Rapid Detection of Meat Spoilage by Measuring Volatile Organic Compounds by Using Proton Transfer Reaction Mass Spectrometry

The evolution of the microbial spoilage population for air- and vacuum-packaged meat (beef and pork) stored at 4°C was investigated over 11 days. We monitored the viable counts (mesophilic total aerobic bacteria, Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, and Enterococcus spp.) by the microbiological standard technique and by measuring the emission of volatile organic compounds (VOCs) with the recently developed proton transfer reaction mass spectrometry system. Storage time, packaging type, and meat type had statistically significant (P < 0.05) effects on the development of the bacterial numbers. The concentrations of many of the measured VOCs, e.g., sulfur compounds, largely increased over the storage time. We also observed a large difference in the emissions between vacuum- and air-packaged meat. We found statistically significant strong correlations (up to 99%) between some of the VOCs and the bacterial contamination. The concentrations of these VOCs increased linearly with the bacterial numbers. This study is a first step toward replacing the time-consuming plate counting by fast headspace air measurements, where the bacterial spoilage can be determined within minutes instead of days.     

Changes in the Microbiology of Vacuum-packaged Beef

The development of the microbial flora on meat stored in vacuum-bags at 0–2° for up to 9 weeks was studied. Although the proportion of lactic acid bacteria increased relative to the aerobic spoilage organisms, the numbers of the latter continued to increase throughout storage. The initial contamination of the meat before vacuum-packaging was important; meat with a very low initial number had lower numbers of bacteria throughout storage for up to 9 weeks and steaks cut from such meat which had been stored always had 1–2 days' additional aerobic shelf life at 4°. Spoilage of these steaks was due either to slime formation and off-odour associated with high counts of presumptive Pseudomonas spp., or by discoloration and souring (lactic acid bacteria). Extract release volume and pH measurements performed on the vacuum-packaged primal joints were only of value in determining the onset of aerobic spoilage when large numbers of Gram negative organisms were present, whereas the titrimetric method of spoilage evaluation of the vacuum-packaged meat showed a correlation with spoilage due to lactic organisms.     

Spoilage-related activity of Carnobacterium maltaromaticum strains in air-stored and vacuum-packed meat

One hundred three isolates of Carnobacterium spp. from raw meat were analyzed by random amplification of polymorphic DNA (RAPD) and PCR and were identified by 16S rRNA gene sequencing. Forty-five strains of Carnobacterium maltaromaticum were characterized for their growth capabilities at different temperatures, NaCl concentrations, and pH values and for in vitro lipolytic and proteolytic activities. Moreover, their spoilage potential in meat was investigated by analyzing the release of volatile organic compounds (VOCs) in meat stored in air or vacuum packs. Almost all the strains were able to grow at 4, 10, and 20°C, at pH values of 6 to 9, and in the presence of 2.5% NaCl. The release of VOCs by each strain in beef stored at 4°C in air and vacuum packs was evaluated by headspace solid-phase microextraction (HS-SPME)-gas chromatography-mass spectrometry (GC-MS) analysis. All the meat samples inoculated and stored in air showed higher numbers of VOCs than the vacuum-packed meat samples. Acetoin, 1-octen-3-ol, and butanoic acid were the compounds most frequently found under both storage conditions. The contaminated meat samples were evaluated by a sensory panel; the results indicated that for all sensory odors, no effect of strain was significant (P > 0.05). The storage conditions significantly affected (P < 0.05) the perception of dairy, spoiled-meat, and mozzarella cheese odors, which were more intense in meat stored in air than in vacuum packs but were never very intense. In conclusion, different strains of C. maltaromaticum can grow efficiently in meat stored at low temperatures both in air and in vacuum packs, producing volatile molecules with low sensory impacts, with a negligible contribution to meat spoilage overall.     

Changes in the spoilage-related microbiota of beef during refrigerated storage under different packaging conditions

The microbial spoilage of beef was monitored during storage at 5 degrees C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O2 and 40% CO2 (MAP2), and (iii) 20% O2 and 40% CO2 (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.     

