Effect of digestion temperature and pH on treatment efficiency and evolution of volatile fatty acids during thermophilic aerobic digestion of model high strength agricultural waste

Thermophilic aerobic digestion (TAD) of a model agricultural waste, potato peel slurry, at soluble chemical oxygen demand (COD) load equivalent to approximately 8.0 gl(-1), was carried out under batch conditions at 0.5 vvm aeration rate. Digestions were carried out at temperatures of 45, 50, 55, 60 and 65 degrees C (or left unregulated) without pH control to study the effect of digestion temperatures on TAD. The effects of digestion pH on the process were studied at pH 6.0, 7.0, 8.0, 9.0 and 9.5 (and in unregulated control) all at 55 degrees C. Except for digestion at 65 degrees C, which was inoculated extraneously using culture of Bacillus strearothermophilus all reactions were carried out using the populations indigenous to the waste. During digestion at different temperatures, the removal of soluble COD increased with temperature to reach a peak at 60 degrees C before declining slightly, removal of soluble solid (SS) followed similar pattern and reached peak at 65 degrees C being the highest temperature studied, while the degradation of TSS and TS (TSS + TS) decreased with an increase in temperature. Digestion at pH 7.0 was more efficient than at other pH values. Acetate was the predominant volatile fatty acid (VFA) in all the reactions and accounted for up to 90% of the total. Digestion at 60 degrees C led to the greatest accumulation of acetate, and this coincided with the period of highest oxygen uptake, and rapid consumption of soluble carbohydrate. Iso-valerate was also produced at all pH values. Digestion at 55 degrees C and also at pH 7.0 led to rapid and efficient processes with least accumulation of VFA and should be of interest in full-scale processes whenever it is practicable to regulate the digestion pH and temperature. The result of digestion at unregulated pH indicates that gradual adaptation may be used to achieve efficient treatment at elevated pH values. This would be of interest in full-scale processes where it is not practicable to tightly regulate digestion pH, and where the waste is produced at a pH value much higher than neutral.     

Protein enrichment of corn cob heteroxylan waste slurry by thermophilic aerobic digestion using Bacillus stearothermophilus

Thermophilic aerobic digestion (TAD) of heteroxylan waste was implemented at waste load of 30gL(-1) with mineral nitrogen supplementation to study effect of the process on waste degradation, protein accretion and quality. Digestions were carried out at 45 50, 55, 60 and 65 degrees C using Bacillusstearothermophilus in a CSTR under batch conditions at 1.0vvm aeration rate, pH 7.0 for a maximum of 120h. Amylase and xylanase activities appeared rapidly in the digest, while basal protease activity appeared early in the digestion and increased towards end of the processes. Highest degradation of volatile suspended solid, hemicellulose and fibre occurred at 55 degrees C while highest degradation of total suspended solid occurred at 60 degrees C. Highest protein accretion (258.8%) and assimilation of mineral nitrogen and soluble protein occurred at 55 degrees C. The % content of amino acids of digest crude protein increased relative to raw waste and with digestion temperature. Quality of digest protein was comparable to the FAO standard for feed use. TAD has potentials for use in the protein enrichment of waste.     

