Cheese Defects Research Articles

Use of fluorescent CTP1L endolysin cell wall-binding domain to study the evolution of Clostridium tyrobutyricum during cheese ripening

Clostridium tyrobutyricum is a bacteria of concern in the cheese industry, capable of surviving the manufacturing process and causing butyric acid fermentation and late blowing defect of cheese. In this work, we implement a method based on the cell wall-binding domain (CBD) of endolysin CTP1L, which detects C. tyrobutyricum, to monitor its evolution in cheeses challenged with clostridial spores and in the presence or absence of reuterin, an anti-clostridial agent. For this purpose, total bacteria were extracted from cheese samples and C. tyrobutyricum cells were specifically labelled with the CBD of CTP1L attached to green fluorescent protein (GFP), and detected by fluorescence microscopy. By using this GFP-CBD, germinated spores were visualized on day 1 in all cheeses inoculated with clostridial spores. Vegetative cells of C. tyrobutyricum, responsible for butyric acid fermentation, were detected in cheeses without reuterin from 30 d onwards, when LBD symptoms also became evident. The number of fluorescent Clostridium cells increased during ripening in the blowing cheeses. However, vegetative cells of C. tyrobutyricum were not detected in cheese containing the antimicrobial reuterin, which also did not show LBD throughout ripening. This simple and fast method provides a helpful tool to study the evolution of C. tyrobutyricum during cheese ripening.

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.

Novel approach to enumerate clostridial endospores in milk

Clostridial contamination of raw milk causes late-blowing, a severe quality defect in cheese. Consequently, milk containing high numbers of cheese-damaging clostridial spores should not be used for the production of certain types of hard and semi-hard cheese. Currently, there is no officially standardised method available to monitor clostridial spore levels in milk, and major drawbacks like long analysis time, labour intensity, uncertainty of results and insufficient selectivity for clostridia exist for usually used conventional MPN (most probable number) techniques. Therefore, an optimised medium in combination with a semi-automated application for the enumeration of clostridia in milk was developed. The aim of this study was to evaluate this new methodology in comparison with a conventional method (using Bryant and Burkey broth) based on the analysis of 84 milk samples. Method inclusivity was further tested using pure clostridial cultures, and selectivity was assessed by molecular identification of isolates obtained from the new assay. The novel approach proved to be suitable for the detection of clostridia in both suppliers’ and processed milk, also indicating that it is superior in selectivity, sensitivity and analysis time compared to conventional techniques.

Hot topic: Geographical distribution and strain diversity of Lactobacillus wasatchensis isolated from cheese with unwanted gas formation

Lactobacillus wasatchensis, an obligate heterofermentative nonstarter lactic acid bacteria (NSLAB) implicated in causing gas defects in aged cheeses, was originally isolated from an aged Cheddar produced in Logan, Utah. To determine the geographical distribution of this organism, we isolated slow-growing NSLAB from cheeses collected in different regions of the United States, Australia, New Zealand, and Ireland. Seven of the cheeses showed significant gas defects and 12 did not. Nonstarter lactic acid bacteria were isolated from these cheeses on de Man, Rogosa, and Sharpe medium supplemented with ribose, a preferred substrate for Lb. wasatchensis. Identification was confirmed with 16S rRNA gene sequencing and the API50CH (bioMérieux, Marcy l'Etoile, France) carbohydrate panel. Isolates were also compared with one another by using repetitive element sequence-based PCR (rep-PCR). Lactobacillus wasatchensis was isolated only from cheeses demonstrating late-gas development and was found in samples from 6 of the 7 cheeses. This supports laboratory evidence that this organism is a causative agent of late gas production defects. The rep-PCR analysis produced distinct genetic fingerprints for isolates from each cheese, indicating that Lb. wasatchensis is found in several regions across the United States and is not a local phenomenon.

Efficient milking hygiene reduces bacterial spore contamination in milk.

