In vitro degradability of feed proteins in the rumen: use of non-rumen proteases

2005 ◽  
Vol 56 (8) ◽  
pp. 797 ◽  
Author(s):  
M. Aslam Mirza ◽  
E. L. Miller
Keyword(s):  
Correlation Coefficient ◽  
Soybean Meal ◽  
Streptomyces Griseus ◽  
Correlation Coefficients ◽  
In Vitro Assays ◽  
Vitro Assay ◽  
Bacterial Protease ◽  
Ruminal Protein Degradability

Various feed proteins were incubated independently with bacterial protease from Streptomyces griseus (SGP), papain (Corica papaya), and ficin (Ficus glabrata) in a simple laboratory assay to predict ruminal protein degradability. The estimates obtained from in vitro assays were compared with those obtained from an in situ analysis using synthetic fibre bags. The rate and extent of degradation in vitro using proteases from non-rumen sources differed among substrates used. A high correlation coefficient (r2 = 0.99) was observed between N-degradability from the in vitro method using SGP and in situ estimates when soybean meal was the substrate. Soybean meal nitrogen (N) was almost completely hydrolysed (0.99) in vitro. The correlation coefficients were low and variable with assays using other enzymes. The correlation coefficient was also high (r2 = 0.77–0.84) with in vitro methods using either SGP, papain, or ficin when incubated with fish meal. The N disappearance from barley in vitro was slow to moderate. The ‘b’ estimate of barley obtained with the in vitro assay was significantly (P < 0.01) lower than that observed in situ. Slower proteolysis observed in barley may possibly be linked to poor accessibility of structural proteins rather than the degradability of N per se. None of the enzymes could rank barley in the same order as the in situ method.

scholarly journals Estimation of Relationship Between In Situ and In Vitro Rumen Protein Degradability of Extruded Full Fat Soybean

2017 ◽  
Vol 5 (10) ◽  
pp. 1237
Author(s):  
Arzu Erol Tunç ◽  
Yusuf Cufadar ◽  
Sema Yaman
Keyword(s):  
Phosphate Buffer ◽  
Streptomyces Griseus ◽  
Correlation Coefficients ◽  
Regression Equations ◽  
Enzymatic Method ◽  
In Vitro Method ◽  
Vitro Protein ◽  
Protein Degradability

The objectives of this study were to estimate the protein degradability of extruded full fat soybean (ESB) by in situ (nylon bag) and in vitro enzymatic method and to develop an equation in order predict in situ degradability from in vitro values. In the study enzymatic technique; hydrolysis after 1 h (INV1) and after 24 h (INV24) by a purified protease extracted from Streptomyces griseus in a borate-phosphate buffer at pH 8 was used as in vitro method. Relationship between in situ effective protein degradability (INSE) and in vitro degradability after 1 and 24 hours incubations (INV1 and INV24) were determined. In situ protein degradability was measured at 0, 2, 4, 8, 16, 24, and 48 and at 72 h incubations in the rumen of 3 Holstein cows. In the study INSE, INV1 and INV24 were determined as 58.05, 20.24 and 41.46% respectively. Despite there were differences between in situ and in vitro protein degradability values, correlation coefficients between in situ and in vitro protein degradability of ESB were high and regression equations for estimation of in situ from in vitro were found significant. As conclusion in vitro enzymatic protein degradability (INV1 and INV24) can be used for estimation of in situ effective protein degradability of extruded full fat soybean.

