Spanish Cheese Research Articles

Occurrence of Shiga toxin-producing Escherichia coli in a Spanish raw ewe's milk cheese

The aim of the present study was to investigate the occurrence of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in ‘Castellano’ cheese, a non-cooked and hard or semi-hard Spanish cheese made from ewe's milk. A total of 83 raw milk cheese samples with different ripening times (2.5, 6 and 12 months) were taken at 30 cheese factories. Samples were examined for the presence of STEC using in the first stage the Association of Official Analytical Chemists (AOAC) official method number 997.11, and then, in the second stage, isolates were tested for virulence genes using genotypic (PCR) methods. Three STEC strains were detected in two samples (2.4%) of ‘Castellano’ cheese, one with 2.5 and the other one with 12 month-ripening period. From those STEC isolates, two were identified as E. coli O14 and the third presented an O-specific polysaccharide not-groupable serologically (ONG). PCR showed that all isolates were characterized by harbouring the Shiga toxin ( stx) stx 1 gene and by the absence of the genes for stx 2, eaeA, and ehxA virulence factors. This study revealed the potential of STEC to survive in long-ripened-hard cheeses.

Phenotypic and molecular identification of yeast species associated with Spanish blue-veined Cabrales cheese

Classification of 74 yeasts isolated during manufacture and ripening of Cabrales cheese was attemped by phenotypic and molecular methods. Phenotypic classification included the use of commercial kits and classical biochemical analyses. Molecular classification was performed by restriction fragment length polymorphism (RFLP) of the ITS1-5.8S-ITS2 region, sequencing and comparison of the sequences in databases. Forty-one isolates were identified by phenotypic tests, including strains of Debaryomyces hansenii (14 isolates) , Kluyveromyces lactis (11 isolates) and Pichia fermentans (9 isolates). The identification was confirmed by the HaeIII and HinfI RFLP profiles and comparison of their corresponding nucleotide sequences. The phenotypic classification failed to identify many other species; 16 isolates assigned to Zygosaccharomyces were genetically related to Pichia (13 isolates) and Saccharomyces (3 isolates). Classification of a few isolates was not possible, either because yeast species are new or a lack of sequences for this region in databases.

Application of SAFES methodology in Manchego-type cheese manufacture

Manchego-type cheese is one of the most popular spanish cheeses. It is a food made up basically of an insoluble matrix, fat and sodium chloride dissolution. The insoluble matrix is mainly constituted by protein (casein) and the NaCl present in dissolution form is consequence of salting operation. Salting was carried out with saturated brine at 10 °C of temperature during 36 h.The aim of salting is to reduce product water activity ( a w), although specific role of salt ions has been described, such as their influence on determined enzyme activity responsible for cheese ripening. Ripening, at 10 °C of temperature and 80–85% of relative humidity during three months, is the other main step in the elaboration process of this type of cheese; during this phase, physical and chemical changes, as well as complex enzymatic reactions give place to certain organoleptic characteristics which are most appreciated by the consumer. SAFES (Systematic Approach to Food Engineering Systems) methodology has been applied to Manchego-type cheese process. In this sense, salting and ripening at 30 and 90 days have been considered as critical points due to the appearance or disappearance of certain components and phases, as water, salt, soluble nitrogen, proteins in solid phase and others, that contribute to the quality of the final product.

Conjugated linoleic acid content and isomer distribution during ripening in three varieties of cheeses protected with designation of origin

Cheeses have been identified as important sources of conjugated linoleic acid (CLA), a mixture of positional and geometric isomers with potential anticarcinogenic activity and other beneficial properties. The objectives of this study were to examine the effects of ripening on the overall CLA content as well as on the isomers profile using GC and Ag +-HPLC. Three Spanish cheeses Protected with Designation of Origin (Mahón, Manchego and Cabrales) were manufactured in different cheesemaking plants and monitored at different times during the ripening period. Total CLA content varied from 3 to 9 mg/g of total fatty acids and rumenic acid (9- cis, 11- trans C18:2, RA) represented more than 75% of total CLA. After RA, 7–9 ( cis/ trans plus trans/ cis), 11- trans, 13- trans and 11- trans, 13- cis C18:2 were the main CLA isomers. The results achieved confirm that the effect of ripening on the total CLA concentration and isomer distribution was negligible.

