**3.3. Identification by recA sequence**

Hence the recA gene has been used as a molecular chronometer in addition to rRNA genes. The 1.2 kb band was gel isolated and subjected to sequencing results and showed that the four strains A, C, E, and G were *B. subtilis* rather than *B. amyloliquefaciens*. Still these results need more confirmation.

## **3.4. Identification by specific-PCR for** *B. subtilis*

To solve this problem, identification using specific-PCR for *B. subtilis* was described. Based on ytcP gene encoding a hypothetical protein, a PCR primer pair ytcF and ytcR were designed for *B. subtilis* species for specific amplification purpose. Using this primer pair, a 0.46 kb fragment was amplified only from *B. subtilis* strains, whereas no similar band was detected from *B. licheniformis or B. amylo*l*iquefaciens* strains. These primers were subjected to the six isolates and PCR resulted in the 0.46 kb band as shown in Fig. 3 and this confirmed that all isolates A, C, E, G, K, M surely belong to *B. subtilis not B. amyloliquefaciens*.

**Figure 3.** PCR using the chromosomal DNA of the 6 Bacillus isolates. Lane 1, 100 bp ladder; lane 2–7, using primers recR × recF; lane 8–13, using B. subtilis specific primers ytcF × ytcR.

### **3.5. Levan and levansucrase production**

The six honey isolates were tested for production of levansucrase, the optimized conditions for the isolates ranged from 8 to 12% (w/v) commercial sucrose, 37–40 ºC, 24– 28 h, 50–100 rpm and pH 6–7.0 (data not shown). Among all the tested isolates, M and K isolates showed the highest levansucrase activities (62 and 59 U/mL). The presence of NaCl (1–4%, w/v) showed great influence in enzyme activity, the enzyme production increased from 2 to 3 folds according to the strain (Fig. 4). Paper chromatography of the product hydrolysate revealed the presence of only fructose and tiny traces of glucose, pointing to the levan nature of the product. Furthermore, the acid hydrolysate of the polysaccharide produced by the isolates was exclusively fructose, as revealed by HPLC. Levan was harvested by precipitation from the culture broth by addition of ethanol. The yield and consistency of the product varied according to the isolate. The final products were a brownish-white gummy material, which could be freeze-dried or vacuum-dried. The highest amount of levan was produced on the medium containing commercial sucrose, followed by beet molasses (Fig. 5). While a small amount of microbial polysaccharide (alcohol precipitate) was also produced when the organism was grown on lactose and glucose, it was worthy to record that no polysaccharides were produced on fructose. The amounts of levan decreased 40–50% in the presence of NaCl. Isolates M, K, A, C, E, and G produced 11, 16.25, 6.60, 1.81, 1.74, 6.6 g/L levan, respectively under the optimized conditions. The levan products consisted of one fraction characterized by high and low molecular masses (40.938, 71.887, 43.487, 154.638, 77.753 and 14.200 kDa for isolates K, M, A, C, E, and G, respectively) (Fig. 6).

204 The Complex World of Polysaccharides

need more confirmation.

*amyloliquefaciens*.

**3.3. Identification by recA sequence** 

**3.4. Identification by specific-PCR for** *B. subtilis*

Hence the recA gene has been used as a molecular chronometer in addition to rRNA genes. The 1.2 kb band was gel isolated and subjected to sequencing results and showed that the four strains A, C, E, and G were *B. subtilis* rather than *B. amyloliquefaciens*. Still these results

To solve this problem, identification using specific-PCR for *B. subtilis* was described. Based on ytcP gene encoding a hypothetical protein, a PCR primer pair ytcF and ytcR were designed for *B. subtilis* species for specific amplification purpose. Using this primer pair, a 0.46 kb fragment was amplified only from *B. subtilis* strains, whereas no similar band was detected from *B. licheniformis or B. amylo*l*iquefaciens* strains. These primers were subjected to the six isolates and PCR resulted in the 0.46 kb band as shown in Fig. 3 and this confirmed that all isolates A, C, E, G, K, M surely belong to *B. subtilis not B.* 

**Figure 3.** PCR using the chromosomal DNA of the 6 Bacillus isolates. Lane 1, 100 bp ladder; lane 2–7,

The six honey isolates were tested for production of levansucrase, the optimized conditions for the isolates ranged from 8 to 12% (w/v) commercial sucrose, 37–40 ºC, 24– 28 h, 50–100 rpm and pH 6–7.0 (data not shown). Among all the tested isolates, M and K isolates showed the highest levansucrase activities (62 and 59 U/mL). The presence of

using primers recR × recF; lane 8–13, using B. subtilis specific primers ytcF × ytcR.

