Differential Real-Time PCR Assay for Enumeration of Lactic Acid Bacteria in Wine

Oenococcus oeni is often employed to perform the malolactic fermentation in wine production, while nonoenococcal lactic acid bacteria often contribute to wine spoilage. Two real-time PCR assays were developed to enumerate the total, and nonoenococcal, lactic acid bacterial populations in wine. Used together, these assays can assess the spoilage risk of juice or wine from lactic acid bacteria.     

LACTIC ACID BACTERIA AS SPOILAGE ORGANISMS OF FRUIT JUICE PRODUCTS

SUMMARY: The spoilage of wines and ciders by lactic acid bacteria is described. The pioneering work on wine bacteriology at the turn of the century is discussed in addition to modern work carried out in Australia and the U.S.A. Similarly, past and present work on the spoilage of ciders by lactic acid bacteria is discussed, special reference being made to their activity against malic acid as well as chlorogenic acid and its derivatives.     

Monitoring of Saccharomyces and Hanseniaspora populations during alcoholic fermentation by real-time quantitative PCR

Real-time, or quantitative, PCR (QPCR) was developed for the rapid quantification of two of the most important yeast groups in alcoholic fermentation (Saccharomyces spp. and Hanseniaspora spp.). Specific primers were designed from the region spanning the internal transcribed spacer 2 (ITS2) and the 5.8S rRNA gene. To confirm the specificity of these primers, they were tested with different yeast species, acetic acid bacteria and lactic acid bacteria. The designed primers only amplified for the intended group of species and none of the PCR assays was positive for any other wine microorganisms. This technique was performed on reference yeast strains from pure cultures and validated with both artificially contaminated wines and real wine fermentation samples. To determine the effectiveness of the technique, the QPCR results were compared with those obtained by plating. The design of new primers for other important wine yeast species will enable to monitor yeast diversity during industrial wine fermentation and to detect the main spoilage yeasts in wine.     

Lactic acid bacteria associated with wine grapes from several Australian vineyards

AIMS: The detection and isolation of lactic acid bacteria by enrichment methods from wine grapes cultivated in vineyards located in New South Wales, Australia. METHODS AND RESULTS: Enrichment cultures in de Man, Rogosa and Sharpe (MRS) broth, MRS + ethanol (5%), MRS broth supplemented with 15% (v/v) tomato juice (MRST), pH 5.5 and 3.5 and autoenrichment in grape juice homogenate were used to detect lactic acid bacteria on wine grapes. Bacteria were isolated from enrichment cultures by plating onto MRS and MRST agar and identified by 16S rDNA sequence analysis and phenotypical methods. A molecular method, PCR-denaturing gradient gel electrophoresis (DGGE) was also used to examine the bacteria that developed in enrichment cultures. Species of Lactobacillus, Enterococcus, Lactococcus and Weissella were detected in enrichments by plating and PCR-DGGE. Other bacteria (Sporolactobacillus, Asaia, Bacillus ssp.) were also found in some enrichment cultures. The principal malolactic bacterium, Oenococcus oeni, was not isolated. CONCLUSIONS: The incidence and populations of lactic acid bacteria on wine grapes were very low. Damaged grape berries showed a greater presence of these bacteria than undamaged berries. The diversity of bacterial species isolated from the grapes was greater than those previously reported and represented both lactic acid bacteria and nonlactic acid bacteria. Some of these bacteria (i.e. Lactobacillus lindneri, Lactobacillus kunkeei) could be detrimental to wine production. Oenococcus oeni was not found on grapes, but its recovery could be obscured by overgrowth from other species. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria are significant in wine production because they conduct the malolactic fermentation and cause stuck or sluggish alcoholic fermentation and wine spoilage. This study investigates wine grapes as a potential source of these bacteria.     

液质发酵食品中微生物群落及与乳酸菌间相互作用研究进展

液质发酵食品发酵过程中微生物组成复杂,复杂的微生物发酵体系会影响微生物的生长和代谢,继而改变微生物的群落结构与功能,最终影响液质发酵食品的品质。乳酸菌在食品发酵中对形成风味物质、提高营养价值、抑制腐败菌生长具有重要的作用。本文主要对近年来食醋、酱油和饮料酒等液质发酵食品中微生物群落及与乳酸菌间相互作用关系进行综述,了解液质发酵食品在发酵过程中微生物群落结构和群落中乳酸菌与其他微生物的相互作用类型,探讨乳酸菌在发酵过程中的功能以及乳酸菌与其他微生物之间的相互作用机制。     

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.     

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.     

