Sacchachitin, a novel chitin-polysaccharide conjugate macromolecule present in Ganoderma lucidum: Purification, composition, and properties

Figures & data

Figure 1.  Flowchart showing the process used for purification of sacchachitin from G. lucidum.

Table 1.  Proximate composition in the fruiting bodies of G. lucidum.

Composition	Content (%w/w)
Moisture	11.0 ± 0.3
Crude ash	1.5 ± 0.1
Crude fat	3.0 ± 0.2
Crude fiber	29.2 ± 0.3
Crude protein	13.6 ± 0.1
Carbohydrate	41.6 ± 0.7

Values are expressed as mean ± SD from triplicate experiments (n = 3).

Table 2.  Comparison of composition in different fractions and the sacchachitins obtained by different processes from the fruiting bodies of G. lucidum.

Fraction	Yield (%w/w)	Carbohydrate (%w/w)	Protein (%w/w)	Hexosamine (%w/w)	Mean MW, (kDa)
L5	0.7 ± 0.1^a	75.1 ± 0.3^f	21.4 ± 0.1^e	6.3 ± 0.1^b	8230 ± 58^e
L6	9.1 ± 0.2^e	52.9 ± 0.2^d	46.9 ± 0.2^g	4.6 ± 0.1^a	1101 ± 55^a
L7	1.5 ± 0.1^b	70.3 ± 0.3^e	28.1 ± 0.1^f	8.6 ± 0.1^d	1198 ± 56^b
L8	7.4 ± 0.2^d	41.8 ± 0.2^c	13.4 ± 0.1^c	8.9 ± 0.1^e	6945 ± 61^d
L9	3.5 ± 0.1^c	80.7 ± 0.3^g	15.6 ± 0.1^d	11.1 ± 0.2^f	4570 ± 44^c
L10: Sacchachitin (this paper)	3.4 ± 0.1^c	32.4 ± 0.2^a	10.1 ± 0.2^b	16.3 ± 0.1^g	8632 ± 62^f
Sacchachitin Mimic Su et al. (1997)	14.7 ± 0.3^f	28.0 ± 0.3^b	9.6 ± 0.1^a	6.6 ± 0.2^c	9756 ± 55^g

L5: ethanol precipitate from hot water extract; L6: the isoelectric point precipitate from 2% NaOH extract; L7: the three-fold volume ethanol precipitate from 2% NaOH extract; L8: the isoelectric point precipitate from 10% KOH extract; L9: the three-fold volume ethanol precipitate from 10% KOH extract; L10 (SGL): Sacchachitin carbohydrate was measured by the phenol-H₂SO₄ method. Protein was determined by the Bradford protein assay. Hexosamine was determined by the Johnson hexosamine assay (Johnson, 1971). The molecular weight (MW) was determined by gel permeation chromatography (GPC). Values in each column with different superscripts indicate significantly different between each other (p < 0.05). Data are expressed in mean ± SD from triplicate experiments.

Table 3.  Assignment of IR absorption for different products and their deacetylation degree.

Product	Bond vibration frequency (cm^–1) found in the IR spectra
Vibration mode	Sacchachitin		Sacchachitosan
	This paper	MSC	This paper	MSC
νO–H	3450.5 b, s	3455.7 b, s	3445.2 b, s	3419.2 b, s
νN–H	3500–3300 (masked)	3500–3300 (masked)	3500–3300 (masked)	3500–3300 (masked)
νC–H	2932.5 w	2932.4 w	2920.7 w	2922.6 w
νC=O	Amide 1678.5	Amide 1678.3	Disappeared	Disappeared
δN–H	Amide I 1550.4	Amide I 1551.4	Disappeared	Disappeared
δC–H	1413.5;	1412.6;	1420.3;	1421.3;
	1381.6;	1376.4;	1384.6;	1373.1;
	1325.8	1324.5		1314.3
νC–C	1152.7	1154.6	1154.2	1158.4
νC–N	1125.9	1121.7	1123.5	1125.6
νC–O	1029.5	1028.7	1081.9	1078.0
δN–H	780.2	779.5	–	–
δC–H	712.3	713.3	–	–
δC=O	690.2	690.5	–	–
Deacetylation degree (%)^a	37.6 ± 2.6	39.4 ± 3.2	ue	ue

Source of the products: MSC was the sacchachitin processed according Su et al. (1997) from G. tsugae.

^aThe method for determination of the degree of deacetylation was following Lin et al. (1992).

b, s: broad and strong; w: weak. ue: unexamined.

Figure 2.  The IR spectra of sacchachitins and sacchachitosans. (A) Our product sacchachitn and MSC (the mimic Su et al., 1997). (B) Our product sacchachitosan and (C) sacchachitosan from MSC. The characteristic absorption reads in (A) (at cm^–1): ν_O–H 3455.7–3450.5, ν_N–H 3500–3300, ν_C–H 2932.5–2932.4, 2300–2400 unassigned, ν_C=O 1736.3–1734.6, amide I δ_N–H 1551.4–1550.4, δ_C–H 1413.5–1324.5, ν_C–C 1154.6–1152.7, ν_C–N 1125.9–1121.7, ν_C–O 1029.5–1028.7, δ_N–H 780.2–779.5, δ_C–H 713.3–712.3, and δ_C=O 690.5-690.2. Similar absorption was seen in (B) and (C), but lacking the bands ν_C=O 1736.3–1734.6 and the amide I bands δ_N–H 1551.4–1550.4.

Table 4.  %T of characteristic IR absorption bands before and after deacetylation.

