Molecular analysis of Oenococcus oeni population dynamics and the effect of aeration and temperature during alcoholic fermentation on malolactic fermentation

The effects of aeration and temperature during alcoholic fermentation (AF) on spontaneous and inoculated malolactic fermentation (MLF) of wine have been analysed by following the population dynamics of Oenococcus oeni strains with multiplex random amplified polymorphic DNA‐polymerase chain reaction. It has enabled us to follow precisely the proportion of different autochthonous strains and the starter strain through fermentations. Lack of aeration led to delays in AF, which meant that autochthonous lactic acid bacteria could develop earlier and prevented the starter strain from developing correctly. Temperature was not found to lead to any differences. Two strains were isolated in the same spontaneous MLF, suggesting that, in some cases, multiple strains might be responsible for the degradation of malic acid in wine. It can be concluded that delays in the AF can negatively affect the control of MLF and that this can be studied by following the development of the different strains of O. oeni.     

Oenococcus oeni and malolactic fermentation – moving into the molecular arena

Malolactic fermentation (MLF) is the bacterial‐driven decarboxylation of L‐malic acid to L‐lactic acid and carbon dioxide, and brings about deacidification, flavour modifications and microbial stability of wine. Pasteur first described the presence of ‘bacteria’ in wine nearly one‐hundred‐and‐fifty years ago and the subsequent elucidation of the bacterial‐driven malolactic reaction was described about fifty years later. Over the following years the occurrence of MLF became apparent in wines worldwide, and eventually Oenococcus oeni was identified as the principal organism involved in the process. O. oeni is remarkable in its ability to tolerate the nutritionally poor and challenging, harsh wine environment; however, it can be a difficult and sometimes unreliable organism to work with in the winery. A greater knowledge of its biology would undoubtedly facilitate the development of strains and practices, with improved performance outcomes. We already know a considerable amount about the biochemistry and physiology of O. oeni, and ironically, although we know little about its genetics, its genome has been sequenced. With this groundwork in place and molecular biology techniques at our disposal we are poised to increase our knowledge and understanding of this organism enormously.     

The influence of malolactic fermentation and Oenococcus oeni strain on glycosidic aroma precursors and related volatile compounds of red wine

Free and glycosidically bound volatile compounds of red wine were measured after malolactic fermentation (MLF) with four different commercial starter cultures of Oenococcus oeni. MLF resulted in a significant decrease in the concentration of total glycosides, expressed as phenol‐free glycosyl glucose. Gas chromatographic analyses of wine enzyme hydrolysates showed that the extent of hydrolysis of glycosides during MLF was dependent on both bacterial strain and chemical structure of the substrate. The highest decrease was observed for glycosidic precursors of primary terpene alcohols. Glycoside‐related aroma compounds such as linalool, farnesol, and β‐damascenone were increased after MLF with all the bacterial strains tested. Two of the strains were also able to release significant amounts of vinylphenols during MLF. Copyright © 2006 Society of Chemical Industry     

Evolution of red wine anthocyanins during malolactic fermentation,postfermentative treatments and ageing with lees

A comparative study was conducted on nine batches of wine, from the same initial wine, subjected to malolactic fermentation and ageing in barrels, under different technological conditions: Malolactic fermentation in barrel or in tank, with or without wine clarification, ageing with or without lees and stirring or no stirring of the lees. Samples were taken of the initial wine, of the wine at the end of malolactic fermentation, of the wines after clarifying treatments, and after 3, 6, 9, 12 and 14 months of ageing in the barrel, making a total of 48 wines. As a result of the anthocyanin analysis of all the wines studied, a total of 21 different anthocyanin compounds were detected, which can be classified into four groups: simple glucosides, acetyl glucosides, cinnamoyl glucosides and pyroanthocyanins. During MLF, it was shown that the effect of the container used seems to be more important than the metabolic activity of the bacteria responsible for the process. From application of the LSD test, significant differences were found in the concentrations of all the anthocyanin compounds identified due to ageing time and significant differences were also revealed for most anthocyanin compounds in relation to the manufacturing method, especially the presence or absence of lees.     