Clostridia in the milk can lead to late blowing, a cheese defect. Clostridia are ubiquitous, deriving from both the farm environment and the feed ingested by the cows, and are transferred into the milk through faecal contamination. Our aim was to investigate the effect of different in-parlour practices on the content of anaerobic spore-forming bacteria in milk, and to monitor the variation in spore content in the feed and environment. The experiment, conducted in an experimental dairy during autumn, was repeated in exactly the same way for two consecutive years. The experimental design applied three different milking routines in three consecutive 7-d periods: forestripping alone (F); forestripping and post-dipping (F+Post); pre-dipping, wiping, forestripping and post-dipping (Pre+F+Post). Teat skin swabs and samples of feed, faeces, bedding materials and milk were collected for microbiological analyses. The dietary forage of the lactating cows included maize silage, which, in both years, was found to have the highest level of clostridial spore contamination. Pre-dipping with a detergent/emollient solution, and drying with a disposable paper towel, proved much more efficient in reducing spore contamination than forestripping alone, both on the teats (1·30 vs. 2·20 log10 MPN/swab; P < 0·001) and in the milk (1·82 vs. 2·47 log10 MPN/L, P < 0·02), while post-dipping had little influence on spore count. The standard plate count in milk was significantly lower with Pre+F+Post treatment than with F (3·80 vs. 4·51 log10 CFU/mL, P < 0·01). The teat preparation procedure did not influence the lactic acid bacterial levels in the milk, which is very positive in that decreased lactic acid bacterial content can lessen raw milk cheese quality.

PRINCIPAIS DEFEITOS EM QUEIJO MINAS ARTESANAL: UMA REVISÃO

A preferência do consumidor por queijos artesanais de leite cru vem crescendo continuamente devido à sua intensidade e variação de sabor se comparado ao queijo de leite pasteurizado. O desenvolvimento do sabor e aroma no queijo de leite cru é regido principalmente pela variada microbiota endógena existente e enzimas naturais do leite. O clima, a altitude, a pastagem nativa e outras características regionais permitem que o queijo Minas artesanal tenha um sabor típico e único. No entanto, a maioria dos queijos artesanais são fabricados de maneira rudimentar, seguindo tecnologias utilizadas pelos seus antepassados e este fato pode propiciar o aparecimento de defeitos, que acarretam perdas econômicas aos produtores. Grande parte dos defeitos pode ter origem na qualidade do leite destinado à fabricação, assim como na qualidade e quantidade dos ingredientes utilizados, técnicas de fabricação e maturação que podem aumentar o número de falhas. Nesta revisão, serão abordados os principais defeitos que surgem especificamente nos queijos artesanais, assim como suas origens, prevenção e reparação.

Factors Affecting Sensory Quality of Goat Milk Cheeses: A Review

The combination of tastes and aromas associated with caprine milk lead to a range of unique flavors and sensory properties that are critical to overall quality of caprine milk and its processed products. Goat milk production has risen dramatically in the past two decades. Since numerous breeds of goats have been raised under different localities, environmental and management conditions in many parts of the world, no single factor can universally describe or identify sensory characteristics of a wide variety of goat cheeses produced and consumed around the world. Rather, there are many factors that influence the flavor and sensory qualities of goat milk cheeses. The flavor of cheese arises from a series of complex reactions involving microbial metabolism and enzymatic reactions which include proteolysis of proteins, lipolysis of fats, and fermentation of carbohydrates. Caprine milk reportedly has more fat, protein and ash, and less lactose than cow milk. The types of pasture fed to an animal not only affect the taste of cheese but the color of the products. Because of the amount of carotene present in large amounts in green forage and its contribution to the yellow color in cheese, cheeses made with spring milk are likely to be more yellowish color than those made with winter milk. Lipolysis of fat causes rancid flavor defect in milk, cheese, and other dairy products, while proteolysis provides a major impact on flavor, aroma, and textural characteristics of most cheese varieties. Much more research needs to be done on the sensory quality of goat milk cheese to show the factors directly causing sensory properties of different varieties of goat milk cheeses. Understanding the complexity of flavors embodied by goat milk and goat milk products further aids in the production of quality products that enhance the consumer acceptability of caprine milk cheese products. This paper explores the current up-to-date reports on various factors associated with sensory qualities of caprine milk cheese products.

