The diversity of dominant lactic acid bacteria population in 12 months ripened Parmigiano Reggiano cheeses was investigated by a polyphasic approach including culture-dependent and independent ...methods. Traditional plating, isolation of LAB and identification by 16S rDNA analysis showed that strains belonging to
Lactobacillus casei group were the most frequently isolated.
Lactobacillus helveticus,
Lactobacillus delbrueckii subsp.
lactis,
Lactobacillus parabuchneri, and
Lactobacillus buchneri species were detected with lower frequency. PCR-denaturing gradient gel electrophoresis (DGGE) applied to DNA extracted directly from cheese samples and sequencing of rDNA amplicons confirmed the complex microbiological pattern of LAB in ripened Parmigiano Reggiano cheeses, with the significant exception of the
Lactobacillus fermentum species, which dominated in several samples, but was not detected by cultivation. The present combination of different approaches can effectively describe the lactic acid bacteria population of Parmigiano Reggiano cheese in advanced stages of ripening, giving useful information for elucidating the role of LAB in determining the final cheese quality.
From each of twenty wheels of Parmigiano–Reggiano cheese of different ages (1–24 months), samples representative of the inner part (I, 10 cm from the round side and 7 cm from the flat side) and of ...the outer part (O, the rest of the cheese after removing the rind) were collected. For each sample, free fatty acids (FFAs, expressed as mg kg
−1 cheese fat) were determined by quantitative capillary gas chromatography. In 18- and 24-month-old cheese, total levels of FFAs (as sum of individual FFAs) were significantly (
P ≤ 0.05) higher in O than I, whereas no differences between regions were observed in 1-, 6- and 12-month-old cheeses. In terms of short (C4–C8) and medium (C10–C14) chain FFAs, levels in the outer regions were higher (
P ≤ 0.05) than inner regions for cheeses from 6- to 24-month-old. The content of long-chain FFAs (C16–C18:2) was higher in O than I in 18- and 24-month-old cheeses.
One hundred forty-one lactobacilli strains isolated from natural whey starter cultures and ripened Grana Padano and Parmigiano Reggiano cheeses were tested for their susceptibility to 13 antibiotics, ...in particular, penicillin G, ampicillin, amoxicillin, oxacillin, cephalotin, cefuroxime, vancomycin, gentamicin, tetracycline, erythromycin, clindamycin, co-trimoxazole, and nitrofurantoin. The strains belonged to the species Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. rhamnosus, and L. casei. The strains of the first two species were isolated from whey starter cultures, and the strains of the last two species were from the ripened cheeses. Significant differences among the strains in their antibiotic resistance were found in relation to the type of cheese and, especially, the strains from Parmigiano Reggiano were more resistant to gentamicin and penicillin G. The strains isolated in the ripened cheese were generally more resistant than those isolated from natural whey starter cultures; in particular, significant differences regarding oxacillin, vancomycin, cephalotin, and co-trimoxazole were observed. Finally, no significant difference in relation to the type of cheese was found among the thermophilic lactobacilli isolated from whey cultures, while the facultatively heterofermentative lactobacilli isolated from Parmigiano Reggiano showed higher resistance toward gentamicin and penicillin G than did the same species isolated from Grana Padano.
A new high performance liquid chromatography with subsequent triple mass spectrometry (HPLC-MS
3) method for the quantitative determination of Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) was applied in a ...series of 44 traditional cheeses. Additionally, inhibition of the angiotensin-converting enzyme (ACE) was measured in vitro by determination of IC
50 values. The correlation coefficients between the IC
50 values and the sum of the two peptides VPP and IPP in hard, semi-hard and in the group of soft cheeses were at
r
=
-
0.797
, −0.580 and −0.357, respectively, suggesting that the high ACE-inhibiting activity found in the water soluble extracts of some investigated hard cheeses was mainly due to the presence of these two tripeptides. Concentrations of VPP and IPP were in the range of 0–224
mg
kg
−1 and 0–95
mg
kg
−1, respectively, indicating that some cheese varieties contain similar concentrations of VPP and IPP to recently developed fermented milk products with blood-pressure lowering capacity.
