MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION IN THE PRODUCTION OF LAMBIC AND GUEUZE

Lambic and Gueuze are special Belgian beers obtained by spontaneous fermentation. Micro-organisms involved in this fermentation were counted and differentiated using several selective growth media. Micro-organisms were also isolated from samples of Lambic of different age and originating from different casks and brews and identified by classical tests. The following general pattern of microbial development was observed. After 3 to 7 days the fermentation started with the development of wort Enterobacteriaceae and strains of Kloeckera apiculata. These organisms were overgrown after 3 to 4 weeks by strains of Saccharomyces cerevisiae and S. bayanus. These were responsible for the main fermentation, lasting for 3 to 4 months. This was followed by a strong bacterial activity. This period was dominated by the growth of strains of Pediococcus cerevisiae. These reached their maximal numbers during the summer months and were responsible for a fivefold increase in lactic acid concentration. In some casks they caused ropiness. After the main fermentation period Lambic is very sensitive to spoilage by acetic acid bacteria of the genus Acetomonas. The presence of air may be the determining factor for their development. After 8 months a new increase in yeast cells was noted. These belonged now mainly to the genus Brettano-myces bruxellensis and Br. lambicus. They caused a further slow decrease in residual extract and the appearance of special flavours. Oxidative yeasts of the genera Candida, Cryptococcus, Torulopsis and Pichia were also detected and may be responsible for the formation of a flim on the beer surface after the main fermentation.     

SYNTHESIS OF AROMA COMPONENTS DURING THE SPONTANEOUS FERMENTATION OF LAMBIC AND GUEUZE

Lambic is a type of Belgian beer obtained by spontaneous fermentation of wort. The fermentation in casks takes almost two years, and after that period the beer may be re-fermented in bottles and is then called gueuze. To gain insight into the many microbiological transformations occurring during this process, a qualitative and quantitative study of several components formed over a two-year period was undertaken. It was found that lambic and gueuze are characterized by high contents of ethyl acetate, ethyl lactate, lactic acid and acetic acid. Two phases of fermentation may be distinguished: a primary phase lasting for about three months during which almost all the ethanol and higher alcohols are formed and a very long secondary phase during which lactic acid is formed first, followed by ethyl lactate. Some lower fatty acids, including all the acetic acid, are made at the very beginning of the first phase. An important part of the ethyl acetate is made during the first phase but the amounts increase slowly to high levels during the second phase. Unusually low amounts of isoamyl acetate are detected. Several components are above the reported threshold levels of taste and are thus probably involved in the genesis of the typical aroma of lambic and gueuze. These results will allow a further study of the relations between the components found and the microbial populations involved in the spontaneous fermentation inherent in the brewing of lambic and gueuze.     

THE SUPPRESSION OF GUSHING BY THE USE OF HOP OIL

The addition to beer of small amounts of hop oil provides a means of suppressing gushing, without adversely affecting foam stability. The hydrocarbon fraction of hop oil is a more effective gushing suppressor than the oxygenated fraction.     

THE RAPID DETECTION OF BREWERY SPOILAGE MICRO-ORGANISMS

Recent developments in the rapid detection of low concentrations of micro-organisms are discussed and their relevance to improved brewery quality control methods is assessed. A method based on the change in pH of a general purpose growth medium appears to offer the advantages of both time and sensitivity over methods currently in use.     

SUCROSE AGAR AND ITS MODIFICATION FOR THE SELECTIVE GROWTH OF DISTILLERY ISOLATES OF THE LACTIC ACID BACTERIA

Modified Sucrose Agar can be rendered selective, for lactic acid bacteria encountered in the malt whisky industry, by the incorporation of the two antibiotics cycloheximide (actidione) and 2-phenylethanol coupled with incubation under anaerobic conditions (atmosphere of CO₂). Under these conditions, growth of Gram Negative bacteria and yeasts is suppressed and Lactobacillus sp. can be differentiated from Pediococcus sp.     

THE SEPARATION AND IDENTIFICATION OF A DIMER OF CATECHIN OCCURRING IN BEER

A dimer of catechin has been separated from a number of ales, stouts and lagers. This compound has been shown to have the same structure as procyanidin B3, which has previously been isolated from plant sources. It can be conveniently synthesized from dihydroquercetin and catechin.     