A Human Volunteer Study on the Prebiotic Effects of HP-Inulin—Faecal Bacteria Enumerated Using Fluorescent In Situ Hybridisation (FISH)

An in vivo study was carried to determine the effect of HP-inulin, a high-molecular-weight fraction of chicory-derived inulin, on the human gut microflora composition. Ten healthy volunteers were allowed a free-living diet whereby they also ingested 8 g/d of maltodextrin for 14 days and this was followed by 8 g/d HP-inulin for 14 days. Nine of the ten volunteers completed the trial. The trial was conducted in a double blind manner and faeces were collected periodically such that predominant groups of gut bacteria i.e. total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium perfringens/histolyticum sub-group and lactobacilli/enterococci could be enumerated. To overcome difficulties with culture-based techniques, the bacteria were enumerated using fluorescent in situ hybridisation (FISH). A small but statistically significant increase in bifidobacteria was observed when data from the volunteers were pooled. Similarly, a statistically significant increase was observed in clostridial numbers, although the magnitude of change in this bacterial group was about ten times less than that seen with bifidobacteria. HP-inulin intake had little or no effect on numbers of total bacteria,Bacteroides spp., or lactobacilli and enterococci present in the gut microflora of the volunteers. This study has confirmed the prebiotic nature of HP-inulin. However, in this trial the effects were most marked in those volunteers with low starting levels of bifidobacteria—indicating that there may be a relationship between prebiotic effect and initial bifidobacterial numbers.     

A note on microbial spoilage of sheep meat at ambient temperature

Shelf-life and microbial spoilage of sheep carcass meat at ambient temperature under commercial conditions were studied. The initial bacterial count of carcasses ranged 5.6-5.8 log/cm2. Staphylococcus spp. (48%) predominated in the initial microflora of carcasses followed by Micrococcus spp. (19%) and Escherichia spp. (12%). Microbial spoilage of carcasses occurred around 20 h when the bacterial count reached 8.0-9.0 log/cm2. Thus the shelf life of carcasses at ambient temperature was 19 h. The predominant micro-organisms at the time of spoilage were Escherichia and 'Acinetobacter-like' organisms. It was also observed that Enterobacter, Pseudomonas and Staphylococcus spp. could form a major part of the final flora. The presence of Escherichia and Staphylococcus spp. in higher percentages on the surface of carcasses at the time of spoilage presents the scope for health hazards.     

A substrate-mediated assay of bacterial proton efflux/influx to predict the degree of spoilage of beef mince stored at chill temperatures

A method was developed to predict spoilage of minced meat at chill temperatures, based on the difference in proton efflux from and influx into bacterial cells. This difference depends on the number of organisms present, the available glucose in the meat sample and the ability of the organisms to metabolize amino acids. The proton efflux/influx of a meat filtrate containing bacteria was measured at 25°C with a pH/ion meter in the presence of peptone with or without glucose. There was a noticeable rate of change of mV h_-1 of the meat filtrate prior to the organoleptic detection of spoilage which may be used semi-predictively to determine the remaining shelf-life of meat at different storage temperatures. The method could be investigated further, encompassing type and relative numbers of organisms, incubation temperature, meat type and composition (i.e. available glucose) to produce a spoilage prediction model. The method does not require sophisticated equipment, only a standard pH/ion meter, is cheap, needing only peptone and glucose, is relatively simple, and takes less than 2 h to perform.     

Carnobacterium divergens - a dominating bacterium of pork meat juice

Nonspoiled food that nevertheless contains bacterial pathogens constitutes a much more serious health problem than spoiled food, as the consumer is not warned beforehand. However, data on the diversity of bacterial species in meat juice are rare. To study the bacterial load of fresh pork from ten different distributors, we applied a combination of the conventional culture-based and molecular methods for detecting and quantifying the microbial spectrum of fresh pork meat juice samples. Altogether, we identified 23 bacterial species of ten different families analyzed by 16S rRNA gene sequencing. The majority of isolates were belonging to the typical spoilage bacterial population of lactic acid bacteria (LAB), Enterococcaceae, and Pseudomonadaceae. Several additional isolates were identified as Staphylococcus spp. and Bacillus spp. originating from human and animal skin and other environmental niches including plants, soil, and water. Carnobacterium divergens, a LAB contributing to the spoilage of raw meat even at refrigeration temperature, was the most frequently isolated species in our study (5/10) with a bacterial load of 10(3) - 10(7) CFU mL(-1). In several of the analyzed pork meat juice samples, two bacterial faecal indicators, Serratia grimesii and Serratia proteamaculans, were identified together with another opportunistic food-borne pathogen, Staphylococcus equorum. Our data reveal a high bacterial load of fresh pork meat supporting the potential health risk of meat juice for the end consumer even under refrigerated conditions.     

