Experimental Planning for Production of β-D-Glucan, Purification and Fluorescence Properties from Basidiomycete Strains

1. Introduction

Coffee and fruit industries produce annually a huge amount of agro-industrial wastes which are highly polluted and a disposal problem in the environment [1,2]. These wastes are very rich in a large variety of bioactive compounds which could be valorised into higher value products either by extraction and purification or by fermentation to produce fine chemicals [3,4]. Basidiomycete mushroom strains have been known, for many centuries, to grow on agro-industrial wastes producing high value products such as enzymes, β-D-glucans, lectins, vitamins, lipids and several secondary metabolites [5,6]. It has been well-established that natural or processed foods containing safe amounts of such bioactive compounds are found to provide clinically validated and documented health benefits by using specific biomarkers [7,8]. These foods have been found to improve human health, lower the risk of chronic bacterial and viral diseases as well as to prevent and management of symptoms of diseases [8,9].

Among these biomolecules, β-D-glucans play a major role as biological response modifiers (BRM) in several clinical disorders such as immunomodulatory properties, antimicrobial and antioxidant activity, and hypocholesterolemic and hypolipidemic effects, HIV, cancer, diabetes, neurological, cardiovascular and immunological disorders [10,11].

Experimental planning is an important strategy for overproduction of mushroom β-D-glucans from agro-industrial wastes as it provides useful information about significant factors and the interactions between the variables [12]. There are a vast majority of agro-industrial wastes that can be used for cultivation of basidiomycete strains such as rice husks, coffee residues, fruit peels and pomaces, agricultural straw and dairy wastes. Several basidiomycete mushrooms have been used in these bioconversion processes such as Pleurotus sp. Agaricus sp., Cordyseps sp. and Hericium sp. [1,2,3,4].

Basidiomycete mushroom strains produce a heterogeneous mixture of several β-D-glucans of different sizes, structures and either bound to proteins or in free forms [13,14]. The purification of such β-D-glucans in homogeneous preparations is of crucial interest to investigate their structure-function relationship [15,16,17]. Therefore, it is of great interest to devise a specific chromatographic technique to purify these biological macromolecules as well as to investigate their structural properties. Immobilized metal affinity chromatography (IMAC) has been widely used to purify proteins with high degree of purity [18]. To our knowledge , this technique has not been used to isolate and purify β-D-glucans from basidiomycete strains. As far as literature is concerned, there are a few reports for purification of β-D-glucans from mushrooms strains by using gel filtration and ion-exchange chromatography [16,17] On the other hand, structural characterization of β-D-glucans is of great scientific interest in order to understand their interaction with cells in vivo [11,13,15] To our knowledge, intrinsic fluorescence techniques have not been used to characterize β-D-glucans from basidiomycete strains although the use of fluorescence probes bound to β-D-glucans has been reported in the literature [19,20] FTIR analysis of β-D-glucans is valuable to obtain structural information about these biological macromolecules and therefore several chromatographic factions of β-D-glucans were analysed by FTIR to investigate their structural properties [16].

The present work involved experimental planning for optimization of production of extracellular β-D-glucans from several basidiomycete strains by using several coffee and fruit agro-industrial wastes. Subsequently, these β-D-glucans were purified by IMAC by using different stationary phases and metal chelates and fluorescence and FTIR properties of these β-D-glucans were investigated in detail.

2. Materials and Methods

2.1. Chemicals

Imidazole, iminodiacetic acid (IDA), 1,4-butanediol diglycidyl ether (BDGE), Sepharose 6B, epichlorohydrin (EPI), Sepharose 4B and 6B, Congo red dye, Alcian blue 8GX, phenol, β-D-1,3-glucan from Euglena gracilis and barley β-D-glucan were obtained from Sigma -Aldrich (USA).

Potato dextrose agar (PDA) medium was supplied by HiMedia Laboratories (Mumbai, India). Milk permeate, orange and apple peels, pine sawdust, yellow lupine, rice husk, apple pomace, yellow lupin and waste coffee grounds were homemade. All other reagents were of analytical grade.

