SCREENING YEAST STRAINS FOR THEIR ABILITY TO PRODUCE PHENOLIC OFF-FLAVOURS: A SIMPLE METHOD FOR DETERMINING PHENOLS IN WORT AND BEER

Amounts of several phenolic compounds in wort and beer were measured by gas-chromatography following extraction with a small volume of chloroform. The procedure described is not specific for phenols and the limit of sensitivity is 0·05–0·1 μg ‘phenol’/ml. However, the precision is reasonable and this relatively rapid method has proved useful for screening yeast strains for their ability to produce phenolic off-flavours. Phenolic-tasting beers contained significant amounts of 4-vinyl guaiacol; whilst this compound may not be the sole cause of the undesirable flavour, its production is symptomatic of the unsuitability of a yeast strain for brewing.     

RESTRICTION MAPPING OF THE POF I GENE

The gene (POFI) which imparts to certain yeasts the ability to decarboxylate phenolic acids to corresponding phenolic compounds has been analysed by restriction mapping. New restriction sites have been used to examine differences between Pof⁺ and Pof^? Saccharomyces cerevisiae strains. Southern Blot analysis of selected yeast strains has demonstrated that the POFI gene sequence is highly conserved between the Pof⁺ strain from which the gene was cloned, two Pof^? lager brewing strains and one Pof⁺ Saccharomyces brewery isolate. However, sequence differences have been found between the original Pof⁺ strain, a Pof^?laboratory strain and a Pof^? ale brewing strain.     

EXPRESSION OF THE STA2 GLUCOAMYLASE GENE OF SACCHAROMYCES CEREVISIAE IN BREWERS' YEAST

Two fragments of DNA containing the Saccharomyces cerevisiae STA2 glucoamylase gene, with differing lengths of 5î non-coding DNA, were separately subcloned into a yeast centromeric plasmid. Of these two subclones, only the shorter one (containing 127 base-pairs of 5î non-coding DNA) was able to confer glucoamylase production on a standard laboratory strain of S. cerevisiae. The longer subclone (containing 465 bp of 5î non-coding DNA) did, however, confer glucoamylase production on a strain of S. cerevisiae lacking a functional STA10 gene (which encodes a repressor of STA2 gene expression). All-yeast plasmids lacking bacterial DNA were constructed from the two STA2 subclones for the transformation of a lager brewing yeast. Only the shorter STA2 subclone conferred glucoamylase activity on this yeast. The level of enzyme activity was comparable to that produced by the same yeast strain containing STA2 expressed from the PGK1 (that is, PGK1) promoter.     

A RAPID,SIMPLE AND RELIABLE METHOD OF DIFFERENTIATING BREWING YEAST STRAINS BASED ON DNA RESTRICTION PATTERNS

DNA was isolated from polyploid brewing ale and lager yeast strains using a simple and rapid procedure which was a modification of a previously described method of Seehaus et al.¹⁴ The isolated DNA was cut with a number of restriction enzymes and subjected to agarose gel electrophoresis. Significant differences in banding patterns were observed between a Saccharomyces cerevisiae ale strain DNA and Saccharomyces uvarum (carlsbergensis) lager strain DNA, particularly with the enzyme Hpal. Differences were also observed between the banding patterns of digests from two ale strains, and from two lager strains. Use of this technique with appropriate restriction enzymes should prove useful for the rapid differentiation of brewing yeast strains.     

A RAPID SPECTROPHOTOMETRIC METHOD TO DETERMINE ESTERASE ACTIVITY OF YEAST CELLS IN AN AQUEOUS MEDIUM

A new rapid method to determine the total esterase enzymatic activity of yeast cells is proposed. In a sodium phosphate buffer a β-naphthol synthetic ester is hydrolized by cells, and the released β-naphthol is coupled with a diazonium salt (Fast Garnet GBC) in the presence of sodium dodecyl sulfate. The whole procedure is carried out in an aqueous buffer medium, and the resulting azo dye is directly evaluated by absorbance measurement at 524 nm. The analytical results from different assays were adjusted to a fixed cell concentration with a statistical procedure. The method shows good repeatability, reproducibility and detectability, and it requires simple equipment and instruments. It is therefore suitable both for routine analysis, as industrial yeast strain screening, and for yeast physiological studies, in order to improve the aromatic quality of fermented drinks.     

