Cyberlindnera fabianii, an Uncommon Yeast Responsible for Gluten Bread Spoilage.

A single strain of yeast was isolated from industrial gluten bread (GB) purchased from a local supermarket. This strain is responsible for spoilage consisting of white powdery and filamentous colonies due to the fragmentation of hyphae into short lengths (dust-type spots), similar to the spoilage produced by chalk yeasts such as Hyphopichia burtonii, Wickerhamomyces anomalus and Saccharomycopsis fibuligera. The isolated strains were identified initially by traditional methods as Wickerhamomyces anomalus, but with genomic analysis, they were definitively identified as Cyberlindnera fabianii, a rare ascomycetous opportunistic yeast species with low virulence attributes, uncommonly implicated in bread spoilage. However, these results demonstrate that this strain is phenotypically similar to Wi. anomalus. Cy. fabianii grew in GB because of its physicochemical characteristics which included pH 5.34, Aw 0.97 and a moisture of about 50.36. This spoilage was also confirmed by the presence of various compounds typical of yeasts, derived from sugar fermentation and amino acid degradation. These compounds included alcohols (ethanol, 1-propanol, isobutyl alcohol, isoamyl alcohol and n-amyl alcohol), organic acids (acetic and pentanoic acids) and esters (Ethylacetate, n-propil acetate, Ethylbutirrate, Isoamylacetate and Ethylpentanoate), identified in higher concentrations in the spoiled samples than in the unspoiled samples. The concentration of acetic acid was lower only in the spoiled samples, but this effect may be due to the consumption of this compound to produce acetate esters, which predominate in the spoiled samples.

Expression of Caseicin from Lacticaseibacillus casei and Lacticaseibacillus zeae Provides Insight into Antilisterial Class IIa Bacteriocins.

In this study, an in silico screening approach was employed to mine potential bacteriocin clusters in genome-sequenced isolates of Lacticaseibacillus zeae UD 2202 and Lacticaseibacillus casei UD 1001. Two putative undescribed bacteriocin gene clusters (Cas1 and Cas2) closely related to genes encoding class IIa bacteriocins were identified. No bacteriocin activity was recorded when cell-free supernatants of strains UD 2202 and UD 1001 were tested against Listeria monocytogenes. Genes encoding caseicin A1 (casA1) and caseicin A2 (casA2) were heterologously expressed in Escherichia coli BL21 (DE3) using the nisin leader peptide cloned in-frame to the C-terminal of the green fluorescent gene (mgfp5). Nisin protease (NisP) was used to cleave caseicin A1 (casA1) and caseicin A2 (casA2) from GFP-Nisin leader fusion proteins. Both heterologously expressed peptides (casA1 and casA2) inhibited the growth of L. monocytogenes, suggesting that casA1 and casA2 are either silent in the wild-type strains or are not secreted in an active form. The minimum inhibitory concentration (MIC) of casA1 and casA2, determined using HPLC-purified peptides, ranged from < 0.2 µg/mL to 12.5 µg/mL when tested against Listeria ivanovii, Listeria monocytogenes, and Listeria innocua, respectively. A higher MIC value (25 µg/mL) was recorded for casA1 and casA2 when Enterococcus faecium HKLHS was used as the target. The molecular weight of heterologously expressed casA1 and casA2 is 5.1 and 5.2 kDa, respectively, as determined with tricine-SDS-PAGE. Further research is required to determine if genes within Cas1 and Cas2 render immunity to other class IIa bacteriocins.

Investigating Safety and Technological Traits of a Leading Probiotic Species: Lacticaseibacillus paracasei.

