Evaluation of amino acid-decarboxylative microbiota throughout the ripening of an Italian PDO cheese produced using different manufacturing practices

Aim:  To investigate the presence of biogenic amines (BAs) in Montasio cheese produced by using different cheese manufacturing practices.
Methods and Results:  Three batches of Montasio cheese were made in the following way: batch A using raw milk and natural milk culture, batch B with thermized milk and natural milk culture and batch C with thermized milk and natural milk culture added of a commercial starter culture. During 120 days of ripening analyses were performed for microbial counts and BA content; indeed, the potential to produce BAs was screened in lactic acid bacteria and Enterobacteriaceae isolates. At the end of ripening, the total BA contents of cheeses from batches A, B and C were 166·3, 207·3 and 29·8 mg kg⁻¹, respectively. Amino acid decarboxylase activity was widespread among isolates.
Conclusions:  The BA content of Montasio cheese from the three batches was below the threshold proposed as potentially toxic. The highest BA content was found in cheese produced using thermized milk and natural milk culture; therefore, the thermal treatment of milk was not enough by itself to reduce the counts of decarboxylase-positive bacteria in cheese. The use of selected starters guaranteed a low BA content in Montasio cheese.
Significance and Impact of the Study:  The study of the effects of some technological processes on the incidence of decarboxylative microbiota in 'protected denomination of origin' cheeses could provide useful information on the hygienic risk related to their production.     

Heterogeneity of Lactobacillus plantarum isolates from feta cheese throughout ripening

Thirty-two Lactobacillus plantarum strains isolated from Feta cheese throughout ripening were studied for their phenotypic characteristics, protein profile of cell-free extracts, enzyme profiles, plasmid profiles, proteolytic and acidifying abilities and ability to grow at low pH and in the presence of bile. Results showed that some biotechnologically important characteristics, such as acidifying and proteolytic activities, can differ between strains. In addition, different plasmid profiles suggest the presence of different Lact. plantarum strains in Feta cheese throughout ripening. The results suggest the possibility of choosing strains with specific biotechnologically interesting properties.     

Acceleration of cheese ripening

The characteristic aroma, flavour and texture of cheese develop during ripening of the cheese curd through the action of numerous enzymes derived from the cheese milk, the coagulant, starter and non-starter bacteria. Ripening is a slow and consequently an expensive process that is not fully predictable or controllable. Consequently, there are economic and possibly technological incentives to accelerate ripening. The principal methods by which this may be achieved are: an elevated ripening temperature, modified starters, exogenous enzymes and cheese slurries. The advantages, limitations, technical feasibility and commercial potential of these methods are discussed and compared.     

The role of fungi in cheese ripening

Fungi are important in the manufacture of two types of cheese—blue-veined cheeses, and Camembert and Brie. Among the former are Roquefort, Gorgonzola and Stilton, dependent on the mold Penicillium roqueforti and the bacterium Streptococcus lactis. Camembert and Brie require Penicillium camemberti and lactic acid- producing streptococci; the mold Oospora lactis and the organism Bacterium linens may also play roles in their manufacture.     

Acceleration of cheese ripening with liposome-entrapped proteinase

Summary Rulactine, a proteinase used for the acceleration of cheese ripening, was entrapped in three types of liposomes and these were added to Saint-Paulin cheese type manufacturing milk. Enzyme entrapment rates ranged from 3 to 9% according to the type of liposomes and liposome retention rates in cheese curd from 35 to 65%. An electrophoretic study of protein breakdown in the cheeses gave correlative data.     

