Italian Journal of Food Safety 2016; volume 5:5501

Evolution of the microbiologi- Introduction Correspondence: Carlo Spanu, Department of cal profile of vacuum-packed Veterinary Medicine, University of Sassari, via salata during Ricotta salata cheese or Ricottone or Ricotta Vienna 2, 07100 Sassari. Toscanella is a salted variety of ricotta tradi- Tel: +39.079.229454 - Fax: +39.079.229458. shelf-life E-mail: [email protected] tionally made in () from the Daniele Casti,1 Christian Scarano,1 remaining after the production of Pecorino Key words: ; ; Composition; Carlo Pala,1 Francesca Cossu,1 Romano protected designation of origin or Shelf life. other . A wide variety of Sonia Lamon,1 Vincenzo Spanu,1 whey cheeses are manufactured from sheep Conflict of interest: the authors declare no poten- Michela Ibba,1 Anna Maria Mocci,1 milk in several European countries, especially tial conflict of interest. Francesco Tedde,1 Gavino Nieddu,2 in the Mediterranean basin: i.e. , 1 Carlo Spanu, and (), Anari Funding: this work was funded by Programma di Enrico Pietro Luigi De Santis1 Sviluppo Rurale Sardegna 2007-2013 Misura 124 (Cyprus), Requesón (Spain), Requeijao Cooperazione per lo Sviluppo di Nuovi Prodotti, 1 Department of Veterinary Medicine, (Portugal), (France), Urdă (Balkans Processi e Tecnologie nei Settori Agricolo University of Sassari, Sassari; region), each with its own distinctive technol- Alimentare e in quello Forestale, project ID: 2Cooperativa Allevatori Ovini, Fenosu ogy. In most of the cases these are fresh and H78F13000050007. The authors are grateful to all (OR), Italy soft or semi-soft traditional products made in the members of the joint dairy industry consor- small-scale operations and sold in local mar- tium Associazione Temporanea di Scopo - Aziende kets. The manufacturing of ricotta salata Casearie Riunite for their cooperation in the rese- arch. cheese in Sardinian industrial cheese-making Abstract plants follows the traditional batch process Received for publication: 25 August 2015. (Laore, 2015). The whey is filtered and pre- Revision received: 9 October 2015. heated at 60-70°C with the use of a plate heat Accepted for publication: 9 October 2015. Ricotta salata cheese is a salted variety of exchanger. The whey can be added with pas- ricotta traditionally made in Sardinia (Italy) teurised cream or milk to enrich the solid con- This work is licensed under a Creative Commons from the whey remaining after the production tent. The blend is then transferred to large Attribution-NonCommercial 4.0 International of Pecorino Romano protected designation of open kettles (1200-1500 L capacity) and heated License (CC BY-NC 4.0). origin or other sheep milk cheeses. Ricotta sala- to temperature above 85°C and held for 30 ta cheese is very critical for the possible growth ©Copyright D. Casti et al., 2016 min. After the flocculated protein rises to the Licensee PAGEPress, Italy of pathogenic and spoilage microorganisms. surface, clots are collected using perforated Italian Journal of Food Safety 2016; 5:5501 Sporadic cases of listeriosis associated with ladles and transferred into plastic cylindrical doi:10.4081/ijfs.2016.5501 ricotta salata cheese have been reported over molds lined with cheese cloth. The curd is then recent years. The objective of the present study pressed to enhance drainage for up to 24 h and was to assess the evolution of spoilage and transferred in cold room (10-12°C) for about Bacillus cereus strains. Outbreaks and spo- pathogen microorganism of vacuum-packed 10 days. Salting can be made by adding directly radic cases of listeriosis associated with ricot- ricotta salata cheese during the entire product sodium chloride to the whey or to the curd dur- ta salata cheese have been reported over shelf-life. The durability study was conducted ing molding or by drying salt during refrigerat- recent years (RASFF, 2008; CDC, 2012). on 18 vacuum-packed ricotta salata cheese sam- ed storage or in brine after molding. Ricotta Evidence that L. monocytogenes contamination ples analysed at the beginning of the shelf-life salata cheese is dried until the moisture con- of ricotta salata cheese origins from the pro- and after 60 and 90 days of refrigerated storage. tent is ca. 50% if intended to be used for grat- cessing environment and that this product Pathogens as Listeria monocytogenes and ing or of ca. 55-60% if used as is. After the pro- supports the growth of the pathogen during Bacillus cereus were never detected. During duction process the mean fat and protein con- refrigerated storage have been documented shelf-life total bacterial counts ranged between tent are respectively 28-33% and 14-23%, the 7.90±0.64 and 9.19±0.58 CFU g-1 on the rind and pH ranges from 6.1 to 6.9, while the water (Spanu et al., 2012, 2015a; Ibba et al., 2013). -1 Despite the frequent contamination of ricotta between 2.95±0.68 and 4.27±1.10 CFU g in the activity (aW) from 0.940 to 0.970 (Spanu et al., inner paste, while Enterobacteriaceae ranged 2012, 2013). The final product is a firm wheel salata cheese with B. cereus and the recovery between 4.22±0.66 and 5.30±0.73 CFU g-1 on of ca. 3 kg weight, which is individually packed of potentially pathogenic strain, no cases of B. the rind and 3.13±1.80 and 2.80±0.88 CFU g-1 in in vacuum bags as a whole or after cutting into cereus human illness have been associated the inner paste. By considering the technology wedges. Ricotta salata cheese is stored at with the consumption of ricotta salata cheese used, the intrinsic properties and the almost refrigeration temperature and the attributed (De Santis et al., 2008). The high moisture total absence of competing microflora, ricotta shelf-life differs from three weeks up to sever- content, the high pH and the availability of salata cheese can support the growth of al months, depending on the food business nutrients considered, such product is very sus- spoilage and pathogen microorganisms origi- operators (Spanu et al., 2015b). Ricotta salata ceptible to microbiological spoilage by nating from the processing environment. The cheese is often used in salads or many other Enterobacteriaceae, yeast, moulds and high level of total bacterial counts and dishes sliced, crumbled or grated without fur- Pseudomonas. The presence of pathogens and Enterobacteriaceae observed both on the rind ther heating. In consideration of its physico- contaminants such as L. monocytogenes and B. and in the inner paste suggests contamination chemical properties and the presence of and cereus has also been observed. However, little of the product from the processing environ- edible rind, ricotta salata cheese is very critical research has been conducted to describe the ment. Therefore, a strict implementation of for the possible growth of pathogenic and evolution of the microbiological profile of hygiene during processing is essential in order spoilage microorganisms. Pathogens of partic- Ricotta salata cheese during the shelf-life to reduce the load of environmental contami- ular concern are those able to grow in ricotta (Spanu et al., 2013). nants that may grow during refrigerated stor- salata cheese at refrigeration temperatures, The objective of the present study was to age. i.e. Listeria monocytogenes and psychrotrophic assess and characterise the evolution of