Curcumin: Natural Antimicrobial and Anti Inflammatory Agent

2021 ◽  
pp. 1-8
Author(s):  
Pehlivanović Belma ◽  
Čaklovica Kenan ◽  
Lagumdžija Dina ◽  
Omerović Naida ◽  
Žiga Smajić Nermina ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Protein Denaturation ◽  
Dose Range ◽  
In Vitro Assays ◽  
Natural Antimicrobial ◽  
In Vivo Models ◽  
Vitro Assay ◽  
Inflammatory Agent ◽  
Anti Inflammatory

The pursuance of novel antimicrobial and anti-inflammatory agents has been expanding due to a significant need for more efficient pharmacotherapy of various infections and chronic diseases. During the last decade, pharmacokinetics, pharmacodynamics and pharmacological properties of curcumin have been extensively studied. The aim of the present study was to evaluate the antibacterial activity of curcumin against both Gram-positive and Gram-negative bacteria as well as its antifungal activity by using in vitro agar well diffusion assay. Moreover, the anti-inflammatory activity of curcumin was determined with in vitro assay of inhibition of protein denaturation. Results demonstrated wide antimicrobial activity of curcumin upon all of the test bacteria and fungi. The strongest activity of curcumin was observed at a concentration of 0.50 mg/ml against S. aureus, L. monocytogenes, E. coli, P. aeruginosa and C. albicans, resulting in a maximum zone of inhibition of 14.7 mm, 14.3 mm, 13.7 mm, 10.7 mm and 10.7 mm, respectively. Findings suggested that the antimicrobial activity of curcuminis dependent upon the concentrations. Furthermore, results demonstrated high effectiveness of curcumin compared to standard acetylsalicylic acid in inhibiting heat-induced protein denaturation, which activity is also depended upon the concentrations. The present study emphasises the potential application of curcumin as a natural antimicrobial and anti-inflammatory agent. However, findings of this study are restricted to in vitro assays and consideration should be given to conducting a study involving wider dose range test substances as well as including further research on in vivo models.

scholarly journals An In Vitro Assessment of Immunostimulatory Responses to Ten Model Innate Immune Response Modulating Impurities (IIRMIs) and Peptide Drug Product, Teriparatide

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7461
Author(s):  
Claire K. Holley ◽  
Edward Cedrone ◽  
Duncan Donohue ◽  
Barry W. Neun ◽  
Daniela Verthelyi ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune Responses ◽  
Innate Immune Response ◽  
Innate Immune ◽  
Cytokine Secretion ◽  
Poly I:C ◽  
In Vitro Assays ◽  
Innate Immune Responses ◽  
Vitro Assay

Understanding, predicting, and minimizing the immunogenicity of peptide-based therapeutics are of paramount importance for ensuring the safety and efficacy of these products. The so-called anti-drug antibodies (ADA) may have various clinical consequences, including but not limited to the alteration in the product’s distribution, biological activity, and clearance profiles. The immunogenicity of biotherapeutics can be influenced by immunostimulation triggered by the presence of innate immune response modulating impurities (IIRMIs) inadvertently introduced during the manufacturing process. Herein, we evaluate the applicability of several in vitro assays (i.e., complement activation, leukocyte proliferation, and cytokine secretion) for the screening of innate immune responses induced by ten common IIRMIs (Bacillus subtilis flagellin, FSL-1, zymosan, ODN2006, poly(I:C) HMW, poly(I:C) LMW, CLO75, MDP, ODN2216, and Escherichia coli O111:B4 LPS), and a model biotherapeutic Forteo™ (teriparatide). Our study identifies cytokine secretion from healthy human donor peripheral blood mononuclear cells (PBMC) as a sensitive method for the in vitro monitoring of innate immune responses to individual IIRMIs and teriparatide (TP). We identify signature cytokines, evaluate both broad and narrow multiplex cytokine panels, and discuss how the assay logistics influence the performance of this in vitro assay.

