STUDY ON ANTIBACTERIAL ACTIVITY OF BEE VENOM

Yeon Jo Ha 1 , Chi Won Noh , Woo Young Bang, Sam Woong Kim and Sang Wan Gal. 1. Department of Pharmaceutical Engineering, Gyeongnam National University of Science and Technology. 2. Gyeongsangnam-do Agricultural Research & Extension Services, Jinju, 52733, South Korea. 3. National Institute of Biological Resources (NIBR), Environmental Research Complex, Incheon 22689, South Korea. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The purpose of this study was to investigate the antimicrobial activity against Salmonella infection which causes intestinal diseases from bee venom which is one of the social insects, and to find a way which use ghost vaccine. The minimum inhibitory concentration (MIC) of bee venom against Salmonella Typhimurium χ3339 was 101.81 ug/ml. Based on the result of MIC, the antimicrobial activity according to amount of the cells showed strong activities below 10 6 CFU/ml, but exhibited no and low activity at 10 8 and 10 7 CFU/ml, respectively. In addition, the antimicrobial activity assay according to the cultural temperature showed the strongest activity at 37℃. The thermal stability of bee venom appeared to be very stable at temperatures below 60℃, but gradually decreased at more than 60℃. The protease inhibitor mixture was shown to greatly enhance the antimicrobial activity of bee venom. Since most of the activity is lost due to the treatment with Proteinase K, it is estimated that the antimicrobial activity of bee venom is induced by peptides. Therefore, we suggest that bee venom can be used as an antimicrobial agent for ghost vaccine production.

…………………………………………………………………………………………………….... Introduction:-
Bee venom is a colorless transparent and viscous liquid to maintain strong aromatic bitter taste, and an off-white or yellowish white mass or powder in dry state. The specific gravity of the bee venom solution is 1.13 and the acidity is in the range of 5.2 ~ 5.5. Bee venom is easy to dissolve in water and acids, but hardly soluble in alcohol. The bee venom solution loses 70% of its liquid weight by quickly drying when exposed to air at room temperature. Bee venom maintains its long-term activity in the frozen state, but tends to be easily destroyed by oxidizing substances (Koh, 1993;Kim, 1996).
Bee venom is composed of about 40 components, which consists of peptide components such as melittin, apamin, and mastcell degranulating (MCD) peptide, enzyme components such as phospholipase A2, hyauronidase, and acid phosphomonoester, and amine components such as histamine, dopamine, and noradrenaline (Chmielewska and Szczęsna, 2004; Kokot and Matysiak, 2009). Among them, melittin is a major component that accounts for about 50% of the dry bee venom, and it is known that it works very well in pain and inflammation (Piek, 1986;Curcio-Vonlanthen V et al., 1997). Melittin was first discovered as a hemolytic element in 1954, and observed by cell membrane activity during erythrocyte lysis in 1972 . Structurally, it consists of 26 amino acids, which strongly inhibits macrophage migration and synergizes with phospholipase A2 during hemolysis to increase mutual activity. In the enzyme action, it stimulates catecholamine and cortisone secretion by stimulating the pituitary and adrenal cortex system, induces an increase of serotonine in the hypothalamus, and has anti-ISSN: 2320-5407 Int. J. Adv. Res. 6(1), 229-235 230 inflammatory action via stabilization of the lysosomal membranes (Kwan et al., 2000;Lee, 2000). There are also dissolution, enzymes, pain induction, radiation-resistant action, and antibacterial action (An et al., 2010).
To date, many AMPs (antimicrobial peptides) have been obtained from nature, but the most promising candidates in recent years have been originated from poisonous animals such as snakes, scorpions, spiders, ants, wasps, bees, and centipede (Wang et al., 2016). Peptide toxins such as androtonin, parbutoporin, opistoporins, TstH, and vpAmp 1.0, which are derived from scorpion venom, show strong antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria or fungi (Hetru et  In this study, the antimicrobial activity against Salmonella Typhimurium χ3339, a causative organism of Salmonella infection, was evaluated by the bee venom and examined for the possibility to apply the venom for preparation of ghost vaccine.

