DIFFERENTIATION OF BREWERY YEAST STRAINS BY DNA FINGERPRINTING

A reliable and reproducible non-radioactive DNA fingerprinting technique has been used to differentiate between twelve production brewery yeast strains. The method comprises total DNA preparation from the yeast strains, enzymatic digestion of the DNA and analysis of the DNA fragments by “probing” with known DNA labelled with the plant steroid digoxigenin. A range of probes and several enzymes have been investigated. A successful combination is the restriction endonuclease Hind III and a probe derived from Ty1 (a yeast transposable element). The main advantages of this method are its unambiguity, reproducibility and suitability for routine brewery QC use.     

APPLICATION OF THE POLYMERASE CHAIN REACTION TO THE RAPID ANALYSIS OF BREWERY YEAST STRAINS

The rapid discrimination of closely-related Saccharomyces cerevisiae strains can pose a significant problem to breweries, in particular where closely related strains are being used simultaneously to manufacture different products. In this study, two PCR approaches have been examined to assess their usefulness for the discrimination of brewery ale and lager yeast strains. PCR using arbitrary primers (RAPD PCR) was found unsuitable for such an application since the DNA profiles generated from brewery strains were generally found to be identical, due presumably to the close genetic relatedness of these yeasts. In contrast, PCR using δ sequence primers could rapidly differentiate between many ale and lager strains and characteristic profiles for these were generated. This method could also be applied directly to yeasts isolated from brewery worts or from active dried yeast preparations. Results of such analyses were available within the working day.     

FERMENTATION PROPERTIES OF BREWING YEAST WITH KILLER CHARACTER

The cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae has been transferred to three different commercially-used brewing yeasts; two ale strains and one lager strain. The ease with which the character can be transferred is very strain dependent. In addition to killer character, mitochondria from the brewing strain have been transferred into the new ‘killer’ brewing strains. Fermentations carried out with the manipulated strains produced beers which were very similar to those produced by the control brewing strains. The beers produced by killer brewing strains containing brewing yeast mitochondria were most like the control beers and could not be distinguished from them in three glass taste tests. In addition to producing good beers the genetically manipulated yeasts killed a range of contaminant yeasts and were themselves immune to the action of Kil-k₁ killer yeasts.     

THE INCIDENCE AND SIGNIFICANCE OF WILD SACCHAROMYCES CONTAMINANTS IN THE BREWERY

The persistence of low levels of contamination by non-brewing Saccharomyces through several batch fermentations establishes the immuno-fluorescent method as a very sensitive procedure for estimating the microbiological purity of pitching yeasts. Trade return figures for draught beers show that in this brewery the principal cause for high rejection rates has, on several occasions, been contamination of pitching yeasts with “wild” Saccharomyces. The recommendation is made that pitching yeasts should be discarded when the level of infection achieves 100 cells of wild Saccharomyces per million cells of brewing yeast.     

THE GENETIC MANIPULATION OF KILLER CHARACTER INTO BREWING YEAST

A procedure is described whereby the cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae is transferred to a brewing yeast strain. Neither preparation of protoplasts of the brewing yeast nor mutation of its nuclear genes are required for this process. The brewing yeast killer strains produced have the advantages over their parent brewing cell that they kill sensitive yeasts and are immune to the killing action of certain killer yeasts. The method described offers significant advantages over the process of transformation as a means of genetically manipulating commercial yeasts.     

