Influence of microbial probiotics on ruminant health and nutrition: sources, mode of action and implications.

Globally, ruminant production contributes immensely to the supply of the highest quality and quantity of proteins for human consumption, sustenance of livelihoods, and attainment of food security. Nevertheless, the phasing out of antibiotics in animal production has posed a myriad of challenges, including poor growth, performance and nutrient utilization, pathogen colonization, dysbiosis, and food safety issues in ruminants. Probiotics (direct-fed microbials), comprising live microbial strains that confer health and nutritional benefits to the host when administered in appropriate quantities, are emerging as a viable, safe, natural and sustainable alternative to antibiotics. Although the mechanisms of action exerted by probiotics on ruminants are not well elucidated, dietary probiotic dosage to ruminants enhances development and maturation, growth and performance, milk production and composition, nutrient digestibility, feed efficiency, pathogen reduction, and mitigation of gastrointestinal diseases. However, the beneficial response to probiotic supplementation in ruminants is not consistent, being dependent on the microbial strain selected, combination of strains, dose, time and frequency of supplementation, diet, animal breed, physiological stage, husbandry practice, and farm management. Nonetheless, several studies have recently reported beneficial effects of probiotics on ruminant performance, health and production. This review conclusively re-iterates the need for probiotics inclusion for the sustainability of ruminant production. Considering the role that ruminants play in food production and employment, global acceptance of sustainable ruminant production through supplementation with probiotics will undoubtedly ensure food security and food safety for the world. © 2021 Society of Chemical Industry.

Chemical composition, ruminal degradation kinetics, and methane production (in vitro) of winter grass species.

BACKGROUND: Information about the nutritive value, dry matter (DM) digestibility, and methane (CH4 ) emission potential of grass species is required for their optimal utilization in ruminant rations. The present study was designed: (i) to quantify the nutrient profile, mineral composition and in vitro dry matter digestibility (IVDMD) of winter grass species commonly available in northern Pakistan; and (ii) to measure the in vitro gas production (IVGP) and CH4 emission of the grass species during 72 h in vitro ruminal fermentation. Seven grass species, namely, Cenchrus ciliaris, Setaria anceps, Panicum antidotale, P. maximum, Pennisetum purpureum, Pennisetum orientale, and Atriplex lentiformis were assessed. RESULTS: A high level of variability (P < 0.001) was observed among grass species for the content of all measured nutrients, IVDMD, IVGP, and CH4 -production. Notably, the content (g kg-1 DM) of crude protein varied from 59.8 to 143.3, neutral detergent fiber from 560.3 to 717.9, IVDMD from 375.1 to 576.2, and 72 h cumulative IVGP from 97.6 to 227.4 mL g-1 organic matter (OM) and CH4 from 48 to 67 mL g-1 OM. Among the grasses, P. antidotale had greater content (g kg-1 DM) of crude protein (CP) (143.3), IVDMD (576.2), and 72 h cumulative IVGP (227.4 mL g-1 OM), and produced the smallest amount of total CH4 (48 mL g-1 OM) during 72 h fermentation. In contrast, A. lentiformis had the lowest content (g kg-1 DM) of CP (59.8), IVDMD (375.1), 72 h cumulative IVGP (97.6 mL g-1 OM), and produced a greater amount of total CH4 (67 mL g-1 OM) during 72 h fermentation. CONCLUSION: The findings of the current study highlight that it is possible to select and further develop grass species with high nutritional value and lower CH4 -production, which can improve livestock productivity, farm profitability, and long-term environment sustainability. © 2020 Society of Chemical Industry.

Relationships between chemical composition and in vitro gas production parameters of maize leaves and stems.

