Postharvest Biologyand Techn(~ogy

ELSEVIER Postharvest Biology and Technology 4 (1994) 99-110

Ageing of the mushroom (Agaricus bisporus) under post-harvest conditions

A. B r a a k s m a *, D . J . S c h a a p , T. d e Vr i je , W . M . E J o n g e n , E . J . W o l t e r i n g

Agrotechnological Research Institute (ATO-DLO), P.O. Box 17, 6700 AA Wageningen, The Netherlands

(Accepted 21 September 1993)

Abstract

Mushroom sporophores (Agaricus bisporus) as closed caps 3-5 cm in diameter were analysed for membrane constituents and properties, and compared with similar tissue from mushrooms stored for 3 days at 20°C. The leakage of non-specific or specific (K +) ions from pileus tissue was unaffected by storage, as was the content and composition of phospholipids and fatty acids. The sterol/phospholipid ratio and the fluidity of liposomes was unaffected by ageing. However, the membrane proteins in pileus and gill tissue decreased, but membrane viscosity remained constant. In contrast to higher plants, mushroom senescence appears to be independent of degradation of specific membrane lipid components.

Key words: Ageing; Agaricus bisporus; Fatty acids; Membrane fluidity; Mushroom; Phospho- lipids; Sterols; Post-harvest

I. Introduct ion

T h e physiology of the spo rophore of the fungus Agaricus bisporus ( the c o m m o n m u s h r o o m ) is d i rec ted towards the deve lopmen t of viable spores. Dur ing post- harves t life, the ma in processes associated with the deve lopmen t of the sporophore , such as e longat ion of the stipe, opening of the pileus, expansion of gill-tissue and spore format ion , p roceed in a way similar to m u s h r o o m s that are still in contac t

* Corresponding author. Fax: (08370) 12260.

Abbreviations: DTI" = dithiothreitol; DPH = 1,6 diphenylhexatriene; GC = gas chromatography; HPTLC -- high performance thin layer chromatography; MSTFA -- 9N-Methyl-N-TMS-Trifluoroacet- amide; PMSF = phenylmethylsulfonyl fluoride; HEPES = 4-[2-hydroxyethyl]-l-piperazineethanesul- fonic acid; EDTA = [ethylenedinitrilo]tetraacetic acid; Tris-HCl = 2-amino-2-[hydroxymethyl]-l,3-pro- panediol; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PS/PI = phosphatidylserine and/or phosphatidylinositol; PA = phosphatidic acid; CL = cardiolipin.

0925-5214/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved. SSDI 0 9 2 5 - 5 2 1 4 ( 9 3 ) E 0 0 5 6 - J

100 A. Braaksma et aL / Postharvest Biology and Technology 4 (1994) 99-110

with the mycelium. These phenomena, together with a general loss of appear- ance, are considered negative quality characteristics. Post-harvest senescence in a variety of horticultural commodities is accompanied by changes in cell membrane characteristics leading to loss of barrier function and loss of turgescence (Mazliak, 1987 and references therein). In mushrooms, softening or loss of firmness during post-harvest life is ascribed to changes in membrane permeability (Beelman et al., 1987). In rose and carnation petals and in senescing leaves membrane composi- tional changes, in particular changes in their sterol/phospholipid ratio, were shown to be associated with senescence (McKersie et al., 1978; Lees and Thompson, 1980; Borochov et al., 1982a, b; Thompson et al., 1982; Sylvestre et al., 1989). Increased sterol/phospholipid ratios are known to decrease membrane fluidity and may affect the conformation and function of proteins (e.g. enzymes), that are membrane- embedded (Adam et al., 1983; Borochov et al., 1986; Duxbury et al., 1991). The increase in sterol/phospholipid ratio is generally ascribed to a decrease in phos- pholipid content, whereas the sterol content remains unchanged (Borochov et al., 1982a; Borochov and Faiman-Weinberg, 1984).

The present study was undertaken to determine whether similar processes are of equal importance during post-harvest development and senescence of the edible fungusAgaricus bisporus. Therefore, we analyzed the electrolyte leakage of different tissues, membrane composition and fluidity of membrane vesicles derived from different tissues from the mushroom immediately after harvest and after 3 days storage at 20°C.

