Materials and reagents

Petri dishes (90 mm × 15 mm) (Nest, catalog number: 752001)
Polypropylene microcentrifuge tubes (2.0 mL) (Eppendorf, Biologix, catalog number: 80-0020)
Paper towel
Conical tubes (50 mL) (Falcon, catalog number: 602002-1)
Erlenmeyer flask (250 mL)
Beakers (10 and 500 mL) (Glassco Aleman, catalog number: 229.202.02, 229.202.08A)
Borosilicate glass bottle with screw cap with rubber/Teflon liner (250 mL) (Glassco, catalog number: 6679.269.245.01)
Nichrome inoculating needle (Citoplus, catalog number: 33210001B)
Filter paper, pore size: 3 μm (Munktell Ahlstrom, catalog number: 391)
Filter paper, pore size: 10 μm (Ahlstrom Munksjo, catalog number: 389)
Pistil (Axygen, Pes15b)
Core samplers (diameter: 5 mm)
Magnetic stir bar (Kartell^TM, catalog number: 0076800)

Biological materials

Schizophyllum radiatum Fr. (Fungario Universidad del Tolima; Laboratory collection, strain 030, voucher: LRD27)
Lentinus crinitus (L.) Fr. (Fungario Universidad del Tolima; Laboratory collection, strain 147, voucher: ZF37)

Reagents

Agar agar (Sigma-Aldrich, catalog number: BCBS4444V, 05040)
Peptone (EMD, Millipore, catalog number: WM879031-906BD)
Yeast extract (OXOID, catalog number: LP0021)
Malt extract (OXOID, catalog number: LP0039)
D-Glucose (EMD Millipore, catalog number: K51794437 004)
D-Sucrose (Fisher Scientific, catalog number: S5-500)
Sodium hydroxide (NaOH) (EMD Millipore, catalog number: B1101198-436)
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Honeywell Specialty Chemicals Seelze, catalog number: 10314903)
Iron (II) sulfate heptahydrate (FeSO₄·7H₂O) (Sigma-Aldrich, catalog number: F7002)
Ammonium sulfate [(NH₄)₂SO₄] (PanReac Química SLU, catalog number: A1032)
Anhydrous dipotassium hydrogen phosphate (K₂HPO₄) (Mallinckrodt Baker, catalog number: 3252-01)
Ethanol, 96% (Fábrica de Licores Tolima)

Solutions

Malt agar medium (see Recipes)
SYAMI culture medium (see Recipes)
Sodium hydroxide (NaOH) 0.1 N (see Recipes)

Recipes

Malt agar medium
Reagent Final concentration
Peptone 3 g/L
Malt extract 30 g/L
Agar-agar 15 g/L

SYAMI culture medium
Reagent Final concentration
MgSO₄·7H₂O 0.05 g/L
K₂HPO₄ 2.0 g/L
FeSO₄·7H₂O 0.2 g/L
(NH₄)₂SO₄ 0.15 g/L
Yeast extract 2.0 g/L
Carbon source (glucose or sucrose) 40 g/L

Sodium hydroxide (NaOH) 0.1 N
Reagent Quantity
NaOH 1 g
H₂O 250 mL

Equipment

Centrifuge (HERMLE Labortechnik GmbH, Z326K, catalog number: 311)
Orbital shaker (IKA LABORTECHNIK, KS250 basic)
Fume hood (Jpinglobal, catalog number: JPCEGH1200-BB-PP)
Laminar flow cabinet, horizontal (Purificación y análisis de fluidos)
Ultra-low temperature freezer, -85 °C (Kaltis, catalog number: 390)
Freeze dryer (Buchi, model: Lyovapor L-200)
Autoclave (All American, model: 1941X)
Refrigerator (COLDLINE, model: FORTE V13-RHC)
Barnstead Smart2Pure UV/UF (Thermo Scientific, type: Smart2Pure 3 UV/UF)
Hot plate magnetic stirrer (Stuart, catalog number: UC152)
Analytical balance (KERN, type: ACS 220-4)
Blender (Recco, model: 242748)
Drying stove (Memmert, catalog number: ref 1470)
Desiccator (Metacrilato 250 mm)
Sieve (Endecotts Ltd., ASTM E-11, catalog number: 65803- TAN AC8-1-1/2, 37,3)
Borosilicate glass filtration system (Glassco, catalog number: COL-764-260.202.01)
Incubator, 28 °C (Binder, catalog number: BD023UL)
pH meter (SI Analytics, type: HandyLab 100)
Water bath (Buchi, catalog number: B-480)

Software and datasets

Microsoft Excel for calculations
RStudio Team, 2015. RStudio: Integrated Development Environment for R. Boston, MA. Available at: http://www.rstudio.com/

Procedure

Production of EPS using submerged cultures
1. S. radiatum and L. crinitus culture preparation.
  1. Inoculate each fungal strain from a 4 °C distilled water stock onto solid malt agar medium in a 90 mm × 15 mm Petri dish and incubate at 28 °C until the plate attains full growth for 4–7 days (see Graphical overview, step 1). It is also possible to use other Petri dish sizes.
  2. After full growth, cut the solid malt agar medium with a sterile core sampler (5 mm diameter) and transfer three discs of each fungal strain to 2.0 mL polypropylene microcentrifuge tubes, using a sterile nichrome inoculating needle.
  3. Add 1 mL of sterile distilled water and disperse the agar discs using a pistil. Another option to disperse is to use a stainless-steel immersion blender for 3 s.
  4. Transfer the mixture to a 250 mL Erlenmeyer containing 50 mL of SYAMI culture medium previously autoclaved at 15 psi for 15 min and incubate by shaking at 150 rpm and 28 °C for 72 h or another specific duration; this time depends on the experimental design used and the study strain. For example, S. radiatum strain can produce EPS at 72 h and its maximum production is reached at 144 h. L. crinitus strain can produce EPS at 144 h. Other fungal spices may require more time for EPS production.
    Note: Work under laminar flow cabinet.

EPS extraction
1. Neutralization and enzyme inactivation.
  1. After the incubation time, add the culture to a 500 mL beaker and measure the pH. The pH can be between 4 and 5.
  2. Add sterile distilled water in a 2:1 ratio (100 mL of H₂O to 50 mL of culture).
  3. Homogenize the cultures (mycelia and liquid) using immersion blender (two pulses).
  4. Neutralize the mixture (pH = 7.0) using NaOH (0.1 N).
  5. Transfer the mixture to a 250 mL borosilicate glass bottle and incubate in a water bath for 30 min at 80 °C.
    Note: Work under fume hood.
2. Weighing mycelial biomass and EPS.
  1. Place 20 mL of the mixture in 50 mL conical tubes. In the following steps, the weight of the mycelial biomass and EPS will be determined.
  2. Centrifuge at 10,610× g for 30 min at 4 °C.
  3. Transfer the supernatant to a fresh 50 mL conical tube. Add cold ethanol (99.8%) to the supernatant in a 1:1 ratio for EPS precipitation. Store this mixture and biomass pellet at 4 °C for 12 h.
  4. Transfer the precipitated EPS and biomass pellets obtained by centrifugation to quantitative filter paper discs and place them on 100 mm × 15 mm Petri dishes that were previously weighed. Place each sample on a different filter/Petri dish.
  5. Incubate the Petri dishes in a drying stove at 100–110 °C for 2 h.
  6. Place the Petri dishes in a desiccator for 30 min.
  7. Subsequently, weigh the Petri dishes for each sample (EPS precipitated and biomass pellet).
  8. Use the following formula to calculate the dry weight of biomass and EPS:
    Dry weight = (C - A)/(B - A) × 100
    where A is the weight of the empty filter paper/Petri dish, B is the weight of the filter paper/Petri dish with the fresh sample, and C is the weight of the filter paper/Petri dish with the dried sample.
3. EPS purification.
  1. Filter 130 mL of the mixture derived from step B1e using a quantitative paper (pore size: 10 μm). Use a borosilicate glass filtration system.
  2. Repeat the filtering step twice using a quantitative paper (pore size: 3 μm). Use a borosilicate glass filtration system. This prevents the passage of small fragments of mycelium.
  3. Transfer the mixture to 50 mL conical tubes.
  4. Centrifuge at 10,610× g for 30 min at 4 °C.
  5. Transfer the supernatant to a 500 mL borosilicate glass bottle.
  6. Add cold ethanol (96%) in a 1:1 ratio for EPS precipitation. Incubate at 4 °C for 12 h (Figure 1a).
    
    Figure 1. Precipitation, lyophilization, and solubilization of exopolysaccharides (EPS) obtained from submerged cultures of macrofungi
  7. Separate the EPS using a 40 μm sieve. The EPS will be suspended on top of the sieve. Use a container under the sieve to collect the ethanol (see Graphical overview, steps 8, 9, and 10). It is also possible to use filter paper of the same pore size.
  8. Add sterile distilled water and repeat steps B3f and B3g.
  9. Place the obtained EPS in an ultra-low temperature freezer (-85 °C, 2 h).
  10. Freeze-dry the EPS (see Table 1).
    
    Table 1. Parameters for lyophilization of macrofungi exopolysaccharides (EPS)
    Dried material Strains Freezing parameters Drying shelf temperature regulation Pressure of the chamber Drying time
    EPS S. radiatum and L. crinitus -50 °C up to 60 °C 0.01 mbar 72 h
  11. Determine the weight of the freeze-dried EPS (Figure 1b).