Exploring the microbiota of the red-brown defect in smear-ripened cheese by 454-pyrosequencing and its prevention using different cleaning systems

Red-brown pigmentation can occasionally form in smeared-ripened cheese such as Fontina during the ripening process. This reaction is due to over-development of the typical microbiota present on the rind. Previous studies have demonstrated the relationship between red-brown pigmentation and the traditional utilization of wooden shelves during cheese ripening. The first part of the paper focuses on the characterisation of yeast and bacterial microbiota: plate counts and 454-pyrosequencing were performed in spoiled (n = 6) and non-spoiled cheeses (n = 6) and on the wooden shelves used during ripening. The second part shows different systems tested for cleaning the wooden shelves and avoiding the development of the red-brown defect in cheese: washing with hot water and ozone treatment. Actinobacteria, dominated on the wooden shelves, suggesting to be responsible for the red-brown pigmentation; they were also found in traces in the defected cheese samples. Galactomyces and Debaryomyces were the main species characterizing the yeast population, with Debaryomyces being the most dominant species on the shelves used during ripening of the red-brown defective cheese. Hot water treatment reduced the microbial contamination of shelves, whereas only the ozone treatment ensured complete elimination of both yeast and bacteria, resulting in the cheese rind not having the red-brown defect.

0548 Determination of treatments to reduce late gassy defect in cheese due to Lactobacillus wasatchensis WDC04 contamination

0548 Determination of treatments to reduce late gassy defect in cheese due to Lactobacillus wasatchensis WDC04 contamination

Influence of raw milk quality on processed dairy products: How do raw milk quality test results relate to product quality and yield?

This article provides an overview of the influence of raw milk quality on the quality of processed dairy products and offers a perspective on the merits of investing in quality. Dairy farmers are frequently offered monetary premium incentives to provide high-quality milk to processors. These incentives are most often based on raw milk somatic cell and bacteria count levels well below the regulatory public health-based limits. Justification for these incentive payments can be based on improved processed product quality and manufacturing efficiencies that provide the processor with a return on their investment for high-quality raw milk. In some cases, this return on investment is difficult to measure. Raw milks with high levels of somatic cells and bacteria are associated with increased enzyme activity that can result in product defects. Use of raw milk with somatic cell counts >100,000cells/mL has been shown to reduce cheese yields, and higher levels, generally >400,000 cells/mL, have been associated with textural and flavor defects in cheese and other products. Although most research indicates that fairly high total bacteria counts (>1,000,000 cfu/mL) in raw milk are needed to cause defects in most processed dairy products, receiving high-quality milk from the farm allows some flexibility for handling raw milk, which can increase efficiencies and reduce the risk of raw milk reaching bacterial levels of concern. Monitoring total bacterial numbers in regard to raw milk quality is imperative, but determining levels of specific types of bacteria present has gained increasing importance. For example, spores of certain spore-forming bacteria present in raw milk at very low levels (e.g., <1/mL) can survive pasteurization and grow in milk and cheese products to levels that result in defects. With the exception of meeting product specifications often required for milk powders, testing for specific spore-forming groups is currently not used in quality incentive programs in the United States but is used in other countries (e.g., the Netherlands).

Concurrence of spores of Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus polymyxa in silage, dairy cow faeces and raw milk

Spores of lactate-fermenting clostridia, known as butyric acid bacteria (BAB), can cause severe quality defects in semi-hard cheeses, called late-blowing. The routine detection method of BAB spores involves most probable number quantification of spores of anaerobic, gas-forming bacteria in a medium containing lactic acid. In this study, BAB spores were detected in 296 samples of soil, maize and grass silage, dairy cow faeces and farm tank milk collected from dairy farms in The Netherlands and the most abundant populations identified. Three major populations were detected, namely Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus spp. Paenibacillus polymyxa was the most abundant species of the latter group. The results indicate that the three spore populations share the same sources and the same contamination route of milk. In contrast to the Clostridium species, P. polymyxa isolates were unable to ferment lactate into butyrate. P. polymyxa spores are presumably unable to cause cheese defects.