酒酒球菌在青梅汁中的生长及苹果酸乳酸发酵特性的研究

以青梅汁为原料,利用酒酒球菌的苹果酸-乳酸发酵（MLF）对青梅汁进行生物降酸的研究。结果表明:接种量为2.0×108CFU/mL,青梅汁的pH≥3.4时,MLF能正常进行,并使样品的酸度降低61.35%以上;接种量为2.6×107CFU/mL时,青梅汁的pH为3.6才能触发MLF,样品的酸度降低了77.60%;接种量为2.3×108CFU/mL,在pH3.6和4.0的青梅汁中,添加1.0g/100g的葡萄糖的样品,与不添加葡萄糖的样品相比,其酸度分别降低了27.04%和34.63%;在柠檬酸-柠檬酸钠缓冲体系中,酒酒球菌使体系酸度降低了26.40%,证实酒酒球菌可利用柠檬酸作为碳源,进行MLF。      

Assessment of the genetic polymorphism and biogenic amine production of indigenous Oenococcus oeni strains isolated from Greek red wines

In the warm climate country of Greece malolactic fermentation (MLF) has received limited attention. Molecular techniques and High Performance Liquid Chromatography (HPLC) were used to study the genetic polymorphism of autochthonous lactic acid bacteria developing towards the end of spontaneous MLF of Greek red wines and for the assessment of their potential to produce harmful biogenic amines. This research revealed that native Oenococcus oeni isolates are very much adapted to specific winery conditions since the majority of spontaneous MLF were driven mostly or exclusively by a single strain of O. oeni. Native O. oeni strains showed only limited dispersion since cluster analysis uncovered only few common genotypes among indigenous isolates from different wineries. The genotype of a frequently used malolactic starter was more than often detected among autochthonous isolates without nevertheless compromising the biodiversity of natural microflora residing in wineries but rather becoming a part of it. For the majority of the wine samples studied, MLF implementation and storage in bottles resulted in negligible changes on the levels of the BA histamine, tyramine, phenylethylamine, cadaverine as well as of ethylamine, methylamine, isobutylamine. We provide evidence that autochthonous O. oeni isolates can only contribute to putrescine accumulation in Greek wines but still the specific trait behaves as strain-specific with a limited dispersion.     

Inducing malolactic fermentation in Chardonnay musts and wines using different strains of Oenococcus oeni

Malolactic fermentations (MLF) were induced in a commercially prepared Washington State Chardonnay must to evaluate the influence of timing of inoculation and pre-culture conditions of Oenococcus oeni strains MCW, EQ-54, and WS-8. The must (pH 3.62, 21.5°Brix) was divided into lots and inoculated with Saccharomyces cerevisiae strain CY3079. Strains of O. oeni were pre-cultured by growing in diluted juice or by re-hydration of freeze-dried strains. Bacteria were inoculated into the musts before (Day 0) or after completion of the alcoholic fermentation (Day 22). Yeast populations exceeded 10⁷cfu/mL in all fermentations that proceeded to dryness. However, the viability of most strains of O. oeni quickly declined after inoculation regardless of the timing of inoculation or the strain used. MLF was induced in the wines inoculated with strains EQ-54 and WS-8 but not with MCW, and the rate depended on the time of inoculation. The method used to prepare bacterial starter cultures had no apparent influence on the completion of MLF. Values for volatile acidity were slightly higher (P< 0.05) in wines inoculated with O. oeni before alcoholic fermentation compared with those inoculated after alcoholic fermentation.     

Effect of phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii

The aim of this work was to investigate the effect of wine phenolic aldehydes, flavonoids and tannins on growth and viability of strains of Oenococcus oeni and Lactobacillus hilgardii. Cultures were grown in ethanol-containing MRS/TJ medium supplemented with different concentrations of phenolic aldehydes or flavonoids and monitored spectrophotometrically. The effect of tannins was evaluated by monitoring the progressive inactivation of cells in ethanol-containing phosphate buffer supplemented with grape seed extracts with different molecular weight tannins. Of the phenolic aldehydes tested, sinapaldehyde, coniferaldehyde, p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde and 3,4,5-trihydroxybenzaldehyde significantly inhibited the growth of O. oeni VF, while vanillin and syringaldehyde had no effect at the concentrations tested. Lact. hilgardii 5 was only inhibited by sinapaldehyde and coniferaldehyde. Among the flavonoids, quercetin and kaempferol exerted an inhibitory effect especially on O. oeni VF. Myricetin and the flavan-3-ols studied (catechin and epicatechin) did not affect considerably the growth of both strains. Condensed tannins (particularly tetramers and pentamers) were found to strongly affect cell viability, especially in the case of O. oeni VF. In general, this strain was found to be more sensitive than Lact. hilgardii 5 to the phenolic compounds studied. This work contributes to the knowledge of the effect of different phenolic compounds on the activity of wine lactic acid bacteria, which, especially in the case of aldehydes and of different molecular weight fractions of tannins, is very scarce.     

Interaction of pH, ethanol concentration and wine matrix on induction of malolactic fermentation with commercial 'direct inoculation' starter cultures

The effect of the interaction between pH (2.9–3.5), alcohol concentration (12.5–14.5% v/v) and wine matrix (Chardonnay, Grenache blend and Cabernet Sauvignon) on malolactic fermentation (MLF) induced by two commercially available directly inoculated Oenococcus oeni starter cultures was determined. Wine matrix had greatest impact on the rate of MLF, followed by pH and alcohol; the matrix effect was significantly modified by pH and bacterial culture. The rate of L-malic acid degradation by starter culture A was 3.3–61.3% in the Grenache blend and 3.3–4.5% in the Chardonnay wine compared to the Cabernet Sauvignon wine, for the pH/alcohol ranges 2.9/14.5 to 3.5/12.5. L-malic acid degradation rate by starter culture B was lower but less affected by wine matrix. The rate of L-malic acid degradation was closely related to culture viability, with a high rate of L-malic acid degradation being associated with bacterial growth and vice versa . Treatment of two wines in which MLF had previously failed to complete with various combinations of nutrients, carbon fining and heating, restored MLF, suggesting that a growth-limiting nutrient was absent and/or a toxic substance not related to pH, alcohol or SO₂ concentration was present. This work demonstrated that the wine matrix may affect culture viability more significantly than either pH or alcohol concentration. A high rate of MLF was shown to depend on factors permitting bacterial growth, however, the two strains studied responded differently to the wine matrix, suggesting that malolactic bacteria vary in tolerance to various stresses in the wine environment.     

乳酸菌肉品发酵剂的发酵特性研究

对从湘西泡菜水中分离出的5株植物乳杆菌（Lactobacillus planterum）,1株肠系膜明串珠菌（Leuconostocmesenteroides）,1株戊糖片球菌（Pediococcuspentosaceus）,进行耐盐和亚硝酸盐能力、生长和产酸能力、硝酸盐还原和氨基酸脱羧能力的研究以及一系列生理生化实验来验证其在肉品发酵上的可用性。结果表明:植物乳杆菌（Lactobacillus planterum-A）和戊糖片球菌（Pediococcuspentosaceus-6）对食盐和亚硝酸盐均具有较好的耐受性,对蛋白质和脂肪不具有明显的分解作用,不具有硝酸盐还原酶和氨基酸脱羧酶活性,对大肠杆菌（Escherichia coli）,沙门氏菌（Salmonellas spp.）等实验菌株有较好的抑制作用;70℃可致死。符合作为肉品发酵剂的要求,可作为下步实验的出发菌株。      

野生猕猴桃酒苹果酸-乳酸发酵优良乳酸菌的筛选与耐受性研究

该研究通过形态观察及生理生化试验,采用酸性番茄（ATB）培养基从自然启动苹果酸-乳酸发酵（MLF）的野生猕猴桃酒中筛选MLF优良乳酸菌,并对其SO2、酒精、pH耐受性进行测定。结果表明,经初筛得到6株乳酸菌,分别编号为R6、R7、R11、R14、R15、R18。其中,菌株R6、R15可耐受SO2 80 mg/L,菌株R7可耐受SO2 100 mg/L;菌株R6、R7可耐受酒精度14%vol,菌株R15可耐受酒精度12%vol;菌株R6、R7可耐受pH 3.2,菌株R15可耐受pH 3.0。综合分析,菌株R6、R7和R15具有较强的SO2、酒精及pH耐受能力,为MLF优良乳酸菌。     

Inhibition of spoilage lactic acid bacteria by lysozyme during wine alcoholic fermentation

