Growth at sub-zero temperatures of black spot fungi from meat

Glycerol can prevent both freezing and desiccation of micro-organisms growing at sub-zero temperatures. On media containing glycerol, at concentrations readily tolerated by the organisms at ambient temperatures, three species of fungi isolated from black spot spoilt meat failed to grow at temperatures much below -5°C. This would, therefore, seem to be the minimum possible growth temperature of these organisms. Although the fungi could grow on frozen media, their rates of growth were such that, on frozen meat, several months would be required for colonies to become barely visible. It therefore seems that significant black spot spoilage will only develop on frozen meat if it is held at temperatures within 2–3° below the freezing point for prolonged periods, or if the meat surface reaches higher temperatures with surface drying inhibiting bacterial growth. There has been little study during the last 50 years of mould spoilage of meat, although it is still of importance in the international trade in frozen meats. Because moulds grow relatively slowly, they only spoil meat if the storage conditions prevent bacterial growth, but there are few firm data on the time and temperature requirements for visible mould growth to develop in the absence of bacterial spoilage. Such data are necessary if the causes of particular outbreaks of fungal spoilage are to be assessed correctly.     

A Note on the Identities of Organisms Causing Black Spot Spoilage of Meat

Four fungal species, Cladosporium cladosporioides, C. herbarum, Penicillium hirsutum and Aureobasidium pullulans were isolated from meat spoiled by black spot and shown to produce black spot colonies when inoculated on to sterile meat slices which were incubated at — 1°C. Penicillium hirsutum grew only on the meat surface but the other species penetrated into the tissues, apparently in response to arid conditions at the surface. It is clear from these observations that the traditional association of black spot spoilage with C. herbarum alone is incorrect.     

Biotoxic activity in the Mucorales

The toxigenicity of representatives of 15 species of Mucorales (Absidia glauca, Actinomucor elegans, Cunninghamella elegans, Helicostylum piriforme, Mortierella isabellina, Mortierella (Mucor) rammaniana, Mucor hiemalis, Mucor mucedo, Mucor spinosus, Phycomyces blakesleeanus, Rhizopus oligosporus, Rhizopus stolonifer, Syncephalastrum racemosum, Thamnidium elegans, Zygorhynchus moelleri) towards the larvae of brine shrimp (Artemia salina) and the growth of pea seedlings (Pisum sativum) and tobacco plants (Nicotiana tabacum) was evaluated. The fungi were cultivated on malt extract agar and aqueous solutions of the cultures were tested.Thamnidium elegans showed a marked toxic action towards brine shrimp (mortality: 74.1%) andPhycomyces, Actinomucor andSyncephalastrum were only weakly toxic. Length and weight of stems of pea seedlings were moderately reduced by extracts ofAbsidia, Cunninghamella, Zygorhynchus andThamnidium and to a lesser degree byMucor spinosus. Cunninghamella andMucor spinosus also inhibited the development of pea hypocotyls. The length of tobacco stems was reduced byMortierella ramanniana, Rhizopus stolonifer andCunninghamella elegans. Wilting or other toxic phenomena were never observed with both test plants. Considering the present results and data from literature it is suggested that species of Mucorales have only a weak toxigenicity.     

南极淡水分离出的菌物

本文报道从南极乔治王岛所采11个水样中分离出的菌物98株,初步鉴定出17属21种,其中6属(Acremonium,Arthrinium,Gliocladium,Phoma,Scopulariopsis,Ulocladium)13种为南极新记录,后者是:桃色顶孢Acremonium persicinum(Nicot)W.Gams,密顶孢A.strictum W.Gams,构巢曲霉Aspergillus nidulans(Eidam)Winter,聚多曲霉Asp.sydowii(Bainier & Sart)Thom & Church,芽枝状枝孢Cladosporium cladosporioides(Fries)de Vries,长孢被孢霉Mortierella elongata Linnemann,桔灰青霉Penicillium aurantiogriseum Dierckx,平滑青霉Pen.glabrum(Wehmer)Westling,鲜绿青霉Pen.viridicatum Westling,白瓶霉Phialophora alba v.Beyma,帚状瓶霉Ph.fastigiata(Lagerberg & Melin)Conant,纸状葡萄穗霉Stachybotrys chartarum(Ehrenberg ex Link)Hughes,和葡萄状单隔霉Ulocladium botrytis Preuss。温度试验表明:这些菌虽多为世界广布种,但已适应南极地区寒冷的气候。     

