Coproduction of colicin V and lactic acid bacteria bacteriocins in lactococci and enterococci strains of biotechnological interest.

AIMS: The aim of this study was the coproduction in a single strain of the Gram-negative bacteriocin colicin V with other bacteriocins from lactic acid bacteria (LAB). METHODS AND RESULTS: Colicin V was expressed in Lactococcus and Enterococcus strains by replacing the colicin V leader peptide by the leader peptide and promoter of d-alanyl-d-alanine carboxypeptidase from Lactobacillus reuteri CECT925 in pNZ8048 (pNZ:LR-colV). The antimicrobial activity of colicin V against the indicator organism Escherichia coli DH5α in transformed strains was checked by agar diffusion assay and SDS-PAGE analysis. CONCLUSIONS: Lactococcus and Enterococcus transformed with pNZ:LR-colV were able to coproduce colicin V at high levels together with other LAB bacteriocins such as nisin A, nisin Z, lacticin 481 or enterocins A and B, obtaining broad-spectrum activity strains with large potential applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The construction showed in this work could be used for the heterologous expression of other bacteriocins active against Gram-negative bacteria or wide-spectrum bacteriocins from LAB.

Bioactivation of Phytoestrogens: Intestinal Bacteria and Health.

Phytoestrogens are polyphenols similar to human estrogens found in plants or derived from plant precursors. Phytoestrogens are found in high concentration in soya, flaxseed and other seeds, fruits, vegetables, cereals, tea, chocolate, etc. They comprise several classes of chemical compounds (stilbenes, coumestans, isoflavones, ellagitannins, and lignans) which are structurally similar to endogenous estrogens but which can have both estrogenic and antiestrogenic effects. Although epidemiological and experimental evidence indicates that intake of phytoestrogens in foods may be protective against certain chronic diseases, discrepancies have been observed between in vivo and in vitro experiments. The microbial transformations have not been reported so far in stilbenes and coumestans. However, isoflavones, ellagitanins, and lignans are metabolized by intestinal bacteria to produce equol, urolithins, and enterolignans, respectively. Equol, urolithin, and enterolignans are more bioavailable, and have more estrogenic/antiestrogenic and antioxidant activity than their precursors. Moreover, equol, urolithins and enterolignans have anti-inflammatory effects and induce antiproliferative and apoptosis-inducing activities. The transformation of isoflavones, ellagitanins, and lignans by intestinal microbiota is essential to be protective against certain chronic diseases, as cancer, cardiovascular disease, osteoporosis, and menopausal symptoms. Bioavailability, bioactivity, and health effects of dietary phytoestrogens are strongly determined by the intestinal bacteria of each individual.

Short communication: Combined antimicrobial activity of reuterin and diacetyl against foodborne pathogens.

Reuterin (ß-hydroxypropionialdehyde) is a broad-spectrum antimicrobial substance produced by some strains of Lactobacillus reuteri during anaerobic fermentation of glycerol. Some of these strains are able to survive and produce reuterin in cheese and yogurt when added as adjuncts to the starter. Similarly, in fermented dairy foods, other inhibitory compounds such as lactic acid and diacetyl are produced during fermentation. In this work, we studied the combined effect of reuterin and diacetyl under different pH conditions against Escherichia coli O157:H7, Salmonella Enteritidis, and Listeria monocytogenes. Results from agar spot assays showed that the antimicrobial activity of reuterin-producing strains against the gram-negative bacteria tested was enhanced as the concentration of diacetyl increased to 50 mg/kg, and was higher under acidic conditions (pH 5.0) for the 3 pathogenic strains. The combination of reuterin and diacetyl had an additive effect against L. monocytogenes only at diacetyl concentrations of 50 mg/kg and pH 5.0. In addition, growth kinetics studies showed that the combination of 1 activity unit (AU)/mL of reuterin with 100mg/kg diacetyl increased the lag time of the 3 pathogens. In milk, synergistic antimicrobial activity was observed with the combination of 1 AU/mL reuterin and 50 or 100 mg/kg of diacetyl on the gram-negative strains tested, and with 1 AU/mL reuterin and 100 mg/kg of diacetyl on L. monocytogenes. The greatest inhibition of the 3 pathogens was achieved in acidified milk at pH 5.0 with reuterin (1 AU/mL) and diacetyl (100 mg/kg). Based on these results, the combination of reuterin and diacetyl in acidified dairy products could be a promising strategy to control food pathogens in these products.

