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Although at present depression is one of the most disabling disorders in our social environment, the understanding of its pathogenesis and the resources for its treatment are still unsatisfactory. The importance of brain asymmetry in the pathogenesis of disorders in brain function, including mood disorders such as depression, is a highly unexplored, sometimes underrated, and even ignored topic. It is important to note that the basal and pathological functional lateralization must have an underlying neurochemical substrate. It is also necessary to indicate that the brain asymmetry extends to a neurovisceral integration whose behavior may also be lateralized. One of the most studied axis from the functional point of view is the brain-heart connection, in whose operation there are observations that suggest an asymmetric behavior in basal conditions that is modified by central and peripheral changes, as well as by pharmacological treatments. There are evidences that connect cardiovascular function, neurochemical asymmetries, and depression. A deep understanding of the bilateral behavior of the brain following pathophysiological changes in blood pressure as well as pharmacologically induced, can provide us with therapeutic suggestions for the treatment of depression. In this article, we analyze remarkable results of some representative selected contributions, with which we discuss our proposal on the relationship between hypertension, depression and neurochemical asymmetry.

This research was supported by the Ministry of Science and Innovation through project no. SAF 2008 04685 C02 01., In the present study, we analyzed the activity of several aminopeptidases (angiotensinases) involved in the metabolism of various angiotensin peptides, in pituitary and adrenal glands of untreated Wistar–Kyoto (WKY) and spontaneously hypertensive rats (SHR) or treated with the antihypertensive drugs captopril and propranolol or with the L-Arginine hypertensive analogue L-NG-Nitroarginine Methyl Ester (L-NAME). Intra- and inter-gland correlations between angiotensinase activities were also calculated. Membrane-bound alanyl-, cystinyl-, and glutamylaminopeptidase activities were determined fluorometrically using aminoacyl- -naphthylamide as substrates. Depending on the type of angiotensinase analyzed, the results reflect a complex picture showing substantial differences between glands, strains, and treatments. Alanyl-aminopeptidase responsible for the metabolism of Ang III to Ang IV appears to be the most active angiotensinase in both pituitary and adrenals of WKY and particularly in SHR. Independently of treatment, most positive correlations are observed in the pituitary gland of WKY whereas such positive correlations are predominant in adrenals of SHR. Negative inter-gland correlations were observed in control SHR and L-NAME treated WKY. Positive inter-gland correlations were observed in captopril-treated SHR and propranolol-treated WKY. These results may reflect additional mechanisms for increasing or decreasing systolic blood pressure in WKY or SHR., Spanish Government SAF 2008 04685 C02 01

A lateralized distribution of neuropeptidase activities in the frontal cortex of normotensive and hypertensive rats has been described depending on the use of some vasoactive drugs and linked to certain mood disorders. Asymmetrical neuroperipheral connections involving neuropeptidases from the left or right hemisphere and aminopeptidases from the heart or plasma have been suggested to play a role in this asymmetry. We hypothesize that such asymmetries could be extended to the connection between the brain and physiologic parameters and metabolic factors from plasma and urine. To assess this hypothesis, we analyzed the possible correlation between neuropeptidases from the left and right frontal cortex with peripheral parameters in normotensive (Wistar Kyoto [WKY]) rats and hypertensive rats (spontaneously hypertensive rats [SHR]) untreated or treated with vasoactive drugs such as captopril, propranolol and L-nitro-arginine methyl ester. Neuropeptidase activities from the frontal cortex were analyzed fluorometrically using arylamide derivatives as substrates. Physiological parameters and metabolic factors from plasma and urine were determined using routine laboratory techniques. Vasoactive drug treatments differentially modified the asymmetrical neuroperipheral pattern by changing the predominance of the correlations between peripheral parameters and central neuropeptidase activities of the left and right frontal cortex. The response pattern also differed between SHR and WKY rats. These results support an asymmetric integrative function of the organism and suggest the possibility of a different neurometabolic response coupled to particular mood disorders, depending on the selected vasoactive drug., This work was supported by the Ministry of Science and Innovation through project no. SAF 2008 04685 C02 01.

The brain renin–angiotensin system (RAS) has been recently involved in the homeostatic regulation of energy. Our goal was to analyse the influence of a diet rich in saturated fatty acids (butter) against one enriched in monounsaturated fatty acids (olive oil) on hypothalamic RAS, and their relationship with the metabolism of fatty acids. Increases in body weight and visceral fat, together with an increase in aminopeptidase A expression and reductions in AngII and AngIV were observed in the hypothalamus of animals fed with the butter diet. In this group, a marked reduction in the expression of genes related to lipid metabolism (LPL, CD36, and CPT-1) was observed in liver and muscle. No changes were found in terms of body weight, total visceral fat and the expression of hepatic genes related to fatty acid metabolism in the olive oil diet. The expressions of LPL and CD36 were reduced in the muscles, although the decrease was lower than in the butter diet. At the same time, the fasting levels of leptin were reduced, no changes were observed in the hypothalamic expression of aminopeptidase A and decreases were noted in the levels of AngII, AngIV and AngIII. These results support that the type of dietary fat is able to modify the hypothalamic profile of RAS and the body energy balance, related to changes in lipid metabolism., University of Jaen PP2009/13/03

