Untreated Casein Research Articles

ORIGINAL ARTICLE: Bioavailability of lysine for kittens in overheated casein is underestimated by the rat growth assay method*,†

Growth assays were performed to determine lysine bioavailability for kittens and rats in untreated and heated casein; these values were compared with estimates obtained with an in vitro method. Body weight, food intake, nitrogen and dry matter digestibility, and plasma lysine were determined during an 80-day growth trial using kittens (n = 16). Body weight and food intake were determined during a 21-day growth trial using weanling rats (n = 80). The growth data showed bioavailable lysine to be 102.4% and 100.2% (for untreated casein) and 66.1% and 51.7% (for heated casein) for kittens and rats, respectively. There was no relationship between plasma lysine and dietary lysine concentrations for kittens. There were no significant differences in nitrogen or dry matter digestibility among diets for kittens. The chemically reactive lysine content of untreated casein was 99.6%, and of heated casein was 67.1%. Heat treatment of casein resulted in significantly decreased lysine bioavailability as estimated by all methods. For untreated casein, both growth assays showed good agreement with the in vitro method for available lysine. For heated casein, the rat growth assay significantly underestimated bioavailable lysine as determined in kittens while the in vitro method closely approximated this value for the cat.

Investigation of surface modification of casein films by rennin enzyme action using micro-beam grazing incidence small angle X-ray scattering

Micro-beam grazing incidence small angle X-ray scattering (μGISAXS) scans were performed to investigate structural changes that occur after deposition of a droplet of rennin on a casein film. The subsequent diffusion of the droplet into the film led to a lateral and vertical concentration gradient of rennin. As a result of the enzyme action, the film surface became roughened and the formation of islands — evident. The altered surface morphology led to a variation in the μGISAXS pattern, more precisely to the occurrence of a second so-called Yoneda peak. As for the untreated casein film, the mean size of the micelle components (casein micelles, substructure and colloidal calcium phosphate) remained constant within the rennin droplet area. In contrast, a broadening of their corresponding size distributions could be detected. Finally, we discuss the potential of GISAXS scanning experiments with micrometer-sized X-ray beams in view of problems with the interpretation of the data obtained from imaging techniques such as atomic force microscopy.

Hydrogen peroxide and ferulic acid-mediated oxidative cross-linking of casein catalyzed by horseradish peroxidase and the impacts on emulsifying property and microstructure of acidified gel

Horseradish peroxidase (HRP, EC 1.11.1.7) was used in this study to catalyze oxidative cross-linking of casein in the presence of hydrogen peroxide and cross-linker ferulic acid. Cross-linking of casein was demonstrated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ultraviolet absorbance analysis. Oxidative cross-linked casein mediated by hydrogen peroxide and ferulic acid was prepared at casein concentration of 5% (w/ w), HRP addition of 3 mkat·g-1 proteins, ferulic acid addition of 6 mmol·l-1, 3% (w/w) H2O2 addition of 1 ml, reaction temperature 37°C and reaction time 3 h. Analysis results showed that the emulsifying activity index and emulsifying stability index of cross-linked casein prepared were enhanced compared to that of untreated casein totally. Microstructure of chemical acid-induced gel of cross-linked casein was observed under scanning electron microscopy and appeared to be more compact and uniform than that of casein. Hydrogen peroxide and ferulic acid-mediated oxidative cross-linking of casein catalyzed by horseradish peroxidase might have a beneficial to the emulsifying property or gelation of casein.

Interactions between Denatured Milk Serum Proteins and Casein Micelles Studied by Diffusing Wave Spectroscopy

The acid-induced aggregation of casein micelles from milk, in the presence of different whey protein preparations from heated and unheated milk, has been studied using diffusing wave spectroscopy (DWS). In particular, the study focused on the turbidity (or l*) parameter obtainable from DWS, which can give information on the interactions between particles in aggregating systems. The experiments provided evidence that the presence of small, soluble, whey protein/kappa-casein aggregates derived from heated milk gave rise to interactions with both heated and unheated casein micelles over a pH range of 5.6 down to 5.2. Comparison of heated and unheated milks, together with milks whose sera had been exchanged, showed that direct interactions were indeed occurring, even between untreated casein micelles and soluble whey protein complexes. Comparison of the behavior of the whey protein aggregates in emulsion preparations where they could not interact with the large particles confirmed that the effect was specific to the presence of casein micelles and could not arise simply from the aggregation of the whey proteins themselves.