GROWTH OF AEROBIC WILD YEASTS

Wild yeasts of the genera Debaryomyces, Hansenula and Pichia are commonly considered to be associated with spoilage only under aerobic conditions. However, in pure cultures in either wort or a synthetic medium of yeast nitrogen base + 10% glucose, yeasts of these genera grew as well as a brewing strain of Saccharomyces cerevisiae under anaerobic conditions. Growth of S. cerevisiae was increased by the addition of unsaturated fatty acid (Tween 80) or ergosterol to the medium for anaerobic culture. No equivalent requirement was observed for the wild yeasts examined. Indeed, growth of the wild yeasts was often reduced by the addition of Tween 80, which as a surfactant prevented formation of the surface film of growth. Even under anaerobic conditions, these yeasts grew best with a surface pellicle. Although capable of good anaerobic growth in pure culture, growth of the wild yeasts was suppressed under anaerobic conditions in mixed culture with S. cerevisiae, simulating a contaminated brewery fermentation. However, the contaminants competed successfully with S. cerevisiae under aerobic conditions. There was no evidence of a “killer” effect, but prevention of pellicle formation, or production of inhibitory levels of pH or ethanol under anaerobic conditions could explain the suppression of wild yeasts under anaerobic fermentation conditions.     

SOME OBSERVATIONS ON YEAST MUTATION DURING CONTINUOUS FERMENTATION

The spontaneous genetic mutation of yeasts, including brewery yeasts, is a well-known phenomenon. By analogy with other micro-organisms it may be suspected that under the peculiar conditions of continuous fermentation mutant forms will tend to accumulate, involving the risk sooner or later of far-reaching qualitative changes of the yeast mass. Since mutations usually imply the loss, rather than the gain, of desirable characters it cannot be excluded that such changes may be detrimental to beer quality. The observations reported here on a typical bottom fermenting strain of brewery yeast, Saccharomyces carlsbergensis, have shown that after cultivation for nine months in a continuous fermentor approximately one half of the cells had mutated. The mutations included loss of flocculence, reduction of fermentation efficiency, reduction of final attenuation, change of growth rate, and production of undesirable flavours. One mutant form of especial taxonomic interest even appeared to have lost its claim to membership of the parental species.     

AMINO ACID BALANCE IN YEAST FERMENTATION WITH A SYNTHETIC AMINO ACID MIXTURE AS NITROGEN SOURCE

Two brewery yeasts, one bottom- and one top-fermenting strain, were allowed to ferment an 8% glucose solution containing as nitrogen source an amino acid mixture simulating that obtained when yeast was autolysed. The amounts given were approximately twice as high as the expected requirements. After completion of fermentation the total amounts of each amino acid in the whole system, i. e., in medium and yeast, were determined. The results show that the yeast had not taken up amino acids according to its own composition. The amino acids previously found to be rapidly absorbed from brewery wort were present in the whole system in considerably smaller amounts than in the original medium, indicating that these acids had been utilized as a nitrogen souce or for other purposes. The acids which are taken up slowly from brewery wort were present in larger amounts than in the original medium, indicating that they had been synthesized despite the excess in the medium. The two strains showed relatively similar behaviour.     

THE USE OF CONTROLLED LEVELS OF ACTIDIONE FOR BREWING AND NON-BREWING YEAST STRAIN DIFFERENTIATION

The variability of results using actidione media for the identification of beer spoilage bacteria has been shown to be due to the heat sensitivity of this reagent. Critical evaluation shows that with a particular brewery yeast, media containing 0·06 p.p.m. allow the growth of a single brewing strain but inhibit others. This permits the rapid classification of yeast strains in this pitching yeast. In a further brewery, media containing 0·16 p.p.m. differentiate brewing yeast strains from beer spoilage yeasts and this permits the quantitative assessment of these for process control at all stages of brewing. In a particular instance where a culture yeast strain is not inhibited at 0·16 p.p.m. but is p-aminobenzoic acid dependent, a differential medium incorporating this low level of actidione and omitting the vitamin allows the identification of beer spoilage yeasts in process control. These results illustrate the potential of this approach for microbiological control in specific brewery problems.     

INHIBITION OF BEER-SPOILAGE LACTIC ACID BACTERIA BY NISIN

149 strains of bacteria, mostly brewery contaminants able to spoil wort or beer, and 12 brewing strains of yeast (8 ale and 4 lager strains) have been screened using a well-test assay for sensitivity to the food preservative, Nisin (E234), Nisin inhibited growth of 92% of the gram-positive strains, predominantly lactic acid bacteria of the genera Lactobacillus and Pediococcus. In contrast, all 32 gram-negative strains tested, except 3 Flavobacter strains, were Nisin-resistant; in addition none of the brewing yeasts showed Nisin-sensitivity. Therefore. Nisin has potential applications in preventing spoilage of worts or beers by lactic acid bacteria.     