Insulin-regulated aminopeptidase (IRAP, cystinyl aminopeptidase, CysAP) and aminopeptidase M (alanyl aminopeptidase, AlaAP) are closely related enzymes involved in cognitive, metabolic, and cardiovascular functions. These functions may be modulated by the type of fat used in the diet. In order to analyze a possible coordinated response of both enzymes we determined simultaneously their activities in frontal cortex, liver, and plasma of adult male rats fed diets enriched with fats differing in their percentages of saturated, mono or polyunsaturated fatty acids such as sesame, sunflower, fish, olive, Iberian lard, and coconut. The systolic blood pressure, food intake, body and liver weight as well as glucose and total cholesterol levels in plasma were measured. The type of fat in the diet influences the enzymatic activities depending on the enzyme and its location. These results suggest cognitive improvement properties for diets with predominance of polyunsaturated fatty acids. Physiological parameters such as systolic blood pressure, food intake, and biochemical factors such as cholesterol and glucose in plasma were also modified depending on the type of diet, supporting beneficial properties for diets rich in mono and polyunsaturated fatty acids. Inter-tissue correlations between the analyzed parameters were also modified depending on the type of diet. If the type of fat used in the diet modifies the behavior and relationship between CysAP and AlaAP in and between frontal cortex, liver and plasma, the functions in which they are involved could also be modified.

The retina acts as an independent clock informing the central pacemaker, the suprachiasmatic nucleus, under environmental light conditions, with consequences of such inputs for the central and peripheral nervous system. Differences in the behavior of the left and right retinas depending on environmental light conditions may influence the information projected to the brain hemispheres. The retina possesses neuropeptides that act as neurotransmitters or neuromodulators. Alanyl-aminopeptidase (AlaAP, EC 3.4.11.2) activity regulates some of these neuropeptides and therefore reflects their function. We analyzed AlaAP activity in the left and right retinas of adult male rats at successive time points under standard (12/12 h light/dark cycle) and nonstandard (constant light) conditions. AlaAP activity was measured fluorometrically using alanyl-beta-naphthylamide as the substrate. Under standard conditions, there were no differences in the left or right retina between time points, with the left retina predominating, particularly in the light period. In contrast, under constant light, no left versus right differences were observed, but significant differences between time points appeared. In comparison with standard conditions, constant conditions led to significantly higher AlaAP activity. Considering all the left retina data in comparison with all the right retina data, no correlation was found between the left and right retinas under standard conditions, but a significant positive correlation was observed under constant light. These results demonstrate an asymmetrical response of retinal AlaAP activity to changes in environmental light conditions, which may affect the functions in which the substrates of AlaAP are involved and the information projected to the brain hemispheres.

Aims Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) differ in their renin-angiotensin system function and sympathetic tone. The metabolism of angiotensins and vasopressin depends on the action of certain aminopeptidases whose activity may be influenced by the autonomic nervous system. Their regulation may differ between WKY and SHR in hypothalamus and plasma according to the sympathetic tone. We analyzed aminopeptidases responsible for the hydrolysis of certain angiotensins, vasopressin, cholecystokinin or enkephalins in hypothalamus and plasma of WKY and SHR in untreated controls rats and under beta-adrenoceptor blockade. Systolic blood pressure, food intake, water intake and diuresis were measured as parameters modulated by the autonomic nervous system and the above mentioned peptides. Main methods Glutamyl-, aspartyl-, cystinyl- and alanyl-aminopeptidase activities were analyzed fluorimetrically in plasma and hypothalamus of control and propranolol-treated (100 mg/kg/day administered in drinking water for 1 month) WKY and SHR, using arylamide derivatives as substrates. Key findings An opposite response of aminopeptidases to propranolol treatment between plasma and hypothalamus was observed in either WKY and SHR. Furthermore, the behavior of aminopeptidases was inversed between WKY and SHR either in hypothalamus and plasma: while the activity increased in hypothalamus and decreased in plasma of WKY, it decreased in hypothalamus and increased in plasma of SHR. Significance These results revealed an inverse response of aminopeptidases between hypothalamus and plasma and also an opposite behavior of these enzymes between WKY and SHR in hypothalamus and plasma. These observations support the involvement of the sympathetic system in the modulation of aminopeptidase activities.

Fat type in diet is responsible for specific changes in gut microbiota (GM). Extra virgin olive oil (EVOO) has been shown to be beneficial for blood pressure and to produce effects on GM. To analyze the cause-effect relationship between intestinal microbial changes and blood pressure, we studied the effect of EVOO on fecal microbiota and systolic blood pressure (SBP) levels in spontaneously hypertensive rats (SHR). SHR were fed either an enriched EVOO diet or a standard diet for a period of 12 weeks. At the end of the experimental period, the microbial profiles in the feces were studied in both groups by using PCR-denaturing gradient gel electrophoresis. Real-time PCR was used to quantify the selected bacterial groups. The results demonstrated significant differences when using Lactobacillus (p

The cardiovascular control involves a bidirectional functional connection between the brain and heart. We hypothesize that this connection could be extended to other organs using endocrine and autonomic nervous systems (ANS) as communication pathways. This implies a neuroendocrine interaction controlling particularly the cardiovascular function where the enzymatic cascade of the renin-angiotensin system (RAS) plays an essential role. It acts not only through its classic endocrine connection but also the ANS. In addition, the brain is functionally, anatomically, and neurochemically asymmetric. Moreover, this asymmetry goes even beyond the brain and it includes both sides of the peripheral nervous and neuroendocrine systems. We revised the available information and analyze the asymmetrical neuroendocrine bidirectional interaction for the cardiovascular control. Negative and positive correlations involving the RAS have been observed between brain, heart, kidney, gut, and plasma in physiologic and pathologic conditions. The central role of the peptides and enzymes of the RAS within this neurovisceral communication, as well as the importance of the asymmetrical distribution of the various RAS components in the pathologies involving this connection, are particularly discussed. In conclusion, there are numerous evidences supporting the existence of a neurovisceral connection with multiorgan involvement that controls, among others, the cardiovascular function. This connection is asymmetrically organized.

