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Influence of diet on growth yields of rumen micro-organisms in vitro and in vivo: influence on growth yield of variable carbon fluxes to fermentation products.

The efficiency of rumen microbial production (EMP) in vitro and in vivo was examined for three roughages (lucerne (Medicago sativa L.) hay, oat (Avenia sativa L.)-berseem clover (Trifolium alexandrinum cultivar BigBee) hay and maize (Zea mays L.) crop residue (MCR)) and for five isonitrogenous (106 g crude protein (Nx6.25)/kg) diets formulated from lucerne hay, oat-berseem clover hay, MCR, soybean meal and maize grain to provide degradable intake protein for the production of 130 g microbial protein/kg total digestible nutrients. EMP in vivo was determined by intestinal purine recovery in sheep and ranged from 240 to 360 g microbial biomass/kg organic matter truly degraded in MCR and in one of the diets respectively (P<0.05). EMP in vitro was estimated by the substrate degraded : gas volume produced thereby (termed partitioning factor, PF (mg/ml)) at times of estimated peak microbial production and after 16.0 and 24.0 h of incubation. For the diets, PF values were significantly related to EMP in vivo at peak microbial production (P=0.04), but not after 16.0 (P=0.08) and 24.0 h (P=0.66). For roughages, PF values were significantly related to EMP in vivo only when measured after 16.0 h (P=0.04). For MCR and diets, a close non-linear relationship was found between PF values at peak microbial production and EMP in vivo (R(2) 0.99, P<0.0001) suggesting a maximum EMP in vivo of 0.39. Low gas production per unit substrate degraded (high PF) was associated with high EMP in vivo. The in vitro study of the products of fermentation, short-chain fatty acids, gases and microbial biomass (by purine analysis) after 16.0 h of incubation showed very strong relationships (R(2)> or =0.89, P<0.0001) between short-chain fatty acids, gases and gravimetrically measured apparent degradability. Except for maize grain, the true degradability of organic matter estimated by neutral-detergent solution treatment agreed with the sum of the products of fermentation (R(2) 0.81, P=0.0004). After 16.0 h of incubation, the synergistic effects of diet ingredient on diets were greater for microbial biomass (18 %) than for short-chain fatty acids and gas production (7 %). It is concluded that measurement of gas production only gives incomplete information about fodder quality; complementation of gas measurements by true degradability measurements is recommended.

Ammonia↗

The curing agent sodium nitrite, used in the production of fermented sausages, is less inhibiting to the bacteriocin-producing meat starter culture Lactobacillus curvatus LTH 1174 under anaerobic conditions.

Curvacin A is a listericidal bacteriocin produced by Lactobacillus curvatus LTH 1174, a strain isolated from fermented sausage. The response of this strain to an added curing agent (sodium nitrite) in terms of cell growth and bacteriocin production was investigated in vitro by laboratory fermentations with modified MRS broth. The strain was highly sensitive to nitrite; even a concentration of 10 ppm of curing agent inhibited its growth and both volumetric and specific bacteriocin production. A meat simulation medium containing 5 ppm of sodium nitrite was tested to investigate the influence of the gas phase on the growth and bacteriocin production of L. curvatus LTH 1174. Aerating the culture during growth had no effect on biomass formation, but the oxidative stress caused a higher level of specific bacteriocin production and led to a metabolic shift toward acetic acid production. Anaerobic conditions, on the other hand, led to an increased biomass concentration and less growth inhibition. Also, higher maximum volumetric bacteriocin activities and a higher level of specific bacteriocin production were obtained in the presence of sodium nitrite than in fermentations under aerobic conditions or standard conditions of air supply. These results indicate that the inhibitory effect of the curing agent is at least partially masked under anaerobic conditions.

Aerobiosis↗

Sodium chloride reduces production of curvacin A, a bacteriocin produced by Lactobacillus curvatus strain LTH 1174, originating from fermented sausage.

Lactobacillus curvatus LTH 1174, a strain originating in fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of salt (sodium chloride) were investigated in vitro during laboratory fermentations using modified MRS medium. A model was set up to describe the effects of different NaCl concentrations on microbial behavior. Both cell growth and bacteriocin activity were affected by changes in the salt concentration. Sodium chloride clearly slowed down the growth of L. curvatus LTH 1174, but more importantly, it had a detrimental effect on specific curvacin A production (k(B)) and hence on overall bacteriocin activity. Even a low salt concentration (2%, wt/vol) decreased bacteriocin production, while growth was unaffected at this concentration. The inhibitory effect of NaCl was mainly due to its role as an a(w)-lowering agent. Further, it was clear that salt interfered with bacteriocin induction. Additionally, when 6% (wt/vol) sodium chloride was added, the minimum biomass concentration necessary to start the production of curvacin A (X(B)) was 0.90 g (cell dry mass) per liter. Addition of the cell-free culture supernatant or a protein solution as a source of induction factor resulted in a decrease in X(B), an increase in k(B), and hence an increase in the maximum attainable bacteriocin activity.

