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.     

Diversity among lactococci isolated from ewes' raw milk and cheese

P. GAYA, M. BABÍN, M. MEDINA and M. NUÑEZ.1999.The technological and genetic characteristics of lactococci present in ewes' raw milk and 1-d-old ewes' raw milk cheeses sampled over a 1-year period were investigated. The proportion of lactic acid bacteria isolates from milk samples able to decrease milk pH by more than 1·25 units after 6 h incubation at 30 °C reached 14·5% in spring vs 10·7% in summer, 8·3% in autumn and 3·0% in winter. In 1-d-old cheese samples, the proportion of lactic acid bacteria able to lower milk pH by more than 1·25 units increased up to 32·3% in spring vs 23·4% in summer, 8·0% in autumn and 10·3% in winter. Fast acid-producing lactic acid bacteria mainly belonged to the genus Lactococcus . Using polymerase chain reaction protocols, fast acid-producing lactococci were grouped as 61  Lactococcus lactis subsp. lactis , 13  L. lactis subsp. cremoris and 14  L. lactis subsp. lactis biovar diacetylactis. Randomly amplified polymorphic DNA (RAPD) fingerprinting of fast acid-producing lactococci, using two primers, resulted in 21 different RAPD patterns for L. lactis subsp. lactis isolates, nine RAPD patterns for L. lactis subsp. cremoris isolates and three RAPD patterns for L. lactis subsp. lactis biovar diacetylactis isolates. Up to 19 different RAPD patterns were found for L. lactis isolates from cheeses made in a particular month.     

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.     

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.     

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.     

The microstructure and distribution of micro-organisms within mature Serra cheese

The distribution of micro-organisms in mature Serra, a traditional Portuguese cheese made from unpasteurised ewes' milk without added starter culture, was examined by light microscopy and electron microscopy. Four populations of micro-organisms were recognized according to their position within the cheese: (i) those present as apparently axenic colonies within the curd matrix; (ii) bacteria growing along curd junctions; (iii) yeasts and bacteria present in the smear on the surface of the cheese and (iv) bacteria found in cracks which penetrated the outer part of the cheese from the rind. Two types of crystals were observed, together with contaminants of vegetable origin and somatic cells originating from the milk.     

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.     

Biotechnological methods to accelerate cheddar cheese ripening

Cheese is one of the dairy products that can result from the enzymatic coagulation of milk. The basic steps of the transformation of milk into cheese are coagulation, draining, and ripening. Ripening is the complex process required for the development of a cheese's flavor, texture and aroma. Proteolysis, lipolysis and glycolysis are the three main biochemical reactions that are responsible for the basic changes during the maturation period. As ripening is a relatively expensive process for the cheese industry, reducing maturation time without destroying the quality of the ripened cheese has economic and technological benefits. Elevated ripening temperatures, addition of enzymes, addition of cheese slurry, attenuated starters, adjunct cultures, genetically engineered starters and recombinant enzymes and microencapsulation of ripening enzymes are traditional and modern methods used to accelerate cheese ripening. In this context, an up to date review of Cheddar cheese ripening is presented.     

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.     

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.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2–3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10⁷ cfu/g.     

Proteinases encapsulated in stimulated release liposomes for cheese ripening

Chymosin, a neutral proteinase from Bacillus subtilis and cardoon cyprosins, were co-encapsulated with phospholipase C in stimulated release liposomes. Encapsulated enzymes were added separately to milk to make cheese. Chymosin and the neutral proteinase accelerated as-casein degradation in comparison with control cheese, whereas b-casein degradation was accelerated by neutral proteinase and cyprosins. Neutral proteinase yielded the highest increase in soluble nitrogen. Cheese flavour intensity was enhanced by the neutral proteinase and cyprosins but not by chymosin.     

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.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2-3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10(7) cfu/g.     

