Chemicals
1,1-diphenyl-2-picryl-hydrazyl (DPPH), peroxidase, H2O2, ABTS (2,2-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid, polyoxyethylenesorbitan monolaurate (Tween-20), Ascorbic acid (vitamin C), butylated hydroxytoluene (BHT), nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), phenazine methosulphate (PMS), sodium nitroprusside (SNP), sulfanilamide, ortho-H3PO4, naphthylethylene diamine dihydrochloride, diammonium salt, 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-1,2,4-triazine (ferrozine), Ferrous chloride, trichloroacetic acid (TCA), potassium ferricyanide, Leuco-2,7-dichlorofluorescien diacetate, hematin, arachidonic acid, Cyclooxygenases enzymes (COX-1 from sheep , EC. 1.14.99.1 or COX-2), acetylcholine esterase enzyme, acetylthiocholine iodide, DTNB, butyryl thio-choline Iodide, butyrylcholinesterase enzyme, kojic acid, tyrosinase enzyme, and L-Dopa were purchased from Sigma-Aldrich, USA. Ammonium thiocyanate was purchased from E. Merck. All chemicals and solvents are analytical grade.
Isolation, Screening and identification of marine bacteria for marine sediments
Sampling
Three samples were collected from marine sediment at different locations from Sahl Hashish, Hurghada Red Sea, Egypt.
Isolation of bacteria
The collected samples (10 g) were placed in 100 mL of sterile seawater and homogenized by shaking at 200 rpm for 20 min, and a serial dilution was performed (Hayakawa and Nonomura 1987). Finally, 50 μL of the supernatant of each dilution was inoculated on marine nutrient agar, and the plates were incubated at 37°C for 24 h. The colonies that appeared per plate of each sample were subjected to purification. EPS production was confirmed by mucoid colony formation followed by precipitation of EPS using alcohol. A string test was also utilised for further confirmation. Fresh cultures of marine isolates were applied on solid agar medium (g/L); glucose 20, CaCO3 0.1, NH4NO3 0.8, K2HPO4 0.6, KH2PO4 0.05, MnSO4.4H2O 0.1, yeast extract 1.0 and agar 15, and incubated separately at 37ºC. Mucoid colony formation was examined by observing the presence of slimy mucoid colonies on the plate until 3 days. Furthermore, loopful colonies were immersed in alcohol to observe the precipitation of EPSs. When a loopful of culture is mixed with pre-chilled absolute ethanol, colonies precipitate out from the solution, confirming the EPSs producer.
Screening for production of EPSs
Isolates were screened for production of EPSs in a modified MY medium with 75% seawater. The medium composed of (g/L): peptone 4.0, yeast extract 2.0, and sucrose 20.0, pH 7 (Jiang et al. 1999). The medium was distributed in Erlenmeyer flask of 250 mL containing 50 mL of working volume was set. The flasks were autoclaved and inoculated using actively growing culture and incubated at 37ºC for 3 days. The fermented broth was collected and centrifuged at 5000 rpm at 4°C for 30 min Sigma-Laborzentrifugen, 2K15 to remove bacteria cells. TCA (5%) was added and left over night at 4°C and centrifuged at 5000 rpm to remove protein (Liu et al. 2011). The pH of clear solution was adjusted to 7.0 with 0.1 M NaOH and the crude EPS was precipitated by the addition of 4-volume of pre-chilled absolute ethanol to the supernatant liquid; this was stored overnight at 4°C. The sample was then centrifuged, after decanting off the ethanol solution the recovered pellet was re-dissolved in deionized water. The dissolved crude EPS was subjected to another precipitation step with pre-chilled absolute ethanol, followed by subsequent centrifugation and decantation of ethanol as described above. The pellet obtained was then re-dissolved in a minimum of deionized water and dialyzed three times (1000 mL´3) against flowing tap-water using dialysis tubing (MWCO 2000) for 24 h. The dialyzed solution was subjected to fractional precipitated by 1, 2, 3 and 4-volumes of pre-chilled absolute ethanol. The yield major fraction obtained by 1-volume of absolute ethanol was dried under vacuum at 40 ºC (Shene et al. 2008). The UV absorption spectrum was recorded using a UV-Vis Spectrophotometer (2401PC Shimadzu, Japan) between 200 and 800 nm, in order to examine the existence of proteins and nucleic acids (Wang and Luo 2007). The main fraction of EPS from 1 volume ethanol (BMEPS) was assay in-vitro against Acetyl cholinesterase inhibition.
Identification of promising isolate
The promising isolate (S16) which produced high amounts of EPS and acetylcholinesterase inhibitor was identified based on biochemical, morphological, and physiological characteristics of the potential producer as determined by adopting standard methods (Cappuccino and Sherman 2004; Collins et al. 1989). The strain was confirmed with 16S ribosomal RNA gene sequence and compared with other bacterial sequences by using NCBI BLAST. Taxonomic affiliation of the sequences was retrieved from classifier program of ribosomal database project (Maidak et al. 1997; Tamura et al. 2007).
