Causal Relationship between Microbial Ecology Dynamics and Proteolysis during Manufacture and Ripening of Protected Designation of Origin (PDO) Cheese Canestrato Pugliese

Pyrosequencing of the 16S rRNA gene, community-level physiological profiles determined by the use of Biolog EcoPlates, and proteolysis analyses were used to characterize Canestrato Pugliese Protected Designation of Origin (PDO) cheese. The number of presumptive mesophilic lactococci in raw ewes'' milk was higher than that of presumptive mesophilic lactobacilli. The numbers of these microbial groups increased during ripening, showing temporal and numerical differences. Urea-PAGE showed limited primary proteolysis, whereas the analysis of the pH 4.6-soluble fraction of the cheese revealed that secondary proteolysis increased mainly from 45 to 75 days of ripening. This agreed with the concentration of free amino acids. Raw ewes'' milk was contaminated by several bacterial phyla: Proteobacteria (68%; mainly Pseudomonas), Firmicutes (30%; mainly Carnobacterium and Lactococcus), Bacteroidetes (0.05%), and Actinobacteria (0.02%). Almost the same microbial composition persisted in the curd after molding. From day 1 of ripening onwards, the phylum Firmicutes dominated. Lactococcus dominated throughout ripening, and most of the Lactobacillus species appeared only at 7 or 15 days. At 90 days, Lactococcus (87.2%), Lactobacillus (4.8%; mainly Lactobacillus plantarum and Lactobacillus sakei), and Leuconostoc (3.9%) dominated. The relative utilization of carbon sources by the bacterial community reflected the succession. This study identified strategic phases that characterized the manufacture and ripening of Canestrato Pugliese cheese and established a causal relationship between mesophilic lactobacilli and proteolysis.     

Population dynamics of lactobacilli in Grana cheese

A survey on the presence, microbial diversity, and population dynamics of lactobacilli in Grana cheese is presented. Evolution of thermophilic rod lactic acid bacteria within the first two days from cheese making and during ripening was different according to different bacterial groups, which were selectively enumerated and identified by molecular methods. Species-specific microbial counts indicated prevalence ofLactobacillus helveticus in both the whey starter and the cheese at moulding, and ofLactobacillus delbrueckii subsp.lactis in cheese after two months of ripening. In more advanced ripening, a decrease of total thermophilic lactobacilli and an increase of mesophilic lactobacilli (mostly belonging toLactobacillus casei/paracasei andLactobacillus rhamnosus) was observed. PCR fingerprinting of lactobacilli, which was performed by PCR-fingerprinting, indicated a marked microbial heterogeneity within theLactobacillus spp. populations, which enabled strain (or group)-specific fingerprints to be observed.     

Impact of two starter cultures on proteolysis in Kashkaval cheese

Summary Kashkaval cheese is produced with a traditional yogurt starter culture S. thermophilus 13a + Lb. delbrueckii ssp. bulgaricus 2–11. The present paper has investigated the contribution of a selected starter culture (Lactococcus lactis ssp. lactis C11 + Streptococcus thermophilus P23 + Lactobacillus casei ssp. casei RP5) for proteolysis of Kashkaval (A) in comparison with a traditional yogurt culture (B) and soluble nitrogen (at pH 4.6 and in 12% TCA) and free amino acids as indicators of Kashkaval ripeness. After the microflora was isolated and differentiated, the largest count of mesophilic cocci (69%) and lower counts of thermophilic cocci (19.8%) and mesophilic lactobacilli (11.2%) were found in cheddared curd A, whereas in cheddared curd B thermophilic cocci (69%) prevailed over thermophilic lactobacilli (30%). Hot-brining of curd at 72 °C for 2 min destroyed 91 and 84.5%, respectively, of the starter microflora A and B and two batches of Kashkaval started ripening with 3.9 × 10⁸ c.f.u. g⁻¹ and 3.4 × 10⁸ c.f.u. g⁻¹. The ripening of Kashkaval cheese occurred mainly under the influence of the starter microflora. Ripening of Kashkaval A occurred with dominating presence of Lactobacillus casei ssp. casei RP5 from 64% at day 30 to 95.2% at day 90. In Kashkaval B the participation of Lactobacillus delbrueckii ssp. bulgaricus 2–11 increased from 36.3 to 39.9%, however it always remained relatively smaller (1.5–2.3-fold) than that of thermophilic cocci. Great activity of transformation of pH 4.6-soluble nitrogen into TCA-soluble nitrogen in Kashkaval A was registered. The high proteolytic activity of starter culture A shortened the ripening process by 30 days (30-day-old Kashkaval A had 17.3% level of proteolysis, 29.9% depth of proteolysis). Kashkaval B reached satisfactory ripeness properties when it was 60 days old (20.2% level of proteolysis, 24.9% depth of proteolysis). The high activity of amino acid release mainly by Lactobacillus casei ssp. casei RP5 and to a lesser extent by the cocci caused accumulation of a considerable amount of free amino acids in ripe Kashkaval A (421.9 mg (100 g)⁻¹ for 30-day-old Kashkaval). In ripe 30-day-old Kashkaval A the concentration of free amino acids was three times higher than in Kashkaval B, with domination of lysine, leucine, phenylalanine, valine and threonine.     

Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis

Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products.     

Proteolytic activity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in frozen-stored Kashkaval cheese

Proteolytic activity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in Kashkaval cheeses of varying aging times, stored at −10 to −12°C for 12 months, was studied. It was established that the proteolysis of Kashkaval cheese induced by the starter culture was significantly delayed by freezing. The noncasein nitrogen (NCN/TN) and nonprotein nitrogen (NPN/TN) as a percentage of total nitrogen increased slightly during frozen storage of Kashkaval. It was found that NCN/TN and NPN/TN values increased to a larger extent in frozen-stored Kashkaval samples with shorter aging time. Enhanced proteolysis was observed during ripening of thawed Kashkaval cheese. There was greater accumulation of noncasein nitrogen in thawed Kashkaval samples compared to the control samples. The enhanced proteolysis during ripening of thawed Kashkaval cheese resulted in larger amounts of high and medium molecular weight peptides and lower amounts of low molecular weight peptides and free amino acids as compared to controls.     

Growth and Proteolytic Activity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in Reduced Sodium Kashkaval Cheese

Summary Reduced sodium Kashkaval cheese was produced from cow’s milk. Mixtures of NaCl, NaCl:KCl (1:1, 2:1) and NaCl:KH₂PO₄ (1:1, 2:1) were used for hot brining and salting of the cheddarized cheese curd. There were no significant differences (P < 0.05) in the count of Lb. delbrueckii ssp. bulgaricus after aging of Kashkaval samples. At the end of the ripening process the counts of Lb. delbrueckii ssp. bulgaricus reached 10⁶ c.f.u./g and the counts of Streptococcus thermophilus varied from 10⁴ to 10⁵ c.f.u./g. Proteolysis during ripening of reduced sodium Kashkaval cheese, initiated by the starter microorganisms Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, was studied through the changes in the levels of non-casein and non-protein nitrogen. It was observed that non-casein and non-protein nitrogen increased significantly (P < 0.05) during ripening. The amounts of non-casein and non-protein nitrogen accumulated in the studied Kashkaval samples were similar. That indicates that the partial replacement of NaCl with KCl or KH₂PO₄ does not cause significant changes in the course of proteolysis of Kashkaval cheese by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.     

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.     

Detection, Growth, and Amine-Producing Capacity of Lactobacilli in Cheese

A differential plating medium was developed to detect decarboxylating lactobacilli in cheese. With this medium, 15 cheeses made from raw milk were investigated for the presence of these bacteria. Five histidine-decarboxylating strains and one tyrosine-decarboxylating strain were isolated. The isolates were identified with the API 50L system. Accordingly, each of the five histidine-decarboxylating strains was identified as Lactobacillus buchneri, whereas the tyrosine-decarboxylating strain is a representative of Lactobacillus brevis. Cheesemaking experiments using a low inoculum concentration of the histidinedecarboxylating L. buchneri strain St2A (0.2 CFU/ml of milk) showed that, under conditions of accelerated proteolysis, histamine may accumulate rapidly; after 3 months of ripening, 410 mg/kg was found. An inoculum concentration of 5 CFU/ml gave rise to the formation of 1,060 mg/kg.     

Manufacture of Cheddar cheese using probiotic Lactobacillus plantarum K25 and its cholesterol-lowering effects in a mice model

The probiotic adjunct Lactobacillus plantarum K25 was inoculated into milk to produce probiotic cheese. The effect of Lb. plantarum K25 on cheese composition, microbiological growth and survival during the manufacturing and ripening period, primary and secondary proteolysis during cheese ripening, and the in vivo cholesterol-lowering ability of the probiotic cheese were investigated. The results showed that the use of adjunct Lb. plantarum K25 in Cheddar cheese did not affect the cheese components including moisture, protein, fat, salt content and the pH value of cheese. During the whole ripening period, the probiotic adjunct maintained its viability, suggesting the effectiveness of Cheddar cheese as a vehicle for delivery of probiotic bacteria. No significant differences were observed in water-soluble nitrogen, 70?% ethanol-soluble nitrogen, 5?% phosphotungstic acid-soluble nitrogen, free amino acids and urea-PAGE patterns between the control and probiotic cheeses. Assessment of the in vivo cholesterol-lowering property of cheese with Lb. plantarum K25 showed that the levels of serum total cholesterol, low-density lipoprotein cholesterol and triglycerides decreased significantly, and the level of serum high-density lipoprotein cholesterol increased in mice fed with the probiotic cheese. The results indicated the potential function as a dietary item of the probiotic cheese with Lb. plantarum K25 to reduce the risk of cardiovascular diseases.     

