Francesco Villani

Changes in the Spoilage-Related Microbiota of Beef during Refrigerated Storage under Different Packaging Conditions

ABSTRACT The microbial spoilage of beef was monitored during storage at 5°C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O2 and 40% CO2 (MAP2), and (iii) 20% O2 and 40% CO2 (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.

Spoilage microbiota associated to the storage of raw meat in different conditions

The spoilage of raw meat is mainly due to undesired microbial development in meat during storage. The type of bacteria and their loads depend on the initial meat contamination and on the specific storage conditions that can influence the development of different spoilage-related microbial populations thus affecting the type and rate of the spoilage process. This review focuses on the composition of raw meat spoilage microbiota and the influence of storage conditions such as temperature, packaging atmosphere and use of different preservatives on the bacterial diversity developing in raw meat. In addition, the most recent tools used for the detection and identification of meat microbiota are also reviewed.

Mesophilic and Psychrotrophic Bacteria from Meat and Their Spoilage Potential In Vitro and in Beef

ABSTRACT Mesophilic and psychrotrophic populations from refrigerated meat were identified in this study, and the spoilage potential of microbial isolates in packaged beef was evaluated by analyzing the release of volatile organic compounds (VOC) by gas chromatography-mass spectrometry (GC/MS). Fifty mesophilic and twenty-nine psychrotrophic isolates were analyzed by random amplified polymorphic DNA-PCR, and representative strains were identified by 16S rRNA gene sequencing. Carnobacterium maltaromaticum and C. divergens were the species most frequently found in both mesophilic and psychrotrophic populations. Acinetobacter baumannii, Buttiauxella spp. and Serratia spp. were identified among the mesophilic isolates, while Pseudomonas spp. were commonly identified among the psychrotrophs. The isolates were further characterized for their growth at different temperatures and their proteolytic activity in vitro on meat proteins extracts at 7°C. Selected proteolytic strains of Serratia proteamaculans, Pseudomonas fragi, and C. maltaromaticum were used to examine their spoilage potential in situ. Single strains of these species and mixtures of these strains were used to contaminate beef chops that were packed and stored at 7°C. At time intervals up to 1 month, viable counts were determined, and VOC were identified by GC/MS. Generally, the VOC concentrations went to increase during the storage of the contaminated meats, and the profiles of the analyzed meat changed dramatically depending on the contaminating microbial species. About 100 volatiles were identified in the different contaminated samples. Among the detected volatiles, some specific molecules were identified only when the meat was contaminated by a specific microbial species. Compounds such as 2-ethyl-1-hexanol, 2-buten-1-ol, 2-hexyl-1-octanol, 2-nonanone, and 2-ethylhexanal were detectable only for C. maltaromaticum, which also produced the highest number of aldehydes, lactones, and sulfur compounds. The highest number of alcohols and ketons were detected in the headspace of meat samples contaminated by P. fragi, whereas the highest concentrations of some alcohols, such as 1-octen-3-ol, and some esters, such as isoamyl acetate, were produced by S. proteamaculans. In conclusion, different microbial species can contribute to meat spoilage with release of different volatile compounds that concur to the overall quality decrease of spoiling meat.

Selection of Lactobacillus strains from fermented sausages for their potential use as probiotics.

A rapid screening method was used to isolate potentially probiotic Lactobacillus strains from fermented sausages after enrichment in MRS broth at pH 2.5 followed by bile salt stressing (1% bile salts w/v). One hundred and fifty acid- and bile-resistant strains were selected, avoiding preliminary and time-consuming isolation steps. Strains were further characterized for survival at pH 2.5 for 3 h in phosphate-buffered saline and for growth in the presence of 0.3% bile salts with and without pre-exposure at low pH. Twenty-eight strains showed a survival >80% at pH 2.5 for 3 h; moreover, most of the strains were able to grow in the presence of 0.3% bile salts. Low pH and bile resistance was shown to be dependent on both the species, identified by phenotypic and molecular methods, and the strain tested. This is the first report on the direct selection of potentially probiotic lactobacilli from dry fermented sausages. Technologically interesting strains may be used in the future as probiotic starter cultures for novel fermented sausage manufacture.

PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802

Aims:  Evaluation of the occurrence of most known staphylococcal enterotoxin (SE) genes, egc (enterotoxin gene cluster) 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.

