Effect of Commercial and Autochthonous Bioprotective Cultures for Controlling Listeria monocytogenes Contamination of Pecorino Sardo Dolce PDO Cheese.

The composition and physicochemical characteristics of short-aged Pecorino Sardo PDO (Protected Designation of Origin) cheese makes it permissive to Listeria monocytogenes growth. The PDO product specification stipulates that this cheese is produced with whole sheep's milk inoculated with cultures from the area of origin. Therefore, the use of bioprotective cultures for the inhibition of pathogens in PDO cheeses is allowed only if autochthonous microorganisms are used. Furthermore, bioprotective cultures are generally used on the cheese surface to prevent the outgrowth of L. monocytogenes, the application of which can be time-consuming and require specialist technical knowledge. In this study, we examine the direct addition of bioprotective cultures to the cheese vat and compare the activity of a commercial bioprotective culture (Lactiplantibacillus plantarum) and an autochthonous lactic acid bacterium with bioprotective properties (Lactobacillus delbruekii sups. sunkii), for the inhibition of L. monocytogenes in Pecorino Sardo PDO cheese. Three types of Pecorino Sardo PDO cheese were made with bioprotective cultures added directly to the cheese milk along with the starter inoculum: PSA, with the commercial bioprotective culture; PSB, with the autochthonous bioprotective culture; and a CTRL cheese with no bioprotective culture. A challenge test was performed on each of these cheeses by artificially contaminating the cheese surface with L. monocytogenes (2 Log10 CFU/g). Three batches of each cheese type were analyzed to enumerate mesophilic and thermophilic lactic acid bacteria and to investigate the growth potential of L. monocytogenes during manufacturing, at the end of ripening, at the end of shelf-life, and after 180 days from cheese production. Both bioprotective cultures tested in this study showed inhibitory action against the pathogen with 0.3-1.8 Log10 CFU/g (colony-forming unit per gram) reduction levels. The autochthonous organism, L. sunkii, was as effective as the commercially supplied culture, and the addition of the bioprotective cultures to the cheese-making procedure offered protection against L. monocytogenes. The direct addition of bioprotective cultures to the making procedure of Pecorino Sardo PDO cheese is a potentially innovative strategy to improve the safety of this product.

Use of commercial protective cultures in portioned sheep milk cheeses to control Listeria monocytogenes.

The main objective of this study was to innovate soft and semi-cooked sheep milk cheese production processes with the use of a commercial protective culture able to control Listeria monocytogenes growth. A freeze-dried commercial culture of Lactobacillus plantarum was tested in DS cheese and PS cheese, two types of pasteurized sheep milk, raw-paste cheeses aged for no less than 20 and 30 days respectively. In the first step, in vitro tests were conducted to identify the most suitable matrix for the growth of L. plantarum in order to create a subculture that could be used at industrial cheese-making plants. During the second phase of the study, L. plantarum culture was introduced in the manufacturing process of the cheeses in a production plant. Finally, a challenge test was conducted on portioned DS and PS cheeses in order to evaluate the activity of the protective culture against L. monocytogenes: the cheeses were portioned, experimentally contaminated with L. monocytogenes strains, vacuum packed and stored at +4°C (correct storage conditions) and at +10°C (thermal abuse). Cheeses were analysed at the end of the shelf-life to evaluate the presence and growth of L. monocytogenes, to enumerate lactic acid bacteria and determine chemicalphysical features. The results confirmed that protective cultures are a useful technological innovation to control L. monocytogenes growth during cheese storage without altering composition, microflora and chemical- physical characteristics of the product. However, the use of protective cultures should be applied as an integration of risk control measures and not as a substitute for preventive actions.

Production of Farmstead Lactose-Free Pecorino di Osilo and Ricotta Cheeses from Sheep's Milk.

