Application of triplex-PCR with an innovative combination of 3 pairs of primers for the detection of milk's animal origin in cheese and yoghurt.

In this research communication we describe an innovative protocol that combines three pairs of primers, two from the literature and one designed in our laboratory, for application in triplex-PCR on somatic cell DNA to enable identification of the species origin (cow, sheep, goat) of cheeses and yogurts with a detection limit of 0.1%. Mislabeling was detected in 15 out of 40 cheeses and in 18 out of 40 yogurts tested. The suggested procedure is a quick and reliable tool for identifying the animal origin of cheeses and yogurts and it can be used to certify product reliability on the domestic and international market. Additionally, in combination with a serological test it can offer a reliable tool for detecting the presence of cow's whey.

Innovación en el desarrollo de queso fundido para lonchear a partir de queso fresco de cabra / Innovation in the Development of Melted Cheese for Slicing from Fresh Goat Cheese

INTRODUCCIÓN: El objetivo principal de este trabajo es desarrollar un derivado lácteo, queso fundido, con unas propiedades sensoriales adecuadas, mediante un proceso tecnológicamente viable para las empresas lácteas artesanales de Andalucía, de forma que se mejore la competitividad de dicho sector, a través de la diversificación de su producción y el aprovechamiento de los recursos. MÉTODOS: Para la elaboración de queso fundido se tomó como materia prima, queso fresco de leche pasteurizada de cabra, de tipo pasta prensada, el cual se molturó y sometió al proceso de fundición. Los métodos analíticos aplicados, son análisis físico - químico, sensorial y estadístico. RESULTADOS: Tras las determinaciones analíticas, se observa que durante el proceso de fundición se incrementa el extracto seco del producto final, así como la disminución del pH por la adición de ácido láctico. En cuanto a los parámetros sensoriales, destacan el 'Olor Mantequilla' y 'Olor Leche Cocida', debido al tratamiento térmico aplicado, así como notas a especias. CONCLUSIONES: Los resultados obtenidos en este trabajo indican la posibilidad, tanto desde un punto de vista tecnológico como sensorial, de desarrollar un queso fundido en bloque a partir de queso fresco de cabra

INTRODUCTION: The main objective of this work is to develop a milk derivative, melted cheese, with sensory properties suitable for consumption, through a technologically viable process for artisanal dairy companies in Andalusia, so as to improve the competitiveness of that sector, through the diversification of its production and the use of productive resources. METHOD: For the elaboration of melted cheese, fresh cheese was made from pasteurized goat milk, pressed type, which was crushed and subjected to the casting process. The analytical methods were physical - chemical, sensory and statistical analysis. RESULTS: After the analytical determinations, it is observed that during the smelting process the dry extract of the final product is increased, as well as the pH by the addition of lactic acid, which is determinant in the structural development. As for the sensory parameters, the 'Odor Butter' and 'Odor Baked Milk' stand out, due to the applied heat treatment, as well as notes to spices. CONCLUSIONS: The results obtained in this work indicate the possibility, both from a technological and sensorial point of view, to develop a block cheese made from fresh goat cheese

δ34 S for tracing the origin of cheese and detecting its authenticity.

Casein δ34 S of 725 samples of cheese from all over the world were measured using IRMS. δ34 S alone made it possible to establish characteristic ranges of values for two types of Italian cheese (Grana Padano PDO and Parmigiano Reggiano PDO) and for the different regions and provinces of both the Grana Padano PDO and the Parmigiano Reggiano PDO zones. Moreover, δ34 S of PDO Italian samples were compared to both Italian (not PDO) and foreign competitive cheese samples. In all the cases, sulfur isotopic ratio analysis was a powerful tool to fix characteristic ranges of values for cheeses coming from different countries and to improve the information given by other isotopic parameters.

Serra da Estrela cheese's free amino acids profiles by UPLC-DAD-MS/MS and their application for cheese origin assessment.

