Behaviour of Listeria monocytogenes in packaged fresh mushrooms (Agaricus bisporus)

AIMS: The aim of this study was to evaluate the potential of Listeria monocytogenes to grow in mushrooms packaged in two different types of PVC films when stored at 4 degrees C and 10 degrees C. METHODS AND RESULTS: Mushrooms were packed in two polymeric films (perforated and nonperforated PVC) and stored at 4 degrees C and 10 degrees C. The carbon dioxide and oxygen content inside the packages, aerobic mesophiles, psychrotrophs, Pseudomonas spp., Listeria monocytogenes, faecal coliforms, Escherichia coli, anaerobic spores and major sensory factors were determined. The mushrooms packaged in nonperforated film and stored at 4 degrees C had the most desirable quality parameters (texture, development stage and absence of moulds). Listeria monocytogenes was able to grow at 4 degrees C and 10 degrees C in inoculated mushrooms packaged in perforated and nonperforated films between 1 and 2 log units during the first 48 h. After 10 d of storage, the populations of L. monocytogenes were higher in mushrooms packaged in nonperforated film and stored at 10 degrees C. CONCLUSIONS: MAP followed by storage at 4 degrees C or 10 degrees C extends the shelf life by maintaining an acceptable appearance, but allows the growth and survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: According to this study additional hurdles must be studied in order to prevent the growth of L. monocytogenes.     

环境因子对双孢蘑菇损伤敏感性的影响（英文）

在机械损伤过程中,白色双孢蘑菇极易被损伤并迅速褐变,使得机械采收的鲜菇质量低下,并且影响机械采收技术的应用和发展。机械采收双孢蘑菇的褐变程度(损伤敏感性)受栽培环境条件的影响。本文应用因子设计分析的方式研究多个因子对双孢蘑菇机械损伤敏感性的影响。以4个菌株为材料测试3个环境因子对双孢蘑菇损伤敏感性的影响,即覆土的厚度(分为2.5cm和5cm两个水平),覆土的湿度(分为干覆土和正常覆土两个水平),以及菇房内的相对湿度(分为80%和87%两个水平)。目的是找出能够在不敏感菌株和敏感菌株间产生最大敏感性差异的环境条件,由此得到的环境条件将应用到后续的群体分离分析中以得到最大的敏感性变异率。根据方差分析结果得出基因型(菌株)和覆土厚度是影响损伤敏感性的显著性因子,因子间相互作用显著。能够在不敏感菌株和敏感菌株间产生最大的损伤敏感性差异的环境条件为正常湿度的5cm厚覆土材料和87%的菇房内相对湿度。     

Direct immobilization of tyrosinase enzyme from natural mushrooms (Agaricus bisporus) on D-sorbitol cinnamic ester

Mushroom tyrosinase was immobilized from an extract onto the totally cinnamoylated derivative of D-sorbitol by direct adsorption as a result of the intense hydrophobic interactions that took place. The immobilization pH value and mass of lyophilized mushrooms were important parameters that affected the immobilization efficiency, while the immobilization time and immobilization support concentration were not important in this respect. The extracted/immobilized enzyme could best be measured above pH 3.5 and the optimum measuring temperature was 55 degrees C. The apparent Michaelis constant using 4-tert-butylcatechol as substrate was 0.38+/-0.02 mM, which was lower than for the soluble enzyme from Sigma (1.41+/-0.20 mM). Immobilization stabilized the extracted enzyme against thermal inactivation and made it less susceptible to activity loss during storage. The operational stability was higher than in the case of the tyrosinase supplied by Sigma and immobilized on the same support. The results show that the use of p-nitrophenol as enzyme-inhibiting substrate during enzyme extraction and immobilization made the use of ascorbic acid unnecessary and is a suitable method for extracting and immobilizing the tyrosinase enzyme, providing good enzymatic activity and stability.     

Compost bacteria and fungi that influence growth and development of Agaricus bisporus and other commercial mushrooms

Applied Microbiology and Biotechnology - Mushrooms are an important food crop for many millions of people worldwide. The most important edible mushroom is the button mushroom (Agaricus bisporus),...     

