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Effect of processing on the content and biological activity of polysaccharides from Pleurotus ostreatus mushroom
LWT - Food Science and Technology 66 (2016) 27e33
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Effect of processing on the content and biological activity of polysaccharides from Pleurotus ostreatus mushroom Wojciech Radzki a, *, Marta Ziaja-Sołtys b, Jakub Nowak c, Jolanta Rzymowska b, ska a, Monika Michalak-Majewska a, Jolanta Topolska b, Aneta Sławin a Marta Zalewska-Korona , Andrzej Kuczumow d a
Department of Fruits, Vegetables and Mushrooms Technology, University of Life Sciences in Lublin, 20-704, Lublin, Poland Department of Biology and Genetics, Medical University of Lublin, 20-093, Lublin, Poland Department of Chemistry, Lublin Catholic University, 20-718, Lublin, Poland d Institute of Environmental Engineering, Lublin Catholic University, 37-450, Stalowa Wola, Poland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 February 2015 Received in revised form 19 August 2015 Accepted 5 October 2015 Available online 8 October 2015
Water soluble polysaccharides (WSP) were isolated from processed and non-processed fruiting bodies of oyster mushroom (Pleurotus ostreatus). The processing methods involved: blanching, boiling and blanching followed by fermenting with a strain of lactic acid bacteria (Lactobacillus plantarum). The yields of WSP ranged from 78.7 ± 1.5 mg/g to 120.1 ± 4.9 mg/g dry weight of sample. Blanching did not affect the content of WSP. Boiling for 15 min, led to the substantial decrease in the amount of WSP (34.7% decline). The isolated samples differed in carbohydrate, protein and phenolics content. FTIR spectroscopy of the WSP samples confirmed the presence of both a- and b-glycosidic linkages. Gel permeation chromatography showed the presence of compounds having the molecular weight of 198.3, 11.9, 3.1 kDa. The samples possessed antioxidant capacity measured by ABTS method (14.14 ± 0.63 to 29.48 ± 1.12 mmoles of Trolox per 1 g dw) and FRAP assay (2.49 ± 0.54 to 16.52 ± 0.55 mmoles of Trolox equivalents per 1 g dw). The antioxidant potential was decreased by the processing. Similarly, antiproliferative activity of WSP towards human breast cell lines (MCF-7 and T-47D) was lower due to the processing. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Oyster mushroom Polysaccharides Antioxidant Antiproliferative
1. Introduction Mushrooms are abundant source of polysaccharides which are the part of their cell walls. They differ greatly in molecular weight, structure, conformation and physical properties (Wasser, 2002). Additionally, some of them are reported to exert beneficial effect on health and can be used in the treatment of some diseases. Biological activity of mushroom polysaccharides is mainly related to their immunomodulating and anticancer properties. Moreover, they are also known to exhibit antiviral effect, lower blood lipids or possess antioxidant and antiproliferative activity (Roupas, Keogh, Noakes, Margetts, & Taylor, 2010; Stachowiak & Reguła, 2012). Pleurotus ostreatus (known as oyster mushroom) is a popular, widely cultivated edible fungus, known for its hypocholesteroloemic
* Corresponding author. E-mail address: [email protected] (W. Radzki). http://dx.doi.org/10.1016/j.lwt.2015.10.016 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
properties. A few different polysaccharides were isolated from fruiting bodies of this species, including pleuran, a high molecular weight b-(1 / 3) (1 / 6)-glucan. These polysaccharides are reported to demonstrate immunomodulating, antioxidant, antiproliferative or prebiotic activity. Number of studies revealed that they vary in their chemical structure in terms of molecular weight, glycosidic bond conformation, branching, tertiary conformation or sugar composition. Moreover, the chemical structure of mushroom polysaccharides affect their biological activity (Zhang, Cui, Cheung, & Wang, 2007). High molecular weight (2200e2900 kDa), b-(1 / 3) (1 / 6)-glucan possessing prebiotic activity was obtained by , Jablonský, Slukova , and Copíkov (2008). Other Synytsya, Mí ckova a authors isolated proteoglycans of lower molecular weight (1e31 kDa) which were capable of inhibiting proliferation of various cancer cell lines (Lavi, Friesem, Geresh, Hadar, & Schwartz, 2006; Martin & Brophy, 2010; Tong et al., 2009). Antiproliferative effect results from the induction of apoptosis of cancer cells. Antioxidant activity of P. ostreatus polysaccharides was demonstrated by
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proteoglycans described by other researchers (Sun & Liu, 2009; Xia, Fan, Zhu, & Tong, 2011). Mushrooms are rarely eaten raw and they require to be processed before the consumption. Most often thermal or hydrothermal treatments are applied. It is well known that processing of food may cause substantial changes in its chemical composition and thus affect nutritional and health properties. However, few studies describe the effect of processing on mushrooms-derived biologically active polysaccharides. Fan, Li, Deng, and Ai (2012) investigated how different drying techniques affect antioxidant activity of polysaccharides that were isolated from the medicinal mushroom Ganoderma lucidum. Thetsrimuang, Khammuang, Chiablaem, Srisomsap, and Sarnthima (2011) compared antioxidant and antiproliferative activities of polysaccharides obtained from fresh and dried Lentinus polychrous fungus. So far, however, no studies focused on the influence of hydrothermal processing on the biologically active mushrooms-derived polysaccharides. Therefore, the present paper aims to verify the impact of some processing methods on the content, chemical composition, antioxidant and antiproliferative activity of water soluble polysaccharides (WSP) obtained from P. ostreatus fruiting bodies. The applied processing used in this work included boiling, blanching and fermenting with lactic acid bacteria (Lactobacillus plantarum). Lactic acid fermentation is a process which allows to develop food probiotic products with improved nutritional quality (Beena Divya, Kulangara Varsha, Madhavan Nampoothiri, Ismail, & Pandey, 2012). 2. Materials and methods 2.1. Mushroom samples Fresh fruiting bodies of P. ostreatus were purchased directly from a producer (in 2013) and belonged to the same crop. After a harvest they were kept at 5 C and were subjected to processing within 5 h. 2.2. Processing of mushrooms The mushrooms were divided into four groups (500 g per group) and were further submitted to processing technologies: blanching in water containing 0.5% (w/v) citric acid (5 min, 95 C); boiling in water (15 min, 100 C); blanching in water (as reported above) and fermenting with lactic acid bacteria (see below). The fourth group was not processed (control). The fermented mushrooms were prepared as follows. Fruiting bodies after blanching were washed with cool water and put tightly into screw-capped plastic vessels (PET). The brine solution was added which consisted of sucrose (10 g/1 kg of blanched mushrooms) and 3% (w/v) NaCl. The mixture was then inoculated with 5 mL of bacterial suspension (106 cfu/mL). The mushrooms were fermented for 10 days at 21e22 C and then stored 20 days at 5 C. The final pH of the product was 3.7. All the samples were then subjected to freeze-drying with an Alpha 1-2LD plus freeze dryer (Christ, Germany) and ground to fine powder. 2.3. Extraction of water soluble polysaccharides Four grams of powdered mushrooms were suspended in 200 mL of 80% ethanol and extracted in a rotary shaker at 80 C for 60 min. The ethanolic extract containing low molecular weight compounds was removed by centrifugation (3755 g, 20 min) and the solid residue was washed twice with 80% ethanol and centrifuged. The alcohol insoluble fraction was then re-suspended in deionised water (ratio 1:50 w/v) and autoclaved at 115 C for 180 min. The obtained slurry was cooled and centrifuged (3755 g, 20 min) and
