Assessment of aflatoxin M1 enrichment factor in cheese produced with naturally contaminated milk.

Aflatoxin M1 (AFM1) is a well-known carcinogenic compound that may contaminate milk and dairy products. Thus, with the regulation 1881/2006, the European Union established a concentration limit for AFM1 in milk and insisted on the importance of defining enrichment factors (EFs) for cheese. In 2019, the Italian Ministry of Health proposed four different EFs based on cheese's moisture content on a fat-free basis (MMFB) for bovine dairy products. This study aimed to define the EFs of cheese with different MFFB. The milk used for cheesemaking was naturally contaminated with different AFM1 concentrations. Results showed that all the EF average values from this study were lower than those of the Italian Ministry of Health. Hence, the current EFs might need to be reconsidered for a better categorization of AFM1 risk in cheese.

Inactivation of Listeria monocytogenes and Salmonella spp. in Milano-Type Salami Made with Alternative Formulations to the Use of Synthetic Nitrates/Nitrites.

During the manufacture of Italian salami, a traditional meat product, a sequence of hurdles like meat fermentation, air-drying, and long ripening processes are generally sufficient to inhibit the growth of most pathogens. Furthermore, Italian salami are traditionally produced by adding synthetic nitrates/nitrites to raw meat with safety and technological aims, even if controversial opinions about their use still remain, particularly in relation to the consumer demand for natural food products. In this context, the aim of the study was to investigate the inactivation of Listeria monocytogenes and Salmonella spp. during the manufacturing process of Milano-type salami made with different formulations to evaluate the contribution of the hurdles and the vegetable or synthetic additives on the inactivation of pathogens. Thus, a challenge study was performed dividing ca. 400 kg of Milano-type salami batter into three batches: Batch (A) without nitrates/nitrites; Batch (B) with vegetable nitrates, and Batch (C) with synthetic nitrates/nitrites. The batches were separately inoculated with L. monocytogenes and Salmonella spp. and the pathogens' survival was evaluated during the fermentation, draining, and 70-day ripening of the Milano-type salami. The pathogen counts decreased in all tested conditions, even though the highest inactivation of L. monocytogenes and Salmonella spp. (p < 0.05) was observed when nitrates or nitrites were added to the batter. This study shows how the safety of these products cannot exclude the aspect of the hurdle technology during the process, which plays a major role in the reduction of pathogens, but additives like nitrates and nitrites allow for a greater margin of safety. Thus, further studies are needed to validate the use of natural compounds as alternatives to conventional preservatives in meat products. These results may provide new information to support food business operators in producing traditional foods with alternative preservatives and competent authorities in verifying the safety of the products made with natural compounds, and to control the process parameters responsible for the synergistic effect against pathogens such as L. monocytogenes and Salmonella spp.

Modeling the behavior of Listeria innocua in Italian salami during the production and high-pressure validation of processes for exportation to the U.S.

A model describing Listeria innocua evolution according to process parameters of 51 Italian salami processes and HPP in 31 companies was developed. A total of 51 challenge tests were performed. During processing a L. innocua reduction of 0.34-4.32 Log10 CFU/g was observed and HPP further reduced the count of 0.48-3.47 Log10 CFU/g; an overall reduction of 1.04-5.68 is reached. PH after acidification/drying process, aw after seasoning, duration of the seasoning and caliber resulted associated (p < 0.05) with L. innocua decrease. HPP efficacy was associated (p < 0.05) with aw and pH of the product: higher the pH and aw after the acidification/drying and seasoning phases, higher resulted the L. innocua reduction after HPP. No significant association was observed between L.innocua and salt, nitrate and starter content and other characteristics of process. The model meets companies and Authorities needs and represents a useful tool to predict L. monocytogenes lethality, giving recommendations to food business operators interested in exportation to the U.S.

Raw donkey milk versus raw cow's milk. A preliminary study to compare the growth of Listeria monocytogenes and Staphylococcus aureus.

