Gram-negative Psychrotrophic Bacteria Research Articles

Enhancing extracellular membrane vesicle productivity of Shewanella vesiculosa HM13, a prospective host for vesiculation-mediated protein secretion, by weakening outer membrane-peptidoglycan linkage

Shewanella vesiculosa HM13, a psychrotrophic gram-negative bacterium isolated from the intestinal contents of horse mackerel, produces abundant extracellular membrane vesicles (EMVs) by budding the outer membrane. The EMVs of this bacterium carry a single major cargo protein, P49, of unknown function, which may be useful as a carrier for the secretory production of heterologous proteins as cargoes of EMVs. In this study, to increase the utility of S.vesiculosa HM13 as a host for EMV-mediated protein production, we improved its EMV productivity by weakening the linkage between the outer membrane and underlying peptidoglycan layer. In gram-negative bacteria, the outer membrane is connected to peptidoglycans predominantly through Braun's lipoprotein (Lpp), and the formation of this linkage is catalyzed by an l,d-transpeptidase (Ldt). We constructed gene-disrupted mutants of Lpp and Ldt and assessed their EMV productivity. The EMVs of the lpp- and ldt-disrupted mutants grown at 18°C were evaluated using nanoparticle tracking analysis, and their morphologies were observed using transmission electron microscopy. As a result, an approximately 2.5-fold increase in EMV production was achieved, whereas the morphology of the EMVs of these mutants remained almost identical to that of the parent strain. In accordance with the increase in EMV production, the mutants secreted approximately 2-fold higher amounts of P49 than the parent strain into the culture broth as the EMV cargo. These findings will contribute to the development of an EMV-based secretory production system for heterologous proteins using S.vesiculosa HM13 as a host.

Identification, proteolytic activity quantification and biofilm-forming characterization of Gram-positive, proteolytic, psychrotrophic bacteria isolated from cold raw milk.

Psychrotrophic bacteria of raw milk face the dairy industry with significant spoilage and technological problems due to their ability to produce heat-resistant enzymes and biofilms. Despite extensive information about Gram-negative psychrotrophic bacteria in milk, little is known about Gram-positive psychrotrophic bacteria in milk, and their proteolytic activity and biofilm-forming characteristics. In the present study, Gram-positive, proteolytic, psychrotrophic bacteria of cold raw milk were identified, and their proteolytic activity and biofilm-forming capacity were quantified. In total, 12 genera and 22 species were represented among the bacterial isolates, however 50% belonged to three genera, namely Staphylococcus (19.4%), Bacillus (16.7%), and Enterococcus (13.9%). Different levels of proteolytic activity were detected in the identified isolates, even among the strains belonging to the same species. In addition, proteolytic activity was significantly higher at 25°C than at 7°C for all isolates. The crystal violet staining assay in polystyrene microtitre plates revealed a high level of variation in the biofilm-forming capacity at 7°C. After 72 hours of incubation, 11.1% of the strains did not produce a biofilm, while 27.8%, 52.8%, and 8.3% produced low, moderate, and high amounts of biofilm on polystyrene, respectively. The psychrotrophic bacteria were also able to produce biofilms on the surface of stainless steel coupons in ultra-high temperature milk after 72 h of incubation at 7°C; the number of attached cells ranged from 1.34 to 5.11 log cfu/cm2. These results expand the knowledge related to the proteolytic activity and biofilm-forming capacity of Gram-positive psychrotrophic milk bacteria.

Assessment of the Spoilage Microbiota during Refrigerated (4 °C) Vacuum-Packed Storage of Fresh Greek Anthotyros Whey Cheese without or with a Crude Enterocin A-B-P-Containing Extract.

Although fresh whey cheeses are prone to rapid deterioration, mainly by psychrotrophic Gram-negative bacteria and lactic acid bacteria (LAB), data on the specific spoilage species in traditional Greek whey cheeses are scarce. Therefore, this study quantified growth and characterized the primary spoilage bacteria in fresh Anthotyros whey cheeses stored at 4 °C in a vacuum for 40 days, without or with an added 5% (v/w) of an enterocin A-B-P crude extract (CEntE). Psychrotrophic Pseudomonas spp., Aeromonas spp., Hafnia spp. and Serratia spp. grew faster than LAB during early storage. However, LAB outgrew the Gram-negative bacteria and prevailed by mid to late storage in all cheese batches, causing a strong or milder batch-dependent natural acidification. Two major non-slime-producing and two minor biotypes of Leuconostoc-like bacteria, all identified as Leuconostoc mesenteroides by 16S rRNA sequencing, dominated the LAB association (76.7%), which also included four subdominant Carnobacterium maltaromaticum biotypes (10.9%), one Leuconostoc lactis biotype (3.3%) and few Lactococcus (1.6%), mesophilic Lactobacillus (0.8%) and Enterococcus (0.8%). Growth and distribution of LAB and Gram-negative species were strongly batch-dependent and plant-dependent. The CEntE neither retarded growth nor altered the whey cheese spoilage association but enhanced LAB growth and the declines of Gram-negative bacteria by late storage.

