Employment of autochthonous microflora in Pecorino Sardo cheese manufacturing and evolution of physicochemical parameters during ripening

The isolation and identification of lactic acid bacteria (LAB) from raw ewes’ milk and traditional Pecorino Sardo cheese made from this milk without the addition of starter culture was carried out to define the autochthonous lactic microflora present in milk and the evolution of LAB during cheese ripening. Isolation of 275 strains belonging to different Lactococcus, Lactobacillus, Streptococcus and Enterococcus species was achieved. Coccal-shaped LAB were found to predominate during cheese fermentation, while lactobacilli were preponderate during the latter phase of ripening. The technological selection of a total of 174 LAB strains belonging to the species Lactococcus lactis, Streptococcus thermophilus, Lactobacillus helveticus and Lb. casei allowed an experimental starter to be prepared, in which a potentially probiotic species, Lb. casei was used. The suitability of the autochthonous starter culture was tested in cheese-making trials, using thermised ewes’ milk, by comparing experimental Pecorino Sardo cheese with a control cheese produced at industrial scale using a whey starter culture from previous batches of manufacture. In particular, microbiological and physicochemical parameters were determined over 210 days of cheese ripening. Although sensory evaluation did not show any significant difference between experimental and control Pecorino Sardo cheeses, the use of the selected autochthonous starter allowed the production of experimental cheese with a significantly higher level of free amino acids, in particular essential amino acids, in comparison with the Pecorino Sardo control cheeses.     

Effect of whey pH at drainage on physicochemical, biochemical, microbiological, and sensory properties of Mozzarella cheese made from buffalo milk during refrigerated storage

The objective of this research was to determine the effect of drainage pH on physicochemical, biochemical, microbiological and sensory properties of Mozzarella cheese made from buffalo milk during refrigerated storage. Four vats of cheese were made at 4 different whey drainage pH (6.2, 5.9, 5.6, and 5.2). Lower drainage pH caused higher pH 4.4-soluble N and pH 4.4-soluble N:total N. Interaction of drainage pH at d 1 and 30 of storage on all soluble nitrogen fractions was significant. Degradation of caseins in samples made at a drainage pH of 6.2 was lower than that of other cheese samples. The decreasing whey drainage pH significantly increased counts of thermophilic and mesophilic lactobacilli of the samples during refrigerated storage. No coliforms or Escherichia coli were detected in the cheeses. The average sensory property scores of all cheese samples were very close, and, as expected, storage time had a negative effect on all sensory scores.     

Hygienic and physicochemical quality characterisation of artisanal and industrial Pecorino Calabrese cheese

Samples of artisanal and industrial Pecorino Calabrese cheese were collected from 20 dairy farms located in Calabria (Italy) and analysed to evaluate the microscopic and microbiological quality. The number of yeasts and moulds, total coliforms and enterococci was significantly higher in the artisanal cheese. The presence of two samples in both farms exceeding the hygienic limit in terms of Escherichia coli count highlights incorrect hygienic procedures in this kind of production. The filth analysis showed that the amount of solid impurities was significantly higher in the artisanal cheese, while only arthropods were more frequent in the industrial one.     

Effect of different salting technologies on the chemical and microbiological characteristics of PDO Pecorino Siciliano cheese

The present work was carried out to evaluate the effect of two salting technologies [dry salting (DS) and the combined dry-brine salting (DBS)] on the chemico-physical and microbiological characteristics of PDO Pecorino Siciliano cheeses of different final weight (6 and 12 kg). Dry matter was significantly influenced by both salting process and final size. Twelve kilogram cheeses treated by DBS showed higher protein content with higher soluble nitrogen per cent than 6 kg cheeses. Salt content was in the range 3.1–4.0% on dry matter. The colour did not show significant differences for any of the factors, but 12 kg cheeses subjected to DS showed higher yellow index than the other cheeses. The resistance at 30% of strain was influenced by cheese size, with 6 kg cheeses showing higher resistances than 12 kg cheeses. All cheeses were dominated by coccus LAB, but pseudomonads and Enterobacteriaceae showed comparable levels of about 10⁵ cfu/g. Significant microbiological differences were evidenced only for enterococci and yeasts concerning the final cheese size. Thirteen species of LAB, belonging to five genera (Enterococcus, Lactobacillus, Lactococcus, Pediococcus and Streptococcus), were identified, but several spoilage/pathogenic species were also identified, especially Pseudomonas putida, Citrobacter freundii and Stenotrophomonas maltophilia. LAB isolates were preliminary evaluated for their physiological characteristics in view of developing autochthonous starters to improve the microbiological quality of PDO Pecorino Siciliano cheese.     

