Reduction of salt content variability of dry-cured ham production using non-invasive technologies in an industrial environment.

Salt content variability of dry-cured ham production is a challenge for the industry since many factors can influence salt uptake during the salting procedure. The aim of this work was to define and evaluate different modifications of the salting procedure to reduce the salt content variability of an industrial dry-cured ham production. Results showed that magnetic induction (MI) is a valid technology for industrial purposes as it can predict in-line the fat and salt contents of hams with a percentage error of 1.75% and 0.38%, respectively. Modifications of the salting process defined according to raw material characteristics obtained in-line reduced the salt content variability (SD) of the global production from 0.337% to 0.283%. Moreover, a 25% reduction of the salt content variability in hams of similar weight and fat content could be achieved when using a reclassification of the defined categories with MI technology after 6 days of salting. Because of the complexity of the salting process, new tools combined with strategies need to be investigated and developed to overcome the variability coming from other sources than weight and the fat content of hams.

Computed tomography and predictive microbiology for non-invasive evaluation of the impact of dry-cured ham production process conditions on the behaviour of Listeria monocytogenes and Clostridium botulinum.

Variability of salt content in dry-cured ham production can pose microbiological food safety issues, especially in salt reduced and/or non-nitrified products. In this regard, computed tomography (CT) could help to non-invasively characterised the product to further adjust the production process and ensure its safety. The aim of this work was to study the application of CT to estimate aw in dry-cured ham to be used by predictive microbiology to evaluate the impact of the production process on the behaviour of Listeria monocytogenes and Clostridium botulinum. Effect of nitrite elimination and fat content of hams was also evaluated. Thirty hams with two different fat content levels were characterised analytically and using CT at different key points in the process. The safety of the process was evaluated by applying predictive microbiology using both analytical and CT data as model inputs. Results showed that nitrite and fat content had an impact on the predicted growth potential of the pathogens evaluated. After the resting period, if no nitrite is added, the time needed for 1 log increase (tinc) of L. monocytogenes would shorten by 26% and 22% in lean and fat hams, respectively. After week 12, important differences on tinc values for C. botulinum were found between both groups of hams (ca. 40% shorter in fat hams). CT can provide reliable pixel-to-pixel information for predictive microbiology to evaluate the growth of relevant pathogens, but further studies are needed to validate this combination as a tool to evaluate the safety of the production process.

Tyramine and histamine risk assessment related to consumption of dry fermented sausages by the Spanish population.

Tyramine and histamine are the main dietary bioactive amines related to acute adverse health effects. Dry fermented sausages can easily accumulate high levels of these hazards and are frequently consumed in Spain. The present work aims to assess the exposure to tyramine and histamine from the consumption of dry fermented sausages by the Spanish population and to assess the risk to suffer acute health effects from this exposure. A probabilistic estimation of the exposure to these hazards was derived combining probability distributions of these amines in dry fermented sausages (n = 474) and their consumption by the Spanish population. The mean dietary exposure to tyramine and histamine was 6.2 and 1.39 mg/meal, respectively. The risk of suffering hypertensive crisis or histamine intoxication by healthy population due to tyramine or histamine intake, respectively, exclusively from dry fermented sausages, can be considered negligible. For individuals under treatment with MAOI drugs, the probability to surpass the safe threshold dose (6 mg/meal) was estimated as 34%. For patients with histamine intolerance, even the presence of this amine in food is not tolerable and it could be estimated that 7000 individuals per million could be at risk to suffer the related symptoms after consuming dry fermented sausages.

Modelling the impact of water activity and fat content of dry-cured ham on the reduction of Salmonella enterica by high pressure processing.

This work aimed to quantify the impact of aw and fat content of dry-cured ham on the Log reduction of Salmonella enterica by high pressure (HP). Dry-cured ham with adjusted aw (0.86-0.96) and fat content (10-50%) was inoculated with S. enterica and pressurised (347-852MPa, 5min/15°C), following a Central Composite Design. Polynomial regression indicated a significant impact of pressure and aw on S. enterica HP-lethality. By lowering aw a clear piezoprotection was observed. At low aw (0.88) the S. enterica reduction was little affected by increasing pressure (e.g. 2.3 to 3.2 Logs at 450 to 750MPa, respectively). At the highest aw the estimated inactivation ranged from 3.3 to 8.9 Logs at 450 to 750MPa, respectively. No significant piezoprotective effect on S. enterica was recorded by the fat content. The relevance of food characteristics on the HP-lethality of S. enterica indicate the need to validate the HP effectiveness on the specific product.

