3.3. Monosaccharide Composition and Linkage
Composition of neutral monosaccharides in the fractions is summarized in
Table 3. Among neutral sugars, glucose predominated in all of the fractions, especially in
F3 and
F4. Glucans of various structure predominate in fungal cell walls [
47], so it is not surprising that the glucose content in all fractions, with the exception of
F1, exceeded 85 mol % and even reached about 92 mol % in the water-insoluble fractions. In contrast, in the fraction
F1, glucose (35.6 mol %) was found together with comparable amounts of galactose (26.9 mol %) and mannose (26.5 mol %). This fraction also contained the highest percentage of fucose (6.7 mol %) and xylose (4.3 mol %). Since mannose and galactose were found in almost equimolar proportions, these sugars are possibly interconnected in the form of mannogalactan (or galactomannan) polysaccharide in a mixture with
d-glucans. A similar monosaccharide composition was achieved with cold water extraction of
Pleurotus ostreatus basidiocarps [
36], where the presence of branched mannogalactan was proved by methylation analysis and correlation NMR. Mannose, fucose, and xylose were found as minor sugars in various proportions (less than 5 mol % each) in all of the other fractions analyzed. Galactose was also found in the fraction
F2, but at content lower than 2%, and the glucose content prevailed. This means that the dominant polysaccharide of this fraction is
d-glucan. This fraction also contained 5.5%
w/w of uronic acids, as determined by photometry. A similar composition was previously determined for
Pleurotus ostreatus and
G. lucidum polysaccharides obtained by microwave-assisted extraction [
78]. Aqueous extracts of fungal cell walls usually contain the most labile structural elements of the outer layer, and these are primarily heteropolysaccharides, although some glucans may also be present. However, the water-soluble glucans are preferably released later during the hot water extraction [
79], so most of the heteropolysaccharides, mainly mannogalactans, were collected by cold water extraction. Subsequent extractions with alkaline solutions usually lead to the release of mainly glucans of various structures. A similar relationship between fungal glucans and mannogalactans was previously observed for various multistep extraction protocols. As described for the
G. lucidum basidiocarps [
34], successive isolation steps including hot 0.9% NaCl, hot water, and alkaline extractions resulted in an increase in glucose, while the contributions of galactose and mannose were decreased, with branching also declining from high to almost negligible.
Sugar linkage analysis made for the more pronounced water-soluble fraction
F2 revealed about 13 types of sugar derivatives, suggesting a complex chemical structure of the polysaccharides (
Table 4). This fraction contained five major types of the fragments in descending order: 1,3-linked, terminal, 1,4-linked, 1,3,6-linked, and 1,6-linked glucosyl units. These fragments indicate the presence of different glucans, possibly a mixture of branched (1→3)(1→6)-
β-
d-glucan and non-branched amylose-like (1→4)-α-
d-glucan. The 1,6-linked glucosyl units can form chains of non-branched (1→6)-
β-
d-glucan or be part of side chains in the branched
β-
d-glucan. Fragments of mannose, galactose, fucose, and xylose were also found, but their proportion was too low to judge the inter-connection between them and predict the structure of the corresponding heteropolysaccharides. Obviously, the composition and linkages in polysaccharides obtained from fungi of the genus
Ganoderma will depend on the type and method of extraction. For example, methylation analysis of polysaccharide isolated from
G. atrum consisted mainly of 1,3-linked, terminal, 1,3,6-linked, 1,4-linked, and 1,6-linked glucosyl units (from 21% to 12% each), but also contained smaller amounts of galacturonic acid, mannosyl and other glucosyl units [
80]. In contrast, two polysaccharides GLC-1 and GLC-2 isolated from
G. lucidum were defined as galactoglucan and glucan, respectively; both composed of 1,6- and 1,3-linked glucosyl units and GLC-1 also contained 1,6-linked galactosyl residues [
81]. In any case, hot water extractable polysaccharides from
Ganoderma are likely to be highly branched and may contain other carbohydrates besides glucose.
3.5. Vibrational Spectra
FTIR and FT Raman spectra of the fractions
F1–
F4 are shown in
Figure 4a,b. The assignment of vibrational bands is given according to the literature [
45,
48,
82,
83,
84,
85]. The spectra of all fractions showed several intense overlapping IR bands in the range of 950–1200 cm
−1, mainly CC and CO stretching vibrations of the pyranose rings, proving the presence of polysaccharides as the main component of the samples analyzed. An IR band around 1157 cm
−1 can be assigned to the stretching vibration of the COC glycosidic bonds. Several Raman bands at 1417, 1276, 1205, 1116, 1093, and 1029 cm
−1 are also characteristic for polysaccharides. A weak IR/Raman band around 897 cm
−1 from C1H is present in all spectra and is characteristic of
β-anomeric unit configurations, which could indicate
β-glucans and, in the case of insoluble part
F4, also chitin. Additional IR bands and shoulders that can be assigned to
β-glucans were found near 1375, 1315, 1157, 1073, and 1040 cm
−1 [
84]. The FTIR spectrum of
F4 has three bands at 1656 cm
−1 (amide I), 1557 cm
−1 (amide II), and 1317 cm
−1 (amide III) that indicate the presence of chitin. In the case of the
F1, the IR bands of amide vibrations around 1648 and 1546 cm
−1 were assigned to proteins [
86]. The presence of chitin and proteins in the mentioned fractions was also confirmed by the significant nitrogen content (see
Table 2).
