Coculture of porcine cumulus-oocyte complexes with porcine luteal cells during IVM: effect on oocyte maturation and embryo development.

Coculture with somatic cells is an alternative to improve suboptimal invitro culture conditions. In pigs, IVF is related to poor male pronuclear formation and high rates of polyspermy. The aim of this study was to assess the effect of a coculture system with porcine luteal cells (PLCs) on the IVM of porcine cumulus-oocyte complexes (COCs). Abattoir-derived ovaries were used to obtain PLCs and COCs. COCs were matured invitro in TCM-199 with or without the addition of human menopausal gonadotrophin (hMG; C+hMG and C-hMG respectively), in coculture with PLCs from passage 1 (PLC-1) and in PLC-1 conditioned medium (CM). In the coculture system, nuclear maturation rates were significantly higher than in the C-hMG and CM groups, but similar to rates in the C+hMG group. In cumulus cells, PLC-1 coculture decreased viability, early apoptosis and necrosis, and increased late apoptosis compared with C+hMG. PLC-1 coculture also decreased reactive oxygen species levels in cumulus cells. After IVF, monospermic penetration and IVF efficiency increased in the PLC-1 group compared with the C+hMG group. After invitro culture, higher blastocysts rates were observed in the PLC-1 group. This is the first report of a coculture system of COCs with PLCs. Our model could be an alternative for the conventional maturation medium plus gonadotrophins because of its lower rates of polyspermic penetration and higher blastocysts rates, key issues in porcine invitro embryo production.

Expression of GnRH receptor and 3ßHSD during meiosis and foliculogénesis in Columba livia (Aves: Columbiformes): Histological and immunohistochemical analysis.

During the ovarian ontogeny in birds, five fundamental events can be recognized: migration and colonization of the primordial germ cells, differentiation and proliferation of oogonies, an organization of germinal nests, beginning of the meiotic process and folliculogenesis. The knowledge of these events is fundamental for the interpretation of the processes involved in the differentiation of female gametes. However, there are only references for some model species such as Gallus gallus domesticus and Coturnix coturnix. In a previous study, the histological structure of embryonic ovaries of Columba livia was revealed. Therefore, the objective of this work is to characterize the processes of meiosis and folliculogenesis C. livia from the analysis of the expression of the GnRH receptor, the 3ßHSD enzyme and the cell proliferation protein PCNA in embryonic and postnatal ovaries. Therefore, the expression of GnRHR, 3ßHSD, and PCNA was revealed in histological testicular and ovarian preparations in embryos (stages 41-43) and neonates (2, 5, 7, 10 and 75 days post-hatching). The present study demonstrates that the fate of germline cells is dictated by their location during gonadal development. Thus, the germline cells located in the cortex of the left gonad enter meiosis, while those in the right gonad and those in the medulla of the left ovary fail to go into meiosis. This indicates that somatic signals, instead of an autonomous cellular mechanism, regulate the entry of the germline cells into meiosis in the C. livia embryo. Future studies will be focused on the analysis of proteins associated with meiotic events and folliculogenesis in embryonic and neonatal ovaries of C. livia, to evaluate the regulation of meiosis in vitro.

Apoptosis of bovine granulosa cells: Intracellular pathways and differentiation.

Follicular atresia in granulosa and theca cells occurs by apoptosis through weak hormonal stimulation. We have previously proposed an in vitro model to study this process by inducing apoptosis in BGC-1, a bovine granulosa cell line, and in primary cultures from ovaries with or without corpus luteum (CPGB+ and CPGB-, respectively), with different doses of gonadotropin releasing hormone (GnRH) analogs (leuprolide acetate (LA) as agonist and antide as antagonist). BGC-1 represent immature granulosa cells, whereas CPGB represent different degrees of luteinization. Our aim was to evaluate the intracellular pathways involved in the GnRH regulation of apoptosis in BGC-1. Treatment with LA 100nM but not with antide led to an increase in BAX over BCL-2 expression, showing antagonism of antide. All treatments inhibited phospholipase-D (PLD) activity compared to control, implying agonist behavior of antide. Progesterone in vitro production and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) expression revealed different degrees of luteinization: BGC-1 were immature, whereas CPGB+ were less differentiated than CPGB-. We concluded that LA-induced apoptosis in BGC-1 occurs by activation of the mitochondrial pathway and by inhibition of PLD activity and that antide might work both as an antagonist of the intrinsic pathway and as an agonist of the extrinsic protection pathway by inhibiting PLD activity.

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.

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.

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.

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.

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.

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.