Biologically active amines in fermented and non-fermented commercial soybean products from the Spanish market.

Biologically active amines were determined in commercial soybean products. The antioxidant polyamines were found in both non-fermented and fermented soybean products. Natto and tempeh showed the highest content of polyamines (75-124 and 11-24 mg/kg of spermidine and spermine, respectively). On the other hand, the bacterial-related biogenic amines, tyramine, histamine, tryptamine and ß-phenylethylamine, were detected in practically all fermented products with a high variability. The highest contents were found in sufu, tamari and soybean paste. Extremely high tyramine and histamine contents, 1700 and 700 mg/kg, respectively, found in some sufu samples could be unhealthy. However, biogenic amines observed in the other soybean products should not be a risk for healthy consumers. However, individuals who take monoamine and diamine oxidase inhibitors drugs should be strongly recommended to avoid this kind of products in order to suffer no adverse health effects. These biogenic amines were not detected in non-fermented soybean products.

Isoflavone profile and protein quality during storage of sterilised soymilk treated by ultra high pressure homogenisation.

The application of ultra high pressure homogenisation (UHPH) treatments is useful to obtain fine and stable soymilk emulsions. Changes of isoflavones, protein digestibility and lysine availability during 4 months of storage at 20 ± 2°C in soymilk treated by UHPH (300 MPa and 75°C of inlet temperature) were studied in comparison to UHT-sterilised soymilk (142°C, 6s). Results indicated that although there was a significantly higher extractability of isoflavones in UHT (about 38%) than in UHPH-treated samples (about 15%), similar total contents were found at the end of storage. The interconversion of isoflavones into ß-glucosides was faster in UHT than in UHPH-treated soymilk. Similar evolution of protein digestibility in both UHPH and UHT-treated soymilks was found, being slightly higher in the initial UHT (88.4%) than in UHPH-treated samples (83.3%). No great differences were observed in the % of blocked lysine among samples after treatments, neither in their evolution throughout storage.

Changes of isoflavones and protein quality in soymilk pasteurised by ultra-high-pressure homogenisation throughout storage.

Ultra high pressure homogenisation (UHPH) is an emerging technology to obtain fine and stable soymilk emulsions. Very little information is available about the stability of this kind of product during storage. Changes of isoflavone profile, protein digestibility and lysine availability in pasteurised-UHPH soymilks were studied for 21 days at 4 °C in comparison to heat-pasteurised soymilks. Neither heat nor UHPH treatments affected the isoflavone profile, as the percentage of each chemical form, in comparison with unprocessed-soymilk. During storage, there was a conversion of isoflavone forms to aglycones, being higher in heat-processed samples. In addition, UHPH samples showed a lesser percentage of blocked lysine during storage. No differences were observed in the evolution of protein digestibility among samples. Therefore, UHPH treatment seems to produce a better product, due to lower amounts of aglycones, and higher protein nutritional quality than heat treatment.

Effect of ultra high pressure homogenization treatment on the bioactive compounds of soya milk.

Ultra high pressure homogenization (UHPH) is a useful novel technology to obtain safe and high-quality liquid foods. The effect of UHPH at 200 and 300 MPa in combination with different inlet temperatures (Tin) (55, 65 and 75 °C) on the bioactive compounds of soya milk was studied. Total phytosterols increased with the higher combination of pressure and temperature. The main phytosterol was ß-sitosterol, followed by stigmasterol and campesterol. Total tocopherols in UHPH-treated soya milks decreased as the temperature and pressure increased. UHPH treatment also affected the different chemical forms of tocopherols. No biogenic amines were detected in any of the analyzed soya milks. Meanwhile, the polyamines SPD and SPM were found in all soya milks, being stable to the UHPH treatment. Total isoflavones increased with the higher combination of pressure and temperature. No differences in the isoflavone profile were found, with ß-glucoside conjugates being the predominant form.

Fast simultaneous determination of free and conjugated isoflavones in soy milk by UHPLC-UV.

Ultra high pressure liquid chromatography (UHPLC) has become a widely used technique in separation science as it can shorten analysis time while maintaining resolution and increasing sensitivity. Here we developed a UHPLC method with UV detection for the simultaneous determination of isoflavone aglycones (daidzein, glycitein and genistein), their corresponding glycoside forms (daidzin, glycitin and genistin), and acetyl and malonyl conjugate forms of glycosides (malonyldaidzin, malonylglycitin, malonylgenistin, acetyldaidzin, acetylglycitin, acetylgenistin) in soy milk. The method allowed reliable determinations of 12 isoflavones in less than 8 min of chromatographic elution. It provided a satisfactory linearity ranging from 0.1 to 5mg/L, except for daidzin and genistin, for which it ranged from 0.1 to 10mg/L. In addition, it showed acceptable chromatographic sensitivity, with a detection limit lower than 0.05 mg/L and a determination limit below 0.2mg/L. The precision, in terms of relative standard deviation, was lower than 5% and the accuracy, as mean recovery, was greater than 95%. Total isoflavone content in commercial soy milk samples (n=8) ranged from 1805.22 to 3195.59 mg/kg dw. Genistin and Daidzin were the major isoflavones in all samples. The proportion of aglycones, ß-glucosides, and acetyl and malonyl glucosides differed among the samples. ß-Glucosides were identified as the predominant forms.