2.2. Mushroom Samples

Pleurotus ostreatus and Ganoderma carnosum were isolated from old growth forests of the Olympic Peninsula in Port Townsend - WA (USA). Pleurotus ostreatus mushroom stems were a generous gift of a mushroom grower in Amsterdam (Netherlands). These mushroom strains, Fomes fomentarius, Ganoderma applanatum, Ganoderma lucidum violeta, Irpex lacteus, Phlebia rufa and Piptoporus betulinus were kindly supplied by Department of Agronomy of University of Trás-os-Montes and Alto Douro (UTAD). These cultures were grown in PDA and maintained at 4 °C. On the other hand, the young fruiting body of Agaricus blazei, Inonotus obliquus, Polyporus umbellatus, Poria cocos, Ganoderma lucidum, Hericium erinaceus, Coriolus versicolor and Lentinula edodes were kindly supplied by Mycology Research Laboratory, Ltd. (United Kingdom).

In order to develop a culture medium for cultivation of mushroom strains for β-D-glucans production, several agro-industrial wastes were tested. The agro-industrial wastes in dry form were corncobs, rice husks, oat husks, sawdust, and lupin seeds which were supplied by the Department of Agronomy of University of Trás-os-Montes and Alto Douro (UTAD). The wet agro-industrial wastes were coffee grounds, pineapple, banana, mango and pear peels.

2.3. Methods

2.3.1. Growth and Maintenance Conditions of Mushroom Strains

All mushroom strains were grown in PDA for 2 weeks at 25 ºC and maintained at 4ºC in PDA.

2.3.2. Production of β-D-glucan from Basidiomycete Mushroom Strains in Culture Media Containing Agro-Industrial Wastes

All mushroom strains were grown in media containing 1 g/L KH₂PO_4, 1 g/L MgSO₄, 1g/L (NH4)₂SO₄, 15 g/L of purified agar and 4 g/L either dry residues or 40 g/L of wet residues at pH 5.6. In a second phase, the basidiomycete cultures that exhibited growth in the media described above were inoculated into liquid medium which had the same composition as the solid media, except it did not include purified agar. The mycelia on the solid medium were scraped off and collected in Eppendorf tubes containing sterile saline. After resuspension of the mycelia, it was used as inoculum for the liquid media. The cultures were incubated at 25 °C in an incubator with orbital shaking at 150 rpm for 14 days.

2.3.3. Isolation of of β-D-Glucan from Basidiomycete Mushroom Strains

After fermentation, the mycelial biomass was separated from the fermentation broth by centrifugation for 30 min at 4,500 rpm at 4 °C. Extracellular β-D-glucans were isolated by precipitation with 4 volumes of 95% ethanol to the supernatant and the mixture was stored overnight at 4 °C. The precipitate was collected after centrifugation for 5 min, 10,000 rpm and 4 °C. Finally, the sediment was dissolved in 50 mM phosphate buffer pH 7.4.

The isolation of intracellular β-D-glucans (IBG) from mycelial biomass pellet from Pleurotus ostreatus, Ganoderma applanatum and Ganoderma carnosum and from young fruiting bodies powder from Agaricus blazei, Ganoderma lucidum, Hericium erinaceus, Coriolus versicolor, Lentinula edodes, Pleurotus ostreatus, Inonotus obliquus, Auricularia auricula, Grifola frondosa, Polyporus umbellatus, Cordyseps sinensis and Poria cocos was performed by multistep water extraction followed by extraction with alkali and acidic solutions as described previously [21]. Briefly, the aqueous extraction from mycelial biomass pellet from P. Briefly, FW1, FW2, FKOH, FHCl, FNaOH represent samples of extraction with cold H2O, hot H2O, KOH, HCl and NaOH, respectively [21].

2.3.4. Total Polysaccharides and Protein Assays

Total polysaccharides in several samples were quantified by the phenol–sulfuric acid method, using polygalacturonic acid as the standard [22] whereas protein quantification was performed by Coomassie blue dye-binding method with BSA as standard [23].

2.3.5. Congo Red Assay for Specific Determination of of β-D-glucan with Triple Helical Structure

Congo red dye colorimetric assay was carried out to quantify the concentration of β-D-glucans in several samples as described previously, using β-D-glucans from barley as standard [21].

2.3.6. Alcian Blue Dye Colorimetric Assay for of β-D-Glucan

Alcian blue dye colorimetric assay was carried out to quantify the concentration of β-D-glucans in several samples as described previously, using β-D-glucans from barley as standard [24].

