Effects of alginates and sodium carrageenates mixed with soy proteins on the coefficient of protein efficiency

Male 21 d-old Wistar rats, were fed for 4 wk with diets containing casein or soybean proteins (10%) with 0.5, 1, 2 or 3% sodium alginate or sodium carrageenan or without any alginate or carrageenan. Daily protein intake and weight gain of casein-fed rats were not significantly different (P less than 0.05) from those of rats fed soybean meal with alginate, whatever the dose received. Rats fed 3% carrageenan in soybean meal had significantly higher feed intake than that of rats fed casein. At the levels studied, alginate had no effect on the Protein Efficiency Ratio (PER), but carrageenan did. The addition of increased quantities of carrageenan to soybean meal followed by heating the mixture led to a progressive and significant decrease in PER at all levels of carrageenan compared to casein feeding. The addition of 3% carrageenan to heated soybean meal, corresponding to 0.62% of meal diet, led to a significant decrease in PER. These results confirm the precipitating role of carrageenans on proteins.     

Production of casein hydrolysates by extracellular acid proteinases of immobilized Humicola lutea cells

Casein hydrolysis was studied during the cultivation of immobilized Humicola lutea cells producing acid proteinases. By monitoring the cultivation with time, various casein hydrolysates could be obtained, from partially modified proteins (yield 80%) with improved emulsion properties to peptones (yield > 50%) with a degree of hydrolysis >40%. The casein from the fermentation medium appeared to be simultaneously a nitrogen source, an inducer of proteinase biosynthesis, and a substrate for the production of casein hydrolysates. Casein (4%) and glucose (2%) ensured optimal cultivation conditions. The fungal cells, immobilized in calcium alginate beads, required a short cultivation time and demonstrated comparable hydrolysis of casein during five to seven reuses in batch mode. Correspondence to: B. Tchorbanov     

Continuous production of high degree casein hydrolysates by immobilized proteases in column reactor

The enzymatic complete hydrolysis of casein was investigated by using immobilized endopeptidase and exopepti dase packed in the jacketed column reactors. The mass transfer efficiency of proteins was improved by using sliced shrimp chitin hull as enzyme support, which formed a network structure inside the column reactor that prevented the formation of protein precipitate and increased the line flow rate of protein solution. The specificity of the protease was of crucial importance for both the hydrolysis degree and the free amino acid content of the hydrolysates. Of the enzymes tested, the immobilized A. oryzae protease was the most effective enzyme in breaking down the casein molecules and releasing the free amino acid from casein hydrolysates. The immobilized pancreatic and kidney exopeptidase could lead to a 20% increase of free amino acids. The free amino acid content of casein hydrolysates was 34.81% after processing and could reach to 64% if the column length was doubled, but 100% hydrolysis was impossible as the reverse reaction was also taking place. The casein hydrolysates was characterized by its high degree of hydrolysis and high content of free amino acids. It can be applied in infant formula, element diet, and as a protein ingredient for food industry.     

Effect of the peptide and vitamin composition of casein hydrolysates on the β-galactosidase activity of Kluyveromyces bulgaricus cells

Instead of yeast extracts, casein hydrolysates from several suppliers were added to culture media and analyzed with respect to their ability to stimulate β-galactosidase activity in Kluyveromyces bulgaricus cells. Four enzymatic casein hydrolysates caused a significantly higher stimulation of enzyme activity, while acid casein hydrolysates clearly reduced the enzyme activity. Enzymatic casein hydrolysates, inducing high and low lactase activity, were analyzed with respect to average peptide length (APL), vitamins (niacin, pathothenate) as well as free and bound amino acids. The molecular weights of these casein hydrolysates were estimated by gel filtration. No correlation was found between the degree of enzyme stimulation and the vitamin contents, the APL values and the free amino acid contents of the casein hydrolysates. Casein hydrolysates stimulating the lactase activity were less soluble in water and, in a gel filtration column, they showed three peaks with slightly lower molecular weights than the three peaks seen in hydrolysates which had no effect on activity. APL values of alcoholic precipitates of casein hydrolysates showed an inverse correlation to lactase activity. The molecular weights of alcoholic precipitates of lactase stimulating digests were also lower compared to non-stimulating ones. Alcoholic precipitates with lactase-stimulating activity were more hydrophilic, as was shown by a smaller proportion absorbed in a C18 column and by amino acid analysis. Our results sugest that alcoholic precipitates could probably be important in the lactase stimulation and their composition should be further investigated. The mechanism is nevertheless complex and may be caused by various simultaneous factors.     

