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.     

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.     

Yeast populations associated with the artisanal cheese produced in the region of Serra da Canastra, Brazil

The aim of this work was to describe the yeast populations present during the manufacturing of Minas cheese of the region of Serra da Canastra, Minas Gerais state, Brazil. Canastra cheese is produced from raw cow’s milk at the farmhouse level using artisanal procedures and natural whey cultures as starters. Samples from 10 farms were studied, and they included: raw milk, natural starter, cheese curd before salting and cheese after 5 days of ripening. The most frequent yeasts in whey, curd and cheese were Debaryomyces hansenii, Kluyveromyces lactis, Kodamaea ohmeri and Torulaspora delbrueckii. Many yeast isolates were able to produce proteases, lipases and β-galactosidades. Production of these enzymes by yeasts in the cheese would contribute to the development of the characteristic flavor and smell during the ripening process.     

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.     

Enhancement of cheese flavors with microbial esterases

The characteristic flavor of hard Italian cheeses is associated with the presence of fatty acids, particularly butyric acid, liberated from milk fat during the ripening process. To ensure proper development and control of flavor, animal pregastric esterases or lipases are routinely added to the milk before coagulation of the curd. Such esterases are also used to generate flavor in enzyme modified cheese and other dairy products. Esterases from microbial sources have been investigated as agents to enhance flavor in cheese. We have found that an esterase from Mucor miehei exhibits the type of lipolytic activity needed for this application. Romano and fontina cheeses of excellent quality have been prepared by the use of this esterase. It has also been used successfully in the preparation of enzyme modified cheese, and, in turn, processed American cheese.     

Studies on Milk Clotting Enzymes Produced by Basidiomycetes

In the course of screening tests of Basidiomycete proteolytic enzymes, it was observed that some strains produced milk clotting enzymes with fairly weak proteolytic activities.

When sucrose-polypeptone and sucrose-corn steep liquor media were used, only 6 strains out of 44 strains tested showed weak milk clotting activities. Cheddar cheese making with culture filtrates of these 6 strains revealed that the culture filtrates of 2 strains, Irpex lacteus Fr. and Fomitopsis pinicola (Fr.) Karst., were able to produce Cheddar cheese of good quality.

On the other hand, when sucrose-distillers solubles media were used, a lot of strains showed high proteolytic activity in addition to high milk clotting activity. The ratio of milk clotting to proteolytic activities (MCA/PA) was assumed to be an important index for the selection of organism, and F. pinicola and Coriolus consors (Berk.) Imaz. were selected as the strain with high MCA/PA ratio.

As the investigation on culture conditions of 3 strains mentioned above showed that F. pinicola and I. lacteus, were richly productive of milk clotting enzymes, the 2 strains except C. consors were used for further studies on cheese making.

Cheddar cheese making with crude enzymes revealed that cheese products produced by the enzyme of F. pinicola had a slightly bitter taste after 5 months’ ripening but that those produced by the enzyme of I. lacteus had good quality.     

The production of mixed cultures containing strains of Lactococcus lactis, Leuconostoc cremoris and Lactobacillus rhamnosus, on commercial starter media

Mixed starters containing Lactococcus lactis, Leuconostoc cremoris and Lactobacillus rhamnosus strains were produced on commercial starter media (MB Complete, Thermolac, Marlac), as well as on milk. With the exception of Marlac, the starters were cultured under pH control. The effect of media and incubation temperature (22 or 32°C) on population ratios, on specific acidifying activities (SAA) of the cultures as well as on their ability to produce aroma compounds in milk was studied. The starters had higher contents in lactobacilli when they were produced at 32°C, whereas a tendency to obtain higher Leuconostoc populations was observed at 22°C. With respect to the lactococci, there was a significant interaction between temperature and growth medium for both strains. Thus, Le. cremoris T2 reached higher populations at 32°C if grown in MB complete and Thermolac, whereas in Marlac and skim milk, viable counts were higher at 22°C. The lactococci represented 50% of the total population of the culture at the beginning of the incubation, but they composed between 80% and 99% of the total population following fermentation. The best medium for growth of Leuconostoc was milk, but populations of only 10⁸ cfu/ml were reached. The lactobacilli did not grow well in MB Complete, and their development was best in the low-phosphate Marlac medium. The cultures grown on Marlac had the highest SAA values, whereas those grown on MB complete had the lowest. Overall, more ethanol and diacetyl were detected in the fermented milks when the starters used to inoculate them were produced at 22°C. Journal of Industrial Microbiology & Biotechnology (2000) 25, 288–297. Received 23 June 2000/ Accepted in revised form 22 September 2000     