Determination of microbial contamination sources for use in quality management of cheese industry: “Dil” cheese as an example

The microbiological quality, safety and shelf-life of cheeses depend on manufacture and handling in an environment that meets basic standards for hygiene and the management of hygiene in the process. In this research contamination sources of “Dil” cheese during production in a local dairy plant in Bursa, Turkey were determined. Eighteen different control points (raw milk, pasteurized milk, heated curd, molded cheese before kneading, kneaded cheese, brine solution for kneading, thermophilic culture, rennet, calcium chloride solution, brine solution for cheese, cheese vat, workers hands, production room air, production room floor, production room wall, packaging material and packaged cheese) have been examined for the enumeration of total aerobic mesophilic bacteria, Staphylococci, Enterobacteriaceae, Salmonella spp., Escherichia coli, lactic acid bacteria, Pseudomonas spp. and yeast-moulds. It was determined that viability of lactic acid bacteria in thermophilic culture was not in high numbers and some contaminations to “Dil” cheese were detected from the starter culture. Brine solutions and rennet were contaminated with Staphylococci. Yeast and moulds in production room air were the major sources of contamination. Pasteurization and kneading in hot brine solution can eliminate some of the microorganisms but that was not sufficient in the production of Dil cheese. Finish cheese should meet specific hygienic standards, with respect to regulations post-contaminations to the cheese must be inhibited and a HACCP plan should be established during production.     

Influence of lamb rennet paste on composition and proteolysis during ripening of Pecorino foggiano cheese

Rennet pastes produced by lambs subjected to three different feeding systems (mother suckling [MS], artificial rearing [AR], and artificial rearing with Lactobacillus acidophilus supplementation [ARLB]) and slaughtered at two different ages (20 and 40 d) were used for the manufacture of Pecorino foggiano cheese. Composition and proteolysis during ripening of Pecorino foggiano cheese (four replicates batches) were analyzed. Proteolysis was greater in cheeses made with rennet pastes from lambs slaughtered at 20 d, as shown by analysis of nitrogen fractions (water-soluble N and proteose peptones). Supplementation of milk substitute with L. acidophilus may have influenced the growth dynamics of lactic acid bacteria in the rennet pastes, with positive effects on levels of lactobacilli in cheese at the beginning of the ripening time. Lower pH values in ARLB cheese during ripening, together with higher cell loads, suggest that supplementation of milk replacer with L. acidophilus resulted in higher proteolytic activity, as also confirmed by the composition of the pH 4.6—insoluble nitrogen fraction. No differences were found in total concentration of free amino acids among the experimental cheeses; phenylalanine, isoleucine, leucine, lysine were found at the highest levels. The addition of probiotic bacteria to milk substitute in lamb rearing appears to give good-quality lamb rennet paste.     

Active coating and modified-atmosphere packaging to extend the shelf life of Fior di Latte cheese

In this work the combination of active coating and modified-atmosphere packaging (MAP) was used to prolong the shelf life of Fior di Latte cheese. The active coating was based on sodium alginate (8% wt/vol) containing lysozyme (0.25 mg/mL) and EDTA, disodium salt (Na₂-EDTA, 50 mM). The MAP was made up of 30% CO₂, 5% O₂, and 65% N₂. The speed of quality loss for the Fior di Latte cheese, stored at 10°C, was assessed by monitoring pH and weight loss, as well as microbiological and sensorial changes. Results showed that the combination of active coating and MAP improved Fior di Latte cheese preservation, increasing the shelf life to more than 3 d. In addition, the substitution of brine with coating could allow us to gain a double advantage: both preserving the product quality and reducing the cost of its distribution, due to the lower weight of the package.     

The influence of Bifidobacterium Bb-12 and lactic acid incorporation on the properties of Minas Frescal cheese

The effects of a probiotic bacterium (Bifidobacterium Bb-12) and lactic acid on the microbiological, physicochemical, rheological and microstructural proprieties of Minas Frescal cheese were evaluated after 1 day and after 28 days of storage (5 ± 1 °C). The cheese was produced with four different treatments: with no lactic acid (C1 and C2), with lactic acid (C3 and C4) and with bifidobacteria (C2 and C3). The cheese formulations containing bifidobacteria were classified as probiotic. The addition of bifidobacteria to the cheese did not influence its yield, protein or lipid levels one day after production. Moreover, after 28 days of storage, the cheese samples with no lactic acid showed lower moisture levels compared to the samples containing lactic acid (P < 0.05). The absence of lactic acid had an influence on the rheological and microstructural behavior, making them more elastic, firm and compact, however all the cheese samples evaluated showed a higher tendency to viscosity than to elasticity. The Minas Frescal cheese with Bifidobacterium Bb-12 and lactic acid showed great potential as a functional food, with possible industrial and commercial application.     

