Microorganisms studied
The microbial strains used in this work were Lactobacillus plantarum FSO1 and Candida pelliculosa L18, isolated in our laboratory from traditional fermented green olive brine [17]. In this traditional process, non-debittered green olives of Moroccan Picholine variety were directly brined and led to undergo a natural fermentation process, allowing the transformation of olives into an edible product.
Chemicals and reagents
All the chemicals used in this work were purchased from Sigma-Aldrich. The solvents (methanol, toluene, and acetic acid) were of analytical grade. The commercial enzyme beta-glucosidase (EC.3.2.1.21) was purchased from Sigma-Aldrich.
Detection of β-glucosidase enzyme
API-ZYM gallery
The enzymatic profile of the strains of L. plantarum FSO1 and C. pelliculosa L18 was determined using the API-ZYM gallery (BioMerieux, France). This gallery (ref 25200) is a simplified semi-quantitative evaluation technique for 19 enzymes [18]. It was used to highlight the enzymatic profile of the strains, including β-glucosidase and esterase.
The 20 wells of the gallery were inoculated with 65 μl of overnight cultures of L. plantarum FSO1 and C. pelliculosa L18, obtained on De Man Rogosa and Sharpe (MRS) and Yeast Extract Glucose (YEG) broth, respectively. The microbial suspensions were standardized to 5-6 Mac Farland. After inoculation, the gallery was incubated at 28 °C for 5 h, then a drop of each reagent ZYM A (Ref 70494) and ZYM B (Ref 70493) was added to each well to develop the stain revealing enzyme activity. After 5 min, a numerical value from 0 to 5 was assigned based on the evaluation of the color developed in each enzyme reaction, using the color card provided by the manufacturer [19, 20].
Esculin-based medium
The esculin-based medium was used to detect the β-glucosidase activity in L. plantarum FSO1 and C. pelliculosa L18, according to the method of [21]. Briefly, the medium composed of casein peptone 8 g/L, esculin sesquihydrate 1 g/L, ferric ammonium citrate 1 g/L, agar 17 g/L, pH 7.4, is autoclaved and then poured in Petri dishes. Overnight cultures (60 μl) of the strains (FSO1 and L18) were drop inoculated on the medium and then incubated at 30 °C for 48 h. Sixty microliters of β-glucosidase (2mg/ml) and distilled water were used as positive and negative controls, respectively. After incubation of the culture assays, made in triplicate, a positive β-glucosidase activity is indicated by a black precipitate around colonies, due to the hydrolysis of esculin to glucose and esculetin. The esculetin reacts with iron III ions to form a black precipitate.
Induction and localization of β-glucosidase activity
The strains (L. plantarum FSO1 and C. pelliculosa L18) were tested for the induction and localization (extracellular, intracellular, or membrane) of β-glucosidase activity. For this, overnight cultures (5 μL) of the strains (FSO1 and L18) were inoculated in 5 mL of normal MRS and modified MRS (MRSm), containing 1% (w/v) of oleuropein (Extrasynthese, Genay, France) as a sole carbon source. After incubation at 30 °C for 7 days, the obtained cultures were centrifuged at 12000 g for 12 min at 4 °C (Hermle Labnet Z216MK). The supernatant (supernatant 1: E) obtained was separated from the pellet (cell cream). The cell cream was then resuspended in 0.1 M phosphate buffer at pH 5, and then the cells were lysed by sonication at 4 °C for 45 min in an ultrasonic bath (Bandelin Sonorex Digitec) with continuous and pulsed modes of 5 s ON/5 s OFF according to the method of Pchelintsev, Adams [22]. The lysed cells were centrifuged, at 12000×g for 12 min at 4 °C, to separate the intracellular and the membrane fractions. After these operations three fractions were obtained for each strain, the extracellular (supernatant 1: E), the intracellular (supernatant 2: I), and the membrane pellet (cell wall suspension: M) fractions. These fractions (E, I, and M) were used to measure the enzymatic activity.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
The fraction E, obtained with oleuropein (1%) as a sole carbon source, was added with (NH4)2 SO4 80% (w/v) to precipitate proteins. After overnight precipitation, the precipitates formed were collected by centrifugation at 10000 × g for 20 min at 4 °C, and the pellets obtained were dissolved in 20 mM sodium acetate buffer pH 5.0. The sodium dodecyl sulfate (SDS) gel was prepared with 0.1% SDS in 15% separating gels and 5.0% stacking gels (final gel concentration). Tris-glycine buffer pH 8.3 containing 0.1% SDS was used as the electrode buffer. Discontinuous SDS-PAGE in reducing conditions was performed according to the procedure of Laemmli [23]. Samples were treated with Laemmli buffer and boiled for 10 min at 95 °C before application to the gel. Electrophoresis was run from cathode to anode at 130 V, 30 mA for 40 min at room temperature in a Mini-Gel Electrophoresis Unit (Bio-Rad). The following proteins were used for calibration: lysozyme (14.6 kDa), esterase (28 kDa), β-glucosidase (60 kDa), bovine serum albumin (66 kDa), glucose oxidase (160 kDa). After running electrophoresis, proteins in the gel were visualized by staining with silver nitrate, according to the method of Wray et al. [24].