Caseinolysis in cheese by Enterobacteriaceae strains of dairy origin

AIMS: To study the effect of Enterobacteriaceae strains of dairy origin on caseins under cheese manufacture and ripening conditions. METHODS AND RESULTS: Strains belonging to the genera Enterobacter, Escherichia, Hafnia and Serratia were isolated from fresh raw milk cheeses. Residual caseins in cheeses made from milk individually inoculated with 10 strains of Enterobacteriaceae were determined by capillary electrophoresis. Hierarchical cluster analysis of strains based on data of residual caseins grouped together strains from the same genus, excepting Hafnia strains, which were separated into two groups. Serratia was the most proteolytic genus in our study. Preferences for degradation of casein fractions differed among the four genera studied. CONCLUSIONS: Enterobacteriaceae strains posses proteolytic systems active on all casein fractions under cheese manufacture and ripening conditions. The effects on caseins were similar for strains belonging to the same genus. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Enterobacteriaceae in cheeses may affect proteolysis during ripening. Assays of Enterobacteriaceae proteolytic activity on milk agar plates may underestimate their caseinolytic activity in cheese.     

Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk

Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml⁻¹. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10³ cfu ml⁻¹ at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3·2 ng g⁻¹ of cheese made with an initial population of 10³–10⁶ cfu ml⁻¹. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.     

Effect of heat treatment on the proteolytic activity of mesophilic bacteria isolated from goats' milk cheese

Strains of mesophilic lactococci and lactobacilli isolated from goats' milk cheese were grown to maximum density in milk at 30°C, pH 6·5. They were subsequently cooled to 12°C and then heated at 50°, 52° and 54°C (holding time, 15 s). The micro-organisms tested were Lactococcus lactis subsp. lactis IFPL 60, IFPL 22 and IFPL 359, Lactobacillus casei subsp. casei IFPL 731 and Lactobacillus plantarum IFPL 3, isolated from raw goats' milk cheese. The heated cells presented lower viability and acidification capacity than unheated cells. After heat treatment at 50°C, all the test strains effected practically no reduction in pH of milk (6 h), except for Lactococcus lactis subsp. lactis IFPL 60, which reduced pH to 5·9 as compared to 4·9 attained by the unheated controls. After treatment, proteolytic, aminopeptidase and dipeptidase activities of cell-free extracts decreased to a lesser extent than the number of viable cells with acidifying ability. The results suggest that these strains, if treated at 50°C, may be suitable as extra sources of important ripening enzymes in cheese making.     

Fate of Listeria monocytogenes during manufacture and ripening of semi-hard cheese

Semi-hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 10⁵ Listeria monocytogenes /ml in cheeses 1 and 2 and 4 times 10³ L. monocytogenes /ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese.     

Persistence of Mycobacterium paratuberculosis during manufacture and ripening of cheddar cheese

Model Cheddar cheeses were prepared from pasteurized milk artificially contaminated with high 10(4) to 10(5) CFU/ml) and low (10(1) to 10(2) CFU/ml) inocula of three different Mycobacterium paratuberculosis strains. A reference strain, NCTC 8578, and two strains (806PSS and 796PSS) previously isolated from pasteurized milk for retail sale were investigated in this study. The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar. The survival of M. paratuberculosis cells was monitored over a 27-week ripening period by plating homogenized cheese samples onto HEYM agar medium supplemented with the antibiotics vancomycin, amphotericin B, and nalidixic acid without a decontamination step. A concentration effect was observed in M. paratuberculosis numbers between the inoculated milk and the 1-day old cheeses for each strain. For all manufactured cheeses, a slow gradual decrease in M. paratuberculosis CFU in cheese was observed over the ripening period. In all cases where high levels (>3.6 log(10)) of M. paratuberculosis were present in 1-day cheeses, the organism was culturable after the 27-week ripening period. The D values calculated for strains 806PSS, 796PSS, and NCTC 8578 were 107, 96, and 90 days, respectively. At low levels of contamination, M. paratuberculosis was only culturable from 27-week-old cheese spiked with strain 806PSS. M. paratuberculosis was recovered from the whey fraction in 10 of the 12 manufactured cheeses. Up to 4% of the initial M. paratuberculosis load was recovered in the culture-positive whey fractions at either the high or low initial inoculum.     

