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 degrees 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.     

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.     

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.     

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 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.     

Development of a Microbial Model for the Combined Effect of Temperature and pH on Spoilage of Ground Meat, and Validation of the Model under Dynamic Temperature Conditions

The changes in microbial flora and sensory characteristics of fresh ground meat (beef and pork) with pH values ranging from 5.34 to 6.13 were monitored at different isothermal storage temperatures (0 to 20°C) under aerobic conditions. At all conditions tested, pseudomonads were the predominant bacteria, followed by Brochothrix thermosphacta, while the other members of the microbial association (e.g., lactic acid bacteria and Enterobacteriaceae) remained at lower levels. The results from microbiological and sensory analysis showed that changes in pseudomonad populations followed closely sensory changes during storage and could be used as a good index for spoilage of aerobically stored ground meat. The kinetic parameters (maximum specific growth rate [μ_max] and the duration of lag phase [λ]) of the spoilage bacteria were modeled by using a modified Arrhenius equation for the combined effect of temperature and pH. Meat pH affected growth of all spoilage bacteria except that of lactic acid bacteria. The “adaptation work,” characterized by the product of μ_max and λ(μ_max × λ) was found to be unaffected by temperature for all tested bacteria but was affected by pH for pseudomonads and B. thermosphacta. For the latter bacteria, a negative linear correlation between ln(μ_max × λ) and meat pH was observed. The developed models were further validated under dynamic temperature conditions using different fluctuating temperatures. Graphical comparison between predicted and observed growth and the examination of the relative errors of predictions showed that the model predicted satisfactorily growth under dynamic conditions. Predicted shelf life based on pseudomonads growth was slightly shorter than shelf life observed by sensory analysis with a mean difference of 13.1%. The present study provides a “ready-to-use,” well-validated model for predicting spoilage of aerobically stored ground meat. The use of the model by the meat industry can lead to effective management systems for the optimization of meat quality.     

Characterization of Bacterial Communities in Feces from Healthy Elderly Volunteers and Hospitalized Elderly Patients by Using Real-Time PCR and Effects of Antibiotic Treatment on the Fecal Microbiota

Fecal bacteria were studied in healthy elderly volunteers (age, 63 to 90 years; n = 35) living in the local community, elderly hospitalized patients (age, 66 to 103; n = 38), and elderly hospitalized patients receiving antibiotic treatment (age, 65 to 100; n = 21). Group- and species-specific primer sets targeting 16S rRNA genes were used to quantitate intestinal bacteria by using DNA extracted from feces and real-time PCR. The principal difference between healthy elderly volunteers and both patient cohorts was a marked reduction in the Bacteroides-Prevotella group following hospitalization. Reductions in bifidobacteria, Desulfovibrio spp., Clostridium clostridiiforme, and Faecalibacterium prausnitzii were also found in the hospitalized patients. However, total 16S rRNA gene copy numbers (per gram of wet weight of feces) were generally lower in the stool samples of the two groups of hospitalized patients compared to the number in the stool samples of elderly volunteers living in the community, so the relative abundance (percentage of the group- and species-specific rRNA gene copies in relation to total bacterial rRNA gene copies) of bifidobacteria, Desulfovibrio spp., C. clostridiiforme, and F. prausnitzii did not change. Antibiotic treatment resulted in further reductions in the numbers of bacteria and their prevalence and, in some patients, complete elimination of certain bacterial communities. Conversely, the numbers of enterobacteria increased in the hospitalized patients who did not receive antibiotics, and due to profound changes in fecal microbiotas during antibiotic treatment, the opportunistic species Enterococcus faecalis proliferated.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

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     

Dissimilatory Iron Reduction and Odor Indicator Abatement by Biofilm Communities in Swine Manure Microcosms

