Process for inhibiting the growth of a culture of lactic acid bacteria, and optionally lysing the bacterial cells, and uses of the resulting lysed culture

The invention provides a process for inhibiting the growth of a culture of lactic acid bacteria, or a product containing such culture e.g. a cheese product, in which in the cells of the lactic acid bacteria a holin obtainable from bacteriophages of Gram-positive bacteria, esp. from bacteriophages of lactic acid bacteria is produced in situ, the gene encoding said holin being under control of a first regulatable promoter, said holin being capable of exerting a bacteriostatic effect on the cells in which it is produced by means of a system, whereby the cell membrane is perforated, while preferably the natural production of autolysin is not impaired. It is preferable that additionally a lysin obtainable from lactic acid bacteria or their bacteriophages is produced in situ in the cells of the lactic acid bacteria, the gene encoding said lysin being under control of a second regulatable promoter, whereby the produced lysin effects lysis of the cells of the lactic acid bacteria. The second regulatable promoter can be the same as the first regulatable promoter and the genes encoding the holin and the lysin, respectively can be placed under the same regulatable promoter in one operon. Preferably the promoters are regulatable by the food-grade ingredients or parameters. Other uses of the invention include preparing a mixture of peptides which are modified by peptidases freed after the lysis, using the lysed culture as a bactericidal agent against spoiling bacteria or pathogenic bacteria for improving the shelf life of a product containing the lysed culture

Process for the lysis of a culture of lactic acid bacteria by means of a lysin, and uses of the resulting lysed culture

The invention provides a process for the lysis of a culture of lactic acid bacteria, or a product containing such culture e.g. cheese, by means of a lysin through the in situ production of a homologous autolysin, or a heterologous autolysin obtainable from Gram-positive bacteria esp. from lactic acid bacteria. The gene encoding said autolysin is controlled by a promoter, preferably regulated by food-grade ingredients or parameters, to achieve an enhanced lysis after induction resulting in an enhanced production of total autolysin compared with the natural production level of the homologous autolysin during fermentation or shortly thereafter. Other uses of the invention include preparing a mixture of peptides which are modified by peptidases freed after the lysis, using the autolysin as a bactericidal agent against spoiling bacteria or pathogenic bacteria for improving the shelf life of a product containing the lysed culture

Complex inducible promoter system derivable from a phage of a lactic acid bacterium (lab), and the use in a lab for production of a desired protein

The invention provides a complex inducible promoter system derivable from a phage of a lactic acid bacterium, especially one having the DNA sequence of SEQ. ID. NO: 3 given in Figure 2, or a DNA sequence essentially corresponding to those sequences, and a modification of (an essential part of) such promoter system in which the mitomycin C induction system is replaced by a food-grade system, e.g. a temperature-initiated induction system or a salt-initiated induction system. Also is provided a recombinant vector and a transformed lactic acid bacterium comprising (an essential part of) such promoter system. Further a process for producing a desired protein by such transformed bacterium is provided, comprising expressing a gene encoding said desired protein or a precursor thereof under control of such promoter system or an essential part thereof. Preferably the transformed lactic acid bacterium is made food-grade due to using food-grade DNA sequences and/or removing non-food-grade DNA sequences. When required, the desired protein can be secreted by the lactic acid bacterium if a DNA sequence fused to the gene encoding the desired protein is present which effects secretion of the desired protein or its precursor. The process can be used in a fermentation process, in which the desired protein causes lysis of the bacterial cells so that the contents of the cells can be released, or in which the desired protein is an enzyme involved in flavour formation, or in which the desired protein has a function in a cheese production process, such as chymosin or a precursor thereof, or an enzyme involved in flavour formation

Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme

The bacteriocin pediocin PA-1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA-1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N-terminal domains of PedD and a subgroup of bacteriocin ABC-transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin