Proteolytic Enzyme Preparation from Pseudomonas fragi: Its Action on Pig Muscle

An extracellular preparation from Pseudomonas fragi with proteolytic enzyme activity was isolated, and its action on meat proteins and meat protein ultrastructure was studied. First, a suitable growth medium for proteolytic enzyme production was determined, and a method for partial purification of the proteolytically active fraction was developed. The enzyme preparation displayed optimal proteolytic activity at neutral pH and 35 C. Proteolytic activity was irreversibly lost by mild heat treatment. The enzyme preparation was tested for its ability to hydrolyze isolated pig muscle proteins. Myofibrillar protein was rapidly degraded, G-actin and myosin were broken down at a slower rate, and the sarcoplasmic proteins were least susceptible to hydrolysis. Electron micrographs of pork muscle showed that the proteolytic enzyme preparation caused a complete loss of dense material from the Z line. Similarities are discussed between the action of P. fragi extracellular proteolytic enzyme(s) on meat and normal bacterial spoilage of meat.     

A study of the relative incidence of different Pseudomonas groups on meat using a computer-assisted identification technique employing only carbon source tests

A computer-assisted probabilistic identification technique employing 18 carbon source utilization tests has been developed and applied to 787 Pseudomonas strains isolated from beef, pork and lamb stored under aerobic conditions. Seven hundred and twelve (89.7%) were identified using these tests alone and a further six (0.8%) with extra tests. Taxa detected were Ps. fragi cluster 2, 390 strains (49.6% of all isolates); Ps. fragi cluster 1, 191 strains (24.9%); meat cluster 3, 87 strains (11.1%); Ps. fluorescens biotype I, 31 strains (3.9%); Ps. fluorescens biotype III, 7 strains (0.9%); and Ps. putida , 1 strain (0.1%). The relative incidence of members of the various taxa was similar on beef, pork and lamb, and was unaffected by storage temperature in the range 0°–10°C. Each taxon was also detected at similar rates before and after spoilage. Meat origin (abattoir) affected the frequency of detection of meat cluster 3 and Ps. fluorescens biotype I strains but did not affect the incidence of detection of either cluster of Ps. fragi.     

A study of the relative incidence of different Pseudomonas groups on meat using a computer-assisted identification technique employing only carbon source tests

A computer-assisted probabilistic identification technique employing 18 carbon source utilization tests has been developed and applied to 787 Pseudomonas strains isolated from beef, pork and lamb stored under aerobic conditions. Seven hundred and twelve (89.7%) were identified using these tests alone and a further six (0.8%) with extra tests. Taxa detected were Ps. fragi cluster 2, 390 strains (49.6% of all isolates); Ps. fragi cluster 1, 191 strains (24.9%); meat cluster 3, 87 strains (11.1%); Ps. fluorescens biotype I, 31 strains (3.9%); Ps. fluorescens biotype III, 7 strains (0.9%); and Ps. putida, 1 strain (0.1%). The relative incidence of members of the various taxa was similar on beef, pork and lamb, and was unaffected by storage temperature in the range 0 degrees-10 degrees C. Each taxon was also detected at similar rates before and after spoilage. Meat origin (abattoir) affected the frequency of detection of meat cluster 3 and Ps. fluorescens biotype I strains but did not affect the incidence of detection of either cluster of Ps. fragi.     

Fish protein hydrolysis by a psychrotrophic marine bacterium isolated from the gut of hake (Merluccius hubbsi)

The aims of this study were to identify a psychrotrophic bacterium, strain CR41, producing a cold adapted protease during growth at low temperatures and to evaluate the ability of the cells to hydrolyze hake fish protein. The strain was isolated from the intestinal tract of hake collected from the San Jorge Gulf (Patagonia, Argentina) and it was identified as Pseudoalteromonas. Growth and fish protein hydrolysis were determined using an aerated simple mineral medium plus 10% fish protein concentrate. Proteolytic activity was measured at 7 and 22 degrees C during culture in the concentrate. Protease production started in the exponential growth phase and reached a maximum during stationary phase. Protease activity at 7 degrees C was lower than at 22 degrees C. After 8 h of incubation, the percentage of hydrolyzed protein was 84% at 7 degrees C and 95% at 22 degrees C. Electrophoresis detection showed that degradation of muscle hake proteins was complete at both temperatures, and in gelatin zymograms extracellular activity showed two proteolytic bands with apparent molecular masses of approximately 31.6 and 62 kDa.     

