Cold-active pectinolytic activity of psychrophilic-basidiomycetous yeast Cystofilobasidium capitatum strain PPY-1

A pectinolytic and psychrophilic yeast was isolated from soil from Abashiri, Hokkaido, Japan. The phenotype and sequencing of the 28S rDNA of the isolated strain (PPY-1) indicated a taxonomic affiliation to the basidiomycetous yeast Cystofilobasidium capitatum. C. capitatum strain PPY-1 was able to grow on two pectic compounds, polygalacturonate and pectin, at below 5 degrees C. Moreover, the extracellular fraction of the strain exhibited pectin methylesterase, pectin lyase and polygalacturonase activities at 5 degrees C. Thus strain PPY-1 may produce novel enzymes that are able to degrade pectin at low temperature, although the strain has isozymes of these enzymes.     

Purification and characterisation of two enzymes related to endogenous formaldehyde in Lentinula edodes

In this study, γ-glutamyl transpeptidase (GGT) and l-cysteine sulphoxide lyase (C-S lyase) were purified from the fruiting body of Lentinula edodes in three steps and then characterised. We found that GGT together with C-S lyase caused the generation of endogenous formaldehyde in L. edodes. GGT was composed of a large subunit of 41 kDa and a small subunit of 25 kDa, and C-S lyase was composed of two identical subunits of 46 kDa, as determined by SDS–PAGE. GGT was stable at pH 8.0–10.0 with an optimum pH of 8.8, and was stable at 20–50 °C with an optimum activity at 37 °C. C-S lyase was stable at pH 8.0–9.0 with an optimum pH of 8.5, and was stable at 20–60 °C with an optimum activity at 40 °C. The present work supports the study of the mechanism of endogenous formaldehyde in L. edodes.     

A second pectin lyase gene (pel2) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene products

A second pectin lyase gene, designated pel2, was isolated from a shoyu koji mold Aspergillus oryzae KBN616 and characterized. The structural gene comprised 1306 bp with three introns. The ORF encoded 375 amino acids with a signal peptide of 19 amino acids. The deduced amino acid sequence showed high similarity to those of A. oryzae Pel1, Aspergillus niger pectin lyases and Glomerella cingulata Pn1A. The pel2 gene was overexpressed under the control of the promoter of the A. oryzae TEF1 gene for purification and enzymatic characterization of its gene product. The gene product exhibited two molecular masses of 48 and 44 kDa due to different degrees of glycosylation. Both proteins had the same pH optimum of 6.0 and temperature optimum of 50 degrees C.     

Pectin lyase and polygalacturonase of Aspergillus niger pathogenic for yam tubers (Diascorea rotundata)

Polygalacturonase and pectin lyase of Aspergillus niger partially purified by ethanol, ammonium sulphate precipitation, DEAE-cellulose and Sephadex G-150 column chromatography were characterized. Polygalacturonase gave optimum activity at pH 4–5, and at 35°C. It was stable at pH 3–7 and at 20–50°C. The molecular weight was 38020. For pectin lyase optimum activity occurred at pH 5 and 45°C. The enzyme was stable at pH 3–4 and at 40–50°C. The molecular weight was 30 900. Yam tissue was optimally macerated at pH 4–5 by the enzymes. At pH 4.5, potassium sorbate (0.6 mg/ml), benzoic acid (0.8 mg/ml) and sodium benzoate (1.0 mg/ml) caused complete inhibition of polygalacturonase activity. With pectin lyase, this effect was achieved with potassium sorbate and benzoic acid each at 0.9 mg/ml, but not with sodium benzoate.     