Lipolysis in Urfa cheese produced from raw and pasteurized goats’ and cows’ milk with mesophilic or thermophilic cultures during ripening

Lipolysis was evaluated in Urfa cheese made from raw and pasteurized goats’ and cows’ milk with mesophilic or thermophilic cultures. The acid degree values (ADVs) of the cows’ milk cheeses were significantly (P < 0.05) higher until 60 d of storage than that of cheese made from goats’ milk. Total free fatty acid (FFA) contents of goats’ milk cheese were significantly (P < 0.001) lower than that of cows’ milk cheese throughout ripening, whereas goats’ milk cheese flavour was higher (P < 0.05) than cows’ milk cheese. Pasteurization of milk prior to cheese-making has a negative influence, not only on the level of lipolysis throughout ripening, but also on the relative amounts of short chain FFAs and sensory properties of the cheeses (P < 0.001). Cheese produced without starter bacteria underwent significantly (P < 0.05) higher lipolysis than cheeses produced with mesophilic or thermophilic starter bacteria, while cheese made with thermophilic starter culture had similar flavour to cheese made without starter culture.     

Ripening and seasonal changes in microbial groups and in physico-chemical properties of the ewes’ cheese Pecorino del Poro

Three batches of Pecorino del Poro, ewes’ cheese made from raw milk, were examined throughout a 28-day ripening time at three different seasons. High logarithmic counts per gram of cheese for mesophilic coccal-shaped lactic acid bacteria (6.70–12.45), mesophilic lactobacilli (4.82–11.73), thermophilic coccal-shaped lactic acid bacteria (2.30–9.90), and thermophilic lactobacilli (2.95–8.15) were found. Coccal-shaped lactic acid bacteria were the dominant microorganisms throughout ripening. The microorganisms used as an indicator of hygiene during manufacture of the cheeses, coliforms and Escherichia coli, were considerably lower, as were enterococci and yeasts. Coliforms and E. coli decreased sharply throughout ripening. Physico-chemical parameters such as pH (5.07–7.03), dry matter (46.34–72.79%), ether extract (31.35–51.84% of dry matter), crude protein (29.93–44.73% of dry matter), and chloride content (2.36–4.11% of dry matter) were also determined. Probably, the use of selected autochthonous mesophilic lactococci as a starter would control or suppress the growth of undesirable microorganisms. The results obtained suggest the need for improvements in milking and dairy conditions.     

Influence of Australian Native Herbs on the Maturation of Vacuum-packed Cheese

The composition, microbiology and biochemistry of semi-hard cheeses flavoured with native mint, lemon myrtle and bush tomato (BT) were compared with unflavoured (control) cheese during a 90-day period of maturation. Moisture, protein and salt levels of all cheeses were similar and did not change during maturation. However, the fat content of control cheese was significantly higher than that of the flavoured cheeses while the pH of cheese flavoured with BT was consistently lower throughout maturation. Total viable organisms, Lactobacillus and Lactococcus counts were between 10⁶ and 10⁷ colony forming units (cfu)/g cheese for all cheeses. Yeast and mould count was <10² cfu/g cheese throughout the maturation of all cheeses except in the cheese flavoured with BT which was >10³ cfu/g cheese. Biochemical indices of proteolysis and lipolysis increased with the extent of maturation in all cheeses but were most pronounced in the BT-flavoured cheese. The capillary electrophoretic profile of this cheese also indicated a more extensive hydrolysis of both α s- and β -caseins. The microbiological quality of BT appeared to have exerted a very significant influence on cheese properties.     