Metabolism of Explosive Compounds by Sulfate-Reducing Bacteria

The metabolism of various explosive compounds—1,3,5-trinitrobenzene (TNB), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX)—by a sulfate-reducing bacterial consortium, Desulfovibrio spp., was studied. The results indicated that the Desulfovibrio spp. used all of the explosive compounds studied as their sole source of nitrogen for growth. The concentrations of TNB, RDX, and HMX in the culture media dropped to below the detection limit (<0.5 ppm) within 18 days of incubation. We also observed the production of ammonia from the nitro groups of the explosive compounds in the culture media. This ammonia served as a nitrogen source for the bacterial growth, and the concentration of ammonia later dropped to <0.5 mg/L. The sulfate-reducing bacteria may be useful in the anaerobic treatment of explosives-contaminated soil. Received: 23 January 1998 / Accepted: 5 March 1998     

Antimicrobial activity of Myrtus communis L. water-ethanol extract against meat spoilage strains of Brochothrix thermosphacta and Pseudomonas fragi in vitro and in meat

The antimicrobial activity of hydro-alcoholic extract of Myrtus communis L. was investigate in vitro and in situ against different meat spoilage biotypes of Pseudomonas fragi and Brochothrix thermosphacta. Water-ethanol myrtle extract (WEME) was prepared from myrtle leaves and used to study the antimicrobial activity, Minimum Inhibitory Concentration (MIC) and Minimum Lethal Concentration (MLC). Naturally contaminated ground beef was used to evaluate in situ antibacterial activity of freeze-dried myrtle extract at different concentrations. In vitro antimicrobial activity of WEME was significantly more effective against B. thermosphacta than P. fragi strains. MIC and MLC values were in the range of 12.5–50 and 25–100 mg DM/ml, respectively, for B. thermosphacta and P. fragi strains. In situ results showed that the microbial population, except for Pseudomonas spp., decreased significantly in ground meat with added 5 % of freeze-dried myrtle extract. In conclusion, the findings demonstrate the effectiveness of myrtle extract to control or prevent the proliferation of meat spoilage bacteria.     

Effect of microbial cell‐free meat extract on the growth of spoilage bacteria

Aims: This study examined the effect of microbial cell‐free meat extract (CFME) derived from spoiled meat, in which quorum sensing (QS) compounds were present, on the growth kinetics (lag phase, and growth rate) of two spoilage bacteria, Pseudomonas fluorescens and Serratia marcescens. Methods and Results: Aliquots of CFME from spoiled meat were transferred to Brain Heart Infusion broth inoculated with 10³ CFU ml^?1 of 18 h cultures of Ps. fluorescens or Ser. marcescens, both fresh meat isolates; CFME derived from unspoiled fresh meat (‘clean’ meat) served as a control. Changes in impedance measurements were monitored for 48 h, and the detection time (Tdet) was recorded. It was found that in the absence of CFME containing QS compounds the Tdet was shorter (P < 0·05) than that in broth samples with added CFME from spoiled meat. The rate of growth of Ps. fluorescens, recorded as the maximum slope rate of conductance changes (MSrCC), after Tdet, was higher (P < 0·05) in samples with CFME containing QS compounds compared to samples without CFME or CFME derived from ‘clean’ meat. Similar results in MSrCC of impedance changes were obtained for Ser. marcescens. Conclusions: The study indicated that the growth rate (expressed in MSrCC units) of meat spoilage bacteria in vitro was enhanced in samples supplemented with CFME containing QS compounds compared to control samples (i.e., without CFME or with CFME from ‘clean’ meat). This behaviour may explain the dominant role of these two bacteria in the spoilage of meat. Significance and Impact of the Study: These results illustrate the potential effect of signalling compounds released during storage of meat on the behaviour of meat spoilage bacteria. Understanding such interactions may assist in the control of fresh meat quality and the extension of its shelf life.     