Complete Lipooligosaccharide Structure from Pseudoalteromonas nigrifaciens Sq02-Rifr and Study of Its Immunomodulatory Activity.

Lipopolysaccharides (LPS) are surface glycoconjugates embedded in the external leaflet of the outer membrane (OM) of the Gram-negative bacteria. They consist of three regions: lipid A, core oligosaccharide (OS), and O-specific polysaccharide or O-antigen. Lipid A is the glycolipid endotoxin domain that anchors the LPS molecule to the OM, and therefore, its chemical structure is crucial in the maintenance of membrane integrity in the Gram-negative bacteria. In this paper, we reported the characterization of the lipid A and OS structures from Pseudoalteromonas nigrifaciens Sq02-Rifr, which is a psychrotrophic Gram-negative bacterium isolated from the intestine of Seriola quinqueradiata. The immunomodulatory activity of both LPS and lipid A was also examined.

Next Generation Sequencing (NGS) for the Determination of Fish Flesh Microbiota

The objective of the study is the assessment of the microbial ecology and safety of fish in Greece using next-generation sequencing (NGS) and the correlation of the species of microbial flora with the production of histamine. Fourteen different fish species were obtained from local fish stores (Greece) within 1 day from capture. The initial microbiota in fish flesh was determined using NGS. The main pathogenic bacterial species identified in the tested fish samples included Vibrio spp., Clostridium spp., Staphylococcus, Flavobacterium and Janthinobacterium representing both native freshwater habitats and contaminants arising from different sources, including sewage and direct contamination by wild animals, livestock, and feed. The initial spoilage microbiota of fish consisted of several psychrotrophic Gram-negative bacteria, such as Pseudomonas, Acinetobacter, Moraxella, Shewanella, Psychrobacter, Lactobacillus, Brochothrix and Photobacterium. The results of the study show the potential of the application and the usefulness of NGS for the determination of microbial flora associated with food-borne diseases and spoilage in fish products. Histamine formation correlated with the valid reads (concentration and number of bacteria) and slightly with the genus of the identified microorganisms.

Identification of natural product compounds as quorum sensing inhibitors in Pseudomonas fluorescens P07 through virtual screening

Pseudomonas fluorescens, a Gram-negative psychrotrophic bacteria, is the main microorganism causing spoilage of chilled raw milk and aquatic products. Quorum sensing (QS) widely exists in bacteria to monitor their population densities and regulate numerous physiological activities, such as the secretion of siderophores, swarming motility and biofilm formation. Thus, searching for quorum sensing inhibitors (QSIs) may be another promising way to control the deterioration of food caused by P. fluorescens. Here, we screened a traditional Chinese medicine (TCM) database to discover potential QSIs with lesser toxicity. The gene sequences of LuxI- and LuxR-type proteins of P. fluorescens P07 were obtained through whole-genome sequencing. In addition, the protein structures built by homology modelling were used as targets to screen for QSIs. Twenty-one compounds with a dock score greater than 6 were purchased and tested by biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136). The results showed that 10 of the compounds were determined as hits (hit rate: 66.67%). Benzyl alcohol, rhodinyl formate and houttuynine were effective QSIs. The impact of the most active compound (benzyl alcohol) on the phenotypes of P. fluorescens P07, including swimming and swarming motility, production of extracellular enzymes and siderophores, N-acylhomoserine lactone (AHLs) content and biofilm formation were determined. The inhibitory mechanism of benzyl alcohol on the QS system of P. fluorescens P07 is further discussed. This study reveals the feasibility of searching for novel QSIs through virtual screening.

