A series of in vitro experiments were completed to evaluate the potential of enzyme extracts, obtained from the white-rot fungi Trametes versicolor (TV1, TV2), Bjerkandera adusta (BA) and Fomes fomentarius (FF), to increase degradation of cell wall components of wheat straw. The studies were conducted as a completely randomized design and analysed using one-way ANOVA. Enzyme activities of the extracts, previously obtained from a liquid culture medium, were characterized in terms of laccase and peroxidase for ligninolytic activity. Carboxymethyl cellulase (CMCase) and avicell digesting cellulase (Avicelase) were used for cellulolytic enzyme assays. Wheat straw samples were incubated with enzyme extracts in a citrate buffer (pH 5.0) in a forced air oven at 25 °C for 6 days. In vitro NDF digestibility (IVNDFD), and the rate and extent of NDF fermentation, without and after incubation with the white-rot enzyme extracts, were determined using a gravimetric microbiological method and a gas production technique, respectively. Results from cell wall chemical composition showed that TV2 and BA enzyme extracts decreased NDF concentration (P<0.05) and that TV1 had higher activity (P<0.05) towards cellulose. There was an increase in IVNDFD (P<0.05), resulting from treatment of wheat straw with enzyme extracts from BA, TV1 and TV2, reaching a difference of 13% for TV2 (P<0.05), versus the non-treated straw control. Treatment with enzyme extract from TV2 caused increased gas production (P<0.05) after the first 20 h of incubation, and also increased the maximum rate of gas production, thus enhancing fermentation kinetics. This study indicates that enzyme extracts from white-rot fungi can be used to develop new approaches to overcome low digestibility of some plant cell walls. Utilization of different substrates to produce enzyme extracts can lead to production of viable ligninolytic complexes which could improve the nutritive value of fibrous feeds.

The objectives were to evaluate effects of phenolic acid content and composition on the digestibility of six meadow hays from Northern Portugal. Digestibility was assessed by gas production, in vitro and in situ degradation methods. Four cows fed diets at energy maintenance were used for in situ incubations and to provide rumen fluid for in vitro incubations. There were no relationships between phenolic acid concentrations and other cell wall components. The dry matter (DM) potential degradation (a+b) was positively related to the etherified fractions of ferulic acid (FAeth, P=0.012) and p-coumaric acid (PCAeth, P < 0.001) and to the total amount of ferulic acid (FAtotal; P=0.033). The insoluble, but potentially degradable, constant (b) of DM had a positive relationship with PCAeth (P=0.008). The in vitro neutral detergent fibre digestibility (IVNDFD), and the estimated asymptotic gas production of the second phase (A(2)), were positively related to the esterified fraction of ferulic acid (FAest, P < 0.05), FAeth (P < 0.01) and FAtotal (P < 0.05). The total amount of p-coumaric acid (PCAtotal) had a positive relationship with IVNDFD (P=0.047). In vitro DM digestibility (IVDMD) was positively correlated with FAeth and FAtotal (P < 0.05). In contrast, lignin (pm) concentrations negatively correlated with DM effective degradability (ED; P=0.005) and the maximum rate of gas production of the second phase (R mG?; P=0.009). Degradation kinetic constants i.e., (c) of DM and NDF tended to be negatively correlated to the phenolic acid concentrations, mainly with the PCAeth fraction (P=0.056 and P=0.006, respectively). The same trend occurred for the fractional rate of fermentation of the second phase (R2; P=0.09). Principal component analysis confirmed that lignin (pm) concentration (principal component 3) is one of the major limiting factors to in vitro DM digestibility of these hays. Thus it seems that for these meadow hays, both lignin (pm) content and cross-linkages between cell wall polymers influenced rate and extent of DM degradation. (c) 2006 Elsevier B.V. All rights reserved.

