Food Structure

Volume 3 Number 2 Article 12

1984

Ultrastructural Studies of Raw and Processed Tissue of the Major Cultivated Mushroom, Agaricus bisporus

E. M. Jasinki

B. Stemberger

R. Walsh

A. Kilara

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Recommended Citation Jasinki, E. M.; Stemberger, B.; Walsh, R.; and Kilara, A. (1984) "Ultrastructural Studies of Raw and Processed Tissue of the Major Cultivated Mushroom, Agaricus bisporus," Food Structure: Vol. 3 : No. 2 , Article 12. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol3/iss2/12

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ULTRASTRUCTURAL STUDIES OF RAW AND PROCESSED TISSUE OF THE MAJOR CULTIVATED MUSHROOM, AGARICUS BISPORUS

E. M. Jasinski. B. Sternberger, R. Walsh, and A. Kilara

Food Science Department, The Pennsylvania State University, 111 Borland Laboratory, University Park, PA 16802

Introduction

Commercial mushroom processors currently lose Shrinkage, the loss of weight during process­ approximately 30 percent of the mushroom weight ing (blanching and canning), continues to plaque due to shrinkage during processing (blanchi ng and the mushroom processing industry. Weight losses canning) , resulting in substantial economic loss­ can range as high as 30 percent, which results in es . Microscopy was used to assess the extent and substantial economic losses to the processor (Eby, type of chemical and structural changes induced by 1975) . Shrinkage results from a loss of water and processing mushrooms and causing shri nkage. Scan­ water soluble solids from the mushroom tissue. ning e l ectron microscopy revealed that the Ho we ver, the cause of this loss is not totally processing operations includi ng vacuum hydration , understood. Microscopy can be used in determining blanching , and thermal treatment do not damage the the severity of mushroom processing or in assess ­ integrity of the tissue. Light microscopy re­ ing the effects of processing and/or handling on vealed that the morphology of the tissue, shape the quality of mushrooms . Therefore , an ultra­ and spacing of cells, appear similar for raw and structural examination of raw and processed processed mushroom tissue. However, the intra­ (blanched and canned) mushroom tissue should pro­ cell ular material remained indistinct for both vide insight into the causes of mushroom shrink­ tissue types, and the processed tissue appeared age. distorted. Transmission electron microscopy re­ vea 1ed that commercia 1 mushroom processing caused co~e~2~;~ ~~s c~~ ~{~;~!d (~~~~~~o~i ~~ ~~e t:~r~~jor intracellular damage to the tissue. The heat {Chang, 1980). Information is available on the treatment caused the coagulation of cytoplasmic structure and ultrastructure of raw mushroom material and the disruption of intracellular mem­ tissue; however, minimal information is available branes, resulting in the loss of water holding on the ultrastructure of processed mushroom capacity of the tissue. Therefore, shrinkage of tissue. The general ultrastructure of fungi and processed mushrooms results from "denaturation" of the organelles and the associated loss of water ~::n ~~~;:~;~~~~~r(Br~~ke~;a~~~~~ ~!~k~~~\~a~~ ., holding capacity by those organelles . 1974; Angeli - Papa and Eyme , 1978 ; Alexopoulus and Mims, 1979). These investigations pertain to the stipe elongation mechanism (Craig et al. , 1977a; Craig et al . , 1979), the cell wall structure (Novaes- Ledieu and Garcia-Mendoza , 1981). basidia and septal pore structures (Craig et al . , 1977b; Flegler et al., 1976), cell mem brane development Initial paper received July 14 1984 . (Eyme and Angel i-Couvy, 1975) , the basidiospore Final manuscript received November 8 1984 . structure (Elliott, 1977; Rast and Hollenstein , Direct inquiries to E.M. Jasinski. 1977}. and nuclear distribution (Wanq and Wu, Telephone number: (814) 863-2961. 1974) . However, the relationship of-mushroom ultrastructure to pr ocessing has not been investi­ gated. Mushroom processing operati ons have two major purposes : a) to provide a steri 1 e product and b) to minimize shrinkage of mushroom tissue, to KEY WORDS: Agar icus bisporus, Ultrastructure of maximize canned product yield. Var i ous processes raw mushroom , Ultrastructure of processed mush­ that limit shrinkage have been reported (Go rmley, room, Mushroom, Canning, Transmission electron 1972; Mc Ard le et al ., 1974; Seel ma n and McArdle, m1croscopy, Scanning electron microscopy, Light 1975; Steinbuch, 1979 ). Gormley (1972) found that microscopy . freez ing unblanched mushrooms produced a higher qua lity product than freezing blanched mushrooms . Steinbuc h (1979} found that vacuum packing of

