Microbiological and physicochemical profile of Italian steak tartare and predicting growth potential of Listeria monocytogenes.

In the present study, growth potential of Listeria monocytogenes in steak tartare samples taken at retail and belonging to 13 brands marketed in Northern Italy was investigated. The samples were submitted to microbiological and chemical-physical characterization. The data obtained were used as inputs for the application of the predictive microbiology software FSSP that allows the estimation of the growth of L. monocytogenes during the shelf-life. Lactic acid bacteria, the main component of the microflora, gave variable counts among the brands (from 3.38 to 6.24 log CFU/g). pH and aw values were always higher than 5.3 and 0.96, respectively, thus they could not be considered as single efficient hurdles to prevent the growth of L. monocytogenes according to the EC Reg. 2073/2005; the same was observed for salt content (constantly <2 %) and nitrites (not quantifiable in all the samples, even if declared in some labels). Nevertheless, the combination of all the hurdles, evaluated by predictive microbiology using critical development factors, resulted in an estimated growth <0.5 log CFU/g throughout the shelf life; this output allowed us to consider all the steak tartare analysed as unfavourable substrate for L. monocytogenes growth. The information obtained could be useful for tartare producers as well as for competent authority to evaluate the effective risk concerning these typology of products.

Preliminary Investigation towards the Use of Infrared Technology for Raw Milk Treatment.

Infrared (IR) technology offers a promising solution for reducing microbiological loads in various food types while preserving their quality traits, such as flavour. However, research on IR's application in complex matrices is limited. Therefore, our preliminary study aimed to evaluate its effectiveness in sanitizing bovine raw milk. We assessed the bacterial count before and after IR treatment by comparing volatile organic compound profiles via headspace extraction and GC/MS analysis. Our findings showed that higher energy levels led to a greater bacterial reduction. IR85 was the most effective in reducing Coliforms and Enterobacteriaceae in non-homogenised samples, with a reduction ranging from -1.01 to >-2.99 and from -1.66 to -3.09 Log CFU/mL, respectively. IR60 and 70 showed no efficacy, while IR80 had intermediate but still satisfactory effect. IR85 notably affected volatile compounds, particularly increasing hexanal (from 0.08 to 4.21 ng g-1) and dimethyl sulphone (from 10.76 to 26.40 ng g-1), while IR80 better preserved the aroma profile. As a result, only IR80 was tested with homogenised raw milk, demonstrating significant bacterial reduction (from >2.39 to 3.06 Log CFU/mL for Coliforms and from 1.90 to >2.45 Log CFU/mL for Enterobacteriaceae) and maintaining the aroma profile quality.

Shelf-life of sheep arrosticini packaged in protective atmosphere.

The aim of this study was to evaluate the shelf-life of pre-packaged sheep's arrosticini produced in a factory located in northern Italy. Samples were divided into two series and packaged in modified atmosphere with specific gas mixtures: conventional (C: 35% O2/15% CO2/50% N2) and experimental (E: 30% CO2/70% N2). All the samples were stored at 4°C for 10 days and subjected, after 5, 8 and 10 days (t5, t8, t10), to triplicate microbiological and chemical- physical (pH, total volatile basic nitrogen, thiobarbituric acid reactive substances) analyses. At the same time, the colorimetric analysis and a sensorial evaluation were carried out (pack tightness, presence of color or odor alterations), assigning a discrete score from 0 to 5. The samples showed initial total bacterial counts close to 5 Log CFU/g: these values gradually increased during storage, exceeding the threshold of 8 Log CFU/g in the C series; lower values were detected in the E series. A similar trend was highlighted for Enterobacteriaceae, with initial loads of about 3 Log CFU/g and an increase until t10, reaching values higher than 6 Log CFU/g in the C series and close to 5 Log CFU/g in the E series (P=0.002). E. coli also showed a similar trend, although with values approximately 1 Log lower than Enterobacteriaceae. Pseudomonas spp. showed initial counts close to 4.5 Log CFU/g, with a different increase in the C series (6.5 Log CFU/g at t10) and the E series (4.95 Log CFU/g) (P= 0.006). The higher growth in the C series was also observed for lactic acid bacteria, with an increase from 3 to 5 Log CFU/g (3.8 Log CFU/g in the E series P=0.016). The other microbiological parameters showed very low counts and, in most cases undetectable counts (<2 Log CFU/g) for the entire period considered. The initial values obtained from the measurement of the colorimetric indices were within the norm for this type of product: however, starting from t5, lower values of red index and lightness in the E series were measured, with an evident greying of meat surface. The results of the sensory evaluation indicated that the product showed optimal sensorial characteristics up to 8 days of shelf-life in the C series, while the use of an oxygenfree atmosphere, despite having a moderate inhibiting effect on the microbial populations, has led to an early modification of the product (5 days of storage), due to the presence of superficial greyish areas. The microbiological characteristics of arrosticini strictly depend on the hygienic conditions of slaughtering and production; even in optimal situations, the product is particularly perishable, and requires careful management of storage temperatures and times, to maintain its quality characteristics.

