Antonella Costantini

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.

Biogenic Amine Production by Contaminating Bacteria Found in Starter Preparations Used in Winemaking

The aim of this work was to investigate if contaminating microorganisms, eventually present in bacteria and yeast preparations used as commercial starters in winemaking, have the ability to produce the biogenic amines histamine, putrescine and tyramine. Thirty commercial starters (14 yeasts Saccharomyces cerevisiae and 16 bacteria Oenococcus oeni) were cultured in synthetic broth and analyzed by TLC to detect amine production. Oenococcus oeni commercial preparations did not contain contaminants, but some yeast preparations resulted contaminated with amine-producing bacteria. Bacterial contaminants were isolated and analyzed for their ability to produce biogenic amines using HPLC and TLC. Decarboxylase genes were identified using PCR analysis followed by sequencing. Fermentations were performed in grape juice with two yeast commercial preparations containing bacterial contaminants to check if the potential biogenic amine production could happen also during winemaking. It was found that this production is possible; in particular, in the conditions used in this work, tyramine production was detected. Therefore, the results of this study have significance in relation to the risk of biogenic amines in wine. Moreover a novel species of Lactobacillus was found to be able to produce histamine.

Application of real-time RT-PCR to study gene expression in active dry yeast (ADY) during the rehydration phase.

During the industrial production of active dry yeast (ADY) and its subsequent use in winemaking, the yeast cell is subjected to drastic environmental changes that force it to undergo extensive metabolic modifications and changes in gene expression. In this study, we describe the use of real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor gene expression in ADY Saccharomyces cerevisiae during rehydration in different media. We used three statistical approaches to investigate the expression stability of eight potential reference genes during the rehydration process. The reference system thus obtained was used to normalize the expression values of three genes codifying for the ammonium transporters -- MEP1, MEP2, and MEP3 -- and two genes involved in the osmotic response-SIP18 and GPD1. The results suggested that for the target genes tested, the yeast reacted immediately to rehydration only when a fermentable carbon source was present in the medium. Furthermore, MEP2 expression was modulated by the ammonium concentration, indicating that nitrogen catabolite repression (NCR) is active during the rehydration phase.

Biochemical Transformations Produced by Malolactic Fermentation

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Degradation of Ochratoxin A by Brevibacterium Species

The ability to degrade ochratoxin A was studied in different bacteria with a well-known capacity to transform aromatic compounds. Strains belonging to Rhodococcus, Pseudomonas, and Brevibacterium genera were grown in liquid synthetic culture medium containing ochratoxin A. Brevibacterium spp. strains showed 100% degradation of ochratoxin A. Ochratoxin α was detected and identified by high-performance liquid chromatography-mass spectrometry (HPLC-MS) as a degradation product in the cell-free supernatants. The degradation of ochratoxin A is of public concern for food and environmental safety, because it could contribute to the development of new biological ochratoxin A detoxification systems in foodstuffs. In this study, the degradation of ochratoxin A by bacteria belonging to the food chain was demonstrated for the first time.

Putrescine production from different amino acid precursors by lactic acid bacteria from wine and cider.

The aim of this work was to study the production of biogenic amines and particularly putrescine in lactic acid bacteria (LAB) related to wine and cider. We applied an analytical protocol that involves the use of PCR and TLC techniques to determine the production of putrescine from different precursors. Moreover, we also studied the ability of the Lactobacillus and Pediococcus tested to produce histamine and tyramine. The results showed that the majority of the Lactobacillus brevis analyzed harbour both AgDI and tdc genes and are tyramine and putrescine producers. Conversely, among the other LAB tested, only one Lactobacillus hilgardii and one Pediococcus pentosaceus produced putrescine. The AgDI gene was also detected in two other LAB (Lactobacillus mali and Pediococcus parvulus), but no putrescine production was observed. Finally, hdc gene and histamine production were found in strains (L. hilgardii 5211, isolated from wine, and Lactobacillus casei 18, isolated from cider) that were not putrescine producers.