2.3.7. Experimental Design to Optimize Extracellular β-D-Glucan Production

The experimental design was conducted using three factors and two levels with duplicates: basidiomycete species (Lentinula edodes and Ganoderma applanatum), agro-industrial waste concentration (40 g/L and 400 g/L for wet waste or 4 g/L and 40 g/L for dry waste), and cultivation time (3 and 7 days). The upper (+) and lower (-) levels were defined based on the preliminary results. The experimental design matrix was obtained with Design Expert version 10 software, and the results were fitted to the following factorial model equation:

$$\mathrm{y}={\mathsf{\beta }}_0+{\mathsf{\beta }}_{\mathrm{A}}{\mathrm{x}}_{\mathrm{A}}+{\mathsf{\beta }}_{\mathrm{B}}{\mathrm{x}}_{\mathrm{B}}+{\mathsf{\beta }}_{\mathrm{C}}{\mathrm{x}}_{\mathrm{C}}+{\mathsf{\beta }}_{\mathrm{A}\mathrm{B}}{\mathrm{x}}_{\mathrm{A}}{\mathrm{x}}_{\mathrm{B}}+{\mathsf{\beta }}_{\mathrm{A}\mathrm{C}}{\mathrm{x}}_{\mathrm{A}}{\mathrm{x}}_{\mathrm{C}}$$

where y represents the concentration of β-D-glucans, β0 is the mean of all responses, β is the regression coefficient and x are the prediction variable. The statistical analysis of the model was evaluated according to the ANOVA methodology.

2.3.8. Preparation of Stationary Phases for IMAC:

The activation of Sepharose 6B and 4B was carried out as described previously [25] by using either epichlorohydrin (EPI) or 1,4-butanediol diglycidyl ether (BDGE) as the spacer arm and either iminodiacetic acid (IDA) or nitrilotriacetic (NTA) as chelating agents.

2.3.9. Chromatographic Behaviour of β-D-glucans from Mushroom Strains on Immobilized Metal Chelates (IMAC):

A rapid batch method carried out in ELISA microtiter plates was used to study the adsorption of β-D-glucans on stationary phases [25]. In the present work, β-D-glucans adsorption to all these stationary phases was considered to be partial when β-D-glucans was detected both at the washing and elution steps. On the other hand, β-D-glucans did not bind to the column when total β-D-glucans applied to the column was recovered in five column volumes of buffer at the washing step, exhibiting a sharp activity peak. 96-well microtiter plates were used to study the adsorption of β-D-glucans from mushroom strains on immobilized metal chelates of Cu(II), Ni(II), Zn(II) and Co(II) at pH 8.0. The culture supernatant containing β-D-glucans (0.1 mg) was diluted in 20 mM sodium phosphate buffer containing 1M NaCl pH 8.0. Desorption of the β-D-glucans from metal chelates was carried out by using 20 mM phosphate buffer containing 1 M NaCl and 500 mM imidazole. All washing and elution fractions were assayed for β-D-glucan by congo red assay method and these experiments were performed in triplicates at room temperature.

2.3.10. Purification of β-D-Glucan from Basidiomycete Mushroom Strains by IMAC

According to the results of the chromatographic behaviour performed on 96-well microtiter plates, a set of experimental conditions were selected for purification of β-D-glucan from some mushroom strains. A column containing 5 mL of sedimented epoxy-activated Sepharose 6B-IDA-Cu(II) (S3) as the stationary phase was washed sequentially with 5 volumes of 50 mM copper sulfate, water and then finally with 20 mL of equilibration buffer at pH 8.0 [26]. The culture supernatant containing β-D-glucan was diluted 1:2 with equilibration buffer and applied to the column at a flow rate of 15 mL/h. The column was then washed with equilibration buffer and the chromatographic fractions were collected and read at A₂₈₀, A₅₁₀ and fluorescence intensity. Once the A₂₈₀ was less than 0.05, β-D-glucan was desorbed from the column with a linear gradient of imidazole (0-500 mM) in the same buffer system at a flow rate of 30 mL/h and fractions were assayed for β-D-glucan and protein content by fluorescence and colorimetric assays.

2.3.11. Intrinsic Fluorescence Measurements of β-D-Glucans

The samples containing β-D-glucans were also analyzed on a spectrofluorimeter (JASCO JP-8300) in quartz cuvettes with a 1 cm optical path length. Spectra Manager software was used for spectral acquisition and processing. The following parameters were set to obtain synchronous fluorescence spectra: excitation wavelength of 250-735 nm; emission wavelength of 260-750 nm; excitation and emission slit widths of 5 nm; variation of delta wavelength Dl of 5, 10, 20 and 30 nm , excitation wavelength increments of 5 nm; emission wavelength increments of 0.5 nm; response of 0.1 s; light source of Xe lamp and scan speed of 10,000 nm/min.

