The Influence And Regulation Of Amino Acids Profile On Fermentation Performance And Flavor Of Lager Yeast

Wort component,mostly consists of carbon sources and nitrogenous compounds,is of considerable significance to yeast physiological status,fermentation properties and metabolism of flavor active substances.Even the proper control of wort carbohydrate has been accomplished by the existing brewing technology;it is still difficult to achieve precise control over yeast fermentation performance and the consistency or representativeness characteristics of beer flavor.In addition,researches of the influence and regulation of amino acids on yeast fermentation performance and flavor active substance metabolism were mainly based on Saccharomyces cerevisiae,which cannot be used to guide the flavor regulation of the allopolyploid Lager yeast.Therefore,it is urgent to carry out researches on the utilization of nitrogen sources and its regulatory mechanism in lager yeast.In this study,the impact of wort nitrogen components on Lager brewing yeast（Saccharomyces pastorianus TT-1）fermentation performance and the regulation of amino acids catabolism were studied.The effect of wort nitrogen level and its amino acids profile on the yeast fermentation characteristics and assimilation of amino acids by lager yeast were studied first and the key amino acids related to fermentation and flavor formation were defined.Furthermore,the Influence of key amino acids on the regulation of yeast nitrogen metabolism was investigated and the mechanism was proposed.The difference of variant Lager yeast in regulation of nitrogen metabolism was researched.At last,the influence of different fermentation conditions on the amino acid assimilation,fermentation performance and nitrogen metabolism regulation of yeast was studied further,which provided the theoretical basis and method guideline for the optimization of nitrogen source for beer brewing.The main results were described as follows:（1）Fluctuation of wort amino acids profile and its influence on beer flavor were studied by using mathematical statistical method based on the wort and fermentation data of industrial production.In addition,the effect of wort nitrogen level on Lager yeast fermentation performance and flavor production were analyzed through fermentation experiments of amino acid addition with different nitrogen conditions in a synthetic medium.The results showed that there were differences in the amino acids profile between different factories,especially in the content of Glu.FAN level in worts of the same factory was stablely controlled between201²20 mg?L^-1,but the amino acids profile and its absorption rate fluctuated greatly,and there was no correlation found in the absorption rate of amino acids with the total amino acids contents.There were also significant differences in the higher alcohols and esters content of the final product.The results of fermentation experiments of amino acid addition with different nitrogen conditions in a synthetic medium showed that the changes in the content of amino acids could influence yeast absorption rate of amino acids and the formation of fermentation products when the of carbohydrate amino acids levels were the same.Therefore,in order to meet the nutritional demand of yeast for nitrogen source and the final flavor production,not only the content of wort FAN needs to be controlled,but also the composition of wort amino acid profile.（2）The Influence of wort amino acids profile on Lager yeast fermentation performance and flavor production were analyzed through a series of fermentation experiments with different nitrogen conditions in a synthetic medium with the application of a two-level factorial Plackett–Burman design.The results showed that the most important amino acids affecting the growth and flavor metabolism were Glutamate acid,proline,lysine and valine.Glutamate acid and proline were identified as important determinants for their negative roles in yeast proliferation and amino acids assimilation.The most significant effect on higher alcohol production was exercised by the content of Valine.Lysine can facilitate yeast growth.（3）We developed a GeXP analyzer-based multiplex polymerase chain reaction assay to simultaneously assess the transcription levels of genes involved in nitrogen catabolism regulation and nitrogen permease of Lager yeast.Further,the regulative mechanisms of different key amino acids on nitrogen catabolism and transportation in lager yeast were proposed.The results showed that different amino acids intensified yeast NCR regulation with different mechanisms and divergent pathways.Glu,Lys and Val,as yeast preferred nitrogen sources,showed consistent NCR regulation throughout the exponential stage of yeast growth.However,Pro as yeast non-preferred nitrogen source,revealed NCR regulation at late-exponential stage.A major difference between the two stages of yeast growth was that NCR was active through NCR activators or repressors at mid-exponential stage,whereas SPS-sensing system and Tor1/2 signal transduction pathway dominated the NCR control at late-exponential stage.Further,we confirmed that the expression of yeast nitrogen transfer permease was differentially inhibited by various amino acids.The transcription of nitrogen permease genes（AGP1,ALP1,HIP1,GNP1,MEP2/3,DIP5 and MUP1）of lager yeast was inhibited by the key amino acids addition,which resulted in restrained amino acids absorption at mid-exponential stage.Lys and Val had the strongest inhibition effect,while Pro showed the weakest inhibition effect.At late-exponential stae,the inhibition by Glu and Pro exhibited was attenuated.（4）Under the same amino acids profiles and fermentation conditions,the effects of 5different industrial Lager yeast strains on the fermentation performance and the amino acid metabolism were studied.The results showed that the regulative pattern of different Lager yeast on nitrogen catabolism maintained in accordance with that of the different key amino aicds,which was regulate through NCR activators,repressors or SPS-sensing system and finally affected wort amino acids absorption.However,there existed significant difference in the fermentation performance and gene transcription of variant Lager yeast.Compared with the flavor production of diacetyl,pentanedione,acetaldehyde and esters,5 different Lager strains showed significantly different in the flavor generation of alcohol.Studies on the regulation of Lager yeast amino acid metabolism by gene transcription showed TT-2,TT-3and TT-4 increased the uptake of wort nitrogen through increasing the expression of nitrogen permease genes by reducing nitrogen metabolism repression（upregulated NCR activators or downregulated repressors and SPS-sensing system）,while TT-5 decreased its amino acid absorption through enhanced NCR（downregulated NCR activators）.（5）Under consistent amino acids profiles,the effect of different fermentation conditions（the pitching rate,wort aeration,pressure,and temperature）on the amino acids metabolism and flavor formation of lager yeast were conducted in pilot scale.Results showed that the fermentation conditions affected the fermentation performance and amino acid metabolism of Lager yeast.There were differences in amino acids absorption rates,transcription of nitrogen metabolism regulation genes and related permease genes,flavor substance formation under different fermentation conditions.The influence of the fermentation pressure was most significant.In addition,upregulation of the activator of NCR（GAT1 and GLN3）could be found in low temperature fermentation and high pressure fermentation,which will facilitate the transcription of nitrogen permease genes.However,increasing the pitching rate would upregulate the expression of repressor genes of NCR（DAL80,GZF3 and URE2）and resulted in the downregulation of transcription of nitrogen permease genes.Furthermore,combined with the wort amino acid profiles of the industrial production,the amount of key amino acids in wort were specified with Lager yeast s.pastorianus TT-1 as Glu:60⁸0 mg?L^-1;Pro:350³90 mg?L^-1;Lys:75¹00 mg?L^-1;Val:110¹40 mg?L^-1,which could be used to guide process improvement.By monitoring the content of key amino acids in the wort,the flavor fluctuation of the finished beer was significantly reduced and the stability of higher alcohol and ester ratio were improved.