Inactivation of animal viruses during sewage sludge treatment

Using a previously developed filter adsorption technique, the inactivation of a human rotavirus, a coxsackievirus B5, and a bovine parvovirus was monitored during sludge treatment processes. During conventional anaerobic mesophilic digestion at 35 to 36 degrees C, only minor inactivation of all three viruses occurred. The k' values measured were 0.314 log10 unit/day for rotavirus, 0.475 log10 unit/day for coxsackievirus B5, and 0.944 log10 unit/day for parvovirus. However, anaerobic thermophilic digestion at 54 to 56 degrees C led to rapid inactivation of rotavirus (k' greater than 8.5 log10 units/h) and of coxsackievirus B5 (k' greater than 0.93 log10 unit/min). Similarly, aerobic thermophilic fermentation at 60 to 61 degrees C rapidly inactivated rotavirus (k' = 0.75 log10 unit/min) and coxsackievirus B5 (k' greater than 1.67 log10 units/min). Infectivity of parvovirus, however, was only reduced by 0.213 log10 unit/h during anaerobic thermophilic digestion and by 0.353 log10 unit/h during aerobic thermophilic fermentation. Furthermore, pasteurization at 70 degrees C for 30 min inactivated the parvovirus by 0.72 log10 unit/30 min. In all experiments the contribution of temperature to the total inactivation was determined separately and was found to be predominant at process temperatures above 54 degrees C. In conclusion, the most favorable treatment to render sludge hygienically safe from the virological point of view would be a thermal treatment (60 degrees C) to inactivate thermolabile viruses, followed by an anaerobic mesophilic digestion to eliminate thermostable viruses that are more sensitive to chemical and microbial inactivations.     

Inactivation of animal viruses during sewage sludge treatment.

Using a previously developed filter adsorption technique, the inactivation of a human rotavirus, a coxsackievirus B5, and a bovine parvovirus was monitored during sludge treatment processes. During conventional anaerobic mesophilic digestion at 35 to 36 degrees C, only minor inactivation of all three viruses occurred. The k' values measured were 0.314 log10 unit/day for rotavirus, 0.475 log10 unit/day for coxsackievirus B5, and 0.944 log10 unit/day for parvovirus. However, anaerobic thermophilic digestion at 54 to 56 degrees C led to rapid inactivation of rotavirus (k' greater than 8.5 log10 units/h) and of coxsackievirus B5 (k' greater than 0.93 log10 unit/min). Similarly, aerobic thermophilic fermentation at 60 to 61 degrees C rapidly inactivated rotavirus (k' = 0.75 log10 unit/min) and coxsackievirus B5 (k' greater than 1.67 log10 units/min). Infectivity of parvovirus, however, was only reduced by 0.213 log10 unit/h during anaerobic thermophilic digestion and by 0.353 log10 unit/h during aerobic thermophilic fermentation. Furthermore, pasteurization at 70 degrees C for 30 min inactivated the parvovirus by 0.72 log10 unit/30 min. In all experiments the contribution of temperature to the total inactivation was determined separately and was found to be predominant at process temperatures above 54 degrees C. In conclusion, the most favorable treatment to render sludge hygienically safe from the virological point of view would be a thermal treatment (60 degrees C) to inactivate thermolabile viruses, followed by an anaerobic mesophilic digestion to eliminate thermostable viruses that are more sensitive to chemical and microbial inactivations.     

Diversity of thermophilic populations during thermophilic aerobic digestion of potato peel slurry

Aims:  To study the diversity of thermophiles during thermophilic aerobic digestion (TAD) of agro-food waste slurries under conditions similar to full-scale processes.
Methods and Results:  Population diversity and development in TAD were studied by standard microbiological techniques and the processes monitored by standard fermentation procedures. Facultative thermophiles were identified as Bacillus coagulans and B. licheniformis, while obligate thermophiles were identified as B. stearothermophilus . They developed rapidly to peaks of 10⁷ to 10⁸ in ≤48 h. Stability of obligate thermophiles increased with process temperatures. Thermophiles were unstable at process pH above or below neutral, but developed rapidly at all aeration rates. Peak populations were higher in the median than at extremes of aeration rates. Obligate thermophiles were unstable at low aeration rates. Process self-heating was higher at lower than at higher aeration rate. Beyond 96 h most thermophiles were present as spores.
Conclusions:  Limited range of indigenous thermophiles drives TAD of slurry. They develop rapidly and are stable at most digestion conditions.
Significance and Impact of the Study:  Development and stability of thermophiles in TAD suggest that the process may be operated in a wide range of conditions; and even at short HRT in continuous processes without compromising efficiency.     

