Formation of biogenic amines in raw milk Hispánico cheese manufactured with proteinases and different levels of starter culture.

Two proteinases, a neutral proteinase from Bacillus subtilis and a cysteine proteinase from Micrococcus sp., were used to accelerate the ripening process of raw cow's milk Hispánico cheese, a semihard variety. Two levels (0.1% and 1%) of a commercial starter culture containing Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were added for cheese manufacture. The influence of both factors, proteinase addition and level of starter culture, on the growth of amino acid-decarboxylating microorganisms and on the formation of biogenic amines during cheese ripening was investigated in duplicate experiments. The population of tyrosine decarboxylase-positive bacteria, which represented less than 1% of the total bacterial population in most cheese samples, and tyrosine decarboxylase-positive lactobacilli was not influenced by proteinase addition or level of starter culture. Tyramine was detected in all batches of cheese from day 30. Its concentration was significantly (P < 0.05) influenced by proteinase addition but not by the level of starter culture and increased with cheese age. After 90 days of ripening, 103 to 191 mg/kg of tyramine was found in the different cheese batches. Histamine was not detected until day 60 in cheese with neutral proteinase and 1% starter culture and until day 90 in the rest of the cheeses. The concentration of this amine did not exceed 20 mg/kg in any of the batches investigated. Phenylethylamine and tryptamine were not found in any of the samples.

Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese.

The influence of two proteinases (Bacillus subtilis neutral proteinase and Micrococcus sp. cysteine proteinase) and two starter culture levels (0.1% and 1%) on biogenic amine formation has been studied in raw ewes' milk Manchego cheese. Amino acid decarboxylating micro-organisms were determined on tyrosine enriched selective media. Biogenic amines were analysed by capillary electrophoresis in citrate buffer at pH 3.6. Addition of proteinases and level of starter culture did not influence the population of micro-organisms with amino acid decarboxylating activity, which represented on average 1% of the bacterial population in 30-day-old cheeses. Tyramine and histamine were detected in all batches of cheese from day 30. Concentrations of tyramine and histamine were higher in cheeses made from milk with neutral proteinase (up to 356 and 284 mg kg(-1), respectively, after 90 days) than in cheeses made from milk with cysteine proteinase (up to 269 and 189 mg kg(-1), respectively) or with no proteinase added (up to 305 and 226 mg kg(-1), respectively). Formation of tyramine and histamine was also favoured in cheeses made with 1% starter culture with respect to cheeses made with only 0.1% starter culture, probably due to the higher pH values of the former cheeses. After 90 days of ripening, concentrations of 10-20 mg kg(-1) phenylethylamine were observed in 9 of the 12 batches, and levels < 10 mg kg(-1) tryptamine were only detected in 3 batches, with no significant relationship between the concentration of these amines and proteinase addition or level of starter culture.

Combined effect of bacteriocin-producing lactic acid bacteria and lactoperoxidase system activation on Listeria monocytogenes in refrigerated raw milk.

The bactericidal activity of three bacteriocin-producing lactic acid bacteria alone and in combination with milk lactoperoxidase (LP) system activation against Listeria monocytogenes in refrigerated raw milk was studied. After 4 d at 4 degrees C, the population of L. monocytogenes in milk inoculated with bacteriocin-producing Lactococcus lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Enterococcus faecalis INIA 4 was reduced by 0.21-0.24 log units. Activation of the LP system did not enhance inhibition at this temperature. After 4 d at 8 degrees C, L. monocytogenes levels in the non-activated LP system milk inoculated with L. lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Ent. faecalis INIA 4 were reduced by 1.87, 1.54 and 1.11 log units compared to control milk, whereas in the activated LP system milk, this reduction was 1.99, 2.10 and 1.06, respectively. The higher nisin production by L. lactis subsp. lactis ESI 515 in milk with activated LP system than in non-activated LP system milk was responsible for the more pronounced decrease of L. monocytogenes counts in the former.

Inhibition of IL-2R and SLA class II expression on stimulated lymphocytes by a suppressor activity found in homogenates of African swine fever virus infected cultures.

Virus free supernatants (VFS) obtained by ultracentrifugation of homogenates of African swine fever (ASF) virus infected cultures inhibited the proliferative response and the expression in peripheral blood mononuclear cells of two activation molecules, the IL-2 receptor (IL-2R) and the swine MHC class II antigens (SLA II), induced by several stimuli (lectins, PMA plus the calcium ionophore A23187 or specific antigen). This inhibition was time dependent: no effect was seen on IL-2R expression when VFS was added after 48 h, when the expression of this molecule reached its maximum. However at this time the proliferative response was still inhibited. The presence of VFS in the cultures was necessary to inhibit both the IL-2R expression and the proliferation of cells. In these conditions the addition of exogenous IL-2 to the cultures failed to restore the IL-2R expression and the proliferation shown by control stimulated cells. Furthermore, the IL-2 activity found in supernatants from cell cultures stimulated with Con A in the presence of VFS was even higher than in cultures stimulated without VFS. The inhibition observed suggests an important impairment of host immunocompetence in ASF infected swine.

Quantification of low levels of rainbow trout immunoglobulin by enzyme immunoassay using two monoclonal antibodies.

An enzyme immunoassay has been developed for quantitation of low levels of trout immunoglobulin (Ig). This assay uses two monoclonal antibodies, one as capture antibody and the other as detector, directed against two non-overlapping epitopes on the heavy chains of trout Ig. The assay shows high reproducibility and can detect 0.12 micrograms trout Ig ml-1. Coefficients of intra- and interassay variation ranged from 3.8 to 7.1% and from 7.9 to 17.4%, respectively. Analysis of 37 healthy trout showed increasing serum Ig concentration with size. The mean Ig concentration was 0.67 mg ml-1 for trout of about 20 g and 9.1 mg ml-1 for trout weighing more than 125 g.

Analysis of T lymphocyte subsets proliferating in response to infective and UV-inactivated African swine fever viruses.

The proliferative response to infective and UV-inactivated African swine fever virus was analyzed in cells from pigs surviving an experimental infection with attenuated virus. All the pigs showed strong dose-dependent proliferative responses to both infective and UV-inactivated virus. This response was also observed when nitrocellulose-bound solubilized virus proteins were used in the assay. Heterologous isolates also induced proliferation, however it was significantly lower than that induced by the isolate used to infect the animals. The response to infective virus was blocked equally by anti-CD4 and anti-CD8 monoclonal antibodies (mAb); the response to UV-inactivated virus was almost abolished by anti-CD4 and 60% inhibited by anti-CD8 mAb. FACS analysis of 28-day T cell lines derived from peripheral blood mononuclear cells demonstrated the progressive increase of the CD8+ subset when the cells were stimulated with infective virus, whereas the stimulation with UV-inactivated virus induced the increase of both CD4+ and CD8+ subsets. In this case, the sum of CD4+ and CD8+ percentages was higher than the total percentage of T cells, suggesting the presence of cells positive for both CD4+ and CD8+.