2. Materials and methods

Experimental material The mushrooms (race U1), cap diameter 3-5 cm, of first flush, used in this study

are harvested at commercial stage, e.g. buttons with a pileus diameter of 3-5 cm. After 3 days (75 h) at 20°C and high relative humidity (>93%), the pileus has fully opened, spore-formation has started and visible symptoms of deterioration are apparent (i.e. browning, appearing of cavities in the stipe).

Measurement of electrolyte leakage Cylindrical segments of 5 mm diameter were excised from a particular tissue

and sliced into 3 mm thin discs. Extreme care was taken to prevent squeezing the tissue in any way. The discs were immediately immersed into 20 ml of demineralized water. The change in total electrical conductivity (conductivity meter PW9505, Philips, Eindhoven, The Netherlands) and the increase of K+-ions (K+-electrode: Orion, 319BN; reference electrode: UX1, Orion Research Inc., USA) in the water was simultaneously measured during 15 min. Following two cycles of freezing and thawing and eventually disrupting remaining tissue with a glass rod, the total conductivity and K + concentration were measured (100% value).

Analysis of membrane components Tissues were frozen in liquid nitrogen and freeze-dried. Lipids were extracted

A. Braaksma et al. /Postharvest Biology and Technology 4 (1994) 99-110 101

under an atmosphere of nitrogen gas according to the method described by Bligh and Dyer (1959) from freeze-dried, powdered tissue. The methanol-chloroform fraction was dried and stored under N2 at -25°C. Alternatively, lipid extracts were made according to the method described by Kates and Eberhardt (1957) using fresh material.

Sterols were first separated from other lipids present in the extract by high performance thin-layer chromatography (HPTLC) on silicagel (Kieselgel 60, Merck, Darmstadt, Germany) with chloroform : diethyl ether : ammonia (25%) = 90 : 10 : 0.7 (v/v) as solvent. Sterols were scraped from the plate and silylated with 9N-methyl- N-TMS-trifluoroacetamide (MSTFA) during 30 min at 120°C in hexane and de- termined by gas chromatography (GC). The GC (Perkin Elmer Sigma 2000) was equipped with a WCOT fused silica 25 m x 0.25 mm ID CP-SIL 5CB column (Chrompack, Middelburg, The Netherlands). The temperature programming was 1.5 min at 240°C, then increasing with 20°C/min up to 300°C. Total run time was 15 min. Betulin was present as an internal standard throughout the procedure. Sterol identification was done by comparing the retention time with commercial standards (Sigma, St.Louis, MO, USA) and by GC-MS (Carlo Erba QMD1000, equipped with the same column as used for GC analysis of the sterols and a temperature program of 250°C, increasing with 5°C/min up to 300°C).

Sterol analysis was also performed using the colorimetric method described by Courchain et al. (1959), following HPTLC separation (without betulin) as described above or after digitonin precipitation to determine the free sterol content. For analysis of the phospholipid composition, the total lipid extract was separated by HPTLC using chloroform : methanol : ammonia (25%) : water = 90 : 54 : 5.5 : 5.5 (v/v) as solvent one and chloroform : methanol : acetic acid : water = 90 : 40 : 12 : 2 (v/v) as solvent two. Identification was carried out by using commercial standards (Sigma, St. Louis, MO, USA). The spots were visualised by iodine vapour, scraped off and phosphate-quantified by the method described by Rouser et al. (1970). The recovery of phosphate on HPTLC was more than 93%.

Lipid-bound fatty acids were determined by GC as described by Muuse and Van der Kamp (1985) by incubating 0.3 ml extract with 0.2 ml 2 M KOH in methanol. After shaking for 1 min, the extract was neutralised with NaHSO4 and dried with Na2SO4. After additional shaking (30 s) phase separation was accelerated by short centrifugation. The lower phase was injected in the GC. Total fatty acid concentration and composition were determined by GC essentially as described by Huijberts et al. (1992). Triglyceride content was estimated with a Carlo Erba HRGC5300 gas chromatograph, equipped with a HTSIMDIST CB column (5 m x 0.5 mm) with a temperature program running from 150 to 360°C with 25°C/min.

