Possible correlation between levansucrase production and probiotic activity of Bacillus sp. isolated from honey and honey bee

  • Abdelhamid A. Hamdy
  • Nouran A. Elattal
  • Magdy A. Amin
  • Amal E. Ali
  • Nahla M. Mansour
  • Ghada E. A. Awad
  • Hassan M. Awad
  • Mona A. Esawy
Original Paper


Five bacterial isolates from honey and bee gut were selected based on their high levansucrase activity and levan yield which were strongly positively correlated. All isolates showed good tolerance to temperature up to 70 °C, to NaCl up to 3 M and to 0.1% H2O2. They maintained over 59 and 64% survival at pH 9.0 and 2.0 respectively, but showed varying tolerance to 0.1% bile salts and pancreatic enzymes. Most isolates were susceptible to widely used antibiotics, but demonstrated diverse antimicrobial activity. Non hemolytic isolates were identified on the basis of 16S rRNA sequencing as Bacillus subtilis HMNig-2 and B. subtilis MENO2 with 97% homology. They exhibited promising probiotic characteristics and achieved highest levansucrase activity of 94.1 and 81.5 U/mL respectively. Both exhibited highest biofilm formation ability in static microtiter plate assay. Also, they achieved 34 and 26% adhesion respectively to Caco-2cells and had highest free radical scavenging activity of 30.8 and 26.2% respectively. The levans of the two isolates showed good antimicrobial activity against some pathogens and exhibited positive prebiotic effect (prebiotic index >1) with Lactobacillus casei and Lactobacillus reuteri. Results suggest a correlation between levansucrase production, levan yield and pre-probiotic activities of the studied strains.


Honey isolates Probiotic bacteria Levansucrase Levan Prebiotic 



This work was supported by National Research Center, Chemistry of Natural and Microbial Products Department by project Grant Number 10050305. Special thanks to Prof. Harold Corke (Shanghai Jiao Tong University, China) for his generous help in revising the paper’s language.


  1. Abdel-Fattah AF, Mahmoud DA, Esawy MA (2005) Production of levansucrase from Bacillus subtilis NRC 33a and enzymic synthesis of levan and fructo-oligosaccharides. Curr Microbiol 51:402–407. doi: 10.1007/s00284-005-0111-1 CrossRefGoogle Scholar
  2. Aebi H (1984) Catalase in vitro. Method Enzymol 105:121–126CrossRefGoogle Scholar
  3. Audisio MC, Torres MJ, Sabate DC, Ibarguren C, Apella MC (2011) Properties of different lactic acid bacteria isolated from Apismellifera L. bee-gut. Microbiol Res 166:1–13. doi: 10.1016/j.micres.2010.01.003 CrossRefGoogle Scholar
  4. Bader J, Albin A, Stahl U (2012) Spore-forming bacteria and their utilization as probiotics. Benef Microbes 3(1):67–75. doi: 10.3920/BM2011.0039 CrossRefGoogle Scholar
  5. Block RJ, Vurrum EL, Zweig U (1955) A manual of paper chromatography and paper electrophoresis. Academic Press, New York, p 127. doi: 10.1002/ange.19550671421 CrossRefGoogle Scholar
  6. Bosch M, Fuentes MC, Audivert S, Bonachera MA, Peiró S, Cuñé J (2014) Lactobacillus plantarum CECT 7527, 7528 and 7529, probiotic candidates to reduce cholesterol levels. J Sci Food Agric 94:803–809. doi: 10.1002/jsfa.6467 CrossRefGoogle Scholar