Dietary intake of natural antioxidants: vitamins and polyphenols.

Oxidative stress is a condition in which oxidant metabolites exert their toxic effect because of an increased production or an altered cellular mechanism of protection; oxidative stress is rapidly gaining recognition as a key phenomenon in chronic diseases. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. Endogenous defence mechanisms are inadequate for the complete prevention of oxidative damage, and different sources of dietary antioxidants may be especially important. This article calls attention to the dietary antioxidants, such as vitamins A, C, and E and polyphenols. Compelling evidence has led to the conclusion that diet is a key environmental factor and a potential tool for the control of chronic diseases. More specifically, fruits and vegetables have been shown to exert a protective effect. The high content of minerals and natural antioxidant as vitamins A, C, and E and polyphenols in fruits and vegetables may be a main factor responsible for these effects.

Updated knowledge about polyphenols: functions, bioavailability, metabolism, and health.

Polyphenols are important constituents of food products of plant origin. Fruits, vegetables, and beverages are the main sources of phenolic compounds in the human diet. These compounds are directly related to sensory characteristics of foods such as flavor, astringency and color. Polyphenols are extensively metabolized both in tissues and by the colonic microbiota. Normally, the circulating polyphenols are glucuronidated and/or sulphated and no free aglycones are found in plasma. The presence of phenolic compounds in the diet is beneficial to health due to their antioxidant, anti-inflammatory, and vasodilating properties. The health effects of polyphenols depend on the amount consumed and their bioavailability. Moreover, polyphenols are able to kill or inhibit the growth of microorganisms such as bacteria, fungi, or protozoans. Some dietary polyphenols may have significant effects on the colonic flora providing a type of prebiotic effect. The anti-nutrient properties of polyphenols are also discussed in this paper. The antioxidant, anti-inflammatory, vasodilating, and prebiotic properties of polyphenols make them potential functional foods.

Response of Lactobacillus casei BL23 to phenolic compounds.

AIMS: To determine the inhibitory effect of phenolic compounds on Lactobacillus casei BL23, the role of two component signal transduction systems (TCS) and the response of Lact. casei BL23 to p-coumaric acid. METHODS AND RESULTS: Growth of Lact. casei BL23 and 17 derivative strains defective in each TCS harboured by this strain in the presence of p-coumaric acid, ferulic acid, caffeic acid or methyl gallate was monitored. Furthermore, changes in the protein content of Lact. casei BL23 when exposed to p-coumaric acid were evaluated by 2D-SDS-PAGE. Eleven proteins differentially expressed in the presence of p-coumaric acid were detected. Six of them could be identified: ClpP and HtrA, involved in protein turnover and folding, acetyl-CoA carboxylase, involved in lipid metabolism, and an arginyl-tRNA synthetase were more abundant, whereas PurL and PurN, involved in purine biosynthesis, were less abundant. CONCLUSIONS: No significant differences were observed between the parental strain and the TCS-defective mutants. p-Coumaric acid elicited a response against membrane and cytoplasmic damages. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitory effect of phenolic compounds on Lact. casei BL23 has been determined. For the first time, cytoplasmic proteins presumably involved in the response of Lact. casei BL23 against p-coumaric acid have been identified.

Regulation of hdc expression and HDC activity by enological factors in lactic acid bacteria.

AIMS: The aim of this work was to study the influence of enological factors on the histidine decarboxylase gene (hdc) expression and on histidine decarboxylase enzyme (HDC) activity in Lactobacillus hilgardii, Pediococcus parvulus and Oenococcus oeni. METHODS AND RESULTS: Cell extracts and whole cells were used. Glucose, fructose, malic acid and citric acid diminished the hdc expression. Ethanol did not increase hdc expression or activity in cells, but increased HDC activity. Temperature and pH had effect on the activity of HDC but not on hdc expression. Tartaric acid and l-lactic acid, and sulphur dioxide (SO(2)) had no effect on enzyme synthesis and activity. Bacterial species differ in the relative enzymatic activity but all the factors affected similarly to L. hilgardii, P. parvulus and O. oeni. CONCLUSIONS: The hdc gene expression was lowered by glucose, fructose, malic acid, and citric acid, whereas ethanol enhanced the HDC enzyme activity. The conditions that normally occur during malolactic fermentation and later on, could favour histamine production. SO(2) could prevent bacterial growth, but does not diminish the HDC enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on hdc expression and HDC activity can contribute to the prevention of histamine formation during wine production and storage.