Objective. Enkephalins are neuropeptides involved in functions such as pain modulation and/ or cognitive processes. It has been reported that dietary fat modifies enkephalins in the brain. Since enkephalins are hydrolyzed by enkephalinases, the study of the influence of dietary fats, differing in their degree of saturation, on brain fatty acids content and enkephalinase activity is important to understand its regulatory role on neuropeptides under different type of diets. Methods. We analyzed enkephalinase activity, assayed with alanine-β-naphthylamide as sub-strate, in frontal cortex of adult male rats fed diets supplemented with fish oil, olive oil or coconut oil, which markedly differed in the saturation of their fatty acids. Results. Rats fed a diet enriched with coconut oil had lower soluble enkephalinase activity than the group fed olive oil (pmembrane-bound enkephalinase activity than the group fed with olive (p Conclusions. Dietary fat modifies enkephalinase activity in the frontal cortex depending on the degree of saturation of the used oil. It is postulated that the functions, in which enkephalins are involved, such as pain modulation or cognitive functions, may also be affected according to the type of oil used in the diet.

A comparative study on potential risks was carried out in a collection of 50 enterococci isolated from faeces of mice fed a standard or a high-fat diet enriched with extra virgin olive oil, refined olive oil or butter, at the beginning, after six weeks and after twelve weeks of experiments. Strains were biochemically assessed and genetically characterized. E. faecalis and E. casseliflavus were the most frequently isolated species in any diet and time points. Apart from the fact of not having isolated any strain from the virgin olive oil group during the last balance, we found statistically significant differences p &lt, 0 . 05 among the diets in the percentage of antibiotic resistance and in the presence of the enterococcal surface protein gene (esp), as well as a tendency p &lt, 0 . 1 for the presence of the tyrosine decarboxylase gene (tdc) to increase over time in the group of isolates from the standard diet. When the resistance of the strains to virgin or refined olive oil was studied, only the group of enterococci from high fat diets showed a significantly higher percentage of resistance to refined olive oil p &lt, 0 . 05 , while both types of oil equally inhibited those isolated from the standard diet p &gt, 0 . 05 .

After millennia of knowledge of opium, it was only recently that endogenous substances called opioids with similar properties to opium and derivatives were discovered. The first to be discovered were enkephalins. In addition to the regulation of their synthesis and expression of receptors, an important mechanism for the regulation of their functions carried out by multiple proteolytic enzymes acting at all levels of their structure is described. The action of such enzymes, known as enkephalinases, is also regulated by endogenous and exogenous factors which ultimately affect the control of the enkephalins's action. For therapeutic purposes, it is not only necessary to develop specific inhibitors but also to acquire a deep knowledge of the influence that such factors exert on their activities. This knowledge could help us to establish adapted therapeutic strategies in the treatment of pain or other processes in which enkephalinases are involved. In this chapter, some of these regulatory factors are discussed, such as regional and subcellular distribution, developmental changes, diurnal variations, hormonal influences, stress, dietary factors or interactions with other neurotransmitters.

Vital functions, such as blood pressure, are regulated within a framework of neurovisceral integration in which various factors are involved under normal conditions maintaining a delicate balance. Imbalance of any of these factors can lead to various pathologies. Blood pressure control is the result of the balanced action of central and peripheral factors that increase or decrease. Special attention for blood pressure control was put on the neurovisceral interaction between Angiotensin II and the enzymes that regulate its activity as well as on nitric oxide and dopamine. Several studies have shown that such interaction is asymmetrically organized. These studies suggest that the neuronal activity related to the production of nitric oxide in plasma is also lateralized and, consequently, changes in plasma nitric oxide influence neuronal function. This observation provides a new aspect revealing the complexity of the blood pressure regulation and, undoubtedly, makes such study more motivating as it may affect the approach for treatment.

Extra virgin olive oil (EVOO) has been reported to have a distinct influence on gut microbiota in comparison to other fats, with its physiological benefits widely studied. However, a large proportion of the population consumes olive oil after a depurative process that not only mellows its taste, but also deprives it of polyphenols and other minority components. In this study, we compare the influence on the intestinal microbiota of a diet high in this refined olive oil (ROO) with other fat-enriched diets. Swiss Webster mice were fed standard or a high-fat diet enriched with EVOO, ROO, or butter (BT). Physiological parameters were also evaluated. At the end of the feeding period, DNA was extracted from feces and the 16S rRNA was pyrosequenced. The group fed ROO behaved differently to the EVOO group in half the families with statistically significant differences among the diets, with higher comparative levels in three families&mdash, Desulfovibrionaceae, Spiroplasmataceae, and Helicobacteraceae&mdash, correlating with total cholesterol. These results are again indicative of a link between specific diets, certain physiological parameters and the prevalence of some taxa, but also support the possibility that polyphenols and minor components of EVOO are involved in some of the proposed effects of this fat through the modulation of the intestinal microbiota