Effect of aeration rate and waste load on evolution of volatile fatty acids and waste stabilization during thermophilic aerobic digestion of a model high strength agricultural waste

Thermophilic aerobic digestion (TAD) is a relatively new, dynamic and versatile low technology for the economic processing of high strength waste slurries. Waste so treated may be safely disposed of or reused. In this work a model high strength agricultural waste, potato peel, was subjected to TAD to study the effects of oxygen supply at 0.1, 0.25, 0.5 and 1.0 vvm (volume air per volume slurry per minute) under batch conditions at 55 degrees C for 156 h on the process. Process pH was controlled at 7.0 or left unregulated. Effects of waste load, as soluble chemical oxygen demand (COD), on TAD were studied at 4.0, 8.0, 12.0 and 16.0 gl(-1) (soluble COD) at pH 7.0, 0.5 vvm and 55 degrees C. Efficiency of treatment, as degradation of total solids, total suspended solids and soluble solid, as well as soluble COD significantly increased with aeration rate, while acetate production increased as the aeration rate decreased or waste load increased, signifying deterioration in treatment. Negligible acetate, and no other acids were produced at 1.0 vvm. Production of propionate and other acids increased after acetate concentration had started to decrease and, during unregulated reactions coincided with the drop in the pH of the slurry. Acetate production was more closely associated with periods of oxygen limitation than were other acids. Reduction in oxygen availability led to deterioration in treatment efficiency as did increase in waste load. These variables may be manipulated to control treated waste quality.     

Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis

Streptococcus faecalis S-48 produces a broad spectrum antibiotic, active against Gram-positive and Gram-negative bacteria. This substance is produced in solid and liquid media and also in a defined basal medium. It is sensitive to protease, pronase, or trypsin, heating at 70 degrees C, and alkaline pH, but resistant to treatment with lipase, lysozyme, alkaline phosphatase, DNAase, RNAase, acidic or neutral pHs, and also lower temperatures (60 degrees C). Several organic solvents cause precipitation, but not inactivation. This antibiotic has been partially purified by gel filtration and further ion-exchange chromatography. Its molecular weight has been estimated close to 2000. The biological activity of this antagonistic substance against the selected indicator strains, Streptococcus faecalis S-47 and Escherichia coli U-9, is bactericidal. The characterization of this substance, initially classified as a bacteriocin, indicates that it is an antibiotic of peptidic nature. The significance of antibiotic occurrence in group D of the genus Streptococcus is also discussed.     

Unfolding rates of globular proteins determined by kinetics of proteolysis

A convenient method for the determination of unfolding rates of small globular proteins under physiological conditions was developed using digestion with proteases. The apparent first-order rate constants for digestion of lysozyme with thermolysin and with Pronase at pH 8 and 50 degrees C were shown to be saturated with increases of concentrations of these proteases. The maximum rate constants extrapolated were identical in digestions with two different proteases, and were found to be equal to the unfolding rate constant of lysozyme. Similarly, the unfolding rate constant of RNase A at pH 8 and 50 degrees C, and those of lysozyme, RNase A and beta-lactoglobulin at pH 8 and 40 degrees C, were determined by the digestion method. Thus, it was shown that digestion by proteases proceeds mainly via the unfolded state of proteins.     

Optimized Batch Fermentation of Cheese Whey-Supplemented Feedlot Waste Filtrate to Produce a Nitrogen-Rich Feed Supplement for Ruminants

A simple and reliable method is described which allows determination of virus inactivation rates during sludge treatment processes in situ. Bacteriophage f2 was adsorbed onto an electropositive membrane filter which was then sandwiched between two polycarbonate membranes with pores smaller than the virus diameter. The resulting sandwich was fixed in an open filter holder, and several such devices were connected before being exposed in sludge-digesting tanks. The device described prevented uncontrolled virus escape, but allowed direct contact of the various inactivating or stabilizing substances present in the environment tested with the virus adsorbed to the carrier membrane. After exposure to an environment, the surviving fraction of virus was eluted from the inner filter and determined by plaque counting. By using polycarbonate membranes without pores for sandwiching, the influence of temperature alone on virus inactivation could be measured. Thermophilic fermentation at 60 degrees C and at 65 kPa pressure led to a bacteriophage f2 titer reduction of 3.5 log10 units per h, whereas during thermophilic digestion at 54.5 degrees C titers decreased 1.2 log10 units per h. During mesophilic digestion an inactivation rate of only 0.04 log10 units per h was observed. Under these latter conditions, temperature had only a minor effect (19%) on virus inactivation, whereas at 54.5 degrees C during thermophilic digestion heat accounted for 32% of the total inactivation, and during thermophilic fermentation at 60 degrees C temperature and pressure were 100% responsible for virus denaturation.     

