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)).

Prophage-mediated modulation of interaction of Streptococcus thermophilus J34 with human intestinal epithelial cells and its competition against human pathogens.

The human intestinal microbiota plays an important role in human health. While adhesion to gastrointestinal mucosa is a prerequisite for colonisation, inhibition of adhesion is a property which may prevent or reduce infections by food borne pathogens. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus represent the two lactic bacteria constituting the yoghurt culture. These starter cultures have been claimed to be probiotic. In our study we compared two S. thermophilus strains (i.e. lysogenic strain J34 and corresponding non-lysogenic [prophage-cured] strain J34-6), with respect to (1) their in vitro adhesion properties to HT29 cells and (2) their cell surface hydrophobicities. Effects of the two strains on inhibition of adhesion of the pathogens Listeria monocytogenes Scott A, Staphylococcus aureus 6732 and Salmonella enteritidis S489 were studied in vitro with HT29 cell cultures. Lysogenic strain J34 was shown to be considerably more effective than the non-lysogenic derivative strain J34-6.

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.

Characterisation of pSMA23, a 3.5 kbp plasmid of Lactobacillus casei, and application for heterologous expression in Lactobacillus.

The complete nucleotide sequence of plasmid pSMA23 isolated from Lactobacillus casei A23 was determined. Plasmid pSMA23 is a 3497bp circular molecule with a G+C content of 38.18%. Four putative open reading frames were identified. Based on homology, two orfs were identified as genes encoding replication initiation (Rep) and mobilisation (Mob) protein, respectively. Various regulatory regions like promoters, ribosome binding site (RBS), transcriptional terminators were deduced from the sequences of rep and mob. The origin of replication (dso) was predicted. Shuttle vectors pL142 and pL157 were constructed for Escherichia coli and Lactobacillus using rep gene and ori of pSMA23 for replication in Lactobacillus, the ori of the commercial vector pBluescript SkII+ for replication in E. coli, and the erythromycin and chloramphenicol resistance genes of pE194 and pC194, respectively, as selection markers. Transformants of E. coli and Lactobacillus were obtained on media containing erythromycin and chloramphenicol, respectively, suggesting expression of the ermC and cat194 genes in both organisms. The shsp gene of plasmid pSt04 of Streptococcus thermophilus encoding a small heat shock protein and the Lactobacillus plantarum cbh gene encoding conjugated bile salts hydrolase were cloned and successfully expressed in the heterologous host Lb. casei LK1 with the aid of pSMA23-derived vectors.

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.

Survival of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the terminal ileum of fistulated Göttingen minipigs.

The ability of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus administered in yogurt to survive the passage through the upper gastrointestinal tract was investigated with Göttingen minipigs that were fitted with ileum T-cannulas. After ingestion of yogurt containing viable microorganisms, ileostomy samples were collected nearly every hour beginning 3 h after food uptake. Living L. delbrueckii subsp. bulgaricus and S. thermophilus were detected in the magnitude of 10(6) to 10(7) per gram of intestinal contents (wet weight) in all animals under investigation. A calculation of the minimum amount of surviving bacteria that had been administered is presented. Total DNA extracted from ileostomy samples was subjected to PCR, which was species specific for L. delbrueckii and S. thermophilus and subspecies specific for L. delbrueckii subsp. bulgaricus. All three bacterial groups could be detected by PCR after yogurt uptake but not after uptake of a semisynthetic diet. One pig apparently had developed an endogenous L. delbrueckii flora. When heat-treated yogurt was administered, L. delbrueckii was detected in all animals. S. thermophilus or L. delbrueckii subsp. bulgaricus was not detected, indicating that heat-inactivated cells and their DNAs had already been digested and their own L. delbrueckii flora had been stimulated for growth.

Probiotic bacteria in fermented foods: product characteristics and starter organisms.