The spectra of the lyophilized raw fraction
F2 and the products of its successive purification including sub-fractions
F2b and
F2c obtained by anionic preparative chromatography are shown in
Figure 5. In all these spectra, the intense highly overlapped bands at 950–1200 cm
−1 confirmed the predominance of polysaccharides. Phenolic compounds and other small molecules were removed by washing with 0.2 mol L
−1 HCl in ethanol, which was confirmed by a decrease in a number of bands related to stretching vibrations of C=C bonds. To remove protein residues, a two-step enzymatic hydrolysis with pepsin and pronase was used. This is because pepsin is capable of cleaving long polypeptide chains into fragments that are still quite large and thus cannot be completely removed. Therefore, for a more complete hydrolysis of proteins, other proteases should be used after pepsin to cleave these fragments into smaller peptides and free amino acids that can be easily removed. The change in the protein content in the fraction can be traced to the intensity of the bands of amide vibrations at about 1647 cm
−1 (amide I) and 1540 cm
−1 (amide II) [
86]. It should be noted here that the amide I band is not well suitable for the detection of the reminder protein due to overlap with the pronounced scissor vibrations of bound water, and therefore, it is better to use the less intense amide II band for this purpose. As a result, after all purification procedures, the band of amide II became insignificant, which confirms the effective removal of proteins. On the other hand, the two vibration bands of the carboxyl groups at 1740 cm
−1 (C=O stretching) and 1240 cm
−1 (CO stretching) remained unchanged after purification, so these groups are most likely part of polysaccharides, possibly in the form of uronic acids [
87]. The spectra of sub-fractions did not contain these two bands; instead, the presence of two bands near 1610 cm
−1 and 1405 cm
−1, which were assigned to the antisymmetric and symmetric stretching vibrations of the carboxylate anions, respectively, confirmed that the sub-fractions, especially
F2c, probably contain uronic acids, but in the form of a salt [
87]. These two carboxylate bands were not strongly pronounced in the FTIR spectrum of
F2b; therefore, this sub-fraction contained fewer uronic acids. The spectra of both sub-fractions have several bands at 1375, 1315, 1155, 1075, 1040, and 895 cm
−1 characteristic for
β-anomeric glucose units and, therefore,
β-glucans [
65,
66,
67,
68,
69].
3.6. NMR Spectra
The assignment of the proton and carbon signals of the main sugar units of the fractions
F1,
F2, and
F3 is summarized in
Table 6. The assignment was made using semi-empirical calculations of Casper software (Future Systems Solutions, Inc., USA) [
88] and using the literature [
47,
48,
89].
The COSY NMR spectrum of the fraction
F1 (
Figure 6a) showed several H1α/H2 cross peaks assigned to, 1,6-linked (
A,
A’) and 1,2,6-linked α-
d-galactosyl (
C,
C’), terminal
β-
d-mannosyl (
B,
B’), and 1,4-linked α-
d-glucosyl (
G) units. These units probably come from two polysaccharides, i.e., branched
O-2-
β-
d-mannosyl-(1→6)-α-
d-galactan, and, in smaller amounts, amylose-like (1→4)-α-
d-glucan. In contrast to mannogalactans previously described for mushrooms of genus
Pleurotus [
36,
90,
91], this mannogalactan obtained from
G. resinaceum was not methylated at the
O-3 position of the backbone galactosyl units because no signals of OCH
3 groups were found. Moreover, several H1
β/H2 cross peaks were assigned to terminal
β-
d-glucosyl (
D), 1,6-linked
β-
d-glucosyl (
D’), 1,3-linked
β-
d-glucosyl (
E), 1,3,6-linked
β-
d-glucosyl (
E’), and 1,4-linked
β-
d-glucosyl (
F,
F’) units; all of them are probably parts of a highly branched (1→3)(1→4)(1→6)-
β-
d-glucan. The HMQC spectrum (
Figure 6b) shows the signals that indicate the presence of the CHOH, CHOR, CH
2OH, and CH
2OR’ residues of the above polysaccharides. The HMQC signal of CH
3 at 1.20 ppm/16.2 ppm and COSY cross peak at 1.20 ppm/4.13 ppm arose from the α-fucosyl units (
H). The HMQC signal at 2.02 ppm/22.9 ppm indicated a small amount of
O-acetyl groups. Therefore, according to the relative intensities of the resonance signals and previous analyses mentioned above,
F1 contains branched mannogalactan as the main product, lower content of branched
β-glucan, and a negligible amount of α-glucan. Water-soluble heterogalactan has been previously isolated from the fruiting bodies of
Ganoderma atrum [
52]. As in the case of the current study, this polysaccharide had a backbone of 1,6-linked α-
d-galactosyl units, but in this case, terminal α-
l-fucosyl and α-
d-mannosyl units and oligosaccharide fragments can act as side chains attached to the
O-6 position of some of the backbone units.