Hygienic quality, lipolysis and sensory properties of Spanish Protected Designation of Origin ewe’s milk cheeses manufactured with lamb rennet paste

A lamb rennet paste preparation was studied to determine its suitability for the manu- facture of the three main Spanish raw ewe's milk cheeses with Protected Designation of Origin (Manchego, Idiazabal and Zamorano) from the hygienic and sensory points of view. Cheeses were manufactured with lamb rennet paste or with commercial rennets (bovine powder rennet for Man- chego cheese and liquid lamb rennet for Zamorano and Idiazabal cheeses) at identical levels of coa- gulating strength. Free fatty acids and partial glycerides were also analysed to study lipolysis levels and to relate them to sensory properties. No statistically significant differences were detected in microbiological counts for any of the species analysed between cheeses manufactured with lamb rennet paste and cheeses manufactured with non-paste commercial rennet. We conclude that the hygienic quality of the cheeses made in this study with lamb rennet paste was comparable with that of cheeses made with non-paste commercial rennet. Cheeses made with lamb rennet paste showed a higher lipolysis level than cheeses made with non-paste commercial rennet. Sensory differences between cheeses made with either type of rennet were mainly detected in Idiazabal cheese, whereas the sensory profile of the cheeses made with lamb rennet paste or with non-paste commercial rennet was rather similar for Manchego and Zamorano cheeses. Idiazabal cheeses manufactured with lamb rennet paste showed the highest scores for odour and flavour intensities, sharp odour, rennet odour and flavour, butyric flavour and pungent character. On the other hand, Idiazabal cheeses made with non-paste commercial rennet had higher scores for milk and buttery odour attributes than cheeses made with lamb rennet paste. lamb rennet paste / cheese / ewe's milk / lipolysis / hygienic quality / sensory properties

Morphotypic and molecular identification of filamentous fungi from Spanish blue-veined Cabrales cheese, and typing of Penicillium roqueforti and Geotrichum candidum isolates

Thirty five white and blue-greenish filamentous fungi, dominant during ripening of Cabrales cheese, were morphotypically and genotypically identified. Morphotypic analysis identified Penicillium spp. (12 isolates), Penicillium roqueforti (11 isolates), Geotrichum candidum (10 isolates), Acremonium charticola (1 isolate), and one isolate that could not be identified. Polymerase chain reaction (PCR) amplicons of their ITS1-5.8S-ITS2 region were subjected to Restriction fragment length polymorphism (RFLP) analysis with six restriction enzymes. Strains morphotypically identified as P. roqueforti all showed identical RFLP profiles with all enzymes, as did all the G. candidum strains; confirming their phenotypic classification. However, molecular analysis showed the Penicillium spp. group to be composed of P. commune (5 isolates) , P. chrysogenum (4 isolates), P. roqueforti (2 isolates), and P. citreonigrum (1 isolate). The genetic diversity found among the different P. roqueforti and G. candidum isolates was assessed by Rapid amplified polymeric DNA-polymerase chain reaction (RAPD-PCR) with two primers. Nine P. roqueforti strains were defined among the 13 isolates, and every G. candidum isolate represented an individual strain.

Identification of ACE-inhibitory peptides in different Spanish cheeses by tandem mass spectrometry

Different Spanish cheeses (Cabrales, Idiazabal, Roncal, Manchego, Mahon and a goat's milk cheese), made with diverse technologies and milk from different species, were studied for their angiotensin converting enzyme (ACE)-inhibitory activity. Among the water-soluble extract WSE<1000-Da of the different samples, Cabrales cheese was the most active with an inhibitory activity of 76.1%. On the contrary, Mahon cheese showed the lowest ACE-inhibitory index (56.6%). In order to identify the peptides involved in this activity, the WSE<1000-Da were analyzed by HPLC-MS/MS and off-line MS/MS. A total of 41 major peptides were identified in the different cheeses. Most of them derived from β-and αs1-casein. Several of these peptides were selected on the basis of the presence of proline as the C-terminal amino acid, and they were chemically synthesized. All peptides showed moderate or low ACE-inhibitory activity. Peptide DKIHP [β-CN f(47–51)], a sequence detected in all samples except Mahon cheese, showed the highest inhibitory activity with an IC50 value of 113.1 μM.

