Stefano Rea

Use of duplex polymerase chain reaction (duplex-PCR) technique to identify bovine and water buffalo milk used in making mozzarella cheese.

Molecular biology techniques have been used for species identification in food of animal origin in relatively recent years. A polymerase chain reaction (PCR) based method, the multiplex PCR, was recently applied to species identification in meat and meat products. It allows co-amplification of separate regions of a single gene or specific fragments, each typical of a different animal species in a single PCR reaction, using different pairs of primers in the same reaction mix. In the present paper, the duplex-PCR technique is proposed to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk). Because of its lower cost, undeclared bovine milk is added to water buffalo milk for making different kinds of mozzarella cheese. The results of this experiment indicate the applicability of this method, which showed an absolute specificity for the two species and a high sensitivity even down to low DNA concentrations (1 pg). In bovine and water buffalo mixtures of both milk and mozzarella cheese, the minimum concentration tested was 1% of bovine in water buffalo milk and water buffalo in bovine milk. The importance of the somatic cell content in raw milk is also discussed with special reference to the evaluation of mixtures (milk or cheese) of the two species.

The occurrence of giant fibres in different muscles of two chicken genotypes

Abstract 1. The occurrence of Giant Fibres (GF) in three muscles (Pectoralis major (PM), Iliotibialis lateralis and Semimembranosus) with different types of energy metabolism was studied in slow- and fast-growing chicken strains. 2. A total of 20 one-day-old Leghorn chicks (slow-growing) and 20 broiler (Ross 508) chicks (fast-growing) were reared to 100 and 45 d, respectively. 3. A small percentage of GF was seen in pre rigor muscle samples even at 3 min post mortem in both genotypes and in all muscle types studied. 4. From 3 min to 24 h post mortem GF increased both in Leghorn and broiler chickens but to a different extent according to muscle type and genotype. 5. The highest GF 24 h post mortem value was found in the PM muscles belonging to the fast-growing broiler line. 6. It was concluded that every type of muscle can develop GF, but this phenomenon is more evident in the PM especially in animals selected for increased growth rate.

Welfare indicators during broiler slaughtering

Abstract The aim of this study was to identify the most relevant welfare indicators for unloading, lairage, stunning, killing and post-mortem inspection in a poultry slaughter plant. Different indicators were unloading duration, lairage time, environmental variables in the lairage facilities, shackling time and electrical variables used in the water bath. Lairage time did not correlate strongly with dead on arrival. Heat stress was limited by means of ventilation systems, correct cage placement and appropriate stocking density per crate. The acceptable shackling period was about 30 s. The presence of a corneal reflex showed that an animal was alive, while spontaneous wing flapping, spontaneous eye blinking and response to a painful stimulus were regarded as indicators of stunning efficiency. It was concluded that the presence of recent traumatic injuries during the post-mortem inspection could be a valid means to establish whether corrective measures concerning the handling, transport and loading procedures should be taken.

Differences in chemical, physical and microbiological characteristics of Italian burrata cheeses made in artisanal and industrial plants of Apulia Region

The burrata cheese is a traditional product from Southern Italy, consisting of an envelope of pasta filata (stretched curd) filled with cream and pasta filata strips (usually leftovers from mozzarella production). Physical [water activity (aw), pH], chemical (moisture, NaCl content) and microbiological [total viable count (TVC), Listeria monocytogenes, Salmonella spp., Yersinia enterocolitica, Bacillus cereus, Escherichia coli, Enterobacteriaceae, coagulase-positive staphylococci] characteristics of burrata cheeses manufactured in artisanal and industrial plants were evaluated. The artisanal burrata showed lower aw values in the filling and the final product. The same was recorded in the filling for the moisture, probably due to differences between the types of cream used in the artisanal and the industrial cheesemaking. The pH value of the filling differed between the two groups but no difference was recorded in the final product. Microbiological differences were also recorded, with higher values for TVC and E. coli in artisanal than industrial burrata. All samples were negative for the other microbial determinations, with the exception of coagulase-positive staphylococci and Y. enterocolitica, which were detected in artisanal burrata. Differences in cheesemaking process were probably responsible for the strong variability of the physical and chemical data between the two cheeses; furthermore, differences in the hygienic features were also recorded. Even though artisanal products showed lower aw and pH values and higher NaCl concentration, the higher E. coli loads highlighted the need for a more accurate compliance with hygienic procedures along the artisanal cheesemaking process.