Gram-positive foodborne pathogens such as Listeria monocytogenes and Staphylococcus aureus can grow in a wide variety of foods, including raw milk. The aim of the study was to compare the growth of L. monocytogenes and S. aureus inoculated in donkey and cow samples of raw milk during a storage time of 11 days at 8 °C. Moreover, the study aimed to evaluate the influence of lactic acid bacteria (LAB) content on the growth of the two microbiological populations considered. LAB content was lower in raw donkey milk than in raw cow's milk during the entire analyses; on the other hand, pH levels were higher in the donkey milk rather than in the cow's milk, although both values showed a decrease at the day 11. S. aureus showed no significant differences in the two types of milk. From day 0 to 11, L. monocytogenes increased from 3.68 ± 0.02 log CFU/mL to 6.31 ± 0.07 log CFU/mL and from 3.64 ± 0.04 log CFU/mL to 4.59 ± 1.04 log CFU/mL, in donkey milk and in cow's milk, respectively. Our results showed that donkey milk is a more favourable matrix to support the growth of L. monocytogenes than cow's milk.

Behavior of Listeria innocua during the manufacturing and pit-ripening of Formaggio di Fossa di Sogliano PDO cheese.

Formaggio di Fossa di Sogliano is a traditional Italian Protected Designation of Origin (PDO) cheese ripened for a minimum of 5 months, with the feature of a ripening of at least 80 to at most 100 days in pits, digged into tuffaceous rocks according to medieval tradition of Italy. In this study, a challenge test using Listeria innocua as a surrogate of Listeria monocytogenes was performed, with the aim of increasing knowledge concerning the impact of the Fossa cheese process, and especially of the traditional ripening process of this PDO, on the behaviour of L. monocytogenes. Pasteurized milk was experimentally inoculated with 4.5 log CFU/mL cocktail by three L. innocua strains, and L. innocua and Mesophilic Lactic Acid Bacteria (LAB) counts as well as the evolution of temperatures, pH and aw values were monitored throughout the manufacturing and ripening processes. Throughout the ripening in maturation room a constant temperature of 8°C was observed reaching a temperature between 10 and 15.5°C during ripening into pit. In the final products data for LAB concentration, pH and aw values were roughly in accordance with literature, even if some differences were, probably due to variability of artisanal cheese productions. The numbers of L. innocua showed a slight decrease but remained stable until the end of ripening in maturation room, whereas a significant reduction of the microorganism was observed in the final product, at the end of the ripening into the pit. The findings give scientific evidence that the process of this PDO prevented the L. innocua growth, allowing us to speculate a similar behaviour of L. monocytogenes. Based on this study, the recommendation to extend as much as possible the ripening into pit (from 80 to 100 days) was provided to food business operators as a risk mitigation strategy to be implemented.

Reduction of Salmonella spp. populations in Italian salami during production process and high pressure processing treatment: Validation of processes to export to the U.S.

This study involved ten enterprises producing Italian salami, 20 different samples of fermented sausages underwent challenge tests to assess and record the following parameters: time, temperature, pH, aw, and Salmonella counts. A linear regression model was used to describe the Salmonella spp. decay: at the end of the process the result of total Salmonella reduction was 0.97-5.84 Log10 CFU/g and it was significantly associated with pH at the end of acidification/drying process, aw at the end of seasoning period, the duration of seasoning, and the caliber of salami respectively. High Pressure Processing (HPP) further reduced the Salmonella level by 2.41-5.84 Log10 CFU/g with an efficacy that resulted inversely associated with aw of salami at the end of seasoning; the objective of 5-Log reduction was always reached in all the cases tested by the production process plus HPP. This model could be a useful tool for enterprises and Authorities to evaluate the efficacy of the processes to reduce Salmonella load for exportation to the U.S.

Preparation of Powdered Infant Formula: Could Product's Safety Be Improved?

The recent outbreak of Salmonella Agona linked to the consumption of infant formula (powdered formula) has rekindled the attention about the correct procedures for preparation and use of these products. International guidelines have already been published so far, particularly in association with Cronobacter sakazakii in early 2000s. FAO/WHO suggested to reconstitute formula with water at no less than 70°C. We therefore contaminated powdered formula with low levels of Salmonella spp and C sakazakii to evaluate the pathogens inactivation during the formula preparation using water at 70°C. In these conditions we observed a survival of both pathogens, indicating that the suggested recommendations may be not enough to guarantee the safety of this product. Higher temperatures are needed to reduce the biological risk, even if it may be not easily realized in actual domestic conditions. Moreover, the impact on the nutritional value of reconstituted formulas should be evaluated.