Influence of Temperature on Bacterial Infection of the Hen''s Egg

Temperature of incubation had a marked effect on infection of eggs in which the air cells had been inoculated with a washed suspension of Serratia marcescens. There was no evidence of bacterial multiplication or spoilage in eggs held at 10 C for 42 days. Multiplication occurred in the shell membranes of eggs held at 30 or at 37 C when the yolk made contact with these membranes, and continued in the contents of the egg, at which time the first signs of spoilage appeared. In a few eggs, very large populations were present in the shell membranes and in the albumen. In eggs inoculated with Pseudomonas fluorescens and held at 10 C, bacterial multiplication occurred in the shell membranes in the first 7 days of incubation. These populations did not appear to change in size in the 7- to 14-day period of incubation. Renewed multiplication and concomitant spoilage of the contents was observed in many of the eggs thereafter.     

Influence of Temperature on Bacterial Infection of the Hen's Egg

Temperature of incubation had a marked effect on infection of eggs in which the air cells had been inoculated with a washed suspension of Serratia marcescens. There was no evidence of bacterial multiplication or spoilage in eggs held at 10 C for 42 days. Multiplication occurred in the shell membranes of eggs held at 30 or at 37 C when the yolk made contact with these membranes, and continued in the contents of the egg, at which time the first signs of spoilage appeared. In a few eggs, very large populations were present in the shell membranes and in the albumen. In eggs inoculated with Pseudomonas fluorescens and held at 10 C, bacterial multiplication occurred in the shell membranes in the first 7 days of incubation. These populations did not appear to change in size in the 7- to 14-day period of incubation. Renewed multiplication and concomitant spoilage of the contents was observed in many of the eggs thereafter.     

Proteolytic activity of Trichoderma viride in mixed culture with Sclerotium rolfsii in soil.

Cultures of Sclerotium rolfsii and Trichoderma viride together in autoclaved soil were assayed at intervals during 8 days of incubation for proteolytic activity (PA) of T. viride. Significant proteolytic activity was detected only in soil containing T. viride (i.e., T. viride alone or S. rolfsii + T. viride); greatest activity occurred between 3 and 4 days after infestation and declined rapidly thereafter. Maximal PA in the mixed-culture soil was accompanied by an increase in soil pH. optimal pH values for PA was 5.5-6.5 with a maximum at 6.0.     

Properties of intracellular ribonuclease utilized for RNA reduction in disintegrated cells of Saccharomyces cerevisiae.

The properties of intracellular RNase in disintegrated cell suspensions of Saccharomyces cerevisiae have been studied. The influence of salt addition and/or incubation of the suspension on the activity of RNase and on the degradation of endogenous RNA was determined. No significant change in the RNase activity in the disintegrated suspensions was obtained by addition of 3% NaCl or by incubation at 50 degrees C with 3% NaCl. During the incubation with NaCl the active RNase was able to degrade endogenous RNA. By incubation without salt the RNase was inactivated. Inactivation also occurred after extraction at alkaline pH. The RNase had an optima at pH 5-6 and temperatures between 50-60 degrees C. The main part of the RNase in the unincubated suspension was soluble also at pH 4.0. No serious protein degradation occurred during the short time incubation needed for RNA reduction. 70% of the protein in the suspensions was recovered in the precipitate at pH 4.0 after 20 min of incubation. The corresponding protein recovery from unincubated suspensions was 77%.     

