Cabrales Cheese Research Articles

Prodigiosin-Producing Serratia marcescens as the Causal Agent of a Red Colour Defect in a Blue Cheese.

Technological defects in the organoleptic characteristics of cheese (odour, colour, texture, and flavour) reduce quality and consumer acceptance. A red colour defect in Cabrales cheese (a traditional, blue-veined, Spanish cheese made from raw milk) occurs infrequently but can have a notable economic impact on family-owned, artisanal cheesemaking businesses. This work reports the culture-based determination of Serratia marcescens as the microbe involved in the appearance of red spots on the surface and nearby inner areas of such cheese. Sequencing and analysis of the genome of one S. marcescens isolate, RO1, revealed a cluster of 16 genes involved in the production of prodigiosin, a tripyrrole red pigment. HPLC analysis confirmed the presence of prodigiosin in methanol extracts of S. marcescens RO1 cultures. The same was also observed in extracts from red areas of affected cheeses. The strain showed low survival rates under acidic conditions but was not affected by concentrations of up to 5% NaCl (the usual value for blue cheese). The optimal conditions for prodigiosin production by S. marscescens RO1 on agar plates were 32 °C and aerobic conditions. Prodigiosin has been reported to possess antimicrobial activity, which agrees with the here-observed inhibitory effect of RO1 supernatants on different bacteria, the inhibition of Enterobacteriaceae, and the delayed development of Penicillium roqueforti during cheesemaking. The association between S. marcescens and the red colour defect was strengthened by recreating the fault in experimental cheeses inoculated with RO1. The data gathered in this study point towards the starting milk as the origin of this bacterium in cheese. These findings should help in the development of strategies that minimize the incidence of pigmenting S. marcescens in milk, the red defect the bacterium causes in cheese, and its associated economic losses.

Candida cabralensis sp. nov., a yeast species isolated from traditional Spanish blue-veined Cabrales cheese

Three yeast strains, 1AD8(T), 3AD15 and 3AD23, belonging to a previously unknown yeast species were isolated from two independent batches of the Spanish blue-veined Cabrales cheese, a traditional cheese manufactured without the addition of starter and mould cultures. Physiological characterization revealed that the unknown yeast is not fermentative and does not assimilate lactose; rather it assimilates dl-lactic acid and ethanol, major end products of lactic acid bacteria metabolism in cheese. The novel yeast is anamorphic. Phylogenetic tree reconstruction based on nucleotide sequence comparison of the D1/D2 region of the 26S rRNA gene showed that Pichia terricola and Pichia fermentans are the closest relatives of the unknown species. The name Candida cabralensis sp. nov. is proposed, and the isolate 1AD8(T) (=CECT 13027(T) =CBS 11679(T)) is the type strain of this novel taxon.

Chemical and physical methods for the control of the mite Acarus farris on Cabrales cheese

The astigmatid mite Acarus farris is the main species of mite infesting Cabrales cheese, a traditional Spanish product. This species may reach population density levels of 260 mobile mites/cm 2, resulting in high economic losses. Different chemical and physical methods that were able to control this pest without affecting the organoleptic quality of Cabrales cheese were tested. The efficacy of four short-chain fatty acids (caproic, caprylic, pelargonic and capric) and two sugar alcohols (ribitol and xylitol) were tested under laboratory conditions against mobile stages of A. farris. Only pelargonic acid significantly increased mortality in comparison with controls (63.4 vs. 26.1%, respectively). Additionally, the contact effect of the monoterpene eucalyptol was assessed in cheese maturing rooms at doses of 2.5 and 1.25 μl/cm 2. The first dose effectively controlled A. farris, but its application negatively affected the organoleptic values of the treated cheeses. Finally, the application of the waxy food coating READOM CBR to Cabrales cheeses in maturing rooms, kept the mite population low but negatively modified the external appearance of the cheese, an important quality parameter. The physical approach was based on lowering the usual cheese maturing temperature (15 °C) to 6, 4 or 2 °C. As temperature decreased, A. farris density was drastically reduced from 174 ± 33 mites/cm 2 at the control temperature (15 °C) to 14, 11 and 1 mites/cm 2 for 6, 4 and 2 °C, respectively, though the maturing time was considerably extended. The feasibility of both chemical and physical methods for the control of A. farris is discussed.