Compliance between Food and Feed Safety: Eight-Year Survey (2013-2021) of Aflatoxin M1 in Raw Milk and Aflatoxin B1 in Feed in Northern Italy.

Aflatoxins (AFs) are fungal metabolites that are found in feed and food. When ruminants eat feed contaminated with aflatoxin B1 (AFB1), it is metabolised and aflatoxin M1 (AFM1) is excreted in the milk. Aflatoxins can result in hepatotoxic, carcinogenic, and immunosuppressive effects. The European Union thus set a low threshold limit (50 ng/L) for presence of AFM1 in milk. This was in view of its possible presence also in dairy products and that quantification of these toxins is mandatory for milk suppliers. In the present study, a total of 95,882 samples of whole raw milk, collected in northern Italy between 2013 and 2021, were evaluated for presence of AFM1 using an ELISA (enzyme-linked immunosorbent assay) method. The study also evaluated the relationship between feed materials collected from the same farms in the same area during the same period (2013-2021) and milk contamination. Only 667 milk samples out of 95,882 samples analysed (0.7%) showed AFM1 values higher than the EU threshold limit of 50 ng/L. A total of 390 samples (0.4%) showed values between 40 and 50 ng/L, thus requiring corrective action despite not surpassing the regulatory threshold. Combining feed contamination and milk contamination data, some feedingstuffs seem to be more effective in defying potential carryover of AFs from feed to milk. Combining the results, it can be concluded that a robust monitoring system that covers both feed, with a special focus on high risk/sentinel matrices, and milk is essential to guarantee high quality and safety standards of dairy products.

Prevalence, Virulence Potential, and Growth in Cheese of Bacillus cereus Strains Isolated from Fresh and Short-Ripened Cheeses Sold on the Italian Market.

This study investigated B. cereus presence in 122 samples belonging to 34 typologies of fresh or short-ripened cheeses made from cow, sheep, goat, or buffalo pasteurized milk, and sold on the Italian market. B. cereus was isolated at a prevalence of 9.8%, with a marked variability among cheese categories, and at low counts (always below 2.26 Log CFU/g). Twelve isolates were identified by MALDI-TOF analysis and typified by RAPD PCR as belonging to different B. cereus strains. All the strains were tested for the production of hemolysin BL, phosphatidylcholine-specific phospholipase C, proteases, and biofilm formation, and for the presence of chromosomal toxin-encoding genes (sph, plcA, cytK, entFM, bcet, nheA, nheB, nheC). Overall, 92% of strains harbored bcet, 75% the three genes nheA, nheB, and nheC, as well as plcA and sph, 67% entFM, and 33% cytK. All strains showed biofilm-forming ability. A chemical-physical characterization of the cheeses was also performed to show their suitability as substrates for B. cereus growth, showing high heterogeneity in terms of pH, aw, salt content, and concentration of organic acids. Finally, the ability to support spore germination and vegetative cell growth of a selected cheese was investigated in spores-inoculated samples maintained at 10 °C and 15 °C, showing the inhibitory effect of low storage temperatures.

Bacillus cereus in Dairy Products and Production Plants.

