PREPARATION OF PROTEASE FROM MIXED RUMEN MICROORGANISMS AND ITS USE FOR THE IN VITRO DETERMINATION OF THE DEGRADABILITY OF TRUE PROTEIN IN FEEDSTUFFS

1987 ◽  
Vol 67 (1) ◽  
pp. 55-64 ◽  
Author(s):  
S. MAHADEVAN ◽  
F. D. SAUER ◽  
J. D. ERFLE
Keyword(s):  
Proteolytic Activity ◽  
Rumen Fluid ◽  
Streptomyces Griseus ◽  
Potassium Phosphate ◽  
Canola Meal ◽  
Rumen Microorganisms ◽  
Degradation Rates ◽  
Feed Protein

Mixed rumen microorganisms present in bovine rumen fluid were extracted with butanol-acetone to provide a dry powder which retained 75–80% of the proteolytic activity of strained rumen fluid (SRF). Sixty percent of the proteolytic activity of the powder was extracted with water and concentrated on an Amicon XM-300 filter to give a protease preparation which had about 30% of the activity present in the SRF. The protease preparation was used for the determination of the rates of feed protein degradation in vitro by incubating at pH 6.8 in 0.1 M potassium phosphate buffer and measuring the rate of amino acid (and ammonia) production by the ninhydrin method. The relative degradation rates of the true proteins from feedstuffs tested were: soybean meal > fish meal > linseed meal and blood meal > canola meal > corn gluten meal. Substituting Streptomyces griseus protease for the rumen protease gave results which were very different from those obtained with the rumen enzyme. The advantages of using the rumen protease over other in vitro methods and the practical applicability of the procedure are discussed. Key words: Rumen, protease, preparation, in vitro, feed protein degradability

The evaluation of Acacia and other tree pods for goats: influence of rumen fluid source and polyethylene glycol addition on in vitro gas production

2002 ◽  
Vol 2002 ◽  
pp. 132-132 ◽  
Author(s):  
V. Mlambo ◽  
F. L. Mould ◽  
T. Smith ◽  
E. Owen ◽  
I. Mueller-Harvey
Keyword(s):  
Polyethylene Glycol ◽  
Prolonged Exposure ◽  
Rumen Fluid ◽  
Gas Production ◽  
Rumen Microorganisms ◽  
Protein Supplements ◽  
In Vitro Gas Production ◽  
Adaptive Mechanisms ◽  
Acacia Pods

After prolonged exposure to tanniniferous diets, it has been reported that some rumen microorganisms acquire defensive mechanisms against tannins (Brooker et al., 2000) or produce tannin-degrading enzymes. Such rumen microorganisms are said to be “tannin resistant” as their fermentation activity is less inhibited by the presence of tannins in the host’s diet. As acacia pods contain tannins their use as protein supplements for goats in the dry season may require that they be first detannified e.g. by using polyethylene glycol (PEG). However, goats with prior exposure to tanniniferous diets may have developed adaptive mechanisms to deal with tannins. This study, therefore, investigated the need for tannin inactivation in feeds given to ‘adapted’ animals by comparing the effect on the in vitro fermentation of tree pods incubated with and without PEG using rumen fluid from adapted and unadapted goats.

scholarly journals Rapid Determination of Lysine in Biological Samples by Isocratic Liquid Chromatography

1999 ◽  
Vol 82 (4) ◽  
pp. 809-813 ◽  
Author(s):  
Mamun M Or-Rashid ◽  
Ryoji Onodera ◽  
Shaila Wadud ◽  
Mohamed-Emad A Nasser ◽  
Mohammad R Amin
Keyword(s):  
Liquid Chromatography ◽  
Biological Samples ◽  
Limit Of Detection ◽  
Rapid Determination ◽  
Rumen Fluid ◽  
Uv Detector ◽  
Rumen Microorganisms ◽  
Microbial Nitrogen ◽  
Limit Of Quantitation

