2014 Seminar, Graduate Institute of Food Science and Biotechnology,
National Chung Hsing University
Title: Characterisation and immunomodulating activities of exo-polysaccharides from submerged
cultivation of Hypsizigus marmoreus
Journal: Food Chemistry
5-Year Impact Factor: 3.867
Date: 2014/12/12
Speaker: Lan Min Lin 林蘭閔 (NO.3) Moderator: 張原華 Advisor: Dr.林美吟
Introduction
Exo-polysaccharides were purified from the fermentation broth of Hypsizigus marmoreus. The
fractions B-I-I and B-II-I have potent complement fixating activity, however, B-N-I, B-I-I, B-II-I and B-II-II
exhibited significant macrophage stimulating activity. Molecular weights of the four
exo-polysaccharides were determined to be 6.3, 120,150 and 11 kDa respectively. Molecular
characterisation showed that B-N-I is basically an α-1→4 glucan; B-I-I is a heavily branched a-mannan
with 1→2. B-II-I and B-II-II, have a backbone of rhamno-galacturonan with 1→2. Structure–activity
relationship analysis indicated that monosaccharide compositions, molecular weight, certain
structural units are the principal factors. Their immunomodulating activities may explain the health
benefits of the mushroom.
Materials and Methods
2.1. Fungi strains and submerged cultivation
The fungus was cultivated on PDA medium. → 250 ml Erlenmeyer flasks →
The fermentation in 500 ml flasks → Centrifugation →Collecting supernatant
2.2. Isolation and purification of the exo-polysaccharides
Isolation: anion exchange Chromatography &Purification: gel filtration
2.3. Complement fixating activity
Based on inhibition of haemolysis in antibody sensitised sheep red blood cells (SRBC).
2.4. Measurement of nitric oxide production Cell line: Raw 264.7
2.5. Determination of the monosaccharide composition Analysed by GC
2.7. Dtermination of the linkages of the polysaccharides Analysed by GC/MS
2.8. NMR analysis of the polysaccharides
NMR experiments were performed at 600.13 MHz (1H) and 150.90 MHz (13C) on a Bruker AV600 and a
Bruker AVII600 NMR spectrometer.10 mg of the sample was dissolved in D2O (99.9%) to a total
volume of 0.5 ml in 5 mm NMR tubes.
Results and Discussion
3.1. Purification and molecular weight determination
The neutral B-N-I was purified from B-N. B-I-I, B-I-II and B-I-III were obtained from the acidic fraction
B-I, while B-II-I and B-II-II, were separated from B-II.
3.2. Complement fixating activity
B-I-I and B-II-I (the high molecular weight) exhibited the strongest complement fixating activities with
ICH50 values of 5.8 and 3.2μg/ml respectively.
3.3. Measurement of nitric oxide (NO) production from macrophages
The B-N-I, B-I-I, B-II-I and B-II-II, showed statistically significant stimulating effects on NO release from
macrophages, at a concentration of 100 μg/ml.
3.4. Carbohydrate compositions of the polysaccharides
3.5.Linkage compositions of the exo-polysaccarides (Table 2)
3.6.NMR spectroscopy (Fig 3)
Conclusion (Four exo-polysaccharides)
The results indicated that B-N-I, an α-1→4 glucan; B-I-I, a heavily branched a-mannan; B-II-I and
B-II-II, containing a backbone of rhamnogalacturonan. Their potent immunomodulating activities
indicate the great pharmaceutical potential of the exo-polysaccharides.
References
1 Zhang, B. Z., Yan, P. S., Chen, H., & He, J. (2012). Optimization of production conditions for
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2 Inngjerdingen, K. T., Coulibaly, A., Diallo, D., Michaelsen, T. E., & Paulsen, B. S. (2006).A complement
fixing polysaccharides from Biophytum pertersianum Klotzsch, a medicinal plant from Mali, West
Africa. Biomacromolecules, 7, 48–53.