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PubMed1995

Distribution of activity of hydrolytic enzymes in the digestive tract of rabbits.

Activities of twelve hydrolytic enzymes in the digestive tract of young rabbits before weaning (4 weeks old) and adult rabbits (3 months old) were measured. The principal digestive enzymes in both groups of rabbits appeared to be amylase (EC 3.2.1.1), maltase (EC 3.2.1.20), pectinase (EC 3.2.1.15) and proteinases. The stomach of young rabbits contained most of the lipolytic activity and 45.7% of the total proteolytic activity of the digestive tract. The highest specific activities (per g digesta) of amylase, maltase and proteinase in young rabbits were found in the small intestine. Total activities (per segment) of amylase and maltase in the small intestine and the caecum were similar. Activities of cellulase (EC 3.2.1.4), inulinase (EC 3.2.1.7) and beta-glucosidase (EC 3.2.1.21) were low and activity of pectinase was fairly high in all segments of the digestive tract. The highest activity of urease (EC 3.5.1.5) was found in the caecum. Enzymic profiles of the colonic chymus resembled those of the caecum. Total hydrolytic activity was lower in the colon than in the caecum. Specific activities of amylase and invertase (EC 3.2.1.26) were lower and those of inulinase and lactase (EC 3.2.1.23) higher in 4-week-old rabbits than in 3-month-old rabbits. Gastric proteinase represented almost half of the total proteolytic activity of the digestive tract, whereas lipolytic activity of gastric contents was not found in measurable quantities in adult rabbits. The caecal contents of adult rabbits contained most of the total activity of lipase (EC 3.1.1.3), cellulase, xylanase (EC 3.2.1.32), pectinase, lactase, invertase, beta-glucosidase and urease present in the digestive tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed2005

Protein profile of pepsin-digested carious and sound human dentine.

The purpose of this study was to describe the protein profile of pepsin-digested carious and sound dentine using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Carious and sound dentine powder was decalcified using 10% EDTA at pH 7.4 for 48 h. The decalcified pellet was digested using pepsin at pH 2 under sequenced conditions: at 4 degrees C for 24 h, a further 24 h at 23 degrees C, and finally for 24 h at 37 degrees C. After every step, the soluble fraction was separated by centrifugation and analyzed in 15% SDS-PAGE. Two bands at 56 and 62 kDa could be observed in carious dentine digests and were considered specific carious bands. Similar bands could be observed in sound dentine samples, but only after pepsin digestion at higher temperatures (23 degrees C and 37 degrees C). Pepsin digests non-helical collagen and the triple helix structure of collagen is lost when the temperature rises. The bands at 56 and 62 kDa in sound dentine specimens thus represent pepsin-cleaved collagen. There is a possibility that the specific carious bands in carious dentine represent collagen decomposed in a manner similar to the way pepsin digests native dentine collagen at 23 degrees C and 37 degrees C.

Collagen↗
PubMed2002

Determination of the digestible energy intake and apparent absorption of macroelements in pasture-fed lactating Thoroughbred mares.

AIM: To measure the nutritive value of pasture in terms of digestible energy (DE) intake (DEI) and dry matter (DM) digestibility, and to determine the apparent absorption of macroelements in lactating Thoroughbred mares grazed on pasture. METHODS: DM intake (DMI) and DEI were determined from daily faecal DM output measured in grazing mares, divided by the DM indigestible fraction (1-digestible DM), measured in a digestibility trial using pasture-fed mares. Eight lactating mares and their foals, that had a mean age of 40 days, were grazed separately on 50x100 m areas of pasture and daily faecal DM outputs were recorded for 8 days. Five mares and their foals were then placed in individual bare 20x20 m corrals containing custom-made feeding stations for 14 days to determine the indigestible DM fraction. DM, gross energy content, crude protein (CP), soluble carbohydrate, acid detergent fibre (ADF), neutral detergent fibre (NDF), lipid, and macroelement composition of the pasture offered and faeces were determined and their digestibility and/or apparent absorption calculated. RESULTS: DM digestibility of the pasture was 0.6 and the DMI and DEI of a grazing 560 kg mare in early lactation nursing a foal growing at 1.34 kg/day was 13.6 (SE 0.8) kg/day and 146.9 (SE 8.4) MJ DE/day, respectively. Apparent absorptions of the macroelements measured were: Ca 0.75, P 0.43, Mg 0.63, Na 0.78, and K 0.72. CONCLUSIONS: Good quality ryegrass-white clover pasture that had a DE content of 10.8 MJ/kg DM, and a macroelement composition (g/kg) of Ca 3.33, P 3.0, Mg 1.67, Na 1.67, and K 24.2, will provide adequate DMI, DEI, and macroelement intake to lactating Thoroughbred mares.