Animal waste odors arising from products of anaerobic microbial metabolism create community relations problems for livestock producers. We investigated a novel approach to swine waste odor reduction: the addition of FeCl₃, a commonly used coagulant in municipal wastewater treatment, to stimulate degradation of odorous compounds by dissimilatory iron-reducing bacteria (DIRB). Two hypotheses were tested: (i) FeCl₃ is an effective source of redox-active ferric iron (Fe³⁺) for dissimilatory reduction by bacteria indigenous to swine manure, and (ii) dissimilatory iron reduction results in significant degradation of odorous compounds within 7 days. Our results demonstrated that Fe³⁺ from FeCl₃ was reduced biologically as well as chemically in laboratory microcosms prepared with prefiltered swine manure slurry and limestone gravel, which provided pH buffering and a substrate for microbial biofilm development. Addition of a 1-g liter⁻¹ equivalent concentration of Fe³⁺ from FeCl₃, but not from presynthesized ferrihydrite, caused initial, rapid solids flocculation, chemical Fe³⁺ reduction, and E_h increase, followed by a 2-day lag period. Between 2 and 6 days of incubation, increases in Fe²⁺ concentrations were accompanied by significant reductions in concentrations of volatile fatty acids used as odor indicators. Increases in Fe²⁺ concentrations between 2 and 6 days did not occur in FeCl₃-treated microcosms that were sterilized by gamma irradiation or amended with NaN₃, a respiratory inhibitor. DNA sequences obtained from rRNA gene amplicons of bacterial communities in FeCl₃-treated microcosms were closely related to Desulfitobacterium spp., which are known representatives of DIRB. Use of iron respiration to abate wastewater odors warrants further investigation.     

Microscale Distribution of Populations and Activities of Nitrosospira and Nitrospira spp. along a Macroscale Gradient in a Nitrifying Bioreactor: Quantification by In Situ Hybridization and the Use of Microsensors

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 μm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a short-term incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitrite-oxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira-like bacteria in situ.     

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.     

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.     

Molecular Fingerprinting of Dairy Microbial Ecosystems by Use of Temporal Temperature and Denaturing Gradient Gel Electrophoresis

Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of ~150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.     

Bacteria and Methanogens Differ along the Gastrointestinal Tract of Chinese Roe Deer (Capreolus pygargus)

The current study provides the insight into the bacteria in the gastrointestinal tract (GIT) and methanogens presented in the rumen and cecum of the Chinese roe deer (Capreolus pygargus). The ruminal, ileal, cecal, and colonic contents, as well as feces, were obtained from each of the three, free-range, roe deer ingesting natural pasture after euthanasia. For the bacterial community, a total of 697,031 high-quality 16S rRNA gene sequences were generated using high-throughput sequencing, and assigned to 2,223 core operational taxonomic units (OTUs) (12 bacterial phyla and 87 genera). The phyla Firmicutes (51.2%) and Bacteroidetes (39.4%) were the dominant bacteria in the GIT of roe deer. However, the bacterial community in the rumen was significantly (P<0.01) different from the other sampled regions along the GIT. Secondly, Prevotella spp., Anaerovibrio spp., and unidentified bacteria within the families Veillonellaceae and Paraprevotellaceae were more abundant in the rumen than in the other regions. Unidentified bacteria within the family Enterobacteriaceae, Succinivibrio spp., and Desulfovibrio spp. were more predominant in the colon than in other regions. Unidentified bacteria within the family Ruminococcaceae, and Bacteroides spp. were more prevalent in the ileum, cecum and fecal pellets. For methanogens in the rumen and cecum, a total of 375,647 high quality 16S rRNA gene sequences were obtained and assigned to 113 core OTUs. Methanobrevibacter millerae was the dominant species accounting for 77.3±7.4 (S.E) % and 68.9±4.4 (S.E) % of total sequences in the rumen and cecum of roe deer, respectively. However, the abundance of Methanobrevibacter smithii was higher in the rumen than in the cecum (P = 0.004). These results revealed that there was intra variation in the bacterial community composition across the GIT of roe deer, and also showed that the methanogen community in the rumen differed from that in the cecum.     

