Fibrolytic enzyme treatment prior to ensiling increased press-juice and crude protein yield from grass silage.

Grass is a versatile raw material for green biorefineries and preserving it as silage provides a year-round feedstock. The objective of the current study was to evaluate the effect of fibrolytic enzyme application on silage as a feedstock for a biorefinery. Two batches of grass (mixture of timothy and meadow fescue) silages were ensiled in pilot scale after fibrolytic enzyme was applied to them at four levels. Enzyme application increased fibre degradation linearly during ensiling and increased lactic and acetic acid concentrations in the silage. Simultaneously, silage fermentation quality improved as indicated by decreasing pH and ammonia values. Press-juice and crude protein yields increased in response to the fibrolytic enzyme application, which is beneficial in a biorefinery concept for retrieving valuable nutrients from grass matrix. Optimized ensiling methodology can be considered as a pretreatment for a biorefinery process.

Hypocholesterolemic Effect of the Lignin-Rich Insoluble Residue of Brewer's Spent Grain in Mice Fed a High-Fat Diet.

Insoluble residue (INS) is a lignin-rich fraction of brewer's spent grain (BSG) that also contains ß-glucan and arabinoxylan, the major constituents of dietary fiber. We investigated the effects of INS in diet-induced obese mice in terms of lipid metabolism and metabolic diseases. Male mice (C57bl6) were fed a high-fat diet (HFD), a HFD + 20% INS, a HFD + 20% cellulose (CEL), a HFD with a combination of 20% INS-CEL (1:1), or a control diet for 14 weeks. Insulin and glucose tolerance tests were performed after 12 weeks. Fasting plasma lipids, bile acid, and fecal bile acid were measured after 14 weeks of feeding, and tissues were collected for gene expression analysis. Body weight gain was significantly reduced with all fibers, but only INS and INS-CEL decreased fasting plasma low-density lipoprotein cholesterol and total cholesterol compared to HFD. CEL and INS-CEL significantly improved insulin resistance. Fecal bile acids were significantly increased by all fibers, but there was no change in plasma bile acid. Clostridium leptum was increased with all fibers, but universal bacterial diversity was only with INS and INS-CEL. In addition, INS significantly increased the abundance of Bacteriodes, while CEL decreased Atopobium and Lactobacillus. INS feeding significantly upregulated various genes of cholesterol and bile acid metabolism, such as Srebp2, Hmgcr, Ldlr, Cyp7a1, Pparα, Fxr, and Pxr, in the liver. INS, INS-CEL, and CEL significantly attenuated liver steatosis. Our results suggest that INS from BSG induced beneficial systemic changes in mice via gut microbiota, bile acids, and gene expression in the liver.

Hydrothermal treatment followed by enzymatic hydrolysis and hydrothermal carbonization as means to valorise agro- and forest-based biomass residues.

The suitability of several abundant but underutilized agro and forest based biomass residues for hydrothermal treatment followed by enzymatic hydrolysis as well as for hydrothermal carbonization was studied. The selected approaches represent simple biotechnical and thermochemical treatment routes suitable for wet biomass. Based on the results, the hydrothermal pre-treatment followed by enzymatic hydrolysis seemed to be most suitable for processing of carbohydrate rich corn leaves, corn stover, wheat straw and willow. High content of thermally stable components (i.e. lignin) and low content of ash in the biomass were advantageous for hydrothermal carbonization of grape pomace, coffee cake, Scots pine bark and willow.

Interactions of Insoluble Residue from Enzymatic Hydrolysis of Brewer's Spent Grain with Intestinal Microbiota in Mice.

Brewer's spent grain (BSG) is the major side-stream from brewing. As BSG is rich in dietary fiber and protein, it could be used in more valuable applications, such as nutritional additives for foods. Our aim was to elucidate whether an insoluble lignin-rich fraction (INS) from BSG is metabolized by mice gut microbiota and how it affects the microbiota. Our results indicated that lignin was partially degraded by the gut microbiota, degradation products were absorbed, and finally excreted in urine. Therefore, they contribute to the phenolic pool circulating in the mammalian body, and may have systemic effects on health. In addition, the effects of the test diets on the microbiota were significant. Most interestingly, diversities of predominant cecal and fecal bacteria were higher after the intervention diet containing INS than after the intervention diet containing cellulose. Since low fecal bacterial diversity has been linked with numerous diseases and disorders, the diversity increasing ability opens very interesting perspectives for the future.

Structure of Brewer's Spent Grain Lignin and Its Interactions with Gut Microbiota in Vitro.

