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 (Dimocarpus longan) is an evergreen subtropical fruit crop and is rich in nutrition. It has been widely planted in Southeast Asia, especially Thailand. Longan flowers are the primary source of honey production in Thailand (Wongsiri et al. 2012) and the main sugars of longan fruit are sucrose, fructose and glucose (Yuncharad et al. 2008). Therefore, using longan fruit as raw material to develop alternative carbohydrate feeding or longan syrup is an important way to improve longan fruit efficiency and increase carbohydrate-rich substrate for feeding honey bees in the rainy season. Nevertheless, while honey bees collect nectar and pollen from plants, they may carry fungus spores. Khuna et al. (2021) reported that fungi in the genus Aspergillus section Nigri were found in agricultural areas in northern Thailand. It is important to correctly identify the Aspergillus species that produce enzymes in order to prevent fungal disease from natural products. Classical methods were followed to investigate several morphological characteristics of fungal cultures and media and are the most widely used tools for fungal identification (Danmek et al. 2014). Aspergillus spp. is the type of mold that produces a wide variety of enzymes that can be found in fruits and agricultural products (Nasser 2017). Aspergillus section Nigri, especially A. niger is considered a dominant invertase enzyme-producing fungus that is thermally resistant and has a wide range of applications compared with a similar enzyme produced by bacteria and yeast (Robledo-Olivo et al. 2009). This strain is normally a non-pathogenic fungus that belongs to the “Generally Recognized as Safe” category and is used to produce enzymes (Schuster et al. 2002).

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 Saccharomyces cerevisiae (Boddy et al. 1993; Romero-Gomez et al. 2000). Invertase production from A. niger has received very little attention despite its potential for producing invert syrup from fruit. Therefore, the purpose of this study was to look at the fungal communities found in longan pollen collected from northern Thailand. Invertase-producing strains of Aspergillus spp. in section Nigri were investigated and characterized concerning the potential for use as a biological reagent for invert syrup production from longan sugar. Furthermore, after feeding honey bees with three different types of longan syrup for 16 weeks, compared to a traditional sugar syrup, the adult honey bee population was evaluated. This research demonstrates the use of invertase produced by Aspergillus spp. in longan syrup processing was safely fed to honey bees.

Sample collection and media preparation

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 Aspergillus flavus and parasiticus agar (AFPA) (Cotty 1994) consisted of 1.0 % (w/v) peptic digest of animal tissue, 2.0 % (w/v) yeast extract, 0.05 % (w/v) ferric ammonium citrate, 1.0 mL of 0.2% (w/v) dichloran in ethanol and 1.5% (w/v) agar. The semi-synthetic medium (SS) (Danmek et al. 2014) consisted of 0.2% (w/v) peptone, 0.1% (w/v) yeast extract, 0.2% (w/v) KH₂PO₄, 0.05% (w/v) MgSO₄, 0.01% (w/v) CaCl₂, 0.2% (v/v) Tween 80 and 0.05 % (w/v) ZnSO₄·7H₂O, 0.05% (w/v) FeSO₄·7H₂O, and 0.05% (w/v) MnSO₄·H₂O were used as culture media.

Isolation of invertase-producing strains of black Aspergillus spp.

Longan pollen was used as a resource to isolate different strains of Aspergillus spp. using serial dilution and spread plate technique on AFPA. All fungi species were identified according to taxonomic schemes proposed by Klich (2002). Naked eye observations of macroscopic characteristics, including colony diameter, colony colors, colony texture, conidial color, sclerotia, reverse color, and soluble pigments were employed. Fine structural characteristics, including seriation, vesicle shape, stipe length, and conidial shapes, size and texture, were observed under a dissecting microscope. On AFPA black aspergilli in the Aspergillus section Nigri were identified based on the slightly yellowish reverse colony and black conidia appearance. In addition, the molecular investigation of isolated black Aspergillus spp. was examined in the laboratory according to the modified method of Danmek et al. (2014); Alshehri and Palanisamy (2020). Isolated black Aspergillus spp. was cultured in an SS medium with 2.0% (w/v) sucrose as the sole carbon source and bromothymol blue as an indicator, followed by incubation at pH 7.0 for 3–5 days and was used to detect invertase activity. Invertase-producing strains that produced a zone of clearance in the medium were transferred to PDA to prepare the homogenous spore suspension (10⁶ spores/mL) by suspending in 0.85% (w/v) sodium chloride.

