María José Benito

Screening of lactic acid bacteria and bifidobacteria for potential probiotic use in Iberian dry fermented sausages

The purpose of this study was to select lactic acid bacteria and bifibobacteria strains as potential probiotic cultures during the processing of Iberian dry fermented sausages. A total of 1000 strains were isolated from Iberian dry fermented sausages (363), and human (337) and pig faeces (300) in different culture media. Around 30% of these strains, mainly isolated from Iberian dry fermented sausages in LAMVAB agar, were pre-selected for testing as potential probiotics by their ability to grow adequately at the pH values and NaCl concentrations of these meat products during the ripening process. Of the in vitro investigations used to predict the survival of a strain in conditions present in the gastro intestinal tract, exposure to pH 2.5 showed itself to be a highly discriminating factor with only 51 out of 312 pre-selected strains resisting adequately after 1.5h of exposure. All acid-resistant isolates identified as lactobacilli originated from human faeces (Lactobacillus casei and Lactobacillus fermentum) and pig faeces (Lactobacillus reuteri, Lactobacillus animalis, Lactobacillus murinus, and Lactobacillus vaginalis). Pediococcus acidilactici strains were isolated from Iberian dry fermented sausages and pig faeces, whereas the greatest number of Enterococcus strains were identified as Enterococcus faecium, with this species being isolated from Iberian dry fermented sausages, and human and pig faeces. Most of these strains are promising probiotic meat culture candidates suitable for Iberian dry fermented sausages.

Characterization of Micrococcaceae isolated from iberian dry-cured sausages

The populations of Micrococcaceae in different types of Iberian dry-cured sausages from central-west Spain were characterized and their technological and antimicrobial properties determined in order to evaluate their suitability as starter cultures in dry-cured sausage manufacture. Of a total of four hundred strains isolated from two manufacturers, one hundred and sixty-six were selected to evaluate nitrate reductase, proteolytic, lipolytic, and antimicrobial activities, and growth at different values of pH and water activity (a(w)). Most of the strains were identified as Staphylococcus except for eight isolates assigned to Kocuria spp. The species most often isolated was Staphylococccus xylosus. Others were, in descending order of abundance, S. aureus, S. lugdunensis, S. saprophyticus, S. sciuri, S. chromogenes, and S. capitis. The distributions of the minority Staphylococcus species were different for the two manufacturers. All the investigated strains were able to grow at pH and a(w) greater than 5.0 and 0.85, respectively, the values usually found in Iberian dry-cured sausages. Five S. xylosus strains showed antimicrobial activity against some indicator strains which were investigated. Seven strains with the best properties were pre-selected and tested for their lipolytic and proteolytic activities against pork fat and myofibrillar and sarcoplasmic pork proteins, respectively, and for their low biogenic amines production. Most of the strains showed proteolytic and lipolytic activities, but none produced histamine, tyramine, phenylethylamine, or spermine. Three strains, identified as Staphylococcus xylosus, possess useful properties which make them candidates for testing as starter cultures in pilot processing of Iberian sausages.

Application of Lactobacillus fermentum HL57 and Pediococcus acidilactici SP979 as potential probiotics in the manufacture of traditional Iberian dry-fermented sausages.

The purpose of this study was to investigate the potential of implanting the probiotics Lactobacillus fermentum HL57 and P. acidilactici SP979 during manufacture of Iberian dry-fermented sausages, and their effect on the sensory properties of these meat products. Microbial counts were studied in sausages inoculated with these strains and compared with a control batch. To detect the presence of probiotic strains at high counts (>7 log CFU/g) in the inoculated sausages, specific PCR and (RAPD)-PCR techniques were carried out. Changes due to probiotic inoculations on parameters and compounds related to fermentation, and nitrogen and lipid fractions were determined, and the impact on sensory quality evaluated by descriptive and hedonic tests. Iberian dry-fermented sausages inoculated with L. fermentum HL57 and P. acidilactici SP979 may be considered as functional products according to the counts of these strains found at the end of processing. However, inoculation with L. fermentum HL57 increased the amount of acetic acid and lipid degradation products such as malonaldehyde in Iberian dry-fermented sausages, resulting in a negative impact on relevant sensory parameters related to colour and taste. On the contrary, P. acidilactici SP979 did not notably modify the physicochemical parameters or the sensory quality of Iberian dry-fermented sausages when compared to the autochthonous microbial population present in the control batch.

Rapid differentiation of lactic acid bacteria from autochthonous fermentation of Iberian dry-fermented sausages

The populations of lactic acid bacteria (LAB) in different types of Iberian dry-fermented sausages from central-west Spain were identified. A simple and rapid electrophoretic method of whole-cell protein profiles was evaluated, correlating it with 16S rRNA gene sequence analysis and biochemical identification by API 50 CHL. A total of 96 isolates were identified by SDS-PAGE showing stable profiles corresponding to 30-45 polypeptides in the range 95-8kDa that were clearly different for the different species and were grouped with those of the 9 reference strains used in this study. The SDS-PAGE method showed that the predominant species were Pediococcus acidilactici (48%) followed by Lactobacillus plantarum (23%) and Lactobacillus brevis (18%). The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database. However, biochemical identifications by API 50 CHL showed different errors at the genus and species level. In sum, the SDS-PAGE analysis showed itself to be a rapid and accurate differentiation method for the most commonly encountered LAB isolates in dry-fermented sausages.

