Cremoris SK11 Research Articles

Effect of two thermoresistant non-starter lactic acid bacteria strains on volatilome profile during Cheddar ripening simulation

Dairy farm management practices can modify milk microbiota and therefore modulate non-starter lactic acid bacteria (NSLAB) found in cheese. These NSLAB can cause organoleptic defects. This study aimed to investigate the impact of two potential NSLAB in Cheddar cheesemaking: Lactiplantibacillus plantarum RKG 2-212 a strain isolated both in corn silage and raw milk, and Lactobacillus delbrueckii RKG R10, a strain isolated after pasteurisation of milk from a farm using grass and legume silage, and corn silage. The whole genome of these two lactobacilli was first sequenced. Then, the thermoresistance was evaluated after treatment at 60 °C for 5 min and compared to reference strains. Both lactobacilli were highly thermoresistant compared to other three lactic acid bacteria which are Lactococcus lactis subsp. cremoris ATCC 19257 and SK11, and L. plantarum ATCC 14917 (P < 0.0001). They lost less than 1 log cfu/mL (Δlog) and their genome contained a great number of copy number of genes coding for heat shock protein. During a Pearce test activity simulating Cheddar cheesemaking, the two lactobacilli did not show interaction with the starter Lcc. lactis subsp. cremoris SK11, and their population remained stable. During a ripening simulation, L. delbrueckii RKG R10 had a slight loss in viability in cheese slurry samples incubated at 30 °C for 12 d. However, L. plantarum RKG 2–212 had considerable growth, from 6.51 to 8.3 log cfu/g. This growth was associated with the acidification of the slurries (P < 0.0001). The presence of the lactobacilli modified the profile of volatile compounds evaluated by gas chromatography–mass spectrometry, accounting for 10.7% of the variation. The strain L. plantarum RKG 2-212 produced volatile compounds in greater quantity that could be associated with organoleptic defects such as acetic acid and 2-methylbutyraldehyde. Therefore, silage can be a vector of thermoresistant lactic acid bacteria for milk which can lead to flavor defects in cheese.

Transcription profiling of interactions between Lactococcus lactis subsp. cremoris SK11 and Lactobacillus paracasei ATCC 334 during Cheddar cheese simulation

The starter cultures (Lactococcus sp.) and non-starter lactic acid bacteria (mostly Lactobacillus spp.) are essential to flavor development of Cheddar cheese. The aim of this study was to elucidate the transcriptional interaction between Lactococcus lactis subsp. cremoris SK11 and Lactobacillus paracasei ATCC 334 in mixed cultures during simulated Cheddar cheese manufacture (Pearce activity test) and ripening (slurry). Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of 34 genes common to both bacteria and for eight genes specific to either L. lactis subsp. cremoris SK11 or L. paracasei ATCC 334. The multifactorial analysis (MFA) performed on fold change results for each gene revealed that the genes linked to stress, protein and peptide degradation as well as carbohydrate metabolism of L. paracasei ATCC 334 were especially overexpressed in mixed culture with L. lactis subsp. cremoris SK11 during the ripening simulation. For L. lactis subsp. cremoris SK11, genes coding for amino acid metabolism were more expressed during the cheese manufacture simulation, especially in single culture. These results show how complementary functions of starter and NSLAB contribute to activities useful for flavor development.

Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712

The widely used plasmid-free Lactococcus lactis strain MG1363 was derived from the industrial dairy starter strain NCDO712. This strain carries a 55.39 kb plasmid encoding genes for lactose catabolism and a serine proteinase involved in casein degradation. We report the DNA sequencing and annotation of pLP712, which revealed additional metabolic genes, including peptidase F, d-lactate dehydrogenase and α-keto acid dehydrogenase (E3 complex). Comparison of pLP712 with other large lactococcal lactose and/or proteinase plasmids from L. lactis subsp. cremoris SK11 (pSK11L, pSK11P) and the plant strain L. lactis NCDO1867 (pGdh442) revealed their close relationship. The plasmid appears to have evolved through a series of genetic events as a composite of pGdh442, pSK11L and pSK11P. We describe in detail a scenario by which the metabolic genes relevant to the growth of its host in a milk environment have been unified on one replicon, reflecting the evolution of L. lactis as it changed its biological niche from plants to dairy environments. The extensive structural instability of pLP712 allows easy isolation of derivative plasmids lacking genes for casein degradation and/or lactose catabolism. Plasmid pLP712 is transferable by transduction and conjugation, and both of these processes result in significant molecular rearrangements. We report the detailed molecular analysis of insertion sequence element-mediated genetic rearrangements within pLP712 and several different mechanisms, including homologous recombination and adjacent deletion. Analysis of the integration of the lactose operon into the chromosome highlights the fluidity of the MG1363 integration hotspot and the potential for frequent movement of genes between plasmids and chromosomes in Lactococcus.