the supernatant was concentrated with a rotary evaporator and precipitated with three volumes of 2-propanol (24 h, at 5 C). The precipitate was then centrifuged, washed twice with 80% methanol, re-dissolved in hot deionised water, lyophilised and weighed. The extraction process was done in triplicate. 2.4. Chemical characteristics of polysaccharides 2.4.1. Determination of total carbohydrate, protein and phenolics content The content of carbohydrates in water soluble polysaccharides was measured with phenol-sulphuric acid method, using glucose as a standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The amount of protein was determined according to the method developed by Bradford (1976), using bovine serum albumin as a standard. Total phenolics content was measured according to the method of Singleton and Rossi (1965) with gallic acid used as a standard. 2.4.2. FTIR spectroscopy FTIR spectra of lyophilised water soluble polysaccharides were recorded on Nicolet NXR 9650 spectrometer (Thermo, USA). The data was collected in the range of 4000e600 cm1 and ATR technique was applied. 2.4.3. Gel permeation chromatography The molecular weight of the isolated polysaccharides was determined with gel permeation chromatography, according to the modified method described by other authors (Malinowska, Krzyczkowski, Łapienis, & Herold, 2009). The samples were dissolved in aqueous solution of NaN3 (0.1%, w/v) and were applied to three TSK-GEL columns: G5000PWXL, G3000PWXL and G2500PWXL (7.8 300 mm, Tosoh, Japan). The chromatographic system was equipped with K-501 pump (Knauer, Germany) and Refracto Monitor IV refractive index detector (LDC Analytical, USA). The flow of the mobile phase (0.1% (w/v) NaN3) was set at 1 mL/ min. Pullulans of different molecular weight were used to construct a standard curve. 2.5. Antioxidant activity 2.5.1. ABTS radical scavenging activity The assay was done according to the method described by Re et al. (1999). ABTS reagent was prepared by incubating 7 mM ABTS solution with 2.45 mM potassium persulfate solution for 16 h at room temperature. The ABTSþ solution was then brought to an absorbance of 0.7 (at 734 nm). The samples (25 mL, 1 mg/mL) were mixed with 975 mL of ABTSþ solution and left to stand for 15 min at room temperature. The absorbance was measured at 734 nm against a blank sample. The calibration curve was done with different concentrations of Trolox (20e200 mM) and the results were expressed as micromoles of Trolox equivalent (TE) per 1 g of mushroom dry weight. 2.5.2. Ferric reducing antioxidant power (FRAP) The ability of polysaccharides to reduce ferric ions was analysed according to the method described by Benzie and Strain (1996). Fresh FRAP reagent was prepared daily by mixing 300 mM acetate buffer (pH 3.6) with 10 mM 2,4,6-tripyridyl-striazine (TPTZ) solution in 40 mM HCl and 20 mM FeCl3$6H2O (10:1:1 ratio). The reagent was incubated at 37 C for 90 min before an analysis. Aliquots of the samples (100 mL) were mixed with FRAP reagent (final volume, 2 mL) and were then incubated at 37 C for 90 min. The change in the absorbance was measured at 593 nm. Trolox aqueous solutions (20e200 mM) were used to construct the calibration curve
W. Radzki et al. / LWT - Food Science and Technology 66 (2016) 27e33
and the results were reported as micromoles of Trolox equivalent (TE) per 1 g of mushroom dry weight.
2.7. Statistical analysis All measurements were carried out in triplicate and the obtained data was expressed as mean ± SD (standard deviation). The data was evaluated using unidirectional analysis of variance ANOVA with a level of significance set at a ¼ 0.05. Statistically different data was compared by Fisher's least significant difference (LSD) test. 3. Results and discussion 3.1. Water soluble polysaccharides content The amount of WSP obtained from non-processed P. ostreatus was 78.7 ± 1.5 mg/g dw, as shown in Fig. 1a. The processing of the fruiting bodies caused marked changes in the content of WSP. Blanching resulted in the increase of WSP (120.1 ± 4.9 mg/g dw). This could be explained by the fact that blanching leads to an extraction of soluble low molecular weight compounds and thus increasing the percentage of the fraction containing high molecular weight compounds. The similar effect was observed in blanched potatoes in which the content of pectin, cellulose and hemicellulose was increased (Gołubowska, 2005). Furthermore, Zivanovic and Buescher (2004) reported that blanching of Agaricus bisporus fruiting bodies caused the increase of total polysaccharides and protein content. Fruiting bodies that were cooked for 15 min also contained higher amount of WSP (100.4 ± 4.7 mg/g dw), comparing to control group. This fact could be still attributed to the change of the proportion of easily soluble substances to difficultly soluble compounds. This has been noted before by Dikeman, Bauer, Flickinger, and Fahey (2005) who observed substantial
Amount of cW SP [mg/g dw fruiting bodies]
Antiproliferative activity was tested on MCF-7 human breast adenocarcinoma cell line (ATCC HTB-22) and T-47D human ductal breast (epithelial tumour) carcinoma cell line (ECACC Catalog, No. 85102201). MCF-7 cell line was grown in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma), 50 mg/mL bovine insulin (Sigma), 100 U/mL penicillin (Polfa, Poland) and 100 mg/mL streptomycin, (Polfa, Poland). The complete growth medium for T47D was RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma) and antibiotics (100 U/mL penicillin (Polfa, Poland)), 100 mg/mL streptomycin, (Polfa, Poland). Both cell lines were grown at 37 C in humidified atmosphere with 5% CO2 (standard conditions). Antiproliferative activity of the tested polysaccharides was measured with MTT assay developed by Mosmann (1983). T47D and MCF-7 cells were seeded on 96-well plates at concentrations of 3 105 cells/mL and 2 105 cells/mL, respectively. After 24 h incubation at standard conditions (37 C, 5% CO2 flow, 95% air humidity), when cells in each well reached about 75%e80% confluence, the growth medium was replaced with dilutions of the tested polysaccharides dissolved in RPMI medium containing 2% FBS (at the concentrations ranging from 25 mg/mL to 250 mg/mL). Cells were incubated with polysaccharides for 24 h, followed by addition of 25 mL of MTT (Sigma) solution (5 mg/mL in PBS) per well. After 3 h incubation at 37 C formazan crystals were solubilised by adding 100 mL of lysis buffer (10% SDS in 0.01 M HCl) per well. Plates were incubated overnight at standard conditions. The absorbance was read at 540 nm with a plate reader (680XR, BioRad), and the mean value for each concentrations was calculated. The percentage of viable cells were calculated from the absorbance.