Prevalence, seasonality, and growth of enterococci in raw and pasteurized milk in Victoria, Australia

This study investigated the prevalence, seasonality, and species variety of enterococci present in raw milk factory silos and pasteurized milk in 3 dairying regions in Victoria, Australia, over a 1-yr period. Additionally, the growth ability of thermoduric enterococci isolated in this study (Enterococcus faecalis, E. faecium, E. hirae, and E. durans) was determined in milk at temperatures likely to occur during storage, transport, and distribution, and before domestic consumption (4 and 7°C). Enterococci were detected in 96% of 211 raw milk samples, with an average count of 2.48 log10 cfu/mL. Counts were significantly lower in winter than summer (average 1.84 log10 cfu/mL) and were different between factories but not regions. Enterococcus faecalis was the most prevalent species isolated from raw milk in every factory, comprising between 61.5 and 83.5% of enterococcal species across each season. Enterococci were detected in lower numbers in pasteurized milk than in raw milk and were below the limit of detection on spread plates (<10 cfu/mL) after factory pasteurization. Residual viable cells were only detected following enrichment using 100-mL samples of milk, with 20.8% of the samples testing positive; this equated to a decrease in the average raw milk enterococci count of >4 log10 cfu/mL following pasteurization. Although E. faecalis predominated in raw milk and E. durans was found in only 2.9% of raw milk samples, E. durans was the most prevalent species detected in pasteurized milk. The detection of enterococci in the pasteurized milk did not correlate with higher enterococci counts in the raw milk. This suggested that the main enterococci populations in raw milk were heat-sensitive and that thermoduric enterococci survived pasteurization in a small numbers of instances. All of the thermoduric enterococci that were assessed for growth at likely refrigeration temperatures were able to grow at both 4 and 7°C in sterile milk, with generation times of 35 to 41h and 16 to 22h, respectively. Thermoduric enterococci were detected in pasteurized milk stored at 4°C for 2 wk (typically 1 to 9 cells/100mL, up to 2.82 log10 cfu/mL), demonstrating the potential of enterococci to survive pasteurization and contribute to milk spoilage at refrigeration temperatures. This is particularly relevant for milk that is aseptically packaged to exclude gram-negative psychrotrophic bacteria and kept above the recommended storage temperature of ≤5°C.

エジプトにおいて生産・製造された生乳, クリームおよびヨーグルトから分離したグラム陰性低温細菌の脂質分解性および蛋白分解性

エジプトにおいて生産・製造された生乳, クリームおよびヨーグルトから分離したグラム陰性低温細菌の脂質分解性および蛋白分解性

Characterization of Gram-negative psychrotrophic bacteria isolated from Italian bulk tank milk.

Eighty psychrotrophic bacterial strains, isolated from different northwest Italian bulk tank milks destined for Grana Padano cheese production, were identified by 16S rRNA gene amplification and partial sequence analysis of the rpoB gene. Pseudomonas spp. were the most commonly occurring contaminants, P. fluorescens being the predominant isolated species, along with Enterobacteriaceae, primarily Serratia marcescens. RAPD-PCR was used to study genetic variability and distinguish closely related strains; a high degree of genetic heterogeneity among the strains was highlighted. All the strains were characterized for their ability to produce proteases, lipases and lecithinases at different temperatures (7, 22, and 30 °C). Forty-one of the psychrotrophic strains were positive for all the enzymatic activities. The highest number of positive strains for all the incubation temperatures was found for lipolytic activity (59), followed by proteolytic (31) and lecithinase (28) activities, and the enzymatic traits varied among the Pseudomonas and Enterobacteriaceae strains. The proteolytic psychrotrophic strains were screened for the presence of the aprX gene, coding for a heat-resistant metalloprotease in Pseudomonas spp. The aprX gene was detected in 19 of 63 Pseudomonas strains, and was widespread in the P. fluorescens strains (14/19). PRATICAL APPLICATION: The study provides new data on the enzymatic activity of Gram-negative psychrotrophic bacteria, useful in developing strategies to control the proteo-lipolytic spoilage of raw and processed milk that causes gelation, off-flavors, and loss of sensory quality and shelf life.