This study investigated the chemical composition (proximate and Van Soest analysis) and in vitro gas production parameters of maize leaves and stems separately, and related the in vitro gas production parameters with the chemical composition, of thirteen maize cultivars. After harvest in September 2016, all plants were separated into two morphological fractions: leaves and stems. The crude protein (CP) content was greater, and the ratio of acid detergent lignin (ADL) to potentially rumen degradable fibre (calculated as the difference between neutral detergent fibre and ADL; ADL:pRDF) was lower in the leaves than in the stems in all 13 cultivars. For the leaves, the cumulative gas production between 3 and 20 hr (A2), representing cell wall fermentation in the rumen fluid, and the cumulative 72-hr gas production (GP72), representing total organic matter (OM) degradation, were moderately to weakly correlated with the chemical composition, including hemicellulose, cellulose, ADL and CP content (R2  < 0.40), whilst the best relationship between the half-time value (B2), representing the rate of cell wall degradation, and chemical composition had an R2 of 0.63. For the stems, the best relationship between A2, B2 and GP72 with chemical composition was greater (R2  ≥ 0.74) and the best relationship included hemicellulose (A2 only), cellulose and ADL (GP72 and A2 only) contents. In conclusion, maize leaves and stems differed in chemical composition, in particular CP content and ADL:pRDF. The A2 and GP72 of the stems, but not of the leaves, were highly correlated with the chemical composition, indicating that the cell wall and OM degradation of maize stems can be better predicted by its chemical composition.

Isolipidic replacement of krabok oil by whole krabok seed reduces in vitro methanogenesis, but negatively affects fermentation.

The background of the current in vitro study involves the issue of methane (CH4 ) production inherent to rumen fermentation. One of the dietary strategies to reduce enteric CH4 production by ruminants involves the supplementation of medium-chain fatty acids in diets. As such, oils containing high amounts of MCFA, such as coconut, palm kernel and krabok oil, are of much interest to formulate energy efficient and environmentally friendly rations for ruminants. Krabok oil (KO) reduces methanogenesis, but the appropriate inclusion level of dietary KO is unclear. We therefore investigated the dose-response relationship between krabok oil and CH4 production. In practice, the use of whole krabok seed (WKS), instead of KO, is easier, but the efficacy of WKS to inhibit methanogenesis was hitherto unknown. Thus, we also investigated whether WKS provides an alternative tool to inhibit CH4 production. The experimental substrates contained either KO, WKS, the residue of WKS after fat extraction residue (FER) or FER + KO. Appropriate amounts of WKS or its derivatives were added to a basal substrate so as to attain either a low, medium or high content of KO, that is, 37-46, 90-94 and 146-153 g/kg dry matter respectively. The experimental substrates were formulated to keep the amounts of incubated fat-free OM, crude protein, neutral detergent fibre and acid detergent fibre constant in order to avoid biased results through potential differences in fermentability between WKS and its derivatives, and the basal substrate. The latter resembled the ingredient composition of a total mixed ration commonly used in Thai dairy cows. Fully automated gas production (GP) equipment was used to measure gas- and CH4 production. Irrespective of the type of substrate (p ≥ .115), both the absolute (ml/g fat-free OM) and relative (% of total GP) CH4 production was reduced at the highest inclusion level of WKS or its derivatives (p ≤ .019). Total GP (ml/g fat-free OM), however, was reduced after incubation of FER, FER + KO, and WKS, but not KO, at the highest inclusion level of the respective substrates (p = .019). Volatile fatty acids were likewise affected (p ≤ .001). Krabok oil can inhibit CH4 production but only when the dietary KO content is at least 9.4% (DM). Supplementation of KO in the form of WKS, however, is considered not opportune because the fat extracted residue of WKS is poorly degraded during fermentation.

Improving ruminal digestibility of various wheat straw types by white-rot fungi.

