at an oven temperature of 40ºC.
Regarding acceptance of honey bees to the longan syrup test, our result show that honey bees accepted boiled and invertase-added longan syrups but rejected fleshly extracted longan syrup. Therefore, we fed adult honey bees with two forms of longan syrup: boiled and invertase-added longan syrup. The adult honey bee population was estimated by using the Burgett and Burikam method (1985). For the experiment, fifteen colonies representing an estimated number of adult honey bees were chosen and separated into three groups for the experiment. A completely randomized design was used and the 2 forms of longan syrup were both used in the study. Each group of honey bee colonies received a different type of syrup. The first group was fed with longan syrup that had been boiled (T1). The second group was fed with invertase-added longan syrup (T2), while the third group of honey bees was fed with sugar syrup at a 1:1 sugar-to-water ratio (T0; as a control).
All the experiments were conducted in triplicate and experimental results were represented as the mean standard deviation. Statistical analysis of variance (ANOVA) was calculated (
The presented study revealed the presence of fungal species in bee pollen samples. The relative densities and frequencies of the isolated fungi are presented in Table 1. Longan pollen was discovered to be associated with several fungal species, of which
Through fermentation process black aspergilli were isolated and then characterized for their invertase production. Sucrose was applied as the sole carbon source and isolated black aspergilli were incubated at 30°C for 7 days. The clearance zones of isolated black
When testing the crude enzyme at 60°C, this fungus presented an invertase activity at 1.05 ± 0.12 U/mL. The effect of pH on the enzyme activity indicates that the invertase activity of the isolated
Our results discovered carbohydrate composition (%) of fructose, glucose, sucrose and a small amount of maltose in all types of the prepared longan syrup. Figure 6 presents the sugar content of the longan syrup we investigated. Fresh longan syrup (Fig. 6A) and boiled longan syrup (Fig. 6B) have similar fructose contents at 1.3%, while invertase added longan syrup (Fig. 6C) has higher fructose at 2.08%. From Figure 6, the data showed no statistically significant differences in sugar content between A and B syrups (
An examination of using longan syrup as an alternative carbohydrate supplement for the honey bees was conducted. Two forms of longan syrup and sugar water were fed to the 15 colonies of honey bees and the number of adult honey bee was evaluated every 2 weeks for 16 weeks (as shown in Fig. 7). The result showed that all forms of longan syrup (T1 and T2) and sugar water (T0) fed to the honey bee did not affect the population sizes of adult honey bees. Our results show an increasing trend in the number of adult honey bees population when fed with both longan syrup and sugar syrup, with no statistically significant differences for longan syrup examinations (T1 and T2) compared with sugar syrup (T0) (
Our investigations into different fungal species isolated from longan pollen showed colony color, margins, texture and colony reverse colors, according to the reports of Klich (2002) and Somchart et al. (2019). Some
Carbohydrates are an essential component of the honey bee colony’s nutrition, and both larvae and adults require them to grow and develop appropriately. Adult bees can survive on glucose, fructose, sucrose, trehalose, maltose, and melezitose, among other carbohydrates (Standifer et al. 1978). The traditional way of using raw materials high in sucrose to make it easier for honey bees to digest is by inverting sugar, obtained by mixing sucrose and water in a 2:1 ratio and boiling with an acidifying agent, including vinegar or lemon juice (Frizzera et al. 2020). However, this process produced high levels of hydroxymethylfurfural (HMF), which has been shown to be toxic to honey bees (Krainer et al. 2016; LeBlanc et al. 2009). For that reason, some researchers are interested in using a combination of low temperature and biological reagents to reduce HMF in syrup processing, such as ascorbic acid (vitamin C), acetic acid (vinegar) and citric acid (lemon juice). It was found that syrup produced using the biological method had less HMF than the traditional method using high acid and temperature (Ceksteryte and Racys 2006). Charistos et al. (2015) examined the long-term effects of sugar syrup feeding over the year and reported that the trend of the adult honey bee population increased from 8,500 bees to 14,500 bees. Our findings indicate that when honey bees were given both longan syrup and sugar syrup, the number of adult honey bees increased. According to Atallah and Naby (1979), when honey bees were fed an aqueous sucrose solution (1:1), commercial invert sugar, and honey, the result showed that the invert sugar helped them survive. Brighenti et al. (2017) discovered that when 0.1 and 0.16 g of citric acid were added to sugar syrup, the mortality rate was reduced when compared to other supplementary. Our data suggests that boiling and invertase-treated longan syrup can be used as alternative food supplements for honey bees when natural food supplies are limited. The use of longan syrup as a sugar substitute may help beekeepers reduce production costs.
We examined the fungal communities of longan pollen in northern Thailand. The results showed that most of the isolated species from longan pollen were
The authors would like to thank Mr.Chainarong Wongsansree, Chiang Rai Animal Nutrition Research and Development Center, Department of livestock, for technical and laboratory supports. We thank anonymous reviewers for their comments on this manuscript.
KD carried out the laboratory and field study, performed the analysis and wrote the manuscript. RR and BC participated in the field study and edited the manuscript. CS and CJ reviewed and edited the manuscript. The final version was read and approved by the authors.
This research has received finding support from the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (grant number B16F640174) and was partially supported by the fundamental fund of the University of Phayao, Thailand (FF64-RIM006/2021).
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
The authors declare that they have no competing interests.