Low-Level Clostridial Spores' Milk to Limit the Onset of Late Blowing Defect in Lysozyme-Free, Grana-Type Cheese.

The growth of clostridial spores during ripening leads to late blowing (LB), which is the main cause of spoilage in Grana Padano Protected Designation of Origin (PDO) cheese and other hard, long-ripened cheeses such as Provolone, Comté, and similar cheeses. This study aimed to verify the cause-effect relationship between the level of clostridial butyric spores (BCS) in milk and the onset of the LB defect. To this end, experimental Grana-type cheeses were produced without lysozyme, using bulk milk with different average BCS content. The vat milk from the so-called "virtuous" farms (L1) contained average levels of BCS of 1.93 ± 0.61 log most probable number (MPN) L-1, while the vat milk from farms with the highest load of spores (L2), were in the order of 2.99 ± 0.69 log MPN L-1. Cheeses after seven months of ripening evidenced a strong connection between BCS level in vat milk and the occurrence of LB defect. In L2 cheeses, which showed an average BCS content of 3.53 ± 1.44 log MPN g-1 (range 1.36-5.04 log MPN g-1), significantly higher than that found in L1 cheeses (p < 0.01), the defect of LB was always present, with Clostridium tyrobutyricum as the only clostridial species identified by species-specific PCR from MPN-positive samples. The L1 cheeses produced in the cold season (C-L1) were free of defects whereas those produced in the warm season (W-L1) showed textural defects, such as slits and cracks, rather than irregular eyes. A further analysis of the data, considering the subset of the cheesemaking trials (W-L1 and W-L2), carried out in the warm season, confirmed the presence of a climate effect that, often in addition to the BCS load in the respective bulk milks (L1 vs. L2), may contribute to explain the significant differences in the chemical composition and some technological parameters between the two series of cheeses. Metagenomic analysis showed that it is not the overall structure of the microbial community that differentiates L1 from L2 cheeses but rather the relative distribution of the species between them. The results of our trials on experimental cheeses suggest that a low-level BCS in vat milk (<200 L-1) could prevent, or limit, the onset of LB in Grana-type and similar cheeses produced without lysozyme.

Characterization and pre-industrial validation of Streptococcus thermophilus strains to be used as starter cultures for Crescenza, an Italian soft cheese.

The aim of this work was to search for new candidate strains to be included in a culture for Crescenza, a rindless soft cheese, today produced mainly at industrial level using selected starter cultures composed of S. thermophilus. Performance testing was applied to 29 pre-selected strains and a scoring approach was developed to identify the most suitable candidates to be employed in Crescenza cheesemaking. Eight S. thermophilus strains fulfilling most of the desired properties (e.g., high phage resistance, fast acidification rate, no growth below 20 °C, NaCl sensibility, no post acidification at 4 °C) were selected. These strains were grouped in pairs to design different starter culture formulations, which were preliminary tested for the production of Crescenza cheeses at laboratory scale. Two couples of binary cultures (designed Phage rotation 1 and Phage rotation 2) were finally designed and used as starters in pilot scale cheesemaking. The combinations, especially those designed in Phage rotation 1, appeared to be suitable for Crescenza production and showed mutual similarity in terms of strain characteristics, technological performance, and cheese quality. The selection method and ranking approach presented in this work may be adapted to other species of LAB showing traits of industrial interest.

Application of Recombined Milk to Produce Crescenza-Type Cheese in Laboratory-Scale Cheesemaking: Implications on Technology and Sensory Properties.

This work evaluated the effect of recombined skimmed milk (RM), mixed in different ratios (40, 60, and 100%) with fresh cow milk, on the processing technology and quality of Crescenza, an industrial soft cheese of the Italian dairy tradition. Crescenza-type cheeses were produced at a laboratory scale, following the industrial process. Control cheese consisted of Crescenza-type cheese produced with 100% whole fresh milk. Compared to control cheese, the substitution of fresh milk with 60-100% of RM deteriorated the coagulation properties and led to a higher moisture retention, whereas, with 40% of RM, the differences were not statistically significant. Cheeses produced with any concentration of RM, although of acceptable quality, differed significantly in terms of sensory properties from control cheese. The addition of colloidal calcium phosphate, or CaCl2 together with a reduction in the size of the curd at cutting, minimized the differences in composition and sensory properties between cheeses produced with 40% RM and control cheese. This study suggested the applicability of 40% RM to obtain Crescenza-type cheese with suitable quality characteristics. The type of product, the technology, the quality, and quantity of the powders are all key factors to be taken into account for a successful application.