Modeling the protective effect of aw and fat content on the high pressure resistance of Listeria monocytogenes in dry-cured ham.

High pressure processing (HPP) is a promising food preservation technology as an alternative to thermal processing for microbial inactivation. The technological parameters, the type of microorganism, and the food composition can greatly affect the microbicidal potential of HPP against spoilage and pathogenic microorganisms. Presently, the number of available models quantifying the influence of food characteristics on the pathogen inactivation is scarce. The aim of this study was to model the inactivation of Listeria monocytogenes CTC1034 in dry-cured ham, as a function of pressure (347-852MPa, 5min/15°C), water activity (aw, 0.86-0.96) and fat content (10-50%) according to a Central Composite Design. The response surface methodology, based on the equation obtained with a stepwise multivariate linear regression, was used to describe the relationship between bacterial inactivation and the studied variables. According to the best fitting polynomial equation, besides pressure intensity, both aw and fat content exerted a significant influence on HP-inactivation of L. monocytogenes. A clear linear piezoprotection trend was found lowering the aw of the substrate within the whole range of tested pressure. Fat content was included in the model through the quadratic term and as interaction term with pressure, resulting in a particular behavior. A protective effect due to the presence of high fat content was identified for pressure treatments above ca. 700MPa. At lower pressure, higher inactivation of L. monocytogenes occurred by increasing the fat content above 30%. The results emphasize the relevant influence of intrinsic factors on the L. monocytogenes inactivation by HPP, making necessary to assess and validate the effectiveness of HPP on specific food products and consequently set process criteria adjusted to each particular food product.

Analysing and modelling the growth behaviour of Listeria monocytogenes on RTE cooked meat products after a high pressure treatment at 400 MPa.

Various predictive models are available for high pressure inactivation of Listeria monocytogenes in food, but currently available models do not consider the growth kinetics of surviving cells during the subsequent storage of products. Therefore, we characterised the growth of L. monocytogenes in sliced cooked meat products after a pressurization treatment. Two inoculum levels (10(7) or 10(4) CFU/g) and two physiological states before pressurization (freeze-stressed or cold-adapted) were evaluated. Samples of cooked ham and mortadella were inoculated, high pressure processed (400 MPa, 5 min) and subsequently stored at 4, 8 and 12 °C. The Logistic model with delay was used to estimate lag phase (λ) and maximum specific growth rate (µmax) values from the obtained growth curves. The effect of storage temperature on µmax and λ was modelled using the Ratkowsky square root model and the relative lag time (RLT) concept. Compared with cold-adapted cells the freeze-stressed cells were more pressure-resistant and showed a much longer lag phase during growth after the pressure treatment. Interestingly, for high-pressure inactivation and subsequent growth, the time to achieve a concentration of L. monocytogenes 100-fold (2-log) higher than the cell concentration prior to the pressure treatment was similar for the two studied physiological states of the inoculum. Two secondary models were necessary to describe the different growth behaviour of L. monocytogenes on ready-to-eat cooked ham (lean product) and mortadella (fatty product). This supported the need of a product-oriented approach to assess growth after high pressure processing. The performance of the developed predictive models for the growth of L. monocytogenes in high-pressure processed cooked ham and mortadella was evaluated by comparison with available data from the literature and by using the Acceptable Simulation Zone approach. Overall, 91% of the relative errors fell into the Acceptable Simulation Zone.

Inactivation of Serratia liquefaciens on dry-cured ham by high pressure processing.