[Italian Journal of Food Safety 2016; 5:5501] [page 57] Article

spoilage and pathogen microorganism of vacu- zero (T0), time 60 (T60) and time 90 (T90) Statistical analysis um-packed ricotta salata cheese under refrig- respectively 60 days and 90 days after the Differences in mean mesophilic bacteria erated storage (4±2°C) during the entire prod- arrival of the samples. In order to avoid tem- counts (log CFU g-1), intrinsic properties uct shelf-life. 10 perature abuse during the storage period, the [mean±standard deviation (SD)] and compo- temperature of the cold room was monitored in sition (%±SD) between sampling times were continuous and recorded daily. Ricotta samples compared using Fisher’s least significant dif- were analysed for the determination of the ference (LSD) test. All statistical analyses Materials and Methods microbiological profile, physicochemical prop- were performed with R Studio software, ver- Ricotta salata cheese samples erties and composition. In Table 1 the number of samples and the analysis performed at each sion 0.98.1103 (RStudio, Inc., Boston, MA, The study was conducted on vacuum-packed sampling time are reported. USA). ricotta salata cheese wheels (Criovac Cook-In HT3000; Sealedair Ltd., St Neots, UK), which Microbiological profile were provided by a local cheese-making plant From each ricotta salata cheese sample, 25 g using the whey remaining after the manufac- aliquots of the rind (2 cm in depth) and of the Results turing of sheep milk cheeses (Table 1). The inner paste were aseptically collected. Aliquots main steps of the process flow of ricotta salata were analysed for the enumeration of total Microbiological profile cheese examined in the present study are viable count (TVC) at 30°C (ISO 4833:2003; Pathogen microorganisms such as L. mono- summarised in the diagram in Figure 1. A total ISO, 2003), L. monocytogenes (ISO 11290- cytogenes and B. cereus were always below the of 18 samples were randomly selected from 3 1:1996; ISO, 1996), B. cereus (EN/ISO detection limit (10 and 100 log CFU g-1, different batches (6 ricotta salata cheese for 10 7932:2004; ISO, 2004b), Enterobacteriaceae each batch) produced within one month. After respectively) both on the rind and in the inner (ISO 21528-2; ISO, 2004a), yeasts and moulds a drying period of approximately 10-15 days in paste. The mean TVC at T0 was ca. 8 log10 CFU (ISO 6611/IDF 94; ISO, 2004c). -1 -1 cold room, ricotta salata cheese samples were g on the rind and ca. 3 log10 CFU g in the sent to the laboratory. Immediately after their Physico-chemical properties and inner paste, no significant increase during the arrival, samples were labelled according to composition storage was observed. The mean their use for the experiment and stored at Enterobacteriaceae count was always greater pH and a were measured using pH meter refrigeration temperature (4±1°C) until analy- W GLP22 (Crison Instruments SA, Barcelona, on the rind as compared to the inner paste, sis was performed. Spain) and water activity meter Aqualab 4 TE with maximum concentration of ca. 5 log10 CFU -1 -1 Experimental design (Decagon, Pullman, WA, USA), respectively. g and 3 log10 CFU g respectively. Yeasts and -1 The durability study was conducted Near infrared transmittance (NIT) composi- moulds ranged between 3 and 5 log CFU g on analysing ricotta salata cheese samples at dif- tional analyser (FOSS, Eden Prairie, MN, USA) the rind and were ca. 2 log CFU g-1 on the inner -1 ferent time during the shelf-life. Sampling was used for the determination of centesimal paste. Table 2 shows the results (log10 CFU g ; times were: within 24 h after the arrival of composition which included analysis of mois- mean±SD) of the targeted microbial groups at ricotta salata cheese wheels defined as time ture, fat, protein, salt and total solids. the different sampling times.

