**Author details**

208 The Complex World of Polysaccharides

The public health and the discovery of new drugs is a main objective of many research activities, however, sometime this type of research activity cost a lot of money. Although, in this proposed research we will be aiming towards the protection of the public health and introduce a new drugs contribute in solve the problem of serious diseases through products that will save a lot of money to our economy. Within this context, six mobile spore-forming, and Gram-positive facultative aerobic bacilli were isolated from different honey samples and identified as *Bacillus* spp. On the base of morphological, and molecular identification, using 16S rRNA sequence method. *B. subtilis* isolates are biologically and commercially important as producers of a great variety of secondary metabolites such as antibiotics, and enzymes (Desai & Banat 1997; Roberts et al., 1996). The16S rRNA sequence method could not identify the Bacillus isolates at the species level where revealed identical to any of three strains *B. subtilis or B. licheniformis or B. amyloliquefaciens.* In fact DNA-based identification methods such as 16S rRNA gene sequencing and 16S–23S intergenic region sequencing have been used widely for the purpose of identification and typing of microorganisms isolated from natural environments including fermented foods (Hansen et al., 2001; Levine, et al., 2005). But identification based on rRNA gene sequences fails to distinguish one species from the other if they share highly similar rRNA genes. This is true for some Bacillus species. It is difficult to distinguish *B. subtilis* from closely related *B. licheniformis* or *B. amyloliquefaciens* by rRNA gene sequences because of no significant differences in their rRNA sequences (Nakamura, 1989; Nakamura et al., 1999). Other genes such as recA (Rodriguez et al., 2007) and dnaJ (Shah et al., 2007) have been employed instead of rRNA genes. It is necessary to compare results from different identification methods as a whole before to reach a conclusion (Bourque et al., 1995). To solve the ambiguity in differentiating them based solely on the 16S rRNA gene, it was turned to 16–23S intergenic region, recA gene, and ended with *B. subtilis* specific primers. It is specifically useful to distinguish organisms with highly similar rRNA genes recA gene was amplified from the chromosomal DNA of bacilli isolates by PCR, using primers (recF and recR) (Payne et al., 2005). When rRNA and recA gene sequences were considered together, it was possible to conclude that the G, M isolates are belonging to *B. subtilis* but the other isolates A, C, E needed further identification. For the accurate distinguish between the 6 isolates, the specific-PCR for *B. subtilis* based on ytcP gene was used and the results revealed that the six strains are belonging to *B. subtilis*. The present results proved high phenotypic and genotypic variability among *B. subtilis* isolates, where they showed different morphological and biological properties suggesting them as new different species of *B. subtilis* with valuable impact in the industry. Many authors reported in the production of levansucrase from *B. subtilis* (Euzenat et al., 1997; Le Gorrec et al., 2002). The various sugars, initial pH, fermentation temperature, and agitation speed affected the levansucrase production by *B. subtilis* (Abdel-Fattah et al., 2005; Shih et al., 2005). The result ensure the halophilc feature of *B. subtilis* levansucrases, this comes from its osmophilic character. Enhancements of levansucrase production in the presence of NaCl were reported (Euzenat et al., 2006; Poli et al., 2009). As far as we are aware no studies were reported on the effect of NaCl on the enzyme production. It seemed that levan production by the isolates was

**4. Discussion** 

Mona A. Esawy\* , Eman F. Ahmed and Wafaa A. Helmy *Department of Chemistry of Natural and Microbial Products, National Research Centre, Dokki, Giza, Egypt* 

Nahla M. Mansour *Gut Microbiology and Immunology Lab, Central of Excellence for Advanced Sciences (CEAS), National Research Centre, Dokki, Giza, Egypt* 

Waled M. El-Senousy *Water Pollution Research Department, National Research Centre, Dokki, Giza, Egypt* 

Mounir M. El-Safty *Central Laboratory for Evaluation of Veterinary Biologics (CLVB), Abbassia, Cairo, Egypt* 

\* Corresponding Author

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