Lacticaseibacillus spp. are genetically close lactic acid bacteria species widely used in fermented products for their technological properties as well as their proven beneficial effects on human and animal health. This study, the first to include such a large collection of heterogeneous isolates (121) obtained from international collections belonging to Lacticaseibacillus paracasei, aimed to characterize the safety traits and technological properties of this important probiotic species, also making comparisons with other genetically related species, such as Lacticaseibacillus casei and Lacticaseibacillus zeae. These strains were isolated from a variety of heterogeneous sources, including dairy products, sourdoughs, wine, must, and human body excreta. After a preliminary molecular characterization using repetitive element palindromic PCR (Rep-PCR), Random Amplification of Polymorphic DNA (RAPD), and Sau-PCR, particular attention was paid to safety traits, evaluating antibiotic resistance profiles, biogenic amine (BA) production, the presence of genes related to the production of ethyl carbamate and diaminobenzidine (DAB), and multicopper oxidase activity (MCO). The technological characteristics of the strains, such as the capability to grow at different NaCl and ethanol concentrations and different pH values, were also investigated, as well as the production of bacteriocins. From the obtained results, it was observed that strains isolated from the same type of matrix often shared similar genetic characteristics. However, phenotypic traits were strain-specific. This underscored the vast potential of the different strains to be used for various purposes, from probiotics to bioprotective and starter cultures for food and feed production, highlighting the importance of conducting comprehensive evaluations to identify the most suitable strain for each purpose with the final aim of promoting human health.

Leuconostoc gelidum Is the Major Species Responsible for the Spoilage of Cooked Sausage Packaged in a Modified Atmosphere, and Hop Extract Is the Best Inhibitor Tested.

Cooked sausages packaged in a modified atmosphere (MAP: 20% CO2, 70% N2, <0.2% O2) with evident yellow stains were analyzed. The aims of this work were to study the microbial cause of the spoilage and to evaluate different antimicrobial compounds to prevent it. Leuconostoc gelidum was identified as the primary cause of the yellow coating in spoiled cooked sausage, as confirmed by its intentional inoculation on slices of unspoiled sausage. Leuconostoc gelidum was the main bacteria responsible for the yellow coating in spoiled cooked sausage, as confirmed by its intentional inoculation on slices of unspoiled sausage. The yellow color was also evident during growth in the model system containing cooked sausage extract, but the colonies on MRS agar appeared white, demonstrating that the food substrate stimulated the production of the yellow pigment. The spoilage was also characterized by different volatile compounds, including ketones, ethanol, acetic acid, and ethyl acetate, found in the spoiled cooked sausage packages. These compounds explained the activity of Leuc. gelidum because they are typical of heterofermentative LAB, cultivated either on food substrates or in artificial broths. Leuc. gelidum also produced slight swelling in the spoiled packages. The efficacy of different antimicrobials was assessed in model systems composed of cooked sausage extract with the antimicrobials added at food product concentrations. The data showed that sodium lactate, sodium acetate, and a combination of sodium lactate and sodium diacetate could only slow the growth of the spoiler-they could not stop it from occurring. Conversely, hop extract inhibited Leuc. gelidum, showing a minimal inhibitory concentration (MIC) of approximately 0.008 mg CAE/mL in synthetic broth and 4 mg CAE/kg in cooked sausage slices. Adding hop extract at the MIC did not allow Leuc. gelidum growth and did not change the sensorial characteristics of the cooked sausages. To our knowledge, this is the first report of the antimicrobial activities of hop extracts against Leuc. gelidum either in vitro or in vivo.

Ancient Roman bacterium against current issues: strain Aquil_B6, Paenisporosarcina quisquiliarum, or Psychrobacillus psychrodurans?

IMPORTANCE: Since 1988, through the United States government's founding, the National Center for Biotechnology Information (NCBI) has provided an invaluable service to scientific advancement. The universality and total freedom of use if on the one hand allow the use of this database on a global level by all researchers for their valuable work, on the other hand, it has the disadvantage of making it difficult to check the correctness of all the materials present. It is, therefore, of fundamental importance for the correctness and ethics of research to improve the databases at our disposal, identifying and amending the critical issues. This work aims to provide the scientific community with a new sequence for the type strain Paenisporosarcina quisquiliarum SK 55 and broaden the knowledge of the Psychrobacillus psychrodurans species, in particular, considering the ancient strain Aquil_B6 found in an ancient Roman amphora.

