The medicinal properties of many species of mushroom. Measurement of β-glucan in Mushrooms and Mycelial Products - PDF

Description
364 McClery & Drg: Journl of AOAC Interntionl Vol. 99, No. 2, 2016 DIETARY SUPPLEMENTS Mesurement of β-glucn in Mushrooms nd Mycelil Products Brry V. McClery 1 nd Ann Drg Megzyme Interntionl Irelnd, Bry

Please download to get full document.

View again

of 10
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Information
Category:

Fashion

Publish on:

Views: 43 | Pages: 10

Extension: PDF | Download: 0

Share
Transcript
364 McClery & Drg: Journl of AOAC Interntionl Vol. 99, No. 2, 2016 DIETARY SUPPLEMENTS Mesurement of β-glucn in Mushrooms nd Mycelil Products Brry V. McClery 1 nd Ann Drg Megzyme Interntionl Irelnd, Bry Business Prk, Southern Cross Rd, Bry, County Wicklow, Irelnd A robust nd relible method hs been developed for the mesurement of β-glucn in mushroom nd mycelil products. Totl glucn (plus free glucose nd glucose from sucrose) ws mesured using controlled cid hydrolysis with H 2 SO 4 nd the glucose relesed specificlly ws mesured using glucose oxidse/peroxidse regent. α-glucn (strch/glycogen) plus free glucose nd glucose from sucrose were specificlly mesured fter hydrolysis of strch/glycogen to glucose with glucomylse nd sucrose to glucose plus fructose with invertse nd the glucose specificlly mesured with GOPOD regent. β-glucn ws determined by the difference. Severl cid nd enzyme-bsed methods for the hydrolysis of the β-glucn were compred, nd the best option ws the method using H 2 SO 4. For most smples, similr β-glucn vlues were obtined with both the optimized HCl nd H 2 SO 4 procedures. However, in the cse of certin smples, specificlly Gnoderm lucidum nd Pori cocus, the H 2 SO 4 procedure resulted in significntly higher vlues. Hydrolysis with 2 N trifluorocetic cid t 120 C ws found to be much less effective thn either of the other two cids evluted. Assys bsed totlly on enzymtic hydrolysis, in generl, yielded much lower vlues thn those obtined with the H 2 SO 4 procedure. The medicinl properties of mny species of mushroom hve been vlued nd used in trditionl Chinese medicine for centuries. More recent studies (1 7) hve demonstrted tht the key ctive compounds re triterpenoids, ergosterol, nd, most importntly, 1,3:1,6-β-glucn. This β-glucn ctivtes the immune system nd might even hve nticrcinogenic properties (1 7). The nticrcinogenic properties of the β-glucn from Lentinul edodes [Lentinn Shiitke (1, 4)], Grifol frondos [Grifoln (2, 3)], Gnoderm lucidum [Reishi (6)], nd others hve been demonstrted throughout the pst 3 to 4 decdes. There is concern within the regultory community regrding helth clims relting to nutritionl supplements s well s the identity nd purity of these products (8), nd this reltes prticulrly to medicinl mushrooms where the key ctive components hve been identified s 1,3:1,6-β-glucn, triterpenoids, nd ergosterol. Received November 12, Accepted by AP December 17, Corresponding uthor s e-mil: DOI: /jocint Fungl nd yest cell wlls re composed of 50% 1,3:1,6-β-glucn, nd numerous structurl components hve been identified (9). The 1,3:1,6-β-glucns of severl mushroom species hve been studied in considerble detil nd the most predominnt structurl feture hs been identified s 1,3-β-glucn bckbone with single d-glucosyl residues linked 1,6-β to every third (9) or fourth d-glucosyl unit in the 1,3-β-glucn bckbone. However, much