Cereus Var Research Articles

Growth behaviour of a spore forming probiotic strain in the gastrointestinal tract of broiler chicken and piglets

The growth behaviour of the probiotic strain Bacillus cereus var. toyoi in the gastrointestinal tract of broiler chicken and suckling piglets was evaluated. The strain germinated rapidly in intestinal samples from both animal species. Less than 10% of spores were recovered from the chicken crop and piglet stomach, respectively. Lumen samples and mucosal tissues from the hind gut of piglets displayed increasing colonization of the probiotic strain throughout the trial period. After oral administration of vegetative cells to broiler chicken and weaned piglets, sporulation was detected in all intestinal samples. The distribution of spore CFU indicated repeated germination and sporulation during the intestinal passage in piglets. B. cereus var. toyoi was not able to colonize the intestinal tract of both animal species. However, the probiotic strain was detected in suckling piglets before uptake of B. cereus var. toyoi supplemented feed. It is concluded that B. cereus var. toyoi germinates rapidly in broiler chicken and piglets, which is a necessary prerequisite for its possible probiotic effects. Germination and in vivo sporulation of vegetative cells indicated that the probiotic strain was metabolically active in the intestine of both animal species.

In vitro studies on the effects of Saccharomyces boulardii and Bacillus cereus var. toyoi on nutrient transport in pig jejunum

The probiotics Saccharomyces boulardii and Bacillus cereus var. toyoi are nonpathogenic microbes which have been shown to affect certain functions of the mucosal barrier in pig jejunum such as electrogenic ion transport capacity and paracellular permeability. The present studies were performed to investigate potential effects of the probiotics on jejunal nutrient transport such as sodium‐dependent glucose transport or proton‐dependent dipeptide transport. For this purpose the in vitro Ussing‐chamber technique was applied in order to examine net electrogenic ion flux rates (short circuit currents, Isc) across isolated intact jejunal epithelia in the absence and presence of either 10 mmol/l glucose (mucosal side) or two‐fold application of 5 mmol/l glycyl‐ l‐sarcosine or glycyl‐ l‐glutamine to the mucosal bathing solution. Brush border membrane vesicles (BBMV) were prepared in order to characterize kinetic parameters (Vmax, Km) of Na‐dependent glucose transport. Intestinal tissues were obtained from growing pigs in a weight range between 23 and 33 kg. All animals were fed twice daily and received 0.8–0.9 kg/day of a standard diet. After a 9‐ to 10‐day adaptation period the diets for treated animals were either supplemented for 8 days with 1.7×107 colony‐forming units (CFU)/g feed of S. boulardii or for 3 weeks with 106 CFU/g feed B. cereus var. toyoi. Under basal conditions Isc values were not affected by different treatment protocols (controls: 0.74 ± 0.04 µeq/cm2 per h, n=9; S. boulardii: 0.74 ± 0.12 µeq/cm2 per h, n=7; B. cereus 0.68 ± 0.09 µeq/cm2 per h, n=5). Irrespective of dietary treatment, the addition of glucose resulted in significant increases of Isc indicating substantial onset of electrogenic net Na/glucose cotransport. Maximal Isc values occurred within 30 min and reached 2.79 ± 0.41 µeq/cm2 per h in control epithelia. This was significantly lower than found in S. boulardii (4.47 ± 0.43 µeq/cm2 per h, p < 0.05) and B. cereus var. toyoi tissues (4.45 ± 0.31 µeq/cm2 per h, p < 0.05). Gt values were 22.4 ± 1.3 mS/cm2 in control animals and were significantly lower as shown in S. boulardii (p < 0.01) and B. cereus var. toyoi (p < 0.01)‐treated animals (28.4 ± 1.3 and 29.9 ± 0.8 mS/cm2, respectively). Vmax values of Na‐dependent glucose uptake into BBMV differed significantly between controls (0.64 ± 0.08 nmol/mg protein per 10 s; n=5), S. boulardii (0.89 ± 0.06 nmol/mg protein per 10 s; n=5, p < 0.05) and B. cereus var. toyoi preparations (1.08 ± 0.05 nmol/mg protein per 10 s; n=3, p < 0.01). Km values were not significantly affected (control: 0.31 ± 0.04 mmol/l, S. boulardii: 0.29 ± 0.05 mmol/l, B. cereus var. toyoi: 0.21 ± 0.01 mmol/l). Irrespective of dietary treatment, application of the dipeptide model substances glycyl‐ l‐sarcosine or glycyl‐ l‐glutamine resulted in significant increases of Isc indicating marked stimulation of electrogenic net H+/dipeptide cotransport. Highest Isc responses occurred in B. cereus var. toyoi preparations and lowest were found in control tissues. However, these differences were not significant. Gt values were not affected by different dietary treatments. The results clearly demonstrate that oral administration of either S. boulardii or B. cereus var. toyoi stimulates Na‐dependent glucose absorption in pig jejunum.

