comparison of two methods for the quantitation of 13-glucans from

Comparison of Two Methodsfor the Quantitation of 13-Glucans

from Shiitake MushroomsDavid Brauer

Tom KimmonsMark Phillips

ABSTRACT. Fungal polysaccharides and glucans, including lentinanfrom shiitakes, have been identified as promoting human health, andconsequently, the sale of mushroom-based health products has increasedsignificantly in recent years. Reliable methods for the quantitation oflentinan must be available if shiitakes or shiitake-derived products areto be promoted as health-promoting food. The objective of this studywas to characterize two methods for the quantitation of 3-glucans fromaqueous extracts of shiitake mushrooms. The first method utilized etha-nol precipitation and batch size exclusion chromatography to isolate afraction enriched in lentinan, called high molecular weight polysaccha-rides, HMWP. Gel filtration chromatography characterization of HMWP

David Brauer is affiliated with Dale Bumpers Small Farms Research Center/Agri-cultural Research Service/U.S. Department of Agriculture, Booneville, AR.

Tom Kimmons and Mark Phillips are affiliated with Shirley Community Develop-ment Corp., 366 Brown Road, Shirley, AR.

Address correspondence to: David Brauer, Dale Bumpers Small Farms ResearchCenter/Agricultural Research Service/U.S. Department of Agriculture, 6883 Highway23, Booneville, AR 72927 (E-mail: [email protected]).

Mentioning of a trade name does not constitute an endorsement by USDA overother similar products. This research was supported in part by funds from USDA Spe-cific Cooperative Agreement #58-6227-4-017 to the Shirley Community DevelopmentCorporation.

Received September 20, 2005.

Journal of Herbs, Spices & Medicinal Plants, Vol. 13(3) 2007Available online at http://jhsmp.haworthpress.com© 2007 by The Haworth Press. All rights reserved.

doi:10. 13005044vl3n03_02 15

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16JOURNAL OF HERBS, SPICES & MEDICINAL PLANTS

fraction indicated that the size distribution of carbohydrates was similarto that of a lentinan standard. Analyses for total starch and glycogen, toa lesser extent, indicate these polysaccharides are less than 10% of thetotal carbohydrates in HMWP. These results suggest that the HMWPfraction had a high level of lentinan. The second method measured theproduction of glucose by enzymes that specifically hydrolyze plant3-glucans. The amount of carbohydrate detected by the enzymatic 13-glucanassay was significantly less than the carbohydrate found in the HMWPfraction. The low content of 3-glucan was not due to a failure of the as-say's enzymes to function. In addition, no glucose was produced when alentinan standard was subjected to the 3-glucan assay. These results in-dicate that the carbohydrate content of HMWP fraction was a better indi-cator of lentinan content of shiitakes compared to the mushroom'sJ3-glucan content. doi: 10.1 300/J044v 1 3n03_02 [Article copies available for a

fee from The Haworth Document Delivery Sen'ice: 1-800-HA WORTH. E-mail ad -dress. <docdelivery@haworthpress. corn> Websire: <http.//www.Hawort/iPress.corn> © 2007 by The Haworth Press. All rights reserved.]

KEYWORDS. Lentinan, polysaccharides, lichenase, 0-glucosidase, eth-anol insoluble, starch, glycogen

INTRODUCTION

Shiitake (Lentinula edodes (Berk.) Pegler) mushroom production in theUnited States has expanded greatly since its start about 30 years ago.Shii take production doubled between 1990 and 1995 to over 2,500,000 ki-lograms annually and current production exceeds 4,000,000 kg (18). Manyhealth benefits have been ascribed to shiitake mushrooms and compoundsisolated from them (3, 8, 10, 17). Worldwide sales of medicinal mushroomproducts in 2000 were estimated to exceed $10 billion (22). One of thehealth benefits ascribed to shiitakes is improved cardiovascular fitness.There is substantial evidence that consumption of shiitake mushrooms willdecrease serum cholesterol (4, 5).

