Soluble Lactobacillus delbrueckii subsp. bulgaricus 92059 PrtB proteinase derivatives for production of bioactive peptide hydrolysates from casein.

The proteinase-encoding prtB gene of Lactobacillus (Lb.) delbrueckii (d.) subsp. bulgaricus 92059 was cloned and sequenced. Two soluble, secreted, C-terminally His-tagged derivatives were constructed and expressed in Lactococcus lactis by means of the NICE® Expression System. In both obtained derivatives PrtBb and PrtB2, the C-terminal, cell wall-binding domain was deleted. In addition, in derivative PrtB2, the C-terminal part of the B domain was deleted and the signal sequence was replaced by a lactococcal export signal. The affinity-purified derivatives were both proteolytically active. Peptide hydrolysates produced from casein with each of the derivatives showed identical peptide composition, as determined by liquid chromatography-mass spectrometry. Comparison of the peptides generated to those generated with living Lb. d. subsp. bulgaricus 92059 cells (Kliche et al. Appl Microbiol Biotechnol 101:7621-7633, 2017) showed that ß-casein was the casein fraction most susceptible to hydrolysis and that some significant differences were observed between the products obtained by either the derivatives or living Lb. d. subsp. bulgaricus 92059 cells. When tested for biological activity, the hydrolysate obtained with PrtBb showed 50% inhibition of angiotensin-converting enzyme at a concentration of 0.5 mg/ml and immunomodulation/anti-inflammation in an in vitro assay of TNF-α induced NFκB activation at concentrations of 5 and 2.5 mg/ml, respectively. The enzymatically obtained hydrolysate did not show any pro-inflammatory or cytotoxic activity.

Screening for proteolytically active lactic acid bacteria and bioactivity of peptide hydrolysates obtained with selected strains.

In a screening for proteolytically active lactic acid bacteria, three strains, Lactobacillus delbrueckii ssp. lactis 92202, Lactobacillus helveticus 92201, and Lactobacillus delbrueckii ssp. bulgaricus 92059, showed the highest activities following growth in milk. All three strains degraded α- and ß-casein, but did not hydrolyse κ-casein. HPLC analysis of skim milk fermentation revealed increasing amounts of peptides after 5 and 10 h with Lb. d. ssp. bulgaricus 92059. Hydrolysates obtained with Lb. d. ssp. lactis 92202 and Lb. d. ssp. bulgaricus 92059 revealed the highest angiotensin-converting enzyme-inhibitory effect. The effect was dose dependent. Almost no effect (<10%) was seen for Lb. helveticus 92201. For Lb. d. ssp. bulgaricus 92059, maximal inhibition of approx. 65% was reached after 25 h of fermentation. In an in vitro assay measuring potential immunomodulation, hydrolysates of the three strains yielded anti-inflammatory activities in the presence of TNF-α. However, the effects were more pronounced at lower hydrolysate concentrations. In the absence of TNF-α, slight pro-inflammatory effects were observed. The hydrolysate of Lb. d. ssp. bulgaricus 92059, when purified by means of solid-phase extraction, exhibited pro-inflammatory activity. Sour whey containing Lb. d. ssp. bulgaricus 92059 cells showed pro-inflammatory activity while cell-free sour whey was clearly anti-inflammatory. In the purified hydrolysate, 20 different α- and ß-casein (CN)-derived peptides could be identified by LC-MS. Most peptides originated from the central and C-terminal regions of ß-casein. Peptide length was between 9 (ß-CN(f 59-67)) and 22 amino acids (ß-CN(f 117-138)).

Fate of Listeria innocua during production and ripening of smeared hard cheese made from raw milk.