Response of Channel Catfish to Diets Containing T-2 Toxin

The T-2 toxin is a trichothecene mycotoxin produced by certain molds of the genus Fusarium that infect the grains, wheat by-products, and oilseed meals used in the production of animal feeds. An aquarium study was conducted with juvenile channel catfish Ictalurus punctatus. Experimental diets were prepared by replacing the untreated casein in a semipurified diet with casein treated with pure T-2 toxin in the amounts necessary to produce five levels of the toxin. Dietary concentrations were 0, 0.625, 1.25, 2.5, and 5.0 mg/kg of diet. A concurrent study with similarly sized channel catfish was conducted to substantiate the toxicity of the T-2 toxin. This study required feeding the control diet at the same levels of daily consumption (pair-feeding) as in the treatments with the three highest levels of T-2 toxin (1.25, 2.5, and 5.0 mg/kg) to compare differences in weight gain, feed conversion, hematocrit, and survivability. After 8 weeks, significant (P < 0.05) reductions in growth were observed for all treatments fed T-2 toxin compared with the control diet. Significantly poorer feed conversion was found only for the highest level of T-2 toxin. Hematocrit values were adversely affected by the inclusion of T-2 toxin at 1.25, 2.5, and 5.0 mg/kg. Pair-fed catfish had a significantly greater increase in body weight than fish fed the highest level of mycotoxin only. Pair-fed treatments had hematocrit values that were very similar to those of the control and significantly higher than those of fish receiving 1.25, 2.5, and 5.0 mg/ kg. The survivability of the fish fed T-2 toxin at 2.5 and 5.0 mg/kg was significantly lower than that of control fish and their pair-fed counterparts. Histopathological anomalies of stomach, head, and trunk kidneys were observed in fish receiving the three highest levels of T-2 toxin. These results demonstrate that this feed-borne mycotoxin is toxic to juvenile channel catfish.

Effects of bovine somatotropin and ruminally undegraded protein on feed intake, live weight gain, and mohair production by yearling Angora wethers.

Yearling Angora wethers (n = 24; 24+/-1.0 kg BW) were used in an experiment with a 2 x 2 factorial arrangement of treatments to investigate effects of bovine somatotropin (bST) treatment and dietary level of ruminally undegraded protein on DMI, ADG, and mohair production. Untreated casein (UC) or casein treated with formaldehyde (TC) was included at 7% DM of a diet containing 11% CP and 46% concentrate. A slow-release bST form was administered weekly to deliver 0 (Control) or 100 microg/ (kg BW.d) of bST. Plasma concentrations of bST and IGF-I were increased (P < .05) during the 7-d period following bST injection. Ruminal fluid ammonia N concentration was lower (P < .01) for TC than for UC before feeding (6.6 vs 7.5 mg/dL) and 4 h later (8.2 vs 12.2 mg/dL), and total VFA concentration was lower (P < .01) for TC than for UC. Treatment with bST decreased (P = .08) DMI with UC (1.15 vs .91 kg/d) and increased (P = .08) DMI with TC (.95 vs 1.06 kg/d). Formaldehyde treatment of casein increased ADG (65, 74, 55, and 91 g/d; P = .03) and clean fleece production (P < .01; 14.1, 17.3, 15.0, and 18.4 g/d for UC-Control, TC-Control, UC-bST, and TC-bST, respectively), with no effect of bST during the 8-wk period of treatment or for the 8 wk thereafter (P > .10). In conclusion, with yearling Angora wethers, bST does not seem useful to enhance mohair production and may not alter effects of dietary level of ruminally undegradable protein on mohair production.

Effect of hydrochloric acid treatment of palm kernel for dehulling on the nutritional quality of kernel protein

The effect of hydrochloric acid treatment for the dehulling of palm kernel on the nutritional quality of its protein was studied and compared with that of untreated kernel protein and casein. Rats fed a treated defatted kernel protein diet had slightly lower gains in body weight (59·5 g) when compared to those fed a casein diet (64·2 g), while the gains in body weight in the animals fed untreated kernel protein diet were considerably lower (42·5 g). The PER of the treated protein was 2·27 (95% of that of casein) while that of the untreated protein was lower; only 1·63. In the case of the treated protein diet, 80% of food nitrogen was absorbed compared to 94% for casein and 65% for untreated protein, indicating improvement in protein digestibility with HCl treatment. The essential amino acid content of the treated kernel protein was comparable to egg protein. Available lysine content was higher in the treated sample than that of the untreated sample. © 1998 SCI.