Characterization of a cold-active lipase from Psychrobacter cryohalolentis K5T and its deletion mutants

A gene coding for cold-active lipase from the psychrotrophic Gram-negative bacterium Psychrobacter cryohalolentis K5(T) isolated from a Siberian cryopeg has been cloned and expressed in Escherichia coli. The recombinant protein Lip1Pc with a 6× histidine tag at its C-terminus was purified by nickel affinity chromatography. With p-nitrophenyl dodecanoate (C12) as a substrate, the purified recombinant protein displayed maximum lipolytic activity at 25°C and pH 8.0. Increasing the temperature above 40°C and addition of various metal ions and organic solvents inhibited the enzymatic activity of Lip1Pc. Most nonionic detergents, such as Triton X-100 and Tween 20, slightly increased the lipase activity, while SDS completely inhibited it. To investigate the functional significance of the Lip1Pc N-terminal domain, we constructed five deletion mutants of this protein. The ND1 and ND2 mutants displayed specific activity reduced by 30-35%, while other truncated proteins were completely inactive. Both mutants demonstrated increased activity towards p-nitrophenyl decanoate (C10) and impaired utilization of C16 substrate. Although optimum reaction temperature of ND2 lowered to 20°C, it displayed enhanced stability by 44% after incubation at 40°C. The results prove that the N-terminal domain of Lip1Pc has a fundamental impact on the activity and stability of the protein.

Effect of inoculation of lactic acid bacteria on proteolytic activity of psychrotrophic Gram-negative bacteria in fresh farmed sea bass (Dicentrarchus labrax) fillets during storage at 4 °C under vacuum-packed conditions

The effect of two strains of lactic acid bacteria (LAB) (Lactococcus lactis and Carnobacterium piscicola) on the proteolytic activity of four strains of Psychrotrophic Gram-negative bacteria [Psy G(−)] (Pseudomonas fluorescens, Aeromonas hydrophila, Pseudomonas putida and Photobacterium damselae) has been determined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), in fresh vacuum-packed farmed sea bass (Dicentrarchus labrax) fillets artificially contaminated, during 21 days of chilled storage. The profiles of sarcoplasmic (SP) and myofibrillar (MP) proteins indicated that the major changes were produced with Pseudomonas fluorescens, Aeromonas hydrophila, and Pseudomonas putida starters. The results also showed that LAB strains presented a weak proteolytic activity against MP and SP proteins in muscle of fresh sea bass. In fact, we noted the less pronounced degradation of protein fractions in samples inoculated with LAB combination. Moreover, a significant bacteriostatic effect of LAB strains was demonstrated against all microflora, particularly mesophilic aerobic plate counts (MAPC) and psychrotrophic bacterial counts (PBC), with fillets remaining unspoiled until the end of storage, against values of 7 and 8 log CFU/g, respectively; control fish fillets exceeded the upper acceptability limit.

Cloning, purification, and characterization of a cold-adapted esterase produced byPsychrobacter cryohalolentisK5Tfrom Siberian cryopeg

A psychrotrophic gram-negative bacterium Psychrobacter cryohalolentis K5(T) was previously isolated from a cryopeg within Siberian permafrost and its genome has been completely sequenced. To clone and characterize potential cold-active lipases/esterases produced by P. cryohalolentis K5(T) , we have identified their potential genes by alignment with amino acid sequences of lipases/esterases from related bacteria. One of the targets, EstPc, was cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant protein was produced with a 6x histidine tag at its C-terminus and purified by nickel affinity chromatography. Purified recombinant protein displayed maximum esterolytic activity with p-nitrophenyl butyrate (C4) as a substrate at 35 °C and pH 8.5. Activity assay conducted at different temperatures revealed that EstPc is a cold-adapted esterase which displayed more than 90% of its maximum activity at 0-5 °C. In contrast to many known cold-active enzymes, it possesses relatively high thermostability, preserving more than 60% of activity after incubation for 1 h at 80 °C. It was activated by Ca(2+) , Mn(2+) , and EDTA whereas Zn(+2) , Cu(+2) , Co(+2) , Ni(+2) , and Mg(+2) inhibited it. Various organic solvents (ethanol, methanol and others) inhibited the enzyme. Most non-ionic detergents, such as Triton X-100 and Tween 20 increased the lipase activity while SDS completely inhibited it.

Identification of cold-inducible inner membrane proteins of the psychrotrophic bacterium, Shewanella livingstonensis Ac10, by proteomic analysis

