Enhancement of β-Glucan Biological Activity Using a Modified Acid-Base Extraction Method from Saccharomyces cerevisiae

Beta glucan (β-glucan) has promising bioactive properties. Consequently, the use of β-glucan as a food additive is favored with the dual-purpose potential of increasing the fiber content of food products and enhancing their health properties. Our aim was to evaluate the biological activity of β-glucan (antimicrobial, antitoxic, immunostimulatory, and anticancer) extracted from Saccharomyces cerevisiae using a modified acid-base extraction method. The results demonstrated that a modified acid-base extraction method gives a higher biological efficacy of β-glucan than in the water extraction method. Using 0.5 mg dry weight of acid-base extracted β-glucan (AB extracted) not only succeeded in removing 100% of aflatoxins, but also had a promising antimicrobial activity against multidrug-resistant bacteria, fungi, and yeast, with minimum inhibitory concentrations (MIC) of 0.39 and 0.19 mg/mL in the case of resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, respectively. In addition, AB extract exhibited a positive immunomodulatory effect, mediated through the high induction of TNFα, IL-6, IFN-γ, and IL-2. Moreover, AB extract showed a greater anticancer effect against A549, MDA-MB-232, and HepG-2 cells compared to WI-38 cells, at high concentrations. By studying the cell death mechanism using flow-cytometry, AB extract was shown to induce apoptotic cell death at higher concentrations, as in the case of MDA-MB-231 and HePG-2 cells. In conclusion, the use of a modified AB for β-glucan from Saccharomyces cerevisiae exerted a promising antimicrobial, immunomodulatory efficacy, and anti-cancer potential. Future research should focus on evaluating β-glucan in various biological systems and elucidating the underlying mechanism of action.


SI.1.2.1 Autolysis of yeast cells
The method of autolysis was based on the documented by Piotrowska and Masek with some modifications (Piotrowska and Masek 2015). Briefly, 100 g of yeast cells were added to 50 mL of 10 mM phosphate buffer (pH 8.0), then the mixture was subjected to 5 min of sonication instead of glass beads shaking for 4h. The next step was centrifugation (5000 × g, 15 min) and washing twice with a phosphate buffer. In order to separate protein substances, 1% of sodium dodecyl sulphate (SDS) was added to the remainder, followed by shaking for 3h at 30 °C. The resulting cell walls preparation was dried in absolute ethanol.

SI.1.2.2 Extraction of β-glucan
The output from the previous step was transferred to a suitable amount of water and autoclaved for 4h according to the proposed method (Freimund, Sauter et al. 2003, Liu, Wang et al. 2008. The insoluble fraction of water was then centrifuged at 6,000 × g for 25 min under cooling, and the collected pellets were air dried at room temperature. The code of this extracted sample was S2.

SI.2.2. High performance liquid chromatography (HPLC)
Protein and carbohydrate contents were analyzed using HPLC using the published methods (Dubois, Gilles et al. 1956, Bradford 1976. HPLC separation was carried out using Luna 5u C18 column (250 x 4.6 mm). The mobile phase used was acetonitrile: water (1:1 v/v) and the flow rate was 0.5 mL/min. The injection volume of both sample and standard were 20 µL and the pH was 5. The detection of β-Glucan was taken place at 305 nm.

SI.3. Aflatoxins removal
The efficiency of the extracted β-glucan to remove aflatoxins (AFS) was tested using batch mode of experiment. Serial concentrations of the extracted β-glucan, acid-base and water extraction, (0.1, 0.2, 0.3, 0.4, 0.5 mg of dry weight) were tested for its ability to remove aflatoxins (1000 ng/mL). The extracted β-glucan samples were dissolved in 0.1 M phosphate buffered saline (PBS) pH 6.2 and then AFS were added. The mixture was incubated with agitation (4 × g) at 30 °C for 24 h. To determine the amount of the residual AFS in the supernatant after 24 h, the samples were centrifuged at 7000× g for 10 min and the residual AFS was determined using TLC method. The control sample consisted of the same mixtures, but without β-glucan.
Thin layer chromatography (TLC) was used for AFS analysis. Briefly, 10 μL of the above residual was spotted on TLC along with the control. The running solvent were: Chloroform: methyl alcohol (97: 3 v/v). Aflatoxins B1 detected as bright blue fluoresces at Rf 0.78 under long waves UV light (Zohri, Aboul-Nasr et al. 2017). Moreover, the efficiency of β-glucan was estimated by the determination of AFS concentration at the beginning and the end of the experiments (Piotrowska and Masek 2015).

