In vitro degradability of mature and immature leaves of tropical forage legumes differing in condensed tannin and non-starch polysaccharide content and composition

A study was designed to examine the relationships between protein, condensed tannin and cell wall carbohydrate content and composition and the nutritional quality of seven tropical legumes (Desmodium ovalifolium, Flemingia macrophylla, Leucaena leucocephala, L pallida, L macrophylla, Calliandra calothyrsus and Clitotia fairchildiana). Among the legume species studied, D ovalifolium showed the lowest concentration of nitrogen, while L leucocephala showed the highest. Fibre (NDF) content was lowest in C calothyrsus, L Leucocephala and L pallida and highest in L macrophylla, which had no measurable condensed tannins. The highest tannin concentration was found in C calothyrsus. Total non-structural polysaccharides (NSP) varied among legumes species (lowest in C calothyrsus and highest in D ovalifolium), and glucose and uronic acids were the most abundant carbohydrate constituents in all legumes. Total NSP losses were lowest in F macrophylla and highest in L leucocephala and L pallida. Gas accumulation and acetate and propionate levels were 50% less with F macrophylla and D ovalifolium as compared with L leucocephala. The highest levels of branched-chain fatty acids were observed with non-tanniniferous legumes, and negative concentrations were observed with some of the legumes with high tannin content (D ovalifolium and F macrophylla). Linear regression analysis showed that the presence of condensed tannins was more related to a reduction of the initial rate of gas production (0-48 h) than to the final amount of gas produced or the extent (144h) of dry matter degradation, which could be due to differences in tannin chemistry. Consequently, more attention should be given in the future to elucidating the impact of tannin structure on the nutritional quality of tropical forage legumes. (C) 2003 Society of Chemical Industry.

Influence of fibrolytic enzymes on the hydrolysis and fermentation of pure cellulose and xylan by mixed ruminal microorganisms in vitro

A series of in vitro studies was, conducted to determine the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixture (1:1 wt/wt) of both. The enzyme product (Liquicell 2500, Specialty Enzymes and Biochemicals, Fresno, CA) was derived from Trichoderma reesei and contained mainly xylanase and cellulase activities. Addition of enzyme (0.5, 2.55 and 5.1 muL/g of DM) in the absence of ruminal fluid increased (P < 0.001) the release of reducing sugars from xylan and the mixture after 20 h of incubation at 20degreesC. Incubations with ruminal fluid showed that enzyme (0.5 and 2.55 muL/g of DM) increased (P < 0.05) the initial (up to 6 h) xylanase, endoglucanase, and beta-D-glucosidase activities in the liquid fraction by an average of 85%. Xylanase and endoglucanase activities in the solid fraction also were increased (P < 0.05) by enzyme addition, indicating an increase in fibrolytic activity due to ruminal microbes. Gas production over 96 h of incubation was determined using a gas pressure measurement technique. Incremental levels of enzyme increased (P < 0.05) the rate of gas production of all substrates, suggesting that fermentation of cellulose and xylan was enzyme-limited. However, adding the enzyme at levels higher than 2.55 muL/g of DM failed to further increase the rate of gas production, indicating that the maximal level of stimulation was already achieved at lower enzyme concentrations. It was concluded that enzymes enhanced the fermentation of cellulose and xylan by a combination of pre- and postincubation effects (i.e., an increase in the release of reducing sugars during the pretreatment phase and an increase in the hydrolytic activity of the liquid and solid fractions of the ruminal fluid), which was reflected in a higher rate of fermentation.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - II. Effects on rate of acidification, fibre degradation during ensiling and rumen fermentation

