Bee's honey is one of the greatest natural gifts to humankind, and Indian Ayurveda emphasises the various applications of honey, viz. obesity, burns, cuts, wounds, dermatitis etc. However, all the applications of honey are based on their chemical composition, but they vary depending upon the botanical, geographical origins, and bee feed. With this background, the current study aimed to evaluate the quality of honey produced by the stimulative diets ingested by honey bees (Apis mellifera). At the onset, stimulative diets were served to honey bees for 1 year, and an adequate volume of honey samples was collected before and after the ingestion of stimulative diets. After that, the honey samples were used to study the antioxidant activity and antibacterial potential as per standard protocol. The results revealed that the antioxidant activity of post-feeding honey samples exhibited a higher rate, i.e., 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Pre-feeding: 88.06 ± 0.214 and post-feeding: 91.79 ± 0.609 μmol/litre), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Pre-feeding: 20.55 ± 0.336 and post-feeding: 29.01 ± 1.114%), and ferric reducing ability of plasma (FRAP) (Pre-feeding: 0.296 ± 0.039 and post-feeding: 0.396 ± 0.001 mg TE/100 gm). Similarly, a high rate of antibacterial efficiency was shown in post-feeding honey samples against E. coli at a concentration of 800 µg/mL. It was shown with considerable antibacterial activity against Salmonella enterica as well. Overall, we have demonstrated honey's antioxidant and antibacterial effects and may have therapeutic potential as honey.
Honey is an appreciated natural gift to humankind, derived entirely from honeybees. Honey is the by-product of nectar collected by bees from flowers, with the addition of some digestive enzymes produced by the honeybees themselves. Honey is a natural antioxidant composed of sugars, water, proteins, vitamins, minerals, flavonoids, polyphenolic compounds, and plant derivatives [1, 2]. Its antioxidant properties help prevent cardiovascular diseases, cancer, cataracts, mutations in DNA structure, skin ulcers, gastrointestinal disorders, and inflammatory processes [3,4,5,6,7]. The antioxidant effects are initiated by the components phenolic acid, polyphenols, enzymes, vitamins, and amino acids . The antioxidant content of honey varies based on its floral origin, climatic conditions, processing method, and handling [9, 10]. The antioxidant activity of honey can be measured using various methods, such as DPPH, FRAP, AEAC, ORAC, and TEAC [4, 6, 11,12,13]. The dark honey showed higher phenolic content and better antioxidant activity than amber and light honey samples . The colour intensity of honey is positively correlated with its antioxidant activity and phenolic content . Honey samples collected from higher altitude regions have higher antioxidant potential than those collected from lower altitude regions [15,16,17,18,19].
Honey has been used since ancient times as a medicine to treat various human diseases, including infections. It has antimicrobial properties due to its low water content, high osmolarity, low pH, and hydrogen peroxide content. The botanical origin of honey may be responsible for its antibacterial properties, and many types of honey are sold with a standardised level of antimicrobial capacity. Manuka honey has been found to have an inhibitory effect on approximately 60 species of bacteria [20,21,22,23,24,25]. Studies have reported the chemical composition of pure honey and its role in antibacterial effects on different bacterial isolates viz. Staphylococcus aureans, Salmonella typhi, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa . There is an urgent need to develop alternative antimicrobials to overcome antimicrobial resistance. This study aims to fill that gap and provide evidence for improving beekeeping practices.
2 Material and methods
2.1 Feeding of stimulative diets
A weighted quantity of formulated diet was fed to bee colonies during the summer dearth period. The formulated diet was provided using the standard top bar method, which is the most widely accepted method of feeding [27,28,29,30,31]. After being wrapped in butter paper, Patties were placed on the top bars of the bee hive (Fig. 1). Paper was punctured at several places from the lower sides of the packet for bees to feed upon the diet. The small amounts of feeding material leak out from the holes of the pack so that the bees get attracted to feed upon the diet containing defatted soybean flour, parched gram, brewer’s yeast, protein hydrolysate powder, sugar and honey.
