Invited review: Hygienic quality, composition, and technological performance of raw milk obtained by robotic milking of cows.

Automatic milking systems (AMS), first introduced on dairy farms in the 1990s, rapidly spread across many countries. This technology is based on the voluntary milking of dairy cattle in a completely automated process, which relies on computer management, with a substantial average increase in milking frequency. Compared with conventional milking, AMS significantly alters herd management, with important implications on economic, technical, and social aspects of farming, on animal physiology, health, and well-being. These aspects are explored in an extensive body of research. In contrast, the effects of AMS adoption on milk quality are often overlooked. This review draws together both positive and negative effects of AMS on the milk production chain, particularly emphasizing the variations of hygienic and compositive characteristics of raw milk and their interplay, as compared with milk obtained with conventional milking. Scattered and sometimes conflicting literature exists on whether and how these variations may influence quality and yield of the derived dairy products. Current scientific knowledge on these crucial aspects is thus reviewed, with particular focus on milk technological suitability for being processed into dairy products having the target characteristics in terms of taste, structure, on-storage stability, and sustainability. Provided the managing conditions are optimized, AMS allow increased milk production, mostly due to more frequent milking, without compromising the milk characteristics that are crucial to food industry for processing. Nevertheless, specific biochemical aspects related to the changed milking interval, which determines the duration of enzyme activities and bacterial growth in milk, need further research.

Impact assessment of traditional food manufacturing: The case of Grana Padano cheese.

The dairy sector is recognised as one of the most impacting agricultural activities. In Italy approximately 24% of cow's milk is destined to Grana Padano, a Protected Designation of Origin long ripening cheese. The Grana Padano production has increased by 10% in the last decade and approximately reached 183,000 t in 2015. Around 38% of this production is exported to Germany, US, France and to the rest of the world. This study evaluated the environmental impact of production of Grana Padano, through a "cradle to cheese factory gate" Life Cycle Assessment. The study involved an Italian cheese factory that produces about 3.6% of the total production of Grana Padano cheese and a group of 5 dairy farms, chosen among the farms that sold all milk produced to the cheese factory. The functional unit was 1 kg of Grana Padano cheese 12-month ripened. Environmental impacts of co-products: whey, cream, butter and buttermilk were also evaluated. Two sensitivity analyses were conducted: the first one had the aim to explore the effect of different allocation methods based on dry matter content, economic or nutritive value of cheese, respectively; the second one considered the variation of the impacts of milk production and its effect on cheese environmental impact. Milk production phase gave the most important contribution to the environmental impact of cheese, with a percentage of 93.5-99.6% depending on the impact category. Excluding milk production from the system boundary, milk transport and use of electricity were the main responsible of the environmental impact of cheese-making process. The climate change impact for the production of 1 kg Grana Padano was 10.3 kg of CO2 eq, using a dry matter allocation method, while 16.9 and 15.2 kg of CO2 eq adopting economic and nutritive value allocation methods, respectively.

A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy.

Non-pathogenic spore-forming Clostridia are of increasing interest due to their application in biogas production and their capability to spoil different food products. The life cycle for Clostridium includes a spore stage that can assist in survival under environmentally stressful conditions, such as extremes of temperature or pH. Due to their size, spores can be investigated by a range of microscopic techniques, many of which involve sample pre-treatment. We have developed a quick, simple and non-destructive fluorescent staining procedure that allows a clear differentiation between spores and vegetative cells and effectively stains spores, allowing recovery and tracking in subsequent experiments. Hoechst 34580, Propidium iodide and wheat germ agglutinin WGA 488 were used in combination to stain four strains of Clostridia at different life cycle stages. Staining was conducted without drying the sample, preventing changes induced by dehydration and cells observed by confocal laser scanner microscopy or using a super-resolution microscope equipped with a 3D-structured illumination module. Dual staining with Hoechst/Propidium iodide differentiated spores from vegetative cells, provided information on the viability of cells and was successfully applied to follow spore production induced by heating. Super-resolution microscopy of spores probed by Hoechst 34580 also allowed chromatin to be visualised. Direct staining of a cheese specimen using Nile Red and Fast Green allowed in situ observation of spores within the cheese and their position within the cheese matrix. The proposed staining method has broad applicability and can potentially be applied to follow Clostridium spore behaviour in a range of different environments.

Microfiltration and ultra-high-pressure homogenization for extending the shelf-storage stability of UHT milk.

Fat separation, gelation or sedimentation of UHT milk during shelf-storage represent instability phenomena causing the product rejection by consumers. Stability of UHT milk is of increasing concern because access to emerging markets currently implies for this product to be stable during shipping and prolonged storage, up to 12 months. The role of microfiltration prior to UHT process in avoiding or retarding the gelation or sediment formation was studied by comparing microfiltered UHT milk to conventional UHT milk. A second trial was set up to study the effects of double ultra-high pressure homogenization in delaying the cream rising and UHT milk homogenized once at lower pressure was taken as control. All milk samples were produced at industrial plant level. Milk packages were stored at 22 °C, opened monthly for visually inspecting the presence of cream layer, gel or sediment and then analysed. Microfiltration markedly delayed the formation of both gel particles and sediment, with respect to the control, and slowed down the proteolysis in terms of accumulation of peptides although no correlation was observed between the two phenomena. The double homogenization, also evaluated at ultra-structural level, narrowed the fat globule distribution and the second one (400 MPa), performed downstream to the sterilization step, disrupted the fat-protein aggregates produced in the first one (250 MPa). The adopted conditions avoided the appearance of the cream layer in the UHT milk up to 18 months. This study contributes important knowledge for developing strategies to delay instability phenomena in UHT milk destined to extremely long shelf storage.