To quantify the inactivation of Serratia liquefaciens exerted by high pressure processing (HPP), slices of dry-cured ham were inoculated and processed combining different levels of technological parameters: pressure (347-852 MPa), time (2.3-15.8 min) and temperature (7.6-24.4 °C) according to a central composite design. Bacterial inactivation, as logarithmic reduction, indicated that S. liquefaciens was relatively sensitive to HPP. Six log reductions were achieved in a total of 10 trials combining pressures of 600 MPa or above with different holding times and temperatures. The inactivation of S. liquefaciens was analysed through the multiple regression analysis to generate a second order polynomial equation. Pressure and time were the two factors which significantly determined the inactivation of S. liquefaciens on dry-cured ham. Temperature did not significantly affect the lethality of the process. The response surface methodology was used to determine optimum process conditions to maximize the inactivation of S. liquefaciens in the experimental range tested. The maximum inactivation of S. liquefaciens in dry-cured ham was achieved by combining a pressure of 650 MPa with a holding time of 8 min. Combinations above these values (i.e. 750 MPa for 13 min) would not significantly improve the lethality of the process.

Influence of technological conditions of sausage fermentation on the aminogenic activity of L. curvatus CTC273.

The influence of technological factors (temperature and relative humidity of the manufacturing process and the diameter of the sausage) on the aminogenic activity of the strain Lactobacillus curvatus CTC273 was evaluated. Inoculation of sausages with L. curvatus CTC273 resulted in the accumulation of large amounts of biogenic amines (higher than 1000 mg/kg dry matter in some samples) during the manufacture of fuet and llonganissa sausages. Sausages produced via process 'A' (3 days at 20-23 °C and 90-95% RH followed by 20 days at 12-14 °C and 70% RH) contained significantly higher amounts (p < 0.05) of biogenic amines than those manufactured via process 'B' (23 days at 12-13 °C and 70-90% RH), specifically tyramine, cadaverine and phenylethylamine in llonganissa and phenylethylamine in fuet. The higher fermentation temperature and relative humidity during the fermentation stage in process 'A' promoted decarboxylase activity in L. curvatus CTC273 and thus favoured amine accumulation. The diameter of the sausages also influenced biogenic amine production. Higher amine levels were found (p < 0.05) in llonganissa than in fuet, regardless of the manufacturing conditions. The effect of the factors considered on the modulation of aminogenic activity is not necessarily linked to the effect of strain growth, but chiefly favouring proteolytic and decarboxylase reactions.

Model for Listeria monocytogenes inactivation on dry-cured ham by high hydrostatic pressure processing.

The aim of the work was to develop and validate a model of the inactivation of Listeria monocytogenes on dry-cured ham by high hydrostatic pressure (HHP) processing, as a function of the technological parameters: intensity, length and fluid temperature. Dry-cured ham inoculated with L. monocytogenes was treated at different HHP conditions (at 347-852 MPa; for 2.3 to 15.75 min; at 7.6 to 24.4 °C) following a central composite design. Bacterial inactivation was assessed in terms of logarithmic reductions of L. monocytogenes counts on selective media. According to the best fitting and most significant polynomial equation, pressure and time were the most important factors determining the inactivation extent. The significance of the quadratic term of pressure and time indicated that little effect was observed below 450 MPa, whereas holding time longer than 10 min did not result in a meaningful reduction of L. monocytogenes counts. Temperature did not show significant influence at the range assayed. The model was validated with results obtained from further experiments and bibliographical data within the range of the experimental domain. The accuracy factor and bias factor were within the proposed acceptable values indicating the suitability of the model for predictive purposes, such as prediction of the process criteria to meet the Food Safety Objectives. The results of this work may help food processors to select optimum processing conditions of HHP.

Technological conditions influence aminogenesis during spontaneous sausage fermentation.

The influence of two manufacturing processes on biogenic amine formation during the manufacture of Spanish dry fermented sausages of different diameters (fuet and llonganissa) was evaluated to elucidate which conditions allow better control of the aminogenic activity of spontaneous microbiota. Technological conditions affected both the amounts and the qualitative profile of biogenic amine accumulated. The higher processing temperature and relative humidity in process A (simulating those applied in industrial manufacture) favoured aminogenesis, since biogenic amine accumulation was faster and higher than in sausages manufactured under the process B (close to those used in traditional practices). The major amine differed depending on the diameter of the sausages, tyramine being the major amine in fuet (2.5 cm diameter sausage), and putrescine in llonganissa (4.5 cm). Moreover, sausages of higher diameter (llonganissa) had higher biogenic amine contents compared with the thinnest sausages (fuet). Conditions would modulate biogenic amine accumulation not only due to its influence on development of the bacterial population but also on its aminogenic activity. From the biogenic amine point of view, when sausages are spontaneously fermented, traditional lower temperatures and relative humidities are more appropriate than those usually applied in industrial processes.