Fiber (NDF), starch, crude protein (CP), crude fat (CF), minerals and fatty acid composition of raw shelled chestnut kernels were determined on dry matter (DM) basis to chemically characterize the fruits of Castanea sativa in three regions of Trasos-Montes (Carrazedo de Montenegro, Vinhais and Vila Real). This study showed that chemical composition depends on cultivar and year, the interaction cultivar x year also being significant. The observed variation in starch and CP was mainly determined by cultivar while the remaining parameters were mostly affected by year and cultivar x year interaction. In general, chestnuts showed either high or medium concentrations of starch and NDF (60.7 +/- 5.3 and 22.5 +/- 5.5 g.100g(-1) DM, respectively). CF was consistently low (1.3 +/- 0.5 g.100g(-1) DM), showing however high percentage of C18:1 (25.7 +/- 4.5% of total fatty acid methyl esters - FAME) and of essential PUFA, namely C18:3 (6.6 +/- 1.7% FAME) and C18:2 (51.5 +/- 4.8% FAME). High levels of P and K (181.6 +/- 58.7 and 1202.7 +/- 351.4 mg.100g(-1) DM, respectively) were noticed in mineral composition. Among the studied cultivars, 'Lada' and 'Benfeita' presented greater nutritional characteristics due to the levels of CP(9.5 +/- 3.2 and 12.0 +/- 3.6 g.100g(-1) DM, respectively) and PUFA, particularly C18:3-omega 3 (8.4 +/- 0.7 and 9.0 +/- 2.3% FAME, respectively).

The effect of cellulose crystallinity on in vitro digestibility (IVD) and fermentation kinetics was investigated in samples of meadow hay and barley, wheat and rice straws. A saturated solution of potassium permanganate was used to isolate the celluloses, and their crystallinity was evaluated in a Fourier transform infrared spectrometer as the ratio of the observed transmittance at two distinct wave frequencies (alpha(1372)(-1)/alpha(2900)(-1)). IVD was determined after 48h of incubation, and the kinetics of fermentation was studied with fully automated gas production equipment. Despite significant differences (P < 0.05), crystallinity showed low variation (0.30-0.42) among celluloses and correlated positively with IVD (P < 0.05). Although positively correlated with maximal gas production (r = 0.91) and rates of fermentation, IVD (averaging 784 g kg(-1) organic matter (OM)) was also low for all samples as compared with the usually referred values for isolated celluloses. The lowest means were observed for meadow hay and rice straw. The cumulative gas production profiles were well described by a monophasic model (r(2) = 0.997, RSD (residual standard deviation) = 8.367), but all the curves had a lag phase varying from 3 to 6h. Cellulose isolated from rice straw showed the lowest maximal gas production (366mlg(-1) OM) and highest times to reach half of the maximal gas production and maximal rate. Transmission electron microscopy (TEM) of two cellulose samples showing considerable gas production at highest rates as compared with cellulose isolated from rice straw that provided the lowest gas production at the lowest rate revealed generalised bacterial colonisation, the presence of glycocalyx fibres and cell erosion in all samples. Although samples collected after 12 h of fermentation appeared to present lower microbial attack as compared with those incubated for 24h, TEM did not explain the observed differences in the data. Since crystallinity, IVD, gas production and rates of fermentation were shown to be associated with the nutritional availability of cellulose, further research dealing with extreme values of cellulose crystallinity may be important. (C) 2003 Society of Chemical Industry.

Kinetics of digestion of 12 forages previously fed ad libitum to mature ewes over a period of 8 weeks was studied in an automatic gas production system. Samples (≈400mg) were incubated in vitro with a mixed solution of rumen fluid for 72h and the volume of gas produced was recorded every 20min. Kinetics of fermentation was described by the exponential equation Y=a+b(1−e−c(t−L)) and by a mono- and a biphasic Michaelis–Menten equation Y=Ai/(1+(Bi/t)Ci). Constants of the exponential model were not significantly correlated to voluntary dry matter intake (DMI) and digestible dry matter intake (DDMI). The DMI and DDMI were best predicted through stepwise regression analysis using the constants of the biphasic Michaelis–Menten equation. The gas production constants A1/(2×B1) and C2 explained 60% of the variation of DMI, while A1/(2×B1), C2, A2/(2×B2) and A2 could account for 94% of the variation of DDMI. In vitro gas production at 8h explained 76 and 48% of the variation observed for DDMI and DMI, respectively.