SYNTHESIS OF AROMA COMPOUNDS BY WORT ENTEROBACTERIA DURING THE FIRST STAGE OF LAMBIC FERMENTATION

Lambic is a special type of Belgian beer obtained by a spontaneous fermentation. The fermentation is initiated by a growth of enterobacteria and non-maltose fermenting yeasts. These organisms die off after one to two months. To gain a clear insight in the relations between the enterobacteria and the aroma compounds formed in wort during this first period, several bacterial isolates were studied with respect to their metabolites formed in a synthetic medium and in two different lambic worts, using aerobic or semi-anaerobic conditions. The results showed that enterobacteria, especially Enterobacter species, are responsible for the production of 2, 3-butanediol, acetic, lactic and succinic acid and lower amounts of ethyl acetate and higher alcohols which are the main aroma compounds found in 1 to 2 months old lambic. Ethanol production is mainly due to yeast activity. The results are in agreement with previous determinations of entero-bacterial species present in first phase lambic¹⁸ .     

THE DETECTION AND IDENTIFICATION OF PEDIOCOCCUS AND MICROCOCCUS IN BREWERIES,USING A SEROLOGICAL METHOD

A commercially-available antiserum for the identification of group D strains of Streptococcus contains antibodies which react with Pediococcus and Micrococcus, and provides a convenient method to detect these genera in yeast, wort and beer. After suitable adsorption it can be used to distinguish between Pediococcus and Micrococcus and, within each genus, to differentiate between groups normally found in a brewery environment and those from other sources.     

THE USE OF 14C-LABELLED POLYPHENOLS TO STUDY HAZE FORMATION IN BEER

Haze formation in beer is claimed to involve the gradual polymerization of polyphenols and their subsequent reaction with proteins to form insoluble complexes. The mechanism of haze formation has now been studied using ¹⁴C-labelled epicatechin and dimeric catechin. Epicatechin does not polymerize to form dimeric or trimeric polyphenols when beer is stored and is only incorporated into beer haze to a small extent. Some of the epicatechin combines with nitrogen-containing compounds during storage to form soluble complexes. However, dimeric catechin does not form soluble complexes with nitrogen-containing compounds and instead there is a substantial incorporation of this dimer into beer haze. Dimeric polyphenols are important haze precursors which form insoluble complexes with one or more of the polypeptides of beer, probably by an oxidative coupling mechanism.     

乳酸片球菌细菌素的活性及特性的研究

试验采用TGE（trypticase glucose yeast extlact）肉汤培养基作为乳酸片球菌的培养基，培养条件为37℃过夜静止培养。利用改变pH的方法，即通过菌体对细菌素的吸附与解吸，对细菌素进行分离纯化，试验表明细菌素对植物乳杆菌，绿脓杆菌，单核细胞增多症李氏杆菌，沙门氏菌，枯草杆菌，金黄色葡萄球菌和大肠杆菌O157均有抑制作用。并且还具有耐热，耐盐的特性．     

气相色谱-质谱法分析啤酒中的风味物质

采用乙醚直接萃取和蒸馏-乙醚萃取2种方法提取,以气相色谱一质谱法对啤酒的风味物质进行定性分析,从啤酒中共分离鉴定出37种成分,其主要成分是甲酸异戊酯、甲酸异丙酯、苯乙醇、异戊醇、2-乙氧基丙醇等酯类和醇类物质,它们的含量约占总量的70%以上.     

反相高效液相色谱法测定酵母RNA降解的5''''-核苷酸

建立了一种利用反相高效液相色谱法同时测定啤酒废酵母提取的核糖核酸（RNA）降解生成的4种核苷酸（5’-AMP、5’-CMP、5’-GMP、5＇-UMP）的方法，应用反相μBondapak C18柱，水：甲醇：冰醋酸：四丁基氢氧化铵（TBAOH，10％）=883．5：100：15：1．5，检测波长254nm，4种核苷酸能一次性分离，回收率均达到96％以上，标准偏差分别为2．74％、0．98％、1．65％和1．23％。     

