Development of a rapid PCR protocol to detect Vibrio parahaemolyticus in clams.

Vibrio parahaemolyticus is part of the natural microflora of estuarine and coastal marine waters and can be also present in seafood, especially shellfish and bivalve molluscs. In this study we compared the reference cultural method ISO 6887-3 with two molecular methods, multiplex PCR and real-time PCR, for the detection of two distinct genetic markers (tlh species-specific gene and tdh virulence gene) of V. parahaemolyticus in bivalve mollusc. The analyses were performed on clams inoculated with V. parahaemolyticus ATCC 43996 at T0 and after a 3 and 6 h of pre-enrichment in alkaline saline peptone water. Counts on agar plates were largely inaccurate, probably due to other Vibrio species grown on the TCBS selective agar. Multiplex PCR assays, performed using primers pairs for tdh and tlh genes, showed a detection limit of 104 CFU/g of shell stock within 6 h of pre-enrichment, respecting however the action level indicated by the National Seafood Sanitation Program guideline. Detection by tdh gene in real-time PCR reached the definitely highest sensitivity in shorter times, 101 CFU/g after 3 h of pre-enrichment, while the sensitivity for the tlh gene was not promising, detecting between 105 and 106 CFU/g after 6 h of pre-enrichment. Our findings provide a rapid routine method of detection of V. parahaemolyticus based on tdh gene by real-time PCR for commercial seafood analysis to identify the risk of gastrointestinal diseases.

High pressure homogenization vs heat treatment: safety and functional properties of liquid whole egg.

This research investigated the potential of multi-pass homogenization treatment for the inactivation of Salmonella enterica serovar Enteritidis inoculated at different levels in liquid whole egg (LWE) comparing the efficacy of this treatment with a traditional thermal one performed at 65 °C. Moreover, the effects of high pressure treatment (HPH) on structural and functional properties such as viscosity, microstructure and foaming abilities of LWE were investigated. The data obtained suggested that the multi-pass high pressure treatment at 100 MPa of S. enterica serovar Enteritidis inoculated in LWE at 7 and 4 log CFU/ml resulted in a first order inactivation kinetic, while the thermal inactivation curves of S. enterica serovar Enteritidis inoculated at 8 and 4 log CFU/ml presented a non-linear behaviour, with a marked tail after 3 min of treatment at 65 °C. Additionally, HPH treatment caused an increase in foaming capacity of LWE, with respect to the untreated samples, passing from values of 26% of the control to 50% of pressure treated samples.

Acid stress-mediated metabolic shift in Lactobacillus sanfranciscensis LSCE1.

Lactobacillus sanfranciscensis LSCE1 was selected as a target organism originating from recurrently refreshed sourdough to study the metabolic rerouting associated with the acid stress exposure during sourdough fermentation. In particular, the acid stress induced a metabolic shift toward overproduction of 3-methylbutanoic and 2-methylbutanoic acids accompanied by reduced sugar consumption and primary carbohydrate metabolite production. The fate of labeled leucine, the role of different nutrients and precursors, and the expression of the genes involved in branched-chain amino acid (BCAA) catabolism were evaluated at pH 3.6 and 5.8. The novel application of the program XCMS to the solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) data allowed accurate separation and quantification of 2-methylbutanoic and 3-methylbutanoic acids, generally reported as a cumulative datum. The metabolites coming from BCAA catabolism increased up to seven times under acid stress. The gene expression analysis confirmed that some genes associated with BCAA catabolism were overexpressed under acid conditions. The experiment with labeled leucine showed that 2-methylbutanoic acid originated also from leucine. While the overproduction of 3-methylbutanoic acid under acid stress can be attributed to the need to maintain redox balance, the rationale for the production of 2-methylbutanoic acid from leucine can be found in a newly proposed biosynthesis pathway leading to 2-methylbutanoic acid and 3 mol of ATP per mol of leucine. Leucine catabolism to 3-methylbutanoic and 2-methylbutanoic acids suggests that the switch from sugar to amino acid catabolism supports growth in L. sanfranciscensis in restricted environments such as sourdough characterized by acid stress and recurrent carbon starvation.

