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Different techniques to study rumen fermentation characteristics of maturing grass and grass silage.

Grass samples were harvested during the 1993 growing season after a precut on April 27, 1993 and were stored frozen or left to ensile in 30-L buckets. Effects on chemical composition and fermentation kinetics of the maturation of the grass and of ensiling were investigated. Chemical composition and fermentation kinetics were determined using the gas production technique, in vitro techniques, and the nylon bag technique. Two silage samples were also investigated in vivo. Maturation caused a decrease in crude protein content and organic matter degradability and an increase in neutral detergent fiber, acid detergent fiber, and lignin contents. Degradation rates were highest for the youngest samples and decreased as grass and silage matured. This result could be observed from the first derivative of the cumulative gas production curves (i.e., the rate of gas production) and from the gas production parameters. The rate of degradation of the nonsoluble fraction of the young samples, determined using the gas production technique, was relatively higher in rumen fluid from a cow fed silage from grass cut at a young stage. More mature samples were degraded relatively faster in rumen fluid from a cow fed mature grass silage, suggesting a specific adaptation of the rumen microorganisms to the grass properties. There was a good relationship among the second phase of gas production (i.e., fermentation of the nonsoluble fraction), maturity of the grass and grass silage samples, degradability determined with the Tilley and Terry technique, and degradability determined after 46 h of incubation in rumen fluid. Results obtained with both of the different in vitro techniques and the nylon bag technique were confirmed by the in vivo experiments involving the two silage samples.

Animals↗

Isolation of ekatetrone, a new metabolite of producing variants of Streptomyces aureofaciens.

From a mixture of substances formed by producing strains of Streptomyces aureofaciens under conditions of submerged fermentation a new metabolite, ekatetrone, was isolated. Its isolation and basic physical and chemical data are described. Ekatetrone is a quinone derivative with a carboxamide group. In tests in vitro with cells of Ehrlich's ascites tumour evidence was provided that ekatetrone inhibits proteo- and nucleosynthesis.

Animals↗

Surface binding of aflatoxin B1 by Saccharomyces cerevisiae strains with potential decontaminating abilities in indigenous fermented foods.

Saccharomyces cerevisiae constitutes one of the most important microorganisms involved in food fermentations throughout the world. Aflatoxin B(1) binding abilities of S. cerevisiae strains isolated from indigenous fermented foods from Ghana, West Africa were tested in vitro. Results show that aflatoxin binding was strain specific with 7 strains binding 10-20%, 8 strains binding 20-40% and 3 strains binding more than 40% of the added aflatoxin B(1) when grown and incubated under standard conditions. Binding by two of the strains was further characterized. Highest binding capacity was seen with cells collected at the exponential growth phase with the strains A18 and 26.1.11 binding 53.0 and 48.8% of the total toxin respectively and the binding reduced towards the stationary phase. Aflatoxin B(1) binding increased steadily when the cells were incubated with 1 to 20 microg/ml of aflatoxin B(1). Binding was not affected by the cells grown at temperatures ranging from 20 to 37 degrees C, but was significantly reduced at 15 degrees C. Binding seems to be a physical phenomenon with cells treated at 52, 55 and 60 degrees C for 5 and 10 min or 120 degrees C for 20 min binding significantly higher quantities (more than 2-fold in 120 degrees C treated cells) of aflatoxin B(1) than their viable counterpart. Similarly, when the cells were treated with 2 M HCl for 1 h, up to 2-fold increase in binding was observed. The results obtained show that some strains of S. cerevisiae, viable or non-viable, are effective aflatoxin binders and these properties should be considered in the selection of starter cultures for relevant indigenous fermented foods where high aflatoxin level is a potential health risk.

Aflatoxin B1↗

Potent in vitro antimalarial activity of metacycloprodigiosin isolated from Streptomyces spectabilis BCC 4785.