Genetic diversity of lactococci and enterococci isolated from home-made Pecorino Sardo ewes' milk cheese

AIMS: To assess the intraspecific genetic diversity of lactococci and enterococci isolated from 24-h, 1- and 2-month-old home-made Pecorino Sardo ewes' milk cheese. METHODS AND RESULTS: Two molecular techniques, plasmid profiling and pulsed-field gel electrophoresis, were used in order to type the isolates at strain level. The present study revealed that the lactococcal and enterococcal microbial populations of home-made Pecorino Sardo cheese were complex, not only 24 h after manufacture, but also after 1 and 2 months of ripening. The genetic diversity at subspecies level ranged from 58 to 80% during the three periods examined. The study also showed that the strains that dominated in the first stage of ripening were not necessarily predominant in the later periods. A high number of strains isolated at 24 h were still present in the mature cheese, but many of the genotypes were only found in the cheese after 1 or 2 months. CONCLUSIONS: The results showed a high intraspecific genetic diversity in the natural microbial population colonizing home-made Pecorino Sardo cheese. Two molecular techniques are necessary for a thorough and precise typing at strain level in order to better distinguish between closely related isolates and between isolates that probably belong to the same clonal lineage. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic complexity observed in the present study is of particular relevance in the preservation of the natural microflora of traditional Protected Designation of Origin raw milk cheeses, as well as in the selection of new starter strains for the dairy industry.     

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.     

Microbiological characteristics of Crottin goat cheese made in different seasons

General profiles and effect of season on microbiological characteristics of Crottin goat's cheese were evaluated in different batches during storage. Microorganisms determined were: mesophilic, proteolytic, halotolerant, psychotrophs, coliforms, yeasts and Staphylococcus aureus. The presence of Escherichia coli, Salmonella spp., Yersinia enterocolitica and pH and moisture content were also analyzed. E. coli, Salmonella spp. and Y. enterocolitica were not detected in the Argentinian soft goat cheese. Cheese made during winter had significant differences in mesophilic, psychrotrophics, halotolerant microorganisms and yeast between batches. In fall cheese, there were significant differences to mesophilic, proteolytic and psychrotrophics. There were not significant differences to the microbial groups in summer cheese. The variability between batches in Crottin cheese occurred during cold months, which may be attributed to the coagulation process of the cheese at environmental temperature (without any control), creating a delay in the lactic flora activity in winter. The results showed a significant influence of the seasons in the microbial population evolution during storage, suggesting that the differences in each microbial group in different seasons could be caused by differences in the predominant strains of each group. Further studies are needed for the composition of the microorganisms, where goat's products have not specific hygienic and quality standards in Argentina.     

Microbiological characteristics of anevato: a traditional greek cheese

Nine batches of Anevato, raw goat milk cheese, were examined throughout a 60 day storage time at three different periods within the lactation season of the goat. High mean log counts per gram of cheese for aerobic bacteria (7·92–9·56), lactic acid bacteria (7·78–9·32), Gram-negative organisms 5·64–9·67), psychrotrophs (7·90–11·79) and proteolytic bacteria (7·57–9·36) were found. Enterobacteriaceae, coliforms and yeasts were considerably lower. Enterobacteriaceae and coliforms in the curd of cheese made in May were lower by approximately 3·0 log₁₀ cfu g⁻¹ than counts in curd made in January, and were lower by about 2·5 log₁₀ cfu g⁻¹ than those in cheese made in March. This coincided with lower pH and higher counts of lactic acid bacteria in cheese made in March and May. Yeast populations were affected by the season and were higher in May than March and/or January. Lactococci dominated in the cheese until 15 days, but lactobacilli became predominant after 30 days. Lactococcus lactis was the most abundant species of lactic acid bacteria found in Anevato cheese. Results suggest the need for improving milk quality and/or using heat-treated milk to produce Anevato cheese; the use of L. lactis as a starter would possibly eliminate or suppress the growth of undesirable organisms.     

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.     

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.