Both fungal homogeneous and heterogeneous polysaccharides havebeen identified as promoting human health (19). One of the suspectedeffects of fungal glucans is immune system stimulation (3, 7, 17). Salesof mushroom-based immune boosters increased about threefold between2002 and 2003 (23). Lentinan has been identified as a health promoting,water-soluble 3-glucan from shiitakes (3, 17). There is evidence that in-travenous injections, as well as ingestion, of lentinan promote human

Brauer, Ki,nnions, and Phillips 17

health (9), and stimulate the immune system (3, 7, 17). However, thehealth effects of lentinan ingestion have not been studied as extensivelyas responses to lentinan injections. Shiitakes may contain other glucanswith health-promoting properties, but specific constituents have notbeen identified yet. Lentinan consists of a 13(1-3) linked D-glucan back-bone with two 13(1-6) linked glucose side chains for every five 13(1-3)linked glucose residues and has a molecular weight of about 400 kilo-daltons (20). Lentinan is readily soluble in water but insoluble in 50%(v/v) ethanol (15, 16).

Reliable methods for the quantitation of lentinan must be available ifshiitake mushrooms or shiitake derived products are to be promoted ashealth aids or functional foods. Several methods for lentinanl13-glucansquantitation have been reported from edible mushrooms. Mizono et al.(16) and Minato et al. (15) reported the development and use of an ELISAassay to detect levels of lentinan in mushrooms. Widespread adoption ofMizono et al. (16) has not occurred, possibly because of the limitedavailability of the lentinan reactive antibody.

Manzi and Pizzoferrato (12) adapted the protocol of McCleary andGlennie-Holmes (14), by which the 13-glucans from barley and malt arefirst digested to glucose by a highly purified 3-glucanase (lichenase)from Bracillus subtilis and 13-D-glucosidase, and then glucose is quanti-fied by changes in absorbance at 510 nm after oxidization by glucoseoxidase and peroxidase. Manzi and Pizzoferrato's modification for fun-gal 3-glucans included adjustments in the amount of added hydrolyticenzymes and assay time. However, Manzi and Pizzoferrato' s (12) didnot report data regarding digestion of lentinan by the lichenase and 13-glucosidase. Therefore, it is not known if lentinan is a component ofthe 13-glucan fraction that the method detects.

Brauer et al. (1) reported a method to quantitate a fraction that in-cluded lentinan. Brauer et al. (1) subjected aqueous extracts of shiitakemushrooms to fractionation by ethanol precipitation and size exclusionchromatography. These authors reported that lentinan recovery dur-ing these 2 steps was nearly quantitative. Total carbohydrate contentof the partially purified fraction was determined colorimetric ally afterreaction with anthrone using the method of Brink et al. (2). Brauer et al.(1) did not establish the purity of the carbohydrate fractions after ethanolprecipitation and gel chromatography and therefore these authors re-ferred to the fraction as high molecular weight polysaccharides (HMWP).Brauer et al. (1) did not examine the possibility that HMWP containedeither starch or glycogen. Potentially HMWP could be contaminatedwith these polysaccharides due to similarities in molecular weight and


solubility in water and ethanol. Dikeman et al. (6) reported that cookedmature and immature shiitake mushrooms contained slightly more than20% of dry weight (DW), as total starch as determined by amylogluco-sidase digestion. Some fungi produce glycogen (21), however data on itsabundance in shiitakes are lacking.

Recently Ko and Lin (ii) published a new method for quantificationof 13(1-3) glucans by fluorescence. This assay is based on the facts thataniline blue fluorescences at 500 to 520 mm when it binds to 13(1-3)glucans and the fluorescence intensity is greater when the helical confor-mation of the glucan is uncoiled under alkaline pH (11). The assay wascalibrated with curdlan, a relatively simple 13(1-3) glucan (11). Ko andLin (11) reported that shiitake mushrooms, purchased from a local freshfood market, contained only 0.37% 13(1-3) glucans on a DW basis or ap-proximately 4 mg/g DW. These values for 13(1-3) glucans are substan-tially less than the lentinan contents reported by the ELISA method (15,16) and HMWP content reported by Brauer et al. (I). It is quite possiblethat the 13(1-3) linkages of lentinan interfere with aniline blue bindingand/or its fluorescence intensity.

The objectives of this study were to: (a) further characterize the Pu-rity of lentinan in the HMWP fraction obtained by the protocol de-scribed by Brauer et al. (1); (b) determine the effectiveness of lichenaseand 13-glucosidase to degrade lentinan; and (c) compare estimates oflentinan content as determined by the Brauer et al. (1) and Manzi andPizzoferrato (12) methods.