Bee venom and bacterial strain:-
The bee venom powder used in this study was purchased from Chung Jin Biotech Co., Ltd (South Korea), and applied as a concentration of 10 ug/ul. The test strain was examined by Salmonella Typhimurium χ3339.

Analysis of minimal inhibitory concentration (MIC):-
MIC analysis of bee venom against Salmonella Typhimurium χ3339 was performed with a microtiter plate method. Bee venom powder was dissolved in an appropriate concentration (10 μg/μl) and used for antimicrobial activity analysis. The used strain was employed for MIC assay by adjusting the precultured broth to 10 6 CFU/ml or an appropriate concentration. The concentrations of bee venom used in the analysis were 0, 25, 50, 100, and 250 μg/ml. Each 200 μl of the reaction solution was added into a microtiter plate, and the antimicrobial activity was observed after culturing at 37℃ for 16 h. The results were measured at 600 nm with an ELISA leader (Multiscan GO, Thermo Scientific Co. Ltd., Rochester, NY, USA).

Antibacterial activity of bee venom according to the amount of bacterial cells:-
The antimicrobial activity of Salmonella Typhimurium χ3339 for the amount of bacterial cells was assayed by a microtiter plate method. Concentration of bee venom according to MIC results obtained from 10 6 CFU/ml were used for the subsequent analysis of antimicrobial activity. The used strains were employed for the evaluation depending on the amount of bacterial cells by adjusting the cultured broth at concentrations of 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , and 10 3 CFU/ml. Each 200 μl of the reaction solution was added into a microtiter plate, and the antimicrobial activity was observed after culturing at 37℃ for 16 h. The results were measured at 600 nm with an ELISA leader (Multiscan GO, Thermo Scientific Co. Ltd., Rochester, NY, USA).

Analysis of antimicrobial activity for bee venom by temperature
The antimicrobial activity of Salmonella Typhimurium χ3339 by temperature was assayed by microtiter plate method. The bee venom was used for the antimicrobial activity analysis according to the MIC result. The used strain was employed for MIC assays by adjusting the precultured broth to 10 6 CFU/ml or an appropriate concentration. Each 200 μl of the reaction solution was added into a microtiter plate, and the antimicrobial activity was observed after culturing at 37℃ for 16 h. The results were measured at 600 nm with an ELISA leader (Multiscan GO, Thermo Scientific Co. Ltd., Rochester, NY, USA).

Evaluation of thermal stability for bee venom:-
Analysis of the thermal stability of bee venom for Salmonella Typhimurium χ 3339 was performed using a microtiter plate method. The bee venom was used for the antimicrobial activity analysis according to the MIC result. The used strain was employed for evaluation of thermal stability by adjusting the precultured broth to 10 6 CFU/ml or an appropriate concentration. The bee venom used in the evaluation was examined after heating for 10 minutes at 0, 30, 40, 50, 60, 70, and 80℃, respectively. Each 200 μl of the reaction solution was added into a microtiter plate, and the antimicrobial activity was observed after culturing at 37℃ for 16 h. The results were measured at 600 nm with an ELISA leader (Multiscan GO, Thermo Scientific Co. Ltd., Rochester, NY, USA).
Antibacterial activity of bee venom according to protease inhibitor mixture:-Antimicrobial activities of Salmonella Typhimurium χ3339 according to protease inhibitor mixture of bee venom were analyzed by microtiter plate method. The bee venom was used for the antimicrobial activity analysis according to the MIC result. The used strains were employed for evaluation of an effect for protease inhibitor mixture by adjusting the precultured broth to 10 8 CFU/ml or an appropriate concentration. The protease inhibitor mixture used in the assay was Protease inhibitor P8465 (Sigma-Aldrich, Milwaukee, WI, USA) and the concentrations were 0, 0.5, 1, 2, 5, and 10 μl. Each 200 μl of the reaction solution was added into a microtiter plate, and the antimicrobial activity was observed after culturing at 37℃ for 16 h. The results were measured at 600 nm with an ELISA leader (Multiscan GO, Thermo Scientific Co. Ltd., Rochester, NY, USA).

