ANTIBACTERIAL AND DIASTASE ENZYME ACTIVITIES OF HONEY Apis mellifera FROM INDONESIA

RESULTS AND DISCUSSION

Water Content

Among the 9 samples analyzed, the water content of honey adhered to predetermined standards. Coffea robusta 2 honey had the lowest water content at 17.50%, while Calliandra calothyrsus had the maximum at 22.00% determined by SNI. The moisture content of Nephelium lappaceum, Melaleuca laucadendron, Ceiba pentandra, Mangifera indica, Coffea robusta 1, Acacia carpa, and Hevea brasiliensisensi were 21.50%, 20.50%, 20.50%, 19.50%, 19.00%, 18.50%, and 18.00%, respectively. The ANOVA statistical analysis supported this result, showing a significant difference in the water content of the 9 monofloral honeys (p-value < 0.05). This showed that differences in plants as a source of nectar had an impact on the water content of honey. The results of Duncan's test (α=0.05) on the water content of 9 monofloral honeys showed 8 groups, namely (1) Calliandra calothyrsus, (2) Nephelium lappaceum L., (3) Melaleuca laucadendron L. and Ceiba pentandra (L.) Gaertn, (4) Mangifera indica, (5) Coffea robusta 1, (6) Acacia carpa, (7) Hevea brasiliensisensi M., and (8) Coffea robusta 2. These groups were categorized based on the proximity of their water content, as shown in Figure 1.

The water content in honey determines the stability of fermentation because it allows easy fermentation over time. Therefore, honey needs to be subjected to heat treatment to prevent fermentation by sugar-tolerant yeasts and maintain its liquid state (Singh and Singh 2018). Relative humidity (Rh) in Indonesia ranges from 60–90%, indicating that the water content in honey is approximately 18.3–33.1%. Honey is hygroscopic, and must be stored in a place that is not permeable to air. According to Pujiarti et al. (2021), signs of a fermentation process are the foam and gas produced by honey. Hasan et al. (2020) reported that the antibacterial activity of multiflora honey from Riau RHU4 has the lowest water content of 16.45% and RHI2 honey of 20.56%.

The fermentation process in yeast can be significantly influenced by the high water content in honey, leading to a decline in its overall quality. Moreover, the shelf life of honey is affected by its water content and a higher water content results in a relatively short shelf life. According to Hasan et al. (2020), honey can absorb up to 33% water by weight in moist air. Good-quality honey has a low water content, indicating that thicker honey has a lower water content (Swari et al. 2019). Honey harvested at an older age contains less water than at a younger age. Generally, at harvest ages < 14 days, the water content of honey is relatively high. According to Nasharuddin et al. (2022), Indonesian honey is often fermented due to its high water content of >26%. The Indonesian National Standard (2018) reported that the quality requirement of water content in honey was a maximum of 22%. A previous study showed a correlation between the water and sugar content of honey, and the honey crystallization process was natural (Nasharuddin et al. 2022).

Diastase Enzyme Activity

The average results of diastase enzyme activity in monofloral honey were shown in Figure 2. The highest diastase enzyme activity in Mangifera indica, Nephelium lappaceum, and Coffea robusta 2 was 20.00 DN, while the lowest was in Calliandra calothyrsus at 4.00 DN. Furthermore, the diastase enzyme activity of Melaleuca laucadendron, Ceiba pentandra, and Hevea brasiliensisensi was 10.00 DN, whereas Acacia carpa and Coffea robusta 1 were 6.67 DN and 5.00 DN, respectively.

These results were supported by ANOVA statistical analysis which showed a significant difference between the 9 monofloral honey (p-value < 0.05) based on their diastase enzyme activity. This showed that differences in plants as a source of nectar affected it. It also showed the impact of the differences in plants as a source of nectar. The results of Duncan's test (α=0.05) on the activity of the diastase enzyme from 9 monofloral honey showed 5 groups, namely (1) Mangifera indica, Nephelium lappaceum L., and Coffea robusta 2, (2) Melaleuca laucadendron L., Ceiba pentandra (L.) Gaertn, and Hevea brasiliensisensi M., (3) Acacia carpa, (4) Coffea robusta 1, and (5) Calliandra calothyrsus M. These monofloral honey awerere categorized based on their proximity to the activity of the diastase enzyme they possess, as shown in Figure 2.