Monitoring of microbial metabolites and bacterial diversity in beef stored under different packaging conditions.

Beef chops were stored at 4°C under different conditions: in air (A), modified-atmosphere packaging (MAP), vacuum packaging (V), or bacteriocin-activated antimicrobial packaging (AV). After 0 to 45 days of storage, analyses were performed to determine loads of spoilage microorganisms, microbial metabolites (by solid-phase microextraction [SPME]-gas chromatography [GC]-mass spectrometry [MS] and proton nuclear magnetic resonance [(1)H NMR]), and microbial diversity (by PCR-denaturing gradient gel electrophoresis [DGGE] and pyrosequencing). The microbiological shelf life of meat increased with increasing selectivity of storage conditions. Culture-independent analysis by pyrosequencing of DNA extracted directly from meat showed that Brochothrix thermosphacta dominated during the early stages of storage in A and MAP, while Pseudomonas spp. took over during further storage in A. Many different bacteria, several of which are usually associated with soil rather than meat, were identified in V and AV; however, lactic acid bacteria (LAB) dominated during the late phases of storage, and Carnobacterium divergens was the most frequent microorganism in AV. Among the volatile metabolites, butanoic acid was associated with the growth of LAB under V and AV storage conditions, while acetoin was related to the other spoilage microbial groups and storage conditions. (1)H NMR analysis showed that storage in air was associated with decreases in lactate, glycogen, IMP, and ADP levels and with selective increases in levels of 3-methylindole, betaine, creatine, and other amino acids. The meat microbiota is significantly affected by storage conditions, and its changes during storage determine complex shifts in the metabolites produced, with a potential impact on meat quality.

Synthesis of cyclopropane fatty acids in Lactobacillus helveticus and Lactobacillus sanfranciscensis and their cellular fatty acids changes following short term acid and cold stresses.

An implemented GC method to separate and quantify the cell cyclopropane fatty acids lactobacillic (C19cyc11) and dehydrosterculic (C19cyc9) was used to study the adaptive response to sublethal acid and cold stresses in Lactobacillus helveticus and Lactobacillus sanfranciscensis. The comparison of the composition of cellular fatty acids of the two strains and their changes after 2 h of stress exposure under micro-aerobic and anaerobic conditions indicated that the aerobic biosynthetic pathway for unsaturated fatty acids is prevalent in L. sanfranciscensis, while the anaerobic pathway is prevalent in L. helveticus. Indeed in the latter strain, in the presence of a source of oleic acid and under micro-aerobic conditions, C18:1n11 and its post-synthetic derivative C19cyc11 accounted for overall proportion ranging from 52 to 28% of the total FAs. On the other hand L. sanfranciscensis synthesizes by aerobic pathway C18:1n9 and transforms it to C19cyc9. However in this species the cumulative level of these two FAs did not exceed 30%. The relevant proportion of dodecanoic acid in the latter species suggests that carbon chain shortening is the principal strategy of L. sanfranciscensis to modulate fluidity or chemico-physical properties of the membranes.

Different fecal microbiotas and volatile organic compounds in treated and untreated children with celiac disease.

This study aimed at investigating the fecal microbiotas of children with celiac disease (CD) before (U-CD) and after (T-CD) they were fed a gluten-free diet and of healthy children (HC). Brothers or sisters of T-CD were enrolled as HC. Each group consisted of seven children. PCR-denaturing gradient gel electrophoresis (DGGE) analysis with V3 universal primers revealed a unique profile for each fecal sample. PCR-DGGE analysis with group- or genus-specific 16S rRNA gene primers showed that the Lactobacillus community of U-CD changed significantly, while the diversity of the Lactobacillus community of T-CD was quite comparable to that of HC. Compared to HC, the ratio of cultivable lactic acid bacteria and Bifidobacterium to Bacteroides and enterobacteria was lower in T-CD and even lower in U-CD. The percentages of strains identified as lactobacilli differed as follows: HC (ca. 38%) > T-CD (ca. 17%) > U-CD (ca. 10%). Lactobacillus brevis, Lactobacillus rossiae, and Lactobacillus pentosus were identified only in fecal samples from T-CD and HC. Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus gasseri were identified only in several fecal samples from HC. Compared to HC, the composition of Bifidobacterium species of T-CD varied, and it varied even more for U-CD. Forty-seven volatile organic compounds (VOCs) belonging to different chemical classes were identified using gas-chromatography mass spectrometry-solid-phase microextraction analysis. The median concentrations varied markedly for HC, T-CD, and U-CD. Overall, the r(2) values for VOC data for brothers and sisters were equal to or lower than those for unrelated HC and T-CD. This study shows the effect of CD pathology on the fecal microbiotas of children.