For steady-state fluorescence assays in a microtiter plate fluorescence reader, 100 µL of sample was pipetted into a well of a NUNC 96 microplate. The samples consisted of extracellular β-D-glucan from Lentinula edodes cultures and commercial barley β-D-glucan for comparative purposes. The samples were analyzed in triplicate, and blank assays consisting solely of deionized water and phosphate buffer were performed for each microplate. Intrinsic fluorescence was read in a microplate reader (FLUOstar OPTIMA-BMG Labtec), using excitation filters corresponding to wavelengths of 380, 400, 420, 430, 485, or 510 nm and emission filters corresponding to 480, 528, 542, 550, or 620 nm.

2.3.12. FTIR Analysis of β-D-Glucans

In order to exploit the structural features of β-D-glucans, FTIR analysis was performed. All samples were previously freeze-dried with a UNICRYO MC2L. Infrared spectra were recorded on a Bruker Vertex 70 (with OPUS 5.5 software) as KBr pellets, a total of 128 scans at a resolution of 2 cm^-1 in a range of 400–4000 cm⁻¹.

2.3.13. Statistical Analysis

SigmaPlot 16.0 (2011-2012 Systat Software inc.) was used to draw graphs in this research work. Experimental results are means of three parallel measurements and the results are presented as mean values ± standard deviation (SD). Correlation and regression analyses were performed with the Excel software 2024 package (Academic License, Microsoft of Portugal). Correlations were considered statistically significant at p < 0.05 according to Tukey HSD and Scheffé test.

3. Results and Discussion

3.1. Optimization of Cultivation Conditions for β-D-Glucan Overproduction

Basidiomycete mushroom strains were inoculated onto solid media supplemented with several agro-industrial wastes, according to the materials and methods. The data in Table 1 revealed the cultivation time required to obtain mycelial biomass across the entire surface of the solid media in the petri dishes with different basidiomycete species and agro-industrial wastes. According to Table 1, the four residues that resulted in the fastest culture growth were yellow lupine, coffee grounds, banana peels and oat hulls. Cultures grown on solid media containing these wastes were then inoculated into liquid medium containing the same agro-industrial residue. From these four agro-industrial residues tested, waste coffee grounds yielded the highest concentrations of β-D-glucan.

The data in Figure 1A have revealed that Lentinula edodes and Ganoderma applanatum exhibited the highest β-D-glucan levels when grown in media supplemented with waste coffee grounds.

Phebia rufa was the only basidiomycete strain that produced β-D-glucan when grown in medium supplemented with yellow lupin (Figure 1 B). However, the levels of β-D-glucan obtained in this medium were lower than those obtained from cultures grown in medium with coffee grounds.

The data in Figure 2 have revealed that the levels of β-D-glucan obtained by the Congo Red dye and phenol-sulfuric acid methods were rather different since the latter is a broad assay for carbohydrate analysis which detected all types of sacharides including proteoglycans, glycoproteins and glycolipds [22]. The highest β-D-glucan levels was observed on 11^th day of culture, whereas highest polysaccharide levels were obtained on the 14^th day of culture. β-D-glucan levels determined by the Alcian Blue dye method, exhibited a similar profile to Congo Red dye method, although with lower values ​​and highest β-D-glucan levels were obtained on 7^th day rather than 11^th day of culture.

According to [21] the Congo Red dye method measured β-D-glucans with a triple helix structure whereas the Alcian Blue dye method was more sensitive and quantified β-D-glucans, regardless of the conformation presented in these biological macromolecules [24]. The levels of extracellular β-D-glucans obtained by using the Alcian Blue dye method for Lentinula edodes supernatant were surprisingly lower than those obtained using Congo Red (Figure 2). Regarding intracellular β-D-glucans from basidiomycetes, the levels reported using Alcian Blue dye were lower than those obtained with Congo Red for only a few samples. These results may be explained by the fact that the alcian blue method detects acidic and sulfated β-D-glucans whose levels may be low in such samples whereas Congo red assay detects neutral β-D-glucans with triple helix conformation [21,24].

The data in Table 2 revealed the comparative analysis of the levels of extracellular and intracellular β-D-glucans produced by these three basidiomycete strains in several agro-industrial wastes.

These data are difficult to compare with published reports on β-D-glucans production because the culture media, mushroom strains and growth conditions are significantly different from the present work.

