Development of a quantitative PCR for detection of Lactobacillus plantarum starters during wine malolactic fermentation

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at 3.6 × 10(6) CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above 10(5)/ml for approx. 10 days, after which cell numbers decreased to levels of 10(3) CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. 1 × 10(2) CFU/ml was detected. The minimum detection level for quantitative PCR in this study was 1 × 10(2) to 1 × 10(3) CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.     

The inhibitory activity of wine yeast starters on malolactic bacteria

Malolactic fermentation is a process that is influenced by various factors that can inhibit the growth of the malolactic bacteria. Inhibitory metabolites produced by yeast may have an important role in the correct development of malolactic fermentation. For these reasons, we have investigated the effects of such metabolites on the growth of malolactic bacteria under different environmental conditions, to aid in our understanding of the significance of these interactions in the wine-making environment. Our screening methods to detect interactions between yeast and malolactic bacteria showed a variable and wide diffusion of yeast inhibitory activity on the growth of the malolactic bacteria. However, this first approach to determine this inhibitory activity of yeast gave an overestimation when compared to the results obtained under actual wine-making conditions. The evaluation of malic acid consumption indicated that under inhibitory conditions a partial L-malic acid degradation was seen, indicating that the malolactic activity continued without bacterial growth. However, these yeast-inhibiting effects in addition to other environmental factors could cause a complete failure of malolactic fermentation.     

Changes of synaptosomal energy metabolism induced by hypoxia during aging

Synaptosomes were isolated from the motor area of the cerebral cortex of normoxic or hypoxic (PaO₂=17–19 mmHg, for 15 min) beagle dogs of different ages. Synaptosomes were incubated in Krebs-Henseleit-Hepes buffer (for 10 min at 24°C) and the energetic state was defined by: the balance of the labile phosphates (ATP, ADP, AMP, and creatine phosphate); the respiratory rate; the redox state of the intramitochondrial NAD-couple. By the present experimental model, it is possible to evaluate the potential damage (induced by the in vivo hypoxic insult) that synaptosomes cannot reverse under optimal incubation. Aging affected the phosphorylation state of the post-hypoxic incubated synaptosomes. The oxygen consumption rate was quite similar in the synaptosomal fractions from the motor area of hypoxic beagle dogs of different ages, but the cytochromec anda contents were lower in the preparations from hypoxic older brains. In dogs of different ages, hypoxia always lowered the respiration of the synaptosomes, but aging affected the oxygen consumption rates only in post-hypoxic synaptosomes incubated with succinate. The synaptosomal energetic state was defined also by the redox state of the intramitochondrial NAD-couple (G_ox-red) and the phosphorylation state of adenine nucleotide system (G_ATP). The free-energy change (G) for the coupled reactions was calculated, too. In synaptosomes isolated from the cerebral cortex of dogs submitted to hypoxia, the equilibrium (calculated for the mitochondrial electron transfer chain and the phosphorylation of adenine nucleotides) was markedly altered as function of aging. The extensive age-related G changes were largely supported by alteration of the phosphorylation state of adenine nucleotides, rather than by modification of the redox state of the electron transfer chain.All present data suggest that the bioenergetic derangement caused by hypoxia and aging may be interpreted also in terms of modification of the biophysical and biochemical mechanisms involving the mitochondrial membranes and particularly the inner mitochondrial membrane.Special Issue Dedicated to Dr. Abel Lajtha.     