The ability of the ficin enzyme technique to predict the in situ degradability of lucerne N

1999 ◽  
Vol 1999 ◽  
pp. 156-156
Author(s):  
G. Gizzi ◽  
E.R. Deaville ◽  
D.I. Givens
Keyword(s):  
Protein Degradation ◽  
Microbial Population ◽  
Reasonable Accuracy ◽  
Complex Process ◽  
Ruminal Protein Degradability ◽  
Protein Degradability

The assessment of protein degradability in the rumen is a complex process. The infinite combination of interaction between the rumen microbial population and the nature of the protein fed to the animal makes the estimation of ruminal protein degradability very arduous. At present the in situ technique is the most popular means of predicting ruminal nitrogen (N) degradation. However this procedure is slow, expensive and relies on the use of numerous surgically prepared animals. A number of studies (Assoumani et al., 1992; Aufrère and Cartailler, 1988) have shown that the use of in vitro methods using proteases can predict with reasonable accuracy the extent of protein degradation. The objective of this experiment was to examine the possibility of replacing the in situ technique with an in vitro procedure based on the use of the ficin protease to predict the extent of N degradation.

scholarly journals Ruminal fermentation and digestion of cattle diets with total and partial replacement of soybean meal by a slow-release urea product

2019 ◽  
Vol 64 (No. 7) ◽  
pp. 294-301
Author(s):  
S Gonzalez-Munoz ◽  
J Sanchez ◽  
S Lopez-Aguirre ◽  
J Vicente ◽  
J Pinos-Rodriguez
Keyword(s):  
Soybean Meal ◽  
Degradation Rate ◽  
Slow Release ◽  
In Vitro Assay ◽  
Partial Replacement ◽  
Vitro Assay ◽  
Dietary Substitution ◽  
Slow Release Urea

One in vitro assay and one in vivo trial with ruminally cannulated Holstein steers were conducted to evaluate the effects of a dietary substitution of soybean meal by a urea and slow-release urea source of fermentation and degradation of diets for cattle. The experimental diets consisted of the total mixed rations defined as the control with soybean meal (SBM), U (urea), SRU (slow-release urea), and SRU+U+AA (0.42% + 0.42% + 1% amino acids methionine and lysine). The dietary substitution of SBM by U or SRU reduced (P &lt; 0.05) the total gas production (V), microbial mass and degradation at 72 h incubation under the in vitro conditions, as well as the degradation rate (c) and the total volatile fatty acids (VFA) in the rumen of the steers; however, when the dietary substitution of SBM was by U+SRU+AA, those values did not decrease. In the steers, the dietary substitution of SBM by U and SRU reduced the ruminal degradation rate and the total VFA, and increased the ammonia N, but when SBM was substituted by U+SRU+AA in the diets, these changes were not observed. No advantage of SRU over U was found. The dietary substitution of SBM by U, SRU, U+SRU+AA did not modify the molar proportion of the VFA in the rumen nor were there changes in the nutrient digestion or excretion. Both the in vitro assay and the in vivo trial indicated that replacing SBM with U or SRU increases the ruminal ammonia N concentrations and reduces the degradation rate in the rumen, although those undesirable findings were not found when the SBM was replaced by U+SRU+AA. Therefore, it is feasible to replace the SBM with a combination of urea, slow-release urea, lysine and methionine in the diet for the ruminants.

Influence of growing conditions on rumen escape protein and chemical composition in grass

2000 ◽  
Vol 2000 ◽  
pp. 56-56
Author(s):  
J.W. Cone ◽  
A.H. van Gelder ◽  
A.A. Kamman ◽  
V.A. Hindle
Keyword(s):  
Chemical Composition ◽  
Protein Quality ◽  
Streptomyces Griseus ◽  
Grass Silage ◽  
Growing Conditions

The amount of rumen escape protein is commonly determined with the nylon bag technique. However, there is also an in vitro technique described using a protease of Streptomyces griseus (Aufrère et al., 1991; Cone et al., 1996), allowing systematical analysis of protein quality in a large number of samples. The aim of this study was to identify the influences of growing conditions on content of rumen escape protein in grass and grass silage and to investigate the relationships between rumen escape protein determined in vitro and in situ and chemical composition.

scholarly journals Leucine-Rich Diet Modulates the Metabolomic and Proteomic Profile of Skeletal Muscle during Cancer Cachexia