Bioassay-guided fractionation of the extract from the fermentation broth of Streptomyces spectabilis BCC 4785 led to the isolation of three principle antimalarial agents, metacycloprodigiosin, bafilomycin A(1), and spectinabilin. Metacycloprodigiosin exhibited potent in vitro activity against Plasmodium falciparum K1, with a 50% inhibitory concentration of 0.0050 +/- 0.0010 microg/ml, while its cytotoxicity was much weaker.

Animals↗

Milky disease bacteria.

A comparative study was made of all available milky-disease species and strains that have been isolated around the world from beetle larvae (family Scarabaeidae). Included in the study were Bacillus popilliae Dutky, B. lentimorbus Dutky, and B. lentimorbus var. maryland from the United States; B. euloomarahae Beard and B. lentimorbus var. australis Beard from Australia; B. fribourgensis Wille from Switzerland; and New Zealand milky disease (Dumbleton). The organisms were classified into three groups: (i) those containing parasporal bodies, including B. popilliae Dutky, B. fribourgensis Wille, and New Zealand milky disease (Dumbleton); (ii) those without a visible parasporal body and with spore morphology similar to B. lentimorbus Dutky, including B. lentimorbus var. australis Beard; and (iii) those with very tiny spores and no parasporal body, including B. euloomarahae Beard and B. lentimorbus var. maryland. All available milky-disease species and strains were cultivated in vitro on Brain Heart Infusion Agar plates. However, the most fastidious organisms-B. euloomarahae and B. lentimorbus var. maryland-could not be grown until they were passed through a life cycle in larvae of a large scarabaeid beetle infesting rotting wood. Then they remained stable for only one or two subcultures. All the milky-disease organisms produced larger cells in vitro than they did in vivo. The pattern of sugar fermentations was similar for all milky-disease species. It appears that there is a very low percentage of strains of B. popilliae, B. lentimorbus, and the other milky-disease organisms that have the inherent genetic makeup to permit them to sporulate on artificial media, if conditions are favorable. Among these conditions are a sufficiently high cell population and a reduced oxygen tension. Spores produced in vitro may have a low virulence via the normal ingestion pathway, even though they show apparent virulence when injected directly into the hemocoel.

Animals↗

Possible mechanism underlying the antiherpetic activity of a proteoglycan isolated from the mycelia of Ganoderma lucidum in vitro.

GLPG (Ganoderma lucidum proteoglycan) was a bioactive fraction obtained by the liquid fermentation of the mycelia of Ganoderma lucidum, EtOH precipitation, and DEAE-cellulose column chromatography.GLPG was a proteoglycan with a carbohydrate: protein ratio of 10.4: 1. Its antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were investigated using a cytopathic inhibition assay. GLPG inhibited cell death in a dose-dependent manner in HSV-infected cells. In addition, it had no cytotoxic effect even at 2 mg/ml. In order to study the mode of action of the antiviral activity of GLPG, cells were treated with GLPG before, during, and after infection, and viral titer in the supernatant of cell culture 48 h post-infection was determined using a TCID((50)) assay. The antiviral effects of GLPG were more remarkable before viral treatment than after treatment. Although the precise mechanism has yet to be defined, our work suggests that GLPG inhibits viral replication by interfering with the early events of viral adsorption and entry into target cells. Thus, this proteoglycan appears to be a candidate anti-HSV agent.

Animals↗

Effects of three benzimidazole anthelmintics on the ruminal fermentation ability of sheep.

The effects of albendazole, fenbendazole and oxfendazole on the fermentation ability of the rumen were assessed in 12 Dorset sheep, using as parameters volatile fatty acid concentration, percentage digestibility of cellulose in vitro and pH. Although the digestibility of cellulose was affected only at high concentrations, volatile fatty acid concentrations were markedly reduced after treatment. Correlation coefficient analysis revealed a significant correlation between digestibility of cellulose and volatile fatty acids, as well as between volatile fatty acids and pH. Despite an evident decrease in volatile fatty acids and digestibility of cellulose, it is suggested that this does not outweigh the advantages derived from the use of these benzimidazoles.

Albendazole↗

Production of human interleukin-8 expressed in Escherichia coli: from a laboratory scale for in vitro tests via a technical scale for animal studies to a process scale for a GMP-compatible production.

