Antimicrobial Properties and Cytotoxicity of Sulfated (1,3)-β-D-Glucan from the Mycelium of the Mushroom Ganoderma lucidum

Abstract

Ganoderma lucidum BCCM 31549 has a long established role for its therapeutic activities. In this context, much interest has focused on the possible functions of the (1,3)-β-D-glucan (G) produced by these cultures in a stirred-tank bioreactor and extracted from their underutilized mycelium. In the existing study, we report on the systematic production of G, and its sulfated derivative (GS). The aim of this study was to investigate G and its GS from G. lucidum in terms of their antibacterial properties and cytotoxicity spectrum against human prostate cells (PN2TA) and human caucasian histiocytic lymphoma cells (U937). ¹H NMR for both G and GS compounds showed β-glycosidic linkages and structural similarities when compared with two standards (laminarin and fucoidan). The existence of characteristic absorptions at 1,170 and 867 cm^-1 in the FTIR (Fourier Transform Infrared Spectroscopy) for GS demonstrated the successful sulfation of G. Only GS exhibited antimicrobial activity against a varied range of test bacteria of relevance to foodstuffs and human health. Moreover, both G and GS did not show any cytotoxic effects on PN2TA cells, thus helping demonstrate the safety of these polymers. Moreover, GS showed 40% antiproliferation against cancerous U937 cells at the low concentration (60 μg/ ml) applied in this study compared with G (10%). Together, this demonstrates that sulfation clearly improved the solubility and therapeutic activities of G. The water-soluble GS demonstrates the potential multifunctional effects of these materials in foodstuffs.

Introduction

Bacterial infection is one of the most significant causes of food degradation, and there is little attention on the role of food producers to prevent this phenomenon. Foodstuffs represent a rich source of nutrients often stored under conditions of permissible temperature and humidity. In addition to food degradation by microorganisms, high levels of multiplying microorganisms present in the food may initiate food poisoning, which can contribute to public health problems [1] and disrupting supply chain issues worldwide. Ideally, improving the safety and spoilage characteristics of foodstuffs by including other naturally occurring products that may possess both antimicrobial and other desirable biological activities (e.g., cytotoxicity on cancer cells, health-giving) potentially offers a route to safer foods with enhanced health-imparting characteristics. This approach makes use of the potential for ‘’bifunctional’’ effects [24] of glucan materials derived from traditional food sources, including some species of mushrooms [21]. These natural foods have been shown to be a relatively unexplored source for improvements in food safety, and preservation while providing extra health benefits [40].

Mushrooms of the genus Ganoderma have been eaten for many centuries in Asia to encourage well-being, durability, and endurance [11,22]. To date, more than 120 species of Ganoderma have been identified across the world. In the last 30 years, there has been significant scientific interest in the species Ganoderma lucidum. This fungus has been lately shown to possess varied health benefits, such as antibacterial effects [14] and antiproliferative effects on cancer cells [19]. In this study, β-glucan produced by the cultures with potential bioactivities was extracted from the mycelia.

The extracted mycelial G. lucidum β-glucan (G) is known to act as a biological response modifier. Therefore, much research has focused on this fungal polysaccharide as a functional foodstuff and source for the development of biomedical drugs [14]. The clinical utilization of β-glucans has one main difficulty in addition to the limited availability referred to above; that is, their comparative absence of solubility in aqueous solution, which leads to difficulties in product analysis, formulation, and delivery. This is usually ascribed to the high number of –OH groups in the β-glucan leading to the native polymer adopting a compact triple stranded helix conformation, which determines their poor solubility in aqueous condition [36]. These demonstrate the failure of existing glucan products, whereas the proposed glucan sulfate would not.

Upon preliminary isolation from G. lucidum, the β-glucan (mainly (1-3)-β-D-glucan) exists as an insoluble microparticulate. Thus, a technique such as sulfation is needed to alter the molecule’s hydrophobicity, thus making it water-soluble and potentially more bioactive in aqueous systems. The proposed sulfation technique has been used as an effective approach to improve the antibacterial, antiproliferative, anti-inflammatory, antitumor, and immunomodulatory activities of a range of other polysaccharides [5,17,38,40]. A previous effort by Williams et al. [37] demonstrated that insoluble (1-3)-β-glucan was able to dissolve in water by a sulfation process, while increasing the positive biological functions [5].