PRECIPITATION TESTS AS AN AID TO THE CLASSIFICATION OF PEDIOCOCCI AND AEROCOCCI

The possibility of using simple precipitation reactions for pediococci and aerococci, viz. the standard ‘ring test’ performed in microcontainers and the gel-diffusion test method, has been examined. With both methods, antigen extracts of the pediococci (13 strains) precipitated 100% with both homologous and heterologous pediococcus antisera and only approx. 15% with aerococcus antisera. Antigen extracts of the aerococci (12 strains) precipitated with homologous antisera at the rate of approx. 91% (ring test) and 82% (gel-diffusion test), whereas the positive reaction with heterologous aerococcus antisera was at the rate of only 10.9% (ring test) and 5.4% (gel-diffusion test). No precipitation was observed between antigen extracts of the aerococci and antisera of the pediococci, and only a few reactions between aerococcus antisera and pediococcus antigens were registered. The results indicate that the pediococci tested represent a serological homogenous bacterial group, whereas the aerococci lack such homogenity.     

THE FRACTIONATION OF POLYPEPTIDES FROM BARLEY AND BEER BY HYDROPHOBIC INTERACTION CHROMATOGRAPHY: THE INFLUENCE OF THEIR HYDROPHOBICITY ON FOAM STABILITY

Polypeptides from beer and from aqueous extracts of barley have been separated by hydrophobic interaction chromatography. The most stable foams are given by polypeptides of greatest hydrophobic character whereas hydrophilic polypeptides give much less stable foams. This effect is most pronounced in polypeptides of molecular size between 5000 and 30,000 and less so in the case of high molecular weight polypeptides (> 50,000).     

基因芯片技术鉴定食源性金黄色葡萄球菌

为了建立快速鉴定食派陛金黄色葡萄球菌的基因芯片技术，采用PCR方法扩增金黄色葡萄球菌16SrRNA基因的DNA片段．序列进行BLAST比较并通过软件设计其特异性探针，采用基因芯片杂交技术鉴定食源性金黄色葡萄球菌样品。结果：对所有样品进行基因芯片杂交技术处理并扫描观察，金黄色葡萄球菌的杂交结果呈阳性，其检测结果与传统方法鉴定结果一致。结论：应用基因芯片鉴定技术可快速、准确地鉴定食源性金黄色葡萄球菌，值得推广应用。     

QUANTITATIVE ASPECTS OF THE CONTROL BY OXYGENATION OF ACETATE ESTER CONCENTRATION IN BEER OBTAINED FROM HIGH-GRAVITY WORT

A single oxygenation of fermenting wort controls the otherwise excessive synthesis of ethyl acetate and iso-amyl acetate which may occur during the fermentation of wort of high specific gravity; ethyl acetate levels are more easily reduced than those of iso-amyl acetate. The effect of oxygen on acetate ester levels can be attributed to (a) the virtual suppression of ester synthesis during treatment and (b) the reduced rate of synthesis in the period following treatment. The latter effect is of greater mangitude if oxygenation is timed correctly. For an initial specific gravity of 1.080, acceptable levels of the acetate esters may be obtained by oxygenating for two hours whilst the specific gravity of the fermenting wort is in the range 1.063–1.044. The quantity of oxygen which must be supplied during this time has been defined in relation to the extent of attenuation which is desired and the effect of changing the rate of oxygen supply has been assessed. The rate of fermentation is greatly increased by oxygenation.     

CONTROL OF THE DIMETHYL SULPHIDE CONTENT OF BEER BY REGULATION OF THE COPPER BOIL

In pilot brewing trials where wort boil time was varied from 15 min up to 2 hours the DMS contents of the resultant beers were greatly altered without substantial effect on many other aspects of beer quality. The worts were separately fermented by two yeast strains and a consistent difference between DMS levels of the pairs of beers was found which was almost independent of the boil time. The breakdown of the malt-derived DMS precursor in boiling wort was found to be a first order reaction having a rate constant of about 0.02 min^?1, implying a half life of about 35 min. The breakdown rate of S-methylmethionine in a boiling sucrose solution at pH 5.3 was similar. Free DMS was found to leave this wort substitute rapidly, again as a first order process, but with a rate constant of 0.18 min^?1 (t_1/2 ～ 4 min) when the evaporation rate was 17%/h.     

THE INSTITUTE OF BREWING ANALYSIS COMMITTEE DETERMINATION OF ZINC IN BEER BY ATOMIC ABSORPTION SPECTROSCOPY

A method for the determination of zinc in beer by atomic absorption spectroscopy is recommended on the basis of a collaborative study.