Indigenous bacteria and bacterial metabolic products in the gastrointestinal tract of broiler chickens

The gastrointestinal tract is a dynamic ecosystem containing a complex microbial community. In this paper, the indigenous intestinal bacteria and the microbial fermentation profile particularly short chain fatty acids (SCFA), lactate, and ammonia concentrations are reviewed. The intestinal bacterial composition changes with age. The bacterial density of the small intestine increases with age and comprises of lactobacilli, streptococci, enterobacteria, fusobacteria and eubacteria. Strict anaerobes (anaerobic gram-positive cocci, Eubacterium spp., Clostridium spp., Lactobacillus spp., Fusobacterium spp. and Bacteroides) are predominating caecal bacteria in young broilers. Data from culture-based studies showed that bifidobacteria could not be isolated from young birds, but were recovered from four-week-old broilers. Caecal lactobacilli accounted for 1.5-24% of the caecal bacteria. Gene sequencing of caecal DNA extracts showed that the majority of bacteria belonged to Clostridiaceae. Intestinal bacterial community is influenced by the dietary ingredients, nutrient levels and physical structure of feed. SCFA and other metabolic products are affected by diet formulation and age. Additional studies are required to know the bacterial metabolic activities together with the community analysis of the intestinal bacteria. Feed composition and processing have great potential to influence the activities of intestinal bacteria towards a desired direction in order to support animal health, well-being and microbial safety of broiler meat.     

The role of oxygen in the microbial spoilage of luncheon meat cooked in a plastic casing

The shelf life at 25°C of chub-packed luncheon meat was inversely related to oxygen (O₂) availability within the casing. With fibrous casings that are freely permeable to O₂, shelf life was less than 3 d. With plastic casings of low O₂ permeability, the shelf life was 7 d when air was trapped in the emulsion during the casing filling process, 14 d when air was not trapped (normal vacuum-stuffing) and greater than 28 d when vacuum-stuffed chubs were stored in hydrogen (H₂). The initial spoilage bacteria, Bacillus spp., grew only at the surface unless air was trapped in the emulsion when growth occurred throughout the luncheon meat. Bacillus spp. failed to grow on luncheon meat stored under H₂. Oxygen availability within the casing determined both the site and rate of microbial spoilage of chub-packed luncheon meat.     

Monitoring of microbial metabolites and bacterial diversity in beef stored under different packaging conditions

Beef chops were stored at 4°C under different conditions: in air (A), modified-atmosphere packaging (MAP), vacuum packaging (V), or bacteriocin-activated antimicrobial packaging (AV). After 0 to 45 days of storage, analyses were performed to determine loads of spoilage microorganisms, microbial metabolites (by solid-phase microextraction [SPME]-gas chromatography [GC]-mass spectrometry [MS] and proton nuclear magnetic resonance [(1)H NMR]), and microbial diversity (by PCR-denaturing gradient gel electrophoresis [DGGE] and pyrosequencing). The microbiological shelf life of meat increased with increasing selectivity of storage conditions. Culture-independent analysis by pyrosequencing of DNA extracted directly from meat showed that Brochothrix thermosphacta dominated during the early stages of storage in A and MAP, while Pseudomonas spp. took over during further storage in A. Many different bacteria, several of which are usually associated with soil rather than meat, were identified in V and AV; however, lactic acid bacteria (LAB) dominated during the late phases of storage, and Carnobacterium divergens was the most frequent microorganism in AV. Among the volatile metabolites, butanoic acid was associated with the growth of LAB under V and AV storage conditions, while acetoin was related to the other spoilage microbial groups and storage conditions. (1)H NMR analysis showed that storage in air was associated with decreases in lactate, glycogen, IMP, and ADP levels and with selective increases in levels of 3-methylindole, betaine, creatine, and other amino acids. The meat microbiota is significantly affected by storage conditions, and its changes during storage determine complex shifts in the metabolites produced, with a potential impact on meat quality.     