Bacterial spoilage of wine and approaches to minimize it

Bacteria are part of the natural microbial ecosystem of wine and play an important role in winemaking by reducing wine acidity and contributing to aroma and flavour. Conversely, they can cause numerous unwelcome wine spoilage problems, which reduce wine quality and value. Lactic acid bacteria, especially Oenococcus oeni, contribute positively to wine sensory characters, but other species, such as Lactobacillus sp. and Pediococcus sp can produce undesirable volatile compounds. Consequences of bacterial wine spoilage include mousy taint, bitterness, geranium notes, volatile acidity, oily and slimy-texture, and overt buttery characters. Management of wine spoilage bacteria can be as simple as manipulating wine acidity or adding sulfur dioxide. However, to control the more recalcitrant bacteria, several other technologies can be explored including pulsed electric fields, ultrahigh pressure, ultrasound or UV irradiation, and natural products, including bacteriocins and lysozyme.     

Which lactic acid bacteria are responsible for histamine production in wine?

AIMS: To quantify the ability of 136 lactic acid bacteria (LAB), isolated from wine, to produce histamine and to identify the bacteria responsible for histamine production in wine. METHODS AND RESULTS: A qualitative method based on pH changes in a plate assay was used to detect wine strains capable of producing high levels of histamine. Two quantitative, highly sensitive methods were used, an enzymatic method and HPLC, to quantify the histamine produced by LAB. Finally, an improved PCR test was carried out to detect the presence of histidine decarboxylase gene in these bacteria. The species exhibiting the highest frequency of histamine production is Oenococcus oeni. However, the concentration of histamine produced by this species is lower than that produced by strains belonging to species of Lactobacillus and Pediococcus. A correlation of 100% between presence of histidine decarboxylase gene and histamine production was observed. Wines containing histamine were analysed to isolate and characterize the LAB responsible for spoilage. CONCLUSIONS: Oenococcus was able to synthesize low concentrations of histamine in wines, while Pediococcus parvulus and Lactobacillus hilgardii have been detected as spoilage, high histamine-producing bacteria in wines. SIGNIFICANCE AND IMPACT OF THE STUDY: Information regarding histamine-producing LAB isolated from wines can contribute to prevent histamine formation during winemaking and storage.     

Lactic acid bacteria of meat and meat products

When the growth of aerobic spoilage bacteria is inhibited, lactic acid bacteria may become the dominant component of the microbial flora of meats. This occurs with cured meats and with meats packaged in films of low gas permeability. The presence of a flora of psychrotrophic lactic acid bacteria on vacuum-packaged fresh chilled meats usually ensures that shelf-life is maximal. When these organisms spoil meats it is generally by causing souring, however other specific types of spoilage do occur. Some strains cause slime formation and greening of cured meats, and others may produce hydrogen sulphide during growth on vacuum-packaged beef. The safety and stability of fermented sausages depends upon fermentation caused by lactic acid bacteria. Overall the presence on meats of lactic acid bacteria is more desirable than that of the types of bacteria they have replaced.     

Lactic acid bacteria of wines: stimulation of growth and malolactic fermentation

After the appearance of “Etudes sur le vin” by Pasteur, in enology lactic acid bacteria have been considered as deteriorating agents for more than 50 years. About 1920, Ferré in Burgundy and Ribéreau-Gayon in Bordeaux demonstrated the enological importance of the transformation of malic to lactic acid. This notion is now generally accepted in most vinicultural areas. Malolactic fermentation is encouraged, especially for red wines, for two reasons: a) it eliminates the taste of malic acid and lowers the acidity of the wine, b) it assures the biological stability of wines conserved with a minimum of sulphurous anhydride. In traditional vinification, malolactic fermentation is the result of bacterial growth. It is spontaneous, that means induced by the endogenous lactic acid bacteria of grapes and winery equipment. In the must, yeasts and bacteria develop simultaneously; in the antagonism between yeasts and bacteria the bacterial population is more often becoming dominant than being suppressed. The grapes are sulphited so that bacterial growth occurs only after complete exhaustion of sugars by the yeasts. Consequently, alteration of the wine, as a result of sugar fermentation by the bacteria, is prevented. In a well-controlled vinification lactic acid bacteria can complete their growth cycle in the wine. Wine, however, is a poor culture medium and the bacteria multiply under restricted nutritional, physical and chemical conditions. As a consequence, malolactic fermentation is difficult to control in practice, in spite of all the research done for more than 30 years. For a long time, one has tried to stimulate malolactic fermentation by inoculating wine with bacteria. Until now, the problem has been to determine the biomass of bacteria, sufficient for fermentation to take place as well as the quality required. The desired physiological state of the bacteria in the inoculum is also not known.     