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.     

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.     

Ability of Thermophilic Lactic Acid Bacteria To Produce Aroma Compounds from Amino Acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an α-keto acid such as α-ketoglutarate (α-KG) as the amino group acceptor, and produced α-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces α-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without α-KG addition. In the presence of α-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced α-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from α-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an α-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the α-keto acid conversion to acids in L. helveticus and S. thermophilus is an α-keto acid dehydrogenase that produces acyl coenzymes A.     

A survey of the microbial and chemical composition of seven semi-ripened Provola dei Nebrodi Sicilian cheeses

AIMS: The microbial and chemical composition of seven different semi-ripened (45 days) Provola dei Nebrodi Sicilian cheese samples were assessed in order to investigate the diversity of the microbial population in cheese made from different geographical areas throughout Sicily. METHODS AND RESULTS: The samples, which were obtained from seven different Provola dei Nebrodi manufacturers, were assessed using selective media. Interestingly, concentrations of presumptive lactobacilli represented over 90% of the total microbial population. In total, 105 presumptive Lactobacillus isolates were characterized to determine the relatedness of the isolates between the seven different cheeses. Randomly amplified polymorphic DNA polymerase chain reaction (RAPD PCR) analysis of the 105 presumptive lactobacilli indicated the presence of 22 distinct isolates. Further investigation of the isolates using pulsed field gel electrophoresis (PFGE) following restriction with the enzyme ApaI revealed the presence of 19 distinct macrorestriction patterns and the presence of between one and four distinct isolates per cheese sample (out of a total of 15 isolates per cheese randomly taken from Lactobacillus selective media plates). Analysis of the 16S rDNA sequence of each genetically distinct isolate demonstrated the dominance of the Lactobacillus casei species in all cheese samples assessed. Lactobacillus delbrueckii and Pediococcus pentosaceus species were also detected. The concentration of free amino acids, used to estimate the extent of proteolysis in each cheese, ranged from 59 to 433 mg 100 g(-1) cheese. CONCLUSIONS: Microbiological assessment of the cheeses demonstrated the dominance of Lactobacillus species after 45 days of ripening with levels ranging from 8.3 to 9.4 log CFU g(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the diversity of lactobacilli within an artisanal Sicilian cheese, enabling the identification of 17 strains of Lact. casei, one strain of Lact. delbrueckii and Ped. pentosaceus through the combined use of RAPD PCR, PFGE and 16S rDNA sequencing.     

The effect of low pressure-homogenized lactic cultures on the development of proteolysis in cheese slurry

Summary The aim of this study was to determine the effect of low pressure-homogenization of lactic acid bacteria (LAB) on the development of proteolysis in the slurry medium. For the slurry, the milk was pasteurized at 65 °C for 30 min, cooled to 32 °C and coagulated. The curd obtained was blended; the dry matter was adjusted to 30% by adding distilled water, placed into the flasks and autoclaved. The LAB Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactobacillus helveticus were used in cheese slurry. Homogenization was performed at 30 MPa and 40 °C. The cheese slurries were incubated with and without homogenized cultures at 9 and 30 °C for up to 72 h. During incubation, the changes in trichloroacetic acid-soluble nitrogen (TCA-SN) and phosphotungstic acid-soluble nitrogen (PTA-SN) as well as pH were monitored. The results showed that pH development was slower in the slurries to which homogenized culture was added. Higher TCA-SN and PTA-SN values were obtained from the slurries incubated at 30 °C. Moreover, higher TCA-SN and PTA-SN values were found in the slurries incubated with homogenized mesophilic culture and Lb. helveticus (P<0.05). The results suggested that homogenization of the cultures was a promising method for the acceleration of cheese ripening.     

Biogenic amine-forming microbial communities in cheese

The aim of this study was to screen two cheese starter cultures and cheese-borne microbial communities with the potential to produce biogenic amines in cheese during ripening. Bacteria of the genera Enterococcus and Lactobacillus and coliform bacteria were isolated from Dutch-type semi-hard cheese at the beginning of the ripening period. Statistically significant counts of bacterial isolates were screened for the presence of specific DNA sequences coding for tyrosine decarboxylase (tyrDC) and histidine decarboxylase (hDC) enzymes. The PCR analysis of DNA from 14 Enterococcus and 3 Lactobacillus isolates confirmed the presence of the targetted DNA sequences. Simultaneously, 13 tyrDC- and 3 hDC-positive isolates were grown in decarboxylase screening medium and this was followed by HPLC analysis of the produced tyramine and histamine. Conventional and molecular taxonomic analyses of the above-mentioned isolates identified the following species: Enterococcus durans (7 strains), Enterococcus faecalis (3 strains), Enterococcus faecium (1 strain), Enterococcus casseliflavus (3 strains), Lactobacillus curvatus (1 strain), Lactobacillus lactis (1 strain) and Lactobacillus helveticus (1 strain). All of the above Enterococcus and two of the Lactobacillus strains originated from contaminating microbial communities. The L. helveticus strain, which was tyrosine decarboxylase-positive and exhibited tyramine production, originated from starter culture 1 used for cheese production. Comparison of partial tyrDC sequences of positive Enterococcus isolates revealed 89% sequence similarity, and that of hDC-positive Lactobacillus isolates revealed 99% sequence similarity.     