Journal Article↗
PubMed2003

Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells.

We investigated whether different protocols for the digestion of adult human articular cartilage influence the cell yield and capacity to attach and proliferate in culture dishes. Chondrocyte yields were expressed as a percentage of the total number of cells in the tissue, determined both histologically (using the dissector method) and biochemically (measuring the DNA content of tissue digests). Human cartilage specimens (n = 79) were digested using different protocols based on combinations of collagenase II (CGN), trypsin/EDTA, hyaluronidase, and tosyllysylchloromethane (TLCM). Yields of viable chondrocytes were the highest within a specific range of CGN concentrations and digestion times, but always < 22% of the total available cells. The combination of CGN with trypsin/EDTA or TLCM accelerated the digestion process but did not significantly increase cell yields. The percentage of viable cells that attached to culture dishes ranged 75-85% (< 19% of the total) and was reduced by TLCM. Doubling times of attached cells were comparable in all experimental groups. Our results indicate that chondrocyte yields and capacity to attach and proliferate are not highly sensitive to the specific isolation protocol used. However, typically used cartilage digestion protocols yield only a small fraction of the total available cells, possibly introducing an uncontrolled selection of certain chondrocyte subpopulations.

Adult↗
PubMed2003

Effects of temperature on post-methanation of digested dairy cow manure in a farm-scale biogas production system.

A post-methanation process that could be adopted at farm-scale, operating at temperatures prevailing in farm manure digester post-storage tanks, was evaluated. Digested manure samples from a farm digester (35 degrees C) and post-storage tank (5-10 degrees C) were incubated in parallel batches at 5-20 degrees C and as reference at 35 and 55 degrees C. Specific methane yields (kg(-1) volatile solids (VS)(added waste)) were 0.20-0.26 m3 at 35-55 degrees C and 0.085-0.09 m3 at 10-20 degrees C for digester material (345 days of incubation) and 0.16-0.21 m3 at 35-55 degrees C, 0.053-0.087 kg(-1) VS(added waste) m3 at 15-20 degrees C and 0.026 m3 at 10 degrees C for post-storage tank material (250 days). Both materials produced less than 0.005 m3 at 5 degrees C. However, an increase in temperature to 35 degrees C (40 days) improved methane production in assays pre-incubated at 5-20 degrees C (9 months). These results suggest that the untapped methane potential of the digested manure cannot effectively be recovered at temperatures prevailing in farm digested manure storage tanks during the winter in Northern latitudes. Nevertheless, as ambient temperatures increase during the late spring, an increase in methanogenesis can be expected.

Animals↗
PubMed1976

[The effect of various pH values on in vitro peptic digestion of proteins in the presence of Cu2 ions].

Studies were carried out to investigate the effect of different pH values on the peptic digestion of soya protein in the presence and in the absence of Cu2+ ions. The studies were performed in vitro at a pH of 2.2 and 3.2, with 2.96 X 10(-5) mole of Cu2+ ions present in 11 of the reaction mixture. The reaction was carried out in a digestion apparatus permitting dialysis of the cleavage products. Different parameters were used as criteria of digestion, viz. the quantity of N contained in the reaction vessel (residue) and in the resulting dialysis products as determined by Kjehldahl microanalysis and automatic amino acid analysis, the proportions of digestion products found in the different molecular ranges after partition of Sephadex G 75 and the composition of amino acids in the cleavage products. From the distribution of the reaction products on the residue and dialysis products and on the different molecular ranges it was found that additions of Cu2+ ions at pH 2.2 produced a considerable inhibition of digestion. With a rise in pH to 3.2 peptic digestion decreased even without the addition of Cu2+. Supplementation of Cu2+ ions produced only a slight additional effect in the molecular range termed "exclusion limit". In the case of the amino acids tyrosine and phenylalanine it was found that an increase in pH changed the composition of peptides within the different molecular ranges. Additions of Cu2+ had no influence on the amino acid composition.

Copper↗
PubMed1950

Crystalline desoxyribonuclease; digestion of thymus nucleic acid; the kinetics of the reaction.