Thyroid disorders affect the hypothalamic-pituitary-adrenal axis with important consequences on the cardiovascular function in which the renin-angiotensin system plays a major role. Hypo and hyperthyroidism influence the classic main components of the renin-angiotensin system. However, the behavior of other elements of the renin-angiotensin system such as Ang III, Ang 2-10, Ang IV, or AT4, regulated by angiotensinase enzymes such as alanyl- (AlaAP), cystinyl- (CysAP), glutamyl- (GluAP), or aspartyl-aminopeptidase (AspAP), has not yet been described. In order to obtain a comprehensive view on the response of the renin-angiotensin system in the hypothalamic-pituitary-adrenal axis of animals with thyroid disorders, these enzyme activities were simultaneously analyzed fluorometrically, using arylamide derivatives as substrates in hypothalamus, anterior and posterior pituitary, adrenals and plasma of euthyroid, hypothyroid, and hyperthyroid rats, and their intra- and inter-tissue correlations were evaluated. The response is depending on the type of enzyme studied, its location and the thyroid status. Anterior pituitary, adrenals and plasma were mainly affected by the thyroid disorders. In the anterior pituitary, GluAP and AspAP increased in hypothyroid rats. In adrenals, AlaAP and CysAP decreased in hypothyroid whereas GluAP and AspAP decreased in hyperthyroid rats. In plasma, while AlaAP increased in hypo- and hyperthyroid rats, CysAP and GluAP decreased only in hyperthyroid. In comparison with euthyroid, intra-tissue correlations decreased in hypothyroid but inter-tissue correlations decreased mainly in hyperthyroid rats. Thyroid disorders also produced a disruption in the pattern of inter-tissue correlations observed in euthyroid. These results suggest that thyroid hormone levels hit components of the renin-angiotensin system and may influence the paracrine and endocrine cross talk between cells.

The type of fat in the diet determinates the characteristics of gut microbiota, exerting a major role in the development of metabolic syndrome. We hypothesize that a diet enriched with extra virgin olive oil (EVOO) has a distinctive effect on the intestinal microbiome in comparison with an enriched butter diet (BT) and this effect is related to the physiological benefits exerted by EVOO. Swiss Webster mice were fed standard (SD) or two high fat diets enriched with EVOO or butter. Hormonal, physiological and metabolic parameters were evaluated. At the end of the feeding period, DNA was extracted from faeces and the 16S rRNA genes were pyrosequenced. Among the main significant differences found, BT triggered the highest values of systolic blood pressure, correlating positively with the percentage of Desulfovibrio sequences in faeces, which in turn showed significantly higher values in BT than in EVOO. EVOO had the lowest values of plasmatic insulin, correlating inversely with Desulfovibrio, and had the lowest plasmatic values of leptin which correlated inversely with Sutterellaceae, Marispirillum and Mucilaginibacter dageonensis, the three showing significantly higher percentages in EVOO. The lowest total cholesterol levels in plasma were detected in SD, correlating positively with Prevotella and Fusicatenibacter, both taxa with significantly greater presence in SD. These results may be indicative of a link between specific diets, certain physiological parameters and the prevalence of some taxa, supporting the possibility that in some of the proposed effects of virgin olive oil the modulation of intestinal microbiota could be involved.

Fermentation of caper berries is a traditional process relying on the indigenous microbiota. In this study, the application of Lactobacillus plantarum Lb9 isolated from spontaneous caper berry fermentation as starter culture was evaluated by means of culture-dependent and independent methods. The results obtained here indicated that L. plantarum Lb9 was able to carry on an accelerated lactic acid fermentation of caper berries, dominating the background microbiota present in the raw material until the end of fermentation even when fermentation broth was added with high salt concentration. Persistence of L. plantarum Lb9 in the starter-added fermentation was confirmed by RAPD-PCR of isolates from viable cell counting during the fermentation. TTGE analysis of the microbial community throughout fermentation process also indicated that lactobacilli were the predominant microbiota, and thus the microbial composition during the controlled fermentation was more homogeneous compared with spontaneous fermentations. Furthermore, the sensorial quality of the starter-added fermented capers was similar to the spontaneous fermented products. In conclusion, L. plantarum Lb9 was an adequate starter culture strain for caper berry fermentation prevailing over other bacteria present in the raw material. This fermentation ensured both the quality and safety of the end product.

The aim of the present work was to improve our knowledge on the mechanisms underlying the beneficial or deleterious effects on testicular function of the so-called Mediterranean and Western diet by analyzing glutamyl aminopeptidase (GluAP), gamma glutamyl transpeptidase (GGT) and dipeptidyl peptidase IV (DPP IV) activities in testis, as enzymes involved in testicular function. Male Wistar rats (6 months old) were fed for 24 weeks with three different diets: standard (S), an S diet supplemented with virgin-olive-oil (20%) (VOO), or a S diet enriched with butter (20%) plus cholesterol (0.1%) (Bch). At the end of the experimental period, plasma lipid profiled (total triglycerides, total cholesterol and cholesterol fractions (HDL, LDL and VDL)) were measured. Enzymatic activities were determined by fluorimetric methods in soluble (sol) and membrane-bound (mb) fractions of testicular tissue using arylamide derivatives as substrates. Results indicated an increase in plasmatic triglycerides, total cholesterol, LDL and VLDL in Bch. A significant increase of mb GluAP and GGT activities was also found in this diet in comparison with the other two diets. Furthermore, significant and positive correlations were established between these activities and plasma triglycerides and/or total cholesterol. These results support a role for testicular GluAP and GGT activities in the effects of saturated fat (Western diet) on testicular functions. In contrast, VOO increased sol DPP IV activity in comparison with the other two diets, which support a role for this activity in the effects of monounsaturated fat (Mediterranean diet) on testicular function. The present results strongly support the influence of fatty acids and cholesterol on testicular GluAP and GGT activities and also provide support that the reported beneficial influence of the Mediterranean diet in male fertility may be mediated in part by an increase of testicular sol DPP IV activity.

