Maria Izquierdo-Pulido

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.

Determination of water-soluble vitamins in infant milk by high-performance liquid chromatography

A rapid, simple and reliable liquid chromatographic method has been developed for the simultaneous determination of nicotinamide, thiamin, riboflavin, pyridoxine, pyridoxal, pyridoxamine, cyanocobalamine and folic acid in liquid and powdered infant milk. Ion-pair chromatography with a reversed-phase C18 column is used. Six vitamins were resolved in a single analysis; total analysis time never exceeded 55 min. A mobile phase of methanol-water (15:85), 5 mM octanesulfonic acid, with 0.5% triethylamine at pH 3.6 and a flow-rate of 1.0 ml/min gave the most satisfactory separation of these vitamins using a UV detector set at different wavelengths. Sample preparation involves acidification to precipitate proteins, and centrifugation followed by gravity filtration. Linearity, precision, recovery and sensitivity were always satisfactory. Detection limits ranged from 0.02 to 0.10 microgram/ml and determination limits ranged from 0.03 to 0.25 microgram/ml.

Flavanol and Flavonol Contents of Cocoa Powder Products : Influence of the Manufacturing Process

Major brands of cocoa powder products present in the Spanish market were analyzed for monomeric flavanols [(+)-catechin and (-)-epicatechin] and flavonols [quercetin-3-glucuronide, quercetin-3-glucoside (isoquercitrin), quercetin-3-arabinoside, and quercetin]. In addition, the influence of the manufacturing process of cocoa powder products, in particular, the alkalinization treatment ( Dutching), on the original content of these flavonoids has been studied. (-)-Epicatechin was in the range of 116.02-730.26 microg/g, whereas (+)-catechin was in the range of 81.40-447.62 microg/g in the commercial cocoa products studied. Among flavonols, quercetin-3-arabinoside and isoquercitrin were the major flavonols in the cocoa powder products studied, ranging from 2.10 to 40.33 microg/g and from 3.97 to 42.74 microg/g, respectively, followed by quercetin-3-glucuronide (0.13-9.88 microg/g) and quercetin aglycone (0.28-3.25 microg/g). To our knowledge, these results are the first quantitative data in relation to the content of individualized flavonol derivatives in commercial cocoa powder products. The alkalinization treatment resulted in 60% loss of the mean total flavonoid content. Among flavanols, (-)-epicatechin presented a larger decline (67%, as a mean percentage difference) than (+)-catechin (38%), probably because of its epimerization into (-)-catechin, a less bioavailable form of catechin. A decline was also confirmed for di-, tri-, and tetrameric procyanidins. In the case of flavonols, quercetin presented the highest loss (86%), whereas quercetin-3-glucuronide, quercetin-3-arabinoside, and isoquercitrin showed a similar decrease (58, 62, and 61%, respectively). It is concluded that the large decrease found in the flavonoid content of natural cocoa powder, together with the observed change in the monomeric flavanol profile that results from the alkalinization treatment, could affect the antioxidant properties and the polyphenol biovailability of cocoa powder products.

Milk Does Not Affect the Bioavailability of Cocoa Powder Flavonoid in Healthy Human

Background: The beneficial effects of cocoa polyphenols depend on the amount consumed, their bioavailability and the biological activities of the formed conjugates. The food matrix is one the factors than can affect their bioavailability, but previous studies have concluded rather contradictory results about the effect of milk on the bioavailability of polyphenols. Aim: The objective was to evaluate the possible interaction of milk on the absorption of (–)-epicatechin ((–)-Ec) from cocoa powder in healthy humans. Methods: 21 volunteers received three interventions in a randomized crossover design with a 1-week interval (250 ml of whole milk (M-c) (control), 40 g of cocoa powder dissolved in 250 ml of whole milk (CC-M), and 40 g of cocoa powder dissolved with 250 ml of water (CC-W)). Quantification of (–)-Ec in plasma was determined by LC-MS/MS analysis prior to a solid-phase extraction procedure. Results: 2 h after the intake of the two cocoa beverages, (–)-Ec-glucuronide was the only (–)-Ec metabolite detected, showing a mean (SD) plasma concentration of 330.44 nmol/l (156.1) and 273.7 nmol/l (138.42) for CC-W and CC-M, respectively (p = 0.076). Conclusion: Cocoa powder dissolved in milk as one of the most common ways of cocoa powder consumption seems to have a negative effect on the absorption of polyphenols; however, statistical analyses have shown that milk does not impair the bioavailability of polyphenols and thus their potential beneficial effect in chronic and degenerative disease prevention.