Diversity of facultatively anaerobic microscopic mycelial fungi in soils

The numbers of microscopic fungi isolated from soil samples after anaerobic incubation varied from tens to several hundreds of CFU per one gram of soil; a total of 30 species was found. This group is composed primarily of mitotic fungi of the ascomycete affinity belonging to the orders Hypocreales (Fusarium solani, F. oxysporum, Fusarium sp., Clonostachys grammicospora, C. rosea. Acremonium sp., Gliocladium penicilloides, Trichoderma aureoviride, T. harzianum, T. polysporum, T. viride. T. koningii, Lecanicillum lecanii, and Tolypocladium inflatum) and Eurotiales (Aspergillus terreus, A. niger, and Paecilomyces lilacimus), as well as to the phylum Zygomycota, to the order Mucorales (Actinomucor elegans, Absidia glauca, Mucor circinelloides, M. hiemalis, M. racemosus, Mucor sp., Rhizopus oryzae, Zygorrhynchus moelleri, Z. heterogamus, and Umbelopsis isabellina) and the order Mortierellales (Mortierella sp.). As much as 10-30% of the total amount of fungal mycelium remains viable for a long time (one month) under anaerobic conditions.     

Czapek Casein 50% Glucose (CZC50G): a new medium for the identification of foodborne Chrysosporium spp.

A new medium Czapek Casein 50% Glucose agar (CZC50G) has been developed, on which the four foodborne Chrysosporium spp., C. xerophilum, C. inops, C. farinicola and C. fastidium can be distinguished by differences in growth rates and colony morphology. Chrysosporium xerophilum and C. inops both produced dense white colonies, but C. xerophilum grew faster than C. inops , 22 mm in 14 d compared to 9–12 mm in 14 d at 25°C. Some isolates produced a yellow or red reverse due to the reaction of ferric ammonium citrate incorporated in the medium with a fungal metabolite. Chrysosporium farinicola and C. fastidium both grew poorly on this medium and produced sparse colonies: C. farinicola grew faster. Electron micrographs of arthroconidia with a cryo-scanning electron microscope showed thickening of the spore walls in C. inops but not in C. xerophilum . The aleurioconidia of C. farinicola and C. fastidium were different in shape. The differences in colony morphology and growth rate on CZC50G reflected these differences and demonstrated that these four species could be distinguished easily on CZC50G.     

Ethanol production in table jelly by two species of Chrysosporium

Different commercial brands of tablet jelly were stored in sealed containers for 12 weeks at an initial water activity of 0·90. The surface of those containing real fruit juice became covered with a dense white mould identified as Chrysosporium inops. That of those without fruit juice did not. This type of spoilage has been observed in commercially stored samples. When spores of this fungus and the closely related species, C. xerophilum , were added to commercial tablet jelly, the fungi grew only on samples containing real fruit juice. It is suggested that the fruit juice may provide some essential nutrient required for growth of these nutritionally fastidious species as well as the fungal spores themselves. Ethanol was detected in the headspace gas of stored samples of mouldy jelly. The production of ethanol by the two Chrysosporium species was confirmed by fermentation of glucose in liquid batch culture: approximately 2% ethanol was formed in 14 d. The ability of C. xerophilum and C. inops to utilize the Embden-Meyerhof-Parnas pathway may explain why these slow-growing xerophilic moulds become dominant after storage at low oxygen tension.     