To date, the cytotoxicity and antimicrobial activity of extracts from G. lucidum mycelia, particularly the glucan sulfate (GS), have not been completely characterized. In the current study, glucan from G. lucidum mycelia was sulfated. Both the glucan (G) and sulfated glucan (GS) structures were matched to known standards and screened antimicrobial and cytotoxic effects. The results showed that GS exhibited significant antimicrobial activities as well as antiproliferative responses while showing no toxic effects and hence could be utilized as a potential additive in food systems. With that, its presence would inhibit both spoilage and pathogenic bacteria, and impart significant health benefits noted in this study.

 

Materials and Methods

Reagents

Gentamicin susceptibility test discs (30 μg of concentration) were supplied by Thermo Scientific Oxoid (Fisher Scientific, UK). In this experiment, human caucasian histiocytic lymphoma (U937) and human prostate normal cells (PNT2A) were obtained from the European Collection of Cell Cultures, supplied by Sigma-Aldrich, (UK). DMEM and TrypLE Express were provided by Gibco (Life Technologies, UK). RPMI without L-glutamine was supplied by Lonza BioWhittaker (Belgium). Hank’s balanced salt solution was provided by Sigma-Aldrich (USA). The 96-well plates, TPP 92096, were provided by TPP (Trasadingen, Switzerland). Cell culture spectroscopy analysis was done using a Wallac, Victor2H20 Multilabel Counter with IR, high-density TR-Fluorometry, robot loading a nd s tacker ( PerkinElmer, MA, USA). A ll s olvents and chemicals used were of analytical grade.

Fungal Material

G. lucidum BCCM 31549 was obtained from the Belgian Coordinated Collections of Microorganisms (BCCM/MUCL), [Agro] Industrial Fungi and Yeast Collection (Belgium). The fungus was subcultured onto potato dextrose agar (PDA, Oxoid Limited, UK) upon receipt from the supplier to avoid any contamination and ensure viability as suggested from previous research [10]. Plates were inoculated and incubated at 30℃ for 7 days and stored at 4℃. The strain was preserved on PDA slants. The fermentation strategy was implemented in a stirred-tank bioreactor and the mycelial pellets were extracted.

Extraction, Isolation, and Sulfation

Distilled water was used to rinse the mycelia (biomass) off the sieves from the fermented culture broth. Through Whatman filter paper, they were filtered and vaporized to 50 ml at 60-80℃. This volume was added to 150 ml of ethanol for macromolecule precipitation, containing the desired polysaccharide-derived β-glucan. A glass rod was used to obtained the product by twirling. Based on the macromolecule precipitation, the precipitate was attached or adsorbed onto the glass rod and harvested from the solution. The glucose, however, may be confined within the extracted precipitate, which was then splashed using 96% (v/v) ethanol. Subsequently, the solution was dialyzed against distilled water for 3 days (MW cut-off = 10,000 Da) using a dialysis tube (Fisher Scientific). The residual glucan was aerated and pre-chill in a -20℃ freezer. After a couple of hours (h), the samples were transferred to a -80℃ freezer for 24 h and then freeze-dried for 48 h. Later on, the build-up moisture surrounding the precipitated glucan was completely evaporated. It was then resuspended in distilled water, freeze-dried in the -80℃ freezer, and evaluated to yield a 1,3-β-D-glucan (G).

The G produced from the bioreactor fermentation process was water-insoluble; therefore, an inevitable process needs to be implemented to increase its solubility in water. Suzuki et al. [32] and Williams et al. [37] carried out sulfation of active 1,3-β-D-glucans to increase their solubility or increase their bioavailability. Hence, sulfation of the current water-insoluble G was executed in this experiment. The improvised G sulfation method of Williams et al. [37] was followed. Soluble 1, 3-β-D-glucan sulfate (GS) was produced as outlined in Fig. 1. First, 1 g of microparticulate G was liquefied in 50 ml of DMSO containing 6 M urea. Then 8 ml of concentrated sulfuric acid was added drop-wise directly erstwhile to heating. In a water bath, the solution was heated at 100℃ and the reaction process continued for 3 to 6 h. By 90 min, a crystalline precipitate (ammonium sulfate) was formed. The mixture solution was then vented at room temperature, and 1 L of ultrapure, pyrogen-free, distilled water (Millipore, MA, USA) was added. The GS solution was then pre-filtered to remove unreacted polymers in G. The GS solution was dialyzed using a Vivaflow 200, using a 10,000 MW cut-off filter (Sartorius Stedim Lab Ltd, UK). The final volume was reduced to 500 ml and lyophilized to dryness.