Factors affecting the production of putrescine from agmatine by Lactobacillus hilgardii XB isolated from wine.

AIMS: To elucidate and characterize the metabolic putrescine synthesis pathway from agmatine by Lactobacillus hilgardii X(1)B. METHODS AND RESULTS: The putrescine formation from agmatine by resting cells (the normal physiological state in wine) of lactic acid bacteria isolated from wine has been determined for the first time. Agmatine deiminase and N-carbamoylputrescine hydrolase enzymes, determined by HPLC and LC-Ion Trap Mass Spectrometry, carried out the putrescine synthesis from agmatine. The influence of pH, temperature, organic acids, amino acids, sugars and ethanol on the putrescine formation in wine was determined. CONCLUSIONS: Resting cells of Lact. hilgardii X(1)B produce putrescine in wine. The putrescine production was carried out from agmatine through the agmatine deiminase system. SIGNIFICANCE AND IMPACT OF THE STUDY: These results have significance from two points of view, wine quality and toxicological and microbiological aspects, taking account that putrescine, which origin is still controversial, is quantitatively the main biogenic amine found in wine.

Comparative survey of putrescine production from agmatine deamination in different bacteria.

This article aims to study putrescine production in Lactobacillus hilgardii strain X(1)B, an agmatine degrader isolated from wine, and to compare it with three other different species, previously reported as putrescine producers from agmatine: Pseudomonas aeruginosa PAO1, Enterococcus faecalis ATCC11700 and Bacillus cereus CECT 148(T). The effect of different biogenic amines, organic acids, cofactors, amino acids and sugars on putrescine production was evaluated. In some cases, a similar effect was found in all the strains studied but the magnitude differed. Arginine, glucose and fructose showed an inhibitory effect, whereas the presence of agmatine induced the production of putrescine in all microorganisms. In other cases, the effect differed between P. aeruginosa PAO1 and the other microorganisms. Histamine and tyramine poorly influenced the utilization of agmatine, although a small increase in putrescine production was observed in P. aeruginosa PAO1. Succinate, spermidine and spermine also led to an increase in putrescine production in P. aeruginosa PAO1, whereas the succinate had no effect in the other microorganisms. Spermine and spermidine always produced a diminution in agmatine deamination. In this work, we have also demonstrated that pyridoxal 5-phosphate, Mg(2+) and Mn(2+) had no effect on putrescine production from agmatine. Results presented in this paper indicate differences in regulation mechanisms of agmatine deiminase pathway among P. aeruginosa PAO1 and L. hilgardii X(1)B, E. faecalis ATCC11700 and B. cereus CECT 148(T). These results are significant from two points of view, first food quality, and second the toxicological and microbiological aspects. It should be taken into account that putrescine, whose origin is still controversial, is quantitatively the main biogenic amine found in food.

The malate sensing two-component system MaeKR is a non-canonical class of sensory complex for C4-dicarboxylates.

Microbial colonization of different environments is enabled to a great extent by the plasticity of their sensory mechanisms, among them, the two-component signal transduction systems (TCS). Here, an example of TCS plasticity is presented: the regulation of L-malate catabolism via malic enzyme by MaeRK in Lactobacillales. MaeKR belongs to the citrate family of TCS as the Escherichia coli DcuSR system. We show that the Lactobacillus casei histidine-kinase MaeK is defective in autophosphorylation activity as it lacks a functional catalytic and ATP binding domain. The cognate response regulator MaeR was poorly phosphorylated at its phosphoacceptor Asp in vitro. This phosphorylation, however, enhanced MaeR binding in vitro to its target sites and it was required for induction of regulated genes in vivo. Elucidation of the MaeR structure revealed that response regulator dimerization is accomplished by the swapping of α4-ß5-α5 elements between two monomers, generating a phosphoacceptor competent conformation. Sequence and phylogenetic analyses showed that the MaeKR peculiarities are not exclusive to L. casei as they are shared by the rest of orthologous systems of Lactobacillales. Our results reveal MaeKR as a non-canonical TCS displaying distinctive features: a swapped response regulator and a sensor histidine kinase lacking ATP-dependent kinase activity.