Changes in carbohydrates,amino acids and proteins in developing seed of chickpea

Developing seeds of chickpea cultivars G-130, L-550 and 850-3/27 grown under field conditions were sampled at different stages of maturity and analysed for soluble sugars, starch, soluble nitrogen, protein nitrogen and amino acids. Fr. wt of seeds of all three cultivars decreased after 28 days of flowering while the dry wt continued to increase. Rapid starch accumulation was observed between 14 and 28 days after flowering. Starch as per cent of seed dry wt started to decrease after 28 days, while starch per seed increased till maturity. The percentage of salt-soluble proteins decreased with maturation of seed. The electrophoretic pattern revealed that deposition of seed storage protein in cotyledons occurred 14 days after flowering. Most of the biochemical activity apparently occurred between 14 and 28 days after flowering.     

The activity of the 20S proteasome is maintained in detached wheat leaves during senescence in darkness

In the present paper, we studied the participation of the 20S proteasome, the proteolytic component of the ubiquitin–proteasome pathway, in the remobilization of bulk proteins in senescing wheat leaves. The detached leaves of 15-d-old plants were incubated in darkness for several days, and various proteolytic activities were analysed in soluble extracts prepared at 0, 48 and 96 h after detachment. The endoproteolytic activity, measured at pH 7.5 and 5.4, increased more than 10-fold and the total peptidasic activity increased up to 5-fold after 96 h of incubation in the dark, when expressed as specific activity. In the same period, the leaf-protein content decreased to less than 50% of that present at the initial time. The 20S proteasome chymotrypsin-like activity remained constant when it was expressed as activity per leaf fresh weight and resulted 2-fold higher in terms of specific activity. The western blot analysis showed that the amount of 20S proteasome protein and ubiquitin–protein conjugates also remained constant until 4 d of incubation in darkness. These results indicate that the ubiquitin–proteasome pathway remains functional until the late phases of senescence suggesting that it may participate in the regulatory aspects of the process rather than in the massive protein breakdown.     

Ageing-Time Dependent Changes of Angiotensin I-Converting Enzyme-Inhibiting Activity of Protein Hydrolysates Obtained from Dry-Cured Pork Loins Inoculated with Probiotic Lactic Acid Bacteria

This study aimed to determine the ageing-time dependent changes of the angiotensin I-converting enzyme (ACE)-inhibition of protein extracts obtained from LAB-inoculated dry-cured pork loins over 360 days of ageing and their hydrolysates obtained after in vitro hydrolysis and absorption. The increasing ageing time was accompanied by a growth in the ACE inhibitory activity of the water-soluble and salt-soluble protein extracts. Based on the hierarchical cluster analysis, 180 days was indicated as the optimal time for ageing of dry-cured pork loins in terms of ACE inhibition. The effect of the strain used on the ACE inhibition varied over long-term aging. The peptides generated during in vitro hydrolysis have between 6 and 22 amino acids in a sequence; the Pro, Ala, Lys, and Glu molecules comprise the largest share. This study demonstrated that pork muscle proteins may lead to production of numerous peptides with ACE inhibitory properties.

     

复合乳酸菌对冷藏海鲈鱼块的保鲜效果

【目的】研究复合乳酸菌对冷藏海鲈鱼块的保鲜效果。【方法】以冷藏海鲈鱼块为对象,筛选出3株能够明显抑制其优势腐败菌(草莓假单胞菌Pseudomonas fragi,腐败希瓦氏菌Shewanella putrefacens)生长的单一乳酸菌,同时也筛选出对其优势腐败菌具有最显著抑制效果的一组复合乳酸菌,再将该复合乳酸菌接种到海鲈鱼块上,在4°C冷藏过程中,通过感官评定、挥发性盐基氮(TVB-N值)的测定和优势腐败菌的计数来评价复合乳酸菌对冷藏海鲈鱼块的保鲜效果。【结果】单一乳酸菌(干酪乳杆菌LC1、植物乳杆菌LP1和乳酸菌L3)对2株冷藏海鲈鱼优势腐败菌的抑制效果明显;复合乳酸菌(干酪乳杆菌LC1+植物乳杆菌LP1+乳酸菌L3)的抑菌效果最为显著;在4°C冷藏过程中,复合乳酸菌能使冷藏海鲈鱼块发生感官变化延缓6 d、使TVB-N值的升高延缓2 d,同时显著抑制优势腐败菌的生长。【结论】复合乳酸菌对冷藏海鲈鱼块具有良好的保鲜作用,能有效延长其货架期。     