Animals↗

Development of a quantitative tool for the comparison of the prebiotic effect of dietary oligosaccharides.

AIMS: To develop a quantitative equation [prebiotic index (PI)] to aid the analysis of prebiotic fermentation of commercially available and novel prebiotic carbohydrates in vitro, using previously published fermentation data. METHODS: The PI equation is based on the changes in key bacterial groups during fermentation. The bacterial groups incorporated into this PI equation were bifidobacteria, lactobacilli, clostridia and bacteroides. The changes in these bacterial groups from previous studies were entered into the PI equation in order to determine a quantitative PI score. PI scores were than compared with the qualitative conclusions made in these publications. In general the PI scores agreed with the qualitative conclusions drawn and provided a quantitative measure. CONCLUSIONS: The PI allows the magnitude of prebiotic effects to be quantified rather than evaluations being solely qualitative. SIGNIFICANCE AND IMPACT OF THE STUDY: The PI equation may be of great use in quantifying prebiotic effects in vitro. It is expected that this will facilitate more rational food product development and the development of more potent prebiotics with activity at lower doses.

Bacteria↗

Influence of complex nutrient source on growth of and curvacin a production by sausage isolate Lactobacillus curvatus LTH 1174.

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of a complex nutrient source were investigated in vitro during laboratory fermentations with modified MRS medium. A modification of the nutrient depletion model was used to fit the data describing growth and bacteriocin production. Both cell growth and bacteriocin activity were influenced by changes in the complex nutrient source concentration. Standard MRS medium clearly limited the growth of L. curvatus LTH 1174. Higher nutrient concentrations, up to a certain degree, led to improved growth, a higher attainable biomass concentration, and a higher bacteriocin activity in the supernatant. A lower concentration of complex nutrient source caused severe growth inhibition, leading to a lower biomass concentration but a much higher specific bacteriocin production. When examining the separate components of the complex nutrient source, a stimulating effect of bacteriological peptone on growth was found without an adverse effect on bacteriocin production, resulting in increased curvacin A activity. Furthermore, specific depletion of the amino acids tyrosine, serine, and asparagine/aspartic acid was observed for this strain.

Anti-Bacterial Agents↗

Modelling growth and bacteriocin production by Lactobacillus curvatus LTH 1174 in response to temperature and pH values used for European sausage fermentation processes.

Lactobacillus curvatus LTH 1174, a strain isolated from fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of temperature (20-38 degrees C) and pH (4.8-7.0) were investigated in vitro during laboratory fermentations using de Man, Rogosa and Sharpe (MRS) medium. A predictive, successfully validated model was set up to describe the influence of temperature and pH on the microbial behavior. Both cell growth and bacteriocin activity were influenced by changes in temperature and pH. The optimum temperature value for cell growth, 34.5 degrees C, did not correspond with the optimum temperature for curvacin A production (20-27 degrees C). Interestingly, the pH range for growth and curvacin A production was broad. Thus, Lb. curvatus LTH 1174 seems to be a promising bacteriocin-producing strain for use in European sausage fermentations that are performed at temperatures near 25 degrees C.

Animals↗

In vitro effects of a thiopeptide and monensin on ruminal fermentation of soluble carbohydrates.

Using a purified diet and a mixed culture of ruminal microorganisms, we studied the in vitro effects of a sulfur-containing peptide (A10255) and monensin on ruminal VFA and lactic acid concentrations. After 22 h of fermentation, total VFA concentration was greater in cultures with A10255 and monensin (2.5 ppm each) than in untreated controls (84.1, 91.9, and 65.2 mM for cultures with A10255, monensin, and control, respectively). The molar proportions of propionate were more than 40% greater, and the molar proportions of acetate were about 15% less, in treated than in untreated cultures. The molar proportions of the remaining VFA were lowest in monensin-treated cultures, intermediate in A10255-treated cultures, and greatest in untreated cultures. Addition of A10255 or monensin also prevented the accumulation of lactate and maintained higher pH than in untreated cultures. In pure culture, growth of Streptococcus bovis was slowed by A10255 at pH 5.5 and 6.5. Growth of S. bovis was reduced markedly with addition of monensin at pH 6.5 and inhibited at pH 5.5. Growth of Lactobacillus acidophilus was inhibited more at pH 5.5 than 6.5 with monensin but was unaffected by A10255. The thiopeptide A10255 and monensin inhibited ruminal lactate production, but sensitivities of lactate-producing bacteria differed between compounds.

