2. Universidad Cardenal Herrera-CEU

The present study reports the natural mycobiota occurring in dry-cured hams, and in particular on the incidence of mycotoxin-producing fungi. A total of 338 fungal colonies were isolated from three stages of production, these being the post-salting, ripening and aging stages in two manufacturing plants. The results show that fungi were more frequently isolated from the aging stage and that the predominant filamentous fungal genus isolated was Penicillium. Seventy-four of the 338 fungal strains were selected for identification at the species level by using morphological criteria and internal transcribed spacers sequencing. Of the 74 fungal strains, 59 were Penicillium strains. Sixteen Penicillium species were identified, with P. commune (24 strains) and P. chrysogenum (13 strains) being the most abundant. The potential ability to produce cyclopiazonic acid (CPA) and ochratoxin A (OTA) was studied by isolating the culture followed by HPLC analysis of these mycotoxins in the culture extracts. The results indicated that 25 (33.7%) of the 74 fungal strains produced CPA. Worth noting is the high percentage of CPA-producing strains of P. commune (66.6%) of which some strains were highly toxigenic. P. polonicum strains were also highly toxigenic. With respect to OTA-producing fungi, a low percentage of fungal strains (9.5%) were able to produce OTA at moderate levels. OTA-producing fungi belonged to different Penicillium species including P. chrysogenum, P. commune, P. polonicum and P. verrucosum. These results indicate that there is a possible risk factor posed by CPA and OTA contamination of dry-cured hams.

Fungi play a key role in dry-cured ham production because of their lipolytic and proteolytic activities. In the present study, 74 fungal strains from dry-cured Teruel hams and air chambers were tested for proteolytic and lipolytic activities, with a view to their possible use as starter cultures. Lipolytic activity of fungi was studied against lauric, palmitic, stearic and oleic acids, whereas proteolytic activity was studied against casein and myosin. Of the 74 fungal strains tested, most of them demonstrated lipolytic activity (94.59 %). Lipolytic activity against lauric and oleic acids was stronger than against palmitic and stearic acids. 39 strains (52.70 %) demonstrated proteolytic activity against casein and the 6 highest proteolytic strains were also tested for pork myosin proteolysis. Some strains belonging to Penicillium commune, Penicillium chrysogenum, Penicillium nalgiovense and Cladosporium cladosporioides were selected because of their significant proteolytic and lipolytic activities and could be suitable to use as starters in dry-cured ham.

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.

En la elaboración del jamón curado los hongos que forman parte su biota superficial desempeñan un papel importante ya que, además de producir reacciones lipolíticas y proteolíticas, algunas especies pueden producir micotoxinas. Este hecho es de especial importancia en procesos de elaboración artesanal, como el del jamón D.O.P. de Teruel, en los que no se emplean cultivos iniciadores, pudiendo desarrollarse en superficie una población fúngica incontrolada que puede afectar a la salubridad y calidad sensorial del producto. El objetivo principal de este trabajo fue caracterizar la biota fúngica del jamón D.O.P. de Teruel, prestando especial atención al estudio de su capacidad toxigénica y enzimática. El estudio se realizó en dos secaderos adscritos a la D.O.P. de Teruel, aislándose un total de 338 cultivos puros de la superficie del jamón y del ambiente de las cámaras de procesado, en 3 etapas de elaboración: postsalado, secado y envejecimiento. Los mayores recuentos fúngicos se obtuvieron en la etapa de envejecimiento, siendo el género mayoritario Penicillium (221 cepas). A continuación se realizó una caracterización molecular de las cepas de Penicillium mediante la técnica de ap-PCR, dando como resultado un gran número de patrones moleculares diferentes. El dendrograma resultante del análisis de los patrones de bandas puso de manifiesto una gran diversidad genética de las cepas de Penicillium aisladas del jamón. También se seleccionaron 74 cepas representantes de la variabilidad morfológica y molecular, para su identificación a nivel de especie mediante la secuenciación de las regiones ITS del ADN ribosomal. Dentro del género Penicillium, el género con mayor número de cepas secuenciadas, se identificaron un total de 16 especies. De ellas, Penicillium commune (32,43%) y Penicillium chrysogenum (17,57%) fueron las más abundantes. El árbol filogenético resultante del análisis de secuencias mostró una variabilidad genética muy pequeña en las secuencias ITS de las cepas de Penicillium analizadas, obteniendo agrupamientos con bajos niveles de significación. Por otro lado, se estudió la capacidad de producción de ácido ciclopiazónico (CPA) y ocratoxina A (OTA) en medio de cultivo de las 74 cepas secuenciadas. Los análisis cromatográficos de dicho estudio indicaron que el 37,84% de las cepas fueron productoras de CPA, siendo P. commune la especie que presentó mayor capacidad de producción (62,5% de las cepas productoras). Respecto a los hongos productores de OTA, el 14,86% de las cepas mostró dicha capacidad aunque en niveles moderados. Las cepas productoras de OTA pertenecían a las siguientes especies: Penicillium verrucosum, P. commune, Penicillium polonicum y P. chrysogenum. Asimismo, se estudió la contaminación por CPA y OTA en 10 muestras comerciales de jamón curado de los dos secaderos muestreados. Se detectó CPA y OTA en 8 y 7 muestras de jamón, respectivamente. Finalmente, se estudió la actividad proteolítica y lipolítica de las 74 cepas secuenciadas, siendo las especies P. commune, P. chrysogenum y Cladosporium cladosporioides las que presentaron mayor capacidad enzimática. Molds play a key role in dry-cured ham production because of their ability to produce mycotoxins and their ability to produce lipolytic and proteolytic activities. Dry-cured Teruel ham is not usually inoculated with starter cultures, so an unwanted fungal population could grow on the surface and reduce their hygienic-sanitary and sensory quality. The aim of the present study was to characterize natural mycobiota occurring in dry-cured Teruel ham, paying special attention to the incidence of mycotoxin-producing fungi and enzymaticproducing fungi. A total of 338 fungal colonies were isolated from three stages of production including postsalting, drying and aging in two manufacturing plants. Fungi were isolated from both the surface of dry-cured hams and the environment of the chambers. The results show that fungi were more frequently isolated from the aging stage and the predominant filamentous fungal genus isolated was Penicillium (221 strains). Then, molecular characterization of Penicillium strains by ap-PCR technique was carried out, obtaining a large number of different molecular patterns. The dendrogram analysis of banding patterns revealed a high genetic diversity of strains of Penicillium isolated. Also, a total of 74 out 338 fungal strains were selected, by morphological and molecular criteria, for identification at species level by ITS sequencing. Within Penicillium, the genus with the largest number of strains sequenced, sixteen Penicillium species were identified being Penicillium commune (32.43%) and Penicillium chrysogenum (17.57%) the most abundant. On the other side, potential ability to produce cyclopiazonic acid (CPA) and ochratoxin A (OTA) in vitro was studied in 74 sequenced strains. Results indicated that 37.84% of fungal strains produced CPA, being P. commune the species that showed higher production ability (62.5% of the producing strains). With respect to OTA-producing fungi, a low percentage of fungal strains (14.86%) were able to produce OTA in moderate levels. OTA-producing fungi belonged to different Penicillium species including Penicillium verrucosum, P. commune, Penicillium polonicum and P. chrysogenum. Otherwise, ten dry-cured ham samples belonged to the last stage of two manufacturing plants included in the study were analyzed allowing to assess the presence of CPA and OTA. CPA was detected in 8 of 10 ham samples and OTA was detected in 7 of 10 ham samples analyzed. Finally, the same 74 strains identified at species level were tested for proteolytic and lipolytic activities. The strains which showed greatest proteolytic and/or lipolytic activity belong to the species P. commune, P. chrysogenum and Cladosporium cladosporioides.