Digestion of Herring by Indigenous Bacteria in the Minke Whale Forestomach

Northeastern Atlantic minke whales (Balaenoptera acutorostrata) have a multichambered stomach system which includes a nonglandular forestomach resembling that of ruminants. Bacteria from the forestomachs of herring-eating whales were enumerated and isolated in an anaerobic rumen-like culture medium (M8W medium). The total viable population of anaerobic bacteria ranged from 73 × 10⁷ to 145 × 10⁸/ml of forestomach fluid (n = 4). Lactobacillus spp. (19.7%), Streptococcus spp. (35.9%), and Ruminococcus spp. (12.8%) were the most common of the bacterial strains (n = 117) isolated by use of M8W medium from the forestomach fluid population of two minke whales. Most of the isolates stained gram positive (93.2%), 62.4% were cocci, and all strains were strictly anaerobic. The population of lipolytic bacteria in one animal, enumerated by use of a selective lipid medium, constituted 89.7% of the viable population. The total viable population of anaerobic bacteria in freshly caught and homogenized herring (Clupea harengus) ranged from 56.7 to 95.0 cells per gram of homogenized prey (n = 3) when M8W medium was used. Pediococcus spp. (30.6%) and Aerococcus spp. (25.0%) were most common of the bacterial strains (n = 72) isolated from the homogenized herring. Most of the bacterial strains were gram positive (80.6%), and 70.8% were cocci. Unlike the forestomach bacterial population, as many as 61.1% of the strains from the herring were facultatively anaerobic. All bacterial strains isolated from the prey had phenotypic patterns different from those of strains isolated from the dominant bacterial population in the forestomach, indicating that the forestomach microbiota is indigenous. Scanning electron microscopic examinations revealed large numbers of bacteria, surrounded by a glycocalyx, attached to partly digested food particles in the forestomach. These data support the hypothesis that symbiotic microbial digestion occurs in the forestomach and that the bacteria are indigenous to minke whales.     

Microbial challenges of poultry meat production

Food safety and shelf-life are both important microbial concerns in relation to broiler meat production. Focus is mainly placed on the absence or control of potentially pathogenic microbes such as Salmonella spp. and Campylobacter spp. but, from the commercial point of view, other spoilage bacteria also play a role as potential threats. Regarding food safety, the primary target should be the production of pathogen-free live animals, thus allowing slaughter plants to keep the processing line free of those microorganisms.Consumers believe that quality of foods from organic production is superior to foods from conventional production. The aim of the present study was to evaluate and compare the bacterial quality of chicken meat from organic and conventional production on the basis of traditional meat quality criteria. Fresh free grazing broiler carcasses were purchased directly from rural households (n = 80) and fresh retail chicken parts from conventional broiler carcasses from the local supermarkets in the region of Epirus (Poultry Producers Association. Arta) (n = 200).The samples were microbiologically tested for the presence of bacteria such as: Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Enterobacteriaceae, Escherichia coli, Campylobacter spp., and C. perfringens. Total count of aerobic mesophilic bacteria was also determined. Bacteriological tests were performed by means of standard methods of isolation and identification of individual species of bacteria according to ISO requirements. API-tests (bioMerieux) and Vitek 2 Identification System (bioMerieux) were used for biochemical determination. High levels of microbial contamination and occurrence of pathogenic bacteria at then fresh free grazing broiler carcasses reflect the poor hygienic quality of the slaughter conditions in the rural households.     

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.     