Aspects of the pharmacokinetics of doxycycline given to healthy and pneumonic East African dwarf goats by intramuscular injection.

The effect of experimentally induced Pasteurella haemolytica pneumonia on the pharmacokinetics of doxycycline (Doxycen Retard) administered intramuscularly was studied in seven East African dwarf goats. The study was conducted in two consecutive phases, separated by a washout period of four weeks. The experimental infection, induced by intratracheal administration of 5 ml of 10(7) to 10(9) cfu/ml of Pasteurella haemolytica, produced a temperature rise, depression and laboured breathing within 6-12 days after inoculation. The concentrations of doxycycline in the serum were determined by a quantitative microbiological assay using an agar-gel diffusion method employing Bacillus cereus var mycoides (ATCC 11778) as the test organism, with a level of detectability of approximately 0.05 micrograms/ml. The concentration-time curve of doxycycline in the serum after intramuscular injection of 20 mg/kg bodyweight of the long-acting formulation before and after experimental infection was adequately described by a one-compartment open model. The maximum serum concentrations (Cmax) of doxycycline were lower in pneumonic goats than in healthy goats (3.87 +/- 0.52 and 5.56 +/- 0.213 micrograms/ml, respectively), suggesting an increased distribution volume in the peripheral compartment. The mean +/- SEM absorption rate (ka) before infection (1.13 +/- 0.02 h-1) was smaller than the after infection (8.23 +/- 3.81 h-1), but the difference was not significant. The apparent elimination half-life (t 1/2 beta) (24.51 +/- 0.02 h) after infection was significantly increased (p < 0.05), while the corresponding rate constant (beta) was decreased (p < 0.01). The absorption half-life (t 1/2(alpha)) (0.137 +/- 0.03 h) was significantly decreased (p < 0.01) after infection. The distribution volume (Vd(beta)) was significantly increased after infection (p < 0.05). It is concluded that, although experimental infection had an effect on the disposition kinetics of doxycycline, this was not sufficiently pronounced to require alteration of the dosage during disease.

Microbiology of activated sludge treating wastewater containing Ni(II) and Cr(VI)

The species of microorganisms dominating a laboratory-scale once-through activated sludge unit treating simulated wastewater with different influent strengths (650 and 1300 mgl−1 COD) and containing 5 to 10 mgl−1 Ni(II) and 1 to 50 mgl−1 Cr(VI) were identified by standard biochemical tests. Within this framework, combinations of metals and influent COD concentrations were tried. Organisms, namely Proteus rettgeri, Acinetobacter sp., Flavobacterium lutescens, Bacillus cereus var. albolactis, Klebsiella sp., Arthrobacter simplex, and Pseudomonas mendocina, were identified at different stages of the experiments.

Some pharmacokinetic parameters of doxycycline in east African goats after intramuscular administration of a long-acting formulation.

A compartmental and non-compartmental study was carried out on five adult goats following intramuscular administration of doxycycline at 20 mg/kg bodyweight. The concentration of the drug in serum was determined by a microbiological assay employing Bacillus cereus var mycoides (ATCC 11778) as the test organism. The mean serum concentration (Cmax) and the time of maximum concentration (Tmax) were 1.87 micrograms/ml and 0.85 h, respectively. Using compartmental analysis, the plasma concentration-time curve of doxycycline best fitted a three-compartment open model with first-order absorption. A three-phase disposition of doxycycline was found, the terminal elimination half-life being approximately 40 h. The statistical moment theory was mainly used for non-compartmental analysis. The value obtained for the mean residence time (MRT) was 16.4 h. The mean values for the volume of distribution at steady state (Vdss), determined by compartmental and non-compartmental analyses, were 8.73 and 13.19 L/kg, respectively. There were no statistically significant differences when the major pharmacokinetic parameters were compared. It was concluded that the pharmacokinetic behaviour of doxycycline in goats after intramuscular administration is characterized by a three-compartment model with a slow terminal elimination phase. Based on current knowledge, this could be due to enterohepatic recycling and/or flip-flop kinetics. The study indicated that a single intramuscular administration of 20 mg/kg of doxycycline may only provide therapeutic concentrations for up to 24 h owing to slow absorption at the injection site.