BACKGROUND: This study investigated the ruminal degradability of various wheat straw types by the white-rot fungi Ceriporiopsis subvermispora (CS) and Lentinula edodes (LE). Different cultivars (CV) of wheat straw at different maturity stages (MS) were treated with the fungi for 7 weeks and assessed for chemical composition and in vitro gas production (IVGP). RESULTS: Both fungi showed a more pronounced degradation of lignin on a more mature straw (MS3; 89.0%) in comparison with the straw harvested at an earlier stage (MS1; 70.7%). Quantitative pyrolysis coupled to gas chromatography and mass spectrometry, using 13 C lignin as an internal standard 13 C-IS Py-GC/MS revealed that lignin in more mature straw was degraded and modified to a greater extent. In contrast, cellulose was less degraded in MS3, as compared to MS1 (8.3% versus 14.6%). There was no effect of different MS on the IVGP of the fungus-treated straws. Among the different straw cultivars, the extent of lignin degradation varied greatly (47% to 93.5%). This may explain the significant (P < 0.001) effect of cultivar on the IVGP of the fungal-treated straws. Regardless of the factors tested, both fungi were very capable of improving the IVGP of all straw types by 15.3% to 47.6%, (as compared to untreated straw), with CS performing better than LE - on different MS (33.6% versus 20.4%) and CVs (43.2% versus 29.1%). CONCLUSION: The extent of lignin degradation caused by fungal treatment was more pronounced on the more mature and lignified straw, while variable results were obtained with different cultivars. Both fungi were capable of improving the IVGP of various straw types. © 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Evaluation of fungal degradation of wheat straw cell wall using different analytical methods from ruminant nutrition perspective.

BACKGROUND: White rot fungi have been used to improve the nutritive value of lignocellulose for ruminants. In feed analysis, the Van Soest method is widely used to determine the cell wall contents. To assess the reliability of this method (Method A) for determination of cell wall contents in fungal-treated wheat straw, we compared a combined monosaccharide analysis and pyrolysis coupled to gas chromatography with mass spectrometry (Py-GC/MS) (Method B). Ruminal digestibility, measured as in vitro gas production (IVGP), was subsequently used to examine which method explains best the effect of fungal pretreatment on the digestibility of wheat straw. RESULTS: Both methods differed considerably in the mass recoveries of the individual cell wall components, which changed on how we assess their degradation characteristics. For example, Method B gave a higher degradation of lignin (61.9%), as compared to Method A (33.2%). Method A, however, showed a better correlation of IVGP with the ratio of lignin to total structural carbohydrates, as compared to Method B (Pearson's r of -0.84 versus -0.69). Nevertheless, Method B provides a more accurate quantification of lignin, reflecting its actual modification and degradation. With the information on the lignin structural features, Method B presents a substantial advantage in understanding the underlying mechanisms of lignin breakdown. Both methods, however, could not accurately quantify the cellulose contents - among others, due to interference of fungal biomass. CONCLUSION: Method A only accounts for the recalcitrant residue and therefore is more suitable for evaluating ruminal digestibility. Method B allows a more accurate quantification of cell wall, required to understand and better explains the actual modification of the cell wall. The suitability of both methods, therefore, depends on their intended purposes. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Preservation of Ceriporiopsis subvermispora and Lentinula edodes treated wheat straw under anaerobic conditions.

BACKGROUND: No attention has been paid so far to the preservation of fungal-treated lignocellulose for longer periods. In the present study, we treated wheat straw (WS) with the white-rot fungi Ceriporiopsis subvermispora and Lentinula edodes for 8 weeks and assessed changes in pH, chemical composition and in vitro gas production (IVGP) weekly. Fungal-treated WS was also stored for 64 days 'as is', with the addition of lactic acid bacteria (LAB) or with a combination of LAB and molasses in airtight glass jars mimicking ensiling conditions. RESULTS: Both fungi significantly reduced the lignin and hemicellulose content of WS, and increased the cellulose content. The IVGP increased with increasing time of incubation, indicating the increase in digestibility. Both fungi lowered the pH of WS under 4.3, which guarantees an initial and stable low pH during anaerobic storage. Minor changes in fibre composition and IVGP were observed for stored L. edodes treated WS, whereas no change occurred for C. subvermispora. CONCLUSION: It is possible to conserve C. subvermispora and L. edodes treated straw under anaerobic condition without additives up to 64 days. This finding is important for practical application to supply fungi-treated feed to ruminant animals for a prolonged period. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Lignin composition is more important than content for maize stem cell wall degradation.