The composition of fresh and stored oyster mushrooms (Pleurotus ostreatus)

Soluble carbohydrate, protein, polysaccharide and cell wall composition were assayed in freshly harvested Pleurotus ostreatus sporophores and those stored for 4 days at 2° or 18°. Mannitol and trehalose were present at 1.8 and 6.5% dry wt respectively in fresh sporophores, and at reduced levels in those stored at 18°. In sporophores stored at 2°, trehalose levels increased by up to 122%. Soluble polysaccharide appeared to be composed of glycogen-like material, which was susceptible to post-harvest breakdown, and components containing mannose and other sugars. The total protein content was 42% dry wt; no protein degradation was seen in sporophores stored at 2°, but about 25% of the protein disappeared during storage at 18°. Cell wall polysaccharide was utilised during storage. Respiration rate was about 8–10 ml CO₂/g dry wt/hr at harvest and declined to about 5 ml/g dry wt/hr after 40 hr storage at 18°.     

Effects of Caenorhabditis elegans (Nematoda: Rhabditidae) on the spread of the bacterium Pseudomonas tolaasii in mushrooms (Agaricus bisporus)

The effects of mass-produced saprobic rhabditid nematodes, Caenorhabditis elegans on the spread of the bacterial blotch pathogen, Pseudomonas tolaasii , were studied in mushroom growth chambers. C. elegans significantly reduced the intensity of blotch on sporophores. Repeated isolations of the bacterial flora from the gut of C. elegans recovered from mushroom sporophores during cropping, revealed the presence of Pseudomonas fluorescens biovar reactans . All the isolates of P. fluorescens biovar reactans isolated from nematodes were antagonists of P. tolaasii .
C. elegans produced much larger populations in monoxenic cultures with P. fluorescens biovar reactans than with P. tolaasii . It is suggested that as C. elegans selects P. fluorescens biovar reactans rather than P. tolaasii as a food substrate it probably spreads the antagonist in the mushroom crop and may contribute to the control of bacterial blotch.     

Effect of spent mushroom compost tea on mycelial growth and yield of button mushroom (Agaricus bisporus)

Preliminary studies suggested that the use of compost tea made from spent mushroom substrate (SMS) may be regarded as a potential method for biologically controlling dry bubble disease in button mushroom. The aim of this study was to assess the effect of SMS compost tea on the host, the button mushroom, to ascertain whether the addition of these water extracts has a toxic effect on Agaricus bisporus mycelium growth and on mushroom yield. In vitro experiments showed that the addition of SMS compost tea to the culture medium inoculated with a mushroom spawn grain did not have an inhibitory effect on A. bisporus mycelial growth. The effect of compost teas on the quantitative production parameters of A. bisporus (yield, unitary weight, biological efficiency and earliness) was tested in a cropping trial, applying the compost teas to the casing in three different drench applications. Quantitative production parameters were not significantly affected by the compost tea treatments although there was a slight delay of 0.8-1.4 days in the harvest time of the first flush. These results suggest that compost teas have no fungitoxic effect on A. bisporus so that they can be considered a suitable biocontrol substance for the control of dry bubble disease.     

Relative contribution of nematodes (Caenorhabditis elegans) and bacteria towards the disruption of flushing patterns and losses in yield and quality of mushrooms (Agaricus bisporus)

Laboratory tests of bacteria isolated from the body surface, or from the gut, of a saprophagous rhabditid nematode Caenorhabditis elegans infesting mushrooms (Agaricus bisporus) showed that some bacteria enhanced nematode reproduction and that others inhibited it. As some bacteria were shown to inhibit mycelial growth of the mushroom, the effects of Acinetobacter calcoaceticus var. anitratus, Enterobacter cloacae and Serratia liquefaciens, either alone or in combination with C. elegans, on the flushing patterns, quality and yield of A. bisporus (strain Horst U3) were studied. Bacteria alone had little effect on flushing patterns whereas C. elegans delayed the onset of mushroom production and significantly disrupted the growth pattern of crops, with mushrooms appearing more regularly and not within obvious flushes. Inoculation with bacteria resulted in ‘browning’ of mushrooms that was even more pronounced in C. elegans treatments. Characteristic distortion of sporophores was observed only in the presence of C. elegans. Nematodes commonly colonised sporophores. Bacteria affected the size of nematode populations both on the sporophores and in the casing. Significant yield loss occurred; up to 10% when bacteria were inoculated, up to 27.8% when C. elegans was inoculated, and up to 35% with both bacteria and nematodes. Synergism between C. elegans and A. calcoaceticus var. anitratus was observed; the combination resulted in significantly greater reduction in mushroom yield than any other treatment. It is concluded that bacteria contribute to yield loss and quality deterioration in A. bisporus but that the effects are far greater in the presence of C. elegans.     

