Primers used for gene amplification
In recent years, significant progress has been made in discovering and developing new bacterial polysaccharides that possess novel and highly functional properties (Baird et al 1983). Although their ubiquitous role in biological processes and their versatility as biocompatible, environmentally friendly materials are beyond doubt, polysaccharides are still considered to be the "sleeping giant" of biotechnology.
Honey contained a great variety of dominant spores and in consequence their dominant spores are expected to be new expolysaccarides sources which could be isolated. This expectation comes from the honey constituents which is mainly fructose (about 38.5%) and glucose (about 31.0%) (Crosby and Alfred 2004). Aerobic spore forming Bacillus were the most frequently encountered microbes on the external surface, crop and intestine of the honey bees and consequently honey (Root, 1993, Esawy et al., 2011).
Most of the researches in the honey field focused on its antimicrobial, antioxidant and anticancer activities, also the identification of the dormant endospore inside it (Sabate, et al., 2009). None till now paid attention to the enzymatic products of these dormant endospores (Esawy et al., 2011). Osmophilic microorganisms survive environmental extremes of desiccation, pressure and acidity, it is expected that their biopolymers will also have some unique properties to adapt to such extreme conditions. This investigation concerned the question of whether honey collect bacteria that are good producers of levansucrase and levan yield. Recently, screening of 16 bacterial honey isolates for levansucrase production showed that all the tested isolates were levansucrase producers despite variations in the degree of activity (data not published yet). Levansucrase, one of the fructosyltransferases or glycansucrases, is produced by various microorganisms (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 (
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 (2→6) 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
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.
2.3. Chromosomal DNA and plasmid extraction
Chromosomal DNA was prepared from overnight culture in LB,using AxyGEN Biosciences DNA extraction kit, according to manufacturer’s instructions. Plasmid extraction was performed using Wizard mini prep. extraction kit (Promega) according to manufacturer’s instructions with slight modification, where 50 L of lysozyme (200 mg/mL) were added to the resuspended buffer and incubated at 37 °C for 1 h then the protocol was carried on as described in the kit (O’Sullivan & Klaenhammer, 1993; Sambrook, et al., 1989).
2.4. PCR amplification for molecular identification
To amplify the 16S rRNA gene, a primer pair hybridizing to two conserved regions in 16S rRNA genes from
2.5. Bacterial strains and growth conditions
Bacillus strains used in this work were cultivated in Luria-Bertani (LB) broth or agar at 37 °C. Defined medium was used for cellular production of levansucrase (Yanase et al., 1992). It had the following composition (g/L): yeast extract, 2.5; commercial sucrose, 80; MgSO4, 0.2 and K2HPO4, 5.5. The medium was completed by the addition of 1 L distilled water and the pH was adjusted to 7.0 before autoclaving. The parameters included initial incubation temperature (25–45 °C); different concentrations of sucrose (80–160 g); incubation time (16–48 h); (50–150) rpm; pH (5-9) and NaCl (1–4% w/v) were studied. The sucrose was substituted with fructose, glucose, lactose (80 g/L) and beet molasses (equivalent to 80 g sucrose) to study their effects on enzyme production.
2.6. Cellular production
Cultivation was carried out in 250 mL Erlenmeyer flasks. Each flask contained 50 mL production medium and was autoclaved for 15 min. The flasks were then inoculated with 2.0 ml inoculum and incubated for 24 h at 30 °C. The culture broth was then centrifuged in a cooling centrifuge (K70; Janektzki, Germany) at 10,397×g to separate the bacterial cells from the supernatant.
2.7. Assay of levansucrase
Levansucrase assay was performed according to the method of Yanase et al. (1992) with some modification. 0.5 ml of culture filtrate was incubated with 1 ml 20% (w/v) sucrose and 0.5 mL 0.1 M acetate buffer at pH 5.2 and incubated at 37°C for min. The decreasing amounts of sugars produced were measured by glucose oxidase kits. One unit of enzyme activity was defined as the amount of enzyme that produced decreasing sugars equivalent to 1 _mol of glucose/min.
