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 .     

FATTY ACIDS AND ESTERS PRODUCED DURING THE SPONTANEOUS FERMENTATION OF LAMBIC AND GUEUZE

Lambic and gueuze are Belgian beers obtained by spontaneous fermentation of wort. During previous studies it was found that they result from the successive development of enterobacteria, Kloeckera and Saccharomyces yeasts, bacteria of the genus Pediococcus, and Brettanomyces yeasts. The beers are characterized by high concentrations of acetic and lactic acid, ethyl acetate and ethyl lactate. This study of the content of the higher fatty acids during a 20 month fermentation period confirms the succession of the different micro-organisms. Pure cultures of isolated yeasts and bacteria produced fatty acids which were also found in the fermenting wort at periods when these organisms were active. Lambic and Gueuze are especially rich in caprylic (C₈) and capric (C₁₀) acids. These are probably produced by Saccharomyces and Brettanomyces. Important amounts of ethyl caprylate and ethyl caprate were also found. As ethyl caprate is almost absent in other beers, it might be considered as another typical aroma component of lambic and gueuze.     

Comparison of Two Extraction Techniques,Solid‐Phase Microextraction Versus Continuous Liquid–Liquid Extraction/Solvent‐Assisted Flavor Evaporation,for the Analysis of Flavor Compounds in Gueuze Lambic Beer

Lambic is a beer style that undergoes spontaneous fermentation and is traditionally produced in the Payottenland region of Belgium, a valley on the Senne River west of Brussels. This region appears to have the perfect combination of airborne microorganisms required for lambic's spontaneous fermentation. Gueuze lambic is a substyle of lambic that is made by mixing young (approximately 1 year) and old (approximately 2 to 3 years) lambics with subsequent bottle conditioning. We compared 2 extraction techniques, solid‐phase microextraction (SPME) and continuous liquid–liquid extraction/solvent‐assisted flavor evaporation (CCLE/SAFE), for the isolation of volatile compounds in commercially produced gueuze lambic beer. Fifty‐four volatile compounds were identified and could be divided into acids (14), alcohols (12), aldehydes (3), esters (20), phenols (3), and miscellaneous (2). SPME extracted a total of 40 volatile compounds, whereas CLLE/SAFE extracted 36 volatile compounds. CLLE/SAFE extracted a greater number of acids than SPME, whereas SPME was able to isolate a greater number of esters. Neither extraction technique proved to be clearly superior and both extraction methods can be utilized for the isolation of volatile compounds found in gueuze lambic beer.     

Microbial acidification,alcoholization, and aroma production during spontaneous lambic beer production

Acidic beers, such as Belgian lambic beers and American and other coolship ales, are becoming increasingly popular worldwide thanks to their refreshing acidity and fruity notes. The traditional fermentation used to produce them does not apply pure yeast cultures but relies on spontaneous, environmental inoculation. The fermentation and maturation process is carried out in wooden barrels and can take up to three years. It is characterized by different microbial species belonging to the enterobacteria, acetic acid bacteria, lactic acid bacteria, and yeasts. This review provides an introduction to the technology and four fermentation strategies of beer production, followed by the microbiology of acidic beer production, focusing on the main microorganisms present during the long process used for the production of Belgian lambic beers. © 2018 Society of Chemical Industry     

WORT ENTEROBACTERIA AND OTHER MICROBIAL POPULATIONS INVOLVED DURING THE FIRST MONTH OF LAMBIC FERMENTATION

Lambic is a special type of Belgian beer obtained by spontaneous fermentation. The micro-organisms occurring during the first month of lambic fermentation were counted using several selective media. During this period a mixed flora is present in which the Enterobacteriaceae predominate. They were isolated from samples of lambic of different ages originating from different breweries and identified using the minitek identification system. Much variation may occur in types of Enterobacteriaceae: Klebsiella aerogenes, Enterobacter cloacae, E. aerogenes, Escherichia coli, Citrobacter freundii, A-D group bacteria and Hafnia alvei are found during the first month of fermentation. E. cloacae is found most frequently followed by K. aerogenes. After 30 to 40 days all Enterobacteria die off because of the activities of yeasts. Lactic acid and acetic acid bacteria are found in very low numbers during the first month of fermentation; Staphylococci and Streptococci are not present.     