Effects of sub-lethal concentrations of hexanal and 2-(E)-hexenal on membrane fatty acid composition and volatile compounds of Listeria monocytogenes, Staphylococcus aureus, Salmonella enteritidis and Escherichia coli.

In recent years, the interest in the possible use of natural alternatives to food additives to prevent bacterial and fungal growth has notably increased. Plants and plant products can represent a source of natural alternatives to improve the shelf-life and the safety of food. Some of these compounds, i.e. hexanal, hexanol, 2-(E)-hexenal and 3-(Z)-hexenol, produced throughout the lipoxygenase pathway have important roles in plant defence with a protective action towards microbial proliferation in wounded areas. Otherwise, hexanal and 2-(E)-hexenal have evidenced a marked antimicrobial activity against food spoilage and pathogenic microbial species both in model and real systems. The precise mechanisms of action of all these antimicrobial compounds are not yet clear. Because the usage of these compounds as antimicrobials in foods has to be supported by the comprehension of mechanisms of action of these compounds, the overall purpose of this work was to study the modifications of the cell membrane and volatile compounds of Listeria monocytogenes, Staphylococcus aureus, Salmonella enteritidis and Escherichia coli during the growth in the presence of sub-lethal doses of these two aldehydes. The results obtained evidenced that the tested molecules induced noticeable modifications of the composition of cell membrane and the volatile compounds produced during the growth. Although specific differences in relation to the species considered were identified, 2-(E)-hexenal and hexanal induced a marked increase of some membrane associated fatty acids, both linear and branched fatty acids as well as unsaturated fatty acids, and released free fatty acids.

Assessment of safety, nutritional, and spoilage characteristics of different lagoon grey mullets (Liza ramada, Liza aurata, and Liza saliens).

Different lagoon grey mullets such as Liza ramada (thinlip mullet), Liza aurata (golden grey mullet), and Liza saliens (leaping grey mullet) were analyzed for their nutritional, microbiological, and safety parameters. The microbiological values never exceeded the lower limits stipulated by the Italian Higher Institute of Health. The pathogenic species frequently associated with seafood (Salmonella, Listeria monocytogenes, Vibrio cholerae, Vibrio parahaemolyticus, and Aeromonas hydrophila) were never detected. The absence of coliforms and of Escherichia coli was noted in all fish species after 4 days of storage in ice. Heavy metals such as cadmium and mercury were always below the detection limits (0.01 mg/kg). All three fish species had low levels of total biogenic amines (80 to 100 mg/kg), and the presence of histamine was sporadic. All Liza species, particularly L. ramada and L. saliens, are a good source of omega3 long-chain polyunsaturated fatty acids.

Role of surface-inoculated Debaryomyces hansenii and Yarrowia lipolytica strains in dried fermented sausage manufacture. Part 1: Evaluation of their effects on microbial evolution, lipolytic and proteolytic patterns.

The aim of this work was to study the effects of Debaryomyces hansenii and Yarrowia lipolytica strains, used with lactic acid starter cultures (Lactobacillus plantarum), on the manufacture of dried fermented sausages to understand their role on sausage microbial evolution, lipolytic and proteolytic patterns. The inoculation of the yeast strains did not markedly affect the sausage's microbial flora. The sausages with the yeast strains showed more marked and earlier water activity (a(w)) reductions. Moreover, the surface inoculation of the yeast strains resulted, at the end of ripening, in more pronounced proteolysis and lipolysis. The lipolytic patterns of the products were affected not only by the yeast strain but also by the level of mincing of the meat mixture used.