Effect of Selected Starter Cultures on Physical, Chemical and Microbiological Characteristics and Biogenic Amine Content in Protected Geographical Indication Ciauscolo Salami.

The aim of the study was to evaluate the biogenic amine (BA) content of Ciauscolo salami made with and without the use of a selected started culture. Two batches of salami were made following the guidelines of the Protected Geographical Indications: with and without adding a commercial starter culture made of Lactobacillus plantarum and Staphylococcus xylosus. Six samples of salami per batch were collected at different ripening times (0, 15, 30, 45 and 60 days) for physical, chemical and microbiological analyses and for the determination of BA content. No differences were recorded for physical, chemical and microbiological analyses except for Staphylococcus spp. count at the time of casing (T0) and total volatile basic nitrogen (TVBN) from 30 days (T2) to the end of the ripening time (60 days, T4). After 60 days of ripening, the use of selected starter culture significantly affected the amount of putrescine (195.15 vs 164.43 mg/100 g in salami without and with starters, respectively), cadaverine (96.95 vs 104.40 mg/100 g in salami without and with starters, respectively), histamine (81.94 vs 69.89 mg/100 g in salami without and with starters, respectively), and spermine (36.88 vs 33.57 mg/100 g in salami without and with starters, respectively). Despite significantly higher values of TVBN, the use of selected starter culture determined no significant effects on the BA content of the products.

Depuration Capacity of Mussels (Mytilus galloprovincialis) in Presence of Marteilia Spp. Parasites

Bivalve molluscs are filter-feeding organisms present in the water column: during their activity, they could retain micro-organisms that are potentially dangerous to human health. For this reason, EU Regulations may require that a purification treatment be performed prior to bivalve trade. The length of the purification process could be affected by stress factors, such as parasitic infections. The purpose of this study was to determine if the presence of Marteilia spp. parasite in shellfish could modify time and efficacy of their microbiological purification treatment, in order to set up specific protocols. Lysosomal membrane stability, phagocytosis capacity, granulocyte/hyalinocyte rate and neutral lipid accumulation are biomarkers used to evaluate shellfish physiological state. These biomarkers were used to exclude any differences caused by stressor factors that could affect the purification results. Mussels were sampled from two different production areas. The presence or absence of parasites was confirmed by cytological test. Both groups of parasitized and non-parasitized mussels were contaminated with E.coli: they were then sampled for microbiological analyses and tested for biomarkers for up to 70 hours of purification. Parasitized and non-parasitized molluscs did not show any differences in levels of E. coli after 12, 24, 36, 48 and 70 hours of depuration. In relation to biomarkers, mussels seem to react to Lysosomal membrane stability in presence of Marteilia. The present study shows that the presence of Marteilia spp. does not affect the purification rate of mussels.

Seasonal variations of Italian Mediterranean Buffalo (Bubalus bubalis) Mozzarella cheese quality.

Water buffalo Mozzarella cheese is more appreciated in the summer, but milk production is higher during wintertime, as water buffalo are seasonally polyoestrous animals. The aim of the study reported in this Research Communication was to evaluate the effect of the reversing of the calving period on Mozzarella cheese composition and quality traits. Ten batches of Mozzarella cheeses produced during the summer and winter periods were analysed for pH, colour, chemical composition. Seasonal differences were observed for cheese yield (26·66% in winter and 25·61% in summer), moisture content (66·54% in winter and 61·18% in summer) colour and consumer evaluation.

