The microbiota of Mozzarella di Bufala Campana PDO cheese: a study across the manufacturing process.

Introduction: Mozzarella di Bufala Campana PDO cheese (MBC) is a globally esteemed Italian cheese. The traditional cheesemaking process of MBC relies on natural whey starter culture, water buffalo's milk, and the local agroecosystem. Methods: In this study, the microbial ecology of intermediate samples of MBC production, coming from two dairies with slightly different cheesemaking technology (dairy M large producer, and dairy C medium-small), was investigated using 16S rRNA amplicon sequencing. This research aimed to provide insights into the dynamics of microbial consortia involved in various cheesemaking steps. Results and discussion: All samples, except for raw buffalo milk, exhibited a core microbiome predominantly composed of Streptococcus spp. and Lactobacillus spp., albeit with different ratios between the two genera across the two MBC producers. Notably, the microbiota of the brine from both dairies, analyzed using 16S amplicon sequencing for the first time, was dominated by the Lactobacillus and Streptococcus genera, while only dairy C showed the presence of minor genera such as Pediococcus and Lentilactobacillus. Intriguingly, the final mozzarella samples from both producers displayed an inversion in the dominance of Lactobacillus spp. over Streptococcus spp. in the microbiota compared to curd samples, possibly attributable to the alleviation of thermal stress following the curd stretching step. In conclusion, the different samples from the two production facilities did not exhibit significant differences in terms of the species involved in MBC cheesemaking. This finding confirms that the key role in the MBC cheesemaking process lies with a small-sized microbiome primarily composed of Streptococcus and Lactobacillus spp.

Freezing as a solution to preserve the quality of dairy products: the case of milk, curds and cheese.

When thinking of the freezing process in dairy, products consumed in frozen state, such as ice creams come to mind. However, freezing is also considered a viable solutions for many other dairy products, due to increasing interest to reduce food waste and to create more robust supply chains. Freezing is a solution to production seasonality, or to extend the market reach for high-value products with otherwise short shelf life. This review focuses on the physical and chemical changes occurring during freezing of milk, curds and cheeses, critical to maintaining quality of the final product. However, freezing is energy consuming, and therefore the process needs to be optimized to maintain product's quality and reduce its environmental footprint. Furthermore, the processing steps leading to the freezing stage may require some changes compared to traditional, fresh products. Unwanted reactions occur at low water activity, and during modifications such as ice crystals growth and recrystallization. These events cause major physical destabilizations of the proteins due to cryoconcentration, including modification of the colloidal-soluble equilibrium. The presence of residual proteases and lipases also cause important modifications to the texture and flavor of the frozen dairy product.

Water status and dynamics of high-moisture Mozzarella cheese as affected by frozen and refrigerated storage.

High-moisture Mozzarella is one of the most exported cheeses worldwide, but affected by short shelf-life. Freezing can help to reduce waste, but its effect on quality needs to be considered. In this study, the physico-chemical changes of Mozzarella occurring during frozen storage and subsequent refrigerated storage (after thawing) were evaluated. Frozen cheeses stored at -18 °C between 1 and 4 months showed microstructural damage and different physical, textural, sensory properties. With NMR relaxometry it was possible to observe freeze-related dehydration of caseins, by measuring the changes in water relaxation times within the matrix. These modifications were confirmed by microstructural observations that showed the formation of larger serum channels in samples subjected to freezing, compared with fresh cheeses. Sensory evaluation showed skin peeling off in frozen samples. By observing the changes at various length scales it was therefore possible to identify the critical points affecting HM Mozzarella cheese quality during frozen storage.

Ripening of Nostrano Valtrompia PDO Cheese in Different Storage Conditions: Influence on Chemical, Physical and Sensory Properties.

Nostrano Valtrompia is a hard, long-ripened, Italian Protected Designation of Origin (PDO) cheese typically produced by applying traditional cheesemaking practices in small dairies. Due to the limited production, this cheese is characterized by an important market price. Nostrano Valtrompia physico-chemical and sensory quality can be influenced by the duration and conditions of ripening. The objectives of this work were to characterize the physico-chemical and sensory characteristics of Nostrano Valtrompia cheese ripened for 12 and 16 months and to study the influence of different ripening warehouses: a temperature conditioned warehouse (TCW) and in a traditional, not conditioned warehouse (TNCW). The moisture gradient from the rind to the center of the cheese influenced texture, moisture, aw and color. Ripening in different warehouses did not affect the overall appreciation of the cheese nor other physico-chemical (color, moisture) or sensory traits. TCW cheeses were characterized by a slightly softer texture, slightly different openings distribution, and a different sensory perception than TNCW cheeses. These minor differences were related to the less variable environmental ripening conditions of TCW than TNCW. The results of this study can be useful to support the management of the ripening conditions of Nostrano Valtrompia PDO cheese and to rationally introduce new, suitable ripening sites.