A study was carried out to determine the influence of fibrolytic enzymes derived from mesophilic or thermophilic fungal sources, added at ensiling, on time-course fermentation characteristics and in vitro rumen degradation of maize silage. The mesophilic enzyme was a commercial product derived from Trichodenna reesei (L), whereas the thermophilic enzyme was a crude extract produced from Thermoascus aurantiacus (Ta) in this laboratory. The fungus was cultured using maize cobs as a carbon source. The resulting fermentation extract was deionised to remove sugars and characterised for its protein concentration, main and side enzymic activities, optimal pH, protein molecular mass and isoelectric point. In an additional study, both enzymes were added to maize forage (333.5 g DM/kg, 70.0, 469.8, 227.1 and 307.5 g/kg DM of CP, NDF, ADF and starch, respectively) at two levels each, normalized according to xylanase activity, and ensiled in 0.5 kg capacity laboratory minisilos. Duplicate silos were opened at 2, 4, 8, 15, and 60 days after ensiling, and analysed for chemical characteristics. Silages from 60 days were bulked and in vitro gas production (GP) and organic matter degradability (OMD) profiles evaluated using the Reading Pressure Technique (RPT), in a completely randomised design. The crude enzyme extract contained mainly xylanase and endoglucanase activities, with very low levels of exoglucanase, which probably limited hydrolysis of filter paper. The extract contained three major protein bands of between 29 and 55 kDa, with mainly acidic isoelectric points. Ensiling maize with enzymes lowered (P < 0.05) the final silage pH, with this effect being observed throughout the ensiling process. All enzyme treatments reduced (P < 0.05) ADF contents. Treatments including Ta produced more gas (P < 0.05) than the controls after 24 h incubation in vitro, whereas end point gas production at 96 h was not affected. Addition of Ta increased (P < 0.01) OMD after 12 h (410 and 416 g/kg versus 373 g/kg), whereas both L and Ta increased (P < 0.05) OMD after 24 h. Addition of enzymes from mesophilic or thermophilic sources to maize forage at ensiling increased the rate of acidification of the silages and improved in vitro degradation kinetics, suggesting an improvement in the nutritive quality. (C) 2003 Elsevier B.V All rights reserved.

Use of fibrolytic enzymes to improve the nutritive value of ruminant diets - A biochemical and in vitro rumen degradation assessment

Two commercial enzyme products, Depol 40 (D) and Liquicell 2500 (L), were characterised from a biochemical standpoint and their potential to improve rumen degradation of forages was evaluated in vitro. Enzyme activities were determined at pH 5.5 and 39 degreesC. Analysis of the enzyme activities indicated that L contained higher xylanase and endoglucanase, but lower exoglucanase, pectinase and alpha-amylase activities than D. The Reading Pressure Technique (RPT) was used to investigate the effect of enzyme addition on the in vitro gas production (GP) and organic matter degradation (OMD) of alfalfa (Medicago sativa L.) stems and leaves. A completely randomised design with factorial arrangement of treatments was used. Both alfalfa fractions were untreated or treated with each enzyme at four levels, 20 h before incubation with rumen fluid. Each level of enzyme provided similar amounts of filter paper (D1, L1), endoglucanase (D2, L2), alpha-L-arabinofuranosidase (D3, L3) and xylanase units (D4, L4) per gram forage DM. Enzymes increased the initial OMD in both fractions, with improvements of up to 15% in leaves (D4) and 8% in stems (L2) after 12 h incubation. All enzyme treatments increased the extent of degradation (96 h incubation) in the leaf fractions, but only L2 increased final OMD in the stems. Direct hydrolysis of forage fractions during the pre-treatment period did not fully account for the magnitude of the increases in OMD, suggesting that the increase in rate of degradation was achieved through a combined effect of direct enzyme hydrolysis and synergistic action between the exogenous (applied) and endogenous (rumen) enzymes. (C) 2003 Elsevier Science B.V. All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - I. Effects of ensiling temperature, enzyme source and addition level

