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PubMed2006

A fermentation assay to evaluate the effectiveness of antimicrobial agents on gut microflora.

The measurement of gas produced as a fermentation end product in vitro was correlated with absorbance as a measure of bacterial growth and was used as a rapid screening procedure to test the antimicrobial activity of certain essential oil and tannin secondary plant metabolites on gastrointestinal microorganisms from chickens. The assay was optimised using Clostridium perfringens and Lactobacillus fermentum, and tested in antimicrobial assays against C. perfringens; the minimum inhibitory concentration for each essential oil and condensed tannin was determined. The effect of penicillin-G on C. perfringens, in both growth and fermentation assays, was similar, and for all secondary metabolites tested, concentrations that inhibited fermentation were also bacteriocidal. The assay was also used to demonstrate the effect of dietary composition and enzyme supplementation on fermentation of mixed gut microflora in vitro; results are compared with in vivo results for the same dietary treatments. The data demonstrate that the effects of bioactive secondary plant products and feed composition on individual organisms or mixed gut microflora can be tested by analysis of fermentative activity in vitro, and that this provides a rapid assay for testing potential poultry feed additives before in vivo trials.

Animals↗

PubMed2001

Colonic fermentation as affected by antibiotics and acidic pH: Application of an in vitro model.

Antimicrobial substances such as vancomycin or metronidazole suppress normal gut flora, thereby preventing physiological fermentation of colonic substrates that may promote mucosal inflammation. This study was designed to establish an in vitro model of microbial metabolism in the colon under control and disturbed conditions (acidic pH) to investigate specific effects of vancomycin and metronidazole on the production of short chain fatty acids (SCFA), which play a pivotal role in maintaining homeostasis in the colon. The experiments were carried out with the colon simulation technique (Cositec) representing an in vitro model for the semi-continuous incubation of defined colon contents. Inocula and fermentable substrates were sampled from cecal contents of fistulated pigs. Disturbed microbial metabolism was generated by reduction of pH in the fermentation vessels from 6.7 to 5.8 and 5.1. In general, application of either vancomycin or metronidazole resulted in a significant decrease of SCFA production rates indicating substantial disturbance of the homeostasis of microbial metabolism. With low doses of vancomycin acetate and butyrate production rates were reduced and with high doses of the antibiotic propionate production was inhibited to a greater extent. Treatment with metronidazole inhibited butyrate production almost completely. Similarly, low pH caused a reduction in total SCFA production, which was mainly due to respective decrease of acetate synthesis. Metronidazole effects were not consistently changed at low pH. The Cositec system provides an excellent facility to test the effects of different antibiotics under defined conditions. In this study, both vancomycin and metronidazole affected microbial metabolism to a considerable extent. Both substances may thus be responsible for disturbances of colon function in vivo.

Acid-Base Equilibrium↗

PubMed2004

Study on furundu, a traditional Sudanese fermented roselle (Hibiscus sabdariffa L.) seed: effect on in vitro protein digestibility, chemical composition, and functional properties of the total proteins.

Furundu, a meat substitute, is traditionally prepared by cooking the karkade (Hibiscus sabdariffa L.) seed and then fermenting it for 9 days. Physicochemical and functional properties of raw and cooked seed and of furundu ferments were analyzed. Furundu preparation resulted in significant changes in karkade seed major nutrients. Total polyphenols and phytic acid were also reduced. The increase in total acidity and fat acidity coupled with a decrease in pH indicates microbial hydrolysis of the major nutrients; proteins, carbohydrates, and fats. In vitro digestibility of the seed proteins reached the maximum value (82.7%) at the sixth day of fermentation, but thereafter it significantly decreased. The effect of furundu preparation on N solubility profiles and functional properties, such as emulsification and foaming properties and other related parameters, is investigated in water and in 1 M NaCl extracts from defatted flour samples. The results show that cooking followed by fermentation affects proteins solubility in water and 1 M NaCl. The foaming capacity (FC) from the flour of raw seed decreased as a result of cooking. Fermentation for 9 days significantly increased the FC of the cooked seed, restoring the inherent value. Foam from fermented samples collapsed more rapidly during a period of 120 min as compared to the foam from raw and cooked karkade seeds; stability in 1 M NaCl was lower as compared to those in water. In water, the emulsion stability (ES) from the fermented samples was significantly higher than that of the raw seed flour. Addition of 1 M NaCl significantly decreased the ES of the fermented samples.