Effector Molecules and Regulatory Proteins: Applications.

Bacteria respond to their external environment by modulating gene expression in the presence of certain effector molecules. The adaptive responses are mediated by transcriptional regulators that, after binding to the DNA, recognize these effector molecules and modify transcription. Some applications of regulatory proteins are reviewed here.

Distribution of CGRP-like immunoreactivity in the chick and quail brain.

Calcitonin gene-related peptide (CGRP)-containing neurones have been implicated in the transmission of visceral sensory information to the cortex and in the control of arterial blood pressure in mammals. However, little is known about its function in other vertebrates. As a first step toward investigating the function of CGRP in birds, its distribution was studied in the domestic chick and quail brain by means of immunocytochemistry, by using antibodies against rat CGRP. The distribution of CGRP immunoreactivity in the chick and quail central nervous system was found to be similar. CGRP-immunoreactive (CGRPi) perikarya were not present in the telencephalon. In the diencephalon, CGRPi perikarya were present mainly in the shell of the thalamic nucleus ovoidalis, the nucleus semilunaris paraovoidalis, the nucleus dorsolateralis posterior thalami, and in the hypothalamic nucleus of the ansa lenticularis. In the brainstem, CGRPi perikarya were present in the nucleus mesencephalicus nervi trigemini, the nucleus tegmenti ventralis, the locus coeruleus, the nucleus linearis caudalis and in the parabrachial region. In addition CGRPi perikarya were found in the motor nuclei of the III, IV, V, VI, VII, IX, X, and XII cranial nerves. The telencephalon contained CGRPi fibres within the paleostriatal complex (mainly in the ventral paleostriatum), parts of the neostriatum and ventral hyperstriatum, parts of the archistriatum, and the septum. In the diencephalon, the densest plexus of CGRPi fibres was observed in the dorsal reticular thalamus. A less dense CGRPi innervation was present in some dorsal thalamic nuclei and in the medial and periventricular hypothalamus. The pretectum and midbrain tegmentum also contained CGRPi fibres, whereas the optic tectum was virtually devoid of immunolabelling. Scattered CGRPi fibres were observed in the central grey and neighbouring pontine areas. Some of the sensory fibres of the trigeminal, vagal, glossopharyngeal, and spinal nerves were also CGRPi. The results of comparative studies indicate that the presence of CGRP in some thalamo-telencephalic projections is a primitive feature of the forebrain of amniotes. Therefore, the brain areas giving rise to and receiving such a projection in different vertebrates, are likely to be homologous.

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.

Improved enzymatic method for the rapid determination of histamine in wine.

Fermented foods are frequently contaminated by histamine generated by microorganisms possessing histidine decarboxylase activity. The ingestion of large amounts of histamine can cause serious toxicological problems in man. Thus, it becomes important to set a reliable method for rapid histamine quantification in foods. The detection of bacteria exhibiting histidine decarboxylase activity is also important to estimate the risk of contamination of food. Previous enzymatic methods used to quantify histamine in fish gave erroneously high values due to interference when applied to wine. A new enzymatic method is described that allows the direct determination of histamine concentrations in this type of sample. It can be used for the detection of histamine in synthetic media, grape must or wine (white, rose, red) without polyphenols or sugar interferences. This new enzymatic method shows a good correlation (R2 = 0.996, p < 0.001) between the histamine concentrations and absorbances in the interval 0.4-160 mg l(-1). Comparison between this enzymatic method and a high-performance liquid chromatography method showed a high correlation (R2 =0.9987, p<0.001). A miniaturized enzymatic method is also proposed, which is particularly useful when high numbers of samples must be analysed.