Commercial ripening starter microorganisms inoculated into cheese milk do not successfully establish themselves in the resident microbial ripening consortia of a South german red smear cheese

Production of smear-ripened cheese critically depends on the surface growth of multispecies microbial consortia comprising bacteria and yeasts. These microorganisms often originate from the cheese-making facility and, over many years, have developed into rather stable, dairy-specific associations. While commercial smear starters are frequently used, it is unclear to what degree these are able to establish successfully within the resident microbial consortia. Thus, the fate of the smear starters of a German Limburger cheese subjected to the "old-young" smearing technique was investigated during ripening. The cheese milk was supplemented with a commercial smear starter culture containing Debaryomyces hansenii, Galactomyces geotrichum, Arthrobacter arilaitensis, and Brevibacterium aurantiacum. Additionally, the cheese surface was inoculated with an extremely stable in-house microbial consortium. A total of 1,114 yeast and 1,201 bacterial isolates were identified and differentiated by Fourier transform infrared spectroscopy. Furthermore, mitochondrial DNA restriction fragment length polymorphism, random amplified polymorphic DNA, repetitive PCR, and pulsed field gel electrophoresis analyses were used to type selected isolates below the species level. The D. hansenii starter strain was primarily found early in the ripening process. The G. geotrichum starter strain in particular established itself after relocation to a new ripening room. Otherwise, it occurred at low frequencies. The bacterial smear starters could not be reisolated from the cheese surface at all. It is concluded that none of the smear starter strains were able to compete significantly and in a stable fashion against the resident microbial consortia, a result which might have been linked to the method of application. This finding raises the issue of whether addition of starter microorganisms during production of this type of cheese is actually necessary.     

The role of lactic acid bacteria in accelerated cheese ripening

Abstract: The ripening of cheese is a slow and consequently an expensive process. The economic advantage of rapid development of more intense cheese flavour in shorter periods of time would be substantial. Lactic acid bacteria play a key role during ripening and can therefore be used as accelerating agents. This review describes the different strategies where lactic acid bacteria or their enzymes were used to reduce the ripening time of cheese. The advantages, limitations and technical feasibility as well as the commercial potential of the different approaches are also considered.     

Diversity and dynamics of lactobacilli populations during ripening of RDO Camembert cheese

The diversity and dynamics of Lactobacillus populations in traditional raw milk Camembert cheese were monitored throughout the manufacturing process in 3 dairies. Culture-dependent analysis was carried out on isolates grown on acidified de Man - Rogosa - Sharpe agar and Lactobacillus anaerobic de Man Rogosa Sharpe agar supplemented with vancomycin and bromocresol green media. The isolates were identified by polymerase chain reaction - temperature gradient gel electrophoresis (PCR-TGGE) and (or) species-specific PCR and (or) sequencing, and Lactobacillus paracasei and Lactobacillus plantarum isolates were characterized by pulsed field gel electrophoresis (PFGE). Milk and cheese were subjected to culture-independent analysis by PCR-TGGE. Presumed lactobacilli were detected by plate counts throughout the ripening process. However, molecular analysis of total DNA and DNA of isolates failed to detect Lactobacillus spp. in certain cases. The dominant species in the 3 dairies was L. paracasei. PFGE analysis revealed 21 different profiles among 39 L. paracasei isolates. Lactobacillus plantarum was the second most isolated species, but it occurred nearly exclusively in one dairy. The other species isolated were Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus helveticus, a Lactobacillus psittaci/delbrueckii subsp. bulgaricus/gallinarum/crispatus group, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, Lactobacillus brevis, Lactobacillus kefiri, and Lactobacillus perolens. Lactobacilli diversity at the strain level was high. Dynamics varied among dairies, and each cheese exhibited a specific picture of species and strains.     

The capacity of Enterobacteriaceae species to produce biogenic amines in cheese

The amino acid decarboxylating activity and production of biogenic amines by 104 cheese-associated Enterobacteriaceae species (58 Enterobacter, 18 Serratia, eight Escherichia, seven Hafnia, six Arizona, four Citrobacter and three Klebsiella) were investigated. All strains could decarboxylate at least two amino acids in M?ller's broth and in Niven's medium, and the decarboxylase activity was strain specific. In a laboratory medium containing all free amino acids, all strains could produce more than 100 ppm cadaverine, putrescine was produced by 96% of strains. Tyramine and histamine were produced in the lowest concentrations. A positive correlation existed between cadaverine concentration and Enterobacteriaceae counts in cheese, that may have caused the increase in decarboxylase content. This study suggests that it is possible to limit the presence of cadaverine in cheese, thereby controlling the Enterobacteriaceae counts, a sign of contamination during cheese making and/or storage.     