more complex structures hve lso been reported (10 14). The structures of mushroom nd fungl β-glucns re different from the cerel β-glucns (so-clled mixed-linkge β-glucns) tht re liner polyscchrides in which d-glucosyl residues re linked 1,3-β- nd 1,4-β-, nd the rtio of these linkge types vries with the source of the β-glucn (e.g., ots, brley, nd whet). Other β-glucns include cellulose (1,4-β-d-glucn) nd curdln (1,3-β-d-glucn). A highly specific enzymtic procedure hs been described for the mesurement of cerel 1,3:1,4-β-d-glucns (15, 16). Enzymtic procedures hve lso been described for the mesurement of 1,3:1,6-β-d-glucns in commercil yest products (17, 18); however, lthough these procedures re useful for this prticulr ppliction, they re less specific thn the method tht hs been developed for the mesurement of cerel β-glucn (15, 16). No quntittive enzymtic procedure hs been described for the mesurement of β-glucn in mushroom fruiting bodies or mycelium. Prk et l. (19) mesured the β-glucn content of Agricus blzei by first extrcting the nonstrch polyscchride frction ccording to the dietry fiber method of Prosky et l. (20, 21) using thermostble α-mylse nd myloglucosidse to hydrolyze strch/glycogen in the mushroom smple nd recovering nonstrch polyscchride by lcohol precipittion, wshing, nd drying. The recovered polyscchride ws subsequently cid hydrolyzed, nd glucose ws determined enzymticlly. Rhee et l. (22), used similr procedure to mesure β-glucn content of Inonotus obliquus (Chg). In this cse, the polyscchride recovered following incubtion of the smple with α-mylse nd myloglucosidse under cid conditions nd the resulting glucose ws determined by HPLC. These uthors lso extrcted polyscchride in n lkline buffer (ph 10). No enzyme tretment ws included to remove α-glucn becuse this mushroom contins very little α-glucn. Synytsy et l. (23) used the Yest nd Mushroom β-glucn kit described in Megzyme technicl booklet K-YBGL, in which totl glucn is mesured by hydrolysis with cid nd α-glucn is specificlly mesured by enzymtic hydrolysis. Glucose ws specificlly mesured with glucose oxidse/peroxidse regent, nd β-glucn is determined by the difference. Other interesting, but nonquntittive, methods hve been described for the mesurement of β-glucn in mushroom products, including the method of Mizuno et l. (24) using n ELISA nd tht of Molleken et l. (25) who used congo red dye. McClery & Drg: Journl of AOAC Interntionl Vol. 99, No. 2, Mnzi nd Pizzoferrto (26) mesured the β-glucn content of rnge of edible mushrooms using lichense nd β-glucosidse to hydrolyze the β-glucn. However, lichense ( specific 1,3:1,4-endo-β-glucnse) hs no ction on 1,3:1,6-β-glucns, nd the β-glucosidse hs limited ction on the polyscchrides, consequently the determined β-glucn vlues were gretly underestimted. Lichense nd β-glucosidse re used in the quntittive mesurement of 1,3:1,4-β-glucns from cerel grins (15, 16). Becuse complete enzymtic hydrolysis of β-glucn in mushroom products is very difficult, not only becuse of the rry of β-glucn linkge types present but lso s result of the linkges to chitin nd protein, the best pproch to quntittive determintion of this polyscchride is complete cid hydrolysis to glucose, with subsequent mesurement of glucose to mesure totl glucn. α-glucn cn either be removed before cid hydrolysis, or it cn be mesured seprtely nd ccounted for. Dllies et l. (27) mesured β-glucn content of the