Optimized batch fermentation of cheese whey-supplemented feedlot waste filtrate to produce a nitrogen-rich feed supplement for ruminants

A simple and reliable method is described which allows determination of virus inactivation rates during sludge treatment processes in situ. Bacteriophage f2 was adsorbed onto an electropositive membrane filter which was then sandwiched between two polycarbonate membranes with pores smaller than the virus diameter. The resulting sandwich was fixed in an open filter holder, and several such devices were connected before being exposed in sludge-digesting tanks. The device described prevented uncontrolled virus escape, but allowed direct contact of the various inactivating or stabilizing substances present in the environment tested with the virus adsorbed to the carrier membrane. After exposure to an environment, the surviving fraction of virus was eluted from the inner filter and determined by plaque counting. By using polycarbonate membranes without pores for sandwiching, the influence of temperature alone on virus inactivation could be measured. Thermophilic fermentation at 60 degrees C and at 65 kPa pressure led to a bacteriophage f2 titer reduction of 3.5 log10 units per h, whereas during thermophilic digestion at 54.5 degrees C titers decreased 1.2 log10 units per h. During mesophilic digestion an inactivation rate of only 0.04 log10 units per h was observed. Under these latter conditions, temperature had only a minor effect (19%) on virus inactivation, whereas at 54.5 degrees C during thermophilic digestion heat accounted for 32% of the total inactivation, and during thermophilic fermentation at 60 degrees C temperature and pressure were 100% responsible for virus denaturation.     

Yield and protein quality of thermophilic Bacillus spp. biomass related to thermophilic aerobic digestion of agricultural wastes for animal feed supplementation

Bacillus spp. responsible for thermophilic aerobic digestion (TAD) of agricultural wastes were studied for their growth rate, yield and protein quality (amino acid profile) under conditions that approximate full-scale waste digestion as pointers to the capacity of TAD to achieve protein enrichment of wastes for reuse in animal feeding. Specific growth rates of the thermophiles varied with temperature and aeration rates. For Bacillus coagulans, the highest specific growth rate was 1.98 muh(-1); for Bacillus licheniformis 2.56 muh(-1) and for Bacillus stearothermophilus 2.63 muh(-1). Molar yield of B. stearothermophilus on glucose increased with temperature to a peak of 0.404 g g(-1) at 50 degrees C before declining. Peak concentration of overflow metabolite (acetate) increased from 10 mmol at 45 degrees C to 34 mmol at 65 degrees C before declining. Accumulation of biomass in all three isolates decreased with increase in temperature while protein content of biomass increased. Highest biomass protein (79%) was obtained in B. stearothermophilus at 70 degrees C. Content of most essential amino acids of the biomass improved with temperature. Amino acid profile of the biomass was comparable to or superior to the FAO standard for SCP intended for use in animal feeding. Culture condition (waste digestion condition) may be manipulated to optimize protein yield and quality of waste digested by TAD for recycling in animal feed.     