Patties were replaced weekly and the leftover residue (if any) was weighed on a high precision Electronic Balance (Model JAX) to calculate the amount of diet consumed by the bees during 1 week time.
2.2 Collection of honey samples
Extraction of honey was done with the help of a honey extractor. Honey was collected from all the colonies including experimental and control at two stages: (i) Pre-feeding (before feeding the bees with stimulative diets) (ii) post-feeding (after feeding the bees with stimulative diets) and kept in tight glass jars.
2.3 Analysis of antioxidant and antibacterial activity of honey
To evaluate the quality of honey produced by the stimulative diets ingested by honey bees (Apis mellifera). We have studied the antioxidant activity and antibacterial potential as per the method described below: -
2.4 Determination of the total antioxidant activity of honey
2.4.1 FRAP (Ferric reducing antioxidant power) assay
The FRAP assay, developed by Benzie and Strain (1996), is a method for measuring the total antioxidant power of biological fluids . It was followed in the present study with little modifications. Two honey samples were treated with warm distilled water (1gm/10 ml warm distilled water). At low pH, the ferric 2, 4, 6-tripyridyl-s-triazine [Fe (III)—TPTZ] complex reduced to the ferrous 2, 4, 6-tripyridyl-s-triazine [Fe (II)-TPTZ] complex and then an intense blue colour developed. That was checked by measuring the change in absorption at 593 nm. The working FRAP reagent was prepared by mixing 10 volumes of 300 mmol/L acetate buffer, pH 3.6, with 1 volume of 10 mmol/L TPTZ in 40 mmol/L HCl and 1 volume of 20 mmol/L FeCl3. 200 µl of honey samples was mixed with 3 ml of freshly prepared FRAP reagent and incubated at 25 °C for 10 min. Then reading was taken at 593 nm wavelength against a blank that was prepared by using distilled water. The standard curve was prepared by using aqueous solutions of ferrous sulfate (100–2000 μM/l concentration) and the results were expressed as micromoles of ferrous equivalent (μmol Fe (II)) per kg of honey.
2.4.2 DPPH radical scavenging activity
The antioxidant properties of honey were also measured by evaluating the free radical-scavenging activity of the DPPH radical. The free radical scavenging activity of honey samples was determined by the DPPH test proposed by Brand- Williams et al. with a slight modification. In this procedure, 200 μl of each honey sample was mixed with 3 ml of DPPH methanol solution (100 mM) in a test tube . The mixture was shaken and incubated for 30 min in the dark. The absorbance of a clear solution was determined at 517 nm using a spectrophotometer. An ethanolic solution of DPPH (100 mM) was used as a control and the percentage of DPPH radical scavenging activity was calculated according to the following equation:
2.4.3 ABTS assay
The ABTS assay of honey samples was determined by the method used by Bouhlali et al. with minor modifications . The stock solution of the 2, 2’–azinobis (3-ethylbenzthiazoline-6-acid) diammonium salt (ABTS) radical was prepared by dissolving 76.8 mg of (ABTS+) in 20 mL of a sodium persulphate solution (2.45 Mm). The solution was allowed to stand in the dark for 12–16 h and then a working solution was obtained by diluting the stock solution with methanol to obtain an absorbance of 0.7 ± 0.005 at 734 nm wavelength. 200 μl of each honey sample mixed with 3 ml of the ABTS radical solution was incubated for 10 min at room temperature and the absorbance was recorded at 734 nm. A standard curve was obtained by using an aqueous solution of Trolox. The total antioxidants were expressed as micromoles of Trolox equivalents per gm of honey.
2.4.4 Determination of the antibacterial activity of honey
Salmonella enterica (MTCC 3219), Escherichia coli (MTCC062), were acquired from the Institute of Microbial Technology (IMTECH), Chandigarh, Punjab, India. Microbial culture media are obtained from Himedia Laboratories Pvt. Ltd, Mumbai, India. In all the experiments, Millipore water (18.2 U) was used (Millipore Applied Systems, USA) and all the strains were sub-cultured every 28 days to maintain cell viability, Salmonella sp by Brain–heart infusion medium (BHI) and Escherichia coli in nutrient agar (NA) and stored at 4 ℃.