Changes in bacterial populations in the ileum of pigs fed low-phosphorus diets supplemented with different sources of fermentable carbohydrates

An experiment was conducted to evaluate the effects of differently fermentable carbohydrates on changes in bacterial populations in the ileum of growing pigs fed low-phosphorus (P) diets. Eight barrows (mean surgery BW 36 ± 0.9 kg) were fitted with simple T-cannulae at the distal ileum and were assigned to one of four dietary treatments: maize-soybean meal based control diet (CD), or 0.75 of CD supplemented with 0.25 lignocellulose, maize starch and high-methylated apple-pectin, respectively. Total bacterial cell counts as well as cell counts of Lactobacillus spp., Lactobacillus reuteri, Lactobacillus amylovorus/Lactobacillus sobrius, Lactobacillus mucosae, Enterococcus spp., Enterococcus faecium, Enterococcus faecalis, bifidobacteria, Clostridium coccoides cluster, Clostridium leptum cluster, Bacteroides–Prevotella–Porphyrmonas group and Enterobacteriaceae were determined by quantitative realtime PCR in DNA extracts of ileal digesta. Denaturing gradient gel electrophoresis (DGGE) of DNA fragments, generated by PCR targeting total or Lactobacillus spp. 16S rDNA, was used to estimate the bacterial diversity in the ileum. Lignocellulose supplementation tended (P<0.1) to increase cell counts of total bacteria in faeces compared with the control. Ileal bacterial populations responded differently to carbohydrate addition. Maize starch supplementation strongly stimulated the growth of total lactobacilli and Lactobacillus species (P≤0.05). Lignocellulose, in turn, enhanced the numbers of bifidobacteria, but reduced those of L. amylovorus compared with the control (P<0.05). Finally, pectin tended to increase the cell numbers of L. amylovorus/L. sobrius and the Bacteroides–Prevotella–Porphyrmonas group compared with the control (P<0.1). DGGE analysis revealed increased band numbers for total bacteria in the ileum of animals fed the lignocellulose and maize starch supplemented diets, while pectin reduced total bacterial (P<0.1) and Lactobacillus spp. diversity (P<0.05) compared with the control, as determined with the Shannon's index. Ileal VFA concentrations were decreased by pectin, while lignocellulose decreased faecal VFA concentrations. In conclusion, ileal bacterial populations and diversity are susceptible to changes in the carbohydrate composition of the diet. However, these changes were not related to major differences in the number of total bacteria in ileal digesta and faeces, but rather to changes in the bacterial species composition and their metabolic activity.     

Bacteria in Crude Oil Survived Autoclaving and Stimulated Differentially by Exogenous Bacteria

Autoclaving of crude oil is often used to evaluate the hydrocarbon-degrading abilities of bacteria. This may be potentially useful for bioaugmentation and microbial enhanced oil recovery (MEOR). However, it is not entirely clear if “endogenous” bacteria (e.g., spores) in/on crude oil survive the autoclaving process, or influence subsequent evaluation of the hydrocarbon-degradation abilities of the “exogenous” bacterial strains. To test this, we inoculated autoclaved crude oil medium with six exogenous bacterial strains (three Dietzia strains, two Acinetobacter strains, and one Pseudomonas strain). The survival of the spore-forming Bacillus and Paenibacillus and the non-spore-forming mesophilic Pseudomonas, Dietzia, Alcaligenes, and Microbacterium was detected using a 16S rRNA gene clone library and terminal restriction fragment length polymorphism (T-RFLP) analysis. However, neither bacteria nor bacterial activity was detected in three controls consisting of non-inoculated autoclaved crude oil medium. These results suggest that detection of endogenous bacteria was stimulated by the six inoculated strains. In addition, inoculation with Acinetobacter spp. stimulated detection of Bacillus, while inoculation with Dietzia spp. and Pseudomonas sp. stimulated the detection of more Pseudomonas. In contrast, similar exogenous bacteria stimulated similar endogenous bacteria at the genus level. Based on these results, special emphasis should be applied to evaluate the influence of bacteria capable of surviving autoclaving on the hydrocarbon-degrading abilities of exogenous bacteria, in particular, with regard to bioaugmentation and MEOR. Bioaugmentation and MEOR technologies could then be developed to more accurately direct the growth of specific endogenous bacteria that may then improve the efficiency of treatment or recovery of crude oil.