Fig. 1.Homogeneous reaction for sulfated (1-3)-β-D-glucan (GS) preparation: process scheme. Adapted from Wang et al. [36].

Elemental Analysis

The content of C, H, O, N, and S was estimated using a Perkin Palmer 2400 Series II CHNS/O Elemental Analysis (MA, USA) device. Based on the recorded results of the elemental analysis, the degree of sulfation (DS) is defined by the following Eq. (1) according to Wang et al. [36].

where s is the mass ratio of S element in the product glucan sulfate (GS). From now on, DS signifies the number of sulfate groups per glucose residue.

Infrared Spectroscopy

FTIR spectra of the G and GS samples were taken using a FTIR 3000 spectrophotometer (Japan) following the method of Shi et al. [29]. For jelly-like specimens (GS), FTIR attenuated total reflectance (Perkin Elmer) was used to acquire the spectrum.

1H NMR Spectroscopy

The NMR spectra of both G and GS were taken using a DXM 500 FT-NMR spectrometer (Switzerland). Both compounds were liquefied in deuterium oxide –d6 at the concentration of about 10 to 30 mg/ml. All spectra were obtained at 80℃, respectively. The scan number was 16, and the chemical shifts (δ) are indicated in parts per million (ppm). Laminarin from Laminaria digitata (Sigma-Aldrich, UK) was used as the comparison standard for G, whereas fucoidan from Fucus vesiculosus (Sigma-Aldrich) was used as the comparison standard for GS.

Bioassay of Antimicrobial Activity

The test bacteria used for antimicrobial sensitivity testing comprised Pseudomonas aeruginosa, Salmonella Enteritidis, Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli that were obtained from the General Microbiology Lab Collections (SIPBS, UK). In addition, Escherichia coli EPIC S17, Salmonella BA54 SL1344 pSsaG, Listeria monocytogenes, Shigella sonnei 20071599, and Methicillin-Susceptible-Staphylococcus aureus ATCC 292123 were kindly supplied by Dr. Jun Yu (SIPBS, UK). At 20℃, the strains were kept in the suitable freshly prepared medium and rejuvenated two times before being applied in the proposed assays. Bacteria were cultured with an oxygen supplied environment at 37℃ in nutrient agar (NA) medium.

Kirby-Bauer disc diffusion assay, MIC, and MBC. Determination of antimicrobial activity was carried out using the Kirby-Bauer disc diffusion a ssay m ethod. F irst, 20 ml o f NA medium was decanted into each Petri dish. All test microorganisms were adjusted to 0.5 McFarland standards using sterile broth medium. Once h ardened, about r oughly 2 00 μl of suspension of the test bacteria was smeared on the prepared agar. The standardized 11 mm sterile discs (blank) (Sigma-Aldrich) with an identical absorbed GS volume were soaked with a known amount of extract. It was positioned moderately onto the agar overlay. The plates were carefully incubated overnight at 37℃ or for 48 h at 30℃ depending on the growth requirement of the bacterium. Gentamicin was applied as the positive control whereas ethanol was the negative control. After the incubation, the diameters (mm) of the inhibition zone were measured. Inhibition zones that were larger than 11 mm were considered positive for antimicrobial reactions.

The minimal inhibitory concentration (MIC) was evaluated by microdilution using 96-well microtiter plates, according to Li et al. [20] with slight modifications. Sterile broth medium in conjunction with 0.5 McFarland standards was used for bacterial suspension adjustment. GS compounds were dissolved in sterile ultrapure water and serially diluted into 200, 100, 20, 10, 8, 5, 3, 2, and 1mg/ml. The final mixture was 25 μl of compounds with 75 μl of a suspension of each bacterium (working volume of 100 μl). Each test culture was pipetted onto the plates and incubated for 24 h at 30℃. Once the incubation time ended, the turbidity or cloudiness was taken as the signal or indication for bacterial growth. The lowest diluted concentration at which the incubated mixture stayed clear after microscopic assessment (under a binocular microscope) was thus selected as the MIC.