2.4.5 Honey sterility in before antibacterial study
At the onset, stored honey samples (pre-feeding and post-feeding sample) were sterile by the method described by James et al. Briefly, full loop (8'') of pre-feeding and post-feeding honey samples were streaked on freshly prepared Brain–heart infusion medium (BHI) agar plate and nutrient agar (NA) plate respectively; thereafter the streaked plates were incubated at 37 ℃ for 24 h.
2.4.6 Evaluation of antibacterial efficacy of honey
To investigate the antibacterial potential of the pre-feeding and post-feeding honey samples were studied by turbidity assay. For this study, in two different 15 mL glass test tubes (150 × 15 mm) containing 10 mL BHI-broth and nutrient broth respectively; thereafter, Salmonella enterica and Escherichia coli were inoculated with ~ 105 CFU/mL of bacterial cells. Then, a different volume of pre-feeding and post-feeding (400, 800 µg/mL) honey samples was added and mixed gently for 10 to 15 min. Subsequently, the solution was incubated in a shaking incubator (Eppendorf, Innova 42) at 37 ℃ for 24 h. After the incubation, the progress of inhibition was observed on the test tube, where the minimum concentration that did not have any turbidity was taken as the MIC (Minimum Inhibitory Concentration) value of pre-feeding and post-feeding honey samples. Moreover, to quantitatively determine the antibacterial activity of the pre-feeding and post-feeding being properly diluted, (serial dilution) and about 100 µL bacterial suspension was speared on the Brain–heart infusion agar plates (BHI-A) and nutrient agar plates (NA). Consequently, the plates were then incubated at 37 ℃ for 24 h. Photographs of Salmonella enterica and Escherichia coli grown on BHI-A/NA plates were obtained of a number of colonies. Then, the lowermost concentration was taken for bacteriological progress on the BHI-A/NA plates and selected for MBC (Minimum Bactericidal Concentrations)  (Heatley, 1944).
3 Results and discussion
3.1 Antioxidant activity
ABTS assay, DPPH radical scavenging assay and FRAP assay were performed on the honey samples to determine the antioxidant activity of honey. Results of the antioxidant activity of honey are presented in Table 1.
3.2 ABTS [2, 2´-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)] assay
In the current research, free radical scavenging activity (ABTS) was recorded at a maximum (91.79 μmol/litre) in post-feeding honey samples. A statistically significant difference was recorded among tested samples, as the lowest (88.06 μmol/litre) scavenging activity was found in the pre-feeding samples (Fig. 2). The results of pre-feeding samples of honey are in line with previous studies, who reported higher radical scavenging activity of honey by the ABTS method [36,37,38,39,40]. However, there is no significant report available in the literature on checking the free radical scavenging activity of honey samples collected after artificial feeding to bee colonies. This increase in free radical scavenging activity the feeding of stimulative diet might be due to presence of defatted soybean flour and parched gram.
3.3 DPPH (2, 2-diphenyl-1-picryl-hydrazyl) assay
Free radical scavenging activity (DPPH) varied significantly (p < 0.05) among both honey samples i.e. pre-feeding and post-feeding honey samples. The highest antioxidant activity was recorded in post-feeding samples of honey (29.01%), whereas it was recorded least in pre-feeding honey samples (20.55%) (Fig. 2). Observations of the present study are similar to the findings of [4, 6, 16, 18, 41]; who reported variability in radical scavenging activity among different samples of honey.
3.4 Ferric reducing antioxidant power (FRAP) assay
The antioxidant content of honey depends upon the botanical origin of honey, climatic conditions, method of processing, handling etc. A similar pattern of results was found in FRAP assay, as higher antioxidant capacity (0.396 ± 0.001 mgTE/100 gm) was recorded in post-feeding samples and least capacity (0.296 ± 0.039 mgTE/100 gm) of antioxidant was found in pre-feeding samples of honey (Fig. 2). However, no significant difference was recorded among both samples. Findings of the present study are endorsed with the previous studies); who reported that all honey samples exhibit a strong antioxidant power [12, 13, 17, 37, 42, 43].