菠萝皮渣糯米果酒发酵过程中主要成分变化研究

以菠萝皮渣和糯米为原料,进行糯米果酒发酵,并采用相关国标检测方法、液相色谱（LC）及气相色谱质谱联用（GC-MS）法,分析菠萝皮渣糯米果酒在发酵过程中可溶性固形物、酒精度、有机酸、挥发酸和香气成分等成分变化。结果表明：发酵过程中糖度迅速降低、酒精度升高后维持稳定,pH值在3.50～3.80之间,呈先降后升趋势,酸度先降低直至陈酿后期轻微升高。有机酸含量在发酵过程中总体呈下降趋势,草酸、酒石酸含量逐渐降低,柠檬酸、苹果酸、丙酮酸、乳酸含量先降后升,琥珀酸和乙酸呈上升趋势。果酒中挥发酸含量不断下降,第60天甲酸、乙酸含量分别为0.30 mg/L和0.26 mg/L。菠萝皮渣糯米果酒中共检测出88种香气成分,其中41种酯类、22种醇类、10种酸类和15种其他类,发酵过程中乳酸乙酯、乙酸异戊酯、2-甲基丁酸乙酯、正己酸乙酯、辛酸乙酯、乙酸苯乙酯、癸酸乙酯、月桂酸乙酯和十六酸乙酯含量较高,相对含量在0.27%～20.57%。     

IDENTIFICATION OF LAMBIC SUPERATTENUATING MICRO-ORGANISMS BY THE USE OF SELECTIVE ANTIBIOTICS

The microbial population present in Iambic beer after one year of spontaneous fermentation consists mainly of Brettanomyces yeasts, lactic acid bacteria and acetic acid bacteria. The density of the wort by that time has decreased to around 3.5° Plato. At that time a period of superattentuation is initiated, resulting in a Iambic with sometimes less than 1° Plato. Such old Iambics are used in the production of gueuze. In order to find out which organisms are really necessary for this process. Iambic attenuated to around 3.5° Plato was pasteurized and re-inoculated with a mixed microbial population obtained from fermenting Iambic. By the addition of the antibiotics actidione, pimaricin, gentamycin, oxytetracycline and nisin it was found that Brettanomyces was the main organism responsible for superattenuation, although this was less pronounced when Pediococcus was absent. Acetic acid bacteria were not involved. Bacteria alone were not really superattenuating. The process with Iambic wort which had reached the 3.5° Plato value by a natural spontaneous fermentation was slower than with a Iambic wort pre-fermented to 3.5° Plato with S. cerevisiae. It was found that Brettanomyces but not Saccharomyces survives well under the conditions normally found for a 1 year old Iambic.     

骏枣果醋发酵过程中营养品质变化及风味成分分析

以骏枣果酒为原料发酵果醋,对发酵过程中营养品质指标变化及发酵前后挥发性成分进行比较研究。结果表明,36～72 h期间为发酵产酸的主要时期,发酵过程中共检测到醋酸、草酸、柠檬酸、乳酸、琥珀酸、苹果酸6种有机酸,发酵后果醋中醋酸和琥珀酸含量分别为发酵前的15倍和3.7倍,乳酸和草酸含量则显著性降低,总酸味强度是果酒的6.17倍。发酵过程中总酚含量不断下降,维生素C含量先下降后趋于稳定,DPPH自由基清除率和总抗氧化能力显著升高。醋酸发酵前后共检测到挥发性成分65种,其中果醋检测到39种,相比果酒减少8种,果醋中酯类物质相对含量最高为37.42%,其中乙酸乙酯和乙酸异戊酯为主要的酯类物质,其次是酸类物质相对含量为30.69%。     

ORGANIC ACIDS AND GLYCEROL IN BEER

The concentrations of pyruvic acid, citric acid, L-malic acid, acetic acid, L-lactic acid and glycerol were determined in beers of different type and origin. Large differences in organic acid and glycerol contents were found, especially between different types of beer. The amounts of acetic acid, lactic acid and glycerol can be used as process parameters. During malting a decrease of the malic acid-citric acid ratio occurred due to the pathway of the TCA-cycle. The acetic acid content and glycerol contents showed increases during fermentation. The latter parameter is useful to indicate if alcohol-free beer has been produced by fermentation. Determination of apparent final attenuation degrees of worts according to the usual laboratory practice resulted in higher glycerol contents than was found for the corresponding beers, making the value of this assay questionable.     

