Malolactic fermentation of wine: study of the influence of some physico-chemical factors by experimental design assays

The ability of selected lactic acid bacteria to carry out malolactic fermentation depends on the level of numerous wine characteristics. A Hadamard's experimental matrix was used to determine the main effects of 11 physico-chemical factors on malolactic activity of three Leuconostoc œnos strains and one Lactobacillus plantarum strain. Ethanol had the greatest inhibitory effect on the achievement of malolactic fermentation for all Leuc. œnos strains. An inhibitory effect of the L-malic acid was also found in the operating conditions. These strains show different degrees of sensitivity to pH. One of these strains was inhibited by SO₂. Malolactic activity of the Lact. plantarum strain is mainly affected by a low pH, and this strain is often less efficient than Leuc. œnos strains. This methodology could be used for the selection of strains for malolactic starters. Further work is in progress using factorial design in order to determine the interactions between influential factors.     

Response of Leuconostoc œnos to environmental changes

Malolactic fermentation, a spontaneous step in vinification, is frequently controlled by use of malolactic bacterial starters of Leuconostoc œnos. Strains isolated from fermenting wines and then submitted to industrial processes to obtain freeze-dried preparations lose their natural adaptation to survive and to grow in wine. However, culture conditions influence their adaptability as well as their survival after exposure to stress. As the plasma membrane was considered as the primary target for the deleterious effects of the environment, the changes of its components after cell heating and incubation in hostile conditions were studied. The ratio of phospholipids to proteins of the membrane decreased significantly when cells were submitted to any kind of stress. Phospholipids decreased while the protein content increased up to fivefold. Only some membrane proteins were over-synthesized compared to control cells. Seventeen protein bands were separated by gel electrophoresis of which three in particular were always over-expressed after the cells were shocked. Some also appeared when the cells entered the stationary phase. These results show that Leuc. œnos responded to environmental stresses by changing its membrane composition. Stress proteins appeared whatever the nature of the shock. This phenomenon may be involved in the acquisition of resistance of Leuc. œnos to harsh conditions in wine.     

Histidine carboxylase of Leuconostoc œnos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene

Histidine decarboxylase (HDC) was purified to homogeneity from Leuconostoc ' nos 9204, a wine lactic acid bacterium. Histidine decarboxylase comprised two subunits, respectively α and β. The hdc gene was cloned and sequenced. The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc. ' nos 9204 HDC was synthesized as a precursor proHDC π₆ (Mr 205 000). A cleavage between Ser-81 and Ser-82 generated the α (Mr 25 380) and β (Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (αβ)₆. At the optimal pH of 4·8, the HDC activity exhibited a simple Michaelis-Menten kinetic ( K _m = 0·33 mmol l⁻¹, V _max = 17·8 μmol CO₂ min⁻¹ mg⁻¹), while at pH 7·6 it was sigmoidal (cooperativity index of 2). Histamine acted as a competitive inhibitor ( K _i = 32 mmol l⁻¹). The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms. These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine. Bacteria carrying the HDC activity must be avoided during selection of strains for the production of malolactic starters.     

Degradation of biogenic amines by vineyard ecosystem fungi. Potential use in winemaking

Aims: To evaluate the ability of grapevine ecosystem fungi to degrade histamine, tyramine and putrescine in synthetic medium and in wines. Methods and Results: Grapevine and vineyard soil fungi were isolated from four locations of Spain and were subsequently identified by PCR. A total of 44 fungi were evaluated for in vitro amine degradation in a microfermentation system. Amine degradation by fungi was assayed by reversed‐phase (RP)‐HPLC. All fungi were able to degrade at least two different primary amines. Species of Pencillium citrinum, Alternaria sp., Phoma sp., Ulocladium chartarum and Epicoccum nigrum were found to exhibit the highest capacity for amine degradation. In a second experiment, cell‐free supernatants of P. citrinum CIAL‐274,760 (CECT 20782) grown in yeast carbon base with histamine, tyramine or putrescine, were tested for their ability to degrade amines in three different wines (red, white and synthetic). The highest levels of biogenic amine degradation were obtained with histamine‐induced enzymatic extract. Conclusion: The study highlighted the ability of grapevine ecosystem fungi to degrade biogenic amines and their potential application for biogenic amines removal in wine. Significance and Impact of Study: The fungi extracts described in this study may be useful in winemaking to reduce the biogenic amines content of wines, thereby preventing the possible adverse effects on health in sensitive individuals and the trade and export of wine.     