PCR–DGGE as a tool for characterizing dominant microbial populations in the Spanish blue-veined Cabrales cheese

The microbial populations of cheese milk and rennet extracts used in the production of traditional, Spanish, blue-veined Cabrales cheese were identified by PCR–DGGE analysis of the V3 region of the bacterial 16S rRNA gene and of the D1 region of the eukaryotic 26S rRNA genes. Ripe cheeses (60 days old) were examined in the same way. The results obtained by this culture-independent technique were compared to others previously obtained by conventional culturing methods. Rennet extracts were dominated by a number of Lactobacillus species, including Lb. plantarum, a non-starter lactic acid bacterium dominant during ripening. Lactococcus lactis was only found in one rennet extract. The cheese milk was clearly dominated by Lactococcus-like bacteria, with Lc. lactis in the greatest number. This bacterium was also dominant in the cheese samples (on both the surface and in the interior), in agreement with results obtained by culturing. The sequences of several bacterial DGGE bands from all samples showed less than 97% homology to known, cultured species. This indicates that unknown species are present in the Cabrales cheese environment and that culture-independent methods are needed to fully characterize this ecosystem.

Seasonal variation of the free fatty acids contents of Spanish ovine milk cheeses protected by a designation of origin: A comparative study

The extent and pattern of lipolysis in Spanish PDO raw ewes’ milk cheeses, Manchego, Zamorano and La Serena, were studied. Semi-cured and cured cheeses were analysed. Significant differences were found among the PDO cheeses in all free fatty acid (FFA) concentrations, except for the medium chain fatty acids (MCFAs; C10:0 to C14:0). All individual FFA concentrations increased significantly with ripening time. Seasonality was also significant: the highest concentrations of total FFAs were found in cured cheeses manufactured in spring. Ratios of short, medium and long chain fatty acids to total FFAs in the different cheeses were indicative of the types of cheeses and of the season in which they were made. Discriminant analysis classified correctly 84% of Manchego cheeses, 93% of La Serena cheeses and 92% of Zamorano cheeses in the respective variety. All of the cured Manchego and Zamorano cheeses were correctly classified for the season of manufacture.

Free fatty acid composition of regionally-produced Spanish goat cheese and relationship with sensory characteristics

A study was made of free fatty acid composition and sensory characteristics (odour and taste) in regionally-produced Spanish goat cheeses. The most abundant FFAs were oleic, palmitic, stearic, capric and myristic acid which together accounted for roughly 85% of total FFAs. These cheeses generally underwent a lower degree of lipolysis than did other goat cheeses. Panellists judged the cheeses as having considerable odour and flavour intensity. However, both total FFA content and sensory attributes varied considerably among cheeses due, in all likelihood, to differences in ripening time and to production by different manufacturers. Principal component analysis generated three principal components (PC) that accounted for 70% of total variance; the variables that best correlated with them were long-chain and medium-chain free fatty acids (PC1), brine odour, bitterness and goat milk odour (PC2) and short-chain free fatty acids (PC3).

Phenotypic and genotypic characterization of lactobacilli isolated from Spanish goat cheeses

Lactic acid bacteria from 18 Spanish goat cheeses produced by seven dairies were isolated to evaluate the genetic diversity of this bacterial community. 136 representative isolates were characterized by phenotyping and Randomly Amplified Polymorphic DNA (RAPD-PCR) analysis. Ten species were identified with predominance of Lactobacillus paracasei subsp. paracasei. The presence of L. curvatus, L. pentosus, L. cellobiosus and L. rhamnosus has not hitherto been reported in Spanish goat cheeses. A high degree of genetic diversity was found for L. paracasei subsp. paracasei, L. curvatus and L. plantarum. Some of the identified strains displayed strong acidifying and proteolytic capacities.

Purification of cynarases from artichoke ( Cynara scolymus L.): enzymatic properties of cynarase A

Aspartic proteinases from flowers of Cynara cardunculus have been extensively studied and long used as coagulants in the manufacture of several traditional Spanish and Portuguese cheeses. These endopeptidases are called cardosins or cynarases, depending on the authors. However, the proteinases of another plant of the genus Cynara, the artichoke ( Cynara scolymus), are less known, probably because the flower of this plant is usually consumed as a vegetable. In the study described here, three proteinases (cynarases A, B and C) with milk-clotting properties were purified from the stigma of artichoke. All three proteinases are glycoproteins and composed of a one large and one small subunit. The enzymatic properties of cynarase A, a glycoprotein containing N-linked high mannose type glycans, which express maximum activity at pH 5.0 and 70 °C, were studied in detail. Catalytic and inhibition studies indicated that this cynarase is of the aspartic acid type. The results indicate artichoke extract could also be used in the milk industry in the same way as the extract obtained from the flower of C. cardunculus.