BACKGROUND: The relationship between the chemical and molecular properties - in particular the (acid detergent) lignin (ADL) content and composition expressed as the ratio between syringyl and guaiacyl compounds (S:G ratio) - of maize stems and in vitro gas production was studied in order to determine which is more important in the degradability of maize stem cell walls in the rumen of ruminants. Different internodes from two contrasting maize cultivars (Ambrosini and Aastar) were harvested during the growing season. RESULTS: The ADL content decreased with greater internode number within the stem, whereas the ADL content fluctuated during the season for both cultivars. The S:G ratio was lower in younger tissue (greater internode number or earlier harvest date) in both cultivars. For the gas produced between 3 and 20 h, representing the fermentation of cell walls in rumen fluid, a stronger correlation (R2 = 0.80) was found with the S:G ratio than with the ADL content (R2 = 0.68). The relationship between ADL content or S:G ratio and 72-h gas production, representing total organic matter degradation, was weaker than that with gas produced between 3 and 20 h. CONCLUSION: The S:G ratio plays a more dominant role than ADL content in maize stem cell wall degradation. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Potential use of cowpea (Vigna unguiculata (L.) Walp.) stover treated with white-rot fungi as rabbit feed.

BACKGROUND: Lignin inhibitory effects within the cell wall structure constitute a serious drawback in maximizing the utilization of fibrous feedstuffs in animal feeding. Therefore treatments that promote efficient delignification of these materials must be applied. This study evaluated the potential of white-rot fungi to upgrade the nutritive value of cowpea stover for rabbit feeding. RESULTS: There was an increase in the crude protein content of all substrates as a result of fungi treatments, reaching a net gain of 13% for Pleurotus citrinopileatus incubation. Overall, net losses of dry and organic matter occurred during fungi treatments. Although the fiber content remained identical, higher consumption of cell wall contents was measured for P. citrinopileatus incubation (between 40 and 45%). The incubation period did not influence lignin degradation for any of the fungi treatments. Differences within the fungal degradation mechanisms indicate that P. citrinopileatus treatment was most effective, enhancing in vitro organic matter digestibility by around 30% compared with the control. CONCLUSION: Treatment of cowpea stover with P. citrinopileatus led to an efficient delignification process which resulted in higher in vitro organic matter digestibility, showing its potential in the nutritional valorization of this feedstuff. © 2017 Society of Chemical Industry.

Nutritive value of maize silage in relation to dairy cow performance and milk quality.

Maize silage has become the major forage component in the ration of dairy cows over the last few decades. This review provides information on the mean content and variability in chemical composition, fatty acid (FA) profile and ensiling quality of maize silages, and discusses the major factors which cause these variations. In addition, the effect of the broad range in chemical composition of maize silages on the total tract digestibility of dietary nutrients, milk production and milk composition of dairy cows is quantified and discussed. Finally, the optimum inclusion level of maize silage in the ration of dairy cows for milk production and composition is reviewed. The data showed that the nutritive value of maize silages is highly variable and that most of this variation is caused by large differences in maturity at harvest. Maize silages ensiled at a very early stage (dry matter (DM) < 250 g kg(-1)) were particularly low in starch content and starch/neutral detergent fibre (NDF) ratio, and resulted in a lower DM intake (DMI), milk yield and milk protein content. The DMI, milk yield and milk protein content increased with advancing maturity, reaching an optimum level for maize silages ensiled at DM contents of 300-350 g kg(-1), and then declined slightly at further maturity beyond 350 g kg(-1). The increases in milk (R(2) = 0.599) and protein (R(2) = 0.605) yields with maturity of maize silages were positively related to the increase in starch/NDF ratio of the maize silages. On average, the inclusion of maize silage in grass silage-based diets improved the forage DMI by 2 kg d(-1), milk yield by 1.9 kg d(-1) and milk protein content by 1.2 g kg(-1). Further comparisons showed that, in terms of milk and milk constituent yields, the optimum grass/maize silage ratio depends on the quality of both the grass and maize silages. Replacement of grass silage with maize silage in the ration, as well as an increasing maturity of the maize silages, altered the milk FA profile of the dairy cows, notably, the concentration of the cis-unsaturated FAs, C18:3n-3 and n-3/n-6 ratio decreased in milk fat. Despite variation in nutritive value, maize silage is rich in metabolizable energy and supports higher DMI and milk yield. Harvesting maize silages at a DM content between 300 and 350 g kg(-1) and feeding in combination with grass silage results in a higher milk yield of dairy cows.