The renin-angiotensin system (RAS) plays a major role in the control of blood pressure (BP) and water balance by coordinating brain, heart and kidney functions, connected with each other by hormonal and neural mechanisms through the autonomic nervous system (ANS). RAS function may be monitored by the study of the enzymes (angiotensinases) involved in the metabolism of its active peptides. In order to study the relationship between the brain-heart-kidney axis and the control of BP and water balance, we analyzed the correlation of angiotensinase activities, assayed as arylamidase activities, between hypothalamus, left ventricle, renal cortex and renal medulla, collected from Wistar-Kyoto and spontaneously hypertensive rats, treated or not treated with L-NAME [N(G)-nitro-L-arginine methyl ester]. This compound not only inhibits the formation of nitric oxide but also disrupts the normal function of the ANS activating the sympathetic nervous system (SNS) to increase BP. In addition, to assess the influence of the SNS, we studied the effect of its blockade by treatment of both strains with propranolol. The present results support the notion that RAS function of the brain-heart-kidney axis, as reflected by the activities of angiotensinases, is reciprocally connected by afferent and efferent mechanisms between these locations, presumably through the ANS. These results reveal new aspects of neuroendocrine regulation possibly involving the ANS.

Quantitative real-time PCR may be a rapid and automated procedure for detection of bacterial pathogens from food samples. Nevertheless, when testing the effects of antimicrobials on the viability of bacterial pathogens in foods, we found that DNA from dead cells interfered greatly in the detection of viable Listeria monocytogenes after treatment with the broad-spectrum bacteriocin enterocin AS-48. To overcome this problem, a quantitative real-time PCR (qRT-PCR) assay based on bacterial mRNA was adapted to quantify viable L. monocytogenes in food after bacteriocin treatments. The procedure allowed a better and faster estimation of viable cells compared to PALCAM viable cell counts when the threshold level was 2 log units/g of food, while PALCAM viable count allowed detection of one log unit/g. This procedure may be useful to verify the efficacy of bacteriocins against L. monocytogenes in foods.

In an ecological study, a collection of Gram-positive bacteria were isolated from Gueddid, an artisanal Tunisian fermented meat. From these, 24 strains showed antimicrobial activity and identified as Enterococcus faecium using molecular methods. The enterococci strains were further investigated regarding their safety aspects and functional properties. All the isolates produce bacteriocins with inhibitory activity against several food spoilage bacteria and food borne pathogens, including Listeria spp. Enterococcus spp. and Staphylococcus aureus. One isolate was active against Escherichia coli CECT 877. The majority of the isolates tested positive upon PCR amplification of structural genes for enterocins A, B and P. Investigation of virulence factors by PCR amplification revealed the presence of genes encoding for gelatinase (gelE), enterococcal antigen (efaAfm), sex pheromone (cpd and ccf) and expression of cytolysin (the haemolytic component cylB) whereas, other presumed virulence genes encoding for (agg, esp, cylM, cylA, and cob) were not detected. The isolates were mostly resistant to erythromycin, rifampicin, ciprofloxacin, lavofloxacin, and nitrofurantoin. All of them showed sensitivity to several antibiotics (ampicillin, penicillin, vancomycin, chloramphenicol, teicoplanin, gentamicin, streptomycin, and quinupristin/dalfopristin). Tyrosine, lysine, ornithine and histidine were not decarboxylated by any enterococcal isolate. Nine of the antagonistic enterococci tested did not show any virulence traits or produced biogenic amines, and still had important technological properties. The safety aspects of these selected strains should be studied in deeper details in order to evaluate their potential for biotechnological applications.

A collection of 25 isolates from foods of animal origin (including mainly milk and cheese, together with meat and ham) was studied. Enterococci were identified at species levels as E. faecalis (9 isolates) and E. faecium (16 isolates). Investigation of virulence factors by PCR amplification revealed incomplete sets of cytolysin genes both in E. faecalis and E. faecium isolates. Among E. faecalis, PCR amplification revealed a high incidence of genes encoding for enterococcal surface protein esp (7/9 isolates), enterococcal antigen efaAfs (6/9), aggregation substance agg (2/9) and sex-pheromone encoding genes ccf, cob, cpd (which were detected in 9, 5 and 3 out of 9 isolates, respectively). By contrast, only esp (7/16 isolates) and efaAfm (10/16) were detected among E. faecium. Antibiotic resistance detected at higher frequencies included rifampicin, nitrofurantoin, ciprofloxacin and levofloxacin. Vancomycin resistance was also detected among E. faecalis and E. faecium. E. faecalis isolates showed decarboxylating activity mostly for tyrosine (5/9 isolates), while E. faecium isolates showed a broader decarboxylating capacity, involving tyrosine (11/16 isolates) ornithine (6/16), lysine (4/16) and histidine (3/16). Six isolates produced bacteriocins, and genes encoding for enterocins A, B, P, L50, and 1071 were detected. Many isolates tested positive for several of the traits investigated, which raises concerns about their possible role as reservoirs for dissemination of antibiotic resistance and virulence traits in foods.