Temperature relationships of fungi isolated at low temperatures from soils and other substrates

A study of the temperature relationships of 20 mycelial and yeast fungi which had been isolated at low temperatures from soils and from abattoirs indicated that few fungi can be regarded as truly psychrophilic. Only 1 species failed to grow at 25C. Although all the species investigated were able to grow at 4C and can therefore be considered as potential spoilage organisms on refrigerated foods, their optimal growth temperature was either 15C or 25C. The 20 species could be divided into 4 groups in relation to their temperature relationships, particularly their optimal temperatures and their ability to grow at 30C.A number of fungi able to grow at 4C has been isolated from soils and from abattoirs in New Zealand (1, 2). Several mycelial fungi can show reasonable growth below 10C, and some even down to –10C, but, when laboratory studies have been performed, most of these fungi prove to have an optimal growth temperature at 25C or above (3). These fungi, however, can be a cause of serious spoilage of a variety of refrigerated foods and even at the temperature currently used for meat storage ( –12C), occasional consignments of meat are encountered which show the characteristic spots of fungal contamination. During investigations into the occurrence of these fungi in an abattoir, (1), 9 mould and 4 yeast species were isolated which could grow at 4C, and further studies into the reservoirs of these fungi in the outside environment resulted in the isolation of a further 8 mould and 2 yeast species (2). This paper reports on investigations into the temperature relationships of 20 of these species.     

Conditions affecting growth and enterotoxin production by Staphylococcus aureus on temperature-abused chicken meat

Growth of Staphylococcus aureus at 15°C, with and without addition of representative spoilage bacteria, was studied in cooked, whole chicken meat and chicken broth. In the absence of competitors, the organism grew better in broth culture than on whole meat, but multiplied more slowly in broth when other organisms were present, even from twice the previous level of inoculum. The presence of competitors had no marked effect on the growth of Staph. aureus on whole meat. Enterotoxin A was not produced at 15°C on either whole meat or in broth, and occurred at 20°C only in pure culture. At 30° and 37°C, toxin was produced whether or not competitors were present. Toxin production by Staph. aureus appeared to be influenced more by growth temperature than by bacterial competition.     

The Development of Aerobic Spoilage Flora on Meat Stored at Chill Temperatures

In meat juice medium, aerobic spoilage bacteria utilized the following substrates in the order shown: Pseudomonos , glucose, amino acids, lactic acid; Acinetobacter , amino acids, lactic acid: Enterobacter , glucose, glucose-6-phosphate, amino acids; Microbacterium thermosphactum , glucose, glutamate. All the bacteria grew at their maximum rate utilizing the first and second substrates, but the growth rates declined when these were exhausted. The growth rate of Acinetobacter was reduced at pH 5·7 and below. All other species grew at their maximum rate within the pH range 5·5–7·0. On meat pseudomonads grew faster than the other species at all temperatures between 2° and 15°C. Interactions between any two species were observed only when one organism had attained its maximum cell density. Substrate exhaustion at the meat surface did not limit bacterial growth and it is suggested that the maximum cell density of aerobic spoilage cultures is determined by oxygen limitation of growth.     

Distribution of fungi on two mite species and their habitats in Egypt

Fungi associated with two mite species,Acotyledon krameri andTyrophagus putrescentiœ, and their habitats were studied using 1% glucose-Czapek's and potato-dextrose agar media at 28±2°C. A total of 54 species and one variety belonging to 25 genera were recovered from different habitats of mite species; 36 species and one variety belonging to 18 genera were associated with mite bodies. Nearly most fungal species isolated from mite bodies were also encountered in their habitats. The most common species wereAspergillus flavus, A. fumigatus, A. niger, Mucor racemosus, Nectria hœmatococca. A. terreus, Scopulariopsis brevicaulis andTrichurus spiralis were isolated only from mite bodies whereasFusarium oxysporum, Penicillium chrysogenum, P. corylophilum, P. funiculosum andRhizopus stolonifer were recovered from the mite habitats.A. krameri individuals survived well onCunninghamella elegans, F. oxysporum andM. racemosus cultured on both type of media, whereasCylindrocarpon destructans was best forT. putrescentiœ survival.     

Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species

Extracellular polysaccharide (EPS) antigens produced by different mould species were purified and partially characterized. Purification included (NH4)2SO4 treatment, Sepharose CL-4B column chromatography and Con A-sepharose chromatography. The EPS of Penicillium digitatum, Mucor racemosus and Cladosporium cladosporioides showed high antigenic capacities. Immunologically the EPS were partially genus-specific, but cross-reactivity was observed. The EPS antigens produced by species of Penicillium, Aspergillus repens and Geotrichum candidum lost their immunological activity upon heating (100 degrees C) at pH 1.8, while the EPS antigen of M. racemosus, Rhizopus oligosporus and C. cladosporioides were stable under the same conditions. The dominant monosaccharides present in the EPS antigen were mannose, galactose and glucose. The EPS obtained from cultures of M. racemosus and R. oligosporus also contained rhamnose. In the EPS produced by Penicillium spp. and A. repens the galactose residues were determined to be immunodominant.     

Mycoflora and trichothecene toxins of paddy grains from Egypt

120 species and 38 genera were collected from 64 samples of paddy grains on glucose- and cellulose-Czapek's agar at 28 °C. The total count of glycophilic and cellulose-decomposing fungi fluctuated between 216–29760; and 124–11320 colonies/g paddy grains on the two media, respectively.On glucose agar, the most common species were Aspergillus niger, A. flavus, A. sydowi, A. terreus, A. fumigatus, A. ochraceus, Penicillium chrysogenum, P. corylophilum, Fusarium oxysporum, Alternaria alternata, Cladosporium cladosporioides, Trichoderma viride and Mucor racemosus.On cellulose agar with pH 5.5 & 8.0, the most prevalent fungi were Stachybotrys chartarum, S. bisybi, Aspergillus niger, A. flavus, Fusarium oxysporum, Alternaria alternata, Drechslera sativus and Acremonium strictum.Extracts from 64 paddy samples were tested against brine shrimp larvae (Artemis salina). Of these 9 displayed varying degrees of toxicity. Trichothecene-toxins were detected in the extracts of three paddy samples only. Diacetoxyscirpenol and T-2 toxin were detected in two samples and only T-2 toxin in the other.     

The effects of temperature on growth of four high Arctic soil fungi in a three-phase system

The effect of temperature on the growth of Chrysosporium pannorum, Cylindrocarpon sp., Penicillium janthinellum, and Phoma herbarum, isolated from tundra soils, was studied. The growth in two systems, glucose-mineral agar plates and sand, moistened with glucose-mineral broth, was compared. All isolates showed an exponential increase in mass (measured as protein increase) in sand and a linear rate of extension on agar. Radial increase on agar was shown not to be a good index of growth in sand. Trends in growth rates in the sand cultures indicated that all four fungi can grow at low temperatures. The growth rate for Penicillium janthinellum at 15 degrees C was higher than at 20 degrees C, and Cylindrocarpon sp. and Phoma herbarum had higher growth rates at 2.5 degrees C than at 5 degrees C. These data suggest that there may be some adaptation by these fungi to growth in Arctic regions.     

Water relations of xerophilic fungi isolated from prunes

The predominant spoilage fungi of dried and high-moisture prunes were members of the Aspergillus glaucus group and Xeromyces bisporus. Chrysosporium spp. were also important. At the mean pH of prune flesh (3.8) and at 25 C, X. bisporus grew at water activities (a_w) down to 0.605, and Chrysosporium fastidium grew to 0.686. Germination was always followed by growth, but within the 120-day incubation period, the minimum a_w permitting asexual sporulation was usually higher than that permitting germination. Sexual sporulation often required an even higher a_w. The water requirements of aspergilli were appreciably greater at this pH than near neutrality, no species germinating below 0.738 a_w. This was probably a consequence of a high spore-death rate during incubation at low a_w and pH.     