Thermal Resistance of Pseudomonas fragi in Milk Containing Various Amounts of Fat

Similar populations of Pseudomonas fragi were grown at 25 C for 20 hr or at 7 C for 7 days in milk containing 0, 10, and 20% fat; they were then heated at 48, 50, and 52 C in milk containing 0, 10, and 20% fat. After inoculation, the heating medium contained 2.1 x 10(6) to 6.9 x 10(6) organisms per milliliter. The P. fragi cells grown in skim milk had greater thermal resistance (D(52) = 3.0 to 3.1) than those grown in milk containing fat (D(52) = 1.9 to 2.5). The organisms grown at 7 C for 7 days in milk containing 10% fat were more resistant (D(52) = 3.0) than those grown in the same medium at 25 C for 20 hr (D(52) = 2.0). The presence of 0 to 20% milk fat in the heating medium had no apparent effect on the thermal resistance.     

Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase

The autodigestive proteolytic activity of extracts of cotyledons of mung beans (Phaseolus aureus Roxb.) increased 4- to 5-fold during germination. A similar increase was found in the ability of these extracts to digest added casein or mung bean globulins. The increase occurred after a 2-day lag during the next 2 to 3 days of germination and coincided with the period of rapid storage protein breakdown. To understand which enzyme(s) may be responsible for this increase in proteolytic activity, the hydrolytic activity of cotyledon extracts toward a number of synthetic substrates and proteins was measured. Germination was accompanied by a marked decline in leucine aminopeptidase, while carboxypeptidase increased about 50%. There were no dramatic changes in either α-mannosidase or N-acetyl-β-glucosaminidase, enzymes which may be involved in the metabolism of the carbohydrate moieties of the reserve glycoproteins. The increase in general proteolytic activity was closely paralleled by a 10-fold increase in endopeptidase activity. This activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide. Studies with inhibitors of proteolytic enzymes showed that reagents which blocked sulfhydryl groups also inhibited the rise in general proteolytic activity. Our results suggest that the appearance of a sulfhydryl-type endopeptidase activity is a necessary prerequisite for the rapid metabolism of the reserve proteins which accompanies germination.     

Detection and Incidence of Specific Species of Spoilage Bacteria on Fish: II. Relative Incidence of Pseudomonas putrefaciens and Fluorescent Pseudomonads on Haddock Fillets

Pseudomonas putrefaciens has been found to constitute one of the major species of spoilage bacteria on haddock fillets. The initial population of this organism on fillets of high bacterial quality is uniformly below 4% and most frequently no greater than 1%. During refrigerated storage, the organism increases at a more rapid rate than the total psychrophilic population, comprising 50 to 90% of the total population when the total count exceeds 10(6)/g of tissue. Fluorescent pseudomonads were shown to constitute a second group of predominant pseudomonads constituting up to 19.3% of the total population after 8 days of refrigerated storage. Of a total of 45 fluorescent pseudomonads isolated from haddock fillets, 14 (31.1%) were found to be potent fish spoilers. The use of a soft-agar-gelatin plating technique showed a parallel increase of proteolytic organisms with total count indicating that proteolytic organisms other than P. putrefaciens and fluorescent pseudomonads increase at a slower rate than these two groups.     

Isolation and initial characterization of a Bacillus subtilis mutant with novel protease secretion capability

A bank of pTV32 (Tn 917 lacZ) - generated Bacillus subtilis mutants were examined on milk agar for the ability to produce proteases at 48 degrees C. A single mutant, BUL786, was isolated, which could hydrolyze casein after overnight incubation at 48 degrees C. This mutant secreted protease 10 fold more at 48 degrees C when compared to 37 degrees C, and part of the activity appears to be 48 degrees C-specific. At high temperatures, other strains of B. subtilis, including hyperprotease secretors, were unable to secrete protease to any significant degree. The BUL786 strain is missing the 97K major heat shock protein. Since a number of other proteins also appear to be secreted at 48 degrees C, this mutant may be a hypersecretor of exported proteins at temperatures greater than 45 degrees C.     

