Wine vinegar production using a noncommercial 100-litre bubble column reactor equipped with a novel type of dynamic sparger

This paper describes batch and semicontinuous acetic acid fermentations for wine vinegar production carried out with Acetobacter pasteurianus, and an industrial strain using a noncommercial 100-L bubble column reactor equipped with a novel type of gas-liquid dynamic sparger. Results showed acetification rates with this fermentor (i.e., an overall acetic acid productivity of 1. 8 g/L/h and yield of 94%) similar to that of the Frings acetator and higher as compared to others fermentors in current industrial use in Spanish wine vinegar factories, and a linear relationship between overall productivity and kLa with different operating conditions and fermentation scales. Copyright 1999 John Wiley & Sons, Inc.     

Spoilage of bottled red wine by acetic acid bacteria

AIMS: To determine the bacterial species associated with an outbreak of spoilage in commercially bottled red wine where the bottles had been stored in an upright vertical compared with horizontal position. METHODS AND RESULTS: Bottled wines comprising Cabernet Sauvignon, Pinot Noir, Shiraz, Merlot and blended red varieties were examined for visible spoilage. Analysis of visibly affected and non-affected wines revealed a spectrum of aroma and flavour defects, ranging from loss of fruity aroma, staleness, oxidized character to overt volatile acidity. Only acetic acid bacteria, and not yeast or lactic acid bacteria, could be isolated from both spoiled and unspoiled wines and were found to grow only on Wallerstein Nutrient (WL) medium supplemented with 10% red wine or 1-2% ethanol. Analysis of the 16S rRNA region and RAPD-PCR analysis showed the isolates to be a closely related group of Acetobacter pasteurianus, but this group was differentiated from the group comprising beer, vinegar and cider strains. CONCLUSIONS: A. pasteurianus was the species considered responsible for the spoilage but the isolates obtained had atypical properties for this species. In particular, they failed to grow on WL nutrient medium without ethanol or wine supplementation. Storage of the bottles of wine containing A. pasteurianus in an upright vertical position specifically induced growth and spoilage in a proportion of the bottles under conditions that were inhibitory for horizontally stored bottles. We hypothesize that the upright position created a heterogeneous environment that allowed the growth of bacteria in only those bottles sealed with cork closures that had upper limit for the natural permeability to oxygen. Such a heterogeneous environment would not exist in horizontally stored bottles as the larger volume of wine adjacent to the cork would strongly compete with the bacteria for the oxygen as it diffuses through the cork closure. SIGNIFICANCE AND IMPACT OF THE STUDY: A low level of bacteria (acetic acid bacteria) in wine can proliferate and cause wine spoilage in bottles stored in an upright vertical as opposed to an horizontal position under conditions that would normally limit bacterial development.     

TASTE GROUP THRESHOLDS AND SENSORY EVALUATION OF SPANISH WINE VINEGARS

Wine vinegar is a product obtained from wine acidification which contains at least 5% by wt. of acetic acid, in general without any additives or colorings.
Aspects studied in this work include: the determination of the taste group thresholds (geometric mean of the individual best-estimate thresholds "BET") of two different acids (citric and acetic acids) in aqueous solution and spanish vinegars produced from table and sherry wines. The results obtained suggest that wine vinegar can be considered something more than just an acidulant agent.
In order to evaluate differences among wine vinegars, discriminant tests for twenty-five spanish vinegars (sherry, table and flavored vinegars) were applied. Six of the twelve attributes freely chosen by assessors allowed grouping of the spanish wine vinegars according to their sensory aspects.     

Influence of Wine Composition on the Heat Resistance of Potential Spoilage Organisms

Pasteurization studies were conducted on 29 yeast and five lactic acid bacteria. In general the yeasts were more heat resistant in wine than were the bacteria. The one exception was a strain of Lactobacillus fructivorans that gave an average D-value of 1.7 min at 60 C. Alcohol was the wine constituent that had the greatest effect on resistance; D-values for all test species were inversely related to the ethanol concentration. The response of organisms to other factors such as pH, sugar, and sulfur dioxide varied with the species.     