2.8. Separation of levan polymer
The levan producing organisms were cultivated on a defined medium as described above. After growth, the culture was centrifuged to remove bacterial cells; the levan was precipitated with two volumes of absolute ethanol. The precipitate was collected and dried under vacuum.
Paper chromatography was performed according to Block et al. (1995). Hydrolysate of products of levan were analyzed by either paper chromatography on whatman No. 1. The mixtures at the end of incubation time were boiled for 3 min to stop the reaction. Chromatographic development was carried out with a solvent system of n-butanol:acetone:water (4:5:1) and detected by spraying with aniline hydrogen phthalate. The acid hydrolysate of the polysaccharide produced by the six isolates was analyzed using high-pressure liquid chromatography (HPLC). A 7.8 mm ×300 mm PL-HI-PLEXPB column was linked to a differential refractometer. The column temperature was maintained at 80 °C. The aqueous mobile phase was delivered at a flow rate of 0.6 ml/min.
2.10. Determination of molecular weight
Different concentrations of levan and oligosaccharide were prepared and the flow time of equal volume for each concentration at 30°C was determined in a U-shaped Ostwald viscometer. Flow time of the same volume of distilled water was also determined as control. Thus, specific viscosity/C (gsp) was estimated. A plot of levan and oligosaccharide concentration (C) against intrinsic viscosity (C) (gsp/C) therefore yielded a straight line.
2.11. Antivirus detection
Two types of viruses were used, highly pathogenic avian influenza H5N1 virus Egyptian isolate, was used at titre of 106 EID50/mL (embryo infective dose per mL) and adenovirus type 40 with different doses 201×104, 1× 105, and 1×106 infectious particles/mL obtained from the Holding Company for Biological Products & Vaccines (VACSERA).
2.12. Specific pathogen free (SPF) eggs
SPF embryonated chicken eggs were used at nine days old and inoculated via the allantoic sac route. SPF eggs (Brown et al., 2007) were obtained from Nile SPF Eggs, Koomoshiem, Fayoum, Egypt.
2.13. Cytotoxicity test
It was done according to Simoes et al., (1999) and Walum et al., (1990). Briefly, All samples (100 mg) were dissolved in 500 _L of water or ethanol. Samples A, E, M, and K were dissolved in ethanol while samples C and G were dissolved in water. Decontamination of samples was done by adding 12 _L of 100x of antibiotic–antimycotic mixture to 500 _L of each sample. Then, bi-fold dilutions were done to 100 _L of original dissolved samples and 100 _L of each dilutions were inoculated in Hep-2 cell line (obtained from the Holding Company for Biological Products & Vaccines VACSERA, Egypt) previously cultured in 96 multi well plates (Greiner-Bio one, Germany) to estimate the non toxic dose of the tested samples. Cytotoxicity assay was done using cell morphology evaluation by inverted light microscope and cell viability test applying trypan blue dye exclusion method.
2.14. Cell morphology evaluation by inverted light microscopy
Hep-2 cell cultures (2× 105 cells/mL) were prepared in 96-well tissue culture plates (Greiner-Bio one, Germany). After 24 h incubation at 37 °C in a humidified 5% (v/v) CO2 atmosphere cell monolayers were confluent, the medium was removed from each well and replenished with 100 _L of bi-fold dilutions of different samples tested prepared in DMEM (GIBCO BRL). For cell controls 100 _L of DMEM without samples was added. All cultures were incubated at 37 °C in a humidified 5% (v/v) CO2 atmosphere for 72 h. Cell morphology was observed daily for microscopically detectable morphological alterations, such as loss of confluence, cell rounding and shrinking, and cytoplasm granulation and vacuolization. Morphological changes were scored (Simoes et al., 1999).
2.15. Cell viability assay
It was done by trypan blue dye exclusion method (Walum et al., 1990). Hep-2 cell cultures (2×105 cells/mL) were grown in 12-well tissue culture plates (Greiner-Bio one, Germany). After 24 h incubation, the same assay described above for tested samples cytotoxicity was followed by applying 100 L of tested samples dilutions (bifold dilutions) per well. After 72 h the medium was removed, cells were trypsinized and an equal volume of 0.4% (w/v). Trypan blue dye aqueous solution was added to cell suspension. Viable cells were counted under the phase contrast microscope.