A REVIEW : Arginine metabolism in wine lactic acid bacteria and its practical significance

This article begins with an introduction to malolactic fermentation in wine, followed by a review of the occurrence of arginine degradation in wine lactic acid bacteria and the pathway of arginine catabolism, the distribution of enzymes responsible, and the formation of products. The bioenergetics of wine lactic acid bacteria and arginine degradation are then reviewed. This is followed by a review of the possible mechanisms of arginine transport, and regulation of arginine metabolism and synthesis of the enzymes for arginine catabolism. Finally, the practical significance of arginine metabolism in wine lactic acid bacteria is reviewed with respect to taxonomic utility, biological significance and oenological implications.     

复合乳酸菌对冷藏海鲈鱼块的保鲜效果

【目的】研究复合乳酸菌对冷藏海鲈鱼块的保鲜效果。【方法】以冷藏海鲈鱼块为对象,筛选出3株能够明显抑制其优势腐败菌(草莓假单胞菌Pseudomonas fragi,腐败希瓦氏菌Shewanella putrefacens)生长的单一乳酸菌,同时也筛选出对其优势腐败菌具有最显著抑制效果的一组复合乳酸菌,再将该复合乳酸菌接种到海鲈鱼块上,在4°C冷藏过程中,通过感官评定、挥发性盐基氮(TVB-N值)的测定和优势腐败菌的计数来评价复合乳酸菌对冷藏海鲈鱼块的保鲜效果。【结果】单一乳酸菌(干酪乳杆菌LC1、植物乳杆菌LP1和乳酸菌L3)对2株冷藏海鲈鱼优势腐败菌的抑制效果明显;复合乳酸菌(干酪乳杆菌LC1+植物乳杆菌LP1+乳酸菌L3)的抑菌效果最为显著;在4°C冷藏过程中,复合乳酸菌能使冷藏海鲈鱼块发生感官变化延缓6 d、使TVB-N值的升高延缓2 d,同时显著抑制优势腐败菌的生长。【结论】复合乳酸菌对冷藏海鲈鱼块具有良好的保鲜作用,能有效延长其货架期。     

Changes in the Microbiology of Vacuum-packaged Beef

The development of the microbial flora on meat stored in vacuum-bags at 0–2° for up to 9 weeks was studied. Although the proportion of lactic acid bacteria increased relative to the aerobic spoilage organisms, the numbers of the latter continued to increase throughout storage. The initial contamination of the meat before vacuum-packaging was important; meat with a very low initial number had lower numbers of bacteria throughout storage for up to 9 weeks and steaks cut from such meat which had been stored always had 1–2 days' additional aerobic shelf life at 4°. Spoilage of these steaks was due either to slime formation and off-odour associated with high counts of presumptive Pseudomonas spp., or by discoloration and souring (lactic acid bacteria). Extract release volume and pH measurements performed on the vacuum-packaged primal joints were only of value in determining the onset of aerobic spoilage when large numbers of Gram negative organisms were present, whereas the titrimetric method of spoilage evaluation of the vacuum-packaged meat showed a correlation with spoilage due to lactic organisms.     

Antifungal lactic acid bacteria with potential to prolong shelf-life of fresh vegetables

AIM: The aim of this study was to isolate and identify antifungal lactic acid bacteria from fresh vegetables, and evaluate their potential in preventing fungal spoilage of vegetables. METHODS AND RESULTS: Lactic acid bacteria from fresh vegetables were enriched in MRS (de Man Rogosa Sharpe) broth and isolated by plating on MRS agar. All the isolates (359) were screened for activity against Aspergillus flavus of which 10% showed antifungal activity. Potent antifungal isolates were identified by phenotypic characters and confirmed by partial 16S rRNA gene sequencing. These were screened against additional spoilage fungi viz. Fusarium graminearum, Rhizopus stolonifer, Sclerotium oryzae, Rhizoctonia solani, Botrytis cinerea and Sclerotinia minor by overlay method. Most of the isolates inhibited wide range of spoilage fungi. When fresh vegetables were inoculated with either cell suspension (10(4) cells ml(-1)) or cell-free supernatant of Lact. plantarum, followed by application of vegetable spoilage fungi (A. flavus and F. graminearum, R. stolonifer, B. cinerea each with 10(4) conidia ml(-1)) the vegetable spoilage was significantly delayed than control. CONCLUSIONS: Fresh vegetables constitute a good source of lactic acid bacteria with ability to inhibit wide range of spoilage fungi. Such bacteria can be applied to enhance shelf-life of vegetables. In the present study, we report for the first time the antifungal activity of Weissella paramessenteroides and Lact. paracollinoides isolated from fresh vegetables, against wide range of food spoilage fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Fresh vegetables can be used as a source of antifungal lactic acid bacteria. Their exploitation as biopreservative will help in prolonging shelf-life of fresh vegetables.     