Influence of ethanol,malate and arginine on histamine production of <Emphasis Type="Italic">Lactobacillus hilgardii</Emphasis> isolated from an Italian red wine

Wine, like other fermented foods, may contain biogenic amines produced by lactic acid bacteria via amino acids decarboxylation. The most relevant amines from the toxicological standpoint are histamine and tyramine. The complexity of fermented substrates makes it difficult to suggest a priori how variables can modulate amine production. Lactobacillus hilgardii ISE 5211 was isolated from an Italian red wine. Besides producing lactate from malate, this strain is also able to convert arginine to ornithine and histidine to histamine. In the present investigation we studied the influence of malate, arginine and ethanol on histamine accumulation by L. hilgardii ISE 5211. Ethanol concentrations above 13% inhibit both histamine accumulation and bacterial growth; concentrations below 9% affect neither growth nor histamine production. However, an ethanol concentration of 11% allows a low but continuous accumulation of histamine to occur. Arginine also delays histamine accumulation, while malate appears to have no effect on histidine-histamine conversion.     

Expression of Lactobacillus brevis IOEB 9809 tyrosine decarboxylase and agmatine deiminase genes in wine correlates with substrate availability

Aims: Lactobacillus brevis IOEB 9809 is able to produce both tyramine and putrescine via tyrosine decarboxylase and agmatine deiminase enzymes, respectively, when cultured on synthetic media. The aims of this study were to assess the expression of L. brevis IOEB 9809 tdc and aguA1 genes, during wine fermentation and to evaluate the effect of substrate availability and pH on tdc and aguA1 expression, as well as on biogenic amine production and L. brevis viability. Methods and Results: The relative expression of L. brevis IOEB 9809 tdc and aguA1 genes was analysed in wine by quantitative real‐time RT‐PCR (qRT‐PCR) during a period of incubation of 30 days. Cell viability, pH values, putrescine and tyramine concentration were monitored throughout the experiments. Conclusions: The wine trials indicated that L. brevis IOEB 9809 is able to produce both tyramine and putrescine during wine fermentation. Increased cell viability was also observed in wine supplemented with tyrosine or agmatine. qRT‐PCR analysis suggests a strong influence of substrate availability on the expression of genes coding for tyrosine decarboxylase and agmatine deiminase in L. brevis IOEB 9809. Less evident is the relationship between putrescine and tyramine production and tolerance to wine pH. Significance and Impact of Study: To our knowledge, this study represents the first assessment of relative expression of L. brevis IOEB 9809 genes involved in biogenic amine production in wine. Furthermore, an effect of biogenic amine production on viability of L. brevis during wine fermentation was established.     

Formation of histamine and tyramine by lactic acid bacteria in decarboxylase assay medium

A modified decarboxylase assay medium (DCA medium) was used for studying the production of biogenic amines by Leuconostoc oenos DSM 20252 and two strains of Lactobacillus buchneri (Lb14 and St2A). The DCA medium contained histidine, lysine, ornithine and tyrosine as precursors of the respective biogenic amines. Under the experimental conditions both strains of Lact. buchneri produced > 90% of the maximum amount of histamine within 24 h. Only tyramine was produced by Leuc. oenos DSM 20252. accountine for 88% of the maximum theoretical amount within 24 h.     

Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test

On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes ( hdc A) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus , oligonucleotides unique to the hdc A genes were synthesized and used in PCR. All histidine-decarboxylating lactic acid bacteria gave a signal with primer set JV16HC/JV17HC in PCR. In addition to this primer set, CL1/CL2 and CL1/JV17HC were also useful for the detection of histamine-forming Leuconostoc œnos strains in PCR. The 150 base pair amplification product of the decarboxylating Leuc. œnos strain generated with primer set CL1/CL2 was sequenced. Alignment studies showed a high degree of relatedness among the hdc A gene products of Gram-positive bacteria.
The amplification products of the hdc A genes from Lact. buchneri and Leuc. œnos were used to serve as a DNA probe in hybridization studies. All histidine-decarboxylating lactic acid bacteria gave a hybridization signal with the DNA probes. In hybridization only one false-positive signal with a Lactobacillus lindneri strain was observed, which was anticipated to contain a truncated hdc A gene.
In addition to these DNA probe tests, a simple and reliable activity test is presented, which can be used during starter selection to test strains for histidine decarboxylase activity.     