Selected lactic acid bacteria as a hurdle to the microbial spoilage of cheese: Application on a traditional raw ewes' milk cheese

To evaluate the efficacy of lactic acid bacteria (LAB) to improve the hygienic safety of a traditional raw milk cheese, the raw ewes' milk protected denomination of origin (PDO) Pecorino Siciliano cheese was used as a model system. Different Pecorino Siciliano curds and cheeses were used as sources of autochthonous LAB subsequently used as starter and non-starter LAB. These were screened for their acidification capacity and autolysis. Starter LAB showing the best performance were genotypically differentiated and identified: two strains of Lactococcus lactis subsp. lactis were selected. From the non-starter LAB, Enterococcus faecalis, Lactococcus garvieae and Streptococcus macedonicus strains were selected. The five cultures were used in individual or dual inocula to produce experimental cheeses in a dairy factory for which production was characterised by high numbers of undesirable bacteria. At 5-month of ripening, the experimental cheeses produced with LAB were characterised by undetectable levels of enterobacteria and pseudomonads and the typical sensory attributes.     

Terminal-restriction fragment length polymorphism and automated ribosomal intergenic spacer analysis profiling of fungal communities in Camembert cheese

Identifying and isolating yeasts and moulds within a fungal community is challenging. The main goal of the present study was to assess a new approach for the detection and identification of fungi involved in Camembert cheese rind formation to replace the use of traditional microbiological techniques. Two molecular methods, terminal-restriction fragment length polymorphism (T-RFLP) and automated ribosomal intergenic spacer analysis (ARISA), were adapted and compared for their potential to determine fungal composition directly from cheese samples. The two techniques in combination with principal component analysis showed differences in the fungal composition of cheeses when comparing surface with centre, different batches, manufacturing processes and ripening times. Moreover, cheese stabilisation induced changes in the flora at the cheese centre, and difference in size (150 g versus 1 kg) modified surface flora. Nine fungal genera were identified in cheese samples: Cladosporium, Debaryomyces, Geotrichum, Kluyveromyces, Mucor, Penicillium, Pichia, Saccharomyces and Yarrowia.     

Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties

Water-soluble extracts of 9 Italian cheese varieties that differed mainly for type of cheese milk, starter, technology, and time of ripening were fractionated by reversed-phase fast protein liquid chromatography, and the antimicrobial activity of each fraction was first assayed toward Lactobacillus sakei A15 by well-diffusion assay. Active fractions were further analyzed by HPLC coupled to electrospray ionization-ion trap mass spectrometry, and peptide sequences were identified by comparison with a proteomic database. Parmigiano Reggiano, Fossa, and Gorgonzola water-soluble extracts did not show antibacterial peptides. Fractions of Pecorino Romano, Canestrato Pugliese, Crescenza, and Caprino del Piemonte contained a mixture of peptides with a high degree of homology. Pasta filata cheeses (Caciocavallo and Mozzarella) also had antibacterial peptides. Peptides showed high levels of homology with N-terminal, C-terminal, or whole fragments of well known antimicrobial or multifunctional peptides reported in the literature: α_S1-casokinin (e.g., sheep α_S1-casein (CN) f22-30 of Pecorino Romano and cow α_S1-CN f24-33 of Canestrato Pugliese); isracidin (e.g., sheep α_S1-CN f10-21 of Pecorino Romano); kappacin and casoplatelin (e.g., cow κ-CN f106-115 of Canestrato Pugliese and Crescenza); and β-casomorphin-11 (e.g., goat β-CN f60-68 of Caprino del Piemonte). As shown by the broth microdilution technique, most of the water-soluble fractions had a large spectrum of inhibition (minimal inhibitory concentration of 20 to 200 μg/mL) toward gram-positive and gram-negative bacterial species, including potentially pathogenic bacteria of clinical interest. Cheeses manufactured from different types of cheese milk (cow, sheep, and goat) have the potential to generate similar peptides with antimicrobial activity.     

A sensitive HPLC method to detect hen's egg white lysozyme in milk and dairy products

Because of the lytic activity on the cell wall of bacteria like Clostridium tyrobutyricum, hen's egg white lysozyme is used in cheese manufacturing to prevent late blowing. A HPLC method capable to quantify as low as 0.8 ppm lysozyme in milk and cheese is proposed. Lysozyme was extracted with 1 m NaCl at pH 6.0 and the extract was deproteinized at low pH values, reaching a recovery up to 90%. Reversed-phase HPLC was performed on a polymeric column and monitoring lysozyme under fluorescence detection (excitation at 280 nm and emission at 340 nm). The repeatability of this determination tested on a 15-month-aged hard cheese and expressed as relative standard deviation was 1.47 (n=6) and no interference of peptides formed during ripening was observed. Four commercial preparations of egg white lysozyme gave a similar fluorescence response and the partitioning over cheese and whey was studied with one of them. About 80% of the lysozyme added to the cheesemilk at concentrations up to 80 ppm was retained in the cheese and the concentration factor of lysozyme from the cheesemilk to the cheese proved to be 8.2 on average. This HPLC method and the microbiological assay using Micrococcus luteus were compared in cheese analysis, proving the former to be more accurate and reliable than the latter. Eighteen commercial samples including both generic cheeses and cheeses having protected designation of origin, all of them not declared to contain lysozyme, showed concentrations of this enzyme ranging from 0 to 111 ppm.     

