Oxygen tension measurement as a means of detecting incipient spoilage of raw milk by psychrotrophic bacteria?

A large fall (89% to 10% saturation) in the oxygen tension of raw milk incubated with constant aeration and agitation at 7°C in a fermenter was observed to occur prior to the onset of extracellular protease and lipase production by microorganisms. This same pattern with respect to oxygen tension and lipase production was also observed when raw milk samples from creamery silos were incubated at 7°C in an orbital incubator. The results are in agreement with earlier fermenter studies in which strains of Pseudomonas fluorescens were grown in a simulated milk medium at 7°C. Measurement of oxygen tension may be able to be used to detect incipient enzyme-mediated spoilage of raw milk by psychrotrophs.     

Survival of Lipase during Manufacture of Nonfat Dry Milk

Extracellular lipases from several strains of Pseudomonas fluorescens were active against milk fat. The activity of P. fluorescens strain B52 lipase in reconstituted NDM using β-naphthyl caprylate as substrate was 70% of that found with skim milk; however, hydration of the NDM at 4°C for 12 h resulted in 85% recovery of activity. As little as .0025% (vol/vol) of P. fluorescens spent media was detected in reconstituted skim milk powder using β-naphthyl caprylate. The process of freeze-drying or spray-drying without heating had little effect on either native or P. fluorescens B52 lipase. The bacterial lipase activity was reduced by 12 to 21% and 55 to 59% in NDM treated with low heat (72°C/16 s) and high heat (110°C/2 min), respectively, while the native lipases were completely inhibited by these treatments. Both native and bacterial enzymes were stable when stored in NDM for extended periods at 20°C. In a limited study of commercial NDM samples, microbial lipase was not detected, suggesting that the quality of the raw milk was sufficiently high to restrict the presence of heat-stable lipolytic enzymes.     

Stimulation by Calcium of the Formation of Active Extracellular Proteinase by Pseudomonas fluorescens spp.

Pseudomonas fluorescens produced extracellular proteinase in mineral salts medium in response to the addition of small molecular weight compounds derived from skim milk. Further fractionation of these compounds on Sephadex G-10 gave a highly stimulatory fraction lacking in peptides or amino acids but high in calcium. Further studies showed that proteinase production in mineral salts medium containing calcium was 29 to 64% of that in skim milk. Zinc, manganese, and magnesium ions failed to substitute for calcium. At 20 and 5°C, 50 and 10 μM calcium, respectively, were required for half maximum synthesis. Several complex media supported variable growth and enzyme synthesis; however, this variability did not seem related to the calcium content of the medium. Calcium was required for the formation of active extracellular proteinase by a number of strains of Pseudomonas fluorescens when growing in mineral salts medium. These results suggest that milk proteins are not essential for proteinase synthesis by Pseudomonas fluorescens.     

Lipid breakdown and sensory analysis of milk inoculated with Chryseobacterium joostei or Pseudomonas fluorescens

The lipolytic activity, lipid oxidation, sensory effect and production of volatile compounds by Chryseobacterium joostei and Pseudomonas fluorescens in full cream milk, incubated at 4 and 25 °C, were investigated. Lipolytic activity, measured by the amount of free fatty acids released, showed high levels for both organisms. Although no significant differences between C. joostei and Ps. fluorescens were observed at 4 °C, C. joostei was significantly (P < 0.001) more lipolytic than Ps. fluorescens at 25 °C. Lipid oxidation was measured by thiobarbituric acid analysis. No significant differences between any of the inoculated milk samples were noted. During sensory analysis, C. joostei produced stronger odours than Ps. fluorescens. Different odours were also described for the two species. Volatile compound analysis determined that C. joostei has the ability to produce the same amount of off-odours during psychrotrophic and mesophilic growth conditions as Ps. fluorescens, but with different odour profiles.     