In Vitro Fermentation Potential of Gut Endogenous Protein Losses of Growing Pigs.

Fermentation of dietary and endogenous protein in the hindgut is generally considered detrimental to the health of pigs. We investigated the in vitro fermentation potential of porcine endogenous protein in ileal digesta and colonic mucus, using a N-free buffer with an excess of fermentable carbohydrates. Urea, whey protein isolate (WPI, positive control), WPI hydrolysate (WPIH), and combinations of the latter two were used to validate the assay. A new biphasic model, including a linear end simulation, fitted to the gas production data over a 48-h period identified the time point when substrate fermentation ended. A higher degree of hydrolysis of WPI resulted in a higher maximum gas production rate (Rmax, P < 0.01). Differences in Rmax and the time required to reach Rmax were observed among ileal digesta samples, with Rmax increasing with the insoluble protein content, and highest Rmax occurring with colonic mucus samples (P < 0.05). The endogenous proteins entering the large intestine of pigs can ferment more rapidly compared to highly soluble and digestible protein sources, with Rmax positively correlated with decreasing solubility of endogenous nitrogenous components.

Stability of fatty acids during wilting of perennial ryegrass (Lolium perenne L.): effect of bruising and environmental conditions.

BACKGROUND: Oxidation of fatty acids (FA) during field wilting of herbage could cause extensive losses of polyunsaturated FA. Recent studies showed a variable effect of wilting on the losses of FA. This suggests that environment and management conditions influence the loss of FA during wilting. The present study investigated the stability of FA in untreated and mechanically bruised perennial ryegrass, wilted under field conditions for 0, 12, 24, 36 and 48 h, or wilted under controlled climate conditions at three temperatures (15, 25 or 35 °C) and two light (dark or light) regimes to dry matter (DM) contents of 425, 525 or 625 g kg(-1). RESULTS: During 48 h of field wilting, the total FA content declined (15.2 to 11.9 g kg(-1) DM) consistently, despite an increase in herbage DM content (197 to 676 g kg(-1)). Under controlled climate conditions, the herbage total FA content declined (15.1 to 11.7 g kg(-1) DM) mainly during the prolonged (56 to 62 h) initial drying to a DM content of 425 g kg(-1) and did not decline with further drying to DM contents of 525 and 625 g kg(-1). The decline in total FA was associated with a parallel decline in C18:3 content under field (9.15 to 6.36 g kg(-1) DM) and controlled (9.12 to 6.15 g kg(-1) DM) conditions. Concomitantly, the proportion of C18:3 in total FA decreased, whilst the proportion of C16:0 and C18:0 increased. Lower losses of FA (P<0.05) were observed at 15 °C compared to 25 and 35 °C. Light did not affect the losses of FA during wilting. CONCLUSIONS: The duration of the wilting period mainly affected the changes in FA content and composition. Stability of FA in herbage could be increased by minimising the duration of wilting.

Changes in fatty acid content and composition in silage maize during grain filling.

BACKGROUND: The stage of maturity at harvest has a major effect on the fatty acid (FA) content and composition of forage plants consumed by dairy cows. The present study investigated the dynamics of FA content and composition in stover (leaves and stem) and ears (cob, shank and husks) of two maize genotypes (G2 and G6) grown on sandy and clay soils and harvested at 14, 42, 56, 70 and 84 days after flowering (DAF). In addition, the FA content and composition of six maize genotypes (G1-G6) grown on the two soil types were compared at the normal harvest time of early genotypes in the Netherlands (70 DAF). RESULTS: The contents of total FAs and major individual FAs in both stover and ears changed significantly (P < 0.001) during the grain-filling period (14-84 DAF). In stover the contents of C16:0, C18:2, C18:3 and total FAs declined (P < 0.001) while those of C18:0 and C18:1 increased (P < 0.001) with progressive grain filling. The rate of decline in C18:3 and total FA contents was slower during 14-56 DAF as compared with 56-84 DAF. In ears, the contents of C16:0, C18:1, C18:2 and total FAs increased up to 56 DAF and then remained more or less constant until 84 DAF. At 70 DAF the content of polyunsaturated fatty acids (PUFAs) in both stover and ears did not differ among the six genotypes. However, the average contents of C16:0, C18:3 and total FAs in stover were higher (P < 0.05) on clay soil, whereas those of C18:0 and C18:1 were higher on sandy soil. CONCLUSION: The results demonstrate that the maximum PUFA content in silage maize is harvested around 56 DAF, in the present study at a T(sum) of 927 °C.d or at an ear dry matter content of 440 g kg(-1) , which is before the onset of rapid senescence. Any further delay in harvesting will cause a rapid decline in C18:3 content in maize silages.