The bacteriocinogenic strain Lactobacillus plantarum 2.9 isolated from the traditional pearl millet-based African fermented food ben saalga was tested for inhibition of food poisoning and pathogenic bacteria in MRS broth and in a malted millet flour slurry. In MRS broth, strain 2.9 completely eliminated Bacillus cereus, Escherichia coli O157:H7 and Salmonella enterica cells within 48 h incubation at 22–30 °C. A much lower inhibition was observed at 15 °C. The inhibitory effect of strain 2.9 on the above-mentioned target bacteria was corroborated in the malted millet flour slurry, reducing viable cell counts below detection levels after 8 h storage for B. cereus or after 24 h for S. enterica and 48 h for E. coli. An Enterobacter aerogenes strain was only moderately inhibited in the slurry. Results from the present study suggest that strain 2.9 could be used as starter culture to improve the microbiological safety of fermented ben saalga.

A collection of enterococci isolated from meat, dairy and vegetable foods from Morocco including 23 Enterococus faecalis and 15 Enterococcus faecium isolates was studied. All isolates were sensitive to ampicillin, penicillin, and gentamicin. Many E. faecalis isolates were resistant to tetracycline (86.95%), followed by rifampicin (78.26% ciprofloxacin (60.87%), quinupristin/dalfopristin (56.52%), nitrofurantoin (43.47%), levofloxacin (39.13%), erythromycin (21.73%), streptomycin (17.39%), chloramphenicol (8.69%), vancomycin (8.69%), and teicoplanin (4.34%). E. faecium isolates showed a different antibiotic resistance profile: a high percentage were resistant to nitrofurantoin (73.33%), followed by erythromycin (66.60%), ciprofloxacin (66.66%), levofloxacin (60.00%), and rifampicin (26.66%), and only a very low percentage were resistant to tetracycline (6.66%). One isolate was resistant to vancomycin and teicoplanin. The incidence of virulence factors was much higher among E. faecalis isolates, especially for genes encoding for sex pheromones, collagen adhesin, enterococcal endocarditis antigen, and enterococcal surface protein. Isolates with multiple factors (both antibiotic resistance and virulence traits) were also more frequent among E. faecalis isolates, in which one isolate cumulated up to 15 traits. By contrast, several isolates of E. faecium had only very few unwanted traits as compared to only two isolates in E. faecalis. The high abundance of isolates carrying virulence factors and antibiotic resistance traits suggests that the sanitary quality of foods should be improved in order to decrease the incidence of enterococci.

A bacteriocin-producing lactic acid bacterium (strain 2.5) isolated from cow’s milk used in cheese production from Northern Morocco was selected for its strong anti-listerial activity. The producer strain was identified as Enterococcus faecalis by molecular methods. Strain 2.5 carried the genetic determinants for the two-peptide enterococcal bacteriocin enterocin 1071, and the active bacteriocin was purified to homogeneity by reversed-phase chromatography from culture broths of the producer strain. Strain 2.5 carried two plasmids (of ∼7 and 40 kb). Characterization of strain 2.5 at biosafety level indicated that this strain is non-haemolytic, and lacks the genetic determinants for most of the virulence factors described in enterococci (cylB, cylM, gelE, ace and agg) although it carried the genetic determinants cylA, efaAfs as well as determinants for the sex pheromone peptides cpd, cob, and ccf. Strain 2.5 was resistant to tetracycline, rifampicin, and ciprofloxacin, but it was sensitive to penicillin, ampicillin, vancomycin, and teicoplanin. Results from the present study support the potential role of strain 2.5 as an anti-listerial agent to be tested in traditional fermented foods.

A collection of 17 enterococci isolates obtained from fermentations of capers (the fruits of Capparis sp.) were investigated for incidence of known virulence determinants, antibiotic resistance and production of biogenic amines. Molecular identification revealed the presence of Enterococcus faecium (nine isolates), Enterococcus faecalis (4), E. avium (3) and Enterococcus casseliflavus/flavescens (1). Alpha-haemolytic activity was detected in two E. avium and one E. faecalis isolates, and beta-haemolytic activity was detected in E. casseliflavus/flavescens. The haemolytic component cylB was detected by PCR amplification in three non-haemolytic isolates and in E. casseliflavus/flavescens. The collagen adhesin ace gene and the endocarditis associated antigen gene efaA(fm) were detected in two isolates each. Genes encoding sex pheromone precursors (cpd, cob, ccf) were detected in E. faecalis and E. casseliflavus/flavescens. Other presumed virulence genes (agg, gelE, cylM, cylA and efaA(fs)) were not detected. All isolates were resistant to rifampicin, erythromycin and ciprofloxacin, and some were also resistant to quinupristin/dalfopristin, tetracycline, levofloxacin, gentamicin and streptomycin. Vancomycin resistance was not detected. Tyrosine decarboxylation was detected in all E. faecium isolates. Given the high resistance of enterococci to environmental conditions, and their implication in opportunistic infections, the incidence of potential virulent enterococci in foods (especially those of a higher risk-like home-made foods) should be carefully studied.

A collection comprising 14 isolates of Pediococcus pentosaceus and one Pediococcus acidilactici from the fermentation of caper fruits was studied. All isolates showed very similar fermentation profiles and produced a limited number of exoenzymes. All isolates carried large plasmids of diverse sizes between 20 and 55 kb, while some also contained smaller plasmids between 10 and 16 kb. Cluster analysis of plasmid profiles revealed four main groups with various degrees of similarities. All amino acid decarboxylation tests were negative, suggesting that pediococci are not involved in generation of biogenic amines. None of the isolates showed hemolytic activity. Antimicrobial resistance tests revealed that all isolates were sensitive to 11 different antimicrobials while being resistant to ciprofloxacin (MIC > or =2 mg/liter) and intrinsically resistant to vancomycin (MIC > or =16 mg/liter) and teicoplanin (MIC > or =16 mg/liter).