VOLATILE CONSTITUENTS OF VINEGAR. IV. FORMATION OF VOLATILES IN THE FRINGS PROCESS AND A CONTINUOUS PROCESS OF MALT VINEGAR MANUFACTURE

Gas liquid chromatography was used to fractionate and measure volatiles formed during the Frings process and during a continuous process of malt vinegar manufacture. Acetaldehyde, ethyl alcohol, iso-amyl alcohol, acetic acid, n-propyl alcohol, sec-butyl alcohol, isobutyl alcohol, n-amyl alcohol, and also sec-butyl acetate, isobutyl acetate and n-amyl acetate were detected during the alcoholic fermentation but propionic acid, isobutyric acid and acetoin were produced during acetification. The increase in the number and concentration of the volatiles of these processes has been compared with those found for the Quick method.     

复合酯化酶在固态法白酒发酵过程中对微量成分影响的研究

研究了复合酯化酶在固态法白酒发酵过程中对基酒中醛、有机酸、酯、醇等呈香呈味物质的影响。结果表明．总酸、乙酸、己酸含量有提高,乳酸、丙酸则呈现出逐步降低的趋势;杂醇类物质下降明显;复合酯化酶在固态法白酒酿造过程对乙酸乙酯的影响不明显,己酸乙酯随复合酯化酶用量的增加而增加显著,当复合酯化酶的用量为2．5％时,己酸乙酯浓度达...     

静态顶空气质联用法测定霞多丽在酒精发酵过程中香气的变化

运用静态顶空-气相色谱质谱联用法,从霞多丽葡萄汁到酒精发酵结束,每隔12h对其香气成分进行检测。检测得到的香气成分通过质谱谱库进行定性,色谱峰面积归一化法进行定量。测定结果显示:霞多丽在酒精发酵过程中共检测出31中香气物质,其中醇类物质有11种,酯类物质有13种,其他物质(酸、醛、酮)有7种。发酵前后香气成分的种类和相对含量变化显著。其中酯类和醇类物质的种类和相对含量随着发酵的进行有所增加,而其他类(酸、醛、酮)物质的种类和相对含量随着发酵的进行急剧下降。发酵结束后,发酵液的主要香气成分为:乙醇、2-甲基丙醇、3-甲基丁醇、2,3-丁二醇、苯乙醇、正丙醇、己醇、乙酸己酯、辛酸乙酯、壬酸乙酯、2-苯基乙酸乙酯、十二酸乙酯、乙酸乙酯、2-甲基乙酸丙酯、3-甲基乙酸丁酯、己酸乙酯、乙酸。     

Microbiological and biochemical profile of cv. Conservolea naturally black olives during controlled fermentation with selected strains of lactic acid bacteria

The effect of controlled fermentation processes on the microbial association and biochemical profile of cv. Conservolea naturally black olives processed by the traditional anaerobic method was studied. The different treatments included (a) inoculation with a commercial starter culture of Lactobacillus pentosus, (b) inoculation with a strain of Lactobacillus plantarum isolated from a fermented cassava product and (c) uninoculated spontaneous process. Microbial growth, pH, titratable acidity, organic acids and volatile compounds were monitored throughout the fermentation. The initial microbiota consisted of Gram-negative bacteria, lactic acid bacteria and yeasts. Inhibition of Gram-negative bacteria was evident in all processes. Both starter cultures were effective in establishing an accelerated fermentation process and reduced the survival period of Gram-negative bacteria by 5 days compared with the spontaneous process, minimizing thus the likelihood of spoilage. Higher acidification of the brines was observed in inoculated processes without any significant difference between the two selected starter cultures (113.5 and 117.6mM for L. plantarum and L. pentosus, respectively). L. pentosus was also determined as the major species present during the whole process of spontaneous olive fermentation. It is characteristic that lactic acid fermentation was also initiated rapidly in the spontaneous process, as the conditions of fermentation, mainly the low salt level (6%, w/v) favored the dominance of lactic acid bacteria over yeasts. Lactic, acetic and propionic were the organic acids detected by HPLC in considerable amounts, whereas citric and malic acids were also present at low levels and degraded completely during the processes. Ethanol, methanol, acetaldehyde, ethyl acetate were the major volatile compounds identified by GC. Their concentrations varied among the different treatments, reflecting varying degrees of microbial activity in the brines. The results obtained from this study could help the Greek table olive industry to improve the existing processing schemes in order to increase product consistency and quality expanding the international market for naturally black olives.     

