2. Universidad Cardenal Herrera-CEU

The population dynamic of constitutive biota on 84 samples belonging to two different types of French fermented dry sausages during the ripening process in a pilot-scale ripening chamber was investigated. Samples were analyzed in three steps of their production: fresh product, first drying stage, and finished product. In addition, 180 strains of lactic acid bacteria were identified using a miniaturized biochemical procedure of characterization. In general, the number of lactic acid bacteria that evolved during the ripening process of French dry sausages increased during the first days of the process after which the number of these organisms remained constant at approximately 8 log CFU/g. Lactobacillus sakei and Pediococcus pentosaceus, bacteria added as starter, were the dominant species. Pediococcus urinaeequi, Pediococcus acidilactici, and particularly Lactobacillus curvatus were also present. Finally, we have to take into account that the controlled conditions of the pilot plant generally contribute to the homogenization of the behavior of the starter biota.

The sensitivity of different methods for the isolation and identification of Campylobacter, Listeria and Salmonella was compared, and their application in food safety control in a poultry slaughterhouse was evaluated. The VIDAS, SimPlate, Reveal and VIP systems were used, together with traditional microbiological methods. The study was carried out in a slaughterhouse and in the poultry carving room. One hundred and twenty samples (40 per microorganism) obtained from carcasses, viscera, different chicken parts, water and the environment were evaluated. For Campylobacter, the VIDAS system performed better than plate confirmation. The traditional method yielded results similar to those obtained with the SimPlate method. For Listeria, the plate count method proved less sensitive than the VIDAS, VIP and Reveal systems, which yielded similar results. For Salmonella, the VIDAS system displayed a detection rate comparable to that of traditional methods, while the Reveal system detected twice as many positive samples (16 in total). The Reveal method performed significantly better than either the plate count method or the VIDAS system. The alternative methods used here could be successfully applied in food safety control in poultry slaughterhouses, providing similar or better results and taking less time to perform. The VIDAS system, in general, and the Reveal system in the case of Listeria and Salmonella, appear to be effective alternatives to traditional methods.

During the processing of dry-cured meat products, sarcoplasmic and myofibrillar proteins undergo proteolysis, which has a marked effect on product flavor. Microbial proteolytic activity is due to the action of mostly lactic acid bacteria (LAB) and to a lesser extent micrococci. The proteolytic capacity of molds in various meat products is of interest to meat processors in the Mediterranean area. Eleven LAB and mold strains from different commercial origins were tested for proteolytic activity against pork myosin, with a view to possible use of these strains as starter cultures for Iberian dry-cured ham. Proteolytic activity was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LAB strains with the highest proteolytic activity were Lactobacillus plantarum (L115), Pediococcus pentosaceus (Saga P TM), and Lactobacillus acidophilus (FARGO 606 TM). The best fungal candidate was Penicillium nalgiovense LEM 50I followed by Penicillium digitatum, Debaryomyces hansenii, and Penicillium chrysogenum.

The study was carried out under field conditions in a commercial farm, and 1,440 as-hatched Ross-308 broilers were included. Broilers were randomly distributed into 24 experimental 4-m2 pens (60 broilers/pen). Pens were randomized to the 3 treatment groups: a) tylvalosin 10 mg/kg of live BW during 2 d, b) positive control (tylosin during 2 d), and c) negative control (no treatment). The drugs were provided in the water supply. Mortality, individual BW, and feed intake were assessed. Clostridium presence was assessed in fecal and cecal samples, coccidian oocyst counts were assessed in fecal samples, and bacterial diversity was assessed in ileal content. Live BW at 42 d old was significantly better in the tylvalosin group than in tylosin and no-treatment groups, with tylvalosin-treated broilers reaching 80 to 100 g higher final live weight. Average daily gain results mirrored BW findings. The improvement of feed conversion rate with tylvalosin amounted to 0.13 and to 0.10 versus tylosin and no-treatment, respectively, with mortality being similar in all groups. Significantly reduced sulfite-reducing Clostridium and Clostridium perfringens counts in tylvalosin and tylosin groups versus the no-treatment group were observed in cecum content samples. In conclusion, according to the present study results, tylvalosin, at doses substantially lower than registered for poultry in Europe, has proven effective in controlling the colonization of the cecum by Clostridium ssp. in broilers, improving some productive performances.