An Escherichia coli K 12 strain has been constructed for efficient expression of recombinant biologically active human IL-8 (Interleukin-8). The development of a fermentation and purification process from the laboratory scale (cells from 15 l fermentation broth) to a production scale (cells from 200 l fermentation broth) is described. Material obtained from the laboratory scale was used for initial in vitro studies and for the development of a biological assay. An upscale purification process starting from 80 l fermentation broth resulted in larger amounts of IL-8 needed for preclinical studies. This process includes a fully automated control of the initial affinity chromatography step. Finally, a production process which differed markedly from the small-scale processes was tailor-made for GMP conformity and economic considerations. It consists of a cell disruption step followed by two crossflow diafiltrations with different molecular weight cut offs and filtration rates, one cation exchange chromatography and a final dialysis step. In order to enhance the overall yield of biologically active IL-8, conditions for a resolubilisation of insoluble IL-8 present in the remaining pellet after cell disruption were worked out.

Bioreactors↗

Sulfur influences on rumen microorganisms in vitro and in sheep and calves.

When continuously cultured ruminal microbes were given orchardgrass hay and sufficient sulfuric acid or hydrochloric acid to maintain a pH of 5.5, fermentation and numbers of protozoa were reduced compared with cultures whose pH was controlled with phosphoric acid. Likewise, when sulfur-deficient, purified diets were supplied to cultures, less methane (mmol X liter-1 X d-1), 3.2 vs 32.6, was produced and fewer cellulolytic bacteria (log10/ml), 5.8 vs 7.2 were present than when cultures were given the same diet supplemented with .3% elemental sulfur. The rumen of sheep fed the .04% sulfur diet had reduced digesta weights (1.69 vs 3.2 kg) compared with sheep fed the diet with .34% sulfur at the same intake. There also was reduced methanogenesis 12.3 vs 25.8 mmol X liter-1 X d-1) and reduced numbers of cellulolytic bacteria (7.4 vs 8.4 log10/ml) in sulfur-deficient sheep in comparison to sulfur-supplemented sheep. In growing calves, the same types of bacteria predominated in the rumen, but more facultative anaerobic bacteria were isolated from calves fed .04% sulfur than from calves fed diets with .34 to 1.72% sulfur. None of the dietary levels of sulfur appeared toxic. Regardless of treatment, volatile fatty acids were more predominant than lactic acid as end-products of fermentation of ruminal microbes in fermenters, sheep and calves. The greater methanogenesis and the greater cellulolytic bacterial numbers of sulfur-supplemented sheep compared with sulfur-deficient in vitro cultures, is interpreted to be the result of recycling of sulfur to the rumen in sheep where it is efficiently scavengered by ruminal bacteria.

Animal Feed↗

Nutrient content of complementary foods based on processed and fermented sorghum, groundnut, spinach, and mango.

BACKGROUND: Many African mothers use gruels made of maize and sorghum as complementary foods for their infants because they cannot afford the cost of nutritionally superior commercial weaning foods. OBJECTIVE: To improve the nutritional quality of traditional sorghum gruels used as complementary foods for children between six months and five years in the northern part of Cameroon. METHODS: Sorghum grains were processed by dehulling, sprouting, dehulling and cooking, and sprouting and cooking, then fermented using Saccharomyces cerevisae and tested for nutritional quality. The processed samples were analyzed for their nutrient composition and the digestibility of proteins and carbohydrates. RESULTS: The resulting products showed a significant reduction in antinutritional factors (e.g., phenolic compounds and phytates). In addition, germination improved the in vitro digestibility of proteins and carbohydrates. These effects were enhanced by fermentation of the resulting sorghum flour, regardless of how grains were pretreated, although the protein and mineral content remained low. Use of a composite flour made from pretreated sorghum, groundnut, spinach, and mango improved the protein, mineral, and vitamin A and C content of the gruels. Fermentation of composite flours resulted in an improvement in the protein content. CONCLUSIONS: This study, conducted under local conditions and using local technology, describes an opportunity for improving the quality of complementary foods using local ingredients.