Occurrence of Clostridium spp. in ewe’s milk: enumeration and identification of isolates

The presence of gas-producing clostridia in ewe’s milk can lead to the occurrence of late-blowing defects in cheeses. However, data on this aspect are limited. In the present study, using the most probable number (MPN) method, clostridial spores were enumerated in 527 ewe’s milk samples collected in the Grosseto Province (Tuscany, Italy) from autumn 2014 to summer 2015. In addition, using polymerase chain reaction (PCR), we identified the species most frequently involved in late-blowing defects in cheese (Clostridium tyrobutyricum, Clostridium butyricum, Clostridium beijerinckii, and Clostridium sporogenes), and of Clostridium perfringens. Gas-producing clostridial spores were detected in 99% of samples. Spore concentrations ranged from 360 to more than 110,000 spores∙L−1. We observed that 86% of samples had a spore load higher than 1000 spores∙L−1. During autumn and summer, spore concentrations were significantly higher than in winter and spring (P < 0.001). A total of 222 isolates obtained from 77 MPN positive tubes from different milk samples were subjected to PCR. Colonies from 63/77 (82%) MPN positive tubes were taxonomically identified. Among the 63 PCR-positive samples, C. perfringens was the most frequently detected species (56%), followed by C. sporogenes (44%), C. tyrobutyricum (7.9%), C. butyricum (1.6%), and C. beijerinkii (1.6%). In addition, in 11% of the MPN positive tubes, at least two clostridial species were found to be present simultaneously. This work highlights the presence of clostridial spores in ovine milk from central Italy (Tuscany) and led to the identification of some of the clostridia species involved in such high spore loads.

Thermus and the Pink Discoloration Defect in Cheese.

A DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch's postulates when the defect was recreated by the reintroduction of a T.thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin.

Genomic approach to studying nutritional requirements of Clostridium tyrobutyricum and other Clostridia causing late blowing defects

Clostridium tyrobutyricum is the main microorganism responsible for the late blowing defect in hard and semi-hard cheeses, causing considerable economic losses to the cheese industry. Deeper knowledge of the metabolic requirements of this microorganism can lead to the development of more effective control approaches. In this work, the amino acids and B vitamins essential for sustaining the growth of C. tyrobutyricum were investigated using a genomic approach. As the first step, the genomes of four C. tyrobutyricum strains were analyzed for the presence of genes putatively involved in the biosynthesis of amino acids and B vitamins. Metabolic pathways could be reconstructed for all amino acids and B vitamins with the exception of biotin (vitamin B7) and folate (vitamin B9). The biotin pathway was missing the enzyme amino-7-oxononanoate synthase that catalyzes the condensation of pimeloyl-ACP and l-alanine to 8-amino-7-oxononanoate. In the folate pathway, the missing genes were those coding for para-aminobenzoate synthase and aminodeoxychorismate lyase enzymes. These enzymes are responsible for the conversion of chorismate into para-aminobenzoate (PABA). Two C. tyrobutyircum strains whose genome was analyzed in silico as well as other 10 strains isolated from cheese were tested in liquid media to confirm these observations. 11 strains showed growth in a defined liquid medium containing biotin and PABA after 6–8 days of incubation. No strain showed growth when only one or none of these compounds were added, confirming the observations obtained in silico. Furthermore, the genome analysis was extended to genomes of single strains of other Clostridium species potentially causing late blowing, namely Clostridium beijerinckii, Clostridium sporogenes and Clostridium butyricum. Only the biotin biosynthesis pathway was incomplete for C. butyricum and C. beijerincki. In contrast, C. sporogenes showed missing enzymes in biosynthesis pathways of several amino acids as well as biotin, folate, and cobalamin (vitamin B12). These observations agree with the results of growth experiments of these species in liquid media reported in the literature. The results of this study suggest that biotin and folate are potential targets for reducing late blowing in cheese and highlight the usefulness of genomic analysis for identifying essential nutrients in bacteria.