Shewanella livingstonensis Ac10 is a psychrotrophic Gram-negative bacterium that grows at temperatures close to 0°C. Previous proteomic studies of this bacterium identified cold-inducible soluble proteins and outer membrane proteins that could possibly be involved in its cold adaptation (Kawamoto et al. in Extremophiles 11:819-826, 2007). In this study, we established a method for separating the inner and outer membranes by sucrose density gradient ultracentrifugation and performed proteomic studies of the inner membrane fraction. The cells were grown at temperatures of 4 and 18°C, and phospholipid-enriched inner membrane fractions were obtained. Two-dimensional polyacrylamide gel electrophoresis and peptide mass fingerprinting analysis of the proteins identified 14 cold-inducible proteins (more than a 2-fold increase at 4°C). Six of these proteins were predicted to be inner membrane proteins. Two predicted periplasmic proteins, 5 predicted cytoplasmic proteins, and 1 predicted outer membrane protein were also found in the inner membrane fraction, suggesting their association with the inner membrane proteins and/or lipids. These cold-inducible proteins included proteins that are presumed to be involved in chemotaxis (AtoS and PspA), membrane protein biogenesis (DegP, SurA, and FtsY), and morphogenesis (MreB). These findings provide a basis for further studies on the cold-adaptation mechanism of this bacterium.

Pseudomonas spp. and Hafnia alvei growth in UHT milk at cold storage

Gram-negative psychrotrophic bacteria isolated from samples of spoiled commercial ultra-heat treated (UHT) milk from Taiwan were comprised of 67% Pseudomonas spp. and 22% Enterobacteriaceae. Whole milk, low-fat milk, and flavored milk samples were tested. The modified Gompertz equation was used to model the data and provided a good fit for the growth of Pseudomonas spp. and Hafnia alvei at 7°C for 14 days in both whole UHT milk and low-fat UHT milk. From this model, the estimated lag time for H. alvei was longer than that for Pseudomonas fluorescens in both whole UHT milk and low-fat UHT milk. The growth rate of H. alvei was higher than that of P. fluorescens. The pH value was significantly reduced by H. alvei than P. spp. after cultured in whole and low-fat UHT milk at 7°C for 14 days.

Novel halotolerant bacterium from cryopeg in permafrost: Description of Psychrobacter muriicola sp. nov.

A novel halotolerant psychrotrophic gram-negative bacterium, strain 2pS, was isolated from lenses of water brine in Arctic permafrost (cryopeg). The optimal growth of the new strain was observed at 16-18 degrees C; the maximal and minimal growth temperatures were 37 degrees C and -2 degrees C, respectively. The pH growth range was 5.8 to 8.5 (optimum 6.5-7.5) and the range of medium salinity was 0 to 100 g/l (optimum 3-8 g/l NaCl). The strain 2pS did not produce acid from carbohydrates and utilized acetate, yeast extract, pyruvate, glutarate, fumarate, caproate, heptanoate, butyrate, malate, DL-lactate, citrate, L-proline, L-tyrosine, butanol, and dulcitol as the sole carbon and energy sources. The major fatty acids of the cell wall at optimal growth temperature were C18:1(omega 7) and C18:1(omega 9). The G + C DNA content was 46.0 mol.%. Phylogenetic analysis of the 16S rRNA gene sequences showed that the studied strain was the closest (97% similarity) to Psychrobacter nivimaris DSM 16093T, a halotolerant psychrotrophic bacterium isolated from the Arctic sea's ice. Genotypic and phenotypic differences of the new bacterium from closely related species lead to the conclusion that strain 2pS belongs to a novel species of the genus Psychrobacter: Psychrobacter muriicola sp. nov.

Eicosapentaenoic Acid Plays a Beneficial Role in Membrane Organization and Cell Division of a Cold-Adapted Bacterium,Shewanella livingstonensisAc10

Shewanella livingstonensis Ac10, a psychrotrophic gram-negative bacterium isolated from Antarctic seawater, produces eicosapentaenoic acid (EPA) as a component of phospholipids at low temperatures. EPA constitutes about 5% of the total fatty acids of cells grown at 4 degrees C. We found that five genes, termed orf2, orf5, orf6, orf7, and orf8, are specifically required for the synthesis of EPA by targeted disruption of the respective genes. The mutants lacking EPA showed significant growth retardation at 4 degrees C but not at 18 degrees C. Supplementation of a synthetic phosphatidylethanolamine that contained EPA at the sn-2 position complemented the growth defect. The EPA-less mutant became filamentous, and multiple nucleoids were observed in a single cell at 4 degrees C, indicating that the mutant has a defect in cell division. Electron microscopy of the cells by high-pressure freezing and freeze-substitution revealed abnormal intracellular membranes in the EPA-less mutant at 4 degrees C. We also found that the amounts of several membrane proteins were affected by the depletion of EPA. While polyunsaturated fatty acids are often considered to increase the fluidity of the hydrophobic membrane core, diffusion of a small hydrophobic molecule, pyrene, in the cell membranes and large unilamellar vesicles prepared from the lipid extracts was very similar between the EPA-less mutant and the parental strain. These results suggest that EPA in S. livingstonensis Ac10 is not required for bulk bilayer fluidity but plays a beneficial role in membrane organization and cell division at low temperatures, possibly through specific interaction between EPA and proteins involved in these cellular processes.

