Polar Lipids of Brewer's Yeasts

Both traditional and DNA‐based methods sometimes fail to differentiate between closely related strains of commercial interest in the brewing industry. The aim of this study was to compare species and sub species of Saccharomyces cerevisiae on the basis of their polar lipid chemistry using chromatographic methods. Six isolates were studied after propagation under batch conditions. Polar lipids were then extracted from lyophilised cultures and analysed by TLC in order to separate phospholipid families. TLC showed that the major phospholipid classes present were PC > PE > PG. Two unidentified phospholipids were found, one only in strain 34/70. The major peaks detected by GLC were identified as methyl esters of palmitic acid and palmitoleic acid. The fatty acid composition of PC varied between strains and novel data on lecithin acyl constituents were observed. The polar lipid method succeeded in differentiating strain 34/70 — one of the most commonly used brewer's lager yeast — from strain 34/78 and other species tested. The presence of unusual polar lipids in Saccharomyces sensu stricto yeasts may be useful in distinguishing between other closely related strains.     

红曲黄酒酿造用曲及传统酿造过程中酵母菌的多样性研究

目的:探讨红曲黄酒酿造用曲中酵母菌多样性以及传统酿造过程中酵母菌菌群结构变化,为我国传统红曲黄酒中酵母菌资源的利用和对传统酿酒的有效控制及现代化酿酒新工艺的建立提供基础数据。方法:收集福建各地区的红曲黄酒酿造用曲20份,从中分离纯化出300株酵母菌,通过ITS1-5.8S-ITS2的PCR-RFLP指纹图谱对酵母菌进行分型,从各个分型类群中随机选取代表菌株,利用26S rDNA基因D1/D2区域序列分析进行分类鉴定;并采用PCR-RFLP快速分型鉴定技术分析红曲黄酒传统酿造过程中酵母菌菌群结构的变化。结果:从红曲黄酒酿造用酒曲中总共分离鉴定出12种类型酵母菌,其中扣囊复膜孢酵母(Saccharomycop-sis fibuligera)、酿酒酵母(Saccharomyces cerevisiae)和弗比恩毕赤酵母(Pichia fabianii)是酒曲中3种主要的酵母菌类型。红曲黄酒传统酿造过程酵母菌群的跟踪分析共鉴定出4种酵母菌,即酿酒酵母、扣囊复膜孢酵母、季也蒙毕赤酵母(Pichia guilliermondii)、粘性红圆酵母(Rhodotorula mucilaginosa)。在酿造前期扣囊复膜孢酵母是优势酵母菌,而在酿造的后期,酿酒酵母完全取代之成为优势菌。结论:红曲黄酒酿造用酒曲中的酵母菌具有丰富的生物多样性,红曲黄酒传统酿造过程酵母菌菌群结构处于动态变化,最终酿酒酵母成为酿造体系的优势酵母菌。     

BREWING YEAST IDENTIFICATION AND CHROMOSOME ANALYSIS USING HIGH RESOLUTION CHEF GEL ELECTROPHORESIS

Contour-clamped homogeneous electric field (CHEF) gel electrophoresis has been used to study the karyotypes of a range of Saccharomyces cerevisiae yeast strains. The time required from sampling yeast cultures to CHEF analysis was achieved within six hours, making this procedure very useful in reference and quality control work in the brewing industry. Regions of the chromosome profiles were closely studied by adjusting electrophoresis conditions to increase resolution between bands. Both ale and lager strains of brewing yeasts were studied alongside haploid laboratory strains. By comparing different regions of the profiles even very closely related strains of lager yeast could be distinguished. Brewing strains consistently had significantly more chromosome bands than haploid laboratory strains. The electrophoretic karyotypes of brewing yeasts were represented as groups of bands on CHEF gels which apparently comigrated with their haploid chromosomal counterparts.     

LIPID COMPOSITION OF AEROBICALLY AND ANAEROBICALLY PROPAGATED BREWER'S BOTTOM YEAST

Aerobically grown pitching yeast is very rich in unsaturated fatty acids and sterol esters compared to traditional, anaerobic yeast. The principal fatty acids in aerobic yeast cells are unsaturated palmitoleic and oleic acids, whereas in anaerobic cells saturated palmitic acid predominates. The difference in fatty acid distribution between aerobic and anaerobic cells is most marked in the sterol esters. The fatty acids of phospho-lipids are more stable, although remarkable differences are observed. The sterols of aerobic cells are almost entirely in esterified form and zymosterol is the principal sterol. During the first hours of fermentation a rapid synthesis of palmitoleic acid is observed when anaerobic yeast is used for pitching and the wort is aerated. The synthesis of oleic acid requires more oxygen and time than is available under normal brewing conditions. When aerobic pitching yeast is used no more unsaturated fatty acids are synthesised and the lipid stores of pitching yeast are distributed among the daughter cells. The decrease in acetate ester production by aerobic pitching yeast is concluded to be due to a decrease in acetyl CoA synthesis, which may be caused by the high proportion of unsaturated fatty acids in membrane lipids.     