The impact of sodium chloride reduction on Grana-type cheese production and quality.

With the aim to reduce the Na content, hard cheeses manufactured using the same technology as for Grana cheese (Grana-type) were salted using three brines containing different amounts of KCl (K-brines) and compared with control cheeses, salted with marine NaCl. A lower weight loss was observed in cheeses salted with K-brines (K-cheeses), whereas the yield and dry matter did not differ significantly between K-cheeses and controls. After 3 months of ripening (T3), the distribution of the Na cations (Na) was centripetal, with a higher Na concentration in the outer (0-3 cm of depth) layer, whereas the K cations (K) seemed to diffuse into the cheese more rapidly and homogeneously. Starting from the 6th month (T6), the distribution of both Na and K was stabilized through the different cheese layers. The use of the brine with the highest concentration of potassium (53.8% K) enabled us to successfully halve the Na content compared to the controls whereas, with the other brines, the reduction of Na was below 30%. At the end of ripening (T9), all the cheeses were without defects and the partial substitution of Na with K did not impact on the chemical composition, microbiological characteristics and ripening process. The sensory evaluation did not show any difference between K-salted and control cheeses in discriminant analysis.

Can the development and autolysis of lactic acid bacteria influence the cheese volatile fraction? The case of Grana Padano.

In this study, the relationship between the dynamics of the growth and lysis of lactic acid bacteria in Grana Padano cheese and the formation of the volatile flavor compounds during cheese ripening was investigated. The microbial dynamics of Grana Padano cheeses that were produced in two different dairies were followed during ripening. The total and cultivable lactic microflora, community composition as determined by length heterogeneity-PCR (LH-PCR), and extent of bacterial lysis using an intracellular enzymatic activity assay were compared among cheeses after 2, 6 and 13months of ripening in two dairies. The evolution of whole and lysed microbiota was different between the two dairies. In dairy 2, the number of total cells was higher than that in dairy 1 in all samples, and the number of cells that lysed during ripening was lower. In addition, at the beginning of ripening (2months), the community structure of the cheese from dairy 2 was more complex and was composed of starter lactic acid bacteria (Lactobacillus helveticus and Lactobacillus delbrueckii) and NSLAB, possibly arising from raw milk, including Lactobacillus rhamnosus/Lactobacillus casei and Pediococcus acidilactici. On the other hand, the cheese from dairy 1 that ripened for 2months was mainly composed of the SLAB L. helveticus and L. delbrueckii. An evaluation of the free-DNA fraction through LH-PCR identified those species that had a high degree of lysis. Data on the dynamics of bacterial growth and lysis were evaluated with respect to the volatile profile and the organic acid content of the two cheeses after 13months of ripening, producing very different results. Cheese from dairy 1 showed a higher content of free fatty acids, particularly those deriving from milk fat lipolysis, benzaldehyde and organic acids, such as pGlu and citric. In contrast, cheese from dairy 2 had a greater amount of ketones, alcohols, hydrocarbons, acetic acid and propionic acid. Based on these results, we can conclude that in the first cheese, the intracellular enzymes that were released from lysis were mainly involved in aroma formation, whereas in the second cheese, the greater complexity of volatile compounds may be associated with its more complex microbial composition caused from SLAB lysis and NSLAB (mainly L. rhamnosus/L. casei) growth during ripening.

Cholesterol removal capability of lactic acid bacteria and related cell membrane fatty acid modifications.

Milk and dairy products play an important role in a healthy diet because of their high nutritional value, even if they represent a source of lipids and cholesterol. Nowadays, some commercially hypocholesterolemic products are available, which contain lactic acid bacteria (LAB). Therefore, the aims of this study were to test and compare the cholesterol removal abilities of different LAB species and to investigate the capacity of the cholesterol to change the cellular fatty acid composition of microorganisms. Fifty-eight strains of dairy LAB were studied for their ability to remove cholesterol during 24 h of growth. Two of them, L. plantarum 885 and L. acidophilus LA-5®, showed the higher reduction capability. For these strains, the cellular fatty acid composition was studied. They showed a different behaviour, which appeared related to the needs of the cells to maintain the characteristics of membrane fluidity, but was dependent upon their original fatty acid composition. Further studies are required to better characterise the LAB strains to be used to develop fermented dairy products with reduced cholesterol content or be able to induce hypocholesterolemic effects. It will also be interesting to investigate the possible modifications of the cell membrane caused by cholesterol and its possible involvement in cell metabolism.