a
d
120 c
100
b a
80
60
40
20
0 8
b
Control
Blanched
c
c
Boiled Blanched and fermented
b Amount of cW SP [mg/g fw fruiting bodies]
2.6. Antiproliferative activity
140
29
6 a
4
2
0 Control
Blanched
Boiled Blanched and fermented
Fig. 1. The content of polysaccharides isolated from P. ostreatus fruiting bodies calculated on dry weight basis (A) and the fresh weight basis (B). Error bars indicate mean ± standard deviation (n ¼ 3). Different letters indicate significant differences among the treatments according to LSD test (p < 0.05).
concentration of the fibre in various mushroom species, following the cooking process. The level of WSP in blanched and fermented samples (88.9 mg/g dw) was higher compared to non-processed fruiting bodies, but lower than in fruiting bodies that were solely blanched or cooked. In order to investigate whether or not the applied processing techniques had a negative impact on the content of WSP, the results were calculated on the fresh weight basis, taking into consideration the loss of the mass (Fig. 1b). As can be seen from the graph, blanching did not affect negatively the content of WSP. However, boiling for 15 min, led to the substantial decrease in the amount of WSP (34.7% decline, comparing to the control). The observed loss could be attributed to the water extraction of the polysaccharides during the cooking procedure. With respect to the blanched and fermented mushrooms, the observed decrease was lower (14.7%). Because blanching alone did not influence the content of WSP it is possible that the presence of bacteria could be responsible for the loss. Additionally, two weeks storage could have caused WSP to diffuse into brine.
3.2. Chemical characteristics of water soluble polysaccharides 3.2.1. Carbohydrate, protein and phenolics content The amount of total carbohydrates, protein and phenolics is
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Table 1 The amount of total carbohydrate, proteins and polyphenolics in the isolated water soluble polysaccharides of P. ostreatus. Mean values ± standard deviation (n ¼ 3) with different letters indicate significant differences among the treatments according to LSD test (p < 0.05). Treatment
Total carbohydrate (% dw)
Control Blanched Boiled Blanched and fermented
58.0 76.3 71.3 89.2
± ± ± ±
3.9a 5.6b 2.9b 3.5c
Protein content (% dw) 7.8 3.9 7.1 3.0
± ± ± ±
0.5c 0.2b 0.5c 0.2a
Total phenolics content (% dw) 0.77 0.41 0.53 0.29
± ± ± ±
0.04d 0.03b 0.06c 0.06a
presented in Table 1. The total carbohydrate content in the WSP extracted from non-processed fruiting bodies was 58.0 ± 3.9% dw. This value was similar to that reported by other authors (62.8 ± 7.7%), who isolated polysaccharides from P. ostreatus using similar method (Mitra, Khatua, & Acharya, 2013). The extracts obtained from blanched, boiled, blanched and fermented mushrooms contained higher level of carbohydrates (76.3 ± 5.6%, 71.3 ± 2.9, 89.2 ± 3.5%, respectively). Regarding to the protein content, its quantity in the control samples reached 7.8 ± 0.5% dw. This amount was much higher comparing to those reported by Zhang, Dai, Kong, and Chen (2012) who found that the protein content ranged from 0.84% to 1.23%, depending on the method of isolation. This huge discrepancy could be attributed to the use of DAE resin by these researchers during the isolation procedure. On the other hand, other studies demonstrated much higher protein content in polysaccharides isolated from P. ostreatus ranging from 15% (Mitra et al., 2013) to 24% (Xia et al., 2011). The processing of mushrooms caused significant change in the protein content of WSP. In the samples isolated from blanched mushrooms its amount decreased approximately two times, relative to the control and reached 3.9 ± 0.2%. Boiling resulted in rather negligible loss of protein level (7.1 ± 0.5%) in relation to control samples. The greatest decline in the protein content was observed in WSP extracted from blanched and fermented mushrooms where its value reached 3.0 ± 0.2%. It could be
possible that the lowest amount of protein in WSP obtained from blanched and fermented mushrooms could result from the presence of bacteria which released enzymes which caused degradation of protein (Dallagnol, Pescuma, DeValdez, & Roll an, 2013). With regard to the phenolics, they were found in the smallest amount (below 1% dw) comparing to other WSP constituents. Total phenolics content of polysaccharides isolated from non-processed fruiting bodies was 0.77 ± 0.04% dw. This data does not support the findings by Vamanu (2012) in which polyphenolics quantity was approximately ten times higher. This huge difference probably resulted from slightly different extraction procedure because the authors have omitted the stage of alcoholic extraction. Besides, the polysaccharides were obtained from mycelium, not from fruiting bodies. The fact that mushroom polysaccharides contain polyphenolic compounds is well established (Klaus et al., 2011). Polyphenolics tend to bind with polysaccharides with hydrogen bounds, hydrophobic interactions or even covalent bounds (Renard, Baron, Guyot, & Drilleau, 2001). Bound polyphenolics contribute to antioxidant activity of polysaccharides and their presence is highly beneficial. They are slowly released from the matrix by intestine microbes and are absorbed by an organism (Saura-Calixto, 2011). The processing of fruiting bodies led to substantial changes in the phenolics content and a similar trend to protein was observed. The rapid, 46% decline was noticed as the result of blanching and 31% in the case of cooking. A possible explanation for this is that hydrothermal processes may lead to solubilisation and releasing of phenolics ndez, Arte s-Herna ndez, Go mez, & Arte s, 2013). (Martínez-Herna Another possible explanation for this effect is thermal degradation rez, Carballo, & Franco, due to the elevated temperature (Martínez, Pe ska, Filipiak-Florkiewicz, & Pisulewski, 2013; Sikora, Cieslik, Leszczyn 2008). Lactic acid fermentation caused the highest (63%) decrease in phenolics content. This could be explained by diffusion of soluble phenolics into brine. This process could possibly have been enhanced ska, 2005). by bacterial enzymes (Ciska, Karamac, & Kosin
3.2.2. FTIR spectroscopy The FTIR spectra of isolated WSP are depicted in Fig. 2. They are
Fig. 2. FTIR spectra of water soluble polysaccharides obtained from non-processed (control) and processed fruiting bodies of P. ostreatus.
W. Radzki et al. / LWT - Food Science and Technology 66 (2016) 27e33
rrez, generally similar to that obtained by other authors (Gutie Prieto, & Martínez, 1996) and they show bands characteristic of the presence of polysaccharides and protein molecules. The broad peak in the region of 3000e3500 cm1 could be attributed to OeH and NeH stretching vibrations, while the band at ~2930 cm1 is the result of CeH stretching. The absorption at ~1645 cm1 (amide I) is indicative of C]O stretching mode, whereas the signal at 1530 cm1 corresponds to the presence of bending vibrations of NeH groups. These two bands indicate the presence of protein. In the region of 1450e1300 cm1 there are signals related to bending vibrations of CH2, OeH and CeOeH (Moha cek-Grosev, Bo zac, & Puppels, 2001). Intense peaks observed at 950e1190 cm1 result from stretching vibrations of CeOeC, COH and CeC (Larkin, 2011). Analysis of 700e950 cm1 region can give information on the type of glycosidic links present in the sample. Signal at ~895 cm1 may suggest the presence of b-glucans (Moha cek-Grosev et al., 2001), while bands at ~930 cm1, ~850 cm1, ~760 cm1 are indicative of a-glucans (Novak et al., 2012; Wiater et al., 2011).