The COSY and HMQC NMR spectra of the fraction
F2 are shown in
Figure 7a,b. As in the case of
F1, branched (1→3)(1→6)-
β-
d-glucan is represented here by 1,3-linked, 1,4-linked, 1,6-linked, 1,3,6-linked, and terminal
β-
d-glucosyl units (
D–F). Furthermore, the presence of mannogalactan was proved by the weak signals of 1,6-linked and 1,2,6-linked
α-
d-galactosyl and terminal
β-
d-mannosyl units (
A–C). A weak HMQC signal at 2.03 ppm/22.96 ppm was assigned to CH
3 of
O-acetyl groups. In addition, 1,4-linked
α-
d-glucosyl (
G) and
α-
l-fucosyl (
H) units were also detected. In general, branched β-glucan predominated in this fraction, while
α-glucan and mannogalactan were present to a lesser extent. Water-soluble highly branched
β-glucans of similar structure have been previously described for some
Ganoderma species [
18]; these polysaccharides were isolated from fruiting bodies, and crowing culture of mycelium [
92] and spores [
93,
94]. For example, water-soluble
β-
d-glucan was previously isolated from basidiocarps of
Ganoderma resinaceum [
95]. It was a highly branched polysaccharide that contained 1,3-linked
β-
d-glucosyl units in the backbone, partially substituted mainly by the side chains of 4-
O-substituted
β-
d-glucosyl units at the
O-6 on average for every two residues of the main chain.
The
1H NMR spectra of the
F2b and
F2c sub-fractions were measured in D
2O at 20 °C and 80 °C and are shown in
Figure 8. For both fractions, when measured at 20 °C, an intense HOD signal overlapped the H1β resonances, and when measured at 80 °C, this intense signal shifted upfield and already overlapped the H6 resonances. The spectra of both sub-fractions are very similar to the spectrum of the initial fraction
F2, and only minor differences were observed between them. Within the fractions, there is certainly a different ratio between the signals of individual glucosyl residues. For example, the signals of terminal
β-
d-glucosyl units
D were more pronounced in the case of sub-fraction
F2c, so this product should have a higher degree of branching.
The COSY and HMQC NMR spectra of the fraction
F3 are represented in
Figure 9. The spectra were measured in Me
2SO-
d6, so there is no proton exchange between the hydroxylic groups and the solvent, and the COSY spectra reveal both CH and OH proton signals (
Figure 9a). The assignment of the proton and carbon signals of the main sugar units is shown in
Table 6. The H1/H2 and OH/CH cross peaks with different intensities found in the COSY spectrum were assigned to the three main glucosyl units. The signals of 1,3-linked
β-
d-glucosyl units (
A) were the most pronounced; the less intense signals were assigned to the 1,3-linked α-
d-glucosyl (
B), terminal
β-glucosyl (
C), and 1,3,6-linked
β-
d-glucosyl (
D) residues. The signals of units
A were also the most intense in the HMQC spectrum (
Figure 9b). These units are evidently inter-connected into the (1→3)-
β-
d-glucan backbone that can be slightly branched at
O-6 of the units
D (branching points) by the terminal
β-
d-glucosyl units
C. Slightly branched (1→3)(1→6)-
β-
d-glucan was isolated by alkaline extraction from the fruiting bodies of
Ganoderma japonicum [
96]. In this polysaccharide, only every 30th unit of the backbone was a branching point carrying a terminal
β-
d-glucosyl attached at the
O-6 position. In contrast, the units
B are rather linked to each other, yielding the (1→3)-
α-
d-glucan, which is less pronounced in
F3. Pure
α-
d-glucan of a similar structure has been previously isolated from
Ganoderma lucidum by alkaline extraction [
89]. The alkaline extract from mycelium of
Ganoderma tsugae contained the mixture of (1→3)-α-
d-glucan and mannoxylan [
97].