Listeria monocytogenes in Gorgonzola cheese: Study of the behaviour throughout the process and growth prediction during shelf life.

As reported on RASFF's portal, in the first 9months of 2016, a total of 13 "alerts/information for attention" were issued concerning the presence of Listeria monocytogenes in mould cheeses throughout Europe. This study analyzes the behaviour of L. monocytogenes in Gorgonzola cheese, a typical Italian soft blue-veined cheese, when contaminated at different time points. In the first challenge test, the pasteurized milk was contaminated and the complete cheese manufacture (cheesemaking, ripening) and shelf life was simulated. After a decrease during the first days of the cheesemaking, the pH remained constant for 35days (5weeks) and then it increased rapidly reaching the final values of 6.8±0.02 in the core and 5.8±0.4 on the rind. At the same time, the pathogen concentration decreased (about 2logCFU/g), although during the last week a rapid pathogen growth was observed after the rise in pH values. When the cheese was stored at thermal abuse condition (8-12°C), the pathogen concentration on the rind was 4.8±0.3 log CFU/g and after 66days (about 9weeks) no significant difference (p>0.05) was observed; whereas, a growth from 5.4±0.4 to 7.1±0.5logCFU/g was observed in the core. A second challenge test was performed using three batches of commercial slices of Gorgonzola cheese inoculated by L. monocytogenes and stored at 8°C. The maximum specific growth rates (µmax, 1/h) of L. monocytogenes estimated ranged from 0.007 to 0.061. The square root model was used to predict the µmax at others temperature and to establish the time necessary to reach the European critical legal limit of 2logCFU/g, in different storage scenarios. The predictions obtained in this study can be applied to any time-temperature profile, and in particular to the conditions to which the product is most likely to be subject in normal use, up to its final consumption. This study can be considered a valuable contribution also aimed at supporting the monitoring surveys carried out by officers of the Regional Veterinary Authority.

Seasonal Variability of Thermophilic Campylobacter Spp. in Raw Milk Sold by Automatic Vending Machines in Lombardy Region.

In temperate climates, a seasonal trend was observed in the incidence of human campylobacteriosis cases, with peaks reported in spring and autumn in some countries, or in summer in others; a similar trend was observed in Campylobacter spp. dairy cattle faecal shedding, suggesting that cattle may play a role in the seasonal peak of human infection. The objectives of this study were to assess if a seasonal trend in thermophilic Campylobacter spp. contamination of raw milk exists and to evaluate a possible relation between this and the increase of human campylobacteriosis incidence in summer months. The results showed a mean prevalence of 1.6% of milk samples positive for thermophilic Campylobacter spp. with a wide range (0.0-3.1%) in different months during the three years considered. The statistical analysis showed a significant difference (P<0.01) of the prevalence of positive samples for thermophilic Campylobacter spp. between warmer and cooler months (2.3 vs 0.6%). The evidence of a seasonal trend in thermophilic Campylobacter spp. contamination of raw milk sold for direct consumption, with an increase of the prevalence in warmer months, may represent one of the possible links between seasonal trend in cattle faecal shedding and seasonal trend in human campylobacteriosis.

Multiple-Strain Approach and Probabilistic Modeling of Consumer Habits in Quantitative Microbial Risk Assessment: A Quantitative Assessment of Exposure to Staphylococcal Enterotoxin A in Raw Milk.

