scholarly journals Stability of feed enzymes in physiological conditions assayed by in vitro methods

1993 ◽  
Vol 2 (2) ◽  
pp. 125-132
Author(s):  
Johan Inborr ◽  
Anne Grönlund
Keyword(s):  
Proteolytic Enzymes ◽  
Wheat Gluten ◽  
Xylanase Activity ◽  
Initial Value ◽  
Enzyme Preparations ◽  
Neutral Ph ◽  
Feed Enzymes ◽  
The Stability ◽  
Upper Gastrointestinal

A series of in vitro incubations were carried out to investigate the stability of two enzyme preparations in conditions similar to those in the upper gastrointestinal tract of monogastric animals. The two enzyme products, one crude xylanase from Trichoderma longibrachiatum (Multifekt K) and the other a specifically manufactured feed enzyme (Avizyme SX®), were subjected to incubations at low and neutral pH with and without proteolytic enzymes (pepsin and pancreatin). Wheat gluten was employed together with the crude xylanase to investigate its potential as a stabilising agent. Due to the buffering effect of Avizyme SX®, incubations were carried out with (pH 2.5) and without (pH 3.2) addition of either citric or hydrochloric acid. Incubation of the crude xylanase at low pH followed by incubation at neutral pH resulted in negligible loss of xylanase activity whereas β-xylosidase recovery fell to 57 per cent of the initial value (P

Screening of fibrolytic enzymes as additives for ruminant diets: relationship between enzyme activities and the in vitro degradation of enzyme-treated forages

2002 ◽  
Vol 2002 ◽  
pp. 210-210 ◽  
Author(s):  
D. Colombatto ◽  
D.P. Morgavi ◽  
A.F. Furtado ◽  
K.A. Beauchemin
Keyword(s):  
Enzyme Activities ◽  
Biochemical Properties ◽  
Ruminal Fermentation ◽  
Maize Silage ◽  
In Vitro Degradation ◽  
Enzyme Preparations ◽  
Commercial Enzyme ◽  
Ruminant Diets ◽  
Feed Enzymes

Results in the literature concerning the efficacy of feed enzymes for ruminant diets have been mixed. Commercial preparations currently used are fermentation extracts containing several enzymic activities. It has been suggested that ruminal fermentation of grass and maize silages is enzyme-limited (Wallace et al., 2001). In order to design better enzyme additives, the enzyme activities likely to affect the animal responses should be identified. This study examined 23 commercial enzyme preparations for their biochemical properties and their ability to influence the in vitro degradation of alfalfa and maize silage.

scholarly journals Supplementation of pig starter diets with carbohydrate-degrading enzymes - stability, activity and mode of action

1994 ◽  
Vol 3 (Supplement) ◽  
pp. 1-21
Author(s):  
Johan Inborr
Keyword(s):  
Broiler Chickens ◽  
Wheat Gluten ◽  
Live Weight ◽  
Gi Tract ◽  
In Vivo Models ◽  
Vitro Assay ◽  
Degrading Enzymes ◽  
The Stability

A total of five experiments were conducted to investigate the stability of feed enzymes to steam pelleting and the proteolytic conditions in the gastrointestinal (GI) tract of pigs and poultry, and to try and elucidate the mechanisms behind the improved performance of pigs fed enzyme-supplemented barley/wheat-based diets. The results of the pelleting stability experiment showed that the commercial feed enzyme employed maintained most of its activity in conditioning temperatures up to 85°C.Furthermore, it became evident that measuring enzyme recovery in pelleted feeds by in-vitro assay methods underestimated the actual activity. For this purpose in-vivo models such as that based on gut viscosity measurements in broiler chickens gives a more accurate estimate. Gut viscosity also correlated highly with live weight gain (r2=0.624) and feed utilisation (r2=0.616) of broiler chickens. The in-vitro incubations using conditions similar to those of the GI tract showed that enzymes are not readily denatured and inactivated in such conditions and indicated that wheat and wheat gluten, and possibly similar feed ingredients, may help to maintain the activity longer either due to their buffering capacity or by providing substrates for the enzymes. This was supported by the results of the in-vivo measurements. In the stomach of pigs, 10-20per cent of the xylanase and β-glucanase activities added to the diets could still be recovered 4 hours after feeding. In the ileum, proportionally more added enzyme activities were recovered between 4 and 6 than 0 and 2 hours after feeding. In broiler chickens fed an enzyme-supplemented barley-based diet, β-glucanase was fully recovered in the proximal part of the small intestine, giving further proof of the stability of the enzymes employed to the conditions of the GI tract. When a mixture of fibre- and starch-degrading enzymes were added to a diet based on wheat and barley, β-glucan, starch and dry matter digestibilities were significantly (P

