Evaluation of the spiral plating method for the enumeration of microorganisms throughout the manufacturing and ripening of a raw goat's milk cheese

A statistical comparison of the spiral plate count (SPLPC) and the standard plate count (SPC) methods for enumeration of microorganisms in raw goat's milk cheese throughout its manufacturing and ripening was carried out. Enumeration of mesophiles, lactic acid bacteria (presumptive lactococci, presumptive leuconostocs, and presumptive lactobacilli), Micrococcaceae, Enterobacteriaceae, and molds and yeasts was carried out for milk, curd, and 2-, 5-, 10-, 17-, and 27-day-old cheeses. Average counts for the SPLPC and SPC methods differed by less than half of a log cycle for all microbial groups studied (range of difference, -0.1386 [mesophiles] to +0.4397 [presumptive lactobacilli]). The results of the SPLPC method compared favorably with the results of the SPC procedure for mesophiles, presumptive lactococci, presumptive leuconostocs, Enterobacteriaceae, and molds and yeasts (the variance between replicate platings was close to 0.005, and correlation coefficients were >0.9). Correlation coefficients were lower for Micrococcaceae (r = 0.824) and presumptive lactobacilli (r = 0.670). Analysis of variance showed that the plating method was a significant factor (P < 0.05) for presumptive lactobacilli counts. In general, results from the SPLPC method compared favorably with results from SPC procedure in the enumeration of microorganisms in goat cheese throughout its manufacturingand ripening processes. However, the suitability of the SPLPC method depends mainly on the microbial group studied.     

Evaluation of biogenic amines and microbial counts throughout the ripening of goat cheeses from pasteurized and raw milk

The effect of the hygienic quality of milk on changes in microbial counts and biogenic amine content was evaluated during ripening of goat cheeses manufactured from pasteurized and raw milks at 1, 14, 30, 60 and 90 d. The original milk, rennet, curd and whey were also included in the study. The pH, salt content and extent of proteolysis in the cheese were also evaluated. Spermidine and spermine were the main amines in raw milk, while they were minor amines in cheeses. Other amines increased markedly during ripening, tyramine being the main amine in cheese made from raw milk and cadaverine and putrescine in those produced from pasteurized milk. Enterobacteriaceae counts decreased during ripening whereas those of lactic acid bacteria increased, especially lactobacilli and enterococci. Cheese made from raw milk showed higher microbial counts during ripening than those made from pasteurized milk, especially for Enterobacteriaceae and enterococci, counts being 2 or 3 log units higher. Raw milk cheese showed remarkably higher biogenic amines compared with pasteurized milk cheeses. Therefore, pasteurization of milk causes a decrease in final biogenic amine content of cheese as a result of the reduction of its microbial counts.     

Proteolysis in goat cheese made from raw,pasteurized or pressure-treated milk

Primary and secondary proteolysis of goat cheese made from raw (RA), pasteurized (PA; 72 °C, 15 s) and pressure-treated milk (PR; 500 MPa, 15 min, 20 °C) were examined by capillary electrophoresis, nitrogen fractionation and HPLC peptide profiles. PA milk cheese showed a more important hydrolysis (P<0.05) of α_s1-casein than RA milk cheese at the first stages of ripening (15 days), while PR milk cheese had a level between those seen in PA and RA milk cheeses. Degradation of β-casein was more important (P<0.05) in PA and PR than in RA milk cheeses at 15 days of ripening. However, from thereon β-casein in PR and RA milk cheeses was hydrolyzed at essentially similar rates, but at lower rates (P<0.05) than in PA milk cheeses. Pressure treatment could induce proteolysis of β-casein in a way, which is different from that produced by heat treatment. There was an increase in 4.6-soluble nitrogen (WSN) and in trichloroacetic acid (TCASN) throughout ripening in cheeses, but higher contents (P<0.05) in PA and PR milk cheeses at the end of ripening were observed. PR milk cheeses contained considerably higher content (P<0.05) of free amino acids than RA or PA milk cheeses. In general, heat and pressure treatments had no significant effect on the levels of hydrophobic and hydrophilic peptides.     