Draft genome sequences of 14 Lacticaseibacillus spp. strains, representatives of a collection of 200 strains.

Lactobacilli have a fundamental role in the food industry as starters and probiotics, therefore, requiring special attention concerning food safety. In this work, 14 strains selected accordingly to their genetic fingerprint and physiologic characteristics are presented as representatives of a collection of 200 strains.

Microbial Spoilage of Traditional Goose Sausages Produced in a Northern Region of Italy.

Recently, during the ripening of goose sausage, a defect consisting of ammonia and vinegar smell was noticed. The producer of the craft facility, located in Lombardia, a Northern region of Italy, asked us to identify the cause of that defect. Therefore, this study aimed to identify the potential responsible agents for the spoilage of this lot of goose sausages. Spoilage was first detected by sensory analysis using the "needle probing" technique; however, the spoiled sausages were not marketable due to the high ammonia and vinegar smell. The added starter culture did not limit or inhibit the spoilage microorganisms, which were represented by Levilactobacillus brevis, the predominant species, and by Enterococcus faecalis and E. faecium. These microorganisms grew during ripening and produced a large amount of biogenic amines, which could represent a risk for consumers. Furthermore, Lev. brevis, being a heterofermentative lactic acid bacteria (LAB), also produced ethanol, acetic acid, and a variation in the sausage colour. The production of biogenic amines was confirmed in vitro. Furthermore, as observed in a previous study, the second cause of spoilage can be attributed to moulds which grew during ripening; both the isolated strains, Penicillium nalgiovense, added as a starter culture, and P. lanosocoeruleum, present as an environmental contaminant, grew between the meat and casing, producing a large amount of total volatile nitrogen, responsible for the ammonia smell perceived in the ripening area and in the sausages. This is the first description of Levilactobacillus brevis predominance in spoiled goose sausage.

Microbial and Physico-Chemical Characterization of Cold Smoked Sea Bass (Dicentrarchus labrax), a New Product of Fishery.

The aim of this study was to investigate the microbial and physico-chemical characteristics of cold smoked sea bass (CSSB), a novel italian fish product. The microbiological analyses showed the presence of bacterial contamination from the raw material, the environment, and the production process. The microbial spoilage population was dominated by lactic acid bacteria (LAB) associated with Gram-negative fermenting bacteria, including Photobacterium phosphoreum and psychrotrophic Enterobacteriaceae. Brochotrix thermospacta and Aeromonas spp. were also present; in contrast, mould and yeast were not detected (<2 CFU/g). High levels (6-7 log CFU/g) of LAB and total bacteria count (TBC) were observed from day 45 of storage; however, their presence does not seem to have influenced the total volatile basic nitrogen (TVB-N), which always remained below 35 mg N/100 g. Consequently, the product is acceptable until day 60 of storage, considering that the malonaldehyde index (TBARS) was lower than 6.5 nmol/g. Pathogenic bacteria such as Salmonella spp. and Listeria monocytogenes were not detected. Currently, there is a growing demand for seafood due to its high quality and nutritional value. Cold smoked sea bass offers a source of macro- and micronutrients essential for the proper functioning of the human body. It is also rich in protein and omega-3 fatty acids. The WHO and FAO evaluated the benefits and risks and concluded that there is convincing evidence of health benefits from fish consumption, such as a reduction in the risk of heart failure and improved neurodevelopment in infants and young children when fish is consumed by the mother before and during pregnancy. The CSSB analysed in this study demonstrated to have health benefits due to long-chain omega-3 PUFAs and other nutrients, such as proteins, minerals, and vitamin D, which are sometimes difficult to obtain from other sources. The results show that CSSB has a high nutritional value and excellent microbial quality.

Thermophilin 13: In Silico Analysis Provides New Insight in Genes Involved in Bacteriocin Production.