A series of experiments was completed to investigate the impact of addition of enzymes at ensiling on in vitro rumen degradation of maize silage. Two commercial products, Depot 40 (D, Biocatalysts Ltd., Pontypridd, UK) and Liquicell 2500 (L, Specialty Enzymes and Biochemicals, Fresno, CA, USA), were used. In experiment 1, the pH optima over a pH range 4.0-6.8 and the stability of D and L under changing pH (4.0, 5.6, 6.8) and temperature (15 and 39 degreesC) conditions were determined. In experiment 2, D and L were applied at three levels to whole crop maize at ensiling, using triplicate 0.5 kg capacity laboratory minisilos. A completely randomized design with a factorial arrangement of treatments was used. One set of treatments was stored at room temperature, whereas another set was stored at 40 degreesC during the first 3 weeks of fermentation, and then stored at room temperature. Silages were opened after 120 days. Results from experiment I indicated that the xylanase activity of both products showed an optimal pH of about 5.6, but the response differed according to the enzyme, whereas the endoglucanase activity was inversely related to pH. Both products retained at least 70% of their xylanase activity after 48 h incubation at 15 or 39 degreesC. In experiment 2, enzymes reduced (P < 0.05) silage pH, regardless of storage temperature and enzyme level. Depol 40 reduced (P < 0.05) the starch contents of the silages, due to its high alpha-amylase activity. This effect was more noticeable in the silages stored at room temperature. Addition of L reduced (P < 0.05) neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents. In vitro rumen degradation, assessed using the Reading Pressure Technique (RPT), showed that L increased (P < 0.05) the initial 6 h gas production (GP) and organic matter degradability (OMD), but did not affect (P > 0.05) the final extent of OMD, indicating that this preparation acted on the rumen degradable material. In contrast, silages treated with D had reduced (P < 0.05) rates of gas production and OMD. These enzymes, regardless of ensiling temperature, can be effective in improving the nutritive quality of maize silage when applied at ensiling. However, the biochemical properties of enzymes (i.e., enzymic activities, optimum pH) may have a crucial role in dictating the nature of the responses. (C) 2003 Elsevier B.V. All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - III. Comparison of enzymes derived from psychrophilic, mesophilic or thermophilic sources

A completely randomised study was completed to examine the influence of fibrolytic enzymes derived from psychrophilic, (F), mesophilic, (L) or thermophilic (Ta) sources, applied at ensiling, on the chemical characteristics and in vitro rumen fermentation of maize silage, assessed using the Reading Pressure Technique (RPT). Treatments, all in triplicate, consisted of untreated maize forage or treated with preparations F, L, Ta or a mixture (1: 1, v/v) of F and L (FL), at two levels each, and ensiled for 210 days in plastic mini-silos. Addition of enzymes L decreased (P < 0.05) silage pH relative to the control, whereas enzyme Ta tended (P < 0.10) to reduce it. Preparations F, L and Ta tended to reduce (P < 0.10) the fibre contents of the silages, with effects being attributable to a decrease in the cellulose fraction. Starch contents were reduced (P < 0.05) in the treatments including enzyme F. End-point (96 h) gas production (GP) values did not differ among treatments, suggesting that enzymes did not change the total amount of fermentable substrate. However, consistent with the decrease in starch contents, adding enzyme F reduced (P < 0.05) GP at most incubation times. Addition of enzymes increased (P < 0.05) the initial (6 h) organic matter degradation (OMD) levels in all but one treatment (F), with increases of 14, 19, and 26% for preparations L, Ta, and FL, respectively, averaged across levels. Furthermore, the addition of enzymes increased (P < 0.05) the soluble OM losses, however, these increases did not fully account for the initial increase in OMD. The latter suggests that enzymes increased solubility and also altered silage structure, making it more amenable to degradation by ruminal microorganisms. As a result of the increase in OMD, without a concomitant increase in GP, the fermentation efficiency was greatly increased (P < 0.05) in enzyme treatments. Addition of enzymes to maize at ensiling, particularly those from the mesophilic and thermophilic sources used here, have the potential to increase the initial rate of silage OMD. (C) 2003 Elsevier B.V. All rights reserved.