Effect of frozen and refrigerated storage on proteolysis and physicochemical properties of high-moisture citric mozzarella cheese.

High-moisture mozzarella is one of the most-exported Italian cheeses worldwide, but its quality is affected by storage. Freezing is regarded as a solution to decrease product waste, extend market reach, and increase convenience, but its effect on quality has to be estimated. In this study, the details related to proteolysis, physicochemical properties, and sensory quality parameters of high-moisture mozzarella as a function of frozen storage (1, 3, and 4 mo) and subsequent refrigerated storage after thawing (1, 3, and 8 d) were evaluated. Frozen cheeses stored at -18°C showed a higher extent of proteolysis, as well as different colorimetric and sensory properties, compared with the fresh, nonfrozen control. Sensory evaluation showed the emergence of oxidized and bitter taste after 1 mo of frozen storage, which supports the proteolysis data. The extent of proteolysis of frozen-stored cheese after thawing was greater than that measured in fresh cheese during refrigerated storage. These results help better understand the changes occurring during frozen storage of high-moisture mozzarella cheese and evaluate possible means to decrease the effect of freezing on the cheese matrix.

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.

Applicability of Confocal Raman Microscopy to Observe Microstructural Modifications of Cream Cheeses as Influenced by Freezing.

Confocal Raman microscopy is a promising technique to derive information about microstructure, with minimal sample disruption. Raman emission bands are highly specific to molecular structure and with Raman spectroscopy it is thus possible to observe different classes of molecules in situ, in complex food matrices, without employing fluorescent dyes. In this work confocal Raman microscopy was employed to observe microstructural changes occurring after freezing and thawing in high-moisture cheeses, and the observations were compared to those obtained with confocal laser scanning microscopy. Two commercially available cream cheese products were imaged with both microscopy techniques. The lower resolution (1 µm/pixel) of confocal Raman microscopy prevented the observation of particles smaller than 1 µm that may be part of the structure (e.g., sugars). With confocal Raman microscopy it was possible to identify and map the large water domains formed during freezing and thawing in high-moisture cream cheese. The results were supported also by low resolution NMR analysis. NMR and Raman microscopy are complementary techniques that can be employed to distinguish between the two different commercial formulations, and different destabilization levels.

Heat Resistance of Listeria monocytogenes in Dairy Matrices Involved in Mozzarella di Bufala Campana PDO Cheese.

The presence of Listeria monocytogenes in Mozzarella di Bufala Campana Protected Designation of Origin cheeses may depend on curd stretching conditions and post contaminations before packaging. To avoid cross-contamination, thermal treatment of water, brines and covering liquid may become necessary. The present study aimed to improve knowledge about L. monocytogenes thermal resistance focusing on the influence of some cheese making operations, namely curd stretching and heat treatment of fluids in contact with cheese after molding, in order to improve the safety of the cheese, optimize efficacy and sustainability of the processes. Moreover, the role that cheese curd stretching plays in L. monocytogenes inactivation was discussed. The 12 tested strains showed a very heterogeneous heat resistance that ranged from 7 to less than 1 Log10 Cfu/mL reduction after 8 min at 60°C. D-values (decimal reduction times) and z-values (thermal resistance constant) calculated for the most heat resistant strain among 60 and 70°C were highly affected by the matrix and, in particular, heat resistance noticeably increased in drained cheese curd. As cheese curd stretching is not an isothermal process, to simulate the overall lethal effect of an industrial process a secondary model was built. The lethal effect of the process was estimated around 4 Log10 reductions. The data provided may be useful for fresh pasta filata cheese producers in determining appropriate processing durations and temperatures for producing safe cheeses.

Ability of a Wild Weissella Strain to Modify Viscosity of Fermented Milk.

Despite the fact that strains belonging to Weissella species have not yet been approved for use as starter culture, recent toxicological studies open new perspectives on their potential employment. The aim of this study was to evaluate the ability of a wild Weissella minor (W4451) strain to modify milk viscosity compared to Lactobacillus delbrueckii subsp. bulgaricus, which is commonly used for this purpose in dairy products. To reach this goal, milk viscosity has been evaluated by means of two very different instruments: one rotational viscometer and the Ford cup. Moreover, water holding capacity was evaluated. W4451, previously isolated from sourdough, was able to acidify milk, to produce polysaccharides in situ and thus improve milk viscosity. The ability of W4451 to produce at the same time lactic acid and high amounts of polysaccharides makes it a good candidate to improve the composition of starters for dairy products. Ford cup turned out to be a simple method to measure fermented milk viscosity by small- or medium-sized dairies.

Physical characterization of whole and skim dried milk powders.