Bacteriocins are a large family of ribosomally synthesised proteinaceous toxins that are produced by bacteria and archaea and have antimicrobial activity against closely related species to the producer strain. Antimicrobial proteinaceous compounds are associated with a wide range of applications, including as a pathogen inhibitor in food and medical use. Among the several lactic acid bacteria (LAB) commonly used in fresh and fermented food preservation, Streptococcus thermophilus is well known for its importance as a starter culture for yoghurt and cheese. Previous studies described the bacteriocin thermophilin 13 exclusively in S. thermophilus SFi13 and the genes encoding its production as an operon consisting of two genes (thmA and thmB). However, the majority of bacteriocins possess a complex production system, which involves several genes encoding dedicated proteins with relatively specific functions. Up to now, far too little attention has been paid to the genes involved in the synthesis, regulation and expression of thermophilin 13. The aim of the present study, using in silico gene mining, was to investigate the presence of a regulation system involved in thermophilin 13 production. Results revealed the dedicated putative bacteriocin gene cluster (PBGC), which shows high similarity with the class IIb bacteriocins genes. This newly revealed PBGC, which was also found within various strains of Streptococcus thermophilus, provides a new perspective and insights into understanding the mechanisms implicated in the production of thermophilin 13.

Lactobacilli, a Weapon to Counteract Pathogens through the Inhibition of Their Virulence Factors.

To date, several studies have reported an alarming increase in pathogen resistance to current antibiotic therapies and treatments. Therefore, the search for effective alternatives to counter their spread and the onset of infections is becoming increasingly important. In this regard, microorganisms of the former Lactobacillus genus have demonstrated the ability to reduce the virulence of pathogens. In addition to the production of bioactive substances, self- and coaggregation, and substrate competition, lactobacilli influence gene expression by downregulating genes associated with the virulence of pathogens. As demonstrated in many in vivo and in vitro trials, lactobacilli counteract and inhibit various virulence factors that favor pathogens, including the production of toxins, biofilm formation, host cell adhesion and invasion, and downregulation of virulence genes linked to quorum sensing. The aim of this review is to summarize current studies on the inhibition of pathogen virulence by lactobacilli, an important microbial group well known in the industrial and medical fields for their technological and probiotic properties that benefit human hosts with the potential to provide an important aid in the fight against pathogens besides use of the current therapies. Further research could lead to the identification of new strains that, in addition to alleviating adverse effects, could improve the efficacy of antibiotic therapies or play an important preventive role by reducing the onset of pathogen infections if regularly taken.

Microbial Characterization of Retail Cocoa Powders and Chocolate Bars of Five Brands Sold in Italian Supermarkets.

A microbial characterization of cocoa powder and chocolate bars of three batches of five different brands sold in Italian markets was performed. The results showed a variable microbial population consisting of mesophilic and thermophilic spore formation in both types of products. The chocolate bars were also contaminated with molds of environmental origin. Bacillus spp. and Geobacillus spp. were found in both products. The chocolate bars were also contaminated by molds belonging to the genera Penicillium and Cladosporium. The sporogenous strains mainly originate from the raw materials, i.e., cocoa beans, as the heat treatments involved (roasting of the beans and conching of the chocolate) are not sufficient to reach commercial sterility. Furthermore, the identified spore-forming species have often been isolated from cocoa beans. The molds isolated from chocolate seem to have an origin strictly linked to the final phases of production (environment and packaging). However, the level of contaminants is limited (<2 log CFU/g); the molds do not develop in both products due to their low Aw (<0.6) and do not affect the safety of the products. However, a case of mold development in chocolate bars was observed. Among the isolated molds, only Penicillium lanosocoeruleum demonstrated a high xero-tolerance and grew under some conditions on chocolate bars. Its growth could be explained by a cocoa butter bloom accompanied by the presence of humidity originating from the bloom or acquired during packaging.

Improving the Shelf-Life of Fish Burgers Made with a Mix of Sea Bass and Sea Bream Meat by Bioprotective Cultures.