Alternative methodologies - stretching the in vitro box

Current gas-based in vitro evaluation systems are extremely powerful research techniques. However they have the potential to generate a great deal more than simple fermentation dynamics. Details from four experiments are presented in which adaptation, and novel application, of an in vitro system allowed widely differing objectives to be examined. In the first two studies, complement methodologies were utilised. In such assays, an activity or outcome is inferred through the occurrence of a secondary event rather than by direct observation. Using an N-deficient incubation medium, the increase in starch fermentation, when supplemented with individual amino acids (i.e., known level of N) relative to that of urea (i.e., known quantity and N availability), provided an estimate of their microbial utilisation. Due to the low level of response observed with some arnino acids (notably methionine and lysine), it was concluded, that they may not need to be offered in a rumen-inert form to escape rumen microbial degradation. In another experiment, the extent to which degradation of plant cell wall components was inhibited by lipid supplementation was evaluated using fermentation gas release profiles of washed hay. The different responses due to lipid source and level of inclusion suggested that the degree of rumen protection required to ameliorate this depression was supplement dependent. That in vitro inocula differ in their microbial composition is of little interest per se, as long as the outcome is the same (i.e., that similar substrates are degraded at comparable rates and end-product release is equivalent). However where a microbial population is deficient in a particular activity, increasing the level of inoculation will have no benefit. Estimates of hydrolytic activity were obtained by examining fermentation kinetics of specific substrates. A number of studies identified a fundamental difference between rumen fluid and faecal inocula, with the latter having a lower fibrolytic activity, which could not be completely attributed to microbial numbers. The majority of forage maize is offered as an ensiled feed, however most of the information on which decisions such as choice of variety, crop management and harvesting date are made is based on fresh crop measurements. As such, an attempt was made to estimate ensiled maize quality from an in vitro analysis of the fresh crop. Fermentation profiles and chemical analysis confirmed changes in crop composition over the growing season, and loss of labile carbohydrates during ensiling. In addition, examination of degradation residues allowed metabolizable energy (ME) contents to be estimated. Due to difficulties associated with starch analysis, the observation that this parameter could be predicted by difference (together with an assumed degradability), allowed an estimate of ensiled maize ME to be developed from fresh material. In addition, the contribution of the main carbohydrates towards ME showed the importance of delaying harvest until maximum starch content has been achieved. (c) 2005 Elsevier B.V. All rights reserved.

In vitro determination of prebiotic properties of oligosaccharides derived from an orange juice manufacturing by-product stream

Fermentation properties of oligosaccharides derived from orange peel pectin were assessed in mixed fecal bacterial culture. The orange peel oligosaccharide fraction contained glucose in addition to rhamnogalacturonan and xylogalacturonan pectic oligosaccharides. Twenty-four-hour, temperature- and pH-controlled, stirred anaerobic fecal batch cultures were used to determine the effects that oligosaccharides derived from orange products had on the composition of the fecal microbiota. The effects were measured through fluorescent in situ hybridization to determine changes in bacterial populations, fermentation end products were analyzed by high-performance liquid chromatography to assess short-chain fatty acid concentrations, and subsequently, a prebiotic index (PI) was determined. Pectic oligosaccharides (POS) were able to increase the bifidobacterial and Eubacterium rectale numbers, albeit resulting in a lower prebiotic index than that from fructo-oligosaccharide metabolism. Orange albedo maintained the growth of most bacterial populations and gave a PI similar to that of soluble starch. Fermentation of POS resulted in an increase in the Eubacterium rectale numbers and concomitantly increased butyrate production. In conclusion, this study has shown that POS can have a beneficial effect on the fecal microflora; however, a classical prebiotic effect was not found. An increase in the Eubacterium rectale population was found, and butyrate levels increased, which is of potential benefit to the host.

