Analysis of Agaricus bisporus tyrosinase activation and phenolics utilization during Pseudomonas tolaasii or tolaasin-induced discolouration

Mycol. Res. 102 (12) : 1497–1502 (1998)

1497

Printed in the United Kingdom

Analysis of Agaricus bisporus tyrosinase activation and phenolics utilization during Pseudomonas tolaasii or tolaasin-induced discolouration

C. S O L E R-R I V A S1, S. J O L I V ET2, D. Y U K S EL1, N. A R P I N2, J. M. O L I V E R3 A N D H. J. W I C H E R S1 " Agrotechnological Research Institute (ATO-DLO). Bornsesteeg 59.6708 PD Wageningen. The Netherlands # Laboratoire de Mycochimie, UnitT eT de Formation et de Recherche de Chimie et Biochimie, UniversiteT Claude Bernard. 43 Bd du 11 Novembre 1918. 69622 Villeurbanne, Cedex, France $ Institut National de la Recherche Agronomique, Station de Recherches sur les Champignons, Centre de Recherches de Bordeaux. BP 81.33883 Villenave d’Ornon, Cedex, France

Discolouration due to senescence differs from blotch-related discolouration, which might indicate differential mechanisms being operational. In samples infected with bacteria or with a partially purified toxin extract, a higher degradation of total tyrosinase than in senescening mushrooms was found. Simultaneously, the active tyrosinase was increasing resulting in an increase in percentage of active tyrosinase. Phenolic substrates of the active tyrosinase were being oxidized, proportionally to the damage detectable on the mushroom cap. γ-Glutaminyl-3,4-dihydroxybenzene was degraded first, followed by γ-glutaminyl-4-hydroxybenzene and later tyrosine. The amount of melanin that was synthesized was larger than the sum of oxidation of the phenols measured. Principal Component Analysis explained 84 % of the variance in symptoms, and it demonstrated the phenol oxidation and active tyrosinase level as the most important parameters for the browning induced by bacteria or a tolaasin preparation treatment.

Bacterial blotch is a common disease for mushrooms, including commercially grown Agaricus bisporus (Olivier, Guillaumes & Martin, 1978 ; Nair & Bradley, 1980 ; Murata & Magae, 1996). Strain-related differences in susceptibility have been reported (Moquet Mamoun & Olivier, 1996 ; Soler-Rivas et al., 1997). The disease reduces crop yields and causes brown and sunken lesions, covering the entire fruit body surface, thus decreasing visual attractiveness (Burton, 1986 ; Miller & Spear, 1995). This disease is caused by Pseudomonas tolaasii, a bacterium endemic from the casing soil (Cutri, Macauley & Roberts, 1984, Cochet, Gillman & Lebeault, 1992). The bacterium exerts its action through a toxin called tolaasin (Nair & Fahy, 1973 ; Malcolm, 1981 ; Rainey, Brodey & Johnstone, 1992). Tolaasin is a lipodepsipeptide (Nutkins et al., 1991 ; Rainey, Brodey & Johnstone, 1991) with detergent-like and surface active properties (Hutchison & Johnstone, 1993). It is able to induce the formation of ion channels in the cell membranes (Brodey et al., 1991). Brown discolouration is a common phenomenon in senescent mushrooms (Burton, 1988 a ; Wichers & van Leeuwen, 1996). In Agaricus bisporus, both the polyphenol oxidases (PPOs) tyrosinase and laccase occur. Laccase is the main PPO in mycelium, whereas tyrosinase is more prominent in fruit bodies (Moore & Flurkey, 1989 ; Flurkey, 1991). The brown colour of the cap surface is produced by the action of tyrosinase on phenolic substances such as tyrosine, γglutaminyl-4-hydroxybenzene (GHB) and γ-glutaminyl-3,4dihydroxybenzene (GDHB) (Boekelheide et al., 1979 ; Stu$ ssi & Rast, 1981 ; Soulier, Foret & Arpin, 1993). In premature, healthy bodies tyrosinase is found almost