Abstract A simple, rapid, and sensitive method was developed for detection and quantitation of lysine (Lys) in various biological samples by isocratic liquid chromatography (LC). Samples containing Lys and other amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC-CI). The mobile phase used for isocratic elution was 50 mmol/L sodium acetate buffer (pH 4.20)-acetonitrile (43 + 57, v/v). Lys was detected with a UV detector at 265 nm. The derivatized Lys eluted from a LiChrospher 100 RP-18 (150× 4.0 mm id) column at a retention time of 5.6 min. The limit of detection was 0.73 μmol/L (signal-to-noise [S/N] ratio, 3:1), and the limit of quantitation was 2.37 μmol/L (S/N ratio, 10:1). Lys recoveries from fortified biological samples were >97.5%. Average Lys contents found in rumen fluid samples collected before the morning feeding and at 2.0,4.0, and 6.0 h after feeding were 4.26,3.34,3.58, and 3.82 μmol/L, respectively. The hydrolysate of a sample of mixed rumen microorganisms collected before the morning feeding was determined to contain 1.372 μmol/mg microbial nitrogen in the form of Lys. The Lys concentrations of human plasma, goat plasma, human urine, and goat urine were 140.0, 102.0,58.0, and 32.0 μmol/L, respectively.

scholarly journals TRYPTOPHAN UTILIZATION BY RUMEN MICROORGANISMS IN VITRO

1970 ◽  
Vol 50 (2) ◽  
pp. 331-335 ◽  
Author(s):  
ELIZABETH CANDLISH ◽  
T. J. DEVLIN ◽  
L. J. LaCROIX
Keyword(s):  
Benzene Ring ◽  
Continuous Flow ◽  
Rumen Fluid ◽  
Rumen Microorganisms

A continuous flow, artificial rumen apparatus was used to investigate the utilization of DL-tryptophan–14C(U)–benzene ring labelled material by rumen microorganisms. Half of the labelled material left the rumen sac in the first six hours. After a 24-hour period, less than 4% of the label was associated with either the protozoal or the bacterial fraction of the rumen fluid.

Study of the effects of PR toxin, mycophenolic acid and roquefortine C on in vitro gas production parameters and their stability in the rumen environment

2014 ◽  
Vol 153 (1) ◽  
pp. 163-176 ◽  
Author(s):  
A. GALLO ◽  
G. GIUBERTI ◽  
T. BERTUZZI ◽  
M. MOSCHINI ◽  
F. MASOERO
Keyword(s):  
Mycophenolic Acid ◽  
Rumen Fluid ◽  
Rumen Fermentation ◽  
Gas Production ◽  
Farm Animals ◽  
Marginal Effect ◽  
Rumen Microorganisms ◽  
Marginal Effects ◽  
Roquefortine C

