Staphylococcus aureus in the Processing Environment of Cured Meat Products.

The presence of Staphylococcus aureus in six dry-cured meat-processing facilities was investigated. S. aureus was detected in 3.8% of surfaces from five facilities. The occurrence was clearly higher during processing (4.8%) than after cleaning and disinfection (1.4%). Thirty-eight isolates were typified by PFGE and MLST. Eleven sequence types (STs) were defined by MLST. ST30 (32%) and ST12 (24%) were the most abundant. Enterotoxin genes were detected in 53% of isolates. The enterotoxin A gene (sea) was present in all ST30 isolates, seb in one ST1 isolate, and sec in two ST45 isolates. Sixteen isolates harbored the enterotoxin gene cluster (egc) with four variations in the sequence. The toxic shock syndrome toxin gene (tst) was detected in 82% of isolates. Regarding antimicrobial resistance, twelve strains were susceptible to all the antibiotics tested (31.6%). However, 15.8% were resistant to three or more antimicrobials and, therefore, multidrug-resistant. Our results showed that in general, efficient cleaning and disinfection procedures were applied. Nonetheless, the presence of S. aureus with virulence determinants and resistance to antimicrobials, particularly multidrug-resistant MRSA ST398 strains, might represent a potential health hazard for consumers.

Effect of high-pressure processing and chemical composition on lipid oxidation, aminopeptidase activity and free amino acids of Serrano dry-cured ham.

Lipid oxidation and proteolysis are essential processes in Serrano dry-cured ham quality. The influence of high pressure processing (HPP) at 600 MPa for 6 min on lipid oxidation, aminopeptidase (AP) activities and free amino acids (FAA) in ripened Serrano hams of different chemical composition after 5 months at 4 °C were studied. HPP increased lipid peroxidation indexes. Composition influenced both indexes, with higher levels in hams of medium or high intramuscular fat (IMF) content and in hams of low or medium salt content or salt-in-lean ratio. HPP lowered AP activities by more than 50%. Composition also affected AP activities, with lower levels in hams of low aw, high IMF content, low salt content or low salt-in-lean ratio. At the end of refrigerated storage, HPP only affected Arg and Tyr levels. Many of the individual FAA reached higher levels in hams of low aw, medium or high IMF content, low or medium salt content, or low or medium salt-in-lean ratio.

Application of recombinant lactic acid bacteria and bifidobacteria able to enrich soy beverage in dihydrodaidzein and dihydrogenistein.

Dihydrodaidzein (DHD) and dihydrogenistein (DHG) are intermediate compounds in the production of equol and 5-hydroxy-equol from daidzein and genistein by certain intestinal bacteria. In this work, we explored the heterologous expression of the daidzein reductase gene from Slackia isoflavoniconvertens DSM22006, responsible for the formation of DHD and DHG, in nine lactic acid bacteria and Bifidobacterium strains under a strong constitutive promoter in the plasmid pNZ:TuR.dzr. All the transformed strains showed high production of DHD and DHG both from pure daidzein and genistein and from the isoflavone glycosides present in soy beverage. In addition, Lactococcus lactis MG1363 pNZ:TuR.dzr, expressing the recombinant daidzein reductase, incremented the production of equol by equol-producing intestinal microbiotas in a colonic environment. Nevertheless, other recombinant strains tested showed the opposite effect to L. lactis MG1363 pNZ:TuR.dzr in the production of equol by intestinal microbiota. Here, we describe for the first time the production of 5-hydroxy-equol by human intestinal microbiota, both from genistein and DHG. The use of DHD and DHG as substrates, compared to daidzein and genistein, resulted in an increment of equol and 5-hydroxy-equol production by intestinal microbiota. The recombinant strains developed would be of value for the development of fermented soy beverage enriched in DHD and DHG with the aim of facilitate equol and 5-hydroxy-equol production by intestinal microbiota.

Effect of Lactococcus lactis expressing phage endolysin on the late blowing defect of cheese caused by Clostridium tyrobutyricum.

