Spanish Cheese Research Articles

Screening Method to Evaluate Amino Acid-Decarboxylase Activity of Bacteria Present in Spanish Artisanal Ripened Cheeses.

A qualitative microplate screening method, using both low nitrogen (LND) and low glucose (LGD) decarboxylase broths, was used to evaluate the biogenic amine (BA) forming capacity of bacteria present in two types of Spanish ripened cheeses, some of them treated by high hydrostatic pressure. BA formation in decarboxylase broths was later confirmed by High Performance Liquid Chromatography (HPLC). An optimal cut off between 10–25 mg/L with a sensitivity of 84% and a specificity of 92% was obtained when detecting putrescine (PU), tyramine (TY) and cadaverine (CA) formation capability, although these broths showed less capacity detecting histamine forming bacteria. TY forming bacteria were the most frequent among the isolated BA forming strains showing a strong production capability (exceeding 100 mg/L), followed by CA and PU formers. Lactococcus, Lactobacillus, Enterococcus and Leuconostoc groups were found as the main TY producers, and some strains were also able to produce diamines at a level above 100 mg/L, and probably ruled the BA formation during ripening. Enterobacteriaceae and Staphylococcus spp., as well as some Bacillus spp. were also identified among the BA forming bacteria isolated.

High Hydrostatic Pressure as a Tool to Reduce Formation of Biogenic Amines in Artisanal Spanish Cheeses.

Two artisanal varieties of cheese made in Spain, one made of ewes’ raw milk and the other of goats’ raw milk were selected to evaluate the effect of a high hydrostatic pressure (HHP) treatment at 400 MPa during 10 min at 2 °C on the formation of biogenic amines (BA). These conditions were applied at the beginning of the ripening (before the 5th day; HHP1) and in the case of ewes’ milk cheeses also after 15th days (HHP15). BA formation was greatly influenced by HHP treatments in both types of cheese. HHP1 treatments significantly reduced the amounts of BA after ripening, being tyramine and putrescine the most affected BA in goats’ milk cheeses and tyramine and cadaverine in ewes’ milk cheeses. The BA reduction in the HHP1 samples could be explained by the significant decrease in microbiological counts, especially in the LAB, enteroccocci and enterobacteria groups at the beginning of ripening. The proteolysis in these samples was also affected reducing the amount of free amino acids. Although proteolysis in ewes’ milk cheeses HHP15 was similar than in control samples a reduction of BA was observed probably because the decrease caused on microbial counts.

Cardoon-based rennets for cheese production.

The use of crude aqueous extracts of Cynara cardunculus flowers as coagulants in the production of high-quality sheep and goat cheeses-as are the cases of several Portuguese and Spanish cheese varieties with Protected Designation of Origin status-has been maintained since ancient times. The unique rheological attributes and sensory properties characteristic of these cheeses have always suggested that this plant coagulant (and, therefore, its isolated milk-clotting proteases) could be used as alternative rennet in the dairy industry, particularly suited for the production of sheep and goat cheeses. However, the lack of standardization of C. cardunculus crude flower extracts, whose quality and performance depends on numerous factors, has always hampered the application of this plant rennet in industrial production scales. To overcome these limitations, and to aim at developing more effective solutions with potential for scalability of production and commercial application, several strategies have been undertaken in more recent years to establish new cardoon-based rennets. This review provides an overview on these developments and on the currently available solutions, which range from producing standardized formulations of native cardoon enzymes, to the optimization of the heterologous production of cardosins and cyprosins to generate synthetic versions of these milk-clotting enzymes. Challenges and emerging opportunities are also discussed.

The influence the serving suggestion displayed on soft cheese packaging has on consumer expectations and willingness to buy

This paper studies the influence that serving suggestions on a popular Spanish soft cheese – known as “queso fresco”—packaging have on consumer expectations regarding certain attributes of the product, its ideal consumption time during the day and the consumer’s willingness to buy. Some 247 people participated in this investigation, evaluating 8 attributes and 5 possible consumption times of 5 different soft cheese packages with different accompaniments displayed on the serving suggestions. The results show that the consumer perceives the soft cheese to have the qualities and ideal consumption time based on the accompaniments shown in the serving suggestion. The study also shows that serving suggestion influences willingness to buy since there is a strong positive relationship between willingness to buy and accompaniments that are healthy and for special diets. These results have important implications for soft cheese producers, marketers, and packaging designers.