酪氨酸酚解酶高产菌株快速筛选模型的建立与应用

以甲酚红作指示剂 ,在加有酪氨酸的固态培养基上 ,根据甲酚红由黄变红所形成的变色圈的大小筛选酪氨酸酚解酶高产突变株是一种平板快速筛选方法。弗氏柠檬酸细菌ATCC80 90菌株经紫外线和亚硝基胍诱变处理后 ,经该法筛选得到突变株C37 30 ,该菌株产酪氨酸酚解酶达 12 5U/ g干菌体 ,较出发菌株提高 2 32 %。连续 5代传代试验表明 ,突变株C37 30产酶稳定     

Immobilization and characterization of C‐S lyase from onion (Allium cepa) bulbs

Cysteine sulfoxide lyase (C‐S lyase; EC 4.4.1.4), the enzyme responsible for flavor potentiation in minced Allium tissues, was immobilized by entrapment within an alginate gel. Both the free and immobilized C‐S lyase had an optimum pH of 7.5 for activity. Also, similar Km values were observed for both forms of the enzyme using (±)‐S‐methyl‐L‐cysteine sulfoxide (± MCSO; 14.2–19.2 mM) and an alk(en)yl‐L‐cysteine sulfoxide extract (0.9–1.8 mM) prepared from onion bulbs. Both forms of the enzyme were stable for 6–8 weeks at 4°C. At 25°C, the immobilized enzyme was fully stable for 5 weeks whereas the free enzyme lost 50% of its activity within 2 weeks. As the alginate content in the gel was increased from 0.25 to 3%, the % yield of activity of the immobilized enzyme system decreased from 65 to 44%. However, as % alginate content was increased, the yield of active enzyme immobilized in the gel increased from 62 to 97%. Reaction of both the free and alginate‐entrapped C‐S lyase with (±) MCSO resulted in the production of a distinctive cabbage‐like aroma. Onion aroma was liberated when either form of the enzyme was recombined with an odorless alk(en)yl‐L‐cysteine sulfoxide extract from onion.     

THE EFFECT OF CRAWFISH PROTEASES ON INACTIVATION AND THE HYDROLYTIC CLEAVAGE OF PECTIC ENZYMES1

Four proteases (CP‐I, ‐II, ‐111 and ‐IV) were isolated and purified from hepatopancreas of crawfish. Based upon their activities with known inhibitors, they resembled trypsin. All were cross‐reactive with polyclonal antibodies raised against CP‐II, indicating they shared structural components. Each of these was able to inactivate orange peel pectinesterase, tomato pectinesterase and pectate lyase C at room temperature under nondenaturing conditions. Using matrix‐assisted‐laser‐desorption time of flight mass spectrometry (MALDl‐TOF MS), an analysis of peptides generated during proteolysis of pectate lyase C from Erwinia chrysenthemi showed similar peptide patterns for the four crawfish proteases, indicating a common specificity for each isozyme. However, the cleavage patterns were different from those obtained by the action of bovine trypsin on pectate lyase C. These studies indicate the four proteases from the hepatopancreas of crawfish are isozymes which may be used for the inactivation of pectinolytic enzymes.     

Effect of ozonated water treatment on microbial control and on browning of iceberg lettuce (Lactuca sativa L.)

We examined the effect of ozonated water treatment on microbial control and quality of cut iceberg lettuce (Lactuca sativa L.). Fresh-cut lettuce was washed in ozonated water (3, 5, and 10 ppm) for 5 min at ambient temperature. The native bacterial population on the lettuce declined in response to a rise in ozone concentration. However, there was no further bacterial reduction (1.4 log CFU/g) above 5 ppm ozone. Although ozonated water treatment increased the phenylalanine ammonia lyase (PAL) activity of the lettuce stored at 10 degrees C compared with the water wash treatment after 1 day of storage, the concentration of ozone did not affect PAL activity. The a* value of the residue of the lettuce methanol extracts, which reflects the extent of browning, increased dramatically in lettuce treated with 10 ppm ozonated water compared with other treatments. Treatment with 3 or 5 ppm ozonated water resulted in more rapid changes in the a* value than after the water treatment. The combined treatment of hot water (50 degrees C, 2.5 min) followed by ozonated water (5 ppm, 2.5 min) had the same bactericidal effect as treatment with ozonated water (5 ppm, 5 min) or sodium hypochlorite (NaOCl, 200 ppm, 5 min), giving a reduction in bacteria numbers of 1.2 to 1.4 log CFU/g. The ascorbic acid content of the lettuce was not affected by these treatments. The combined treatment of hot water followed by ozonated water greatly inhibited PAL activity for up to 3 days of storage at 10 degrees C. Treatment with this combination greatly suppressed increases in the a* value, thus retarding the progress of browning compared with other treatments throughout the 6-day storage. NaOCl treatment also inhibited browning for up to 3 days of storage. Bacterial populations on the lettuce treated with sanitizers were initially reduced but then showed rapid growth compared with that of the water wash treatment, which did not reduce bacterial counts initially.     

