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Brewing Chemistry Analysis –                                                                                              Quality Assurance –

                    Profile of Metabolic Enzyme                                                                                               CO2 Determination



                    Material production using microorganisms such as   Yeast strain S288C and single gene deletion mutants of strain          Quality assurance is only one of the attributes for brand name   bottles, carbon dioxide ensures that the necessary pressure is
                    Saccharomyces Cerevisiae has been applied to fermented food   BY4742 were cultured in media containing non-labeled or C-13   products such as beer. And quality is dependent on the quality   maintained. This is an important factor for guaranteeing shelf
                    such as beer. These microbial cell factories have been   labeled glucose for metabolic enzyme analysis. Strain S288C      of the starting materials, strict production processes as well as   life and fresh-tasting beer. A constant concentration of carbon
                    metabolically engineered by regulating the expression levels of   grown in heavy C-13 labeled glucose acted as an internal   specialists whose keen eyes oversee the entire production.   dioxide also ensures a steady taste and consistent quality of the
                    enzymes. To understand these metabolic pathways, it is   standard for comparison to the light C-12 mutated growth         Finally, millions of consumers are testing their favorite brand of   beer. Beer contains 4 – 6 g/L CO2.
                    important to monitor the abundance of enzymes. Metabolic   strains*. Information regarding glycerol biosynthesis, ethanol   beer every day.                                  Carbon dioxide in the world of Total Organic Carbon (TOC)
                    enzymes can be subjected to trypsin digestion, and   pathway, tricarboxylic acid (TCA) cycle, polysaccharides, and        An important player in the production process and a condition   analyzers is referred to as Inorganic Carbon (IC). The IC method
                    representative tryptic peptides selected for analysis. LC-MS/MS is   amino acids was determined by LC-MS/MS analysis. In one   for quality is invisible: carbon dioxide (CO2), formed during the   is a suitable alternative to the classical Corning reference
                    a highly sensitive and specific analytical technique used for   example, the LCMS-8050 measured 303 peptides of 137 proteins   fermenting process. After filling the beer into barrels and   method for measuring CO2*.
                    quantitation of specific peptides in biological samples. Unlike   by monitoring 2856 MRM transitions#. The library* for
                    ELISA, LC/MS-based MRM (Multiple Reaction Monitoring)   metabolic enzymes in yeast contains 3,584 MRM transitions,
                    approaches do not require an antibody for each     including stable isotopes. It covers all 498 trypsin digested
                    protein/peptide.                                   peptides of 228 types of enzymes derived from budding yeast.




                                    12
                     Light: Culture with  C  Glucose
                                      6
                                                                                                 Tryptic
                           Yeast A                                                               peptide
                                     Cultivation
                                    12
                                    ( C Glucose)  cell collection  extraction    digestion
                     Heavy: Culture with  C  Glucose                                                               Light                      Inorganic Carbon (IC) measured as CO 2 in beer by a TOC analyzer*
                                     13
                                       6                                                                                                                                                         Shimadzu TOC-L Total Organic Carbon analyzer for the analysis of CO 2
                                                                                                                   Heavy
                                                                                                 Tryptic   MRM analysis
                           Yeast B                                                               peptide
                                     Cultivation
                                    13
                                    ( C Glucose)  cell collection  extraction    digestion                                                                         6
                                                                                                                                                                                                                Corning  TOC
                                                                                                                                                                  5.5
                                                                                                                                                                   5
                                                                                                                                                                 Co2 [g/L]  4.5
                            Pdc1     Pdc6      Adh1     Adh3
                                                                                                                                                                   4
                                                                                                                                                                  3.5
                                                                                                                                                                   3
                           Adh6      Ald4      Ald5     Ald6                                                                                                           beer1  alcohol-free  beer2  alcohol-free  beer3  beer4  unfiltrated
                                                                                                                                                                              beer1           beer2                   beer4
                                                                                                                                                                   CO2 concentration measured by TOC and Corning reference method for various beers*


                    Metabolic profile of Ethanol pathway enzyme levels in wild type (black)   Shimadzu LCMS-8050 for measuring metabolic enzymes in yeast
                    and three deletion strains (greys) of S. cerevisiae. #












                    * Shimadzu Brochure C146-E275: “LC/MS/MS MRM Library for Metabolic Enzymes in Yeast”
                    #   Shimadzu Corporation Application Data Sheet: “Quantitative proteomics of metabolic enzymes in S. cerevisiae with UFMS technologies”

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