Serra da Estrela cheese is a high-value Portuguese Protected Designation of Origin cheese, produced with raw ewe milk. Thus, information regarding its composition is of utmost relevance for both consumers and certified producers. In this work, the chromatographic profiles of free amino acids in cheeses (45 days of maturation, 6 producers located in 5 municipalities and produced from November 2017 to March 2018) were established by UPLC-DAD-MS/MS. The proposed method allowed detecting 19 free amino acids and cystine with overall limits of detection and quantification lower than 44 µmol/L (1.4 mg/100 g cheese, wet matter) and than 134 µmol/L (4.2 mg/100 g cheese, wet matter), respectively. In all cheeses, 17 free amino acids were quantified including 8 essential amino acids (histidine, leucine-isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) and 9 non-essential amino acids (arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, proline, serine and tyrosine). The amounts of the free amino acids, essential free amino acids, branched chain free amino acids (leucine, isoleucine and valine) plus the free amino acids ratios (mg/g protein) were further used to identify the producer of Serra da Estrela cheeses. Linear discriminant analysis coupled with the simulated annealing variable selection algorithm was used allowing the correct classification of 96% and 90 ±â€¯8% of the samples, for leave-one-out and repeated K-fold cross-validation procedures, respectively. The satisfactory predictive performance pointed out the possibility of using cheeses' amino acids profiles as origin biomarkers for authenticity control, warranting the correctness identification of the cheese producer/brand, which is quite relevant for ensuring the consumer confidence and satisfaction when purchasing this high-value dairy food.

Terpenoid traceability of commercial sheep cheeses produced in mountain and valley farms: From pasture to mature cheeses.

Mountain cheeses are characterized by their strong link to the territory and are testimonial of the cultural heritage. However, there is a lack of traceability and authenticity indicators for most mountain cheeses produced in Europe even though their terroir value is demanded by cheese-makers, consumers and regulatory bodies. The present study investigated the potential and reliability of terpenoids as traceable compounds in Idiazabal PDO cheeses made with raw milk from commercial sheep flocks grazed on lowlands or mountain grasslands. Terpenoids were analysed in individual pasture plants and ripened cheeses. Ingested diets were estimated by microscopic examination of plant residues in sheep faeces, and terpenoid composition of pastures and sheep diets were calculated from isolated plant species analysis. About 100 individual terpenoids were detected in botanical species collected from pastures and 40 compounds in mature cheeses. Important differences were found in the abundance of terpenoids identified in lowland and highland pastures mainly depending on the botanical family and the contribution of each botanical species to both type of grasslands. Estimated sheep diet composition of lowland and highland flocks was different and, in consequence, ingested terpenoids calculated from the estimated diet composition and individual plant terpene analysis were different. The multivariate approach provided robustness in the terpenoid traceability from pasture to cheese selecting individual terpenoids, particularly sesquiterpenoids such as γ-cadinene and aromadendrene, which strongly contributed to discriminate between mountain and valley cheeses. These results can help regulatory bodies to implement effective traceability and authentication procedures to identify mountain cheeses and to protect added-value dairy products.

Traditional Colonial-type cheese from the south of Brazil: A case to support the new Brazilian laws for artisanal cheese production from raw milk.

Artisanal Colonial-type cheese is made from raw milk and is the main cheese produced by rural families of the southern region of Brazil. The aim of this study was to investigate, identify problems, and propose solutions for the current situation of small family farms producing and informally selling artisanal Colonial-type cheese located in the western part of Santa Catarina State in Southern Brazil. A semistructured questionnaire was employed in 12 rural properties to analyze the mode of production. Physical-chemical and microbiological analyses of water, raw milk, and cheese were performed, and it was found that 92, 50, and 100% of the samples, respectively, were outside of the current Brazilian regulatory parameters. None of the cheesemakers involved in this study met the requirements, as established by law, for artisanal cheese production from raw milk. This study concluded that technical support and changes in public policy are needed to ensure the preservation of this artisanal cheese, considering the historical importance and cultural traditions of these local communities and the socioeconomic importance of cheesemaking to family farming. Furthermore, more research on the safety of the cheese produced from raw milk is needed as well as the development of specific microbiological standards for artisanal Brazilian cheeses. Public policies aimed at guaranteeing food safety that formalize the commercialization of these cheeses will increase food security in those communities that currently produce artisanal cheese informally.

Identification of texture parameters influencing commercial cheese matrix disintegration and lipid digestion using an in vitro static digestion model.

Cheese characteristics, such as composition or textural properties, can impact the matrix degradation rate which could modulate the bioaccessibility of fatty acids during digestion. The aim of this study was to identify texture parameters influencing cheese degradation in a gastrointestinal environment. A static in vitro digestion model has been used on nine commercial cheeses: young and aged cheddar, regular and light cream cheese, parmesan, feta, camembert, mozzarella, and sliced processed cheese. At the end of gastric digestion, camembert and mozzarella presented the lowest matrix disintegration whereas aged cheddar, regular and light cream cheeses showed the highest. For all cheeses, the fatty acid release was fast during the first 30 min of duodenal digestion and slowed down afterwards. A partial least square regression revealed that springiness, cohesiveness, and hardness were negatively correlated to the rate of cheese disintegration during gastric digestion. In addition, textural parameters were not correlated with free fatty acid release. By modulating cheese texture, it could be possible to influence matrix disintegration during gastrointestinal digestion which could have an impact on lipids release.

Caracterização sensorial de queijo colonial por perfil flash e analise descritiva quantitativa / Sensory characterization of colonial cheese by flash profile and quantitative descriptive analysis

O queijo colonial é tradicional da região sul, pouco estudado, e com escassez de dados descritivos a seu respeito. Dentro do mercado atual, métodos simples e rápidos para descrição de produtos, como o perfil flash, tem sido demandados, surgindo como alternativa para métodos tradicionais, como a analise descritiva quantitativa. Dessa forma, o presente estudo visou correlacionar o método usual de analise com o de perfil flash para caracterizar sensorialmente queijos coloniais do sudoeste paranaense. Três marcas comerciais de queijo colonial foram avaliadas. O queijo foi caracterizado por cor amarela, características ácidas e sabor salgado principalmente. O método perfil flash mostrou ser eficiente na avaliação do produto, com coeficiente de correlação multivariado RV de 0,989, apresentando simplicidade e rapidez na avaliação.

Caracterização de queijos coalho e utilização de análise multivariada na identificação de origem de queijos produzidos em diferentes mesorregiões da Bahia / Characterization of coalho cheese and use of multivariate analysis in identification of origin of cheese produced in different mesoregions of Bahia

O queijo coalho é um dos produtos mais tradicionais da região Nordeste do Brasil. Considerando a escassez de registros da caracterização dos queijos coalho produzidos na Bahia, objetivou-se com este trabalho caracterizar queijos coalho de diferentes mesorregiões da Bahia como etapa para identificação de origem, utilizando análise multivariada. Umidade, pH, cinzas, gordura e proteína estavam de acordo com a legislação. Queijos obtidos do Extremo-Oeste se diferiram dos produzidos no Sul e Centro-Sul e a maioria dos parâmetros analisados associou-se a altos coeficientes de variação, possivelmente, devido a particularidades regionais e falta de padronização do processamento. Foi possível caracterizar os queijos das mesorregiões analisadas e diferenciá-los com base nas suas identificações de origem. Com a análise multivariada observou-se a separação das amostras do Extremo-Oeste das demais e os parâmetros gordura, umidade, EST e derretimento contribuíram significativamente para tal.

Identificação de queijos Minas Artesanais produzidos na região do Estado de Minas utilizando técnicas multivariadas / Identification of cheeses Hand Mines produced in the region of the State of Mines using multivariate techniques

Minas Gerais conta com sete regiões reconhecidas como produtoras de queijos artesanais, no qual cada região possui uma combinação de características que conferem aos queijos atributos inigualáveis. Objetivou-se identificar queijos Minas Artesanais produzidos nas regiões da Serra da Canastra, Serra do Salitre e Serro utilizando análises multivariadas. Foram adquiridas 15 amostras de queijos da Serra da Canastra, Serra do Salitre e Serro e analisados os teores de umidade, cinzas, gordura, pH, acidez e cor e realizadas análises de componentes principais e análise de agrupamento. Os componentes principais 1 e 2 descreveram 94,63% da variância dos dados. Na análise de agrupamento houve a formação de dois grupos. A aplicação das técnicas multivariadas pode ser uma estratégia na identificação de queijos Minas Artesanais na prevenção de fraudes.

Estimation of somatic cell count levels of hard cheeses using physicochemical composition and artificial neural networks.

This study addresses the prediction of the somatic cell counts of the milk used in the production of sheep cheese using artificial neural networks. To achieve this objective, the neural network was designed using 33 parameters of the physicochemical composition of the cheeses obtained after they have been matured for 12 mo as input data. The physicochemical analysis of the cheeses revealed that the somatic cell count level of the cheese has a significant influence on the amount of protein, fat, dry extract, and fatty acids. When properly set up, the neural network allows the correct classification of the cheeses (100% of correct results in both training and test phases) and therefore their samples in each of the 3 nominal output variables (low, average, and high somatic cell counts). The fatty composition of the cheeses, individual fatty acids, and fat acidity are the variables that most affect the correct operation of the neural network.

Profiling of bacterial and fungal communities of Mexican cheeses by high throughput DNA sequencing.

Cheese is a live food whose preparation involves procedures and microbial communities playing an important role for the final product. We characterized the bacterial and fungal diversity of seventeen different Mexican cheeses by high-throughput DNA sequencing of 16S/18S rDNA libraries. We propose the existence of bacterial and fungal core communities, where at genera level, bacteria include Streptococcus spp., Lactococcus spp., Lactobacillus spp., Aerococcus spp., and Weisella spp. while at species level, the fungal community includes Galactomyces reessii, Scheffersomyces stipitis, Saccharomyces cerevisiae (baker's yeast), and S. cerevisiae_rm11-1a. In addition to the bacterial and fungal core communities, we found members of the cheese microbiota that could be associated to other factors of the cheese manufacturing process. Co-occurrence analysis made in this work, indicates that bacterial and fungal communities maintain positive and negative interactions which are important to shape the resident microbial communities in cheeses. This work is a contribution to the description of the microbial diversity found in some Mexican cheeses.

Untargeted metabolomics reveals differences in chemical fingerprints between PDO and non-PDO Grana Padano cheeses.

The purpose of this preliminary study was to discriminate the chemical fingerprints of Protected Designation of Origin (PDO) Grana Padano cheeses from non-PDO "Grana-type" cheeses by means of an untargeted metabolomic approach based on ultra-high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC/QTOF-MS). Hierarchical cluster analysis and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) allowed discriminating PDO vs. non-PDO cheeses. Lipids (fatty acids and their derivatives, phospholipids and monoacylglycerols), amino acids and oligopeptides, together with plant-derived compounds were the markers having the highest discrimination potential. It can be postulated that Grana Padano value chain, as strictly defined in the PDO production specification rules, can drive the biochemical processes involved in cheese making and ripening in a distinct manner, thus leaving a defined chemical signature on the final product. These preliminary findings provide the basis for further authenticity studies, aiming to protect the designation of origin of PDO Grana Padano cheese by applying a comprehensive foodomics-based approach.

Acquired (dis)liking of natural cheese in different repeated exposure environment.

This study investigated the development of (dis)liking of various types of natural cheese with repeated exposures among Korean young females in two environment settings. Six types of natural cheese with varying flavor and texture characteristics were selected: Brie, Emmental, Gorgonzola, Gouda, Parmigiano-Reggiano and sharp Cheddar. The subjects were randomly divided into two groups (individual evaluation vs. social interaction) and exposed to all six types of cheeses eight times for 4 weeks. The acceptance scores for these cheeses were monitored at the beginning (first taste test) and end (second taste test) of the exposure test as well as 1 month after the completion of exposure test (third taste test). The results showed that the acceptance of cheese was affected by the cheese type and the number of test trials but not by tasting environment. The acceptance levels were higher for Brie, Gouda and sharp Cheddar than for Emmental, Gorgonzola and Parmigiano-Reggiano. When the subjects were exposed to Brie, Emmental, Gouda, and sharp Cheddar repeatedly, their acceptance level increased significantly. This study demonstrated that the liking of natural cheese can occur through repeated exposure to the cheese, but with the outcome varying with the type of cheese.

Measuring temporal liking simultaneously to Temporal Dominance of Sensations in several intakes. An application to Gouda cheeses in 6 Europeans countries.