Identification of a novel enzymatic activity from lactic acid bacteria able to degrade biogenic amines in wine

The main objectives of this study were the search for enzymatic activities responsible for biogenic amine (BA) degradation in lactic acid bacteria (LAB) strains isolated from wine, their identification, and the evaluation of their applicability for reducing BAs in wine. Fifty-three percent of the 76 LAB cell extracts showed activity against a mixture of histamine, tyramine, and putrescine when analyzed in-gel. The quantification of the degrading ability for each individual amine was tested in a synthetic medium and wine. Most of the bacteria analyzed were able to degrade the three amines in both conditions. The highest percentages of degradation in wine were those of putrescine: up to 41 % diminution in 1 week. Enzymes responsible for amine degradation were isolated and purified from Lactobacillus plantarum J16 and Pediococcus acidilactici CECT 5930 strains and were identified as multicopper oxidases. This is the first report of an efficient BA reduction in wine by LAB. Furthermore, the identity of the enzymes involved has been revealed.     

Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods

Aims: To develop rapid methods allowing enumeration of lactic acid bacteria producing biogenic amines in wines and to analyse wine samples by the methods. Methods and Results: Methods based on quantitative PCR targeting bacterial genes involved in histamine, tyramine and putrescine production were developed and applied to detect and quantify the bacteria producing these biogenic amines in wine. Analysis of 102 samples revealed low populations of the targeted bacteria in grape must samples, an increased bacteria biomass in wine samples after alcoholic fermentation, reaching the highest population levels (above 10⁶ cells ml^?1) during spontaneous malolactic fermentation. A minimum of 10³ ml^?1 producing cells was required for production of more than 1 mg l^?1 of biogenic amines. Accumulation of putrescine in wine was correlated with the presence of bacteria carrying an ornithine decarboxylation pathway. Trials of winemaking showed that the use of selected bacteria for inducing malolactic fermentation was efficient to limit the proliferation of undesirable bacteria and the production of biogenic amines. Conclusion: Methods using quantitative PCR are efficient to enumerate biogenic amines‐producing cells in wine. Significance and Impact of the Study: The methods can help to better control and to improve winemaking conditions in order to avoid biogenic amine production.     

Biogenic amine production by lactic acid bacteria isolated from cider

AIMS: To study the occurrence of histidine, tyrosine and ornithine decarboxylase activity in lactic acid bacteria (LAB) isolated from natural ciders and to examine their potential to produce detrimental levels of biogenic amines. METHODS AND RESULTS: The presence of biogenic amines in a decarboxylase synthetic broth and in cider was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Among the 54 LAB strains tested, six (five lactobacilli and one oenococci) were biogenic amine producers in both media. Histamine and tyramine were the amines formed by the LAB strains investigated. Lactobacillus diolivorans were the most intensive histamine producers. This species together with Lactobacillus collinoides and Oenococcus oeni also seemed to produce tyramine. No ability to form histamine, tyramine or putrescine by Pediococus parvulus was observed, although it is a known biogenic amine producer in wines and beers. CONCLUSIONS: This study demonstrated that LAB microbiota growing in ciders had the ability to produce biogenic amines, particularly histamine and tyramine, and suggests that this capability might be strain-dependent rather than being related to a particular bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of biogenic amines by food micro-organisms has continued to be the focus of intensive study because of their potential toxicity. The main goal was to identify the microbial species capable of producing these compounds in order to control their presence and metabolic activity in foods.     

Tyrosine-containing peptides are precursors of tyramine produced by lactobacillus plantarum strain IR BL0076 isolated from wine