Temperature modulates the production and activity of a metalloprotease from Pseudomonas fluorescens 07A in milk

This work evaluated the expression and activity of a metalloprotease released by Pseudomonas fluorescens 07A in milk. Low relative expression of the protease by the strain was observed after incubation for 12 h at 25°C while the strain was in the logarithmic growth phase. After 24 h, protease production significantly increased and remained constant for up to 48 h, a time range during which the strain remained in the stationary phase. Conversely, at refrigeration temperatures, at 12 h the strain was still in the lag phase and expressed the protease at higher levels than when the logarithmic phase was reached. Casein fractions were highly degraded by P. fluorescens 07A, the purified protease, and the bacterial pellet on d 7 of incubation at 25°C and to a lesser extent at 10°C for the sample incubated with the bacterium. Heat treatment at 90°C for 5 min completely inactivated the proteolytic activity of the purified protease and the bacterial pellet. This work contributes to the knowledge about the conditions of milk storage that influence the production and activity of this extracellular metalloprotease. The results demonstrate the need to find alternative strategies to control the synthesis and activity of proteolytic enzymes in the dairy industry to ensure the quality of processed products.     

Inhibition of Lipolytic Activity in Milk by Polysaccharides,

The effect of gums on the activity of milk lipase and a Pseudomonas lipase in milk was investigated. Gums were hydrated in water and mixed with whole milk. Lipase was added to the gum-milk mixture and hydrolysis was determined after 48 h at 4°C by the acid degree value method. Of the gums tested, the anionically charged λ-, ι- and κ-carrageenan, furcellaran, and sodium alginate significantly inhibited milk lipase activity by 93.7, 81.2, 46.8, 50.6, and 62.1%, respectively. Furthermore, λ-carrageenan was 87.6% effective in inhibiting lipolysis by a purified Pseudomonas fluorescens MC50 lipase in milk. The other gums tested, tragacanth, carboxymethyl cellulose, locust bean, propylene glycol alginate, xanthan, microcrystalline cellulose, guar, and arabic did not significantly inhibit milk lipase. Commonly used stabilizers can inhibit lipolytic activity in milk.     

Isolation and characterisation of a heat-resistant peptidase from Pseudomonas panacis withstanding general UHT processes

A secreted peptidase from Pseudomonas panacis was identified and purified. Genome sequencing of the producer strain allowed identification of the peptidase as AprA based on a comparison to peptide sequences of mass spectra obtained from the purified enzyme. The amino acid sequence of the 49.4 kDa peptidase was 98% similar to the metallopeptidase AprX from a Pseudomonas fluorescens strain. The peptidase showed maximum activity at pH 8 and 40 °C and withstood general ultra-high temperature (UHT) processing (138 °C for 18 s) in skim milk, with 88.0 ± 7.7% of the initial enzyme activity remaining after heating. The peptidase showed considerable enzyme activity under storage conditions of UHT milk. The potential for spoilage of milk might during storage was verified by adding very low enzyme activities to UHT-treated milk. The addition of 1 pkat mL⁻¹ peptidase activity resulted in a destabilisation of the milk during four weeks storage.     

Kinetic models for pulsed electric field and thermal inactivation of Escherichia coli and Pseudomonas fluorescens in whole milk

Inactivation of Escherichia coli ATCC 11775 and Pseudomonas fluorescens ATCC 948 in UHT whole (4% fat) milk during thermal processing at 56–62 °C and pulsed electric field (PEF) processing at 30 or 35 kV cm⁻¹ at approximately 30, 40 or 50 °C was investigated. E. coli ATCC 11775 was more heat-resistant than P. fluorescens ATCC 948, but more susceptible to PEF processing. All inactivation kinetics showed strong deviations from log-linearity. Thus, a simplified logistic (log-decay) regression model was used to accurately predict thermal and PEF inactivation of E. coli ATCC 11775 and P. fluorescens ATCC 948 under various treatment conditions. This is a useful tool for identifying processing conditions to inactivate pathogenic and spoilage microorganisms in whole milk at sub-pasteurisation temperatures.     

Development of a novel loop-mediated isothermal amplification assay for the detection of lipolytic Pseudomonas fluorescens in raw cow milk from north China