Effect of temperature on the microstructure of fat globules and the immunoglobulin-mediated interactions between fat and bacteria in natural raw milk creaming.

Natural creaming of raw milk is the first step in production of Grana Padano and Parmigiano Reggiano Protected Denomination of Origin cheeses. This process decreases the fat content and plays an important role in the removal of clostridia species that may cause late-blowing defects in ripened cheeses. Partial coalescence of fat globules-that may influence fat behavior in cheese making and affect the microstructure of fat in the final cheese product-was observed at creaming temperatures higher than 22°C by confocal laser scanning microscopy. The widespread practice of heating of milk at 37°C before creaming at 8°C resulted in important changes in the size distribution of fat globules in raw milk, potentially altering the ability of fat to entrap clostridia spores. We investigated the role of immunoglobulin classes in both the clustering of fat globules and the agglutination of Clostridium tyrobutyricum to fat globules during creaming. Immunogold labeling and transmission electron microscopy showed that IgA and IgM but not IgG were involved in both clustering and agglutination. Both vegetative cells and spores were clearly shown to agglutinate to fat droplets, a process that was suppressed by thermal denaturation of the immunoglobulins. The debacterization of raw milk through natural creaming was improved by the addition of purified immunoglobulins. Overall, these findings provide not only a better understanding of the phenomena occurring during the natural creaming but also practical insights into how the process of creaming may be optimized in cheese production plants.

New insight on crystal and spot development in hard and extra-hard cheeses: Association of spots with incomplete aggregation of curd granules.

Chemical composition and structure of different types of macroparticles (specks, spots) and microparticles (microcrystals) present in hard and extra-hard cheeses were investigated. Light microscopy revealed that the small hard specks had the structure of crystalline tyrosine, as confirmed by amino acid analysis. Spots showed a complex structure, including several curd granules, cavities, and microcrystals, and were delimited by a dense protein layer. Spots contained less moisture and ash than the adjacent cheese area, and more protein, including significantly higher contents of valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine. Microcrystals were observed by light and electron microscopy and analyzed by confocal micro-Raman. Among others, calcium phosphate crystals appeared to consist of a central star-shaped structure immersed in a matrix of free fatty acids plus leucine and phenylalanine in free form or in small peptides. A hypothetical mechanism for the formation of these structures has been formulated.

Lysozyme affects the microbial catabolism of free arginine in raw-milk hard cheeses.

Lysozyme (LZ) is used in several cheese varieties to prevent late blowing which results from fermentation of lactate by Clostridium tyrobutyricum. Side effects of LZ on lactic acid bacteria population and free amino acid pattern were studied in 16 raw-milk hard cheeses produced in eight parallel cheese makings conducted at four different dairies using the same milk with (LZ+) or without (LZ-) addition of LZ. The LZ-cheeses were characterized by higher numbers of cultivable microbial population and lower amount of DNA arising from lysed bacterial cells with respect to LZ + cheeses. At both 9 and 16 months of ripening, Lactobacillus delbrueckii and Lactobacillus fermentum proved to be the species mostly affected by LZ. The total content of free amino acids indicated the proteolysis extent to be characteristic of the dairy, regardless to the presence of LZ. In contrast, the relative patterns showed the microbial degradation of arginine to be promoted in LZ + cheeses. The data demonstrated that the arginine-deiminase pathway was only partially adopted since citrulline represented the main product and only trace levels of ornithine were found. Differences in arginine degradation were considered for starter and non-starter lactic acid bacteria, at different cheese ripening stages.

Mechanisms of Clostridium tyrobutyricum removal through natural creaming of milk: A microscopy study.

Clostridium tyrobutyricum is the main spoilage agent of late blowing defect (LBD) in Grana Padano and Parmigiano-Reggiano cheeses; LBD is characterized by openings and holes and is sometimes accompanied by cracks and an undesirable flavor. Even a very few spores remaining in the cheese curd may cause LBD; thus, it is essential to eradicate them during milk natural creaming. By this process, most of the bacteria, somatic cells, and spores rise to the top of the milk, together with the fat globules, and are removed with the cream. Previous studies suggested that milk immunoglobulins mediate the interactions between fat globules and bacteria that occur upon creaming but no direct evidence for this has been found. Moreover, other physical chemical interactions could be involved; for example, physical entrapment of spores among globule clusters. To maximize the efficiency of the natural creaming step in removing Cl. tyrobutyricum, it is essential to understand the nature of spore-globule interactions. With this aim, raw milk was contaminated with spores of Cl. tyrobutyricum before going to creaming overnight at 8°C, after which spore and bacteria removal was >90%. The obtained cream was analyzed by light interference contrast and fluorescence microscopy and by transmission electron microscopy (TEM). Results showed that most of the vegetative cells and spores, which were stained with malachite green before addition to milk, adhered tightly to the surface of single fat globules, the membranes of which appeared heterogeneous when stained with the fluorescent dye DilC18(3)-DS. Using the same dye, we observed transient and persistent interactions among globules, with formation of clusters of different sizes and partial coalescence of adhering membranes. Transmission electron microscopy examination of replicates of freeze-fractured cream allowed us to observe tight adhesion of spores to fat globules. Ultrathin sections revealed that this adhesion is mediated by an amorphous, slightly electron-opaque material, sometimes granular in appearance. Bacteria also adhered to different fat globules, linking them together, which suggests that adhesion was strong enough to maintain a stable contact. Although we cannot exclude physical entrapment of bacteria among fat globule clusters, we show for the first time that most of the bacteria are adhered to fat globules by an electron-opaque material whose nature has yet to be determined. Immunoglobulins are certainly the best candidates for adhesion but other compounds may be involved.