The diastase enzyme is one of many different types found in every variety of honey and contributes to its nutritional value. Bees introduce the diastase enzyme during the ripening of the honey. This enzyme breaks the glucosidic bonds in starch and other oligo- and polysaccharides into simple sugars. According to Singh and Singh (2018), adequate heating and storage are crucial to retaining the market value of honey since the diastase enzyme is extremely sensitive to heat.

According to Ichsan et al. (2022), in the chemical test with the diastase enzyme, 17 out of 22 samples were found to have good quality because they met the SNI requirement. The Diastase Number (DN) value ranged from 0.64 to 23.11, while for fake honey, the value of DN was 0, indicating that it was undetectable. The absence of the diastase enzyme in a honey sample can serve as an indicator of potential adulteration, suggesting that the sample may be classified as fake honey. Such samples solely consist of sugars without the presence of the diastase enzyme. Furthermore, Tulandi (2019) showed that storage temperature greatly affected the quality of honey. There was a significant difference in diastase enzyme activity in each treatment at storage temperature. The best storage temperature for honey was 26˚C (room temperature) compared to hot (50˚C) and cold (±5˚C) temperatures.

The activity of this enzyme decreased with storage and heating time, as indicated by its measurement through DN. It is observed that diastase activity decreases as the temperature rises, and the decline becomes more significant with higher temperature increments. The diastase activity decreases quickly during isothermal heating at a constant temperature but increases once again as the number of heating cycles increases (Singh and Singh 2018). According to Harjo et al. (2015), diastase activity in rubber and rambutan honey was 11.89 DN and 11.58 DN, respectively. The presence of the diastase enzyme in honey can be attributed to the bee's ability to produce saliva. Consequently, a higher value of the diastase enzyme indicates a greater concentration of bee saliva contained in honey.

A decrease in the value of the diastase enzyme activity can occur in the storage process before testing. Therefore, it is advisable to conduct diastase enzyme testing shortly after the honey is harvested to ensure accurate assessment. The activity of the diastase enzyme is an indicator of the quality of honey because it can indicate its processing and storage time. According to Pujiarti et al. (2021), storage can significantly affect the inactivation of enzymes. Honey products usually do not meet requirements for commercialization after they have been heated, reheated, or stored for a long time, because the diastase activity is below the quality requirements. According to the Indonesian National Standard (2018), the quality requirements for the value of honey diastase activity are more than 3 DN.

Antibacterial Activity

The smallest clear zone for Staphylococcus aureus was 14.73 mm in Mangifera indica, while the largest was 19.47 mm in Coffea robusta 2. The clear zones for Acacia carpa, Melaleuca laucadendron, Coffea robusta 1, Ceiba pentandra, Hevea brasiliensisensi, Nephelium lappaceum, and Calliandra calothyrsus were 18.53 mm, 17.73 mm, 17.03 mm, 16.12 mm, 16.10 mm, 16.03 mm, and 15.73 mm, respectively, as shown in Figure 3. The smallest clear zone for Escherichia coli was 21.53 mm in Nephelium lappaceum, while the largest was 27.93 mm in Ceiba pentandra. The values for Coffea robusta 2, Acacia carpa, Coffea robusta 1, Melaleuca laucadendron, Mangifera indica, Hevea brasiliensisensi, and Calliandra calothyrsus were 26.13 mm, 24.60 mm, 24.53 mm, 22.53 mm, 22.07 mm, 21.90 mm, and 21.60 mm, respectively, as shown in Figure 4.