Lipases secreted by psychrotrophic bacteria are known to be heat resistant and can remain active even after the thermal processing of milk products. Such enzymes are able to destabilize the quality of milk products by causing a rancid flavor. Rapid detection of a small amount of heat-resistant lipase-producing psychrotrophic bacteria is crucial for reducing their adverse effects on milk quality. In this study, we established and optimized a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Pseudomonas fluorescens in raw cow milk, as the most frequently reported heat-resistant lipase-producing bacterial species. Pseudomonas fluorescens-specific DNA primers for LAMP were designed based on the lipase gene sequence. Reaction conditions of the LAMP assay were tested and optimized. The detection limit of the optimized LAMP assay was found to be lower than that of a conventional PCR-based method. In pure culture, the detection limit of the LAMP assay was found to be 4.8 × 10¹ cfu/reaction of the template DNA, whereas the detection limit of the PCR method was 4.8 × 10² cfu/reaction. Evaluation of the performance of the method in P. fluorescens-contaminated pasteurized cow milk revealed a detection limit of 7.4 × 10¹ cfu/reaction, which was 10² lower than that of the PCR-based method. If further developed, the LAMP assay could offer a favorable on-farm alternative to existing technologies for the detection of psychotrophic bacterial contamination of milk, enabling improved quality control of milk and milk products.     

Purification and Characterization of Four Extracellular Proteases Isolated from Raw Milk Psychrotrophs

Extracellular proteases from psychrotrophic strains of Bacillus coagulans (LY 9), Bacillus sp. (LY 10), Bacillus subtilis (LY 11), and Pseudomonas fluorescens (LY 13) were purified and characterized. The molecular weight of the purified protease from Pseudomonas fluorescens LY 13 was 4.50 × 10⁴, and from the three Bacillus species ranged from 3.35 × 10⁴ to 3.90 × 10⁴. The proteases from LY 10 and LY 13 were monomeric proteins, whereas the protease from LY 9 was in a polymeric form that contained up to 14 subunits. Only the protease from Pseudomonas fluorescens LY 13 showed trypsin-like activity. All four proteases were inhibited by ethylenediaminetetraacetate and would be classified as metallo proteases. Casein was the preferred substrate for these proteases. Susceptibility of casein fractions to attack by these proteases varied with the enzyme source. Maximum enzyme activity was between pH 6.5 and 7.5. The protease from Pseudomonas fluorescens LY 13 retained more activity after heating at 63°C for 30 min than the proteases from the three Bacillus species. Calcium ion showed a protective effect by decreasing heat denaturation of the proteases from LY 9 and LY 11. This protective effect tended to be greater in the presence of Tris-HC1 buffer (.05 M, pH 7.5) plus 10% skim milk than in buffer only.     

Microbial competition: effect of Pseudomonas fluorescens on the growth of Listeria monocytogenes

Listeria monocytogenes Scott A was cultured alone and in coculture with Pseudomonas fluorescens ATCC 33231 to characterize quantitatively the effects of microbial competition on the growth of this psychrotrophic pathogen. The bacteria were cultured in brain–heart infusion broth (BHI), using a 3×3×3×2 complete factorial design to assess the impact of temperature (4, 12, 19°C), initial pH (5·0, 6·0, 7·0), and sodium chloride content (5, 25, 45 gl⁻¹) on the interaction between the two micro-organisms. Samples were periodically plated on BHI agar and Vogel Johnson agar to obtain total counts and L. monocytogenes counts, respectively. Growth curves were generated by fitting the data to the Gompertz equation, and the derived growth kinetics were compared. WhenP. fluorescens did influence the growth of L. monocytogenes, the primary effect was a suppression of the maximum population density (MPD) reached by the pathogen. Suppression of L. monocytogenes was generally associated with low incubation temperatures (4°C) and sodium chloride levels (5 and 25 gl⁻¹). Slight increases (<1·0 log cfu ml⁻¹) in the MPD attained by L. monocytogenes were observed when grown in the presence of P. fluorescens at higher temperatures (12 and 19°C) and sodium chloride levels (25 and 45 gl⁻¹) when the pH was 5·0. The current study supports earlier work that indicates that reliance on microbial competition as a barrier to control L. monocytogenes in refrigerated foods will require detailed knowledge of how the interaction between the pathogen and the microflora is affected by environmental and food characteristics such as storage temperature, pH, and water activity.     