A study was made of the enzymatic properties of crystalline desoxyribonuclease. The general effect of the crystalline enzyme on its specific substrate, thymus nucleic acid, was found to be essentially the same as described by previous workers for the digestive action of crude preparations of the enzyme. The digestive action consists mainly in splitting thymus nucleic acid into fragments approaching the size of tetranucleotides. The digested nucleic acid is diffusible through collodion or cellophane membranes and is non-precipitable with strong acid, alcohol, or proteins. The digestion of thymus nucleic acid by desoxyribonuclease is accompanied by the liberation of one atom equivalent of free acid per four atoms of nucleic acid phosphorus. Crystalline desoxyribonuclease acts very slowly, if at all, in the absence of magnesium (or manganese) ions. The optimal concentration of magnesium ion required increases with the increase in concentration of the substrate but is independent of the enzyme concentration. The optimal pH range for the action of crystalline desoxyribonuclease is 6.0 to 7.0. A study was made of the kinetics of the digestion of thymus nucleic acid as manifested mainly by the gradual formation of acid-soluble split products. At low concentrations of nucleic acid, the process approximates closely a reaction of the first order, the unimolecular constant being independent of the concentration of desoxyribonuclease in the digestion mixture. At relatively higher concentrations of substrate, however, the initial rate of reaction decreases rapidly with the increase in concentration of substrate, and the reaction as a whole is represented by non-symmetric S-shaped curves apparently too complicated for a simple rational interpretation.

Deoxyribonucleases↗
PubMedDate not supplied

Fiber digestion and energy utilization of fat sand rats (Psammomys obesus) consuming the chenopod Anabasis articulata.

Most herbivorous rodents consume a variety of plants and, when available, select ones low in fiber content. In contrast, the fat sand rat (Psammomys obesus), a diurnal gerbillid rodent that is wholly herbivorous, is able to survive while consuming only the halophytic chenopod Anabasis articulata. We hypothesized that these gerbils are able to digest chenopods efficiently and that fiber digestion contributes substantially to their energy budget. Digestibility of total fibers (NDF), hemicellulose, and cellulose was 51.6%, 66.4%, and 44.0%, respectively. Energy derived from fiber digestion was 211.7 kJ kg(-0.75) d(-1) and resulted mostly from hemicellulose digestion. Average daily metabolic rate was 658.4 kJ kg(-0.75) d(-1), and basal metabolic rate (BMR) was determined to be close to 192.6 kJ kg(-0.75) d(-1). Therefore, fiber digestion provided 32% of maintenance requirements and 110% of BMR requirements, one of the highest values reported thus far for placental mammals. The efficiency of utilization of the chenopod was 0.29, a low value compared to other dietary items. We concluded that A. articulata fulfills all the energy and nutrient requirements of fat sand rats, even though energy yields and efficiency of utilization of its energy is low.

Adaptation, Physiological↗
PubMed1997

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.

DNA↗
PubMed2003

Methods to determine metabolizable energy and digestibility of feed ingredients in the domestic pigeon (Columba livia domestica).

The influence of length of excreta collection period (1, 3, 6, 10, 14 d) and prefeeding protocol (7 d either individual feeding in collection cages or group feeding in housing cages) on AMEn, nitrogen retention (NR), and apparent DM, organic matter and ether extract digestibility of corn and peas were evaluated in domestic pigeons (Columba livia domestica). In addition, the use of internal markers [acid-insoluble ash (AIA) and acid detergent lignin (ADL)] to determine AMEn, NR, and apparent digestibility was compared with the method of measuring total feed input and excreta output. A quadratic (y = a + bx + cx2) trend in the CV for AMEn, NR, and apparent digestibility coefficients found over collection periods with corn presented evidence that excreta collection for a period of 3 d will produce a CV of 5% less than the minimum CV. Although no trend could be detected in CV for peas, a 3-d excreta collection period resulted in relatively low variation. Both AIA and ADL, when used as internal markers, resulted in AMEn, NR, and digestibility values below (P < 0.05) those obtained with total collection with corn. However, values between markers were comparable (P > 0.05) for all components evaluated. The ADL was unsuccessful as marker with peas. Group prefeeding of pigeons in housing cages resulted in lower feed intake, excreta output, NR, and apparent digestibility than when birds were adapted individually to collection cages. This study presents evidence that the method of measuring total feed intake and excreta output for a period of 3 d, with individual adaptation of birds to collection cages, resulted in the most reliable values for AMEn, NR, and apparent digestibility of DM, organic matter and ether extract of feed ingredients in pigeons.

Animal Feed↗
PubMed1990

A simple method for the release of islets by controlled collagenase digestion of the human pancreas.

