More than a million tons of soybean meal is imported from South America to Denmark every year to use as feed.
To reduce the import and to reduce transportation, alternative locally grown crops are researched. A potential
protein source is perennial legumes. Green biorefinery concept is a concept that converts different green
substrates into multiple products including a protein concentrate. While this concept extracts some of the
protein present in the substrate, there are still structure bound proteins left. This project focus on optimizing
enzyme production and enzymatic hydrolysis, which is used to break the lignocellulosic structure and extract the
proteins. Four different experimental parts are performed. Alfalfa press cake is used as the main substrate in
three of the experimental parts and fresh clover grass is used in the last part. The first experimental part is the
substrate composition, where moisture, ash, protein, sugar, and lignin content are measured. The second
experimental part is the optimization of enzyme production, where the parameters time of incubation, light
availability, inoculation temperature, initial moisture content, and inoculum size are analyzed together with
testing four different substrates. The third experimental part is the optimization of enzymatic hydrolysis, where
the parameters incubation time, enzyme units per substrate, pH of inoculum, and incubation temperature are
tested together with analyzing the potential of using diluted brown juice as a buffer. The last experimental part
is the effect of enzymatic hydrolysis, where the created enzyme cocktail is used on fresh substrate and the effect
of the enzymatic hydrolysis is tested by analyzing the supernatant and solids using different methods. Alfalfa
press cake contains a dry matter on 34.6 ± 0.7 g/100g substrate, with a protein content on 13.8 ± 0.08 g/100gDM substrate and a cellulose concentration on 31.7 ± 0.4 g/100g substrate. The optimal parameters for enzyme
production are incubation in darkness for 9 days at 25-30°C with a moisture content on 75% and at least 5*106
spores/g-DM substrate. Of the tested substrates, alfalfa press cake is the substrate producing the least amount
of FPA activity. The optimal parameters for enzymatic hydrolysis are 7.5 FPU/g-DM substrate at 50°C and pH in
the range of 4-5.5. Incubation at 24 hours yields 8% of total released sugars. It is possible to use diluted brown
juice as buffer. In the effect of enzymatic hydrolysis, substrate is degraded. Unfortunately, the protein
concentration results do not indicate a higher protein extraction after an enzymatic hydrolysis. It is therefore
unclear if the hydrolysis affects the extracted protein concentration.