Determination of β-glucosidase activity
The β-glucosidase activity of the fractions (E, I, and M) obtained from the strains (FSO1 and L18) was determined according to the method of Norkrans [25], by measuring the hydrolysis of para-nitrophenyl-β-d-glucopyranoside (p-NPG) (Merck) used as substrate. The reaction mixture contained 0.9 mL of p-NPG (5 mM) in a 50 mM citrate buffer (pH 4.8) and 0.1 mL of the enzymatic fraction (E, I, or M), obtained in presence of oleuropein (1%) as a sole carbon source. The reaction was carried out at 50 °C for 10 min and then stopped by the addition of 2 mL of Na2CO3 (1M) and 10 mL of distilled water. The amount of para-nitrophenol (p-NP) released was determined by measuring its absorbance at 405 nm. One unit of enzymatic activity (U) was defined as the amount of enzyme which produced 1 μmol of p-NP (para-nitrophenol) per min under the conditions of the experiment. A calibration curve was prepared using p-NP. All the tests were carried out in triplicate.
Antioxidant activity
The antioxidant activity of the extracellular fraction, of the strains FSO1 and L18, and their combination (FSO1/L18) obtained on MRSm after 7 days of incubation at 30 °C, was evaluated by measuring the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging effect, according to the method of [26], with some modifications. Briefly, 50 μl of the sample (extracellular fraction) was added to 1.95ml of freshly prepared DPPH (25 mg/l in methanol). After 40 min of incubation in dark at room temperature, the absorbance was measured at 517 nm against a blank, consisting of 200 μl of DPPH solution and 100 μl of methanol. Ascorbic acid (0-100 μg/ml) was used as control. The antioxidant activity (AA, %) was calculated using the following equation: AA (%) = ((A0 − A1)/A0) × 100, where A0 was the absorbance of the blank, and A1 was the absorbance of the sample of extracts and standards. Three replicates were taken for each assay.
β-Glucosidase production conditions
Effect of incubation time
The in vitro monitoring of β-glucosidase production was determined by measuring the hydrolysis rate of p-NPG, to β-d-glucose and para-nitrophenol, by β-glucosidase of the fractions E of the strains FSO1, L18 and their combination (FSO1/L18). For this, 10 μL of overnight cultures of each strain (5 μL each of each strain when combined, FSO1/L18) were inoculated in 10 mL of modified MRSm broth, containing oleuropein (1%, w/v) as a sole carbon source. The cultures, made in triplicate, were incubated at 30 °C from one day to 9 days. Samples were harvested from the cultures and analyzed for their β-glucosidase activity and biomass content. The β-glucosidase activity was measured as described above. The biomass was measured by enumeration of L. plantarum FSO1 on MRS agar and C. pelliculosa L18 on Potato Dextrose Agar (PDA). Cycloheximide (0.01%, w/v) was added to MRS to prevent the growth of yeast, while gentamicin (4%, w/v) was added to PDA to prevent the growth of L. plantarum.
