Sara Bover-Cid

Improved screening procedure for biogenic amine production by lactic acid bacteria

An improved screening plate method for the detection of amino acid decarboxylase-positive microorganisms (especially lactic acid bacteria) was developed. The suitability and detection level of the designed medium were quantitatively evaluated by confirmation of amine-forming capacity using an HPLC procedure. The potential to produce the biogenic amines (BA) tyramine, histamine, putrescine, and cadaverine, was investigated in a wide number of lactic acid bacteria (LAB) of different origin, including starter cultures, protective cultures, type strains and strains isolated from different food products. Also, several strains of Enterobacteriaceae were examined. Modifications to previously described methods included lowering glucose and sodium chloride concentrations, and increasing the buffer effect with calcium carbonate and potassium phosphate. In addition, pyridoxal-5-phosphate was included as a codecarboxylase factor for its enhancing effect on the amino acid decarboxylase activity. The screening plate method showed a good correlation with the chemical analysis and due to its simplicity it is presented as a suitable and sensitive method to investigate the capacity of biogenic amine production by LAB. Tyramine was the main amine formed by the LAB strains investigated. Enterococci, carnobacteria and some strains of lactobacilli, particularly of Lb. curvatus. Lb. brevis and Lb. buchneri, were the most intensive tyramine formers. Several strains of lactobacilli, Leuconostoc spp., Weissella spp. and pediococci did not show any potential to produce amines. Enterobacteriaceae were associated with cadaverine and putrescine formation. No significant histamine production could be detected for any of the strains tested.

Amino acid-decarboxylase activity of bacteria isolated from fermented pork sausages.

The occurrence of amino acid-decarboxylase activity in 92 strains of lactic acid bacteria, coagulase-negative staphylococci, and Enterobacteriaceae isolated from Spanish fermented pork sausages was investigated. The presence of biogenic amines in a decarboxylase synthetic broth was determined by ion-pair high performance liquid chromatography with o-phtalaldehyde post-column derivatization. Among the 66 lactic acid bacteria strains tested, 21 lactobacilli (in particular, Lactobacillus curvatus) and all 16 enterococci were amine producers. Tyramine was the main amine produced by these bacteria, although they also produced phenylethylamine, tryptamine, and/or the diamines putrescine and cadaverine. None of the lactic acid bacteria produced histamine. Coagulase-negative staphylococci were found to be negative amine-producers. Aromatic monoamines, apart from histamine, were not formed by Enterobacteriaceae. This family was responsible for cadaverine and putrescine production. The results obtained for biogenic amine production by bacteria in a synthetic medium suggest that amino acid-decarboxylase activity is strain dependent rather than being related to specific species.

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.

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.

Changes in biogenic amine and polyamine contents in slightly fermented sausages manufactured with and without sugar

The effect of sugar omission on biogenic amine contents of slightly fermented sausages during ripening and storage was evaluated. Two batches of spontaneously fermented sausages were produced with and without sugars in two different trials at pilot-plant scale. Ripened sausages were stored at 4 and 19°C for a further 20 days. Tyramine and cadaverine were the main amines formed during ripening, their contents being significantly higher in batches without sugar. High counts of LAB and Enterobacteriaceae could be associated with the production of tyramine and cadaverine, respectively. The occurrence of putrescine depended on the trial and batch. Sausages without sugar contained more putrescine than those with sugar in trial 1, but this was not repeated in trial 2, in which a high production of agmatine occurred. Tryptamine and phenylethylamine were only detected in the later stages of ripening, their contents also being higher in sausages without sugar. Biogenic amine contents generally rose during storage at 19°C, the increase being especially important for cadaverine and tyramine in sausages without sugar. Levels of spermidine and spermine remained constant during ripening and decreased slightly during storage. Sugar omission is not recommended because it might increase biogenic amine accumulation during the manufacture and storage of slightly fermented sausages.

Effect of proteolytic starter cultures of Staphylococcus spp. on biogenic amine formation during the ripening of dry fermented sausages

The effect of proteolytic starter cultures of Staphylococcus carnosus and Staphylococcus xylosus on biogenic amine production was examined during the fermentation process of dry sausages. Microbial counts (lactic acid bacteria, Micrococcaceae and Enterobactenaceae), pH, moisture and proteolysis-related parameters were also studied. The polyamines spermine and spermidine were the main amines found in the raw material and they only showed slight fluctuations during the fermentation. The four elaborated batches presented a significant (P < 0.001) formation of tyramine and putrescine. The main rate of amine production was during the first three days, when a sharp pH decrease and the development of lactic acid bacteria occurred. Sausages fermented with starters had lower amounts of tyramine than naturally fermented sausages (control), but differences in the Micrococcaceae counts were only significant during the first week of the ripening process. A slight formation of diaminopropane, cadaverine, agmatine, tryptamine and phenylethylamine was observed. The amounts of histamine were constant and remained below 0.5 mg/kg of dry matter, while serotonin, octopamine and dopamine were not detected. The sausages with Staphylococcus as starter culture showed strong proteolysis that was correlated with higher pH values than those of the control sausages. However, no positive correlation was found between the proteolysis index and biogenic amine production. Since proteolysis was stronger during the second half of the ripening process, the release of free amino acids as amine precursors occurred later than the early amine production.