三种酒曲酿造的崇明老白酒的挥发性风味物质分析及比较

分别采用八二酒曲、纯种米根霉曲及酿酒酵母曲、纯种米根霉曲和粘红酵母曲按崇明老白酒工艺酿造老白酒,应用顶空固相微萃取-气相色谱质谱联用法对样品进行挥发性物质的检测,并对其进行醇酯比、香气活力值(OAVs)的计算及主成分分析,筛选出较佳的酒曲,得出崇明老白酒的主体香气.辛酸乙酯、1-辛烯-3-醇、3-甲基丁酸乙酯、己酸乙酯和乙酸异戊酯的OAVs大于10,是崇明老白酒的主要呈香物质.由八二酒曲酿造的酒液挥发性物质总量最高,醇酯比为5.89∶1,呈香物质以具蘑菇香的1-辛烯-3-醇为主.米根霉和酿酒酵母曲酿造的酒液酯类、芳香族化合物种类最多,醇酯比3.24∶1,香气以丁酸乙酯的甜香、苯乙醛、苯乙醇和乙酸苯乙酯的玫瑰花香为主要特征.米根霉和粘红酵母曲酿造的酒液挥发性化合物总量最低,醇酯比为2.86,主要香气特征为辛酸乙酯的甜香和己酸乙酯的水果香.纯种曲酿造的酒液的香气类型、风味种类比例、呈香物质的丰富程度均优于八二酒曲酿造的酒液.     

Hydrolases as markers of wine aging

Indigenous wine hydrolytic enzymes come from both grape berries and the microorganisms involved in the fermentation process. The levels of these enzymes in wine vary according to the elaboration period and to wine aging; among them invertase and polygalacturonase are enzymes fairly resistant to denaturation and with utility to determine wine aging. Invertase may be employed as a marker of wine aging and it may be also used for differentiating wines from different regions of origin and different grape varieties. Polygalacturonase is a good indicator useful to differentiate the year of harvesting for wines elaborated with the same grape variety. Both enzymes may possibly be used as indicators of potentially fraudulent “aging-accelerated” processes.     

Sex differences in survival and mitochondrial bioenergetics during aging in Drosophila

The goal of this study is to test the role of mitochondria and of mitochondrial metabolism in determining the processes that influence aging of female and male Drosophila. We observe that Drosophila simulans females tended to have shorter lifespan, higher levels of hydrogen peroxide production and significantly lower levels of catalase but not superoxide dismutase compared to males. In contrast, mammalian females tend to be longer lived, have lower rates of reactive oxygen species production and higher antioxidant activity. In both Drosophila and mammals, mitochondria extracted from females consume a higher quantity of oxygen when provided with adenosine diphosphate and have a greater mtDNA copy number than males. Combined, these data illustrate important similarities between the parameters that influence aging and mitochondrial metabolism in Drosophila and in mammals but also show surprising differences.     

Ultrastructural changes of sparkling wine lees during long-term aging in real enological conditions

Ultrastructural changes of lees of three series of sparkling wines produced using the traditional method during long-term aging (4?years) were assessed by high-pressure freezing in combination with transmission electron microscopy. The stratified structure of the cell wall disappeared throughout aging. After 18?months, the microfibrous material of the cell wall appeared more diffuse and the amorphous midzone of the inner wall layer was progressively degraded. From 30?months onward, the cell wall consisted of a tangled structure of fibers. In spite of these changes, the cell wall of yeasts remained unbroken at 48?months of wine aging. Cell membrane breakage was observed for the first time in lees of Saccharomyces cerevisiae. An increase in the thickness of the periplasmic space owing to plasmolysis and of the number of cells with less cytoplasmic content was observed during aging. Morphological evidence of microautophagy was detected for the first time in S.?cerevisiae in enological conditions.     

Yeast adapted to wine: nitrogen compounds released during induced autolysis in a model wine

As important as the blend of base wines before bottling, one of the most important steps in the champagne-making process is the long ageing on lees. Two yeast strains of Saccharomyces cerevisiae MC001 and MC002, used in champagne wine production, were allowed to autolyse. After 8 days of autolysis, active dry yeasts adapted to wine released 1.7- to 1.8-fold more nitrogen compounds than nonadapted active dry yeast. The nitrogen content (total, proteins, peptides and amino) present in autolysates was measured for yeasts adapted to wine. The composition of free amino acids and amino acids constituting peptides showed no difference between the two strains of yeast used. Studies of intracellular proteolytic activity and release of peptides showed no correlation between these two phenomena. These results indicate that yeasts adapted to wine give results similar to those that occur in wine during ageing. Journal of Industrial Microbiology & Biotechnology (2002) 29, 134–139 doi: 10.1038/sj.jim.7000291 Received 19 December 2001/ Accepted in revised form 14 June 2002     

Evolution of colour vision in mammals

Colour vision allows animals to reliably distinguish differences in the distributions of spectral energies reaching the eye. Although not universal, a capacity for colour vision is sufficiently widespread across the animal kingdom to provide prima facie evidence of its importance as a tool for analysing and interpreting the visual environment. The basic biological mechanisms on which vertebrate colour vision ultimately rests, the cone opsin genes and the photopigments they specify, are highly conserved. Within that constraint, however, the utilization of these basic elements varies in striking ways in that they appear, disappear and emerge in altered form during the course of evolution. These changes, along with other alterations in the visual system, have led to profound variations in the nature and salience of colour vision among the vertebrates. This article concerns the evolution of colour vision among the mammals, viewing that process in the context of relevant biological mechanisms, of variations in mammalian colour vision, and of the utility of colour vision.     

Nitrogen compounds in wine during its biological aging by two flor film yeasts: An approach to accelerated biological aging of dry sherry-type wines

Urea, ammonium, and free amino acid contents were quantified in biological aging of a young wine under two flor film forming yeast strains, Saccharomyces cerevisiae race capensis and S. cerevisiae race bayanus, and compared. Cell viability in the film was different for the two yeast strains. Thus, capensis maintained a much greater number of viable cells per surface area than bayanus and hence used greater amount of nitrogen compounds. The main source of nitrogen for the yeasts during the biological aging process was L-proline. The two yeast strains also differed in the amounts of assimilable nitrogen they utilized, in their preferences for amino acid consumption, and kinetics. To accelerate the aging process, the effect of controlled monthly aeration of the wine aged with capensis strain was investigated. The results revealed that short aeration did not appreciably increase the overall consumption of assimilable nitrogen, but consumption of some nitrogen compounds was accelerated (particularly L-proline, L-tryptophan, L-glutamic acid, ammonium ion, L-lysine, and L-arginine); the use of L-ornithine was inhibited; and GABA, L-methionine, and urea were depletes. Probably the aeration increases the aroma compounds, thereby producing wines with improved sensory properties. (c) 1997 John Wiley & Sons, Inc.     

剪叶损伤与昆虫取食对兴安落叶松(Larix gmelinii)针叶中缩合单宁诱导作用的差异

单宁是植物内与抗虫性相关的重要次生代谢物质.为了研究昆虫取食及剪叶诱导对兴安落叶松(Larix gmelinii)针叶内缩合单宁含量的影响,用剪叶法和落叶松毛虫(Dendrolimus superans)幼虫取食处理5年生兴安落叶松苗,以香草醛-盐酸法测定其剩余健康针叶内缩合单宁含量的变化.结果表明:(1)处理后1～10d,剪叶4枝75%,8枝50%、75%,12枝25%、50%、75%及虫伤4枝75%的兴安落叶松健康针叶内的缩合单宁含量均明显高于对照(P<0.01),15d时,剪叶8枝75%,12枝50%的兴安落叶松针叶内的缩合单宁含量仍高于对照(P<0.05),说明剪叶与虫伤处理均能诱导缩合单宁含量增加.(2)剪叶4枝75%,8枝50%,12枝25%、75%的处理之间,诱导的缩合单宁含量差异不显著(P>0.05).剪叶8枝75%,12枝50%诱导缩合单宁含量增加幅度比其它剪叶处理大,且诱导持续时间长.说明剪叶程度虽能影响缩合单宁含量,但二者并不呈线性关系.(3)剪叶4枝75%在5d时诱导缩合单宁含量最高,虫伤4枝75%在10d时诱导缩合单宁含量最高,说明剪叶诱导的缩合单宁含量高峰早于虫伤处理.但处理后1～10d,剪叶及虫伤4枝75%的落叶松针叶内缩合单宁含量均明显高于对照(P<0.01或P<0.05),之后与对照差异不显著(P>0.05),说明在受到诱导处理后,兴安落叶松产生应激反应,使其针叶内缩合单宁含量在一定时间内先增加,后逐渐恢复到正常水平.由上可见,可以采取适当的损伤处理取得与昆虫取食相似的兴安落叶松抗性反应.     

Identification and characterization of filamentous fungi isolated from fermentation starters for Hong Qu glutinous rice wine brewing

Hong Qu glutinous rice wine is one of the most popular traditional rice wines in China. Traditionally, this wine is brewed from glutinous rice with the addition of wine fermentation starters (Hong Qu (also called red yeast rice) and White Qu). The objective of this study was to investigate the variability of filamentous fungi associated with traditional fermentation starters through a traditional culture-dependent method and a molecular identification approach. In this study, forty-three filamentous fungi were separated by traditional culture-dependent means (macro- and microscopic characteristics) from 10 fermentation starters and classified into 16 different species based on morphological examination and the internal transcribed spacer (ITS) sequences analysis. It was observed that the genus Aspergillus had the highest number (14 isolates) of isolates followed by Rhizopus (11 isolates), Monascus (5 isolates) and Penicillium (4 isolates). The species R. oryzae, A. niger, A. flavus and M. purpureus were frequently found in wine starter samples, among which R. oryzae was the most frequent species. The enzyme-producing properties (glucoamylase, α-amylase and protease) of all fungal isolates from different starters were also evaluated. A. flavus, R. oryzae and M. purpureus were found to be better glucoamylase producers. A. flavus, R. oryzae and A.oryzae exhibited higher activity of α-amylase. A. flavus and A. oryzae had higher protease activity. However, some fungal isolates of the same species exhibited a significant variability in the production levels for all determined enzyme activity. This study is the first to identify filamentous fungi associated with the starter of Hong Qu glutinous rice wine using both traditional and molecular methods. The results enrich our knowledge of liquor-related micro-organisms, and can be used to promote the development of the traditional fermentation technology.     

黄酒贮存酸败关键微生物的分离鉴定

【背景】贮存陈酿是黄酒生产的重要工艺环节,但由于黄酒中营养物质含量丰富,贮存陈酿过程中时常出现酸败变质的现象,尤其是在大罐的陈酿过程中酸败的发生对黄酒行业造成较大的经济损失。【目的】解析黄酒贮存陈酿过程中造成黄酒酸败的关键微生物,为黄酒贮存酸败微生物的控制提供依据。【方法】采用高通量测序技术分析不同来源酸败黄酒中的主要微生物种类;设计针对性培养基分离培养酸败黄酒中的难培养微生物;设计微生物特异性引物,对16S r RNA基因高度相似的酸败微生物进行区分鉴定;将分离的黄酒酸败微生物接入到未发生酸败的黄酒中验证其生酸能力。【结果】高通量测序技术分析结果显示,酸败黄酒中污染微生物主要为乳酸杆菌属(丰度95%)微生物,在种水平上分析比对结果显示两种难培养微生物[耐酸乳杆菌(Lactobacillus acetotolerans)和食果糖乳杆菌(Lactobacillus fructivorans)]的相对丰度达到了82%以上;采用改进的分离培养基分离出酸败黄酒中的难培养污染微生物36株;利用耐酸乳杆菌rec A基因和食果糖乳杆菌tuf基因的特异性引物准确鉴定出这些微生物菌株为28株耐酸乳杆菌和8株食果糖乳杆菌;将两种主要酸败微生物接入到未发生酸败的黄酒中,培养2周后能够显著提高黄酒酸度。【结论】基于高通量测序的未培养技术可作为食品中难培养污染微生物快速分析的有效方法。基于未培养技术和可培养技术相结合,首次解析并验证黄酒贮存过程中造成黄酒酸败的主要微生物为耐酸乳杆菌和食果糖乳杆菌。     

Syndrome of accelerated aging induced by carcinogenic environmental factors

The available data on effect of various environmental carcinogenic factors (chemical mutagens, polycyclic aromatic hydrocarbons, nitroso compounds, aromatic amines, tobacco smoking, ionizing radiation, constant illumination, alimentary obesity) upon the organisms suggest that it induces standard pattern of changes at different levels of integration (molecular, cellular, systemic) similar to characteristics of accelerated aging. These changes are favorable to development of age-associated diseases, including cardiovascular those, malignancies, diabetes mellitus type 2, metabolic syndrome, decrease in resistance to stress, immunodepression which lead to life span reduction and premature death.