Reproduction, longevity and life table parameters of Tyrophagus neiswanderi (Acari: Acaridae) at constant temperatures

The astigmatid mite Tyrophagus neiswanderi Johnston and Bruce is mainly considered a pest of ornamental and horticultural crops. However, this mite has been found infesting Cabrales cheese in Spain, though its population density is low compared to Acarus farris, the prevalent species of astigmatid mite encountered in Cabrales cheese maturing caves. One of the factors that might be influencing this differential abundance is temperature. In the present study the effect of temperature on reproductive parameters and longevity of T. neiswanderi was examined at six constant temperatures, ranging from 10 to 31 degrees C, and a relative humidity of 90 +/- 5%. Preoviposition period, fecundity and daily fecundity were adversely affected by extreme temperatures while the oviposition period increased as temperature was reduced. Male and female longevity increased as temperature decreased, but males showed significantly greater longevity than females. Additionally, this difference was greater as temperature decreased. The effect of temperature on the intrinsic rate of natural increase of T. neiswanderi populations was described by the non-linear Lactin model. The optimum temperature for development was predicted at 26.6 degrees C. At this temperature, the population doubling time is 2.8 days. The lower and upper thresholds for T. neiswanderi populations were calculated at 7.4 and 31.7 degrees C, respectively. According to these results, the influence of temperature on the low population density of this mite found in Cabrales maturing caves compared with A. farris is discussed.

Evaluation of low humidity treatments to control Acarus farris (Acari: Acaridae) in Cabrales cheese

Laboratory experiments were conducted to establish the best combination of relative humidity (r.h.) and time of exposure to be applied on Cabrales cheeses infested with Acarus farris. Laboratory assays revealed that less than 30 h were required to obtain 90% mortality of mites at 50% and 60% r.h. Males were more susceptible than females to low relative humidity, since their lethal period values (LP) were lower than those obtained for females at the same relative humidity. Moreover, the response within sexes to low moisture treatments changed as exposure time increased, since the LP50 obtained for each sex at 50 and 60% r.h. were statistically different whereas the LP90 showed no significant differences within sexes. Accordingly, two modifications of the traditional maturing process were established to assess the efficacy of low moisture treatments to control A. farris on infested cheeses. The first modification consisted of one single exposure of 48 h at 50% r.h. and the second one consisted of two exposures of 48 h at 50% r.h. separated by a time interval of 15 days. No significant differences in final population density were observed for both low moisture treatments compared to control cheeses. Therefore, low humidity treatments are not effective to control A. farris in Cabrales cheese, despite the good results obtained in laboratory assays.

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.

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.

Microbial diversity and succession during the manufacture and ripening of traditional, Spanish, blue-veined Cabrales cheese, as determined by PCR-DGGE

The diversity and dynamics of the dominant microbial communities arising during the manufacture and ripening of four batches of naturally fermented Cabrales cheese were investigated by the PCR-DGGE culture-independent technique. Total microbial DNA was extracted from cheese milk, curd and cheese samples and used as template material in PCR experiments to amplify the V3 region of the bacterial 16S rRNA gene, plus the D1 region of the eukaryotic 26S rRNA gene. These regions were then analysed using DGGE. Eukaryotic and bacterial bands were identified by isolation, reamplification and sequencing. The results were compared to those obtained in a previous microbial characterization of the same four batches using classical culturing methods. Great variability was recorded between batches by the PCR-DGGE technique. This was also shown by culturing, and underlines the uniqueness of artisanal products. Lactocococcus lactis subsp. lactis was dominant from the cheese milk stage until the end of ripening, whereas populations of certain Lactobacillus species appeared during ripening. Populations of species never isolated by culturing were found to be numerous by the PCR-DGGE method, in particular Lactococcus garvieae and Lactococcus raffinolactis. Other, completely unknown lactococci were also detected. The dominant eukaryotic populations from day 15 onwards were those of Penicillium roqueforti and Geotrichum candidum.

