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.     

啤酒酵母的基因改良研究动态

近年来 ,利用基因工程进行酵母的育种在发酵广谱碳水化合物、提高糖化效率 ,改良酵母凝聚特性和改善啤酒风味方面取得了很大成绩。基因重组菌株将逐步应用到生产实践中。     

德国啤酒酵母和国内啤酒酵母发酵性能的研究

酵母菌种是啤酒酿造的关键，不同的菌种可用来酿造不同类型的啤酒。本试验对德国酵母和国内酵母的发酵性能进行了对比研究，总结出了二者之间的差异。     

ENTEROBACTER AGGLOMERANS—A NEW BACTERIAL CONTAMINANT ISOLATED FROM LAGER BEER BREWERIES

From a large number of bacterial strains isolated from all stages of the brewing process in four different beer breweries in South Africa, fifty-five bacterial strains were identified as Enterobacteriaceae by phenotypic analysis. All enterobacterial species previously reported in brewery samples, as well as eighteen strains of Enterobacter agglomerans were found. E. agglomerans strains were isolated from pitching yeast and fermenting wort samples. The occurrence of E. agglomerans in pitching yeast is significant and indicates that these bacteria survive the brewing process. E. agglomerans is regarded as a potential beer spoilage contaminant in lager beer breweries.     

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.     

DETECTION OF WILD YEASTS IN THE BREWERY EFFICIENCY OF DIFFERENTIAL MEDIA

The efficiency of five differential media in the detection of wild yeasts was compared. On the basis of the types of wild yeast they are able to detect, these differential media were classified into two groups. Group I consists of actidione medium and lysine medium, and is suitable for the detection of non-Saccharomyces wild yeasts. In this group, the lysine medium detected more species and a higher percentage of wild yeasts than the actidione medium. Group II consists of crystal violet medium, SDM, and Lin's medium, and is suitable for the detection of Saccharomyces wild yeasts. In this group, Lin's medium is superior to crystal violet medium and SDM for wild yeast detection. It is suggested that lysine medium and Lin's medium be employed together for the detection of wild yeasts in the brewery.     

Beer enemy number one: genetic diversity,physiology and biofilm formation of Lactobacillus brevis

Lactobacillus brevis is the most significant beer spoilage bacteria worldwide. It is found as a contaminant at all stages of brewing, including during primary and secondary fermentation, storage, filtration and the packaging process. In production with flash pasteurisation and subsequent hygienic filling, avoiding and tracing secondary contaminations is the key to a microbiologically stable product. However, L. brevis strains vary in their spoilage potential and can grow in many different beer types. This study presents a physiological test scheme for growth potential and biofilm formation in various media. It was determined that a large number of L. brevis strains can form biofilms as a first coloniser. The identification of the species alone is therefore not enough to be sure of the spoilage risk, which shows the need for a more in depth differentiation. DNA fingerprint techniques are crucial to differentiate isolates of this species at strain level. The rep‐PCR fingerprint system (GTG)₅ was used to differentiate a selected collection of 20 isolates, which were characterised in growth and biofilm formation in various media. The data showed a high variation within the selected isolates. As second step, generated fingerprint clusters of L. brevis were traced back to contamination sources in a German brewery, revealing a high number of isolates with potentially varying growth, spoilage and biofilm potential. L. brevis being the demonstrator species, the PCR system used is a powerful and compatible tracing and troubleshooting tool for all kinds of spoilage bacteria in the brewing industry. © 2019 The Institute of Brewing & Distilling     

CHARACTERIZATION OF BREWING YEAST STRAINS BY NUMERICAL ANALYSIS OF TOTAL SOLUBLE CELL PROTEIN PATTERNS

Total soluble cell proteins from 33 yeast strains from the brewing industry were extracted and subjected to polyacrylamide gel electrophoresis. Yeast strains were grouped by computerized numerical analysis of protein banding patterns. Three clusters were obtained at r>0.90. Cluster I contained 21 Saccharomyces cerevisiae lager beer strains. Cluster II comprised two strains isolated from beer with a phenolic off flavour and a third strain used for lager beer brewing. Cluster III consisted of two bottom ale yeasts. Protein patterns of yeast strains within each cluster corresponded closely or were identical. However, the intensity of certain bands often varied and the number of peaks recorded was not identical. These minor differences were reproducible and regarded as characteristic for the specific strains. Protein patterns can therefore be used to characterize or fingerprint individual yeast strains.     

Application of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry as a monitoring tool for in‐house brewer's yeast contamination: a proof of concept

Contamination of brewer's pitching yeast cultures with wild‐type yeasts or bacteria is unwanted as it can corrupt the fermentation outcome and causes huge economic losses for the brewing industry. The applicability of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a fast tool to monitor the purity of brewer's yeast cultures was investigated. This proof of concept was examined for a brewer's yeast strain contaminated with wild‐type yeast and for bottled beer produced by fermentation with that particular contaminated brewer's yeast strain. The data demonstrated that MALDI‐TOF MS is very suitable to discriminate between brewing and non‐brewing yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

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.     