**3.5. Levan and levansucrase production** 

**Figure 4.** The effect of absence and presence of different concentrations of NaCl on levansucrase production from honey isolate. Note: NaCl conc (0–4%) represented from left to right.

**Figure 5.** The difference between amounts of levan produced in sucrose medium (series 1) and in molasses medium (series 2)

**Figure 6.** The levan molecular weights from *Bacillus subtilis* isolates

## **3.6. Cytotoxicity test**

206 The Complex World of Polysaccharides

molasses medium (series 2)

**Figure 5.** The difference between amounts of levan produced in sucrose medium (series 1) and in

**Figure 6.** The levan molecular weights from *Bacillus subtilis* isolates

The non toxic doses for samples C and G were 0.5 mg/mL in water. On the other hand, the non toxic doses for samples A, E, and M were 0.2 mg/mL in ethanol while the nontoxic dose of sample K was 0.1 mg/ml in ethanol.

## **3.7. Anti-adenovirus type 40 assay**

The samples (C, E, G and K) had weak effect on adenovirus 40 which did not exceed 10%. The two samples A and M revealed antiviral effect on adenovirus type 40 ranged from 50 to 60% as shown in Table 2.


**Table 2.** Effect of levan A and levan M on the infectivity of enteric adenovirus type 40 (DNA virus). viral infectivity.

#### **3.8. Anti-H5N1 virus assay**

All the embryos of the positive controls died and the allantoic fluid of each was positive for haemagglutination assay (HA), while all the embryo of negative control were not died and the allantoic fluid of each was negative for HA. Three levans of code K, M, E showed antiviral against HPAI H5N1. Each of these samples showed antiviral effect when inoculated with H5N1 virus 1 h before inoculation into nine days old ECEs, while they had no effect on the virus when inoculated simultaneously with the virus just after mixing or after the virus inoculation for 1 h (Table 3). These results also revealed that simultaneous inoculation of the levans product or even after infection was of no value.


**Table 3.** Showed that three levans of code K, M, E had antiviral against HPAI- H5N1 NDE: Number dead Haemagglutination assay

#### **4. Discussion**

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 dependent mainly on commercial sucrose media, where the use of beet molasses, glucose, and lactose led to noticeable reduction in levan synthesis. Beet molasses was used as lowcost substitutes for sucrose in commercial levan yield (Han & Watson, 1992). The decrease in levan yield in the molasses medium (2.533 g/L) when compared to the commercial sucrose (21.685 g/L) was also reported (De Oliveira et al., 2007). Although higher Halomonas sp. AAD6 biomass concentrations were observed when glucose, maltose, fructose and galactose were used as carbon sources, levan levels were very low comparing with sucrose (Poli et al., 2009). The six isolates produced different levan weights, with wide range of molecular mass. On the other hand, it was reported that halophilic Halomonas sp. AAD6 cells grown in the presence of sucrose afforded the highest levan production levels (1.073 g/L) (Poli et al., 2009). Also, *B. polymyxa* produced about 40 g/L extracellular polysaccharide per liter in sucrose medium, which was about three times that produced by familiar levan producers (Han, 1989). Levan antitumor activity was reported by many authors (Yoon, Yoo, Cha, & Lee, 2004,) but as far as we are aware nothing was reported on antiviral activity of this fructose polymer. The present findings showed antiviral effects of K, M, and E levan products on H5N1 virus, While, A and M levan products showed antiviral effects on adenovirus type-40. It was obvious that the product M was entirely effective against both respiratory RNA virus (H5N1) and enteric adenovirus type 40 (DNA virus). It was apparent that each of effective levan showed antiviral effect when inoculated with H5N1 virus 1 h before inoculation into nine days old ECEs, while they had no effect on the virus when inoculated simultaneously with the virus just after mixing or after 1 h of inoculation.

The outcome of this study is the probable suitability of some types of levan as a safe and cheap natural product in antiviral treatments with applying the known roles concerning the use of these compounds. In addition, this article affords honey micro flora as a new and important sources of levansucrase enzymes, could be have biotechnological applications in pharmaceutical industries.
