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The antimicrobial activity of hydro-alcoholic extract of Myrtus communis L. was investigate in vitro and in situ against different meat spoilage biotypes of Pseudomonas fragi and Brochothrix thermosphacta. Water-ethanol myrtle extract (WEME) was prepared from myrtle leaves and used to study the antimicrobial activity, Minimum Inhibitory Concentration (MIC) and Minimum Lethal Concentration (MLC). Naturally contaminated ground beef was used to evaluate in situ antibacterial activity of freeze-dried myrtle extract at different concentrations. In vitro antimicrobial activity of WEME was significantly more effective against B. thermosphacta than P. fragi strains. MIC and MLC values were in the range of 12.5–50 and 25–100 mg DM/ml, respectively, for B. thermosphacta and P. fragi strains. In situ results showed that the microbial population, except for Pseudomonas spp., decreased significantly in ground meat with added 5 % of freeze-dried myrtle extract. In conclusion, the findings demonstrate the effectiveness of myrtle extract to control or prevent the proliferation of meat spoilage bacteria.

Ninety three isolates of Brochothrix spp. from raw meat were analysed by PCR-based molecular fingerprinting and identified by species-specific Real-Time (RTi)-PCR and 16S rRNA gene sequencing. Thirty three strains were characterized for their growth capability in sarcoplasmic extract with and without glucose, for in vitro lipolytic activity, in vitro and in situ (in beef) proteolytic activity and for amino-decarboxylase activity. Moreover, spoilage potential of seven selected strains in sterile and non-sterile meat was investigated by analyzing the release of volatile organic compounds (VOCs) during storage in air. All the strains analysed were unable to hydrolyze sarcoplasmic proteins in vitro and in situ and they did not show lipolytic activity at 4. °C or 20. °C. Almost all were able to grow in the presence of sarcoplasmic extract with glucose and produced histamine. The release of VOCs by each strain in sterile and non sterile beef stored at 4. °C in air was evaluated by HS-SPME-GC/MS analysis. Acetoin and 1-octen-3-ol and 3-methyl-1-butanol were the major compounds isolated from sterile and non-sterile meat samples inoculated with single strains of Brochothrix (B.) thermosphacta and high concentration of acetoin was found in all inoculated meat samples. The role of B. thermosphacta as meat spoiler does not seem to be influenced by indigenous microbiota of meat while its development in meat is associated to a significant increase (P.

Lactobacillus curvatus 54M16 was isolated from traditional fermented sausages of Campania region (Italy) and identified by 16S rRNA and hsp60 gene sequencing and by SDS-PAGE of whole cell proteins. The strain produced more than one bacteriocin, carrying the genes for sakacin X, T and P as demonstrated by PCR studies. The ability of L. curvatus 54M16 to produce multiple bacteriocins is confirmed by molecular mass spectrometry analysis and N-terminal amino acid sequencing. Among the bacteria tested, the pathogens Listeria monocytogenes and Bacillus cereus and the meat spoilage Brochotrix thermosphacta were sensitive to the bacteriocins. No inhibition of tested Gram-negative bacteria, some strains of Lactobacillus and of strains of Staphylococcus aureus was observed. All the in vitro conditions tested for the production of the bacteriocins (temperature, pH and NaCl), indicate that the strain of L. curvatus 54M16 is able to grow and to produce the antagonistic substances at pH 4.5 and in the presence of 4% NaCl. The strain has good acidifier capability and it is able to hydrolyse sarcoplasmic but not myofibrillar proteins, lipids and to reduce nitrates. Moreover it shown high values of SOD, while the aminopeptidase activity is restricted to whole cells that hydrolyse at high rates the amino acids l -arginine, l -valine, l -phenylalanine and l -lysine. The suitability of L. curvatus 54M16 as starter culture was assayed during sausage fermentation. Its use can improve the quality and safety of the traditional fermented sausages prepared without antimicrobial additives.