Effect of single strains of Lactococci on manufacture and chemical quality of fresh Beyaz peynir, Turkish white-brined cheese

Beyaz peynir, Turkish white-brined cheese, was produced with six different strains of Lactococcus and ripened at 6–8 °C. Two of these were Lc. lactis subsp. lactis UC317 and NCDO763, while four of them were Lc. lactis subsp. cremoris SK11, HP, Wg2 and AM2. To determine their performance in manufacture of Beyaz cheesemaking, the activity of six different strains of Lactococcus were monitored during cheese production by determining the curd acidity. Also, their proteolytic and lipolytic activities were tested by determining nitrogen fractions and free fatty acid contents, respectively, after 3 days of ripening. These cheeses made with each strain of Lactococcus did not differ significantly in pH decline during cheesemaking. The gross compositions of the fresh cheeses were found to be similar; however, the cheeses made with HP and Wg2 had the lowest and highest levels of salt-in-moisture content, respectively. Water-soluble nitrogen, 12% trichloroacetic acid soluble nitrogen, total free amino acid and total ...

Effect of probiotic bacteria on prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses in vitro.

The proliferation of yeasts in the mixed bacterial and fungal biofilms colonising silicone rubber voice prostheses in laryngectomised patients is the main cause of malfunctioning of the valve mechanism on the oesophageal side of the prostheses. Indwelling voice prostheses usually have to be replaced every 3-4 months. The consumption of probiotic bacteria is largely motivated by health claims related to the urogenital and lower digestive tract, but not to the upper digestive tract. The present study examined the influence of probiotic bacteria on the prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses, as formed in a modified Robbins device. Exposure of oropharyngeal biofilms on voice prostheses to suspensions of Bifidobacterium infantis 420 or Enterococcus faecium 603 did not significantly reduce the number of yeasts in the biofilm. However, suspensions of Lactobacillus fermentum B54, L. rhamnosus 744 or L. lactis cremoris SK11 led to a reduction in the number of yeasts harvested from the voice prostheses. Suspensions of L. casei Shirota and Streptococcus thermophilus B significantly reduced the number of yeasts in the biofilm to 39% and 33%, respectively. The reduction brought about in yeast prevalence in the mixed biofilm was greatest by exposure to a suspension of L. lactis 53, with yeast prevalence only 4% of the control. In conclusion, the study demonstrated that the prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses might be controlled by consumption of probiotic bacteria.

Structural changes and interactions involved in the Ca(2+)-triggered stabilization of the cell-bound cell envelope proteinase in Lactococcus lactis subsp. cremoris SK11.

The cell-bound cell envelope proteinase (CEP) of the mesophilic cheese-starter organism Lactococcus lactis subsp. cremoris SK11 is protected from rapid thermal inactivation at 25 degrees C by calcium bound to weak binding sites. The interactions with calcium are believed to trigger reversible structural rearrangements which are coupled with changes in specific activity (F. A. Exterkate and A. C. Alting, Appl. Env. Microbiol. 65:1390-1396, 1999). In order to determine the significance of the rearrangements for CEP stability and the nature of the interactions involved, the effects of the net charge present on the enzyme and of different neutral salts were studied with the stable Ca-loaded CEP, the unstable so-called "Ca-free" CEP and with the Ca-free CEP which was stabilized nonspecifically and essentially in its native conformation by the nonionic additive sucrose. The results suggest that strengthening of hydrophobic interactions is conducive to stabilization of the Ca-free CEP. On the other hand, a hydrophobic effect contributes significantly to the stability of the Ca-loaded CEP; a phased salting-in effect by a chaotropic salt suggests a complex inactivation process of this enzyme due to weakening of hydrophobic interactions and involving an intermediate enzyme species. Moreover, a Ca-triggered increase of a relatively significant hydrophobic effect in the sucrose-stabilized Ca-free CEP occurs. It is suggested that in the Ca-free CEP the absence of both local calcium-mediated backbone rigidification and neutralization of negative electrostatic potentials in the weak Ca-binding sites, and in addition the lack of significant hydrophobic stabilization, increase the relative effectiveness of electrostatic repulsive forces on the protein to an extent that causes the observed instability. The conditions in cheese seem to confer stability upon the cell-bound enzyme; its possible involvement in proteolysis throughout the ripening period is discussed.

