Potential of wine-associated lactic acid bacteria to degrade biogenic amines

Some lactic acid bacteria (LAB) isolated from fermented foods have been proven to degrade biogenic amines through the production of amine oxidase enzymes. Since little is known about this in relation to wine micro-organisms, this work examined the ability of LAB strains (n = 85) isolated from wines and other related enological sources, as well as commercial malolactic starter cultures (n = 3) and type strains (n = 2), to degrade histamine, tyramine and putrescine. The biogenic amine-degrading ability of the strains was evaluated by RP-HPLC in culture media and wine malolactic fermentation laboratory experiments. Although at different extent, 25% of the LAB isolates were able to degrade histamine, 18% tyramine and 18% putrescine, whereas none of the commercial malolactic starter cultures or type strains were able to degrade any of the tested amines. The greatest biogenic amine-degrading ability was exhibited by 9 strains belonging to the Lactobacillus and Pediococcus groups, and most of them were able to simultaneously degrade at least two of the three studied biogenic amines. Further experiments with one of the strains that showed high biogenic amine-degrading ability (L. casei IFI-CA 52) revealed that cell-free extracts maintained this ability in comparison to the cell suspensions at pH 4.6, indicating that amine-degrading enzymes were effectively extracted from the cells and their action was optimal in the degradation of biogenic amines. In addition, it was confirmed that wine components such as ethanol (12%) and polyphenols (75 mg/L), and wine additives such as SO₂ (30 mg/L), reduced the histamine-degrading ability of L. casei IFI-CA 52 at pH 4.6 by 80%, 85% and 11%, respectively, in cell suspensions, whereas the reduction was 91%, 67% and 50%, respectively, in cell-free extracts. In spite of this adverse influence of the wine matrix, our results proved the potential of wine-associated LAB as a promising strategy to reduce biogenic amines in wine.     

Malolactic Fermentation and Secondary Metabolite Production by Oenoccocus oeni Strains in Low pH Wines

Abstract: Different wine varieties, including some with low pH, were studied to determine the ability to grow and produce secondary metabolites of a previously selected autochthonous Oenococcus oeni strain (C22L9), compared with a commercial strain. Monitoring of malolactic fermentation (MLF) was carried out by microbiological and chemical analysis of wines. The concentration of some major volatile compounds and biogenic amines in wines before and after malolactic fermentation was also determined. The results showed major differences in MLF duration both between wines and strains, although the differences between strains were slight for most of the analyzed compounds. Statistically significant differences in citric acid degradation were found in all wine varieties and it was confirmed that O. oeni C22L9 is a poor degrader of citric acid; this means that MLF can be prolonged without the risk of producing high concentrations of diacetyl and acetoin. Sensory analysis of wines after MLF showed similar characteristics in wines from both strains. This study thus shows that O. oeni C22L9 possesses even better sensory and fermentation properties than the commercial strain and can be used in wines with different characteristics, which makes it highly valuable for industrial use. Practical Application: The increasingly use of grape varieties of low pH in winemaking and the higher alcohol content of wines, as a consequence of the climatic change, make interesting the study of the behavior during MLF of O. oeni strains in order to determine their ability to grow, when growth conditions are not optimal, and to produce secondary metabolites. A comparative study was conducted using an autochthonous O. oeni strain (C22L9) and a commercial O. oeni strain and 4 wine varieties.     

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.     

Biogenic amines formation in Streptococcus thermophilus isolated from home-made natural yogurt

Twelve different biogenic amines formation in 58 isolates of Streptococcus thermophilus from home-made natural yogurt were investigated in histidine (HDB) and lysine decarboxylase broth (LDB). All S. thermophilus isolates had an ability to produce twelve different biogenic amines in HDB and LDB. Most of the S. thermophilus isolates formed low amounts of histamine (1–50 mg/L) from histidine. Apart from one isolate, S. thermophilus produced tyramine at low (47 isolates) and medium (10 isolates) levels. The amount of each specific biogenic amine produced by S. thermophilus was generally lower than 100 mg L⁻¹. Also, the presence of hdcA gene was investigated using PCR technique and relation between gene and histamine production was conducted in S. thermophilus isolates. This study showed that most of the S. thermophilus isolates have the ability to form biogenic amines, especially histamine, and tyramine, which is an important consideration when selecting strains as starter cultures.     

Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine

Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using gene-specific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress.     

酿酒工艺对葡萄酒中生物胺的影响

探讨了不同酿酒工艺对葡萄酒中生物胺(组胺、苯乙胺、酪胺、色胺、腐胺、尸胺、精胺和亚精胺)含量的影响。结果显示,葡萄酒酿造过程中生物胺主要在酒精发酵和苹果酸-乳酸发酵过程产生。酒精发酵过程生成量较少,主要产生腐胺和精胺,果胶酶的使用、酵母接种量、发酵温度等因素可以调节酒精发酵过程生物胺的生成量;乳酸菌是葡萄酒生物胺最主要来源,在苹果酸-乳酸发酵过程中会产生大量的组胺和色胺。     

Formation of biogenic amines throughout the industrial manufacture of red wine

Changes in biogenic amines (histamine, methylamine, ethylamine, tyramine, phenylethylamine, putrescine, and cadaverine) were monitored during the industrial manufacture of 55 batches of red wine. The origin of these amines in relation to must, alcoholic fermentation, malolactic fermentation, sulfur dioxide addition, and wine aging and the interactions between amines and their corresponding amino acids and pH were statistically evaluated in samples from the same batches throughout the elaboration process. Some amines can be produced in the grape or the musts (e.g., putrescine, cadaverine, and phenylethylamine) or can be formed by yeast during alcoholic fermentation (e.g., ethylamine and phenylethylamine), although quantitatively only very low concentrations are reached in these stages (less than 3 mg/liter). Malolactic fermentation was the main mechanism of biogenic amine formation, especially of histamine, tyramine, and putrescine. During this stage, the increase in these amines was accompanied by a significant decline in their amino acid precursors. Significant correlations between biogenic amine formation and the disappearance of their corresponding amino acids were observed, which clearly supports the hypothesis that malolactic bacteria are responsible for accumulation of these amines in wines. No increase in the concentration of biogenic amines was observed after SO2 addition and during wine aging, indicating that sulfur dioxide prevents amine formation in subsequent stages.     

Genetic diversity of Oenoccoccus oeni isolated from wines treated with phenolic extracts as antimicrobial agents

Molecular techniques have been applied to study the evolution of wine-associated lactic acid bacteria from red wines produced in the absence and presence of antimicrobial phenolic extracts, eucalyptus leaves and almond skins, and to genetically characterize representative Oenococcus oeni strains. Monitoring microbial populations by PCR-DGGE targeting the rpoB gene revealed that O. oeni was, as expected, the species responsible for malolactic fermentation (MLF). Representative strains from both extract-treated and not-treated wines were isolated and all were identified as O. oeni species, by 16S rRNA sequencing. Typing of isolated O. oeni strains based on the mutation of the rpoB gene suggested a more favorable adaptation of L strains (n = 63) than H strains (n = 3) to MLF. Moreover, PFGE analysis of the isolated O. oeni strains revealed 27 different genetic profiles, which indicates a rich biodiversity of indigenous O. oeni species in the winery. Finally, a higher number of genetic markers were shown in the genome of strains from control wines than strains from wines elaborated with phenolic extracts. These results provide a basis for further investigation of the molecular and evolutionary mechanisms leading to the prevalence of O. oeni in wines treated with polyphenols as inhibitor compounds.     