Changes in organic acids during ripening of cheeses made from raw, pasteurized or high-pressure-treated goats’ milk

Organic acids of cheeses made from raw (RA), pasteurized (PA; 72°C, 15 s) or pressure-treated (PR; 500 MPa, 15 min, 20°C) goats’ milk were qualitatively and quantitatively assessed during ripening. Nine organic acids (citric, pyruvic, malic, lactic, formic, acetic, uric, propionic and butyric) were analysed in each sample by HPLC.Milk treatment did not affect the total organic acids content of 1-day-old cheeses, which increased steadily from day 1 to day 60. At the end of ripening, RA and PR milk cheeses both exhibited higher concentration of organic acids than in those made from PA milk.Lactic acid was found in higher concentration in PR milk cheese from 30 days of ripening. The RA milk cheese, that showed the highest nonstarter lactic acid bacteria counts, were characterized by an elevated amount of propionic and acetic acids. These cheeses also were negatively correlated with both pyruvic and citric acid contents. The PA milk cheese showed a high level of malic acid, and was clearly differentiate from RA and PR milk cheeses by its low level of butyric acid.     

On the microbiology of Serra da Estrela cheese: geographical and chronological considerations

Serra da Estrela cheeses, produced following artisanal practices from raw ewe's milk in two consecutive years in five selected dairies, were sampled throughout a 60-day ripening period. The viable numbers of the major microbial groups (Enterobacteriaceae, staphylococci and yeasts, as well as such lactic acid bacteria as lactobacilli, lactococci and enterococci) were determined and statistical significance of the results was assessed. Significant differences in the viable counts of lactococci, lactobacilli, enterococci, Enterobacteriaceae and staphylococci were found for the various geographical locations, whereas the year of manufacture played a significant role only upon the viable numbers of yeasts and staphylococci. These data back up the claim that those microbial families usually implicated with health hazards are prone to a wide variability, which reflects the intrinsic heterogeneity associated with production and handling of raw milk in traditional cheesemaking.     

Changes in textural, microstructural, and colour characteristics during ripening of cheeses made from raw, pasteurized or high-pressure-treated goats’ milk

Goats’ milk cheeses were made from raw (RA), pasteurized (PA; 72°C, 15 s) or pressure-treated (PR; 500 MPa, 15 min, 20°C) milk to compare textural, microstructural, and colour characteristics in relation to ripening time. Texture, microstructure and colour were evaluated by uniaxial compression and stress relaxation tests, confocal laser scanning microscopy and Hunter colorimetry, respectively.Raw and PR cheeses were firmer and less fracturable than PA cheese, but differences became less notable toward the end of ripening. PA and PR cheeses were less cohesive than RA cheese. Although cheeses exhibited a loss of elastic characteristics with ageing, PR cheese showed the most elastic behaviour initially. Confocal laser scanning micrographs displayed PR cheese with a regular and compact protein matrix, with small and uniform fat globules resembling the structure of RA cheese. Finally, colour evaluation demonstrated significant differences between cheeses due to milk treatments and ripening time.     

Characterization of microflora in homemade semi-hard white Zlatar cheese

The Zlatar cheese belongs to the group of traditionally homemade cheeses, which are produced from nonpasteurized cow's milk, without adding of any bacterial starter culture. Changes were followed in lactic acid bacteria population and chemical composition during the ripening period of cheese up to 60 days. Results showed that the percentage of lactic acid cocci was higher in raw milk and one day old cheese and their percentage was gradually decreasing, whereas the number of lactobacilli was increasing. After 30 days of cheese ripening the number of cocci increased again, reaching the number of lactobacilli. The results of API 50 CH system and rep-PCR analysis showed that Lactobacillus paracasei subsp. paracasei, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcuus faecium and Enterococcus faecalis were the main groups present during the ripening of Zlatar cheese. Results revealed that in older cheeses (45 and 60 days old) enterococci were the main group present. It was also demonstrated that 57 isolates showed antimicrobial activity. The number of bacteria showing antimicrobial activity slowly decreased during the ripening period and in samples of 60 days old cheese producers of antimicrobial activities were not detected.     