Fast detection and quantification of four dairy propionic acid bacteria in milk samples using real-time quantitative polymerase chain reaction

Propionibacterium freudenreichii is added to vat milk to create the characteristic eyes and typical nutty flavour of Emmentaler Protected Designation of Origin (PDO) cheese, but leads to serious quality defects in other raw milk cheeses from Switzerland. To trace propionic acid bacteria (PAB) in raw milk, we developed and validated a fast quantitative polymerase chain reaction (qPCR)-based method for P. freudenreichii, Propionibacterium thoenii, Propionibacterium jensenii, and Propionibacterium acidipropionici. qPCR-standard curves were linear over five log units down to 101 copies per reaction (R ≥ 0.997); efficiencies ranged from 0.83 to 0.97. In spiking experiments, the lower limits of quantification were 101–102 cfu mL−1 raw milk. Fifty one vat milk samples were analysed with plate count method and qPCR in parallel. Compared with the classic plate count method, the newly developed qPCR method gave faster and species specific determination of four dairy PAB in milk and yielded comparable quantitative results.

Low-sodium Cheddar cheese: Effect of fortification of cheese milk with ultrafiltration retentate and high-hydrostatic pressure treatment of cheese

Low-sodium cheeses often exhibit an acidic flavor due to excessive acid production during the manufacturing and the initial stage of ripening, which is caused by ongoing starter culture activity facilitated by the low salt-in-moisture levels. We proposed that this excessive starter-induced acidity could be prevented by the fortification of cheese milk with ultrafiltration (UF) retentates (to increase curd buffering), and by decreasing microbial activity using the application of high-hydrostatic pressure (HHP) treatment (that is, to reduce residual starter numbers). Camel chymosin was also used as a coagulant to help reduce bitterness development (a common defect in low-sodium cheeses). Three types of low-Na (0.8% NaCl) Cheddar cheeses were manufactured: non-UF fortified, no HHP applied (L-Na); UF-fortified (cheese milk total solids=17.2±0.6%), no HHP applied (L-Na-UF); and UF-fortified, HHP-treated (L-Na-UF-HHP; 500 MPa for 3 min applied at 1 d post-cheese manufacture). Regular salt (2% NaCl) non-UF fortified, non-HHP treated (R-Na) cheese was also manufactured for comparison purposes. Analysis was performed at 4 d, 2 wk, and 1, 3, and 6 mo after cheese manufacture. Cheese functionality during ripening was assessed using texture profile analysis and dynamic low-amplitude oscillatory rheology. Sensory Spectrum and quantitative descriptive analysis was conducted with 9 trained panelists to evaluate texture and flavor attributes using a 15-point scale. At 4 d and 2 wk of ripening, L-Na-UF-HHP cheese had ~2 and ~4.5 log lower starter culture numbers, respectively, than all other cheeses. Retentate fortification of cheese milk and HHP treatment resulted in low-Na cheeses having similar insoluble calcium concentrations and pH values compared with R-Na cheese during ripening. The L-Na-UF cheese exhibited significantly higher hardness values (measured by texture profile analysis) compared with L-Na cheese until 1 mo of ripening; however, after 1 mo, all low-Na cheeses exhibited similar hardness values, which were significantly lower than R-Na cheese. Pressure treatment significantly increased maximum loss tangent (meltability) from rheology testing and decreased melt temperature. Sensory results indicated only very slight bitterness (<2.5 out of 15-point scale) was detected in all cheeses during the 6 mo of ripening. The L-Na-UF-HHP cheese did not significantly differ in bitterness and acidity from R-Na cheese during ripening. Pressures treatment of cheese at 500 MPa and cheese milk retentate fortification could be used to improve the quality of low-Na cheese.