The lack of updated knowledge about the physical properties of milk powders aimed us to evaluate selected physical properties (water activity, particle size, density, flowability, solubility and colour) of eleven skim and whole milk powders produced in Europe. These physical properties are crucial both for the management of milk powder during the final steps of the drying process, and for their use as food ingredients. In general, except for the values of water activity, the physical properties of skim and whole milk powders are very different. Particle sizes of the spray-dried skim milk powders, measured as volume and surface mean diameter were significantly lower than that of the whole milk powders, while the roller dried sample showed the largest particle size. For all the samples the size distribution was quite narrow, with a span value less than 2. The loose density of skim milk powders was significantly higher than whole milk powders (541.36 vs 449.75 kg/m3). Flowability, measured by Hausner ratio and Carr's index indicators, ranged from passable to poor when evaluated according to pharmaceutical criteria. The insolubility index of the spray-dried skim and whole milk powders, measured as weight of the sediment (from 0.5 to 34.8 mg), allowed a good discrimination of the samples. Colour analysis underlined the relevant contribution of fat content and particle size, resulted in higher lightness (L*) for skim milk powder than whole milk powder, which, on the other hand, showed higher yellowness (b*) and lower greenness (-a*). In conclusion a detailed knowledge of functional properties of milk powders may allow the dairy to tailor the products to the user and help the food processor to perform a targeted choice according to the intended use.

Invited review: Microbial evolution in raw-milk, long-ripened cheeses produced using undefined natural whey starters.

The robustness of the starter culture during cheese fermentation is enhanced by the presence of a rich consortium of microbes. Natural starters are consortia of microbes undoubtedly richer than selected starters. Among natural starters, natural whey starters (NWS) are the most common cultures currently used to produce different varieties of cheeses. Undefined NWS are typically used for Italian cooked, long-ripened, extra-hard, raw milk cheeses, such as Parmigiano Reggiano and Grana Padano. Together with raw milk microbiota, NWS are responsible for most cheese characteristics. The microbial ecology of these 2 cheese varieties is based on a complex interaction among starter lactic acid bacteria (SLAB) and nonstarter lactic acid bacteria (NSLAB), which are characterized by their different abilities to grow in a changing substrate. This review aims to summarize the latest findings on Parmigiano Reggiano and Grana Padano to better understand the dynamics of SLAB, which mainly arise from NWS, and NSLAB, which mainly arise from raw milk, and their possible role in determining the characteristics of these cheeses. The review is presented in 4 main sections. The first summarizes the main microbiological and chemical properties of the ripened cheese as determined by cheese-making process variables, as these variables may affect microbial growth. The second describes the microbiota of raw milk as affected by specific milk treatments, from milking to the filling of the cheese milk vat. The third describes the microbiota of NWS, and the fourth reviews the knowledge available on microbial dynamics from curd to ripened cheese. As the dynamics and functionality of complex undefined NWS is one of the most important areas of focus in current food microbiology research, this review may serve as a good starting point for implementing future studies on microbial diversity and functionality of undefined cheese starter cultures.

Synthetic peptides as substrate for assaying the proteolytic activity of Lactobacillus helveticus.

Four Lactobacillus helveticus strains were studied for proteolytic capacity and general aminopeptidase (AP) and X-Pro dipeptidyl aminopeptidase (DAP) activity. The rate of hydrolysis and the activity against synthetic substrates with N-terminal residues of Arg, Lys, Leu, Glu or Pro, varied markedly among the strains. The X-Pro DAP activity was consistently high. The crude cell-wall and cytoplasm extracts from strain Lb. helveticus ISLC59 were analysed thoroughly for their proteolysis ability by using four synthetic peptide substrates, including alpha(s)1-CN(f1-23). Peptides formed during in vitro hydrolysis of the synthetic substrates by cell wall and cytoplasm preparations were identified by LC-ESI/MS. In doing so, it was possible to infer a prevalent endopeptidase activity splitting Lys7-His8 and Gln13-Glu14 bonds in the cytoplasm, and to deduce a secondary activity, which hydrolysed Glu14-Val15, Leu16-Asn17, Glu18-Asn19 and Lys3-His4 bonds lacking in the cell-wall. The presence of exopeptidases, as mainly AP, DAP, and carboxypeptidase (CPase) was deduced from the formation of several N- and C-terminally truncated peptides sets. The AP activity was higher in the cell-wall layer, where CPase activity was absent. The in vitro assays with cell extracts of the Lb. helveticus ISLC59 strain revealed extensive exopeptidase and endopeptidase activities. In several cases, the hydrolytic system of Lb. helveticus that splits in vitro alpha(s)1-CN(f1-23) peptide bonds was similar to that of Lactococcus lactis. The effects were also compared with those occurring in vivo in hard cheese such as Grana Padano.