Improvement of volatile composition of wines by controlled addition of malolactic bacteria

The effect of malolactic fermentation (MLF) on the volatile composition of red wines was studied by inoculation with selected lactic acid bacteria. Four wines were inoculated with different Oenococcus oeni (syn. Leuconostoc oenos) strains, the major malolactic species found in wines, and one was inoculated with a Lactobacillus sp. strain. A non inoculated wine was also analyzed to act as a control. Malolactic fermentation and evolution of non volatile compounds were followed by HPLC and after the depletion of the malic acid present in wine the volatile compounds were extracted and analyzed by gas chromatography with flame ionization and mass spectrometry. Wines which had undergone the MLF showed a significant increment in total higher alcohols, esters and acids that are important in the sensory properties and quality of wine.     

Ester synthesis and hydrolysis in an aqueous environment,and strain specific changes during malolactic fermentation in wine with Oenococcus oeni

Previous work has shown that Oenococcus oeni produces esterases that are capable of hydrolysing artificial substrates. Using SPME–GCMS, this study provides evidence that purified O. oeni esterases have the ability to both synthesise and hydrolyse esters. Two purified esterases (EstA2 and EstB28) synthesised ethyl butanoate and ethyl hexanoate to varying degrees. Both purified esterases hydrolysed ethyl butanoate, ethyl hexanoate and ethyl octanoate. Once this dual activity was confirmed, malolactic fermentation (MLF) trials were conducted in wine with O. oeni strains that had been previously observed to have either high or low esterase activity against artificial substrates. Strain specific differences were observed and strains with low esterase hydrolysis activity against artificial substrates had a higher level of total esters measured after MLF. The results demonstrate the impact that O. oeni has on wine aroma and relates this to the ester hydrolysis and synthesis abilities of O. oeni strains.     

Biogenic amines and the importance of starter cultures for malolactic fermentation

Biogenic amines are compounds produced mainly by microorganisms from the decarboxylation of some amino acids which may cause several health issues in susceptible people. In the winemaking process, one of the factors with the greatest impact on the final concentration of biogenic amines in wine is the malolactic fermentation, which mainly involves the action of lactic acid bacteria. This article gathers scientific literature on the importance of the use of inoculation in winemaking, especially for the potential formation of biogenic amines in spontaneous fermentations, with emphasis on malolactic fermentation. It establishes the set of attributes that influence the formation of biogenic amines in winemaking and aspects related to their toxicity, especially histamine and tyramine. With the growing interest of the market in products with less chemical appeal, the safety of wines, whether from non‐conventional or traditional winemaking, must increasingly be observed. Therefore, the importance of using cultivated starter cultures is discussed, along with the safety of organic wines and the presentation of articles that seek to reconcile the production of organic wine with the preservation of the ‘microbiological terroir’ in a safe way, using selected wild yeasts and lactic acid bacteria.     

Production of biogenic amines by lactic acid bacteria: screening by PCR, thin-layer chromatography, and high-performance liquid chromatography of strains isolated from wine and must

Biogenic amines are frequently found in wine and other fermented food. We investigated the ability of 133 strains of lactic acid bacteria isolated from musts and wines of different origins to produce histamine, tyramine, and putrescine. We detected the genes responsible for encoding the corresponding amino acid decarboxylases through PCR assays using two primer sets for every gene: histidine decarboxylase (hdc), tyrosine decarboxylase (tdc), and ornithine decarboxylase (odc); these primers were taken from the literature or designed by us. Only one strain of Lactobacillus hilgardii was shown to possess the hdc gene, whereas four strains of Lactobacillus brevis had the tdc gene. None of the Oenococcus oeni strains, the main agents of malolactic fermentation, was a biogenic amine producer. All PCR amplicon band-positive results were confirmed by thin-layer chromatography and high-performance liquid chromatography analyses.     