The microscopic growth range was then pipetted (100 μl) to the NA. Sterile L-spreaders were used to make the spreading even. Following that, the concentration indicating the MIC and at least two of the more concentrated dilutions were plated and enumerated to determine viable colonies specifically for minimum bactericidal concentration (MBC) determination. The media were cultured at 30℃ for 24 h to observe for any microorganism growth. For the MBC, the minimum or lowest concentration in the medium that had less than five colonies was used.

The method of the Strathclyde Institute of Drug Research was used for the antimicrobial test on the bacteria Klebsiella pneumoniae ATCC 13883 and Mycobacterium marinum ATCC BAA 535, using 96-well microtiter plates [4,18]. These tests were in triplicate, and the GS was supplied at 10mg/ml. Gentamicin was used as a positive control for the bacteria, and DMSO as the negative control.

Bioassay of Cytotoxicity

On normal cells. Cell lines were grown in the appropriate freshly prepared complete medium in a cell culture incubator (gaseous composition 95% air, 5% CO2) at 37℃. The PN2TA normal human prostate cell line was sustained in a complete medium comprising RPMI, 5 ml of penicillin-streptomycin, 50 ml of f etal b ovine serum, 5ml o f L-glutamine, and pH at 7.4. AlamarBlue assay determined the cytotoxic effect of both G and GS. Initially, 96-well microtiter plates were seeded with the PN2TA cells at 2 × 104 cells/ml for each well. Cells were permitted to cultivate one day before being introduced to GS: 500, 300, 50, 30, 5, and 3 μg/ml. For the negative control group, 4% (v/v) Triton-X was added to the medium. After incubation for the indicated hours, 10% (working volume per well) of alamarBlue reagent was decanted to each well and incubated for an extra 6 h in a humidified incubator. Thereafter, the resazurin in the alamarBlue undergoes oxidation-reduction change in response to cellular metabolic change. The reduced form resazurin is pink and extremely fluorescent, and the strength of fluorescence produced is proportional to some living cells that have undergone respiration. The wavelength of 570 nm was used for absorbance reading. For analysis, cytotoxic activity was calculated based on cell survival ratio (%).

On cancer cells. The cytotoxicity of both G and GS were also tested on the cancerous cell U937 by 96-well microtiter plate using alamarBlue assay. The U937 cells at a density of 3 × 105 cells/well were exposed to 60, 50, 30, and 10 μg/ml of both G and GS at day 1 prior to incubation. As for the control group, an identical volume of complete sterile medium was applied (positive control) whereas Triton X (4%) was the negative control. After incubation for the designated period, 10% of alamarBlue reagent was pipetted to each well and incubated for an extra 6 h in a humidified incubator. The alamarBlue reagent initiats resazurin to undergo oxidation-reduction change in response to the cellular metabolic modification. The wavelength of 570 nm was used for absorbance reading. For analysis, cytotoxic activity was calculated based on cell survival ratio (%).

Statistical Analysis

All analyses were carried out in triplicate, and the respective mean ± SD was determined using the software GraphPad Prism 5 (ver. 5.01) and shown as error bars. If the error bars do not appear, then they are less than the size of the icon or symbol.

 

Results and Discussion

Glucan Solubility

In this study, the method for solubilization of G employs DMSO to dissolve initially the water-insoluble G preceding sulfation [11]. The DMSO and other reaction products were removed from G by extensive dialysis to ensure the purity of the GS produced. The solubility of the GS in water was measured post-sulfation to assess the effects of sulfation on G. In ultrapure distilled water, the final solubility of G was below 5% (w/v), but that of its GS was above 95% (w/v). Table 1 recaps the solubility and yields of the insoluble G and soluble GS. Furthermore, the GS was readily dissolved without heating, whereas the G needs 0.1 M of NaOH at 80℃ to assist dilution in water. The improved tractability of GS about G represents a significant aid in developing and implementing assays.

Table 1.Values are means of four batches.

The introduction of the sulfate group has several purposes. Based on the present study’s findings, the aqueous solubility of the extracted G from the fermenter was as poor as that of G prepared from other procedures. Astonishingly, this was mentioned in the literature that G was less suitable for medicinal applications [13]. In terms of the commercial importance of bioactive glucan, the water-insoluble G shows slight bioactivity, although G by-products such as pullulan sulfate, lentinan sulfate, and dextran sulfate have been suggested to display high anti-HIV activities and small anticoagulant activities [36]. Wang and Zhang [34] also revealed that the sulfation process on the fruiting bodies of G. lucidum producing G have led to enhanced antitumor and antiviral activities [23,27]. However, comparable studies on the antimicrobial activity and cytotoxicity of mycelial-sourced GS are limited.