Characterization of invertase producing strains

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 µmol of reducing sugar per 1.0 minute.

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.

Longan syrup feeding to honey bee

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).

Data analysis

All the experiments were conducted in triplicate and experimental results were represented as the mean standard deviation. Statistical analysis of variance (ANOVA) was calculated (p < 0.05) employing SPSS version 26.0 (IBM Co., Armonk, NY, USA).

Separation of fungal species

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 Aspergillus was the most common genus. The taxonomy of Aspergillus spp. has always been complex due to the large number of species with minor distinctions. Aspergillus spp. produces slightly yellowish colonies and a brighter, reverse colony appearance (Fig. 1). The result indicated that macroscopic and microscopic techniques are appropriate for identifying Aspergillus spp. For example, the Aspergillus section Flavi was recognized morphologically based on its yellow-green conidial color. The black Aspergillus spp. in the Aspergillus section Nigri was morphologically identified based on its black colony, while the Aspergillus section Terrei was recognized by its tan to brown colony. Among the isolated Aspergillus spp., the highest frequency was observed in section Nigri (15.63%), followed by section Flavi (12.50%) and section Terrei (6.25%), while the most common isolated species were Trichoderma spp. (12.50%), Rhizopus spp. (9.38%) Neurospora spp. (9.38%), Chaetomium spp. (6.25%) Fusarium spp. (6.25%) and Penicillium spp. (7.54%) (Fig. 2). However, due to insufficient mycelia characteristics, 14.34% of unknown species were unable to be identified.

Table 1 . The percentage of occurrence of fungal species isolated from longan pollen.

Fungal	Occurrence (%)
Aspergillus section Nigri	15.63
Aspergillus section Flavi	12.50
Aspergillus section Terrei	6.25
Trichoderma spp.	12.50
Rhizopus spp.	9.38
Neurospora spp.	9.38
Chaetomium spp.	6.25
Fusarium spp.	6.25
Penicillium spp.	7.54
Unknown species without conidia	14.34

Figure 1. Morphological characteristic of Aspergillus spp. (A. flavus) with a yellow reverse colony on AFPA (A = reverse colony and B = colony) and green sporulation on PDA (C). AFPA: Aspergillus flavus and parasiticus agar; PDA: potato dextrose agar.

Figure 2. Colonies of major isolated fungi from longan pollen were grown on PDA at 30°C for 7 days (A = A. niger, B = Aspergillus section Terrei, C = A. flavus, D = Rhizopus spp., E = Trichoderma spp., F = Fusarium spp., G = Neurospora spp., H = Chaetomium spp., and I = Penicillium spp.). PDA: potato dextrose agar.

Invertase production

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 Aspergillus spp. on the SS agar plate ranged between 5.04 ± 1.72 to 8.01 ± 1.11 cm, while the other isolates of Aspergillus spp. did not produce the zone of clearance. The best invertase-producing black Aspergillus spp. was identified as A. niger from the LP5 sample (Fig. 3).

Figure 3. A. niger LP5 grown on PDA for 7 day at 30°C (A), conidiophore (B), and conidia (C). PDA: potato dextrose agar.

Enzyme activity

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 A. niger LP5 was active in the pH range 4–6. The pH level of 6.0 showed significantly higher activity than other treatments (p < 0.05) (Fig. 4). When the effect of temperature on the invertase activity was investigated, A. niger LP5 showed the highest activity of 1.13 ± 0.10 U/mL at 60°C when compared with the other treatments (p < 0.05). A decrease in enzyme activity was found when the temperature was over 60°C (Fig. 4). The results of the interaction between temperature and pH revealed that increasing both temperatures (> 60°C) and pH (> 6.0) led to a decrease in enzyme activity. In Figure 5, the effects of metal ions and compounds are summarized. Invertase activity in A. niger LP5 was not stimulated by any of the metal ions or compounds. A more than 50% reduction in relative activity in the presence of the Cu ion was discovered. The elevated Cu ion concentration may affect invertase inhibition (0.1 M final concentration).