The positive control on both bacteria showed that a clear zone was formed. This is consistent with the ANOVA statistical analysis, showing that the 9 monofloral honey were significantly different (p-value < 0.05) based on their antibacterial activity. The results of Duncan's test (α=0.05) on the inhibition zone of Staphylococcus aureus bacteria from 9 monofloral honey showed 6 groups, namely (1) Coffea robusta 2 and Acacia carpa, (2) Acacia carpa and Melaleuca laucadendron L., (3) Melaleuca laucadendron L. and Coffea robusta 1, (4) Coffe robusta 1, Ceiba pentandra (L.) Gaertn, and Nephelium lappaceum L., (5) Ceiba pentandra (L.) Gaertn, Hevea brasiliensisensi M., Nephelium lappaceum L., and Calliandra calothyrsus M., and (6) Mangifera indica. These monofloral honey were grouped based on the proximity of their inhibition zones, as shown in Figure 3. Furthermore, Duncan's test at α=0.05 on the inhibition zone of Escherichia coli bacteria showed that 4 groups, namely (1) Ceiba pentandra (L.) Gaertn, (2) Coffe robusta 1, (3) Acacia carpa and Coffea robusta 2, and (4) Melaleuca laucadendron L., Mangifera indica, Hevea brasiliensisensi M., Calliandra calothyrsus M., and Nephelium lappaceum L. They were groups based on the proximity of their inhibition zones, as shown in Figure 4.

The clear zone value is determined by subtracting the diameter of the disc used from the measured clear zone diameter. The effectiveness of bacterial inhibition by honey is influenced not only by physicochemical characteristics but also by its bioactive compounds. Furthermore, the content of bioactive compounds is obtained from nectar sources that are taken and processed by honey bees (Hasan et al. 2020).

The monofloral honey antibacterial test was carried out on two test bacteria, namely Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). Furthermore, antibacterial activity was determined based on the diameter of the clear zone formed after incubation. This test used the antibiotic ciprofloxacin (100 ppm) as the positive control. According to the Clinical Laboratory Standard Institute (CLSI) (2013), antibacterial activity is classified as weak or lacking when the resulting inhibition zone is ≤12 mm. Similarly, it is classified as medium and strong when the inhibition zone is 13-17 mm and ≥18mm, respectively. Based on this classification, the test results showed that monofloral honey had moderate to strong antibacterial activity against Staphylococcus aureus. This was because the diameter of the resulting inhibition zone was in the range of 14.73 mm and 19.47 mm. The antibacterial activity of monofloral honey against Escherichia coli was classified as strong because the diameter of the resulting inhibition zone was greater than 18 mm, ranging from 21.53 mm to 27.93 mm.

According to Hasan et al. (2020), the smallest inhibition zone of Staphylococcus aureus for multi-floral honey from Riau was 7.84 mm for 20% RHI1 honey, while the largest for RHI2 80% honey was 19.25 mm. The smallest inhibition zone of Escherichia coli in RHI1 honey with a concentration of 40% was 1.22 mm, while the largest inhibition zone in PLW1 60% honey was 4.58 mm. According to Kaligis et al. (2020), the minimum inhibitory concentration (MIC) of forest honey toward Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa is 12.5%, 12.5%, and 25%, respectively. In this study, there was no minimum bactericidal concentration (MBC) in either forest or black honey. Furthermore, the antibacterial effect of forest honey is superior to that of black honey by a small margin.

Gram-negative bacteria tend to be more sensitive to the antibacterial substances contained in honey than Gram-positive. They also have hydrophilic groups that easily bind to polar compounds, making the antibacterial activity of honey more effective against Gram-negative bacteria than Gram-positive (Wadi 2022). Furthermore, the study conducted with monofloral honey from Indonesia showed that Escherichia coli was the most influential bacteria. This can be attributed to osmosis, facilitated by the high sugar content of honey, causing water to flow out of the cells, such that the resulting pure and undiluted honey prevents the growth of germs. According to Almasaudi (2021), Dehydration also causes cells to shrink, which hinders their ability to survive in a hypertonic sugar solution, thereby reducing the antibacterial potential.