Refeeding after ingestion of diets containing 10% soybean protein associated with various concentrations of alginate or sodium carrageenan. Effects on growth and lipid parameters in the rat]

Male 21-d-old Wistar rats were fed over 3 experimental periods. During the first period of 4 wk, diets contained 10% casein or defatted soy flour proteins, with or without 0.5, 1, 2 or 3% sodium alginate or sodium carrageenan, and were heated. During the second period, they were fed a standard diet for 16 wk with 17% proteins, and during the third period, they received the same diet as in the first period, but with 20% proteins. Rat body weights were measured throughout the study period; plasma lipid levels were then determined after fasting. Presence of sodium alginate in the diet had no effect on growth, but rats fed carrageenan presented growth retardation at the end of the experimental period, which was not altered by refeeding the standard diet. Sodium alginate did not modify rat triglyceridemia, except at the 1% level. Carrageenan had a hypotriglyceridemic effect. Alginate and carrageenan had no effect on blood cholesterol. Compared to soybean protein, casein intake did not increase plasma cholesterol levels as generally described. The effect of carrageenans on growth and plasma triglyceride levels could be a result of their physico-chemical properties.     

Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 x 10(8) purified phage per ml for 24 h at 37 degrees C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10(10) and 10(12) phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.     

Autolytic degradation of chicken intestinal proteins

Data on the exhaustive degradation of chicken intestinal proteins by endogenous proteases, which could be utilized as a means to prepare protein hydrolysate, is reported in the present paper. Chicken intestine possesses proteolytic activities (cathepsin B, D, H, L, aminopeptidases and alkaline proteases) comparable to that in organ tissues like liver and spleen, which could degrade the tissue proteins extensively. The autolytic degradation was found to be optimum at pH 2.5 and 60 degrees C. Analysis by SDS-PAGE showed a time dependent degradation of proteins to low molecular weight (<10 kDa) products. Kinetic studies employing specific inhibitors indicated that the degradation (90-94%) of proteins at acidic pH is governed largely by pepstatin sensitive proteases. The acidic extract of the tissue was found to hydrolyse albumin, casein and soybean proteins efficiently. Results point to the possible application of tissue autolysis for obtaining protein hydrolysates from chicken intestine. Chicken intestine could also serve as a potential source of much needed proteolytic enzymes for food and pharmaceutical applications.     

Malondialdehyde production and erythrocyte membrane resistance to free radicals, in function of adequate or inadequate protein intake, associated with different oils (sunflower, soybean, coconut, salmon)]

Requirements in polyunsaturated fatty acids (PUFA) of series n-3 and n-6 may be amplified and their metabolism, transport, and utilization may be impaired in the long term, by protein depletion. The aim of this study was to evaluate, in young rats, malondialdehyde (MDA) production and erythrocyte membrane antioxidative defense, when they were fed balanced (20% casein) or depleted (2% casein) protein diet associated with various oils (sunflower, soybean, coconut or salmon). Over a short period (28 days), eight groups of 10 male Wistar rats were fed eight different diets: TOC (20% casein + 5% sunflower oil), TOd (2% casein + 5% soybean oil), SOC (20% casein + 5% soybean oil), SOd (2% casein + 5% soybean oil), COC (20% casein + 5% coconut oil), COd (2% casein + 5% coconut oil), SAC (20% casein + 5% salmon oil), SAd (2% casein + 5% salmon oil). Blood was removed, MDA was assessed in plasma (reaction with thiobarbituric acid). Washed erythrocytes were subjected to organic free radical generator (Kit KGRL 400 Spiral R.D., Couternon, 21560 France). The haemoglobin released was analysed by spectrophotometry. The total anti-radical defense status was expressed as the length of time to reach 50% hemolysis (T 50% in min). Plasma of deficient groups (2% casein) exhibited low concentrations of protein, particularly with coconut and salmon oils; phospholipid and total cholesterol, excepted with diet containing coconut oil; triacylglycerol; and VLDL. Malondialdehyde. In groups fed balanced protein diets, the lowest values were obtained with salmon and coconut oils. MDA contents of groups TOd, COd and SAd were higher than those of their respective control groups, but significantly only in group COd. Antiradical defense status. Total anti-radical defence status in erythrocytes was not modified in the short term by balanced or depleted protein diets which ever oil was used, despite deep changes in fatty acid composition of membrane phospholipids. In particular, phospholipid contents in eicosapentaenoic, docosahexaenoic acids were greatly enhanced by the consumption of salmon oil compared to the values obtained with coconut oil.     