Figure 4. Relative invertase activity of the A. niger LP5 (A) and temperature conditions (B).

Figure 5. Relative invertase activity analysis of various metal ions. Mg: MgSO4; Ca: CaCl2; Mn: MnSO4; Fe: FeSO4; Co: CoCl2; Cu: CuSO4; Zn: ZnSO4; EDTA: ethylenediaminetetraacetic acid.

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 (p ≥ 0.05). However, the glucose content of C syrup (3.45%) was significantly higher than both A (3.01%) and B (3.12%) syrups (p < 0.05). On the other hand, the sucrose content of C syrup (3.08%) was significantly lower than both A (4.25%) and B (4.02%) syrups, respectively (p < 0.05). Maltose content was lower than 0.2% in all syrup samples, and lactose content was not detected in any type of syrup. However, all types of prepared longan syrup contain an approximate 8.54% of carbohydrate.

Figure 6. The sugar content of freshly extracted longan syrup (A), boiled syrup at 100°C for 15 minutes (B), and enzymatically treated invert syrup incubated at 60°C for 60 minutes (C).

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) (p ≥ 0.05). Our findings indicate that both boiled longan syrup (T1) and invertase-added longan syrup (T2) can be used as food supplements for honey bees when natural food sources are insufficient.

Figure 7. Adult worker bee population fed with syrup for 16 weeks. Sugar syrup (control: T0), boiled syrup at 100°C for 15 minutes (T1), and enzymatically treated invert syrup by adding ten units of invertase per liter, incubated at 60°C for 60 minutes (T2).

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 Aspergillus spp. strains have similar morphological characteristics, making the challenges of identification (Somchart et al. 2019) despite the fact that identifying Aspergillus spp. based on morphological characteristics is one of the oldest and most widely used methods. Our results are similar to those obtained by Somchart et al. (2019) and indicate that the most common fungi that occurred in bee pollen were Aspergillus spp., Penicillium spp., Rhizopus spp., and Trichoderma spp. The data in the presented study regarding fungal genera are in general consistent with the previous research. In Europe, 80% of the Aspergillus spp. were found in commercially available bee pollen (González et al. 2005). Most fungi isolated from bee pollen were related to those usually present in grains and cereals, which are also known as “field” and “storage” fungi (Logrieco et al. 2003). Even though molecular techniques are necessary to characterize fungal species that do not produce spores, the conventional method based on morphological characteristics continues to be used, and the adventages of this method include its inexpensiveness and simplicity (Danmek et al. 2014; Klich 2002; Somchart et al. 2019).

Aspergillus spp., Penicillium spp., and Fusarium spp. are the primary groups of toxigenic field fungal genera. Al-Hagar et al. (2015) and Romero-Gemez et al. (2000) reported that Aspergillus section Nigri including A. niger was the dominant fungal species producing high values of invertase. Sirisansaneeyakul et al. (2012) reported that invertase favored a pH range of 4 to 9 and a temperature range of 30°C to 80°C. Oliveira-Alves et al. (2013) reported optimal invertase conditions for other Aspergillus spp., including A. nidulans, incubated at 54°C–62°C and a pH of 4.8–5.6. In support of this result, Sirisansaneeyakul et al. (2012) stated that both A. niger showed the best invertase activities at a pH range of between 4.0–5.0 and a temperature range of 50°C–60°C.

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 Aspergillus spp. In our study, morphological and microscopic methods are equally crucial for the complete identification and confirmation of important Aspergillus spp. and other fungi. Furthermore, discovering an invertase-producing strain of A. niger LP5 has enabled its application for enzyme utilization in the invert sugar preparation process. We conclude that adding ten units per liter of invertase from A. niger LP5 to longan syrup and incubating at 60°C for 60 minutes helps increase the concentration of fructose and glucose. Thus, longan syrup could increase the number of honey bees by supplying an inverted diet, which is vital for feeding and maintaining honey bee populations. A further approach is to investigate the effects of mycotoxin contamination in bee pollen and syrup on the number and lifespan of honey bees.

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.

Not applicable.

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.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.