PCR Identification of Lysogenic Lactococcus lactis Strains

Lactococcus lactis plays an essential role as a starter in the dairy industry. Unfortunately this species is susceptible to bacteriophage infections that result in fermentation failures and subsequent economic losses. This paper reports a PCR screening method that detects prophages in the genomes of wild lactococcal strains isolated from Cabrales cheese, a traditional, Spanish, blue-veined cheese, which is not inoculated. Approximately, one sixth of the samples tested carried a prophage in the genome. PCR positive strains were treated with mitomycin C to induce the lytic cycle and to determine the viability of the detected prophages. 66.6% of the PCR-positive strains lysed after induction, showing the threat these viruses pose to the dairy industry. The results of this work show that PCR could be routinely used as a rapid screening method for the detection of lysogenic Lactococcus strains.

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.

Development and survival of the cheese mites, Acarus farris and Tyrophagus neiswanderi (Acari: Acaridae), at constant temperatures and 90% relative humidity

Two species of acarid mites, Acarus farris and Tyrophagus neiswanderi, have been identified infesting Cabrales cheese in an Asturian maturing cave, the former being the prevalent species. The developmental rate and survival of immature stages of these mites were examined at constant temperatures, ranging from 7 to 29.7 °C for A. farris, and 10 to 31 °C for T. neiswanderi, and a relative humidity (r.h.) of 90±5%. The larval stage of A. farris was particularly susceptible to low and high temperatures with 81.7% and 95.2% mortality at 7 and 29.7 °C, respectively. Tyrophagus neiswanderi larvae also showed the greatest mortality at extreme temperatures among immature stages, though at a lower level than for A. farris (8.6% and 25.6% at 10 and 31 °C, respectively). The optimal temperature for development appeared to be 27–28 °C for both species and the developmental rates were higher for A. farris than T. neiswanderi within the range of the cooler temperatures prevalent in the cheese-maturing caves. The nonlinear Logan type-III model provided the best fit for the relationship between developmental rates and temperature ( R a 2>0.99) for all immature stages of A. farris, whereas the development of T. neiswanderi was better described by the Lactin model ( R a 2>0.97). The lower and upper developmental threshold temperatures predicted for each stage of A. farris were 3–4 °C lower than those predicted for T. neiswanderi. The differential temperature-development rate for each species might explain the greater abundance of A. farris compared to T. neiswanderi. Furthermore, manipulation of temperature based on modeling predictions may well be used to control mite populations during the cheese maturing process.

Microbial, chemical and sensorial variables of the Spanish traditional blue-veined Cabrales cheese, as affected by inoculation with commercial Penicillium roqueforti spores

This paper reports the technological effects of inoculating Cabrales cheese (a traditional, Spanish, blue-veined cheese) with Penicillium roqueforti spores. Three batches of inoculated Cabrales cheese were manufactured and a number of their microbial and biochemical variables recorded. The results were compared with those obtained for three batches of control cheese made using traditional technology (i.e., adding neither starter cultures nor fungal spores). Although mould and yeast populations grew more quickly in the inoculated cheeses, their normally dominant and representative microbial populations were not affected neither was their gross biochemical composition changed. The variations observed were thought to be caused by the uncontrolled environmental conditions of manufacture and ripening. The development of free amino acids and volatile compounds was significantly increased at 30 days in the inoculated cheeses, although the values for both types of cheese were almost identical at 90 days. The inoculated cheeses obtained higher scores in a hedonistic sensorial evaluation. Thus, inoculation improved the standardization and quality of the cheese.