Effect of non-ionic tensides on the growth of some soil bacteria

The effect of some nonylphenyl-ethylene oxide polymers on the growth of Bacillus megaterium, B. cereus var. mycoides, B. polymyxa, B. subtilis, Pseudomonas fluorescence and Azotobacter chroococcum was investigated in the concentration range 20–800 ppm with the agar diffusion method. The zones of inhibition, restricted growth and eventual stimulation were determined with a Shimadzu C-930 dual wavelength TLC scanner. The data matrix was evaluated by principal component analysis. A. chroococcum was insensitive to each tenside at each concentration. The growth of the other microorganisms was inhibited by the tensides. With B. megaterium and B. cereus var. mycoides stimulation was also observed. The effect of the non-ionic tensides decreased with increasing length of the hydrophilic ethylene oxide chain. This phenomenon can be explained by the assumption that the activity of tensides depends on their membrane-damaging effect. The bulky nonylphenyl group inserts between the apolar fatty acid chains disorganizing the membrane structure. The longer hydrophilic ethylene oxide chain modifies the distribution of tenside between the apolar and polar regions of the membrane, preferring the aqueous phase. This results in the decrease or loss of biological activity.

Quantitative determination of oxytetracycline in honey by cylinder plate microbioassay

A cyclinder plate microbioassay is described for the presumptive detection of oxytetracycline (OTC) in honey, using Bacillus cereus var. mycoidesas the test organism. The inhibition of this organism due to naturally occurring inhibitory substances (NOIS) in honey has been reduced to &lt;0.09 8g/g OTC equivalents. In honey with low NOIS level, this procedure detected 0.04 8g OTC/g honey. Most of the NOIS effect in honey was due to its acidic pH. Confirmation of OTC was made using the OTC resistant organism, Staphylococcus epidermidis. The use of the Difco B. cereus spore suspension method proved to be more reproducible and more sensitive than the B. cereus var. mycoicles vegetative cell suspension method. The microbioassay detects OTC in honey with greater sensitivity than other methods presently used.

Physiology of Sporolactobacillus strains isolated from different habitats and the indication of in vitro antagonism against Bacillus species

In an ecological study only low numbers of Sporolactobacillus were found in habitats such as the faeces of herbivores, the rumen of cattle and the final waste water of an abattoir. Their presence in the final waste water of an abattoir indicates their possible association with food, and, more specifically, with meat. Differences were found in some physiological characteristics. One isolate (L2404) differed from the authentic Sporolactobacillus ATCC 15538 by its inability to ferment inulin, its growth in presence of 6.5% NaCl and in 0.2% tellurite, by the isomer(s) of lactic acid produced and the mol% G + G in the DNA. One Sporolactobacillus isolate (L2407) showed antagonism against Bacillus cereus, Bacillus cereus var. mycoides, Bacillus megaterium and Bacillus subtilis.

Toxigenic Bacillus cereus var. fluorescens in Human Milk

Human milk with high bacterial load of B. cereus var. fluorescens is reported. The organism was identified by morphological, cultural, and biochemical characteristics. Lecithinase and haemolysin were produced in rabbit and bovine blood. The toxigenic nature of the organism was confirmed by production of necrosis on rabbit skin and fluid accumulation in rabbit ileal loop. An antibiotic resistogram also has been included.

A novel Bacillus pullulanase. Its properties and application in the glucose syrups industry.