ABSTRACT: BACKGROUND: Biogenic amines are molecules with allergenic properties. They are found in fermented products and are synthesized by lactic acid bacteria through the decarboxylation of amino acids present in the food matrix. The concentration of biogenic amines in fermented foodstuffs is influenced by many environmental factors, and in particular, biogenic amine accumulation depends on the quantity of available precursors. Enological practices which lead to an enrichment in nitrogen compounds therefore favor biogenic amine production in wine. Free amino acids are the only known precursors for the synthesis of biogenic amines, and no direct link has previously been demonstrated between the use of peptides by lactic acid bacteria and biogenic amine synthesis. RESULTS: Here we demonstrate for the first time that a Lactobacillus plantarum strain isolated from a red wine can produce the biogenic amine tyramine from peptides containing tyrosine. In our conditions, most of the tyramine was produced during the late exponential growth phase, coinciding with the expression of the tyrDC and tyrP genes. The DNA sequences of tyrDC and tyrP in this strain share 98% identity with those in Lactobacillus brevis consistent with horizontal gene transfer from L. brevis to L. plantarum. CONCLUSION: Peptides amino acids are precursors of biogenic amines for Lactobacillus plantarum strain IR BL0076.     

Formation of biogenic amines as criteria for the selection of wine yeasts

This work deals with biogenic amine production by yeast strains isolated from grapes and wines. A total of 50 strains were tested for their capacity to produce biogenic amines in wine. In general, all the species produced very low or non-detectable amounts of histamine, whereas methylamine and agmatine were formed by all the species considered. The highest concentration of total biogenic amines was formed by Brettanomyces bruxellensis, with an average value of 15 mg/l, followed by Saccharomyces cerevisiae with an average of 12.14 mg/l. The other species formed less than 10 mg of total biogenic amines per litre. Wines fermented with the most fermentative strains of S. cerevisiae species had the highest contents of ethanolamine, from 2.3 to 16 mg/l, and of agmatine, from 3.1 to 7.5 mg/l. The strains of the other species, which exhibited a low fermentative ability, Kloeckera apiculata, B. bruxellensis and Metschnikowia pulcherrima, varied in the production of agmatine and phenylethylamine. A significant variability in the production of cadaverine was characteristic of Candida stellata strains, which varied also in ethanolamine production. Our results emphasize the importance of using selected strains of S. cerevisiae, not only for the expression of desirable technological traits, but also to avoid potentially negative effects on human health. Therefore, the characterization of strains of S. cerevisiae for the 'production of biogenic amines' becomes of applicative interest.     

Evolution of microbial populations and biogenic amine production in dry sausages produced in Southern Italy

AIMS: To evaluate the occurrence and evolution of biogenic amines during ripening of fermented sausages and their relationship with physico-chemical and microbiological properties of the product. METHODS AND RESULTS: Salsiccia and Soppressata were obtained from artisanal and industrial plants in Basilicata and pH, aW, microbial counts and biogenic amine content were measured. A high variability in amine content was observed. 2-Phenylethylamine and histamine were rarely found, while the tyramine, putrescine and cadaverine content increased during ripening. No correlation was found between individual biogenic amine content, microbial counts or physico-chemical parameters. CONCLUSION: Starter cultures did not necessarily prevent the production of biogenic amines whose total contents were usually higher in Soppressata, a product with a larger diameter and aW compared with Salsiccia. SIGNIFICANCE AND IMPACT OF THE STUDY: Literature findings on biogenic amine content and the evolution of microbial populations were confirmed. Normal ranges for amine content in Salsiccia and Soppressata are reported.     

Study of histamine effects on phagocytosis and enzyme secretion of Tetrahymena pyriformis

1. The biogenic amine histamine develops effects not only in mammalian cells and tissues but in ciliated unicellular Tetrahymena as well. In addition to binding and internalization of labelled histamine, low concentrations can stimulate the phagocytosis of cells in inorganic salt solution. 2. In inorganic solution Tetrahymena cells secrete acid hydrolases to the medium. High concentration of histamine (10 mM) decreases the secretion of three investigated acid hydrolases in a different manner. We think that in this process the primary determinant is the alkaline character of histamine. 3. The effect of histamine on phagocytosis differs from the effect on secretion since the low, physiological concentration of histamine stimulates phagocytosis, the higher concentrations inhibit it. In the background of these effects possibly the hormone character is dominant. It is supported by the fact that histamine antagonists influence the process differently.     