exclusively in an inactive form. During the development of the fruit body, tyrosinase becomes actived (Yamaguchi, Hwang & Campbell, 1970). In an earlier study, it was found that a tolaasin containing extract was able to induce the activation of the tyrosinase, probably by a non-proteolytic mechanism (Soler-Rivas et al., 1997). Senescence-related and blotch-related discolouration would then share the same ability to activate latent tyrosinase, albeit at a much higher rate in the latter case (Yamaguchi et al., 1970 ; Soler-Rivas et al., 1997). Bacterial blotch discolouration might, therefore be considered to be a strongly accelerated version of the senescence-related browning process. In this study, a detailed analysis of the discolouration produced by the tyrosinase activation, the activation process and phenolics utilization during P. tolaasii-infection or tolaasin treatment is presented in order to compare both senescence-related and blotch-related browning processes. The relation between types and duration of infection or treatment was investigated by principal component analysis.

M A T E R I A L S A N D M E T H O DS The mushroom strain used in this investigation was Agaricus bisporus (J. E. Lange) Imbach U1 (Somycel). The Mushroom Experimental Station, Horst, The Netherlands, supplied colonized beds a few days before the onset of fruit body production. Fruit bodies were grown at 18 mC and 80–85 % r.h. The bacterial strain used was the smooth, pathogenic, white

Brown blotch discolouration process in A. bisporus line test positive (Wong & Preece, 1979) Pseudomonas tolaasii (S. G. Paine) SPC 8907. This strain was a subculture of P. tolaasii 112 S (Preece) (NCPPB no. 3148). Bacterial suspension and tolaasin extract preparation P. tolaasii suspension was prepared according to the procedure described in Soler-Rivas et al. (1997). Mushrooms were infected with ca 9n5i10( cells ml−". Toxin extract was prepared following the cultivation and toxin purification procedure described by Nutkins et al. (1991). 2n5 mg ml−" partially purified toxin (PT) from an extract before the HPLC purification step was used. Inoculation procedure Mushrooms of approx.p30–40 mm of cap diam., in stage 2 or 3 (classification according to Hammond & Nichols, 1976) from the first flush, were harvested, their complete stalk removed and their caps placed in a box as in Olivier et al. (1978). Caps were inoculated with water (control), with P. tolaasii or with a partially purified tolaasin extract (PT) following the procedure described by Soler-Rivas et al. (1997) and incubated at 12m during 50 h. Six mushrooms were used per datapoint. Change of colour (as browning level), enzyme activity (tyrosinase activity), substrates (tyrosine, GDHB and GHB) and product (melanin) for the enzyme were monitored. Browning level and tyrosinase activity Cap colour was measured with a Chroma Meter (Minolta CR200, 1 cm slot). The changes of colour were measured as in Soler-Rivas et al. (1997) using the L, a, b scale. ∆E was defined as N[∆L#j∆a#j∆b#]. Samples were prepared as described in Soler-Rivas et al. (1997) to measure the tyrosinase activity as -DOPA oxidation activity following the procedure of Wichers et al. (1984). The colour measurements were the average of eight samples, the tyrosinase activity was measured in triplicate. Extraction of phenols and HPLC analysis Ten mg of lyophilized mushroom powder from each sample were mixed with 750 µl of 0n5 % (w\v) sodium bisulphite in 1 % (v\v) acetic acid solution (Jolivet et al., 1995). The mixture was shaken in a Vortex for 1 min and centrifuged for 2 min at 12 000 rpm. The supernatant containing the phenols was collected in another Eppendorf tube and the pellet was used for a second extraction with 750 µl of the solution. After the second centrifugation of the pooled supernatants, a 100 µl aliquot was injected for HPLC analysis. Phenol quantification was carried out on a reversed phase HPLC column (Hypersil BDS C18 5 µm 250i4n6 mm, Alltech) using as mobile phase : 1n8 % (v\v) tri-fluoroacetic acid (TFA) (A) and 10 % (v\v) acetonitrile in 1n8 % (v\v) TFA (B) following a gradient : from 0 to 20 min and from 5 to 46 % B (flow rate 1n0 ml min−"). Detection was performed by