Fermentation of resistant starches: influence of in vitro models on colon carcinogenesis

Resistant starch type 2 (RS2) and type 3 (RS3) containing preparations were digested using a batch (a) and a dynamic in vitro model (b). Furthermore, in vivo obtained indigestible fractions from ileostomy patients were used (c). Subsequently these samples were fermented with human feces with a batch and a dynamic in vitro method. The fermentation supernatants were used to treat CAC02 cells. Cytotoxicity, anti-genotoxicity against hydrogen peroxide (comet assay) and the effect on barrier function measured by trans-epithelial electrical resistance were determine. Dynamically fermented samples led to high cytotoxic activity, probably due to additional compounds added during in vitro fermentation. As a consequence only batch fermented samples were investigated further. Batch fermentation of RS resulted in an anti-genotoxic activity ranging from 9-30% decrease in DNA damage for all the samples, except for RS2-b. It is assumed that the changes in RS2 structures due to dynamic digestion resulted in a different fermentation profile not leading to any anti-genotoxic effect. Additionally, in vitro batch fermentation of RS caused an improvement in integrity across the intestinal barrier by approximately 22% for all the samples. We have demonstrated that batch in vitro fermentation of RS2 and RS3 preparations differently pre-digested are capable of inhibiting the initiation and promotion stage in colon carcinogenesis in vitro.

Safety evaluation of oligofructose: 13 Week rat study and in vitro mutagenicity

Oligofructose (OF), comprised of fructose oligomers with a terminal glucose unit, is a family Of oligosaccharides derived from the hydrolysis of inulin. Consumption of OF in animals and humans increases colonic bifidobacteria levels. The present study evaluates the safety of OF in both a 13 week rat feeding Study and Using in Vitro mutagenicity tests. Fecal bifidobacteria levels were also determined by in situ hybridization to assess a biological function of OF. Rats received either a control diet OF diets containing one of four doses of OF. Total, HDL, and LDL-cholesterol levels were significantly lower at several time points during the study in groups receiving OF compared to controls with the largest effects Occurring in the high dose male animals. Weight gain in the male high dose group was significantly lower at early time points compared to controls but]lot Significantly different at the end of study. As expected, cecal weights increased in a dose-related manner and fecal bifidobacteria levels also demonstrated a dose-related increase. There were no consistent differences in gross pathology or histopathology related to dietary OF. OF did not induce a positive response in the Ames test or chromosomal aberration test with CHO cells. These results demonstrate no adverse effects of OF. (C) 2008 Elsevier Ltd. All rights reserved.

In vitro fermentation of sugar beet arabinan and arabino-oligosaccharides by the human gut microflora

Aims: To determine the fermentation profiles by human gut bacteria of arabino-oligosaccharides of varying degree of polymerization. Materials and Methods: Sugar beet arabinan was hydrolyzed with a commercial pectinase and eight fractions, of varying molecular weight, were isolated by gel-filtration chromatography. Hydrolysis fractions, arabinose, arabinan and fructo-oligosaccharides were fermented anaerobically by gut bacteria. Total bacteria, bifidobacteria, bacteroides, lactobacilli and the Clostridium perfringens/histolyticum sub. grp. were enumerated using fluorescent in situ hybridization. Results: Bifidobacteria were stimulated to different extents depending on molecular weight, i.e. maximum increase in bifidobacteria after 48 h was seen on the lower molecular weight fractions. Lactobacilli fluctuated depending on the initial inoculum levels. Bacteroides numbers varied according to fraction; arabinan, arabinose and higher oligosaccharides (degree of polymerization, dp > 8) resulted in significant increases at 24 h. Only carbohydrate mixtures with dp of 1-2 resulted in significant increases at 48 h (log 8.77 +/- 0.23). Clostridia decreased on all substrates. Conclusions: Arabino-oligosaccharides can be considered as potential prebiotics. Significance and Impact of the Study: Arabinan is widely available as it is a component of sugar beet pulp, a co-product from the sugar beet industry. Generation of prebiotic functionality from arabinan would represent significant added value to a renewable resource.