SI.4. Antimicrobial activity SI.4.1. Isolation of Microorganisms
All bacterial isolates were kindly provided from the department of Medical Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt. These clinical isolates were obtained from clinical cases of infections admitted to Assiut University hospital, Assiut, Egypt, as diarrhea, urinary tract infections, keratitis, bacterial and fungal pneumonia, otomycosis, and thrush and wound infections. The clinical isolates were proved by using the VITEK 2 automated microbiology system (BioMérieux). Fungal strains were isolated in our lab during the PhD study of Dr. Enas M.
Amer. Yeast isolate has been kindly provided by Dr. Mohamed Farhan from his PhD study (Farhan, Moharram et al. 2019).
The clinical bacterial isolates used were multidrug resistant strains, they were resistant to βlactam (amoxicillin and oxacillin), cephalosporins (cefazolin, ceftriaxone and cefepime) and macrolides

SI.4.2. Initial evaluation of the β -glucan antibacterial and antifungal activities
The antimicrobial activities of the S1 and S2 β-glucan extracted were evaluated by agar well diffusion assay (Ginovyan, Keryan et al. 2015). Suspensions of the tested microbial strains were adjusted to turbidity of 0.5 McFarland Standard, which equals to 1-2 x10 6 CFU /mL for bacteria and 1-100 mg/mL and controls (100 mg/mL) were dispensed into the wells. Vancomycin (50 μg/ mL), gentamicin (10 μg/ mL) and fluconazole (25 μg /mL) as positive control were used for Gram positive bacteria, Gram negative bacteria, and fungi, respectively. The negative control agent was 1% DMSO and water. The plates were incubated for 24 h at 37 °C for bacteria and C. albicans at 28°C for A. flavus and A. niger. The diameters of inhibition zones (ZOI) around the wells were measured in mm. All experiments were independently repeated three times and the obtained data were statistically evaluated using GraphPad prism 5.03 software (GraphPad Software, Inc.; USA).

SI.5.1. Cell culture
Human monocytes cells (HMCs) were maintained in RPMI media containing HEPES (10 mM), and FBS (10 %) at cell concentration of lower than 10 6 cells/mL. β-glucans were added at the cell's concentration of 10 µg/mL for 16 h. A biological dye, phorbol myristate acetate (PMA), was used as a positive control.

SI.5.2. Cytokine Assays
Supernatants were collected from the treated cells. Naïve cells at the same numbers were used as negative control. IL-2, IL-6, TNFα and IFN-ɤ were measured by ELISA (R&D kits, USA) which is agreeing to the manufacturer's guidelines. The levels of these cytokines were calculated from the standard curves generated using the provided recombinant proteins according the manufacturer's instructions.

SI.6.3. Apoptosis and necrosis using FACS analysis
The apoptotic and necrotic cell death induced by water extracted β-glycan were measured using Annexin V and propidium iodide (PI). In brief, WI-38, MDA-MB-23, and HePG-2 cells were cultured in 10 tissue culture dishes with initial number of 4 x 10 5 cell/ Ml in RPMI growth media and treated with (0.0 and 250 µg/mL) of β-glycan and incubated for 24 h. Briefly, the cells washed once with PBS and suspended in 100 µL of 1X Annexin V as binding buffer and add 5µL FITC and incubated for 15 minutes at room temperature then 5 µL of PI were added to each tube. Finally, 400 µL of 1X Annexin V were added to each tube and analyzed using Becton Dickinson FACS.

SI.6.4. Data analysis
All experiments were repeated three times and the obtained data were expressed as the mean ± SD and compared using a two-tailed student's t-test. Results were analyzed using GraphPad Prism 5.03 software (GraphPad Software, Inc.; USA) and considered significant if p values were less than 0.05.