Viricidal Effects of Lactobacillus and Yeast Fermentation

The survival of selected viruses in Lactobacillus- and yeast-fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Five viruses, including Newcastle disease virus, infectious canine hepatitis virus, a porcine picornavirus, frog virus 3, and bovine virus diarrhea, were inoculated into a mixture of ground food waste (collected from a school lunch program) containing Lactobacillus acidophilus. Mixtures were incubated at 20, 30, and 40°C for 216 h. In a second trial, four viruses, including Newcastle disease virus, infectious canine hepatitis virus, frog virus 3, and a porcine picornavirus, were inoculated into similar edible waste material containing Saccharomyces cerevisiae. Mixtures were incubated at 20 and 30°C for 216 h. Samples were obtained daily for quantitative (trial 1) and qualitative (trial 2) virus isolation. Temperature, pH, and redox potential were monitored. Controlled pH and temperature studies were also done and compared with the inactivation rates in the fermentation processes. In trial 1 (Lactobacillus fermentation), infectious canine hepatitis virus survived the entire test period in the fermentation process but was inactivated below pH 4.5 in the controlled studies. Newcastle disease virus was inactivated by day 8 in the fermentation process and appeared to be primarily heat sensitive and secondarily pH sensitive in the controlled studies. The porcine picornavirus survived the fermentation process for 8 days at 20°C but was inactivated more rapidly at 30 and 40°C. The controlled studies verified these findings. Frog virus 3 was inactivated by day 3 in the fermentation process and appeared to be sensitive to low pH in the controlled studies. Bovine virus diarrhea was rapidly inactivated in the fermentation process (less than 2 h) and was pH and temperature sensitive. In trial 2 (yeast fermentation), infectious hepatitis virus survived the entire test period in the fermentation process. Newcastle disease virus was inactivated by day 7 at 20°C and day 6 at 30°C. The porcine picornavirus was inactivated by day 7 at 30°C but survived the entire test period at 20°C. Frog virus 3 was inactivated by day 3 at 20°C and day 2 at 30°C.     

Isolation, Purification, and Characterization of a Killer Protein from Schwanniomyces occidentalis

The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. The protein was stable between pH 2.0 and 5.0 and inactivated at temperatures above 40°C. The killer protein was chromosomally encoded. Mannan, but not β-glucan or laminarin, prevented sensitive yeast cells from being killed by the killer protein, suggesting that mannan may bind to the killer protein. Identification and characterization of a killer strain of S. occidentalis may help reduce the risk of contamination by undesirable yeast strains during commercial fermentations.     

Purification and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic Bacillus sp.

Two xylanases, designated XylA and XylB, were purified from the culture supernatant of the alkaliphilic Bacillus sp. strain AR-009. The molecular masses of the two enzymes were estimated to be 23 kDa (XylA) and 48 kDa (XylB) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pHs for activity were 9 for XylA and 9 to 10 for XylB. The temperature optima for the activity of XylA were 60°C at pH 9 and 70°C at pH 8. XylB was optimally active at 75°C at pH 9 and 70°C at pH 8. Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65°C in pH 8 and 9 buffers.     

Characterization of a Protease from a Psychrotroph, Pseudomonas fluorescens 114

A psychrotrophic bacterium isolated from river sediment was identified as Pseudomonas fluorescens 114. It grew at 0°C and optimally at 20°C. The bacterium produced a protease with a molecular weight of 47,000, which was stable in the pH range of 5 to 9 and worked optimally between pH 6.5 and 10. Activity was optimal at 35°C and was lost immediately at 50°C and after 5 min at 45°C. At 0, 10, and 20°C, 24, 38, and 57% of optimal activity were observed, respectively.     

Modeling the Recovery of Heat-Treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 Spores at Suboptimal Temperature and pH Using Growth Limits

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.     

STUDIES ON THE LACTASE OF ESCHERICHIA COLI

A "lactase solution" was prepared from Escherichia coli. The mechanism of its action has been studied and changes in the rate of hydrolysis under various conditions investigated. The hydrolysis of lactose by the enzyme approximates the course of reaction of the integrated Michaelis-Menten equation. One molecule of enzyme combines with one molecule of substrate. E. coli lactase is readily inactivated at pH 5.0, and its optimal activity at 36°C. is reached between pH 7.0 and pH 7.5. The optimal temperature for its action was found to be 46°C. when determinations were carried out after an incubation period of 30 minutes. Its inactivation by heat follows the course of a first order reaction, and the critical thermal increment between the temperatures of 45°C. and 53°C. was calculated to be 56,400 calories per mol. The enzyme is activated by potassium cyanide, sodium sulfide, and cysteine, and irreversibly inactivated by mercuric chloride, silver nitrate, and iodine. After inactivation with copper sulfate partial reactivation is possible, while the slight inhibition brought about by hydrogen peroxide is completely reversible. The possible structure of the active groups of E. coli lactase as compared with other enzymes has been discussed.     