Characteristics of lactococci cultures produced in commercial media

Strains ofLactococcus lactis ssplactis andL. lactis sspcremoris were propagated on milk, three commercial highly buffered media (HB media), and four commercial media designed for external pH control (EC media). With milk and HB media, fermentation was allowed to proceed until a pH of 4.9 was reached. With EC media, pH was maintained at 6.0 with 5 N NH₄OH. The cultures were analyzed for chain length, viable population, specific acidifying activity (SAA) and specific proteolytic activity (SPA). The starters were stored at 4° C for 3 days, and analyses for chain length, viable population and SAA were repeated. It was more difficult to standardize medium composition with the rehydrated commercial blends, as their titratable acidities had greater proportional variations than milk. As a rule, chain length was longer in fresh cultures than in the stored starters, andL. lactis sppcremoris cultures had longer chains thanL. lactis ssplactis. All commercial media produced starters with total populations at least as high as that obtained in milk. With the EC media, populations could be five times greater than with milk; increases were less important in HB media. The increase in population in EC and HB media was more marked withL. lactis ssplactis than forL. lactis sspcremoris strains. Storage at 4° C for 3 days did not significantly reduceL. lactis populations, but mortality (up to 70%) was observed withL. lactis sspcremoris. The overall SAA ofL. lactis ssplactis cultures in EC media was 35% lower than milk- or HB media-grown starters, but the greater populations reached in EC media enabled a significant reduction in inoculation rate. Some statistically significant correlations were obtained between SAA and SPA (positive) as well as with chain length (negative), but the coefficients of determination were generally very low. The drop in pH during storage at 4° C was less with HB media than in milk, and was in relation to their buffering capacity.     

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.     

Spatial Distribution of Lactococcus lactis Colonies Modulates the Production of Major Metabolites during the Ripening of a Model Cheese

In cheese, lactic acid bacteria are immobilized at the coagulation step and grow as colonies. The spatial distribution of bacterial colonies is characterized by the size and number of colonies for a given bacterial population within cheese. Our objective was to demonstrate that different spatial distributions, which lead to differences in the exchange surface between the colonies and the cheese matrix, can influence the ripening process. The strategy was to generate cheeses with the same growth and acidification of a Lactococcus lactis strain with two different spatial distributions, big and small colonies, to monitor the production of the major ripening metabolites, including sugars, organic acids, peptides, free amino acids, and volatile metabolites, over 1 month of ripening. The monitored metabolites were qualitatively the same for both cheeses, but many of them were more abundant in the small-colony cheeses than in the big-colony cheeses over 1 month of ripening. Therefore, the results obtained showed that two different spatial distributions of L. lactis modulated the ripening time course by generating moderate but significant differences in the rates of production or consumption for many of the metabolites commonly monitored throughout ripening. The present work further explores the immobilization of bacteria as colonies within cheese and highlights the consequences of this immobilization on cheese ripening.     

Dynamics of Bacterial Communities during the Ripening Process of Different Croatian Cheese Types Derived from Raw Ewe's Milk Cheeses

Microbial communities play an important role in cheese ripening and determine the flavor and taste of different cheese types to a large extent. However, under adverse conditions human pathogens may colonize cheese samples during ripening and may thus cause severe outbreaks of diarrhoea and other diseases. Therefore in the present study we investigated the bacterial community structure of three raw ewe''s milk cheese types, which are produced without the application of starter cultures during ripening from two production sites based on fingerprinting in combination with next generation sequencing of 16S rRNA gene amplicons. Overall a surprisingly high diversity was found in the analyzed samples and overall up to 213 OTU₉₇ could be assigned. 20 of the major OTUs were present in all samples and include mostly lactic acid bacteria (LAB), mainly Lactococcus, and Enterococcus species. Abundance and diversity of these genera differed to a large extent between the 3 investigated cheese types and in response to the ripening process. Also a large number of non LAB genera could be identified based on phylogenetic alignments including mainly Enterobacteriaceae and Staphylococcacae. Some species belonging to these two families could be clearly assigned to species which are known as potential human pathogens like Staphylococcus saprophyticus or Salmonella spp. However, during cheese ripening their abundance was reduced. The bacterial genera, namely Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Brevibacterium, Corynebacterium, Clostridium, Staphylococcus, Thermoanerobacterium, E. coli, Hafnia, Pseudomonas, Janthinobacterium, Petrotoga, Kosmotoga, Megasphaera, Macrococcus, Mannheimia, Aerococcus, Vagococcus, Weissella and Pediococcus were identified at a relative low level and only in selected samples. Overall the microbial composition of the used milk and the management of the production units determined the bacterial community composition for all cheese types to a large extend, also at the late time points of cheese ripening.     

Biosynthesis of flavors by Penicillium roqueforti

The development of the unique flavor of blue type cheese depends on the concerted action of numerous enzymes of Penicillium roqueforti involved in protein and lipid metabolism. Protease(s) by degrading casein modify the texture and background flavor of the ripening cheese. Lipase by hydrolyzing milk triglycerides provides flavorful fatty acids and precursors of methyl ketones. The enzyme complex involved in the partial oxidation of free fatty acids and the properties of β-ketoacyl decarboxylase which generates the major flavor components of blue cheese are discussed. Fermentation of P. roqueforti for the rapid production of methyl ketones is briefly reviewed.     