1498 monitoring absorbance at 224 and 245 nm with a uv detector (Pharmacia). Tyrosine, GHB and GDHB were analysed. Peaks were identified on the basis of their retention times compared to those of standards (6n8, 10n0 and 13n1 min for GDHB, GHB and tyrosine (Serva) respectively). GDHB and GHB were purified according to Jolivet et al. (1995). Obtained values were the average of eight replicates per data point. Quantification of melanin Melanin was extracted from 10 mg of lyophilized mushroom powder from each sample according to Lotan & Lotan (1980). Melanin was analysed by spectrophotometry measuring the absorbance at 400 nm. The melanin concentration was calculated with a standard curve of synthetic melanin (Sigma). Samples were measured in duplicate. Analysis by PCA The brown blotch discolouration data were analysed by Principal Component Analysis (PCA) using The Unscrambler2 v. 6n0., a multipurpose statistical software package (Camo AS, Trondheim, Norway). PCA calculates linear combinations of variables (components) that describe as much of the variance of the original data as possible. This allows the original multidimensional matrix to be simplified without substantial loss of information, and so eases the interpretation of complex data matrices. Results of PCA can be graphically displayed as two sets of plots. In the first, correlation of variables with successive components can be plotted to aid interpretation of components ; in the second, sample scores can be plotted to show relationships between samples (Unscramble2 user’s manual, 1996). Treatment type and time of incubation made up the samples. Nine variables (∆L, ∆a, ∆b, total and active tyrosinases, tyrosine, GHB, GDHB and melanin concentrations) were studied. RESULTS Control mushrooms, inoculated with water, did not suffer any visual damage in the cap so changes in the measured parameters were assumed to be the result of natural senescenceinduced phenomena. Browning level Discolouration of the samples is shown in Fig. 1. In control mushrooms, the ∆E curve was strongly correlated to the ∆a parameter. Turning red was, therefore, the most important change in the colour of control mushrooms. ∆b and ∆L remained stable during the observation period. In infected or PT-treated mushrooms, ∆E was more closely correlated with the evolution of ∆L, so in this case getting darker was the dominant change. The ∆b parameter increased more than the ∆a parameter in infected mushrooms. In PTtreated mushrooms the ∆a parameter even decreased while the ∆b parameter remained constant. The total change of colour, measured as ∆E in response to

C. Soler-Rivas and others 10

1499

Control

P. tolaasii

PT extract

40

40

30

30

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10

Browning level

8

6

4

2

0

0 0

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0 0

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0

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Time (h)

Fig. 1. Colour development during 50 h of incubation with water as control, Pseudomonas tolaasii and a partially purified tolaasin extract (PT) on Agaricus bisporus caps. The ∆E parameter is represented as (— —), ∆L parameter as (– –4– –), ∆a as (– k=– –) and ∆b as (—#—). (I) indicates the standard deviation. 6

Control

5

20

25

6

P. tolaasii

5

20

10

0

0 0

10

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30

40

3 10

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2 5

Active tyrosinase (%)

Tyrosinase activity (µkat g–1)

25

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Time (h)

Fig. 2. Tyrosinase activity development during 50 h of incubation with water, Pseudomonas tolaasii and PT-extract on Agaricus bisporus caps. The curves represent : total tyrosinase (— —) and percentage of active tyrosinase (– –4– –). (I) indicates the standard deviation. Table 1. Degradation of phenolic compounds and melanin formation during 50 hrs of incubation with water, Pseudomonas tolaasii and PT extracts on Agaricus bisporus caps (levels in mg g−" dw). Control