Variability of the lipolytic activity in Yarrowia lipolytica strains in pork fat.

This work studied the variability in lipolytic activity in 35 strains of Yarrowia lipolytica inoculated in pork fat after 7 and 21 days of storage at 15 °C. The strains were able to generate three different hydrolysis profiles. In particular, the strains PO10, PO14, RO1, RO5, Y15, Y16A, Y20, B5, 7B, 7B3, 16B and 21C caused an increase with time in concentrations of C16:0, C18:0, C18:1, C18:1(Δ11) and C18:2 which were the predominant free fatty acids (FFAs). On the contrary, the strains PO1, PO19, PO23, RO22, Y12, B4, B74, GB, 5B, 5D, 27D and W29 showed an opposite trend, while the remaining ones induced no change. Because the released FFAs can be considered precursors for flavour development, the results suggest the potential use of some Y. lipolytica strains in sausage making to improve the overall aroma.

Variability of the lipolytic activity and volatile molecules production by a strain of Yarrowia lipolytica in pork fat and its dependence on environmental conditions.

The effects of selected variables, i.e. temperature, water activity and yeast inoculation level, on the lipolytic pattern and volatile production by Yarrowia lipolytica Y16A (chosen on the basis of a previous screening) were assessed. The variables were varied according to a central composite design and the models obtained enabled evaluation and weighting of the effects of the independent variables on the free fatty acids (FFAs) and volatile profiles in pork fat based medium. The polynomial models showed the levels temperature, water activity of the pork fat based system and yeast strain inoculation were able to maximize the release of specific FFAs or molecules of sensory importance.

Potentialities of high-pressure homogenization to inactivate Zygosaccharomyces bailii in fruit juices.

UNLABELLED: This experimental work was aimed to evaluate the effects of repeated high-pressure homogenization (HPH) treatments at 100 MPa on the inactivation and regrowth of Zygosaccharomyces bailii inoculated in apricot and carrot juices. Thus, the spoilage yeast was inoculated in both the juices at level of about 5 log CFU/g and the 2 systems were treated with a lab-scale Panda homogenizer for 8 passes at 100 MPa. Microbiological and chemico-physical analyses were performed immediately after the treatment and during the juice storage at room temperature. Microbial data highlighted that yeast inactivation increased with the number of passes applied. Eight passes at 100 MPa allowed yeast inactivation higher than 2.5 log CFU/mL regardless of the juice considered. On the contrary, the juice type affected the yeast fate (growth or death) over the storage at 25 degrees C. In fact, Z. bailii was able to attain the spoilage threshold (6 log CFU/mL) in apricot juice, although with growth kinetics dependent of the survivor levels after HPH treatment. In carrot juice this microorganism was unable to recover over the storage in the most severely treated samples. The HPH treatment had a significant effect on apricot juice pH and viscosity, while no significant effect was observed in carrot juice. The viscosity measurements showed that the application of one pass at 100 MPa resulted in the triplication of apricot viscosity index. No further significant viscosity increase (P > 0.05) was observed increasing the number of passes at 100 MPa. PRACTICAL APPLICATION: The results obtained in the present study and the proposed technology could be exploited by the industries of the beverage sector to increase the shelf life of these kinds of products. Moreover, from a technological point of view, the increase of viscosity, following the high-pressure homogenization treatment, represents a tool to expand the product gamma without the use of gelling additives or thermal treatments, which are detrimental for the sensorial and nutritional properties of this kind of products.

Effect of alpha-linolenic, capric and lauric acid on the fatty acid biosynthesis in Staphylococcus aureus.