Compositional Analysis

Elemental analysis. Elemental analysis was accomplished to attain the composition of the GS and, therefore, its degree of sulfation. Basic examination of lyophilized GS gave a composition of (w/w) 24.5% C, 5.72% H, 49.92% O, 9.85% S, and 10.01% N (Table 2). When compared with standard fucoidan, GS had the same C and H values but was slightly different in H, O, and S. This was due to different sulfation techniques applied on each GS and fucoidan, respectively, and this might generate different molecular weights.

Table 2.Laminarin is a standard for (1,3)-β-D-glucan from Laminaria digitata. Fucoidan is a standard for sulfated-(1,3)-β-D-glucan from Fucos vesiculosus. Values were in triplicate and are presented as the mean ± SD. The p value is <0.05.

Based on the composition of GS, the DS of GS is thus 0.90, indicating 90 sulfate groups are present on every 100 glucose subunits within the polysaccharide on average. When compared with the previous DS value (0.94) of sulfated polysaccharide (S-GL) of G. lucidum reported by Wang and Zhang [34], the current DS value of GS (0.90) was broadly similar.

IR spectroscopy. Table 3 and Fig. 2 summarize the results of using FTIR spectroscopy to assess the structural characteristics of the G and GS. Both molecules showed the typical IR absorptions of polysaccharides at 1,250 and 1,650 cm-1: 1,170 and 1,651 cm-1, respectively. These IR absorptions as well as those in the “anomeric region” at 950–700 cm-1 allow us to differentiate β from α glucans spectroscopically [35]. Overall, the D-glucosidic linkage arrangement is β-type both prior to and following the sulfation.

Table 3.aValues represent the mean ± SD (p < 0.05) for triplicate experiments. G indicates gram-positive (G+) or gram-negative (G-) bacteria. bSterile disc size was 11 mm, indicating negative reactions, and positive reactions were more than 11 mm. cEthanol was used as the negative control and gentamicin (GENT) was used as the positive control. dThe minimum inhibiting concentration (MIC) (as mg/ml). eThe minimum bactericidal concentration (MBC) (as mg/ml).

Fig. 2.Comparison of β-glucan IR spectra. A: glucan (G); B: glucan sulfate (GS), derived from extended batch cultures of G. lucidum BCCM 31549 mycelium.

In the functional group region of the G spectra, there were significant absorptions at 3,400, 1,077, 2,925, 1,374, 1,647 , 1,246, 1,540, 1,077, and 892 cm-1, which resembles the elongating absorption bands of poly -OH, C=O=C, -CH2, -CH3, C=0, amide, pyranose ring, and β-configuration of D-glucose units. As compared with the previous work by Wang et al. [36] and Liu et al. [22], the specific absorption of G at 892.9 cm-1 demonstrates that the compound is a β-glucan. The characteristic peak of the β-configuration at 892.9 cm-1 was also noted in the spectra of GS with two new absorption peaks at 1,170 and 867 cm-1 also present (Fig. 2), which match to the S=0 asymmetrical stretching and C-S-C symmetrical vibration [35]. These confirmed that the GS had been efficiently synthesized from G.

NMR spectroscopy. As can be seen in Figs. 3 and 4, 1H NMR spectroscopic analysis of the G and GS from G. lucidum was conducted at 80℃ using D20-d6 as a solvent. Using ppm as the standardized unit for NMR studies, 1H NMR spectra of the G were compared with the standard laminarin (β-1,3-D-glucan) from L. digitata, whereas the GS spectra were compared with the standard fucoidan (sulfated-β-1,3-D-glucan) from F. vesiculosus. The spectrum chemical shifts of δ 3.9 to 5.4 ppm and δ 2.6 to 5.5 ppm indicate that both compounds were glucans, as can be observed in Figs. 3 and 4, respectively. The current work is comparable to previous research by Ji et al. [16], which analyzed laminarin and sulfated laminarin in the area of the 1H NMR spectrum of δ 4.49-5.5 ppm. Thus, these spectra indicate that the glycosidic bonds in both G (Fig. 3) and GS (Fig. 4) were β-type.