SUMMARYMoulds belonging to Penicillium section roqueforti are common contaminants of feedstuffs and produce several mycotoxins that can cause health hazards when ingested by farm animals. Among these, PR toxin (PR), mycophenolic acid (MY) and roquefortine C (RC) have been frequently detected in forages, particularly silages. The aims of the current trials were to study the effects of the presence of pure mycotoxins on in vitro rumen fermentation parameters and to assess their stability in the rumen environment. Two successive in vitro gas production experiments were carried out: a central composite design with four replications of central point (CCD) and a completely randomized design with a fully factorial arrangement of treatments (FFD). In CCD, the effects of PR, MY and RC concentrations in diluted rumen fluid (i.e. 0·01, 0·30, 1·01, 1·71 and 2·00 μg of each mycotoxin/ml) were tested. Gas volume produced after 48 h of incubation (Vf) decreased linearly as concentrations of RC and MY in diluted rumen fluid increased, with marginal effects similar for two mycotoxins, being respectively −14·6 and −13·4 ml/g organic matter (OM) for each 1·0 μg/ml of increment in mycotoxin concentration. Similarly, total volatile fatty acid (VFA) production decreased quadratically as concentrations of RC and MY increased, with marginal effects about two times higher for MY than RC, being −4·22 and −2·62 mmol/l for each 1·0 μg/ml of increment in mycotoxin concentration. With respect to maximum Vf (i.e. 410·6 ml/g OM) and VFA (98·06 mmol/l) values estimated by the model, decreases of 13·6 and 15·2% were obtained when incubating the highest RC and MY concentrations, respectively. The PR did not interfere with rumen fermentation pattern and it was not recovered after 48 h of incubation, whereas the stabilities of MY and RC in rumen fluid were similar and on average equal to about 50%. On the basis of CCD results, a second experiment (FFD) was carried out in which only effects of MY and RC concentrations (i.e. 0, 0·67, 1·33 and 2·00 μg of each mycotoxin/ml of diluted rumen fluid) were tested. Data from FFD showed Vf decreased linearly when concentrations of MY and RC increased, with marginal effect two-folds higher for MY than for RC (−11·1 ml/g OM and −6·7 ml/g OM, respectively). Similar marginal effects of MY and RC in decreasing VFA production were recorded: −2·38 and −2·86 mmol/l for each 1·0 μg/ml of increment in mycotoxin concentration, respectively. At the highest RC and MY tested concentrations, Vf and VFA decreased by 8·7 and 10·7%, respectively, over maximum estimated values. In FFD, the average amounts of MY and RC recovered in rumen fluid after 48 h of incubation were 79·0 and 40·6%, respectively. In conclusion, the MY and RC from standards interfered with rumen microorganisms at relatively low levels and were partially stable in the rumen environment after 48 h of incubation. These findings suggested that MY and RC could interfere with digestive processes and might represent a potential risk for ruminants fed diets containing feeds contaminated by mycotoxins produced by P. roqueforti.

scholarly journals Optimization of Diet for Post Larvel/Juvenile Sea Bass and Hybrid Stripped Bass Based on Enzymatic Profiles of their Digestive Tracts

1995 ◽  
Author(s):  
Sheenan Harpaz ◽  
Steven G. Hughes ◽  
Pinhas Lindner
Keyword(s):  
Proteolytic Activity ◽  
Proteolytic Enzymes ◽  
Juvenile Fish ◽  
Research Work ◽  
Sea Bass ◽  
Original Research ◽  
Young Fish ◽  
Feed Formulation ◽  
Feed Protein

The overall goal of this research work was to identify the main proteolytic activities which take place in the digestive tracts of young bass fish, and use the knowledge acquired in order to improve feed protein utilization in juvenile fish based on their digestive capacity. The results of the work clearly showed that the young fish possess the entire profile of proteolytic enzymes which is found in adult fish. Yet, in the young fish the level of activity is substantially lower per gram tissue (or gram protein) as compared with the activity found in the digestive tracts of the same fish at an older (larger) age. In addition it was found that the main proteolytic enzyme in these fish is chymotrypsin which accounts for almost 80% of the proteolytic activity. An effort aimed at enhancing this activity has lead to the interesting finding that alcohol substantially enhances the proteolytic activity of fish intestines. Fish intestinal homogenates were used in order to evaluate the suitability of various feeds for the fish. Potential feed proteins were subjected to the proteolytic activity of the fish enzymes in vitro, in a manner simulating the natural process. The proteolytic activity was monitored by the valuation of the products, i.e. amino acid released. This method has proven to be a powerful tool which enables us to predict with a very high degree of accuracy the potential of a feed to promote growth. Selection of feed based on the proteolytic capacity of the fish degestive tracts can now be implemented in feed formulation, as anticipated in the original research proposal.

scholarly journals Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and in batch cultures of rumen microorganisms

2015 ◽  
Vol 153 (6) ◽  
pp. 1097-1106 ◽  
Author(s):  
I. MATEOS ◽  
M. J. RANILLA ◽  
C. SARO ◽  
M. D. CARRO
Keyword(s):  
Bacterial Diversity ◽  
Rumen Fluid ◽  
Similarity Index ◽  
Bacterial Strains ◽  
Rumen Microorganisms ◽  
Batch Cultures ◽  
Alfalfa Hay ◽  
Grass Hay ◽  
Forage Type