The idea of having untrained consumers performing Temporal Dominance of Sensations (TDS) and dynamic liking in the same session was recently introduced (Thomas, van der Stelt, Prokop, Lawlor, & Schlich, 2016). In the present study, a variation of the data acquisition protocol was done, aiming to record TDS and liking simultaneously on the same screen in a single session during multiple product intakes. This method, called Simultaneous Temporal Drivers of Liking (S-TDL), was used to describe samples of Gouda cheese in an international experiment. To test this idea, consumers from six European countries (n=667) assessed 4 Gouda cheeses with different ages and fat contents during one sensory evaluation session. Ten sensory attributes and a 9-point hedonic scale were presented simultaneously on the computer screen. While performing TDS, consumers could reassess their liking score as often as they wanted. This new type of sensory data was coded by individual average liking scores while a given attribute was perceived as dominant (Liking While Dominant; LWD). Although significant differences in preference were observed among countries, there were global preferences for a longer dominance of melting, fatty and tender textures. The cheese flavour attribute was the best positive TDL, whereas bitter was a strong negative TDL. A cluster analysis of the 667 consumers identified three significant liking clusters, each with different most and least preferred samples. For the TDL computation by cluster, significant specific TDL were observed. These results showed the importance of overall liking segmentation before TDL analysis to determine which attributes should have a longer dominance duration in order to please specific consumer targets.

Changes in stable isotope ratios in PDO cheese related to the area of production and green forage availability. The case study of Pecorino Siciliano.

RATIONALE: No study has investigated the variations in stable isotope ratios (SIRs) of bioelements within a Protected Designations of Origin (PDO) cheese and few studies have focused on the dietary background of animals. For traceability purposes, it is important to know how and whether these issues affect SIRs in a PDO cheese. METHODS: Thirty-six Pecorino Siciliano cheese samples were collected in three east-Sicilian areas in seasons in which green herbage was present or absent in the diet of the animals. The determination of C, N and S SIRs was performed using an isotope ratio mass spectrometer coupled with an elemental analyser. The H and O SIRs were measured using an isotope ratio mass spectrometer equipped with a thermal conversion elemental analyser pyrolysis unit. RESULTS: The C, N, H, O and S SIRs measured in defatted cheeses were subjected to a multivariate stepwise discriminant analysis to verify if cheeses could be distinguished based on their geographical origin and on the animals' feeding regimen. Sulfur and nitrogen SIRs allowed the best discrimination among the three areas (97.2% correct classification of the cheeses). The discrimination of the feeding system, to check the presence or not of fresh forage in the diet of the animals, correctly classified 86.1% of the cheeses. The C and O SIRs were the most effective parameters. CONCLUSIONS: This study demonstrates that the variability in C, H, O, N and S SIRs can allow discrimination between cheeses produced in a narrow geographical region within a PDO area. This may lead to the search for new tools in authentication studies such as the creation of specific isoscapes. Moreover, this study confirms that SIR determination can also discriminate cheeses obtained from animals fed in stalls from those produced when animals have access to green forage, with benefit in terms of environmental impact, animal welfare and product quality. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneously tracing the geographical origin and presence of bovine milk in Italian water buffalo Mozzarella cheese using MALDI-TOF data of casein signature peptides.