3.3. Antioxidant capacity Water soluble polysaccharides isolated from both processed and non-processed fruiting bodies displayed ABTS scavenging activity ranging from 14.14 ± 0.63 to 29.48 ± 1.12 mmoles of Trolox per 1 g dw (Fig. 4). The control sample showed the highest value (29.48 ± 1.12 mmoles of Trolox per 1 g dw), which was rapidly decreased due to the processing. As the result of blanching approximately 26% decline was noticed, whereas boiling led to approximately 12% decrease. The highest drop in activity (approximately 52%) demonstrated the samples that were obtained from the blanched and fermented mushrooms. Our data are in qualitative agreement with other studies that investigated antioxidant capacity of fermented A. bisporus and P. ostreatus fruiting bodies and reported substantial decline in ABTS radical scavenging activity (approximately 60% and 90%, respectively) (Ska˛ pska et al., 2008). With respect to the FRAP assay, the activity values were lower comparing with ABTS assay and varied from 2.49 ± 0.54 to 16.52 ± 0.55 mmoles of Trolox equivalents per 1 g dw (Fig. 4). However, the similar trend was observed in terms of the decrease of the antioxidant activity. The highest value was observed in the control samples (16.52 ± 0.55 mmoles of Trolox equivalents per 1 g dw) and the processing methods had a negative impact on the
Control
10 5 0 Blanched
5
Refractive index [mV]
3.2.3. Gel permeation chromatography Gel permeation chromatography was performed to identify molecular weight of main WSP components. All the spectra showed sharp, symmetrical peak at 18.1e19.1 mL. It corresponds to a compound having molecular weight of 198.3 kDa (Fig. 3). The chromatogram of the extracted WSP from the control samples possessed also intense signals at 22.4 mL and 24.68 mL which can be attributed to the compounds of the lower molecular mass (11.9 and 3.1 kDa, respectively). These signals were not recorded in the samples obtained from the processed mushrooms. The presence of 198.3 kDa fraction is in agreement with the results of other authors who extracted P. ostreatus polysaccharides (using similar isolation method) which had molecular mass 187 kDa (Maity et al., 2011). Other researchers obtained the fractions of the lower molecular weight: 1e10 kDa (Lavi et al., 2006) and 10.5 kDa (Synytsya et al., 2009). The processing of fruiting bodies, regardless of the method applied, caused 3.1 kDa fraction to disappear. Probably it could be eluted during the applied processes. Additionally, blanching led to diminishing of 11.9 kDa fraction. Further studies should be undertaken to investigate the chemical composition of each fraction and the role they play in exerting antioxidant and antiproliferative activities.
15
31
0 Boiled
5
0 20 15
Blanched and fermented
10 5 0 16 17 18 19 20 21 22 23 24 25 26 Retention volume [mL] Fig. 3. Gel permeation chromatography of water soluble polysaccharides extracted from non-processed (control) and processed fruiting bodies.
activity. Blanching resulted in approximately 60% decrease, while boiling caused approximately 40% decline. The highest decrease (approximately 85%) was noticed for WSP isolated from blanched and fermented fruiting bodies. Previous studies has shown that hydrothermal treatments leads to the decrease of total antioxidant potential of mushrooms (Nguyen, Nagasaka, & Ohshima, 2012; Soler-Rivas, RamírezAnguiano, Reglero, & Santoyo, 2009). The current study found that the applied processing altered antioxidant activity of the obtained
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W. Radzki et al. / LWT - Food Science and Technology 66 (2016) 27e33
lower comparing to MCF-7 line. The lowest cell viability was found in non-processed samples (73.4 ± 2.3%) and similarly to MCF-7 line, the processing led to the statistically relevant decrease in the activity. Current literature does not provide information on the impact of hydrothermal processing on antiproliferative activity of foodderived polysaccharides. However, some studies were conducted on rich in phenolics methanolic extracts. Chatthongpisut, Schwartz, and Yongsawatdigul (2015) showed that cooking procedures leads to substantial decline in antiproliferative activity of purple rice. Also other authors reported that lotus roots and white onions cooked for 10 min possessed lower antiproliferative potential (Im et al., 2012). 4. Conclusions
Fig. 4. Antioxidant capacity of water soluble polysaccharides extracted from nonprocessed (control) and processed mushrooms. Error bars indicate mean ± standard deviation (n ¼ 3). Different letters indicate significant differences among the treatments according to LSD test (p < 0.05).
100 c
90 b
b
80
Cell viability [%]
70
a
c b
a
a
60 50 40 30 20 10
Water soluble polysaccharides were extracted from both processed and non-processed mushrooms, giving different yields. The processing (boiling and blanching followed by fermenting) led to the decrease of polysaccharides content. Therefore, the real intake of these macromolecules can differ between raw and processed form. Moreover, all the processes caused substantial changes in chemical composition of polysaccharides (protein and phenolics contents were decreased). Along with the changes of chemical composition caused by the processing, antioxidant and antiproliferative activities were decreased which may affect nutritional and health values. Therefore, hydrothermal processing, especially long, should not be applied in order to fully retain health promoting properties of P. ostreatus. Further work would be suggested to investigate the impact of different processing techniques like microwave cooking, steaming or frying on bioactive polysaccharides from P. ostreatus. Moreover, since mushrooms polysaccharides are known to exert immunomodulating effect, more research is required to determine the impact of processing on this effect. References
Control Blanched Boiled Blanched and fermented
0 MCF-7
T-47D
Fig. 5. Cell viability (%) of MCF-7 and T-47D human adenocarcinoma cell line after treatment with water soluble polysaccharides (250 mg/mL) obtained from nonprocessed (control) and processed P. ostreatus fruiting bodies. Error bars indicate mean ± standard deviation (n ¼ 3). Different letters indicate significant differences among the treatments according to LSD test (p < 0.05).
polysaccharides. As a consequence, this may affect their healthpromoting properties.