Identification of a gene cluster encoding Krebs cycle oxidative enzymes linked to the pyruvate carboxylase gene in Lactococcus lactis ssp. lactis C2

We identified a 14-kb pyruvate carboxylase gene-containing fragment from a lactococcal C2-lambda phage genomic library. Downstream of the pyruvate carboxylase gene-containing fragment, a gene cluster coding for open reading frames displaying extensive homology to citrate synthase, aconitase, and a truncated isocitrate dehydrogenase was identified. However, the truncation was shown to have occurred during the cloning by two noncontiguous Sau3AI fragments ligating together. The lactococcal citrate synthase gene consisted of 1323 bp and encoded a 441-amino acid citrate synthase protein. The lactococcal aconitase gene was 2544 bp and encoded an 848-amino acid protein. Corresponding to the complete citrate synthase gene, citrate synthase activity was detected in Lactococcus lactis ssp. lactis C2. Isocitrate dehydrogenase activity was found to be missing in Lactococcus lactis C2, suggesting that the gene may be incomplete or is not expressed, resulting in a requirement for glutamic acid in lactococci.     

The effect of nisin on growth kinetics from activated Bacillus cereus spores in cooked rice and in milk

The growth kinetics of germinated cells from activated spores of Bacillus cereus in cooked white rice and in milk were evaluated at different temperatures for control samples and for samples with 25 microg of nisin per ml added. Nisin was applied in the form of Nisaplin (10(6) IU/g), which contained 25,000 microg of nisin per g. The length of the lag phase for cooked white rice controls was 120 h at 10 degrees C, 8 h at 25 degrees C, and 2.5 h at 33 degrees C. The generation times for cooked rice were 327.7 min at 10 degrees C, 59.0 min at 25 degrees C, and 42.3 min at 33 degrees C; those for milk without nisin were 297.0 min at 20 degrees C, 31.2 min at 30 degrees C, 28.6 min at 35 degrees C, and 33.7 min at 40 degrees C; and those for milk with nisin added were 277.2 min at 20 degrees C, 66.9 min at 30 degrees C, and 66.4 min at 35 degrees C. No development of B. cereus was observed for milk with nisin added at 40 degrees C for 12 h, in which germinated cells decreased by a decimal reduction time (D) of 4.7 h. A temperature of 45 degrees C was shown to be harmful to B. cereus, decreasing the germinated cells in both formulations with D-values of 4.3 to 4.6 h. Similar inhibition of cell growth at 40 degrees C was not observed with lower nisin concentrations.     

Some Pectinolytic and Cellulolytic Enzyme Activities of Fungi Causing Rots of Cocoyams

Representative isolates of Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum and F solani recovered from rotten cocoyams were studied for the production of pectinolytic and cellulolytic enzymes. All the isolates elaborated high levels of hydrolase, lyase and pectinesterase in cocoyam tissue medium and lyase and pectinesterase in pectin medium. There were no significant differences in the overall levels of lyase and pectinesterase activities produced by all the isolates in both media. The level of hydrolase, lyase and pectinesterase activities individually produced by A niger, B theobromae and C rolfsii in both media was significantly higher than that of any other isolate. The highest hydrolase activity was produced by C rolfsii in cocoyam medium while A niger produced the highest lyase activity in pectin medium. Maximum pectinesterase activity was obtained from B theobromae and C rolfsii in pectin medium. All the test isolates produced cellulase in a medium containing carboxymethyl cellulose with C rolfsii showing significantly high activity followed by F oxysporum and A niger. © 1997 SCI.     