基于多元统计学分析红曲米醋醋酸发酵阶段挥发性风味特征组分

为探究红曲米醋酿造过程中挥发性风味及特性组分变化规律,以红曲米醋醋酸发酵阶段为研究对象,采用电子鼻、气相色谱-质谱（gas chromatography-mass spectrometry,GC-MS）联用和气相色谱-嗅闻（gas chromatography-olfactometry,GC-O）联用技术对挥发性风味物质进行分析,并结合聚类分析、主成分分析和偏最小二乘判别法的多元统计方法进行不同发酵阶段的风味物质差异性分析,最终筛选出特征性组分。电子鼻分析可用于区分不同发酵时期的红曲米醋。通过GC-MS和GC-O识别出发酵过程中共有54 种挥发性风味化合物。经多元统计学方法筛选得到挥发性风味特征组分,醋酸发酵早期为正辛醇、异丁醇和戊酸乙酯;中期为苯甲酸、棕榈酸乙酯、正己醇、2,4-二叔丁基苯酚和乳酸乙酯;中后期为乙酸丙酯、乳酸乙酯和乙酸异丁酯;末期为L(＋)-2,3-丁二醇和庚酸乙酯。本研究结果为红曲米醋香气的调控和风味改善提供重要理论依据。     

MICROBIOLOGICAL ASPECTS OF A MIXED YEAST—BACTERIAL FERMENTATION IN THE PRODUCTION OF A SPECIAL BELGIAN ACIDIC ALE

The evolution of the microbial population during the industrial fermentation of a Belgian acidic ale was followed, and the most important micro-organisms were identified. In essence, the process can be considered to be a tandem fermentation, as a period of ethanolic fermentation is followed by a period of lactic acid fermentation. The process consists of three stages. The first stage is a seven day fermentation mainly by Saccharomyces; the second is a four to five week fermentation in which Lactobacilli become important; in the third stage a twenty to twenty-four month fermentation in cask, Brettanomyces species, Lactobacilli, Pediococcus parvulus and Acetic Acid bacteria are all present. The evolution of the Brettanomyces-Pediococcus coculture during the tertiary fermentation shows a remarkable similarity with the evolution during a gueuze fermentation.     

Investigation of the main parameters during the fermentation of Chinese Luzhou‐f lavour liquor

The aim of this study was to investigate the evolution of some physicochemical properties of fermented Zaopei throughout the fermentation. For each layer of Zaopei, the temperature gradually increased to a maximum after about 2 weeks, and then remained stable for about 15 days before it began to decrease; acidity tended to increase during the entire fermentation, while the starch content decreased throughout the fermentation. The reducing sugar content for all layers reached a maximum after one week and decreased afterwards. Ethanol accumulated with time and reached a peak after 5 weeks for each layer, then tended to decrease but rose again slightly after 7 weeks. The moisture content in the top and middle layers increased gradually and reached saturation after 5 weeks, then remained almost unchanged, while the moisture of the bottom layer increased throughout the fermentation. The ethyl caproate, ethyl acetate and ethyl butyrate content of all layers remained almost constant for the first 2 weeks, then increased gradually and reached a maximum after 6 weeks, and subsequently declined slightly. The ethyl lactate of each layer increased throughout the fermentation. The caproic acid, acetic acid, butyric acid and lactic acid in all of the layers reached the highest level on the sixth week, followed by a continuous decrease until week 8, thereafter followed by a slight increase in trend. Additionally, the qualities of the raw liquor distilled from the different layers of Zaopei were investigated and the results demonstrated that the bottom layer possessed the best quality. Copyright © 2015 The Institute of Brewing & Distilling     