3.4. Antiproliferative activity The extracted water soluble polysaccharides were analysed in terms of their antiproliferative activity against MCF-7 and T-47D breast cancer cell lines. The results showed that WSP displayed slight inhibitory action on both cells lines (Fig. 5). The effect was most evident at the concentrations of 250 mg/mL. The strongest activity was displayed by the samples derived from non-processed fruiting bodies (cell viability reached 68.8 ± 1.7%). As the result of processing, the drop of the activity was noticed and the cell viability amounted to 77.9 ± 3.0%, 72.1 ± 4.1% and 80.0 ± 2.8% for blanched, boiled, blanched and fermented samples, respectively. With regard to the T-47D cell line, the antiproliferative activity was generally
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Effect of processing on the content and biological activity of polysaccharides from Pleurotus ostreatus mushroom Wojciech Radzki a, *, Marta Ziaja-Sołtys b, Jakub Nowak c, Jolanta Rzymowska b, ska a, Monika Michalak-Majewska a, Jolanta Topolska b, Aneta Sławin a Marta Zalewska-Korona , Andrzej Kuczumow d a
Department of Fruits, Vegetables and Mushrooms Technology, University of Life Sciences in Lublin, 20-704, Lublin, Poland Department of Biology and Genetics, Medical University of Lublin, 20-093, Lublin, Poland Department of Chemistry, Lublin Catholic University, 20-718, Lublin, Poland d Institute of Environmental Engineering, Lublin Catholic University, 37-450, Stalowa Wola, Poland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 February 2015 Received in revised form 19 August 2015 Accepted 5 October 2015 Available online 8 October 2015
Water soluble polysaccharides (WSP) were isolated from processed and non-processed fruiting bodies of oyster mushroom (Pleurotus ostreatus). The processing methods involved: blanching, boiling and blanching followed by fermenting with a strain of lactic acid bacteria (Lactobacillus plantarum). The yields of WSP ranged from 78.7 ± 1.5 mg/g to 120.1 ± 4.9 mg/g dry weight of sample. Blanching did not affect the content of WSP. Boiling for 15 min, led to the substantial decrease in the amount of WSP (34.7% decline). The isolated samples differed in carbohydrate, protein and phenolics content. FTIR spectroscopy of the WSP samples confirmed the presence of both a- and b-glycosidic linkages. Gel permeation chromatography showed the presence of compounds having the molecular weight of 198.3, 11.9, 3.1 kDa. The samples possessed antioxidant capacity measured by ABTS method (14.14 ± 0.63 to 29.48 ± 1.12 mmoles of Trolox per 1 g dw) and FRAP assay (2.49 ± 0.54 to 16.52 ± 0.55 mmoles of Trolox equivalents per 1 g dw). The antioxidant potential was decreased by the processing. Similarly, antiproliferative activity of WSP towards human breast cell lines (MCF-7 and T-47D) was lower due to the processing. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Oyster mushroom Polysaccharides Antioxidant Antiproliferative
1. Introduction Mushrooms are abundant source of polysaccharides which are the part of their cell walls. They differ greatly in molecular weight, structure, conformation and physical properties (Wasser, 2002). Additionally, some of them are reported to exert beneficial effect on health and can be used in the treatment of some diseases. Biological activity of mushroom polysaccharides is mainly related to their immunomodulating and anticancer properties. Moreover, they are also known to exhibit antiviral effect, lower blood lipids or possess antioxidant and antiproliferative activity (Roupas, Keogh, Noakes, Margetts, & Taylor, 2010; Stachowiak & Reguła, 2012). Pleurotus ostreatus (known as oyster mushroom) is a popular, widely cultivated edible fungus, known for its hypocholesteroloemic
* Corresponding author. E-mail address: [email protected] (W. Radzki). http://dx.doi.org/10.1016/j.lwt.2015.10.016 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
properties. A few different polysaccharides were isolated from fruiting bodies of this species, including pleuran, a high molecular weight b-(1 / 3) (1 / 6)-glucan. These polysaccharides are reported to demonstrate immunomodulating, antioxidant, antiproliferative or prebiotic activity. Number of studies revealed that they vary in their chemical structure in terms of molecular weight, glycosidic bond conformation, branching, tertiary conformation or sugar composition. Moreover, the chemical structure of mushroom polysaccharides affect their biological activity (Zhang, Cui, Cheung, & Wang, 2007). High molecular weight (2200e2900 kDa), b-(1 / 3) (1 / 6)-glucan possessing prebiotic activity was obtained by , Jablonský, Slukova , and Copíkov (2008). Other Synytsya, Mí ckova a authors isolated proteoglycans of lower molecular weight (1e31 kDa) which were capable of inhibiting proliferation of various cancer cell lines (Lavi, Friesem, Geresh, Hadar, & Schwartz, 2006; Martin & Brophy, 2010; Tong et al., 2009). Antiproliferative effect results from the induction of apoptosis of cancer cells. Antioxidant activity of P. ostreatus polysaccharides was demonstrated by
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proteoglycans described by other researchers (Sun & Liu, 2009; Xia, Fan, Zhu, & Tong, 2011). Mushrooms are rarely eaten raw and they require to be processed before the consumption. Most often thermal or hydrothermal treatments are applied. It is well known that processing of food may cause substantial changes in its chemical composition and thus affect nutritional and health properties. However, few studies describe the effect of processing on mushrooms-derived biologically active polysaccharides. Fan, Li, Deng, and Ai (2012) investigated how different drying techniques affect antioxidant activity of polysaccharides that were isolated from the medicinal mushroom Ganoderma lucidum. Thetsrimuang, Khammuang, Chiablaem, Srisomsap, and Sarnthima (2011) compared antioxidant and antiproliferative activities of polysaccharides obtained from fresh and dried Lentinus polychrous fungus. So far, however, no studies focused on the influence of hydrothermal processing on the biologically active mushrooms-derived polysaccharides. Therefore, the present paper aims to verify the impact of some processing methods on the content, chemical composition, antioxidant and antiproliferative activity of water soluble polysaccharides (WSP) obtained from P. ostreatus fruiting bodies. The applied processing used in this work included boiling, blanching and fermenting with lactic acid bacteria (Lactobacillus plantarum). Lactic acid fermentation is a process which allows to develop food probiotic products with improved nutritional quality (Beena Divya, Kulangara Varsha, Madhavan Nampoothiri, Ismail, & Pandey, 2012). 2. Materials and methods 2.1. Mushroom samples Fresh fruiting bodies of P. ostreatus were purchased directly from a producer (in 2013) and belonged to the same crop. After a harvest they were kept at 5 C and were subjected to processing within 5 h. 2.2. Processing of mushrooms The mushrooms were divided into four groups (500 g per group) and were further submitted to processing technologies: blanching in water containing 0.5% (w/v) citric acid (5 min, 95 C); boiling in water (15 min, 100 C); blanching in water (as reported above) and fermenting with lactic acid bacteria (see below). The fourth group was not processed (control). The fermented mushrooms were prepared as follows. Fruiting bodies after blanching were washed with cool water and put tightly into screw-capped plastic vessels (PET). The brine solution was added which consisted of sucrose (10 g/1 kg of blanched mushrooms) and 3% (w/v) NaCl. The mixture was then inoculated with 5 mL of bacterial suspension (106 cfu/mL). The mushrooms were fermented for 10 days at 21e22 C and then stored 20 days at 5 C. The final pH of the product was 3.7. All the samples were then subjected to freeze-drying with an Alpha 1-2LD plus freeze dryer (Christ, Germany) and ground to fine powder. 2.3. Extraction of water soluble polysaccharides Four grams of powdered mushrooms were suspended in 200 mL of 80% ethanol and extracted in a rotary shaker at 80 C for 60 min. The ethanolic extract containing low molecular weight compounds was removed by centrifugation (3755 g, 20 min) and the solid residue was washed twice with 80% ethanol and centrifuged. The alcohol insoluble fraction was then re-suspended in deionised water (ratio 1:50 w/v) and autoclaved at 115 C for 180 min. The obtained slurry was cooled and centrifuged (3755 g, 20 min) and