Chemical Phenomena↗

PubMed2003

[Inhibition of Cryptoporus volvatus ferment substance on release of leukotriene B4, C4 and D4 from neutrophils in rats in vitro].

OBJECTIVE: To study inhibitory the effects of Cryptoporus volvatus ferment substance(CVFS) on leukotriene production in vitro from neutrophils in rats. METHODS: Neutrophil aggregation was induced by intraperitoneal injection of glycogen in rats. After 16 h, intraperitoneal lavage fluid(PLF) was collected and neutrophils were removed. Neutrophils were stimulated by calcium ionophore A23187 in vitro to produce leukotriene B(4), C(4), D(4). The concentrations of leukotriene B(4), C(4) and D(4) were measured by reversed-phase high-performance liquid chromatography(HPLC). RESULT: CVFS at 0.25, 1, 4 mg x L(-1)decreased leukotriene B(4), C(4), D(4) release from neutrophils in a concentration-dependent manner. Inhibitory rate of CVFS 0.25, 1, 4 mg x L(-1 )on A23187-induced leukotriene B(4) production was 27.4%, 54.2% and 78.8%(P<0.05), respectively. Inhibitory rate of leukotriene C(4) production was 65.1%, 74.3 and 79.0%(P<0.05), respectively. Inhibitory rate of leukotriene D(4) production was 55.6%, 60.9% and 72.8%(P<0.05), respectively. CONCLUSION: The results suggest that suppression of leukotriene release may be a mechanism of the anti-inflammation and anti-asthma effects of CVFS.

Animals↗

PubMed2000

Antibiosis between ruminal bacteria and ruminal fungi.

Cellulose digestion, bacterial numbers, and fungal numbers were monitored over time in vitro by using a purified cellulose medium with and without antibiotics (penicillin and streptomycin). All fermentations were inoculated with a 1:10 dilution of whole rumen contents (WRC). Without antibiotics, cellulose digestion was higher (P < 0.01) at 24, 30, 48, and 72 h; fungi had almost disappeared by 24 h, while bacterial concentrations increased over 100-fold in 24 h and then decreased gradually up to 72 h. In those fermentations with added antibiotics, fungal concentrations increased 4-fold by 30 h and up to 42-fold at 72 h; bacterial concentrations were markedly reduced by 24 h and remained low through 72 h. Similar results were obtained with ground alfalfa as a substrate. In further studies, the in vitro fermentation of purified cellulose without antibiotics was stopped after 18 to 20 h, and the microbial population was killed by autoclaving. Antibiotics were added to half of the tubes, and all tubes were reinoculated with WRC. After 72 h, extensive cellulose digestion had occurred in those tubes without antibiotics, as compared to very low cellulose digestion with added antibiotics. The extent of this inhibition was found to increase in proportion to the length of the initial fermentation period, suggesting the production of a heat-stable inhibitory factor or factors. The inhibitory activity was present in rumen fluid, could be extracted from lyophilized rumen fluid (LRF) with water, and was stable in response to proteolytic enzymes. In addition, the water-extracted residue of LRF was found to contain growth factor activity for rumen fungi in vitro.

Animals↗

PubMed1996

Effect of glucose fermentation on fiber digestion by ruminal microorganisms in vitro.