Corn stover usage and farm profit for sustainable dairy farming in China.

OBJECTIVE: This study determined the optimal ratio of whole plant corn silage (WPCS) to corn stover (stems+leaves) silage (CSS) (WPCS:CSS) to reach the greatest profit of dairy farmers and evaluated its consequences with corn available for other purposes, enteric methane production and milk nitrogen efficiency (MNE) at varying milk production levels. METHODS: An optimization model was developed. Chemical composition, rumen undegradable protein and metabolizable energy (ME) of WPCS and CSS from 4 cultivars were determined to provide data for the model. RESULTS: At production levels of 0, 10, 20, and 30 kg milk/cow/d, the WPCS:CSS to maximize the profit of dairy farmers was 16:84, 22:78, 44:56, and 88:12, respectively, and the land area needed to grow corn plants was 4.5, 31.4, 33.4, and 30.3 ha, respectively. The amount of corn available (ton DM/ha/yr) for other purposes saved from this land area decreased with higher producing cows. However, compared with high producing cows (30 kg/d milk), more low producing cows (10 kg/d milk) and more land area to grow corn and soybeans was needed to produce the same total amount of milk. Extra land is available to grow corn for a higher milk production, leading to more corn available for other purposes. Increasing ME content of CSS decreased the land area needed, increased the profit of dairy farms and provided more corn available for other purposes. At the optimal WPCS:CSS, MNE and enteric methane production was greater, but methane production per kg milk was lower, for high producing cows. CONCLUSION: The WPCS:CSS to maximize the profit for dairy farms increases with decreased milk production levels. At a fixed total amount of milk being produced, high producing cows increase corn available for other purposes. At the optimal WPCS:CSS, methane emission intensity is smaller and MNE is greater for high producing cows.

Transfer of pyrrolizidine alkaloids from ragwort, common groundsel and viper's bugloss to milk from dairy cows.

To investigate the transfer of pyrrolizidine alkaloids (PAs) from feed to milk, rumen-cannulated dairy cows were intra-ruminally fed with 200 g/day of dried plant material of either ragwort (mixture of Jacobaea vulgaris and Senecio inaequidens), common groundsel (Senecio vulgaris) or viper's bugloss (Echium vulgare) for a period of 4 days. PA levels in the plant materials were 3767, 2792 and 1674 µg g-1 respectively. Feed intake, milk yield and several blood parameters indicative for liver function were not influenced by the treatment. When fed ragwort, increased levels of PAs were detected in the milk, in particular jacoline and an unidentified cyclic diester, possibly a hydroxylated metabolite from retrorsine. The latter was the most important PA in milk from cows fed common groundsel. For viper's bugloss, echimidine was the most abundant identified PA but in addition several hydroxylated PA metabolites were detected. For ragwort, the overall PA transfer was estimated at 0.05% and 1.4% for jacoline (N-oxide). Transfer rates were similar for viper's bugloss (0.05%) but lower for common groundsel (0.01%). Only a small portion of the administered PAs was quantified in milk, urine and faeces, with an overall balance of 4.5%, 2.9% and 5.8%, for ragwort, common groundsel and viper's bugloss, respectively. Samples taken from the rumen indicated that the N-oxides were converted into the free bases, which was confirmed by in vitro studies with the same plant species incubated with ruminal fluid. These results confirm that the transfer of PAs to milk is relatively low but may be of concern for human health regarding the genotoxic and carcinogenic properties of these compounds. The transfer rate depends on the type of PAs present in the weeds. The incomplete balance of input vs output stresses the need to further investigate the metabolism and the potential transfer of metabolites into edible products.