In order to optimize the overproduction of extracellular β-D-glucans from submerged fermentation of basidiomycete strains, a systematic study was developed to analyze several factors that affect the production of these compounds. The experimental design was conducted using a 2³-way full factorial design, with a total of eight duplicate experiments. After the experimental runs, statistical significance, the effect of each variable, and multivariate interactions on β-D-glucan concentration were evaluated (Table 3).

The data in Table 3 summarized the statistical analysis of variance (ANOVA). The F-value of 1.34x10⁴ indicated that the model is significant and that there was only a 0.01% probability of such an F-value being due to noise. The coefficient of determination R² = 0.9999 indicated that there was a statistical correlation between the response and the variables considered and that only 0.01% of the total variation was not explained by the model. The statistical analysis shows that the significant factors for β-D-glucan production are all those considered: the basidiomycete species (variable A), the agro-industrial waste concentration (variable B), the cultivation time (variable C), and the interactions between the variables AB, AC, BC, and ABC. However, the basidiomycete species, the waste concentration, and the interaction between the species and the agro-industrial waste exhibited the greatest effect. The following empirical equation was obtained to estimate the concentration of β-D-glucans:

y = 39,77-26,57xA+26,18xB-2,97xC-28,53xAB+1,27xAC-1,66xBC+14,21xABC

where: y is the concentration of β-D-glucans, A is the species of basidiomycete, B is the concentration of agro-industrial waste and C is the cultivation time. This equation was used to facilitate plotting the response surfaces which are represented in Figure 3A and Figure 3B.

The data in Figure 3A and Figure 3B exhibited the effect the optimized factors on β-D-glucan production during submerged fermentation.

The culture of Lentinula edodes in a culture medium with 400 g/L of coffee grounds produced the highest concentration of extracellular β-D-glucans (141.16 mg/L) on the third day of fermentation and was therefore the culture selected for subsequent studies (Figure 3B). These data on experimental planning are difficult to perform a comparative analysis with published reports as there are no work reported in the literature about mushroom β-D-glucans.

3.2. Intrinsic Fluorescence Spectroscopy

Since the literature search revealed lack of publications on the intrinsic fluorescence of β-D-glucans from medicinal basidiomycete strains without the use of extrinsic fluorochromes, we decided to investigate the intrinsic fluorescence of these β-D-glucans. Therefore, the sensitivity, or gain, of the microplate reader was initially evaluated. In these tests, a sample of extracellular β-D-glucans extracted from a culture of Lentinula edodes grown in 400 g/L of waste coffee grounds was used as described in the Materials and Methods section. The instrument gain/sensitivity was monitored in the range of 1800 and 2200, and the results were exhibited in Figure 4A. These data revealed an increase in fluorescence intensity as a function of sensitivity or gain with the highest peak at 528 nm and 2200 (Figure 4A).

The data in Figure 4B exhibited the highest fluorescence emission of the extracellular β-D-glucans from Lentinula edodes at 528 nm with excitation at 420 nm.

Additionally, these samples were analyzed by synchronous fluorescence spectroscopy by using a spectrofluorometer. The data in Figure 5 A exhibited synchronous fluorescence spectra for commercial barley β-D-glucan at 10 nm Dl at different concentrations by obtaining peaks at 492 and 540 nm.

Similarly, we also investigated synchronous fluorescence spectra of β-D-glucan from several mushroom strains as shown in Figure 5B. These data also revealed two peaks at 492 and 540 nm which are in agreement with barley β-D-glucan spectra.

Regarding these fluorescence data on mushroom β-D-glucan, it is not possible to perform a comparative analysis with published report as there are no work reported in the literature about intrinsic fluorescence of β-D-glucan.

3.3. Immobilized Metal Affinity Chromatography

Several chromatographic gels containing different immobilized transition metals, spacer arms and stationary phases were tested at pH 8 and the results are shown in Table 4. The chromatographic behavior of extracellular β-D-glucans from Lentinula edodes, grown in a medium supplemented with 400 g/L of waste coffee grounds, exhibited adsorption of these β-D-glucans to IMAC columns which was affected by several factors, such as the size of the spacer arm, chelating agent and metal ion (Table 4).