Two in vitro digestion trials were performed to determine whether the negative effect on fiber digestion when pH was maintained at > 6.2 was attributable to glucose alone or to end products of glucose fermentation. In some treatments, glucose was depleted by a previous 6-h incubation; the supernatant from this incubation was used as the buffer source for treatments using the fermented glucose medium. In trial 1, mixed cultures were grown on cellulose, soybean hulls, and corn bran in fresh media with 0 (control) or 25 mM glucose, in media previously fermented for 6 h with 0 (control) or 25 mM glucose, or in fermented control medium plus 25 mM lactic acid. The rate of NDF digestion was decreased with fermented glucose medium but not with fresh glucose medium or lactic acid medium. Concentrations of lactate, propionate, and butyrate did not appear to affect NDF digestion directly. In trial 2, six treatment media were used: control and glucose media that were either fresh or previously fermented for 6 h and fermented control and glucose media treated with a protease. Rate of NDF digestion was slower in cultures with fermented glucose medium that was treated with protease than in fermented control medium without protease. When treated with protease, rate of NDF digestion was not different between the fermented control medium and the fermented glucose medium. Thus, the negative effect on fiber digestion appeared to be attributable partially to a proteinaceous inhibitor that was produced in culture media containing a rapidly fermented sugar.

Animals↗

PubMed1994

Endemic necrotizing enterocolitis: lack of association with a specific infectious agent.

We conducted a comprehensive analysis of bacterial, parasitic and viral agents present in stool samples of 23 necrotizing enterocolitis cases and 23 matched and 10 random controls. Enterococcus spp., Staphylococcus epidermidis, and Escherichia coli were the most common aerobic bacterial species isolated. Astrovirus was identified in a stool sample from one control. Eight infants were bacteremic; in 7 of 8 the same organism was also present in the stool. No one bacterial species or strain (as identified by plasmid profile analysis) was associated with occurrence of illness. Plasmid analysis further suggested that each infant was colonized with his or her own distinctive aerobic bacterial flora. With the exception of isolates from two control patients which hybridized with a probe for diffuse adherence, no diarrheagenic E. coli was identified. Five (45%) of 11 case infants were colonized with coagulase-negative staphylococci (all S. epidermidis) that produced delta-hemolysin in vitro, as compared with 13 (87%) of 15 control infants. Necrotizing enterocolitis was not associated with an increased ability to ferment carbohydrate, as measured by in vitro beta-galactosidase activity. Our data do not support the hypothesis that endemic necrotizing enterocolitis in our institution is caused by a single infectious agent, nor was there evidence that previously proposed virulence mechanisms such as production of delta-hemolysin or increased in vitro carbohydrate fermentation play a critical role in disease occurrence.

Bacteriological Techniques↗

PubMed2002

Application of metabolic engineering to improve both the production and use of biotech indigo.

A fermentation process was developed for production of indigo from glucose using recombinant Escherichia coli. This was achieved by modifying the tryptophan pathway to cause high-level indole production and adding the Pseudomonas putida genes encoding naphthalene dioxygenase (NDO). In comparison to a tryptophan-over-producing strain, the first indigo-producing strain made less than half of the expected amount of indigo. Severe inactivation of the first enzyme of aromatic biosynthesis, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (the aroGfbr gene product), was observed in cells collected from indigo fermentations. Subsequent in vitro experiments revealed that DAHP synthase was inactivated by exposure to the spontaneous chemical conversion of indoxyl to indigo. Indigo production was thereafter improved by increasing the gene dosage of aroGfbr or by increasing substrate availability to DAHP synthase in vivo by either amplifying the tktA (transketolase) gene or inactivating both isozymes of pyruvate kinase. By combining all three strategies for enhancing DAHP formation in the cell, a 60% increase in indigo production was achieved. Metabolic engineering was then further applied to eliminate a byproduct of the spontaneous conversion of indoxyl to indigo, thereby solving a serious problem with the use of bio-indigo in the final denim dyeing application.

Bioreactors↗

PubMed2002

Iron fortification of wheat flour: bioavailability studies.

Bioavailability refers to that fraction of nutrients which is utilized by the body out of the total indigested amount. Various direct and indirect methods for the determination of bioavailability are available. We determined the bioavailability of iron from fortified wheat flour using both in vitro and in vivo methods. The bioavailability data will be used to make the recommendation for a fortification strategy in Pakistan. The in vitro bioavailability of iron from fortified wheat flour was determined using in vitro enzymatic digestion and fermentation by simulating the condition of the small intestine and colon in the laboratory. Different products were prepared from the fortified ferrous sulfate (FeSO4) and unfortified wheat flour. The total iron of the samples was measured by the wet-digestion method and analyzed on an atomic absorption spectrometer (AAS). To obtain the percentage of iron released, the samples were subjected to pepsin digestion and dialysis. The dialysate was collected at 3, 6, 9, and 12 hours and read on an AAS. The retentates from the above were subjected to the fermentation condition of the colon by inoculating it with human fecal inoculum and incubating it for 24 hours at 37 degrees C under anerobic conditions. The dialysate was collected at 3- and 6-hour intervals and read on an AAS. More iron was released from fortified wheat flour (4.6%), leavened chapati (6.8%), and Nan (15.1%) than from the unfortified control samples. Fermentation and leavening resulted in a better release, which was evident from in vitro digestion results.

