A selective medium for the rapid detection by an impedance technique of Pseudomonasspp. associated with poultry meat

A new medium for detecting and enumerating Pseudomonas spp. associated withpoultry meat spoilage by a rapid impedance technique was developed, after testing potentialgrowth promoters for eight Pseudomonas strains and inhibitors against eight competingstrains (Enterobacteriaceae) able to grow on the medium of Mead and Adams (1977). Four basalmedia (brain heart infusion, brucella broth, Shaedler broth and Whitley impedance broth (WIB))and a synthetic medium were evaluated. Whitley impedance broth was the best basal medium fordetecting variations in impedance in relation to Pseudomonas growth. The efficiency ofWIB was improved by adding compounds which enhanced the growth of Pseudomonas onthe synthetic medium. Among the incubation temperatures tested, 22°C proved to be the bestcompromise between growth of Pseudomonas associated with poultry meat spoilage andinhibition of competitors. Among the 15 inhibitory substances evaluated against Pseudomonas competitors, five were chosen for inclusion in the final medium : metronidazole,carbenicilline, cetrimide, cycloheximide and diamide (MCCCD medium). Preliminary resultsobtained from experiments with beef and pork meat showed that this medium could also be usedwithout diamide and at an incubation temperature of 25°C. The impedance technique usingMCCCD medium was then compared with an official method which uses the medium of Meadand Adams (1977) on 106 samples of poultry neck skin. The linear regression coefficient betweenthe two techniques was approximately r = 0·85. Impedance was able to detect 10³ Pseudomonas g⁻¹ within less than 19 h making it a promisingtechnique for predicting poultry meat spoilage.     

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.     

Temperature function integration and the development and metabolism of poultry spoilage bacteria

The rate of spoilage of chicken tissues, the development of spoilage bacteria, and the utilization of amino acids by spoilage bacteria as a function of temperature were more accurately described by the general spoilage curve of Olley and Ratkowsky (Food Technol. Aust. 25:66-73, 1973; Food Technol. N.Z. 8:13-17, 1973) than by the linear equation of Spencer and Baines (Food Technol. [Chicago] 18:175-179, 1964). Remaining shelf life of poultry tissues may be predicted at temperatures up to 16 degrees C by using a temperature function integrator which incorporates the general spoilage curve.     

Temperature function integration and the development and metabolism of poultry spoilage bacteria.

The rate of spoilage of chicken tissues, the development of spoilage bacteria, and the utilization of amino acids by spoilage bacteria as a function of temperature were more accurately described by the general spoilage curve of Olley and Ratkowsky (Food Technol. Aust. 25:66-73, 1973; Food Technol. N.Z. 8:13-17, 1973) than by the linear equation of Spencer and Baines (Food Technol. [Chicago] 18:175-179, 1964). Remaining shelf life of poultry tissues may be predicted at temperatures up to 16 degrees C by using a temperature function integrator which incorporates the general spoilage curve.     

THE EFFECT OF THE TETRACYCLINE COMPOUNDS ON THE STORAGE LIFE AND MICROBIOLOGY OF CHILLED EVISCERATED POULTRY

SUMMARY: Chlortetracycline, oxytetracycline and tetracycline, incorporated at 10 p/m in the ice slush used for cooling, were equally efficient in extending the storage life of eviscerated poultry which was subsequently frozen, thawed and stored at 1°. There was complete correlation between the sensory assessment of 'off'odour and the total numbers of micro-organisms present when these were determined by taking bulk samples of skin from the area under the wing and of the surface tissue of the visceral cavity near the vent. Direct microscopical counts confirmed that the latter area was the most heavily contaminated part of the visceral cavity. Non-pigmented strains of Pseudomonas were the main spoilage organisms of untreated chickens but Achromobacter strains and yeasts were responsible for the spoilage of the chlortetracycline treated chickens. Fluorescence was only observed in spoiled chickens where pigmented strains of Pseudomonas occurred in significant numbers. Spoilage in both control and antibiotic treated poultry was accompanied by a rise in pH in the contaminated muscles, but for various reasons this change could not be used as a direct measure of the extent of the microbial contamination.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5·8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15°C in (1) aerobic conditions; (2) 30% CO₂+ air; (3) 30% CO₂+ N₂; and (4) 100% CO₂. When samples were held at 1°C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6°C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1°C rather than 37°C. In CO₂ atmospheres growth of L. monocytogenes was inhibited on meat held at 6°C, especially under 100% CO₂. By contrast, storage at 15°C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5.8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15 degrees C in (1) aerobic conditions; (2) 30% CO2 + air; (3) 30% CO2 + N2; and (4) 100% CO2. When samples were held at 1 degree C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6 degrees C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1 degree C rather than 37 degrees C. In CO2 atmospheres growth of L. monocytogenes was inhibited on meat held at 6 degrees C, especially under 100% CO2. By contrast, storage at 15 degrees C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