  7. Brown SM, Howell ML, Vasil ML, Anderson AJ, Hassett DJ (1995) Cloning and characterization of the KatB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase, purification of KatB, cellular localization and demonstration that it is essential for optimal resistance to hydrogen peroxide. J Bacteriol 177:6536–6544CrossRefGoogle Scholar
  8. Burits M, Bucar F (2000) Antioxidant activity of Nigella sativa essential oil. Phytother Res 14:323–328CrossRefGoogle Scholar
  9. Bush K, Fisher JF (2011) Epidemiological expansion, structural studies, and clinical challenges of new β-lactamases from gram-negative bacteria. Annu Rev Med 65:455–478. doi: 10.1146/annurev-micro-090110-102911 Google Scholar
  10. Chaiyawan N, Taveeteptaiku P, Wannissorn B, Itsaranuwat P (2010) Characterization and probiotic properties of Bacillus strains isolated frombroiler. Thai J Vet Med 40:207–2014Google Scholar
  11. Clinical and Laboratory Standards Institute (2014) Performance standards for antimicrobial susceptibility testing; 24th informational supplement CLSI M100-S24. Clinical and Laboratory Standards Institute, WayneGoogle Scholar
  12. Coconnier MH, Klaenhammer TR, Kerneis S (1992) Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture. Appl Environ Micro 58:2034–2039Google Scholar
  13. Conway PL, Gorbach SL, Goldin BR (1987) Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. J Dairy Sci 70:1–12. doi: 10.3168/jds.S0022-0302(87)79974-3 CrossRefGoogle Scholar
  14. Corthesy B, Gaskins HR, Mercenier A (2007) Cross-talk between probiotic bacteria and the host immune system. J Nutr 137:781S–790S. doi: 10.1111/jam.12521 Google Scholar
  15. Cuendet M, Hostettmann K, Potterat O (1997) Iridoidglucosides with free radical scavenging properties from Fragreablumei. Helv Chim Acta 80:1144–1151. doi: 10.1002/hlca.19970800411 CrossRefGoogle Scholar
  16. Dal Bello F, Walter J, Hertel C, Hammes WP (2001) In vitro study of prebiotic properties of levan-type exopolysaccharides from Lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis. Syst Appl Microbiol 24:232–237. doi: 10.1078/0723-2020-00033 CrossRefGoogle Scholar
  17. Esawy MA, Ahmed EF, Helmy WA, Mansour NM, El-Senousy WM, El-Safty MM (2011) Production of levansucrase from novel honey Bacillus subtilis isolates capable of producing antiviral levans. Carbohyd Polym 36:823–830. doi: 10.1016/j.carbpol.2011.05.035 CrossRefGoogle Scholar
  18. Esawy MA, Awad GEA, Ahmed EF, Danial EN, Mansour NM (2012a) Evaluation of honey as new reservoir for probiotic bacteria. Adv Food Sci 34:72–81Google Scholar
  19. Esawy MA, Mansour SH, Ahmed EF, Hassanein NM, El Enshasy HA (2012b) Characterization of extracellular dextranase from a novel halophilic Bacillus subtilis NRC-B233b a mutagenic honey isolate under solid state fermentation. J Chem 9:1494–1510. doi: 10.1155/2012/860619 Google Scholar
  20. Esawy MA, Amer H, Gamal-Eldeen AM, El Enshasy HA, Helmy WA, Abo-Zeid MA, Malek R, Ahmed EF, Awad GE (2013) Scaling up, characterization of levan and its inhibitory role in carcinogenesis initiation stage. Carbohyd Polym 95:578–587. doi: 10.1016/j.carbpol.2013.02.079 CrossRefGoogle Scholar
  21. Esawy MA, Helal MI, Hassan ME, Hassanein NM, Hashem AM (2016) Enzymatic synthesis using immobilized Enterococcus faecalis Esawy dextransucrase and some applied studies. Int J Biol Macromol 92:56–62. doi: 10.1016/j.ijbiomac.2016.06.041 CrossRefGoogle Scholar
