Extraction and determination of total polyphenols in residual BSY (Brewing Spent Yeast) from the craft beer process

BALUSSI MACARENA; PEREZ ETHEL E.; CONSTENLA DIANA M

Buenos AIres

Congreso; 11th World Congress of Chemical Engineering; 2023

Asociación Argentina de Ingenieros Químicos

Brewing Spent Yeast (BSY) is the second largest residue from brewing process, obtained at the end of fermentatlon stage. lt is rich in carbohydrates, proteins, minerals, vitamins, and polyphenols (PP) that precipitate together with the exhausted yeasts. This fact suggests that BSY is a source of bioactive compounds that could become additives in the food, cosmetic and pharmaceutical industries. The objective of this work was to standardize a method for the determination of PP extractad from BSY, eliminating the interference that could be produced by the proteins and carbohydrates present in the extract. The BSY used in this study was provided by a local craft brewery.Extraction of PP. An amount of 0.5 g of dry BSY, and 1OO. ml of MilliQ-water (MQw) were mixed for 5 min, at 25°C with magnetic stirring. The mixture was centrifugad 20 min at 3000 g. Supematant was recovered, stored at -20°C (extract).Protein Precipitation. Trichloroacetic acid 50% (w/v) was added to extracts, in a relation of 2.5 ml:10ml. After 12 h at 40 °C,the acidified extract was centrifuged (1O min, 15000 g). The supernatant (protein free extract, PFE) was neutralized by adding 1M NaOH [1].Sugar removal. 1O mlof extract was evaporated to dryness in a rotary evaporator and taken up with 1O ml of methanol. Sep-Pak C1B cartridges were conditioned with B ml methanol, 4 ml MQw, and 5 ml 0.01M HCI, sample loaded and washed with 1O mL of MQw. Finally, the sample was eluted with 12 mL of methanol. The eluate was dried by N2 stream, adjusted to 1O mL with MQw (carbohydrate free extract, CFE), and analyzed for total polyphenol content (TPC).TPC analysis. A volume of 0.5-2.0 mL of extract/PFE/CFE, 50 microL Folin Ciocalteu reactive, and 5.0 ml of MQw, were place in a 1O mL amber volumetric flask and mixed vigorously. After standing B min at 25°C, 1.5 mL of 20 % Na2CO3 and MQw to a final volume of 1O mL were added (pH•10). After about 2 h at 25°C, the absorbance at 760 nm in a spectrophotometer was measured. Blank (PP free sample) was made in the same way without including extract [2]. Samples should develop a bluish color. Gallic acid (30-80 mg L·1) in MQw was used for calibration curves.lt was verified that the proteins present in the aqueous extracts produced an increase in absorbance of up to 50%. On the other hand, the carbohydrates present in the extract did not interfere in the TPC determination, so this purification step can be avoided.In conclusion, it was possible to develop a method for the determination of TP in BSY avoiding the interferences that could be caused by other substances present in the sample. lt is very important to set the final pH of samples between 9.8-10.0 before measuring the absorbance. lt is also important to respect the reaction times and temperatures.