Probiotic bacteria are sold mainly in fermented foods, and dairy products play a predominant role as carriers of probiotics. These foods are well suited to promoting the positive health image of probiotics for several reasons: 1) fermented foods, and dairy products in particular, already have a positive health image; 2) consumers are familiar with the fact that fermented foods contain living microorganisms (bacteria); and 3) probiotics used as starter organisms combine the positive images of fermentation and probiotic cultures. When probiotics are added to fermented foods, several factors must be considered that may influence the ability of the probiotics to survive in the product and become active when entering the consumer's gastrointestinal tract. These factors include 1) the physiologic state of the probiotic organisms added (whether the cells are from the logarithmic or the stationary growth phase), 2) the physical conditions of product storage (eg, temperature), 3) the chemical composition of the product to which the probiotics are added (eg, acidity, available carbohydrate content, nitrogen sources, mineral content, water activity, and oxygen content), and 4) possible interactions of the probiotics with the starter cultures (eg, bacteriocin production, antagonism, and synergism). The interactions of probiotics with either the food matrix or the starter culture may be even more intensive when probiotics are used as a component of the starter culture. Some of these aspects are discussed in this article, with an emphasis on dairy products such as milk, yogurt, and cheese.

Safety considerations of DNA in food.

Recombinant DNA techniques are capable of introducing genetic changes into food organisms that are more predictable than those introduced through conventional breeding techniques. This review discusses whether the consumption of DNA in approved novel foods and novel food ingredients derived from genetically modified organisms (GMOs) can be regarded as being as safe as the consumption of DNA in existing foods. It concludes that DNA from GMOs is equivalent to DNA from existing food organisms that has always been consumed with human diets. Any risks associated with the consumption of DNA will remain, irrespective of its origin, because the body handles all DNA in the same way. The breakdown of DNA during food processing and passage through the gastrointestinal tract reduces the likelihood that intact genes capable of encoding foreign proteins will be transferred to gut microflora. The review does not specifically address food safety issues arising from the consumption of viable genetically modified microorganisms but it shows that the likelihood of transfer and functional integration of DNA from ingested food by gut microflora and/or human cells is minimal. Information reviewed does not indicate any safety concerns associated with the ingestion of DNA per se from GMOs resulting from the use of currently available recombinant DNA techniques in the food chain.

Identification of lactobacillus delbrueckii and subspecies by hybridization probes and PCR.

Three methods addressing two different target sites were compared for identification and differentiation of the subspecies Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis/delbrueckii. A PCR method - three primer pairs that enable direct identification of the species and the two subspecies, respectively - was derived from a DNA fragment showing significant similarities to parts of the addAB genes of Bacillus sutbtilis. In addition, two oligonucleotide probes for the two subspecies were designed from that DNA region. Further, two oligonucleotide probes targeting the 16S rDNA were developed for subspecies differentiation by a one base-pair difference following identification of the species. Moreover, these probes were demonstrated to be applicable for in situ hybridization experiments. The results obtained by the different methods were in good agreement.

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.

Bacillus subtilis develops competence for uptake of plasmid DNA when growing in milk products.

Transformation with plasmid DNA of naturally competent cells of Bacillus subtilis 168 in milk products was studied. Plasmid pMG36enpr, a broad host-range lactococcal vector carrying an erythromycin resistance and the B. subtilis npr gene encoding neutral protease, was taken up by B. subtilis cells grown in UHT chocolate milk. Under these conditions competence was optimal during transition from exponential to stationary growth phase, resulting in 9 x 10(1) transformants per 0.01 microgram DNA. No manipulation of the cells was necessary for competence to develop. When cells were pregrown in synthetic medium, higher transformation rates were obtained in assays, where the subsequent transformation experiments were either done in chocolate milk diluted 1:1 (v/v) with synthetic growth medium (up to 8 x 10(2) transformants) or in undiluted chocolate milk (1 x 10(2) transformants). The number of transformants was reduced to 4 x 10 (1), when diluted milk or flavored milks were used. No transformants were obtained in diluted yoghurt. Controls, in which both the preculturing and the transformation assays were done in synthetic medium, gave the maximum number of transformants (4 x 10(3) transformants per 0.01 microgram DNA).

Comparison of the lysogeny modules from the temperate Streptococcus thermophilus bacteriophages TP-J34 and Sfi21: implications for the modular theory of phage evolution.