Distribution of aminogenic activity among potential autochthonous starter cultures for dry fermented sausages.

Any bacterial strain to be used as starter culture should have suitable characteristics, including a lack of amino acid decarboxylase activity. In this study, the decarboxylase activity of 76 bacterial strains, including lactic acid bacteria and gram-positive, catalase-positive cocci, was investigated. These strains were previously isolated from European traditional fermented sausages to develop autochthonous starter cultures. Of all the strains tested, 48% of the lactic acid bacteria strains and 13% of gram-positive, catalase-positive cocci decarboxylated one or more amino acids. Aminogenic potential was strain dependent, although some species had a higher proportion of aminogenic strains than did others. Thus, all Lactobacillus curvatus strains and 70% of Lactobacillus brevis strains had the capacity to produce tyramine and beta-phenylethylamine. Some strains also produced other aromatic amines, such as tryptamine and the diamines putrescine and cadaverine. All the enterococcal strains tested were decarboxylase positive, producing high amounts of tyramine and considerable amounts of beta-phenylethylamine. None of the staphylococcal strains had tyrosine-decarboxylase activity, but some produced other amines. From the aminogenic point of view, Lactobacillus plantarum, Lactobacillus sakei, and Staphylococcus xylosus strains would be the most suitable for use as autochthonous starter cultures for traditional fermented sausages.

Histamine, cadaverine, and putrescine produced in vitro by enterobacteriaceae and pseudomonadaceae isolated from spinach.

A total of 364 bacterial isolates, obtained from spinach leaves, were assayed in a decarboxylase broth containing histidine, lysine, and ornithine to check their ability to produce biogenic amines, and then quantified by high-performance liquid chromatography. Among these isolates, 240 formed cadaverine, 208 formed putrescine, and 196 formed histamine, in widely varying amounts. They frequently produced more than one biogenic amine. Klebsiella pneumoniae subsp. pneumoniae and Morganella morganii were the main histamine producers, with mean values of 1,600 and 2,440 mg/liter, respectively, followed by Pantoea spp. 3 (1,710 mg/liter) and Hafnia alvei (2,500 mg/liter). Enterobacter amnigenus and Enterobacter cloacae produced particularly high amounts of putrescine, with mean values of 2,340 and 2,890 mg/liter, respectively. The strongest cadaverine formation was shown by Serratia liquefaciens (3,300 mg/liter), Serratia marcescens (3,280 mg/liter), and Stenotrophomonas maltophilia (1,000 mg/liter).

Validation of an ultra high pressure liquid chromatographic method for the determination of biologically active amines in food.

Biologically active amines include the so called biogenic amines, such as histamine, tyramine and cadaverine, and polyamines such as spermidine and spermine. Ultra high pressure liquid chromatography (UHPLC) is a new generation of separation techniques that takes full advantage of chromatographic principles to increase speed flow which drastically reduce analysis time. The aim of the present work was to validate a rapid method of UHPLC to detect the presence of biogenic amines and polyamines in food. Different food matrixes (wine, fish, cheese, and dry fermented sausage) were used in order to test the versatility of the method. The UHPLC method described in this article has been demonstrated as a reliable procedure to determine 12 biogenic amines and polyamines in less than 7min of chromatographic elution. The method provides a satisfactory linearity and chromatographic sensitivity with a detection limit lower than 0.2mg/L and a determination limit falling below 0.3mg/L for all amines. The precision, in terms of relative standard deviation, was lower than 5% and the accuracy, as mean recovery, was between 93% and 98%, depending on the food matrix.

Thin-layer chromatography for the identification and semi-quantification of biogenic amines produced by bacteria.