191 E. 1~. Jasinski, B. Sternberger, R. Walsh, et al. unblanched frozen mushrooms showed promise in im­ to 3% glutaraldehyde in the same buffer, post proving mushroom quality and reducing shrinkage. fixed in 1% osmium tetroxide, and treated with Seelman and McArdle (1975) found that post-harvest 0.5% uranyl acetate in 0.1 t~ sodium acetate over­ night. The samples were dehydrated with acetone g~s~rf~~ ~~s~~~o~~ ~~ ~~~~ai~1l~:~~e~~t~~:~ing and embedded in Spurr firm epoxy resin (Spurr, (PSU-3S- Process) or vacuum hydration (SSV - Process) 1969). Samples were sectioned, stained with 1% prior to blanching, increased the canned product toluidine blue for light microscopy or with 3% yield (decreased shrinkage). Increased yields due uranyl acetate in 7. 5% methanol, 0. 2% lead c i­ to storage at elevated temperatures supported the trate (Venable and Coggeshall , 1965), and Reynolds hypothesis that chemical changes within the mush­ lead stain (Reynolds , 1963) for transmission elec­ room tissue caused an increase in water retention tron microscopy. A Leitz- Ortholux 1 ight micro­ by the tissue (Beelman and McArdle, 1975). The scope and an Hitachi HUllE electron microscope higher storage temperatures resulted in an in­ were used . crease in the mushroom tissue's ability to absorb more water before processing and retain more water Results and Discussion during processing (Beelman and McArdle , 1975). These results indicate that a loss of water hold­ Typical conm1ercial mushroom processing 1n ing capacity or water binding capacity of the the United States (F ig. 1) involves washing the mushroom tissue during processing causes shrink­ mushrooms, then cold storage for up to 48 hours, age. and applying a vacuum hydration treatment, which Chemical changes in the protein structure of completes the pre-processing steps . The remain­ the mushroom tissue could produce the increase in ing steps of the process include blanching, fill ­ water holding capacity during storage (Beelman inq, brining, closing, thermal processing, cool­ et al. , 1973). However, the specific chemical and ing, and storage. The major

192 Ultrastructural Studies of Agaricus bisporus

CXJI I I NF OF CQ MMFR C!AI Ml !SHROOM PROCESSED PR:JCESS I NG OPERATION

CAP OUTSIDE- SURFACE 1---11 00pm

Fig . 1. Flow chart of a typical comnercial mush ­ Fig. 4. SEM micrograph of the outside surface of room processing operation. raw (a) and processed (b) mushroom cap. Arrows indicate gaps large enough for bacteria to enter.

RAW PROCESSED

STEM OUTSIDE - SURFACE 1--1100pm GILLS H 100pm OUTSIDE- SURFACE

Fig. 2. Scanning electron micrograph of the outside surface of raw (a) and processed (b) mush­ Fig. 5. SEM micrograph of the outside surface of room stem . raw (a) and processed (b) mushroom gills.

RAW PROCESSED RAW

STEM GILLS 1---11 00pm CROSS SECTION OUTSIDE-SURFACE H10pm

Fig - 3. SEf1 micrograph of the cross- secti on of raw Fig . 6. SEM micrograph of the outside surface of (a) and processed (b) mushroom stem . Arrows raw (a) and processed (b) mushroom gills. i ndicate septa .

193 E. M. Jasinski, B. Sternberger , R. Walsh, et al. processing (Figs. 2-6). This indicates that the the cells (Figs. Sa and 9c), which oress the cyto­ large amount of water forced into the mushroom plasmic material near the cell wall (Fig. 9a). tissue durinq vacuum hydration does not damage the Therefore, the majority of coagulated electron external cellular structure . The loss of water dense material remains necr the cell wall in the from the tissue during blanching and thenna l pro­ processed tissue (Fig . 8b ). The gills contain cessing results in the shriveled and distorted ap­ the greatest amount of i nt racellular material pearance of the tissue. The second portion of (F ig. lOa) due to the gills being the reproductive this investigation used light microscopy to deter­ region of the mushroom and these regulate other mine if the general morpho1ogy changed during pro­ functions such as stipe growth (Hagimoto and cessing. Konishi, 1972). In this case, the electron dense Light microscopy revealed several character­ material generally appears in the center of the istics present in all sections of the mushroom cell leaving gaps between it and the cell wall tissue. First, the morphology of the tissue, (Fig. !Db). These gaps may permit the gill tissue shape and spacing of cells, appear similar for the to lose more water than t he cap or stem during raw and processed mushroom tissue. Septate hyphae processing . compose the cap and the stem with random crossing Second, the outer layer of the cell wall at hyphae in the cap and longitudinal hyphae in the the tips of the basidia appear broken and sepa­ stem. The gills contain three layers of cells rated from the rest of the cell wall (Fig. lOb, (Craig et al., 1977b), which include trama cells arrows). This phenomenon did not appear in any (T), subhymenial cells (SH), and hymenial cells other section of the mushroom tissue. The broken (H) (Fig. 7). Second, the intracellular material cell wall may allow more ~later to leave the of the raw and processed tissue remained indis­ tissue. tinct (Fig. 7). Finally, all of the processed Finally, mushroom processing causes distor­ tissue appeared distorted although the organiza­ tion in the processed tissue (Figs. 8b,9b,l0b). tion remained the same as in the raw tissue The distortion, most apparent in the gill tissue (Fig. 7). The final portion of this investigation (Fi~. lOb), appears as irregular shaped cells with used transmission electron microscopy to determine gaps or channels between cells. Commercial pro­ if intracellular damage occurred as a result of cesses allow the absorption of water into the mushroom processing . mushroom tis sue. However, whether the water is The transmission electron micrographs (Figs. absorbed directly into the cells or into the gaps 8-10) revealed that comme rcial mushroom processing between the cells has not been determined. In results in damage to the intracellular material any case, processinq releases all of the absorbed (Figs. 8b,9b,!Db). The raw mushroom tissue (Figs. water plus part of the original water from the 8a,9a,l0a) contained identifiable intracellular tissue, causing the distortion of cells and chan­ material characteristic of basidiomycetes (Bracker, neling between cells seen in the processed tissue 1967; Beckett et al., 1974; Eyme and Angeli-Couvy, (Figs. 8b , 9b,!Db). 1975; Craig et al., 1977a; Alexopoulus and Mims, 1979; Craig et al ., 1979). The structures include Summary and Conclusions cell walls (W), cellular membranes (CM), nucleus (N) , nuclear membranes (NM) , mitochondria (M), Commercial mushroom processing appreciably concentric lamellae membranes (CL), electron dense alters the intracellular organization of the mush­ bodies (DB), lipid droplets (D), and vacuoles (V). room tissue causing shrinkage . Scanning electron The damaged processed mushroom tissue lacks iden­ micrographs showed that processing did not destroy tifiable cellular material except for remnants of the surface s true tura 1 features of the mushroom the cell walls (W), cellular membranes (CM), tissue. However , the processed tissue appeared dolipore septum (S), and vacuoles (V). There­ shriveled and distorted, though intact. light maining intracellular material appears as clusters micrographs revealed similar cell types for raw of precipitated electron dense material (D) and processed tissue, but the processed cells throughout the cells (Figs. 8b,9b,!Db). appeared distorted. Transmiss ion electron micro­ Severa l of the consequences of commercial graphs showed that processing caused intracellular mushroom processing appear in the transmission damage to the mushroom tissue. All cellular or­ electron micrographs (Figs. 8-10). First, as ganelles became coagulated electron dense material previously mentioned, the intracellular material resulting in a loss of water holding capacity of appears as electron dense clusters rather than the organelles. This results in shrinkage of the as distinct organelles (Figs. 8b,9b,!Db). The tissue due to a loss of water and water soluble heat treatment applied during processing caused solids. Therefore, the loss in canned product the coagulation of cytoplasmic material such as yield of processed mushrooms results from proteins and the disruption of the compartmental­ "denaturation" of the organelles and the associat­ izing intracellular membranes {Figs. 8b,9b,1Db). ed loss of water holding capacity by those organ­ The coagulation and the disruption of intracellu­ elles. lar membranes result in the loss of water holding capacity of the tissue so that the tissue shrinks Acknowledgement to 80 percent of its original weight. However, the intact cell wall and coagulated material traps Published as Paper No. 6738 Journal Series of The the remaining water in the tissue. Pennsylvania AgriculturarExperiment Station, Two types of tissue are present in the mush­ University Park, PA 16802 room, the stem and cap, and the gills. The stem and cap contain large vacuoles in the center of