Relationship between biogenic amine contents and the size of dry fermented sausages.

Three trials were carried out to study the influence of the diameter on biogenic amine contents and related parameters (pH, humidity and proteolysis) in fermented sausages. The first trial was done on three groups of Spanish dry fermented sausages with different diameter. In the second, two sections (centre and edge) of salchichón sausages were examined. The last trial consisted in the study of the ripening of two batches of sausages fermented under the same conditions but with two different diameters. Biogenic amine contents varied among the different type of products as well as among the same type of samples. Generally, amine levels in the biggest diameter sausages were higher than in the thinnest sausages. Amine contents were higher in the central part of the sausages than in the edge. During the ripening, larger tyramine amounts were formed in sausages with the biggest diameter. Statistical correlations were found among the diameter, the pH, the proteolysis and some amines. The results of the three trials agree with the hypothesis that the diameter is a factor that may affect the formation of biogenic amines during sausage fermentation.

Ion-pair high-performance liquid chromatographic determination of biogenic amines and polyamines in wine and other alcoholic beverages.

An optimised ion-pair reversed-phase high-performance liquid chromatographic method with automatic o-phthalaldehyde post-column derivatization and spectrofluorometric detection for the same-run separation and quantification of 12 biogenic amines and polyamines in alcoholic beverages has been validated. The reliability of the method was satisfactory in terms of linearity (from 0.5 to 15 mg/l), precision (relative standard deviation below 5%), recovery (from 98.7 to 101.1%), and sensitivity (detection limit between 0.03 and 0.06 mg/l). The automatic accomplishment of the derivatization step reduces time and effort of analysis, especially thanks to the easy preparation of the sample.

Influence of hygienic quality of raw materials on biogenic amine production during ripening and storage of dry fermented sausages

The effect of the hygienic status of raw materials on biogenic amine production during ripening and storage (at 4 and 15 degrees C) of fermented sausages was studied. Two portions of fresh lean and back fat pork were stored for 5 days at -20 degrees C (treatment A) and at 4 degrees C (treatment B), respectively. Raw materials of treatment A maintained their hygienic quality high and low amine content. Raw materials of treatment B showed from 1 to 3 log (CFU/g) higher microbial counts and a biogenic amine index near 50 mg/kg, indicating poorer hygienic quality. The quality of raw materials influenced the composition and the concentration of biogenic amines produced during the ripening sausages. Sausages of treatment A (A-sausages) showed a large accumulation of tyramine (up to 100 mg/kg dm) followed by putrescine and cadaverine (<15 mg/kg). In contrast, B-sausages resulted in earlier and much greater amine production, and cadaverine, tyramine, and putrescine levels were 50-, 2.6-, and 6.5-fold higher than those of A-sausages. Other biogenic amines, such as octopamine, tryptamine, phenylethylamine, and histamine were also produced in B-sausages. The higher proteolysis and the lower pH of B-sausages might have favored the decarboxylase activity of microorganisms. Biogenic amine contents of sausages during storage depended on the raw materials used and storage temperature. No significant modification on the amine contents was observed during the storage of A-sausages at either temperature. Greater changes occurred in B-sausages stored at 15 degrees C than in those stored at 4 degrees C. Higher temperatures favored proteolytic and decarboxylase reactions, resulting in increased amine concentrations after storage.

High hydrostatic pressure and biopreservation of dry-cured ham to meet the Food Safety Objectives for Listeria monocytogenes

This work aimed to evaluate the effect of nisin application (biopreservation) combined with high hydrostatic pressure processing (HHP) on the behavior of Listeria monocytogenes CTC1034 intentionally inoculated (at ca. 10(7)cells/g) onto the surface of ready-to-eat (RTE) sliced dry-cured ham. Two types of dry-cured ham, which had different water activities and fat contents were studied (a(w) of 0.92 and 14.25% fat and a(w) of 0.88 and 33.26% fat). Three batches were prepared for each type of product: (C) control, without nisin; (N) nisin directly applied (200 AU/cm(2)) and (F) nisin applied through active packaging, polyvinyl alcohol films with 200 AU/cm(2). Half of the samples were pressurized at 600 MPa for 5min. Counts of L. monocytogenes were periodically monitored throughout 60 days of storage at 8°C. The physico-chemical characteristics of the products enabled the survival of L. monocytogenes, but it was significantly reduced by the presence of nisin. The effect of biopreservation was greater when applied directly to the surface and in the product with lower water activity in comparison with the active packaging and the high water activity products, respectively. The immediate inactivation of L. monocytogenes by HHP ranged from 1.82 to 3.85 Log units, depending on the type of dry-cured ham. The lower the water activity, the less was the inactivation induced by HHP, both immediately and during storage. The reduction of L. monocytogenes immediately after HHP and during storage was more evident in batches with nisin applied directly to the surface of the product. The pathogen was not detected in some samples from day 5 of storage in the product with higher water activity. The effect of nisin applied through active packaging was lower than the direct application. The results of the present study indicated that HHP, as post-processing listericidal treatment, is more effective (both immediately and long term) than the use of nisin as an antimicrobial measure. However, the both hurdles combined (i.e. biopreservation and HHP) provided a wider margin of safety in the control of L. monocytogenes during the storage of RTE cured meat products.