Quantitative microbial risk assessment (QMRA) models are extensively applied to inform management of a broad range of food safety risks. Inevitably, QMRA modeling involves an element of simplification of the biological process of interest. Two features that are frequently simplified or disregarded are the pathogenicity of multiple strains of a single pathogen and consumer behavior at the household level. In this study, we developed a QMRA model with a multiple-strain approach and a consumer phase module (CPM) based on uncertainty distributions fitted from field data. We modeled exposure to staphylococcal enterotoxin A in raw milk in Lombardy; a specific enterotoxin production module was thus included. The model is adaptable and could be used to assess the risk related to other pathogens in raw milk as well as other staphylococcal enterotoxins. The multiplestrain approach, implemented as a multinomial process, allowed the inclusion of variability and uncertainty with regard to pathogenicity at the bacterial level. Data from 301 questionnaires submitted to raw milk consumers were used to obtain uncertainty distributions for the CPM. The distributions were modeled to be easily updatable with further data or evidence. The sources of uncertainty due to the multiple-strain approach and the CPM were identified, and their impact on the output was assessed by comparing specific scenarios to the baseline. When the distributions reflecting the uncertainty in consumer behavior were fixed to the 95th percentile, the risk of exposure increased up to 160 times. This reflects the importance of taking into consideration the diversity of consumers' habits at the household level and the impact that the lack of knowledge about variables in the CPM can have on the final QMRA estimates. The multiple-strain approach lends itself to use in other food matrices besides raw milk and allows the model to better capture the complexity of the real world and to be capable of geographical specificity.

Behaviour of Listeria Monocytogenes and Escherichia Coli O157:H7 During the Cheese Making of Traditional Raw-Milk Cheeses from Italian Alps.

The behaviour of Listeria monocytogenes and Escherichia coli O157:H7 was studied during the manufacture and ripening of two traditional Italian Alps cheeses. Each cheese type was manufactured in a pilot plan from raw cow milk (without the addition of starter cultures) artificially inoculated with L. monocytogenes and E. coli O157:H7 to a final concentration of about 4 log CFU/mL. The pathogens were enumerated throughout the cheese making and ripening processes to study their behaviour. When the milk was inoculated with 4 Log CFU/mL, the pathogens counts increased in the first time during the manufacturing process and then remained constant, until the end of ripening, or decreased significantly. Results indicate that the environment and nature of food borne pathogens affected the concentration of the bacteria during the manufacturing and ripening process. Thus, the presence of low cells numbers of L. monocytogenes and E. coli O157:H7 in milk destined for the production of raw milk cheeses characterized by a cooking of the curd less than 48°C can constitute a hazard for the consumer.

Human campylobacteriosis related to the consumption of raw milk sold by vending machines in Italy: Quantitative risk assessment based on official controls over four years.

A quantitative risk assessment (RA) model was developed to describe the risk of campylobacteriosis linked to consumption of raw milk sold in vending machines in Italy. Exposure assessment was based on the official microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2011, microbial growth during storage, destruction experiments, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time-temperature field handling conditions detected. Two separate RA models were developed, one for the consumption of boiled milk and the other for the consumption of raw milk, and two different dose-response (D-R) relationships were considered. The RA model predicted no human campylobacteriosis cases per year either in the best (4°C) storage conditions or in the case of thermal abuse in case of boiling raw milk, whereas in case of raw milk consumption the annual estimated campylobacteriosis cases depend on the dose-response relationships used in the model (D-R I or D-R II), the milk time-temperature storage conditions, consumer behaviour and age of consumers, namely young (with two cut-off values of ≤5 or ≤6 years old for the sensitive population) versus adult consumers. The annual estimated cases for young consumers using D-R II for the sensitive population (≤5 years old) ranged between 1013.7/100,000 population and 8110.3/100,000 population and for adult consumers using D-R I between 79.4/100,000 population and 333.1/100,000 population. Quantification of the risks associated with raw milk consumption is necessary from a public health perspective and the proposed RA model represents a useful and flexible tool to perform future RAs based on local consumer habits to support decision-making on safety policies. Further educational programmes for raw milk consumers or potential raw milk consumers are required to encourage consumers to boil milk to reduce the associated risk of illness.

Quantitative risk assessment of human salmonellosis and listeriosis related to the consumption of raw milk in Italy.