Fig. 3.1H NMR spectra of (1-3)-β-D-glucan (G) derived from extended batch cultures of G. lucidum BCCM 31549 mycelium and laminarin (Laminaria digitata) standard in D20-d6 at 80℃.

Fig. 4.1H NMR spectra of sulfated (1-3)-β-D-glucan (GS) derived from extended batch cultures of G. lucidum BCCM 31549 mycelium and fucoidan (Fucus vesiculosus) standard in D20-d6 at 80℃.

Evaluation of the “anomeric region” of 1H NMR spectra in this study with those described previously specifies that they are of similar pattern [22,33,36]. For the G (Fig. 3) 1H NMR spectra, the signals at δ 5.08, 4.50, and 4.40 were assigned to OH-2, OH-6, and OH-4 when compared with the reported work by Wagner et al. [33]. The GS (Fig. 4) 1H NMR spectra also exhibited similarity to G with the signals at δ 5.21, 4.52, and 4.40. When compared, the anomeric signals for both compounds in the present study (G and GS) were at δ 4.5 ppm and δ 4.2 ppm, respectively, indicating β-configuration for glucopyranosyl units as reported by Liu et al. [22].

Moreover, the 1H NMR spectrum of GS displayed that the chemical shift of hydrogen usually stimulated downfield relative to G, which showed that most of the hydroxyl groups in G had been sulfated and similarly specified that GS had β-glycosidic bonds. From the IR and 1H NMR analyses, it is possible to conclude that the G compound was composed of (1-3)-β-D-linkages, which gave the polymer structure apparently as a 1,3-β-D-glucan.

Assessment of Antimicrobial Activity

The antimicrobial effect of the GS from G. lucidum was tested against 10 species of bacteria as G was not evident. Their strength was measured quantitatively and qualitatively by the absence or presence of inhibition zones, zone diameters, and MBC and MIC values. The findings of these tests are summarized in Table 3 (inhibition zone diameters). Among the bacterial strains tested in Table 3, when the GS reached 500 mg/ml, the diameters (mm) of the inhibition zone were 34 ± 3.2, 24 ± 2.6, 32 ± 1.0, 25 ± 2.6, 23 ± 2.8, 27 ± 1.5, 28 ± 0.5, 26 ± 1.0, 30 ± 1.0, and 30 ± 3.1 for E. coli EPIC S17, E. coli, L. monocytogenes, Shigella sonnei 20071599, P. aeruginosa, S. Enteritidis, Salmonella BA54 SL 1344 (pSsaG), Staph. aureus, Staph. epidermidis, and Methicillin-Susceptible Staph. aureus ATCC 292123, respectively. The inhibition zone diameters increased with increasing GS prepared concentrations (Table 3). These reactions displayed that the antimicrobial effect of GS was dose-dependent and that the gentamicin positive control was clearly effective against all the test bacteria.

Furthermore, the MIC concentrations for bacterial strains were in the range of 1-5 mg/ml and the MBC concentration range was 5-10 mg/ml, except for the resilient Shigella sonnei 20071599 (Table 3, No. 3). Among four species of gram-positive bacteria verified, the greatest active antimicrobial activity of GS was shown against Staph. aureus (Table 3, No. 8), and its MIC was 2 mg/ml. Meanwhile, the antimicrobial activity of GS was verified against six species of gram-negative microbial strains. GS exhibited fairly strongest antimicrobial activity against E. coli (Table 3, No. 2) (MIC = 1 mg/ml), and seven species of microbial strains were shown to have MIC concentrations at respective 3 mg/ml and the most resistant bacterium was Staph. epidermis (Table 3, No. 9) (MIC = 5 mg/ml).

When compared with other studies where derivatized fungal polymers have been examined as food preservatives and their antimicrobial activity has been assessed [8], it showed that SC2 sulfated-polysaccharide (chitosan) has MIC values higher than 2 mg/ml [28] for Staph. aureus, L. monocytogenes, Vibrio parahaemolyticus, P. aeruginosa, Shigella dysenteriae, V. cholera, Aeromonas hydrophila and S. Typhimurium. SC2 shows a much higher MICs against gram-positive than gram-negative bacteria. Devlieghere [7], Muzzarelli et al. [26], and Hernandez-Lauzardo et al. [15] also tested the antimicrobial activity of chitosan as a food preservative and gave results for MICs at or above 2.5 mg/ml. The closest comparison to the present study involved an assessment of MIC values of an ethanolic extract of G. atrum sourced from powdered fruiting bodies varying from 1.6 to 6.25 mg/ml for the common bacterial food contaminants [20], which was also reported by Ferreira et al. [11]. Thus, the MICs recorded for the GS in the present study are broadly similar to those reported in other studies for fungal-derived polymers.