MATERIALS AND METHODS

Mushroom Samples, Mycelium Extract and Lentinan. The Lentinulaedodes mycelium extract was obtained from Pack Kwang C & S, Co.Ltd. (Sung Man City, South Korea). Purified lentinan stabilized withmannitolwas obtained from Eureka Biochemicals (South Melbourne,Australia). Contents of the ampoule were dissolved in water. Lentinanwas precipitated by the addition of an equal volume of ethanol and cen-trifuging at 10,000 g for 60 mm. The resulting pellet was dissolvedin water to create a stock solution of lentinan standard with a totalcarbohydrate content of 1 mg per ml. Dried substrate grown shiitakecaps were obtained locally from Wal-Mart (World Variety Products, Inc.,Los Angles, CA, USA).

The Shirley Community Development Corporation produced SHVDand SHCH powders. These two acronyms were chosen to identify two

Brauer, Kimmons, and Phillips 19

different preparations from shiitake mushrooms. Shiitake caps (strainSnowcap) were harvested from oak at veil break and frozen immedi-ately. SHVD powder was produced by lyophilizing frozen caps in alarge commercial scale freeze drier, grinding and packaging the result-ing powder in vacuum sealed, plastic commercial food bags. SHCHpowder was produced in a similar fashion except that the caps werelyophilized in a research-type freeze drier. Four samples of log-grownshiitake caps grown at Booneville, AR were included in these studies.One sample consisted of caps harvested at or just after veil break fromwhite oak (Quercus alba L.) logs inoculated with the Westwind strain.This sample (Westwind on oak logs) was included because previousstudy (1) indicated its HMWP content was high. The other three shiitakemushroom samples, designated as EP2, EP9, and EP12, included capsfrom several species of logs each inoculated with a different strain ofshiitake, thus representing an average of several shiitake strains and treespecies combination. Caps in all four samples were frozen immediatelyafter harvesting and then lyophilized. After freeze-drying, caps wereground, and stored at —20°C.

Aqueous extracts of shiitake mushroom samples and mycelium ex-tracts were prepared as described previously (1). Briefly, finely groundmushroom caps and mycelium extract were suspended in 40°C using25 ml of water per g DW and extracted overnight (12 to 16 h) on a rotaryshaker running at 150 rpm. Extracts were filtered through Whatman#42 paper and the residue was leached with an additional 10 ml of 40°Cwater. Filtrates were centrifuged at 12,000 g for 30 min at 15°C to re-move fine particulate material and the supernatant was used as the crudeextract.

3-Glucan Method. The method of Manzi and Pizzoferrato (12) wasfollowed as closely as possible. Glucose assay kits were obtained fromMegazyme, International Ireland Limited (Wicklow, Ireland). Lichenasesolution was diluted with 10 ml 20 mM sodium phosphate buffer (pH 6.5)to produce a 100 Units/ml stock solution. Up to 1 ml aliquots of mush-room extracts were diluted to 3 ml with 20 mM sodium phosphate buffer(pH 6.5) and 0.1 ml of the 100 Units Lichenase/mI stock were added. Af-ter 60 min at 40°C, 12 ml of water were added to the lichenase digestionsolution, the solution was filtered and 0.3 ml aliquots of filtrate weresubjected to 3-glucosidase digestion. The 3-glucosidase solution wasdiluted 10-fold with 50 mM sodium acetate buffer (pH 4.0) to achievea stock solution with 4 Units/ml. The 3-glucosidase reaction was a!-towed to proceed for 60 min at 40°C. At the end of the incubation pe-riod, 3 ml of the glucose oxidase/peroxidase reagent were added per


3-glucosidase digestion mixture. Color development was allowed toproceed for 20 min at 40°C before glucose content was determined byabsorbance at 510 nm. Highly purified glucose was used as standards.Where indicated, a standardized barley flour sample (4% 13-glucan contentby DW, Megazyme, International Ireland Limited, Wicklow, Ireland)was used. Typically, 0.2 g of the barley flour sample was suspended in Iml of 50% ethanol in water. The suspended barley flour was diluted with5 ml of sodium phosphate buffer (pH 6.5) and incubated for 2 min inboiling water to insure dissolution.