The fate of 2 different Listeria innocua strains was analyzed during the production and ripening of smeared raw milk Greyerzer cheese (Gruyère). These strains were used as surrogates for the pathogenic Listeria monocytogenes, as they are physiologically very similar. Bacterial cells were added to the cheese milk at levels of 105 cfu/mL. During the first 24 h of cheese making, the number of the test strains decreased to a level of below 102 cfu/g. Obviously, the cooking temperature of 56°C and the subsequent slight temperature decrease to 50°C within 70 min contributed to a distinct reduction of Listeria counts. The counts in the cheese cores did not exceed 103 cfu/g within 12 wk of cheese ripening and Listeria was not detectable after 24 wk. In contrast to the cores of the cheeses of the 4 batches in this study, their rinds always contained a high listerial load of approximately 106 to 108 cfu/g throughout the entire ripening period. The smeared surface showed an increase of pH to alkaline values, corresponding to smear microbiota development. Coryneforms and Staphylococcus counts were stable at >107 cfu/cm2 over 175 d, whereas yeast counts decreased to about 105 cfu/cm2 at the end of ripening. The study shows that the smear culture had no noticeable anti-listerial potential. When removing the rind or portioning such smeared cheese loaves with a cutting device, a postprocess contamination of the core might occur, thus presenting a major hygienic risk.

Fate of Shiga toxin-producing and generic Escherichia coli during production and ripening of semihard raw milk cheese.

The fate of 5 different Escherichia coli strains, including 3 Shiga toxin-producing E. coli (STEC) strains, was analyzed during the production and ripening of semihard raw milk cheese. The strains, which were previously isolated from raw milk cheese, were spiked into raw milk before cheese production at 2 different levels (approximately 10(1) and 10(3) cfu/mL, respectively). Two cheese types were produced, which differed in cooking temperatures (40 and 46°C). The cheeses were sampled during manufacture and the 16-wk ripening period. An increase in E. coli counts of approximately 3.5 log(10) cfu/g occurred from raw milk to fresh cheese at d 1, which was attributed to a concentration effect during cheese production and growth of the strains. During ripening over 16 wk, a slow, continuous decrease was observed for all strains. However, significant differences were found between the E. coli strains at the applied spiking levels, whereas the inactivation was similar in the 2 different cheese types. The 2 generic E. coli strains survived at higher counts than did the 3 STEC strains. Nevertheless, only 1 of the 3 STEC strains showed significantly weaker survival at both spiking levels and in both cheese types. Six of 16 cheeses made from raw milk at a low spiking level contained more than 10 cfu/g of STEC at the end of the 16-wk ripening process. After enrichment, STEC were detected in almost all cheeses at both spiking levels. Particularly because of the low infectious dose of highly pathogenic STEC, even low colony counts in raw milk cheese are a matter of concern.

Preservation of functionality of Bifidobacterium animalis subsp. lactis INL1 after incorporation of freeze-dried cells into different food matrices.

The aim of this work was to investigate how production and freeze-drying conditions of Bifidobacterium animalis subsp. lactis INL1, a probiotic strain isolated from breast milk, affected its survival and resistance to simulated gastric digestion during storage in food matrices. The determination of the resistance of bifidobacteria to simulated gastric digestion was useful for unveiling differences in cell sensitivity to varying conditions during biomass production, freeze-drying and incorporation of the strain into food products. These findings show that bifidobacteria can become sensitive to technological variables (biomass production, freeze-drying and the food matrix) without this fact being evidenced by plate counts.

Identification and characterisation of lactobacilli isolated from Kimere, a spontaneously fermented pearl millet dough from Mbeere, Kenya (East Africa).

The objective of the study was to isolate potential probiotic lactobacilli from Kimere, a pearl millet dough prepared in the Mbeere community of Kenya, East Africa, by fermentation for 18-24 hours. Kimere samples, collected from 11 different homesteads in Mbeere, showed average pH values of 3.63±0.29. Counts of presumptive lactobacilli were 8.52±0.02 log10 colony forming units per gram, respectively. 48 presumptive Lactobacillus isolates were characterised and identified by biochemical and molecular methods. Lactobacillus fermentum (46 isolates) was the dominant Lactobacillus species detected. Analysis of strain diversity with pulsed-field gel electrophoresis indicated relatively large biodiversity among L. fermentum isolates. All L. fermentum isolates were able to grow in MRS medium containing 0.3% ox gall. Twelve of them were able to grow in the presence of 3% ox gall, and of these 60% survived incubation at pH 3 in the presence of 2 mg pepsin per ml for three hours.