Two quantitative risk assessment (RA) models were developed to describe the risk of salmonellosis and listeriosis linked to consumption of raw milk sold in vending machines in Italy. Exposure assessment considered the official microbiological records monitoring raw milk samples from vending machines performed by the regional veterinary authorities from 2008 to 2011, microbial growth during storage, destruction experiments, consumption frequency of raw milk, serving size, and consumption preference. Two separate RA models were developed: one for the consumption of boiled milk and the other for the consumption of raw milk. The RA models predicted no human listeriosis cases per year either in the best or worst storage conditions and with or without boiling raw milk, whereas the annual estimated cases of salmonellosis depend on the dose-response relationships used in the model, the milk storage conditions, and consumer behavior in relation to boiling raw milk or not. For example, the estimated salmonellosis cases ranged from no expected cases, assuming that the entire population boiled milk before consumption, to a maximum of 980,128 cases, assuming that the entire population drank raw milk without boiling, in the worst milk storage conditions, and with the lowest dose-response model. The findings of this study clearly show how consumer behavior could affect the probability and number of salmonellosis cases and in general, the risk of illness. Hence, the proposed RA models emphasize yet again that boiling milk before drinking is a simple yet effective tool to protect consumers against the risk of illness inherent in the consumption of raw milk. The models may also offer risk managers a useful tool to identify or implement appropriate measures to control the risk of acquiring foodborne pathogens. Quantification of the risks associated with raw milk consumption is necessary from a public health perspective.

Microbiological and Physico-Chemical Changes During Manufacture of an Italian Goat Cheese Made from Raw Milk.

The aim of this work was to study the microbiological and physico-chemical changes throughout three cheesemaking replicates of Italian Formaggelle di capra cheese made from raw goat milk. Therefore, during the process, three samples of milk, curd and cheese at 3, 7, 11, 14, 21 and 30 days of ripening old cheese were taken from three cheesemaking replicates. The average of total mesophilic bacteria and Enterobacteriaceae count in raw milk was 5.27±0.57 and 3.8±1.02 Log cfu/mL, respectively. Lactic acid bacteria was the predominant bacterial group during the process, and they developed in different ways in each of the media used (M17 and MRS agar). Variability of microbial concentrations was observed between three cheesemaking replicates. A correlation between the presence of higher levels of Enterobacteriaceae in milk and the presence of other contaminants bacteria such as Escherichia coli ß-glucuronidase-positive and coagulase-positive staphylococci was observed. In cheesemaking replicate n. 2, E. coli level was 5.07±0.03 Log cfu/mL and increased by about 1 log until the last week of ripening, when the level decreased to 5.69±0.2 Log cfu/mL. The milk used for the cheesemaking replicate n. 2 was found to be contaminated also by coagulase-positive staphylococci (3.18±0.06 Log cfu/mL), but the behaviour of this group appeared to be very variable. In this study a first step of process control and microbial groups study was performed and the cheesemaking process was registered in the website www.ars-alimentaria.it, the Italian site supported by the Italian Board of Health.

Reduction of Escherichia Coli O157:H7 During Manufacture and Ripening of Italian Semi-Dry Salami.

In order to simulate a contamination at the processing plant, one batch of freshly-processed salami batter (20 kg) was inoculated (1% v:w) with 5 log colony forming unit (CFU)/g of a multi-strain cocktail of two strains of Escherichia coli O157:H7 (registered and wild strain). Another batch was inoculated (1% v:w) with sterile physiological saline solution and used to check the lactic acid bacteria (Lab) behaviour and the changes of physicochemical parameters (pH and aw ). Both batches were then processed to obtain a semi-dry salami (Hungarian-style): microbiological and physico-chemical properties were monitored during 94 days of ripening. During the manufacturing process, the levels of pathogen decreased of about 2.18 log CFU/g with respect to the initial inoculated levels. The behaviour of the indigenous bacteria such as Lab and the physico-chemical properties can help to determine the fate of pathogens throughout processing.

Study of Growth Potential of Listeria Monocytogenes in Low Fat Salami: An Innovative Italian Meat Product.