  22. FAO/WHO (2001) Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Report of a Joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria 1–4, Oct. American Córdoba Park Hotel, Córdoba, p 6Google Scholar
  23. Foligne B, Peys E, Vandenkerckhove J, Van Hemel J, Dewulf J, Breton J, Pot B (2012) Spores from two distinct colony types of the strain Bacillus subtilis PB6 substantiate anti-inflammatory probiotic effects in mice. Clin Nutr 31:987–994. doi: 10.1016/j.clnu.2012.05.016 CrossRefGoogle Scholar
  24. Gilliland SE, Staley TE, Bush LJ (1984) Importance of bile tolerance of Lactobacillus acidophilus used as dietary adjunct. J Dairy Sci 67:3045–3051. doi: 10.3168/jds.S0022-0302(84)81670-7 CrossRefGoogle Scholar
  25. Harely JP, Prescott L (1996) Laboratoryexercises in microbiology. McGraw Hill, New YorkGoogle Scholar
  26. Hashem AM, Gamal AA, Hassan ME, Hassanein NM, Esawy MA (2016) Covalent immobilization of Enterococcus faecalis Esawy dextransucrase and dextran synthesis. Int J BiolMacromol 82:905–912. doi: 10.1016/j.ijbiomac.2015.09.076 CrossRefGoogle Scholar
  27. Hirayama K, Rafter J (2000) The role of probiotic bacteria in cancer prevention. Microbes Infect 2:681–686. doi: 10.1016/S1286-4579(00)00357-9 CrossRefGoogle Scholar
  28. Hoque MA, Banu MN, Nakamura Y, Shimoishi Y, Murata Y (2008) Proline and glycinebetaine enhance antioxidant defense and methylglyoxal detoxification systems and reduce NaCl-induced damage in cultured tobacco cells. J Plant Physiol 165:813–824. doi: 10.1016/j.jplph.2007.07.013 CrossRefGoogle Scholar
  29. Huang Y, Adams MC (2004) In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria. Int J Food Microbiol 91:253–260. doi: 10.1016/j.ijfoodmicro.2003.07.001 CrossRefGoogle Scholar
  30. Hussein MM, Kheiralla ZM, Helal MM, Saker EA (2010) The prebiotic activities of oligosaccharides derived from partial hydrolysis of commercial Algal polysaccharides. Egypt Pharm 9:1–23Google Scholar
  31. Kimoto H, Kurisaki J, Tsuji NM, Ohmomo S, Okamoto T (1999) Lactococci as probiotic strains: adhesion to human enterocyte-like Caco-2 cells and tolerance to low pH and bile. Lett Appl Microbiol 29:313–316.doi: 10.1046/j.1365-2672.1999.00627.x CrossRefGoogle Scholar
  32. Kwon GH, Lee HA, Park JY, Kim JS, Lim J, Park CS, Kwon DY, Kim YS, Kim JH (2009) Development of a RAPD-PCR method for identification of Bacillus species isolated from cheonggukjang. Int J Food Microbiol 129:282–287. doi: 10.1016/j.ijfoodmicro.2008.12.013 CrossRefGoogle Scholar
  33. Laue H, Schenk A, Li H, Lambertsen L, Neu TR, Molin S, Ullrich MS (2006) Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae. Microbiology 152:2909–2918. doi: 10.1099/mic.0.28875-0 CrossRefGoogle Scholar
  34. Li S, Zhao Y, Zhang L, Zhang X, Huang L, Li D, Niu CH, Yang Z, Wang Q (2012) Antioxidant activity of Lactobacillus plantarumstrains isolated from traditional Chinese fermented foods. Food Chem 135:1914–1919. doi: 10.1016/j.foodchem.2012.06.048 CrossRefGoogle Scholar
  35. Limoli DH, Jones CJ, Wozniak DJ (2015) Bacterial extracellular polysaccharides in biofilm formation and function. Microbiol Spectr 3:1–19. doi: 10.1128/microbiolspec.MB-0011-2014 Google Scholar
  36. Manhar AK, Bashir Y, Saikia D, Nath D, Gupta K, Konwar BK, Kumar R, Namsa ND, Mandal M (2016) Cellulolytic potential of probiotic Bacillus subtilis AMS6 isolated from traditional fermented soybean (Churpi): an in vitro study with regards to application as an animal feed additive. Microbiol Res 186:62–70. doi: 10.1016/j.micres.2016.03.004 CrossRefGoogle Scholar