yest, Scchromyces cerevisie, by hydrolysis of the polyscchride to glucose using procedure described by Selvendrn et l. (28) for plnt cell wlls, in which smples were first suspended in 72% w/w sulfuric cid t room temperture nd then hydrolyzed t ~100 C in 2 M sulfuric cid ccording to Semn (29). Under these conditions, the polyscchride ws completely hydrolyzed, nd there ws miniml loss of glucose through further degrdtion. In the current study, cid hydrolysis nd enzymtic procedures for hydrolysis of β-glucn in mushroom smples were compred, nd quntittive method for mesurement of β-glucn in these products ws developed. Mterils nd Methods Mterils () Chemicls. Sulfuric cid, 95 98% (18.4 M; Ct. No L-D), hydrochloric cid, 37% (12 M, Ct. No L-D), trifluorocetic cid (TFA, Ct. No G-D), potssium hydroxide, 85% (Ct. No KG-D), nd lyticse (Ct. No. L KU; SLBL7091V) were obtined from Sigm-Aldrich (St. Louis, MO). Brley β-glucn (Ct. No. P-PGBM), yest β-glucn (Ct. No. P-YBGL), curdln (Ct. No. P-CURDL), exo-1,3-β-glucnse (100 U/mL) plus β-glucosidse (100 U/mL) (Ct. No. E-EXBGOS, Lot ), Totl Strch ssy kit (Ct. No. K-TSTA), Yest nd Mushroom β-glucn ssy kit (Ct. No. K-YBGL), enzymtic Yest β-glucn ssy kit (Ct. No. K-EBHLG), nd Sucrose/ Glucose ssy kit (K-SUCGL) were obtined from Megzyme Interntionl (Bry, County Wicklow, Irelnd). Sulfuric cid (72% w/w, ~12 M) ws prepred by crefully dding 650 ml of concentrted sulfuric cid (98%, sp. gr ) to 300 ml of distilled wter. The volume ws then djusted to 1 L. TFA (2 M) ws prepred by dding 25 ml of concentrted cid to 162 ml of distilled wter. Sodium cette buffer (200 mm, ph 5) ws prepred by dding 11.6 ml of glcil cetic cid (1.05 g/ml) to 900 ml of distilled wter nd djusting the ph to 5.0 using 4 M (16 g/100 ml) sodium hydroxide solution. The volume ws djusted to 1 L. (b) Mushrooms nd mycelil products. All of the pure mushroom fruiting bodies nlyzed in this study were supplied by Jeff Chilton (Nmmex, Gibsons, BC, Cnd; Tble 1). Tble 1. Detils of the pure mushroom fruiting bodies supplied by Nmmex Commercil cpsules contining mushroom nd mycelil products (fruiting bodies nd mycelium) were purchsed vi the internet from Amzon.com. Detils of these smples re provided in Tble 2. The A. niger β-glucn control used in these studies ws obtined from the Yest nd Mushroom β-glucn ssy kit (Megzyme Ct. No. K-YBGL). The concentrtion of this β-glucn ws determined using both this kit nd lso enzymticlly using the kit K-EBHLG. Methods Bsidiomycete species Lot number Product code 1 Polyporus umbelltus (lumpy brcket) 2 Trmetes versicolor (turkey til) 3 Inonotus obliquus (Chg mushroom) 4 Gnoderm lucidum (Reishi or Lingzhi) MZ-PKPu1304 MZ-PKTv1412 MZ-FGlo1401 MZ-XTYGI PuMuWp00 TvMuWp00 IoMuWp00 GIMuWp00 5 Agricus blzei MZ-ZFPAb1405 ABMuWp00 6 Grifol frondos (hen of the woods) 7 Gnoderm lucidum (Reishi or Lingzhi) MZ-QYZGf MZ-QYZGI GfMuWp00 GIMuWp00 8 Pori cocos powder (pori) MZ-QYZPc PcMuWp11 9 Lentinul edodes powder (Shiitke) 10 Cordyceps militris (scomycete) 11 Hericium erinceus (Lion s mne mushroom) 12 Agricus bisporus (button mushroom) 13 Pleurotus ostretus (oyster mushroom) 14 Tremell fuciformis (white jelly mushroom) 15 Grifol frondos (hen of the woods) MZ-ZFPLe1309-P MZ-QYZCm1406 MZ-PKHe1304 MZ-PKAbb1412 MZ-JCPo14112 MZ-PKTf1304 MZ-PKGf1304 LeMuWp11 CmMuWp00 HeMuWp11 AbbMuWp00 PoMuWp00 TfMuWp00 GfMuWp00 16 Lentinul edodes (Shiitke) MZ-PKLe1412 LeMuWp00 17 Pleurotus eryngii (king