一株番茄表面着生醋酸菌的分离鉴定及产酸条件优化

醋酸菌是食醋酿造过程中的关键菌种,性能优良的菌种对于产品品质的提升意义重大。以分离自番茄表面的产醋酸菌为研究对象,通过生理生化指标鉴定、16S rRNA编码基因比对及系统发育树构建等方法对其种类进行鉴定,并通过单因素实验、正交实验对鉴定为醋酸菌的菌株进行培养条件优化。结果表明,所分离的3株醋酸生产菌中,BQ-1被鉴定为醋酸杆菌属(Acetobacteraceae),在以酵母粉为主要氮源,蔗糖为主要碳源的培养基中,其最高产酸量为1823 g·L-1。由于该菌株在番茄表面具有很强的生长能力,因此有望应用于番茄果醋的酿造。     

Acetobacter senegalensis isolated from mango fruits: Its polyphasic characterization and adaptation to protect against stressors in the industrial production of vinegar: A review

It has been more than a decade since Acetobacter senegalensis was isolated, identified and described as a thermotolerant strain of acetic acid bacteria. It was isolated from mango fruits in Senegal and used for industrial vinegar production in developing countries, mainly in sub-Saharan Africa. The strain was tested during several spirit vinegar fermentation processes at relatively high temperatures in accordance with African acclimation. The upstream fermentation process had significant stress factors, which are highlighted in this review so that the fermentation process can be better controlled. Due to its high industrial potential, this strain was extensively investigated by diverse industrial microbiologists worldwide; they concentrated on its microbiological, physiological and genomic features. A research group based in Belgium proposed an important project for the investigation of the whole-genome sequence of A. senegalensis. It would use a 454-pyrosequencing technique to determine and corroborate features that could give this strain significant diverse bio-industrial applications. For instance, its application in cocoa bean fermentation has made it a more suitable acetic acid bacterium for the making of chocolate than Acetobacter pasteurianus. Therefore, in this paper, we present a review that summarizes the current research on A. senegalensis at its microbial and genomic levels and also its specific bio-industrial applications, which can provide economic opportunities for African agribusiness. This review summarizes the physiological and genomic characteristics of Acetobacter senegalensis, a thermotolerant strain isolated from mango fruits and intended to be used in industrial vinegar fermentation processes. It also explores other bio-industrial applications such as cocoa fermentation. Vinegar fermentation is usually performed with mesophilic strains in temperate regions of the world. Developing countries, such as Senegal, import vinegar or make ‘fake’ vinegar by diluting acetic acid obtained from petrochemicals. The use of a thermotolerant Acetobacter senegalensis strain as a solid functional starter culture, as well as the design of a new adapted bioreactor, has significantly contributed to food security and the creation of small- to medium-sized enterprises that produce mango vinegar in West Africa.     

用生料制备功能性双歧醋生产工艺的实验室研究

目的将双歧杆菌、醋酸菌、酵母菌和粉碎的制醋原料及麸曲共同发酵,通过生料制醋的方法来制备功能性双歧醋。方法将粉碎的玉米与麸曲、酵母液、麸皮和水搅拌均匀,使其经过液态糖化和酒精发酵后,接入醋酸菌和双歧杆菌(二者比例为1∶1),同时加入辅料,进行醋酸发酵,当检测到醋酸酸度为5.0%~7.5%时,加入食盐终止发酵,经过过滤,除菌澄清得到功能性双歧醋。结果双歧醋的最终醋酸度为3.2%,外观红棕色,光泽度好,清澈透明,无沉淀和悬浮物。总菌数:醋酸菌为3.3×1011/m l,双歧杆菌为1.9×107/m l;活菌数:醋酸菌为1.7×1011/m l,双歧杆菌为6.8×106/m l;大肠菌群数3个/100 m l;致病菌:不得检出。结论双歧杆菌及其代谢物可以在双歧醋中存活,生料固态发酵制备双歧醋的方法可行。     

Induction of apoptosis in human leukemia cells by naturally fermented sugar cane vinegar (kibizu) of Amami Ohshima Island