I 94 Ultrastructural Studi es of Agaricus bisporus

RAW PROCESSED

GILLS CROSS - SECTION

Fig. 9. TEM micrograph of the cross- section of the Fig. 7. light micrograph of the cross-section of raw (a) and processed {b) mush room stem . Nucleus the gills for raw (a) and processed (b) mushroom. (N), cell walls (W) , cellular membranes (CM) , Trama (T), subhymenia l (SH) , and hymenial (H) cells mitochondria (M) , and electron dense material {0) are visualized . are observed.

RAW PROCESSED RAW PROCESSED

..

Fig. 8. Transmiss i on electron micrograph of Fig. 10 . TEM mic rograph of the cross-section of the l ongitudina l cut of the raw (a) and processed the gills for raw (a) and processed (b) mushroom (b) mushroom stem . Nucleus (N), cell walls (W) , contains nucleus (N), 1 ipid droplets (0), vacuoles mitochondria (M) , concentric lamellae (CL), dolipore (V), concentric lamellae (Cl) , and electron dense septum {5), vacuo les (V) , and electron dense mat­ materia l (D). The arrows indicate partially erial (D) are observed. broken cell wall.

References See lman RB, McArdle FJ. (1975). Influence of post-harvest storage temperatures and soaking on Alexopoulus CJ, Mims CW. (1979). Introductory yield and quality of canned mushrooms, J . Food Mycology, John Wiley and Sons , NY, 414-428, 446- Sci., ~ . 669-671. 469. Bonner JT, Kane KK , Levy RH . (1956). Studies on Angeli-Papa J , Eyme J. (1978). Ultrastructural the mechanics of growth in the common mushroom , changes during development of Agaricus bisporul Agaricus campestris , Mycologia, .1§_. 13- 19. and Agaricus sylvicola, in: The Biology and Cu ti­ vation of Edible Mushrooms, S. T. Chang and W. A. Bracker CE. (1967). Ultrastructure of Fungi. Hayes {eds. ), Academ ic Press , NY, 53-81. Ann . Rev. Phytopathology, .?_, 343- 374 . Beckett A, Heath IB, Mclaughlin OJ . (1974) . An Chang ST. ( 1980). Mushrooms as human food. Bi o­ At l as of Fungal Ultrastructure, Longman Group science, 1Q, 399- 401. Limited, London, 181-209. Craig GO, Gull K, Wood OA. (1977a). Stipe elon­ Seelman RB, Kuhn GO, McArdle FJ. (1973). Influ­ gation in Agaricus bisporus. J. Ge n. Micro., l.Q£, ence of post-harvest storage and soaking treat­ 337- 347. ments on the yiel d and quality of canned mushrooms, Craig GO, Newsman RJ, Gull K, Wood OA. (1977 b). J. Food Sci ., ~ . 951-953. Subhymenial branching and dolipore septation in