Lignin is part of dietary fiber, but its conversion in the gastrointestinal tract is not well understood. The aim of this work was to obtain structural information on brewer's spent grain (BSG) lignin and to understand the behavior of the polymeric part of lignin exposed to fecal microbiota. The original BSG and different lignin fractions were characterized by pyrolysis-GC/MS with and without methylation. Methylation pyrolysis proved that the ratio between guaiacyl and syringyl units was similar in all lignin samples, but the ratio between p-coumaric and ferulic acids varied by the isolation method. Combined pyrolysis results indicated higher acylation of γ-OH groups in syringyl than in guaiacyl lignin units. The polymeric lignin structure in the alkali-soluble fraction after enzymatic hydrolysis was slightly altered in the in vitro colon fermentation, whereas lignin in the insoluble residue after enzymatic treatments remained intact.

Release of small phenolic compounds from brewer's spent grain and its lignin fractions by human intestinal microbiota in vitro.

Brewer's spent grain (BSG), the major side-stream from brewing, is rich in protein, lignin, and nonstarch polysaccharides. Lignin is a polyphenolic macromolecule considered resilient toward breakdown and utilization by colon microbiota, although some indications of release of small phenolic components from lignin in animals have been shown. The aim of this study was to investigate if the human intestinal microbiota can release lignans and small phenolic compounds from whole BSG, a lignin-enriched insoluble fraction from BSG and a deferuloylated fraction, in a metabolic in vitro colon model. The formation of short-chain fatty acid (SCFA) was also investigated. More lignin-related monomers and dilignols were detected from the lignin-enriched fraction than from BSG or deferuloylated BSG. SCFA formation was not suppressed by any of the fractions. It was shown that small lignin-like compounds were released from these samples in the in vitro colon model, originating most likely from lignin.

Interactions of a lignin-rich fraction from brewer's spent grain with gut microbiota in vitro.

Lignin is a constituent of plant cell walls and thus is classified as part of dietary fiber. However, little is known about the role of lignin in gastrointestinal fermentation. In this work, a lignin-rich fraction was prepared from brewer's spent grain and subjected to an in vitro colon model to study its potential bioconversions and interactions with fecal microbiota. No suppression of microbial conversion by the fraction was observed in the colon model, as measured as short-chain fatty acid production. Furthermore, no inhibition on the growth was observed when the fraction was incubated with strains of lactobacilli and bifidobacteria. In fact, the lignin-rich fraction enabled bifidobacteria to survive longer than with glucose. Several transiently appearing phenolic compounds, very likely originating from lignin, were observed during the fermentation. This would indicate that the gut microbiota was able to partially degrade lignin and metabolize the released compounds.

Pre-hydrolysis with carbohydrases facilitates the release of protein from brewer's spent grain.

Brewer's spent grain (BSG) is the most abundant side-stream from brewing. It is food-grade being rich in dietary fibre and protein and thus having potential as their source for both food and non-food applications. Initial treatment of milled BSG with a carbohydrase cocktail from Humicola insolens significantly enhanced the subsequent solubilisation of protein from the residual biomass. When treated with an alkaline protease, 76% of BSG protein was solubilized, whereas the yields were significantly lower with neutral or acidic proteases. In alkaline conditions significant amount of protein (53%) as predominantly low molecular weight protein was solubilized even without any protease addition. The degree of protein solubilisation was influenced by the time of exposure of modified BSG to the alkaline environment. The non-enzymatic protein solubilisation was, however, only observed when BSG had been initially treated with the carbohydrase, suggesting the protein is surrounded by cell wall polysaccharides restricting its initial release.

Characterization of lipids and lignans in brewer's spent grain and its enzymatically extracted fraction.

Brewer's spent grain (BSG), the major side stream of brewing, consists of the husks and the residual parts of malts after the mashing process. BSG was enzymatically fractionated by a two-step treatment with carbohydrate- and protein-degrading enzymes, which solubilized 66% of BSG. BSG contained 11% lipids, which were mostly triglycerides, but also a notable amount of free fatty acids was present. Lipids were mostly solubilized due to the alkaline pH applied in the protease treatment. The main fatty acids were linoleic, palmitic, and oleic acids. Several lignans were identified in BSG, syringaresinol and secoisolariciresinol being the most abundant, many associated with the cell wall matrix and released by the alkaline-protease treatment.

Effect of a milling pre-treatment on the enzymatic hydrolysis of carbohydrates in brewer's spent grain.

Millions of tonnes of brewer's spent grain (BSG) are annually produced worldwide as a by-product of the brewing industry. BSG has the potential to be a valuable source of food, chemicals and energy if cost-efficient fractionation methods can be developed. A 2-fold improvement in carbohydrate solubilisation could be achieved through the introduction of a milling step prior to enzymatic hydrolysis. Course and fine milled fractions were characterized by particle size distribution and light microscopy. Fine milling decreased particle size down to the micron level and this in turn improved the carbohydrate solubility yield by a multi-enzyme mixture from 23% up to 45%. Carbohydrate solubilisation could be further increased through the supplementation of this enzyme preparation with additional cellulases. The physical degradation caused by the milling also liberated soluble carbohydrates without the requirement of any enzymatic treatment.