SUMMARYThe objective of the current study was to assess how closely batch cultures (BC) of rumen microorganisms can mimic the dietary differences in fermentation characteristics found in the rumen, and to analyse changes in bacterial diversity over the in vitro incubation period. Four ruminally and duodenally cannulated sheep were fed four diets having forage : concentrate ratios (FCR) of 70 : 30 or 30 : 70, with either alfalfa hay or grass hay as forage. Rumen fluid from each sheep was used to inoculate BC containing the same diet fed to the donor sheep, and the main rumen fermentation parameters were determined after 24 h of incubation. There were differences between BC and sheep in the magnitude of most measured parameters, but BC detected differences among diets due to forage type similar to those found in sheep. In contrast, BC did not reproduce the dietary differences due to FCR found in sheep for pH, degradability of neutral detergent fibre and total volatile fatty acid (VFA) concentrations. There were differences between systems in the magnitude of most determined parameters and BC showed higher pH values and NH3–N concentrations, but lower fibre degradability and VFA and lactate concentrations compared with sheep. There were significant relationships between in vivo and in vitro values for molar proportions of acetate, propionate and butyrate, and the acetate : propionate ratio. The automated ribosomal intergenic spacer analysis (ARISA) of 16S ribosomal deoxyribonucleic acid showed that FCR had no effect on bacterial diversity either in the sheep rumen fluid used as inoculum (IN) or in BC samples. In contrast, bacterial diversity was greater with alfalfa hay diets than those with grass hay in the IN, but was unaffected by forage type in the BC. Similarity index between the bacterial communities in the inocula and those in the BC ranged from 67·2 to 74·7%, and was unaffected by diet characteristics. Bacterial diversity was lower in BC than in the inocula with 14 peaks out of a total of 181 detected in the ARISA electropherograms never appearing in BC samples, which suggests that incubation conditions in the BC may have caused a selection of some bacterial strains. However, each BC sample showed the highest similarity index with its corresponding rumen IN, which highlights the importance of using rumen fluid from donors fed a diet similar to that being incubated in BC when conducting in vitro experiments.

scholarly journals Lovastatin-Enriched Rice Straw Enhances Biomass Quality and Suppresses Ruminal Methanogenesis

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Mohammad Faseleh Jahromi ◽  
Juan Boo Liang ◽  
Rosfarizan Mohamad ◽  
Yong Meng Goh ◽  
Parisa Shokryazdan ◽  
...  
Keyword(s):  
Rice Straw ◽  
Rumen Fluid ◽  
Methanogenic Archaea ◽  
Primary Objective ◽  
Gas Production ◽  
Cellulolytic Bacteria ◽  
Hmg Coa Reductase ◽  
Rumen Microorganisms ◽  
Coa Reductase

The primary objective of this study was to test the hypothesis that solid state fermentation (SSF) of agro-biomass (using rice straw as model); besides, breaking down its lignocellulose content to improve its nutritive values also produces lovastatin which could be used to suppress methanogenesis in the rumen ecosystem. Fermented rice straw (FRS) containing lovastatin after fermentation withAspergillus terreuswas used as substrate for growth study of rumen microorganisms usingin vitrogas production method. In the first experiment, the extract from the FRS (FRSE) which contained lovastatin was evaluated for its efficacy for reduction in methane (CH4) production, microbial population, and activity in the rumen fluid. FRSE reduced total gas and CH4productions (P<0.01). It also reduced (P<0.01) total methanogens population and increased the cellulolytic bacteria includingRuminococcus albus,Fibrobacter succinogenes(P<0.01), andRuminococcus flavefaciens(P<0.05). Similarly, FRS reduced total gas and CH4productions, methanogens population, but increasedin vitrodry mater digestibility compared to the non-fermented rice straw. Lovastatin in the FRSE and the FRS significantly increased the expression of HMG-CoA reductase gene that produces HMG-CoA reductase, a key enzyme for cell membrane production in methanogenic Archaea.