Production of an Amylase-Sensitive Bacteriocin by an Atypical Leuconostoc paramesenteroides Strain

An atypical Leuconostoc paramesenteroides strain isolated from retail lamb produced a bacteriocin, leuconocin S, that was inactivated by α-amylase, trypsin, α-chymotrypsin, protease, and proteinase K but not by lipase or heat treatment at 60°C for 30 min. Supernatants from culture broths produced two glycoprotein bands on sodium dodecyl sulfate-polyacrylamide gels; these had molecular weights of 2,000 and 10,000 and activity against Lactobacillus sake ATCC 15521. The crude bacteriocin preparation was bacteriostatic and dissipated proton motive force. Bacteriocin activity was produced over a wide pH range (5.2 to 7.9) on buffered agar medium, with an optimum pH of pH 6.15. The optimum pH for production in broth was 6.5 to 7.0.     

The inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.     

Isolation, Purification, and Some Properties of Penicillium chrysogenum Tannase

Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40°C. The enzyme was stable up to 30°C and within the pH range of 4.0 to 6.5. The K_m value was found to be 0.48 × 10⁻⁴ M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels.     

Comparison of the Thermostability Properties of Three Acid Phosphatases from Molds: Aspergillus fumigatus Phytase, A. niger Phytase, and A. niger pH 2.5 Acid Phosphatase

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90°C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases of A. fumigatus and A. niger were both denatured at temperatures between 50 and 70°C. After heat denaturation at temperatures up to 90°C, A. fumigatus phytase refolded completely into a nativelike, fully active conformation, while in the case of A. niger phytase exposure to 55 to 90°C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases, A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of ≥80°C. In feed pelleting experiments performed at 75°C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85°C, however, the recovery of enzymatic activity was considerably higher for A. fumigatus phytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80°C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may favorably affect its pelleting stability.     

Inactivation of Ascaris Eggs in Source-Separated Urine and Feces by Ammonia at Ambient Temperatures

Sustainable management of toilet waste must prevent disease transmission but allow reuse of plant nutrients. Inactivation of uterus-derived Ascaris suum eggs was studied in relation to ammonia in source-separated urine without additives and in human feces to which urea had been added, in order to evaluate ammonia-based sanitation for production of safe fertilizers from human excreta. Urine was used concentrated or diluted 1:1 and 1:3 with tap water at 4, 14, 24, and 34°C. Fecal material, with and without ash, was treated with 1% or 2% (wt/wt) urea at 24 and 34°C. At 34°C eggs were inactivated in less than 10 days in urine and in amended feces. At 24°C only feces with 2% (wt/wt) urea or 1% (wt/wt) urea at high pH (10) inactivated all eggs within 1 month, and no inactivation was observed after 75 days in urine diluted 1:3 (18 ± 11 mM NH₃). At temperatures of ≥24°C, NH₃ proved to be an efficient sanitizing agent in urine and feces at concentrations of ≥60 mM. Treating fecal material at 34°C can give a 6-log₁₀ egg inactivation within 1 month, whereas at 24°C 6 months of treatment is necessary for the same level of egg inactivation. At temperatures of 14°C and below, inactivation rates were low, with viable eggs after 6 months even in concentrated urine.     

Purification of rabbit kidney cytokinase and a comparison of its properties with human urokinase

1. The cytokinase (tissue activator of plasminogen) content of several mammalian tissues was evaluated by a quantitative casein hydrolysis method. 2. An alkaline (pH10·5) extraction of cytokinase from rabbit kidney lysosome–microsome fraction, followed by chromatography on DEAE-cellulose at pH7·6 with stepwise or linear increase in concentration of phosphate buffer, gave an 86-fold purification of the enzyme. The purified material was non-proteolytic against casein and heated fibrin and was freeze-dried without significant loss of activity or solubility. 3. Cytokinase is a protein with E^0·1%_1cm.=0·87 at 280mμ, and does not possess sufficient hexose or sialic acid to be classified as a glycoprotein. It has S_20,w 2·9–3·1s and molecular weight 50000 when measured on a calibrated Sephadex G-100 column. It has an isoelectric point between pH8 and pH9, and is maximally active and stable at pH8·5. It is inactivated by heat at 78°. 4. Cytokinase and human urokinase have the same K_m value and are inhibited in a partially competitive manner by -aminohexanoic acid and aminomethylcyclohexanecarboxylic acid. They are also inhibited by cysteine and arginine, but are unaffected by iodoacetamide and p-chloromercuribenzoate. 5. On the basis of this and other evidence it is suggested that rabbit kidney cytokinase and human urokinase are similar, if not identical, enzymes.     