Bacteria infected

PT extract treated

Hours

Tyr

GHB

GDHB

Melanin

Tyr

GHB

GDHB

Melanin

Tyr

GHB

GDHB

Melanin

0n25 0n5 1 2 3 20 30 40 50

— — — — 0n54p0n08 0n49p0n09 0n46p0n07 0n46p0n05 0n46p0n10

— — — — 0n80p0n14 0n71p0n11 0n71p0n11 0n67p0n11 0n67p0n13

— — — — 0n65p0n15 0n46p0n10 0n43p0n10 0n40p0n13 0n41p0n12

— — — — 0n00p0n00 0n00p0n00 0n00p0n00 0n68p0n10 1n06p0n25

— — — — 0n59p0n10 0n40p0n09 0n33p0n08 0n27p0n07 0n25p0n06

— — — — 0n73p0n14 0n37p0n11 0n28p0n08 0n25p0n13 0n20p0n12

— — — — 0n72p0n11 0n51p0n10 0n24p0n13 0n05p0n06 0n02p0n03

— — — — 0n37p0n05 0n87p0n20 1n23p0n11 2n42p0n25 3n27p0n28

0n57p0n11 0n53p0n18 0n47p0n11 0n44p0n10 0n43p0n11 0n32p0n10 0n27p0n09 0n19p0n06 0n16p0n08

0n75p0n10 0n69p0n14 0n65p0n14 0n53p0n12 0n47p0n13 0n25p0n10 0n20p0n07 0n19p0n05 0n18p0n09

0n71p0n14 0n68p0n13 0n53p0n13 0n49p0n08 0n45p0n08 0n20p0n12 0n06p0n04 0n02p0n01 0n00p0n00

0n06p0n00 0n07p0n00 0n07p0n05 0n07p0n02 0n07p0n30 2n91p0n10 4n65p0n23 5n32p0n41 6n15p0n35

Brown blotch discolouration process in A. bisporus PC2

1·0

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X-loadings

Tyrosinase activity

(a )

Aa

0·8

0·6

0·4

Ab

0·2

GDHB Tot. tyrase

Act. tyrase

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Tyr

GHB

–0·2 Melanin

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– 0·3

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X-expl: 70%, 14% 4

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Scores

(b)

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–1

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P0·25 B30 A3 B20 B3

Phenols and melanin : substrates and product of tyrosinase

PC1

–2 –5

–4

–3

–2

–1

0

1

2

3

Concomitant with discolouration, the tyrosinase activity changed. In control, injected and treated mushrooms, a decrease of total tyrosinase was found (Fig. 2). In control samples, the active tyrosinase had a constant value of 0n14p0n01 µkat g−" dw. No significant increase in the percentage of activation compared to infected samples was found. In infected or PT-treated mushrooms (Fig. 2) the total tyrosinase activity decreased at a higher rate than the control, in particular when mushrooms showed more severe discolouration, as was the case for the PT-treated samples. In these samples, the main degradation occurred up to 20 h of incubation ; in later stages degradation was not very pronounced. The degradation of tyrosinase correlated with the level of damage. The degradation rate in the infected mushrooms was not as high as in PT-treated mushrooms (in the latter case approx. 70 % of the total tyrosinase was degraded while for infected samples this was approx. 30 % of the total enzyme). In infected mushrooms, the levels of active tyrosinase during the incubation time changed from 0n19p0n03 µkat g−" dw at an early stage (3 h) to 0n28p0n07 µkat g−" dw after 50 h. In PT-treated mushrooms, samples harvested at 15 min contained almost the same level of active tyrosinase as the control (0n13p0n00 µkat g−" dw). The level increased further, however, and at 3 h it was already higher than in the infected mushrooms (0n23p0n03 µkat g−" dw). Eventually, it increased to 0n42p0n03 µkat g−" dw, at 50 h of incubation (not shown). In infected mushrooms, where a lower damage was observed than in PT-treated samples, the level of active tyrosinase increased almost linearly, to 2 % after 50 h (Fig. 2). In PT treated samples this value increased to 5n6 % at the end of the observation time. This increase confirmed earlier observations (Soler-Rivas et al., 1997) for tolaasin extract treated mushrooms.