Bacteria, Anaerobic↗

PubMed1991

Supplemental corn grain for steers grazing native rangeland during summer.

Effects of supplemental corn grain on forage OM intake (FOMI), digesta kinetics, ruminal fermentation patterns, in vitro OM digestibility (IVOMD), and in situ OM digestion were examined in steers grazing summer blue grama rangeland in northeastern New Mexico during July and August 1988. Sixteen ruminally cannulated steers (average BW 507 kg) were allotted to four treatments and individually fed whole-shelled corn at 0, .2, .4, and .6% of BW in a complete random design with repeated measurements over time. Forage OMI decreased linearly (P = .02) with increasing levels of supplemental corn; however, a tendency toward greater FOMI, as well as faster particulate and fluid passage, was observed when corn was fed at .2% of BW compared with 0, .4, or .6% of BW. Molar proportions of butyrate increased (P less than .10) but molar proportions of acetate and propionate, ruminal pH, and total VFA concentration did not change (P greater than .10) with added corn. Added corn linearly decreased (P less than .10) ruminal ammonia N concentrations in July, but patterns were inconsistent in August. A cubic response (P less than .05) for in situ OM disappearance with added corn was noted after 24, 72, and 96 h of incubation. Supplemental whole corn fed at .2% of BW had no detrimental effects and tended to increase FOMI. However, supplemental corn fed at .4 or .6% of BW decreased FOMI compared with 0 or .2% of BW.

Ammonia↗

PubMed2002

Stability of ingested methylcellulose in the rat determined by polymer molar mass measurements by light scattering.

Methylcellulose (MC) is ingested by humans in food and pharmaceutical formulations. The functional properties of MC like those of other linear polymers depend primarily on polymer length or molar mass for largely linear polymers. Although many studies in animals and humans have shown complete excretion of MC, in vitro human fecal fermentation studies indicate that MC can be degraded and presumably lose some of its functionality. In this study, MC polymer distribution in the feces from rats fed a diet containing 8% methylcellulose were compared to the fed MC. The water-soluble polymers in the feces were separated by a size exclusion chromatography (SEC) and the polymer distributions determined by multiple angle laser light scattering (MALLS). Detection of the fluorescent MC-calcofluor complex was used to confirm the identity of the eluting MC peak. All dietary MC was recovered in the feces. There is a small shift (P < 0.06) in the weight-averaged molecular weight of polymer distribution of MC extracted from the feces to 2.71 +/- 0.15 x 10(5) g/mol from 3.15 +/- 0.02 x 10(5) g/mol in the standard. There is also an increase in the polydispersity from 1.21 in the standard to 1.8 in the fecal extract. The distribution of the substituted methoxylated glucose monomers by gas chromatography also confirms the stability of MC fed to rats. The amount of actual hydrolysis is estimated to be about 0.1 glycosidic linkage/molecule. MC is not easily determined by standard dietary fiber methods, and SEC with MALLS and/or fluorescence may be a useful alternative.

Animals↗

PubMed2004

Extrusion conditions affect chemical composition and in vitro digestion of select food ingredients.

An experiment was conducted to determine the effects of extrusion conditions on chemical composition and in vitro hydrolytic and fermentative digestion of barley grits, cornmeal, oat bran, soybean flour, soybean hulls, and wheat bran. Extrusion conditions altered crude protein, fiber, and starch concentrations of ingredients. Organic matter disappearance (OMD) increased for extruded versus unprocessed samples of barley grits, cornmeal, and soybean flour that had been hydrolytically digested. After 8 h of fermentative digestion, OMD decreased as extrusion conditions intensified for barley grits and cornmeal but increased for oat bran, soybean hulls, and wheat bran. Total short-chain fatty acid production decreased as extrusion conditions intensified for barley grits, soybean hulls, and soybean flour. These data suggest that the effects of extrusion conditions on ingredient composition and digestion are influenced by the unique chemical characteristics of individual substrates.