The efficacy of lysozyme against indigenous lactic acid bacteria (LAB) and four inoculated spoilage LAB cultures was investigated in laboratory scale Chardonnay winemaking trials (at pH 3.8). These LAB cultures included Lactobacillus kunkeei, Lactobacillus brevis, Pediococcus parvulus , and Pediococcus damnosus . Three concentrations of lysozyme were used: 0, 125 and 250 mg/L. Alcoholic fermentation of the grape juice was carried out at 20±0.5°C using Saccharomyces cerevisiae . Lysozyme did not have any negative impact on yeast growth and sugar reduction. This enzyme was found to be very effective in inhibiting the growth of all four LAB cultures investigated. Under the given experimental conditions, as high as an 8 log cell reduction was obtained for some of the strains. The acetic acid production by L. brevis and L. kunkeei was significantly reduced in the treatments with 125 and 250 mg/L lysozyme added ( P < 0.01). The effect of lysozyme on the cells of the LAB cultures was examined under a scanning electron microscope. It is evident that lysozyme had a detrimental impact on the cells of these cultures. Based on these observations, it is concluded that lysozyme may be a useful tool for winemakers to control the growth of spoilage LAB and to reduce the production of volatile acids. The addition of lysozyme may also prevent the increase of volatile acidity during stuck/sluggish alcoholic fermentation. This tool is particularly useful in high pH wines where SO₂ is less effective.     

乳酸菌发酵剂高密度培养的研究

研究了乳酸菌生长繁殖的环境条件(温度、接种量、起始pH等)和培养基组成(氮源、碳源、缓冲盐等),优化确定了乳酸菌发酵剂的适宜培养条件为:起始pH值为6.5,培养温度为37℃,培养基配比为麦芽糖∶乳糖(1∶1)2%、牛肉膏1.0%、缓冲盐A0.5%、NaCl0.25%、MgSO40.1%,接种量4%,进一步探索了半连续法进行高密度培养,结合优化的培养条件,可使乳酸菌的液体发酵活菌密度至1.1×1012CFU/mL。     

新疆传统乳制品中优良乳酸菌的筛选及多菌株发酵剂的研究

从新疆地区采集的46份传统乳制品中分离获得的612株乳酸菌,根据菌株来源、培养条件,形态特征、凝乳状态和产酸特性,选择其中20株乳酸菌进行生理生化鉴定和酸奶发酵试验,结果表明,分离株中3株为链球菌,4株为乳球菌,13株为乳杆菌,平均发酵产酸速率在8.11~12.43oT/h,不同菌株在酸乳冷藏期间表现出不同的后酸化活性,冷藏15d滴定酸度提高了6.3%~55.7%。不同菌株凝乳时黏度在272~1085cp,多数菌株发酵乳在冷藏期间黏度呈上升趋势,球菌的产黏能力普遍低于杆菌。气相色谱分析结果显示不同菌株发酵乳中丁二酮在3.51~68.74mg/L,乙醛在3.51~68.74mg/L,丙酮在0.87~48.35mg/L,乙酸乙酯在0~103.68mg/L,异戊醇都未检出。经过混合菌株发酵生产酸乳比较,发现以S.thermophilusX1、S.thermophilusX5、L.plantarumY5和L.delbrueckii bulgaricussubsp.Y10按1∶1∶1∶1比例组合作为发酵剂,生产的酸乳组织状态较好、风味独特、后酸化弱。     

Lactic Acid Bacteria in Spanish Red Rose and White Musts and Wines under Cellar Conditions

The dynamics of the lactic acid bacterial (LAB) population in different cellars, fermentation conditions and musts were analyzed. The number of LAB in fresh musts diminished quickly in the first few days, but an increase of these bacteria occurred during the later stages of the alcoholic fermentation. The species found in fresh musts were Lactobacillus plantarum, L. hilgardii, L. brevis, L. confusus, and Leuc. onostoc paramesenteroides. At the end of fermentation only L. hilgardii, Leuc. oenos and L. fructivorans were isolated. The API 50 CHL system was not useful to identify strains of Leuc. oenos species. All the isolated strains degraded malic acid in synthetic medium but only two strains belonging to Leuconostoc oenos species did it in wine.     

酒酒球菌高密度培养条件的优化

酒酒球菌（Oenococcus oeni）主导的苹果酸-乳酸（MLF）发酵是优质葡萄酒生产的重要工艺环节,制备高活菌数的酒酒球菌发酵剂是保障该环节顺利进行的重要前提之一。研究发现,一定量的L-苹果酸可以有效促进酒酒球菌的生长,并通过高密度培养条件优化提高酒酒球菌菌体密度。结果表明,通过正交试验得出的最佳高密度培养条件为初始pH值5.1、接种量3%、L-苹果酸添加量1 g/L。在此优化条件下,酒酒球菌ES-1的菌体密度较高,为（7.33±0.40）×109 CFU/mL;进一步结合化学中和法和半连续培养法,可使最终菌体密度达（1.67±0.11）×1010 CFU/mL,是对照组的11倍。该研究获得的高密度培养优化方案可为制备优质本土酒酒球菌发酵剂奠定基础。