Solubilization
1. Preparation of concentrated EPS solution.
  1. Weigh 100 mg of lyophilized EPS in a 50 mL beaker.
  2. Add 1.25 mL of NaOH (0.1 N) and 10 mL of sterile distilled water.
  3. Dissolve the mixture using a magnetic stirrer for 1–2 h. Do not heat.
  4. Add sterile distilled water to a final volume of 25 mL.
  5. The final concentration is 4 mg/mL.
  6. The final concentration of NaOH is 0.005 N.
  7. You can prepare solutions at different concentrations as required by assays.
  8. Consider the volume of NaOH (0.1 N) used because it can modify the EPS structure. In this case, it is recommended to use the smallest volume possible and not exceed the final concentration of 0.005 N.
  9. The prepared EPS solution can be stored at 4 °C (Figure 1c).

Data analysis

Establish an experimental design for EPS production using a submerged culture. Use a central or factorial design and analyze the data using analysis of variance or response surface methodology (RSM) (Cui and Zhang, 2012; Supramani et al., 2019; Yu et al., 2022).
Consider that the production of EPS is affected by several variables, such as inoculum properties, time, and culture conditions (Hamidi et al., 2022).
Use a negative control of the culture medium (without inoculum) for later use in the design and for calculating productive parameters.
Carbon sources are among the most crucial variables for EPS production. It is important to utilize at least two different carbon sources when producing EPS, as the production can vary significantly. Glucose or sucrose are proposed in this protocol; however, other conventional and unconventional sources can be used. The concentration of the carbon source varies among different fungal species. The use of 40 g/L is proposed in this protocol; however, other authors reported values up to 120 g/L (Mahapatra and Banerjee, 2013).
During the purification process, filtration is crucial for separating biomass residues. EPS can be used to test cell lines or other biological activity assays.

Calculate the dry EPS, dry biomass, volumetric productivity, specific productivity, practical yield, and recovery efficiency (Fariña et al., 1998; Q. Meng et al., 2021) (Table 2).

Table 2. Fermentation parameters corresponding to the exopolysaccharides (EPS) production using submerged culture

Parameters	Equation
Dry EPS	(g/L)
Dry biomass	(g/L)
Volumetric productivity (VP)	Dry EPS (g/L)/Fermentation time (h)
Specific productivity	VP (g/L/h)/Dry biomass (g/L)
Practical yield	Dry EPS (g/L/)/Initial carbon source substrate (g/L)
Recovery efficiency	Lyophilized EPS (g/L)/Dry EPS (g/L)

Analyze the data using RStudio or MATLAB. The data correspond to the determination of the equations in Table 2.
Determine the purity of the EPS by quantifying total polysaccharides, phenols, and proteins. The methodologies for these determinations are available in Parimelazhagan (2016).

Validation of protocol

Results for dry EPS are presented in Table 3. The results are reported as mean ± standard deviation, n = 3 for this work.

Table 3. Comparison of dry exopolysaccharides (EPS) corresponding to its production by macrofungi strains using submerged culture

Macrofungi	Reference	Condition culture	Dry EPS (g/L)
Schizophyllum radiatum Fr	This work	pH 5.5, SYAMI medium, 40 g/L of sucrose, 7 days, 150 rpm	10.58 ± 3.28
Lentinus crinitus (L.) Fr	This work	pH 5.5, SYAMI medium, 7 days, 40 g/L sucrose, 150 rpm	4.61 ± 1.30
Schizophyllum radiatum Fr	Meng et al., 2021	pH-, seed medium, 6 days, 40 g/L of glucose, 1.0 g/L of Tween 80	3.77
Lentinus tigrinus (Bull.) Fr.	He et al., 2016	Lactose 60 g/L, yeast extract 4 g/L; pH = 5.0, 12 days, 0.2 % Tween 80.	4.10 ± 0.27

General notes and troubleshooting

The experimental procedures necessary to carry out the protocol involve the use of chemical substances with a low risk of ignition and irritation by contact with the skin, eyes, and mucous membranes, so all protection elements and safety measures must be used to avoid and prevent accidents.

Acknowledgments

This study was supported by Project No. 58653, Alianza Colombia Científica, Gobierno Nacional de Colombia y Banco Mundial, Proyecto Betaglucanes, Contrato FP448442-211-2018 of 2018. This protocol was adapted from the work carried out by Fariña et al. (2001). We thank Dr. Julia Fariña, PROIMI, CABBIO and MINCIENCIAS for the course entitled: “Mycoprospecting: from nature to biotechnological product. Production in bioreactors and downstream processing.”

Competing interests

The authors declare that they have no conflicts of interest.

Ethical considerations

The specimens used in this study were collected under a permit conceded for access to biological resources for non-commercial purposes (Permiso Marco de Recolección, Resolución 2191 de 2018, Universidad del Tolima) and contract acknowledged for access to genetic resources and derivate products for non-commercial purposes (Contrato Marco de Acceso a Recursos Genéticos y sus Productos Derivados No. 294 de 2020. Expediente RGE0353-3, Universidad del Tolima).

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