Presence of acylated homoserine lactones (AHLs) and AHL-producing bacteria in meat and potential role of AHL in spoilage of meat

Quorum-sensing (QS) signals (N-acyl homoserine lactones [AHLs]) were extracted and detected from five commercially produced vacuum-packed meat samples. Ninety-six AHL-producing bacteria were isolated, and 92 were identified as Enterobacteriaceae. Hafnia alvei was the most commonly identified AHL-producing bacterium. Thin-layer chromatographic profiles of supernatants from six H. alvei isolates and of extracts from spoiling meat revealed that the major AHL species had an R(f) value and shape similar to N-3-oxo-hexanoyl homoserine lactone (OHHL). Liquid chromatography-mass spectrometry (MS) (high-resolution MS) analysis confirmed the presence of OHHL in pure cultures of H. alvei. Vacuum-packed meat spoiled at the same rate when inoculated with the H. alvei wild type compared to a corresponding AHL-lacking mutant. Addition of specific QS inhibitors to the AHL-producing H. alvei inoculated in meat or to naturally contaminated meat did not influence the spoilage of vacuum-packed meat. An extracellular protein of approximately 20 kDa produced by the H. alvei wild-type was not produced by the AHL-negative mutant but was restored in the mutant when complemented by OHHL, thus indicating that AHLs do have a regulatory role in H. alvei. Coinoculation of H. alvei wild-type with an AHL-deficient Serratia proteamaculans B5a, in which protease secretion is QS regulated, caused spoilage of liquid milk. By contrast, coinoculation of AHL-negative strains of H. alvei and S. proteamaculans B5a did not cause spoilage. In conclusion, AHL and AHL-producing bacteria are present in vacuum-packed meat during storage and spoilage, but AHL does not appear to influence the spoilage of this particular type of conserved meat. Our data indicate that AHL-producing H. alvei may induce food quality-relevant phenotypes in other bacterial species in the same environment. H. alvei may thus influence spoilage of food products in which Enterobacteriaceae participate in the spoilage process.     

Changes in the Spoilage-Related Microbiota of Beef during Refrigerated Storage under Different Packaging Conditions

The microbial spoilage of beef was monitored during storage at 5°C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O₂ and 40% CO₂ (MAP2), and (iii) 20% O₂ and 40% CO₂ (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.     

The influence of bacterial inoculants on the microbial ecology of aerobic spoilage of barley silage.

The aerobic decomposition of barley silage treated with two inoculants (LacA and LacB) containing mixtures of Lactobacillus plantarum and Enterococcus faecium was investigated over a 28-day period. Initially, yeast and bacterial populations were larger in silage inoculated with LacA than in silage treated with LacB or water alone (control). Differences in the succession of yeasts in silage treated with LacA were observed relative to the other two treatments. From silage treatment with LacA, Issatchenkia orientalis was the most prevalent yeast taxon over all of the sample times, and the filamentous fungus Microascus brevicaulis was also frequently isolated at later sample dates (> or = 14 days). In contrast, Saccharomyces exiguus was the most prominent yeast recovered from silage treated with LacB and water alone on days 2 and 4, although it was supplanted by I. orientalis at later sample times. Successional trends of bacteria were similar for all three treatments. Lactobacillus spp. were initially the most prevalent bacteria isolated, followed by Bacillus spp. (primarily Bacillus pumilus). However, the onset of Bacillus spp. prominence was faster in LacA silage, and Klebsiella planticola was frequently recovered at later sample times (> or = 14 days). More filamentous fungi were recovered from LacA silage on media containing carboxylmethylcellulose, pectin, or xylan. The most commonly isolated taxa were Absidia sp., Aspergillus flavus, Aspergillus fumigatus, Byssochlamys nivea, Monascus ruber, Penicillium brevicompactum, Pseudoallescheria boydii, and M. brevicaulis. The results of this study indicated that the two bacterial inoculants incorporated into barley at the time of ensilage affected the microbial ecology of silage decomposition following exposure to air. However, neither of the microbial inoculants effectively delayed aerobic spoilage of barley silage, and the rate of decomposition of silage treated with one of the inoculants (LacA) was actually enhanced.     