Animals↗

Expression of acylphosphatase in Saccharomyces cerevisiae enhances ethanol fermentation rate.

Previous experiments in vitro have demonstrated the ability of acylphosphatase to increase the rate of glucose fermentation in yeast. To evaluate the possibility of increasing fermentation in vivo also, a chemically synthesized DNA sequence coding for human muscle acylphosphatase was expressed at high level in Saccharomyces cerevisiae. Ethanol production was measured in these engineered strains in comparison with a control. Acylphosphatase expression strongly increased the rate of ethanol production both in aerobic and anaerobic culture. This finding may be potentially important for the development of more efficient industrial fermentation processes.

Acid Anhydride Hydrolases↗

Acarbose raises serum butyrate in human subjects with impaired glucose tolerance.

The fermentation of starch in vitro produces a higher proportion of butyrate than the fermentation of most other substrates. The alpha-glucosidase inhibitor acarbose increases the amount of starch entering the colon, and has been shown to increase faecal butyrate in humans. It is generally considered that colonic butyrate is quantitatively removed by the colonic mucosa and liver and does not appear in peripheral blood. However, studies in animals suggest that a small proportion of colonic butyrate reaches peripheral blood. Thus, we hypothesised that an increase in colonic butyrate production would result in a rise in serum butyrate in human subjects. To test this, subjects with impaired glucose tolerance were randomly treated in a double-blind fashion with placebo (n 11) or acarbose (n 11) (100 mg three times per day). Serum short-chain fatty acid concentrations were measured twelve times over 12 h with subjects eating a standard diet before randomization and after 4 months of therapy. At baseline, 12 h mean serum butyrate concentrations were similar in the placebo and acarbose groups (2.8 (SE 0.7) and 3.3 (SE 0.6) microM, respectively). After 4 months on placebo, mean serum butyrate (2.6 (SE 0.5) microM) was no different from baseline. However, after 4 months on acarbose, serum butyrate had increased to 4.2 (SE 1.0) microM, a value which differed significantly from both the baseline value in the acarbose group and the treatment value in the placebo group. We conclude that acarbose increased serum butyrate in subjects with impaired glucose tolerance. These results support the hypothesis that increased colonic butyrate production in human subjects can be detected by an increase in serum butyrate.

Acarbose↗

Effect of sheep rumen fermentation and methane inhibition on the toxicity of Senecio jacobaea.

Senecio jacobaea (SJ) was incubated in sheep rumen fluid-buffer mixtures to determine if metabolism and(or) detoxication of pyrrolizidine alkaloids (PA) was occurring. The nontoxic reduction metabolite, 7 beta-hydroxy-l-methylene-8 alpha-pyrrolizidine, was not detected when SJ-rumen fluid incubation extracts were subjected to high performance liquid chromatography and mass spectrographic analysis. Rats were used as assay animals in another experiment to assess the toxicity of SJ incubated in rumen fluid. Incubation treatments were: Rumen fluid (RF) from a sheep not fed SJ (RF-0); RF autoclaved before incubation (RF-0A); RF from sheep fed 50% SJ for 1 wk (RF-1); RF from a sheep fed 50% SJ for 5 wk (RF-5), and RF-5 with 5 microM iodoform in the incubation medium (RF-5I). The SJ treatments were included in rat diets at the 10% level. Dietary treatment and mean rat survival times (days) were: control, no mortality; 10% untreated SJ, 43; RF-0, 53; RF-0A, 55; RF-1, 44; RF-5, 56; RF-5I, 44, There were no significant differences in survival time. This indicates that SJ was not detoxified as a result of incubation in sheep RF in vitro, and suggests that rumen detoxification does not account for resistance of sheep to SJ. The pH and volatile fatty acid concentrations of the incubation mixtures were measured before and after incubation. Acetate/propionate and pH following incubation were respectively: RF-0A, 7.3, 4.3; RF-0, 4.2, 4.4; RF-1, 2.7, 4.5; RF-5, 2.6, 4.5; RF-5I, 2.4, 4.5. These data show that although pretreatment of the rumen fluid donor with dietary SJ and addition of iodoform to the incubation mixture favor reductive rumen metabolism, detoxification of PA from SJ does not occur during in vitro sheep rumen fermentation.