Arachis↗

Influence of cultures of Aspergillus oryzae on rumen and total tract digestibility of dietary components.

Three trials were conducted to evaluate the effect of dried cultures of Aspergillus oryzae on nutrient utilization by mature Holstein cows fitted with ruminal and duodenal cannulas. In trial 1, four cows (two dry and two lactating) were used to test Aspergillus oryzae (3 g/d) and a control treatment at two forage amounts in a 4 x 4 Latin square. Trial 2 compared control, A. oryzae, and Saccharomyces cerevesiae using six lactating cows in a repeated 3 x 3 Latin square design. For trial 3, four lactating cows were used in a switchback design to compare control to A. oryzae treatment. In trials 2 and 3, diets contained 70 and 61% concentrate. A fourth in vitro trial was conducted in conjunction with trial 3 in which rumen fluid was obtained from cows adapted or unadapted to A. oryzae. On all trials except high forage in trial 1, A. oryzae increased rumen and total tract digestibility of fiber fractions. Rumen VFA and ammonia were not affected by fungal cultures. Aspergillus oryzae increased rate of rumen fermentation of alfalfa but not of milo or wheat straw. In vitro disappearance of DM from alfalfa, milo, and wheat straw was increased by A. oryzae, and previous adaptation was not required to stimulate in vitro DM digestibility. These results indicate that a primary effect of A. oryzae is stimulation of fiber digestion by rumen microbes.

Ammonia↗

Effects of short-chain fatty acids on in vitro bacterial growth of Bacteroides fragilis and Escherichia coli.

Short-chain fatty acids (SCFA) are produced in the large bowel of nonruminant mammals by bacterial anaerobic fermentation. The aim of the present study was to investigate the effects of SCFA on the in vitro growth of Bacteroides fragilis and Escherichia coli. B. fragilis and E. coli isolated from fresh human clinical samples and a reference strain for each species were incubated in a meat infusion broth with increasing amounts of SCFA and grown under anaerobic conditions at a temperature of 37 degrees C. Bacterial growth was estimated by spectrophotometry. Rate of growth was calculated from the logarithmic growth phase. SCFA, in concentrations normally found in the human colon, had a significant (p<0.01) inhibitory effect of the in vitro growth rate for E. coli, while they were without effect on the in vitro growth rate of B. fragilis. It may be concluded that under in vitro conditions SCFA had growth-inhibitory effects on E. coli, while they had no effect on B. fragilis.

Bacteroides fragilis↗

Binding properties of lactic acid bacteria from 'Idly' towards food-borne mutagens.

Lactic acid bacteria from the fermented non-dairy food 'Idly' were examined in vitro for their ability to bind food-borne mutagens. They were subjected to high pressures and temperatures to investigate alterations in their binding potential. All the isolates tested exhibited good binding activity towards the amino acid pyrolysates 3-amino-1-4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-d]indole and a moderate level of binding of 2-amino-6-methyl-dipyrido[1,2-3',2'-d]imidazole. No significant binding activity was observed with other mutagens, such as aflatoxins. High-pressure and heat treatment did not substantially alter the binding activity of the bacteria.

Biomass↗

Stephacidin A and B: two structurally novel, selective inhibitors of the testosterone-dependent prostate LNCaP cells.

Two novel antitumor alkaloids, Stephacidin A and B, were isolated from the solid fermentation of Aspergillus ochraceus WC76466. Both alkaloids exhibit in vitro cytotoxicity against a number of human tumor cell lines; however, stephacidin B demonstrated more potent and selective antitumor activities, especially against prostate testeosterone-dependent LNCaP cells with IC50 value of 60 nM. The structures of stephacidin A and B were established on the basis of the NMR data and X-ray crystallography. With 15 rings and 9 chiral centers, stephacidin B represents one of the most structurally complex and novel alkaloids occurring in nature.

Antineoplastic Agents↗

Changes in pig serum lipids, nutrient digestibility and sterol excretion during cecal infusion of propionate.