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1880 ◽  
Author(s):  
Bread Cruz ◽  
André Oliveira ◽  
Lais Rosa Viana ◽  
Leisa Lopes-Aguiar ◽  
Rafael Canevarolo ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cancer Cachexia ◽  
Energy Generation ◽  
In Vitro Assays ◽  
Proteomic Profile ◽  
Adult Rats ◽  
Vitro Assay ◽  
Tumour Bearing

Background: Cancer-cachexia induces a variety of metabolic disorders, including skeletal muscle imbalance. Alternative therapy, as nutritional supplementation with leucine, shows a modulatory effect over tumour damage in vivo and in vitro. Method: Adult rats distributed into Control (C), Walker tumour-bearing (W), control fed a leucine-rich diet (L), and tumour-bearing fed a leucine-rich diet (WL) groups had the gastrocnemius muscle metabolomic and proteomic assays performed in parallel to in vitro assays. Results: W group presented an affected muscle metabolomic and proteomic profile mainly related to energy generation and carbohydrates catabolic processes, but leucine-supplemented group (WL) recovered the energy production. In vitro assay showed that cell proliferation, mitochondria number and oxygen consumption were higher under leucine effect than the tumour influence. Muscle proteomics results showed that the main affected cell component was mitochondria, leading to an impacted energy generation, including impairment in proteins of the tricarboxylic cycle and carbohydrates catabolic processes, which were modulated and improved by leucine treatment. Conclusion: In summary, we showed a beneficial effect of leucine upon mitochondria, providing information about the muscle glycolytic pathways used by this amino acid, where it can be associated with the preservation of morphometric parameters and consequent protection against the effects of cachexia.

scholarly journals Enterotoxins production, biofilm formation and antimicrobial resistance of Staphylococcus aureus strains isolated from refrigerated raw cow milk

2019 ◽  
Vol 42 ◽  
pp. e45231
Author(s):  
Camila Lampugnani ◽  
Maike Taís Maziero Montanhini ◽  
Maria Emilene Martino Campos‐Galvão ◽  
Luis Augusto Nero ◽  
Luciano dos Santos Bersot
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Resistance ◽  
Biofilm Formation ◽  
Raw Milk ◽  
Cow Milk ◽  
In Vitro Assays ◽  
Vitro Assay ◽  
Milk Samples ◽  
Adherence Ability

This study aimed to isolate Staphylococcus aureus in refrigerated raw cow milk, and identify the presence of enterotoxin-expression genes, enterotoxin production and adherence ability, and antimicrobial resistance potential of the isolated strains. Fifty raw milk samples obtained in different dairy farms were analyzed for S. aureus and evaluated in the isolates the presence of genes associated with the production of major staphylococcal enterotoxins and biofilm formation. In vitro assays were also performed to evaluate the production of enterotoxins and adherence ability, and the antimicrobial resistance. One half (25/50) of raw milk samples presented coagulase-positive staphylococci and 95.2% of the isolates were confirmed to be S. aureus. Among them, 42.4% were carrying genes for enterotoxins production; however, only one isolate was able to produce enterotoxins. All S. aureus isolates were carrying at least two genes associated with biofilm formation and 95.2% isolates was able to adhere upon the in vitro assay. All isolates demonstrated antimicrobial resistance potential to one or more of the tested antibiotics.

Assessment of Inhibition of Bovine Hepatic Cytochrome P450 by 43 Commercial Bovine Medicines Using a Combination of In Vitro Assays and Pharmacokinetic Data from the Literature

2020 ◽  
Vol 13 (2) ◽  
pp. 123-131
Author(s):  
Steven X. Hu ◽  
Chase A. Mazur ◽  
Kenneth L. Feenstra
Keyword(s):  
Liver Microsomes ◽  
In Vitro Assay ◽  
In Vitro Assays ◽  
Pharmacokinetic Studies ◽  
Pharmacokinetic Data ◽  
Vitro Assay ◽  
Administration Routes ◽  
Ic50 Values