Preparation of liposomes and microsomal vesicles Liposomes were prepared from dry lipid films of the total lipid extract containing

5/zmol phospholipid. The lipid films were hydrated in 1 ml of 100 mM HEPES (pH 7.2) and 100 mM NaC1 and sonicated five times for 30 s with 30 s intervals on ice with a Vibracell sonicator (Sonics and Materials Inc., Danbury, CT, USA), at 40% output energy. A subsequent centrifugation step (10,000 g) yielded no pellet.

102 A. Braaksma et at /Postharvest Biology and Technology 4 (1994) 99-110

For preparation of microsomal membrane vesicles, 10-13 g of fresh tissue was ground in two volumes of 50 mM HEPES (pH 7.4), 2 mM EDTA, 1 mM 1,4-dithio- treitol (DTF), 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 250 mM sucrose at 0°C. A clear supernatant was obtained by two centrifugation steps (30,000 g, 10 min). The microsomal vesicles were pelleted by ultra-centrifugation (100,000 g, 60 min) and redissolved in the same buffer solution without PMSE

Fluidity measurements and protein determination Fluidity measurements were performed on liposomes and membrane vesicles at a

concentration of 0.5 mM phospholipid and 10/xM of the lipophilic fluorescent probe 1,6-diphenylhexatriene (DPH) in the above-mentioned buffer after an incubation of 30 min at 37°C. Fluorescence polarization of the DPH-labelled membranes was measured in a device previously described by Visser and Lee (1980).

Protein determinations were carried out as described by Bradford (1976) with bovine serum albumin as a standard. The outcome of the analyses of tissue from day 0 and day 3 were tested statistically by using Student t-test, at 95% confidence.

3. Results

Electrolyte leakage The increases in K + leakage and in total conductivity in eluates from fresh and

three-days old pileus tissue are shown in Fig. 1. Following an initial rapid phase of K + leakage during the first 2 min, a steady loss of K + over time was observed. The rapid initial phase is ascribed to washout from damaged cells. The much slower, almost linear phase, is ascribed to efflux from the cytoplasmic compartment of undamaged ceils. The data of the leakage experiments with tissue from day 0 and day 3 were not statistically significantly different. The rate of K + leakage was similar for fresh and aged tissues. A similar pattern was observed from the total electrolyte leakage data which were measured simultaneously. Again, leakage was similar in fresh and aged tissues. Also in discs excised from the stipe, both K + and total electrolyte leakage were similar for fresh and 3 days old tissues (data not shown). Excessive wounding during isolation of gills made it impossible to perform leakage experiments in these tissues. The data on leakage in stipe and pileus tissues indicate that ageing is not accompanied by increased membrane permeability. Using a calibration curve, it was calculated that K + leakage was responsible for most of the increase in total conductivity.

Weight changes During post-harvest development, changes in fresh and dry weight were observed

due to continued transpiration/respiration and respiration, respectively. The de- crease in fresh weight over a 3-day period was 6.9-4-1.2% for whole mushrooms. We determined the relative contribution of the pileus, gills and stipe to the total fresh and dry weight of fresh and aged mushrooms (Table 1). Judging from the Student t-test (90% significance) the contribution of the pileus and gills to the total fresh weight slightly changed during the 3-day post-harvest development. The

A. Braaksma et aL / Postharvest Biology and Technology 4 (1994) 99-110 103

~.. 100 o~ w 80 < v

60 w ..J w > 40 I -

..i w 20 t~

0

v

LU 80 <

< 60 w _J w -> 40 I--- < ...i w 20 n"

DAY 0

DAY 3

Total

6 2 4 6 8 10 12 14

T IME (MIN)

Fig. 1. Changes in total conductivity ( l ) and K+-ions (,x) in eluates from fresh (day 0) and three days old pileus tissue discs. Vertical bars represent 2 x SD (n = 5).