Growth potential of Listeria monocytogenes strains in mixed ready-to-eat salads

In this study, a microbiological challenge test in three artificially contaminated retail mixed mayonnaise-based ready-to-eat salads stored at refrigerator temperatures (3 °C and 7 °C) for 48 h was carried out. Shrimp-tomato salad, smoked ham salad and garlic cheese salad were separately contaminated by a suspension of particular Listeria monocytogenes strains. The number of L. monocytogenes, Enterobacteriaceae, staphylococci and total plate count (CFU/g) was determined. Listeria monocytogenes growth potential in the salads was calculated and evaluated.A significant increase in total plate count and L. monocytogenes count throughout storage of all three investigated salads was found. Enterobacteriaceae levels were high at the beginning in all salads but significantly (p < 0.05) decreased throughout the experiment depending on the temperature.All investigated L. monocytogenes strains demonstrated growth at both temperatures but expressed different growth potential. Especially garlic cheese salad and smoked ham salad were able to support the growth of Listeria. Shrimp-tomato salad supported growth the least. The growth potential increased with the increasing temperature and exceeded 0.5 log₁₀ CFU/g in many cases. If the potential for growth is > 0.5 log₁₀ CFU/g, food products can potentially endanger human health. Reference strain (ATCC 7644) showed the least growth potential almost in all cases in comparison with strains isolated from frozen pollock loins and from thermally treated specialty sausage containing preservatives. To eliminate the occurrence of microbiological risks, the shelf-life of the studied salads was estimated.     

Microbial diversity and dynamics of Pecorino di Filiano PDO,a traditional cheese of Basilicata region (Southern Italy)

The evolution of microbial populations of ‘Pecorino di Filiano’ (PF) cheese was investigated during ripening in natural cave and storeroom. 62.5% of isolates grow at 45 and 15 °C and 77.7% showed high salt concentrations tolerance. Brevibacterium linens was dominant in surface samples. Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus paracasei subsp. paracasei were more frequently isolated both surface and core samples, while Leuconostoc lactis and Leuconostoc mesenteroides subsp. mesenteroides prevailed among Leuconostoc isolates. Our results suggest the importance of the ripening environment of cheeses and how a biological ecosystem affects and produces the typical features of artisanal products.     

Production of biogenic amines during the ripening of Pecorino Abruzzese cheese

The production of biogenic amines (BA) during the manufacturing and ripening of sheep milk Pecorino Abruzzese cheeses prepared from raw milk without starter culture (A) and from pasteurized milk with added starter (B) were compared. At the end of ripening (60 days), the total BA contents of cheeses of batches A and B were 697 and 1086 mg kg⁻¹, respectively; the dominant BA were different. Single isolates of enterococci, pseudomonads and Enterobacteriaceae were screened for their potential to produce BA. Qualitative tests indicated a large spread of BA-forming cultures among the members of the Enterobacteriaceae and lactic acid bacteria (LAB). Differences among the levels of BA produced in UHT milk by representative isolates of coliforms, Pseudomonas and LAB were observed in relation to the microbial group or the isolate. The results emphasize the need to improve the general hygienic conditions of Pecorino Abruzzese cheese manufacture and control the indigenous bacterial population.     

Evaluation of the antioxidant properties and oxidative stability of Pecorino cheese made from the raw milk of ewes fed Rosmarinus officinalis L. leaves

The aim of this work was to study the effect of rosemary leaf dietary supplementation on the antioxidant activities and total phenolic content of Pecorino cheese. Three hundred and twenty‐four sheep were randomly assigned to two dietary groups, which received a standard diet based on lucerne hay and concentrate (400 g per day). The concentrate of the rosemary supplemented group contained 2.50% dried rosemary leaves. The trial lasted 7 weeks. Cheesemaking was performed 3, 5 and 7 weeks from the start of the trial. The Pecorino cheese antioxidant activity was modified by the diet. Rosemary supplementation increased the total phenolic content, enhanced the antioxidant properties and decreased the lipid oxidation of cheese. A slight decrease in flavour was detected in cheeses after 7 weeks of rosemary administration.