The screening TLC method described enables the simultaneous identification and semi-quantification of eight biogenic amines with a large number of samples handled in a short period of time. The dansylation process time was drastically reduced from 80 to 18 min by increasing the reaction temperature to 100 degrees C. Bacteria could be classified as no, low, moderate or powerful amine producers when no spots were noticeable in TLC plates, less than 50 mg/L, from 50 to 500 mg/L, or more than 500 mg/L of amines were present in the decarboxylase broth medium, respectively. This TLC method is a simpler, faster and less expensive alternative to other methods, such as differential culture media, HPLC and even more sensitive than other TLC procedures.

Relationships between microbial population dynamics and putrescine and cadaverine accumulation during dry fermented sausage ripening.

AIMS: To evaluate the concomitant effects of three technological variables (fermentation temperature, NaCl and glucose added to the meat batter) on diamines (cadaverine, putrescine and histamine) accumulation and microbial changes during ripening of dry fermented sausages. METHODS AND RESULTS: The variables were modulated according to an experimental design and predictive mathematical models were obtained. The models indicated that the sausages were characterized by low histamine amount independently on the applied conditions. In contrast, putrescine and cadaverine accumulation was considerable and significantly affected by the three variables. The microbial population dynamics suggest that lactic acid bacteria (LAB) and microstaphylococci are favoured by increasing glucose concentration until 0.7 g kg(-1), while Enterobacteriaceae are negatively influenced by NaCl concentration and, to a lesser extent, by fermentation temperature. CONCLUSIONS: Data obtained showed a relationship between Enterobacteriaceae growth and cadaverine and putrescine accumulation in sausages during ripening. The conditions more favourable for LAB and microstaphylococci induced a reduced growth of Enterobacteriaceae with a consequent reduced accumulation of putrescine and cadaverine. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of systematic experimental designs allows to individuate the technological conditions suitable to keep the aminogenic microflora under control, thus reducing the risk of diamines production during traditional fermented food manufacture.

Aminogenesis control in fermented sausages manufactured with pressurized meat batter and starter culture.

The application of high hydrostatic pressure (200MPa) to meat batter just before sausage fermentation and the inoculation of starter culture were studied to improve the safety and quality of traditional Spanish fermented sausages (fuet and chorizo). Higher amounts of biogenic amines were formed in chorizo than in fuet. Without interfering with the ripening performance in terms of acidification, drying and proteolysis, hydrostatic pressure prevented enterobacteria growth but did not affect Gram-positive bacteria significantly. Subsequently, a strong inhibition of diamine (putrescine and cadaverine) accumulation was observed, but that of tyramine was not affected. The inoculated decarboxylase-negative strains, selected from indigenous bacteria of traditional sausages, were resistant to the HHP treatment, being able to lead the fermentation process, prevent enterococci development and significantly reduce enterobacteria counts. In sausages manufactured with either non-pressurized or pressurized meat batter, starter culture was the most protective measure against the accumulation of tyramine and both diamines.

Diversity of microorganisms in the environment and dry fermented sausages of small traditional French processing units.

Naturally fermented sausages produced in nine traditional French processing units and their environmental surfaces were characterised by microbial and physico-chemical analyses. Salmonella and Staphylococcus aureus were not detected in the environment whereas Listeria monocytogenes was detected in four samples. Staphylococcus/Kocuria, lactic acid bacteria (LAB), Enterobacteriaceae, Pseudomonas, yeasts/moulds and enterococci contaminated the surfaces of two processing units, indicating insufficient cleaning and disinfection procedures. The final sausages did not present any health risk in seven of the processing units. In two of the processing units, the final sausages were contaminated with S. aureus and L. monocytogenes, respectively, at levels exceeding the maximum tolerable limit. Staphylococcus/Kocuria and LAB grew well in the products. Biogenic amines were found in the majority of the final products. Their occurrence was associated with high numbers of lactic acid bacteria and enterococci. The study outlined the processing and microbial diversities of French naturally fermented sausages.

Safety properties and molecular strain typing of lactic acid bacteria from slightly fermented sausages.