Seafood products are one of the most perishable foods, and their shelf life is limited by enzymatic and microbial spoilage. Developing methods to extend the shelf life of fresh fish could reduce food waste in the fishery industry, retail stores, and private households. In recent decades, the application of lactic acid bacteria (LAB) as bioprotective cultures has become a promising tool. In this study, we evaluated the use of four starter cultures, previously selected for their properties as bioprotective agents, for sea bass and sea bream burgers biopreservation. Starter cultures impacted the microbial populations, biochemical parameters (pH, TVB-N), and sensory properties of fish burgers, during 10 days of storage at 4 °C and then 20 days at 8 °C in modified atmosphere packaging (MAP). Also, storage time influenced the microbial and physicochemical characteristics of all the tested samples, except for TVB-N values, which were significantly higher in the uninoculated burgers. The volatilome changed in the different treatments, and in particular, the samples supplemented with starter presented a profile that described their rapid growth and colonization, with the production of typical molecules derived from their metabolism. The addition of bioprotective cultures avoided bloating spoilage and improved the sensory parameters of the burgers. The shelf life of the fish burgers supplemented with starter cultures could be extended up to 12 days.

Draft Genome Sequences of Eight Bacilli Isolated from an Ancient Roman Amphora.

Paleomicrobiology, the study of ancient microbiological material, allows us to understand different evolutionary phenomena in bacteria. In this study, eight bacilli isolated from an ancient Roman amphora, which dates to the IV to V sec. AD, were sequenced and functionally annotated.

Validation of a Standard Protocol to Assess the Fermentative and Chemical Properties of Saccharomyces cerevisiae Wine Strains.

This paper reports on a common experiment performed by 17 Research Units of the Italian Group of Microbiology of Vine and Wine (GMVV), which belongs to the Scientific Society SIMTREA, with the aim to validate a protocol for the characterization of wine strains of Saccharomyces cerevisiae. For this purpose, two commercial S. cerevisiae strains (EC 1118 and AWRI796) were used to carry out inter-laboratory-scale comparative fermentations using both synthetic medium and grape musts and applying the same protocol to obtain reproducible, replicable, and statistically valid results. Ethanol yield, production of acetic acid, glycerol, higher alcohols, and other volatile compounds were assessed. Moreover, the Fourier transform infrared spectroscopy was also applied to define the metabolomic fingerprint of yeast cells from each experimental trial. Data were standardized as unit of compounds or yield per gram of sugar (glucose and fructose) consumed throughout fermentation, and analyzed through parametric and non-parametric tests, and multivariate approaches (cluster analysis, two-way joining, and principal component analysis). The results of experiments carried out by using synthetic must showed that it was possible to gain comparable results from three different laboratories by using the same strains. Then, the use of the standardized protocol on different grape musts allowed pointing out the goodness and the reproducibility of the method; it showed the main traits of the two yeast strains and allowed reducing variability amongst independent batches (biological replicates) to acceptable levels. In conclusion, the findings of this collaborative study contributed to the validation of a protocol in a specific synthetic medium and in grape must and showed how data should be treated to gain reproducible and robust results, which could allow direct comparison of the experimental data obtained during the characterization of wine yeasts carried out by different research laboratories.

Evaluation of Different Techniques, including Modified Atmosphere, under Vacuum Packaging, Washing, and Latilactobacillus sakei as a Bioprotective Agent, to Increase the Shelf-Life of Fresh Gutted Sea Bass (Dicentrarchus labrax) and Sea Bream (Sparus aurata) Stored at 6 ± 2 °C.

Fish meat is very perishable because of indigenous and microbial enzymes, which determine spoilage and shelf life. The deterioration processes, which lead to an important, sequential, and progressive modification of the initial state of freshness, are fast and depend on rearing, harvesting, slaughtering, handling, and storage conditions. Usually, the shelf life of gutted fish stored at 4 ± 2 °C under vacuum packaging (VP-1.0 bar) and modified atmosphere packaging (MAP, 70% N2, <1% O2, 30% CO2) is approximately 9 days. The aim of this work was to improve the shelf life and preserve the microbiological and sensory quality of farmed gutted sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) using different methods, including VP, MAP, and bioprotective culture containing Latilactobacillus sakei, until 12-14 days. Microbiological, physicochemical, and sensory quality indices were monitored to confirm the effectiveness of biopreservation on product quality during proper refrigeration (4 ± 2 °C) or abuse (6 ± 2 °C, simulating supermarkets and consumer fridges) storage period. Considering the quality indexes represented by Enterobacteriaceae, total volatile nitrogen (TVB-N), and malonaldehyde concentrations (TBARS) and the sensorial analysis, the VP samples were more acceptable than the MAP fish, even though the shelf-life of the VP and MAP fish was similar at about 12 days. The second phase of the work was to evaluate the shelf-life of both VP fish stored at 6 ± 2 °C, which simulates the normal abuse temperature of supermarkets or consumer fridges. Data confirmed the previous results and demonstrated, despite the abuse temperature of storage, a shelf-life of about 12 days. Finally, the third phase consisted of prolonging the shelf life until 14 days of storage at 6 ± 2 °C by washing the gutted sea bass and sea bream in a suspension of bioprotective starter (7 log CFU/mL) with or without the addition of dextrose (0.1%) and by VP packaging. The bioprotective culture reduced the growth of spoilage microorganisms. Consequently, the total volatile nitrogen (TVB-N) concentration in both fish species was low (<35 mg N/100 g). Nonprofessional and untrained evaluators confirmed the acceptability of the inoculated samples by sensorial analysis.

Antibiotic resistance and virulence factors in lactobacilli: something to carefully consider.

Lactobacilli are a ubiquitous bacteria, that includes many species commonly found as part of the human microbiota, take part in the natural food fermentation processes, are used as probiotics, and in the food sector as starter cultures or bio-protectors. Their wide use is dictated by a long history of safe employ, which has allowed them to be classified as GRAS (General Recognized As Safe) microorganisms by the US Food and Drug Administration (FDA) and QPS (Qualified Presumption of Safety) by the European Food Safety Authority (EFSA, 2007; EFSA, 2021). Despite their classification as safe microorganisms, several studies show that some members of Lactobacillus genus can cause, especially in individuals with previous pathological conditions, problems such as bacteremia, endocarditis, and peritonitis. In other cases, the presence of virulence genes and antibiotic resistance, and its potential transfer to pathogenic microorganisms constitute a risk to be considered. Consequently, their safety status was sometimes questioned, and it is, therefore, essential to carry out appropriate assessments before their use for any purposes. The following review focuses on the state of the art of studies on genes that confer virulence factors, including antibiotic resistance, reported in the literature within the lactobacilli, defining their genetic basis and related functions.

Zygosaccharomyces rouxii is the predominant species responsible for the spoilage of the mix base for ice cream and ethanol is the best inhibitor tested.

A mix base for ice cream (MBIC) is used to produce artisanal or industrial ice creams and desserts and consists of a mixture of different ingredients, including sugar, egg yolk, natural flavors, starch and milk proteins. MBICs, which have chemical-physical characteristics that include a pH of 5.61 and an activity water (Aw) less than or equal to 0.822, are packaged in tin boxes and stored at ambient temperature. Despite the low Aw, MBIC can support osmotolerant and osmophilic yeast growth. The aim of our work was to study the behavior of Zygosaccharomyces rouxii, the main microorganisms responsible of MBIC spoilage, either in the vivo or in a model system in order to inhibit its growth by the selection of antimicrobial agents. Different osmotolerant yeasts belonging to the genus Zygosaccharomyces were isolated and identified from spoiled and unspoiled lots of MBICs. In particular, Z. rouxii was the predominant species responsible for the spoilage, which depended on the high temperature of storage (>20 °C) and was highlighted by the presence of alcohol, esters, acids and gas (CO2), which blew open the tin boxes. To stop spoilage, different antimicrobial compounds were tested: sulfur dioxide, sorbic and benzoic acids and ethanol. However, only 2% v/v ethanol was required to achieve the total inhibition of the Z. rouxii cocktails tested in this work. The use of other antimicrobials cannot be recommended because they were not able to stop yeast spoilage and changed the color and flavor of the products. Conversely, the use of ethanol is suggested because of its extreme effectiveness against osmotolerant yeasts, and the added amount was less than or equal to the taste threshold limit. The MBICs, treated with ethanol, were stable till the end of their shelf-life (6 months).