The antimicrobial activity of alpha-linolenic, capric and lauric acids on Staphylococcus aureus was studied in relation to their effect on the de novo fatty acid biosynthesis. Labelled acetate was used as integrated carbon source and traced in the de novo fatty acid by using a GC-Mass spectrometer and the single ion monitoring (SIM) technique. The detection of the incorporation of the labelled carbon into the individual cell fatty acids (FAs) provided an insight into the different effects of alpha-linolenic, capric and lauric acids on the FA biosynthesis. The results suggested that FAs pathway is the major target of alpha-linolenic acid and that other enzymes in addition to FabI are involved in S. aureus response mechanism when medium chain fatty acids are present.

Effect of high pressure homogenization on Saccharomyces cerevisiae inactivation and physico-chemical features in apricot and carrot juices.

This experimental work aimed to evaluate the potential of high pressure homogenization (HPH) to inactivate Saccharomyces cerevisiae 635 inoculated both in apricot and carrot juices. The sensitivity of the yeast was evaluated both in relation to its initial inoculum level (about 3.0 and 6.0 Log(10) cfu/ml) and number of passes applied at 100 MPa. Moreover, the effects of repeated pressure treatments at 100 MPa were evaluated for pH, water activity and viscosity of carrot and apricot juices. Data obtained showed that repeated high pressure homogenization passes at 100 MPa allowed a significant inactivation of the spoilage yeast inoculated in both juices. However, as expected, the inactivation of the considered strain was greatly affected by the food matrix. In fact, a higher viability loss of S. cerevisiae 635 was observed in carrot juice than in apricot juice. Concerning the recovery, data obtained showed that the decrease of the inoculum level to 3.0 Log(10) cfu/ml prevented (at least for 6 days) cell proliferation in the samples of apricot and carrot juices treated with more than four and seven passes, respectively. Also the refrigeration of the treated samples prevented cell recovery and, in some cases, induced a further decrease in cell viability also in the samples inoculated with 6.0 Log(10) cfu/ml allowing a further increase in the juice shelf-life. An interesting and promising result was the enhanced viscosity of apricot juices treated up to five passes at 100 MPa.

Effect of a synbiotic food consumption on human gut metabolic profiles evaluated by (1)H Nuclear Magnetic Resonance spectroscopy.

The capacity of human lactobacilli and bifidobacteria to produce metabolites under conditions that may prevail in the human intestine has been studied "in vitro". However, the effect of systematic probiotic consumption on human metabolic phenotype has not been investigated in faeces. This paper shows the potential for the use of (1)H Nuclear Magnetic Resonance ((1)H NMR) spectroscopy for studying the changes of the metabolic profiles of human faecal slurries. Faeces of 16 subjects, characterized by different natural levels of lactobacilli and bifidobacteria were recovered before and after 1 month of supplementation with a synbiotic food based on Lactobacillus acidophilus, Bifidobacterium longum and fructooligosaccharides, and analyzed by (1)H NMR. Multivariate statistical approach has been applied to the data obtained and particularly Canonical Discriminant Analysis of Principal Coordinates (CAP). More than 150 molecules belonging to short chain fatty acids, organic acids, esters, alcohols and amino acids were detected and quantified in the samples considered. The number and the extent of these molecules in faecal slurries were strongly affected by the synbiotic food consumption and gave rise to characteristic metabolic signature. In particular, the short chain fatty acid concentrations significantly increased while the amino acids contents decreased. The comparison of the data indicated that the intake of the synbiotic food alters the host metabolism in a measure dependent on the initial level of lactobacilli and bifidobacteria detected in the faecal specimens. The analysis of (1)H NMR profiles with CAP allowed a separation of faecal samples of the subjects on the basis of the synbiotic food intake. The multivariate statistical approach used demonstrated the potential of NMR metabolic profiles to provide biomarkers of the gut-microbial activity related to dietary supplementation of probiotics.

Suitability of high pressure-homogenized milk for the production of probiotic fermented milk containing Lactobacillus paracasei and Lactobacillus acidophilus.