Proof of concept: predicting the onset of meat spoilage by an integrated oxygen sensor spot in MAP packages

During storage of modified atmosphere packaged (MAP) meat, the initial microbiota grows to high cell numbers, resulting in perceptible spoilage after exceeding a specific threshold level. This study analyses, whether elevated oxygen consumption in the headspace of MA-packages would enable a prediction method for meat spoilage. We monitored the growth of single spoiling species inoculated on high-oxygen MAP beef and poultry, performed sensorial analysis and determined oxygen concentrations of the headspace via a non-invasive sensor spot technology. We detected microbial headspace oxygen consumption occurring prior to perceptible meat spoilage for certain species inoculated on beef steaks. However, headspace oxygen consumption and cell counts at the onset of spoilage were highly species-dependent, which resulted in a strong (Brochothrix thermosphacta) and moderate (Leuconostoc gelidum subspecies) decrease of the headspace oxygen content. No linear decrease of the headspace oxygen could be observed for Carnobacterium divergens and Carnobacterium maltaromaticum inoculated on poultry meat. We demonstrate the applicability of an incorporated oxygen sensor spot technology in MAP meat packages for detection of spoilage in individual packages prior to its perceptible onset. This enables individual package evaluation and sorting within retail, and consequently reduces meat disposal as waste.     

Influence of antimicrobial agents on the spoilage of a meat-based entomophage diet

The microbial decomposition of a meat-based entomophage diet presented in Parafilm packets was investigated. Considerable bacteria but not fungi were associated with components used to prepare the diet (i.e., hens' eggs, liver, and ground beef). At the initial sampling time, there were no differences among diet treatments in the size of bacterial or fungal populations. Bacterial populations in diets not containing antibacterial agents rapidly increased and reached an asymptote by 24 h (approximately 10(10) colony-forming units per gram). Bacterial populations also increased in diets containing antibacterial agents, but they were significantly smaller than in diets not containing antibacterial agents. The most prevalent bacteria isolated were Carnobacterium piscicola, Carnobacterium divergens, Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides, and Enterococcus spp., regardless of the antibacterial treatment used. The proliferation of fungi was delayed relative to bacteria, but significant differences were observed among the diet treatments. Fungi were most inhibited by sorbic acid and propionic acid in the absence of antibacterial agents. The most common fungi isolated were the yeasts Candida zeylanoides, Torulaspora globosa, and Yarrowia lipolytica. The pH of diets not containing antibacterial agents decreased rapidly and was highly correlated with increases in bacteria but not fungi. The results of this study demonstrate that antimicrobial agents significantly inhibit spoilage microorganisms in a meat-based diet and that alternative management strategies to delay the decomposition of such diets presented in Parafilm packets should target lactic acid spoilage bacteria, particularly Carnobacterium and Lactobacillus species.     

Induction of conidiation by endogenous volatile compounds in Trichoderma spp

Light and starvation are two principal environmental stimuli inducing conidiation in the soil micromycete Trichoderma spp. We observed that volatiles produced by conidiating colonies of Trichoderma spp. elicited conidiation in colonies that had not been induced previously by exposure to light. The inducing effect of volatiles was both intra- and interspecific. Chemical profiles of the volatile organic compounds (VOCs) produced by the nonconidiated colonies grown in the dark and by the conidiating colonies were compared using solid-phase microextraction of headspace samples followed by tandem GC-MS. The conidiation was accompanied by increased production of eight-carbon compounds 1-octen-3-ol and its analogs 3-octanol and 3-octanone. When vapors of these compounds were applied individually to dark-grown colonies, they elicited their conidiation already at submicromolar concentrations. It is concluded that the eight-carbon VOCs act as signaling molecules regulating development and mediating intercolony communication in Trichoderma.