the supernatant was concentrated with a rotary evaporator and precipitated with three volumes of 2-propanol (24 h, at 5 C). The precipitate was then centrifuged, washed twice with 80% methanol, re-dissolved in hot deionised water, lyophilised and weighed. The extraction process was done in triplicate. 2.4. Chemical characteristics of polysaccharides 2.4.1. Determination of total carbohydrate, protein and phenolics content The content of carbohydrates in water soluble polysaccharides was measured with phenol-sulphuric acid method, using glucose as a standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The amount of protein was determined according to the method developed by Bradford (1976), using bovine serum albumin as a standard. Total phenolics content was measured according to the method of Singleton and Rossi (1965) with gallic acid used as a standard. 2.4.2. FTIR spectroscopy FTIR spectra of lyophilised water soluble polysaccharides were recorded on Nicolet NXR 9650 spectrometer (Thermo, USA). The data was collected in the range of 4000e600 cm1 and ATR technique was applied. 2.4.3. Gel permeation chromatography The molecular weight of the isolated polysaccharides was determined with gel permeation chromatography, according to the modified method described by other authors (Malinowska, Krzyczkowski, Łapienis, & Herold, 2009). The samples were dissolved in aqueous solution of NaN3 (0.1%, w/v) and were applied to three TSK-GEL columns: G5000PWXL, G3000PWXL and G2500PWXL (7.8 300 mm, Tosoh, Japan). The chromatographic system was equipped with K-501 pump (Knauer, Germany) and Refracto Monitor IV refractive index detector (LDC Analytical, USA). The flow of the mobile phase (0.1% (w/v) NaN3) was set at 1 mL/ min. Pullulans of different molecular weight were used to construct a standard curve. 2.5. Antioxidant activity 2.5.1. ABTS radical scavenging activity The assay was done according to the method described by Re et al. (1999). ABTS reagent was prepared by incubating 7 mM ABTS solution with 2.45 mM potassium persulfate solution for 16 h at room temperature. The ABTSþ solution was then brought to an absorbance of 0.7 (at 734 nm). The samples (25 mL, 1 mg/mL) were mixed with 975 mL of ABTSþ solution and left to stand for 15 min at room temperature. The absorbance was measured at 734 nm against a blank sample. The calibration curve was done with different concentrations of Trolox (20e200 mM) and the results were expressed as micromoles of Trolox equivalent (TE) per 1 g of mushroom dry weight. 2.5.2. Ferric reducing antioxidant power (FRAP) The ability of polysaccharides to reduce ferric ions was analysed according to the method described by Benzie and Strain (1996). Fresh FRAP reagent was prepared daily by mixing 300 mM acetate buffer (pH 3.6) with 10 mM 2,4,6-tripyridyl-striazine (TPTZ) solution in 40 mM HCl and 20 mM FeCl3$6H2O (10:1:1 ratio). The reagent was incubated at 37 C for 90 min before an analysis. Aliquots of the samples (100 mL) were mixed with FRAP reagent (final volume, 2 mL) and were then incubated at 37 C for 90 min. The change in the absorbance was measured at 593 nm. Trolox aqueous solutions (20e200 mM) were used to construct the calibration curve
W. Radzki et al. / LWT - Food Science and Technology 66 (2016) 27e33
and the results were reported as micromoles of Trolox equivalent (TE) per 1 g of mushroom dry weight.
2.7. Statistical analysis All measurements were carried out in triplicate and the obtained data was expressed as mean ± SD (standard deviation). The data was evaluated using unidirectional analysis of variance ANOVA with a level of significance set at a ¼ 0.05. Statistically different data was compared by Fisher's least significant difference (LSD) test. 3. Results and discussion 3.1. Water soluble polysaccharides content The amount of WSP obtained from non-processed P. ostreatus was 78.7 ± 1.5 mg/g dw, as shown in Fig. 1a. The processing of the fruiting bodies caused marked changes in the content of WSP. Blanching resulted in the increase of WSP (120.1 ± 4.9 mg/g dw). This could be explained by the fact that blanching leads to an extraction of soluble low molecular weight compounds and thus increasing the percentage of the fraction containing high molecular weight compounds. The similar effect was observed in blanched potatoes in which the content of pectin, cellulose and hemicellulose was increased (Gołubowska, 2005). Furthermore, Zivanovic and Buescher (2004) reported that blanching of Agaricus bisporus fruiting bodies caused the increase of total polysaccharides and protein content. Fruiting bodies that were cooked for 15 min also contained higher amount of WSP (100.4 ± 4.7 mg/g dw), comparing to control group. This fact could be still attributed to the change of the proportion of easily soluble substances to difficultly soluble compounds. This has been noted before by Dikeman, Bauer, Flickinger, and Fahey (2005) who observed substantial
Amount of cW SP [mg/g dw fruiting bodies]
Antiproliferative activity was tested on MCF-7 human breast adenocarcinoma cell line (ATCC HTB-22) and T-47D human ductal breast (epithelial tumour) carcinoma cell line (ECACC Catalog, No. 85102201). MCF-7 cell line was grown in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma), 50 mg/mL bovine insulin (Sigma), 100 U/mL penicillin (Polfa, Poland) and 100 mg/mL streptomycin, (Polfa, Poland). The complete growth medium for T47D was RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma) and antibiotics (100 U/mL penicillin (Polfa, Poland)), 100 mg/mL streptomycin, (Polfa, Poland). Both cell lines were grown at 37 C in humidified atmosphere with 5% CO2 (standard conditions). Antiproliferative activity of the tested polysaccharides was measured with MTT assay developed by Mosmann (1983). T47D and MCF-7 cells were seeded on 96-well plates at concentrations of 3 105 cells/mL and 2 105 cells/mL, respectively. After 24 h incubation at standard conditions (37 C, 5% CO2 flow, 95% air humidity), when cells in each well reached about 75%e80% confluence, the growth medium was replaced with dilutions of the tested polysaccharides dissolved in RPMI medium containing 2% FBS (at the concentrations ranging from 25 mg/mL to 250 mg/mL). Cells were incubated with polysaccharides for 24 h, followed by addition of 25 mL of MTT (Sigma) solution (5 mg/mL in PBS) per well. After 3 h incubation at 37 C formazan crystals were solubilised by adding 100 mL of lysis buffer (10% SDS in 0.01 M HCl) per well. Plates were incubated overnight at standard conditions. The absorbance was read at 540 nm with a plate reader (680XR, BioRad), and the mean value for each concentrations was calculated. The percentage of viable cells were calculated from the absorbance.