High pressure homogenization (HPH) is one of the most promising alternatives to traditional thermal treatment for food preservation and diversification. In order to evaluate its potential for the production of fermented milks carrying probiotic bacteria, four types of fermented milks were manufactured from HPH treated and heat treated (HT) milk with and without added probiotics. Microbiological, physicochemical and organoleptic analyses were carried out during the refrigerated period (35 d at 4 degrees C). HPH application to milk did not modify the viability of the probiotic cultures but did increase the cell loads of the starter cultures (ca. 1 log order) compared with traditional products. The coagula from HPH-milk was significantly more compacted (P<0.05) (higher firmness) than that obtained with HT-milk, and it had the highest values of consistency, cohesiveness and viscosity indexes compared with fermented milks produced without HPH treatment. All the samples received high sensory analysis scores for each descriptor considered. HPH treatment of milk can potentially diversify the market for probiotic fermented milks, especially in terms of texture parameters.

Two 2[5H]-furanones as possible signaling molecules in Lactobacillus helveticus.

Two 2[5H]-furanones, in association with medium-chain fatty acids, were released in whey by Lactobacillus helveticus exposed to oxidative and heat stresses. This species plays an important role in cheese technology, particularly for Swiss-type cheeses and Grana cheese. Moreover, it significantly contributes to cheese ripening by means of an early autolysis and the release of enzymes during processing. Experimental evidence of the involvement of the two 2[5H]-furanones, detected by a gas chromatography-mass spectrometry/solid-phase microextraction technique, in the autolysis phenomenon has been obtained. Zymograms performed by using renaturing sodium dodecyl sulfate-polyacrylamide gels were used to detect the bioactivity of the supernatants containing the two furanones on fresh cells of the same strain. In addition to bands corresponding to known autolysins, new autolysins were detected concomitant with the exposure of Lactobacillus helveticus to the supernatants, which can be regarded as conditioned media (CM), and to a commercial furanone, 5-ethyl-3-hydroxy-4-methyl-2[5H]-furanone (HEMFi), having spectral data similar to those of the newly described 2[5H]-furanones. Morphological changes were observed when fresh cells were exposed to CM containing the two 2[5H]-furanones and HEMFi. The two furanones produced by Lactobacillus helveticus, which met a number of criteria to be included in cell-cell signaling molecules, have a presumptive molecular mass lower than those of already known 3[2H]-furanones having an autolytic activity and being produced by gram-negative bacteria. Moreover, they present a different chemical structure with respect to the furanones already identified as products of Lactococcus lactis subsp. cremoris or to those identified in some cheeses with Lactobacillus helveticus as a starter culture.

Alteration in cellular fatty acid composition as a response to salt, acid, oxidative and thermal stresses in Lactobacillus helveticus.

The fundamental question in this study is concerned with whether the increase of unsaturated fatty acids in the cell membrane is a general response of certain thermotolerant strains or species when exposed to superoptimal temperatures, and in combination with other stresses, especially oxidative stress. A strain of Lactobacillus helveticus, a species widely used as a starter in the dairy industry and able to tolerate high temperature and NaCl concentrations as well as acidic conditions, was chosen for this study. Cells of strain CNBL 1156, grown in its natural medium (i.e. milk whey), were exposed for 100 min to sublethal combinations of temperature, NaCl, H(2)O(2) and pH, modulated according to a Central Composite Design. The fatty acid composition of cell lipid extract was identified by GC/MS. Polynomial equations, able to describe the individual interactive and quadratic effects of the independent variables on cell fatty acid composition, were obtained. The results and the mathematical models relative to the individual fatty acids indirectly suggest that desaturase activation or hyperinduction play an important role in the response to heat stress. In fact, the relative proportions of oleic, linoleic and palmitic acids increased with temperature in a range between 38 and 54 degrees C. The fatty acid profiles included vernolic acid (up to 37% of total fatty acids), an epoxide of linoleic acid not previously reported in microbial cells. In particular, this epoxide was present in cells exposed to low pH in combination with high temperatures and oxidative stress. In conclusion, these results provide experimental support to the hypothesis that the increase of an oxygen-consuming desaturase system, with a consequent increase in fatty acid desaturation, is a cellular response to environmental stresses able to protect the cells of this anaerobic micro-organism from toxic oxygen species and high temperatures.