Heat resistance of Yersinia enterocolitica grown at different temperatures and heated in different media.

In the range of 4-20 degrees C, growth temperature did not influence the heat resistance at 54-66 degrees C for Yersinia enterocolitica at pH 7 in citrate phosphate buffer. However, when cells were grown at 37 degrees C. the D62 increased from 0.044 to 0.17 min. This increase was constant at all heating temperatures tested (z = 5.7-5.8). Growth temperature did not influence the proportion of heat-damaged cells after a heat treatment, as measured by their response to a 2% of sodium chloride added to the recovery medium. The sensitivity of heat treated cells to nisin or lysozyme depended on growth temperature: Whereas the number of cells grown at 4 degrees C surviving heat treatment was the same regardless of the presence of 100 IU/ml of nisin or 100 microg/ml of lysozyme in the recovery medium, that of cells grown at 37 degrees C was, in these media, lower. The pH of maximum heat resistance in citrate phosphate buffer was pH 7 for cells grown at 37 degrees C, but pH 5 for those grown at 4 degrees C. In both suspensions the magnitude of the effect of pH on heat resistance was constant at all heating temperatures. For cells grown at 4 degrees C the heat resistance at 54-66 degrees C, in skimmed milk or pH 7 buffer, was the same. For cells grown at 37 degrees C this also applied for heat treatment at 66 degrees C but at 56 degrees C the heat resistance in skimmed milk was higher.     

Alicyclobacillus in orange juice: occurrence and heat resistance of spores.

Spore suspensions of a pure culture of Alicyclobacillus acidoterrestris DSM 2498 were submitted to different heat treatments (60 degrees C for 60 min, 60 degrees C for 30 min, 70 degrees C for 20 min, 80 degrees C for 5 min, 80 degrees C for 10 min, 80 degrees C for 30 min, and boiling for 5 min) to determine the best activation conditions in orange juice. The best treatment for spore activation was shown to be 70 degrees C/20 min. Seventy-five samples of concentrated orange juice from 11 different suppliers were examined for the presence of thermophilic acid-tolerant spore formers by the most probable number technique using Bacillus acidocaldarius medium (BAM broth) and incubation at 44 degrees C for 5 days after a prior spore activation. After incubation, isolation was carried out using BAM agar medium incubating at 44 degrees C for 5 days. Typical colonies were submitted to a microscopic examination, evaluation for the presence of spores, and various biochemical tests. Of the orange juice samples examined, 14.7% were found to be positive for Alicyclobacillus. The thermal death time open tube method was used to determine the heat resistance of the spores of strains confirmed as being Alicyclobacillus. The D-values determined were in the range from 60.8 to 94.5 min at 85 degrees C, 10.0 to 20.6 min at 90 degrees C, and 2.5 to 8.7 min at 95 degrees C. The z-values were between 7.2 degrees C and 11.3 degrees C. The results demonstrated the occurrence of Alicyclobacillus in orange juice and the high heat resistance of the spores that could survive the heat treatments normally applied in the processing of orange juice.     

Standard plate counts of drinking water: a comparison between incubation temperatures of 20 and 30 degrees C