Functional and Immunological Properties of Casein Hydrolysate Produced from a Two‐Stage Membrane System

ABSTRACTA uniform hydrolysate with a relatively low free amino acid content (&lt;5%) was produced from bovine casein with a commercial protease (Alcalase) in a two‐stage membrane reactor. The trichloroacetic acid‐soluble nitrogen was 98.68% and degree of hydrolysis was 23.2% for the second‐stage hydrolysate. The retained immunologically active casein was 2.32% and whey protein was 1.09% in the hydrolysate as determined by ELISA. The second‐stage protein hydrolysate was completely soluble at 2% (w/v) from pH 3.0 to 9.0. Water sorption increased two to four times at water activities of 0.33 to 0.95 as compared to untreated casein.

Effect of treatment temperature on dietary quality of ammonia fiber explosion (AFEX) treated casein for rats

Dietary quality of casein treated by the ammonia fiber explosion (AFEX) process was evaluated in weanling Sprague-Dawley rats. Two 19 day trials were conducted, in which four animals received one of the four (first trial) or five (second trial) treatment diets. Animal performance, nitrogen digestion characteristics and biological value were determined for diets (protein content 18%) containing untreated casein or casein AFEX treated at 40, 60, 80, or 100°C. AFEX treatment at temperatures of 40–100°C did not detrimentally effect ( P > 0.10) feed intake, weight gain, or nitrogen intake. AFEX treatment at 60°C increased ( P < 0.05) feed efficiency by 7% over controls. Higher ( P < 0.05) fecal (0.23 vs. 0.15 g per 5 days) and urinary nitrogen (1.26 g vs. 1.12 g per 5 days), as well as reduced ( P < 0.05) apparent nitrogen digestibility (91.4% vs. 94.2%), nitrogen retention (43.6% vs. 49.4%), and biological value (47.8% vs. 52.4%) were observed for diets containing casein treated at 60°C and higher temperatures vs. controls. Results from this study suggest decreased casein digestion and absorption, and decreased utilization of absorbed nitrogen with AFEX treatments of 60°C and higher.

The effect of methanal (formaldehyde) treatment of casein on its digestion in vivo and in vitro

AbstractThe effect of methanal (HCHO) treatment of casein on its in vitro and in vivo digestion was investigated. As the quantity of HCHO bound to casein increased, the in vitro rumen degradability decreased but no further reduction occurred above 10.6 g HCHO kg−1 treated casein DM. The proportion of the original HCHO (25.4 g HCHO kg−1 treated casein DM) remaining bound to casein at pH values 3.0 to 6.0 was approximately 0.5; the proportion remaining bound at pH 2.0 was significantly less (0.39). N solubility and in vitro digestion of casein by pepsin/HCl and/or intestinal proteolytic enzymes was significantly reduced by treatment with HCHO (10.6 g HCHO kg−1 treated casein DM). When fed to rats the dry matter (DM) and true N digestibilities decreased significantly as the ratio HCHO;casein increased; the digestibility of the N‐free component of the diet remained unchanged. The quantity of available lysine in the diets and their true biological value (BV) decreased as the level of bound HCHO increased; there was significant linear relationship between BV and available lysine. The apparent disappearances of amino acids from the small intestine of sheep, when untreated or HCHO treated caseins (12.3 g HCHO kg−1 DM) were infused into the duodenum, were not affected by the pH of the infusate (pH 2.5 or 6.8) but were significantly reduced by HCHO treatment. It is concluded that exposure of HCHO‐treated casein to low pH values does not completely release bound HCHO from casein and that the intestinal digestion of casein to which HCHO remains bound is reduced when compared to untreated casein. All amino acids determined showed similarly reduced apparent intestinal absorptions due to HCHO treatment. It is suggested that the lower BV of HCHO‐treated caseins was due to reduced metabolic availability of lysine.