Complementing DIGE proteomics and DNA subarray analyses to shed light on Oenococcus oeni adaptation to ethanol in wine-simulated conditions

UNLABELLED Direct addition of Oenococcus oeni starters into wine can cause viability problems. In the present study, the influence of ethanol in wine-simulated conditions on O. oeni has been evaluated by complementing microarray techniques and DIGE proteomics. Two different ethanol concentrations were studied. In 12% ethanol, pyrimidine anabolism was stimulated, but in 8% ethanol some energy-consuming biosynthetic pathways were limited. The most significant result was the stress response induced by alcohol that concerned both the cell-envelope and specific stress proteins. Interestingly, 8% and 12% ethanol triggered different stress responses: in mild ethanol stress (8%), chaperones with prevalent refolding activity (like HSP20) were over-expressed, whereas at higher alcohol concentration (12%), together with HSP20 and the refolding DNAJ/K, also chaperones having proteolytic activity (like ClpP) were induced. Furthermore the stress response repressor HrcA was downregulated only at 12% ethanol, suggesting that it controls stress pathways, which are different from those active at 8% alcohol. This result confirms that the HrcA system is operative in O. oeni where the CtrS system is prevalent. BIOLOGICAL SIGNIFICANCE The use of malolactic starter cultures has become widespread to control the MLF process and to prevent off-flavors. There is significant interest in understanding the molecular mechanisms that O. oeni uses to adapt to harsh wine conditions. The overall results highlight that the alcohol-induced stress response involves not only biosynthesis of stress proteins but also envelope-linked mechanisms. From a practical point of view this research underlines the importance of starters acclimation to induce responses that would allow better adaptation to the wine. As a consequence, a well adapted starter can complete malolactic fermentation and improve the final wine quality.

Development of a New Method for Detection and Identification of Oenococcus oeni Bacteriophages Based on Endolysin Gene Sequence and Randomly Amplified Polymorphic DNA

ABSTRACT Malolactic fermentation (MLF) is a biochemical transformation conducted by lactic acid bacteria (LAB) that occurs in wine at the end of alcoholic fermentation. Oenococcus oeni is the main species responsible for MLF in most wines. As in other fermented foods, where bacteriophages represent a potential risk for the fermentative process, O. oeni bacteriophages have been reported to be a possible cause of unsuccessful MLF in wine. Thus, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages would be advisable. However, currently, no methods have been described to identify phages infecting O. oeni. In this study, two factors are addressed: detection and typing of bacteriophages. First, a simple PCR method was devised targeting a conserved region of the endolysin (lys) gene to detect temperate O. oeni bacteriophages. For this purpose, 37 O. oeni strains isolated from Italian wines during different phases of the vinification process were analyzed by PCR for the presence of the lys gene, and 25 strains gave a band of the expected size (1,160 bp). This is the first method to be developed that allows identification of lysogenic O. oeni strains without the need for time-consuming phage bacterial-lysis induction methods. Moreover, a phylogenetic analysis was conducted to type bacteriophages. After the treatment of bacteria with UV light, lysis was obtained for 15 strains, and the 15 phage DNAs isolated were subjected to two randomly amplified polymorphic DNA (RAPD)-PCRs. By combining the RAPD profiles and lys sequences, 12 different O. oeni phages were clearly distinguished.

Quantitative expression analysis of mleP gene and two genes involved in the ABC transport system in Oenococcus oeni during rehydration

Oenococcus oeni is recognized as the principal microorganism responsible for malolactic fermentation, and the control of its activity is of primary importance in winemaking. The aim of this study was to quantify the levels of expression of the malate transporter gene (mleP) and of two genes putatively involved in the ATP-binding cassette transport system (oeoe_1651, oeoe_0550) to better understand the physiological response of bacteria during rehydration. These genes coding for transporters were studied in different rehydration media. Initially, three different statistical algorithms were used to identify suitable reference genes to be used for the normalization of expression data in O. oeni during rehydration, and to this purpose, the best genes found were ddl and gyrB. The results showed that the genes for transporters of malate and sugar (mleP, oeoe_1651) were activated immediately after a few minutes of rehydration, when specific medium compositions were used.

Identification of reference genes suitable for normalization of RT-qPCR expression data in Saccharomyces cerevisiae during alcoholic fermentation

Expression data from RT-qPCR (reverse transcription quantitative PCR) needs to be normalized to account for experimental variability among samples caused by differential yields of the transcripts in RNA extraction or in the reverse transcription. The most common method is to normalize against one or more reference genes (RG). We have selected RGs suitable for normalization of RT-qPCR raw data in Saccharomyces cerevisiae during alcoholic fermentation. The RGs were evaluated by three different statistical methods. The suitability of the selected RG sets was compared with ACT1, a commonly used non-validated single RG, by normalizing the expression of two target genes. Expression profiles of the target genes revealed the risk of misleading interpretation of expression data due to an unreliable RG.