Two-dimensional gel analysis of the proteome of lager brewing yeasts

Modern lager brewing yeasts used in beer production are hybrid strains consisting of at least two different genomes. To obtain information on the identity of the parental strains that gave rise to industrial lager yeasts, we used two-dimensional (2-D) gel electrophoresis and analysed the proteomes of different Saccharomyces species isolated from breweries. We found that the proteome of lager brewing yeasts and of the type strains of S. carlsbergensis, S. monacensis and S. pastorianus can be interpreted as the superimposition of two elementary patterns. One originates from proteins encoded by a S. cerevisiae-like genome. The other corresponds to a divergent Saccharomyces species whose best representative is a particular S. pastorianus strain, NRRL Y-1551. A map of industrial lager brewing yeasts has been established, with the individual origin of proteins and with identification of protein spots by comparison to known S. cerevisiae proteins. This 2-D map can be accessed on the Lager Brewing Yeast Protein Map server through the World Wide Web. This study provides the first example of the use of proteome analysis for investigating taxonomic relationships between divergent yeast species.     

啤酒酿造中的微生物污染

由于啤酒酿造的环境奈件,如营养丰富的麦芽汁、发酵过程中酵母产生的生长因子以及较长的发酵时间等非常适宜于微生物的生长,所以在啤酒酿造中许多环节都存在微生物污染的可能性。啤酒酿造过程中的微生物污染不仅会影响产品的质量,严重时还会给啤酒生产厂家造成一定的经济损失。介绍了啤酒酿造过程中的污染微生物的种类、来源以及对啤酒质量的影响。通过了解啤酒酿造中微生物的污染情况,可以有助于我们生产高质量的啤酒产品.     

LIPIDS IN THE BREWERY—A MATERIAL BALANCE

Lipids, measured as total long-chain fatty acids, have been determined on samples taken from all stages in a pilot-scale brewery. This exercise has identified both the materials which supply the lipids in brewing, and the processes responsible for their removal. The balance demonstrates areas where significant quantities of lipid are generated or lost. Very little of the lipid present in the raw materials survives the brewing process: of the 277 g lipid in 8800 g malt, only 0·1 g (0·03%) remains in the finished beer. Spent grains, hot break and cropped yeast are all effective means of removing lipids.     

Enhancing the Microbiological Stability of Malt and Beer — A Review

While beer provides a very stable microbiological environment, a few niche microorganisms are capable of growth in malt, wort and beer. Growth of mycotoxin‐producing fungi during malting, production of off‐flavours and development of turbidity in the packaged product due to the growth and metabolic activity of wild yeasts, certain lactic acid bacteria (LAB) and anaerobic Gram negative bacteria, impact negatively on beer quality. It follows that any means by which microbial contamination can be reduced or controlled would be of great economic interest to the brewing industry and would serve the public interest. There has been an increasing effort to develop novel approaches to minimal processing, such as the exploitation of inhibitory components natural to raw materials, to enhance the microbiological stability of beer. LAB species, which occur as part of the natural barley microbiota, persist during malting and mashing, and can play a positive role in the beer‐manufacturing process by their contribution to wort bioacidification or the elimination of undesirable microorganisms. Other naturally occurring components of beer that have been valued for their preservative properties are hop compounds. It may be possible to enhance the antimicrobial activities of these compounds during brewing. Some yeast strains produce and excrete extracellular toxins called zymocins, which are lethal to sensitive yeast strains. Yeast strains resistant to zymocins have been constructed. Imparting zymocinogenic activity to brewing yeast would offer a defence against wild yeasts in the brewery. Thus, the antimicrobial properties of naturally occurring components of raw materials can be exploited to enhance the microbial stability of beer.     

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.     

A note on the use of conductimetry in brewery microbiological control

The growth in beer of 15 brewery strains of Lactobacillus was detected using the automatic conductance measuring technique afforded by the Malthus 8 Growth Analyser. 0·15% ammonium chloride was added to the beer as an electrolyte and sodium acetate was used to raise the pH from ca 4·0 to 4·2 or above. No measurable responses were obtained using unmodified beer. The value of the method for development in routine brewery microbiological control is briefly discussed.     

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.     

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.     

Investigation of beer‐spoilage ability of Dekkera/Brettanomyces yeasts and development of multiplex PCR method for beer‐spoilage yeasts

There have been many beer‐spoilage incidents caused by wild yeasts. Saccharomyces cerevisiae, Dekkera anomala and D. bruxellensis have been recognized as beer‐spoilage yeasts in the brewing industry. In contrast, the beer spoilage ability of Brettanomyces custersianus has not been well characterized, although this species was isolated from beer. In this study, the beer‐spoilage ability of currently described Dekkera/Brettanomyces yeast species was investigated. As a consequence, D. anomala, D. bruxellensis and B. custersianus were shown to grow in commercial beers. On the other hand, the remaining two Brettanomyces species, B. naardenensis and B. nanus, did not grow in beer. These results indicate that B. custersianus should be recognized as a beer‐spoilage species, in addition to S. cerevisiae, D. anomala, and D. bruxellensis. Therefore we developed multiplex polymerase chain reaction (PCR) for the simultaneous detection and identification of B. custersianus and the other beer‐spoilage yeast species. For this purpose, PCR primers were designed in the internal transcribed spacer region or 26S rDNA, and each PCR product was made in different sizes to easily discriminate the species from electrophoretic results. Specificity, reactivity and sensitivity of the designed primers were evaluated. As a result, the developed multiplex PCR method was shown to have high specificity and reactivity, and therefore was considered as an effective tool to identify beer‐spoilage yeast species. This tool can contribute to microbiological quality assurance in breweries. Copyright © 2015 The Institute of Brewing & Distilling     

STRAINS OF YEAST LETHAL TO BREWERY YEASTS

Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.