The production of antimicrobial substances was studied among 195 bacterial isolates from retail table olives. A total 86 isolates tested positive, and they clustered in 10 groups according to their inhibitory spectra. Many isolates (38.37%) produced strong inhibition against all bacteria tested (Listeria innocua, Lactococcus lactis, Bacillus cereus, B. megaterium, Staphylococcus aureus, Micrococcus luteus, Enterococcus faecalis, and Escherichia coli). The selected bacterial isolates were Gram-positive bacteria with rod morphology (62.67%), short rods (26.65%) or cocci (10.67%). Isolates producing antimicrobial substances may be useful as starters to enhance control of table olive fermentation and improve the safety of retail table olives.

The antimicrobial effect of the broad-spectrum bacteriocin enterocin AS-48 against the toxicogenic psychrotrophic strain Bacillus cereus LWL1 has been investigated in a model food system consisting of boiled rice and in a commercial infant rice-based gruel dissolved in whole milk stored at temperatures of 37 degrees C, 15 degrees C and 6 degrees C. In food samples supplemented with enterocin AS-48 (in a concentration range of 20-35 mug/ml), viable cell counts decreased rapidly over incubation time, depending on the bacteriocin concentration, the temperature of incubation and the food sample. Enterotoxin production at 37 degrees C was also inhibited. Heat sensitivity of endospores increased markedly in food samples supplemented with enterocin AS-48: inactivation of endospores was achieved by heating for 1 min at 90 degrees C in boiled rice or at 95 degrees C in rice-based gruel. Activity of enterocin AS-48 in rice gruel was potentiated by sodium lactate in a concentration-dependent way.

Fermentation of capers (the fruits of Capparis sp.) was studied by molecular and culture-independent methods. A lactic acid fermentation occurred following immersion of caper berries in water, resulting in fast acidification and development of the organoleptic properties typical of this fermented food. A collection of 133 isolates obtained at different times of fermentation was reduced to 75 after randomly amplified polymorphic DNA (RAPD)-PCR analysis. Isolates were identified by PCR or 16S rRNA gene sequencing as Lactobacillus plantarum (37 isolates), Lactobacillus paraplantarum (1 isolate), Lactobacillus pentosus (5 isolates), Lactobacillus brevis (9 isolates), Lactobacillus fermentum (6 isolates), Pediococcus pentosaceus (14 isolates), Pediococcus acidilactici (1 isolate), and Enterococcus faecium (2 isolates). Cluster analysis of RAPD-PCR patterns revealed a high degree of diversity among lactobacilli (with four major groups and five subgroups), while pediococci clustered in two closely related groups. A culture-independent analysis of fermentation samples by temporal temperature gradient electrophoresis (TTGE) also indicated that L. plantarum is the predominant species in this fermentation, in agreement with culture-dependent results. The distribution of L. brevis and L. fermentum in samples was also determined by TTGE, but identification of Pediococcus at the species level was not possible. TTGE also allowed a more precise estimation of the distribution of E. faecium , and the detection of Enterococcus casseliflavus (which was not revealed by the culture-dependent analysis). Results from this study indicate that complementary data from molecular and culture-dependent analysis provide a more accurate determination of the microbial community dynamics during caper fermentation.

The bacteriocinogenic strain RJ16 isolated from goat cheese has been identified as Enterococcus faecium by species-specific PCR, DNA–rRNA hybridization and rDNA sequencing. Purified bacteriocin from strain RJ16 is a carboxypeptidase A-resistant peptide with a molecular mass (7125 Da) very close to the cyclic peptide enterocin AS-48. Bacteriocin from strain RJ16 and AS-48 show identical antibacterial spectra, although the former is slightly less active on strains of Listeria monocytogenes and Bacillus cereus. Producer strains show cross-immunity. PCR amplification of total DNA from strain RJ16 with primers for the AS-48 structural gene and sequencing of the amplified fragment revealed an almost identical sequence (99.5%), except for a single mutation that predicts the change of Glu residue at position 20 of AS-48 to Val. Therefore, bacteriocin produced by E. faecium RJ16 should be considered a variant of AS-48, which we call AS-48RJ. PCR amplification revealed that strain RJ16 contains the complete as-48 gene cluster. Hybridization with probes for as-48 gene cluster revealed a chromosomal location of as-48 genes in strain RJ16, being the first example of a chromosomal location of this bacteriocin trait. Strain RJ16 produced enzymes of interest in food processing (esterase, esterase lipase and phytase activities), and did not decarboxylate amino acids precursors for biogenic amines. Strain RJ16 did not exhibit haemolytic or gelatinase activities, and PCR amplification revealed the lack of genes encoding for known virulence determinants (aggregation substance, collagen adhesin, enterococcal surface protein, endocarditis antigens, as well as haemolysin and gelatinase production). Strain RJ16 was resistant to ciprofloxacin ( MIC > 2 mg l - 1 ) and levofloxacin ( MIC > 4 mg l - 1 ) and showed intermediate resistance to nitrofurantoin and erythromycin, but was sensitive to ampicillin, penicillin, streptomycin, gentamicin, rifampicin, chloramphenicol, tetracycline, quinupristin/dalfopristin, vancomycin and teicoplanin. Altogether, results from this study suggest that this broad-spectrum bacteriocin-producing strain may have a potential use in food preservation.