Comparison of the volatile composition and sensory characteristics of Spanish PDO cheeses manufactured from ewes’ raw milk and animal rennet

Abstract Idiazabal, Manchego, Roncal and Zamorano varieties are the Spanish cheeses manufactured under Protected Denomination of Origin (PDO) from ewes’ raw milk and coagulated with animal rennet. Two batches of each cheese variety were ripened for six months and the cheeses were compared for sensory characteristics and composition of volatile compounds. Seventy-six volatile compounds were identified by GC-MS analysis of headspaces of the cheeses. The volatile profile of the four PDO cheeses differed significantly. Acids were the most abundant volatile compounds in Idiazabal, Roncal, and Zamorano cheeses, whereas alcohols were the main volatile compounds in Manchego cheese due to the large percentage of 2-butanol. Aldehydes, ketones and esters were minor compounds in all the cheese varieties, whereas terpenes and unsaturated hydrocarbons were only found in Manchego cheese. The sensory profiles, of the four PDO cheeses also differed significantly. The highest scores for sharp, brine and rennet odours, and rancid and rennet flavours were assessed in Idiazabal and Zamorano cheeses, whereas milky and buttery odour scores were higher in Manchego, Roncal and Zamorano cheeses than in Idiazabal cheese. Principal component analysis was applied to sensory attributes and volatile composition of the cheeses. Differences in flavour and odour attributes were correlated with differences in the volatile compounds. Two principal components correlating sensory attributes and volatile compounds were defined as “strength factor” and “sweetness factor”. The “strength factor” distinguished among the four cheese varieties, except between Roncal and Manchego cheeses, and the “sweetness factor” distinguished the Zamorano cheese from the Idiazabal, Manchego and Roncal cheeses.

Application of multielement stable isotope ratio analysis to the characterization of French, italian, and spanish cheeses.

The stable isotope ratios (delta13C, delta15N, and delta34S of casein and delta13C and delta18O of glycerol) measured by IRMS of French, Italian, and Spanish cheeses are presented and discussed. Variability factors such as animal-feeding regimen, geographical origin, and climatic and seasonal conditions were studied to check the possibilities of cheese characterization offered by each isotopic parameter. Delta13C values of both casein and glycerol appeared to be strongly correlated to the amount of maize in the animal diet. Delta15N and delta34S of casein proved to be mostly influenced by the geoclimatic conditions of the area (aridity, closeness to the sea, altitude). Delta18O of glycerol was more dependent on the geographical origin of the cheeses and on climatic/seasonal parameters. By applying a multivariate stepwise canonical discriminant analysis, good discrimination possibilities for the different European cheeses were obtained, confirmed by the classification analysis, when >90% of the samples were correctly reclassified.

Growth and metabolic behaviour of Lactococcus lactis subsp. lactis IPLA�947 in anaerobic lactose-limited chemostat cultures

Lactococcus lactis subsp. lactis IPLA 947 is a starter strain isolated from a Spanish farmhouse acid-coagulated cheese. To use this strain as starter culture in Afuega’l Pitu cheese manufacture, anaerobic lactose-limited chemostat cultures were used to optimise the active biomass production in a simple medium (BRFS). Growth, lactose consumption and metabolite production were measured at different pH values (5.5, 6.0 and 6.5) and dilution rates (0.06 to 0.96 h−1). The influence of both variables on the quoted parameters was described by means of mathematical models. These provided a satisfactory representation of the experimental data. The bacterial response surface showed a significant increase in the biomass when the pH increased and the dilution rate decreased. As dilution rate increased, biomass yield increased but only at the lowest pH. The lactose consumption and the ratio of metabolic end products were also influenced by both variables. As dilution rate decreased, the residual lactose decreased and increased amounts of metabolites other than lactate (acetate, formate, ethanol) were produced. The minimum lactose consumption was obtained at pH 5.5 and 0.96 h−1 dilution rate. A significant rise of acetate and formate levels occurred as pH increased. A maximum lactate level, along with an acetate, formate and ethanol production close to the minimum, was predicted by mathematical modelling at 0.5173 h−1 dilution rate and pH 6.054.

Development of Lactobacillus plantarum LL441 and its plasmid-cured derivatives in cheese.

A wild Lactobacillus plantarum strain and two of its plasmid-cured derivatives were separately used as adjunct cultures in the manufacture of a Gouda-like traditional Spanish cheese. The wild strain, LL441, harbours seven plasmids and produces a lantibiotic-like bacteriocin. The LL441-B2 derivative has lost plasmids of 40 and 80 kb and the bacteriocin-producing capability. The LL441-B11 derivative has lost in addition a 70 kb plasmid encoding active alpha- and beta-galactosidases. All three strains could be used as adjunct cultures as none of the technological and biochemical parameters of the cheeses was affected. Both the wild-type and the two derivatives were recovered from experimental cheeses up to 30 days after manufacture at similar rates of nearly 20%. Thus, the phenotypic traits under examination were not essential for L. plantarum to grow into the cheese matrix.