Spore-forming Bacillus cereus is a common contaminant of dairy products. As the microorganism is widespread in the environment, it can contaminate milk at the time of milking, but it can also reach the dairy products in each phase of production, storage and ripening. Milk pasteurization treatment is not effective in reducing contamination and can instead act as an activator of spore germination, and a potential associated risk still exists with the consumption of some processed foods. Prevalences and concentrations of B. cereus in milk and dairy products are extremely variable worldwide: in pasteurized milk, prevalences from 2% to 65.3% were reported, with concentrations of up to 3 × 105 cfu/g, whereas prevalences in cheeses ranged from 0 to 95%, with concentrations of up to 4.2 × 106 cfu/g. Bacillus cereus is also well known to produce biofilms, a serious concern for the dairy industry, with up to 90% of spores that are resistant to cleaning and are easily transferred. As the contamination of raw materials is not completely avoidable, and the application of decontamination treatments is only possible for some ingredients and is limited by both commercial and regulatory reasons, it is clear that the correct application of hygienic procedures is extremely important in order to avoid and manage the circulation of B. cereus along the dairy supply chain. Future developments in interventions must consider the synergic application of different mild technologies to prevent biofilm formation and to remove or inactivate the microorganism on the equipment.

Characterization of a Bacillus cereus strain associated with a large feed-related outbreak of severe infection in pigs.

AIMS: Bacillus cereus is often responsible for foodborne diseases and both local and systemic infections in humans. Cases of infection in other mammals are rather rare. In this study, we report a B. cereus feed-related outbreak that caused the death of 6234 pigs in Italy. METHODS AND RESULTS: Massive doses of a Gram-positive, spore-forming bacterium were recovered from the animal feed, faeces of survived pigs and intestinal content of dead ones. The B. cereus MM1 strain was identified by MALDI-TOF MS and typified by RAPD-PCR. The isolate was tested for the production of PC-PLC, proteases, hemolysins and biofilm, for motility, as well as for the presence of genes encoding tissue-degrading enzymes and toxins. Antimicrobial resistance and pathogenicity in Galleria mellonella larvae were also investigated. Our results show that the isolated B. cereus strain is swimming-proficient, produces PC-PLC, proteases, hemolysins, biofilm and carries many virulence genes. The strain shows high pathogenicity in G. mellonella larvae. CONCLUSIONS: The isolated B. cereus strain demonstrates an aggressive profile of pathogenicity and virulence, being able to produce a wide range of determinants potentially hazardous to pigs' health. SIGNIFICANCE AND IMPACT OF STUDY: This study highlights the proficiency of B. cereus to behave as a devastating pathogen in swine if ingested at high doses and underlines that more stringent quality controls are needed for livestock feeds and supplements.

Evaluation of beef in purified sea water: microbiological and chemical-physical aspects.

In this study the influence of the maintenance of beef (Bavarian heifer meat submitted to dry aging for 1 month) in purified sea water was evaluated considering microbiological and physical-chemical parameters. The treated samples were perforated on their upper and lower surfaces, subsequently placed in sterile plastic containers, and covered with purified sea water while the control samples were wrapped in a plastic film; all the samples were stored at 2°C and sample analyses were performed on the day of receipt (t0) and after five (t5), seven (t7) and ten (t10) days of storage. The initial bacterial population was close to 6 log CFU/g, as expected in meat subjected to a long ageing. After 5 and 10 days of storage the treated samples showed significantly lower counts for most parameters (Total Bacterial Count, Enterobacteriaceae, Lactic Acid Bacteria and Pseudomonas spp.) if compared to the control samples. Halotolerant bacteria, yeasts and moulds showed no differences between the two series. During the test, the exposed surface of treated meat samples underwent a partial protein denaturation. Analyses show a moderate acidification of the meat during the first part of storage (from t0 to t5), followed by a substantial stabilization in the second part of the trial. The meat maintained in purified sea water acquired a significantly and constantly higher water retention due to the absorption of salt by the meat. No significant difference was detected in terms of tenderness between the two series.

Collection and analysis of post mortem inspection outcomes (liver lesions) from different cattle slaughtering plants located in Northern and Southern Italy.