4

X-expl: 70%, 14%

Fig. 3. PCA analysis of all parameters measured in the samples described in the methods. (a) Distribution of loadings or parameters (the browning parameters were : ∆L (AL), ∆a (Aa) and ∆b (Ab)), the enzymatic parameters were : total tyrosinase (Tot.tyrase) and active tyrosinase (Act.tyrase), and the substrate parameters were : Tyrosine (Tyr), GHB and GDHB, and melanin. Note that the PC1 axis scale is non-linear. (b) Distribution of scores of control (B), infected (A) and PT-treated (P) samples. The number represents time of incubation (h).

infection or treatment was hyperbolic. An initial fast discolouration phase was followed by a second phase of slower discolouration (Fig. 1). In infected mushrooms this slower phase started (after approx. 45 h) later than in PTtreated mushrooms (after 20 h) but also the intensity of browning was lower (in infected samples 30 U and PT-treated 42 U for ∆E).

In control mushrooms, tyrosine, GHB and GDHB levels did not decrease significantly during the incubation time (Table 1). GHB was found in the highest concentration (on average 0n70p0n12 mg g−"), followed by tyrosine (0n48p 0n08 mg g−") and GDHB (0n47p0n12 mg g−"). The obtained values for control mushrooms were similar to those found by Jolivet et al. (1995) for A. bisporus S609 in the pileipellis and trama tissues. Melanin could not be measured until 30 h of incubation. In infected mushrooms, the substrates were consumed much faster than in the control. GDHB levels were depleted fastest. GHB seemed to be more rapidly oxidized than tyrosine. In infected samples, after 3 h of incubation, the concentrations were still similar to the control. After 20 h, 52 % of the initial concentration (compared to the control) of GHB and 83 % of the initial concentration of tyrosine were still not degraded. At the end of the observation time only 28 % of GHB and 51 % of tyrosine remained. In PT-treated samples, a similar but faster process occurred. After 3 h of

C. Soler-Rivas and others incubation, only 65 % of GHB remained and 87 % of tyrosine was still present. After 20 h, only 35 % of the initial concentration of GHB and 66 % of the tyrosine was still not degraded and at the end of the observation time only 24 % of GHB and 32 % of tyrosine remained. The sum of the amounts of phenolics that were used was less than the amount of melanin that was formed. Perhaps influx of phenolic compounds from other metabolic processes, for instance tyrosine from protein degradation, may account for the strong increase in melanin. Statistical study of infection during an incubation time of 50 h PCA explained 84 % of variation by the first two principal components (PC). 70 % for PC1 and 14 % for PC2. This indicates that with the parameters that were measured, the infection-related discolouration process could be almost explained. The loadings of the brown blotch discolouration parameters (Fig. 3 a) were clearly divided on PC1. They were positioned in two groups on the positive and negative axis respectively of PC1. The phenolic compounds and the total tyrosinase were loaded opposite to the browning level (∆L and ∆b colours), the active tyrosinase and the amount of melanin formed, thus having high negative correlations. The parameter ∆a showed a high loading on the positive side of PC2. The position of loadings in this model proposed the distribution of sample scores in the way shown in Fig. 3 b. The samples that were incubated for a short period were positioned in the positive part for first principal component and negative for second principal component. All samples corresponding to the control remained closely together having higher scores for phenol concentrations (GHB, GDHB, and tyrosine) and total tyrosinase activity. The other two types of samples were distributed following incubation time-related curves, shifting from the positive to the negative part of PC1. DISCUSSION Senescence-related colour changes as measured with the (L, a, b) system are, in healthy mushrooms, characterized by an increase in the a and b parameters (increasing redness and yellowness) and a decrease in the L parameter (decreasing brightness) (Smith, Love & Burton, 1993 ; Sapers et al., 1994). Water-treated samples used as control followed the same discolouration values as non-inoculated senescent mushrooms. In these samples, the dominant colour change is to red. Samples that were infected with P. tolaasii or treated with a tolaasin-containing extract showed very similar discolouration patterns. In both, the dominant change was ‘ turning dark ’ as reflected by the decreasing -parameter. Total tyrosinase was found to decrease in both control and infected samples. In the control, this initial degradation could be due to the melanization process, to the lability of the enzyme or a decline in synthesis (i.e. turnover) (Wykes, Dunnill & Lilly, 1971), or due to mushroom proteases liberated in the harvest process (Burton, 1988 b ; Burton et al., 1997). In PT-treated samples, this degradation was not mainly