Evaluation of carcass hygiene in sheep subjected to gas de-pelting with different skinning procedures

The aim of the study was to evaluate the hygienic status of sheep carcasses skinned with two different procedures, the pulling down and Y cut methods, with and without the use of compressed filtered air inflation. Five sheep carcasses per day for each of the four skinning methods considered were sampled on ten different slaughtering days using wet and dry swab techniques at a local abattoir specialised in ovine slaughtering. A pool of four different sampling sites (brisket, shoulder, thorax and rump) was considered for each animal. Furthermore, ten animals were also randomly selected on different slaughtering days for each of the four skinning techniques and the four sampling sites were separately swabbed and analysed in each animal. The total viable count (TVC) and Enterobacte riaceae count were performed and the presence of Salmonella spp. was also tested. The daily average mean value of each parameter was in compliance with limits set by Regulation (EC) 1441/2007, falling into satisfactory or acceptable category for Enterobacteriaceae and within the acceptable level range for TVC for both the methods used with and without air de-pelting. For both TVC and Enterobacteriaceae count, no statistically significant differences (P>0.05) were recorded between samples obtained from carcasses skinned with and without air inflation for either of the skinning methods used and any of the sites sampled. No Salmonella spp. were detected in any of the tested samples. Nonetheless, no improvement in the carcass hygiene was detected either and, for this reason, other aspects should be taken into consideration when considering adopting the gas de-pelting method.

Animal welfare evaluation at a slaughterhouse for heavy pigs intended for processing

The Council Regulation (EC) No. 1099/2009 requires slaughterhouse managers to implement specific standard operating procedures for all pre-slaughter stages considered at risk, aimed at achieving adequate levels of animal welfare. This survey was aimed at testing the applicability to an abattoir for heavy pigs of an assessment system of animal welfare through animal-based measures. In the monitoring of handling operations, the number of animals fallen/slipped and prodded, and that of vocalising pigs were recorded. In the monitoring of the immobilisation stage, carried out on the same pigs, vocalisations were recorded at the entrance to the box and falls/slips occurring inside it. Animal welfare assessment during the stunning-sticking-bleeding steps, was carried out by recording the head-only electrical stunning basic parameters set by legislation, vocalisations resulting from hot wanding, and clinical signs of consciousness, sensibility and certain death. Except for immobilisation, the percentage of occurrence of these events above acceptability limits was detected in all other pre-slaughter steps. The most critical stages were: handling in the unloading area and along the single-file chute, stunning and especially bleeding, where 84.13% of animals showed one or more signs of consciousness and/or sensibility recovery. Wrong placement of electrodes observed in 53.98% of the animals, insufficient voltage and low amperage may explain why a high percentage of pigs recovered consciousness and/or sensibility before death. Some simple restructuring of unloading area, slowdown of slaughter line speed, increase of personnel involved in pre-slaughter management and regular calibration of the electrical stunning device could be effectively corrective measures aimed at raising the animal welfare level at the slaughterhouse under study.

Influence of rainfall on E. coli concentrations in clams: results of collaboration between competent health authority and producers’ association in the province of Fermo (Italy)

The Area Vasta di Fermo (the competent health authority of the Marche Region), in collaboration with the local producers’ Association conducted a series of studies on the presence of E. coli as an indicator of faecal contamination in the environment, in shellfish and in fishery waters, and on the factors that can affect their presence. These studies, carried out from 2008 to 2011, included an assessment of the currents along the coast, of the precipitations, and data from the monitoring of E. coli on shellfish harvested in the collection areas were examined. The results showed that in most cases, small concentrations of microorganisms in shellfish corresponded to little or no precipitations, while an increase in the levels of E. coli was preceded by more or less abundant rainfalls. The conclusions suggest that it is advisable to carry out a more detailed risk analysis which should take into account the above-mentioned factors. Furthermore, monitoring alone based on the determination of indicator organisms, especially when carried out as a single analysis or with a small number of E. coli determinations, does not provide a satisfactory indication of safety. The regional surveillance plans should be applied timely and rigorously, together with onsite investigations aimed at identifying changes that can affect the presence of E. coli in shellfish. Food business operators themselves could implement good manufacturing practices to verify whether the microbiological parameters are within the prescribed limits after rainfalls, especially if heavy.