Isolation of Cellulolytic Anaerobic Extreme Thermophiles from New Zealand Thermal Sites

Avicel enrichment cultures from 47 thermal-pool sites in the New Zealand Rotorua-Taupo region were screened for growth and carboxymethyl cellulase activity at 75°C. Eight anaerobic cellulolytic cultures were obtained. The effect of temperature on carboxymethyl cellulase activity was measured, and bacteria were isolated from the five best cultures. Bacteria from two sources designated TP8 and TP10 grew at 75°C, accumulated reducing sugar in the growth medium and gave free cellulases with avicelase activity. Bacteria from sources designated Tok4, Tok8, and Wai21 grew at 75°C, accumulated no free sugars in the medium, and gave free carboxymethyl cellulases with virtually no avicelase activity. All were obligate anaerobic nonsporeforming rods which stained gram negative, grew on pentoses as well as hexoses, and gave ethanol and acetate as major fermentation end products. The isolated strain which produced the most active and stable cellulases (trivially designated TP8.T) had lower rates of free endocellulase accumulation at 75°C than did Clostridium thermocellum at 60°C, but its cellulase activity against avicel and filter paper in culture supernatants was comparable. Tested at 85°C, TP8.T carboxymethyl cellulases included components which were very stable, whereas C. thermocellum carboxymethyl cellulases were all rapidly inactivated. The TP8.T avicelase activity was relatively unaffected by Triton X-100, EDTA, and dithiothreitol. Evidence was obtained for the existence of unisolated, cellulolytic extreme thermophiles producing cellulases which were more stable and active than those from TP8.T.     

Growth of Candida ingens on Supernatant from Anaerobically Fermented Pig Waste: Effects of Temperature and pH

Candida ingens, a pellicle-forming yeast utilizing volatile fatty acids, grew over a pH range of 4.1 to 6.0 on nonsterile supernatants from anaerobically fermented pig wastes; growth was inconsistent between pH 4.1 and 4.6. When ambient temperature above the pellicle was 21°C and the temperature of the medium was 29 to 32°C, a pH range of 4.8 to 5.0 gave yields of 1.90 to 3.31 g of dry matter per liter, and 0.059 to 0.065 mol of volatile fatty acids was utilized per liter. There was no advantage in utilization of volatile fatty acids and yield of dry matter in keeping the pH constant during a 24-h growth period. C. ingens grew at pH 4.8 and 5.0 when both ambient and medium temperatures were 30°C. When ambient temperature was 10°C, maximum yield and utilization of volatile fatty acids occurred at a medium temperature of 28 to 30°C.     

Characterization of an endo-Acting Amylopullulanase from Thermoanaerobacter Strain B6A

A thermoanaerobe (Thermoanaerobacter sp.) grown in TYE-starch (0.5%) medium at 60°C produced both extra- and intracellular pullulanase (1.90 U/ml) and amylase (1.19 U/ml) activities. Both activities were produced at high levels on a variety of carbon sources. The temperature and pH optima for both pullulanase and amylase activities were 75°C and pH 5.0, respectively. Both the enzyme activities were stable up to 70°C (without substrate) and at pH 4.5 to 5.0. The half-lives of both enzyme activities were 5 h at 70°C and 45 min at 75°C. The enzyme activities did not show any metal ion activity, and both activities were inhibited by β- and γ-cyclodextrins but not by α-cyclodextrin. A single amylolytic pullulanase responsible for both activities was purified to homogeneity by DEAE-Sepharose CL-6B column chromatography, gel filtration using high-pressure liquid chromatography, and pullulan-Sepharose affinity chromatography. It was a 450,000-molecular-weight glycoprotein composed of two equivalent subunits. The pullulanase cleaved pullulan in α1,6 linkages and produced multiple saccharides from cleavage of α-1,4 linkages in starch. The K_ms for pullulan and soluble starch were 0.43 and 0.37 mg/ml, respectively.     