Contagem, isolamento e caracterização de bactérias psicrotróficas contaminantes de leite cru refrigerado

Com os objetivos de quantificar, isolar e caracterizar bactérias psicrotróficas contaminantes de leite cru refrigerado, produzido na região da Zona da Mata de Minas Gerais e Sudeste do Rio de Janeiro, foram analisadas amostras de leite coletadas de 20 tanques coletivos e 23 tanques individuais. As contagens de bactérias psicrotróficas nas amostras de leite para os dois tipos de tanques de refrigeração variaram entre 10² e 10(7) Unidades Formadoras de Colônias (UFC) ml-1, porém, um maior número de tanques coletivos apresentou contagens acima de 1 x 10(5) UFC ml-1. Foi verificada a predominância de bactérias psicrotróficas gram-negativas (81,2%), que foram identificadas pelos sistemas API 20E e API 20NE nos gêneros: Aeromonas, Alcaligenes, Acinetobacter, Burkholderia,Chryseomonas, Enterobacter, Ewingella, Klebsiella, Hafnia, Methylobacterium, Moraxella, Pantoea, Pseudomonas, Serratia, Sphingomonas e Yersinia. As bactérias gram-positivas (18,8%) foram identificadas com API 50 CH, API Coryne e API Staph, nos gêneros: Bacillus, Brevibacterium, Cellum/Microbacterium, Kurthia e Staphylococcus. Os sistemas API utilizados não identificaram todos os isolados bacterianos. Pseudomonas foi o gênero mais isolado e P. fluorescens foi a espécie predominante. A maioria dos isolados bacterianos apresentou atividade proteolítica e/ou lipolítica a temperaturas de refrigeração de 4°C, 7°C e 10°C, evidenciando seu alto potencial de deterioração do leite e dos produtos lácteos. Os resultados ressaltam que maior atenção deve ser dada aos procedimentos que impeçam a contaminação do leite por esses microrganismos.

Detection of acylated homoserine lactones in gram-negative proteolytic psychrotrophic bacteria isolated from cooled raw milk

Through a mechanism called quorum sensing, bacteria are able to express specific genes in response to population density. Cell-to-cell communication in bacteria is mediated by signal molecules such as acylated homoserine lactones (AHLs). This work aimed to detect AHL production in Gram-negative psychrotrophic bacteria isolated from raw milk. A total of 84.9% of the bacteria were identified as AHL producers eliciting a diversity of responses in the AHL-monitor systems. These results demonstrate that AHL-production is common among psychrotrophic bacteria isolated from milk, and indicate that quorum sensing may play an important role in the spoilage of this product.

The p-nitroaniline test to asses the bacterial microbiota of raw ground meat aerobically stored

The previously developed p-nitroaniline test for assessing the microbial load of meat surfaces has been now adapted to determine the microbial quality of raw ground meat. A good correlation ( r = 0.91) between bacterial count determined by the pour plate method and the p-nitroaniline test was obtained. The sensitivity of the new method was of the order of magnitude of 10 4 cfu/g. This method allows the assay of ground meat in approximately 2.5 h, it does not require expensive equipment and the results can be interpreted both spectrophotometrically and visually. Additionally, it has been proven that the method is useful in estimating the microbial quality of raw meat irrespective of the species of Gram-negative psychrotrophic bacteria prevailing in the meat during refrigerated storage.

A rapid method for the estimation of the microbiological quality of refrigerated raw milk based on the aminopeptidase activity of Gram-negative bacteria

A rapid method for the estimation of the microbiological quality of refrigerated raw milk was developed based on the aminopeptidase activity of Gram-negative psychrotrophic bacteria, the dominant microbial population in refrigerated foods. Two versions of the test for measuring the aminopeptidase activity are reported (direct and indirect). A good correlation (r=0.93–0.95) between bacterial counts estimated by conventional methods and the aminopeptidase activity determined in milk was obtained. The sensitivity of the indirect assay was 2×104 cfu mL−1. This sensitivity limit complies with the level of detection required to satisfy regulations in many countries. The aminopeptidase test allows the assessment of raw milk quality in approximately 2.5 h, does not require either high-cost equipment or specialised operators, and results can be interpreted both spectrophotometrically and visually.