CINNAMIC ACID AS THE BASIS OF A MEDIUM FOR THE DETECTION OF WILD YEASTS

Cinnamic acid (100 μg ml^?1) incorporated in a solid medium was found to inhibit the growth of brewing strains (Pof^?) of yeast while permitting the growth of Pof+ wild yeast contaminants. Typically, colonies of Saccharomyces cerevisiae var. diastaticus (Pof+) mixed with brewing yeast (S. cerevisiae NCYC 240) were visible after 5d incubation at 25°C. The incubation time required to detect a selection of brewery wild yeast isolates was found to vary from 3–12 d.     

Determination and fractionation of metals in beer: A review

Major, minor and trace metals are important in beer fermentation since they supply the appropriate environment for yeast growth and influence yeast metabolism. A real concern is the content of copper (Cu) and iron (Fe), which are involved in beer conditioning and ageing through reactions resulting in formation of reactive oxygen species. The reactive oxygen species readily oxidize organic compounds present in beer, changing the quality of foaming and the flavour stability of beer. In view of brewing technology and beer processing, knowledge regarding functions of metals and their speciation in brewing liquors and beer is of special significance. Metals in beer also have a certain nutritional importance, but their actual effect related to beer consumption depends on the type of species they form with low and high molecular mass organic ligands which naturally occur in beer. This review covers the determination and fractionation of metals in beer using atomic spectrometry methods. Special attention is drawn to the role of metals in beer and brewing, possible metal associations, methods of beer preparation before analysis on the total metal content, and approaches to metal partitioning in beer.     

Determination and fractionation of metals in beer: a review.

Major, minor and trace metals are important in beer fermentation since they supply the appropriate environment for yeast growth and influence yeast metabolism. A real concern is the content of copper (Cu) and iron (Fe), which are involved in beer conditioning and ageing through reactions resulting in formation of reactive oxygen species. The reactive oxygen species readily oxidize organic compounds present in beer, changing the quality of foaming and the flavour stability of beer. In view of brewing technology and beer processing, knowledge regarding functions of metals and their speciation in brewing liquors and beer is of special significance. Metals in beer also have a certain nutritional importance, but their actual effect related to beer consumption depends on the type of species they form with low and high molecular mass organic ligands which naturally occur in beer. This review covers the determination and fractionation of metals in beer using atomic spectrometry methods. Special attention is drawn to the role of metals in beer and brewing, possible metal associations, methods of beer preparation before analysis on the total metal content, and approaches to metal partitioning in beer.     

Determination and fractionation of metals in beer: a review

Major, minor and trace metals are important in beer fermentation since they supply the appropriate environment for yeast growth and influence yeast metabolism. A real concern is the content of copper (Cu) and iron (Fe), which are involved in beer conditioning and ageing through reactions resulting in formation of reactive oxygen species. The reactive oxygen species readily oxidize organic compounds present in beer, changing the quality of foaming and the flavour stability of beer. In view of brewing technology and beer processing, knowledge regarding functions of metals and their speciation in brewing liquors and beer is of special significance. Metals in beer also have a certain nutritional importance, but their actual effect related to beer consumption depends on the type of species they form with low and high molecular mass organic ligands which naturally occur in beer. This review covers the determination and fractionation of metals in beer using atomic spectrometry methods. Special attention is drawn to the role of metals in beer and brewing, possible metal associations, methods of beer preparation before analysis on the total metal content, and approaches to metal partitioning in beer.     

STEROL SYNTHESIS IN RELATION TO GROWTH AND FERMENTATION BY BREWING YEASTS INOCULATED AT DIFFERENT CONCENTRATIONS

The sterol contents of brewing yeasts harvested from an anaerobic fermentation and inoculated into air-saturated wort rise rapidly from ca. 1 mg/g dry yeast to ca. 10 mg/g and remain at this level until the oxygen is absorbed. After all oxygen is removed from the medium, sterol synthesis ceases and the concentration in the cells declines as growth occurs until a limiting value is reached, when reproductive growth ceases. In the presence of oxygen, sterols are present primarily as sterol esters; free sterols accumulate after oxygen uptake is complete. The total quantity of sterol which is synthesized in the presence of oxygen determines the extent of subsequent yeast growth. It is shown that the magnitude of sterol synthesis in the yeast population as a whole is influenced by the pitching rate.     