Naturally fermented vinegar such as Kibizu (sugar cane vinegar in Amami Ohshima, Japan), Kurozu (black rice vinegar in Kagoshima, Japan), Kouzu (black rice vinegar in China) and red wine vinegar in Italy had potent radical-scavenging activity analyzed by DPPH method. For the elucidation of food factor for cancer prevention contained in naturally fermented vinegar, the induction of apoptosis in human leukemia cell HL-60 was investigated with sugar cane vinegar Kibizu. Fraction eluted by 40% methanol from Amberlite XAD 2 chromatography of sugar cane vinegar showed potent radical scavenging activity. The fraction also showed the activity repressing growth of typical human leukemia cells such as HL-60, THP-1, Molt-4, U-937, Jurkat, Raji and K-562. On the other hand, the fraction did not have any growth inhibition activity against human fetal lung cell TIG-1. The most potent radical-scavenging activity and the growth repression activity of the leukemia cell were observed in the same chromatographic fraction of methanol 40%. From cell sorting FACS analyses, electron microscopic observations and cytochemical staining of chromatin and nuclear segments in human leukemia cell HL-60 treated with the active fraction, it was concluded that apoptosis was induced in the leukemia cell by the fraction of sugar cane vinegar and resulted in the repression of growth of the human leukemia cells. Chromatographic fraction of sugar cane juice eluted by 20% methanol showed potent activities of radical-scavenging and growth repression of HL-60. These results led us the consideration that active components in sugar cane juice could be converted to more lipophilic compounds with activity to induce apoptosis in HL-60 by microbial fermentation with yeast and acetic acid bacteria.     

The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine

AIMS: The objective of this study was to investigate the effects of free molecular and bound forms of sulphur dioxide and oxygen on the viability and culturability of a selected strain of Acetobacter pasteurianus and a selected strain of Brettanomyces bruxellensis in wine. METHODS AND RESULTS: Acetic acid bacteria and Brettanomyces/Dekkera yeasts associated with wine spoilage were isolated from bottled commercial red wines. One bacterium, A. pasteurianus strain A8, and one yeast, B. bruxellensis strain B3a, were selected for further study. The resistance to sulphur dioxide and the effect of oxygen addition on these two selected strains were determined by using plating and epifluorescence techniques for monitoring cell viability in wine. Acetobacter pasteurianus A8 was more resistant to sulphur dioxide than B. bruxellensis B3a, with the latter being rapidly affected by a short exposure time to free molecular form of sulphur dioxide. As expected, neither of these microbial strains was affected by the bound form of sulphur dioxide. The addition of oxygen negated the difference observed between plate and epifluorescence counts for A. pasteurianus A8 during storage, while it stimulated growth of B. bruxellensis B3a. CONCLUSIONS: Acetobacter pasteurianus A8 can survive under anaerobic conditions in wine in the presence of sulphur dioxide. Brettanomyces bruxellensis B3a is more sensitive to sulphur dioxide than A. pasteurianus A8, but can grow in the presence of oxygen. Care should be taken to exclude oxygen from contact with wine when it is being transferred or moved. SIGNIFICANCE AND IMPACT OF THE STUDY: Wine spoilage can be avoided by preventing growth of undesirable acetic acid bacteria and Brettanomyces/Dekkera yeasts through the effective use of sulphur dioxide and the management of oxygen throughout the winemaking process.     

Bacteriocinogenic Potential of <Emphasis Type="Italic">Enterococcus faecium</Emphasis> Isolated from Wine

A total of 145 lactic acid bacteria isolated from a variety of Turkish red wines during malolactic fermentation were screened to find bacteriocin-producing strains. Among them, 14 isolates of Enterococcus faecium were identified to produce bacteriocins. PCR screening revealed that some isolates harbored entA and entB genes while some harbored entA, entB and entP genes. An isolate designated as Ent. faecium H46 was selected to characterize its bacteriocins. The bacteriocins were purified to homogeneity from culture supernatant by Amberlite XAD-16, cation-exchange and reverse-phase chromatography. MALDI-TOF mass spectrometry analysis identified the bacteriocins as enterocin A and enterocin B. The presence of Ent. faecium is noteworthy since it is not associated with wine fermentation. However, it has been reported as an important wine spoilage organism due to its potential to produce tyramine. Although species of Enterococcus is not known as wine bacteria, contamination by Ent. faecium may arise from grapes or wineries equipments used for wine production.     

Development of a sequence-characterized amplified region marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation

Control over malolactic fermentation (MLF) is a difficult goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starters (MLS) in a stressful environment such as wine. In this study, we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as a model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based randomly amplified polymorphic DNA (RAPD) band. The 5' and 3' flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled strain-specific detection without cross amplification of other O. oeni strains or wine species of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detected as few as 2.2 × 10(2) CFU per ml of red wine with good quantification effectiveness, as shown by the correlation of QPCR and plate counting results. Therefore, the cultivation-independent monitoring of a single O. oeni strain in wine based on a SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF.     

Cultivation and preservation of vinegar bacteria

Ten strains of acetic acid bacteria were investigated for their characteristics of growth and metabolism. The strains were identified as those presently in use for industrial vinegar production in southern Germany. At the time of isolation from industrial acetators the total concentrations, i.e. acetic acid (w/v) plus ethanol (v/v), of the fermenting vinegars were 6.1–14.9%. The applicability of a previously described method for starter preparation was examined for the various isolates as well as for the type strains of species of the genera Gluconobacter and Acetobacter. Isolates from cider or wine vinegar fermentations grew readily in RAE-medium to total counts of >1×10⁹ cells ml⁻¹. For the cultivation of strains isolated from spirit vinegar fermentations AE-medium proved most suitable. Cultures of these strains exhibited lag phases of 2–5 days and grew up to total counts of <1×10⁹ cells ml⁻¹. All type strains could be grown on RAE-agar. The use of 20% malt extract as cryo-protectant was effective for the preservation of all strains. Upon revitalization the cultures were suitable as inoculum for starting fermentations in pilot acetators. 16S rRNA-targeted oligonucleotide probes were constructed which were species specific for Gluconobacter oxydans or Acetobacter aceti or group specific for Acetobacter europaeus/Acetobacter xylinum. The probes hybridized with the DNA of the respective type strains. Four isolates were allotted to A. europaeus/A. xylinum applying the group specific probe. The DNA of six of the Acetobacter sp. hybridized with none of the probes.     

Bovine Bile Resistance Increases Leuconostoc oenos 44.40 Viability upon Lyophilization

A screening method based on the selection of strains of Leuconostoc oenos 44.40 resistant to bovine bile was developed to obtain strains of the organism more resistant to lyophilization damage. These strains could be used as starter cultures in the malolactic fermentation of wine. The strain resistant to bovine bile was 20% more viable after lyophilization than strains not resistant to bovine bile. This was confirmed in both laboratory-scale production (100 ml) and pilot-scale production (100 liters). Lyophilized cells of strains sensitive and resistant to bovine bile were inoculated into wine, and the malate metabolism by the organism was monitored in the wine. Resistance to bovine bile did not change the malate metabolism characteristic of the organism. A comparison was made of the fatty acid compositions of the two strains. There was a difference in the fatty acid distribution pattern for these two strains. The bovine bile-resistant strain contained more dodecanoic, hexadecanoic, and octadecanoic acid and less tetradecanoic and hexadecanoic acid than did the bovine bile-sensitive strain. Both strains contained high levels of C-19 cyclopropane fatty acid.     

Plasmid analysis of high acetic acid-resistant bacterial strains by two-dimensional agarose gel electrophoresis and insights into the phenotype of plasmid pJK2-1

Plasmid profiling can be used for quick molecular characterization of bacteria. In the study reported here, this method was used to compare the plasmid profiles of strains of Gluconacetobacter europaeus, one of the dominant species in industrial vinegar production. Further analysis of three selected strains by two-dimensional agarose gel electrophoresis showed that the plasmid profiles are composed of different forms of plasmids of the same size. One of these plasmids, pJK2-1, was introduced into Gluconacetobacter oboediens JK3 as a chimeric plasmid (pJT2) with pUC18. The recombinant strain showed a shorter lag phase in a medium with 3 and 5 % (v/v) acetic acid. Deletion of a part of plasmid pJK2-1 allowed a region that contributes to this novel phenotype of G. oboediens JK3 pJT2 to be identified. Non-problematic handling of G. oboediens JK3 warrants further study in elucidating the function of plasmids involved in the production of vinegar.     

耐乙酸乳杆菌的筛选及其产乳酸特性

【背景】耐受乙酸的乳酸菌是传统谷物醋醋酸发酵过程中产生乳酸及其风味衍生物的重要功能微生物。【目的】从镇江香醋醋醅中分离鉴定具有耐乙酸特性的乳酸菌,并评价不同条件下该菌株的产乳酸能力。【方法】利用4%(体积比)乙酸含量的MRS培养基分离耐乙酸乳酸菌;对其进行16S rRNA基因鉴定、基因组测序、形态观察以及生理生化特性研究;考察不同乙酸浓度、葡萄糖浓度、发酵温度和时间对菌株产乳酸能力的影响。【结果】分离得到一株可耐受6%乙酸的乳杆菌Lactobacillus sp. JN500903;在厌氧静置、接种量5%、乙酸浓度5%、葡萄糖浓度40 g/L、发酵温度37°C、发酵时间10 d条件下,该菌株乳酸产量为16.1 g/L。【结论】乳杆菌JN500903能够耐受6%乙酸浓度,具有在酸性环境下合成乳酸的能力,有一定的应用潜力。     