a
d
120 c
100
b a
80
60
40
20
0 8
b
Control
Blanched
c
c
Boiled Blanched and fermented
b Amount of cW SP [mg/g fw fruiting bodies]
2.6. Antiproliferative activity
140
29
6 a
4
2
0 Control
Blanched
Boiled Blanched and fermented
Fig. 1. The content of polysaccharides isolated from P. ostreatus fruiting bodies calculated on dry weight basis (A) and the fresh weight basis (B). Error bars indicate mean ± standard deviation (n ¼ 3). Different letters indicate significant differences among the treatments according to LSD test (p < 0.05).
concentration of the fibre in various mushroom species, following the cooking process. The level of WSP in blanched and fermented samples (88.9 mg/g dw) was higher compared to non-processed fruiting bodies, but lower than in fruiting bodies that were solely blanched or cooked. In order to investigate whether or not the applied processing techniques had a negative impact on the content of WSP, the results were calculated on the fresh weight basis, taking into consideration the loss of the mass (Fig. 1b). As can be seen from the graph, blanching did not affect negatively the content of WSP. However, boiling for 15 min, led to the substantial decrease in the amount of WSP (34.7% decline, comparing to the control). The observed loss could be attributed to the water extraction of the polysaccharides during the cooking procedure. With respect to the blanched and fermented mushrooms, the observed decrease was lower (14.7%). Because blanching alone did not influence the content of WSP it is possible that the presence of bacteria could be responsible for the loss. Additionally, two weeks storage could have caused WSP to diffuse into brine.
3.2. Chemical characteristics of water soluble polysaccharides 3.2.1. Carbohydrate, protein and phenolics content The amount of total carbohydrates, protein and phenolics is
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Table 1 The amount of total carbohydrate, proteins and polyphenolics in the isolated water soluble polysaccharides of P. ostreatus. Mean values ± standard deviation (n ¼ 3) with different letters indicate significant differences among the treatments according to LSD test (p < 0.05). Treatment
Total carbohydrate (% dw)
Control Blanched Boiled Blanched and fermented
58.0 76.3 71.3 89.2
± ± ± ±
3.9a 5.6b 2.9b 3.5c
Protein content (% dw) 7.8 3.9 7.1 3.0
± ± ± ±
0.5c 0.2b 0.5c 0.2a
Total phenolics content (% dw) 0.77 0.41 0.53 0.29
± ± ± ±
0.04d 0.03b 0.06c 0.06a
presented in Table 1. The total carbohydrate content in the WSP extracted from non-processed fruiting bodies was 58.0 ± 3.9% dw. This value was similar to that reported by other authors (62.8 ± 7.7%), who isolated polysaccharides from P. ostreatus using similar method (Mitra, Khatua, & Acharya, 2013). The extracts obtained from blanched, boiled, blanched and fermented mushrooms contained higher level of carbohydrates (76.3 ± 5.6%, 71.3 ± 2.9, 89.2 ± 3.5%, respectively). Regarding to the protein content, its quantity in the control samples reached 7.8 ± 0.5% dw. This amount was much higher comparing to those reported by Zhang, Dai, Kong, and Chen (2012) who found that the protein content ranged from 0.84% to 1.23%, depending on the method of isolation. This huge discrepancy could be attributed to the use of DAE resin by these researchers during the isolation procedure. On the other hand, other studies demonstrated much higher protein content in polysaccharides isolated from P. ostreatus ranging from 15% (Mitra et al., 2013) to 24% (Xia et al., 2011). The processing of mushrooms caused significant change in the protein content of WSP. In the samples isolated from blanched mushrooms its amount decreased approximately two times, relative to the control and reached 3.9 ± 0.2%. Boiling resulted in rather negligible loss of protein level (7.1 ± 0.5%) in relation to control samples. The greatest decline in the protein content was observed in WSP extracted from blanched and fermented mushrooms where its value reached 3.0 ± 0.2%. It could be
possible that the lowest amount of protein in WSP obtained from blanched and fermented mushrooms could result from the presence of bacteria which released enzymes which caused degradation of protein (Dallagnol, Pescuma, DeValdez, & Roll an, 2013). With regard to the phenolics, they were found in the smallest amount (below 1% dw) comparing to other WSP constituents. Total phenolics content of polysaccharides isolated from non-processed fruiting bodies was 0.77 ± 0.04% dw. This data does not support the findings by Vamanu (2012) in which polyphenolics quantity was approximately ten times higher. This huge difference probably resulted from slightly different extraction procedure because the authors have omitted the stage of alcoholic extraction. Besides, the polysaccharides were obtained from mycelium, not from fruiting bodies. The fact that mushroom polysaccharides contain polyphenolic compounds is well established (Klaus et al., 2011). Polyphenolics tend to bind with polysaccharides with hydrogen bounds, hydrophobic interactions or even covalent bounds (Renard, Baron, Guyot, & Drilleau, 2001). Bound polyphenolics contribute to antioxidant activity of polysaccharides and their presence is highly beneficial. They are slowly released from the matrix by intestine microbes and are absorbed by an organism (Saura-Calixto, 2011). The processing of fruiting bodies led to substantial changes in the phenolics content and a similar trend to protein was observed. The rapid, 46% decline was noticed as the result of blanching and 31% in the case of cooking. A possible explanation for this is that hydrothermal processes may lead to solubilisation and releasing of phenolics ndez, Arte s-Herna ndez, Go mez, & Arte s, 2013). (Martínez-Herna Another possible explanation for this effect is thermal degradation rez, Carballo, & Franco, due to the elevated temperature (Martínez, Pe ska, Filipiak-Florkiewicz, & Pisulewski, 2013; Sikora, Cieslik, Leszczyn 2008). Lactic acid fermentation caused the highest (63%) decrease in phenolics content. This could be explained by diffusion of soluble phenolics into brine. This process could possibly have been enhanced ska, 2005). by bacterial enzymes (Ciska, Karamac, & Kosin
3.2.2. FTIR spectroscopy The FTIR spectra of isolated WSP are depicted in Fig. 2. They are
Fig. 2. FTIR spectra of water soluble polysaccharides obtained from non-processed (control) and processed fruiting bodies of P. ostreatus.
W. Radzki et al. / LWT - Food Science and Technology 66 (2016) 27e33
rrez, generally similar to that obtained by other authors (Gutie Prieto, & Martínez, 1996) and they show bands characteristic of the presence of polysaccharides and protein molecules. The broad peak in the region of 3000e3500 cm1 could be attributed to OeH and NeH stretching vibrations, while the band at ~2930 cm1 is the result of CeH stretching. The absorption at ~1645 cm1 (amide I) is indicative of C]O stretching mode, whereas the signal at 1530 cm1 corresponds to the presence of bending vibrations of NeH groups. These two bands indicate the presence of protein. In the region of 1450e1300 cm1 there are signals related to bending vibrations of CH2, OeH and CeOeH (Moha cek-Grosev, Bo zac, & Puppels, 2001). Intense peaks observed at 950e1190 cm1 result from stretching vibrations of CeOeC, COH and CeC (Larkin, 2011). Analysis of 700e950 cm1 region can give information on the type of glycosidic links present in the sample. Signal at ~895 cm1 may suggest the presence of b-glucans (Moha cek-Grosev et al., 2001), while bands at ~930 cm1, ~850 cm1, ~760 cm1 are indicative of a-glucans (Novak et al., 2012; Wiater et al., 2011).