Background: There has been a lack of information about the inhibition of bovine medicines on bovine hepatic CYP450 at their commercial doses and dosing routes. Objective: The aim of this work was to assess the inhibition of 43 bovine medicines on bovine hepatic CYP450 using a combination of in vitro assay and Cmax values from pharmacokinetic studies with their commercial doses and dosing routes in the literature. Methods: Those drugs were first evaluated through a single point inhibitory assay at 3 μM in bovine liver microsomes for six specific CYP450 metabolisms, phenacetin o-deethylation, coumarin 7- hydroxylation, tolbutamide 4-hydroxylation, bufuralol 1-hydroxylation, chlorzoxazone 6-hydroxylation and midazolam 1’-hydroxylation. When the inhibition was greater than 20% in the assay, IC50 values were then determined. The potential in vivo bovine hepatic CYP450 inhibition by those drugs was assessed using a combination of the IC50 values and in vivo Cmax values from pharmacokinetic studies at their commercial doses and administration routes in the literature. Results: Fifteen bovine medicines or metabolites showed in vitro inhibition on one or more bovine hepatic CYP450 metabolisms with different IC50 values. Desfuroylceftiour (active metabolite of ceftiofur), nitroxinil and flunixin have the potential to inhibit one of the bovine hepatic CYP450 isoforms in vivo at their commercial doses and administration routes. The rest of the bovine medicines had low risks of in vivo bovine hepatic CYP450 inhibition. Conclusion: This combination of in vitro assay and in vivo Cmax data provides a good approach to assess the inhibition of bovine medicines on bovine hepatic CYP450.

Potential of solubility, enzymatic methods and NIRS to predict in situ rumen escape protein

1997 ◽  
Vol 45 (2) ◽  
pp. 291-306 ◽  
Author(s):  
J.L. De Boever ◽  
B.G. Cottyn ◽  
J.M. Vanacker ◽  
C.V. Boucque
Keyword(s):  
Phosphate Buffer ◽  
Near Infrared ◽  
Streptomyces Griseus ◽  
Maize Silage ◽  
Near Infrared Reflectance ◽  
In Vitro Method ◽  
Rumen Degradation ◽  
The Difference

The percentage of feed protein escaping rumen degradation was measured by the in situ method (%EPsitu) for 29 compound feeds, untreated and formaldehyde-treated soyabean meal and 12 forages: 3 grass silages, 2 maize silages, fresh grass, grass hay, fodder beets, fresh potatoes, ensiled beet pulp, chopped ear-maize silage and brewers' grains. Loss of particles through bag pores was determined by the difference between the washable fraction (W) and the fraction soluble in borate-phosphate buffer at pH 6.7 (S). W - S was most pronounced for compound feeds (on average 14.4 percentage units), for brewers' grains and maize silages. A correction of %EPsitu, assuming that W - S degrades like the potentially degradable fraction, was not appropriate. Solubility in borate-phosphate buffer after 1 h, enzymic degradability by protease from Streptomyces griseus or ficin after 1, 6 and 24 h and near infrared reflectance spectroscopy (NIRS) (for compound feeds alone) were examined as a routine method to predict %EPsitu. With the buffer and S. griseus the effect of pH (6.7 vs. 8.0) and at pH 8.0 the effect of amount of substrate (500-mg sample vs. 20 mg N) were tested. With ficin, 500-mg samples were incubated at pH 6.7. Predictions were better when compound feeds and forages were considered separately. However, the best in vitro method was different for the 2 feed categories, being solubility in buffer for the compound feeds and enzymic degradation of a constant amount of protein with S. griseus at pH 8.0 for forages. NIRS showed potential to predict %EPsitu of compound feeds, but needs more reference samples. The Dutch feed tables appeared more accurate than the best in vitro method for compound feeds, but was too inaccurate for some forages like fodder beets, maize silage and ear-maize silage.

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