Clostridium tyrobutyricum has been identified as a major species associated with the late blowing defect (LBD) of semi-hard and hard cheeses, due to undesirable butyric acid fermentation. To find new strategies to control this spoilage bacterium, we investigated the delivery of a bacteriophage endolysin by a cheese starter culture. The nisin producer Lactococcus lactis subsp. lactis INIA 415 was engineered to produce the CTP1L endolysin, encoded by the virulent bacteriophage ΦCTP1 of C. tyrobutyricum and with a demonstrated lytic activity in vitro, to the cheese matrix. The presence of the nisRK two-component regulatory system in the host strain allowed constitutive expression of the endolysin under the control of the nisA promoter (PnisA), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. Engineered lysins with a second cell wall binding domain were also tested and shown to have improved lytic activity. Transformation of L. lactis subsp. lactis INIA 415 with endolysin delivery plasmids had a detrimental effect on its ability to produce nisin in milk, but did not affect its acidifying capacity. Transformed L. lactis subsp. lactis INIA 415 were evaluated as starters in cheeses contaminated with spores of C. tyrobutyricum. Evolution of microbiological parameters, pH and dry matter of cheeses were studied, and Clostridium metabolism and LBD in cheeses were monitored by sensory and instrumental analyses during ripening. Cheese made with the parental strain L. lactis subsp. lactis INIA 415 delayed LBD by one month, attributable to the activity of the nisin, but it was not sufficient to arrest the growth of C. tyrobutyricum during ripening completely. The use of the endolysin-producing strains in cheese manufacture as single cultures also delayed the appearance of LBD by one month, attributable to the activity of the endolysin produced in situ during ripening, because nisin activity in these cheeses was very low at day 1 and undetectable from 15 days onwards. Endolysin was more effective than nisin in inhibiting Clostridium growth, since cheeses made with the CTP1L or the chimeric derivative producers only as starters showed lower LBD symptoms, higher lactic acid levels and lower concentrations of propionic and butyric acids (associated with off-flavours) than cheese made with the parental strain. Investigation of different promoters to maximise endolysin production may help to implement CTP1L as a tool to control C. tyrobutyricum by L. lactis cheese starter and reduce LBD even further.

Expression of a ß-glucosidase in bacteria with biotechnological interest confers them the ability to deglycosylate lignans and flavonoids in vegetal foods.

Lignans and flavonoids are found in plants in their glycosylated forms and need to be hydrolyzed to aglycones to become bioavailable. Putative ß-glucosidase genes from Lactobacillus mucosae INIA P508 were inserted into the plasmid pNZ:TuR. The strain Lactococcus lactis MG1363 harboring the plasmid pNZ:TuR.glu913 showed high ß-glucosidase activity and was able to transform secoisolariciresinol diglucoside (SDG) into secoisolariciresinol (SECO). Lactic acid bacteria and Bifidobacterium strains harboring pNZ:TuR.glu913 were incubated with a soy beverage supplemented with flax seed extracts. SDG was almost completely consumed by the transformed strains, while concentration of SECO greatly increased. Moreover, these strains showed high deglycosylation of the isoflavone glycosides daidzin and genistin. In addition, other lignan and flavonoid aglycones were produced, i.e. matairesinol, pinoresinol, quercetin, and eriodyctiol. These deglycosylase activities were maintained when this glucosidase gene was cloned in a food grade vector, pLEB590, and transformed into L. lactis MG1363. This is the first report of the use of a food grade plasmid that confers the ability to efficiently catalyze the deglycosylation of lignans, isoflavonoids, flavones, and flavanones. The recombinant bacteria of this study would be of value for the development of fermented vegetal foods enriched in bioavailable forms of lignans and flavonoids.

Production of O-desmethylangolensin, tetrahydrodaidzein, 6'-hydroxy-O-desmethylangolensin and 2-(4-hydroxyphenyl)-propionic acid in fermented soy beverage by lactic acid bacteria and Bifidobacterium strains.