Analysis of antimicrobial activity of bee venom by simultaneous treatment with Proteinase K:-
The antimicrobial activities of Salmonella Typhimurium χ3339 by simultaneous treatment with Proteinase K of bee venom were analyzed by microtiter plate method. The used strain was employed for evaluation of an effect for protease by adjusting the precultured broth to 10 6 CFU/ml or an appropriate concentration. Proteinase K (Sigma-Aldrich, Milwaukee, WI, USA) was used by 80 ug/ml concentration for the analysis. Each 200 μl of the reaction solution was added into a microtiter plate, and the antimicrobial activity was observed after culturing at 37℃ for 16 h. The results were measured at 600 nm with an ELISA leader (Multiscan GO, Thermo Scientific Co. Ltd., Rochester, NY, USA). Proteinase K (Sigma-Aldrich, Milwaukee, WI, USA) was used by for the analysis.

Results and Discussion:-
Antimicrobial activity against S. Typhimurium:-This study was done to evaluate the efficacy of commercially available bee venom through S. Typhimurium. It is known that the antibacterial activity of commercial bee venom is 20 to 40 ug / ml (Leandro et al., 2015). However, it was presumed that some activities were changed during addition of excipient and drying process in the process of powdering bee venom. As a result of this study with the purchased bee venom, the minimal inhibitory concentration (MIC) for S. Typhimurium 3339 was observed by 101.8085 ug/ml (Fig. 1). This result is 2.5 ~ 5 times lower than the previous research results. Therefore, it is judged that the excipient is mixed with the corresponding concentration or some activity is lost in the drying process. Fig. 1:-Evaluation of antibacterial activity for bee venom. Salmonella Typhimurium χ3339 strain was diluted to 10 6 CFU/ml and applied for the study. Bee venom was used by concentration of 10 μg/μl and added at concentrations of 0, 25, 50, 100, and 250 μg/ml. Xand Y-axes indicate optical density at 600 nm and concentration of bee venom, respectively.
One of the purposes of this study is to produce Salmonella ghost vaccine with treatment of bee venom. Therefore, we investigated whether the bee venom activity is a good function in high population of the strain. The antimicrobial activity was evaluated by the amount of MIC value obtained from 10 6 CFU/ml to investigate the antimicrobial activity against the high concentration of the bacterial mass. As shown in Fig. 2, higher activity was observed below 10 6 CFU/ml, whereas the activity was markedly lowered at 10 7 CFU/ml and little activity at 10 8 CFU/ml. Therefore, it is suggested that a higher content of bee venom must be used in order to exhibit high antimicrobial activity at 10 8 CFU/ml. Although it is a disadvantage of using a high concentration of bee venom, it is assumed that bee venom powder employed in this study is a possibility of application as a raw material for production of ghost vaccine. Salmonella Typhimurium χ3339 strain was diluted to 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , and 10 3 CFU/ml, respectively, and employed for the study. Bee venom was used as a stock at a concentration of 10 μg/μl, and added at the concentration of the MIC of 10 6 CFU/ml. X-and Y-axes indicate optical density at 600 nm and amount of the cells, respectively.