Growth stimulation of Brevibacterium sp. by siderophores.

AIMS: To assess which types of siderophores are typically produced by Brevibacterium and how siderophore production and utilization traits are distributed within this genus. METHODS AND RESULTS: During co-cultivation experiments it was found that growth of B. linens Br5 was stimulated by B. linens NIZO B1410 by two orders of magnitude. The stimulation was caused by the production of hydroxamate siderophores by B. linens NIZO B1410 that enabled the siderophore-auxotrophic strain Br5 to grow faster under the applied iron-limited growth conditions. Different patterns of siderophore production and utilization were observed within the genus Brevibacterium. These patterns did not reflect the phylogenetic relations within the group as determined by partial 16S rDNA sequencing. Most Brevibacterium strains were found to utilize hydroxamate siderophores. CONCLUSIONS: Brevibacteria can produce and utilize siderophores although certain strains within this genus are siderophore-auxotrophic. SIGNIFICANCE AND IMPACT OF THE STUDY: It is reported for the first time that brevibacteria produce and utilize siderophores. This knowledge can be utilized to stimulate growth of auxotrophic strains under certain conditions. Enhancing the growth rate of Brevibacterium is of importance for the application of this species, for example, for cheese manufacturing or for industrial production of enzymes or metabolites.

Molecular identification and differentiation of Staphylococcus species and strains of cheese origin.

Amplified Ribosomal-DNA Restriction Analysis (ARDRA) was used to differentiate among 12 species and 4 subspecies of the genus Staphylococcus. With a universal primer pair a 2.4 kbp PCR-product was amplified, including the 16S rDNA, the 16S-23S rDNA interspacer region, and about 500 bp of the 23S rDNA. Species-specific restriction patterns were found using the restriction enzymes HindIII and XmnI separately. Cheese related staphylococci were clearly differentiated. ARDRA results were in good agreement with results of partial sequencing of the 16S rDNA. ARDRA could fully replace the biochemical identification with ID32 Staph (BioMerieux) which was less reliable when staphylococci of cheese origin were analysed. Genomic restriction digests of cheese-related S. equorum strains by SmaI and SacI gave unique strain-specific restriction patterns which can be used to identify starter staphylococci in a complex microbial environment such as the surface of Red-Smear cheeses.

Identification and differentiation of species and strains of Arthrobacter and Microbacterium barkeri isolated from smear cheeses with Amplified Ribosmal DNA Restriction Analysis (ARDRA) and pulsed field gel electrophoresis (PFGE).

ARDRA (Amplified Ribosomal-DNA Restriction Analysis) was used to differentiate among species and genera of Arthrobacter and Microbacteria. Species-specific restriction patterns of PCR-products were obtained with NciI for Arthrobacter citreus (DSM 20133T), A. sulfureus (DSM 20167T), A. globiformis (DSM 20124T) and A. nicotianae strains (DSM 20123T, MGE 10D, CA13, CA14, isolate 95293, 95294, and 95299), A. rhombi CCUG 38813T, and CCUG 38812, and Microbacterium barkeri strains (DSM 30123T, MGE 10D, CA12 and CA15, isolate 95292, and isolate 95207). All yellow pigmented coryneforme bacteria isolated from the smear of surface ripened cheeses were identified as either A. nicotianae or M. barkeri strains. Using pulsed field gel electrophoresis (PFGE) strain specific restriction pattern for all Arthrobacter species and Microbacteria tested were obtained with restriction enzymes AscI and SpeI.

Sporulation of Penicillium camemberti in submerged batch culture.