Risk Assessment of the Intake of Pb, Cd and Hg by Consumption of Spanish Cheeses

The presence of heavy metals in foods may pose a risk to human health. The main objective of this paper is to carry out a risk assessment study of lead, cadmium and mercury intake by consumption of Spanish cheeses. In general, the concentration levels found were far from the Provisional Tolerable Weekly Intake (PTWI) for these elements except for mercury in Gata-Hurdes cheese indicating a possible contamination by a unknown source. On the other hand, the concept of toxic density is a powerfull tool which can help to initially assess potential risk, focusing on those cheeses exceeding 100% toxic density.

Rheological, textural, colour and sensory characteristics of a Spanish blue cheese (Valdeón cheese)

The aim of this work was to study the rheological, textural, colour and sensory characteristics of Valdeón blue-veined cheese during ripening. Eight batches of cheese were manufactured and 48 cheeses were analysed. All parameters studied were affected by ripening time. 120-day-old cheese was characterized by higher G′, G″ and G* values, showing a more elastic disposition, as well as, lower fracturability, gumminess and chewiness values and higher adhesiveness values. Moreover, as time increased cheeses were characterized by a decrease in L * and a * values. Finally, the sensory attributes that most influenced the perception of Valdeón cheese were: saltiness and pungenty; sour and pungent odours; adhesiveness and granularity; and taste strength.

Simultaneous quantification of trihalomethanes and haloacetic acids in cheese by on-line static headspace gas chromatography–mass spectrometry

Trihalomethanes (THMs) and haloacetic acids (HAAs) are the two most prevalent classes of disinfection by-products (DBPs) that are present in treated water. Four THMs and six HAAs are regulated by several countries in drinking waters but no regulation for these DBPs has been established in foods. THMs are volatile species that can easily be determined by static headspace (SHS)-GC–MS, but HAAs require a derivatisation step to make them suitable for GC due to their polar and hydrophilic nature. This paper describes the first analytical method that performs the simultaneous determination of 10 THMs and 13 HAAs (chlorinated, brominated and iodinated) in cheeses by SHS in one unique GC–MS run. Parameters controlling leaching, centrifugation, derivatisation and volatilisation were optimised taking into account the high volatility of THMs and the thermal instability of HAAs. To increase sensitivity, 3g of cheese was extracted with 10mL of water at pH 4.5–7.7, and after centrifugation the supernatant (∼8mL) was introduced into an HS vial for the derivatisation (HAAs) and volatilisation (HAA esters and THMs) of the species in an automatic SHS unit coupled to GC–MS. Detection limits within the range of 0.05–0.50 and 0.15–0.85μg/kg for THMs and HAAs, respectively, were obtained, and the relative standard deviation was lower than 10% for all the target analytes. Recoveries throughout the whole method were between 85–90% and 92–97% for THMs and HAAs, respectively. The SHS-GC–MS method was applied for the determination of THMs and HAAs in 3 groups of Spanish cheeses, which can be contaminated through contact with treated water during the manufacturing steps. Up to 2 THMs and 4 HAAs were found at μg/kg levels in the samples analysed.

Alternative to decrease cholesterol in sheep milk cheeses

The presence of cholesterol in foods is of nutritional interest because high levels of this molecule in human plasma are associated with an increasing risk of cardiovascular disease and nowadays consumers are demanding healthier products. The goal of this experiment was to diminish the cholesterol content of Manchego, the most popular Spanish cheese manufactured from ewes milk. For this purpose three bulk milks coming from dairy ewe fed with 0 (Control), 3 and 6% of linseed supplement on their diet were used. Nine cheeses (3 per bulk milk) were manufactured and ripened for 3months. Cholesterol of ewes milk cheese from 6% to 12% linseed supplemented diets decreased by 9.6% and 16.1% respectively, therefore supplying a healthier profile. In a second experiment, different sources of unsaturated fatty acids (rich in oleic, linoleic and α-linolenic acids) were supplemented to dairy ewes and no significant differences were found on cheese cholesterol levels.

A comparative biochemical study of two industrially produced short-ripened cow's milk cheeses with PDO status: Rennet-curd Tetilla cheese and acid-curd Cebreiro cheese

The aim of this study was to characterise industrial Tetilla and Cebreiro Protected Designation of Origin (PDO) Spanish cheeses from a biochemical point of view and to relate some of the biochemical parameters to the cheesemaking process. Some differences in the amounts of organic acids and free amino acids between the two types of cheese were attributed to milk characteristics and the processing technology. The most abundant volatile compounds in both types of cheese were ethanol, isopropanol and diacetyl. The simple and nonspecific volatile profiles of the cheeses appear to be related to the commercial mesophilic DL-starters used in the manufacturing process.

Lactose-mediated carbon catabolite repression of putrescine production in dairy Lactococcus lactis is strain dependent

Lactococcus lactis is the lactic acid bacterial (LAB) species most widely used as a primary starter in the dairy industry. However, several strains of L. lactis produce the biogenic amine putrescine via the agmatine deiminase (AGDI) pathway. We previously reported the putrescine biosynthesis pathway in L. lactis subsp. cremoris GE2-14 to be regulated by carbon catabolic repression (CCR) via glucose but not lactose (Linares et al., 2013). The present study shows that both these sugars repress putrescine biosynthesis in L. lactis subsp. lactis T3/33, a strain isolated from a Spanish artisanal cheese. Furthermore, we demonstrated that both glucose and lactose repressed the transcriptional activity of the aguBDAC catabolic genes of the AGDI route. Finally, a screening performed in putrescine-producing dairy L. lactis strains determined that putrescine biosynthesis was repressed by lactose in all the L. lactis subsp. lactis strains tested, but in only one L. lactis subsp. cremoris strain. Given the obvious importance of the lactose-repression in cheese putrescine accumulation, it is advisable to consider the diversity of L. lactis in this sense and characterize consequently the starter cultures to select the safest strains.

Free amino acid profile of Spanish artisanal cheeses: Importance of gamma-aminobutyric acid (GABA) and ornithine content

The content of 18 free amino acids in 34 Spanish artisanal cheeses was measured. The amino acids were determined by high performance liquid chromatography (HPLC) with pre-column derivatization, reversed-phase separation, and UV detection. The total content of free amino acids in the cheese samples ranged from 0.19g/kg to 67.95g/kg. The amount of lactic acid bacteria (LAB), the main microflora in cheese, is also reported. Considerable amounts of free amino acids, such as gamma-aminobutyric acid (GABA; average 330±50mg/kg) and ornithine (Orn; 450±60mg/kg), were found in some samples, thus indicating specific microbial activities. GABA concentrations were not influenced by the kind of ripening or milk origin (p<0.05); however, the concentration of Orn in cheese made from cow's milk was higher (p<0.05) than that found in cheese made from goat's milk, regardless of whether these products were ripened or semi-ripened. These amino acids are considered potentially bioactive because of their various physiological activities which are beneficial for health.

Microbiological, physico-chemical and proteolytic changes in a Spanish blue cheese during ripening (Valdeón cheese)

The aim of this work was to study the microbiological, physico-chemical and proteolytic changes in Valdeón blue-veined cheese during ripening. Eight replicas of cheese were produced and a total of 48 cheeses were analysed. Lactic acid bacteria, mainly lactococci, were the predominant flora during the early stages of ripening, gradually being replaced by moulds and yeasts (8 log units). Enterococci and Enterobacteriaceae counts were very low or zero. This variety was characterised by a total solids content of 61.80g per 100g−1 of cheese, a salt/moisture ratio of 8.92g salt per 100g−1 moisture, a pH of 6.4–7.6 and a water activity of 0.917. At the end of ripening, primary and secondary proteolysis were very high, which resulted in an almost total degradation of αs1- and β-casein (approximately 90%). The peptide profile of the aqueous soluble extracts at pH 4.6 showed great complexity during ripening.