Characterization of Hydrogen Sulfide-Producing Bacteria Isolated from Meat and Poultry Plants

A survey of the types of aerobic organisms able to produce H2S on peptone iron agar (Levin, 1968), and commonly occurring in meat and poultry plants, revealed that these could be divided into four distinct groups. The ability of representative strains of each type to grow at low temperatures and cause off-odors on chicken muscle was examined. The results are discussed in relation to the role of these organisms in the psychrophilic spoilage of meat and meat products.     

Overexpressing ovotransferrin and avian β-defensin-3 improves antimicrobial capacity of chickens and poultry products

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian β-defensin-3 (AvβD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvβD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

     

Production of Off Odours by Isolates from Poultry Skin with Particular Reference to Volatile Sulphides

Poultry skin free from psychrotrophic micro-organisms was used to assess the abilities of isolates from poultry skin to produce off odours. Pseudomonas group I and II strains were the major off odour producers and the predominant volatile produced was methanethiol. Results obtained using skin as a spoilage substrate were similar to those obtained with leg muscle but a higher proportion of strains produced volatile sulphides from methionine and cystine supplemented media. Addition of glucose to the methionine medium inhibited methanethiol production.     

Molecular identification and biodiversity of potential allergenic molds (Aspergillus and Penicilium) in the poultry house: first report

The production of poultry has proved to be a significant source of fungi. Penicillium and Aspergillus are among the most important species, because they can cause irritations, infections, allergies, spoilage of food and beverages and are able to produce dangerous mycotoxins. Detection and identification of the species in these genera is very significant, because it provides relevant information about the properties of the responsible strains. Therefore, their taxonomic was determined using a phenotypic and molecular (ITS and partial β-tubulin sequences) methods. Investigations were conducted in the poultry house located near Wroc?aw in Lower Silesia (Poland). Using morphological analyses, 215 fungal strains were identified. Among them, 56 belonged to the Penicilium and Aspergillus genera. The results obtained from sequence analysis corresponded well with the morphological identification. Classification at species level was possible in most cases using the sequence data. Most isolates belong to Penicilium genus, with the follow dominating species (P. chrysogenum, P. polonicum and P. olsonii).     

Multilocus Sequence Typing of Leuconostoc gelidum subsp. gasicomitatum,a Psychrotrophic Lactic Acid Bacterium Causing Spoilage of Packaged Perishable Foods

Leuconostoc gelidum subsp. gasicomitatum is a psychrotrophic lactic acid bacterium (LAB) that causes spoilage of a variety of modified-atmosphere-packaged (MAP) cold-stored food products. During the past 10 years, this spoilage organism has been increasingly reported in MAP meat and vegetable products in northern Europe. In the present study, the population structure within 252 L. gelidum subsp. gasicomitatum strains was determined based on a novel multilocus sequence-typing (MLST) scheme employing seven housekeeping genes. These strains had been isolated from meat and vegetable sources over a time span of 15 years, and all 68 previously detected pulsed-field gel electrophoresis (PFGE) genotypes were represented. A total of 46 sequence types (STs) were identified, with a majority of the strains (>60%) belonging to three major STs, which were grouped into three clonal complexes (CCs) and 17 singletons by Global Optimal eBURST (goeBURST). The results by Bayesian analysis of population structure (BAPS) mostly correlated with the grouping by goeBURST. Admixture analysis by BAPS indicated a very low level of exchange of genetic material between the subpopulations. Niche specificity was observed within the subpopulations: CC1 and BAPS cluster 1 consisted mostly of strains from a variety of MAP meats, whereas vegetable strains grouped together with strains from MAP poultry within CC2 and BAPS cluster 2. The MLST scheme presented in this study provides a shareable and continuously growing sequence database enabling global comparison of strains associated with spoilage cases. This will further advance our understanding of the microbial ecology of this industrially important LAB.     

Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, α-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 °C for 15, 30 and 60 min, or 121 °C for 15 min. Lactococcin R remained active after storage at −20 and −70 °C for 3 months and after exposure to a pH of 2–9. The molecular weight of lactococcin R was about 2·5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml⁻¹) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0·5% glucose, at 6·5–7·0 initial pH values, 30 °C temperature and 18–24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6–7 (95%). Crude lactococcin R at 1280 AU ml⁻¹ was bactericidal, reducing colony counts of Listeria monocytogenes by 99·98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2·5 kDa vs 3·4 kDa, and in being sensitive to pepsin and α-chymotrypsin to which nisin is resistant.     

Amplified Fragment Length Polymorphism Fingerprinting of Pseudomonas Strains from a Poultry Processing Plant

Molecular typing has been used previously to identify and trace dissemination of pathogenic and spoilage bacteria associated with food processing. Amplified fragment length polymorphism (AFLP) is a novel DNA fingerprinting technique which is considered highly reproducible and has high discriminatory power. This technique was used to fingerprint 88 Pseudomonas fluorescens and Pseudomonas putida strains that were previously isolated from plate counts of carcasses at six processing stages and various equipment surfaces and environmental sources of a poultry abattoir. Clustering of the AFLP patterns revealed a high level of diversity among the strains. Six clusters (clusters I through VI) were delineated at an arbitrary Dice coefficient level of 0.65; clusters III (31 strains) and IV (28 strains) were the largest clusters. More than one-half (52.3%) of the strains obtained from carcass samples, which may have represented the resident carcass population, grouped together in cluster III. By contrast, 43.2% of the strains from most of the equipment surfaces and environmental sources grouped together in cluster IV. In most cases, the clusters in which carcass strains from processing stages grouped corresponded to the clusters in which strains from the associated equipment surfaces and/or environmental sources were found. This provided evidence that there was cross-contamination between carcasses and the abattoir environment at the DNA level. The AFLP data also showed that strains were being disseminated from the beginning to the end of the poultry processing operation, since many strains associated with carcasses at the packaging stage were members of the same clusters as strains obtained from carcasses after the defeathering stage.     

Isolation of a Hop-Sensitive Variant of Lactobacillus lindneri and Identification of Genetic Markers for Beer Spoilage Ability of Lactic Acid Bacteria

We have isolated a hop-sensitive variant of the beer spoilage bacterium Lactobacillus lindneri DSM 20692. The variant lost a plasmid carrying two contiguous open reading frames (ORF s) designated horB_L and horC_L that encode a putative regulator and multidrug transporter presumably belonging to the resistance-nodulation-cell division superfamily. The loss of hop resistance ability occurred with the loss of resistance to other drugs, including ethidium bromide, novobiocin, and cetyltrimethylammonium bromide. PCR and Southern blot analysis using 51 beer spoilage strains of various species of lactic acid bacteria (LAB) revealed that 49 strains possessed homologs of horB and horC. No false-positive results have been observed for nonspoilage LAB or frequently encountered brewery isolates. These features are superior to those of horA and ORF 5, previously reported genetic markers for determining the beer spoilage ability of LAB. It was further shown that the combined use of horB/horC and horA is able to detect all 51 beer spoilage strains examined in this study. Furthermore sequence comparison of horB and horC homologs identified in four different beer spoilage species indicates these homologs are 96.6 to 99.5% identical, which is not typical of distinct species. The wide and exclusive distribution of horB and horC homologs among beer spoilage LAB and their sequence identities suggest that the hop resistance ability of beer spoilage LAB has been acquired through horizontal gene transfer. These insights provide a foundation for applying trans-species genetic markers to differentiating beer spoilage LAB including previously unencountered species.     

Asaia sp., an Unusual Spoilage Organism of Fruit-Flavored Bottled Water

A gram-negative bacillus was isolated from a batch of fruit-flavored bottled water, which had spoiled as a result of bacterial overgrowth (>10⁶ CFU/ml). The spoilage organism was extremely difficult to identify phenotypically and was poorly identified as Pasturella sp. (78.7% identification profile) employing the API 20NE identification scheme, which gave the profile 5040000. Molecular identification through PCR amplification of a partial region of the 16S rRNA gene followed by direct automated sequencing of the PCR amplicon allowed identification of the organism. Due to the sequence identity (100%) between the spoilage organism and a reference strain in GenBank, the spoilage isolate was considered to be an Asaia sp., a recently described genus and member of the acetic acid bacteria. This is the first report of Asaia sp. causing spoilage of a foodstuff and highlights the benefits of molecular identification techniques based on 16S rRNA gene sequences in the identification of unusual spoilage organisms.     