One hundred three isolates of Carnobacterium spp. from raw meat were analyzed by random amplification of polymorphic DNA (RAPD) and PCR and were identified by 16S rRNA gene sequencing. Forty-five strains of Carnobacterium maltaromaticum were characterized for their growth capabilities at different temperatures, NaCl concentrations, and pH values and for in vitro lipolytic and proteolytic activities. Moreover, their spoilage potential in meat was investigated by analyzing the release of volatile organic compounds (VOCs) in meat stored in air or vacuum packs. Almost all the strains were able to grow at 4, 10, and 20°C, at pH values of 6 to 9, and in the presence of 2.5% NaCl. The release of VOCs by each strain in beef stored at 4°C in air and vacuum packs was evaluated by headspace solid-phase microextraction (HS-SPME)-gas chromatography-mass spectrometry (GC-MS) analysis. All the meat samples inoculated and stored in air showed higher numbers of VOCs than the vacuum-packed meat samples. Acetoin, 1-octen-3-ol, and butanoic acid were the compounds most frequently found under both storage conditions. The contaminated meat samples were evaluated by a sensory panel; the results indicated that for all sensory odors, no effect of strain was significant ( P > 0.05). The storage conditions significantly affected ( P < 0.05) the perception of dairy, spoiled-meat, and mozzarella cheese odors, which were more intense in meat stored in air than in vacuum packs but were never very intense. In conclusion, different strains of C. maltaromaticum can grow efficiently in meat stored at low temperatures both in air and in vacuum packs, producing volatile molecules with low sensory impacts, with a negligible contribution to meat spoilage overall.

The microbial ecology of “soppressata of Vallo di Diano,” a traditional dry fermented sausage from southern Italy, was studied by using both culture-dependent and culture-independent approaches. The ripened fermented sausages were characterized by high microbial loads of both staphylococci and lactobacilli. Using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) targeting the variable V3 and V1 regions of the 16S rRNA gene and direct DNA sequencing, it was possible to identify Staphylococcus xylosus , S. succinus , and S. equorum among the staphylococci and Lactobacillus sakei and L. curvatus within the lactobacilli. Moreover, Debaryomyces hansenii was the main yeast species found by targeting the yeast 26S rRNA gene by PCR-DGGE. Selected strains of S. xylosus , L. sakei , and L. curvatus were characterized for their technological properties in the ripening conditions of the fermented sausages so as to select an autochthonous starter formulation. The selection included the determination of nitrate reductase, lipolytic, and antioxidant activity and proteolysis with myofibrillar and sarcoplasmic protein fractions. Such properties were evaluated in both in vitro and in situ assays; the latter were performed by using each strain as a starter in the laboratory-scale manufacture of soppressata of Vallo di Diano and by monitoring the microbiological and chemical changes at the end of ripening. The results show differences between the in vitro and in situ selection results and indicate that in situ evaluation of the technological performance of specific strains is better suited to selecting autochthonous starter cultures for fermented-meat products than in vitro evaluation.

Microbial spoilage of meat is a complex event to which many different bacterial populations can contribute depending on the temperature of storage and packaging conditions. The spoilage can derive from microbial development and consumption of meat nutrients by bacteria with a consequent release of undesired metabolites. The volatile organic compounds (VOCs) that are generated during meat storage can have an olfactory impact and can lead to rejection of the product when their concentration increase significantly as a result of microbial development. The VOCs most commonly identified in meat during storage include alcohols, aldehydes, ketones, fatty acids, esters and sulfur compounds. In this review, the VOCs found in fresh meat during storage in specific conditions are described together with the possible bacterial populations responsible of their production. In addition, on the basis of the data available in the literature, the sensory impact of the VOCs and their dynamics during storage is discussed to highlight their possible contribution to the spoilage of meat.

The aim of this study was to design species-specific PCR assays for rapid and reliable identification and differentiation of Staphylococcus ( S. ) carnosus and S. simulans strains. Two different sets of primers, targeting the manganese-dependent superoxide dismutase ( sod A) gene of S. carnosus and S. simulans , respectively, were designed. Species-specificity of both sets of primers was evaluated by using 93 strains, representing 26 different species of the genus Staphylococcus , 3 species of the genus Kocuria ( K .), 1 species of the genus Micrococcus ( Mic .) and 1 species of the genus Macrococcus ( Mac .) as reference. By using primers simF and simR the expected PCR fragment was obtained only when purified DNA from S. simulans strains was used. Amplification performed by using primers carF and carR produced a PCR fragment of the expected length, when DNA from strains of S. carnosus and S. condimenti were used as template. Nevertheless, Dra I digestion of the carF/carR PCR fragment allowed a clear differentiation of strains of these two species. Species-specific PCR assays designed during this study, overcoming many of the limitations of the traditional identification procedures, can be considered a valid strategy for detection and identification of S. carnosus and S. simulans strains. The rapidity (about 4 h from DNA isolation to results), the reliability and low cost of the PCR procedures established suggests that the methods may be profitably applied for specific detection and identification of S. carnosus , S. condimenti and S. simulans strains in starter cultures and meat products.