Aflatoxin and aflatoxicosis

A wide range of fungi, amounting to fifty-six species belonging to twenty-two genera, have been recovered from animal feeds and foodstuffs.The most frequent fungi were Aspergillus niger, and A. flavus, followed by Mucor racemosus, Alternaria alternata, Rhizopus stolonifer, Penicillium corylophilum and P. notatum. Three genera were found to be of moderate occurrence, namely, Mucor, Rhizopus and Alternaria. The three following genera were of low occurrence: Cladosporium, Fusarium, and Neurospora.The fluorescence method of detecting aflatoxin-producing strains demonstrated that only one isolate of A. flavus possesses this property. Certain species of Penicillium and Aspergillus produced fluorescent substances (metabolites) similar in color to B and G aflatoxin. These substances were subsequently proved not to be aflatoxin by (TLC) chromatography.The animal and public health significance from such toxins was also discussed.     

Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species

Extracellular polysaccharide (EPS) antigens produced by different mould species were purified and partially characterized. Purification included (NH₄)₂SO₄ treatment, Sepharose CL-4B column chromatography and Con A-sepharose chromatography. The EPS of Penicillium digitatum, Mucor racemosus and Cladosporium cladosporioides showed high antigenic capacities. Immunologically the EPS were partially genus-specific, but cross-reactivity was observed. The EPS antigens produced by species of Penicillium, Aspergillus repens and Geotrichum candidum lost their immunological activity upon heating (100C) at pH 18, while the EPS antigen of M. racemosus, Rhizopus oligosporus and C. cladosporioides were stable under the same conditions. The dominant monosaccharides present in the EPS antigen were mannose, galactose and glucose. The EPS obtained from cultures of M. racemosus and R. oligosporus also contained rhamnose. In the EPS produced by Penicillium spp. and A. repens the galactose residues were determined to be immunodominant.     

The Development of the Anaerobic Spoilage Flora of Meat Stored at Chill Temperatures

In meat juice medium under anaerobic conditions, spoilage bacteria utilized the following substrates: Microbacterium thermosphactum , glucose; Enterobacter , glucose and glucose-6-phosphate; Lactobacillus , glucose and arginine. On meat stored anaerobically. Lactobacillus grew faster than the other species at all temperatures between 2° and 15 °C. Enterobacter had a greater affinity for glucose than M. thermosphactum. As a result, high numbers of Enterobacter inhibited growth of M. thermosphactum under the glucose limiting conditions at the meat surface. High numbers of Lactobacillus inhibited growth of both competing species, apparently by producing an inhibitory substance.     

Relationship between spore germination kinetics and lag time during growth of Mucor racemosus

AIMS: Spore germination requires microscopic observation whereas fungal growth results in a macroscopic examination. This paper aims at establishing a relationship between the percentage of germinated spores and parameters easily available from visible development. METHODS AND RESULTS: About 225 spores of Mucor racemosus were inoculated on PDA medium and incubated at 15 degrees and 25 degrees C. Germination kinetics were modelled by a logistic function. Fungal development provided two parameters, a growth rate, micro, and a lag period, lambda, defined as the slope of the straight line of the graph radius (mm) vs time (h) and the intercept of this line with the X-axis, respectively. CONCLUSIONS: It was found that the lag period coincided with the completion of the germination process, although the number of spores inoculated should be controlled carefully. SIGNIFICANCE AND IMPACT OF THE STUDY: Providing that this result can be generalized, this procedure would constitute a significant breakthrough for predicting food spoilage by moulds.     

Locating nisin-producing Lactococcus lactis in a fermented meat system

S.C. STRINGER, C.E.R. DODD, M.R.A. MORGAN AND W.M. WAITES. 1995. Antibody-linked probes were used to locate nisin in a fermented meat system. Free nisin or nisin bound to susceptible cells or food components was not detected. Colonies of nisin-producing Lactococcus lactis were stained at all times during growth. The position of nisin-producing L. lactis colonies was noted and compared with the location of spoilage organisms or the distribution of areas with a fermented meat appearance. No relationship between the distribution of starter culture and the location of spoilage organisms or areas of fermentation was observed. In addition to the presence of L. lactis , a rapidly fermentable sugar was also required to obtain a fermented appearance and to reduce the levels of spoilage organisms.