Table 1 Contribution of the different parts of the mushroom to the total fresh and dry weight in three experiments

Part Time Mean fresh weight Mean dry weight (d) (%) (%)

Pileus 0 55.6 (1.2) 6.5 (0.3) 3 49.5 (4.5) 5.8 (0.2)

Gills 0 22.5 (0.8) 7.9 (0.3) 3 26.8 (0.2) 7.5 (0.3)

Stipe 0 22.0 (0.2) 7.0 (0.4) 3 23.8 (4.3) 5.2 (0.4)

Each experiment consists of 10 mushrooms and each experiment is a different batch. Fresh weight is expressed in % relative to the total weight. Dry weight is expressed in % of fresh weight. Figures in brackets represent the SD over the three experiments.

104 A. Braaksma et aL / Postharvest Biology and Technology 4 (1994) 99-110

contribution to the fresh weight of the pileus decreased and the contribution to the fresh weight of the gills increased. A decrease in dry weight of about 18% was observed in the stipe. The pileus showed also a (statistical significant) decrease but to a lesser extent, whereas in the gills no significant change was apparent.

Membrane composition Lipids were extracted under an atmosphere of nitrogen from freeze-dried powder

according to the method of Bligh and Dyer (1959). This extraction method was compared with the extraction method of Kates and Eberhardt (1957) which uses fresh material. This latter method is presumed to inhibit phospholipase activity immediately. In test samples these methods yielded essentially similar results with respect to phospholipid content and composition. Thereafter all analyses were done in extracts made from freeze-dried powder.

The phospholipid and sterol contents of the different tissues are shown in Table 2. No significant changes in phospholipid content as a result of ageing were observed. The relative phospholipid content in the gills was slightly higher as compared to other tissues.

Identification of phospholipids was done by two dimensional HPTLC using commercial standards. The phospholipid composition in the different tissues was comparable (Fig. 2). The relative masses were PC : PE : PS/PI : PA: CL = 42 : 42 : 11 : 2.5 : 1.5. Ageing did not affect the phospholipid composition (Fig. 2).

The free sterol content in the lipid extract was measured colorimetrically (Table 2). No significant changes in free sterol content as a result of ageing were observed. The sterol/phospholipid ratio was approximately 0.1 and no consistent change as a result of ageing was observed. The free sterol content was about 10% of the total sterol content and the ratio of bound/free sterol did not change during the 3 days of post-harvest development (data not shown). However, we did notice that the sterol spot on HPTLC (visualised by iodine vapour) was always more intense in extracts from 3 days old tissues compared with extracts from fresh (day 0) tissues.

Table 2 Phospholipid and free sterol contents of different parts of the mushroom measured directly after harvest (day 0) and after 3 days at 20°C

Part Time Phospholipid Sterol Sterol/phospholipid (d) (/xmol/g DW) (/zmol/g DW) ratio

Pileus 0 25.5 (0.6) 2.52 (0.03) 0.099 (0.004) 3 26.0 (2.6) 3.57 (0.48) 0.137 (0.012)

Gills 0 38.1 (3.6) 3.30 (1.30) 0.089 (0.040) 3 34.7 (9.4) 2.04 (0.41) 0.062 (0.013)

Stipe 0 26.5 (3.0) 2.46 (0.60) 0.092 (0.012) 3 27.5 (3.8) 2.66 (1.97) 0.089 (0.054)

Sterol and phospholipid determinations were done in triplicate. Values are means of three experiments. Figures in brackets represent SD. The ratio is the mean of the three separate calculated ratios from each batch.

A. Braaksma et aL / Postharvest Biology and Technology 4 (1994) 99-110 105

60 PILEUS GILLS STIPE

50

4o

~- 30

o , PC I PE I CL ~S)PI I PA I PC I pE I CL

PS/PI PA PC PE CL PS/PI PA

PHOSPHOLIPID COMPOSITION Fig. 2. Phospholipid composition of different parts of fresh (day 0, black bars) and old mushrooms (day 3, white bars). PC = phosphatidyicholine; PS/PI = phosphatidylserine/phosphatidylinositol; PE = phosphatidylethanolamine; PA = phosphatidic acid; CL = cardiolipine. Vertical bars in the figure represent 1 x SD for the 3 batches examined. Each batch was determined three times and the standard deviation of the mean from one batch was less than 5%.