The present work was aimed to define and validate farmstead production of lactose-free Pecorino di Osilo cheese, fresh ricotta cheese, and salted and smoked ricotta cheese (Ricotta mustia). The enzymatic activity of the commercial preparation containing lactase (1.1 g/mL), preliminarily tested using a spectrophotometric titration, showed activity equal to 4950±40 neutral lactase unit/g. The amount of lactase required to obtain the lactose-free milk was then established in triplicate laboratory trials, by adding the enzyme at concentrations of 0.7, 0.9 and 1.1 g/L in flasks containing 160 mL of raw sheep's milk. Samples were incubated under conditions expected during milk storage and cheese-making. The residual lactose content in milk was determined by enzymatic method. The addition of lactase at concentration of 1.1 g/L of milk reduced the lactose concentration below the limit of detection (LOD) of 0.06 g/L. The procedure was validated at a dairy farm, using three different batches of bulk raw sheep's lactose-free milk that were transformed into Pecorino di Osilo cheese. The resulting whey was used to produce fresh ricotta and Ricotta mustia cheese. Raw milk and whey samples were always below lactose detection limit. The residual lactose was measured in Pecorino di Osilo cheese, after 24 hours and 30 days from production; in fresh ricotta cheese, after 48 hours; in Ricotta mustia cheese after 7 days. The determination of lactose content in cheese samples was conducted by a gas chromatography-flame ionization detection method, which showed a LOD and limit of quantification respectively of 1.8 and 5.6 mg/kg for cheese, and 1.35 and 4.2 mg/kg for both ricotta cheeses. The lactose concentration was always below the relevant LOD values in all samples. The mean concentration of galactose and glucose were respectively 13,000±2000 and 11,000±2000 mg/kg in fresh Pecorino di Osilo, 1100±300 and 1200±300 mg/kg in fresh ricotta, and 950±400 and 750±250 mg/kg in Ricotta mustia. The results of the present study showed that the production of farmstead lactose-free Pecorino di Osilo cheese and ricotta cheeses from raw sheep's milk is easily achievable. The main issue for farmstead production of artisanal lactose-free products is the implementation of permanent procedures based on hazard-analysis and critical control principles aimed at guaranteeing the effectiveness of the process and at acquiring analytical evidences to demonstrate the fulfilment of law requirements for labelling.

Evolution of the Microbiological Profile of Vacuum-Packed Ricotta Salata Cheese During Shelf-Life.

Ricotta salata cheese is a salted variety of ricotta traditionally made in Sardinia (Italy) from the whey remaining after the production of Pecorino Romano protected designation of origin or other sheep milk cheeses. Ricotta salata cheese is very critical for the possible growth of pathogenic and spoilage microorganisms. Sporadic cases of listeriosis associated with ricotta salata cheese have been reported over recent years. The objective of the present study was to assess the evolution of spoilage and pathogen microorganism of vacuum-packed ricotta salata cheese during the entire product shelf-life. The durability study was conducted on 18 vacuum-packed ricotta salata cheese samples analysed at the beginning of the shelf-life and after 60 and 90 days of refrigerated storage. Pathogens as Listeria monocytogenes and Bacillus cereus were never detected. During shelf-life total bacterial counts ranged between 7.90±0.64 and 9.19±0.58 CFU g-1 on the rind and between 2.95±0.68 and 4.27±1.10 CFU g-1 in the inner paste, while Enterobacteriaceae ranged between 4.22±0.66 and 5.30±0.73 CFU g-1 on the rind and 3.13±1.80 and 2.80±0.88 CFU g-1 in the inner paste. By considering the technology used, the intrinsic properties and the almost total absence of competing microflora, ricotta salata cheese can support the growth of spoilage and pathogen microorganisms originating from the processing environment. The high level of total bacterial counts and Enterobacteriaceae observed both on the rind and in the inner paste suggests contamination of the product from the processing environment. Therefore, a strict implementation of hygiene during processing is essential in order to reduce the load of environmental contaminants that may grow during refrigerated storage.

Occurrence and behavior of Bacillus cereus in naturally contaminated ricotta salata cheese during refrigerated storage.

The present study shows the fate of Bacillus cereus in refrigerated ricotta salata cheese during shelf-life. 144 ricotta salata cheese belonging to nine naturally contaminated batches were stored refrigerated and analyzed at 24 h, 30, 60 and 90 days of storage. Total bacterial count, B. cereus spores and vegetative forms, intrinsic properties and composition were determined. The presence of spores was sporadic while the prevalence and the level of B. cereus vegetative cells decreased respectively from 83.3 % to 4.65 ± 0.74 cfu g(-1) at the beginning of the observation period to 33.3 % and 1.99 ± 0.55 cfu g(-1) after 90 days. No information is currently available on the fate of B. cereus in ricotta salata. The production process of ricotta salata includes steps such as whey heating followed by slow cooling of clots, which expose to the risk of spore germination and successive growth to levels compatible with toxins production. The prolonged refrigerated storage was not favorable to sporulation, explaining the successive death of vegetative cells. The present study demonstrate the potential risk of food poisoning as consequence of pre-formed emetic toxins in ricotta salata. Food safety of ricotta salata relies on the rapid refrigeration of the product during critical phases for cereulide production.