Evaluation of thermal stability:-
To investigate the suitable temperature for bee venom to be used for ghost vaccination, we evaluated the antimicrobial activity according to each cultural temperature of S. Typhimurim. As shown in Fig. 3, high activity was observed at 25℃ and 37℃, but remarkably low activity was observed at 42℃. Bee venom treated at 25℃ showed high antimicrobial activity, but overall growth was significantly reduced. Therefore, it is suggested that treatment at 37℃ was appropriate for ghost formation of S. Typhimurium. Fig. 3:-Examination of antimicrobial activity according to cultural temperature. Salmonella Typhimurium χ3339 strain was diluted to 10 6 CFU/ml and applied for the study. Bee venom was used by concentration of 10 μg/μl, and added at the concentration of the MIC of 10 6 CFU/ml. X-and Y-axes indicate optical density at 600 nm and cultural temperature, respectively. C; control without bee venom, T; treatment with bee venom.
In order to increase the possibility of commercial application, the used AMPs must have high thermal stability. Therefore, in this study, heat stability was evaluated at each temperature for 10 minutes. As shown in Fig. 4, the activity was remained by treatment at 0℃, 30℃, 40℃, and 50℃ for 10 minutes, but the activity was gradually decreased when the temperature was further increased. Therefore, it is presumed that most activity of the bee venom is retained when it is treated at a temperature of 60℃ or lower in the processing process for powder. Fig. 4:-Evaluation of thermal stability of bee venom. Salmonella Typhimurium χ3339 strain was diluted to 10 6 CFU/ml and applied for the study. Bee venom was used by concentration of 10 μg/μl, and added at the concentration of the MIC of 10 6 CFU/ml. Bee venom was added after heating for 10 minutes at 0 ~ 80℃. X-and Y-axes indicate optical density at 600 nm and cultural temperature, respectively. Con; control without bee venom.

Analysis of association between antimicrobial activity and peptide:-
Treatment with high-concentration of Salmonella at MIC value of 10 6 CFU/ml showed a tendency to significantly decrease activity at an amount of the cells higher than 10 7 CFU/ml (Fig. 1). Therefore, to overcome these drawbacks, we analyzed the activity with a protease inhibitor mixture. It is well known that protease inhibitor plays a role in inhibiting the activity of protease in the cultural medium and correcting the MIC value (Ha et al., 2017).
Protease inhibitor mixture was added in each concentration to analyze its activity. As a result, the treatment with protease inhibitor mixture depending on concentration showed that the activity of the mixture was increased by 50% or more when 1 μl of the mixture was treated and increased to the maximum value at 2 μl ( Salmonella Typhimurium χ3339 strain was diluted to 10 8 CFU/ml and applied for the study. Bee venom was used by concentration of 10 μg/μl, and added at the concentration of the MIC of 10 6 CFU/ml. Protease inhibitor mixture was added in an amount of 0 μl, 0.5 μl, 1 μl, 2 μl, 5 μl, and 10 μl. Xand Y-axes indicate optical density at 600 nm and concentration of protease inhibitor mixture, respectively. We treated proteinase K in order to confirm that the antimicrobial activity of bee venom is derived from the peptide. As a result, high activity was observed when proteinase K was not treated, but proteinase K treatment showed low activity regardless of activation and inactivation states of proteinase K (Fig. 6). The MIC of melittin and phospholipase A2 were 4 ~ 40 and 400 ug/ml, respectively, whereas in the case of treatment at the same time, the MIC was 6 ~ 80 ug/ml (Leandro et al., 2015). Therefore, it is assumed that most of the antimicrobial activity of bee venom is derived from the peptide melittin and phospholipase A2. Fig. 6:-Analysis of antimicrobial activity according to simultaneous treatment with proteinase K. Salmonella Typhimurium χ3339 strain was diluted to 10 6 CFU/ml and applied for the study. Bee venom was used by concentration of 10 μg/μl, and added at the concentration of the MIC of 10 6 CFU/ml. X-and Y-axes indicate optical density at 600 nm and concentration of protease inhibitor mixture, respectively. Con; non treated.
In summary, these results indicate that bee venom purchased from the market is slightly lower than the general activity, but it can be used for the production of ghost vaccine when mixed with a protease inhibitor mixture. In addition, since it has a temperature stability up to 60℃, it has been found that there is no problem in making the powder into industrial use.