Dietary protein and fatty acid composition of liver lipids in the rat

In order to compare the effects of different sources of dietary protein on the fatty acid composition of phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylinositols (PI), cholesteryl esters and triacylglycerols, male rats were fed for a 4-week period on cholesterol-free, or cholesterol-containing, diets based on casein, or soybean protein and olive oil. The most conspicuous difference observed was the occurrence of significantly higher levels of 5,8,11-eicosatrienoic acid, 20:3 (n - 9), in the different lipid classes of casein-fed, compared with soybean protein-fed, animals. In the PI fraction of livers from the groups of rats fed casein diet, this fatty acid amounted to between 9.9 and 13.3% by weight of the total fatty acids. Phospholipids from livers of casein-fed rats contained increased levels of oleic acid, 18:1 (n - 9) (in PC and PE) and reduced levels of stearic acid (18:0). Moreover, in this group of rats PI contained a reduced level of arachidonic acid, 20:4 (n - 6). A casein-related decrease in the linoleic acid, 18:2 (n - 6), content of PC and PE was observed only in the rats fed on cholesterol-free diet. Effects on the fatty acid composition were also observed in the triacyglycerol and cholesteryl ester fractions, in which the rats fed casein diet showed higher levels of palmitoleic acid, 16:1 (n - 7) (cholesterol-supplemented diet) and lower values for linoleic acid, than the soybean protein-fed rats.     

Reduction in Exopolysaccharide Viscosity as an Aid to Bacteriophage Penetration through Pseudomonas aeruginosa Biofilms

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 × 10⁸ purified phage per ml for 24 h at 37°C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10¹⁰ and 10¹² phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.     

Susceptibility to trypsinolysis of esterified milk proteins

Methyl-, ethyl- and propyl-esters of beta-lactoglobulin, alpha-lactalbumin and beta-casein were prepared and then hydrolyzed with trypsin in various conditions. Resulting hydrolysates were analysed by SDS electrophoresis and RP-HPLC. The degree of hydrolysis of esterified samples was generally lower than those of the non-modified proteins. The highest degrees of hydrolysis were obtained at pH 7--8 with native and esterified protein samples. beta-Lactoglobulin propyl ester and beta-casein methyl ester yielded the lowest degrees of hydrolysis. Ethyl- and propyl-esters of beta-casein showed high resistance towards tryptic attack, even after 20 h of hydrolysis. SDS electrophoretic patterns of tryptic hydrolysates of native proteins showed bands corresponding to low molecular weights. Tryptic hydrolysates of esterified proteins showed bands with higher sizes. RP-HPLC profiles of tryptic hydrolysates of esterified samples showed peaks with longer elution times than those obtained with native proteins, indicating the presence of more hydrophobic peptide populations. A peptic pre-treatment improved tryptic action on esterified proteins. It resulted in a better resolution of RP-HPLC profiles and in a complete disappearance of the protein after 20 h tryptic hydrolysis.     

Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue

Protein kinase CKII (i.e. casein kinase II, CKII, NII) is expressed at a higher level in rapidly proliferating tissues and in solid human tumours (e.g. colorectal carcinomas) when compared to the corresponding non-neoplastic colorectal mucosa. This could be shown by (a) Western blotting of cellular extracts from solid tumours followed by immunostaining with an anti-CKII polyclonal antibody, (b) immunohistochemical staining of cells from tissue sections and (c) by activity measurements using the CKII-specific synthetic peptide (RRRDDDSDDD). The maximum observed activity in the colorectal carcinomas investigated was up to eightfold higher in the tumour specimens than in the non-neoplastic tissue (i.e. colorectal mucosa). The activity range was between 33-350 U/mg protein and in the case of colorectal mucosa 13-106 U/mg protein. The amount of CKII determined in the individual tumours was in the range 0.4-1.6 nmol/g tissue.     

Physicochemical characterisation of enzymatically hydrolysed derivatives of acetylated starch

Potato starch modified to different degrees by substitution with acetyl groups was the subject of this study undertaken to determine the influence of conditions of enzymatic hydrolysis on the surface-active properties of hydrolysates of acetylated starch. The effect of acetylation of starch preparation on its susceptibility to enzymatic hydrolysis in the membrane reactor was also considered. All hydrolysates of acetylated starch samples investigated were found to bring a decrease in the surface/interfacial tension, both at the air/water and the toluene/water interfaces. For binary hydrolysate-surfactant systems, the surface mole fractions in the mixed adsorbed monolayer at the air/water interface were estimated. For mixed systems, the synergism in reducing the surface tension at the air/water interface was observed. The experimentally obtained dynamic surface tension data for the aqueous solution of acetylated starch hydrolysates were used to estimate the diffusion coefficients. Particle size distributions of the hydrolysates formed in the aqueous solutions were compared to those of commercial maltodextrin.     

