Optimizing of in situ enzyme production and hydrolysis to enhance protein extraction from fresh clover grass

Jørgensen, Helene Kjerulf

4. term

Sustainable Biotechnology, Master

2021

2021-06-03

71 pages

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.

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