Evaluation of two antimicrobial packaging films against Escherichia coli O157:H7 strains in vitro and during storage of a Spanish ripened sheep cheese (Zamorano)

Abstract The antimicrobial activity of two packaging films (polypropylene – PP- and polyethylene terephthalate – PET-) coated with different concentrations (0, 4, 6 and 8%) of essential oil from Origanum vulgare (OR) and Ethyl Lauroyl Arginate HCl (LAE) was tested against two Escherichia coli O157:H7 strains using in vitro systems and a raw milk sheep cheese model (Zamorano). The influence of the antimicrobials on the sensory attributes of cheese was also evaluated. For both strains, the MBC (minimum bactericidal concentration) values were identical to their respective MIC (minimum inhibitory concentration) values and lower for LAE (25 mg/l) than for OR (200–400 mg/l). PP and PET films coated with OR were tested by a vapour phase assay and the Japanese Standard method (JIS Z 2801:2000). Both films coated with LAE were tested by an agar diffusion method. Overall, in vitro tests were effective against both strains. The inhibitory activity depended on the active compound concentration, the target strain and the packaging material, PET being more effective than PP. For inoculated cheese slices, OR and LAE PP films did not effectively decrease E. coli O157:H7 counts after 7-days cold storage. PET films incorporating 6 and 8% of OR and LAE significantly (p 0.05) affect sensorial characteristics of Zamorano cheese. Packaging with PET films coated with ≥6% LAE concentrations might be useful in reducing E. coli O157:H7 numbers in sheep cheese.

Peptidomic study of Spanish blue cheese (Valdeón) and changes after simulated gastrointestinal digestion.

It is increasingly evident that digestion can affect the biological activity of cheese by the release of new active peptides from their precursors or, on the contrary, giving rise to fragments without activity. The characterization of the peptidome of a Spanish blue cheese, Valdeón, has been conducted before and after gastrointestinal digestion, and the digests have been compared to those obtained from pasteurized skimmed milk powder (SMP) using a bioinformatics platform. Peptidomic profiling of digests revealed several regions that are especially resistant to digestion (among them β-casein 60-93, 128-140, and 193-209). Some of them correspond to well-conserved regions between species (human, cow, sheep, and goat) and include peptide sequences with reported bioactivity. The great peptide homology found between both digests, cheese and SMP, suggests that the gastrointestinal digestion could bring closer the profile of products with different proteolytic state. Although most of the biologically active peptides found in cheese after digestion were also present in SMP digest, there were some exceptions that can be attributed to the absence of the relevant precursor peptide before digestion.

Molecular identification and quantification of tetracycline and erythromycin resistance genes in Spanish and Italian retail cheeses.

Large antibiotic resistance gene pools in the microbiota of foods may ultimately pose a risk for human health. This study reports the identification and quantification of tetracycline- and erythromycin-resistant populations, resistance genes, and gene diversity in traditional Spanish and Italian cheeses, via culturing, conventional PCR, real-time quantitative PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). The numbers of resistant bacteria varied widely among the antibiotics and the different cheese varieties; in some cheeses, all the bacterial populations seemed to be resistant. Up to eight antibiotic resistance genes were sought by gene-specific PCR, six with respect to tetracycline, that is, tet(K), tet(L), tet(M), tet(O), tet(S), and tet(W), and two with respect to erythromycin, that is, erm(B) and erm(F). The most common resistance genes in the analysed cheeses were tet(S), tet(W), tet(M), and erm(B). The copy numbers of these genes, as quantified by qPCR, ranged widely between cheeses (from 4.94 to 10.18log⁡10/g). DGGE analysis revealed distinct banding profiles and two polymorphic nucleotide positions for tet(W)-carrying cheeses, though the similarity of the sequences suggests this tet(W) to have a monophyletic origin. Traditional cheeses would therefore appear to act as reservoirs for large numbers of many types of antibiotic resistance determinants.