Indices of protein kinase activity and the problem of biochemical diagnosis of malignant growth

Protein kinase, cAMP-dependent and casein kinase activities and the ratio of these activities were determined in normal and neoplastic tissues of the colonic mucosa. Substantial activation of casein kinase and an appropriate decrease in the activity ratio of the enzymes were revealed in the malignant tissue. Protein kinase activity changed in a similar fashion in the histologically intact mucosa located 25-30 cm from the center of the tumor. The data obtained are suggestive of the possibility of using the above-indicated findings for the biochemical diagnosis of malignant neoplasms.     

Effects of peptides derived from dietary proteins on mucus secretion in rat jejunum

The hypothesis that dietary proteins or their hydrolysates may regulate intestinal mucin discharge was investigated in the isolated vascularly perfused rat jejunum using an enzyme-linked immunosorbent assay for rat intestinal mucins. On luminal administration, casein hydrolysate [0.05-5% (wt/vol)] stimulated mucin secretion in rat jejunum (maximal response at 417% of controls). Lactalbumin hydrolysate (5%) also evoked mucin discharge. In contrast, casein, and a mixture of amino acids was without effect. Chicken egg albumin and its hydrolysate or meat hydrolysate also did not modify mucin release. Interestingly, casein hydrolysate-induced mucin secretion was abolished by intra-arterial TTX or naloxone (an opioid antagonist). beta-Casomorphin-7, an opioid peptide released from beta-casein on milk ingestion, induced a strong mucin secretion (response at 563% of controls) that was inhibited by naloxone. Intra-arterial beta-casomorphin-7 also markedly increased mucin secretion (410% of controls). In conclusion, two enzymatic milk protein hydrolysates (casein and lactalbumin hydrolysates) and beta-casomorphin-7, specifically, induced mucin release in rat jejunum. The casein hydrolysate-induced mucin secretion is triggered by a neural pathway and mediated by opioid receptor activation.     

Absorption of oligo-L-[35S]methionine after feeding of a low casein or a low soybean protein isolate diet in rats.

We studied the absorptive properties of oligo-L-methionine (OM), which is an enzymatically synthesized and slowly digestible peptide. Previously, we demonstrated that when OM was added to a low casein diet, the improvement of the body weight gain was higher than when OM was added to a low soybean protein isolate (SPI) diet and we suggested that the difference in the supplementary effect of OM depends on its absorptive rate. In the present study, the OM absorption estimated by the portovenous difference in radioactivity derived from 35S-labeled OM was higher in the casein diet than in the SPI diet in early stages of feeding after fasting. Absorbed OM was quantified by subtracting the radioactivity of [35S]OM remaining in the whole gut from the ingested [35S]OM, 90 and 180 min after feeding casein and SPI diets containing 3% [35S]OM. We also estimated the absorptive efficiencies by subtracting the amount of radioactivity remaining in the intestines from the amount of [35S]OM emptied from the stomach as percentages of the emptied OM. Both the amount of absorbed OM and absorptive efficiencies of OM were higher in the casein group than in the SPI group, and the higher absorptive efficiency in the casein group indicates a higher digestibility for OM when rats are fed a 3% OM diet after fasting. The digestibility of [35S]OM measured by fecal excretion of radioactivity of OM during normal feedings for diets containing 0.3% [35S] OM for 7 days was about 80% in the casein group and 60% in the SPI group. We conclude that the different supplementary effects of OM in the low casein and SPI diets depend on the difference in OM digestibility. The difference in the digestibility of OM may partly depend on the faster absorption rate of OM in the early stages of feeding.     