The study evaluated the outcomes of cattle liver postmortem examination (64,766 animals) obtained from three slaughterhouses (two located in Lombardy, Northern Italy, and one in Puglia, Southern Italy) through the period 2016-2020. The frequency of specific lesions determining liver condemnation was calculated and the influence of several factors (animal age/category, geographical area, season, plant) was considered. A mean prevalence of 8% was observed, with a significant difference among the plants (range 6.4-12.8%). A significant difference was observed among the animal categories and age classes, with higher condemnation rates in animals aged more than 30 months (mainly cows). Steatosis was the most frequent lesion observed in cows (about half of the total), whereas liver abscesses were the most diffused in younger animals (half of the total in young bulls). Other frequently observed lesions were distomatosis and perihepatitis. A different pattern was observed between the two geographical areas, with a higher prevalence of steatosis in Lombardy (mainly intensive dairy farming), and of distomatosis in Puglia (animals mainly grazed on pasture). The season influenced the prevalence of lesions, and especially of steatosis (higher in summer). A different lesion prevalence was also observed between the two plants on similar animal populations, suggesting a difference in the notification and classification procedure among the plants. This study highlights the importance of a proper sharing procedure of the information obtained from the post mortem inspection in order to facilitate an optimal use of Food Chain Information and a useful feedback for farmers.

Growth potential of Listeria monocytogenes in veal tartare.

In the present study the growth potential of Listeria monocytogenes in veal tartare was evaluated. A challenge test was performed on three tartare batches at 8°C, aiming to evaluate the growth potential of the pathogen. The data indicated the absence of a significant growth (δ<0.5 log cfu/g) during the entire period. When considering intermediate sampling times, an increase of 0.56 log cfu/g was detected after five days of storage in one of the batches. Microflora of veal tartare was dominated by lactic acid bacteria, that increased gradually during the trial, reaching counts up to 7 Log CFU/g in two of the three batches considered. Spoilage bacteria were present (especially Pseudomonas spp., yeasts and Enterobacteriaceae) but in very low counts and with a limited increase during the period considered. Finally, daily maximum tolerable L. monocytogenes counts were calculated to highlight the maximum acceptable load to avoid the overcoming of the legal limit of 100 CFU/g: a total increase of 0.95 log cfu/g in 12 days of shelf-life was estimated, obtaining a "safety initial concentration" at t0 of 10 CFU/g of the pathogen.

Evolution of Food Safety Features and Volatile Profile in White Sturgeon Caviar Treated with Different Formulations of Salt and Preservatives during a Long-Term Storage Time.

Caviar is a semi-preserved fish preparation in which cold storage (around 0 °C) and packaging under anaerobic conditions are fundamental to guarantee adequate safety parameters. Consumers seem to prefer caviar prepared with food salt only, but according to the needs of the different distribution channels, some preservatives are used in order to prolong its shelf life and to allow less restrictive storage conditions. Traditionally, the most common preservative was sodium tetraborate (borax), a salt that contributes to the sensory profile of caviar. However, due to its toxicity, borax has been banned in many countries, and the current trend is to reduce or eliminate its use. In this study, we evaluated the evolution of food safety parameters (pH, water activity, microbiological parameters) and the volatile profile during 14 months of storage in caviar samples treated with three different preservatives: I. exclusively NaCl, II. a mixture of borax and NaCl, and III. a mixture of organic acids and salts. Microbial presence was studied by means of plate counts; volatile organic compounds were identified on the sample headspace by means of solid phase microextraction with gas-chromatography and mass spectrometry. Results showed relevant differences among the three treatments investigated, with salt samples characterized by the highest viable counts and the greatest presence of volatile products driven by oxidative and spoilage processes, mainly occurring toward lipid and amino acids. On the contrary, the mixture of organic acids and salts showed the best response during the entire storage period. Finally, the employment of a multiparametric statistic model allowed the identification of different clusters based on the time of ripening and the preservative treatments used.

Pseudomonas spp.: Are Food Grade Organic Acids Efficient against These Spoilage Microorganisms in Fresh Cheeses?