Influence of low pH on the metabolic activity of Lactobacillus buchneri and Lactobacillus parabuchneri strains in Tilsit-type model cheese

Round-shaped and uniformly distributed eyes are important quality features for several Swiss semi-hard cheese varieties such as Tilsit. Recently, the growth of histamine-producing strains of Lactobacillus parabuchneri has been associated with cheese defects, such as crack formation and burning taste. In this paper, the influence of pH on the metabolic activity of added strains of Lactobacillus buchneri and L. parabuchneri, which possess various CO2-producing activities, was studied in Tilsit-type model cheeses. Two different pH values were obtained by modifying the cheese-making process (curd washing and type of ripening). Lactate, free amino acids, free short-chain fatty acids and 1,2-propanediol were determined in the ripened cheeses. In the acidic cheeses (average pH = 5.40), significantly more 1,2-propanediol was produced, presumably from lactate. Lactobacillus parabuchneri FAM 21731, a histamine-producing strain, produced small amounts of 1,2-propanediol (0.2 mmol.kg−1) and high amounts of histamine (3.3 mmol.kg−1). Ornithine was produced by all the studied strains, with the highest amount of 9.0 mmol.kg−1 produced by L. parabuchneri FAM 21835 in the acidic cheeses. Standard cheese making representing the high pH group (curd washed and smear ripened, average pH = 5.70), and the addition of a glutamate decarboxylase-positive L. buchneri, resulted in higher amounts of γ-aminobutyric acid (GABA) (8.1 compared to 3.3 mmol.kg−1 in the control standard cheeses). Irrespective of the strain, the GABA level was much higher in all the acidic cheeses than in the standard cheeses (14.7 compared to 4.6 mmol.kg−1, respectively). The study clearly demonstrates the importance of the cheese pH in the metabolic activity of the added strains during cheese ripening.

Effect of jenny milk addition on the inhibition of late blowing in semihard cheese

The occurrence of late blowing defects in cheese produces negative effects on the quality and commercial value of the product. In this work, we verified whether the addition of raw jenny milk to bulk cow milk reduced the late blowing defects in semihard cheeses. During cheesemaking, different aliquots of jenny milk were poured into 2 groups of 4 vats, each containing a fixed amount of cow milk. A group of cheeses was created by deliberately contaminating the 4 vats with approximately 3 log10 cfu/mL milk of Clostridium tyrobutyricum CLST01. The other 4 vats, which were not contaminated, were used for a second group of cheeses. After 120 d of ripening, some physical, chemical, and microbiological parameters were evaluated on the obtained semihard cheeses. Differences in sensory properties among cheeses belonging to the uncontaminated group were evaluated by 80 regular consumers of cheese. Our results showed that the increasing addition of jenny milk to cow milk led to a reduction of pH and total bacterial count in both cheese groups, as well as C. tyrobutyricum spores that either grew naturally or artificially inoculated. We observed a progressive reduction of the occurrence of late blowing defects in cheese as consequence of the increasing addition of jenny milk during cheese making. Moreover, the addition of jenny milk did not affect the acceptability of the product, as consumers found no difference among cheeses concerning sensorial aspects. In conclusion, the important antimicrobial activity of lysozyme contained in jenny milk has been confirmed in the current research. It is recommend for use as a possible and viable alternative to egg lysozyme for controlling late blowing defects in cheese.

Occurrence of sensory defects in semi-hard ewe’s raw milk cheeses

Sensory quality is one of the most important attributes of cheeses, as consumers demand homogeneous and unaltered products. The presence of sensory defects in the final product causes financial losses and consequently has a great economic impact on cheese makers. Therefore, a study was conducted to find out main defects that affect sensory quality: eyes, paste, rind, flavour, texture and shape of commercial semi-hard ewe’s raw milk cheeses. The frequency of occurrence of relevant sensory defects in cheeses with different ripening times and those manufactured in different seasons was also investigated. Samples were collected along 5 years during the annual sensory quality control from different cheese makers. Although flavour is a major determinant of consumer acceptance, most common defects were those related to the internal appearance, especially cracks, which were more frequent in summer cheeses, and caverns, which were more frequent in spring and winter cheeses. White paste, soft texture and acid flavour defects were more frequent in cheeses with short-ripening period. Medium- and long-ripened cheeses presented a higher percentage of an excess of rind halo with a darker paste colour, animal flavour and marks in the rind. Due to compositional milk changes throughout cheesemaking seasons, cheese makers should adapt their manufacturing practises to those changes and try to achieve uniform cheeses. The results found in this study may be useful for the quality improvement of semi-hard ewe’s raw milk cheeses.

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.