trumpet mushroom) 18 Flmmulin velutipes (velvet shnk) 19 Agricus bisporus (Portobello) MZ-PKPe1412 MZ-PKFv1412 MZ-PKAbp1412 PeMuWp00 FvMuWp00 AbpMuWp00 () Mesurement of α-glucn (strch/glycogen). Mushroom smples were milled to pss 1.0 mm screen. Approximtely 100 mg (weighed ccurtely) of the smple ws dded to mm Fisher Brnd culture tube, nd the tube ws tpped to ensure tht the entire smple fell to the bottom of the tube. A mgnetic stirrer br (5 15 mm) nd 2.0 ml of ice-cold 2 M KOH ws dded to ech tube, nd the tube contents were stirred using mgnetic stirrer in n ice wter bth for 20 min to dissolve the strch/glycogen. Eight milliliters of 1.2 M sodium 366 McClery & Drg: Journl of AOAC Interntionl Vol. 99, No. 2, 2016 Tble 2. Detils of mushroom products obtined commercilly in cpsule form Smple No. Bsidiomyces species Product detils from bottle lbels 1 Gnoderm lucidum Reishi Mushroom (Gnoderm lucidum) (fruiting bodies) 1.2 g/2 tblets. Other ingredients include: microcrystlline cellulose (plnt fiber) species blend Mitke (Grifol frondos) mycelium; Chg (Inonotus obliquus) mycelium; Reishi (Gnoderm lucidum vr. resinceum s.l.) mycelium; Cordyceps (Cordyceps sinensis s.l.) mycelium; Royl sun Blzei (Agricus brsillensis f. blzei) mycelium; Enokitke (Flmmulin volutipes) mycelium; Mesim (Phellinus linteus) mycelium; Turkey Tils (Trmetes versicolor) mycelium; Zhu Ling (Polyporus umbelltus) mycelium; Lions Mne (Hericlum erinceus) mycelium; Mitke (Grifol frondos) fruitbodies; Artists Conk (Gnoderm pplntum s.l.) mycelium; Oregon Reishi (Gnoderm oregonense s.l.) mycelium; Agrikon (Fomitopsis officinlis) mycelium; Amdou (Fomes fomentrius) mycelium; Shitke (Lentinul edodes) mycelium; Birch Polypore (Piptoporus betulinus) mycelium; Split Gill Polypore (Schizophyllum commune) mycelium. 3 7 species blend Royl Sun Blzei (Agricus brsiliensis) mycelium; Cordyceps (Cordyceps sinesis s.l.) mycelium; Reishi (Gnoderm lucidum s.l.) mycelium; Mitke (Grifol frondos) mycelium; Lion s Mne (Hericlum erinceus) mycelium; Chg (Inonotus obliquus) mycelium; Mesim (Phellinus linteus) mycelium. Other ingredients freeze-dried mycelium, brown rice, pulluln. 4 Gnoderm lucidum Reishi Mushroom fruiting body extrct, 120 mg. Reishi mushroom myceli powder 880 mg. Reishi mushroom fruiting body extrct, 120 mg (stndrdized for 10% polyscchrides). Other ingredients; vegetble cellulose, rice flour, clcium silicte. 5 Gnoderm lucidum Reishi mushroom powder without ny dditives. 480 mg cpsules. 6 Gnoderm lucidum/ Lentinul edodes Reishi Mushroom Extrct Powder (10:1) (Gnoderm lucidum) 90 mg; Reishi mushroom powder (Gnoderm lucidum) 300 mg; Shitke Mushroom Extrct Powder (4:1) (Lentinul edodes). 270 mg; per 2 cpsules. 7 Cordyceps sp. (scomycete) Orgnic Cordyceps (mycelium). 1.5 g/2 cpsules. 8 Cordyceps sp. (scomycete) Pure Cordyceps cpsules. 525 mg ech, 100% orgnic. Full Spectrum. Cordyceps sinensis in nonorgnic vegetrin cpsule, nothing more, nothing less. 9 Gnoderm lucidum Red Reishi. 100% Orgnic Gnoderm lucidum mg. 10 Cordyceps sinensis (scomycete) Cordyceps sinensis (deep lyer cultivted myceli extrct) 1.2 g. Other ingredients include microcrystlline cellulose (plnt fiber). 11 Cordyceps sinensis (scomycete) Cordyceps dried extrct (mycelium) mg. 10% cordycepic cid. Other ingredients; rice powder. 