Effect of pH
The effect of pH on the production of β-glucosidase, by the strains FSO1 and L18 and their combination, was determined by measuring the β-glucosidase activity of their culture obtained on MRSm broth adjusted to different initial pHs (4, 5, and 6), and inoculated with 10 μl of overnight cultures of the strains (FSO1, L18, and FSO1/L18), as described above. After 7 days of incubation at 30 °C, the β-glucosidase activity was measured on the extracellular fraction, using the same protocol described above. Two controls were used in this experiment, citrate buffer (50 mM at pH 4.8) as negative control and commercial β-glucosidase (EC 3.2.1.21, Sigma-Aldrich) as a positive control. All the experiments were performed in triplicate.
Effect of NaCl
The effect of NaCl on the production of β-glucosidase by the strains studied (FSO1, L18, and FSO1/L18) was determined by measuring the β-glucosidase activity of their cultures obtained on MRSm broth adjusted to different NaCl concentrations (0, 2, 4, 6, 8, and 10%, w/v), and inoculated with overnight cultures as described above. After 7 days of incubation at 30 °C, the β-glucosidase activity was measured on the extracellular fraction using the same protocol described above. Two controls were used in this experiment, citrate buffer (50 mM at pH 4.8) as negative control and commercial β-glucosidase (EC 3.2.1.21) as a positive control. All the experiments were performed in triplicate.
Confirmation of enzymatic activity by TLC
To confirm the biodegradation of oleuropein by the strains, cultures of L. plantarum FSO1, C. pelliculosa L18, and their combination (FSO1/L18) were carried out on MRSm broth. After 7 days of incubation at 30 °C, the phenolic compounds of the microbial cultures were extracted three times with ethyl acetate (8:2, v/v). After decantation, the organic phase was harvested and left in the dark for 30 min in the presence of disodium sulfate, and then dry evaporated at 50 °C. The residue obtained was dissolved in 1 mL of methanol. The phenolic extracts obtained were dissolved in 1 ml of methanol and then subjected to Silica-gel thin layer chromatography (TLC) (Silica gel/TLC-cards, Fluka-60778) to separate the phenolic compounds resulting from the degradation of oleuropein. The phenolic extracts and the standards (oleuropein and hydroxytyrosol), prepared in methanol, were deposited on the TLC cards using a glass capillary. The eluent used was composed of toluene/methanol/acetic acid (15/5/0.5) and (15/5/1) [27]. The observation was realized, when the solvent front reached the upper line (about 10 min), under UV light at 254 nm and 365 nm after drying the plates.
β-Glucosidase activity conditions
Effect of pH
The β-glucosidase activity was measured on the extracellular fraction resulting from the optimal conditions of β-glucosidase production by the strains studied, including pH (pH5 for FSO1, pH6 for L18 and FSO1/L18), and NaCl concentration (0% of NaCl for all strains). The effect of pH on β-glucosidase activity was measured in 50 mM citrate buffer at pH values of (4, 5, and 6), using 5 mM of p-NPG as substrate. The activity of β-glucosidase was measured according to the method described above. All the experiments were made in triplicate.
Effect of NaCl
The effect of NaCl on the β-glucosidase activity of the extracellular fraction of the strains cultures obtained on MRSm was measured in the 50 mM citrate buffer (at pH 5) at 50 °C added with different concentrations of NaCl (0, 2, 4, 6, and 8%, w/v), and using 5 mM of p-NPG as substrate. The activity of the β-glucosidase was measured as described above. All the experiments were made in triplicate.
Effect of temperature
The effect of temperature on β-glucosidase activity of the extracellular fraction of strains cultures obtained on MRSm was measured in 50mM citrate buffer (at pH 5), using the p-NPG (5mM) as substrate. The mixture was maintained at temperature values of 6, 15, 25, 35, 45, and 50 °C for 10 min. The residual activity of β-glucosidase was measured as described above. All the experiments were made in triplicate.
Statistical analysis
The data obtained from the replicate assays were presented as the means ± standard deviation. The means were compared, with a significant difference at p < 0.05, using one-way ANOVA. The results were plotted using Graph Pad Prism, version 8 for Windows, GraphPad Software, San Diego, California, USA.