Globally, animal feed protein is a key factor for production of meat for human consumption. Protein for animal feed is in many parts of the world not available in sufficient amounts; demand is met only through import of feed protein. Such protein deficit can be minimized through optimized use of local protein resources based on upgrade from e.g. green plant biomass. In present work we consider different strategies for protein recovery from white clover and ryegrass screw press pulps, using aqueous extraction, as well as carbohydrases and proteases enhanced extraction. Protein recovery in these studies was determined as a yield of solubilized protein with regard to the total protein in a screw press pulp. Aqueous extraction at pH 8.0 resulted in approx. 40 % protein recovery, while proteases application (Savinase 16.0L, Novozymes) enabled twice higher protein yield. Application of plant cell wall degrading enzymes (Cellic CTec2 and Cellic HTec2, Novozymes) did not provide detectable protein recovery, while consecutive proteases treatment resulted in approx. 95 % protein yield. RuBisCO peptides were demonstrated by amino acid analysis to be the major component of white clover and ryegrass pulp proteolyzates, generated by Savinase 16.0L protease.

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This study examines enzymatic production of linear xylooligosaccharides (XOS) and branched arabinoxylooligosaccharides (AXOS) from monocotyledonous biomass, wheat straw and ryegrass, and compares the in vitro effects of these XOS and AXOS on pig gut microbiota. XOS and AXOS were obtained from the biomass by treatment with different endo-1,4-β-xylanases. XOS of DP2-6 from wheat straw, obtained after treatment with Aspergillus niger endo GH11, suppressed growth of Clostridium perfringens and resulted in a high level of lactic acid production when fermented in vitro by pig fecal microbiota. Analogously, XOS ryegrass produced in the same way also suppressed Cl. perfringens growth, and more so than the corresponding ryegrass AXOS, but AXOS exhibited a more pronounced stimulation of lactic acid bacteria growth than XOS. The prebiotic potential, i.e., suppression of Cl. perfringens and stimulation of lactic acid bacteria, for the ryegrass oligosaccharides was as follows: XOS, produced by A. niger endo-1,4-β-xylanase (GH 11) ≥ AXOS, produced by Thermotoga maritima and Cellvibrio mixtus endo-1,4-β-xylanase s (GH10) > AXOS, produced by Trichoderma viride and Aspergillus aculeatus endo-1,4-β-xylanase s (GH11). These results indicate that wheat straw as well as green grass biomass such as ryegrass have potential as new sources of putative prebiotics for pig feed.

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Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K(m) and k(cat) values are 0.4 mM and 15 sec(-1) for p-nitrophenyl-β-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galactomannans (GM) of various structures. The K(m) and k(cat) values are 1.3 mg/ml and 67 sec(-1) for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K(m) and k(cat) values are 0.08 mM and 35 sec(-1) for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.

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