Purification and properties of a heat-stable enzyme of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Rm12 from raw milk

Pseudomonas fluorescens Rm12 is a kind of Psychrotrophic bacteria growing in cold raw milk. It produced an extracellular heat resistant protease with an estimated molecular weight of 45 kDa by size exclusion chromatography and SDS-PAGE under both reducing and non-reducing conditions. The enzyme, designated Ht13, was purified to electrophoretic homogeneity from the culture supernatant by sequentially using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography and size exclusion chromatography. The specific activity of the enzyme increased 115.5-folds. The optimum pH value and temperature of Ht13 were 7.5 and 40 °C, respectively. Based on its biochemical characteristics, Ht13 can be included in the group of metalloproteases, which was inhibited by 1, 10-phenanthroline and EDTA but not by pepstatin A, chymostatin, STI, E-64, BBI, PMSF and pAPMSF. Mn²⁺ has positive effect on activity and can increased the heat resistance capability, while Ca²⁺ had a negligible effect. For the hydrolysis of azocasein, the Km was 0.012 mg mL⁻¹. The enzyme showed typical heat-stable behavior. After treatment of 160 °C 20 s, the residual activity was 9%. The half-life of the enzyme at 160 °C in buffer with Mn²⁺ was approximately 12 s. Among several main milk proteins, Ht13 can cleave α_s-casein, β-casein and κ-casein. The sequence of 1st–16th amino acids of N-terminal was MSKVKDKAIVSAAQAS, which was same as those proteases excreted from some other P. fluorescens. However, their molecular weights, the activation ion and amino acid composition were different, suggesting Ht13 from P. fluorescens Rm12 is a novel protease.     

Combined Action of Lipase and Microbial Phospholipase C on a Model Fat Globule Emulsion and Raw Milk

Phospholipase C from Pseudomonas fluorescens hydrolyzed phospholipids adsorbed to fat globules in a model emulsion composed of butteroil emulsified with crude soy lecithin. Modification of the membrane by this enzyme enhanced lipolysis when steapsin was introduced to the emulsion contained in a Bio-fiber beaker-type dialysis system. Following an initial period of induction, the velocity of lipolysis in the model emulsion was greater in the presence of phospholipase C than in its absence. Phospholipase C also enhanced the activity of milk lipase in raw milk. The magnitude of enhancement was much greater in milk than in the model emulsion. Presumptive data suggested that lipolysis by phospholipase C varied with intrinsic differences among various animals.     

The production characteristics of volatile organic compounds and their relation to growth status of Staphylococcus aureus in milk environment

In our previous research, 3-methyl-butanal and 3-methyl-butanoic acid were identified as representative and specific volatile organic compounds released by Staphylococcus aureus in broth. In this study, we explored the production of the 2 volatiles and their correlation to Staph. aureus growth in milk under different conditions. We found significant correlations between the production of 3-methyl-butanoic acid and cell counts of 5 Staph. aureus strains in sterile milk, and there were no obvious differences for its production among 5 tested strains. The intensities of the 2 volatiles were similar and positively correlated with bacterial counts in cultures at 25°C and 37°C despite delayed production of volatiles at 25°C; however, neither compound could be detected at 4°C. The production of 3-methyl-butanoic acid was strongly correlated with growth of Staph. aureus mixed with Streptococcus agalactiae, Escherichia coli O157:H7, and Shigella flexneri, whereas correlations for 3-methyl-butanal were not statistically significant. Compared with the monoculture of Staph. aureus, in mixed cultures, production of 3-methyl-butanal was decreased and that of 3-methyl-butanoic acid was comparatively higher. In pasteurized and raw milks, production of 3-methyl-butanoic acid was correlated with growth of Staph. aureus, and 3-methyl-butanoic acid could be detected when Staph. aureus populations reached 10⁶ to 10⁷ cfu/mL in pasteurized milk and 10⁵ to 10⁶ cfu/mL in raw milk; the correlations for 3-methyl-butanal were not statistically significant. Our results suggest that 3-methyl-butanoic acid is a more suitable marker for high counts of Staph. aureus in milk, whereas 3-methyl-butanal is a transient metabolite and easily depressed by environmental factors.     

Colonization of Beef Muscle Surfaces by Pseudomonas fluorescens and Pseudomonas fragi

Attached and unattached cell densities were determined for Pseudomonas fluorescens and Pseudomonas fragi growing on the surface of beef muscle stored at 4 and 25°C, in presence of NaCl, KCl, and CaCl₂. A mechanical rinsing procedure was developed for this purpose. Both species colonized the surface at both temperatures and were enhanced at low (4°C) temperature. Attached cells represented up to 90% of the total until a density of 10⁵-10⁶ CFU cm^?2 was reached. At that point, a proportion of attached cells to unattached cells declined but colonization of the surface continued. In presence of CaCl₂, ratios of attached to unattached cells did not decline, suggesting a significant role for the calcium ion in colonization. Ability to colonize meat surfaces may be a significant competitive advantage for meat spoilage bacteria such as Pseudomonas fragi and Pseudomonas fluorescens.     