G. MAURIELLO, D. ERCOLINI, A. LA STORIA, A. CASABURI AND F. VILLANI. 2004. Aims: The aims of this work were to (i) use a bacteriocin produced by Lactobacillus curvatus 32Y active against Listeria monocytogenes to activate polythene films by different methods, (ii) implement a large-scale process for antilisterial polythene films production and (iii) verify the efficacy of the developed films in inhibiting the growth of L. monocytogenes during the storage of meat products. Methods and Results: The film was made active by using the antilisterial bacteriocin 32Y by Lact. curvatus with three different procedures: soaking, spraying and coating. The antimicrobial activity of the activated films was tested in plate assays against the indicator strain L. monocytogenes V7. All the used procedures yielded active polythene films although the quality of the inhibition was different. The coating was therefore employed to develop active polythene films in an industrial plant. The antimicrobial activity of the industrially produced films was tested in experiments of food packaging involving pork steak and ground beef contaminated by L. monocytogenes V7 at roughly 10 3 CFU cm )2 and gram respectively. The results of the challenge tests showed the highest antimicrobial activity after 24 h at 4� C, with a decrease of about 1 log of the L. monocytogenes population. Conclusions: Antimicrobial packaging can play an important role in reducing the risk of pathogen development, as well as extending the shelf life of foods. Significance and Impact of the Study: Studies of new food-grade bacteriocins as preservatives and development of suitable systems of bacteriocin treatment of plastic films for food packaging are important issues in applied microbiology and biotechnology, both for implementing and improving effective hurdle technologies for a better preservation of food products.

The functional diversity of a population of sixty-five different strains of P. fragi isolated from fresh and spoiled meat was studied in order to evaluate the population heterogeneity related to meat spoilage potential. The strains were characterized for the proteolytic activity at 4 degrees C on beef sarcoplasmic proteins and only 9 strains were found to be proteolytic. An iron-dependent growth behaviour was shown when each strain was grown in citrate medium containing either myoglobin, haemoglobin or iron chloride as iron sources. Increase of maximum population and mu(max) in presence of different iron sources was registered. The release of volatile organic compounds (VOC) by each strain in beef during aerobic storage at 4 degrees C was evaluated by GC-MS. A considerable variability of occurrence of each molecule in the GC-MS profiles obtained by the different strains was observed ranging from 3% to 79% although the strains showed a high degree of similarity. In particular, ethylhexanoate, ethyloctanoate, ethylnonenoate, ethyldecanoate, 1-octen-3-ol, 3-octanone, 4-methylthiophenol, and 2-pentylfurane were produced by more than 50% of the strains. Representative strains were used to spoil meat in the same conditions used for the VOC analysis and the samples were evaluated by a sensory panel. The results of the sensory analysis indicated that the different strains could significantly affect the odour of meat and strains characterized by production of esters gave fruity odours to the spoiled meat. However, the similarity of strains based on the sensory profiles does not necessarily match the similarity shown in VOC profiles. P. fragi has a significant role in the microbial ecology of meat and the influence of meat-related sources of iron on the growth behaviour of many different strains suggests that meat can be an ecological niche for P. fragi. Regardless of the proteolytic and lipolytic capacities shown in vitro, different molecular types of P. fragi can release odour active volatile molecules and play a similar overall role as spoilage agents of meat.