12 Inonotus obliquu Chg Mushroom (mycelium) 400 mg. Other ingredients include: brown rice flour Bottled products contining encpsulted mycelium/mushroom powder. Bottles were purchsed from online retilers. Most of the products studied re mycelium propgted on grin. cette buffer (ph 3.8) ws dded to ech tube with mixing on vortex stirrer. A totl of 0.2 ml of mixture of myloglucosidse (1630 U/mL) plus invertse (500 U/mL) (from Megzyme ssy kit, Ct. No. K-YBGL) ws immeditely dded, the contents were mixed well, nd the tubes were incubted t 40 C for 30 min. For smples contining 10% strch, this solution (10.3 ml finl volume) ws nlyzed directly. For smples contining % strch/glycogen, the tube contents were quntittively trnsferred to 100 ml volumetric flsk nd djusted to volume with deionized wter, nd the contents were mixed thoroughly. In both cses, 1.0 ml of the solution ws centrifuged t g for 3 min in microfuge, nd 0.1 ml of the superntnt solutions ws nlyzed for glucose with glucose oxidse/peroxidse regent. (b) Mesurement of totl glucn. (1) Hydrolysis with sulfuric cid. Mushroom smples were milled to pss 1.0 mm screen. Approximtely 100 mg (weighed ccurtely) of the smple ws dded to mm Fisher Brnd culture tube, nd the tube ws tpped to ensure tht the entire smple fell to the bottom of the tube. A totl of 2.0 ml of ice-cold 12 M sulfuric cid ws dded to ech tube, nd the tubes were cpped nd stirred on vortex mixer. Tubes were plced in n ice wter bth nd left for 2 h. During this time, the tube s contents were vigorously stirred (for s) severl times on vortex mixer to ensure complete dissolution/dispersion of the smple. Twelve milliliters of wter ws dded to ech tube, nd the tubes were cpped nd vigorously stirred on vortex mixer for 10 s. The cps were loosened nd the tubes were plced in boiling-wter bth (~100 C). After 5 min, the cps were tightened nd the incubtion ws continued t 100 C for 2 h. The tubes were cooled to room temperture, nd the cps were crefully loosened. Six milliliters of 10 M KOH ws dded, nd the tube contents were mixed well. The contents of ech tube were quntittively trnsferred to 100 ml volumetric flsks using wsh bottle contining 200 mm sodium cette buffer (ph 5), nd the volume ws djusted to 100 ml with 200 mm sodium cette buffer (ph 5). The contents were mixed thoroughly, nd n liquot (~1.2 ml) of the solution ws centrifuged t g for 3 min in microfuge; lterntively, 5 ml liquot of the solution ws centrifuged t ~1500 g for 10 min in bench centrifuge. The content of glucose in the solutions ws nlyzed by incubting n liquot (0.1 ml) of the superntnt with 3.0 ml of GOPOD regent t 40 C for 20 min. Absorbnce ws mesured t 510 nm. Concurrently, 0.1 ml liquot of glucose stndrd solution (1 mg/ml), ws incubted in qudruplicte (stndrd) with GOPOD regent; lso, 0.1 ml of cette buffer (200 mm, ph 5) ws incubted with 3.0 ml of GOPOD regent (regent blnk). Alterntively, 0.1 ml of the smple solution ws incubted with 0.1 ml of mixture of exo-1,3-β-glucnse (20 U/mL) plus β-glucosidse (4 U/mL) t 40 C for 60 min, nd the glucose ws determined with GOPOD regent s previously described (ll of the regents used re vilble in the Megzyme test kit Ct. No. K-YBGL). McClery & Drg: Journl of AOAC Interntionl Vol. 99, No. 2, In preliminry experiments, smples were stored in 12 M sulfuric cid for 30, 60, nd 120 min; effect of stirring or intermittent shking in the 12 M sulfuric cid ws evluted; effect of smple size ( mg) ws lso evluted. The effect of time of incubtion t 100 C on determined glucn content nd stbility of relesed glucose ws determined by