Vibrating bond	Frequency^a (cm^–1)	Sacchachitin		Sacchachitosan^c
		(This paper)	MSC^b	(This paper)	(MSC)
νO–H	3650–3300	3450.5	3455.7	3445.2	3419.2
%T	–	27.0	36.5	37.0	3.5
νC=O	Amide 1680–1630	1678.5	1678.3	Disappeared	Disappeared
%T	–	37.0	44.0
δN–H	1640–1550	1645.9	1637.3	1645.9	1637.3
%T	–	40.5	47.0	52.5	17.3
δN–H	Amide I 1570–1515	1550.4	1551.4	Disappeared	Disappeared
%T	–	41.0	47.5	–	–

^aCited frequency range.

^bMSC: the mimic sacchachitin (Su et al., 1997).

Saccachitosans were prepared from corresponding sacchachitins according to Lin et al. (1992) in this paper.

Table 5.  Pattern of monosaccharide composition in different fractions obtained from the fruiting bodies of G. lucidum.

Fraction	Mole% in each fraction or in different sacchachitins
Sugar	L5	L6	L7	L8	L9	Sacchachitin (this paper)	MSC
Rhamnose	11.36 ± 0.07^e	11.15 ± 0.07^e	25.71 ± 0.12 ^a	6.41 ± 0.05^b	8.51 ± 0.07^c	4.49 ± 0.02^a	9.42 ± 0.07^d
Fucose	3.58 ± 0.02^f	3.15 ± 0.02^e	3.46 ± 0.02^f	0.14 ± 0.01^a	1.41 ± 0.01^d	0.59 ± 0.01^b	1.06 ± 0.02^c
Ribose	Trace	Trace	Trace	Trace	Trace	1.68 ± 0.02^b	0.86 ± 0.02^a
Arabinose	4.83 ± 0.02^d	5.83 ± 0.02^e	5.57 ± 0.02^e	0.35 ± 0.01^a	3.52 ± 0.02^c	6.12 ± 0.04^f	0.59 ± 0.02^b
Xylose	0.48 ± 0.02^b	3.08 ± 0.05^d	6.15 ± 0.10^f	0.31 ± 0.01^a	3.31 ± 0.06^e	3.31 ± 0.02^e	0.78 ± 0.01^c
Allose	1.58 ± 0.01^b	ud	ud	ud	0.79 ± 0.02^a	7.75 ± 0.11^d	3.32 ± 0.03^c
Talose	33.28 ± 0.12^e	32.68 ± 0.07^d	6.92 ± 0.10^a	6.83 ± 0.10^a	19.78 ± 0.10^b	29.41 ± 0.14^c	33.82 ± 0.13^e
Mannose	1.93 ± 0.02^c	0.88 ± 0.04^b	2.35 ± 0.01^d	0.86 ± 0.04^b	0.47 ± 0.01^a	43.92 ± 0.22^e	47.90 ± 0.27^f
Galactose	2.66 ± 0.04^e	2.64 ± 0.04^e	2.91 ± 0.03^f	0.69 ± 0.01^a	1.45 ± 0.02^d	0.71 ± 0.01^b	0.77 ± 0.01^c
Glucose	31.50 ± 0.23^c	35.38 ± 0.13^d	46.44 ± 0.28^e	73.48 ± 0.14^g	52.39 ± 0.13^f	1.87 ± 0.12^b	0.69 ± 0.11^a
myo-Inositol	8.60 ± 0.02^d	5.03 ± 0.01^c	ud	10.58 ± 0.12^f	9.30 ± 0.14^e	0.15 ± 0.01^a	0.70 ± 0.01^b

L5: ethanol precipitate from hot water extract; L6: the isoelectric point precipitate from 2% NaOH extract; L7: the three-fold volume ethanol precipitate from 2% NaOH extract; L8: the isoelectric point precipitate from 10% KOH extract; L9: the three-fold volume ethanol precipitate from 10% KOH extract; L10: sacchachitin (this paper). ud: undetected. MSC: sacchachitin mimic Su et al (1997) from G. lucidum.

Figure 3.  The scavenging capability of different polysaccharide fractions of G. lucidum on DPPH radicals (A), the superoxide anions (B), and the hydroxyl free radicals (C). Values are expressed in means ± SD (n = 3). Fractions L5–L10 are indicated in the flow chart in Figure 1.

Figure 4.  Scavenging capability of the two different sacchachitins isolated by different processes from the fruiting bodies of G. lucidum. For the DPPH radicals (A); for the superoxide anions (B); and for the hydroxyl free radicals (C). Values are expressed in means ± SD (n = 3).

Table 6.  Antioxidative IC₅₀ values of different polysaccharide fractions obtained from the fruiting bodies of G. lucidum.

Fraction/sacchachitin	IC50 for DPPH• (mg/mL)	IC50 for •OH (mg/mL)	IC50 for •O2^− (mg/mL)
L5	ud	ud	0.73 ± 0.02
L6	1.99 ± 0.01	ud	0.08 ± 0.01
L7	5.32 ± 0.02	0.50 ± 0.01	0.07 ± 0.01
L8	ud	0.90	0.35 ± 0.02
L9	6.54 ± 0.13	5.14 ± 0.02	0.09 ± 0.01
Sacchachitin (this paper)	ud	4.50 ± 0.04	1.00 ± 0.02
MSC	ud	8.50 ± 0.03	3.30 ± 0.02

L5: Ethanol precipitate of hot water extract; L6: the isoelectric point precipitate from 2% NaOH extract; L7: the three-fold volume ethanol precipitate from 2% NaOH extract; L8: the isoelectric point precipitate from 10% KOH extract; L9: the three-fold volume ethanol precipitate from 10% KOH extract; L10: sacchachitin (this paper). ud: undetected. MSC: sacchachitin mimic Su et al. (1997) from G. lucidum.

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