Characterizations of exopolysaccharide
Monosaccharide, uronic acid and sulfate composition analysis
The partial purified BMEPS (50 mg) was subjected to hydrolysis with 6 N HCl for 4 h at 100 °C in a sealed tube. The HCl was removed on a water bath at 40 °C and (1 mL×3) codistilled with water (Sudhamani et al. 2004). Uronic acids were estimated at 525 nm using the m-hydroxybiphenyl procedure. Sulfate in the hydrolysate was evaluated according to the Dodgson method (Dodgson and Price 1962). The hydrolysate mono-sugars were analyzed by the Agilent HPLC model 1100 series (Agilent USA) (El-Newary et al. 2017).
UV and FT-IR spectra analysis
UV–Vis spectroscopy was conducted on the 2401PC UV–Vis spectrophotometer Shimadzu, Japan, in the wavelength range of 190–700 nm. FTIR was measurements in the range of 400–4000 cm−1 on a Vector 22 Spectrophotometer Bucker (Ibrahim et al. 2014).
Molecular weight determination
The Mw and Mn of the BMEPS were determined by Agilent 1100 HPLC according to (Juneja et al. 2006).
Biological evaluation of polysaccharides
Antioxidant activities
Reduction of Ferric ions (Fe3+) power
The Fe3+reducing power of BMEPs was assayed according to the method of Oyaizu (1986) and was compared with BHT and Ascorbic Acid as reference materials. Successive concentration from polysaccharide and standard materials; butylated hydroxytoluene (BHT) and Ascorbic Acid (LAA) were prepared as 100, 250, 500, 750, and 1000µg/ ml in methanol. The activity expressed as IC50 that was calculated by log-probit analysis.
Ferrous ions (Fe2+) chelating capacity
The Fe2+chelating activity of BMEPs was estimated according to the method of Dinis et al. (1994) and was evaluated by comparing with two standard compounds (BHT and Ascorbic Acid at the same conditions). The percentage of inhibition of ferrozine-Fe2+complex formation was given by the formula: Inhibition (%) = [(A0-A1)/ A0] × 100.
Where A0 was the absorbance of the control, and A1 was the absorbance in the presence of the sample of polysaccharide and standards.
Free radical scavenging characters
DPPH radical scavenging activity
According to the method of Yamaguchi et al. (1998), the free radical scavenging activity of BMEPswas determined using 1,1-diphenyl-2-picryl-hydrazil (DPPH•). According to following equation DPPH radical scavenging activity was calculated:
DPPH• scavenging effect (%) = 100 − [(A0-A1)/A0) ×100].
Where A0 was the absorbance of the control and A1 was the absorbance in the presence of polysaccharide.
ABTS radical cation scavenging activity
Based on the method was described by Miller and Rice-Evans (1997) and was modified by Arnao et al. (2001), ABTS radical cation scavenging activity of BMEPs was estimated. The ABTS radical cation scavenging activity was calculated as follows:
ABTS radical cation scavenging activity (%) = [1- (A sample/A control)] × 100.
Lipid Peroxidation-Ammonium Thiocyanate
The ability of BMEPs to inhibit lipid peroxidation was determined according to the method of Gülçin et al. (2004). The inhibition of lipid peroxidation in percentage was calculated by the following equation:
Lipid Peroxidation Inhibition (%) = [1-(A1-A3)/A2-A4)] ×100
Where A1, A2, A3, andA4 was the absorbance of the sample at 1st day, 2nd, 3rd, and 4th days.
ROS scavenging capacity
Superoxide anion scavenging activity
Measurement of superoxide anion (O2_) scavenging activity of BMEPs was based on the method described by Liu et al. (1997). O2_ scavenging was calculated using the following formula:
The O2_ scavenging % = [(A0-A1)/A0] × 100.
Where A0 was the absorbance of the control, and A1 was the absorbance of polysaccharide or standards samples.
Nitric Oxide radical scavenging activity
Using sodium nitroprusside (SNP), the NO· radical scavenging activity of BMEPs was determined. NO· generated from SNP in aqueous solution at physiological pH to produce nitrite ions which were measured by the Greiss reagent (Marcocci et al., 1994).
Anti-inflammatory activity:
The cyclooxygenase inhibition activity of BMEPs was performed according to Larsen et al. (1996) and Celecoxib was used as standard compound.
Cholinesterase inhibitory effect
Acetylcholinesterase (AchE) and Butyrylcholinesterase inhibitory assessment
Enzymatic activity of BMEPs was analyzed using the method of Ingkaninan 2003. The percent inhibition was calculated using the formula:
{(control absorbance – sample absorbance)/control absorbance} × 100.
Tyrosinase inhibition activity assessment
Tyrosinase inhibition assay of BMEPs was performed according to the methods of Liu et al. 2009. The anti-tyrosinase ability of BMEPswas compared to kojic acid (Sigma, St. Louis, MO, USA). The percentage of tyrosinase inhibition was calculated as follows:
[(Acontrol- Asample)/ Acontrol]×100
Statistical analysis
The data were presented as median ± SE. In-vitro antioxidant data were analyzed by t-test in one-way ANOVA (n=3 replicates) using MIB-SPSS, 25.0 software. P value was less than 0.05.