A simple technique for the controlled collagenase digestion of the human pancreas is described. The pancreas is distended with collagenase, and a biopsy taken and divided into 5 pieces that are placed in Universals containing minimal essential medium and dithizone at 39 degrees C. The pancreas itself is incubated in MEM at 39 degrees C. Starting at 5 min and at intervals thereafter, a Universal is removed from the water bath, shaken for 30 sec, and the contents examined by microscopy. As soon as free cleaved islets are seen, the pancreas is placed into one compartment of a kidney-bowl divided in half by a 1-mm mesh. The pancreas is gently teased apart and fluid digest in the empty half of the bowl aspirated and passed through a 500-micron mesh into ice-cold MEM containing 20% newborn calf serum. This process is repeated until the digestion process has ceased. Using this technique on 20 consecutive pancreata, median wt. (range) 53.9 (45.2-72.9) g, we have counted 131,672 (43,516-400,000) islets in the digest, equivalent to 2394 (715-8000) islets/g pancreas. The volume of islet tissue in the digest was 299 (26-1341) mm3 equivalent to 5.81 (0.36-26.81) mm3/g pancreas. In conclusion, we have found this simple technique to be an effective method for the controlled collagenase digestion of the human pancreas.

Adolescent↗
PubMed1993

Storage of porcine pancreatic digest prior to islet purification. The benefits of UW solution and the roles of its individual components.

The aim of this study was to establish the beneficial effect of storage of pancreatic digest in University of Wisconsin solution on porcine islet purification, the mechanism of this effect, and the components of UW responsible. Ten porcine pancreata were collagenase-digested, and samples of digest were washed and stored for 1 hr in either UW or minimum essential medium at 4 degrees C, prior to separation on continuous linear density gradients of bovine serum albumin. Samples of digest from a further ten pancreata were similarly treated, comparing storage in MEM, UW, and five solutions varying in lactobionate:chloride ratio and raffinose content. The purity of the islet preparations and the densities of islets and exocrine tissue were determined from insulin and amylase assay of aliquots aspirated from these gradients. Washing and storage of digest in UW markedly improved islet purity, compared with MEM, due to an increase in the density of exocrine tissue. Exocrine tissue density following storage was dependent upon the control of acinar cell volume, rather than exocrine enzyme discharge, and was determined primarily by the chloride:lactobionate ratio of the storage solution. Raffinose was of little additional benefit, while the beneficial effect of UW was greater than that due to its lactobionate and raffinose content alone. In conclusion, inadequate purification of islets results from exocrine tissue swelling. This swelling is reduced by storage of the pancreatic digest in UW solution, due primarily to the replacement of chloride by lactobionate in UW.

Adenosine↗
PubMed2001

Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% (18)O water.

Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of (18)O into peptides during peptic digestion of disulfide-linked proteins in 50% (18)O water resulted in isotope patterns and increases in average masses that facilitated identification and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% (16)O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be identified and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.

Amino Acid Sequence↗
PubMed1993

Survival of pathogenic bacteria during mesophilic anaerobic digestion of animal waste.

The survival of pathogenic bacteria was investigated during the operation of a full-scale anaerobic digester which was fed daily and operated at 28 degrees C. The digester had a mean hydraulic retention time of 24 d. The viable numbers of Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes and Campylobacter jejuni were reduced during mesophilic anaerobic digestion. Escherichia coli had the smallest mean viable numbers at each stage of the digestion process. Its mean T90 value was 76.9 d. Yersinia enterocolitica was the least resistant to the anaerobic digester environment; its mean T90 value was 18.2 d. Campylobacter jejuni was the most resistant bacterium; its mean T90 value was 438.6 d. Regression analysis showed that there were no direct relationships between the slurry input and performance of the digester and the decline of pathogen numbers during the 140 d experimental period.

Anaerobiosis↗
PubMed1993

The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria.

The decline in viable numbers of Salmonella typhimurium, Yersinia enterocolitica and Listeria monocytogene in beef cattle slurry is temperature-dependent; they decline more rapidly at 17 degrees C than at 4 degrees C. Mesophilic anaerobic digestion caused an initial rapid decline in the viable numbers of Escherichia coli, Salm. typhimurium, Y. enterocolitica and L. monocytogenes. This was followed by a period in which the viable numbers were not reduced by 90%. The T90 values of E. coli, Salm. typhimurium and Y. enterocolitica ranged from 0.7 to 0.9 d during batch digestion and 1.1 to 2.5 d during semi-continuous digestion. Listeria monocytogenes had a significantly higher mean T90 value during semi-continuous digestion (35.7 d) than batch digestion (12.3 d). Anaerobic digestion had little effect in reducing the viable numbers of Campylobacter jejuni.