Flavor enhancement of reduced fat cheddar cheese using an integrated culturing system.

Mild cheese flavor in reduced fat Cheddar cheese was enhanced by using an integrated starter culture system. Three cultures, Lactococcus lactis subsp. cremoris SK11, L. lactis subsp. lactis biovar. diacetylactis JVI, and Lactobacillus casei 7A, were carefully selected to obtain a nonbitter, mildly acid, buttery flavored cheese. Cheeses were produced from all possible combinations of these cultures with the constraint that L. lactis subsp. cremoris SK11 was used as the primary acid-producing culture. Cheeses made with SK11 were compared to cheeses produced using an L. lactis subsp. cremoris commercial starter culture. Cheeses were ripened for 150 days and periodically sampled for chemical, microbiological, and sensory analysis. Cheeses produced with L. lactis subsp. cremorisSK11 had substantially lower bitterness intensity than the cheeses produced with commercial starter culture. L. lactis subsp. lactis biovar. diacetylactis JVI significantly increased diacetylacetoin and acetate concentrations. Sensory results indicate that these cheeses had increased buttery (diacetyl) flavor.

Production of cheddar cheese using a Lactococcus lactis ssp. Cremoris SK11 derivative with enhanced aminopeptidase activity

A Lactobacillus helveticus CNRZ32 DNA fragment encoding aminopeptidase N (PepN) was cloned into pIL253 to construct pSUW34. The cell-free extract general aminopeptidase activity of Lactococcus lactis ssp. cremoris SK11 transformed with the pSUW34 construct was increased approximately 100-fold relative to L. lactis ssp. cremoris SK11 wild type and pIL253 transformants. Cheddar cheese was manufactured with L. lactis ssp. cremoris HP in combination with either SK11(pIL253) or SK11(pSUW34). A 99% reduction in viable SK11 derivatives occurred within 2 weeks of ripening. The SK11(pSUW34)/HP cheese had approximately 1000-fold greater aminopeptidase activity and elevated levels of TCA and PTA soluble nitrogen. The increased accumulation of particular free amino acids in the SK11(pSUW34)/HP cheese varied between 15% (methionine) and 230% (lysine). Both the SK11(pSUW34)/HP and SK11(pIL253)/HP cheeses were non-bitter and had no significant sensory differences. Thus, the increased free amino acid pool in the SK11(pSUW34)/HP cheese did not result in accelerated flavor development.

Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonidentity between plasmid and chromosomal copies of ISS 1-like sequences in Lactococcus lactis subsp. lactis CNRZ270 and their possible role in chromosomal integration of plasmid genes †

The nucleotide sequence of an insertion sequence (IS) observed during mating experiments using the lactose-protease plasmid, pUCL22, of Lactococcus (Lc.) lactis subsp. lactis CNRZ270, was found to be similar to that of ISS 1 from Lc. lactis subsp. lactis ML3. The IS was named ISS 1RS. The chromosome of this strain contains several copies of ISS 1-like IS as assessed by hybridization. One of these copies was cloned and named ISS 1CH. Its sequence differs from that of the plasmid-borne copy, and appears to be more closely related to ISS 1N from Lc. lactis subsp. cremoris SK11. This suggests independent introduction of both ISS 1 elements. Moreover, the observation of plasmid genes integrated in the CNRZ270 chromosome near ISS 1CH suggests that their presence is the result of integration by a Campbell mechanism using both IS homologies. ISS 1-like sequences were also found on plasmids of numerous Lc. lactis strains, as well as one out of seven Lactobacillus (Lb.) casei and one out of three Lb. plantarum strains examined.