Table 1. Number of ricotta salata cheese wheels and analysis performed at each testing time. Analysis Sampling time

T0 T60 T90 Microbiological profile 6 6 6 Intrinsic properties and composition 6 6 6

T0, 0 days after the arrival of the samples (packaging day); T60, 60 days after the arrival of the samples; T90, 90 days after the arrival of the samples. Number of samples per each time refers to the three batches.

Table 2. Mean log microbial counts of ricotta salata cheese units during shelf-life.

Parameters Samples T0 T60 T90 TVC Rind 7.90±0.64a (n=6/6) 7.79±2.21a (n=6/6) 9.19±0.58a (n=6/6) Paste 2.95±0.68a (n=5/6) 4.51±1.66a (n=6/6) 4.27±1.10a (n=6/6) Enterobacteriaceae Rind 4.22±0.66b (n=4/6) 5.16±0.59ab (n=5/6) 5.30a±0.73 (n=6/6) Paste nd 3.13±1.80a (n=3/6) 2.80±0.88a (n=2/6) Yeasts and moulds Rind 3.02±0.36b (n=3/6) 4.99±0.81a (n=4/6) 4.01±1.03ab (n=4/6) Paste 2.00±0.00a (n=2/6) 1.67±0.58a (n=3/6) 2.30a (n=1/6)

T0, 0 days after the arrival of the samples (packaging day); T60, 60 days after the arrival of the samples; T90, 90 days after the arrival of the sam- ples; TVC, total viable counts; nd, not detected (below the detection limit of the method). Values are expressed as mean±standard deviation Figure 1. Process flow of the analysed -1 a,b (log10 colony forming units g ). Values within brackets indicate the prevalence of positive samples. Means in the same row with different ricotta salata cheese samples. superscript letters are significantly different (P<0.05).