Dynamics of whole and lysed bacterial cells during Parmigiano-Reggiano cheese production and ripening

Microbial succession during Parmigiano-Reggiano cheesemaking was monitored by length heterogeneity PCR (LH-PCR), considering the intact and lysed cells at different stages of cheese production and ripening. When starter species underwent autolysis, species coming from milk were able to grow. For the first time, the LH-PCR technique was applied to study a fermented food.     

Influence of ripening container on the lactic acid bacteria population in Tulum cheese

The objective of the present study was to investigate the influence of container material (plastic or goat-skin bag) on the growth of lactic acid bacteria in Tulum cheese during 9 months of ripening. The lactic acid bacteria in Tulum cheeses were periodically counted on MRS and M17 agars throughout ripening. Results showed that the highest counts of lactic acid bacteria on MRS or M17 were observed at the beginning of ripening and their counts decreased during later stages of ripening. The cheese samples ripened in plastic bags exhibited higher numbers of LAB on MRS and M-17 agars than those ripened in goat-skin bags. A total of 112 strains of lactic acid bacteria were isolated from Tulum cheeses ripened in plastic or goat-skin bags during ripening. The lactic acid bacteria present in the cheese were classified by Microbial Identification System (MIS) based on a comparison of the fatty acid methyl ester profiles. Different species including Enteroccocus, Lactobacillus, Streptococcus, Lactococcus and Pediococcus genera were found in unripened cheese. As ripening proceeded, the species Streptococcus and Lactococcus disappeared and the percentages of the species Enterococcus was unchanged in both containers. There were slight differences between the cheeses ripened in plastic or goat-skin bags in terms of the profiles of lactic acid bacteria isolated. Some species including L. brevis, L. mesenteroides subsp. dextranicum, P. damnosus and E. mundtii were isolated only in the cheeses ripened in plastic bags; however, L. coryniformis and L. malafermentans were isolated only in the cheeses ripened in goat-skin bags at 6 or 9 months of ripening. Also the numbers of E. faecalis isolates were higher in the cheeses ripened in plastic containers than cheeses ripened goat-skin bags at the 6 or 9 months of ripening. The results showed that Lactobacillus and Enterococcus were the predominant species in matured Tulum cheeses in both ripening containers. It seemed possible to produce Tulum cheese with similar characteristics from both the containers used.     

The effect of a commercial starter culture addition on the ripening of an artisanal goat's cheese (Cameros cheese)

The evolution of physicochemical parameters, and the most important microbial groups, were determined for the following three batches of 'Cameros' goat's milk cheese during ripening: Batch R elaborated with raw milk, Batch RS elaborated with raw milk and with the addition of a starter culture, and Batch PS elaborated with pasteurized milk and with the addition of the same culture. No differences in total solids (TS) or in the content of NaCl, fat and total nitrogen (expressed as percentages of TS) were found during the ripening. The pH, fat acidity and non-protein nitrogen (NPN, expressed as a percentage of TN) showed significant differences between the batches. The inoculated batches showed the fastest drop in pH at the beginning of the ripening period, but the cheeses of Batch R showed a higher degree of lipolysis and proteolysis. The addition of a starter influenced the microbiological quality of the cheeses. Differences in the counts of Enterobacteriaceae and faecal coliforms were found between Batches R and RS after 15 days. Staphylococcus aureus increased in number during the early period of ripening and attained a population above 6 log cfu g-1 in Batch R in the period from 5 to 10 days. However, enterotoxins were not detected in this Batch. Batch R showed lower values of lactic acid bacteria at the beginning of the ripening period, but no significant differences were found between batches in the period from 5 to 15 days of ripening. At the beginning of the ripening, Lactococcus was the main lactic acid bacteria, with L. lactis lactis being predominant. After 15 days, the lactic acid bacteria counts decreased in the three batches, especially in the cheeses of Batch PS (only 2.2 log cfu g-1 was found at 60 days), as lactococci (the only lactic acid bacteria present in Batch PS) are incapable of growing under the conditions found in cheeses at the end of their ripening period. At this time, Lactobacillus was the predominant genus in Batches R and RS, with L. plantarum predominant. No lactococci were found from day 30 in Batch R and from day 40 in Batch RS. The cheeses of Batch RS received the most favourable scores from the tasting panel for all attributes judged: cut appearance, colour, aroma, taste, texture and general acceptance.     