LONG-CHAIN FATTY ACID COMPOSITION AS A CRITERION FOR YEAST DISTINCTION IN THE BREWING INDUSTRY

The diversity of yeasts isolated from brewing plants and its role on beer quality makes yeast distinction a major concern in industrial microbiological control. Several approaches have been tried to develop rapid and simple methods to perform such tasks. Among these, stands the utilization of long-chain fatty acid composition of total yeast biomass. In this paper results are reported showing the potential of this technique to characterize yeast flora isolated from industrial plants. Fatty acid profiles of brewing species are clearly differentiated from those of non-Saccharomyces strains using statistical data treatment by principal component analysis (PCA). Distinction between brewing and wild strains of Saccharomyces spp. was not apparent. In comparison, fatty acid profiling showed higher discriminating ability than growth on lysine medium for non-Saccharomyces strains. For distinction of S. cerevisiae var. diastaticus from other Saccharomyces strains, growth on starch medium showed to be necessary.     

Molecular species of phosphatidylethanolamine from continuous cultures of Saccharomyces pastorianus syn. carlsbergensis strains

Saccharomyces pastorianus syn. carlsbergensis strain 34/70 is well known to be the most used strain for lager beer production. The difference between this strain and very closely related strain 34/78 is the latter's greater flocculating character. This single physiological trait can cause technical difficulties in beer production. The aim of this study was to determine whether lipid analysis by a combination of thin layer chromatography (TLC) with electrospray ionization mass spectrometry (ESI-MS) could be used as a strain-typing technique in order to distinguish S. pastorianus syn. carlsbergensis strain 34/70 from strain 34/78. Both strains (34/70 and 34/78) were harvested after continuous culture under standard conditions. Polar lipids were then extracted from lyophilized cultures and analysed by TLC in order to separate phospholipid families. Phosphatidylethanolamine (PE) was extracted and investigated using ESI-MS, to gain further information on individual molecular species. Using TLC analysis, lipids were separated corresponding to standards for PE, phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA) and sphingomyelin (SM). ESI-MS of the PE band, separated by TLC, showed that electrospray mass spectra were highly reproducible for repeat cultures. Novel findings were that both brewing strains displayed major phospholipid peaks with m/z 714, PE (34 : 2) m/z 742, PE (36 : 2) and m/z 758, PE (37 : 1). However, strain 34/78 had additional peaks of m/z 700, PE (33 : 2) and m/z 728, PE (35 : 2). Strain 34/70 had an extra peak with m/z 686 PE (32 : 2). We conclude that combined TLC/ESI-MS can distinguish between S. pastorianus syn. carlsbergensis 34/70 and 34/78 and may be a useful typing technique for differentiation of closely related yeast strains. This novel approach may aid quality assurance and could be suitable for yeast collections and larger industrial companies.     

PCR‐Based Differentiation and Homology of Brewing and Type Strains of the Genus Saccharomyces

Restriction fragment length polymorphism (RFLP) patterns of PCR‐amplified ribosomal RNA gene fragments (rDNA) and randomly amplified polymorphic DNA (RAPD) were applied for the analysis of 15 brewing and 6 related yeast strains of the genus Saccharomyces. One five‐base (ScrFI) and two four‐base cutting (HaeIII, MspI) restriction enzymes were used. The primers 21 and M13 core sequence were selected for RAPD analysis. PCR‐RFLP rDNA analysis with HaeIII, ScrFI and MspI differentiated the strains tested into four, five and four types of patterns, respectively and the analyses of the profiles showed 100% homology, between the yeast strains. One strain was an exception. Homological groups were observed for strains used in breweries globally, from a local production strain and from the isolates identified as S. cerevisiae. Using RAPD analysis, and according to discrete differences in the profiles, it was possible to divide twenty one strains into 15 and 20 groups with primer 21 and M13 respectively. RFLP‐PCR rDNA analysis was used to show similarities in closely related brewing strains, while RAPD analysis was used for differentiation of strains.     

