**2. Experimental**

196 The Complex World of Polysaccharides

levansucrase, dextranase and.lipase.

gradually increase in the various fields (Kang et al., 2009).

(Iizuka et al.,1991; Hernandez, et al 1995; Kojima et al.,1993; Ben Ammar et al., 2002, Esawy et al., 2008). Bacterial levansucrases catalyze at least three different reactions: hydrolysis of sucrose, polymerization of fructose derived from sucrose and hydrolysis of levan. It is reported that levansucrase activity is involved in a variety of processes including survival of bacteria in soil (*B. subtilis*), phytopathogenesis (*Erwinia* and *Pseudomonas* species) and symbiosis (*Paenibacillus polymyxa*) of plant interactive bacteria (Hettwer et al.,1995). *Bacillus subtilis,* known as the hay bacillus or grass bacillus, is a Gram-positive, catalase positive bacterium commonly found in soil (Madigan & Martinko, 2005). Recently, Esawy et al 2011, Esawy a et al 2012 and Esawy b et al 2012 reported in novel *Bacillus subtilis* honey isolates as new sources of very important enzymes such as

Levan is one of two main types of fructans, which are natural homopolymers of fructose (Arvidson et al 2006). It is a naturally occurring polymer of β-D-fructofuranose with β (26) linkages between repeating five-member fructofuranosyl rings and branching at C-1 (Arvidson et al 2006, Barone and Medynets., 2007). Levans produced by different organisms differ in their molecular weight and degree of branching. Levans from plants generally have molecular weights about 2000 - 33.000Da (Rhee et al., 2002). The molecular weight of levan, and the fraction of residues incorporated in side chains, depends on both the source and the growth conditions, with plant levan and microbially-produced levan having very different characteristics (Arvidson et al 2006; Kasapis, and Morris 1994; Kasapis et al., 1994; Newbrun 1971; Stivala, and Bahary 1978; Huber et al., 1994). Recently it was reported in the *B. subtilis* NRC1aza levansucrase, the unique feature of this isolate its ability to produce two types of levan with different molecular weights (El Fattah et al, 2012). Bacterial levans are much larger than those produced by plants, with multiple branches and molecular weights (2-100 million Da) (Pontis and Del Campillo 1985 ; Keith et al., 1991). Levan is non-gelling, non-swelling in water, (Kasapis et al., 1994; Stivala, and Bahary 1978; Huber et al., 1994) and an unusual polysaccharide due to its relatively low intrinsic viscosity compared to other molecules of similarly high molecular weights. Levan can be used as food or a feed additive with prebiotic and hypocholesterolemic effects (Sanders, et al., 2003). Subsequently, there are a variety of potential industrial applications for levan such as a surfactant for household use due to its excellent surfaceactive properties, a glycol/levan aqueous two-phase system for the partitioning of proteins, etc. In addition, in vitro anti-tumor activity of levan produced from *Microbacterium laevaniformans, Rahnella aquatilis* and *Zymomonas mobilis*, has been shown against eight different tumor cell lines (Urdaci, et al 2004;Yoo,et al 2004; Yoon et al.,2004; Liu et al., 2012; El Fattah et al, 2012). Recently, Liu et al., (2012) and El Fattah et al, 2012 reported in the antioxidant activity of native levan and their derivatives. Dahech et al., (2011) reported that polysaccharide levan is efficient in inhibiting hyperglycemia and oxidative stress induced by diabetes and suggests that levan supplemented to diet may be helpful in preventing diabetic complications in adult rats. The market for levan will

## **2.1. Sources of honey and microorganisms**

Three different honey samples were purchased; local honey bee collecting nectar from clover flower; Kashmiry honey, honey bee collecting nectar from desert flower (Saudi Arabian); and Gably honey, a honey bee collecting nectar from desert flower (Libya).

### **2.2. Isolation of bacterial strains from honey samples**

One hundred micro liters of honey samples was spread on nutrient agar plates (g/L): beef ext., 1.0; yeast ext., 2.0; peptone, 5.0 and agar, 25.0. After drying for 20 min in a laminar flow hood, the plates were incubated at 50 °C to avoid the growth of any pathogenic spores for 24 h or until the colonies size was sufficient (approximately larger than 3–5 mm in diameter). The bacterial isolates were streaked onto agar plates and preserved at 4 °C. The purity of the isolates was assessed by colony morphology and microscopy.