A 7.6-kb DNA segment covering the putative lysogeny module of the pac-site-containing temperate Streptococcus thermophilus bacteriophage TP-J34 was sequenced. Sequence alignment with the lysogeny module from the cos-site-containing S. thermophilus bacteriophage phiSfi21 revealed areas of high sequence conservation (e.g., over the int gene), interspersed with regions of low or no sequence similarity (e.g., over the cro gene). Four of the six sharp transition zones from high to low sequence conservation were found within open reading frames coding for the CI repressor, the Anti-repressor, the Immunity protein, and a protein of unknown function. The transition points in the cI and ant genes appear to separate gene segments coding for distinct functional domains of these proteins. In addition, these two transition points were located at or near the deletion sites observed in spontaneous phage phiSfi21 deletion mutants, thus suggesting these transition points as recombinational hotspots. Furthermore, the sequence at the transition point in the cI gene resembles the attachment site of the phage, suggesting the involvement of the phage integrase in at least some of the exchange reactions. Contrary to the initial formulation of the modular theory of phage evolution the unit of the evolutionary exchange in streptococcal phages is not a group of functional genes, but can be as small as a single gene. Exchange reactions can also occur within genes, possibly between gene segments encoding distinct protein domains.

Identification of the receptor-binding regions of pb5 proteins of bacteriophages T5 and BF23.

The receptor-binding protein pb5(T5) of bacteriophage T5, when expressed from the oad gene cloned in pVK88 under the control of the phage T7 promoter/polymerase system, has been shown to bind to its FhuA receptor on the surface of E. coli, where it blocks FhuA for subsequent adsorption of T5 (Mondigler et al., FEMS Microbiol. Lett., 130, 293-300, 1995). In the present study the blocking assay has been applied to analyze the effects of several mutations within oad on the FhuA-binding properties of corresponding pb5 derivatives. Three classes of mutations were tested: (i) oad deletion derivatives, (ii) the oad mutation known to interfere with FhuA-binding of T5 (Heller and Bryniok, J. Virol., 49, 20-25, 1984), and (iii) linker-insertion mutations at a site very close to the oad mutation. Of the corresponding pb5 derivatives only one, a deletion derivative lacking the 153 C-terminal amino acids, was as active in the blocking assay as wild-type pb5(T5). All other derivatives were inactive or almost inactive. Isolation and molecular characterization of phenotypic revertants of T5oad showed that all revertants were true genotypic revertants of the oad mutation. The oad mutation has been identified as a G to T exchange resulting in a substitution of Gly for Trp at position 166 of pb5(T5). DNA sequencing of the hrs gene of bacteriophage BF23 and comparing the deduced amino acid sequence of pb5(BF23) with that of pb5(T5) revealed distinct regions of similarity and nonsimilarity. We propose that the receptor-binding region of pb5(T5) (pb5(BF23)) is formed by the region of nonsimilarity extending from amino acid position 89 (88) to position 305 (283).

Mutual inhibition of cobalamin and siderophore uptake systems suggests their competition for TonB function.

Vitamin B12 (CN-Cbl) and iron-siderophore complexes are transported into Escherichia coli in two energy-dependent steps. The first step is mediated by substrate-specific outer membrane transport proteins and the energy-coupling TonB protein complex, and the second step uses separate periplasmic permeases for transport across the cytoplasmic membrane. Genetic and biochemical evidence suggests that the TonB-dependent outer membrane transporters contact TonB directly, and thus they might compete for limiting amounts of functional TonB. The transport of iron-siderophore complexes, such as ferrichrome, causes a partial decrease in the rate of CN-Cbl transport. Although CN-Cbl uptake does not inhibit ferrichrome uptake in wild-type cells, in which the amount of the outer membrane ferrichrome transporter FhuA far exceeds that of the cobalamin transporter BtuB, CN-Cbl does inhibit ferrichrome uptake when BtuB is overexpressed from a multicopy plasmid. This inhibition by CN-Cbl is increased when the expression of FhuA and TonB is repressed by growth with excess iron and is eliminated when BtuB synthesis is repressed by CN-Cbl. The mutual inhibition of CN-Cbl and ferrichrome uptake is overcome by increased expression of TonB. Additional evidence for interaction of the Cbl and iron transport systems is provided by the strong stimulation of the BtuB- and TonB-dependent transport of CN-Cbl into a nonexchangeable, presumably cytoplasmic pool by preincubation of cells with the iron(II) chelator 2,2'-dipyridyl. Other metal ion chelators inhibited CN-Cbl uptake across the outer membrane. Although the effects of chelators are multiple and complex, they indicate competition or interaction among TonB-dependent transport systems.