DETECTION OF WILD YEASTS IN THE BREWERY

The potential of an established culture medium, Wallerstein Laboratories' nutrient agar, for the detection of wild yeasts has been evaluated and recommended for microbiological quality control in brewery laboratories. Wild yeast strains, including Saccharomyces species, can be differentiated from strains of Saccharomyces cerevisiae by the colour, form and rate of growth of their colonies on this medium within 2–3 days. The sensitivity of the method is such that one wild yeast can be detected in a Saccharomyces cerevisiae population of the order of 10° cells.     

APPLICATION OF PULSED FIELD CHROMOSOME ELECTROPHORESIS IN THE STUDY OF CHROMOSOME XIII AND THE ELECTROPHORETIC KARYOTYPE OF INDUSTRIAL STRAINS OF SACCHAROMYCES YEASTS

Pulsed field chromosome electrophoresis is a powerful new technique in yeast genetics which permits the resolution of intact yeast chromosomes in an agarose gel matrix. We utilized contour-clamped homogeneous electric field electrophoresis (CHEF) to survey representative strains of Saccharomyces yeasts from the brewing, baking, distilling, sake and wine industries for their electrophoretic karyotypes. All of the strains tested were found to have a unique chromosomal profile, indicating the potential of this technology for “fingerprinting” prototrophic strains of Saccharomyces yeasts. By employing an ILV2 gene probe specific for chromosome XIII, we determined that all of the industrial strains of Saccharomyces yeasts possessed a chromosome XIII which migrated in an identical fashion to chromosome XIII from a reference haploid strain of Saccharomyces cerevisiae. While one lager yeast strain, Saccharomyces carlsbergensis M244, was found to contain two alleles of ILV2 when digested genomic DNA was probed with ILV2, the presence of a novel independently migrating chromosome XIII could not be detected. A homeologous chromosome XIII in this yeast will therefore have to be determined by genetic analysis. Pulsed field chromosome electrophoresis is concluded to be a technology with immediate application to Quality Control and Research and Development programs in industries using Saccharomyces yeasts.     

嗜杀酿酒酵母对海南山兰甜酒发酵品质的影响

以海南坡稻山兰为主要原料,在传统手工酿造山兰甜酒的工艺基础上添加嗜杀酿酒酵母作为发酵剂能保持山兰甜酒原始的酒醇甘甜微苦风味,维持独特的米脂芳香。控制和净化发酵醪中的酵母品质,在发酵过程中保持酒味纯正,使糖化与发酵得到同步,发酵力得到加强,酒味更加醇厚。同时发酵环境得到净化,发酵时间缩短,生产效率提高。嗜杀酵母发酵剂令山兰甜酒出现苦味物质的时间提前,同时相对传统工艺生成较高浓度的高级醇,可采取定时移除酒液的方法解决。     

The Occurrence of Beer Spoilage Lactic Acid Bacteria in Craft Beer Production

Beer is one of the world's most ancient and widely consumed fermented alcoholic beverages produced with water, malted cereal grains (generally barley and wheat), hops, and yeast. Beer is considered an unfavorable substrate of growth for many microorganisms, however, there are a limited number of bacteria and yeasts, which are capable of growth and may spoil beer especially if it is not pasteurized or sterile‐filtered as craft beer. The aim of this research study was to track beer spoilage lactic acid bacteria (LAB) inside a brewery and during the craft beer production process. To that end, indoor air and work surface samples, collected in the brewery under study, together with commercial active dry yeasts, exhausted yeasts, yeast pellet (obtained after mature beer centrifugation), and spoiled beers were analyzed through culture‐dependent methods and PCR‐DGGE in order to identify the contaminant LAB species and the source of contamination. Lactobacillus brevis was detected in a spoiled beer and in a commercial active dry yeast. Other LAB species and bacteria ascribed to Staphylococcus sp., Enterobaceriaceae, and Acetobacter sp. were found in the brewery. In conclusion, the PCR‐DGGE technique coupled with the culture‐dependent method was found to be a useful tool for identifying the beer spoilage bacteria and the source of contamination. The analyses carried out on raw materials, by‐products, final products, and the brewery were useful for implementing a sanitization plan to be adopted in the production plant.     