Proteolysis in rennet-coagulated spanish hard cheeses made from milk preserved by refrigeration and addition of carbon dioxide.

On-farm refrigeration of milk reduces the growth rate of mesophilic bacteria so allowing longer storage time of raw milk before processing. However, refrigeration creates a selective pressure favouring the multiplication of psychrotrophic bacteria present as normal contaminants in raw milk. Psychrotrophs are killed by most of the currently employed pasteurization and sterilization treatments of milk but they can produce extracellular heat-stable proteinases and lipases, which are capable of degrading various milk components, affecting the storage-life of heat-processed milk and the quality of dairy products (Champagne et al. 1994; Shah, 1994).

Biochemical changes throughout the ripening of a traditional Spanish goat cheese variety (Babia-Laciana)

The physico-chemical characteristics, proteolysis (classical nitrogen fractions, caseins and their degradation products and free amino acids), and lipolysis (fat acidity and free fatty acids) were studied throughout the ripening of three batches of Babia-Laciana cheese, a Spanish traditional variety made from raw goats’ milk. The main compositional characteristics of this cheese at the end of the ripening are its high content of total solids (TS) (78.0±2.4 g 100 g−1 of cheese) and fat (61.1±1.2 g 100 g−1 of TS), the presence of residual lactose (1.6±0.8 g 100 g−1 of TS) and its low content of sodium chloride (1.1±0.7 g 100 g−1 of TS) and ash (2.8±0.5 g 100 g−1 of TS). Its pH values (4.44±0.72) are extraordinarily low. The evolution and final values of the different nitrogen fractions show that this cheese undergoes a very slight proteolysis, a fact which was corroborated when the caseins and their degradation products were quantified: β-casein did not undergo any modification throughout ripening, while only 21% of the αs-caseins were degraded. Free amino acids content increased by a factor of about 7 throughout ripening, resulting in a high content of γ-amino butyric acid and a low content of glutamic acid at the end of the process. Fat acidity increased very slightly, approximately 4.5 times, during ripening, reaching final values of 3.5±2.2 mg KOH g−1 of fat. The total free fatty acids content showed a similar evolution to fat acidity. At the end of the ripening process, the main free fatty acid was C18:1, followed by C16 and C10.

Composition of the Water-Soluble Fraction of Different Cheeses

Volatile and nonvolatile compounds present in the water-soluble fraction (WSF) and water-soluble fraction with molecular weight lower than 1000 Da (WSF < 1000 Da) of six Spanish cheeses, Cabrales, Idiazábal, Mahón, Manchego, Roncal, and a goat's milk cheese, were analyzed. Different nitrogen fractions (determined by Kjeldahl method), caseins (by capillary electrophoresis), peptides and amino acids (by HPLC), and volatile components (by dynamic headspace coupled to GC-MS) as well as mineral content in the cheese fractions were analyzed and compared. The different nitrogen and volatile compounds identified in the WSF were characteristic of each cheese variety. Cabrales cheese displayed the highest content of free amino acids and the highest quantity and variety of volatile compounds. The WSF < 1000 Da fraction was less representative, especially for volatile compounds, as some of the components were lost in the ultrafiltration. Alcohols were better recovered than ketones and esters.

Histamine and tyramine-forming microorganisms in Spanish traditional cheeses

Histamine and tyramine-forming micro-organisms were studied in 44 samples of different varieties of Spanish cheeses. Isolates (694) were obtained and tested for histidine and tyrosine-decarboxylase activity. Tyramine-forming activity was more frequent in the microorganisms isolated from most of the selective culture media except in those for enterobacterias and moulds. Tyramine-forming identified isolates were mostly Gram positive bacteria (enterococci and some lactic acid bacteria), whereas histamine was formed mainly by enterobacteria, but by some lactic acid bacteria as well. Most of the tyramine-forming lactic acid bacteria strains were isolated from cheeses containing the highest tyramine amounts. Some of these cheeses also showed the highest counts of enterococci. However, histamine-forming lactic acid bacteria were isolated from samples generally containing less than 100 mg/kg of histamine. The amount of tyramine found in the samples was significantly higher than histamine. One sample of Cabrales cheese (a mould-matured cheese) presented the highest amounts of both histamine (928 mg/kg) and tyramine (1807 mg/kg).