Water buffalo (WB) casein (CN) and curd samples from indigenous Italian and international breeds were examined with the objective of identifying signature peptides that could function as an indicator to determine the origin of their milk products. CN in complex mixtures were digested with trypsin, and peptide fragments were subsequently identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The unique presence of a ß-CN A variant and an internally deleted αs1-CN (f35-42) variant in international WB milk samples was ascertained by identifying signature tryptic peptides from either dephosphorylated or native CN. Four signature unphosphorylated peptides derived from ß-CN A, i.e. (f49-68) Asn(68) (2223.6 Da), (f1-28) Ser(10) (3169.4 Da), (f1-29) Ser(10) (3297.4 Da) and (f33-48) Thr(41) (1982 Da) and two from αs1-CN (f35-42) deleted fragments, i.e. (f23-34) Met(31) (1415.7 Da) and (f43-58) Val(44) (1752.7 Da), were identified. Two signature casein phosphopeptides (CPPs), i.e. ß-CN (f1-28) 4P (3489.1 Da) and ß-CN (f33-48) 1P (2062.0 Da), were identified in the tryptic hydrolysate of native casein or curd and cheese samples using in-batch hydroxyapatite (HA) chromatography. All these fragments functioned as analytical surrogates of two αs1- and ß-casein variants that specifically occur in the milk of international WB breeds. Furthermore, the bovine peptide ß-CN (f1-28) 4P had a distinct and lower molecular mass compared with the WB counterpart and functioned as a species-specific marker for all breeds of WB. Advantages of this analytical approach are that (i) peptides are easier to separate than proteins, (ii) signature peptide probes originating from specific casein variants allow for the targeting of all international WB milk, curd and cheese samples and (iii) bovine and WB casein in mixtures can be simultaneously determined in protected designation of origin (PDO) "Mozzarella di Bufala Campana" cheese. This analytical method enabled the specific detection of international WB and bovine casein with a sensitivity threshold of 2 and 0.78 %, respectively. Graphical Abstract Monitoring of prototypic tryptic CPPs by MALDI-TOF analysis in Mediterranean (A), Romanian (B), Indian (C), Polish (D) and Canadian (E) curd samples to guarantee the authenticity of the PDO "Mozzarella di Bufala Campana" cheese.

Development and validation of an APCI-MS/GC-MS approach for the classification and prediction of Cheddar cheese maturity.

Headspace techniques have been extensively employed in food analysis to measure volatile compounds, which play a central role in the perceived quality of food. In this study atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS), coupled with gas chromatography-mass spectrometry (GC-MS), was used to investigate the complex mix of volatile compounds present in Cheddar cheeses of different maturity, processing and recipes to enable characterisation of the cheeses based on their ripening stages. Partial least squares-linear discriminant analysis (PLS-DA) provided a 70% success rate in correct prediction of the age of the cheeses based on their key headspace volatile profiles. In addition to predicting maturity, the analytical results coupled with chemometrics offered a rapid and detailed profiling of the volatile component of Cheddar cheeses, which could offer a new tool for quality assessment and accelerate product development.

Potential use of scotta, the by-product of the ricotta cheese manufacturing process, for the production of fermented drinks.

In the present work, the use of scotta as substrate for bacterial fermentation was studied with the objective of obtaining a drink from transformation of this by-product. Scotta retains most of the lactose of the milk and it is normally colonized by a natural microbiota. A treatment was devised to reduce the autochthonous microbial populations in order to reduce competition towards the inoculated bacterial strains. Nine lactic acid bacteria (LAB) were assessed for their capability to develop in scotta. They evidenced different behaviors regarding growth rate, acidification capability and nitrogen consumption. A co-inoculum of three LAB, namely a Streptococcus thermophilus, a Lactobacillus delbrueckii subsp. bulgaricus and a Lb. acidophilus strains, chosen among those giving the best performances in single-strain fermentation trials, gave abundant (close to 10(9) cfu/ml) and balanced growth and lowered pH to 4.2, a value similar to that of yogurt. These results show that scotta may have potential as a substrate for bacterial growth for the production of a fermented drink. Further studies are needed to optimize the organoleptic aspects of the final product.

A rapid high-performance liquid chromatography-tandem mass spectrometry assay for unambiguous detection of different milk species employed in cheese manufacturing.

The aim of the study was to investigate the possibility to differentiate the 4 most important species in Italian dairy industry (cow, buffalo, sheep, and goat), applying a bottom-up proteomic approach to assess the milk species involved in cheese production. Selective peptides were detected in milk to use as markers in cheese products. Trypsin-digested milk samples of cow, sheep, goat, and buffalo, analyzed by HPLC-tandem mass spectrometry provided species-specific peptides, some of them recognized by Mascot software (Matrix Science Ltd., Boston, MA) as derived from well-known species specific proteins. A multianalyte multiple reaction monitoring method, built with these specific peptides, was successfully applied to cheeses with different composition, showing high specificity in detection of species involved. Neither aging nor production method seemed to affect the response, demonstrating that chosen peptides well act as species markers for dairy products.