A FLUOROMETRIC ASSAY FOR PROTEINASE A IN BEER AND ITS APPLICATION FOR THE INVESTIGATION OF ENZYMATIC EFFECTS ON FOAM STABILITY

A previously developed fluorometric assay using synthetic substrate, Succinyl-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, for yeast proteinase A (PrA) was modified for the accurate and quick determination for the activity in unpasteurized beer. Employing simple HPLC for the determination of 7-amino-4-methylcoumarine (AMC), a final degradation product on this assay, the activity of PrA in beer was measured without the interference of the fluorogenic and photosensitive substance present in beer. The assay for common unpasteurized beers was completed within 5 hours without any concentration procedure. Its linearity and reproducibility were satisfactory for quantitative purposes. Using a purified PrA from brewer's yeast, the effect of the PrA activity on foam stability during storage was furthermore clarified. The exclusive effect of PrA on foam stability was also demonstrated by proteinase inhibitor test.     

KINETIC STUDIES OF THE DECONTAMINATION OF YEAST SLURRIES WITH PHOSPHORIC ACID AND ACIDIFIED AMMONIUM PERSULPHATE AND A METHOD FOR THE DETECTION OF SURVIVING BACTERIA INVOLVING SOLID MEDIUM REPAIR IN THE PRESENCE OF CATALASE

Destruction of Obesumbacterium proteus in pitching yeast slurries is more efficiently accomplished using an acidified ammonium persulphate wash (0·75% w/v; pH 2·8) than by using phosphoric acid at pH 2·1. The effectiveness of both methods against the lactic acid bacteria, acetic acid bacteria and Enterobacter agglomerans is similar. Material cost savings of up to 60% can be achieved through the use of the acidified ammonium persulphatewash. Economy in the use of materials and optimum process efficiency is achieved by determination of the quantity of acid required to adjust the pH of the yeast slurry before washing. Bacteria surviving the yeast wash can be detected reliably using a plating technique involving solid medium repair in the presence of catalase. A buffered diluent must be used to prepare the inoculum.     

A CONVENIENT DOMINANT SELECTION MARKER FOR GENE TRANSFER IN INDUSTRIAL STRAINS OF SACCHAROMYCES YEAST: SMRI ENCODED RESISTANCE TO THE HERBICIDE SULFOMETURON METHYL

The SMR1-410 gene of S. cerevisiae, encoding resistance to the herbicide sulfometuron methyl (SM), was used as a dominant selection marker in yeast replicating and yeast integrating vectors for the transformation of wild type strains of baking, brewing (ale and lager), distilling, wine and sake Saccharomyces yeasts. Transformation of lithium treated cells by a YEp vector resulted in transformation frequencies ranging from 200 to 8,000 transformants per 10 ug of DNA. Utilizing a yeast integrating vector with SMR1–410 as the only yeast DNA sequences, it was demonstrated that a single copy of SMR1–410 is sufficient to confer stably inherited SM resistance. Thus the SMR1–410 sequence has the unique ability to act as a selectable marker and to also provide a site for chromosomal integration. Since transformants were resistant to levels at least seven fold higher than wild type strains the resistance phenotype was clearly expressed and easily scored in all industrial strains tested. Unlike other selection markers derived from mammalian or bacterial cells, SMR1–410 is derived from S. cerevisiae. Thus industrial utilization of this marker as a means of genetically improving food and beverage strains of Saccharomyces yeasts by recombinant DNA technology is enhanced, as government regulatory agencies are likely to view it in a more favourable light.