Effects of cultural conditions on the mycelial growth of healthy and virus-infected cultivated mushroom, Agaricus bisporus

There was a significant inverse correlation (P= 0=001) between concentrations of mushroom viruses 1 and 2 in sporophores and amounts of mycelial growth on malt agar of isolates taken from them. Increasing virus concentrations decreased linear growth of one mushroom strain from 76 mm (healthy) to 35 mm (mildly infected) and 7 mm (severely infected) when incubated at 25°C for 3 wk. Mycelial growth rates of isolates from healthy and from virus-infected mushrooms were compared on eleven agar media. All media clearly differentiated between healthy and severely infected isolates, but fewer separated healthy from mildly infected isolates. Those that did contained maltose, sucrose or starch as carbon source. Media containing peptone usually gave better differentiation than those with other sources of nitrogen, but the best differentiation was obtained with malt agar. Growing healthy and infected isolates on a range of media affected their subsequent growth on malt agar, the growth of some isolates apparently being changed permanently after 2 months on some of the different media. Whereas none of the infected isolates grew less rapidly after this treatment, the growth of some of the mildly infected isolates improved to such an extent that they could no longer be distinguished from healthy isolates. After heat-treatment (1–6 wk at 33°C), mycelial growth rates of infected isolates were increased, but viruses 1 and 2 were not always eliminated unless the heat-treatment was begun immediately after subculture. Mycelial growth rate and colony characters are not infallible criteria of the presence or absence of virus, a feature of particular significance when checking the health of mushroom spawn.     

Evaluation of indigenous potent mushroom growth promoting bacteria (MGPB) on Agaricus bisporus production

Mushrooms such as Agaricus bisporus, are cultivated for food worldwide. Fruit body initiation in Agaricus bisporus is a phase change from the vegetative to the reproductive stage which depends on the presence of a casing layer with particular physical, chemical and microbiological properties. The phase change is achieved practically by environmental manipulation and the presence of naturally occurring bacteria such as Pseuodomonas putida. In this study, 274 individual bacterial isolates were collected by screening the casing layer of 14 edible mushroom farms. The isolates were analysed with respect to biochemical properties, organic and inorganic phosphate solubilization, production of siderophore and growth in the presence of volatile compound of 1-octen-3-ol. It was found that approximately 97% of the strains were able to grow in the presence of 1-octen-3-ol and 36% were able to solubilize phosphorus. Among the isolates, 23 strains were selected as potent mushroom growth promoting bacteria (MGPB) for inoculation of the casing layer. Field experiments using these strains showed various promoting effects on production of mushroom. Finally, 2 strains (strains Bt4 and Ps7) showing the highest increase in A. bisporus production, were characterized as Pseuodomonas putida by molecular methods and identified as the best suited growth promoting inoculants for application in production farms for increasing the mushroom yield.     

Effects of Caenorhabditis elegans (Nematoda: Rhabditidae) on yield and quality of the cultivated mushroom Agaricus bisporus

Thirteen species of saprobic rhabditid nematodes (11 genera) were identified from samples of compost and casing material collected from mushroom farms in the British Isles. Caenorhabditis elegans, the most frequently found saprobe, was mass-produced monoxenically and its effects on the cultivated mushroom, Agaricus bisporus (strain U3) were studied. C. elegans did not multiply in well-prepared, pasteurised, spawned compost, whereas casing material proved to be a highly suitable environment for its reproduction. An initial casing inoculum of 10⁶ nematodes/crate of compost (7.5 kg), caused a significant reduction in mushroom yield. Losses in total mushroom yields of 11%, 20% and 26% were caused by initial inoculum rates of 10⁶, 10⁷and 2 × 10⁷ nematodes/crate, respectively. Yields were negatively correlated with the initial nematode inoculation level and regression equations were derived. The nematode treatments caused fewer mushrooms to be produced and an absence of the usual distinctive flushing patterns. C. elegans caused considerable deterioration in mushroom quality and characteristic distortion of mushrooms. Individual sporophores were mis-shapen, notched and had brown or violet coloured grills. Up to 3.8%, 6.7% and 10.8% of total weight and 3.5%, 5.4% and 8% of total numbers of mushrooms were distorted at the three highest nematode inoculum rates tested. Weights and numbers of distorted mushrooms were positively correlated with the initial nematode population. C. elegans commonly colonised sporophores.     