Characterisation of Urda whey cheese: Evolution of main biochemical and microbiological parameters during ripening and vacuum packaged cold storage

In this study, the main biochemical and microbiological characteristics of Urda, a traditional Greek whey cheese, were determined during ripening at 19 ± 2 °C for 25 days followed by vacuum packaging and storage at 5 °C until day 360. Few differences in pH, water activity, acid degree value, moisture, salt, protein and fat contents were observed between Urda cheeses produced from sheep or goat milk whey at all sampling days (1, 25, 90, 180 and 360). Cheese microbiota was dominated by mesophilic lactic acid bacteria, but high numbers of enterococci, aerobic gram-negative bacteria and Enterobacteriaceae were also present. Increases in all the soluble nitrogen fractions of cheeses occurred, primarily during cold storage. Ketones and terpenes were the most abundant volatile compounds in fresh (1-day) sheep and goat cheeses, respectively, whereas free fatty acids were the most abundant compounds in mature (180-day) cheeses, followed by ketones.     

Specific effect of high-pressure treatment of milk on cheese proteolysis

The extent of primary and secondary proteolysis of cheeses made from raw (RA), pasteurized (PA, 72 degrees C, 15 s) or pressure-treated (PR, 500 MPa, 15 min, 20 degrees C) goats' milk was assessed. Modifications in cheese-making technology were introduced to obtain cheeses with the same moisture content, and thus studied per se the effect of milk treatment on cheese proteolysis.The PR milk cheese samples were differentiated from RA and PA milk cheeses by their elevated beta-lg content, and by the faster degradation of alphas1-, alphas2- and beta-CN throughout ripening. Non-significant differences were found in either pH 4.6 soluble-nitrogen or trichloracetic acid soluble-nitrogen contents of cheeses. However, the pasteurization of milk decreased the free amino acid production in cheese. The RA milk cheeses had the highest amount of proline and the lowest concentrations of serine, tyrosine, arginine and alpha-aminobutyric acid, whereas PR milk cheese showed higher levels of arginine.     

Phenotypic and PCR-based characterization of the microflora in Präst cheese during ripening

Microbiological sampling of Präst cheese from three cheese factories was done during ripening. The evolution of total bacterial counts, lactococci, lactobacilli, enterococci presumptive leuconostoc and pediococci was investigated after 30, 90, 180 and 270 days of ripening. Isolates (140) of non-starter lactic acid bacteria (NSLAB) from 12 Präst cheeses after 90, 180 and 270 days of ripening were examined. The isolates were tested by physiological and biochemical assays, species-specific PCR and 16S rDNA sequencing. The predominant NSLAB species was Lactobacillus paracasei. The development and evolution of the NSLAB microflora in Präst varied according to dairy plant and ripening time.     

Ripening and seasonal changes in microbial groups and in physico-chemical properties of the ewes’ cheese Pecorino del Poro

Three batches of Pecorino del Poro, ewes’ cheese made from raw milk, were examined throughout a 28-day ripening time at three different seasons. High logarithmic counts per gram of cheese for mesophilic coccal-shaped lactic acid bacteria (6.70–12.45), mesophilic lactobacilli (4.82–11.73), thermophilic coccal-shaped lactic acid bacteria (2.30–9.90), and thermophilic lactobacilli (2.95–8.15) were found. Coccal-shaped lactic acid bacteria were the dominant microorganisms throughout ripening. The microorganisms used as an indicator of hygiene during manufacture of the cheeses, coliforms and Escherichia coli, were considerably lower, as were enterococci and yeasts. Coliforms and E. coli decreased sharply throughout ripening. Physico-chemical parameters such as pH (5.07–7.03), dry matter (46.34–72.79%), ether extract (31.35–51.84% of dry matter), crude protein (29.93–44.73% of dry matter), and chloride content (2.36–4.11% of dry matter) were also determined. Probably, the use of selected autochthonous mesophilic lactococci as a starter would control or suppress the growth of undesirable microorganisms. The results obtained suggest the need for improvements in milking and dairy conditions.     