Effect of Penicillium chrysogenum and Debaryomyces hansenii on the volatile compounds during controlled ripening of pork loins

During ripening of meat products such as dry-cured ham, the moulds and yeasts that proliferate on the surface may contribute to flavour development. However, their contribution to volatile components of dry-cured meat products is not known. One strain each of Penicillium chrysogenum and Debaryomyces hansenii, selected from dry-cured ham by their proteolytic activity, were tested to determine their effect on the volatile compounds during ripening. Sterile pork loins were inoculated and ripened for 106 days. Volatile compounds collected with a Solid Phase Micro-Extraction (SPME) fibre were analysed by GC/MS. Inoculation of pork loins with P. chrysogenum lead to a decrease in compounds attributed to lipid oxidation and to an increase of compounds derived from free amino acids. Inoculation with D. hansenii seemed to favour the formation of complex alcohols.

Safety and functional aspects of pre-selected lactobacilli for probiotic use in Iberian dry-fermented sausages.

The purpose of this study was to investigate lactobacilli for potential probiotic use in Iberian dry-fermented sausages. A total of 18 strains, previously selected, isolated from different sources were evaluated according to safety and functional characteristics including biogenic amines and d-lactic acid production, antibiotic susceptibility, haemolysis, cell adhesion, and antimicrobial activity against food-borne pathogens. The strains Lactobacillus reuteri PL519, L. reuteri PL542, and Lactobacillus fermentum HL57 were able to establish their self on the intestinal epithelium, and an inhibition of such pathogenic bacteria as Listeria monocytogenesin vitro. These strains were also considered safe to be used with regard to their low aminogenic potential and d-lactic acid production, antibiotic resistance pattern and haemolytic activity. Thus, they were selected as a potential probiotic meat starter culture suitable for manufacture of dry-fermented Iberian sausages.

Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

ABSTRACT An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.

Differentiation of Staphylococci from Iberian dry fermented sausages by protein fingerprinting.

The Staphylococci populations in different types of Iberian dry fermented sausages from central-west Spain were identified. A simple electrophoretic method of whole-cell proteins and extracellular protein profiling was evaluated for speed of identification. This study was correlated with a 16S rRNA gene sequence analysis and biochemical identification by API Staph. A total of 81 isolates were identified by SDS-PAGE of the whole-cell proteins. These showed stable profiles in the range 99-14kDa that were clearly different for the different species, and were grouped into clusters together with the profiles of the eight reference strains. SDS-PAGE of the extracellular protein extracts provided additional characteristic banding patterns for the characterization of the Staphylococcus species present. The whole-cell SDS-PAGE showed that the predominant species was Staphylococcus saprophyticus (61.7%) followed by Staphylococcus aureus (19.7%). The identifications were confirmed by the 16S rRNA gene sequencing and by a BLAST search of the GenBank database. However, the API Staph biochemical identifications were frequently erroneous at the species level. In sum, SDS-PAGE analysis showed itself to be rapid and accurate in identifying the most commonly encountered Staphylococcus isolates in dry fermented sausages.

Safety and Functional Aspects of Preselected Enterococci for Probiotic Use in Iberian Dry-Fermented Sausages

The purpose of this study was to investigate enterococci for potential probiotic use in Iberian dry-fermented sausages. A total of 15 strains isolated from Iberian dry-fermented sausages, human feces, and pig feces were evaluated for their safety and functional characteristics including biogenic amine (BA) production, antibiotic susceptibility, hemolysis, virulence determinants, cell adhesion, and antimicrobial activity against foodborne pathogens. The strain Enterococcus faecium SE906 was able to establish itself on the intestinal epithelium, inhibiting such pathogenic bacteria as Listeria monocytogenes in vitro. This strain was also considered safe to be used for its low aminogenic potential, and its antibiotic resistance pattern and virulence determinants, being identified as a potential probiotic meat starter culture suitable for manufacture of dry-fermented Iberian sausages.

Identification of fungal contamination and determination of mycotoxigenic molds by micellar electrokinetic capillary chromatography in smoked paprika

The purpose of this work was to analyze the fungal contamination in smoked and unsmoked paprika processed from different cultivars of pepper and to investigate the ability of these and other mycotoxigenic molds to grow and synthesize mycotoxins in smoked paprika. Eighteen mycotoxins were evaluated using micellar electrokinetic capillary chromatography. No relevant differences were found in fungal contamination between smoked and unsmoked paprika. The number of yeasts obtained was low, ranging from 0.4 to 3.29 log CFU g(-1); most of the yeast strains were identified as Cryptococcus spp. followed by Candida spp. All mold counts were <4 log CFU g(-1). Aspergillus, Cladosporium, Penicillium, and Fusarium were the predominant hyphomycete genera. Six mycotoxins were identified in the extracts of several strains isolated from paprika and incubated on malt extract agar. Penicillium expansum followed by Penicillium citrinum and Penicillium raistrickii were the dominant mycotoxigenic fungi isolated. Most of themycotoxin-producing fungi produced detectable amounts of mycotoxins when grown on paprika agar.