Control of biogenic amines in fermented sausages: role of starter cultures.

Biogenic amines show biological activity and exert undesirable physiological effects when absorbed at high concentrations. Biogenic amines are mainly formed by microbial decarboxylation of amino acids and thus are usually present in a wide range of foods, fermented sausages being one of the major biogenic amine sources. The use of selected starter cultures is one of the best technological measures to control aminogenesis during meat fermentation. Although with variable effectiveness, several works show the ability of some starters to render biogenic amine-free sausages. In this paper, the effect of different starter culture is reviewed and the factors determining their performance discussed.

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.

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.

Volatile and biogenic amines, microbiological counts, and bacterial amino acid decarboxylase activity throughout the salt-ripening process of anchovies (Engraulis encrasicholus).

Chemical and microbiological parameters were studied during the industrial production of salt-ripened anchovies (Engraulis encrasicholus). Gradual acidification and increases in the proteolysis index and in total volatile basic nitrogen were observed. At the end of the maturing process, the values reached pH 5.55 +/- 0.03, 21.33 +/- 5.82%, and 44.06 +/- 12.47 mg/ 100 g, respectively. In the three studied anchovy batches, the biogenic amines tyramine, histamine, putrescine, cadaverine, and agmatine increased during ripening. The highest values were found in the batch where initial microbial load was highest (batch 1), especially for enterobacteria and enterococci. Tyramine was the most abundant amine, reaching values from nondetectable to 90 mg/kg, whereas histamine did not surpass 20 mg/kg. Among the microorganisms isolated, Enterobacter cloacae, Aerococcus viridans, Kocuria varians, and Staphylococcus chromogenes were able to decarboxylate amino acids and produce biogenic amines in vitro. Most (70.59%) of the microorganisms identified were able to produce histamine, 23.53% were able to produce the diamines putrescine and cadaverine, and only 11.76% were able to produce tyramine, although this substance was the major biogenic amine found in anchovy samples.

Influence of starter and nonstarter on the formation of biogenic amine in goat cheese during ripening.

Two commercial starters were investigated for their potential ability to decarboxylate amino acids during goat cheese ripening. Two batches of goat cheese were produced with identical pasteurized milk but different starter cultures. One of them contained Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris and the other Lactococcus lactis subsp. lactis. The amine contents, microbial counts, proteolysis-related parameters, pH, total solids and salt content were studied in raw materials and cheeses. In raw materials, polyamines were the prevailing amines, whereas the main amines in cheeses were putrescine, tryptamine and, in particular, tyramine (94.59 mg/kg). Aerobic mesophilic microorganisms and Lactococcus counts increased throughout ripening, while Enterobacteriaceae were no longer detectable in cheese after 30 days of ripening. Amine concentration rose during cheese ripening in both batches. Moreover, the decarboxylase activity of microorganisms isolated from samples during cheese ripening was assayed and discussed.

Trimethylamine and total volatile basic nitrogen determination by flow injection/gas diffusion in Mediterranean hake (Merluccius merluccius).

The reliability of flow injection/gas diffusion (FIGD) methods to determine trimethylamine (TMA-N) and total volatile basic nitrogen (TVB-N) in hake was studied in order to find an alternative and accurate, simple, cheap, and rapid method for non-protein nitrogen determination. FIGD methods involved extracting volatile amines with 7.5% trichloroacetic acid, followed by the injection of the extracts into the FIGD manifold, previously adjusted for TMA-N or TVB-N determinations. Each determination took approximately 2 min. Reliability was satisfactory in linearity, precision, recovery, and sensitivity. There was good correlation (p < 0.001) between FIGD and the classic official methods, for both TMA-N and TVB-N determinations, and also between FIGD and the gas chromatographic procedure described for TMA-N. These results proved that FIGD methods are simpler, cheaper, and faster than current official procedures. To check the suitability of FIGD procedures over a wide range of analyte concentrations, changes of both TMA-N and TVB-N and the P ratio values throughout the ice storage of hake were monitored. The usefulness of each of these potential freshness indicators for hake is discussed.

Biogenic amines and polyamines in milks and cheeses by ion-pair high performance liquid chromatography.