Amino acids and biogenic amines in red varietal wines: the role of grape variety, malolactic fermentation and vintage

This work studies for the first time the amino acid and biogenic amine composition of Rioja red wines made with the red minority varieties Vitis vinífera cv. Monastel and Maturana Tinta de Navarrete, using Tempranillo as a reference variety. The role of malolactic fermentation and vintage on these compounds was also analysed, and discriminate analyses were applied to achieve a possible differentiation of the wines. Amino acid composition allowed a differentiation of wines according to grape variety. Monastel was characterised by the highest value in β-alanine and Maturana Tinta de Navarrete by its highest value in OH-proline. However, biogenic amines were no able to classify varietal wines. The malolactic fermentation had significant changes on the amino acid and biogenic amine content, and allowed distinguishing wines that underwent this process from wines without malolactic fermentation. No correlation was found between total amino acids and total biogenic amines after malolactic fermentation, suggesting that a higher initial concentration of amino acids in the medium did no affect the concentration of biogenic amines after malolactic fermentation. Vintage influenced the amino acid and biogenic amine pattern, obtaining a clear separation of wines by vintages. Monastel and Maturana Tinta de Navarrete wines showed a minor varietal character and were more influenced by the climatic conditions of each vintage than Tempranillo wines. All the wines showed histamine levels below the human physiological threshold and implemented regulations.     

龙眼干白葡萄酒发酵过程中生物胺和氨基酸含量的变化

本研究利用高效液相色谱技术分析了龙眼干白葡萄酒酒精发酵和苹乳发酵过程中8种生物胺和22种氨基酸含量的变化。结果表明,在酒精发酵过程中8种生物胺含量均很低,而在苹乳发酵过程中迅速增加。发酵结束时干白葡萄酒中亚精胺和乙醇胺含量最高,其次为组胺和羟色胺。这4种生物胺的前体氨基酸在苹乳发酵阶段含量没有明显变化,而其它大部分氨基酸在酒精发酵过程中含量下降,在苹乳发酵过程中含量呈现不同程度升高。因此,我们认为：葡萄酒发酵过程中氨基酸含量变化与其对应生物胺的变化之间没有相关性,葡萄酒中生物胺的组成比例可能主要取决于乳酸菌中氨基酸脱羧酶的种类和活性。     

新疆赤霞珠葡萄酒发酵过程中生物胺含量的变化

利用高效液相色谱技术分析葡萄酒发酵过程中8种生物胺含量的变化。结果表明,葡萄酒发酵过程中8种生物胺总含量整体呈上升趋势。色胺含量最高,最高可达到98.03 mg/L;腐胺和组胺含量较低,含量均不超过2.48 mg/L;精胺和亚精胺含量变化相对平稳,波动范围在1.81～3.07 mg/L之间,除色胺外,其他生物胺含量均不超过4.50 mg/L。苯乙胺、尸胺、酪胺等在酒精发酵后期和苹果酸-乳酸发酵初期大幅上升,在苹-乳发酵后期,酪胺和亚精胺含量有所下降,其他生物胺含量均有一定程度上升。     

Release of wine monoterpenes from natural precursors by glycosidases from Oenococcus oeni

It is now well established that wine-related lactic acid bacteria (LAB), especially Oenococcus oeni, possess glycosidase activities that positively contribute to wine aroma through the hydrolysis of grape-derived aroma precursors. In our recent studies, we have identified and characterised several LAB glycosidases with potential in these terms. Here, we report that both a glucosidase and an arabinosidase from O. oeni can release high amounts of monoterpenes from natural substrates under optimal conditions, indicating that these intracellular enzymes might play a significant role in the hydrolysis of aroma precursors during malolactic fermentation. The enzymes from O. oeni exhibited broad substrate specificities (release of both primary/tertiary terpene alcohols) and were even active in grape juice. Further, a sensory panel clearly preferred enzyme-treated Riesling wines over the controls and affirmed that the glycosidases from O. oeni could improve the typical Riesling aroma.     

