Study Of The Effects Of Nitrogen Composition In High Gravity Wort On The Assimilation Of Amino Acids By Lager Yeast And Fermentation Control

Nitrogen limitation, particularly prevailing in the case of high gravity beer brewing,results in poor yeast viability and even stuck or sluggish fermentations. Although wortcontains abundant proteins and longer chain peptides, brewer’s yeast does not assimilate themdue to the fact that cells hardly secrete proteases during fermentation. In the present study,effects of nitrogen levels on the physiological characteristics and fermentation performance oflager yeast was studied firstly. Basis on this, the feasibility of improving fermentationperformance by increasing FAN level of high gravity wort supplemented with exogenousproteases was analyzed. The differences of assimilation pattern of amino acids by lager yeastbetween normal and high gravity fermentations were studied and the key amino acids forimproving fermentation performance were defined. The influences of key amino acidssupplementations during high gravity fermentation on the nitrogen metabolism of lager yeastwere also investigated. Finally, changes in nitrogen assimilation and fermentationperformance of lager yeast under different carbon sources were studied. The new findings ofthis study would be beneficial to high gravity brewing for the optimization of nitrogencomposition and the selection of adjunct types. The main research contents and results are asfollows:(1). Normal gravity wort and high gravity wort with different nitrogen levels were usedto examine their effects on the physiological characteristics and fermentation performance oflager yeast. Results showed that increased wort gravity or decreased nitrogen level coulddecrease cell growth rate, viability, flocculation, consumption rates of carbon and nitrogensources, and gene expression (ATF1, BAT1, BAP2, HSP12and TDH) of brewer’s yeast. Inaddition, high gravity wort with lower nitrogen level showed a decline in wort fermentabilityand ethanol production and an increase in uptake of group B amino acids.(2). The possibility for utilizing unavailable nitrogen from high gravity (20°P) and veryhigh gravity wort (24°P) by adding three food-grade commercial proteases (Neutrase,Flavorzyme and Protamex) at the beginning of fermentations, respectively, was analyzed.Results showed that proteases supplementation significantly increased the FAN level(increased by20mg/L at least) and yeast growth rate, improved fermentation performance,especially for Flavorzyme. Compared with the control,20°P high gravity wort supplementedwith Flavorzyme increased wort fermentability and ethanol production both by12%;24°Pvery high gravity wort supplemented with Flavorzyme increased wort fermentability andethanol production by8%and6%, respectively. Furthermore, proteases supplementation significantly increased the formation of higher alcohols and esters, while the ratio of higheralcohol to ester was relatively constant. The foam of final beers produced by adding proteaseswas as stable as that of the control at each of the corresponding gravities.(3). Changes in free amino acids composition by supplementing three commercialproteases (Neutrase, Flavorzyme and Protamex) at the beginning of wort mashing, and theeffects on the assimilation pattern of amino acids and fermentation performance of lager yeastduring normal and high gravity fermentations were investigated. Results showed thatproteases supplementations significantly improved the extract yield (increased by3%at least)and FAA level (increased by45%at least) of mashed worts. Normal gravity worts treatedwith Flavorzyme and Neutrase exhibited higher yeast growth and better fermentationperformance compared to the control wort, while these beneficial effects were observed inhigh gravity worts treated with Protamex and Neutrase. The reason for the above results isproposed to be the change in the assimilation pattern of amino acids by lager yeast withincreased wort gravity, especially for the improved assimilation ratios of Leu, Arg, Phe, His,Asp and Val. Moreover, in normal gravity fermentations, there were strong correlationsbetween the assimilation amounts of Lys, Leu, Arg and His and wort fermentability. While inhigh gravity fermentations, these good correlations were found with only Lys and His.(4). Influences of Lys, His and their mixture supplementations on the fermentationperformance and nitrogen metabolism in lager yeast during high gravity fermentation werefurther investigated. Results showed that Lys and His supplementations improved yeastgrowth, wort fermentability, ethanol yield and the formation of flavor volatiles. Lyssupplementation up-regulated SPS-regulated genes (LYP1, HIP1, BAP2and AGP1)dramatically, whereas His supplementation repressed NCR-sensitive genes (GAP1and MEP2)significantly throughout the fermentation. Lys and His supplementations increased theconsumption of Glu and Phe, and decreased the consumption of Ser, Trp and Arg. Moreover,Lys and His supplementations exhibited similar effects on the fermentation performance, andwere more effective than their mixture supplementation when the same dose was kept.(5). The effects of different carbon sources (glucose, sucrose and maltose) in high gravity(18°P) and very high gravity wort (24°P) on the cell tolerance, assimilation of amino acidand fermentation performance were studied. Results showed that during high gravityfermentation, glucose and sucrose additions significantly improved fermentation performanceof lager yeast compared to maltose, especially for sucrose, which increased wortfermentability and ethanol production by6%and8%, respectively, while yeast growth wasnot influenced. During very high gravity fermentation, glucose and sucrose additions showed better fermentation performance in the initial stage of fermentation, while maltose additionimproved fermentation performance significantly in the later stage of fermentation. Comparedwith sucrose, maltose addition increased wort fermentability and ethanol production by14%and10%, respectively. Higher wort gravity decreased cell viability but increased the synthesisof intracellular trehalose. Glucose and sucrose additions were not good for the accumulationof intracellular trehalose and thus reduced cell viability. At very high gravity fermentation,maltose addition significantly promoted the excretion of Gly and Ala, and the absorption ofPro in the later stage of fermentation. Increasing wort gravity from18°P to24°P coulddecrease the level of high alcohols but increase the content of esters. Glucose additionsignificantly improved the formation of flavor compounds.