Mechanistic insight in the selective delignification of wheat straw by three white-rot fungal species through quantitative 13C-IS py-GC-MS and whole cell wall HSQC NMR.

BACKGROUND: The white-rot fungi Ceriporiopsis subvermispora (Cs), Pleurotus eryngii (Pe), and Lentinula edodes (Le) have been shown to be high-potential species for selective delignification of plant biomass. This delignification improves polysaccharide degradability, which currently limits the efficient lignocellulose conversion into biochemicals, biofuels, and animal feed. Since selectivity and time efficiency of fungal delignification still need optimization, detailed understanding of the underlying mechanisms at molecular level is required. The recently developed methodologies for lignin quantification and characterization now allow for the in-depth mapping of fungal modification and degradation of lignin and, thereby, enable resolving underlying mechanisms. RESULTS: Wheat straw treated by two strains of Cs (Cs1 and Cs12), Pe (Pe3 and Pe6) and Le (Le8 and Le10) was characterized using semi-quantitative py-GC-MS during fungal growth (1, 3, and 7 weeks). The remaining lignin after 7 weeks was quantified and characterized using 13C lignin internal standard based py-GC-MS and whole cell wall HSQC NMR. Strains of the same species showed similar patterns of lignin removal and degradation. Cs and Le outperformed Pe in terms of extent and selectivity of delignification (Cs ≥ Le >> Pe). The highest lignin removal [66% (w/w); Cs1] was obtained after 7 weeks, without extensive carbohydrate degradation (factor 3 increased carbohydrate-to-lignin ratio). Furthermore, though after treatment with Cs and Le comparable amounts of lignin remained, the structure of the residual lignin vastly differed. For example, Cα-oxidized substructures accumulated in Cs treated lignin up to 24% of the total aromatic lignin, a factor two higher than in Le-treated lignin. Contrarily, ferulic acid substructures were preferentially targeted by Le (and Pe). Interestingly, Pe-spent lignin was specifically depleted of tricin (40% reduction). The overall subunit composition (H:G:S) was not affected by fungal treatment. CONCLUSIONS: Cs and Le are both able to effectively and selectively delignify wheat straw, though the underlying mechanisms are fundamentally different. We are the first to identify that Cs degrades the major ß-O-4 ether linkage in grass lignin mainly via Cß-O-aryl cleavage, while Cα-Cß cleavage of inter-unit linkages predominated for Le. Our research provides a new insight on how fungi degrade lignin, which contributes to further optimizing the biological upgrading of lignocellulose.

Evaluation of an in vitro fibre fermentation method using feline faecal inocula: inter-individual variation.

The present study aimed to evaluate the inter-individual variability in fermentation of standard fibrous substrates by faecal inocula from ten healthy adult female cats. Substrates were citrus pectin (CP), fructo-oligosaccharides (FOS), guar gum (GG), sugar beet pulp (SBP) and wheat middlings (WM). Each substrate was incubated with faecal inoculum from each cat. Gas production was measured continuously during the 48 h incubation and SCFA and organic matter disappearance (only SBP and WM) after incubation. Out of ten cats, nine produced faeces on the days of inoculum preparation. The substrates contrasted in terms of fermentation parameters measured. The inter-individual variability was in general lower for the more simple and pure substrates (CP, FOS, GG) than for the more complex substrates containing mixtures of fibres (SBP, WM). Furthermore, for total SCFA and gas produced, inter-individual variability was lower than for proportions of butyrate and of branched-chain fatty acids and for the parameters of gas production kinetics. It is concluded that the variability in in vitro fermentation parameters is associated with the complexity of fibrous substrates. The presented data are instrumental for the calculation of number of faecal donors required for precise in vitro characterisation of the fermentability of dietary fibres. In addition, the number of faecal donors should be adjusted to the specific fermentation parameter(s) of interest.

Evaluation of an in vitro fibre fermentation method using feline faecal inocula: repeatability and reproducibility.