The majority of starches used in the manu-facture of glucose syrups contain 75-85% amylopectin.1) Amylopectin is a highly branched polysaccharide consisting of linear chains of 1, 4-α-linked D-glucose residues, joined together by 1, 6-α-glucosidic linkages. The branch points occur on average every 20-25 D-glucose units, so that amylopectin contains 4-5% of 1, 6-α-glucosidic linkages.2-4) The 1, 6-α-glucosidic linkages act as a kind of barrier to the action of exo-acting, saccharif ying amylases such as glucoamylases or maltogenic /3-amylases. Endo-acting α-amylases are able to by-pass the branch points, 5) but in general are not capable cf hydrolyzing the 1, 6-α-glucosidic linkage. Recent work by Kobayashi and co-workers has shown that at least one α-amylase (from Thermoactinomyces vulgaris) can hydro-lyze 1, 6-α-glucosidic linkages, in addition to 1, 4-α-glucosidic linkages.6) Glucoamylases can slowly hydrolyze 1, 6-α-glucosidic linkages in amylopectin and partially hydrolyzed amylopectin, 7) but the action of maltogenic exo-amylases ceases as a branch point is approached.8) It is therefore obvious that the efficiency of the saccharification reaction could be improved by incorporating a specific amylopectin debranchina enzyme in the system. Debranching enzyme such as isoamylase [EC 3.2.1.68, glycogen 6-glucanohydrolase] and pullulanase [EC 3.2.1.41, pullulan 6-glucanohy-drolase] have been known for many years, 9) but their use in the glucose syrups industry is far from widespread. Pullulanases from Klebsiella pneumoniae, 10, 11) Streptomyces sp., 12) and Bacillus cereus var, mycoides13) and isoamylases from Pseudomonas amyloderamosa14, 15) and Cy-tophaga sp.16) are not sufficiently thermostable to be used at 60°C. Moreover the Pseudomonas isoamylase was the only debranching enzyme sufficiently acidophilic to be used at a pH of around 4. 5.17, 18) After an extensive screening programme, our research laboratories succeeded in isolating a species of Bacillus which produced a thermostable, acidophilic pullulanase which was free from glucoamylase, β-amylase and a-amylase side activities. Some of the properties of this enzyme will now be described.

Effect of Oxytetracycline and Benzimidazole Treatments on Blight-affected Citrus Trees1

Abstract Citrus blight is a vascular wilt disease of unknown etiology. Blight-affected ‘Pineapple’ sweet orange [Citrus sinensis (L.) Osb.] trees on rough lemon (C. jambhiri Lush.) rootstock in early stages of decline were treated with benzimidazole fungicides or with oxytetracycline (OTC) by trunk injection, soil drench, or injection plus drench. The distribution and persistence of the materials were monitored by bioassay using Bacillus cereus var. mycoides for OTC and Penicillium expansum L. for benzimidazoles. Both materials were well-distributed in the canopy following trunk injections, in the root systems following soil drenches, and though most of the tree after injection plus drench treatments. Benzimidazoles and OTC persisted within the trees for several months and relatively high levels of activity were maintained for over a year by 3 applications. OTC persisted in soil for more than 6 months; whereas, benomyl disappeared from the soil in 3 months or less. Although high levels of bactericidal or fungicidal activity were maintained in the treated trees, most treated trees declined as rapidly as the untreated control trees. The injection plus drench group treated with OTC showed a slight improvement after 2 years. None of the treatments increased tree growth or resulted in increased water uptake. High levels of zinc in trunk wood, an internal symptom of blight, were unaffected by the benzimidazole treatments, but injection plus drench treatment with OTC significantly reduced zinc levels in trunk wood. Since neither OTC nor benzimidazoles completely reversed symptoms of blight, we were unable to conclusively confirm or refute proposed bacterial or fungal etiologies for citrus blight.

Nutrition carbonée et étude taxonomique de Bacillus megaterium et B. cereus

Ten of the 14 strains of Bacillus cereus studied were isolated from pasteurized soil samples by a new selective technique which consists of the use of anaerobic enrichment cultures in peptone medium containing 0.5% KNO3. The 19 strains of B. megaterium use the following 21 sources of carbon and energy: D-glucose, D-galactose, D-fructose, D-mannose, D-glucosamine, D-gluconate, D-ribose, sucrose, cellobiose, maltose, raffinose, D-mannitol, glycerol, glycerate, pyruvate, fumarate, trans-aconitate, DL-aspartate, asparagine, L-glutamate, and L-glutamine. The 14 strains of B. cereus use the following 11 compounds: D-glucose, D-fructose, D-mannose, D-glucosamine, D-ribose, maltose, trehalose, glycerol, acetate, pyruvate, and L-glutamine. The eight strains of B. cereus var. mycoides use the following nine compounds: D-glucose, salicin, D-ribose, cellobiose, maltose, trehalose, glycerol, acetate, and pyruvate. Numerical taxonomy based on 151 characters shows that the 41 strains form two distinct groups. Group A includes the 19 strains of B. magateriurn; group B includes the 14 strains of B. cereus and the 8 strains of B. cereus var. mycoides. The guanine + cytosine content of the DNA of each strain was determined. The following mean values were obtained: B. megaterium, 40.6% ± 0.8; B. cereus, 38% ± 0.9; B. cereus var. mycoides, 38.4% ± 1.1.