Physiological and oenological traits of different Dekkera/Brettanomyces bruxellensis strains under wine-model conditions

Contamination of wine by Dekkera/Brettanomyces bruxellensis is mostly due to the production of off-flavours identified as vinyl- and especially ethyl-phenols, but these yeasts can also produce several other spoiling metabolites, such as acetic acid and biogenic amines. Little information is available about the correlation between growth, viability and off-flavour and biogenic amine production. In the present work, five strains of Dekkera bruxellensis isolated from wine were analysed over 3 months in wine-like environment for growth, cell survival, carbon source utilization and production of volatile phenols and biogenic amines. Our data indicate that the wine spoilage potential of D. bruxellensis is strain dependent, being strictly associated with the ability to grow under oenological conditions. 4-Ethyl-phenol and 4-ethyl-guaiacol production ranged between 0 and 2.7 and 2 mg L(-1), respectively, depending on the growth conditions. Putrescine, cadaverine and spermidine were the biogenic amines found.     

Biogenic amine production by wild lactococcal and leuconostoc strains

Two qualitative and one quantitative HPLC methods were evaluated for the detection of biogenic amine producers among wild dairy lactococcal and leuconostoc strains. High tyramine producers ranging from 370 to 807 mg l⁻¹ were detected by qualitative methods and confirmed by HPLC analysis. Tyramine levels detected throughout the incubation time depended on the concentration of the amino acid precursor available and no tyramine production was observed when strains were grown in milk. However, increasing amounts of tyramine were detected in cultures grown in milk supplemented with different concentrations of tyrosine. Qualitative methods failed to detect weak producers so that tryptamine production (<7 mg l⁻¹) could only be determined by HPLC. None of the tested strains was able to produce histamine. Simultaneous production of different amines was observed by HPLC although no colour change was observed in the specific decarboxylase media. Thus, it was concluded that the amine forming ability should be taken into account when selecting starters for milk fermentations. Qualitative methods could be used as a first screening step to eliminate the highest amine producers while the quantitative methods would detect any producing strain.     

Multiplication and Fermentation of Saccharomyces cerevisiae under Carbon Dioxide Pressure in Wine

Conditions for rapid fermentation of sugar in wine under pressure were sought for use in continuous production of naturally fermented sparkling wine. Wine yeast growth and fermentation were measured under CO(2) pressure. The medium was white wine with added glucose. Pressure was very inhibitory to growth, especially at low pH or high alcohol concentration. Use of various strains of wine yeast, cultures of various ages, or cells adapted to wine did not give more rapid growth. Addition of nutrients increased growth, but under no conditions was growth rapid enough to bring about sufficiently rapid fermentation rates. Conditions for rapid fermentation were sought by use of high levels of cells as inocula. Fermentation rates in wine also were inhibited by pressure, and were dependent on pH and alcohol levels. Addition of nutrients did not increase the fermentation rate, but rapid fermentation rates were obtained, under pressure, by inoculation with high levels of cells adapted several weeks to the base wine. Thus, continuous sparkling-wine production might be practical with proper amounts of adapted cells used as inocula, or perhaps with reuse of the fermentation culture.     

Factors affecting tyramine production in <Emphasis Type="Italic">Enterococcus durans</Emphasis> IPLA 655

The decarboxylation of tyrosine by certain lactic acid bacteria leads to the undesirable presence of tyramine in fermented foods. Tyramine is the most frequent biogenic amine found in cheese and is also commonly found in other fermented foods and beverages. The tyramine-producing strain Enterococcus durans IPLA 655 was grown in a bioreactor under different conditions to determine the influence of carbon source, tyrosine and tyramine concentrations, and pH on tyramine production. The carbon source appeared to have no significant effect on the production of tyramine. In contrast, tyrosine was necessary for tyramine production, while the presence of tyramine itself in the growth medium inhibited such production. pH showed by far the greatest influence on tyramine synthesis; tyramine was produced in the greatest quantities at pH 5.0, although this was accompanied by a reduced growth rate.     

The inverse relationship between the activity of pyridoxal kinase and the level of biogenic amines in rabbit brain

In a series of experiments, an inverse relationship was found to exist between the activity of pyridoxal kinase and the concentrations of brain norepinephrine, dopamine, and serotonin. When the biogenic amine content of the brain is lowered, the activity of pyridoxal kinase rises. Conversely, when the concentration of brain biogenic amines is elevated, a depression occurs in the activity of pyridoxal phosphokinase. This relationship, which resembles an in vivo allosteric inhibition phenomenon, not only is highly suggestive of a definite relation between the brain vitamin B₆ content and the metabolism of the biogenic amines, but also suggests that the concentration of brain biogenic amines might control the production of coenzyme pyridoxal phosphate.