Dietary water-soluble fiber supplementation elicits hypolipidemic effects. Propionate, the 3-carbon short-chain fatty acid derived from colonic fiber fermentation, has previously exhibited inhibition of cholesterol synthesis in vitro and may contribute to serum lipid lowering. This study examines the effect of colonic propionate absorption on serum lipids, sterol excretion and nutrient digestibility. Nine barrows were surgically cannulated at the distal ileum and cecum. Pigs received for 16 d continuous cecal infusions of either propionate (36 mmol/kg 0.75 daily) or saline (control) in a crossover design. Propionate infusion did not lower serum lipids, but increased total serum cholesterol by 15% (P less than 0.05) and LDL cholesterol by 15% (P less than 0.05). Differences in sterol excretion, ileal and fecal nutrient digestibilities, and weight gain were not detected between infusion treatments. The results suggest that propionate absorption does not result in decreased serum lipids and is not responsible for the serum lipid-lowering effects of water-soluble fibers.

Animals↗

Demonstration of aflatoxin inhibitory activity in a cotton seed coat xylan.

An inhibitor of aflatoxin biosynthesis localized in the seed coats of developing cotton was partially purified and characterized. Aqueous extracts from 25-day postanthesis seed coat tissue inhibited aflatoxin (B(inf1)) production in liquid cultures of Aspergillus flavus AF13. Inhibition was concentration dependent, with a 50% effective dose of 173 (mu)g of crude extract per ml of medium. The inhibitor was neutral in charge. Two active fractions were obtained from crude preparations by gel filtration chromatography (BioGel P-100). The purest fraction eluted in the void volume. Carbohydrate composition analysis of this void volume inhibitor indicated a composition of xylose (>90%) and mannose. Aflatoxin production in vitro was inversely related to inhibitor concentration in the fermentation medium (log of aflatoxin versus log of [inhibitor]; r(sup2) = 0.82; P < 0.002). The void volume inhibitor had a 50% effective dose of 6.2 (mu)g/ml, a 28-fold purification of the inhibitor material. These data support the hypothesis that seed coat inhibitory activity is associated with a cottonseed-specific xylan.

Journal Article↗

Absorption and excretion of the 8-hydroxydaidzein in rats after oral administration and its antioxidant effect.

8-Hydroxydaidzein (8-OHD), which is produced during the processing of fermented soybean products, has a potent antioxidant activity in vitro. There is no information regarding the absorption and excretion of this isoflavone, including its antioxidant effect in vivo. In this study, rats were administered a single oral dose of 8-OHD (20 mg/kg body weight), and the blood, liver, kidney and urine were collected at specific intervals up to 18 h after dosing. Free 8-OHD in each tissue was directly determined by using HPLC with electrochemical detection, while its conjugates were detected after the treatment with beta-glucuronidase and sulfatase. The total 8-OHD in liver reached a high level (9.4 nmol/g) at 1 h after dosing, and maintained the relatively high concentration up to 10 h. Most of the 8-OHD was present in free form in liver, while the majority of 8-OHD in plasma was conjugated. This suggests that free 8-OHD in liver is successively converted to glucuronide and/or sulfate and the conjugated 8-OHD is released into the blood. The maximum level of total 8-OHD in plasma or kidney was observed within the first 2 h after the oral administration. The level of 8-OHD in these tissues gradually decreased within the further experiments. Excretion of the 8-OHD in urine began to rise at 1-2 h interval. The mean urinary excretion rate of 8-OHD showed a higher level at 2-4 h and 4-6 h intervals, while the 8-OHD levels at these intervals in plasma or kidney more rapidly decreased. The cumulative recovery of 8-OHD in the urine over the 0-18 h interval was about 36% of the dose. In addition, the liver homogenate from rats killed at 1 h and 2 h after dosing, which contained a higher level of free 8-OHD, showed a significantly lower susceptibility to lipid peroxidation induced by AAPH or Cu2+ than that at 0 h (pre-administered rats). These results suggest that 8-OHD was relatively easily absorbed into rats and might exert its biological activities in vivo, including the antioxidant effect.

Absorption↗