| Abstract [eng] |
Bioactive compounds obtained from lignocellulosic wastes and produced by food industry become attractive in case of cheap material, simple logistics and high value-added products, mostly, valuable compounds collected by different extraction methods, whose extract mixtures or pure compounds are adapted in cosmetics, pharmacy or other fields. Since the raw material is mostly lignocellulose, researchers started using commercial enzyme mixtures, which are characterized by high carbohydrase activity, to get better yields of bioactive molecules. The aim of this work was to determine optimal fermentation conditions by using commercial enzyme mixture Viscozyme L (102 CMC IU) from hemp seed hurds. Optimal fermentation conditions were used when performing enzyme assisted hydrolysis with the biomass of hemp seed hurds. Non-treated and by different combinations of SFE-CO2, PLE, UAE extraction methods pretreated hemp substrate was hydrolyzed by enzyme-assisted extraction. The hardware scheme of the most effective technological process of obtaining bioactive compounds was composed. Three variables (pH, E/S, exposition time) were used to compose Central Composite Rotatable Plan (CCRD) for optimization plan execution with statistical values estimating program Design Expert. Based on 20 experiments plan comprised by the program, bottles with samples were incubated by variable conditions obtained in accordance with the plan. Bottles were shaken with orbital shaker at 250 rpm/min and 50 °C. Reducing sugars were measured by DNS method in liquid phase samples after fermental hydrolysis. Measured response signal values were collected and analyzed using Design Expert program. Optimal conditions were determined: pH=5.54, E/S=6.7, exposition time – 6 hours and 12 minutes using fixed 50 °C temperature and 250 rpm/min velocity of orbital shaker. Hemp seed hurds were pretreated with SFE-CO2, PLE (acetone, 70 % ethanol, 140 °C water), UAE (acetone, 70 % ethanol, 50 mmol/L sodium citric buffer) different extraction methods combinations. The highest extract yield (21.30 %) of soluble compounds were obtained by SFE-CO2, PLE (acetone, 70 % ethanol, 140 °C water) extractions with hemp seed hurds biomass. Also, UAE (acetone) and UAE (70 % ethanol) were used to treat SFE-CO2 pretreated hemp seed hurds material which after UAE were respectively extracted by PLE (acetone) and PLE (70 % ethanol). It was determined that mentioned sequence of extraction combination raised extraction yields of soluble compounds by 11.56 % and 22.50 % respectively comparing with extraction sequences: SFE-CO2, PLE (acetone) or SFE-CO2, PLE (70 % ethanol). Pretreated hemp seed hurds by different extraction combinations were fermented by optimal conditions. It was determined that fermentation with SFE-CO2, PLE (acetone), PLE (70 % ethanol), PLE (140 °C water) pretreated material increases bioactive compound yields from 5 to 8 times comparing with the same material extracted by buffer maintaining the same incubation conditions. Results show that fermentation works best with material, which was pretreated with more additional extractions when higher polar and non-polar compounds were extracted. In this case, the enzymes, which decompose carbohydrates, can reach and hydrolyze lignocellulosic matrix better. In addition, inhibition possibility of bioactive compounds inside the vegetal material decreases. Optimal fermentation conditions were adapted with buckwheat hurds pretreated by different extraction methods. It was determined that fermentation using commercial enzyme mixture Viscozyme L increases soluble compounds in liquid phase more than 10 times comparing with control samples which were incubated using the same conditions but without enzymes. It was also determined that the majority of buckwheat hurds consists of polar compounds, because extraction yield obtained by PLE (140 °C water) was 5.77±0,36 % while extraction yield obtained by the combination of SFE-CO2, PLE (acetone) and PLE (70 % ethanol) was just above 1 %. |