Mediterranean mussel (Mytilus galloprovincialis) is a species of bivalve mollusk that has a high capacity in filtering the water nearby and concentrate the virus and bacteria present in it. This can constitute a microbiological risk when they are consumed afterwards. Thus, depending on water pollution, mussels suffer a depuration process, usually controlled using Escherichia coli as indicator. The aim of this study was to determine the microbiological quality of mussels from various sources after purifying, and to consider the utility of the use of Clostridium perfringens as quality indicator. For this, microbiological analysis were performed in fifteen lots of mussel samples obtained from Spain and Italy. Aerobic mesophilic microorganisms, Enterobacteriaceae, E. coli, Vibrio spp., Listeria monocytogenes, Cl. perfringens, and the presence Salmonella spp. were measured. The results showed the presence of Salmonella spp. in four batches of mussels from various locations and Cl. perfringrens in thirteen of them. All samples were within the legal limits for E. coli. In conclusion, the results propose that the use of E. coli as an indicator microorganism for the mussels' depuration process is not sufficient to assure the microbiological quality of the product. Thus, it would be appropriate to include of Cl. perfringens, as an additional indicator, in the control carried out during and after the refinement is done. Finally, the results emphasized the necessity to properly cook this food product prior to consumption, as it allows the destruction of any microorganisms present.

The behaviour of two combined starter cultures and their influence on the microbiological and physicochemical characteristics of dry-cured ham have been evaluated. Three lots of dry-cured hams were tested during processing (0, 9, 48, 74, 112, 142, 166 and 211 days). Lot 1 had no added starter culture. Lot 2 contained a starter culture of Penicillium chrysogenum, Penicillium digitatum, Penicillium nalgiovense, Debaryomyces hansenii, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus and Micrococcus varians was and lot 3 had L. plantarum, L. acidophilus, P. pentosaceus and M. varians. The use of a selected starter culture based on a combination of lactic acid bacteria (LAB) and fungal strains with a demonstrated proteolytic activity such as P. chrysogenum and D. hansenii (lot 2) did not affect the main characteristics of dry-cured ham processing, even enhancing some desirable aspects, like its non-protein nitrogen contents. LAB strains were not significantly affected by combining them with fungal starter, and better counts were found with respect to control. A higher thiobarbituric acid reactive substances content was described in lot inoculated only with LAB (lot 3). Potentially pathogenic microorganisms were not detected in any of the lots studied. The starter culture used in lot 2 showed a potential interest for use in dry-cured ham production.

Nonthermal methods are more efficient at preserving various biological properties of human milk, as compared with holder pasteurization (HoP), which is the most common preservation method. This study was performed to assess the effects of nonthermal processing on bactericidal activity against Escherichia coli in human milk. Milk samples obtained from the Regional Human Milk Bank in Warsaw at Holy Family Hospital were processed by HoP, irradiated with ultraviolet-C (UV-C) for 5, 10, and 15 minutes (6720 J/L each minute), subjected to 2 variations of high-pressure processing (HPP): 450 MPa for 15 minutes and 200 MPa for 10 minutes + 400 MPa for 10 min, with a 10-minutes break. The samples were then evaluated by a bactericidal assay (raw untreated human milk was used as a control). The bactericidal capacity after HoP was preserved in 12.1% of samples, showing a significant reduction in bactericidal properties compared with in raw milk (P < 0.05). The differences between samples preserved by nonthermal methods and raw milk were not significant (P > 0.05). Nonthermal methods of human milk treatment better preserve the bactericidal capacity compared with holder pasteurisation. Those alternative technologies to HoP can be proposed after further investigation for milk processing for Human Milk Banks facilities.