Experimental Studies on the Volatile Nitrogen Compounds Produced by Pseudomonas Fragi in Fish Extracts

The decomposition of nitrogenous compounds of extracts of cooked halibut meat due to the growth at 4°C and 17°C of Pseudomonas fragi, strain F 111, was followed with determinations of the total volatile nitrogen (TVN) and of trimethylamine (TMA). The steam-distillation method according to Bethea & Hillig (1965) and the Conway-microdiffusion-method according to Farber & Ferro (1956) were used for these determinations. When fish extract was inoculated with the strain F 111 and stored at 4°C for 5 days or at 17°C for 3½ days an increase of TVN was started. This increase of TVN was slower at 4°C than at 17°C. It was shown that in the extract B, which was prepared from fish meat of poor but acceptable commercial quality, the initial TVN was higher, the increase of TVN caused by the action of the strain F 111 was slower, and the TVN maximum was lower than the corresponding values representing extract A. The last mentioned extract was prepared from halibut meat of good commercial quality. The correlation between the increase of TVN and that of pH of the inoculated fish extract was poor. This indicates that the initial increase of pH was not caused by volatile basic compounds. It was shown that the exclusion of air after 1 or more days of incubation at 17°C could delay the onset of the TVN increase but did not prevent it. The final TVN value of the sample, which was layered with paraffin oil 24 hrs. after the inoculation of the strain F 111, was approximately the same as that of the fish extract sample layered after 14 days of incubation at 17°C. In inoculated fish extract samples, which were sterile-filtered on the day when the extract was layered with paraffin oil, no further increase of TVN was observed. It was confirmed that Pseudomonas fragi caused no increase of TMA in the extract of cooked halibut.     

The influence of pH and temperature on initiation of growth of Salmonella spp.

The ability of 13 strains of Salmonella , representing 12 serotypes, to grow in a tryptone-yeast extract-glucose medium, acidified with HC1 to pH values between 3.80 and 5.60 at intervals of 0.20 units, has been investigated. During incubation at 30°C, growth occurred at minimum pH values of 3.8–4.0 in 1–3 d. At 20°C, growth occurred at minimum pH values of 3.8–4.2 in 3–5 d. In tests incubated at 10°C, growth occurred at minimum pH values of 4.4–4.8 in between 10 and 19 d.     

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

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

The effects of phosphate supply on uptake of potassium ions, 2,4-D and atrazine by wheat and maize

The germination percentage of peach [ Prunus persica (L.) Batsch cv. Halford] seeds at 20°C was low (< 20%) after incubation at 5°C for as long as 35 days, but then increased considerably (> 40%) when the seeds were maintained at 5°C for longer than 42 days. Four zones of gibberellin-like activity were found in partially purified seed extracts. Gibberellin-like activity remained low in seeds incubated at 5°C for as long as 28 days, but increased significantly in three of these zones after 35 days, and in the fourth zone after 49 days. The increase in gibberellin-like activity was evident prior to the transfer of the seeds to 20°C. Moreover, seeds maintained at 5°C germinated at this temperature after 63 days. For seeds incubated and germinated at 20°C, both the germination percentage and the gibberellin-like activity remained low throughout the experimental period. Application of the growth retardant paclobutrazol to seeds after 28 days of a 49 day total incubation period at 5°C did not substantially reduce seed germination, although the increase in gibberellin-like activity was prevented. Seeds did, however, require a longer time to germinate after transfer to 20°C and were dwarfed in appearance. Application of GA₃ to seeds prior to stratification increased the percentage germination of seeds only when they had been incubated at 5°C for at least 35 days. The major changes in gibberellin-like activity are, therefore, associated not so much with the processes which allow germination to take place in peach, but more with those processes which allow normal growth and development of the seedling.