Standard plate counts of 5085 drinking water samples gathered in the Region of Basle were carried out over a period of 9 years (1977 to 1985). Two conditions of incubation were evaluated: 20 degrees C and 30 degrees C for 72 h. In ground water samples (3048 samples) colony forming units (cfu) at 30 degrees C were found to be higher than counts at 20 degrees C incubation, 45% of the samples contained greater than or equal to 2 cfu/ml at 30 degrees versus 35% at 20 degrees C. The median was 1 cfu/ml at both temperatures. In spring water samples (2036 samples) bacterial counts at 20 degrees C were found to be higher than counts at 30 degrees C incubation, 61% of the samples contained greater than 10 cfu/ml at 20 degrees C versus 51% at 30 degrees C. The median was 19 cfu/ml at 20 degrees C incubation versus 11 cfu/ml at 30 degrees C. These differences were statistically significant with p less than 0.001 (Wilcoxon matched-pairs signed-rank test). No correlation was found between bacterial counts at 20 degrees C and bacterial counts at 30 degrees C, nor between bacterial counts and original water temperatures. It appears that incubation temperatures of 20 degrees C and 30 degrees C favor the growth of different populations of bacteria and temperature is not the only factor. However, from a practical point of view the use of only one incubation temperature seems to be justified for the purpose of judging the sanitary quality of drinking water.     

Heat treatment for the control of Bacillus cereus spores in foods

The effect of heat treatments on food products related to soybean curd contaminated with Bacillus cereus spores was investigated for the purpose of preventing food-poisoning outbreaks by B. cereus. In the case of B. cereus strains isolated from foods, heating foods inoculated with the spores for 20 min at 70 degrees C, 5 min at 75 degrees C, 2 min at 100 degrees C, or 10 sec with a microwave oven plus reheating after standing for 2 hr at 25 degrees C reduced the number of surviving cells within the foods to less than one-hundredth. In the case of a heat-resistant strain isolated from a food poisoning outbreak, heating for 20 min at 70 degrees C plus reheating after standing for 1.5-2 hr at 35 degrees C, heating for 10 min at 75 degrees C plus reheating after standing for 1.5-2 hr at 35 degrees C, or heating for 2 min at 100 degrees C plus reheating after standing for 4 hr at 25 degrees C was effective. There was not much difference between the flavor components in foods with and without heat treatment at 70 degrees C, as analyzed by gas chromatography-mass spectrometry. These results indicate that these heat treatments are available to control B. cereus spores, without affecting the sensory quality of the foods.     

Effect of temperature on cell population balance in Dexter's culture of murine bone marrow hematopoietic cells with stromal cells

The effect of temperature in the range from 25 to 37 degrees C on the population balance of stromal and multiple-lineage hematopoietic cells from murine bone marrow at various stages of differentiation in Dexter's culture was investigated. The length of time required for stromal cells to reach confluence after inoculation of both cell types harvested from murine bone marrow was shorter at higher temperatures. On the other hand, the hematopoietic cell concentration initially decreased more rapidly at higher temperatures until the confluence of stromal cells, which should then support the proliferation of hematopoietic cells. The concentration of hematopoietic cells began to increase after 2 weeks incubation at 33 and 37 degrees C and after 4 weeks at 29 degrees C. However, the growth of hematopoietic cells was not stable at 37 degrees C, and neither stromal nor hematopoietic cells showed significant growth at 25 degrees C. The specific growth rate of hematopoietic cells at 29 degrees C after 4 weeks was comparable or higher than that at 33 degrees C, while the final concentration of hematopoietic cells was maximal at 33 degrees C. Using a cultivation method in which hematopoietic cells were recharged onto a confluent layer of stromal cells prepared at 33 degrees C, the maintenance and growth of hematopoietic cells were better at 29 degrees C than at higher temperatures. The content of progenitor cells among the hematopoietic cells increased prior to the increase in the hematopoietic cell concentration, and the progenitor cell was greater content at the lower temperature. These results suggest that cultivation at 29 degrees C might be superior to 33 degrees C for the maintenance and growth of hematopoietic cells with a prepared confluent layer of stromal cells.     