Susceptibility of chemostat-grown Yersinia enterocolitica and Klebsiella pneumoniae to chlorine dioxide

The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two enteric bacteria, Yersinia enterocolitica and Klebsiella pneumoniae, to chlorine dioxide was investigated. These organisms were grown in a defined medium in a chemostat and the influence of growth rate, temperature, and cell density on the susceptibility was studied. All inactivation experiments were conducted with a dose of 0.25 mg of chlorine dioxide per liter in phosphate-buffered saline at pH 7.0 and 23 degrees C. The results indicated that populations grown under conditions that more closely approximate natural aquatic environments, e.g., low temperatures and growth at submaximal rates caused by nutrient limitation, were most resistant. The conclusion from this study is that antecedent growth conditions have a profound effect on the susceptibility of bacteria to disinfectants, and it is more appropriate to use the chemostat-grown bacteria as test organisms to evaluate the efficacy of a certain disinfectant.     

Susceptibility of chemostat-grown Yersinia enterocolitica and Klebsiella pneumoniae to chlorine dioxide.

The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two enteric bacteria, Yersinia enterocolitica and Klebsiella pneumoniae, to chlorine dioxide was investigated. These organisms were grown in a defined medium in a chemostat and the influence of growth rate, temperature, and cell density on the susceptibility was studied. All inactivation experiments were conducted with a dose of 0.25 mg of chlorine dioxide per liter in phosphate-buffered saline at pH 7.0 and 23 degrees C. The results indicated that populations grown under conditions that more closely approximate natural aquatic environments, e.g., low temperatures and growth at submaximal rates caused by nutrient limitation, were most resistant. The conclusion from this study is that antecedent growth conditions have a profound effect on the susceptibility of bacteria to disinfectants, and it is more appropriate to use the chemostat-grown bacteria as test organisms to evaluate the efficacy of a certain disinfectant.     

Why does ribonuclease irreversibly inactivate at high temperatures?

The mechanism of irreversible thermoinactivation of bovine pancreatic ribonuclease A in the pH range relevant to enzymatic catalysis has been elucidated. At 90 degrees C and pH 4, the enzyme inactivation is caused by hydrolysis of peptide bonds at aspartic acid residues (the main process) and deamidation of asparagine and/or glutamine residues. At 90 degrees C and neutral pH (pH 6 and 8), the enzyme inactivation is caused by a combination of disulfide interchange (the main process), beta-elimination of cystine residues, and deamidation of asparagine and/or glutamine residues. These four processes appear to demarcate the upper limit of thermostability of enzymes.     

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 degrees 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 degrees 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 degrees C but was inactivated more rapidly at 30 and 40 degrees 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 degrees C and day 6 at 30 degrees C. The porcine picornavirus was inactivated by day 7 at 30 degrees C but survived the entire test period at 20 degrees C. Frog virus 3 was inactivated by day 3 at 20 degrees C and day 2 at 30 degrees C.     

Pyridoxal phosphate protects against an irreversible temperature-dependent inactivation of hepatic delta-aminolevulinic acid synthase

The stability of hepatic delta-aminolevulinic acid synthase (ALAS), the first and rate-limiting enzyme of the heme biosynthetic pathway, was investigated. Incubation of the mitochondrial matrix fraction obtained from either control or allylisopropylacetamide-induced rats at 37 degrees C in Tris-Cl, pH 7.4, EDTA, and dithiothreitol resulted in a rapid decrease in ALAS activity such that 50-70% of the activity was lost after 30 min. Similar decreases in ALAS activity were observed when a cytosolic fraction from the induced animals was incubated at 37 degrees C. Addition of 0.1 mM pyridoxal-P, the cofactor of ALAS, to the preincubation medium completely prevented the observed loss of activity; however, dialysis of the inactive matrix fraction against several changes of buffer containing pyridoxal-P did not restore activity, suggesting that the inactivation was irreversible. These decreases in ALAS activity in the absence of pyridoxal-P were temperature dependent, as a 55% loss of ALAS activity was observed after a 60-min incubation at 30 degrees C, while the enzyme was completely stable when preincubated at 22 degrees C for 60 min. This inactivation of ALAS does not appear to involve proteolytic digestion, as addition of a wide spectrum of protease inhibitors to the preincubation medium in the absence of pyridoxal-P did not protect against the inactivation. The suggestion is made that the cofactor, pyridoxal-P, may dissociate from the enzyme during the preincubation and, consequently, the apoenzyme may be irreversibly inactivated at temperatures above 22 degrees C.     