The antimicrobial activity on the bacteria K. pneumoniae ATCC 13883 and M. marinum ATCC BAA 535 were tested on 96-well microtiter plates to assess the antimicrobial effects of GS. Overall, the results showed some clear inhibition of growth for both these test species (Table 4). The gram-negative K. pneumoniae ATCC 13883 exhibited a survival of 52.8 ± 5.66% (at 500 μg/ml and 24 h incubation) while the acid-fast bacteria M. marinum ATCC BAA 535 gave a survival value of 65.0 ± 3.39% (at 100 μg/ml and 24 h incubation) compared with positive growth controls. Owing to the significant and increasing occurrence of nosocomial infections and destructive changes to human lungs, as mentioned by Daligault et al. [6], by antibiotic-resistant K. pneumoniae ATCC 13883, the possibility of using novel antimicrobials from processed natural sources such as GS extracted from G. lucidum merits further investigation and refinement. Meanwhile, GS might have some potential in controlling the occurrence of common granulomatous diseases arising from M. marinum ATCC BAA 535 that affect individuals who work with fish or keep aquaria, as described by Slany et al. [31].

Table 4.aValues represent averages ± SD (p < 0.05) for triplicate experiments. bA lower percentage of control value means a greater antibacterial effect.

Overall, at present it is not entirely clear what the mechanism(s) of the antimicrobial activity of a sulfated polysaccharide such as GS is likely to be, as there are few studies in this area. Meanwhile, the G was negative in terms of antibacterial impact (results not shown). The steric and repulsive electrostatic properties of sulfate groups and how these might alter the spatial construction of the glucan were proposed by Ji et al. [16] as a possible contributor to the observed behavior of GS. Others suggested that changes in the flexibility of the polysaccharide backbone, and the altered water solubility could lead to variations in biotic response [3,9,30], which may also include the antimicrobial effects. The mechanisms of sulfation on the structure G were proposed for these positive reactions by GS. Consequently, it is essential to further studies in order investigate glucan structure-activity relationships, which might deliver a detailed foundation for their development and improvement.

Slany et al. [31] discussed the impact of sulfation on the structure and biological activity. In general, the sugar chain conformation becomes modified by the process of sulfation such that noncovalent bonds form more readily when the –OH groups in a β-glucan element are replaced with sulfate groups. Similarly, repulsions between the anionic groups lead to elongation of the sugar chain. They propose that these events result in the polymer developing an active conformation, thus initiating the bioactivity surge.

In the last 20 years, there have been insufficient reports on antimicrobial activities of biopolymers from Ganoderma species [6,14,20,39]. This genus has been commonly considered for its therapeutic properties, but less widely explored as a source of novel antibacterial agents [12,14]. However, certain polysaccharides from Ganoderma species employ antibacterial activity by hindering the growth of bacteria and, in some events, by eliminating pathogenic bacteria [30]. Nearly all antibacterial investigations on Ganoderma species have been accomplished on the fruiting body and not on extracts from the liquid cultivated mycelium, a point that is made strongly in the recent review by Ferreira et al. [11]. Meanwhile, most of the positive antibacterial compounds were from alcoholic extracts, hot-water extracts, and triterpenoids of fruiting bodies. The current work is the first to show positive results using GS extracted from G. lucidum mycelium produced in the bioreactor.

Assessment of Cytotoxicity Activity

The current extracted and processed GS from G. lucidum was reactive against pathogenic bacteria. Yet, to ensure whether these compounds might have clinical impact on healthy patient cells and before their introduction as new antimicrobial drugs, some preliminary assessment of the impact of such biomolecules upon normal host cells is of interest. Likewise, assessment of the effects of such derivatized polymers on tumors is of value given the widely reported impact of other fungal macromolecules on such cell types. Accordingly, in the present study, cytotoxicity assays using alamarBlue reagent were carried out on healthy human prostate cells (PN2TA). The in vitro effects of both G and its GS from G. lucidum on PN2TA were studied in the current work (Fig. 5).