3.3. Antioxidant capacity Water soluble polysaccharides isolated from both processed and non-processed fruiting bodies displayed ABTS scavenging activity ranging from 14.14 ± 0.63 to 29.48 ± 1.12 mmoles of Trolox per 1 g dw (Fig. 4). The control sample showed the highest value (29.48 ± 1.12 mmoles of Trolox per 1 g dw), which was rapidly decreased due to the processing. As the result of blanching approximately 26% decline was noticed, whereas boiling led to approximately 12% decrease. The highest drop in activity (approximately 52%) demonstrated the samples that were obtained from the blanched and fermented mushrooms. Our data are in qualitative agreement with other studies that investigated antioxidant capacity of fermented A. bisporus and P. ostreatus fruiting bodies and reported substantial decline in ABTS radical scavenging activity (approximately 60% and 90%, respectively) (Ska˛ pska et al., 2008). With respect to the FRAP assay, the activity values were lower comparing with ABTS assay and varied from 2.49 ± 0.54 to 16.52 ± 0.55 mmoles of Trolox equivalents per 1 g dw (Fig. 4). However, the similar trend was observed in terms of the decrease of the antioxidant activity. The highest value was observed in the control samples (16.52 ± 0.55 mmoles of Trolox equivalents per 1 g dw) and the processing methods had a negative impact on the
Control
10 5 0 Blanched
5
Refractive index [mV]
3.2.3. Gel permeation chromatography Gel permeation chromatography was performed to identify molecular weight of main WSP components. All the spectra showed sharp, symmetrical peak at 18.1e19.1 mL. It corresponds to a compound having molecular weight of 198.3 kDa (Fig. 3). The chromatogram of the extracted WSP from the control samples possessed also intense signals at 22.4 mL and 24.68 mL which can be attributed to the compounds of the lower molecular mass (11.9 and 3.1 kDa, respectively). These signals were not recorded in the samples obtained from the processed mushrooms. The presence of 198.3 kDa fraction is in agreement with the results of other authors who extracted P. ostreatus polysaccharides (using similar isolation method) which had molecular mass 187 kDa (Maity et al., 2011). Other researchers obtained the fractions of the lower molecular weight: 1e10 kDa (Lavi et al., 2006) and 10.5 kDa (Synytsya et al., 2009). The processing of fruiting bodies, regardless of the method applied, caused 3.1 kDa fraction to disappear. Probably it could be eluted during the applied processes. Additionally, blanching led to diminishing of 11.9 kDa fraction. Further studies should be undertaken to investigate the chemical composition of each fraction and the role they play in exerting antioxidant and antiproliferative activities.
15
31
0 Boiled
5
0 20 15
Blanched and fermented
10 5 0 16 17 18 19 20 21 22 23 24 25 26 Retention volume [mL] Fig. 3. Gel permeation chromatography of water soluble polysaccharides extracted from non-processed (control) and processed fruiting bodies.
activity. Blanching resulted in approximately 60% decrease, while boiling caused approximately 40% decline. The highest decrease (approximately 85%) was noticed for WSP isolated from blanched and fermented fruiting bodies. Previous studies has shown that hydrothermal treatments leads to the decrease of total antioxidant potential of mushrooms (Nguyen, Nagasaka, & Ohshima, 2012; Soler-Rivas, RamírezAnguiano, Reglero, & Santoyo, 2009). The current study found that the applied processing altered antioxidant activity of the obtained
32
W. Radzki et al. / LWT - Food Science and Technology 66 (2016) 27e33
lower comparing to MCF-7 line. The lowest cell viability was found in non-processed samples (73.4 ± 2.3%) and similarly to MCF-7 line, the processing led to the statistically relevant decrease in the activity. Current literature does not provide information on the impact of hydrothermal processing on antiproliferative activity of foodderived polysaccharides. However, some studies were conducted on rich in phenolics methanolic extracts. Chatthongpisut, Schwartz, and Yongsawatdigul (2015) showed that cooking procedures leads to substantial decline in antiproliferative activity of purple rice. Also other authors reported that lotus roots and white onions cooked for 10 min possessed lower antiproliferative potential (Im et al., 2012). 4. Conclusions
Fig. 4. Antioxidant capacity of water soluble polysaccharides extracted from nonprocessed (control) and processed mushrooms. Error bars indicate mean ± standard deviation (n ¼ 3). Different letters indicate significant differences among the treatments according to LSD test (p < 0.05).
100 c
90 b
b
80
Cell viability [%]
70
a
c b
a
a
60 50 40 30 20 10
Water soluble polysaccharides were extracted from both processed and non-processed mushrooms, giving different yields. The processing (boiling and blanching followed by fermenting) led to the decrease of polysaccharides content. Therefore, the real intake of these macromolecules can differ between raw and processed form. Moreover, all the processes caused substantial changes in chemical composition of polysaccharides (protein and phenolics contents were decreased). Along with the changes of chemical composition caused by the processing, antioxidant and antiproliferative activities were decreased which may affect nutritional and health values. Therefore, hydrothermal processing, especially long, should not be applied in order to fully retain health promoting properties of P. ostreatus. Further work would be suggested to investigate the impact of different processing techniques like microwave cooking, steaming or frying on bioactive polysaccharides from P. ostreatus. Moreover, since mushrooms polysaccharides are known to exert immunomodulating effect, more research is required to determine the impact of processing on this effect. References
Control Blanched Boiled Blanched and fermented
0 MCF-7
T-47D
Fig. 5. Cell viability (%) of MCF-7 and T-47D human adenocarcinoma cell line after treatment with water soluble polysaccharides (250 mg/mL) obtained from nonprocessed (control) and processed P. ostreatus fruiting bodies. Error bars indicate mean ± standard deviation (n ¼ 3). Different letters indicate significant differences among the treatments according to LSD test (p < 0.05).
polysaccharides. As a consequence, this may affect their healthpromoting properties.
3.4. Antiproliferative activity The extracted water soluble polysaccharides were analysed in terms of their antiproliferative activity against MCF-7 and T-47D breast cancer cell lines. The results showed that WSP displayed slight inhibitory action on both cells lines (Fig. 5). The effect was most evident at the concentrations of 250 mg/mL. The strongest activity was displayed by the samples derived from non-processed fruiting bodies (cell viability reached 68.8 ± 1.7%). As the result of processing, the drop of the activity was noticed and the cell viability amounted to 77.9 ± 3.0%, 72.1 ± 4.1% and 80.0 ± 2.8% for blanched, boiled, blanched and fermented samples, respectively. With regard to the T-47D cell line, the antiproliferative activity was generally
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