Background: Neonatal infections with Cronobacter sakazakii have recently been associated with the consumption of expressed human milk. Study Aims: (1) To evaluate whether human milk has antimicrobial capacity against C. sakazakii and (2) to determine the stability of its capacity when it is subjected to various treatments. Methods: The antimicrobial capacity of human milk against C. sakazakii was evaluated using an observational, cross-sectional, comparative design. Mature human milk samples (N = 29) were subjected to different treatments. After incubation at 37°C for 72 hr, samples were compared with fresh milk on the stability of their antimicrobial capacity. Two-way analysis of variance (ANOVA) was performed. Results: In fresh milk, counts of C. sakazakii were reduced by 47.26% (SD = 6.74) compared to controls. In treated milk, reductions were: refrigeration at 4°C for 72 hr (M = 33.84, SD = 13.84), freezing at –20°C for 1, 2, and 3 months (M = 40.31, SD = 9.10; M = 35.96, SD = 9.39; M = 26.20, SD = 13.55, respectively), Holder pasteurization (M = 23.56, SD = 15.61), and human milk bank treatment with (M = 14.37, SD = 18.02) and without bovine fortifier (M = 3.70, SD = 23.83). There were significant differences (p < .05) between fresh and treated milk. Conclusions: Human milk has antimicrobial capacity against C. sakazakii. However, its capacity is negatively influenced by common preservation and hygienization methods. Milk should be stored refrigerated for a maximum of 72 hr or frozen for a short period of time.

Milk supplied to neonates in neonatal units is kept at room temperature for some time, which could influence microbial growth. This study aims to evaluate the growth of Escherichia coli in HM and PIF under various treatments and conditions, as well as to determine the influence of different thawing methods on microbial growth in HM. The number of E. coli generations appearing over a 4 h period at 22 °C in HM (frozen; frozen and pasteurized; and frozen, pasteurized, and fortified) and in PIF (four brands) was determined. E. coli counts in HM inoculated and thawed using different methods were also compared. In frozen HM and in pasteurized and frozen HM, significant differences were found after 2.5 h and 1.5 h, respectively. In PIF, differences were found between 1.5 and 3 h. With regard to the thawing process, the lowest microorganism counts were obtained at 4 °C overnight; thus, it seems advisable to store milk at room temperature for a maximum of 1 h during administration in neonatal units. Thawing HM at 4 °C overnight should be the method of choice.

Listeria monocytogenes, a contaminant of raw milk, includes hypervirulent clonal complexes (CC) like CC1, CC4, and CC6, highly overrepresented in dairy products when compared to other food types. Whether their higher prevalence in dairy products is the consequence of a growth advantage in this food remains unknown. We examined growth kinetics of five L. monocytogenes isolates (CC1, CC4, CC6, CC9, and CC121) at 37 and 4 °C in ultra-high temperature (UHT) milk and raw milk. At 4 °C, hypovirulent CC9 and CC121 isolates exhibit better growth parameters in UHT milk compared to the hypervirulent CC1, CC4, and CC6 isolates. CC9 isolate in raw milk at 4 °C exhibited the fastest growth and the highest final concentrations. In contrast, hypervirulent isolates (CC1, CC4, and CC6) displayed better growth rates in UHT milk at 37 °C, the mammalian host temperature. Proteomic analysis of representative hyper- (CC1) and hypovirulent (CC9) isolates showed that they respond to milk cues differently with CC-specific traits. Proteins related to metabolism (such as LysA or different phosphotransferase systems), and stress response were upregulated in both isolates during growth in UHT milk. Our results show that there is a Listeria CC-specific and a Listeria CC-common response to the milk environment. These findings shed light on the overrepresentation of hypervirulent L. monocytogenes isolates in dairy products, suggesting that CC1 and CC4 overrepresentation in dairy products made of raw milk may arise from contamination during or after milking at the farm and discard an advantage of hypervirulent isolates in milk products when stored at refrigeration temperatures.