To gain knowledge on the precision of an in vitro method for characterisation of the fermentability of dietary fibres, this study aimed to evaluate the repeatability and reproducibility of such a method. Substrates used were citrus pectin (CP), fructo-oligosaccharides (FOS), guar gum (GG), sugar beet pulp (SBP) and wheat middlings (WM). Each substrate was incubated with faecal inoculum from five cats with three replicates for each substrate-cat combination. Gas production was measured continuously during the 48 h incubation and SCFA and organic matter disappearance (only SBP and WM) were determined after incubation. Four consecutive runs were performed. The within-run variability (repeatability) was generally lower for the more simple and pure substrates (CP, FOS, GG) than for the more complex substrates containing mixtures of fibres (SBP, WM). Replicates showed high variability, in particular for SCFA profiles and parameters of gas production kinetics. The between-run CV (reproducibility) for the measured parameters were, in general, below 10 % for CP, FOS and GG and higher values were obtained for SBP and WM. It is concluded that for precise dietary fibre characterisation, the number of replicates should be multiple and adjusted according to the variability of the parameters of interest and the complexity of fibres. The method yielded reproducible results with some variation in absolute values obtained, which may have an impact on the significance level of the differences among substrates.

The effect of particle size and amount of inoculum on fungal treatment of wheat straw and wood chips.

BACKGROUND: The aim of this study was to optimize the fungal treatment of lignocellulosic biomass by stimulating the colonization. Wheat straw and wood chips were treated with Ceriporiopsis subvermispora and Lentinula edodes with various amounts of colonized millet grains (0.5, 1.5 or 3.0 % per g of wet weight of substrate) added to the substrates. Also, wheat straw and wood chips were chopped to either 0.5 or 2 cm. Effectiveness of the fungal treatment after 0, 2, 4, 6, or 8 wk of incubation was determined by changes in chemical composition, in vitro gas production (IVGP) as a measure for rumen degradability, and ergosterol content as a measure of fungal biomass. RESULTS: Incomplete colonization was observed for C. subvermispora treated wheat straw and L. edodes treated wood chips. The different particle sizes and amounts of inoculum tested, had no significant effects on the chemical composition and the IVGP of C. subvermispora treated wood chips. Particle size did influence L. edodes treatment of wheat straw. The L. edodes treatment of 2 cm wheat straw resulted in a more selective delignification and a higher IVGP than the smaller particles. Addition of 1.5 % or 3 % L. edodes inoculum to wheat straw resulted in more selective delignification and a higher IVGP than addition of 0.5 % inoculum. CONCLUSION: Particle size and amount of inoculum did not have an effect on C. subvermispora treatment of wood chips. At least 1.5 % L. edodes colonized millet grains should be added to 2 cm wheat straw to result in an increased IVGP and acid detergent lignin (ADL) degradation.

Selective ligninolysis of wheat straw and wood chips by the white-rot fungus Lentinula edodes and its influence on in vitro rumen degradability.

BACKGROUND: The present work investigated the influence of lignin content and composition in the fungal treatment of lignocellulosic biomass in order to improve rumen degradability. Wheat straw and wood chips, differing in lignin composition, were treated with Lentinula edodes for 0, 2, 4, 8 and 12 wk and the changes occurring during fungal degradation were analyzed using pyrolysis-gas chromatography-mass spectrometry and detergent fiber analysis. RESULTS: L. edodes preferentially degraded lignin, with only limited cellulose degradation, in wheat straw and wood chips, leaving a substrate enriched in cellulose. Syringyl (S)-lignin units were preferentially degraded than guaiacyl (G)-lignin units, resulting in a decreased S/G ratio. A decreasing S/G ratio (wheat straw: r = -0.72, wood chips: r = -0.75) and selective lignin degradation (wheat straw: r = -0.69, wood chips: r = -0.88) were correlated with in vitro gas production (IVGP), a good indicator for rumen degradability. CONCLUSIONS: L. edodes treatment increased the IVGP of wheat straw and wood chips. Effects on IVGP were similar for wheat straw and wood chips indicating that lignin content and 3D-structure of cell walls influence in vitro rumen degradability more than lignin composition.