Mushroom (Agaricus bisporus) compost quality factors for predicting potential yield of fruiting bodies

A quality model has been developed from parameters determining the interactions of physical, chemical, and biological factors during the preparation of mushroom compost for growing Agaricus bisporus. Our results show that a partial least square model based on the combination of pH, dry matter, ammonia, carbon, hydrogen, ash, Cu, Fe, and Na could explain nearly 90% of the variation in mushroom yield obtained from four compost comparative trials. The yields in the data base for generating the model ranged from 138 to 305 kg per ton of compost. The validity of the yield model has been confirmed in a trial carried out in collaboration with experienced commercial growers. This has significant implications for compost producers, as production efficiencies can be maintained by targeting the important parameters.     

Inhibitory Effects of Hexylresorcinol and Dodecylresorcinol on Mushroom (Agaricus bisporus) Tyrosinase

The effects of hexylresorcinol and dodecylresorcinol on the monophenolase and diphenolase activity of mushroom tyrosinase have been studied. The results show that hexylresorcinol and dodecylresorcinol can inhibit both monophenolase and diphenolase activity of the enzyme. The lag period of the enzyme was obviously lengthened, and the steady-state activity of the enzyme decreased sharply. Two microM of hexylresorcinol and dodecylresorcinol can lengthen the lag period from 98 s to 260 and 275 s, respectively. Both hexylresorcinol and dodecylresorcinol can lead to reversible inhibition of the enzyme. The IC50 values of hexylresorcinol and dodecylresorcinol were estimated as 1.24 and 1.15 microM for monophenolase and as 0.85 and 0.80 microM for diphenolase, respectively. A kinetic analysis shows that hexylresorcinol and dodecylresorcinol are competitive inhibitors. The apparent inhibition constant for hexylresorcinol and dodecylresorcinol binding with free enzyme has been determined to be 0.443 and 0.405 microM for diphenolase, respectively.     

Cell water balance of white button mushrooms (Agaricus bisporus) during its post-harvest lifetime studied by quantitative magnetic resonance imaging

A combination of quantitative water density and T2 MRI and changes therein observed after infiltration with 'invisible' Gd-DTPA solution was used to study cell water balances, cell water potentials and cell integrity. This method was applied to reveal the evolution and mechanism of redistribution of water in harvested mushrooms. Even when mushrooms did not lose water during the storage period, a redistribution of water was observed from stipe to cap and gills. When the storage condition resulted in a net loss of water, the stipe lost more water than the cap. The water density in the gill increased, probably due to development of spores. Deterioration effects (i.e. leakage of cells, decrease in osmotic water potential) were found in the outer stipe. They were not found in the cap, even at prolonged storage at 293 K and R.H.=70%. The changes in osmotic potential were partly accounted for by changes in the mannitol concentration. Changes in membrane permeability were also indicated. Cells in the cap had a constant low membrane (water) permeability. They developed a decreasing osmotic potential (more negative), whereas the osmotic potential in the outer stipe increased, together with the permeability of cells.     

Properties of peat-based casing soils and their influence on the water relations and growth of the mushroom (Agaricus bisporus)

Different combinations of peat and chalk or lime sources with differing moisture contents were used to determine how specific physical and chemical properties of the casing soil relate to the growth and water relations of the mushroom. The peat types varied in terms of decomposition and extraction method; the lime addition varied in terms of rate and type (chalk or sugar beet lime). During the colonisation of the casing soil before fruiting, the extension growth rate of mushroom mycelium was most closely correlated (negatively) with the volumetric moisture content of the casing soil. Scanning electron microscopy showed that mycelium growing at a lower casing soil matric potential (Ψ_m) had a much finer and branched structure than mycelium growing at a higher Ψ_m. Across all the peat and lime source treatments, a relationship was found between the mean Ψ_m of the casing soil and mushroom yield, with an optimum Ψ_m of -7.9 to -9.4 kPa. Mushrooms are produced in ‘flushes’ at about 8-day intervals and during the development of each flush of mushrooms, there was a significant decrease in casing soil Ψ_m . This decrease (to below -40 kPa) was greatest in the second flush, which was the highest yielding. There were no relationships between mushroom yield and casing soil osmotic potential Ψ_π within the range -93 to -154 kPa or any of the other chemical properties and water and air holding characteristics of the casing soils which were determined. Across different casing soil treatments, mushroom dry matter content was negatively correlated with mushroom yield and positively correlated with mushroom tissue osmotic potential. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gibberellin-like Substances in the Sporophores of Agaricus bisporus (Lange) Imbach