Sporulation of Penicillium camemberti was studied in submerged batch fermentation. A defined medium was used with glucose and ammonium as C- and N-sources. Temperature was set to 25 degrees C at pH 5.6. Essential for submerged sporulation was the presence of calcium (14 mM) which was adsorbed to the cell walls in all sporulating strains and inhibited mycelial growth. Acetate led to highly branched conidiophores and was the second main factor for efficient sporulation. The chelating properties of citrate were necessary for keeping calcium and phosphate in solution. Fermentation conditions allowed high spore yields after 96 h (1.6 x 10(8) spores/ml). Cyclopiazonic acid, the mycotoxin common for P. camemberti was produced during fermentation. The levels observed (0.5-4 ppm at 96 h) were strain specific and not related to spore yield.

Bioactive peptides encrypted in milk proteins: proteolytic activation and thropho-functional properties.

The bioactivities of peptides encrypted in major milk proteins are latent until released and activated by enzymatic proteolysis, e.g. during gastrointestinal digestion or food processing. The proteolytic system of lactic acid bacteria can contribute to the liberation of bioactive peptides. In vitro, the purified cell wall proteinase of Lactococcus lactis was shown to liberate oligopeptides from beta- and alpha-caseins which contain amino acid sequences present in casomorphins, casokinines, and immunopeptides. The further degradation of these peptides by endopeptidases and exopeptidases of lactic acid bacteria could lead to the liberation of bioactive peptides in fermented milk products. However, the sequences of practically all known biologically active peptides can also be cleaved by peptidases from lactic acid bacteria. Activated peptides are potential modulators of various regulatory processes in the body: Opioid peptides are opioid receptor ligands which can modulate absorption processes in the intestinal tract, angiotensin-I-converting enzyme (ACE)-inhibitory peptides are hemodynamic regulators and exert an antihypertensive effect, immunomodulating casein peptides stimulate the activities of cells of the immune system, antimicrobial peptides kill sensitive microorganisms, antithrombotic peptides inhibit aggregation of platelets and caseinophosphopeptides may function as carriers for different minerals, especially calcium. Bioactive peptides can interact with target sites at the luminal side of the intestinal tract. Furthermore, they can be absorbed and then reach peripheral organs. Food-derived bioactive peptides are claimed to be health enhancing components which can be used for functional food and pharmaceutical preparations.

Purification and characterization of the lytic activity induced by the prolate-headed bacteriophage P001 in Lactococcus lactis.

The lytic activity induced by the lactococcal bacteriophage P001 was isolated from phage lysates of Lactococcus lactis by a four-step purification procedure. Two proteins lytic for L. lactis were identified with molecular weights of 28 kDA and 8 kDa, respectively. The N-terminal amino acid sequences of the two proteins were determined and degenerated oligonucleotide probes corresponding to these sequences were synthesized. DNA hybridization experiments with phage P001-DNA and lactococcal DNA revealed that both proteins were apparently encoded by a single lysin gene located on the phage P001 genome. This was confirmed by alignment of the determined N-terminal amino acid sequences with nucleotide sequences which were deduced from cloned Lactococcus bacteriophage lysin genes.

Purification and Characterization of an Extracellular Proteinase from Brevibacterium linens ATCC 9174.

An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8.5 and 50(deg)C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg(sup2+) and Ca(sup2+) activated the proteinase, as did NaCl; however, Hg(sup2+), Fe(sup2+), and Zn(sup2+) caused strong inhibition. The sequence of the first 20 N-terminal amino acids was NH(inf2)-Ala-Lys-Asn-Asp-Ala-Val-Gly-Gly-Met-Gly-Tyr-Leu-Ser-Met-Ile-Pro-Se r-Gln-Pro-Gly.

[Optimal ocular correction for computer operators]. / Optymalna korekcja okularowa na komputerowym stanowisku pracy.

Different methods of correction of refractive errors with glasses were discussed in the paper. The problem of the use of antireflection coating and tinting or color coating of the lenses was also presented.

Effect of X-Prolyl Dipeptidyl Aminopeptidase Deficiency on Lactococcus lactis.

