Optimizing of in situ enzyme production and hydrolysis to enhance protein extraction from fresh clover grass
Author
Jørgensen, Helene Kjerulf
Term
4. term
Education
Publication year
2021
Submitted on
2021-06-03
Pages
71
Abstract
Danmark importerer hvert år over en million ton sojaskrå fra Sydamerika til foder. For at mindske import og transport undersøges lokalt dyrkede alternativer, herunder flerårige bælgplanter som potentiel proteinkilde. I et grønt bioraffinaderi omdannes frisk, grøn biomasse til flere produkter, blandt andet et proteinkoncentrat, men noget protein forbliver bundet i plantens lignocellulosiske struktur (den seje blanding af cellulose og lignin i cellevægge). Dette projekt fokuserer på at optimere enzymproduktion og enzymatisk hydrolyse for at bryde denne struktur og frigive mere protein. Arbejdet bestod af fire dele. I tre dele blev lucerne-presskage anvendt som hovedsubstrat, og i den sidste del blev frisk kløvergræs brugt. Først blev substraternes sammensætning analyseret (fugt, aske, protein, sukker og lignin). Lucerne-presskage havde ca. 35% tørstof, omkring 14% protein (af tørstof) og cirka 32% cellulose. Dernæst blev enzymproduktionen optimeret ved at variere inkubationstid, lys, inokulationstemperatur, initial fugt og inokulumstørrelse samt ved at teste fire forskellige substrater. De bedste betingelser var inkubation i mørke i 9 dage ved 25–30°C med 75% fugt og mindst 5*106 sporer pr. g tørstof. Blandt de testede substrater gav lucerne-presskage den laveste FPA-aktivitet (en standardmåling af cellulaseaktivitet). Tredje del optimerede selve den enzymatiske hydrolyse ved at undersøge inkubationstid, enzymdosering (7,5 FPU pr. g tørstof), pH (4–5,5) og temperatur (50°C) samt muligheden for at bruge fortyndet brown juice som buffer; efter 24 timer blev ca. 8% af de samlede sukkerarter frigivet, og det var muligt at anvende fortyndet brown juice som buffer. Til sidst blev effekten af hydrolysen vurderet ved at bruge den udviklede enzymcocktail på frisk biomasse og analysere både supernatant (væskefraktionen) og faste fraktioner. Substratet blev nedbrudt, men målingerne viste ikke en tydelig stigning i udvundet proteinkoncentration efter hydrolyse. Det er derfor uklart, om hydrolysen øger proteinudbyttet.
Denmark imports more than one million tons of soybean meal from South America each year for animal feed. To reduce imports and transport, locally grown alternatives are being explored, including perennial legumes as a potential protein source. In a green biorefinery, fresh green biomass is converted into several products, including a protein concentrate, but some protein remains bound in the plant’s lignocellulosic structure (the tough mix of cellulose and lignin in cell walls). This project focuses on optimizing enzyme production and enzymatic hydrolysis to break that structure and release more protein. The work comprised four parts. Alfalfa press cake was the main substrate in three parts, and fresh clover grass was used in the last. First, substrate composition was measured (moisture, ash, protein, sugars, and lignin). Alfalfa press cake contained about 35% dry matter, around 14% protein (on a dry-matter basis), and roughly 32% cellulose. Next, enzyme production was optimized by varying incubation time, light, inoculation temperature, initial moisture, and inoculum size, and by testing four different substrates. Optimal conditions were incubation in darkness for 9 days at 25–30°C with 75% moisture and at least 5*106 spores per g dry matter. Among the tested substrates, alfalfa press cake produced the lowest FPA activity (a standard measure of cellulase activity). The third part optimized the enzymatic hydrolysis itself by testing incubation time, enzyme dose (7.5 FPU per g dry matter), pH (4–5.5), and temperature (50°C), and assessing whether diluted brown juice could serve as a buffer; after 24 hours about 8% of total sugars were released, and diluted brown juice could be used as a buffer. Finally, the effect of hydrolysis was evaluated by applying the enzyme cocktail to fresh biomass and analyzing both the supernatant (liquid fraction) and solids. The substrate was degraded, but protein concentration measurements did not indicate a clear increase in extracted protein after hydrolysis. It therefore remains unclear whether hydrolysis improves protein yield.
[This summary has been rewritten with the help of AI based on the project's original abstract]
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