Overproduced and purified receptor binding protein pb5 of bacteriophage T5 binds to the T5 receptor protein FhuA.

A promotor-less oad gene of bacteriophage T5, encoding the receptor binding protein pb5, was cloned into pT7-3 under the control of phage T7 promoter phi 10. Induction with IPTG resulted in enhanced production of pb5. Upon fractionation of the producing cells, most of the overproduced pb5 was found in the membrane fraction, which was most likely due to aggregation of the protein. The minor, soluble fraction of pb5 specifically inhibited adsorption of T5 to its FhuA receptor protein. Inhibition was also seen with trace amounts of pb5, and binding of pb5 to FhuA appeared to be almost irreversible. Purification of pb5 from the cytosolic fraction was performed by FPLC using a MonoQ column. pb5, which did not bind to the matrix of the column, was obtained in almost pure form. The purified protein also inhibited T5 adsorption.

Lytic conversion of Escherichia coli by bacteriophage T5: blocking of the FhuA receptor protein by a lipoprotein expressed early during infection.

The nucleotide sequence of the region between the oad gene, encoding the host specificity protein, and the right-terminal repetition of bacteriophage T5 DNA was determined. Five small open reading frames, the first of which was called llp, were detected, which apparently formed an operon transcribed from a promoter that overlapped the oad promoter. Both promoters were confirmed by primer extension assays. Using mRNA isolated at different times after T5 infection, the llp and oad promoters were identified as early and late promoters, respectively. The N-terminus of the llp gene product possess a signal sequence and a processing site characteristic of lipoproteins. After subcloning and expression of llp, its product Llp was identified as a 7.8 kDa polypeptide. Acylation of Llp was confirmed by addition of globomycin, which resulted in the accumulation of the unprocessed precursor form. FhuA+ cells synthesizing Llp were resistant to phage T5. Resistance was caused by inhibition of adsorption of T5 to its FhuA receptor protein. Resistance could be overcome by derepression of fhuA transcription, suggesting a blocking of FhuA by direct interaction with Llp. Since Llp-mediated T5 resistance has several aspects in common with the phenomenon of lysogenic conversion, we suggest that it should be called lytic conversion.

Molecular characterization of the Enterobacter aerogenes tonB gene: identification of a novel type of tonB box suppressor mutant.

The tonB gene of Enterobacter aerogenes was cloned, sequenced, and expressed in Escherichia coli. It complemented an E. coli tonB mutant as efficiently as E. coli tonB, except for colicin B and D sensitivities. However, colicin B and D sensitivities were complemented by a derivative in which the aspartate at position 165 was replaced by a glutamine (TonBD-165-->Q) by site-directed mutagenesis. In E. coli, the corresponding amino acid is a glutamine (Q-160) which is known to be altered in most mutants showing suppression of the btuB451 mutation. Fourteen independent btuB451 suppressor mutations in E. aerogenes tonB which all had suffered the same point mutation resulting in a change from glycine to valine at position 239 (G-239-->V) of the C-terminal end of the protein were isolated. The mutation was located within a region which is nonessential for function of E. aerogenes TonB as well as E. coli TonB. A constructed double mutation, expressing a D-165-->Q/G-239-->V derivative, no longer acted as a btuB451 suppressor. However, it restored colicin B and D sensitivities even more efficiently than the D-165-->Q derivative. Corresponding mutations constructed in E. coli tonB, giving rise to Q-160-->D, G-234-->V, and Q-160-->D/G-234-->V derivatives, showed phenotypes comparable to the E. aerogenes mutations. We take this as evidence that at least a functional interaction between the D-165 (Q-160 in E. coli) and the G-239 (G-234 in E. coli) region is necessary for TonB function. The implications of this interaction for functional instability of TonB are discussed.