The proposed chromatographic method provides a complete resolution of twelve amines in a single run in milks and unripened cheeses, avoiding the losses of resolution linked to fluctuations in working temperature. We also propose an alternative chromatographic gradient, which can be useful for samples that have undergone long ripening periods, like ripened cheeses. According to the results of the reliability study, the method described was precise, accurate, and sensitive. The method was applied to several samples of milks and cheeses and the results showed that the biogenic amine profiles varied greatly, not only between different types of samples but also among the samples from the same kind of products. In unripened cheeses, milks, and yogurts, spermidine and spermine were the prevailing amines, but in ripened cheeses the major amine was tyramine, followed by putrescine and cadaverine.

Stability of vitamins during the storage of liquid infant milks.

The stability of the water-soluble B vitamins and the fat-soluble vitamins A and E of three liquid infant milks was followed throughout 12 months of storage at 20, 30 and 37 degrees C. Two samples were 'follow-on' milks from two different manufacturers and the other a 'junior' milk from one of these. The levels of water-soluble vitamins and vitamin E remained constant during storage at the three temperatures. In contrast, vitamin A decreased in samples from one manufacturer at all storage temperatures, and in samples from the other only at 37 degrees C. A kinetic equation was derived that made it possible to predict the influence of storage temperature on the shelf life of these kinds of products, together with the storage period during which levels of vitamin A recommended as adequate would remain.

Progress of browning reactions during storage of liquid infant milks.

Changes in furfural compounds and reactive lysine were monitorized in three commercial liquid infant milks during 9 months of storage at 20, 30, and 37 degrees C. Samples consisted of two ultrahigh temperature (UHT)-treated milks and one conventionally sterilized milk. Reactive lysine remained constant throughout storage at the three temperatures, whereas, in general an increase in furfural compounds was observed. The heat treatment used in the manufacture of milk is an important factor that influences the levels of furfural compounds although the composition of the milk is also a critical factor. Finally, a study was conducted to find the kinetic equations describing furfural compounds changes and allowing for the prediction of the influence of time and temperature of storage on those changes.

Determination of available lysine in infant milk formulae by high-performance liquid chromatography.

A high-performance liquid chromatography (HPLC) method to determine available lysine is proposed. Available lysine was measured by an optimised fluorodinitrobenzene method on the basis of the reactivity of the free epsilon-amino group of the lysine. The classical acid hydrolysis has been improved and shortened from the usual time of 12 h to 2 h 30 min using an oil bath. Optimal resolution and quantitation of Nn-dinitrophenyllysine was obtained with a Nova-Pak C18 column using an isocratic elution with 35% methanol and 65% 0.01 M sodium acetate buffer (pH 4.5) and a flow-rate of 1 ml/min. Satisfactory results were obtained for the reliability of the method in terms of linearity from 0.1 to 5.0 mg/l of lysine-free base, precision (R.S.D. values between 4.3% and 7.8%), recovery (91.5%) and sensitivity (detection limit of 0.02 mg/l). The proposed method has also been checked for lack of interferences from other dinitrophenyl-amino acids.

Determination of vitamins A and E in infant milk formulae by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method was developed for the determination, in one run, of retinol and alpha-tocopherol in infant milk formulae. The method involved saponification at room temperature and a later extraction of vitamins with n-hexane. The vitamins were resolved with a C18 reversed-phase column and they were detected by UV spectrophotometry. Linearity, precision, recovery and sensitivity were satisfactory. The main advantage of the method proposed is the simultaneous determination of both vitamins using a common extraction procedure and UV detection with a variable-wavelength spectrophotometer.

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.

Changes in Biogenic Amines during the Manufacture and Storage of Semipreserved Anchovies †.

Biogenic amines were determined by using high-pressure liquid chromatography during spoilage of raw Engraulis encrasicholus (anchovies), during ripening and shelf life. Histamine, cadaverine, tyramine, and putrescine were extensively formed at the beginning of spoilage, while ß-phenylethylamine and tryptamine were formed only at advanced stages of spoiling. Serotonin, spermine, and spermidine levels were constant throughout spoilage. Seventeen lots of anchovies were evaluated through the typical ripening process. Each ripening process lasted 18 to 22 weeks, Only a slight formation of biogenic amines was observed during the first period of ripening (2 to 4 weeks). According to our results, ripening had little influence on the formation of amines, and therefore the amount of amines in the final products depends primarily on the levels of these substances in the raw material. During shelf-life studies, anchovies packed in brine were more stable, from the point of view of biogenic amine formation, than anchovies packed in oil. High formation of tryptamine and histamine was observed in both products when they were stored at room temperature. Refrigerated storage prevents amine formation.