HMWP Method. Protocol followed that of Brauer et al. (1). Equalvolumes of crude extract and ethanol were combined, incubated at 20 to25°C for 10 min and then centrifuged at 12,000 g for 30 min at 15°C. Theresulting pellet was dissolved in 10 ml of water 40°C per g DW extracted.The resuspended material is referred to as the ethanol precipitate. A 0.75 mlaliquot of the ethanol precipitate was subjected to size exclusion chroma-tography on a 1 cm X 7 cm column of Sepharcyl S-300-HR. Fractionscorresponding to the void volume were collected and pooled to obtain aHMWP fraction. Analytical size exclusion chromatography was con-ducted on a Sephracyl S-500-HR gel as described previously (1).

Starch Assays. Total starch in shiitakes was determined by AOACmethod 996.11 and AACC method 76.13 after et-amylase and amyloglu-cosidase hydrolysis (13). For aqueous extractions, 0.2 ml of 80% (v/v)ethanol was added to 0.1 g of finely ground caps and then mixed. Threeml containing 300 Units of thermostable a-amylase were added to thedispersed sample and placed in a boiling water bath for 6 mm. Next,samples were diluted with 4.1 ml of 200 mM acetate butter (pH 4.5)containing 20 Units of amyloglucosidase. After 60 min at 40°C, sam-ples were diluted to 100 ml. Glucose content in 0.1 ml aliquots was de-termined colormetrically using glucose oxidase and peroxidase reagentsas described above. For dimethylsulphoxide (DMSO) extractions forresistant starch, 2 ml of DMSO were added to the ground caps dispersedwith 0.2 ml 80% (v/v) ethanol, and the mixture was incubated in a boil-ing water bath for 5 mm. After this step, samples were processed as de-scribed above for aqueous extracts. The DW starch content of a maizestarch standard (98% pure, Megazyme, International Ireland Limited,Wicklow, Ireland) was found experimentally to be 102.6 ± 1.2 (stan-dard deviation, SD) %, and 102.4 ± 1.8 (SD) % by aqueous and DMSOextractions, respectively. Approximately 67% of the DW of glycogen(Type IX, from bovine liver, Sigma-Aldrich Company, St. Louis, MO)was converted to glucose when subjected to total starch and resistant

0.20CC0°o 15

10.10

005

0.00


starch analyses, consistent with the fact that a-amylase hydrolyzesglycogen.

Total starch content of the HMWP was determined as follows. TheHMWP fractions prepared as above were resuspended in 2 nil 40°Cwater. Aliquots (1 ml) were diluted with 3 ml containing 300 Units ofthermostabie a-amylase and incubated in a boiling water bath for 6 mm.Next, samples were diluted with 4.1 ml of 200 mM acetate buffer (pH4.5) containing 20 Units of amyloglucosidase and incubated at 40°C for60 mm. Glucose content of 0.1 ml aliquots was determined color-metrically using glucose oxidase and peroxidase reagents as describedabove. Quantitative recovery of maize starch (101.3 ± 1.2 (SD) %) wasfound experimentally when such samples were subjected to this protocol.

RESULTS

Characteristics of the HMWP Fraction. The vast majority of the car-bohydrates in the purified lentinan standard eluted as a fairly symmetri-cal peak between fractions 24 and 32 (Figure 1). There was a small peak

FIGURE 1. Distribution of carbohydrates when either a lentinan standard orhigh molecular weight polysaccharide (HMWP) fraction from shiitake mush-rooms were subjected to size exclusion chromatography on Sepharcyl S-500column.

05101520253035Fraction number


of carbohydrate coinciding with the exclusion volume (fractions 12-14)with the lentinan standard. There also was a small amount of carbohy-drate eluting in fractions 20-23, just prior to the peak carbohydrate-con-taining fractions. All totaled these two peaks (fractions 12-14 and20-23) contained less than 5% of the total carbohydrate applied to thecolumn. The distribution of carbohydrates in the HMWP fraction wassimilar to that of the lentinan standard because most of the carbohydrateeluted in fractions 24 and 32; however, there were differences in the elu-tion pattern between the HMWP fraction and the lentinan standard. Thepresence of very high molecular weight carbohydrates eluting with thevoid volume in fractions 12-14 was less with the HWMP fraction. Car-bohydrates with a slightly greater molecular weight than the bulk ofthe carbohydrate appeared to elute as two separate carbohydrates thatwere only partially resolved from each other and bulk of the carbohy-drate. The amount of carbohydrate in the higher molecular weight poly-saccharide of these two components eluting in fractions 20-23 wassimilar in quantity to that in the lentinan standard. The carbohydrateseluting as a shoulder to the bulk of the carbohydrate in fractions 24-26were greater in quantity in the HMWP fraction than the lentinan standard.