Two studies were undertaken to evaluate the effects of cellulase and endoxylanase enzymes on the chemical composition and the fermentation characteristics of grass silage, using the gas production technique. Perennial ryegrass was ensiled in 1 litre glass containers for 90 days with cellulase (0·2 g/kg grass on fresh weight basis) and endoxylanase in the concentrations of 0·05 g/kg grass (Endox 0·05) and 0·2 g/kg grass (Endox 0·2). Subsequently, dried samples, ground through a 1 mm screen, were used in chemical analysis and gas production measurements. In Expt 1, the enzyme treatment significantly decreased silage NDF (P < 0·001), ADF (P < 0·001) and acetic acid (P < 0·01) concentrations and increased lactic acid (P < 0·001) production. In Expt 2, lower concentrations of NDF (P < 0·001) and ADF (P < 0·001) in treated silages resulted in increased sugar concentration (P < 0·001). In this experiment, butyric acid was detected. Addition of cellulase and Endox 0·05 enzymes did not alter silage digestibility. In both studies, cellulase and Endox 0·2 treatments tended to increase the rate of gas production within 10 hours of inoculation with rumen fluid whereas Endox 0·05 had no effect. The volume of gas produced was however greater for the untreated silage than for the enzyme-treated silage samples after 48 h of incubation. The strategy of applying cellulase and endoxylanase to the herbage in the ensiling process proved to be effective in modifying the chemical composition and increasing sugar concentration and the rate of gas production of the silages. Further research on the factors determining enzyme effectiveness is therefore suggested to elucidate the mechanisms leading to higher utilization of the released sugars.

Lipids from 23 shelled chestnut kernel samples, corresponding to 7 different cultivars of Castanea sativa Mill. from 3 geographical origins situated in the region of Tras-os-Montes (Portugal), were extracted and fractionated into polar and nonpolar classes. Fatty acid methyl esters of each class were determined by gas chromatography.For the total of the samples, unsaturated fatty acids averaged 72.2 % and 50.7 % of the polar and nonpolar lipid fractions, respectively. Linoleic (51.8 %), oleic (14.6 %) and palmitic (26.7 %) were the main fatty acids in the polar lipids, while the nonpolar lipids contained essentially about 34.3 % lauric, 14.6 % palmitic, 19.3 % oleic and 26.4 % linoleic acids. However, significant differences (P<0.05) among cultivars were observed for the fatty acid composition of both polar and nonpolar lipids.

Studies on triticale as a cereal for grain have been carried out since 1975 in the Northeast of Portugal. Because of its high biomass production, triticale has also been studied as a forage crop since 1979. These studies were designed to investigate whether rye, the major cereal in that region which sometimes is grown as a forage crop or oats, sown in cereal-legume mixtures, could advantageously be replaced by triticale.According to previous data, the line UTAD 36/85, from the UTAD triticale breeding programme, and the Polish cultivar Presto were selected for studying the effect of the seeding rate on forage yield and quality. The regional rye population Vila Pouca and the Portuguese oats cultivar Boa-Fe were used as testers.Dry matter yield, forage quality (in vitro organic dry matter digestibility and crude protein content) and number of stems, m(-2) were evaluated at the seeding rates of 175, 350, 525 and 700 viable seeds.m(-2). Over all variables, triticale line UTAD 36/85 had the highest mean of dry matter yield (8715 kg.ha(-1)) while the regional rye population Vila Pouca had the lowest (7315 kg.ha(-1)). At the rates of 175 and 350 viable seeds.m(-2) the average number of stems. m(-2) for all genotypes was similar but it increased with higher rates. The highest in vitro organic matter digestibility was observed on triticale cv. Presto, 66.6% at the seeding rate of 175 viable seeds.m(-2), and the lowest on oats cv. Boa-FC, 52.8% at the same seeding rate. The highest crude protein content was observed for all seeding rates on the rye population while the oats cultivar showed the lowest one.

The effect of increased osmolarity on bacterial adhesion was studied in samples of natural meadow hay and cellulose previously submitted to in vitro digestion. The samples were collected for transmission electron microscopy after undergoing an in vitro digestion process in which the inoculum osmolarity was increased from 240 mmol (control) to 422 +/- 30 mmol by adding sodium acetate, bicarbonate and chloride.Transmission electron microscopy showed generalized microbial adhesion of the bacteria to the cell wall and cellulose for all the situations studied. The presence of glycocalyx fibres and wide zones of erosion were evident. No synergistic or depressive effects of the sodium salts were observed. Since some evidence of the difficulty of bacterial attack on well packed structures was provided, this ultrastructural study seems to favour the hypothesis of low cellulose digestibility as a result of the structural arrangement of this polymer.