Optimization of nutritional and environmental conditions for pyocyanin production by urine isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a highly pathogenic bacteria involved in numerous diseases among which, are urinary tract infections (UTIs). The pyocyanin secreted as a virulence factor by this bacterium has many beneficial applications but its high cost remains an obstacle for its widespread use. In this study, a total of fifty urine isolates were identified as P. aeruginosa. All strains produced pyocyanin pigment with a range of 1.3–31 µg/ml. The highest producer clinical strain P21 and the standard strain PA14 were used in optimization of pyocyanin production. Among tested media, king’s A fluid medium resulted in the highest yield of pyocyanin pigment followed by nutrient broth. Growth at 37 °C was superior in pyocyanin production than growth at 30 °C. Both shaking and longer incubation periods (3–4 days) improved pyocyanin production. The pyocyanin yield was indifferent upon growth of P21 at both pH 7 and pH 8. In conclusion, the optimum conditions for pyocyanin production are to use King’s A fluid medium of pH 7 and incubate the inoculated medium at 37 °C with shaking at 200 rpm for a period of three to four days.     

Effects of Temperature on Bacterial Communities and Metabolites during Fermentation of Myeolchi-Aekjeot,a Traditional Korean Fermented Anchovy Sauce

Myeolchi-aekjeot (MA) in Korea is produced outdoors without temperature controls, which is a major obstacle to produce commercial MA products with uniform quality. To investigate the effects of temperature on MA fermentation, pH, bacterial abundance and community, and metabolites were monitored during fermentation at 15°C, 20°C, 25°C, and 30°C. Initial pH values were approximately 6.0, and pH values increased after approximately 42 days, with faster increases at higher temperatures. Bacterial abundances increased rapidly in all MA samples after quick initial decreases during early fermentation and then they again steadily decreased after reaching their maxima, which were significantly greater at higher temperatures. Bacterial community analysis revealed that Proteobacteria and Tenericutes were predominant in all initial MA samples, but they were rapidly displaced by Firmicutes as fermentation progressed. Photobacterium and Mycoplasma belonging to Proteobacteria and Tenericutes, respectively, which may include potentially pathogenic strains, were dominant in initial MA, but decreased with the growth of Chromohalobacter, which occurred faster at higher temperatures––they were dominant until 273 and 100 days at 15°C and 20°C, respectively, but not detected after 30 days at 25°C and 30°C. Chromohalobacter also decreased with the appearance of subsequent genera belonging to Firmicutes in all MA samples. Tetragenococcus, halophilic lactic acid bacteria, appeared predominantly at 20°C, 25°C, and 30°C; they were most abundant at 30°C, but not detected at 15°C. Alkalibacillus and Lentibacillus appeared as dominant genera with the decrease of Tetragenococcus at 25°C and 30°C, but only Lentibacillus was dominant at 15°C and 20°C. Metabolite analysis showed that amino acids related to tastes were major metabolites and their concentrations were relatively higher at high temperatures. This study suggests that high temperatures (approximately 30°C) may be appropriate in MA fermentation, in the light of faster disappearance of potentially pathogenic genera, higher amino acids, growth of Tetragenococcus, and faster fermentation.     

Characterization of an Invertase with pH Tolerance and Truncation of Its N-Terminal to Shift Optimum Activity toward Neutral pH

Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.     

Effects of pH and temperature on the wild-type and a mutant form of Neurospora glutamate dehydrogenase

1. We confirm the observation of Bürk (1965) that Neurospora crassa NADP-linked glutamate dehydrogenase normally exists in an inactive form below pH7·0 and in a fully active form above pH8·0 in either tris or orthophosphate buffer. At pH7·4 the enzyme is about half activated at 25°. 2. The variety of the enzyme produced by the mutant am^2l shows a similar behaviour except that the transition is shifted about one pH unit in the alkaline direction. 3. The am^2l enzyme has previously been reported to be activated by brief warming to 30° in phosphate buffer at pH8·0. The wild-type enzyme shows a similar effect at pH7·0. In tris buffer this effect is much less pronounced. 4. The am^2l enzyme is extremely unstable at 47° at pH7·0; its stability is somewhat greater at lower pH, and is markedly increased by increasing the pH in the range 7·0–8·7. The wild-type enzyme also shows an indication of a stability minimum at pH7·0, but a temperature of 60° is needed for a measurable rate of inactivation. 5. The inactive form of the enzyme is much more subject to thermal irreversible denaturation than is the active form.     

Inactivation of a Norovirus by High-Pressure Processing

Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20°C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log₁₀ PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5°C; a 5-min pressure treatment of 350 MPa at 30°C inactivated 1.15 log₁₀ PFU of virus, while the same treatment at 5°C resulted in a reduction of 5.56 log₁₀ PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5°C and 20°C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5°C was sufficient to inactivate 4.05 log₁₀ PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.