In the last years, consequently to EC Regulation no. 1924/2006 on nutrition and health claims made on foods, some Italian food businnes operators (FBOs) leaders in the meat sector, invested in research to develop innovative products such as low fat salami, containing up to 30% less fat than the traditional one. For FBOs it is essential to demonstrate for each production process whether the substrate allows the growth of L. monocytogenes and whether L. monocytogenes could reach or exceed the limit of 100 cfu g-1 at the end of the shelf life, as stated by EC Regulation no. 2073/2005. In the present study, the growth potential of L. monocytogenes during the shelf life of low fat salami packed in modified atmosphere was evaluated. The results show that the product is unable to support the growth of pathogen, even if the storage temperature is between 8 and 12°C.

Growth Potential of Listeria Monocytogenes in Sliced Turkey Bresaola Packed in Modified Atmosphere.

According to EC Regulation No 2073/2005, for food business operators that produce ready-to-eat (RTE) product, it is crucial to be able to demonstrate if the product supports the growth of Listeria monocytogenes. The objective of the study was therefore to evaluate the behaviour of L. monocytogenes in sliced RTE turkey bresaola (made by cured turkey breast 4.5% NaCl, 1% sodium lactate, sodium nitrite 150 ppm and flavouring) during the shelf life of the product, simulating a contamination during the slicing operation. Considering a shelf life of 90 days, as defined by manufacturer, the packages of sliced bresaola were stored at 5°C for 7 days and at 8°C for the remaining storage time (83 days). L. monocytogenes count decreased during storage test from 1.43/1.98 log cfu/g in the three batches tested to 1.03 log cfu/g in one batch and to undetectable levels in the other two batches. The results show that the investigated product is unable to support the growth of L. monocytogenes.

Behaviour of Escherichia Coli O157:H7 During the Manufacture and Ripening of an Italian Traditional Raw Goat Milk Cheese.

Formagelle di capra is a raw goat cheese produced from whole chilled goat milk; traditional technology involving unpasteurised milk and indigenous lactic starter cultures is employed for its production in Italy. The purpose of this study was to assess the behaviour of Escherichia coli O157:H7 during the manufacturing and ripening of this raw goat milk cheese. Raw milk was experimentally inoculated with E. coli O157:H7 in a laboratory scale plant and the count was monitored during production and 30 days of ripening required for this cheese. Results showed that E. coli O157:H7 count increased to more than 1.5 Log cfu g-1 during cheese production and remained constant until the end of ripening. The evidence that E. coli O157:H7 is able to survive during the manufacturing and ripening process suggests that the 30-day ripening period alone is insufficient to eliminate levels of viable E. coli O157:H7 in Formaggelle di capra cheese and that the presence of low numbers of E. coli O157:H7 in milk destined for the production of raw goat milk cheeses could represent a potential source of infection for humans and a threat for consumers.

Four-year monitoring of foodborne pathogens in raw milk sold by vending machines in Italy.

Prevalence data were collected from official microbiological records monitoring four selected foodborne pathogens (Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, and Campylobacter jejuni) in raw milk sold by self-service vending machines in seven Italian regions (60,907 samples from 1,239 vending machines) from 2008 to 2011. Data from samples analyzed by both culture-based and real-time PCR methods were collected in one region. One hundred raw milk consumers in four regions were interviewed while purchasing raw milk from vending machines. One hundred seventy-eight of 60,907 samples were positive for one of the four foodborne pathogens investigated: 18 samples were positive for Salmonella, 83 for L. monocytogenes, 24 for E. coli O157:H7, and 53 for C. jejuni in the seven regions investigated. No significant differences in prevalence were found among regions, but a significant increase in C. jejuni prevalence was observed over the years of the study. A comparison of the two analysis methods revealed that real-time PCR was 2.71 to 9.40 times more sensitive than the culture-based method. Data on consumer habits revealed that some behaviors may enhance the risk of infection linked to raw milk consumption: 37% of consumers did not boil milk before consumption, 93% never used an insulated bag to transport raw milk home, and raw milk was consumed by children younger than 5 years of age. These results emphasize that end-product controls alone are not sufficient to guarantee an adequate level of consumer protection. The beta distribution of positive samples in this study and the data on raw milk consumer habits will be useful for the development of a national quantitative risk assessment of Salmonella, L. monocytogenes, E. coli O157, and C. jejuni infection associated with raw milk consumption.