Effect of proteolytic enzyme on the antibacterial property of egg albumen.

Egg albumen was hydrolyzed by proteolytic enzyme, and its antibacterial property was studied by the paper disc method. Egg albumen and its enzymatic hydrolysate formed an inhibition zone for Bacillus subtilis and Streptococcus lactis. The antibacterial property of hydrolyzed egg albumen varied with the degree of hydrolysis and increased two-fold of egg albumen by bromelin or trypsin treatment. Pronase E produced a large quantity of soluble or formol nitrogen, but its hydrolysate had low antibacterial property. The hydrolyzed egg albumen did not have antibacterial properties for B. cereus and B. cereus var. mycoides, but had antibacterial property for B. coagulans and B. licheniformis.

Disappearance Pattern of Chlortetracycline from the Liver, Bile and Tibia Bone of Growing Chicks

After feeding a diet containing 8, 000ppm equivalent of chlortetracycline (CTC) for 4 weeks and withdrawing CTC from the diet, 5 chicks each of male meat-type chicks were sacrificed at 0, 0.125, 0.25, 0.375, 1, 2, 3, 4, 6 and 8 days after the withdrawal to obtain samples of the liver, bile and left tibia bone. Contents of CTC in the liver and bile were determined microbiologically, using Bacillus cereus var. mycoides ATCC 11778, and that in the bone spectrophotofluorometrically as described by BUYSKE et al.Following Equation 4 was found suitable to describe the change in contents of CTC in the liver and bile of growing chicks, y=A1e-3.942T+A2e-0.3531T……(4)where, y is the content of CTC (ppm equivalent) and T is days after the withdrawal of dietary CTC, A1 and A2 are constants, which are 0.626 and 0.150 for the liver, respectively, and 7.67 and 2.33 for the bile, respectively. The disappearance pattern expressed by Equation 4 can be understood by two-compartment model shown in Fig. 1. The disappearance pattern of CTC from the tibia bone can be described by simple expotential curve of Equation 5, y=43.82e-0.1281T……(5)where, y is CTC content in the bone and T is as in Equation 4.Difference in disappearance patterns from the liver, bile and egg white between CTC and OTC, and between growing chicks and laying hens is discussed. It is also discussed that the bone is suspected to be the storage site in two-compartment model and that part of CTC released from the bone is deposited again in the bone making recycling shunt in two-compartment model as shown in Fig. 3. Similarity of individual error variance of CTC and OTC contents after logarithm transformation is discussed.

Residue and Disappearance of Dietary Oxytetracycline in the Blood, Muscle, Liver and Bile of Growing Chicks

Diets containing various levels from 0 to 4, 000ppm equivalent of oxytetracycline (OTC) were fed to meat-type male chicks and after 8 weeks of age dietary OTC was withdrawn. Chicks were sacrificed at 2, 4, 6 and 8 weeks of age during feeding OTC, and at 0, 3, 6 and 9 hours and 1-8 days after the withdrawal of OTC for sampling the blood, breast muscle, liver and bile. Content of OTC in the organs was determined microbiologically using Bacillus cereus var. mycoides ATCC 11778.No residue was detected in the blood, breast muscle and lived of the chicks given OTC at the level of 20ppm.Residue of OTC in the blood, breast muscle and liver on the dietary levels over 500ppm was in proportion to the dietary level of OTC. More OTC residue was found at 2 weeks of age than at older age.Disappearance pattern from the liver and bile of the chicks after the withdrawal of 4, 000ppm of OTC can be described well by the following equation, y=A1e-c1T+A2e-c2Twhere, y is residue in the organ, T is days after the withdrawal of dietary OTC, A1 and A2 are constant, c1 and c2 are constants indicating the initial and later speed of disapperance. The constants, A1, A2, c1 and c2 were estimated from the data obtained. Application on two-compartment model to explain the disappeance pattern was discussed.