Biochemical and microbiological characterization of artisan kid rennet extracts used for Cabrales cheese manufacture

Four samples of artisanal kid rennet extracts from four different Cabrales cheese producers were biochemically and microbiologically characterized. Most samples had a very acidic pH (around pH 4.0), which may condition their biochemical and microbial variables. The milk-clotting activity of the extracts was assessed using a Formagraph apparatus. The properties of these artisanal rennets were found to be comparable to a 1/10 dilution of 1:10,000 commercial calf rennet; their enzymatic potentials, measured with a semi-quantitative commercial system (API ZYM), showed only very slight differences. A large population of lactobacilli was found in all artisanal kid rennet samples, whereas coliforms, enterococci, staphylococci and leuconostocs were only occasionally encountered. Sixty-four representative colonies were classified by PCR amplification and the sequencing of a stretch of their 16S rDNA genes. Strains of Lactobacillus plantarum completely dominated one of the extracts. In all others samples, strains of this homofermentative species and of the heterofermentative Lactobacillus brevis were present in similar amounts.

Antimicrobial susceptibility of lactic acid bacteria isolated from a cheese environment

In the production of the Spanish traditional blue-veined Cabrales cheese, lactic acid bacteria strains free of antibiotic resistance that have a transferrable capacity are necessary as components of a specific starter. To select for these bacteria, the minimum inhibitory concentration (MIC) of 12 antibiotics and 2 mixtures (containing beta-lactamase inhibitor and penicillin) were determined by microbroth and agar dilution techniques in 146 strains belonging to the genera Lactococcus, Enterococcus, Lactobacillus, and Leuconostoc. The antibiotic-resistance profiles of Lactococcus and Enterococcus species were different from those of Lactobacillus and Leuconostoc, but clear genus- or species-associated patterns were not observed. Cefoxitin and metronidazole were not effective against bacteria of these genera. The MICs of beta-lactam antibiotics for lactobacilli and leuconostoc isolates were higher than those for lactococci and enterococci, but no strain was clinically resistant. All lactobacilli and leuconostoc isolates were resistant to high levels of vancomycin, a type of resistance not seen among the tested members of the genera Lactococcus and Enterococcus. The majority of the observed resistance appeared to be either intrinsic or nonspecific, although some strains of Lactococcus lactis, Enterococcus spp., and Lactobacillus spp. were resistant to antibiotics, such as chloramphenicol, erythromycin, clindamycin, or tetracycline.

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).

Thermogravimetry (TG) applied to the analysis of Cabrales cheese

TG was used in the study of Cabrales cheese in order to obtain information on the changes linked to a weight loss in the substance. In the same way the water activity in the samples was obtained.

Proteolysis of Cabrales cheese and other European blue vein cheese varieties

SummaryThe extent and level of proteolysis were evaluated in Cabrales and five other European blue vein cheese varieties, Bleu de Bresse, Danablu, Edelpilzkäse, Gorgonzola and Roquefort, by analysing their different N-containing constituents and determining the degree of casein hydrolysis. The electrophoretic patterns of the caseins in the cheeses confirmed their strong proteolysis, components featuring the lowest electrophoretic mobility offering the greatest resistance to subsequent hydrolysis. Component αs1 — I was detected in all varieties studied with the exception of Roquefort cheese.

Overall composition, nitrogen fractions and fat characteristics of Cabrales cheese during ripening

Changes in the global composition, textural parameters, nitrogen fractions, fats characteristics and casein breakdown in Cabrales cheese were studied over a maturation period of four months. Extensive proteolysis and lipolysis occurred. Approximately 88 % of the total nitrogen in the mature cheese was water-soluble and nearly 79% of the water-soluble nitrogen was non-protein nitrogen. Degradation of αs- ands-caseins was complete at the end of ripening, and at this stage the total free fatty acid (FFA) content was 33 153 mg/kg. Correlation coefficients were calculated for all variables and stepwise discriminant analysis was applied in order to classify Cabrales cheeses according to ripening time.

Effects of changes during ripening and frozen storage on the physicochemical and sensory characteristics of Cabrales cheeses

SummaryThe physicochemical and organoleptic characteristics of two batches of Cabrales cheese, stored at −20°C for 4 and 8 months, respectively, were studied during subsequent ripening. Frozen storage did not result in significant alterations in overall compositional, rheological and sensory properties or the level of lipolysis. The extent of proteolysis was slightly lower in the cheeses frozen prior to ripening.