Anaerobiosis↗
PubMed1985

Digestion of elastin by porcine pancreatic elastase I and elastase II.

Elastin was fully solubilized by digestion with elastase I or elastase II. Each digest was separated into high-molecular weight and low-molecular weight fractions that were characterized by the correspondence to their amino acid content, N-terminal sequence and C-terminal amino acids. It was found that although the relative amount of amino acids in the low-molecular weight fraction obtained by digestion with elastase I was lower than in digestion with elastase II, no major difference in the type of bonds cleaved in the low- or high-molecular weight fractions of each digest could be seen. There is, however, a remarkable difference in the type of bond cleaved by the two enzymes. While elastase I cleaves mostly Ala-Ala and also Ala-Gly bonds, elastase II hydrolyzes Leu-Ala, Leu-Gly, Phe-Ala, Phe-Gly and Tyr-Ala, Tyr-Gly bonds. Theoretical calculations led us to suggest both digests are composed of cross-linked peptides that vary not only in the molecular size but also in the number of cross-links found in peptides of the same size.

Amino Acid Sequence↗
PubMed1987

Difference in sporogenous bacterial populations in thermophilic (55 degrees C) and mesophilic (35 degrees C) anaerobic sewage digestion.

Spores, sporeforming vegetative cells, and asporogenous populations were enumerated in two semicontinuous anaerobic fermentors digesting municipal primary sludge at 35 and 55 degrees C for more than 87 days. In the 35 degrees C fermentor, the anaerobic total population was 312.5 X 10(6)/ml, with 25.0 X 10(6)/ml being sporogenous. The populations that digest casein, starch, pectin, and cellulose were 23.1 X 10(6), 59.2 X 10(6), 26.2 X 10(6), and 7.3 X 10(6)/ml, respectively, with 2.8 X 10(6), 6.7 X 10(6), 3.4 X 10(6), and 1.5 X 10(6)/ml being sporogenous, respectively. The sporeformers accounted for 8.0 to 20.0% of each of the respective populations. In the 55 degrees C fermentor, the anaerobic total population was 512.5 X 10(6)/ml, with 336.6 X 10(6)/ml being sporogenous. The populations that digest casein, starch, pectin, and cellulose were 97.7 X 10(6), 190.7 X 10(6), 75.8 X 10(6), and 11.2 X 10(6)/ml, respectively, with 47.8 X 10(6), 110.6 X 10(6), 43.3 X 10(6), and 5.1 X 10(6)/ml, respectively, being sporogenous. The sporeformers represented 45.5 to 65.7% of each of the respective populations. The numbers of thermophilic sporeforming vegetative cells in the 55 degrees C fermentor were 9.0 to 19.8 times higher than their counterparts in the 35 degrees C fermentor. Most sporeformers were in the vegetative state in the 35 and 55 degrees C fermentors. After 18 days of fermentation at 55 degrees C, sporeformers carried out most of the digestion; however, the digestion was shared by both sporeformers and asporogenous bacteria after 87 days of fermentation. In the 35 degrees C fermentor, asporogenous bacteria digested most of the sludge. During the 18- and 87-day experimental periods, sporeformers were never predominant.

Bacteria, Anaerobic↗
PubMed1988

Effect of anaerobic digestion on oocysts of the protozoan Eimeria tenella.

The effect of anaerobic digestion of poultry waste on oocysts of the protozoan Eimeria tenella, a common enteric pathogen that causes coccidiosis in poultry, was investigated in this study. Thermophilic (50 degrees C) and mesophilic (35 degrees C) anaerobic digestors, with poultry manure as the substrate, were inoculated with the oocysts. The oocysts were damaged during anaerobic digestion, as determined by morphological change and loss of their ability to sporulate. The recovered oocysts were tested for their infectivity in young chicks, as measured by body weight gain, mortality, and cecal lesions. Oocysts lost all their infectivity during thermophilic digestion, while oocysts subjected to mesophilic digestion remained moderately infective in comparison with untreated oocysts, which produced severe coccidiosis, high mortality, and low body weight gain in chicks. Oocysts were inactivated at 50 degrees C when they were suspended in digestor fluid or saline. Inactivation at 35 degrees C was significantly stronger in the digestor fluid than in the saline, which implied that factors other than temperature were involved in the lethal effect of anaerobic digestion on protozoan oocysts. In this study we demonstrated that the treatment of animal waste by anaerobic digestion, especially at a thermophilic temperature, has the benefits of pathogen control and protection of human and animal health in a farm environment.

Anaerobiosis↗