Avena↗

PubMed1990

Potential dental effects of infants' fruit drinks studied in vitro.

Eighteen different infants' drinks from five manufacturers were examined for their carbohydrate, calcium, phosphorus and acid contents, and their attack on tooth mineral. Seven of the drinks were compared with nine varieties of adults' soft drinks, and demineralization was studied with and without the presence of a cariogenic oral streptococcus. The influence of the acids already in the drinks in dissolving Ca and P outstripped that of any acid generated in these studies in vitro by microbial fermentation of the sugars they contained, giving an indication of their relative erosiveness rather than their cariogenic action. Various other features of the drinks relevant to dental health were identified. Titratable acid was a better guide than pH to their dental properties. Although there were considerable differences between the various infants' drinks, taken as a group, their acidity levels and demineralizing powers were generally lower than those of the adults' drinks.

Adult↗

PubMed1992

Guar, but not psyllium, increases breath methane and serum acetate concentrations in human subjects.

Guar and psyllium are fermented by human fecal bacteria in vitro. To see if they were fermented in vivo, eight subjects were studied over 3 separate days, in random order, while eating a polysaccharide-free diet. Twenty grams guar or psyllium, taken at breakfast, had no effect on breath hydrogen levels over 14 h. Mean breath methane and serum acetate concentrations after guar, 37 +/- 1 ppm and 93 +/- 6 mumol/L, respectively, were significantly greater than after control, 20 +/- 2 ppm (P less than 0.05) and 62 +/- 4 mumol/L (P less than 0.01), and psyllium, 20 +/- 2 ppm (P less than 0.05) and 78 +/- 6 mumol/L (P less than 0.05). Serum acetate increased after guar (area under curve 193 +/- 56 mumol.h/L; P less than 0.02) but decreased after psyllium and control. We conclude that guar is fermented in the human colon, producing rises in breath methane and serum acetate but not hydrogen. Although psyllium had no effect on hydrogen, methane, or acetate, this does not prove that it is not fermented.

Acetates↗

PubMed1994

Variability among atoxigenic Aspergillus flavus strains in ability to prevent aflatoxin contamination and production of aflatoxin biosynthetic pathway enzymes.

Five strains of Aspergillus flavus lacking the ability to produce aflatoxins were examined in greenhouse tests for the ability to prevent a toxigenic strain from contaminating developing cottonseed with aflatoxins. All atoxigenic strains reduced contamination when inoculated into developing bolls 24 h prior to the toxigenic strain. However, only one strain, AF36, was highly effective when inoculated simultaneously with the toxigenic strain. All five strains were able to inhibit aflatoxin production by the toxigenic strain in liquid fermentation. Thus, in vitro activity did not predict the ability of an atoxigenic strain to prevent contamination of developing bolls. Therefore, strain selection for competitive exclusion to prevent aflatoxin contamination should include evaluation of efficacy in developing crops prior to field release. Atoxigenic strains were also characterized by the ability to convert several aflatoxin precursors into aflatoxin B1. Four atoxigenic strains failed to convert any of the aflatoxin biosynthetic precursors to aflatoxins. However, the strain (AF36) most effective in preventing aflatoxin contamination in developing bolls converted all tested precursors into aflatoxin B1, indicating that this strain made enzymes in the aflatoxin biosynthetic pathway.

Aflatoxins↗

PubMed1987

Comparison of sweet white lupin seeds with soybean meal as a protein supplement for lactating dairy cows.