Spanish cheese screening and selection of lactic acid bacteria with high gamma-aminobutyric acid production

Gamma-aminobutyric acid (GABA) is a non-protein four-carbon amino acid which is considered a bioactive component known for its physiological functions, including a regulator of blood pressure, neurotransmitter, diuretic and anti-stress effects. Its use in foods might confer health benefits. Microorganisms such as yeast, fungi or bacteria can produce GABA naturally. Among them, the lactic acid bacteria are being studied for the potential development of fermented foods because their physiological activities and their designation of generally recognized as safe (GRAS). The objective of this study was to evaluate the GABA-production capacity in a whole wheat flour medium of lactic acid bacteria strains that showed a high conversion of glutamic acid to GABA in a screening conducted in 58 Spanish artisanal cheeses. Synthesis of GABA by these strains in a non-optimized whole wheat flour in water solution (1:5) was quantified by High-Performance Liquid Chromatography. The 4 strains showing the highest GABA production were genotypically and phenotypically characterized. Results indicated an interesting fermentative variability between strains. The addition of these isolated lactic acid strains in fermented food products could allow a potentially functional food for regulating hypertension.

Safranal transference from ewe's milk to cheese and whey and antifungal properties of fortified whey

Growth of undesirable moulds is one of the major causes of defects in modern cheese production, and it is necessary to discover natural antifungal substances that are non-toxic to humans. Saffron spice has been successfully used as a new ingredient in some Italian or Spanish cheeses, but transference of safranal (the main compound responsible for saffron aroma) during cheesemaking has not yet been studied. In this study, the distribution of safranal in cheese, whey and requeson (Spanish whey cheese) and the antifungal potential of whey fractions against the mould Penicillium verrucosum were studied. To this end, a duplicate fabrication of cheeses was performed with raw ewe's milk with the addition of 40 μg safranal.kg−1 or control milk (no safranal addition). Safranal was more concentrated in cheese and requeson (78.91 and 76.95 μg.kg−1, respectively) than in whey and requeson whey (35.19 and 34.64 μg.kg−1, respectively). These liquid fractions showed good antifungal properties and inhibited mould with a safranal concentration of around 35 μg.kg−1, while a solution of 80 μg.kg−1 was required for a standard safranal solution to obtain a similar inhibition. These results reveal that whey has a synergistic effect with safranal in preventing cheese spoilage by moulds, which encourages making full use of this problematic by-product.

Combined monodimensional chromatographic approaches to monitor the presence of d-amino acids in cheese

The presence of d-amino acids (d-AAs) as a consequence of natural or artificial interventions such as ageing, microorganism action, preservative and conservative processes (alkali or heat treatment), is a scarcely treated aspect from the scientific community. It is also fully documented that even a minor degree of racemisation on the proteins' AAs is the cause of a reduced digestion of such proteins. Besides interfering with the regular metabolism of l-AAs, d-AAs can also contribute to the development of pathological conditions in humans. So far, nearly all the most important chromatographic techniques were applied to quantify d-AAs in foodstuffs. However, most of them rely upon pre- or post-column derivatization procedures, often combined with sophisticated analytical equipments. Differently, in this paper we propose an easy-to-set up combination of monodimensional chromatographic methods to monitor the variation of the d-Ala, d-Asp and d-Glu content in two commercially available Spanish cheese samples prepared from the same milk mixture and characterized by a different maturity time: no ripening and six months ripening. After the free amino acid mixture was extracted from the two cheese samples, an ion-pairing RP-HPLC achiral protocol was firstly optimized with the objective to avail of a method enabling the complete distinction of Ala, Asp, and Glu from all the other aminoacidic species in the two extracts. An ion-exchange-based chromatographic method was also optimized, thus allowing a profitable fractionation of the two aminoacidic mixtures. With such a procedure, less complex samples to be analyzed with a chiral ligand-exchange chromatography (CLEC) stationary phase based on S-trityl-l-cysteine (l-STC) units were obtained.The optimized CLEC conditions were then applied to the previously identified Ala, Asp and Glu containing fractions as well as to those including all the remaining species. For all the three compounds the enantiomeric excess (ee) was found to decrease passing from the ripened to the fresh cheese. As expected, the largest difference was found for Ala (ee value from 83.0% down to 20.5%), followed progressively by Asp (ee value from 90.5 to 75.0%) and Glu (ee value from 99.0 to 91.8%).