2.16. Haemagglutinating activity assay
This was applied for the allantoic fluids of the inoculated eggs and measured by micro technique of haemagglutination (HA) test (Takatsy, 1955).
2.17. Evaluation for antiviral activity
Three experiments were conducted.
2.17.1. Experiment 1
One hundred and five emberyonated chicken eggs (ECEs) were examined; equal volumes of HPAI H5N1 virus and original extracts were separately used at three levels:
2.17.2. Experiment 2
One hundred and five SPF ECEs were used in this experiment; equal volumes of HPAI H5N1 virus and the original samples were mixed with equal volume of the original samples and inoculated directly into the allontoic sac of five ECEs for each product sample at a dose of 0.20 mL/ECE for each product sample at a dose of 0.2 mL/ECE. Five ECEs were inoculated with equal volume of the HPAI H5N1 virus and saline at dose of 0.2 mL/ECE (positive control). Another five ECEs were inoculated with 0.20 mL/ECE of saline alone (negative control). All the ECEs were incubated at 37 °C and controlled every 2 h till the ECEs of the positive control died
2.17.3. Experiments 3
One hundred and five SPF ECEs of nine days old were used in this experiment. 0.10 mL of the HPAI H5N1 virus was inoculated via the allontoic sac of each ECE into 100 ECEs and then the inoculated ECEs were incubated for 1 h at 37
2.18. Antiviral effect of tested samples on adenovirus type 40
Seventy five microliters of non toxic dilutions were mixed with 75 L of different doses 1 × 104, 1 × 105, and 1 × 106 infectious viral particles/mL of adenovirus type 40 provided by American Type Culture Collection (ATCC). Then the mixture was incubated overnight at 4 °C. Inoculation of 100 L of 10 fold dilutions of treated and untreated adenovirus was done into Hep-2 cell line in 12 multi well-plates. After 1 h incubation for adsorption at 37 °C, 1 mL medium (DMEM) was added to each well. The cell line was observed daily for one week then, three times freezing and thawing for tested plates were done. Nested PCR was done for confirmation of adenovirus (presence/absence) in each well (Puig et al., 1994).
3.1. Molecular identification of the levansucrase producers strains
3.1.1. 16S rRNA sequences and their analogical electrophoresis
Six levansucrase producers’ bacteria were isolated from different honey sources. The isolates resembled each other in cell morphology where cells were rod-shaped, Gram-positive, motile and spore-forming. Colonies were circular, creamy, and no pigment was formed. They were identified as
The DNA of the isolates was extracted as described in Section 2 and the 1.5 kb 16S rRNA gene was amplified for each DNA by PCR using primers bac-F and bac-R. The PCR amplification, purification and sequencing were performed as described previously. The 1.5 kb obtained sequences were aligned and clustered with sequences from the NCBI database. 16S rRNA gene sequence analysis indicated that the six isolates (K, M, A, C, E, and G) were
3.2. Identification by 16–23S intergenic region
To distinguish between these three closely related strains the 16–23S intergenic region was amplified by primers L516SF × L523SR, and then the sequence was determined for the six isolates. The homology results for the 16–23S intergenic region showed that: K and M strains showed 100% similarity to
|16–23S intergenic region||L516SF|
|Yoon et al., 2001|
|Gun-Hee Kwon et al., (2009)|
|Gun-Hee Kwon et al., (2009)|
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
3.4. Identification by specific-PCR for
To solve this problem, identification using specific-PCR for
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).
3.6. Cytotoxicity test
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.
|Percentage of reduction||Final viral doses||Initial viral doses||Sample|
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.
|Code of sample||Experiment 1||Experiment 2||Experiment 3|
|Level 1||Level 2||Level 3||NDE||+HA||NDE||+HA|
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
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
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.