Data were from 45 Holstein cows (23 multiparous, 22 primiparous) assigned by calving date and parity within groups to one of two isonitrogenous (16% crude protein) diets. The diets were 50% forages (corn silage, alfalfa silage) and 50% concentrate, dry basis. In diet A, soybean meal supplied 34.2% of total crude protein; in diet B, ground sweet white lupin seeds provided 37.9% of total crude protein. Cows were fed once daily during the experimental period (d 4 to 116 postpartum). Cows fed lupins consumed significantly less dry matter, produced 1.8 kg/d less milk (but not significantly different), and had lower milk protein percent. Milk fat and total solids percents were similar. Reasons for reduced intake of cows fed lupins were not evident. Traces of alkaloids (.005% dry basis) were present in diet B. Combined results of in vitro continuous culture fermentation and in situ degradation measurements indicated that crude protein from lupins was more degradable than that of soybean meal. Poor performances of cows fed lupins could be partly due to a reduced true protein supply to the small intestine.

Animal Feed↗

PubMed2004

Effects of DL-malate on in vitro forage fiber digestion by mixed ruminal microorganisms.

The objective of this study was to evaluate the effects of 0, 4, 8, and 12 mM DL-malate on the in vitro mixed ruminal microorganism fermentation of alfalfa hay and Coastal bermudagrass hay. When alfalfa hay was the substrate, 4 and 8 mM DL-malate numerically increased propionate concentration, and 12 mM DL-malate increased (P < 0.10) propionate. All three concentrations of DL-malate decreased (P < 0.05) the acetate:propionate ratio. In Coastal bermudagrass hay fermentations, all three DL-malate concentrations increased (P < 0.05) propionate and decreased (P < 0.05) the acetate:propionate ratio, while 4 and 12 mM DL-malate numerically increased in vitro dry matter disappearance. When mixed ruminal microorganisms were incubated with 6.25 mM DL-lactic acid and alfalfa hay, 8 and 12 mM DL-malate increased (P < 0.05) final pH, and 12 mM DL-malate increased (P < 0.10) propionate and decreased (P < 0.10) the acetate:propionate ratio. DL-Malate treatment had little effect on in vitro dry matter disappearance. Addition of 8 and 12 mM DL-malate to Coastal bermudagrass hay plus DL-lactic acid fermentations increased (P < 0.05) final pH, and 8 mM DL-malate increased (P < 0.10) in vitro dry matter disappearance. Even though DL-malate treatment consistently increased final pH values in fermentations that included DL-lactic acid, there was not a corresponding increase in in vitro dry matter disappearance of either alfalfa hay or Coastal bermudagrass hay in the 48-h batch culture incubations.

Animals↗

PubMed2004

Studies of the in vitro intestinal metabolism of isoflavones aid in the identification of their urinary metabolites.

Soy isoflavones have recently gained considerable interest due to their possible health benefits. However, detailed studies on the metabolism of isoflavones are lacking. The aims of the investigation presented here were (1) to study the in vitro intestinal metabolism of isoflavones and their hydroxylated analogues 3'-OH-daidzein, 6-OH-daidzein, 8-OH-daidzein, and 3'-OH-genistein and (2) to characterize the structures of some earlier identified urinary metabolites of soy isoflavones, for which no authentic reference compounds have been available. Isoflavone standards (1-2 mg) were fermented with human fecal flora (16.7%) for 24 h. Metabolites formed during the fermentation were tentatively identified by interpretation of the mass spectra of trimethylsilylated compounds obtained by GC-MS. Compounds having hydroxyl groups at 5-position (i.e., genistein and 3'-OH-genistein) were completely converted to metabolites that could not be detected by the methods used in this study. The metabolism of daidzein and its hydroxylated analogues, 3'-OH-daidzein, 6-OH-daidzein, and 8-OH-daidzein, occurred to a much lesser extent. Minor amounts of reduced metabolites (i.e., isoflavanones and alpha-methyldeoxybenzoins) of these compounds were tentatively identified in fermentation extracts. The retention times and the mass spectra of reduced isoflavone metabolites, obtained from in vitro fermentations of pure compounds, were utilized to identify unknown urinary metabolites of soy isoflavones. Four novel isoflavone metabolites were identified in human urine collected after soy supplementation: 3' '-OH-O-desmethylangolensin, 3',4',7-trihydroxyisoflavanone, 4',7,8-trihydroxyisoflavanone, and 4',6,7-trihydroxyisoflavanone.

Feces↗