Replication and temperature-sensitive maintenance functions of lactose plasmid pSK11L from Lactococcus lactis subsp. cremoris

The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. cremoris) SK11, was isolated on a 14.8-kbp PvuII fragment by shotgun cloning into an Escherichia coli vector encoding erythromycin resistance and selection for erythromycin-resistant transformants of L. lactis subsp. lactis (L. lactis) LM0230. Deletion analysis and Tn5 mutagenesis of the resulting plasmid (pKMP1) further localized the replication region to a 2.3-kbp ScaI-SpeI fragment. DNA sequence analysis of this 2.3-kbp fragment revealed a 1,155-bp open reading frame encoding the putative replication protein, Rep. The replication origin was located upstream of rep and consisted of an 11-bp imperfect direct repeat and a 22-bp sequence tandemly repeated three and one-half times. The overall organization of the pSK11L replicon was remarkably similar to that of pCI305, suggesting that pSK11L does not replicate by the rolling-circle mechanism. Like pSK11L, pKMP1 was unstable in L. lactis LM0230. Deletion analysis allowed identification of several regions which appeared to contribute to the maintenance of pKMP1 in L. lactis LM0230. pKMP1 was significantly more stable in L. cremoris EB5 than in L. lactis LM0230 at all of the temperatures compared. This stability was lost by deletion of a 3.1-kbp PvuII-XbaI fragment which had no effect on stability in L. lactis LM0230. Other regions affecting stability in L. cremoris EB5 but not in L. lactis LM0230 were also identified. Stability assays conducted at various temperatures showed that pKMP1 maintenance was temperature sensitive in both L. lactis LM0230 and L. cremoris EB5, although the plasmid was more unstable in L. lactis LM0230. The region responsible for the temperature sensitivity phenotype in L. lactis LM0230 was tentatively localized to a 1.2-kbp ClaI-HindIII fragment which was distinct from the replication region of pSK11L. Our results suggest that the closely related L. lactis and L. cremoris subspecies behave differently regarding maintenance of plasmids.

Cloning and expression of the Lactococcus lactis subsp. Cremoris SK11 gene encoding an extracellular serine proteinase

The Lactococcus lactis subsp. cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades αs 1-, β- and κ-casein, was identified in a λEMBL3 gene library in Escherichia coli using immunological methods. The complete prtp gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtp gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L. lactis subsp. lactis 712 and L. lactis subsp. cremoris SK11, respectively. The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtp gene was identical to that encoded by the wt proteinase plasmid, pSK111. The expression of recombinant plasmids containing 3′ and 5′ deletions of prtp was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtp were localized and overproduction was obtained in L. lactis subsp. lactis. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtp was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade αs 1-, β- and κ-casein.

Growth and Activity of Mesophilic Lactic Acid Streptococci in Ultrafiltered Skim Milk and in Reconstituted Nonfat Dry Milk of Differing Total Solids Contents

The activity of mesophilic starter cultures Streptococcus lactis C2, Streptococcus cremoris SK11, and Streptococcus diacetylactis DRC3 in 8.3 and 15% total solids reconstituted skim milk was compared with growth of these organisms in 8.9% total solids skim milk (unconcentrated control) and in UF concentrated skim milk of 12 and 13% total solids. The starter cultures were inoculated into the different milk samples and incubated at 30°C for 15 to 16h. Samples were taken periodically for determination of pH, percentage titratable acidity, and cell populations. Cultures grown in UF concentrated milk consistently exhibited higher cell numbers and developed greater acidity than when grown in reconstituted skim milk of similar solids content. Streptococcus cremoris SK11 was the most active culture in acid development in the various media. Growth was not related to solids level or milk type, that is, retentate or reconstituted skim milk. A model for predicting developed acidity from solids content and from type of milk was determined by multiple regression analysis. The results suggest that UF concentrated skim milk is superior to reconstituted skim milk as a substrate for lactic acid bacteria and that skim milk retentates have potential for use as media for starter cultures.

Whey Permeate as a Medium for Mesophilic Lactic Acid Streptococci

Streptococcus lactis C8 was inoculated into whey permeate, reconstituted skim milk permeate, two times (vol/vol) concentrated ultrafiltered whey, reconstituted skim milk, and reconstituted whey to compare growth and acid production. Permeate from reconstituted whey with .1% added yeast extract (BBL) was used as a growth medium for three different Streptococcus lactis ssp. diacetylactis strains and three Streptococcus cremoris strains. Acid development, generation time, and growth were determined for each culture in this medium during the 18-h incubation at 30°C.Streptococcus lactis C8 and Streptococcus cremoris SK11 were each propagated in permeate from reconstituted whey (3.5% total solids) and in RSM (9.0% total solids) over 7 d. Growth and acid production by the organisms in the two media were compared. All of the media in which S. lactis C8 was grown supported growth of the culture. Variations in acid development were observed among the different types of media and appeared to be related to the levels of solids and growth factors present. Permeate from reconstituted whey with .1% yeast extract added supported varying degrees of growth and activity by the six mesophilic lactic streptococci cultures that were inoculated into this medium. The results of propagating S. cremoris SK11 and S. lactis C8 in permeate from reconstituted whey and in RSM showed that growth increases by each culture were similar in the two types of media. The pH remained higher in the permeate than in RSM. The results of this study indicate that whey permeate may be suitable as a growth medium for mesophilic lactic acid streptococci.