This indicates that either the free sterol content in older tissues is higher (this was already ruled out by our data) or that a change in sterol molecular species had occurred. The sterol composition was determined by GC-MS. In fresh tissue (day 0) only mono-ergosterol (ergost-7-en-3-ol) was present, whereas in 3 days old tissue besides mono-ergosterol, also di-ergosterol (ergosta-7,22-dien-3-ol) and traces of triergosterol (ergosta-5,8,22-trien-3-ol) were present. The di-ergosterol content was tentatively estimated to be about one third of the total sterol content. The increase in double bond content in 3 days old tissue preparations explains the higher intensity in iodine staining, since iodine binds to the double bonds.

The above-mentioned data on sterol and phospholipid contents are expressed per gram dry weight. As during ageing dry weight decreases in pileus and stipe (Table 1), the absolute content of phospholipids and sterols also decreases in these tissues.

Total (lipid-bound and free) fatty acids were analyzed by GC. Linoleic acid was most abundant in all samples analyzed. No significant differences in phospholipid fatty acid composition were observed between the different parts of the mushroom and no significant changes due to ageing were apparent (Fig. 3). In gill tissue of fresh mushrooms approximately 10% of the total linoleic acid content was present as free fatty acid. This 10% accounted for more than 90% of the total free fatty acid concentration in this tissue. In aged gill tissue the amount of free linoleic acid had decreased to approximately 3%. During ageing the triglycerid content in the gills, as

106 A. Braaksma et al. / Postharvest Biology and Technology 4 (1994) 99-110

100 PILEUS GILLS STIPE

~ 60

40 >-

0 ' 1 ' 1 ' 1 ' l ' 1 ' I ' l I r l o o . o o o

~- , ~ ~ . . . . . . . ~ - t , , . , ~ ~ ~ ,

o 6 6 6 6 6 6 d ~ d d d d d o ~ d d S o -- "7 6 o

Fig. 3. Fatty acids of total lipid extract isolated from different parts of fresh (day 0, black bars) and old mushrooms (day 3, white bars). The bars represent 1 x SD (n = 3).

judged from GC data, showed a marked increase (data not shown), suggesting that linoleic acid may be used to synthesize triglycerides for spore reserves. In the other mushroom tissues the concentration of free fatty acid was low throughout the 3 days of post-harvest development.

Fluidity measurements Polarization values for liposomes prepared from total lipid extracts from different

mushroom parts labelled with DPH did not change during ageing (Table 3). This

Table 3 Anisotropy measurement upon liposomes and microsomal membrane vesicles from different mushroom tissues directly after harvest (day 0) and after 3 days storage at 20°C

Part Time Anisotropy Anisotropy membrane (d) liposomes vesicles

Pileus 0 0.131 (0.002) 0.124 (0.002) 3 0.129 (0.003) 0.114 (0.007)

Gills 0 0.132 (0.003) 0.104 (0.003) 3 0.134 (0.001) 0.097 (0.005)

Stipe 0 0.135 (0.011) 0.112 (0.003) 3 0.137 (0.001) 0.108 (0.007)

Figures in brackets represent SD (n = 5).

A. Braaksma et al. / Postharvest Biology and Technology 4 (1994) 99-110 107

Table 4 Membrane protein and phospholipid content of different mushroom tissues directly after harvest (day 0) and after 3 days storage at 20°C

Part T i m e Phospholipid/protein (d) ratio

Pileus 0 0.48 (0.12) 3 1.08 (0.05)

Gills 0 0.48 (0.06) 3 0.62 (0.02)

Stipe 0 0.73 (0.15) 3 0.74 (0.09)

Figures in brackets represent SD (n = 3).

is in accordance with the data on membrane lipid composition. Protein content of microsomal membranes showed a significant decrease during ageing in pileus and gill tissue resulting in increased phospholipid/protein ratios in these tissues (Table 4). To examine whether this may affect membrane fluidity, we measured the polarization values for microsomal membrane vesicles of fresh and aged tissues. The results indicate that the bulk lipid fluidity was not affected by a lower protein content in the vesicles (Table 3).