The aim of this work was to characterize protease and esterase activities of staphylococci in order to establish if they could contribute to the release of amino acids and short-chain fatty acids during ripening of fermented sausages. Eighteen Staphylococcus strains belonging to the species Staphylococcus xylosus (5), S. saprophyticus (3), S. equorum (4), S. carnosus (4) and S. simulans (2), previously isolated from different types of Southern Italian fermented sausages, were screened for proteinase, aminopeptidase and esterase activities. Most of the staphylococci strains lacked detectable levels of proteinase activity against casein-fluorescein isothiocynate. In the active strains, this activity was extracellular or cell-envelope associated. The studied staphylococci strains also showed low levels of aminopeptidase activities, which preferentially hydrolysed substrates containing L-methionine, L-leucine and L-phenylalanine as N-terminal residue. In contrast, all staphylococcal strains possessed significant activity against short-chain fatty acid esters. The maximum esterase activities were detected in whole-cell suspensions and cell-free extracts and to a lesser extent in the extracellular medium. The substrates preferentially hydrolysed were rho-nitrophenyl (rho-NP) butyrate and rho-NP caprylate and, secondly, rho-NP palmitate. The extracellular extracts of most of the strains were only active against rho-NP butyrate except for those of S. equorum (SI3, SI4) and S. simulans (Ssm12, Ssm21), which also hydrolysed rho-NP caprylate and rho-NP palmitate. The cell-free extracts and whole cells were mainly active against rho-NP butyrate and rho-NP caprylate, showing activity levels from 1760 U/mg of proteins to 54 U/mg of proteins and from 12,200 U/mg of proteins to 133 U/mg of proteins respectively. These activities were especially high in the strains that belonged to S. xylosus and S. equorum species. The diversity of the studied metabolic properties and, especially, the esterase activities in different staphylococcal species and even strains of the same species emphasize the relevance of these in vitro characterization studies for a rational selection of new starter cultures.

The aim of this study was to determine the technological properties of 2 strains of Staphylococcus simulans (Ssm12, Ssm21) and 4 strains of S. carnosus (SC28, SC31, SC54 and SC55) for the selection of a potential starter cultures to employ in the processing of dry fermented sausages. The strains were studied to evaluate nitrate reductase, proteolytic, lipolytic, decarboxylase and antioxidant activities as well as growth ability at different temperatures, pH and NaCl concentrations. Nitrate reductase activity was determined at 15, 20 and 30 °C. By spectrophotometric method all the strains were able to reduce nitrate to nitrite at the different temperatures but these results were not confirmed by the agar plate method. Antioxidant and lipolytic activities were evaluated by spectrophotometric assay. All the strains showed antioxidative enzymes superoxide dismutase (SOD) and catalase whereas all appeared unable to hydrolyse pork fat. Proteolytic activity was determined by agar plate method, spectrophotometric assay (OPA) and sodium dodecyl sulphate gel-electrophoresis (SDS–PAGE) and all strains appeared to be able to hydrolyse sarcoplasmic proteins but not myofibrillar proteins. Finally, all the strains grew at 15 and 20 °C, in presence of 10%, 15% and 20% of NaCl and at pH 5.0 and 5.5 and were unable to produce histamine, cadaverine and putrescine. The results showed that all strains studied possess useful technological activities that would make them eligible as a good starter cultures for fermented sausages.

The aims of this study were to characterize the population of Micrococcaceae in different types of fermented sausages of Southern Italy and to determine the technological properties of Staphylococcus strains in order to evaluate the suitability of selected strains as starter cultures in the processing of dry fermented pork sausages. Ninety-six strains were studied to evaluate nitrate reductase, proteolytic, lipolytic and antioxidant activities as well as growth ability at different temperatures, pH's and NaCl concentrations. All the strains were classified as Staphylococcus except for one isolate assigned to Kocuria spp. The species most often isolated were S. saprophyticus, S. xylosus and S. equorum, although they were not equally distributed within the different sausages. Other species isolated were, in descending order of abundance, S. succinus, S. warneri, S. lentus, S. vitulus, S. pasteuri, S. epidermidis, and S. haemolyticus. In general, the S. xylosus strains exhibited the best technological properties that would make them eligible as good starter cultures for fermented meat products. However, strains belonging to other species also showed good technological properties. Finally, all strains grew at 10, 15 and 20 °C, in the presence of 10% and 15% of NaCl and at pH 5.0 and 5.5. The results showed that it is possible to formulate a broad variety of staphylococcal starter cultures, adaptable to different technological conditions and sausage manufacture practices.