Animals↗

Fermentability of various fiber sources by human fecal bacteria in vitro.

Certain beneficial effects of fiber in the human diet may be mediated by short-chain fatty acids (SCFAs) produced during anaerobic fermentation in the colon. Two studies, both involving in vitro incubations with human fecal bacteria as inoculum, were conducted to assess fermentation of various fiber sources and to quantitate the SCFAs produced. In experiment 1, substrate fermentability based on total SCFA production ranked as follows: citrus pectin greater than soy fiber greater than sugarbeet fiber greater than pea fiber greater than oat fiber. Fermentation of soy fiber led to higher proportions of propionate and butyrate than did fermentation of other substrates. In experiment 2, fermentation of gum arabic, a mixture of arabic and guar, and apple pectin resulted in greater SCFA production than did fermentation of either oat fiber or corn bran. Fermentation of gums led to more propionate and butyrate production than did that of apple pectin. It may be possible to select fiber sources capable of supporting stipulated amounts of both total and individual SCFA production in the human colon.

Bacteria↗

In vitro study of the effect of different ionophore antibiotics and of certain derivatives on rumen fermentation and on protein nitrogen degradation.

An in vitro study was carried out to evaluate the effect of different ionophore antibiotics and some of their derivatives on rumen fermentation and on the degradation of peanut meal nitrogen. The increase in the production of propionic acid at the expense of acetic acid, observed with lonomycin, nigericin, cationomycin and lysocellin, was identical to that noted with monensin. The decrease in methanogenesis observed in the presence of monensin was also found with cationomycin and lysocellin. With the exception of lysocellin, which greatly reduced protein degradation of peanut meal, and of nigericin, which had no effect on this parameter, the 2 other molecules presented the same action as monensin. The negative effect of monensin on microbial ammonia uptake was demonstrated with the same intensity in the presence of cationomycin; it was slightly higher with nigericin and particularly accentuated with lonomycin and lysocellin. Three ester derivatives of monensin (monensin acetate, monensin propionate and monensin butyrate) had a similar action to that of monensin on the orientation of rumen fermentations. The monensin isobutyrate derivative appeared to be more active than monensin and only weakly altered microbial ammonia uptake. The oxolonomycin and hydroxolonomycin derivatives behaved identically to lonomycin with respect to microbial metabolism and protein nitrogen degradation. Unlike the molecules from which they derive, the deacylated cationomycin and nigericic acid had no effect on the orientation of rumen fermentations. Of the compounds tested and presenting a potential 'growth-promoting action' at least comparable to that of monensin, and which demonstrated lower toxicity on mice, three molecules (oxolonomycin, lysocellin and cationomycin) appeared to present a zootechnical interest as feed additives for growing cattle.

Animals↗

Fleroxacin (Ro 23-6240): activity in vitro against 355 enteropathogenic and non-fermentative gram-negative bacilli and Legionella pneumophila.

The antibacterial activity of fleroxacin (Ro 23-6240, AM-833), a new 6-fluoroquinolone, was determined against 149 strains of enteropathogenic bacteria (17 species) and 191 strains (28 species) of glucose non-fermentative Gram-negative rods (excluding Pseudomonas aeruginosa), and against 15 strains of Legionella pneumophila. The cumulative susceptibility of these groups of bacteria to Ro 23-6240 at the 2 mg/l level were 99.2%, 80.1 and 100% of tested strains, respectively.

Anti-Bacterial Agents↗

Effect of sarsaponin on ruminal fermentation with particular reference to methane production in vitro.