The microbiological quality, safety, and composition of mixtures of ewe's and goat's milk (90:10) used for cheesemaking were evaluated before and after thermization at 60 and 67°C for 30 s. Such mild ...thermal treatments are commonly applied to reduce natural contaminants of raw milk before processing for traditional hard Greek cheeses. Raw milk samples had an average total bacterial count of 7.3 log CFU/ml; most of these bacteria were lactic acid bacteria (LAB) and pseudomonads. The LAB flora of raw milk was dominated by enterococci (40.8%), followed by lactococci (20.4%), leuconostocs (18.4%), and mesophilic lactobacilli (10.2%). Enterococcus faecalis (30.1%) and Enterococcus faecium (13.7%) were the most common LAB isolates, followed by Enterococcus durans, Lactococcus lactis subsp. lactis, Lactobacillus plantarum, and Leuconostoc lactis. Thermization at 60°C for 30 s was effective for reducing raw milk contamination by enterobacteria (5.1 log CFU/ml), coagulase-positive staphylococci (3.3 log CFU/ml), and Listeria (present in 25-ml samples) to safe levels, but it also reduced mesophilic lactococci, leuconostocs, lactobacilli, and selected enterococci (72.0%) in thermized milk. Thermization at 67°C for 30 s had a major inactivation effect on all bacterial groups. Two nisin-producing L. lactis subsp. lactis strains (M78 and M104) were isolated from raw milk, but neither nisin-producing nor other bacteriocin-producing LAB strains were isolated from thermized milk. Thus, thermization treatments control harmful bacteria but also may have a negative impact on milk quality by reducing desirable LAB and the biodiversity of raw milk bacteria overall, inactivating potentially protective LAB strains and enhancing the ability of potentially pathogenic enterococci to grow in fresh cheese curds.
The effect of elevated ripening temperature on physicochemical, biochemical and sensory characteristics in Reggianito Argentino cheese was evaluated to determine the optimal time for cheese ripening ...at 18
°C that ensures typical cheese characteristics. Cheeses ripened at 12 or 18
°C and 85% relative humidity were analysed at 2, 4 and 6
months. Seventy-eight variables (as determined by urea-PAGE, RP-HPLC of the water-soluble at pH 4.6 fraction, free amino acids, free fatty acids and sensory analysis) were considered for the principal component analysis. The statistical analysis allowed determination of the optimal time for ripening Reggianito Argentino cheese at 18
°C, which was ranged between 2 and 3
months. In conclusion, the results obtained were not only useful in characterising the ripening of an Argentinean hard cheese, but also in evaluating the effect of an increase of ripening temperature on the main physicochemical, biochemical and sensory changes of Reggianito Argentino cheese.
For the first time, the identification of
N-γ-glutamyl-,
N-pyroglutamyl- and
N-lactoyl-amino acids in Parmigiano-Reggiano cheese is reported. Their structures were assigned by electrospray mass ...spectrometric analysis and demonstrated for several compounds by the synthesis of authentic specimens and sample spiking. The comparison with the synthesised compounds also allowed the determination of their absolute stereochemistry (
l,
l), which indicated a common enzymatic origin. All these molecules were shown to accumulate in Parmigiano-Reggiano during ripening, probably because of a resistance to proteolysis due to their unusual structures. Thus, these compounds can be used as markers of ripening time and may be involved in the development of the pleasant flavour of aged cheeses. Their presence in other cheeses was also demonstrated.
Traditional artisanal Pecorino Siciliano (PS) cheeses, and two experimental PS cheeses were manufactured using either raw or pasteurised ewes' milk with the addition of starter cultures. The ...bacterial diversity and dynamics of the different cheese types were evaluated both by culturing and characterisation of isolates, and a culture-independent approach based on the 16S ribosomal RNA (rRNA) gene.