Dynamics of lactic acid bacteria populations in Rioja wines by PCR-DGGE, comparison with culture-dependent methods

Lactic acid bacteria populations of red wine samples from industrial fermentations, including two different vinification methods were studied. For this investigation, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis was employed to supplement previous results that were obtained by culture-dependent methods. PCR-DGGE was aimed to study two targeted genes, 16S ribosomal DNA (rDNA) and rpoB, and the results were useful to evaluate the microbial populations in wine samples. Moreover, an improvement of a detection limit determined so far for DGGE analysis was obtained with the method described in this study, what made possible to identify lactic acid bacteria populations below 10¹ colony-forming unit/mL. The species Oenococcus oeni was the most frequently detected bacterium, but identifications close to species Oenococcus kitaharae and Lactococcus lactis that are not often found in wine were firstly identified in samples of this research. PCR-DGGE allowed to detect 9 out of 11 lactic acid bacteria species identified in this study (nine by PCR-16S rDNA/DGGE and four by PCR-rpoB/DGGE), while five species were detected using the modified de Man, Rogosa and Sharpe agar. Therefore, the two methods were demonstrated to be complementary. This finding suggests that analysis of the lactic acid bacteria population structure in wine should be carried out using both culture-dependent and culture-independent techniques with more than one primer pair.     

Discrimination of wine lactic acid bacteria by Raman spectroscopy

Species of Lactobacillus, Pediococcus, Oenococcus, and Leuconostoc play an important role in winemaking, as either inoculants or contaminants. The metabolic products of these lactic acid bacteria have considerable effects on the flavor, aroma, and texture of a wine. However, analysis of a wine’s microflora, especially the bacteria, is rarely done unless spoilage becomes evident, and identification at the species or strain level is uncommon as the methods required are technically difficult and expensive. In this work, we used Raman spectral fingerprints to discriminate 19 strains of Lactobacillus, Pediococcus, and Oenococcus. Species of Lactobacillus and Pediococcus and strains of O. oeni and P. damnosus were classified with high sensitivity: 86–90 and 84–85%, respectively. Our results demonstrate that a simple, inexpensive method utilizing Raman spectroscopy can be used to accurately identify lactic acid bacteria isolated from wine.     

rpoB gene: a target for identification of LAB cocci by PCR-DGGE and melting curves analyses in real time PCR

Lactic acid bacteria (LAB) are essential in the quality of many fermented beverages like beer, cider and wine. In the two later cases, they convert malic acid into lactic acid during the malolactic fermentation. After fermentation, microbial stabilization is needed to prevent the development of spoilage bacteria species. Among them, cocci lead to different alterations: Pediococcus sp., and some strains of Leuconostoc mesenteroides and Oenococcus oeni can produce exopolysaccharides which modify wine viscosity and lead to ropiness. They also can produce acetic acid, biogenic amine, ethyl carbamate and volatile phenols. Therefore detection and identification are crucial. Results of phenotypic tests and DNA-DNA probes are not accurate enough. 16S RNA gene which is currently used for bacterial species identification presents intraspecies heterogeneity. The rpoB gene is an alternative to this limitation. However previous PCR targeting partial sequence of rpoB gene could not delimit cocci species. Therefore we compared the rpoB gene sequence of the six main cocci species found in fermented beverages: P. damnosus, P. dextrinicus, P. parvulus, P. pentosaceus, L. mesenteroides and O. oeni. The most discriminating partial sequence of the rpoB gene was chosen for designing primers. By PCR-DGGE the reliability of these primers was verified. It was controlled in a mixture of several cocci and other lactic acid bacteria (Lactobacillus sp.). Then we adapted the primers and the PCR conditions in order to achieve the identification of cocci species by real time PCR program including the fluorescent dye SYBR Green I, which gives faster results. PCR melt curves were established and a specific T(m) was attributed to each species.     

Growth of yeasts, lactic and acetic acid bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana

AIM: To investigate the microbiological and biochemical changes which occur in palm wine during the tapping of felled oil palm trees. METHODS AND RESUlts: Microbiological and biochemical contents of palm wine were determined during the tapping of felled oil palm trees for 5 weeks and also during the storage. Saccharomyces cerevisiae dominated the yeast biota and was the only species isolated in the mature samples. Lactobacillus plantarum and Leuconostoc mesenteroides were the dominated lactic acid bacteria, whilst acetic acid bacteria were isolated only after the third day when levels of alcohol had become substantial. The pH, lactic and acetic acid concentrations during the tapping were among 3.5-4.0%, 0.1-0.3% and 0.2-0.4% respectively, whilst the alcohol contents of samples collected within the day were between 1.4% and 2.82%; palm wine which had accumulated over night, 3.24% to 4.75%; and palm wine held for 24 h, over 7.0%. CONCLUSION: Accumulation of alcohol in palm wine occurs in three stages during the tapping and marketing with the concurrent lactic and acetic acid fermentation taking place as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts, lactic and acetic acid bacteria are all important in the fermentation of palm wine and influence the composition of the product.