AIM: To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR, plasmid profiling and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize 250 lactic acid bacteria (LAB) isolated from 21 low acid Spanish fermented sausages. Lactobacillus sakei was the predominant species (74%) followed by Lactobacillus curvatus (21.2%) and Leuconostoc mesenteroides (4.8%). By plasmid profiling and RAPD-PCR 144 different strains could be differentiated, 112 belonging to Lact. sakei, 23 to Lact. curvatus and 9 to Leuc. mesenteroides. Ion-pair high performance liquid chromatography was used to detect biogenic amine production. Tyramine and phenylethylamine were produced by 14.4 and 12.4% of the isolates, respectively, all belonging to the species Lact. curvatus. The production of tyramine was stronger than that of phenylethylamine. Partial sequencing of the tyrosine decarboxylase gene from Lact. curvatus was achieved. A specific PCR assay to detect the Lact. curvatus tyramine-producers was designed. The disc diffusion test was used to detect antibiotic resistance among the isolates. Most isolates displayed resistance to vancomycin and gentamicin. Only four strains were resistant to most of the antibiotics tested. None of the isolates were resistant to erythromycin. CONCLUSIONS: Lactobacillus sakei would be the species of choice for further use as starter culture in fermented sausage production. Strain typing and characterization of biogenic amine production together with antibiotic susceptibility testing for the selection of starter cultures could help to increase the quality and safety of the products. SIGNIFICANCE AND IMPACT OF THE STUDY: Species-specific PCR, RAPD and plasmid profiling proved to be efficient at typing LAB at species and strain level. Information on biogenic amine production and transferable antibiotic resistance is important in order to avoid selection of strains with undesirable properties as starter cultures.

Molecular, technological and safety characterization of Gram-positive catalase-positive cocci from slightly fermented sausages.

The population of Gram-positive catalase-positive cocci from slightly fermented sausages was characterized at species and strain level by molecular techniques and some technological and hygienic aspects were also considered. Staphylococcus xylosus was the predominant species (80.8%) followed by Staphylococcus warneri (8.3%), Staphylococcus epidermidis (5.8%) Staphylococcus carnosus (4.6%), and Kocuria varians (0.4%). Proteolytic activity was observed in 23% of the isolates. The species with the highest percentage of proteolytic strains was S. warneri. Lipolytic activity was found in 45.8% of the isolates and S. xylosus was the species with the highest percentage of lipolytic isolates. Biogenic amine production was not widely distributed (only 14.6% of the isolates). Tyramine was the most intense amine produced, although by only 4.6% of the isolates. Phenylethylamine was more frequently detected (10.8% of isolates) but at lower levels. Some strains also produced putrescine (3.3%), cadaverine (2.9%), histamine (1.3%) and tryptamine (0.4%). All isolates were susceptible to linezolid and vancomicin and over 70% were resistant to penicillin G, ampicillin and sulphonamides. Most of the mecA+ strains (only 4.6% of isolates) also displayed resistance to multiple antibiotics. A reduced enterotoxigenic potential was found. Only 3.3% of isolates showed staphylococcal enterotoxins genes, all identified as entC gene. The combination of RAPD-PCR and plasmid profiling allowed the discrimination of 208 different profiles among the 240 Gram-positive catalase-positive cocci characterized, indicating a great genetic variability.

Biogenic amine index for freshness evaluation in iced Mediterranean hake (Merluccius merluccius).

Biogenic amine accumulation was studied during the ice storage of Mediterranean hake. Sensory analysis and counts of Shewanella, Pseudomonas, enterobacteria, psychrotrophic, and mesophilic bacteria provided complementary information on hake spoilage. Putrescine and cadaverine were the main amines accumulated, whereas histamine and tyramine were minor amines but had qualitative interest from the hygienic point of view. Although all biogenic amines were less abundant than in pelagic fish, they may also be used as indicators of freshness and/or spoilage in hake. Cadaverine was the amine best correlated with Shewanella, which was the specific spoilage organism. Therefore, cadaverine may be regarded as the specific spoilage biogenic amine for hake stored at chilling temperatures. However, the biogenic amine index, which considers cadaverine, putrescine, histamine, and tyramine, has several advantages as an indicator of hake quality. Taking into account sensory data, an acceptability limit of the biogenic amine index could be established in 15 to 20 microg/g.