incubting series of smples [glucose, whet strch, brley β-glucn, Actigum (scleroglucn), purified yest β-glucn, nd Cordiceps militris mushroom smple] in 12 M sulfuric cid for 2 h t ~0 C followed by incubtion in 2 M sulfuric cid t 100 C for 0, 30, 60, 90, nd 120 min. (2) Hydrolysis with hydrochloric cid. Mushroom smples were milled to pss 1.0 mm screen. Approximtely 100 mg (weighed ccurtely) of the smple ws dded to mm Fisher Brnd culture tube, nd the tube ws tpped to ensure tht the entire smple fell to the bottom of the tube. A totl of 1.5 ml of 37% v/v (12 M) hydrochloric cid ws dded to ech tube, nd the tubes were cpped nd stirred on vortex mixer. The tubes were plced in wter bth t 30 C for 60 min nd the contents were stirred for 15 s on vortex mixer. Ten milliliters of wter ws dded to ech tube, the tubes were cpped, nd the contents were vigorously stirred on vortex mixer for 10 s. The cps were loosened nd the tubes were plced in boiling-wter bth (~100 C). After 5 min, the cps were tightened nd the incubtion ws continued for 2 h. The tubes were cooled to room temperture, nd the cps crefully loosened. Ten milliliters of 2 M KOH ws dded, nd the contents were mixed well. The contents of ech tube were quntittively trnsferred to 100 ml volumetric flsks using wsh bottle contining 200 mm sodium cette buffer (ph 5), nd the volume ws djusted to 100 ml with 200 mm sodium cette buffer (ph 5). The contents of ech flsk were mixed thoroughly. Smples were then treted in the sme mnner s those from H 2 SO 4 hydrolysis. (3) Hydrolysis with TFA. Mushroom smples were milled to pss 1.0 mm screen. Approximtely 100 mg (weighed ccurtely) of the smple ws dded to mm Fisher Brnd culture tube, nd the tube ws tpped to ensure tht the entire smple fell to the bottom of the tube. Five milliliters of 2 M TFA ws dded to ech tube, the tubes were cpped, nd the contents were stirred vigorously on vortex mixer. The cps were loosened, nd the tubes plced in n oil bth t 120 C. After 2 min, the cps were tightened nd incubtion ws continued for 40 min. The contents of the tubes were stirred fter 10 min intervls for pproximtely 10 s. After 40 min, the tubes were cooled to room temperture nd the cps crefully loosened. Five milliliters of 2 M KOH ws dded, nd the contents were mixed well. The contents of ech tube were quntittively trnsferred to 100 ml volumetric flsks using wsh bottle contining 200 mm sodium cette buffer (ph 5), nd the volume ws djusted the to 100 ml with 200 mm sodium cette buffer (ph 5). The contents of the flsks were mixed thoroughly. Smples were then treted in the sme wy s those from H 2 SO 4 hydrolysis. Enzymtic Methods for Mesurement of β-glucn in Mushroom Smples () Glucn enzymtic method (GEM) ssy. This ssy ws performed s described by Dnielson et l. (17). Under these conditions, lyticse incubtion ws performed t ph 5, which is not optiml (ph optim for lyticse is ). (b) Modified GEM ssy. In modified incubtion, mushroom smple (~20 mg, weighed ccurtely) ws suspended in 0.4 ml of 2 M KOH nd stirred for 20 min in n ice wter bth. The solution ws neutrlized by dding 1.2 ml of 0.6 M sodium cette buffer (ph 3.8). Tris buffer (1 ml, 10 mm, ph 7.1) contining 1 mm EDTA, 20 mm NCl, nd 6000 U of lyticse (units s defined by Sigm Chemicl Co.) ws dded, nd the suspension ws incubted t 50 C for 16 h. The incubtion mixt
Related Search
Similar documents
View more...
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks