Comparative Chemical and Bioactivity Studies of Intra- and Extracellular Metabolites of Endophytic Bacteria, Bacillus subtilis NCIB 3610

  • Azza Abdelmageed Matloub
  • Eman Zakaria GomaaEmail author
  • Amira Ali Hassan
  • Marwa Mahmoud Elbatanony
  • Waled Morsy El-Senousy


Endophytic bacteria are able to produce unique bioactive compounds for various biotechnological applications. The intracellular and extracellular extracts of the endophytic bacterium, Bacillus subtilis NCIB 3610 were investigated for chemical composition as well as evaluated for antiviral, antimicrobial, antifungal and cytotoxic activities. The GC/MS analysis of intracellular and extracellular hexane extracts revealed that 2,6-di-t-butyl-4-methyl phenol was found as main compound in intracellular hexane extract, while pentacosane and hexacosane as well as 14-methyl hexadecanoic methyl ester were found as major metabolites in extracellular hexane extract. The intracellular and extracellular polysaccharides were isolated from aqueous extract which characterized as heteropolysaccharides bounded with protein. The yield of intracellular polysaccharide fraction (FC1) (42.17% ± 1.21) was higher than that of the extracellular polysaccharides fractions (FM1, FM2 and FM3) (12.68%). The GLC analysis of fractions revealed the presence of 8, 8, 6 and 9 monosaccharides, respectively, the fractions FC1, FM2 and FM3 composed mainly of galactose, mannose and glucose with weight-average molecular weight (Mw) 295.5, 165.5 and 129.8 kDa, respectively. The amino acid analysis of protein bounded to intra- and extracellular polysaccharide fractions revealed the presence of 17 amino acids. Glutamic acid and alanine were found as predominant amino acids in FC1, while glycine, glutamic and aspartic acids were existed as dominant amino acids in exopolysaccharide fractions. The anti-bacterial activity displayed that intracellular polysaccharide (FC1) has a promising antibacterial activity against Staphylococcus aureus and Streptococcus pneumoniae which gave 51.6 and 37% relative inhibition activity of Doxycyclin Hydrchloride. Moreover, the most fractions of intracellular and extracellular showed broad spectrum antifungal activity. The evaluation of cytotoxic activity of intracellular and extracellular fractions on HCT116, HepG2 and MCF7 human cell lines showed that intracellular chloroform, ethyl acetate and methanol fractions have a potent cytotoxic activity. Overall, the bioactive fractions obtained from Bacillus subtilis NCIB 3610 can be useful for biotechnological and pharmaceutical application as they showed cytotoxic and antimicrobial activities.


Bacillus subtilis NCIB 3610 Antimicrobial Cytotoxicity Polysaccharides 


Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10989_2019_9856_MOESM1_ESM.docx (401 kb)
Supplementary material 1 (DOCX 401 kb)


  1. Abdelnasser SM, Yahya SMM, Mohamed WF, Asker MM, Abu Shady HM, Mahmoud MG, Gadallah MA (2017) Antitumor exopolysaccharides derived from novel marine Bacillus: isolation, characterization aspect and biological activity. Asian Pac J Cancer Prev 18(7):1847–1854Google Scholar
  2. Abdo SM, Hetta MH, El-Senousy WM, Salah El Din RA, Ali GH (2012) Antiviral activity of freshwater algae. J Appl Pharm Sci 2:21–25Google Scholar
  3. Ahmad MH, Mustafa S, Che Man YB (2015) Microbial polysaccharides and their modification approaches: a Review. Int J Food Prop 18(2):332–347CrossRefGoogle Scholar
  4. Altschul SF, Thomas LM, Alejandro AS, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402CrossRefGoogle Scholar
  5. Baldwin MV, Sonia DA, Sindhu TJ, Chanran M, Bhat AR, Krishnakumar K (2014) A review of biological potential of pyrazine and related heterocyclic compounds. World J Pharm Pharm Sci 3(2):1124–1132Google Scholar
  6. Barbosa TM, Serra CR, Ragione RML, Woodward MJ, Henriques AO (2005) Screening for Bacillus isolates in the broiler gastrointestinal tract. Appl Environ Microbiol 71:968–978CrossRefGoogle Scholar
  7. Barry AL (1980) Procedure for testing antimicrobial agents in agar media. In: Lorin V (ed) Antibiotics in laboratory medicine. Williams Wilkins Co., Baltimore, pp 1–23Google Scholar
  8. Bastos JCS, Kohn LK, Fantinatti-Garboggini F, Padilla MA, Flores EF, Pereira da Silva B, de Menezes CBA, Arns CW (2013) Antiviral activity of Bacillus sp. isolated from the marine Sponge Petromica citrina against bovine viral diarrhea virus, a surrogate model of the Hepatitis C Virus. Viruses 5(5):1219–1230CrossRefGoogle Scholar
  9. Bernard EI, Stanley IR, Grace OI, Ebere PA, Abraham OA, Ibe KE (2018) Toxicity and bioremediation of heavy metals contaminated ecosystem from tannery wastewater: a review. J Toxicol. Google Scholar
  10. Bhoonobtong A, Sawadsitang S, Sodngam S, Mongkolthanaruk W (2012) Characterization of endophytic bacteria, Bacillus amyloliquefaciens for antimicrobial agents production. Int Conf Biol Life Sci IPCBEE. 40:6–11Google Scholar
  11. Chalasani AG, Dhanarajan G, Nema S, Sen R, Roy U (2015) An antimicrobial metabolite from Bacillus sp: significant activity against pathogenic bacteria including multidrug-resistant clinical strains. Front Microbiol 15(6):1335–1342Google Scholar
  12. Cote CK, Heffron JD, Bozue JA, Welkos SL (2015) Bacillus anthracis and other Bacillus species. Mol Med Microbiol, Chapter 102 (Second Edition) 3: 1789–1844Google Scholar
  13. Dickschat JS, Wickel S, Bolten CJ, Nawrath T, Schulz S, Wittmann C (2010) Pyrazine biosynthesis in Corynebacterium glutamicum. Eur J Org Chem 2010:2687–2695CrossRefGoogle Scholar
  14. Esawy MA, Ahmed EF, Helmy WA, Mansour NM, El-Senousy WM, El-Safty MM (2011) Production of a halophilic levansucrase from novel honey Bacillus subtilis isolates capable of producing antiviral levans. Carbohydr Polym 86:823–830CrossRefGoogle Scholar
  15. Feio SS, Barbosa AB, Cabrita M, Nunes L, Esteves A, Roseiro JC, Curto MJM (2004) Antifungal activity of Bacillus subtilis 355 against wood-surface contaminant fungi. J Ind Microbiol Biotechnol 31:199–203CrossRefGoogle Scholar
  16. FujitaY Matsuoka H, Hirooka K (2007) Regulation of fatty acid metabolism in bacteria. Mol Microbiol 66(4):829–839CrossRefGoogle Scholar
  17. Gálvez A, Abriouel H, López RL, Omar NB (2007) Bacteriocin-based strategies for food bio-preservation. Int J Food Microbiol 120:51–60CrossRefGoogle Scholar
  18. Ghoneim MA, Hassan AI, Mahmoud MG, Asker MS (2016) Effect of polysaccharide from Bacillus subtilis sp. on cardiovascular diseases and atherogenic indices in diabetic rats. BMC Comp Alt Med. 16:112–123CrossRefGoogle Scholar
  19. Gouda S, Das G, Sen SK, Shin H, Patra JK (2016) Endophytes: a treasure house of bioactive compounds of medicinal importance. Front Microbiol 7:1538–1551CrossRefGoogle Scholar
  20. Govindarajan M, Kwon S, Weon H (2007) Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiella sp. GR9. World Microbiol Biotechnol 23:997–1006CrossRefGoogle Scholar
  21. Hassan S (2017) Plant growth-promoting activities for bacterial and fungal endophytes isolated from medicinal plant of Teucrium polium L. J Adv Res 8:687–695CrossRefGoogle Scholar
  22. Huang X, Lu Z, Zhao H, Bie X, FengXia LuF, Yang S (2006) Antiviral activity of antimicrobial lipopeptide from Bacillus subtilis fmbj against pseudorabies virus, porcine parvovirus, newcastle disease virus and infectious bursal disease virus in vitro. Int J Pep Res Therapeut 12(4):373–377CrossRefGoogle Scholar
  23. Hurst A (1981) Nisin. Adv Appl Microbiol 27:85–123CrossRefGoogle Scholar
  24. Janczura E, Perkins HR, Rogers HJ (1960) Teichuronic acid: a mucopolysaccharide present in wall preparations from vegetative cells of Bacillus subtilis. Biochem J 80:82–92CrossRefGoogle Scholar