Bacterial communities of the traditional raw ewe's milk cheese “Torta del Casar” made without the addition of a starter

The objective of this work was to study the growth and development of fortuitous flora and food pathogens in the traditional Spanish cheese “Torta del Casar”. Sixteen batches of “Torta del Casar” were manufactured from the same unpasteurized Merino ewe's milk, using Cynara cardunculus L. rennets, corresponding to 16 plants, one for each batch. The cheeses were classified based on their texture spreadability analysis data (TSA) into four clusters, from very creamy to not creamy cheeses.Although there were no differences between the counts of different microbial groups in the different clusters of cheese, the microorganism isolated did differ between clusters, especially between the Group 1 cluster (less creamy cheese) and Group 4 (creamiest cheese), with the most notable isolates in the latter being a LAB Lactococcus lactis subsp. cremoris, and the enterobacteria Serratia proteamaculans and Enterococcus devriesei. Pathogen development was similar in all four groups of cheeses. Small differences were found for some pathogens (Listeria spp. and Salmonella spp.) which may have been due to interference with the different autochthonous microbiota. Indeed, the different autochthonous microbiota appears to influence the texture of the cheeses and the development of pathogens in them.

Characterization of functional, safety, and gut survival related characteristics of Lactobacillus strains isolated from farmhouse goat's milk cheeses

A set of 80 Lactobacillus strains (36 Lactobacillus plantarum and 44 Lactobacillus paracasei) isolated from Spanish farmhouse cheeses have been studied as to their functional and safety properties and their survival under gut-related conditions. None of these 80 Lactobacillus strains were able to hydrolyse starch. A high percentage of L. plantarum and L. paracasei strains were, however, capable of hydrolysing casein (86.1% and 68.2% respectively). For the other characteristics investigated, L. plantarum strains generally had more positive responses than L. paracasei. The latter strains tested negative for most of these characteristics, with the exception of stachyose hydrolysis, which was positive in six strains of L. paracasei. A high percentage (91.7%) of L. plantarum produced haemo-dependent catalase. Phytase was present in 10 L. plantarum and in 2 L. paracasei. Most L. plantarum (83.3%) but no L. paracasei hydrolysed bile salts. All strains were completely resistant to a challenge of pH3, but many showed a loss of viability after a subsequent exposure to 0.3% oxgall; in fact, only one L. paracasei strain and 33 L. plantarum strains (91.67%) were tolerant to both stresses. L. plantarum Mb25 and L. plantarum Mb26 were the most adherent to Caco-2 cells (adherence percentages of 36 and 7% respectively). These two strains were also the most adherent to HeLa 229 cells, with 19.3 and 16.0% adhesion respectively. The Mb26 strain inhibited the adhesion of Listeria monocytogenes to Caco-2 cells when added simultaneously to Listeria and also when added 1h before the pathogen (21.0% and 51.6% adhesion inhibition, respectively). Production of H2O2 was detected in 38.9% of L. plantarum strains and in 9.1% of L. paracasei. Twelve L. plantarum and eight L. paracasei strains produced bacteriocin-like inhibitors. PCR amplifications of several plantaricin genes suggest that all the bacteriocinogenic strains may produce plantaricin E/F and some may also manufacture the plantaricin J/K. The nine L. plantarum strains assayed for antibiotic resistance were resistant to ciprofloxacin (MIC>2μg/ml), vancomycin (MIC>16μg/ml), and teicoplanin (MIC>16μg/ml). Moreover, some strains showed intermediate resistance to penicillin, tetracycline, rifampicin, and levofloxacin. We conclude that farmhouse cheeses are good sources of biotechnologically relevant lactobacilli and that the L. plantarum species shows better biotechnological properties than L. paracasei. This can be deduced from the finding of a high percentage of strains of L. plantarum that exhibit remarkable functional and inhibitory properties and high abilities to survive in gut-related conditions, which can be further developed for biotechnological applications.