The genetic determinant (pepXP) of an X-prolyl dipeptidyl aminopeptidase (PepXP) has recently been cloned and sequenced from both Lactococcus lactis subsp. cremoris (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and L. lactis subsp. lactis (M. Nardi, M.-C. Chopin, A. Chopin, M.-M. Cals, and J.-C. Gripon, Appl. Environ. Microbiol. 57:45-50, 1991). To examine the possible role of the enzyme in the breakdown of caseins required for lactococci to grow in milk, integration vectors have been constructed and used to specifically inactivate the pepXP gene. After inactivation of the gene in L. lactis subsp. lactis MG1363, which is Lac and Prt, the Lac Prt determinants were transferred by conjugation by using L. lactis subsp. lactis 712 as the donor. Since growth of the transconjugants relative to the PepXP strains was not retarded in milk, it was concluded that PepXP is not essential for growth in that medium. It was also demonstrated that the open reading frame ORF1, upstream of pepXP, was not required for PepXP activity in L. lactis. A marked difference between metenkephalin degradation patterns was observed after incubation of this pentapeptide with cell extracts obtained from wild-type lactococci and pepXP mutants. Therefore, altered expression of the pepXP-encoded general dipeptidyl aminopeptidase activity may change the peptide composition of fermented milk products.

Molecular cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. cremoris.

Lactococcus lactis subsp. cremoris P8-2-47 contains an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). A mixed-oligonucleotide probe prepared on the basis of the N-terminal amino acid sequence of the purified protein was made and used to screen a partial chromosomal DNA bank in Escherichia coli. A partial XbaI fragment cloned in pUC18 specified X-PDAP activity in E. coli clones. The fragment was also able to confer X-PDAP activity on Bacillus subtilis. The fact that none of these organisms contain this enzymatic activity indicated that the structural gene for X-PDAP had been cloned. The cloned fragment fully restored X-PDAP activity in X-PDAP-deficient mutants of L. lactis. We have sequenced a 3.8-kb fragment that includes the X-PDAP gene and its expression signals. The X-PDAP gene, designated pepXP, comprises 2,289 nucleotide residues encoding a protein of 763 amino acids with a predicted molecular weight of 87,787. No homology was detected between pepXP and genes that had been previously sequenced. A second open reading frame, divergently transcribed, was present in the sequenced fragment; the function or relationship to pepXP of this open reading frame is unknown.

[Suggestions for the design of a modern anomaloscope]. / Konstruktionsvorschläge für ein modernes Anomaloskop.

Three variants of a new anomaloscope principle are described. This principle is distinguished by the fact that for generation of the mixed color and the reference color three interference filters with narrow band widths are used instead of an expensive dispersion prism. In the first variant the brightness of the mixed color field and the reference field is determined by three detectors (silicon diodes) and kept constant taking the relative spectral response into consideration. The degree of anomalous color vision present is indicated digitally by a microprocessor whose interface also permits data processing equipment to be connected. The second variant uses only one detector which sequentially pulses the three light sources and distributes the results to different signal channels for further processing. In the third variant, measurement is performed in the same way as in the second variant but with only one light source. Fiber optic bundles illuminate of the mixed color and reference fields and also permit adaptation of the eye to neutral. In this case additional optical attenuators are required to keep the luminance of the fields constant. An advantage shared by all three variants is that they have virtually no moving parts, employing monochromatic filters and cemented prism blocks with high-quality electronics. This has made it possible to produce a compact, rugged and efficient new-generation anomaloscope, which renders the considerable calibration and maintenance work previously necessary superfluous.

[A new procedure for keratometry (author's transl)]. / Ein neues Verfahren zur Keratometrie.

The principle of construction of a keratometer is explained which shows digitally the radii of curvature of the cornea obtained by automatic optical measurement and processed by an inbuilt computer.

[The new polaroid positive/negative film type 105 for ophthalmology (author's transl)]. / Der neue Polaroid-Positiv/Negativ-Film der Augenphotographie

Description of the new Polaroid positive/negative film type 105. This film produces within 30 seconds a brilliant positive and a negative for high quality prints. The new camera-back 405 for the use of 8.5 x 10.5 cm Polaroid packfilms fits to any 4 x 5 inch cameras with international back.