The percentage of the total carbohydrates from the HWMP fraction re-covered in the fractions 24-32 was calculated assuming the carbohydratesin the shoulder would elute as a symmetric peak. As such, a lentinan pu-rity of 78.2% for the HMWP fraction was obtained for the extract (Fig-ure 1). Similar fractionations were conducted for HMWP fractions froma variety of shiitake samples varying in the amount of carbohydrate inthe HMWP fraction from 9 to 60 mg carbohydrate per, g DW. The per-centage of carbohydrate eluting in the' fraction 24-32 was independentof the amount of carbohydrate in the ethanol precipitate and averaged77.3 ± 1.2(SD)%(n=6).

Total starch analyses of shiitake caps detected significant quantities ofstarch (Table 1), ranging from 20 to 70 mg starch per g DW. Values forresistant starch, that is, DMSO extractable levels, tended to be 40 to 100%greater than values obtained by aqueous extraction. The starch values re-ported here are less than the 20% DW reported for cooked shiitakes (6).

The amount of starch in the HMWP ranged from 2 to 8 mg per g DWof mushrooms extracted or about one-tenth of the total carbohydratesin HMWP. Therefore, starch was only a minor component of HMWPfraction.

Characteristics of the /3-Glucan Method. Over 95% of the 13-glucanin a standardized barley flour sample was detected when subjected to


TABLE 1. Starch content of shiitakes and HMWP fraction.

Shiitake cap Total starch HMWP fraction

sampleAqueousDMSO1 Total Totalextractextractcarbohydratesstarch

(mg/g dry weight)

EP242.7 ± 3 . 2268.2 ± 6.147.4 ± 3.23.6 ± 1.1

EP922.5 ± 2.145.8 ± 2.424.1 ± 1.22.0 ± 0.4

EP1245.2 ± 2.263.2 ± 2.176.1 ± 8.17.8--2.1

1 DMSO, DimethylsulphoXide.213ata are the average plus/minus standard deviation of 6 determinations.

the method of Manzi and Pizzoferrato (12). The average percentage of3-glucans detected for 6 separate analyses was 96.7 ± 1.2 (SD) %.These results indicate that the protocol was capable of detecting barley3-glucans.

The amount of 3-glucan in crude extracts from shiitakes was lessthan the HMWP content as determined by the Brauer et al. (1). A sam-ple of shiitake caps was found to contain 11.1 ± 1.2 (SD) mg HMWPper g DW (n = 6), while the 3-glucan content of crude aqueous extractswere found to be only 2.6 ± 0.4 mg 3-g1ucan per g DW (n = 6). The3-glucan content of the ethanol precipitate was less than that of thecrude extract, averaging 2.0 ± 0.8 mg 3-g1ucan per g DW (n = 6).

3-glucan assays of shiitake crude extracts and ethanol precipitateswere conducted in the presence and absence of a standardized barleyflour sample to ensure'that the enzymes of the 3-glucan assay were fully.functional. In such systems, 95.9 ± 1.3% (n = 5) of the 3-glucans in thestandardized barley flour sample was detected when added to ethanolprecipitates prepared from shiitakes. Also, 94.7 ± 1.7% (n = 5) of3-glucan added as a standardized barley flour sample was detectedwhen added to crude extracts from shiitakes. These results indicate thatthe enzymes used to detect 3-g1ucans were fully functional in crude ex-tracts and ethanol precipitates prepared from shiitake mushrooms. There-fore, the lower content levels of 3-glucan detected by the Manzi andPizzoferrato (12) method, as compared to the HMWP content as deter-mined by Brauer et al. (1) method, was not due to inactivation of en-zymes added as components of the 3-g1ucan assay.