Preliminary characterization of wine lactobacilli able to degrade arginine

Lactobacillus strains able to degrade arginine were isolated and characterized from a typical red wine. All the strains were gram-positive, catalase-negative and produced both D- and L-lactate from glucose. Strains L2, L3, L4, and L6 were able to produce CO₂ from glucose; however, production of CO₂ from glucose was not observed in strains L1 and L5, suggesting that they belong to the homofermentative wine lactic acid bacteria (LAB) group. All of the lactobacilli were tested for their ability to ferment 49 carbohydrates. The sugar fermentation profile of strain L1 was unique, suggesting that this strain belonged to Lactococcus lactis ssp. cremoris, a non-typical wine LAB. Furthermore, a preliminary typing was performed by using a random amplified polymorphic DNA analysis (RAPD-PCR analysis).     

Aerobic submerged fermentation by acetic acid bacteria for vinegar production: Process and biotechnological aspects

Strictly aerobic acetic acid bacteria (AAB) have a long history of use in fermentation processes, and the conversion of ethanol to acetic acid for the production of vinegar is the most well-known application.At the industrial scale, vinegar is mainly produced by submerged fermentation, which refers to an aerobic process in which the ethanol in beverages such as spirits, wine or cider is oxidized to acetic acid by AAB. Submerged fermentation requires robust AAB strains that are able to oxidize ethanol under selective conditions to produce high-titer acetic acid. Currently submerged fermentation is conducted by unselected AAB cultures, which are derived from previous acetification stocks and maintained by repeated cultivation cycles.In this work, submerged fermentation for vinegar production is discussed with regard to advances in process optimization and parameters (oxygen availability, acetic acid content and temperature) that influence AAB activity. Furthermore, the potential impact arising from the use of selected AAB is described.Overcoming the acetification constraints is a main goal in order to facilitate innovation in submerged fermentation and to create new industry-challenging perspectives.     

沙棘果酒酿造中优良酵母菌的筛选及鉴定

从成熟的沙棘果皮中筛选得到9株酵母茵,通过产气性能、产酒精能力和发酵力测试试验及对发酵所得的沙棘果酒的感官评价,选出R1为最佳的沙棘果酒酿酒用茵株.该茵株适合在酸度较高的沙棘果汁中进行正常的酒精发酵,起酵快、发酵能力强,发酵所得沙棘果酒品质优,而且酒体澄清透明、色泽红润,具有沙棘果酒的典型风味,适用于果酒生产以及下一步...     

Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; beta- d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone alpha-factor secretion signal (MFalpha1(S)) and the phosphoglycerate-kinase-1 gene promoter (PGK1(P)) and terminator (PGK1(T)). The PGK1(P)- MFalpha1(S)- gox- PGK1(T) cassette (designated GOX1) was introduced into a laboratory strain (Sigma1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2.0% less alcohol. This was probably due to the production of d-glucono-delta-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.     

Two new arsenate/sulfate-reducing bacteria: mechanisms of arsenate reduction

Two sulfate-reducing bacteria, which also reduce arsenate, were isolated; both organisms oxidized lactate incompletely to acetate. When using lactate as the electron donor, one of these organisms, Desulfomicrobium strain Ben-RB, rapidly reduced (doubling time = 8 h) 5.1 mM arsenate at the same time it reduced sulfate (9.6 mM). Sulfate reduction was not inhibited by the presence of arsenate. Arsenate could act as the terminal electron acceptor in minimal medium (doubling time = 9 h) in the absence of sulfate. Arsenate was reduced by a membrane-bound enzyme that is either a c-type cytochrome or is associated with such a cytochrome; benzyl-viologen-dependent arsenate reductase activity was greater in cells grown with arsenate/sulfate than in cells grown with sulfate only. The second organism, Desulfovibrio strain Ben-RA, also grew (doubling time = 8 h) while reducing arsenate (3.1 mM) and sulfate (8.3 mM) concomitantly. No evidence was found, however, that this organism is able to grow using arsenate as the terminal electron acceptor. Instead, it appears that arsenate reduction by the Desulfovibrio strain Ben-RA is catalyzed by an arsenate reductase that is encoded by a chromosomally-borne gene shown to be homologous to the arsC gene of the Escherichia coli plasmid, R773 ars system. Received: 18 March 1999 / Accepted: 27 September 1999