Effects of temperature on the elimination of benzocaine and acetylated benzocaine residues from the edible fillet of rainbow trout (Oncorhynchus mykiss)

The effect of temperature (7 degrees C and 16 degrees C) on the extent of accumulation and the elimination of benzocaine (BNZ) and its metabolite, acetylated benzocaine (AcBNZ), in the fillet tissue of rainbow trout was investigated. Residues were measured after bath exposure to an anesthetizing concentration of benzocaine (30 mg/l for 5 min) followed by a maintenance concentration (15 mg/l for 30 min). Immediately after exposure, the BNZ concentration in fillet tissue was approximately 27 micrograms/g at both temperatures; AcBNZ was 0.3 microgram/g at 7 degrees C and 0.6 microgram/g at 16 degrees C. The rates for elimination (alpha and beta) of BNZ and AcBNZ were not significantly different between the two temperatures. Terminal half-lives of elimination for BNZ were 1.62 h at 7 degrees C and 1.63 h at 16 degrees C; half-lives for AcBNZ were 2.36 h at 7 degrees C and 2.77 h at 16 degrees C.     

Effect of pH, NaCl content, and temperature on growth and survival of Arcobacter spp

Growth and survival of six human isolates of the pathogenic Arcobacter spp. in the presence of selected environmental factors were studied. Four strains of Arcobacter butzleri and two strains of Arcobacter cryaerophilus were exposed to pH levels of 3.5 to 8.0. Most strains grew between pH 5.5 and 8.0, with optimal growth of most A. butzleri and A. cryaerophilus strains at pH 6.0 to 7.0 and 7.0 to 7.5, respectively. The 24-h optimal growth range in the presence of NaCl was 0.5 to 1.0% for A. cryaerophilus. However, after 96 h, the optimum was between 0.5 and 2.0% NaCl. The optimum range for growth of A. butzleri strains was 0.09 to 0.5% NaCl after 96 h. The upper growth limits were 3.5 and 3.0% NaCl for A. butzleri and A. cryaerophilus, respectively. Survival at 25 degrees C in up to 5% NaCl was noted for A. butzleri 3556 and 3539 and A. cryaerophilus 3256. Decimal reduction times (D-values) at pH 7.3 in phosphate-buffered saline for three A. butzleri strains were 0.07 to 0.12 min at 60 degrees C, 0.38 to 0.76 min at 55 degrees C, and 5.12 to 5.81 min at 50 degrees C. At pH 5.5, decreased thermotolerance was observed, with D-values of 0.03 to 0.11 min at 60 degrees C, 0.30 to 0.42 min at 55 degrees C, and 1.97 to 4.42 min at 50 degrees C. Calculated z-values ranged from 5.20 to 6.28 degrees C. D-values of a three-strain mixture of A. butzleri in raw ground pork were 18.51 min at 50 degrees C and 2.18 min at 55 degrees C. Mild heat (50 degress C) followed by cold shock (4 or 8 degrees C exposure) had a synergistic lethal effect, reducing more cells than with an individual 50 degrees C treatment or with cold shock temperatures of 12 or 16 degrees C.     

Influence of temperature on growth of Legionella pneumophila biofilm determined by precise temperature gradient incubator

Bacterial growth is influenced by several different culture conditions. Temperature is one of an essential component which regulates bacterial growth and their morphology. The influence of temperature on the length of bacteria was investigated in broth and on agar in a temperature range from 30.0 degrees C to 47.0 degrees C in 0.5 degrees C steps using a newly developed temperature gradient incubator. The incubator is able to reach a set temperature within 2 h and maintain temperature as accurate as +/-0.1 degrees C of the set temperature. Three Legionella pneumophila serotype 1 strains were incubated for 48 h in BCYE-alpha agar at various temperatures ranging from 30.0 degrees C to 48.0 degrees C and length of bacteria grown at each temperature was microscopically measured. Ability of bacteria to multiply at a given temperature was also determined. L. pneumophila serotype 1 strains ATCC 33152, a clinical isolate Okinawa 02-001 were going to elongate to longer than 100 mum when cultured higher than at 39.5 degrees C and at 41.5 degrees C, respectively. Each strain was unable to multiply when cultured higher than at 44.2 degrees C (ATCC 33152) or at 44.0 degrees C (Okinawa 02-001). Those data would provide insights for establishing regulations in terms of maintaining hot water temperature in a facility where a circulating hot water supply-system is available and contamination with Legionella spp. is likely to happen.