A maturation protein is essential for production of active forms of Lactococcus lactis SK11 serine proteinase located in or secreted from the cell envelope

The complete nucleotide sequence of a gene located immediately upstream of the Lactococcus lactis subsp. cremoris SK11 prtP gene encoding the cell envelope-attached proteinase was determined. This gene, designated prtM, was found to be transcribed from the same promotor region as was the proteinase gene but in the opposite direction. The prtM gene directed the expression in Escherichia coli of a protein with a size similar to the expected value of 33 kilodaltons, as deduced from the nucleotide sequence data. The derived amino acid sequence of the PrtM protein indicated the presence of a consensus lipoprotein signal sequence at the N terminus, which suggested that PrtM is a lipoprotein. Plasmids containing the prtM gene, the prtP gene, or both were constructed. Expression studies of L. lactis clones containing these plasmids showed that the prtM gene encodes a trans-acting activity involved in the maturation of cell envelope-located and -secreted forms of the SK11 proteinase.

Thermoinducible Lysis of Temperature-Sensitive Streptococcus cremoris Strains

Temperature-sensitive Streptococcus cremoris SK11 appeared to harbor a temperate phage that was induced at the maximum cooking temperatures used in Cheddar cheese manufacture, i.e., 38 to 40°C. When incubated at 30°C in M17-lactose broth, followed by a shift to 40°C, lysis of the culture occurred within 2h. Results were similar when S. cremoris SK11 was propagated in M17-lactose broth through a simultated Pearce activity test. In 11% solids reconstituted NDM, this strain also exhibited decreased cell numbers approximately 2h after the temperature had reached 40°C in the Pearce activity test. To support the hypothesis that lysis of the cells was due to induction of phage, electron microscopy of the temperature-induced lysate revealed the presence of particles resembling phage heads and tails but only a few intact phage-like particles. Examination of other temperature-sensitive or temperature-insensitive strains of S. cremoris also suggested that lysis of temperature-sensitive strains occurred after cells were incubated at 40°C, but no lysis was observed for temperature-insensitive strains.

Plasmid encoded bacteriophage resistance inStreptococcus cremorisSK11

In Streptococcus cremoris SK11, different permutations of a total of 8 plasmids were observed within and between cultures of various origins. All showed similar growth rates in milk. Those variants which carried a 34-MDa plasmid, pSK112, were resistant to bacteriophage oSK11G, whereas those from which the plasmid was absent or had been cured were sensitive to this phage. Plasmid pSK112 was shown to confer resistance by reduced phage adsorption. These observations have important potential for the development of phage-resistant dairy cultures.

Lysogenic Strains of Group N Lactic Streptococci

A temperate bacteriophage, designated r(1)t, was inducible from the group N lactic streptococcus, Streptococcus cremoris R(1), by ultraviolet irradiation or mitomycin C treatment. Induced lysates produced plaques on lawns of three closely related S. cremoris strains, AM(1), SK(11), and US(3). Strain SK(11) was readily lysogenized. S. cremoris AM(1) was the most reliable indicator strain, although the age of the culture used for seeding plates was critical. Zones of lysis but no plaque formation were observed on lawns of nine additional S. cremoris strains. Phage r(1)t could not be detected in filtrates of stationary-phase R(1) cultures and was near the limits of detection in logarithmically growing cultures. Phage levels were still very low (1 plaque-forming unit on AM(1) per 10 induced cells) in induced lysates of R(1) cultures. These low levels of detectable phage may be attributable to an inadequate indicator, lysogenization of the indicator, adsorption of induced phage to cellular debris, concurrent induction of other undetectable phages, or the production of high proportions of defective phages. Electron micrographs of induced R(1) lysates revealed a high incidence of incomplete phage particles, fragments, and ghosts.