Monitoring of Microbial Metabolites and Bacterial Diversity in Beef Stored under Different Packaging Conditions

ABSTRACT Beef chops were stored at 4°C under different conditions: in air (A), modified-atmosphere packaging (MAP), vacuum packaging (V), or bacteriocin-activated antimicrobial packaging (AV). After 0 to 45 days of storage, analyses were performed to determine loads of spoilage microorganisms, microbial metabolites (by solid-phase microextraction [SPME]-gas chromatography [GC]-mass spectrometry [MS] and proton nuclear magnetic resonance [1H NMR]), and microbial diversity (by PCR–denaturing gradient gel electrophoresis [DGGE] and pyrosequencing). The microbiological shelf life of meat increased with increasing selectivity of storage conditions. Culture-independent analysis by pyrosequencing of DNA extracted directly from meat showed that Brochothrix thermosphacta dominated during the early stages of storage in A and MAP, while Pseudomonas spp. took over during further storage in A. Many different bacteria, several of which are usually associated with soil rather than meat, were identified in V and AV; however, lactic acid bacteria (LAB) dominated during the late phases of storage, and Carnobacterium divergens was the most frequent microorganism in AV. Among the volatile metabolites, butanoic acid was associated with the growth of LAB under V and AV storage conditions, while acetoin was related to the other spoilage microbial groups and storage conditions. 1H NMR analysis showed that storage in air was associated with decreases in lactate, glycogen, IMP, and ADP levels and with selective increases in levels of 3-methylindole, betaine, creatine, and other amino acids. The meat microbiota is significantly affected by storage conditions, and its changes during storage determine complex shifts in the metabolites produced, with a potential impact on meat quality.

Microbial succession during ripening of Naples-type salami, a southern Italian fermented sausage

Studies were carried out on the microbiological and physico-chemical changes which occurred during the ripening of five batches of Naples-type salami, manufactured without starter cultures. Salami were sampled internally and externally, and the following microbial groups were studied: lactic acid bacteria, Micrococcaceae and yeasts. The results obtained indicated that lactobacilli constituted the predominant flora, both on the surface and in the interior of the pieces throughout the ripening period. Micrococcaceae and yeasts were also found in considerable number in both locations. Characterisation of 191 lactic isolates indicated that the salami microflora was dominated by homofermentative lactobacilli; approximately 63% of them could be identified as Lactobacillus sake; 40% showing the traits of a racemase negative variant of this species, once referred to Lactobacillus bavaricus. Yeast population mainly comprised Debaryomyces strains. All the colonies grown on mannitol salt and Kranep agar were catalase-positive cocci; novobiocin-resistant staphylococci were the only Micrococcaceae found. The API Staph identification system did not prove to be reliable: 82% of the isolates remained unidentified. To achieve improved characterisation, cluster analysis was subsequently performed on this group, corroborating the existence of a fairly homogeneous group representing an intermediate variety between Staphylococcus xylosus and Staphylococcus saprophyticus that was isolated during the whole ripening process.

Development of polythene films for food packaging activated with an antilisterial bacteriocin from Lactobacillus curvatus 32Y

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.

Bacterial populations and the volatilome associated to meat spoilage

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.

Spoilage-related microbiota associated with chilled beef stored in air or vacuum pack

In order to study the spoilage-related microbiota of beef at species level, a combination of culture-independent and culture-dependent methods was used to analyse nine different beef samples stored at 4°C in air or in vacuum pack. Plate counts on selective agars after 0, 7 and 20 days of storage showed that vacuum packaging reduced the viable counts of Brochothrix thermosphacta, Pseudomonas spp. and Enterobacteriaceae, whereas the growth of lactic acid bacteria (LAB) was unaffected. Storage in vacuum pack mainly affected viable counts and not necessarily the species diversity of microbial populations on meat. Such populations were studied by PCR-DGGE of DNA directly extracted from meat and from bulk cells from culture media, followed by sequencing of DGGE fragments. Pseudomonas spp., Carnobacterium divergens, B. thermosphacta, Rahnella spp. and Serratia grimesii, or close relatives were detected in the meat at time zero. The use of the culture-independent method highlighted the occurrence of species that were not detected by plating. Photobacterium spp. occurred in most meat samples stored in air or in vacuum pack, which indicates this organism probably has a role in spoilage. In contrast, culture-dependent analysis allowed detection of bacterial species that were not found in DNA extracted directly from meat. This was the case for several species of Serratia or Rhanella among the enterobacteria, and Leuconostoc spp. among the LAB. Besides advancing our knowledge of the species involved in the spoilage of vacuum-packaged meat, this study shows the benefits of combining culture-based and direct approaches to enhance understanding of populations of spoilage bacteria.