Activity of bacterial lipases at chilling temperatures

Two Pseudomonas aeruginosa, two Pseudomonas fluorescens, three Aeromonas hydrophila, one Aeromonas caviae, two Staphylococcus aureus and two Serratia marcescens strains were tested for their ability to digest tributyrin at temperatures ranging from −2°C to +7°C over a period of 38 days using a modified agar-diffusion test. Hydrolysis was observed at all of the temperatures investigated, with the exception ofS. marcescens lipases at −2°C. A decrease in storage temperature was associated with a significant reduction of enzyme activity but no complete inactivation — even at −2°C. Initial reactions occurred after 3 days. For practical applications, it can be concluded that during storage of chilled products, lipolytic activity can occur provided that enzyme synthesis has taken place before chilling has started.     

Lipase-catalyzed transesterification synthesis of citronellyl acetate in a solvent-free system and its reaction kinetics

The lipase-catalyzed citronellol synthesis of citronellyl acetate via transesterification was investigated in a solvent-free system. After screening several lipases, the lipase from Pseudomonas fluorescens was identified as the optimal enzyme for the system. The optimal reaction temperature was 40?°C. The external diffusion limitation could be greatly reduced by increasing the agitation speed to 200?rpm. A linear relationship between the initial reaction rate and an enzyme load of up to 6?mg?mL^?1 demonstrated that the internal diffusion limitations could be minimized. Substrate inhibition was absent when the substrate concentration was below 500?mmol?L^?1, but the experimental results indicated that the product inhibition effect should be considered. The results from the reaction kinetics analysis showed that the reaction obeys the ping-pong bi–bi mechanism that is inhibited by citronellyl acetate. Matlab was used to simulate the model parameters. The experimental values could be satisfactorily fitted to the simulated values with a relative error of 11.98?%.     

The occurrence and growth of yeasts in dairy products

Yeasts were isolated, identified and enumerated from 161 samples of retail dairy products. Highest yeast populations (up to 10⁶–10⁷ cells/g) were found in yogurt and cheese samples while lower counts occurred in samples of pasteurized milk, cream, butter and ice cream. Candida famata, Kluyveromyces marxianus, Candida diffluens and Rhodotorula glutinis were the most frequency isolated species. The growth of these and other species was demonstrated during the refrigerated storage of cream, butter and cheese samples and by their inoculation and incubation in milk and yogurt samples. The predominance and growth species was probably related to their production of protein and fat hydrolysing enzymes and the ability to grow at 5°C.     

Kinetics of destruction ofEscherichia coliandPseudomonas fluorescensinoculated in ewe's milk by high hydrostatic pressure

Ewe's1999199919991999 Academic PressAcademic PressAcademic PressAcademic Pressmilk standardized to 6% fat was inoculated with Escherichia coliand Pseudomonas fluorescensat a concentration of 10⁷and 10⁸ cfu ml⁻¹respectively, and treated by high hydrostatic pressure. Treatments consisted of combinations of pressure (50-300 MPa), temperature (2, 10, 25 and 50°C), and time (5, 10 and 15 min). Violet red bile agar and crystal-violet-tetrazolium count were used to determineE. coliandP. fluorescensrespectively. Pressurization at low and moderately high temperatures produced higher P. fluorescensinactivation than treatments at room temperature, while pressurization at only moderately high temperature produced high E. coliinactivation; low and room temperatures produced similar reductions. On E. coli,reductions of 6.055 log units were produced with 300 MPa for 15 min at 50°C, while on P. fluorescens,reductions of 5.059 log units were produced with 250 MPa for 15 min at 50°C. Both micro-organisms showed a first-order kinetics of destruction in the range 0-30 min, with D-values (at different temperatures and pressures from 150 to 300 MPa) between 2.055 and 18.058 min for E. coli,and 2.058-23.053 min for P. fluorescens.Abaroprotective effect of ewe's milk (6% fat) on both micro-organisms was observed, in comparison with other studies using different means and similar pressurization conditions.     

Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and α_S-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.