Determination of rumen microbial growth in vitro from32P-labelled phosphate incorporation

1977 ◽  
Vol 38 (1) ◽  
pp. 101-114 ◽  
Author(s):  
C. J. Van Nevel ◽  
D. I. Demeyer
Keyword(s):  
Microbial Growth ◽  
Specific Activity ◽  
Rumen Fluid ◽  
List Type ◽  
Substrate Protein ◽  
Isotope Method ◽  
Total Growth ◽  
N Incorporation

1.The extracellular phosphate pool in incubations of rumen fluid or washed cell suspensions of mixed rumen bacteria (WCS) was labelled with32P. From the constant extracellular phosphate pool specific activity and the amount of radioactivity incorporated during incubation, the amount of P incorporated in the microbial fraction was calculated. From the value for nitrogen: P determined in microbial matter, the amount of N incorporated was calculated as a measure of microbial growth.2.Incorporation of soluble non-protein-N in incubations devoid of substrate protein was 50 and 80 % of the values obtained using the isotope method for rumen fluid and WCS respectively. It is suggested that results obtained using the former method reflect 'net growth' of micro-organisms which is the result of simultaneous growth and degradation. The isotope method measures 'total growth', as isotope incorporation is not affected by degradation of non-growing cells.3.Incorporation of32P in P-containing microbial components (mainly nucleic acids) was compared with net synthesis of these components in incubations of WCS. The results showed different specific rates of synthesis and degradation for all components studied. It is concluded that the composition of microbial matter changed during growth.4.When N incorporation, calculated from results obtained using the isotope method in incubations with rumen fluid, was compared with the amount of carbohydrate substrate fermented and the type of fermentation, values between 18.3 and 44.6 g N incorporated/kg of organic matter fermented were obtained. Low values were associated with large proportions of the substrate being fermented to lactate and the use of glucose instead of disaccharides as substrate. Part of the variation could also be attributed to differences in incubation period, reflected in different proportions of polysaccharide formed.5.The use of isotopes for determination of rumen microbial growth in vitro is critically discussed.

Influence of acetohydroxamic acid on some activities in vitro of the rumen microbiota

1968 ◽  
Vol 14 (4) ◽  
pp. 409-416 ◽  
Author(s):  
G. A. Jones
Keyword(s):  
Volatile Fatty Acids ◽  
Urease Activity ◽  
Rumen Fluid ◽  
Selective Medium ◽  
Colony Development ◽  
Acetohydroxamic Acid ◽  
Rumen Microorganisms ◽  
Rumen Microbiota

When acetohydroxamic acid was incubated with washed suspensions of bovine rumen microorganisms the urease activity of the suspensions was depressed; activity could not be restored by the addition of divalent cations which, in the absence of acetohydroxamic acid, stimulated the urease activity of the cells. Acetohydroxamic acid was slowly degraded by the rumen microbiota. When the compound was incorporated into a non-selective medium for the enumeration of rumen bacteria it completely prevented visible colony development by some components of the inoculum and retarded the rate of multiplication of others. Acetohydroxamic acid inhibited the production of volatile fatty acids from added cellulose in strained rumen fluid and modified the molar proportions of acetate, propionate, and butyrate produced from the substrate; whereas in the absence of acetohydroxamic acid propionate production was favored at the expense of acetate; in its presence the acetate:propionate ratio remained constant. The effect of acetohydroxamic acid upon rumen microbial activities in vitro was therefore not limited to inhibition of rumen urease. It was impossible, however, to infer from the results obtained whether the potential value of the compound as a urease inhibitor in vivo would be diminished for this reason; this is because the influence of acetohydroxamic acid on the rumen microbiota in vivo is probably subject to modification by factors, such as the composition of the animal's diet, which were not investigated.

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