Aliquots for the lentinan standard were subjected to 3-g1ucanquantitation as per Manzi and Pizzoferrato (12) protocol. No glucosewas detected when lentinan was subjected to the 3-glucan assay in four


different trials. When standardized barley flour sample was added, 94.5 ±1.1% of the added 3-glucan was detected (n = 4). These results indicatethat the enzymes used to degrade 13-glucans from cereal grains were in-effective in degrading the purified lentinan standard.

Comparison of HMWP and f3-Glucan Assays. The above results indi-cate that the 13-glucan method is probably ineffective in detecting lentinan;however the amounts of carbohydrates in the 3-glucan fraction detected byManzi and Pizzoferrato (12) could be related to lentinan levels. To investi-gate such a possibility, the HMWP and [3-glucan content of 5 differentshiitake mushroom samples were compared. HMWP content varied from9.1 to 58.2 mg carbohydrate per g DW (Table 2). As observed previously(1), highly processed, substrate grown shiitakes had the lowest HMWPcontent, whereas caps grown on logs had higher HMWP content. The13-glucan content varied from 1.6 to 5.6 mg per g DW. There was no lin-ear relationship between 3-glucan and HMWP contents. The regressioncoefficient, r, for equation generated from regressing 13-glucan contentagainst HMWP content was 0.34, which was not significant at P = 0.20.

DISCUSSION

The 3-glucan content for shiitakes reported here was similar to thatreported previously by Manzi and Pizzoferrato (12). These authors (12)reported an average 3-glucan content of 2 mg per g DW and rangedfrom 2 to 5 mg per g DW. The average 3-glucan content reported herewas similar (Table 1). Dikeman et al. (6) reported a 13-glucan content forcooked shiitakes of 0.1% of DW (approximately I mg per g) using theManzi and Pizzoferranto (12) method.

TABLE 2. High molecular weight polysaccharide (HMWP) and 3-glucan con-tent of five shiitake samples.

Shiitake sample 1HMWP content 3-glucan contentMeanSD1MeanSD1

Mycelium extract 39.626.25.00.6Dried substrate grown caps9.14.13.00.7SHCH powder 17.22.92.90.5SHVD powder 15.52.85.60.8Westwind caps on oak logs58.28.41.60.71n=62Mean ± SD

Wr

Brauer, Kimnwns, and Phillips 25

This study, and the study of Manzi and Pizzoferrato (12) reportedthat the 3-glucan content of shiitake was less if carbohydrates were sub-jected to precipitation by 50% (v/v) ethanol. A decrease in 3-glucancontent with ethanol precipitation tends to indicate that the 3-g1ucansdetected by the Manzi and Pizzoferrato (12) method does not includelentinan, since lentinan is insoluble in 50% ethanol (15, 16).

The low 3-glucan content of shiitakes reported here was not related toinactivation of the enzymes used in the assay since the recovery of glucanadded in a standardized barley flour sample was over 95%. In addition,no glucose was detected when a lentinan standard was subjected to the as-say. In total, these results indicate that the 3-glucan method describedby Manzi and Pizzoferrato (12) does not detect glucose produced from thebreakdown of lentinan. The inability of the protocol of Manzi andPizzoferrato (12) to digest lentinan and thus be useful in its quantifica-tion probably results from differences in the linkages of j3-glucans frombarley and shiitakes. The 3-glucans from barley had (1-3) and (1-4)linkages (14), whereas (1-3) and (1-6) linkages are found in lentinan(20). McCleary and Glennie-Holmes (11) used hydrolytic enzymesthat could degrade the 13(1-3) and 13(1-4) linkages found in barley3-glucans, not the 13(1-6) linkages found inlentinan.

The distribution of carbohydrates in the HMWP fraction was similarto that of carbohydrates in lentinan (Figure 1). A majority of the carbo-hydrates in both types of samples eluted just above the column's inter-nal volume, an elution consistent with a molecular weight of 400kilodaltons. The percentage of carbohydrate in these fractions was simi-lar over a fairly wide range of HMWP content. Starch was less than 10%of the total carbohydrates in the HMWP fraction (Table 1). These re-suits strongly suggest that HMWP content of shiitake will approximatetheir lentinan content, and is superior to the enzymatic f3-glucan methodfor the quantitation of lentinan.

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