Relation of Cheddar Cheese Ripening to Bacterial Lipolysis

This investigation was undertaken to find the relationship between fat hydrolysis and lipolytic activities of lactic acid bacteria participated in Cheddar cheese ripening. Increases in titratable acidities due to lactic fermentation were completed at early stage of ripening. Ripening indices (ratio of water-soluble nitrogen to total nitrogen) increased rapidly until 90 days and thereafter gradually up to 150 days. Considerable amounts of free fatty acids were released from cheese fat throughout the ripening period. Cheese bacteria were enumerated on the media of tomato-glucose-agar and acetate-agar. About 70% of bacteria isolated from cheese at age of 150 days were classified into Lactobacillus casei and L. plantarum. Lipolytic activities of lactobacilli isolated were detected definitely on double-layered agar plates containing Victoria blue-stained olive oil. Lipase activities were determined in cheese extracts during ripening.     

Effects of copper supplement on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture

Aims: To determine the effects of supplemented copper (Cu²⁺) on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture. Methods and Results: Thirteen strains belonging to Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus rhamnosus, Streptococcus thermophilus or Propionibacterium freudenreichii species were exposed to various copper concentrations in the proper growth medium at relevant growth temperatures, and the effects of supplemented copper on bacterial growth and cell viability were determined by optical density and pH measurements, also by platings. Among the species considered, L. delbrueckii was the most copper resistant and S. thermophilus the most sensitive to copper. Anaerobic conditions increased this sensitivity significantly. There was also a considerable amount of variation in copper resistance at strain level. Conclusions: Copper resistance is both a species- and strain-dependent property and may reflect variability in copper-binding capacities by cell wall components among species and strains. In addition, the chemical state of copper may be involved. Significance and Impact of the Study: This study revealed that copper resistance is a highly variable property among starter and adjunct strains, and this variability should be considered when strains are selected for Emmental cheese manufacture.     

Optimal culture conditions for producing conjugated linoleic acid in skim-milk by co-culture of different Lactobacillus strains

The ability of different Lactobacillus strains to produce conjugated linoleic acid (CLA) was determined. Three species—Lactobacillus plantarum (Lp), Lactobacillus acidophilus (La) and Streptococcus thermophilus (St)—were co-cultured in a medium containing skim-milk supplemented with hydrolyzed safflower oil. This study was aimed at future applications in dairy products. The optimal operation parameters were established by response surface methodology. More CLA was produced by co-culture than by single strain culture. The CLA produced by co-culture of La and Lp (La–Lp) was more than that produced by La and St (La–St). Maximum CLA production of 316.52 μg/mL was obtained with La–Lp co-culture using a substrate concentration of 5.0 %, inoculum size of 5.0 %, an initial medium pH of 6.4 and a temperature of 36.4 °C for 48 h. To our knowledge, this is the first report in the literature of the use of co-cultures of La–St and La–Lp to produce CLA.     

Microbiological,physical–chemical and sensory evaluation of a traditional Brazilian cheese during the ripening process

The ripening process of the traditional Serra do Salitre cheese, a Brazilian Minas cheese variety, was studied on five farms during 60 days. Faecal coliforms decreased and were not detected after ripening for this period. The contamination by coagulase-negative Staphylococcus was reduced at the end of the ripening period only on farms 3 and 4. Staphylococcus aureus was detected only in fresh cheese on farm 5 and decreased in number until it disappeared. No S. aureus was observed in the fresh cheeses from farms 2 and 4, but these were contaminated, probably by the hands of the manipulators, during the ripening process. The humidity decreased and the proteolysis increased during ripening. No correlation of the salt content and the humidity with the degree of proteolysis was observed. The degree of acceptance in the sensory evaluation was higher for cheese that had ripened for 1 and 15 days.     

Biodiversity of the Bacterial Flora on the Surface of a Smear Cheese

The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B. linens BL2) cheese were investigated. The results show that, contrary to accepted belief, B. linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates). In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species. C. mooreparkense and C. casei grew at pH values below 4.9 in the presence of 8% NaCl, while M. gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl. B. linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.