Use of thermophilic lactic starters in the dairy industry

The use of thermophilic lactic starters in the dairy industry is discussed. The functions of the thermophilic lactic starters in cooked cheese production and its ripening, the bacteria of the starter cultures and various types of starters are described.     

Strategy for Manipulation of Cheese Flora Using Combinations of Lacticin 3147-Producing and -Resistant Cultures

The aim of the present study was to develop adjunct strains which can grow in the presence of bacteriocin produced by lacticin 3147-producing starters in fermented products such as cheese. A Lactobacillus paracasei subsp. paracasei strain (DPC5336) was isolated from a well-flavored, commercial cheddar cheese and exposed to increasing concentrations (up to 4,100 arbitrary units [AU]/ml) of lantibiotic lacticin 3147. This approach generated a stable, more-resistant variant of the isolate (DPC5337), which was 32 times less sensitive to lacticin 3147 than DPC5336. The performance of DPC5336 was compared to that of DPC5337 as adjunct cultures in two separate trials using either Lactococcus lactis DPC3147 (a natural producer) or L. lactis DPC4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious nonstarter lactic acid bacteria in cheddar cheese. Lacticin 3147 was produced and remained stable during ripening, with levels of either 1,280 or 640 AU/g detected after 6 months of ripening. The more-resistant adjunct culture survived and grew in the presence of the bacteriocin in each trial, reaching levels of 10⁷ CFU/g during ripening, in contrast to the sensitive strain, which was present at levels 100- to 1,000-fold lower. Furthermore, randomly amplified polymorphic DNA-PCR was employed to demonstrate that the resistant adjunct strain comprised the dominant microflora in the test cheeses during ripening.     

Proteolytic enzyme activities in Cheddar cheese juice made using lactococcal starters of differing autolytic properties

AIMS: To determine proteolytic enzyme activities released in Cheddar cheese juice manufactured using lactococcal starter strains of differing autolytic properties. METHODS AND RESULTS: The activities of residual chymosin, cell envelope proteinase and a range of intracellular proteolytic enzymes were determined during the first 70 days of ripening when starter lactococci predominate the microbial flora. In general, in cell free extracts (CFE) of the strains, the majority of proteolytic activities was highest for Lactococcus lactis HP, intermediate for L. lactis AM2 and lowest for L. lactis 303. However, in cheese juice, as ripening progressed, released proteolytic activities were highest for the highly autolytic strain L. lactis AM2, intermediate for L. lactis 303 and lowest for L. lactis HP. CONCLUSIONS: These results indicate that strain related differences in autolysis influence proteolytic enzyme activities released into Cheddar cheese during ripening. No correlation was found between proteolytic potential of the starter strains measured in CFE prior to cheese manufacture and levels of activities released in cheese juice. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings further support the importance of autolysis of lactococcal starters in determining the levels of proteolytic activities present in cheese during initial stages of ripening.     

Microbial Diversity of a Camembert-Type Cheese Using Freeze-Dried Tibetan Kefir Coculture as Starter Culture by Culture-Dependent and Culture-Independent Methods

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it''s necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese.     

Fate of Mycobacterium avium subsp. paratuberculosis in Swiss Hard and Semihard Cheese Manufactured from Raw Milk

Raw milk was artificially contaminated with declumped cells of Mycobacterium avium subsp. paratuberculosis at a concentration of 10⁴ to 10⁵ CFU/ml and was used to manufacture model hard (Swiss Emmentaler) and semihard (Swiss Tisliter) cheese. Two different strains of M. avium subsp. paratuberculosis were tested, and for each strain, two model hard and semihard cheeses were produced. The survival of M. avium subsp. paratuberculosis cells was monitored over a ripening period of 120 days by plating out homogenized cheese samples onto 7H10-PANTA agar. In both the hard and the semihard cheeses, counts decreased steadily but slowly during cheese ripening. Nevertheless, viable cells could still be detected in 120-day cheese. D values were calculated at 27.8 days for hard and 45.5 days for semihard cheese. The most important factors responsible for the death of M. avium subsp. paratuberculosis in cheese were the temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening. Since the ripening period for these raw milk cheeses lasts at least 90 to 120 days, the D values found indicate that 10³ to 10⁴ cells of M. avium subsp. paratuberculosis per g will be inactivated.     

Effect of lactic starter cultures on the organic acid composition of milk and cheese during ripening—analysis by HPLC

The major function of lactic starter cultures in cheese making is to produce lactic and other organic acids from the carbohydrates present in milk. The activity of six starter cultures consisting of two Lactococcus lactis ssp. lactis , two Lactococcus lactis ssp. lactis biovar. diacetylactis and two Leuconostoc strains, was tested by monitoring the evolution of the organic acid composition of milk by a modified HPLC method. In addition, their performance as cheese starters was also tested. The HPLC method developed proved to be a precise tool to monitor the organic acid content. Thus, it can be used to follow the fermentation ability of starter cultures, providing information about the type of fermentation. The use of any of the six starters assayed is suggested for manufacturing Afuega'l Pitu cheese.