Analysis of the Bioprotective Potential of Different Lactic Acid Bacteria Against Listeria monocytogenes in Cold-Smoked Sea Bass, a New Product Packaged Under Vacuum and Stored at 6 ± 2°C.

The aim of the work was to monitor the presence of Listeria monocytogenes in cold-smoked fish products (trout, salmon, and sea bass) marketed in Italy. Cold-smoked sea bass is a new product that has not yet been commercialized and was collected from the production facility. Monitoring data have shown that cold-smoked products can be contaminated by L. monocytogenes, the presence of which has been highlighted mainly by enrichment culture (presence in 25 g). The isolated Listeria were serotyped and belonged mainly to low-virulence serotypes (1/2c), followed by serotypes 1/2a, 1/2b, and 4b. Furthermore, considering the ability of L. monocytogenes to grow in these products due to their chemical-physical characteristics (pH > 6.0, Aw > 0.97) and long shelf life at 4°C, an additional aim was to verify the activity of different bioprotective starters, including Lactilactobacillus sakei (LAK-23, Sacco srl, Via Alessandro Manzoni 29/A, 22071 Cadorago, CO, Italy), Carnobacterium spp., Lacticaseibacillus casei (SAL 106), and Lacticaseibacillus paracasei (SAL 211), in cold-smoked sea bass. All starters were bacteriocin producers. For this experiment, smoked sea bass samples were intentionally inoculated with a mixture of three different strains of L. monocytogenes and of each starter culture. After inoculation, the smoked sea bass were vacuum-packed and stored at 6 ± 2°C for 60 days, simulating the typical abuse storage temperature of markets and home refrigerators. At 0, 15, 30, 45, and 60 days, the sea bass samples were analyzed to evaluate the effectiveness of the starters against L. monocytogenes. Listeria monocytogenes growth was prevented only by the addition of the LAK-23 starter. Indeed, at the end of the shelf life, the amount of L. monocytogenes observed was similar to that in the inoculum. Consequently, the use of this starter can allow the inclusion of cold-smoked sea bass or smoked fish products in category 1.3 of Regolamento CE 2073/2005, which are products that do not support the growth of this microorganism. Finally, the activity of the LAK-23 starter did not produce an off flavor or off odor in the smoked sea bass.

A survey of a blown pack spoilage produced by Clostridium perfringens in vacuum-packaged wurstel.

Three hundred Clostridium strains were isolated from spoiled wurstels and were identified by traditional and molecular methods as Clostridium perfringens. The phenotypic characteristics of the strains were studied. All the strains produced acetic and butyric acids and enterotoxin. C. perfringens grew in the spoiled wurstels because it was present in raw meat (Lot 150) at a level of 3.2 log CFU/g due to an unchecked cooling phase that took 28 h to decrease the temperature of the wurstels from 60 to 9-10 °C, which is the lower limit for C. perfringens growth. During the 28 h of cooling, the concentration of C. perfringens increased to 6.5 CFU/g. It was concluded that its presence and the long cooling time were the main factors responsible for the spoilage. Wurstels intentionally made with contaminated meat (3 log CFU/g) but cooled after cooking for 17 h to 9 °C did not support C. perfringens growth; consequently, these wurstels remained unspoiled. The packages of the spoiled wurstels were blown, and the products were soft (soggy), textureless and had the odour of acetic acid, ethanol and sulfur.