Determination of ATP related compounds in fresh and canned tuna fish by HPLC

Abstract Reliability of two HPLC methods to determine ATP, ADP, AMP, IMP, inosine and hypoxanthine in fish was studied after modifying mobile phase composition to improve resolution. Both methods included the same extraction procedure, and used a C18 column and UV detection. Mobile phases were neutral phosphate buffer in both methods but one of them used tetrabutylammonium hydrogen sulphate as ionic supressor. Both procedures gave acceptable results, although the addition of the ionic suppressor led to various advantages, such as improved recovery, precision, and sensitivity. Furthermore, chromatographic procedure without ionic suppressor showed a lack of specificity in canned tuna samples, since an unknown peak appeared at ATP retention time. In addition, IMP, Ino and Hx stability in fish samples and in fish samples extracts stored at -18 °C were studied and no changes on their contents were observed throughout 26 weeks.

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.

Comprehensive identification of walnut polyphenols by liquid chromatography coupled to linear ion trap-Orbitrap mass spectrometry.

Epidemiologic studies and clinical trials have demonstrated consistent benefits of walnut consumption on coronary heart disease risk and other chronic diseases. Walnuts (Juglans regia L.) have been described previously as a rich source of polyphenols with a broad array of different structures. However, an accurate screening of its complete phenolic profile is still lacking. In the present work, liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole-Orbitrap mass spectrometry (LC-LTQ-Orbitrap) was applied for a comprehensive identification of phenolic compounds in walnuts. A total of 120 compounds, including hydrolysable and condensed tannins, flavonoids and phenolic acids were identified or tentatively identified on the base of their retention times, accurate mass measurements and subsequent mass fragmentation data, or by comparing with reference substances and literature. The peak area of each signal in mass chromatograms was used to provide semiquantitative information for comparison purposes. The most abundant ions were observed for ellagitannins, ellagic acid and its derivatives. Furthermore, the high-resolution MS analysis revealed the presence of eight polyphenols that have never been reported in walnuts: stenophyllanin C, malabathrin A, eucalbanin A, cornusiin B, heterophylliin E, pterocarinin B, reginin A and alienanin B.

The effects of milk as a food matrix for polyphenols on the excretion profile of cocoa ( − )-epicatechin metabolites in healthy human subjects

The effect of different food matrices on the metabolism and excretion of polyphenols is uncertain. The objective of the study was to evaluate the possible effect of milk on the excretion of (2)-epicatechin metabolites from cocoa powder after its ingestion with and without milk. Twenty-one volunteers received the following three test meals each in a randomised cross-over design with a 1-week interval between meals: (1) 250 ml whole milk as a control; (2) 40 g cocoa powder dissolved in 250 ml whole milk (CC-M); (3) 40 g cocoa powder dissolved in 250 ml water (CC-W). Urine was collected before consumption and during the 0-6, 6-12 and 12-24 h periods after consumption. (2)-Epicatechin metabolite excretion was measured using liquid chromatography-MS. One (2)-epicatechin glucuronide and three (2)-epicatechin sulfates were detected in urine excreted after the intake of the two cocoa beverages (CC-M and CC-W). The results show that milk does not significantly affect the total amount of metabolites excreted in urine. However, differences in metabolite excretion profiles were observed; there were changes in the glucuronide and sulfate excretion rates, and the sulfation position between the period of excretion and the matrix. The matrix in which polyphenols are consumed can affect their metabolism and excretion, and this may affect their biological activity. Thus, more studies are needed to evaluate the effect of these different metabolite profiles on the body.