Differentiation of Australian wine isolates of Oenococcus oeni using random amplified polymorphic DNA (RAPD)

Intraspecific variation of Oenococcus oeni , the preferred lactic acid bacteria species for inducing malolactic fermentation in wine, was studied using the randomly amplified polymorphic DNA (RAPD) strain fingerprinting technique. Ten of fifteen isolates of O. oeni from Australian wineries situated in different wine regions could be distinguished by the RAPD technique. Strains of O. oeni which originated from the same winery were either indistinguishable or closely related to each other. Six different commercially available O. oeni strains could be differentiated with the four RAPD primers used and their genetic similarity determined. Analysis of O. oeni present in wines from a single source of fruit (Cabernet Sauvignon, vintage 2002) that underwent spontaneous malolactic fermentation revealed wide genetic variation amongst the isolates. Each fermentor contained several different O. oeni strains, which were present throughout alcoholic and malolactic fermentation. These data highlight the sensitivity of RAPDs when suitable primers are applied to O. oeni of unknown genetic origin, thus enabling O. oeni strains with desirable sensory and fermentation properties to be genetically analysed.     

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 effects of freezing and freeze-drying of Oenococcus oeni upon induction of malolactic fermentation in red wine

Summary The use of Oenococcus oeni starter cultures for the induction of malolactic fermentation (MLF) in wine permits control over the timing of the process and the quality of the wine. Successful inoculation of bacterial starter cultures into wine depends on the selection of suitable strains and on the preparation and conservation of those cultures. Medium for Leuconostoc oenos (MLO) is the best medium for easy and rapid growth of O. oeni cultures under laboratory controlled conditions for isolation and identification. However, this study showed that O. oeni cells inoculated in MLO failed to induce MLF in wine while cells grown in Medium of Preculture (MP) or wine, stored at −20 °C or freeze-dried retained the ability to induce MLF when inoculated in wine. Our results suggest that the use of freeze-dried cultures of O. oeni previously grown in MP is the best choice for industrial application.     

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     

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.     

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.     

Influence of different pH values and inoculation time on the growth and malolactic activity of a strain of Oenococcus oeni

The present study was undertaken to evaluate the influence of medium pH and inoculation time on the growth and malolactic activity of an Oenococcus oeni culture. Samples of a commercial white grape juice adjusted to pH 3.2, 3.4 or 3.6, and inoculated with Saccharomyces cerevisiae strain CY3079, were inoculated with a malolactic culture ( Oenococcus oeni strain 31) at the beginning, middle, and end of alcoholic fermentation. The results obtained from this single case study show that it is possible to inoculate the bacterial culture at the three different times during alcoholic fermentation without slowing down or stopping alcoholic fermentation or causing failure of MLF. However, pH and timing of bacterial inoculation were critical to how rapidly MLF starts. At pH 3.2 a lowering of bacterial viability was observed, but a more important reduction was recorded at all tested pH levels when the bacteria were inoculated halfway through alcoholic fermentation. When inoculation was carried out at the end of alcoholic fermentation, the presence of yeast seemed to favour bacterial viability and activity and bacteria performed MLF even in difficult conditions such as pH values around 3. In all wines malolactic fermentation was accompanied by total degradation of malic and citric acids and production of L-lactic acid, D-lactic and acetic acids.     

Molecular characterization of lactic acid bacteria and in situ amylase expression during traditional fermentation of cereal foods

Lactic acid bacteria play an important role in traditional fermented foods consumed in different countries. Study of their taxonomic structure and diversity is necessary for starter culture selection, improved safety and nutritional enhancement. To achieve these objectives, microbial genomic typing methods were used to study genetic differences of autochthonous bacteria and their distribution in two traditional African fermented cereal foods. A total of 85 predominant bacterial species were isolated from ogi and kunu-zaki obtained from Northern and Southern geographical region of Nigeria. They were identified using combination of 16S rRNA gene sequencing, multilocus sequence analysis (MLSA) based on rpoA, pheS and atpA genes as well as M13-PCR gel fingerprints. The results showed that Lactobacillus fermentum was the most frequently isolated species in ogi (71.4%) and kunu-zaki (84.5%). Other species of lactic acid bacteria (LAB) identified were Lactobacillus plantarum, Streptococcus gallolyticus subsp. macedonicus and Pediococcus pentosaceus. Non lactic acid bacteria isolated from these foods were species belonging to the Bacillus and Staphylococcus. Non-metric multidimensional scaling (nMDS) analysis of the M13-PCR fingerprints for LAB strains showed clonal diversity among strains of the same species. In vitro and in situ expression of amylase gene during fermentation by amylolytic L. plantarum ULAG11 was detected, indicating the potential usefulness of such species for development of starter cultures and for controlled fermentation processes.     