  25. Kaaria P, Matiru V, Ndungu M (2012) Antimicrobial activities of secondary metabolites produced by endophytic bacteria from selected indigenous Kenyan plants. Afr J Microbiol Res 6(45):7253–7258Google Scholar
  26. Kaneda T (1977) Fatty acids of the genus Bacillus: an example of branched-chain preference. Bacteriol Rev 41:391–418Google Scholar
  27. Kefi A, Slimene IB, KarkouchI Rihouey C, Azaeiz S, Bejaoui M, Belaid R, Cosette P, Jouenne T (2015) Characterization of endophytic Bacillus strains from tomato plants (Lycopersicon esculentum) displaying antifungal activity against Botrytis cinerea Pers. World J Microbiol Biotechnol 31:1967–1976CrossRefGoogle Scholar
  28. Kiran GS, Priyadharsini S, Sajayan A, Amrudha Ravindrana A, Selvin J (2018) An antibiotic agent pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro isolated from a marine bacteria Bacillus tequilensis MSI45 effectively controls multi-drug resistant Staphylococcus aureus. RSC Adv 8:17837–17846CrossRefGoogle Scholar
  29. Kodali VP, Sen R (2008) Antioxidant and free radical scavenging activities of an exopolysaccharide from a probiotic bacterium. Biotechnol J 3:245–251CrossRefGoogle Scholar
  30. Mardanova AM, Hadieva GF, Lutfullin MT, Khilyas IV, Minnullina LF, Gilyazeva AG, Bogomolnaya LM, Sharipova MR (2017) Bacillus subtilis strains with antifungal activity against the phytopathogenic fungi. Agric Sci 8:1–20Google Scholar
  31. Matloub AA, El-Sherbini M, Borai IH, Magda KE, Rizk MZ, Aly HF, Fouad GI (2013) Assessment of anti-Hyperlipidemic effect and physco-chemical characterization of water soluble polysaccharides from Ulva fasciata Delile. J Appl Sci Res 9(4):2983–2993Google Scholar
  32. Matloub AA, El-Souda SS, El-Senousy WM, Hamed M, Aly H, Ali SA (2015) In vitro antiviral, cytotoxic, antioxidant and hypolipidemic activities of polysaccharide isolated from marine algae. Int J Pharm Phytochem Res 7(5):1099–1111Google Scholar
  33. Mosmann T (1983) Rapid colorimetric assays for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Method 65:55–63CrossRefGoogle Scholar
  34. Nalisha I, Muskhazli M, Nor Farizan T (2006) Production of Bioactive Compounds by Bacillus subtilis against Sclerotium rolfsii. Mal J Microbiol 2(2):19–23Google Scholar
  35. Oshima M, Ariga T (1975) Cyclohexyl fatty acids in acidophilic thermophilic bacteria. J Biol Chem 250(17):6963–6968Google Scholar
  36. Osta Oliveira BE, Cury JA, Filho APR (2017) Biofilm Extracellular polysaccharides degradation during starvation and enamel demineralization. PLoS ONE 12(7):181Google Scholar
  37. Oyedele AO, Ogunbanwo TS (2014) Antifungal activities of Bacillus subtilis isolated from some condiments and soil. Afr J Microbiol Res 8(18):1841–1849CrossRefGoogle Scholar
  38. Phuong TV, Han PN, Diep CN (2018) Bioactive compounds from marine bacterium Bacillus subtilis strain HD16b by gas chromatography-mass spectrometry. Pharm Chem J 5(2):110–118Google Scholar
  39. Qiu Z, Lu X, Li N, Zhang M, Qiao X (2018) Characterization of garlic endophytes isolated from the black garlic processing. Microbiol Open 7:547–557CrossRefGoogle Scholar
  40. Saeed M, Scheel TKH, Gottwein JM, Marukian S, Dustin LB, Bukh J (2012) Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells. Antimicrob Agent Chem 56(10):5356–5373CrossRefGoogle Scholar
  41. Schulz S, Dickschat JS (2007) Bacterial volatiles: the smell of small organisms. Nat Prod Rep 24:814–842CrossRefGoogle Scholar
  42. Sharmila PS, Vidya AK (2015) Characterization and antibacterial activity of bacteriocin producing Bacillus subtilis isolated from raw milk. Int J Appl Bioengin 9:1–6Google Scholar
  43. Vijaybaskar P, Babinastarlin S, Shankar T, Sivakumar T, Anandapandian KTK (2011) Quantification and characterization of exopolysaccharides from Bacillus subtilis (MTCC 121). Adv Biol Res 5(2):71–76Google Scholar
  44. Wang X, Hu W, Zhu L, Yang Q (2017) Bacillus subtilis and surfactin inhibit the transmissible gastroenteritis virus from entering the intestinal epithelial cells. Biosci Rep 37:1–10Google Scholar
  45. Zinniel DK, Lambrecht P, Harris NB, Feng Z, Kuczmarski D, Higley P, Ishimaru CA, Arunakumari A, Barletta RG, Vidaver AK (2003) Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants. Appl Environ Microbiol 68:2198–2208CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Azza Abdelmageed Matloub
    • 1
  • Eman Zakaria Gomaa
    • 2
    Email author
  • Amira Ali Hassan
    • 3
  • Marwa Mahmoud Elbatanony
    • 1
  • Waled Morsy El-Senousy
    • 4
  1. 1.Pharmacognosy DepartmentNational Research CentreGizaEgypt
  2. 2.Department of Biological and Geological Sciences, Faculty of EducationAin Shams UniversityCairoEgypt
  3. 3.Department of Chemistry of Natural and Microbial ProductsNational Research CentreGizaEgypt
  4. 4.Water Pollution Research Department (Virology Lab)National Research CentreGizaEgypt

Personalised recommendations