195 E. M. Jasinski, B. Sternberger, R. Walsh, et al.

Agaricus bisporus, Tran. Br. Mycol. Soc., £2., 337- Venable JH, Coggeshall R. (1965). Simplified 344. lead citrate stain for use in electron microscopy, J. Cell Biol., 407-408. Craig GO, Newsman RJ, Gull K, Wood DA. (1979). £, An ultrastructural and autoradiographic study of Wang HH , Wu JY. , (1974). Nuclear distribution in stipe elongation in Agaricus bisporus, Protoplasma, hyphal system of Agaricus bisporus, Mushroom Sci. 2§., 15-29. IX, 23-29. Eby DL. (1975). Post-harvest s torage of the cul­ Webster J . (1970) . Introduction to Fungi, Cam­ tivated mus hroom (Agaricus bisporus) and its in­ bridge University Press, Cambridge, 279- 310. fluence on qualitative protein changes related to canned product yield, M.S. Thesis, Pennsylvania Discussion with Reviewers State University, University Park, PA, 1-15, 32-51. O.A. Wood· Why "'ere only the beginning and end Elliott, TJ. (1977). Basidiospore number in stages of the process examined since the changes Agaricus bisporus (Lange) Imbach, J. Bacteriology, observed may have occurred during one of several m. 525-526. processes? --­ Eyme MJ, Angeli-Couvy J. (1975) . Cytologie des Authors : The raw mushroom tissue was impregnated hyphes d'Agaricus sylvicola etA. bi sporus : evolu­ with glutaraldehyde under conditions similar to tion des systemes membranaires cytoplasmiques au the vacuum hydration process (see Materials and cours du developpement. Comptes Rendus Acad. Sci. Methods section). Since all cellular structures Paris, 281, Ser D, 771-774. were observed in the raw tissue, the vacuum hy­ dration process d.oes not cause the change in the Falk RH . (1980). Preparati on of plant tissue for tissue. The thermal treatment accorded to the SEM, Scanning Electron Microsc. 1980; II: 79-87. tissue during blanching is less severe than Flegler SL, Hooper GR, Fields WG. (1976). Ultra­ thermal processing . Also, most of the ma terial structural and cytochemical changes in the basid­ lost from the mus hroom during blanching was water iomycete dolipore septum associated with fruiting, impregnated into the tissue during vacuum hydra­ Can. J. Bot., ~. 2243-2253. tion {Beelman and McArdle, 1975). Therefore, we felt that the significant change in the ultra­ Gabriel BL . (1982). Biological Electron Micros­ structure of the tissue results from thermal copy, Van Nostrand Reinhold Co. Inc., NY, 79-149. processi ng; hence, the reason for examining the Gormley TR. (1972) . Quality evaluation of frozen beginning and end stages of the process. mushrooms, Mushroom Sci. VIII, 209-219. D. A. Wood· Cou l d you produce these changes merely Hagimoto H, Konishi M. {1972). Studies on the by therma 1 processing? growth of fruit body of Fungi I. Existence of a Authors: Yes. hormone active to the growth of fruit body in Agaricus bisporus (Lange) Sing, Bot. Mag. Tokyo, F. Ingratta: In what way do the authors feel the 855, 36!-366. freezing method o f proces~ing mushrooms would af­ McArdle FJ, Kuhn GO, Seelman RB. (1974). Influ­ fect the cell ultrastructure? ence of vacuum soaking on yield and quality of Authors. Integrated Quick Blanching (IQB) and canned mushrooms , J . Food Sci ., 12.• 1026-1028. Integrated Quick Freezing (IQF) methods currently used by the frozen mushroom industry, insure the Monacha MS. {1965). Fine structure of the Agaricus carpophore, Can. J. Bot. , 1}. 1329-1334. creation of smal l uniform ice crystals in the frozen product. Assuming minimal thermal shock Novaes-Ledieu M, Garcia-Mendoza C. (1981). The during storage, ~" e would speculate that the ultra­ cell walls of Agaricus bisporus and Agaricus structure of f ro:oz:en mushroom tissue would be campetris fruiting body hyphae, Can. J. Micro., similar to that of raw mushroom tissue. If ther­ I?_, 779-787. ma l shock becomes a factor leading to the forma­ tion of large ice crystals, resulting from the Rast 0, Hollenstein GO. (1977). Architecture of the Agaricus bisporus spore wall, Can. J. Bot., migration of int1racellular water to intercellular water, the rupturre of cell membranes would be §2. 2251-2262. expected. However, we would not necessarily ex­ Reynolds ES. (1963). The use of lead citrate at pect to visualize the electron dense material that high pH as an e l ectron opaque stain in electron was seen as a result of thermal coagulation. microscopy, J. Cell Biol., .!2• 208- 2!2. D.A. Wood· What is the chemical composition of Spurr AR . (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ult. the solids lost from the mushroom tissue during processing? Res . , ~. 31-43. Authors: The water absorbed by the mushroom tis­ Steinbuch E. (1978). Factors affecting quality sue during vacuum hydration is the major compo nent and shrinkage losses of processed mushrooms, Mush­ lost during processing (Seelman and McArdle , 1975; room Sci. X (part II), 759-776. Steinbuch, 1978) . This water contains all of the water soluble pr

196