Fig. 5.Cytotoxicity effects of both glucan (G) and glucan sulfate (GS) derived from extended batch cultures of G. lucidum BCCM 31549 mycelium in a normal human prostate cell line (PN2TA). After the cells were incubated with G and GS treatments (Control, 500, 300, 50, 30, 5, and 3 μg/ml, and Triton X), the viability was measured by alamarBlue assay. Both G and GS were morphologically observed under the microscope at 10× magnification. Data are presented as the mean ± SD, and the p value was > 0.05 when compared with the control. If the error bars do not appear, then they are less than the size of the icon or symbol.

In this study, a series of dose-response assays were implemented to define the cytotoxic reactions in PN2TA. Once the cells were exposed to different concentrations (3, 5, 30, 50, 300, and 500 μg/ml) of GS and G for 24 h, the alamarBlue reagent assay displayed no loss of cell viability. Morphological observations of the treated cells were the same as the control cells; therefore, these data indicated that GS and G did not exhibit cytotoxicity in PN2TA normal human cells. When compared with the previous work by Li et al. [20], the β-glucan from G. atrum did not react on the viability of healthy cells, thus confirming the clinical safety of G. lucidum β-glucan extracts from the current strain.

The cytotoxicity of G and GS against the development of cancer cells (U937) was examined using the alamarBlue reagent in this study. As revealed in Figs. 6 and 7, GS displayed a dose-dependent antiproliferative reaction within the value range of 10-60 μg/ml and exhibited stronger antiproliferation than G. GS showed the most potent antiproliferative effect at 60 μg/ml with approximately 40% antiproliferation compared with 10% for G, as Fig. 7 shows the fewest cell growth with ascending growth towards lower concentrations. As reported, it demonstrates that the antiproliferative activity of cancer cell growth was enriched by the sulfation process (GS) as matched with the unprocessed glucan (G) [2,35].

Fig. 6.Cytotoxicity effects of glucan (G) derived from extended batch cultures of G. lucidum BCCM 31549 mycelium against cancerous human Caucasian histiocytic lymphoma cell line (U937) from a 37-year-old male patient. After the cells were incubated with G treatments (Control, 60, 50, 30, and 10 μg/ml, and Triton X), the viability was measured by alamarBlue assay. G was morphologically observed under the microscope at 10× magnification. Data are presented as the mean ± SD, and the p value was < 0.05 when compared with the control. If the error bars do not appear, then they are less than the size of the icon or symbol.

Fig. 7.Cytotoxicity effects of glucan sulfate (GS) derived from glucan (G) against cancerous human Caucasian histiocytic lymphoma cell line (U937) from a 37-year-old male patient. After the cells were incubated with GS treatments (Control, 60, 50, 30, and 10 μg/ml, and Triton X), the viability was measured by alamarBlue assay. GS was morphologically observed under the microscope at 10× magnification. Data are presented as the mean ± SD, and the p value was < 0.05 when compared with the control. If the error bars do not appear, then they are less than the size of the icon or symbol.

The current concentration of GS (60 μg/ml) applied is considerably lower than that used in the earlier study on of sulfated glucan (sourced from Hypsizigus marmoreus), which showed only 39% of antiproliferative activity at 1,000 μg/ml [2], and thus further concentration increment for the current work would be highly beneficial. As reported, the molecular weight, chemical configuration, degree of branching, and structure of the polymeric backbone were crucial for antiproliferative activity stimulation for both G and GS [25]. Therefore, the biochemical aspects and mechanism of the antiproliferative reactions stimulated by GS from mycelium of G. lucidum is still not fully understood and requests further study.

In summary, it has been shown that the compounds extracted from these mycelial cultures were polysaccharides with a proposed structure of β-1,3-D-glucan when compared with both standards, laminarin and fucoidan. The antimicrobial activity of the GS from G. lucidum was effective against tested microbes in the assays. Moreover, the cytotoxicity of GS was evaluated with normal human prostate cells and no such effects were noted at the levels tested in this study. The GS may also have potential in antiproliferative work based on its cytotoxicity of human Caucasian histiocytic lymphoma cancer cells (U937). These GS activities indicate that sulfate substitution on G not only improved its solubility, but also had an impact on its therapeutic activities, suggesting that sulfation was an effective way to enhance these activities. In relation to this, the GS might have a role as a natural additive in many foods, with multi functional benefits (preservative, antiproliferative, immune stimulation). Further examination of these functions for such polymers and their derivatives will be required.