3.5 Antibacterial activity of honey
Antibacterial efficacy of post-feeding and pre-feeding honey samples and varying degrees of antibacterial activity against Salmonella enterica and Escherichia coli. In our study, we observed the highest antibacterial efficacy for post-feeding samples of honey against E. coli at a concentration of 800 µg/mL and noted some activity against Salmonella enterica as well (Fig. 3). However, we did not observe any considerable antibacterial activity in the pre-feeding honey samples at any of the concentrations used in our study. So far, very limited studies reported the antibacterial efficacy of honey produced after the ingestion of stimulative diets; noteworthy, Cilia et al. reported the antibacterial efficacy of honey against E. coli and Salmonella enteric . On the other hand, there may be studies that need to be explored to investigate the chemical composition of honey after ingestion of stimulative diets and their role in the antibacterial efficacy of honey.
Some studies reported that supplementary feed improved the health of honey bees and increased the quality of honey [45, 46]. One study reported that the administration of saccharose syrup (0.8 L in a 1:1 ratio of saccharose/water) increased the color and consistency of honey . Another study on sugar supplementation in honey bees colonies increased the proline, and improved antioxidants in terms of total phenol (~ 160 mg GAE/100 g), total flavonoid (4.67–6.25 mg CE/100 g), DPPH-RSA (30.65–35.97%), and showed the antimicrobial study . Applying syrup to Apis mellifera L. colony improved the brood cells, increased the collection of honey and pollen, and longevity of honeybees in winter [49,50,51]. Proteinaceous diets on honey bees enhanced brood rearing and increased newly emerged worker bees . Another research found that feeding fermented diets as a protein supplement, including fermented gluten meal, gluten meal, fermented soybean meal, soybean meal, pollen, and sugar syrup separately on the colonies increased brood rearing and improved the health of honey bees [53, 54]. All these studies indicated that different types of supplement applications to honey bee colonies increased the quality of health of honey bees after enhancing their morphological characteristics, which enabled them to increase the collection of honey and pollen. The worker bees also enhance their brood-rearing activity. All these activities may lead to an increase in the collection of honey in the comb. Therefore, this process may suddenly increase the quality of honey, which is also reflected in all the above research findings. In our study, we found that supplement administration increases the quality of honey in terms of antioxidant activity antimicrobial activity. Therefore, the possible reason for all these enhancements in honey quality is syrup supplements, which may have the inductive activity to increase the health of worker bees. Improved quality of honey due to the multiple ingredients might have increased the antibacterial activities.
In our study, post-feeding samples of honey showed maximum antioxidant and free radical scavenging activity and lower levels in the samples collected before feeding diet formulations. Also, a statistically significant difference was observed between the DPPH and ABTS values of both samples of honey (pre- and post-feeding samples). However, no statistically significant difference was noted among the FRAP values of these samples. Antibacterial activity, especially with E. coli and Salmonella enterica, suggests that honey has potential antimicrobial properties and may be used to treat bacterial infection. The results obtained from this study confirmed that the quality of honey is not affected after feeding stimulative diets to honeybees during the dearth period of the year. However, further studies need to be devoted to study the field experiments at a large scale to evaluate the commercial aspects of this formulation; based on the study’s outcome, this formulation may be recommended to beekeepers during the dearth period.
The full dataset in an Excel file is available from the corresponding author on request.
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The authors are highly grateful to Himachal Pradesh Council for Science Technology and Environment, Shimla (India) and Arni University, for providing financial support to carry out this study.
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Kumari, I., Hajam, Y.A., Thiyagarajan, K. et al. Evaluation of antioxidant and antibacterial potential of honey produced from stimulative diet fed bee colonies. Discov Sustain 4, 21 (2023). https://doi.org/10.1007/s43621-023-00135-9