Effects of formaldehyde-treated and untreated casein supplements on performance of dairy cows offered ryegrass-clover pasture

Abstract Four pairs of monozygous twin cows (Friesian and Friesian × Jersey) were offered freshly harvested ryegrass-white clover pasture ad libitum from 3 to 7 weeks after calving. A 1 kg casein supplement was mixed with the pasture daily. One member of each pair received untreated casein and the other received casein protected against rumen degradation by formaldehyde treatment. There were no significant (P > 0.05) differences between dietary groups in intakes of digestible dry matter, digestible nitrogen, or yields of milk, milkfat, and milk protein. Nitrogen balance data were similar for both diets. Mean plasma concentrations of urea, insulin, and growth hormone did not differ significantly between the 2 groups. These results indicate that the supply of amino acids reaching the small intestine from a high quality pasture diet was unlikely to limit milk synthesis.

Influence of diet on postweaning malabsorption and diarrhoea in the pig

Five-day-old pigs challenged with 10(3) pathogenic Escherichia coli (nalidixic acid resistant) showed no clinical signs of disease until subsequently weaned at three weeks. Dietary manipulation was shown to influence xylose malabsorption, diarrhoea and bacterial proliferation after weaning. Brief, but not continuous, contact with the diet before weaning markedly increased the severity of subsequent disease after weaning. Immunogenicity of the weaning diet was critical for the development of the disease. Two diets, identical except that in one the protein source (casein) had previously been enzymatically hydrolysed, were compared. Pigs fed the predigested diet showed no clinical signs of post weaning diarrhoea whereas those fed the untreated casein all developed diarrhoea.

Administration of lysinoalanine-containing proteins to rats and the characterization of their small intestinal contents.

Gluten and casein, 100 mg each, were dissolved in 4 ml of 0.2Ν NaOH and treated at 70°C for 1 hr. The amounts of lysinoalanine (LAL) formed were 4.0 μmol in gluten and 12.8 μmol in casein. Each of the diets prepared with the LAL-containing proteins, i.e., LAL gluten and LAL casein, was administered to rats by meal feeding. The rats of the LAL gluten diet group and those of the LAL casein diet group were sacrificed at 4 hr and 2 hr, respectively, after the start of the meal feeding and their small intestinal contents investigated. LAL gluten was found less digestible than the untreated gluten used as a control. A similar result was obtained when LAL casein and untreated casein were compared for digestibility. Both in the LAL gluten diet group and in the LAL casein diet group, the small intestinal LAL was located in a peptide fraction with a molecular weight of 1000∼3000 daltons; no free LAL was detected in the small intestinal contents in either case. These rults indicate that gluten and casein treated wit...

Formaldehyde-treated proteins for dairy cows--effects on blood hormone concentrations.

Seven Friesian heifers in mid-lactation were used in an experiment to measure the effects of including casein, formaldehyde-treated casein or formaldehyde-treated soya-bean meal in the diet on performance and on the concentrations of certain metabolites and hormones in the blood. 2. Milk yield and the concentration of fat, protein and lactose in milk were not affected by the treatments. 3. The concentration of growth hormone (GH) in jugular venous blood was higher with both formaldehyde-treated proteins than it was with untreated casein (P less than 0.05). Diet had no effect on insulin, prolactin or thyroxine concentrations in blood. 4. Urea concentration tended to be higher and non-esterified fatty acids lower in blood from heifers offered the diet containing untreated casein, but these differences were not statistically significant. Blood glucose concentration was not affected by dietary treatment. 5. It was concluded that blood GH concentration can be increased by offering protein supplements which will increase total amino acid supply to the intestines in lactating cattle. This appears to be a direct effect on GH status independent of effects on milk yield.

Effect of different levels of casein, with or without formaldehyde treatment, on carbohydrate metabolism between mouth and duodenum of steers.