Use of a polymerase chain reaction for specific detection of the malolactic fermentation bacterium Oenococcus oeni (formerly Leuconostoc oenos) in grape juice and wine samples

A PCR method has been developed that enables rapid and direct identification of the malolactic bacterium Oenococcus oeni from grape must or wine samples. Two primers, based on unique, highly conserved regions within the 16S rRNA gene of O. oeni , were used to amplify a 995 bp fragment which is specific for O. oeni . Other species of bacteria from Lactobacillus, Pediococcus and Acetobacter which may be found in grape must or wine were not detected using this technique. This diagnostic test is able to specifically detect in the order of 10³colony forming units per mL of O. oeni in a wine sample, and can be used for monitoring bacterial growth during malolactic fermentation.     

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.     

Molecular and physiological characterization of natural microbial communities isolated from a traditional Southern Italian processed sausage

The development of the natural microbial populations during traditional processing of the "Salame di Senise", a typical little known sausage produced in the South of Italy, was investigated by using molecular and physiological techniques for taxonomic identification and technological characterization of strains. The application of RAPD-PCR over more than 90 colonies made it possible to isolate 18 bacterial and two yeast biotypes identified by partial rDNA sequencing as belonging mainly to three species of Bacillus, three species of Lactobacillus, three species of Staphylococcus and Debaryomyces hansenii. The physiological analyses revealed that the isolates belonging to Lactobacillus genus were the most acidifying, whereas Staphylococcus strains did not develop significant proteolytic and lipolytic activities. Interestingly, some Bacillus strains produced the highest values of proteolytic and lipolytic activities. The results for the technological properties of Bacillus strains isolated from this Southern Italian sausage, made without a selected starter, suggest that Bacillus strains, always present in meat curing, could play a role in the development of texture and organoleptic characteristics of the sausages.     

Transformation of isoflavone phytoestrogens during the fermentation of soymilk with lactic acid bacteria and bifidobacteria

In the present study, soymilk is fermented with lactic acid bacteria (Streptococcus thermophilus BCRC 14085, Lactobacillus acidophilus BCRC 14079) and bifidobacteria (Bifidobacterium infantis BCRC 14633, B. longum B6) individually, and in combination. The change in the content of various isoflavones (aglycones, glucoside, acetyl- and malonyl-glucosides) and the beta-glucosidase activity in soymilk during fermentation is investigated. It is observed that fermented soymilk contains a lower total isoflavone content (81.94-86.61 microg/ml) than soymilk without fermentation (87.61 microg/ml). Regardless of starter organism employed, fermentation causes a major reduction in the contents of glucoside, malonylglucoside and acetylglucoside isoflavones along with a significant increase of aglycone isoflavones content. The level of change in the content of various isoflavones and beta-glucosidase activity after fermentation varies with the starter organism. Among all the fermented soymilks tested, soymilk fermented with S. thermophilus showed the highest beta-glucosidase activity and the greatest increase in the contents of aglycones. The percentage of daidzein, genistein and glycitein to total isoflavone content in S. thermophilus-fermented soymilk increases from an initial 14.24%, 6.89% and 2.45%, respectively, to 36.20%, 28.80% and 12.44% after 24h of fermentation. Finally, the increase of aglycones and decrease of glucoside isoflavones during fermentation coincides with the increase of beta-glucosidase activity observed in fermented soymilk.     

发酵条件对酸樱桃发酵汁挥发性风味物质的影响探究

目的 考察发酵条件对酸樱桃发酵汁中风味物质的影响。方法 利用3株乳酸菌发酵酸樱桃汁, 以pH为指标明确最佳发酵菌种和稀释比例。随后利用气相色谱-离子迁移谱法(gas chromatography-ion mobility spectrometry, GC-IMS)探究糖添加量、pH、氮源、稀释比例对发酵挥发性风味物质的影响。结果 最佳发酵菌为鼠李糖乳杆菌(Lactobacillus GG, LGG), 最佳稀释比例为1:3, 氮源对挥发性风味物质几乎没有影响; 而不同添加量的碳源(葡萄糖)导致发酵液乙偶姻、双乙酰等含量差异; 随着pH增加, 乙偶姻、双乙酰、2-戊酮等含量显著升高; 稀释比例通过影响发酵原汁的浓度导致发酵后风味物质乙偶姻含量显著增加、2-戊酮含量显著降低。进一步差异比对3株乳酸菌酸樱桃发酵汁的风味, 发现LGG代谢脂肪酸可产生较多的2-庚酮及2-戊酮。结论 菌种、糖添加量、稀释比例、起始pH均可导致发酵液中乙偶姻、2-庚酮及2-戊酮等挥发性风味物质产生显著影响, 本研究为酸樱桃的加工利用提供理论指导。     

木薯酒发酵过程中的有机酸变化及对其品质的影响分析

以新鲜甜木薯为原料,跟踪监测木薯酒发酵过程中的理化指标,并采用高效液相色谱法分析木薯酒发酵期间的有机酸组成及动态变化情况,进一步分析酒发酵过程中的酸味强度。结果表明,发酵过程中,木薯酒中的还原糖含量先快速上升至最高点最后趋于平稳（54.67 mg/mL）;酒精度先上升后平稳（7.11%vol）;总酸含量先缓慢减少至2.21 mg/mL,再缓慢增加,后快速增加,与pH值变化趋势相反。酒石酸、乳酸、乙酸、柠檬酸、琥珀酸是木薯酒发酵中重要有机酸,分别占木薯酒总有机酸含量的2.66%～12.31%,41.50%～63.53%,18.21%～23.82%,1.22%～27.77%,3.17%～15.89%,其他有机酸是酒中的辅助酸味特征成分。乳酸和乙酸是木薯酒的主体酸,其味道强度值（TAV）占总TAV值的65.82%～95.84%,发酵84 h的木薯酒有最高的味道强度值,其可达到223.87。     

Inhibitory effects of lactic acid and lauricidin on spoilage organisms of chicken breast during storage at chilled temperature

Different concentrations of lauricidin (LU, containing 1% lactic acid) and lactic acid alone (LA) were evaluated for their effectiveness in reducing naturally occurring microflora of raw chicken breasts. Chicken breasts were dipped in 0 (control), 0.5, 1.0, 1.5, and 2.0% solutions of LU (w/v) or LA (v/v) for 10, 20, and 30 min and stored at 4 °C for 14 d. Total Plate Counts (TPC) and populations of Pseudomonas spp. and Enterobacteriaceae were determined before and after dipping and after storing for 1, 3, 7, 10, and 14 d. Additionally, Hunter L, a, and b values and pH of the chicken breast were also determined. From the obtained results, TPC on chicken breast treated with LU was found to be decreased by 0.92 to 1.2 log CFU/g from a mean initial log 5.69 CFU/g, while those dipped in LA decreased by 0.53 to 2.36 log CFU/g. Pseudomonas population on chicken breast dipped in LU decreased by 0.79 to 1.77 log CFU/g from an initial 3.90 log CFU/g, while in LA treated it decreased by 0.39 to 1.82 log CFU/g. Enterobacteriaceae counts were also found to be reduced by 0.14 to 1.14 log CFU/g on chicken breast dipped in LU, while the reduction was from 0.59 to 2.18 log CFU/g in chicken breast dipped in LA. The major bacterial types isolated from LU treated chicken breast belonged to the Enterobacteriaceae group, which included: Enterobacter, E. coli and Citrobacter. Whereas, in the LA treated breast it belonged to: Pseudomonas, E. coli, and Kocuria rhizophila (formerly Micrococcus luteus). Dipping chicken breast in LU and LA caused a significant decrease (p ≤ 0.05) in their pH values. Also, treatment with LU and LA caused a slight darkening in color (decreased Hunter L value), increase in redness (increased Hunter a value), and increase in yellowness (increased Hunter b value). Based on the results obtained in the present study, Lactic acid and Lauricidin showed high potential to be used as a sanitizer in reducing the population of spoilage microorganisms naturally occurring on raw chicken, and can be explored commercially for extension of their shelf life.     

不同温度条件下湘西苞谷酸发酵过程主要营养品质变化

湘西苞谷酸是一种以玉米和新鲜红辣椒为主要原料加工而成的风味独特的发酵食品,该研究对3种不同温度模式下苞谷酸发酵过程营养品质变化进行测定,并对3种温度条件下的产品进行感官评分。结果表明,3种温度模式下,随着发酵时间的延长,苞谷酸pH、淀粉逐渐降低,亚硝酸盐含量先增加后减少,总游离氨基酸含量呈现逐渐增加的趋势,发酵18 d后pH下降趋缓,游离氨基酸含量增加趋缓;25～30 ℃更利于湘西苞谷酸品质的形成,此温度条件下发酵36 d,苞谷酸pH3.8、淀粉含量33.3 g/100 g、蛋白质含量5.71 g/100 g、乳酸菌数对数值为5.03、亚硝酸盐3.8 g/kg、还原糖0.43 g/100 g、总游离氨基酸1.4 g/100 g。     

Effect of substrate composition and inoculum on the fermentation kinetics and flavour compound profiles of potentially non-dairy probiotic formulations

Most of the commercialized probiotic formulations are dairy-based, and the development of non-dairy probiotic products could be a key area for new functional products. It has been proved that probiotic microorganisms can grow on cereal-based substrates, but flavour development in these has not been studied. In this work nine prospective cereal-based probiotic beverages were produced by inoculating oats, barley and malt substrates with single cultures of Lactobacillus acidophilus (NCIMB 8821), Lactobacillus plantarum (NCIMB 8826), and Lactobacillus reuteri (NCIMB 11951). Volatile, and non-volatile flavour compounds, as well as cell growth, pH, free amino nitrogen (FAN), and total reducing sugars (TRS) profiles were monitored during fermentation. The malt beverage inoculated with L. plantarum had the highest amounts of acetaldehyde and diacetyl with concentrations of 6.21 mg/L and 0.38 mg/L respectively. The greatest amount of lactic acid (4.2 g/L) was measured in the malt beverage inoculated with L. acidophilus. In most of the beverages the maximum cell growth was reached after 8 h of fermentation (6.4–8.2 Log₁₀ CFU/mL). It is clear from this work that each microorganism develops different flavours in the three media tested, and the right combination of microorganisms and substrates could lead to palatable probiotic products.