Following cultivation, artisanal and experimental cheese types showed similar microbial counts, and isolates belonging to
Lactococcus lactis,
Streptococcus thermophilus,
Enterococcus faecalis and
Leuconostoc mesenteroides were identified by phenotypic characterisation and comparison of the restriction fragment length polymorphism (RFLP) of the 16S rRNA gene to that of reference species. The culture-independent fingerprinting technique PCR and denaturing gradient gel electrophoresis (DGGE) of V6 to V8 regions of the 16S rRNA gene of samples taken during artisanal PS cheese manufacture, from raw milk to the ripened cheese, indicated relevant shifts in the microbial community structure.
The dominance of
Streptococcus bovis and
Lactococcus lactis species in the traditional artisanal PS was revealed by 16S rRNA gene sequencing. Comparison of DGGE profiles of samples from milk to ripened cheese, derived from artisanal procedure and the two experimental PS cheeses during production showed similar trends with the presence of intense bands in common. Nevertheless, the profiles of several artisanal cheeses from different farms appeared more diverse, and these additional species are probably responsible for the generally superior flavour and aroma development of traditional PS cheese.
The aim of this study was to evaluate the milk properties and the yield and sensory properties of Cantal cheese made with milk from Holstein or Montbéliarde cows milked once or twice daily. ...Sixty-four grazing cows 32 Holstein (H) and 32 Montbéliarde (M) cows in the declining phase of lactation (157 d in milk) were allocated to 1 of 2 equivalent groups milked once daily (ODM) or twice daily (TDM) for 7 wk. The full-fat raw milk collected during 24h from the 4 groups of cows (M-TDM, M-ODM, H-TDM, and H-ODM) was pooled and processed into Cantal cheese 4 times during the last 4 wk of the experimental period. In all, 16 cheeses were made (2 milking frequencies×2 breeds×4 replicates) and analyzed after a ripening period of 15 and 28 wk. The results showed that for both breeds, the pooled milk content of fat, whey protein, casein, total protein, and phosphorus as well as rennet clotting time and curd firming time were significantly higher with ODM cows, whereas the casein-to-total protein ratio was lower, and lactose, urea, calcium, and free fatty acids contents of milk remained unchanged. The acidification and draining kinetics of the cheese as well as cheese yields and the chemical and rheological properties of the ripened cheese were not significantly modified by milking frequency. For both breeds, the cheeses derived from ODM cows had a slightly yellower coloration but the other sensory attributes, except for pepper odor, were not significantly affected by milking frequency, thereby demonstrating that ODM does not have an adverse effect on the sensory properties of Cantal cheese. Compared with that of Holstein cows, milk from Montbéliarde cows resulted in a higher cheese yield (+1.250 kg/100 kg of milk) and ripened cheeses with lower pH, dry matter, calcium, sodium chloride, and water-soluble nitrogen concentrations. These cheeses had also a less firm and more elastic texture, a more acidic taste, and a yogurt/whey aroma.
Late blowing, caused by the outgrowth of clostridial spores present in raw milk and originating from silage, can create considerable product loss, especially in the production of hard and semi-hard ...cheeses. The conventional method for the isolation of Clostridium spp. from cheeses with late-blowing symptoms is very complicated and the identification of isolates is problematic. The aim of this work was the development of a multiplex PCR method for the detection of the main dairy-related clostridia such as: Cl. beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum. Samples derived from silage, raw milk and hard cheese were analysed by the most probable number (MPN) enumeration. Forty-four bacterial strains isolated from gas positive tubes were used to check the reliability of the multiplex PCR assay. The specificity of the primers was tested by individually analysing each primer pair and the primer pair combined in the multiplex PCR. It was interesting to note that the samples not identified by the multiplex PCR assay were amplified by V2–V3 16S rRNA primer pair and the sequencing revealed the aligned 16S rRNA sequences to be Paenibacillus and Bacillus spp. This new molecular assay provides a simple promising alternative to traditional microbiological methods for a rapid, sensitive detection of clostridia in dairy products.
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