Psychrotolerant Pseudomonas spp. are among the most common spoilage agents in fresh, soft and semi-soft cheeses; therefore, hurdles inhibiting their growth are in strong demand by producers. This study aimed to establish Minimal Inhibiting Concentrations (MICs) of lactic and acetic acid towards P. fluorescens and to evaluate the efficacy of a cheese surface treatment with these two organic acids. MICs were determined in Brain Heart Infusion broth at 30 °C: the inhibition was achieved at a concentration of 49.96 mM and 44.40 mM of acetic and lactic acid, respectively. Two series of inhibition tests were performed on fresh "Primo sale" cheese, inoculated with P. brenneri MGM3, then dipped into different acid solutions (acetic acid: 49.96, 99.92 and 149.88 mM; lactic acid: 44.40, 88.80 and 133.20 mM) and stored at 6 °C. P. brenneri MGM3 were enumerated, including a control series. A significantly lower growth was revealed at the highest concentrations tested, both for acetic (p < 0.01) and lactic acid (p < 0.05) if compared to control samples. A conditioning of "Primo sale" surface with organic acid solutions could be a useful hurdle for Pseudomonas inhibition and shelf-life extension; it should be applied in combination with other mild interventions to fight spoilage and maintain the original product characteristics.

Evaluation of effect of chilling steps during slaughtering on the Campylobacter sp. counts on broiler carcasses.

Campylobacter spp. play an increasing role as foodborne pathogens, with poultry representing the main vehicle of infection, and control measures at the slaughterhouse have been implemented in the last years. In this study, 2 trials were performed, evaluating the effect of the chilling phases currently applied in an industrial slaughterhouse on the Campylobacter sp. contamination of broiler carcasses. In the first trial, neck skin samples were taken from 13 flocks before and after the on-chain air chilling and submitted to analysis of Campylobacter sp. count; in the second trial, 63 carcasses or cuts stored in the chilling room for variable times, with or without skin, were submitted to analysis of Campylobacter sp. count. A selection of 75 isolates was identified by PCR. All carcass skin samples taken from the first trial showed Campylobacter sp. counts higher than 0.7 log cfu/g. A wide variability in the counts (about 3 logs) was detected, showing a high correlation between the counts obtained before and after chilling. A slight decrease (P = 0.011) was observed after chilling (mean difference of about 0.3 log cfu/g), also if variability was observed among the flocks; the number of samples with high Campylobacter sp. counts (≥3 log cfu/g) was reduced (P = 0.010). In the second trial, low counts were generally detected (almost all lower than 3 log cfu/g). An evident decreasing trend was observed during storage, but the survival rate of Campylobacter on the cuts with skin was higher. All the isolates were identified as Campylobacter jejuni (72%) or Campylobacter coli. The data obtained were compared with the threshold limit set by EC Regulation 2073/2005, evidencing the impact of the sampling point on the counts. Our results highlighted the importance of applying a hurdle strategy including on-chain chilling and strict respect of the cold chain, allowing the food business operator to fulfill the process hygiene criteria and avoiding the delivery of highly contaminated meats.

Occurrence of Listeria spp. and Listeria monocytogenes Isolated from PDO Taleggio Production Plants.

The present study evaluated the presence of Listeria spp. and L. monocytogenes in four plants producing PDO Taleggio cheese. A total of 360 environmental samples were collected from different areas during production. The sampling points were identified as Food Contact Surfaces (FCS), transfer-Non Food Contact Surfaces (tr-NFCS), and non-transfer-NFCS (non-tr-NFCS). Fifty-nine ingredients/products were also analyzed. Listeria spp. was found in all the plants with a mean prevalence of 23.1%; plants that included a ripening area showed significantly higher prevalence if compared to the other plants. The positivity rate detected on FCS was moderate (~12%), but significantly lower if compared to NFCS (about 1/4 of the samples, p < 0.01). Among the FCS, higher prevalence was revealed on ripening equipment. Listeria spp. was never detected in the ingredients or products. A total of 125 Listeria spp. isolates were identified, mostly as L. innocua (almost 80%). L. monocytogenes was detected only from two FCS samples, in an area dedicated to the cutting of ripened blue cheeses; strain characterization by whole genome sequencing (WGS) evidenced a low virulence of the isolates. The results of the present study stress the importance of Listeria spp. management in the dairy plants producing PDO Taleggio and similar cheeses, mainly by the application of strict hygienic practices.