Synthesis and biological in vitro evaluation of novel PEG-psoralen conjugates

Psoralens are well-known photosensitizers, and 8- methoxypsoralen and 4,5',8-trimethylpsoralen are widely used in photomedicine as "psoralens plus UVA therapy" (PUVA), in photopheresis, and in sterilization of blood preparations. In an attempt to improve the therapeutic efficiency of PUVA therapy and photopheresis, four poly(ethylene glycol) (PEG)-psoralen conjugates were synthesized to promote tumor targeting by the enhanced permeability and retention (EPR) effect. Peptide linkers were used to exploit specific enzymatic cleavage by lysosomal proteases. A new psoralen, 4-hydroxymethyl-4', 8-dimethylpsoralen (6), suitable for polymer conjugation was synthesized. The hydroxy group allowed exploring different strategies for PEG conjugation, and linkages with different stability such ester or urethanes were obtained. PEG (5 kDa) was covalently conjugated to the new psoralen derivative using four different linkages, namely, (i) direct ester bond (7), (ii) ester linkage with a peptide spacer (8), (iii) a carbamic linker (9), and (iv) a carbamic linker with a peptide spacer (12). The stability of these new conjugates was assessed at different pHs, in plasma and following incubation with cathepsin B. Conjugates 7 and 8 were rapidly hydrolyzed in plasma, while 9 was stable in buffer and in the presence of cathepsin B. As expected, only the conjugates containing the peptide linker released the drug in presence of cathepsin B. In vitro evaluation of the cytotoxic activity in the presence and absence of light was carried out in two cell lines (MCF-7 and A375 cells). Conjugates 7 and 8 displayed a similar activity to the free drug (probably due to the low stability of the ester linkage). Interestingly, the conjugates containing the carbamate linkage (9 and 12) were completely inactive in the dark (IC50 > 100 mu M in both cell lines). However, antiproliferative activity become apparent after UV irradiation. Conjugate 12 appears to be the most promising for future in vivo evaluation, since it was relatively stable in plasma, which should allow tumor targeting and drug release to occur by cathepsin B-mediated hydrolysis.

In vitro evaluation of the microbiota modulation abilities of different sized whole oat grain flakes.

Epidemiological studies and healthy eating guidelines suggest a positive correlation between ingestion of whole grain cereal and food rich in fibre with protection from chronic diseases. The prebiotic potential of whole grains may be related, however, little is known about the microbiota modulatory capability of oat grain or the impact processing has on this ability. In this study the fermentation profile of whole grain oat flakes, processed to produce two different sized flakes (small and large), by human faecal microbiota was investigated in vitro. Simulated digestion and subsequent fermentation by gut bacteria was investigated using pH controlled faecal batch cultures inoculated with human faecal slurry. The different sized oat flakes, Oat 23’s (0.53–0.63 mm) and Oat 25’s/26’s (0.85–1.0 mm) were compared to oligofructose, a confirmed prebiotic, and cellulose, a poorly fermented carbohydrate. Bacterial enumeration was carried out using the culture independent technique, fluorescent in situ hybridisation, and short chain fatty acid (SCFA) production monitored by gas chromatography. Significant changes in total bacterial populations were observed after 24 h incubation for all substrates except Oat 23’s and cellulose. Oats 23’s fermentation resulted in a significant increase in the Bacteroides–Prevotella group. Oligofructose and Oats 25’s/26’s produced significant increases in Bifidobacterium in the latter stages of fermentation while numbers declined for Oats 23’s between 5 h and 24 h. This is possibly due to the smaller surface area of the larger flakes inhibiting the simulated digestion, which may have resulted in increased levels of resistant starch (Bifidobacterium are known to ferment this dietary fibre). Fermentation of Oat 25’s/26’s resulted in a propionate rich SCFA profile and a significant increase in butyrate, which have both been linked to benefiting host health. The smaller sized oats did not produce a significant increase in butyrate concentration. This study shows for the first time the impact of oat grain on the microbial ecology of the human gut and its potential to beneficially modulate the gut microbiota through increasing Bifidobacterium population.