桤叶唐棣果醋发酵工艺优化及有机酸和挥发性成分分析

以桤叶唐棣为原料,经半固态发酵酿制桤叶唐棣果醋,以总酸作为评价指标,优化桤叶唐棣果醋发酵工艺,并对发酵前后有机酸和挥发性成分进行比较。结果表明,最佳发酵条件为初始酒精度9%vol,果酒与果渣质量比1.0:1.6,醋酸菌添加量2.0%,在30 ℃条件下发酵18 d,测得桤叶唐棣果醋总酸含量为4.75 g/100 mL。桤叶唐棣果醋中以醋酸（5.00 mg/mL）、丙二酸（0.41 mg/mL）、乳酸（0.25 mg/mL）为主。主要挥发性物质为3-羟基-2-丁酮（15.86%）、乙酸（35.62%）、乙酸乙酯（24.32%）、苯甲醇（17.60%）、苯甲酸（6.60%）。     

不同乳酸菌接种发酵泡菜风味的研究

以新鲜红皮萝卜为原料,分别接种分离自自然发酵泡菜中的乳明串株菌、乳酸乳球菌、食窦魏斯氏、发酵乳杆菌、植物乳杆菌进行纯种乳酸菌发酵制作泡菜。通过测定泡菜的乳酸菌菌相、总酸度、感官评价、挥发性成分及有机酸,对分离自自然发酵泡菜中不同乳酸菌纯种发酵泡萝卜的风味进行研究。结果表明,发酵达到终点时,接种不同乳酸菌发酵的泡菜中大部分乳酸菌的活菌数相差不大,都在7.0lg CFU/m L左右,泡菜液中的乳酸菌几乎全为植物乳杆菌;大部分乳酸菌纯种发酵泡菜中总酸含量>4g/kg;不同乳酸菌发酵的泡菜挥发性成分及有机酸的种类差异不显著。      

Dynamics of volatile and non-volatile compounds in cocoa (Theobroma cacao L.) during fermentation and drying processes using principal components analysis

Different volatile and non-volatile compounds produced during the fermentation-drying process are considered as indicatives of cocoa beans quality. We found thirty-nine different compounds identified by SPME-HS/GC-MS and related to the desirable notes and off-flavor that have been reported. Volatile and non-volatile compounds were associated with acidity and changes of pH, such as acetic and lactic acid. Using the principal component analysis (PCA), relations between compounds and fermentation and drying day were associated with dynamics of these compounds. The identification of principal compound produced during the fermentation and drying processes can be helpful in searching for off-flavor indicator and as a fermentation index, such as isobutyric, isovaleric and propionic acids. Oxidation of 3-methyl-1-butanol-to-3-methyl-1-butanol acetate can be of use in evaluating the degree of fermentation. At drying, the compounds with the highest levels were acetic and isobutyric acid, ethyl and 3-methyl-1-butanol acetate, pentanal and 2,3-pentanedione, and 1,3-butanediol and 2,3-butanediol. Therefore, acetic acid and isobutyric acid, due to their high levels and their low threshold value could play an important role in the aromatic quality of cacao drying.     

啤酒中有机酸含量的RP-HPLC检测条件优化及其酿造过程中动态分析

该研究对反相高效液相色谱（RP-HPLC）法测定啤酒中有机酸含量的方法进行优化,最终确定了RP-HPLC法测定啤酒中草酸、乳酸、酒石酸、乙酸、苹果酸、α-酮戊二酸、抗坏血酸、柠檬酸、琥珀酸9种有机酸的检测条件,并结合发酵机理对啤酒酿造过程中的有机酸含量动态变化进行分析。结果表明,RP-HPLC优化条件为：检测波长215 nm、流动相缓冲液0.10 mol/L KH2PO4、pH值3.0、流速0.6 mL/min。有机酸在质量浓度0.2～400.0 mg/L范围内线性关系良好（R2均＞0.99）,加标回收率79.3%～110.0%,精密度试验结果相对标准偏差（RSD）为0.3%～11.5%,表明该方法精密度高、准确性良好。在啤酒酿造过程中有机酸总量及乙酸、琥珀酸、苹果酸、乳酸、柠檬酸、α-酮戊二酸含量呈现先增长后平稳的趋势,草酸、抗坏血酸、酒石酸含量变化相对平稳。