Characterization of the lactic acid bacteria in ewe's milk and cheese from northwest Argentina

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.     

Phenotypic and PCR-based characterization of the microflora in Norvegia cheese during ripening

Microbiological sampling of Norvegia cheese from three cheese factories was done during ripening. The evolution of aerobic mesophilic bacteria, lactococci, lactobacilli, enterococci, presumptive leuconostoc and pediococci was investigated after 30, 90, 180 and 270 days of ripening. Isolates (135) of non-starter lactic acid bacteria (NSLAB) from nine Norvegia cheeses after 90, 180 and 270 days of ripening were examined. The isolates were tested by physiological and biochemical assays, species-specific PCR and 16S rDNA sequencing. After 90 days of ripening Leuconostoc spp., most probably from the starter, and the NSLAB specie Lactobacillus paracasei dominated among the isolates, however, after longer ripening Lb. paracasei dominated. The development and evolution of the microflora in Norvegia varied according to dairy and ripening time.     

Microbiological changes in ‘San Simón’ cheese throughout ripening and its relationship with physico-chemical parameters

Changes in the microbial flora of San Simón cheese, manufactured from raw milk without the addition of starter cultures, were studied during the manufacture and ripening, with the object of contributing to the characterization or typifying of this variety.Microbial counts from cheese surface were, on the whole, 1 log unit lower with respect to the ones taken in the core of the cheese, with the exception of the counts in MSA and OGYEA media.Lactic acid bacteria (major microbial group) reached their maximum counts in 4-week-old cheese from core samples, although on the surface of the cheese the maximum count of lactococci and leuconostoc were obtained after 2 weeks. Other microbial groups (Micrococcaceae, Enterococci and Enterobacteriaceae) reached their maximum counts in 1-week-old cheese and decreased slightly afterwards during ripening. The decrease of the Enterobacteriaceae was a little more striking, although they did not disappear completely at the end of the ripening process.The evolution of major physico-chemical parameters investigated during manufacture and ripening of San Simón cheese, contribute to the slight decrease of all microbial groups, since the parameter values during ripening were not markedly dysgenic for any of the microbial groups investigated.     

Identification of Lactic Acid Bacteria Isolated from Tulum Cheese During Ripening Period

The fresh Tulum cheese was manufactured and then ripened at 10 ± 2°C for 3 months. Isolation and identification of lactic acid bacteria (LAB) was carried out during the ripening period of 90 days. Lactic acid bacteria were isolated from PCA, M17, and MRS agar. The strains isolated were identified using morphology, colony pigmentation, production of carbon dioxide from glucose, growth at 4 and 40°C, salt tolerance, starch hydrolysis, and sugar fermentation with the API system methods. A total of 253 strains isolated during the storage. Identifying strains belonged to genera of the lactobacilli (133), pediococci (44), enterococci (29), leuconostocs (27), and lactococci (8). 12 of 253 strains were not also identified. Lactobacilli (75.2%) were frequently determined on MRS medium. In the results of this article, lactobacilli were found at high frequencies, while enterococci, lactococci, leuconostocs, and pediococci were found at low frequencies in Tulum cheese. Lactobacilli increased during the ripening period, but the others did not change a significant amount.     