组合酶法快速酿造蓝莓酒新工艺的研究

以蓝莓果为酿造原料,在酿造过程中,对复合酶的种类和最佳配比进行了试验,确定了各种酶的用量和最佳工艺条件。经分离筛选得到了适用于蓝莓酒发酵的酿酒一号酵母菌株,对形态与生理特征进行了鉴定,并测试了酵母菌种的产酒力、耐SO2能力、耐乙醇能力及耐酸力。蓝莓果经生物酶解、灭酶、澄清处理,用酿酒一号酵母菌株发酵、陈酿、过滤、调配等工艺酿造的蓝莓半甜酒和蓝莓甜酒,果香浓郁,清澈明亮,营养丰富,口味纯正。该工艺提高出汁率10%,改变了传统红酒生产的工艺过程。酿造周期由传统工艺的三年以上缩短为六个月,为红酒的生产开辟了一条新途径。     

酵母菌高糖胁迫机制的研究进展

耐高糖酵母在高糖的发酵环境下仍能有效发酵,这与酵母的高糖胁迫机制密切相关。深刻了解耐高糖酵母的胁迫机制,对定向改造酵母菌的性能和酿酒工业具有重要意义。该文综述了耐高糖酵母的筛选和与酵母菌高糖胁迫机制相关的代谢组学、转录组学、蛋白质组学和基因组学的研究进展,为酵母菌高糖胁迫机理的进一步研究提供参考。     

Chemical constituents of some basidiomycetes

The sterol, fatty acid and free amino acid (FAA) contents of some Basidiomycetes were determined. The overall sterol distribution was similar to that of other mushrooms, with ergosterol as the predominant sterol accompanied by the other closely related sterols. The mushrooms examined also contained high levels of fatty acids of which linoleic is the most prominent. Glutamic acid, valine and proline were the dominant FAA.     

绍兴黄酒淋饭酒母酿制过程生物化学成份变化研究

为了对绍兴黄酒淋饭酒母酿制过程中微生物和化学成分的变化规律进行研究,通过对酒精、还原糖、总酸、挥发酸、pH值、酵母数、球菌数和杆菌数等生化指标的定期观察、检测,总结出了淋饭酒母酿制过程中微生物和化学成分的变化规律,得知淋饭酒母中优良微生物来源于酒药和麦曲,同时阐述了乳酸杆菌和乳酸球菌在淋饭酒母制作过程中的重要作用,是保障淋饭酒母制作正常顺利进行的关键菌,在淋饭酒母中正常和有益的乳酸杆菌有6种左右,以植物乳杆菌居多。     

Microbiological Analyses of Dry and Slurry Yeasts for Brewing

Classical microbiological methods in association with molecular methods (DNA amplification, Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) were used. These methods, developed to rapidly analyze microbial communities on the basis of sequence‐specific separation of DNA amplicons, allowed the detection of DNA differences in the amplicons tested and the identification of the strains analyzed by the comparison of unknown sequences with sequences of known species. TGGE allowed the comparison of the different Saccharomyces cerevisiae strains used in brewing while DGGE allowed the identification of lactic acid bacteria (LAB) in beer. These methods are a reliable tool for fast comparison of strains of Saccharomyces cerevisiae collected from different craft breweries where they were used as starters to check the presence of possible yeast contaminants in the brewing process and for rapid LAB identification.     

Overview of craft brewing specificities and potentially associated microbiota

The brewing process differs slightly in craft breweries as compared to industrial breweries, as there are fewer control points. This affects the microbiota of the final product. Beer contains several antimicrobial properties that protect it from pathogens, such as low pH, low oxygen and high carbon dioxide content, and the addition of hops. However, these hurdles have limited power controlling spoilage organisms. Contamination by these organisms can originate in the raw materials, persist in the environment, and be introduced by using flavoring ingredients later in the process. Spoilage is a prominent issue in brewing, and can cause quality degradation resulting in consumer rejection and product waste. For example, lactic acid bacteria are predominately associated with producing a ropy texture and haze, along with producing diacetyl which gives the beer butter flavor notes. Other microorganisms may not affect flavor or aroma, but can retard fermentation by consuming nutrients needed by fermentation yeast. Quality control in craft breweries today relies on culturing methods to detect specific spoilage organisms. Using media can be beneficial for detecting the most common beer spoilers, such as Lactobacillus and Pediococci. However, these methods are time consuming with long incubation periods. Molecular methods such as community profiling or high throughput sequencing are better used for identifying entire populations of beer. These methods allow for detection, differentiation, and identification of taxa.     

DIFFERENTIATION OF BREWING YEAST

Current methods for the differentiation of brewing yeasts are described and their performance critically discussed. Although these methods are widely used they are non-specific and require confirmation by other tests. Emergent approaches such as pyrolysis gas chromatography or the use of gene cloning techniques are considered and their application reviewed. It is concluded that development of such methods could offer a unique ‘fingerprint’ for each brewing yeast based on a single test.     

Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known ‘wild’ yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA‐encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non‐Saccharomyces yeast species as well as for brewing and non‐brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non‐introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub‐grouped depending on restriction profiles generated with HinfI.