Casein turnover in rabbit mammary explants in organ culture

1. Explants of mammary gland from mid-pregnant rabbits were cultured in medium 199 containing insulin, prolactin and cortisol, and specific anti-casein immunoglobulin G was used to measure the amount, rate of synthesis and rate of degradation of casein in the explants in the presence of hormones and after removal of hormones from previously stimulated tissue. 2. The amount of casein in particle-free supernatants prepared from mammary explants was measured by ;rocket' immunoelectrophoresis. 3. The rate of incorporation of l-[4,5-(3)H]leucine into casein was measured after isolation of the casein by immunoadsorbent chromatography and polyacrylamide-gel electrophoresis in the presence of urea and sodium dodecyl sulphate. 4. Casein accumulates in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in a decrease in the rate of accumulation of casein in the explants. 5. Casein-synthetic rate increases in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in continued casein synthesis at approx. 30% of the rate in the presence of hormones. The synthetic rate does not decrease to values observed in explants cultured throughout in the absence of hormones. 6. Casein is not degraded in mammary explants during a phase of rapid casein accumulation (36-72h) in the presence of hormones. Furthermore casein is not degraded when hormones are removed from the tissue after between 36 and 72h in culture. 7. Casein is glycosylated in mammary explants; the extent of glycosylation parallels the rate of synthesis. The glycosylated protein is rapidly secreted from the tissue. 8. The results are consistent with the notion that after hormonal stimulation mammary explants from mid-pregnant rabbits synthesize, glycosylate and rapidly secrete casein. Removal of hormones decreases the synthetic rate of casein, but does not cause the accumulation of a pool of degradable casein in the lobuloalveolar cells.     

Ovomucoid rendered insoluble by heating with wheat gluten but not with milk casein

The effect of wheat gluten, soybean protein and milk casein on the heat-induced in solubilization of egg white ovomucoid was investigated by using ELISA inhibition and immunoblotting analysis. Heat treatment at 180 degree C for 10 min of egg white mixed with wheat gluten specifically accelerated the heat-induced change in ovomucoid. Such an effect was weakly brought about by soybean protein, but not by casein.     

Intestinal fat suppresses protein-induced exocrine pancreatic secretion in chronically bile-pancreatic juice-diverted rats

Previously, we showed that the increase in pancreatic enzyme secretion was lower after feeding a casein diet containing fat than that after feeding a fat-free casein diet in chronically bile-pancreatic juice (BPJ)-diverted rats. In the present study, we determined whether the suppressive effects of fats on flow volume of BPJ and pancreatic enzyme secretion depend on delaying gastric emptying and examined the characteristics of the suppression with intraduodenal instillation of soybean oil or lecithin in BPJ-diverted rats. The study was conducted as three separate experiments using conscious rats with chronic BPJ diversion by means of a common bile-pancreatic duct catheter. The flow volume of BPJ and the secretion of pancreatic amylase and trypsin were determined after intraduodenal instillation of the test solution. Exocrine pancreatic secretion was strongly stimulated by administration of guanidinated casein hydrolysate (HGC, 150 mg/ml) in chronic BPJ-diverted rats. However, pancreatic secretion after administration of an emulsion containing HGC with either soybean oil (100 mg/ml) or mixed fat (50 mg/ml soybean oil + 50 mg/ml lecithin) was much lower than that after administration of HGC alone. In contrast, administration of the soybean oil emulsion without HGC resulted in a small, but significant increase in the volume of BPJ. The suppressive effects of soybean oil (100 mg/ml) on the increases in the BPJ flow and enzyme secretion were similar to those of sodium taurocholate (10 mg/ml), and there was no additive effect of soybean oil on taurocholate suppression. In conclusion, duodenally instilled soybean oil suppressed increases in flow volume of BPJ and pancreatic enzyme secretion induced by HGC in chronic BPJ-diverted rats, showing that the suppressive effect of the fat does not depend on delaying gastric emptying.     

Regulation of neurotoxin and protease formation in Clostridium botulinum Okra B and Hall A by arginine.

Supplementation of a minimal medium with high levels of arginine (20 g/liter) markedly decreased neurotoxin titers and protease activities in cultures of Clostridium botulinum Okra B and Hall A. Nitrogenous nutrients that are known to be derived from arginine, including proline, glutamate, and ammonia, also decreased protease and toxin but less so than did arginine. Proteases synthesized during growth were rapidly inactivated after growth stopped in media containing high levels of arginine. Separation of extracellular proteins by electrophoresis and immunoblots with antibodies to toxin showed that the decrease in toxin titers in media containing high levels of arginine was caused by both reduced synthesis of protoxin and impaired proteolytic activation. In contrast, certain other nutritional conditions stimulated protease and toxin formation in C. botulinum and counteracted the repression by arginine. Supplementation of the minimal medium with casein or casein hydrolysates increased protease activities and toxin titers. Casein supplementation of a medium containing high levels of arginine prevented protease inactivation. High levels of glucose (50 g/liter) also delayed the inactivation of proteases in both the minimal medium and a medium containing high levels of arginine. These observations suggest that the availability of nitrogen and energy sources, particularly arginine, affects the production and proteolytic processing of toxins and proteases in C. botulinum.