Assessment of viral inactivation during pH 3.3 pepsin digestion and caprylic acid treatment of antivenoms

Antivenoms are manufactured by the fractionation of animal plasma which may possibly be contaminated by infectious agents pathogenic to humans. This study was carried out to determine whether pre-existing antivenom production steps, as carried out by EgyVac in Egypt, may reduce viral risks. Two typical manufacturing steps were studied by performing down-scaled viral inactivation experiments: (a) a pH 3.3 pepsin digestion of diluted plasma at 30 degrees C for 1h, and (b) a caprylic acid treatment of a purified F(ab')2 fragment fraction at 18 degrees C for 1h. Three lipid-enveloped (LE) viruses [bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), and vesicular stomatitis virus (VSV)] and one non-lipid-enveloped (NLE) virus [encephalomyocarditis virus (EMC)] were used as models. Kinetics of inactivation was determined by taking samples at 3 time-points during the treatments. The pH 3.3 pepsin digestion resulted in complete clearance of PRV (>7.0 log(10)) and in almost complete reduction of VSV (>4.5 but < or =6.4 log(10)), and in a limited inactivation of BVDV (1.7 log(10)). EMC inactivation was > or =2.5 but < or =5.7 log(10). The caprylic acid treatment resulted in complete inactivation of the 3 LE viruses tested: BVDV (>6.6 log(10)), PRV (>6.6 log(10)), and VSV (>7.0 log(10)). For EMC no significant reduction was obtained (0.7 log(10)). Cumulative reduction was >13.6, >11.5, >8.3 and > or =2.5 for PRV, VSV, BVDV and EMC, respectively. Therefore the current manufacturing processes of at least some animal antisera already include production steps that can ensure robust viral inactivation of LE viruses and moderate inactivation of a NLE virus.     

Use of hydrostatic pressure for inactivation of microbial contaminants in cheese

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30 degrees C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10 degrees C and by 6-log-unit cycles at temperatures of 20 and 30 degrees C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20 degrees C and >400 MPa at 30 degrees C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20 degrees C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.     

Autolytic enzyme system of Streptococcus faecalis. V. Nature of the autolysin-cell wall complex and its relationship to properties of the autolytic enzyme of Streptococcus faecalis

Cell walls from exponential-phase cultures of Streptococcus faecalis ATCC 9790 contain an autolysin (a beta-N-acetylmuramide glycanhydrolase, E.C. 3.2.1.17) which has been isolated from trypsin-speeded wall autolysates. The autolysin, which was excluded from Bio-Gel P-60, was further fractionated by diethylaminoethyl (DEAE)-cellulose chromatography or filtration on Bio-Gel P-200. After DEAE-cellulose chromatography, which removed most of the wall polysaccharide, autolysin activity was extremely labile and was rapidly lost at -20 C, even in the presence of albumin. The P-60-excluded enzyme was rapidly bound by walls at both 37 C (50% bound in about 1 min) and 0 C (50% bound in less than 4 min). Wall-bound autolysin could not be removed by 1.0 m ammonium acetate (pH 6.9). Autolysin was also bound by walls that had been extracted with 10% trichloroacetic acid or treated with 0.01 n periodate, suggesting that the nonpeptidoglycan wall polymers are not important for binding. Wall-bound autolysin was more stable than the soluble enzyme to proteinase digestion, acetone (40%), 8 m urea (at 0 C), or to inactivation at 56 C. Two bacterial neutral proteinases (which do not hydrolyze ester bonds) activated latent wall-bound autolysin, suggesting that activation results from the cleavage of one or more peptide bonds. The group A streptococcal proteinase activated latent autolysin but differed from the other proteinases in that it did not inactivate soluble autolysin. The results suggest that the autolysin is not covalently linked to the wall. The high affinity of the walls for the autolysin appears to be responsible for the firm, not easily reversed binding.