The effect of variation of thermal processing on the microbial spoilage of chub-packed luncheon meat

Process pasteurization values for reference temperature 70°C (P₇₀) were calculated from the temperature profiles of 250 g luncheon meat chubs cooked under experimental conditions. A simple equation relating Process P₇₀-value and the time and temperature of cooking was derived. With minimal cooking (P₇₀= 40) the surviving microflora (10³/g) was dominated by species of Lactobacillus, Brochothrix and Micrococcus. These organisms were destroyed by more intensive cooking (P₇₀= 105), leaving a flora (10²/g) composed of Bacillus and Micrococcus species. The spoilage that developed after 14 d storage at 25°C reflected the severity of the heat treatment received by each chub: with P₇₀ between 40 and 90, a Streptococcus spoilage sequence occurred; with P₇₀ between 105 and 120, a Bacillus/Streptococcus spoilage sequence occurred; with P₇₀ of 135 and above, a Bacillus spoilage sequence occurred. Cooking to a P₇₀= 75 was adequate to reduce the surviving microflora to the 10²/g level associated with current good manufacturing practice.     

Microbial Spoilage of Fresh Refrigerated Beef Liver

The types and numbers of micro-organisms involved in the spoilage of refrigerated beef liver were studied together with pH, hydration and organoleptic changes of the material. Fresh liver harboured a mixed population ( c . 1 × 10⁵ organisms/g) of Gram positive cocci, chromogens and non-chromogens, sporeformers, presumptive coliforms and Gram negative rods. When samples were rejected organoleptically, after 7–10 days at 5°, the contamination attained levels of c . 7–8 × 10⁷ organisms/g. Spoilage was due to souring; the pH fell from 6·3 (fresh liver) to c . 5·9. Lactic acid bacteria were predominant and Gram negative bacteria did not exceed 1·0 × 10⁶ organisms/g. This type of spoilage is explained by the carbohydrate content of c . 5% in liver. The value of pH appears to be a reliable indicator of liver freshness, with a pH of 6·1 indicating incipient spoilage.     

Asaia sp., an unusual spoilage organism of fruit-flavored bottled water

A gram-negative bacillus was isolated from a batch of fruit-flavored bottled water, which had spoiled as a result of bacterial overgrowth (>10(6) CFU/ml). The spoilage organism was extremely difficult to identify phenotypically and was poorly identified as Pasturella sp. (78.7% identification profile) employing the API 20NE identification scheme, which gave the profile 5040000. Molecular identification through PCR amplification of a partial region of the 16S rRNA gene followed by direct automated sequencing of the PCR amplicon allowed identification of the organism. Due to the sequence identity (100%) between the spoilage organism and a reference strain in GenBank, the spoilage isolate was considered to be an Asaia sp., a recently described genus and member of the acetic acid bacteria. This is the first report of Asaia sp. causing spoilage of a foodstuff and highlights the benefits of molecular identification techniques based on 16S rRNA gene sequences in the identification of unusual spoilage organisms.     

Use of a Titrimetric Method to Assess the Bacterial Spoilage of Fresh Beef

A new method of determining bacterial spoilage in fresh beef is presented. The technique is based upon the fact that as beef undergoes refrigerator spoilage, there is a gradual increase in the production of alkaline substances by the spoilage flora. The level of these substances was measured by titrating meat homogenates to a pH 5.00 end point, employing 0.02 n HCl and an autotitrator. When 23 samples of ground beef from retail stores were tested, an average of 1.32 ml of acid was required for titration of 1 g of fresh beef to pH 5.00, whereas 2.58 ml was required for the same meat at the onset of spoilage. Preliminary data indicate that beef which requires more than 2 ml of 0.02 n HCl/g to lower its pH to 5.00 under the conditions of the test is in some state of incipient spoilage. The statistical correlation between titration values, log bacterial numbers, and extract-release volume was high (P < 0.001). The technique is simple to execute and is highly reproducible, and duplicate samples can be run within 15 min.