Reduced biogenic amine contents in sauerkraut via addition of selected lactic acid bacteria

The contents of free amino acids and biogenic amines in spontaneously fermented sauerkraut, inoculated or not with specific lactic acid bacterium strains, were monitored throughout 45 d of storage. The strains tested were Lactobacillus plantarum 2142, Lactobacillus casei subsp. casei 2763 and Lactobacillus curvatus 2771. In both the control and the experiments, the total amino acid contents increased with time – and the predominant ones were aspartic acid, arginine and glutamic acid. However, upon inoculation with either strain, the total biogenic amine contents remained considerably lower than those of the control (especially in the cases of L. casei subsp. casei and L. curvatus); every single biogenic amine was actually below the 100 ppm-threshold. The dominant biogenic amines in the control were putrescine – and tyramine and histamine, to a lesser extent; the putrescine content was 10-fold lower if inoculation had taken place with either lactobacillus tested; and histamine and tyramine were essentially absent during storage, whereas they ranked above 200 ppm in the control by 45 d. Hence, an efficient food-grade biological tool was made available that constrains buildup of dangerous biogenic amines in fermented vegetables during storage.     

RNA fingerprinting analysis of Oenococcus oeni strains under wine conditions

Oenococcus oeni is a lactic acid bacterium of economic interest used in winemaking. This bacterium is the preferred species for malolactic fermentation (MLF) due its adaptability to the chemically harsh wine environment. MLF enhances the organoleptic properties and ensures deacidification of wines.     

Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineae

Discovery, characterisation and use of novel yeast strains for winemaking is increasingly regarded as a way for improving quality and to provide variation, including subtle characteristic differences in fine wines. The objective of this work was to evaluate the use of a native apiculate strain, selected from grapes, Hanseniaspora vineae (H. vineae) 02/5A. Fermentations were done in triplicate, working with 225 L oak barrels, using a Chardonnay grape must. Three yeast fermentation strategies were compared: conventional inoculation with a commercial Saccharomyces cerevisiae strain, ALG 804, sequential inoculation with H. vineae and then strain ALG 804 and spontaneous fermentation. Yeast strain identification was performed during fermentation, in which the apiculate strain was found to be active, until 9% of alcohol in volume, for the co-fermentation and the spontaneous fermentation was completed by three native S. cerevisiae strains. Basic winemaking parameters and some key chemical analysis, such as concentration of glycerol, biogenic amines, organic acids, and aroma compounds were analysed. Sensory analysis was done using a trained panel and further evaluated with professional winemakers. Sequential inoculation with H. vineae followed by S. cerevisiae resulted in relatively dry wines, with increased aroma and flavour diversity compared with wines resulting from inoculation with S. cerevisiae alone. Wines produced from sequential inoculations were considered, by a winemaker’s panel, to have an increased palate length and body. Characteristics of wines derived from sequential inoculation could be explained due to significant increases in glycerol and acetyl and ethyl ester flavour compounds and relative decreases in alcohols and fatty acids. Aroma sensory analysis of wine character and flavour, attributed to winemaking using H. vineae, indicated a significant increase in fruit intensity described as banana, pear, apple, citric fruits and guava. GC analysis of the relative accumulation of 23 compounds to significantly different concentrations for the three fermentation strategies is discussed in relation to aroma compound composition.     

Characterization and amino acid metabolism performances of indigenous Oenococcus oeni isolated from Chinese wines

Oenococcus oeni is a multiple physical stress-tolerant lactic acid bacterium that plays an important role in wine making. It is often added as a starter culture to carry out malolactic fermentation (MLF). In this study, a total of 22 out of 127 lactic acid bacteria, isolated from Chinese wines undergoing MLF, were identified as O. oeni by species-specific PCR and 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis showed that all strains could be typed under these conditions, and three main groups were determined by cluster analysis, which showed intraspecific homology higher than 69 %. Eight strains, representative of SE-AFLP clusters, were tested for malolactic activity. Significant differences were observed among strains with regard to the amount of malic acid consumed. Seventeen amino acids in different wines that were inoculated by 4 O. oeni strains, respectively, were analyzed before and after MLF. The results indicated that the amino acid metabolism of the 4 strains was significantly different between each strain.