According to literature search, there are no reports on the use of IMAC for separation and purification of mushroom β-D-glucans. It has been shown that longer spacer arms, such as 1,4 butanediol diglycidyl ether (BDGE) increase the adsorption of monoclonal antibodies as well as contaminating proteins [25] . As far as β-D-glucans are concerned, Cu(II) immobilized chelates in the matrix exhibited the highest adsorption on S1 compared to S2, which contains a EPI as spacer arm and is therefore shorter. However, the opposite result was obtained when comparing S3 with S4 and S5 with S6. During this present investigation, two chelating agents IDA (tridentate) and NTA (tetradentate) were analyzed. From the structural point of view, IDA and NTA differ by the presence of an additional carboxymethyl group on NTA which makes it a stronger coordinator of metal ions because it has 4 valencies available compared to the 3 of IDA. Although both chelating agents have 2 valencies available to interact with the imidazole group of histidine residues, NTA has higher affinity for metal ions compared to IDA (Table 4).

The data in Table 4 has revealed β-D-glucans adsorption occurred in the following order : Cu²⁺ > Zn²⁺ > Ni²⁺ > Co²⁺, β-D-glucans from Lentinula edodes exhibited adsorption to the Sepharose 4B-BDGE-EPI-Cu2+ stationary phase (pH 8).

Among all stationary phases tested, S3 stationary phase of Sepharose 4B-BDGE-IDA-Cu(II) was the best chromatographic gel for purification of β-D-glucans with high recovery although it exhibited a partial adsorption of β-D-glucans (Figure 6 A).

The data shown in Figure 6A exhibited two β-D-glucans peaks at washing and desorption steps assayed by congo red method which correlated well with fluorescence data at 528 nm.

Since we were interested to identify protein-bound β-D-glucans in Lentinula edodes, a chromatographic profile was obtained for protein (A₂₈₀) and β-D-glucan assays as well as imidazole gradient (Figure 6 B).

The data in Figure 6 B have revealed two protein and β-D-glucans peaks at the washing and desorption steps suggesting a partial adsorption of these biological macromolecules. Therefore, these β-D-glucans were either bound to proteins or some free contaminating proteins were present in these column fractions. IMAC has been widely used for purification of native and recombinant proteins containing available histidine residues on their surfaces [27,28]. As far as adsorption of β-D-glucans to immobilized metal chelates is concerned, the molecular mechanism may be due to hydrophobic interactions between the solute and long spacer arm of the stationary phase [26].

3.4. FTIR Spectroscopy

The purified fractions of β-D-glucans from IMAC of Lentinula edodes were analysed by FTIR spectroscopy. The data in Figure 7 have revealed some absorption bands characteristic of β-D-glucans.

Figure 6. - FTIR spectra of selected IMAC fractions of extracellular Beta-D- glucan from L. edodes..

Figure 6. - FTIR spectra of selected IMAC fractions of extracellular Beta-D- glucan from L. edodes..

The strong band at 3474 cm^-1 is due to the extension vibration of the O-H bond whereas this broad band centered near 3300 cm^-1 is specific for carbohydrates [16,29]. It has been described a broad band at 3000-3500 cm^-1 in the spectrum of Ganoderma apllanatum, corresponding to the O-H extension vibration in hydrogen bonds and the N-H vibration [29]. Moreover, some bands were also observed in the spectra at 1638 cm^-1, corresponding to the stretching vibration of the C=O group of amide I, and at 1619 cm^-1 due to deformation of the NH bond and the C-N extension of amide II, which suggest the presence of proteins. There are also absorption bands characteristic of the C-O-C deformation (1180 cm^-1), the anomeric C vibration of carbohydrates (1080 cm^-1) and the β conformation of carbohydrates (865 cm^-1), which demonstrate the existence of β-D-glucans in all fractions [16,29].

4. Conclusions and Future Work

To author’s knowledge, this is the first report about the use of experimental planning for β-D-glucan production from mushroom strains. This work involved the optimization of culture media containing agro-industrial wastes to maximize the productivity of intra- and extracellular β-D-glucans from basidiomycete strains. The highest β-D-glucans concentration (141.16 mg/L), determined by the Congo red dye method, was obtained after extracting β-D-glucans from the fermentation broth of Lentinula edodes cultures in a medium containing 400 g/L of coffee grounds. To author’s knowledge, this is the first report about the use of IMAC for purification of β-D-glucans from L. edodes which was influenced by the size of the spacer arm, chelating agent and metal ion. To author’s knowledge, this is the first report about the intrinsic fluorescence analysis of β-D-glucans from mushroom strains. The purified fractions were analysed by fluorescence as well as by FTIR spectroscopy in order to obtain additional structural information about these biological macromolecules. However, the characterization of fractionated β-D-glucans should be complemented with future studies by using ELISA, HPLC, GC-MS, FTIR, Fluorescence spectroscopy and NMR.