This experiment was designed to investigate the effects of different concentrations (0, 1.2, 1.8, 2.4, and 3.2 g/L) of sarsaponin on ruminal microbial methane production using the substrates soluble potato starch, cornstarch, or hay plus concentrate (1.5:1). Ruminal fluid was collected from a dairy cow, mixed with phosphate buffer (1:2) and incubated (30 ml) anaerobically at 38 degrees C for 6 and 24 h with or without sarsaponin. Excluding the lower level of sarsaponin, pH of the medium was slightly decreased. Ammonia-N concentration and numbers of protozoa were decreased in a dose-dependent manner. Total volatile fatty acids and total gas production were increased. Molar proportion of acetate was decreased and propionate was increased with a corresponding decrease in acetate:propionate ratio. Hydrogen production was decreased. As the concentration of sarsaponin increased from 1.2 to 3.2 g/L, fermentation of soluble potato starch, cornstarch, or hay plus concentrate decreased methane production from 20 to 60% (6 h) and 17 to 50% (24 h), 21 to 58% (6 h) and 18 to 52% (24 h), and 23 to 53% (6 h) and 15 to 44% (24 h), respectively. Excluding the lower dose concentration (1.2 g/L) of sarsaponin, in vitro disappearance of dry matter of hay plus concentrate was decreased after 24 h. In conclusion, these results show that sarsaponin stimulated the mixed ruminal microorganism fermentation as well as to inhibit methane production in vitro.

Acetic Acid↗

[Extent and effect of fermentation in the large intestine of swine. 2. Extent of fermentation, pattern of volatile fatty acids and in vitro formation rate].

After adaptation to 8 extremely different rations 40 pigs with 108 to 125 kg LW and 6 lactating sows with 183 kg were slaughtered to obtain digesta of the hindgut. From digesta of caecum and three sections of the colon microbial metabolites and in vitro-VFA-production rates were determined. pH-values increased from 5.5 to 6.3 from caecum to the end of colon. VFA-concentrations and -production-rates decreased along the bowel to one third. Highest fermentations came from rations with 50% dried beet pulp. Daily VFA-production per animal were placed between 799 and 3,134 mmol. Concerning VFA-Mol-%, ileal influx of cellulose increased acetate, high intakes raised butyrate and valeriate. By several reasons the IMOTO-method of in vitro-measuring was censured.

Animal Feed↗

[Influences on fermentation and thiamine metabolism in bovine rumen fluid (in vitro)--part 3: Effects of artificially contaminated hay with Cladosporium herbarum and Fusarium graminearum, respectively].

The influence of contaminated hay [Cladosporium herbarum (CL) and Fusarium graminearum (FU), respectively] on fermentation and thiamine metabolism of bovine rumen content was investigated using the longterm rumen simulation technique (RUSITEC). Six investigation periods 25 days long each were carried out. A nine days feeding period with normal hay was followed by the testphase I (five days) with a mixture of normal and mouldy hay and testphase II (five days) with additive an 0.3 mg thiamine per reaction vessel. The last four days served as regeneration time with normal hay. The following marginal effects of mouldy hay on rumen fermentation patterns could be noted. A) During testphase I: cellulase activity: +10.0% (FU); alternation of the thiamine derivate pattern, but no effect on total thiamine content (CL, FU). B) During testphase II the results were more obvious: bacterial protein synthesis: -22.6% (CL), -24.4% (FU). Alternation of the fatty acid pattern: propionate (-7.30% FU), n-butyrate (-3.90% CL, +3.49% FU), n-valerate (-8.5% CH, FU). Cellulase activity: -17.0% (CL, FU). But no effect on total thiamine (CL, FU); alternation of the thiamine derivate pattern: more non phosphorylate thiamine. The noted effects on rumen fermentation and thiamine metabolism were not severe enough to be responsible for the development of a CCN.

Animal Feed↗

Hindgut fermentation in the wombats: two marsupial grazers.

The wombats Vombatus ursinus and Lasiorhinus latifrons have a capacious proximal colon with only a vestigial caecum. The pattern of microbial fermentation in the hindgut of both species was studied in captive animals fed a pelleted straw diet and in wild wombats feeding on their natural winter diets. Digesta pH was low in the stomach but near neutrality along the hindgut, indicating effective absorption and/or buffering of the colonic contents. Initial proportions and production rates of short chain fatty acids in vitro reflected the fermentation of plant cell walls. Proportions of isobutyrate, isovalerate and n-valerate increased towards the distal colon indicating proteolysis and subsequent fermentation of amino acids. The low ammonia content of digesta fluid suggested that ammonia released from these amino acids was absorbed and utilized by the wombats and their gut microbes. Wild wombats had higher concentrations and production rates of short chain fatty acids than captive animals, which was consistent with the higher apparent digestibility of their natural diet. The energy from short chain fatty acids in captive animals was 30-33% of digestible intake. Energy intakes were low and similar to resting metabolic rates estimated for marsupials. Actual resting metabolic rates of the wombats are probably lower than these estimates, and the proportion of energy derived from fermentation substantially higher than the 53-61% estimated in wild wombats. The energy from fermentation clearly enables wombats to utilize diets high in fibre.

Animals↗