Indigenous raw milk microbiota influences the bacterial development in traditional cheese from an alpine natural park

Nostrano di Primiero is a 6-month ripened cheese produced from raw milk collected in the Paneveggio-Pale di San Martino Natural Park area in the Italian Dolomites. In summer, this cheese is made using milk collected from two different areas, Passo Rolle and Vanoi, in the Paneveggio Natural Park. During the experiment, the milk from the two areas was separately processed, and cheeses were made in the same cheese factory using the same technological process. The microbiota of raw milk and cheeses of the two areas was isolated and the dominant population was monitored by RAPD analysis and identified by 16S rRNA sequence. The milk of the Passo Rolle area was mainly composed of mesophilic strains, thermophilic Streptococcus thermophilus, and low amounts of enterococci were also found; the milk of the Vanoi area was dominated by mesophilic microbiota mostly Lactococcus lactis ssp. cremoris and ssp. lactis and Lactobacillus paracasei ssp. paracasei. The plating of the natural starter culture revealed the presence of a relevant community of thermophilic cocci and lower amounts of enterococci. The dynamic population analysis showed the importance of the natural starter culture in the first 2 days of cheese ripening in both cheeses. Moreover, the large biodiversity observed in the raw milks was also detected in the cheeses during ripening. The Vanoi cheese was dominated by Enterococcus faecium and Streptococcus macedonicus in the first two days and mesophilic 21 Lb. paracasei ssp. paracasei became the most represented population after 15 days of ripening. In the first few days, the Rolle cheese was characterized by being mainly composed of thermophilic S. macedonicus and S. thermophilus and secondarily by mesophilic cocci. During ripening, the microbiota composition changed, and at 15 days, mesophilic lactobacilli were the dominant population, but later, this was mainly composed of mesophilic cocci and lactobacilli. The taxonomical identification by 16S rRNA sequence confirmed a large biodiversity related to raw milk microbiota and only five strains of S. macedonicus, Lactobacillus plantarum, 21 Lb. paracasei ssp. paracasei, Lactobacillus fermentum and E. faecium were detected in both cheeses.     

Monitoring and identification of bacteria associated with safety concerns in the manufacture of São Jorge, a Portuguese traditional cheese from raw cow's milk

The aim of this study was to assess the hygienic quality of raw milk used in the manufacture of S?o Jorge, a Protected Denomination of Origin Portuguese semihard cheese, as well as to ascertain the sanitary conditions prevailing during its processing. Viable counts of Enterobacteriaceae and Micrococcaceae were accordingly obtained, pertaining to 21 independent batches (including samples of raw milk, curd, and cheeses after 1, 3, and 4 months of ripening), from 7 dairy farms. Standard plate counts (log CFU per milliliter or per gram) ranged from 6.1 to 8.6 in raw milk, whereas they ranged from 7.0 to 8.0 in 4-month-old cheeses. Viable counts of Enterobacteriaceae ranged between 5.9 and 7.0 in raw milk and between 0.0 and 1.3 in 4-month-old cheeses. Species identified within this family encompassed Klebsiella oxytoca, Klebsiella cloacae, Klebsiella pneumoniae, Enterobacter sakazakii, and Escherichia coli; Klebsiella ornithinolytica, Klebsiella terrigena, and Serratia odorifera were detected only in raw milk. No Salmonella whatsoever could be detected in any of the samples. Viable counts of Micrococcaceae ranged between 4.7 and 5.9 and between 1.3 and 3.3 in raw milk and 4-month-old cheeses, respectively. Species identified within this family encompassed Staphylococcus sciuri, Staphylococcus epidermidis, Staphylococcus saprophyticus (which was found mainly in ripened cheeses), and Staphylococcus aureus (which was not detected in 4-month-old cheeses). Accompanying physicochemical analyses included determination of moisture, salt, and pH. Statistical analyses revealed a negative correlation between salt content and viable numbers of Enterobacteriaceae in cheese, whereas in the case of Micrococcaceae, a more negative correlation was found between viable numbers and moisture content than between viable numbers and pH. The results of our study indicate, in general, poor milk handling conditions in all farms, given that the indicators total mesophile and Enterobacteriaceae counts were high, between 100- and 1,000-fold those enforced by international standards pertaining to the matrices in question. However, by the time of regular consumption (i.e., after 4 months of ripening), S?o Jorge cheeses exhibit low levels of contamination by Enterobacteriaceae and S. aureus, as well as absence of Salmonella.     