Identification and Pathogenic Potential of Bacillus cereus Strains Isolated from a Dairy Processing Plant Producing PDO Taleggio Cheese.

Low levels of contamination by Bacillus cereus at the cheese farm is essential for reducing any opportunity for growth prior consumption. In this study, B. cereus distribution in a plant producing Protected Designation of Origin Taleggio cheese was investigated and the virulence potential of the isolates was evaluated. Seventy-four samples were collected from Food and Non Food Contact Surfaces (FCS, NFCS), saline curd, and Taleggio. The eleven isolates were identified, typified, and clustered. Strains were tested for the production of hemolysins, hemolysin BL (HBL), phosphatidylcholine-specific phospholipase C (PC-PLC), proteases, and biofilm, and for the presence of chromosomal toxin-encoding genes (sph, plcA, cytK, entFM, bcet, entS, nheA, nheB, nheC). B. cereus was detected on NFCS, FCS, and curd, but not in Taleggio. The isolates were grouped into six clusters, and all produced PC-PLC, hemolysins, and proteases, and most of them HBL (66.7%). All the clusters harbored the nheA, sph, plcA, entFM, and cytK genes, and some also nheB (83.3%), nheC (66.7%), bcet (50.0%), and entS (66.7%). All strains showed biofilm-forming ability. Our data reveal possible contamination of production plants and cheese curd by potentially virulent B. cereus, but bacterial absence in Taleggio highlights the efficacy of a proper management of the production phases in assuring consumer's protection.

Comparison of Chemical Composition and Safety Issues in Fish Roe Products: Application of Chemometrics to Chemical Data.

Processed fish roes are acquiring considerable importance in the modern food market, entering more and more often as an ingredient in food preparation and as caviar substitutes. In this study, we defined quality, traceability and safety issues related to processed fish roe products from different species. The results obtained allowed to distinguish eggs originated from different fish species and to discriminate between fish roes and caviar samples obtained from four different sturgeons species. We observed that roes showed a trend of grouping according to ecological and reproductive habits of fish species. We highlighted the differences between eggs originated by farmed and freshwater fish, enriched in n6 polyunsaturated fatty acids (PUFAs), and all the others, in which n3 PUFAs were prevalent. In addition, we evaluated processed fish roes under a food safety point of view, combining microbiological analysis with the determination of organic acids, used in some products as authorized preservatives. Microbiological characterization has proved a general good hygienic level for these products. Organic acids determination showed values in compliance with European Union (EU) regulations in almost of samples; in some cases, we found a mismatch between the organic acids detected and what was reported in labels. Processed fish roes could be considered a safe product that can provide to human nutrition a valuable content of essential fatty acids.

Biopreservation as a potential hurdle for Bacillus cereus growth in fresh cheese.

This study aimed to evaluate the possible inhibitory effect of natural lactic acid bacteria on the growth of 2 Bacillus cereus strains. First, we evaluated the behavior of spores of B. cereus GPe2 and D43 when inoculated before cheesemaking using pasteurized or raw milk; no statistical differences were observed between cheese produced with the 2 types of milk. Then, lactic acid bacteria (LAB) were isolated from cheese at the last sampling time, identified, and tested in vitro for their antagonistic activity and organic acid production by using an HPLC method, showing antimicrobial potential. The LAB that produced larger inhibition halos (>9 mm) against B. cereus strains (LAB 3, 6, 9, 10: Lactococcus lactis ssp. lactis; LAB 7: Lactococcus lactis ssp. cremoris) were selected to produce a LAB mixture for subsequent tests. Spores of B. cereus GPe2 and D43 were inoculated in pasteurized milk before cheesemaking with or without addition of the LAB mixture at a high dosage. Bacillus cereus grew more slowly when LAB were added to the dairy matrix (with differences from 2.36 to 2.66 log cfu/g in B. cereus GPe2 and D43 growth).

Wet bone-in ageing and effect on beef quality technological parameters.