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Intrinsic properties and composition Contaminants may reach the product by means the inner paste during the entire shelf-life. The pH of ricotta salata cheese during of aerosol, condenses, dripping or inappropri- This further confirms the importance of the refrigerated storage under vacuum packing ate practices of operators (Wiedmann, 2003). strict implementation of hygiene during the The batch production of ricotta salata cheese production and storage steps (Tompkin, 2002; decreased from an initial level (T0) of is an open system which exposes the product Carpentier and Cerf, 2011). The pathways of 6.54±0.05 to a final level (T90) of 5.68±0.16 to environmental contamination. The enumer- contamination of ricotta salata cheese with (P<0.05). The aw was stable for the entire duration of the study at level between ation of total viable count showed a total bacte- sporeformers differs with respect to L. monocy- 0.97±0.01 and 0.98±0.01 (P>0.05). Minor rial load ca. 4 log greater on the rind as com- togenes and other environmental contami- changes in composition (moisture, fat, pro- pared to the inner paste. This is the result of nants. In fact, spore forming bacteria enter the teins, salt and total solids) were observed dur- the exposition of the rind to the processing dairy chain mainly with raw milk (Heyndrickx, ing storage. Table 3 shows the intrinsic proper- environment. This is confirmed by the high 2011). The spores present in milk can resist to ties (mean±SD) and composition (%±SD) contamination with Enterobacteriaceae espe- heat applied to milk such as thermisation and cially on the rind. Although no microbiological evolution during storage (T0, T60, T90) of ricotta pasteurisation treatments (Scheldeman et al., salata cheese wheels. criteria is defined by Regulation (EC) No 2006). High temperature reached during whey 2073/205 (European Commission, 2005) for heating (>80-85°C) activates the germination Enterobacteriaceae in whey cheeses that of residual spores, which are facilitated in undergone heat treatment, the presence of their growth by the absence of other competi- Discussion these important indicator microorganisms tive microflora eliminated by heat treatments reveals that in the cheese-making plants good (Griffiths and Phillips, 1990). Despite it is The durability of ricotta salata cheese hygienic and good manufacturing practices almost impossible to avoid the contamination defined by food business operator is generally should be improved. In particular, the presence of spores from the dairy industry, cases of of several months under refrigerated storage. of Enterobacteriaceae in the inner paste indi- human illness associated to the consumption The shelf-life of ricotta salata cheese depends cates poor hygienic conditions in the phase of of dairy products contaminated with B. cereus upon a number of interacting factors other ricotta salata cheese molding. This is a very is a rare finding (EFSA, 2015). B. cereus than storage temperature such as packaging critical step in ricotta production, where man- strains are generally not able to grow at tem- conditions, product composition, presence of ual manipulation can transfer contaminants perature <10°C, only few psychrotrophic preservatives and competitive microflora. The from the processing environment into the strains grow at temperature as low as 4-5°C. combination of production technology (whey inner product. With reference to pathogenic Therefore, temperature abuse is necessary heating which inactivates natural microflora microorganisms it is demonstrated that L. during storage to lead multiplication of the and absence of starter cultures), intrinsic monocytogenes contamination origins from the vegetative cells. In a product such as ricotta processing environment. Food contact and properties (high pH and elevated aW), deter- salata cheese, characterised by an extended mine the ability of the product to support the non-food contact surfaces represent the main shelf-life, sporeformers have the time to reach growth of pathogen and spoilage microorgan- vehicles of contamination, mainly due to concentration potentially dangerous to human isms. The origin of contamination can be from manipulation of the product after the produc- health (ICMSF, 1996). An essential strategy for raw materials or from the environment and tion process. This explains the frequent recov- the control of sporeformes is the strict cold- differs from one microorganism to another ery of L. monocytogenes on ricotta salata chain maintenance for the entire shelf-life of (Almeida et al., 2013). The production of ricot- cheese rind (Spanu et al., 2012, 2015a; Ibba et the product. During the 90 days of refrigerated ta salata cheese, especially in the traditional al., 2013). Contamination of the inner paste is storage B. cereus was never detected, indicat- batch manufacturing system, includes manual a rare finding and is generally associated with ing that a combination of the quality of raw manipulation of the curd after floating, the poor hygienic procedures during manual mold- material and temperature control during stor- exposition to environmental contamination ing (Spanu et al., 2015b). Previous studies age can contribute to the safety of ricotta sala- from several food contact surfaces (drainage reported a prevalence of contamination rang- ta cheese. tables, plastic moulds, pressing equipment, ing from 0.0% up to 30.0% of the samples (Pilo drying shelves) and non-food contact surfaces et al., 2007; Spanu et al., 2015a). In the present (floors, drains, walls and ceiling). study L. monocytogenes was below the detec- tion limit (<0.04 CFU g-1) both on the rind and Conclusions

The batch production system adopted in Sardinian traditional sheep ricotta salata Table 3. Intrinsic properties and composition evolution during storage of ricotta salata cheese production and the presence of manual cheese wheels. manipulation make this product particularly exposed to environmental contamination. In Parameters T T T 0 60 90 addition, the absence of a competitive pH 6.54±0.03a 6.04±0.14b 5.68±0.18c microflora, the richness in nutrient and the a a a intrinsic properties are favourable conditions aW 0.973±0.005 0.977±0.008 0.978±0.005 for growth of spoilage and pathogen microor- Moisture (%) 57.9±1.85a 56.3±0.88a 57.2±1.81a ganisms. The strategies for the control of envi- a a a Total solids (%) 42.13±1.84 44.65±3.75 43.33±5.01 ronmental contaminants are based on their Fat (%) 19.55±2.08a 21.24±0.94a 20.08±1.74a prevention through the strict implementation Protein (%) 14.84±0.81a 15.18±0.52a 15.17±1.03a of hygienic conditions during production Salt (%) 3.42±0.24a 3.10±0.34a 3.38±0.34a process. Superficial contamination of the prod- ucts can be reduced by the adoption of post- T0, 0 days after the arrival of the samples (packaging day); T60, 60 days after the arrival of the samples; T90, 90 days after the arrival of the sam- ples. Values are expressed as mean or % (where specified)±standard deviation. a-cMeans in the same row with different superscript letters lethality treatment (i.e. heat treatment) in vac- are significantly different (P<0.05). uum-packed product, allowing to extend ricotta

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