Solid-State 13 C CP MAS NMR Spectroscopy as a Tool for Detection of ( 1 → 3 , 1 → 6 )-β-D-Glucan in Products Prepared from Pleurotus ostreatus

The CCP/MASNMR solid-state NMR technique was used to detect the presence of β-D-glucan and trace compounds in samples prepared fromdried, naturally grownoystermushroom (Pleurotus ostreatus) and commercially available products of dried, specially cultivated oystermushroomandβ-D-glucan isolated from thismushroom.TheNMR spectra of all samples displayed signals typical for (1→ 3, 1→ 6)-β-D-glucan; however, signals which could be assigned to other trace compounds—(1→ 3)-α-glucan, chitin, and proteins—were also observed in the spectra.The amount of trace compounds was negligible in the commercially available products.

NMR spectroscopy is considered to be a unique method for the study of structure and molecular dynamics in solids.Modern solid-state NMR techniques such as magic angle spinning (MAS) and cross-polarisation (CP) enable conformation study of solid materials [21].According to our knowledge, only a few papers [8,10,12,16,17] deal with NMR study of -D-glucans in the solid state, despite the fact that solid-state NMR spectroscopy is a nondestructive technique that preserves the 3D structure of the analyzed polymer [16,21].Measured 13 C CP/MAS NMR spectra of powdered (1 → 3, 1 → 6)--D-glucan isolated from Pleurotus ostreatus (pleuran) are consistent with those published in [8,11].The presence of compounds in the sample of (1 → 3, 1 → 6)--D-glucan can be recognized by resonance signals outside the range of 50-110 ppm.As signals not coming from -D-glucan are present in the measured 13 C CP/MAS NMR spectrum, another two samples of Pleurotus ostreatusa dietary supplement sold in pharmacies and a natural sample-were also studied.Hydration of polysaccharides gives rise to conformational stabilization which is reflected in spectra by narrowing and splitting of resonance lines [12,17,22,23].Hence, 13 C CP/MAS NMR experiments were also performed on a hydrated sample of (1 → 3, 1 → 6)--D-glucan isolated from Pleurotus ostreatus.

Materials and Methods
The sample S1 of (1 → 3, 1 → 6)--D-glucan isolated from Pleurotus ostreatus and the sample S2 powdered Pleurotus ostreatus are commercial products sold in pharmacies as dietary supplements.The cultivation of Pleurotus ostreatus for their production is specially controlled.To achieve better resolution of the NMR spectrum the sample S1 was hydrated for a week placed on a glass dish in a desiccator with water.The sample S3 was prepared from dried and ground natural edible mushroom Pleurotus ostreatus.
The solid-state 13 C CP/MAS NMR measurements were carried out on the 400 MHz Varian solid-state NMR spectrometer in the Centre for Solid-State NMR Spectroscopy of the Slovak National NMR Centre at the Technical University of Košice.The high-resolution 13 C CP/MAS NMR spectra were recorded at the resonance frequency of approximately 100 MHz with the use of 4 mm rotors and MAS frequency of 12000 Hz and /2 pulse duration of 1.9 s.In the CP experiments the Hartmann-Hahn condition was achieved with the radio frequency field strength of 58 kHz, a contact time of 1 ms, and relaxation delay of 4 s between two consecutive scans.A high-power proton-decoupling field of 92 kHz was applied during data acquisition.The spectra were obtained at room temperature averaging over 5000-33000 scans.
The chemical shifts were referenced to the TMS using adamantane as an external standard.

Conclusions
Powder samples of oyster mushrooms and of (1 → 3, 1 → 6)--D-glucan isolated from Pleurotus ostreatus were studied using 13 C CP/MAS NMR.The NMR spectrum of (1 → 3, 1 → 6)--D-glucan revealed the occurrence of some other compounds in the sample; the asymmetry of the C1 peak can be caused by the presence of (1 → 3)--glucan.Resonance lines of chitin and other compounds containing carbonyl and aliphatic carbons remaining after isolation were also observed in the spectrum.Traces of some proteins appeared in the sample of Pleurotus ostreatus specially cultivated for the production of food supplements.The sample of natural Pleurotus ostreatus contained some other compounds in addition to those present in the cultivated sample.Lower content of (1 → 3, 1 → 6)--D-glucan in the naturally grown mushroom than in the cultivated one was inferred from the shape of the 13 C CP/MAS NMR spectra.
13 C CP/MAS NMR spectrum of natural Pleurotus ostreatus (sample S3) (Figure3(b)) has similar features to that of sample S2 (Figure 3(a)), except that the signals outside the range of 45-110 ppm, in which signals of -(1 → 3, 1 → 6)-D-glucan are observed, and the signal near 93 ppm are enhanced.Additional minor broad peaks with chemical shifts near 158, 119, and 40 ppm also appear in the spectrum of sample S3, revealing the presence of other compounds not mentioned previously.This means that in the sample of natural Pleurotus ostreatus the content of -(1 → 3, 1 → 6)-Dglucan is lower than that in the sample of Pleurotus ostreatus specially cultivated for the production of food supplements.
are observed.