The vast majority of microbes normally grow in neutral environments with pH values between 6.5 and 7.5. One of the most distinguishing characteristics of the antibacterial qualities of honey is its acidity, which ranges between pH 3.2 and 4.5. Its acidity is caused by the presence of a significant organic acid (gluconic) that is present in the sample at a concentration of 0.5% (w/v). The natural glucose oxidase enzyme converts glucose into glycogen acid, which is a particularly effective antibacterial agent. Furthermore, low pH in pure and undisturbed honey can add to its antibacterial capabilities, when diluted in food or other body fluids (Almasaudi 2021).

Cianciosi et al. (2018) reported that flavonoids and phenolic acids were among the broad group of compounds known as polyphenols and distinguished by their phenolic structures. These biological components are converted from nectar to honey and play a significant role in the health benefits of honey. The presence of numerous phenolic chemicals in honey provides a plausible explanation for its antibacterial properties (Gunes et al. 2017).

Several studies explored the antibacterial activity of phenolic acids in honey. Among these acids, caffeic was found to induce oxidative stress and chlorogenic acid increases membrane permeability, resulting in cytoplasmic and nucleotide leakage (Gorniak et al. 2019). Furthermore, gallic acid induces intracellular leakage as a result of the breakdown of cell membranes and increased hole formation (Shi et al. 2016). P-coumaric acid disrupts cell membranes and bacterial DNA brushing, and syringic acid causes cell membrane dysfunction (Borges et al. 2013). The antibacterial mechanisms of flavonoids found in honey include apigenin and kaemferol, which can inhibit DNA gyrase. Galanin inhibits peptidoglycan synthesis from ribosomes and luteolin inhibits FAS-I in mycobacteria and inhibits DNA helicase DnaB and RecBCD. On the other hand, myricetin inhibits DNA B helicase, pinocembrin induces cell lysis (Collins et al. 2019), and catechins contribute to the generation of H2O2 (Das et al. 2015).

Honey Clusterization

The results of the score plot on the antibacterial activity of Staphylococcus aureus and Escherichia coli and diastase activity show 4 main clusters of monofloral honey from Indonesia with different nectar sources (Figure 5). The first cluster (Figure 5a) consists of Hevea brasiliensisensi from Jember. The second cluster (Figure 5b) consists of Mangifera indica from Pasuruan and Nephelium lappaceum from Subang. The third cluster (Figure 5c) consists of Acacia carpa from Tanjung Jabung and Calliandra calothyrsus from Sukabumi. Finally, the fourth cluster (Figure 5d) consists of Melaleuca laucadendron honey from Sragen, Ceiba pentandra from Pati, Coffea robusta 1 from Malang, and Coffea robusta 2 from Jember.

The clustering of monofloral honey derived from 9 nectar-sourced plants in Indonesia was determined using the multivariate PCA method. This analysis considered both the antibacterial activity and diastase enzymes. The results showed that there are groups with similarities and some exhibit dissimilarities. The PCA method is used to simplify data and enhance readability by transforming the data linearly to form a new coordinate with maximum variance (Ghani et al. 2016).

The arrangement of taxonomy is based on phylogenetic and chemotaxonomy. In this context, plants that share a close kinship tend to possess similar types and homologies of secondary metabolite content. The identified chemical compounds have antibacterial activity. For example, cluster 1 is the genus Hevea, which contains flavonoids (Evahelda et al. 2017). Cluster 2 is of the Sapindales order, and it contains acetophenone, butanoic acid, and benzyl alcohol (Rohman 2017). Cluster 3 belongs to the Fabaceae family, containing protocatechuic, gallic, caffeic, and ascorbic acid (Obisitioiu et al. 2021). Cluster 4 belongs to the Magnoliopsida class, which is characterized by aromatic compounds, such as sesquiterpenes, isoflavonoids, and benzylisoquinone alkaloids (Chassagne et al. 2021).

The differences in honey characteristics, such as taste, aroma, color, and composition, can be attributed to the diverse range of plants whose nectar is collected by bees for honey production. These variations subsequently influence the antibacterial activity of honey. Furthermore, several factors contribute to the antibacterial activity of honey, namely the types of flowers that take their nectar from different areas and their high sugar content (Arumsari et al. 2019).