scholarly journals The Presence of Chemical Cross-Linking Stabilises HIV-1 Envelope Glycoprotein Trimer Antigens in a Model of Intramuscular Immunisation

2021 ◽  
Author(s):  
Luiza Farache Trajano ◽  
Rebecca Moore ◽  
Quentin Sattentau
Keyword(s):  
Human Serum ◽  
Envelope Glycoprotein ◽  
Proteolytic Enzymes ◽  
Neutrophil Infiltration ◽  
Inhibitory Effect ◽  
Cross Linking ◽  
The Stability ◽  
Antibody Epitopes ◽  
Hiv 1

Background: The HIV-1 envelope glycoprotein (Env) is the target of antigen design for antibody- based vaccination. In 2019, four trimeric Env vaccines entered an experimental trial: ConM, ConS, and their cross-linked counterparts. The trimers were formulated with MPLA adjuvant. Studies have demonstrated that adjuvants trigger neutrophil infiltration. Neutrophils activate and degranulate releasing proteases, namely elastase and cathepsinG. Aims: To assess the stability and immunogenicity of these vaccines in the presence of adjuvant- recruited neutrophils and their proteolytic enzymes. Methods: Trimers were incubated with commercially-sourced proteases. To analyse stability, samples were reduced, denatured and separated using gel electrophoresis. To assess antibody binding, a trimer-protease incubation was followed by an ELISA. To establish more physiologically relevant conditions, harvested neutrophils were exposed to various adjuvants. The supernatant, shown to contain elastase, was incubated alongside the vaccines. The reducing and denaturing gels, as well as the ELISA, was repeated. Results: Gel analysis revealed that un-crosslinked trimers underwent significant digestion whereas cross-linking conferred enhanced stability. In the presence of neutrophil-sourced protease-containing-supernatant, trimers displayed resistance to digestion. The differential stability profile of Env trimers when exposed to commercially sourced compared to supernatant- derived proteases may be due to the inhibitory effect of human serum on elastase. Antibody epitopes were maintained in vitro. Conclusion: The vaccine antigens are sensitive to enzymatic degradation. This is reduced by cross-linking and human serum.

scholarly journals Proteolysis by Sourdough Lactic Acid Bacteria: Effects on Wheat Flour Protein Fractions and Gliadin Peptides Involved in Human Cereal Intolerance

2002 ◽  
Vol 68 (2) ◽  
pp. 623-633 ◽  
Author(s):  
Raffaella Di Cagno ◽  
Maria De Angelis ◽  
Paola Lavermicocca ◽  
Massimo De Vincenzi ◽  
Claudio Giovannini ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Protein Extraction ◽  
Proteolytic Enzymes ◽  
Lactobacillus Brevis ◽  
Enzyme Preparations ◽  
Wide Range ◽  
Gliadin Peptides ◽  
Hydrolysis Of

ABSTRACT Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination of K 562 (S) cells with respect to the whole 31-43 fragment of A-gliadin.