This study aimed to evaluate the effect of wet ageing on chemical-physical characteristics of sirloin and bone-in rib eye steaks obtained from 18 months old young bulls and 27 months old heifers. A positive beef tenderization effect of wet ageing was observed with lower values constantly detected in the rib-eye samples and significantly lower shear force values showed in heifer steaks if compared to bulls. The pH values detected both in pre- and post-ageing samples fell in the normal range for meats. The water holding capacity of sirloin samples increased with ageing while in rib eye steaks, no significant differences were detected among samples taken before and after ageing. Meat ageing resulted in a decrease of cooking loss, while meat colour parameters were not influenced by ageing and fell within the normal range. An evident improvement of the sensorial quality of bone-in wet aged beef, especially in tenderness, was achieved.

ß-hydroxyacyl-CoA-dehydrogenase activity differentiates unfrozen from frozen-thawed Yellowfin tuna (Thunnus albacares).

Changes in the ß-hydroxyacyl-CoAdehydrogenase (HADH) activity of fresh and frozen-thawed Yellowfin tuna were examined. A statistical approach to HADH activities determined in press juice allowed to set a critical value to differentiate fresh from frozen-thawed Yellowfin tuna: the threshold value was 3.7 U mL-1 at the probability level of 1%. The analysis of 37 tuna (not ready to eat) sampled on retail revealed the unconformity to labelling of 4 samples. A simple statistical algorithm was built to get probabilities from observed values on tuna of being or not frozen/thawed.

Non-thermal inactivation of Listeria spp. in a typical dry-fermented sausage: "Bergamasco" salami.

Aim of the present study was the evaluation of the growth potential of Listeria spp. inoculated in the typical North Italian dry fermented sausage "Bergamasco" salami during its production. As it was necessary to carry out the challenge test in the production line of the industry, according to the guidelines of the European Reference Laboratory for Listeria monocytogenes, a non-pathogenic "surrogate" microorganism was used: for the inoculum, two strains of Listeria innocua (1 ATCC, 1 strain isolated from a similar substrate) were used. The inoculation of the samples occurred during grinding and mixing of the sausage mass, before the filling. To avoid cross-contamination, the control samples were produced before the contaminated ones. After the dripping, salamis were subjected to the normal production process (drying and maturation in five steps at specific temperatures and humidity rates). The inoculated products were subjected to the enumeration of Listeria spp. at T0 (day of inoculation) and at T4 (post-drying), and every 10 days during curing (T10, T20, T30, T40, T50, T60, T70, T80 and T90), as this salami is generally sold as whole piece with varying levels of curing (from T20 to T90). Since the product may be cut in half and vacuumpacked, at each of the times starting from T20, half salami was vacuum-packed and stored for 30 days at 12°C, at the end of the which Listeria spp. enumeration was performed again. At all times and for each type of samples of each of the three batches, the enumeration of the natural microflora (Total Viable Count, lactic acid bacteria, Pseudomonas spp., Enterobacteriaceae) and the determination of water activity and pH were performed on control samples. The product was characterized by a high concentration of microflora (8-8.5 Log UFC/g), consisting mainly of lactic acid bacteria, added to the mixture at the beginning of the production process. The pH showed a decrease over time, expected for this type of products, due to the development of lactic acid bacteria (final pH: 5.42-5.55). The water activity reached values able to inhibit the development of Listeria spp. (final aw: 0.826-0.863). Listeria counts in the tested batches of "Bergamasco" salami showed the absence of significant growth in the product with a reduction of loads if compared to T0, between -0.59 and -1.04 Log CFU/g. Even in the samples subjected to vacuum packaging and storage at 12°C, the absence of significant increase of lactic acid bacteria in the product was highlighted with further decrease of bacterial loads (-0.70/-0.79 Log CFU/g if compared to T20). Considering the worst case scenario (thus the batch with the highest growth potential), in the products stored in the curing room at 14-16°C, at humidity of 80% and in the samples stored at 12°C and vacuum packaged, the threshold indicated by the EURL Lm guidelines (+0.5 Log CFU/g) for the growth of Listeria spp. was not reached, allowing to classify "Bergamasco" salami in the category 1.3 of the EC Reg. 2073/2005 as "Ready-to-eat food unable to support the growth of Listeria monocytogenes".