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The late blowing defect in Grana Padano PDO cheese: effects of milk natural creaming and cheese making conditions
No full textThis PhD research project aims at achieving a better understanding of the cheese late blowing, the most invasive defect affecting hard cheeses like Grana Padano and primarily promoted by Clostridium tyrobutyricum. Phenomena that characterize the natural creaming of raw milk will be studied in view of maximizing the entrapment of bacterial cells and spores into the cream. Furthermore, agents and conditions inducing spore germination will be investigated in both model systems and real cheese makings. This information will give scientific basis for optimization of the critical steps in cheese making in view of preventing the defect without using antimicrobial additives
THE LATE BLOWING DEFECT IN GRANA PADANO CHEESE: THE MECHANISMS OF MILK HEALING THROUGH NATURAL CREAMING AND THE EFFECTS OF CHEESE MAKING CONDITIONS IN INDUCING CLOSTRIDIUM SPORE GERMINATION
Full text linkThis thesis project was designed to explore the technological, chemical and microbiological factors involved in the late blowing defect (LBD) of Grana Padano (GP) with the aim of setting up every possible precaution to overcome this economically important flaw. LBD is a cheese defect caused by some Clostridium species, mostly Clostridium tyrobutyricum, and characterized by holes, cracks as well as an unpleasant flavour. Natural creaming of milk is the first and most critical step in GP making, during which C. tyrobutyricum spores are risen to the top of milk together with fat globules, thus being eliminated from partially skimmed milk. This step has been thoroughly investigated with particular reference to the interaction type between spores and fat globules. Transmission Electron Microscopy (TEM) revealed an electron-dense material sticking bacteria to fat globules. This material has been identified as immunoglobulins, mainly IgA and to a lesser extend IgM, by immunogold labelling. Many IgA molecules, but not IgM and IgG, were localized on C. tyrobutyricum cell wall suggesting the presence of anti-IgA receptors on it. Following this finding, immunoglobulins were purified from colostrum and their ability to enhance bacteria rising during natural creaming of milk investigated. Indeed, the addition of immunoglobulins led to a significant increase in spore rising with respect to untreated control. Specific natural creaming trials were also carried out to study the effect of creaming temperature on fat microstructure. Fat supramolecular organization, the unwanted fat globule structural rearrangement, was observed when creaming was carried out at 22 and 40 °C, as well as in sample tempered at 37 °C prior to the normal creaming at 8 °C. This suggests that the often-adopted thermal treatment before creaming is deleterious for fat microstructure.
The behaviour of C. tyrobutyricum throughout the further steps of GP making and ripening was also investigated by an innovative approach. Vegetative bacteria cells and spores, independently sealed within dialysis tubes, were kept in the vat for the entire cheese making and then into the cheese over 6-month ripening. They were sampled and counted at milk renneting, curd cooking, curd extraction, in-mould acidification, brine salting, 3- and 6-month ripening. Furthermore, their morphological changes were monitored by TEM and the free amino acid utilization evaluated. Vegetative bacteria cells died during curd cooking up to 54 °C and then were no longer cultivable. However, 2 x 102 CFU/mL of these cells were sporulated in the curd at extraction and a higher number of spores was found at the end of 6-month ripening. Few spores germinated during curd acidification, when lactate was available. C. tyrobutyricum proved to convert arginine to citrulline and then to ornithine throughout the cheese ripening. This capability was confirmed in both milk and cheese model systems, indicating the vitality and metabolic activity of bacterial cells and, possibly, their ability of modifying GP sensorial properties.
Lysozyme (LZ) is used in GP as well as in other cheese varieties to prevent the LBD. Thus, it has been considered important to verify its effects on lactic acid bacteria population and cheese free amino acid pattern. This study was carried out 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. 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 L. fermentum proved to be the species mostly affected by LZ. The total content of free amino acids indicated that the proteolysis extent is characteristic of each dairy, regardless to the presence of LZ. Instead, in LZ+ cheeses, microbial degradation of arginine into citrulline and ornithine was always promoted. As, the amount of ornithine was quantitatively lower than citrulline it is likely that the arginine-deiminase pathway was only partially adopted.
Working with C. tyrobutyricum spores gave rise to the need of a practical and effective staining method that could both facilitate spore detection and easily discriminate spore from vegetative bacterial cells. A fast and robust protocol for fluorescent staining of spores and vegetative cells of C. tyrobutyricum was set up, by using Hoechst 34580 and Propidium Iodide (HO/PI) stains that allowed to distinguish viable, dead and sporulated cells of C. tyrobutyricum. The HO/PI staining protocol proved to be suitable for other three Clostridia that can cause LBD: C. butyricum, C. sporogenes and C. beijerinkii. Furthermore, Hoechst 34580 dye was successfully used, together with Nile Red and Fast Green, to observe spores into the cheese matrix. In this case, spores were experimentally added to milk during cheese making and the triple staining was performed on slices of cheese after a month ripening.
Finally, during the investigations related to this thesis project, it was possible to shed light on other inclusions present in GP matrix during and after ripening, such as specks, spots and microcrystals. 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 surrounded by a dense protein layer. Spots contained significantly less moisture and ash than the adjacent cheese area, and were significantly richer in protein, including significantly higher levels of valine, methionine, isoleucine, leucine, tyrosine and phenylalanine. Microcrystals were observed by light and electron microscopy and analysed 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 besides leucine and phenylalanine, in free form or in small peptides. A hypothetical mechanism for the formation of these structures was also formulated
Changes in the soluble nitrogen fraction of milk throughout PDO Grana Padano cheese-making
Full text linkThe behaviour of soluble nitrogen compounds during Grana Padano cheese-making was studied at eight dairies. Raw milk, skimmed milk, sweet whey and the derived natural whey culture, collected from 24 processes, were analysed for soluble whey proteins (α-lactalbumin and β-lactoglobulin), proteose-peptones (PP), small peptides (SP), caseinomacropeptides (CMPs), and free amino acids (FAAs). The PP fraction increased during milk natural creaming, then part of it was selectively retained in the curd and the rest degraded in the first few hours of whey fermentation, together with α-lactalbumin, CMPs and part of SP. Features outlined for the whey culture were confirmed on 30 samples collected at six different dairies. A time course study of the whey fermentation showed that degradation of α-lactalbumin began when the pH dropped below 4, whereas β-lactoglobulin content did not change. Uptake of specific FAAs was shown to support the initial growth of lactic acid bacteria in whey
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
New insight on crystal and spot development in hard and extra-hard cheeses : Association of spots with incomplete aggregation of curd granules
Full text linkChemical 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
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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