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.     

The ecology of killer yeasts: Interference competition in natural habitats

Killer yeasts are ubiquitous in the environment: They have been found in diverse habitats ranging from ocean sediment to decaying cacti to insect bodies and on all continents including Antarctica. However, environmental killer yeasts are poorly studied compared with laboratory and domesticated killer yeasts. Killer yeasts secrete so-called killer toxins that inhibit nearby sensitive yeasts, and the toxins are frequently assumed to be tools for interference competition in diverse yeast communities. The diversity and ubiquity of killer yeasts imply that interference competition is crucial for shaping yeast communities. Additionally, these toxins may have ecological functions beyond use in interference competition. This review introduces readers to killer yeasts in environmental systems, with a focus on what is and is not known about their ecology and evolution. It also explores how results from experimental killer systems in laboratories can be extended to understand how competitive strategies shape yeast communities in nature. Overall, killer yeasts are likely to occur everywhere yeasts are found, and the killer phenotype has the potential to radically shape yeast diversity in nature.     

IFA在酿酒污染野生酵母快速检测中的应用

用荧光抗体技术进行快速检测,方法简便、灵敏、快速。用三种混合血清可以检测出常见全部污染野生酵母。该技术在工厂中应用结果表明,酿酒种酵母中野生酵母污染现象是存在的,而且随着种酵母使用代数的增加而越趋严重。     

GENE TECHNOLOGY FOR INDUSTRIAL YEASTS

Yeast genetics is now available as a practical tool for the development of brewing industry practices. The contribution of Brewing Research Foundation work (1978–84) to recent advances is illustrated by the construction of brewing strains with superattenuating (amylolytic) or anti-contaminant properties. Approaches based on hybridisation (by rare mating) or recombinant DNA technology have been evaluated. Techniques developed for (i) gene transfer to brewing strains, (ii) ensuring stable inheritance of novel characteristics and (iii) exploiting the secretory ability of yeast strains, can be widely applied not only with brewing, distilling, baking or wine yeasts, but also in the use of yeasts to produce novel biotechnical products. ‘Spin-off’ from these studies includes valuable methods for differentiating or enumerating wild yeasts in brewery quality control.     

Determination of killer activity in yeasts isolated from the elaboration of seasoned green table olives

In this work 51 yeasts strains isolated from seasoned green table olives and belonging to the Candida, Debaryomyces, Kluyveromyces, Pichia, and Saccharomyces genera were characterized by their killer activity in different conditions. Killer activity of isolates was analyzed in a medium with different pH's (3.5 to 8.5) and NaCl concentrations (5, 8, and 10%). At every pH tested, all the genera studied had killer strains, although the smallest percentages of killer yeasts were found at the highest pH (8.5). The presence of 5 and 8% NaCl increased the detected killer percentage, but the highest salt concentration (10%) decreased it. The interaction between the reference killer yeasts and yeasts isolated from olives was analyzed. Most isolates were killer-sensitive to one or more killer reference strains. Only 2 of the 51 strains tested were considered killer-neutral. Cross-reaction trials between isolates and spoilage yeasts showed that, of the isolates, nine killer strains, belonging to Debaryomyces hansenii, Kluyveromyces marxianus, Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae, had the broadest spectra of action against yeasts that cause spoilage. These killer yeasts and the toxins that they produce are candidates for further investigation as suppressors of indigenous olive table yeast growth. The results confirmed the highly polymorphic expression of the killing activity, with each strain showing different killer activities. This method may thus be very useful for simple and rapid characterization of yeast strains of industrial interest.