4. Discussion

Ageing in plants or excised plant parts is often accompanied by changes in the composition of the membranes. Generally, ageing is accompanied by a decrease in membrane phospholipid content resulting in an increased sterol/phospholipid ratio. This ratio is considered to be an important factor in determining membrane fluidity and permeability. Ageing-associated changes in fluidity and sterol/phospholipid ra- tios have been demonstrated in ageing petals, cotyledons, leaves and fruit (McKersie and Thompson, 1978; McKersie et al., 1978; Lees and Thompson, 1980; Thompson et al., 1982; Borochov et al., 1982a, b; Legge et al., 1986; Brown et al., 1987, 1991; Sylvestre and Paulin, 1987; Paliyath et al., 1987; Itzhaki et al., 1990).

In mushroom tissues the sterol/phospholipid ratio did not show a significant change during ageing while membrane fluidity, and K + and total electrolyte leakage were also not affected (Fig. 1, Tables 2 and 3). It may therefore be concluded that ageing in mushrooms is not accompanied by drastic changes in membrane properties. This is somewhat surprising because under post-harvest conditions mushrooms show clear symptoms of deterioration such as discoloration, browning, softening and appearance of cavities in the stipe (Murr and Morris, 1975; Minamide et al., 1985; Beelman et al., 1987; own observations).

The increase in sterol/phospholipid ratio observed in senescing carnation and rose petals was shown to be the result of a decreased phospholipid content rather than an increased sterol content (Borochov et al., 1982a; Borochov and Faiman- Weinberg, 1984). Experimental data on phospholipid and sterol content are usually

108 A. Braaksma et al. / Postharvest Biology and Technology 4 (1994) 99-110

expressed on the basis of membrane proteins (Itzhaki et al., 1990; Brown et al., 1991). In mushrooms, harvest induces proteolytic activity and subsequent protein breakdown (Burton, 1988; own observations). Since we have already shown that phospholipid content is relative stable during the post-harvest phase (Table 2), this leads to an increased phospholipid/protein ratio (Table 4). It is clear that, if expressed on a protein basis, the phospholipid content would increase. Our data expressed on the basis of dry weight do not show a decrease in phospholipid content. Since in pileus and stipe a decrease in dry weight occurs, it is evident that an absolute decrease in phospholipid content in the stipe and in the pileus takes place during ageing. The data therefore indicate that the occurrence of cavities in the stipe is not a process where selective breakdown of membrane constituents occurs. Ageing in harvested mushrooms is not accompanied by the same processes that are observed in ageing plant tissues.

Brown et al. (1991) showed that although the phospholipid composition may not change, specific modifications may occur in acyl chains during phospholipid catabolism. In mushroom preparations we did not find any significant change in the fatty acid content or composition (Fig. 3), indicating that a comparable situation does not take place in mushrooms. The fatty acid content and composition are in agreement with previous published data (L6sel, 1988).

In rose and carnation an overall decrease in protein content and modifications in the polypeptides in petal membranes was observed during ageing (Borochov et al., 1990). In the mushroom tissues investigated a pronounced decrease in membrane protein content was observed resulting in a changed phospholipid/protein ratio (Ta- ble 4). Although it may be hypothesized that this may affect overall functioning of the cells, the decline in membrane protein relative to phospholipid content appar- ently does not affect membrane microviscosity and permeability (Table 3), which is in agreement with work of Faragher et al. (1987) and Lynch and Thompson (1984).

In summary, the ageing-associated changes in membrane composition, mem- brane fluidity and permeability observed in most plant tissues were absent in the investigated mushroom tissues.

Acknowledgements

The authors thank T.C. de Rijk for the GC-MS measurements and G. Huijberts and B.G. Muuse for their help and advice with the fatty acid analysis. The authors thank the CNC, the Dutch Mushroom Growers Organisation for their financial contribution.

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