Sporophores of cultivated Agaricus bisporus (Lange) Imbach,were shown to contain a gibberellin-like substance active inthe dwarf maize (d-5), -amylase and other bioassays. Ethyl-acetateextraction followed by paper, column, and thin-layer chromatographyrevealed the presence of one major active substance. Ficin hydrolysisof dried sporophore powder, after the complete removal of freesubstances, released more gibberellin-like substances, one ofwhich appeared identical to the free compound. The free substance was predominantly in the lamellae and residualpileus tissue. The major active substance released by ficinoccurred mostly in the lamellae but also in substantial equalamounts in both stipes and pilei. No activity was found in extractsof dikaryotic vegetative mycelium on malt agar. The level ofactivity in extracts from sporophores stored at – 20 °Cfell sharply after 7 d, and then remained constant over a periodof 6 weeks. The content of gibberellin-like substances in youngand old whole sporophores showed wide variation between experiments.In most cases young 2-d tissue had higher levels than old, 11-dtissue on a fresh-weight basis. Purified sporophore extractsand authentic gibberellins had no stimulating effect on growthof sporophores or of cultured vegetative mycelium. The inhibitorsof diterpene biosynthesis, CCC, and AMO-1618 induced a smallincrease in mycelial growth rate. Ethyl-acetate extraction ofhorse-straw compost prior to inoculation with Agaricus bisporusshowed the presence of gibberellin-like activity in significantamounts.     

Purification and characterization of four isolectins of mushroom (Agaricus bisporus)

Four lectins were purified from a mushroom (Agaricus bisporus) by ammonium sulfate fractionation, anion-exchange chromatography, affinity chromatography on bovine submaxillary mucin-Sepharose 4B and preparative isoelectric focusing. They were designated as ABA-I (pI 6.70), II (pI 5.98), III (pI 5.69) and IV (pI 5.53). Polyacrylamide gel electrophoresis of each lectin in the presence of sodium dodecyl sulfate gave a single band with an apparent molecular mass of 16 000 Da. Sedimentation equilibrium analysis suggested that each lectin is a tetramer of subunits. The four lectins were found to have quite similar carbohydrate-binding specificities. The hemagglutination activities of the lectins were effectively inhibited by bovine and porcine submaxillary mucins (BSM and PSM), and NH2-terminal glyco-octapeptides obtained by cyanogen bromide cleavage of human erythrocyte glycophorin A. In addition, desialylated PSM-glycopeptides were more potent inhibitors than untreated PSM-glycopeptides. Among monosaccharides and their glycosides, only methyl N-acetyl-alpha-galactosaminide inhibited lectin binding at a high concentration, but a synthetic oligosaccharide, O-beta-galactopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-alpha-D- galactopyranosyl)-N-tosyl-L-serine, was a strong inhibitor.     

Primordia initiation of mushroom (Agaricus bisporus) strains on axenic casing materials

The mushroom (Agaricus bisporus) has a requirement for a "casing layer" that has specific physical, chemical and microbiological properties which stimulate and promote the initiation of primordia. Some of these primordia then may develop further into sporophores, involving differentiation of tissue. Wild and commercial strains of A. bisporus were cultured in axenic and nonaxenic microcosms, using a rye grain substrate covered by a range of organic and inorganic casing materials. In axenic culture, A. bisporus (commercial strain A15) was capable of producing primordia and mature sporophores on charcoal (wood and activated), anthracite coal, lignite and zeolite, but not on bark, coir, peat, rockwool, silica or vermiculite. Of six strains tested, only the developmental variant mutant, B430, produced rudimentary primordia on axenic peat-based casing material. However, none of these rudimentary primordia developed differentiated tissues or beyond 4 mm diameter, either on axenic casing material in the microcosms or in larger-scale culture. In larger-scale, nonaxenic culture, strain B430 produced severely malformed but mature sporophores in similar numbers to those of other strains. Typically, 3-6% of primordia developed into mature sporophores, but significant differences in this proportion, as well as in the numbers of primordia produced, were recorded between 12 A. bisporus strains.     

Inhibitory effects of phloridzin dihydrate on the activity of mushroom (Agaricus bisporus) tyrosinase

The inhibitory effects of phloridzin dihydrate on the activity of mushroom tyrosinase have been studied. The results show that phloridzin can inhibit the diphenolase activity of the enzyme and the inhibition displays to be reversible. The IC(50) value was estimated as 110microM. The kinetic analysis showed that the inhibition of phloridzin on the diphenolase activity of the enzyme is of competitive type, and the inhibition constant (K(I)) was determined to be 64.3microM. The inhibitory effects of the different concentrations of phloridzin on the monophenolase activity were also studied. There were almost no changes in the lag period and the steady-state rate, while the plateaus in the inhibitory curve lowered with increasing the concentration of phloridzin when using tyrosine as a substrate.