Effect of lactobacillus adjunct cultures on the microbiological and physicochemical characteristics of Roncal-type ewes'-milk cheese

The effect of two different experimental adjunct cultures composed of native facultatively heterofermentative lactobacilli (FHL) on the development of various groups of micro-organisms in Roncal-type ewes' milk cheese was studied. Four cheese batches were manufactured from raw milk (C), pasteurized milk (P), pasteurized milk and an adjunct culture of Lactobacillus paracasei (PP); and pasteurized milk and adjunct culture of Lactobacillus paracasei plus Lactobacillus plantarum (PPP). Retention of the two adjunct cultures in the cheeses was good, and population levels remained constant at around 10(7) cfu g(-1) of cheese throughout ripening. Levels of Enterobacteriaceae and enterococci fell off more abruptly in the batches made with the Lactobacillus adjunct cultures, suggesting competition between the added lactobacilli and those groups of micro-organisms. The inhibitory effect was greater for the adjunct culture composed of L. paracasei plus L. plantarum. Lactococcal levels were higher in the batches made with added FHL, which may be indicative of a synergistic effect between these two groups.     

Possible implications of milk pasteurization on the manufacture and sensory quality of ripened cheese

Cheese made from raw milk represents an important proportion of the traditional cheeses, particularly in South European countries. Besides destruction of pathogenic bacteria, the most significant changes in milk relevant to cheesemaking, which are induced by pasteurization are:

• a partial elimination of the milk microorganisms which may grow in cheese during ripening,

• a partial or total activation or inhibition of the plasmin/plasminogen complex, cathepsin D, lipoprotein lipase and alkaline phosphatase. Enzymes from psychrotrophic bacteria, acid phosphatase and xanthine oxidase, which may be active during ripening, withstand pasteurization.,

• a slight (7%) denaturation of serum proteins and little or no modification of the cheesemaking properties (coagulation, acidification by lactic acid bacteria).

From experimental work carried out on several cheese varieties, comparing pasteurized or microfiltered milk and raw milk cheeses, it was found that facultatively heterofermentative lactobacilli, Micrococcaceae, enterococci, and propionibacteria in Swiss-type cheese, are found at higher levels in raw milk cheese. The main biochemical modification of cheese during ripening concerns the nature and extent of proteolysis. Although there is no clear trend in the breakdown of _s1- and β-caseins, milk pasteurization leads to a significant decrease of the amount of small peptides and free amino acids and to different HPLC profiles. Experiments carried out with sensory analysis show that, in all cases, pasteurized or microfiltered milk cheeses have received lower flavour intensity scores than raw milk cheeses. From this review, it is concluded that the indigenous milk microflora, with its diversity of species and strains, appears to be mainly responsible of the specific sensory properties of raw milk cheeses.     

Influence of the starter culture on the microbiological and sensory characteristics of ewe's cheese

Changes which take place in the sensory characteristics of cheeses during ripening are influenced by different factors, involving rennet, starter culture and adventitious contamination of the cheese by non-starter lactic acid bacteria. The objective of this work was to study the influence of the starter on sensory and microbiological ewe's cheese properties during ripening time. Four batches (two with starter added and two without) were manufactured. Milk and cheeses at different stages of ripening were analysed. Cheeses manufactured without adding starter showed a significantly higher level of mesophilic aerobic microflora, lactobacilli, facultatively heterofermentative lactobacilli and enterococci (indigenous microflora) than cheeses manufactured with starter. This study has also shown that adding or not adding starter affects the flavour profile of the cheese. Cheeses with starter added showed greater intensity of the following attributes: refreshing, astringent, sweet; and received lower scores on bitterness. With respect to texture, the said cheeses develop a more homogenous texture and greater elasticity throughout ripening.