In vitro hatching of Hymenolepis diminuta eggs in Tenebrio molitor extracts and in defined enzyme preparations

Parasitology ◽  
1972 ◽  
Vol 64 (3) ◽  
pp. 389-400 ◽  
Author(s):  
R. C. Lethbridge
Keyword(s):  
Proteolytic Enzymes ◽  
Tenebrio Molitor ◽  
Defined Medium ◽  
Hymenolepis Diminuta ◽  
Enzyme Preparations

The results of several experiments that included variation of the pH and concentration of the extracts and analysis of the role of enzymes separated from midgut fluid by electrophoresis indicated that hatching involved two or more proteolytic enzymes that digested the cytoplasmic layer and embryophore. Amylases from T. molitor and other sources were shown to play no part in hatching. A defined medium based on analyses of midgut fluid and containing trypsin, chymotrypsin and peptidases was shown to digest the embryophore and effect the release of hexacanths in a time comparable to that obtained with T. molitor extracts. The specificity of the enzymes attacking the cytoplasmic layer remains unknown.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

The rod-to-coccoid body transformation in cultures of Helicobacter pylori

1990 ◽  
Vol 48 (3) ◽  
pp. 626-627
Author(s):  
A. R. Crooker ◽  
W. G. Kraft ◽  
T. L. Beard ◽  
M. C. Myers
Keyword(s):  
Helicobacter Pylori ◽  
Growth Cycle ◽  
Negative Bacterium ◽  
Body Formation ◽  
Coccoid Form ◽  
Spiral Form ◽  
Prolonged Culture ◽  
H Pylori ◽  
Upper Gastrointestinal

Helicobacter pylori is a microaerophilic, gram-negative bacterium found in the upper gastrointestinal tract of humans. There is strong evidence that H. pylori is important in the etiology of gastritis; the bacterium may also be a major predisposing cause of peptic ulceration. On the gastric mucosa, the organism exists as a spiral form with one to seven sheathed flagella at one (usually) or both poles. Short spirals were seen in the first successful culture of the organism in 1983. In 1984, Marshall and Warren reported a coccoid form in older cultures. Since that time, other workers have observed rod and coccal forms in vitro; coccoid forms predominate in cultures 3-7 days old. We sought to examine the growth cycle of H. pylori in prolonged culture and the mode of coccoid body formation.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

In-vitro- und In-vivo-Wirkung von Schilddrüsenhormon auf das Wachstum von Neuroblastomzellen

1990 ◽  
Vol 29 (03) ◽  
pp. 120-124
Author(s):  
R. P. Baum ◽  
E. Rohrbach ◽  
G. Hör ◽  
B. Kornhuber ◽  
E. Busse
Keyword(s):  
Cell Differentiation ◽  
Neuroblastoma Cells ◽  
Control Group ◽  
Initial Value ◽  
Initial Rise ◽  
Biphasic Effect ◽  
Contrast Microscopy ◽  
Morphological Sign

The effect of triiodothyronine (T3) on the differentiation of cultured neuroblastoma (NB) cells was studied after 9 days of treatment with a dose of 10-4 M/106 cells per day. Using phase contrast microscopy, 30-50% of NB cells showed formation of neurites as a morphological sign of cellular differentiation. The initial rise of the mitosis rate was followed by a plateau. Changes in cyclic nucleotide content, in the triphosphates and in the activity of the enzyme ornithine decarboxylase (ODC) were assessed in 2 human and 2 murine cell lines to serve as biochemical parameters of the cell differentiation induced by T3. Whereas the cAMP level increased significantly (3 to 7 fold compared with its initial value), the cGMP value dropped to 30 to 50% of that of the control group. ATP and GTP increased about 200%, the ODC showed a decrease of about 50%. The present studies show a biphasic effect of T3 on neuroblastoma cells: the initial rise of mitotic activity is followed by increased cell differentiation starting from day 4 of the treatment.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Sign in / Sign up

Export Citation Format

Share Document