Scanning electron microscopy of bacterial colonies.

A technique is described for observing bacterial colony growth. Bacillus cereus, B. subtilis, and B. cereus var. mycoides were grown on strips of dialysis membrane layered on nutrient agar. Microcolonies of the organisms on strips were fixed in Formalin vapor in situ; the strips then were removed from the agar and secured to scanning microscope specimen stubs without markedly disturbing the cellular arrangement. Scanning electron micrographs clearly depict morphology of individual cells, as well as the spatial orientation of cells within the colony. This technique is reproducible, adaptable, and simple.

Scanning Electron Microscopy of Bacterial Colonies

A technique is described for observing bacterial colony growth. Bacillus cereus, B. subtilis, and B. cereus var. mycoides were grown on strips of dialysis membrane layered on nutrient agar. Microcolonies of the organisms on strips were fixed in Formalin vapor in situ; the strips then were removed from the agar and secured to scanning microscope specimen stubs without markedly disturbing the cellular arrangement. Scanning electron micrographs clearly depict morphology of individual cells, as well as the spatial orientation of cells within the colony. This technique is reproducible, adaptable, and simple.

Detection of Residual Antibiotics from Slaughtered Swine Carcases

Detection of residual antibiotics, such as penicillin (PC), streptomycin (SM), and oxytetracyclin (OTC) in slaughtered swine carcases was made by the standard bioassay technique, using Sarcina lutea ATCC 9341, Bacillus subtilis ATCC 6633, and Bacillus cereus var. mycoides ATCC 11778, respectively.Specimens obtained for the examination were neck muscle, liver and kidney of 29 pigs which had been treated with the above mentioned antibiotics. In addition to this field survey, an experimental study on the reliability of disk method, for the detection of penicillin incorporated in pork extract, was also carried out comparing with the standard cup method.As the results it was found that 1 (3.7%) out of 27 muscles, 6 (29.0%) of 21 livers, and 4 (20.0%) kidney contained 0.16-1.1IU/g of PC, while 3 of 21 (14.2%) liver and 2 of 21 (9.5%) kidney contained 0.15-0.75μg/g of SM. However, none of the specimen tested contained any detectable amount of OTC.As for the reliability of the disk method, it was found that almost the same sensitivity with that of the cup method could be achieved, especially at a lower concentration of the antibiotics. Therefore, it is recommeded to use disk method as a routine screenig procedure for the detection of residual penicillin in animal tissues.

Microorganisms associated with immature Prionus laticollis (Coleoptera: Cerambycidae)

Alcaligenes sp., Bacillus cereus var. mycoides, Bacillus circulans, Bacillus sp., Escherichia intermedia, Enterobacter aerogenes, Flavobacterium sp., Streptomyces sp., and 2 unidentified gram-negative rods were isolated from the digestive tracts of 24 field-collected larvae of Prionus laticollis kept in the laboratory for 1 week or more. B. cereus var. mycoides, E. aerogenes, E. intermedia, and the unidentified rods were isolated from each larva examined within 24 hr of collection. Other types of micro-organisms were not isolated on the culture media used. Attempts to culture anaerobic bacteria from 12 larvae gave no results. B. cereus var. mycoides and a species of Cephalosporium were isolated from dead pupae and Paecilomyces (= Isaria) farinosa was found on dead last stage larvae (prepupae).

Studies on Biologically Active Substances Formed by Bacilli

The products of several Bacillus strains were investigated on rabbit serum calcium decreasing, oxytocic and toad heart function promoting activities. These products were obtained from the clear supernatant fluid of the culture medium after the cells were removed by centrifugation.For the production of rabbit serum calcium decreasing substance, Bacillus subtilis K and Bacillus natto No. 8 were found to be usefull, Bacillus megaterium KM, Bacillus cereus var. mycoides and Bacillus subtilis K produced oxytocic principle. Bacillus subtilis K, Bacillus brevis and Bacillus megaterium KM also produced toad heart function promoting factor.A procedure was developed to obtain the electrophoretically homogenous rabbit serum calcium decreasing substance from culture filtrate of Bacillus subtilis K. The crude substance was obtained as isoelectric precipitate by adjusting the culture filtrate to pH 3.0. The crude substance was purified by gel filtration on a Sephadex G-75 column, isoelectric fractionation and chromatog...