Aims: Activity of the bacteriocin EJ97 produced by Enterococcus faecalis EJ97 against strains of ‘Bacillus macroides/B. maroccanus’ isolated from spoiled zucchini puree was investigated. Methods and Results: The influence of several factors like bacteriocin concentration, incubation temperature, pH, growth medium and chemical perservatives on bacteriocin activity was investigated. Enterocin EJ97 [2 arbitrary units (AU) per millilitre] had a marked bactericidal effect on strain INRA P53-2 after 4 h of incubation at 37°C, 24 h at 15°C or 48 h at 4°C. Activity was markedly reduced at pH values of 5·0 and 9·0, but was potentiated by sodium nitrite, sodium benzoate, sodium lactate and sodium tripolyphosphate. Inhibition of strain INRA P53-2 in a commercial vegetable puree required a 10-fold higher bacteriocin concentration. Strain EJ97 was able to grow and produce bacteriocin on vegetable puree, but no inhibition of strain INRA P53-2 was detected. Conclusions: The concentration-dependent bactericidal activity of enterocin EJ97 against strain INRA P53-2 was higher at 37°C and neutral pH, and was potentiated by chemical preservatives. Although enterocin EJ97 was less active in vegetable puree, the concentrations providing bactericidal activity in this food matrix are practical for commercial use. Significance and Impact of the Study: Enterocin EJ97 may have a potential for use in the prevention of food spoilage caused by ‘B. macroides/B. maroccanus’.

Summary The incidence and diversity of enterococci in retail food samples of meat, dairy and vegetable origin was investigated. Enterococci were present, at concentrations of 10 1 to 10 4 CFU/g. Fifty selected isolates from food samples grouped in two separate clusters by RAPD analysis. Cluster G1 (72% of the isolates) contained the E. faecium CECT 410 T type strain, and also showed a high degree of genetic diversity. Cluster G2 (28% of the isolates) contained the E. faecalis CECT 481 T type strain and was genetically more homogeneous. Virulence traits (haemolysin, gelatinase or DNAse activities, or the presence of structural genes cylL , ace, asa1 and esp ) were not detected. All isolates were sensitive to the antibiotics ampicillin, penicillin, gentamicin, streptomycin and chloramphenicol. A high pecentage of isolates were resistant to erythromycin and rifampicin. Many isolates showed intermediate sensitivity to several antibiotics (tetracycline, ciprofloxacin, levofloxacin, or quinupristin/dalfopristin). Vancomycin and teicoplanin resistance was detected in one strain, but vanA, vanB, vanC1, vanC2 or vanC3 genes were not detected. Many of the isolates showed functional properties of food or health relevance. Production of antimicrobial substances was detected in 17 of the isolates, and 14 of them carried structural genes for enterocins A, B and/or P.

Enterocin EJ97 showed a concentration-dependent bactericidal activity against vegetative cells of Bacillus coagulans CECT 12. Highest activity was obtained at incubation temperatures of 37°C and 15°C, but almost no activity was detected during incubation of cells at 4°C. Enterocin EJ97 showed highest activity against B. coagulans CECT 12 at pH 7.0. Some decrease of activity was detected at pH 5.0, and a very low activity was detected during incubation of cells at pH 9.0. Enterocin activity was not modified by sodium benzoate or by sodium chloride, but it was enhanced to some extent by sodium tripolyphosphate, and was highly potentiated by sodium nitrite at concentrations above 50 μg/ml.

A soil bacterium able to undergo multicellular development and a coordinated gliding in swarms, requires an accurate regulatory network of phosphorelay proteins. Inorganic phosphate is a limiting nutrient in soil and its importance in regulation is critical. As a step towards studying phosphate regulation and its influence in the developmental process in this bacterium, we screened a Myxococcus xanthus library for clones with phosphatase activity, and found four different ones. The deduced sequence of one of the cloned inserts is similar to that of the classic transmembrane histidine protein kinase of the sensor family of the two-component signal transduction systems with a high sequence similarity to the sensor kinase in the Pho regulon of Bacillus subtilis PhoR. This gene has been named phoR1 and its deduced amino acid sequence consists of 455 residues with a predicted molecular mass of 48.5 kDa. The M. xanthus PhoR1 deduced sequence contains all the characteristic histidine protein kinase motifs in the same order and with the same spacing. A hydropathy profile indicates two membrane-spanning segments located at the extreme N-terminus, according to the putative sensor role of this domain. A gene-disrupted mutant is unable to produce normal mature fruiting bodies and produces fewer spores.

Production of laccase activity by the hyphomycete Chalara (syn. Thielaviopsis ) paradoxa CH32 reached highest levels at the idiophase, but was not enhanced by putative laccase inducers. Laccase activity from the extracellular fluid was purified and characterised, consisting of a single protein of 67 kDa, sensitive to heat and to acidic pH. Laccase activity showed an optimum pH of 6.5 for most of the substrates, while some substrates were oxidised only at pH 4.5. The optimum temperature for enzyme activity was 30 °C. Laccase activity was inhibited by metal cations, especially by Hg 2+ . It was also inhibited by reducing agents, EDTA, potassium cyanide and sodium azide. The enzyme retained a high percentage of activity in the presence of some organic solvents (methanol, ethanol and iso -propyl alcohol). The substrate specificity of the purified laccase was greatly influenced by the nature and the position of the substituting groups in the phenolic ring.