AbstractSteers, fitted with simple rumen and duodenal cannulas, were given a basal diet of approximately equal parts of straw and flaked maize [containing 12 g N kg−1 dry matter (DM)] or similar diets in which part of the flaked maize was replaced by different amounts of untreated (UT) casein or formaldehyde‐treated (FT) casein to give low (L), medium (M) or high (H) intakes of nitrogen (19, 26 and 34 g N kg−1 DM, respectively). Rumen bacteria, separated from samples of rumen digesta taken about 4 h after giving a feed of the basal diet, contained approximately 160 g α‐dextran‐glucose (‘starch’) and 300 g crude protein (CP) kg−1 DM. Bacteria from steers receiving the highest level of FT or UT, contained approximately 140 and 70 g ‘starch’ and 320 and 580 g CP kg−1 DM, respectively. It was calculated that bacteria contributed approximately 65, 50 and 30 g ‘starch’ day−1 at the duodenum of steers receiving basal, FT casein and UT casein diets, respectively. Of the total carbohydrates entering the small intestine, all the rhamnose and ribose and approximately 50–65% of the mannose, galactose and ‘starch’ were contributed by the bacteria, the remainder being dietary in origin. Virtually all the xylose, arabinose and cellulose‐glucose were contributed by surviving dietary materials. For steers receiving the basal ration, mean coefficients of digestibility between mouth and duodenum (corrected for bacterial contribution) were 0.91, 0.97, 0.79, 0.43, 0.22 and 0.41 for mannose, starch‐glucose, galactose, arabinose, xylose and cellulose‐glucose, respectively. Coefficients of digestibility for diets supplemented with FT caseins were not appreciably different from those obtained with basal diets, but for diets supplemented with UT caseins those of arabinose, xylose and cellulose‐glucose were significantly increased to 0.66, 0.55 and 0.56, respectively.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis of Purified Casein Fractions Treated with Milk-Clotting Enzymes

Acid casein was fractionated into αs-, β-, and κ-casein and was treated separately with Mucor miehei and Endothia parasitica enzymes and calf chymosin. The enzyme treated casein fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the electrophoretic patterns were compared with those of untreated casein fractions. All three enzymes showed proteolytic activity, but Endothia parasitica enzyme degraded αs-casein most.

Interactions between dietary protein, ovulation rate and follicle stimulating hormone level in the ewe

Ovulation rate (OR) was studied in two experiments using mature Border Leicester × Merino ewes in which oestrous cycles were synchronized using a prostaglandin analogue. In both experiments a basal ration of 500 g of lucerne/barley pellets was provided. In the first experiment, ewes were fed individually iso-energy supplements of 500 g of either peas, lupins, soybean pellets or lucerne/barley pellets. The ovulation studied at laparoscopy occurred approximately 34 days after starting the supplementary feeding. Ewes fed lupins or soybean pellets had higher ( P < 0.05) OR's than the ewes fed the other diets. In the second experiment, ewes were fed either iso-protein supplements of peas or lupins or casein supplement (170 or 100 g of protein) either formalin treated or untreated. The ovulations studied at laparoscopy occurred approximately either 17 or 34 days after the first feeding of the supplement. Follicle stimulating hormone (FSH) levels in plasma were measured over the 8 days prior to the second ovulation. There were no differences ( P < 0.05) in OR's at the first ovulation. However, by the second, ewes fed peas had the highest ( P < 0.05) OR while those fed lupins or protected casein had similar OR's. These tended to be higher than in the ewes fed untreated casein. FSH levels were generally higher from 8 days to 3 days prior to ovulation in ewes which were to have twin ovulations compared to those having single ovulations. The results confirm that feeding high energy or high protein will increase OR. There are independent effects of energy and protein. The results suggested that the ovulation rate may be related to FSH levels.

Effect of protected casein supplements on pasture intake, milk yield and composition of cows in early lactation

Thirty dairy cows in early lactation were individually fed in stalls on high quality pasture (Lolium peuenne, Dactylis glomerata and Trifoliium repens) and given either formaldehyde-treated casein or untreated casein at 1000 g/day. Nitrogen content and apparent digestibility of herbage nitrogen was 2.8 and 70.4% respectively. Treated casein significantly increased the yield of milk by 13 % and milk protein by 15 % although neither supplement affected milk composition. High-producing cows showed a greater response to formaldehyde-treated casein, with increases in milk yield of 0.5 �0.14 kg per kg increase in level of milk production. Increases in milk synthesis were associated with increased efficiency in utilization of nutrients and not with changes in pasture intake. The results support the hypothesis that formaldehyde-treated casein provided more protein for duodenal digestion and thereby increased the supply of essential amino acids which were limiting milk production. It is concluded that milk synthesis in cows fed solely on high quality pasture in early lactation is limited by the amount of protein absorbed post-ruminally.

Protein nutrition of growing lambs. 1. Responses in growth and rumen function to supplementation of a low-protein-cellulosic diet with either urea, casein or formaldehyde-treated casein.

1. The effects of supplementation of a cellulose-based diet with either urea, casein or formaldehydetreated (HCHO)-casein were studied in growing lambs. Responses were measured in terms of growth rate, food intake and food conversion ratio.2. In Expt 1 lambs were given free access to a basal diet containing (g/kg) oat-hulls (700) and solka floc (300) (containing 5 g nitrogen/kg dry matter (DM)) supplemented (g/kg basal diet) with either urea (25), untreated casein (75), HCHO-casein (75) or combinations of these. Food intake was increased on average by 27% above that on the basal diet by the addition of either urea, casein, HCHO-casein plus urea. Urea plus HCHO-casein when given as a combined supplement further increased food intake on average by 60% above that on the basal diet. Supplements of either urea, casein, HCHO-casein or casein or casein plus urea changed a mean live-weight loss of 40 g/d on the basal diet to a mean live-weight gain of 56 g/d. Urea plus HCHO-casein further increased lamb growth to 112 g/d.3. In Expt 2 lambs were given free access to the basal diet (plus 25 g urea/kg diet) used in Expt I. In this experiment the content of insoluble and soluble casein in the diets was varied by the addition of HCHO-casein and untreated casein of 0, 150; 50, 100; 100, 50 and 150, o g/kg basal diet respectively. Maximum lamb growth (141 g/d) was obtained with a supplement of 25 g urea plus 100 g HCHO-casein and 50 g casein/kg.4. The growth responses to these supplements suggest a requirement for soluble N by the rumen microorganisms to maximize rumen fermentation, and for maximum growth rate on this diet a further requirement for amino acids produced by protein which has escaped degradation in the rumen.5. Fermentation and the absorption of nutrients were examined in Expt 3 in lambs fitted with simple ‘T’-shaped cannula in the duodenum and ileum, and fed ad lib. one of the diets: a basal diet of oat hulls and solka floc, or the basal diet supplemented (g/kg) with either urea (25), urea plus casein (150), or urea (25) plus HCHO-casein (150). The rates of production of volatile fatty acids (VFA), methane and microbial cells were measured using isotope-dilution techniques. The apparent absorption of nutrients was determined by differences in the quantity of those nutrients in digesta at the duodenum and ileum.6. Supplements of urea, urea plus casein and urea plus HCHO-casein increased organic matter (OM) intake in lambs by 65% above that on the basal diet. OM digestibility was unchanged by the from of nitrogen supplementation. The rates of production of all fermentation end-products varied directly with voluntary food intake.7. Rumen methane production remained constant at 0.09 mol methane/MJ metabolizable energy (ME) intake on all diets, which represented an 11% loss of digestible energy (DE). Hindgut methane production was highest on the urea-supplemented diet.8. The rate of VFA production (mol/MJ ME intake) in the rumen was highest on the diet supplemented with urea in comparison with the basal, urea plus casein and urea plus HCHO-casein diets (which were not significantly different). The molar proportions of the individual VFA in rumen fluid were not significantly different between diets except for the branched chain and higher fatty acids which were highest in proportion with the urea plus casein diet.9. The loss of energy in the faeces, urine or as methane in expired air was not influenced by the form of N supplementation. DE and ME were greater on the supplemented diets, as a result of the increased OM intake of these diets.10. There was no effect of the form of N supplementation on OM digested in the rumen, small intestine or large intestine. Of an increase in OM intake, apparently 55% was digested in the rumen (of which 19% was incorporated into rumen micro-organisms) and 26% disappeared in the small intestines. The apparent digestibility of OM for all diets was 0.67.

IN VITRO DIGESTIBILITY OF SUCCINYLATED PROTEIN BY PEPSIN AND PANCREATIC PROTEASES

ABSTRACTSuccinylated and untreated casein were hydrolyzed with pepsin and pancreatin. Neither Nɛ‐succinyllysine nor lysine was detected in the hydrolysate of succinylated casein, although the formation of lysine from untreated casein was detected. The composition of other amino acids from the hydrolysate was similar to that found in the untreated casein. The succinylation of casein suppressed tryptic hydrolysis, but had no influence on chymotryptic hydrolysis. Nα‐tosyl‐lysine‐methylester and its Nɛ‐succinylated derivative inhibited the activity of chymotrypsin to the same extent. Neither carboxy‐peptidases A nor B released Nɛ‐succinyl‐lysine from t‐butyloxycarbonyl‐glycyl‐Nɛ‐succinyl‐lysine. These results reveal that the lysyl bonds of succinylated proteins are not hydrolyzed by proteases present in digestive glands.