1501 due to proteases produced by P. tolaasii, because the PT extract was found not to contain proteolytic activity (SolerRivas et al., 1997). In this situation, therefore, tyrosinase degradation might be ascribed to mushroom proteases activated by the decompartmentalization resulting from the effects of tolaasin (Rainey et al., 1991 ; Hutchison & Johnstone, 1993). The PT preparation contained probably more tolaasin than the bacterial suspension, resulting in stronger membrane disruption and degradation of the enzyme. When comparing water-treated and infected or PT-treated samples, differences were observed in the active tyrosinase and levels of phenolic compounds. No activation of tyrosinase, and no degradation of phenols were found in control samples on the time-scale of the experiment. In infected or PT-treated samples, the percentage of active tyrosinase increased during the incubation time and appeared related to the severity of the damage produced on the cap. Lower damage, as in infected samples, resulted in a lower percentage of activation while in PT-treated samples, a higher increase was detected. In these samples, the activation of tyrosinases provoked the oxidation of phenolic compounds into melanin. The first oxidized substrate was GDHB, followed by GHB and last by tyrosine. The decrease of phenol concentrations was not strictly correlated to the increase in melanin concentration, which was found to increase more. These differences might indicate that at the beginning of infection the firstly oxidized substrates were mainly the ones quantified (tyrosine, GHB and GDHB), for which tyrosinase shows a high affinity (Boekelheide et al., 1979 ; Stu$ ssi & Rast, 1981 ; Soulier et al., 1993). At a later stage, when the discolouration process was more severe, other substrates may have been transformed into melanin, melanoproteins or other products. PCA analysis showed that the level of phenols and the total tyrosinase activity were oppositely correlated to the colour parameters ∆L and ∆b, and to the melanin and active tyrosinase. Browning (∆L) and active tyrosinase were very closely related. So the level of active tyrosinase appeared closely involved in the browning process. The amount of phenols seemed equally important in the browning process as was active tyrosinase, because the absolute values of the loadings of both parameters on the PC1 were nearly the same (0n4 and k0n4). Melanin content was shown as a very important parameter in the samples incubated for 50 h. Differences between the development in time of symptoms for PT-treated and infected samples were found. The PTtreated samples turned more red than the infected samples at the earlier stages of incubation (reflected by a high score in PC2 where ∆a is positioned). Later, both curves seemed to follow a similar tendency towards the brown colour. The datapoints A40, A50 and P20 were very close. This indicates that they had similar values which could mean that PT extract caused very similar symptoms in only 20 h of incubation as did bacteria in 40–50 h. The parameters measured in this general overview appeared to be strongly related to the discolouration process in A. bisporus U1 fruit bodies due to brown blotch disease. The PT treatment effect was slightly different from that of infection, but only for the initial phases of discolouration. Senescence related discolouration (controls) appears to differ from blotch

Brown blotch discolouration process in A. bisporus related discolouration. Senescent mushrooms turned reddish. No increase in the activation of tyrosinase and no degradation of phenols was detected in the time-scale of experimentation. Melanin was only formed at the end of the incubation time. Mushrooms with brown blotch symptoms turned dark rapidly. Activation of tyrosinase was induced, and, as consequence, phenolic compounds were degraded. A very high amount of melanin was synthesized. This work was supported by a grant (AIR 1 CT 94 – 5959) from the European Commission under the framework of the Agriculture, Agro-industry and Fisheries (AIR) program.

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