Isoflavones intake is associated with health benefits. The metabolism of isoflavones by bacteria plays a key role in their biotransformation. Therefore, commercial soy drink was fermented by 11 lactic acid bacteria (LAB) and 9 bifidobacteria strains. The majority of the strains showed deglycosylation of the isoflavone glycosides present in soy drink and appearance of the aglycones daidzein, genistein and glycitein. Moreover, we observed the further transformation of daidzein into O-desmethylangolensin (O-DMA) and tetrahydrodaidzein, alongside with dihydrodaidzein (DHD) and a putative isomer of DHD. On the other hand, genistein was transformed by nearly all strains into 6-hydroxy-O-desmethylangolensin (6-hydroxy-O-DMA), but no dihydrogenistein production was registered. A high concentration of 2-(4-hydroxyphenyl)-propionic acid was observed, suggesting the degradation of O-DMA and 6-hydroxy-O-DMA. The potential of LAB and Bifidobacterium strains to produce functional soy drink enriched with bioactive isoflavones is demonstrated in this work.

Inactivation of Listeria monocytogenes during dry-cured ham processing.

The effect of Serrano and Iberian dry-cured ham processing and ripening on Listeria monocytogenes inactivation at the surface of whole hams was investigated. Salted hams were surface inoculated (6.5 log CFU) with a cocktail of 4 L. monocytogenes strains isolated from environment and products of a meat industry. Serrano and Iberian hams were ripened for 16 and 24 months, respectively. A decrease of at least 4.6 log units on the surface of Serrano ham was recorded after 4 months for L. monocytogenes counts, which remained under the detection limit thereafter. L. monocytogenes declined by >5 log units on the surface of Iberian ham during the first 9 months and was not detected afterwards. The higher nitrite content of Serrano ham might have accelerated the decrease of the pathogen. This study validates the inactivation of L. monocytogenes on the surface of whole dry-hams during extended ripening.

Technological Properties of Bifidobacterial Strains Shared by Mother and Child.

Technological processes in the dairy industry and the further passage through the gastrointestinal tract could impair viability and functionality of probiotic bifidobacteria. In the present work, the growth in milk of nine bifidobacterial strains shared by mother and child, their survival to freeze-drying and cold storage, and their behavior in a model cheese were investigated. All the strains exhibited high stability to the technological conditions studied when compared with two commercial strains. Bifidobacterium breve INIA P734 and Bifidobacterium bifidum INIA P671 as adjunct cultures maintained high stability during manufacture and ripening of cheese. Both strains showed, at the end of ripening period, resistance to simulated gastrointestinal conditions. Moreover, their presence did not affect negatively the quality of cheese. B. breve INIA P734 and B. bifidum INIA P671 could be considered as potential candidates for their use in cheese as adjunct cultures.

Influence of different lignan compounds on enterolignan production by Bifidobacterium and Lactobacillus strains.

Enterolignans, i.e. enterodiol and enterolactone, are polyphenols derived from the microbial metabolism of dietary lignans. They are considered phytoestrogens because of their estrogenic/antiestrogenic activity, which confers them benefits to human health when they reach sufficient levels in plasma. Hence, there is a great interest in studying the bacteria involved in enterolignan production. In the present study, three bifidobacterial strains (Bifidobacterium bifidum INIA P466, Bifidobacterium catenulatum INIA P732 and Bifidobacterium pseudolongum INIA P2) were found capable of producing low levels of enterodiol (2-11 µM) from lignan extracts; while another one (Bifidobacterium pseudocatenulatum INIA P946) was found to produce an important increment of the lignan secoisolariciresinol (SECO). Subsequently, the three enterodiol-producing bifidobacteria and another three Lactobacillus strains previously identified as enterolignans producers (Lactobacillus gasseri INIA P508, Lactobacillus salivarius INIA P448 and Lb. salivarius INIA P183), were tested on pure lignans yielding both enterodiol and enterolactone from secoisolariciresinol (SECO), while they did not metabolised the other lignan tested (i.e. matairesinol). B. catenulatum INIA P732 and Lb. gasseri INIA P508 were the strains that transformed the greatest percentage of SECO, yielding enterolactone concentrations above 2 mM. In addition, the formation of the intermediate compound dihydroxyenterodiol was observed as part of SECO transformation by all the strains. In this work, we have demonstrated for the first time how strains of Bifidobacterium and Lactobacillus are capable of carrying out the complete enterolignan metabolism, transforming a purified lignan (SECO) into enterodiol and enterolactone. The isolation and characterization of bacteria able to metabolize lignans and produce enterolignans, especially belonging to Bifidobacterium and Lactobacillus genera, is of biotechnological interest, because of their potential application in functional foods and as probiotics.