G. BLAIOTTA, D. ERCOLINI, C. PENNACCHIA, V. FUSCO, A. CASABURI, O. PEPE AND F. VILLANI. 2004. Aims: Evaluation of the occurrence of most known staphylococcal enterotoxin (SE) genes, egc (enterotoxin genecluster) and TSST1 (toxic shock syndrome toxin 1) gene in both coagulase-positive (CPS) and coagulase-negative(CNS) staphylococcal strains isolated from meat and dairy products.Methods and Results: Specificity and reliability of the PCR detection methods used were ascertained by usingnine reference strains of Staphylococcus (S. aureus) harbouring SE genes (seAtoseE; seG, seH, seI, seM, seJ, seN andseO) and egc (containing the following sequence of genes: seO, seM, seI, uent1, uent2, seN and seG). Of 109 wildStaphylococcus spp. strains analysed, only 11 S. aureus strains were SE and/or TSST1 PCR-positive. The last 11strains also appeared to harbour the egc. Restriction endonuclease analysis of part of the egc of both reference andwild strains showed that different variants of the egc exist. Moreover, nucleotide sequences of seG and seI indicatethat the egc of the strain AB-8802 is characterized by the presence of variants of these enterotoxins (seGv and seIv).Conclusions: The occurrence of SE genes in CNS and other non-S. aureus species isolated from Napoli-typesalami, raw water buffalo milk and natural whey cultures used for mozzarella cheese manufacturing is very rare.Significance and Impact of the Study: During this study it was shown that at least five different egc may exist inS. aureus. A thorough study of egc polymorphism should provide further insight into the phylogenetics of the egc.Keywords: coagulase-negative staphylococci, enterotoxin gene cluster (egc), food, seGv (seG variant), seIv (seIvariant), staphylococcal enterotoxin (se) genes, Staphylococcus aureus.

Aims: The aim of this study was to determine the proteolytic activities of Staphylococcus xylosus strains on sarcoplasmic and myofibrillar proteins in order to evaluate the suitability of selected strains as starter cultures in the processing of a dry fermented pork sausage. Methods and Results: The proteolytic activity of 27 strains of Staphylococcus xylosus on sarcoplasmic and myofibrillar proteins was determined by agar plate method, o-phtaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Four strains were selected for the formulation of six starter cultures to use in the production of ‘Naples type’ salami. The proteolytic contribution of starters was determined by SDS-PAGE, comparing the protein profile of inoculated sausages with that of uninoculated sausages after 0, 15 and 33 days of ripening. The results showed that the proteolytic activity of some strains, determined by the agar plate method, were not confirmed by electrophoretic and spectrophotometric assays. In fact, of 24 strains of Staphylococcus xylosus able to hydrolyse muscle protein extracts on agar plate, only 12 strains were shown to change SDS-PAGE profile of pork proteins. The SDS-PAGE profile of sarcoplasmic proteins extracted from all sausages showed that the major changes were produced with starters S3, S4 and S5 after 15 days of ripening. Also myofibrillar proteins undergo major changes after 15 days of ripening and the protein profiles showed the same pattern in all samples, except for the sausages produced with starter S4. Conclusions: The results of this work showed that the muscle protein extracts hydrolysis test is suitable for preliminary screening of Staphylococcus xylosus strains on the basis of their proteolytic activity. However, evaluation of muscle protein hydrolysis in a food model system could then be more appropriate for selecting micro-organisms for use as starter cultures for fermented sausages. Significance and Impact of the Study: The potential of the findings is discussed with reference to the formulation of starter cultures for the dry fermented sausages production.

Halobacterial representatives were isolated from salted fish, naturally occurring salt pans as well as artificial saline pools in the Mediterranean area. The isolation techniques experimented proved successful, allowing halophilic archaea to be retrieved in almost 72% of analysed samples. About 65% of strains could be presumptively ascribed to the species Haloarcula marismortui by RFLP (Restriction Fragment Length Polymorphism) grouping and 16S rRNA gene sequence comparison. Nevertheless, cluster analysis of RAPD-PCR patterns revealed a wide heterogeneity among isolated strains. Biochemical features of technological interest, such as proteolytic, lipolytic and decarboxylase activity, were investigated in order to elucidate the role of archaea during ripening of salted anchovies. Proteolytic activity was only evidenced on the sarcoplasmic fraction extracted from fresh anchovies and for only four strains out of 21. No strain revealed either lipolytic capability on cod liver oil or proteolytic activity on fresh anchovies myofibrillar extract. Finally, only one strain exhibited decarboxylase activity, minimizing the potential responsibility for the spoilage of salted anchovies, since little direct evidence of the degradation process is provided. However, the possible pro-technological contribution of proteolytic strains to improve the quality of salted anchovies needs further investigation.