  37. Mishra V, Prasad DN (2005) Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics. Int J Food Microbial 103:109–115. doi: 10.1016/j.ijfoodmicro.2004.10.047 CrossRefGoogle Scholar
  38. Saunders S, Bocking A, Challis J, Reid G (2007) Effect of Lactobacillus challenge on Gardnerella vaginalis biofilms. Colloids Surf B 55:138–142. doi: 10.1016/j.colsurfb.2006.11.040 CrossRefGoogle Scholar
  39. Sorokulova I (2008) Preclinical testing in the development of probiotics, a regulatory perspective with Bacillus strains as an example. Clin Infect Dis 46:S92–S95. doi: 10.1086/523334 CrossRefGoogle Scholar
  40. Succi M, Tremonte P, Reale A, Sorrentino E, Grazia L, Pacifico S, Coppola R (2005) Bile salt and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. FEMS Microbiol Lett 244:129–137. doi: 10.1016/j.femsle.2005.01.037 CrossRefGoogle Scholar
  41. Takahashi N, Xiao J-Z, Miyaji K, Yaeshiima T, Hiramatsu A, Iwatsuki K, Kokubo S, Hosono A (2004) Selection of acid tolerant bifidobacteria and evidence for a lowpH-inducible acid tolerance response in Bifidobacterium longum. J Dairy Res 71:340–345. doi: 10.1017/S0022029904000251 CrossRefGoogle Scholar
  42. Tanaka T, Oi S, lizuka M, Yamamoto T (1978) Levansucrase of Bacillus subtilis. Agric Biol Chem 42:323–326. doi: 10.1080/00021369.1978.10862976 Google Scholar
  43. Terraf MC, JuarezTomás MS, Nader-Macías ME, Silva C (2012) Screening of biofilm formation by beneficial vaginal lactobacilli and influence of culture media components. J Appl Microbiol 113:1517–1529. doi: 10.1111/j.1365-2672.2012.05429.x CrossRefGoogle Scholar
  44. Thompson JD, Higgins DG, Gibson TJ, Clustal W (1994) Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  45. Wang M, Zhao W-Z, Xu H, Wang Z-W, He S-Y (2015) Bacillus in the guts of honey bees. (Apismellifera; Hymenoptera, Apidae mediate changes in amylase values. Eur J Entomol 112:619–624. doi: 10.14411/eje.2015.095 Google Scholar
  46. Woodford N, Turton JF, Livermore DM (2011) Multiresistant gram-negative bacteria, the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol Rev 35:736–755. doi: 10.1111/j.1574-6976.2011.00268.x CrossRefGoogle Scholar
  47. Yanase H, Iwata M, Nakahigashi R, Kita K, Kato N, Tonomura K (1992) Purification, crystallization, and properties of extracellular levansucrase from Zymomonas mobilis. J Biosens Biotechnol Bioch 56:1335–1337. doi: 10.1271/bbb.56.1335 CrossRefGoogle Scholar
  48. Ziemer CJ, Gibson GR (1998) An overview of probiotics, prebiotics and synbiotcs in the functional food concept: perspectives and future strategies. Int Dairy J 8:473–479.  10.1016/S0958-6946(98)00071-5 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Abdelhamid A. Hamdy
    • 1
  • Nouran A. Elattal
    • 1
  • Magdy A. Amin
    • 2
  • Amal E. Ali
    • 2
  • Nahla M. Mansour
    • 1
  • Ghada E. A. Awad
    • 1
  • Hassan M. Awad
    • 1
  • Mona A. Esawy
    • 1
  1. 1.Pharmaceutical Industries Division, Chemistry of Natural and Microbial Products DepartmentNational Research CentreGizaEgypt
  2. 2.Department of Microbiology and Immunology, Faculty of PharmacyCairo UniversityCairoEgypt

Personalised recommendations