Survival of surface ripening cultures during storage and monitoring their development on cheese

AIMS: To study the survival of bacteria isolated from the surface of smear cheese and monitor their development during cheese ripening. METHODS AND RESULTS: The storage of five potential bacterial surface-ripening cheese cultures, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variable, Microbacterium gubbeenense and Staphylococcus saprophyticus, in maximum recovery diluent (MRD), containing 0.85% w/v or 5% w/v NaCl, at 21 or 4 degrees C for 40 days, was investigated. All five strains studied survived well with a maximum decrease of c. 2.5 log(10) CFU ml(-1) after storage for 40 days at 4 degrees C in 0.85% or 5% w/v NaCl. Survival, especially of C. variable, was less at 21 degrees C. The development of defined ripening cultures containing C. casei and Debaryomyces hansenii on two farmhouse cheeses was also evaluated. Using pulsed-field gel electrophoresis (PFGE) for the bacteria and mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) for the yeast, it was shown that the ripening cultures could be re-isolated in high numbers, 10(8) CFU cm(-2) for C. casei and 10(6) CFU cm(-2) for D. hansenii, from the cheese surface after 2.5 weeks of ripening. CONCLUSIONS: Ripening strains of surface ripening cultures can be stored in MRD containing 5% w/v salt at 4 degrees C for at least 40 days. Such cultures are recovered in high numbers from the cheese during ripening. SIGNIFICANCE AND IMPACT OF STUDY: This study has provided a low-cost and efficient way to store bacteria that could be used as ripening cultures for smear cheese. Such cultures can be recovered in high numbers from the cheese surface during ripening.     

Unveiling the fungal mycobiota present throughout the cork stopper manufacturing process

A particular fungal population is present in the main stages of the manufacturing process of cork discs. Its diversity was studied using both dependent (isolation) and independent culture methods (denaturing gel gradient electrophoresis and cloning of the ITS1-5.8S-ITS2 region). The mycobiota in the samples taken in the stages before and after the first boiling seems to be distinct from the population in the subsequent manufacturing stages. Most isolated fungi belong to the genera Penicillium, Eurotium and Cladosporium. The presence of uncultivable fungi, Ascomycota and endophytes in raw cork was confirmed by sequencing. The samples taken after the first boiling contained uncultivable fungi, but in a few samples some isolated fungi were also detected. The main taxa present in the following stages were Chrysonilia sitophila, Penicillium glabrum and Penicillium spp. All applied techniques had complementary outcomes. The main factors driving the shift in cork fungal colonization seem to be the high levels of humidity and temperature to which the slabs are subjected during the boiling process.     

Enterobacteriaceae as indicators of good manufacturing practices in rendering plants

Finished products and samples from the environment of the production line in rendering plants were checked for Enterobacteriaceae and salmonellae. Improvements in hygiene and measures taken to limit multiplication of microorganisms in the dry area of the production lines resulted in reduction of both numbers of Enterobacteriaceae in environmental samples and frequency of their occurrence in finished products. Simultaneously, there was an equivalent reduction of salmonellae positives in environmental samples and finished products. Consequently, the determination of Enterobacteriaceae can be used as an effective tool to assess the improvements in good manufacturing practices.Based on a paper presented at the Symposium on Food Microbiology, The Hague, September 1981.